A MOLECULAR PHYLOGENY OF THE SPARIDAE (PERCIFORMES

Transcription

A MOLECULAR PHYLOGENY OF THE SPARIDAE (PERCIFORMES: PERCOIDEI)
_____________
A Dissertation
Presented to
The Faculty of the School of Marine Science
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Doctor of Philosophy
____________
Copyright
Thomas Martin Orrell
2000
APPROVAL SHEET
This dissertation is submitted in partial fulfilment of
the requirements for the degree of
Doctor of Philosophy
___________________________
Thomas M. Orrell
Approved 9 November 2000
___________________________
John A. Musick, Ph.D.
Committee Co-Chairman/Advisor
___________________________
John E. Graves, Ph.D.
Committee Co-Chairman/Advisor
___________________________
Kimberly S. Reece, Ph.D.
___________________________
Peter A. Van Veld, Ph.D.
___________________________
Kent E. Carpenter, Ph.D.
Department of Biology
Old Dominion University, Norfolk, VA
___________________________
G. David Johnson, Ph.D.
Division of Fishes, National Museum of Natural History
Smithsonian Institution, Washington, D.C.
DEDICATION
I am dedicating this dissertation to the memory of Emo. J. Benvenuti, my grandfather,
who died on 13 March 1998. He was a quiet teacher who had all the patience in the
world.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
CHAPTER ONE. A MOLECULAR PHYLOGENY OF THE FAMILY
SPARIDAE (PERCOIDEI: PERCIFORMES) INFERRED FROM THE
MITOCHONDRIAL CYTOCHROME B GENE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
CHAPTER TWO. A MOLECULAR PHYLOGENY OF THE FAMILY
MITOCHONDRIAL 16S RIBOSOMAL RNA GENE . . . . . . . . . . . . . . . . . . . . . . . . . 126
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
CHAPTER THREE. COMBINED ANALYSIS OF
CYTOCHROME B AND 16S MITOCHONDRIAL SEQUENCES . . . . . . . . . . . . . . 195
iii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
CHAPTER FOUR. RECONSTRUCTING SPARID RELATIONSHIPS WITH A
SINGLE-COPY NUCLEAR DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
VITA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
iv
ACKNOWLEDGMENTS
I would like to thank my dissertation committee for their constant support
throughout the duration of this study. I am especially indebted to my co-major advisors
Drs. Jack Musick and John Graves for giving me encouragement, advice, guidance and
funding during my time at VIMS. Dr. Kimberly Reece provided invaluable advice in the
laboratory and was an unending source of molecular knowledge. I am very grateful to my
outside committee members. Dr. Kent Carpenter inspired this study by suggesting that
we work together on a phylogeny of the Sparidae. He generously collected the majority
of the samples used in this study and afforded me numerous opportunities to collect
samples as well. He graciously provided hours of advice for the completion of this
project and his critical review of this dissertation contributed countless suggestions for
improvement Dr. G. David Johnson furnished the framework of many of the
relationships surveyed in this study. His encouragement and critical eye kept me honest.
Dr. Peter Van Veld critically reviewed this dissertation and gave me many helpful
suggestions over the years.
I would like to thank the following individuals and organizations for their
assistance in collecting specimens, without whose help this work would not have been
possible: S. Almatar, L. Beckley, B.B. Collette, F. Crock, N. DeAngelis, M. DeGravelle,
D. Etnier, H. Ishihara, J. Gelsleichter, A. Graham, R. Grubbs, K. Harada, Y. Iwatsuki, J.
Jenke, R. Kraus, E. Massuti, K. Matsuura, L. Ter Morshuizen, P. Oliver, A.W. Paterson,
J. Paxton, J. Scialdone, D. Scherrer, G. Sedberry, M. Smale, W. F. Smith-Vaniz, K.
Utsugi, G. Yearsley, T. Wasaff, J.T. Williams, and the VIMS Trawl Survey. I would like
v
to acknowledge the following people for laboratory assistance: J. McDowell, D. Carlini,
V. Buonaccorsi, C. Morrison and K. Macdonald and all members of the VIMS fisheries
genetics laboratory during my time at VIMS. I would also like to thank E.O. Wiley for
numerous suggestions during this study. This research was supported by grants from: the
Food and Agriculture Organization of the United Nations, Lerner Gray Fund for Marine
Research from the American Museum of Natural History, an E.C. and Charlotte E. Raney
Award of the American Society of Ichthyologists and Herpetologists, and by the VIMS
Dean of Graduate Studies.
vi
LIST OF TABLES
Table
Page
1.
Annotated list of the valid genera of the Sparidae . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.
Subfamilies of the Sparidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.
Collection data for specimens used in cytochrome b study . . . . . . . . . . . . . . . . . 46
4.
Multiple alignment of nucleotide sequences of the cytochrome b gene . . . . . . . 48
5.
Cytochrome b nucleotide sequence characteristics . . . . . . . . . . . . . . . . . . . . . . . 72
6.
Pairwise values of mean % sequence divergence derived from uncorrected “p”
genetic distance for all taxa, ingroup taxa and outgroup taxa . . . . . . . . . . . . . . . 73
7.
Pairwise values of mean % sequence divergence derived from uncorrected “p”
genetic distance within and between subfamilies . . . . . . . . . . . . . . . . . . . . . . . . 74
8.
Base compositional bias calculated across all, ingroup, and outgroup taxa
9.
Multiple alignment of amino acid residues translated from the nucleotide
. . . 75
sequences of the cytochrome b gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
10.
FAO area assignments for the Sparidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
11.
Matrix of FAO areas and characters used during analysis of vicariance
biogeography - cytochrome b data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
12.
Collection data for specimens used in 16S study . . . . . . . . . . . . . . . . . . . . . . . . 149
13.
Multiple alignment of nucleotide sequences of the partial 16S gene from the
Sparidae and outgroup species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
14.
Constant, uninformative and informative characters for stems and loops . . . . . 167
15.
Non-conserved areas in multiple alignment of 16S gene . . . . . . . . . . . . . . . . . 167
16.
Mean pairwise values of percent sequence divergence based on uncorrected “p”
vii
genetic distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
17.
Contribution of loops and stems to the overall sequence divergence . . . . . . . . 169
18.
Matrix of FAO areas and characters used during analysis of vicariance
biogeography - 16S data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
19.
Incongruence Length Distance values between different data partitions . . . . . . 216
20.
Matrix of FAO areas and characters from dependent cytochrome b (Top) and16s
(bottom) trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
21.
Collection data for specimens used in Tmo-4c4 study . . . . . . . . . . . . . . . . . . . 241
22.
Primers and Sequences used to amplify the Tmo-4c4 locus. . . . . . . . . . . . . . . 242
23.
Tmo-4c4 clone numbers sequenced for each taxon those samples direct sequenced
are indicated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
24.
Multiple alignments of nucleotide sequences of the nuclear Tmo-4c4 locus . . 244
viii
LIST OF FIGURES
Figure
Page
1.
Suggested phylogeny of spariform relationships redrawn from Akazaki (1962) . 10
2.
Hypothesized relationships of the Sparoidea (Sparidae, Centracanthidae,
Lethrinidae and Nemipteridae) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.
Polymerase chain reaction primers used in the cytochrome b study . . . . . . . . . . 88
4.
Total substitutions at all codon positions plotted as a function of pairwise percent
sequence divergence for ingroup taxa only - cytochrome b gene . . . . . . . . . . . . 90
5.
Total substitutions at the first codon position plotted as a function of pairwise
percent sequence divergence for ingroup taxa only . . . . . . . . . . . . . . . . . . . . . . . 92
6.
Total substitutions at the second codon position plotted as a function of pairwise
7.
Total substitutions at the third codon position plotted as a function of pairwise
8.
Total substitutions at the third codon position and pooled first and second codon
positions plotted as a function of pairwise percent sequence divergence for
ingroup taxa only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
9.
A ratio of transitions/total substitutions and transversions/total substitutions from
third codon plotted as a function of pairwise percent sequence divergence for
ingroup taxa only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
10.
Transitional substitutions, separated into nitrogenous base type, plotted as a
functions of pairwise percent sequence divergence of ingroup taxa only . . . . . 102
11.
Frequencies of each of four bases of the cytochrome b gene for 62 taxa at all
ix
codon positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
12.
Frequencies of each of four bases of the cytochrome b gene for 62 taxa at the first
codon position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
13.
Frequencies of each of four bases of the cytochrome b gene for 62 taxa at the
second codon position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
14.
Frequencies of each of four bases of the cytochrome b gene for 62 taxa at the third
codon position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
15.
Four equally parsimonious trees - unweighted data . . . . . . . . . . . . . . . . . . . . . . 112
16.
Strict consensus of four equally parsimonious trees - unweighted data . . . . . . 114
17.
Two equally parsimonious trees - weighted data . . . . . . . . . . . . . . . . . . . . . . . . 116
18.
A strict consensus of two equally parsimonious trees from the - weighted data
(transversions only third codon) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
19.
A strict consensus of 699 equally parsimonious trees from amino acid residue
translations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
20.
Numbered ancestral nodes of the Sparidae clade from the weighted cytochrome b
consensus tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
21.
Clade of biogeographic relationships overlaid on map of the world . . . . . . . . . 124
22.
Total number of substitutions plotted as a functions of uncorrected sequence
divergence -16S gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
23.
Total number of stem substitutions plotted as a functions of uncorrected sequence
divergence - 16S gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
24.
Total number of loop substitutions plotted as a functions of uncorrected sequence
divergence - 16S gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
25.
Loop transitions were separated into functional nucleic acid classes and plotted as
a function of uncorrected sequence divergence - 16S gene . . . . . . . . . . . . . . . 177
26.
Non conserved loop and conserved loop transitions plotted as a function of
x
uncorrected sequence divergence - 16S gene . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
27.
Adjusted loop and stem distances plotted as a function of uncorrected sequence
divergence -16S gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
28.
Mean values and ranges of percent base composition for all, stem, loop, and nonconserved loop characters of the 16S mtDNA fragment . . . . . . . . . . . . . . . . . . 183
29.
A strict consensus of 8 equally parsimonious trees from the analysis of all
columns of data from t 16S sequence data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
30.
A strict consensus of 3952 equally parsimonious trees from a weighted analysis
of the 16S sequence data. All loop transitional substitutions were given a weight
of 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
31.
A strict consensus of 9828 equally parsimonious trees from a weighted analysis of
the 16S sequence data. Non-conservative loop transitional substitutions were
given a weight of 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
32.
Numbered ancestral nodes of the Sparidae clade from the unweighted 16S
consensus tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
33.
Clade of 16S biogeographic relationships overlaid on map of the world . . . . . 193
34.
Sequence divergence of each data partition plotted as a function of total sequence
divergence for all pairwise comparisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
35.
A strict consensus of three equally parsimonious trees from a heuristic search of
all cytochrome b and 16S nucleotide characters . . . . . . . . . . . . . . . . . . . . . . . . 220
36.
A single most parsimonious tree from a heuristic search of the weighted
cytochrome b nucleotides (3rd codon transitions =0) and all 16S nucleotides . . 222
37.
A strict consensus of 35 equally parsimonious trees from a heuristic search of all
cytochrome b amino acid residue and 16S nucleotide sequences . . . . . . . . . . . 224
38.
A strict consensus from a heuristic search of all unweighted sequences resulted in
11 equally parsimonious trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
xi
39.
A single most parsimonious tree from vicariance biogeographic analysis of the
combined cytochrome b and 16S dependent data matrices yielded . . . . . . . . . . 228
40.
Total substitutions plotted as a function of sequence divergence for putative copy
1 (top) and copy 2 (bottom) of the Tmo-4c4 locus . . . . . . . . . . . . . . . . . . . . . . 251
41.
A strict consensus of 81 equally parsimonious trees from parsimony analysis of
all clone sequences and characters of the Tmo-4c4 locus . . . . . . . . . . . . . . . . . 253
42.
Tree from neighbor-joining analysis of all clone sequences and characters of the
Tmo-4c4 locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
xii
ABSTRACT
Sparids are a diverse group of over 110 marine species whose putative six
subfamilies have been defined primarily on the basis of dentition and feeding type.
Monophyly of the subfamilies has not been tested, nor have the phylogenetic relationships
of all sparid genera been hypothesized.
I used mitochondrial DNA sequences from the complete cytochrome b gene
(1140bp) and the partial 16S gene (621bp) in independent and combined analyses to test
the monophyly of the Sparidae, elucidate the inter-relationships of the 33 recognized
genera of the Sparidae, test the validity of the six subfamilies of the Sparidae, and test the
monophyly of the Sparoidea. The cytochrome b (cyt b) analyses included 40 sparid
species, ten closely related species, ten basal percoids, and two non-perciform outgroup
species. A subset of these taxa was used in the independent 16S mtDNA analyses and
combined analyses.
The Sparidae were monophyletic in all analyses from independent and combined
data sets. The centracanthid Spicara was consistently found within the sparid clade and is
considered a member of the Sparidae. The monophyly of the Centracanthidae is not
supported because Spicara was polyphyletic within the sparids in all analyses. These data
suggest that a revision of Pagrus, Pagellus, Dentex is in order because these genera were
not monophyletic in any analysis. Two mitochondrial lineages were reconstructed in all
analyses, but the previously proposed six sparid subfamilies (Boopsinae, Denticinae,
Diplodinae, Pagellinae, Pagrinae, and Sparinae) were not monophyletic in any analysis.
This suggests that the feeding types that these subfamilies are based were independently
derived multiple times within sparid fishes. There was support from the weighted cyt b
analysis for a monophyletic Sparoidea, and in all cyt b analyses the Lethrinidae were
sister to the Sparidae. However, the Sparoidea were not monophyletic in the independent
16S or combined analyses. Evidence from the weighted cyt b, 16S and combined
analyses suggests a sister relationship between Moronidae and Lateolabrax.
Biogeographic analysis revealed that there were two areas of sparid evolution: the
eastern Indian Ocean - western Pacific and the western Indian Ocean Mediterranean/Atlantic. Sparids in these two areas probably arose from a Tethyan
ancestor.
xiii
A MOLECULAR PHYLOGENY OF THE SPARIDAE (PERCIFORMES: PERCOIDEI)
GENERAL INTRODUCTION
The Sparidae are a diverse group of over 110 mostly neritic species whose putative six
subfamilies have been defined primarily on the basis of dentition and feeding type (Smith,
1938; Akazaki, 1962). The monophyly of these subfamilies has not been tested, nor have
the phylogenetic relationships of all sparid genera been hypothesized. Smith (1938) and
Smith and Smith (1986) initially delineated the 33 sparid genera (Table 1) into four
subfamilies based mostly on dentition (Table 2). The Boopsinae have compressed outer
incisiform teeth and are typically herbivores or feed on small invertebrates. The
Denticinae are typical piscivores with enlarged canines in front and smaller conical teeth
behind. The Pagellinae lack canines, have small conical outer teeth, small inner molars
and are usually carnivorous on small invertebrates. The Sparinae have jaws with bluntly
rounded molars posteriorly, enlarged front teeth and are carnivorous, feeding on
crustaceans, mollusks, and small fish. Akazaki (1962) erected two new subfamilies
(Table 2), also largely defined by dentition. He moved the genera Diplodus,
Archosargus, and Lagodon from the Sparinae into the Diplodinae and he moved Pagrus,
Argyrops, and Evynnis from the Sparinae into the Pagrinae. Akazaki defined the
Diplodinae as having six to eight anterior teeth in the jaws and obliquely projecting
incisors, and the Pagrinae as those with four canines on the upper jaw, four to six canines
on the lower jaw, scales on the head extending to the interorbital region, molar teeth in
two series, and a reddish body.
2
3
The Sparidae have historically been a heterogenous group of fishes, often
associated with the Lethrinidae, Nemipteridae, Lutjanidae, Caesionidae, and Haemulidae
(Jordan and Fesler, 1893; Schultz 1953). Akazaki (1962) used osteology to define
"spariform" fishes that included the Nemipteridae, Sparidae, and Lethrinidae (Figure 1).
Akazaki suggested that the spariform fishes had three "stems": the primitive
Nemipteridae-stem; the intermediate Sparidae-stem; and the highly specialized
Lethrinidae-stem. Johnson (1980) proposed the superfamily Sparoidea to include
Akazaki’s three spariform families and the Centracanthidae. He added the
Centracanthidae based on maxillary-premaxillary distal articulation and other osteological
characters. Johnson disagreed with Akazaki's placement of the Sparidae between the
Nemipteridae and Lethrinidae and presented preliminary anatomical and osteological
evidence that the Nemipteridae and Lethrinidae were more closely related to each other
than they were to either the sparids or to the centracanthids (Figure 2).
There has been only limited morphological analysis of the Sparidae since Regan
(1913) gave the first practical definition of the family to include those species, “ in that
the distal end of the praemaxillary ramus overlaps the maxillary externally”. The
majority of morphological works have been regional in scope. Smith (1938) attempted to
reinforce the taxonomy of South African sparids, but recognized that the:
relationships between the species have never been properly investigated. Generic limits
have been sadly lacking in uniformity; in some cases monotypic genera have been
defined within the limits which are very narrow by contrast with others which have
embraced forms so widely divergent and polymorphous as to be almost without parallel
in any other families.
Akazaki (1962) provided the most complete morphological analysis of sparid
relationships to date, although his treatise did not include all genera. His results inferred
phylogenetic relationships of the sparids based on traditional, non-quantitative, methods.
4
Johnson (1980) better defined the relationships of the families often associated with the
Sparidae. However, he was unable to examine all of the genera of sparids and the generic
relationships remain untested.
Similarly, few molecular studies have examined the evolutionary relationships of
the Sparidae and none has employed cladistic analysis to investigate the evolutionary
history of all sparid genera. Taniguchi et al. (1986) investigated 18 isozyme loci from
skeletal muscle, liver, and heart tissues to infer the genetic relationships of ten species
from six genera of Japanese sparids. Their results established a close genetic relationship
of Japanese sparids; a genetic distance (Nei, 1978) of less than 0.01 between Japanese
members of the genera Pagrus, Evynnis, Argyrops, and Dentex. A greater genetic
distance (>0.013) was found between these four genera and Sparus and Acanthopagrus.
Basaglia (1991) analyzed six isozymes from seven different tissues of 15 sparid species to
infer phylogenetic relationships based on an “index of divergence”. Basaglia and
Marchetti (1991) examined white muscle proteins in the same 15 species analyzed by
Basaglia (1991) and presented a more quantitative analysis based on pairwise similarity
coefficients that clustered the sparids into respective subfamilies - Boopsinae, Diplodinae,
and Pagellinae. The Pagellinae Lithognathus mormyrus, clustered with the Sparinae as
did the Denticinae, Dentex dentex. Garrido-Ramos et al. (1994, 1995, and 1999) used
centromeric satellite DNA to elucidate the relationships of Mediterranean sparids. The
later study sampled 10 taxa from four genera to infer phylogenetic relationships based on
neighbor-joining and distance (UPGMA) analyses. Jean et al. (1995) examined three
mitochondrial regions, the displacement loop, tRNAPhe, and 12S rRNA gene, of five taxa
from two genera of Taiwanese sparids. Their resulting phylogenetic tree was based on
Tamura-Nei genetic distances. Hanel and Sturmbauer (2000) used 16S rDNA sequences
to examine the evolution of trophic types in Northeastern Atlantic and Mediterranean
sparids. Based on an analysis of 24 taxa from 10 sparid genera, they concluded that
5
trophic types evolved more than once in sparid fishes.
In this dissertation I present a rigorous phylogenetic analysis of sparid
relationships. I include representatives of all valid sparid genera and use parsimony
analysis of mitochondrial molecular data to infer relationships. In the first chapter I
present the results of phylogenetic analyses of the complete mitochondrial cytochrome b
gene for 40 sparid species, ten closely related species, ten basal percoids, and two nonperciform outgroup species. In the second chapter I show results of phylogenetic
analyses of the partial mitochondrial 16S rRNA gene for 40 sparid taxa and 16 closely
related percoid taxa.
The third chapter provides a phylogenetic analysis from combined mitochondrial data sets
using character congruence. I also investigate the biogeographic relationships of the
Sparidae using the method of vicariance biogoegraphy (Brooks 1985; Wiley 1988a, b;
Wiley et al., 1991). In each chapter, I have used the resulting phylogenies as dependent
data to estimate the biogeographic aspect of evolution of the sparids.
I have chosen to use parsimony analysis as a method to reconstruct phylogeny
because it minimizes ad hoc assumptions of evolution. Under parsimony, the tree with
the shortest number of steps is chosen as an estimator of phylogeny. The shortest tree has
the least number of evolutionary changes required by the data to produce that tree and
has the least amount of homoplastic character changes. The minimal tree should have the
greatest number of shared, derived homologous characters (synapomorphies) as evidence
for relationships. Outgroup species are used to determine homology of characters. In
this study I have used a wide range of outgroup species to infer phylogeny. Outgroup
species include those that have been classically associated with the Sparidae (Lethrinidae,
Nemipteridae, Haemulidae, Lutjanidae), basal percoids (Moronidae, Lateolabrax and
Centropomus) and taxa outside of Perciformes (Cyprinidae). The wide range of
outgroups allowed for the testing of multiple hypotheses from these data.
6
Two types of clade support were used throughout this dissertation; nonparametric
bootstrap (Felsenstein 1985) as well as total value and partitioned Bremer values (Bremer
1988). Nonparametric bootsrap employs psuedoreplicate data matrices to provide an
estimate of phylogenetic signal at each node. Decay indices are a measure of support for
the nodes in a given tree. The shortest unconstrained tree (or strict consensus of equally
parsimonious trees) is found. A constraint statement is assigned for each node in the
shortest tree or strict consensus tree. All trees, inconsistent with each constraint
statement, are found. The indices are calculated for each node by comparing the length of
the original shortest tree (or strict consensus) to the length of the inconsistent tree/s length
at each constrained node. The additional number of steps between the original tree and
the constrained tree/s becomes the index number. Additionally decay indices can be
calculated for partitioned data following the method of Baker and Desalle (1997) and
Baker, et al. (1998). The partitioned indices examine how each part of a partitioned dataset contributes to the decay value at a particular node. The partition value can be either
positive or negative (negative values conflicting support for a node) with the sum of each
node’s partitions equaling the overall decay value for that node (Sorensen 1999).
In this dissertation I infer phylogenies from mitochondrial DNA, including the
complete cytochrome b gene, the partial 16S gene, and from both genes in combination
to: 1) test the monophyly of the Sparidae; 2) elucidate the inter-relationships of the 33
recognized genera of the Sparidae; 3) test the validity of the six subfamilies of the
Sparidae; and, 4) to test the monophyly of the Sparoidea. In addition, in a final chapter
to this dissertation, I give results of an attempt to infer phylogeny based on a single copy
nuclear locus TMO-4c4.
7
TABLE 1. ANNOTATED LIST OF THE VALID GENERA OF THE SPARIDAE
(PERCIFORMES: PERCOIDEI)
Acanthopagrus Peters 1855:242 (as subgenus of Chrysophrys)
Synonym: Mylio Commerson in Lacepede 1802
Archosargus Gill 1865:266
Argyrozona Smith 1938:300 (as subgenus of Polysteganus)
Argyrops Swainson 1839:171 (as subgenus of Chrysophrys)
Synonym: Parargyrops Tanaka 1916
Boops Cuvier 1814:91
Synonym: Box Valenciennes in Cuvier & Valenciennes 1830
Boopsoidea Castelnau 1861:25
Calamus Swainson 1839:171 (as subgenus of Chrysophrys)
Synonym: Aurata Catesby 1771
Synonym: Grammateus Poey:1872
Cheimerius Smith 1938:292 (as subgenus of Dentex)
Chrysoblephus Swainson 1839:171 (as subgenus of Chrysophrys)
Crenidens Valenciennes in Cuvier & Valenciennes 1830:377
Cymatoceps Smith 1938:259
Dentex Cuvier 1814:92
Synonym: Synagris Klein in Walbum 1792
Synonym: Taius Jordan & Thompson 1912
Diplodus Rafinesque 1810:26
Synonym: Denius Gistel 1848
Synonym: Sargus Cuvier 1816
Synonym: Sargus Klein 1775
Evynnis Jordan & Thompson 1912:573
Gymnocrotaphus Gunther 1859:413
Lagodon Holbrook 1855:56
Synonym: Sphenosargus Fowler 1940 (as subgenus of Salema)
Lithognathus Swainson 1839:172 (as subgenus of Pagellus)
Oblada Cuvier 1829:185
Pachymetopon Gunther 1859:413
Synonym: Simocantharus Fowler 1933 (as subgenus of Spondyliosoma)
Pagellus Valenciennes in Cuvier & Valenciennes 1830:169
Synonym: Nudipagellus Fowler 1925 (as subgenus of Pagellus)
Pagrus Cuvier 1816:272
Synonym: Semapagrus Fowler 1925 (as subgenus of Pagrus)
Synonym: Sparidentex Munro 1948
Petrus Smith 1938:302
Polyamblyodon Norman 1935:21
Polysteganus Klunzinger 1870:763 (as subgenus of Dentex)
8
TABLE 1 (Continued). ANNOTATED LIST OF THE VALID GENERA OF THE
SPARIDAE (PERCIFORMES: PERCOIDEI)
Synonym: Axineceps Smith 1938 (as subgenus of Polysteganus)
Porcostoma Smith 1938:270
Pterogymnus Smith 1938:257
Rhabdosargus Fowler 1933:175 (as subgenus of Diplodus)
Synonym: Austrosparus Smith 1838
Synonym: Prionosparus Smith 1942 (as subgenus of Austrosparus)
Sarpa Bonaparte 1831:171 (as subgenus of Box [Boops])
Objective Synonym: Eusalpa Fowler 1925
Sparodon Smith 1938:249
Sparidentex Munro 1948:276
Sparus Linnaeus 1758:277
Objective Synonym: Aurata Oken (ex Cuvier) 1817
Synonym: Caeso Gistel 1848
Objective Synonym: Chryseis Schinz 1822
Synonym: Chrysophris Cuvier 1829
Objective Synonym: Daurada Stark 1828
Objective Synonym: Dorada Jarocki 1822
Synonym: Dulosparus Fowler 1933 (as subgenus of Sparus)
Synonym: Eudynama Gistel 1848
Synonym: Pagrichthys Gill 1893:97
Spondyliosoma Cantor 1848:1032
Objective Synonym: Cantharus Cuvier 1816
Objective Synonym: Cantharusa Strand 1928
Objective Synonym: Caranthus Barnard 1927
Stenotomus Gill 1865:266
Synonym: Mimocubiceps Fowler 1944
9
TABLE 2. SUBFAMILIES OF SPARIDAE FOLLOWING AKAZAKI (1962)* AND
SMITH AND SMITH (1986)
Boopsinae
Boops Cuvier 1814:91
Crenidens Valenciennes in Cuvier & Valenciennes 1830:377
Gymnocrotaphus Gunther 1859:413
Oblada Cuvier 1829:1851933 (as subgenus of Spondyliosoma)
Pachymetopon Gunther 1859:413
Polyamblyodon Norman 1935:21
Sarpa Bonaparte 1831:171 (as subgenus of Box [Boops])
Spondyliosoma Cantor 1848:1032
Denticinae
Argyrozona Smith 1938:300 (as subgenus of Polysteganus)
Cheimerius Smith 1938:292 (as subgenus of Dentex)
Dentex Cuvier 1814:92
Petrus Smith 1938:302
Polysteganus Klunzinger 1870:763 (as subgenus of Dentex)
Sparidentex Munro 1948:276
Diplodinae*
Archosargus Gill 1865:266
Diplodus Rafinesque 1810:26
Lagodon Holbrook 1855:56
Pagellinae
Boopsoidea Castelnau 1861:25
Pagellus Valenciennes in Cuvier & Valenciennes 1830:169
Lithognathus Swainson 1839:172 (as subgenus of Pagellus)
Pagrinae*
Argyrops Swainson 1839:171 (as subgenus of Chrysophrys)
Evynnis Jordan & Thompson 1912:573
Pagrus Cuvier 1816:272
Sparinae
Acanthopagrus Peters 1855:242 (as subgenus of Chrysophrys)
Calamus Swainson 1839:171 (as subgenus of Chrysophrys)
Chrysoblephus Swainson 1839:171 (as subgenus of Chrysophrys)
Cymatoceps Smith 1938:259
Porcostoma Smith 1938:270
Pterogymnus Smith 1938:257
Rhabdosargus Fowler 1933:175 (as subgenus of Diplodus)
Sparodon Smith 1938:249
Sparus Linnaeus 1758:277
Stenotomus Gill 1865:266
10
Figure 1. Suggested phylogeny of spariform relationships redrawn from Akazaki (1962).
Akazaki’s phylogeny was based on non-empirical analysis and should not be considered
quantitative. Branch lengths in this tree do not represent degree of relatedness between
taxa.
Petapodus
Nemipteridae
Scolopsis
Nemipterus
Denticiniae
Sparidae
Cheimerius
Dentex
Cantharus
Sarpa
Boops
Boopsinae
Pagrinae
Pagellinae
Sparinae
Diplodinae
Pagrus
Argyrops
Evynnis
Pagellus
Stenotomus
Sparinae
Acanthopagrus
Calamus
Archosargus
Diplodus
Lagodon
Puntazzo
Monotaxis
Lethrinidae
Gymnocranius
Gnathodentex
Lethrinus
12
Figure 2. Hypothesized relationships of the Sparidae, Centracanthidae, Lethrinidae and
Nemipteridae of Akazaki (1962) top and Johnson (1980) bottom.
Akazaki 1962
Nemipteridae
Sparidae
Lethrinidae
Johnson 1980
Nemipteridae
Lethrinidae
Sparidae + Centracanthidae
CHAPTER ONE. A MOLECULAR PHYLOGENY OF THE FAMILY
MITOCHONDRIAL CYTOCHROME B GENE
15
INTRODUCTION
Mitochondrial DNA (mtDNA) has proven to be useful in molecular phylogenetic
studies because evolutionary relationships can be inferred among higher levels, between
recently divergent groups, populations, species and even individuals (Avise, 1994). The
mtDNA molecule is double stranded, circular, clonally (maternally) inherited. Most
substitutions are point mutations although insertions/deletions are not rare (Avise, 1994).
The mtDNA genome is relatively small (approximately 16,500 base pairs in fishes), has a
high nucleotide substitution rate at synonymous sites and lacks recombination (Brown et
al., 1979; Rand, 1994; Cantatore et al., 1994). The mitochondrial DNA of perciform fish
is comprised of 13 protein-coding genes, two ribosomal RNA (rRNA) genes and 22
transfer RNA (tRNA) genes (Meyer, 1993).
Within the mitochondrial genome, different regions evolve at different rates .
Cummings et al. (1995) explored the ability of single mtDNA genes to recover the same
tree/s found when using whole mtDNA genomes; assuming that whole genomic
phylogenies are a better measure of the “true” phylogeny. Their findings indicated that no
one gene represented all of the information expressed by the whole genome. In an ideal
phylogenetic study, random or directed sampling of base pairs from across all
mitochondrial genes would deduce the same tree as resulted in the analysis of the total
genome; however, many constraints exclude wide-range gene sampling, and sequencing
of the entire mtDNA genome from a wide-range of taxonomic representatives is even
more daunting. Because each protein coding region, rRNA gene and tRNA has different
functional and structural constraints, each mtDNA gene region is unique in its intrinsic
16
rate of substitution. Therefore, analysis of only single genes, partial gene regions, or
both in combination has become a common approach in molecular phylogenetics. It is
difficult to evaluate the usefulness of a particular gene a priori and often a pilot study
must be conducted to realize a gene’s potential for inferring a phylogeny for a particular
group. Examination of published sequences and phylogenies often reflect a gene’s
strength and limitation, and can provide a basis for gene selection.
Cytochrome c oxidase subunit I (COI) has been found to be one of the more
highly conserved genes of the mitochondrial genome (Brown, 1985). For a comparison
of taxonomic representatives of the order Perciformes or at the family level (less
divergence time) this gene may be too conserved (not variable enough) and therefore,
yield little phylogenetic information. To date COI has not been analyzed widely in fishes.
The large (16S) and small (12S) subunits of mitochondrial ribosomal DNA are
reasonably conserved within perciform fish and are typically used for higher level
(subfamily, family, superfamily, suborder, order) analyses (Hillis and Dixon, 1991,
Wiley et al., 1998). The control region or displacement loop (D-loop) of mtDNA has
highly variable non-coding sites positioned between very conserved functional areas.
Because reduced selection constraints in regions of D-loop, the control region is used for
analysis of closely related taxa where increased variation may reflect recent evolutionary
events (Lockhart et al., 1995). Other mtDNA genes such as ATPase6, ATPase8,
cytochrome b or NADH(n) may be preferable for phylogenetic comparisons for taxa of
intermediate divergence (generic and family level) because these gene regions are more
variable than both rRNAs or COI, but are less variable than the D-loop.
Of mtDNA protein-coding genes, cytochrome b (cyt b) has proven to be a robust
evolutionary marker, revealing phylogenies at various taxonomic levels in fishes.
Cytochrome b codes for a functionally conserved protein and can be phylogenetically
informative on interspecific and intraspecific studies, but is probably best suited for
17
closely related taxa because nucleotide sequence variation is less saturated by multiple
substitutions (Meyer 1993). Cytochrome b was informative in actinopterygian
phylogenetic relationships (Lydeard et al., 1995; Lydeard and Roe, 1997; Schmidt et al.,
1998). Cantatore et al. (1994) used cyt b sequence analysis to survey the phylogenetic
relationships of five widely diverse families of perciform fishes and observed that the rate
of divergence was similar to that of sharks (Martin et al., 1992; Martin and Palumbi
1993), yet slower than that of mammals and birds. Finnerty and Block (1995)
demonstrated cyt b to be useful in resolving phylogenetic relationships of the perciform
suborder Scombroidei and Song et al. (1998) used cyt b to assess the phylogenetic
relationships among percid fishes. Zardoya and Meyer (1996) classified cyt b as a good
“phylogenetic performer” among phylogenetically distant relatives.
Much is known about the structure and function of cytochrome b (Esposti et al.,
1993). The translated product is a transmembrane protein that forms the central catalytic
subunit of ubiquinol:cytochrome c oxidase. This enzyme is a ligand that contains a heme
prosthetic group. The central iron ion in the heme acts as the primary electron transport
during mitochondrial respiration (Esposti et al., 1993).
The cytochrome b molecule is bi-polar with five negatively charged proton input
regions, four positively charged proton output regions and eight transmembrane regions
(Esposti et al., 1993, Lydeard and Roe 1997). Previous studies including Irwin et al.
(1991) and Lydeard and Roe (1997) have found the most variable amino acid residues in
the negative side followed by the transmembrane region and the positive output region
being least variable. The conservation of the positive side is likely due to a functional
constraint as the positive terminus associates with an iron-sulfur subunit during ubiquinol
oxidation (Esposti et al., 1993, Lydeard and Roe 1997).
Cytochrome b was chosen as a molecular marker for this study because it is a
coding gene likely to provide useful phylogenetic information at many taxonomic levels.
18
Functional constraints balance stochastic mutations at nucleotide positions across cyt b
and variable rates of synonymous and nonsynonymous substitutions among codon
positions contribute to the gene’s utility as an evolutionary marker.
In this chapter, I present the results of phylogenetic analyses of the complete
mitochondrial cytochrome b (cyt b) gene (1140 bp) for 40 sparid species, ten closely
related species, ten basal percoids, and two non-perciform outgroup species. I used
parsimony analyses from cyt b nucleotide and amino acid sequences to test the
monophyly of the Sparidae, the validity of the six subfamilies of the Sparidae, the
evolutionary relationships of the 33 genera of the Sparidae, and the monophyly of
Sparoidea. A resulting phylogeny was used as the basis for analysis of the biogeographic
aspects of sparid evolution.
19
.
MATERIALS AND METHODS
Sampling - Following the classification of Akazaki (1962) and Smith (1986),
representatives of the six subfamilies (Boopsinae, Denticinae, Diplodinae, Pagellinae,
Pagrinae and Sparinae) and 33 genera of the family Sparidae were collected. Taxonomic
representatives were also collected for other members of the superfamily Sparoidea
(Centracanthidae, Nemipteridae and Lethrinidae), and for possible close outgroups in the
Percoidei ( Haemulidae, Lutjanidae, and Caesionidae). The basal percoids Moronidae +
Lateolabrax were used to root the Sparidae and related families within the perciformes.
Sequences of two ostariophysins, Luxilus and Cyprinus were used as distant outgroups in
this study. Collection data for each sample are provided in Table 3. When possible,
specimen identifications were validated through voucher specimens deposited at
museums in country-of-origin, at the National Museum of Natural History in Washington,
DC (USNM), Virginia Institute of Marine Science, Gloucester Point, VA (VIMS) or at
Old Dominion University, Norfolk, VA. (ODU). Museum catalog numbers and GenBank
accession numbers are provided in Table 3.
Specimen Preservation - Gill tissue or white muscle tissue was collected from fresh
samples or from frozen samples acquired at markets or in museum collections. Tissue
was removed from frozen samples and not allowed to thaw before adding to buffer.
Tissues were placed into a buffer solution of 0.25 M disodium ethylenediaminetetraacetate (EDTA), 20% dimethyl sulfoxide (DMSO), saturated sodium chloride (NaCl), pH
8.0 (Seutin et al., 1990) and stored at room temperature.
20
DNA Extraction - High molecular weight DNA was isolated from gill tissue or white
muscle tissue. Approximately 0.05-0.1g of tissue was removed using sterile scalpel
blades and forceps. Forceps were rinsed in 10% bleach and two changes of de-ionized
water (diH20), dipped in 95% ethyl alcohol and flame sterilized between samples.
Samples were placed in a 1.5ml microfuge tubes followed by protein digestion,
phenol:chloroform extraction, and ethanol precipitation following Sambrook, et al.
(1989) or using the tissue protocol of QIAamp® System DNA extraction kits (QIAGEN
Inc, USA, Valencia CA).
Primers - The following primer pairs were used for PCR amplification in this study and
were mapped against the equivalent sequence positions on the mitochondrial genome of
Cyprinus carpio (GenBank Accession X61010): CYTbUnvL - L15242 (Kocher et al.,
1989)/CYTbUnvH - H16458 (Cantatore et al., 1994); CYTbGludgL L15249/CYTBThrdgH - H16465 (Palumbi et al., 1991); and CYTB4xdgL - L14249
(modified from Palumbi et al., 1991)/CYTb4xdgH - H16435 (derived from consensus
sequences during this study). See Figure 3 for primer sequences, location illustration and
map alignment. Primers were ordered from Genosys (Genosys Biotechnologies, Inc., The
Woodlands, TX). Primers sites were located within the transfer ribonucleic acids (tRNA)
genes that flank either end of the mtDNA cytochrome b gene (tRNAGlu and tRNAThr).
Amplification - A 50 µl PCR amplification of cyt b was performed with 5-10 ng of each
template DNA. The following reagents from the PCR Reagent System (GIBCO BRL
Life Technologies, Gaithersburg, MD) were used in each reaction: 5 µl 10X PCR Buffer
plus Mg [200mM Tris-HCL (pH8.4), 500 mM KCL, 15 mM MgCl2]; 1µl 10mM dNTP
Mix [10mM each dATP, dCTP, dGTP, dTTP]; 50 pmols of each primer, 0.25 µl Taq
DNA polymerase [5U/µl]. Either a Perkin Elmer Cetus (Norwalk, CT ) or an MJ
21
Research PTC-200 (Watertown. MA) thermocycler was used for PCR amplification with
the following cycle parameters: initial denaturation [94EC for 4.0 min]; 35 cycles of
[denaturation 94EC for 1.0 min, annealing 48EC-51EC (depending on sample) for 1.0
min; extension 72EC for 3.0 min]; final extension [70EC for 5 min]; icebox [4EC
indefinitely].
Cloning and Sequencing - Once target sequences were selected and successfully
amplified, sequence reactions were performed. The polymerase chain reaction products
were cloned using the Invitrogen TA Cloning® Kit, (Invitrogen Corporation, San Diego,
CA). Ligated PCR product was transformed and cloned into competent Escherichia coli.
Transformed colonies were grown overnight on LB-agar plates in the presence of
ampicillin and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (Xgal). Colonies with inserts
were streaked on new LB-agar plates in the presence of ampicillin and Xgal. White
colonies were screened for appropriate inserts using the quick screening methods from
Sambrook et al. (1989). Colonies with target insert were grown in 3 mL preparations of
terrific broth + ampicillin overnight (Tartof and Hobbs, 1987). Purified plasmid DNA
was obtained by either standard plasmid preparation protocols (Seutin et al., 1990) or by
using a PERFECTprep® kit (5Prime ÿ 3Prime, Inc. Boulder, CO) and suspended in 65Fl
diH20.
Sequencing was performed following the dideoxynucleotide chain termination
method (Sanger et al., 1977), using manual and automated sequencing techniques.
Initially, a 357 base pair fragment of the cyt b gene was amplified for 20 taxa using the
primers UCYTB144F-tgggsncaratgtcntwytg and UCYTB272R-gcraanagraartaccaytc (J.
Quattro - unpublished), cloned and manually sequenced. During manual reactions both
strands of approximately 5Fg of plasmid DNA were sequenced using the Sequenase
Version 2.0 Kit (United States Biochemical, Cleveland, OH). Primers that flank each end
22
of the plasmid insert, the M13 Reverse Primer (New England BioLabs, Beverly, MA) and
the T7 Forward Promotor (GIBCO BRL Life Technologies) were used to sequence 357
base pairs of cytochrome b. Approximately 6.25 ci of the radiolabeled marker, 35S-(athio)-deoxynucleoside triphospate (New England Nuclear, Boston, MA.), was
incorporated during reactions to visualize sequences on a resulting autoradiogram.
Reactions were electrophoresed through a 6% polyacrylamide gel. The gel was
transferred to 3 MM chromatography paper, vacuum-dried and exposed to Kodak
BioMax autoradiograph film. The film was developed within 1 week following initial
exposure and sequences were read directly from the autoradiogram and recorded by hand.
Separate results are not included for the manual sequencing. The 357 base pair fragment
explored by manual sequencing was internal to the entire gene and was re-sequenced
during automated sequencing. This information was used to survey the utility of
cytochrome b across selected members of the Sparidae and outgroups. Once the utility of
cytochrome b was established for this group, the entire gene was sequenced for all
samples using a LiCor automated sequencer.
All complete sequences reported in this study were sequenced using automated
techniques (no sequences reported are from manual techniques). Plasmid DNA was
quantified using a DyNAQuant 200 flourometer (Amersham Pharmacia Biotech,
Buckinghamshire, England) and approximately 300 fmol of plasmid DNA was used in
each cycle sequencing reaction. Both forward and reverse IRD800 flourescently labeled
M13 primers were used (Li-Cor, Inc. Lincoln, NE). A heat stable DNA polymerase,
Thermo Sequenase TM (Amersham Pharmacia Biotech) was used to incorporate the
IRD800 flourescently labeled primer during cycle sequencing. To relax structural stops
during electrophoreses, 7-deaza-dGTP was used during chain building. The flourescently
labeled termination reactions were then electrophoresed through a 66cm, 0.25mm thick,
4% LongRanger (FMC BioProducts, Rockland, Maine) acrylamide gel on a Li-Cor 4000L
23
automated sequencer. The resulting electronic gel image was analyzed using BaseImage
V2.3 software (Li-Cor).
Sequence Alignment - Both light and heavy strand sequences were obtained for all taxa.
Light strand sequences were inverted (reversed and complimented) with the heavy strand
sequence. A consensus sequence from combined light and heavy strands was made for
all taxa. Cytochrome b nucleic acid sequences were aligned by eye and by the Clustal
feature of Gene Jockey II (Biosoft, Cambridge UK). Two cytochrome b sequences from
GenBank: accession numbers X81567 (Sparidae: Boops boops) and X8156 (Moronidae
Dicentrarchus labrax) were used to aid alignment. The resulting alignment introduced
no gaps due to deletions or insertions and there were no inconsistent alignments between
taxa. Once sequences were aligned they were assigned codon positions and the reading
frames were proofed for frame-shifts. Ambiguities were referenced against the
sequencing gel image and corrected as necessary. Nucleotide sequences were translated
into amino acid residues using the extended mitochondrial translation table in
MacCLADE 3.07 (Madisson and Madisson, 1993).
Sequence Divergence and Mutation Analysis - Mean uncorrected pairwise genetic
distance (uncorrected “p” distance) was calculated using PAUP* ver 4.0b4a (Swofford,
1998) between all taxa, between ingroup taxa and between outgroup taxa. Because of
the large difference in sequence divergence between ingroup and outgroup taxa, mutation
analysis was restricted to ingroup taxa only. Scatter plots of transitions (Ts = AøG,
CøT) and transversions (Tv = AøC, AøT, CøG, GøT) as a function of mean
sequence divergence were calculated for combined codon positions and for individual
codon position from ingroup taxa only.
24
Base Compositional Bias - Base compositional bias (Irwin et al., 1991) was calculated
for all, ingroup and outgroup taxa, for combined codon positions, and for individual
codon positions. Base compositional bias was calculated using the following formula:
4
C = (2 / 3) ∑ | ci − 0.25|,
i =1
where C is the compositional bias and ci is the frequency of the ith base (Irwin et al.,
1991). The bias measurements for each nucleotide base were then summed across taxa
and divided by the total number of taxa for each group measured (all, ingroup, outgroup).
Phylogenetic Analysis - Parsimony analysis was performed using PAUP4.0b2*
(Swofford, 1998). The most parsimonious tree (MPT), or equally parsimonious trees
(EPTs) and strict consensus were obtained for each analysis. The number of constant
characters, parsimony-uninformative and parsimony informative characters, tree length,
consistency index and the retention index were determined. Bootstrap replicate support
was conducted using PAUP* and decay values (Bremer 1988) were calculated for clade
support using the program TreeRot.v2 (Sorensen 1999).
Biogeographic Analysis - A quantitative analysis of the biogeographic relationships of the
Sparidae was conducted using analysis of vicariance biogeography (Brooks 1985; Wiley
1988a, b; Wiley et al., 1991). Parsimony analysis was used to reconstruct the
biogeographic relationships inferred from the cytochrome b phylogeny. During
biogeographic analysis, a matrix of independent data (areas of occurrence) and dependent
data (phylogeny of the Sparidae) was constructed. All nodes on the original tree were
labeled (terminal taxa, internal nodes=ancestral states). A list of areas of occurrence was
then prepared for each taxon. To define distributional data as objectively as possible,
25
“FAO areas” were used for each taxon (FAO, 1995). The FAO areas were established by
FAO (1995) as a means to provide defined distributional areas for fisheries statistical
purposes. Each node was assessed for each of the areas. A binary data-matrix was
developed based on the presence (1) or absence (0) of each dependent variable (node) for
each independent variable (area). The data matrix was converted to a NEXUS file and
analyzed with PAUP*. The resulting tree was then overlaid onto a map of the world.
26
RESULTS
Sequence Characteristics - The full 1140 nucleotide basepairs of the cytochrome b gene
was sequenced for 57 taxa. Nucleotide sequences for all taxa included in this study are
given in Table 4. Of the 1140 characters sampled across all taxa, 483 (42%) were
constant, 115 (10%) variable characters were parsimony uninformative and 542 (48%)
variable characters were parsimony informative (Table 5). Of all informative characters,
69% came from the third codon position. Third codon position bases were more variable
(2 constant, 2 uninformative, 376 informative) than first codon position bases (203
constant, 49 uninformative, 128 informative) and second codon position bases (276
constant, 66 uninformative, 38 informative). Informative characters were primarily
transitions in the third codon position (Ts= 68% of all substitutions) and first codon
position (Ts=66% of all substitutions). Less of the informative characters were
transitions in the second codon position (Ts=59%, Tv=41%).
Sequence Divergence - Mean uncorrected pairwise genetic distance (uncorrected “p”
distance) is given in Table 6. between all, ingroup, and outgroup taxa. The mean
uncorrected pairwise sequence divergence among all taxa was 20.22%. The mean
pairwise sequence divergence between outgroup taxa was 22.73%, and the mean
pairwise sequence divergence between ingroup taxa was 16.27%. Mean pairwise
uncorrected sequence divergence was calculated among and between the six subfamilies
of the Sparidae (Table 7). The smallest within group (subfamily) uncorrected sequence
divergence was found within the subfamily Diplodinae (11.14%) and the greatest
divergence was found within the subfamily Sparinae (16.58%). The smallest uncorrected
27
divergence was between the subfamilies Denticinae and Pagellinae (14.23%) and the
greatest uncorrected sequence divergence was between Boopsinae and Denticinae
(17.66%).
Mutation Analysis - Scatter plots of transitions and transversions as a function of mean
sequence divergence were calculated for combined codon positions and for individual
codon position from ingroup taxa only (Figures 4-7). Combined codon positional
transitions reached a maximum number of substitutions ( > 160) at approximately 20%
sequence divergence (Figure 4; Ts-All - 2nd order polynomial y = -0.3212x2 + 12.339x +
13.82, R2 = 0.2725). Combined codon positional transversions continued to increase with
sequence divergence in a linear relationship (Figure 4; Tv-All - 2nd order polynomial y =
-0.0176x2 + 4.0373x - 3.6503, R2 = 0.2038 ) across the entire range of sequence
divergence. In graphs from codon positions one and two (Figures 5 and 6) there
appeared to be a linear increase in the total number of substitutions as a function of
sequence divergence. However, second order polynomial trend lines fitted only a small
percentage of the variance in the data (Figures 5 and 6; 2nd order polynomials). In the first
codon position, there were 30 transitions at 20% sequence divergence, but only 15
transversions at 20% sequence divergence. In the second codon position, there were very
few substitutions overall (<9), and there was nearly an equal number of transitions and
transversions at the maximum sequence divergence of 20%. Third positional transitions
appeared to asymptote as sequence divergence increased (Figure 7, Ts-3rd). A second
order polynomial fitted to the data had the equation y= -0.2707x2 + 11.417x -4.4675,
R2=0.4766. This relationship might signify transitional, third codon position site
saturation, because the number of transitional substitutions leveled off after 15%
sequence divergence. When the total number of substitutions from the third codon
position and the total number of substitutions from pooled first and second codon
28
positions are plotted as a function of sequence divergence (Figure 8), it is apparent that
the majority of all substitutions were found in the third codon position. Because third
positional changes accounted for the majority of all substitutions, the contribution of
transitions and transversions to the total number of third position substitutions was
plotted as a function of sequence divergence (Figure 9). The contribution of transitions as
a percentage of total substitutions decreased with increasing sequence divergence, and the
converse relationship was true for transversions (although the relative number of total
substitutions was contributed from transitions). As sequence divergence increased, the
contribution of transversions to the total number of substitutions increased. Because,
transitions are saturated, they are less informative than transversions at uncorrected
sequence divergence greater than 15%. At greater than 15% uncorrected sequence
divergence, transversions provided the majority of phylogenetic signal. Therefore,
transversions should become increasingly more important in deriving phylogenetic
relationships from third codon position characters as sequence divergence increases.
Transitional substitutions were separated into nitrogenous base type, purines (AøG) and
pyrimidines (CøT) and plotted against sequence divergence ( Figure 10). Purines
reached saturation at a lower sequence divergence (just above 10% ) than did pyrimidines
(>15%).
Base Compositional Bias - The overall positional bias was 0.133 (13%). The highest bias
was found in the third codon position that had a bias of 0.274 (27%) and there was a
strong anti-guanine bias in the third codon position with a subsequent shift to procytosine (Table 8). Cumulative frequencies of each of the four base pairs was calculated
for all 62 taxa and a chi-square test of base heterogeneity were calculated for all codon
positions, and for codon positions one, two and three (Figures 11 to 14). The overall
codon bias was not significant (X2=166.9, df=183 p=0.798), but had a lower p value than
29
chi-square test from individual codons, reflecting the anti-guanine bias. The first codon
position (X2=34.22, df=183, p > 0.995) and second codon position (X2=9.68, df=183, p >
0.995) did not demonstrate significant heterogeneity among taxa in base composition.
The frequencies of the four bases in the third codon position was strongly unequal (antiguanine, pro-cytosine) and the chi-square test demonstrated significant heterogeneity
among taxa in third codon position base frequency (X2=477.6, df=183, P < 0.001).
Amino Acid Translations - Nucleotide base pairs were translated into amino acid residues
(Table 9) . Of 380 total residues, 190 (50%) were constant across all taxa and of the
variable characters 93 (24.5%) were parsimony uninformative and 97 (25.5%) were
parsimony informative across all taxa.
Phylogenetic Relationships
Unweighted Data - A heuristic search of 1000 random addition replicates of the
cytochrome b nucleotide data set resulted in four equally parsimonious trees (Figure 15,
tree length = 6416, CI = 0.1900; RI = 0.4258, starting seed 796159554; all characters
unordered and equal weight of 1; outgroup taxa Cyprinus carpio and Luxilus zonatus;
branch-swapping = stepwise addition; swapping algorithm = tree bisection-reconnection;
no topological restraints; character-state optimization = accelerated transformations).
Bootstrap support (1000 replicates) and partitioned Bremer decay values (20, unrestricted
random addition sequences per node) were calculated and are shown on a strict consensus
of four equally parsimonious trees (Figure 16). In all EPTs (Figure 15) the family
Sparidae formed a fully resolved clade with strong bootstrap (99%) and decay support
(22) for sparid monophyly. Based on the partitioned Bremer values, 75% of the support
is based on third positional changes and 25% was based on first positional changes.
Included in the monophyletic Sparidae clade are two species of the centracanthid genus
30
Spicara. Pending further evidence and for the purpose of this analysis, Spicara will be
considered a member of the Sparidae (see discussion for more details). Within the
monophyletic Sparidae + Spicara clade, two distinct clades are educed. A Bremer decay
support of 3 separates the sparid taxa in “Group I” ((((Boops boops, Crenidens
crenidens), (((Oblada melanura, ((Diplodus argenteus, (Diplodus bermudensis, Diplodus
holbrooki)), Diplodus cervinus)), (Lithognathus mormyrus, Pagellus bogaraveo)),
((Acanthopagrus berda, Sparidentex hasta), ((Rhabdosargus thorpei, Sparodon
durbanensis), Sparus auratus)))), (((Gymnocrotaphus curvidens, (Pachymetopon aeneum,
Polyamblyodon germanum)), Boopsoidea inornata), (Sarpa salpa, (Spondyliosoma
cantharus, Spicara maena)))), ((Archosargus probatocephalus, Lagodon rhomboides),
(Calamus nodosus, Stenotomus chrysops))) from a second clade, “Group II”, with decay
support (8) for the sparid taxa (((Argyrozona argyrozona, ((Petrus rupestris,
Polysteganus praeorbitalis), ((Chrysoblephus cristiceps, Cymatoceps nasutus),
Porcostoma dentata))), Pterogymnus laniarius), (((((Cheimerius nufar, Dentex dentex),
Pagrus auriga), (Pagellus bellottii, Pagrus pagrus)), (Argyrops spinifer, (Evynnis
japonica, Pagrus auratus))), (Dentex tumifrons, Spicara alta)))). The sister group to the
Sparidae are the Lethrinidae (Lethrinus ornatus, Lethrinus rubrioperculatus). However,
neither Bremer decay nor bootstrap values support the lethrinids as separate from other
percoids. The Nemipteridae were included with the Moronidae + Lateolabrax in an
unresolved clade described here from the strict consensus tree (((Dicentrarchus labrax,
Dicentrarchus punctatus), ((Morone americanus, Morone mississippiensis), Morone
chrysops, Morone saxatilis)), ((Lateolabrax japonicus, Lateolabrax japonicus2),
Lateolabrax latus), (Scolopsis ciliatus, Nemipterus marginatus))). The haemulids and
lutjanids + caesionids form an unresolved dichotomy with a weak support (decay value 3)
separating them from other percoids ((Haemulon sciurus, Pomadasys maculatus),
(Caesio cuning, Lutjanus decussatus)). Strong decay support (9) and bootstrap values
31
separate Centropomus undecimalis from other percoid groups. Cyprinus carpio and
Luxilus zonatus were clearly outside of the Perciformes.
Weighted Data: Transversions only Third - Saturation in the third codon position
transitions occurred at the approximate mean pairwise sequence divergence within the
Sparidae. Transitions at a saturated site may not reflect homologous changes between
taxa and evolutionary relationships inferred from saturated sites are less likely to be
reflective of phylogenetic history. Down-weighting or eliminating the contribution of
saturated transitions within the analysis might decrease systematic error due to homoplasy
(Swofford, Olsen, Waddell and Hillis in Hillis, et al., 1996). Although not all third codon
position transitions were saturated, there is no means to separate those that are saturated
from those that are unsaturated. For that reason, all third position transitions were
eliminated in this analysis (even though some informative data were sacrificed). A step
matrix was developed that weighted all third position transitions 0 and transversions 1
and was applied to third positions under the “Set Character Types” of the Data option of
PAUP4.0b2* . A heuristic search of 1000 random addition replicates yielded two EPTs
(Figures 17, tree length = 2536, CI = 0.2504, RI = 0.5816 starting seed 303516572,
cytochrome b nucleotide data set using all changes at first and second position and only
transversions in third position, all other parameters of this analysis as in the previous
analysis). A strict consensus of both tree is shown in Figure 18 and revealed a
monophyletic Sparidae and a monophyletic Sparoidea (Sparidae & Centracanthidae +
Lethrinidae + Nemipteridae ) although no bootstrap support and minimal decay support
(2) for the Sparoidea node was observed. The Sparoidea were sister to Lutjanidae &
Caesionidae followed by Haemulidae and a clade containing Moronidae & Lateolabrax .
The later clade established Lateolabrax sister to Moronidae. To date no cladistic study
has established the Moronidae sister to Lateolabrax, although the relationship has been
32
hypothesized based on scale anatomy by McCully (1962) and based on morphology by
Waldman (1986). Within the Sparidae, Calamus, Stenotomus and Archosargus &
Lagodon were removed from their previous placement and are basal to all other Sparidae.
The two distinct clades rendered in the previous analysis remained, but the relative
location of certain taxa was not stable. Notably, Boops shifted in placement from Boops
& Crenidens in the previous tree to (Boops & Sarpa) + (Spondyliosoma & Spicara) and
Crenidens shifted to a clade with Lithognathus.
Nucleotide Translations - A heuristic search of 100 random addition replicates of the
cytochrome b amino acid residues translated from the nucleotide data set resulted in 699
equally parsimonious trees (tree length = 637, CI = 0.5620, RI = 0.7082, starting seed
785084916; all characters unordered and equal weight of 1; outgroup taxa Cyprinus
carpio and Luxilus zonatus; branch-swapping = stepwise addition; swapping algorithm =
tree bisection-reconnection; no topological restraints; character-state optimization =
accelerated transformations). Bootstrap support (100 replicates, restricted to 5000 trees
searched/replicate of length > 400) and Bremer decay values (20, restricted random
addition sequences per node 1000 trees searched/replicate of length > 400 ) were
calculated and are shown on a strict consensus of the 699 equally parsimonious trees
(Figure 19). The Sparidae were monophyletic in the consensus of all EPTs and contained
two distinct clades, similar to both the nucleotide analyses. As with the unweighted
nucleotide tree, Calamus nodosus was basal to all other sparids. Notable in the consensus
tree was the removal of Lagodon from other western Atlantic sparids and the subsequent
inclusion of Lagodon to a Spondyliosoma-Spicara clade. As expressed from the
nucleotide data, the amino acids support Lethrinidae as sister to the Sparidae. The
placement of the Centropomus undecimalis translation within the Nemipteridae was
curious. Lateolabrax placed basal to all other percoids and was not sister to the Morone-
33
Dicentrarchus clade as supported by the nucleotide data. Clade support for sparid
monophyly was strong, but there was no bootstrap support for the two distinct clades
found within the sparids. Three clades (Boops boops & Sarpa salpa), (Acanthopagrus
berda & Sparidentex hasta) and (Rhabdosargus thorpei & Sparodon durbanensis) had
minimal support within the Sparidae and the only other structuring was limited to the
clade of (Pachymetopon aeneum & Polyamblyodon germanum).
Biogeography - The monophyletic Sparidae clade within the unweighted cytochrome b
phylogeny (Figure 18) was used as the source of dependent data for biogeographic
analysis. The Sparidae clade was pruned from the original tree and all ancestral states
were numbered (Figure 20). The geographic distribution was defined for terminal taxa
using FAO areas (Table 10) and these areas were treated as independent data. Areas of
occurrence were determined relative to each terminal taxa or ancestral node and coded
into a binary matrix; presence=1, absence=0 (Table 11).
Parsimony analysis of the data matrix resulted in a single most parsimonious tree
and is overlaid on a world-wide map in Figure 21 (tree length = 102; CI =0.7549; RI =
0.8377; of 82 total characters; all characters unordered and equal weight, 5 characters
were constant, 7 variable characters were parsimony uninformative, 70 variable
characters were parsimony informative; 1000 replicates of random stepwise addition;
100 trees held at each step; starting seed = 1524546659; character-state optimization:
Delayed transformation).
All taxa were found in more than one FAO area, except the Bermuda endemic
Diplodus bermudensis and the Japanese endemic Evynnis japonica. Many of the taxa
were assigned to multiple areas (for example, Acanthopagrus berda). The tree was
rooted to the node of southwestern Pacific Ocean and eastern Indian Ocean + western
central Pacific Ocean + northwestern Pacific Ocean. The western Indian
34
Ocean/southeastern Atlantic clade was sister to eastern Atlantic/Mediterranean-Black Sea
species. These were in turn sister to western Atlantic species. All Atlantic Sparidae + the
western Indian Ocean/Atlantic species were sister to Pacific Ocean/eastern Indian Ocean
clade. The eastern Indian Ocean - western central and northwestern Pacific taxa formed
an unresolved polytomy.
35
DISCUSSION
Sequence Divergence and Codon Base Composition Bias
The majority of sequence divergence was due to third codon position substitutions
as seen in the plot of total substitutions from the third position as a function of
uncorrected sequence divergence (Figure 7). Third positional transitional substitutions
accounted for the most of the homoplasy in the cytochrome b data set. These characters
were not informative for resolving relationships of more distant species in the unweighted
analysis. Third positional transitions appeared to reach saturation at <15% sequence
divergence and provided the majority of homoplastic characters to the analysis. Site
saturation occurs when the accumulation of multiple substitutions at a site confounds the
estimation of the total number of substitutions. For example, a site that started with the
nucleotide “A” mutated to “G” and then reverted to “A” would constitute two
substitution events. During phylogenetic analysis, those taxa with the original “A” at that
position would be paired with those taxa with the reverted “A” at that position because
there is no way to separate when a base pair has reverted or changed multiple times.
During analysis the multiple substitution event that occurred at this site would be masked.
In fact those taxa with the reverted “A” are actually recent descendants of those taxa with
the “G” substitution and not of those with the original “A”. This systematic error is
intrinsic to all nucleotide analyses and there is no means by which to separate all multiple
substitutions at saturated positions. Because of saturation, third positional transitions
were weighted to a value of 0 in the weighted analysis in order to estimate phylogenetic
relationships. The sequence divergence and third positional substitution saturation
reported here were equivalent to other cytochrome b studies that include percoid fishes
36
(for example, Lydeard and Roe, 1997; Song et al., 1999 and others).
Base compositional bias was comparable to other cytochrome b studies of fishes
(Cantatore et al., 1994; Lydeard and Roe, 1997 and Song et al., 1998). The bias across
all codons was 13% for all taxa compared. The smallest bias value was found in the first
codon position, with a value of 4.2%, and the highest value was found in the third codon
position, with a value of 27%. There was a strong anti-guanine bias in the third codon
position with a shift to pro-cytosine. This has been reported in perciform fishes (Meyer,
1993; Cantatore, et al., 1994; Lydeard and Roe, 1997 and Song et al., 1999) and is not
uncommon in other vertebrates. The taxa in this study had nearly equivalent variation in
base compositional bias as reflected in the low standard deviation values (Table 8). The
highest standard deviations were found in the third codon position. High levels of
variation between taxa in base compositional bias can lead to potentially unreliable
phylogenies (Lydeard and Roe, 1997). The variations in this data set were comparable to
other cyt b studies and not considered high enough to mislead phylogenetic inference.
(Lydeard and Roe, 1997).
The Sparidae were monophyletic with the inclusion of the Centracanthidae in all
phylogenies derived from the cyt b sequence data or subsequent amino acid residue
translations. Thus, the centracanthids are here considered members of the Sparidae. This
study included members of Spicara but did not include Centracanthus. Therefore overall
placement of the Centracanthidae could not be verified. For the sake of discussion
brevity, the Sparidae + Spircra will be referred to as the “Sparidae”. The Centracanthidae
were considered members of the Sparidae by Jordan and Fesler (1893) and very closely
related to the Sparidae based on jaw morphology by Regan (1913) and Smith (1938).
Johnson (1980) noted the close relationship of the Centracanthidae to the Sparidae, but he
37
retained the family status of the Centracanthidae “pending a more complete
understanding of sparoid interrelationships”. The molecular evidence reported in this
study support the provisional insertion of Spicara within the sparids, but these same data
question the monophyly of the Centracanthidae because there was no support for a
monophyletic Spicara; it was polyphyletic in the unweighted and weighted nucleotide
analyses.
The sister group to the Sparidae was the Lethrinidae in both nucleotide analyses
and also in the amino acid translations. There was support for the superfamily Sparoidea
(Sparidae, Centracanthidae, Lethrinidae and Nemipteridae) in both the weighted
nucleotide and amino acid analyses but no support was found in the unweighted
nucleotide analysis. Lack of support for the Sparoidea from the unweighted data-set may
be a result of unresolvable third positional substitutions that render the third codon highly
homoplastic. The Sparidae were derived within the Sparoidea. The Lethrinidae were
sister to the Sparidae and the Nemipteridae were basal within the Sparoidea. In the
amino acid residue phylogeny, Centropomus undecimalis was included with the
Nemipteridae. This was probably due to an artifact of inadequate taxonomic sampling of
the snooks. No other centropomid cyt b sequence was available during this study and
although the amino acid translation of Centropomus undecimalis is similar to the
nemipterids, it would likely fall out into a unique centropomid clade if another sequence
were available.
The Sparidae appear derived within the Sparoidea. The Lethrinidae were sister to
the Sparidae and the Nemipteridae were basal to the Lethrinidae + Sparidae. The cyt b
nucleotide data did not support a strong relationship between the nemipterids and the
lethrinids as proposed by Johnson (1980). The sequence data did support an overall sister
relationship between the Sparidae and Lethrinidae (unweighted, weighted, and amino
acid), but there was little clade support for placement of the Nemipteridae sister to the
38
Lethrinidae in the weighted analysis and the nemipterids were sister to the Moronidae +
Lateolabrax in the unweighted tree. A moderate decay value=3 supported the Sparoidea
in the weighed analysis, but there is no support for the monophyly of the Sparoidea in the
unweighted or amino acid residue trees.
In the weighted analysis the relationship of the Sparoidea to other percoids was
better defined than in the unweighted analysis. The lutjanoids (Lutjanus + Caesio) placed
sister to Sparoidea, and the Haemulidae were sister to the lutjanoids + Sparoidea. The
Moronidae + Lateolabrax were basal (decay=7) to the Haemulidae + lutjanoids +
Sparoidea. Centropomus undecimalis was sister to all other percoids.
Phylogenetic relationships of the sparid Subfamilies
Of the four subfamilies of Sparidae (Boopsinae, Denticinae, Pagellinae, Sparinae)
proposed by Smith (1938) and Smith and Smith (1986) and the two (Pagrinae and
Diplodinae) proposed by of Akazaki (1962), none was found to be monophyletic based on
cytochrome b nucleotide data or subsequent amino acid translations. Since these
subfamilies are defined mostly by trophic type, these results suggests that different
feeding modes have evolved multiple times within the Sparidae. This is similar to what
was concluded by Hanel and Sturmbauer (2000) based on a phylogeny of sparid 16S
sequences. It is also possible that the cytochrome b phylogenies reflect past introgression
or hybridization events. Such events would confound phylogenies because they would
no longer reflect true descendent relationships but instead would reflect recent (or past)
hybridization events. Therefore, members of different genera (from the same area) might
appear more closely related to each other than to other members of their own genus.
There is no conclusive evidence for hybridization within natural populations of sparid
fishes. However, Dentex dentex and Pagrus major are known to hybridize in captivity
(Kraljevic, and Dulcic, 1999).
39
The Boopsinae, as defined by Smith (1986), comprise Boops, Crenidens,
Gymnocrotaphus, Oblada, Pachymetopon, Polyamblyodon, Sarpa and Spondyliosoma.
In both of the nucleotide trees, all members of the Boopsinae were contained within
Group I of the family Sparidae (Figure 16, clade I and Figure 18, clade I) but are rendered
paraphyletic within the group. The southeastern Atlantic/western Indian Ocean Sarpa
salpa was sister to a clade containing the eastern Atlantic Spondyliosoma cantharus, and
the eastern Atlantic/Mediterranean centracanthid Spicara maena. These were sister to a
group of western Indian Ocean fishes - the boopsines Pachymetopon aeneum +
Polyamblyodon germanum and Gymnocrotaphus curvidens and the pagelline,
Boopsoidea inornata. Most of these species have trophic similarities that may explain
their evolutionary relatedness, as well as shared geographic rage.
Most genera of the Denticinae (Argyrozona, Cheimerius, Dentex, Petrus,
Polysteganus and Sparidentex) were found within sparid Group II (Figure 16, clade II and
Figure 18, clade II) except for Sparidentex hasta, which was found in Group I . Within
the Denticinae, Dentex was not monophyletic. In the unweighted and weighted analysis,
Dentex dentex was sister to Cheimerius nufar and Dentex tumifrons was sister to the
centracanthid Spicara alta. The placement of Dentex tumifrons with Spicara alta is
novel, but enigmatic without a more complete understanding of the placement of the
other members of Centracanthidae. Petrus rupestris, a southeastern Atlantic/western
Indian Ocean species and the western Indian Ocean Polysteganus praeorbitalis formed a
robust clades in both nucleotide analyses. Furthermore, Argyrozona argyrozona, a
western Indian Ocean denticine was basal to Petrus + Polysteganus. Yet these fish were
sister to a clade containing three western Indian Ocean Sparinae (Chrysoblephus,
Cymatoceps and Porcostoma) as opposed to other denticines. Pterogymnus laniarius, a
southeastern Atlantic/western Indian Ocean sparine fell outside of this whole group.
Sparidentex hasta is problematic within the denticines. Munro (1948:276) placed it
40
within the Denticinae based on dentition, although it belongs outside of the Denticinae
based on jaw morphology (K. Carpenter, unpublished data). In both nucleotide clades
Sparidentex hasta was sister to Acanthopagrus berda. This may appear as an unlikely
relationship, but Sparus hasta (senior synonym of Sparidentex hasta) was synonymized
with Acanthopagrus berda (Forsskål 1775) by Bauchot & Skelton 1986:331, although
they clearly are not congeners (K. Carpenter, unpublished data).
The Diplodinae (Archosargus, Diplodus and Lagodon) were restricted to sparid
Group I in the unweighted nucleotide clade, but they were polyphyletic in the weighted
nucleotide tree. Diplodus formed a well supported, monophyletic clade in the
unweighted and weighted trees. Diplodus holbrooki was sister to the Bermuda endemic
species, Diplodus bermudensis and both were sister to D. argenteus and the South
African D. cervinus. Oblada melanura a Mediterranean/Eastern Atlantic monotypic
sparid, clustered with Diplodus in the unweighted and weighted analyses. The placement
of the genus outside of Boopsinae is novel. In the unweighted analysis, Archosargus
probatocephalus formed a stable node with Lagodon rhomboides but had little affinity for
other genera in the Diplodinae of Akazaki (1962). The mtDNA placed four genera of
Western Atlantic Sparidae together; the diplodines, A. probatocephalus and L.
rhomboides, were sister to the sparines Stenotomus chrysops and Calamus nodosus. The
weighted analysis supported the Archosargus + Lagodon clade, but placed it outside
Groups I + II and Stenotomus, and placed Calamus basal to all other Sparidae.
The subfamily Pagellinae (Boopsoidea, Pagellus and Lithognathus) was
polyphyletic within the Sparidae. The genus Pagellus were polyphyletic in the
unweighted and weighted data analyses. Pagellus was the only sparid genus that was
found in both Group I and Group II. The Mediterranean Pagellus bellottii was sister to
the western Atlantic Pagrus pagrus in Group II, and Pagellus bogaraveo was sister to
Lithognathus mormyrus in the unweighted analysis, but there was no support for this
41
relationship in the weighted clade. Pagellus appears to be a polyphyletic assemblage. As
previously mentioned, Boopsoidea inornata fell within the boopsines and not with
pagellines.
The Pagrinae (Argyrops, Evynnis, Pagrus) were paraphyletic within sparid Group
II. Within the Pagrinae the genus Pagrus was problematic. Pagrus was paraphyletic
within Group II of the Sparidae. The Western Pacific genera of Pagrinae (Pagrus
auratus, Evynnis japonica and Argyrops spinifer) formed a well supported clade in the
unweighted analysis. The genetic relationship of Pagrus auratus to its geographic
siblings rather than to other members of Pagrus is enigmatic and is supported by the close
genetic distance found among Japanese sparids of Taniguchi et al. (1986). Either the
taxonomic placement of Evynnis and Argyrops outside of Pagrus is in error, or Pagrus is
not a monophyletic group.
In the unweighted and weighted analyses, the subfamily Sparinae (Acanthopagrus,
Calamus, Chrysoblephus, Chrysophrys, Porcostoma, Pterogymnus, Rhabdosargus,
Sparodon, Sparus and Stenotomus) was distributed across both sparid Group I and II. As
mentioned above, Calamus nodosus and Stenotomus chrysops formed a stable node that
was sister to a clade containing other western Atlantic sparids in the unweighted tree.
However, both Calamus and Stenotomus were basal to other Sparidae in the weighted
analysis. Within Sparid Group II of both the unweighted and weighted analyses, there
was a strong grouping of western Indian Ocean (WIO) and southeastern Atlantic-WIO
sparids that included four members of the Sparinae (Cymatoceps nasutus, Chrysoblephus
cristiceps, Porcostoma dentata and Pterogymnus laniarius) and three Denticinae
(Argyrozona argyrozona, Petrus rupestris and Polysteganus praeorbitalis), suggesting an
attraction of South African native (P. dentata and C. cristiceps) or South African
distributed Sparidae. In Group I from the unweighted data, the sparines including
Acanthopagrus berda + Sparidentex hasta , were sister to Rhabdosargus thorpei +
42
Sparodon durbanensis and Sparus auratus. These sparids had a broader distribution
(WIO, Indo-Pacific and eastern Atlantic) and did not reflect the geographic similarity as
found in the Sparinae from Group II. The placement of Sparidentex hasta with sparines
primarily from the western Indian Ocean (except S. auratus from the eastern Atlantic) is
possible evidence that Sparidentex hasta belongs outside of the Denticinae.
Phylogenetic Relationships From the Amino acid residue data
The Sparidae formed a well supported monophyletic group in the clade derived
from the amino acid residue data, yet there was little support for phylogenetic structuring
within the Sparidae; only three sister relationships were supported: Pachymetopon
aeneum + Polyamblyodon germanum, Acanthopagrus berda + Sparidentex hasta, and
Rhabdosargus thorpei + Sparodon durbanensis. Additionally, there was only weak
support for independent groups in this data set. Notable within the Sparidae clade,
Diplodus cervinus, the only non-Western Atlantic form, was removed from other
Diplodus, and this may reflect an isolation event that allowed for the co-evolution of
sister groups with no genetic mixing across the Atlantic. In agreement with the weighted
analysis, Calamus nodosus was basal to all other Sparidae. Weak evidence (decay=2)
supported the Lethrinidae sister to the Sparidae in the amino acid residues, but there is no
support for Sparoidea.
Biogeography - The results of biogeographic analysis suggested a strong vicariant
explanation to the structuring of genera within the Sparidae. There were two areas of
sparid evolution, eastern Indian Ocean - western Pacific and western Indian Ocean Mediterranean/Atlantic. These species probably had a Tethyan Sea common ancestor.
However, little more can be drawn from this analysis, because the vicariance
biogeography clade was based on a single tree example and this is considered equivocal
43
to a single transformation series explaining a clade (Wiley et al., 1991). It would be
preferable to have multiple dependent data sets (phylogenies from other species) to test
the biogeographic relationships of a group. Therefore, the power of this analysis is little
more than can be derived from purely descriptive biogeography.
Phylogenetic Conclusions
The results of parsimony analyses based on the cytochrome b nucleotide data
generated during this study: 1) established a close phylogenetic relationship of species,
such as Polyamblyodon germanum and Polysteganus praeorbitalis; 2) established the
monophyly of the family Sparidae; 3) estimated the validity of subfamilies within the
Sparidae; and lastly 4) clarified the relationships of sparoid fishes within the percoids.
There is a limit to the ability of these data to elucidate relationships without considering
the subtle characteristics of nucleotide substitution within cyt b. The phylogenetic
structuring, displayed in the consensus tree from the unweighted analysis, was dominated
by changes in the third codon position (transitions > transversions). The partitioned
Bremer values showed the majority of the support for a given clade came from the third
codon position (Figure 16). In the weighted analysis, where all changes were considered
from the first and second codon, and only transversions from the third position
(transitions weight=0), there was an obvious reduction in the contribution of third
positional support for clades and a subsequent increase in the proportion of support from
first and second positions (Figure 18). The overall effect of down-weighting transitions
during the weighted analysis was increased resolution for intermediate relationships. In
the unweighted clade, the signal of intermediate percoid relationships was shaded by site
saturation (accumulation of multiple substitutions at a site) in the third codon position.
This resulted in unresolvable phylogenetic information for taxa that had divergences of
the same magnitude as that of site saturation. By removing the saturated data, the
44
phylogenetic signal was derived from the more conserved codon positions, one and two,
and from the less frequent transversional changes of codon position three. Conserved
data possibly contained useful information of past evolutionary events that were
invaluable in resolving intermediate perciform relationships.
The most conserved data, derived from this study, was that of amino acid residue
translations of the cytochrome b nucleotide data. The translations were informative in
constructing a monophyletic Sparidae that was sister to the Lethrinidae, but were not
informative in resolving percoid relationships.
The two distinct groups deduced within the Sparidae revealed the inability of the
current classification to define sub-structuring within the family. These groups are similar
to the “Lineages 2 & 3" found in Hanel and Strumbauer’s (2000, Fig. 1.) 16S phylogeny
of northeastern Atlantic and Mediterranean sparid. Clearly the subfamilies, as currently
proposed, are not valid. Within each group, derived from cytochrome b, no distinct
pattern could be visualized that reflects the current criteria for sub-structuring. For
example, sparid Clade I contains all members of the current Boopsinae (herbivores with
compressed outer incisiform teeth) and but also members of Sparinae, Pagellinae and
Diplodinae. There is no external morphological character to unite members of this group,
nor do they all belong to one feeding type (herbivore, carnivore and omnivore). There is
no evidence overall for geographic similarity to unite all these fish, although there is
some structuring within certain nodes based on geography. It is suggested from these
data that feeding types evolved multiple times within the Sparidae.
The phylogenetic relationships derived from the cytochrome b nucleotide data
and the subsequent amino acid translations corroborate the Sparidae as monophyletic.
However, none of the phylogenetic trees from these data support the division of the
Sparidae into the previously defined subfamilies; the currently defined subfamilies are
inadequate to explain the structuring produced from these data. Although, there were two
45
groups within the Sparidae, there is no unifying character/s within these groups to support
defining them as subfamilies. Clearly a revision of the Sparidae is needed to redefine
structuring between genera. The nucleotide data supported the Lethrinidae sister to the
Sparidae and there was support, however minimal, for the Sparoidea..
46
TABLE 3. COLLECTION DATA FOR SPECIMENS USED. GENBANK
ACCESSION NUMBER, MUSEUM COLLECTION NUMBERS AND COLLECTION
LOCALITY ARE GIVEN. MUSEUM ACRONYMS ARE FOLLOWING (LEVITON
ET AL., 1985 AND 1988).
All
TAXA
Outgroup Taxa
Centropomidae
Centropomus undecimalis
Cyprinidae
Cyprinus carpio
Luxilus zonatus
Haemulidae
Haemulon sciurus
Pomadasys maculatus
Lethrinidae
Lethrinus ornatus
Lethrinus rubrioperculatus
Lutjanidae
Caesio cuning
Lutjanus decussatus
Moronidae + (Lateolabrax)
Dicentrarchus labrax
Dicentrarchus punctatus
Lateolabrax japonicus
Lateolabrax japonicus2
Lateolabrax latus
Morone americanus
Morone chrysops
Morone mississippiensis
Morone saxatilis
Nemipteridae
Nemipterus marginatus
Scolopsis ciliatus
Ingroup Taxa
SPARIDAE
Boopsinae
Boops boops
Crenidens crenidens
Gymnocrotaphus curvidens
Oblada melanura
Pachymetopon aeneum
Polyamblyodon germanum
Sarpa salpa
Spondyliosoma cantharus
Denticinae
Argyrozona argyrozona
Cheimerius nufar
Dentex dentex
GenBank
Accession#
Museum
Catalog#
AF240739 No Voucher
X61010
U66600
Collection
Locality
Florida (Collected by J. Gelschlecter)
Sequences From GenBank
AF240747 No Voucher
AF240748 USNM uncat
Florida, Big Pine Key, W. of Bridge
Philippines, Manila Market
AF240751 USNM 345259 Philippines, Bolinao, Luzon
AF240752 USNM Uncat
Australia, W. Australia CSIRO SS 8/95/45
AF240749 USNM 345193 Philippines, Iloilo Panay, Market
AF240750 USNM 346695 Philippines., Northern Negros, Market
X81566
AF240740
AF250741
AF240742
AF240743
AF240744
AF240745
AF045362
AF240746
No Voucher
fish market, Spain
VIMS 10381
Picture Voucher, Japan Market Sample
VIMS 10381
MTUF 27451
Sasebo, Nagasaki Prefecture, Japan
No Voucher
VIMS Trawl Survey Chesapeake Bay
UT 85.91
Cherokee Reservoir, Grainger Co, TN
VIMS Uncat
AF240754 USNM 345202 Philippines, Luzon, Manila, Market
AF240753 USNM 346853 Philippines, Guimaras Island, JTW 95-1
X81567
AF240699
AF240700
AF240701
AF240702
AF240703
AF240704
AF240705
No Voucher
Qatif Market, eastern Saudi Arabia
RUSI 49447
Kenton-on-Sea, South Africa
No Voucher
Spain, Azohia, Bay of Cartagena
RUSI 49672
RUSI 49690
RUSI 49456
ODU 2782
Fiumicino Fish Market, Italy
AF240706 RUSI 58449
Durban Fish Market, South Africa
AF240707 RUSI 49443
AF143197 Sequences From GenBank
47
Dentex tumifrons
Petrus rupestris
Polysteganus praeorbitalis
Sparidentex hasta
Diplodinae
Archosargus probatocephalus
Diplodus argenteus
Diplodus bermudensis
Diplodus cervinus
Diplodus holbrooki
Lagodon rhomboides
Pagellinae
Boopsoidea inornata
Lithognathus mormyrus
Pagellus bogaraveo
Pagellus bellottii
Pagrinae
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Sparinae
Acanthopagrus berda
Calamus nodosus
Chrysoblephus cristiceps
Cymatoceps nasutus
Porcostoma dentata
Pterogymnus laniarius
Rhabdosargus thorpei
Sparodon durbanensis
Sparus auratus
Stenotomus chrysops
Centracanthidae
Spicara alta
Spicara maena
AF240708
AF240709
AF240710
AF240734
AMS I.36450-002 Nelson Bay, Australia
AF240716
AF240721
AF240722
AF240723
AF240724
AF240726
VIMS 010192
NSMT-P 48013
No Voucher
RUSI 49680
ODU 2789
VIMS Uncat
Chesapeake Bay
Sea Life Park Tokyo, Origin: Argentina.
Bermuda
Atlantic, South Carolina
Florida Keys, Bahia Honda Ocean Side
AF240711
AF240712
AF240713
AF240714
ODU 2791
ODU 2784
ODU 2785
ODU 2792
St. Sebastian Bay, South Africa
Fiumicino Market, Italy
R/V African, Station 17491, South Africa
AF240717
AF240725
AF240727
AF240728
AF240729
AMS I.36447-001 N. Territory, Australia
NSMT-P 47497
No Voucher
ODU 2786
ODU 2790
Miyazaki, Kyushu Prefecture, Japan
New Zealand, Sydney Market
V. Emmanul Market, Rome, Italy
AF240715
AF240718
AF240719
AF240720
AF240730
AF240731
AF240732
AF240733
AF240735
AF240736
USNM 345989
No Voucher
RUSI 49441
RUSI 49445
RUSI 58450
KENT SAPL1
RUSI 49683
RUSI 49673
ODU 2787
VIMS Uncat
Philippines, Manila, Market.
Atlantic S.E. Charleston, SC
Durban, South Africa
Plettenberg Bay, South Africa
Ponta do Ouro, Mozambique
Chesapeake Bay
RUSI 49684
RUSI 49686
NSMT-P Uncat Shuwaik Market, Kuwait City, Kuwait
AF240737 ODU 2793
AF240738 ODU 2788
Carpenter, K.E.
MTUF-Museum of the Tokyo University of Fisheries, Tokyo, Japan; NSMT-P National Science Museum
(Zoological Park) Tokyo, Japan; ODU- Old Dominion University, Norfolk, VA; RUSI- Rhodes University;
J.L.B. Smith Institute of Ichthyology, Grahamstown, South Africa; UT University of Tennessee,
Knoxville, TN; USNM- United States National Museum of Natural History, Washington, DC; VIMSVirginia Institute of Marine Science, Gloucester Point, VA
48
TABLE 4. MULTIPLE ALIGNMENT OF NUCLEOTIDE SEQUENCES OF THE
CYTOCHROME B GENE FROM THE SPARIDAE AND OUTGROUP SPECIES.
TAXONOMIC NAMES WITH A SUFFIX OF “GB” ARE FROM GENBANK AND
ARE INCLUDED WITH ORIGINAL SEQUENCES FOR EASY COMPARISON.
49
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Dicentrarchus labraxGB
Lateolabrax latus
Morone americanus
Morone chrysops
Morone mississippiensisGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
ATGGCAAGCCTTCGAAAAACACACCCCCTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACTCACCCCCTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACCCACCCCCTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACCCACCCCCTATTAAAAATTGCTAACCATGC
ATGGCTAGCCTTCGAAAAACGCACCCCCTATTAAAAATTGCTAATCACGC
ATGGCAAGCCTTCGAAAGACACACCCGCTATTAAAAATTGTTAATCATGC
ATGACAAATCTTCGAAAGACTCACCCCCTCCTGAAAATTGCCAATCACGC
ATGGCAAGCCTCCGAAAAACCCACCCACTATTAAAGATTGCTAATCACGC
ATGGCTAGCCTTCGAAAGACTCACCCCCTATTAAAAATTGCTAATCACGC
ATGGCAAGTCTCCGAAAAACGCACCCCCTATTAAAAATCGCTAACCACGC
ATGACAAGCCTTCGAAAAACTCACCCCCTATTAAAAATTGCTAATCATGC
ATGGCAAGCCTCCGGAAAACCCACCCACTACTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAAACCCAGCCCTTACTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAAACACACCCCCTATTAAAAATCGCCAACCACGC
ATGGCAAGCCTTCGAAAAACACACCCCCTATTAAAAATCGCTAACCACGC
ATGGCAAGCCTTCGAAAAACACACCCCCTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTACGAAAAACACACCCCCTATTAAAAATCGCCAACCACGC
ATGGCAAGCCTTCGAAAGACCCACCCCTTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACACACCCCCTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAAACCCACCCCCTATTAAAAATCGCTAACCACGC
ATGGCAAGCCTACGAAAAACACACCCCCTGCTAAAAATCGCTAACCACGC
ATGGCAAGCCTTCGAAAAACACACCCCTTGTTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACGCACCCCCTACTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAAACGCACCCCCTATTAAAAATTGCTAATCACGC
ATGGCAAGCCTCCGGAAGACTCACCCCTTATTAAAAATTGCTAATCATGC
ATGGCAAGCCTTCGAAAGACCCACCCCTTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTCCGAAAAACCCATCCATTATTAAAAATTGCTAATCACGC
ATGGCAAGCCTCCGAAAGACTCACCCCTTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTTCGAAAGACCCACCCCCTATTAAAAATTGCTAATCACGC
ATGGCAAGCCTTCGAAAGACCCACCCCTTGTTAAAAATTGCTAATCACGC
ATGGCAAGCCTTCGAAAGACGCACCCCCTATTAAAAATTGCTAACCACGC
ATGGCTAGCCTCCGAAAAACTCACCCCCTCTTAAAAATCGCCAATCATGC
ATGGCAAGCCTCCGAAAAACGCACCCCCTATTAAAAATTGCTAACCATGC
ATGGCAAGCCTTCGAAAAACGCATCCTTTATTAAAAATTGCTAACCACGC
ATGGCAAGTCTTCGAAAAACTCACCCCCTCTTAAAAATCGCCAATCACGC
ATGGCAAGCCTTCGTAAGACACACCCCCTCTTAAAAATCGCTAATCACGC
ATGGCAAGCCTTCGAAAGACACACCCCCTATTAAAAATTGCGAACCACGC
ATGGCAAGCCTCCGAAAGACTCACCCCCTACTAAAAATTGCTAACCACGC
ATGGCCAGCCTTCGAAAGACGCACCCCTTATTAAAAATTGCTAACCACGC
ATGACAAGCCTTCGAAAGACACACCCTTTATTAAAAATTGCTAACCACGC
ATGGCAAGCCTACGAAAAACACACCCTCTCATTAAAATCGCTAACGACGC
ATGGCAAGCCTACGAAAAACCCACCCACTGATAAAAATCGCTAATGGCGC
ATGGCAAGCCTACGAAARNNCCACCCCCTCCTAAAAATCGCAAACGACGC
ATGGCCGCCCTCCGTAAAACACACCCCCTATTAAAAATCGCAAATCATGC
ATGAGCGCCCTCCGTAAAACACACCCCCTACTAAAGATTGCAAATCACGC
ATGGCAAGCCTTCGAAAAACCCACCCCCTACTAAAAATCGCAAACGACGC
ATGGCAAGCCTTCGAAAAACCCACCCCCTGCTAAAAATCGCAAACGACGC
ATGGCAAGCCTTCGAAAAACCCACCCCCTGCTAAAAATCGCAAACGACGC
ATGGCCTCGCTTCGTAAATCGCACCCACTGCTTAAAATCGCAAACAACGC
ATGGCCGCCCTTCGTAAAACACATCCCCTACTAAAAATCGCAAATGACGC
ATGGCCTCGCTTCGTAAATCGCACCCACTACTTAAAATCGCAAACAACGC
ATGGCCGCCCTTCGTAAAACGCACCCGCTACTAAAAATCGCAAACGACGC
ATGGCCAACCCCCGAAAAACTCACCCCCTACTAAAGATTGCGAATGACGC
ATGGCAAACCTTCGTAAAACCCATCCATTATTAAAGATCGCAAACGATGC
ATGGCAAGCCTACGCAAAACCCACCCACTACTAAAAATTGCAAACGACGC
ATGGCAAGCCTACGCAAAACCCACCCACTACTAAAAATTGCTAACGACGC
ATGGCTTGCTTACGCAAAACGCACCCCCTCCTAAAAATTGCAAACGACGC
ATGGCTAGCTTACGCAAGACCCATCCCCTCCTAAAAATTGCTAACGATGC
ATGGCCAGCCTTCGCAAGACGCCCCCCCTCCTAATAATTGCTAACAACGC
ATGGCCAGCCTTCGCAAAACTCATCCTCTCCTTAAAATCGCAAATGACGC
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
50
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
AGTAGTTGACCTACCTGCACCCTCCAACATTTCCGTTTGATGAAATTTTG
ACTAGTTGACCTGCCCGCACCCTCCAACATTTCCGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCATCAAATATTTCTGTCTGATGAAATTTCG
AGTAGTTGACCTACCTGCGCCCTCCAANATTTCTGTTTGATGAAATTTTG
ATTAGTTGATCTCCCTGCACCATCCAATATTTCCGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCCTCCAACATTTCCGTCTGATGAAATTTTG
ACTAGTCGACCTACCCGCCCCTTCCAATATTTCTGTTTGATGAAATTTCG
AGTAGTTGACCTACCTGCACCCTCTAATATTTCTGTCTGATGAAATTTTG
AGTAGTTGATCTACCTGCGCCTTCCAACATTTCCGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCCTCCAATATTTCAGTCTGATGAAACTTTG
GGTAGTTGATCTACCGGCACCCTCCAACATTTCTGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCCTCTAATATTTCTGTCTGATGAAATTTTG
AGTAGTTGACTTACCTGCACCCTCCAATATCTCTGTTTGATGAAACTTCG
AGTAGTCGACCTACCTGCACCTTCCAATATTTCCGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCTTCCAATATTTCTGTTTGATGAAATTTTG
AGTAGTAGACCTACCTGCGCCATCGAATATTTCTGTCTGATGAAATTTTG
AGTAGTCGACCTTCCTGCACCTTCCAATATTTCCGTCTGATGAAATTTTG
ACTAGTCGACCTGCCCGCACCCTCCAATATTTCCGTTTGATGAAATTTTG
AGTAGTTGACCTACCTGCACCATCCAATATTTCCGTTTGATGAAATTTTG
AGTAGTCGATCTACCTGCACCTTCCAACATTTCCGTCTGATGAAACTTTG
AGTAGTCGATCTACCTGCACCTTCTAACATTTCCGTCTGGTGGAACTTCG
AGTAGTTGACCTACCTGCACCTTCCAATATTTCTGTCTGATGAAACTTTG
AGTAGTTGACCTGCCTGCACCCTCTAATATTTCCGTTTGATGAAATTTTG
ACTAGTTGACCTGCCTGCACCATCGAATATTTCTGTCTGATGAAATTTTG
AGTAGTTGATCTACCTGCACCCTCTAATATTTCTGTCTGATGAAATTTTG
AGTAGTAGACCTACCTGCACCCTCCAATATTTCTGTTTGATGAAATTTTG
ACTAGTTGACCTACCTGCGCCCTCAAACATTTCTGTATGATGAAATTTTG
AGTAGTTGATCTACCTGCACCCTCCAACATTTCTGTCTGATGAAATTTTG
AGTAGTTGACCTACCTGCGCCTTCCAACATTTCAGTATGATGAAATTTTG
AGTAGTTGACCTACCTGCACCTTCTAACATCTCTGTTTGATGGGACTTCG
ACTAGTTGACCTACCTGCGCCCTCAAACATTTCTGTATGATGAAATTTTG
GGTAGTGGACCTTCCTGCCCCCTCCAATATTTCAGTCTGATGAAATTTTG
ACTAGTTGATCTCCCTGCGCCCTCTAATATTTCCGTCTGATGAAATTTTG
AGTAGTCGACCTACCTGCGCCCTCCAATATTTCCGTCTGATGAAATTTTG
AGTAGTAGACCTACCTGCCCCCTCCAACATTTCGGTCTGATGAAACTTTG
AGTAATTGATCTACATGCACCCTCCAATATTTCCGTCTGATGAAATTTTG
ACTAGTTGACCTCCCCGCACCCGCTAACATTTCTGTCTGATGAAATTTTG
ACTCGTCGACCTACCTGCACCCTCCAACATTTCCGTCTGATGAAATTTTG
CCTGGTGGATCTGCCTGCACCCTCCAATATTTCTGTTTGATGAAATTTTG
ACTAGTTGACCTCCCCGCACCCTCTAACATCTCTGTCTGATGAAATTTTG
ACTAGTTGACCTACCAACACCATCCAACATCTCAGCATGATGAAACTTTG
ACTGGTTGACCTTCCAACACCATCAAACATCTCAGCGCTATGAAACTTCG
ACTAATTGACCTCCCAGCCCCCTCCAACATCTCAGCATGATGGAACTTCG
ACTTGTTGACCTGCCGGCCCCTTCAAATATTTCAGTTTGATGAAATTTCG
ACTTGTTGATCTACCAGCCCCCTCCAACATTTCAGTTTGATGAAATTTCG
ACTAGTCGACCTCCCTGCCCCCTCAAACATCTCAGTCTGATGAAATTTTG
ACTGGTCGACCTCCCTGCTCCCTCAAACATCTCGGTCTGATGAAATTTTG
ATTGGTAGACCTCCCTGCCCCCTCGAATATCTCAGTTTGATGAAACTTCG
ACTCGTTGACTTACCTGCCCCCTCAAATATCTCTGTTTGATGAAACTTTG
ACTTGTTGACCTGCCCGCCCCCTCAAACATCTCTGTTTGATGAAACTTTG
ACTCATTGACTTACCTGCCCCCTCAAATATCTCTGTTTGATGAAACTTTG
ACTTGTTGACTTACCTGCCCCTTCAAACATCTCTGTTTGATGAAATTTTG
ACTAGTTGACCTCCCAGCCCCATCCAATATTTCTGTATGATGAAACTTTG
ATTAATTGACCTCCCTGCCCCCTCCAACATCTCCGTATGATGAAATTTTG
ACTAGTTGATCTCCCCGCACCCTCCAATATTTCAGTATGATGAAACTTTG
ACTAGTTGACCTCCCCGCACCCTCTAATATTTCAGTATGATGAAACTTTG
AGTCCTTGACCTTCCAGCCCCTTCAAACATCTCAGTTTGATGAAACTTCG
AGTAGTCGACTTACCAGCCCCCTCTAACATTTCAGTATGATGAAACTTTG
CCTCATTGATCTCCGCGCGCCCTCCAATATCTCAGCCTGATGAAACTTCG
CCTAGTTGACCTACCCGCCCCCGCCAATATTTCTGCATGATGAAATTTTG
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
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[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
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[100]
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51
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GGTCTCTCCTCGGTCTCTGCTTAATCTCCCAACTTCTCACAGGACTATTT
GATCCCTACTTGGCCTTTGTTTAATTTCTCAACTTCTCACGGGCCTATTC
GCTCCCTCCTCGGCCTCTGCCTAATCTCTCAGCTCCTTACAGGACTCTTC
GTTCCCTGCTCGGCCTTTGCCTAATCTCTCAACTTCTCACAGGCCTGTTC
GTTCCCTGCTTGGCCTCTGTCTTATTTCCCAGCTCCTTACAGGGCTATTC
GATCCCTACTTAGCCTCTGTTTAATTTCTCAACTCCTTACAGGACTATTC
GATCTCTCCTTGGTCTCTGTTTAATTTCCCAGCTCCTTACAGGCCTTTTC
GCTCCCTACTGGGTCTCTGCCTAATTTCTCAACTTCTCACAGGGCTGTTC
GCTCCCTGCTCGGCCTCTGCCTAATCTCCCAACTCCTCACAGGATTATTC
GATCCCTACTTGGCCTTTGTTTAGTCTCCCAACTACTTACAGGACTATTC
GCTCTCTGCTCGGCCTCTGTCTAATCTCTCAACTCCTCACAGGGCTATTT
GCTCCCTGCTCGGCCTCTGCTTAATTTCTCAAATCCTCACAGGACTGTTC
GTTCCCTCCTCGGCCTCTGCCTGATTTCCCAACTCCTCACAGGCCTATTC
GATCCTTACTTGGTCTCTGCTTAATTTCTCAACTCCTTACAGGACTTTTC
GATCCTTACTTGGTCTCTGCTTAATTTCTCAACTTCTTACAGGACTTTTT
GATCCTTACTCGGTCTCTGCTTAATTTCTCAACTCCTTACAGGACTATTT
GATCCTTACTTGGTCTCCGCTTAATTTCCCAACTTCTTACAGGACTTTTT
GCTCCCTACTTGGCCTCTGCCTAATTTCTCAGCTCCTCACAGGACTGTTC
GATCCCTACTTGGTCTCTGTTGAATTTCCCAACTCCTCACAGGACTATTT
GGTCCCTACTTGGCCTCTGCTTAATCTCCCAACTCCTTACAGGCCTATTC
GATCCCTACTTGGTCTCTGCCTCATCTCTCAACTTCTTACAGGGTTATTC
GATCTTTACTCGGTCTCTGCCTAATTTCTCAACTCCTTACAGGACTATTT
GATCCCTCCTTGGCCTCTGCTTAATCTCCCAACTCCTCACAGGACTATTC
GATCCCTTCTTGGTCTCTGCCTAATCTCTCAACTCCTTACAGGACTATTT
GATCCCTGCTCGGCCTTTGTTTAATCTCCCAACTCCTAACAGGATTATTC
GCTCTCTACTCGGCCTCTGCCTAATCTCTCAGATCCTCACAGGACTATTC
GCTCCTTACTCGGTCTCTGCCTAATTTCTCAACTACTCACAGGTCTTTTT
GTTCCCTACTTGGCCTTTGCCTGATCTCCCAACTCCTAACAGGACTATTC
GCTCCCTGCTCGGTCTTTGCCTAATCTCTCAAATCCTCACGGGGCTATTC
GTTCCCTGCTCGGCCTCTGCCTAATCTCCCAACTCCTTACAGGCTTATTC
GCTCCCTGCTCGGCCTTTGCCTAATCTCCCAGCTCCTCACAGGATTGTTC
GATCCCTCCTTGGCCTCTGCTTAATTTCCCAGCTCCTCACAGGACTATTC
GCTCCCTGCTCGGTCTTTGCCTAATCTCTCAAATCCTCACGGGGCTATTC
GGTCCTTGCTGGGTCTATGCCTAATCTCGCAACTCCTAACAGGCCTATTT
GATCTTTACTTGGTCTTTGCCTGATCTCCCAGCTCCTCACAGGACTATTT
GGTCCCTCCTCGGTCTCTGCTTAATCTCCCAACTTCTTACAGGACTATTT
GGTCCTTGCTCGGTCTATGCTTAATCTCTCAGCTCCTAACAGGGCGGTTT
GATCCCTCCTCGGTCTCTGTCTAATTTCTCAGCTTCTGACAGGGCTATTC
GGTCCCTACTTGGTCTTTGCCTAATTTCTCAACTCCTCACAGGACTTTTC
GATCCCTCCTCGGCCTTTGTTTAATTTCCCAACTTCTTACAGGCCTGTTC
GCTCCCTGCTCGGCCTTTGCCTAATCTCCCAACTCCTTACAGGCCTATTC
GGTCCCTACTTGGTCTCTGTTTAATTTCCCAACTCCTCACAGGACTCTTC
GATCCCTCCTAGGACTATGCTTAATTACCCAAATTTTAACCGGCCTATTC
GATCCCTTCTAGGGTTGTGCTTAATCACTCAAATCCTCACCGGATTATTC
GCTCCCTCCTAGGCCTCTGCTTAATTGCCCAAATTCTTACAGGCCTATTT
GTTCGCTCTTAGGCCTATGCTTGATTTCCCAAATTCTTACAGGTCTATTT
GCTCGCTCTTAGGCCTATGTTTGATCTCCCAAATCCTTACAGGCCTGTTT
GTTCCCTTCTTGGCCTTTGCTTGATTACTCAAATCCTCACAGGGCTATTC
GCTCTCTTCTTGGCCTCTGCTTGATCACTCAGATCCTCACAGGATTATTC
GCTCTCTTCTAGGCCTCTGCTTGTTCACACAGATCATTACTGGGCTATTC
GCTCACTCCTGGGCCTCTGCTTAATTTCCCAAATTCTCACAGGCCTATTT
GCTCACTCTTGGGCCTATGTTTAATTTCTCAGATCCTTACAGGGCTATTC
GCTCACTCCTTGGCCTCTGTTTGATCTCCCAAATCCTCACAGGCCTATTT
GCTCACTCTTGGGCCTATGTCTAATTTCCCAAATCCTTACAGGCCTATTC
GCTCCCTCCTAGGCCTCTGCCTCATTTCACAGATCGTCACCGGCCTTTTC
GCTCCCTACTAGGACTTTGTCTCATTTCCCAAATCGTTACGGGACTATTC
GCTCTCTACTTGGCCTTTGCTTAATTGCCCAACTCCTAACAGGACTCTTC
GCTCTCTACTTGGCCTTTGCTTAATTGCCCAAATCCTAACAGGACTATTC
GCTCCCTCCTTGGTCTCTGCTTAATTGCCCAAATCTTAACCGGGCTTTTC
GTTCTCTTCTGGGTCTTTGCTTAATCGCTCAAATCCTAACAGGCCTGTTC
GGTCTCTACTAGGTCTTTGCTTAGCCGCCCAAATTTTAACAGGCCTGTTC
GGTCTCTTCTAGGCCTTTGTCTGATTGCACAACTTCTAACAGGCCTATTT
[150]
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[150]
[150]
[150]
[150]
52
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CTTGCTATACATTATACTTCCGATATTGCCACAGCCTTCTCCTCCGTAGC
CTCGCTATACACTACACCTCAGATATCGCCACAGCATTTTCTTCTGTTGC
CTTGCTATACACTACACCTCCGATATTGCCACAGCCTTCTCCTCTGTTGC
CTCGCCATACACTACACCTCAGACATCGCCACAGCCTTTTCTTCTGTCGC
CTCGCCATGCACTATACCTCCGATATCGCCACAGCCTTCTCTTCCGTTGC
CTTGCTATACACTATACTTCAGATATCGCCACGGCCTTTTCTTCTGTCGC
CTTGCTATACATTATACCCCCGACATCGCTACAGCATTTTCTTCTGTTGC
CTTGCCATACATTACACCTCAGACATCGCCACAGCTTTTTCTTCCGTTGC
CTTGCCATACACTACACCTCAGATATTGCCACAGCTTTTTCTTCTGTCGC
CTCGCCATACACTATACCTCCGACATCGCCACAGCCTTTTCTTCGGTTGC
CTTGCCATACACTATACTTCAGATATTGCCACAGCCTTTTCTTCCGTCGC
CTTGCTATGCATTACACCTCGGACATTGCTACAGCCTTTTCTTCTGTCGC
CTCGCTATACACTACACCTCAGACATTAACACAGCCTTCTCTTCCGTTGC
CTTGCTATACACTACACCTCTGATATCGCCACAGCCTTTTCTTCCGTAGC
CTTGCTATGCACTACACCTCTGATATCGCCACAGCCTTTTCTCCTGTAGC
CTTGCTATACACTACACCTCTGATATCGCCACAGCCTTTTCTTCCGTAGC
CTTGCTATACACTACACCTCTGGTATCGCCACAGCCTTTTCTTCTGTAGC
CTTGCCATACATTATACCTCGGACATTGCCACAGCCTTTTCTTCTGTTGC
CTTGCTATACATTATACCTCAGATATTGCCACAGCCTTTTCTTCCGTTGC
CTCGCTATACACTACACTTCAGATATCGCTACAGCATTCTCCTCTGTTGC
CTTGCTATGCATTATACCTCCGACATCGCCATAGCTTTCTCCTCCGTAGC
CTCGCTATACACTACACCTCTGACATCGCCACGGCCTTTTCTTCTGTAGC
CTTGCTATGCACTATACTTCAGATATTGCCACAGCCTTTTCTTCCGTTGC
CTTGCTATACATTACACCTCCGATATTGCCACAGCCTTTTCCTCTGTCGC
CTTGCCATACACTACACCTCAGACATTGCCACAGCCTTTTCCTCCGTCGC
CTTGCCATACACTATACCTCAGACATTGCTACAGCCTTTTCTTCCGTTGC
CTTGCCATACATTATACCTCAGACATTGCTACAGCCTTTTCCTCTGTTGC
CTTGCCATACACTATACTTCAGATATCGCCACGGCTTTTTCTTCTGTTGC
CTTGCTATACACTACACCTCAGATATTGCCACTGCCTTTTCTTCCGTCGC
CTTGCCATACACTATACCTCAGACATTGCCACAGCCTTTTCTTCTGTTGC
CTTGCCATACATTATACCTCAGATATCGCTACAGCCTTTTCTTCTGTCGC
CTTGCTATGCACTATACTTCAGATATCGCCACAGCCTTTTCTTCCGTTGC
CTTGCTATACACTACACCTCAGATATTGCCACTGCCTTTTCTTCCGTCGC
CTTGCTATACACTACACTTCCGACATCGCCACAGCCTTTTCTTCTGTTGC
CTCGCCATGCATTACACCTCAGACATCGCCACAGCCTTTTCTTCTGTCGC
CTTGCTATACATTACACTTCCGATATCGCCACAGCTTTTTCTTCCGTAGC
CTTGCAATACACTACACCTCCGACATCGCCACAGCCTTTTCATCCGTTGC
CTCGCTATGCACTACACTTCCGATATCGCCACAGCCTTCTCTTCCGTAGC
CTTGCCATACATTATACTTCAGACATTGCCACAGCCTTTTCTTCTGTTGC
CTCGCTATGCACTATACTTCAGATATCGCAACAGCATTCTCTTCTGTTGC
CTCGCCATACATTACACCTCAGACATTGCCACAGCCTTCTCTTCCGTCGC
CTTGCTATACACTACACTTCGGATATTGCCACAGCCTTTTCTTCTGTTGC
CTAGCCATACACTACACCTCAGACATTTCAACCGCATTCTCATCTGTTAC
CTAGCTATACATTACACCTCTGACATCTCAACCGCATTTTCATCCGTCAC
CTAGCCATACACTATACATCCGACATCAACATAGCATTCACCTCTGTCGC
TTAGCTATACATTATACTTCAGATATCGCAACAGCCTTCTCCTCCATCGC
TTGGCAATACACTACACCTCAGATATCGCTACAGCCTTCTCTTCTATCGC
CTTGCAATACACTACACCTCAGATGTTGCCACCGCCTTCTCGTCCGTAGC
CTTGCAATACACTACACCTCAGATGTTGCCACCGCCTTCTCGTCCGTAGC
CTTGCAATACACTACACCTCGGATGTGGCAACTGCCTTTTCATCCGTGGC
CTGGCTATACACTATACCTCGGACATTGCCACAGCCTTCTCTTCTGTTGC
CTAGCCATGCACTATACCTCTGATATTGCCACAGCCTTCTCCTCCGTTGC
CTGGCCATACACTATACCTCGGACATTGCCACAGCCTTCTCTTCTGTTGC
CTAGCTATACATTATACCTCAGATATCGCTACAGCCTTCTCCTCCGTCGC
CTAGCCATGCACTATACCTCTGACATCGCCACAGCCTTCTCATCCGTTGC
CTTGCTATACACTACACCTCCGACATCGCTACAGCTTTCTCATCTGTTGC
CTCGCCATACACTACACCTCCGACATTAGCATGGCCTTCTCATCTGTCGC
CTCGCCATACACTATACATCCGACATTACCATAGCCTTCTCATCCGTCGC
CTCGCAATACATTACACTTCCGACATTGCCACCGCTTTCTCCTCCGTTGC
CTTGCAATACATTACACTTCTGATATCGCCACAGCATTCTCCTCTGTCGC
CTTGCAATACATTACACATCTGACATTGCAACAGCATTTTCTTCCGTCGC
CTTGCCATACACTATACTTCCGATATTGCAACAGCTTTCTCTTCAGTCGC
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[200]
[200]
[200]
[200]
53
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CCATATTTGCCGAGATGTAAATTATGGGTGGCTAATCCGAAACCTTCACG
TCACATCTGCCGGGACGTTAATTACGAATGACTTATCCGAAACCTCCACG
CCACATCTGCCGAGACGTAAACTACGGCTGACTAATCCGTAATCTTCACG
TCACATTTGTCGAGATGTAAACTACGGCTGACTTATCCGCAACCTTCATG
CCACATCTGCCGAGATGTAAACTATGGCTGACTCATCCGAAACCTACATG
CCACATTTGCCGAGATGTAAACTACGGGTGACTCATCCGAAATCTTCACG
CCATATTTGCCGAGACGTAAACTATGGATGACTTATTCGCAATCTCCACG
TCACATTTGTCGAGACGTAAACTACGGTTGACTTATCCGCAACCTCCATG
CCACATCTGTCGAGACGTAAATGATGGCTGACTCATCCGCAACCTTCATG
TCACATCTGCCGAGATGTTAACTATGGGTGACTAATCCGTAACCTCCACG
CCACATCTGTCGAGACGTAAATTACGGCTGACTTATCCGAAACCTTCATG
CCATATTTGTCGAGACGTAAACTACGGCTGACTTATCCGCAATCTCCATG
ACACATCTGTCGAGACGTAAATTACGGATGACTTATCCGCAACCTCCATG
CCACATCTGCCGAGACGTAAACTACGGATGACTAATCCGAAACCTCCACG
CCATATCTGCCGGGACGTAAACTACGGATGGCTTATCCGAAATCTCCATG
CCATATCTGTCGAGACGTAAACTACGGCTGACTTATCCGCAATCTCCATG
CCACATCTGCCGAGATGTGAACTACGGGTGGCTCATCCGTAACCTTCATG
CCACATCTGTCGTGACGTAAATTACGGATGACTAATCCGAAACCTCCACG
CCACATCTGTCGGGACGTAAACTACGGCTGACTTATCCGTAACCTCCACG
CCACATCTGCCGAGATGTAAACTACGGATGGCTGATCCGAAACCTCCACG
CCACATCTGTCGAGACGTGAACTACGGGTGACTCATCCGAAACCTTCACG
CCACATCTGTCGAGACGTAAACTACGGATGGCTAATCCGAAATCTCCACG
ACATATCTGTCGAGACGTAAACTACGGCTGACTTATCCGTAACCTCCATG
CCATATCTGCCGAGACGTAAACTACGGCTGACTTATCCGCAATCTCCATG
TCACATTTGTCGAGACGTAAACTACGGCTGACTTATCCGCAACCTTCATG
ACACATTTGTCGAGACGTAAACTACGGCTGACTTATCCGTAATCTTCATG
CCACATCTGTCGAGACGTAAACTATGGATGACTTATCCGCAACCTCCATG
TCACATCTGCCGAGACGTAAATTATGGATGACTAATCCGCAACCTTCATG
CCACATCTGTCGAGACGTAAACTACGGCTGACTTATCCGCAACCTTCATG
CCACATCTGCCGAGACGTAAACTACGGATGACTCATCCGAAACCTCCACG
CCACATCTGTCGAGACGTAAACTATGGATGACTTATCCGCAACCTCCATG
CCATATCTGCCGGGACGTCAACTACGGATGGCTCATCCGAAACCTCCATG
ACACATTTGTCGAGACGTGAATTACGGCTGACTCATCCGGAATCTCCACG
CCATATCTGCCGAGATGTTAACTACGGCTGACTAATCCGAAACCTTCACG
CCACATCTGTCGAGACGTGAACTACGGATGGCTTATCCGAAACCTTCATG
CCACATCTGCCGAGATGTAAATTACGGATGGCTCATCCGAAACCTTCACG
ACACATTTGCCGAGATGTAAATTATGGCTGACTCATCCGAAATCTTCACG
TCATATTTGCCGAGACGTAAATTATGGATGACTTATCCGTAATCTTCACG
ACACATCTGCCGAGACGTAAATTACGGGTGGCTCATCCGAAATCTCCACG
ACACATCTGTCGAGATGTAAATTACGGCTGACTCATCCGAAATCTTCACG
CCACATCTGCCGAGACGTAAATTACGGCTGACTAATCCGTAATGTACACG
GCACATTTGCCGGGACGTTAACTATGGCTGACTTATCCGGAACATGCACG
TCACATCTGCCGAGATGTAAACTACGGATGGCTTATCCGAAACCTCCACG
ACACATTTGTCGAGATGTTAACTATGGCTGACTTATTCGTAATCTTCACG
ACACATTTGTCGAGATGTCAACTATGGTTGACTTATCCGCAATCTTCACG
ACATATTTGCCGCGACGTAAACTACGGCTGACTAATTCGAAATGTTCACG
ACACATTTGCCGCGACGTAAACTACGGTTGACTAATTCGTAATATTCACG
ACACATCTGCCGTGACGTGAACTACGGCTGACTAATTCGAAATGTCCACG
ACACATTTGCCGAGACGTAAACTACGGCTGGTTAATTCGTAACCTTCATT
ACACATCTGCCGAGATGTAAATTACGGCTGACTTATTTGCAACCTCCACG
ACACATTTGCCGAGATGTAAACTATGGTTGATTAATTCGTAATCTTCATT
ACACATTTGCCGAGATGTAAATTATGGATGACTAATTCGTAACCTTCACG
CCATATCTGCCGAGACGTGAACTATGGCTGACTCATCCGAAACCTCCACG
CCATATTTGCCGAGATGTAAACTTCGGCTGACTTATTCGTAATCTACATG
CCACATCTGCCGAGATGTAAACTACGGTTGACTAATCCGTAATCTCCACG
ACACATCTGCCGAGACGTAAACTACGGATGGCTCATCCGTAACCTACATG
CCACATTTGCCGAGACGTCAACTACGGCTGGCTCATCCGCAACCTTCATG
ACACATTTGCCGAGACGTTAACTATGGTTGGCTCATCCGCAACCTCCACG
CCACATCTGCCGAGACGTAAATTACGGCTGACTTATCCGAAATCTTCATG
CCACATCTGCCGAGACGTAAACTATGGCTGACTGATCCGCAATCTGCACG
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54
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CCAACGGAGCATCTTTTTTCTTCATCTGTATTTATCTTCACATTGGGCGG
CCAACGGAGCATCGTTCTTCTTTATTTGCATTTATTTTCACATTGGACGA
CCAATGGAGCATCCTTCTTTTTTATCTGCATTTACCTTCACATCGGACGA
CCAACGGAGCATCTTTCTTCTTCATCTGCATTTATCTCCACATCGGTCGA
CCAACGGAGCATCTTTCTTCTTCATCTGCATTTACCTTCACATCGGCCGA
CCAACGGAGCATCTTTCTTTTTTATTTGTATTTACCTTCATATCGGGCGA
CCAACGGGGCTTCCTTCTTTTTCATTTGTATTTATCTTCACATTGGACGA
CCAACGGAGCATCCTTCTTTTTCATCTGCATTTACCTCCACATCGGTCGA
CCAACGGAGCATCTTTCTTCTTCATTTGTATTTATCTGCACATTGGACGA
CTAACGGAGCATCCTTCTTTTTTATCTGCATTTACCTCCACATCGGACGA
CTAACGGAGCATCTTTCTTCTTCATTTGTATTTATCTTCACATCGGACGG
CTAATGGAGCATCTTTTTTCTTCATCTGCATTTACCTTCACATCGGACGA
CCAACGGAGCCTCTTTCTTCTTTATTTGTATTTACCTTCACATCGGGCGA
CCAACGGAGCATCTTTCTTCTTTATTTGTATTTACCTTCACATCGGGCGA
CCAACGGAGCATCTTTCTTCTTTATCTGTATTTACCTTCACATCGGGCGA
CTAACGGAGCATCTTTCTTCTTTATCTGTATTTACCTCCACATCGGACGA
CCAACGGAGCATCTTTCTTCTTTATCTGTATTTACCTTCACATCGGGCGA
CCAATGGAGCATCTTTCTTTTTCATCTGCATCTACCTTCACATCGGACGG
CCAACGGAGCATCTTTCTTTTTTATTTGTATTTATCTTCATATCGGACGA
CTAACGGAGCATCATTCTTCTTTATTTGTATCTACCTTCACATCGGACGA
CAAACGGAGCATCCTTCTTTTTTATTTGTATTTACCTCCATATCGGTCGG
CCAACGGAGCATCCTTCTTCTTCATCTGTATTTATCTCCACATCGGACGA
CCAACGGAGCATCTTTCTTTTTTATTTGTATTTATGTTCATATCGGACGA
CTAATGGAGCATCTTTCTTCTTTATCTGTATTTACCTTCACATCGGACGG
CTAATGGAGCATCCTTCTTCTTTATTTGCATCTACCTCCATATCGGACGG
CTAATGGAGCATCTTTCTTTTTCATCTGCATCTACCTTCACATCGGACGA
CCAACGGAGCATCCTTCTTTTTCATCTGCATTTACCTCCACATCGGACGA
CTAACGGAGCATCCTTCTTTTTCATCTGCATCTACCTCCACATTGGCCGA
CCAACGGAGCATCCTTTTTCTTCATCTGCATCTATCTTCACATCGGAGGA
CCAACGGAGCATCTTTCTTCTTCATTTGCATTTATCTTCACATCGGGCGA
CCAACGGAGCCTCTTTCTTCTTCATTTGCATCTACCTTCACATTGGACGA
CTAACGGAGCATCTTTCTTTTTTATTTGTATTTTCCTCCATATCGGACGA
CCAACGGAGCATCCTTTTTCTTCATCTGCATCTATCTTCACATCGGACGA
CCAACGGAGCATCTTTCTTTTTCATTTGCATTTATTTACACATTGGCCGA
CCAACGGAGCATCGTTCTTTTTCATTTGCATTTATCTTCACATTGGACGA
CTAACGGAGCTTCTTTTTTCTTCATCTGTATTTACCTCCACATCGGACGA
CCAATGGAGCATCCTTCTTTTTTATCTGCATTTATCTGCACATCGGCCGA
CCAACGGAGCATCTTTCTTTTTTATTTGTATTTACCTCCATATCGGACGA
CCAACGGGGCATCTTTCTTTTTTATTTGCATTTACCTTCACATCGGACGA
CCAACGGAGCATCCTTCTTCTTCATCTGTATTTACCTTCACATTGGACGA
CCAACGGTGCATCCTTCTTCTTTATTTGCATCTACCTTCACATTGGGCGG
CCAACGGAGCATCTTTCTTTTTTATTTGTATTTACCTTCATATTGGGCGA
CCAACGGAGCATCATTCTTCTTCATTTGCATCTACATACACATCGCCCGA
CCAACGGAGCATCATTTTTCTTCATCTGTATTTACATGCACATTGCTCGT
CTAACGGCGCCTCTTTCTTCTTCATCTGCATGTACCTCCACATCGGCCGA
CCAATGGTGCATCTTTCTTCTTTATTTGTATTTATCTTCACATTGGCCGA
CCAATGGCGCATCTTTCTTCTTTATTTGTATCTATCTTCATATTGGCCGA
CCAACGGCACATCCTTCTTCTTCATCTGCATTTACATACATATTGGGCGG
CCAACGGCACATCCTTCTTCTTCATTTGCATCTACATACATATCGGACGA
CCAACGGCGCATCCTTCTTCTTCATTTGCATCTACATGCATATCGGGCGA
CTAATGGCGCATCTCTCTTCTTCATCTGTATTTACCTTCATATCGGCCGA
CCAACGGCGCATCTCTCTTTTTTATCTGCATCTACCTCCACATTGGCCGA
CTAATGGCGCATCTCTCTTCTTCATCTGCATTTATCTTCATATTGGTCGA
CCAACGGTGCATCTTTCTTTTTCATCTGTATTTATCTTCACATTGGCCGA
CTAACGGCGCATCCTTCTTTTTCATCTGCATCTACCTCCACATCGGACGA
CTAATGGTGCATCCTTCTTCTTTATTTGTATCTACCTTCACATCGGACGA
CCAACGGTGCCTCCTTCTTCTTCATCTGCATCTACCTCCACATCGGCCGA
CCAACGGTGCCTCCTTCTTCTTTATTTGCATCTACCTCCACATCGGCCGA
CAAACGGAGCCTCCTTCTTCTTCATCTGCATCTACCTTCACATCGGCCGA
CAAATGGAGCCTCCTTCTTCTTCATCTGCATCTACCTCCATATTGGCCGC
CTAATGGAGCATCTTTCTTCTTTATTTGCATCTACCTTCATATCGGCCGG
CTAACGGAGCCTCCTTTTTCTTTATCTGCATCTACCTCCACATCGGCCGA
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[300]
[300]
[300]
[300]
[300]
[300]
[300]
55
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GGACTCTACTACGGCTCCTACCTTTACAAAGAAACCTGAAATATTGGAGT
GGGTTGTACTACGGCTCTTACCTCTACAAAGAGACATGAAACATTGGTGT
GGGCTCTATTACGGCTCCTATCTTTATAAAGAAACATGAAACATCGGTGT
GGCTTATACTATGGTTCCTACCTCTACAAAGAGACATGAAACATTGGTGT
GGACTTTACTACGGCTCGTACCTTTACAAAGAAACCTGAAATATTGGGGT
GGGCTTTATTACGGCTCATACCTTTATAAAGAAACATGAAATATCGGAGT
GGACTATACTACGGCTCCTATCTCTACAAAGAGACATGAAATATCGGAGT
GGCCTTTACTATGGCTCTTACCTCTACAAAGAGACATGAAACATTGGTGT
GGTCTCTACTATGGCTCCTACCTCTACAAAGAAACATGAAATATCGGTGT
GGCCTTTACTATGGGTCTTATCTTTACAAAGAAACATGAAACATTGGAGT
GGCCTATACTACGGCTCCTATCTCTACAAATGGACATGAAACATTGGTGT
GGCCTCTACTATGGCTCCTACCTCTACAAAGAAACATGAAACATTGGCGT
GGACTCTACTACGGCTCCTACCTCTACAAAGAAACATGAAACATCGGCGT
GGACTTTATTATGGCTCATACCTCTATAAAGAGACATGAAACATCGGAGT
GGACTTTATTATGGCTCATACCTCTACAAAGAGACATGAAACATCGGAGT
GGCCTTTACTATGGCTCTTATCTCTATAAAGAGACATGAAACATTGGTGT
GGACTTTATTACGGCTCGTACCTCTACAAAGAAACATGAAATATTGGAGT
GGACTTTACTATGGCTCCTACCTCTACAAAGAAACATGAAACATTGGAGT
GGACTTTATTACGGCTCCTACCTCTACAAAGAAACATGAAATATTGGTGT
GGACTTTATTACGGCTCTTATCTCTATAAAGAAACATGAAACATTGGAGT
GGACTCTATTATGGCTCGTATCTTTACAAAGAAACATGAAACATTGGAGT
GGACTCTATTACGGCTCCTATCTTTACAAAGAAACATGAAATATCGGAGT
GGCCTTTATTACGGCTCTTACCTTTATAAAGAAACATGAAACATTGGTGT
GGCCTCTACTACGGCTCTTATCTCTATAAAGATACATGAAATATTGGTGT
GGTCTCTACTACGGCTCTTATCTCTATAAAGAGACATGAAACATTGGGGT
GGCCTCTATTACGGGTCCTACCTCTATAAAGAAACGTGAAATATTGGTGT
GGCCTCTACTATGGTTCCTACCTGTACAAGGAAACATGAAATATTGGTGT
GGGCTCTACTATGGCTCTTACCTCTACAAAGAGACATGAAACATTGGTGT
GGCCTCTACTATGGTTCTTATCTCTACAAAGAAACGTGAAACATTGGCGT
GGACTCTATTATGGCTCCTATCTTTACAAAGAGACATGAGACATCGGAGT
GGCCTCTACTATGGTTCCTACCTGTACAAGGAAACATGAAATATTGGTGT
GGACTTTACTACGGTTCTTACCTATATAAAGAGACATGAAACATCGGGGT
GGACTTTATTACGGCTCCTATCTCTACAAAGAAACATGAAATATCGGAGT
GGGCTTTATTACGGCTCCTACCTCTACAAAGAAACTTGAAACATCGGAGT
GGACTTTACTACGGCTCATACCCTTATAAGGTAACATGAAACATTGGAGT
GGGCTCTACTACGGCTCTTATCTCTATAAAGATACATGAAACATCGGAGT
GGGCTTTACTATGGCTCATATCTCTATAAAGAAACATGAAACATCGGAGT
GGACTGTACTATGGCTCCTACCTCTATAAAGAAACATGAAACATCGGTGT
GGGCTCTATTACGGCTCTTACCTCTACAAAGAAACATGAAACATTGGTGT
GGTCTCTACTATGGCTCTTATCTCTATAAAGAGACATGAAACATCGGAGT
GGCCTATACTACGGATCATACCTTTACAAAGAAACCTGAAACATTGGTGT
GGCCTTTACTACGGGTCCTACCTTTATAAAGAAACCTGAAACGTTGGAGT
GGCCTTTATTACGGCTCCTACCTTTACAAAGAGACCTGAAATATCGGAGT
GGCCTGTACTACGGCTCATACCTGTATAAAGAAACATGAAACATCGGGGT
GGCCTATATTACGGGTCATACTTATATAAAGAAACATGAAATATTGGAGT
GGTCTTTACTACGGCTCCTACCTTTACAAAGAGACCTGAAACATCGGAGT
GGTCTTTACTACGGCTCTTACCTGTACAAAGAGACCTGAAACATCGGAGT
GGCCTTTACTATGGTTCCTACCTCTACAAAGAAACATGAAACGTCGGGGT
GGTCTCTATTATGGTTCCTACCTATATAAAGAGACATGAAACATTGGGGT
GGCCTTTATTACGGCTCCTATTTATACAAAGAGACATGGAACATTGGGGT
GGTCTCTACTATGGCTCCTACCTATATAAAGAGACGTGAAACATTGGGGT
GGTCTCTACTATGGCTCCTACTTGTATAAAGAAACGTGAAACATTGGAGT
GGTTTATACTACGGCTCATACCTCTATAAAGAGACATGAAACATCGGAGT
GGACTCTATTACGGCTCATACCTATATAAAGAAACATGAAACATCGGAGT
GGCCTTTACTACGGCTCGTACCTCTACAAAGAGACATGAAACATTGGAGT
GGCCTCTACTACGGATCATACCTTTACAAAGAGACATGAAACATTGGAGT
GGTCTGTACTATGGCTCCTACCTCTACAAAGAAACTTGAAACATCGGAGT
GGCCTATACTATGGATCTTACCTTTACAAAGAAACTTGAAATATCGGGGT
GGCCTATATTATGGATCCTATCTCTACATAGAGACATGAAACATCGGGGT
GGACTATACTACGGCTCTTACCTTTATAAAGAGACATGAAATATCGGTGT
[350]
[350]
[350]
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[350]
[350]
[350]
[350]
[350]
[350]
[350]
[350]
[350]
[350]
[350]
[350]
56
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CGTTCTCCTTCTCCTAGTTATAGCAACAGCCTTCGTAGGGTATGTACTCC
CGTTCTCCTTCTCCTAGTAATAGCAACCGCCTTCGTAGGCTACGTCCTTC
CGTCCTTCTCCTCCTTGTAATAGCAACAGCCTTCGTAGGCTATGTCCTTC
CATTCTTCTTCTTCTCGTAATAGCAACAGCCTTTGTGGGCTACGTCCTCC
CGTTCTCCTCCTCCTAGTTATAGGAACCGCCTTCGTAGGCTATGTTCTCC
CATCCTCCTCCTCCTAGTTATAGGAACCGCCTTCGTAGGCTACGTTCTTC
AATTCTCCTCCTCCTGGTAATAATAACTGCCTTCGTAGGCTACGTTCTCC
TATCCTTCTCCTTCTTGTAATGGCAACAGCCTTTGTAGGCTACGTCCTCC
TATTCTCCTCCTCCTCGTAATAGCAACAGCCTTCGTGGGCTATGTCCTCC
CGTTCTTCTTCGCCTAGTTATAGGAACTGCCTTCGTAGGATACGTTCTTC
CATTCTACTTCTTCTCGTGATAGCAACAGCCTTCGTAGGCTACGTCCTCC
TATCCTTCTTCTTCTTGTAATAGCAACAGCCTTCGTAGGCTACGTTCTCC
TGTCCTTCTCCTCCTTGTAATAGCGACAGCTTTCGTAGGCTACGTTCTCC
CGTCCTTCTTCTCCTAGTTATGGGAACTGCTTTCGTCGGCTACGTCCTTC
CGTCCTTCTCCTCCTGGTCATAGGAACTGCTTTCGTCGGCTACGTCCTTC
CGTCCTCCTCCTCCTTGTAATAGCAACAGCCTTCGTAGGCTACGTCCTTC
TATTCTTCTTCTCCTGGTTATAGGAACTGCCTTTGTAGGCTACGTCCTCC
TGTCCTCCTCCTCTTAGTTATAGCAACCGCTTTTGTAGGCTACGTTCTCC
TGTCCTCCTCCTTCTAGTTATAGGAACTGCCTTCGTAGGCTACGTCCTTC
TGTCCTCCTGCTCCTAGTTATAGGAACTGCTTTCGTAGGTTACGTTCTTC
TATTCTTCTTCTCCTAGTTATAGGAACTGCTTTCGTGGGCTACGTCCTTC
TGTTCTCCTTCTTTTAGTTATAGGAACTGCCTTCGTGGGCTATGTACTCC
TGTCCTCCTCCTCCTTGTAATAGCTACAGCCTTCGTAGGCTACGTTCTTC
CGTCCTCCTCCTCCTTGTGATAGCAACAGCCTTCGTAGGCTACGTTCTTC
AATCCTTCTTCTTCTTGTAATAGCAACAGCCTTTGTAGGCTACGTTCTTC
TGTCCTCCTCCTCCTTGTAATAGCAACAGCCTTCGTAGGTTACGTCCTTC
CATCCTCCTCCTCCTTGTGATAGCAACAGCCTTCGTAGGCTATGTCCTCC
TATTCTCCTCCTCCTCGTGATAGCCACAGCCTTCGTAGGGTACGTCCTCC
CATTCTCCTTCTCCTCGTAATAGCCACAGCTTTCGTAGGCTATGTTCTCC
CATTCTTCTTCTCCTAGTCATAGGAACTGCTTTCGTAGGCTACGTCCTTC
CATCCTCCTCCTCCTTGTGATAGCAACAGCCTTCGTAGGCTATGTCCTCC
AGTTCTTTTACTCTTAGTAATAGGATCTGCCTTCGTTGGCTATGTCCTTC
CGTTCTCCTCCTCCTAGTCATAGGAACTGCCTTCGTAGGCTATGTCCTCC
TGTCCTCCTCCTCCTAGTTATAGCAACAGCCTTTGTAGGTTACGTTCTCC
TGTTCTCCTTCTTTTAGTTATAGGAACTGCCTTCGTGGGCTACGTACTCC
TGTCCTCCTCCTATTAGTTATAGGAACTGCTTTCGTAGGTTACGTACTCC
CATTCTTCTCTTACTCGTTATAGGAACCGCCTTTGTGGGCTATGTCCTCC
CGTTCTTCTTCTTCTGGTTATAGCAACTGCCTTCGTAGGCTACGTCCTCC
CGTCCTTCTCCTCCTCGTAATAGCAACAGCCTTCGTAGGCTACGTCCTCC
TGTACTTCTTCTCCTAGTTATAGCAACCGCCTTCGTAGGCTACGTCCTCC
AGTCCTTCTACTACTAGTCATGATAACAGCCTTCGTTGGCTATGTTCTTC
CGTACTACTCCTTCTAGTCATGATGACAGCCTTTGTGGGTTATGTACTCC
AATCCTCCTACTACTAGTAATAATAACCGCATCCGTCGGCTATGTCCTCC
AATCCTTCTCCTCTTAGTAATAATGACAGCCTTCGTAGGCTATGTGTTGC
AGTGCTACTCCTCTTAGTAATAATAACAGCCTTTGTAGGTTACGTATTAC
AGTCCTGCTCCTATTAGTTATAATGACTGCCTTCGTGGGCTACGTCCTCC
AATTCTCCTCCTCCTAGTTATAATGACTGCCTTCGTGGGCTACGTCCTCC
AGTCCTGCTCCTTTTAGTCATGATAACCGCCTTTGTAGGCTACGTCCTCC
TGTTCTCCTCCTATTAGTAATAATGACAGCTTTCGTAGGCTACGTCCTAC
GGTTCTTCTCCTTTTAGTAATAATAACAGCCTTCGTGGGCTACGTCCTAC
CATTCTCCTTCTCCTAGTAATAATAACAGCTTTCGTAGGTTACGTCTTAC
AGTTCTTCTCCTCTTAGTAATAATAACAGCTTTCGTAGGCTACGTCCTAC
TGTACTCCTCCTCCTAGTTATGATAACCGCATTCGTAGGCTACGTCCTGC
TATCCTCCTCCTTCTAGTAATAATAACCGCATTCGTAGGCTACGTCCTGC
CGTCCTTCTTCTCCTAGTGATAGCAACTGCGTTCGTAGGCTACGTCCTAC
CGTCCTGCTCCTCCTAGTAATAGCAACTGCCTTCGTAGGCTATGTACTCC
CGTCCTACTCCTTCTAGTAATGATGACCGCCTTTGTAGGGGATGTCCTTC
TGTCCTTCTTCTTTTAGTTATAATGACCGCCTTTGTAGGGTACGTCCTCC
AATCCTGCTATTATTAGTGATAATAACAGCATTCGTCGGTTACGTCCTAC
TATTCTTCTTCTTCTGGTGATAATAACAGCCTTTGTAGGTTACGTCCTCC
[400]
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[400]
[400]
[400]
[400]
[400]
[400]
[400]
[400]
[400]
[400]
[400]
57
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CTTGAGGTCAAATATCCTTTTGAGGAGCTACCGTAATTACCAACCTCCTA
CATGAGGACAAATATCCTTCTGAGGGGCAACCGTCATTACTAACCTTCTA
CATGAGGACAAATATCATTCTGAGGGGGTACTGTTATTACCAACCTTCTT
CCTGAGGACAAATATCCTTCTGAGGGGCTACCGTCATTACTAACCTCCTT
CATGAGGACAAATGTCCTTCTGAGGAGCGACTGTCATTACCAACCTCCTA
CATGAGGACAAATATCTTTCTGAGGAGCAACCGTCATTACCAACCTCTTA
CATGAGGACAGATATCCTTCTGAGGAGCAACTGTCATCACTAACCTCCTA
CATGAGGACAAATATCATTCTGAGGGGCTACCGTCATCACGAATCTTCTT
CCTGAGGACAAATATCCTTCTGAGGGGCCACTGTTATTACCAACCTCCTT
CCTGAGGACAAATATCTTTTTGAGGGGCAACTGTAATCACTAACCTCCTC
CCTGAGGGCAAATATCCTTCTGAGGGGCCACCGTCATTACTAACCTCCTC
CATGAGGACAAATATCATTCTGAGGAGCTACCGTCATCACCAATCTTCTC
CATGAGGACAAATGTCCTTCTGAGGGGCCACCGTCATTACCAACCTCCTC
CATGAGGACAAATGTCCTTTTGAGGAGCAACCGTTATTACCAACCTCCTG
CATGAGGACAAATATCCTTTTGAGGAGCAACCGTTATTACCAACCTCCTG
CATGAGGACAAATGTCCTTTTGAGGAGCAACCGTTATTACCAACCTCCTA
CATGAGGACAAATGTCCTTTTGAGGAGCAACCGTTATTACCAACCTCCTG
CTTGAGGACAAATGTCATTCTGAGGGGCCACTGTCATTACCAACCTCCTT
CATGAGGACAAATATCCTTCTGAGGAGCAACTGTCATCACTAATCTCTTA
CATGAGGGCAGATATCCTTCTGAGGAGCGACCGTCATTACCAACCTCCTA
CATGAGGACAAATGTTCTTCTGAGGGGCAACCGTCATCACCAACCTACTT
CATGAGGACAAATATCCTTCTGAGGGGCAACCGTCATTACTAACCTCCTC
CATGAGGACAAATATCCTTCTGAGGGGCAACTGTTATTACCAAACTCTTA
CATGAGGACAAATGTCCTTCTGAGGGGCCACCGTCATTACTAACCTCCTG
CATGAGGACAAATATCATTCTGAGGGGCTACTGTCATTACCAACCTTCTC
CTTGAGGACAAATATCCTTCTGAGGAGCCACTGTCATCACTAACCTCCTT
CATGAGGGCAAATATCGTTCTGAGGGGCCACCGTCATTACTAATCTCCTC
CGTGAGGACAAATATCATTCTGAGGAGCCACCGTCATCACCAACCTCCTT
CGTGAGGGCAAATATCCTTGTGAGGCGCGACTGTCATTACCAACCTCGTT
CTTGAGGACAAATATCCTTCTGAGGAGCCACTGTTATTACCAACCTCCTT
CCTGAGGACAAATATCCTTCTGAGGGGCCACCGTTATTACCAACCTACTC
CATGAGGACAAATATCCTTCTGAGGGGCGACTGTTATTACCAGCCTTTTA
CCTGAGGACAAATATCATTCTGAGGGGCCACTGTTATTACCAACCTCCTC
CATGAGGACAGATATCTTTCTGAGGGGCAACCGTCATCACCAACCTTCTA
CATGAGGGCAAATATCCTTCTGAGGGGCAACCGTCATCACCAACCTCTTA
CTTGAGGGCAAATATCCTTTTGAGGGGCAACCGTTATTACTAACCTCTTG
CATGAGGACAAATGTCTTTCTGAGGGGCTACCGTTATTACCAACCTTCTG
CATGAGGACAAATATCTTTCTGAGGGGCAACTGTTATTACCAACCTTCTT
CCTGAGGACAAATGTCATTTTGGGGAGCAACCGTCATTACTAACCTCCTC
CATGAGGACAAATATCCTTTTGAGGAGCAACCGTCATTACTAACCTCCTA
CATGGGGACAAATATCCTTCTGAGGCGCTACCGTCATTACCAACCTCCTT
CCTGAGGACAAATGTCATTTTGAGGGGCAACCGTCATTACTAACCTCCTT
CATGAGGACAAATATCCTTTTGAGGCGCCACAGTAATCACAAACCTCCTA
CATGGGGCCAAATATCCTTCTGAGGTGCTACCGTTATTACAAATCTTCTA
CCTGAGGACAAATATCATTCTGAGGTGCTACCGTTATCACCAACCTCCTC
CCTGAGGACAAATATCTTTTTGAGGCGCTACAGTTATTACTAATCTATTA
CCTGAGGACAAATATCCTTCTGAGGGGCTACAGTTATTACTAATTTATTA
CATGAGGTCAAATATCCTTCTGAGGGGGCACCGTCATCACCAACCTCCTG
CATGAGGGCAGATATCCTTCTGAGGGGGCACCGTCATCACTAACCTCCTG
CGTGAGGCCAAATGTCTTTCTGGGGGGCCACCGTCATCACCAACCTTCTA
CGTGAGGTCAAATGTCTTTCTGAGGAGCAACAGTCATCACCAATTTATTA
CCTGAGGCCAAATATCTTTTTGAGGGGCAACAGTTATTACCAATTTATTA
CATGAGGCCAAATGTCCTTCTGAGGGGCAACAGTCATCACCAATTTATTA
CCTGAGGTCAGATATCTTTCTGAGGGGCAACAGTCATTACTAATTTATTA
CGTGAGGACAAATGTCCTTCTGAGGTGCCACCGTCATCACAAACCTCCTC
CATGAGGACAAATGTCCTTTTGAGGTGCTACCGTCATCACAAACCTACTC
CATGAGGACAAATGTCCTTCTGAGGTGCTACCGTCATTACCAACCTCCTC
CCTGAGGACAAATATCATTCTGAGGAGCCACCGTTATTACCAACCTGCTT
CATGAGGACAAATGTCTTTCTGAGGGGCCACAGTCATTACAAATCTCCTA
CATGAGGACAAATATCTTTCTGAGGGGCTACCGTAATTACAAACCTCCTC
CATGAGGCCAAATGTCATTCTGAGGCGCCACCGTAATTACAAACCTTCTT
CCTGAGGCCAAATGTCATTCTGAGGTGCAACCGTAATCACTAACCTTTTA
[450]
[450]
[450]
[450]
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[450]
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[450]
[450]
[450]
[450]
[450]
[450]
[450]
[450]
[450]
[450]
[450]
[450]
58
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
TCCGCCGTCCCCTATATTCGCGGAACACTCGTCCAATGAATTTGAGGTGG
TCCGCTGTCCCTTACGTTGGAGGCACCCTAGTCCAATGAATTTGAGGAGG
TCCGCTGTCCCTTATGTAGGCGGTACCCTTGTTCAATGAATTTGAGGGGG
TCTGCCGTCCCATATGTAGGCGGCACCCTTGTACAATGAATTTGAGGAGG
TCCGCTGTCCCCTACGTCGGAGGGACCCTCGTTCAATGAATCTGGGGTGG
TCCGCTGTCCCTTACATTGGCGGTACCCTCGTCCAATGAATCTGAGGGGG
TCGGCTGTACCATATGTCGGGAGCACCCTAGTTCAATGAATCTGGGGAGG
TCCGCCGTCCCATATGTAGGCGGCACCCTCGTTCAATGAATCTGAGGGGG
TCCGCCGTTCCATATGTAGGCGGCACCCTTGTCCAATGAATCTGAGGAGG
TCCGCTGTCCCCTACGTCGGCGGCACCTTCGTCCAATGAATTTGAGGCGG
TCCGCCGTTCCATACGTAGGTGGCACCCTCGTCCAATGAATCTGAGGGGG
TCCGCTGTCCCATATGTAGGTGGTACCCTAGTTCAATGAATTTGAGGGGG
TCCGCTGTTCCCTACGTAGGCGGCACCCTTGTCCAATGAATTTGAGGGGG
TCCGCCGTTCCCTACGTAGGAGGAACTCTCGTTCAATGGATCTGAGGCGG
TCCGCCGTTCCCTACGTAGGAGGAACTCTCGTTCAATGAATCTGAGGCGG
TCCGCCGTTCCCTACGTAGGGGGAACTCTAGTTCAATGAATCTGAGGGGG
TCCGCCGTTCCCTACGTAGGAGGAACTCTCGTTCAATGGATCTGAGGCGG
TCTGCCGTCCCCTATGTAGGTGGCACCCTTGTTCAATGGATCTGAGGAGG
TCCGCTGTTCCCTACGTTGGTGGCACTCTCGTCCAATGAATCTGAGGAGG
TCCGCTGTTCCCTACATTGGAGGCACCCTAGTCCAATGAATTTGAGGAGG
TCCGCCGTCCCTTATGTTGGTGGCACCCTCGTACAATGGATCTGAGGTGG
TCTGCCGTCCCCTACGTCGGAGGGACCCTCGTCCAATGGATCTGAGGGGG
TCCGCTGTTCCCTACGTTGGCGGCACCCTCGTCCAATGGATCTGAGGAGG
TCTGCTGTCCCCTACGTCGGTGGAACCCTCGTTCAATGAATCTGAGGCGG
TCCGCCGTCCCATACGTGGGCGGCACTCTCGTTCAATGAATCTGGGGCGG
TCTGCCGTTCCATATGTAGGTGGCACCCTTGTTCAATGGATTTGAGGAGG
TCTGCTGTCCCATACGTGGGCGGCACCCTCGTCCAATGAATCTGAGGGGG
TCCGCCGTTCCCTACGTAGGCGGTACTCTCGTTCAATGGATTTGAGGAGG
TCCGCCGTCCCATATGTAGGCGGTACCCTCGTCCAATGAATTTGAGGGGG
TCCGCCGTCCCATATGTGGGCGGCACCCTTGTCCAGTGAATTTGAGGTGG
TCTGCCGTCCCATACGTCGGCGGCACCCTTGTCCAGTGAATTTGAGGGGG
TCCGCTGTTCCCTACGTTGGCAGCACCCTCGTCCAATGAATCTGAGGAGG
TCCGCCGTACCATACGTGGGTGGCACTCTCGTACAATGGATTTGAGGGGG
TCCGCCGTACCCTACATCGGTGGTACTCTTGTCCAATGATTCTGAGGTGG
ACCGCTGTCCCCTACGTTGGCGGCACCCTTGTCCAATGAATCTGGGGAGG
TCCGCCGTCCCCTATGTTGGCGGAACACTTGTCCAATGAATTTGAGGGGG
TCCGCCGTACCCTACGTCGGCGGCACTCTTGTCCAATGAATTTGGGGGGG
TCCGCCGTCCCCTATGTTGGAGGCACTCTTGTCCAATGAATTTGAGGAGG
TCCGCGGTTCCCTACGTCGGGGGCACTCTTGTGCAATGAATCTGAGGAGG
TCAGCCGTTCCCTACGTTGGAGGCACCCTAGTTCAGTGGATCTGGGGAGG
TCTGCCGTCCCATATGTAGGCGGCACCCTTGTACAATGAATCTGAGGGGG
TCCGCTGTTCCTTACGTTGGAGGCACTCTTGTACAATGAATCTGAGGGGG
TCTGCCGTACCATACATGGGAGACATGTTAGTCCAATGAATCTGAGGTGG
TCAGCAGTGCCTTATATAGGGGACACCCTTGTACAGTGGATTTGAGGCGG
TCCGCCGTACCCTACGTAGGAGACATCCTAGTCCAATGAATCTGAGGAGG
TCCGCCGTACCTTATGTAGGTAATACACTAGTTCAGTGGATTTGAGGGGG
TCCGCCGTACCTTATGTAGGCAACACACTAGTTCAGTGAATTTGAGGAGG
TCCGCTGTACCCTACGTAGGAAACACTCTCGTCCAATGAATCTGAGGCGG
TCCGCTGTTCCATATGTAGGCAACACCCTGGTCCAATGAATCTGAGGCGG
TCCGCCGTCCCCTACATCGGTAACACCCTTGTCCAATGGATTTGGGGCGG
TCTGCCGTCCCCTATGTAGGAAACACCCTGGTTCAATGAATCTGGGGCGG
TCCGCTGTCCCCTATGTAGGGAACACCCTAGTTCAATGAATCTGAGGAGG
TCCGCCGTCCCCTATGTAGGAAACACCCTAGTTCAATGGATCTGAGGTGG
TCCGCTTTGCCCTATGTAGGAAACACCCTAGTTCAATGAATCTGGGGCGG
TCTGCCGTTCCCTACGTCGGAAACACACTAGTCCAATGAATCTGAGGGGG
TCAGCCGTCCCCTACGTTGGTAACACTCTCGTTCAATGAATCTGAGGGGG
TGTGCGATCCCCTACGTGGGCAACACCCTAGTCCAATGAGTCTGAGGAGG
TCTGCCATTCCATACGTCGGCAACACCCTTGTCCAATGAATCTGGGGCGG
TCCGCCGTACCTTACGTTGGTAACACACTAGTTCAATGAATCTGAGGGGG
TCCGCCGTCCCATACGTAGGCAACACCCTAGTTCAATGAATCTGAGGGGG
TCTGCCGTCCCGTATGTCGGAAACACACTAGTTCAATGGATCTGAGGCGG
TCCGCAGTCCCGTATGTCGGAAACATACTAGTCCAATGAATTTGAGGAGG
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[500]
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[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
[500]
59
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
ATTTTCAGTTGACAATGCAACCCTAACGCGCTTCTTCGCTTTCCACTTCC
CTTCTCGGTAGACAACGCAACCTTAACCCGATTCTTCGCCTTCCACTTCC
TTTCTCAGTAGACAATGCAACCCTAACCCGATTCTTTGCTTTCCATTTTC
CTTTTCAGTAGACAACGCCACCCTCACTCGATTTTTTGCCTTCCACTTCC
CTTCTCGGTAGACAATGCAACTCTAACCCGCTTCTTTGCCTTCCACTTCC
ATTCTCAGTAGACAATGCAACCCTAACTCGCTTCTTTGCCTTCCATTTTC
GTTTTCAGTAGACAATGCAACCTTAACCCGATTCTTTGCCTTCCACTTCC
CTTTTCAGTAGACAACGCCACCCTAACCCGATTTTTTGCCTTCCATTTCC
CTTCTCGGTCGATAACGCCACCCTCACACGATTCTTCGCCTTCCACTTTC
GTTTTCAGTCGACAATGCAACTCTCACCCGTTTCTTTGCCTTCCACTTTC
CTTTTCAGTAGATAACGCCACTCTCACACGATTCTTTACCTTCCACTTCC
CTTCTCGGTAGATAATGCCACCTTAACCCGATTTTTTGCCTTCCACTTCC
CTTCTCTGTAGACAACGCTACCTTAACCCGGTTCTTTGCCTTCCACTTCC
ATTTTCAGTAGACAATGCAACCCTAACCCGATTCTTCGCCTTCCACTTCC
GTTTTCAGTAGACAATGCAACCCTAACCCGATTCTTCGCCTTCCACTTCC
GTTTTCAGTAGACAATGCGACCCTGACCCGCTTCTTTGCCTTCCACTTCC
GTTTTCAGTAGACAATGCAACCCTAACCCGATTCTTCGCCTTCCACTTCC
CTTTTCAGTAGACAACGCCACCTTAACTCGGTTTTTTGCCTTCCACTTCC
GTTTTCAGTAGATAATGCAACACTAACCCGCTTCTTTGCCTTCCATTTTC
CTTTTCAGTAGACAACGCAACCCTAACCCGGTTCTTTGCCTTCCACTTCC
ATTCTCAGTAGACAATGCAACTTTAACCCGCTTCTTTGCCTTCCACTTCC
ATTTTCGGTAGATAACGCCACCCTAACCCGCTTCTTTGCCTTCCACTTTC
ATTTTCAGTAGATAACGCCACCTTAACCCGCTTCTTTGCCTTCCACTTTC
CTTCTCAGTTGACAATGCAACCCTAACTCGCTTCTTTGCTTTCCACTTCC
CTTTTCAGTAGACAACGCTACCCTAACCCGATTCTTCGCCTTCCACTTCC
CTTTTCAGTAGACAATGCCACCTTAACTCGGTTCTTTGCCTTCCACTTCC
CTTCTCAGTAGATAACGCCACCCTAACCCGATTTTTTGCCTTCCACTTTC
CTTCTCAGTAGATAACGCTACCCTAACCCGATTCTTTGCCTTCCACTTCC
CTTTTCGGTAGATAACGCCACTCTCACACGATTCTTTGCCTTCCACTTCC
CTTTTCAGTAGATAACGCCACTCTCACACGATTCTTTGCCTTCCACTTCC
CTTTTCAGTAGACAACGCCACCCTAACCCGATTTTTTGCCTTCCACTTCC
ATTTTCAGTTGATAACGCCACCTTAACCCGCTTCTTTGCCTTCCACTTTC
GTTTTCAGTAGATAACGCCACCCTCACACGATTCTTCGCCTTCCACTTCC
CTTTTCAGTTGACAACGCAACCCTTACCCGCTTCTTTGCCTTCCATTTCC
GTTGTCAGTTGACAACGCAACACTAACCCGCTTCTTTGCCTTCCACTTTC
GTTTTCAGTTGACAACGCAACCCTAACCCGCTTCTTCGCTTTCCACTTCC
CTTTTCAGTCGACAACGCAACCCTAACCCGCTTCTTTGCCTTCCACTTCC
GTTTTCAGTTGATAATGCAACCCTGACCCGCTTCTTTGCCTTCCATTTCC
TTTTTCAGTAGACAATGCAACCCTAACCCGTTTCTTTGCCTTCCACTTCC
TTTCTCAGTCGACAACGCAACCTTAACCCGATTCTTTGCCTTCCACTTCC
CTTCTCAGTAGATAACGCCACCCTAACCCGATTCTTTGCCTTCCACTTCC
GTTCTCAGTAGACAATGCAACCTTAACCCGCTTCTTCGCCTTCCACTTCC
GTTCTCAGTAGACAATGCAACACTAACACGATTCTTCGCATTCCACTTCC
CTTTTCAGTAGATAACGCCACGTTAACACGATTCTTCGCCTTCCACTTCC
CTTCTCAGTTGACAACGCAACCCTCACCCGATTCTTTGCCTTCCACTTCC
CTTTTCAGTAGATAACGCCACTCTTACACGGTTCTTCGCGTTCCACTTCC
GTTTTCAGTAGATAACGCCACCCTCACACGGTTCTTCGCATTCCACTTCC
CTTTTCAGTAGACAATGCCACCCTTACCCGCTTCTTCGCCTTCCACTTTT
TTTTTCAGTAGATAACGCCACCCTTACCCGCTTTTTCGCTTTCCACTTTC
GTTTTCAGTAGATAACGCCACCCTTACCCGTTTCTTCGCTTTCCACTTCC
CTTTTCAGTCGATAACGCTACACTCACACGATTCTTTGCTTTCCACTTCC
CTTCTCAGTTGATAATGCCACACTCACACGATTCTTCGCTTTCCACTTCC
CTTTTCAGTTGATAACGCCACACTCACACGATTCTTTGCTTTCCACTTCC
CTTCTCAGTTGATAACGCCACACTCACACGATTCTTCGCTTTCCACTTCC
CTTCTCTGTAGACAATGCAACGCTAACTCGCTTCTTTGCCTTCCATTTCC
TTTCTCCGTTGACAACGCCACCCTCACTCGATTCTTTGCCTTCCACTTCC
CTTTTCGGTAGATAACGCCACCCTCACCCGATTCTTCGCATTCCACTTCC
CTTCTCAGTAGACAACGCCACCCTAACCCGCTTCTTCGCATTCCACTTCC
ATTCTCAGTAGACAACGCAACACTAACCCGCTTCTACGCCCTCCACTTCC
CTTTTCGGTTGACCACGCAACCCTAACCCGATTCTTCGCCTTCCACTTCT
ATTCTCAGTAGATAATGCCACCCTCACCCGATTCTTTGCATTCCACTTCC
CTTCTCAGTTGACCACGCCACACTCACCCGTTTCCTTACCTTCCACTTCC
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
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[550]
[550]
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[550]
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[550]
[550]
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[550]
[550]
[550]
[550]
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[550]
[550]
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[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
[550]
60
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
TCCTCCCTTTTATTGTAGCCGCTATAACTATACTCCACCTCCTCTTCCTA
TCCTTCCATTCATCGTAGCAGCAATAACTATGCTCCACCTCCTATTCCTG
TCCTCCCCTTTATTGTTGCAGCCATAACTATACTTCACCTTCTCTTCTTA
TTCTGCCCTTTATTGTTGCAGCCGTAACTATACTTCACCTTCTTTTCCTA
TCCTCCCCTTCGTCGTTGCAGCCATGACCATGCTTCACCTCCTCTTCCTA
TTCTTCCCTTTGTTGTCGCAGCCATAACCATGCTTCACCTCCTATTCCTG
TTTTCCCCTTTGTTGTTGCAGCTATAACTATGCTGCACCTCCTTTTCCTA
TCCTTCCCTTTATTGTCGCAGCCGTAACTATACTCCACCTTCTCTTCCTG
TCCTGCCCTTTATTGTTGCAGCCATAACCATGCTTCATCTTCTTTTCTTA
TCCTCCCCTTTATCGTTGCAGCTATAACTATACTCCACCTACTGTGCCTT
TTCTACCCTTTATCGTTGCAGCTATAACTATACTTCACCTCCTTTTCTTA
TCCTCCCCTTTATTGTTGCGGCCGTAACTATGCTCCACCTTCTTTTTCTG
TTCTGCCCTTCATTGTAGCAGCCATAACCATACTTCATCTTCTTTTCTTA
TTCTCCCCTTCATTGTCGCCGCCATAACCATGCTTCACCTCTTATTCCTG
TTCTCCCCCTCGTTGTCGCCGCCATAACCATGCTTCACCTCTTATTCCTG
TTCTTCCCTTCATTATTGCTGCCATGACCATGCTTCACCTCTTATTCCTG
TTCTTCCCTTCGTTGTCGCCGCCATAACCATGCTTCACCTCTTATTCCTG
TCTTCCTCTTTATTGTTGCAGCCATAATCATACTTCATCTTCTTTTCTTA
TTCTTCCCTTTGTTGTCGCAGCCATAACCTTACTTCACCTACTGTTCCTG
TCCTTCCATTCATCGTAGCAGCAATAACAATACTTCACCTTCTATTCCTA
TCCTTCCCTTCATTGTTGCCGCTATGACAATGCTCCATCTGCTATTTCTT
TCCTTCCCTTTATTGTTGCCGCCATAACTATGCTCCACCTCCTATTTTTA
TCCTTCCCTTTGTTGTCGCAGCCATAACCATACTTCACCTACTATTCTTA
TCCTACCCTTCGTCGTAGCCGCTATAACCATACTGCACCTCTTATTCCTT
TCCTGCCCTTCATTGTTGCAGCCATAACCATACTACATCTTCTCTTCTTA
TTCTACCTTTTATTGTTGCAGCCATGACTATACTTCACCTTCTTTTCTTA
TCCTGCCTTTCATTGTTGCAGCCGTAACCATACTCCATCTTCTTTTCTTA
TCCTTCCCTTTATTGTTGCAGCCATAACTATGCTTCACCTTCTTTTCCTA
TCCTGCCCTTTATTGTTGCAGCCATGACCATGCTTCACCTTCTTTTCTTA
TCCTGCCCTTTATTGTTGCAGCCATAACCATGCTTCACCTTCTTTTCTTA
TCCTGCCCTTTATCGTCGCAGCCATAACCATACTTCACCTTCTTTTCCTC
TCCTCCCCTTTGTTGTCGCAGCCATAACCATACTTCACCTACTATTCCCG
TCCTACCATTTATCGTCGCAGCTATAACCATACTCCACCTTCTTTTCTTA
TCCTCCCCTTTATTGTTGCAGCCATAACTATGCTTCACCTCCTATTCCTT
TCCTTCCCTTCGTCGTTGCGGCTATAACCATACTTCATCTCCTTTTCCTG
TCCTCCCCTTTATTGTTGCCGCCATGACTATACTCCACCTCCTCTTCCTA
TCCTCCCCTTTATTGTTGCAGCCATGACTATGCTACACCTTCTATTCCTT
TTCTCCCCTTCGTCATTGCAGCCATAACCATACTGCATCTTCTGTTCCTC
TTCTTCCCTTCATTGTTGCAGCTATGACCATACTTCACCTCTTATTCCTA
TCCTCCCCTTTATTGTTGCAGCAATAACTATGCTCCACCTCCTATTCCTA
TTCTGCCCTTTATTGTAGCAGCCATAACTATACTTCACCTTCTTTTCTTA
TCCTTCCTTTCATTGTTGCAGCCATAACCATACTTCACCTCTTATTCCTG
TACTACCATTTGTTATTGCCGCCGCAACCATCATCCACCTGCTGTTCCTC
TGTTCCCATTCGTCATCGCCGGCGCAACTGTTCTCCACTTACTATTTTTA
TACTTCCCTTTGTAGTCGCAGCCATAATAATCCTCCATCTCTTATTCCTA
TATTCCCATTCGTAATCGCTGGTGCCACAATACTACACCTCCTTTTTCTT
TCTTTCCATTCGTAATTGCAGGTGCCACCCTTCTGCACCTTCTTTTCCTC
TATTCCCCTTCATTATTGCGGGGGCAACCGTCATCCATCTGCTTTTCCTC
TGTTCCCCTTCGTTATTGCGGGAGCAACCCTCATCCATCTGATTTTCCTC
TATTCCCCTTCGTCATTGCGGGTGCAACGTTTATTCACCTGCTTTTCCTT
TTTTCCCATTCATCATTGCCGCCGCCACCCTCTTACACCTCCTCTTTCTC
TCTTCCCATTTGTCATCGCTGCTGCCACCGTCTTACACCTTTTGTTCCTC
TTTTCCCATTCATCATTGCCGCTGCCGCTATCTTACACCTCCTCTTCCTC
TCTTCCCGTTCGTCATTGCTGCTGCCACCATTTTACACCTCCTTTTCCTT
TTCTCCCCTTCATCATCGCCGCCGCAACGGTCATCCACCTTCTTTTCCTC
TTCTTCCACTTATCGTTACAGCTGCAACCCTAATTCACCTCTTATTCCTC
TTCTACCCTTTATCATCGCAGCAGTAACCATACTCCACCTCCTATTCCTG
TCCTCCCGTTCATCATTGCAGCCGTTACAATACTACACCTGCTTTTCCTC
TCTCTCCATTCGTAATTGCAGCAGCCACAACACCTCACCTCCGGTTCCTA
TATTCCCCTTCGTCATTGCAGCAGCCACTATACTTCACCTTCTTTTCCTC
TATTCCCATTTGTCATTGCCGCTATAACCCTCCTACATTTGCTTTTCCTA
TCTTCCCGTTTGTAATTGCAGCTGCTACCCTCCTTCACCTTCTGTTCCTC
[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
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[600]
[600]
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[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
[600]
61
Aanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CATGAAACAGGCTCAAACAATCCTCTAGGTTTAAACTCCGACACGGACAA
CACGAAACAGGATCAAACAACCCCCTCGGCCTAAACTCCGACACAGACAA
CACGAAACGGGCTCAAATAATCCTCTGGGCCTAAACTCAGACACAGATAA
CATGAAACTGGCTCAAATAACCCCCTCGGGCTAAACTCAGACGCAGATAA
CACGAAACAGGCTCAAACAACCCAATCGGCCTAAACTCTGACACAGACAA
CATGAAACAGGCTCAAACAACCCCCTTGGTCTAAACTCTGACACAGACAA
CACGAAACAGGCTCAAATAACCCCCTCGGCCTAAACTCCGACACAGATAA
CACGAAACAGGCTCAAACAACCCCCTTGGCCTAAACTCAGACACAGACAA
CATGAGACAGGTTCAAATAACCCTCTCGGCCTAAACTCAGACACAGACAA
CACGAAACAGGCTCAAACAACCCCCTCGGCCTGAACTCTGACACAGACAA
CATGAAACAGGCTCAAACAACCCCCTTGGTTTAAACTCAGACACAGACAA
CACGAAACAGGCTCAAACAACCCCCTTGGCCTAAACTCCGACACGGACAA
CACGAAACAGGCTCAAACAATCCCCTCGGCCTAAACTCAGACACAGACAA
CATGAAACAGGCTCAAACAACCCCCTTGGCCTAAATTCTGACACAGACAA
CACGAAACAGGCTCAAACAACCCCCTTGGCCTAAATTCTGATACAGACAA
CATGAAACAGGCTCATATAATCCCCTCGGGGTAAATTCAGACACAGACAA
CATGAAACAGGCTCAAACAACCCCCTTGGTCTAAACTCTGATACAGACAA
CACGAAACAGGATCAAACAATCCCCTCGGCCTAAACTCCGACACAGATAA
CACGAAACAGGCTCAAACAACCCCCTCGGTCTCAACTCCGACACAGATAA
CATGAAACAGGCTCAAATAACCCCCTTGGCCTAAACTCTGACACAGACAA
CATGAAACAGGCTCAAACAACCCCCTTGGTCTTAACTCTGATACAGATAA
CATGAAACAGGTTCAAACAATCCACTCGGCCTAAATTCTGATACAGACAA
CATGAAACAGGATCAAACAACCCCTTAGGCCTAAACTCAGACACAGACAA
CACGAAACAGGCTCAAATAACCCTCTCGGCTTGAACTCAGATACAGACAA
CACGAGACAGGCTCAAACAATCCCCTAGGTTTAAACTCAGATACAGACAA
CACGAAACAGGCTCAAACAACCCCCTCGGCTTAAACTCAGACACAGACAA
CACGAAACAGGCTCAAACAACCCACTCGGCTTAAACTCAGACGCGGACAA
CATGAAACAGGCTCAAACAATCCCCTCGGTCTAAACTCAGACACAGACAA
CACGAGACAGGTTCAAACAACCCGCTCGGCTTAAACTCAGACACAGATAA
CATGACACAGGCTCAAACAACCCCCTTGGCCTTAATTCTGACACAGATAA
CATGAAACAGGCTCAAACAACCCCCTAGGACTGAACTCAGACGCAGACAA
CATGAAACAGGCTCTAATAACCCCCTCGGACTAAATTCTGACACAGACAA
CACGAAACTGGTTCAAATAACCCCCTCGGCCTAAACTCCGACACAGATAA
CACGAAACAGGCTCCAACAACCCCCTCGGCCTGAACTCCGACACAGACAA
CATGAAACAGGCTCTAACAACCCCCTTGGTCTAAACTCTGACACGGACAA
CATGAAACAGGCTCTAACAACCCCCTCGGCCTAAATTCTGACACAGATAA
CACGAAACTGGCTCAAACAACCCCCTTGGCCTAAACTCTAACACAGACAA
CATGAAACAGGCTCAAACAATCCCCTCGGCCTAAATTCTGACACAGACAA
CACGAAACAGGCTCAAACAACCCTCTCGGCCTAAATTCAGACACAGACAA
CACGAAACCGGGTCTAACAACCCCCTCGGCCTAAACTCTGACACAGACAA
CACGAAACAGGATCAAACAACCCGATCGGACTAAACTCAGACGCAGACAA
CACGAAACAGGCTCGAACAACCCTGCCGGGTTAAACTCCGACGCCGATAA
CACGAAACAGGCTCAAACAACCCAATAGGCCTAAACTCCAACGTAGACAA
CATCAAACGGGCTCCAATAACCCCTTAGGCCTTAACTCAGATGTAGATAA
CACCAGACCGGCTCCAACAACCCCCTGGGCCTGAACTCAGACGTGGACAA
CACGAAACAGGATCCAACAACCCCCTTGGCCTTAACTCCGACGCGGACAA
CACGAAACAGGATCCAACAACCCCCTTGGGCTTAACTCCGAAGCGGACAA
CATGAAACAGGGTCCAACAACCCCCTTGGCCTCAACTCCGACGCAGATAA
CATGAGACAGGCTCCAACAATCCTCTAGGCCTCAACTCCGATGTGGATAA
CATGAAACAGGGTCCAACAACCCCTTAGGCCTTAACTCTGATGTAGACAA
CACGAAACAGGGTCTAATAATCCCCTAGGCCTCAACTCTGATATAGATAA
CATGAGACGGGGTCCAATAATCCCTTAGGCCTCAACTCTGATGTAGATAA
CACCAAACAGGCTCGAACAATCCCCTCGGCCTAAACTCAGACGCAGACAA
CACGAAACAGGATCAAACAATCCCCTTGGACTGAACTCAGACGCCGACAA
CACGAAACTGGGTCAAACAACCCTCTCGGCCTAAACTCAGACGCGGACAA
CACGAAACAGGGTCTAACAACCCTCTAGGCCTAAACTCAGACGTAGACAA
CACGAAACCGGGTCAAACAACCCCCTGGGCCTAAACTCAGACTCAGATAA
CATGAGACTGGCTCAAATAACCCTTTGGGACTAAACTCAGACTCGGACAA
CACGAAACAGGCTCCAACAACCCCCTAGGCCTCTCATCAGACACAGATAA
CACGAAACCGGCTCAAATAACCCCCTCGGACTTAATTCTGATACAGATAA
[650]
[650]
[650]
[650]
[650]
[650]
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[650]
[650]
[650]
[650]
[650]
[650]
[650]
[650]
[650]
[650]
[650]
62
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
AATTTCTTTCCACCCATACTTCTCTTACAAAGACCTTCTAGGTTTCGCTG
AATTTCATTCCACCCTTACTTCTCATACAAAGACCTCCTGGGATTCGCAG
AATTTCCTTCCACCCGTACTTCTCTTACAAAGACCTACTGGGATTTGCAG
AATTTCCTTCCACCCATATTTCTCTTATAAAGACTTGCTCGGGTTTGCAG
AATTTCTTTCCACCCATACTTCTCTTACAAAGATTTGCTAGGATTTGCAG
AATTTCTTTTCACCCATACTTCTCTTACAAAGACCTCTTAGGATTTGCAG
AATCTCCTTCCATCCATACTTTTCTTACAAAGATCTCCTAGGATTTGCGG
AATTGCTTTCCACCCCTACTTCTCTTACAAGGACCTACTTGGTTTCGCAG
GATTTCCTTTCACCCTTACTTTTCTTACAAAGACTTACTTGGATTTGCAG
AATTTCCTTCCACCCCTATTTCTCATACAAAGACCTTCTGGGCTTTGCAG
AATTTCCTTCCACCCTTACTTTTCTTACAAAGACTTACTTGGATTTGCAG
AATTTCTTTCCACCCTTACTTCTCTTACAAAGACCTCCTTGGATTTGCAG
AATTTCTTTCCACCCATACTTTTCTTACAAAGACTTACTTGGGTTCGCAG
AATTTCTTTCCACCCATACTTCTCCTACAAAGACCTTCTAGGGTTTGCAG
AATTTCTTTCCACCCATACTTCTCCTATAAAGACCTTCTAGGGTTTGCAG
AATTTCTTTCCACCCATACTTTTCCTACAAAGACCTTCTAGGATTTGCAG
AATTTCCTTCCACCCATACTTCTCCTATAAAGACCTTCTAGGCTTTGCAG
AATCTCTTTCCACCCATACTTATCTTACAAAGACCTGCTTGGTTTTGCAG
AATTTCTTTTCACCCATACTTTTCTTATAAAGACGTTTTAGGATTTGCAG
AATTTCATTCCACCCATACTTCTCATACAAAGACCTCCTAGGATTCGCAG
AATTTCATTCCACCCATACTTTTCTTACAAGGACCTTTTAGGCTTTGCGG
AATTTCTTTCCACCCATATTTCTCCTACAAAGACCTTTTAGGATTCGCAG
AATTTCTTTTCACCCATATTTTTCTTACAAAGACCTTTTAGGATTTGCAG
AATTTCTTTTCACCCATACTTCTCTTACAAAGACCTACTAGGATTTGCAG
AATTTCCTTCCACCCATACTTTTCTTACAAAGACCTACTCGGCTTCGCAG
AATCTCTTTCCACCCATATTTTTCCTACAAAGACCTGCTCGGTTTCGCAG
AATCTCCTTCCACCCTTATTTCTCTTATAAAGACCTACTTGGGTTTGCAG
AATCTCCTTCCACCCATATTTCTCTTATAAAGACCTACTTGGATTCGCAG
GATTTCTTTCCACCCATACTTTTCTTACAAAGACTTACTTGGATTTGCAG
AATTTCTTTCCACCCTTACTTTTCTTACAAGGACTTACTTGGGTTTGCAG
AATTTCTTTCCACCCATACTTCTCTTACAAAGACCTGCTTGGATTTGCAG
AATCTCTTTTCACCCATACTTTTCTTATAAAGACCTTTTAGGATTTGCAG
AATCTCTTTTCATCCCTACTTTTCCTATAAAGACCTACTTGGGTTTGCAG
AATTTCGTTCCACCCATACTTTTCTTATAAAGACCTCCTTGGATTTGCAG
AATGTCATTCCATCCATACTTTTCTTACAAAGACCTGCTAGGGTTTGCAG
AATTTCTTTCCACCCATACTTCTCTTATAAAGACCTCCTAGGATTTGCAG
AATCTCGTTCCACCCATACTTTTCTTACAAAGATCTTCTTGGATTTGCAG
AATTTCTTTCCACCCATACTCCTCATATAAAGACCTTCTTGGATTCGCAG
AATTTCCTTTCACCCATACTTCTCTTACAAAGATCTGCTAGGGTTTGCAG
AATTTCTTTCCACCCATACTTCTCTTACAAGGATTTATTAGGGTTCGCAG
AATTTCTTTCCACCCATATTTTTCTTACAAAGACCTGCTTGGATTTGCAG
AATTTCTTTCCACCCATACTTCTCTTACAAAGACCTATTGGGATTTGCAG
AGTCTCTTTCCACCCGTACTTCTCATACAAAGACCTCCTTGGGTTCGTAA
AATCTCCTTCCACCCTTACTTCTCCTATAAAGACCTCCTTGGCTTCGTTC
AATCCCATTTCACCCCTACTTCTCCTACAAAGACCTCCTCGGCTTCGTAG
AATCTCATTCCACCCCTACTTCTCATACAAAGATCTCCTAGGGTTCGCAA
AATTTCATTCCACCCTTACTTCTCATACAAAGACCTTCTCGGCTTCGCAA
AATCCCCTTCCACCCGTACTTCTCCTATAAAGACCTGCTCGGCTTTGCGG
AATCCCTTTCCACCCGTATTTCTCCTACAAAGACCTGCTAGGATTTGCAG
AATCCCCTTCCACCCGTACTTTTCCTACAAAGACCTGCTAGGGTTCGCAG
AATCCCATTCCACCCCTATTTTTCATACAAAGACCTCCTAGGAGCCACAG
AATCCCATTTCACCCCTACTTTTCATACAAAGACATTTTAGGCTTCGCAG
AATCCCATTCCACCCCTACTTTTCATACAAAGACCTCCTAGGGTTCACAG
AATTCCATTCCACCCCTATTTCTCATACAAAGATCTTTTAGGGTTCGCAG
AATCTGATTCCACCCATATTTCTCATATAAAGACCTCCTAGGCTTTGCAG
AATCTCATTCCACCCCTATTTGTCCTACAAAGAGGTGTTAGGCTTTGTAG
AATGTCTTTCCACCCCTACTTCTCATACAAAGACCTCCTAGGCTTCGTAG
AATCTCCTTCCACCCTTACTTCTCCTATAAAGACCTACTAGGCTTCGTGG
AATCTCCTTCCACCCCTACTTCTCCTACAAAGACCTCCTAGGATTTGCAG
AATTTCTTTCCACCCTTACTTCTCCTATAAAGACCTGCTAGGATTTGCAG
AATCTCTTTCCATCCATACTTCTCCTACAAGGACCTTCTGGGCTTTGCAG
AATCTCTTTCCACCCATACTTCTCATACAAAGACCTTATCGGCTTCGCAG
[700]
[700]
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[700]
[700]
[700]
[700]
[700]
[700]
[700]
[700]
[700]
63
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GTGTGATTATTCTACTAACTTGTCTTGCACTATTCGCCCCCAATCTTCTT
GTGTAATTATTTTACTAACCTGCCTCGCATTATTCGCCCCCAGCCTCTTA
GTGTGATTATCCTATTAACTTGCCTCGCATTATTTGCCCCTAACCTCCTA
GGGTGCTCATCTTACTAACCTGCCTCGCACTATTTTCTCCTAACCTCTTA
GCGTAATTATTCTACTCACTTGCCTTGCATTATTCGCCCCCAACCTTCTA
CTGTAATCATTTTACTAACCTGCCTCGCACTATTTGCCCCCAACCTCCTG
GGGTAATCATTTTACTAACCTGCCTCGCACTATTTTCCCCCAACCTCTTA
GCGTAATTATCTTATTAACCTGCCTCGCACTATTTGCCCCCAACCTGCTA
GAGTAATTATTTTATTAACCTGCCTTGCACTATTTGCCCCCAACCTCTTA
GAGTAATCATTTTACTAACCTGCCTCGCGCTATTCGCCCCCAACCTCTTA
GAGTAATTATCTTATTAACCTGCCTCGCACTATTTGCTCCCAACCTCCTA
GCGTCATTATCTTACTAACCTGTCTCGGACTATTTGCCCCTAACCTCTTA
GCGTAATTATCTTGTTAACCTGCCTCGCACTATTTGCCCCCAATCTCCTA
GTGTAATCATTCTATTAACCTGTCTTGCACTATTTGCCCCCAACCTTCTC
GTGTAATTATTCTATTAACCTGCCTTGCACTATTTGCCCCCAACCTCCTC
GCGTAATTATTTTATTAACCTGTCTAGCACTATTTGCTCCAAACCTCCTA
GCGTGATCATTCTATTGACTTGCCTGGCATTATCTGCCCCCAACCTCCTA
GCGTAATCATTCTACTAACCTGCCTCGCACTATTTGCCCCCAATCTCTTA
GAGTAATTATTCTGTTAACCTGCCTGGCACTGTTTGCCCCCAACCTCCTT
GCGTAATCATCTTACTGACCTGTCTTGCACTATTTGCCCCCAACCTTCTT
GGGTAATCATTCTACTAACTTGCCTTGCATTATTTGCCCCCAACCTCCTA
GCGTAATCATTCTATTAACCTGCCTTGCACTATTTGCCCCTAACCTTCTC
GCGTAATTATTCTACTAACTTGTCTTGCACTATTTGCCCCTAACCTCTTG
CCGTGATCATTTTATTAACTTGCCTTGCACTATTCACCCCGAACCTGCTA
GCGTAGTCATTCTACTAACCTGCCTTGCATTATTTGCCCCTAACATCTTA
GCGTAATTATTCTATTAACCTGTCTTGCACTATTCGCCCCCAACCTCCTG
GAGTAATTATTTTATTAACCTGCCTTGCACTATTTGCCCCCAACCTCCTA
GGGTAATTATCTTACTAACCTGCCTCGCACTATTTGCCCCCAACCTCTTG
GCGTAATCATCCTACTAACCTGCCTTGCACTATTTGCCCCCAACCTGTTA
GGGTAATCATTCTACTGACTTGCCTTGCATTATTTGCTCCCAACCTCCTG
GGGTAATCATCCTATTAACCTGCCTTGCACTATTTGCCCCCAACCTCCTG
GCGTAATTATGTTATTAACCTGCCTCGCTTTATTTGCCCCCAACCTACTT
GCGTAATCATTTTATTAACCTGCCTTGCACTGTTTGCCCCCAACCTCCTA
GCGTAATTATTCTATTAACTTGCTTAGCATTATTTGCCCCCAACCTGCTT
GCGTAATTATCCTTCTCACTTGCCTTGCCTGATTTGCCCCAAACCTGCTC
CTGTAATTATCTTATTAACTTGTCTTGCCCTATTCGCCCCTAATCTCCTA
GCCTGCTTATTTTATTAACCTGCCTCGCATTATTCGCCCCCAACCTCCTG
GCGTAAATATTTTACTAACCTGCCTCGCACTATTTGCCCCCAACCTCCTG
GCGTGATTATACTACTAACCTGCCTCGCACTATTCGCCCCCAACCTCTTA
CTGTAATTATCCTATTAACCTCCCTTGCTCTATTTGCCCCCAACCTTCTA
TTATACTCCTAGCTCTTACACTACTAGCACTATTCTCCCCTAACTTACTA
TGTTATTGCTGGCCCTCACCTCTCTAACGTTTTTCTCCCCCACCCTGCTC
TTCTACTCTTCACCCTCACCTCCCTGGCCCTATTCCTGCCAAACCTCTTA
TTGTTCTAATTGGATTAACTAGCCTCGCACTGTTTTCCCCTAACCTCCTA
TCGTTTTAATTGGCCTAGCTAGCCTCGCACTGTTCTCCCCCAACCTGCTG
TTCTTCTAACCGCACTCGCCTCGCTAGCACTATTCTCCCCCAACCTCCTG
TTCTTCTAACCGCACTTGCCTCGCTAGCGCTATTCTCCCCTAACCTCCTC
TCCTTTTAACCGCACTCGCCGCACTAGCGCTCTTTTCTCCGAACCTCTTA
CCGTTCTAATTGGCCTCACCTCCCTCGCCTTATTCTCCCCCAACCTCCTA
CCGTCCTAGTTGGCCTGACTTCTCTCGCCCTGTTCTCCCCAAACATCTTA
CCGTCCTAATTAGCCTCACCTCCCTCGCCTTATTTTCTCCTAACCTCCTG
CCGTCCTAATTGGTCTCACCTCTCTTGCCTTATTCTCCCCTAACCTCTTA
TTCTACTTATTGCCCTCACATGCCTGGCCCTCTTCTCCCCCAACCTCCTC
TACTCCTCATTGCACTCGCATGCCTAGCCCTCCTTTCCCTTAACCTGCTA
TCGTACTGATCGCACTAGTCTGCCTGGCATTATTTGCCCCCAACCTTCTA
TCGTTCTTATCGCACTAACCTCCCTAGCACTATTCTCACCCAACCTTCTT
CAGTGTTAATTGCCCTGACCTCTCTTGCTCTCTTCTCACCCAATTTGCTA
CAGTACTAATTGCTCTTACCTCCTTAGCCCTATTCTCTCCTAACCTCCTT
CCGTCATCATCTTTCTTACATGCTTAGCACTATTTTCCCCCAACCTCTTA
CCATTCTTATTACCCTTACTTGCCTTGCTCTCTTCTCCCCTAATCTCCTT
[750]
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[750]
[750]
[750]
[750]
[750]
[750]
[750]
[750]
[750]
64
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GGAGACCCAGACAACTTCACCCCCGCAAACCCTCTAGTTACCCCACCCCA
GGAGACCCAGACAACTTTACCCCTGCAAACCCACTAGTTACCCCACCCCA
GGAGACCCAGATAACTTCACACCTGCAAATCCCCTCGTCACTCCCCCTCA
GGAGATCCAGACAACTTTACCCCTGCAAATCCTCTAGTCACCCCTCCACA
GGAGACCCAGACAACTTCACCCCCGCAAACCCGCTAGTCACACCACCCCA
GGAGACCCCGACAACTTTACCCCAGCAAACCCACTGGTCACTCCACCCCA
GGCGACCCAGATAACTTTACACCTGCAAATCCTCTAGTCACCCCTCCTCA
GGAGATCCAGACAATTTCACGCCTGCAAACCCATTAGTCACTCCCCCCCA
GGAGATCCAGACAATTTCACTCCTGCAAACCCACTAGTTACCCCTCCCCA
GGAGACCCAGACAACTTCACCCCAGCGAATCCCCTAGTCACCCCTCCCCA
GGAGACCCTGACAATTTTACCCCTGCAAACCCACTAGTTACCCCTCCCCA
GGAGACCCAGACAATTTTACACCTGCAAACCCACTAGTCACACCCCCTCA
GGCGACCCGGACAACTTCACCCCCGCAAACCCATTAGTTACTCCTCCCCA
GGGGACCCAGACAACTTCACCCCAGCTAATCCCCTAGTCACACCACCACA
GGGGACCCAGACAACTTCACCCCAGCTAATCCCCTAGTCACACCACCACA
GGAGACCCAGACAACTTCACCCCAGCCAACCCTTTAGTCACACCACCACA
GGGGACCCAGACAACTTCACCCCAGCTAATCCCCTAGTCACACCGCCACA
GGAGACCCAGATAATTTCACCCCTGCAAACCCTCTAGTCACTCCCCCTCA
GGAGACCCAGACAACTTCACCCCAGCAAACCCACTAGTTACACCACCCCA
GGAGACCCGGACAACTTCACCCCTGCAAACCCCCTTGTCACCCCACCCCA
GGCGACCCAGATAATTTTACCCCAGCAAACCCTCTAGTTACCCCACCCCA
GGGGACCCGGATAACTTCACCCCAGCAAATCCTTTAGTCACTCCGCCCCA
GGAGACCCAGACAACTTCACCCCTGCGAATCCATTAGTTACCCCTCCCCA
GGAGACCCAGACAACTTCACCCCAGCAAACCCCCTAGTCACCCCTCCCCA
GGAGACCCAGACAATTTTACACCAGCCAACCCACTAGTTACCCCTCCTCA
GGAGACCCAGACAATTTCACCCCCGCGAACCCCCTAGTCACTCCCCCTCA
GGAGACCCAGACAATTTTACGCCTGCAAACCCATTAGTTACCCCCCCCCA
GGAGATCCGGACAATTTTACGCCTGCGAACCCACTGGTTACACCCCCTCA
GGAGATCCAGACAACTTCACCCCTGCAAACCCCCTAGTCACCCCTCCCCA
GGAGACCCAGACAATTTCACTCCTGCAAACCCGCTAGTTACCCCTCCCCA
GGAGACCCAGACAATTTCACCCCTGCAAATCCATTAGTCACCCCTCCCCA
GGAGACCCAGACAACTTCACCCCTGCAAATCCACTAGTTACCCCTCCCCA
GGAGACCCAGACAATTTCACCCCTGCAAACCCCCTGGTCACCCCCCCCCA
GGAGACCCAGATAACTTCACCCCTGCAAACCCCTTAGTTACCCCTCCCCA
GGAGACCCCGACAACTTCACCCCAGCAAATCCTCTAGTTACCCCGCCCCA
GGAGACCCAGATAACTTCACCCCCGCAAACCCATTAGTCACCCCTCCCCA
GGGGACCCTGAGAATTTTACCCCGGCAAACCCCCTAGTCACCCCTCCCCA
GGGGACCCAGACAATTTCACCCCGGCAAATCCTCTAGTTACCCCTCCTCA
GGAGACCCTGACAACTTCACGCCTGCAAACCCCTTGGTCACCCCCCCCCA
GGAGACCCCGATAATTTTACCCCTGCAAACCCACTAGTTACTCCACCCCA
GGAGACCCGGACAACTTCACCCCCGCAAACCCCCTAGTTACCCCTCCCCA
GGAGACCCCGACAATTTTACACCTGCAAATCCTTTAGTTACCCCCCCCCA
GGAGACCCAGAAAACTTCACCCCCGCAAACCCTCTAGTTACACCACCCCA
GGCGACCCAGAGAACTTCACCCCGGCGAACCCGCTAGTTACCCCACCGCA
GGAGACCCCGACAACTTCACCCCCGCAAACCCACTAGTTACCCCACCCCA
GGAGACCCAGACAATTTTACACCAGCCAATCCGCTGGTAACCCCTCCCCA
GGGGACCCGGACAACTTTACACCCGCCAACCCACTCGTAACCCCGCCCCA
GGTGATCCGGACAATTTCACCCCCGCAAACCCGCTAGTTACGCCCCCACA
GGTGACCCGGACAATTTCACCCCTGCAAACCCATTAGTTACTCCCCCACA
GGTGACCCGGATAATTTCACCCCAGCGAACCCGCTAGTCACTCCCCCACA
GGGGACCCAGACAACTTCACGCCCGCCAACCCACTCGTGACGCCCCCTCA
GAGCACCAAGATAACTTCACGCCAGCCAACCCCCTTGTGACTCCTCCTCA
GGGGACCCAGACAACTTCACGCCTGCCAACCCACTCGTAACACCCCCTCA
GGGGATCCAGATAACTTCACACCAGCCAACCCACTCGTAACGCCCCCGCA
GGAGACCCAGACAATTTCACACCAGCCAACCCCTTAGTTACCCCACCTCA
GGAGACCCAGACAACTTCACTCCCGCCAACCCCCTAGTGACACCACCTCA
GGCGACCCAGACAACTTCACCCCAGCCAACCCCCTAGTGACTCCCCCTCA
GGCGACCCAGACAACTTCACCCCCGCCAACCCCCTAGTGACACCCCCACA
GGGGACCCAGACAATTTCACGCCCGCCAACCCACTAGTCACCCCTCCCCA
GGTGACCCAGACAACTTCACCCCCGCCAACCCCCTGGTTACTCCGCCCCA
GGAGACCCAGACAATTTCACCCCTGCAAACCCGCTAGTCACCCCACCCCA
GGAGACCCCGACAATTTTACCCCAGCCAACCCGCTAGTTACCCCTCCACA
[800]
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[800]
[800]
[800]
[800]
[800]
[800]
[800]
[800]
[800]
65
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CATTAAACCCGAATGATATTTCCTATTTGCATACGCTATCCTACGCTCAA
CATTAAACCCGAATGATACTTCCTATTTGCATACGCAATTCTCCGTTCCA
TATTAAACCCGAGTGATACTTCCTGTTTGCATATGCAATTCTGCGCTCAA
TATTAAGCCCGAATGATACTTTTTATTTGCATACGCAATTCTCCGCTCTA
CATTAAGCCTGAGTGATACTTCCTGTTTGCCTATGCCATTCTACGCTCAA
CATTAAACCCGAGTGGTACTTCTTATTTGCATACGCAATTTTACGCTCAA
CATCAAGCCTGAATGATATTTCCTGTTTGCATACGCAATTCTACGATCGA
TATTAAACCTGAATGATACTTCCTGTTTGGTTACGCAATTCTCCGCTCAA
TATTAAACCCGAATGATACTTCCTATTTGCATACGCAATTCTCCGCTCTA
CATTAAGCCTGAATGATACTTCTTATTCGCATACGCGATCCTACGCTCAA
TATTAAACCCGAATGATATTTTCTATTTGCTTACGCAATTCTCCGCTCTA
TATCAAGCCTGAATGATATTTCCTATTTGCATACGCAATTCTCCGGTCAA
CATTAAGCCTGAGTGATATTTCCTATTTGCTTATGCAATCCTCCGCTCAA
TATCAAGCCTGAATGAAACTCCCTATTTGCGTACGCGATTCTACGCTCAA
TATCAAGCCTGAATGATACTTCCTATTTGCGTACGCGATTCTGCGCTCAA
TATCAAGCCAGAATGATACTTCCTGTTTGCGTACGCAATTCTACGCTCGA
TATCAAGCCTGAATGATACTTCCTATTTGCGTACGCGATTCTGCGCTCAA
TATTAAGCCCGAATGGTATTTCCTATTTGCATACGCGATTCTACGCTCGA
TATTAAGCCCGAATGATATTTCTTATTCGCGTACGCAATTCTGCGCTCAA
CATTAAACCCGAATGGTACTTCCTATTCGCATACGCAATTCTCCGCTCAA
TATCAAGCCTGAATGATACTTCCTGTTTGCCTACGCAATTCTACGCTCGA
TATTAAGCCTGAATGATACTTCCTGTTTGCGTACGCAATTCTACGCTCAA
CATTAAACCCGAATGATACTTCCTATTTGCATACGCAATTCTACGCTCAA
TATTAAACCTGAATGATATTTCTTATTTGCATACGCAATCCTCCGCTCAA
TATTAAACCCGAATGATACTTCCTATTTGCATACGCCATCCTGCGCTCAA
TATCAAGCCCGAATGATACTTCCTATTTGCGTACGCAATTCTACGCTCAA
TATCAAGCCTGAATGATATTTCTTATTTGCGTACGCAATTCTACGCTCAA
TATTAAACCCGAATGATACTTCCTATTTGCGTATGCAATTCTACGCTCAA
TATTAAGCCCGAATGGTATTTTCTGTTTGCATACGCAATTCTCCGCTCTA
TATTAAACCCGAATGGTACTTTCTGTTTGCATACGCAATTCTCCGCTCTA
CATTAAGCCCGAATGATATTTCTTATTCGCATACGCCATCCTTCGCTCAA
CATTAAACCCGAGTGATACTTTCTATTTGCATACGCAATTCTACGCTCAA
TATTAAACCCGAATGATACTTCCTGTTTGCGTACGCAATTCTCCGCTCTA
CATTAAACCCGAATGATACTTCCTATTTGCCTATGCAATCTTACGCTCAA
CATTAAACCTGAATGATACTTCTTATTTGCGTATGCTATTCTACGCTCAA
TATTAAACCCGAATGGTACTTCTTATTTGCGTATGCCATCTTACGTTCAA
CATCAAGCCCGAGTGATATTTCCTGTTTGCCTACGCAATCTTGCGCTCAA
CATTAAGCCCGAGTGATATTTCTTATTTGCCTACGCAATTTTACGCTCAA
TATTAAGCCCGAGTGATATTTTTTGTTTGCGTACGCTATTCTTCGCTCAA
TATCAAGCCCGAATGATACTTCCTGTTTGCATACGCAATTCTACGCTCAA
CATTAAGCCCGAATGATACTTCTTATTTGCCTATGCAATTCTCCGTTCAA
TATTAAACCCGAATGATATTTCCTGTTTGCATACGCCATTCTTCGATCAA
CATCAAACCAGAATGATACTTCCTATTTGCCTACGCCATCCTACGATCAA
CATTCAACCCGAGTGGTACTTCTTGTTCGCCTACGCTATTATCCGGTCTA
CATCAAGCCCGAATGATACTTCCTATTTGCCTACGCCATTCTCCGCTCCA
TATTAAGCCCGAGTGATACTTTTTATTTGCCTACGCTATTCTTCGCTCAA
TATTAAACCCGAATGATACTTTTTATTTGCTTACGCTATTCTTCGCTCCA
TATTAAACCAGAGTGATATTTCCTATTTGCTTACGCCATTCTCCGATCAA
TATCAAGCCAGAGTGATATTTCCTGTTTGCTTACGCCATTCTCCGATCAA
CATCAAGCCAGAGTGATACTTTTTATTTGCTTACGCCATCCTACGATCAA
CATTAAACCAGAATGATATTTCCTATTTGCCTACGCCAATCTTCGGTCAA
CATCAAGCCAGAATGATACTTCCTGTTTGCCTATGCCATCCTTCGATCAA
CATCAAGCCAGAATGATACTTCCTATTTGCCTACGCTATCCTTCGGTCAA
CATTAAACCAGAATGATATTTCCTATTTGCCTACGCCATTCTTCGATCAA
CATTAAGCCCGAATGATATTTCCTGTTCGCATACGCCATTCTCCGCTCAA
CATTAAGCCAGAATGATATGTCCTATTCGCATACGCCATTCTCCGTTCAA
TGTCAAGCCTGAATGATACTTCCTGTTTGCTTACGCCACCCTCCGGTCAA
CATCAAGCCCGAATGATACTTCCTATTCGCATACGCCATTCTACGTTCGA
CATTAAGCCTGAGTGATATTTCCTCTTTGCCTACGCCATTCTACGTTCAA
TATTAAACCTGAATGATACTTCCTCTTTGCATACGCTATCCTTCGATCAA
TATTAAGCCGGAGTGATATTTCCTATTTGCATATGCCATTCTACGGTCAA
TATTAAGCCAGAGTGATACTTCCTCTTTGCGTACGCAATCCTACGATCGA
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
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[850]
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[850]
[850]
[850]
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[850]
[850]
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[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
[850]
66
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
TTCCTAATAAACTAGGAGGAGTCCTTGCCCTCTTGGCATCTATCTTAGTT
TCCCTAATAAACTAGGAGGTGTCCTAGCCCTCCTAGCCTCCATTCTAGTC
TTCCAAACAAACTAGGAGGTGTCCTTGCCCTCCTAGCTTCCATCCTTGTG
TTCCCAACAAACTAGGGGGAGTCCTGGCCCTTCTAGCCTCCATCCTAGTC
TTCCCAACAAACTAGGAGGCGTCCTAGCCCTTTTAGCTTCTATTTTAGTC
TTCCCAACAAACTAGGAGGAGTCCTCGCCGTTTTAGCCTCAATTCTAGTT
TTCCTAATAAACTAGGGGGCGTACTAGCACTTCTAGCCTCAATCCTTGTT
TTCCAAATAAACTAGGAGGAGTCCTTGCCCTCCTAGCTTCTATTCTAGTA
TCCCTAACAAATTAGGGGGAGTCCTGGCCCTCCTTGCCTCTATTCTAGTC
TTCCTAACAAGCTCGGAGGAGTCCTTGCCCTCTTAGCCTCCATTCTTGTC
TTCCAAATAAGCTGGGAGGAGTCCTCGCTCTCCTCGCCTCTATTCTAATC
TTCCAAATAAACTAGGCGGAGTCCTGGCCCTCCTAGCTTCTATTCTGGTC
TTCCGAATAAATTAGGCGGTGTCCTCGCCCTCCTCGCCTCTATTCTAGTA
TCCCCAACAAACTAGGGGGAGTCCTTGCCCTCCTTGCCTCCATGCTAGTC
TCCCCAACAAACTGGGGGGAGTTCTTGCCCTCCTTGCCTCCATTCTAGTC
TCCCTAATAAACTAGGAGGAGTTCTTGCCCTCCTTGCCTCTATCCTAGTC
TCCCCAACAAACTAGGAGGAGTTCTTGCCCTCCTTGCCTCCATTCTAGTC
TTCCAAACAAACTAGGGGGAGTCCTAGCCCTTCTAGCCTCTATCCTTGTA
TTCCTGACAAACTAGGGGGAGTCCTCGCTCTCCTGGCCTCCATTCTAGTT
TTCCTAATAAACTAGGGGGCGTTTTAGCCCTCCTAGCCTCTATCCTAGTC
TTCCTAACAAACTGGGAGGGGTCCTTGCCCTTCTGGCCTCTATCCTGGTC
TTCCGAATAAACTAGGAGGAGTTCTTGCCCTCCTTGCCTCCATCCTAGTT
TTCCCAACAAATTAGGAGGGGTCCTTGCCCTCCTTGCCTCCATCCTAGTC
TTCCCAATAAACTCGGAGGGGTCCTTGCCCTCTTAGCCTCCATCCTAGTT
TCCCAAACAAACTAGGCGGAGTCCTCGCCCTCCTAGCCTCTATTCTAGTA
TTCCAAACAAACTAGGAGGAGTCCTGGCTCTTCTAGCCTCTATCCTCGTA
TTCCAAATAAACTAGGTGGAGTCTTAGCCCTCCTAGCCTCCATCCTAGTA
TTCCAAATAAACTGGGCGGAGTCCTAGCCCTCCTAGCCTCTATTCTAGTA
TTCCAAACAAGCTGGGAGGAGTCCTGGCCCTCTTAGCCTCTATTCTAGTC
TTCCAAACAAACTAGGAGGGGTCCTGGCCCTCCTTGCCTCCATCCTAGTC
TCCCCAATAAACTAGGAGGAGTCCTAGCCCTCCTGGCCTCTATCTTAGTC
TTCCTAACAAATTAGGAGGAGTCCTTGCCCTCCTTGCCTCCATCCTAGTC
TCCCAAACAAACTAGGGGGAGTCCTAGCCCTCCTCGCATCTATTCTGGTC
TTCCTAACAAGCTAGGTGGGGTACTTGCCCTATTGGCCTCCATTCTAGTC
TTCCTAATAAACTAGGGGGCGTACTAGCCCTCCTAGCCTCCATTCTAGTC
TTCCTAACAAGCTAGGAGGAGTTCTTGCCCTCCTGGCCTCCATCCTAGTC
TCCCTAACAAACTAGGAGGAGTTCTTGCCCTACTGGCCTCAATTTTAGTC
TCCCTAACAAGCTAGGAGGGGTCCTTGCCCTCTTGGCCTCTATTCTAGTC
TTCCTAACAAACTAGGAGGCGTCCTAGCCCTCCTAGCTTCAATCCTAGTC
TTCCAAACAAACTAGGAGGTGTCCTGGCCCTTCTGGCTTCTATCCTAGTC
TTCCAAACAAACTGGGGGGAGTCCTAGCCCTTCTAGCCTCCATCCTAGTC
TCCCGAATAAACTAGGAGGTGTTCTGGCCCTGCTAGCTTCAATCCTGGTC
TTCCAAACAAACTCGGAGGTGTCCTTGCACTCCTATTCTCCATTCTGGTA
TTCCAAACAAGCTAGGGGGAGTCCTAGCGCTATTATTCAGTATTCTAGTA
TCCCAAACAAACTAGGAGGAGTACTCGCACTCCTGTCCTCGATCCTAGTC
TCCCAAACAAACTAGGCGGGGTGTTGGCATTACTAGCATCTATTCTAGTA
TTCCGAACAAGCTAGGCGGAGTATTGGCACTACTAGCATCTATTTTAGTA
TTCCGAACAAGCTAGGTGGTGTACTAGCCCTTCTATTCTCCATCTTAGTA
TTCCTAACAAACTAGGTGGTGTTTTAGCCCTCCTATTCTCCATTTTAGTG
TTCCTAACAAACTAGGCGGCGTTTTAGCCCTGCTGTTTTCCATCTTAGTA
TCCCTAATAAACTGGGAGGAGTTCTAGCACTACTGGCATCTATCTTAGTG
TCCCCAATAAATTAGGAGGAGTGCTAGCATTACTAGCATCCATTTTAGTA
TCCCTAATAAACTGGGAGGGGTTCTAGACTTACTGGCATCTATCTTAGTG
TTCCTAACAAATTAGGGGGAGTGTTAGCATTACTAGCATCTATTTTAGTA
TTCCGAATAAACTAGGAGGAGTTCTTGCCCTCCTGGCCTCCATCCTAGTT
TGCCAAACAAACTCGGAGGAGTTTTAGCCCTACTCGCCTCAAGTCTTGTC
TTCCCAACAAACTCGGAGGCGTCCTAGCCCTGCTCGCCTCAATCCTCGTG
TTCCCAACAAACTAGGAGGCGTCCTAGCCCTCCTCGCCTCAATCCTAGTA
TTCCCAACAAACTAGGGGGTGTCCTAGCTCTACTTGCCTCTATCCTAGTT
TCCCCAATAAACTAGGGGGTGTCCTGGCCCTACTTGCTTCCATCTTAGTC
TTCCAAATAAGCTGGGCGGGGTGCTCGCTTTATTAGCCTCAATCCTCGTC
TTCCAAATAAACTTGGAGGTGTCTTAGCCCTTCTAGCCTCTATCCTAGTA
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
[900]
67
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CTTATGGTTGTTCCCATACTCCACACTTCCAAACAACGAAGCCTAACCTT
CTCATAGTTGTCCCCATCCTTCACACCTCTAAACAACGAAGCCTAACTTT
CTTATAGTCGTCCCGATCCTCCACACATCCAAACAACGAAGTCTCACATT
CTAATACTCGTCCCCTTCCTTCACACATCCAAACAACGAAGTCTCACATT
CTTATAGTAGTCCCACTCCTCCACACTTCTAAACAACGAAGCCTAACCTT
CTTATAGTTGTCCCTATTCTTCACACCTCTAAACAACGAAGCCTAACCTT
CTCATGATTGTACCAATTCTCCACACCTCTAAGCAACGAAGCCTAACCTT
CTTATGGTTGTCCCCATCCTCCACACATCTAAACAACGAAGCCTTACATT
CTAATAGTTGTTCCCATCCTCCACACATCTAAACAACGAAGCCTCACATT
CTTATGGTTGTCCCAATCCTGCACACCTCTAAACAACGAAGCCTGACCTT
CTAATACTTGTCCCCCTCCTTCACACATCTAAACAACGAAGCCTCACATT
CTAATAGTTGTTCCCATCCTCCACACATCTAAACAACGAAGCCTTACATT
TTGATGGTAGTTCCTATCCTTCACACCTCTAAGCAACGAAGCCTTACATT
CTCATAGTGGTCCCCAGCCCCCATACCCCTAAACAACGAAGCCTTACCAG
CTCATAGTTGTCCCCATCCTCCATACCTCTAAACAACGAAGCCTTACCTT
CTTATAGTCGTCCCCATCCTCCACACCTCTAAACAACGAAGCCTAACCTT
CTCATAGTTGTCCCCATCCTCCACACCTCTAAACAACGAAGCCTTACCTT
CTAATGGTTGTTCCTATTCTCCACACATCTAAACAACGAAGCCTCACATT
CTAATGGTTGTCCCCATTCTTCACACCTCTAAACAACGAAGCCTAACCTT
CTTATGATTGTGCCCATTCTTCATACCTCTAAACAACGAAGTCTGACCTT
CTTATAGTTGTACCAATCCTTCACACCTCTAAACAACGGAGCCTCACCTT
CTTATGGTCGTCCCCATCCTCCACACCTCTAAACAACGAAGCCTAACTTT
CTGATAGTTGTTCCTATCCTACATACTTCTAAGCAACGCAGCCTAACCTT
CTCATGGTTGTACCCATCCTTCACACCTCCAAACAACGGAGCTTAACCTT
CTCATAGTTGTACCTATTCTTCATACATCCAAACAACGAAGTCTGACCTT
CTAATGGTCGTCCCCATCCTCCACACATCTAAACAGCGAAGCCTCACATT
TTAATAGTTGTTCCCATTCTTCACACATCTAAGCAACGAAGCCTTACATT
CTCATGGTCGTTCCCATTCTTCATACATCCAAACAACGAAGTCTAACATT
CTAATAGCCGTCCCCATCCTTCACATATCTAAGCAACGAAGCCTTACATT
CTGATAGTTGTCCCCATCCTCCACACATCTAAACAACGAAGCCTCACATT
CTGATAGTTGTCCCCATCCTCCACACATCTAAGCAACGAAGCCTTACGTT
CTGATAGTTGTTCCTACCCTACACACTTCTAAACAACGCAGCCTAACCTT
CTAATAGTTGTCCCCATCCTACACACATCCAAACAGCGAAGCCTCACATT
CTCATAATTGTCCCCATCCTTCACACCTCTAAACAACGAAGCCTCACTTT
CTAATAGTTGTTCCCCTCCTCCACACCTCTAAACAACGAAGCCTAACCTT
CTCATGGTTGTTCCCATACTCCACACTTCCAAACAACGAAGTCTAACCTT
CTCGTAGTTGTCCCTGTCCTCCACACCTCCAAGCAGCGGAGCCTGACCTT
CTAATGGTTGTCCCCATTCTCCACACCTCTAAACAACGAAGCCTCACCTT
TTTTTGGTCGTACCTATTTTTCACACCTCTAAACAACGAAGCTTAACTTT
CTTATGGTCGTCCCAATCCTGCACACCTCCAAACAACGAAGCTTAACTTT
TTAATGGTTGTCCCTATTCTCCACACATCTAAACAACGAAGCCTAACATT
CTCATGGTTGTGCCCATTCTTCACACCTCTAAACAACGGAGCCTAACCTT
TTAATAGTAGTACCACTACTACACACCTCAAAACAACGAGGACTAACATT
CTATTGGTGGTCCCCATTTTACATACCTCAAAGCAACGAGGACTAACCTT
CTCATACTAGTACCCCTTCTCCACACCTCAAAACAACGAGGCCTAATATT
CTTATAGTAGTACCCTATTTACATACATCAAAACAACGAAGTATAACATT
CTTATAGTAGTACCCTTTTTACATACATCCAAACAACGAAGTATAACATT
CTTATACTGGTCCCAATCCTCCACACATCAAAACAACGAGCCCTAACCTT
CTTATACTAGTCCCCATCCTCCACACGTCAAAACAACGAGCTTTGACCTT
CTTATACTAGTCCCGATCCTTCACACATCGAAACAACGAGCCCTAACTTT
CTCATAACAGTACCTTTTCTCCACACATCTAAACAACGAAGCTTAACATT
CTCATAGCAGTGCCTTTCCTACACACATCGAAACAACGAAGTTTAACATT
CTCTTAACAGTACCTTTTCTGCATACATCCAAACAACGAAGCTTAACATT
CTCATAGTAGTACCCTTTCTACATACATCAAAACAACGAAGTCTAACATT
CTGATGGTAGTCCCCATCCTTCACACATCTAAACAACGAAGCCTCACCTT
CTCATAGTCGTCCCCTTCCGTCATACATCTAAACAACGAAGCCTTACCTT
CTCATAGTCGTACCAATCCTCCACACCTCCAAACAACGAGGACTAACGTT
CTTATGGTCGTTCCCATCCTCCACACCTCTAAACAACGAGGCCTAACATT
CTTATAGTGGTGCCAATTCTACACACCTCAAAACAACGAAGCCTCACATT
CTTATAGTAGTTCCGATCCTCCACACCTCCAAACAACGAAGCCTTACATT
CTCATGCTGGTCCCACTTCTCCACACATCCAAACAACGAAGCCTCACCTT
CTTATACTAGTTCCTATCCTCCACACTTCTAAGCAACGAAGTCTAACATT
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
[950]
68
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
CCGACCATTTACTCAATTCCTATTCTGAGCACTCATTGCAAATGTGGTAA
CCGACCCCTTACCCAATTCCTATTCTGAGCACTCATTGCAAACGTAGCCA
TCGTCCCATCACTCAGTTCCTATTCTGAGCACTCATTGCAAACGTAGCCA
CCGGCCAATTTCTCAATTTCTATTCTGAATACTTATTGCTAATGTGGCCA
CCGACCCGTGACCCAATTCCTATTTTGAGCACTTATTGCAAACGTAGCAA
CCGACCTGCCACTCAATTCCTATTCTGAACACTCATTGCAAACGTAGCAA
CCGACCTCTGACTCAGTTCTTATTCTGAGCACTTATCGCAAACGTGGTAG
CCGGCCTGTCACTCAATTCCTGTTCTGAGCACTCATTGCAAATGTAGCTA
CCGACCAGTAACCCAATTCCTATTTTGAGCGCTCATCGCAAATGTAGCGA
CCGACCAGTTACCCAGTTCCTATTCTGAGCACTAATTGCAAACGTAGCAA
CCGACCAGTGACCCAATTCCTATTCTGAACGCTCACTGCGAATGTAGCAG
CCGACCTATAACTCAATTCCTGTTTTGAGCACTCATTGCAAACGTAGCAA
CCGGCCATTAACCCAATTCCTGTTCTGACTGCTCATTGCAAACGTAGCAA
CCGACCAGTAACCCAGATCCTATTCTGAGCACTTATTGCAAACGTAGCTA
CCGACCAGTAACCCAGTTCCTATTCTGAGCACTTATTGCAAACGTAGCTA
CCGACCAGTGACCCAATTCCTATTCTGAACACTTATTGCAAACGTGGCTA
CCGGCCAGTAACCCAGTTCCTATTCTGAGCACTTATTGCAAACGTAGCTA
TCGACCTATAACTCAATTCCTATTTTGAGCACTCATCGCAAACGTAGCAA
CCGACCTGTCACCCAATTCCTGTTCTGAGCACTCATTGCAAACGTGGCAA
CCGACCCCTCACCCAATTCTTATTCTGAACACTCATTGCAAATGTAGCCA
CCGACCAGTGACCCAATTCTTGTTCTGAGCTCTCATTGCAAACGTAGCCA
CCGACCAGTTACCCAATTCCTATTCTGAGCACTCATTGCAAACGTAGCCA
CCGACCTATCACCCAGTTCCTATTCTGGGCACTCACTGCAAACGTAGCAA
CCGGCCAGTTACTCAATTCTTGTTCTGAGCACTTATTGCAAACGTTGCAA
CCGGCCCATAACCCAATTCCTGTTCTGAGCACTTATTGCAAACGTAGCTA
TCGACCTGTCACCCAGTTCCTGTTTTGAGCACTCATTGCAAATGTAGCAA
TCGACCCATAACTCAATTCCTGTTCTGAGCGCTCATTGCAAACGTAGCAA
CCGACCCATAACTCAGTTCCTGTTTTGAGCGCTCATTGCAAATGTAGCAA
CCGGCCAATCACCCAATTCCTTTTCTGAACGCTCATTGCAAATGTAGCAA
CCGACCAATGACCCAATTCCTGTTCTGAGCACTCATGGCAAATGTAGCAA
CCGGCCCGTAACCCAATTCCTATTTTGAGCACTAATTGCAAACGTAGCAA
CCGGCCTATCACCCAGTTCCTATTCTGGGCACTCATCGCAAACGTAGCAA
CCGACCAATAACTCAATTCCTATTCTGAGCACTAATTGCAAATGTAGCAA
CCGACCAGCCACTCAATTCCTATTCTGGACGCTCATCGCCAACGTAGCAA
CCGACCCATAACCCAATTCCTATTCTGAGCACTCATTGCAAACGTAGCAA
CCGACCCGTTACTCAATTCCTATTCTGAGCACTTATTGCAAACGTGGCAA
CCGGCCAGTTACCCAATTCCTGTTTTGAACACTCATCGCCAACGTAGCTA
CCGACCAGTCACCCAATTCCTATTCTGAGCACTCGTTGCAAACGTAGCAA
CCGGCCCCTAACCCAATTCTTATTCTGAACACTAATTGCAAATGTTGCAA
CCGACCTATAACCCAATTCTTATTCTGAGCACTTATTGCAAACGTAGCCA
TCGACCCGTGACTCAATTCCTCTTCTGAGCACTCATTGCAAACGTAGCAA
CCGACCCGTGACCCAATTCTTATTCTGAACACTAGTCGCGAACGTAGCAA
CCGCCCCATCACCCAATTCCTATTCTGAACCCTAGTAGCGGACATAATTA
CCGGCCAATCACTCAGTTTTTATTCTGAACCTTAGTGGCAGATATAGTTA
CCGACCCGCCTCACAACTCCTATTCTGAGTTCTCGTSGCAGACGTAGCCA
CCGACCCGTAACACAGTTTTTATTCTGGGCTCTCGTCGCTGATGTTATAA
TCGCCCCCTGACACAATTTCTATTTTGAACCCTTGTTGCGGATGTTATGG
CCGACCTATTACCCAATTCCTTTTCTGAACACTCATTGCGGACGTTGCCA
CCGACCAATCACGCAATTCCTCTTCTGAACACTCATCGCAGATGTCGCCA
CCGGCCTATCACTCAGTTCCTCTTCTGGACGCTAATCGCAGATGTTGCCA
CCGTCCATTAACCCAGCTTCTATTTTGAACCCTTATTGCAGATGTAGTAA
CCGCCCGTTAACCCAGCTTCTATTTTGGGCCCTAATTGCAGACGTAGCAA
CCGTCCATTAACCCAATTTCTGTTTTGAACCCTTATTGCAGATGTAATAA
CCGCCCGTTAACCCAGCTTCTATTCTGAACCCTCATTGCAGATGTAGCAA
CCGCCCGGTCACACAGTTCCTCTTCTGAACACTCATTGCAGACGTTGCAA
CCGACCACTATCCCAACTCCTATTCTGAACACTCGTTGCCGATGTTGCCA
CCGACCTGTAACTCAGTTCCTATTCTGGACCCTAATTGCAAACGTCGCCA
CCGGCCCGTCACTCAATTCTTATTCTGAACCTTAATCGCAAACGTTGCCA
CCGACCCTTAACCCAGTTCCTATTCTGAACACTAATTGCTAACGTCGCAA
CCGGCCTTTGACCCAATTCCTGTTTTGGACCTTAATCGCCAACGTTGCCA
CCGACCAATCTCCCAGTTCTTGTTTTGAGTGCTGATTGCGGACGTCGCCA
CCGGCCAATTTCTCAATTCCTATTCTGAACGCTAATCGCAGACGTTGCCA
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
[1000]
69
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
TTCTCACATGAATTGGAGGTATGCCAGTTGAAGAACCTTACATTATTATT
TCCTAACATGAATCGGAGGAATGCCAGTTGAAGAACCATACATTATCATC
TCCTCACATGAATTGGCTTTTTGCTCGTTGAAGACCCATATATTATTATT
TTCTCACATGAATTGGTGGTATGCCCGTCGAAGATCCGTATATCATTATT
TCCTAACCTGAATTGGAGGAATGCCAGTCGAAGAACCTTATATTATTATT
TCCTGACATGAATTGGAGGAATACCAGTTGAAGAGCCCTATATCATCATT
TTTTAACATGAATTGGGGGCATGCCGGTCGAAGACCCATATATTATTATT
TCCTTACATGAATTGGCGGAATGCCCGTTGAAGACCCATACATTATTATT
TTCTTACATGAATTGGCGGAATACCCGTCGAAGACCCATACATCATTATT
TTCTTACATGAATCGGCGGGATACCAGTTGAAGAACCATACATCATCATC
TTCTTACATGAATTGGCGGCATACCCGTCGAAGACCCCTATATTATCATT
TCCTTACTTGAATTGGCGGAATGCCCGTTGAAGACCCATACATCATTATC
TCCTTACATGAATTGGTGGAATGCCCGTTGAAGATCCATATATTATTATT
TCCTCACATGAATCGGAGGAATGCCAGCCGAAGAACCTTACAAAATTATT
TCCTCACATGAATCGGAGGAATACCAGTTGAAGAACCTTACATTATTATT
TCCTCACATGAATCGGAGGGATACCAGTTGAAGACCCTTACATTATTATT
TCCTCACATGAATCGGAGGAATACCAGTTGAAGAACCTTATATTATTATT
TCCTCACATGAATCGGCGGAATGCCCGTTGAGGACCCATACATCGTTATT
TCCTGACATGAATCGGAGGGATACCAGTCGAAGGGCCCTATATTATTATC
TCCTAACATGAATTGGAGGGATACCAGTTGAAGACCCTTATATTATCATC
TCCTCACATGAATTGGCGGAATACCTGTTGAAGAGCCTTATATCATTATT
TCCTCACATGAATCGGAGGAATACCAGTCGAAGAACCTTACATTATTATC
TTTTAACATGAATTGGGGGAATGCCAGTTGAAGAACCTTATATCATTATC
TCCTCACATGAATTGGAGGAATACCCGTTGAAGAGCCTTACATTATTATT
TCCTTACGTGAATTGGAGGAATACCAGTCGAAGATCCGTACATCGTCATT
TCCTCACATGAATCGGCGGAATGCCCGTTGAAGACCCGTACATCATTATT
TCCTCACATGAATTGGTGGAATACCAGTTGAAGACCCGTACATCATTATC
TTCTTACATGAATTGGTGGAATGCCCGTTGAAGACCCATACATCATTATT
TCCTTACATGAATTGGTGGAATGCCCGTCGAAGACCCATACATTGTCATT
TCCTCACATGAATTGGCGGAATGCCTGTCGAAGACCCTTATATTGTTATT
TTCTCACATGAATCGGCGGAATACCCGTTGAAGACCCTTACATTGTTATT
TTTTAACATGAATTGGTGGAATGCCAGTTGAGGAACCTTATATCATTATC
TTCTTACATGAATCGGAGGCATACCAGTTGAAGACCCATACATTATCATT
TCCTTACATGAATTGGAGGAATGCCCGTCGAAGAACCTTACATCATTATT
TTTTAACTTGAATTGGAGGAATACCAGTTGAAGAACCCTACATCATCATT
TTCTCACATGAATTGGAGGCATACCAGTTGAAGAGCCTTACATTATCATT
TTCTCACATGAATTGGGGGAATACCAGTCGAAGAGCCCTATATTATTATT
TTCTCACATGAATCGGAGGAATGCCAGTCGAAGAGCCTTACATCATTATT
TCCTGACCTGAATCGGAGGAATACCAGTTGAAGACCCCTACATTATGATT
TCCTAACATGAATTGGAGGGATGCCAGTTGAAGAACCATACATTATTATC
TCCTCACATGAATTGGGGGCATACCCGTCGAAGACCCATATATTGTCATC
TTCTCACCTGAATCGGAGGCATACCAGTTGAAGACCCCTACATTATGATT
TCCTAACATGAATTGGAGGCATACCAGTAGAGCATCCCTTCATCATTATT
TTCTGACATGAATTGGGGGTATACCTGTAGAACACCCATACATTATTATT
TCCTAACCTGAATCGGCGGAATACCGGTTGAACACCCCTATATCATTGTT
TTCTAACTTGAATTGGAGGCATGCCCGTTGAGCACCCTTTTATTATTATC
TTCTCACTTGAATTGGGGGTATGCCAGTTGAACACCCCTTTATTATTATT
TTCTCACTTGAATCGGAGGCATGCCTGTTGAACACCCATTCATTATTATT
TTCTCACGTGAATCGGGGGCATACCAGTTGAGCACCCATTCATTATTATT
TTCTCACCTGGATCGGAGGTATGCCAGTCGAACACCCATTTATTATCATC
TTCTCACTTGAATTGGAGGAATGCCTGTTGAACACCCATTTATTATTATT
TCCTCACTTGAATTGGAGGAATGCCTGTCGAACATCCTTTCATTATTATT
TTCTTACTTGGATTGGCGGAATGCCTGTCGAACACCCCTTCATTATTATT
TTCTCACTTGAATCGGAGGAATGCCTGTTGAACACCCCTTTATTATCATC
TCCTCACATGAATTGGAGGCATGCCTGTCGAACACCCCTTCATTATTATT
GTCTTACATGAATCGGTGGCATGCCTGTAGAACACCCCTACATCATCATC
TCCTCACTTGAATTGGCGGAATACCCGTCGAGCATCCATTCATCATCATC
TCCTTACCTGAATCGGCGGAATACCCGTCGAACATCCATTCATCATCATC
TTCTCACGTGAATCGGGGGAATACCCGTCGAGCACCCCTTTATCATTATC
TCCTTACATGGATTGGAGGAATGCCTGTTGAGCATCCATTCATTATTATC
TTCTTACATGAATTGGAGGAATGCCAGTAGAAGACCCCTACATTATCATT
TTCTTACATGGATCGGGGGAATGCCCGTAGAACATCCATACATTATTATT
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
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[1050]
[1050]
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[1050]
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[1050]
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[1050]
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[1050]
[1050]
[1050]
[1050]
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[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
[1050]
70
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GGTCAAATCGCCTCTCTCACCTACTTCTCCCTCTTCCTAGTTATTATCCC
GGCCAAGTTGCCTCCCTAACCTACTTCTCCCTCTTCCTAATTATTATTCC
GGTCAAATTGCATCCCTAACCTACTTCGCCCTCTTCTTACTTATCATACC
GGCCAAATTGCATCTCTCACCTACTTCGCACTCTTCCTGTTTATCATTCC
GGCCAAATTGCATCCCTAACCTACTTCTCGCTCTTCTTACTTATTATTCC
GGCCAAATTGCATCCCTAACCTATTTTTCCATCTTCCTAATTATTATCCC
GGTCAAATCGCATCACTAACCTATTTCTCCCTCTTCTTAATTATTATTCC
GGCCAAATTGCTTCCCTCACCTATTTTGCTCTCTTCCTGCTAATTATACC
GGTCAAATTGCATCCCTCACCTACTTTGCTCTCTTCCTATTTATCATCCC
GGTCAAGTTGCATCCCTAACTTACTTCTCCCTCTTCCTAATTATTATCCC
GGGCAAATTGCATCCCTCACCTACTTCGCTCTTTTCCTATTTATTAATAC
GGCCAGATCGCTTCCCTCACTTACTTTGCGCTTTTCCTGTTTATCTTCCC
GGCCAGATCGCATCCCTTACGTACTTCGCCCTTTTCCTAATTATTATTCC
GGCCAAATCGCATCCCTCACCAACATCTCCCTCTTCCTAGTTGTTAACCC
GGCCAAATCGCATCCCTCACCTACTTCTCCCTCTTCCTAGTTGTTATCCC
GGCCAAATTGCATCCCTCACCTACTTCTCCCTTTTCCTAGTCATTATCCC
GGCCAAATCGCATCCCTCACCTACTTCTCCCTCTTCCTAGTTGTTATCCC
GGTCAAATTGCATCCCTTACTTACTTTGCTCTTTTCTTGCTTATCATCCC
GGCCAAATTGCATCCCTAACCTACTTTTCCCTCTTCCTAGTCATTATCCC
GGCCAGATTGCTTCCCTGACCTACTTCTCCCTCTTCCTAATTATTATCCC
GGTCAAATTGCATCCCTAACCTATTTCTCCCTCTTCCTTGTAGTCATACC
GGCCAAATCGCATCCCTCACCTATTTCTCCCTCTTCCTAGTAATCATCCC
GGCCAAATTGCGTCTTTAACCTACTTTTCCCTCTTTCTAATCATTATCCC
GGCCAAGTCGCATCCCTAACCTACTTTTCCCTCTTTTTAGTTATTATCCC
GGTCAAATCGCATCCCTTACATACTTTGCTCTCTTCCTGCTTATTATCCC
GGTCAAATTGCATCCCTGACTTATTTTGCCCTTTTCCTACTTATCATACC
GGCCAGATTGCCTCCCTTACCTACTTTGCTCTCTTTCTGTTCATCATCCC
GGCCAAATCGCATCCCTTACCTACTTCGCCCTTTTCCTGCTTATTATTCC
GGCCAAATTGCATCCCTCACCTACTTCGCCCTTTTCCTCTTTATTATTCC
GGTCAAGTTGCATCCCTCACCTACTTCGCTCTTTTCCTGTTTATTATTCC
GGCCAAATTGCATCTCTTACCTACTTCGCCCTATTCCTATTCATTATCCC
GGTCAAATTGCGTCCTTAACTTACTTCTCCCTCTTTCTAATCATTATCCC
GGACAAATTGCATCTCTTACCTACTTCGCTCTTTTCCTATTTATTATACC
GGCCAAGTGGCATCCCTATCTTACTTCTCCCTCTTCCTAATTATCATGCC
GGCCAAGTCGCATCCCTAACCTACTTTTCACTATTCCTAGTTATTATTCC
GGCCAAATCGCATCTCTTACCTATTTCTCCCTCTTCCTAGTTATCATCCC
GGCCAGGTTGCATCTCTAACGTACTTCTCCCTCTTCCTCATTATCATACC
GGCCAAGTTGCATCACTAACCTACTTCTCTCTTTTCCTAGTCATTATTCC
GGCCAAATTGCATCCGTGACGTACTTATCCCTCTTCCTAATTATTATCCC
GGCCAAATCGCATCCTTAACCTACTTCGCCCTCTTCCTAATTATTATCCC
GGACAAGTCGCATCCCTAACTTATTTCGCTCTCTTCTTACTCATTATCCC
GGTCAAGTCGCATCCCTAACCTACTTCTCCTTATTTCTCATTATCATCCC
GGACAAATTGCATCCGTCCTATACTTCGCACTATTCCTCATTTTTATGCC
GGCCAAGTCGCCTCAGTTCTGTACTTTGCATTATTCCTCCTCCTTGCCCC
GGACAAATCGCATCCCTCCTCTACTTCCTACTATTCTTAGTGCTCATACC
GGCCAAGTTGCTTCCTTACTGTATTTCCTCTTGTTCCTTGTCTTCATCCC
GGCCAAGTTGCCTCCTTATTGTATTTCCTCTTATTCCTGGTCCTCATCCC
GGACAAATCGCTTCTCTACTCTACTTCCTTATTTTTTTAGTACTATTCCC
GGACAAATCGCTTCTCTACTCTACTTCCTTATTTTTCTAGTCCTCTTTCC
GGACAATTAGCTTTTTTGTTTTATTTCTTTATCTTCTTAGTGTTATTCCC
GGCCAAATCGCCTCGCTCTTATATTTTCTTCTTTTCCTCGTGCTCATACC
GGCCAAATCGCCTCTCTCCTGTACTTTCTTCTCTTTCTTGTTTTTATACC
GGCCAAATCGCCTCCCTCTTATATTTTCTTCTTTTTCTTGTATTTATACC
GGCCAAGTCGCCTCACTCTTATACTTCCTTCTCTTCCTTGTTTTCATACC
GGACAAGTCGCCTCCTTCCTGTACTTCTTCCTATTCCTAGTCTTCACGCC
GGCCAAATTGCCTCTTTTCTGTACTTCTCCCTATTTTTAGTCCTGTTCCC
GGACAAATCGCCTCCGTCCTGTACTTCTTGTTATTCCTAGTCCTCACCCC
GGACAAATCGCCTCCGTTCTATACTTCCTACTATTCCTAGTGTTCGCCCC
GGCCAAATCGCCTCCCTGCTCTACTTCTCCCTCTTCCTAATCATCACCCC
GGCCAGATTGCCTCACTGCTTTACTTTTCACTCTTCCTAATCATCACACC
GGCCAAGTCGCATCTGTCCTCTATTTCTCCATTTTCTTGGTCTTCATGCC
GGCCAAATCGCATCAGTACTTTATTTCTCTATCTTCCTCCTCCTAATACC
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
[1100]
71
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
TGCAGCGGCAACAGTGGAAAACAAGGTACTAGGCTGACAA
TACAGTAGCCATAGTAGAAAACAAAGTATTAGGTTGACAA
TGCGACAGCATTAGTAGAAAATAAAGTCTTAGGCTGACAA
TGTAGCAGCACTCGTAGAAAACAAAGTGCTGGGCTGTAAC
TATAGCAGCAACTTTAGAAAACAAAGTACTAGGCTGACAA
CGCAACAGCAATTTTAGAGAACAAAGTACTAAACTGGTAA
TGTAACAGCCACAGTGGAAAATAAGGTGCTAGGCTGACAA
TGTTGCCGCATTAGCAGAAAACAAGGTATTAGGCTGACAA
TGCAGCAGCATTAGCAGAGAACAAAGTACTAGGCTGACAA
TACAGCAGCAGCTGTAGAGAACAAAGTCCTAGGCTGACAA
TGCAGCAGCACTAGTAGAAAACAAAATGCTAGGCTGACAA
TGTCACAGCATTAGTAGAAAACAAAGTATTAGGCTGACAA
CACAACAGCGTTAGCAGAAAATAAAATATTAAGTTTACAA
AGCCGCAGCAGTTTTAGAAAACAAGGTGTTAGGCTGACAA
TGCCGCAGCAGTTTTAGAAAACAAAGTGTTAGGCTGACAA
TGCTGCAGCAACTATAGAAAACAAGGTATTAGGCTGACAG
TGCCGCAGCAGTTTTAGAAAACAAAGTGTTAGGCTGACAA
CGCAACAGCATTAGTAGAGAACAAAGTACTTGGCTGACAA
CGCAGCAGCAACTATGGAAAACAAAGTGCTAGGCTGGCAA
CATAGCAGCCACGGTGGAAAATAAAATACTAAGCTGACAA
CGCGGCTGCAGCCATTGAAAACAAAGTACTAGGCTGACAG
TGCCGCAGCAGTGATAGAAAACAAAGTATTAGGCTGACAA
CGCAACAGCGACCATTGAAAATAAAATGCTAGGCTGACAG
CGCAGCAGCAACGATGGAAAATAAAGTGTTAGGCTGACAG
CGCCACAGCACTAGCAGAAAACAAAGTGTTAGGCTGACAA
CACAGCAGCATTAGTAGAAAACAAAGTGCTAGGTTGACAA
TGTCACAGCAATAGCAGAGAACAAAGTACTAGGCTGACGA
TGCCACAGCATTAGTAGAAAATAAAGTGTTAGGCTGACAA
TACAGCAGCACTGGCAGAAAACAAAATGCTAGGCTTACAA
TGCAGCAGCACTAGCAGAAAATAAAGTGCTAGGCTGACAA
CGCAGCAGCACTAGCAGAAAACAAAGTGCTAGGCTGACAA
TGCAACAGCAACCATTGAAAATAAAATGCTAGGCTGACAA
TGCAGCAGCACTAGTAGAAAACAAAGTCCTAGGCTGACAA
AGCCGCAGCAACTTTAGAAAATAAAGTCCTAGGCTGACGG
TGCAGCAGCAACTTTAGAAAATAAAGTATTAGGCTGACAA
TACAGCAGCAACTATAGAGAACAAAGTGTTAGGCTGACAA
AGCTGCAGCAACCCTGGAAAATAAGGCCCTAGGCTGGCAG
CGCCGCAGCCACTATGGAAAACAAAGTCTTAGGCTGACAA
CACAGCAGCAACCTTAGAAAATAAAGTTTTAGGCTGGCAA
CGCGACAGCCACAGTAGAAAACAAAGTGTTAGGCTGATAA
CACAGCAGCGTTAGCAGAAAACAAAGTTTTAGGCTGGAAA
TACAGCAGCAACTTTAGAAAATAAAGTCTTAGGCTGACGA
ACTAGCAGGATGGTTAGAAAATAAAGCACTAAAATGAGCT
ACTTGCAGGATGAGCAGAGAACAAGGCATTGAAATGAGCT
ACTGGCAGGCTGATGGGAAAATAAACTCTTAAACTGGCAA
CGTTGTTGGAGAATTAGAGAACAAAGCTTTAGAGTGGCTT
TGTTGTCGGGGAGCTAGAAAACAAAGCTTTAGAGTGACTT
GCTAGCGGGCTGACTAGAAAATAAAGCTCTCGGATGAACT
CGTGGCGGGCTGATTAGAAAATAAGGCTCTCGGATGAACT
TCTAGCAGGCTGATTAGAAAATAAAGCTCTTGGATGAACT
CATGGCCGGCGAATTAGAAAACAAAGCTCTAGGGTGACTT
CATTGCAGGGGAACTAGAGAATAAAGCCCTAGAATGACTT
TATTGCCGGCGAATTAGAAAACAAGGCCCTAGGATGATCC
CATTGTAGGCGAACTAGAAAATAAGGCTCTAGAATGACTT
ACTAGCAAGCTGGCTTGAGAACAAAGCACTCGGATGAGCC
GCTAGCAGGCTGACTCGAGAACAAAGTAATAGGCTGGTCC
CTTAGCAGGATGACTAGAAAACAAAGCCCTCGGATGAGTC
ACTAGCAGGATGACTAGAAAACAAAGCCCTTGGATGGCTT
GGCTGCAGGCTGATTCGAAAACAAGTCACTAGGATGACGA
CGCCGCAGGCTGATTTGAAAACAAGTCACTAGGATGGCGA
TCTCGTAGGAGCAGTAGAAAATAAAGTTATAGGCTGAACA
CTTGCAGGATGAGCAGGGAATAAAATCCTTCGCTGAGCAT
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
[1140]
72
TABLE 5. CYTOCHROME B NUCLEOTIDE SEQUENCE CHARACTERISTICS
Total Characters=1140
1st Codon
2nd Codon
3rd Codon
Totals
Characters
380
380
380
1140
Constant Characters
203
276
2
483 (42%)
Uninformative Characters
49
66
2
115 (10%)
Informative Characters
128
38
376
542 (48%)
% of Informative - Ts
66%
59%
68%
% of Informative - Tv
34%
41%
32%
73
TABLE 6. PAIRWISE VALUES OF MEAN % SEQUENCE DIVERGENCE
DERIVED FROM UNCORRECTED “P” GENETIC DISTANCE FOR ALL TAXA,
INGROUP TAXA AND OUTGROUP TAXA
Groups
All
Ingroup Taxa
Outgroup Taxa
Sparidae -vs- All
Lutjanidae -vs- All
Haemulidae -vs- All
Nemipteridae -vs- All
Lethrinidae -vs- All
Centracanthidae -vs- Lutjanidae
Centracanthidae -vs- Haemulidae
Centracanthidae -vs- Nemipteridae
Centracanthidae -vs- Lethrinidae
Haemulidae -vs- Lutjanidae
Haemulidae -vs- Nemipteridae
Haemulidae -vs- Lethrinidae
Lethrinidae -vs- Lutjanidae
Lethrinidae -vs- Nemipteridae
Lutjanidae -vs- Nemipteridae
Sparidae -vs- Centracanthidae
Sparidae -vs- Haemulidae
Sparidae -vs- Lethrinidae
Sparidae -vs- Lutjanidae
Sparidae -vs- Nemipteridae
Lutjanidae
Haemulidae
Lethrinidae
Nemipteridae
Sparidae
Uncorrected
%Divergence Number of
Standard
Distance
Comparisons
Deviation
0.2022
20.22
N=1891
0.1627
16.27
N=861
0.2273
22.73
N=190
0.2306
23.06
N=880
0.2120
21.20
N=121
0.2249
22.49
N=121
0.2366
23.66
N=121
0.2252
22.52
N=121
0.1993
19.93
N=4
0.2213
22.13
N=4
0.2287
22.87
N=4
0.2239
22.39
N=4
0.2002
20.02
N=4
0.2377
23.77
N=4
0.2248
22.48
N=4
0.1982
19.82
N=4
0.2327
23.27
N=4
0.2296
22.96
N=4
0.1646
16.46
N=84
0.2238
22.38
N=84
0.2244
22.44
N=84
0.2098
20.98
N=84
0.2338
23.38
N=84
0.1228
12.28
N=1
0.1833
18.33
N=1
0.1702
17.02
N=1
0.2281
22.81
N=1
0.1627
16.27
N=861
0.0419
0.0209
0.0187
0.0274
0.0169
0.0153
0.0117
0.0118
0.0120
0.0099
0.0144
0.0072
0.0141
0.0082
0.0161
0.0063
0.0113
0.0061
0.0158
0.0122
0.0076
0.0111
0.0097
N/A
N/A
N/A
N/A
0.0209
74
TABLE 7. PAIRWISE VALUES OF MEAN % SEQUENCE DIVERGENCE
DERIVED FROM UNCORRECTED “P” GENETIC DISTANCE WITHIN AND
BETWEEN SUBFAMILIES
Boopsinae
Denticinae
Diplodinae Pagellinae
Pagrinae
Sparinae
Boopsinae
Mean % Divergence 14.95
Standard Deviation
2.194
Pairwise
28
Denticinae
14.22
Standard Deviation
2.187
2.115
Pairwise
48
15
Diplodinae
16.94
11.14
Standard Deviation
2.987
3.653
2.588
Pairwise
48
36
15
14.23
15.14
16.02
Standard Deviation
1.920
2.099
3.764
0.387
Pairwise
32
24
24
6
14.65
17.31
16.62
13.61
Standard Deviation
2.110
1.647
3.972
2.045
1.280
Pairwise
40
30
30
20
10
16.49
16.28
16.77
16.96
16.58
Standard Deviation
1.951
2.612
3.026
2.0216
2.397
2.353
Pairwise
88
66
66
44
55
55
Pagellinae
Pagrinae
Sparinae
75
TABLE 8. FOLLOWING (IRWIN ET AL., 1991) BASE COMPOSITIONAL BIAS,
THE UNEQUAL PROPORTIONS OF THE FOUR BASES (A, C, G, AND T), WAS
CALCULATED ACROSS ALL, INGROUP, AND OUTGROUP TAXA
ALL Codons
First Codon
Second Codon
Third Codon
Taxon
A
C
G
T
A1
C1
G1
T1
A2
C2
G2
T2
A3
C3
G3
T3
Acantho berd
25.0
29.5
14.9
30.6
23.9
27.4
26.3
22.4
20.0
25.5
13.9
40.5
31.1
35.5
4.5
28.9
Archosarg pro
25.9
31.4
14.7
28.0
23.9
26.3
26.3
23.4
20.0
25.3
13.9
40.8
33.7
42.6
3.9
19.7
Argyrops spin
24.5
30.0
15.1
30.4
23.7
27.1
26.3
22.9
20.0
25.0
13.9
41.1
29.7
37.9
5.0
27.4
Argyroz argy
23.4
30.3
15.5
30.8
23.2
27.4
26.3
23.2
20.0
25.0
13.9
41.1
26.9
38.5
6.3
28.2
Boops boops
24.5
31.4
15.5
28.6
23.7
27.1
26.3
22.9
20.0
25.3
14.2
40.5
29.7
41.8
6.1
22.4
Boopsoid ino
26.1
29.2
14.6
30.0
25.5
25.5
25.5
23.4
20.3
25.5
13.7
40.5
32.6
36.6
4.7
26.1
Calamus nod
25.1
28.6
15.6
30.7
25.0
26.8
25.0
23.2
20.3
25.0
13.7
41.1
30.0
33.9
8.2
27.9
Cheimer nuf
24.1
30.4
15.7
29.7
23.4
27.4
27.4
21.8
20.0
25.3
14.2
40.5
28.9
38.7
5.5
26.8
Chrysobl cris
24.5
30.5
15.2
29.8
23.7
26.3
27.1
22.9
20.0
25.8
13.9
40.3
29.7
39.5
4.5
26.3
Crenid crenid
24.6
31.4
15.9
28.2
23.4
26.6
27.4
22.6
20.0
25.5
14.7
39.7
30.3
42.1
5.5
22.1
Cymatoc nas
24.9
30.3
14.7
30.1
24.7
27.4
25.3
22.6
20.0
25.8
14.2
40.0
30.0
37.6
4.7
27.6
Dentex dent
24.4
29.9
15.4
30.3
23.9
26.8
26.6
22.6
20.0
25.0
14.2
40.8
29.2
37.9
5.5
27.4
Dentex tumif
24.3
31.1
15.3
29.3
25.0
25.8
25.3
23.9
20.3
25.3
13.7
40.8
27.6
42.4
6.8
23.2
Diplodus arg
25.5
31.7
15.9
26.9
24.5
27.1
27.4
21.1
20.5
26.1
14.7
38.7
31.6
41.8
5.5
21.1
Diplodus berm
24.6
31.0
15.6
28.8
22.9
27.4
27.6
22.1
20.0
25.3
14.2
40.5
31.1
40.3
5.0
23.7
Diplodus cerv
25.0
30.3
15.5
29.2
24.5
26.6
26.3
22.6
20.0
25.5
14.2
40.3
30.5
38.7
6.1
24.7
Diplodus holb
24.6
31.1
15.7
28.6
22.9
27.1
27.6
22.4
19.7
25.3
14.5
40.5
31.1
41.1
5.0
22.9
Evynnis japo
24.3
28.9
15.9
30.9
23.4
26.3
27.1
23.2
20.0
25.0
13.9
41.1
29.5
35.5
6.6
28.4
Gymno curvid
24.6
28.7
16.4
30.3
23.4
26.1
27.4
23.2
19.7
25.8
14.7
39.7
30.8
34.2
7.1
27.9
Lagod rhomb
26.1
31.8
14.5
27.6
25.5
27.1
24.7
22.6
20.0
25.5
13.9
40.5
32.9
42.6
4.7
19.7
Lithogna mor
23.4
30.5
16.5
29.6
23.4
27.1
27.4
22.1
20.0
25.0
14.2
40.8
26.8
39.5
7.9
25.8
Oblada melan
24.7
30.8
15.7
28.8
23.7
26.3
27.1
22.9
20.0
25.3
14.2
40.5
30.5
40.8
5.8
22.9
Pachy aenum
24.9
29.0
15.6
30.4
25.0
25.5
26.1
23.4
20.0
25.8
14.2
40.0
29.7
35.8
6.6
27.9
Pagellus acar
24.6
29.7
15.5
30.1
23.4
25.8
27.4
23.4
20.0
25.5
14.2
40.3
30.5
37.9
5.0
26.6
Pagellus bell
25.1
30.6
15.3
29.0
23.7
26.8
26.8
22.6
20.0
25.8
13.9
40.3
31.6
39.2
5.0
24.2
Pagrus aurat
24.4
30.3
15.6
29.7
24.2
26.8
26.3
22.6
20.0
25.8
13.7
40.5
28.9
38.2
6.8
26.1
Pagrus auriga
24.9
28.9
15.5
30.7
24.2
25.3
26.8
23.7
19.7
25.5
14.2
40.5
30.8
35.8
5.5
27.9
Pagrus pagru
24.7
29.7
15.5
30.0
23.9
27.4
26.6
22.1
20.0
25.5
13.9
40.5
30.3
36.3
6.1
27.4
Petrus rupest
23.9
31.0
16.2
28.9
24.5
26.3
26.6
22.6
20.0
25.8
13.7
40.5
27.4
40.8
8.4
23.4
Polyam germ
24.5
30.0
15.4
30.1
24.7
26.1
26.1
23.2
19.5
26.1
14.5
40.0
29.2
37.9
5.8
27.1
Polysteg pra
25.3
31.2
15.6
27.9
23.7
27.9
26.6
21.8
20.0
25.5
13.9
40.5
32.1
40.3
6.3
21.3
Porcost denta
23.7
30.6
16.1
29.6
23.4
27.1
27.1
22.4
20.0
25.8
13.9
40.3
27.6
38.9
7.4
26.1
Pterogy laniri
24.1
31.9
15.7
28.2
23.4
26.6
27.1
22.9
20.0
25.8
13.9
40.3
28.9
43.4
6.1
21.6
Rhabdo thorp
23.9
30.4
15.7
30.0
23.7
25.5
26.1
24.7
19.7
25.8
14.5
40.0
28.2
40.0
6.6
25.3
Sarpa salpa
25.0
30.4
15.7
28.9
23.4
26.8
26.8
22.9
20.0
25.5
14.2
40.3
31.6
38.9
6.1
23.4
Spariden hast
24.2
30.5
15.4
29.8
24.2
25.5
26.6
23.7
20.0
25.8
13.9
40.3
28.4
40.3
5.8
25.5
Sparod durb
22.5
31.1
17.8
28.6
23.4
26.8
27.1
22.6
19.7
26.1
14.7
39.5
24.2
40.5
11.6
23.7
Sparus auratus
24.0
30.0
15.8
30.2
23.7
26.3
27.1
22.9
20.3
25.8
13.9
40.0
28.2
37.9
6.3
27.6
76
Spondyl can
24.4
29.4
15.8
30.4
24.5
25.8
25.3
24.5
20.0
25.5
14.2
40.3
28.7
36.8
7.9
26.6
Stenotom chr
25.4
30.3
15.4
28.9
24.5
26.1
26.3
23.2
20.3
25.8
13.9
40.0
31.6
38.9
6.1
23.4
Spicara alta
24.2
32.0
16.0
27.8
23.4
26.6
27.1
22.9
20.0
25.8
13.9
40.3
29.2
43.7
6.8
20.3
Spicara smar
24.5
29.7
15.4
30.4
24.2
25.8
26.1
23.9
19.7
26.3
13.7
40.3
29.5
37.1
6.3
27.1
Cyprinus carp
29.7
30.0
14.1
26.1
25.3
26.6
25.3
22.9
20.0
25.0
13.2
41.8
43.9
38.4
3.9
13.7
Luxilus zonat
24.1
29.0
18.0
28.9
23.7
24.2
26.3
25.8
19.7
26.1
13.7
40.5
28.9
36.8
13.9
20.3
Centropo und
25.8
34.9
14.2
24.7
22.6
29.7
25.3
22.1
21.1
22.9
13.4
42.4
33.7
52.1
3.9
9.7
Dicentra labr
24.8
26.0
16.4
32.8
23.2
24.5
26.3
26.1
20.5
23.9
13.7
41.8
30.8
29.5
9.2
30.5
Dicentra pun
24.3
27.8
16.6
31.3
22.6
25.5
26.3
25.5
20.3
23.7
13.9
42.1
30.0
34.2
9.5
26.3
Lateolab jap1
23.5
32.3
16.1
28.1
23.7
27.6
26.1
22.6
19.7
25.3
13.9
41.1
27.1
43.9
8.4
20.5
Lateolab jap2
23.0
31.7
16.7
28.7
24.2
26.8
25.8
23.2
19.7
25.3
13.9
41.1
25.0
42.9
10.3
21.8
Lateolab latus
22.3
31.0
17.7
29.0
22.9
25.0
26.6
25.5
19.7
25.3
13.7
41.3
24.2
42.6
12.9
20.3
Morone ameri
24.0
30.2
15.6
30.2
23.2
27.1
25.3
24.5
20.3
25.5
13.4
40.8
28.7
37.9
8.2
25.3
Morone chrys
24.1
29.6
16.1
30.2
22.4
25.3
27.1
25.3
20.8
25.3
12.9
41.1
29.2
38.2
8.4
24.2
Morone missi
24.2
29.6
14.9
31.3
24.2
25.8
23.9
26.1
20.3
25.3
13.4
41.1
28.2
37.6
7.4
26.8
Morone saxa
24.9
28.3
15.4
31.4
22.4
25.3
26.6
25.8
20.3
25.0
13.2
41.6
32.1
34.7
6.3
26.8
Haemul sciur
23.3
34.5
16.0
26.2
23.2
28.2
26.3
22.4
20.0
25.5
13.9
40.5
26.8
49.7
7.6
15.8
Pomad macu
24.5
31.3
15.4
28.9
22.6
27.6
26.3
23.4
20.0
24.7
14.5
40.8
30.8
41.6
5.3
22.4
Caesio cunin
23.8
34.4
16.5
25.4
22.4
28.2
27.6
21.8
19.7
24.2
14.5
41.6
29.2
50.8
7.4
12.6
Lutjanus decu
25.1
34.2
14.9
25.8
23.2
28.4
26.6
21.8
19.7
24.7
13.7
41.8
32.4
49.5
4.5
13.7
Lethrinus orna
23.9
33.6
16.1
26.4
23.4
27.4
25.8
23.4
20.0
27.1
14.2
38.7
28.4
46.3
8.2
17.1
Lethrinus rubr
23.2
31.1
16.0
29.8
23.4
26.1
25.8
24.7
19.7
26.3
13.9
40.0
26.3
40.8
8.2
24.7
Scolops ciliat
24.0
30.6
15.1
30.3
24.2
29.2
24.7
21.8
21.1
25.5
13.2
40.3
26.8
37.1
7.4
28.7
Nemipt margi
24.1
30.2
16.6
29.1
23.7
25.0
26.3
25.0
18.9
25.5
13.9
41.6
29.7
40.0
9.5
20.8
MEAN
24.5
30.6
15.7
29.2
23.8
26.6
26.4
23.3
20.0
25.4
14.0
40.6
29.7
39.7
6.7
23.8
SD
1.02
1.56
0.74
1.56
0.75
1.05
0.80
1.17
0.32
0.62
0.39
0.69
2.75
4.07
2.03
4.32
ALL BIAS
0.133
0.042
0.215
0.274
IN BIAS
0.132
0.042
0.212
0.267
OUT BIAS
0.134
0.041
0.220
0.288
All Codons
First Codon
Second Codon
Third Codon
77
TABLE 9. MULTIPLE ALIGNMENT OF AMINO ACID RESIDUES TRANSLATED
FROM THE NUCLEOTIDE SEQUENCES OF THE CYTOCHROME B GENE FROM
THE SPARIDAE AND OUTGROUP SPECIES. TAXONOMIC NAMES WITH A
SUFFIX OF “GB” ARE FROM GENBANK AND ARE INCLUDED WITH ORIGINAL
SEQUENCES FOR EASY COMPARISON.
78
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIVNHAVVDLPAP1NI1VWWNFG1LL2LCLI1QLLTGLF
MTNLRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLV1QLLTGLF
MT2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MA2LRKTQPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLRLI1QLLTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCWI1QLLTGLF
MA2LRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWDFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHAVVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGRF
MA2LRKTHPLLKIANHAVIDLHAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLLKIANHALVDLPAPANI1VWWNFG1LLGLCLI1QLLTGLF
MT2LRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QLLTGLF
MA2LRKTHPLIKIANDALVDLPTP1NI1AWWNFG1LLGLCLITQILTGLF
MA2LRKTHPLMKIANGALVDLPTP1NI1ALWNFG1LLGLCLITQILTGLF
MA2LRKTHPLLKIANDALIDLPAP1NI1AWWNFG1LLGLCLIAQILTGLF
MAALRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
M2ALRKTHPLLKIANHALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MA2LRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLITQILTGLF
MA2LRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLITQILTGLF
MA2LRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLFTQIITGLF
MA1LRK1HPLLKIANNALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MAALRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MA1LRK1HPLLKIANNALIDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MAALRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLI1QILTGLF
MANPRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLI1QIVTGLF
MANLRKTHPLLKIANDALIDLPAP1NI1VWWNFG1LLGLCLI1QIVTGLF
MA2LRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLIAQLLTGLF
MA2LRKTHPLLKIANDALVDLPAP1NI1VWWNFG1LLGLCLIAQILTGLF
MACLRKTHPLLKIANDAVLDLPAP1NI1VWWNFG1LLGLCLIAQILTGLF
MA2LRKTHPLLKIANDAVVDLPAP1NI1VWWNFG1LLGLCLIAQILTGLF
MA2LRKTPPLLMIANNALIDLRAP1NI1AWWNFG1LLGLCLAAQILTGLF
MA2LRKTHPLLKIANDALVDLPAPANI1AWWNFG1LLGLCLIAQLLTGLF
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
[50]
79
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNYEWLIRNLHANGA1FFFICIYFHIGR
LAMHYTPDIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNDGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DINTAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIATAF1PVAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1GIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIAMAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYVHIGR
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGG
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLHANGA1FFFICIFLHIGR
LAMHYT1DI1TAF11VTHICRDVNYGWLIRNVHANGA1FFFICIYMHIAR
LAMHYT1DI1TAF11VTHICRDVNYGWLIRNMHANGA1FFFICIYMHIAR
LAMHYT1DINMAFT1VAHICRDVNYGWLIRNLHANGA1FFFICMYLHIGR
LAMHYT1DIATAF11IAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DIATAF11IAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DVATAF11VAHICRDVNYGWLIRNVHANGT1FFFICIYMHIGR
LAMHYT1DVATAF11VAHICRDVNYGWLIRNIHANGT1FFFICIYMHIGR
LAMHYT1DVATAF11VAHICRDVNYGWLIRNVHANGA1FFFICIYMHIGR
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLH1NGA1LFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNYGWLICNLHANGA1LFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNYGWLIRNLH1NGA1LFFICIYLHIGR
LAMHYT1DIATAF11VAHICRDVNFGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DI2MAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
LAMHYT1DITMAF11VAHICRDVNYGWLIRNLHANGA1FFFICIYLHIGR
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
80
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GLYYG1YLYKETWNIGVVLLLLVMATAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMATAFVGYVLPWGQM1FWGGTVITNLL
GLYYG1YLYKETWNIGVILLLLVMATAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMGTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVILLLLVMGTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVILLLLVMMTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLRLVMGTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKWTWNIGVILLLLVMATAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMGTAFVGYVLPWGQMFFWGATVITNLL
GLYYG1YLYKETWNIGVILLLLVMGTAFVGYVLPWGQM1FWGATVITKLL
GLYYG1YLYKDTWNIGVVLLLLVMATAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVILLLLVMATAFVGYVLPWGQM1LWGATVITNLV
GLYYG1YLYKETWDIGVILLLLVMGTAFVGYVLPWGQM1FWGATVIT2LL
GLYYG1YLYKETWNIGVVLLLLVMG1AFVGYVLPWGQM1FWGATVITNLL
GLYYG1YPYKVTWNIGVVLLLLVMGTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKDTWNIGVVLLLLVMGTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMMTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNVGVVLLLLVMMTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVILLLLVMMTA1VGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMMTAFVGYVLPWGQM1FWGGTVITNLL
GLYYG1YLYKETWNIGVILLLLVMMTAFVGYVLPWGQM1FWGGTVITNLL
GLYYG1YLYKETWNVGVVLLLLVMMTAFVGYVLPWGQM1FWGATVITNLL
GLYYG1YLYKETWNIGVVLLLLVMMTAFVGDVLPWGQM1FWGATVITNLL
GLYYG1YLYMETWNIGVILLLLVMMTAFVGYVLPWGQM1FWGATVITNLL
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
[150]
81
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
1AVPYIRGTLVQWIWGGF1VDNATLTRFFAFHFLLPFIVAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFIVAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFIVAAVTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFVVAAMTMLHLLFL
1AVPYIGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFVVAAMTMLHLLFL
1AVPYVG2TLVQWIWGGF1VDNATLTRFFAFHFLFPFVVAAMTMLHLLFL
1AVPYVGGTFVQWIWGGF1VDNATLTRFFAFHFLLPFIVAAMTMLHLLCL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFTFHFLLPFIVAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPLVVAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFIIAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLFLFIVAAMIMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFVVAAMTLLHLLFL
1AVPYIGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFIVAAMTMLHLLFL
1AVPYVG2TLVQWIWGGF1VDNATLTRFFAFHFLLPFVVAAMTMLHLLFP
1AVPYIGGTLVQWFWGGF1VDNATLTRFFAFHFLLPFIVAAMTMLHLLFL
TAVPYVGGTLVQWIWGGL1VDNATLTRFFAFHFLLPFVVAAMTMLHLLFL
1AVPYVGGTLVQWIWGGF1VDNATLTRFFAFHFLLPFVIAAMTMLHLLFL
1AVPYMGDMLVQWIWGGF1VDNATLTRFFAFHFLLPFVIAAATIIHLLFL
1AVPYMGDTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAGATVLHLLFL
1AVPYVGDILVQWIWGGF1VDNATLTRFFAFHFLLPFVVAAMMILHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAGATMLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAGATLLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFIIAGATVIHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAGATLIHLIFL
1AVPYIGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAGATFIHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFIIAAATLLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAAATVLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFIIAAAAILHLLFL
1ALPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAAATILHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLLPFIIAAATVIHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLLPLIVTAATLIHLLFL
CAIPYVGNTLVQWVWGGF1VDNATLTRFFAFHFLLPFIIAAVTMLHLLFL
1AIPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLLPFIIAAVTMLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFYALHFL1PFVIAAATTPHLRFL
1AVPYVGNTLVQWIWGGF1VDHATLTRFFAFHFLFPFVIAAATMLHLLFL
1AVPYVGNTLVQWIWGGF1VDNATLTRFFAFHFLFPFVIAAMTLLHLLFL
1AVPYVGNMLVQWIWGGF1VDHATLTRFLTFHFLFPFVIAAATLLHLLFL
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[200]
[200]
[200]
82
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLALFAP2LL
HETG1NNPLGLN1DADKI1FHPYF1YKDLLGFAGVLILLTCLALF1PNLL
HETG1NNPIGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAAVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLALF1PNLL
HETG1NNPLGLN1DTDKIAFHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLGLFAPNLL
HETG1YNPLGVN1DTDKI1FHPYL1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDVLGFAGVIILLTCLAL1APNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAAVIILLTCLALFTPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVVILLTCLALFAPNIL
HETG1NNPLGLN1DADKI1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HDTG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DADKI1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIMLLTCLALFAPNLL
HETG1NNPLGLN1DTDKM1FHPYF1YKDLLGFAGVIILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIILLTCLAWFAPNLL
HETG1NNPLGLN1DTDKI1FHPY11YKDLLGFAAVIILLTCLALFAPNLL
HETG1NNPLGLN1NTDKI1FHPYF1YKDLLGFAGLLILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVNILLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAGVIMLLTCLALFAPNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLLGFAAVIILLT1LALFAPNLL
HETG1NNPIGLN1DADKV1FHPYF1YKDLLGFVIMLLALTLLALF1PNLL
HETG1NNPAGLN1DADKI1FHPYF1YKDLLGFVLLLLALT1LTFF1PTLL
HETG1NNPMGLN1NVDKIPFHPYF1YKDLLGFVVLLFTLT1LALFLPNLL
HQTG1NNPLGLN1DVDKI1FHPYF1YKDLLGFAIVLIGLT2LALF1PNLL
HQTG1NNPLGLN1DVDKI1FHPYF1YKDLLGFAIVLIGLA2LALF1PNLL
HETG1NNPLGLN1DADKIPFHPYF1YKDLLGFAVLLTALA1LALF1PNLL
HETG1NNPLGLN1EADKIPFHPYF1YKDLLGFAVLLTALA1LALF1PNLL
HETG1NNPLGLN1DADKIPFHPYF1YKDLLGFAVLLTALAALALF1PNLL
HETG1NNPLGLN1DVDKIPFHPYF1YKDLLGATAVLIGLT1LALF1PNLL
HETG1NNPLGLN1DVDKIPFHPYF1YKDILGFAAVLVGLT1LALF1PNIL
HETG1NNPLGLN1DMDKIPFHPYF1YKDLLGFTAVLI2LT1LALF1PNLL
HETG1NNPLGLN1DVDKIPFHPYF1YKDLLGFAAVLIGLT1LALF1PNLL
HQTG1NNPLGLN1DADKIWFHPYF1YKDLLGFAVLLIALTCLALF1PNLL
HETG1NNPLGLN1DADKI1FHPYL1YKEVLGFVVLLIALACLALL1LNLL
HETG1NNPLGLN1DADKM1FHPYF1YKDLLGFVVVLIALVCLALFAPNLL
HETG1NNPLGLN1DVDKI1FHPYF1YKDLLGFVVVLIALT1LALF1PNLL
HETG1NNPLGLN1D1DKI1FHPYF1YKDLLGFAAVLIALT1LALF1PNLL
HETG1NNPLGLN1D1DKI1FHPYF1YKDLLGFAAVLIALT1LALF1PNLL
HETG1NNPLGL11DTDKI1FHPYF1YKDLLGFAAVIIFLTCLALF1PNLL
HETG1NNPLGLN1DTDKI1FHPYF1YKDLIGFAAILITLTCLALF1PNLL
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[250]
[250]
[250]
[250]
[250]
[250]
83
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLA1ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLAVLA1ILV
GDPDNFTPANPLVTPPHIKPEWYFLFGYAILR1IPNKLGGVLALLA1ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLA1ILI
GDPDNFTPANPLVTPPHIKPEWN1LFAYAILR1IPNKLGGVLALLA1MLV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPDKLGGVLALLA1ILV
GDPENFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLA1ILV
GDPENFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLF1ILV
GDPENFTPANPLVTPPHIQPEWYFLFAYAIIR1IPNKLGGVLALLF2ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALL11ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLF1ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYANLR1IPNKLGGVLALLA1ILV
EHQDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLALLA1ILV
GDPDNFTPANPLVTPPHIKPEWYFLFAYAILR1IPNKLGGVLDLLA1ILV
GDPDNFTPANPLVTPPHIKPEWYVLFAYAILR1MPNKLGGVLALLA12LV
GDPDNFTPANPLVTPPHVKPEWYFLFAYATLR1IPNKLGGVLALLA1ILV
[300]
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[300]
[300]
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[300]
[300]
[300]
[300]
84
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
LMVVPMLHT1KQR2LTFRPFTQFLFWALIANVVILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPLTQFLFWALIANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPITQFLFWALIANVAILTWIGFLLVEDPYIII
LMLVPFLHT1KQR2LTFRPI1QFLFWMLIANVAILTWIGGMPVEDPYIII
LMVVPLLHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPATQFLFWTLIANVAILTWIGGMPVEEPYIII
LMIVPILHT1KQR2LTFRPLTQFLFWALIANVVVLTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEEPYIII
LMLVPLLHT1KQR2LTFRPVTQFLFWTLTANVAVLTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPMTQFLFWALIANVAILTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPLTQFLFWLLIANVAILTWIGGMPVEDPYIII
LMVVP2PHTPKQR2LT2RPVTQILFWALIANVAILTWIGGMPAEEPYKII
LMVVPILHT1KQR2LTFRPVTQFLFWTLIANVAILTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPMTQFLFWALIANVAILTWIGGMPVEDPYIVI
LMVVPILHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEGPYIII
LMIVPILHT1KQR2LTFRPLTQFLFWTLIANVAILTWIGGMPVEDPYIII
LMVVPILHT1KQR2LTFRPITQFLFWALTANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPMTQFLFWALIANVAILTWIGGMPVEDPYIVI
LMAVPILHM1KQR2LTFRPITQFLFWTLIANVAILTWIGGMPVEDPYIVI
LMVVPTLHT1KQR2LTFRPITQFLFWALIANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPMTQFLFWALMANVAILTWIGGMPVEDPYIVI
LMVVPILHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEDPYIVI
LMIVPILHT1KQR2LTFRPATQFLFWTLIANVAILTWIGGMPVEEPYIII
LMVVPLLHT1KQR2LTFRPMTQFLFWALIANVAILTWIGGMPVEEPYIII
LMVVPMLHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEEPYIII
LVVVPVLHT1KQR2LTFRPVTQFLFWTLIANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPVTQFLFWALVANVAILTWIGGMPVEEPYIII
FLVVPIFHT1KQR2LTFRPLTQFLFWTLIANVAILTWIGGMPVEDPYIMI
LMVVPILHT1KQR2LTFRPMTQFLFWALIANVAILTWIGGMPVEEPYIII
LMVVPILHT1KQR2LTFRPVTQFLFWALIANVAILTWIGGMPVEDPYIVI
LMVVPILHT1KQR2LTFRPVTQFLFWTLVANVAILTWIGGMPVEDPYIMI
LMVVPLLHT1KQRGLTFRPITQFLFWTLVADMIILTWIGGMPVEHPFIII
LLVVPILHT1KQRGLTFRPITQFLFWTLVADMVILTWIGGMPVEHPYIII
LMLVPLLHT1KQRGLMFRPA1QLLFWVLVADVAILTWIGGMPVEHPYIIV
LMVVPYLHT1KQR2MTFRPVTQFLFWALVADVMILTWIGGMPVEHPFIII
LMVVPFLHT1KQR2MTFRPLTQFLFWTLVADVMVLTWIGGMPVEHPFIII
LMLVPILHT1KQRALTFRPITQFLFWTLIADVAILTWIGGMPVEHPFIII
LMTVPFLHT1KQR2LTFRPLTQLLFWTLIADVVILTWIGGMPVEHPFIII
LMAVPFLHT1KQR2LTFRPLTQLLFWALIADVAILTWIGGMPVEHPFIII
LLTVPFLHT1KQR2LTFRPLTQFLFWTLIADVMILTWIGGMPVEHPFIII
LMVVPFLHT1KQR2LTFRPLTQLLFWTLIADVAILTWIGGMPVEHPFIII
LMVVPILHT1KQR2LTFRPVTQFLFWTLIADVAILTWIGGMPVEHPFIII
LMVVPFRHT1KQR2LTFRPL1QLLFWTLVADVA2LTWIGGMPVEHPYIII
LMVVPILHT1KQRGLTFRPVTQFLFWTLIANVAILTWIGGMPVEHPFIII
LMVVPILHT1KQRGLTFRPVTQFLFWTLIANVAILTWIGGMPVEHPFIII
LMVVPILHT1KQR2LTFRPLTQFLFWTLIANVAILTWIGGMPVEHPFIII
LMVVPILHT1KQR2LTFRPLTQFLFWTLIANVAILTWIGGMPVEHPFIII
LMLVPLLHT1KQR2LTFRPI1QFLFWVLIADVAILTWIGGMPVEDPYIII
LMLVPILHT1KQR2LTFRPI1QFLFWTLIADVAILTWIGGMPVEHPYIII
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85
Acanthopagrus berda
Argyrops spinifer
Boops boopsGB
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentexGB
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
GQIA1LTYF1LFLVIIPAAATVENKVLGWQ
GQVA1LTYF1LFLIIIPTVAMVENKVLGW3
GQIA1LTYFALFLLIMPATALVENKVLGWQ
GQIA1LTYFALFLFIIPVAALVENKVLGCN
GQIA1LTYF1LFLLIIPMAATLENKVLGWQ
GQIA1LTYF1IFLIIIPATAILENKVLNWQ
GQIA1LTYF1LFLIIIPVTATVENKVLGWQ
GQIA1LTYFALFLLIMPVAALAENKVLGWQ
GQIA1LTYFALFLFIIPAAALAENKVLGWQ
GQVA1LTYF1LFLIIIPTAAAVENKVLGWQ
GQIA1LTYFALFLFINTAAALVENKMLGWQ
GQIA1LTYFALFLFIFPVTALVENKVLGWQ
GQIA1LTYFALFLIIIPTTALAENKML2LQ
GQIA1LTNI1LFLVVNPAAAVLENKVLGWQ
GQIA1LTYF1LFLVVIPAAAVLENKVLGWQ
GQIA1LTYF1LFLVIIPAAATMENKVLGWQ
GQIA1LTYF1LFLVVIPAAAVLENKVLGWQ
GQIA1LTYFALFLLIIPATALVENKVLGWQ
GQIA1LTYF1LFLVIIPAAATMENKVLGWQ
GQIA1LTYF1LFLIIIPMAATVENKML2WQ
GQIA1LTYF1LFLVVMPAAAAIENKVLGWQ
GQIA1LTYF1LFLVIIPAAAVMENKVLGWQ
GQIA1LTYF1LFLIIIPATATIENKMLGWQ
GQVA1LTYF1LFLVIIPAAATMENKVLGWQ
GQIA1LTYFALFLLIIPATALAENKVLGWQ
GQIA1LTYFALFLLIMPTAALVENKVLGWQ
GQIA1LTYFALFLFIIPVTAMAENKVLGWR
GQIA1LTYFALFLLIIPATALVENKVLGWQ
GQIA1LTYFALFLFIIPTAALAENKMLGLQ
GQIA1LTYF1LFLIIIPATATIENKMLGWQ
GQIA1LTYFALFLFIMPAAALVENKVLGWQ
GQVA1LTYFALFLFIIPAAALAENKVLGWQ
GQIA1LTYFALFLFIIPAAALAENKVLGWQ
GQVA1L1YF1LFLIIMPAAATLENKVLGWR
GQVA1LTYF1LFLVIIPAAATLENKVLGWQ
GQIA1LTYF1LFLVIIPTAATMENKVLGWQ
GQVA1LTYF1LFLIIMPAAATLENKALGWQ
GQVA1LTYF1LFLVIIPAAATMENKVLGWQ
GQIA1VTYL1LFLIIIPTAATLENKVLGWQ
GQIA1LTYFALFLIIIPATATVENKVLGWQ
GQVA1LTYFALFLLIIPTAALAENKVLGWK
GQVA1LTYF1LFLIIIPTAATLENKVLGWR
GQIA1VLYFALFLIFMPLAGWLENKALKWA
GQVA1VLYFALFLLLAPLAGWAENKALKWA
GQIA1LLYFLLFLVLMPLAGWWENKLLNWQ
GQVA1LLYFLLFLVFIPVVGELENKALEWL
GQVA1LLYFLLFLVLIPVVGELENKALEWL
GQIA1LLYFLIFLVLFPLAGWLENKALGWT
GQIA1LLYFLIFLVLFPVAGWLENKALGWT
GQLAFLFYFFIFLVLFPLAGWLENKALGWT
GQIA1LLYFLLFLVLMPMAGELENKALGWL
GQIA1LLYFLLFLVFMPIAGELENKALEWL
GQIA1LLYFLLFLVFMPIAGELENKALGW1
GQVA1LLYFLLFLVFMPIVGELENKALEWL
GQVA1FLYFFLFLVFTPLA2WLENKALGWA
GQIA1FLYF1LFLVLFPLAGWLENKVMGW1
GQIA1VLYFLLFLVLTPLAGWLENKALGWV
GQIA1VLYFLLFLVFAPLAGWLENKALGWL
GQIA1LLYF1LFLIITPAAGWFENK1LGWR
GQIA1LLYF1LFLIITPAAGWFENK1LGWR
GQVA1VLYF1IFLVFMPLVGAVENKVMGWT
GQIA1VLYF1IFLLLMPLQDEQGMK1FAEH
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
[380]
86
TABLE 10. FAO AREA ASSIGNMENTS FOR THE SPARIDAE USED IN THIS STUDY.
FAO AREAS WERE ESTABLISHED BY FAO (1995)
TAXON
FAO Area
Acanthopagrus berda
Argyrops spinifer
Boops boops
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentex
Dentex tumifrons
Diplodus argenteum
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellottii
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Petrus rupestris
Porcostoma dentata
Sarpa salpa
Sparidentex hasta
Sparus auratus
Spicara maena
Spicara alta
Stenotomus chrysops
8, 7, 10, 12
3, 6, 5
4, 8, 7, 10, 12
4, 8
2, 1, 9, 4
4, 8
3, 6
4, 8
4, 8
4, 8
4, 8
2, 1, 9
7, 10, 12
6, 5
6
2, 1, 9, 4
3, 6
10
4, 8
3, 6
2, 1, 9, 4, 8
2, 1, 9, 4
4, 8
2, 1, 9
1, 9, 4
7, 10, 12, 11
2, 1, 9, 4
3, 2, 6, 1, 9, 5
4,8
4,8
4, 8
4, 8
4, 8
4, 8
2, 1, 9, 4, 8
4, 8
8, 7
2, 1, 9
2, 1, 9
1, 4
2, 1, 9, 4
3, 6
1-Atlantic, Eastern Central; 2-Atlantic, Northeast; 3-Atlantic, Northwest; 4-Atlantic, Southeast;
5-Atlantic, Southwest; 6-Atlantic, Western Central; 7-Indian Ocean, Eastern; 8-Indian Ocean,
Western; 9-Mediterranean and Black Sea; 10-Pacific, Northwest; 11-Pacific, Southwest; 12-Pacific,
Western Central
87
TABLE 11. MATRIX OF FAO AREAS AND CHARACTERS. FAO AREAS WERE TREATED AS INDEPENDENT DATA
AND TAXA PLUS ANCESTRAL NODES WERE TREATED AS DEPENDENT DATA DURING PARSIMONY ANALYSIS.
CHARACTERS ARE DEFINED BELOW (AN=ANCESTRAL NODE).
AREAS
.........1.........2.........3.........4.........5.........6.........7.........8..
1234567890123456789012345678901234567890123456789012345678901234567890123456789012
Atlantic, Eastern Central
0000100000010001000011011011000000100111101011100110110001000000111111101101111111
Atlantic, Northeast
0000100000010001000011010011000000100110101011100110110001000000001111101101111111
Atlantic, Northwest
0100001000000000100100000001000000000000010000011110110001000000000011101111111000
Atlantic, Southeast
0011110111100001001011101010111111110001101111100110111111111111111111101101111111
Atlantic, Southwest
0100000000000100000000000001000000000000000000001110110001000000000011101111111000
Atlantic, Western Central
0100001000000110100100000001000000000000010000011110110001000000000011101111111000
Indian, Ocean Eastern
1010000000001000000000000100000000001000000000000001110001000000110000111101111011
Indian, Ocean Western
1011010111100000001010100000111111111000001111100001111111111111011101101101111011
Mediterranean/Black Sea
0000100000010001000011011011000000100110101011100110110001000000001111101101111100
Pacific, Northwest
1010000000001000010000000100000000000000000000000001110001000000110000111101111000
Pacific, Southwest
0000000000000000000000000100000000000000000000000000000000000000000000011101110000
Pacific, Western Central
1010000000001000000000000100000000000000000000000001110001000000110000111101111000
CHARACTERS
1 Acanthopagrus berda; 2 Archosargus probatocephalus; 3 Argyrops spinifer; 4 Argyrozona argyrozona; 5 Boops boops; 6 Boopsoidea inornata; 7 Calamus
nodosus; 8 Cheimerius nufar; 9 Chrysoblephus cristiceps; 10 Crenidens crenidens; 11 Cymatoceps nasutus; 12 Dentex dentex; 13 Dentex tumifrons; 14 Diplodus
argenteus; 15 Diplodus bermudensis; 16 Diplodus cervinus; 17 Diplodus holbrooki; 18 Evynnis japonica; 19 Gymnocrotaphus curvidens; 20 Lagodon rhomboides;
21 Lithognathus mormyrus; 22 Oblada melanura; 23 Pachymetopon aeneum; 24 Pagellus bogaraveo; 25 Pagellus bellottii; 26 Pagrus auratus; 27 Pagrus auriga;
28 Pagrus pagrus; 29 Petrus rupestris; 30 Polyamblyodon germanum; 31 Polysteganus praeorbitalis; 32 Porcostoma dentata; 33 Pterogymnus laniarius; 34
Rhabdosargus thorpei; 35 Sarpa salpa; 36 Sparodon durbanensis; 37 Sparidentex hasta; 38 Sparus auratus; 39 Spicara maena; 40 Spicara alta; 41 Spondyliosoma
cantharus; 42 Stenotomus chrysops; 43 AN1; 44 AN2; 45 AN3; 46 AN4; 47 AN5; 48 AN6; 49 AN7; 50 AN8; 51 AN9; 52 AN10; 53 AN11; 54 AN12; 55 AN13;
56 AN14; 57 AN15; 58 AN16; 59 AN17; 60 AN18; 61 AN19; 62 AN20; 63 AN21; 64 AN22; 65 AN23; 66 AN24; 67 AN25; 68 AN26; 69 AN27; 70 AN28; 71
AN29; 72 AN30; 73 AN31; 74 AN32; 75 AN33; 76 AN34; 77 AN35; 78 AN36; 79 AN37; 80 AN38; 81 AN39; 82 AN40
88
FIGURE 3. PCR primers and their relative sequence map alignment relative to the
mitochondrial genome of Cyprinus carpio (GenBank Accession X61010) are given. The
graphic represents the location of these primers on the heavy and light strand of tRNA Glu
and tRNA Thr that flank Cytochrome b gene in perciform fishes. The following primer
pairs were used for PCR: CYTbUnvL (L15242)/CYTbUnvH (H16458); CYTbGludgL
(L15249)/CYTBThrdgH (H16465); and CYTB4xdgL (L14249)/CYTb4xdgH (H16435).
CYTbGludgL-TGACTTGAARAACCAYCGTTG-L15249
CYTb4XdgL-TGAYWTGAARAACCAYCGTTG-L15249
CYTbUnvL-CGAACGTTGATATGAAAAACCATCGTTG-L15242
5'AATTCTTGCTCAGACTTTAACCGAGACCAATGACTTGAAGAACCACCGTTGTTATTCAACTACAAGAACCAC3'
|
L15219 (TRNA -GLU Cyprinuis carpio 15219-15287)
CYTbGludgL
CYTb4xdgL
CYTbUnvL
tRNA-Glu
Cytochrome b
tRNA-Thr
CYTB4XdgR
CYTbUnvH
CYTbThrdg-H
H16434(TRNA-THR Cyprinus carpio 16434-16505)
|
3'GCGCTAGGGAGGAATTTAACCTCCGATCTTCGGATTACAAGACCGATGCTTTTAGGCTAAGCTACTAGGGCA5'
CYTB4xdgH-TGRVNCTGAGCTACTASTGC-H1643 5
CYTBUnvH-ATCTTCGGTTTACAAGACCGGTG-H16458
CYTBThrdgH-CTCCAGTCTTCGRCTTACAAG-H16565
90
FIGURE 4. Total substitutions at all codon positions plotted as a function of pairwise
percent sequence divergence for ingroup taxa only. All data derived from cytochrome b
nucleotide sequences. Second order polynomials were fitted to the data (Ts: y =
-0.3212x2 + 12.339x + 13.82, R2 = 0.2725 and Tv: y = -0.0176x2 + 4.0373x - 3.6503, R2
= 0.2038).
Key:
Ts-All =Transitions all codon positions
Tv-All =Transversions all codon positions
Poly. (Ts-All) = polynomial line fitted to transitions from all codon positions
Poly. (Tv-All) = polynomial line fitted to transversions from all codon positions
180
160
Total Substitutions
140
120
100
80
60
40
20
0
0
5
10
15
20
%Sequence Divergence (Uncorrected)
Ts-All
Tv-All
Poly. (Ts-All)
Poly. (Tv-All)
25
92
FIGURE 5. Total substitutions at the first codon position plotted as a function of pairwise
percent sequence divergence for ingroup taxa only. All data derived from cytochrome b
nucleotide sequences. Second order polynomials were fitted to the data (Ts: y =
-0.0082x2 + 1.0262x + 3.3566, R2 = 0.1291 and Tv: y = 0.0203x2 - 0.2574x + 3.6401, R2
= 0.0678).
Key:
Ts-1st = Transitions 1st codon position
Tv-1st = Transversions 1st codon position
Poly. (Ts-1st) = polynomial line fitted to transitions from 1st codon position
Poly. (Tv-1st) = polynomial line fitted to transversions from 1st codon position
35
30
Total Substitutions
25
20
15
10
5
0
0
5
10
15
20
% Sequence Divergence (Uncorrected)
Ts-1st
Tv-1st
Poly. (Ts-1st)
Poly. (Tv-1st)
25
94
FIGURE 6. Total substitutions at the second codon position plotted as a function of
pairwise percent sequence divergence for ingroup taxa only. All data derived from
cytochrome b nucleotide sequences. Second order polynomials were fitted to the data (Ts:
y = 0.0121x2 - 0.2146x + 3.1507, R2 = 0.0396 and Tv: y = 0.0764x + 0.478, R2 =
0.0127).
Key:
Ts-2nd = Transitions 2nd codon position
Tv-2nd = Transversions 2nd codon position
Poly. (Ts-2nd) = polynomial line fitted to transitions from 2nd codon position
Poly. (Tv-2nd) = polynomial line fitted to transversions from 2nd codon position
9
8
Total Susbtitutions
7
6
5
4
3
2
1
0
0
5
10
15
20
Ts-2nd
Tv-2nd
Poly. (Ts-2nd)
Linear (Tv-2nd)
25
96
FIGURE 7. Total substitutions at the third codon position plotted as a function of
pairwise percent sequence divergence for ingroup taxa only. All data derived from
cytochrome b nucleotide sequences. Second order polynomials were fitted to the data
(Ts: y = -0.2707x2 + 11.417x - 4.4675, R2 = 0.4766 and Tv: y = 0.1607x2 + 1.0435x 9.5465, R2 = 0.7008).
Key:
Ts-3rd = Transitions 3rd codon position
Tv-3rd = Transversions 3rd codon position
Poly. (Ts-3rd) = polynomial line fitted to transitions from 3rd codon position
Poly. (Tv-3rd) = polynomial line fitted to transversions from 3rd codon position
160
140
Total Substitutions
120
100
80
60
40
20
0
0
5
10
15
20
% Sequence Divegence (Uncorrected)
Ts-3rd
Tv-3rd
Poly. (Ts-3rd)
Poly. (Tv-3rd)
25
98
FIGURE 8. Total substitutions at the third codon position and pooled first and second
codon positions plotted as a function of pairwise percent sequence divergence for ingroup
taxa only. All data derived from cytochrome b nucleotide sequences. Second order
polynomials were fitted to the data (Ts-Tv3rd: y = -0.11x2 + 12.46x - 14.014, R2 =
0.9402 and Ts-Tv1st+2nd: y = 0.0261x2 + 0.5738x + 11.025, R2 = 0.1537).
Key:
Ts-Tv1st+2nd = All substitutions 1st and 2nd codon positions
Ts-Tv3rd = All substitutions 3rd codon position,
Poly. (Ts-Tv1st+2nd) = polynomial line fitted to all substitutions from the 1st and 2nd
codon positions
Poly. (Ts-Tv3rd) = polynomial line fitted to all substitutions from the 3rd codon position
250
Substitutions
200
150
100
50
0
0
5
10
15
20
Ts+Tv 1st+2nd
Ts+Tv3rd
Poly. (Ts+Tv3rd)
Poly. (Ts+Tv 1st+2nd)
25
100
FIGURE 9. A ratio of transitions/total substitutions and transversions/total substitutions
from third codon plotted as a function of pairwise percent sequence divergence for
ingroup taxa only. All data derived from cytochrome b nucleotide sequences.
Key:
Ts/Ts+Tv = Ratio of transitions to total substitutions from the 3rd codon position
Tv/Ts+Tv = Ratio of transversions to total substitutions from the 3rd codon position
Poly. (Ts/Ts+Tv) =Polynomial line fitted to the ratio of transitions to total substitutions
from the 3rd codon position
Poly. (Tv/Ts+Tv) =Polynomial line fitted to the ratio of transitions to total substitutions
from the 3rd codon position
1
0.9
% of Total Substitutions
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
Ts/Ts+Tv
Tv/Ts+Tv
Poly. (Ts/Ts+Tv)
Poly. (Tv/Ts+Tv)
25
102
FIGURE 10. Transitional substitutions, separated into nitrogenous base type, purines
(AøG) and pyrimidines (CøT) plotted as a functions of pairwise percent sequence
divergence of ingroup taxa only. All data derived from cytochrome b nucleotide
sequences.
120
100
Transitions
80
60
40
20
0
0
5
10
15
AG
CT
20
25
104
FIGURE 11. Frequencies of each of four bases of the cytochrome b gene for 62 taxa at
all codon positions. The relative frequency of each base was not equal and a chi-squared
test of heterogeneity among taxa from all codon positions was slightly significant (X2 =
166.9 df==183 P=0.798).
Acanthopagrus berda
Argyrops spinifer
Boops boops
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentex
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellotti
Pagrus auratus
Pagrus auriga
A
Pagrus pagrus
Taxon
Petrus rupestris
C
G
Porcostoma dentata
T
Sarpa salpa
Sparidentex hasta
Sparus auratus
Spicara alta
Spicara maena
Stenotomus chrysops
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
MEAN
0%
20%
40%
60%
Cumulative Frequency
80%
100%
106
the first codon position. The relative frequency of each base was not equal, but a chisquared test did not demonstrate heterogeneity among taxa in the first codon position base
frequencies X2 =34.22, df=183, P > 0.995).
Acanthopagrus berda
Argyrops spinifer
Boops boops
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentex
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellotti
Pagrus auratus
Pagrus auriga
Pagrus pagrus
A1
Taxon
Petrus rupestris
C1
G1
Porcostoma dentata
T1
Sarpa salpa
Sparidentex hasta
Sparus auratus
Spicara alta
Spicara maena
Stenotomus chrysops
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
MEAN
0%
20%
40%
60%
80%
100%
108
Figure 13. Frequencies of each of four bases of the cytochrome b gene for 62 taxa at the
second codon position. The relative frequency of each base was not equal (anti-guanine,
pro-thymine), but a chi-squared test did not demonstrate heterogeneity among taxa in the
second codon position base frequencies X2=9.68, df=183, P >0.995).
Acanthopagrus berda
Argyrops spinifer
Boops boops
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentex
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellotti
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Taxon
Petrus rupestris
A2
C2
Porcostoma dentata
G2
T2
Sarpa salpa
Sparidentex hasta
Sparus auratus
Spicara alta
Spicara maena
Stenotomus chrysops
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
MEAN
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110
the third codon position. The relative frequency of each base was not equal (anti-guanine,
pro-cytosine) and a chi-squared test demonstrated significant heterogeneity among taxa in
the third codon position base frequencies (X2=477.59, df=183, P< 0.001
Acanthopagrus berda
Argyrops spinifer
Boops boops
Boopsoidea inornata
Calamus nodosus
Cheimerius nufar
Crenidens crenidens
Cymatoceps nasutus
Dentex dentex
Dentex tumifrons
Diplodus argenteus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Oblada melanura
Pachymetopon aeneum
Pagellus bogaraveo
Pagellus bellotti
Pagrus auratus
Pagrus auriga
Pagrus pagrus
Taxon
Petrus rupestris
A3
C3
Porcostoma dentata
G3
T3
Sarpa salpa
Sparidentex hasta
Sparus auratus
Spicara alta
Spicara maena
Stenotomus chrysops
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
MEAN
0%
20%
40%
60%
80%
100%
112
FIGURE 15. Four equally parsimonious trees, unweighted data
Tree One
Tree Three
Cyprinus carpio
Luxilus zonatus
Caesio cuning
Lutjanus decussatus
Haemulon sciurus
Pomadasys maculatus
Scolopsis ciliatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Lethrinus ornatus
Petrus rupestris
Porcostoma dentata
Cymatoceps nasutus
Dentex tumifrons
Spicara alta
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Lagodon rhomboides
Calamus nodosus
Stenotomus chrysops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Boops boops
Crenidens crenidens
Acanthopagrus berda
Sparidentex hasta
Sparus auratus
Pagellus bogaraveo
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
Cyprinus carpio
Luxilus zonatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Lethrinus ornatus
Petrus rupestris
Porcostoma dentata
Cymatoceps nasutus
Dentex tumifrons
Spicara alta
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Lagodon rhomboides
Calamus nodosus
Stenotomus chrysops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Boops boops
Crenidens crenidens
Acanthopagrus berda
Sparidentex hasta
Sparus auratus
Pagellus bogaraveo
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
Tree Two
Tree Four
Cyprinus carpio
Luxilus zonatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Lethrinus ornatus
Petrus rupestris
Porcostoma dentata
Cymatoceps nasutus
Dentex tumifrons
Spicara alta
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Lagodon rhomboides
Calamus nodosus
Stenotomus chrysops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Boops boops
Crenidens crenidens
Acanthopagrus berda
Sparidentex hasta
Sparus auratus
Pagellus bogaraveo
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
Cyprinus carpio
Luxilus zonatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Lethrinus ornatus
Petrus rupestris
Porcostoma dentata
Cymatoceps nasutus
Dentex tumifrons
Spicara alta
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Lagodon rhomboides
Calamus nodosus
Stenotomus chrysops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Boops boops
Crenidens crenidens
Acanthopagrus berda
Sparidentex hasta
Sparus auratus
Pagellus bogaraveo
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
114
FIGURE 16. Strict consensus of four equally parsimonious trees from the unweighted
data. A heuristic search of 1000 random addition replicates on the cytochrome b
nucleotide data set resulted in four equally parsimonious trees. (tree length=6416,
CI=0.1900, RI=0.4258). Subfamilies are labeled as follows (BO=Boopsinae,
DE=Denticinae, DI=Diplodinae,PA=Pagrinae,PE=Pagellinae, and SP=Sparinae).
Figure key is as follows:
“N” = Node
D
= Total Decay Value at “N”
1st = Partitioned Decay at 1st Codon at “N”
2nd = Partitioned Decay at 2nd Codon at “N”
3rd = Partitioned Decay at 3rd Codon at “N”
B
= Bootstrap Support Value at “N”
17(
98
3.0
3.9
10.1
)
2.5
4.0 )
5( -1.5
100
8.2
9(
1.0
1.5
6.5
4.5 )
23(10.2
14(
74
)
23(
0.9
2.4
19.8
)
1.3
0.4
12.3
)
9.5
3.0
13.5
)
26(
100
98
100
68
1.5
3 (-2.0)
3.5
73
3.5
0.0 )
21(17.5
6.5
17( 4.0 )
100
95
6.5
0.0
0.0 )
5( 5.0
70
0.0
0.0 )
3( 3.0
88
-0.1
98
55
1.0
)
3(-1.0
3.0
8( 0.5
)
7.6
-0.5
2.0 )
< 12( 10.5
<
1.0
3(-1.0
)
3.0
54
-4.2
)
4(-1.0
9.2
9(
6(
6.0
22( -0.4 )
-1.0
1.0
7.0
99
)
-3.0
0.0
12.0
)
<
69
2.0
0.0 )
11( 9.0
-1.0
5(
-0.7
0.0
5.7
)
16.4
7( 0.0 )
<
85
1.0
7( 1.0
)
5.0
2(
-2.0
0.0
4.0
)
)
11.5
0.0 )
4( 2.2
65
69
<
1.8
)
<
53
-3.2
1.2
6.0
0.0 )
9( -2.5
-0.5
1.7
1.8
87
8.0
4(
3(
98
3.0
0.0 )
16( 13.0
99
81
-2.5
0.0 )
59
8( 10.5
-0.5
) 9.0 100
2( 2.0
0.5
0.0 )
28( 19.0
-0.4
2(
4(
-1.2
0.0
2.8
)
-1.2
0.0
3.2
<
9.4
< 10( 1.0
)
4.8
95
83
0.0 )
9( 4.2
12(
99
2.0
0.0
10.0
)
-1.5
)
3( 0.0
4.5
0.3
)
63
<
) 0.0 98
5(-0.8
5.4
100
10( -1.0 )
11.0
1.6
20( 0.0 ) 0.4 70
18.4 2 ( 0.0 )
1.6
Group II
8.5
3.7 )
17(4.8
100
Group I
88
4.5
9( 0.5
)
4.0
Cyprinus carpio
Luxilus zonatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Lethrinus ornatus
SP Pterogymnus laniarius
DE Argyrozona argyrozona
DE Petrus rupestris
DE Polysteganus praeorbitalis
SP Porcostoma dentata
SP Chrysoblephus cristiceps
SP Cymatoceps nasutus
DE Dentex tumifrons
Spicara alta
PA Argyrops spinifer
PA Evynnis japonica
PA Pagrus auratus
PE Pagellus bellottii
PA Pagrus pagrus
PA Pagrus auriga
DE Cheimerius nufar
DE Dentex dentex
DI Archosargus probatocephalus
DI Lagodon rhomboides
SP Calamus nodosus
SP Stenotomus chrysops
BO Sarpa salpa
BO Spondyliosoma cantharus
Spicara maena
PE Boopsoidea inornata
BO Gymnocrotaphus curvidens
BO Pachymetopon aeneum
BO Polyamblyodon germanum
BO Boops boops
BO Crenidens crenidens
SP Acanthopagrus berda
DE Sparidentex hasta
SP Sparus auratus
SP Rhabdosargus thorpei
SP Sparodon durbanensis
PE Lithognathus mormyrus
PE Pagellus bogaraveo
BO Oblada melanura
DI Diplodus cervinus
DI Diplodus argenteus
DI Diplodus bermudensis
DI Diplodus holbrooki
116
FIGURE 17. Two equally parsimonious trees - weighted data
Tree One
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lethrinus ornatus
Calamus nodosus
Stenotomus chrysops
Lagodon rhomboides
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Dentex tumifrons
Spicara alta
Porcostoma dentata
Cymatoceps nasutus
Petrus rupestris
Boops boops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Crenidens crenidens
Pagellus bogaraveo
Sparus auratus
Acanthopagrus berda
Sparidentex hasta
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
Tree Two
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
icentrarchus punctatus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lethrinus ornatus
Calamus nodosus
Stenotomus chrysops
Lagodon rhomboides
Argyrops spinifer
Evynnis japonica
Pagrus auratus
Pagellus bellottii
Pagrus pagrus
Pagrus auriga
Cheimerius nufar
Dentex dentex
Dentex tumifrons
Spicara alta
Porcostoma dentata
Cymatoceps nasutus
Petrus rupestris
Boops boops
Sarpa salpa
Spicara maena
Boopsoidea inornata
Pachymetopon aeneum
Crenidens crenidens
Pagellus bogaraveo
Sparus auratus
Acanthopagrus berda
Sparidentex hasta
Oblada melanura
Diplodus cervinus
Diplodus argenteus
Diplodus holbrooki
118
FIGURE 18. A strict consensus of two equally parsimonious trees from the weighted
data (transversions only third codon). A heuristic search of 1000 random addition
replicates resulted in two equally parsimonious trees. (Tree length = 2536, CI = 0.2504,
RI = 0.5816). Subfamilies are labeled as follows (BO=Boopsinae, DE=Denticinae,
DI=Diplodinae, PA=Pagrinae, PE=Pagellinae, and SP=Sparinae).
“N” = Node
D
1st = Partitioned Decay at 1st Codon at “N”
2nd = Partitioned Decay at 2nd Codon at “N”
3rd = Partitioned Decay at 3rd Codon at “N”
B
27 (
100
)
2.3
5.0
14.7
22 (
0.2
)
24 (
100
7 (-3.0
9.8 )
12.9
3.5
7.6
)
4.5
)
10 (0.0
5.5
8.1
1.6
-0.7
9(
)
2(-2.5
3.4
1.1
3 (-1.0
4.0
0.0
)
2.0
)
3.0
)
4(0.0
1.0
)
<
3.0
11.4
100
7.2
1.0 )
2(-6.2
6(
-5.3
0.0
11.3
<
0.2
)
6.3
0.8)
3(-4.2
90
9(-0.1)
89
56
16(-1.0
)
14.0
)
14 ( 2.6
0.0
100
100
11 (0.0
9.0
<
100
91
1.5
)
8 (-0.3
6.8
69
76
0.5
)
2 (0.0
1.5
100
89
70
8.9
-3.0
)
2 (-0.1
5.1
<
<
-2.5
4 ( 1.0 ) 0.4
5.5
2 (1.0
0.6
)
-3.3
)
2 ( 0.0
5.3
0.5
2(
3.8
0.4
-2.2
)
3(0.0
<
2.5
0.5
4 (0.5
3.0
)
68
4(0.0
2.0
2.0
)
5.2
)
8.8
0.0
51
54
)
14(0.0
)
3( 0.7
2.3
<
55
7.8
1.0 )
4(-4.8
77
100
53
4.5
100
0.2 )
15 (10.2
2(
-1.0
0.0
3.0
)
53
4(
-0.3
0.7
3.5
)
73
56
-1.0 89
) 4.0 100
6( 0.0
7.0
0.0 )
17(13.0
<
-3.0
2 ( 0.0 )
5.0
56
-0.1
1.1)
9( 8.0
90
81
-1.5
4 ( 0.0 )
97
5.5
0.0
6 (-1.0
) 2.0 100
7.0
6 (0.0 )
60
4.0
Group II
6.5
1.0
19.5
)
Group I
100
8.1
2.8
16.1
27(
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Morone chrysops
Morone saxatilis
Morone americanus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Scolopsis ciliatus
Lethrinus ornatus
SP Calamus nodosus
PA Evynnis japonica
PA Pagrus auratus
PA Pagrus pagrus
PA Pagrus auriga
DE Cheimerius nufar
DE Dentex dentex
DE Dentex tumifrons
Spicara alta
DE Petrus rupestris
BO Boops boops
BO Sarpa salpa
Spicara maena
SP Sparus auratus
BO Oblada melanura
120
FIGURE 19. A strict consensus of 699 equally parsimonious trees from the amino acid
translations. A heuristic search of 100 random addition replicates on the cytochrome b
amino acid residue translated from the nucleotide data set resulted 699 equally
parsimonious trees (tree length = 637, CI = 0.5620; RI = 0.7082). Subfamilies are
labeled as follows (BO=Boopsinae, DE=Denticinae, DI=Diplodinae, PA=Pagrinae,
PE=Pagellinae, and SP=Sparinae).
“N”
“N” = Node
D
B
D
B
6
99
2
100
4
97
100
61
6
4
89
53
5
98
99
2
98
Group II
8
2
53
2
59
2
55
85
Group I
8
87
Cyprinus carpio
Luxilus zonatus
Lateolabrax latus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Morone saxatilis
Morone chrysops
Morone americanus
Scolopsis ciliatus
Lethrinus ornatus
SP Calamus nodosus
PA Evynnis japonica
PA Pagrus pagrus
Spicara alta
PA Pagrus auratus
DE Dentex tumifrons
DE Petrus rupestris
DE Cheimerius nufar
DE Dentex dentex
PA Pagrus auriga
Spicara smaris
BO Oblada melanura
PE Pagellus acarne
SP Sparus auratus
BO Boops boops
BO Sarpa salpa
122
FIGURE 20. The clade containing Sparidae was copied from the weighted cytochrome b
tree (Figure 18.) and ancestral nodes were numbered so that they could be assigned FAO
areas during analysis of vicariance biogeography.
33
34
30
32
27
31
28
35
26
25
29
23
24
22
21
36
20
19
18
17
39
40
38
15
37
14
13
1
16
5
4
3
2
11
10
10
9
8
7
6
SP Calamus nodosus
PA Pagrus auratus
PA Evynnis japonica
PA Pagrus auriga
PA Pagrus pagrus
DE Dentex dentex
DE Cheimerius nufar
DE Dentex tumifrons
Spicara alta
DE Petrus rupestris
BO Boops boops
BO Sarpa salpa
Spicara maena
SP Sparus auratus
BO Oblada melanura
124
FIGURE 21. Clade of biogeographic relationships overlaid on map of the world.
A consensus of two MPTs overlaid on a map of the world. No distances are implied by
branch lengths or by inter-nodal spaces.
Atlantic Northeast
Mediterranean Black Sea
Pacific Southwest
Pacific Northwest
Pacific Western Central
Indian Ocean Eastern
Indian Ocean Western
Atlantic Southeast
Atlantic Eastern Central
Atlantic Southwest
Atlantic Western Central
Atlantic Northwest
CHAPTER TWO. A MOLECULAR PHYLOGENY OF THE FAMILY
MITOCHONDRIAL 16S RIBOSOMAL RNA GENE
127
INTRODUCTION
The vertebrate mitochondrial genome has two genes that code for the ribosomal
RNA, the small subunit (12S) and the large subunit (16S). The small subunit is about
819-975 bp and the large subunit is about 1571-1640 in vertebrates (Meyer, 1993).
Based on the complete mtDNA genomic sequence of Cyprinus carpio Chang et al.
(1994), the12S molecule is coded from approximately 950 base pairs and the 16S
molecule is coded from approximately1680 base pairs. Secondary structure models exist
for the encoded functional RNAs of both genes (Gutell et al, 1985; Gutell and Fox,
1988; Vawter and Brown, 1993) and these models have been used to infer homology
between taxa (for example, Vawter and Brown, 1993; Horovitz and Meyer, 1995; Ortí et
al., 1996; Wiley et al., 1998). Secondary structure comprises loop structures (loops and
bulges), stem structures and strings of unpaired regions (often forming incomplete
internal loops). These structures have been found to have different rates of mutation
based on physical, functional or compensatory restraints (for example, Vawter and
Brown, 1993; Meyer 1993). Substitutions in mitochondrial rRNA genes are dominated
by transitions (between closely related taxa) and transversions that become increasingly
more prevalent with increased sequence divergence (Meyer, 1993).
The mtDNA ribosomal genes evolve at a faster rate than do nuclear ribosomal
genes (Hillis and Dixon, 1991, Meyer 1993). Both the large and small subunits have
been hypothesized to be potentially informative in phylogenetic relationships among
vertebrate ingroup taxa that diverged since the Mesozoic period - within the last 150
million years (Mindell and Honeycutt, 1990). Based on comparisons of relatively few
128
vertebrate organisms, Hillis and Dixon (1991) found that 12S/16S could be used for most
Cenozoic comparisons and they hypothesized that mitochondrial rDNA may be useful in
comparisons of vertebrate taxa that have diverged as recently as 20 million years.
Ribosomal DNA has been used to infer phylogenies from a number of vertebrate
species, most recently, but not limited to, primates (Horovitz and Meyer), lizards (Fu
1999) and warblers (Cibios et al. 1999). Numerous studies have used 12S/16SmtDNA
sequences to resolve evolutionary relationships in fishes. Ortí et al. (1996) used
fragments of both 12S and 16S genes to examine evolution in serrasalmin piranhas and
ribosomal DNA was found to give a, “robust estimate of serrasalmin relationships”.
Additionally and notably, Ortí et al. (1996) presented useful teleost structure models for
the12S and 16S RNA fragments that were based on the models derived for vertebrates by
Gutell and Fox (1988) and Gutell et al. (1993). The phylogenetic relationships of
lampridiform fishes were investigated using 12S/16S mtDNA and morphological
characters (Wiley et al., 1998). The combined ribosomal data corroborated previously
published morphological lampridiform relationships and the 16S mtDNA data set proved
to resolve relationships in a manner “logically consistent” with those obtained when
ribosomal mtDNA and morphological data were combined.
Many studies have examined the evolutionary histories and relationships of
Perciform fishes using mitochondrial ribosomal DNA. Ritchie, et al. (1996) used
12S/16S fragments to examine the phylogeny of the trematomid fishes (Nototheniidae.
Perciformes); Tang et al. (1999) used 12S/16S to infer a phylogeny of the Acanthuroidei;
Bernardi and Bucciarelli (1999) used cytochrome b and 16S sequences in a phylogeny of
the Embiotocidae; Tringali et al. (1999) analyzed allozymes and 16S in the phylogenetics
of the Centropomidae; and Farias et al. (1999) used a fragment of 16S to infer a
phylogeny of neotropical cichlids. Hanel and Sturmbauer (2000) used 16S sequences to
estimate a phylogeny of trophic types in Northeastern Atlantic and Mediterranean
129
sparids. They examined a 486 bp fragment for 24 sparid fishes and used the
centracanthid Spicara as outgroup. Their findings showed that there was no support for
the current subfamilies associated with the taxon they sampled.
In this chapter the results of phylogenetic analyses of the partial mitochondrial
16S rRNA gene are reported for 40 sparid taxa and 16 closely related percoid taxa.
Parsimony analysis of the nucleotide sequences was used to test 1) the monophyly of the
Sparidae; 2) the validity of the six subfamilies of the Sparidae; 3) the evolutionary
relationships of the 33 genera of the Sparidae; 4) the monophyly of Sparoidea; and, 5) the
relationship of Sparoid fishes to other classically related percoids. Replicate bootstrap
support and Bremer decay indices were used as indicators of clade support.
Additionally, the resulting 16S phylogeny was used as a basis to estimate the
biogeographic aspects of sparid evolution.
130
MATERIAL AND METHODS
Collection Data - Collection data are as in Chapter One, although a reduced number of
taxa was used in this chapter. Sequences from GenBank were used for two outgroups
species, Cyprinus carpio and Morone chrysops. A complete taxa list is shown in Table
12 and is organized to reflect taxon sets (All, Ingroup and Outgroup) discussed
throughout this chapter. Catalog numbers of vouchers are given in Table 12.
Primers - The primer sequences used for PCR amplification in this study were the 16Sar5' (Palumbi in Hillis et al., 1996) and 16Sbr-3' (Kocher and White, 1989). Primers were
ordered from Genosys (Genosys Biotechnologies, Inc. The Woodlands, TX).
Amplification - A 50 µl PCR amplification of the partial 16S mtDNA consisted of 5-10
ng of each template DNA, 5 µl 10X PCR Buffer (Invitrogen Corporation) [100mM TrisHCL (pH8.3), 500 mM KCL, 25 mM MgCl2, 0.01% gelatin]; 1µl 10mM dNTP Mix from
the PCR Reagent System (GIBCO BRL) [10mM each dATP, dCTP, dGTP, dTTP]; 50
pmols each of primers, 0.50 µl Platinum Taq DNA polymerase (GIBCO BRL Life)
[5U/µl] and 42.00µl diH2O. All amplifications were performed on a MJ Research PTC200 thermocycler (Watertown. MA) with the following cycle parameters: initial
denaturation [95EC for 4.0 min]; 35 cycles of [denaturation 94EC for 1.0 min, annealing
45EC-47EC (depending on sample) for 1.0 min; extension 65EC for 3.0 min]; final
extension [65EC for 10 min]; icebox [4EC indefinitely].
Cloning and Sequencing - All samples were cloned and sequenced on a Li-Cor L4000
automated sequencer as outlined in the Materials and Methods section of Chapter One.
131
Sequence Alignment - Both light and heavy strand sequences were sequenced for all
individuals. Light strand sequences were inverted (reversed and complimented) with the
heavy strand sequence. A consensus of both strands was used for this study. The 16S
nucleic acid sequences were aligned by the Clustal feature of Gene Jockey II (Biosoft,
Cambridge UK) and by CLUSTAL W (Thompson et al., 1994). Sequences were further
aligned based on putative stem and loop structure following Wiley, et al. (1998).
Putative stem structures were those where base pairing might be expected. All other
areas (loops, bulges and non-paired regions) were classified as putative loops. Gaps were
minimized by aligning sequences in relation to secondary structure and each stem or loop
area was organized into a column of data within a PAUP* NEXUS file. A teleost model
of the 16S fragment (Ortí. et al.,1996) and a primate structural model (Horovitz and
Meyer 1995) were used to locate conserved loops and stems and then secondary-structure
was folded by eye. Stem and loop regions were examined for base-pair complementarity
and gaps were added to the alignment between distantly related taxa.
Character Sets - Within the NEXUS file, each column of data was designated either stem
or loop , and loops were further divided into conserved and non-conserved positions.
Gaps greater than two nucleotides were designated as “highly variable” and assigned nonconserved. Step-matrices were developed to separate substitutions into transitions (Ts =
AøG, CøT) and transversions (Tv = AøC, AøT, CøG, GøT). Step-matrices were
used in conjunction with character sets to correct for site saturation during parsimony
analysis.
Sequence Characteristics and Mutation Analysis - Uncorrected sequence divergence was
determined for pairwise comparisons of all taxa, ingroup taxa and outgroup taxa. Gaps
were treated as missing data during calculations of sequence divergence, because distance
132
calculations can not treat gaps as a fifth character. The average contribution of loop (CL)
and stem (CS) characters to the overall sequence divergence from pairwise comparisons
was calculated using the following formulas where,
( LDi × TL)
Tc
CL = ∑ i =1 (
) n
( LDi × TL) ( SDi × TS )
+
Tc
Tc
n
/
( SDi × TS )
Tc
CS = ∑ i =1 (
) n
( LDi × TL) ( SDi × TS )
+
Tc
Tc
n
/
n=Total number of pairwise comparisons; L Di = Distance Loops (sans gaps) at each
comparison; SDi =Distance Stems (sans gaps) at each comparison; TL= Total Number of
Loop Characters (sans gaps); TS = Total Number of Stem Characters (sans gaps); Tc =
Total Number of Characters (sans gaps).
Stem and loop structures were examined for site saturation by plotting: 1) the total
number of mutations as a function sequence divergence; 2) stem and loop transitions as a
function of sequence divergence, and; 3) stem and loop transversions as a function
sequence divergence. Transitions were further separated into nitrogenous base type,
purines and pyrimidines, and plotted as a function of uncorrected sequence divergence.
Loop structures were divided between conserved loop characters and non-conserved loop
characters. Non-conserved loop characters were those where a multiple number of gaps
(>2) were needed to align sequences between taxa.
Phylogenetic Analysis - Parsimony analysis was performed using PAUP4.0b4a*
(EPT) and strict consensus were obtained for each analysis. The number of constant
consistency index (CI) and the retention index (RI) were recorded for each analysis.
133
Bootstrap replicate support was conducted using PAUP* and decay (Bremer 1988) and
partitioned decay values (Baker and Desalle, 1997 and Baker, et al., 1998) were
calculated for clade support using the program TreeRot.v2 (Sorensen 1999). Gaps were
treated as a 5th character state in the data block.
Biogeographic Analysis - A quantitative analysis of the biogeographic relationships of the
Sparidae was conducted using the method of vicariance biogeography (Brooks 1985;
Wiley 1988a, b; Wiley et al., 1991). Parsimony analysis was used to reconstruct the
biogeographic relationships inferred from the 16S unweighted phylogeny. See material s
and methods in Chapter One for more detail on vicariance biogeography.
134
RESULTS
Sequence Characteristics - Sequencing of the 16S mtDNA gene produced an average of
574 nucleotide base pairs per taxon (Table 13). Multiple alignments resulted in a
consensus with 621 positions (base pairs and gaps). Loop and stem structures were
identified, and all characters were assigned as either loops or stems (S or L above
alignments in Table 13). On average more columns of data were designated to the loop
category (381 columns) than to the stem category (240 columns) . Most of all characters
(columns of data) were highly conserved in the16S data set (Table 14). Of the 621
positions, 40% were constant and 22% were parsimony uninformative (autapomorphic
changes - not shared by more than one taxon). The number of constant
characters/uninformative characters was split nearly equally across stems and loops
(Table14). The majority (80%) of all parsimony informative variable characters were
from loops regions (Table 14). Six areas contained multiple gaps after all taxa were
aligned (Table 15), five of which were located within loop structures. All five of these
highly variable loops were designated “non-conserved”.
Sequence Divergence - The largest sequence divergence was 26% between Centropomus
undecimalis and Scolopsis ciliatus and the smallest sequence divergence was 1.2%
between Pachymetopon aeneum and Polyamblyodon germanum. Mean pairwise
sequence divergence between all pairwise comparisons of taxa was 9.70% (Table 16).
Average divergence between comparisons of outgroup taxa was 14.49% and between
pairwise comparisons of ingroup taxa was 6.21%. On average, and between each taxa set
(All, Ingroup and Outgroup), loop characters contributed 4 times as much as stem
135
characters to overall sequence divergence (Table 17). Across each taxon set the total
contribution of loops to overall sequence divergence was nearly constant at >80%. There
was a slight increase to 83% in loop contribution in outgroup species (Outgroup; Table
17).
Mutation Analysis - A plot of all substitutions as a function of uncorrected sequence
divergence showed that a maximum of approximately 70 substitutions was reached at
greater than 25% sequence divergence (Figure 22). Transversional substitutions were
not saturated and a second order polynomial fitted to transversional data had the
following characteristics: y = 638.15x2 + 138x - 1.8914, R2 = 0.9372. Transitional
substitutions were found to be saturated at nearly 15% sequence divergence. A second
order polynomial was fitted to transitional data ( y = -631.81x2 + 374.19x + 1.9608, R2 =
0.899). The total number of stem substitutions was plotted as a functions of uncorrected
sequence divergence (Figure 23). Neither transversions nor transitions were saturated as
sequence divergence increased and all stem substitutions reached a maximum of
approximately 20 at greater than 25% sequence divergence. Second order polynomials
were fitted to the data and had the following properties: transitions (y = 68.676x2 +
53.276x + 0.9629; R2 = 0.7379) and transversions (y = 464.67x2 - 59.195x + 2.9619, R2 =
0.6269). Loop substitutions plotted as a functions of uncorrected sequence divergence
revealed that transitions saturated at nearly 15% sequence divergence (Figure 24). A
second order polynomial strongly fitted the data: y = -700.48x2 + 320.91x + 0.9979, R2 =
0.8066. Transversions in loop regions were nearly twice as frequent as those in stems,
peaking at nearly 50 substitutions at 25% sequence divergence. Because loop transitions
were saturated, they were examined more closely. Loop pyrimidine substitutions (CøT)
and purine substitutions (AøG) were plotted as a function of uncorrected sequence
divergence (Figure 25). Both types of nucleic acid substitutions were equally saturated.
136
Loop pyrimidine transitions were slightly more frequent than purine substitutions and
second order polynomials fitted to the data had nearly equal values; CT ( y = -375x2 +
177.44x + 0.016, R2 = 0.6942) and AG (y = -325.48x2 + 143.47x + 0.9819, R2 = 0.673).
Non-conserved and conserved loop transitions were plotted as a function of uncorrected
sequence divergence (Figure 26). Non-conserved loop transitions were saturated, and
represented the full signal of all transitional saturated characters (2nd order polynomial y
= -610.06x2 + 220.37x + 2.1544 R2 = 0.5817). However, conserved loop transitions were
not saturated and proved to be much less frequent than non-conserved loop transitions.
Because conserved loop transitions were not saturated, they were phylogenetically
informative across the full range of pairwise sequence divergence.
Loop and stem distances were converted to a fraction of total sequence divergence
and plotted as a function of uncorrected sequence divergence.(Figure 27). Loop or stem
distances were converted by multiplying each respective pairwse distance by the total
number of loop or stem characters (sans gaps) and then dividing by the total number of
characters (sans gaps).
In the resulting plot, distances between loop characters
accounted for the majority of the total sequence divergence from each pairwise
comparison.
Base Composition - Mean values and ranges (one standard deviation) of percent base
composition for all, stem, loop, and non-conserved loop characters of the 16S mtDNA
fragment are presented in Figure 28. Over all structures and taxa, there was bias
towards the use of adenine and a slight bias for the use of cytosine. There were no
significant differences in base composition values between or within ingroup and
outgroup taxa. There was a significant difference in the base usage of putative loop and
stem structures. Loop characters were heavily pro-adenine and anti-guanine. Stem
characters were heavily pro-guanine, slightly pro-cystine and anti-guanine.
137
Unweighted Data - Of the 621 columns of data (base pairs and gaps), 251 were constant,
137 variable characters were parsimony uninformative and 233 variable characters were
parsimony informative. A simple heuristic search resulted in eight equally parsimonious
trees (tree length=1527, CI=0.4224, RI=0.5444; 10,000 trees held at each stepwise
addition, branch swapping = tree bisection-reconnection; outgroup taxa Cyprinus carpio;
no topological restraints). Bootstrap support (bs) of 1000 replicates and partitioned
Bremer decay values (20, unrestricted random addition sequences per node) were
calculated and are shown on a strict consensus of eight equally parsimonious trees (Figure
29). The Sparidae + Spicara formed a well resolved, monophyletic clade. Bootstrap and
decay support for the sparid node was strong (bs= 81, decay=5); nearly all of the decay
value for this node (4.2) came from stem characters. Within the Sparidae, a clade
comprised of Boops boops + Sarpa salpa (decay=12) fell basal to all other sparids. The
support for B. boops and S. salpa basal to other Sparidae was weak (decay=2). All other
Sparidae+Spicara were in one of two groups: Sparidae Group I (Figure 29) - and
Sparidae Group II (Figure 29). Support for the node defining Group I was weak
(decay=2). Within Group I four terminal nodes had strong support: Spondyliosoma
cantharus + Spicara smaris (decay=9, bs=99); Acanthopagrus berda + Sparidentex hasta
(decay=11, bs=98); and Pachymetopon aeneum + Polyamblyodon germanum (decay=14,
bs=100). A weak decay value of 2 separated the western Atlantic Stenotomus, Calamus
and Lagodon species from other Group I taxa.
There was no support from this analysis for the Sparoidea; neither the lethrinids
nor nemipterids were sister to the sparids. Rather, a clade of “other” percoids,
comprising the moronids + Lateolabrax, haemulids, and lutjanids was sister to the
Sparidae. The lethrinids and nemipterids formed an unsupported clade that was basal to
the “other” percoids and the Sparidae. Neither decay values nor bootstrap values were
138
robust for percoid inter-relationships; less than 2 steps were needed to find a tree
inconsistent with any given constrained node and bootstrap analysis collapsed internal
percoid nodes into an unresolved polytomy. However, Centropomus undecimalis
consistently fell basal to all other percoids.
No Transitional Substitutions All Loops - Of 621 total columns of data, 240 columns
were treated as stem characters and defined as unordered. The remaining 381 characters
were designated as loops. A step matrix was developed that weighted all loop transitions
0 and transversions 1 and was applied to loop positions under the “Set Character Types”
of the Data option of PAUP*. Of the 621 characters, 251 were constant, 165 variables
sites parsimony-uninformative and 205 were parsimony-informative. A heuristic search,
of 1000 random addition replicates, resulted in 3952 equally parsimonious trees (tree
length=755, CI=0.5325, RI=0.6491, 100 trees held at each step, branch swapping=tree
bisection-reconnection; starting seed = 1815351626; outgroup taxa Cyprinus carpio).
The consensus tree (Figure 30) contained a monophyletic Sparidae + Spicara (decay=9,
bs=81). Within the Sparidae, generic relationships were less clearly defined than when
all characters were included. The taxa that comprised “Group I” were unresolved within
the Sparidae forming a polytomy. Oblada remained allied to Diplodus, but Diplodus
cervinus fell basal to all other Diplodus + Oblada. Group II taxa were distinct from other
Sparidae, but there was little resolution for relationships within the group. Calamus
nodosus was distinct from other Group II species.
No Transitional Substitutions Non Conserved Loops - The same step matrix, described
above, was used to weight “non- conserved” loops regions as designated in Table 15. Of
the 621 columns of data, 429 were of type unordered and 192 were user defined with the
step matrix. After applying this weighting, of the 621 total characters, 251 were constant,
139
159 were parsimony-uninformative and 211 were parsimony informative. A 1000
random addition replicates resulted in 9826 equally parsimonious trees (tree length=Tree
length = 951, CI=0.5047, RI = 0.6032, 100 trees held at each step, branch swapping=tree
bisection-reconnection; starting seed=462434539; outgroup taxa Cyprinus carpio). A
strict consensus of all trees is shown in Figure 31. The Sparidae + Spicara were
monophyletic in the resulting tree, but within the Sparidae there was very little generic
resolution. Group II taxa, as defined in the two previous analyses, remained distinct from
other Sparidae, and within Group II (Cheimerius nufar, (Pagellus bellottii + Pagrus
pagrus), Argyrops spinifer, (Evynnis japonica + Pagrus auratus), Pagrus auriga) were
distinct from other taxa. However, there was no support for this node. Relationships for
the majority of other sparid species were not clearly defined, resulting in a polytomy.
Species that had been robustly supported in other trees remained strong in this analysis,
including: Acanthopagrus berda + Sparidentex hasta, Pachymetopon aeneum +
Polyamblyodon germanum and Rhabdosargus thorpei, Sparodon durbanensis, Sparus
auratus. Diplodus cervinus did not clade with other Diplodus & Oblada, but was part of
the unresolved polytomy in Group I species. Archosargus probatocephalus fell basal to a
clade containing the boopsines Boops boops + Sarpa salpa and Spondyliosoma cantharus
+ Spicara maena.
Biogeographic Analysis - The monophyletic Sparidae clade within the unweighted 16S
phylogeny (Figure 29) was used as the source of dependent data for biogeographic
analysis because it had the best resolution of generic relationships. The Sparidae clade
was pruned from the original tree and all ancestral states were numbered (Figure 32). The
geographic distribution was defined for terminal taxa using FAO areas (Table 18) and
these areas were treated as independent data. Areas of occurrence were determined
relative to each terminal taxa or ancestral node and coded into a binary matrix;
140
presence=1, absence=0 (Table 18). Parsimony analysis of the data matrix resulted a
single most parsimonious tree and is overlaid on a world-wide map in Figure 33 (tree
length = 83; CI =0.7470; RI = 0.8333; of 75 total characters; all characters unordered and
equal weight, 13 characters were constant, 3 variable characters were parsimony
uninformative, 59 variable characters were parsimony informative; 1000 replicates of
random stepwise addition; 100 trees held at each step; starting seed = 1996341824;
character-state optimization: delayed transformation)
As shown in Chapter One, all taxa were found in more than one FAO area, except
the Bermuda endemic Diplodus bermudensis and the Japanese endemic Evynnis japonica.
The tree was rooted to the southwestern Pacific Ocean clade. The western Indian
Ocean/southeastern Atlantic clade was sister to eastern Atlantic/Mediterranean-Black Sea
species. However, the southwestern Atlantic terminal node was sister to the Pacific
Southwestern node instead of with other Atlantic areas. In the16S tree, used as
dependant data, Pagrus pagrus clustered with P. auratus, Evynnis japonicus and
Argyrops argyrops and this, in part, contributed to the placement of southwestern
Atlantic node. With the exception of the southwestern Atlantic the Atlantic Sparidae +
the western Indian Ocean/Atlantic species were sister to Pacific Ocean/eastern Indian
Ocean clade. The eastern Indian Ocean - western central and northwestern Pacific taxa
formed an unresolved polytomy that were sister to the southwestern Pacific Ocean and
southwestern Atlantic species.
141
DISCUSSION
Sequence Characteristics, Sequence Divergence and Mutation Analysis - The majority
(81%) of parsimony informative characters was found within putative loop structures;
only 19% putative stem characters were informative. Of the small number of
informative stem characters, some were possibly parsimony-misleading due to
compensatory mutational dependence. Ribosomal RNA contains know secondary
structure that is dependent on Watson-Crick and wobble base-paring between noncontinuous rRNA bases (Dixon and Hillis, 1993). Bases that comprise structural
elements of stems and occasionally loops are often mutation-constrained. A mutation at
one site may or may not have an effect on base-pairing up or down stream from that
mutation. In fact, it is possible that stem regions evolve as a paired region. Dixon and
Hillis (1993) addressed this problem by weighting of “compensatory” mutations and
concluded that stem characters are phylogenetically informative, but at the expense of
reduced character independence. In fact, they found that all complementary bases are not
”inextricably linked”.
A phylogenetic study based on rDNA might contend with compensatory
mutations by weighting all characters equally (assign a weight of 1.0 - at the expense of
reduced character independence), down-weighting characters that violate character
independence (some weight less than 1.0), or ignoring all non-independent data
(weighting 0 - at the expense of removing phylogenetically informative data).
There was no empirical way to determine an exact value for down-weighting stem
bases to compensate for compensatory mutations. Dixon and Hillis (1993) suggest using
a value between a weight of 0.5 and 1.0. A value of 0.5 would be used if all stem
142
characters “evolve as pairs” with each base representing half of a character. Because not
all stem mutations are linked, a value of 0.5 might overcompensate for interdependence
and Dixon and Hillis (1993) suggested a “compromise” of 0.8.
I made no attempt to correct for compensatory mutations in the analyses
presented in results. (Although no results were given, I weighted stem characters by both
0.5 and 0.8 and there was little or no topological difference, respectfully, between these
trees and those generated with all stem characters weighted 1.0). Rather, all stem
substitutions were given equal weight at the possible expense of reduced character
independence. Only a small number of putative stem characters were parsimony
informative, so they contributed little to the overall phylogenetic signal. Therefore, a
weight less than of 1.0 (0.8 or 0.5) did not drastically effect the overall phylogenetic
relationships. Additionally, secondary structure models were folded by eye, and due to
gaps between distantly related taxa, it was very difficult to accurately identify
compensatory stem structure mutations for all taxa. Similarly, Dixon and Hillis (1993)
found, “no obvious difference between results of the analysis with all characters equally
weighted and the results of the analysis with stem characters weighted 0.8.”
Pairwise sequence divergences were similar to those reported for other teleosts.
Farias et al. (1999) found a maximum uncorrected pairwise divergence of 20% (average ~
17%) in cichlids; Wiley et al. (1998) found a maximum 0f >25% Tamura-Nei distance in
acanthomorph relationships and Tang et al. (1999) found < 20% Tamura-Nei distance in
the Acanthuroidei. On average, mean sequence divergence for all pairwise comparisons
of taxa in this study (9.7%) were well below the level at which loop substitutions
appeared to reach saturation. Putative loop mutations deviated from a linear relationship
when genetic distance approached approximately 15% sequence divergence. The average
pairwise divergence for ingroup species (6.21%) was well below the apparent level of
loop saturation. Because neither stems nor loops were saturated for ingroup species, all
143
characters were potentially informative and all characters were treated with equal weight.
Loop transitions appeared to reach saturation between outgroup taxa in this study,
but transversions were not saturated for either loops or stems. In previous phylogenetic
studies using 16S (Mindell and Honeycutt, 1990; Ortí et al., 1996; Wiley et al., 1998;
Tang et al., 1999), plots of loop substitutions as a function of sequence divergence
revealed that loop transitions were saturated for some taxa. In some cases, loop
transversions were also saturated between some more distantly related taxa.
Average mean pairwise divergence for outgroup species was 14.49%, nearly at the
same level at which apparent saturation occurred in loops. Because, on average, sequence
divergence of ingroup species was far below the level at which loops appeared to reach
saturation, loop substitutions were not uninformative for ingroup comparisons. However,
the same was not true for pairwise comparisons between ingroup taxa and outgroup taxa
and between pairwise comparisons of outgroup taxa.
The difference between ingroup loop transitions (not saturated) and outgroup loop
transitions (saturated) complicated the determination of character weights. With these
data, all substitutions were potentially informative for ingroup species. Therefore, no
weighting of any character was necessary to infer a phylogeny of the Sparidae from the
16S data. An equal weight of all characters was consistent with sparid relationships but
potentially inconsistent with outgroup relationships. Clearly, the outgroup taxa were
saturated for loop transitions, and the relationships inferred from these characters could
be potentially misinformative. I tried to account for this inconsistency in two ways. I
inferred a phylogeny of these taxa with all loop substitutions weighted 0. This removed
any potentially misinformative data and I believed that this would help define outgroup
relationships. However, in weighting all loops 0, I removed some potentially informative
conserved loop areas. Therefore, I inferred a phylogeny of these taxa weighting only
144
highly variable loop transitions 0, so that I would not lose conserved information.
It was not possible to assign different character weight to individual taxon groups.
For any given character, the same weight must be applied to each taxa during parsimony
analysis (the same being true for neighbor joining, maximum likelihood or distance
methods). Although multiple variable values can be assigned to characters when these
values vary with characters, each taxa must be assigned the same value for each
respective character. If there are different substitution between groups of taxa, as was
seen with the ingroup and outgroup in this study, only a single rate can be compensated
during any given analysis. A single rate compensation limits any analysis where more
than one potential substitution rate is present and, therefore, forces a compromise in
results. One either accepts possible inconsistent results (lack of support for Sparoidea in
the unweighted analysis) or accepts possible lack of resolution (loss of node definition
when loop transitions were weighted 0 for the weighted analyses).
Base Composition - Base compositional bias was similar to that found in teleosts (Ortí et
al., 1996) and a cytosine and adenine bias has been reported in other fishes (Otrí et al.,
1996 and Farias et al., 1999). A guanine/cytosine bias in stems has been observed in
fishes and other vertebrates (Vawter and Brown 1993; Ortí et al., 1996; Farias et al.,
1999; Cibois et al., 1999). Vawter and Brown (1993) reported that “structural regions
requiring base pairings” (i.e. stems) might be biased towards guanine/cytosine (G/C)
because G/C interactions are a more stable conformation for paired regions. The bias
towards adenine in unpaired regions has been attributed to possible hydrophobic
interaction of adenine with proteins (Gutell et al., 1985) . Adenine was the least polar of
the four bases and, therefore, adenine-rich areas might act as preferred protein binding
sites.
145
Phylogenetic Analysis and Phylogenetic Relationships - The 16S gene resulted in
intermediate resolution for the sparid genera but there was little resolution between the
Sparidae and other percoid families. The Sparidae + Spicara formed a monophyletic
group in all 16S analyses (see Chapter One for details on the placement of
Centracanthidae relative to Sparidae). Results from the16S mtDNA did not support the
current subfamily designations for genera of the Sparidae (Boopsinae, Denticinae,
Diplodinae, Pagellinae, Pagrinae and Sparinae). Based on all 16S sequence analyses, the
sparid genus Pagrus was paraphyletic. The sparid genus Pagellus and the centracanthid
genus Spicara were polyphyletic. None of these analyses supported the Lethrinidae as
sister to the Sparidae and there was no support, based on the 16S mtDNA, for a
monophyletic Sparoidea (Sparidae + Spicara, Lethrinidae and Nemipteridae).
Relationships of Genera of the Sparidae
Within the monophyletic Sparidae + Spicara the inter-generic relationships were
not robust. There was very little overall support for the relationships of Group I and
Group II genera as described in Chapter One. Decay and bootstrap values were robust for
sister group relationships (for example Pachymetopon aeneum and Polyamblyodon
germanum or Diplodus holbrooki and Diplodus bermudensis), but nearly absent for other
relationships. The Diplodus + Oblada clade was well supported in the unweighted tree,
however, the Indian Ocean D. cervinus was removed from other members of the group as
loop substitutions were down-weighted. Many of the genera that placed in Group I and
Group II of the unweighted tree (Figure 29) are in agreement with the placement of
genera in “Lineage 3" and “Lineage 2" of Hanel and Sturmbauer (2000, Fig.1.).
Relationships of the subfamilies of the Sparidae
There was no support from the16S sequence data for any of the subfamilies
146
proposed by Smith (1938), Akazaki (1962) and Smith and Smith (1986). The boopsines
were polyphyletic within the Sparidae and B. boops and S. salpa fell basal to all other
sparids in the unweighted analysis. The pagellines and sparines were also polyphyletic
within the Sparidae. The diplodines were paraphyletic but restricted to Group I and the
denticines and pargrines were paraphyletic but restricted to Group II. Clearly, based on
16S sequence data, the subfamilies as currently defined are artificial, and in agreement
with the findings of Hanel and Sturmbauer 2000. Despite the regional nature of their
study, the authors had a broad enough sampling of sparid species to conclude that trophic
types evolved more than once in sparid fishes. However, Hanel and Sturmbauer used
differential weighting based on a “sliding window analysis” of their data set to infer a
phylogeny. The authors did not use known secondary structure models to aid in the
alignment of their sequences or for the identification of potential areas of saturation.
Since the rates of substitution for 16S have been shown to be directly related to secondary
structure, it is not clear from their analysis how their differential weighting was related to
actual areas of rapid substitution or saturation. It is important to understand where
increased substitutions occur. Secondary structure models of 16S mtDNA often predict
that very saturated nucleotides neighbor very conserved nucleotides. The method of
Hanel and Strumbauer may not have been sensitive to this fact and saturated characters
might have been incorrectly weighted. This may have lead to artificially strong node
support for relationships in their tree.
Relationships of the Sparoidea
These data did not support Akazaki’s Spariform fishes or Johnson’s Sparoidea.
However, Johnson’s (1980) hypothesis that the Lethrinidae and Nemipteridae are closely
related is supported in all 16S analyses; these families form an independent clade that is
basal to other percoids and the Sparidae + Spicara. The 16S sequence analyses were
147
unable to recover the Sparoidea and there was little support outgroup relationships, do in
part, to the unreliable transitional saturation of loop characters.
Biogeography - The biogeographic analysis suggested a considerable structuring for
neighboring area relationships based on the dependent 16S phylogeny. Within the
unweighted 16S phylogeny, many taxa clustered together that had shared, overlapping
distributions. The hypothesized biogeographic clade should reflect these relationships. If
taxa in the dependent tree formed a clade due to common ancestral history (as opposed to
shared geographic range) then the biogeographic tree would likely not reflect strong area
relationships; terminal nodes corresponding to neighboring geographic areas might be
less likely to be sister. Such is the case in the 16S data with Pagrus pagrus. P. pagrus
from the western Atlantic claded with Pagrus, Evynnis and Argyrops from the
southwestern Pacific. This relationship is contrary to that found during biogeographic
analysis using the cytochrome b phylogeny as dependant data. The affinity of P. pagrus
to other pagrine members was based on shared evolutionary history and not geographic
relatedness. The resulting biogeographic tree had southwestern Atlantic and
southwestern Pacific areas sister, reflecting the signal of P. pagrus from the dependent
data. If, however, taxa were clustered together in the dependent tree based on shared
geographic areas, the corresponding biogeographic clade might strongly support
geographic neighbors as sister. This is not to say that shared evolutionary histories
should not reflect shared geographic histories. In fact, they should. This analysis
possibly shows that due to the plasticity of classification, or the multiple evolution of
trophic types, there was a strong relationship for taxa from the same area to form clades
in all analysis. Either the current classification has failed to unite species that are similar
based on shared evolution, or the genetic phylogeny here reflects a history not completely
based on shared evolution (hybridization or homoplastic characters due to saturation).
148
From these data it appears that there were two areas of sparid evolution, eastern
Indian Ocean - western Pacific and western Indian Ocean - Mediterranean/Atlantic.
These species probably had a Tethyan Sea common ancestor. As with the cytochrome b
analysis in the previous chapter, little more can be drawn from this analysis, because the
vicariance biogeography clade was based on a single tree example and this is considered
equivocal to a single transformation series explaining a clade (Wiley et al., 1991).
Phylogenetic Conclusions
The 16S sequence data was of minimal use in resolving phylogenetic relationships
of the Sparidae and related taxa. The partial gene had many variable positions associated
with loops, yet the rate of substitution was not fast enough for significant, well supported
resolution of most generic relationships. For more distantly related taxa, such as between
outgroups, 16S was not particularly informative. The saturation of loop characters
beyond 15% and the very conservative stem characters left few informative characters for
resolution of distantly related taxa.
Analyses of the partial 16S gene showed that there was no support for the
currently defined sparid subfamilies and that two major evolutionary groups of sparids
exist. These data are supported by Hanel and Strumbauer (2000) and by analysis of the
cyt b gene in the previous chapter. Additionally, biogeographic analysis using 16S
dependent data show significant evolutionary structuring based on area relationships
within the Sparidae.
149
TABLE 12. COLLECTION DATA FOR SPECIMENS USED. GENBANK
ACCESSION NUMBER, MUSEUM COLLECTION NUMBERS AND COLLECTION
LOCALITY ARE GIVEN. MUSEUM ACRONYMS ARE FOLLOWING (LEVITON
ET AL., 1985 AND 1988).
All
GenBank
Museum
Collection
TAXA
Outgroup Taxa
Centropomidae
Cyprinidae
Cyprinus carpio
Haemulidae
Haemulon sciurus
Pomadasys maculatus
Lethrinidae
Lethrinus ornatus
Lutjanidae
Caesio cuning
Lutjanus decussatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Nemipteridae
Scolopsis ciliatus
Ingroup Taxa
SPARIDAE
Boopsinae
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Denticinae
Cheimerius nufar
Dentex tumifrons
Petrus rupestris
Sparidentex hasta
Diplodinae
Accession#
Catalog#
AF247437 No Voucher
X61010
Locality
AF247442 No Voucher
AF247443 USNM Uncat
Florida, Big Pine Key, W. of Bridge,
Philippines, Manila Fish Market
AF247446 USNM 345259 Philippines, Bolinao, Luzon
AF247447 USNM Uncat
Australia, W. Australia CSIRO SS 8/95/45
AF247444 USNM 345193 Philippines, Iloilo Panay, Fish Market
AF247445 USNM 346695 Philippines., Northern Negros, Fish Market
AF247437
AF247438
AF247439
AF247440
AF055610
AF247441
No Voucher
fish market, Spain
VIMS 10381
MTUF 27451
Sasebo, Nagasaki Prefecture, Japan
No Voucher
VIMS Uncat
AF247448 USNM 345202 Philippines, Luzon, Manila, Fish Market
AF247449 USNM 346853 Philippines, Guimaras Island, JTW 95-1
AF247396
AF247397
AF247398
AF247399
AF247400
AF247401
AF247402
AF247403
MED180
No Voucher
RUSI 49447
OM1
RUSI 49672
RUSI 49690
RUSI 49456
ODU 2782
AF247404
AF247405
AF247406
AF247407
AF247408
AF247431
RUSI 58449
RUSI 49443
Qatif Market, eastern Saudi Arabia
Durban Fish Market, South Africa (KSA1)
AMS I.36450-002 Nelson Bay, Australia
RUSI 49684
RUSI 49686
NSMT-P Uncat Shuwaik Market, Kuwait City, Kuwait
AF247414 VIMS 010192
AF247419 No Voucher
Chesapeake Bay
Bermuda
150
Diplodus cervinus
AF247420 RUSI 49680
Diplodus holbrooki
AF247421 ODU 2789
Lagodon rhomboides
AF247423 VIMS Uncat
Pagellinae
Boopsoidea inornata
AF247409 ODU 2791
St. Sebastian Bay, South Africa
AF247410 ODU 2784
Pagellus bogaraveo
AF247411 ODU 2785
Pagellus bellottii
AF247412 ODU 2792
R/V African , Station 17491, South Africa
Pagrinae
Argyrops spinifer
AF247415 AMS I.36447-001 N. Territory, Australia
Evynnis japonica
AF247422 NSMT-P 47497 Miyazaki, Kyushu Prefecture, Japan
Pagrus auratus
AF247424 No Voucher
New Zealand, Sydney Fish Market,
Pagrus auriga
AF247425 ODU 2786
V. Emmanul Fish Market, Rome, Italy
Pagrus pagrus
AF247426 ODU 2790
Sparinae
Acanthopagrus berda
AF247413 USNM 345989 Philippines, Manila, Market.
Calamus nodosus
AF247416 No Voucher
AF247417 RUSI 49441
Cymatoceps nasutus
AF247418 RUSI 49445
Porcostoma dentata
AF247427 RUSI 58450
AF247428 KENT SAPL1 Plettenberg Bay, South Africa
AF247429 RUSI 49683
Ponta do Ouro, Mozambique
AF247430 RUSI 49673
Sparus auratus
AF247432 KENT 21
Stenotomus chrysops
AF247433 VIMS Uncat
Chesapeake Bay
Centracanthidae
Spicara alta
AF247434 ODU 2793
Carpenter, K.E.
Spicara maena
AF247435 ODU 2788
MTUF-Museum of the Tokyo University of Fisheries, Tokyo, Japan; NSMT-P National Science Museum
(Zoological Park) Tokyo, Japan; ODU- Old Dominion University, Norfolk, VA; RUSI- Rhodes University;
J.L.B. Smith Institute of Ichthyology, Grahamstown, South Africa; UT University of Tennessee,
Knoxville, TN; USNM- United States National Museum of Natural History, Washington, DC; VIMSVirginia Institute of Marine Science, Gloucester Point, VA
151
TABLE 13. MULTIPLE ALIGNMENT OF NUCLEOTIDE SEQUENCES OF THE
PARTIAL 16S GENE FROM THE SPARIDAE AND OUTGROUP SPECIES.
TAXONOMIC NAMES WITH A SUFFIX OF “GB” ARE FROM GENBANK AND
ARE INCLUDED WITH ORIGINAL SEQUENCES FOR EASY COMPARISON.
152
Argyrops spinifer
Boopsoidea inornata
Sparadon durbanensis
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
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10
20
|
|
789012345678901
L..............
GCAA-AATCAACGAA
GCAA-GACCAATGAA
GCAA-GACCAATGAA
GCAA-AATCAACGAA
GCAAGACTTAATGAA
GCAA-AACTAATGAA
GCAA-GACCAATGAA
GCAA-AACCAATGAA
GCAA-GACCAATGAA
GCAA-GACCAATGAA
GCAA-AAACAATGGA
GCAA-GACCAATGAA
GCAA-A-TCAATGAA
GCAA-AATCAACGAA
GCAA-A-TTAACGAA
GCAA-AGTTAACGAA
GCAA-AATCAATGAA
GCAA-GACCAATGAA
GCAA-A-TTAACGAA
GCAA-A--TAATGAA
GCAA-AACCAATGAA
GCAA-AATCAATGTA
GCAA-AATTAATGAA
GCAA-AATTAACGAA
GCAA-GACCAATGAA
GCAA-GACCAATGAA
GCAA-GACCAATGAA
GCAA-AATCAACGAA
GCAA-TACTAAAGAA
GCAATAATCGATGAA
GCAA-AATTAACGAA
GCAA-AATTAACGAA
GCAA-AATCAACGAA
GCAA-AATCAACGAA
GCAA-AATTCACGAA
GCAA-A--TAATGAA
GCAA-GACCAATGAA
GCAA-AATCAATGTA
GCAA-AATCAATGAA
GCAA-GACCAATGAA
GCAA-CGAACCAAGT
GTAA-AGCAAAA--GCAA-ACTTA-TGTA
GCAA-AACTA-TGAA
GCAA-AACTA-CGAA
GCAA-ACTTA-TGAA
GCAA-ACTTA-TGAA
GCAA-ACTTA-TGAA
GCAA-AATCAATGAA
GCAATAATTGCTGTA
GCAA-AATCAACGAA
GCAA-AATCAATAAA
GCAA-AATCAATGAA
GCAA-AACCAACGAA
GCAA-CTAT-AAGAA
GCAA-AAC--ACACA
2345678
S......
TAAGAGG
TAAGAGG
TAAGAGG
TAATAAG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAATAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAATATG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
ATAGGAG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAAG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
TAAGAGG
30
|
90123
L....
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-TC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CG
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-TC
TC-CC
TC-CC
GT-CC
TC-CT
TC-CA
TC-CC
TC-CC
TC-TT
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
TC-CC
4567
S...
GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCTCCGCCGCCGCCGCCGCCGCCGCCGCCGCCCGCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCAGCC
GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCC-
8
L
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
40
|
9012 3
S....L
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCT- C
CCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
TGCC C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- T
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
GCC- C
153
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
4
.
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
T
G
T
T
T
T
T
T
T
T
T
T
G
A
56789
S....
GTGAC
GTGAC
GTGAC
GTAAC
GTGAC
GTGAC
GTGTC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTTAC
GTCAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGCG
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
GTGAC
50
|
012345
L.....
TATATTATATTATACTATATTATATTATATTATATTATATTATACTGTACTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTACATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTATATTACAAAAACCTATATTATAATATAATATATTATATTATATTATATTGTATTGTATTATAATATATTATATTATATTGTTT-
678
S..
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
CTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
CCT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
GTT
60
|
90 12345
L. S....
TA ACGGC
TA ACGGC
TA ACGGC
TA ACAGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
CA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGAC
TA ACGGC
TA ACGGC
C- AACGG
CT CAACG
TA ACGGC
CA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
TA ACGGC
AA ACGGC
AA GCGGC
70
|
6789012345
S.........
CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGACGGT--CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CCCGGCGGTT
GCCGCGGT-CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT---CGCGGT----
80
|
678901
L.....
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTA
ATTTTG
ATTTTG
ATTTTG
ATTCTG
ATTCTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTT
ATTTTG
ATTTTG
ATTCTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATCTTA
ATTTTG
ATTTTG
ATCTTA
ATCTTA
ATCTTA
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATTTTG
ATACTG
ATCCTG
2345
S...
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
ACCG
154
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
67
..
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
TG
89
L.
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CA
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CA
CG
CG
CA
CT
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CC
CC
90
100
|
|
012 345678901234
S... L..........
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGTAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGTAATCA
AAG GTAGCGCAATCA
AAG GTAGCGTAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
AAG GTAGCGTAATCA
AAG GTAGCGTAATCA
AAG GTAGCGCAATCA
AAG GTAGCGCAATCA
567
S..
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
TTT
CTT
CCT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
CTT
110
|
89012
S....
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTTTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTTTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCTT
GTCCT
GTCTT
GTCTT
GTCTC
GTCTC
GCCCT
GCCCT
3456789
L......
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATA
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATA
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATA
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
TTAAATG
120
|
01234
S....
GAGAC
GAGAC
AAGAC
GAGAC
GAGAC
AGGAC
GAGAC
AAGAC
GAGAC
GAGAC
AAGAC
GAGAC
GAGAC
AGGAC
GAGAC
GAGAC
GAGAC
AAGAC
GAGAC
AAGAC
GAGAC
GAGAC
GAGAC
GAGAC
AAGAC
GAGAC
AAGAC
GAGAC
GAGAC
GAGAC
GAGAC
GAGAC
GAGAC
GAGAC
AAGAC
GAGAC
AAGAC
GAGAC
GAGAC
GAGAC
GAGAC
AAGAC
AAGAC
GAGAC
GAGAC
GGGAC
GAGAC
GAGAC
GGGAC
AGGAC
GAGAC
GAGAC
GGGAC
GGGAC
GGGGC
GGGGC
567
L..
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CCG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CCG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CTG
CCG
CTG
CTG
CTG
CTG
CCG
CCG
155
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
130
|
8901234
.......
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAC
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAC
TATGAAT
TATGAAT
TATGAAT
TATGAAC
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
TATGAAT
567
S..
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
GGC
140
150
|
|
8901 234567890 1234
L... S........ L...
ATCA CGAGGGCTT AACACCA CGAGGGCTT AACATCA CGAGGGCTT AGCATCA CGAGGGCTT AACATCA CGAGGACTT AGCACCA CGAGGGCTT AGCACCA CGAGGGCTT AGCACCA CGAGGGCTT AACACCA CGAGGGCTT AGCATCA CGAGGGCTT AGCATCA CGAGGGCTT AGCACCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AGCATCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AACACCA CGAGGGCTT AACATCA CGAGGGCTT AGCACCA CGAGGGCTT AACACCA CGAGGGCTT AGCATCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AACACCA CGAGGGCTT AACACCA CGAGGGCTT AACACCA CGAGGGCTT AACATCA CGAGGGCTT AACACCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AACATCA CGAGGGCTT AGCATCA CGAGGGCTT AACATCA CGAGGGCTT AACACCA CGAGGGCTT AGCATCA CGAGGGCTT AGCACCA CGAGGGCTT GGCATCA CGAGGGCTT AGCTCCA CGAGGGCTT AGCTAAA CGAGGGCTT AACTCCA CGAAGGCTT AACATCA CGAGGGCTT GACACGA CGAGGGCTT GACACGA CGAGGGCTT GACATAA CGAGGGCTT GACATAA CGAGGGCTT AACATAA CGAGGGCTT GACATAA CGAGGGCTT AGCATAA CGAGGGCTT ATCATAA CGAGGGCTT AACATAA CGAGGGCTT AGCATAA CGAGGG-TT CAAC
ATAA CGAGGG-TT CAAC
ATTA CGAGGGCTA ATCACCA CGAGGGCTA AGC-
56789
S....
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
TGTCT
160
|
0 12345678 9
L S........L
C CCCTCCCC A
C CCCCTCCC A
C CCCCTCCC A
C CTCTCCCC A
C CCCCTTCC A
C CCTCTCCC A
C CCTCTCCC A
C CCTCTCCC A
C CCTTTCCC A
C CCTTTCCC A
C TCCCTTCC A
C CCCCTCCC A
C CCCCTCCC A
C CCCTCCCC A
C CCCTCCCC A
C CCCCCCCC G
C CCTCTCCC A
C CCTCTCCC A
C CTTTCCCC A
C CCTCTCCC A
C CCCCTCCC A
C CCCTCCCC A
C CCCTCCCC A
C CCCTCCCC A
C CCTCTCCC A
C CCTCTCCC A
C CCTCTCCC A
C CCCTCCCC A
C CCTCCCCC A
C CCCTCCCC A
C CTCTCCCC A
C CCCTCCCC A
C CTCTCCCC A
C CCCTCCCC A
C CCTCTCCC A
C CCTCTCCC A
C CCTCTCCC A
C CCCCTCCC A
C CCCTCCCC A
C CCCCTCCC A
C CCCTTTCA A
C CTTTTTCA A
C CTTCCCCT A
C CTTTTTCC A
C CTTTTTCA G
C CTCTCTCA A
C CTTTCTCA A
C CTTTCTCA A
C CTTTTTCA A
C CTTTTTCC A
C CTCTTTCA A
C CTCTTTCC A
C CTCTCTCA A
C CTCTCTCG A
C CTTTTTCC A
C CTTTTTCA G
156
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
170
|
012
S..
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
GTC
180
|
3456789012345
L............
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATT
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATT
AATGAAATTGATT
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATCAAATTGATT
AATGAAATTGATC
AATGAAATTGATC
AGTGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAGTTGATT
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATT
AATGAAATTGATT
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATT
AATGAAATTGATC
AATGAAATTGATC
AGTGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
GATGAAATTGATC
AATGAAGTTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
AGTTAAATTGATC
AGTTAAATTGATC
AATGAAATTGATC
AATGAAATTGATC
190
|
678901 23456789
S..... L.......
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
CCCCCG TGCCGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCTCCG TGCAGAAG
CCCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
CCCCCG TGCAGAAA
TCTCCG TGCAGAAG
TTCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TACCCG TGCAGAAG
TCCCCG TGCAAAAG
TCCCCG TGCAGAAG
TCTCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
CCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
TCCCCG TGCAGAAG
200
|
012345
S.....
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGAGGA
CGGGGA
CGGGGA
CGGGGA
CGGAGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CCGGGA
CGGGGA
CGGGAA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGAGA
CGGGGA
CGGGGA
CGGGGA
CGGG-A
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGTA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
CGGGGA
210
|
6789012
L.......
TTATTAC
TAAATAC
TAAAGAC
TACTAAC
TAAGCAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAGAAC
TAAGAAC
TAAATAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TGAGAAC
TAACAAC
TAAAAAC
TAATAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAAAAAC
TAATAAC
TAAAAAC
TAGTACC
TAAAAAC
TAAAAAC
TAGCAAC
TGAAAAC
TAAAAAC
TAAAAAC
TAAAGAC
TAAAAAC
TAACAAC
TAATAAC
TAATACT
TACACTC
TACACAC
TACCAAC
TACTTAC
TTAATAC
TTAACAC
TTAACAC
TAAGACC
TAACCAC
TGCACAC
TACGTAC
TAGCACC
TGACCCC
TAGACCC
TATAATC
157
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
34567
S....
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
TTAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAC
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ACAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
ATAAG
220
|
890123456
L........
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGGGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
ACGAGAAGA
230
|
7890 12
S... L.
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT TT
CCCT AT
CCCT GT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT GT
CCCT GT
CCCT AT
CCCT AT
CCCT AT
CCCT AT
CCCT GT
CCCT GT
3456789
S......
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGTTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
GGAGCTT
240
250
|
|
012345678901234567
L.................
TAGACGTCAAGGCAGCTC
AAGACGTCAAGACAGCTC
AAGACGCCAGGCCTGCTC
TAGACGTCAGAGCAGCCC
AAGACGCCAGGGCAGCTC
AAAACGCCAGGACAGCTC
AAGACGCCAGGACAGCTC
TAGACGCCAGGACAGCCT
AAGACGCCAGAACAGCCC
AAGACGCCAGAACAGCCC
AAGACGCCAGGGCAGCTC
TAGGCGCCAGGGCAGCTC
TAGACGTCAGAGCAGCCC
TAGGCGTTAGAGCAGCTC
TAGACGTCAGAGCAGCTC
TAGACGTCGGAGCAGCTC
AAGGCGCCAGGACAGCTC
AAGACGCCAAGGCAGCTC
AAGACGTAAGAGCAGCTC
AAGGCGCCAGGACAGCTC
AAGACGCCAGAACAGCTC
AAGATACCAAGACAGCTC
TAGACGTCAGAGCAGCTC
TAGACGTCAGAGCAGCTC
AAGACGCCAGAACAGCCC
AAGACGCCAGAGCAGCCC
AAAACGCCAGAACAGCCC
TAGACGTCAGAGCAGCTC
AAGACACCAGAGTAGCCC
TAGACGTCAGAGCAGCCC
TAGACGTCAGAGCAGCTC
TAGACGTCAAAGCAGCTC
TAGACGTAAGAGCAGCTC
TAGACGTCAGAGCAGCCC
AAGGCGCCAGAGCAGCTC
AAGACGCCAGGACAGCTC
AAGGCGCCAGAACAGCTC
AAGACGCCAGAGCAGCTC
TAGACGTCAGAGTAGCTC
AAGGCGCCAGGGCAGCCC
AAGGTACAAAACTCAACC
TAGATGTTACAACAAGCA
TAGACA-CAAGAGAGACT
TAGACGCCAGGACAGACC
TAGACACCAGGACAGACC
TAGACA-CAAGACAGATT
TAGACAT-AAGATAGATT
TAGACA-CAAGACAGATT
TAGACACCAAGGTAGATC
TAGACGCCAGGGCAGACC
TAGACACCAAAGCAGACC
TAGACACCAAAGCAGACC
TAGACACCAAGGTAGATT
TAGACACCAAGGCAGACT
TAGATATTAAGATTGCCC
TAGACTTTAAGACTGACC
158
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
260
270
280
290
300
|
|
|
|
|
89012345678901234567890123456789012345678901234567
..................................................
ATGTAAA-GCACCCC-TAAAC--AAAGGAA---GAAACCAAATGAA-ACC
ACGTTAA-ACACCCC--AAAC--AAAGGGA---AAAACCAAGTGAA-TCC
ACGTTAA-ACACCCT-CTCAC--AAGAGAG--ATAAACCGAGTGAA-CCC
ATGTAAA-GCACCCC-TAAAC--AAAGGGA--TAAAACCAAATGAA-ATC
ACGTTAA-ACGCCCC-TAATA--AAGGAAT---AAAACCTAGTGAA-TCC
ATGTTAA-GCACCCC-TAAAT--AAAGGAG---TAAACCAAGCGAA-ACC
ACGTTAA-ACACCTC-CAAAT--AAAGGAA--ATAAACCAAGTGAA-TCC
ACGTTAA-ACACCCC-AAAAT--AAAGGAA--AAAA-CCAAGTAAA-TCC
ACGTCAA-GTACCTC-CTAAT--AAAGAAA--ATAAACCAAGTGGA-TCC
ACGTCAA-GTACCTC-TTAAT--AAAGAAA--ATAAACCAAGTGGA-TCC
ACGTTAA-ACACCCC-TAATA--AAGGAAT---TCAACCAAGCGAA-TCC
ACGTTAA-ACATCCT-GAATA--AAGGGAA----GAACCAAGTGAA-CCC
ATGTTAA-ACACCCC-CAAAC--AAGGGGA---AAAACCAAATGAA-GCC
ATGTTAA-ACACCCC-CAAAC--AAAGGGA---TAAACCAAATGAA-CCC
ATGTTAA-ACACCCCCTAAAC--AAAGGAA---ATAACCAAGTGAA-ACC
ATGTTAA-ACACCCC-TAAAC--AAAGGAA---AAAACCAAATGAA-CCC
ACGTTAA-ACACCCC-TTCAT--AAAGGAA---AAAACCAAGTGAA-TCC
ACGTTAA-ACACTCC-CAAAT--AAGGGGA---CAAACCAAGTGAACCTC
ATGTAAA-GCACCCC-TAAAC--AAAGGAA---AAAACCAAATGAAGTCC
ATGTTAA-ACACTCC-AAAAT--AAAGGAA--ATAAACTGATTGAAGCCC
ATGTTAA-ACACCCC-TAAAT--AAAGGAA---AAAACCAAATGAA-CCC
ATGTTAA-GCACCCC-AAAAT--AAAAGAA--TAAAACCAAATGAA-CTC
ATGTTAA-ACACCCC-TAGAC--AAAGGAA---AAAACCAAATGAA-GCC
ATGTTAA-GCACCCCCTGAAC--AAAGGAA---AAAACAAAATGGA-TCC
ACGTTAA-ACACCCC-GAAAT--AAAGGAA--AAAA-CCAAATGGA-CCC
ACGTTAA-ACACCCT-AAAAC--AAAAG-C--TAAAACAAAGTGGA-CCC
ACGTTAA-ACACCCC-AAAAT--AAAGGAA--AAAA-CCAAGTGGA-CCC
ATGTAAA-GCACCCC-TTAAC--AAAGGAA---AAAACCAAATGAA-ACC
ATGTTAA-ACACCCC-TAAAT--AAAGGGA--AAAAACCAAATGGA-CCC
ATGTAAA-GCACCCC-TTAAC--AAAGGAA---AAAACCAAATGAA-GCC
ATGTAAA-GCACCCC-TAAAC--AAAGGGA---AAAACCAAATGTA-ACC
ATGTTAA-ACACCCC-TAAAC--AAAGGAA---AAAACCAAATGAA-ATC
ATGTAAA-GCACCCC-TGAAC--AAAGGAA---AAAACCAAATGAA-ACC
ATGTTAA-ACATCCC-TAAACTTAAAGGAA---AAAACCAAATGAA-GCC
ACGTTAA-ACACCCT-CATAC--AAGAAGA--ATAAACTTAGTGAA-CCC
ACGTTAA-ACACTCC-TGGAC--AAAGGAA--ATAAACTGAGTGAAACCC
ACGTCAA-ACACCCC-CGCAT--AAAGGGA--ATAAACCAAGTGGA-CCC
ATGTTAA-ACACCCC-CAGAT--AAAGGGA--AAAAACCAAATGAA-CCC
ATGTAAA-GCACCCC-TAAAC--AAAGGGA---TAAACCAAATGAA-CCC
ACGTTAA-ACACCCC-TAGTT--AAAGGAA---TGAACCAAGTGAA-CCC
ACGTTAA-GCAACTC---AATAAAAAGCAA----AAACCTTGTGGA-TCA
TTATGTG-CTATTCC-CAAAC--AAAGGGG---ATAACCACTAGC--CCC
GCCTTCA-TTAACCC-AAAAT--AAAGGGC--CAAAGCTAAAGGTA-GCC
ATGTCAA-GCAACCC-CTGAC--AA-GGGT---CAAACCAAATGCA-CCC
ATGTCAA-ACAACCC-CGGAC--AACGGGC---TAAACCAAATGCA-CCC
ACCTCTT-CTAACCC-AATAC--AGAAGAC--GAAA-TTAGAGGTA-GAC
ACCTTAA-TCAACCC-AGAAC--AAAGGGC--GGAA-CCAGAGGTA-AAC
ATCTTAA-TCAACCC-AAAAT--AAAGGGC--GGAA-CTAGAGATA-AAC
ATGTTAA-GCACCCC-GAAAC--AAAGGGT---CAAACTAGATGAG-CCC
ACGTTAA-GCACCCC-TAAAT--ACAGGGC---TAAACCACATGAC-CCC
ATGTTAA-AAACCCT-TAAAC--AAAAGGC---CAAACCAAATGAA-CCC
ATGTTAA-ACACCCC-AAAAC--AAAGGAC---CAAACCAAATGAT-CCC
TATGTTTAACCTTCT-TCGAC--AAGAGAAG-AAAAACTAAATAAACCCC
TATGTTTAACCTTCT-CCAAT--ACGAGAAG-AAAAACTAAATAAGCTCC
CTCTT--AACATGCTCAAGAT--AAAGACAT-CT-AAATAAGGGA--CAC
CATTC----CCCAC-CTTGAT--AACCCCTGGCTGAAACATGGGA--TAC
159
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
310
|
8901234567
..........
TGCCCCAATG
TGCCCTAATG
TGCTCTAGTG
TGTCCTAATG
TGCTCTAATG
TGCCCTAGTG
TGCCCTAGTG
TGTTCTAGTG
TGCCCTAATG
TGCCCTAATG
TGCTCTAATG
TGCCCCAGTG
TGCCCTAATG
TGCTTTCATG
TGTCCTAATG
CGCCCTAATG
TGCCCTAGTG
TGCCCTAATG
TGCCCTAATG
TGTCCTAGTG
TGCTCTAATG
TGTCTTAATG
TGCCCTAATG
TGCCCTAGTG
TGTTTTAGTG
CGCTCTAGTG
TGCTCTAGTG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCTCCAGTG
TGCCCTAATG
TGCTCTAGTG
TGTCCTAATG
TGCCCTAATG
TGCCCCAGTG
TGAGATTTTA
TGCCCAAACA
CTCTCCAGCG
TGTCCCAATG
TGCCCTAATG
CTTTCCAGCG
CTTTCCAACG
CTTTCCAGCG
TGCCCTCATG
TGCCCCTATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
TGCCCTAATG
AATCCGACTA
CGTCTGACTA
320
|
89012
S....
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TTTTT
TCTTT
TCTTT
TCTTT
TTTTT
TCTTT
TTTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TTTTT
TCTTT
TTTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TTTTT
CCTTC
TCTTC
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTC
TCTTC
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
TCTTT
34567
S....
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
GGTTG
330
|
890 12345
L.. S....
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGGCC
GGG CGACC
GGG CGACGGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG TGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
GGG CGACC
67
L.
GC
GC
GC
GC
AC
GC
GC
GC
GC
GC
AC
GC
GT
GC
GC
GC
GC
GC
GC
AC
GC
GC
GC
GC
GC
GC
GC
GC
GC
GC
GC
GC
GC
GC
AC
AC
GC
GA
GC
GC
AC
AT
GT
AC
GC
GT
GT
GT
AT
AT
GC
GC
GT
GT
GC
GT
340
|
89012
S....
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGGGA
GGAGG
GGGAA
GGGGT
GGGGA
GGGGA
GGGGT
GGGGT
GGGGT
GGGGT
GGGGT
GGGGA
GGGGA
GGGGC
GGGGC
GGGGA
GGGGA
350
I
34567890
L.......
AATACAAA
AAAATAAA
AAAATTTA
AATACAAA
ATCATAAA
AAAATAAA
AAAATAAA
AAAACTTA
AAAATGAA
AAAATGAA
AGCATAAA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AAAACAAA
AAAATTAA
AATACAAA
AAAATTTA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AAAACTTA
AAAACTTA
AAAACTTA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AATACAAA
AGAATTCA
AAAACTTA
AAAATTAA
AATACAAA
AATACAAA
AATACAAA
AAAGAAAA
ACAAGAAA
AAAACAAA
ACTACAAA
AATACGAA
AAGACAAA
AAAACAAA
AAAACAAA
AAAAT-AA
AAAACAAA
AACACAAA
AACACAAA
A-TACAAA
A-CACAAA
TTAAAAGA
TTAACAGA
160
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
1
.
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
C
G
A
A
A
A
A
A
C
A
A
A
A
A
A
23456
S....
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCTCC
TCCCA
CTCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
CCCCC
360
|
78901 23
L.... S.
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ACGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ACGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ACGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GA
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ACGT GG
-ATGA GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ATGT GG
-ACGT GG
-ATGT GG
-AGGT GG
-CGTC TC
-ACGT GG
-ACGT GG
-ACGT GG
-ATGA GG
-ATGT GG
-ATGA GG
CACGC GG
-ACGT GG
-ACGT GG
-ACGT GG
-ACGT GG
-ATGT GG
-ACGT GG
-ATGT GG
370
380
390
|
|
|
456789012345678901234567890123456
L................................
ATTGGGAACA----CAC--------------AT
AGCAGGAACA----CTA-------------TGT
AACGGGAACA----CTA-------------TGT
AGTAGGAACA----CAT--------------GT
AATGGGAGCA----CCA-------------CAC
AATAAGAATA----CCA-------------TAT
AATAGAAATA----CTA-------------TAT
AGTAAGAGTA----CTA-------------TAC
AATGGGAGTA----CTA-------------TGC
AATGGGAGTA----CTA-------------TGC
AATAGGAACA---CTTC--------------AC
AACAGGAACA----CTA-------------AGT
AGTAGGAACA----CGT--------------TT
AATGGGCGCG----CAC--------------AC
AGCAGGAATA----CGT--------------AT
AGTAGGAACA----CAT--------------AT
AGTAGGAACA----CTA-------------TGT
AATAGGAGTA----CTA-------------CGC
ACTAGGAATA----CAC--------------AT
AATAGGAATA----CTA-------------TTT
AATAGGAATA----CTA-------------TGT
AGTAGGAGTA----CAA-------------TAT
AGTAGGAATA----CGT--------------AT
AGTGGGAATA----CAC--------------AT
AATAGGAGTA----CTA-------------TAT
AATAGGAGTA----CTA-------------TAC
AATAGGAGTA----CTA-------------TAC
AGTAGGAATA----CAC--------------AT
AAAAGGAATA----CTA-------------TGT
AGTAGGAATA----CAT--------------AT
AGTAAGAACA----CAG--------------AT
AGTAGGAATA----CAT--------------AT
AGTAGGAACA----CAT--------------AT
AATAGGAACA----CAT--------------GT
AACAGGGACA----CTA-------------TGT
AATAGGAATA----CTA-------------TTT
AACAGGAACA----CTA-------------TAT
AATAGGAACA----CTA-------------TGT
AATAGGAGCA----CAA--------------GC
ACCAGGAACA----CTA-----------CATGT
ACTGGGAAAA---------------------CC
TGTATTGGAATCCTTATTTAAACCCAAACAAAC
AATAGGAGCA----TCC--------------TC
AATGGGAACA----CCC---------------AATGGGGACA----CCC---------------C
AAAAGGAGCA----CCC---------------C
AATAGGAGCA----CC---------------TC
AATAGGAGCA----CCC---------------C
AAAGGGAGAA----CCT-------------TCC
AACGGGAGTA----CTT-------------TCC
AACGAGGA-A---CACC-------------TCC
AATGAG-A-A---CACC-------------TCC
AGCAGGAGCA---CATC-------------TAC
AGCAGGAGCA---CAAT-------------TAC
ACCGGGGATA-AAAGCTTACCCC-------TAT
ACCGAG-TG----CCTA-------------CGC
161
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
400
410
420
430
|
|
|
|
7890123456789012345678901234567890123456
........................................
TCCCAGACCCAAGAGCTCCCGCTCTAATGAA-CAGAAATT
TCCTAAATCCAAGAGCTCCCGCTCTAATAAG-CAGAACTT
TCCTTAATCCGAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTAAACCCAAGAGCTCCCGCTCTAATACA-CAGAAATT
TCCTAAACCCAAGAGCTTCCGCTCTAATGAA-CAGAACTT
TCTTAGATCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TTCTAAATCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCTTAGATCCGAGAGCTCCCGCTCTAATAAA-CAGAATTT
TCCTAGTTCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTAACTCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTAAACCCAAGAGCCTCCGCTCTAACAAA-CAGAACTT
TCCCAAACTCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTAAACTCAAGAGCTCCCGCTCTAACGAA-CAGAAATT
TCCTAAACCCAAGAGCTCCCGCTCTAATAAA-CAGAAATT
TCCTAGATCCAAGAGCTCCCGCTCTAATAAA-CAGAAATT
TCCTAAACCCAAGAGCTCCCGCTCTAATGAA-CAGAAATT
TCCTAGATCTAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTACATCCAAGAGCTCCCGCTCTAATAAA-CAGAATTT
TCCCATACCCAAGAGTTCCCGCTCTAATAAA-CAGAGCTT
TCCTAAATCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCCAAATTCAAGAGTTCCCACTCTAATAAA-CAGAATTT
TCCTAAACCCAAGAGCTACCGCTCTAATGAA-CAGAAATT
TCTTAAATCCAAGAGCTCCCGCTCTAATAAA-CAGAATTT
TCTCAAATCCAAGAGCTCCCGCTCTAATAAA-CAGAATTT
TCTTAAATCCAAGAGCTCCCGCTCTAATAAA-CAGAATTT
TCCTAGACCCAAGAGCTCCCGCTCTAATAAA-CAGAAATT
TCCCAAATCCAAGAGCTCCCGCTCTAGCAAA-CAGAACTT
TCTTAAACCCAAGAGCTCCCGCTCTAATGAA-CAGAAATT
TCCTAAACTCAAGAGCTCCCGCTCTAATGAA-CAGAAATT
TCCTAAATCCAAGAGCTCCCGCTCTAATGAA-CAGAAATT
TCCTCAATTCCAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTAGACCTAAGAGCTCCCGCTCTAATGAA-CAGAACTT
TCCTTAATCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTACATCCAAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCCAAACCCGAGAGCTACCGCTCTAATAAA-CAGAACTT
TCCTAAAACCAAGAGAGACATCTCTAAGCAC--AGAACAT
TTCTTCAAACGAGAGCCACCACTCTAATTCAACAGAATTT
CTTCA--ACCAAGAGCTCCCGCTCTAAGGAA-CAGAACTT
TCCTACAACTGAGAGCTTCCGCTCTAGTGAA-CAGAAATT
CTCTACAACCAAGAGCTTCCGCTCTAATTAA-CAGAAATT
TCCTTCAACCCAGAGTTCCCACTCTAAGGAA-CAGAATTT
TCCTTCAACCTAGAGCTCCCGCTCTAAGGAA-CAGAATTT
TCCTTCAACCTAGAGCTCCCGCTCTAAGGAA-CAGAAATT
TCCTACAACCCAGAGCTCCCGCTCTAATAAA-CAGAACTT
TCCTCAAACCCAGAGCCTCCGCTCTAATTAG-CAGAACCT
TCTCACAGCCAAGAGCTCCCGCTCTAGTAAA-CAGAAATT
TCTCACAACCAAGAGCTCCCGCTCTACTGAA-CAGAAATT
TCCTACAGCTAAGAGCTACCGCTCTAATAAG-CAGAATTT
TCCCACAGCTAAGAGCTACCGCTCTAATAAG-CAGAATTT
CCCTACAATCAAGAGTTACCACTCTAAATAA-CAGAATTT
ACTCAAAACCAAGAGTTACCACTCTAGTTAA-CAGAACTT
78
S.
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CC
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
TT
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT
440
|
901234
L.....
GACCAA
GACCAA
GACCAA
GACCAA
GGCCAT
GACCAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCAT
GACCAA
GACCAA
GACCAA
GACCAG
GACCAA
GACTAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCTA
GACCAA
GACCAA
GACCAA
GACCAA
GACCAG
GACCAA
GACCAA
GACGAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCTA
GACCAA
GACCAA
GACCAA
GACCAG
GACCAA
GGCCAC
GACCAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCAA
GACCTA
GACCTA
GACCAA
GACCAA
GACCAA
GACCAA
GACCAA
GACTTA
162
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
450
|
567890123
.........
CAA--GATC
CAA--GATC
CAA--GATC
TAA--GATC
ATTA-GATC
CAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
TTTA-GATC
CTTA-GATC
TAA--GATC
CAA--GATC
CAA--GATC
TAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
AATG-GATC
CTA--GATC
TAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
TTG--GATC
TAA--GATC
TAA--GATC
CAA--GATC
CAA--GATC
CAA--GATC
CGA--GATC
ATT--GGAT
CAA--GATC
CTA--GATC
CAA--GATC
CTAA-GATC
ATAT-GATC
CGAT-GCAA
TCAGTGATC
CAA--GATC
TAA--GATC
TTAATGATC
TTAATGATC
TTAATGATC
AA---GATC
TA---GACC
CTA--GACC
TCA--GATC
CAAT-GATC
TAAT-GATC
-AAATGATC
CAAATGATC
4567
S...
CGGC
CGGC
CGCA
CGGC
CGGT
CGGC
CGGC
CGGC
CGGC
CGCA
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGCA
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CGGC
CCGG
CGGC
CGCA
CGGC
GGCA
CGGC
AGCC
CGGC
CGGC
CGCG
CGGC
CGGC
CG-G
--GC
CGGT
CGGC
CGGC
CGAC
CGAC
CGAC
CGAC
460
|
890123456
L........
-ACAT----AAT------AC-----ACAT----AAA-----AAA-----AAT-----AGT-----AAT-----ATG-----TAC-----TAT-----ACAT----ACAT----ACAT----ATAT----AAT-----AAT-----ACAT----AAT-----AAT-----AAT-----ACAT----ATAT----AAT-----AAT------AT-----ATAT----AAT-----ACAC----ATAT----ACAT----ACAC----ACAT----AAA-----CAAT----AAC------AT-----ACCT----GTG----TAACACATA
-GAT-----ATC-----AAC------AT-----ACC-----AAT-----CATT----AAA-----AAA-----AAG-----ATA-----AAC-----AAC-----AAA-----AAA-----
470
480
|
|
7890 123456789012
S... L...........
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
ACCG ATC-AACGAACC
GCCG ATC-AACGGACC
GCG- ATCCAACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
CCG- ATC-AACGGACC
ACCG ATC-AACGGACC
GCGA ATC-AACGGAAC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGAACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGAACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
GCGA ATC-AACGGACC
GCCG ATC-AACGGACC
GCGA ATCCAACGGACC
GCCG ATC-AACGGACC
GCCG ATC-AACGGACC
CCG- ATC-AACGGAAC
GCCG ATC-AACGAACC
---- -T--AACGAACC
GCCG ATC-AACGAACC
GCGA ATT-AACGGACC
GCCG ATC-AACGGACC
GCCG ATT-AACGAACT
GCCG ATC-AACGGACC
GCCG ATC-AACGAACC
GCCG ATC-AACGGACC
ACCG ATC-AACGAACC
ACCG ATC-AACGGACC
AGCG ATC-AACGGACC
GTCG ATC-AACGAACC
GTCG ATC-AACGAACC
GTCG ATC-AACGGACC
GTCG ATC-AACGAACC
34567 8
S.....L
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
AAGTT A
AAGTT A
GAGTT A
GAGTT A
GAGTT A
AAGTT A
GAGTT A
GAGTT A
AAGTT A
AAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
GAGTT A
163
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
490
|
90 12
S. L.
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CT
CC CC
CC CT
CC CT
CC CT
CC CT
CC CT
CC CC
CC CC
CC CT
CC CT
CC CT
CC CT
CC CC
CC CC
3456
S...
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
500
|
7890123
L......
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
ATAACAG
45
S.
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
6
L
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
510
|
78901234
S.......
AATCCCCT
AATCCCCT
AATCCTCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCTCCT
AATTCCCT
AATCCCCT
AATCCCCT
AATCCCCT
AGTCCCCT
AATCCTCT
AATCCTCT
AATCCCCT
AATCCCCT
AATCTCCT
AATCTCCT
AATCTCCT
AATCCTCT
5678
L...
TAAA
TAAA
TAAA
TAAA
TAAA
TAGA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
TAAA
CCCA
TTCA
TCTA
TTTA
TTTA
TTAA
TTTA
TTTA
TTTA
TTTA
TTTA
TTTA
TTTA
TCTA
TCAA
TTGA
520
|
90 1234567
S. L.......
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GTTCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCTCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GTCCCTA
GA GCCCTTA
GA GCCCTTA
GA GCCCCTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GTCCCTA
GA GCCCTTA
GA GCCCTTA
GA GCCCTTA
GA GTCCTTA
GA GTCCATA
GA GCCCATA
GA GCCCTTA
GA GCCCATA
GA GCCCATA
GA GCCCATA
GA GCCCATA
GA GCCCATA
GA GCCCATA
GA GCCCATA
GA GGCCATA
GA GGCCATA
GA GTCCATA
GA GTCCCTA
GA GTTCTTA
GA GTTCATA
164
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
89
S.
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
530
|
0123
L...
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
AACA
AACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACG
GATA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
540
|
45678901 23
S....... L.
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGAGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGAGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGCT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGAGGGTT TA
AGAGGGTT TA
AGGGGGTT TA
AGGGGGTT TA
AGGAGGTT TA
AGGAGGTT TA
AGGGGGTT TA
AGGGGGTT TA
45
S.
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
CG
550
|
678901234
L........
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACTTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
ACCTCGATG
560
|
567890123456
S...........
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TCGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGG?TCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
TTGGATCAGGAC
7
L
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
89
S.
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TT
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
TC
165
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
570
|
01 2345678
L. S......
CT AGTGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AAAGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CC AATGGTG
CT AATGGTG
-T AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT AATGGTG
CT ATTGGCG
CC AATGGCG
580
|
9012
L...
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAAC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
TAGA
CAGC
3456789
S......
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
-GCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
AGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
CGCTATT
AGCTATT
AACTATT
590
|
012
L..
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
AAG
34
S.
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GG
GGG
GG
GG
GG
GG
600
|
567890 12345
S..... L....
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
GTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGA- -GTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTTC
TTCGTT TGTGC
TTCGTT TGTTC
TTCGTT TGTTC
GTCGTT TGTTC
TTCGTT TGTTC
610
I
67890
S....
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
AACGA
166
Argyrops spinifer
Boopsoidea inornata
Cheimerius nufar
Boops boops
Crenidens crenidens
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Dentex tumifrons
Petrus rupestris
Pagellus bogaraveo
Pagellus bellottii
Acanthopagrus berda
Calamus nodosus
Cymatoceps nasutus
Diplodus cervinus
Diplodus holbrooki
Evynnis japonica
Lagodon rhomboides
Pagrus auratus
Pagrus auriga
Porcostoma dentata
Pagrus pagrus
Sparidentex hasta
Sparus auratus
Stenotomus chrysops
Spicara alta
Spicara maena
Cyprinus carpioGB
Lateolabrax latus
Morone americanus
Morone chrysopsGB
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
123456
L.....
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TGAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTGA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTA???
TTAA-A
TTAA-A
TTAATA
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
TTAA-A
620
|
78901
S....
ATCGT
GACCT
GTCAT
GTCCC
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
?????
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
GTCCT
167
TABLE 14. CONSTANT, UNINFORMATIVE AND INFORMATIVE CHARACTERS
- STEMS AND LOOPS
Constant
Uninformative Informative
Total
no.(% of row
total)
no.(% of row
total)
no.(% of row
total)
Characters
All
251(40%)
137(22%)
233(38%)
621
Stems
125(52%)
70(29%)
45(19%)
240
Loops
126(33%)
67(18%)
188(49%)
381
TABLE 15. NON-CONSERVED AREAS IN MULTIPLE ALIGNMENT
Base Positions
Number Bases
Longest Run of Gaps
Structural
Element
07-21
15
3
Loop
66-75
10
4
Stem
240-317
78
4
Loop
364-430
67
21
Loop
439-453
15
3
Loop
458-466
9
5
Loop
168
TABLE 16. MEAN PAIRWISE VALUES OF PERCENT SEQUENCE DIVERGENCE
BASED ON UNCORRECTED “P” GENETIC DISTANCE
All Taxa
Mean % sequence divergence
9.70
Standard Deviation
0.048
Number of Pairwise
Comparisons
1540
Ingroup taxa (including Centracanthidae)
6.21
Standard Deviation
0.019
Number of Pairwise
Comparisons
780
Outgroup taxa
14.49
Standard Deviation
0.052
Number of Pairwise
Comparisons
120
169
TABLE 17. CONTRIBUTION VALUES FOR LOOPS AND STEMS CL AND CS
GIVEN AS A PERCENTAGE. AVERAGE CONTRIBUTION OF LOOPS AND
STEMS TO THE OVERALL SEQUENCE DIVERGENCE BASED ON PAIRWISE
COMPARISONS FOR ALL, INGROUP AND OUTGROUP TAXA.
Loops
All Taxa
Ingroup Taxa
Outgroup Taxa
81.2%
80.6%
82.6%
All Taxa
Ingroup Taxa
Outgroup Taxa
18.8%
19.4%
17.4%
Stems
170
TABLE 18. MATRIX OF FAO AREAS AND CHARACTERS. FAO AREAS WERE TREATED AS INDEPENDENT DATA
AND TAXA PLUS ANCESTRAL NODES WERE TREATED AS DEPENDENT DATA DURING PARSIMONY ANALYSIS.
CHARACTERS ARE DEFINED BELOW (AN=ANCESTRAL NODE).
CHARACTERS
AREAS
.........1.........2.........3.........4.........5.........6.........7......
1234567890123456789012345678901234567890123456789012345678901234567890123456
0000100000000100001101101100000010011110000001111111111100111111111111111111
Atlantic, Northeast
0000100000000100001101001100000010011010000001111111111100111111111111111111
Atlantic, Northwest
0100001000000010010000000100000000000001000011110010101111100111111111111110
Atlantic, Southeast
0011110111100100101110101011111111000110011101111111111100111011111111111111
Atlantic, Southwest
0100000000000000000000000100000000000000000000000000000100100111111111111110
0100001000001010010000000100000000000001000011110010101111100111111111111110
Indian Ocean, Eastern
1010000000010000000000010000000000100000110000000010101100100111111111111110
Indian Ocean, Western
1011010111100000101010000011111111100000111100001111101100011011111111111111
Mediterranean/Black Sea
0000100000000100001101101100000010011010000001111111111100111111111111111111
Pacific, Northwest
1010000000010001000000010000000000000000110000000010101100100111111111111110
Pacific, Southwest
0000000000000000000000010000000000000000000000000000000000000111111111111110
1010000000010000000000010000000000000000110000000010101100100111111111111110
1 Acanthopagrus berda; 2 Archosargus probatocephalus; 3 Argyrops spinifer; 4 Argyrozona argyrozona; 5 Boops boops; 6 Boopsoidea inornata; 7 Calamus nodosus;
8 Cheimerius nufar; 9 Chrysoblephus cristiceps; 10 Crenidens crenidens; 11 Cymatoceps nasutus; 12 Dentex tumifrons; 13 Diplodus bermudensis; 14 Diplodus
cervinus; 15 Diplodus holbrooki; 16 Evynnis japonica; 17 Gymnocrotaphus curvidens; 18 Lagodon rhomboides; 19 Lithognathus mormyrus; 20 Oblada melanura; 21
Pachymetopon aeneum; 22 Pagellus bogaraveo; 23 Pagellus bellottii; 24 Pagrus auratus; 25 Pagrus auriga; 26 Pagrus pagrus; 27 Petrus rupestris; 28 Polyamblyodon
germanum; 29 Polysteganus praeorbitalis; 30 Porcostoma dentata; 31 Pterogymnus laniarius; 32 Rhabdosargus thorpei; 33 Sarpa salpa; 34 Sparodon durbanensis; 35
Sparidentex hasta; 36 Sparus auratus; 37 Spicara maena; 38 Spicara alta; 39 Spondyliosoma cantharus; 40 Stenotomus chrysops; 41 AN 57; 42 AN 58; 43 AN 59; 44
AN 60; 45 AN 61; 46 AN 62; 47 AN 63; 48 AN 64; 49 AN 65; 50 AN 66; 51 AN 67; 52 AN 68; 53 AN 69; 54 AN 70; 55 AN 71; 56 AN 72; 57 AN 73; 58 AN 74; 59
AN 75; 60 AN 76; 61 AN 77; 62 AN 78; 63 AN 79; 64 AN 80; 65 AN 81; 66 AN 82; 67 AN 83; 68 AN 84; 69 AN 85; 70 AN 86; 71 AN 87; 72 AN 88; 73 AN 89; 74
AN 90; 75 AN 106; 76 AN 107
171
Figure 22- Total number of substitutions plotted as a functions of uncorrected sequence
divergence. Transitions - 2nd Order Polynomial y = -631.81x2 + 374.19x + 1.9608, R2 =
0.899 and Transversions - 2nd Order Polynomial y = 638.15x2 + 138x - 1.8914,
R2 = 0.9372.
Key:
Ts = Transitions all characters
Tv = Transversions all characters
Poly. (Ts) = polynomial line fitted to transitions from all characters
Poly. (Tv) = polynomial line fitted to transversions from all characters
90
80
70
Substitutions
60
50
40
30
20
10
0
0
5
10
15
20
Ts
Tv
Poly. (Ts)
Poly. (Tv)
25
30
173
Figure 23- Total number of stem substitutions plotted as a functions of uncorrected
sequence divergence. Transitions - 2nd Order Polynomial y = 68.676x2 + 53.276x +
0.9629; R2 = 0.7379 and Transversions - 2nd Order Polynomial y = 464.67x2 - 59.195x
+ 2.9619 R2 = 0.6269.
Key:
Ts Stems = Transitions stem characters
Tv Stems = Transversions stem characters
Poly. (Ts Stems) = polynomial line fitted to transitions from stem characters
Poly. (Tv Stems) = polynomial line fitted to transversions from stem characters
25
Substitutions
20
15
10
5
0
0
5
10
15
20
Ts Stems
Tv Stems
Poly. (Ts Stems)
Poly. (Tv Stems)
25
30
175
Figure 24- Total number of loop substitutions plotted as a functions of uncorrected
sequence divergence. Transitions - 2nd Order Polynomial y = -700.48x2 + 320.91x +
0.9979 R2 = 0.8066 and Transversions - 2nd Order Polynomial y = 173.48x2 + 197.2x 4.8533 R2 = 0.9235.
Key:
Ts Loops = Transitions loop characters
Tv Loops = Transversions loop characters
Poly. (Ts Loops) = polynomial line fitted to transitions from loop characters
Poly. (Tv Loops) = polynomial line fitted to transversions from loop characters
70
60
Substitutions
50
40
30
20
10
0
0
5
10
15
20
25
Ts Loops
Tv Loops
Poly. (Ts Loops)
Poly. (Tv Loops)
30
177
Figure 25- Loop transitions were separated into functional nucleic acid classes as either
pyrimidine substitutions (CøT) or purine substitutions (AøG) and plotted as a function
of uncorrected sequence divergence. CT- 2nd Order Polynomial y = -375x2 + 177.44x +
0.016 R2 = 0.6942 and AG y = -325.48x2 + 143.47x + 0.9819 R2 = 0.673.
Key:
AG = Purine transitions loop characters
CT = Pyrimidine transitions loop characters
Poly. (AG) = polynomial line fitted to purine transitions from loop characters
Poly. (CT) = polynomial line fitted to pyrimidine transitions from loop characters
40
35
30
Substitutions
25
20
15
10
5
0
0
5
10
15
20
AG
CT
Poly. (AG)
Poly. (CT)
25
30
179
Figure 26- Non conserved loop and conserved loop transitions plotted as a function of
uncorrected sequence divergence. Conserved loop 2nd Order Polynomial y = -21.75x2 +
153.82x - 0.1936 R2 = 0.855 and Non-Conserved loop 2nd Order Polynomial
y =-610.06x2 + 220.37x + 2.1544 R2 = 0.5817.
Key:
Ts Conserved Loops = Transitions conserved loop characters
Ts Non-Conserved Loops = Transitions non-conserved loop characters
Poly. (Ts Loops) = polynomial line fitted to transitions from conserved loop characters
Poly. (Tv Loops) = polynomial line fitted to transitions from non-conserved loop
characters
50
45
40
Transitions
35
30
25
20
15
10
5
0
0
5
10
15
20
25
%Sequence Divergence (Uncorrected)
Ts Conserved Loops
Ts Non-conserved Loops
Poly. (Ts Con Loops)
Poly. (Ts Non-con Loops)
30
181
Figure 27. Adjusted loop and stem distances plotted as a function of uncorrected
sequence divergence. Loop or stem distances were adjusted by multiplying each
respective pairwse distance by the total number of loop or stem characters and then
dividing by the total number of characters.
Sequence Divergence of Loops and Stems
0.25
0.2
0.15
0.1
0.05
0
0
5
10
15
20
Loop Divergence
Stem Divergence
25
30
183
Figure 28. Mean values and ranges (one standard deviation) of percent base composition
for all, stem, loop, and non-conserved loop characters of the 16S mtDNA fragment.
0.5
16S all
16S loops
16S stems
16S non-conserved loops
0.45
0.4
0.35
Percentage
0.3
0.25
0.2
0.15
0.1
0.05
0
A
G
C
T
185
Figure 29. A strict consensus of 8 equally parsimonious trees from the analysis of all
columns of data from the 16S sequence data. Subfamilies are labeled as follows
(BO=Boopsinae, DE=Denticinae,
DI=Diplodinae, PA=Pagrinae, PE=Pagellinae,
and SP=Sparinae).
“N” = Node
D
Stems= Partitioned Decay from Stems at “N”
Loops= Partitioned Decay from Loops at “N”
B
5.1
22(16.9
)
5.3
27(21.7
)
78
( 3.0 )
100
88
5 2.0
6( 0.1
5.9 )
5(-0.5
5.5 )
-2.5
16(18.5
)
( 0.0 )
<
91
( )
2 0.0
2.0
0.2
12(11.8
)
( )
92
100
5 5.0
2(-1.0
3.0 )
94
2(-0.7
2.7 )
78
63
99
62
2-0.5
2.5
81
2(1.0
1.0 )
Group II
5( 4.2
0.8 )
<
2(0.0
2.0 )
<
9( 2.5
6.5 )
2(0.9
1.1 )
99
<
3.7 )
11(7.3
0.1
14(13.9
)
5( 2.7
2.3 )
98
100
66
<
2( 1.7
0.3 )
89
0.9
5(4.1
)
< 56
Group I
5( 2.5
2.5 )
100
Cyprinus carpio
Lethrinus ornatus
Scolopsis ciliatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
BO Boops boops
BO Sarpa salpa
DE Dentex tumifrons
Spicara alta
DE Petrus rupestris
DE Cheimerius nufar
PA Pagrus auriga
PA Evynnis japonica
PA Pagrus auratus
PA Pagrus pagrus
SP Calamus nodosus
Spicara maena
SP Sparadon durbanensis
SP Sparus auratus
BO Oblada melanura
187
Figure 30. A strict consensus of 3952 equally parsimonious trees from a weighted
analysis of the 16S sequence data. All loop transitional substitutions were given a weight
of 0. Subfamilies are labeled as follows
(BO=Boopsinae, DE=Denticinae, DI=Diplodinae,
PA=Pagrinae, PE=Pagellinae, and SP=Sparinae).
“N” = Node
D
B
)
8.2)
21(12.8
13( 5.4
7.6 )
6 ( 0.3
5.7 )
5 ( 2.0
3.0 )
3 ( 0.1
2.9 )
58
10( 1.3
8.7 )
82
96
82
98
0.4)
4 (- 4.4
88
2 ( 0.1
1.9 )
0.5
3( 2.5
)
9 ( 2.0
7.0 )
100
75
85
81
6 ( 1.1
4.9 )
90
57
52
4( 0.3
3.7 )
Group II
5 ( 4.4
0.6 )
100
Group I
3(
3.1
-0.1
Cyprinus carpio
Lethrinus ornatus
Scolopsis ciliatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lateolabrax latus
Morone chrysops
Morone americanus
Morone saxatilis
Spicara maena
BO Boops boops
BO Sarpa salpa
SP Sparus auratus
BO Oblada melanura
SP Calamus nodosus
Spicara alta
DE Cheimerius nufar
DE Dentex tumifrons
PA Pagrus auriga
PA Pagrus pagrus
PA Evynnis japonica
PA Pagrus auratus
DE Petrus rupestris
79
62
189
Figure 31. A strict consensus of 9828 equally parsimonious trees from a weighted
analysis of the 16S sequence data. Non-conservative loop transitional substitutions were
given a weight of 0. All other characters were assigned a weight of 1. Subfamilies are
labeled as follows (BO=Boopsinae,
DE=Denticinae, DI=Diplodinae, PA=Pagrinae,
“N” = Node
D
B
3
23
6
100
100
87
93
5
5
11
99
5
83
2
6
8
3
7
71
75
82
93
63
3
54
2
6
2
95
65
Group II
5
92
80
Group I
13
Cyprinus carpio
Lethrinus ornatus
Scolopsis ciliatus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lateolabrax latus
Morone chrysops
Morone americanus
Morone saxatilis
SP Calamus nodosus
BO Oblada melanura
SP Sparus auratus
BO Boops boops
BO Sarpa salpa
Spicara maena
DE Dentex tumifrons
DE Petrus rupestris
Spicara alta
DE Cheimerius nufar
PA Pagrus auriga
PA Pagrus pagrus
PA Evynnis japonica
PA Pagrus auratus
77
4
2
<
191
FIGURE 32. The clade containing Sparidae was copied from the unweighted 16S tree
(Figure 29.) and ancestral nodes were numbered so that they could be assigned FAO areas
during analysis of vicariance biogeography.
36
33
34
32
31
37
30
29
28
22
21
27
35
26
25
24
23
19
18
20
14
17
12
16
2
1
13
4
3
11
10
9
8
7
6
5
BO Boops boops
BO Sarpa salpa
DE Dentex tumifrons
Spicara alta
DE Petrus rupestris
DE Cheimerius nufar
PA Pagrus auriga
PA Evynnis japonica
PA Pagrus auratus
PA Pagrus pagrus
SP Calamus nodosus
Spicara maena
SP Sparus auratus
BO Oblada melanura
193
FIGURE 33. Clade of 16S biogeographic relationships overlaid on map of the world.
A single most parsimonious tree is overlaid on a map of the world. No distances are
implied by branch lengths or by inter-nodal spaces.
Mediterranean and Black Sea
Atlantic Southwest
Pacific Southwest
Pacific Northwest
Atlantic Southeast
Atlantic Northeast
Atlantic Northwest
CHAPTER THREE. COMBINED ANALYSIS OF
CYTOCHROME B AND 16S MITOCHONDRIAL SEQUENCES
196
INTRODUCTION
This study produced two molecular data sets that were used for phylogenetic
analysis of the Sparidae. Each data set was independently analyzed in the previous two
chapters. In this chapter, cytochrome b and 16S sequences were combined for an
unpartitioned analyses of sparid evolution.
Two competing methods of phylogenetic inference, taxonomic congruence and
character congruence, have been a recent area of controversy to systematists (Mickevich
and Farris, 1981; Miyamoto, 1985; Kluge, 1989; Barrett et al., 1991; Chippindale and
Wiens, 1994; Bull et al., 1993; Eernisse and Kluge, 1993; de Queiroz,1993; Kluge and
Wolf, 1993; Farris et al., 1994; Miyamoto and Fitch, 1995; Wheeler, 1995; Cunningham,
1997). Taxonomic congruence derives a consensus phylogeny from data sets that are
analyzed separately. Character congruence (also know as “total evidence”) uses all
available data, unpartitioned, to produce a phylogenetic hypothesis. Intermediate to both
methods is that of prior agreement. Prior agreement involves making a priori
determination of the appropriateness of combining data in an analysis. (Bull et al., 1993;
Huelsenbeck et al., 1994; Huelsenbeck, et al., 1996; Siddall, 1997). If data are found to
be significantly incongruent, then they may not be combined. However, if deemed
congruent, the data can be combined without further evidence.
Under taxonomic congruence, each individual dat set is assumed to have
information that might otherwise be lost if data were combined prior to analysis. Each
data partition is presumed to be an independent test of the phylogenetic hypothesis, and
the congruence of trees from these independent data is thought to provide a better
197
estimate of the “true phylogeny”(Miyamoto and Fitch, 1995). The underlying assumption
of taxonomic congruence is that data have independent partitions that are related to
differences in evolutionary processes. Bull et al. (1993) called these “process partitions”
and defined them as sets of data that evolve under different sets of rules. Miyamoto and
Fitch (1995) proposed that characters sets may be considered independent process
partitions when, “...genes are not genetically linked, ...the products of their genes do not
interact with each other, ...the genes do not specify the same function, ...the gene products
are not components of a common pathway (e.g., electron transport system), ...or the gene
products do not regulate the expression of loci in other partitions.” Under taxonomic
congruence, independent process partitions would be analyzed separately, and would not
be combined because their independence improves the relevance of consensus of
phylogenies.
Opponents of taxonomic congruence suggest that phylogenies derived from
partitioned data are more susceptible to sampling error, because characters are drawn
from original trees which represents a smaller sample than if all characters were
combined (Huelsenbeck et al., 1996). Additionally, taxonomic congruence ignores the
possibility that combined data might reveal relationships not resolved by individual
analysis (Kluge, 1989, Eernisse and Kluge, 1993, Kluge and Wolf, 1993, Huelsenbeck et
al., 1996). Kluge and Wolf (1993) criticized taxonomic congruence, because the
congruence tree is considered to be “sub-optimal” to a tree based on all the data, and
Barrett et al., (1991) found that taxonomic congruence can “positively contradict” the
most parsimonious tree found from the pooled data-set.
Proponents of character congruence believe there to be no natural partitions to
data. Partitions are thought to be artifacts of scientific discovery and that all data should
be considered when estimating a phylogeny (Kluge, 1989). However, character
congruence shares a common tenet with taxonomic congruence - the independence of
198
characters. Under character congruence, each synapomorphy, within a data set, is
considered a separate, independent, confirmation or rejection of taxonomic relationships
(Kluge, 1989). Therefore, character congruence maximizes the “informativeness” and
“explanatory power” of the data used in the analysis (Kluge, 1989).
Systematists opposed to character congruence believe that when different data
partitions are combined, a partition with more characters will “swamp” a partition with
fewer characters, resulting in a loss of information from the smaller data partition.
Additionally, character congruence is believed to give erroneous phylogenies when there
is significant heterogeneity between data partitions. Bull et al., (1993) showed that in
extreme cases, where there was at least 80% incongruent characters in a combined data
set, the chances of finding a “true phylogeny” were near zero - even if more characters
were added to the matrix.
Prior agreement uses statistical analysis of the data partitions to select whether or
not they should be combined. If statistically significant heterogeneity is present between
data sets, then prior agreement would not combine these data without an attempt to
homogenize the incongruence (weighting or removal of data). If there is no significant
heterogeneity between data sets, then they can be combined for analysis without further
justification. Prior agreement would appear to be a simple concept, however the
statistical methods for testing for heterogeneity are not capable of determining which
characters in a data set are incongruent. Siddall (1997) discovered that a significant
measure of incongruence between data sets might be misleading. If data are incongruent,
those characters in conflict may be from a very small percentage of the data, and, in fact,
the majority of all characters may be in agreement. Rejecting to combine data, based on a
significant heterogeneity, might be at the expense of losing valuable information for
estimating a phylogeny. Therefore, significant heterogeneity may have little to do with
the overall combinability of characters. Prior agreement as a test of data combinability
199
may be arbitrary in execution because it is based on a priori definitions of data partitions
that are themselves arbitrary (Siddall, 1997).
Examining all the data available for a combined analysis sometimes involves
novel techniques. Agosti, Jacobs and DeSalle (1996) proposed including amino acid
residue sequences and nucleic acid sequences in the same analysis. They concluded that
amino acid and nucleic acid sequences are, “simply two different ways of character
coding the same information”. Their approach was to include both sequences, for the
same gene, in one analysis. Agosti et al. (1996) and Birstein and DeSalle (1998) showed
that both types of sequences were not completely dependent on each other and that in
combination, the amino acid sequences gave weight to some of the nucleic acid
characters that were important to building phylogenetic hypothesis. Functionally, adding
the amino acids to the analysis was considered a priori weighting of the data set (Agosti
et al., 1996 and Birstein and DeSalle 1998). I have used this method when combing all
data sets. Silent third and first position changes are invaluable to the substitutions that
help define the phylogenies for the more closely related data. However, many
relationships of the taxa in this study are intermediate in resolution. It is believed that the
inclusion of both the nucleic acid sequences and the amino acid residue translations in
combination with the 16S data, will help resolve the intermediate relationships.
All methods of data analysis are subject to criticism and one need not be paralyzed
by conjecture in deciding what method to use to estimate phylogenetic relationships from
data. I have chosen an approach that makes no judgement on whether congruence or
incongruence should be assumed of my data a priori. Instead, I have simply elected to
report known congruence or incongruence between data-sets based on statistical analysis
(incongruence length difference tests/partition homogeneity test). I have chosen to
combine the cytochrome b and 16S data so the maxima of available characters can be
used to estimate phylogenetic relationships. I will show conflict between data sets by
200
giving partitioned Bremer support values for each node in resulting cladograms (Baker
and DeSalle, 1997 and Baker, et al., 1998). I will present data based on; 1) unweighted
data from the cytochrome b and 16S nucleotide sequences - all positions have equal
weight (assumed weighting based on the number of characters from each data set); 2)
weighted parsimony, where the cytochrome b third codon transitions are weighted 0 and
all characters are included from the 16S data set; 3) cytochrome b amino acid residues
and 16S nucleotides; 4) appended amino acid weighting - the amino acid residue
sequences and the nucleotide sequences from cytochrome b and all nucleotide characters
from the 16S data are added together.
201
Collection and data matrix - Collection data are as in Chapters One and Two. The total
taxa used here are as in Chapter Two; Chapter One taxa were reduced to the same taxa
Chapter Two. A data matrix was developed that included three partitions. The first
partition was the cyt b nucleotide data, the second partition was the cyt b amino acid
residue translations and the third partition was 16S nucleic acid data. The total matrix
was 56 taxa by 2141 characters. Sequences from GenBank were used for two outgroups
species for the16S data matrix (Cyprinus carpio and Morone chrysops) and for two
species for the cytochrome b data (Cyprinus carpio and Boops boops).
Data Analysis
Sequence Characteristics - Pairwise sequence divergences were calculated for all 56 taxa
for the combined data and for each data partition (cyt b nucleotides, cyt b amino acids,
and 16S). The pairwise sequence divergences for each data partitions were plotted as a
function of total sequence divergence to compare the evolutionary rates of each partition.
Incongruence Length Test - Incongruence length difference (ILD) tests (Mickevich and
Farris 1981; Farris et al., 1994) were conducted on all partitioned data using the partition
homogeneity function in PAUP. Farris et al. (1994) developed the IDL statistic to test the
null hypothesis of congruence (that two populations [partitions] do not give significantly
different phylogenies). Incongruence length difference is the deviation between the
numbers of steps from individual analysis and the number of steps from combined
analysis. The test is conducted by measuring the distance of the observed data partitions,
202
and the distances for a number of randomized partitions, drawn from the original data
partitions. If some number (W) of the randomly selected distances (S) are smaller than
the observed distance (D), then a statistic (Type I error rate) can be estimated for rejecting
the null hypothesis:1- S/(W+1) (Farris et al., 1994). The null hypothesis is rejected when
the Type I error rate value falls below a pre-set threshold (at 5% significance level).
Ideally, 1000 randomly selected replicates are generated during the ILD test. Because of
significant computing power needed to complete ILD tests between some partitions, not
all test could be conducted for 1000 replicates. These are noted in the results.
(EPTs), and strict consensus were obtained for each analysis. The number of constant
consistency index and the retention index were determined. Bootstrap replicate support
was conducted using PAUP* and decay values (Bremer 1988) were calculated for clade
support using the program TreeRot.v2 (Sorensen 1999).
Four analyses were conducted. In the first analysis “CytbNuc16S”, the cyt b
nucleotide characters and 16S nucleotide partitions were used (the cyt b amino acid
translations were excluded). All positions from both partitions were included and of
equal weight. In the second analysis “CytbNuc3ds16S”, the cyt b nucleotide and 16S
partition were included, but third codon transitions from the cyt b data were weighted 0
(the cyt b amino acid translations were excluded). All other characters were included and
of equal weight. The third analysis “CytbProt16S” used the cyt b amino acid translations
in combination with the 16S nucleotide partition (the cyt b nucleotides were excluded).
All character were included and of equal weight. In last analysis, the “Total” all data
partitions were used and all characters were included and of equal weight (“weighting”
203
was based on the method of appended amino acids of Agosti et al., (1996)). Except
where noted in results a heuristic search of 1000 random addition sequences were
conducted. In some analyses, 100 random addition sequences were used due to
computing restraints.
Biogeographic Analysis - Parsimony analysis was used to reconstruct the biogeographic
relationships inferred from the cytochrome b and 16S sequence data simultaneously
(Brooks 1985; Wiley 1988a, b; Wiley et al., 1991). Data from Chapters One and Two
were used to analyze the biogeographic aspect of sparid evolution. The matrices from
Chapter One, Table 11 and Chapter Two, Table 18 were combined into one matrix and
then used as the basis for vicariance biogeography analysis.
204
RESULTS
Sequence Divergence - The mean sequence divergence between all pairwise comparisons
of the combined data partitions of 2141 characters was 16.6 ± 3.9%. Mean sequence
divergence for pairwise comparisons from the cyt b nucleotide partition was 19.7 ± 3.9%,
from the cyt b amino acid partition was 8.0 ± 4.1% and from the 16S data partition was
9.7 ± 4.8%. The sequence divergence of each data partition was plotted as a function of
total sequence divergence for all pairwise comparisons (Figure 34). The cyt b nucleotide
partition exhibited the greatest sequence divergence, followed by the 16S partition and
then by the cyt b amino acid residue partition. The cytochrome b partition accounted for
the majority of sequence divergence at low total sequence divergence and increased
linearly until approximately 20%. The 16S and cyt b amino acid residue partitions had
very similar plots until approximately 20% sequence divergence. After 20% the 16S
gene had a sharp increase in divergence.
Incongruence Length Difference - Results of ILD test are given in Table 19. No
incongruence was found between any of the data partitions tested. For each test, most of
the randomly selected distances were smaller than or equal to the length of the observed
combined partitions. The null hypothesis was not rejected for any of the test performed,
signifying that the data partitions were not significantly incongruent. Therefore, the data
partitions could be combined, in any permutation, without further tests of incongruence or
justification. The most conflict between partitions (p = 0.51) was found between cyt b
nucleotides (third codon transitions=0) and 16S nucleotides. The least amount of conflict
205
was between cyt b nucleotides and cyt b amino acid residues (p = 0.94) and between all
partitions (p = 093).
Phylogenetic Analyses
Combined Data: CytbNuc16S - A heuristic search of 1000 random addition replicates
yielded three equally parsimonious trees (strict consensus, Figure 35, tree length = 7451,
CI = 0.2439, RI = 0.4225; 100 trees held at each stepwise addition; starting seed =
949570787; all characters unordered and equal weight of 1; outgroup taxa Cyprinus
carpio; branch-swapping = stepwise addition; swapping algorithm = tree bisectionreconnection no topological constraints; character state optimization = accelerated
transformations). Of 1761 total characters, 753 (43%) characters were constant, 251
(14%) variable characters were parsimony uninformative and 757 (43%) variable
characters were parsimony informative characters. The Sparidae + Spicara were
monophyletic in all trees found, and two distinct groups (Group I and Group II , Figure
35) were found within the sparids. Most nodes were fully resolved, with the exception of
the clade containing primarily South African native sparids. There was an unresolved
tricotomy between Argyrozona argyrozona, Pterogymnus laniarius and a clade
containing Petrus rupetris + Polysteganus praeorbitalis and a tricotomy formed by
Chrysoblephus cristiceps, Cymatoceps nasutus and Porcostoma dentata. As was found
in all independent analysis of each data partition (Chapters One and Two), Pagellus and
Spicara were polyphyletic within the sparids and Pagrus was paraphyletic within Group I
taxa. Most of the node support was contributed from the cyt b partition. Of the 37 nodes
within the sparid clade, 20 had negative decay values from the 16S partition, one had
negative support from the cyt b partition and 16 had positive support from both partitions.
Of those positive nodes, the 16S partition was the major contributor to only four.
There was no support in this analysis for any of the six sparid subfamilies.
206
Likewise, there was no support for the Sparoidea; the placement of the Lethrinidae and
Nemipteridae was different in this analysis than in independent analyses of the cyt b or
16S data partitions. A clade containing the haemulids and the lutjanids/caesionids fell
sister to the Sparidae. This relationship was not seen during independent analysis of data
partitions. Nodes corresponding to outgroup species were constrained and analyzed with
each data partition, independently. There was no support (decay=0) from independent
analysis of either partition for a sister relationship between the Sparidae and the
haemulids + lutjanids/caesionids. Similarly, in this analysis, the lethrinids were sister to
sparids + haemulids + lutjanid/caesionid clade. There was no support (decay=0) from the
constrained analysis of independent partitions for this relationship. The nemipterids fell
basal to all other percoids, except Centropomus undecimalis, which has fallen basal to all
Perciformes in all molecular analyses. There was support (decay=5 16S and decay=10
cyt b) from constrained analysis of independent partitions for this node. Overall, most of
the decay support for outgroup nodes came from the cyt b data (9 of 14 nodes) and there
was positive contribution from the 16S partition to most nodes (11 of 14 nodes).
Combined Data: CytbNuc3ds16S - A heuristic search of 1000 random addition replicates
yielded a single most parsimonious tree (Figure 36, tree length = 3900 , CI = 0.3197, RI =
0.5406;100 trees held at each stepwise addition; starting seed = 600819088; cytochrome b
nucleotide data set using all changes at first and second position and transversions in
third position weighted 1, transitions third codon weighted 0; all characters from the16S
data weighted 1; outgroup taxa Cyprinus carpio; all other parameters as in previous
analysis). Of 1761 total characters, 753 (43%) were constant, 268 (15%) variable were
parsimony uninformative and 740 (42%) were parsimony informative. The Sparidae +
Spicara were monophyletic in this analysis. There was full resolution to all terminal
nodes within the Sparidae and all taxa placed in Group I and II as in the previous analysis.
207
However, the relative placement of many genera was different than in the previous
analysis. Within Group I taxa (Group I, Figure 36) the cyt b partition supported the
western Atlantic clade (A. probatocephalus + L. rhomboides and C. nodosus and S.
chrysops) basal to all other Group I species, but there was strong negative support from
the 16S partition for this arrangement (-5.7 from 16S, 6.7 from cyt b). The boopsines,
Boops boops and Sarpa salpa were basal to other Group I sparids in the previous
analysis, but grouped with Spondyliosoma cantharus and Spicara maena. The cyt b
partition supported this relationship, but there was no overall decay support for this
relationship. Crenidens crenidens and Lithognathus mormyrus formed a weakly
supported clade (-2.0 from 16S, 4.0 from cyt b). Boopsoidea inornata, Gymnocrotaphus
curvidens and Pachymetopon aeneum + Polyamblyodon germanum were supported
intermediate within Group I species by a moderate decay value (-2.0 from 16S, 5.0 from
cyt b).
In the previous analysis, Dentex tumifrons and Spicara alta were basal to other
Group II species, but in this analysis, they placed mostly with pagrine species. A clade
containing South African sparines and denticines was well resolved within Group II
species, with the almost all decay support coming from the cyt b partition.
There was no support in this analysis for the six sparid subfamilies. Outgroup
species were similar to the previous analysis and there was no support for the Sparoidea
in this tree. The lethrinids moved intermediate to the Sparidae + other percoids and the
nemipterids.
Combined Data: CytbProt16S - A heuristic search of 1000 random addition replicates
resulted in 35 equally parsimonious trees (strict consensus Figure 37, tree length = 2142,
CI = 0.4542, RI = 0.5683; 100 trees held at each stepwise addition; starting seed =
1264550021; all characters were unordered and of equal weight, gaps were treated as
208
additional state; outgroup taxa Cyprinus carpio; all other parameters as in the first
analysis). Of 1001 total characters, 452 (45%) characters were constant, 230 (23%)
characters were parsimony uninformative, 319 (32%) characters were
parsimony-informative. The sparids were monophyletic in all 35 equally parsimonious
trees and both Group I and Group II species were supported (strict consensus, Figure 37).
However, Calamus nodosus was basal to all sparids and formed an unresolved tricotomy
with the Group I and Group II clades. Calamus nodosus fell basal to all other sparids in
the consensus tree derived from cyt b amino acid residue translations in Chapter One
(Figure 19). Generic relationships of the sparids were not clearly resolved as in the
previous two analyses, possibly due to the introduction of the conserved amino acid
residue translations into this analysis. Most of the decay support for nodes within the
sparid clade came from the 16S partition. There was no support in this analysis for any of
the six sparid subfamilies. The outgroup species in this analysis were unresolved; a
polytomy was formed of all percoid species (with the exception of C. undecimalis which
placed outside of other percoids). Based on the lack of resolution, any of the percoid
groups sampled were equally likely to be sister to the sparids. The node support for
external outgroup relationships was dominated by the 16S partition. There was no
support from these data for a monophyletic Moronidae + Lateolabrax.
Combined Data: Total - A heuristic search of 100 random addition sequences resulted in
11 equally parsimonious trees (strict consensus Figure 38; tree length = 8066; CI =
0.2659; RI = 0.4375; all characters unordered and of equal weight; starting seed =
550461514; number of trees held at each step during stepwise addition = 100, outgroup
taxa = Cyprinus carpio; all other parameters as in the first analysis). Of 2141 total
characters 954 (45%) were constant; 344 (16%), were parsimony-uninformative; 843
(39%) were parsimony-informative. In this analysis, the Sparidae + Spicara were
209
monophyletic. By including cyt b nucleotide and amino acid residue sequences with the
16S nucleotide sequences, less supported nodes (seen in previous analyses) collapsed,
leaving mainly external nodes that were well supported. A number of unresolved internal
nodes were found in the sparid clade. For example, the internal nodes of Group I species
collapsed to a polytomy, with no support for the basal placement of Boops boops and
Sarpa salpa to other Group I species. Most decay support for external nodes came from
the cyt b nucleotides. In Group II species a polytomy containing South African native
sparids was well supported distinct from other Group II taxa. The decay value for this
node (8) came entirely from the cytochrome b gene. Only the cyt b nucleotide partition
supported the clade of western Pacific, Argyrops sister to Evynnis and Pagrus auratus,
but both the 16S and cyt b nucleotide partitions supported the relationship between
Evynnis and Pagrus auratus. In addition, both the 16S and cyt b nucleotide partitions
contributed to the decay value (7) for the clade of Cheimerius nufar + Pagrus auriga and
Pagellus bellottii + Pagrus pagrus. There was no support for subfamilies in this analysis
and the Sparoidea were not monophyletic. The outgroup relationships in this analysis
were identical to those found in the cytochrome b nucleotide/16S (CytbNuc16S) analysis
and node support was dominated by the cyt b nucleotide partition.
Biogeographic Analysis - The matrix used for this analysis is presented in Table 20. The
topology of the combined vicariance biogeography tree was the same as the cyt b
biogeographic tree in Chapter One (Figure 21). Of 158 total characters, 18 were constant,
10 were parsimony-uninformative and 130 were parsimony-informative. A single most
parsimonious tree was found. (Figure 39, tree length = 189, CI = 0.7407, RI = 0.8275.
1000 replicates of random stepwise addition; 100 trees held at each step; starting seed =
394372018; character-state optimization: Delayed transformation, tree rooted to the
Indian Ocean, Pacific northwest, Pacific west central + Pacific southwest node). Both
210
data partitions contributed nearly equally to decay values, except for the Atlantic
southwest and Atlantic northwest + Atlantic western central clade. Here, the 16S
dependent data and cytochrome b dependent data were in conflict (partitioned decay = -2
from 16S and 3 from cyt b). In the 16S independent tree the Atlantic southwest terminal
node was sister to the Pacific southwest area (Figure 33), but the placement of Atlantic
southwest area in the independent cytochrome b tree is identical to this tree (Figure 21).
This conflict is further reflected in the node uniting all Atlantic, Mediterranean, and
Indian Ocean western areas, where all node support came from the cyt b dependent data.
211
DISCUSSION
The use of character congruence resulted in increased support for many sparid
relationships. However, combined analysis was not very informative in resolving
outgroup relationships. Although there was no significant heterogeneity between any of
the data partitions tested, there was still conflict between data partitions at many of the
nodes. It was clear from the plot of sequence divergence for each data partition as a
function of total sequence divergence that cytochrome b nucleotides sequences were most
divergent of the three data partitions. Based on the plot, cyt b nucleotide sequences
might, therefore, contain more substitutions useful for resolving relationships. This could
explain why the cytochrome b nucleotide data dominated decay support for most nodes in
the three analysis where it was used (CytbNuc16S, CytbNuc3ds16S , and Total). Yet,
that is not to say that each data partition is not capable of contributing to the overall
phylogeny or for resolution of specific nodes. Each partition’s intrinsic rate of
substitution was valuable in contributing to the overall phylogeny. Although support for
a particular node might be dominated by one partition, other partitions typically
contributed to the total nodal support.
The incongruence length test is an indicator of overall homogeneity (or
heterogeneity) of the data partitions; a statistical measure of data conflict. However,
regardless of the overall homogeneity, there maybe considerable resolving power of
combined partitions for nodes of a given tree. The method of reporting partitioned decay
values (Baker and DeSalle, 1997 and Baker, et al., 1998) serves as an objective method
for understanding how conflict between partitions is resolved throughout a tree. Conflict
is shown, but no action is taken as a result of conflict, a posteriori. For example, if one
were to accept only those clades that were supported by multiple partitions (as might be
212
done in a censuses method), then data would be lost. Multiple partitions will often agree
or disagree at a particular node, correctly or incorrectly supporting the “true phylogeny”.
By rejecting those nodes that are in conflict, then one is left with a phylogeny with
reduced explanatory power. To reject a node that is conflict is equivalent to knowing the
true phylogeny. A method should be questioned if it selects one partition over another
partition as a better estimator of the “true phylogeny”, because the true phylogeny can
never be known. (Of course there is reason to reject data if there is a prior knowledge of
character mis-information, i.e. saturation, non-homologous alignments or comparing nonorthologous or paralogous sequences.) Hypotheses of evolutionary history are equally
valid when they are based on data partitions that contain synapomorphies. Under
parsimony, each synapomorphy, within a data set is considered a separate, independent
confirmation or rejection of taxonomic relationships; the shortest tree obtained should
have the best explanatory power of all the data (available). Therefore, the shortest tree
(or strict consensus) will have the minimum amount of conflict between data partitions.
Unless there is complete agreement between data partitions (in which case there is no
need for data combination) there will be some conflict for relationships between
partitions. I’ve chosen to report conflict and not reject hypotheses because of conflict,
leaving the explanation of relationships to parsimony.
Phylogenetic relationships
Combined analysis of cyt b and 16S nucleotide sequences - The best resolution of sparid
relationships was in the unweighted (CytbNuc16S) and weighted ( CytbNuc3rds16S)
analyses, based on the combined analyses of the data partitions, the Sparidae + Spicara
were a monophyletic group. These results are not surprising, since all independent
estimates of phylogeny in Chapters One and Two found this group to be monophyletic.
213
The stability of these trees was verified by the large number of nodes that had decay
values > 1. Another measure of stability in these trees was in the consistency and
retention indices. The unweighted tree had a CI=0.2439 and RI=0.4225 and these
numbers increased to a CI=0.3197 and RI=0.5406 in the weighted tree. However, the
ILD test between the cyt b (third codon transitions weighted 0) and 16S partitions showed
the most conflict (p = 0.51) between all partitions tested. Because the CI and RI are
actual measures of synapomorphies and homoplasy that occur in a tree and the ILD test is
a measure of net character conflict between data partitions, they may not be agreement
between the measures. Although CI and RI index numbers might seem low, relative to
the large size of the data matrix, they indicate a stable tree. The increase in the weighted
tree values justified the removal of the saturated third codon transitions because it
increased the stability of the tree. Here the cyt b and 16S partitions were in better
agreement in estimating sparid evolution than in the unweighted tree, despite having the
lowest ILD value.
The relationship between the Sparidae and outgroup species was different in the
combined analysis than in independent estimates of phylogeny. Although there was
strong agreement between the data partitions for most external nodes, there was conflict
between the data partitions in support for internal nodes that defined relationships
between outgroups and the Sparidae. Most nodes were dominated by the cyt b partition,
yet the outgroup relationships are different than when the cyt b partition was analyzed
independently. The relationship of the Lethrinidae and Nemipteridae in the weighted
analysis was similar to the placement in the independent analysis of the 16S partition. As
mentioned in Chapter Two, the 16S loop characters are transitionally saturated in
outgroups species and are potentially misleading because they represent homoplasy.
These characters could not be removed without removing informative characters from
ingroup species (not saturated for loop transitions). The conflict between saturated 16S
214
characters and the cyt b sequences has possibly resulted in reduced resolution for
outgroup relationships.
Combined analysis of cyt b amino acid residues and 16S nucleotide sequences - It was
hoped that there would be increased resolution for outgroup nodes by including the
conserved cyt b amino acid residue translations with the 16S nucleotide sequences.
Because the 16S loop transitions are potentially saturated for more distantly related
species, the inclusion of conserved data might have bolstered support for relationships
masked by the noise of saturation. However, this was not the case. There was no
resolution at all for outgroup species. I think that given the mis-informative substitutions
in loop characters, a polytomy based on these data is acceptable. I would rather loose
resolution than to accept possibly misleading phylogenetic relationships.
Combined analysis of all data: Total - All forms of weighting are based on the assumption
that an applied weight will result in a better supported tree than if no weight were applied.
Amino acid weighting attempts to give more support to nucleic acid characters that,
“impart structure to a phylogenetic hypothesis” (Agosti, et al., 1996). This method is
valuable for closely related taxa that have variable amino acid sequences. Because the
amino acid translations are probably not entirely independent of the nucleic acid
sequences, then equal weight should be given to nucleotides and amino acids as not to
amplify character-independence problems. (Agosti, et al., 1996). Although previous
analyses showed an increase in tree support by removing 3rd codon transitions, I decided
to only use a single weighting scheme in this analysis (amino acids) and not to down weight third codon transitions.
The overall effect of including the amino acids and all other characters in one
analysis was that some less supported internal nodes collapsed while the majority of
215
nodes were well supported. The Sparidae + Spicara were monophyletic, but were no
longer fully resolved. However, the majority of relationships that remain were well
supported, including both sparid lineages. Group I sparids, overall, had better support
for nodes from all data partitions, but Group II sparids were structured primarily from the
cyt b nucleotides. The outgroup relationships were identical to that of the unweighted
analysis of cyt b and 16S nucleotides. This is not surprising, since all nucleotide
characters were included, and there was no resolution in outgroup species when the
amino acid residues were combine with the 16S sequences. The amino acids imparted
negative support for the three nodes defining outgroup relationships.
Biogeographic Analysis - The combined analysis of sparid biogeography resulted in tree
topology identical to that of the independent cyt b analysis. Conflict in the tree was
evident where the 16S independent tree and the cyt b independent tree disagreed. The
placement of Atlantic southwest node in this tree was only supported by the cyt b data,
but the evidence from that partition strongly supports it sister to other Atlantic areas. The
alternative hypothesis, of from the 16S data is not strongly supported.
216
Table 19. ILD values between different data partitions
Partitions
D
S
W+1
p value
cyt b nucleotides and 16S
7416
39
1000
0.61
cyt b nucleotides (3rds=0) and 16S
3872
487
1000
0.51
cyt b amino acids and 16S
2111
6
126
0.64
cyt b nucleotides and cyt b amino acids
6472
6
100
0.94
All partitions
8000
7
100
0.93
If a number (W) of the randomly selected distances (S) is smaller than the observed
distance (D), then a statistic (Type I error rate) can be estimated for rejecting the null
hypothesis:1- S/(W+1) (Farris et al., 1994).
217
TABLE 20. MATRIX OF FAO AREAS AND CHARACTERS FROM CYT B (TOP) AND 16S (BOTTOM)
Characters
Areas
.........1.........2.........3.........4.........5.........6.........7.........8..
1234567890123456789012345678901234567890123456789012345678901234567890123456789012
0000100000010001000011011011000000100111101011100110110001000000111111101101111111
Atlantic, Northeast
0000100000010001000011010011000000100110101011100110110001000000001111101101111111
Atlantic, Northwest
0100001000000000100100000001000000000000010000011110110001000000000011101111111000
Atlantic, Southeast
0011110111100001001011101010111111110001101111100110111111111111111111101101111111
Atlantic, Southwest
0100000000000100000000000001000000000000000000001110110001000000000011101111111000
0100001000000110100100000001000000000000010000011110110001000000000011101111111000
1010000000001000000000000100000000001000000000000001110001000000110000111101111011
1011010111100000001010100000111111111000001111100001111111111111011101101101111011
Mediterranean and Black
0000100000010001000011011011000000100110101011100110110001000000001111101101111100
Pacific, Southwest
1010000000001000010000000100000000000000000000000001110001000000110000111101111000
Pacific, Northwest
0000000000000000000000000100000000000000000000000000000000000000000000011101110000
1010000000001000000000000100000000000000000000000001110001000000110000111101111000
0000100000000100001101101100000010011110000001111111111100111111111111111111
Atlantic, Northeast
0000100000000100001101001100000010011010000001111111111100111111111111111111
Atlantic, Northwest
0100001000000010010000000100000000000001000011110010101111100111111111111110
Atlantic, Southeast
0011110111100100101110101011111111000110011101111111111100111011111111111111
Atlantic, Southwest
0100000000000000000000000100000000000000000000000000000100100111111111111110
0100001000001010010000000100000000000001000011110010101111100111111111111110
1010000000010000000000010000000000100000110000000010101100100111111111111110
1011010111100000101010000011111111100000111100001111101100011011111111111111
Mediterranean Black Sea
0000100000000100001101101100000010011010000001111111111100111111111111111111
Pacific, Southwest
1010000000010001000000010000000000000000110000000010101100100111111111111110
Pacific, Northwest
0000000000000000000000010000000000000000000000000000000000000111111111111110
1010000000010000000000010000000000000000110000000010101100100111111111111110
218
Figure 34. The sequence divergence of each data partition was plotted as a function of
total sequence divergence for all pairwise comparisons. The cytochrome b partition had
the greatest mean pairwise sequence divergence (19.7 ± 3.9%). The cyt b amino acid
partition and 16S data partition had similar mean pairwise sequence divergences of was
8.0 ± 4.1% and 9.7 ± 4.8%, respectively.
30
% Sequence Divergence Per Partition
25
20
15
10
5
0
0
5
10
15
20
25
Total % Uncorrected Sequnce Divergence
Cytochrome b Nucleotides
Cytochrome b amino acids
16S
30
220
Figure 35. Combined analysis: CytbNuc16S - A heuristic search of all cyt b and 16S
nucleotide characters yielded 3 equally parsimonious trees. A strict consensus is shown
(tree length = 7451, CI = 0.2439, RI = 0.4225). Subfamilies are labeled as follows
(BO=Boopsinae, DE=Denticinae, DI=Diplodinae, PA=Pagrinae, PE=Pagellinae, and
SP=Sparinae).
“N” = Node
D
A
= Decay Value From 16S Partition
B
= Decay Value From Cyt b Nucleotide
Partition
Group II
Group I
Cyprinus carpio
Scolopsis ciliatus
(22.8, 21.2) 44
30
(11.0, 19.0)
Lateolabrax latus
(3.0, 2.0) 5
(11.2, 6.8) 18
Morone americanus
(17.0, 21.0) 38
Morone chrysops
(4.1, 9.9) 14
4
(-1.0, 5.0)
Morone saxatilis
Lethrinus ornatus
43
(29.5,
13.5)
(5.0, -4.0) 1
Haemulon sciurus
21
(2.0, 19.0)
Pomadasys maculatus
(5.6, -0.6) 5
Caesio cuning
(3.1, 24.9) 28
Lutjanus decussatus
DE Dentex tumifrons
(-1.8, 4.8) 3
(-0.5, 4.5) 4
Spicara alta
(-1.1, 6.1) 5
DE Petrus rupestris
(-1.5, 11.5) 10
(-1.5, 8.5) 7
(-0.2, 4.2) 4
(2.4, 0.6) 3
(-1.5, 2.5)
1
(2.5, -0.5) 2
PA Evynnis japonica
(-2.0, 12.0) 10
PA Pagrus auratus
(3.0, 9.0) 12
DE Cheimerius nufar
(1.5, 7.5) 9
PA Pagrus auriga
(3.5, 4.5) 8
(11.0, 24.0) 35
(-1.2, 5.8) 7
(2.0. 9.0) 11
PA Pagrus pagrus
BO Boops boops
13
(10.0. 3.0)
BO Sarpa salpa
(-4.3, 11.1) 7
(1.5, 0.5) 2
(18.8, 25.2) 44
(-1.5, 2.5) 1
(-2.6, 12.6) 10
(9.3, 21.7) 31
Spicara maena
(-1.2, 2.2) 1
(-1.5, 4.5) 3
(-2.2, 5.2) 3
SP Calamus nodosus
(1.0, 11.0) 12
(-1.2, 2.2) 1
(13.3, 10.7) 24
(-1.9, 2.9) 1
SP Sparus auratus
(-1.6, 2.6) 1
(7.5, 3.5) 11
13
(-1.1, 14.1)
(0.7, 1.3) 2
(-3.0, 4.0) 1
BO Oblada melanura
(-2.2, 3.2) 1
(0.8, 8.2) 9
(3.0, 9.0) 12
(-1.0, 24.0) 23
222
Figure 36. Combined analysis: CytbNuc3rds16S - A heuristic search of the weighted cyt
b nucleotides (3rd codon transitions =0) and all 16S nucleotides yielded a single most
parsimonious tree (tree length = 3900 , CI = 0.3197, RI = 0.5406). Subfamilies are
“N” = Node
D
A
B
= Decay Value From Cyt b Nucleotide
Partition
41
(18.8, 22.2)
16
24
(5.0, 19.0)
2
(-2.6, 3.6)
(-2.0, 3.0)
1
38
(18.0, 20.0)
12
(7.0, 5.0)
(2.0, 1.0)
(-0.8, 1.8)
3
1
11
(8.0, 3.0)
18
(2.8, 15.2)
(-1.0, 2.0)
1
(-0.5, 3.5)
3
(-1.0, 4.0)
3
2
(-1.7, 3.7)
(0.0, 1.0)
(1.0, 1.0)
1
(2.3, 0.7)
3
(-1.4, 12.4)
11
2
(-3.4, 8.4)
1
(-3.7, 4.7)
(-1.6, 2.6)
1
2
(0.0, 2.0)
2
(-3.5, 5.5)
(2.3, 0.7)
(2.3, 21.7)
3
4
(2.3, 1.7)
24
(1.0, 1.0)
2
2
(1.0, 1.0)
5
(-4.7, 9.7)
(1.5, 1.5)
3
3
(2.3, 0.7)
4
(12.5, -0.6)
(-1.2, 5.2)
(-5.7, 6.7)
1
(-1.7, 5.7)
23
4
3
(-0.1, 3.1)
(15.0, 16.0)
31
3
(-2.0, 5.0)
2
(-2.0, 4.0)
(-2.2, 3.2)
1
(-4.0, 8.0)
(-1.3, 2.3)
12
1
(6.8, 16.2)
(-5.7, 6.7)
5
4
12
(5.0, 7.0)
(-0.7, 16.7)
1
(9.0, 10.0)
(1.0, 0.0)
16
19
1
(3.0, 5.0)
8
8
(2.0, 6.0)
(1.0, 7.0)
8
Group II
(7.0, 9.0)
Group I
28
(30.8, -2.8)
Cyprinus carpio
Scolopsis ciliatus
Lethrinus ornatus
Lateolabrax latus
Morone americanus
Morone chrysops
Morone saxatilis
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
DE Petrus rupestris
DE Dentex tumifrons
Spicara alta
PA Evynnis japonica
PA Pagrus auratus
DE Cheimerius nufar
PA Pagrus auriga
PA Pagrus pagrus
SP Calamus nodosus
BO Boops boops
BO Sarpa salpa
Spicara maena
SP Sparus auratus
BO Oblada melanura
224
Figure 37. Combined analysis: CytbProt16S - A heuristic search of all cyt b amino acid
residue and 16S nucleotide sequences resulted in 35 equally parsimonious trees. A strict
consensus is shown (tree length = 2142, CI = 0.4542, RI = 0.5683). Subfamilies are
“N” = Node
D
A
B
= Decay Value From Cyt b Amino Acid
Partition
(6.1, 0.9)
7
(9.2, 2.8)
(23.7, 1.3)
(29.9, 2.1)
12
25
32
15
(7.4, -1.4)
(17.5, -2.5)
(7.0, -1.0)
6
6
(1.5, 0.5)
(4.1, -3.1)
(3.8, 0.2)
1
14
(4.5, -2.5)
1
2
1
(0.1, 0.9)
1
(-0.7, 1.7)
(-0.7, 1.7)
(2.0, -1.0)
(-1.0, 2.0)
1
(1.4, -0.4)
(3.3, -2.3)
1
4
(-0.5, 1.5)
(-0.8, 2.8)
(12.7, 1.3)
2
1
1
1
1
1
(1.8, -0.8)
(14.3, -1.3)
(2.5, -1.5)
(5.3, -4.3)
1
(5.3, -4.3)
1
13
(6.9, 3.1)
10
(7.5, 2.5)
10
1
4
(5.3, -1.3)
(-1.3, 4.3)
(0.2, 1.8)
3
2
(-1.8,2.8)
1
(15.8, 1.2)
(4.3, -0.3)
4
(0.3, 1.7)
17
2
Group II
10
Group I
(9.2, 0.8)
Cyprinus carpio
Centropomis undecimalis
Lateolabrax latus
Haemulon sciurus
Pomadasys maculatus
Caesio cuning
Lutjanus decussatus
Lethrinus ornatus
Scolopsis ciliatus
Morone americanus
Morone chrysops
Morone saxatilis
SP Calamus nodosus
DE Dentex tumifrons
Spicara alta
PA Pagrus auratus
DE Cheimerius nufar
PA Pagrus auriga
DE Petrus rupestris
PA Pagrus pagrus
PA Evynnis japonica
BO Boops boops
BO Sarpa salpa
Spicara maena
SP Sparus auratus
BO Oblada melanura
226
Figure 38. Combined analysis: Total - A heuristic search of all unweighted sequences
resulted in 11 equally parsimonious trees. A strict consensus is shown (tree length =
8066; CI = 0.2659; RI = 0.4375). Subfamilies are labeled as follows (BO=Boopsinae,
DE=Denticinae, DI=Diplodinae, PA=Pagrinae, PE=Pagellinae, and SP=Sparinae).
“N” = Node
D
A
B
= Decay Value From Cyt b Amino Acid
Partition
C
= Decay Value From Cyt b Nucleotide Partition
Group II
Group I
Cyprinus carpio
Centropomis undecimalis
Scolopsis ciliatus
(35.7, 0.1, 13.2) 49
(6.7, 3.9, 24.4) 35
Lateolabrax latus
2
(2.5, -1.9, 1.4)
17
36
(6.7, 0.9, 9.4)
Morone americanus
(16.7, -2.1, 21.4)
14
Morone chrysops
(7.3, -0.4, 7.1)
3
Morone saxatilis
(0.0, -3.9, 6.9)
Lethrinus ornatus
45
1
(20.7, 1.4, 22.9)
(5.7, -0.6, -4.1)
Haemulon sciurus
22
(3.7, 0.4, 17.8)
Pomadasys maculatus
3
(-0.0, -2.6, 5.6)
Caesio cuning
30 Lutjanus decussatus
(6.7, -0.6, 23.9)
DE Dentex tumifrons
(0.7, -3.2, 5.5) 3
3
(2.7, -1.1, 1.4)
Spicara alta
11
8
(0.1, 1.3, 9.6)
3
(-3.3, 2.4, 5.9)
(0,0, -3.9, 6.9)
Petrus rupestris
10 DE
(-4.3, 5.4, 8.9)
1
(2.3, -0.6, -0.7)
(-3.3, -0.6, 13.9) 10
PA Evynnis japonica
10 PA Pagrus auratus
(3.9, -1.0, 7.1)
(-1.3, -0.6, 8.9) 7
DE Cheimerius nufar
39
(-0.9, 4.0, 8.0) 11
(10.7, 4.4, 23.9)
PA Pagrus auriga
(3.2, -0.7, 4.5) 7
11 PA Pagrus pagrus
(2.4, 0.4, 8.2)
BO Boops boops
14
(8.9, 2.4, 2.6)
BO Sarpa salpa
33
(8.7, 1.9, 22.5)
Spicara maena
9
(-2.3, -1.1, 12.4)
(-4.4, 1.3, 10.1) 7
Pachymetopon aeneum
46 BO
(17.1, 2.7, 26.2)
(-2.2, 0.5, 4.7) 3
1
(0.7, -0.5, 0.7)
(-0.8, -3.1, 8.9) 5
SP Calamus nodosus
11
(1.2, -0.7, 10.4)
Acanthopagrus berda
26 SP
(12.0, 2.8, 11.2)
1
SP Sparus auratus
(-1.1, 0.2, 1.9)
10
(4.2, 1.4, 4.4)
16
(0.7, -0.3, 0.6) 1
(-1.3, 2.9, 14.4)
1
(-1.1, -0.1, 2.2)
1
(-1.2, 0.2, 2.0)
BO Oblada melanura
9
(1.1, 0.6, 7.2)
11
(3.0, -1.0, 9.0)
bermudensis
26 DI Diplodus
(0.5, 2.9, 22.6)
228
Figure 39. Vicariance biogeographic analysis of the combined cyt b and 16S dependent
data matrices yielded a single most parsimonious tree (tree length = 189, CI = 0.7407, RI
= 0.8275).
(7,4)
(-2, 3)
(1,1)
(3.5, 2.5)
(0,7)
(10.6, 20.4)
(5,6)
(7.1, 8.9)
root
Mediterranean and Black Sea
Pacific Southwest
Pacific Northwest
Atlantic Southeast
Atlantic Northeast
Atlantic Southwest
Atlantic Northwest
CHAPTER FOUR. RECONSTRUCTING SPARID RELATIONSHIPS WITH A
SINGLE-COPY NUCLEAR DNA
231
INTRODUCTION
In this chapter I present results from an attempt to estimate sparid evolution based on a
single-copy nuclear locus. The gene selected was problematic and the results were
inadequate to resolve sparid relationships. Possible reasons for the inability of this locus
to estimate sparid evolution are given in the discussion
Nuclear DNA provides an alternative to mitochondrial DNA as source for genes
from which to infer independent phylogenies. Although there are many nuclear genes
available for phylogenetic analyses, their use can be challenging. Nuclear genes often
exist as multiple copies and distinguishing between orthologous genes (homologous gene
sequences from a speciation event ) from paralogous genes (homologous gene sequences
from a duplication event) can be difficult. Additionally, concerted evolution of gene
copies increases the complexity of identifying orthologous genes. Furthermore, many
nuclear genes have been affected by recombination and insertion/deletion events (indels)
and introns. Inserted DNA often complicates the recovery of the entire gene during PCR
amplification. Inserted DNA and introns can interrupt the coding region of the gene and
often inserted DNA or introns can greatly increase the size of the amplified product.
Indels can also inhibit the amplification of gene if they interrupt primer binding sites.
Low copy number of nuclear coding genes (compared to the high copy number of genes
in mitochondrial DNA or chloroplast DNA) also contributes to reduced PCR product
(Hillis, Moritz and Mable, 1996).
There are nuclear genes available for DNA phylogenetic analyses, but few have
been found to be informative for teleost relationships. Many coding genes are strongly
conserved and are only appropriate for distantly related teleost taxa (growth hormone,
232
actin, 5S, 18S, 28S, creatine kinase) and of these genes several have multiple gene copies.
Few nuclear genes have been found to evolve very quickly in fishes. The recombination
activating genes (RAG1, RAG2) have been found potentially useful for relationships in
mormyrid fishes (John Sullivan, pers. com.) and in beloniforms (Nathan Lovejoy,
unpublished dissertation 1999). Ideally, a coding gene is preferred over a rRNA for
molecular phylogenies because the translated reading frame facilitates determination of
gene orthology. Also, a single copy of a gene would simplify amplification and analysis
because no determination of paralogy is necessary. Therefore, of all nuclear genes, a
single-copy, coding gene with sufficient variation between taxa would be ideal for
estimating phylogenetic relationships.
Streelman and Karl (1997) successfully amplified a locus-specific primer pair
(Tmo-4C4) in 17 labroid fishes. This locus is an open-reading frame and extended to 511
nucleotides in all fishes sampled. Only a single copy of the locus was amplified in the
study. A BLAST ( Altschul et al. 1990) search of GenBank databases determined that no
sequence matched the locus. Although, low probability matches were made to muscle
specific proteins titin and connectin (Streelman and Karl, 1997).
In this chapter I present the results from an analysis of the Tmo-4c4 single copy
nuclear locus to infer relationships of the Sparidae. This locus was problematic for these
fishes, and the phylogenetic relationships presented are valid only for determining the
usefulness of the gene. For this reason the results and discussion are brief.
233
Collection Data - Collection data and voucher catalog numbers are shown in Table 21.
Representatives of all six sparid subfamilies but only 15 members of the 33 sparid genera
were sequenced
Primers - The primer sequences used for PCR amplification in this study were the Tmo4c4 locus specific primers of Streelman and Karl (1997). To direct sequence the Tmo4c4 locus, an additional primer set was developed that added a M13 primer site to the 5'
end of the Streelman and Karl primers. Primer sequences are listed in Table 22 and were
ordered from Genosys (Genosys Biotechnologies, Inc. The Woodlands, TX).
Amplification - A 50 µl PCR amplification of the Tmo-4c4 locus was prepared with 5-10
ng of each template DNA. The following reagents were used in each reaction: 5 µl 10X
PCR Buffer (Invitrogen Corporation) [100mM Tris-HCL (pH8.3), 500 mM KCL, 25 mM
MgCl2, 0.01% gelatin]; 1µl 10mM dNTP Mix from the PCR Reagent System (GIBCO
BRL) [10mM each dATP, dCTP, dGTP, dTTP]; 50 pmols each of primers, 0.50 µl
Platinum Taq DNA polymerase (GIBCO BRL Life) [5U/µl] and 42.00µl diH2O. All
amplifications were performed on a MJ Research PTC-200 thermocycler (Watertown.
MA) with the following cycle parameters: initial denaturation [97EC for 3.0 min]; 34
cycles of [denaturation 95EC for 1.0 min, annealing 54-55.5EC (depending on sample) for
1.0 min; extension 72EC for 30 seconds]; final extension [72EC for 7 min]; icebox [4EC
indefinitely].
234
Cloning and Sequencing - Most samples were cloned and sequenced on a Li-Cor L4000
automated sequencer as outlined in the Materials and Methods section of Chapter One.
Two samples (indicated in Table 23) were direct sequenced using M13-Tmo-4c4 primers
with “tails” that corresponded to the M13 universal priming sites attached to the 5' end of
the primer sequence. Once PCR reactions were run, inserts had both forward and reverse
IRD800 flourescently labeled M13 primers sites and could be used directly in sequencing
reactions. The PCR products were purified prior to sequencing by adding 4 ul 3M
NaOaAC and precipitated with 2 volumes ethanol. Sequencing proceeded as in Chapter
One.
Sequence Alignment - Both light and heavy strand sequences were obtained for all taxa.
Light strand sequences were inverted (reversed and complimented) with the heavy strand
sequence. A consensus of both strands was used for this study. The resulting alignment
introduced no gaps due to deletions or insertions and there were no inconsistent
alignments between taxa.
Sequence Divergence and Mutation Analysis - Mean uncorrected pairwise genetic
distance (uncorrected “p” distance) was calculated using PAUP* between all taxa,
between ingroup taxa and between outgroup taxa. Scatter plots of transitions and
transversions as a function of mean sequence divergence were calculated for combined
codon positions and between putative gene copies (paralogous genes).
(EPT) and strict consensus were obtained for each analysis. The number of constant
235
consistency index (CI) and the retention index (RI) were recorded for each analysis. A
neighbor-joining tree was generated in PAUP to examine the genetic distance between
putative gene copies.
236
RESULTS
Sequencing resulted in 511 base pairs per sample. There were no indel events
between taxa and no gaps were necessary to align sequences. In total, 42 clones were
sequenced for 26 taxa. Blast searches of all 26 sequences against published GenBank
sequences confirmed a highly probable match to only the Tmo-4c4 locus submissions of
Streelman and Karl (1997). Sequencing resulted in two distinct groups of sequences, that
represent putative copies of the gene. These two copies were identifiable by eye and
verified during phylogenetic analysis (see Phylogenetic Analysis below for more detail).
Table 24 presents the multiple alignments of all clones sequenced. Sample names
prefixed by “2" were visually different from other clone sequences and are hereby
considered putative copy 2 (copy 2). All other sequences are considered putative copy 1
(copy 1).
Mean pairwise genetic distance for copy 1 sequences was 5.5 ± 3.3 % (range 0.2%
to 12.5%) and for copy 2 sequences was 2.3 ± 1.5% (range 0.2 % to 6.2%). Problematic
with copy 1 sequences were pairwise genetic distances of 0 between two different sets of
clones. The sequence of clone Spondyliosoma cantharus C17-30-IV was identical to the
sequence of clone Morone saxatilis C6-22-III and the sequence of clone Polysteganus
praeorbitalis C10-22-IV was identical to the sequence of clone Lateolabrax japonicus
C1-6-III.
Plots of substitutions as a function of sequence divergence (Figure 40) revealed
that transitions were more common than transversions for both putative gene copies, and
that neither transitions or transversions were saturated in either putative gene copy.
Phylogenetic Analyses
237
Parsimony analysis of all clone sequences and characters was conducted. All of
the 511 total characters were unordered and of equal weight; 352 (69%) were constant, 62
(12%) were variable and parsimony uninformative and 97 (19%) were variable and
parsimony informative. A heuristic search of 1000 random addition sequences resulted in
81 equally parsimonious trees (strict consensus Figure 41, tree length = 270, CI = 0.7185,
RI = 0.9110). In the resulting tree, a node containing all copy 2 sequences was separate
from all other sparid copy one sequences (Figure 2, heavy stroke-weight clade). Clones
of three taxa were found in both copy 1 and copy 2 clades: Porcostoma dentata, Calamus
nodosus, and Polysteganus praeorbitalis. The nemipterid sequences were basal to copy 2
sequences. There were only sparid sequences within the copy 2 node (including Spicara)
and no outgroup species were found to have copy 2, although the nemipterids were sister
to this group.
The node containing all copy 1 sequences was split into two clades. One clade
represented all Atlantic fishes, with the exception of the clone Spondyliosoma cantharus
C17-30-IV, but this clone was problematic in that it was identical in sequence to the clone
of Morone saxatilis C6-22-III. The other major clade within copy 1 taxa represented all
other sequences. These taxa correspond to non-Atlantic species, with the exception of
Diplodus bermudensis that was sister to the clone of Sarpa salpa. In both major clades of
copy 1 species outgroup species were mixed with ingroup species.
The sparids based on these sequences are not monophyletic. Problematic in the
both copy 1 and copy 2 clades are that separate clones of the same taxa do not group
together (for example, Oblada melanura C1-22-IV and C2-22-IV in copy 1 clade and
Polysteganus praeorbitalis SA34-C9-22-III and SA34-C8-22-III in copy 2 clade). A
distance based tree was produced to examine branch lengths between taxa. Neighborjoining analysis of these data produced a tree based on genetic distance (Figure 41). For
copy 1 sequences, many of the terminal branches showed very little genetic distance
238
between species (for example Morone chrysops, Lagodon rhomboides and Calamus
nodosus). The same trend was found in terminal nodes of copy 2 sequences, but a long
internal branch separated copy 2 sequences from the nemipterids. Overall, most taxa
were separated by short terminal nodes.
239
DISCUSSION
It would appear that there are more than one copy of the Tmo-4c4 locus in sparids based
on the analyses of sequences generated in this study. The sequences of the two putative
copies could be distinguished by eye. For example, in the clone Calamus nodosus-C130-III the first three base pairs were ATT, but in the clone 2Calamus nodosus-C2-30-III
the first three base pairs were ATA. These sequences are the same for all putative copy 1
and copy 2 genes, respectively. Similarly, copy 2 sequences share a string of five
thymines from base 34 through 39. Blast analysis confirmed that copy 1 clones were
extremely similar to GenBank sequences of the Tmo-4c4 locus (Streelman and Karl,
1997) and the copy 2 sequences were novel, but still very similar to GenBank Tmo-4c4
locus sequences (Steelman and Karl, 1998). Both parsimony analysis and neighborjoining analysis indicated that putative copy 1 and copy 2 sequences were different from
each other.
The unexpected result of two different sequence types might be the result of
contamination. The Tmo-4c4 locus might have been sequenced from a containment
rather than from the intended sample. Contamination of the original DNA or DNA
preparations might cause an unwanted product (such as a fungus or bacteria) to be
amplified during PCR. If this product were then cloned and sequenced, two different
“copies” of DNA might appear, one copy representing sequences from fish, and the other
from the contaminant. However, if this were the case, one would expect the contaminant
sequences to be nearly identical. This was not the case. Divergences between copy 2
species ranged from range 0.2 % to 6.2%. No contaminant was amplified in negative
PCR controls, and a numerous tests of PCR chemicals revealed no containment.
240
This is not to say that a contaminant was not present in all (or some) DNA tubes
or subsequent isolations. Re-isolations of fresh DNA using different isolation chemicals
from those samples where two copies existed still produced two putative copies. Plasmid
contamination was also considered. If a plasmid of a particular sequence contaminates
PCR or other steps, the contaminant plasmid is often preferentially amplified or cloned
over the target of interest. However, I don’t think this was the case for two reasons.
First, a plasmid contaminant would have very nearly identical sequences, so there would
be very little genetic distance between taxa. This is clearly not the case for most clones in
this study. (It might explain the similarity of Clone Spondyliosoma cantharus C17-30IV with Morone saxatilis C6- 22-III and clone Polysteganus praeorbitalis C10-22-IV
with clone Lateolabrax japonicus C1-6-III). Second, I direct sequenced two samples
(Pterogymnus laniarius and Cymatoceps nasutus) of the copy 2 sequence before the
Tmo-4c4 locus was cloned. Therefore, it would be unlikely for Tmo-4c4 plasmid
contamination to effect direct sequencing, because no plasmid in the lab corresponded to
that locus.
I think that it is likely that there are multiple copies of the Tmo-4c4 locus in the
sparids. No second copy was found in any of the outgroup species. Because the sparids
are very closely related, it is possible that the second copy represents a recent duplication
event and there has been little time for mutation to effect the gene.
The Tmo-4c4 locus did not have a useful substitution rate in the sparids to resolve
relationships. There was very low sequence divergence between copy 1 gene sequences
and even less between copy 2 gene sequences. The low sequence divergence in this gene
might partially explain why outgroup species were included with ingroup species in the
parsimony tree.
Neither putative copy of the Tmo-4c4 gene was useful for resolving phylogenetic
relationships due to low sequence divergence. In all trees the branch lengths of most
241
terminal nodes were very short. This was an indication that there was not enough genetic
difference between taxa to construct an informative phylogenetic tree. If there are few
substitutions between taxa, then not enough synapomorphies exist for phylogenetic
resolution. This could cause closely related taxa to be split apart based on only a few
substitutions. Consequently, only a couple of nucleotide substitutions might have been
enough divergence to prevent different clones from the same sample from grouping
together. In an uninformative tree, small differences due to sequencing error or to
parental copy can cause tree structuring, but are phylogenetically misinformative because
they do not reflect synapomorphies. Due to these problems, no further analyses of these
data was warranted.
242
TABLE 21. COLLECTION DATA FOR SPECIMENS USED. MUSEUM
COLLECTION NUMBERS, COLLECTION LOCALITY ARE GIVEN. MUSEUM
ACRONYMS ARE FOLLOWING (LEVITON ET AL., 1985 AND 1988).
All
Museum
Collection
TAXA
Outgroup Taxa
Centropomidae
Lutjanidae
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
Nemipteridae
Scolopsis ciliatus
Ingroup Taxa
SPARIDAE
Boopsinae
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Denticinae
Cheimerius nufar
Diplodinae
Lagodon rhomboides
Pagellinae
Pagellus bogaraveo
Pagrinae
Pagrus auratus
Sparinae
Calamus nodosus
Cymatoceps nasutus
Porcostoma dentata
Centracanthidae
Spicara alta
Catalog#
Locality
No Voucher
USNM 346695
Philippines., Northern Negros, Fish Market
No Voucher
VIMS 10193
VIMS Uncat
UT 85.91
VIMS Uncat
fish market, Spain
Cherokee Reservoir, Grainger Co. TN
USNM 345202
USNM 346853
Philippines, Luzon, Manila, Fish Market
Philippines, Guimaras Island, JTW 95-1
OM1
RUSI 49672
RUSI 49457
KENT 22
RUSI 49443
RUSI 49686
RUSI 49688
VIMS 010192
No Voucher
VIMS Uncat
Chesapeake Bay
Bermuda (BBC)
KENT 176
KENT 46
New Zealand, Sydney Fish Market
05960006
RUSI 49445
KENT SA8
KENT SAPL1
Plettenberg Bay, South Africa
KENT 24BSA
Carpenter, K.E.
243
Table 22. PRIMERS AND SEQUENCES USED FOR THE TMO-4C4 LOCUS.
Tmo-4c4F
CCTCCGGCCTTCCTAAAACCTCTC
Tmo-4c4R
CATCGTGCTCCTGGGTGACAAAGT
M13+Tmo4c4F
cacgacgttgtaaaacgacCCTCCGGCCTTCCTAAAACCTCTC
M13+Tmo4c4R
ggataacaatttcacacaggCATCGTGCTCCTGGGTGACAAAGT
244
TABLE 23. CLONE NUMBERS SEQUENCED FOR EACH TAXON ARE GIVEN.
TWO SAMPLES INDICATED WERE DIRECT SEQUENCED.
All
TAXA
Outgroup Taxa
Centropomidae
Lutjanidae
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
Nemipteridae
Scolopsis ciliatus
Ingroup Taxa
SPARIDAE
Boopsinae
Oblada melanura
Pachymetopon aeneum
Sarpa salpa
Denticinae
Cheimerius nufar
Diplodinae
Lagodon rhomboides
Pagellinae
Pagellus bogaraveo
Pagrinae
Pagrus auratus
Sparinae
Calamus nodosus
Cymatoceps nasutus
Porcostoma dentata
Centracanthidae
Spicara alta
Clone Numbers
Direct Sequence
Clone:C10A 29 IV
No
Clone: C15 22 III
No
Clone: C14 15 III
Clone: C1 6 III
Clone: C7 6 III
Clone: C6 15 III
Clone: C6 22 III
No
No
No
No
No
Clone C23 15 III
Clone: C11 6 III
No
No
Clones: C1 22 IV, C2 22 IV
Clones: C12 05 VI, C14 05 V
No
No
No
No
Clone: C8 5 VI
Clones: C9 22 III, C8 22 III
Clone: C10 22 IV
No
No
No
Clones: C2 05 VI, C1 05 VI
Clones: C8 30 III , C7 30 III
No
No
No
No
No
Clones: C1 30 III, C2 30 III, C3 30 III
Clone: C2 15 III
No
Yes
No
Yes
Clone: C13 29 IV
No
245
Table 24. MULTIPLE ALIGNMENTS OF NUCLEOTIDE SEQUENCES OF THE
NUCLEAR LOCUS Tmo-4c4. THOSE TAXA WITH A PREFIX OF “2" ARE
CONSIDERED PUTATIVE COPY 2
246
Centropomus undecimalis-C10A-29-IV
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
Oblada melanura-C1-22-IV
Pachymetopon aeneum-C13-30-IV
Sarpa salpa-C14-05-VI
Spondyliosoma cantharus-C18-30-IV
Polysteganus praeorbitalis-SA36-C10-22-IV
Pagellus bogaraveo-C21-30-IV
Archosargus probatocephalus-C1-05-VI
Diplodus bermudensis-C9-30-III
Lagodon rhomboides-C8-30-III
Pagrus auratus-C19-30-III
Porcostoma dentata-C11-30-III
Calamus nodosus-C1-30-III
2Calamus nodosus-C2-30-III
2Porcostoma dentata-C12-30-III
2Cymatoceps nasutus-C9-15-III
2Cymatoceps nasutus-direct
2Polysteganus praeorbitalis-SA34-C8-22-III
2Pterogymnus lanarius-direct
2Pterogymnus lanarius-C2-15-III
2Cheimerius nufar-C8-5-VI
2Spicara alta-C13-29-IV
ATTAAGAAAAGGGTGTTTGAAAATGACTCGCTGACATTTTGCGCCGAAGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCCCTAACATTTTATGCTGAGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCTCTAACATTTTACGCTGAGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTTGCTAATGTTTAACGCTGAGGT
ATTAAGAWAAGAGTGTTTGAAAATGACTTGCTAATGTTTAACGCTGAGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCCTTATCATTTTATGCTGAGGT
ATTAAGATAAGAGTGTTTGAAAATGACTCCTTATCATTTTATGCTGAGGT
ATTAAGAAAAGAGTGTTTGARAATGACTTGCTAATGTTTAACGCTGAGGT
ATTAAGAAAAGAATGTTTGAAAATGACTTGCTAATGTTTAACGCTGAGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCCTTATCATTTTATGCTGTGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCCTTATCATTTTATGCTGTGGT
ATTAAGAAAAGAGTGTTTGAAAATGACTCTTTATCATTTTATGCTGAGGT
ATTAAGAAAAGAGTGTTTGATAATGACTCCTTATCATTTTATGCTGAGGT
ATTAAGAAAAAGATGTTTGAAAATGACTTGCTAATGTTTAACGCTGAGGT
ATAAAGAAAAGAGTGTTCGAAAATGACTCTCTCACATTTTTTGCCGAGGT
ATAAAGAAAAGAGTGTTCGAAAATGACTCTCTCACATTTTTTGCCGAGGT
ATAAAGAAAAGAGTGTTCGAAAATGACTCTCTAACATTTTTTGCCGAGGT
ATAAAGGGAAGAGTGTTCGAAGATGACTCTCTAACATTTTTTGCCGAGGT
[
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Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
2Pterogymnus lanarius-direct
2Pterogymnus laniarius-C2-15-III
GTTTGGCCTACCTTCACCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGCCTACCCTCCCCTGAAGTAAAGTGGTTCTGCAACAAATCCCAAC
GTTTGGCCTGCCTTCCCCCGAAGTAAAGTGGTTCTGCAACAAATCCCAAC
GTTTGGCCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGCCTACCGTCTCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGCCTACCGTCTCCTGAGGTGAAGTGGTTCTGCTACATAACCCAAC
GTTTGGCCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCGAC
GTTTGGCCTACCTTCCCCTGACGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGCCTACCTTCCCCTGACGTGAAGTGGTTCTGCAACAAAACCCAAC
ATTTGGGCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
ATTTGGGCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGGCTGCCTTCACCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGGCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
GTTTGGGCTACCTTCCCCTGAGGTGAAGTGGTTCTGCAACAAAACCCARC
GTTTGGGCTACCTTCCCCTGCGGTGAAGTGGTTCTGCGACAAATCCCAAC
GTTTGGGCTACCTTCTCCTGAGGTGAAGTGGTTCTGCAACAAAACCCAAC
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247
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
2Pterogymnus laniarius-direct
TGGTGGCAGACAACAGAGTTAAAATGGGGAGAGATGGTGATACTATCTCA
TGGAAGAGGATGACAGAATTAAAATGGAAAGAGATGGTGATAGCATCTCA
TGGAAGCAGACGACAGAATTAAAATGGAGAGAGACGGTGATAGCATCTCA
TGGTGGCAGACGACAGAGTTAAAATGGAGAGAGATGGCGATAGCATCTCA
TGGTGGCAGACGACAGAGTTAAAATGGAGAGAGATGGCGATA?CATCTCA
TGGTTGCAGATGACAGAATTAAAATGGAGAGAGATGGGGATAGCATTTCG
TGGTGGCAGATGACAGAATTAAAATGGAGAGAGATGGGGATAGCATCTCG
TGGTGGCAGATGACAGATTTATTATGGAGAGAGATGGGGATAGCATCTCG
TGGTGGCAGACGACAGAGTTAAAATGGAGAGAGATGGCGATAGCATCCCA
TGGTGGCAGACGACAGACTTAAAATGGAGAGAGATGGCGATAGCATCCCA
TGGTGGCAGACGACAGACTTAAAATGGAGASACATCGCGATAGCATCTCA
TGGTGGCGGATGACAGAATTAAAATGGAGAGAGATGGGGATAGCATCTCG
TGGTGGCGGATGACAGAATTAAAATGGAGAGAGATGGGGATAGCATCTCG
TGGTGGCAGATGATAGAATTAAAATGGAGAGAGATGGGGATAGCATCTCG
TGGTGGCAGACGACAGAGTTAAAATGGAGAGACATGGCGATAGCATCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCATTTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCATTTCA
TAGTGGCAGATGACAGAGTGAGAATGGAGAGAGATGGTGATAGCATCTCA
TCGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCATCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAAAGAGATGGTGATAGCATCTCA
TCGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCCTCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTRATAGCATCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCATCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGATGGTGATAGCATCTCA
TAGTGGCAGATGACAGAGTTAAAATGGAGAGAGACGGTGATAGCATCTCA
[
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150]
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
CTAACAGTTCACAATGTCACTAAAGCAGATCAGGGAGAGTATATCTGCGA
CTGACAATTCACAATGTTACTAAAGCCGATCAGGGAGAATATATCTGCGA
CTGACAATTCACAACGTCACTAAAGCCGACCAGGGAGAGTATATCTGCGA
CTAACAATTCACAATGTTACTAAAGCTGACCAGGGAGAGTATATCTGTGA
CTAACAATTCACAATGTTACTAAAGCCGACCAAGGCGAGTATATCTGTGA
CTAACAATTCACAATGTCACTAAAGCCGACCAGGGCGAGTATATCTGTGA
CTAACMATTCACAATGTCACTAAAGCCGACCAGGGCGAGTATATCTGTGA
CTAACAATTCACAATGTCACTAAAGCCGGCCAGGGCGAGTATATCTGTGA
CTAACAATTCACAATGTTACTAAAGCSGACCAGGGCGAGTATATCTGTGA
CTGACAATTCACAATGTCACTAAAGCTGATCAGGGAGAATATATCTGCGA
CTGACAATTCACAATGTCACTAAAGCTGATCAGGGAGAATATATCTGCGA
CTAACAATTCACAATGTCACTAAAGCCGATCAGGGAGAGTATATCTGCGA
CTAACAATTCACAATGTCACTAAAGCTGATCAGGGAGAGTATATCTGCGA
CTAACAATTCACAATATCACTAAAGCCGATCAGGGAGAGTATATCTGCGA
CTAACAATTCACAATGTCACTAAAGCTGATCCGGGAGAGTCTATCTGCGA
CTAACAATTCACAATGTCACTAAAGCCGATCAGGGAGAGTATATCTGTGA
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248
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
GGCTGTGAACTATGTCGGGGAAGCCAGAAGTGTTGCTCTGGTGGTAGTTG
GGCTATGAATTATGTCGGGGAAGCCAGAAGTGTTGCTTCAGTGGTAGTTG
GGCTATGAATTATGTTGGGGAAGCAAGGAGTGTTGCTTTAGTGGTTGTTG
GGCTGTAAACTATGTTGGGGAAGCCAGAAGTGTTGCTTTAGTGGTAGTTG
GGCTGTGAACTATGTTGGGGAAGCCAGAAGTGCTGCTTTAGTGGTAGTTG
GGCTGTGAACTATGTTGGGGAAGCCAGAAGTGTTGCTTTAGTGGTAGTTG
GGCTGTGAACTATGTTGGGGAAGCCAGATGTGTTGCTTTAGCGGTAGTTG
GGCTGTGAACTATGTTGGGGAAGCCAGAAGTGTTGCTTTAGTGGTAGTCG
GGCTGTCAATTACGTCGGGGAAGCCAGAAGTGTTGCTTTAGTTGTTGTTG
AGCTGTCAATTACGTCGGGGAAGCCAGAAGTGTTGCTTTAGTTGTTGTTG
GGCTGTCAATTACGTCGGGGAAGCCAGAAGCGTTGCTTTAGTTGTTGTTG
GGCTGTCAATTATGTCGGGGAAGCCAGAAGTGTTGCTTTAGTTGTTGTTG
GGCTGTCAATTACGTCGGGGAAGCCAGAAGCGTTGCTTTAGTTGTTGTTG
GGCTGTMAATTACGTCGGGGAAGCCAGAAGTGTTGCTTTAGTTGTTGTTG
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250]
250]
250]
250]
250]
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
TATCACAGGAAGTAAGGTTCATGCCTGCCCCACCTGCTGTCACTCATCAA
TATCACAGGAAGTGAGGTTCTTGCCTGCTCCACCTGCTGTCACCCATCAG
TGTCACAGGAAGTGAGGTTCTTGCCTGCCCCGCCTGCTGTCACCCATCAG
TATCGCAGGAAGTGAGGTTCATGCCTGCTCCACCTGCTGTCACCCACCAG
TATCGCAGGAAGTGAGGTTCATGCCTGCCCCACCTACTGTCACCCATCAG
TATCGCAGGAAGTGAGGTTCATGCCTGCTCCACCTACTGTCACCCATCAG
TATCGCAGGAAGTGAGGTTCATGCCTGCACCACCTACTGTCACCCATCAG
TATCGCAAGATGTGAGGTTCATGCCTGCTCCACCTGCTGTCACCCTCCGG
TATCGCAAGAAGTGAGGTTCATGCCTGCTCCACCTGCTGTCACCCACCAG
TATCGCAGGAAGTGAGGTTCATGCCTGCTCCACCTGCTGTCACCCACCAC
TATC?CAGGAAGTGAGGTTCATGCCTGCTCCACCTACTGTCACCCATCAG
TATCGCAAGAAGTGAGGTTCATGCCTGCTCCACCTGCTGTAACCCACCAA
TGTCACAGGAAGTTAGGTTCTTGCCTGCCCCACCTGCTGTCACCTGTCAG
TGTCACAGGAAGTTAGGTTCTTGCCTGCCCCACCTGCTGTCACCTGTCAG
TGTCACAAGAAGTGAGGTTCATGCCTGCCCCACCTGCTGTCACCCATCAG
TGTCACAAGAAGTAAGGTTCATGCCTGCCCCACCTGCTGTCACCCATCAG
TGTCACAAGAAGTGAGGTTCCTGCCTGCCCCACCTGCTGTCACCCATCAG
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249
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
CATGTGATGGAGTTTGATGTGGAGGAAGACGACTCCTCTCGTTCTCCATC
CATGTCATGGAGTTTGATGTTGAGGAGGATGACTCGTCTCGCTCTCCTTC
CATGTCATGGAGTTTGATGTTGAGGAGGACGACTCATCACGCTCTCCTTC
CATGTGATGGAGTTTGATGTGGAGGAAGATGACTCCTCTCGTTCTCCATC
CATGTAATGGAGTTTGATGTGGAGGAAGATGACTCATCTCGTTCTCCCTC
CATGTAATGGAGTTTGATGTGGAGGAAGATGACTCCTCTCGTTCTCCTTC
CATGTAATGGAGTTTGATGTGGAGGAAGATGACTCATCTCGTTCTCCTTC
CATGTGATGGAGTTTGGTGTGGAGGAAGATGACTCCTCTCGTTCTCCATC
CACGTGATGGAGTTTGATGTGGAGGAAGATGACTCCCCTCGTTCTCCATC
CTTGTGATGGAGTTTGATGTGGAGGAAGATGACTCATCTCGTTCTCCCTC
CACGTGATGGAGTTTGATGTGGAGGAAGATGACTCCCCTCGTTCTCCATC
CATGTGATGGAGTTTGATGTGGAGGAAGATGACTCGTCTCGCTCTCCTTC
CATGTGATGGAGTTTGATGTGGAGGAGGATGATTCGTCTCGCTCTCCTTC
CATGTGATGGAGTTTGATGTGGAGGAAGATGATTCGTCTCGCTCTCCTTC
CATGTGATGGAGTTTGATGTGGAGGAAGATRACTCKTCTCGMTCTCCTTC
CATGTGATGGAGTTTGATGTGGAGGAAGACGACTCGTCTCGCTCTCCTTC
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Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
CCCTCAGGAGATTCTGCTTGAAGTAGAGTTGGATGAAAATGAGGTGAAAG
TCCTCAGGAGATTCTGCTTGAAGTCGAACTGGATGAAAACGAAGAGAAAG
CCCTCAGGAGATCCTGCTTGAAGTAGAACTAGATGAAAACGAGGTGAAAG
CCCTCTAGAGATTCTGCTCGAAGTTGAATTAGATGAAAACGAGGTGAAAG
CCCTCAAGAGATTCTGCTCGAAGTTGAATTAGATGAAAACGAGGTGAAAG
CCCTCAGGAGATTCTGCTTGAAGTAGAATTGGATGAAAATGAGGTGAAAG
CCCTCAAGAGATTCTGCTCGAAGTTGAATTAGATGAAAACGAGGTGAATG
CCCTCAGGAGATTCTGCTTGAAGTAGAATTGGATGAAAACGAGGTGAAAG
ACCTCAGGAGATTCTGCTCGAAGTCGAATTGGATGAAAATGAGGTGAAAG
ACCTCAGGAGATTCTGCTCGAAGTCGAATTGGATGAAAATGAGGTGAAAG
CCCTCAGGAGATTCTGCTCGAAGTGGAATTGGATGAAAATGAGGTGAAAG
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250
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
AATTTGAGAAACAGGTTAAGATCATCACCATTCCCGAGTACACGGCTGAC
AATTTGAGAAACAGGTTAAGATTATCACCATCCCTGAGTACACTGCTGAC
AATTTGAGAAACAGGTTAAGATTATCACCATTCCCGAGTACACTGCTGAC
AATTTGAGAAACAGGTTAAGATCATCACTATTCCTGAGTACACAGCCGAC
AATTTGAGAAACAAGTTAAGATCATTACCATTCCTGAGTACACAGCCGAC
AATTTGAGAAACAGGTTAAGATCATTACCATTCCTGAGTACACAGCCGAC
AATTTGAGAAACAGGTTAAGATCATTACCATTCCT-AGTACACAGCCGAC
AATTTGAGAAACAGGTTAAGATCATTAMCATTCCTGAGTACACAGCCGAC
AATTTGAGAAACAGGTTAAGATCATCACGATTCCAGAGTACACAGCAGAC
AGTTTGAGAAACAGGTTAAGATCATCACGATTCCAGAGTACACAGCAGAC
AATTTGAGAAACAGGTTAAGATCATCACGATTCCCGAGTACACAGCGGAC
AATTTGAGAAACAGGTTAAGATCATCACGATTCCCGAGTACACAGCAGAC
AATTTGAGAAACAGGTTAAGATCATCACAATTCCCGAGTACACAGCGGAC
AATTTGAGAAACAGGTTAAGATCATCACAATTCCCGAGTACACAGCGGAC
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Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
AACAAGAGCATGATCATATCTTTGGATGTGTTACCAAGTATTTATGAGGA
AACAAGAGCATGATCATATCTTTGGATGTGTTGCCAAGTATCTATGAGGA
AACAAGAGCATGATCATATCTTTGGATGTGTTGCCAAGTATTTATGAGGA
AACAAGAGCATGATCATATCTTTGGATGTGTTACCGAGTATTTATGAGGA
AACAAGAGCATGATCATTTCTTTGGATGTGTTACCAAGTATTTATGAGGA
AACAAGAGCATGATAATTTCTTTGGATGTATTACCGAGTATTTATGAGGA
AACAAGAGCATGATAATTTCTTTGGATGTGTTACCGAGTATTTATGAGGA
AACAAGAGCATGATCATATGTTTGGATGTGTTACCGAGTATTTATGAGGA
AACAACAGCATGATCATATCTTTGGATGTGTTACCGAGTATTTATGTGGA
AACAAGAGCATGATCATATCTTTGGATATGTTACCGAG?ATTTATGAGGA
AACAAGAGCATGATAATTTCTTTGGATGTGTTACCGAGTATTTATGAGGG
AACAAGAGCATGATCATATCTTTGGATGTATTACCGAGTGTTTATGAGGA
AACAAGAGCATGATCATATCTTTGGATGTATTACCGAGTGTTTATGAGGT
AACAAGAGCATGATCATATCTTTGGATGTATTACCGAGTATTTATGAGGA
AACAAGAGCATGATCATATCTTTGGATGTATTACCAAGTATTTATGAGGA
AACAAGA?CATGATCATATCTTTGGATGTATTACCGA?TATTTATGA?GA
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251
Scolopsis ciliatus
Lutjanus decussatus
Morone americanus
Morone chrysops
Morone saxatilis
GGGTGCTGTGG
GGGAACTGTGG
GGGCACTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGCACTGTGG
GGGCACTGTGG
GGGCACTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCAGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
GGGTGCTGTGG
AGGCACTGTGG
AGGCACTCTGG
AGGCACTGTGG
AGGCACTGTGG
AGGCACTGTGG
AGGCACTGTGG
AGGCACTGTGG
GGGTGCTGTGG
A?GCACTGTGG
AGGCACTGTGG
AGGCACTGTGG
AGGCACTGTGG
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Figure 40. Total substitutions plotted as a function of sequence divergence for putative
copy 1 (top) and copy 2 (bottom) of the Tmo-4c4 locus. C1=copy 1, C2=copy 2,
Ts=Transition, Tv=Transversion.
60
50
Total Substitutions
40
30
20
10
0
0
0.05
0.1
0.15
0.2
Uncorrected Sequence Divergence
C1 Ts
C2 Tv
20
Total Substitutions
15
10
5
0
0
0.01
0.02
0.03
0.04
Uncorrected Sequence Divergence
C2 Ts
C2 Tv
0.05
0.06
0.07
254
Figure 41. Parsimony analysis of all clone sequences and characters resulted in 81
equally parsimonious trees (strict consensus, tree length = 270, CI = 0.7185, RI =
0.9110). Clone number follows taxon name. Putative copy 2 sequence clade has heavystroke weight lines.
Centropomus undecimalis (C10A 29 IV)
Scolopsis ciliatus
Polysteganus praeorbitalis (SA34 C8 22 III)
Porcostoma dentata (C12 30 III)
Cheimerius nufar (C8 5 VI)
Pterogymnus laniarius direct
Pterogymnus laniarius (C2 15 III)
Spicara alta (C13 29 IV)
Calamus nodosus (C2 30 III)
Cymatoceps nasutus (C9 15 III)
Cymatoceps nasutus (direct)
Archosargus probatocephalus (C1 05 VI)
Morone americanus
Lagodon rhomboides (C7 30 III)
Morone chrysops
Morone saxatilis
Diplodus bermudensis (C10 30 III)
Spondyliosoma cantharus (C17 30 IV)
Sarpa salpa (C12 05 VI)
Pagrus auratus (C20 30 III)
Pachymetopon aeneum (C15 30 IV)
Pagellus bogaraveo (C21 30 IV)
Lutjanus decussatus
Polysteganus praeorbitalis (SA36 C10 22 IV)
Oblada melanura (C1 22 IV)
256
Figure 42. Neighbor-joining tree of all clone sequences and characters. Clone number
follows taxon name. Putative copy 2 sequence clade has heavy-stroke weight lines.
Lutjanus decussatus
Polysteganus praeorbitalis (SA36 C10 22 IV)
Morone saxatilis
Morone chrysops
Morone americanus
Scolopsis ciliatus
Cymatoceps nasutus (direct)
Spicara alta (C13 29 IV)
Cheimerius nufar (C8 5 VI)
Pterogymnus laniarius direct
Pterogymnus laniarius (C2 15 III)
Centropomus undecimalis (C10A 29 IV)
0.01
258
CONCLUSIONS
To summarize the results of phylogenetic hypotheses tested in this dissertation:
1.
The Sparidae were monophyletic in all analyses, and were inclusive of the
centracanthid Spicara.
2.
There were two distinct groups of sparids in all analyses.
3.
There was no support from any of these analyses for the monophyly of any of the
six recognized subfamilies of sparids (Boopsinae, Dentecinae, Diplodinae,
Pagellinae, Pagrinae or Sparinae).
4.
The Sparoidea were monophyletic in the weighted cytochrome b nucleotide
analysis, but there was no support for a monophyletic Sparoidea from the 16S or
combined data. The Lethrinidae were sister to the Sparidae in all cytochrome b
analyses.
Monophyly of the Sparidae
The monophyly of the Sparidae was well supported in all analyses from all datasets. The
centracanthid Spicara was found within the sparid clade in all phylogenies derived and the
centracanthids were considered members of the Sparidae. The overall placement of the
centracanthids could not be verified, because this study included members of Spicara but did not
include Centracanthus. The molecular evidence support the provisional insertion of Spicara
within the sparids, but these same data question the monophyly of the Centracanthidae because
259
there was no support for a monophyletic Spicara; it was polyphyletic in the unweighted and
weighted nucleotide analyses.
Generic Relationships within the Sparidae
In all phylogenies, there was strong support for Acanthopagrus berda sister to
Sparidentex hast and there was robust support in all analyses for a sister relationship between
Pachymetopon aeneum and Polyamblyodon germanum. There was strong support from the 16S
data to unite Boops boops and Sarpa salpa, but less support from the cyt b data as only the
weighted and amino acid trees support the clade. In all analyses, the western Atlantic Diplodus
holbrooki was sister to the Bermuda endemic Diplodus bermudensis and there was support from
cyt b for Diplodus argenteus sister to D. holbrooki and D. bermudensis (D. argenteus was not
sequenced for the 16S gene). Based on all analyses, Pagrus, Pagellus, Dentex, and the
centracanthid Spicara are not monophyletic and that a revision of these genera is in order.
Subfamilies of the Sparidae
There was no support in any of the analyses from independent or combined datasets for
the monophyly of any of the subfamilies attributed to the Sparidae. Two mitochondrial lineages
were consistently reconstructed in all analyses. These lineages were not structured on trophic
type, as each lineage has multiple trophic types and neither of the lineages was structured solely
on geographic range as similar geographic range was repeated within both lineages. It is evident
from these data that the current subfamilies are artificial and are not valid. Different trophic
types have evolved more than once in the Sparidae and a classification based on dentition and
feeding types does not reflect the evolutionary relationships of these species. Subfamily
designations might be reconsidered to reflect the two lineages found in this study.
The Sparoidea
260
The superfamily Sparoidea was supported from the weighted cyt b data, but not from the
unweighted analyses. The Sparoidea were not monophyletic in the cyt b amino acid residue
analysis because Centropomus undecimalis grouped with the Nemipteridae, although the
Lethrinidae fell intermediate to the Sparidae and Nemipteridae as in the weighted cytochrome b
analysis. There was no support from 16S nucleotides or from combined analyses for a
monophyletic Sparoidea. The evidence for Sparoidea from the cytochrome b unweighted
analysis supports an hypothesis alternative to those of Akazaki (1962) and Johnson (1980). The
molecular data support the Lethrinidae intermediate to a basal Nemipteridae and a derived
Sparidae + Spicara.
Biogeographic Relationships
Biogeographic analysis of sparid evolution revealed that there were two areas of sparid
evolution: the eastern Indian Ocean - western Pacific and the western Indian Ocean Mediterranean/Atlantic. These two areas probably derived from a common Tethyan Sea
ancestor. Little more can be drawn from this analysis, because all vicariance biogeography
clades were based on a single tree example and this is considered equivocal to a single
transformation series explaining a clade (Wiley et al., 1991).
Other Relationships
The Moronidae were sister to Lateolabrax in the weighted cyt b analysis, all16S analyses
and all combined analyses with the exception of the analysis of cyt b amino acids combined
with16S nucleotides. The sister relationship between the moronids and Lateolabrax was
hypothesized by McCully (1962) and Waldman (1986). The overall support for the nodes was
not strong in individual analyses, but there was robust support in the combined analyses. The
Sparidae have been classically associated with the haemulids and lutjanids/caesionids, although
there is no evidence that unite them. These groups were sister to the Sparidae in the combined
261
analyses although the relationships are not robust. In all analyses, except from the cyt b amino
acid residues, Centropomus undecimalis was basal to other percoids.
Future Research
The phylogenetic hypotheses presented in this dissertation are based on mitochondrial
DNA only. Although a nuclear locus was sequenced to reconstruct sparid relationships, no
useable phylogenetic conclusions could be drawn from the data. The relationships presented in
this dissertation should be tested by an independent nuclear locus or multiple nuclear loci. To
date there are few nuclear loci available that are phylogenetically informative for generic level
relationships of perciform fishes. Future research should involve surveying as many loci as
possible to test these relationships and to include as much data as possible in an character
congruence analysis.
These data support a monophyletic Sparidae, but there are questions as to the monophyly
of Pagrus, Pagellus, Dentex and the centracanthid Spicara. Complete revision of these genera
are needed as well as an in depth analyses of the interrelationships of sparid genera based on
morphological analysis. The current subfamilies, as defined, are inadequate to explain the
phylogenetic relationships of the genera and should not be considered valid. A total evidence
approach to sparid relationships that includes as many independent data sources as possible is
needed to help define relationships.
262
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VITA
THOMAS MARTIN ORRELL
Born in Sharon, Connecticut, 6 May 1965. Graduated from Salisbury School,
Salisbury Connecticut in 1983. Earned B.A. in Biology, University of Richmond,
Richmond, Virginia in 1987. Worked as a museum specialist for the Division of Fishes,
National Museum of Natural History, Smithsonian Institution, Washington, D.C. from
1987-1993. Entered the Master of Arts Program, College of William and Mary, School
of Marine Science in 1993. Bypassed Master of Arts Degree and entered the doctoral
program College of William and Mary, School of Marine Science in1996.
.

A MOLECULAR PHYLOGENY OF THE SPARIDAE (PERCIFORMES

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