Phylogenetic relationships among Boleosoma darter species

Molecular Phylogenetics and Evolution 53 (2009) 249–257
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Phylogenetic relationships among Boleosoma darter species (Percidae: Etheostoma)
K.L. Heckman a, T.J. Near a,b, S.H. Alonzo a,*
a
b
Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, CT 06511, USA
Peabody Museum of Natural History, Yale University, New Haven, CT, USA
a r t i c l e
i n f o
Article history:
Received 12 February 2009
Revised 26 May 2009
Accepted 27 May 2009
Available online 31 May 2009
Keywords:
Hybridization
Molecular phylogeny
Breeding color
a b s t r a c t
Darters represent a species rich group of North American freshwater fishes studied in the context of their
diverse morphology, behavior, and geographic distribution. We report the first molecular phylogenetic
analyses of the Boleosoma darter clade that includes complete species sampling. We estimated the relationship among the species of Boleosoma using DNA sequence data from a mitochondrial (cytochrome b)
and a nuclear gene (S7 ribosomal protein intron 1). Our analyses discovered that the two Boleosoma species with large geographic distributions (E. nigrum and E. olmstedi) do not form reciprocally monophyletic
groups in either gene trees. Etheostoma susanae and E. perlongum were phylogenetically nested in E.
nigrum and E. olmstedi, respectively. While analysis of the nuclear gene resulted in a phylogeny where
E. longimanum and E. podostemone were sister species, the mitochondrial gene tree did not support this
relationship. Etheostoma vitreum was phylogenetically nested within Boleosoma in the mitochondrial
DNA and nuclear gene trees. Our analyses suggest that current concepts of species diversity underestimate phylogenetic diversity in Boleosoma and that Boleosoma species likely provide another example
of the growing number of discovered instances of mitochondrial genome transfer between darter species.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
Darters are a species rich clade of North American freshwater
fishes. Extensive work on the taxonomy of darters has produced
a classification that comprises 17 or 18 subgenera and as many
as five genera (Keck and Near, 2008; Near and Keck, 2005; Page,
2000). Over the past several years, DNA sequence data have been
used to investigate species-level relationships within darter clades
(Keck and Near, 2008; Lang and Mayden, 2007; Near, 2002; Near
et al., 2000; Porter et al., 2002; Porterfield et al., 1999). In some
cases, relationships estimated in molecular phylogenies are incongruent with traditional taxonomic arrangements that are based on
overall morphological similarities (Lang and Mayden, 2007; Near,
2002). While a growing number of molecular studies have investigated the evolutionary relationships among darters, there are no
published studies that provide molecular phylogenies of Boleosoma
that sample all the species in the clade.
Boleosoma is an interesting darter clade for molecular phylogenetic analysis, because a clear consensus does not exist regarding
species level diversity in the clade (e.g., Cole, 1965; Cole, 1967;
Shute, 1984; Starnes and Starnes, 1979). In addition, there are
striking differences among these species in male nuptial coloration,
a character routinely used in the diagnosis and differentiation of
darter taxa (Page, 1983). Etheostoma nigrum, E. susanae, and E. olms* Corresponding author. Fax: +1 203 432 3854.
E-mail addresses: [email protected] (K.L. Heckman), thomas.near@
Yale.edu (T.J. Near), [email protected] (S.H. Alonzo).
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2009.05.027
tedi males darken during the reproductive season, while in E. perlongum, E. longimanum, and E. podostemone reproductive males
exhibit bright nuptial coloration (Lindquist et al., 1981; Page,
1983 pp. 89, 92). Boleosoma species also vary in the sizes of their
geographic ranges. Etheostoma nigrum and E. olmstedi are found
throughout a large portion of eastern North America (Figs. 1 and 2),
while the other four Boleosoma species have relatively restricted
geographic distributions (Figs. 1 and 2). A phylogenetic analysis
using discretely coded morphological characters resulted in monophyly of the five sampled Boleosoma species; and within this clade
were two monophyletic groups, a clade containing E. longimanum
and E. podostemone, and a clade containing E. nigrum, E. olmstedi,
and E. perlongum (Simons, 1992).
There have been various opinions regarding the species level
diversity in Boleosoma (Bailey and Gosline, 1955; Cole, 1957; Collette, 1965; Winn, 1958). Five species were recognized in an evaluation of variation in external morphological characters (e.g., scale
row and fin element counts, patterns of squamation), and these
species were distributed among three species groups: (1) Etheostoma olmstedi and E. nigrum, (2) E. longimanum and E. podostemone,
and (3) E. perlongum (Cole, 1957). Based on the analysis of allozyme
data and external morphological characters, E. perlongum has been
argued to represent an ecomorph of E. olmstedi and not a distinct
species (Shute, 1984); however, E. perlongum does exhibit disparate male breeding coloration and life history characteristics when
compared to E. olmstedi (Lindquist et al., 1981; Shute, 1984).
Etheostoma susanae is another Boleosoma species with a
complicated taxonomic history. Jordan and Swain (1883) described
250
K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
E. nigrum
E. susanae
E. perlongum
25
50
100
Scale of Kilometers
Fig. 1. Sampling locations of E. nigrum, E. susanae, and E. perlongum specimens. Species distributions are shaded and each circle represents a sampled location.
Boleosoma susanae from the Cumberland River drainage above the
Cumberland Falls in Kentucky. The species was later considered a
subspecies of Etheostoma nigrum (Kuhne, 1939). The subspecific
status of E. susanae was supported by some systematists (Burr
and Warren, 1986, p. 310; Cole, 1967, 1972; Etnier and Starnes,
1993, pp. 510–512; Page, 1983, pp. 84–85), but others did not accept that E. susanae was sufficiently divergent from E. nigrum to
warrant subspecific status (Clay, 1975, p. 350; Jenkins et al.,
1972, p. 102). Examination of external morphological characters
led to the observation that relative to E. nigrum populations in
the Ohio River drainage, E. susanae was more similar to E. nigrum
populations in the upper Kentucky River system that is geographically adjacent to the upper Cumberland River (Starnes and Starnes, 1979). This morphological similarity was interpreted as
evidence of ‘‘intergradation” resulting from hybridization between
E. susanae and E. nigrum, and used to support the subspecific classification of the former (Starnes and Starnes, 1979). A study of
mitochondrial DNA haplotype variation concluded that Etheostoma
susanae is a distinct species when compared to E. nigrum (Strange,
1998).
Complicating the systematics of Boleosoma further is the reported hybridization and introgression between E. nigrum and
E. olmstedi in areas where the two species are sympatric (Chapleau
and Pageau, 1985; Cole, 1965; Jenkins and Burkhead, 1994, p. 846;
McAllister et al., 1972; Stone, 1947). The two species are morphologically similar with differentiation requiring close inspection of
the number of pores of the infraorbital and preoperculomandibular
canals, completeness of infraorbital canal, degree of squamation of
the cheek, nape and breast, number of rays in the second dorsal fin,
and qualitative assessment of the shape of the pectoral and pelvic
fins (Chapleau and Pageau, 1985; Cole, 1965, 1967; Jenkins and
Burkhead, 1994, p. 846; McAllister et al., 1972; Menhinick, 1991,
p. 172; Stone, 1947). While the frequency of hybrid specimens is
generally low (Chapleau and Pageau, 1985; Cole, 1965; McAllister
et al., 1972), intermediacy of external morphological traits has
been the basis for the hypothesis that E. nigrum and E. olmstedi
are hybridizing in the middle portions of the James and Roanoke
River systems (Clark, 1978; Jenkins and Burkhead, 1994, p. 846);
however, analyses of allozyme variation did not find evidence of
introgression between these two species in the James River system
(Falls, 1982).
The darter species Etheostoma vitreum has long been classified
in the monotypic subgenus Ioa (Bailey and Etnier, 1988; Bailey
and Gosline, 1955; Jenkins and Burkhead, 1994; Page, 1983). Evidence from the distribution of breeding tubercles (Collette,
1965), allozyme variation (Wood and Mayden, 1997), and external
morphology (Bailey and Etnier, 1988) has hinted at a close relationship between E. vitreum and Boleosoma. Phylogenetic analyses
of both mitochondrial DNA and nuclear gene sequences that sampled a single Boleosoma species and E. vitreum resulted in a strongly
supported monophyletic group relative to the other sampled darter
species (Lang and Mayden, 2007; Sloss et al., 2004; Song et al.,
1998). Phylogenetic analyses that sampled two Boleosoma species
and E. vitreum discovered that Boleosoma was paraphyletic with respect to E. vitreum (Mayden et al., 2006; Shaw et al., 1999).
In this study, we present a molecular phylogenetic analysis
based on a mitochondrial and a nuclear gene to investigate the
phylogenetic relationship among species of Boleosoma. We focused
specifically on determining (1) the status and phylogenetic affinities of E. susanae and E. perlongum, (2) whether the widely distrib-
K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
85
80
75
70
15
10
35
30
85
80
75
Fig. 2. Sampling locations of Etheostoma olmstedi, E. podostemone, and E. longimanum specimens. Species distributions are shaded and each circle represents a
sampled location.
uted species E. olmstedi and E. nigrum form reciprocally monophyletic groups, (3) the phylogenetic relationships of E. longimanum
and E. podostemone, and (4) the monophyly of Boleosoma, relative
to E. vitreum. We sampled all six Boleosoma species, multiple populations of E. nigrum and E. olmstedi throughout their large geographic distributions, and E. vitreum. In addition, we sampled
gene sequences from species in other darter clades to serve as outgroups in the phylogenetic analyses.
2. Materials and methods
Specimens of all Boleosoma species and Etheostoma vitreum
were sampled throughout their respective geographic ranges (Figs.
1 and 2, Table 1). Multiple collection sites were sampled for E. nigrum (23 sites) and E. olmstedi (13 sites). Specimens were anesthetized using MS-222 and tissues for DNA isolation were collected as
pectoral fin clips and stored in 95–100% ethanol. After tissue
biopsy, specimens were fixed in 10% formalin for approximately
10 days, rinsed in distilled water for five days, and transferred to
70% ethanol for long-term preservation. Voucher specimens were
deposited in the Yale University Peabody Museum of Natural History fish collection (Moore and Boardman, 1991). In regions where
251
species identification has proven difficult, especially between E. nigrum and E. olmstedi, completeness of the infraorbital canal, number of second dorsal fin rays, and the number of infraorbital pores
were examined for species identification (Jenkins and Burkhead,
1994, p. 846; Menhinick, 1991, p. 172; Page, 1983; Stone, 1947).
DNA was extracted from tissues using the Qiagen DNeasy Blood
and Tissue Kit. Targeted genes were amplified from isolated template DNA using PCR. The mitochondrial encoded cytochrome b
(cytb) and nuclear encoded ribosomal protein S7 ribosomal protein
intron 1 (S7) were amplified using previously published primer sequences and thermal cycling conditions (Chow and Hazama, 1998;
Near et al., 2000). Amplified PCR products were prepared for cycle
sequencing using Qiagen PCR Purification kit. Sequencing was performed at the DNA Analysis Facility on Science Hill at Yale University. Sequence fragments were edited in Sequencher 4.6 and
aligned manually in MacClade 4.05 (Maddison and Maddison,
2000). Individuals that exhibited heterozygous nucleotide sites in
the nuclear encoded S7 intron were analyzed with PHASE software
that implements a Bayesian algorithm for haplotype reconstruction (Stephens and Donnelly, 2003; Stephens et al., 2001), and
the two haplotypes identified in heterozygous individuals were
treated as distinct operational taxonomic units in the phylogenetic
analyses.
Phylogenetic analyses were performed on aligned gene sequences sampled from Boleosoma species and Etheostoma vitreum.
Additional gene sequences from other darter species in the clades
Ammocrypta, Etheostoma and Percina were collated from the Genbank NCBI database and used as outgroup taxa (Table 1). A Bayesian method that implements a Metropolis-coupled Markov Chain
Monte Carlo algorithm was used to estimate phylogenetic relationships using the computer program Mr. Bayes version 3.1.2 (Ronquist and Huelsenbeck, 2003). Models of substitution were
chosen using the Akaike Information Criterion (AIC), as implemented in the computer program Modeltest 3.7 (Posada and
Crandall, 1998). The model of nucleotide substitution used for each
analysis was general-time-reversible with a proportion of sites that
are invariable and among site rate variation (GTR+I+G). Each codon
position was treated as a separate partition in the cytb phylogenetic analysis. There were no partitions applied to the S7 intron
alignment. MrBayes was run for three million generations, excluding the initial 100,000 generations as burnin as assessed by Markov
chain convergence. Optimal Bayesian phylogenies were inferred
for each of the two genes. Posterior trees were visualized using FigTree 1.1 (Drummond and Rambaut, 2007).
3. Results
Nucleotide sequences were generated for two genes, cytb
(1119bp) and S7 (527bp) for 78 specimens (Table 1). The two genes
differed in the number of phylogenetically informative sites; with
the mitochondrial cytb containing more than four times more than
nuclear encoded S7 (218 vs. 35 within Boleosoma). In both the cytb
and S7 Bayesian phylogenies (Figs. 3 and 4), all Boleosoma species
formed a clade and E. vitreum was nested within Boleosoma. The
nodes representing the most recent common ancestor of Boleosoma
and E. vitreum were supported with significant Bayesian posterior
probabilities. The placement of E. vitreum differed in the cytb and
S7 phylogenies. In the cytb tree, E. vitreum shared a common ancestor with a clade containing specimens sampled from northern E.
olmstedi populations, E. longimanum, and E. nigrum sampled from
the Upper James River Drainage; however, this node was not supported with a significant Bayesian posterior probability (Fig. 3). In
the S7 phylogeny, E. vitreum shared a common ancestor with E. nigrum and E. olmstedi sampled from the James River Drainage, and E.
olmstedi sampled from the Savannah and Waccamaw River sys-
252
K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
Table 1
Specimen information including specimen code, tissue collection catalogue numbers (YFTC, Yale Fish Tissue Collection; KU University of Kansas), museum voucher catalogue
numbers (YPM, Yale Peabody Museum of Natural History, Yale University; UT, University of Tennessee Research Collection of Fishes; MCZ, Museum of Comparative Zoology,
Harvard University; INHS, Illinois Natural History; KU, University of Kansas), sampling locations, Peabody Natural History Museum and NCBI Genbank accession numbers. For
four specimens only nuclear data was available while for six other specimens only mitochondrial data was available. Their exclusion has no qualitative effect on the results.
Species
Specimen code
Tissue catalogue
Museum voucher
Stream, county, state
Genbank cytb
Genbank S7 intron
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
C
D
E
A
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
S
T
U
V
W
X
Y
Z
AA
AB
AC
AD
AE
AF
AG
AH
GG
CC
DD
EE
FF
B
C
D
E
F
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
AA
BB
CC
DD
EE
FF
A
C
D
C
D
E
C
D
YFTC 2236
KUI 842
KUI 897
YFTC 263
YFTC 1194
YFTC 1195
KUI 839
KUI 847
YFTC 10417
YFTC 692
YFTC 693
YFTC 10218
YFTC 10232
YFTC 10699
YFTC 10707
YFTC 10744
YFTC 6777
YFTC 8939
YFTC 10816
YFTC 11174
YFTC 9971
YFTC 6999
YFTC 6287
YFTC 7008
YFTC 8393
YFTC 8363
YFTC 8385
YFTC 834
YFTC 9331
YFTC 9332
YFTC 9334
YFTC 9324
YFTC 9326
YFTC 9328
YFTC 9330
YFTC 11173
KUI 869
YFTC 8669
YFTC 8695
YFTC 8705
KUI 5963
No catalogue
No catalogue
YFTC 9257
YFTC 8511
YFTC 8609
YFTC 8748
YFTC 8749
YFTC 8750
YFTC 8761
YFTC 8758
YFTC 8757
YFTC 8716
YFTC 8717
YFTC 8728
YTFC 8732
YFTC 6164
YFTC 5978
YFTC 8781
YFTC 6017
YFTC 8008
YFTC 8009
YFTC 6080
YFTC 8737
No voucher
YFTC 8006
YFTC 8007
YFTC 8689
YFTC 8664
YFTC 8706
YFTC 8347
YFTC 8354
No voucher
KU 23142
KU 23142
INHS 39507
INHS 47437
INHS 47437
KU 23143
KU 23143
YPM18408
INHS 64378
INHS 64378
No voucher
No voucher
YPM 18400
YPM 18400
YPM 18381
No voucher
YPM 15960
YPM 18268
YPM 17303
YPM 17085
UT 91.7547
UT 91.7286
UT 91.7547
No voucher
YPM 16204
YPM 16204
INHS 42974
YPM 18234
YPM 18234
YPM 18234
YPM 18234
YPM 18234
YPM 18234
YPM 18234
YPM 17303
KU 29892
YPM 15883
YPM 16374
YPM 16374
MCZ 163579
No voucher
No voucher
YPM 16162
YPM 15989
YPM 17506
YPM 17063
YPM 17063
YPM 17063
YPM 17063
YPM 17063
YPM 17063
YPM 16363
YPM 16363
YPM 16363
YPM 16363
No voucher
UT 91.7223
YPM 16091
YPM 15865
No voucher
No voucher
No voucher
YPM 16363
No voucher
No voucher
No voucher
YPM 16374
YPM 15883
YPM 16374
YPM 15843
YPM 15843
Catawba Creek, Roanoke, VA
Catawba Creek, Roanoke, VA
Catawba Creek, Roanoke, VA
Lake Andrusia, Beltrami, MN
Wisconsin River, Sauk, WI
Wisconsin River, Sauk, WI
East Branch of Mill Creek, Wabaunsee, KS
East Branch of Mill Creek, Wabaunsee, KS
South Prong Little Black River, Ripley, MO
Lost Creek, Warren, MO
Lost Creek, Warren, MO
Bourbeuse River, Franklin, MO
Bourbeuse River, Franklin, MO
Cub Creek, Garland, AR
Cub Creek, Garland, AR
Ouachita River Montgomery, Arkansas
Cane Creek, Colbert, AL
Beaver Creek, Wilcox, AL
Pumpkin Creek, Lafayette, MS
Wakatoneta Creek, Licking, OH
Elk River, Kanawha, WV
Dix River, Lincoln, KY
Buck Fork at Flat Rock Road, Todd, KY
Dix River, Lincoln, KY
South Fork Kentucky River, Clay, KY
Red Bird River, Clay, KY
Red Bird River, Clay, KY
Middle Fork Kentucky River, Leslie, KY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Oatka Creek, Monroe, NY
Wakatoneta Creek, Licking, OH
Catawba Creek, Roanoke, VA
South Fork Roanoke River, Franklin, VA
Blackwater River, Franklin, VA
Blackwater River, Franklin, VA
Lamoille River, Chittenden, VT
Ash Swamp Brook, Providence, RI
Naugatuck River, Litchfield, CT
Hiller Brook, Dutchess, NY
Dead River, Somerset, NJ
Conowingo Creek, Cecil, MD
Appomattox River, Appomattox, VA
Appomattox River, Appomattox, VA
Appomattox River, Appomattox, VA
Appomattox River, Appomattox, VA
Appomattox River, Appomattox, VA
Appomattox River, Appomattox, VA
Holiday Creek, Appomattox, VA
Holiday Creek, Appomattox, VA
Holiday Creek, Appomattox, VA
Holiday Creek, Appomattox, VA
Briar Creek, Richmond, GA
Green River, Polk, NC
Orange Creek, Putnam, FL
Bush River, Newberry, SC
Camp Swamp Creek, Columbus, NC
Camp Swamp Creek, Columbus, NC
Hollow Creek, Aiken, GA
Holiday Creek, Appomattox, VA
Lake Waccamaw, Columbus, NC
Lake Waccamaw, Columbus, NC
Lake Waccamaw, Columbus, NC
Blackwater River, Franklin, VA
South Fork Roanoke River, Franklin, VA
Blackwater River, Franklin, VA
Laurel Creek, McCreary, KY
Laurel Creek, McCreary, KY
GQ183639
GQ183640
GQ183641
GQ183642
GQ183643
GQ183644
GQ183645
GQ183646
GQ183647
GQ183648
GQ183649
GQ183650
GQ183651
GQ183652
GQ183653
GQ183654
GQ183655
GQ183656
GQ183657
GQ183658
GQ183659
GQ183660
GQ183661
GQ183662
GQ183663
GQ183664
GQ183665
GQ183666
GQ183667
GQ183668
GQ183669
GQ183670
GQ183671
GQ183672
GQ183673
–
GQ183674
GQ183675
GQ183676
GQ183677
GQ183678
GQ183679
GQ183680
GQ183681
GQ183682
GQ183683
GQ183684
GQ183685
GQ183686
GQ183687
GQ183688
GQ183689
GQ183690
GQ183691
GQ183692
GQ183693
GQ183694
GQ183695
GQ183696
GQ183697
GQ183698
GQ183699
GQ183700
–
–
GQ183704
GQ183705
–
GQ183702
GQ183703
GQ183635
GQ183636
GQ183713
GQ183710
GQ183711
GQ183714
–
GQ183715
GQ183716
GQ183717
–
–
GQ183718
GQ183719
GQ183720
GQ183721
GQ183722
GQ183723
–
GQ183724
GQ183725
GQ183726
GQ183727
GQ183728
GQ183729
GQ183730-1
GQ183732
GQ183733
GQ183734
GQ183735
GQ183736
GQ183738-9
GQ183740
GQ183741
GQ183742
GQ183743-4
GQ183745-6
GQ183749
–
GQ183747
GQ183748
GQ183750
GQ183751
GQ183752
GQ183753
GQ183754
GQ183756-7
GQ183758-9
GQ183760-1
GQ183762-3
GQ183764-5
GQ183766-7
GQ183768-9
GQ183770-1
GQ183772-3
GQ183774-5
GQ183776
GQ183777
GQ183778
GQ183779
GQ183780
GQ183781
GQ183782-3
GQ183784
GQ183785
GQ183786-7
GQ183791
–
GQ183792-3
GQ183789
GQ183790
GQ183755
GQ183794
GQ183795
longimanum
longimanum
longimanum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
nigrum
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
olmstedi
perlongum
perlongum
perlongum
podostemone
podostemone
podostemone
susanae
susanae
253
K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
Table 1 (continued)
Species
Specimen code
Tissue catalogue
Museum voucher
Stream, county, state
Genbank cytb
Genbank S7 intron
E.
E.
E.
E.
E.
E
C
D
E
F
YFTC 8358
KUI 833
KUI 834
YFTC 8678
YFTC 8682
YPM 15843
KU 24389
KU 23144
YPM 16372
YPM 16372
Laurel Creek, McCreary, KY
Little Dan River, Patrick, VA
Blackwater River, Franklin, VA
Blackwater River, Franklin, VA
Blackwater River, Franklin, VA
GQ183637
GQ183706
GQ183707
GQ183708
GQ183709
GQ183796
GQ183797
GQ183798
GQ183799-00
GQ183801
AF412536
AY374272
AY964706
AF386535
AF183940
AF183941
AF183943
–
–
AF412570
EF035538
EF035553
AY573272
EF035491
EF035492
EU094725
EF035497
AY517756
susanae
vitreum
vitreum
vitreum
vitreum
Outgroup species
E. virgatum
E. punctatum
E. zonale
Ammocrypta beanii
Ammocrypta bifascia
Ammocrypta clara
Ammocrypta pellucida
Percina aurantiaca
Percina. maculata
tems, and this node was supported with a significant Bayesian posterior probability (Fig. 4).
Sampled individuals of E. olmstedi and E. nigrum were not reciprocally monophyletic in either the cytb or the S7 Bayesian phylogenies. Monophyly of sampled E. nigrum haplotypes was
confounded by the nesting of E. podostemone and E. susanae haplotypes in a large clade containing all E. nigrum haplotypes sampled
from the Mississippi, Mobile, and Great Lakes Drainages (Fig. 3).
Individuals of E. nigrum sampled from the Kentucky River system
grouped in two clades. Those from the lower Kentucky River were
more closely related to specimens sampled from other Ohio River
drainage tributaries; however, specimens from the upper Kentucky
River system shared common ancestry with the upper Cumberland
endemic E. susanae (Fig. 3). Haplotypes sampled from E. nigrum in
the upper portions of the James and Roanoke rivers systems were
closely related to E. olmstedi from northern Atlantic Ocean tributaries. Etheostoma nigrum sampled from the Great Lakes Drainage in
the Genesee River system contained mitochondrial haplotypes that
were phylogenetically disparate, with five of seven haplotypes closely related to other E. nigrum populations sampled from the Ohio
River drainage (Fig. 3). The other two Great Lakes E. nigrum haplotypes were nested within a clade of E. olmstedi haplotypes sampled
from northern Atlantic Ocean tributaries (Fig. 3).
Sampled E. olmstedi haplotypes were distributed in two major
clades, one containing specimens sampled from northern Atlantic
Ocean tributaries that extend from the Connecticut River system
south to the lower James Rivers system. The other clade E. olmstedi
clade was paraphyletic, relative to E. perlongum, and contained
haplotypes sampled from specimens collected in Atlantic Ocean
tributaries south of the James River in Virginia that extended from
the Waccamaw River in North Carolina, south to the St. Johns River
in Florida (Fig. 3). Haplotypes sampled from Waccamaw River E.
olmstedi shared common ancestry with those sampled from E. perlongum in Lake Waccamaw, and uncorrected pairwise sequence
divergence at cytb was approximately 2.3% between Waccamaw
River E. olmstedi and Lake Waccamaw E. perlongum.
The S7 Bayesian phylogeny was much less resolved than the
cytb tree, but like the cytb phylogeny the sampled alleles from E.
nigrum or E. olmstedi were not reciprocally monophyletic (Fig. 4).
Despite lacking resolution when compared to the mitochondrial
phylogeny, the S7 nuclear gene tree provides several interesting insights on Boleosoma relationships. The phylogenetic relationships
of E. longimanum and E. podostemone were discordant between
the cytb and S7 Bayesian phylogenies. In the cytb phylogeny E. podostemone was nested in a clade containing E. nigrum haplotypes,
and E. longimanum was closely related to the southern E. olmstedi
clade and E. nigrum sampled from the upper James River system
(Fig. 3). In the S7 phylogeny E. podostemone and E. longimanum
were sister species, as predicted in previous morphological and
allozyme analyses (Fig. 4, Cole, 1957, 1972; Shute, 1984; Simons,
1992). In addition, the S7 alleles sampled from E. nigrum in the
Great Lakes all clustered together in the nuclear gene tree, but
the node was not supported with a significant Bayesian posterior
probability.
4. Discussion
Molecular phylogenetics has the potential to provide novel insights into species relationships within and among darter lineages
that differ dramatically from inferences based on overall similarity
of external morphological traits (Mayden et al., 2006; Near, 2002).
This is especially true for darter lineages containing species with
subtle morphological distinctions (Hollingsworth and Near, 2009;
Page et al., 2003). In this study, the phylogenies estimated from
mitochondrial and nuclear gene trees provided unique insights
and novel hypotheses regarding the relationships among Boleosoma species. These inferences include strong support for the monophyly of Boleosoma and E. vitreum, lack of reciprocal monophyly in
both E. nigrum and E. olmstedi, resolution of the phylogenetic relationships of E. perlongum and E. susanae, and support for the sister
species relationship of E. podostemone and E. longimanum. In addition to the specific inferences regarding relationships among Boleosoma species, the phylogenetic analyses of the mitochondrial and
nuclear genes highlight problems of species delimitation and identify patterns consistent with introgression resulting from hybridization between Boleosoma species.
Incongruence between gene trees can occur for a number of reasons, including lineage sorting, hybridization, and introgression.
Incongruence due to lineage sorting is more likely when lineages
diversify within a short period of time or when the ancestral species has a large effective population size (Maddison, 1997; Nei,
1987). Hybridization produces a pattern where alleles are shared
between species that may not be each other’s closest relative,
while introgression results when there is a permanent incorporation of alleles into a population that originate from another species.
Differentiating between ancestral polymorphism and introgression
can be difficult (Holder et al., 2001). This issue is exacerbated when
lineage sorting is incomplete and species share ancestral polymorphisms, which is likely for nuclear genes that have an effective
population size that is four times larger than mtDNA genes (Hudson and Coyne, 2002; Hudson and Turelli, 2003). In this study, it
appears that the S7 phylogeny lacks resolution due to ancestral
polymorphism, because the backbone of the tree is poorly sup-
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K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
Fig. 3. Fifty percent majority rule consensus tree of Bayesian posterior distribution generated using cytochrome b (mtDNA), estimated using substitution model GTR+I+G
partitioned by codon position in Mr. Bayes 3.1.2 (mean ln likelihood = 8271). Nodes with a Bayesian posterior probability value P0.95 are marked with an asterisk.
ported and relationships among some of the species are poorly resolved (Fig. 4).
Phylogenetic analyses of both the mitochondrial and nuclear
genes produce trees consistent with previous hypotheses that
Boleosoma and E. vitreum are closely related (Cole, 1972; Lang
and Mayden, 2007; Near et al., 2000; Shaw et al., 1999; Sloss
et al., 2004; Song et al., 1998; Wood and Mayden, 1997). Etheostoma vitreum was nested within Boleosoma in both gene trees,
but relationships of E. vitreum were less resolved in the mitochondrial gene tree (Figs. 3 and 4). Our taxon sampling included only a
few other darter species as outgroups (Table 1), perhaps casting a
degree of uncertainty on this result. However, the most recent
common ancestor of the Boleosoma–E. vitreum clade was supported
with significant Bayesian posterior probabilities in both gene trees
(Figs. 3 and 4), indicating that increased taxon sampling of more
darter species is unlikely to change this result.
In the James and Roanoke River systems, E. nigrum is distributed
in the upper reaches that are at higher elevation with a piedmont
topography, and E. olmstedi is distributed in the lower reaches of
these systems (Cole, 1957, 1972; Jenkins and Burkhead, 1994;
Zorach, 1971). Jenkins and Burkhead (1994 pp. 846–847) identify
regions in these two river systems where E. nigrum and E. olmstedi
are indistinguishable, because morphological characters appear
intermediate between the two species. It is possible that populations classified as E. nigrum in the upper James, Roanoke, Tar, and
Neuse rivers systems (the last two were not sampled in our analy-
K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
255
Fig. 4. Fifty percent majority rule consensus tree of Bayesian posterior distribution generated using S7 ribosomal protein intron 1 (nDNA), estimated using substitution model
GTR+I+G in Mr. Bayes 3.1.2 (mean ln likelihood = 2398.97). Nodes with a Bayesian posterior probability value P0.95 are marked with an asterisk. When two alleles were
sampled from a single specimen, the second allele is differentiated with a ‘‘b”.
ses) are closely related to E. olmstedi (populations from the lower
James River system and north), and not related to E. nigrum. The
only characters that can distinguish E. nigrum and E. olmstedi are
the morphology of the infraorbital canal, the modal count of second dorsal fin rays, and a qualitative assessment of pectoral and
pelvic fin shape (Chapleau and Pageau, 1985; Cole, 1965, 1967;
Jenkins and Burkhead, 1994, p. 846; McAllister et al., 1972; Menhinick, 1991, p. 172; Stone, 1947). In addition, lateral line scale
counts have been used as evidence to assign populations in the
upper James, Roanoke, Tar, and Neuse rivers systems (Atlantic
Slope) as E. nigrum. In a study of 63 populations, the mean lateral
line scale count in E. olmstedi ranged from 37.9 to 55.5, but most
values were between 48 and 50 (Cole, 1967), Mississippi River
drainage E. nigrum exhibited slightly lower counts with mean values ranging between 45.1 and 52.4 (Cole, 1972), and Atlantic Slope
E. nigrum mean lateral line scale counts were the lowest and ranged between 39.2 and 43.8 (Cole, 1972). Etheostoma nigrum from
the Mississippi Drainage and E. nigrum from the Atlantic Slope
were considered conspecific, because they both exhibit interrupted
infraorbital canals and lower lateral line scale counts, relative to
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K.L. Heckman et al. / Molecular Phylogenetics and Evolution 53 (2009) 249–257
E. olmstedi. Based on the patterns present in our phylogenetic analyses (Figs. 3 and 4), it is quite possible that these morphological
traits do not accurately delimit species diversity currently recognized as E. nigrum and E. olmstedi.
The issue of incongruence between morphological characters
and delimited species is also germane to the phylogenetic relationships of E. susanae discovered in the mitochondrial gene tree
(Fig. 3). Previous analyses revealed substantial morphological similarity between E. susanae and E. nigrum populations in the upper
Kentucky River system, relative to E. nigrum in other Ohio River
drainage tributaries that included the lower Kentucky River system
(Starnes and Starnes, 1979). Our phylogenetic analyses are consistent with this observation. In the mitochondrial phylogeny, E. susanae is resolved as the sister species of E. nigrum sampled in the
upper Kentucky River system, and this clade is the sister lineage
of all other sampled E. nigrum from the Mississippi River drainage,
Great Lakes, and Mobile drainage (Fig. 3). Etheostoma nigrum sampled from the lower Kentucky River were distantly related to E. susanae and E. nigrum from the upper Kentucky River system. The
results from morphological analyses and the mitochondrial phylogeny leads to the possibility that E. susanae may include the E. nigrum populations in the upper Kentucky River system, or these
upper Kentucky River E. nigrum populations may represent a new
and undescribed species. Future analyses of molecular phylogenies
and morphological variation should shed light on this important
problem in Boleosoma species delimitation.
The molecular phylogenies are consistent with a hypothesis
that E. perlongum shares common ancestry with E. olmstedi in the
Waccamaw River (Shute, 1984). This result indicates that speciation of E. perlongum was relatively recent and in this short time
it has diverged substantially in ecology, reproductive behavior,
and male nuptial coloration (Lindquist et al., 1981; Shute, 1984;
Shute et al., 1982). In the process of diversification, E. perlongum
may have been subjected to a novel set of selective pressures associated with the lacustrine habitat of Lake Waccamaw that resulted
in subsequent speciation. Previous studies have suggested that
predation pressures, population bottlenecks due to climate change,
and ecological specialization were factors in evolution of E. perlongum (Hubbs and Raney, 1946; Shute, 1984).
The mitochondrial and nuclear gene trees reveal patterns of
introgression that appear both ancient and contemporaneous. Ancient mitochondrial transfer between species appears to partially
explain the substantial incongruence between the mitochondrial
and nuclear gene trees with regard to relationships of E. podostemone and E. longimanum. Assessment of overall morphological
similarity (Cole, 1972), phylogenetic analysis of discretely coded
morphological characters (Simons, 1992), and allopatric distribution in adjacent river systems all support the hypothesis that E.
podostemone and E. longimanum are sister species. This is also supported in the nuclear S7 gene tree (Fig. 4), but the mitochondrial
haplotypes observed in E. podostemone are phylogenetically nested
in a clade of E. nigrum haplotypes sampled from the Mississippi
River drainage, Great Lakes, and Mobile Basin (Fig. 3). This appears
to be another case of mitochondrial DNA transfer between darter
species resulting from hybridization (Bossu and Near, 2009; Piller
et al., 2008; Ray et al., 2008; Williams et al., 2007). In all of the discovered cases of mitochondrial transfer between darter species,
the recipient species is fixed for the mitochondrial genome of the
donor species, and the recipient and donor species are sympatric
(Bossu and Near, 2009; Ray et al., 2008). Based on our limited sampling of E. podostemone, we assume that the species is fixed for a
mitochondrial genome originating from E. nigrum; however, what
is unique is that the Mississippi–Great Lakes–Mobile E. nigrum donor lineage is not sympatric with E. podostemone, and populations
of E. nigrum in sympatry with E. podostemone in the upper Roanoke
system are distantly related to E. podostemone and the Mississippi–
Great Lakes–Mobile E. nigrum donor lineage (Figs. 3 and 4). Thus,
the pattern in the gene trees is consistent with mitochondrial
introgression from E. nigrum into E. podostemone from a lineage
of E. nigrum that was previously distributed in tributaries of the
Atlantic Ocean and sympatric with E. podostemone, but are no longer distributed in this area.
The other example of mitochondrial transfer between Boleosoma species involves E. nigrum and E. olmstedi in the Genesee River
system (Great Lakes drainage). Hybrid specimens of E. nigrum and
E. olmstedi have been reported in this area, and in other Great Lakes
tributaries (Chapleau and Pageau, 1985; Cole, 1965; Jenkins and
Burkhead, 1994, p. 846; McAllister et al., 1972; Stone, 1947). Our
samples of specimens from this area were morphologically consistent with E. nigrum; however, mitochondrial haplotypes observed
in these specimens were phylogenetically disparate. Most were
phylogenetically nested in the Mississippi–Great Lakes–Mobile E.
nigrum clade, as expected. Haplotypes from two of the seven specimens were phylogenetically nested in the northern E. olmstedi
clade (Fig. 3). There was no observed introgression of S7 alleles, because in the nuclear gene tree all of the sampled alleles were closely related (Fig. 4). This result substantiates previous
assessments of hybridization between these two species that were
based on observations of intermediacy of external morphological
traits and is consistent with observations in other darter clades
of mitochondrial introgression with no observed introgression of
nuclear alleles (Bossu and Near, 2009; Piller et al., 2008; Ray
et al., 2008).
The phylogenetic analyses presented in this study provide several important insights on phylogenetic relationships within Boleosoma. In particular, the gene trees strongly support the inclusion of
E. vitreum in Boleosoma, provide molecular phylogenetic evidence
for the sister species relationship of E. longimanum and E. podostemone, and illuminate the phylogenetic affinities of E. susanae and E.
perlongum. The gene trees also provide a guide to future species
delimitation in Boleosoma, as the molecular phylogenies highlight
various inconsistencies between morphological variation, clades
present in the phylogenies, and recognized taxonomic species.
The information obtained from our molecular phylogenetic analysis of Boleosoma is not limited to issues of phylogeny and species
diversity, but also includes important observations that hybridization between species has been a factor in the evolutionary history
of this very interesting clade of darter species.
Acknowledgments
The University of Kansas Ichthyology Collection supplied tissue
samples for a number of specimens. Many people have participated
in the collection of specimens in the last several years: C. Bossu, R.
Carlson, R. Harrington, P. Hollingsworth, E. Karagliou, B. Keck, B.
Kendrick, J. Klein, N. Kelly, A. Near. Yale University Animal Care
and Use Committee approved this work. Funding was provided
by National Science Foundation (IOS-0343417 to SHA, DEB0716155 to TJN) and Yale University.
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