Mating patterns and mechanisms of isolation in Adriatic

Transcription

Hybridization vs. reproductive isolation:
Mating patterns and mechanisms of isolation in Adriatic pipefish
(Syngnathidae)
Master Thesis
Florian Moser
January 2013
Institute of Evolutionary Biology and Environmental Studies
University of Zurich
Supervisor: Prof. Dr. Tony Wilson
Coordinator: Prof. Dr. Peter Linder
“The role of hybridization in evolution has been one of the most controversial topics in the whole
field of evolutionary study.” (Stebbins 1963)
Acknowledgements
Without the following persons, my thesis would not have turned out the way it did. Therefore many thanks to:
Prof. Dr. Tony B. Wilson, for supervision and advice in all matters and his help in the beginning of my fieldwork
Prof. Dr. Maria B. Rasotto, for hosting me in the Stazione Idrobiologica Umberto D’Ancona
in Chioggia and some inspiring discussions
Wilson group:
Camilla Whittington, for advice and support in all matters and always giving her best to create a good atmosphere
Alexander Nater, for advice and support in lab work and statistics
Stefan Sommer, for precious statistical advice
Jan Knott, for advice and support for everything concerning aquaculturing of pipefish, from
the setup of the system to animal care advice, and for his help with sampling pipefish
and in breaking down and returning all the equipment safely from Chioggia back to
Zurich
Alexandra Wegmann, for advice and support in administrative manners and help with the
planning of my fieldwork
Team at the Stazione Idrobiologia Umberto d’Ancona in Chioggia:
Carlotta Mazzoldi, for help and advice during fieldwork
Lisa Locatello, for help and advice during fieldwork and support with Italian Authorities
Federica Poli, for help and advice during fieldwork and long discussions about experimental
setup, results and other concerns
Andrea Sambo, for his ability to fix every problem, his help with connecting my tanks to the
system and his support during various sampling trips
Verena Riedl, for help during sampling and nerves as strong as steel in unexpected situations
Anamarija Vrbatović, for help with sampling pipefish on early mornings
Vito Sabia, for his support during sampling and his delicious lunches
Further Elisa Cenci, Emilio Riginella, Linda Pasolli, Valentina Melli, …
i
Further:
Offices in Italy (Pierpaolo Penzo at the Servizio Caccia e Pesca di Venezia and Dr. Maria V.
Gravina at the Servizio Pesca di Rovigo), for sampling permissions
Swiss Academy of Sciences, for their financial support of my fieldwork
Daniel Heersink and Steven Geinitz, for their statistical advice concerning GLM
Glauco Camenisch und Eliane Escher, for their help with genotyping
Linda Rudin, for her Tetris-skills while packing the car
My parents, Doris and Konrad Moser, for their financial support during my education and
for their backing during this year
My girlfriend, Anja Ziegler, for her support with all my smaller and larger problems during
this year and for being a source of relaxation beyond the life at University
ii
Table of Contents
Acknowledgements
i
List of Figures and Tables
v
1
Summary
1
1
Zusammenfassung
3
2
Introduction
4
2.1
Main consequences of hybridization
4
2.1.1
Reinforcement of isolating mechanisms
4
2.1.2
Merging of taxa
5
2.1.3
Introgression
5
2.1.4
Development of novel multi-gene-complexes
5
2.2
Isolating mechanisms and mating systems
6
2.2.1
Sex-roles
6
2.2.2
Partner preferences
6
2.2.3
Multiple mating
7
2.3
Study organisms
7
3
Aim of this study
10
4
Materials and Methods
12
4.1
Samples
12
4.2
Aquaculture facility
13
4.3
Experimental design
13
4.3.1
Mating pattern in S. taenionotus and Hybridization in the wild
13
4.3.2
Preference experiments
15
4.3.3
No-choice hybridization experiment and viability of hybrid offspring
18
4.4
5
Statistical analyses
18
4.4.1
Genetic data
18
4.4.2
Preference experiments
18
Results
5.1
20
Mating pattern in S. taenionotus
20
5.1.1
Allelic richness
20
5.1.2
Multiple mating in S. taenionotus
20
5.2
Preexperimental observations
22
5.2.1
Body measurements
22
5.2.2
Mating behavior
23
iii
5.3
25
5.3.1
Body measurements
25
5.3.2
Time measurements
25
5.3.3
Preference for larger partners
26
5.3.4
Variables influencing the large preference index
27
5.4
Experiment 2: Preference between con- and interspecific partners
27
5.4.1
Body measurements
27
5.4.2
Time measurements
28
5.4.3
Preference for conspecific partners
30
5.4.4
Variables influencing the conspecific preference index
30
5.5
No-choice mating experiment
31
5.5.1
Body measurements
31
5.5.2
Behavior
32
5.6
6
Experiment 1: Size dependent preference in S. taenionotus
Hybridization in wild populations
Discussion
6.1
Mating systems
33
36
36
6.1.1
Mating behavior
36
6.1.2
Sex-roles
37
6.1.3
Preference for large partners
38
6.1.4
Multiple mating
39
6.2
Hybridization in the Adriatic pipefish species S. taenionotus and S. typhle
40
6.2.1
Body measurements
40
6.2.2
Species recognition
42
6.2.3
Experimental hybridization
43
6.2.4
Hybridization in wild populations
44
7
Conclusions
46
8
References
47
9
Appendix
52
iv
List of Figures and Tables
Figure 4.1
Map of the sampling sites
12
Figure 4.2
Setup of the experimental tanks
16
Figure 4.3
Experiment 1: Experimental setup for size-based preference in S.
taenionotus
Standard length (SL) of pipefish
17
17
Figure 5.3
Experiment 2: Experimental setup for the interspecific preference
experiment
Developmental stage of the embryos of pregnant male S.
taenionotus collected in 2012
Correlation between standard length [cm] and wet weight [g] in
the experimental pipefish
Length of all sampled S. typhle in Venice in three summers
Figure 5.4
Experiment 1: Boxplot of choice time [s] per sex
25
Figure 5.5
26
Figure 5.6
Experiment 1: The preference for large partners ranked by increasing SL
Experiment 1: Boxplot of lpi per sex
Figure 5.7
Experiment 2: Boxplot of cpi per species and sex
29
Figure 5.8
Experiment 2: Boxplot of choice time [s] per species and sex
29
Figure 5.9
31
Figure 5.10
Experiment 2: Relation between cpi and the size difference between the SL of stimuli [cm]
No-choice experiment 1: Size distribution
Figure 5.11
No-choice experiment 2: Size distribution
32
Figure 5.12
Female S. taenionotus a) displaying the ornaments and
b) not displaying the ornaments
Structure plot for all six analyzed microsatellite loci in adult pipefish
Structure plot for all six analyzed microsatellite loci in the embryos
33
Figure 4.4
Figure 4.5
Figure 5.1
Figure 5.2
Figure 5.13
Figure 5.14
17
21
22
23
27
32
35
35
v
Table 4.1
Variables included in the Generalized Linear Models (GLM)
19
Table 5.1
20
Table 5.2
Number of alleles of the embryos of every clutch and
heterozygosity of each locus
Minimal number of mothers contributing eggs per clutch
Table 5.3
Experiment 1: Measurements for focal females
26
Table 5.4
Experiment 1: Measurements for focal males
26
Table 5.5
Experiment 1: GLM table
27
Table 5.6
Experiment 2: Standard length (SL), choice time and cpi
measurements for S. taenionotus and S. typhle
Experiment 2: GLM table
28
31
Table 5.9
No-choice experiment: SL measurements for S. taenionotus and S.
typhle
No-choice experiment: Observed courtship and mating behavior
Table 5.10
FST- and P-values between different sampling sites and years
34
Table 5.7
Table 5.8
21
30
32
vi
Hybridization vs. reproductive isolation: Mating patterns and mechanisms of isolation in Adriatic pipefish
Summary
1 Summary
Interspecific hybridization is an important mechanism in evolutionary processes which
may result in both the gain or loss of biodiversity. Hybridization has received increasing
attention due to the detection of hybrid swarms and the breakdown of isolating mechanisms
in natural populations, events which can lead to speciation reversal and to the loss of
biodiversity.
In this study I investigated the importance of hybridization in the evolutionary history of
the Adriatic pipefish species Syngnathus typhle and S. taenionotus. This system is an especially interesting model of hybridization, as previous research suggests a hybrid origin of S.
taenionotus as the result of crosses between S. rostellatus and S. typhle ca. 0.25 mya.
Whereas morphological and ecological similarities between S. taenionotus and S. typhle suggest that contemporary hybridization between these species may still be possible, S.
rostellatus is no longer found in the Adriatic. While considerable data are already available
on the mating behavior of S. typhle, nothing is known about the mating system of S.
taenionotus. Because many premating isolating mechanisms are associated with courtship
behavior, I examined key aspects of reproductive ecology in S. taenionotus, including sexroles, multiple mating and courtship behavior, and compared those findings with those of S.
typhle. I also carried out a series of preference experiments, aimed at detecting the factors
responsible for reproductive isolation between S. taenionotus and S. typhle in natural populations.
Behavioral observations revealed interspecific differences in courtship behavior, including sex-specific differences in the initiation of courtship. In S. typhle courting is initiated by
females, while reproductive behavior in S. taenionotus is initiated by males. While major
components of courtship behavior were similar between the two species, they were performed in different spatial orientations. Whereas S. typhle remained in a vertical position
within the eelgrass, S. taenionotus were often horizontal and close to the ground. The experiments and genetic data together revealed sex-role reversal, multiple mating and a preference for large partners in S. taenionotus males, patterns that have been previously found in
S. typhle.
Reciprocal no-choice mating experiments resulted in a low frequency of matings between S. typhle males and S. taenionotus females, but no matings between S. taenionotus
males and S. typhle females. Successful matings involved the transfer of small numbers of
1
Summary
eggs, none of which survived to maturity, suggesting possible gametic incompatibility. As the
no-choice experiments and genetic data of wild-caught males failed to reveal evidence for
viable hybrid offspring, reproductive isolation between the two species appears to be complete. Despite superficial similarities in courtship behavior between the two species, both
pre- and post-mating isolating mechanisms appear to be sufficient to limit the frequency of
interspecific matings, and to maintain species separation.
2
Zusammenfassung
1 Zusammenfassung
Kreuzungen zwischen verschiedenen Arten können evolutionsbiologisch sowohl kreative
als auch destruktive Folgen haben und somit zum Gewinn, Erhalt und Verlust von Biodiversität führen. Seit Kurzem wird der Hybridisierung wieder vermehrt Aufmerksamkeit geschenkt,
da vermehrt Hybridzonen, in welchen sich Arten vermischen, entdeckt wurden. Dies kann im
Extremfall zur Artenverschmelzung und somit zum Verlust von Biodiversität führen.
In dieser Studie untersuchte ich, welche Rolle Hybridisierung in der evolutionären Vergangenheit der beiden Seenadelarten Syngnathus typhle und S. taenionotus gespielt hat und
noch heute spielt. Dieses System ist besonders interessant, da vorangegangene Studien entdeckten, dass S. taenionotus durch hybride Artbildung aus den Arten S. typhle und S. rostellatus hervorging. Während S. taenionotus und S. typhle in der Adria noch koexistieren, und
somit Hybridisierung möglich wäre, findet sich zwischen den Vorkommen von S. rostellatus
und von S. taenionotus keine Überlappung mehr. Das Paarungsverhalten von S. typhle wurde
bereits sehr gut erforscht, während über das Paarungssystem von S. taenionotus so gut
nichts bekannt ist. Da die meisten Isolationsmechanismen, Gegenspieler der Hybridisierung,
an das Paarungssystem der involvierten Arten gebunden sind, erforschte ich die wichtigsten
Eigenschaften des Paarungssystems von S. taenionotus um sie mit denjenigen von S. typhle
zu vergleichen. Zusätzlich führte ich verschiedene Preferenzexperimente durch, um weitere
Informationen über reproduktive Isolationsmechanismen zwischen den beiden Arten zu erhalten.
Nebst vertauschten Geschlechterrollen, Polygynie und Präferenz für grosse Paarungspartner in S. taenionotus-Männchen, alles Merkmale die auch in S. typhle zu finden sind,
entdeckte ich Unterschiede im Paarungsverhalten der beiden Arten. In S. typhle initiierte das
Weibchen die Balz, während in S. taenionotus die Männchen dieselbe initiierten. Ein weiterer Unterschied war die Lage, in welcher das ansonsten gleiche Paarungsverhalten gezeigt
wurde. S. typhle blieb dabei stets, dem Seegras gleich, in vertikaler Lage, während S. taenionotus die meisten Verhaltensmuster horizontal und in der Nähe des Grundes vollzog.
Da ich weder in den No-choice Versuchen hybriden Nachwuchs erzeugen konnte, obwohl unidirektional einige wenige Eier übertragen wurden, noch die genetischen Analysen
Hinweise auf lebende Hybride gaben, komme ich zum Schluss, dass sich diese beiden Arten
untereinander nicht mehr fortpflanzen und dass diese reproduktive Isolation auf verhaltenstechnische und postkopulative Isolationsmechanismen zurückzuführen ist.
3
Introduction
2 Introduction
Interspecific hybridization, defined as the interbreeding of organisms of different species, is a widespread phenomenon in plants and animals. Among animals, hybridization has
long been thought to be a destructive force, due to the inviability or infertility of most hybrids (Darwin 1859; Mayr 1963). Currently the importance of hybridization in evolutionary
processes is widely accepted, and both destructive and constructive effects of hybridization
have been detected (Hubbs and Hubbs 1932; Anderson 1936; Anderson and Hubricht 1938;
Hubbs 1955). The four most common consequences of hybridization are thought to be the
reinforcement of isolating mechanisms, the merging of taxa, introgression, and the development of novel multi-gene-complexes (leading to hybrid speciation), as reviewed by Scribner et al. (2000). Hybridization thus can be an important mechanism for both the gain and
loss of biodiversity (Dowling and Secor 1997; Seehausen 2004).
2.1 Main consequences of hybridization
2.1.1 Reinforcement of isolating mechanisms
When hybridization was described by Darwin (1859), he mentioned that “Hybrids, ... ,
have their reproductive organs functionally impotent“. A similar conclusion was reached by
Mayr (1963): ‘‘The majority of ... hybrids are totally sterile. ... Even those hybrids that produce normal gametes in one or both sexes are nevertheless unsuccessful in most cases and
do not participate in reproduction ... when they do backcross to the parental species, they
normally produce genotypes of inferior viability that are eliminated by natural selection.”
The opinion that hybrids are infertile, or not even viable, has been demonstrated in many
systems. When hybrid offspring are inviable or infertile, their parents’ fitness is reduced
compared to individuals mating conspecifically. Under such circumstances natural selection
is expected to act against hybrid matings, favoring individuals mating with conspecific mating partners and having reproductive isolating mechanisms to prevent interspecific mating
(Dobzhansky 1937). Selection against hybridizing individuals is thus expected to lead to a
relative increase and accumulation of both pre- and postmating isolating mechanisms.
Premating isolation can be caused by temporal (differences in mating season and/or timing),
ecological (different distributions or niches), and behavioral or mechanical (incompatibility
due to anatomical reasons) isolation whereas postmating isolating mechanisms include con4
Introduction
specific sperm precedence (Hewitt et al. 1989; Howard et al. 1998), gamete incompatibility
(Lessios and Cunningham 1990; Alipaz et al. 2001) and hybrid infertility (Dobzhansky 1937;
Mayr 1963; Coyne 2004).
2.1.2 Merging of taxa
As hybridization leads to the exchange of genetic material between species, it can lead
to the breakdown of isolating mechanisms and may ultimately reverse speciation
(Fredrickson and Hedrick 2006; Seehausen et al. 2008; Grobler et al. 2011). This consequence has recently gained considerable attention, because of the detection of hybrid
swarms in which reproductive barriers have completely broken down (Bettles et al. 2005).
The loss of reproductive isolation is often caused by the loss of environmental heterogeneity
(Seehausen et al. 1997; Crispo et al. 2011) or the introduction of new species (reviewed in
Rhymer and Simberloff 1996).
2.1.3 Introgression
Introgression, the exchange of genes between evolutionary distinct lineages, generally
leads to increased genetic diversity within a species and can therefore be an important
source of genetic variability (Anderson 1948). As proposed by the sexual selection hypothesis, introgression often results in the exchange of genetic material from a common to a rare
species (Moyer 1981; Wirtz 1999; Grant and Grant 2008). Introgression appears to be the
most common consequence of hybridization if hybrids are viable and fertile (Scribner, Page,
Bartron 2000).
2.1.4 Development of novel multi-gene-complexes
If hybrids are viable and fertile, the development of novel multi-gene-complexes in hybrid offspring may occur, an event which can lead to hybrid speciation. This happens if hybrids mate with one another instead of backcrossing to parental species. Hybrids consequently diverge from their parental species until they become reproductively isolated and
independent species. This outcome is possible if hybrids are intermediate to their parents
(Hablützel 2009) as well as if they transgress the range of their parental species (Lexer et al.
2003). In both cases backcrossing must be somehow restricted, which can be achieved via
the rapid development of sexual isolating mechanisms such as distinct coloration patterns
5
Introduction
involved in mate choice (Stelkens and Seehausen 2009), or the occupation of a niche that
the parental species are unable to access (Uzzell and Darevsky 1975; Lexer et al. 2003).
2.2 Isolating mechanisms and mating systems
Reproductive isolating mechanisms among species are typically separated into pre- and
postmating isolating mechanisms, reflecting separate episodes in the reproductive process
(Arnold 1997). Both factors can impede or promote the formation and persistence of hybrids
(Coyne 2004). In most systems of teleost fish premating isolating mechanisms are stronger
than postmating isolating mechanisms (Blum et al. 2010).
Most premating isolating mechanisms are linked to the mating system of the two possible parental species (reviewed in Arnold 1997). Therefore knowledge about the mating system of potentially hybridizing taxa is essential to understand factors influencing hybridization intensity. If mating systems are very similar between two potential congeners, premating isolation may be incomplete, and hybridization may occur.
Sexual selection plays an important role in reproductive divergence, and not surprisingly,
the most rapidly developing and strongest differences among recently diverged taxa are often differences in mating and courtship behavior (Mayr 1963; Mendelson 2003). Small
changes in behavior or slightly different preferences are often sufficient to prevent interspecific hybridization (reviewed in Turelli et al. 2001).
2.2.1 Sex-roles
The majority of species have conventional sex-roles, in which males play the more active
part during courtship and mating, resulting in increased intrasexual competition and therefore stronger sexual selection in males (Bateman 1948). While less common, sex-role reversed species are also found, in which females actively court males and males are choosy.
Different sex-roles in potentially hybridizing species may reduce the probability of hybridization in at least one direction, because when potential mating partners of both species
discriminate during mating, the likelihood of interspecific reproduction may be reduced.
2.2.2 Partner preferences
In species with conventional sex-roles, males often develop secondary sexual traits,
which are thought to indicate male quality (described in Darwin 1871). Mate choice is fre6
Introduction
quently based on such quality traits. Strong species-specific preference for such traits may
reinforce intraspecific matings and lead to reproductive isolation. If traits preferred within a
species are even more highly developed in a sister species, this can lead to a conflict between sexual traits and species recognition in the choosy sex, and therefore increase the
chance of hybridization (Hankison and Morris 2002).
2.2.3 Multiple mating
Multiple mating is a widespread pattern in many taxa and the causes and consequences
of polygamy have been well studied. The numerous hypotheses for the evolution of multiple
mating (Berglund et al. 1988; Jennions and Petrie 2000) typically assume that the search for
mating partners and mating itself is costly, and that multiple mating must thus provide benefits which exceed the costs of additional matings. Benefits could include the inheritance of
good genes, which enhance the viability of offspring or increase sexual attractiveness (Fisherian sexy sons) and genetic “bet-hedging”, reducing the negative effects of mating with low
quality partners (Olsson and Shine 1997; Lorch and Chao 2003). If hybrid offspring reduce
the fitness of the parents in species which cannot clearly differentiate between own and
closely-related species, multiple mating may be beneficial, as it may help to “dilute” the
negative effect of mating with interspecific partners (Yasui 1998; Lorch and Chao 2003).
2.3 Study organisms
Bony fish have the highest rate of hybridization among vertebrates (Wirtz 1999), offering a great opportunity to assess the causes and consequences of hybridization. Members of
the genus Syngnathus, which show extraordinary parental care, with males protecting and
brooding the offspring in specialized brood pouches, seem especially well suited to address
questions concerning how mating behavior influences hybridization, because eggs are fertilized after their transfer from the female (Fiedler 1954) ensuring paternity of the brooding
male (Berglund et al. 1986a). Therefore stochastic hybridization, such as that sometimes
detected in open substrate spawners (Stewart 1966; Cooper 1980), can be excluded.
The first evidence of interspecific mating in Syngnathus pipefish was provided by Wilson
(2006a), who detected the occurrence of unidirectional hybridization between two Californian Syngnathus species, S. leptorhynchus and S. auliscus. A genetic analysis of wild-caught
pregnant males revealed hybrid offspring in 2/7 (28.6%) S. leptorhynchus clutches, but no
7
Introduction
hybrids in an equal number of S. auliscus broods. Interestingly, despite the relatively high
frequency of interspecific matings between S. leptorhynchus males and S. auliscus females,
no free-living hybrid adults were found in the adult population, and all hybrid individuals
were early-stage embryos, suggesting that S. leptorhynchus x S. auliscus hybrids are inviable
(Wilson 2006a).
Analyses of Mediterranean pipefish by Hablützel (2009), using both genetic and morphological data, determined that S. taenionotus is a species of hybrid origin, which was produced by interspecific matings between S. typhle, a widespread European species, and S.
rostellatus, a pipefish species that is no longer found in the Adriatic Sea, as parental species.
Close affinities of mtDNA haplotypes from S. taenionotus and S. typhle haplotypes, led
Hablützel (2009) to infer “massive mitochondrial introgression to S. taenionotus” from S.
typhle. A molecular clock analysis suggests that S. typhle and S. taenionotus separated 0.25 –
1.11 million years ago (mya, Hablützel 2009). Considering that interspecific mating occurs
between the more distantly related Californian Syngnathus species (divergence time = 1.45 ±
0.42 mya), which differentiated earlier than S. typhle and S. taenionotus (Wilson 2006a), and
the coexistence of S. taenionotus with one of its parental species, interspecific hybridization
in Mediterranean populations may be influencing the population genetic structure of these
two relatives.
The distribution of S. taenionotus and S. typhle broadly overlap in the Adriatic. S. typhle
is most often found in eelgrass beds with Zostera spp., whereas S. taenionotus prefer sandy
habitats with some Ulva spp. or Gracilaria spp. (Franzoi et al. 1993; Franco et al. 2006;
Hablützel 2009) for shelter. The reproductive season of the two species also differ, with S.
taenionotus reproducing from February to August, while S. typhle mates between April and
October. Despite differences, hybridization between the two species is possible during the
four months in which both species are reproductively active (Franzoi et al. 1993; Rispoli
2007).
Mating behavior and mate choice is well studied in S. typhle but remains poorly understood in S. taenionotus. Size-dependent mate choice has been detected in male S. typhle
(Berglund et al. 1986a; Billing et al. 2007; Sundin et al. 2010), and active courtship behavior
and intrasexual competition in females, including dances and ornament displays, consistent
with sex-role reversal, have been observed (Fiedler 1954; Berglund et al. 1986a; Vincent et
al. 1995). Similar data for S. taenionotus would be useful to assess the likelihood of interspe8
Introduction
cific hybridization between these two species. As S. taenionotus is significantly smaller than
S. typhle (16.5 cm (N = 75) for S. taenionotus vs. 20.7 cm (N = 63) for Venetian S. typhle),
size-based mating preference could have significant impact on the frequency and direction
of hybridization.
9
Aim of this study
3 Aim of this study
The aim of this study is to determine the frequency of contemporary hybridization
between S. typhle and S. taenionotus and to what extent interspecific mating is influencing
the genetic structure of the two species. The fact that S. taenionotus appears to be of hybrid
origin, with S. typhle as one of its parental species (Hablützel 2009), makes this system
especially interesting, as genetic divergence since the original hybridization event reflects
the early stage of the early development of pre- and postreproductive isolating mechanisms.
While the reproductive behavior of S. typhle has been well characterized, very little is
known about mating behavior in S. taenionotus, as an aspect of life history critical to
understanding the likelihood of interspecific reproduction between these two species. As
part of a general effort to investigate interspecific hybridization between these two species,
this thesis investigates the following questions:
(1) Is there evidence for sex-role reversal in Syngnathus taenionotus as it has been found
in S. typhle? Differences in sex-roles between the two species would reduce the
likelihood of hybridization in at least one direction, because of the low likelihood of
interspecific mating between two putatively choosy individuals.
(2) Do S. taenionotus males and/or females show a preference for large mating
partners? A preference for large mating partners in S. taenionotus could lead to high
rates of hybridization with S. typhle, due to the larger size of the latter species.
(3) Does S. taenionotus mate multiply? Multiple mating is thought to be a mechanism to
dilute costly mating in species unable to differentiate among costly and beneficial
mating partners. If S. taenionotus mate multiply, the genetic consequences of
hybridization may be reduced.
(4) Do S. typhle and S. taenionotus hybridize under no-choice conditions in the
laboratory? Do such hybrid matings produce viable and fertile offspring? The
frequency of hybridization in laboratory populations provides an indication of the
extent to which premating isolating mechanisms prevent hybridization in the wild. If
10
Aim of this study
hybrid offspring from laboratory-crosses are viable and fertile, they could potentially
be having an important impact on natural populations.
(5) Is there ongoing hybridization between S. taenionotus and S. typhle in nature?
Genetic evidence of clutches of mixed parentage would indicate the contemporary
frequency of hybridization in natural populations of the two species.
(6) Do S. taenionotus and S. typhle prefer conspecific partners over heterospecific
partners? Conspecific mating preference would provide clear evidence of premating
isolation between the two species.
Due to the hybrid origin of S. taenionotus and its distribution overlap with S. typhle, the
Adriatic pipefish provide an extraordinarily interesting system to investigate the process of
reproductive divergence. This study is expected to contribute to a better understanding of
the early stages of reproductive isolation and may also be relevant for understanding how
hybridization may potentially lead to the reversal of speciation (Fredrickson and Hedrick
2006; Seehausen 2006; Seehausen et al. 2008; Grobler et al. 2011).
11
4 Materials and Methods
4.1 Samples
S. taenionotus specimens were caught at the entrance to the Sacca degli Scardovari
(44°50.4' N 12°27.0' E, Figure 4.1), located south of the Po Delta. Samplings were performed
between 12th and 20th May using a Zodiac trawling a seine with a squared opening of 1 m2
and a mesh size of 4 mm resulting in 62 adult males and 34 females. For an additional
sampling on the 13th July, an 8 m hand-trawled beach seine was used, with which 12 males
and six females were caught. Beach seine sampling was restricted to low tide, as the algal
habitat preferred by Syngnathus spp. was not accessible during high tide. The sampled
habitat was generally sandy, partly covered by macroalgae, mainly Gracilaria, Ulva and
Enteromorpha. Eelgrass was not found in this area. The success of each haul ranged from 0 18 adult S. taenionotus with both methods. S. abaster were frequently caught in the seine as
well as other marine species including fish (e.g. Pleuronectiformes, Gobidae, Atherina sp.,
Mugil sp.), crabs, shrimps and cuttlefish. Additionally a single juvenile S. typhle individual
was collected during beach seining in July.
Figure 4.1: Map of the sampling sites: right top: Sampling area for S. typhle, right bottom: Sampling
area for S. taenionotus
12
S. typhle were caught in the southern part of the Lagoon of Venice near Chioggia
(45°13.8′N 12°16.5′E, Figure 4.1). Samplings were performed from mid May until 20th July by
trawling (see above details for methodology) resulting in 41 adult males and 37 females. S.
abaster and Nerophis ophidion were caught in high abundance as well as few S. acus and
two Hippocampus sp. Other fish, crustacean, jelly- and cuttlefish species were also caught
regularly. The sampled habitats were eelgrass meadows containing Zostera marina or Z. nolti
and sparsely some Ulva sp. and Enteromorpha sp.
Despite efforts to collect pipefish to the east of Pila (44°58.5’N 12°29.8’E), located
between the Lagoon and the Sacca, we were unable to identify a site at which both species
co-occur. Consequently, animals used in the subsequent experiments were all collected
allopatrically.
4.2 Aquaculture facility
All experiments were performed at the Stazione Idrobiologica “Umberto
D’Ancona” of the University of Padua in Chioggia, Italy. Animals were held in plastic
tanks (60 x 40 x 30 cm3) with three darkened sides (one short side was left clear for
observation) for at least nine days prior to experiments. All tanks contained artificial
eelgrass for shelter and an air stone to promote gas exchange. Stock tanks were
located in the same air-conditioned (20°C) room as experimental tanks (for
description see part 4.3.3) and attached to the same flow-through system. Water
conditions followed natural variation except for fluctuations in temperature (19-25°C)
which were reduced due to air-conditioning in the room. Photoperiod was kept
constant (13.5 h light, 10.5 h dark). Pipefish were held separated by sex and species
and fed ad libidum with frozen gamma blister mysis shrimp (Tropical Marine Centre)
twice daily.
4.3 Experimental design
4.3.1 Mating pattern in S. taenionotus and Hybridization in the wild
Previous parentage analyses in S. typhle collected across the species range revealed
multiple mating in 76% of males, with a maximum of five (2.7 ± 1.2) females per clutch. The
males of the Venetian population, however, mated monogamously in 88% of cases (7/8,
13
Rispoli and Wilson 2008), indicating broad differences among populations. To investigate the
existence of polygyny and to determine whether there is evidence for interspecific
hybridization in natural populations in S. taenionotus, seven pregnant males were sampled
for parentage analyses. Animals were euthanized with an overdose of MS222 (Ethyl-3aminobenzoate, methansulfonate acid salt, 98%). Four additional pregnant males caught in
summer 2008 were also included in this analysis. Two random adult S. typhle collected from
the Venice Lagoon and the S. typhle individual from the Sacca degli Scardovari were
genotyped at the same loci for comparison. All samples were stored at -20°C at the Institute
of Evolutionary Biology and Environmental Studies at the University of Zurich prior to genetic
analyses.
4.3.1.1
DNA extraction
Embryos were removed from male brood pouches and stored individually in 96-well polymerase chain reaction (PCR) plates to maintain pouch order. DNA was extracted from all
fathers and every fifth embryo of each clutch (1+N*5), systematically sampling of the male
brood. Adult DNA was extracted from tail tissue (5.7 – 11.7 mg) with DNeasy 96 Tissue Kits
(QIAGEN, Basel, Switzerland) following the manufacturer’s recommendations except for the
final step, where DNA was diluted in 95 µl AE Buffer. Embryo DNA was extracted following
the extraction protocol outlined in Gloor and Engels (1992).
4.3.1.2
Microsatellites
Microsatellites are highly variable repetitive sequences of two to six base pairs of DNA
(Jarne and Lagoda 1996). Alleles differ in the number of repeat units of the repetitive sequences and therefore in length. Due to their high mutation rate and their inheritance, microsatellites are well-established genetic markers for studies of closely related species. For
parental analyses, I used six microsatellite loci which have been shown to amplify successfully in S. typhle and S. taenionotus (Hablützel 2009), four of which were originally developed
for S. leptorhynchus (Slep06, Slep10, Slep12, Slep13; Wilson 2006b), and two (Styph12 and
Styph44) developed for S. typhle (Jones et al. 1999).
14
4.3.1.3
PCR amplification and genotyping
Microsatellites were PCR-amplified using a DNA Engine Tetrad® 2, Peltier Thermal Cycler
(MJ Research, Waltham, MA, USA), following the protocol outlined in Hablützel (2009).
For genotyping, 1 µl of the PCR product was added to a solution containing 0.08 µl
GeneScan 500 LIZ genotyping standard (Applied Biosystems), 9.92 µl of 99.5 % deionized
HiDi Formamide (Sigma) and 10 µl ddH2O, and incubated for 10 minutes at 95°C before snap
cooling on ice for at least five minutes.
Fragment separation was performed on an automated sequencer (ABI 3730, Applied
Biosystems) and output data were analyzed using GeneMapper® v4.0 (Applied Biosystems).
4.3.2 Preference experiments
Preference experiments were performed in four glass tanks measuring 55 x 55 x 75 cm 3
(l x w x h). These tanks were subdivided into three sections, two smaller compartments (22.3
x 22.3 x 75 cm3) containing stimulus animals, and a larger third compartment (22.3 x 55 x 75
cm3) containing the focal individual (Figure 4.2). Dividers between the stimulus and focal
compartment were transparent (to include visual cues) and porous (to include olfactory
cues), and were opaque and solid between the stimulus compartments to exclude
intrasexual competition. Each corner of the experimental tank was equipped with artificial
eelgrass for shelter. All individuals were transferred into experimental tanks before 5pm on
the evening before the experiments were performed for acclimatization. During
acclimatization, all compartments were separated by opaque dividers to prevent water flow.
The opaque divider between the focal and stimulus compartments was removed at the start
of each experiment, allowing visual and chemical communication between stimulus and
focal individuals.
Each trial ran for 60 minutes. The total time the focal individual spent in each preference
area (two choice areas, highlighted in yellow in Figure 4.3) was recorded for further analyses.
All trials were recorded by two cameras (Abus, ProfiLine, TV7043 and TV7047), one
recording from top to determine preference according to the time spent in the confined
areas, and the other camera recording from the front to detect courtship behavior
associated with reproductive preference. During the acclimatization period and experiments,
animals were not fed.
15
A preference index (lpi = large preference index or cpi = conspecific preference index)
was calculated for every trial using the following formulae:
Figure 4.2: Setup of the experimental tanks (55 x 55 x 75 cm3)
4.3.2.1
Size dependent preference experiment in S. taenionotus (Experiment 1)
The goal of this experiment was to investigate whether S. taenionotus shows size
associated preferences, a pattern observed in S. typhle males (Berglund et al. 1986a; Billing
et al. 2007; Sundin et al. 2010). Standard length (= SL; Figure 4.4) of each individual was
measured before each experiment.
The two stimulus animals differed by at least 1.3 cm in female focal experiments (2.3 ±
1.1 cm) and by at least 0.8 cm in SL in male focal experiments (1.8 ± 1.0 cm), with the
placement of the stimulus individuals randomized to prevent spurious correlation. The focal
individual was of the opposite sex and of random size. 20 replicates were performed for
each sex.
16
♀
♂
♂
♀
♂
♀
Legend:
yellow = choice area
= transparent and
porous
= opaque and solid
Figure 4.3: Experimental setup for the size-based preference in S. taenionotus (Exp. 1)
Figure 4.4: Standard length (SL) in pipefish (by Hablützel 2009)
4.3.2.2
Preference between con- and interspecific individuals (Exp. 2)
A second experiment was designed to test for conspecific preferences in males and
females of both S. taenionotus and S. typhle (Figure 4.5). Because S. typhle are typically
larger than S. taenionotus (Dawson 1986), the S. typhle stimulus was larger than the S.
taenionotus stimulus (8.0 ± 3.7 cm) in 81/82 trials. Again here, the placement of stimulus
animals was randomized during each trial.
Experiments were conducted with males and females of both species as focal animals (S.
taenionotus male (N=20), S. taenionotus female (N=20), S. typhle male (N=22), S. typhle
female (N=20)) to detect preferences for conspecifics. SL (Figure 4.4) and wet weight was
measured for every individual at the conclusion of the first experimental trial of each fish.
Each individual was used a maximum of one time as focal, conspecific and interspecific
individual. Animals were held for a minimum of two days (2 – 18 days) after the conclusion
of an experiment before use in a subsequent trial.
1
♀
♂ 3
♂ ♀
♀
2
♀
♂
♂
Legend:
red = S. taenionotus
blue = S. typhle
yellow = choice area
= transparent and
porous
= opaque and solid
♀
♀
♂
♂
Figure 4.5: Experimental setup for the interspecific preference experiment (Exp. 2)
17
4.3.3 No-choice hybridization experiment and viability of hybrid offspring
To investigate whether hybridization is possible between S. typhle and S. taenionotus, a
reciprocal no-choice mating experiment was carried out, with 10 S. taenionotus females
together with 10 S. typhle males in a single tank (55 x 55 x 75 cm3) and 10 S. taenionotus
males together with 10 S. typhle females in a second tank of equal size. Both tanks contained
artificial eelgrass for shelter. Pipefish were sampled to cover their natural size range to
simulate natural mating conditions as closely as possible. Animals were held together for
seven days (08/07 – 15/07). Behavioral observations of interspecific interactions were
carried out on the first day of the experiment, and video recordings throughout the entire
experimental period provided additional data on individual behavior.
To investigate the viability of hybrid offspring and the number of eggs present in male
brood pouches, transferred eggs were counted weekly. Pregnant males were held
individually in plastic tanks of 60 x 40 x 30 cm3 (lwh), equipped with artificial eelgrass.
After the conclusion of the experiments, three randomly selected, non-mated males of
each species were placed to mate with the ten conspecific females for 24 hours to assess
reproductive activity.
4.4 Statistical analyses
4.4.1 Genetic data
To infer the genetic mating pattern in S. taenionotus, genetic data were analyzed using
Cervus version 3.0.3 (Field Genetics Ltd., Tristan Marshall 1998-2007) for allele counts and
heterozygosity, and GERUD2 (Jones 2005), which calculates the minimal number of mothers
contributing eggs to a clutch by using multi-locus data. FST-analyses were performed using
Microsatellite-analyzer software (Weir and Cockerham 1984; MSA, Dieringer and Schlotterer
2003) version 4.05, using 10’000 permutations of genotypes among populations to test the
reliability of FST-values (Schafer et al. 2006; Mirol et al. 2007). This conservative procedure
allows for linkage among the loci without the assumption of Hardy-Weinberg equilibrium.
Bonferroni correction was applied to account for multiple testing.
4.4.2 Preference experiments
Analyses were performed using R version 2.13.2 (© 2011 The R Foundation for Statistical
Computing) and graphical outputs were produced using R and MICROSOFT EXCEL 2007.
18
Two sided one sample t-tests, concerning the null hypothesis of no preference (i.e.
lpi/cpi = 0.5) were used to test for preference for each sex and species independently.
Pearson correlation analyses were performed to test for correlation between two variables (SL against wet weight and several variables against the two preference indexes). To
test for differences among fixed factors (time of day, tank, sex and species of the focal fish)
Kruskall-Wallis and Mann-Whitney tests were performed in R.
Comparisons between regressions between sexes were tested with the smatr-package in
R (Warton et al. 2012).
Levene tests, which test for the null hypothesis of homoscedasticity between two groups
of samples, were performed in R to test for difference in variance among the different focal
fish types for the preference indexes and total choice time.
Finally, Generalized Linear Models (GLM) were used to test the combined effects of the
variables listed in Table 4.1. Two-way interactions among all variables were included in the
initial models, and factors and covariates of the model were then reduced stepwise based on
the Akaike Information Criterion (AIC). All abiotic factors were excluded from the GLM of
experiment 1 due to the lack of Pearson correlation with the preference indexes (date: r2 =
0.136, P = 0.423) or the lack of difference among groups (time of day of experiment: KruskalWallis Chi-squared H = 0.001, P = 0.973, tank: H = 17.57, P = 0.484). Abiotic factors were
similarly excluded from experiment 2 (date: r2 = 0.085, P = 0.469 (Pearson correlation); tank:
H (Kruskall-Wallis) = 2.224, P = 0.5272; time of day: H = 0.336, P = 0.0562).
Table 4.1: Variables included in the Generalized Linear Models (GLM)
Dependent variable
Fixed factors
Covariates
lpi
Sex of focal fish
cpi
Sex of focal fish
Species of focal fish
SL of focal fish [cm]
∆SL between stimuli [cm]
SL of focal [cm]
∆SL between stimuli [cm]
19
Results
5 Results
5.1 Mating pattern in S. taenionotus
5.1.1 Allelic richness
For each microsatellite locus, 1 - 12 alleles were detected in the eleven clutches (Table
5.1). The most variable microsatellite was Slep12 with a total of 30 alleles and an observed
heterozygosity of 0.84, whereas Styph44 was essentially fixed with a single allele of high
frequency in each species (134 bp for S. taenionotus and 143 bp for S. typhle, Table 9.1 in the
Appendix) with an observed heterozygosity of 0.05 (Table 5.1).
Table 5.1: Number of alleles detected in each clutch and heterozygosity of each locus
Father
SCA08_086
SCA08_097
SCA08_098
SCA08_100
SCA12_1
SCA12_2
SCA12_3
SCA12_4
SCA12_5
SCA12_6
SCA12_7
Total
Observed
heterozygosity
Expected
heterozygosity
Slep06
5
6
5
7
6
4
7
5
5
8
4
13
0.836
Slep10
2
3
2
2
5
1
3
3
3
2
3
7
0.276
Slep12
9
7
7
12
9
6
6
6
5
12
5
30
0.839
Slep13
4
6
4
5
6
4
5
3
4
6
6
16
0.741
Styph12
6
8
3
7
5
5
6
6
5
7
4
18
0.868
Styph44
2
4
2
2
1
1
1
1
2
2
1
8
0.053
0.834
0.290
0.916
0.822
0.855
0.262
5.1.2 Multiple mating in S. taenionotus
All embryos had pigmented eyes, and at a late stage of development (stage 7-10 of 11;
Sommer et al. 2012; Table 5.2 and Figure 5.1). Microsatellite amplifications were typically
more successful in clutches with embryos of an earlier developmental stage (Table 9.2 in the
appendix), which is opposed to previous genetic analyses of S. typhle embryos (Rispoli
2007).
Microsatellite analyzes using GERUD2 (Jones, 2005) revealed multiple mating in S.
taenionotus with a minimum of 3 mothers (4.3 ± 1.25 mothers) contributing eggs to a
complete clutch and at least 2 mothers in the incomplete clutch of SCA12_7 (Table 5.2).
20
Results
The number of eggs a mother contributed was very variable as well, ranging from 1 – 12
analyzed embryos, indicating a transfer of up to 64 eggs, as only every fifth embryos was
analyzed.
A positive linear correlation was detected between SL of the father and number of
embryos (r2 = 0.892, df = 4, t = 3.948, P = 0.017), but no correlation between number of
embryos and the minimum number of mothers per clutch (r2 = 0.469, df = 4, t = 1.061, P =
0.348).
Table 5.2: Minimal number of mothers contributing eggs per clutch, * = pouch only half filled, SCA =
Sacca degli Scardovari
Sampling area and
month of collection
Father ID
Paternal SL [cm]
# of embryos
# of analyzed
embryos
# of analyzed loci
Min. # of mothers
Developmental
stage of embryos
(Sommer 2012)
SCA June 2008
SCA July 2012
086 097 098 100
1
2
3
4
5
6
7*
n.a. n.a. n.a. n.a. 17.6 17.0 15.1 15.1 13.9 16.8 14.6
140 135 104 154 150 105 75
64
70 135 37
15
18
14
24
17
19
14
10
12
19
6
6
5
4
4
6
3
6
>6
6
4
6
4
6
5
3
3
6
3
6
5
6
2
10
10
10
10
10
8
7
10
8
10
8
Figure 5.1: Developmental stage of the embryos of pregnant male S. taenionotus collected in 2012
21
Results
5.2 Preexperimental observations
5.2.1 Body measurements
SL of S. taenionotus ranged from 12 - 19.7 cm (15.35 ± 2.09 cm) with a wet weight
between 0.5 g and 2.8 g (1.31 ± 0.54 g). S. typhle SL ranged from 15.8 - 30.1 cm (23.78 ± 3.42
cm) with wet weights ranging from 1.6 - 10.8 g (5.69 ± 2.47 g). SL and wet weight were
positively correlated in both species (S. taenionotus: df = 35,t = 9.37, r2 = 0.85, P < 0.001; S.
typhle: df = 36,t = 14.7, r2 = 0.93, P < 0.001; Figure 5.2). Sex-specific differences were not
detected (S. taenionotus: lr < 0.001, b = 0.251, df = 1, P = 0.995; S. typhle: lr = 0.016, b =
0.708, df = 1, P = 0.898). Therefore SL was used in further analyses as an estimate of body
size.
Figure 5.2: Correlation between standard length [cm] and wet weight [g] in the experimental
pipefish; orange = S. taenionotus females (N = 20), red = S. taenionotus males (N = 17), green = S.
typhle females (N = 19) and blue = S. typhle males (N = 19), red line = all S. taenionotus, blue line = all
S. typhle
Although all adult S. typhle were collected in the Lagoon of Venice, the individuals
collected for this study were significantly larger than those collected in previous sampling
seasons (2006 vs. 2012: t = -8.82, df = 44.5, P < 0.001; 2008 vs. 2012: t = -6.04, df = 23.5, P <
0.001). Adult S. typhle collected from the Lagoon of Venice in June 2006 (Rispoli and Wilson
2008) reached an average female SL of 21.1 ± 3.6 cm (N = 4), while males reached an
average SL of 14.2 ± 1.0 cm (N = 19, Figure 5.3)). Similarly, pipefish sampled in the Lagoon in
June 2008 (Wegmann 2009) reached an average total length (TL = SL + length of the tail fin)
22
Results
of 14.3 ± 3.6 cm for females (N = 12) and 14.9 ± 3.3 cm for males (N = 10, Figure 5.3). The
animals collected for the present study reached an average SL of 23.0 ± 3.0 cm for females
(N = 19) and 24.4 ± 3.8 cm for males (N = 19, Figure 5.3).
35
Length [cm]
30
25
20
15
10
5
0
Individual
Figure 5.3: Length of all sampled S. typhle in Venice; red = 2006 (SL), green = 2008 (total
length – 1 cm, as an approximation to SL), blue = 2012 (SL)
5.2.2 Mating behavior
Detailed observation of reproductive activity during mating in experimental tanks containing a single male and female S. taenionotus, which performed four egg transfers within
the observed eight hours, revealed that courtship always started with the horizontal approach of the male to the female from behind, which was repeated several times without
the female showing any reaction. The male performed a cycling movement so that his approach is always from behind. When the female reacted, she exposed her ventral surface to
the male (ventral exposure) by swimming ahead of him until her ventral surface was even
with the male’s head. The male consistently reacted to the female’s behavior by flicking, a
short rotating or shaking movement along the anteroposterior axis. This flicking-behavior
was shown before, during and/or after dancing, when male and female synchronously swam
in circles facing one another in a horizontal orientation. Following several bouts of dancing,
the mating pair switched to a vertical orientation, connected to one another and passively
but quickly shot to the surface, while the eggs were transferred. While the male and female
returned to the bottom of the tank, the male performed shaking movements, which, according to Fiedler (1945), may be associated with egg fertilization and packing within the pouch.
Repeated approaches occurred after a break of variable length (here: 10, 18 and 10
minutes), during which the male promenaded or remained in an S-shaped posture with the
tail laying on the ground. One of these mating events took place during the dark portion of
23
Results
the light cycle, indicating that mating behavior in S. taenionotus is not strictly limited to daytime.
In the male-biased mixed tank (three males and ten females, four observed egg transfers
within 160 min), it was more difficult to assess which behavior acted as the initiator to subsequent egg transfer. In one of four cases the process occurred as outlined above, whereas
in another case reproductive behavior started in similar fashion, but the first flicking of the
male attracted a second female and the dance was performed in a group of three, until one
female retired and egg transfer was performed as described above. In the other two cases,
the male joined two females already performing dance-like competition. These groups then
danced as a group of three, until one female returned to the ground while the other one
transferred eggs, while shooting to the surface. Postcopulatory behaviors did not differ from
the description above.
Observation of mating behavior in a single pair of S. typhle (two egg transfers over two
hours) revealed some notable differences from the pattern exhibited by S. taenionotus. In
contrast to S. taenionotus, mating behavior in S. typhle was initiated by the female with both
individuals aligned vertically (head up) in the eelgrass (vertical approach). The female always
approached from above the male, with her ventral surface at the height of the male’s head.
In the absence of a male response to this ventral display the female reapproached the male
in a similar fashion. The female then placed herself close to the male and performed a short
shaking movement. The male reacted with a shaking behavior along his anteroposterior axis,
identical to the flicking behavior described for male S. taenionotus. This flicking of the male
was repeated several times before the male and female connected and moved passively, but
quickly to the surface during the egg-transfer. Similar to the pattern observed in S.
taenionotus, male S. typhle exhibit shaking behavior after egg transfer while moving to the
bottom of the tank, a behavior which is, according to Fiedler (1945), associated with egg
fertilization and packing in the pouch. The female then continued to exhibit courtship behavior of increased intensity, no longer moving beyond the male, leading to a dance-like behavior with the fish in vertical orientation, wherein the male frequently performed the S-shaped
posture. The dance lasted until the male started flicking once more and the next egg transfer
took place.
24
Results
5.3 Experiment 1: Size dependent preference in S. taenionotus
In this experiment focal female SL ranged from 12.6 – 18.9 cm (16.4 ± 1.2 cm; Table 5.3).
Male focal fish were slightly larger, measuring between 13.2 - 18.9 cm SL with a mean of
16.6 ± 1.2 cm (Table 5.4).
5.3.2 Time measurements
There were significant differences in how much time focal animals spent in either choice
area (= choice time) in these trials. This pattern was especially pronounced for focal females,
which spent from none (choice time = 0 s) up to the entire experimental period (choice time
= 3600 s) in the choice areas (Table 5.3). In general, female focal fish spent slightly but not
significantly (U = 141, P = 0.385) more time in the choice-area than did males (mean: 1915 ±
1320 s vs. 1498 ± 1210 s; Figure 5.4). The trials in which the focal animal did not spend any
time in the choice areas (females: N = 3, males: N = 0) were excluded from further analyses.
Figure 5.4: Box-whisker plot (median/quartiles/range) of choice time [s] per sex
25
Results
Table 5.3: Measurements for focal females in
experiment 1
Focal females (N = 20)
ID SL focal Choice∆ SL
lpi
[cm]
time [s] stimuli
[cm]
28
12.6
7
1.4
1
13
15.0
3533
3.1
0.604
4
15.4
0
1.5
7
15.5
3368
1.7
1
18
15.6
1051
1.5
0.986
20
15.6
111
1.7
0
30
15.9
260
5.1
0.877
26
16.0
424
3.0
1
33
16.0
906
1.4
0
9
16.1
3106
1.9
0.231
19
16.4
3099
5.5
0.122
5
16.6
3599
1.9
0.621
8
16.6
1163
2.3
1
12
17.1
0
2.2
16
17.1
2607
2.6
0.857
6
17.3
3156
1.9
0.608
29
17.3
2534
2.0
0.750
23
17.4
2373
1.8
0.753
21
17.5
1199
2.4
0.061
22
18.3
0
1.3
-
Table 5.4: Measurements for focal males in
experiment 1
Focal males (N = 20)
ID SL focal Choice∆ SL
lpi
[cm]
time [s] stimuli
[cm]
40
13.2
1416
1.5
0.336
31
14.8
607
1.5
0.787
10
15.0
3418
1.5
0.899
35
15.7
2405
2.4
0.230
11
16.1
1122
5.3
0.525
38
16.1
2248
1.8
0.976
37
16.2
172
1.3
0.669
2
16.7
192
1.7
0.990
24
16.9
41
1.8
1
32
16.9
1006
1.3
0.694
1
17.0
718
1.4
0.585
34
17.1
85
2.4
1
17
17.2
968
1.3
0.654
36
17.2
3218
2.4
0.886
42
17.2
3406
0.8
0.816
15
17.4
789
1.4
0.286
27
17.4
1472
2.9
1
3
17.6
3441
1.8
0
14
17.6
582
1.3
0.911
41
18.9
2664
1.0
0.983
5.3.3 Preference for larger partners
A two sided, one sample t-test, concerning the null hypothesis of no size-dependent
partner preference (lpi = 0.5) revealed no significant preference in females (mean = 0.616,
T16 = 1.247, P = 0.231, Figure 5.5a) but highly significant preference for large partners in
males (mean = 0.711, T19 = 3.152, P = 0.005, Figure 5.5b)
a)
b)
Figure 5.5: Size preferences for a) female and b) male focal fish ranked by increasing SL on the x-axis
26
Results
5.3.4 Variables influencing the large preference index
Sex (P < 0.001) and size (P < 0.001) of the focal fish and their interaction (P < 0.001) had
a significant impact on the lpi (Table 5.5). Size difference between the two stimuli did not
affect lpi, either alone or in interaction with either of the other two variables.
Table 5.5: GLM table for experiment 1
Coefficients:
(Intercept)
Sex of the focal fish
Size of the focal fish
Stimuli size difference
Interaction of sex and size of focal
interaction of size of focal and
stimuli size difference
Estimate
9.17440
-10.00190
-0.52533
-1.61488
0.63580
Df
31
1
1
1
1
Std. Error
2.07171
1.15629
0.12795
0.98585
0.07072
z-value
4.428
-8.650
-4.106
-1.638
8.990
Pr(>|z|)
9.49e-06
< 2e-16
4.03e-5
0.101
< 2e-16
0.09480
1
0.06087
1.557
0.119
The effect of sex of the focal fish is shown in the figure 5.6, where a stronger preference
for large partners was detected in male experiments. Lpi values of focal males were also
more clustered and less scattered than those of the focal females. Although differences in lpi
were not significant (t = -0.834, df = 30.1, P = 0.411), the difference in variance was (Levene
Test Statistic W = 0.307, P = 0.012).
Figure 5.6: Box-whisker plot (median/quartiles/range) of lpi per sex
5.4 Experiment 2: Preference between con- and interspecific partners
S. typhle used in this experiment were significantly larger than S. taenionotus individuals
(t = 12.75, df = 61.9, P < 0.001), reflecting species-specific size dimorphism (see Intro27
Results
duction). SL of S. taenionotus ranged from 12.0 - 19.7 cm (15.4 ± 2.1 cm) and from 15.8 30.1 cm (23.8 ± 3.4 cm) for S. typhle (Table 5.6).
5.4.2 Time measurements
The conspecific preference index (cpi) indicates the focal individual’s preference for an
individual of its own species relative to the total time spent in the preference zones of both
species (choice time). All four groups (each sex and species as focal animal) showed a broad
range in both choice time and cpi (Table 5.6, Figures 5.7 and 5.8). Trials in which focal animals spent no time in the choice zones (N = 6) were excluded from further analyses.
Generally S. typhle show slightly higher preference for conspecific partners than do S.
taenionotus (t = 1.38, df = 67.7, P = 0.172). Whereas the cpi values of S. typhle cluster around
0.8, the values for S. taenionotus cluster at the two extremes. This difference in variance
among species is highly significant (W = 7.721, P = 0.007, Figure 5.8).
Table 5.6: Measurements for experiment 2 for a) focal female S. taenionotus, b) focal male S.
taenionotus, c) focal females S. typhle, d) focal male S. typhle
a) focal female S. taenionotus (N = 20)
focal SL choice
cpi
∆ SL stim[cm]
time [s]
uli [cm]
12.0
1243
0.049
5.2
12.5
2213
0.134
7.3
12.9
3572
0.964
9.8
13.3
228
0.346
7.9
13.4
476
0.897
12.9
13.4
374
0.751
11.3
13.7
2533
0.666
16.9
14.0
347
0.813
17.0
14.5
678
0.935
11.5
14.6
81
0.222
7.7
15.5
1032
0.057
1.3
15.5
3600
0.003
2.0
15.6
807
0.093
9.9
15.7
2371
0.183
7.4
16.5
3229
1
11.3
16.6
316
0.532
7.5
17.2
2279
0.297
8.6
17.5
2553
0.914
10.6
17.9
1385
0.796
4.8
19.7
1385
0.929
5.2
b) focal male S. taenionotus (N = 20)
focal SL choice
cpi
∆ SL stim[cm]
time [s]
uli [cm]
12.6
826
0.168
4.5
13.1
3586
0
6.0
13.2
484
0.847
7.3
13.3
1984
0.862
5.1
13.4
0
10.7
13.5
3600
1
15.5
13.8
3585
0.008
12.0
13.8
546
0.341
10.9
14.5
18
1
10.6
14.6
1243
0.978
14.2
15.9
0
6.9
16.0
3048
1
6.8
16.5
281
0.456
6.6
16.5
1065
0.151
6.1
16.8
2921
0.507
4.9
17.7
2491
0.536
2.7
18.0
1718
0.782
3.8
18.0
281
0.1
7.3
18.8
3292
0.334
8.5
19.2
1830
0.089
8.4
28
Results
c) focal female S. typhle (N = 20)
focal SL choice
cpi
∆ SL stim[cm]
time [s]
uli [cm]
17.4
2471
0.153
8.3
20.2
2097
0.728
9.8
20.3
2158
0.565
5.3
20.5
2027
0.683
6.1
20.6
2001
0.125
4.8
21.6
1829
0.579
8.7
22.0
425
1
5.2
22.1
1957
0.427
5.2
22.1
2783
0.944
10.2
22.5
1434
0.85
10.6
22.8
2505
0.317
11.9
22.9
1886
0.019
13.0
24.0
2632
0.687
1.3
24.0
2187
0.697
11.3
24.0
643
0.764
7.2
25.1
2495
0.596
6.1
27.4
1975
0.459
10.8
28.5
2832
0.951
16.3
29.5
3383
0.749
12.9
d) focal male S. typhle (N = 20)
focal SL choice
cpi
∆ SL stim[cm]
time [s]
uli [cm]
15.8
1576
0.895
7.6
16.8
590
0.832
11.4
20.7
1388
0.576
6.1
21.1
1827
0.794
5.6
23.0
3551
0.566
10.2
23.2
2332
0.816
15.4
24.0
3285
0.939
0.8
24.0
2426
0.955
7.0
24.2
1600
0.679
6.1
25.2
1172
0.872
4.1
25.3
2566
0.552
5.6
25.3
2041
0.991
8.3
25.7
987
0
6.0
26.7
1639
0.051
0.8
26.7
1760
0.338
5.8
27.5
2409
0.597
6.6
27.8
1841
0.843
9.2
29.5
2247
0.768
8.4
30.1
598
0.452
7.4
In S. typhle, males showed higher preference for conspecific partners (t = -1.06, df =
35.9, P = 0.298) and were less active than females (t = 0.84, df = 35.5, P = 0.406, Figure 5.7
and 5.8), whereas in S. taenionotus, males showed reduced preference for conspecifics (t =
0.40, df = 34.1, P = 0.690) and were more active than females (t = -0.98, df = 33.3, P = 0.332,
Figure 5.7 and 5.8).
Figure 5.7: Box-whisker plot of choice time [s]
per species and sex; taf = S. taenionotus female,
tam = S. taenionotus male, tyf = S. typhle female,
tym = S. typhle male
Figure 5.8: Box-whisker plot of cpi per
species and sex; taf = S. taenionotus female,
tam = S. taenionotus male, tyf = S. typhle
female, tym = S. typhle male
29
Results
5.4.3 Preference for conspecific partners
A two sided, one sample t-test, revealed a significant preference for conspecific partners
in male S. typhle (mean = 0.66, t = 2.41, df = 18, P = 0.027). No evidence for species
preference could be detected in either sex of S. taenionotus (female: mean = 0.53, t = 0.35,
df = 19, P = 0.730; male: mean = 0.48, t = -0.23, df = 16, P = 0.825) and female S. typhle
(mean = 0.56, t = 0.99, df = 18, P = 0.336).
5.4.4 Variables influencing the conspecific preference index
A significant effect of the interaction of the sex of the focal fish and size difference
between stimuli was found (P = 0.045), along with a minor effect of the sex of the focal fish
(P = 0.062) and the interaction of focal individual sex and size (P = 0.064) on the cpi (Table
5.7).
Table 5.7: GLM table for experiment 2
Coefficients:
(Intercept)
Sex of focal fish
Species of focal fish
Size difference between the stimuli
Size of focal fish
Interaction of focal sex and stimuli size
difference
Interaction between sex and size of
focal fish
Interaction of focal size and stimuli
size difference
Estimate
-4.61526
6.71585
1.93092
-0.26828
0.19463
Df
67
1
1
1
1
Std. Error
2.885474
3.598167
1.369417
0.175177
0.157273
z-value
-1.599
1.866
1.410
-1.531
1.238
Pr(>|z|)
0.1097
0.0620
0.1585
0.1256
0.2159
0.20293
1
0.101073
2.008
0.0447
-0.33893
1
0.183215
-1.850
0.0643
0.00388
1
0.008322
0.466
0.6415
Focal males and females differed in their response to size differences between stimuli,
with males showing stronger preference when stimuli differed more greatly in size (t = 1.69,
df = 34, r2 = 0.28, P = 0.100) and females showing the opposite pattern (t = -0.90, df = 37, r2 =
-0.15, P = 0.37). When considering the species separately, both sexes of S. taenionotus
showed stronger conspecific preference when size differences were high (female: t = -2.97,
df = 18, r2 = -0.57, P = 0.008; male: t = -0.71, df = 15, r2 = -0.18, P = 0.488; Figure 5.9a) and
female and male S. typhle differed in their response, with males showing stronger
conspecific preference when size differences were high (t = 1.35, df = 17, r 2 = 0.31, P =
0.196), and females showing a stronger conspecific preference when the two stimuli were of
30
Results
similar size (t = -0.78, df = 17, r2 = -0.19, P = 0.448; Figure 5.9b). The relationship between
stimulus size and cpi was only significant for female S. taenionotus.
a)
b)
∆
∆
Figure 5.9: Relation between cpi and stimulus SL difference [cm] for focal a) S. taenionotus and b) S.
typhle; red = male S. taenionotus, orange = female S. taenionotus, blue = male S. typhle, green =
female S. typhle
5.5 No-choice mating experiment
Pipefish used in this experiment ranged between 12.4 - 28 cm SL (Table 5.8). Overlap in
size between species was possible in the experiments involving S. typhle females and S.
taenionotus males, but all S. typhle males were larger in size than S. taenionotus females
(Figures 5.10 and 5.11). In both experiments, the two species were significantly different in
size (male S. typhle and female S. taenionotus: t = 6.21, df = 10.4, P < 0.001; male S.
taenionotus and female S. typhle: t = -4.23, df = 17.9, P < 0.001).
Table 5.8: SL measurements for S. taenionotus and S. typhle in the no-choice experiments
SL [cm]
Min. – Max.
Median
Mean ± SD
S. taenionotus
females
15.3 – 18.0
16.4
16.49 ± 0.94
S. taenionotus
males
12.4 – 20.4
17.45
17.33 ± 2.17
S. typhle
females
17.2 – 25.5
21.8
21.61 ± 2.36
S. typhle
males
18.6 – 28.0
23.35
23.23 ± 3.30
31
30
30
25
25
20
20
SL [cm]
SL [cm]
Results
15
10
15
10
5
5
0
0
Figure 5.10: SL [cm] distribution in the no-choice
experiment 1; red = S. taenionotus males, blue =
S. typhle females
Figure 5.11: SL [cm] distribution in the nochoice experiment 2; red = S. taenionotus females, blue = S. typhle males
5.5.2 Behavior
Behavioral interactions among species could be observed regularly, throughout the
experimental period, and male flicking was observed in both trials (Table 5.9). Interestingly,
several S. typhle specimens of both sexes were horizontally aligned along the bottom of the
experimental tanks, a behavior atypical for this species (see 5.2.2).
In addition to courtship behavior, intrasexual competitive behavior could be observed in
both sexes of the two species, including chasing behavior, dance-like paired or grouped
swimming and ornament display in females (Figure 5.12).
Table 5.9: Observed courtship and mating behavior; taf = S. taenionotus females, tam = S.
taenionotus males, tyf = S. typhle females, tym = S. typhle males
Behavior
Approaching
the partner
Ventral
display
Flicking
Dancing
Going up to
the surface
Shaking
S. taenionotus
Male
(horizontal,
on the
ground)
Female
S. typhle
Female
(vertical, in
the
eelgrass)
Female
Observed in the no-choice experiment
tam /tyf
taf /tym
tam approaching tyf, taf approaching tym,
tyf laying on the
tym laying on the
ground (unusual for
ground (unusual for
S. typhle)
S. typhle)
No
No
Male
Horizontal
Both
Male
Vertical
Both
Yes
No
No
Yes
No
No
Male
Male
No
No
32
Results
a)
b)
Figure 5.12: Female S. taenionotus a) displaying the ornaments and b) not displaying the ornaments
After one week of free mating in the no-choice experiment, two S. typhle males carried
eggs, though the number of eggs detected was low, with two eggs in one male and four eggs
brooded by the second individual. After the first week of pregnancy, the first male had
already lost both of his eggs, whereas the other individual still carried four eggs, none of
which showed further development or growth. A week later there were still four bulges
visible in this individual, but still no growth or development detectable. After the third week
(06.07.2012) the second male was no longer pregnant.
In subsequent intraspecific mating trials, 1/3 (33%) of S. typhle and 2/3 (66%) of S.
taenionotus males successfully mated within 24 hours.
5.6 Hybridization in wild populations
Microsatellite data show clear differentiation between S. taenionotus and S. typhle
alleles at at least two of the six analyzed microsatellites (personal data and data from
Hablützel 2009; Table 9.1 in the appendix). These two loci (Slep12 and Styph44) are
33
Results
subsequently useful for identifying hybrids between the two species. S. taenionotus show a
broad range of alleles at Slep12, with alleles ranging in length between 188 bp and 238 bp
(25 alleles). S. typhle, in contrast show less variability (7 alleles) and are almost fixed to the
194 bp-allele (72.1 %). Similarly, Styph44 differed between the two species, essentially fixed
for a single allele of high frequency in each species (134 bp for S. taenionotus (96,3%) and
143 bp for S. typhle (92.3%); Table 9.1 in the Appendix) and an observed heterozygosity of
0.05 (Table 5.1). The S. typhle individual caught in the Sacca degli Scardovari was heterozygous for this locus, with one allele of 143 bp and the other one of 134 bp.
FST-analyses revealed the smallest differences (FST < 0.115) within species collected in
different years (Table 5.10), and indicated that the differences between the S. typhle
specimens caught near Scardovari (SCA) and the S. taenionotus specimens collected at this
site (FST = 0.204 – 0.212) were smaller than those detected between S. typhle of Venice
(VEN) and S. taenionotus of Scardovari (FST = 0.288 – 0.310). Interestingly, the lowest interspecific FST values were detected between the S. typhle collected at the Sacca degli
Scardovari and the S. taenionotus from the Lagoon of Venice (FST = 0.144) a value only
slightly larger than that detected between S. taenionotus from Venice and those from
Scardovari (Table 5.10).
Table 5.10: FST- (below axis) and P-values (above axis) between different sampling sites and years;
B
= significant after Bonferroni-correction
S. taenionotus
S. typhle
P-values:
FSTSCA 2008 SCA 2012
VEN
VEN 2008 VEN 2012
SCA
values:
(N = 41)
(N = 7)
(N = 2)
(N = 43)
(N = 2)
(N = 2)
2008
0
0.213
0.065
< 0.001B
< 0.001B
0.002B
S. taenionotus 2012
0.008
0
0.015
< 0.001B
< 0.001B
0.024
B
VEN
0.055
0.114
0
< 0.001
0.344
0.357
2008
0.288
0.308
0.289
0
0.088
0.026
S. typhle
2012
0.297
0.310
0.246
0.052
0
0.325
SCA
0.204
0.212
0.144
0.092
0.053
0
An individual-based analysis of hybridization revealed clear differentiation among the
two species, but a significant number of individuals of mixed genetic ancestry could be
detected (Figure 5.13). S. taenionotus individuals caught in the Lagoon of Venice (Hablützel
2009) show a pattern similar to that detected in the Sacca degli Scardovari population.
Interestingly one of the two S. typhle specimens caught near Scardovari in 2012 (SCAty12) is
of mixed ancestry (Figure 5.13), showing indications of hybrid ancestry.
34
Results
The S. taenionotus clutches, analyzed from the Sacca population showed little evidence
of hybrid ancestry, and the single outlier individual may be an artifact as data were only
VENta08
SCAty12
SCAty08
VENty08
SCAta08
VENty12
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
SCAta12
assignment probability
available from 3 markers for this individual.
Figure 5.13: Newhybs plot for six analyzed microsatellite loci in adult pipefish, red = S. taenionotus,
blue = S. typhle, purple = F1-Hybrids, light purple = F2-Hybrids, orange = Backcrosses to S.
taenionotus, green = backcrosses to S. typhle, VEN = Lagoon of Venice, SCA = Sacca degli Scardovari,
ta = S. taenionotus, ty = S. typhle, 12 = caught in 2012, 08 = caught in 2008 (samples of 2008 provided
by Hablützel 2009)
assignment probability
100%
80%
60%
40%
20%
0%
1
2
3
2012
4
5
6
7
86
97
98
100
2008
Figure 5.14: Newhybs plot for six analyzed microsatellite loci in the embryos, red = S. taenionotus,
blue = S. typhle, purple = F1-Hybrids, light purple = F2-Hybrids, orange = Backcrosses to S.
taenionotus, green = backcrosses to S. typhle, VEN = Lagoon of Venice, SCA = Sacca degli Scardovari,
x-axes: identity of the fathers
35
Discussion
6 Discussion
The experimental analyses carried out here provided evidence of multiple mating and a
significant preference for large females in S. taenionotus, both patterns previously found in
S. typhle. Observation of courtship behavior revealed minor differences between the two
species, including sex-specific differences in the initiation of courtship and in courtship orientation. While courtship was initiated by males in S. taenionotus, and mating behavior was
exhibited mainly in horizontal orientation close to the bottom, courtship in S. typhle was
initiated by females and was performed in vertical orientation.
Reciprocal no-choice experiments between S. taenionotus and S. typhle resulted in a low
frequency of matings between S. typhle males and S. taenionotus females, but no reciprocal
matings. Successful matings involved the transfer of small numbers of eggs, none of which
survived to maturity. Genetic analysis of wild-caught pregnant males revealed no evidence
for interspecific gene exchange. Taken together these results suggest reproductive isolation
of the two species due to differences in courtship behavior and postmating barriers.
6.1 Mating systems
6.1.1 Mating behavior
Courtship and mating behaviors observed in S. taenionotus are generally similar to behaviors exhibited by other members of the genus Syngnathus (described in Fiedler 1954).
However minor differences were detected which may be relevant for understanding the lack
of evidence for contemporary hybridization between S. taenionotus and S. typhle. For example, the sex initiating mating appears to differ between the two species in single pair
matings. Additionally, all behaviors prior to egg transfer, including dancing, are performed in
different orientations; vertically in the eelgrass in S. typhle and horizontally close to the floor
in S. taenionotus. Interestingly, results for S. typhle from previous observations of mating
behavior differ among populations and studies. Fiedler (1954) described the active search
for mating partners in both sexes of S. typhle, followed by dancing behavior in a horizontal
orientation similar to that observed in S. taenionotus here. His description of female trunk
bending and “fake” dodging (“sprödes” Ausweichen) by the male are similar to the dancing
behavior described in this study and previously by Vincent et al. (1995). In contrast to
Fiedler’s (1954) observation of mainly horizontal courtship, Vincent et al. (1995), analyzing
courtship in the wild, observed vertical courtship, as observed here in S. typhle, after a hori36
Discussion
zontal search by males for receptive females. Vincent (1995) also described egg transfer in S.
typhle following female-female competition without obvious courtship behavior, a pattern
similar to that observed in S. taenionotus, where egg transfer was twice observed after a
male joined two dancing females. Mating behavior in S. typhle thus appears to be variable,
depending on populations and the abundance of potential mating partners, competitors and
ecological conditions (Fiedler 1954; Ahnesjö 1995; Vincent et al. 1995; Rispoli and Wilson
2008). Further analyses of geographical variation in mating behavior would be of interest in
order to determine the extent to which the pattern observed for Venice pipefish is generalizable to other populations in the region.
6.1.2 Sex-roles
Intrasexual competition was observed among males and females of both species during
the no-choice experiment, and dance-like competitive behavior was also exhibited in both
sexes of the two species. Fiedler (1954) observed the same intrasexual behavior in both male
and female S. typhle, but interpreted this as courtship rather than competition. He suggested that intrasexual interactions reflected the inability of S. typhle to differentiate between
the two sexes when aroused. Coloration-specific competition as described in S. typhle females (Berglund and Rosenqvist 1993; Bernet et al. 1998) was restricted to females of both
species, suggesting stronger sexual selection on females and possible sex-role reversal in
both species. Sex-specific activity levels differed between the two experiments, with females
showing higher activity during the intraspecific experiment (female: 1915 ± 1320 s vs. male:
1499 ± 1210 s) and males showing higher activity when presented with both S. taenionotus
and S. typhle females (male: 1927 ± 1240 s vs. female: 1535 ± 1167 s). While this differences
likely reflects stochastic variation, further investigation of sex-specific courtship activity
would be worthwhile.
Although activity during preference experiments and competitive behavior during nochoice mating experiments failed to provide clear indications of the dominant sex-roles in S.
taenionotus, previous studies have provided evidence of sex-role reversal in several
Syngnathus spp (Roelke and Sogard 1993; Jones and Avise 1997; Watanabe et al. 2000; Jones
et al. 2001) including S. typhle (Berglund et al. 1986a; Berglund et al. 1988). Further investigations of intrasexual competition and choosiness in S. taenionotus will be needed to see if it
shows sex-roles consistent with the other species in the genus Syngnathus.
37
Discussion
6.1.3 Preference for large partners
Pipefish exhibit indeterminate growth. As such, body size provides an estimate of individual age, possibly reflecting genetic quality, and acting as an indicator mechanism during
courtship (Andersson and Simmons 2006). Individuals which live longer are successful in
predation avoidance, foraging and potentially have a higher quality immune system preventing illness or parasitism (Folstad and Karter 1992). Mating with larger females also provides
direct benefits in Syngnathus spp. because larger females typically both produce larger eggs,
leading to larger embryos and therefore better survival rates, and transfer more eggs per
mating (Berglund et al. 1986a; Berglund et al. 1988).
In this study, preferences for large partners were detected in male S. taenionotus, but
not in females, a pattern expected to lead to larger body size in females due to sexual selection. However, there is no evidence of sexual size dimorphism in this species, in contrast to
the female-biased sexual size dimorphism detected in some populations of S. typhle (Rispoli
and Wilson 2008). This may indicate that additional selective forces may act to limit female
size in S. taenionotus and S. typhle populations in which males and females are similar in size
(Berglund et al. 1986a; Svensson 1988; Rispoli and Wilson 2008).
Although a number of studies have investigated preferences for large mating partners in
S. typhle, the results of these studies differ. Whereas most studies detected significant preferences for large mating partners in males (Berglund et al. 1988; Billing et al. 2007; Sundin et
al. 2010), Sandvik et al. (2000) found no such preference. The experimental design of the
Sandvik (2000) study may be prone to errors, as the placement of shelters in the choice areas may have induced focal fish to stay in the choice areas longer than they otherwise would.
In addition, Sandvik (2000) scored mating activity only every 30 minutes, which may not adequately reflect natural mating behavior, considering the large number of movements of the
focal fish detected in the experiments described here. Such point sampling could lead to a
non-representative pattern differing from that for the full experimental period. The experimental design of Sundin et al. (2010) is most similar to that carried out here, and is perhaps
the best comparison. The Sundin et al. (2010) study detected a highly significant size preference for large mating partners in male S. typhle.
Convincing data for size preference in S. typhle females are not yet available, as previous
studies revealed contradicting results (Berglund et al. 1986a; Sandvik et al. 2000) or failed to
investigate female preference (Billing et al. 2007; Sundin et al. 2010). The present study
38
Discussion
found no preference for large partners in female S. taenionotus. Interestingly, lpis were neither randomly distributed nor did they cluster around 0.5 as would be expected if individuals
were uninterested in the trait of interest. The fact that females’ values cluster around zero
and one (Figure 5.5) suggest that there may be some size associated preferences in females,
although these preferences may be individual specific. To isolate the effects of size from
other preference cues, the use of video-playback in mate choice experiments could be helpful (Robinson-Wolrath 2006).
Data for Hippocampus abdominalis from an identical experimental setup (Mattle 2007)
shows a very similar pattern to that observed here. Both S. taenionotus and H. abdominalis
show higher preference for large partners in males (S. taenionotus: mean = 0.71 ± 0.30; H.
abdominalis: 0.71 ± 0.27) than in females (S. taenionotus: 0.62 ± 0.38; H. abdominalis: 0.61 ±
0.39), and female variance is higher in both studies (S. taenionotus: females: 0.147; males:
0.090; H. abdominalis: females: 0.149; males: 0.071). The results of these studies indicate
that preferences for large mating partners in males but not in females may be a widespread
phenomena in syngnathids.
6.1.4 Multiple mating
Multiple mating in males is common in Syngnathus spp. (Wilson et al. 2003; reviewed in
Coleman and Jones 2011). Only S. scovelli (Jones and Avise 1997; Jones et al. 2001), some S.
floridae (Mobley and Jones 2009) and Italian populations of S. typhle (Rispoli 2007) exhibit
frequent monogamy. The present study supports the general pattern of multiple mating in
Syngnathus males.
With an average of more than 4 females contributing eggs to each clutch, S. taenionotus
shows one of the highest rates of multiple mating in the genus. These numbers may be
underestimates, since GERUD2 calculates the minimum number of mothers required to
produce the observed allele combinations. As only every fifth embryo was analyzed, females
which contributed a small number of eggs may not have been sampled in the analysis (Jones
et al. 1999). Since all of the males (11/11) analyzed here mated multiply, the frequency of
multiple mating in S. taenionotus appears to be close to 100%.
Multiple mating is also common in S. typhle, and has been detected in both males and
females (Berglund et al. 1988; Rispoli and Wilson 2008). Whereas the parentage analysis
carried out here does not allow for inferences concerning polyandry in S. taenionotus, I ob39
Discussion
served four mating events of the same pair of fish within eight hours, indicating that females
do not transfer all ripe eggs at once and are thus physiologically capable of multiple mating.
Multiple mating by males may occur due to physiological constraints, including limits in
the number of mature eggs produced by females (Berglund et al. 1988; Franzoi et al. 1993),
or may be an adaptive tactic to increase fitness. Female S. typhle produce more than enough
eggs to fill male brood pouches (Berglund et al. 1989; Berglund and Rosenqvist 1990), but
the large brood pouch of male S. taenionotus is capable of holding all ripe eggs from ≥ 2 females (Franzoi et al. 1993).
Multiple mating for genetic “bet-hedging” is most important in species which are not
able to clearly differentiate between beneficial and costly mating partners (Jennions and
Petrie 2000). Mating partners can be costly due to intragenomic conflicts, sterility or kin, all
of which generally reduce the fitness of the parents (Lorch and Chao 2003). The lack of clear
species recognition in preference trials suggests that S. typhle and S. taenionotus individuals
may be unable to distinguish conspecifics before commencing courtship. As such, multiple
mating in this two species could be selected for, as it may lead to higher average fitness due
to the dilution of costly mating. Therefore “bet-hedging” may be one of the benefits advancing multiple mating in Syngnathus spp. Furthermore, multiple mating is inevitable for S.
taenionotus males looking to make use of their large brood pouch capacity.
6.2 Hybridization in the Adriatic pipefish species S. taenionotus and S. typhle
The S. typhle specimens collected during this study, were significantly larger than the S.
taenionotus individuals, and significantly larger (SL = 23.8 ± 3.4 cm) than previous field collections of S. typhle. In the well-studied Swedish population, specimens rarely reach SL of
more than 20 cm (Berglund et al. 1986, Berglund et al. 1988, Sandvik et al. 2000, Sundin et
al. 2010). Other populations of S. typhle reach an average total length (TL = SL + length of the
tailfin) between 13.4 cm (Askö, Sweden) and 24.5 cm (France; Morey et al. 2003; Gurkan and
Taskavak 2007; Rispoli and Wilson 2008).
Rispoli (2007) sampled adult S. typhle from a number of populations in Europe and revealed significant interpopulational size differences. Specimens from the Lagoon of Venice
reached an average SL of 21.1 ± 3.8 cm (N = 4) for females and 14.2 ± 1.0 cm (N = 19) for
40
Discussion
males. Subsequent sampling in Venice by Wegmann (2009) yielded an average total length
of 14.3 ± 3.6 cm for females (N = 12) and 14.9 ± 3.3 cm for males (N = 10).
Since S. typhle exhibits indeterminate growth, differences between studies may indicate
that younger adults were collected in previous surveys relative to the individuals sampled
here. When considering all S. typhle collected in Rispoli (2007), Wegmann (2009) and the
present study (Figure 5.3), animals appear to fall into a number of groups of similar size. Assuming that these groupings reflect annual cohorts, individuals younger than a year can
reach SL up to 17 cm, those between one and two years of age may reach SL up to 26 cm
and larger animals are most likely to be older than 2 years.
Sampling dates alone do not explain the major size differences of the Venetian populations collected in different years. Both of previous samplings were carried out at the end of
June in 2006 (Rispoli) and in 2008 (Wegmann), close to the middle of the sampling period of
the present study (mid of May until end of July 2012). One potential explanation for the absence of small and young adult S. typhle here is a possible shift in the mating season in 2012
due to an extremely cold winter, with two days of water temperature below 0°C in February
2012 and an ice layer on the lagoon (data from the hydrobiological station in Chioggia, see
Figures 9.1 and 9.2 in the Appendix), something which delayed reproduction in other fish
species in the lagoon (i. e. Zosterissessor ophiocephalus; Poli, personal communication). A
temporal shift in the mating season in 2012 due to climatic conditions could explain the absence of small adults in this summer, but not the apparent absence of large individuals in
previous studies. Possible reasons therefore include temporal migration of adult pipefish
(Vincent et al. 1994), variable size-specific selection pressure related to predation pressure
and/or food availability, and/or differences in sampling methods. Whereas previous studies
used a boat driven by a 15 PS motor, the use of a 55 PS motor in 2012 allowed for a more
rapid trawling. If larger individuals are able to swim faster than smaller pipefish, they may
have been able to evade capture during earlier sampling.
S. taenionotus collected in 2012 reached an average SL of 15.3 ± 2.1 cm, slightly smaller
than those collected by Hablützel (2009; TL = 18.0 ± 1.3 cm), leading to an unexpectedly
large interspecific size difference (difference of means: 8.5 cm), something which led to a
significant size bias in the experiments involving both species (experiment 2 and no-choice
experiments). As a consequence at least two classes of factors in experiment 2, speciesspecific and size-dependent, likely influenced preferences of focal individuals. Because males
41
Discussion
of both species exhibit preference for large partners (Berglund et al. 1986a; Sundin et al.
2010, this study) a bias to the generally larger S. typhle females would be expected in the
male trials of both species. As S. typhle were sometimes more than double the size of the S.
taenionotus in the same trials, size sometimes may have acted as a species-recognition cue,
with individuals falling far outside the range of conspecifics being avoided. The potential
interaction between size- and species-recognition cues makes it difficult to interpret the results of this experiment. However, as this interspecific size dimorphism reflects the situation
in wild populations, no efforts were taken to reduce size differences, i.e. by excluding the
largest S. typhle and the smallest S. taenionotus individuals. To check whether size differences prevent animals from mating, intraspecific mating experiments with different sized
mating partners could be helpful.
6.2.2 Species recognition
The concept of species recognition is often used in studies investigating the origin and
maintenance of species (Mendelson and Shaw 2012). Mendelson and Shaw (2012) defined
species recognition as “a measurable difference in behavioral response toward conspecifics
as compared to heterospecifics”. Species able to distinguish between conspecifics and other
species are expected to show stronger responses to conspecific stimuli relative to these from
other species. Since no such pattern could be detected for S. taenionotus and female S.
typhle, their ability to clearly identify conspecifics is doubtful. Female S. typhle failed to exhibit any clear difference. In S. taenionotus, however, some individuals showed clear preferences for one or the other stimulus, but such preferences were not evident at the population level. To check whether the individual preferences detected here reflect real preferences, or whether they occurred by chance, repeated trials with the same focal animal
would be helpful. If the same preference is shown in most trials, individual preference may
be assumed, otherwise these patterns may reflect a stochastic artifact.
S. typhle, and possibly S. taenionotus (see 5.1.2), appear to be sex-role reversed
(Berglund et al. 1986b). As such, males of both species are expected to be more choosy, and
therefore should perform better in species recognition to reduce costly interspecific interactions. The fact that male S. typhle showed stronger response to conspecific stimuli than did
S. taenionotus males, can be explained by the preference for large sized individuals in both
species (Berglund et al. 1988; Billing et al. 2007; Sundin et al. 2010, this study). In male S.
42
Discussion
typhle, species recognition and preference for large partners are expected to reinforce one
another, as larger S. typhle females should be preferred both due to species and to size. These two signals are in conflict in S. taenionotus males, which would be expected to prefer S.
taenionotus females due to species recognition, but S. typhle due to their larger size. The
high variance in preferences shown by S. taenionotus males thus suggests that individual
males may differ in their response to size- and species-based cues. A similar conflict between
sexual selection and species recognition has been observed in several hybridizing fish taxa
(Hankison and Morris 2002; Rosenfield and Kodric-Brown 2003; Rosenthal and Ryan 2011).
The absence of a significant preference for conspecific partners in females may reflect
the lower degree of female choosiness in these two species (Berglund and Rosenqvist 1993).
Because S. typhle females showed no preference (cpi ca. 0.5), they are either not able to
discriminate between con- and interspecifics, or they simply do not mind with whom they
mate. The pattern found in S. taenionotus females was similar to that observed in males,
with no clear overall preference, but individual preferences for either the large or the conspecific stimulus, reflected in higher female variance, similar to the pattern detected in experiment 1. This may indicate some degree of choosiness in at least some female S.
taenionotus. As a consequence, there may be a sexual trait preference independent of size
in female S. taenionotus that is competing with species recognition. Further experiments to
investigate sexual-traits and species recognition-cues would allow a better understanding of
mate choice in S. taenionotus.
6.2.3 Experimental hybridization
Few eggs were transferred from S. taenionotus to S. typhle during the no-choice experiments, and no S. taenionotus males mated at all during the trials. These results conflict with
the results from the preference experiments in indicating strong premating isolation between the two species. In cases of successful mating, all transferred eggs failed to develop,
indicating that postmating isolation is also strong in this system. Observations of intraspecific
matings in both species at the same time of day suggest that temporal barriers to mating
likely are not present in this system. Post-experimental tests led to intraspecific mating in
less than24 h in 2/3 S. taenionotus and 1/3 S. typhle, indicating that individuals were reproductively receptive during the trials.
43
Discussion
The absence of strong conspecific preferences in experiment 2 suggests that both species do not discriminate in terms of potential mating partners. The exhibition of intrasexual
competition and the beginning of courtship behavior, i.e. flicking, reinforces this assumption,
as well as the exhibition of S. taenionotus-behavior in S. typhle, displaying in horizontal orientation. Nonetheless, there were few successful matings, indicating that interspecific differences in early courtship behavior may act to limit mating between the two species. Mechanical barriers to egg transfer may also exist, as the eggs of S. typhle are larger (2.0 mm)
than these of S. taenionotus (1.4 mm). As such, egg transfer from S. typhle females to S.
taenionotus males may be restricted. The experimental design used here may have also limited opportunities for mating due to high densities, which may have increased competitive
interactions. Additional no-choice mating experiments using variable densities and sex ratios
would provide valuable additional data on the potential for hybridization between the two
species under laboratory conditions.
The precise form of postmating isolation responsible for the inviability of S. taenionotus
eggs brooded by S. typhle males is unclear (Lessios and Cunningham 1990; Alipaz et al.
2001). Another possibility is the absence of a species-specific fertilization stimulus either
during egg transfer, which would lead to successful transfer of unfertilized eggs, or after egg
transfer, which triggers sperm release and fertilization of the transferred eggs.
6.2.4 Hybridization in wild populations
Perhaps unsurprisingly given the results of the laboratory trials, FST and structure analyses revealed clear differentiation of the two species in natural populations. FST values between temporal samples of the same populations were low, indicating stable allele frequencies over time. Interspecific differences, in contrast, were significantly higher. The individuals
collected from sites where they occur at low densities (i.e. S. taenionotus in the Lagoon of
Venice and S. typhle in the Sacca degli Scardovari) were less distinct, but still closer to their
own species than to the sympatric population of the heterospecifics. These results indicate
limited gene flow between populations north and south of the Po, and a lack of contemporary gene flow between the two species.
Structure analyses provided some evidence of ancestral hybridization, but the absence
of F1 or F2 hybrids, suggests that these results could reflect the hybrid origin of S.
taenionotus (Hablützel 2009). This suggestion is further reinforced by the fact that the spe44
Discussion
cies remain genetically distinct, even when collected in areas where they occur in low densities relative to heterospecifics, something which has been suggested to increase hybridization frequency in the less frequent species (Moyer 1981; Arnold et al. 1993; Wirtz 1999).
Parentage analyses revealed a similar pattern, with few embryos providing some evidence of
ancestral hybridization.
The only wild-caught individual which showed evidence of backcrossing to both species,
and therefore could not be assigned with confidence to a single species based on genetic
data, was the Scardovari S. typhle specimen caught in 2012, which carried an allele common
in S. taenionotus. Again, this counterintuitive pattern may simply reflect incomplete lineage
sorting since the ancient hybridization event 0.25 – 1.11 mya. Larger collection of such individuals would be necessary to infer contemporary mating between the two species.
The pre- and postmating isolating mechanisms explored here are likely reinforced by
ecological isolating mechanisms in natural populations. Despite efforts to identify a site at
which both species occur at high densities, such a location has not yet been found. As a consequence, the S. taenionotus and S. typhle individuals used here were collected from sites
where they largely interact with conspecifics. As both of the S. typhle specimens collected
near Scardovari were juveniles, a resident population of S. typhle may not be found in the
region, and juvenile pipefish may be transferred beyond their natural range by currents
(Giani et al. 2012). Previous studies have detected high densities of S. typhle and low densities of S. taenionotus in the Lagoon of Venice with a ratio of ca. 150 : 1 (Riccato et al. 2003;
Franco et al. 2006), and high densities of S. taenionotus and low densities of S. typhle in the
Sacca degli Scardovari, with a ratio of ca. 100 : 1 (Franzoi et al. 1993; Hablützel 2009). Detailed spatial surveys of the region would be necessary to identify whether the two species
occur sympatrically at sites along the Italian coastline. Such an area would be the most likely
location for hybridization, and would provide for more realistic tests of conspecific preference, which would be expected to be especially highly developed in mixed populations. Even
if the two species do co-occur spatially, they may be ecologically segregated, because S.
typhle mainly occur in eelgrass beds, which they only leave sparsely, whereas S. taenionotus
are mainly found in areas with sandy bottoms partly covered with macroalgae. Such habitat
segregation may act to minimize species interactions, and further limit the possibility for
hybridization.
45
Conclusions
7 Conclusions
The present study provided insights into the mating system of S. taenionotus and S.
typhle. Both species show evidence for sex-role reversal, with female-female competition
and increased sexual selection on females. Males of both species show a preference for large
mating partners. The species exhibit different levels of multiple mating: Venetian S. typhle
males mated with fewer females than S. taenionotus males. Despite the general similarities
in reproductive behavior, the two species show sex-specific differences in the initiation of
courtship and differ in their courtship orientation. Whereas S. taenionotus perform their
courtship close to the ground and in horizontal orientation, S. typhle exhibit mating behavior
in a vertical orientation, mimicking eelgrass.
To detect contemporary hybridization, reciprocal no-choice mating experiments between S. taenionotus and S. typhle were carried out, which resulted in a low frequency of
matings between S. typhle males and S. taenionotus females, but not vice-versa. Successful
matings involved the transfer of small numbers of eggs, none of which survived to maturity.
Additional genetic analyses of adult pipefish did not reveal evidence for contemporary hybridization in wild populations, even though S. typhle females and both sexes of S.
taenionotus did not discriminate between species in experiment 2.
Thus the mating systems of the two Adriatic pipefish species S. taenionotus and S. typhle
are very similar, apart from minor differences in courtship behavior. As no evidence for successful hybridization could be detected, either in wild populations or in no-choice mating
experiments, I postulate that the above mentioned minor behavioral differences are sufficient to limit the frequency of unsuccessful and therefore costly interspecific matings, and
thus maintain species separation.
46
References
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51
Appendix
9 Appendix
Table 9.1: Genotypes of adult pipefish:
SCA = Scardovari, VEN = Lagoon of Venice, ta = S. taenionotus, ty = S. typhle, 12 = 2012, 08 = 2008
Locus Names
SCAta12_1
SCAta12_2
SCAta12_3
SCAta12_4
SCAta12_5
SCAta12_6
SCAta12_7
SCAta08_1
SCAta08_2
SCAta08_3
SCAta08_4
SCAta08_5
SCAta08_6
SCAta08_7
SCAta08_8
SCAta08_9
SCAta08_10
SCAta08_11
SCAta08_12
SCAta08_13
SCAta08_14
SCAta08_15
SCAta08_16
SCAta08_17
SCAta08_18
SCAta08_19
SCAta08_20
SCAta08_21
SCAta08_22
SCAta08_23
SCAta08_24
SCAta08_25
SCAta08_26
SCAta08_27
SCAta08_28
SCAta08_29
SCAta08_30
SCAta08_31
SCAta08_32
SCAta08_33
SCAta08_34
SCAta08_35
SCAta08_36
SCAta08_37
SCAta08_39
SCAta08_40
SCAta08_41
SCAta08_42
VENty12_1
VENty12_2
VENty08_1
VENty08_2
VENty08_3
VENty08_4
VENty08_5
VENty08_8
VENty08_9
VENty08_10
VENty08_11
VENty08_12
VENty08_13
VENty08_14
VENty08_15
VENty08_16
VENty08_17
Slep06
197
199
194
194
194
194
194
194
195
192
194
194
196
202
194
204
194
196
196
190
190
192
196
194
194
194
190
194
194
194
198
192
190
196
194
198
194
192
192
196
196
194
190
196
194
196
192
194
197
203
192
192
192
192
198
192
192
194
198
192
192
192
192
192
198
199
203
199
197
201
203
199
205
212
196
201
200
200
202
200
206
194
210
208
198
198
200
196
200
194
198
192
200
198
198
198
194
196
208
202
200
204
192
194
198
196
194
194
198
204
212
192
198
201
205
198
202
200
198
200
194
200
200
200
208
198
194
208
198
200
Slep10
271
271
267
271
271
271
271
271
271
271
271
271
269
269
271
271
271
269
271
271
271
267
271
271
267
271
271
271
271
271
275
271
271
275
271
271
275
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
275
271
271
271
271
271
271
271
269
271
271
271
271
271
271
271
271
271
271
271
267
275
271
271
271
271
271
271
271
271
271
271
271
271
275
271
271
271
271
275
271
271
269
271
269
269
273
269
269
269
269
269
271
269
271
269
273
277
273
273
271
269
275
271
271
271
273
271
271
271
273
273
273
Slep12
204
208
192
208
208
216
205
208
200
208
198
198
204
204
194
208
198
192
233
208
188
196
196
200
206
223
223
227
233
198
198
198
208
198
219
196
208
208
213
208
194
192
200
213
223
200
208
233
194
194
194
194
194
194
194
194
194
194
194
194
194
194
194
194
194
221
219
216
216
208
221
238
216
213
238
218
217
229
213
208
208
213
196
233
229
190
225
200
227
225
231
223
233
233
211
219
198
208
198
227
196
215
208
227
208
221
233
208
221
223
213
208
233
194
194
198
194
194
194
194
194
194
194
196
194
194
198
198
194
198
Slep13
350
350
352
350
352
350
344
352
350
348
350
350
352
348
339
354
341
352
337
350
350
348
346
353
352
335
339
350
352
352
344
354
350
350
335
344
352
352
350
348
335
352
354
350
352
350
348
350
345
353
345
347
347
352
346
352
347
347
347
345
350
348
352
345
346
352
357
352
354
354
357
353
354
356
356
350
350
352
350
346
356
355
357
350
352
350
352
352
357
352
357
350
353
357
353
357
357
352
350
335
344
354
352
350
352
335
352
355
352
352
357
356
352
349
353
346
357
352
353
355
357
353
352
349
351
352
352
355
352
345
Styph12
155
149
153
176
155
163
170
155
163
153
163
163
153
165
149
172
145
153
153
178
163
165
153
180
172
165
176
163
184
157
176
176
153
188
153
178
153
186
180
188
163
143
163
252
149
172
143
155
157
149
161
163
163
172
163
153
153
163
153
153
176
159
163
174
155
172
163
186
170
184
172
165
153
151
163
157
149
180
172
174
153
172
161
157
186
163
170
153
184
157
163
184
178
174
169
172
161
172
195
178
172
165
165
172
163
157
167
174
170
167
172
172
159
167
Styph44
134
134
134
134
134
134
134
127
127
127
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
143
143
143
143
143
143
143
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
143
143
143
143
143
143
143
143
143
143
143
143
143
143
130
143
143
143
143
143
143
143
143
143
143
52
Appendix
VENty08_18
VENty08_19
VENty08_20
VENty08_21
VENty08_22
VENty08_23
VENty08_24
VENty08_25
VENty08_26
VENty08_27
VENty08_28
VENty08_29
VENty08_30
VENty08_31
VENty08_32
VENty08_33
VENty08_34
VENty08_35
VENty08_36
VENty08_37
VENty08_38
VENty08_39
VENty08_40
VENty08_41
VENty08_42
VENty08_43
VENty08_44
VENty08_45
SCAty12_1
SCAty08_1
VENta08_1
VENta08_2
192
192
192
192
192
198
192
192
192
192
194
200
194
192
192
192
192
192
192
198
192
192
192
192
194
192
192
198
194
204
208
194
200
194
208
198
198
198
202
206
238
194
200
198
196
202
202
192
202
202
198
200
206
200
200
200
200
194
192
198
0
269
0
273
269
269
269
273
271
273
271
271
269
269
269
271
269
273
269
269
273
273
273
271
269
269
269
271
279
273
273
273
273
271
269
269
269
273
273
269
273
271
267
271
271
270
194
194
194
194
194
194
194
198
194
192
194
192
190
194
194
194
198
194
194
194
194
194
194
194
194
192
194
194
194
194
196
196
211
196
194
194
196
194
194
211
225
194
196
198
194
198
194
194
198
198
194
194
194
194
198
194
194
198
194
198
198
194
196
213
345
345
351
345
352
347
348
345
330
352
348
348
330
330
325
353
347
347
352
353
353
347
350
347
352
353
330
353
346
345
349
354
353
353
353
348
353
352
352
347
353
352
353
348
354
330
345
354
353
350
353
353
354
353
351
355
357
353
330
357
354
352
350
354
159
169
170
164
163
157
167
154
169
159
168
170
178
178
165
169
163
170
165
167
165
157
165
157
165
167
174
170
169
151
163
171
153
170
157
165
194
155
153
153
169
192
184
190
172
163
186
172
197
170
172
163
174
201
169
192
165
201
194
155
180
169
136
143
134
143
143
143
129
143
143
143
143
143
143
143
143
143
143
143
143
132
143
143
143
143
143
143
143
143
143
143
143
143
143
143
143
143
143
143
136
143
143
143
143
143
143
143
143
143
134
143
134
134
143
143
143
143
134
134
53
Appendix
Table 9.2: Genotypes of S. taenionotus embryos:
SCA = Scardovari, fat = father
SCA1_000
SCA1_001
SCA1_006
SCA1_011
SCA1_016
SCA1_021
SCA1_026
SCA1_031
SCA1_036
SCA1_041
SCA1_046
SCA1_051
SCA1_056
SCA1_061
SCA1_066
SCA1_071
SCA1_076
SCA1_081
SCA1_086
SCA1_091
SCA1_096
SCA1_101
SCA1_106
SCA1_111
SCA1_116
SCA1_121
SCA1_126
SCA1_131
SCA1_136
SCA1_141
SCA1_146
SCA2_000
SCA2_001
SCA2_006
SCA2_011
SCA2_016
SCA2_021
SCA2_026
SCA2_031
SCA2_036
SCA2_041
SCA2_046
SCA2_051
SCA2_056
SCA2_061
SCA2_066
SCA2_071
SCA2_076
SCA2_081
SCA2_086
SCA2_091
SCA2_096
SCA2_101
SCA3_000
SCA3_001
SCA3_006
SCA3_011
SCA3_016
SCA3_021
SCA3_026
SCA3_031
SCA3_036
SCA3_041
SCA3_046
SCA3_051
SCA3_056
SCA3_061
SCA3_066
Slep06
197
194
199
197
194
199
199
197
197
194
197
194
197
194
194
194
194
197
197
194
194
192
199
201
199
199
199
199
197
199
197
197
199
197
197
197
197
199
199
199
192
197
194
199
194
197
194
199
203
199
199
197
197
199
194
194
203
199
203
194
201
201
194
199
194
194
199
201
201
199
199
194
194
194
194
194
194
194
203
203
203
203
203
203
203
199
203
203
199
203
199
199
199
199
199
203
199
199
194
194
199
194
199
194
194
199
192
199
199
199
205
194
205
201
201
201
201
201
201
194
107
207
203
Slep10
271
267
271
271
271
275
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
277
271
271
271
275
273
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
267
271
271
271
271
271
271
271
271
271
271
271
267
267
267
271
271
271
271
271
271
271
271
271
275
275
271
275
271
271
275
271
271
275
271
275
271
275
Slep12
204
204
204
204
221
204
221
221
204
204
221
221
221
204
204
204
221
204
204
221
221
204
204
204
204
204
204
208
208
213
204
208
196
208
196
196
198
198
198
198
198
219
208
219
198
208
198
198
198
219
198
208
208
192
192
192
216
192
194
216
192
192
192
192
216
192
196
196
221
208
208
208
240
240
240
221
221
231
231
231
231
204
221
204
221
204
231
221
233
221
221
221
208
210
221
221
221
221
214
219
208
218
208
208
208
208
208
219
208
235
235
235
208
235
219
219
208
235
208
235
219
216
192
216
216
200
216
216
216
215
215
196
216
196
216
216
Slep13
350
350
352
352
Styph12
155
163
350
352
153
163
346
342
352
345
352
352
352
352
350
352
352
352
350
352
352
350
352
354
352
352
350
352
352
352
153
153
155
163
155
163
155
136
188
188
352
350
352
350
155
155
155
155
155
155
155
155
163
188
155
163
155
188
163
155
188
163
352
346
350
352
352
352
352
350
350
357
357
357
352
352
352
352
353
353
350
352
350
350
352
350
350
350
350
350
353
352
352
352
352
352
335
344
344
344
344
344
340
350
350
344
352
352
350
352
352
352
352
357
357
357
357
357
357
357
357
357
357
357
352
357
352
352
357
353
353
353
353
353
357
352
352
352
352
352
352
352
352
352
352
352
352
352
352
352
153
163
155
163
153
153
153
149
149
157
149
149
165
149
149
149
149
165
165
165
165
149
165
149
165
165
149
165
165
153
153
153
153
153
149
149
153
153
153
149
153
153
153
153
163
163
163
163
163
155
163
165
157
165
153
153
174
174
174
165
165
174
174
165
165
174
174
165
174
165
174
174
165
153
180
190
180
190
153
153
188
188
188
153
172
172
172
180
Styph44
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
54
Appendix
SCA3_071
SCA4_000
SCA4_001
SCA4_006
SCA4_011
SCA4_016
SCA4_021
SCA4_026
SCA4_031
SCA4_036
SCA4_041
SCA4_046
SCA4_051
SCA4_056
SCA4_061
SCA5_000
SCA5_001
SCA5_006
SCA5_011
SCA5_016
SCA5_021
SCA5_026
SCA5_031
SCA5_036
SCA5_041
SCA5_046
SCA5_051
SCA5_056
SCA5_061
SCA5_066
SCA6_000
SCA6_001
SCA6_006
SCA6_011
SCA6_016
SCA6_021
SCA6_026
SCA6_031
SCA6_036
SCA6_041
SCA6_046
SCA6_051
SCA6_056
SCA6_061
SCA6_066
SCA6_071
SCA6_076
SCA6_081
SCA6_086
SCA6_091
SCA6_096
SCA6_101
SCA6_106
SCA6_111
SCA6_116
SCA6_121
SCA6_126
SCA6_131
SCA7_000
SCA7_001
SCA7_006
SCA7_011
SCA7_016
SCA7_021
SCA7_026
SCA7_031
SCA7_036
SCA086_000
SCA086_001
SCA086_006
SCA086_011
SCA086_016
194
194
197
197
197
194
197
194
194
207
197
203
199
199
201
203
203
203
194
197
194
194
194
194
197
197
194
192
194
192
192
192
194
192
192
192
192
192
194
194
194
194
194
201
194
194
194
194
194
194
203
194
203
194
194
194
194
194
194
194
194
194
194
197
201
197
203
197
201
201
201
205
201
205
201
201
194
194
194
194
194
194
194
203
203
194
194
203
203
194
194
197
203
194
194
205
203
205
205
205
203
203
199
199
207
207
197
203
194
190
194
194
194
194
194
194
194
194
194
194
199
199
199
199
197
199
201
194
194
194
196
196
194
205
194
205
205
205
271
271
271
271
271
271
271
271
271
275
275
271
271
271
275
271
275
271
275
273
275
275
275
273
275
271
271
275
271
271
271
271
271
271
263
271
263
263
271
271
271
271
271
271
271
271
271
271
271
271
271
273
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
275
271
271
275
275
275
271
271
271
269
271
271
271
271
275
275
271
275
271
271
269
271
192
208
216
194
216
192
208
192
192
216
204
204
204
216
216
208
208
208
208
208
208
208
208
198
198
198
208
198
208
208
216
204
204
204
208
208
196
208
221
216
216
208
208
221
216
216
208
216
208
198
221
216
221
221
221
213
221
221
205
225
225
225
225
204
214
205
214
208
204
216
204
198
196
216
221
216
221
208
216
208
208
216
208
216
216
216
216
208
238
238
227
238
227
238
238
208
208
208
218
208
218
218
221
221
221
221
221
216
221
221
231
229
223
221
221
223
223
223
221
223
221
221
221
227
227
227
227
221
233
233
238
238
238
238
238
238
238
214
238
216
216
231
216
208
350
350
352
354
345
350
153
176
163
176
176
172
180
176
180
180
134
134
134
134
134
134
134
134
161
155
180
180
134
134
155
176
134
134
134
126
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
126
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
127
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
352
346
346
346
346
346
346
346
350
352
350
352
350
352
352
350
350
350
350
350
350
350
354
354
352
354
354
352
354
354
354
354
354
354
354
352
352
357
357
350
352
357
357
350
165
155
155
155
155
165
155
165
155
155
155
155
153
155
153
153
163
163
172
163
163
163
165
176
172
165
165
165
172
165
172
165
176
176
176
172
176
172
172
165
172
180
172
165
180
165
350
350
352
350
348
348
348
348
348
352
352
350
350
350
350
350
352
350
350
350
344
344
344
344
345
344
344
353
344
352
352
354
354
352
356
357
357
352
357
357
357
357
357
357
357
356
354
350
350
356
357
352
352
350
353
352
352
353
353
354
354
357
357
354
358
354
358
354
153
165
155
163
163
163
163
155
163
163
163
165
163
153
153
153
153
163
163
163
170
153
153
153
153
163
163
163
163
155
163
153
153
153
163
176
165
165
163
163
163
165
163
163
165
172
172
163
163
165
165
165
163
163
176
170
170
170
170
170
170
170
170
163
190
163
163
163
55
Appendix
SCA086_021
SCA086_026
SCA086_031
SCA086_036
SCA086_041
SCA086_046
SCA086_051
SCA086_056
SCA086_061
SCA086_066
SCA086_071
SCA086_076
SCA086_081
SCA086_086
SCA086_091
SCA086_096
SCA086_101
SCA086_106
SCA086_111
SCA086_116
SCA086_121
SCA086_126
SCA086_131
SCA086_136
SCA097_000
SCA097_001
SCA097_006
SCA097_011
SCA097_016
SCA097_021
SCA097_026
SCA097_031
SCA097_036
SCA097_041
SCA097_046
SCA097_051
SCA097_056
SCA097_061
SCA097_066
SCA097_071
SCA097_076
SCA097_081
SCA097_086
SCA097_091
SCA097_096
SCA097_101
SCA097_106
SCA097_111
SCA097_116
SCA097_121
SCA097_126
SCA097_131
SCA098_000
SCA098_001
SCA098_006
SCA098_011
SCA098_016
SCA098_021
SCA098_026
SCA098_031
SCA098_036
SCA098_041
SCA098_046
SCA098_051
SCA098_056
SCA098_061
SCA098_066
SCA098_071
SCA098_076
SCA098_081
SCA098_086
SCA098_091
194
194
194
196
199
194
205
205
194
194
194
194
199
205
194
201
205
194
205
205
194
194
205
201
194
201
205
194
199
199
205
205
199
205
205
205
205
205
205
205
205
205
205
205
195
212
201
212
269
271
271
269
271
271
271
271
271
271
269
271
271
271
271
271
271
271
269
271
271
271
269
271
271
271
269
271
271
271
271
271
271
271
201
212
271
275
197
212
267
271
271
271
205
212
195
199
271
267
267
271
271
271
271
271
195
199
195
192
196
196
192
196
201
196
196
205
205
196
196
196
192
196
196
192
196
192
192
192
192
194
192
192
205
205
196
196
196
196
197
205
196
196
197
196
194
197
271
271
275
271
271
275
271
271
271
271
275
275
275
275
271
271
271
271
271
271
271
271
275
275
271
271
271
275
204
204
198
208
216
208
216
208
352
354
352
350
352
358
358
354
204
216
208
208
208
216
204
208
204
204
208
204
202
204
208
204
204
208
200
213
200
200
198
200
208
216
227
208
223
216
216
223
208
208
223
208
208
216
216
216
208
233
213
223
223
231
200
231
352
350
350
350
350
350
354
350
350
350
350
350
350
350
350
350
354
352
350
335
335
350
350
354
354
352
352
354
354
352
354
354
352
354
354
352
352
354
354
352
354
354
356
350
356
352
350
356
200
200
213
213
213
192
192
192
208
200
192
208
192
200
192
213
213
200
198
198
213
208
198
198
198
204
198
198
204
198
204
198
198
204
198
208
208
208
227
208
194
231
231
231
231
231
200
200
200
213
208
200
213
213
208
200
213
213
200
200
213
213
238
208
198
198
204
208
198
208
208
204
238
208
204
198
238
238
227
227
208
218
350
350
350
350
350
335
335
345
335
345
335
345
345
335
345
350
350
350
354
354
350
350
350
350
356
356
356
350
350
356
350
350
350
350
352
352
348
350
352
356
356
356
348
348
348
348
348
348
350
348
350
350
348
348
348
348
352
348
348
352
348
356
348
356
348
348
356
348
356
356
348
356
356
352
356
348
350
356
163
157
163
153
163
163
157
163
163
153
163
163
163
155
155
155
163
163
163
155
155
163
155
163
163
163
163
163
173
163
163
163
163
163
163
163
163
163
163
163
163
165
163
163
155
165
165
165
163
163
165
163
163
184
176
173
176
184
155
155
163
163
163
173
153
157
157
180
157
180
157
163
157
157
157
155
155
155
155
163
155
153
153
153
153
157
157
157
153
153
153
153
153
153
153
153
157
153
157
157
153
184
184
184
184
184
184
163
180
163
184
163
163
163
163
163
173
163
157
163
163
163
163
163
163
163
163
163
163
163
163
163
157
163
157
157
163
163
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
127
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
138
134
134
134
134
134
134
134
134
134
134
134
134
134
127
134
127
134
134
134
134
134
134
134
134
127
127
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
136
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
56
Appendix
SCA098_096
SCA098_101
SCA100_000
SCA100_001
SCA100_005
SCA100_011
SCA100_016
SCA100_021
SCA100_026
SCA100_031
SCA100_036
SCA100_041
SCA100_046
SCA100_051
SCA100_056
SCA100_061
SCA100_066
SCA100_071
SCA100_076
SCA100_081
SCA100_086
SCA100_091
SCA100_095
SCA100_101
SCA100_106
SCA100_111
SCA100_116
SCA100_121
SCA100_126
SCA100_131
SCA100_136
SCA100_141
SCA100_146
SCA100_151
194
192
194
201
196
194
194
194
194
194
201
194
194
201
194
194
201
194
194
194
201
194
190
190
194
194
194
194
197
197
194
197
194
201
196
196
201
201
201
201
201
194
201
201
201
201
201
201
201
201
201
201
201
201
201
201
194
194
196
203
205
205
201
201
197
201
197
205
271
271
271
271
271
271
271
269
271
271
271
271
271
271
271
271
271
269
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
271
208
208
198
198
198
198
194
198
212
216
194
194
198
194
198
194
194
198
198
194
194
194
212
198
198
218
216
198
208
208
218
216
200
218
218
198
218
218
198
198
204
198
108
218
218
234
208
218
218
218
218
212
212
218
218
224
348
348
350
350
350
350
350
339
350
350
350
350
350
350
350
350
350
339
350
350
350
350
350
348
352
350
358
352
350
356
350
350
350
350
352
356
352
352
352
356
350
352
352
356
352
350
350
356
218
198
218
198
198
227
224
224
224
198
350
350
350
350
352
352
153
153
163
153
157
163
155
157
173
163
155
155
155
155
155
155
155
155
149
149
149
155
155
153
153
153
153
157
163
155
163
157
153
157
163
157
176
163
163
176
163
176
176
173
176
176
176
163
176
176
163
163
176
163
176
176
163
163
176
176
163
163
176
163
176
176
163
176
134
134
134
134
134
125
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
134
57
Appendix
Table 9.3: Raw data of experiment 1: m = male, f = female, lpi = large preference index
Trial
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
26
27
28
29
30
31
32
33
Date
29.05.
29.05.
29.05.
30.05.
30.05.
30.05.
31.05.
02.06.
02.06.
03.06.
03.06.
04.06.
04.06.
05.06.
05.06.
06.06.
06.06.
07.06.
07.06.
08.06.
08.06.
09.06.
09.06.
10.06.
11.06.
11.06.
13.06.
13.06.
14.06.
14.06.
15.06.
15.06.
time
10:24
08:58
08:57
08:56
08:59
10:37
09:25
09:03
09:04
10:30
10:29
09:04
09:05
09:08
09:08
09:23
09:24
09:03
09:03
09:10
09:10
10:35
10:35
09:06
08:56
08:57
10:31
10:32
09:05
09:06
08:12
08:12
sex
m
m
m
f
f
f
f
f
f
m
m
f
f
m
m
f
m
f
f
f
f
f
f
m
f
m
f
f
f
m
m
f
Focal
SL [cm]
17.0
16.7
17.6
15.4
16.6
17.3
15.5
16.6
16.1
15.0
16.1
17.1
15.0
17.6
17.4
17.1
17.2
15.6
16.4
15.6
17.5
18.3
17.4
16.9
16.0
17.4
12.6
17.3
15.9
14.8
16.9
16.0
Stimulus left
SL [cm]
time [s]
17.6
420
17.7
190
15.5
3441
15.0
0
18.0
2269
16.9
1918
17.4
3368
13.3
0
15.1
2387
17.1
3074
17.7
589
17.5
0
16.9
2135
17.6
530
16.6
226
17.1
374
15.5
335
18.6
1036
12.1
2721
17.6
0
17.5
1126
16.8
0
17.1
1786
15.6
0
16
0
15.5
0
17.4
7
16.5
634
12.2
32
15.7
129
15.7
308
18.0
0
Stimulus right
SL [cm]
time [s]
16.2
298
16.0
2
17.3
0
16.5
0
16.1
1384
15.0
1238
15.7
0
15.6
1163
17.0
719
15.6
344
12.4
533
15.3
0
13.8
1398
16.3
52
15.2
563
19.7
2233
16.8
633
17.1
15
17.6
378
15.9
111
19.9
73
18.1
0
15.3
587
17.4
41
19.0
424
18.4
1472
16.0
0
18.5
1900
17.3
228
17.2
478
17.0
698
16.6
906
∆ SL [cm]
1.4
1.7
1.8
1.5
1.9
1.9
1.7
2.3
1.9
1.5
5.3
2.2
3.1
1.3
1.4
2.6
1.3
1.5
5.5
1.7
2.4
1.3
1.8
1.8
3.0
2.9
1.4
2.0
5.1
1.5
1.3
1.4
lpi
0.585
0.990
0
0.621
0.608
1
1
0.231
0.899
0.525
0.604
0.911
0.286
0.857
0.654
0.986
0.122
0
0.061
0.753
1
1
1
1
0.750
0.877
0.787
0.306
0
58
Appendix
Table 9.4: Raw data of experiment 2:
Spec = species, SL = standard length, w = wet weight, ta = S. taenionotus, ty = S. typhle, m = male, f =
female, cpi = conspecific preference index
Focal fish
Date
26.06.
26.06.
27.06.
27.06.
28.06.
28.06.
29.06.
29.06.
30.06.
01.07.
01.07.
03.07.
03.07.
03.07.
04.07.
04.07.
04.07.
04.07.
05.07.
05.07.
05.07.
06.07.
06.07.
06.07.
06.07.
07.07.
07.07.
07.07.
08.07.
08.07.
08.07.
08.07.
09.07.
09.07.
10.07.
10.07.
10.07.
11.07.
11.07.
12.07.
12.07.
14.07.
14.07.
14.07.
15.07.
15.07.
15.07.
16.07.
16.07.
17.07.
17.07.
17.07.
18.07.
18.07.
18.07.
18.07.
19.07.
19.07.
20.07.
20.07.
20.07.
20.07.
21.07.
21.07.
time
08:43
08:44
09:00
09:01
08:57
09:02
09:03
09:04
09:04
08:52
08:52
09:02
09:03
10:21
08:47
08:49
10:19
10:20
10:30
09:00
10:32
08:58
10:35
09:01
10:37
10:33
10:34
08:58
10:37
09:02
09:08
10:38
08:58
08:59
08:58
10:24
09:07
09:00
09:01
08:56
08:57
08:57
09:03
10:36
09:03
09:04
10:54
09:06
10:31
08:58
08:58
10:30
08:47
10:20
08:48
10:21
09:04
09:04
10:25
09:05
09:05
10:26
08:58
10:33
name tank
tam1
a
tam2
b
taf1
a
taf2
b
tam3
a
tym1
b
tyf1
a
tyf2
b
tam4
b
tym2
a
taf3
b
tyf3
a
tam5
b
taf4
c
tym3
a
tym4
c
tyf4
b
taf5
d
tyf6
a
tyf5
b
taf6
c
taf7
a
tym6
b
tyf7
c
tym7
d
tyf8
b
tam6
c
taf8
d
tam8
a
tyf9
b
tam7
c
tyf10
d
tyf11
a
tym9
b
taf9
b
tam9
c
taf10
d
tyf13
a
tyf14
b
tam10
a
tym10
b
taf11
a
tam11
c
tam12
b
tym11
b
tam13
c
tam14
a
taf12
b
tym13
c
tyf15
a
tym14
b
tam15
c
tyf16
a
tym15
b
tym16
c
taf13
d
tym17
a
taf14
c
tyf17
a
tym18
b
taf15
c
tym19
d
tam16
a
tym20
b
spec
ty/ta
ta
ta
ta
ta
ta
ty
ty
ty
ta
ty
ta
ty
ta
ta
ty
ty
ty
ta
ty
ty
ta
ta
ty
ty
ty
ty
ta
ta
ta
ty
ta
ty
ty
ty
ta
ta
ta
ty
ty
ta
ty
ta
ta
ta
ty
ta
ta
ta
ty
ty
ty
ta
ty
ty
ty
ta
ty
ta
ty
ty
ta
ty
ta
ty
sex
m/f
m
m
f
f
m
m
f
f
m
m
f
f
m
f
m
m
f
f
f
f
f
f
m
f
m
f
m
f
m
f
m
f
f
m
f
m
f
f
f
m
m
f
m
m
m
m
m
f
m
f
m
m
f
m
m
f
m
f
f
m
f
m
m
m
SL
cm
13.3
16.0
17.2
17.5
16.8
20.7
22.8
22.5
17.7
27.5
15.7
17.4
18.8
17.9
25.2
25.7
20.6
12.5
22.0
24.0
15.6
15.5
25.3
21.6
24.0
20.2
15.9
15.5
12.6
22.9
18.0
24.0
20.5
24.0
16.6
18.0
16.5
25.1
22.1
16.5
29.5
19.7
14.5
19.2
24.2
13.8
16.5
13.7
15.8
27.4
25.3
13.8
22.1
16.8
27.8
12.0
21.1
14.5
24.0
30.1
13.4
26.7
13.4
23.2
W
g
0.83
1.08
1.19
1.32
2.02
3.41
4.26
5.14
1.78
7.9
0.98
2.09
1.7
2.17
5.5
6.76
2.6
0.53
3.66
6.95
1.13
0.96
6.01
3.84
5.41
2.64
1.41
0.93
0.57
4.58
2.07
4.85
3.25
6.06
1.33
1.95
1.22
6.15
5.1
1.87
10.3
2.8
1.35
2.67
5.81
1.07
1.58
0.89
1.62
6.18
9.05
1.06
5.17
1.97
10.8
0.80
3.43
1.15
4.85
9.11
1.06
8.82
1.06
5.74
left
sec
273
3048
677
2333
1480
799
1711
215
1340
1438
1938
2093
1100
1102
1022
987
1751
295
425
152
75
59
18
770
201
570
0
12
139
1850
1345
1808
643
2317
168
253
0
1009
836
128
522
98
0
163
1086
3555
161
1686
1411
906
1417
360
155
491
1552
1182
1450
634
1524
270
49
84
0
1903
right
sec
1711
0
1602
220
1441
589
794
1219
1151
971
433
378
2192
283
150
0
250
1918
0
491
732
973
2023
1059
3084
1527
0
3588
687
36
373
824
1384
109
148
28
3229
1486
1121
153
1725
1287
18
1667
514
30
904
847
165
1069
1149
186
2628
99
289
61
377
44
663
328
427
1555
0
429
cpi
0.862
1
0.297
0.914
0.507
0.576
0.317
0.850
0.536
0.597
0.183
0.153
0.334
0.796
0.872
0
0.125
0.134
1
0.764
0.093
0.057
0.991
0.579
0.939
0.728
0.003
0.168
0.019
0.782
0.687
0.683
0.955
0.532
0.100
1
0.596
0.427
0.456
0.768
0.929
1
0.089
0.679
0.008
0.151
0.666
0.895
0.459
0.552
0.341
0.944
0.832
0.843
0.049
0.794
0.935
0.697
0.452
0.897
0.051
0.816
stimulus
stimulus
left compartment rigth compartment
spec
SL
w
spec
SL
w
ty/ta
cm
g
ty/ta
cm
g
ty
20.6 2.6
ta
15.5 0.96
ta
15.7 0.98
ty
22.5 4.26
ta
15.6 1.84
ty
24.2 5.81
ta
16.9 1.56
ty
27.5 7.9
ta
12.5 0.53
ty
17.4 2.09
ty
21.6 3.84
ta
15.5 0.93
ta
13.3 0.83
ty
25.2 5.5
ta
16.9 1.56
ty
27.5 7.9
ta
17.9 2.17
ty
20.6 2.6
ty
22.0 3.66
ta
15.4 1.7
ty
20.7 3.41
ta
13.3 0.83
ta
18.4 1.7
ty
26.7 8.82
ta
15.5 0.93
ty
24.0 6.95
ta
19.2 2.67
ty
24.0 6.06
ty
21.6 3.84
ta
17.5 1.32
ta
16.5 1.22
ty
22.5 4.26
ta
15.9 1.41
ty
20.7 3.41
ta
18.0 1.95
ty
25.3 6.01
ty
24.0 6.06
ta
18.8 1.7
ta
17.7 1.78
ty
24.9 7.12
ta
16.8 2.02
ty
26.7 8.82
ta
14.5 1.35
ty
15.8 1.62
ta
15.7 0.98
ty
24.0 4.85
ta
16.5 1.87
ty
25.2 5.5
ta
16.6 1.33
ty
17.4 2.09
ta
18.0 1.95
ty
27.8 10.8
ty
22.5 5.14
ta
15.6 1.13
ta
18.8 1.7
ty
16.8 1.97
ta
17.5 1.32
ty
22.0 3.66
ta
16.5 1.58
ty
29.5 10.3
ta
16.5 1.22
ty
20.3 3.17
ty
15.8 1.62
ta
14.5 1.35
ta
19.2 2.67
ty
25.3 6.01
ty
22.5 5.14
ta
15.5 0.96
ta
16.5 1.87
ty
24.0 5.41
ty
22.9 4.58
ta
15.6 1.13
ty
27.8 10.8
ta
16.5 1.58
ta
19.2 2.67
ty
25.3 9.05
ty
23.2 5.74
ta
18.0 2.07
ta
16.6 1.33
ty
23.2 4.42
ta
15.6 1.13
ty
24.0 4.85
ty
21.1 3.43
ta
15.9 1.41
ta
14.5 1.15
ty
25.1 6.15
ta
13.7 0.89
ty
22.1 5.1
ty
22.9 4.58
ta
13.3 0.89
ty
24.0 4.85
ta
12.0 0.8
ta
14.4 1.02
ty
20.5 3.25
ta
12.6 0.57
ty
29.5 10.3
ty
22.1 5.1
ta
14.5 1.15
ty
24.2 5.81
ta
13.4 1.06
ty
25.3 5.12
ta
19.7 2.8
ty
24.2 4.68
ta
13.3 0.89
ta
13.8 1.07
ty
24.0 5.41
ty
25.1 6.15
ta
13.7 0.89
ty
23.2 4.42
ta
14.0 1
ty
23.2 5.74
ta
18.0 2.07
ty
20.2 2.64
ta
14.6 1.17
ta
13.8 1.06
ty
25.3 9.05
ty
24.9 5.55
ta
13.6 1.15
ty
20.3 3.17
ta
12.9 0.69
ty
26.7 7.75
ta
13.8 1.07
ty
20.5 4.58
ta
19.7 2.8
ta
14.6 1.17
ty
25.3 5.12
ty
27.4 6.18
ta
12.0 0.8
∆ SL
stimuli
5.1
6.8
8.6
10.6
4.9
6.1
11.9
10.6
2.7
6.6
7.4
8.3
8.5
4.8
4.1
6.0
4.8
7.3
5.2
7.2
9.9
1.3
8.3
8.7
0.8
9.8
6.9
2.0
4.5
13
3.8
1.3
6.1
7.0
7.5
7.3
11.3
6.1
5.2
6.6
8.4
5.2
10.6
8.4
6.1
12.0
6.1
16.9
7.6
10.8
5.6
10.9
10.2
11.4
9.2
5.2
5.6
11.5
11.3
7.4
12.9
0.8
10.7
15.4
59
Appendix
21.07.
21.07.
22.07.
22.07.
22.07.
22.07.
23.07.
23.07.
23.07.
23.07.
24.07.
24.07.
24.07.
24.07.
09:02
10:34
10:25
09:03
10:26
09:04
09:05
10:25
10:26
09:05
10:32
09:01
10:33
09:02
taf16
tam17
tam18
tym21
tyf18
tyf19
taf17
taf18
tam19
tyf20
tam20
taf19
tym22
taf20
c
d
a
b
c
d
a
b
c
d
a
b
c
d
ta
ta
ta
ty
ty
ty
ta
ta
ta
ty
ta
ta
ty
ta
f
m
m
m
f
f
f
f
m
f
m
f
m
f
13.3
13.1
13.2
23.0
29.5
28.5
13.4
14.6
14.6
20.3
13.5
12.9
26.7
14.0
0.89
1.09
0.81
4.61
10.7
8.76
1.00
1.17
1.43
3.17
1.09
0.69
7.75
1.53
149
0
74
1542
849
976
93
18
1216
938
3600
3444
1165
65
79
3586
410
2009
2534
1856
281
63
27
1220
0
129
595
282
0.346
0
0.847
0.566
0.749
0.951
0.751
0.222
0.978
0.565
1
0.964
0.338
0.813
ty
ta
ty
ta
ta
ty
ty
ta
ta
ta
ta
ta
ta
ty
22.5
13.4
20.2
14.0
13.8
30.1
24.9
13.4
13.2
18.1
14.0
13.2
13.6
30.1
4.02
1
2.64
1.53
1.06
9.11
5.55
1.06
0.89
2.58
1
0.81
1.11
9.11
ta
ty
ta
ty
ty
ta
ta
ty
ty
ty
ty
ty
ty
ta
14.6
19.4
12.9
24.2
26.7
13.8
13.6
21.1
27.4
23.4
29.5
23.0
19.4
13.1
1.43
3.74
0.69
4.68
7.75
1.06
1.15
3.43
6.18
5.75
10.7
4.61
3.74
1.09
7.9
6.0
7.3
10.2
12.9
16.3
11.3
7.7
14.2
5.3
15.5
9.8
5.8
17.0
40
Air Temperature
[°C]
35
30
Water
Temperature [°C]
25
20
Salinity [g/kg]
15
10
pH
5
0
Oxygen [ml/l]
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
20
11
20
12
Figure 9.1: Hydrobiological data of the Lagoon in Venice from 2002 – 2012 measured in Chioggia
based on monthly averages (Data provided by the hydrobiological station in Chioggia:
http://chioggia.scienze.unipd.it/ita/ParametriLaguna.html)
Temperature [°C]
35
30
25
20
15
10
5
0
Sep Nov Jan Mar Mai Jul
05 05 06 06 06 06
07 07 08 08 08 08
11 11 12 12 12 12
Figure 9.2: Water and air temperature in Chioggia in the sampling years; red = water temperature,
blue = air temperature, grey = sampling period (Data provided by the hydrobiological station in
Chioggia: http://chioggia.scienze.unipd.it/ita/ParametriLaguna.html)
60

Mating patterns and mechanisms of isolation in Adriatic

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