Reproductive isolation following hybrid speciation in Mediterranean pipefish (Syngnathus spp.)

Animal Behaviour 161 (2020) 77e87

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Reproductive isolation following hybrid speciation in Mediterranean pipefish (Syngnathus spp.) Florian N. Moser a, Anthony B. Wilson a, b, c, * a

University of Zurich, Institute of Ecology and Environmental Sciences, Zurich, Switzerland Brooklyn College, Department of Biology, Brooklyn, NY, U.S.A. c The Graduate Center, City University of New York, NY, U.S.A. b

a r t i c l e i n f o Article history: Received 29 July 2019 Initial acceptance 28 August 2019 Final acceptance 22 November 2019 MS. number: A19-00513R Keywords: hybridization mate choice multiple mating parentage reproductive isolation

The development of reproductive isolation is a crucial step in the speciation process. Premating isolation is often implicated in traditional models of divergence with gene flow, but the evolution of reproductive isolating mechanisms has been poorly explored in species resulting from hybrid speciation. We investigated the mechanisms of reproductive isolation between two closely related Adriatic pipefish species, Syngnathus taenionotus and Syngnathus typhle, that show a complex genetic history consistent with introgressive hybridization. We studied the genetic mating system of S. taenionotus in situ, quantified differences in the reproductive behaviour of the two species and carried out a series of behavioural experiments aimed at identifying the factors responsible for the maintenance of species integrity in natural populations. We identified subtle differences in courtship behaviour between the two species and evidence of a preference for large mating partners in reproductive males of both species. Individual preferences were equivocal in conspecificeheterospecific preference trials, and S. typhle males were the only group that showed a significant preference for conspecifics. Reciprocal no-choice mating experiments resulted in a low frequency of heterospecific matings between S. typhle males and S. taenionotus females, all of which failed to produce viable offspring, indicating the presence of both strong premating and postmating isolation in this system. Our results suggest that the development of reproductive isolating mechanisms between species produced by homoploid hybridization may differ from that expected under standard models of divergence with gene flow. © 2020 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

To protect organismal biodiversity, we must understand the processes that shape and maintain it, namely the mechanisms that result in the formation and persistence of species. The increasing prevalence of genomic tools for a wide range of study systems has provided a new body of evidence supporting the importance of heterospecific hybridization as an engine of species diversity (Genner & Turner, 2012; Lamichhaney et al., 2018; Meier et al., 2017; Payseur & Rieseberg, 2016). Heterospecific hybridization is widespread in both plants and animals, and can act as an important driver of both the gain and loss of biodiversity (Arnold, 1997; Dowling & Secor, 1997; Grobler et al., 2011; Meier et al., 2017; Seehausen, 2004). The interplay between reproductive isolation and hybridization during the early stages of speciation can strongly influence the direction and magnitude of evolutionary change (Arnold, 1997; Coyne & Orr, 2004). However, it remains unclear

* Correspondence: A. B. Wilson, Brooklyn College, Department of Biology, 2900 Bedford Avenue, Brooklyn, NY, 11210, U.S.A. E-mail address: [email protected] (A. B. Wilson).

how reproductive isolation in such hybrid or admixed populations evolves. Reproductive isolation can occur prior to mating, through temporal or spatial segregation of reproduction and/or behavioural changes associated with courtship, or after mating, through gametic incompatibility, hybrid inviability or reduced hybrid fitness. Premating isolation barriers are often the first to evolve, followed by extrinsic postmating barriers and eventually intrinsic postmating barriers, ultimately resulting in irreversibly isolated species (Arthur & Dyer, 2015; Baack, Melo, Rieseberg, & OrtizBarrientos, 2015; Coyne & Orr, 1997; Grant & Grant, 1996; Lackey & Boughman, 2017; Schemske, 2010; Seehausen, 2015). Ray-finned fish have the highest rate of hybridization among vertebrates (Wirtz, 1999), offering a great opportunity to assess its causes and consequences. While the detrimental effects of hybridization in wild fish populations have been well documented (Halas & Simons, 2014; Seehausen, Takimoto, Roy, & Jokela, 2008; Taylor & Piercey, 2017; Vonlanthen et al., 2012), there is an increasing body of evidence showing that hybridization can result in increased species diversity and may play an important role in

https://doi.org/10.1016/j.anbehav.2020.01.006 0003-3472/© 2020 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87

adaptive radiation (Barrier, Baldwin, Robichaux, & Purugganan, 1999; Kautt, Machado-Schiaffino, & Meyer, 2016; Kolbe et al., 2007; Lamichhaney et al., 2018; Meier et al., 2017; Nadeau et al., 2012; Richards & Martin, 2017). The majority of studies investigating hybridization in fish have been performed in freshwater systems, where premating isolation is widespread and often based on assortative mating, but postmating isolation is frequently rare or absent (Mendelson, 2003; Schumer et al., 2017; Seehausen, van Alphen, & Witte, 1997). Members of the marine pipefish genus Syngnathus show an extraordinarily highly developed form of paternal care, with males protecting and brooding offspring in specialized brood pouches, and are thus a unique model in which to address questions concerning how mating behaviour influences hybridization. Because syngnathid eggs are fertilized after their transfer from the female (Fiedler, 1954), paternity of the brooding male is assured (Berglund, Rosenqvist, & Svensson, 1986). Successful reproduction in syngnathid fishes involves complex behavioural sequences prior to mating and requires active participation on the part of both sexes €, Vincent, (Bahr, Sommer, Mattle, & Wilson, 2012; Wilson, Ahnesjo & Meyer, 2003). Despite complex courtship behaviour and intrapouch fertilization, heterospecific interactions, courtship and matings have been observed in natural populations of syngnathid fishes (Hablützel, 2009; Otero-Ferrer et al., 2015; Vincent, Ber€, 1995; Wilson, 2006b). glund, & Ahnesjo The first genetic evidence of historical hybridization in Mediterranean Syngnathus was provided by Hablützel (2009), who found evidence for hybrid ancestry in several species of Mediterranean pipefish based on molecular and morphological analyses. Syngnathus taenionotus, an endemic pipefish restricted to the Adriatic Sea, appears to be the product of homoploid hybridization between Syngnathus typhle and Syngnathus rostellatus. While S. rostellatus is not currently found in the Mediterranean (Hablützel & Wilson, 2011), S. typhle has a broad geographical distribution, and co-occurs with S. taenionotus in the Adriatic (Dawson, 1986), where it has an overlapping reproductive season between April and August (Franzoi, Maccagnani, Rossi, & Ceccherelli, 1993). Shared mtDNA haplotypes between S. taenionotus and S. typhle are consistent with massive mitochondrial introgression to S. taenionotus following the original hybridization event ca. 0.25 e 1.1 million years ago (Hablützel, 2009). In this study, we performed genetic analyses and behavioural observations to investigate the mating system of S. taenionotus, the product of hybridization between S. typhle and S. rostellatus. Genetic analyses of parentage in natural populations were complemented with laboratory preference and mating experiments between the sympatrically occurring sister species pair S. typhle and S. taenionotus, to better understand the mechanisms of reproductive isolation following hybrid speciation.

METHODS Sample Collection Sexually mature S. taenionotus specimens were collected from the Sacca degli Scardovari (44
50.40 N, 12
27.00 E) between May and July 2012 by trawl and beach seine. The sampled habitat was generally sandy and partly covered by macroalgae (Gracilaria, Ulva and Enteromorpha). While S. typhle are rarely encountered at the Sacca degli Scardovari site, a single S. typhle was collected during our July 2012 collection. Sexually mature S. typhle were also collected in the southern region of the Lagoon of Venice (45
13.80 N, 12
16.50 E) from May to July 2012 by trawling. The sampled habitats were eelgrass

meadows containing Zostera marina or Zostera nolti and sparsely some Ulva and Enteromorpha. Syngnathus typhle co-occurs with several Syngnathus species including S. taenionotus in the Venice Lagoon (Franco, Franzoi, Malavasi, Riccato, & Torricelli, 2006; Franzoi, Franco, & Torricelli, 2010). Field collection permits were issued by the provinces of Venice and Rovigo, for the Venice Lagoon and Sacca degli Scardovari sites, respectively. Standard length (SL; tip of the snout to the base of the caudal fin) and wet weight of animals were recorded, and animals were transported to the Statione Idrobiologica Umberto d’Ancona of the University of Padua in Chioggia (IT) for behavioural trials and genetic sampling, using battery-powered bubblers and regular water changes during transport in order to ensure adequate aeration. Experimental animals were kept separated by sex and species and quarantined for a minimum of 9 days prior to behavioural trials, and were fed ad libidum with frozen mysid shrimp (Tropical Marine Centre) twice per day. Stock tanks contained artificial eelgrass for shelter and an airstone to promote gas exchange and were located in the same air-conditioned (20
C) room as experimental tanks. Experimental and stock tanks were attached to the same flowthrough system, and water temperature (19 e 25
C) and photoperiod (13.5:10.5 h light:dark cycle) were maintained throughout the experimental period. All animals not used for genetic analysis of parentage were released at the point of collection following the conclusion of the behavioural trials.

Genetic Analyses Eleven brooding S. taenionotus males were sampled for genetic analysis of parentage from animals collected in 2008 (Hablützel, 2009) and 2012 (present study). Animals were euthanized with an overdose of MS222 (ethyl-3-aminobenzoate, methansulfonate acid salt, 98%), pouch fullness was estimated by eye and embryos were removed from brood pouches and stored individually in 96well plates, maintaining pouch order. Pipefish embryos are clustered within the male's pouch based on their order of deposition, facilitating the analysis of parentage (Jones & Avise, 2001). DNA was extracted from fathers and every fifth embryo of each clutch (e.g. Rispoli & Wilson, 2008). Adult DNA was extracted from tail tissue with DNeasy 96 Tissue Kits (QIAGEN, Basel, Switzerland) following the manufacturer's recommendations, and was eluted in 95 ml of AE Buffer. Embryo DNA was extracted following the protocol outlined in Gloor and Engels (1992). Six microsatellite loci were used for parentage analyses, four originally developed for Syngnathus leptorhynchus (Slep06, Slep10, Slep12, Slep13; Wilson, 2006a), and two (Styph12 and Styph44) developed for S. typhle (Jones, Rosenqvist, Berglund, & Avise, 1999). All microsatellites with the exception of Styph44 are variable in both S. typhle and S. taenionotus (4e38 alleles per locus) and differentiate between the two species (Appendix, Table A1). Microsatellites were PCR-amplified using a DNA Engine Tetrad 2, Peltier Thermal Cycler (MJ Research, Waltham, MA, U.S.A.), following the protocol outlined in Wilson and Eigenmann Veraguth (2010). Fragment separation was performed on an automated sequencer (ABI 3730, Applied Biosystems, ABI Prism, Foster City, CA, U.S.A.) and genetic data were analysed using GeneMapper v.4.0 (Applied Biosystems). Genetic data were analysed using GERUD3 (Jones, 2005), which calculates the minimum number of parents contributing to a clutch of offspring. Monte Carlo simulations (1000 iterations) were performed in GERUD3Sim (Jones, 2005) to evaluate the statistical power of our parentage analysis, using empirical allele frequencies for the four most variable microsatellite loci (Table A1), and assuming the sampling of 20 offspring from a total clutch of 120 individuals derived from four mothers.

F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87

Mating and Courtship Behaviour Observation To investigate potential species-level differences in courtship and mating behaviour and to compare with previously published data on pipefish reproductive behaviour (Fiedler, 1954; Vincent et al., 1995), conspecific mating pairs of S. typhle and S. taenionotus were placed separately in 210-litre glass tanks (55  55  75 cm) equipped with artificial eelgrass for shelter (1 tank per species). Animals were recorded on digital video from overhead and front-view cameras (ABUS, Wetter, Germany), allowing animals to interact without external disturbance. Courtship and mating behaviours were assessed from video records and used to produce reproductive ethograms for both species (see Results, Table 2). Courtship behaviours were also observed in mixed tanks of males and females of each species (1 tank per species; 3 males and 10 females per tank) to explore how intrasexual interactions influence courtship and mating. Behavioural Preference Experiments Conspecific and heterospecific preference experiments were conducted in 210-litre glass tanks subdivided into three sections, with an opaque and solid divider (light grey in Fig. 1a) separating the two stimulus sections (27  27  75 cm), and a clear and porous divider separating the two stimulus compartments from the focal chamber (55  27  75 cm; Fig. 1). Dividers allowed the exchange of chemical and visual cues between stimulus and focal animals, but prevented interaction between the two stimulus individuals. Water flow from the stimulus compartments to the focal chamber enhanced the transfer of potential chemical cues from the stimulus animals (e.g. Bahr et al., 2012). Each corner of the experimental tank was equipped with artificial eelgrass for shelter

79

(Fig. 1). Pipefish were transferred into experimental tanks the evening before each experiment for acclimitization, and all compartments were separated by opaque dividers until the onset of the experimental trial. The experiment started the next morning between 0800 and 1030 hours with the removal of the opaque divider between compartment of the focal fish and the stimuli. Animals were not fed during the acclimitization and experimental periods. Experiments were run in one of four identical experimental tanks. Each experiment ran for 60 min, and was recorded on digital video from overhead and front-view cameras. Preference zones (25  23  75 cm each) included the space immediately in front of each stimulus chamber (dark grey in Fig. 1), and the total time the head of the focal animal was present in each preference zone was recorded as an indication of preference. A zone of 4 cm separated the two preference zones. The placement of stimulus individuals was randomized for each trial, and each animal was used a maximum of one time as a focal, conspecific and heterospecific individual. Experimental animals were held for a minimum of 2 days (2 e 18 days) before their use in a subsequent trial. Experiment 1: Size-dependent Preference in S. taenionotus Previous research has demonstrated that both male and female S. typhle show a preference for large-bodied partners (Berglund et al., 1986; but see Sandvik, Rosenqvist, & Berglund, 2000; Sundin, Berglund, & Rosenqvist, 2010). Our first experiment tested for the presence of size-based preferences in male and female S. taenionotus. We offered each focal individual a choice between two conspecific individuals of the opposite sex differing in standard length (SL). Size differences between stimulus animals were a minimum of 1.3 cm in female trials (mean DSL ± SD ¼ 2.31 ± 1.11 cm, N ¼ 20) and at least 0.8 cm in male trials

Table 1 Summary statistics for S. taenionotus males assayed for genetic analysis of parentage ID

Year

SL (cm)

Pouch filled (%)

No. of embryos (genotyped)

Loci

Minimum no. of mates

SCAta08_086 SCAta08_097 SCAta08_098 SCAta08_100 SCAta12_1 SCAta12_2 SCAta12_3 SCAta12_4 SCAta12_5 SCAta12_6 SCAta12_7 Average ± SD

2008 2008 2008 2008 2012 2012 2012 2012 2012 2012 2012 e

16.7 17.3 15.4 19.3 17.6 17.0 15.1 15.1 13.9 16.8 14.6 16.3 ± 1.6

100 100 100 100 100 100 100 100 100 100 50a e

140 (19) 135 (21) 104 (17) 154 (23) 150 (17) 105 (19) 75 (14) 64 (9) 70 (13) 135 (18) 37 (6) 113 ± 34.3 (17.0 ± 4.1)

6 4 6 6 6 5 6 3 5 6 6 5.3 ± 1.1

4 5 3 6 5 2 4 3 2 5 2 3.9 ± 1.4

SL: standard length. a SCAta12_7 was excluded from parentage calculations, as its pouch was not completely filled.

Table 2 Ethogram of courtship and mating behaviour observed in conspecific and heterospecific mating experiments S. taenionotus (N ¼ 5 observations)

S. typhle (N ¼ 2 observations; Vincent et al., (1995)

No-choice experimenta S. taenionotus male/S. typhle female

S. typhle male/S. taenionotus female

Approach

Male (horizontal on ground)

Female (vertical in eelgrass)

Ventral display Flicking Dancing Surface swim Shaking

Female (horizontal) Male (horizontal on ground) Horizontal Both Male

Female (vertical in eelgrass) Male Vertical Both Male

Males approach females; females on ground (unusual for S. typhle) Female Male Only intrasexual Not observed Not observed

Females approach males; males lying on ground Female Male Only intrasexual Not observed Not observed

Behaviour

a

Details in Appendix, Table A4.

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Figure 1. Schematic view of the experimental design of the behavioural mate preference experiments from (a) the front and (b) the top. Tanks were separated into three sections, two for the stimuli and one for the focal individual (front). The two stimuli were divided by an opaque divider (light grey). The choice zone in the focal compartment is highlighted in dark grey. In (b), the green dots indicate the position of the artificial eelgrass, and the dashed line represents the clear and porous divider between the focal animal and the stimuli.

(1.84 ± 0.94 cm, N ¼ 20). The size difference between stimuli was not significantly different between the male and the female trials (t37 ¼ 1.405, P ¼ 0.168). Experiment 2: Conspecific/Heterospecific Preference in S. taenionotus and S. typhle In our second experiment, we offered focal animals a choice between a conspecific and heterospecific individual of the opposite sex, using an identical experimental design to that outlined for experiment 1, above. Experiments were conducted with males and females of both species as focal animals (S. taenionotus male, N ¼ 20; S. taenionotus female, N ¼ 20; S. typhle male, N ¼ 19; S. typhle female, N ¼ 19). Experiment 3: No-Choice Mating Experiment Syngnathus pipefish reproduce readily under laboratory conditions and produce clutch sizes in line with those of wild-caught individuals (e.g. Paczolt & Jones, 2010; Sagebakken, Ahnesjo, Goncalves, & Kvarnemo, 2011; Sommer, Whittington, & Wilson, 2012), something that has made this group an important model for the study of mating competition and reproductive ecology €, 1995; Aronsen, Berglund, Mobley, Ratikainen, & (Ahnesjo Rosenqvist, 2013; Sundin et al., 2010). To investigate whether heterospecific hybridization is possible between S. typhle and S. taenionotus, we carried out a reciprocal no-choice mating experiment, placing 10 individuals of each species together with 10 heterospecifics of the opposite sex (M  F; F  M). Tanks contained artificial eelgrass for shelter. Pipefish were sampled to cover their natural size range and were held together for 7 days. We conducted 2 h behavioural observations of heterospecific interactions on the first day of the experiment, followed by daily 10 min point observations of mating behaviour on days 2 e 5 of the 7-day experimental trial to assess reproductive behaviour in heterospecific interactions. At the end of the experiment, we held pregnant males individually in 70-litre plastic tanks equipped with artificial eelgrass and counted transferred eggs noninvasively in the pouches of

pregnant males (e.g. Sagebakken et al., 2011). We regularly monitored eggs and embryos for developmental stage and viability. Following the conclusion of experiment 3, we placed experimental animals from the heterospecific trials (N ¼ 3 per species) together with conspecifics of the opposite sex for 24 h to assess reproductive activity and to gain additional insight into conspecific courtship behaviour. Animal Welfare Note All animals used in the experiments outlined here were collected with approval from the Venice and Rovigo regional governments (Collection permits 24142 and 19532, respectively). Pipefish were collected by beach seining and trawling. To keep handling stress for the animals to a minimum during collections, beach seines were kept partially submerged in water during the subsampling of pipefish. Trawling was performed at low speed (< 5 km/h) and over short distances (< 100 m per trawl) to reduce collection stress for experimental animals. Experiments were carried out locally at the Statione Idrobiologica of the University of Padua in Chioggia (IT), using a flow-through aquarium system with water provided from the Lagoon of Venice. Water temperatures were maintained with a climate-control system, and lighting was adjusted to match local summer conditions (13.5:10.5 h light:dark). Animals were fed ad libidum with frozen mysid shrimp twice per day, provided with artificial eelgrass for shelter and permitted to move freely during preference and mating trials. Every effort was taken to ensure that the animals experienced minimum stress in holding aquaria and in experimental aquaria. Statistical Analyses We performed statistical analyses of morphological and behavioural data in R v.3.6.0 (R Development Core Team, 2016). We performed Pearson correlation analyses to test for a correlation between SL and wet weight. For the behavioural trials, we calculated a preference index as the time spent in front of the larger stimulus (LPI) or the conspecific stimulus (CPI), divided by the total time spent in either choice area (0 e 1 scale). We used two-sided

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one-sample t tests, concerning the null hypothesis of no preference (i.e. preference index ¼ 0.5) to independently test for preference for each sex and species. Levene tests were run to test for differences in the variance of the preference indexes among the different focal fish groups. We used generalized linear mixed-effects models (GLME) to test for the effects of predictors on the preference indices with the ‘lme4’ package, using a binomial distribution with a logit link function, weighting for the total time spent in the two choice areas using the offset term in ‘glmer’. For experiment 1, we included sex and SL of the focal fish, the size difference of the stimulus fish and the size of the larger stimulus, and two-way interactions between all variables and the sex of the focal animal as independent variables, to test for sex-specific effects of LPI predictors. Time of day and experimental tank were included as random effects. Nonsignificant interactions were removed in a stepwise fashion from the fully parameterized model to produce a minimal model. For experiment 2, we performed an initial GLME including CPI as the dependent variable, species and sex as categorical predictors and size of the focal animal, conspecific stimulus and size difference between the stimuli (SL of the conspecific  SL of the heterospecific stimulus) as continuous predictors. Time of day and tank were again included as random effects. We constructed a second set of GLMEs to explore sexspecific differences in CPI predictors within species, as for experiment 1. Fully parameterized GLME models were stepwise reduced using the procedure outlined for experiment 1 above. RESULTS Body Measurements Standard length (SL) of field-collected S. taenionotus ranged from 12.0 cm to 19.7 cm (mean ± SD ¼ 15.3 ± 2.1 cm) with a wet weight between 0.5 g and 2.8 g (mean ± SD ¼ 1.3 ± 0.5 g). Syngnathus typhle SL ranged from 15.8 cm to 30.1 cm (mean ± SD ¼ 23.7 ± 3.4 cm) with wet weights ranging from 1.6 g to 10.8 g (mean ± SD ¼ 5.6 ± 2.5 g). SL and wet weight were positively correlated in both species (Pearson correlation: S. taenionotus: r38 ¼ 0.84, P < 0.001; S. typhle: r36 ¼ 0.93, P < 0.001). SL was used as a measure of body size in subsequent analyses. Genetic Mating Structure The 11 analysed S. taenionotus clutches contained 64e154 embryos (Table 1). Clutch size was positively correlated with male SL (Pearson correlation: r8 ¼ 0.882, P < 0.001). A significant correlation was also found between male SL and the number of mothers contributing to each clutch (Pearson correlation: r8 ¼ 0.687, P ¼ 0.028). The six-locus microsatellite array used for parentage analysis in S. taenionotus produced a cumulative exclusion probability of 0.998 (Appendix, Table A1) and Monte Carlo simulations indicated that a parentage analysis based on our four most variable microsatellite loci (Slep6, Slep12, Slep13, Styph12) would accurately infer the correct number of maternal genotypes in >93% of cases, indicating significant statistical power. We found evidence of multiple mating in S. taenionotus males, with an average of 3.9 females contributing eggs to each clutch (Table 1). Each female contributed 6 e 88 eggs per clutch, and all females were identified as conspecifics. Syngnathus taenionotus shows a high level of polygyny compared to S. typhle, which shows high levels of polygyny in northern populations in Scandinavia (up to 3.7 ± 0.2 mothers) but low levels of polygyny in populations inhabiting the Adriatic Sea (1.3 ± 0.3 mothers; Rispoli & Wilson, 2008).

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Comparative Analyses of Courtship Behaviour Preliminary observations of reproductive activity during mating in an experimental tank containing a male and female S. taenionotus, which performed four egg transfers over a period of 8 h, revealed that courtship commenced with a horizontal ‘approach’ of the male to the female from behind (Table 2), which was repeated several times, resulting in a cycling movement of the male. When the female reacted, she swam ahead of the male until her ventral surface was even with his head (‘ventral display’). The male consistently reacted to the female's display by ‘flicking’, a short rotating or shaking movement along the anteroposterior axis. This flicking behaviour was shown before, during and/or after ‘dancing’, when the male and female swam synchronously 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, as eggs were transferred (‘surface swim’). As the male and female returned to the bottom of the tank, the male performed ‘shaking’ movements, which are typically associated with egg fertilization and packing within the pouch (Fiedler, 1954). Repeated approaches occurred after a break of variable length (here: 10, 18 and 10 min), during which the male promenaded or remained in an S-shaped posture with his tail touching the bottom of the tank. In the higher-density conspecific tank of S. taenionotus following experiment 3 (three males and 10 females, four observed egg transfers over 160 min), it was more difficult to assess which behaviour acted as the initiator to subsequent egg transfer. In one of four cases the process occurred as outlined above, while in a second case, reproductive behaviour started by a horizontal approach, but the flicking of the male attracted a second female, resulting in a dance involving the male and both females, until one female retired and egg transfer was performed as described above. In the final two cases, the male joined two females already performing dance-like competition. Postcopulatory behaviours were identical in all observations. The mating behaviour of S. typhle has been well characterized elsewhere (Vincent et al., 1995), and our observations are consistent with these earlier reports. Briefly, observation of mating behaviour in S. typhle (two egg transfers over 120 min between a single reproductive pair) revealed notable differences from the pattern exhibited by S. taenionotus. In contrast to S. taenionotus, mating behaviour in S. typhle was initiated by the female, with both individuals aligned vertically (head up) in the eelgrass (‘vertical approach’, Table 2). The female always approached from above the male, with her ventral surface at the height of the male's head (‘ventral display’). In the absence of a male response to this display, the female reapproached the male in a similar fashion repeatedly. The female then placed herself close to the male and performed a short shaking movement. The male reacted with a shaking behaviour along his anteroposterior axis, identical to the flicking behaviour described for male S. taenionotus. This ‘flicking’ of the male was repeated several times before the male and female connected and moved quickly to the surface during the egg transfer (‘surface swim’). Similar to the pattern observed in S. taenionotus, male S. typhle exhibited ‘shaking’ behaviour after egg transfer while returning to the bottom of the tank. Experiment 1. Size-dependent Preference in S. taenionotus Focal female SL in experiment 1 ranged from 12.6 cm to 18.3 cm (mean ± SD ¼ 16.3 ± 1.2 cm). Male focal fish measured between 13.2 and 18.9 cm (mean ± SD ¼ 16.6 ± 1.2 cm) in SL. Focal fish size did not differ between the sexes (t38 ¼ -0.89, P ¼ 0.380). All 20 focal males and 17/20 focal females left shelter and entered the

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preference zone at least once. A two-sided, one-sample t test revealed no significant size preference in females (mean LPI (large preference index) ¼ 0.62, t16 ¼ 1.25, P ¼ 0.231) but a significant preference for large partners in male S. taenionotus (mean LPI ¼ 0.71, t19 ¼ 3.15, P ¼ 0.005; Fig. 2), consistent with the pattern previously documented in S. typhle (Berglund et al., 1986; Sandvik et al., 2000). While females showed evidence of a more bimodal distribution in LPI, variance in LPI was not significantly different between the two sexes (Levene test: F1,35 ¼ 1.465, P ¼ 0.234; Fig. 2). None of the predictors included in the GLME significantly influenced the LPI (reduced model: Table 3; full model: Appendix, Table A2), indicating that while males showed significant evidence of a preference for large mating partners, male and female LPI did not significantly differ.

Experiment 2: Conspecific/Heterospecific Preference Standard length of S. taenionotus in experiment 2 ranged from 12.0 cm to 19.7 cm (mean ± SD ¼ 15.3 ± 2.1 cm) and S. typhle SL ranged from 15.8 cm to 30.1 cm (mean ± SD ¼ 23.7 ± 3.4 cm). Syngnathus typhle were significantly larger than S. taenionotus (t62 ¼ 12.7, P < 0.001), and S. typhle stimulus individuals were larger in 77 of 78 trials (mean DSL ± SD ¼ 8.0 ± 3.7 cm), consistent with size distributions found in natural populations of the two species. Three of 20 focal S. taenionotus males did not enter the choice area at any time during the experiment and were excluded from further analyses. A two-sided, one-sample t test revealed a significant conspecific preference in male S. typhle (mean ¼ 0.66; t test: t18 ¼ 2.41, P ¼ 0.027), but not in females (mean ¼ 0.56; t18 ¼ 0.99, P ¼ 0.336; Fig. 3a). No evidence of conspecific preference was detected in either sex of S. taenionotus (male: mean ¼ 0.48; t test: t16 ¼ -0.22, P ¼ 0.825; female: mean ¼ 0.53; t19 ¼ 0.35, P ¼ 0.730; Fig. 3a). Interestingly, while S. typhle showed a strong and consistent preference for conspecifics, with the majority of individuals showing a conspecific preference index (CPI) ~0.8, preference values for S. taenionotus clustered into two major groups at CPI ~0.1 and CPI ~0.9 (Fig. 3b). This difference in variance between the two species was significant (Levene test: F1,73 ¼ 8.21, P ¼ 0.005).

Large preference index (LPI)

1

0.8

0.6

0.4

0.2

0

Table 3 Reduced GLME for experiment 1 (size preference in S. taenionotus; AIC ¼ 55.5)

Intercept Sex of focal SL of focal SL of larger stimulus SL difference between stimuli

Estimate

SE

Z

P

1.885 0.436 0.168 -0.224 -0.087

9.543 0.858 0.362 0.504 0.369

0.198 0.508 0.465 -0.445 -0.235

0.843 0.611 0.642 0.656 0.814

SL: standard length.

The reduced GLME (AIC ¼ 107.6) revealed a significant difference between species in CPI (P ¼ 0.040; Table 4) and a marginal effect (P ¼ 0.060) of the size difference between the stimuli on CPI. All other predictors did not have a significant impact on CPI (Table 4). In the species-specific GLMEs, reduced models revealed a significant effect of the size difference between the stimuli (P ¼ 0.045), and its interaction with the sex of the focal fish (P ¼ 0.048) on CPI in S. taenionotus, and a significant effect of the interaction of sex and size difference between the stimuli on CPI in S. typhle (P ¼ 0.029; Table 5) e the fully parameterized model is shown in the Appendix (Table A3). The stimulus size effect found in S. taenionotus was chiefly driven by focal females, which displayed a preference for heterospecifics when the SL difference between the two stimuli was small, but they showed an increasing preference for conspecifics as the size difference between the two stimuli increased (Fig. 3c). The stimulus size effect found in S. typhle, on the other hand, was driven primarily by males, all of whom preferred the conspecific stimulus when the difference between the two stimuli exceeded 8 cm (Fig. 3c). Experiment 3: No-choice Mating Experiment Behavioural interactions among S. typhle and Syngnathus taeniontous were observed regularly in no-choice mating experiments (Appendix, Table A4). Male flicking was observed in both species, but more regularly by S. typhle males (Table 2, Appendix, Table A4), and flicking behaviour was most frequently observed in close proximity of other males. Interestingly, several S. typhle specimens of both sexes were horizontally aligned along the bottom of the experimental tanks, a behaviour atypical in conspecific interactions of this species (Table 2). In addition to courtship behaviour, intrasexual competitive behaviour was observed in both sexes of the two species, including chasing behaviour, dance-like paired or grouped swimming and female display (Appendix, Table A4). After 1 week of free interaction in the no-choice experiment, 2/ 10 (20%) of S. typhle males carried small numbers of eggs (male 1: 2 eggs; male 2: 4 eggs), significantly fewer eggs than are typical in conspecific matings of this species (Rispoli & Wilson, 2008). No S. taenionotus males carried eggs at the conclusion of the experiment. The course of brooding in the two carrying males was monitored over 3 weeks. Eggs showed no signs of development and were reabsorbed by the males by the end of the observation period. In subsequent intraspecific mating trials, 1/3 (33%) of S. typhle and 2/3 (66%) of S. taenionotus males mated successfully within 24 h, indicating that both species were reproductively receptive during the experimental period. DISCUSSION

Female

Male S. taenionotus

Figure 2. Bean plot of the large preference index (LPI) for each sex in S. taenionotus. The thick lines represent mean preference scores. The dashed horizontal line indicates the null expectation of no preference (LPI ¼ 0.5). The size of the dots indicates the difference in standard length between the two stimuli for each trial.

Species integrity in mixed populations of closely related species can be compromised by a lack of strong reproductive isolation. While premating isolating mechanisms are often the first to evolve during divergence with gene flow and are a key step in the speciation process, hybrid species result from the failure of reproductive

F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87 S. taenionotus

(b) 0.8

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.2

0.2

0

0 0

0.2 0.4 0.6 0.8

0 5

10

15

S. typhle

Female S. typhle

CPI

1 0.5

0.8

0.8

0.6

0.6

Male

Female

S. taenionotus

0

Male

0.2 0.4 0.6 0.8

0 5

1

10

15

Δ SL between stimuli

CPI

S. typhle

0.4 0.2

0

0

15

Male S. typhle 1

0.4

10

Δ SL between stimuli

1

0.2

Female

5

Δ SL between stimuli

1.5

0

0.4 0.2

1

CPI

0.4

CPI

1

0.6

0.2

Male S. taenionotus

1

CPI

0.8

Female S. taenionotus

(c)

1

CPI

Density

1

Density

Conspecific preference index (CPI)

(a)

83

5

10

15

Δ SL between stimuli

Figure 3. (a) Bean plot of conspecific preference index (CPI) for each species and sex. The thick lines represent mean preference scores. The size of the dots indicates the difference in standard length (SL) between the two stimuli for each trial. (b) Density plots of conspecific preference in S. taenionotus (both sexes combined) and for S. typhle (both sexes combined). (c) Relationship between stimulus size difference and CPI for S. taenionotus and S. typhle. Dashed horizontal lines in (a) and (c) indicate the null expectation of no preference (CPI ¼ 0.5).

The Mating System of S. taenionotus

isolation between parental species, and may differ in their ability to recognize and avoid heterospecifics as potential mates. In this study, we show that while premating isolation is well developed between S. taenionotus and S. typhle, it is insufficient to entirely maintain the separation of the two species in sympatry. While differences in size and courtship behaviours between the two species reduce the frequency of heterospecific matings in laboratory trials, postzygotic isolation is well developed and hybrid inviability maintains complete reproductive isolation of the two species when they do successfully mate. Heterospecific matings involved the transfer of small numbers of eggs, none of which survived to maturity, similar to the pattern observed in sympatric populations of southern Californian pipefish (Wilson, 2006b).

Multiple mating by males is common in Syngnathus spp. (reviewed in Coleman & Jones, 2011; Wilson et al., 2003) and influenced by local ecological conditions (Mobley & Jones, 2007; Rispoli & Wilson, 2008; Wilson, 2009). Syngnathus taenionotus shows one of the highest rates of multiple mating yet documented in Syngnathus spp. (reviewed in Coleman & Jones, 2011). While researchers have debated the potential value of multiple mating in natural systems (Jennions & Petrie, 2000), multiple mating may be beneficial when prezygotic reproductive barriers between species are incomplete. Under such conditions, mating with multiple partners may lead to higher average fitness due to the dilution of costly heterospecific matings (Lipshutz, 2018). A preference for large mating partners has been documented for a multitude of species. In syngnathids, male preferences for largebodied females are common (Berglund et al., 1986; Billing, Rosenqvist, & Berglund, 2007; Sundin et al., 2010), and our observation of preferences for large partners in male, but not female, S. taenionotus are consistent with the general pattern observed in this group. In syngnathids, larger females tend to produce larger eggs, leading to larger embryos with higher survival rates (Berglund et al., 1986; Berglund, Rosenqvist, & Svensson, 1988), providing direct fitness benefits to males. Body size in species with indeterminate growth also indicates age, possibly reflecting genetic quality, and therefore may act as a quality signal during courtship (Andersson & Simmons, 2006; Folstad & Karter, 1992).

Table 4 Overall GLME for experiment 2 (conspecific preference; AIC ¼ 107.6)

Intercept Species of focal Sex of focal SL of focal SL difference between stimuli SL of conspecific stimulus

Estimate

SE

Z

P

-3.787 2.838 0.443 -0.055 -0.187 0.164

3.444 1.383 0.561 0.092 0.100 0.152

-1.100 2.053 0.790 -0.592 -1.882 1.080

0.272 0.040 0.430 0.554 0.060 0.280

SL: standard length. Significant P values are shown in bold.

Table 5 Final species-specific GLME analyses for experiment 2 (conspecific preference) S. taenionotus (AIC ¼ 56.7)

Intercept Sex of focal SL of focal SL difference between stimuli SL of conspecific stimulus Focal sex*focal SL Focal sex*SL difference between stimuli

S. typhle (AIC ¼ 50.4)

Estimate

SE

Z

P

Estimate

SE

Z

P

-16.159 16.266 0.590 -0.637 0.102 -0.719 0.670

8.408 8.581 0.368 0.317 0.263 0.436 0.340

-1.922 1.895 1.604 -2.008 0.388 -1.649 1.975

0.055 0.058 0.109 0.045 0.698 0.099 0.048

-9.492 19.916 0.235 -0.544 0.356 -0.970 0.856

8.465 13.187 0.225 0.307 0.293 0.553 0.393

-1.121 1.510 1.041 -1.770 1.216 -1.755 2.178

0.262 0.131 0.298 0.077 0.224 0.079 0.029

SL: standard length. Significant P values are shown in bold.

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F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87

Courtship and mating behaviours of S. taenionotus differ subtly from the stereotypical behaviours common in other species in this genus (described in Fiedler, 1954), a factor that may be relevant for understanding the lack of contemporary hybridization between S. taenionotus and S. typhle. The most striking differences observed in laboratory trials are that courtship is often initiated by the male in S. taenionotus, and that all behaviours prior to egg transfer, including dancing, are performed in a horizontal orientation. Interestingly, S. typhle itself shows evidence of intraspecific variation in courtship. Fiedler (1954) described dancing behaviour in a horizontal orientation for Mediterranean S. typhle, similar to the reproductive behaviour observed in S. taenionotus. In contrast, Vincent et al. (1995) observed vertical courtship in Swedish S. typhle, consistent with the pattern observed in S. typhle in our study. Mating behaviour in S. typhle thus appears to be variable, depending on the population and the abundance of potential €, mating partners, competitors and ecological conditions (Ahnesjo 1995; Fiedler, 1954; Rispoli & Wilson, 2008; Vincent et al., 1995), behavioural flexibility that may contribute to reproductive isolation when species occur in sympatry. More extensive sampling of behavioural variation in isolation and at sites at which the two species co-occur could help to clarify the extent to which interspecific interactions influence the development of local behavioural phenotypes.

Species Recognition, Hybridization and Reproductive Isolation Both male S. typhle and S. taenionotus show a preference for large mating partners in conspecific trials (Berglund et al., 1986; this study). However, our preference experiments involving heterospecifics revealed that only male S. typhle show a significant preference for conspecific mating partners. The observation of higher variance and a bimodal distribution of CPI in S. taenionotus, the smaller of the two studied species, suggests that size-based preference cues may compete with species recognition cues in this species, confounding mating decisions in mixed populations. Such conflicts have been observed in other hybridizing fish taxa (Hankison & Morris, 2002; Rosenfield & Kodric-Brown, 2003; Rosenthal & Ryan, 2011). Despite a lack of species differentiation in heterospecific mate preference experiments, no free-living hybrids have been found between S. typhle and S. taenionotus in contemporary populations (Hablützel, 2009; Rispoli & Wilson, 2008; this study), indicating strong reproductive isolation 250 000 years after hybrid speciation (Hablützel, 2009). Our reciprocal no-choice experiments revealed additional mechanisms that may limit hybridization between these two species. First, both instances of heterospecific mating involved the transfer of eggs from the smaller of the two species (S. taenionotus females) to the larger (S. typhle males), and thus may reflect mechanical barriers to egg transfer. Syngnathus typhle eggs are significantly larger than those of S. taenionotus (1.8 mm versus 1.4 € , & Kvarnemo, 2011; Franzoi et al., 1993), mm; Goncalves, Ahnesjo and may be too large to fit within the pouch of S. taenionotus males. Heterospecific matings between Californian pipefish also involve the transfer of eggs from the smaller of the two species (Syngnathus auliscus) to the larger species (S. leptorhynchus) (Wilson, 2006b), suggesting that egg size and oviposition may be important barriers to heterospecific mating in syngnathid species. In contrast to the pattern found in Pacific coast pipefish, where large numbers of eggs were transferred between heterospecifics in free-living populations (Wilson, 2006b), both cases of heterospecific mating between S. typhle males and S. taenionotus females involved the transfer of small numbers of eggs, and all transferred eggs failed to develop, indicating that postmating isolation is well

developed in this system. Notably, recent data suggest that heterospecific matings in Syngnathus pipefish may be beneficial for males even when they do not produce viable offspring, as males can n & Kvarnemo, absorb nutrients contained within eggs (Andre 2014; Sagebakken, Ahnesjo, Mobley, Goncalves, & Kvarnemo, 2010), and may gain additional benefits through increased attracn & Kvarnemo, 2014; Jamieson, tiveness to other females (e.g. Andre €m & Kangas, 1996; Ridley & 1995; Kraak & Weissing, 1996; Lindstro Rechten, 1981). Future studies will be necessary to identify whether the inviability of transferred eggs is the result of defects in fertilization, spermeegg incompatibilities and/or developmental abnormalities caused by genetic divergence between the two species. Our study provides strong evidence of both pre- and postmating isolating mechanisms in closely related, sympatric pipefish species following hybrid speciation (Hablützel, 2009). The development of premating isolating mechanisms during species divergence has been found in many sympatric species pairs (Alexander & Breden, 2004; Fuller, McGhee, & Schrader, 2007; Hatfield & Schluter, 1999; Lackey & Boughman, 2017; Mendelson, 2003; Seehausen, 2015), but in the majority of these studies, postmating barriers are weak or absent, allowing for the production of hybrid offspring under laboratory conditions. Whereas most well-documented hybrid speciation events involve polyploidy, a process that may spontaneously generate genetic incompatibilities (Maheshwari & Barbash, 2011), our understanding of the evolution of reproductive isolation during homoploid hybrid speciation is incomplete (Schumer, Rosenthal, & Andolfatto, 2014). Most studies of homoploid hybrid speciation emphasize the importance of prezygotic reproductive isolation in the early stages of the speciation event (Abbott, Hegarty, Hiscock, & Brennan, 2010; Schumer et al., 2014), driven by new phenotypic combinations of olfactory (Christophe & Baudoin, 1998), acoustic rez et al., 2006; (Lamichhaney et al., 2018) and/or visual (Mava Melo, Salazar, Jiggins, & Linares, 2009) mate choice cues. Interestingly, the few studies on homoploid speciation that have investigated postzygtic reproductive isolation have reported evidence for rez at least partial postzygotic isolation (Abbott et al., 2010; Mava et al., 2006; reviewed for birds in Ottenburghs, 2018). These studies and our findings together illustrate that postzygotic reproductive isolation between hybrids and their parental species may play an important role in hybrid speciation, especially when prezygotic isolation is incomplete, indicating that the development of reproductive isolating mechanisms between species of hybrid origin may differ from that expected under standard models of speciation. Data Availability Raw data and an R script used to carry out the statistical analyses described here are provided as Supplementary material and are available at doi: https://doi.org/10.1016/j.anbehav.2020.01.006 Funding This work was supported by the University of Zurich and the Swiss National Science Foundation via grant 114117 to A.B.W. Acknowledgments We thank Maria Berica Rasotto from the University of Padova and her team (especially Andrea Sambo, Federica Poli, Verena Riedl, Lisa Locatello and Anamarija Vrbatovic) at the field station in Chioggia for providing access to field station facilities, and for logistical advice and assistance during fieldwork. We also thank

F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87

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Appendix

Table A1 Microsatellite summary statistics (after Habluetzel, 2009) S. typhle (N ¼ 51)

Locus

Slep6 Slep10 Slep12 Slep13 Styph12 Styph44 a

S. taenionotus (N ¼ 52)

Alleles

Range (bp)

Heterozygosity (observed/expected)

Exclusion probability

Alleles

Range (bp)

Heterozygosity (observed/expected)

Exclusion probability

16 5 8 13 27 4

192e246 270e282 190e224 331e357 151e231 129e143

0.784/0.863 0.745/0.685 0.529/0.511 0.902/0.864 0.941/0.927 0.098/0.095

0.727 0.428 0.312 0.735 0.853 0.049 0.996

16 4 38 17 22 1

190e215 268e276 192e276 335e359 145e197 134

0.788/0.868 0.288/0.261 0.942/0.952 0.788/0.837 0.942/0.855 e

0.739 0.141 0.903 0.690 0.734 e 0.998

a

a

Cumulative exclusion probability. Table A2 Results of the fully parameterized GLME for experiment 1 (size preference in S. taenionotus; AIC ¼ 59.7)

Intercept Sex of focal SL of focal SL of larger stimulus SL difference between stimuli Focal sex*focal SL Focal sex*SL difference between stimuli Focal sex*SL of larger stimulus SL: standard length.

Estimate

SE

Z

P

7.729 -28.542 -0.259 -0.157 -0.148 0.852 0.149 0.853

10.947 33.257 0.533 0.515 0.451 0.730 0.823 1.683

0.706 -0.858 -0.486 -0.305 -0.327 1.167 0.181 0.507

0.480 0.391 0.627 0.760 0.744 0.243 0.857 0.613

F. N. Moser, A. B. Wilson / Animal Behaviour 161 (2020) 77e87

87

Table A3 Results for the fully parameterized species-specific GLME models separated by species for experiment 2 (conspecific preference) S. taenionotus (AIC ¼ 58.7)

Intercept Sex of focal SL of focal SL difference between stimuli SL of conspecific stimulus Sex of focal*SL of focal Sex of focal*SL difference between stimuli Sex of focal*SL of conspecific stimulus

S. typhle (AIC ¼ 52.7)

Estimate

SE

Z

P

Estimate

SE

Z

P

-16.321 16.818 0.592 -0.638 0.111 -0.726 0.677 -0.026

9.473 11.669 0.373 0.333 0.393 0.445 0.376 0.529

-1.723 1.441 1.587 -1.919 0.282 -1.631 1.800 -0.049

0.085 0.150 0.112 0.055 0.778 0.103 0.072 0.961

-5.627 15.018 0.178 -0.433 0.215 -0.880 0.724 0.153

9.604 17.511 0.228 0.331 0.347 0.533 0.538 0.622

-0.586 0.858 0.778 -1.309 0.620 -1.652 1.345 0.245

0.558 0.391 0.436 0.190 0.535 0.099 0.179 0.806

SL: standard length.

Table A4 Summary of courtship-associated behavious observed in experiment 3 (no-choice experiment) during day 1 (2 h observation) and during 10 min observations on days 2, 3, 4 and 5 of the 7-day experiment S. typhle female  S. taenionotus male

Male approaches female Female approaches male Ventral display of females Flicking male Flicking female Dancing males only Dancing females only Surface swim Shaking

S. taenionotus female  S. typhle male

Day 1

Day 2

Day 3

Day 4

Day 5

Day 1

Day 2

Day 3

Day 4

Day 5

9 43 2 5 0 3 1 0 0

1 2 1 0 0 0 1 0 0

1 1 1 0 0 1 0 0 0

1 2 2 2 0 1 2 0 0

0 3 1 0 0 2 1 0 0

21 39 4 17 0 5 2 0 0

1 1 0 0 0 1 1 0 0

0 0 0 1 0 2 0 0 0

3 4 0 1 0 3 2 0 0

2 1 2 6 0 2 1 0 0

No courtship behaviours were observed on days 6 or 7.