During adaptation to different habitat types, both morphological and behavioral traits can undergo divergent selection. Males often ﬁght for status in dominance hierarchies and rank positions pre- dict reproductive success. Ecotypes with reduced ﬁghting abilities should have low reproductive success when migrating into habitats that harbor ecotypes with superior ﬁghting abilities. Livebearing ﬁshes in the Poecilia mexicana-species complex inhabit not only regular freshwater environments, but also independently colonized sulﬁdic (H S-containing) habitats in three river drainages. In the current study, we found ﬁghting intensities in staged contests to be considerably lower in some but not all sulﬁdic surface ecotypes and the sulﬁdic cave ecotype compared with populations from non-sulﬁdic surface sites. This is perhaps due to selection imposed by H S, which hampers oxygen uptake and transport, as well as cellular respiration. Furthermore, migrants from sulﬁdic habitats may lose ﬁghts even if they do not show overall reduced aggressiveness, as phys- iological performance is likely to be challenged in the non-sulﬁdic environment to which they are not adapted. To test this hypothesis, we simulated migration of H S-adapted males into H S-free 2 2 waters, as well as H S-adapted cave-dwelling males into sulﬁdic surface waters. We found that intruders established dominance less often than resident males, independent of whether or not they showed reduced aggressiveness overall. Our study shows that divergent evolution of male aggressive behavior may also contribute to the maintenance of genetic differentiation in this sys- tem and we call for more careful evaluation of male ﬁghting abilities in studies on ecological speciation. Key words: ecological speciation, extremophile teleost, local adaptation, Poecilia, premating isolation, selection against migrants. Ecological speciation describes the process during which repro- 2011). Consequently, previous studies on ecological speciation ductive isolation arises as a consequence of adaptation to differ- have often considered components of intersexual selection, that ent ecological selection pressures (Schluter 2001; Hendry 2004; is, mate choice. Mate choice can contribute to reproductive isola- Nosil et al. 2005; Rundle and Nosil 2005). It is hypothesized that tion through assortative mating (Endler and Houde 1995; evolution of reproductive isolation is more likely if ecological Rundle et al. 2005; Svensson et al. 2006; Langerhans et al. 2007; traits under divergent selection also play a role in sexual selection Grant and Grant 2008; Seehausen et al. 2008; Tobler et al. 2009; (i.e., “magic traits”), as is the case for traits that function as sig- Ronald et al. 2012; Plath et al. 2013; Harris and Siefferman nals during mate attraction (Feulner et al. 2009; Servedio et al. 2014). V C The Author(s) (2017). Published by Oxford University Press. 125 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 126 Current Zoology, 2018, Vol. 64, No. 1 Although divergent abiotic environments have the potential to dominant males may monopolize females from different ecotypes directly impact the expression of energetically expensive behaviors, and thus foster hybridization events (Qvarnstro ¨ m et al. 2012). It has been hypothesized that sulfide-adapted males may lose such as aggression, the role of divergent expression (and evolution) fights with resident males when migrating into non-sulfidic habitats of aggressive behavior in fostering reproductive isolation remains (Bierbach et al. 2012). Fish from H S-containing waters have lower much less well-studied (Parzefall 1969, 1974, 1979; Peters et al. body condition and fat stores (Tobler et al. 2006; Plath et al. 2007c; 1973; Burchards et al. 1985; Langecker et al. 1995; Espinasa et al. Riesch et al. 2010b, 2011), which may be a consequence of low 2005; Bierbach et al. 2012; Qvarnstro ¨ m et al. 2012; Kowalko et al. food availability in such extreme habitats: Fishes in these low- 2013). In systems in which dominance hierarchies play a central role oxygen environments spend more time (and energy) at the surface, in determining male reproductive success, reduced aggressiveness where water oxygen concentration is higher, reducing the time spent should translate into male inferiority in mate competition upon foraging (Plath et al. 2007c). In addition, physiological costs associ- encounter of different behavioral phenotypes (Plath and Strecker ated with H S-detoxification likely play very important roles 2008; Bierbach et al. 2012). This could partly restrict gene-flow (Grieshaber and Vo ¨ lkel 1998; Tobler et al. 2016, 2017). between different locally adapted populations; particularly, if males Previous studies reported a reduction of male sexual activity in from different ecotypes fair worse in aggressive encounters in the extremophile P. mexicana populations (Plath et al. 2003; habitat type they are not locally adapted to (see Winkelmann et al. Plath 2008). Assuming that this is due to physiological limitations 2014). In this scenario, reduced gene-flow would also depend on on energy expenditure, it is likely that these fish will show a reduc- selection for either divergent mate choice or strong female habitat tion in male aggressiveness. This should reduce the reproductive suc- fidelity to prevent hybridization among migratory females and cess of immigrant (sulfide-adapted) males in non-sulfidic stream locally adapted males (van Doorn et al. 2009). sections through both mate choice and male competition. In theory, Livebearing fishes in the Poecilia mexicana species-complex a pattern of sulfide-adapted males being inferior in mate competi- have independently adapted to toxic hydrogen sulfide (H S) in at tion with males from non-sulfidic habitats could emerge even when least three river systems in Chiapas and Tabasco in southern Mexico sulfide-adapted males are not less aggressive overall in their sulfidic (Palacios et al. 2013; Riesch et al. 2015). Hydrogen sulfide-rich habitats but show impaired physiological performance in non- aquatic habitats provide extreme conditions for extremophile popu- sulfidic water (see Plath et al. 2010, 2013). lations, as H S depletes the water of freely available oxygen, ham- An additional ecological variable in our study system is the pres- pers oxygen uptake and transport in the blood, and blocks ence or absence of light: In the Tacotalpa drainage, P. mexicana components of cellular oxidative phosphorylation (Pfenninger et al. colonized two limestone caves, the sulfidic Cueva del Azufre and the 2014; Tobler et al. 2014,2017). Early studies in this system have non-sulfidic Cueva Luna Azufre (Tobler et al. 2008b; Plath and identified a number of adaptive traits that allowed successful colo- Tobler 2010). Cave populations have reduced but still functional nizers to exploit sulfidic habitats (Tobler et al. 2008a, 2017; Riesch eyes, which are, however, slightly smaller than those of surface- et al. 2010b, 2011, 2015; Tobler and Plath 2011; Pfenninger et al. dwelling fish (Tobler et al. 2008a; Plath and Tobler 2010). A pre- 2014; Kelley et al. 2016). Besides other traits (see the “Discussion” vious study demonstrated that males from the sulfidic cave show section), H S-adapted populations convergently evolved enlarged reduced aggression compared with males from sulfidic surface and heads. It is proposed that the larger buccal cavity allows passage of non-sulfidic surface populations in that drainage, and that at least a larger volumes of water over larger gill surfaces, thereby increasing part of those behavioral population differences are heritable potential for oxygen uptake in oxygen deplete environments (Bierbach et al. 2012). (Tobler et al. 2011). Ecotypes that lack specialized adaptations to In our present study, we tested whether sulfidic ecotypes from cope with H S-related toxicity experience high mortality when three different river drainages show reduced aggression compared migrating into sulfidic waters (Tobler et al. 2009; Plath et al. 2013). with males from adjacent non-sulfidic populations. In the Tacotalpa On the other hand, sulfide-adapted fish have the ability to venture drainage, we also confirmed that males from the sulfidic cave popu- into surrounding non-sulfidic habitats at least for short periods of lation are less aggressive compared with sulfidic as well as non- time, and both sulfide-adapted and not sulfide-adapted ecotypes can sulfidic surface populations. Given the unidirectional migration be found interacting in non-sulfidic water at the confluence of from sulfidic habitats to non-sulfidic ones as well as from the cave sulfidic creeks and (larger) non-sulfidic streams (Sommer-Trembo to sulfidic surface and from there to non-sulfidic surface habitats, et al. 2016). we furthermore asked whether migrating males would lose fights in Population genetic studies have identified low levels of gene-flow aggressive encounters with resident males, thus fostering reproduc- among P. mexicana populations from different habitat types but tive isolation. clearly detected evidence for unidirectional migration from sulfidic to non-sulfidic habitats (Plath et al. 2013). Even though female Materials and Methods choice is thought to determine the outcome of male mate competi- tion to a large extent as female poeciliids have a preferences for Study system and test animals large, dominant males (Sommer-Trembo et al. 2016), and females Wild-caught mollies (Poecilia spp.) were collected near the city of (at least from non-sulfidic habitats) preferentially mate with males Teapa in southern Mexico (federal state of Tabasco, United States from their own population (Tobler et al. 2009; Plath et al. 2010, of Mexico). In this region, the mountains of the Sierra Madre de 2013; Greenway et al. 2016), dominance hierarchies among males Chiapas meet the wide floodplains of northern Tabasco. Several sul- and use of coercive mating tactics (Evans et al. 2003; Plath et al. fide spring complexes of volcanic origin (Rosales-Lagarde et al. 2007b; Magurran 2011; Bierbach et al. 2013a) could, in theory, 2008) are distributed across three major tributaries of the Rı ´o impede the evolution of reproductive isolation. At the interface Grijalva (from east to west: Rı ´os Tacotalpa, Puyacatengo, and between habitats where different ecotypes frequently meet, Pichucalco; Figure 1). At least four spring complexes were Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Bierbach et al. Male competition enhances reproductive isolation 127 independently colonized by poeciliid fishes (P. mexicana, 200 and 500 ka ago; Pfenninger et al. 2014), which show strong P. sulphuraria, Gambusia eurystoma, Pseudoxiphophorus bimacula- phylogenetic affinity to present Northern Mexican P. mexicana tus, and Xiphophorus hellerii; Palacios et al. 2013; Plath et al. 2013; limantouri (Tobler et al. 2011; Palacios et al. 2013; Plath et al. Greenway et al. 2014; Riesch et al. 2016). Non-sulfidic and sulfidic 2013; Pfenninger et al. 2014). Invasion of H S-springs in the (H S-containing) habitats within each drainage are interconnected Tacotalpa and Puyacatengo drainages was more recent (approx- and not separated by physical barriers that would prevent migra- imately 100 ka ago) and those sulfide-adapted ecotypes each are tion. In the Tacotalpa drainage also two limestone caves were closely related to P. mexicana mexicana from adjacent non-sulfidic colonized by P. mexicana, the sulfidic Cueva del Azufre and the habitats (Tobler et al. 2011; Plath et al. 2013; Pfenninger et al. non-sulfidic Cueva Luna Azufre (Tobler et al. 2008b; Plath and 2014; Kelley et al. 2016). In the case of the sulfide cave-dwellers, Tobler 2010). Thus, the study system provides four distinct ecotypes fish were collected from a front chamber of the Cueva del Azufre (surface: sulfidic and non-sulfidic; cave: sulfidic and non-sulfidic). (chamber II; after Gordon and Rosen 1962), which receives some However, in the current study, we were not able to include fish from light through cracks in the cave roof. the non-sulfidic cave Cueva Luna Azufre due to a flooding event Focal fish (only males; see Tables 1 and 2 for sample sizes) were that prevented access to this cave. In the Rı ´o Pichucalco drainage, caught with a seine (4 m long, 4 mm mesh-width) and immediately the sulfide ecotype has been described as a distinct species, transported in aerated coolers to a field station in Teapa (Centro de P. sulphuraria (Alvarez 1948), which is endemic to sulfide spring Investigacio´ n e Innovacio´ n para la Ensenanza ~ y el Aprendizaje). complexes at the Ban~os del Azufre and Rancho La Gloria Details on collection sites can be found in Figure 1. To avoid (Tobler et al. 2008c; Palacios et al. 2013; Greenway et al. 2014). ambiguity of species assignment, we did not collect fish in the mix- Phylogenetic analyses suggest that colonization of H S-springs 2 ing zones of sulfidic and non-sulfidic waters. Upon capture, all speci- occurred first in the Pichucalco drainage by P. sulphuraria (between mens were individually inspected and assigned to ecotypes using distinctive external morphological features (Tobler et al. 2011). Prior to the experiments, fish were acclimated to laboratory condi- tions in aerated 42-L tanks and in case of sulfide-adapted fish to nor- moxic (non-sulfidic) conditions for 3 days, which is within the range of previously used acclimation periods (see Plath et al. 2013; Sommer-Trembo et al. 2016). This was done to allow sulfide- adapted ecotypes to gradually habituate to normoxic and sulfide- free conditions, as previous attempts to conduct the experiments under sulfidic conditions failed. We initially held fish in water from the collection site but gradually exchanged the water on a daily basis and replaced it by sulfide-free stream water. We provided ad libitum amounts of TetraMin flake food during the course of the experiment. General testing procedure To test whether sulfide-adapted surface as well as cave-dwelling populations show an overall reduction in male aggressiveness com- pared with their counterparts from non-sulfidic habitats, we staged Figure 1. Map of the study area. (1) Ban~os del Azufre (sulﬁdic, black arrow); contests of size-matched males from the same ecotypes (sulfidic (2) Rı´o Ixtapangajoya (non-sulﬁdic, clear arrow); (3) Puyacatengo bridge (non- surface, non-sulfidic surface, as well as sulfidic cave). Fighting sulﬁdic); (4) Puyacatengo springs (sulﬁdic); (5) Cueva del Azufre (sulﬁdic intensity was scored as the number of aggressive interactions per cave); (6) El Azufre creek (sulﬁdic); and (7) Arroyo Bonita (non-sulﬁdic). Cities are indicated with dots in light gray while river systems are underlined black. pair. We decided to sum aggressions per pair in intra-ecotype pairs Table 1. Overview of populations used to establish mean aggressiveness of different ecotypes in the Poecilia mexicana-species complex; numbers of sampling sites follow Figure 1 (see main text) Population N Light H S Mean pair size Mean SL difference tdf P Pichucalco drainage P. sulphuraria (1) 9 þþ 27.3 6 0.7 1.7 6 0.3 0.86 8 0.42 P. mexicana (2) 7 þ 34.6 6 1.9 1.3 6 0.4 1.47 6 0.19 Puyacatengo drainage P. mexicana (4) 10 þþ 25.0 6 0.4 0.8 6 0.6 2.25 9 0.05 P. mexicana (3) 4 þ 36.2 6 2.6 1.5 6 0.3 2.1 3 0.12 Tacotalpa drainage P. mexicana (7) 11 þ 32.3 6 0.9 1.0 6 0.3 2.01 10 0.06 P. mexicana (6) 13 þþ 27.3 6 0.5 0.9 6 0.2 0.00 10 1.00 P. mexicana (5) 14 þ 28.2 6 0.6 1.9 6 0.3 1.58 4 0.19 Notes: Listed are the numbers of ﬁghts analyzed, relevant ecological habitat parameters [light absent () or present (þ); H S absent () or present (þ)], as well as mean dyad body size (standard length, SL) and mean body size difference [both in (mm)]. Paired t-tests were used to compare the body size of winners and losers in ﬁghts where one male established dominance. Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 128 Current Zoology, 2018, Vol. 64, No. 1 Table 2. Outcome of staged ﬁghts between males from different ecotypes (see Figure 1 in the main text for sampling site codes) Ecotype pairs N Resident SL Intruder SL tdf P Resident won Intruder won v P Pichucalco drainage P. mexicana (2) versus P. sulphuraria (1) 17 25.1 6 0.4 24.6 6 0.5 0.89 16 0.39 14 1 11.27 0.001 Puyacatengo drainage P. mexicana (2) versus P. mexicana (4) 10 26.2 6 0.5 25.8 6 0.5 1.18 9 0.27 7 1 4.50 0.034 Tacotalpa drainage P. mexicana (7) versus P. mexicana (6) 15 31.5 6 0.8 30.8 6 0.8 1.09 14 0.30 11 2 6.21 0.013 P. mexicana (6) versus P. mexicana (5) 12 26.4 6 0.9 25.7 6 0.8 1.87 11 0.09 7 1 4.50 0.034 Notes: We deﬁned the not sulﬁde-adapted ecotype as “resident” male, while the sulﬁde- or cave-adapted male was deﬁned as “intruder.” Listed are the numbers of ﬁghts, mean body sizes of resident and intruder males (SL in mm), and results from paired t-tests comparing male body size of winners and losers. Numbers of ﬁghts won by both resident and intruder males are given, along with the results of v -tests. as we showed in a previous study that fight dynamics in dyads of been kept in different tanks prior to the experiment and were thus size-matched males are very even until dominance is established not familiar to each other. (Bierbach et al. 2012). This seems to be an inherent feature of fights Observations took place on the next day between 09:00 and 13:00. At the beginning of the experiment, we removed the partition in poeciliids (Bierbach et al. 2013a). separating the two males and recorded behavioral interactions for a To address the question of whether males migrating from sulfidic surface to non-sulfidic surface habitats as well as from the cave to maximum of 10 min, starting with the first interaction. We sepa- rated both contestants immediately after dominance was established the surface could compete with resident males, or whether they have to avoid serious injuries. Apart from the loss of single scales, no a disadvantage in intrasexual competition in their ancestral habitats severe injuries and no mortality occurred during these experiments. (Bierbach et al. 2012), we simulated possible migration events by If dominance was not established after 10 min of observation we ter- staging fights between size-matched males of the resident ecotype minated the contest and scored fight outcome as “no winner dis- and migrating males in all three drainages and assessed the likeli- cernible.” Even though dyads were matched for body size, males hood of migrating males establishing dominance over resident ones. differed in aspects of their fin and body coloration and were thus Here, we scored aggressive behaviors for each individual in a pair easily distinguishable. We focused on three aggressive behaviors that instead of summing up aggressive behaviors per pair. This was done occur frequently in Poecilia spp. (Parzefall 1969; Bierbach et al. to see whether fight dynamics may have differed between males of 2012, 2013a). (1) S-position: This threat display usually initiates a different ecotypes. Due to a lack of males from non-sulfidic sites in fight. Males swim in a parallel or anti-parallel position and bend the Rı ´o Puyacatengo, we staged contests between sulfide-adapted their bodies in an S-shaped manner while all unpaired fins are males from the Rı ´o Puyacatengo sulfidic springs and males from the erected. (2) Tail-beats: The aforementioned behavior is often fol- nearby, non-sulfidic Rı ´o Ixtapangajoya (Table 1 and Figure 1). The lowed or superimposed by tail-beats, which involve fast movements Rı ´o Ixtapangajoya/Rı ´o Teapa is a confluent to the Rı ´o Puyacatengo, of head and tail in opposing directions that either touch the oppo- and overall aggressiveness of males from these two non-sulfidic sites nent’s body or sent shock waves toward the opponent. (3) Bites: did not differ (Figure 2A; independent-samples t-test: t ¼ 0.28, We defined all incidences of ramming and attack with the mouth P¼ 0.79). into the direction of the opponent as bites, because these behaviors We could not stage fights in H S-containing water, which would occur too fast and are too similar to be distinguishable by the human provide sulfide-adapted ecotypes which their natural conditions and eye. Poecilia mexicana males are known to exhibit homosexual would have simulated migration from the clear-water habitats into behavior; cave-dwelling populations in particular tend to respond to sulfidic ones for three reasons: (i) fish from non-sulfidic habitats had aggressive behavior by showing sexually motivated behaviors exceedingly low survival rates when translocated into sulfidic habi- (Parzefall 1969; Tobler et al. 2005). Although we observed several tats (Tobler et al. 2011), (ii) genetic analyses found little migration homosexual interactions in fights involving cave-adapted males, fre- in this direction (Plath et al. 2013), and (iii) maintaining constant quencies were low (mean6 SEM: 0.96 1.6 homosexual behaviors H S concentrations during experimentation is difficult since H S 2 2 per fight) and so these behaviors were excluded from our analyses. degasses very quickly. Contest outcome was evaluated based on behavioral differences between the competitors (Morris et al. 1992, 1995). Folded fins, Behavioral assessment of aggressiveness head-down posture, and a position at the periphery of the tank typi- and dominance cally characterized the loser of the contest. Winners, on the other Plexiglas mouse cages (23 15 16.5 cm), filled with river water, hand, constantly chased and further attacked losers with spread fins were used to stage dyadic encounters between size-matched males. while occasionally displaying S-positions. We are aware of other Males were separated overnight by an opaque plastic divider placed methods to establish aggression in fishes (e.g., mirror tests; Balzarini in the center of the test tank. The outer sides were covered with gray et al. 2014) but decided to use staged-contests throughout our study paper to minimize external disturbance. In both experiments, male as these can also be applied to study contests between different contestants were closely size-matched (see t-tests on body size differ- ecotypes. After a contest, we measured body size (standard length, ences in Tables 1 and 2). An air pump provided well-oxygenated SL) of both contestants to the nearest millimeter by briefly water during this period, but the air stone was removed from the laying the fish flat on plastic foil-covered millimeter paper tanks directly prior to the testing phase. Males within each dyad had (see Tables 1 and 2). Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Bierbach et al. Male competition enhances reproductive isolation 129 Figure 2. General aggressiveness and intruder inferiority in staged ﬁghts involving males from sulﬁde-adapted and not sulﬁde-adapted ecotypes. (A) Contests of males from the same ecotype; shown are mean (6SEM) numbers of aggressive behaviors per ﬁght. (B) Intruder inferiority in all three drainages. Shown are frac- tions of ﬁghts won by the “resident” male (not sulﬁde-adapted ecotype) or by the intruder, and ﬁghts with no clear winner. In all three drainages, resident males were signiﬁcantly more likely to win contests (v -tests, see Table 2). (C) Details of ﬁghts between resident and intruder males in all three drainages. Shown are mean (6SEM) numbers of aggressive behaviors per ﬁght shown by both types of males. Asterisks indicate signiﬁcant differences in post hoc t-tests (see main text). Statistical analyses However, both covariates and their interactions with the main fac- All analyses were performed using SPSS 23 (IBM). To achieve tor “ecotype” were excluded from the final models as they had no approximate Gaussian distribution, all dependent data were log- statistically significant effects (F< 0.8, P> 0.5). We used transformed prior to statistical analyses. We compared frequencies independent-samples t-tests for post hoc comparisons of overall aggressiveness (log-transformed sum of all aggressive behaviors) of aggressive behaviors (using log-transformed numbers of S-posi- tions, tail beats, and bites/ramming per fight as dependent variables) between ecotypes in all three drainages (sulfidic versus non-sulfidic) during fights of males from the same ecotypes by means of multi- and Fisher’s least significant differences (LSD) for comparing the three ecotypes in the Tacotalpa drainage. variate general linear models (GLM; F-ratios calculated from Wilk’s For our cross-ecotype fights, we employed v -tests to compare lambda). We ran two separate analyses: First, we analyzed only the subset of fights of sulfide-adapted and not sulfide-adapted surface numbers of fights won by males from either ecotype. Moreover, we compared numbers of aggressive behaviors shown by individual ecotypes, while including the main factor “ecotype” (nested within males from both ecotypes using paired-samples t-tests. drainage). Second, we analyzed the subset of ecotypes in the Rı ´o Tacotalpa drainage including the cave ecotype, again using “ecotype” as a main factor: (1) the non-sulfidic creek Arroyo Results Bonita, (2) the sulfidic creek El Azufre, and (3) the sulfidic cave Cueva del Azufre. This approach was chosen as cave ecotypes from Aggressiveness of sulfide-adapted and not sulfide- the Tacotalpa drainage have no cave-dwelling counterparts in the adapted surface ecotypes two other drainages, and a comparison with fish from other drain- When comparing numbers of aggressive behaviors in dyadic fights ages is thus biologically irrelevant. Poecilia mexicana can differ dra- between males from sulfidic and non-sulfidic surface habitats from matically in adult male body size (Bierbach et al. 2012, 2013a) all three drainages investigated herein, we found a significant effect which might affect aggressive behavior. Thus, although we staged of the main factor “ecotype (nested within drainage)” (multivariate fights between size-matched males, the mean body size of male GLM: F ¼ 4.00, P< 0.001), suggesting that ecotypes differed 15,127.4 dyads differed between trials and so we initially included “mean in at least one drainage under investigation. Post hoc independent- dyad body size” as a covariate in our models. Furthermore, we samples t-tests comparing the aggressiveness of ecotypes from accounted for minute differences in body size within pairs and thus sulfidic and non-sulfidic habitats within drainages found the sulfide- initially also included “body size difference” as another covariate. adapted ecotype in the Rı ´o Tacotalpa drainage to show significantly Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 130 Current Zoology, 2018, Vol. 64, No. 1 Figure 3. General aggressiveness and intruder inferiority in the Tacotalpa drainage. (A) Comparison of aggressiveness among ecotypes in contests of males from the same ecotype. Shown are mean (6SEM) numbers of aggressive behaviors per ﬁght. Asterisks indicate signiﬁcant differences in post hoc LSD tests. (B) Intruder inferiority in staged encounters of “migrating” males of the cave-dwelling ecotype into adjacent sulﬁdic surface waters, or of males from the surface- dwelling sulﬁde-adapted ecotype into adjacent non-sulﬁdic surface waters. Shown are fractions of ﬁghts won by resident or intruder males, as well as ﬁghts in which no winner was discernible. In both cases, resident males were signiﬁcantly more likely to establish dominance (v -tests, see Table 2). (C) Details of staged ﬁghts between resident (surface-dwelling sulﬁde-adapted ecotype) and intruder males (sulﬁdic cave-ecotype). Shown are mean (6SEM) numbers of aggressive behaviors shown by both types of males per ﬁght. Details on contests between males from the surface-dwelling not sulﬁde-adapted ecotype and the surface- dwelling sulﬁde-adapted ecotype (El Azufre) are shown in Figure 2C. Asterisks indicate signiﬁcant differences in post hoc paired t-tests. reduced aggression (t ¼ 4.66, P< 0.001; Figure 2A), while eco- ecotype (El Azufre) displayed significantly more S-positions when types did not differ significantly in mean aggressiveness in the other paired with males of the sulfidic cave ecotype than vice versa two drainages (Rı ´o Pichucalco: t ¼ 0.98, P¼ 0.34; Rı ´o (t ¼ 2.25, P¼ 0.045; Figure 3C). 14 11 Puyacatengo: t ¼ 0.98, P¼ 0.34; Figure 2A). Discussion Intruder inferiority in fights between sulfide-adapted To test the hypothesis that harsh conditions in hydrogen sulfide and not sulfide-adapted surface males (H S)-containing and/or perpetually dark (i.e., cave) environments In all staged contests that simulated migration of males from sulfidic select for the reduction of energetically costly behaviors, we com- into non-sulfidic surface habitats, resident (i.e., not sulfide-adapted) pared male aggressiveness among different locally adapted ecotypes males were significantly more likely to establish dominance in the P. mexicana species-complex (Peters et al. 1973; Parzefall (Figure 2B and Table 1). In congruence with the results from the 1974, 2001; Bierbach et al. 2012). Reduced aggressiveness of the staged contests of males from the same ecotype (see above), we surface-dwelling sulfide-adapted ecotype compared with the closely found pronounced differences in numbers of aggressive behaviors related surface-dwelling ecotype from non-toxic waters was between resident and migrant males in the Tacotalpa drainage, and observed in only one of three drainages investigated here (Tacotalpa males from the non-sulfidic Arroyo Bonita directed significantly drainage). The sulfidic cave-dwelling ecotype from the same drain- more bites toward sulfide-adapted El Azufre males than vice versa age also showed reduced aggressiveness. Independent of whether or (paired-samples t-test: t ¼ 2.73, P¼ 0.016; Figure 2C). not males showed reduced aggressive behavior overall, males from sulfidic surface and cave habitats were significantly less likely to Aggressiveness of surface- and cave-dwelling ecotypes establish dominance when competing with males from habitats In the Tacotalpa drainage, male aggressiveness differed significantly without those physico-chemical stressors. We suggest that this pat- among ecotypes (multivariate GLM: F ¼ 9.25, P< 0.001). 6,66.0 tern could result in inferiority in mate competition for extremophile Males of the non-sulfidic surface ecotype (Arroyo Bonita) were sig- males (i.e., sulfidic surface and sulfidic cave ecotypes) that migrate nificantly more aggressive than males of the surface-dwelling sul- into more benign habitats, which represents the only possible routes fide-adapted ecotype (El Azufre; Fisher’s LSD: P¼ 0.001) and males of migration in those systems (Tobler et al. 2011; Plath et al. 2013). of the sulfidic cave-dwelling ecotype (P< 0.001). Moreover, males We found no general reduction of aggressive behavior in surface- from the sulfidic surface habitat (El Azufre) were more aggressive dwelling sulfide-adapted ecotypes from the Pichucalco and than males of the cave-dwelling ecotype (P¼ 0.002; Figure 3A). Puyacatengo drainages, but males from these river systems were still inferior in aggressive competition when paired with males of the not Intruder inferiority in fights between cave- and surface- sulfide-adapted ecotype from their respective drainage. It seems, dwelling ecotypes therefore, that adapting to life in H S-rich environments does not Similar to the results of staged contests between surface-dwelling necessarily result in reduced aggressiveness even when tested under males from sulfide-adapted and not sulfidic-adapted ecotypes (see non-sulfidic conditions but inevitably lowers males’ resource hold- above), our analyses of ecotypes from the Tacotalpa drainage that ing potential (RHP, sensu Parker 1974) when competing with fish also included the cave ecotype found resident males to establish from non-sulfidic waters. This could have several reasons: First, dominance over intruders at significantly higher frequencies changes in body morphology of sulfide-adapted ecotypes include (Figure 3B). Males from the surface-dwelling sulfide-adapted larger heads (Tobler et al. 2008a; Fontanier and Tobler 2009; Downloaded from https://academic.oup.com/cz/article-abstract/64/1/125/4669816 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Bierbach et al. Male competition enhances reproductive isolation 131 Tobler et al. 2011), which could negatively affect swimming ability introgression of allelic variants stemming from the cave ecotype that (Van Wassenbergh et al. 2015), and ultimately result in poor fight- may be only weakly (or not at all) counter-selected in the sulfidic ing performance. Second, H S-adapted ecotypes show altered phys- surface habitat. As an alternative explanation, one might also argue iological pathways along with differential gene expression patterns that extreme habitats in the Tacotalpa drainage were colonized by compared with not sulfide-adapted ecotypes (Tobler et al. 2014, less aggressive (and competitively inferior) fish from the ancestral 2017). Antioxidant enzymes, in particular, are down-regulated in population (Winkelmann et al. 2014). Thus, future studies should sulfidic, hypoxic water (Kelley et al. 2016; Passow et al. 2017; address the question of whether male aggression was involved in ini- Tobler et al. 2017), which could hamper physiological performance tial species divergence, or just in maintaining or shaping of species upon transfer into sulfide-free waters. More generally speaking, var- boundaries and co-existence of divergent phenotypes. ious physiological pathways may not have had enough time to In our study system, natural selection acts mostly through H S- adjust to the new conditions (normoxia, no H S) in our experimen- toxicity with high mortality rates of not sulfide-adapted fish in sulfi- tal design. Additionally, access to the water surface affects short- dic waters (and vice versa also in the Tacotalpa drainage, see Plath term survivability in the toxic environments (Plath et al. 2007c; et al. 2013). Furtermore, predation by the aquatic heteropteran Tobler 2009b), and Poecilia spp. in sulfidic habitats spend a consid- Belostoma cf. bakeri at the interface of cave and surface habitats erable amount of their time budget conducting aquatic surface respi- was found to act against migrants, as surface-dwelling ecotypes are ration (Tobler 2009b). This behavior, however, comes at a cost of more likely to fall victim to predation under dark conditions, while constraining time available for benthic foraging, as evidenced by cave ecotypes suffer greater predation under light conditions [Tobler behavioral observations in the wild and from the examination of gut (2009a), see also Riesch et al. (2010a) for avain predation]. fullness (Tobler 2009b; Roach et al. 2011). Our tests, however, did Regarding the contribution of sexual selection, several studies have not allow us to uncover the mechanism behind contest inferiority of shown strong preferences in females from non-sulfidic habitats for H S-adapted ecotypes and future experiments, especially using mating with males from their own population, whereas females common-garden raised fish from H S habitats (i.e., individuals from sulfidic environments show no such ecotype-based mating reared under normoxic and non-sulfidic conditions), are urgently biases (Tobler et al. 2009; Plath et al. 2010, 2013; Sommer-Trembo needed. Staging fights between different ecotypes that are all et al. 2016), which can be ascribed to a large extent to adaptive, common-garden reared would, for example, allow us to tell apart divergent evolution of male body shape (Greenway et al. 2016). the effects of current acclimation (or a lack thereof) to non-sulfidic Divergent evolution of body coloration in cave ecotypes allows conditions that fish in our tests (and migrants in the wild) will surface-dwelling females to choose males from their own ecotype, experience. A different pattern was observed in the Tacotalpa drainage, in while cave-dwelling females do not show a preference for males which both sulfide-adapted surface and cave ecotypes showed from their own ecotype (Bierbach et al. 2013b). However, in a mat- reduced aggressiveness compared with the ecotype from non-sulfidic ing system where dominant males monopolize small groups of surface waters. Bierbach et al. (2012) examined male aggressiveness females (Bierbach et al. 2014) and typically only those males father in laboratory-reared fish from all four distinct ecotypes in the the majority of offspring then (but see Schartl et al. 1993), male– Tacotalpa system (non-sulfidic surface, sulfidic surface, sulfidic male competition could actually play an even stronger role than cave, and non-sulfidic cave) and reported that not only selection mate choice. from H S but also from darkness led to reduced aggressiveness. This Similar patterns as reported here are likely to also occur in other confirmed earlier studies in which cave fish from the sulfidic cave systems in which reproductive isolation emerges as a consequence of (Cueva del Azufre) showed reduced aggressive behavior, and cross- local adaptation. Our study highlights that inferences about the breeding experiments revealed polygenic Mendelian inheritance of putative outcome of male contests between different ecotypes can- behavioral population differences (Peters et al. 1973; Parzefall 1974, not be made based on comparisons of mean aggressiveness assessed 1979). Similar patterns of reduced aggressive behavior are found in during contests of opponents from the same ecotype. Even when cer- other cave fishes (see Parzefall and Trajano 2010), even though tain ecotypes do not show reduced aggressiveness overall, cross- some species, like Pimelodella kronei, can be highly aggressive ecotype contests need to be conducted to evaluate the role played by (Trajano 1991; Hoenen and Trajano 1995). Nevertheless, fights male competition for the emergence of reproductive isolation. between males from the same population reported in Bierbach et al. (2012) were staged with laboratory-reared individuals that had been reared under light conditions for at least two generations. Our cur- Acknowledgments rent study, using wild-caught fish, confirmed a reduction in aggres- We thank the people of Tapijulapa and Teapa for their hospitality during our sive behaviors in both sulfidic surface and sulfidic cave ecotypes. visits. The present study was supported by the DFG (PL 470/3-1; BI 1828/2- The exact selective mechanisms (or triggers of plastic changes) 1), DAAD, and the Leibniz Competition (SAW-2013-IGB-2). Fish were col- leading to reduced aggressiveness in the sulfide-adapted cave and lected under the authorization of the Mexican government (SGPA/DGVS- surface ecotypes in the Tacotalpa drainage but not in both others 04315/11, PRMN/DGOPA-009/2015, and PRMN/DGOPA-012/2017, issued remain elusive, but we argue in favor of independent evolutionary by SEMARNAT, SAGARPA-CONAPESCA-DGOPA). We also like to thank trajectories (Pfenninger et al. 2014, 2015). Gene flow from cave- Alexandra Tyers and two anonymous reviewers for their valuable comments dwelling P. mexicana out of the Cueva del Azufre into the popula- on earlier versions of our manuscript. tion inhabiting the adjacent sulfidic surface habitat in front of the cave (El Azufre) is detectable, while gene flow in the opposite direc- Funding tion is virtually absent (Plath et al. 2007a; Tobler et al. 2008a, 2009). Thus, it seems possible that reduced aggressiveness—as seen The present study was ﬁnancially supported by the DFG (PL 470/3-1; BI in both sulfide-adapted surface-dwelling males and cave-dwelling 1828/2-1), the German Academic Exchange Sevice (DAAD), and the Leibniz males in this particular drainage—is merely a consequence of Association (SAW-2013-IGB-2). 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