Integrative analyses of convergent adaptation in sympatric extremophile fishes.

The evolution of independent lineages along replicated environmental transitions frequently results in convergent adaptation, yet the degree to which convergence is present across multiple levels of biological organization is often unclear. Additionally, inherent biases associated with shared ancestry and variation in selective regimes across geographic replicates often pose challenges for confidently identifying patterns of convergence. We investigated a system in which three species of poeciliid fishes sympatrically occur in a toxic spring rich in hydrogen sulfide (H2S) and an adjacent nonsulfidic stream to examine patterns of adaptive evolution across levels of biological organization. We found convergence in morphological and physiological traits and genome-wide patterns of gene expression among all three species. In addition, there were shared signatures of selection on genes encoding H2S toxicity targets in the mitochondrial genomes of each species. However, analyses of nuclear genomes revealed neither evidence for substantial genomic islands of divergence around genes involved in H2S toxicity and detoxification nor substantial congruence of strongly differentiated regions across population pairs. These non-convergent, heterogeneous patterns of genomic divergence may indicate that sulfide tolerance is highly polygenic, with shared allele frequency shifts present at many loci with small effects along the genome. Alternatively, H2S tolerance may involve substantial genetic redundancy, with non-convergent, lineage-specific variation at multiple loci along the genome underpinning similar changes in phenotypes and gene expression. Overall, we demonstrate variability in the extent of convergence across organizational levels and highlight the challenges of linking patterns of convergence across scales.

Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in the Poecilia mexicana species complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

Evolutionary Rate Shifts in Coding and Regulatory Regions Underpin Repeated Adaptation to Sulfidic Streams in Poeciliid Fishes.

Adaptation to extreme environments often involves the evolution of dramatic physiological changes. To better understand how organisms evolve these complex phenotypic changes, the repeatability and predictability of evolution, and possible constraints on adapting to an extreme environment, it is important to understand how adaptive variation has evolved. Poeciliid fishes represent a particularly fruitful study system for investigations of adaptation to extreme environments due to their repeated colonization of toxic hydrogen sulfide-rich springs across multiple species within the clade. Previous investigations have highlighted changes in the physiology and gene expression in specific species that are thought to facilitate adaptation to hydrogen sulfide-rich springs. However, the presence of adaptive nucleotide variation in coding and regulatory regions and the degree to which convergent evolution has shaped the genomic regions underpinning sulfide tolerance across taxa are unknown. By sampling across seven independent lineages in which nonsulfidic lineages have colonized and adapted to sulfide springs, we reveal signatures of shared evolutionary rate shifts across the genome. We found evidence of genes, promoters, and putative enhancer regions associated with both increased and decreased convergent evolutionary rate shifts in hydrogen sulfide-adapted lineages. Our analysis highlights convergent evolutionary rate shifts in sulfidic lineages associated with the modulation of endogenous hydrogen sulfide production and hydrogen sulfide detoxification. We also found that regions with shifted evolutionary rates in sulfide spring fishes more often exhibited convergent shifts in either the coding region or the regulatory sequence of a given gene, rather than both.

Thermal tolerance in an extremophile fish from Mexico is not affected by environmental hypoxia.

The thermal ecology of ectotherm animals has gained considerable attention in the face of human-induced climate change. Particularly in aquatic species, the experimental assessment of critical thermal limits (CTmin and CTmax) may help to predict possible effects of global warming on habitat suitability and ultimately species survival. Here we present data on the thermal limits of two endemic and endangered extremophile fish species, inhabiting a geothermally heated and sulfur-rich spring system in southern Mexico: The sulfur molly (Poecilia sulphuraria) and the widemouth gambusia (Gambusia eurystoma). Besides physiological challenges induced by toxic hydrogen sulfide and related severe hypoxia during the day, water temperatures have been previously reported to exceed those of nearby clearwater streams. We now present temperature data for various locations and years in the sulfur spring complex and conducted laboratory thermal tolerance tests (CTmin and CTmax) both under normoxic and severe hypoxic conditions in both species. Average CTmax limits did not differ between species when dissolved oxygen was present. However, critical temperature (CTmax=43.2°C) in P. sulphuraria did not change when tested under hypoxic conditions, while G. eurystoma on average had a lower CTmax when oxygen was absent. Based on this data we calculated both species' thermal safety margins and used a TDT (thermal death time) model framework to relate our experimental data to observed temperatures in the natural habitat. Our findings suggest that both species live near their thermal limits during the annual dry season and are locally already exposed to temperatures above their critical thermal limits. We discuss these findings in the light of possible physiological adaptions of the sulfur-adapted fish species and the anthropogenic threats for this unique system.

Selection on standing genetic variation mediates convergent evolution in extremophile fish.

Hydrogen sulfide is a toxic gas that disrupts numerous biological processes, including energy production in the mitochondria, yet fish in the Poecilia mexicana species complex have independently evolved sulfide tolerance several times. Despite clear evidence for convergence at the phenotypic level in these fishes, it is unclear if the repeated evolution of hydrogen sulfide tolerance is the result of similar genomic changes. To address this gap, we used a targeted capture approach to sequence genes associated with sulfide processes and toxicity from five sulfidic and five nonsulfidic populations in the species complex. By comparing sequence variation in candidate genes to a reference set, we identified similar population structure and differentiation, suggesting that patterns of variation in most genes associated with sulfide processes and toxicity are due to demographic history and not selection. But the presence of tree discordance for a subset of genes suggests that several loci are evolving divergently between ecotypes. We identified two differentiation outlier genes that are associated with sulfide detoxification in the mitochondria that have signatures of selection in all five sulfidic populations. Further investigation into these regions identified long, shared haplotypes among sulfidic populations. Together, these results reveal that selection on standing genetic variation in putatively adaptive genes may be driving phenotypic convergence in this species complex.

Multispecies collective waving behaviour in fish.

Collective behaviour is widely accepted to provide a variety of antipredator benefits. Acting collectively requires not only strong coordination among group members, but also the integration of among-individual phenotypic variation. Therefore, groups composed of more than one species offer a unique opportunity to look into the evolution of both mechanistic and functional aspects of collective behaviour. Here, we present data on mixed-species fish shoals that perform collective dives. These repeated dives produce water waves capable of delaying and/or reducing the success of piscivorous bird attacks. The large majority of the fish in these shoals consist of the sulphur molly, Poecilia sulphuraria, but we regularly also found a second species, the widemouth gambusia, Gambusia eurystoma, making these shoals mixed-species aggregations. In a set of laboratory experiments, we found that gambusia were much less inclined to dive after an attack as compared with mollies, which almost always dive, though mollies dived less deep when paired with gambusia that did not dive. By contrast, the behaviour of gambusia was not influenced by the presence of diving mollies. The dampening effect of less responsive gambusia on molly diving behaviour can have strong evolutionary consequences on the overall collective waving behaviour as we expect shoals with a high proportion of unresponsive gambusia to be less effective at producing repeated waves. This article is part of a discussion meeting issue 'Collective behaviour through time'.

Fish waves as emergent collective antipredator behavior.

The collective behavior of animals has attracted considerable attention in recent years, with many studies exploring how local interactions between individuals can give rise to global group properties.1-3 The functional aspects of collective behavior are less well studied, especially in the field,4 and relatively few studies have investigated the adaptive benefits of collective behavior in situations where prey are attacked by predators.5,6 This paucity of studies is unsurprising because predator-prey interactions in the field are difficult to observe. Furthermore, the focus in recent studies on predator-prey interactions has been on the collective behavior of the prey7-10 rather than on the behavior of the predator (but see Ioannou et al.11 and Handegard et al.12). Here we present a field study that investigated the anti-predator benefits of waves produced by fish at the water surface when diving down collectively in response to attacks of avian predators. Fish engaged in surface waves that were highly conspicuous, repetitive, and rhythmic involving many thousands of individuals for up to 2 min. Experimentally induced fish waves doubled the time birds waited until their next attack, therefore substantially reducing attack frequency. In one avian predator, capture probability, too, decreased with wave number and birds switched perches in response to wave displays more often than in control treatments, suggesting that they directed their attacks elsewhere. Taken together, these results support an anti-predator function of fish waves. The attack delay could be a result of a confusion effect or a consequence of waves acting as a perception advertisement, which requires further exploration.

Parallel shifts of visual sensitivity and body coloration in replicate populations of extremophile fish.

Visual sensitivity and body pigmentation are often shaped by both natural selection from the environment and sexual selection from mate choice. One way of quantifying the impact of the environment is by measuring how traits have changed after colonization of a novel habitat. To do this, we studied Poecilia mexicana populations that have repeatedly adapted to extreme sulphidic (H2 S-containing) environments. We measured visual sensitivity using opsin gene expression, as well as body pigmentation, for populations in four independent drainages. Both visual sensitivity and body pigmentation showed significant parallel shifts towards greater medium-wavelength sensitivity and reflectance in sulphidic populations. Altogether we found that sulphidic habitats select for differences in visual sensitivity and pigmentation. Shifts between habitats may be due to both differences in the water's spectral properties and correlated ecological changes.

Acoustic and visual stimuli combined promote stronger responses to aerial predation in fish.

Bird predation poses a strong selection pressure on fish. Since birds must enter the water to catch fish, a combination of visual and mechano-acoustic cues (multimodal) characterize an immediate attack, while single cues (unimodal) may represent less dangerous disturbances. We investigated whether fish could use this information to distinguish between non-threatening and dangerous events and adjust their antipredator response to the perceived level of risk. To do so, we investigated the antipredator behavior of the sulphur molly (Poecilia sulphuraria), a small freshwater fish which is almost exclusively preyed on by piscivorous birds in its endemic sulfide spring habitat. In a field survey, we confirmed that these fish frequently have to distinguish between disturbances stemming from attacking birds (multimodal) and those which pose no (immediate) threat such as bird overflights (unimodal). In a laboratory experiment, we then exposed fish to artificial visual and/or acoustic stimuli presented separately or combined. Sensitivity was high regardless of stimulus type and number (more than 96% of fish initiated diving), but fish dove deeper, faster, and for longer when both stimuli were available simultaneously. Based on the system's high rates of bird activity, we argue that such an unselective dive initiation with subsequent fine-tuning of diving parameters in accordance to cue modality represents an optimal strategy for these fish to save energy necessary to respond to future attacks. Ultimately, our study shows that fish anticipate the imminent risk posed by disturbances linked to bird predation through integrating information from both visual and acoustic cues.

Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment.

Convergent evolution of conserved mitochondrial pathways underlies repeated adaptation to extreme environments.

Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

An interaction mechanism for the maintenance of fission-fusion dynamics under different individual densities.

Animals often show high consistency in their social organisation despite facing changing environmental conditions. Especially in shoaling fish, fission-fusion dynamics that describe for which periods individuals are solitary or social have been found to remain unaltered even when density changed. This compensatory ability is assumed to be an adaptation towards constant predation pressure, but the mechanism through which individuals can actively compensate for density changes is yet unknown. The aim of the current study is to identify behavioural patterns that enable this active compensation. We compared the fission-fusion dynamics of two populations of the live-bearing Atlantic molly (Poecilia mexicana) that live in adjacent habitats with very different predator regimes: cave mollies that inhabit a low-predation environment inside a sulfidic cave with a low density of predatory water bugs (Belostoma sp.), and mollies that live directly outside the cave (henceforth called "surface" mollies) in a high-predation environment. We analysed their fission-fusion dynamics under two different fish densities of 12 and 6 fish per 0.36 m2. As expected, surface mollies spent more time being social than cave mollies, and this difference in social time was a result of surface mollies being less likely to discontinue social contact (once they had a social partner) and being more likely to resume social contact (once alone) than cave mollies. Interestingly, surface mollies were also less likely to switch among social partners than cave mollies. A random walk simulation predicted each population to show reduced social encounters in the low density treatment. While cave mollies largely followed this prediction, surface mollies maintained their interaction probabilities even at low density. Surface mollies achieved this by a reduction in the size of a convex polygon formed by the group as density decreased. This may allow them to largely maintain their fission-fusion dynamics while still being able to visit large parts of the available area as a group. A slight reduction (21%) in the area visited at low densities was also observed but insufficient to explain how the fish maintained their fission-fusion dynamics. Finally, we discuss potential movement rules that could account for the reduction of polygon size and test their performance.

Functional consequences of phenotypic variation between locally adapted populations: Swimming performance and ventilation in extremophile fish.

Natural selection drives the evolution of traits to optimize organismal performance, but optimization of one aspect of performance can influence other aspects of performance. Here, we asked how phenotypic variation between locally adapted fish populations affects locomotion and ventilation, testing for functional trade-offs and trait-performance correlations. Specifically, we investigated two populations of livebearing fish (Poecilia mexicana) that inhabit distinct habitat types (hydrogen-sulphide-rich springs and adjacent nonsulphidic streams). For each individual, we quantified different metrics of burst swimming during simulated predator attacks, steady swimming and gill ventilation. Coinciding with predictions, we documented significant population differences in all aspects of performance, with fish from sulphidic habitats exhibiting higher steady swimming performance and higher ventilation capacity, but slower burst swimming. There was a significant functional trade-off between steady and burst swimming, but not between different aspects of locomotion and ventilation. Although our findings about population differences in locomotion performance largely parallel the results from previous studies, we provide novel insights about how morphological variation might impact ventilation and ultimately oxygen acquisition. Overall, our analyses provided insights into the functional consequences of previously documented phenotypic variation, which will help to disentangle the effects of different sources of selection that may coincide along complex environmental gradients.

Expression analyses of cave mollies (Poecilia mexicana) reveal key genes involved in the early evolution of eye regression.

Eye regression occurs across cave-dwelling populations of many species and is often coupled with a decrease or loss in eye function. Teleost fishes are among the few vertebrates to undergo widespread colonization of caves and often exhibit eye regression with blindness. Cave populations of the poeciliid fish Poecilia mexicana (cave molly) exhibit reduced-albeit functional-eyes, offering the opportunity to investigate partial eye regression. We sequenced eye transcriptomes of cave and surface populations of P. mexicana to identify differentially expressed genes that potentially underlie eye regression in cave mollies. We identified 28 significantly differentially expressed genes, 20 of which were directly related to light sensitivity, eye structure and visual signaling. Twenty-six of these genes were downregulated in cave compared to surface populations. Functional enrichment analysis revealed eye-related gene ontologies that were under-represented in cave mollies. In addition, a set of co-expressed genes related to vision and circadian rhythm was correlated with habitat type (cave versus surface). Our study suggests that differential gene expression plays a key role in the beginning evolutionary stages of eye regression in P. mexicana, shedding further light on regressive evolution in cavefish.

Correlated divergence of female and male genitalia in replicated lineages with ongoing ecological speciation.

Divergence of genital traits among lineages has the potential to serve as a reproductive isolating barrier when copulation, insemination, and fertilization are inhibited by incompatibilities between female and male genitalia. Despite widespread evidence for genital trait diversity among closely related lineages and coevolution of female and male genitalia within lineages, few studies have investigated genital evolution during the early stages of speciation. We quantified genital variation in replicated population pairs of Poecilia mexicana with ongoing ecological speciation between sulfidic (H2 S containing) and nearby nonsulfidic habitats. These analyses revealed rapid and correlated divergence of female and male genitalia across evolutionarily independent population pairs exposed to divergent selection regimes. Both sexes exhibited convergent evolution of genital traits among populations inhabiting similar habitat types. Our results demonstrate that genital evolution can occur during the early stages of speciation-with-gene-flow, potentially as a result of variation in the intensity of sexual conflict among populations. Our results suggest genitalia may contribute to early stages of divergence and challenge the generality of previously suggested mechanisms of genital evolution in poeciliids.

Bacterial Diversity in Replicated Hydrogen Sulfide-Rich Streams.

Extreme environments typically require costly adaptations for survival, an attribute that often translates to an elevated influence of habitat conditions on biotic communities. Microbes, primarily bacteria, are successful colonizers of extreme environments worldwide, yet in many instances, the interplay between harsh conditions, dispersal, and microbial biogeography remains unclear. This lack of clarity is particularly true for habitats where extreme temperature is not the overarching stressor, highlighting a need for studies that focus on the role other primary stressors (e.g., toxicants) play in shaping biogeographic patterns. In this study, we leveraged a naturally paired stream system in southern Mexico to explore how elevated hydrogen sulfide (H2S) influences microbial diversity. We sequenced a portion of the 16S rRNA gene using bacterial primers for water sampled from three geographically proximate pairings of streams with high (> 20 µM) or low (~ 0 µM) H2S concentrations. After exploring bacterial diversity within and among sites, we compared our results to a previous study of macroinvertebrates and fish for the same sites. By spanning multiple organismal groups, we were able to illuminate how H2S may differentially affect biodiversity. The presence of elevated H2S had no effect on overall bacterial diversity (p = 0.21), a large effect on community composition (25.8% of variation explained, p < 0.0001), and variable influence depending upon the group-whether fish, macroinvertebrates, or bacteria-being considered. For bacterial diversity, we recovered nine abundant operational taxonomic units (OTUs) that comprised a core H2S-rich stream microbiome in the region. Many H2S-associated OTUs were members of the Epsilonproteobacteria and Gammaproteobacteria, which both have been implicated in endosymbiotic relationships between sulfur-oxidizing bacteria and eukaryotes, suggesting the potential for symbioses that remain to be discovered in these habitats.

Concordant Changes in Gene Expression and Nucleotides Underlie Independent Adaptation to Hydrogen-Sulfide-Rich Environments.

The colonization of novel environments often involves changes in gene expression, protein coding sequence, or both. Studies of how populations adapt to novel conditions, however, often focus on only one of these two processes, potentially missing out on the relative importance of different parts of the evolutionary process. In this study, our objectives were 1) to better understand the qualitative concordance between conclusions drawn from analyses of differential expression and changes in genic sequence and 2) to quantitatively test whether differentially expressed genes were enriched for sites putatively under positive selection within gene regions. To achieve this, we compared populations of fish (Poecilia mexicana) that have independently adapted to hydrogen-sulfide-rich environments in southern Mexico to adjacent populations residing in nonsulfidic waters. Specifically, we used RNA-sequencing data to compare both gene expression and DNA sequence differences between populations. Analyzing these two different data types led to similar conclusions about which biochemical pathways (sulfide detoxification and cellular respiration) were involved in adaptation to sulfidic environments. Additionally, we found a greater overlap between genes putatively under selection and differentially expressed genes than expected by chance. We conclude that considering both differential expression and changes in DNA sequence led to a more comprehensive understanding of how these populations adapted to extreme environmental conditions. Our results imply that changes in both gene expression and DNA sequence-sometimes at the same loci-may be involved in adaptation.

Female Choice Undermines the Emergence of Strong Sexual Isolation between Locally Adapted Populations of Atlantic Mollies (Poecilia mexicana).

Divergent selection between ecologically dissimilar habitats promotes local adaptation, which can lead to reproductive isolation (RI). Populations in the Poecilia mexicana species complex have independently adapted to toxic hydrogen sulfide and show varying degrees of RI. Here, we examined the variation in the mate choice component of prezygotic RI. Mate choice tests across drainages (with stimulus males from another drainage) suggest that specific features of the males coupled with a general female preference for yellow color patterns explain the observed variation. Analyses of male body coloration identified the intensity of yellow fin coloration as a strong candidate to explain this pattern, and common-garden rearing suggested heritable population differences. Male sexual ornamentation apparently evolved differently across sulfide-adapted populations, for example because of differences in natural counterselection via predation. The ubiquitous preference for yellow color ornaments in poeciliid females likely undermines the emergence of strong RI, as female discrimination in favor of own males becomes weaker when yellow fin coloration in the respective sulfide ecotype increases. Our study illustrates the complexity of the (partly non-parallel) pathways to divergence among replicated ecological gradients. We suggest that future work should identify the genomic loci involved in the pattern reported here, making use of the increasing genomic and transcriptomic datasets available for our study system.

Intrasexual competition enhances reproductive isolation between locally adapted populations.

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 predict 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 (H2S-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 H2S, 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 physiological 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 H2S-adapted males into H2S-free waters, as well as H2S-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 system and we call for more careful evaluation of male fighting abilities in studies on ecological speciation.

Insights into the Social Behavior of Surface and Cave-Dwelling Fish (Poecilia mexicana) in Light and Darkness through the Use of a Biomimetic Robot.

Biomimetic robots (BRs) are becoming more common in behavioral research and, if they are accepted as conspecifics, allow for new forms of experimental manipulations of social interactions. Nevertheless, it is often not clear which cues emanating from a BR are actually used as communicative signals and how species or populations with different sensory makeups react to specific types of BRs. We herein present results from experiments using two populations of livebearing fishes that differ in their sensory capabilities. In the South of Mexico, surface-dwelling mollies (Poecilia mexicana) successfully invaded caves and adapted to dark conditions. While almost without pigment, these cave mollies possess smaller but still functional eyes. Although previous studies found cave mollies to show reduced shoaling preferences with conspecifics in light compared to surface mollies, it is assumed that they possess specialized adaptations to maintain some kind of sociality also in their dark habitats. By testing surface- and cave-dwelling mollies with RoboFish, a BR made for use in laboratory experiments with guppies and sticklebacks, we asked to what extent visual and non-visual cues play a role in their social behavior. Both cave- and surface-dwelling mollies followed the BR as well as a live companion when tested in light. However, when tested in darkness, only surface-dwelling fish were attracted by a live conspecific, whereas cave-dwelling fish were not. Neither cave- nor surface-dwelling mollies were attracted to RoboFish in darkness. This is the first study to use BRs for the investigation of social behavior in mollies and to compare responses to BRs both in light and darkness. As our RoboFish is accepted as conspecific by both used populations of the Atlantic molly only under light conditions but not in darkness, we argue that our replica is providing mostly visual cues.