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.

Reproductive individuality of clonal fish raised in near-identical environments and its link to early-life behavioral individuality.

Recent studies have documented among-individual phenotypic variation that emerges in the absence of apparent genetic and environmental differences, but it remains an open question whether such seemingly stochastic variation has fitness consequences. We perform a life-history experiment with naturally clonal fish, separated directly after birth into near-identical (i.e., highly standardized) environments, quantifying 2522 offspring from 152 broods over 280 days. We find that (i) individuals differ consistently in the size of offspring and broods produced over consecutive broods, (ii) these differences are observed even when controlling for trade-offs between brood size, offspring size and reproductive onset, indicating individual differences in life-history productivity and (iii) early-life behavioral individuality in activity and feeding patterns, with among-individual differences in feeding being predictive of growth, and consequently offspring size. Thus, our study provides experimental evidence that even when minimizing genetic and environmental differences, systematic individual differences in life-history measures and ultimately fitness can emerge.

Social competence improves the performance of biomimetic robots leading live fish.

Collective motion is commonly modeled with static interaction rules between agents. Substantial empirical evidence indicates, however, that animals may adapt their interaction rules depending on a variety of factors and social contexts. Here, we hypothesized that leadership performance is linked to the leader's responsiveness to the follower's actions and we predicted that a leader is followed longer if it adapts to the follower's avoidance movements. We tested this prediction with live guppies that interacted with a biomimetic robotic fish programmed to act as a 'socially competent' leader. Fish that were avoiding the robot were approached more carefully in future approaches. In two separate experiments we then asked how the leadership performance of the socially competent robot leader differed to that of a robot leader that either approached all fish in the same, non-responsive, way or one that did change its approach behavior randomly, irrespective of the fish's actions. We found that (1) behavioral variability itself appears attractive and that socially competent robots are better leaders which (2) require fewer approach attempts to (3) elicit longer average following behavior than non-competent agents. This work provides evidence that social responsiveness to avoidance reactions plays a role in the social dynamics of guppies. We showcase how social responsiveness can be modeled and tested directly embedded in a living animal model using adaptive, interactive robots.

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'.

Leveraging big data to uncover the eco-evolutionary factors shaping behavioural development.

Mapping the eco-evolutionary factors shaping the development of animals' behavioural phenotypes remains a great challenge. Recent advances in 'big behavioural data' research-the high-resolution tracking of individuals and the harnessing of that data with powerful analytical tools-have vastly improved our ability to measure and model developing behavioural phenotypes. Applied to the study of behavioural ontogeny, the unfolding of whole behavioural repertoires can be mapped in unprecedented detail with relative ease. This overcomes long-standing experimental bottlenecks and heralds a surge of studies that more finely define and explore behavioural-experiential trajectories across development. In this review, we first provide a brief guide to state-of-the-art approaches that allow the collection and analysis of high-resolution behavioural data across development. We then outline how such approaches can be used to address key issues regarding the ecological and evolutionary factors shaping behavioural development: developmental feedbacks between behaviour and underlying states, early life effects and behavioural transitions, and information integration across development.

The emergence and development of behavioral individuality in clonal fish.

Behavioral individuality is a ubiquitous phenomenon in animal populations, yet the origins and developmental trajectories of individuality, especially very early in life, are still a black box. Using a high-resolution tracking system, we mapped the behavioral trajectories of genetically identical fish (Poecilia formosa), separated immediately after birth into identical environments, over the first 10 weeks of their life at 3 s resolution. We find that (i) strong behavioral individuality is present at the very first day after birth, (ii) behavioral differences at day 1 of life predict behavior up to at least 10 weeks later, and (iii) patterns of individuality strengthen gradually over developmental time. Our results establish a null model for how behavioral individuality can develop in the absence of genetic and environmental variation and provide experimental evidence that later-in-life individuality can be strongly shaped by factors pre-dating birth like maternal provisioning, epigenetics and pre-birth developmental stochasticity.

Live fish learn to anticipate the movement of a fish-like robot.

The ability of an individual to predict the outcome of the actions of others and to change their own behavior adaptively is called anticipation. There are many examples from mammalian species-including humans-that show anticipatory abilities in a social context, however, it is not clear to what extent fishes can anticipate the actions of their interaction partners or what the underlying mechanisms are for that anticipation. To answer these questions, we let live guppies (Poecilia reticulata) interact repeatedly with an open-loop (noninteractive) biomimetic robot that has previously been shown to be an accepted conspecific. The robot always performed the same zigzag trajectory in the experimental tank that ended in one of the corners, giving the live fish the opportunity to learn both the location of the final destination as well as the specific turning movement of the robot over three consecutive trials. The live fish's reactions were categorized into a global anticipation, which we defined as relative time to reach the robot's final corner, and a local anticipation which was the relative time and location of the live fish's turns relative to robofish turns. As a proxy for global anticipation, we found that live fish in the last trial reached the robot's destination corner significantly earlier than the robot. Overall, more than 50% of all fish arrived at the destination before the robot. This is more than a random walk model would predict and significantly more compared to all other equidistant, yet unvisited, corners. As a proxy for local anticipation, we found fish change their turning behavior in response to the robot over the course of the trials. Initially, the fish would turn after the robot, which was reversed in the end, as they began to turn slightly before the robot in the final trial. Our results indicate that live fish are able to anticipate predictably behaving social partners both in regard to final movement locations as well as movement dynamics. Given that fish have been found to exhibit consistent behavioral differences, anticipation in fish could have evolved as a mechanism to adapt to different social interaction partners.

Cascading indirect genetic effects in a clonal vertebrate.

Understanding how individual differences arise and how their effects propagate through groups are fundamental issues in biology. Individual differences can arise from indirect genetic effects (IGE): genetically based variation in the conspecifics with which an individual interacts. Using a clonal species, the Amazon molly (Poecilia formosa), we test the hypothesis that IGE can propagate to influence phenotypes of the individuals that do not experience them firsthand. We tested this by exposing genetically identical Amazon mollies to conspecific social partners of different clonal lineages, and then moving these focal individuals to new social groups in which they were the only member to have experienced the IGE. We found that genetically different social environments resulted in the focal animals experiencing different levels of aggression, and that these IGE carried over into new social groups to influence the behaviour of naive individuals. These data reveal that IGE can cascade beyond the individuals that experience them. Opportunity for cascading IGE is ubiquitous, especially in species with long-distance dispersal or fission-fusion group dynamics. Cascades could amplify (or mitigate) the effects of IGE on trait variation and on evolutionary trajectories. Expansion of the IGE framework to include cascading and other types of carry-over effects will therefore improve understanding of individual variation and social evolution and allow more accurate prediction of population response to changing environments.

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.

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.

Two Locomotor Traits Show Different Patterns of Developmental Plasticity Between Closely Related Clonal and Sexual Fish.

The capacity to compensate for environmental change determines population persistence and biogeography. In ectothermic organisms, performance at different temperatures can be strongly affected by temperatures experienced during early development. Such developmental plasticity is mediated through epigenetic mechanisms that induce phenotypic changes within the animal's lifetime. However, epigenetic modifiers themselves are encoded by DNA so that developmental plasticity could itself be contingent on genetic diversity. In this study, we test the hypothesis that the capacity for developmental plasticity depends on a species' among-individual genetic diversity. To test this, we exploited a unique species complex that contains both the clonal, genetically identical Amazon molly (Poecilia formosa), and the sexual, genetically diverse Atlantic molly (Poecilia mexicana). We predicted that the greater among-individual genetic diversity in the Atlantic molly may increase their capacity for developmental plasticity. We raised both clonal and sexual mollies at either warm (28°C) or cool (22°C) temperatures and then measured locomotor capacity (critical sustained swimming performance) and unforced movement in an open field across a temperature gradient that simulated environmental conditions often experienced by these species in the wild. In the clonal Amazon molly, differences in the developmental environment led to a shift in the thermal performance curve of unforced movement patterns, but much less so in maximal locomotor capacity. In contrast, the sexual Atlantic mollies exhibited the opposite pattern: developmental plasticity was present in maximal locomotor capacity, but not in unforced movement. Thus our data show that developmental plasticity in clones and their sexual, genetically more diverse sister species is trait dependent. This points toward mechanistic differences in how genetic diversity mediates plastic responses exhibited in different traits.

Male Sexual Preference for Female Swimming Activity in the Guppy (Poecilia reticulata).

Mate choice that is based on behavioural traits is a common feature in the animal kingdom. Using the Trinidadian guppy, a species with mutual mate choice, we investigated whether males use female swimming activity-a behavioural trait known to differ consistently among individuals in many species-as a trait relevant for their mate choice. In the first experiment, we assessed male and female activity in an open field test alone (two repeated measures) and afterwards in heterosexual pairs (two repeated measures). In these pairs, we simultaneously assessed males' mating efforts by counting the number of sexual behaviours (courtship displays and copulations). Male and female guppies showed consistent individual differences in their swimming activity when tested both alone and in a pair, and these differences were maintained across both test situations. When controlling for male swimming behaviour and both male and female body size, males performed more courtship displays towards females with higher swimming activity. In a second experiment, we tested for a directional male preference for swimming activity by presenting males video animations of low- and high-active females in a dichotomous choice test. In congruence with experiment 1, we found males to spend significantly more time in association with the high-active female stimulus. Both experiments thus point towards a directional male preference for higher activity levels in females. We discuss the adaptive significance of this preference as activity patterns might indicate individual female quality, health or reproductive state while, mechanistically, females that are more active might be more detectable to males as well.

Size Selective Harvesting Does Not Result in Reproductive Isolation among Experimental Lines of Zebrafish, Danio rerio: Implications for Managing Harvest-Induced Evolution.

Size-selective mortality is common in fish stocks. Positive size-selection happens in fisheries where larger size classes are preferentially targeted while gape-limited natural predation may cause negative size-selection for smaller size classes. As body size and correlated behavioural traits are sexually selected, harvest-induced trait changes may promote prezygotic reproductive barriers among selection lines experiencing differential size-selective mortality. To investigate this, we used three experimental lines of zebrafish (Danio rerio) exposed to positive (large-harvested), negative (small-harvested) and random (control line) size-selective mortality for five generations. We tested prezygotic preferences through choice tests and spawning trials. In the preference tests without controlling for body size, we found that females of all lines preferred males of the generally larger small-harvested line. When the body size of stimulus fish was statistically controlled, this preference disappeared and a weak evidence of line-assortative preference emerged, but only among large-harvested line fish. In subsequent spawning trials, we did not find evidence for line-assortative reproductive allocation in any of the lines. Our study suggests that size-selection due to fisheries or natural predation does not result in reproductive isolation. Gene flow between wild-populations and populations adapted to size-selected mortality may happen during secondary contact which can speed up trait recovery.

Fixation of allelic gene expression landscapes and expression bias pattern shape the transcriptome of the clonal Amazon molly.

The Amazon molly is a unique clonal fish species that originated from an interspecies hybrid between Poecilia species P. mexicana and P. latipinna It reproduces by gynogenesis, which eliminates paternal genomic contribution to offspring. An earlier study showed that Amazon molly shows biallelic expression for a large portion of the genome, leading to two main questions: (1) Are the allelic expression patterns from the initial hybridization event stabilized or changed during establishment of the asexual species and its further evolution? (2) Is allelic expression biased toward one parental allele a stochastic or adaptive process? To answer these questions, the allelic expression of P. formosa siblings was assessed to investigate intra- and inter-cohort allelic expression variability. For comparison, interspecies hybrids between P. mexicana and P. latipinna were produced in the laboratory to represent the P. formosa ancestor. We have identified inter-cohort and intra-cohort variation in parental allelic expression. The existence of inter-cohort divergence suggests functional P. formosa allelic expression patterns do not simply reflect the atavistic situation of the first interspecies hybrid but potentially result from long-term selection of transcriptional fitness. In addition, clonal fish show a transcriptional trend representing minimal intra-clonal variability in allelic expression patterns compared to the corresponding hybrids. The intra-clonal similarity in gene expression translates to sophisticated genetic functional regulation at the individuum level. These findings suggest the parental alleles inherited by P. formosa form tightly regulated genetic networks that lead to a stable transcriptomic landscape within clonal individuals.

Group-level patterns emerge from individual speed as revealed by an extremely social robotic fish.

Understanding the emergence of collective behaviour has long been a key research focus in the natural sciences. Besides the fundamental role of social interaction rules, a combination of theoretical and empirical work indicates individual speed may be a key process that drives the collective behaviour of animal groups. Socially induced changes in speed by interacting animals make it difficult to isolate the effects of individual speed on group-level behaviours. Here, we tackled this issue by pairing guppies with a biomimetic robot. We used a closed-loop tracking and feedback system to let a robotic fish naturally interact with a live partner in real time, and programmed it to strongly copy and follow its partner's movements while lacking any preferred movement speed or directionality of its own. We show that individual differences in guppies' movement speed were highly repeatable and in turn shaped key collective patterns: a higher individual speed resulted in stronger leadership, lower cohesion, higher alignment and better temporal coordination of the pairs. By combining the strengths of individual-based models and observational work with state-of-the-art robotics, we provide novel evidence that individual speed is a key, fundamental process in the emergence of collective behaviour.

Guppies Prefer to Follow Large (Robot) Leaders Irrespective of Own Size.

Body size is often assumed to determine how successfully an individual can lead others with larger individuals being better leaders than smaller ones. But even if larger individuals are more readily followed, body size often correlates with specific behavioral patterns and it is thus unclear whether larger individuals are more often followed than smaller ones because of their size or because they behave in a certain way. To control for behavioral differences among differentially-sized leaders, we used biomimetic robotic fish (Robofish) of different sizes. Live guppies (Poecilia reticulata) are known to interact with Robofish in a similar way as with live conspecifics. Consequently, Robofish may serve as a conspecific-like leader that provides standardized behaviors irrespective of its size. We asked whether larger Robofish leaders are preferentially followed and whether the preferences of followers depend on own body size or risk-taking behavior ("boldness"). We found that live female guppies followed larger Robofish leaders in closer proximity than smaller ones and this pattern was independent of the followers' own body size as well as risk-taking behavior. Our study shows a "bigger is better" pattern in leadership that is independent of behavioral differences among differentially-sized leaders, followers' own size and risk-taking behavior.

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.

Eye fluke (Tylodelphys clavata) infection impairs visual ability and hampers foraging success in European perch.

Visual performance and environmental conditions can influence both behavioral patterns and predator-prey interactions of fish. Eye parasites can impair their host's sensory performance with important consequences for the detection of prey, predators, and conspecifics. We used European perch (Perca fluviatilis) experimentally infected with the eye fluke Tylodelphys clavata and evaluated their feeding behavior and competitive ability under competition with non-infected conspecifics, in groups of four individuals, for two different prey species (Asellus aquaticus and Daphnia magna). To test whether the effect of T. clavata infection differs at different light conditions, we performed the experiments at two light intensities (600 and 6 lx). Foraging efficiency of perch was significantly affected by infection but not by light intensity. The distance at which infected fish attacked both prey species was significantly shorter in comparison to non-infected conspecifics. Additionally, infected fish more often unsuccessfully attacked A. aquaticus. Although the outcome of competition depended on prey species, there was a general tendency that non-infected fish consumed more of the available prey under both light intensities. Even though individual prey preferences for either A. aquaticus or D. magna were observed, we could not detect that infected fish change their prey preference to compensate for a reduced competitive foraging ability. As infection of T. clavata impairs foraging efficiency and competitive ability, infected fish would need to spend more time foraging to attain similar food intake as non-infected conspecifics; this presumably increases predation risk and potentially enhances transmission success to the final host.

Size-selective harvesting fosters adaptations in mating behaviour and reproductive allocation, affecting sexual selection in fish.

The role of sexual selection in the context of harvest-induced evolution is poorly understood. However, elevated and trait-selective harvesting of wild populations may change sexually selected traits, which in turn can affect mate choice and reproduction. We experimentally evaluated the potential for fisheries-induced evolution of mating behaviour and reproductive allocation in fish. We used an experimental system of zebrafish (Danio rerio) lines exposed to large, small or random (i.e. control) size-selective mortality. The large-harvested line represented a treatment simulating the typical case in fisheries where the largest individuals are preferentially harvested. We used a full factorial design of spawning trials with size-matched individuals to control for the systematic impact of body size during reproduction, thereby singling out possible changes in mating behaviour and reproductive allocation. Both small size-selective mortality and large size-selective mortality left a legacy on male mating behaviour by elevating intersexual aggression. However, there was no evidence for line-assortative reproductive allocation. Females of all lines preferentially allocated eggs to the generally less aggressive males of the random-harvested control line. Females of the large-harvested line showed enhanced reproductive performance, and males of the large-harvested line had the highest egg fertilization rate among all males. These findings can be explained as an evolutionary adaptation by which individuals of the large-harvested line display an enhanced reproductive performance early in life to offset the increased probability of adult mortality due to harvest. Our results suggest that the large-harvested line evolved behaviourally mediated reproductive adaptations that could increase the rate of recovery when populations adapted to high fishing pressure come into secondary contact with other populations.

Naturally clonal vertebrates are an untapped resource in ecology and evolution research.

Science requires replication. The development of many cloned or isogenic model organisms is a testament to this. But researchers are reluctant to use these traditional animal model systems for certain questions in evolution or ecology research, because of concerns over relevance or inbreeding. It has largely been overlooked that there are a substantial number of vertebrate species that reproduce clonally in nature. Here we highlight how use of these naturally evolved, phenotypically complex animals can push the boundaries of traditional experimental design and contribute to answering fundamental questions in the fields of ecology and evolution.