Lake Malawi Cichlid Species Research Articles

Ontogeny and social context regulate the circadian activity patterns of Lake Malawi cichlids

Activity patterns tend to be highly stereotyped and critical for executing many different behaviors including foraging, social interactions, and predator avoidance. Differences in the circadian timing of locomotor activity and rest periods can facilitate habitat partitioning and the exploitation of novel niches. As a consequence, closely related species often display highly divergent activity patterns, suggesting that shifts from diurnal to nocturnal behavior, or vice versa, are critical for survival. In Africa’s Lake Malawi alone, there are over 500 species of cichlids, which inhabit diverse environments and exhibit extensive phenotypic variation. We have previously identified a substantial range in activity patterns across adult Lake Malawi cichlid species, from strongly diurnal to strongly nocturnal. In many species, including fishes, ecological pressures differ dramatically across life-history stages, raising the possibility that activity patterns may change over ontogeny. To determine if rest-activity patterns change across life stages, we compared the locomotor patterns of six Lake Malawi cichlid species. While total rest and activity did not change between early juvenile and adult stages, rest-activity patterns did, with juveniles displaying distinct activity rhythms that are more robust than adults. One distinct difference between juveniles and adults is the emergence of complex social behavior. To determine whether social context is required for activity rhythms, we next measured locomotor behavior in group-housed adult fish. We found that when normal social interactions were allowed, locomotor activity patterns were restored, supporting the notion that social interactions promote circadian regulation of activity in adult fish. These findings reveal a previously unidentified link between developmental stage and social interactions in the circadian timing of cichlid activity.

Phylogenomic Analyses Show Repeated Evolution of Hypertrophied Lips Among Lake Malawi Cichlid Fishes.

Cichlid fishes have repeatedly evolved an astounding diversity of trophic morphologies. For example, hypertrophied lips have evolved multiple times in both African and Neotropical cichlids and could have even evolved convergently within single species assemblages such as African Lake Malawi cichlids. However, the extremely high diversification rate in Lake Malawi cichlids and extensive potential for hybridization has cast doubt on whether even genome-level phylogenetic reconstructions could delineate if these types of adaptations have evolved once or multiple times. To examine the evolution of this iconic trait using protein-coding and noncoding single nucleotide polymorphisms (SNPs), we analyzed the genomes of 86 Lake Malawi cichlid species, including 33 de novo resequenced genomes. Surprisingly, genome-wide protein-coding SNPs exhibited enough phylogenetic informativeness to reconstruct interspecific and intraspecific relationships of hypertrophied lip cichlids, although noncoding SNPs provided better support. However, thinning of noncoding SNPs indicated most discrepancies come from the relatively smaller number of protein-coding sites and not from fundamental differences in their phylogenetic informativeness. Both coding and noncoding reconstructions showed that several “sand-dwelling” hypertrophied lip species, sampled intraspecifically, form a clade interspersed with a few other nonhypertrophied lip lineages. We also recovered Abactochromis labrosus within the rock-dwelling “mbuna” lineage, starkly contrasting with the affinities of other hypertrophied lip taxa found in the largely sand-dwelling “nonmbuna” component of this radiation. Comparative analyses coupled with tests for introgression indicate there is no widespread introgression between the hypertrophied lip lineages and taken together suggest this trophic phenotype has likely evolved at least twice independently within-lake Malawi.

Of bars and stripes: A Malawi cichlid hybrid cross provides insights into genetic modularity and evolution of modifier loci underlying colour pattern diversification.

Understanding the origins of phenotypic diversity among closely related species remains an important largely unsolved question in evolutionary biology. With over 800species, Lake Malawi haplochromine cichlid fishes are a prominent example of extremely fast evolution of diversity including variation in colouration. Previously, a single major effect gene, agrp2 (asip2b), has been linked to evolutionary losses and gains of horizontal stripe patterns in cichlids, but it remains unknown what causes more fine-scale variation in the number and continuity of the stripes. Also, the genetic basis of the most common colour pattern in African cichlids, vertical bars, and potential interactions between the two colour patterns remain unknown. Based on a hybrid cross of the horizontally striped Lake Malawi cichlid Pseudotropheus cyaneorhabdos and the vertically barred species Chindongo demasoni we investigated the genetic basis of both colour patterns. The distribution of phenotypes in the F2 generation of the cross indicates that horizontal stripes and vertical bars are independently inherited patterns that are caused by two sets of genetic modules. While horizontal stripes are largely controlled by few major effect loci, vertical bars are a highly polygenic trait. Horizontal stripes show substantial variation in the F2 generation that, interestingly, resemble naturally occurring phenotypes found in other Lake Malawi cichlid species. Quantitative trait loci (QTL) mapping of this cross reveals known (agrp2) and unknown loci underlying horizontal stripe patterns. These findings provide novel insights into the incremental fine-tuning of an adaptive trait that diversified through the evolution of additional modifier loci.

Diversity in rest-activity patterns among Lake Malawi cichlid fishes suggests a novel axis of habitat partitioning.

ABSTRACTAnimals display remarkable diversity in rest and activity patterns that are regulated by endogenous foraging strategies, social behaviors and predator avoidance. Alteration in the circadian timing of activity or the duration of rest–wake cycles provide a central mechanism for animals to exploit novel niches. The diversity of the >3000 cichlid species throughout the world provides a unique opportunity to examine variation in locomotor activity and rest. Lake Malawi alone is home to over 500 species of cichlids that display divergent behaviors and inhabit well-defined niches throughout the lake. These species are presumed to be diurnal, though this has never been tested systematically. Here, we measured locomotor activity across the circadian cycle in 11 Lake Malawi cichlid species. We documented surprising variability in the circadian time of locomotor activity and the duration of rest. In particular, we identified a single species, Tropheops sp. ‘red cheek’, that is nocturnal. Nocturnal behavior was maintained when fish were provided shelter, but not under constant darkness, suggesting that it results from acute response to light rather than an endogenous circadian rhythm. Finally, we showed that nocturnality is associated with increased eye size after correcting for evolutionary history, suggesting a link between visual processing and nighttime activity. Together, these findings identify diversity of locomotor behavior in Lake Malawi cichlids and provide a system for investigating the molecular and neural basis underlying variation in nocturnal activity.

Ecomorphological divergence and habitat lability in the context of robust patterns of modularity in the cichlid feeding apparatus

BackgroundAdaptive radiations are characterized by extreme and/or iterative phenotypic divergence; however, such variation does not accumulate evenly across an organism. Instead, it is often partitioned into sub-units, or modules, which can differentially respond to selection. While it is recognized that changing the pattern of modularity or the strength of covariation (integration) can influence the range or rate of morphological evolution, the relationship between shape variation and covariation remains unclear. For example, it is possible that rapid phenotypic change requires concomitant changes to the underlying covariance structure. Alternatively, repeated shifts between phenotypic states may be facilitated by a conserved covariance structure. Distinguishing between these scenarios will contribute to a better understanding of the factors that shape biodiversity. Here, we explore these questions using a diverse Lake Malawi cichlid species complex, Tropheops, that appears to partition habitat by depth.ResultsWe construct a phylogeny of Tropheops populations and use 3D geometric morphometrics to assess the shape of four bones involved in feeding (mandible, pharyngeal jaw, maxilla, pre-maxilla) in populations that inhabit deep versus shallow habitats. We next test numerous modularity hypotheses to understand whether fish at different depths are characterized by conserved or divergent patterns of modularity. We further examine rates of morphological evolution and disparity between habitats and among modules. Finally, we raise a single Tropheops species in environments mimicking deep or shallow habitats to discover whether plasticity can replicate the pattern of morphology, disparity, or modularity observed in natural populations.ConclusionsOur data support the hypothesis that conserved patterns of modularity permit the evolution of divergent morphologies and may facilitate the repeated transitions between habitats. In addition, we find the lab-reared populations replicate many trends in the natural populations, which suggests that plasticity may be an important force in initiating depth transitions, priming the feeding apparatus for evolutionary change.

An allelic series at pax7a is associated with colour polymorphism diversity in Lake Malawi cichlid fish

Despite long-standing interest in the evolution and maintenance of discrete phenotypic polymorphisms, the molecular genetic basis of such polymorphism in the wild is largely unknown. Female sex-associated blotched colour polymorphisms found in cichlids of Lake Malawi, East Africa, represent a highly successful polymorphic phenotype, found and maintained in four genera across the geographic expanse of the lake. Previously, we identified an association with an allelic variant of the paired-box transcription factor gene pax7a and blotched colour morphs in Lake Malawi cichlid fishes. Although a diverse range of blotched phenotypes are present in Lake Malawi cichlid species, they all appeared to result from an allele of pax7a that produces increased levels of transcript. Here, we examine the developmental and genetic basis of variation among blotched morphs. First, we confirm that pax7a-associated blotch morphs result primarily from modulation of melanophore development and survival. From laboratory crosses and natural population studies, we identify at least three alleles of pax7a associated with discrete subtypes of blotched morphs, in addition to the ancestral pax7a allele. Genotypes at pax7a support initial evolution of a novel pax7a allele to produce the blotched class of morphs, followed by subsequent evolution of that pax7a blotched allele to produce additional alleles associated with discrete colour morphs. Variant alleles of pax7a produce different levels of pax7a transcript, correlating with pigmentation phenotype at the cellular level. This naturally selected allelic series should serve as a case study for understanding the molecular genetic control of pax7a expression and the evolution of sex-associated alleles.

Interspecific variation in Rx1 expression controls opsin expression and causes visual system diversity in African cichlid fishes.

The mechanisms underlying natural phenotypic diversity are key to understanding evolution and speciation. Cichlid fishes are among the most speciose vertebrates and an ideal model for identifying genes controlling species differences. Cichlids have diverse visual sensitivities that result from species expressing subsets of seven cichlid cone opsin genes. We previously identified a quantitative trait locus (QTL) that tunes visual sensitivity by varying SWS2A (short wavelength sensitive 2A) opsin expression in a genetic cross between two Lake Malawi cichlid species. Here, we identify Rx1 (retinal and anterior neural fold homeobox) as the causative gene for the QTL using fine mapping and RNAseq in retinal transcriptomes. Rx1 is differentially expressed between the parental species and correlated with SWS2A expression in the F2 progeny. Expression of Rx1 and SWS2A is also correlated in a panel of 16 Lake Malawi cichlid species. Association mapping in this panel identified a 413-bp deletion located 2.5-kb upstream of the Rx1 translation start site that is correlated with decreased Rx1 expression. This deletion explains 62% of the variance in SWS2A expression across 53 cichlid species in 29 genera. The deletion occurs in both the sand and rock-dwelling cichlid clades, suggesting that it is an ancestral polymorphism. Our finding supports the hypothesis that mixing and matching of ancestral polymorphisms can explain the diversity of present day cichlid phenotypes.

Evolvability of the Cichlid Jaw: New Tools Provide Insights into the Genetic Basis of Phenotypic Integration

Phenotypic integration is a phenomenon that manifests itself as the covariation among traits, and is thought to substantially influence how evolution unfolds, both in terms of rate and direction, which ultimately determines evolvability. To date little is known about how integration may change across an adaptive radiation, nor do we have a way of determining its genetic basis. Here we sought to test the hypotheses that (1) higher magnitudes of integration are associated with a greater degree of eco-morphological divergence, and (2) integration has a tractable genetic basis. To this end, we first evaluated the magnitude of integration at the population level in the lower jaws of two Lake Malawi cichlid species that exhibit different degrees of trophic specialization. We find that the more eco-morphologically divergent species does indeed exhibit a significantly higher magnitude of integration, which is consistent with our first hypothesis. Next, we developed a new statistical approach based on jackknife pseudovalues to produce a quantitative trait representative of inter-individual variation in the magnitude of integration. This metric was successfully applied to map the genetic basis of integration in the lower jaws of F2 hybrids derived from the two parental species that exhibited differences in the magnitude of integration. We detected three QTLs and two epistatic interactions that contribute to variation in integration within the cichlid mandible. We also detected a single QTL for lower jaw shape. None of the single QTLs for integration identified here overlapped with the interval for lower jaw shape, although one of the epistatic loci for integration did overlap with shape QTL. These results underscore a complex relationship between integration and shape, but suggest largely distinct genetic bases for these two traits. In all, our results show that phenotypic integration has a tractable, yet complex, genetic basis and that we now have the tools available to shed new light on the mechanisms that both promote and limit craniofacial diversity.

THE GENETIC BASIS OF A COMPLEX FUNCTIONAL SYSTEM

The relationship between form and function can have profound effects on evolutionary dynamics and such effects may differ for simple versus complex systems. In particular, functions produced by multiple structural configurations (many-to-one mapping, MTOM) may dampen constituent trade-offs and promote diversification. Unfortunately, we lack information about the genetic architecture of MTOM functional systems. The skulls of teleost fishes contain both simple (lower jaw levers) as well as more complex (jaws modeled as 4-bar linkages) functional systems within the same craniofacial unit. We examined the mapping of form to function and the genetic basis of these systems by identifying quantitative trait loci (QTL) in hybrids of two Lake Malawi cichlid species. Hybrid individuals exhibited novelty (transgressive segregation) in morphological components and function of the simple and complex jaw systems. Functional novelty was proportional to the prevalence of extreme morphologies in the simple levers; by contrast, recombination of parental morphologies produced transgression in the MTOM 4-bar linkage. We found multiple loci of moderate effect and epistasis controlling jaw phenotypes in both the simple and complex systems, with less phenotypic variance explained by QTL for the 4-bar. Genetic linkage between components of the simple and complex systems partly explains phenotypic correlations and may constrain functional evolution.

Constraint and Opportunity: The Genetic Basis and Evolution of Modularity in the Cichlid Mandible

Modular variation, whereby the relative degree of connectivity varies within a system, is thought to evolve through a process of selection that favors the integration of certain traits and the decoupling of others. In this way, modularity may facilitate the pace of evolution and determine evolvability. Alternatively, conserved patterns of modularity may act to constrain the rate and direction of evolution by preventing certain functions from evolving. A comprehensive understanding of the potential interplay between these phenomena will require knowledge of the inheritance and the genetic basis of modularity. Here we explore these ideas in the cichlid mandible by investigating patterns of modularity at the clade and species levels and through the introduction of a new approach, the individual level. Specifically, we assessed patterns of covariation in Lake Malawi cichlid species that employ alternate "biting" and "suction-feeding" modes of feeding and in a hybrid cross between these two ecotypes. Across the suction-feeding clade, patterns of modularity were largely conserved and reflected a functionally based pattern. In contrast, the biting species displayed a pattern of modularity that more closely matched developmental modules. The pattern of modularity present in our F2 population was very similar to the pattern exhibited by the biter, suggesting a role for dominant inheritance. We demonstrate that our individual-level metric of modularity (IMM) is a valid quantitative trait that has a nonlinear relationship with shape. IMMs for each model were used as quantitative characters to map quantitative trait loci (QTL) that underlie modularity. Our QTL analysis offers new insights into the genetic basis of modularity in these fishes that may eventually lead to the discovery of the genetic processes that delineate particular modules. In all, our findings suggest that modularity is both a constraining and an evolvable force in cichlid evolution, as distinct patterns occur between species and variation exists among individuals.

Functional and Genetic Integration in the Skulls of Lake Malawi Cichlids

The level of integration present among organismal traits is thought to influence evolutionary potential, and this potential should be affected by the type or types of integration displayed (e.g., functional, developmental, or genetic). Morphological integration is generally high among functionally related traits, but whether this is predominantly determined by genetic architecture, or is instead a result of biomechanical remodeling during development remains poorly understood. We examine this question in Lake Malawi cichlid fishes by combining a finite-element analysis (FEA) of bite force transmission with quantitative genetic analyses of skull morphology in order to test the hypothesis that functionally coupled traits share a common genetic basis. FEA modeling indicates that the profile of the neurocranium affects its ability to resist forces transmitted from the jaws during biting, and suggests a novel role for skull shape in fish feeding mechanics. Quantitative trait loci mapping demonstrates that the functional integration between jaw and neurocranial shape has a genetic basis, and that this association is being driven by alleles inherited from the specialized biting species. Notably, the co-inheritance of these two functionally related traits in our F2 matches patterns of covariation within and between Lake Malawi cichlid species. Across species, jaw and neurocranial shapes covary, but the trend appears strongest among biting species. Similarly, within populations of biting species, the dimensions of the jaw and neurocranium are tightly linked, whereas this correlation disappears within populations of omnivorous and suction feeding fish. These data suggest (1) that either pleiotropy, or physical linkage maintained by selection, underlies the phenotypic integration of these two functionally related traits, and (2) that this pattern of integration may have influenced the radiation of craniofacial morphology in Lake Malawi cichlids.

Hybridization Promotes Evolvability in African Cichlids: Connections Between Transgressive Segregation and Phenotypic Integration

Hybridization is a potential source of novel variation through (1) transgressive segregation, and (2) changes in the patterns and strength of phenotypic integration. We investigated the capacity of hybridization to generate novel phenotypic variation in African cichlids by examining a large F2 population generated by hybridizing two Lake Malawi cichlid species with differently shaped heads. Our morphometric analysis focused on the lateral and ventral views of the head. While the lateral view exhibited marked transgressive segregation, the ventral view showed a limited ability for transgression, indicating a difference in the genetic architecture and selective history between alternate views of the head. Moreover, hybrids showed a marked reduction in integration, with a lower degree of integration observed in transgressive individuals. In all, these data offer novel insights into how hybridization can promote evolvability, and provide a possible explanation for how broad phenotypic diversity may be achieved in rapidly evolving groups.

A role for male bower size as an intrasexual signal in a Lake Malawi cichlid fish

Disruptive sexual selection through female mate choice has been proposed as a key mechanism driving the evolution of haplochromine cichlid fish diversity. Here we investigated if male secondary sexual traits employed by females in direct mate choice have an additional function in male competition. We conducted a field experiment on aggression among males of a lekking Lake Malawi cichlid species that build volcano-shaped spawning craters, known as bowers, in their breeding territories. Males placed on a short artificial bower received significantly more aggression from adjacent resident males than when placed on a tall artificial bower. This supports the hypothesis that bower size provides information about male competitive ability, and suggests an additional function for this extended phenotypic trait besides a role in attracting females.

Hybridization produces novelty when the mapping of form to function is many to one

BackgroundEvolutionary biologists want to explain the origin of novel features and functions. Two recent but separate lines of research address this question. The first describes one possible outcome of hybridization, called transgressive segregation, where hybrid offspring exhibit trait distributions outside of the parental range. The second considers the explicit mapping of form to function and illustrates manifold paths to similar function (called many to one mapping, MTOM) when the relationship between the two is complex. Under this scenario, functional novelty may be a product of the number of ways to elicit a functional outcome (i.e., the degree of MTOM). We fuse these research themes by considering the influence of MTOM on the production of transgressive jaw biomechanics in simulated hybrids between Lake Malawi cichlid species.ResultsWe characterized the component links and functional output (kinematic transmission, KT) of the 4-bar mechanism in the oral jaws of Lake Malawi cichlids. We demonstrated that the input and output links, the length of the lower jaw and the length of the maxilla respectively, have consistent but opposing relationships with KT. Based on these data, we predicted scenarios in which species with different morphologies but similar KT (MTOM species) would produce transgressive function in hybrids. We used a simple but realistic genetic model to show that transgressive function is a likely outcome of hybridization among Malawi species exhibiting MTOM. Notably, F2 hybrids are transgressive for function (KT), but not the component links that contribute to function. In our model, transgression is a consequence of recombination and assortment among alleles specifying the lengths of the lower jaw and maxilla.ConclusionWe have described a general and likely pervasive mechanism that generates functional novelty. Simulations of hybrid offspring among Lake Malawi cichlids exhibiting MTOM produce transgressive function in the majority of cases, and at appreciable frequency. Functional transgression (i) is a product of recombination and assortment between alleles controlling the lengths of the lower jaw and the maxilla, (ii) occurs in the absence of transgressive morphology, and (iii) can be predicted from the morphology of parents. Our genetic model can be tested by breeding Malawi cichlid hybrids in the laboratory and examining the resulting range of forms and functions.

Species differences in courtship acoustic signals among five Lake Malawi cichlid species (Pseudotropheus spp.)

Male courtship acoustic signals from five Lake Malawi cichlid fish species of the Pseudotropheus zebra complex were recorded and compared. Sounds made by males of P. zebra, Pseudotropheus callainos and the undescribed species known as Pseudotropheus ‘zebra gold’ from Nkhata Bay, and Pseudotropheus emmiltos and Pseudotropheus faizilberi from Mphanga Rocks, differed significantly in the number of pulses and in pulse period. The largest differences in acoustic variables were found among the sympatric Mphanga Rocks species that, in contrast to the other three species, show relatively minor differences in male colour and pattern. These findings suggest that interspecific mate recognition is mediated by multimodal signals and that the mass of different sensory channels varies among sympatric species groups. This study also showed that sound peak frequency was significantly negatively correlated with male size and that sound production rate increased significantly with courtship rate.

Amplified Fragment Length Polymorphism markers reveal population structuring in Dimidiochromis kiwinge (Teleostei: Cichlidae), a commercially-exploited species from Lake Malawi

The understanding of between- and within-population genetic variation and its partitioning on the basis of geographic origin is crucial in designing efficient fishing and conservation strategies of populations and/or species. However, for Lake Malawi's cichlid species, such population genetic studies are hampered by a large amount of genetic variation being shared among populations. To circumvent this problem, it is necessary to use highly polymorphic molecular markers. In this study, Amplified Fragment Length Polymorphism (AFLP) markers were used to assess the genetic diversity within, and among, Dimidiochromis kiwinge populations. AFLP analysis generated a total of 686 bands across all populations, of which 625 (91.1%) were polymorphic. AMOVA (Analysis of Molecular Variance) revealed significant genetic variation, the majority of which (78.7%) resided within populations. All but one of the pairwise fixation indices were significantly different from zero, suggesting limited or no gene flow among populations (overall Fst = 0.2135). Such limited gene flow among populations has vital implications for the management and conservation of this important food resource.

The genetic architecture of male colour differences between a sympatric Lake Malawi cichlid species pair

The genetic basis of traits involved in reproductive isolation is a key parameter in models of sympatric speciation by sexual selection, a potential mechanism driving the explosive radiation of East African cichlids. Analysis of hybrid crosses between two sympatric Lake Malawi cichlid species, representing the extremes of the extant colour distribution, generated Castle-Wright estimates of four to seven loci controlling colour differences. Segregation patterns deviated from a purely additive model with a significant contribution from dominance, and possibly also epistasis. Evidence was found for a strong influence of autosomal loci. As departures from simple additive variation could effect the operation of models of sympatric speciation, dominance and epistasis should not be neglected.

Female preference for conspecific males based on olfactory cues in a Lake Malawi cichlid fish

Research on reproductive isolation in African cichlid fishes has largely focused on the role of nuptial colours, but other sensory modes may play an important role in mate choice. Here, we compare the relative importance of visual and olfactory cues in mate recognition by females of a Lake Malawi cichlid species. Female Pseudotropheus emmiltos were given a choice of spawning next to a conspecific male or a male of the closely-related sympatric Pseudotropheus fainzilberi. Significant preference for conspecific males only occurred when olfactory cues were present. This suggests that divergence of olfactory signals may have been an important influence on the explosive radiation of the East African species flock.

On the origin of Lake Malawi cichlid species: a population genetic analysis of divergence.

The cichlid fishes of Lake Malawi are famously diverse. However, phylogenetic and population genetic studies of their history have been difficult because of the great amount of genetic variation that is shared between species. We apply a recently developed method for fitting the "isolation with migration" divergence model to a data set of specially designed compound loci to develop portraits of cichlid species divergence. Outgroup sequences from a cichlid from Lake Tanganyika permit model parameter estimates in units of years and effective population sizes. Estimated speciation times range from 1,000 to 17,000 years for species in the genus Tropheops. These exceptionally recent dates suggest that Malawi cichlids as a group experience a very active and dynamic diversification process. Current effective population size estimates range form 2,000 to near 40,000, and to >120,000 for estimates of ancestral population sizes. It appears that very recent speciation and gene flow are among the reasons why it has been difficult to discern the phylogenetic history of Malawi cichlids.

Differences in sounds made by courting males of three closely related Lake Malawi cichlid species

Courtship sounds made by three sympatric cichlid species, Pseudotropheus zebra, P. callainos and an undescribed species known as P. ‘zebra gold’ were recorded and compared to investigate the potential role of acoustic signals in mate choice. Sounds were emitted during ‘quiver’ and ‘circle’ components of the male courtship display and consisted of rapidly repeated pulse units. Some sound variables differed significantly among species with P. callainos generally being separated from the other two species. This species produced sounds with higher peak frequency (for a given length) and lower number of pulses than P. ‘zebra gold’ and higher pulse durations than P. zebra. In addition, standard length was inversely related to peak frequency in both P. ‘zebra gold’ and P. callainos(this relation was not tested in P. zebra due to the small sample size). These differences might indicate different regimes of intraspecific sexual selection among the three species.