Genomic architecture of ecologically divergent body shape in a pair of sympatric crater lake cichlid fishes

Determining the genetic bases of adaptations and their roles in speciation is a prominent issue in evolutionary biology. Cichlid fish species flocks are a prime example of recent rapid radiations, often associated with adaptive phenotypic divergence from a common ancestor within a short period of time. In several radiations of freshwater fishes, divergence in ecomorphological traits — including body shape, colour, lips and jaws — is thought to underlie their ecological differentiation, specialization and, ultimately, speciation. The Midas cichlid species complex (Amphilophus spp.) of Nicaragua provides one of the few known examples of sympatric speciation where species have rapidly evolved different but parallel morphologies in young crater lakes. This study identified significant QTL for body shape using SNPs generated via ddRAD sequencing and geometric morphometric analyses of a cross between two ecologically and morphologically divergent, sympatric cichlid species endemic to crater Lake Apoyo: an elongated limnetic species (Amphilophus zaliosus) and a high‐bodied benthic species (Amphilophus astorquii). A total of 453 genome‐wide informative SNPs were identified in 240 F₂ hybrids. These markers were used to construct a genetic map in which 25 linkage groups were resolved. Seventy‐two segregating SNPs were linked to 11 QTL. By annotating the two most highly supported QTL‐linked genomic regions, genes that might contribute to divergence in body shape along the benthic–limnetic axis in Midas cichlid sympatric adaptive radiations were identified. These results suggest that few genomic regions of large effect contribute to early stage divergence in Midas cichlids.     

Crater lake cichlids individually specialize along the benthic–limnetic axis

A common pattern of adaptive diversification in freshwater fishes is the repeated evolution of elongated open water (limnetic) species and high‐bodied shore (benthic) species from generalist ancestors. Studies on phenotype‐diet correlations have suggested that population‐wide individual specialization occurs at an early evolutionary and ecological stage of divergence and niche partitioning. This variable restricted niche use across individuals can provide the raw material for earliest stages of sympatric divergence. We investigated variation in morphology and diet as well as their correlations along the benthic‐limnetic axis in an extremely young Midas cichlid species, Amphilophus tolteca, endemic to the Nicaraguan crater lake Asososca Managua. We found that A. tolteca varied continuously in ecologically relevant traits such as body shape and lower pharyngeal jaw morphology. The correlation of these phenotypes with niche suggested that individuals are specialized along the benthic‐limnetic axis. No genetic differentiation within the crater lake was detected based on genotypes from 13 microsatellite loci. Overall, we found that individual specialization in this young crater lake species encompasses the limnetic‐ as well as the benthic macro‐habitat. Yet there is no evidence for any diversification within the species, making this a candidate system for studying what might be the early stages preceding sympatric divergence.     

REPRODUCTIVE CHARACTER DISPLACEMENT OF MALE STICKLEBACK MATE PREFERENCE: REINFORCEMENT OR DIRECT SELECTION?

Reproductive isolation can evolve between species as a byproduct of adaptation to different niches, through reinforcement, and by direct selection on mating preferences. We investigated the potential role of direct selection in the reproductive isolation between sympatric species of threespine sticklebacks. Each sympatric pair consists of a small-bodied limnetic species and large-bodied benthic species. We compared the mate preferences and courtship behavior of males from one sympatric limnetic population and two allopatric populations. We used limnetic-like allopatric populations to control for the effects of ecological character displacement and adaptation to different niches on mate preferences. The sympatric limnetic males preferred the small limnetic females, whereas the allopatric limnetic-like males preferred the large benthic females, suggesting that adaptation to the limnetic niche does not automatically confer a preference for small limnetic females. This reproductive character displacement of male preference is consistent with the predictions of both reinforcement and direct selection on mate preferences. To test for direct selection, we assessed a prediction of one proposed mechanism: predation by benthic females on eggs guarded by limnetic males. The allopatric males come from populations in which there is no egg predation. Sympatric limnetic males were more aggressive toward benthic females than toward limnetic females, whereas the allopatric limnetic-like males did not treat the two types of females differently. The contrast in male behavior suggests that egg predation has shaped male preferences. Direct selection is potentially more effective than indirect selection via reinforcement, and it is likely that it has been important in building up reproductive isolation between limnetic and benthic sticklebacks.     

Incipient sympatric speciation in Midas cichlid fish from the youngest and one of the smallest crater lakes in Nicaragua due to differential use of the benthic and limnetic habitats?

Understanding how speciation can occur without geographic isolation remains a central objective in evolutionary biology. Generally, some form of disruptive selection and assortative mating are necessary for sympatric speciation to occur. Disruptive selection can arise from intraspecific competition for resources. If this competition leads to the differential use of habitats and variation in relevant traits is genetically determined, then assortative mating can be an automatic consequence (i.e., habitat isolation). In this study, we caught Midas cichlid fish from the limnetic (middle of the lake) and benthic (shore) habitats of Crater Lake Asososca Managua to test whether some of the necessary conditions for sympatric speciation due to intraspecific competition and habitat isolation are given. Lake As. Managua is very small (<900 m in diameter), extremely young (maximally 1245 years of age), and completely isolated. It is inhabited by, probably, only a single endemic species of Midas cichlids, Amphilophus tolteca. We found that fish from the limnetic habitat were more elongated than fish collected from the benthic habitat, as would be predicted from ecomorphological considerations. Stable isotope analyses confirmed that the former also exhibit a more limnetic lifestyle than the latter. Furthermore, split‐brood design experiments in the laboratory suggest that phenotypic plasticity is unlikely to explain much of the observed differences in body elongation that we observed in the field. Yet, neutral markers (microsatellites) did not reveal any genetic clustering in the population. Interestingly, demographic inferences based on RAD‐seq data suggest that the apparent lack of genetic differentiation at neutral markers could simply be due to a lack of time, as intraspecific competition may only have begun a few hundred generations ago.     

Comparison of the prolonged swimming performances of closely related, morphologically distinct three‐spined sticklebacks Gasterosteus spp.

Prolonged swimming performances of two as yet unnamed species of three‐spined stickleback, Gasterosteus spp., were compared. The two fishes (not yet formally described, referred to here as benthic and limnetic) inhabit different niches within Paxton Lake, Texada Island, British Columbia, Canada, and are recent, morphologically distinct species. Limnetics had longer endurance during prolonged swimming than did benthics. The mean regression of the log₁₀ of fatigue time (F_t, s) on swimming speed (U, standard length, L_S s^?1) for limnetics (log₁₀F_t = 7·03 ? 0·46U) had a similar slope, but a significantly higher intercept than that for benthics (log₁₀F_t = 5·55 ? 0·43U). Adult benthics were larger, heavier and deeper‐bodied fish than limnetics. Limnetics, however, had a significantly greater pectoral fin edge:base ratio (mean ± s .e .: limnetics, 4·58 ± 0·43; benthics, 3·63 ± 0·27). In addition, limnetics had significantly lower drag coefficients (C_D) than benthics (limnetics, log₁₀C_D = ?0·49log₁₀R_e + 0·66; benthics, log₁₀C_D = ?0·26log₁₀R_e ? 0·30) where R_e is the Reynolds number [(L_SU (ν^?1), where U and ν are swimming velocity and the kinematic viscosity of the water, respectively]. Compared to their ancestral form, the anadromous three‐spined stickleback Gasterosteus aculeatus L., limnetics and benthics had significantly longer and shorter endurance times, respectively. In addition, both these fishes had significantly higher fast‐start velocities than their ancestral form. Selection due to differential resource use may have lead to divergence of body form, and, therefore, of steady swimming performance. Therefore predation may be the selective force for the similar high escape performance in these two fishes.     

A test of hybrid growth disadvantage in wild, free-ranging species pairs of threespine stickleback (Gasterosteus aculeatus) and its implications for ecological speciation

Ecological speciation is the evolution of reproductive isolation as a direct or indirect consequence of divergent natural selection. Reduced performance of hybrids in nature is thought to be an important process by which natural selection can favor the evolution of assortative mating and drive speciation. Benthic and limnetic sympatric species of threespine stickleback (Gasterosteus aculeatus) are adapted to alternative trophic niches (bottom browsing vs. open water planktivory, respectively) and reduced feeding performance of hybrids is thought to have contributed to the evolution of reproductive isolation. We tested this “hybrid‐disadvantage hypothesis” by inferring growth rates from otoliths sampled from wild, free‐ranging benthic, limnetic, and hybrid sticklebacks in two lakes. There were significant differences in growth rate between lakes, life‐history stages, and among years (maximum P = 0.02), as well as interactions between most factors, but not between hybrid and parental species sticklebacks in most comparisons. Our results provide little evidence of a growth disadvantage in hybrid sticklebacks when free‐ranging in nature. Although trophic ecology per se may contribute less to ecological speciation than envisioned, it may act in concert with other aspects of stickleback biology, such as interactions with parasites, predators, competitors, and/or sexual selection, to present strong multifarious selection against hybrids.     

Genomic signatures of divergent selection and speciation patterns in a ‘natural experiment’, the young parallel radiations of Nicaraguan crater lake cichlid fishes

Divergent selection is the main driving force in sympatric ecological speciation and may also play a strong role in divergence between allopatric populations. Characterizing the genome‐wide impact of divergent selection often constitutes a first step in unravelling the genetic bases underlying adaptation and ecological speciation. The Midas cichlid fish (Amphilophus citrinellus) species complex in Nicaragua is a powerful system for studying evolutionary processes. Independent colonizations of isolated young crater lakes by Midas cichlid populations from the older and great lakes of Nicaragua resulted in the repeated evolution of adaptive radiations by intralacustrine sympatric speciation. In this study we performed genome scans on two repeated radiations of crater lake species and their great lake source populations (1030 polymorphic AFLPs, n ～ 30 individuals per species). We detected regions under divergent selection (0.3% in the crater lake Xiloá flock and 1.7% in the older crater lake Apoyo radiation) that might be responsible for the sympatric diversifications. We find no evidence that the same genomic regions have been involved in the repeated evolution of parallel adaptations across crater lake flocks. However, there is some genetic parallelism apparent (seven out of 51 crater lake to great lake outlier loci are shared; 13.7%) that is associated with the allopatric divergence of both crater lake flocks. Interestingly, our results suggest that the number of outlier loci involved in sympatric and allopatric divergence increases over time. A phylogeny based on the AFLP data clearly supports the monophyly of both crater lake species flocks and indicates a parallel branching order with a primary split along the limnetic‐benthic axis in both radiations.     

Inter- and intra-morph patterns in helminth communities of sympatric whitefish morphs

Infection patterns of trophically transmitted helminth parasites were compared with feeding ecology in two sympatric whitefish Coregonus lavaretus morphs from two lake systems in northern Norway. In both lakes, the pelagic morph was an obligate zooplanktivore, while the benthic morph utilized both the benthivore and zooplanktivore trophic niches. The differences in niche utilization between the two morphs were associated with differences in trophic morphology (gill raker numbers), suggesting that they were genetically dissimilar and reproductively isolated. The benthic morph had the highest number of helminth species, probably because they exhibited a broader niche width compared to the pelagic morph. In both lakes, the species composition and intensities of helminths reflected the trophic diversification of the whitefish ecotypes with respect to different habitat choice (benthic v . pelagic) and dietary specialization (benthivore v . zooplanktivore feeding strategies within the benthic whitefish morph). Zooplanktivorous fish from both morphs acquired parasites mainly from pelagic copepods and in almost equal quantities. The benthivore feeders within the benthic morph had the highest proportion of parasites with transmission stages from benthic organisms. Host feeding behaviour seemed to be a major determinant of the helminth community structure, and helminths appeared to be useful indicators of long-term trophic specialization of whitefish ecotypes.     

SEXUAL SELECTION AGAINST HYBRIDS BETWEEN SYMPATRIC STICKLEBACK SPECIES: EVIDENCE FROM A FIELD EXPERIMENT

Sexual selection against viable, fertile hybrids may contribute to reproductive isolation between recently diverged species. If so, then sexual selection may be implicated in the speciation process. Laboratory measures of the mating success of hybrids may underestimate the amount of sexual selection against them if selection pressures are habitat specific. Male F₁ hybrids between sympatric benthic and limnetic sticklebacks (Gasterosteus aculeatus complex) do not suffer a mating disadvantage when tested in the laboratory. However, in the wild males choose different microhabitats and parental females tend to be found in the same habitats as conspecific males. This sets up the opportunity for sexual selection against male hybrids because they must compete with parental males for access to parental females. To test for sexual selection against adult F₁ hybrid males, we examined their mating success in enclosures in their preferred habitat (open, unvegetated substrate) where limnetic males and females also predominate. We found significantly reduced mating success in F₁ hybrid males compared with limnetic males. Thus, sexual selection, like other mechanisms of postzygotic isolation between young sister species, may be stronger in a wild setting than in the laboratory because of habitat-specific selection pressures. Our results are consistent with, but do not confirm, a role for sexual selection in stickleback speciation.     

Colour plasticity and background matching in a threespine stickleback species pair

Examining differences in colour plasticity between closely‐related species in relation to the heterogeneity of background colours found in their respective habitats may offer important insight into how cryptic colour change evolves in natural populations. In the present study, we examined whether nonbreeding dorsal body coloration has diverged between sympatric species of stickleback along with changes in habitat‐specific background colours. The small, limnetic species primarily occupies the pelagic zone and the large, benthic species inhabits the littoral zone. We placed benthic and limnetic sticklebacks against extremes of habitat background colours and measured their degree of background matching and colour plasticity. Benthics matched the littoral background colour more closely than did the limnetics, although there was no difference between species in their resemblance to the pelagic background colour. Benthics were able to resemble both background colours by exhibiting greater directional colour plasticity in their dorsal body coloration than limnetics, which may be an adaptive response to the greater spectral heterogeneity of the littoral zone. The present study highlights how habitat‐specific spectral characteristics may shape cryptic coloration differences between stickleback species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 902–914.     

Swimming efficiency and the influence of morphology on swimming costs in fishes

Swimming performance is considered a main character determining survival in many aquatic animals. Body morphology highly influences the energetic costs and efficiency of swimming and sets general limits on a species capacity to use habitats and foods. For two cyprinid fishes with different morphological characteristics, carp (Cyprinus carpio L.) and roach (Rutilus rutilus (L.)), optimum swimming speeds (U _mc) as well as total and net costs of transport (COT, NCOT) were determined to evaluate differences in their swimming efficiency. Costs of transport and optimum speeds proved to be allometric functions of fish mass. NCOT was higher but U _mc was lower in carp, indicating a lower swimming efficiency compared to roach. The differences in swimming costs are attributed to the different ecological demands of the species and could partly be explained by their morphological characteristics. Body fineness ratios were used to quantify the influence of body shape on activity costs. This factor proved to be significantly different between the species, indicating a better streamlining in roach with values closer to the optimum body form for efficient swimming. Net swimming costs were directly related to fish morphology.     

Measuring Ucrit and endurance: equipment choice influences estimates of fish swimming performance

This study compared the critical swimming speed (U_crit) and endurance performance of three Australian freshwater fish species in different swim‐test apparatus. Estimates of U_crit measured in a large recirculating flume were greater for all species compared with estimates from a smaller model of the same recirculating flume. Large differences were also observed for estimates of endurance swimming performance between these recirculating flumes and a free‐surface swim tunnel. Differences in estimates of performance may be attributable to variation in flow conditions within different types of swim chambers. Variation in estimates of swimming performance between different types of flumes complicates the application of laboratory‐based measures to the design of fish passage infrastructure.     

EXPERIMENTAL CONFIRMATION THAT BODY SIZE DETERMINES MATE PREFERENCE VIA PHENOTYPE MATCHING IN A STICKLEBACK SPECIES PAIR

Mate choice by phenotype matching, whereby individuals prefer a mate whose phenotype is similar to their own, should facilitate speciation with gene flow. This is because the genes that control mate signal (the phenotype being matched) also determine the preferred mate signal (“mate preference”). Speciation is made even easier if phenotype matching is based on a trait under divergent natural selection. In this case, assortative mating should readily evolve as a byproduct of divergent selection on the trait. Previous observational studies of assortative mating between sympatric, hybridizing threespine stickleback species (Gasterosteus aculeatus complex) suggested that phenotype matching might occur by body size, a trait under divergent natural selection. To test this, we used experimental manipulation of body size to rule out the effects of confounding variables. We found that size‐manipulated benthic and limnetic stickleback females prefer mates whose body size more closely matches their own. It is thus likely that assortative mating by phenotype matching has facilitated the origin and persistence of benthic and limnetic threespine sticklebacks in the face of gene flow.     

Effect of repeated exercise fatigue on the swimming performance of juvenile rock carp (Procypris rabaudi,Tchang)

The swimming performance of juvenile rock carp (Procypris rabaudi, Tchang) subjected to repeated fatigue exercise was studied using a flume-type respirometer at 20°C. The critical swimming speed (U_crit) and oxygen consumption rate (MO₂) of juvenile rock carp were measured during two successive stepped velocity tests, following a 60 min rest interval. U_crit of rock carp was giving a recovery ratio (R_r) of 92.64%, and exertion exercise decreases U_crit. When MO₂ was plotted as a linear function of U, the slope for trial 1 was 1.06 and 1.50 for trial 2, indicating a decreasing in swimming efficiency. The maximum metabolic rate (MMR) increased from 17.06 ± 1.14 mmol O₂/(kg·hr) to 19.14 ± 1.23 mmol O₂/(kg·hr), and the exercise post oxygen consumption rate (EPOC) increased from 9.00 to 9.65 mmol O₂/kg. Repeated fatiguing exercise increased both the aerobic and anaerobic cost of reaching U_crit, but anaerobic metabolism accounted for a larger proportion in the trial 2. The data investigation on the swimming performance and the physiological response to fatigue provide important design criteria for fishways.     

Classification of threespine stickleback along the benthic-limnetic axis

Many species of fish display morphological divergence between individuals feeding on macroinvertebrates associated with littoral habitats (benthic morphotypes) and individuals feeding on zooplankton in the limnetic zone (limnetic morphotypes). Threespine stickleback (Gasterosteus aculeatus L.) have diverged along the benthic-limnetic axis into allopatric morphotypes in thousands of populations and into sympatric species pairs in several lakes. However, only a few well known populations have been studied because identifying additional populations as either benthic or limnetic requires detailed dietary or observational studies. Here we develop a Fisher's linear discriminant function based on the skull morphology of known benthic and limnetic stickleback populations from the Cook Inlet Basin of Alaska and test the feasibility of using this function to identify other morphologically divergent populations. Benthic and limnetic morphotypes were separable using this technique and of 45 populations classified, three were identified as morphologically extreme (two benthic and one limnetic), nine as moderately divergent (three benthic and six limnetic) and the remaining 33 populations as morphologically intermediate. Classification scores were found to correlate with eye size, the depth profile of lakes, and the presence of invasive northern pike (Esox lucius). This type of classification function provides a means of integrating the complex morphological differences between morphotypes into a single score that reflects the position of a population along the benthic-limnetic axis and can be used to relate that position to other aspects of stickleback biology.     

Molecular genetic examination of the polymorphic Arctic charr Salvelinus alpinus of Thingvallavatn, Iceland

Thingvallavatn, Iceland contains two sympatric morphotypes (benthic and limnetic) of Arctic charr Salvelinus alpinus. Each morphotype is composed of two morphs and these differ markedly in ecology, behaviour and life history. We used molecular genetic approaches to test whether (i) genetic heterogeneity exists among morphs and (ii) if morphs arose in allopatry and came into secondary contact or arose sympatrically within the lake through genetic segregation and/or phenotypic plasticity. Direct sequencing of 275 bp of the mitochondrial DNA (mtDNA) control region, mtDNA restriction fragment length polymorphisms and single locus minisatellite analyses detected insufficient variation to test our hypotheses. Analysis of multilocus minisatellite band sharing detected no significant differences between morphs within the same morphotype. However, significant differences among morphs belonging to different morphotypes suggest some genetic heterogeneity in Thingvallavatn charr. Limnetic charr from Thingvallavatn were more similar to sympatric benthic charr than to allopatric limnetics from two other Icelandic lakes. This suggests that the Thingvallavatn morphs arose sympatrically within the lake rather than in allopatry followed by secondary contact.     

Three carnivorous plant species (Drosera spp.) in European Russia: peaceful coexistence?

In spite of the recent improvements in the understanding of carnivorous plants’ biology, some questions have remained unanswered. In this study, the segregation of food niches (i.e. specialization on different categories of prey) for three sympatric carnivorous temperate Drosera species with different shapes of trapping leaves is tested. Potentially available prey was also taken into account, by using artificial traps. Almost all the prey trapped by the three Drosera species and by passive traps belonged to four insect orders: Diptera, Hymenoptera, Coleoptera, Homoptera, as well as Araneae. Diptera specimens were the main prey for all the species. This study demonstrates that arthropods caught by the three temperate sympatric sundew species (D. rotundifolia, D. obovata and D. anglica), belong to the same orders. The proportions of prey from different orders, caught by different sundew species did not differ significantly. The result does not necessarily imply the absence of interspecific competition for prey: arthropods were identified only to order, and competition may have resulted in specialization on prey taxa of lower rank.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION DURING SPECIATION‐WITH‐GENE‐FLOW IN LAKE WHITEFISH SPECIES PAIRS ASSESSED BY RAD SEQUENCING

During speciation‐with‐gene‐flow, effective migration varies across the genome as a function of several factors, including proximity of selected loci, recombination rate, strength of selection, and number of selected loci. Genome scans may provide better empirical understanding of the genome‐wide patterns of genetic differentiation, especially if the variance due to the previously mentioned factors is partitioned. In North American lake whitefish (Coregonus clupeaformis), glacial lineages that diverged in allopatry about 60,000 years ago and came into contact 12,000 years ago have independently evolved in several lakes into two sympatric species pairs (a normal benthic and a dwarf limnetic). Variable degrees of reproductive isolation between species pairs across lakes offer a continuum of genetic and phenotypic divergence associated with adaptation to distinct ecological niches. To disentangle the complex array of genetically based barriers that locally reduce the effective migration rate between whitefish species pairs, we compared genome‐wide patterns of divergence across five lakes distributed along this divergence continuum. Using restriction site associated DNA (RAD) sequencing, we combined genetic mapping and population genetics approaches to identify genomic regions resistant to introgression and derive empirical measures of the barrier strength as a function of recombination distance. We found that the size of the genomic islands of differentiation was influenced by the joint effects of linkage disequilibrium maintained by selection on many loci, the strength of ecological niche divergence, as well as demographic characteristics unique to each lake. Partial parallelism in divergent genomic regions likely reflected the combined effects of polygenic adaptation from standing variation and independent changes in the genetic architecture of postzygotic isolation. This study illustrates how integrating genetic mapping and population genomics of multiple sympatric species pairs provide a window on the speciation‐with‐gene‐flow mechanism.     

The physiology of rapid ecological specialization: A look at the Midas cichlids

Understanding the process of speciation is a primary goal of evolutionary biology, yet the question of whether speciation can reach completion in the presence of gene flow remains controversial. For more than 50 years, the cichlids of Africa, and more recently those in South and Central America, have served as model systems for the study of speciation in nature. Cichlids are distinguished by their enormous species richness, their diversity of behavioural and trophic adaptations, and their rapid rate of divergence. In both Africa and South and Central America, the repeated interaction of geology, new founder events and adaptive evolution has created a series of natural experiments with speciation occurring both within and between waterbodies of differing ages. In the “From the Cover” paper in this issue of the Journal of Molecular Ecology, Raffini, Schneider, Franchini, Kautt and Meyer move beyond the question of which mechanisms drive speciation, and instead show that divergent morphologies and physiologies translate into adaptive traits. They investigate differences in physiology and gene expression profiles in a benthic/limnetic species pair of Midas cichlidsin a 24,000‐year‐old Nicaraguan crater lake. While recently diverged, these two species demonstrate significant ecological, but limited genetic differentiation. The authors find that the distinct morphotypes translate into relevant differences in swimming performance and metabolic rates that correspond to differential gene expression profiles. Hence, the authors take an integrative approach examining the impacts of morphological differences on performance and niche partitioning: an approach that can advance our understanding of the drivers of morphological and physiological divergence during speciation.