Standing genetic variation as a potential mechanism of novel cave phenotype evolution in the freshwater isopod,Asellus aquaticus

Novel phenotypes can come about through a variety of mechanisms including standing genetic variation from a founding population. Cave animals are an excellent system in which to study the evolution of novel phenotypes such as loss of pigmentation and eyes. Asellus aquaticus is a freshwater isopod crustacean found in Europe and has both a surface and a cave ecomorph which vary in multiple phenotypic traits. An orange eye phenotype was previously revealed by F₂ crosses and backcrosses to the cave parent within two examined Slovenian cave populations. Complete loss of pigmentation, both in eye and body, is epistatic to the orange eye phenotype and therefore the orange eye phenotype is hidden within the cave populations. Our goal was to investigate the origin of the orange eye alleles within the Slovenian cave populations by examining A. aquaticus individuals from Slovenian and Romanian surface populations and Asellus aquaticus infernus individuals from a Romanian cave population. We found orange eye individuals present in lab raised surface populations of A. aquaticus from both Slovenia and Romania. Using a mapping approach with crosses between individuals of two surface populations, we found that the region known to be responsible for the orange eye phenotype within the two previously examined Slovenian cave populations was also responsible within both the Slovenian and the Romanian surface populations. Complementation crosses between orange eye Slovenian and orange eye Romanian surface individuals suggest that the same gene is responsible for the orange eye phenotype in both surface populations. Additionally, we observed a low frequency phenotype of eye loss in crosses generated between the two surface populations and also in the Romanian surface population. Finally, in a cave population from Romania, A. aquaticus infernus, we found that the same region is also responsible for the orange eye phenotype as the Slovenian cave populations and the Slovenian and Romanian surface populations. Therefore, we present evidence that variation present in the cave populations could originate from standing variation present in the surface populations and/or transgressive hybridization of different surface phylogenetic lineages rather than de novo mutations.     

Parallel speciation in Astyanax cave fish (Teleostei) in Northern Mexico

We investigated differentiation processes in the Neotropical fish Astyanax that represents a model system for examining adaptation to caves, including regressive evolution. In particular, we analyzed microsatellite and mitochondrial data of seven cave and seven surface populations from Mexico to test whether the evolution of the cave fish represents a case of parallel evolution. Our data revealed that Astyanax invaded northern Mexico across the Trans-Mexican Volcanic Belt at least three times and that populations of all three invasions adapted to subterranean habitats. Significant differentiation was found between the cave and surface populations. We did not observe gene flow between the strongly eye and pigment reduced old cave populations (Sabinos, Tinaja, Pachon) and the surface fish, even when syntopically occurring like in Yerbaniz cave. Little gene flow, if any, was found between cave populations, which are variable in eye and pigmentation (Micos, Chica, Caballo Moro caves), and surface fish. This suggests that the variability is due to their more recent origin rather than to hybridization. Finally, admixture of the young Chica cave fish population with nuclear markers from older cave fish demonstrates that gene flow between populations that independently colonized caves occurs. Thus, all criteria of parallel speciation are fulfilled. Moreover, the microsatellite data provide evidence that two co-occurring groups with small sunken eyes and externally visible eyes, respectively, differentiated within the partly lightened Caballo Moro karst window cave and might represent an example for incipient sympatric speciation.     

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish

Background

How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression.

Results

Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN.

Conclusions

We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

     

Toxic hydrogen sulfide and dark caves: phenotypic and genetic divergence across two abiotic environmental gradients in Poecilia mexicana

Divergent natural selection drives evolutionary diversification. It creates phenotypic diversity by favoring developmental plasticity within populations or genetic differentiation and local adaptation among populations. We investigated phenotypic and genetic divergence in the livebearing fish Poecilia mexicana along two abiotic environmental gradients. These fish typically inhabit nonsulfidic surface rivers, but also colonized sulfidic and cave habitats. We assessed phenotypic variation among a factorial combination of habitat types using geometric and traditional morphometrics, and genetic divergence using quantitative and molecular genetic analyses. Fish in caves (sulfidic or not) exhibited reduced eyes and slender bodies. Fish from sulfidic habitats (surface or cave) exhibited larger heads and longer gill filaments. Common-garden rearing suggested that these morphological differences are partly heritable. Population genetic analyses using microsatellites as well as cytochrome b gene sequences indicate high population differentiation over small spatial scale and very low rates of gene flow, especially among different habitat types. This suggests that divergent environmental conditions constitute barriers to gene flow. Strong molecular divergence over short distances as well as phenotypic and quantitative genetic divergence across habitats in directions classic to fish ecomorphology suggest that divergent selection is structuring phenotypic variation in this system.     

Convergent Adaptations to Cave Life in the Rhamdia Laticauda Catfish Group (Pimelodidae, Teleostei)

The nocturnal Mexican catfish Rhamdia laticauda (Pimelodidae, Teleostei) is the surface sister species of a number of cave species. Comparison between two of them, R. zongolicensis and R. reddelli, reveals an intermediate state of reduction of the biologically functionless eyes, melanin pigmentation and the negative phototactic behavior. The surface species is perfectly adapted to life in darkness. Therefore only few constructive adaptations are developed in the cave species. For survival under conditions of low food supply in the caves the barbels are elongated to improve the senses of taste and touch and more fat can be deposited in the cave fish tissue. Due to convergent evolution the two cave species are morphologically much alike and show only minor diagnostic meristic differences. From geological data it can be concluded that the two cave species started troglobitic evolution at the end of Pleistocene.     

Evolution of Eye Regression in the Cavefish Astyanax: Apoptosis and the Pax-6 Gene

SYNOPSIS. The eye is an extraordinary organ in terms of itsdevelopment and evolution. In cave animals, the eye is sometimesreduced or eliminated as a consequence of adaptation to lifein perpetual darkness. We have used the characid teleost Astyanaxmexicanus as a model system to investigate the mechanisms ofeye degeneration during the evolution of a cave vertebrate.Eyed surface populations of Astyanax entered caves during thePleistocene, and their descendants lost their eyes and pigmentation.Astyanax populations exhibiting various degrees of eye regressionhave been reported in 29 Mexican caves. Surface populationswith characteristics of the ancestral stock still exist in thevicinity of these caves. Thus, Astyanax represents one of thefew instances in which the ancestral (surface fish) and thederived (cavefish) developmental modes are extant and availablefor comparative studies. The cavefish embryo develops an opticprimordium consisting of a lens vesicle and optic cup but therudimentary eye arrests in development and degenerates. Herewe report that eye degeneration is accompanied by extensiveapoptosis and downregulation of the Pax-6 gene in the developinglens. The results suggest that alterations in lens developmentare important factors in eye regression during cavefish evolution.     

Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation

More than 20 populations of the cave-dwelling characid Astyanax occur within a restricted karst area in Mexico. The fish possess reduced eyes without lenses and visual cells. It is still an open question as to whether this condition evolved convergently after multiple entries of the surface ancestor into the different caves or whether a single cave ancestor, already characterized by reduced eyes, spread secondarily within them. In the crosses between specific populations, specimens appear that deviate considerably from those of the parents. They possess larger and better-developed eyes with histologically intact lenses and visual cells; they thus have the structural potential for vision. This indicates that in different cave populations, different mutations in the eye gene system have occurred. In cases where these non-functional rudimentary genes are recombined in hybrid specimens, gene expression may be restored. This is the result of separate evolution of several Astyanax cave populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 545−554.     

Parallels between two geographically and ecologically disparate cave invasions by the same species,Asellus aquaticus (Isopoda,Crustacea)

Caves are long‐known examples of evolutionary replications where similar morphologies (troglomorphies) evolve independently as the result of strong natural selection of the extreme environment. Recently, this paradigm has been challenged based on observations that troglomorphies are inconsistent across taxa and different subterranean habitats. We investigated the degree of replicated phenotypic change in two independent cave invasions by the freshwater isopod Asellus aquaticus; the first in a sulphidic aquifer in Romania, the second in a sinking river in the Dinaric Karst in Slovenia. Both ancestral surface populations still live alongside the subterranean ones. Phylogenetic analyses show independence of the two colonization events, and microsatellite analysis shows no evidence of ongoing genetic exchange between surface and subterranean ecomorphs. The overall morphology has changed dramatically at both sites (50 of 62 morphometric traits). The amount of phenotypic change did not reflect differences in genetic diversity between the two ancestral populations. Multivariate analyses revealed divergent evolution in caves, not parallel or convergent as predicted by the current paradigm. Still, 18 traits changed in a parallel fashion, including eye and pigment loss and antennal elongation. These changes might be a consequence of darkness as the only common ecological feature, because Romanian caves are chemoautotrophic and rich in food, whereas Slovenian caves are not. Overall, these results show that morphologically alike surface populations can diverge after invading different subterranean habitats, and that only about one‐third of all changing traits behave as troglomorphies in the traditional sense.     

Adaptive evolution of lateral plates in three‐spined stickleback Gasterosteus aculeatus: a case study in functional analysis of natural variation

The three‐spined stickleback Gasterosteus aculeatus is a model species for studying questions in ecology and evolution. The rapid diversification of G. aculeatus in post‐glacial freshwater environments, combined with recently developed molecular tools, provides a unique opportunity to study the functional basis of fitness variation in natural populations. In derived freshwater populations, a number of morphological traits have diverged in parallel from the marine ancestral state, including the number of lateral armour plates. Evolution of reduced armour in freshwater populations is due to positive selection from both abiotic and biotic mechanisms. The major effect gene (ectodysplasin‐A or Eda), along with several minor effect genetic regions, has recently been shown to control lateral plate variation. Field experiments have further determined the fitness consequences of allelic variation at the major effect locus. This work helps elucidate the mechanisms connecting genetic variation with phenotypic variation and fitness in the wild, a synthesis that should be applicable to many other phenotypic traits and species of fishes.     

Potential rapid evolution of foot morphology in Italian plethodontid salamanders (Hydromantes strinatii) following the colonization of an artificial cave

How organisms respond to environmental change is a long‐standing question in evolutionary biology. Species invading novel habitats provide an opportunity to examine contemporary evolution in action and decipher the pace of evolutionary change over short timescales. Here, we characterized phenotypic evolution in the Italian plethodontid salamander, Hydromantes strinatii, following the recent colonization of an artificial cave by a forest floor population. When compared with a nearby and genetically related population in the natural forest floor and a nearby cave population, the artificial cave population displayed significant differences in overall foot shape, with more interdigital webbing relative to the other populations. Further, this population evolved significantly larger feet, which corresponded more closely to those found in other cave populations than to forest floor populations to which the cave population is closely related. Finally, we quantified the rate of evolution for both foot shape and foot area, and found that both traits displayed large and significant evolutionary rates, at levels corresponding to other classic cases of rapid evolution in vertebrates. Together, these findings reveal that the response to novel environmental pressures can be large and rapid and that the anatomical shifts observed in the artificial cave population of H. strinatii may represent a case of rapid evolution in response to novel environmental pressures.     

Rapid evolution in response to introduced predators II: the contribution of adaptive plasticity

Background  

Introductions of non-native species can significantly alter the selective environment for populations of native species, which can respond through phenotypic plasticity or genetic adaptation. We examined phenotypic and genetic responses of Daphnia populations to recent introductions of non-native fish to assess the relative roles of phenotypic plasticity versus genetic change in causing the observed patterns. The Daphnia community in alpine lakes throughout the Sierra Nevada of California (USA) is ideally suited for investigation of rapid adaptive evolution because there are multiple lakes with and without introduced fish predators. We conducted common-garden experiments involving presence or absence of chemical cues produced by fish and measured morphological and life-history traits in Daphnia melanica populations collected from lakes with contrasting fish stocking histories. The experiment allowed us to assess the degree of population differentiation due to fish predation and examine the contribution of adaptive plasticity in the response to predator introduction.     

Phenotyping approaches for physiological breeding and gene discovery in wheat

Conceptual models of drought‐adaptive traits have been used in breeding to accumulate complementary physiological traits (PT) in selected progeny, resulting in distribution of advanced lines to rain‐fed environments worldwide by the International Maize and Wheat Improvement Center (CIMMYT). Key steps in PT breeding at CIMMYT include characterisation of crossing block lines for stress adaptive mechanisms, strategic crossing among parents that encompass as many target traits as possible and early generation selection (EGS) of bulks for canopy temperature (CT). The approach has been successful using both elite × elite crosses as well as three way crosses involving stress adapted landraces. Other EGS techniques that are amenable to high throughput include measurement of spectral reflectance indices and stomatal aperture‐related traits. Their genetic‐ and cost‐effectiveness are supported by realisation of genetic yield gains in response to trait selection, and by economic analysis, respectively. Continual reselection within restricted gene pools is likely to lead to diminishing returns, however, exotic parents can be used to introduce new allelic diversity. Examples include landraces from the primary gene pool, and products of inter‐specific hybridisation with the secondary gene pool consisting of closely related wheat genomes. Both approaches have been successful in introducing stress‐adaptive traits. The main problem with knowing which genetic resource to use in wide‐crossing is the uncertainty with which phenotypic expression can be extrapolated from one genome/genepool to another because of their unimproved or undomesticated genetic backgrounds. Nonetheless, their PT expression can be measured and used as a basis for investing in crossing or wide crossing. Discovering the genetic basis of PT is highly complex because putative QTLs may interact with environment and genetic background, including genes of major effect. Detection of QTLs was improved in mapping populations where flowering time was controlled, while new mapping populations have been designed by screening potential parents that do not contrast in the Rht, Ppd and Vrn alleles. Association genetics mapping is another approach that can be employed for gene discovery using exclusively agronomically improved material, thereby minimising the probability of identifying yield QTLs whose alleles have been already improved by conventional breeding.     

A New Blind Cave Fish Population of Genus Astyanax: Geography, Morphology and Behavior

A new population of blind, cave dwelling tetra fish of the genus Astyanax was discovered in Granadas Cave, in the Balsas drainage, southern México. All blind Mexican tetras previously described are from Tampico and San Luis Potosí, northern México. The discovery of a new blind morph thus represents an independent colonization and convergent adaptation to the cave environment by this fish. Individuals of this population display variability of their troglomorphic features. Some individuals presented asymmetrical degeneration of the eyes, where one was normal, but the other somewhat reduced in size and complexity. Loss of pigmentation and eye reduction, although sometimes correlated, were not always linked; reduced eyes were found on pigmented fish and unpigmented fish often possessed normal eyes. Some individuals had reduced lens size or an absence of lens altogether. Retina is highly modified with photoreceptors sometimes absent. Eye reduction was correlated with a diminished size of the optic lobes and an increase of the prosencephalon. Modifications of the skull involve closing in of the circumorbital series of bones. Certain aspects of behavior are also modified.     

Parasite‐mediated selection in a natural metapopulation of Daphnia magna

Parasite‐mediated selection varying across time and space in metapopulations is expected to result in host local adaptation and the maintenance of genetic diversity in disease‐related traits. However, nonadaptive processes like migration and extinction‐(re)colonization dynamics might interfere with adaptive evolution. Understanding how adaptive and nonadaptive processes interact to shape genetic variability in life‐history and disease‐related traits can provide important insights into their evolution in subdivided populations. Here we investigate signatures of spatially fluctuating, parasite‐mediated selection in a natural metapopulation of Daphnia magna. Host genotypes from infected and uninfected populations were genotyped at microsatellite markers, and phenotyped for life‐history and disease traits in common garden experiments. Combining phenotypic and genotypic data a Q_ST–F_ST‐like analysis was conducted to test for signatures of parasite mediated selection. We observed high variation within and among populations for phenotypic traits, but neither an indication of host local adaptation nor a cost of resistance. Infected populations have a higher gene diversity (Hs) than uninfected populations and Hs is strongly positively correlated with fitness. These results suggest a strong parasite effect on reducing population level inbreeding. We discuss how stochastic processes related to frequent extinction‐(re)colonization dynamics as well as host and parasite migration impede the evolution of resistance in the infected populations. We suggest that the genetic and phenotypic patterns of variation are a product of dynamic changes in the host gene pool caused by the interaction of colonization bottlenecks, inbreeding, immigration, hybrid vigor, rare host genotype advantage and parasitism. Our study highlights the effect of the parasite in ameliorating the negative fitness consequences caused by the high drift load in this metapopulation.     

Phenotypic plasticity and karyotype conservation in allopatric populations of Astyanax aff. fasciatus (Teleostei,Characiformes) at the Upper Paraná river basin

Genus Astyanax is known by its wide genetic and morphological diversity. Among the South America's fish, it is one of the most studied groups and with greatest number of characterized population. At the present work, the combined approach of morphological and genetic data analysis was applied. Cytogenetic and morphometric studies were performed in populations of Astyanax aff. fasciatus originating from Paranaíba river and São João river, both from the hydrographic basin of Paranaíba river. Fish originating from a third place at the Mogi‐Guaçu river (Tietê river's basin) and with 2n = 46 chromosomes also went through morphometric analysis. The morphologic data evidenced the populational structuring, following the geographical distribution of the subjects. Cytogenetic data disclosed the same diploid number and karyotype formula for both populations. The other cytogenetic markers comply with the literature. These data show that the two populations of A. aff. fasciatus do not differ among themselves concerning the chromosomal structure, but they structure themselves regarding morphometry. Aspects of evolution of the group are discussed facing this study.     

Phenotypic integration between antipredator behavior and camouflage pattern in juvenile sticklebacks

Predation is a strong selective force that promotes the evolution of antipredator behaviors and camouflage in prey animals. However, the independent evolution of single traits cannot explain how observed phenotypic variations of these traits are maintained within populations. We studied genetic and phenotypic correlations between antipredator behaviors (shoaling and risk‐taking) and morphology traits (pigmentation and size) in juvenile three‐spined sticklebacks by using pedigree‐based quantitative genetic analysis to test phenotypic integration (or complex phenotype) as an evolutionary response to predation risk. Individuals with strongly melanized (i.e., camouflaged) phenotype and genotype were less sociable to conspecifics, but bolder during foraging under predation risk. Individuals with faster growing phenotype and genotype were bolder, and those with lager eyes were more fearful. These phenotypic integrations were not confounded with correlated plastic responses to predation risk because the phenotypes were measured in naïve fish born in the laboratory, but originated from a natural population with predation pressure. Consistent selection for particular combinations of traits under predation pressure or pleiotropic genes might influence the maintenance of the genetic (co)variations and polymorphism in melanin color, growth trajectory, and behavior patterns.     

Diversifying selection drives parallel evolution of gill raker number and body size along the speciation continuum of European whitefish

Adaptive radiation is the evolution of ecological and phenotypical diversity. It arises via ecological opportunity that promotes the exploration of underutilized or novel niches mediating specialization and reproductive isolation. The assumed precondition for rapid local adaptation is diversifying natural selection, but random genetic drift could also be a major driver of this process. We used 27 populations of European whitefish (Coregonus lavaretus) from nine lakes distributed in three neighboring subarctic watercourses in northern Fennoscandia as a model to test the importance of random drift versus diversifying natural selection for parallel evolution of adaptive phenotypic traits. We contrasted variation for two key adaptive phenotypic traits correlated with resource utilization of polymorphic fish; the number of gill rakers and the total length of fish, with the posterior distribution of neutral genetic differentiation from 13 microsatellite loci, to test whether the observed phenotypic divergence could be achieved by random genetic drift alone. Our results show that both traits have been under diversifying selection and that the evolution of these morphs has been driven by isolation through habitat adaptations. We conclude that diversifying selection acting on gill raker number and body size has played a significant role in the ongoing adaptive radiation of European whitefish morphs in this region.