Genetics and hybridization in surface and cave Astyanax (Teleostei): a comparison of regressive and constructive traits

Change in ecological conditions, as seen in surface and cave populations of Astyanax (Teleostei), has caused the divergent evolution of a large number of traits like eyes, coloration, taste, lateral line, and different kinds of behaviour like schooling, sleep or feeding posture. Because of the interfertility of surface and cave forms these fish are an exceptional object to study the morphological and genetic basis of the evolution of such complex regressive and constructive traits. Classical crossing analyses and genomic studies are contributing to growing understanding. Both kinds of traits mostly rely on multiple genetic bases and the phenotypic manifestation in the various crosses is similar. The gene effect underlying the phenotypic manifestation may exhibit an exponential increase at differing amounts in the various traits and crosses. Missing or presence of such genetic interaction helps determine whether the variability of eyes or pigmentation exhibited by Astyanax cave fish populations like Micos, is due to a more recent origin or to secondary hybridization with the surface fish. Neither crossing analysis nor QTL mapping revealed that eye reduction is pleiotropically antagonistically related to the increase of taste buds or lateral line sense. Independent inheritance of traits suggests that Astyanax cave fish are subjected to mosaic evolution.     

Developmental constraints in regressive evolution: studies of the expression of the γs-crystallin gene in the developing lens of cave-dwelling Astyanax fasciatus (Cuvier, 1819) (Teleostei, Characidae) by in situ hybridization

The early morphogenesis of the lens and the expression of the γs-crystallin gene was studied in epigean Astyanax fasciatus and its cave-dwelling derivative. At early stages, the lens of the cave fish develops in a way that is similar to the epigean form. Later, the developmental timing is delayed and growth ceases in the cave-fish lens. With the beginning of cytodifferentiation, the development of the lens breaks down. Crystallin lens fibres are not produced at any time and the γs-crystallin gene, which is transcribed during a limited period in the lens of epigean fishes, is not active in cave specimens. This study confirms earlier immunofluorescence observations that demonstrated the lack of crystallin proteins in the cave-fish lens, but is in contrast to results on the blind mole rat, which showed a persistence of functioning crystallins in the degenerated lens of this species. The significance of developmental constraints in regressive evolution is discussed.     

Regressive evolution in the Mexican cave tetra, Astyanax mexicanus

The evolutionary forces driving the reduction of eyes and pigmentation in cave-adapted animals are unknown; Darwin famously questioned the role of natural selection in eye loss in cave fishes: "As it is difficult to imagine that eyes, although useless, could be in any way injurious to animals living in darkness, I attribute their loss wholly to disuse"[1]. We studied the genetics of eye and pigmentation regression in the Mexican cave tetra, Astyanax mexicanus, by mapping and quantitative trait loci (QTL) analysis. We also mapped QTL for the putatively constructive traits of jaw size, tooth number, and numbers of taste buds. The data suggest that eyes and pigmentation regressed through different mechanisms. Cave alleles at every eye or lens QTL we detected caused size reductions, consistent with evolution by natural selection but not with drift. QTL polarities for melanophore number were mixed, however, consistent with genetic drift. Arguments against a role for selection in the regression of cave-fish eyes cited the insignificant cost of their development [2, 3], but we argue that the energetic cost of their maintenance is sufficiently high for eyes to be detrimental in the cave environment. Regression can be caused either by selection or drift.     

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.     

Cavefish as a model system in evolutionary developmental biology

The Mexican tetra Astyanax mexicanus has many of the favorable attributes that have made the zebrafish a model system in developmental biology. The existence of eyed surface (surface fish) and blind cave (cavefish) dwelling forms in Astyanax also provides an attractive system for studying the evolution of developmental mechanisms. The polarity of evolutionary changes and the environmental conditions leading to the cavefish phenotype are known with certainty, and several different cavefish populations have evolved constructive and regressive changes independently. The constructive changes include enhancement of the feeding apparatus (jaws, taste buds, and teeth) and the mechanosensory system of cranial neuromasts. The homeobox gene Prox 1, which is expressed in the expanded taste buds and cranial neuromasts, is one of the genes involved in the constructive changes in sensory organ development. The regressive changes include loss of pigmentation and eye degeneration. Although adult cavefish lack functional eyes, small eye primordia are formed during embryogenesis, which later arrest in development, degenerate, and sink into the orbit. Apoptosis and lens signaling to other eye parts, such as the cornea, iris, and retina, result in the arrest of eye development and ultimate optic degeneration. Accordingly, an eye with restored cornea, iris, and retinal photoreceptor cells is formed when a surface fish lens is transplanted into a cavefish optic cup, indicating that cavefish optic tissues have conserved the ability to respond to lens signaling. Genetic analysis indicates that multiple genes regulate eye degeneration, and molecular studies suggest that Pax6 may be one of the genes controlling cavefish eye degeneration. Further studies of the Astyanax system will contribute to our understanding of the evolution of developmental mechanisms in vertebrates.     

One eye but no vision: cave fish with induced eyes do not respond to light

One of the most intriguing questions in evolutionary biology is the degree to which behavior is a necessary consequence of morphology. We explore this issue by examining phototactic behavior in epigean (eyed surface-dwelling) and troglomorphic (blind cave) forms of the teleost Astyanax fasciatus whose eyes were modified during embryogenesis by removing one or both lens vesicles from the epigean form or by transplanting the lens vesicle from an epigean fish into the optic cup of a blind cave form. Lens removal results in eye degeneration and blindness in adult epigean fish, whereas lens transplantation stimulates growth of the eye, inducing the development of optic tissues in the normally eyeless adult cave fish. Photoresponsiveness was examined by placing fish in an aquarium with one half illuminated and the other half dark and scoring their presence in the illuminated or dark half. Both the eyeless epigean fish and cave fish with induced eyes are indifferent to the illumination whereas the surface forms are scotophilic, suggesting that optic development and phototactic behavior are decoupled.     

A review of Morphological and Behavioural Changes in the Cave Molly, Poecilia Mexicana, from Tabasco, Mexico

The poeciliid fish Poecilia mexicana successfully colonised a sulfur cave in Tabasco, Mexico. The eye size, melanin pigmentation and scale tuberances as well as aggressive and schooling behaviour are reduced in the cave fish. Besides these regressive characters some constructive ones have been found. The genital region of the female is enlarged to a genital pad and in both sexes the number of taste buds is considerably increased on the upper side of the head. The cephalic lateral line shows a hyperdevelopment with an incomplete covering of some channels. Nearly all of these characters studied form a genetically based gradient from the beginning to the end of the cave, which seems to be the result of gene flow from the outside to the inner part of the cave. A special behavioural adaptation was found among females which prefer bigger males during courtship. In darkness, only the cave females show this preference.     

Mapping a cave fish genome: polygenic systems and regressive evolution

We used random amplified polymorphic DNA (RAPD) fingerprinting to generate anonymous DNA markers in the fish Astyanax mexicanus, a species with both surface and cave populations. Surface individuals are eyed and pigmented; troglobitic forms are blind and depigmented. We hybridized surface fish and Pachon population cave fish and produced a RAPD genomic map 1064 cM in length (about half the total length of the genome) that was used to screen for quantitative trait loci (QTL) for troglomorphic traits. Three QTL for reduced eye size, two for decreased numbers of melanophores, two for condition factor, and the locus for the unifactorial trait, albinism, were mapped. These factors account for an average of 46% of the variance in these traits in the backcross. The results are the first direct demonstration that troglomorphic changes in this population are multifactorial. Two closely linked pairs of QTL were found. Each consisted of a regressive and a constructive trait QTL. These close linkages are unlikely to have occurred by chance (P <.05 for each) and suggest that troglomorphic evolution might be facilitated by pleiotropy or by genetic hitchhiking.     

Losing Sight of Regressive Evolution

When we teach evolution to our students, we tend to focus on “constructive” evolution, the processes which lead to the development of novel or modified structures. Most biology students are familiar with the subjects of finches’ beaks, giraffes’ necks, and hair in mammals. Of course, there is nothing inherently wrong with a constructivist approach to teaching evolution, but if it is our only focus, we may overlook the flip side of the coin. By the flip side of the coin, of course, we are referring to regressive evolution: the loss or degeneration of a trait. Regressive evolution does not often make its way into biology textbooks, but it is of great relevance nonetheless. In all likelihood, when a new trait evolves or an existing one is modified, something is sacrificed in return. In order to develop a flipper, a marine mammal must sacrifice individual digits. You may be familiar with one or more of the following familiar characters lost through regressive evolution: teeth in birds, scales in mammals, and tails in higher primates. For aficionados of cave biology like us, one of the most interesting examples of regressive evolution concerns cave fish: Why do cave fish lose their eyes?     

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.     

Comparative Ultrastructural Studies on the Pineal Organ of the Mexican Catfish Rhamdia laticauda Heckel, 1858 and One of its Cave-dwelling Derivates (Pimelodidae,Teleostei)

The pineal organ of the epigean catfish Rhamdia laticauda and of its cave-dwelling derivate from Cueva del Tunel in Zongolica, Mexico, was examined ultrastructurally. The pineal of the epigean form is relatively small but ultrastructurally well differentiated and characterized as a metabolically active, photoneuroendocrine organ. The outer segments of the sensory cells are composed of 32–55 very regularly arranged sacculi which are obviously functioning in photoreception. Furthermore, the presence of electron-dense vesicles, free ribosomes, conspicuously well-developed Golgi bodies, and large amounts of rough and smooth endoplasmic reticulum in the cytoplasm provide evidence for a secretory activity of the sensory cells. The only regressive traits in the pineal of the cave form regard the outer segments: there are few disintegrated, whorl-like outer segments and the number of sacculi exhibits an increased variability. Compared with other morphological structures dependent on light, for example the eyes, the degree of regression is very low. It is suggested that this is due to a remaining secretory role of the sensory cells, which prevents the functionless, photoreceptive outer segments from rapid degeneration by developmental constraints.     

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic 'hotspots' of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.     

Niche partitioning in orbweaving spiders Meta menardi and Metellina merianae (Tetragnathidae)

Hypogean habitats are relatively simple exhibiting low diversity, low production and relative constancy of environmental factors, and are therefore appropriate for studying species coexistence in situ. We investigated the coexistence of two closely related, similarly sized orb-weaving spider species, Meta menardi and Metellina merianae, living syntopically in a Slovenian cave. We studied the annual dynamics of both species within a mixed population, and the impact of the ambient temperature, relative humidity, airflow and illumination, and compared their trophic niches to legacy data on prey of both species from 55 caves in Slovenia. We predicted a large overlap in their spatial niches and substantial differences in their temporal and trophic niches. We found that their spatial niches overlap greatly with few exceptions, mostly on the dates of notable meteorological changes in the cave but that their temporal niches differ significantly with r-strategy resembling epigean annual dynamic in M. merianae and a steady low abundance course in M. menardi within the cave. We also found that different predatory strategies significantly segregate their trophic niches: M. merianae uses a typical orb-weaving hunting strategy, while M. menardi combines web hunting with off-web hunting. Our findings suggest that both the diverse dynamics and trophic niches enable the coexistence of M. menardi and M. merianae despite their similar spatial niches, and that M. menardi, in particular, is optimally adapted to the epigean/hypogean ecotone.     

Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus

A diverse group of animals has adapted to caves and lost their eyes and pigmentation, but little is known about how these animals and their striking phenotypes have evolved. The teleost Astyanax mexicanus consists of an eyed epigean form (surface fish) and at least 29 different populations of eyeless hypogean forms (cavefish). Current alternative hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species. If the latter is true, the unique phenotypes of different cave-dwelling populations may result from convergence of form, and different genetic changes and developmental processes may have similar morphological consequences. Here we report an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene among different surface fish and cavefish populations. The results identify a minimum of two genetically distinctive cavefish lineages with similar eyeless phenotypes. The distinction between these divergent forms is supported by differences in the number of rib-bearing thoracic vertebrae in their axial skeletons. The geographic distribution of ND2 haplotypes is consistent with roles for multiple founder events and introgressive hybridization in the evolution of cave-related phenotypes. The existence of multiple genetic lineages makes A. mexicanus an excellent model to study convergence and the genes and developmental pathways involved in the evolution of the eye and pigment degeneration.     

Cave-adapted evolution in the North American amblyopsid fishes inferred using phylogenomics and geometric morphometrics

Cave adaptation has evolved repeatedly across the Tree of Life, famously leading to pigmentation and eye degeneration and loss, yet its macroevolutionary implications remain poorly understood. We use the North American amblyopsid fishes, a family spanning a wide degree of cave adaptation, to examine the impact of cave specialization on the modes and tempo of evolution. We reconstruct evolutionary relationships using ultraconserved element loci, estimate the ancestral histories of eye-state, and examine the impact of cave adaptation on body shape evolution. Our phylogenomic analyses provide a well-supported hypothesis for amblyopsid evolutionary relationships. The obligate blind cavefishes form a clade and the cave-facultative eyed spring cavefishes are nested within the obligate cavefishes. Using ancestral state reconstruction, we find support for at least two independent subterranean colonization events within the Amblyopsidae. Eyed and blind fishes have different body shapes, but not different rates of body shape evolution. North American amblyopsids highlight the complex nature of cave-adaptive evolution and the necessity to include multiple lines of evidence to uncover the underlying processes involved in the loss of complex traits.     

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.     

Relaxed purifying selection of rhodopsin gene within a Chinese endemic cavefish genus <Emphasis Type="Italic">Sinocyclocheilus</Emphasis> (Pisces: Cypriniformes)

The cyprinid fish genus Sinocyclocheilus, as the most cavefish rich genus, includes many species showing striking adaptation to caves and convergent reduction or even loss of eyes and pigmentation. RH1 is responsible for dim vision. In order to explore the evolution of RH1 gene in this genus, we sequenced the complete gene from 28 individuals of 16 representative species of Sinocyclocheilus, with cave and surface species included. Phylogenetic analyses supported the monophyly of Sinocyclocheilus and polyphyly of the cave species. Codon models implemented in PAML were used to infer the evolution of RH1. We found that Sinocyclocheilus had a significantly higher evolutionary rate for amino acids than other cyprinid fishes compared, which might be the result of relaxation of purifying selection and could be ascribed to cave habit of this genus. In contrast to previous hypotheses, both cave and surface lineages exhibited a similar rate of molecular evolution, so the RH1 of cave species may still be functional, although these species were highly adapted to cave environment. Two amino acid substitutions (D83G and E122V) that were not reported before were found, which may be useful for site-directed mutagenesis in the future. Handling editor: Christian Sturmbauer     

Anophthalmia and elongation of body appendages in cave scale worms (Annelida: Aphroditiformia)

This study addresses whether cave dwelling annelids exhibited similar reductive and constructive traits equally as strong as those of arthropods and vertebrates inhabiting caves. Known as troglomorphism, these adaptations bring about striking morphologies across invertebrates and vertebrates from both aquatic and terrestrial cave habitats, and include varying degrees of eye and pigmentation loss, as well as hypertrophy of body appendages and sensorial structures. Employing phylogenetic comparative methods and ancestral character reconstructions on a worldwide data set of a group of annelids, the scale worms (Aphroditiformia), we investigate the behavioural and morphological traits of species living in marine caves in comparison with those species living outside caves. Our work demonstrated that cave scale worms respond similar to arthropods in cave environments, showing a significant elongation of sensory parapodial cirri, while lacking eyes and pigmentation. However, whereas elongation of sensory appendages likely occurred in correlation to cave colonization, eyes were plausibly lost in correlation with specialization and colonization of deep‐sea habitats.     

Shared and unique patterns of embryo development in extremophile poeciliids

Background

Closely related lineages of livebearing fishes have independently adapted to two extreme environmental factors: toxic hydrogen sulphide (H₂S) and perpetual darkness. Previous work has demonstrated in adult specimens that fish from these extreme habitats convergently evolved drastically increased head and offspring size, while cave fish are further characterized by reduced pigmentation and eye size. Here, we traced the development of these (and other) divergent traits in embryos of Poecilia mexicana from benign surface habitats (“surface mollies”) and a sulphidic cave (“cave mollies”), as well as in embryos of the sister taxon, Poecilia sulphuraria from a sulphidic surface spring (“sulphur mollies”). We asked at which points during development changes in the timing of the involved processes (i.e., heterochrony) would be detectible.

Methods and Results

Data were extracted from digital photographs taken of representative embryos for each stage of development and each type of molly. Embryo mass decreased in convergent fashion, but we found patterns of embryonic fat content and ovum/embryo diameter to be divergent among all three types of mollies. The intensity of yellow colouration of the yolk (a proxy for carotenoid content) was significantly lower in cave mollies throughout development. Moreover, while relative head size decreased through development in surface mollies, it increased in both types of extremophile mollies, and eye growth was arrested in mid-stage embryos of cave mollies but not in surface or sulphur mollies.

Conclusion

Our results clearly demonstrate that even among sister taxa convergence in phenotypic traits is not always achieved by the same processes during embryo development. Furthermore, teleost development is crucially dependent on sufficient carotenoid stores in the yolk, and so we discuss how the apparent ability of cave mollies to overcome this carotenoid-dependency may represent another potential mechanism explaining the lack of gene flow between surface and cave mollies.