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The forces driving regression of biologically functionless traits remain disputed. There is ongoing debate regarding whether selection, as opposed to disuse and neutral mutations, is involved in this process. Cave species are of particular relevance for study in this regard because in continuous darkness all traits that depend on information from light, such as eyes, dark pigmentation and certain behaviours, abruptly lose their function. Recently, strong selection driving reduction has again been proposed, which relied on modelling analyses based on assumptions such as immigration of surface alleles into the cave forms or no fitness difference existing between Astyanax surface and cave fish. The validity of these assumptions, often applied to reject neutral processes in functionless traits, is questioned in this review. Morphological variation in a trait resulting from genetic variability is typical of biologically functionless traits and is particularly notable in phylogenetically young cave species. It is the most evident indicator of loss of selection, which normally enforces uniformity to guarantee optimal functionality. Phenotypic and genotypic variability in Astyanax cave fish eyes does not derive from genetic introgression by the surface form, but from regressive mutations not being eliminated by selection. This matches well with the principles of Kimura’s neutral theory of molecular evolution.

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.

A hot spot of biodiversity of aquatic cave species of very different biogeographic and systematic origins is located around the Gulf of Mexico. In continental Mexico, Neotropic and Nearctic cave faunas overlap north as well as south of the trans-Mexican volcanic belt and may even co-occur in the same cave. These species may also be associated with marine relics, closely related species that inhabit caves of peri-Carribean islands like Cuba and the Bahamas or of the Yucatan peninsula. Other cave species occurring in this area derive from deep sea forms or show close relationships to species occurring in subterranean water on the volcanic Eastern Atlantic island of Lanzarote. Representatives of the thermosbaenaceans and the remipedes, the latter having developed their centre of radiation in the peri-Carribean Islands, exclusively live in caves but exhibit circumtropical Tethyan distribution.

The surface and cave forms of the characid Astyanax have become a model system of outstanding importance in evolutionary research. It is extraordinary, because this diurnal day-active fish only exhibits minimal preadaptive traits allowing life in continuous darkness. In Astyanax, transition into the new environment took place abruptly and without a phase of gradual adaptation, as is usual when night-active, so-called troglophile surface forms start colonizing caves. Because of the specific characteristic of the surface fish, a large discrepancy and a large number of different traits have evolved in the cave form. The comparatively quick change into a drastically different environment is probably one of the reasons why surface and cave fish are still interfertile and can be genetically analyzed and phenotypically compared. Furthermore, cave colonization has been performed, at least in part, in parallel at different geographically separate locations and at different times. As a result, variously advanced stages of phenotypic evolution and their genetic bases can be studied.

The Neotropic large-eyed and well-pigmented diurnal characid fish Astyanax has developed a series of cave populations in Northeastern Mexico. These divide morphologically into a group of strongly eye- and pigment-reduced (SEP) cave populations and another one characterized by variable eye size and pigmentation (VEP cave populations). Molecular and biogeographic data imply that they derive from the Neotropic Astyanax surface fish, which were able to invade North America up to the Rio Grande drainage after the closure of the Central American land bridge. Its recent distributional pattern is strongly influenced by Pleistocene climatic changes and is characterized by regional extinction and recolonization from the warmer south and/or survival in climatically buffered refuges. An example of this are the SEP cave fish populations, which according to cytochrome b analysis do not cluster with the surface fish from neighboring rivers and creeks but with fish from a remote location about 500 km away in the Central Mexican Plateau. In line with this, they do not group with either the VEP cave fish or with surface fish from the cave area, and based on microsatellites and SNP studies, they exhibit relation to populations from southern Mexico and Belize. The SEP cave fish and some relic surface fish populations from isolated locations all over Mexico derive from the oldest invasion. In contrast, based on cytochrome b studies, the VEP cave populations cluster with the recent surface fish from the cave area, which is widespread in Northern Mexico. The VEP cave populations derive from a more recent invasion of surface fish into Northern Mexico. In particular, the differing degree of eye reduction between SEP and VEP cave fish reflects the different times of cave entry. Cave colonization in VEP and SEP cave populations took place in parallel and resulted in multiple convergent evolutions.

In the face of the neodarwinian paradigm of selection as an agent exclusively claimed to explain evolutionary processes in recent days, almost countless efforts have been made to prove that selection plays an important role in rudimentation processes in cave animals. Efforts mostly focus on the eye and very often pleiotropy is looked upon as being responsible. For example, eye reduction is claimed to antagonistically drive the improvement of taste or the lateral line sense by pleiotropy. However, this could not be confirmed by crossing analysis or by quantitative trait loci mapping. Energy savings have been suggested as another selection factor. This hypothesis implies that all cave species would have to suffer from food limitation. This attempt ignores the fact that the majority of tropical caves, and even some subtropical ones, abound in energy supply. Nonetheless, the traits, having become functionless in the respective cave species occurring in these habitats, regress. Thus, energy limitation is not able to explain regressive evolution of biologically functionless traits in general, or in particular that of the eye in cave species. In fact, independent inheritance of traits suggests that Astyanax cave fish are subjected to mosaic evolution.

The haplotype distribution of the Astyanax surface fish and the origin of its cave forms is strongly influenced by Pleistocene climatic change, due to which large-scale extinction and recolonization by the surface fish took place. Because of this history and the persisting interfertility, the relationship between the Astyanax strongly eye- and pigment-reduced (SEP) and variably eye- and pigment-reduced (VEP) cave populations as well as the recent surface populations is very complex. Although based on nuclear genes clustering with the phylogenetically old SEP, the Pachon and Yerbaniz cave populations are exceptional and show close relationship to both the recent surface and the phylogenetically young VEP cave fish due to mitochondrial capture. Furthermore, the variability of eye size observed in the VEP, especially in the Chica cave population, was often explained by hybridization. This contrasts with the finding that no gene flow exists in the SEP Yerbaniz and VEP Micos cave fish populations, where cave and starved surface fish co-occur. This is explained by the surface fish being washed into the caves and not being able to compete with the well adapted SEP or VEP cave fish in darkness. These observations are in accordance with Gause’s Law which predicts that two ecomorphs competing for the same resource cannot coexist in the same niche. In the cave habitat the cave fish are at an advantage, which will finally lead to the extinction of the surface specimens competing for food. This process is reinforced in the Astyanax caves every year by periodic low food supply outside the rainy seasons. Bottlenecking obviously regularly occurs, which is demonstrated by the low variability of several genetic markers compared with the surface fish. However, mitochondrial introgression in the SEP cave populations like Pachon and Yerbaniz has nonetheless occurred. It is suggested that the introgressed modern haplotypes in the SEP Pachon and Yerbaniz cave fish derive from VEP cave populations, which, like the recent VEP Micos cave fish, were well adapted to cave life and therefore able to co-exist and also to hybridize with the SEP cave populations after the cave systems merged due to karst erosion. The recent surface fish on the one hand and the SEP and VEP cave populations on the other no longer hybridize in nature. In accordance with the Biological Species Concept, the SEP and VEP cave populations therefore can be denominated as a species on their own. The cave fish speciation process provides one of the rare examples of parallel speciation.

The Corona lava tube on the Canarian island of Lanzarote is a unique subterranean ecosystem comprising both dry and submerged cave sections with a total length of almost 8 km. Here, we present the results of a diving exploration of the lava tube that took place from 11 to 25 March 2008. Environmental characteristics are given for ecologically disparate sections of the cave, including the Cueva de los Lagos, the Jameos del Agua, and the Tunel de la Atlantida. Moreover, we compare various habitats within the lava tube, and discuss the origin of the diverse hypogean fauna, including new species of remipede crustaceans and polychaete worms discovered during the expedition.

The process of speciation of most cave living animals starts within the distribution area of their ancestral forms. Fishes from the phylogenetically young Micos cave population of the Mexican characid Astyanax fasciatus (Pisces) may even be found associated with large numbers of its con-specific epigean ancestor in the natural cave biotope. Although the individuals of the epigean and hypogean populations are still interfertile, no hybridization occurs in the cave. In competition for the reduced energy supply in the cave the epigean individuals are inferior and suffer starvation. This has been shown by comparison of the condition factors of epigean and hypogean individuals of A. fasciatus from several populations. The origin of cavernicolous populations depends on a general preadaptation for cave life. The habitat change, which is characteristic of cave colonization, is coupled with radical changes in some environmental factors. The loss of light and a reduced food supply work as disruptive selection factors: Only a few individuals of the epigean form are able to found a cave population of its own under these circumstances. They are individuals characterized by a more than average preadaptation, which according to genetic analysis may be based on only one gene. This gene may improve an advantageous feature like the gustatory equipment or the feeding behaviour by a threshold effect in one step by a considerable amount. In the cave biotope the normal epigean genotypes are now displaced in competition. Thus a speciation process may start based on only one gene which can cause a threshold effect. In this initial phase of speciation there is still a reduced gene flow. Thus more advantageous genes out of the epigean gene pool become concentrated in the newly founded cave population. On account of the energy economy being only slightly improved, the population density is low. Genetic isolation is only achieved when further adaptations to cave life are postadaptively acquired and improved. By better mechanisms for finding and more efficiently using the energy sources and by increasing population size all epigean genotypes are finally displaced. The origin of cave species is discussed as an example of sympatric speciation. Zusammenfassung Sympatrische Artbildung bei Hohlenfischen? Untersuchung an einer Mischpopulation ober- und unterirdischer Astyanax (Characidae, Pisces) Der Speziationsprozes der meisten hohlenbewohnenden Tiere beginnt innerhalb des Verbreitungsareals ihrer obenrdischen Ursprungsarten. Fische der phylogenetisch jungen Micos Hohlenpopulation des mexikanischen Characiden Astyanax fasciatus konnen sogar vergesellschafret mit einer grosen Anzahl ihres conspezifischen oberirdischen Verwandten im naturlichen Hohlenbiotop leben. Obwohl die Individuen der ober- und unterirdischen Populationen noch interfertil sind, findet keine Hybridisierung in der Hohle statt. In der Konkurrenz urn das begrenzte Nahrungsangebot in der Hohle sind die oberirdischen Fische unterlegen und zeigen Hungersymptome. Dieses wurde durch den Vergleich der Konditionsfaktoren der ober- und unterirdischen Individuen verschiedener Populationen von A. fasciatus nachgewiesen. Die Entstehung von Hohlenpopulationen hangt von einer generellen Praadaptation fur das Hohlenleben ab. Der Habitatwechsel, welcher charakteristisch fur die Hohlenbesiedlung ist, ist verknupft mit einschneidenden anderungen einier Umweltfaktoren. Die Dunkelheit und ein vermindertes Nahrungsangebot wirken als disruptive Selektionsfaktoren: Nur wenigge Individuen der oberirdischen Form sind fahig, unter diesen Umstanden von sich aus eine Hohlenpopulation zu grunden. Es sind dies Individuen, die sich durch uberdurchschnittliche Praadaptation auszeichnen, was genetischen Analysen zufolge wahrscheinlich nur auf einem einzigen Gen basiert. Dieses Gen kann durch einen Schwelleneffekt ein vorteilhaftes Merkmal wie den Geschmacksapparat oder das Futtersuchverhalten in einem einzigen Schritt betrachtlich verbessern. Unter Konkurrenz im Hohlenbiotop werden nun die normalen oberirdischen Genorypen verdrangt. Auf diese Weise kann ein Speziationsprozes durch ein einziges Gen, das einen Schwelleneffekt verursacht, beginnen. In dieser Anfangsphase der Speziation existiert noch ein verminderter Genflus. So konnen mehrere vorteilhafte Gene aus dem oberirdischen Genpool in der neu gegrundeten Hohlenpopulation konzentriert werden. Aufgrund der immer noch relativ geringen Energieersparnis ist die Populationsdichte gering. Genetische Isolation ist erst erreicht, wenn weitere Adaptationen an das Hohlenleben postadaptiv erworben und verbessert worden sind. Durch verbesserte Mechanismen zur Auffindung und Ausnutzung der Energieressourcen und durch eine anwachsende Populationsgroōse werden alle oberirdischen Genotypen schlieslich verdrangt. Die Entstehung von Hohlentierarten wird als ein Beispiel fur sympatrische Speziation diskutiert.

One epigean and five cave populations of Astyanax fasciatus were compared with respect to egg and embryo size as well as yolk volume 12 h and 24 h after fertilization. All cave fish have significantly bigger eggs and embryos with more yolk than the epigean ancestor. There are no differences in the chemical composition of cave and surface fish eggs. The genetic analysis of yolk content reveals an additive polygenic mode of inheritance. Zusammenfassung Dotterreichere Eier bei unterirdisch lebenden Astyanax fasciatus (Characidae, Pisces) Eine oberirdische und funf unterirdische Populationen von Astyanax fasciatus wurden in Hinsicht auf Ei- und Embryogrose und auf Dottervolumen 12 h und 24 h nach der Befruchtung unter-sucht. Alle Hohlenfische haben signifikant grosere Eier und Embryonen mit mehr Dotter als der oberirdische Verwandte. Dagegen bestehen keine Unterschiede in der chemischen Zusammenset-zung der Eier ober- und unterirdischer Fische. Die genetische Analyse zeigt, das das Dottervolumen additiv polygen vererbt wird.

The West Indian Ophidiiform fish are characterized by the occurrence of several troglobitic species. They are Ogilbia (= Typhliasina) pearsei (Yucatan), Lucifuga subterraneus, L. dentatus and L. simile (Cuba), and L. spelaeotes (Bahama Islands). The surface relatives, e. g. Ogilbia spp., prefer lightpoor zones. This is shown by a large lens and a retina almost exclusively consisting of rods. L. dentatus, L. subterraneus, and O. pearsei possess extremely reduced eyes. The eye of L. spelaeotes has morphologically an intermediate position. In correlation to varying eye size a dif­ ferent number of rods may be developed. Furthermore in some specimens the lens starts de­ generating. Contrary to the extremely reduced cave forms all specimens of L. spelaeotes can still perceive light with their eyes. The different degrees of reduction in the West Indian Ophidiiform cave fish probably reflect varying phylogenetic ages. A possible date of origin of L. spelaeotes could be at the beginning or the end of the last glaciation. The eyes of neonate individuals of the live-bearing L. dentatus and O. pearsei possess a lens and a retina rudiment. No fundamental structural and histological differences between both species were found at this stage.

Two hypotheses exist to explain ontogenetic eye reduction in Astyanax cave fish: first, after lens induction by the primordial eye cup, the lens plays the role of a central regulator of eye and retina regression or, second, the retina itself is an independent unit of eye development. A comparative study of five blind cave fish populations and their surface sister form was performed to investigate the differences of ontogenetic eye regression between the cave populations during different stages of development. The study revealed that, in addition to the initial formation of smaller primordia, eye regression is also caused during later ontogeny by different relative growth and specific histological characteristics. Whereas the cave fish lens never properly differentiates, the regressive process of the retina is transitorily interrupted by ongoing differentiation. In the newly-discovered Molino cave population, even visual cells with well-organized outer segments develop, which are secondarily reduced at a later ontogenetic stage. This result shows that the retina and lens are independent developmental units within the eye ball. Presumably, the genetic systems responsible for both show independent inheritance, which is also corroborated by hybrids of F2-crosses between the cave and surface fish, in which lens and retina development do not correlate. During ontogeny, the eye size differs between the cave populations. In Pachon cave fish, the relatively large eye size correlates with an ancient introgression from a surface population, which may have delayed eye regression. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92, 287–296.

Astyanax fasciatus has become a model organism for the study of regressive and adaptive evolution in cave animals. To fully understand these processes, it is important to have background information on the systematics and phylogeography of surface and cave populations of this species. Here we investigate the phylogeography of A. fasciatus in North and Central America and also the historical biogeography of this region. Phylogenetic analysis of part of the mtDNA cytochrome b gene from 26 surface and nine cave A. fasciatus populations revealed seven major clades, which, in principle, represent geographical patterns of distribution. However, the four strongly eye and pigment reduced cave populations, Piedras, Sabinos, Tinaja, and Curva, form a separate cluster, which is not sister group to the surface populations from the same locality. Similarly the Belizean populations do not cluster with their geographic neighbors from the Yucatan. The analyses indicate that there have been recurrent invasions of surface Astyanax from the south, that were most likely influenced by major climate changes during the Pleistocene. During this period, ancestors of the strongly eye and pigment reduced cave populations were able to survive underground as thermophilic relics when the surface populations became extinct. The high level of genetic divergence among the different clades shows that differing haplotype lineages must have reinvaded the surface waters from the south and/or back-colonized them from residual habitats and also penetrated into the caves. Nested clade analyses show that recurrent gene flow as well as historic processes like past fragmentation and range expansion have influenced current populations of A. fasciatus in Central and North America. Different haplotype clades of the phylogeny are not compatible with the present taxonomy of Astyanax and, therefore, we propose the application of a single systematic unit, called A. fasciatus.

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.

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.

The early morphogenesis of the degenerate eyes of the Mexican cave fish Astyanax fasciatus and of its conspecific epigean ancestor has been studied comparatively using light- and electron-microscopical techniques; the transcription of the opsin gene has been analysed during early ontogeny in both populations by in situ hybridization. The opsin protein is an integral component of the disk membranes of the photoreceptor cells. In epigean specimens, its expression is correlated with the beginning of outer segment formation on the third day of development. Morphogenesis of the cave fish eye is similar to that of the epigean eye until end of the third day. However, eye growth and morphogenesis of the retina are delayed and sporadic cell death occurs in all retinal layers at the beginning of the second day. Retinal cytodifferentiation breaks off at the point of outer segment formation. Cave specimens are not able to develop regular outer segments at any stage, but the opsin gene is nevertheless expressed in the outer nuclear layer of the developing retina for a limited period of time. On the basis of the comparative morphology and transcriptional studies of epigean and cave specimens, it is suggested that the eye regression of cave fish is primarily the result of mutations of developmental control genes and not of structural genes.

The genetic basis of vertebrate morphological evolution has traditionally been very difficult to examine in naturally occurring populations. Here we describe the generation of a genome-wide linkage map to allow quantitative trait analysis of evolutionarily derived morphologies in the Mexican cave tetra, a species that has, in a series of independent caves, repeatedly evolved specialized characteristics adapted to a unique and well-studied ecological environment. We focused on the trait of albinism and discovered that it is linked to Oca2, a known pigmentation gene, in two cave populations. We found different deletions in Oca2 in each population and, using a cell-based assay, showed that both cause loss of function of the corresponding protein, OCA2. Thus, the two cave populations evolved albinism independently, through similar mutational events.

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.

In the present study we have analyzed the integrity and expression of the alphaA-crystallin gene, that codes for a major structural component of the lens, in a blind cave form of the teleostean fish, Astyanax fasciatus. This is the first alphaA-crystallin gene cloned from a teleostean fish. Sequence comparison of this cave-form gene with its epigean conspecific and with homologs of distantly related taxa has illustrated conservation of regulatory and coding regions. Although no crystallin proteins are produced in the lens of the cave form, and the mRNA of this gene could not be detected by in situ hybridization of different developmental stages, the promoter region of cave-fish alphaA-crystallin is functionally intact. The deduced amino-acid sequence of the alphaA-crystallin gene of the cave form differs from that of its epigean conspecific at only one position (139). This is within an important, small heat-shock protein-related region, HCR2. A comparison of the 5'-flanking regions of the A. fasciatus alphaA-crystallin gene with the chicken homolog revealed the high conservation of lens-specific regulatory sequences and further demonstrates the evolutionary conservation of this gene. 1988 Elsevier Science B.V.

The Pax-6 gene encodes a transcription factor essential for eye development in a wide range of animal phyla. In order to elucidate a possible role of Pax-6 in the eye regression of a blind cave form of the freshwater fish Astyanax fasciatus (Characidae, Teleostei) we investigated the expression of Pax-6 in eyes and brains of different larval stages by in situ hybridization. Pattern, strength, and time course of Pax-6 expression were not altered in the tissues of the cave form when compared to the epigean form. Pax-6 was even expressed in the highly degenerated eyes of late larval stages of the cave form. Comparative sequence analysis of Pax-6 cDNA clones of both forms of Astyanax fasciatus showed the complete integrity of cave fish Pax-6 mRNA. These results suggest that Pax-6 is not involved in the evolutionary process of eye degeneration in this model system of cave-living fishes. Comparison of the Astyanax Pax-6 cDNA with the other available fish Pax-6 sequence from zebrafish revealed putative fish-specific regions of homology. A stretch of 19 N-terminal amino acids is nearly identical on the nucleotide and amino acid levels in both fish species but not present in all other known Pax-6 sequences.

Notable progress in the biological sciences is occasionally made possible by the discovery of a species that exhibits a specialized way of life, but nevertheless will thrive and breed in the laboratory. In the study of cavernicolous animals, the Mexican characid fish Astyanax fasciatus has come to play a role equivalent to that of the fruit fly, Drosophila, in genetics.