Richard Borowsky

Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism

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.

A novel role for Mc1r in the parallel evolution of depigmentation in independent populations of the cavefish Astyanax mexicanus.

The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown phenotype in F2 individuals derived from two independent cave populations: Pachón and the linked Yerbaniz and Japonés caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype arose prior to the fixation of albinism in Pachón cave individuals, it is unclear whether the brown mutation arose before or after the fixation of albinism in the linked Yerbaniz/Japonés caves. Using a QTL approach combined with sequence and functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen independently in geographically separate caves, mediated through different mutations of the same gene. This example of parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species.

Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.

Eye to Eyeless To what extent does adaptation rely on de novo mutation, as opposed to preexisting variation? It has been proposed that heat shock protein 90 (HSP90) can act to maintain cryptic variation by correcting misfolded proteins, until the system is taxed under stress conditions. Focusing on the cavefish Astyanax mexicanus, Rohner et al. (p. 1372) provide evidence that this mechanism contributed to morphological evolution in a natural setting where cryptic variation in eye size was masked by HSP90 in the ancestral river but revealed when the fish were reared and selected in caves. Preexisting but “hidden” variations in eye size provide a substrate for natural selection in fish reared in the dark. In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting.

Multi-trait evolution in a cave fish, Astyanax mexicanus

SUMMARY When surface species colonize caves, a characteristic suite of traits eventually evolves over time, regardless of species. The genetic basis of the inevitable appearance of these very similar phenotypes was investigated through quantitative trait loci (QTL) mapping of 12 traits that differ significantly between the recently evolved (<1 Myr). Mexican cave tetra and its surface conspecific. The traits were a representative set, including eye size, pigment cell numbers, chemical sensitivity, body and skull morphology, standard length, and metabolism. We used both single‐ and multi‐trait models for QTL mapping. QTL effects of these traits were significantly clustered in the genome. We mapped 13 regions in the genome with QTL effects on from three to nine traits. These clusters could be multigenic or could represent single locus with pleiotropic alleles. Given the relatively short time available to construct clusters from unlinked genes through genomic rearrangement, and the counterintuitive polarities of some of the substitution effects, we argue that at least some of the clusters must have a pleiotropic basis.

The cavefish genome reveals candidate genes for eye loss

Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.

Evolutionary Convergence on Sleep Loss in Cavefish Populations

Patterns of sleep vary widely among species, but the functional and evolutionary principles responsible for this diversity remain unknown. The characin fish, Astyanax mexicanus, has eyed surface and numerous blind cave populations. The cave populations are largely independent in their origins, and the species is ideal for studying the genetic bases of convergent evolution. Here we show that this system is also uniquely valuable for the investigation of variability in patterns of sleep. We find that a clearly defined change in ecological conditions, from surface to cave, is correlated with a dramatic reduction in sleep in three independently derived cave populations of A. mexicanus. Analyses of surface × cave hybrids show that the alleles for reduced sleep in the Pachón and Tinaja cave populations are dominant in effect to the surface alleles. Genetic analysis of hybrids between surface and Pachón cavefish suggests that only a small number of loci with dominant effects are involved. Our results demonstrate that sleep is an evolutionarily labile phenotype, highly responsive to changes in ecological conditions. To our knowledge, this is the first example of a single species with a convergence on sleep loss exhibited by several independently evolved populations correlated with population-specific ecologies.

Social inhibition of maturation in natural populations of Xiphophorus variatus (Pisces: Poeciliidae)

According to analyses of field samples, social inhibition of maturation is at work in natural populations of the variable platyfish, Xiphophorus variatus. In the laboratory, adult males inhibit the maturation of juveniles; the inhibition is overcome as the juveniles increase in size. The proportion of maturing males in any field collection is related to the number of adult males present and the size of the juveniles. The more adults, the fewer maturing males are present; the larger the average juvenile, the greater the number of males maturing. The evolution of this system is best understood in terms of individual selection, but consequences of the system buffer the population against the effects of predation.

Social control of adult size in males of Xiphophorus variatus

As in other species of poeciliid fish, male Xiphophorus variatus (Pisces, Poeciliidae) virtually cease growth at maturity. The size of an adult male, then, is determined by its average growth rate before it reaches maturity and the age at which this happens. The latter factor is under social control in this species. When I raised juvenile males in groups, they usually started to mature in sequence, largest first, and subsequently maturing males did not usually reach maturity until they were longer than all previously matured males. As a result, the juvenile size order was negatively correlated with adult size order. When the fish in a group were isolated from one another such patterns disappeared indicating that some degree of social interaction was necessary for their maintenance1.

Synteny and candidate gene prediction using an anchored linkage map of Astyanax mexicanus

The blind Mexican cave tetra, Astyanax mexicanus, is a unique model system for the study of parallelism and the evolution of cave-adapted traits. Understanding the genetic basis for these traits has recently become feasible thanks to production of a genome-wide linkage map and quantitative trait association analyses. The selection of suitable candidate genes controlling quantitative traits remains challenging, however, in the absence of a physical genome. Here, we describe the integration of multiple linkage maps generated in four separate crosses between surface, cave, and hybrid forms of A. mexicanus. We performed exhaustive BLAST analyses of genomic markers populating this integrated map against sequenced genomes of numerous taxa, ranging from yeast to amniotes. We found the largest number of identified sequences (228), with the most expect (E) values <10−5 (95), in the zebrafish Danio rerio. The most significant hits were assembled into an “anchored” linkage map with Danio, revealing numerous regions of conserved synteny, many of which are shared across critical regions of identified quantitative trait loci (QTL). Using this anchored map, we predicted the positions of 21 test genes on the integrated linkage map and verified that 18 of these are found in locations homologous to their chromosomal positions in D. rerio. The anchored map allowed the identification of four candidate genes for QTL relating to rib number and eye size. The map we have generated will greatly accelerate the production of viable lists of additional candidate genes involved in the development and evolution of cave-specific traits in A. mexicanus.

Patterns of mating in natural populations of Xiphophorus (Pisces: Poeciliidae). I. X. maculatus from Belize and Mexico

Poeciliid fishes are one of the dominant groups in Middle America and the West Indies (Rosen and Bailey, 1963). They are relatively small forms with a highly developed sexual dimorphism. Interspecific differences between males are very pronounced, but females differ relatively little. Some species (e.g., Xiphophorus maculatus) often occur in dense aggregates in small ditches or pools. These fishes are ovoviviparous and females may store sufficient sperm to fertilize the ova of several successive broods at monthly intervals (Dzwillo, 1959; Hildemann and Wagner, 1954; van Oordt, 1928; Zander, 1962). Males of many species exhibit incessant and highly visible courtship activity, but virtually nothing is known about the mating system of these forms under natural conditions. In general it is believed to be promiscuous. Hubbs (1964) reported that in a population of Poecilia latipinna and P. formosa virtually all females were gravid, although females outnumbered males by a ratio of 100: 1. Laboratory observation and some progeny analyses of gravid females from natural populations indicate that males of the guppy, Poecilia reticulata, and platyfish, Xiphophorus maculatus, may mate with more than one female and females mate with more than one male (Gandolfi, 1971; Gordon, 1947; Haskins et al., 1961; Kallman, 1965, 1970; Rosenthal, 1952). Territoriality has been reported for only one poeciliid, Poeciliopsis lucida, and its mating system is basically polygynous (Moore and McKay, 1971). Observations on natural populations of X. variatus, however, indicate that males of this species are not territorial and that the mating system is promiscuous (Borowsky, 1969 and unpubl.). In this paper we present the results of an indirect analysis of reproductive patterns in natural populations of the platyfish, Xiphophorus maculatus. This species is highly polymorphic for color patterns the genetic basis of which is known in some detail (Kallman, 1970; Kallman and Atz, 1966). Gravid females were collected and brought to the laboratory for progeny analyses. From the color patterns of offspring and P1 females, we determined the number and genotypes of inseminating males, the existence of sperm succession, fitness differences among genotypes, and whether mating is at random with respect to color patterns. We believe this information to be of interest both to evolutionists and to students of poeciliid fish.