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.

Summary How cave animals adapt to life in darkness is a poorly understood aspect of evolutionary biology [1]. Here we identify a behavioral shift and its morphological basis in Astyanax mexicanus, a teleost with a sighted surface dwelling form (surface fish) and various blind cave dwelling forms (cavefish) [2–4]. Vibration attraction behavior (VAB) is the ability of fish to swim toward the source of a water disturbance in darkness. VAB was typically seen in cavefish, rarely in surface fish, and advantageous for feeding success in the dark. The potential for showing VAB has a genetic component and is linked to the mechanosensory function of the lateral line. VAB was evoked by vibration stimuli peaking at 35 Hz, blocked by lateral line inhibitors, appeared after developmental increases in superficial neuromast (SN) number and size [5–7], and was significantly reduced by bilateral ablation of SN. We conclude that VAB and SN enhancement co-evolved to compensate for loss of vision and help blind cavefish find food in darkness.

Hedgehog (Hh) proteins are responsible for critical signalling events during development but their evolutionary roles remain to be determined. Here we show that hh gene expression at the embryonic midline controls eye degeneration in blind cavefish. We use the teleost Astyanax mexicanus, a single species with an eyed surface-dwelling form (surface fish) and many blind cave forms (cavefish), to study the evolution of eye degeneration. Small eye primordia are formed during cavefish embryogenesis, which later arrest in development, degenerate and sink into the orbits. Eye degeneration is caused by apoptosis of the embryonic lens, and transplanting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye. Here we show that sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh) gene expression is expanded along the anterior embryonic midline in several different cavefish populations. The expansion of hh signalling results in hyperactivation of downstream genes, lens apoptosis and arrested eye growth and development. These features can be mimicked in surface fish by twhh and/or shh overexpression, supporting the role of hh signalling in the evolution of cavefish eye regression.

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 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 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.

Astyanax mexicanus is a teleost with eyed surface-dwelling and eyeless cave-dwelling forms. Eye formation is initiated in cave fish embryos, but the eye subsequently arrests and degenerates. The surface fish lens stimulates growth and development after transplantation into the cave fish optic cup, restoring optic tissues lost during cave fish evolution. Conversely, eye growth and development are retarded following transplantation of a surface fish lens into a cave fish optic cup or lens extirpation. These results show that evolutionary changes in an inductive signal from the lens are involved in cave fish eye degeneration.

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations, and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our dataset. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5–7mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161k - 191k generations ago. The favored demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

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.

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.