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.

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 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.

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.

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.

BackgroundThe loss of phenotypic characters is a common feature of evolution. Cave organisms provide excellent models for investigating the underlying patterns and processes governing the evolutionary loss of phenotypic traits. The blind Mexican cavefish, Astyanax mexicanus, represents a particularly strong model for both developmental and genetic analyses as these fish can be raised in the laboratory and hybridized with conspecific surface form counterparts to produce large F2 pedigrees. As studies have begun to illuminate the genetic bases for trait evolution in these cavefish, it has become increasingly important to understand these phenotypic changes within the context of cavefish origins. Understanding these origins is a challenge. For instance, widespread convergence on similar features renders morphological characters less informative. In addition, current and past gene flow between surface and cave forms have complicated the delineation of particular cave populations.ResultsPast population-level analyses have sought to: 1) estimate at what time in the geological past cave forms became isolated from surface-dwelling ancestors, 2) define the extent to which cave form populations originated from a common invasion (single origin hypothesis) or several invasions (multiple origin hypothesis), and 3) clarify the role of geological and climatic events in Astyanax cavefish evolution. In recent years, thanks to the combined use of morphological and genetic data, a much clearer picture has emerged regarding the origins of Astyanax cavefish.ConclusionsThe consensus view, based on several recent studies, is that cave forms originated from at least two distinct ancestral surface-dwelling stocks over the past several million years. In addition, each stock gave rise to multiple invasions of the subterranean biotope. The older stock is believed to have invaded the El Abra caves at least three times while the new stock separately invaded the northern Guatemala and western Micos caves. This renewed picture of Astyanax cavefish origins will help investigators draw conclusions regarding the evolution of phenotypic traits through parallelism versus convergence. Additionally, it will help us understand how the presence of cave-associated traits in old versus young cave populations may be influenced by the time since isolation in the cave environment. This will, in turn, help to inform our broader understanding of the forces that govern the evolution of phenotypic loss.

The genetic regulators of regressive craniofacial morphologies are poorly understood. To shed light on this problem, we examined the freshwater fish Astyanax mexicanus, a species with surface-dwelling and multiple independent eyeless cave-dwelling forms. Changes affecting the skull in cavefish include morphological alterations to the intramembranous circumorbital bones encircling the eye. Many of these modifications, however, have evolved separately from eye loss, such as fragmentation of the third suborbital bone. To understand the genetic architecture of these eye-independent craniofacial alterations, we developed and scored 33 phenotypes in the context of an F 2 hybrid mapping pedigree bred from Pachón cavefish and surface fish. We discovered several individuals exhibiting dramatic left-right differences in bone formation, such as extensive fragmentation on the right side only. This observation, along with well-known eye size asymmetry in natural cave-dwelling animals, led us to further evaluate left-right genetic differences for the craniofacial complex. We discovered three phenotypes, inclusive of bone fragmentation and fusion, which demonstrated a directional heritable basis only on one side. Interestingly, the overall areas of affected bones were genetically symmetric. Phenotypic effect plots of these novel craniofacial QTL revealed that cave alleles are associated with abnormal conditions such as bony fusion and fragmentation. Moreover, many linked loci overlapped with other cave-associated traits, suggesting regressive craniofacial changes may evolve through linkage or as antagonistic pleiotropic consequences of cave-associated adaptations. These novel findings illuminate significant craniofacial changes accompanying evolution in complete darkness and reveal complex changes to the skull differentially influenced by genetic changes affecting the left and right sides.

Recent years have witnessed renewed interest in defining the embryonic cell populations that directly contribute to the bony skull. This question lies at the intersection of several important developmental, clinical and evolutionary interests. Until recently, our collective understanding of the embryonic origin of the vertebrate osteocranium has been based on a small number of reports published solely using avian models. As data gradually accumulates from other, distantly related species (e.g., mouse and frog), we can begin to evaluate long-standing assumptions regarding the behavior of osteogenic (bone-forming) neural crest cells within a wider phylogenetic and comparative context. In this review, we summarize data collected to date in three major vertebrate taxa: amphibians, birds and mammals. We highlight three largely unexplored topics within the field of osteogenic neural crest development: 1) disagreements in bone tissue origin within and across current model systems; 2) whether the pattern of neural crest cell contribution to skull bone is evolutionarily conservative or labile; and 3) how our understanding of development and morphology will benefit from fate maps using currently unexamined animal models.