Quantitative trait loci concentrate in specific regions of the Mexican cavefish genome and reveal key candidate genes for cave-associated evolution.

A major goal of modern biology is connecting phenotype with its underlying genetic basis. The Mexican cavefish (Astyanax mexicanus), a characin fish species comprised of a surface ecotype and a cave-derived ecotype, is well suited as a model to study the genetic mechanisms underlying adaptation to extreme environments. Here we map 206 previously published quantitative trait loci (QTL) for cave-derived traits in A. mexicanus to the newest version of the surface fish genome assembly, AstMex3. These analyses revealed that QTL cluster in the genome more than expected by chance, and this clustering is not explained by the distribution of genes in the genome. To investigate whether certain characteristics of the genome facilitate phenotypic evolution, we tested whether genomic characteristics associated with increased opportunities for mutation, such as highly mutagenic CpG sites, are reliable predictors of the sites of trait evolution but did not find any significant trends. Finally, we combined the QTL map with previously collected expression and selection data to identify 36 candidate genes that may underlie the repeated evolution of cave phenotypes, including rgrb, which is predicted to be involved in phototransduction. We found this gene has disrupted exons in all non-hybrid cave populations but intact reading frames in surface fish. Overall, our results suggest specific regions of the genome may play significant roles in driving adaptation to the cave environment in Astyanax mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish.

Loci associated with cave-derived traits concentrate in specific regions of the Mexican cavefish genome.

A major goal of modern evolutionary biology is connecting phenotypic evolution with its underlying genetic basis. The Mexican cavefish (Astyanax mexicanus), a characin fish species comprised of a surface ecotype and a cave-derived ecotype, is well suited as a model to study the genetic mechanisms underlying adaptation to extreme environments. Here we map 206 previously published quantitative trait loci (QTL) for cave-derived traits in A. mexicanus to the newest version of the surface fish genome assembly, AstMex3. This analysis revealed that QTL cluster in the genome more than expected by chance, and this clustering is not explained by the distribution of genes in the genome. To investigate whether certain characteristics of the genome facilitate phenotypic evolution, we tested whether genomic characteristics, such as highly mutagenic CpG sites, are reliable predictors of the sites of trait evolution but did not find any significant trends. Finally, we combined the QTL map with previously collected expression and selection data to identify a list of 36 candidate genes that may underlie the repeated evolution of cave phenotypes, including rgrb which is predicted to be involved in phototransduction. We found this gene has disrupted exons in all non-hybrid cave populations but intact reading frames in surface fish. Overall, our results suggest specific "evolutionary hotspots" in the genome may play significant roles in driving adaptation to the cave environment in Astyanax mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish.

Phylogeographic relationships and morphological evolution between cave and surface Astyanax mexicanus populations (De Filippi 1853) (Actinopterygii, Characidae).

The Astyanax mexicanus complex includes two different morphs, a surface- and a cave-adapted ecotype, found at three mountain ranges in Northeastern Mexico: Sierra de El Abra, Sierra de Guatemala and Sierra de la Colmena (Micos). Since their discovery, multiple studies have attempted to characterize the timing and the number of events that gave rise to the evolution of these cave-adapted ecotypes. Here, using RADseq and genome-wide sequencing, we assessed the phylogenetic relationships, genetic structure and gene flow events between the cave and surface Astyanax mexicanus populations, to estimate the tempo and mode of evolution of the cave-adapted ecotypes. We also evaluated the body shape evolution across different cave lineages using geometric morphometrics to examine the role of phylogenetic signal versus environmental pressures. We found strong evidence of parallel evolution of cave-adapted ecotypes derived from two separate lineages of surface fish and hypothesize that there may be up to four independent invasions of caves from surface fish. Moreover, a strong congruence between the genetic structure and geographic distribution was observed across the cave populations, with the Sierra de Guatemala the region exhibiting most genetic drift among the cave populations analysed. Interestingly, we found no evidence of phylogenetic signal in body shape evolution, but we found support for parallel evolution in body shape across independent cave lineages, with cavefish from the Sierra de El Abra reflecting the most divergent morphology relative to surface and other cavefish populations.

Genetic mapping of craniofacial traits in the Mexican tetra reveals loci associated with bite differences between cave and surface fish.

BACKGROUND: The Mexican tetra, Astyanax mexicanus, includes interfertile surface-dwelling and cave-dwelling morphs, enabling powerful studies aimed at uncovering genes involved in the evolution of cave-associated traits. Compared to surface fish, cavefish harbor several extreme traits within their skull, such as a protruding lower jaw, a wider gape, and an increase in tooth number. These features are highly variable between individual cavefish and even across different cavefish populations. RESULTS: To investigate these traits, we created a novel feeding behavior assay wherein bite impressions could be obtained. We determined that fish with an underbite leave larger bite impressions with an increase in the number of tooth marks. Capitalizing on the ability to produce hybrids from surface and cavefish crosses, we investigated genes underlying these segregating orofacial traits by performing Quantitative Trait Loci (QTL) analysis with F2 hybrids. We discovered significant QTL for bite (underbite vs. overbite) that mapped to a single region of the Astyanax genome. Within this genomic region, multiple genes exhibit coding region mutations, some with known roles in bone development. Further, we determined that there is evidence that this genomic region is under natural selection. CONCLUSIONS: This work highlights cavefish as a valuable genetic model for orofacial patterning and will provide insight into the genetic regulators of jaw and tooth development.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

An Adult Brain Atlas Reveals Broad Neuroanatomical Changes in Independently Evolved Populations of Mexican Cavefish.

A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus, is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant, ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus, and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate brain atlases of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions that have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an enlargement of the hypothalamus in all cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and prethalamus. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior.

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

BACKGROUND: Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. RESULTS: Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. CONCLUSION: The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.

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 data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured 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.

A local duplication of the Melanocortin receptor 1 locus in Astyanax.

In this study, we report evidence of a novel duplication of Melanocortin receptor 1 (Mc1r) in the cavefish genome. This locus was discovered following the observation of excessive allelic diversity in a ∼820 bp fragment of Mc1r amplified via degenerate PCR from a natural population of Astyanax aeneus fish from Guerrero, Mexico. The cavefish genome reveals the presence of two closely related Mc1r open reading frames separated by a 1.46 kb intergenic region. One open reading frame corresponds to the previously reported Mc1r receptor, and the other open reading frame (duplicate copy) is 975 bp in length, encoding a receptor of 325 amino acids. Sequence similarity analyses position both copies in the syntenic region of the single Mc1r locus in 16 representative craniate genomes spanning bony fish (including Astyanax) to mammals, suggesting we discovered tandem duplicates of this important gene. The two Mc1r copies share ∼89% sequence similarity and, within Astyanax, are more similar to one another compared to other melanocortin family members. Future studies will inform the precise functional significance of the duplicated Mc1r locus and if this novel copy number variant may have adaptive significance for the Astyanax lineage.

Contrasting demographic and genetic estimates of dispersal in the endangered Coahuilan box turtle: a contemporary approach to conservation.

The evolutionary viability of an endangered species depends upon gene flow among subpopulations and the degree of habitat patch connectivity. Contrasting population connectivity over ecological and evolutionary timescales may provide novel insight into what maintains genetic diversity within threatened species. We employed this integrative approach to evaluating dispersal in the critically endangered Coahuilan box turtle (Terrapene coahuila) that inhabits isolated wetlands in the desert-spring ecosystem of Cuatro Ciénegas, Mexico. Recent wetland habitat loss has altered the spatial distribution and connectivity of habitat patches; and we therefore predicted that T. coahuila would exhibit limited movement relative to estimates of historic gene flow. To evaluate contemporary dispersal patterns, we employed mark-recapture techniques at both local (wetland complex) and regional (intercomplex) spatial scales. Gene flow estimates were obtained by surveying genetic variation at nine microsatellite loci in seven subpopulations located across the species' geographical range. The mark-recapture results at the local spatial scale reveal frequent movement among wetlands that was unaffected by interwetland distance. At the regional spatial scale, dispersal events were relatively less frequent between wetland complexes. The complementary analysis of population genetic substructure indicates strong historic gene flow (global F(ST) = 0.01). However, a relationship of genetic isolation by distance across the geographical range suggests that dispersal limitation exists at the regional scale. Our approach of contrasting direct and indirect estimates of dispersal at multiple spatial scales in T. coahuila conveys a sustainable evolutionary trajectory of the species pending preservation of threatened wetland habitats and a range-wide network of corridors.