Environmental perturbations often lead to the evolution of multiple traits. Determining whether shared genetic factors underlie multi-trait evolution is a central question in evolutionary biology. In the Mexican tetra, Astyanax mexicanus, cave-dwelling populations have repeatedly evolved multiple traits. The repeated evolution of these traits, paired with robust environmental differences between the surface and cave habitats, provide an opportunity to investigate the genetic basis of multi-trait evolution. Here, we investigate the extent to which shared genetic mechanisms underlie the repeated evolution of multiple traits in cavefish. Across cave populations, we find evidence for shared and distinct genetic mechanisms contributing to the evolution of individual traits. Further, multiple traits covary in cave-surface F2 hybrids. Moreover, many of the same pairs of traits covary in independently evolved cave populations. Finally, multiple traits differ between pigmented and albino F2 fish. Quantification of these traits in surface fish with mutations in the albinism gene oculocutaneous albinism 2 (oca2) reveals that mutations in oca2 reduce bottom-dwelling behaviour in A. mexicanus. Together, these findings suggest that multi-trait evolution occurs repeatedly through shared genetic factors across A. mexicanus cave populations. These results are consistent with pleiotropy and/or linkage playing a large role in multi-trait evolution in this species.

Similar traits repeatedly evolve across independent populations in response to similar environmental conditions. For many repeatedly evolved traits it is unknown if populations evolve similar traits through the same or different genetic mechanisms. To address this question, we leveraged the Mexican tetra fish, Astyanax mexicanus , which has evolved repeatedly through altering many traits including reduced sleep duration, eye degeneration, and metabolic shifts to accommodate limited nutrient availability. We defined whether shared or independent genetic architecture govern the repeated evolution of sleep loss, increased food consumption, early onset adipose deposition, and eye loss in different evolutionary origins of the cavefish phenotype by using Quantitative Trait Locus (QTL) mapping across three cave x surface F2 mapping populations. We found that, among the traits evaluated, eye loss exhibits the most genetic repeatability, with ~43% of QTL shared across lineages. Sleep loss and metabolic traits (i.e., feeding, adiposity) were genetically less repeatable, with only ~25-33% of QTL shared across lineages. Next, we explored whether QTL for metabolism, eye loss, and sleep traits in cavefish co-localize in the cavefish genome and could be inherited together to facilitate potential cavefish adaptation. Although these traits have repeatedly co-evolved in cave populations, we did not find evidence for extensive genetic linkage among them. Overall, we found that genetic repeatability is a common feature in the repeated evolution of cave traits, the extent of genetic repeatability varies across cave traits, and that there is little evidence for widespread co-localization of sleep, eye loss, and metabolic traits within the genome.

Similar traits repeatedly evolve across independent populations in response to similar environmental conditions. For many repeatedly evolved traits, it is unknown if populations evolve similar traits through the same or different genetic mechanisms. To address this question, we leveraged the Mexican tetra fish, Astyanax mexicanus, which has repeatedly evolved many traits including reduced sleep duration, eye degeneration, and metabolic shifts to accommodate limited nutrient availability. We defined whether shared or independent genetic architecture governs the repeated evolution of sleep loss, increased food consumption, early onset adipose deposition, and eye loss in different evolutionary origins of the cavefish phenotype by using Quantitative Trait Locus (QTL) mapping across three cave x surface F2 mapping populations. We found that, among the traits evaluated, eye loss exhibits the most genetic repeatability, with ∼43% of QTL shared across lineages. Sleep loss and metabolic traits (i.e., feeding, adiposity) were genetically less repeatable, with only ∼25-33% of QTL shared across lineages. Next, we explored whether QTL for metabolism, eye loss, and sleep traits in cavefish co-localize in the cavefish genome and are inherited together to facilitate potential cavefish adaptation. Although these traits have repeatedly co-evolved in cave populations, we did not find evidence for extensive genetic linkage among them. Overall, we found that genetic repeatability is a common feature in the repeated evolution of cave traits, the extent of genetic repeatability varies across cave traits, and there is little evidence for widespread colocalization of sleep, eye loss, and metabolic traits within the genome.

Structural variants (SVs) are increasingly recognized as key contributors to adaptive evolution, yet they remain underexplored compared with single-nucleotide variation. To understand how large-scale genomic changes shape repeated evolution, we leveraged multiple levels of sequence data across the powerful evolutionary model system of the Mexican tetra fish (Astyanax mexicanus). We constructed one of the first pangenome graphs from a naturally evolving vertebrate, enabling comprehensive discovery of SVs among 120 fish from 11 populations. We discover substantial amounts of structural variation and explore the roles of genomic biases and selection in shaping the distribution of these variants. More than 2400 high-confidence cave-specific deletions are enriched in biological pathways involved in vision, metabolism, and behavior and cluster nonrandomly in quantitative trait loci linked to cavefish traits. Additionally, 67 genes harbor unique deletions between independent cavefish lineages. These reused genes show evidence of population-specific selection (99% contain selective sweeps compared with 8%-15% in genes lacking SVs), indicating that deletions likely rose in frequency through repeated positive selection rather than drift. Together, these results reveal that recurrent deletion events have repeatedly contributed to the evolution of cave-adapted phenotypes and highlight deletions as underexplored contributors of adaptive evolution in extreme environments.

Determining the genetic factors contributing to trait evolution is critical for understanding how and why traits evolve; however, establishing which genes underlie the evolution of complex traits remains challenging. The freshwater fish Astyanax mexicanus, a species that includes blind, cave-dwelling and eyed, surface-dwelling fish, is a powerful model for evolutionary genetics. While genetic mapping studies in this species previously identified genomic regions associated with cave-derived traits, few causative genes and genetic changes have been identified. Here, we develop methods to identify and rapidly functionally assess candidate genes in A. mexicanus, focusing on a defining trait of cave animals, eye loss. Candidate genes were identified based on whether they fell within an eye-related quantitative trait locus, were differentially expressed between surface and cave eyes, and showed evidence of positive selection in cavefish. Single-nucleus RNA-sequencing revealed that these candidate genes were expressed in multiple cell types during development, including those in different tissues of the eye. CRISPR-Cas9-based mutagenesis demonstrated that disruption of nine of these candidate genes in surface fish resulted in altered eye size. Perturbation of one of these genes, fibulin-7 (fbln7), revealed changes in eye size across multiple stages of eye development. Together, this work identified multiple genes associated with the evolution of eye degeneration in A. mexicanus. Further, this study represents a roadmap for rapid identification and functional assessment of candidate genes implicated in the evolution of traits in cavefish that can be applied to other evolutionary genetic models.

Understanding the phenotypic consequences of naturally occurring genetic changes, as well as their impact on fitness, is fundamental to understanding how organisms adapt to an environment. This is critical when genetic variants have pleiotropic effects, as determining how each phenotype impacted by a gene contributes to fitness is essential to understand how and why traits have evolved. Here, we characterized the effects of mutations in the oca2 gene, which underlie albinism and reductions of sleep in the blind Mexican cavefish Astyanax mexicanus, on larval prey capture. We found that when surface A. mexicanus with engineered mutations in oca2 are hunting, they use cave-like, wide-angle strikes to capture prey. However, unlike cavefish or surface fish in the dark, which utilize the lateral line when hunting, oca2 mutant (oca2Δ2bp/Δ2bp) surface fish can use vision when striking at prey from wide angles. We found that when raised under lighted conditions, pigmented surface fish outcompete albino oca2Δ2bp/Δ2bp surface fish when hunting in lighted conditions. In contrast, when surface fish are reared in darkness, oca2Δ2bp/Δ2bp surface fish outcompete their wild type siblings in the dark. This raises the possibility that albinism is detrimental to larval feeding in a surface-like lighted environment, but may confer an advantage to fish in cave-like, dark environments. Together, these results demonstrate that oca2 plays a role in larval feeding behavior in A. mexicanus, and expand our understanding of the pleiotropic phenotypic consequences of oca2 in cavefish evolution.

The regulation of bone size is a poorly understood and complex developmental process. Evolutionary models can enable insight through interrogation of the developmental and molecular underpinnings of natural variation in bone size and shape. Here, we examine the Mexican tetra (Astyanax mexicanus), a species of teleost fish comprising of an extant river-dwelling surface fish and obligate cave-dwelling fish. These divergent morphs have evolved for thousands of years in drastically different habitats, which have led to diverse phenotypic differences. Among many craniofacial aberrations, cavefish harbor a wider gape, an underbite, and larger jaws compared to surface-dwelling morphs. Morphotypes are inter-fertile, allowing quantitative genetic analyses in F2 pedigrees derived from surface × cavefish crosses. Here, we used quantitative trait locus (QTL) analysis to determine the genetic basis of jaw size. Strikingly, we discovered a single genomic region associated with several jaw size metrics. Future work identifying genetic lesions that explain differences in jaw development will provide new insight to the mechanisms driving bone size differences across vertebrate taxa.

Loss-of-function alleles are a pertinent source of genetic variation with the potential to contribute to adaptation. Cave-adapted organisms exhibit striking loss of ancestral traits such as eyes and pigment, suggesting that loss-of-function alleles may play an outsized role in these systems. Here, we leverage 141 whole genome sequences to evaluate the evolutionary history and adaptive potential of single nucleotide premature termination codons (PTCs) in Mexican tetra. We find that cave populations contain significantly more PTCs at high frequency than surface populations. We also find that PTCs occur more frequently in genes with inherent relaxed evolutionary constraint relative to the rest of the genome. Using SLiM to simulate PTC evolution in a cavefish population, we show that the smaller population size and increased genetic drift is sufficient to account for the observed increase in PTC frequency in cave populations without positive selection. Using CRISPR-Cas9, we show that mutation of one of these genes, pde6c, produces phenotypes in surface Mexican tetra that mimic cave-derived traits. Finally, we identify a small subset of candidate genes that contain high-frequency PTCs in cave populations, occur within selective sweeps, and may contribute to beneficial traits such as reduced energy expenditure, suggesting that a handful of PTCs may be adaptive. Overall, our work provides a rare characterization of PTCs across wild populations and finds that they may have an important role in loss-of-function phenotypes, contributing to a growing body of literature showing genome evolution through relaxed constraint in subterranean organisms.

Sleep is an evolutionarily ancient behavior, yet multiple cave-dwelling populations of the Mexican tetra, Astyanax mexicanus, have converged on sleep loss compared to surface fish. However, most of the 34 cave populations remain unstudied, and sleep in natural habitats is largely unknown. To address this, we measured sleep and activity in 15 representative populations of surface, cave, and hybrid populations. All cavefish and hybrid populations tested exhibited drastically reduced sleep, including hybrid populations with diverse eye and pigmentation phenotypes. Mapping behavior onto the A. mexicanus phylogeny revealed that reduced sleep and elevated locomotor activity evolved independently multiple times. Field experiments confirmed that wild fish also exhibit sleep loss, paralleling laboratory findings. These results demonstrate deep evolutionary convergence on sleep loss across cavefish lineages and suggest that sleep reduction is a primary trait contributing to adaptation in subterranean environments.

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.

Hibernation has evolved several times in mammals to overcome harsh winter climates and food scarcity. During hibernation, animals undergo extreme shifts in metabolic rate, heart rate, respiration, and body temperature. These changes reduce energy consumption and allow animals to survive solely on their fat reserves. Understanding the mechanisms for these extreme shifts has long been recognized as a model for translational medicine as hibernators do not exhibit the same adverse effects of extended immobility that non-hibernating mammals suffer. Though work on individual species has illuminated important mechanisms of these functional changes, the genomic basis of this phenotype remains largely unknown, and few studies have drawn on comparative work to elucidate commonalities across diverse hibernating mammals. Synthesizing both single species and comparative approaches, we use metabolomic data from active and denning black bears (Ursus americanus) to guide bioinformatic analyses of genes using tests of selection and evolutionary rate convergence across independent lineages of hibernating mammals. We identify several genes with significant signatures of selection and evolutionary rate convergence in hibernators that represent both previously known and novel genetic mechanisms of the hibernation phenotype. These data provide novel insights into the genetic basis of this adaptation and serve to direct clinical research in hibernation-based therapies. ### Competing Interest Statement The authors have declared no competing interest.

Eye loss is a hallmark trait of animals inhabiting perpetual darkness, such as caves. The Mexican tetra (Astyanax mexicanus) provides an unparalleled model for studying the genetic basis of eye loss. There are two interfertile morphs of the Mexican tetra, sighted surface fish and multiple independently evolved eyeless, blind cavefish populations. Despite decades of research on eye loss in this species, our understanding of the precise genetic basis remains sparse. Here, we focused on the unique Caballo Moro cave, where there is a karst window collapse that introduced sunlight and coexistence of both eyed and eyeless cavefish of similar genetic background. This unique genetic mosaic allowed us to pinpoint coding mutations in Connexin 50 (Cx50), also known as gap junction protein alpha 8b (Gja8b), as critical in the genetic basis of eye loss. CRISPR based knockouts of Cx50 in surface fish result in small or absent eyes as young as 48 hours post-fertilization. Further, we identified similar mutations in Cx50 that alter predicted protein structure among other cave-dwelling fish and even subterranean mammals, indicating a conserved evolutionary mechanism of Cx50 mutations. We introduced a mutation (CX50-S89K) in mice, which resulted in cataracts, smaller eyes, and smaller lenses. Mutations in CX50 mimic those identified in human congenital cataracts. We additionally leveraged phenotypic variation in a hybrid cavefish population to demonstrate that eyes provide fish with a metabolic advantage, providing a mechanism by which loss of eyes could be favored by selection. This unique hybridization event allowed us to identify novel alleles that contribute to the convergent evolution of eye loss, providing profound insights into the genetic underpinnings of one of nature's most fascinating adaptive traits.

Sleep is an evolutionarily ancient and nearly universal behavior throughout the animal kingdom. Multiple cave-dwelling populations of the Mexican tetra, Astyanax mexicanus, have converged on sleep loss compared to river dwelling surface fish. However, the extent to which sleep has been lost across cave populations, and whether sleep loss occurs in wild fish, is poorly understood. We have measured sleep and locomotor activity in 15 distinct populations of A. mexicanus representing multiple lineages that are broadly representative of the 34 cavefish populations identified to date. Strikingly, sleep was largely lost in all cave and hybrid populations that were tested. Hybrid populations retained intermediate eye and pigmentation phenotypes, suggesting sleep loss precedes cave-associated morphological changes. Mapping behavioral changes onto the phylogeny of A. mexicanus populations revealed that independent loss of sleep and elevated locomotor activity has occurred at least three times. Analysis of sleep in the wild confirms that the sleep loss phenotype observed in lab-reared fish is also present in the natural environment. Together, these findings reveal deep evolutionary convergence on sleep loss in cavefish and provide evidence for sleep loss as a primary trait contributing to cave adaptation. ### Competing Interest Statement The authors have declared no competing interest.

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 clusters 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 A. mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish.

Astyanax mexicanus is an emerging model system used to study development, evolution, and behavior of multiple cavefish populations that have repeatedly evolved from conspecific surface fish. Although surface and cavefish live and breed in the laboratory, there are no rapid methods for distinguishing between different cavefish populations. We present 2 methods for genotyping fish for a total of 16 population-specific markers using methods that are easy and inexpensive to implement in a basic molecular biology laboratory. This resource will help researchers maintain independent stocks within the laboratory and distinguish between fish from different populations.

Recent colonization of extreme environments provides unique opportunities to study the early steps of adaptation and the potential for rapid convergent evolution. However, phenotypic shifts during recent colonization may also be due to plasticity in response to changes in the rearing environment. Here, we analyzed a suite of morphological and behavioral traits in paired surface, subterranean, and facultatively subterranean Mexican tetras ( Astyanax mexicanus) from recent introductions in two separate watersheds outside of their native range. We found a variety of phenotypic and behavioral shifts between subterranean and surface populations that are similar to those observed in relatively ancient populations in Mexico. Despite this rapid morphological divergence, we found that most of these trait differences were due to plasticity in response to rearing environments. While most trait assays in common-garden, lab-raised fish indicated that phenotypic shifts in wild fish were the result of plasticity, we also found evidence of genetic control in several traits present in subterranean populations. Interestingly, wall-following behavior, an important subterranean foraging behavior, was greater in lab-born subterranean fish than in lab-born surface fish, suggesting rapid divergence of this trait between subterranean and surface populations. Thus, this study sheds light on the early steps of subterranean evolution, identifies potential rapid behavioral evolution, and suggests that plasticity in traits involving exploratory behavior may facilitate subterranean colonization.

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.

Gene loss is an important mechanism for evolution in low-light or cave environments where visual adaptations often involve a reduction or loss of eyesight. The plaat gene family are phospholipases essential for the degradation of organelles in the lens of the eye. They translocate to damaged organelle membranes, inducing them to rupture. This rupture is required for lens transparency and is essential for developing a functioning eye. Plaat3 is thought to be responsible for this role in mammals, while plaat1 is thought to be responsible in other vertebrates. We used a macroevolutionary approach and comparative genomics to examine the origin, loss, synteny, and selection of plaat1 across bony fishes and tetrapods. We show that plaat1 (likely ancestral to all bony fish + tetrapods) has been lost in squamates and is significantly degraded in lineages of low-visual acuity and blind mammals and fish. Our findings suggest that plaat1 is important for visual acuity across bony vertebrates, and that its loss through relaxed selection and pseudogenization may have played a role in the repeated evolution of visual systems in low-light-environments. Our study sheds light on the importance of gene-loss in trait evolution and provides insights into the mechanisms underlying visual acuity in low-light environments.

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.