Mutations in the albinism gene oca2 alter vision-dependent prey capture behavior in the Mexican tetra.

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. A striking example of a pleiotropic gene contributing to trait evolution is the oca2 gene, coding mutations in which underlie albinism and reductions of sleep in the blind Mexican cavefish, Astyanax mexicanus . Here, we characterize the effects of mutations in the oca2 gene on larval prey capture. We find that when conspecific surface fish with engineered mutations in the oca2 allele are hunting, they use cave-like, wide angle strikes to capture prey. However, unlike cavefish or surface fish in the dark, which rely on lateral line mediated hunting, oca2 mutant surface fish use vision when striking at prey from wide angles. Finally, we find that while oca2 mutant surface fish do not outcompete pigmented surface siblings in the dark, pigmented fish outcompete albino fish in the light. This raises the possibility that albinism is detrimental to larval feeding in a surface-like lighted environment, but does not have negative consequences for fish in cave-like, dark environments. Together, these results demonstrate that oca2 plays a role in larval feeding behavior in A. mexicanus . Further, they expand our understanding of the pleiotropic phenotypic consequences of oca2 in cavefish evolution.

Gene loss and relaxed selection of plaat1 in vertebrates adapted to low-light environments.

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 encodes phospholipases essential for the degradation of organelles in the lens of the eye. These phospholipases 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 showed that plaat1 (probably 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 fishes. 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.

Astyanax mexicanus surface and cavefish chromosome-scale assemblies for trait variation discovery.

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and non-coding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared to other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared to a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species.

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.

Defining the Unseen: Population-Specific Markers for Astyanax mexicanus Blind 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.

Astyanax mexicanus surface and cavefish chromosome-scale assemblies for trait variation discovery.

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and non-coding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared to other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared to a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species.

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.

Developmental environment contributes to rapid trait shifts among newly colonized subterranean habitats.

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.

Selection-driven trait loss in independently evolved cavefish populations.

Laboratory studies have demonstrated that a single phenotype can be produced by many different genotypes; however, in natural systems, it is frequently found that phenotypic convergence is due to parallel genetic changes. This suggests a substantial role for constraint and determinism in evolution and indicates that certain mutations are more likely to contribute to phenotypic evolution. Here we use whole genome resequencing in the Mexican tetra, Astyanax mexicanus, to investigate how selection has shaped the repeated evolution of both trait loss and enhancement across independent cavefish lineages. We show that selection on standing genetic variation and de novo mutations both contribute substantially to repeated adaptation. Our findings provide empirical support for the hypothesis that genes with larger mutational targets are more likely to be the substrate of repeated evolution and indicate that features of the cave environment may impact the rate at which mutations occur.

Gene loss and relaxed selection of plaat1 in vertebrates adapted to low-light environments.

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.

Widespread convergent evolution of alpha-neurotoxin resistance in African mammals.

Convergent evolution is central to the study of adaptation and has been used to understand both the limits of evolution and the diverse patterns and processes which result in adaptive change. Resistance to snake venom alpha-neurotoxins (αNTXs) is a case of widespread convergence having evolved several times in snakes, lizards and mammals. Despite extreme toxicity of αNTXs, substitutions in its target, the nicotinic acetylcholine receptor (nAChR), prevent αNTX binding and render species resistant. Recently, the published meerkat (Herpestidae) genome revealed that meerkats have the same substitutions in nAChR as the venom-resistant Egyptian mongoose (Herpestidae), suggesting that venom-resistant nAChRs may be ancestral to Herpestids. Like the mongoose, many other species of feliform carnivores prey on venomous snakes, though their venom resistance has never been explored. To evaluate the prevalence and ancestry of αNTX resistance in mammals, we generate a dataset of mammalian nAChR using museum specimens and public datasets. We find five instances of convergent evolution within feliform carnivores, and an additional eight instances across all mammals sampled. Tests of selection show that these substitutions are evolving under positive selection. Repeated convergence suggests that this adaptation played an important role in the evolution of mammalian physiology and potentially venom evolution.

Assessing genomic and ecological differentiation among subspecies of the rough-footed mud turtle, Kinosternon hirtipes.

Combining genetic and ecological measures of differentiation can provide compelling evidence for ecological and genetic divergence among lineages. The rough-footed mud turtle, Kinosternon hirtipes, is distributed from the Trans-Pecos region of Texas to the highlands of Central Mexico and contains 6 described subspecies, 5 of which are extant. We use ddRAD sequencing and species distribution models to assess levels of ecological and genetic differentiation among these subspecies. We also predict changes in climatically suitable habitat under different climate change scenarios and assess levels of genetic diversity and inbreeding within each lineage. Our results show that there is strong genetic and ecological differentiation among multiple lineages within K. hirtipes, and that this differentiation appears to be the result of vicariance associated with the Trans-Mexican Volcanic Belt. We propose changes to subspecies designations to more accurately reflect the evolutionary relationships among populations and assess threats to each subspecies.

CaveCrawler: an interactive analysis suite for cavefish bioinformatics.

The growing use of genomics in diverse organisms provides the basis for identifying genomic and transcriptional differences across species and experimental conditions. Databases containing genomic and functional data have played critical roles in the development of numerous genetic models but most emerging models lack such databases. The Mexican tetra, Astyanax mexicanus exists as 2 morphs: surface-dwelling and cave-dwelling. There exist at least 30 cave populations, providing a system to study convergent evolution. We have generated a web-based analysis suite that integrates datasets from different studies to identify how gene transcription and genetic markers of selection differ between populations and across experimental contexts. Results of diverse studies can be analyzed in conjunction with other genetic data (e.g. Gene Ontology information), to enable biological inference from cross-study patterns and identify future avenues of research. Furthermore, the framework that we have built for A. mexicanus can be adapted for other emerging model systems.

Hybridization underlies localized trait evolution in cavefish.

Introgressive hybridization may play an integral role in local adaptation and speciation (Taylor and Larson, 2019). In the Mexican tetra Astyanax mexicanus, cave populations have repeatedly evolved traits including eye loss, sleep loss, and albinism. Of the 30 caves inhabited by A. mexicanus, Chica cave is unique because it contains multiple pools inhabited by putative hybrids between surface and cave populations (Mitchell et al., 1977), providing an opportunity to investigate the impact of hybridization on complex trait evolution. We show that hybridization between cave and surface populations may contribute to localized variation in traits associated with cave evolution, including pigmentation, eye development, and sleep. We also uncover an example of convergent evolution in a circadian clock gene in multiple cavefish lineages and burrowing mammals, suggesting a shared genetic mechanism underlying circadian disruption in subterranean vertebrates. Our results provide insight into the role of hybridization in facilitating phenotypic evolution.

Whole-genome variation of transposable element insertions in a maize diversity panel.

Intact transposable elements (TEs) account for 65% of the maize genome and can impact gene function and regulation. Although TEs comprise the majority of the maize genome and affect important phenotypes, genome-wide patterns of TE polymorphisms in maize have only been studied in a handful of maize genotypes, due to the challenging nature of assessing highly repetitive sequences. We implemented a method to use short-read sequencing data from 509 diverse inbred lines to classify the presence/absence of 445,418 nonredundant TEs that were previously annotated in four genome assemblies including B73, Mo17, PH207, and W22. Different orders of TEs (i.e., LTRs, Helitrons, and TIRs) had different frequency distributions within the population. LTRs with lower LTR similarity were generally more frequent in the population than LTRs with higher LTR similarity, though high-frequency insertions with very high LTR similarity were observed. LTR similarity and frequency estimates of nested elements and the outer elements in which they insert revealed that most nesting events occurred very near the timing of the outer element insertion. TEs within genes were at higher frequency than those that were outside of genes and this is particularly true for those not inserted into introns. Many TE insertional polymorphisms observed in this population were tagged by SNP markers. However, there were also 19.9% of the TE polymorphisms that were not well tagged by SNPs (R2 < 0.5) that potentially represent information that has not been well captured in previous SNP-based marker-trait association studies. This study provides a population scale genome-wide assessment of TE variation in maize and provides valuable insight on variation in TEs in maize and factors that contribute to this variation.

The utility of genomic prediction models in evolutionary genetics.

Variation in complex traits is the result of contributions from many loci of small effect. Based on this principle, genomic prediction methods are used to make predictions of breeding value for an individual using genome-wide molecular markers. In breeding, genomic prediction models have been used in plant and animal breeding for almost two decades to increase rates of genetic improvement and reduce the length of artificial selection experiments. However, evolutionary genomics studies have been slow to incorporate this technique to select individuals for breeding in a conservation context or to learn more about the genetic architecture of traits, the genetic value of missing individuals or microevolution of breeding values. Here, we outline the utility of genomic prediction and provide an overview of the methodology. We highlight opportunities to apply genomic prediction in evolutionary genetics of wild populations and the best practices when using these methods on field-collected phenotypes.

Pleiotropic function of the oca2 gene underlies the evolution of sleep loss and albinism in cavefish.

Adaptation to novel environments often involves the evolution of multiple morphological, physiological, and behavioral traits. One striking example of multi-trait evolution is the suite of traits that has evolved repeatedly in cave animals, including regression of eyes, loss of pigmentation, and enhancement of non-visual sensory systems.1,2 The Mexican tetra, Astyanax mexicanus, consists of fish that inhabit at least 30 caves in Mexico and ancestral-like surface fish that inhabit the rivers of Mexico and southern Texas.3 Cave A. mexicanus are interfertile with surface fish and have evolved a number of traits, including reduced pigmentation, eye loss, and alterations to behavior.4-6 To define relationships between different cave-evolved traits, we phenotyped 208 surface-cave F2 hybrid fish for numerous morphological and behavioral traits. We found differences in sleep between pigmented and albino hybrid fish, raising the possibility that these traits share a genetic basis. In cavefish and other species, mutations in oculocutaneous albinism 2 (oca2) cause albinism.7-12 Surface fish with mutations in oca2 displayed both albinism and reduced sleep. Further, this mutation in oca2 fails to complement sleep loss when surface fish harboring this engineered mutation are crossed to independently evolved populations of albino cavefish with naturally occurring mutations in oca2. Analysis of the oca2 locus in wild-caught cave and surface fish suggests that oca2 is under positive selection in 3 cave populations. Taken together, these findings identify oca2 as a novel regulator of sleep and suggest that a pleiotropic function of oca2 underlies the adaptive evolution of albinism and sleep loss.

Repeated evolution of circadian clock dysregulation in cavefish populations.

Circadian rhythms are nearly ubiquitous throughout nature, suggesting they are critical for survival in diverse environments. Organisms inhabiting largely arrhythmic environments, such as caves, offer a unique opportunity to study the evolution of circadian rhythms in response to changing ecological pressures. Populations of the Mexican tetra, Astyanax mexicanus, have repeatedly invaded caves from surface rivers, where individuals must contend with perpetual darkness, reduced food availability, and limited fluctuations in daily environmental cues. To investigate the molecular basis for evolved changes in circadian rhythms, we investigated rhythmic transcription across multiple independently-evolved cavefish populations. Our findings reveal that evolution in a cave environment has led to the repeated disruption of the endogenous biological clock, and its entrainment by light. The circadian transcriptome shows widespread reductions and losses of rhythmic transcription and changes to the timing of the activation/repression of core-transcriptional clock. In addition to dysregulation of the core clock, we find that rhythmic transcription of the melatonin regulator aanat2 and melatonin rhythms are disrupted in cavefish under darkness. Mutants of aanat2 and core clock gene rorca disrupt diurnal regulation of sleep in A. mexicanus, phenocopying circadian modulation of sleep and activity phenotypes of cave populations. Together, these findings reveal multiple independent mechanisms for loss of circadian rhythms in cavefish populations and provide a platform for studying how evolved changes in the biological clock can contribute to variation in sleep and circadian behavior.

Genetic mapping of metabolic traits in the blind Mexican cavefish reveals sex-dependent quantitative trait loci associated with cave adaptation.

BACKGROUND: Despite a longstanding interest in understanding how animals adapt to environments with limited nutrients, we have incomplete knowledge of the genetic basis of metabolic evolution. The Mexican tetra, Astyanax mexicanus, is a species of fish that consists of two morphotypes; eyeless cavefish that have adapted to a low-nutrient cave environment, and ancestral river-dwelling surface fish with abundant access to nutrients. Cavefish have evolved altered blood sugar regulation, starvation tolerance, increased fat accumulation, and superior body condition. To investigate the genetic basis of cavefish metabolic evolution we carried out a quantitative trait loci (QTL) analysis in surface/cave F2 hybrids. We genetically mapped seven metabolism-associated traits in hybrids that were challenged with a nutrient restricted diet. RESULTS: We found that female F2 hybrids are bigger than males and have a longer hindgut, bigger liver, and heavier gonad, even after correcting for fish size. Although there is no difference between male and female blood sugar level, we found that high blood sugar is associated with weight gain in females and lower body weight and fat level in males. We identified a significant QTL associated with 24-h-fasting blood glucose level with the same effect in males and females. Differently, we identified sex-independent and sex-dependent QTL associated with fish length, body condition, liver size, hindgut length, and gonad weight. We found that some of the genes within the metabolism QTL display evidence of non-neutral evolution and are likely to be under selection. Furthermore, we report predicted nonsynonymous changes to the cavefish coding sequence of these genes. CONCLUSIONS: Our study reveals previously unappreciated genomic regions associated with blood glucose regulation, body condition, gonad size, and internal organ morphology. In addition, we find an interaction between sex and metabolism-related traits in A. mexicanus. We reveal coding changes in genes that are likely under selection in the low-nutrient cave environment, leading to a better understanding of the genetic basis of metabolic evolution.