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.

Dispatches from the neighborhood watch: Using citizen science and field survey data to document color morph frequency in space and time.

Heritable color polymorphisms have a long history of study in evolutionary biology, though they are less frequently examined today than in the past. These systems, where multiple discrete, visually identifiable color phenotypes co-occur in the same population, are valuable for tracking evolutionary change and ascertaining the relative importance of different evolutionary mechanisms. Here, we use a combination of citizen science data and field surveys in the Great Lakes region of North America to identify patterns of color morph frequencies in the eastern gray squirrel (Sciurus carolinensis). Using over 68,000 individual squirrel records from both large and small spatial scales, we identify the following patterns: (a) the melanistic (black) phenotype is often localized but nonetheless widespread throughout the Great Lakes region, occurring in all states and provinces sampled. (b) In Ohio, where intensive surveys were performed, there is a weak but significantly positive association between color morph frequency and geographic proximity of populations. Nonetheless, even nearby populations often had radically different frequencies of the melanistic morph, which ranged from 0% to 96%. These patterns were mosaic rather than clinal. (c) In the Wooster, Ohio population, which had over eight years of continuous data on color morph frequency representing nearly 40,000 records, we found that the frequency of the melanistic morph increased gradually over time on some survey routes but decreased or did not change over time on others. These differences were statistically significant and occurred at very small spatial scales (on the order of hundreds of meters). Together, these patterns are suggestive of genetic drift as an important mechanism of evolutionary change in this system. We argue that studies of color polymorphism are still quite valuable in advancing our understanding of fundamental evolutionary processes, especially when coupled with the growing availability of data from citizen science efforts.

Substrate Promiscuity of a Paralytic Shellfish Toxin Amidinotransferase.

Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins, and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.

Developmental Transcriptomic Analysis of the Cave-Dwelling Crustacean, Asellus aquaticus.

Cave animals are a fascinating group of species often demonstrating characteristics including reduced eyes and pigmentation, metabolic efficiency, and enhanced sensory systems. Asellus aquaticus, an isopod crustacean, is an emerging model for cave biology. Cave and surface forms of this species differ in many characteristics, including eye size, pigmentation, and antennal length. Existing resources for this species include a linkage map, mapped regions responsible for eye and pigmentation traits, sequenced adult transcriptomes, and comparative embryological descriptions of the surface and cave forms. Our ultimate goal is to identify genes and mutations responsible for the differences between the cave and surface forms. To advance this goal, we decided to use a transcriptomic approach. Because many of these changes first appear during embryonic development, we sequenced embryonic transcriptomes of cave, surface, and hybrid individuals at the stage when eyes and pigment become evident in the surface form. We generated a cave, a surface, a hybrid, and an integrated transcriptome to identify differentially expressed genes in the cave and surface forms. Additionally, we identified genes with allele-specific expression in hybrid individuals. These embryonic transcriptomes are an important resource to assist in our ultimate goal of determining the genetic underpinnings of the divergence between the cave and surface forms.

Genetic analysis reveals candidate genes for activity QTL in the blind Mexican tetra, Astyanax mexicanus.

Animal models provide useful tools for exploring the genetic basis of morphological, physiological and behavioral phenotypes. Cave-adapted species are particularly powerful models for a broad array of phenotypic changes with evolutionary, developmental and clinical relevance. Here, we explored the genetic underpinnings of previously characterized differences in locomotor activity patterns between the surface-dwelling and Pachón cave-dwelling populations of Astyanax mexicanus. We identified multiple novel QTL underlying patterns in overall levels of activity (velocity), as well as spatial tank use (time spent near the top or bottom of the tank). Further, we demonstrated that different regions of the genome mediate distinct patterns in velocity and tank usage. We interrogated eight genomic intervals underlying these activity QTL distributed across six linkage groups. In addition, we employed transcriptomic data and draft genomic resources to generate and evaluate a list of 36 potential candidate genes. Interestingly, our data support the candidacy of a number of genes, but do not suggest that differences in the patterns of behavior observed here are the result of alterations to certain candidate genes described in other species (e.g., teleost multiple tissue opsins, melanopsins or members of the core circadian clockwork). This study expands our knowledge of the genetic architecture underlying activity differences in surface and cavefish. Future studies will help define the role of specific genes in shaping complex behavioral phenotypes in Astyanax and other vertebrate taxa.

Characterization and comparison of activity profiles exhibited by the cave and surface morphotypes of the blind Mexican tetra, Astyanax mexicanus.

Departure from normal circadian rhythmicity and exposure to atypical lighting cues has been shown to adversely affect human health and wellness in a variety of ways. In contrast, adaptation to extreme environments has led many species to alter or even entirely abandon their reliance upon cyclic environmental inputs, principally daily cycles of light and darkness. The extreme darkness, stability and isolation of cave ecosystems has made cave-adapted species particularly attractive systems in which to study the consequences of life without light and the strategies that allow species to survive and even thrive in such environments. In order to further explore these questions, we have assessed the rhythmicity of locomotion in the blind Mexican tetra, Astyanax mexicanus, under controlled laboratory conditions. Using high-resolution video tracking assays, we characterized patterns in locomotor activity and spatial tank usage for members of the surface and Pachón cave populations. Here we demonstrate that cavefish have a higher overall level of activity and use the space within the trial tank differently than surface fish. Further, Pachón cavefish show circadian rhythmicity in both activity and spatial tank usage under a 12:12 light/dark cycle. We provide further evidence that these cavefish retain a weakly light-entrainable, endogenous circadian oscillator with limited capability to sustain rhythms in activity, but not spatial tank usage, in the absence of photic cues. Finally, we demonstrate a putative behavioral "masking effect" contributing to behavioral rhythms and provide evidence that exposure to constant darkness during development may alter behavioral patterns later in life.

Natural bone fragmentation in the blind cave-dwelling fish, Astyanax mexicanus: candidate gene identification through integrative comparative genomics.

Animals that colonize dark and nutrient-poor subterranean environments evolve numerous extreme phenotypes. These include dramatic changes to the craniofacial complex, many of which are under genetic control. These phenotypes can demonstrate asymmetric genetic signals wherein a QTL is detected on one side of the face but not the other. The causative gene(s) underlying QTL are difficult to identify with limited genomic resources. We approached this task by searching for candidate genes mediating fragmentation of the third suborbital bone (SO3) directly inferior to the orbit of the eye. We integrated positional genomic information using emerging Astyanax resources, and linked these intervals to homologous (syntenic) regions of the Danio rerio genome. We identified a discrete, approximately 6 Mb, conserved region wherein the gene causing SO3 fragmentation likely resides. We interrogated this interval for genes demonstrating significant differential expression using mRNA-seq analysis of cave and surface morphs across life history. We then assessed genes with known roles in craniofacial evolution and development based on GO term annotation. Finally, we screened coding sequence alterations in this region, identifying two key genes: transforming growth factor ß3 (tgfb3) and bone morphogenetic protein 4 (bmp4). Of these candidates, tgfb3 is most promising as it demonstrates significant differential expression across multiple stages of development, maps close (<1 Mb) to the fragmentation critical locus, and is implicated in a variety of other animal systems (including humans) in non-syndromic clefting and malformations of the cranial sutures. Both abnormalities are analogous to the failure-to-fuse phenotype that we observe in SO3 fragmentation. This integrative approach will enable discovery of the causative genetic lesions leading to complex craniofacial features analogous to human craniofacial disorders. This work underscores the value of cave-dwelling fish as a powerful evolutionary model of craniofacial disease, and demonstrates the power of integrative system-level studies for informing the genetic basis of craniofacial aberrations in nature.

A high-density linkage map for Astyanax mexicanus using genotyping-by-sequencing technology.

The Mexican tetra, Astyanax mexicanus, is a unique model system consisting of cave-adapted and surface-dwelling morphotypes that diverged >1 million years (My) ago. This remarkable natural experiment has enabled powerful genetic analyses of cave adaptation. Here, we describe the application of next-generation sequencing technology to the creation of a high-density linkage map. Our map comprises more than 2200 markers populating 25 linkage groups constructed from genotypic data generated from a single genotyping-by-sequencing project. We leveraged emergent genomic and transcriptomic resources to anchor hundreds of anonymous Astyanax markers to the genome of the zebrafish (Danio rerio), the most closely related model organism to our study species. This facilitated the identification of 784 distinct connections between our linkage map and the Danio rerio genome, highlighting several regions of conserved genomic architecture between the two species despite ~150 My of divergence. Using a Mendelian cave-associated trait as a proof-of-principle, we successfully recovered the genomic position of the albinism locus near the gene Oca2. Further, our map successfully informed the positions of unplaced Astyanax genomic scaffolds within particular linkage groups. This ability to identify the relative location, orientation, and linear order of unaligned genomic scaffolds will facilitate ongoing efforts to improve on the current early draft and assemble future versions of the Astyanax physical genome. Moreover, this improved linkage map will enable higher-resolution genetic analyses and catalyze the discovery of the genetic basis for cave-associated phenotypes.

Complex craniofacial changes in blind cave-dwelling fish are mediated by genetically symmetric and asymmetric loci.

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 F2 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.

An integrated transcriptome-wide analysis of cave and surface dwelling Astyanax mexicanus.

Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptome analysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F(2) pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected in surface fish. This resource will enable powerful genetic and genomic analyses in the future that will better clarify the heritable genetic changes governing adaptation to the cave environment.