The spatiotemporal and genetic architecture of extraoral taste buds in Astyanax cavefish.

Intense environmental pressures can yield both regressive and constructive traits through complex evolutionary mechanisms. Although regression is well-studied, the biological bases of constructive features are less well understood. Cave-dwelling Astyanax fish harbor prolific extraoral taste buds on their heads, which are absent in conspecific surface-dwellers. Here, we present novel ontogenetic data demonstrating extraoral taste buds appear gradually and late in life history. This appearance is similar but non-identical in different cavefish populations, where patterning has evolved to permit taste bud re-specification across the endoderm-ectoderm germ layer boundary. Quantitative genetic analyses revealed that spatially distinct taste buds on the head are primarily mediated by two different cave-dominant loci. While the precise function of this late expansion on to the head is unknown, the appearance of extraoral taste buds coincides with a dietary shift from live-foods to bat guano, suggesting an adaptive mechanism to detect nutrition in food-starved caves. This work provides fundamental insight to a constructive evolutionary feature, arising late in life history, promising a new window into unresolved features of vertebrate sensory organ development.

Gill morphology adapted to oxygen-limited caves in Astyanax mexicanus.

Sensing and acquiring dissolved oxygen is crucial for nearly all aquatic life. This may become even more vital as dissolved oxygen concentrations continue to decline in many aquatic environments. While certain phenotypes that enable fish to live in low oxygen have been characterized, adaptations that arise following sudden, drastic reductions in dissolved oxygen are relatively unknown. Here, we assessed the blind Mexican cavefish, Astyanax mexicanus, for alterations to gill morphology that may be adaptive for life in hypoxic caves. The Astyanax system provides the unique opportunity to compare gill morphology between stereotypical "surface" adapted morphotypes and obligate cave-dwelling conspecifics. While the surface environment is well-oxygenated, cavefish must cope with significantly reduced oxygen. We began by quantifying traditional morphological gill traits including filament number and length as well as lamellar density and height in surface fish and two distinct cave populations, Pachón and Tinaja. This enabled us to estimate total lamellar height, a proxy for gill surface area. We then used immunohistochemical staining to label 5-HT-positive neuroepithelial cells (NECs), which serve as key oxygen sensors in fish. We discovered an increase in gill surface area for both cavefish populations compared to surface, which may enable a higher capacity of oxygen acquisition. Additionally, we found more NECs in Pachón cavefish compared to both surface fish and Tinaja cavefish, suggesting certain selective pressures may be cave-specific. Collectively, this work provides evidence that cavefish have adapted to low oxygen conditions via alterations to gill morphology and oxygen sensing, and informs evolutionary mechanisms of rapid adaptation to dramatic, chronic hypoxia.

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.

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.

An analysis of lateralized neural crest marker expression across development in the Mexican tetra, Astyanax mexicanus.

The biological basis of lateralized cranial aberrations can be rooted in early asymmetric patterning of developmental tissues. However, precisely how development impacts natural cranial asymmetries remains incompletely understood. Here, we examined embryonic patterning of the cranial neural crest at two phases of embryonic development in a natural animal system with two morphotypes: cave-dwelling and surface-dwelling fish. Surface fish are highly symmetric with respect to cranial form at adulthood, however adult cavefish harbor diverse cranial asymmetries. To examine if lateralized aberrations of the developing neural crest underpin these asymmetries, we used an automated technique to quantify the area and expression level of cranial neural crest markers on the left and right sides of the embryonic head. We examined the expression of marker genes encoding both structural proteins and transcription factors at two key stages of development: 36 hpf (∼mid-migration of the neural crest) and 72 hpf (∼early differentiation of neural crest derivatives). Interestingly, our results revealed asymmetric biases at both phases of development in both morphotypes, however consistent lateral biases were less common in surface fish as development progressed. Additionally, this work provides the information on neural crest development, based on whole-mount expression patterns of 19 genes, between stage-matched cave and surface morphs. Further, this study revealed 'asymmetric' noise as a likely normative component of early neural crest development in natural Astyanax fish. Mature cranial asymmetries in cave morphs may arise from persistence of asymmetric processes during development, or as a function of asymmetric processes occurring later in the life history.

Reinterpreting the work of Charles Breder: Sensory neuromasts and orbital skeleton variation in eyeless Astyanax cavefish.

Charles Breder, a pioneering researcher of blind Mexican cavefish was the first to note extreme variation in the facial skeleton of this intriguing subterranean-dwelling organism. Using a system of polar coordinate plots, he identified substantial dysmorphic changes affecting bones of the orbital skeleton. A complication of his landmark publication from 1944 was an error in the number of orbital bones depicted for this species. Intriguingly, however, he proposed an unknown "organizing force" likely influences final bone position and associated dysmorphia. At the time this was merely hypothetical. Roughly eight decades since its publication, however, insights into sensory influences on facial bone development may explain dysmorphia and variation in bone numbers for Astyanax cavefish. A morphological association between mechano-sensory neuromasts of the lateral line and dermal bones of the facial skeleton had been appreciated in the classical literature, but the polarity of this interaction has long remained unclear. Here, we propose that sensory-skeletal integration between sensory neuromasts and bones explain the incomplete numbers of bones, and dysmorphic features such as fusion between neighboring elements. We propose that in closely-related surface fish (and most teleost fish) this developmental coupling enables the sensory and skeletal systems to become integrated into a functional unit over the course of life history. In this opinion article, we discuss the relevance of this (poorly understood) phenomenon as a potential evolutionary source of variation in the facial bone structures of taxa across deep geologic time. We provide three potential explanations for the error in Breder's drawings, that may be explained by natural developmental variation documented in other related species. Moreover, we argue that the natural variation in this "evolutionary" model system is useful for explaining diverse cranial features by uniting aberrations occurring during embryogenesis with long-term adult dysmorphia.

Sensory evolution in a cavefish radiation: patterns of neuromast distribution and associated behaviour in Sinocyclocheilus (Cypriniformes: Cyprinidae).

The genus Sinocyclocheilus, comprising a large radiation of freshwater cavefishes, are well known for their presence of regressive features (e.g. variable eye reduction). Fewer constructive features are known, such as the expansion of the lateral line system (LLS), which is involved in detecting water movements. The precise relationship between LLS expansion and cave adaptation is not well understood. Here, we examine morphology and LLS-mediated behaviour in Sinocyclocheilus species characterized by broad variation in eye size, habitat and geographical distribution. Using live-staining techniques and automated behavioural analyses, we examined 26 Sinocyclocheilus species and quantified neuromast organ number, density and asymmetry within a phylogenetic context. We then examined how these morphological features may relate to wall-following, an established cave-associated behaviour mediated by the lateral line. We show that most species demonstrated laterality (i.e. asymmetry) in neuromast organs on the head, often biased to the right. We also found that wall-following behaviour was distinctive, particularly among eyeless species. Patterns of variation in LLS appear to correlate with the degree of eye loss, as well as geographical distribution. This work reveals that constructive LLS evolution is convergent across distant cavefish taxa and may mediate asymmetric behavioural features that enable survival in stark subterranean microenvironments.

Alterations to cavefish red blood cells provide evidence of adaptation to reduced subterranean oxygen.

Animals inhabiting extreme environments allow the powerful opportunity to examine adaptive evolution in response to diverse pressures. One such pressure is reduced oxygen, commonly present at high-altitude and subterranean environments. Cave-dwelling animals must also deal with darkness and starvation, both of which have been rigorously studied as key forces driving the evolution of cave-associated traits. Interestingly, hypoxia as an environmental pressure has received less attention. Here we examined putatively adaptive phenotypes evolving in a freshwater teleost fish, Astyanax mexicanus, which includes both surface- and cave-dwelling forms. This model system also provides the opportunity to identify convergent responses to hypoxia, owing to the presence of numerous natural and independently-colonised cave populations, alongside closely-related surface conspecifics. The focus of this study is hemoglobin, an essential molecule for oxygen transport and delivery. We found that multiple cave populations harbor a higher concentration of hemoglobin in their blood, which is coincident with an increase in cave morph erythrocyte size compared to surface fish. Interestingly, both cave and surface morphs have comparable numbers of erythrocytes per unit of blood, suggesting elevated hemoglobin is not due to overproduction of red blood cells. Alternatively, owing to an increased cell area of erythrocytes in cavefish, we reason that they contain more hemoglobin per erythrocyte. These findings support the notion that cavefish have adapted to hypoxia in caves through modulation of both hemoglobin production and erythrocyte size. This work reveals an additional adaptive feature of Astyanax cavefish, and demonstrates that coordinated changes between cellular architecture and molecular changes are necessary for organisms evolving under intense environmental pressure.

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.

Evolution in Sinocyclocheilus cavefish is marked by rate shifts, reversals, and origin of novel traits.

BACKGROUND: Natural model systems are indispensable for exploring adaptations in response to environmental pressures. Sinocyclocheilus of China, the most diverse cavefish clade in the world (75 species), provide unique opportunities to understand recurrent evolution of stereotypic traits (such as eye loss and sensory expansion) in the context of a deep and diverse phylogenetic group. However, they remain poorly understood in terms of their morphological evolution. Therefore, we explore key patterns of morphological evolution, habitat utilization and geographic distribution in these fishes. RESULTS: We constructed phylogenies and categorized 49 species based on eye-related condition (Blind, Micro-eyed, and Normal-eyed), habitat types (Troglobitic-cave-restricted; Troglophilic-cave-associated; Surface-outside caves) and existence of horns. Geometric-morphometric analyses show Normal-eyed morphs with fusiform shapes segregating from Blind/Micro-eyed deeper bodied morphs along the first principal-component axis; second axis accounts for shape complexity related to horns. The body shapes showed a significant association with eye-related condition and horn, but not habitat types. Ancestral reconstructions suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to their ancestral Normal-eyed morphs; Sinocyclocheilus are also pre-adapted for cave dwelling. Our geophylogeny shows an east-to-west diversification spanning Pliocene and Pleistocene, with early-diversifying Troglobitic species dominating subterranean habitats of karstic plains whereas predominantly Surface forms inhabit hills to the west. Evolutionary rates analyses suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 5-20 fold increase in rate of eye regression, possibly explained by limited resource availability. Body size and eye size have undergone reversals, but not horns, a trait entailing considerable time to form. CONCLUSIONS: Sinocyclocheilus occupied cave habitats in response to drying associated with aridification of China during late Miocene and the Pliocene. The prominent cave-adaptations (eye-regression, horn-evolution) occur in clades associated with the extensive subterranean cave system in Guangxi and Guizhou provinces. Integration of morphology, phylogeny, rate analyses, molecular-dating and distribution show not only several remarkable patterns of evolution, but also interesting exceptions to these patterns signifying the diversification of Sinocyclocheilus as an invaluable model system to explore evolutionary novelty.

Fur Color and Nutritional Status Predict Hair Cortisol Concentrations of Dogs in Nicaragua.

This study examined the relationships between hair cortisol concentrations (HCC) and sex, age, nutritional status (as determined by body condition scores, or BCS), and body mass (geometric mean calculated from morphometric measurements), as well as the potential influence of hair pigmentation (light, dark, or agouti/mixed) on HCC in dogs of the Bosawas Biosphere Reserve, Nicaragua. The dogs examined in this study live in a marginal environment where disease, malnutrition, and mortality rates are high. For fur color, HCC was significantly higher in light fur than in than dark and mixed fur (p < 0.001). In addition, BCS scores were found to have a negative effect on HCC (p < 0.001). Measures of sex and body size exhibited inconclusive effects on HCC, and when compared to adult dogs, juvenile dogs did not exhibit significantly different HCC. Repeated measures of dogs over time reveal a moderate intra-class correlation, suggesting that there are unmeasured sources of individual-level heterogeneity. These findings imply a need to account for fur color in studies of HCC in dogs, and the study suggests an overlooked relationship between cortisol and body condition scores in undernourished dogs in diverse settings.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

Dark-rearing uncovers novel gene expression patterns in an obligate cave-dwelling fish.

Extreme environments often result in the evolution of dramatic adaptive features. The Mexican tetra, Astyanax mexicanus, includes 30 different populations of cave-dwelling forms that live in perpetual darkness. As a consequence, many populations have evolved eye loss, reduced pigmentation, and amplification of nonvisual sensory systems. Closely-related surface-dwelling morphs demonstrate typical vision, pigmentation, and sensation. Transcriptomic assessments in this system have revealed important developmental changes associated with the cave morph, however, they have not accounted for photic rearing conditions. Prior studies reared individuals under a 12:12 hr light/dark (LD) cycle. Here, we reared cavefish under constant darkness (DD) for 5+ years. From these experimental individuals, we performed mRNA sequencing and compared gene expression of surface fish reared under LD conditions to cavefish reared under DD conditions to identify photic-dependent gene expression differences. Gene Ontology enrichment analyses revealed a number of previously underappreciated cave-associated changes impacting blood physiology and olfaction. We further evaluated the position of differentially expressed genes relative to QTL positions from prior studies and found several candidate genes associated with these ecologically relevant lighting conditions. In sum, this work highlights photic conditions as a key environmental factor impacting gene expression patterns in blind cave-dwelling fish.

Parallel evolution of regressive and constructive craniofacial traits across distinct populations of Astyanax mexicanus cavefish.

Life in complete darkness has driven the evolution of a suite of troglobitic features in the blind Mexican cavefish Astyanax mexicanus, such as eye and pigmentation loss. While regressive evolution is a hallmark of obligate cave-dwelling organisms, constructive (or augmented) traits commonly arise as well. The cavefish cranium has undergone extensive changes compared with closely-related surface fish. These alterations are rooted in both cranial bones and surrounding sensory tissues such as enhancements in the gustatory and lateral line systems. Cavefish also harbor numerous cranial bone asymmetries: fluctuating asymmetry of individual bones and directional asymmetry in a dorsal bend of the skull. This asymmetry is mirrored by the asymmetrical patterning of mechanosensory neuromasts. We explored the relationship between facial bones and neuromasts using in vivo fluorescent colabeling and microcomputed tomography. We found an increase in neuromast density within dermal bone boundaries across three distinct populations of cavefish compared to surface-dwelling fish. We also show that eye loss disrupts early neuromast patterning, which in turn impacts the development of dermal bones. While cavefish exhibit alterations in cranial bone and neuromast patterning, each population varied in the severity. This variation may reflect observed differences in behavior across populations. For instance, a bend in the dorsal region of the skull may expose neuromasts to water flow on the opposite side of the face, enhancing sensory input and spatial mapping in the dark.

A geographical cline in craniofacial morphology across populations of Mesoamerican lake-dwelling fishes.

Together, the complex geological history and climatic diversity of Mesoamerica create a rich source of biodiversity from which evolutionary processes can be studied. Here, we discuss highly divergent morphs of lake-dwelling fishes distributed across Mexico and Central America, originally recognized as members of different genera (Astyanax and "Bramocharax"). Recent phylogenetic studies, however, suggest these morphs group within the same genus and readily hybridize. Despite genetic similarities, Bramocharax morphs exhibit stark differences in cranial shape and dentition. We investigated the evolution of several cranial traits that vary across morphs collected from four lakes in Mexico and Nicaragua and discovered an ecomorphological cline from northern to southern lakes. Northern populations of sympatric morphs exhibit a similar cranial shape and tooth morphology. Southern populations of Bramocharax morphs, however, showed a larger disparity in maxillary teeth, length and frequency of unicuspid teeth, an elongated snout, and a streamlined cranium compared to Astyanax morphs. This divergence of craniofacial morphology likely evolved in association with differences in trophic niches. We discuss the morphological differences across the four lake systems in terms of geological history and trophic dynamics. In summary, our study suggests that Bramocharax morphs are likely locally adapted members derived from independent Astyanax lineages, highlighting an interesting parallel evolutionary pattern within the Astyanax genus.

An Asymmetric Genetic Signal Associated with Mechanosensory Expansion in Cave-Adapted Fish.

A key challenge in contemporary biology is connecting genotypic variation to phenotypic diversity. Quantitative genetics provides a powerful technique for identifying regions of the genome that covary with phenotypic variation. Here, we present a quantitative trait loci (QTL) analysis of a natural freshwater fish system, Astyanax mexicanus, that harbors two morphs corresponding to a cave and surface fish. Following their divergence ~500 Kya, cavefish have adapted to the extreme pressures of the subterranean biome. As a consequence, cavefish have lost numerous features, but evolved gains for a variety of constructive features including behavior. Prior work found that sensory tissues (neuromasts) present in the "eye orbit" region of the skull associate with sensitivity to vibrations in water. This augmented sensation is believed to facilitate foraging behavior in the complete darkness of a cave, and may impact on evolved lateral swimming preference. To this point, however, it has remained unclear how morphological variation integrates with behavioral variation through heritable factors. Using a QTL approach, we discovered the genetic architecture of neuromasts present in the eye orbit region, demonstrating that this feature is under genetic control. Interestingly, linked loci were asymmetric-signals were detected using only data collected from the right, but not left, side of the face. This finding may explain enhanced sensitivity and/or feedback of water movements mediating a lateral swimming preference. The locus we discovered based on neuromast position maps near established QTL for eye size and a facial bone morphology, raising the intriguing possibility that eye loss, sensory expansion, and the cranial skeleton may be integrated for evolving adaptive behaviors. Thus, this work will further our understanding of the functional consequence of key loci that influence the evolutionary origin of changes impacting morphology, behavior, and adaptation.

A Natural Animal Model System of Craniofacial Anomalies: The Blind Mexican Cavefish.

Natural model systems evolving under extreme environmental pressures provide the opportunity to advance our knowledge of how the craniofacial complex evolves in nature. Unlike traditional models, natural systems are less inbred, and, therefore, better model the complex variation of the human population. Owing to the nature of certain craniofacial aberrations in blind Mexican cavefish, we suggest that this organism can provide new insights to a variety of craniofacial changes. Diverse cranial features have evolved in natural cave-dwelling Astyanax fish, which have thrived in the unforgiving darkness and nutrient-poor environment of the cave for countless generations. While the genetic and environmental underpinnings of various cranial anomalies have been investigated for decades, a comprehensive characterization of their molecular and developmental origins remains incomplete. Cavefish provide numerous advantages given the availability of genomic resources, developmental and molecular tools, and the presence of a normative surface-dwelling "ancestral" surrogate for comparative studies. By leveraging the frequency of abnormal and asymmetric cranial features in cavefish, we anticipate advances in our knowledge of the etiologies of irregular cranial features. Extreme adaptations in cavefish are expected to offer new insights into the complex and multifactorial nature of craniofacial disorders and facial asymmetry. Anat Rec, 2018. © 2018 American Association for Anatomy.

In-Frame Indel Mutations in the Genome of the Blind Mexican Cavefish, Astyanax mexicanus.

Organisms living in the subterranean biome evolve extreme characteristics including vision loss and sensory expansion. Despite prior work linking certain genes to Mendelian traits, the genetic basis for complex cave-associated traits remains unknown. Moreover, it is unclear if certain forms of genetic variation (e.g., indels, copy number variants) are more common in regressive evolution. Progress in this area has been limited by a lack of suitable natural model systems and genomic resources. In recent years, the Mexican tetra, Astyanax mexicanus, has advanced as a model for cave biology and regressive evolution. Here, we present the results of a genome-wide screen for in-frame indels using alignments of RNA-sequencing reads to the draft cavefish genome. Mutations were discovered in three genes associated with blood physiology (mlf1, plg, and wdr1), two genes associated with growth factor signaling (ghrb, rnf126), one gene linked to collagen defects (mia3), and one gene which may have a global epigenetic impact on gene expression (mki67). With one exception, polymorphisms were shared between Pachón and Tinaja cavefish lineages, and different from the surface-dwelling lineage. We confirmed the presence of mutations using direct Sanger sequencing and discovered remarkably similar developmental expression in both morphs despite substantial coding sequence alterations. Further, three mutated genes mapped near previously established quantitative trait loci associated with jaw size, condition factor, lens size, and neuromast variation. This work reveals previously unappreciated traits evolving in this species under environmental pressures (e.g., blood physiology) and provides insight to genetic changes underlying convergence of organisms evolving in complete darkness.

Wholemount In Situ Hybridization for Astyanax Embryos.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.