Search

A major goal of modern biology is connecting phenotype with its underlying genetic basis. The Mexican cavefish (Astyanax mexicanus), a characin fish species comprised of a surface ecotype and a cave-derived ecotype, is well suited as a model to study the genetic mechanisms underlying adaptation to extreme environments. Here we map 206 previously published quantitative trait loci (QTL) for cave-derived traits in A. mexicanus to the newest version of the surface fish genome assembly, AstMex3. These analyses revealed that QTL cluster in the genome more than expected by chance, and this clustering is not explained by the distribution of genes in the genome. To investigate whether certain characteristics of the genome facilitate phenotypic evolution, we tested whether genomic characteristics associated with increased opportunities for mutation, such as highly mutagenic CpG sites, are reliable predictors of the sites of trait evolution but did not find any significant trends. Finally, we combined the QTL map with previously collected expression and selection data to identify 36 candidate genes that may underlie the repeated evolution of cave phenotypes, including rgrb, which is predicted to be involved in phototransduction. We found this gene has disrupted exons in all non-hybrid cave populations but intact reading frames in surface fish. Overall, our results suggest specific regions of the genome may play significant roles in driving adaptation to the cave environment in Astyanax mexicanus and demonstrate how this compiled dataset can facilitate our understanding of the genetic basis of repeated evolution in the Mexican cavefish., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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.

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., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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 F 2 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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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.

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., (© 2023. The Author(s).)

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 ( α NTXs) is a case of widespread convergence having evolved several times in snakes, lizards and mammals. Despite extreme toxicity of α NTXs, substitutions in its target, the nicotinic acetylcholine receptor (nAChR), prevent α NTX 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 α NTX 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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

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., Competing Interests: The authors declare no competing interests., (© 2022.)

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 Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Animals respond to sleep loss with compensatory rebound sleep, and this is thought to be critical for the maintenance of physiological homeostasis. Sleep duration varies dramatically across animal species, but it is not known whether evolutionary differences in sleep duration are associated with differences in sleep homeostasis. The Mexican cavefish, Astyanax mexicanus, has emerged as a powerful model for studying the evolution of sleep. While eyed surface populations of A. mexicanus sleep approximately 8 hr each day, multiple blind cavefish populations have converged on sleep patterns that total as little as 2 hr each day, providing the opportunity to examine whether the evolution of sleep loss is accompanied by changes in sleep homeostasis. Here, we examine the behavioral and molecular response to sleep deprivation across four independent populations of A. mexicanus. Our behavioral analysis indicates that surface fish and all three cavefish populations display robust recovery sleep during the day following nighttime sleep deprivation, suggesting sleep homeostasis remains intact in cavefish. We profiled transcriptome-wide changes associated with sleep deprivation in surface fish and cavefish. While the total number of differentially expressed genes was not greater for the surface population, the surface population exhibited the highest number of uniquely differentially expressed genes than any other population. Strikingly, a majority of the differentially expressed genes are unique to individual cave populations, suggesting unique expression responses are exhibited across independently evolved cavefish populations. Together, these findings suggest sleep homeostasis is intact in cavefish despite a dramatic reduction in overall sleep duration., (© 2020 Wiley Periodicals, Inc.)

Different parts of the genome can vary widely in their evolutionary histories and sequence divergence from other species. Indeed, some of the most interesting biology (e.g., hybridization, horizontal gene transfer, variable mutation rates across the genome) is revealed by the discordant relationships between taxa across the genome. The goal for much of evolutionary genetics is centred on understanding the evolutionary processes by which such varied signatures arise and are maintained. Many evolutionary genetics studies seek to identify signatures of positive selection between two closely related ecotypes or taxa by delineating regions with particularly high divergence relative to a genome-wide average, often termed "divergence outliers." In a From the Cover article in this issue of Molecular Ecology, Booker et al. take a major step forward in showing that recombination rate differences are sufficient to create false positive divergence outliers, even under neutrality. They demonstrate that the variance of genome scan metrics is especially high in regions with low recombination rates, consistent with previous work. Furthermore, they show that both relative and absolute measures of divergence (F ST and D XY , respectively) as well as other commonly used statistics in genome scans (e.g., π W , Tajima's D and H12) all have similar covariance between variance and local recombination rate. Finally, Booker et al. show that low recombination regions will tend to produce more outliers if genome-wide averages are used as cut-offs to define genomic outliers. Booker et al.'s results suggest that recombination rate variation, even under neutral conditions, can shape genome scans for selection, and this important variable can no longer be ignored., (© 2020 John Wiley & Sons Ltd.)

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., (© 2020 Wiley Periodicals LLC.)

Background: Advances in sequencing technologies have led to the release of reference genomes and annotations for multiple individuals within more well-studied systems. While each of these new genome assemblies shares significant portions of synteny between each other, the annotated structure of gene models within these regions can differ. Of particular concern are split-gene misannotations, in which a single gene is incorrectly annotated as two distinct genes or two genes are incorrectly annotated as a single gene. These misannotations can have major impacts on functional prediction, estimates of expression, and many downstream analyses., Results: We developed a high-throughput method based on pairwise comparisons of annotations that detect potential split-gene misannotations and quantifies support for whether the genes should be merged into a single gene model. We demonstrated the utility of our method using gene annotations of three reference genomes from maize (B73, PH207, and W22), a difficult system from an annotation perspective due to the size and complexity of the genome. On average, we found several hundred of these potential split-gene misannotations in each pairwise comparison, corresponding to 3-5% of gene models across annotations. To determine which state (i.e. one gene or multiple genes) is biologically supported, we utilized RNAseq data from 10 tissues throughout development along with a novel metric and simulation framework. The methods we have developed require minimal human interaction and can be applied to future assemblies to aid in annotation efforts., Conclusions: Split-gene misannotations occur at appreciable frequency in maize annotations. We have developed a method to easily identify and correct these misannotations. Importantly, this method is generic in that it can utilize any type of short-read expression data. Failure to account for split-gene misannotations has serious consequences for biological inference, particularly for expression-based analyses.

A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus , is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant, ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus , and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate brain atlases of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions that have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an enlargement of the hypothalamus in all cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and prethalamus. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior., (Copyright © 2019 Loomis, Peuß, Jaggard, Wang, McKinney, Raftopoulos, Raftopoulos, Whu, Green, McGaugh, Rohner, Keene and Duboue.)

Background: Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems., Results: Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish., Conclusion: The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap., (© 2019 Wiley Periodicals, Inc.)

RNA sequencing is a popular next-generation sequencing technique for assaying genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field-based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome-wide expression patterns in 30-day-old fry collected from the same brood at the same time point that were flash-frozen in liquid nitrogen and stored at -80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash-freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field-based studies are substantial and should not be ignored., (© 2018 John Wiley & Sons Ltd.)

Cancer is a threat to multicellular organisms, yet the molecular evolution of pathways that prevent the accumulation of genetic damage has been largely unexplored. The p53 network regulates how cells respond to DNA-damaging stressors. We know little about p53 network molecular evolution as a whole. In this study, we performed comparative genetic analyses of the p53 network to quantify the number of genes within the network that are rapidly evolving and constrained, and the association between lifespan and the patterns of evolution. Based on our previous published data set, we used genomes and transcriptomes of 34 sauropsids and 32 mammals to analyze the molecular evolution of 45 genes within the p53 network. We found that genes in the network exhibited evidence of positive selection and divergent molecular evolution in mammals and sauropsids. Specifically, we found more evidence of positive selection in sauropsids than mammals, indicating that sauropsids have different targets of selection. In sauropsids, more genes upstream in the network exhibited positive selection, and this observation is driven by positive selection in squamates, which is consistent with previous work showing rapid divergence and adaptation of metabolic and stress pathways in this group. Finally, we identified a negative correlation between maximum lifespan and the number of genes with evidence of divergent molecular evolution, indicating that species with longer lifespans likely experienced less variation in selection across the network. In summary, our study offers evidence that comparative genomic approaches can provide insights into how molecular networks have evolved across diverse species., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution., (© 2018 John Wiley & Sons Ltd.)

Tandem duplicate genes are proximally duplicated and as such occur in similar genomic neighborhoods. Using the maize B73 and PH207 de novo genome assemblies, we identified thousands of tandem gene duplicates that account for ∼10% of the annotated genes. These tandem duplicates have a bimodal distribution of ages, which coincide with ancient allopolyploidization and more recent domestication. Tandem duplicates are smaller on average and have a higher probability of containing LTR elements than other genes, suggesting origins in nonhomologous recombination. Within relatively recent tandem duplicate genes, ∼26% appear to be undergoing degeneration or divergence in function from the ancestral copy. Our results show that tandem duplicates are abundant in maize, arose in bursts throughout maize evolutionary history under multiple potential mechanisms, and may provide a substrate for novel phenotypic variation., (Copyright © 2018 Kono et al.)

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.

Background: An essential question in evolutionary biology is whether shifts in a set of polygenic behaviors share a genetic basis across species. Such a behavioral shift is seen in the cave-dwelling Mexican tetra, Astyanax mexicanus. Relative to surface-dwelling conspecifics, cavefish do not school (asocial), are hyperactive and sleepless, adhere to a particular vibration stimulus (imbalanced attention), behave repetitively, and show elevated stress hormone levels. Interestingly, these traits largely overlap with the core symptoms of human autism spectrum disorder (ASD), raising the possibility that these behavioral traits are underpinned by a similar set of genes (i.e. a repeatedly used suite of genes)., Result: Here, we explored whether modification of ASD-risk genes underlies cavefish evolution. Transcriptomic analyses revealed that > 58.5% of 3152 cavefish orthologs to ASD-risk genes are significantly up- or down-regulated in the same direction as genes in postmortem brains from ASD patients. Enrichment tests suggest that ASD-risk gene orthologs in A. mexicanus have experienced more positive selection than other genes across the genome. Notably, these positively selected cavefish ASD-risk genes are enriched for pathways involved in gut function, inflammatory diseases, and lipid/energy metabolism, similar to symptoms that frequently coexist in ASD patients. Lastly, ASD drugs mitigated cavefish's ASD-like behaviors, implying shared aspects of neural processing., Conclusion: Overall, our study indicates that ASD-risk genes and associated pathways (especially digestive, immune and metabolic pathways) may be repeatedly used for shifts in polygenic behaviors across evolutionary time.

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

Maize is a diverse paleotetraploid species with considerable presence/absence variation and copy number variation. One mechanism through which presence/absence variation can arise is differential fractionation. Fractionation refers to the loss of duplicate gene pairs from one of the maize subgenomes during diploidization. Differential fractionation refers to non-shared gene loss events between individuals following a whole-genome duplication event. We investigated the prevalence of presence/absence variation resulting from differential fractionation in the syntenic portion of the genome using two whole-genome de novo assemblies of the inbred lines B73 and PH207. Between these two genomes, syntenic genes were highly conserved with less than 1% of syntenic genes being subject to differential fractionation. The few variably fractionated syntenic genes that were identified are unlikely to contribute to functional phenotypic variation, as there is a significant depletion of these genes in annotated gene sets. In further comparisons of 60 diverse inbred lines, non-syntenic genes were six times more likely to be variable than syntenic genes, suggesting that comparisons among additional genome assemblies are not likely to result in the discovery of large-scale presence/absence variation among syntenic genes., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Background: Previous studies examining post-feeding organ regeneration in the Burmese python (Python molurus bivittatus) have identified thousands of genes that are significantly differentially regulated during this process. However, substantial gaps remain in our understanding of coherent mechanisms and specific growth pathways that underlie these rapid and extensive shifts in organ form and function. Here we addressed these gaps by comparing gene expression in the Burmese python heart, liver, kidney, and small intestine across pre- and post-feeding time points (fasted, one day post-feeding, and four days post-feeding), and by conducting detailed analyses of molecular pathways and predictions of upstream regulatory molecules across these organ systems., Results: Identified enriched canonical pathways and upstream regulators indicate that while downstream transcriptional responses are fairly tissue specific, a suite of core pathways and upstream regulator molecules are shared among responsive tissues. Pathways such as mTOR signaling, PPAR/LXR/RXR signaling, and NRF2-mediated oxidative stress response are significantly differentially regulated in multiple tissues, indicative of cell growth and proliferation along with coordinated cell-protective stress responses. Upstream regulatory molecule analyses identify multiple growth factors, kinase receptors, and transmembrane receptors, both within individual organs and across separate tissues. Downstream transcription factors MYC and SREBF are induced in all tissues., Conclusions: These results suggest that largely divergent patterns of post-feeding gene regulation across tissues are mediated by a core set of higher-level signaling molecules. Consistent enrichment of the NRF2-mediated oxidative stress response indicates this pathway may be particularly important in mediating cellular stress during such extreme regenerative growth.

The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades.

Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.

Fine scale meiotic recombination maps have uncovered a large amount of variation in crossover rate across the genomes of many species, and such variation in mammalian and yeast genomes is concentrated to <5kb regions of highly elevated recombination rates (10-100x the background rate) called "hotspots." Drosophila exhibit substantial recombination rate heterogeneity across their genome, but evidence for these highly-localized hotspots is lacking. We assayed recombination across a 40Kb region of Drosophila pseudoobscura chromosome 2, with one 20kb interval assayed every 5Kb and the adjacent 20kb interval bisected into 10kb pieces. We found that recombination events across the 40kb stretch were relatively evenly distributed across each of the 5kb and 10kb intervals, rather than concentrated in a single 5kb region. This, in combination with other recent work, indicates that the recombination landscape of Drosophila may differ from the punctate recombination pattern observed in many mammals and yeast. Additionally, we found no correlation of average pairwise nucleotide diversity and divergence with recombination rate across the 20kb intervals, nor any effect of maternal age in weeks on recombination rate in our sample.

Background: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing., Results: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented., Conclusions: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Premature termination codon (PTC) mutations can have dramatic effects--both adaptive and deleterious--on gene expression and function. Here, we examine the number and selective effects of PTC mutations within the Drosophila pseudoobscura subclade using 18 resequenced genomes aligned to the reference genome. We located and characterized 1,679 PTC mutations in 605 genes across each of these genomes relative to the D. pseudoobscura reference genome, and use RT-PCR to confirm transcription of a subset of these genes containing PTC mutations. We confirm previous findings that genes containing PTC mutations are less selectively constrained and less broadly expressed than non-PTC-containing genes, suggesting that the most of these mutations are at least mildly deleterious. Further, we find highly significant codon usage bias in regions downstream of the PTC in 38 of these PTC-containing genes, suggesting that some of these PTC mutations--if not alternatively spliced out of the transcript--have neutral effects. Ultimately, these analyzes support the view that the PTC mutations are mostly detrimental, but are nonetheless common enough in genomes that a subset could be effectively neutral.

Environmental temperature plays a crucial role in determining a species distribution and abundance by affecting individual physiological processes, metabolic activities, and developmental rates. Many studies have identified clinal variation in phenotypes associated with response to environmental stresses, but variation in traits associated with climatic adaptation directly attributed to sequence variation within candidate gene regions has been difficult to identify. Insect heat shock genes are possible agents of thermal tolerance because of their involvement in protein folding, traffic, protection, and renaturation at the cellular level in response to temperature stress. Previously, members of the Drosophila small heat shock protein (sHSP) complex (Hsp23, Hsp26, Hsp27, Hsp67Ba) have been implicated as candidate climatic adaptation genes; therefore, this research examines sequence variation at these genes in 2 distant populations of Drosophila pseudoobscura. Flies from Tempe, AZ (n = 30) and Cheney, WA (n = 17) were used in the study. We identify high differentiation in the heat-shock complex (F(ST) : 0.219**, 0.262*, 0.279***, 0.166 not significant) as compared with neighboring genes and Tajima's D values indicative of balancing selection (Mann-Whitney U = 38, n(1) = 10 n(2) = 4, P < 0.05 two-tailed), both of which are suggestive of such climatic adaptation.

Chromosomal inversions impact genetic variation and facilitate speciation in part by reducing recombination in heterokaryotypes. We generated multiple whole-genome shotgun sequences of the parapatric species pair Drosophila pseudoobscura and Drosophila persimilis and their sympatric outgroup (Drosophila miranda) and compared the average pairwise differences for neutral sites within, just outside and far outside of the three large inversions. Divergence between D. pseudoobscura and D. persimilis is high inside the inversions and in the suppressed recombination regions extending 2.5 Mb outside of inversions, but significantly lower in collinear regions further from the inversions. We observe little evidence of decreased divergence predicted to exist in the centre of inversions, suggesting that gene flow through double crossovers or gene conversion is limited within the inversion, or selection is acting within the inversion to maintain divergence in the face of gene flow. In combination with past studies, we provide evidence that inversions in this system maintain areas of high divergence in the face of hybridization, and have done so for a substantial period of time. The left arm of the X chromosome and chromosome 2 inversions appear to have arisen in the lineage leading to D. persimilis approximately 2 Ma, near the time of the split of D. persimilis-D. pseudoobscura-D. miranda, but likely fixed within D. persimilis much more recently, as diversity within D. persimilis is substantially reduced inside and near these two inversions. We also hypothesize that the inversions in D. persimilis may provide an empirical example of the 'mixed geographical mode' theory of inversion origin and fixation, whereby allopatry and secondary contact both play a role.

One of the most influential observations in molecular evolution has been a strong association between local recombination rate and nucleotide polymorphisms across the genome. This is interpreted as evidence for ubiquitous natural selection. The alternative explanation, that recombination is mutagenic, has been rejected by the absence of a similar association between local recombination rate and nucleotide divergence between species. However, many recent studies show that recombination rates are often very different even in closely related species, questioning whether an association between recombination rate and divergence between species has been tested satisfactorily. To circumvent this problem, we directly surveyed recombination across approximately 43% of the D. pseudoobscura physical genome in two separate recombination maps and 31% of the D. miranda physical genome, and we identified both global and local differences in recombination rate between these two closely related species. Using only regions with conserved recombination rates between and within species and accounting for multiple covariates, our data support the conclusion that recombination is positively related to diversity because recombination modulates Hill-Robertson effects in the genome and not because recombination is predominately mutagenic. Finally, we find evidence for dips in diversity around nonsynonymous substitutions. We infer that at least some of this reduction in diversity resulted from selective sweeps and examine these dips in the context of recombination rate., Competing Interests: The authors have declared that no competing interests exist.

Selection is expected to maintain primary sex ratios at an evolutionary equilibrium. In organisms with temperature-dependent sex determination (TSD), targets of sex-ratio selection include the thermal sensitivity of the sex-determining pathway (hereafter, sex determination threshold) and nest-site choice. However, offspring sex may be canalized for nests located in thermally extreme environments; thus, genetic variance for the sex determination threshold is not expressed and is invisible to direct selection. The concept of 'effective heritability' accounts for this dependence and provides a more realistic prediction of the expected evolutionary response to selection in the wild. Past estimates of effective heritability of the sex determination threshold, which were derived from laboratory data, suggested that the potential for the sex determination threshold to evolve in the wild was extremely low. We re-evaluated original estimates of this parameter by analysing field-collected measures of nest temperatures, vegetation cover and clutch sex ratios from nests in a population of painted turtles (Chrysemys picta). We coupled these data with measurements of broad-sense heritability of the sex determination threshold in C. picta, using an experiment that splits clutches of eggs between a constant temperature (i.e. typical laboratory incubation) and a daily fluctuating temperature (i.e. similar to natural nests) with the same mean. We found that (i) the effective heritability of the sex determination threshold appears to have been historically underestimated and the effective heritability of nest-site choice has been overestimated and (ii) significant family-by-incubation treatment interaction exists for sex for C. picta between constant- and fluctuating-temperature regimes. Our results suggest that the thermal sensitivity of the sex-determining pathway may play a larger, more complex role in the microevolution of TSD than traditionally thought., (© 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.)

Nesting behaviour is critical for reproductive success in oviparous organisms with no parental care. In organisms where sex is determined by incubation temperature, nesting behaviour may be a prime target of selection in response to unbalanced sex ratios. To produce an evolutionary change in response to sex-ratio selection, components of nesting behaviour must be heritable. We estimated the field heritability of two key components of nesting behaviour in a population of painted turtles (Chrysemys picta) with temperature-dependent sex determination by applying the 'animal model' to a pedigree reconstructed from genotype data. We obtained estimates of low to non-detectable heritability using repeated records across all environments. We then determined environment-specific heritability by grouping records with similar temperatures for the winter preceding the nesting season, a variable known to be highly associated with our two traits of interest, nest vegetation cover and Julian date of nesting. The heritability estimates of nest vegetation cover and Julian date of nesting were qualitatively highest and significant, or nearly so, after hot winters. Additive genetic variance for these traits was not detectable after cold winters. Our analysis suggests that the potential for evolutionary change of nesting behaviour may be dependent on the thermal conditions of the preceding winter, a season that is predicted to be especially subject to climate change.

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