Current advances in primate genomics: novel approaches for understanding evolution and disease.

Primate genomics holds the key to understanding fundamental aspects of human evolution and disease. However, genetic diversity and functional genomics data sets are currently available for only a few of the more than 500 extant primate species. Concerted efforts are under way to characterize primate genomes, genetic polymorphism and divergence, and functional landscapes across the primate phylogeny. The resulting data sets will enable the connection of genotypes to phenotypes and provide new insight into aspects of the genetics of primate traits, including human diseases. In this Review, we describe the existing genome assemblies as well as genetic variation and functional genomic data sets. We highlight some of the challenges with sample acquisition. Finally, we explore how technological advances in single-cell functional genomics and induced pluripotent stem cell-derived organoids will facilitate our understanding of the molecular foundations of primate biology.

Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges.

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. We investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). We leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, we detected ∼36% more REs than short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were "unclassified" following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. We highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.

Nascent transcription reveals regulatory changes in extremophile fishes inhabiting hydrogen sulfide-rich environments.

Regulating transcription allows organisms to respond to their environment, both within a single generation (plasticity) and across generations (adaptation). We examined transcriptional differences in gill tissues of fishes in the Poecilia mexicana species complex (family Poeciliidae), which have colonized toxic springs rich in hydrogen sulfide (H2S) in southern Mexico. There are gene expression differences between sulfidic and non-sulfidic populations, yet regulatory mechanisms mediating this gene expression variation remain poorly studied. We combined capped-small RNA sequencing (csRNA-seq), which captures actively transcribed (i.e. nascent) transcripts, and messenger RNA sequencing (mRNA-seq) to examine how variation in transcription, enhancer activity, and associated transcription factor binding sites may facilitate adaptation to extreme environments. csRNA-seq revealed thousands of differentially initiated transcripts between sulfidic and non-sulfidic populations, many of which are involved in H2S detoxification and response. Analyses of transcription factor binding sites in promoter and putative enhancer csRNA-seq peaks identified a suite of transcription factors likely involved in regulating H2S-specific shifts in gene expression, including several key transcription factors known to respond to hypoxia. Our findings uncover a complex interplay of regulatory processes that reflect the divergence of extremophile populations of P. mexicana from their non-sulfidic ancestors and suggest shared responses among evolutionarily independent lineages.

Toward a genome sequence for every animal: Where are we now?

In less than 25 y, the field of animal genome science has transformed from a discipline seeking its first glimpses into genome sequences across the Tree of Life to a global enterprise with ambitions to sequence genomes for all of Earth's eukaryotic diversity [H. A. Lewin et al., Proc. Natl. Acad. Sci. U.S.A. 115, 4325-4333 (2018)]. As the field rapidly moves forward, it is important to take stock of the progress that has been made to best inform the discipline's future. In this Perspective, we provide a contemporary, quantitative overview of animal genome sequencing. We identified the best available genome assemblies in GenBank, the world's most extensive genetic database, for 3,278 unique animal species across 24 phyla. We assessed taxonomic representation, assembly quality, and annotation status for major clades. We show that while tremendous taxonomic progress has occurred, stark disparities in genomic representation exist, highlighted by a systemic overrepresentation of vertebrates and underrepresentation of arthropods. In terms of assembly quality, long-read sequencing has dramatically improved contiguity, whereas gene annotations are available for just 34.3% of taxa. Furthermore, we show that animal genome science has diversified in recent years with an ever-expanding pool of researchers participating. However, the field still appears to be dominated by institutions in the Global North, which have been listed as the submitting institution for 77% of all assemblies. We conclude by offering recommendations for improving genomic resource availability and research value while also broadening global representation.

Epigenetic inheritance of DNA methylation changes in fish living in hydrogen sulfide-rich springs.

Environmental factors can promote phenotypic variation through alterations in the epigenome and facilitate adaptation of an organism to the environment. Although hydrogen sulfide is toxic to most organisms, the fish Poecilia mexicana has adapted to survive in environments with high levels that exceed toxicity thresholds by orders of magnitude. Epigenetic changes in response to this environmental stressor were examined by assessing DNA methylation alterations in red blood cells, which are nucleated in fish. Males and females were sampled from sulfidic and nonsulfidic natural environments; individuals were also propagated for two generations in a nonsulfidic laboratory environment. We compared epimutations between the sexes as well as field and laboratory populations. For both the wild-caught (F0) and the laboratory-reared (F2) fish, comparing the sulfidic and nonsulfidic populations revealed evidence for significant differential DNA methylation regions (DMRs). More importantly, there was over 80% overlap in DMRs across generations, suggesting that the DMRs have stable generational inheritance in the absence of the sulfidic environment. This is an example of epigenetic generational stability after the removal of an environmental stressor. The DMR-associated genes were related to sulfur toxicity and metabolic processes. These findings suggest that adaptation of P. mexicana to sulfidic environments in southern Mexico may, in part, be promoted through epigenetic DNA methylation alterations that become stable and are inherited by subsequent generations independent of the environment.

Feeding during hibernation shifts gene expression toward active season levels in brown bears (Ursus arctos).

Hibernation in bears involves a suite of metabolical and physiological changes, including the onset of insulin resistance, that are driven in part by sweeping changes in gene expression in multiple tissues. Feeding bears glucose during hibernation partially restores active season physiological phenotypes, including partial resensitization to insulin, but the molecular mechanisms underlying this transition remain poorly understood. Here, we analyze tissue-level gene expression in adipose, liver, and muscle to identify genes that respond to midhibernation glucose feeding and thus potentially drive postfeeding metabolical and physiological shifts. We show that midhibernation feeding stimulates differential expression in all analyzed tissues of hibernating bears and that a subset of these genes responds specifically by shifting expression toward levels typical of the active season. Inferences of upstream regulatory molecules potentially driving these postfeeding responses implicate peroxisome proliferator-activated receptor gamma (PPARG) and other known regulators of insulin sensitivity, providing new insight into high-level regulatory mechanisms involved in shifting metabolic phenotypes between hibernation and active states.

Pathways to polar adaptation in fishes revealed by long-read sequencing.

Long-read sequencing is driving a new reality for genome science in which highly contiguous assemblies can be produced efficiently with modest resources. Genome assemblies from long-read sequences are particularly exciting for understanding the evolution of complex genomic regions that are often difficult to assemble. In this study, we utilized long-read sequencing data to generate a high-quality genome assembly for an Antarctic eelpout, Ophthalmolycus amberensis, the first for the globally distributed family Zoarcidae. We used this assembly to understand how O. amberensis has adapted to the harsh Southern Ocean and compared it to another group of Antarctic fishes: the notothenioids. We showed that selection has largely acted on different targets in eelpouts relative to notothenioids. However, we did find some overlap; in both groups, genes involved in membrane structure, thermal tolerance and vision have evidence of positive selection. We found evidence for historical shifts of transposable element activity in O. amberensis and other polar fishes, perhaps reflecting a response to environmental change. We were specifically interested in the evolution of two complex genomic loci known to underlie key adaptations to polar seas: haemoglobin and antifreeze proteins (AFPs). We observed unique evolution of the haemoglobin MN cluster in eelpouts and related fishes in the suborder Zoarcoidei relative to other Perciformes. For AFPs, we identified the first species in the suborder with no evidence of afpIII sequences (Cebidichthys violaceus) in the genomic region where they are found in all other Zoarcoidei, potentially reflecting a lineage-specific loss of this cluster. Beyond polar fishes, our results highlight the power of long-read sequencing to understand genome evolution.

Selection on standing genetic variation mediates convergent evolution in extremophile fish.

Hydrogen sulfide is a toxic gas that disrupts numerous biological processes, including energy production in the mitochondria, yet fish in the Poecilia mexicana species complex have independently evolved sulfide tolerance several times. Despite clear evidence for convergence at the phenotypic level in these fishes, it is unclear if the repeated evolution of hydrogen sulfide tolerance is the result of similar genomic changes. To address this gap, we used a targeted capture approach to sequence genes associated with sulfide processes and toxicity from five sulfidic and five nonsulfidic populations in the species complex. By comparing sequence variation in candidate genes to a reference set, we identified similar population structure and differentiation, suggesting that patterns of variation in most genes associated with sulfide processes and toxicity are due to demographic history and not selection. But the presence of tree discordance for a subset of genes suggests that several loci are evolving divergently between ecotypes. We identified two differentiation outlier genes that are associated with sulfide detoxification in the mitochondria that have signatures of selection in all five sulfidic populations. Further investigation into these regions identified long, shared haplotypes among sulfidic populations. Together, these results reveal that selection on standing genetic variation in putatively adaptive genes may be driving phenotypic convergence in this species complex.

Convergent evolution of conserved mitochondrial pathways underlies repeated adaptation to extreme environments.

Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

Impacts of heavy metal pollution on the ionomes and transcriptomes of Western mosquitofish (Gambusia affinis).

Our understanding of the mechanisms mediating the resilience of organisms to environmental change remains lacking. Heavy metals negatively affect processes at all biological scales, yet organisms inhabiting contaminated environments must maintain homeostasis to survive. Tar Creek in Oklahoma, USA, contains high concentrations of heavy metals and an abundance of Western mosquitofish (Gambusia affinis), though several fish species persist at lower frequency. To test hypotheses about the mechanisms mediating the persistence and abundance of mosquitofish in Tar Creek, we integrated ionomic data from seven resident fish species and transcriptomic data from mosquitofish. We predicted that mosquitofish minimize uptake of heavy metals more than other Tar Creek fish inhabitants and induce transcriptional responses to detoxify metals that enter the body, allowing them to persist in Tar Creek at higher density than species that may lack these responses. Tar Creek populations of all seven fish species accumulated heavy metals, suggesting mosquitofish cannot block uptake more efficiently than other species. We found population-level gene expression changes between mosquitofish in Tar Creek and nearby unpolluted sites. Gene expression differences primarily occurred in the gill, where we found upregulation of genes involved with lowering transfer of metal ions from the blood into cells and mitigating free radicals. However, many differentially expressed genes were not in known metal response pathways, suggesting multifarious selective regimes and/or previously undocumented pathways could impact tolerance in mosquitofish. Our systems-level study identified well characterized and putatively new mechanisms that enable mosquitofish to inhabit heavy metal-contaminated environments.

The life aquatic: advances in marine vertebrate genomics.

The ocean is hypothesized to be where life on earth originated, and subsequent evolutionary transitions between marine and terrestrial environments have been key events in the origin of contemporary biodiversity. Here, we review how comparative genomic approaches are an increasingly important aspect of understanding evolutionary processes, such as physiological and morphological adaptation to the diverse habitats within the marine environment. In addition, we highlight how population genomics has provided unprecedented resolution for population structuring, speciation and adaptation in marine environments, which can have a low cost of dispersal and few physical barriers to gene flow, and can thus support large populations. Building upon this work, we outline the applications of genomics tools to conservation and their relevance to assessing the wide-ranging impact of fisheries and climate change on marine species.

The biogeographic history of eelpouts and related fishes: Linking phylogeny, environmental change, and patterns of dispersal in a globally distributed fish group.

Modern genetic data sets present unprecedented opportunities to understand the evolutionary origins of diverse taxonomic groups. When the timing of key events is known, it is possible to investigate biogeographic history in the context of major phenomena (e.g., cooling of a major ocean). In this study, we investigated the biogeographic history of the suborder Zoarcoidei, a globally distributed fish group that includes species inhabiting both poles that produce antifreeze proteins to survive chronic subfreezing temperatures. We first generated a multi-locus, time-calibrated phylogeny for the group. We then used biogeographic modeling to reconstruct ancestral ranges across the tree and to quantify the type and frequency of biogeographic events (e.g., founder, dispersal). With these results, we considered how the cooling of the Southern and Arctic Oceans, which reached their present-day subfreezing temperatures 10-15 million years ago (Mya) and 2-3 Mya, respectively, may have shaped the group's evolutionary history, with an emphasis on the most speciose and widely distributed family, eelpouts (family Zoarcidae). Our phylogenetic results clarified the Zoarcoidei taxonomy and showed that the group began to diversify in the Oligocene ~31-32 Mya, with the center of origin for all families in north temperate waters. Within-area speciation was the most common biogeographic event in the group's history (80% of all events) followed by dispersal (20%). Finally, we only found evidence, albeit limited, for ocean cooling underpinning diversification of eelpouts living in the high Antarctic over the last 10 million years.

microRNA expression variation as a potential molecular mechanism contributing to adaptation to hydrogen sulphide.

microRNAs (miRNAs) are post-transcriptional regulators of gene expression and can play an important role in modulating organismal development and physiology in response to environmental stress. However, the role of miRNAs in mediating adaptation to diverse environments in natural study systems remains largely unexplored. Here, we characterized miRNAs and their expression in Poecilia mexicana, a species of small fish that inhabits both normal streams and extreme environments in the form of springs rich in toxic hydrogen sulphide (H2 S). We found that P. mexicana has a similar number of miRNA genes as other teleosts. In addition, we identified a large population of mature miRNAs that were differentially expressed between locally adapted populations in contrasting habitats, indicating that miRNAs may contribute to P. mexicana adaptation to sulphidic environments. In silico identification of differentially expressed miRNA-mRNA pairs revealed, in the sulphidic environment, the downregulation of miRNAs predicted to target mRNAs involved in sulphide detoxification and cellular homeostasis, which are pathways essential for life in H2 S-rich springs. In addition, we found that predicted targets of upregulated miRNAs act in the mitochondria (16.6% of predicted annotated targets), which is the main site of H2 S toxicity and detoxification, possibly modulating mitochondrial function. Together, the differential regulation of miRNAs between these natural populations suggests that miRNAs may be involved in H2 S adaptation by promoting functions needed for survival and reducing functions affected by H2 S. This study lays the groundwork for further research to directly demonstrate the role of miRNAs in adaptation to H2 S. Overall, this study provides a critical stepping-stone towards a comprehensive understanding of the regulatory mechanisms underlying the adaptive variation in gene expression in a natural system.

Can offsetting the energetic cost of hibernation restore an active season phenotype in grizzly bears (Ursus arctos horribilis)?

Hibernation is characterized by depression of many physiological processes. To determine if this state is reversible in a non-food caching species, we fed hibernating grizzly bears (Ursus arctos horribilis) dextrose for 10 days to replace 53% or 100% of the estimated minimum daily energetic cost of hibernation. Feeding caused serum concentrations of glycerol and ketones (ß-hydroxybutyrate) to return to active season levels irrespective of the amount of glucose fed. By contrast, free fatty acids (FFAs) and indices of metabolic rate, such as general activity, heart rate, strength of heart rate circadian rhythm, and insulin sensitivity were restored to approximately 50% of active season levels. Body temperature was unaffected by feeding. To determine the contribution of adipose to the metabolic effects observed after glucose feeding, we cultured bear adipocytes collected at the beginning and end of the feeding and performed metabolic flux analysis. We found a ∼33% increase in energy metabolism after feeding. Moreover, basal metabolism before feeding was 40% lower in hibernation cells compared with fed cells or active cells cultured at 37°C, thereby confirming the temperature independence of metabolic rate. The partial depression of circulating FFAs with feeding likely explains the incomplete restoration of insulin sensitivity and other metabolic parameters in hibernating bears. Further depression of metabolic function is likely to be an active process. Together, the results provide a highly controlled model to examine the relationship between nutrient availability and metabolism on the hibernation phenotype in bears.

Big Data in Conservation Genomics: Boosting Skills, Hedging Bets, and Staying Current in the Field.

A current challenge in the fields of evolutionary, ecological, and conservation genomics is balancing production of large-scale datasets with additional training often required to handle such datasets. Thus, there is an increasing need for conservation geneticists to continually learn and train to stay up-to-date through avenues such as symposia, meetings, and workshops. The ConGen meeting is a near-annual workshop that strives to guide participants in understanding population genetics principles, study design, data processing, analysis, interpretation, and applications to real-world conservation issues. Each year of ConGen gathers a diverse set of instructors, students, and resulting lectures, hands-on sessions, and discussions. Here, we summarize key lessons learned from the 2019 meeting and more recent updates to the field with a focus on big data in conservation genomics. First, we highlight classical and contemporary issues in study design that are especially relevant to working with big datasets, including the intricacies of data filtering. We next emphasize the importance of building analytical skills and simulating data, and how these skills have applications within and outside of conservation genetics careers. We also highlight recent technological advances and novel applications to conservation of wild populations. Finally, we provide data and recommendations to support ongoing efforts by ConGen organizers and instructors-and beyond-to increase participation of underrepresented minorities in conservation and eco-evolutionary sciences. The future success of conservation genetics requires both continual training in handling big data and a diverse group of people and approaches to tackle key issues, including the global biodiversity-loss crisis.

Mountain stoneflies may tolerate warming streams: Evidence from organismal physiology and gene expression.

Rapid glacier recession is altering the physical conditions of headwater streams. Stream temperatures are predicted to rise and become increasingly variable, putting entire meltwater-associated biological communities at risk of extinction. Thus, there is a pressing need to understand how thermal stress affects mountain stream insects, particularly where glaciers are likely to vanish on contemporary timescales. In this study, we measured the critical thermal maximum (CTMAX ) of stonefly nymphs representing multiple species and a range of thermal regimes in the high Rocky Mountains, USA. We then collected RNA-sequencing data to assess how organismal thermal stress translated to the cellular level. Our focal species included the meltwater stonefly, Lednia tumana, which was recently listed under the U.S. Endangered Species Act due to climate-induced habitat loss. For all study species, critical thermal maxima (CTMAX  > 20°C) far exceeded the stream temperatures mountain stoneflies experience (<10°C). Moreover, while evidence for a cellular stress response was present, we also observed constitutive expression of genes encoding proteins known to underlie thermal stress (i.e., heat shock proteins) even at low temperatures that reflected natural conditions. We show that high-elevation aquatic insects may not be physiologically threatened by short-term exposure to warm temperatures and that longer-term physiological responses or biotic factors (e.g., competition) may better explain their extreme distributions.

Long-distance dispersal, ice sheet dynamics and mountaintop isolation underlie the genetic structure of glacier ice worms.

Disentangling the contemporary and historical factors underlying the spatial distributions of species is a central goal of biogeography. For species with broad distributions but little capacity to actively disperse, disconnected geographical distributions highlight the potential influence of passive, long-distance dispersal (LDD) on their evolutionary histories. However, dispersal alone cannot completely account for the biogeography of any species, and other factors-e.g. habitat suitability, life history-must also be considered. North American ice worms ( Mesenchytraeus solifugus) are ice-obligate annelids that inhabit coastal glaciers from Oregon to Alaska. Previous studies identified a complex biogeographic history for ice worms, with evidence for genetic isolation, unexpectedly close relationships among geographically disjunct lineages, and contemporary migration across large (e.g. greater than 1500 km) areas of unsuitable habitat. In this study, we analysed genome-scale sequence data for individuals from most of the known ice worm range. We found clear support for divergence between populations along the Pacific Coast and the inland flanks of the Coast Mountains (mean FST = 0.60), likely precipitated by episodic ice sheet expansion and contraction during the Pleistocene. We also found support for LDD of ice worms from Alaska to Vancouver Island, perhaps mediated by migrating birds. Our results highlight the power of genomic data for disentangling complex biogeographic patterns, including the presence of LDD.

Expression analyses of cave mollies (Poecilia mexicana) reveal key genes involved in the early evolution of eye regression.

Eye regression occurs across cave-dwelling populations of many species and is often coupled with a decrease or loss in eye function. Teleost fishes are among the few vertebrates to undergo widespread colonization of caves and often exhibit eye regression with blindness. Cave populations of the poeciliid fish Poecilia mexicana (cave molly) exhibit reduced-albeit functional-eyes, offering the opportunity to investigate partial eye regression. We sequenced eye transcriptomes of cave and surface populations of P. mexicana to identify differentially expressed genes that potentially underlie eye regression in cave mollies. We identified 28 significantly differentially expressed genes, 20 of which were directly related to light sensitivity, eye structure and visual signaling. Twenty-six of these genes were downregulated in cave compared to surface populations. Functional enrichment analysis revealed eye-related gene ontologies that were under-represented in cave mollies. In addition, a set of co-expressed genes related to vision and circadian rhythm was correlated with habitat type (cave versus surface). Our study suggests that differential gene expression plays a key role in the beginning evolutionary stages of eye regression in P. mexicana, shedding further light on regressive evolution in cavefish.

Bacterial Diversity in Replicated Hydrogen Sulfide-Rich Streams.

Extreme environments typically require costly adaptations for survival, an attribute that often translates to an elevated influence of habitat conditions on biotic communities. Microbes, primarily bacteria, are successful colonizers of extreme environments worldwide, yet in many instances, the interplay between harsh conditions, dispersal, and microbial biogeography remains unclear. This lack of clarity is particularly true for habitats where extreme temperature is not the overarching stressor, highlighting a need for studies that focus on the role other primary stressors (e.g., toxicants) play in shaping biogeographic patterns. In this study, we leveraged a naturally paired stream system in southern Mexico to explore how elevated hydrogen sulfide (H2S) influences microbial diversity. We sequenced a portion of the 16S rRNA gene using bacterial primers for water sampled from three geographically proximate pairings of streams with high (> 20 µM) or low (~ 0 µM) H2S concentrations. After exploring bacterial diversity within and among sites, we compared our results to a previous study of macroinvertebrates and fish for the same sites. By spanning multiple organismal groups, we were able to illuminate how H2S may differentially affect biodiversity. The presence of elevated H2S had no effect on overall bacterial diversity (p = 0.21), a large effect on community composition (25.8% of variation explained, p < 0.0001), and variable influence depending upon the group-whether fish, macroinvertebrates, or bacteria-being considered. For bacterial diversity, we recovered nine abundant operational taxonomic units (OTUs) that comprised a core H2S-rich stream microbiome in the region. Many H2S-associated OTUs were members of the Epsilonproteobacteria and Gammaproteobacteria, which both have been implicated in endosymbiotic relationships between sulfur-oxidizing bacteria and eukaryotes, suggesting the potential for symbioses that remain to be discovered in these habitats.