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Abstract Phased genomes and pangenomes are enhancing our understanding of genetic variation. Accurate phasing and assembly in repetitive regions of the genome remain challenging, however. Addressing this obstacle is crucial for studying structural genomic variation, such as copy number variations (CNVs) common to repetitive regions. Polar fishes, for example, evolved repetitive tandem arrays of antifreeze protein (AFP) genes that facilitate adaptation to freezing and expanded in copy number in colder environments. AFP CNVs remain poorly characterized in polar fishes and may be illuminated by haplotype-aware approaches. We performed long-read sequencing for two polar fishes in the suborder Zoarcoidei and leveraged additional published long-read data to assemble phased genomes. We developed a workflow to measure haplotype diversity in CNV while controlling for misassembly and switch errors—a change from one parental haplotype to another in a contiguous assembly. We presentgfa_parser, which computes and extracts all possible contiguous sequences for phased or primary assemblies from graphical fragment assembly (GFA) files, andswitch_error_screen, which flags potential switch errors.gfa_parserrevealed that assembly uncertainty was ubiquitous across AFP array haplotypes and that standard processing of graphical fragment assemblies can bias measurement of haplotype CNVs. We detected no switch errors in AFP arrays. After controlling for misassembly and switch error, we detected haplotype diversity of AFP CNVs in all studied polar Zoarcoidei species and in 60% of AFP arrays. Intraindividual haplotype diversity spanned differences of 3–16 copies. Our workflow revealed intraspecific genomic diversity in zoarcoids that likely fueled the evolution of AFP copy number across temperature. 

Abstract Microsatellites are powerful markers for tracking genetic variation in wildlife populations due to their high polymorphism and genome-wide abundance. While polymerase chain reaction (PCR)-based fragment size analysis has been the standard for genotyping microsatellites, high-throughput sequencing offers greater resolution and the opportunity to sync historical datasets with modern analyses. We evaluated how genotypes from whole-genome sequencing align with PCR data for 15 microsatellite loci in 11 North American brown bears (Ursus arctos). Brown bear populations in the 48 contiguous United States have declined from approximately 50,000 to fewer than 2,000 over the past decades. Their endangered status has prompted extensive research and genetic monitoring, yielding large, multiyear microsatellite datasets upon which future conservation efforts can build. We achieved an overall microsatellite genotype concordance rate of 94.5% comparing high-throughput sequencing results to PCR based-fragment size results. All discrepancies occurred at complex loci containing multiple insertions and/or deletions (indels). Physically linked indels or single nucleotide polymorphisms (SNPs) occurring within the loci were misinterpreted as independent insertions, underscoring the need for genotyping tools that incorporate phasing when genotyping. To evaluate coverage effects, we downsampled high-throughput sequence data from 30x to 2x. Concordance remained high at 20 to 30x but dropped sharply at 10x, with 5x and 2x having discordant genotypes or insufficient coverage for genotyping. Accurate genotyping required both sufficient depth and number of reads spanning the entire repeat regions. Our results show that short-read whole-genome sequencing can recover microsatellite genotypes with high accuracy when paired with careful variant interpretation. By aligning historical PCR datasets with modern sequencing data, we can preserve decades of genetic insight and strengthen long-term monitoring of at-risk populations. 

Hibernation is a fascinating adaptation to food-scarce winters, characterized by significant physiological and behavioral changes, including fasting, inactivity, and insulin resistance. While hibernation is critical for the survival of many species, hibernation-related traits are often considered pathological in humans. Hibernation has been studied from a genomic perspective, especially with respect to transcription across multiple tissues. These studies have identified the differential activity of signaling pathways related to metabolism, tissue protection, and other mechanisms likely underlying hibernation phenotypes. Bears, in particular, are an interesting model for physiological and genomic studies of hibernation due to their large size and unique mode of hibernation compared to other small mammalian hibernators. Investigating the intricate molecular mechanisms underlying bear hibernation may therefore provide insight into fundamental biological processes with potential translational implications for human health, particularly with respect to metabolic disorders such as type II diabetes. This review focuses on recent advances and outstanding questions related to the exploration of bear hibernation from a genomic perspective. 

ABSTRACT Epigenetic modifications to DNA are proposed to underpin plastic responses to environmental change, and the manner in which DNA methylation contributes to plasticity likely differs among tissues. However, few studies have investigated tissue‐specific DNA methylation responses to ecologically relevant environmental stressors in natural settings. Here, we used reduced representation bisulfite sequencing to examine the influence of in situ microhabitats on DNA methylation in gill and foot of juvenile California mussels (Mytilus californianus), a foundation species with widespread dispersal and little evidence of genetic population structure. We examined mussels from a one‐month reciprocal transplant experiment between a cool, wave‐exposed and a warm, wave‐protected microhabitat. These manipulations—which were previously shown to alter juvenile mussels' heat tolerance—led to significant and tissue‐specific changes in CpG methylation, including within a number of genes with roles in stress response pathways. Differentially methylated genes were involved in processes including heat shock response, proteolysis, and temperature sensing. In gills, differentially methylated CpGs were more likely to occur in introns relative to other inter‐ and intragenic features. This study expands on previous research that examined environmentally driven shifts in DNA methylation by documenting plastic and tissue‐specific changes in DNA methylation between microhabitats in a natural setting. 

Abstract Multiple lineages in the family Poeciliidae have independently adapted to hydrogen-sulfide-rich springs. The independent colonizations of such springs mean that there are naturally replicated lineages that provide a powerful model for studying adaptation and convergent evolution. However, there are limited genomic resources for many genera and species across Poeciliidae. Here, we present six high-quality, chromosome-level, annotated genome assemblies for Poecilia and Gambusia populations, five of which are the first for the species or ecotype, and the remaining assembly improved the current reference genome contiguity by more than 100-fold. Using these new assemblies, we compare repeat content and model historical changes in effective population size. 

Salinity gradients act as strong environmental barriers that limit the distribution of aquatic organisms. Changes in gene expression associated with transitions between freshwater and saltwater environments can provide insights into organismal responses to variation in salinity. We used RNA-sequencing (RNA-seq) to investigate genome-wide variation in gene expression between a hypersaline population and a freshwater population of the livebearing fish speciesLimia perugiae(Poeciliidae). Our analyses of gill gene expression revealed potential molecular mechanisms underlying salinity tolerance in this species, including the enrichment of genes involved in ion transport, maintenance of chemical homeostasis, and cell signaling in the hypersaline population. We also found differences in gene expression patterns associated with cell-cycle and protein-folding processes between the hypersaline and freshwaterL.perugiae. Bidirectional freshwater-saltwater transitions have occurred repeatedly during the diversification of fishes, allowing for broad-scale examination of repeatable patterns in evolution. Therefore, we compared transcriptomic variation inL.perugiaewith other teleosts that have made freshwater-saltwater transitions to test for convergence in gene expression. Among the four distantly related population pairs from high- and low-salinity environments that we included in our analysis, we found only ten shared differentially expressed genes, indicating little evidence for convergence. However, we found that differentially expressed genes shared among three or more lineages were functionally enriched for ion transport and immune functioning. Overall, our results—in conjunction with other recent studies—suggest that different genes are involved in salinity transitions across disparate lineages of teleost fishes. 

Abstract Reconstructions of evolutionary history can be restricted by a lack of high-quality reference genomes. To date, only four of the eight species of bears (family Ursidae) have chromosome-level genome assemblies. Here, we present assemblies for three additional species—the sun, sloth, and Andean bears—and use a whole-genome alignment of all bear species and other carnivores to reconstruct the evolution of Ursidae. Multiple divergence dating approaches suggest that the six Ursine bears likely diversified in the last 5 Ma, but that divergence times within Ursinae are significantly impacted by gene tree heterogeneity. Consistent with this, we observe that nearly 50% of gene trees conflict with our highly supported species tree, a pattern driven by a significant early hybridization event within Ursinae. We also find that the karyotype of Ursinae is largely similar to the ancestral karyotype of all bears twenty million years prior. In contrast to this conservation of structure, dozens of chromosomal fissions and fusions associated with LINE/L1 retrotransposons dramatically restructured the genomes of the giant panda and Andean bear. Finally, we leverage these genomes to identify species-specific evidence for positive selection on genes associated with color, diet, and metabolism. One of these genes, TCPN2, has a role in pigmentation and shows a series of amino acid mutations in the polar bear over the last 0.5 Ma. Collectively, these new genomic resources enable improved reconstruction of the complex evolutionary history of bears and clarify how this enigmatic group diversified. 

Abstract Antifreeze proteins (AFPs) have enabled teleost fishes to repeatedly colonize polar seas. Four AFP types have convergently evolved in several fish lineages. AFPs inhibit ice crystal growth and lower tissue freezing point. In lineages with AFPs, species inhabiting colder environments may possess more AFP copies. Elucidating how differences in AFP copy number evolve is challenging due to the genes’ tandem array structure and consequently poor resolution of these repetitive regions. Here, we explore the evolution of type III AFPs (AFP III) in the globally distributed suborder Zoarcoidei, leveraging six new long-read genome assemblies. Zoarcoidei has fewer genomic resources relative to other polar fish clades while it is one of the few groups of fishes adapted to both the Arctic and Southern Oceans. Combining these new assemblies with additional long-read genomes available for Zoarcoidei, we conducted a comprehensive phylogenetic test of AFP III evolution and modeled the effects of thermal habitat and depth on AFP III gene family evolution. We confirm a single origin of AFP III via neofunctionalization of the enzyme sialic acid synthase B. We also show that AFP copy number increased under low temperature but decreased with depth, potentially because pressure lowers freezing point. Associations between the environment and AFP III copy number were driven by duplications of paralogs that were translocated out of the ancestral locus at which AFP III arose. Our results reveal novel environmental effects on AFP evolution and demonstrate the value of high-quality genomic resources for studying how structural genomic variation shapes convergent adaptation.