The eutherian placenta exhibits rapid morphological evolution and is a hotspot for the emergence of reproductive incompatibilities between closely related species. These evolutionary patterns are thought to be a consequence of rapid divergence in gene expression driven by maternal-fetal conflict over resource allocation. However, it remains unclear how the diversity of placental functions shapes gene specialization and expression divergence. We generated genome-wide gene expression and DNA methylation data from fetal and maternal placental tissues of three closely related mouse lineages (Mus musculus musculus, M.'m.'domesticus, M. spretus) and integrated single-cell expression data to investigate how tissue specialization influences gene expression evolution in the rodent placenta. Comparisons among placental regions within M.'m.'musculus revealed significant differences in patterns of functional enrichment, imprinting, and X-linked expression across placental layers. The labyrinth zone, the primary site of nutrient exchange, showed strong enrichment for parent-of-origin expression of both autosomal and X-linked genes. Cross-species comparisons of gene expression within each placental layer revealed increased expression level divergence at the maternal-fetal interface. We also identified a subset of genes with maternally biased expression that are spatially associated with the maternal-fetal interface. Parent-of-origin DNA methylation was dominated by epigenetic modification of the maternal genome and interspecific comparisons of parent-of-origin expression revealed overall conservation punctuated by changes in imprinting status of two genes. These findings unveil important links between core elements of placental biology and the evolution of placental gene expression, demonstrating how tissue specialization has influenced parent-of-origin effects and interspecific expression divergence.

Failures of the lysosome-autophagy system are a hallmark of ageing and many disease states. As a consequence, interventions that enhance lysosome function are of keen interest in the context of drug development. Throughout the biomedical literature, evolutionary biologists have found cases in which challenges faced by humans in clinical settings have been resolved by non-model organisms adapting to wild environments. Here, we used a primary cell culture approach to survey lysosomal characteristics in species of the genus Mus. We found that fibroblasts from M. spretus, a wild Mediterranean mouse, exhibited elevated lysosomal mass and enzyme activity along with reduced activity of β-galactosidase, a classical marker of cellular senescence, compared with those from M. musculus, a related species adapted to human-associated environments. We propose that classic laboratory models of lysosome function and senescence may reflect characters that diverge from the phenotypes of wild mice. The M. spretus phenotype may ultimately serve as a blueprint for interventions that ameliorate lysosomal dysfunction under conditions of stress and disease.

The snowshoe hare (Lepus americanus) is considered a critical keystone species of the boreal and temperate forests of North America. Although their population ecology and evolutionary dynamics have been studied extensively, a lack of high-quality genomic resources has limited molecular insights into their biology. Here, we combined cytogenetic karyotype analysis, PacBio HiFi long-read sequencing (~33× coverage), and chromosome conformation capture (Hi-C) sequencing to generate a phased chromosome-level genome assembly from a male snowshoe hare specimen. The primary haploid assembly is 2.75 Gb, with the 24 largest scaffolds corresponding to individual chromosomes (2n = 48 chromosomes, scaffold N50 = 131 Mb) and containing only 213 gaps. Assembly completeness was high, with 99.6% of mammalian Benchmarking Universal Single-Copy Orthologs recovered. Comparative analysis of this genome with other Lepus species revealed conservation of chromosomal architecture within the genus, while alignment with the European rabbit (Oryctolagus cuniculus) genome identified chromosomal rearrangements consistent with known karyotypic divergence between hares and rabbits. This genome assembly establishes a critical reference for future genomic studies in snowshoe hares and related taxa.

Unraveling how adaptive traits originate and evolve is key to understanding the mechanisms shaping species' diversity and their adaptive potential. Seasonal color molts, from summer-brown to winter-white, evolved in at least 21 mammals and birds to maintain camouflage in environments with seasonal snow, but the occurrence of winter-brown morphs reflects seemingly convergent local adaptation to distinct snow conditions. In the least weasel (Mustela nivalis), alternative winter morphs map to the pigmentation gene MC1R, but the evolutionary history and functional basis of this variation remained unknown. Using in vitro cellular assays, we show that winter-brown coats are caused by a derived protein-coding amino acid substitution that reduces MC1R affinity to its ligands, ASIP and α-MSH. Using targeted enrichment and sequencing, we find that this mutation arose de novo within the species, around one million years ago, and was maintained across the geographically structured populations formed during its evolution in Europe. Using simulations, we show that genetic drift is unlikely to explain the long-term maintenance of this variant at intermediate frequencies, which can be driven by spatially varying selection, anchoring local adaptive responses. Our results underscore how long-standing adaptive variation can fuel recurrent adaptation to heterogeneous environments through time.

Andean leaf-eared mice (Phyllotis vaccarum) live at the highest elevations of any mammal, and they also have the broadest elevational range, from sea level to mountain summits of >6700 meters. Highland populations have evolved an enhanced thermogenic capacity in hypoxia relative to lowland conspecifics, and this improved physiological performance is associated with an increased mitochondrial respiratory capacity in skeletal muscle. Population genomic analyses identified mechanisms of hypoxia adaptation and revealed an unanticipated dimension of environmental adaptation in P. vaccarum because selection on biotransformation pathways suggests an evolved capacity to metabolize plant-derived dietary toxins. The world's highest-dwelling mammal has adapted to habitats at both the low- and high-elevation limits of its range, and much of the elevation-related selection relates to previously unappreciated aspects of feeding ecology.

ABSTRACT Island populations are at heightened risk of inbreeding due to reduced mating opportunities with unrelated conspecifics. Extensive inbreeding can result in inbreeding depression (reduced fitness of individuals with related parents). Alexander Archipelago wolves ( Canis lupus ligoni ) are a geographically isolated subspecies that occur in the Southeast Alaskan panhandle, USA, and coastal British Columbia, Canada. Wolves on the Prince of Wales Island complex (POW) in Southeast Alaska are expected to have lower levels of resiliency because they are a small, insular population that has experienced habitat fragmentation and cycles of moderate to heavy harvest. To understand the extent of population structure and inbreeding in Alexander Archipelago wolves, we designed a DNA hybridization capture for wolves and sequenced captured DNA from 58 individuals sampled from across Southeast Alaska during 2002–2016. Estimates of the proportion of the genome in runs of homozygosity ( F ROH ) regardless of run length, revealed that POW wolves were most inbred compared to wolves in other areas of Southeast Alaska. Wolves on POW also had more long (≥ 10 Mb) runs of homozygosity than the other populations we assessed, indicating more frequent mating between individuals with recent common ancestors (1–10 generations ago). This pattern indicates a smaller population size for POW wolves in the recent past compared to other Southeast Alaskan populations. Wolves on POW exhibit an extent of inbreeding similar to that observed in Isle Royale National Park wolves, a population that has exhibited severe inbreeding depression. Our work demonstrates the utility of using genomic capture data to infer individual inbreeding so that proactive management (e.g., setting population targets and harvest quotas, curtailing habitat alteration, etc.) can be considered to ensure the long‐term sustainability of small, isolated populations.

A species tree is a central concept in evolutionary biology whereby a single branching phylogeny reflects relationships among species. However, the phylogenies of different genomic regions often differ from the species tree. Although tree discordance is widespread in phylogenomic studies, we still lack a clear understanding of how variation in phylogenetic patterns is shaped by genome biology or the extent to which discordance may compromise comparative studies. We characterized patterns of phylogenomic discordance across the murine rodents-a large and ecologically diverse group that gave rise to the laboratory mouse and rat model systems. Combining recently published linked-read genome assemblies for seven murine species with other available rodent genomes, we first used ultraconserved elements (UCEs) to infer a robust time-calibrated species tree. We then used whole genomes to examine finer-scale patterns of discordance across ∼12 million years of divergence. We found that proximate chromosomal regions tended to have more similar phylogenetic histories. There was no clear relationship between local tree similarity and recombination rates in house mice, but we did observe a correlation between recombination rates and average similarity to the species tree. We also detected a strong influence of linked selection whereby purifying selection at UCEs led to appreciably less discordance. Finally, we show that assuming a single species tree can result in substantial deviation from the results with gene trees when testing for positive selection under different models. Collectively, our results highlight the complex relationship between phylogenetic inference and genome biology and underscore how failure to account for this complexity can mislead comparative genomic studies.

Principal component analysis (PCA) is routinely used in population genetics to assess genetic structure. With chromosomal reference genomes and population-scale whole genome-sequencing becoming increasingly accessible, contemporary studies often include characterizations of the genomic landscape as it varies along chromosomes, commonly termed genome scans. While traditional summary statistics like FST and dXY remain integral to characterizing the genomic divergence profile, PCA fundamentally differs by providing single-sample resolution, thereby making results intuitively interpretable to help identify polymorphic inversions, introgression and other types of divergent sequence. Here, we introduce WinPCA, a user-friendly package to compute, polarize and visualize genetic principal components in windows along the genome. To accommodate low-coverage whole genome-sequencing datasets, WinPCA can optionally make use of PCAngsd methods to compute principal components in a genotype likelihood framework. WinPCA accepts variant data in either VCF or BEAGLE format and can generate rich plots for interactive data exploration and downstream presentation.

Hybrid incompatibilities are a critical component of species barriers and may arise due to negative interactions between divergent regulatory elements in parental species. We used a comparative approach to identify common themes in the regulatory phenotypes associated with hybrid male sterility in two divergent rodent crosses, dwarf hamsters and house mice. We investigated three potential characteristic gene expression phenotypes in hybrids including the propensity of transgressive differentially expressed genes toward over or underexpression, the influence of developmental stage on patterns of misexpression, and the role of the sex chromosomes on misexpression phenotypes. In contrast to near pervasive overexpression in hybrid house mice, we found that misexpression in hybrid dwarf hamsters was dependent on developmental stage. In both house mouse and dwarf hamster hybrids, however, misexpression increased with the progression of spermatogenesis, although to varying extents and with potentially different consequences. In both systems, we detected sex chromosome-specific overexpression in stages of spermatogenesis where inactivated X chromosome expression was expected, but the hybrid overexpression phenotypes were fundamentally different. Importantly, misexpression phenotypes support the presence of multiple developmental blocks to spermatogenesis in dwarf hamster hybrids, including a potential role of meiotic stalling or breakdown early in spermatogenesis. Collectively, we demonstrate that while there are some similarities in hybrid expression phenotypes of house mice and dwarf hamsters, there are also clear differences that point toward unique mechanisms underlying hybrid male sterility. Our results highlight the potential of comparative approaches in helping to understand the causes and consequences of disrupted gene expression in speciation.

Leaf-eared mice (genus Phyllotis) are among the most widespread and abundant small mammals in the Andean Altiplano, but species boundaries and distributional limits are often poorly delineated due to sparse survey data from remote mountains and high-elevation deserts. Here, we report a combined analysis of mitochondrial DNA variation and whole-genome sequence (WGS) variation in Phyllotis mice to delimit species boundaries, to assess the timescale of diversification of the group, and to examine evidence for interspecific hybridization. Estimates of divergence based on cytb data suggest that most diversification of Phyllotis occurred during the past 3 million years. Consistent with the Pleistocene Aridification hypothesis, our results suggest that diversification of Phyllotis largely coincided with climatically induced environmental changes in the mid- to late-Pleistocene. Contrary to the Montane Uplift hypothesis, most diversification in the group occurred well after the major phase of uplift of the Central Andean Plateau. Species delimitation analyses revealed surprising patterns of cryptic diversity within several nominal forms, suggesting the presence of much undescribed alpha diversity in the genus. Results of genomic analyses revealed evidence of hybridization between the sister species P. limatus and P. vaccarum, suggesting that the contemporary zone of range overlap between the two species represents a hybrid zone.

Failures of the lysosome-autophagy system are a hallmark of aging and many disease states. As a consequence, interventions that enhance lysosome function are of keen interest in the context of drug development. Throughout the biomedical literature, evolutionary biologists have discovered that challenges faced by humans in clinical settings have been resolved by non-model organisms adapting to wild environments. Here, we used a primary cell culture approach to survey lysosomal characteristics in selected species of the genus Mus. We found that cells from M. musculus, mice adapted to human environments, had weak lysosomal acidification and high expression and activity of the lysosomal enzyme β-galactosidase, a classic marker of cellular senescence. Cells of wild relatives, especially the Mediterranean mouse M. spretus, had more robustly performing lysosomes and dampened β-galactosidase levels. We propose that classic laboratory models of lysosome function and senescence may reflect characters that diverge from the phenotypes of wild mice. The M. spretus phenotype may ultimately provide a blueprint for interventions that ameliorate lysosome breakdown in stress and disease.

Gestational hypoxia reduces fetal growth and birth weight across mammals, including humans. Evolutionary adaptation to high-elevation hypoxia mitigates these negative effects, and identifying these protective mechanisms may offer insight into how environmental factors interact with gestational physiology to influence health outcomes. We know that gestational hypoxia modifies development of the placenta, which mediates maternal-fetal exchange, but little is known about how high-altitude adaptation interacts with this developmental plasticity to influence placental exchange capacity. We tested the hypothesis that hypoxia-dependent remodelling of the placental exchange surface is protective for fetal growth and thus will be exaggerated in highland-adapted individuals by using a model rodent system, the North American deer mouse. We acclimated lowland- and highland-ancestry deer mice to normoxia or hypoxia (12.3% O2) during gestation and found that lowland-ancestry deer mice expand their placenta and maternal blood spaces in the placenta in response to environmental hypoxia. Highland-ancestry deer mice produce even larger placentas and maternal blood spaces, suggesting that these hypoxia-driven responses may benefit fetal growth by increasing total exchange capacity. Notably, we also found that the fetal blood spaces in highland-ancestry placentas have increased perimeter (a proxy for surface area) per unit area occupied by blood. Similar changes to fetal vasculature have been observed in high-elevation-adapted human populations, which is suggestive of convergent adaptation. Our results demonstrate that the hypoxia-sensitive development of placental vasculature is remodelled by adaptation to environmental hypoxia and that some of these processes may be points for convergent adaptation across species despite distinct placental architectures. KEY POINTS: Evolutionary adaptation to high elevations provides protection against hypoxia-dependent fetal growth restriction. The placenta is a key determinant of fetal growth because it defines the total surface area available for nutrient and gas exchange between the gestational parent and offspring. We tested the hypothesis that evolutionary adaptation to high elevations protects fetal growth by increasing placental surface area for exchange using acclimation experiments in a model rodent system, the North American deer mouse. As we predicted, high-elevation ancestry increased the size of maternal blood spaces in the placenta, especially under gestational hypoxia; however, highland ancestry was also associated with narrower fetal blood spaces, which could increase exchange efficiency. The patterns observed in deer mice resemble developmental plasticity observed in placentas from humans with high-elevation ancestry, pointing to potential for convergent adaptation across species with distinct placental architectures.

In the world's highest mountain ranges, uncertainty about the upper elevational range limits of alpine animals represents a critical knowledge gap regarding the environmental limits of life and presents a problem for detecting range shifts in response to climate change. Here we report results of mountaineering mammal surveys in the Central Andes, which led to the discovery of multiple species of mice living at extreme elevations that far surpass previously assumed range limits for mammals. We live-trapped small mammals from ecologically diverse sites spanning >6700 m of vertical relief, from the desert coast of northern Chile to the summits of the highest volcanoes in the Andes. We used molecular sequence data and whole-genome sequence data to confirm the identities of species that represent new elevational records and to test hypotheses regarding species limits. These discoveries contribute to a new appreciation of the environmental limits of vertebrate life.

It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass (RTM), presumably reflecting reduced sperm competition. Several sperm traits were associated with RTM, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller RTM due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction and that broad patterns of molecular evolution may help identify genes that contribute to male fertility.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPβ. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

The genetic basis of adaptive traits has rarely been used to predict future vulnerability of populations to climate change. We show that light versus dark seasonal pelage in white-tailed jackrabbits (Lepus townsendii) tracks snow cover and is primarily determined by genetic variation at endothelin receptor type B (EDNRB), corin serine peptidase (CORIN), and agouti signaling protein (ASIP). Winter color variation was associated with deeply divergent alleles at these genes, reflecting selection on both ancestral and introgressed variation. Forecasted reductions in snow cover are likely to induce widespread camouflage mismatch. However, simulated populations with variation for darker winter pelage are predicted to adapt rapidly, providing a trait-based genetic framework to facilitate evolutionary rescue. These discoveries demonstrate how the genetic basis of climate change adaptation can inform conservation.

SUMMARYOur understanding of the limits of animal life is continually revised by scientific exploration of extreme environments. Here we report the discovery of numerous mummified cadavers of leaf-eared mice,Phyllotis vaccarum, from the summits of three different Andean volcanoes at elevations 6029-6233 m (19,780-20,449 ft) above sea level in the Puna de Atacama (Chile-Argentina). Such extreme elevations were previously assumed to be completely uninhabitable by mammals. In combination with a live-captured specimen of the same species from the nearby summit of Volcán Llullaillaco (6739 m [=22,110 ft]), the 13 summit mummies represent the highest physical records of mammals in the world. We report a chromosome-level genome assembly forP. vaccarumin combination with a whole-genome re-sequencing analysis and radiocarbon dating analysis that provide insights into the provenance and antiquity of the summit mice. We test alternative hypotheses to explain the existence of mouse graveyards on the summits of Atacama volcanoes. Radiocarbon data indicate that the most ancient of the mummies were at most a few centuries old. Genomic polymorphism data revealed a high degree of continuity between the summit mice and conspecifics from lower elevations in the surrounding Altiplano. Genomic data also revealed equal numbers of males and females among the summit mice and evidence of close kinship between some individuals from the same summit groups. These findings bolster evidence for self-sustaining populations ofPhyllotisat elevations >6000 m and challenge assumptions about the environmental limits of vertebrate life and the physiological tolerances of small mammals.

The global impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to considerable interest in detecting novel beneficial mutations and other genomic changes that may signal the development of variants of concern (VOCs). The ability to accurately detect these changes within individual patient samples is important in enabling early detection of VOCs. Such genomic scans for positive selection are best performed via comparison of empirical data to simulated data wherein evolutionary factors, including mutation and recombination rates, reproductive and infection dynamics, and purifying and background selection, can be carefully accounted for and parameterized. While there has been work to quantify these factors in SARS-CoV-2, they have yet to be integrated into a baseline model describing intra-host evolutionary dynamics. To construct such a baseline model, we develop a simulation framework that enables one to establish expectations for underlying levels and patterns of patient-level variation. By varying eight key parameters, we evaluated 12,096 different model-parameter combinations and compared them to existing empirical data. Of these, 592 models (∼5%) were plausible based on the resulting mean expected number of segregating variants. These plausible models shared several commonalities shedding light on intra-host SARS-CoV-2 evolutionary dynamics: severe infection bottlenecks, low levels of reproductive skew, and a distribution of fitness effects skewed towards strongly deleterious mutations. We also describe important areas of model uncertainty and highlight additional sequence data that may help to further refine a baseline model. This study lays the groundwork for the improved analysis of existing and future SARS-CoV-2 within-patient data. Significance Statement Despite its tremendous impact on human health, a comprehensive evolutionary baseline model has yet to be developed for studying the within-host population genomics of SARS-CoV-2. Importantly, such modeling would enable improved analysis and provide insights into the key evolutionary dynamics governing SARS-CoV-2 evolution. Given this need, we have here quantified a set of plausible baseline models via large-scale simulation. The commonly shared features of these relevant models - including severe infection bottlenecks, low levels of progeny skew, and a high rate of strongly deleterious mutations - lay the foundation for sophisticated analyses of SARS-CoV-2 evolution within patients using these baseline models.

The mammalian placenta is a hotspot for the evolution of genomic imprinting, a form of gene regulation that involves the parent-specific epigenetic silencing of one allele. Imprinted genes are central to placental development and are thought to contribute to the evolution of reproductive barriers between species. However, it is unclear how rapidly imprinting evolves or how functional specialization among placental tissues influences the evolution of imprinted expression. We compared parent-of-origin expression bias across functionally distinct placental layers sampled from reciprocal crosses within three closely related lineages of mice ( Mus ). Using genome-wide gene expression and DNA methylation data from fetal and maternal tissues, we developed an analytical strategy to minimize pervasive bias introduced by maternal contamination of placenta samples. We corroborated imprinted expression at 42 known imprinted genes and identified five candidate imprinted genes showing parent-of-origin specific expression and DNA methylation. Paternally-biased expression was enriched in the labyrinth zone, a layer specialized in nutrient transfer, and maternally-biased genes were enriched in the junctional zone, which specializes in modulation of maternal physiology. Differentially methylated regions were predominantly determined through epigenetic modification of the maternal genome and were associated with both maternally- and paternally-biased gene expression. Lastly, comparisons between lineages revealed a small set of co-regulated genes showing rapid divergence in expression levels and imprinted status in the M. m. domesticus lineage. Together, our results reveal important links between core functional elements of placental biology and the evolution of imprinted gene expression among closely related rodent species.

Our understanding of the limits of animal life is continually revised by scientific exploration of extreme environments. Here we report the discovery of mummified cadavers of leaf-eared mice, Phyllotis vaccarum, from the summits of three different Andean volcanoes at elevations 6,029-6,233 m above sea level in the Puna de Atacama in Chile and Argentina. Such extreme elevations were previously assumed to be completely uninhabitable by mammals. In combination with a live-captured specimen of the same species from the nearby summit of Volcán Llullaillaco (6,739 m)1, the summit mummies represent the highest altitude physical records of mammals in the world. We also report a chromosome-level genome assembly for P. vaccarum that, in combination with a whole-genome re-sequencing analysis and radiocarbon dating analysis, provides insights into the provenance and antiquity of the summit mice. Radiocarbon data indicate that the most ancient of the mummies are, at most, a few centuries old. Genomic polymorphism data revealed a high degree of continuity between the summit mice and conspecifics from lower elevations in the surrounding Altiplano. Genomic data also revealed equal numbers of males and females among the summit mice and evidence of close kinship between some individuals from the same summits. These findings bolster evidence for resident populations of Phyllotis at elevations >6,000 m and challenge assumptions about the environmental limits of vertebrate life and the physiological tolerances of small mammals.