Molecular evolution of male reproduction across species with highly divergent sperm morphology in diverse murine rodents.

Sperm competition can drive rapid evolution of male reproductive traits, but it remains unclear how variation in sperm competition intensity shapes phenotypic and molecular diversity across clades. Old World mice and rats (subfamily Murinae) comprise a rapid radiation and exhibit incredible diversity in sperm morphology and production. We combined phenotype and sequence data to model the evolution of reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, a trait reflective of reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution likely selects for convergent traits related to sperm competitive ability. Molecular evolutionary rates of spermatogenesis proteins also correlated with relative testes mass, but in an unexpected direction. We predicted that sperm competition would result in rapid divergence among species with large relative testes mass, but instead found that many spermatogenesis genes evolve more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved under positive selection, relaxed selection played a greater role underlying rapid evolution in small testes species. Our work demonstrates that sexual selection can impose strong purifying selection shaping the evolution of male reproduction.

Genomic resolution of cryptic species diversity in chipmunks.

Discovery of cryptic species is essential to understand the process of speciation and assessing the impacts of anthropogenic stressors. Here, we used genomic data to test for cryptic species diversity within an ecologically well-known radiation of North American rodents, western chipmunks (Tamias). We assembled a de novo reference genome for a single species (Tamias minimus) combined with new and published targeted sequence-capture data for 21,551 autosomal and 493 X-linked loci sampled from 121 individuals spanning 22 species. We identified at least two cryptic lineages corresponding with an isolated subspecies of least chipmunk (T. minimus grisescens) and with a restricted subspecies of the yellow-pine chipmunk (Tamias amoenus cratericus) known only from around the extensive Craters of the Moon lava flow. Additional population-level sequence data revealed that the so-called Crater chipmunk is a distinct species that is abundant throughout the coniferous forests of southern Idaho. This cryptic lineage does not appear to be most closely related to the ecologically and phenotypically similar yellow-pine chipmunk but does show evidence for recurrent hybridization with this and other species.

Molecular Evolution of Ecological Specialisation: Genomic Insights from the Diversification of Murine Rodents.

Adaptive radiations are characterized by the diversification and ecological differentiation of species, and replicated cases of this process provide natural experiments for understanding the repeatability and pace of molecular evolution. During adaptive radiation, genes related to ecological specialization may be subject to recurrent positive directional selection. However, it is not clear to what extent patterns of lineage-specific ecological specialization (including phenotypic convergence) are correlated with shared signatures of molecular evolution. To test this, we sequenced whole exomes from a phylogenetically dispersed sample of 38 murine rodent species, a group characterized by multiple, nested adaptive radiations comprising extensive ecological and phenotypic diversity. We found that genes associated with immunity, reproduction, diet, digestion, and taste have been subject to pervasive positive selection during the diversification of murine rodents. We also found a significant correlation between genome-wide positive selection and dietary specialization, with a higher proportion of positively selected codon sites in derived dietary forms (i.e., carnivores and herbivores) than in ancestral forms (i.e., omnivores). Despite striking convergent evolution of skull morphology and dentition in two distantly related worm-eating specialists, we did not detect more genes with shared signatures of positive or relaxed selection than in a nonconvergent species comparison. Although a small number of the genes we detected can be incidentally linked to craniofacial morphology or diet, protein-coding regions are unlikely to be the primary genetic basis of this complex convergent phenotype. Our results suggest a link between positive selection and derived ecological phenotypes, and highlight specific genes and general functional categories that may have played an integral role in the extensive and rapid diversification of murine rodents.

Human brain region-specific variably methylated regions are enriched for heritability of distinct neuropsychiatric traits.

BACKGROUND: DNA methylation dynamics in the brain are associated with normal development and neuropsychiatric disease and differ across functionally distinct brain regions. Previous studies of genome-wide methylation differences among human brain regions focus on limited numbers of individuals and one to two brain regions. RESULTS: Using GTEx samples, we generate a resource of DNA methylation in purified neuronal nuclei from 8 brain regions as well as lung and thyroid tissues from 12 to 23 donors. We identify differentially methylated regions between brain regions among neuronal nuclei in both CpG (181,146) and non-CpG (264,868) contexts, few of which were unique to a single pairwise comparison. This significantly expands the knowledge of differential methylation across the brain by 10-fold. In addition, we present the first differential methylation analysis among neuronal nuclei from basal ganglia tissues and identify unique CpG differentially methylated regions, many associated with ion transport. We also identify 81,130 regions of variably CpG methylated regions, i.e., variable methylation among individuals in the same brain region, which are enriched in regulatory regions and in CpG differentially methylated regions. Many variably methylated regions are unique to a specific brain region, with only 202 common across all brain regions, as well as lung and thyroid. Variably methylated regions identified in the amygdala, anterior cingulate cortex, and hippocampus are enriched for heritability of schizophrenia. CONCLUSIONS: These data suggest that epigenetic variation in these particular human brain regions could be associated with the risk for this neuropsychiatric disorder.

X chromosome-dependent disruption of placental regulatory networks in hybrid dwarf hamsters.

Embryonic development in mammals is highly sensitive to changes in gene expression within the placenta. The placenta is also highly enriched for genes showing parent-of-origin or imprinted expression, which is predicted to evolve rapidly in response to parental conflict. However, little is known about the evolution of placental gene expression, or if divergence of placental gene expression plays an important role in mammalian speciation. We used crosses between two species of dwarf hamsters (Phodopus sungorus and Phodopus campbelli) to examine the genetic and regulatory underpinnings of severe placental overgrowth in their hybrids. Using quantitative genetic mapping and mitochondrial substitution lines, we show that overgrowth of hybrid placentas was primarily caused by genetic differences on the maternally inherited P. sungorus X chromosome. Mitochondrial interactions did not contribute to abnormal hybrid placental development, and there was only weak correspondence between placental disruption and embryonic growth. Genome-wide analyses of placental transcriptomes from the parental species and first- and second-generation hybrids revealed a central group of co-expressed X-linked and autosomal genes that were highly enriched for maternally biased expression. Expression of this gene network was strongly correlated with placental size and showed widespread misexpression dependent on epistatic interactions with X-linked hybrid incompatibilities. Collectively, our results indicate that the X chromosome is likely to play a prominent role in the evolution of placental gene expression and the accumulation of hybrid developmental barriers between mammalian species.

The Legacy of Recurrent Introgression during the Radiation of Hares.

Hybridization may often be an important source of adaptive variation, but the extent and long-term impacts of introgression have seldom been evaluated in the phylogenetic context of a radiation. Hares (Lepus) represent a widespread mammalian radiation of 32 extant species characterized by striking ecological adaptations and recurrent admixture. To understand the relevance of introgressive hybridization during the diversification of Lepus, we analyzed whole exome sequences (61.7 Mb) from 15 species of hares (1-4 individuals per species), spanning the global distribution of the genus, and two outgroups. We used a coalescent framework to infer species relationships and divergence times, despite extensive genealogical discordance. We found high levels of allele sharing among species and show that this reflects extensive incomplete lineage sorting and temporally layered hybridization. Our results revealed recurrent introgression at all stages along the Lepus radiation, including recent gene flow between extant species since the last glacial maximum but also pervasive ancient introgression occurring since near the origin of the hare lineages. We show that ancient hybridization between northern hemisphere species has resulted in shared variation of potential adaptive relevance to highly seasonal environments, including genes involved in circadian rhythm regulation, pigmentation, and thermoregulation. Our results illustrate how the genetic legacy of ancestral hybridization may persist across a radiation, leaving a long-lasting signature of shared genetic variation that may contribute to adaptation. [Adaptation; ancient introgression; hybridization; Lepus; phylogenomics.].

Transcriptomic regulation of seasonal coat color change in hares.

Color molts from summer brown to winter white coats have evolved in several species to maintain camouflage year-round in environments with seasonal snow. Despite the eco-evolutionary relevance of this key phenological adaptation, its molecular regulation has only recently begun to be addressed. Here, we analyze skin transcription changes during the autumn molt of the mountain hare (Lepus timidus) and integrate the results with an established model of gene regulation across the spring molt of the closely related snowshoe hare (L. americanus). We quantified differences in gene expression among three stages of molt progression-"brown" (early molt), "intermediate," and "white" (late molt). We found 632 differentially expressed genes, with a major pulse of expression early in the molt, followed by a milder one in late molt. The functional makeup of differentially expressed genes anchored the sampled molt stages to the developmental timeline of the hair growth cycle, associating anagen to early molt and the transition to catagen to late molt. The progression of color change was characterized by differential expression of genes involved in pigmentation, circadian, and behavioral regulation. We found significant overlap between differentially expressed genes across the seasonal molts of mountain and snowshoe hares, particularly at molt onset, suggesting conservatism of gene regulation across species and seasons. However, some discrepancies suggest seasonal differences in melanocyte differentiation and the integration of nutritional cues. Our established regulatory model of seasonal coat color molt provides an important mechanistic context to study the functional architecture and evolution of this crucial seasonal adaptation.

An Annotated Draft Genome of the Mountain Hare (Lepus timidus).

Hares (genus Lepus) provide clear examples of repeated and often massive introgressive hybridization and striking local adaptations. Genomic studies on this group have so far relied on comparisons to the European rabbit (Oryctolagus cuniculus) reference genome. Here, we report the first de novo draft reference genome for a hare species, the mountain hare (Lepus timidus), and evaluate the efficacy of whole-genome re-sequencing analyses using the new reference versus using the rabbit reference genome. The genome was assembled using the ALLPATHS-LG protocol with a combination of overlapping pair and mate-pair Illumina sequencing (77x coverage). The assembly contained 32,294 scaffolds with a total length of 2.7 Gb and a scaffold N50 of 3.4 Mb. Re-scaffolding based on the rabbit reference reduced the total number of scaffolds to 4,205 with a scaffold N50 of 194 Mb. A correspondence was found between 22 of these hare scaffolds and the rabbit chromosomes, based on gene content and direct alignment. We annotated 24,578 protein coding genes by combining ab-initio predictions, homology search, and transcriptome data, of which 683 were solely derived from hare-specific transcriptome data. The hare reference genome is therefore a new resource to discover and investigate hare-specific variation. Similar estimates of heterozygosity and inferred demographic history profiles were obtained when mapping hare whole-genome re-sequencing data to the new hare draft genome or to alternative references based on the rabbit genome. Our results validate previous reference-based strategies and suggest that the chromosome-scale hare draft genome should enable chromosome-wide analyses and genome scans on hares.

The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Neuronal brain-region-specific DNA methylation and chromatin accessibility are associated with neuropsychiatric trait heritability.

Epigenetic modifications confer stable transcriptional patterns in the brain, and both normal and abnormal brain function involve specialized brain regions. We examined DNA methylation by whole-genome bisulfite sequencing in neuronal and non-neuronal populations from four brain regions (anterior cingulate gyrus, hippocampus, prefrontal cortex, and nucleus accumbens) as well as chromatin accessibility in the latter two. We find pronounced differences in both CpG and non-CpG methylation (CG-DMRs and CH-DMRs) only in neuronal cells across brain regions. Neuronal CH-DMRs were highly associated with differential gene expression, whereas CG-DMRs were consistent with chromatin accessibility and enriched for regulatory regions. These CG-DMRs comprise ~12 Mb of the genome that is highly enriched for genomic regions associated with heritability of neuropsychiatric traits including addictive behavior, schizophrenia, and neuroticism, thus suggesting a mechanistic link between pathology and differential neuron-specific epigenetic regulation in distinct brain regions.

Adaptive introgression underlies polymorphic seasonal camouflage in snowshoe hares.

Snowshoe hares (Lepus americanus) maintain seasonal camouflage by molting to a white winter coat, but some hares remain brown during the winter in regions with low snow cover. We show that cis-regulatory variation controlling seasonal expression of the Agouti gene underlies this adaptive winter camouflage polymorphism. Genetic variation at Agouti clustered by winter coat color across multiple hare and jackrabbit species, revealing a history of recurrent interspecific gene flow. Brown winter coats in snowshoe hares likely originated from an introgressed black-tailed jackrabbit allele that has swept to high frequency in mild winter environments. These discoveries show that introgression of genetic variants that underlie key ecological traits can seed past and ongoing adaptation to rapidly changing environments.

Mountain hare transcriptome and diagnostic markers as resources to monitor hybridization with European hares.

We report the first mountain hare (Lepus timidus) transcriptome, produced by de novo assembly of RNA-sequencing reads. Data were obtained from eight specimens sampled in two localities, Alps and Ireland. The mountain hare tends to be replaced by the invading European hare (Lepus europaeus) in their numerous contact zones where the species hybridize, which affects their gene pool to a yet unquantified degree. We characterize and annotate the mountain hare transcriptome, detect polymorphism in the two analysed populations and use previously published data on the European hare (three specimens, representing the European lineage of the species) to identify 4 672 putative diagnostic sites between the species. A subset of 85 random independent SNPs was successfully validated using PCR and Sanger sequencing. These valuable genomic resources can be used to design tools to assess population status and monitor hybridization between species.

The transcriptional landscape of seasonal coat colour moult in the snowshoe hare.

Seasonal coat colour change is an important adaptation to seasonally changing environments but the evolution of this and other circannual traits remains poorly understood. In this study, we use gene expression to understand seasonal coat colour moulting in wild snowshoe hares (Lepus americanus). We used hair colour to follow the progression of the moult, simultaneously sampling skin from three moulting stages in hares collected during the peak of the spring moult from white winter to brown summer pelage. Using RNA sequencing, we tested whether patterns of expression were consistent with predictions based on the established phases of the hair growth cycle. We found functionally consistent clustering across skin types, with 766 genes differentially expressed between moult stages. "White" pelage showed more differentially expressed genes that were upregulated relative to other skin types, involved in the transition between late telogen (quiescent stage) and the onset of anagen (proliferative stage). Skin samples from transitional "intermediate" and "brown" pelage were transcriptionally similar and resembled the regressive transition to catagen (regressive stage). We also detected differential expression of several key circadian clock and pigmentation genes, providing important means to dissect the bases of alternate seasonal colour morphs. Our results reveal that pelage colour is a useful biomarker for seasonal change but that there is a consistent lag between the main gene expression waves and change in visible coat colour. These experiments establish that developmental sampling from natural populations of nonmodel organisms can provide a crucial resource to dissect the genetic basis and evolution of complex seasonally changing traits.

Widespread triploidy in Western North American aspen (Populus tremuloides).

We document high rates of triploidy in aspen (Populus tremuloides) across the western USA (up to 69% of genets), and ask whether the incidence of triploidy across the species range corresponds with latitude, glacial history (as has been documented in other species), climate, or regional variance in clone size. Using a combination of microsatellite genotyping, flow cytometry, and cytology, we demonstrate that triploidy is highest in unglaciated, drought-prone regions of North America, where the largest clone sizes have been reported for this species. While we cannot completely rule out a low incidence of undetected aneuploidy, tetraploidy or duplicated loci, our evidence suggests that these phenomena are unlikely to be significant contributors to our observed patterns. We suggest that the distribution of triploid aspen is due to a positive synergy between triploidy and ecological factors driving clonality. Although triploids are expected to have low fertility, they are hypothesized to be an evolutionary link to sexual tetraploidy. Thus, interactions between clonality and polyploidy may be a broadly important component of geographic speciation patterns in perennial plants. Further, cytotypes are expected to show physiological and structural differences which may influence susceptibility to ecological factors such as drought, and we suggest that cytotype may be a significant and previously overlooked factor in recent patterns of high aspen mortality in the southwestern portion of the species range. Finally, triploidy should be carefully considered as a source of variance in genomic and ecological studies of aspen, particularly in western U.S. landscapes.