Dan Vanderpool

Unlocking the vault: next-generation museum population genomics

Natural history museum collections provide unique resources for understanding how species respond to environmental change, including the abrupt, anthropogenic climate change of the past century. Ideally, researchers would conduct genome‐scale screening of museum specimens to explore the evolutionary consequences of environmental changes, but to date such analyses have been severely limited by the numerous challenges of working with the highly degraded DNA typical of historic samples. Here, we circumvent these challenges by using custom, multiplexed, exon capture to enrich and sequence ~11 000 exons (~4 Mb) from early 20th‐century museum skins. We used this approach to test for changes in genomic diversity accompanying a climate‐related range retraction in the alpine chipmunks (Tamias alpinus) in the high Sierra Nevada area of California, USA. We developed robust bioinformatic pipelines that rigorously detect and filter out base misincorporations in DNA derived from skins, most of which likely resulted from postmortem damage. Furthermore, to accommodate genotyping uncertainties associated with low‐medium coverage data, we applied a recently developed probabilistic method to call single‐nucleotide polymorphisms and estimate allele frequencies and the joint site frequency spectrum. Our results show increased genetic subdivision following range retraction, but no change in overall genetic diversity at either nonsynonymous or synonymous sites. This case study showcases the advantages of integrating emerging genomic and statistical tools in museum collection‐based population genomic applications. Such technical advances greatly enhance the value of museum collections, even where a pre‐existing reference is lacking and points to a broad range of potential applications in evolutionary and conservation biology.

Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales

BackgroundTo date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species.ResultsWe selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.ConclusionsOur study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

Basidiomycete yeasts in the cortex of ascomycete macrolichens

Lichens assemble in three parts Lichen growth forms cannot be recapitulated in the laboratory by culturing the plant and fungal partners together. Spribille et al. have discovered that the classical binary view of lichens is too simple. Instead, North American beard-like lichens are constituted of not two but three symbiotic partners: an ascomycetous fungus, a photosynthetic alga, and, unexpectedly, a basidiomycetous yeast. The yeast cells form the characteristic cortex of the lichen thallus and may be important for its shape. The yeasts are ubiquitous and essential partners for most lichens and not the result of lichens being colonized or parasitized by other organisms. Science, this issue p. 488 Complete functioning lichen thalli have three partners: alga and ascomycete, plus a basidiomycete yeast. For over 140 years, lichens have been regarded as a symbiosis between a single fungus, usually an ascomycete, and a photosynthesizing partner. Other fungi have long been known to occur as occasional parasites or endophytes, but the one lichen–one fungus paradigm has seldom been questioned. Here we show that many common lichens are composed of the known ascomycete, the photosynthesizing partner, and, unexpectedly, specific basidiomycete yeasts. These yeasts are embedded in the cortex, and their abundance correlates with previously unexplained variations in phenotype. Basidiomycete lineages maintain close associations with specific lichen species over large geographical distances and have been found on six continents. The structurally important lichen cortex, long treated as a zone of differentiated ascomycete cells, appears to consistently contain two unrelated fungi.

Negligible nuclear introgression despite complete mitochondrial capture between two species of chipmunks

The idea that species boundaries can be semipermeable to gene flow is now widely accepted but the evolutionary importance of introgressive hybridization remains unclear. Here we examine the genomic contribution of gene flow between two hybridizing chipmunk species, Tamias ruficaudus and T. amoenus. Previous studies have shown that ancient hybridization has resulted in complete fixation of introgressed T. ruficaudus mitochondrial DNA (mtDNA) in some populations of T. amoenus, but the extent of nuclear introgression is not known. We used targeted capture to sequence over 10,500 gene regions from multiple individuals of both species. We found that most of the nuclear genome is sorted between these species and that overall genealogical patterns do not show evidence for introgression. Our analysis rules out all but very minor levels of interspecific gene flow, indicating that introgressive hybridization has had little impact on the overall genetic composition of these species outside of the mitochondrial genome. Given that much of the evidence for introgression in animals has come from mtDNA, our results underscore that unraveling the importance introgressive hybridization during animal speciation will require a genome‐wide perspective that is still absent for many species.

The composite regulatory basis of the large X-effect in mouse speciation.

The disruption of meiotic sex chromosome inactivation (MSCI) has been proposed to be a major developmental mechanism underlying the rapid evolution of hybrid male sterility. We tested this idea by analyzing cell-specific gene expression across spermatogenesis in two lineages of house mice and their sterile and fertile reciprocal hybrids. We found pervasive disruption of sex chromosome gene expression in sterile hybrids at every stage of spermatogenesis. Failure of MSCI was developmentally preceded by increased silencing of autosomal genes, supporting the hypothesis that divergence at the hybrid incompatibility gene, Prdm9, results in increased rates of autosomal asynapsis which in turn triggers widespread silencing of unsynapsed chromatin. We also detected opposite patterns of postmeiotic overexpression or hyper-repression of the sex chromosomes in reciprocal hybrids, supporting the hypothesis that genomic conflict has driven functional divergence that leads to deleterious X-Y dosage imbalances in hybrids. Our developmental timeline also exposed more subtle patterns of mitotic misregulation on the X chromosome, a previously undocumented stage of spermatogenic disruption in this cross. These results indicate that multiple hybrid incompatibilities have converged on a common regulatory phenotype, the disrupted expression of the sex chromosomes during spermatogenesis. Collectively, these data reveal a composite regulatory basis to hybrid male sterility in mice that helps resolve the mechanistic underpinnings of the well-documented large X-effect in mice speciation. We propose that the inherent sensitivity of spermatogenesis to X-linked regulatory disruption has the potential to be a major driver of reproductive isolation in species with chromosomal sex determination.

Range expansion underlies historical introgressive hybridization in the Iberian hare

Introgressive hybridization is an important and widespread evolutionary process, but the relative roles of neutral demography and natural selection in promoting massive introgression are difficult to assess and an important matter of debate. Hares from the Iberian Peninsula provide an appropriate system to study this question. In its northern range, the Iberian hare, Lepus granatensis, shows a northwards gradient of increasing mitochondrial DNA (mtDNA) introgression from the arctic/boreal L. timidus, which it presumably replaced after the last glacial maximum. Here, we asked whether a south-north expansion wave of L. granatensis into L. timidus territory could underlie mtDNA introgression, and whether nuclear genes interacting with mitochondria (“mitonuc” genes) were affected. We extended previous RNA-sequencing and produced a comprehensive annotated transcriptome assembly for L. granatensis. We then genotyped 100 discovered nuclear SNPs in 317 specimens spanning the species range. The distribution of allele frequencies across populations suggests a northwards range expansion, particularly in the region of mtDNA introgression. We found no correlation between variants at 39 mitonuc genes and mtDNA introgression frequency. Whether the nuclear and mitochondrial genomes coevolved will need a thorough investigation of the hundreds of mitonuc genes, but range expansion and species replacement likely promoted massive mtDNA introgression.

Contrasting Levels of Molecular Evolution on the Mouse X Chromosome

The mammalian X chromosome has unusual evolutionary dynamics compared to autosomes. Faster-X evolution of spermatogenic protein-coding genes is known to be most pronounced for genes expressed late in spermatogenesis, but it is unclear if these patterns extend to other forms of molecular divergence. We tested for faster-X evolution in mice spanning three different forms of molecular evolution—divergence in protein sequence, gene expression, and DNA methylation—across different developmental stages of spermatogenesis. We used FACS to isolate individual cell populations and then generated cell-specific transcriptome profiles across different stages of spermatogenesis in two subspecies of house mice (Mus musculus), thereby overcoming a fundamental limitation of previous studies on whole tissues. We found faster-X protein evolution at all stages of spermatogenesis and faster-late protein evolution for both X-linked and autosomal genes. In contrast, there was less expression divergence late in spermatogenesis (slower late) on the X chromosome and for autosomal genes expressed primarily in testis (testis-biased). We argue that slower-late expression divergence reflects strong regulatory constraints imposed during this critical stage of sperm development and that these constraints are particularly acute on the tightly regulated sex chromosomes. We also found slower-X DNA methylation divergence based on genome-wide bisulfite sequencing of sperm from two species of mice (M. musculus and M. spretus), although it is unclear whether slower-X DNA methylation reflects development constraints in sperm or other X-linked phenomena. Our study clarifies key differences in patterns of regulatory and protein evolution across spermatogenesis that are likely to have important consequences for mammalian sex chromosome evolution, male fertility, and speciation.

Know your farmer: Ancient origins and multiple independent domestications of ambrosia beetle fungal cultivars

Bark and ambrosia beetles are highly specialized weevils (Curculionidae) that have established diverse symbioses with fungi, most often from the order Ophiostomatales (Ascomycota, Sordariomycetes). The two types of beetles are distinguished by their feeding habits and intimacy of interactions with their symbiotic fungi. The tree tissue diet of bark beetles is facilitated by fungi, while ambrosia beetles feed solely on fungi that they farm. The farming life history strategy requires domestication of a fungus, which the beetles consume as their sole food source. Ambrosia beetles in the subfamily Platypodinae originated in the mid‐Cretaceous (119–88 Ma) and are the oldest known group of farming insects. However, attempts to resolve phylogenetic relationships and the timing of domestication events for fungal cultivars have been largely inconclusive. We sequenced the genomes of 12 ambrosia beetle fungal cultivars and bark beetle associates, including the devastating laurel wilt pathogen, Raffaelea lauricola, to estimate a robust phylogeny of the Ophiostomatales. We find evidence for contemporaneous diversification of the beetles and their associated fungi, followed by three independent domestication events of the ambrosia fungi genus Raffaelea. We estimate the first domestication of an Ophiostomatales fungus occurred ~86 Ma, 25 million years earlier than prior estimates and in close agreement with the estimated age of farming in the Platypodinae (96 Ma). Comparisons of the timing of fungal domestication events with the timing of beetle radiations support the hypothesis that the first large beetle radiations may have spread domesticated “ambrosia” fungi to other fungi‐associated beetle groups, perhaps facilitating the evolution of new farming lineages.

The Evolution of Polymorphic Hybrid Incompatibilities in House Mice

Reproductive barriers are often assumed to arise from fixed genetic differences between species, despite frequent individual variation in the strength of reproductive isolation between populations. Larson et al. report polymorphism... Resolving the mechanistic and genetic bases of reproductive barriers between species is essential to understanding the evolutionary forces that shape speciation. Intrinsic hybrid incompatibilities are often treated as fixed between species, yet there can be considerable variation in the strength of reproductive isolation between populations. The extent and causes of this variation remain poorly understood in most systems. We investigated the genetic basis of variable hybrid male sterility (HMS) between two recently diverged subspecies of house mice, Mus musculus domesticus and Mus musculus musculus. We found that polymorphic HMS has a surprisingly complex genetic basis, with contributions from at least five autosomal loci segregating between two closely related wild-derived strains of M. m. musculus. One of the HMS-linked regions on chromosome 4 also showed extensive introgression among inbred laboratory strains and transmission ratio distortion (TRD) in hybrid crosses. Using additional crosses and whole genome sequencing of sperm pools, we showed that TRD was limited to hybrid crosses and was not due to differences in sperm motility between M. m. musculus strains. Based on these results, we argue that TRD likely reflects additional incompatibilities that reduce hybrid embryonic viability. In some common inbred strains of mice, selection against deleterious interactions appears to have unexpectedly driven introgression at loci involved in epistatic hybrid incompatibilities. The highly variable genetic basis to F1 hybrid incompatibilities between closely related mouse lineages argues that a thorough dissection of reproductive isolation will require much more extensive sampling of natural variation than has been commonly utilized in mice and other model systems.

The genome and microbiome of a dikaryotic fungus (Inocybe terrigena, Inocybaceae) revealed by metagenomics: The genome and microbiome of Inocybe terrigena

Recent advances in molecular methods have increased our understanding of various fungal symbioses. However, little is known about genomic and microbiome features of most uncultured symbiotic fungal clades. Here, we analysed the genome and microbiome of Inocybaceae (Agaricales, Basidiomycota), a largely uncultured ectomycorrhizal clade known to form symbiotic associations with a wide variety of plant species. We used metagenomic sequencing and assembly of dikaryotic fruiting-body tissues from Inocybe terrigena (Fr.) Kuyper, to classify fungal and bacterial genomic sequences, and obtained a nearly complete fungal genome containing 93% of core eukaryotic genes. Comparative genomics reveals that I. terrigena is more similar to ectomycorrhizal and brown rot fungi than to white rot fungi. The reduction in lignin degradation capacity has been independent from and significantly faster than in closely related ectomycorrhizal clades supporting that ectomycorrhizal symbiosis evolved independently in Inocybe. The microbiome of I. terrigena fruiting-bodies includes bacteria with known symbiotic functions in other fungal and non-fungal host environments, suggesting potential symbiotic functions of these bacteria in fungal tissues regardless of habitat conditions. Our study demonstrates the usefulness of direct metagenomics analysis of fruiting-body tissues for characterizing fungal genomes and microbiome.