Disparate population and holobiont structure of pocilloporid corals across the Red Sea gradient demonstrate species-specific evolutionary trajectories.

Global habitat degradation heightens the need to better understand patterns of genetic connectivity and diversity of marine biota across geographical ranges to guide conservation efforts. Corals across the Red Sea are subject to pronounced environmental differences, but studies so far suggest that animal populations are largely connected, excepting evidence for a genetic break between the northern-central and southern regions. Here, we investigated population structure and holobiont assemblage of two common pocilloporid corals, Pocillopora verrucosa and Stylophora pistillata, across the Red Sea. We found little evidence for population differentiation in P. verrucosa, except for the southernmost site. Conversely, S. pistillata exhibited a complex population structure with evidence for within-reef and regional genetic differentiation, in line with differences in their reproductive mode (P. verrucosa is a broadcast spawner and S. pistillata is a brooder). Analysis for genomic loci under positive selection identified 85 sites (18 of which were in coding sequences) that distinguished the southern P. verrucosa population from the remainder of the Red Sea population. By comparison, we found 128 loci (24 of which were residing in coding sequences) in S. pistillata with evidence for local adaptation at various sites. Functional annotation of the underlying proteins revealed putative roles in the response to stress, lipid metabolism, transport, cytoskeletal rearrangement, and ciliary function (among others). Microbial assemblages of both coral species showed pervasive association with microalgal symbionts from the genus Symbiodinium (former clade A) and bacteria from the genus Endozoicomonas that exhibited significant differences according to host genotype and environment. The disparity of population genetic and holobiont assemblage patterns even between closely related species (family Pocilloporidae) highlights the need for multispecies investigations to better understand the role of the environment in shaping evolutionary trajectories. It further emphasizes the importance of networks of reef reserves to achieve conservation of genetic variants critical to the future survival of coral ecosystems.

Inference of recent admixture using genotype data.

The inference of biogeographic ancestry (BGA) has become a focus of forensic genetics. Misinference of BGA can have profound unwanted consequences for investigations and society. We show that recent admixture can lead to misclassification and erroneous inference of ancestry proportions, using state of the art analysis tools with (i) simulations, (ii) 1000 genomes project data, and (iii) two individuals analyzed using the ForenSeq DNA Signature Prep Kit. Subsequently, we extend existing tools for estimation of individual ancestry (IA) by allowing for different IA in both parents, leading to estimates of parental individual ancestry (PIA), and a statistical test for recent admixture. Estimation of PIA outperforms IA in most scenarios of recent admixture. Furthermore, additional information about parental ancestry can be acquired with PIA that may guide casework.

Horizontal gene transfer-mediated bacterial strain variation affects host fitness in Drosophila.

BACKGROUND: How microbes affect host fitness and environmental adaptation has become a fundamental research question in evolutionary biology. To better understand the role of microbial genomic variation for host fitness, we tested for associations of bacterial genomic variation and Drosophila melanogaster offspring number in a microbial Genome Wide Association Study (GWAS). RESULTS: We performed a microbial GWAS, leveraging strain variation in the genus Gluconobacter, a genus of bacteria that are commonly associated with Drosophila under natural conditions. We pinpoint the thiamine biosynthesis pathway (TBP) as contributing to differences in fitness conferred to the fly host. While an effect of thiamine on fly development has been described, we show that strain variation in TBP between bacterial isolates from wild-caught D. melanogaster contributes to variation in offspring production by the host. By tracing the evolutionary history of TBP genes in Gluconobacter, we find that TBP genes were most likely lost and reacquired by horizontal gene transfer (HGT). CONCLUSION: Our study emphasizes the importance of strain variation and highlights that HGT can add to microbiome flexibility and potentially to host adaptation.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Common structuring principles of the Drosophila melanogaster microbiome on a continental scale and between host and substrate.

The relative importance of host control, environmental effects and stochasticity in the assemblage of host-associated microbiomes is being debated. We analysed the microbiome among fly populations that were sampled across Europe by the European Drosophila Population Genomics Consortium (DrosEU). In order to better understand the structuring principles of the natural D. melanogaster microbiome, we combined environmental data on climate and food-substrate with dense genomic data on host populations and microbiome profiling. Food-substrate, temperature, and host population structure correlated with microbiome structure. Microbes, whose abundance was co-structured with host populations, also differed in abundance between flies and their substrate in an independent survey. This finding suggests common, host-related structuring principles of the microbiome on different spatial scales.

Off-target phenotypes in forensic DNA phenotyping and biogeographic ancestry inference: A resource.

With recent advances in DNA sequencing technologies it has become feasible and cost effective to genotype larger marker sets for forensic purposes. Two technologies that make use of the larger marker sets have come into focus in forensic research and applications; inference of biogeographic ancestry (BGA) and forensic DNA phenotyping (FDP). These methods hold the promise to reveal information about a yet unknown perpetrator from a DNA sample. In contrast, DNA-profiling, that is a standard practice in case work, relies on matching DNA-profiles between crime scene material and suspects on a database of DNA-profiles. Markers for DNA-profiling were developed under the premise to reveal as little additional information about the human source of the profile as possible, the rationale being that personal privacy rights have to be balanced against the public interest in solving a crime. The same argument holds for markers used in BGA and FDP; these markers might also reveal information on off-target phenotypes (OTPs), that go beyond BGA and the phenotypes targeted in FDP. In particular, health related OTPs might shift the balance between privacy protection and public interest. However, to our knowledge, there is currently no convenient resource available to incorporate knowledge on OTPs in BGA and FDP assay design and application. In order to provide such a resource, we performed a systematic search for OTPs associated with a comprehensive set of markers (1766 SNPs) used or suggested to be used for BGA inference and FDP. In this set, we identified a relatively small number of 27 SNPs (1.53%) that convey information on diverse health related OTPs such as cancer risk, induced asthma, or risk of alcoholism. Some of these SNPs are commonly used for FDP and BGA across different marker sets. We conclude that the effects of SNP markers used in FDP and BGA on OTPs are currently limited, with few exceptions that should be considered in a balanced decision on assay design and application.

Ecological specificity of the metagenome in a set of lower termite species supports contribution of the microbiome to adaptation of the host.

BACKGROUND: Elucidating the interplay between hosts and their microbiomes in ecological adaptation has become a central theme in evolutionary biology. A textbook example of microbiome-mediated adaptation is the adaptation of lower termites to a wood-based diet, as they depend on their gut microbiome to digest wood. Lower termites have further adapted to different life types. Termites of the wood-dwelling life type never leave their nests and feed on a uniform diet. Termites of the foraging life type forage for food outside the nest and have access to other nutrients. Here we sought to investigate whether the microbiome that is involved in food substrate breakdown and nutrient acquisition might contribute to adaptation to these dietary differences. We reasoned that this should leave ecological imprints on the microbiome. RESULTS: We investigated the protist and bacterial microbiomes of a total of 29 replicate colonies from five termite species, covering both life types, using metagenomic shotgun sequencing. The microbiome of wood-dwelling species with a uniform wood diet was enriched for genes involved in lignocellulose degradation. Furthermore, metagenomic patterns suggest that the microbiome of wood-dwelling species relied primarily on direct fixation of atmospheric nitrogen, while the microbiome of foraging species entailed the necessary pathways to utilize nitrogen in the form of nitrate for example from soil. CONCLUSION: Our findings are consistent with the notion that the microbiome of wood-dwelling species bears an imprint of its specialization on degrading a uniform wood diet, while the microbiome of the foraging species might reflect its adaption to access growth limiting nutrients from more diverse sources. This supports the idea that specific subsets of functions encoded by the microbiome can contribute to host adaptation.

Individual variation of natural D.melanogaster-associated bacterial communities.

Drosophila melanogaster has become an important model organism to study host-microbe interaction in the laboratory. However, the natural microbial communities that are associated with D. melanogaster have received less attention. Especially, information on inter-individual variation is still lacking, because most studies so far have used pooled material from several flies. Here, we collected bacterial 16S rRNA gene community profiles from a set of 32 individuals from a single population. We simulated pools from the individual data (i) to assess how well the microbiome of a host population is represented by pools, and (ii) to compare variation of Drosophila microbiomes within and between populations. Taxon richness was increased in simulated pools, suggesting that pools paint a more comprehensive picture of the taxa associated with a host population. Furthermore, microbiome composition varied less between pools than between individuals, indicating that differences even out in pools. Variation in microbiome composition was larger between populations than between simulated pools from a single population, adding to the notion that there are population-specific effects on the Drosophila microbiome. Surprisingly, samples from individuals clustered into two groups, suggesting that there are yet unknown factors that affect the composition of natural fly-associated microbial communities and need further research.

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes across a range of taxa. Here, we use immune defence in natural populations of Drosophila melanogaster to understand the rate of evolution in natural populations and the genetics underlying rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect along eastern North America (Massachusetts, Pennsylvania and Virginia). There are pronounced and repeatable changes in the immune response over the approximately 10 generations between spring and autumn collections, with a significant but less distinct difference observed among geographical locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles underlie the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defence in natural populations.

Differential Ecological Specificity of Protist and Bacterial Microbiomes across a Set of Termite Species.

The gut microbiome of lower termites comprises protists and bacteria that help these insects to digest cellulose and to thrive on wood. The composition of the termite gut microbiome correlates with phylogenetic distance of the animal host and host ecology (diet) in termites collected from their natural environment. However, carryover of transient microbes from host collection sites are an experimental concern and might contribute to the ecological imprints on the termite gut microbiome. Here, we set out to test whether an ecological imprint on the termite gut microbiome remains, when focusing on the persistent microbiome. Therefore, we kept five termite species under strictly controlled dietary conditions and subsequently profiled their protist and bacterial gut microbial communities using 18S and 16S rRNA gene amplicon sequencing. The species differed in their ecology; while three of the investigated species were wood-dwellers that feed on the piece of wood they live in and never leave except for the mating flight, the other two species were foragers that regularly leave their nests to forage for food. Despite these prominent ecological differences, protist microbiome structure aligned with phylogenetic relatedness of termite host species. Conversely, bacterial communities seemed more flexible, suggesting that microbiome structure aligned more strongly with the foraging and wood-dwelling ecologies. Interestingly, protist and bacterial community alpha-diversity correlated, suggesting either putative interactions between protists and bacteria, or that both types of microbes in the termite gut follow shared structuring principles. Taken together, our results add to the notion that bacterial communities are more variable over evolutionary time than protist communities and might react more flexibly to changes in host ecology.

Noninvasive analysis of microbiome dynamics in the fruit fly Drosophila melanogaster.

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.

Host species and environmental effects on bacterial communities associated with Drosophila in the laboratory and in the natural environment.

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory-reared Drosophila species. We find no evidence for host species effects in lab-reared flies; instead, lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila-associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild-caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore, we were able to identify candidate pathogens in natural fly isolates.

Genome patterns of selection and introgression of haplotypes in natural populations of the house mouse (Mus musculus).

General parameters of selection, such as the frequency and strength of positive selection in natural populations or the role of introgression, are still insufficiently understood. The house mouse (Mus musculus) is a particularly well-suited model system to approach such questions, since it has a defined history of splits into subspecies and populations and since extensive genome information is available. We have used high-density single-nucleotide polymorphism (SNP) typing arrays to assess genomic patterns of positive selection and introgression of alleles in two natural populations of each of the subspecies M. m. domesticus and M. m. musculus. Applying different statistical procedures, we find a large number of regions subject to apparent selective sweeps, indicating frequent positive selection on rare alleles or novel mutations. Genes in the regions include well-studied imprinted loci (e.g. Plagl1/Zac1), homologues of human genes involved in adaptations (e.g. alpha-amylase genes) or in genetic diseases (e.g. Huntingtin and Parkin). Haplotype matching between the two subspecies reveals a large number of haplotypes that show patterns of introgression from specific populations of the respective other subspecies, with at least 10% of the genome being affected by partial or full introgression. Using neutral simulations for comparison, we find that the size and the fraction of introgressed haplotypes are not compatible with a pure migration or incomplete lineage sorting model. Hence, it appears that introgressed haplotypes can rise in frequency due to positive selection and thus can contribute to the adaptive genomic landscape of natural populations. Our data support the notion that natural genomes are subject to complex adaptive processes, including the introgression of haplotypes from other differentiated populations or species at a larger scale than previously assumed for animals. This implies that some of the admixture found in inbred strains of mice may also have a natural origin.

Expression of the blood-group-related glycosyltransferase B4galnt2 influences the intestinal microbiota in mice.

Glycans on mucosal surfaces have an important role in host-microbe interactions. The locus encoding the blood-group-related glycosyltransferase ß-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) is subject to strong selective forces in natural house-mouse populations that contain a common allelic variant that confers loss of B4galnt2 gene expression in the gastrointestinal (GI) tract. We reasoned that altered glycan-dependent intestinal host-microbe interactions may underlie these signatures of selection. To determine whether B4galnt2 influences the intestinal microbial ecology, we profiled the microbiota of wild-type and B4galnt2-deficient siblings throughout the GI tract using 16S rRNA gene pyrosequencing. This revealed both distinct communities at different anatomic sites and significant changes in composition with respect to genotype, indicating a previously unappreciated role of B4galnt2 in host-microbial homeostasis. Among the numerous B4galnt2-dependent differences identified in the abundance of specific bacterial taxa, we unexpectedly detected a difference in the pathogenic genus, Helicobacter, suggesting Helicobacter spp. also interact with B4galnt2 glycans. In contrast to other glycosyltransferases, we found that the host intestinal B4galnt2 expression is not dependent on presence of the microbiota. Given the long-term maintenance of alleles influencing B4galnt2 expression by natural selection and the GI phenotypes presented here, we suggest that variation in B4galnt2 GI expression may alter susceptibility to GI diseases such as infectious gastroenteritis.

A test of the neutral model of expression change in natural populations of house mouse subspecies.

Changes in expression of genes are thought to contribute significantly to evolutionary divergence. To study the relative role of selection and neutrality in shaping expression changes, we analyzed 24 genes in three different tissues of the house mouse (Mus musculus). Samples from two natural populations of the subspecies M. m. domesticus and M. m. musculus were investigated using quantitative PCR assays and sequencing of the upstream region. We have developed an approach to quantify expression polymorphism within such populations and to disentangle technical from biological variation in the data. We found a correlation between expression polymorphism within populations and divergence between populations. Furthermore, we found a correlation between expression polymorphism and sequence polymorphism of the respective genes. These data are most easily interpreted within a framework of a predominantly neutral model of gene expression change, where only a fraction of the changes may have been driven by positive selection. Although most genes investigated were expressed in all three tissues analyzed, significant changes of expression levels occurred predominantly in a single tissue only. This adds to the notion that enhancer-specific effects or transregulatory effects can modulate the evolution of gene expression in a tissue-specific way.

Emergence of a new gene from an intergenic region.

It is generally assumed that new genes would arise by gene duplication mechanisms, because the signals for regulation and transcript processing would be unlikely to evolve in parallel with a new gene function. We have identified here a transcript in the house mouse (Mus musculus) that has arisen within the past 2.5-3.5 million years in a large intergenic region. The region is present in many mammals, including humans, allowing us to exclude the involvement of gene duplication, transposable elements, or other genome rearrangements, which are typically found for other cases of newly evolved genes. The gene has three exons, shows alternative splicing, and is specifically expressed in postmeiotic cells of the testis. The transcript is restricted to species within the genus Mus and its emergence correlates with indel mutations in the 5' regulatory region of the transcript. A recent selective sweep is associated with the transcript region in M. m. musculus populations. A knockout in the laboratory strain BL6 results in reduced sperm motility and reduced testis weight. Our results show that cryptic signals for transcript regulation and processing exist in intergenic regions and can become the basis for the evolution of a new functional gene.