The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which species share a conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which mimicry is produced in poison frogs. We assembled a 6.02-Gbp genome with a contig N50 of 310 Kbp, a scaffold N50 of 390 Kbp and 85% of expected tetrapod genes. We leveraged this genome to conduct gene expression analyses throughout development of four colour morphs of Ranitomeya imitator and two colour morphs from both R. fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes differentially expressed throughout development, many of them related to melanophores/melanin, iridophore development and guanine synthesis. We also identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species, and identify several plausible candidates for colouration in vertebrates (e.g. cd36, ep-cadherin and perlwapin). Finally, we hypothesise that keratin genes (e.g. krt8) are important for producing different structural colours within these frogs.

A holobiont view of island biogeography: Unravelling patterns driving the nascent diversification of a Hawaiian spider and its microbial associates.

The diversification of a host lineage can be influenced by both the external environment and its assemblage of microbes. Here, we use a young lineage of spiders, distributed along a chronologically arranged series of volcanic mountains, to investigate how their associated microbial communities have changed as the spiders colonized new locations. Using the stick spider Ariamnes waikula (Araneae, Theridiidae) on the island of Hawai'i, and outgroup taxa on older islands, we tested whether each component of the "holobiont" (spider hosts, intracellular endosymbionts and gut microbial communities) showed correlated signatures of diversity due to sequential colonization from older to younger volcanoes. To investigate this, we generated ddRAD data for the host spiders and 16S rRNA gene amplicon data from their microbiota. We expected sequential colonizations to result in a (phylo)genetic structuring of the host spiders and in a diversity gradient in microbial communities. The results showed that the host A. waikula is indeed structured by geographical isolation, suggesting sequential colonization from older to younger volcanoes. Similarly, the endosymbiont communities were markedly different between Ariamnes species on different islands, but more homogeneous among A. waikula populations on the island of Hawai'i. Conversely, the gut microbiota, which we suspect is generally environmentally derived, was largely conserved across all populations and species. Our results show that different components of the holobiont respond in distinct ways to the dynamic environment of the volcanic archipelago. This highlights the necessity of understanding the interplay between different components of the holobiont, to properly characterize its evolution.

Temporal genomic contrasts reveal rapid evolutionary responses in an alpine mammal during recent climate change.

Many species have experienced dramatic changes in their abundance and distribution during recent climate change, but it is often unclear whether such ecological responses are accompanied by evolutionary change. We used targeted exon sequencing of 294 museum specimens (160 historic, 134 modern) to generate independent temporal genomic contrasts spanning a century of climate change (1911-2012) for two co-distributed chipmunk species: an endemic alpine specialist (Tamias alpinus) undergoing severe range contraction and a stable mid-elevation species (T. speciosus). Using a novel analytical approach, we reconstructed the demographic histories of these populations and tested for evidence of recent positive directional selection. Only the retracting species showed substantial population genetic fragmentation through time and this was coupled with positive selection and substantial shifts in allele frequencies at a gene, Alox15, involved in regulation of inflammation and response to hypoxia. However, these rapid population and gene-level responses were not detected in an analogous temporal contrast from another area where T. alpinus has also undergone severe range contraction. Collectively, these results highlight that evolutionary responses may be variable and context dependent across populations, even when they show seemingly synchronous ecological shifts. Our results demonstrate that temporal genomic contrasts can be used to detect very recent evolutionary responses within and among contemporary populations, even in the face of complex demographic changes. Given the wealth of specimens archived in natural history museums, comparative analyses of temporal population genomic data have the potential to improve our understanding of recent and ongoing evolutionary responses to rapidly changing environments.

Evolutionary Genomics at the Human-Environment Interface in Africa.

We report on the first meeting of SMBE in Africa. SMBE Malawi was initiated to bring together African and international researchers who use genetics or genomics to study natural systems impacted by human activities. The goals of this conference were 1) to reach a world-class standard of science with a large number of contributions from Africa, 2) to initiate exchange between African and international researchers, and 3) to identify challenges and opportunities for evolutionary genomics research in Africa. As repored, we think that we have achieved these goals and make suggestions on the way forward for African evolutionary genomics research.

The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which multiple species share the same conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which vivid colour and pattern are produced in a Müllerian mimicry complex of poison frogs. We did this by first assembling a high-quality de novo genome assembly for the mimic poison frog Ranitomeya imitator. This assembled genome is 6.8 Gbp in size, with a contig N50 of 300 Kbp R. imitator and two colour morphs from both Ranitomeya fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes that are differentially expressed throughout development, many of them related to melanocyte development, melanin synthesis, iridophore development and guanine synthesis. Polytypic differences within species may be the result of differences in expression and/or timing of expression, whereas convergence for colour pattern between species does not appear to be due to the same changes in gene expression. In addition, we identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species. Finally, we hypothesize that genes in the keratin family are important for producing different structural colours within these frogs.

Divergence, gene flow, and the origin of leapfrog geographic distributions: The history of colour pattern variation in Phyllobates poison-dart frogs.

The geographic distribution of phenotypic variation among closely related populations is a valuable source of information about the evolutionary processes that generate and maintain biodiversity. Leapfrog distributions, in which phenotypically similar populations are disjunctly distributed and separated by one or more phenotypically distinct populations, represent geographic replicates for the existence of a phenotype, and are therefore especially informative. These geographic patterns have mostly been studied from phylogenetic perspectives to understand how common ancestry and divergent evolution drive their formation. Other processes, such as gene flow between populations, have not received as much attention. Here, we investigate the roles of divergence and gene flow between populations in the origin and maintenance of a leapfrog distribution in Phyllobates poison frogs. We found evidence for high levels of gene flow between neighbouring populations but not over long distances, indicating that gene flow between populations exhibiting the central phenotype may have a homogenizing effect that maintains their similarity, and that introgression between 'leapfroging' taxa has not played a prominent role as a driver of phenotypic diversity in Phyllobates. Although phylogenetic analyses suggest that the leapfrog distribution was formed through independent evolution of the peripheral (i.e. leapfrogging) populations, the elevated levels of gene flow between geographically close populations poise alternative scenarios, such as the history of phenotypic change becoming decoupled from genome-averaged patterns of divergence, which we cannot rule out. These results highlight the importance of incorporating gene flow between populations into the study of geographic variation in phenotypes, both as a driver of phenotypic diversity and as a confounding factor of phylogeographic inferences.

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog.

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin.

ngsTools: methods for population genetics analyses from next-generation sequencing data.

SUMMARY: Next-generation sequencing technologies produce short reads that are either de novo assembled or mapped to a reference genome. Genotypes and/or single-nucleotide polymorphisms are then determined from the read composition at each site, which become the basis for many downstream analyses. However, for low sequencing depths, e.g. , there is considerable statistical uncertainty in the assignment of genotypes because of random sampling of homologous base pairs in heterozygotes and sequencing or alignment errors. Recently, several probabilistic methods have been proposed to account for this uncertainty and make accurate inferences from low quality and/or coverage sequencing data. We present ngsTools, a collection of programs to perform population genetics analyses from next-generation sequencing data. The methods implemented in these programs do not rely on single-nucleotide polymorphism or genotype calling and are particularly suitable for low sequencing depth data. AVAILABILITY: Programs included in ngsTools are implemented in C/C++ and are freely available for noncommercial use at https://github.com/mfumagalli/ngsTools. CONTACT: mfumagalli82@gmail.com SUPPLEMENTARY INFORMATION: Supplementary materials are available at Bioinformatics online.

Nuclear environmental DNA resolves fine-scale population genetic structure in an aquatic habitat.

There is considerable potential for nuclear genomic material in environmental DNA (eDNA) to inform us of population genetic structure within aquatic species. We tested if nuclear allelic composition data sourced from eDNA can resolve fine scale spatial genetic structure of the cichlid fish Astatotilapia calliptera in Lake Masoko, Tanzania. In this ∼35 m deep crater lake the species is diverging into two genetically distinguishable ecomorphs, separated by a thermo-oxycline at ∼15 m that divides biologically distinct water masses. We quantified population genetic structure along a depth transect using single nucleotide polymorphisms (SNPs) derived from genome sequencing of 530 individuals. This population genetic structure was reflected in a focal set of SNPs that were also reliably amplified from eDNA - with allele frequencies derived from eDNA reflecting those of fish within each depth zone. Thus, by targeting known genetic variation between populations within aquatic eDNA, we measured genetic structure within the focal species.

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.

BACKGROUND: To 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Multiple Selection Signatures in Farmed Atlantic Salmon Adapted to Different Environments Across Hemispheres.

Domestication of Atlantic salmon started approximately 40 years ago, using artificial selection through genetic improvement programs. Selection is likely to have imposed distinctive signatures on the salmon genome, which are often characterized by high genetic differentiation across population and/or reduction in genetic diversity in regions associated to traits under selection. The identification of such selection signatures may give insights into the candidate genomic regions of biological and commercial interest. Here, we used three complementary statistics to detect selection signatures, two haplotype-based (iHS and XP-EHH), and one FST-based method (BayeScan) among four populations of Atlantic salmon with a common genetic origin. Several regions were identified for these techniques that harbored genes, such as kind1 and chp2, which have been associated with growth-related traits or the kcnb2 gene related to immune system in Atlantic salmon, making them particularly relevant in the context of aquaculture. Our results provide candidate genes to inform the evolutionary and biological mechanisms controlling complex selected traits in Atlantic salmon.

Genome-wide signatures of complex introgression and adaptive evolution in the big cats.

The great cats of the genus Panthera comprise a recent radiation whose evolutionary history is poorly understood. Their rapid diversification poses challenges to resolving their phylogeny while offering opportunities to investigate the historical dynamics of adaptive divergence. We report the sequence, de novo assembly, and annotation of the jaguar (Panthera onca) genome, a novel genome sequence for the leopard (Panthera pardus), and comparative analyses encompassing all living Panthera species. Demographic reconstructions indicated that all of these species have experienced variable episodes of population decline during the Pleistocene, ultimately leading to small effective sizes in present-day genomes. We observed pervasive genealogical discordance across Panthera genomes, caused by both incomplete lineage sorting and complex patterns of historical interspecific hybridization. We identified multiple signatures of species-specific positive selection, affecting genes involved in craniofacial and limb development, protein metabolism, hypoxia, reproduction, pigmentation, and sensory perception. There was remarkable concordance in pathways enriched in genomic segments implicated in interspecies introgression and in positive selection, suggesting that these processes were connected. We tested this hypothesis by developing exome capture probes targeting ~19,000 Panthera genes and applying them to 30 wild-caught jaguars. We found at least two genes (DOCK3 and COL4A5, both related to optic nerve development) bearing significant signatures of interspecies introgression and within-species positive selection. These findings indicate that post-speciation admixture has contributed genetic material that facilitated the adaptive evolution of big cat lineages.

Quantifying population genetic differentiation from next-generation sequencing data.

Over the past few years, new high-throughput DNA sequencing technologies have dramatically increased speed and reduced sequencing costs. However, the use of these sequencing technologies is often challenged by errors and biases associated with the bioinformatical methods used for analyzing the data. In particular, the use of naïve methods to identify polymorphic sites and infer genotypes can inflate downstream analyses. Recently, explicit modeling of genotype probability distributions has been proposed as a method for taking genotype call uncertainty into account. Based on this idea, we propose a novel method for quantifying population genetic differentiation from next-generation sequencing data. In addition, we present a strategy for investigating population structure via principal components analysis. Through extensive simulations, we compare the new method herein proposed to approaches based on genotype calling and demonstrate a marked improvement in estimation accuracy for a wide range of conditions. We apply the method to a large-scale genomic data set of domesticated and wild silkworms sequenced at low coverage. We find that we can infer the fine-scale genetic structure of the sampled individuals, suggesting that employing this new method is useful for investigating the genetic relationships of populations sampled at low coverage.