Full-Likelihood Genomic Analysis Clarifies a Complex History of Species Divergence and Introgression: The Example of the erato-sara Group of Heliconius Butterflies.

Introgressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species make estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ the multispecies coalescent (MSC) model with and without gene flow to infer the species phylogeny and cross-species introgression events using genomic data from six members of the erato-sara clade of Heliconius butterflies. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of Heliconius telesiphe, a likely hybrid speciation origin for Heliconius hecalesia, and gene flow between the sister species Heliconius erato and Heliconius himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the MSC makes it possible to extract rich historical information of species divergence and gene flow from genomic data. [3s; bpp; gene flow; Heliconius; hybrid speciation; introgression; inversion; multispecies coalescent].

Synteny-Based Genome Assembly for 16 Species of Heliconius Butterflies, and an Assessment of Structural Variation across the Genus.

Heliconius butterflies (Lepidoptera: Nymphalidae) are a group of 48 neotropical species widely studied in evolutionary research. Despite the wealth of genomic data generated in past years, chromosomal level genome assemblies currently exist for only two species, Heliconius melpomene and Heliconius erato, each a representative of one of the two major clades of the genus. Here, we use these reference genomes to improve the contiguity of previously published draft genome assemblies of 16 Heliconius species. Using a reference-assisted scaffolding approach, we place and order the scaffolds of these genomes onto chromosomes, resulting in 95.7-99.9% of their genomes anchored to chromosomes. Genome sizes are somewhat variable among species (270-422 Mb) and in one small group of species (Heliconius hecale, Heliconius elevatus, and Heliconius pardalinus) expansions in genome size are driven mainly by repetitive sequences that map to four small regions in the H. melpomene reference genome. Genes from these repeat regions show an increase in exon copy number, an absence of internal stop codons, evidence of constraint on nonsynonymous changes, and increased expression, all of which suggest that at least some of the extra copies are functional. Finally, we conducted a systematic search for inversions and identified five moderately large inversions fixed between the two major Heliconius clades. We infer that one of these inversions was transferred by introgression between the lineages leading to the erato/sara and burneyi/doris clades. These reference-guided assemblies represent a major improvement in Heliconius genomic resources that enable further genetic and evolutionary discoveries in this genus.

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.

Introgression drives repeated evolution of winter coat color polymorphism in hares.

Changing from summer-brown to winter-white pelage or plumage is a crucial adaptation to seasonal snow in more than 20 mammal and bird species. Many of these species maintain nonwhite winter morphs, locally adapted to less snowy conditions, which may have evolved independently. Mountain hares (Lepus timidus) from Fennoscandia were introduced into the Faroe Islands in 1855. While they were initially winter-white, within ∼65 y all Faroese hares became winter-gray, a morph that occurs in the source population at low frequency. The documented population history makes this a valuable model for understanding the genetic basis and evolution of the seasonal trait polymorphism. Through whole-genome scans of differentiation and single-nucleotide polymorphism (SNP) genotyping, we associated winter coat color polymorphism to the genomic region of the pigmentation gene Agouti, previously linked to introgression-driven winter coat color variation in the snowshoe hare (Lepus americanus). Lower Agouti expression in the skin of winter-gray individuals during the autumn molt suggests that regulatory changes may underlie the color polymorphism. Variation in the associated genomic region shows signatures of a selective sweep in the Faroese population, suggesting that positive selection drove the fixation of the variant after the introduction. Whole-genome analyses of several hare species revealed that the winter-gray variant originated through introgression from a noncolor changing species, in keeping with the history of ancient hybridization between the species. Our findings show the recurrent role of introgression in generating winter coat color variation by repeatedly recruiting the regulatory region of Agouti to modulate seasonal coat color change.

The genomic impact of historical hybridization with massive mitochondrial DNA introgression.

BACKGROUND: The extent to which selection determines interspecific patterns of genetic exchange enlightens the role of adaptation in evolution and speciation. Often reported extensive interspecific introgression could be selection-driven, but also result from demographic processes, especially in cases of invasive species replacements, which can promote introgression at their invasion front. Because invasion and selective sweeps similarly mold variation, population genetics evidence for selection can only be gathered in an explicit demographic framework. The Iberian hare, Lepus granatensis, displays in its northern range extensive mitochondrial DNA introgression from L. timidus, an arctic/boreal species that it replaced locally after the last glacial maximum. We use whole-genome sequencing to infer geographic and genomic patterns of nuclear introgression and fit a neutral model of species replacement with hybridization, allowing us to evaluate how selection influenced introgression genome-wide, including for mtDNA. RESULTS: Although the average nuclear and mtDNA introgression patterns contrast strongly, they fit a single demographic model of post-glacial invasive replacement of timidus by granatensis. Outliers of elevated introgression include several genes related to immunity, spermatogenesis, and mitochondrial metabolism. Introgression is reduced on the X chromosome and in low recombining regions. CONCLUSIONS: General nuclear and mtDNA patterns of introgression can be explained by purely demographic processes. Hybrid incompatibilities and interplay between selection and recombination locally modulate levels of nuclear introgression. Selection promoted introgression of some genes involved in conflicts, either interspecific (parasites) or possibly cytonuclear. In the latter case, nuclear introgression could mitigate the potential negative effects of alien mtDNA on mitochondrial metabolism and male-specific traits.

The hidden history of the snowshoe hare, Lepus americanus: extensive mitochondrial DNA introgression inferred from multilocus genetic variation.

Hybridization drives the evolutionary trajectory of many species or local populations, and assessing the geographic extent and genetic impact of interspecific gene flow may provide invaluable clues to understand population divergence or the adaptive relevance of admixture. In North America, hares (Lepus spp.) are key species for ecosystem dynamics and their evolutionary history may have been affected by hybridization. Here we reconstructed the speciation history of the three most widespread hares in North America - the snowshoe hare (Lepus americanus), the white-tailed jackrabbit (L. townsendii) and the black-tailed jackrabbit (L. californicus) - by analysing sequence variation at eight nuclear markers and one mitochondrial DNA (mtDNA) locus (6240 bp; 94 specimens). A multilocus-multispecies coalescent-based phylogeny suggests that L. americanus diverged ~2.7 Ma and that L. californicus and L. townsendii split more recently (~1.2 Ma). Within L. americanus, a deep history of cryptic divergence (~2.0 Ma) was inferred, which coincides with major speciation events in other North American species. While the isolation-with-migration model suggested that nuclear gene flow was generally rare or absent among species or major genetic groups, coalescent simulations of mtDNA divergence revealed historical mtDNA introgression from L. californicus into the Pacific Northwest populations of L. americanus. This finding marks a history of past reticulation between these species, which may have affected other parts of the genome and influence the adaptive potential of hares during climate change.