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 within and among species.
Methods
We generated new genome-wide resequencing data targeting 207,691 exonic and non-coding regions [totaling 61.7 Megabases (Mb) total] from 14 hare species (30 individuals)and the outgroup pygmy rabbit (Brachylagus idahoensis; 2 individuals). We combined these data with published whole exomes from four snowshoes hares (Lepus americanus; NCBI Sequence Read Archive BioProject PRJNA420081) and extracted data from the reference genome of European rabbit (Oryctolagus cuniculus; OryCun2.0) to use as a second outgroup. Our total sample of 15 hare species (34 individuals, 1 to 4 individuals per species) and 2 outgroup species (3 individuals) included species from all major regions of the Lepus native distribution. A smaller subset of 32 hare individuals (15 hare species), 1 pygmy rabbit individual and the European rabbit reference were used for subsequent analyses (check Supplementary Table S1 and Materials and Methods).

We reconstructed the evolutionary history of Lepus using maximum likelihood with a concatenated alignment and multispecies coalescent approaches, with ASTRAL and SVDquartets. We also studied phylogenetic incongruence among exome regions underlying the species tree. We infered hybridization among extant and ancestral branches of the species tree using network based inferences (PhyloNet and TreeMix) and summary statistics (minimum D-statistic, 'f-branch' statistic, admixture proportion and fraction of admixture).