Effect of Myxoma Virus Species Jump on Iberian Hare Populations.

The myxoma virus species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis) has raised concerns. We assess the decline suffered by Iberian hare populations on the Iberian Peninsula and discuss the association between the effect of myxomatosis and the average abundance index, which we estimated by using hunting bags.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

The evolutionary pathways for local adaptation in mountain hares.

Understanding the evolution of local adaptations is a central aim of evolutionary biology and key for the identification of unique populations and lineages of conservation relevance. By combining RAD sequencing and whole-genome sequencing, we identify genetic signatures of local adaptation in mountain hares (Lepus timidus) from isolated and distinctive habitats of its wide distribution: Ireland, the Alps and Fennoscandia. Demographic modelling suggested that the split of these mountain hares occurred around 20 thousand years ago, providing the opportunity to study adaptive evolution over a short timescale. Using genome-wide scans, we identified signatures of extreme differentiation among hares from distinct geographic areas that overlap with area-specific selective sweeps, suggesting targets for local adaptation. Several identified candidate genes are associated with traits related to the uniqueness of the different environments inhabited by the three groups of mountain hares, including coat colour, ability to live at high altitudes and variation in body size. In Irish mountain hares, a variant of ASIP, a gene previously implicated in introgression-driven winter coat colour variation in mountain and snowshoe hares (L. americanus), may underlie brown winter coats, reinforcing the repeated nature of evolution at ASIP moulding adaptive seasonal colouration. Comparative genomic analyses across several hare species suggested that mountain hares' adaptive variants appear predominantly species-specific. However, using coalescent simulations, we also show instances where the candidate adaptive variants have been introduced via introgressive hybridization. Our study shows that standing adaptive variation, including that introgressed from other species, was a crucial component of the post-glacial dynamics of species.

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. [Adaptation; ancient introgression; hybridization; Lepus; phylogenomics.].

Genetics of randomly bred cats support the cradle of cat domestication being in the Near East.

Cat domestication likely initiated as a symbiotic relationship between wildcats (Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density (i.e., rodents). Humans benefited from the cats' predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor's (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species.

The evolutionary history of the Cape hare (Lepus capensis sensu lato): insights for systematics and biogeography.

Inferring the phylogeography of species with large distributions helps deciphering major diversification patterns that may occur in parallel across taxa. Here, we infer the evolutionary history of the Cape hare, Lepus capensis sensu lato, a species distributed from southern Africa to Asia, by analyzing variation at 18 microsatellites and 9 DNA (1 mitochondrial and 8 nuclear) sequenced loci, from field and museum-collected samples. Using a combination of assignment and coalescent-based methods, we show that the Cape hare is composed of five evolutionary lineages, distributed in distinct biogeographic regions-north-western Africa, eastern Africa, southern Africa, the Near East and the Arabian Peninsula. A deep phylogenetic break possibly dating to the Early Pleistocene was inferred between the African and Asian L. capensis groups, and the latter appear more closely related to other Eurasian hare species than to African Cape hares. The inferred phylogeographic structure is shared by numerous taxa distributed across the studied range, suggesting that environmental changes, such as the progressive aridification of the Saharo-Arabian desert and the fluctuations of savannah habitats in Sub-Saharan Africa, had comparable impacts across species. Fine-scale analyses of the western Sahara-Sahel populations showed rich fragmentation patterns for mitochondrial DNA but not for microsatellites, compatible with the environmental heterogeneity of the region and female philopatry. The complex evolutionary history of L. capensis sensu lato, which possibly includes interspecific gene flow, is not reflected by taxonomy. Integrating evolutionary inference contributes to an improved characterization of biodiversity, which is fundamental to foster the conservation of relevant evolutionary units.

Integrative approaches to guide conservation decisions: Using genomics to define conservation units and functional corridors.

Climate change and increasing habitat loss greatly impact species survival, requiring range shifts, phenotypic plasticity and/or evolutionary change for long-term persistence, which may not readily occur unaided in threatened species. Therefore, defining conservation actions requires a detailed assessment of evolutionary factors. Existing genetic diversity needs to be thoroughly evaluated and spatially mapped to define conservation units (CUs) in an evolutionary context, and we address that here. We also propose a multidisciplinary approach to determine corridors and functional connectivity between CUs by including genetic diversity in the modelling while controlling for isolation by distance and phylogeographic history. We evaluate our approach on a Near Threatened Iberian endemic rodent by analysing genotyping-by-sequencing (GBS) genomic data from 107 Cabrera voles (Microtus cabrerae), screening the entire species distribution to define categories of CUs and their connectivity: We defined six management units (MUs) which can be grouped into four evolutionarily significant units (ESUs) and three (putatively) adaptive units (AUs). We demonstrate that the three different categories of CU can be objectively defined using genomic data, and their characteristics and connectivity can inform conservation decision-making. In particular, we show that connectivity of the Cabrera vole is very limited in eastern Iberia and that the pre-Pyrenean and part of the Betic geographic nuclei contribute the most to the species genetic diversity. We argue that a multidisciplinary framework for CU definition is essential and that this framework needs a strong evolutionary basis.

A genomic map of clinal variation across the European rabbit hybrid zone.

Speciation is a process proceeding from weak to complete reproductive isolation. In this continuum, naturally hybridizing taxa provide a promising avenue for revealing the genetic changes associated with the incipient stages of speciation. To identify such changes between two subspecies of rabbits that display partial reproductive isolation, we studied patterns of allele frequency change across their hybrid zone using whole-genome sequencing. To connect levels and patterns of genetic differentiation with phenotypic manifestations of subfertility in hybrid rabbits, we further investigated patterns of gene expression in testis. Geographic cline analysis revealed 253 regions characterized by steep changes in allele frequency across their natural region of contact. This catalog of regions is likely to be enriched for loci implicated in reproductive barriers and yielded several insights into the evolution of hybrid dysfunction in rabbits: (i) incomplete reproductive isolation is likely governed by the effects of many loci, (ii) protein-protein interaction analysis suggest that genes within these loci interact more than expected by chance, (iii) regulatory variation is likely the primary driver of incompatibilities, and (iv) large chromosomal rearrangements appear not to be a major mechanism underlying incompatibilities or promoting isolation in the face of gene flow. We detected extensive misregulation of gene expression in testis of hybrid males, but not a statistical overrepresentation of differentially expressed genes in candidate regions. Our results also did not support an X chromosome-wide disruption of expression as observed in mice and cats, suggesting variation in the mechanistic basis of hybrid male reduced fertility among mammals.

The Microtus voles: Resolving the phylogeny of one of the most speciose mammalian genera using genomics.

Sequential rapid radiations pose some of the greatest difficulties in phylogenetics, especially when analysing only a small number of genetic markers. Given that most of the speciation events occur in quick succession at various points in time, this creates particular challenges in determining phylogenetic relationships, i.e. branching order and divergence times. With the development of high throughput sequencing, thousands of markers can now readily be used to tackle these issues. Microtus is a speciose genus currently composed of 65 species that evolved over the last 2 million years. Although it is a well-studied group, there is still phylogenetic uncertainty at various divergence levels. Building upon previous studies that generally used small numbers of mitochondrial and/or nuclear loci, in this genomic-scale study we used both mitochondrial and nuclear data to study the rapid radiation within Microtus, using partial mitogenomes and genotyping-by-sequencing (GBS) on seven species representing five Microtus subgenera and the main biogeographic ranges where this group occurs. Both types of genome (mitochondrial and nuclear) generated similar tree topologies, with a basal split of the Nearctic (M. ochrogaster) and Holarctic (M. oeconomus) species, and then a subdivision of the five Palearctic species into two subgroups. These data support the occurrence of two European radiations, one North American radiation, and a later expansion of M. oeconomus from Asia to both Europe and North America. We further resolved the positioning of M. cabrerae as sister group of M. agrestis and refute the claim that M. cabrerae should be elevated to its own genus (Iberomys). Finally, the data support ongoing speciation events, especially within M. agrestis, with high levels of genetic divergence between the three Evolutionarily Significant Units (ESUs) previously identified. Similar high levels of divergence were also found among ESUs within M. oeconomus and M. arvalis.

The transcriptional landscape of seasonal coat colour moult in the snowshoe hare.

Seasonal coat colour change is an important adaptation to seasonally changing environments but the evolution of this and other circannual traits remains poorly understood. In this study, we use gene expression to understand seasonal coat colour moulting in wild snowshoe hares (Lepus americanus). We used hair colour to follow the progression of the moult, simultaneously sampling skin from three moulting stages in hares collected during the peak of the spring moult from white winter to brown summer pelage. Using RNA sequencing, we tested whether patterns of expression were consistent with predictions based on the established phases of the hair growth cycle. We found functionally consistent clustering across skin types, with 766 genes differentially expressed between moult stages. "White" pelage showed more differentially expressed genes that were upregulated relative to other skin types, involved in the transition between late telogen (quiescent stage) and the onset of anagen (proliferative stage). Skin samples from transitional "intermediate" and "brown" pelage were transcriptionally similar and resembled the regressive transition to catagen (regressive stage). We also detected differential expression of several key circadian clock and pigmentation genes, providing important means to dissect the bases of alternate seasonal colour morphs. Our results reveal that pelage colour is a useful biomarker for seasonal change but that there is a consistent lag between the main gene expression waves and change in visible coat colour. These experiments establish that developmental sampling from natural populations of nonmodel organisms can provide a crucial resource to dissect the genetic basis and evolution of complex seasonally changing traits.

An update on the rabbit hemorrhagic disease virus (RHDV) strains circulating in Portugal in the 1990s: earliest detection of G3-G5 and G6.

Rabbit hemorrhagic disease virus (RHDV) causes devastating effects on European rabbit (Oryctolagus cuniculus) populations in the Iberian Peninsula. According to the information available, only genogroup 1 strains were circulating in Iberian wild rabbits until 2011; the antigenic variant G6 has been sporadically detected in rabbitries since 2007. Here, we show for the first time that G3-G5 strains were already present in mainland Portugal in 1998 and that G6 has been circulating since at least 1999. Moreover, we report a G3-G5 strain from the Azores collected in 1998, which is the likely ancestor of Azorean G3-G5like strains. These observations improve the current knowledge on RHDV epidemiology in the Iberian Peninsula and the Azores.

LaGomiCs-Lagomorph Genomics Consortium: An International Collaborative Effort for Sequencing the Genomes of an Entire Mammalian Order.

The order Lagomorpha comprises about 90 living species, divided in 2 families: the pikas (Family Ochotonidae), and the rabbits, hares, and jackrabbits (Family Leporidae). Lagomorphs are important economically and scientifically as major human food resources, valued game species, pests of agricultural significance, model laboratory animals, and key elements in food webs. A quarter of the lagomorph species are listed as threatened. They are native to all continents except Antarctica, and occur up to 5000 m above sea level, from the equator to the Arctic, spanning a wide range of environmental conditions. The order has notable taxonomic problems presenting significant difficulties for defining a species due to broad phenotypic variation, overlap of morphological characteristics, and relatively recent speciation events. At present, only the genomes of 2 species, the European rabbit (Oryctolagus cuniculus) and American pika (Ochotona princeps) have been sequenced and assembled. Starting from a paucity of genome information, the main scientific aim of the Lagomorph Genomics Consortium (LaGomiCs), born from a cooperative initiative of the European COST Action "A Collaborative European Network on Rabbit Genome Biology-RGB-Net" and the World Lagomorph Society (WLS), is to provide an international framework for the sequencing of the genome of all extant and selected extinct lagomorphs. Sequencing the genomes of an entire order will provide a large amount of information to address biological problems not only related to lagomorphs but also to all mammals. We present current and planned sequencing programs and outline the final objective of LaGomiCs possible through broad international collaboration.

Reference-free population genomics from next-generation transcriptome data and the vertebrate-invertebrate gap.

In animals, the population genomic literature is dominated by two taxa, namely mammals and drosophilids, in which fully sequenced, well-annotated genomes have been available for years. Data from other metazoan phyla are scarce, probably because the vast majority of living species still lack a closely related reference genome. Here we achieve de novo, reference-free population genomic analysis from wild samples in five non-model animal species, based on next-generation sequencing transcriptome data. We introduce a pipe-line for cDNA assembly, read mapping, SNP/genotype calling, and data cleaning, with specific focus on the issue of hidden paralogy detection. In two species for which a reference genome is available, similar results were obtained whether the reference was used or not, demonstrating the robustness of our de novo inferences. The population genomic profile of a hare, a turtle, an oyster, a tunicate, and a termite were found to be intermediate between those of human and Drosophila, indicating that the discordant genomic diversity patterns that have been reported between these two species do not reflect a generalized vertebrate versus invertebrate gap. The genomic average diversity was generally higher in invertebrates than in vertebrates (with the notable exception of termite), in agreement with the notion that population size tends to be larger in the former than in the latter. The non-synonymous to synonymous ratio, however, did not differ significantly between vertebrates and invertebrates, even though it was negatively correlated with genetic diversity within each of the two groups. This study opens promising perspective regarding genome-wide population analyses of non-model organisms and the influence of population size on non-synonymous versus synonymous diversity.

Candidate genes underlying heritable differences in reproductive seasonality between wild and domestic rabbits.

Reproductive seasonality is a trait that often differs between domestic animals and their wild ancestors, with domestic animals showing prolonged or even continuous breeding seasons. However, the genetic basis underlying this trait is still poorly understood for most species, and because environmental factors and resource availability are known to play an important role in determining breeding seasons, it is also not clear in most cases to what extent this phenotypic shift is determined by the more lenient captive conditions or by genetic factors. Here, using animals resulting from an initial cross between wild and domestic rabbits followed by two consecutive backcrosses (BC1 and BC2) to wild rabbits, we evaluated the yearly distribution of births for the different generations. Similar to domestic rabbits, F1 animals could be bred all year round but BC1 and BC2 animals showed a progressive and significant reduction in the span of the breeding season, providing experimental evidence that reduced seasonal breeding in domestic rabbits has a clear genetic component and is not a simple by-product of rearing conditions. We then took advantage of a recently published genome-wide scan of selection in the domesticated lineage and searched for candidate genes potentially associated with this phenotypic shift. Candidate genes located within regions targeted by selection include well-known examples of genes controlling clock functions (CRY1 and NR3C1) and reproduction (PRLR).

Sequencing of Sylvilagus VDJ genes reveals a new VHa allelic lineage and shows that ancient VH lineages were retained differently in leporids.

Antigen recognition by immunoglobulins depends upon initial rearrangements of heavy chain V, D, and J genes. In leporids, a unique system exists for the VH genes usage that exhibit highly divergent lineages: the VHa allotypes, the Lepus sL lineage and the VHn genes. For the European rabbit (Oryctolagus cuniculus), four VHa lineages have been described, the a1, a2, a3 and a4. For hares (Lepus sp.), one VHa lineage was described, the a2L, as well as a more ancient sL lineage. Both genera use the VHn genes in a low frequency of their VDJ rearrangements. To address the hypothesis that the VH specificities could be associated with different environments, we sequenced VDJ genes from a third leporid genus, Sylvilagus. We found a fifth and equally divergent VHa lineage, the a5, and an ancient lineage, the sS, related to the hares' sL, but failed to obtain VHn genes. These results show that the studied leporids employ different VH lineages in the generation of the antibody repertoire, suggesting that the leporid VH genes are subject to strong selective pressure likely imposed by specific pathogens.

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.

Conservation implications of the evolutionary history and genetic diversity hotspots of the snowshoe hare.

With climate warming, the ranges of many boreal species are expected to shift northward and to fragment in southern peripheral ranges. To understand the conservation implications of losing southern populations, we examined range-wide genetic diversity of the snowshoe hare (Lepus americanus), an important prey species that drives boreal ecosystem dynamics. We analysed microsatellite (8 loci) and mitochondrial DNA sequence (cytochrome b and control region) variation in almost 1000 snowshoe hares. A hierarchical structure analysis of the microsatellite data suggests initial subdivision in two groups, Boreal and southwestern. The southwestern group further splits into Greater Pacific Northwest and U.S. Rockies. The genealogical information retrieved from mtDNA is congruent with the three highly differentiated and divergent groups of snowshoe hares. These groups can correspond with evolutionarily significant units that might have evolved in separate refugia south and east of the Pleistocene ice sheets. Genetic diversity was highest at mid-latitudes of the species' range, and genetic uniqueness was greatest in southern populations, consistent with substructuring inferred from both mtDNA and microsatellite analyses at finer levels of analysis. Surprisingly, snowshoe hares in the Greater Pacific Northwest mtDNA lineage were more closely related to black-tailed jackrabbits (Lepus californicus) than to other snowshoe hares, which may result from secondary introgression or shared ancestral polymorphism. Given the genetic distinctiveness of southern populations and minimal gene flow with their northern neighbours, fragmentation and loss of southern boreal habitats could mean loss of many unique alleles and reduced evolutionary potential.

Detection of RHDV strains in the Iberian hare (Lepus granatensis): earliest evidence of rabbit lagovirus cross-species infection.

Rabbit hemorrhagic disease virus (RHDV) is a highly lethal Lagovirus, family Caliciviridae, that threatens European rabbits (Oryctolagus cuniculus). Although a related virus severely affects hares, cross-species infection was only recently described for new variant RHDV in Cape hares (Lepus capensis mediterraneus). We sequenced two strains from dead Iberian hares (Lepus granatensis) collected in the 1990s in Portugal. Clinical signs were compatible with a Lagovirus infection. Phylogenetic analysis of the complete capsid gene positioned them in the RHDV genogroup that circulated on the Iberian Peninsula at that time. This is the earliest evidence of RHDV affecting a species other than European rabbits.

Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity.

Sequencing of modern Lepus VDJ genes shows that the usage of VHn genes has been retained in both Oryctolagus and Lepus that diverged 12 million years ago.

Among mammals, the European rabbit (Oryctolagus cuniculus) has a unique mechanism of generating the primary antibody repertoire. Despite having over 200 VH genes, the VH1 gene, the most D-proximal VH gene, is used in 80-90 % of VDJ rearrangements, while the remaining 10-20 % is encoded by the VHn genes that map at least 100 Kb upstream of VH1. The maintenance of the VHn genes usage in low frequency in VDJ rearrangements has been suggested to represent a relic of an ancestral immunologic response to pathogens. To address this question, we sequenced VDJ genes for another leporid, genus Lepus, which separated from European rabbit 12 million years ago. Approximately 25 VDJ gene sequences were obtained for each one of three Lepus europaeus individuals. We found that Lepus also uses the VHn genes in 5-10 % of its VDJ rearrangements. Our results show that the VHn genes are a conserved ancestral polymorphism that has been maintained in the leporids genome and is being used for the generation of VDJ rearrangements by both modern Lepus and Oryctolagus.