Mountain hare transcriptome and diagnostic markers as resources to monitor hybridization with European hares

Abstract

We report the first mountain hare (Lepus timidus) transcriptome, produced by de novo assembly of RNA-sequencing reads. Data were obtained from eight specimens sampled in two localities, Alps and Ireland. The mountain hare tends to be replaced by the invading European hare (Lepus europaeus) in their numerous contact zones where the species hybridize, which affects their gene pool to a yet unquantified degree. We characterize and annotate the mountain hare transcriptome, detect polymorphism in the two analysed populations and use previously published data on the European hare (three specimens, representing the European lineage of the species) to identify 4 672 putative diagnostic sites between the species. A subset of 85 random independent SNPs was successfully validated using PCR and Sanger sequencing. These valuable genomic resources can be used to design tools to assess population status and monitor hybridization between species.

Figure 1. Approximate mountain and European hare distribution.

Approximate distributions of the mountain hare, Lepus timidus, and the European hare, L. europaeus, in Eurasia with indication of the areas of contact and of broad geographic overlap between the species (distribution ranges were adapted from IUCN Spatial Data Resources; IUCN 2016). Circles indicate the mountain hare sampling locations for this work (open circle—Ireland; closed circle—Alps).

Figure 2. Methodological workflow.

Flowchart of the RNA-sequencing setup and data analysis steps. Commands used in the analytical steps shown in bold are detailed in Table 1 (available online only).

Figure 3. Annotation summary.

Number of transcripts annotated with different combinations of methods and databases: all transcripts; transcripts annotated with crb-blast against rabbit transcriptome; transcripts annotated with a unidirectional BLASTx against rabbit transcriptome; transcripts annotated with crb-blast against the Swiss-Prot database; and transcripts annotated with a unidirectional BLASTx against the Swiss-Prot database.

Figure 4. Characterization of inferred SNPs in the sampled populations and species.

(a) Relative proportion of the 41 182 SNPs mapped to the mountain hare transcriptome, summarized as polymorphic within each species and fixed or shared between L. timidus (mountain hare) and L. europaeus (European hare). The proportion is shown considering the complete L. timidus dataset (i) and only the Irish (ii) and Alpine (iii) populations. (b) STRUCTURE analysis to evaluate cluster membership and admixture proportions. Individuals are sorted by population and species. Mountain hare populations are shown in blue and European hare individuals in orange. (c) Principal Component Analysis (PCA) plot using one SNP per contig. The first principal component (PC1) splits species and the second (PC2) the sampled populations.