Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse

Abstract

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

Figure 1. Genomic Structure among Late Pleistocene Horses in Blue, DHs in Red, and PHs in Green

(A) Exome-based ML tree. Nodes show bootstrap support ≥ 95%. AQH, American Quarter Horse. (B) PSMC profiles. Thin lines represent 100 bootstrap replicates. kya, thousand years ago. (C) PCA based on genotype likelihoods. See also Figures S1 and S2 and Tables S1 and S2.

Figure 2. DH and PH Demographic Models Explored in Dadi and Projection Analyses

(A) Best two-population model supported by dadi. (B) Dadi model Log(likelihood) for varying starting dates of isolation between DHs (Franches-Montagnes) and PHs (TEG). (C) Best two-population model supported by projection analyses. (D) Model LSS when considering varying dates for the change in migration dynamics (TMC) for the genome projections based on the DH (Franches-Montagnes, red) and PH (black) reference panels. See also Table S3.

Figure 3. Heterozygosity and Inbreeding Estimates

(A) Average genome-wide heterozygosity for autosomes, disregarding transitions. (B) FHMM inbreeding coefficient and inbreeding coverage across modern and historical PHs and DHs. X denotes not estimated.

Figure 4. DH Introgression into PHs

(A) Genetic components estimated in NGSAdmix (eight clusters). The gray and red gradients represent the PH and DH components, respectively. (B) D statistic heatmaps. The tree topology (Outgroup,(PH,(DH,Paratype))) is tested for each DH and PH combination, disregarding transitions. Darker red colors indicate the admixture component in significant tests; darker gray shades indicate non-significant tests. The proportion of DH introgression within each PH genome, estimated by the average of f₄ ratios involving Franches-Montagnes horses, is indicated next to the PH sample labels. (C) Chromosome painting of DH (red) and PH (gray) ancestry blocks identified along chromosome 5. Asterisk denotes A-line PHs. See also Figure S3 and Tables S4 and S5.