Genomic evidence for two phylogenetic species and long-term population bottlenecks in red pandas

Abstract

The red panda (Ailurus fulgens), an endangered Himalaya-endemic mammal, has been classified as two subspecies or even two species - the Himalayan red panda (A. fulgens) and the Chinese red panda (Ailurus styani) - based on differences in morphology and biogeography. However, this classification has remained controversial largely due to lack of genetic evidence, directly impairing scientific conservation management. Data from 65 whole genomes, 49 Y-chromosomes, and 49 mitochondrial genomes provide the first comprehensive genetic evidence for species divergence in red pandas, demonstrating substantial inter-species genetic divergence for all three markers and correcting species-distribution boundaries. Combined with morphological evidence, these data thus clearly define two phylogenetic species in red pandas. We also demonstrate different demographic trajectories in the two species: A. styani has experienced two population bottlenecks and one large population expansion over time, whereas A. fulgens has experienced three bottlenecks and one very small expansion, resulting in very low genetic diversity, high linkage disequilibrium, and high genetic load.

Fig. 1. Distinguishing morphological differences between two red panda species.

(A and C) The Chinese red panda. (B and D) The Himalayan red panda. (A and B) The face coat color of the Chinese red panda is redder with less white on it than that of the Himalayan red panda. (C and D) The tail rings of the Chinese red panda are more distinct than those of the Himalayan red panda, with the dark rings being more dark red and the pale rings being more whitish. Photo credit: (A) Yunfang Xiu, Straits (Fuzhou) Giant Panda Research and Exchange Center, China; does not require permission. (B) Arjun Thapa, Institute of Zoology, Chinese Academy of Sciences. (C) Yibo Hu, Institute of Zoology, Chinese Academy of Sciences. (D) Chiranjibi Prasad Pokheral, Central Zoo, Jawalkhel, Lalitpur, Nepal; does not require permission.

Fig. 2. Population genetic structure based on whole genomes, Y chromosome SNPs, and mitochondrial genomes of red pandas.

(A) The geographic distribution of wild red panda samples under the background of habitat suitability. Red, QL population; purple, XXL-LS population; blue, SLL population; pink, EH-GLG; dark red, MH. (B) Maximum likelihood phylogenetic tree based on whole-genome SNPs, with the ferret as the outgroup. The values on the tree nodes indicate the bootstrap support of ≥50%. (C) ADMIXTURE result based on whole-genome SNPs with K = 2 to 7. (D) PCA result based on whole-genome SNPs. (E) Network map based on eight Y chromosome SNP haplotypes. (F) Network map based on 41 mitochondrial genome haplotypes.

Fig. 3. Demographic history, divergence, and admixture of two red panda species and their populations.

(A) PSMC analysis revealed different demographic histories of the two species, with a generation time (g) of 6 years and a mutation rate (μ) of 7.9 × 10⁻⁹ per site per generation. The time axis is logarithmic transformed. (B) Fastsimcoal2 simulation reconstructed the divergence, admixture, and demographic history of red panda species and populations. The time axis is logarithmic transformed, and the number of migrants per year between two adjacent populations is shown beside each arrow. (C) TreeMix analysis detected significant gene flow from the EH-GLG to XXL-LS populations. s.e., standard error.

Fig. 4. Genetic variation, genetic load, and natural selection of red pandas.

(A) Genetic variations (nucleotide diversity) of different species and populations based on whole-genome SNPs, mitochondrial genomes, and Y chromosome SNPs. (B) LD of the four populations. (C) Ratios of homozygous derived deleterious or LoF variants to homozygous derived synonymous variants for different populations. The horizontal bars denote population means. (D) Distribution of θ_π ratios (non-MH/MH) and Z(F_ST) values. Data points located to the left of the left vertical dashed lines and the right of the right vertical dashed lines (corresponding to the 5% left and right tails of the empirical θ_π ratio distribution, respectively) and above the horizontal dashed line [the 5% right tail of the empirical Z(F_ST) distribution] were identified as selected regions for the MH (the Himalayan red panda, green points) and non-MH (the Chinese red panda, blue points) populations.