Ancient DNA unravels species identification from Laosicheng site, Hunan Province, China, and provides insights into maternal genetic history of East Asian leopards

Abstract

was generally similar to that of P. pardus and P. uncia, but conspicuously larger than that of Neofelis and Felini species. Notably, the auditory bulla of the skulls was inflated, with a relatively small ectotympanic chamber, a feature distinct from P. uncia. Furthermore, the mastoid process was relatively weak, and the distant postorbital constriction supported a closer affinity to P. pardus than to P. uncia. For accurate species identification, photographs displaying the dorsal view of skulls from 23 extant Felidae species, including 15 Felini species, six Pantherini species, and the two Laosicheng samples, were collected and used for principal component analysis (PCA) employing a variance-covariance matrix of eight landmark measurements (Supplementary Tables S1–S3). Results demonstrated that the Laosicheng samples formed a loose cluster, positioned between the Felini and Pantherini species (Supplementary Figure S2), without providing a definitive species identification. Although the morphological evidence suggested that the two samples belonged to the family Felidae, characterized by distinctive features (e.g., auditory bulla and mastoid process) (Supplementary Figure S3), the damaged state of the skulls presented challenges in specific species determination. Therefore, we attempted to recover endogenous aDNA sequences for a more accurate identification. Previous studies have established the effectiveness of aDNA analysis in the taxonomic identification of ancient animals, with genetic data also allowing for a more comprehensive understanding of population genetic history (Zhu et al., 2022). Here, high-quality mitogenome sequences were successfully obtained for the Laosicheng samples (Supplementary Figures S4). Utilizing a dataset combining the two Laosicheng samples with 114 other individuals representing all genera of the family Felidae (Supplementary Table S4), pairwise genetic distance analysis indicated that the Laosicheng samples share a closer genetic affiliation with the Panthera genus than any other (FST<0.01) (Supplementary Figures S5, S6 and Table S5). Further genetic distance comparisons between the two Laosicheng samples and 79 individuals within the Panthera genus (including P. pardus (n=50), P. leo (n=5), P. uncia (n=15), P. onca (n=4), and P. tigris (n=5)) (Supplementary Figures S5, S6 and Table S5) placed the Laosicheng samples closest to