Supplemental Experimental Procedures

Biological samples were obtained from captive animals of known geographic origin (n = 42), from captive born animals from parents of known geographic origin (n = 38), and from museum specimens (n = 29) (Figure 1). For outgroup comparison, fifteen broadly distrib-uted individuals of known geographic origin from species of Pan-thera (P. tigris, P. pardus, P. uncia, and P. onca) were selected along with ten individuals from each of six well-studied lion (P. leo) popula-tions, along with two domestic cats (Felis catus) (Tables S1 and S2). DNA was extracted from white blood cells, skin fibroblast cells, or tissues according to a phenol-chloroform protocol [S1]. Museum samples of hide, hair, or bone were extracted with a modified sil-ica-based, guanidium extraction method [S2–S4]. Cell lines for DNA extraction and cytogenetic analyses were established accord-ing to standard methods [S5]. Mitochondrial and Nuclear Gene Amplification and Phylogenetic Analysis Sequence variation among clouded leopards and outgroup species was assessed for portions of four mitochondrial (mtDNA) gene seg-ments, one autosomal gene segment (HK1), and four X-linked gene segments (ATP7A, BGN, IDS and PLP). Gene fragments were ampli-fied by PCR of genomic DNA in 96-well-plate format with 76 clouded leopards and two samples from each Panthera genus species and two domestic cats. For the mtDNA genes, 139 bp of ATPase-8 (ATP-8) was amplified with primers ATP-8 [F] 50-ACAACTAGATACATCCACCTGA-30 and [R] 50-GGCGAATAGATTTTCGTTCA-30, 223 bp of Sodium Dehydro-genase-5 (ND5) with primers (F) 50-GTGCAACTCCAAATAAAAG-3