Morphological variation in natural populations is a genomic test bed for studying the interface between molecular evolution and population genetics, but some of the most interesting questions involve non-model organisms that lack well annotated reference genomes. Many felid species exhibit polymorphism for melanism but the relative roles played by genetic drift, natural selection, and interspecies hybridization remain uncertain. We identify mutations of Agouti signaling protein (ASIP) or the Melanocortin 1 receptor (MC1R) as independent causes of melanism in three closely related South American species: the pampas cat (Leopardus colocolo), the kodkod (Leopardus guigna), and Geoffroy’s cat (Leopardus geoffroyi). To assess population level variation in the regions surrounding the causative mutations we apply genomic resources from the domestic cat to carry out clone-based capture and targeted resequencing of 299 kb and 251 kb segments that contain ASIP and MC1R, respectively, from 54 individuals (13–21 per species), achieving enrichment of ~500–2500-fold and ~150x coverage. Our analysis points to unique evolutionary histories for each of the three species, with a strong selective sweep in the pampas cat, a distinctive but short melanism-specific haplotype in the Geoffroy’s cat, and reduced nucleotide diversity for both ancestral and melanism-bearing chromosomes in the kodkod. These results reveal an important role for natural selection in a trait of longstanding interest to ecologists, geneticists, and the lay community, and provide a platform for comparative studies of morphological variation in other natural populations.

Genomic sequence dataThe sequence data file is provided as a ZIP archive containing 3 subfolders, one for each of the three analyzed species (i.e. Lco - Leopardus colocolo; Lge - Leopardus geoffroyi ; Lgu - Leopardus guigna). For each of these species the following sequence files are provided in the fasta format: - unmasked genomic consensus sequence computed for each and all the Asip and Mc1r loci fragments (i.e. for Leopardus colocolo the unmasked consensus sequence file is labeled Lco.fa); - genomic consensus sequence computed for each and all of the Asip and Mc1r loci fragments and masked for repeats and low complexity sequence using RepeatMasker (i.e. for Leopardus colocolo the masked consensus sequence file is labeled Lco_rm.fa); - haplotype sequence data for each of the genomic fragments and each of the analyzed samples, one file per fragment (i.e. Lco_AsipA for AsipA fragment) containing two haplotypic sequences per sample. These sequences are labeled by indicating the genomic fragment, the species, the individual and finally the haplotype (i.e. AsipA_Lco-030_h1 [organism=Leopardus colocolo] ASIP locus segment A). The samples are labeled using the same identifiers as those provided in the manuscript (see Table S1 for details).datadryad.zip