The red leopard (Panthera pardus) colour morph is a colour variant that occurs only in South Africa, where it is confined to the Central Bushveld bioregion. Red leopards have been spreading over the past 40 years, which raises the speculation that the prevalence of this phenotype is related to low dispersal of young individuals owing to high off-take in the region. Intensive selective hunting tends to remove large resident males from the breeding population, which gives young males the chance to mate with resident females that are more likely to be their relatives, eventually increasing the frequency of rare genetic variants. To investigate the genetic mechanisms underlying the red coat colour morph in leopards, and whether its prevalence in South Africa relates to an increase in genetic relatedness in the population, we sequenced exons of six colour coat associated genes and 20 microsatellite loci in twenty wild-type and four red leopards. The results were combined with demographic data available from our study sites. We found that red leopards own a haplotype in homozygosity identified by one non-synonymous SNP and a 1 bp deletion that causes a frameshift in the Tyrosinase Related Protein 1 (TYRP1), a gene known to be involved in the biosynthesis of melanin. Microsatellite analyses indicate clear signs of a population bottleneck and a relatedness of 0.11 among all pairwise relationships, eventually supporting our hypothesis that a rare colour morph in the wild has increased its local frequency due to low natal dispersal. This was backed by a high human-induced mortality rate (40%).

Tissue samples were opportunistically obtained from leopards that were found dead in the field, and hair samples were collected when animals were immobilized for relocation or radio-collaring. DNA was isolated using the NucleoSpin Tissue kit applying the support protocol for tissue samples. We selected 20 nuclear microsatellite loci that are known to be polymorphic in South African leopards. Primers were 5’-labelled with 6-FAM, HEX, Tamra or TET fluorescent dyes. Polymerase Chain Reactions (PCRs) were performed in 10 µL volumes containing 3 µL (H20), 5 µL (1x) Platinum Multiplex PCR Master Mix (Applied Biosystems), 0.5 µL (25 nM) of each primer, and 1 µL (~ 20 ng) genomic DNA. Amplifications were carried out in a Multigene Optimax Thermal Cycler (Labnet) with the following PCR cycling protocol: 3 min of initial activation at 93°C, 10 annealing cycles consisting of 94°C for 15 s, 52-56°C for 15 s and 72°C for 30 s, 20 annealing cycles consisting of 89°C for 15 s, 52-56°C for 15 s, 72°C for 30 s, and a final elongation step of 72°C for 30 min.

For three-digit microsatellite scores, please contact lauratensen@uni-koblenz.de