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. 2023 Jul 13;18(7):e0269967.
doi: 10.1371/journal.pone.0269967. eCollection 2023.

A unique single nucleotide polymorphism in Agouti Signalling Protein (ASIP) gene changes coat colour of Sri Lankan leopard (Panthera pardus kotiya) to dark black

M G C Sooriyabandara  1 , A U Bandaranayake  2 , H A B M Hathurusinghe  3 , S M Jayasundara  3 , M S R R P Marasinghe  1 , G A T Prasad  1 , V P M K Abeywardana  1 , M A Pinidiya  1 , R M R Nilanthi  1 , P C G Bandaranayake  3
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A unique single nucleotide polymorphism in Agouti Signalling Protein (ASIP) gene changes coat colour of Sri Lankan leopard (Panthera pardus kotiya) to dark black

M G C Sooriyabandara et al. PLoS One. .

Abstract

The Sri Lankan leopard (Panthera pardus kotiya) is an endangered subspecies restricted to isolated and fragmented populations in Sri Lanka. Among them, melanistic leopards have been recorded on a few occasions. Literature suggests the evolution of melanism several times in the Felidae family, with three species having distinct mutations. Nevertheless, the mutations or other variations in the remaining species, including Sri Lankan melanistic leopard, are unknown. We used reference-based assembled nuclear genomes of Sri Lankan wild type and melanistic leopards and de novo assembled mitogenomes of the same to investigate the genetic basis, adaptive significance, and evolutionary history of the Sri Lankan melanistic leopard. Interestingly, we identified a single nucleotide polymorphism in exon-4 Sri Lankan melanistic leopard, which may completely ablate Agouti Signalling Protein (ASIP) function. The wild type leopards in Sri Lanka did not carry this mutation, suggesting the cause for the occurrence of melanistic leopords in the population. Comparative analysis of existing genomic data in the literature suggests it as a P. p. kotiya specific mutation and a novel mutation in the ASIP-gene of the Felidae family, contributing to naturally occurring colour polymorphism. Our data suggested the coalescence time of Sri Lankan leopards at ~0.5 million years, sisters to the Panthera pardus lineage. The genetic diversity was low in Sri Lankan leopards. Further, the P. p. kotiya melanistic leopard is a different morphotype of the P. p. kotiya wildtype leopard resulting from the mutation in the ASIP-gene. The ability of black leopards to camouflage, along with the likelihood of recurrence and transfer to future generations, suggests that this rare mutation could be environment-adaptable.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The geographic distribution and habitat association of melanistic leopard morphotypes in Sri Lanka.
Fig 2
Fig 2. A visual morphological differences between Sri Lankan regular and black leopards.
(a) The side view of wild type leopard (PPK-W) and the view of its spot and rosette pattern. (b) The side view of the Sri Lankan melanistic leopard (PPK-B) and the view of its spot and rosette pattern. The spots appear to be still visible in the black background. The cross section of the skin of (PPK-B) with visible fur colouration.
Fig 3
Fig 3. Amino acid alignment of ASIP, including the novel P. p. kotiya sequences.
(a) Codon Variation in the ASIP gene Associated with Melanism in the Sri Lankan leopard P..p. kotiya. Amino acid alignment of ASIP Exon 4, including the novel PPK-W P. p. kotiya- wild and PPK-B P. p. kotiya- melanistic. ERR5671301 wild type P. p. kotiya fossil JX845175 Panthera pardus. Dots indicate the identity of the top sequence; amino acid positions are shown at the end of each line. Dashes represent insertion/deletion (indel) variants. Numbers 1–10 refer to the 10 conserved cysteine residues present in the C-terminal domain. The non-synonymous mutation in melanistic PPK-B is indicated in bold and red. (b) Protein structure of the ASIP gene in the Sri Lankan melanistic leopard.
Fig 4
Fig 4. The mitogenome of Panthera pardus kotiya and melanistic leopard.
Genes on the inside of the circle are transcribed clockwise and those on the outside are transcribed counterclockwise. The different colors represent functional groups, and the darker gray in the inner circle indicates the GC content while the light gray corresponds to the AT content.
Fig 5
Fig 5
(a) Maximum likelihood (b) Bayesian NADH-5 gene tree of 13 Asian leopards along with the P. tigris and P. leo outgroups. The bootstrap values and posterior probabilities are displayed next to the nodes, respectively. The origins of the samples are given inside the brackets. LK–Sri Lanka, IN—India.
Fig 6
Fig 6. Median joining network analysis of the Asian leopards using NADH-5 gene.
The network includes 6 sequences of Sri Lankan leopards and 7 sequences from Indian leopards. Each node represents a unique haplotype. Haplotypes are colour-coded based on the geographical location/species. Size of the circles represents the number of sequences with the same haplotype. Size of the node reflects the frequency of each haplotype.
Fig 7
Fig 7. Mitochondrial phylogenetic tree of Pantherinae and Felinae.
Node bars represent the 95% highest posterior density (HPD). Tree tips are labeled with the species name, accession number, and origin.
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Grants and funding

The Eco-System Conservation Management Project (ESCAMP) of the World Bank provided the funds to the Department of Wildlife Conservation (DWC), Sri Lanka to conduct the research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.