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, 16 (23), 2371-6

Molecular Evidence for Species-Level Distinctions in Clouded Leopards


Molecular Evidence for Species-Level Distinctions in Clouded Leopards

Valerie A Buckley-Beason et al. Curr Biol.


Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).


Figure 1
Figure 1
Geographic and Phylogenetic Depiction of Individuals Used in This Study (A) Geographic range of the four currently recognized clouded leopard subspecies based upon historical descriptions. The numbers depicted before the slash mark indicate the number of samples that amplified robustly and were used in the study. Those numbers depicted after the slash marks indicate the total number of samples that were collected for analysis. Ancient, or museum, samples are listed separately from modern samples in Tables S1 and S2. The three available N. n. diardi samples were born in the wild in Borneo (Table S1). (B) Phylogenetic relationships among haplotypes of Neofelis and Panthera genera were rooted with the domestic cat and based on analysis of the ATP-8 (139 bp) mtDNA gene segment. An asterisk denotes less than 60% bootstrap support. (C) Combined mtDNA and nuclear gene segments (3.9 Kb). Depicted phylogenetic trees were constructed by minimum evolution (ME) with neighbor-joining (NJ) algorithm and Kimura 2 distances. Maximum parsimony (MP) and maximum likelihood (ML) phylogenetic trees were also constructed and showed similar topology. Bootstrap values from 100 iterations are listed above the lines at major nodes for each of the three methods (NJ/MP/ML). Below the branches is the number of steps/number of homoplasies. To the right of each haplotype (in parentheses) is the number of clouded leopard individuals with this haplotype (for mtDNA) or genotype (for nuclear genes). (D) Unrooted neighbor-joining phylogram constructed by Dps (Proportions of Shared Alleles) based upon each individual's composite micro-satellite genotype. Bootstrap support (100 iterations) is indicated.
Figure 2
Figure 2
G Band Karyotype of Chromosome F3 from Three N. n. nebulosa Metaphase Preparations and Three N. n. diardi Preprations Note that N. n. diardi are all acrocentric. The N. n. nebulosa F3 chromosomes are larger and polymorphic for the amount of extrachromatin material, either above or below the centromere. The extended F3 chromosome was present in nine N. n. nebulosa individuals but not in the three N. n. diardi individuals examined.

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