Neofelis Diardi) (Mammalia: Carnivora: Felidae) with the Description of a New Subspecies from Borneo ⇑ Andreas Wilting A, ,1, Per Christiansen B,1, Andrew C
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Molecular Phylogenetics and Evolution 58 (2011) 317–328 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Geographical variation in and evolutionary history of the Sunda clouded leopard (Neofelis diardi) (Mammalia: Carnivora: Felidae) with the description of a new subspecies from Borneo ⇑ Andreas Wilting a, ,1, Per Christiansen b,1, Andrew C. Kitchener c,d,1, Yvonne J.M. Kemp e, Laurentius Ambu f, Jörns Fickel a a Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, 10315 Berlin, Germany b University of Aalborg, Department of Biotechnology, Chemistry, and Environmental Engineering, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark c Department of Natural Sciences, National Museums Scotland, Chambers Street, Edinburgh EH1 1JF, UK d Institute of Geography, School of Geosciences, University of Edinburgh, Drummond Street, Edinburgh EH8 9XP, UK e VU University Amsterdam, Institute of Ecological Science, Department of Animal Ecology, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands f Sabah Wildlife Department, Block B, Wisma MUIS, 88000 Kota Kinabalu, Sabah, Malaysia article info abstract Article history: Recent morphological and molecular studies led to the recognition of two extant species of clouded leop- Received 3 August 2010 ards; Neofelis nebulosa from mainland southeast Asia and Neofelis diardi from the Sunda Islands of Borneo Revised 19 October 2010 and Sumatra, including the Batu Islands. In addition to these new species-level distinctions, preliminary Accepted 3 November 2010 molecular data suggested a genetic substructure that separates Bornean and Sumatran clouded leopards, Available online 11 November 2010 indicating the possibility of two subspecies of N. diardi. This suggestion was based on an analysis of only three Sumatran and seven Bornean individuals. Accordingly, in this study we re-evaluated this proposed Keywords: subspecies differentiation using additional molecular (mainly historical) samples of eight Bornean and 13 Biogeography Sumatran clouded leopards; a craniometric analysis of 28 specimens; and examination of pelage mor- Holotype Pleistocene phology of 20 museum specimens and of photographs of 12 wild camera-trapped animals. Molecular Sunda shelf (mtDNA and microsatellite loci), craniomandibular and dental analyses strongly support the differentia- Taxonomy tion of Bornean and Sumatran clouded leopards, but pelage characteristics fail to separate them com- Toba volcanic eruption pletely, most probably owing to small sample sizes, but it may also reflect habitat similarities between the two islands and their recent divergence. However, some provisional discriminating pelage characters are presented that need further testing. According to our estimates both populations diverged from each other during the Middle to Late Pleistocene (between 400 and 120 kyr). We present a discussion on the evolutionary history of Neofelis diardi sspp. on the Sunda Shelf, a revised taxonomy for the Sunda clouded leopard, N. diardi, and formally describe the Bornean subspecies, Neofelis diardi borneensis, including the designation of a holotype (BM.3.4.9.2 from Baram, Sarawak) in accordance with the rules of the Interna- tional Code of Zoological Nomenclature. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction two separate species, N. nebulosa (mainland southeast Asia), and N. diardi (Borneo and Sumatra, including the Batu Islands). In Clouded leopards are the most elusive of pantherine felids and 2008 this taxonomic revision was adopted by the IUCN Red List even today very little is known about their ecology and status in of Threatened Species and both species are now listed separately the wild (e.g. Grassman et al., 2005; Wilting et al., 2006). Recently, as Vulnerable in the current assessment (Sanderson et al., 2008 analyses of molecular (Buckley-Beason et al., 2006; Wilting et al., for N. nebulosa, Hearn et al., 2008a for N. diardi). 2007a) and morphological data (Christiansen, 2008; Kitchener In a previous study mtDNA and microsatellite genotype differ- et al., 2006) demonstrated that Bornean and Sumatran clouded ences between Bornean and Sumatran clouded leopards suggested leopards are clearly distinct from those on the continental main- the possible distinction of two subspecies of N. diardi (Wilting land. This led to a taxonomic revision of clouded leopards into et al., 2007a,b). This was provisionally supported by craniomandib- ular and dental analyses (Christiansen, 2008), but the small num- ⇑ Corresponding author. ber of individuals, especially in the molecular analysis E-mail address: [email protected] (A. Wilting). (NBorneo =7,NSumatra = 3), was not sufficient to further substantiate 1 Contributed equally. this suggestion. Moreover, the proposed names for the two 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.11.007 318 A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 subspecies did not follow the rules of the International Code of a = 0.011 (2 gamma rate categories). The phylogenetic reconstruc- Zoological Nomenclature (ICZN), henceforth the Code, and hence tions based on these parameters were then performed applying the a formal description is required to establish a valid scientific name maximum likelihood (ML) (Swofford, 2001) approach under a heu- for the Bornean clouded leopard. ristic search scenario with TBR branch swapping and the neighbor In this paper we present a new, extended phylogenetic analysis joining (NJ) method (Saitou and Nei, 1987) both implemented in with additional individuals from Borneo and Sumatra, combined PAUP (v. 4.0b10; Swofford, 2001). Support for nodes was assessed with analyses of morphological (craniomandibular, dental, and by a reliability percentage after 100 (ML), respectively 1000 (NJ) pelage) diversity within populations of Neofelis diardi. The aim of bootstrap iterations. Sequences were also analysed using Bayesian this study was to provide a thorough understanding of geographi- Inference (BI) as implemented in MrBayes (v.3.1.2; Huelsenbeck cal variation in Neofelis diardi, some insights into its evolutionary and Ronquist, 2001). Posterior probabilities for the BI were deter- history and a systematic revision, including the potential for the mined by running three heated chains (default temperature set- formal recognition of subspecies. ting: 0.2) and one cold chain for 1 million generations (Ronquist and Huelsenbeck, 2003). The parameters of the optimal model se- lected by jModelTest were specified as priors. Each analysis was 2. Materials and methods run twice and trees were sampled every 100 generations. Stability of likelihood convergence was determined using the hsumpi com- 2.1. Molecular analysis mand in MrBayes, leading to the exclusion of the first 30,000 sam- ples as burn-in when convergence diagnostics were calculated. In addition to the samples used in Wilting et al. (2007a), we col- Posterior probabilities for nodes were based on the remaining lected 22 additional epithelial (from skulls or skins) or maxillo-tur- topologies. The domestic cat (Felis catus) sequence served as out- binal bone tissue samples from Neofelis spp. (18 N. diardi and 4 N. group (Accession number U20753). nebulosa) from natural history museums, and three fecal samples We computed genetic diversity within and among the different from wild-born Bornean clouded leopards kept in the Lokawi Wild- groups (N. nebulosa, N. diardi (Borneo) and N. diardi (Sumatra) by life Park in Sabah, Malaysia (see Appendix 1). We extracted DNA in estimating pairwise population FST–values (Cockerham and Weir, an isolated ‘ancient DNA laboratory’. Epithelia (30–50 mg) were 1993) and applied different hierarchical analyses of molecular var- minced and turbinates (20–40 mg) were fragmented. For the iance (AMOVA; Excoffier et al., 1992) to estimate the amount of extraction we followed the protocol of Wisely et al. (2004), but population genetic structure. Both tests are implemented in Arle- precipitated the DNA with isopropanol, followed by a washing step quin v.3.5 (Excoffier et al., 2005). Because we used concatenated with 70% ethanol. The dried pellets were dissolved in 200 ll deion- data sets (see above) with potentially differing selective pressures ized sterile water and DNA concentrations were measured spectro- among the various mitochondrial loci (Lopez et al., 1997), we car- photometrically at 260 nm (ND1000, Peqlab GmbH, Erlangen, ried out Tajima’s D test (Tajima, 1989) to investigate whether the Germany). concatenated data set could be treated as a selectively neutrally evolving unit or not. 2.1.1. Mitochondrial DNA (mtDNA) analysis The demographic history of clouded leopards was inferred by We amplified segments of control region (426 bp), ATPase-8 several approaches. Firstly, we used the mismatch distribution (134 bp) and Cyt-b (286 bp), previously used in Wilting et al. (distribution of numbers of site differences j between each pair (2007a), but modified the ATPase-8 forward primer to [50–ATGCCA- of sequences in the populations; Li, 1977, Rogers, 1995, Rogers CAGCTAGATACATCC–3]. PCR reactions were performed in 20 ll, and Harpending, 1992) to calculate the time t since potential containing 4 ll5Â GoTaq PCR Buffer (Promega GmbH, Mannheim, step-wise expansion of a relatively small, but constant population Germany), 2 mM MgCl2, 0.2 mM dNTPs, 1 lM of each primer, 1 at size h0 to a large population at size h1 over t generations