Molecular Phylogenetics and Evolution 58 (2011) 317–328
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Molecular Phylogenetics and Evolution
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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 ll5 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 (muta- unit of GoTaq polymerase (Promega) and 50–100 ng of genomic tional units) in the past with t = s/2l (s: age of expansion, l: muta- DNA. The reactions were performed with an initial denaturation tion rate). We applied a generalized non-linear least-square step at 95 °C for 3 min, followed by 45 cycles of denaturation at approach implemented in Arlequin v.3.5. (Excoffier et al., 2005) 94 °C for 30 s, annealing at 56 °C for 45 s, elongation at 72 °C for and the simplifying assumption of post-expansion population size 45 s, and were completed with a final elongation step at 72 °C for being infinite (Rogers, 1995). The sum of squared deviations (SSD; 10 min. Excess oligonucleotide primers and dNTPs were removed Schneider and Excoffier, 1999) and Harpending’s raggedness index by incubation with exonuclease I and calf-intestine alkaline phos- (Harpending, 1994) were computed to test goodness-of-fit of the phatase (ExoCIAP, Fermentas GmbH, St. Leon-Rot, Germany). observed mismatch distribution to that expected under the sudden Amplicons were directly sequenced bidirectionally using the Big- population expansion model. Parametric bootstrapping (10,000 DyeÒ Terminator kit (v.1.1) and analysed on an A3130xl automated permutations) was applied to obtain confidence intervals around sequencer (both Applied Biosystems Deutschland GmbH, Darms- all estimated parameters (Excoffier et al., 2005, Schneider and tadt, Germany). Excoffier, 1999). Displayed graphically, recent sudden population Sequences were assembled, aligned and edited using ClustalX2 expansions or bottlenecks will generate unimodal graphs, whereas software (Larkin et al., 2007). Sequences from all three mtDNA stable or slowly declining populations will generate a variety of fragments were concatenated and trimmed to identical lengths multimodal distributions, reflecting the highly stochastic shape (849 bp) to suit the lengths of additional sequences from Genbank of gene-trees within populations at demographic equilibrium (see Wilting et al., 2007a for accession numbers). The entire data (Rogers and Harpending, 1992). The mutation rate l, used to calcu- set consisted of 90 sequences (mainland: N = 58, Borneo: N = 15, late the age of expansion, was 2.2 10 9/site year (Kumar and Sumatra: N = 16, outgroup: N = 1). To select the best-fitting nucle- Subramanian, 2002). As a second approach to explore the demo- otide substitution model for the full data set, we used the hierar- graphic history of clouded leopard populations, we computed chical likelihood ratio test approach implemented in the software Fu’s FS statistics (Fu, 1997; 1000 simulations) to detect an excess jModelTest (v.0.1.1; Posada, 2008). The model selected for the data of low-frequency alleles in a growing population as compared with set was the K81 model (Kimura, 1981) with an allowance both for the expected number of alleles in a stationary population, whereby invariant sites (I) and a gamma (G) distribution shape parameter significantly large negative values indicate population expansion (a) for among-site rate variation (K81 +I+G). Parameter values (Fu, 1997, Excoffier and Schneider, 1999). Because assessment of for the model selected were: -lnL = 1790.7657, I = 0.183, and demographic history based on mismatch distribution may be A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 319
biased in samples with high Nhaplotypes/Nindividuals ratios (R)(Fic- from Kitchener et al. (2006), although not all pelages yielded scores kel et al., 2008), we also estimated the divergence time by dividing for all characters. Of these, coloration was scored as three subcat- the net-between-clade-means genetic distances calculated using egories for the presence/absence of yellow, grey and tawny. The Mega v.4.1 (Tamura et al., 2007) following the jModelTest recom- scores for each pelage character were summed for each pelage to mendations (Table 3). As calibration point we used the split be- give a total pelage score (Borneo N = 6; Sumatra N = 14). In addi- tween Panthera and Neofelis at 6.37 million years ago (Johnson tion, similar data were scored, where possible, from camera-trap et al., 2006). The estimated net-between-clade-means distance of photographs of both Sumatran (N = 4) and Bornean (N = 8) clouded 0.29972 translated into a mutation rate of 29.972% (SE = 9.255%). leopards, but owing to limited viewing angles, data were incom- plete for most specimens. Therefore complete data were available 2.1.2. Microsatellite analysis for totals of 16 Sumatran and seven Bornean clouded leopards To genotype individuals we used 12 felid dinucleotide microsat- based on both pelages and camera-trap photographs. Differences ellite primers (FCA8, FCA23, FCA43, FCA45, FCA77, FCA82, FCA105, in mean pelage character scores and total pelage scores for FCA126, FCA132, FCA144, FCA261, FCA310; Menotti-Raymond Bornean and Sumatran samples were assessed using two-sample et al., 1999). One primer of each pair was 50-labeled with a fluores- t-tests with a probability of significance of p = 0.05, using the PAST cent dye (6-FAM or HEX). Based on allele size range and fluorescent statistical package (v.1.99; Hammer et al., 2001). dye used we were able to run three multiplex PCRs with primers for four loci (set1: FCA8, 126, 132 and 261; set2: FCA23, 82, 144, 3. Results 310; set3: FCA43, 45, 77 and 105). Amplification products were sized on an A3130xl automated sequencer, using the ROX500 inter- 3.1. Mitochondrial DNA analysis nal sizing standard (both Applied Biosystems). Only DNA samples that produced congruent results in two independent PCR reactions A total of 22 additional individuals were included in the mtDNA and successfully amplified at least at nine out of the 12 loci were analysis (1 N. nebulosa,8N. diardi – Borneo, 13 N. diardi – Sumatra, included in the final microsatellite analysis. Tests for genotypic dis- the latter including the neotype for N. diardi (see Christiansen, equilibrium between loci, analysis of molecular variance (AMOVA) 2009) and one specimen from the Batu Islands). Besides known and the estimation of RST-values (Slatkin, 1995) were performed Neofelis haplotypes (Wilting et al., 2007a), we identified seven using the software package Arlequin v.3.5 (Excoffier et al., 2005). new haplotypes for clouded leopards (1 for N. nebulosa, 1 for N. dia- A Bayesian clustering method, implemented in STRUCTURE rdi – Borneo and 5 for N. diardi – Sumatra) (Genbank accession (v.2.3, Pritchard et al., 2000), was used to infer population structure numbers HM748835 – HM748855). and assignment of individuals to populations based on allelic geno- The enlarged sample of N. diardi confirmed 38 out of 39 diag- types. A series of tests was conducted assuming different numbers nostic sites between N. nebulosa and N. diardi (Wilting et al., of population clusters (K = 1–9) to guide an empirical estimate of 2007a) and discarded only one site (position 802 of Wilting et al., the number of identifiable populations, assuming an admixture 2007a; where the new haplotype DIB6 from Borneo shared an model with correlated allele frequencies. To determine the appro- adenosine [A] with N. nebulosa). The four nucleotide differences be- priate burn-in and run lengths for reliable parameter estimates of P tween Bornean and Sumatran clouded leopards described in Wilt- and Q, we set K = 1 and watched for the likelihoods to converge un- ing et al. (2007a) at position 204, 234 (Cyt-b), 420 (ATPase-8) and der various burn-in and run lengths. The final burn-in and run 610 (control region) remained fixed. lengths were then 100,000 and 200,000 Markov chains, respec- MtDNA diversity among the three clouded leopard populations tively. We ran ten independent runs for each K and its associated (mainland, Borneo and Sumatra) revealed a very low nucleotide parameter set to verify the consistency of estimates across runs. diversity for N. nebulosa (p = 0.00057) compared with N. diardi For presentation of the assignments, we used the mean of the (p = 0.0037) and moderate nucleotide diversities of p = 0.00126 ten runs and the standard deviation (SD). A factorial correspon- and p = 0.00142 for the populations in Borneo and Sumatra, dence analysis (Clausen, 1998; Greenacre and Degos, 1977) was respectively. AMOVA demonstrated that 98.2% of the genetic vari- carried out to examine the relationship between clouded leopards ability was due to the variance among populations, and only 1.8% and allele frequencies using the software package GENETIX was accounted for by differences within the three populations. (v.4.05.2, Belkhir et al., 1996–2004) under the ‘3D by populations’ Pairwise FST comparisons (Table 1) showed that each of the three setting. populations was significantly differentiated from each of the other two (p < 0.0001). 2.2. Morphological analysis Phylogenetic analysis of mtDNA haplotypes, using neighbor joining (NJ), maximum likelihood (ML) and Bayesian inference 2.2.1. Craniomandibular and dental analysis (BI) approaches, generated congruent topologies that strongly sup- Craniomandibular and dental diversity within Neofelis diardi ported the monophyletic status of N. diardi with high bootstrap was assessed using a sample of 28 adult specimens, of which 18 values (100% NJ, 99% ML, 0.96 BI) (Fig. 1). Furthermore, the mono- came from Sumatra (10#;8$) and 10 from Borneo (5#;5$). For phyletic statuses of Bornean and Sumatran clouded leopards were each specimen 64 craniomandibular and dental measurements also supported with high bootstrap values (Fig. 1). The neotype of and three angular variables were analysed (see Christiansen, N. diardi shared its haplotype (DIS3) with four other Sumatran indi- 2008). The samples were analysed with multivariate analyses viduals and with the specimen from the Batu Islands. (Principal Components Analysis [PCA]; and step-wise Discriminant Analysis [DA] with subsequent jack-knifed classification analysis) on raw variables, and with bivariate comparisons of craniomandib- Table 1 ular and dental ratios. Ratios were arcsine transformed prior to Pairwise FST (mtDNA) and RST (microsatellite loci) values among the three clouded leopard populations; N. nebulosa (Mainland), N. diardi (Borneo) and N. diardi analysis to restore normality (Sokal and Rohlf, 1995), but angular (Sumatra). variables were analysed without transformation. RST/FST Mainland Borneo Sumatra 2.2.2. Pelage analysis Mainland – 0.986 0.986 Samples of pelages of Sumatran (N = 15) and Bornean (N =8) Borneo 0.721 – 0.804 Sumatra 1.018 0.202 – clouded leopards were scored according to eight pelage characters 320 A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328
Fig. 1. Phylogenetic relationships among clouded leopards inferred from mtDNA haplotypes from the concatenated 849 bp mitochondrial sequences. Trees for each of the three methods (NJ/ML/BI) had similar topologies. Numbers above the branches represent bootstrap support, only values >50% are shown. Numbers in parentheses represent the number of individuals sharing the same haplotype. Haplotype codes are shown in Appendix 1. NEB 1 – 6, DIB 1 – 5, and DIS 1 and 2 have been described previously (Wilting et al. 2007a). ⁄Haplotype of the Neotype of N. diardi and the Batu Island specimen; # Haplotype of the specimens from Sarawak.
Mismatch distributions, generated for all three distinct popula- (CI95%: 60–640 kyr) and 396 kyr (CI95%: 160–700 kyr), respectively tions (N. nebulosa, N. diardi – Borneo and N. diardi – Sumatra), re- (Table 2). Using the calibration date derived from Johnson et al. vealed smooth, unimodal distributions (statistics in Table 2), (2006), we estimated a Bornean and Sumatran populations split indicating sudden population expansion (Rogers and Harpending, at approximately 117 kyr (Table 3). 1992). However, analyses which contained more than one popula- tion, Neofelis spp. or N. diardi, showed multimodal patterns 3.2. Microsatellite analysis (Fig. 2A). These multimodal patterns would generally be inter- preted as a stable or slowly declining population. In our case it is Composite genotypes from at least nine of the 12 felid-specific clearly a result of the inclusion of different distinct populations microsatellite loci could only be obtained for a subset of 21 in the same analysis. The different peaks can be explained as differ- clouded leopard samples (five mainland specimens, seven Bornean ences between the populations, as the separate analyses of each specimens and nine Sumatran specimens, including the neotype population showed unimodal patterns. These results are a good for N. diardi and one individual from the Batu Islands; see Appendix example of where the amalgamation of distinct populations biases 1). There was no significant linkage disequilibrium among loci, the result of the mismatch distribution analysis, which may lead to indicating their independent inheritance. The microsatellite AMO- incorrect conclusions about the stability of populations. Therefore, VA demonstrated that 37% of the genetic variability was attribut- we suggest that prior to applying a mismatch distribution analysis, able to variance among populations, and 63% was accounted for a check should be made to ensure that all individuals belong to a by differences within the three populations. Each of the three pair- single population. wise population comparisons showed highly significant population
Based on the mismatch distribution (Rogers, 1995), the approx- genetic differentiation (p < 0.0001) by pairwise RST’s (Table 1). imate divergence time (t = s/2l) of the Bornean and Sumatran pop- The STRUCTURE analysis yielded the following mean values ulations of N. diardi was estimated to be 331 thousand years [kyr] (across 10 runs) of posterior probabilities for the number of A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 321
Table 2 Analyses performed on clades of Neofelis.
Species Neofelis nebulosa N. diardi N. d. borneensis N. d. diardi Parameter Number of individuals N 58 31 15 16 Fragment length/ number of usable sites [nc]a 849/849 849/846 849/846 849/849 Diversity indices Number of haplotypes h 71367 Number of segregating sites S 61154 Transition/ transversion ratio 5/1 10/1 4/1 4/0 Nucleotide diversity p (SD) 0.00057 (0.00055) 0.0037 (0.00219) 0.00126 (0.00099) 0.00142 (0.00112) Haplotype diversity Hd (SD) 0.227 (0.073) 0.877 (0.032) 0.762 (0.096) 0.833 (0.072) Ratio R = h/N 0.121 0.419 0.4 0.375 Tajima’s test of selective neutrality Tajima’s D 1.826 0.438 1.033 0.18 c p (Dsimulated < Dobserved) [1000 simulations] 0.01 0.723 0.158 0.621 Mismatch distribution Average number of nucleotide differences j 0.339 3.131 1.067 1.275 Variance of j 0.658 4.856 0.621 0.672 s 3.00 5.795 1.234 1.469 Test of goodness-of-fit Sum of squared deviation (SSD) 0.002 0.048 0.024 0.025 d p (SSDsimulated P SSDobserved) 0.47 0.2 0.18 0.13 Harpending’s raggedness index 0.378 0.094 0.175 0.17 d p (Ragsimulated P Ragobserved) 0.62 0.33 0.13 0.07 Age of clade based on s [kyr] 803 1556 331 396
CI95% [kyr] 100 – 1090 27 – 2690 62 – 640 158 – 692 Sudden population expansion b b b FS 4.523 2.005 2.61 3.204
p (FS) 0.004 0.203 0.016 0.011 nc: nucleotides; SD: standard deviation; s (tau): units of mutational time; t = s/2l; mutation rate l = 2.2 10 9 per site and year (Kumar and Subramanian 2002); n.c.: not calculable, CI95%: confidence interval at a = 0.05; FS: Fu’s statistics (1000 simulations), kyr: 1000 years. a Uncertain positions (‘‘N’’) were removed prior to analyses. b FS-values are significant (95% level) at p < 0.02 (Fu 1997). c Null-hypothesis H0 is hnon-neutralityi. d H0 is hno fiti.
Fig. 2. Hierarchical model of the clouded leopard populations. (A) Mismatch distributions computed for the concatenated 849 bp of mitochondrial sequences. Solid line: observed distribution of pairwise differences; dashed line: expected distribution under the model of sudden demographic expansion. Numbers in parentheses represent the number of individuals analysed. (B) Genotypic assignment of the individual clouded leopards to 2–4 clusters (populations). Posterior probabilities for the number of populations, given as ln Pr(X|K) for K = 2–4, and admixture coefficients (q) are shown. ID codes are shown in Appendix 1, NDD5 = Neotype N. diardi, NDD9 from Batu Islands, NDB5 and NDB7 from Sarawak. NDD9 q = 0.72. Numbers in parentheses represent the number of individuals analysed in each cluster. 322 A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328
Table 3 Coalescent times based on net genetic distances.
Split Pantheraa/Neofelis N. nebulosa/N. diardi N. d. borneensis/N. d. diardi Parameter Substitution model used Tamura–Nei Tamura–Nei Tamura–Nei Gamma distribution parameter a 0.011 0.1 0.011 d (%) 29.972 8.748 0.550 SE (%) 9.255 2.563 0.300 Time (kyr) 6370b 1859 117
CI95% (kyr) 2436–10,300 769–2948 0–244
a Comprises of Panthera tigris, P. pardus, P. onca; d: net distance between clades, calculated by subtracting the mean intra-clade distances
(dX, dY) from the mean inter-clade distances (dXY) using the equation: d = dXY 1/2(dX + dY)(Nei 1975; Wilson et al. 1985; Nei 1987); CI95%: confidence interval (at 95%) = d ±2 SE. b Calibration date from Johnson et al. (2006); kyr: thousand years. Mutation rates were 29.972% per million years. populations (K): ln Pr(X|K)= 782 (SD = 0.77), 638 (SD = 0.26), formed a single common cluster. Both the neotype for N. diardi 587 (SD = 0.35), 567 (SD = 0.77), 598 (SD = 15.01), 612 and the sample from the Batu Islands clustered with the Sumatran (SD = 4.32), 635 (SD = 13.66), 651 (SD = 22.75), and 675 samples. (SD = 20.03), for K = 1–9, respectively. The probabilities were high- est for K = 3 and K = 4 (underlined). For all K > 4, posterior probabil- 3.3. Craniomandibular and dental analysis ities also varied largely among the ten runs, resulting in large standard deviations. Based on these results we designed a hierar- In PCA analysis, N. diardi males from Borneo and Sumatra chical model with an increasing K from 2 to 4 (Fig. 2B). We showed a distinct degree of separation (Fig. 4a), but for females assumed a separation of the two clouded leopard species there was no discernible separation of the populations on either N. nebulosa and N. diardi at K = 2. This assumption was supported PC1 or PC2 (Fig. 4b). In traditional classification analyses, all in- by the analysis and we observed high admixture coefficients of cluded specimens were assigned correctly to their respective taxa. q > 0.96 and q > 0.99 for N. nebulosa and N. diardi, respectively. At K = 3, not only N. nebulosa and N. diardi were separated, but also the two populations in Borneo and Sumatra, and the high admix- ture coefficients of q > 0.96 (in all but one run) indicated no to very little gene flow between groups. At K = 4, Sumatran clouded leop- ards were further separated in two subgroups, indicating a sub- structure below the proposed subspecies level. The lower admixture coefficients of q > 0.75 indicated the existence (albeit low) of gene flow between the structural units (populations) (Fig. 2B). The samples from the Batu Islands (NDD9) and the neo- type for N. diardi (NDD6) mingled with the Sumatran individuals with high admixture coefficients. The results of the factorial correspondence analysis yielded two factorial components, of which component 1 explained 68% of the variation among the clouded leopard populations and component 2 the remaining 32% (Fig. 3). Neofelis nebulosa and N. diardi were clearly distinct from each other, as were, to a lesser extent, Bornean and Sumatran clouded leopards. Each of the three populations
Fig. 4. Plots of the first two principal components from analyses of 64 cranioman- dibular and dental measurements and three angular variables. (a) Neofelis diardi males; (b) Neofelis diardi females. Symbols: B, specimens of Neofelis diardi from Borneo; S, specimens of Neofelis diardi from Sumatra. The arrows indicate type specimens of Neofelis diardi spp.: RMNH1981 (#; National Museum of Natural Fig. 3. Factorial correspondence analysis of genotype distributions of clouded History, Leiden), neotype of Neofelis diardi and holotype of Neofelis diardi diardi; leopard populations based on allele frequencies of 12 microsatellite loci. FC = Fac- BM.3.4.9.2 ($; Natural History Museum, London), holotype of Neofelis diardi torial Component. borneensis. A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 323
However, the more robust jack-knifed procedure implied that only quent and more prominent in the pelages of Bornean animals, one sample was identified with 100% accuracy, owing to the mor- but this did not separate all animals. The mean total pelage score phology of the subspecies being distinct but not markedly so. In for Bornean animals was 16.71 (N = 7; standard error (SE) = DA, N. diardi males proved non-significantly different albeit nearly 0.680) compared with 16.19 (N = 16; SE = 0.540) for Sumatran so (Wilks’ k = 0.032; F = 7.892; p = 0.0543), but nonetheless, a jack- clouded leopards; there was no significant difference in total pel- knifed classification analysis classified Bornean males with 100% age scores between the two islands (two-sampled t-test, accuracy, whereas four males from Sumatra were misclassified t = 0.564, N = 23, p = 0.579). Although in strict terms of these char- (60% correct). Females were also non-significantly different, albeit acters there was no clear separation between Bornean and Suma- nearly so (Wilks’ k = 0.014; F = 14.186; p = 0.0576). One of the Bor- tran clouded leopards, careful examination of the sample of nean females was misclassified (80% correct), and three of the pelages plus the camera-trap photographs suggested that there Sumatran females were also misclassified (63% correct). Accord- may be pelage differences, although these are not 100% diagnostic ingly, multivariate studies indicate that the Bornean and Sumatran for each population. populations of N. diardi are craniodentally distinguishable with high classification accuracy. Bivariate analyses on ratio variables 4. Discussion (see Christiansen, 2008) corroborated the above. Of 125 analysed craniomandibular and dental ratios, 11 were significantly different The consistent results of molecular (mtDNA and microsatellite (p < 0.05) between the populations of N. diardi from Borneo and loci), craniomandibular and dental data strongly endorse the previ- Sumatra (Table 4). ously suggested distinction of Bornean and Sumatran clouded leopards into two populations with separate evolutionary histo- 3.4. Pelage analysis ries. In the mtDNA tree topology and in the hierarchical model of the microsatellite genotypes the degree of separation between Bor- Only one mean individual pelage character score between Bor- nean and Sumatran clouded leopards should be regarded as the le- nean and Sumatran clouded leopards was significantly different vel of different subspecies. Accordingly, the Bornean clouded (Table 5). Spots within clouds showed a higher mean score in Bor- leopard should be recognised as subspecifically distinct from the nean (N = 16; mean = 2.75) than Sumatran (N = 19; mean = 2.26) Sumatran clouded leopard. The analysis of a single specimen from clouded leopard pelages (t = 2.219; p = 0.034) (other, non-signifi- the Batu Islands revealed no genetic traits specific to these islands. cant results not presented here). These spots do appear more fre- This indicates either past continuous gene flow between the Batu Islands and mainland Sumatra, or that clouded leopards had only Table 4 recently spread to these islands, when land bridges connected Bivariate comparisons of craniomandibular and dental ratios in Neofelis diardi (populations from Borneo and Sumatra); see Christiansen (2008) for a detailed them with mainland Sumatra until 10 kyr ago (Voris, 2000). description of characters. Underlined values are 0.100 P p P 0.050, and values in In contrast to the hierarchical models used in the molecular bold are p < 0.050. Abbreviations: AP, anteroposterior diameter; CBL, condylobasal analysis, the comparative approach used in the morphological skull length. studies yielded no systematic predictions as such. Inference of sub- Character species status to allopatric populations is always going to involve t p certain amounts of ambiguity. However, the populations from Bor- neo and Sumatra were clearly distinguishable in bivariate and mul- Nasal width at narial aperture/CBL (0.072) tivariate studies. The reported overlaps are acceptable within the Nasal width at narial aperture/snout width (0.003) traditionally advocated amount of overlapping in subspecies, the Postorbital constriction/postorbital processes (0.085) Palate length/CBL (0.027) so-called 75% rule (Mayr and Ashlock, 1991). Accordingly, based Width of pterygoid palate/CBL (0.094) on craniomandibular and dental analysis alone, the suggestion of Width of pterygoid palate/width of incisors (0.005) subspecies status to Bornean and Sumatran populations of N. diardi Width of pterygoid palate/width between C1 (0.003) appears justified. 3 Width of pterygoid palate/ width of palate at P (0.087) No significant difference was found between total pelage scores 4 Width of pterygoid palate/ width of palate at P (0.033) of Bornean and Sumatran clouded leopards, and only cloud spots Width of incisors/palate length (0.015) Mastoid width/CBL (0.048) showed a significant difference among individual pelage charac- P4 paracone length/P4 length (0.032) ters, with Bornean animals having a higher mean score, but the 4 P protocone AP/metastyle length (0.059) sample sizes were very small. Given that some overlap in pelage P4 protocone AP/P4 length (0.012) characters might be expected between subspecies according to 4 4 Width of P at protocone/ P length (0.063) the 75% rule (Mayr and Ashlock, 1991), the high degree of overlap P4 protoconid length/P4 length (0.016) observed here could be due to sampling bias alone. There is a sug- P protoconid height/P length (0.049) 4 4 gestion that Bornean clouded leopards may have larger, more angular cloud-like blotches with thicker black borders, and thicker neck and shoulder stripes, as well as more frequent, bolder spots. Table 5 However, many of the camera-trap photographs were derived from t-tests on mean scores for pelage characters of Neofelis diardi from Borneo and Sabah and these differences may have been local. Therefore, these Sumatra. Only cloud spots showed a significant difference, with Bornean animals potential differences need to be tested on much larger samples displaying more cloud spots on average than Sumatran animals. from throughout Borneo in order to provide sufficient data for Character tpN– Borneo N – Sumatra any potential discriminatory pelage characters. Cloud spots 2.219 0.034 16 19 Lightness 1.245 0.222 16 19 4.1. Evolutionary history of N. diardi Brightness 0.380 0.706 16 19 Tawny 0.672 0.506 16 19 Grey 0.269 0.789 16 19 The mismatch distribution analysis and the very low FS-values Nape stripes 0.163 0.872 7 17 revealed a rapid and recent population expansion for the proposed Shoulder pattern 1.470 0.155 8 17 subspecies of N. diardi. Although our molecular data did not allow Dorsal stripes 0.699 0.490 13 18 definite conclusions regarding both the origin and the direction of 324 A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328
island colonization, the higher FS-values for Sumatra provided a ulosa to colonize Sumatra, because the ‘‘savanna corridor’’ (if at all hint that the expansion in Sumatra may have been more recent. present; see Cannon et al., 2009) had a wider extension (up to In addition, most of the Bornean samples included in the molecular 150 km in width) between Borneo and Sumatra (Bird et al., 2005; analysis originated from the northern, Malaysian part of Borneo Heaney, 1991), and the contemporary Malacca river running be- and the only specimens from Kalimantan (Indonesian, southern tween the land masses of Peninsular Malaysia and Sumatra is sup- part of Borneo) included here yielded a previously unrecorded hap- posed to be discontinuous through the Straits of Malacca (Bird lotype (DIB6). Therefore, we might have underestimated the genet- et al., 2005, Heaney, 1991; Meijaard, 2003; Voris, 2000). Although ic diversity of the Bornean population. In this case the scenario seemingly contradictory, the observed geographical distribution of would be that Bornean clouded leopards populated Sumatra during clouded leopards may be explained by the close proximity of Pen- periods of low sea levels in the Pleistocene and were later sepa- insular Malaysia to Sumatra. The consequences of the Toba erup- rated from their source population by rising sea levels. For tion were more severe, indicated by thick ash layers (Rose and 100,000 years of the last 250,000 years (Voris, 2000), the sea level Chesner, 1987), in Peninsular Malaysia than in Borneo, and it can was at least 40 m below its present level, thereby exposing a land be assumed that this region was also emptied by the eruption. Con- bridge between Borneo and Sumatra. sequently, the refugia of N. nebulosa would have been located Based on the mtDNA data, we estimated that the two clouded much further north from the Toba eruption, potentially some- leopard subspecies became genetically separated in the Middle to where in northern Indochina, similar to those of other species Late Pleistocene, between 400 and 100 kyr depending on the meth- (Brandon-Jones, 1996; Luo et al., 2004). This hypothesis is sup- od applied. The use of mtDNA carries some restrictions in node res- ported by our molecular data (very low FS-value and nucleotide olution (Johnson et al., 2006), depending on locus use. However, diversity and unimodal mismatch distribution), which indicated Tajima’s D indicated that the concatenated dataset used in our that the mainland clouded leopard as well had gone through a se- study could be treated as a neutrally evolving unit. The discrepancy vere population bottleneck and only very recently showed a rapid in our estimates of divergence time between Bornean and Suma- population expansion. A recent expansion from this refugial retreat tran clouded leopards in our two models emphasize that the num- would explain why, in contrast to numerous other species in bers should be evaluated carefully. Nonetheless, even the more Southeast Asia (reviewed in Hughes et al., 2003 for birds, Woodruff recent estimate is similar or longer than estimated subspecies and Turner, 2009 for mammals), we could not detect any molecular divergence times in other pantherine cats (72–108 kyr for tigers: or morphological differences between clouded leopards north and Luo et al., 2004, 170–300 kyr for leopards: Uphyrkina et al., 2001). south of the known transition zones, i.e. the Isthmus of Kra or fur- Considering the confidence intervals around the divergence ther north where the peninsula joins the mainland. Thus, the arri- time estimates, the split of the two subspecies of N. diardi corre- val of N. nebulosa in Peninsular Malaysia might have occurred sponds roughly with the catastrophic ‘‘super-eruption’’ of the Toba within the last 10 kyr, after the land bridges between the continent volcano in Sumatra around 73.5 kyr (e.g. Rampino and Self, 1992). and Sumatra had submerged again (Voris, 2000). This was the second largest explosive eruption known in the Phan- In contrast, the prevailing southeasterly winds that blew during erozoic history (Ambrose, 1998) and an order of magnitude larger the time after the Toba eruption would have led to less severe conse- than any other known Quaternary volcanic eruption (Dawson, quences in Borneo, and, consistent with this, no Youngest Toba Tuffs 1992; Huff et al., 1992; Rose and Chesner, 1990). It is assumed that (YTT) have been found there (Pattan et al., 2001). Therefore, a larger the darkness caused by the dust injected in the stratosphere and population of N. diardi could have survived in the Bornean refugium the associated volcanic winter (Rampino and Ambrose, 2000; (indicated by the greater nucleotide diversity of N. diardi compared Rampino and Shelf, 1992, 1993) had a major impact on flora and to N. nebulosa, Table 2). The slightly wetter climate just prior to fauna in the Sunda Shelf, in particular in Sumatra. Large carnivores, and after the volcanic eruption, 74–47 kyr (van der Kaars and Dam, occurring in low population densities with large home-ranges, are 1995), likely resulted in an expansion of forests and would have facil- especially prone to extinction, even more so if they have restricted itated the geographical expansion of N. diardi to Sumatra. geographical distributions (e.g., Cardillo et al., 2004; Schmidt et al., This scenario begs the question as to why N. diardi did not con- 2009; Terborgh, 1974). Although the effects of the Toba eruption tinue its expansion to Peninsular Malaysia. A hypothesis of initial have been discussed controversially (see Louys, 2007; Oppenheimer, colonization of the peninsula by N. diardi and a subsequent 2002), and no large mammal species extinction has so far been replacement by N. nebulosa is testable only with fossil remains, linked to the eruption (Louys, 2007), post-catastrophe recoloniza- which are currently wanting. A larger sample from Peninsular tion events in Sumatra may have obscured local extinctions. Such Malaysia (only one specimen was included in this study, haplotype a situation is conceivable for the evolutionary history of the Sunda NEB3) would allow a search for introgression between N. diardi and clouded leopard, a species with low population densities and large N. nebulosa, and thus could reveal if the two species ever hybri- home-ranges (Hearn et al., 2008a, Mohamed et al., 2009, Wilting dised. So far, pelage (Kitchener et al., 2006), craniomandibular et al., 2006). and dental data (Christiansen, 2008) fail to provide support for A possible scenario is that Sumatra, emptied by the eruption, such hybridisation. was, as the vegetation recovered, recolonized by an expanding The similarity of habitats in Borneo and Sumatra, with mainly clouded leopard population surviving the catastrophic event in a evergreen rainforests, might explain the similarity of pelage pattern refugium. But what was the center of origin for the recoloniza- between Sumatran and Bornean clouded leopards (Allen et al., tion of Sumatra, Peninsular Malaysia or Borneo? Although Peninsu- 2010). N. nebulosa, which also lives in more open habitats such as lar Malaysia lies much closer to Sumatra than Borneo and a dry and deciduous forests and up to the foothills of the Himalayas recolonization from this area has been reported for example for (up to 3000 m), shows in contrast a distinct pelage pattern with the common palm civet Paradoxurus hermaphroditus (Patou et al., lighter and larger cloud shape markings (Kitchener et al., 2006). 2010), all recent analyses on clouded leopards show unanimously However, the preliminary observation of some pelage differences that Sumatran clouded leopards are much more closely related to between animals from Sabah and Sumatra need to be tested on a Bornean clouded leopards (both N. diardi) than to their continental wider geographical sample, but might indicate an example of Glog- relatives (N. nebulosa). Several reasons, for instance a more sea- er’s Rule (Gloger, 1833), where there is a greater degree of melanism sonal and drier habitat between the Peninsular Malaysia and in wetter environments. The craniodental differences between the Sumatra, or a large river running through the Straits of Malacca, subspecies observed in this study might be an indication that can be excluded as explanations for the failure of mainland N. neb- clouded leopards in Sumatra had to specialise in terms of their A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 325 ecological niche (e.g. prey choice), owing to sympatry with potential because clouded leopards are absent from this island, although they competitors such as tigers, Asian golden cats (Catopuma temminckii) were present in the Neolithic Java (Hemmer and von Koenigswald, and dholes (Cuon alpinus), while in Borneo other larger carnivores 1964). In 1827 Coenraad Jacob Temminck published a brief are absent (the Bornean bay cat Catopuma badia is smaller). description of Stamford Raffles’ Arimau Dahan, which he named F. macrocelis, and he correctly noted that it was present not only 4.2. Systematics of N. diardi on Sumatra, but also on Borneo (Temminck, 1827). Temminck did in fact discuss the identity of these two forms (1827: p. 103) Description and diagnosis for the species N. diardi follows Kitch- and was the first to conclude that his Felis macrocelis was not pres- ener et al. (2006) and Christiansen (2008) for pelage and osteoana- ent on Java, and that the two, accordingly, must be different spe- tomy, respectively. The vernacular name of a species is in cies (Temminck, 1827). Cuvier’s specimen was probably from phylogenetics and taxonomy often considered to be irrelevant, Sumatra instead, as has been suggested by Ellerman and Morri- but many species, in particular mammals and birds, are discussed son-Scott (1951), Corbet and Hill (1992), Kitchener et al. (2006), mainly by their vernacular names and not by their scientific equiv- Christiansen (2009), among others. Thus, Felis macrocelis Tem- alent. As such, a vernacular name for a big cat is not irrelevant, but minck is a junior synonym of Felis diardi G. Cuvier (ICZN, 2000: arti- the Code has no authority on assignments of vernacular names cle 23, in particular 23.3). Wilting et al. (2007a) suggested, that if (Article 1.3.5). For N. diardi different vernacular names have been two subspecies of N. diardi were formally recognized, the Sumatran suggested, such as Sundaland clouded leopard (Wilting et al., subspecies should be called N. d. sumatrensis. Since no holotype 2007a) and Diard’s clouded leopard (Christiansen, 2008), the latter was designated, this name is a nomen nudum and not available based on historical precedence (e.g., Audoin et al., 1823; Jardine, according to the Code (Article 15.1). This was amended in Wilting 1834; Ripley and Dana, 1858). However, we advocate formal adop- et al. (2007b). Christiansen (2009) recently designated a neotype tion of the name Sunda clouded leopard, as also used by the IUCN for N. diardi from Palembang, Sumatra (RMNH.1981). The subspe- Red List, because this is the most commonly used name for this cies to be given the same trinomen as the species name is fixed species in southeast Asia. Also, it refers accurately to the species’ by the type specimen (Article 47.1); as such, Neofelis diardi diardi origin in the Sunda region, and it enhances awareness by local peo- is hereby officially adopted for the Sumatran subspecies of N diardi, ple of the importance of this threatened species, which will in turn as fixed by the species’ neotype specimen. In accordance with the hopefully strengthen conservation efforts for this species and other Code (Article 16, specifically 16.4), the type specimen for the sub- Sundaic endemics. species N. d. diardi is hereby fixed as the neotype of the species- Two subspecies of N. diardi are formally recognised. taxon, Neofelis diardi, as RMNH1981. The molecular sequences of the mitochondrial DNA analysis of 4.2.1. Neofelis diardi diardi (Cuvier, 1823) the neotype specimen are available under the Genbank accession Distribution range: Sumatra and Batu Islands. numbers HM748837 (control region), HM748844 (Cyt-b) and The original name of Felis diardi is attributable to Cuvier (1823) HM748851 (ATPase-8) (mtDNA haplotype DIS3, and NDD6 in the based on a specimen allegedly from Java, but this is erroneous microsatellite analysis).
Fig. 5. Holotype of Neofelis diardi borneensis, adult female BM.3.4.9.2 (Natural History Museum, London; Sarawak, north-central Borneo); cranium in (a) lateral; (b) dorsal; (c) ventral view; (d) mandible in lateral view. Scale bar equals 5 cm. 326 A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328
Fig. 6. Pelage of the holotype of Neofelis diardi borneensis ssp. nov. (BM.3.4.9.2) (A) dorsal and (B) ventral.
4.2.2. Neofelis diardi borneensis ssp. nov the ICZN (Article 16.4, specifically 16.4.1, 16.4.2, and Recommen- Distribution range: Borneo. dation 16C - F). Our findings prompt a holotype designation of Etymology: Described after its origin in Borneo. the Bornean subspecies. Here we designate specimen BM.3.4.9.2, Diagnosis: Differs from nominal subspecies, N. d. diardi, in the a skull (Fig. 5) and a skin (Fig. 6) of an adult female, collected following craniomandibular and dental characters; greater width May 14th 1902 by Charles Hose at Baram, Sarawak, Borneo, housed across the nasal aperture and mastoid processes, and shorter pter- at the Natural History Museum in London. This specimen was se- ygoid palate relative to condylobasal skull length; pterygoid palate lected as a holotype, because no specimen in the database of narrow; shorter paracone length and narrower across the proto- molecular data included a skin, skull and mandible. However, cone relative to P4 length than in N. d. diardi; and longer and taller two specimens with molecular data also originate from Sarawak p4 protoconid relative to p4 length than N. d. diardi. Pelage diagno- (Appendix 1): mtDNA sequences (haplotype DIB1) for one of these sis is provisional; more frequent and bolder, cloud spots, larger, two specimens are available under the Genbank accession num- more angular cloud-like blotches than in N. d. diardi, which partic- bers EF440645 (control region), EF437579 (Cyt-b) and EF437572 ularly in shoulder region are intermediate in size between those of (ATPase-8). In the microsatellite analysis the two specimens carry- N. d. diardi and N. nebulosa. Cloud-like blotches tend to have thicker ing genotypes NDB5 and NDB7 originated from Sarawak. The tri- black borders, and neck and shoulder stripes tend to be thicker nominal name of Neofelis diardi borneensis is hereby officially than in N. d. diardi. Ground color tends towards grey with yellow- recognised for the Bornean subspecies according to Article 15.1 ish tinge, whereas Sumatran animals have a tendency towards and 16.4 of the ICZN. tawny too. Within the analysed 849 bp of mitochondrial DNA, four fixed nucleotide differences distinguish it from N. d. diardi. At posi- 5. Conclusion tions 8788 (ATPase-8), 15241 and 15271 (Cyt-b) and 16,957 (con- trol region) [referring to the domestic cat mitochondrial genome, Following molecular and morphological results presented here, GenBank accession number U20753] clouded leopards from Bor- two subspecies of Neofelis diardi are recognised, namely Neofelis neo carry an [C], [T], [T] and [C] respectively, whereas N. d. diardi diardi diardi from Sumatra and the Batu Islands, and N. d. borneensis has [T], [C], [C], and [T] respectively. ssp. nov. from Borneo. Both subspecies should be protected and Holotype: BM.3.4.9.2 female skin and skull collected 14 May managed separately to preserve the integrity of their gene pools 1902 by Charles Hose at Baram, Sarawak, Borneo (see Appendix and their morphological distinctiveness. The IUCN already recog- 2 for a description of the holotype). nizes these subspecies based on the first suggestions by Wilting Referred specimens: ZMB 56310 female skull and skeleton col- et al. (2007a,b) and lists both as Endangered on the current IUCN lected 1907 by Pagel in Lahad Datu, Sabah, Borneo. The molecular Red List (Hearn et al., 2008b – N. d. borneensis, Sunarto et al., sequences of the mitochondrial DNA analysis of the paratype spec- 2008 – N. d. diardi). This classification and the recognition of differ- imen are available under the Genbank accession numbers ent subspecies is of utmost importance for conservation and man- EF440645 (control region), EF437579 (Cyt-b) and EF437572 (ATP- agement purposes, as these different conservation units represent ase-8) (haplotype DIB1 and NDB6 in the microsatellite analysis). an important component of the evolutionary legacy of this threa- Other referred specimens are listed in Appendix 1 (specimens in- tened pantherine felid species. cluded in the molecular analysis), Appendix 3 (craniodental analy- sis) and Appendix 4 (pelage analysis). Wilting et al. (2007a) conditionally proposed the name Neofelis Acknowledgments diardi borneensis for the Bornean subspecies, but this is a nomen nu- dum, because there was no formal description including designa- AW and JF thank all institutions and persons listed in Appendix tion of a holotype and therefore it was not in accordance with 1 that supplied the biological specimens this work is based upon. A. Wilting et al. / Molecular Phylogenetics and Evolution 58 (2011) 317–328 327
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