Evolutionary history and conservation significance of

Journal of Zoology. Print ISSN 0952-8369

Evolutionary history and conservation significance of the Javan leopard Panthera pardus melas ~ aloza1, S. Kramer-Schadt1, A. Wilting1, R. Patel1, H. Pfestorf1,2, C. Kern3, K. Sultan4, A. Ario5, F. Pen 1 1 1,6 V. Radchuk , D. W. Foerster & J. Fickel 1 Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 2 Institute for Biochemistry and Biology, Plant Ecology and Nature Conservation, Potsdam University, Potsdam, Germany 3 Tierpark Berlin, Berlin, Germany 4 Taman Safari Indonesia, Bogor, West Java, Indonesia 5 Conservation International Indonesia, Jakarta Selatan, Indonesia 6 Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany

Keywords biogeography; evolutionary history; Felidae; Southeast Asia; Toba eruption; One Plan Approach; Pleistocene; Javan leopard. Correspondence Andreas Wilting, Leibniz Institute for Zoo and Wildlife Research, Junior Research Group: Biodiversity and Biogeography of Southeast Asia, Alfred-Kowalke-Str. 17, D-10315 Berlin. Tel: +49 (0) 30 5168 619; Fax: +49 (0) 30 5128 104 Email: [email protected] Editor: Andrew Kitchener Received 14 August 2015; revised 3 March 2016; accepted 9 March 2016 doi:10.1111/jzo.12348

Abstract The leopard Panthera pardus is widely distributed across Africa and Asia; however, there is a gap in its natural distribution in Southeast Asia, where it occurs on the mainland and on Java but not on the interjacent island of Sumatra. Several scenarios have been proposed to explain this distribution gap. Here, we complemented an existing dataset of 68 leopard mtDNA sequences from Africa and Asia with mtDNA sequences (NADH5 + ctrl, 724 bp) from 19 Javan leopards, and hindcasted leopard distribution to the Pleistocene to gain further insights into the evolutionary history of the Javan leopard. Our data confirmed that Javan leopards are evolutionarily distinct from other Asian leopards, and that they have been present on Java since the Middle Pleistocene. Species distribution projections suggest that Java was likely colonized via a Malaya-Java land bridge that by-passed Sumatra, as suitable conditions for leopards during Pleistocene glacial periods were restricted to northern and western Sumatra. As fossil evidence supports the presence of leopards on Sumatra at the beginning of the Late Pleistocene, our projections are consistent with a scenario involving the extinction of leopards on Sumatra as a consequence of the Toba super volcanic eruption (~74 kya). The impact of this eruption was minor on Java, suggesting that leopards managed to survive here. Currently, only a few hundred leopards still live in the wild and only about 50 are managed in captivity. Therefore, this unique and distinctive subspecies requires urgent, concerted conservation efforts, integrating in situ and ex situ conservation management activities in a One Plan Approach to species conservation management.

Introduction For more than 150 years, the evolutionary history of the Southeast Asian fauna has fascinated scientists because of the unique geological and geographical history of the Sunda Shelf. Fluctuating sea levels alternatingly exposed and submerged large parts of the shelf, thereby creating periodical land bridges between the islands and mainland Southeast Asia (Fig. 1a) (Voris, 2000). The existence of such land bridges led several authors to conclude that their presence during the Late Pliocene and Pleistocene was sufficient to explain the current distribution of taxa on the Sunda Shelf (e.g. Heaney, 1986). The application of molecular tools, however, revealed a deep history of vicariant evolution [e.g. Brandon-Jones (1996) for Asian Colobinae; Gorog, Sinaga & Engstrom (2004) for rodents; Patou et al. (2010) for common palm civets Journal of Zoology (2016) – ª 2016 The Zoological Society of London

Paradoxurus hermaphroditus; Wilting et al. (2007, 2011) for clouded leopards Neofelis spp.]. Further, a surprisingly close relationship exists between the mammal faunas of Mentawai, a chain of small islands off the Sumatran west coast, and Borneo, with a discontinuous distribution for several species due to their absence on Sumatra (Wilting et al., 2012). Wallace (1876) already recognized that several species occurred on Java and mainland Southeast Asia, but not on Sumatra. This odd distribution pattern is generally explained by climatic similarities between the more seasonal environments in Indochina, India and Java versus the more tropical Sundaic region of Borneo, Sumatra and the Thai/Malay Peninsula (e.g. Meijaard, 2004). The three most prominent theories explaining how species may have crossed the Sundaic region to reach Java are: (1) a Plio- and Pleistocene Malaya-Java land bridge which bypassed Sumatra via the Riau-Lingga Archipelago and the 1

Conservation significance of Javan leopards

A. Wilting et al.

Figure 1 (a) Map of the exposed Sunda Shelf during the Last Glacial Maximum (LGM) ~22 kya. Sea levels were 116 m below present day level. The map is based on data from Voris, 2000 and on the ETOPO1 digital elevation model (Amante & Eakins, 2009). (b–f) Projected South and Southeast Asian leopard distribution for (b) the Middle Pleistocene (~600 kya) using the LGM projections; (c) Late Pleistocene (~74 kya) using the Mid-Holocene projections. The thickness of the Young Toba Tuffs (YTT) were superimposed on the projection to indicate the severity of impact of the Toba super volcanic eruption. In (b) we added the pre-Toba fossil site Lida Ajer (80 kya or 128–118 kya, see Louys, 2012) in Sumatra, and in (f) we indicated the species distribution according to the IUCN Red List of Threatened Species as used for the modelling with a hatched pattern while areas outside of the natural (pre-human) distribution range were shaded in light blue. Black arrows in (b) and (d) indicate inferred dispersal directions of leopards.

islands Bangka and Belitung (Van Bemmelen, 1970; Meijaard, 2004); (2) species occurred throughout large parts of the Sunda Shelf but went extinct on Sumatra due to unstable environmental conditions and high extinction risk, for example, as a consequence of the Toba super volcanic eruption ~74 kya (Wilting et al., 2012), and (3) Javan populations/species are 2

allochthonous, having been introduced by Indian settlers or traders (Bergmans & van Bree, 1986). The leopard Panthera pardus is the only pantherine cat still roaming on Java, after the Sunda clouded leopard Neofelis diardi went extinct during the Holocene (Hemmer, 1976) and the Javan tiger Panthera tigris sondaica in the 1970s Journal of Zoology (2016) – ª 2016 The Zoological Society of London

A. Wilting et al.

(Seidensticker, 1987). Subject to the same anthropogenic pressures as the tiger, the leopard subspecies on Java, P. pardus melas, has undergone significant population decline and is listed as Critically Endangered (Ario, Sunarto & Sanderson, 2008) on The IUCN Red List of Threatened Species. Morphological data [pelage pattern (Hemmer, 1967); craniodental data (Meijaard, 2004) and first molecular data (Miththapala, Seidensticker & O’Brien, 1996; Uphyrkina et al., 2001)] showed that Javan leopards are distinct from mainland Asian leopards, thereby corroborating the subspecific status of Javan leopards and their indigenous occurrence on Java. However, the limited sampling of Javan leopards in previous molecular studies (based on the same two individuals kept in European zoos: Miththapala et al., 1996; Uphyrkina et al., 2001) necessitated further sampling to gain a better understanding of the evolutionary history of Javan leopards. The aim of this study was to reassess the evolutionary history of Javan leopards by adding genetic samples to an existing dataset and to deduce its biogeographical history by projecting potential leopard distribution throughout the Late Pleistocene. We further compiled information about the current in situ and ex situ conservation efforts to provide a broader perspective into the conservation needs of this highly threatened subspecies.

Materials and methods Samples We used 16 archival samples (epithelial tissue from skulls or skins, or maxillo-turbinal bones), two faecal samples (from zoo animals), and one tissue sample (pathological reference sample collection of the IZW) of the Javan leopard (Table 1). DNA was extracted from archival samples [minced epithelia (30– 50 mg) and fragmented turbinates (20–40 mg)] using a phenolchloroform extraction protocol (Wisely et al., 2004) with modifications (Wilting et al., 2011). In case the extraction failed, we repeated the extraction procedure using the Qiagen DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) with an overnight lysis and 15 min incubation at 37°C during the elution. Molecular work on museum samples was always carried out in isolated facilities dedicated to working with archival material. DNA extractions from faecal samples were carried out using the First DNA-all tissue kit (GEN-IAL, Troisdorf, Germany) in an isolated DNA extraction hood dedicated to the work with faecal samples. Extractions of fresh tissue samples were carried out using the Qiagen DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) in a separate hood dedicated to fresh tissue DNA extraction. For all sample types, extraction negatives were always treated like their corresponding samples. They were, as well as PCR negative controls, always included in the PCR set-ups.

Molecular work We amplified segments of the mitochondrial NADH5 gene (611 bp) and the control region (ctrl; 113 bp) that were also used in previous studies (Uphyrkina et al., 2001). As archival Journal of Zoology (2016) – ª 2016 The Zoological Society of London


and faecal samples usually contain degraded DNA (i.e. short DNA fragments), we divided the targeted portion of NADH5 into four short, overlapping fragments using F/RL2 and RL4 from Uphyrkina et al. (2001) as a first forward and fourth reverse primer, respectively (PPA-1R: 50 -TTGTCCGGAGGA AACGAATA-30 , 197 bp PPA-2F: 50 -CTCTTACGCCTTCACC ATCAG-30 ; PPA-2R: 50 -GGGTCTGTGTGTATGTATCATATT GAG-30 , 215 bp PPA-3F: 50 -TTCATCCCTGTAGCGCTTTT30 ; PPA-3R: 50 -CGTCTGCTCGACCATATCAT-30 , 226 bp; PPA-4F: 50 -CGGATGATGATATGGTCGAG-30 164 bp). The portion of the ctrl was amplified using a single primer pair (Uphyrkina et al., 2001). PCR reactions and sequencing were carried out as previously described (Wilting et al., 2011), with at least two replicates of PCR and sequencing reactions for archival and faecal samples. Sequences from the two mtDNA loci were concatenated (NADH5 + ctrl: 724 bp) and aligned with previously published sequences (Uphyrkina et al., 2001; GenBank accession numbers AY035227-92). The resulting alignment consisted of 87 leopard mtDNA sequences (Java n = 21, mainland Asia n = 50, Africa n = 15 and Arabia n = 1), and included a total of 37 distinct haplotypes (Appendix S1).

Phylogenetic and phylogeographic analyses PartitionFinder v.1.1.0 (Lanfear et al., 2012) was used to find the best substitution model and partition scheme for the dataset using the BIC criterion (HKY+I, no subdivision of data). Bayesian inference (BI), maximum-likelihood (ML) and neighbour-joining (NJ) methods were used for phylogeny reconstruction. The NJ analysis was conducted using MEGA v.6.0 (Tamura et al., 2013) with 1000 bootstrap replicates. The ML trees were computed using RAxML (Stamatakis, 2014) with 1000 bootstrap replicates. BI estimates of divergence times and extended Bayesian skyline plots (EBSP) to visualize the historical demography of leopards in different geographical regions (Africa+Arabia, Asia+SriLanka, Java, Sri Lanka) were carried out using BEAST v.1.8.1 (Drummond et al., 2012;). To estimate divergence times, we followed a two-step approach. First, as no calibration age priors were available, the molecular clock rate was estimated using outgroup taxa of the same genus (Panthera leo NC_018053 and Panthera onca NC_022842). A Yule Speciation process was used for species level phylogeny and an uncorrelated lognormal relaxed clock model was implemented to allow for rate heterogeneity among lineages. Tree priors for P. leo (2.51 ma) and P. onca (3.11 ma) were set as normally distributed priors (Davis, Li & Murphy, 2010) and two independent analyses were conducted using Markov Chain Monte Carlo (MCMC) chain lengths of 10 million iterations, logging every 1000th tree. LogCombiner v.1.8.1 (implemented in BEAST v.1.8.1) was used to combine logs from the independent runs. We estimated a molecular clock rate of 0.0206 0.0037 (St.Dev.) per site per Myr, which is consistent with those estimated from other studies on pantherine felids (tigers: 0.0229 per site per Myr, Luo et al., 2004; leopards: 0.0142–0.024 per site per Myr, Uphyrkina et al., 2001). In a second step, we used this molecular clock rate to calculate the age of internal nodes; for this, the root model height prior 3

4

PPA-23 PPA-27

PPA-28

PPA-29

PPA-MEL2 3328

912

911F

PPA-17

ZMB14842

PPA-22

PPA-16

ZMB7710

156284

PPA-13

15739

PPA-21

PPA-12

15740

PPA-MEL1

PPA-11

SMNS18948

PPA-20

PPA-8

SMNS18949

ZMB1220

PPA-7

SMNS31917

PPA-18

PPA-5

SMNS31915

ZMB17498

Sample No

Museum or Zoo ID

MEL1

MEL1

MEL5 MEL1

MEL3

MEL1

MEL1

MEL1

MEL1

MEL2

MEL1

MEL1

MEL1

MEL1

MEL1

MEL1

mtDNA Haplotype

AY035259

AY035259

JN811046 AY035259

JN811044

AY035259

AY035259

AY035259

AY035259

JN811043

AY035259

AY035259

AY035259

AY035259

AY035259

AY035259

NADH5

Table 1 Javan leopard samples included in this study

AY035292

AY035292

AY035292 AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

AY035292

ctrl

juv.

M juv.

M

F

F

F

F juv.

M

M

F juv.

M

Sex

Java

Java

Java

Java

Java

Java East-Java

Nasal bonesa

Scraped tissue from skulla

Nasal bonesa


Hidea

Scraped tissue from skulla Scraped tissue from skulla

Java Amboine, Molucca treated as Java Java

Java

Nasal bonesa

Scraped tissue from skeletona

Pelaboean Ratoe, Java

Scraped tissue from skeletona

Faeces Scraped tissue from skeletona

Java

Blood

Hidea

Preanger Mountains, Java Java

Java



Locale of origin

Source

W

W

Captive W

W

Captive

W

W

W

W

W

W

W

W

W

Birth status

1877

1889

2008 1877

1909

1891

1887

1843

1859

1873

1878

1869

Year

€rike, Dr. Doris Mo €r Staatliches Museum fu Naturkunde Stuttgart €rike, Staatliches Dr. Doris Mo Museum f€ ur Naturkunde Stuttgart €rike, Staatliches Dr. Doris Mo €r Naturkunde Museum fu Stuttgart €rike, Staatliches Dr. Doris Mo €r Naturkunde Museum fu Stuttgart Forschungsinstitut & Naturmuseum Senckenberg, Frankfurt Forschungsinstitut & Naturmuseum Senckenberg, Frankfurt Dr. Frieder Mayer, Naturkundemuseum Berlin Dr. Frieder Mayer, Naturkundemuseum Berlin Dr. Frieder Mayer, Naturkundemuseum Berlin Dr. Frieder Mayer, Naturkundemuseum Berlin Tierpark Berlin, Pathological collection Institute for Zoo and Wildlife Research Kristofer M. Helgen, National Museum of Natural History, Smithsonian Institution Zoological Garden Berlin Dr. Georges Lenglet, Royal Belgian Institute of Natural Science, Brussels Dr. Georges Lenglet, Royal Belgian Institute of Natural Science, Brussels Dr. Georges Lenglet, Royal Belgian Institute of Natural Science, Brussels

Origin contact

Conservation significance of Javan leopards A. Wilting et al.

Journal of Zoology (2016) – ª 2016 The Zoological Society of London

was determined from the first step and was found to be normally distributed around 0.89 (SD 0.165). Coalescent Constant Size was selected as tree prior in this step, as it concerned intraspecific variation. Two independent runs were conducted with MCMC chain lengths of 10 million iterations, logging every 1000th tree and independent runs were combined using LogCombiner v.1.8.1. TreeAnnotator v.1.8.1 (implemented in BEAST v.1.8.1) was used to generate a maximum-clade-credibility tree discarding 10% of the logged trees as burn-in. For the construction of EBSP, the time to most recent ancestor (see Table 2, Fig. 2) was used for each population as tree root height prior with a normal distribution. Each run was conducted for 10 million generations and evaluated using Tracer v1.6 (implemented in BEAST v.1.8.1). Results were plotted using the R package ggplot2 (Wickham, 2009). To visualize the relationships among haplotypes, we generated a median-joining (MJ) network using NETWORK V.4.6.1.3 (Bandelt, Forster & R€ ohl, 1999). Nucleotide (p) and haplotype (h) diversities were calculated in DnaSP v.5.10.01 (Librado & Rozas, 2009). We then examined the partitioning of molecular variance using analysis of molecular variance (AMOVA), implemented in ARLEQUIN v.3.5.1.3 (Excoffier, Laval & Schneider, 2005) examining two scenarios: (1) Javan leopards form a common clade with other Asian leopards (reflecting a more recent split of Javan leopards) and (2) Javan leopards are separated from both Asian and African leopards (reflecting a deeper divergence of Javan leopards). We also examined mtDNA sequence differentiation among populations using pairwise FST and tested for significance by permutating sequences among populations, also implemented in ARLEQUIN.

Java Faeces

Projection of Pleistocene leopard distribution

AY035292 AY035259 Archival samples.

MEL1 PPA-24 PPA-MEL2

We used the current distribution range shapefile of leopards provided by the IUCN/SSC Red List of Threatened Species (Henschel et al., 2008) in the region east of 73–130° and between 22° north to 15° south to ensure that the species distribution modelling is linked to the climatic conditions in South- and Southeast Asia. Furthermore, the Oceanian and Australian zoogeographical realms were excluded from the study area (Holt et al., 2013). The obtained shapefile was converted into raster grids with a resolution of 2.5 arc min. Of these raster cells, 10% (5190 occurrences) were randomly selected to serve as occurrences for model-fitting procedures, and pseudo-absences (n = 5200) were then sampled from the whole remaining study area. We used the bioclimatic variables provided by worldclim.org as environmental predictors (Hijmans et al., 2005) to fit models for the current conditions (interpolations of observed data, representative of 1950–2000). To avoid multicollinearity, only environmental predictors with |r| < 0.7 (Pearson’s product-moment correlation) were retained for model building, resulting in five predictors used (Max Temperature of Warmest Month, Min Temperature of Coldest Month, Temperature Annual Range, Precipitation of Driest Month, Precipitation of Wettest Quarter). Models were built using the

a

PPA-31 911

MEL4

JN811045

AY035292

M

Java Scraped tissue from skeletona

W

Java Scraped tissue from skeletona

Captive

2008

Dr. Georges Lenglet, Royal Belgian Institute of Natural Science, Brussels Dr. Georges Lenglet, Royal Belgian Institute of Natural Science, Brussels Tierpark Berlin 1877

Year Locale of origin Source

juv. AY035292 AY035259

NADH5

PPA-30 911E

mtDNA Haplotype Sample No

MEL1

ctrl

Sex

Birth status


Museum or Zoo ID

Table 1 Continued.

W

Origin contact

A. Wilting et al.


5


A. Wilting et al.

Figure 2 Phylogenetic relationships inferred from 724 bp concatenated mitochondrial sequences (NADH5 + control region). Trees for each of the three methods (BI/ML/NJ) had similar topologies. Roman numbers indicate nodes for which at least one method had a support of >80% (MJ/NJ) or >0.95 (BI). Support values and estimated divergence dates for these nodes are provided in Table 2. For the most ancient splits, support values are indicated in the figure (below branches). Haplotype codes are shown in Table 1 and Appendix S1. All haplotypes were from Uphyrkina et al. (2001), except MEL-2 – MEL-5 which are from this study. Bars next to the tree are colour-coded according to the origin of samples: green for Africa and Arabia, shades of blue for Continental Asia, Java and Sri Lanka (SL).

biomod2 package (Thuiller et al., 2009), and were fitted using maximum entropy (MAXENT) and generalized boosted regression (GBM) algorithms. To account for stochasticity, we generated three sets of pseudo-absences and, prior to model runs, split the data to provide a basis for cross-validation and evaluation of model outcomes (20% of the data kept for evaluation). Models were evaluated via receiver operating characteristic (ROC) and true skill statistic (TSS, e.g. Allouche, Tsoar & Kadmon, 2006), with three runs of crossvalidation. We then built one single ensemble model by averaging the probabilities predicted from 18 models. These 6

models were based on three random presence sets fitted with the two algorithms (MAXENT and GBM) and with three cross-validation runs each. To project potential past leopard distributions, we used environmental data for the Mid-Holocene (about 6000 ya; three global climate models (GCM): GCM4, and MIROCESM, MPI-ESM-P; http://www.worldclim.org/paleo-climate) and the Last Glacial Maximum (LGM; about 22 000 ya; the same three GCMs as used for Mid-Holocene). To account for uncertainty in the GCMs, one ensemble model was built across the predictions of the three GCMs and two modelling Journal of Zoology (2016) – ª 2016 The Zoological Society of London

A. Wilting et al.


algorithms (MAXENT and GBM) for each past time period. The ensemble model was built by averaging the probabilities obtained with each model, analogously to the way described above. To depict the potential negative influence of the Toba super volcano eruption (~74 kya) on the distribution of leopards, we digitized a map showing the thickness of Youngest Toba Tuff (YTT) in the study region (Costa et al., 2014). Because climatic conditions at the outbreak of Toba were similar to the conditions in the Mid-Holocene (Brathauer & Abelmann, 1999), we superimposed our projections for this period with the modelled ash cloud scenario. To hindcast leopard distribution prior to the eruption, we used the projections of the LGM distribution, considering similarities of the climatic conditions in the Middle Pleistocene (600 kya) and the LGM (Brathauer & Abelmann, 1999). All statistical analyses were carried out using R v.3.2.0 (R Core Development Team, 2015). For shapefile and raster manipulations within R, we used the packages maptools (Bivand & Lewin-Koh, 2015), rgdal (Bivand, Keitt & Rowlingson, 2015), and raster (Hijmans & van Etten, 2012).

Results We added 19 mtDNA sequences for P. pardus melas, substantially increasing the sample size for this subspecies from the previous available data (two sequences, (Uphyrkina et al., 2001); Appendix S1). Fifteen of them, including the one labelled as being from Amboina [=Ambon], Moluccas, shared the known haplotype MEL1 (AY035292, Uphyrkina et al., 2001). As the Moluccas are outside of the known distributional range, this specimen was likely mislabelled. The four remainTable 2 Estimated divergence dates with their 95% confidence intervals (CI) and bootstrap supports of phylogenetic tree nodes which had a support of >80% (MJ/NJ) or >0.95 (BI) (see Fig. 2). Phylogenetic relationships were inferred from mtDNA haplotypes from the concatenated 724 bp mitochondrial sequences Node

Time (kya)

I 932 Asia II 622 III 416 Java IV 126 Mainland Asia V 199 VI 69 VII 45 Sri Lanka VIII 70 Mainland Asia IX 37 Africa X 270 XI 174 XII 440 a

95% CI (kyr)

BI/ML/NJ

724–1152

1/67/97

393–886 256–629 41–247

0.93/80/85 0.96/a/a 1/88/91

75–375 6–188 4–115

0.97/a/a 0.95/70/a 1/74/a

12–165

1/77/a

12–115

1/a/90

118–467 82–292 265–651

1/78/74 1/a/a 0.99/83/74

Nodes which had no support >50% (MJ/NJ) or >0.5 (BI).


ing samples had new haplotypes (MEL2 – MEL5; Genbank acc. JN811043 - JN811046). MtDNA nucleotide diversity (p) among Javan leopards was very low compared with African or other (non-Javan) Asian leopards (Table 3). AMOVAs with Javan leopards and mainland Asian leopards combined (reflecting a more recent split of Javan leopards from Asian mainland leopards) portioned less variance among groups than the AMOVA that considered Javan leopards separately (reflecting a deeper divergence of Javan leopards; Table 3). For both scenarios, population pairwise FST values were high (range 0.46– 0.7) and highly significant (P < 0.0001; Table 3). BI, ML and NJ generated essentially congruent phylogenetic tree topologies that strongly supported the monophyletic status of P. pardus melas (BI 0.93; ML 80%; NJ 85%; Fig. 2) as a sister clade to the other Asian leopards. The MJ network further supported the distinct position of Javan leopards, as this subspecies was separated from its Asian relatives by at least eight mutations (Fig. 3a). We estimated that Asian and African leopards diverged approximately 932 kya (CI95: 724–1152 kyr) with a subsequent split of Javan leopards from the other Asian leopards around 622 kya (CI95: 393–885 kyr). The EBSPs suggested a larger stable population for Asian leopards (excluding Java; Fig. 3b) and a small population for Javan leopards (Fig. 3b). The recent population expansions indicated by the EBSPs for Javan and Sri Lankan leopards has to be viewed cautiously as it may be an artefact caused by the low number of sequences analysed (see Grant, 2015). The Pleistocene distribution models projected suitable leopard habitat on the exposed Malaya-Java land bridge during the Middle Pleistocene (Fig. 1b) when climatic conditions and sea levels were more similar to the last LGM (Brathauer & Abelmann, 1999). Our projections estimated the presence of potential suitable leopard habitat in northern and western Sumatra during glacial periods. The reconstruction of the Toba ash cloud based on the YTT showed that the impact of the Toba eruption was most severe in northern Sumatra and Peninsular Malaysia (Fig. 1c).

Discussion Evolutionary history In contrast to Sunda tigers, which are closely related to their continental relatives (Wilting et al., 2015), our molecular data are consistent with earlier studies (Uphyrkina et al., 2001; Meijaard, 2004) and confirmed that Javan leopards are clearly distinct from all other Asian leopards, revealing a deep history of vicariant evolution. We have dispelled doubts regarding the natural origin of leopards on Java (Bergmans & van Bree, 1986), as Javan leopards diverged from other Asian leopards approximately 600 kya. Our study therefore supports the subspecific classification of Javan leopards as P. pardus melas. Javan leopards show a phylogenetic pattern that is distinct from that of Sri Lankan leopards, which are phylogenetically much younger and nested within the mainland Asia clade, suggesting Late Pleistocene/Holocene immigration to Sri Lanka. For Javan leopards, the prolonged period of low sea levels 7


A. Wilting et al.

Table 3 Analyses performed on clades of Panthera pardus Group Parameter NADH5 + ctrl Number of individuals (N) Fragment length (nc) Diversity indices Number of haplotypes (h) Number of segregating sites (S) Transition/transversion ratio Nucleotide diversity p (SD) Haplotype diversity (Hd) (SD) Ratio R =h/N a

PPAa All

PPA Africa

PPA Asia

PPA Java

PPA Sri Lanka

87 724

15 724

50 724

21 724

10 724

37 66 63/6 0.0155 (0.0079) 0. 9341 (0.0146) 0.425

12 33 30/4 0.0152 (0.0082) 0.9619 (0.0399) 0.8

19 30 29/1 0.0070 (0.0038) 0.9045 (0.0225) 0.38

5 5 5/0 0.0007 (0.0007) 0.3524 (0.1314) 0.238

3 2 2/0 0.0008 (0.0008) 0.3778 (0.1813) 0.3

PPA, Panthera pardus.

around 600 kya (Brathauer & Abelmann, 1999) would have permitted migration from mainland Asia to Java via a MalayaJava land bridge, by-passing Sumatra and Borneo (Fig. 1b; Van Bemmelen, 1970; Meijaard, 2004). The drier and more open climate may have further facilitated the southward population expansion of leopards, a species well adapted to open habitats such as savannahs and deciduous forests (Santiapillai & Ramono, 1992; Meijaard, 2004; see also projected distribution during the LGM, Fig. 1b). The lack of leopard fossils from Borneo is consistent with our hypothesis of leopards having by-passed this island. However, the general absence of any fossil remains from the southern part of Borneo hampers final conclusions about the potential presence of leopards there, which according to our distribution projections may have provided suitable leopard habitat during glacial periods. The presence of leopard fossils from Sumatra has been discussed controversially. Some authors state that there is no fossil evidence for leopards on Sumatra (De Vos, 1983; Meijaard, 2004), which would provide even stronger support for the Malaya-Java land bridge (Meijaard, 2004), while others report specimens from the Lida Ajer cave (West Sumatra; Fig. 1; 80 kya or 128–118 kya, see Louys, 2012) as coming from leopards (Tougard, 2001). This controversy most likely arose because the original paper describing the Dubois collection from the Lida Ajer cave (De Vos, 1983) did not list P. pardus specimens, whereas in a later paper de Vos and his co-workers included ‘a few’ leopard specimens for Lida Ajer (Long, De Vos & Ciochon, 1996). De Vos confirmed that in the 1930s or 1940s L. D. Brongersma had assigned Sumatran fossil specimens to both Panthera species (P. pardus and P. tigris), but that these findings were never published and only became accessible to de Vos after his initial description of the Dubois collection in 1983 (J. de Vos, pers. comm.). Our distribution projections for the Pleistocene support a potential presence of leopards on Sumatra, but such a Sumatran presence would then have been independent of the colonization of Java and does not contradict earlier suggestions that leopards by-passed Sumatra while expanding their range to Java (Van Bemmelen, 1970; Meijaard, 2004). The possible former presence of leopards in at least some parts of Sumatra during the Pleistocene raises the question as

8

to why leopards went extinct there. A potential reason is the low prey biomass in tropical evergreen forests (compared with deciduous forests and tall grasslands), a forest type which expanded on Sumatra during interglacials (Meijaard, 2004). In the tropical evergreen forests of Peninsular Malaysia, however, all three larger pantherine cats (tigers, leopards and clouded leopards Neofelis nebulosa) occur sympatrically and in west African rainforests leopards are known to prey on smaller prey species compared to savannah leopards (Jenny & Zuberb€ uhler, 2005). The projections of the potential leopard distribution suggested at least some suitable habitat zones on Sumatra, similar to Peninsular Malaysia, during the LGM, the Mid-Holocene and under the present climate. Thus, the expansion of evergreen forests after the LGM, linked with a lower prey biomass, was most likely not the reason for the extinction of leopards on Sumatra. The thickness of the YTTs indicated a devastating impact of the Toba volcano super eruption (~74 kya; Costa et al., 2014) and it is conceivable that this eruption could have caused the extinction of leopards on Sumatra (Fig. 1c). The wetter and warmer climates (Van der Kaars & Dam, 1997) in the aftermath of this volcanic event (70–55 kyr) and the low habitat suitability projected for the exposed Strait of Malacca during the LGM (Fig. 1d) may have hindered leopards from re-colonizing Sumatra. In contrast to Sumatra, Malay Peninsula was probably recolonized from the north after the Toba eruption, which is supported by the fact that all leopard samples from south of the Isthmus of Kra share the same haplotype, which is also present in Indochina (Luo et al., 2014). This haplotype distribution indicates that after the Middle Pleistocene expansion of leopards from continental Asia to Java, a second, very recent expansion of leopards occurred from Indochina to the Malay Peninsula (Fig. 1d). Such a recent expansion and the selection of individuals with black fur may also explain the near fixation of melanism in this population (Kawanishi et al., 2010).

Conservation implications Our study emphasizes the need for greater conservation efforts for this critically endangered leopard subspecies (Ario et al., 2008), as its extinction would greatly reduce the natural diverJournal of Zoology (2016) – ª 2016 The Zoological Society of London

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Figure 3 Median-joining (MJ) network and extended Bayesian skyline plots (EBSP) for leopards, constructed using concatenated mitochondrial sequences (724 bp). Haplotypes in MJ networks (a) and confidence intervals in EBSPs (b) are colour-coded according to origin of samples: green for Africa and Arabia, shades of blue for Continental Asia, Java and Sri Lanka. In the MJ network (a) numbers above bars connecting haplotypes correspond to number of mutational steps (if >1); circle diameter is proportional to number of occurrences of that haplotype. Haplotype details in Table 1 and Appendix S1.

sity of leopards. Although leopards currently still occur throughout Java, it is estimated that only 5% of the island still contains suitable habitat for this species due to anthropogenic changes (Ministry of Forestry of Indonesia, 2014), and it is very likely that less than 500 individuals remain (Ario, 2010). As the suitable habitat is scattered, all remaining populations Journal of Zoology (2016) – ª 2016 The Zoological Society of London

are small and thus vulnerable to local extinction – the largest existing population occurs in the Gunung Halimun Salak National Park and has only 40-50 individuals (Sakaguchi, Sinaga & Syahrial, in press). Particularly, central Java has seen the loss of leopards from many areas (Gunawan et al., 2009) and national parks and nature reserves are the remaining 9


strongholds (Ario, 2010). Java is densely populated and anthropogenic pressures on remaining natural habitats will continue to increase, which in turn necessitates the protection of leopard habitats from deforestation. The loss of habitat also results in more frequent contacts between leopards and people, as leopards now more often enter agricultural areas. Between 2008 and 2015, 22 such contacts resulted either in the death or the translocation of the leopard to a rescue centre (Ario unpublished data). In most cases, this conflict is indirect, with leopards preying on livestock or pets, such as goats, chickens and dogs (Santiapillai & Ramono, 1992). Thus, further in situ conservation efforts need to address the human–leopard conflict by raising awareness in communities and by stronger law enforcement. Additionally, wildlife corridors will be needed (1) to reduce the likelihood that dispersing leopards enter agricultural or urban areas, and (2) to avoid inbreeding depression in the remaining fragmented populations (Frankham, 2015). A vulnerable wild population, such as the Javan leopard population, requires a meta-population management and thus the integration of the breeding programme in the strategic conservation action plan (Ministry of Forestry of Indonesia, 2014). For decades, no coordinated breeding programme has existed, although several Indonesian zoos or rescue centres house Javan leopards (Gippoliti & Meijaard, 2007). In 2014, an International studbook coordinated by Taman Safari Indonesia and Tierpark Berlin, Germany was established. By the end of 2014, a total of 52 (30 males and 22 females) Javan leopards from 14 institutions (11 in Indonesia and three in Europe) were registered. With 35 leopards (21 males and 14 females) the majority of the captive population consists of wild-born, rescued individuals. Although such a high number of founder individuals could provide a solid basis for a sustainable captive breeding programme, the breeding of wild-born leopards is challenging and special expertise is needed. Currently only two of the 11 Indonesian institutions (both managed by Taman Safari Indonesia) have successfully bred Javan leopards. In order to establish a genetically diverse and demographically healthy captive breeding population, an increased effort is required (1) to achieve breeding success in institutions with currently non-breeding individuals; (2) to establish more unrelated breeding lines; and (3) to provide better breeding facilities within Indonesia, but in the long term also outside of the country. The Javan leopard is one of the oldest pantherine subspecies and, with a few hundred individuals remaining, this subspecies is also one of the most threatened. Over the last decades conservation activities for the Javan leopard have been outcompeted by initiatives for the Sumatran tiger, and Sumatran and Javan rhinos. The data presented here emphasize the urgent need for a more concerted conservation effort, integrating both in situ and ex situ programmes in a One Plan Approach (Byers et al., 2013). The workshop held in 2014 on the status, risks and protection of the Javan leopard, organized jointly by the Ministry of Forestry and the Taman Safari Indonesia group, and the establishment of an international studbook represent the first steps for such an integrated approach. Now subsequent measures are required to ensure that the last pantherine species on Java will not face the same fate as its two relatives. 10

A. Wilting et al.

Acknowledgement A.W., R.P. and J.F. thank all institutions and persons listed in Appendix S1 who supplied the biological specimens this work is based upon. The molecular analysis was funded by the German Research Foundation (DFG grant Fi-698/5-1) and the species distribution modelling by the SAW-2013-IZW-2 grant. A.W. thanks Deike Hesse for valuable comments on earlier drafts of this manuscript, and J€ urgen Niedballa for his assistance with the GIS analysis. C.K., K.S., A.W. and J.F. thank Andreas Knieriem (Berlin Zoos), Tony Sumampau and Bongot Huaso Mulia (Taman Safari Indonesia) for their support with the international studbook and the captive breeding programme.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. List of NCBI data used for the leopard analysis.
