How do terrestrial wildlife communities respond to small-scale Acacia plantations embedded in harvested tropical forest?

To offset the declining timber supply by shifting towards more sustainable forestry practices, industrial tree plantations are expanding in tropical production forests. The conversion of natural forests to tree plantation is generally associated with loss of biodiversity and shifts towards more generalist and disturbance tolerant communities, but effects of mixed-landuse landscapes integrating natural and plantation forests remain little understood. Using camera traps, we surveyed the medium-to-large bodied terrestrial wildlife community across two mixed-landuse forest management areas in Sarawak, Malaysia Borneo which include areas dedicated to logging of natural forests and adjacent planted Acacia forests. We analyzed data from a 25-wildlife species community using a Bayesian community occupancy model to assess species richness and species-specific occurrence responses to Acacia plantations at a broad scale, and to remote-sensed local habitat conditions within the different forest landuse types. All species were estimated to occur in both landuse types, but species-level percent area occupied and predicted average local species richness were slightly higher in the natural forest management areas compared to licensed planted forest management areas. Similarly, occupancy-based species diversity profiles and defaunation indices for both a full community and only threatened and endemic species suggested the diversity and occurrence were slightly higher in the natural forest management areas. At the local scale, forest quality was the most prominent predictor of species occurrence. These associations with forest quality varied among species but were predominantly positive. Our results highlight the ability of a mixed-landuse landscape with small-scale Acacia plantations embedded in natural forests to retain terrestrial wildlife communities while providing an alternate source of timber. Nonetheless, there was a tendency towards reduced biodiversity in planted forests, which would likely be more pronounced in plantations that are larger or embedded in a less natural matrix.

Genomic Adaptations and Evolutionary History of the Extinct Scimitar-Toothed Cat, Homotherium latidens.

Homotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [1-4]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [5]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [6-8]. However, mitochondrial phylogenies can be misled by hybridization [9], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [10], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [10]. To examine the evolutionary history of Homotherium, we generated a ∼7x nuclear genome and a ∼38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (∼22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [5]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [3, 4, 11-14]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage.

Habitat degradation and indiscriminate hunting differentially impact faunal communities in the Southeast Asian tropical biodiversity hotspot.

Habitat degradation and hunting have caused the widespread loss of larger vertebrate species (defaunation) from tropical biodiversity hotspots. However, these defaunation drivers impact vertebrate biodiversity in different ways and, therefore, require different conservation interventions. We conducted landscape-scale camera-trap surveys across six study sites in Southeast Asia to assess how moderate degradation and intensive, indiscriminate hunting differentially impact tropical terrestrial mammals and birds. We found that functional extinction rates were higher in hunted compared to degraded sites. Species found in both sites had lower occupancies in the hunted sites. Canopy closure was the main predictor of occurrence in the degraded sites, while village density primarily influenced occurrence in the hunted sites. Our findings suggest that intensive, indiscriminate hunting may be a more immediate threat than moderate habitat degradation for tropical faunal communities, and that conservation stakeholders should focus as much on overhunting as on habitat conservation to address the defaunation crisis.

Camera-trap evidence that the silver-backed chevrotain Tragulus versicolor remains in the wild in Vietnam.

In an age of mass extinctions, confirming the survival of lost species provides rare second chances for biodiversity conservation. The silver-backed chevrotain Tragulus versicolor, a diminutive species of ungulate known only from Vietnam, has been lost to science for almost three decades. Here, we provide evidence that the silver-backed chevrotain still exists and the first photographs of the species in the wild, and urge immediate conservation actions to ensure its survival.

An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies.

BACKGROUND: The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FINDINGS: Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. CONCLUSIONS: Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

Human-mediated introduction of introgressed deer across Wallace's line: Historical biogeography of Rusa unicolor and R. timorensis.

In this study we compared the phylogeographic patterns of two Rusa species, Rusa unicolor and Rusa timorensis, in order to understand what drove and maintained differentiation between these two geographically and genetically close species and investigated the route of introduction of individuals to the islands outside of the Sunda Shelf. We analyzed full mitogenomes from 56 archival samples from the distribution areas of the two species and 18 microsatellite loci in a subset of 16 individuals to generate the phylogeographic patterns of both species. Bayesian inference with fossil calibration was used to estimate the age of each species and major divergence events. Our results indicated that the split between the two species took place during the Pleistocene, ~1.8 Mya, possibly driven by adaptations of R. timorensis to the drier climate found on Java compared to the other islands of Sundaland. Although both markers identified two well-differentiated clades, there was a largely discrepant pattern between mitochondrial and nuclear markers. While nDNA separated the individuals into the two species, largely in agreement with their museum label, mtDNA revealed that all R. timorensis sampled to the east of the Sunda shelf carried haplotypes from R. unicolor and one Rusa unicolor from South Sumatra carried a R. timorensis haplotype. Our results show that hybridization occurred between these two sister species in Sundaland during the Late Pleistocene and resulted in human-mediated introduction of hybrid descendants in all islands outside Sundaland.

An expanded mammal mitogenome dataset from Southeast Asia.

Southeast (SE) Asia is 1 of the most biodiverse regions in the world, and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia's genetic diversity is still poorly characterized. The growing interest in using environmental DNA to assess and monitor SE Asian species, in particular threatened mammals-has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples. Approximately 55 gigabases of raw sequence were generated. From this data, we assembled 72 complete mitogenome sequences, with an average depth of coverage of ×102.9 and ×55.2 for modern samples and historical samples, respectively. This dataset represents 52 species, of which 30 species had no previous mitogenome data available. The mitogenomes were geotagged to their sampling location, where known, to display a detailed geographical distribution of the species. Our new database of 52 taxa will strongly enhance the utility of environmental DNA approaches for monitoring mammals in SE Asia as it greatly increases the likelihoods that identification of metabarcoding sequencing reads can be assigned to reference sequences. This magnifies the confidence in species detections and thus allows more robust surveys and monitoring programmes of SE Asia's threatened mammal biodiversity. The extensive collections of historical samples from SE Asia in western and SE Asian museums should serve as additional valuable material to further enrich this reference database.

Connecting Earth observation to high-throughput biodiversity data.

Understandably, given the fast pace of biodiversity loss, there is much interest in using Earth observation technology to track biodiversity, ecosystem functions and ecosystem services. However, because most biodiversity is invisible to Earth observation, indicators based on Earth observation could be misleading and reduce the effectiveness of nature conservation and even unintentionally decrease conservation effort. We describe an approach that combines automated recording devices, high-throughput DNA sequencing and modern ecological modelling to extract much more of the information available in Earth observation data. This approach is achievable now, offering efficient and near-real-time monitoring of management impacts on biodiversity and its functions and services.

Genetic Structure and Phylogeography of the Leopard Cat (Prionailurus bengalensis) Inferred from Mitochondrial Genomes.

The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species' distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms).

Phylogeography of red muntjacs reveals three distinct mitochondrial lineages.

BACKGROUND: The members of the genus Muntiacus are of particular interest to evolutionary biologists due to their extreme chromosomal rearrangements and the ongoing discussions about the number of living species. Red muntjacs have the largest distribution of all muntjacs and were formerly considered as one species. Karyotype differences led to the provisional split between the Southern Red Muntjac (Muntiacus muntjak) and the Northern Red Muntjac (M. vaginalis), but uncertainties remain as, so far, no phylogenetic study has been conducted. Here, we analysed whole mitochondrial genomes of 59 archival and 16 contemporaneous samples to resolve uncertainties about their taxonomy and used red muntjacs as model for understanding the evolutionary history of other species in Southeast Asia. RESULTS: We found three distinct matrilineal groups of red muntjacs: Sri Lankan red muntjacs (including the Western Ghats) diverged first from other muntjacs about 1.5 Mya; later northern red muntjacs (including North India and Indochina) and southern red muntjacs (Sundaland) split around 1.12 Mya. The diversification of red muntjacs into these three main lineages was likely promoted by two Pleistocene barriers: one through the Indian subcontinent and one separating the Indochinese and Sundaic red muntjacs. Interestingly, we found a high level of gene flow within the populations of northern and southern red muntjacs, indicating gene flow between populations in Indochina and dispersal of red muntjacs over the exposed Sunda Shelf during the Last Glacial Maximum. CONCLUSIONS: Our results provide new insights into the evolution of species in South and Southeast Asia as we found clear genetic differentiation in a widespread and generalist species, corresponding to two known biogeographical barriers: The Isthmus of Kra and the central Indian dry zone. In addition, our molecular data support either the delineation of three monotypic species or three subspecies, but more importantly these data highlight the conservation importance of the Sri Lankan/South Indian red muntjac.

Phylogeography of the Small Indian Civet and Origin of Introductions to Western Indian Ocean Islands.

The biogeographic dynamics affecting the Indian subcontinent, East and Southeast Asia during the Plio-Pleistocene has generated complex biodiversity patterns. We assessed the molecular biogeography of the small Indian civet (Viverricula indica) through mitogenome and cytochrome b + control region sequencing of 89 historical and modern samples to (1) establish a time-calibrated phylogeography across the species' native range and (2) test introduction scenarios to western Indian Ocean islands. Bayesian phylogenetic analyses identified 3 geographic lineages (East Asia, sister-group to Southeast Asia and the Indian subcontinent + northern Indochina) diverging 3.2-2.3 million years ago (Mya), with no clear signature of past demographic expansion. Within Southeast Asia, Balinese populations separated from the rest 2.6-1.3 Mya. Western Indian Ocean populations were assigned to the Indian subcontinent + northern Indochina lineage and had the lowest mitochondrial diversity. Approximate Bayesian computation did not distinguish between single versus multiple introduction scenarios. The early diversification of the small Indian civet was likely shaped by humid periods in the Late Pliocene-Early Pleistocene that created evergreen rainforest barriers, generating areas of intra-specific endemism in the Indian subcontinent, East, and Southeast Asia. Later, Pleistocene dispersals through drier conditions in South and Southeast Asia were likely, giving rise to the species' current natural distribution. Our molecular data supported the delineation of only 4 subspecies in V. indica, including an endemic Balinese lineage. Our study also highlighted the influence of prefirst millennium AD introductions to western Indian Ocean islands, with Indian and/or Arab traders probably introducing the species for its civet oil.

Two species of Southeast Asian cats in the genus Catopuma with diverging histories: an island endemic forest specialist and a widespread habitat generalist.

Background. The bay cat Catopuma badia is endemic to Borneo, whereas its sister species the Asian golden cat Catopuma temminckii is distributed from the Himalayas and southern China through Indochina, Peninsular Malaysia and Sumatra. Based on morphological data, up to five subspecies of the Asian golden cat have been recognized, but a taxonomic assessment, including molecular data and morphological characters, is still lacking. Results. We combined molecular data (whole mitochondrial genomes), morphological data (pelage) and species distribution projections (up to the Late Pleistocene) to infer how environmental changes may have influenced the distribution of these sister species over the past 120 000 years. The molecular analysis was based on sequenced mitogenomes of 3 bay cats and 40 Asian golden cats derived mainly from archival samples. Our molecular data suggested a time of split between the two species approximately 3.16 Ma and revealed very low nucleotide diversity within the Asian golden cat population, which supports recent expansion of the population. Discussion. The low nucleotide diversity suggested a population bottleneck in the Asian golden cat, possibly caused by the eruption of the Toba volcano in Northern Sumatra (approx. 74 kya), followed by a continuous population expansion in the Late Pleistocene/Early Holocene. Species distribution projections, the reconstruction of the demographic history, a genetic isolation-by-distance pattern and a gradual variation of pelage pattern support the hypothesis of a post-Toba population expansion of the Asian golden cat from south China/Indochina to Peninsular Malaysia and Sumatra. Our findings reject the current classification of five subspecies for the Asian golden cat, but instead support either a monotypic species or one comprising two subspecies: (i) the Sunda golden cat, distributed south of the Isthmus of Kra: C. t. temminckii and (ii) Indochinese, Indian, Himalayan and Chinese golden cats, occurring north of the Isthmus: C. t. moormensis.

Complete mitochondrial genomes of the Laotian Rock Rat (Laonastes aenigmamus) confirm deep divergence within the species.

Mitochondrial genomes of five Laotian Rock Rat (Laonastes aenigmamus) samples from Vietnam and Laos were sequenced using an Illumina platform. After de novo assembly, 13 protein-coding genes and two rRNA (12S and 16S) of the five genomes were aligned and analyzed with those from other related species under maximum likelihood and Bayesian inferences. Both methods revealed congruent tree topologies, which support two independently evolving clades of L. aenigmamus from Laos and Vietnam. The relaxed time calibration analysis showed that the two major lineages of the Laotian Rock Rat split about 8 million years ago, which was consistent with the results from previous studies using only cytochrome b sequences. Such a deep divergence time suggests the recognition of two rock rat species, but further nuclear DNA and morphological data are needed to solve the taxonomy of this taxon.

Planning tiger recovery: Understanding intraspecific variation for effective conservation.

Although significantly more money is spent on the conservation of tigers than on any other threatened species, today only 3200 to 3600 tigers roam the forests of Asia, occupying only 7% of their historical range. Despite the global significance of and interest in tiger conservation, global approaches to plan tiger recovery are partly impeded by the lack of a consensus on the number of tiger subspecies or management units, because a comprehensive analysis of tiger variation is lacking. We analyzed variation among all nine putative tiger subspecies, using extensive data sets of several traits [morphological (craniodental and pelage), ecological, molecular]. Our analyses revealed little variation and large overlaps in each trait among putative subspecies, and molecular data showed extremely low diversity because of a severe Late Pleistocene population decline. Our results support recognition of only two subspecies: the Sunda tiger, Panthera tigris sondaica, and the continental tiger, Panthera tigris tigris, which consists of two (northern and southern) management units. Conservation management programs, such as captive breeding, reintroduction initiatives, or trans-boundary projects, rely on a durable, consistent characterization of subspecies as taxonomic units, defined by robust multiple lines of scientific evidence rather than single traits or ad hoc descriptions of one or few specimens. Our multiple-trait data set supports a fundamental rethinking of the conventional tiger taxonomy paradigm, which will have profound implications for the management of in situ and ex situ tiger populations and boost conservation efforts by facilitating a pragmatic approach to tiger conservation management worldwide.

Defining habitat covariates in camera-trap based occupancy studies.

In species-habitat association studies, both the type and spatial scale of habitat covariates need to match the ecology of the focal species. We assessed the potential of high-resolution satellite imagery for generating habitat covariates using camera-trapping data from Sabah, Malaysian Borneo, within an occupancy framework. We tested the predictive power of covariates generated from satellite imagery at different resolutions and extents (focal patch sizes, 10-500 m around sample points) on estimates of occupancy patterns of six small to medium sized mammal species/species groups. High-resolution land cover information had considerably more model support for small, patchily distributed habitat features, whereas it had no advantage for large, homogeneous habitat features. A comparison of different focal patch sizes including remote sensing data and an in-situ measure showed that patches with a 50-m radius had most support for the target species. Thus, high-resolution satellite imagery proved to be particularly useful in heterogeneous landscapes, and can be used as a surrogate for certain in-situ measures, reducing field effort in logistically challenging environments. Additionally, remote sensed data provide more flexibility in defining appropriate spatial scales, which we show to impact estimates of wildlife-habitat associations.

iDNA from terrestrial haematophagous leeches as a wildlife surveying and monitoring tool - prospects, pitfalls and avenues to be developed.

Invertebrate-derived DNA (iDNA) from terrestrial haematophagous leeches has recently been proposed as a powerful non-invasive tool with which to detect vertebrate species and thus to survey their populations. However, to date little attention has been given to whether and how this, or indeed any other iDNA-derived data, can be combined with state-of-the-art analytical tools to estimate wildlife abundances, population dynamics and distributions. In this review, we discuss the challenges that face the application of existing analytical methods such as site-occupancy and spatial capture-recapture (SCR) models to terrestrial leech iDNA, in particular, possible violations of key assumptions arising from factors intrinsic to invertebrate parasite biology. Specifically, we review the advantages and disadvantages of terrestrial leeches as a source of iDNA and summarize the utility of leeches for presence, occupancy, and spatial capture-recapture models. The main source of uncertainty that attends species detections derived from leech gut contents is attributable to uncertainty about the spatio-temporal sampling frame, since leeches retain host-blood for months and can move after feeding. Subsequently, we briefly address how the analytical challenges associated with leeches may apply to other sources of iDNA. Our review highlights that despite the considerable potential of leech (and indeed any) iDNA as a new survey tool, further pilot studies are needed to assess how analytical methods can overcome or not the potential biases and assumption violations of the new field of iDNA. Specifically we argue that studies to compare iDNA sampling with standard survey methods such as camera trapping, and those to improve our knowledge on leech (and other invertebrate parasite) physiology, taxonomy, and ecology will be of immense future value.

Anticipated climate and land-cover changes reveal refuge areas for Borneo's orang-utans.

Habitat loss and climate change pose a double jeopardy for many threatened taxa, making the identification of optimal habitat for the future a conservation priority. Using a case study of the endangered Bornean orang-utan, we identify environmental refuges by integrating bioclimatic models with projected deforestation and oil-palm agriculture suitability from the 1950s to 2080s. We coupled a maximum entropy algorithm with information on habitat needs to predict suitable habitat for the present day and 1950s. We then projected to the 2020s, 2050s and 2080s in models incorporating only land-cover change, climate change or both processes combined. For future climate, we incorporated projections from four model and emission scenario combinations. For future land cover, we developed spatial deforestation predictions from 10 years of satellite data. Refuges were delineated as suitable forested habitats identified by all models that were also unsuitable for oil palm - a major threat to tropical biodiversity. Our analyses indicate that in 2010 up to 260,000 km(2) of Borneo was suitable habitat within the core orang-utan range; an 18-24% reduction since the 1950s. Land-cover models predicted further decline of 15-30% by the 2080s. Although habitat extent under future climate conditions varied among projections, there was majority consensus, particularly in north-eastern and western regions. Across projections habitat loss due to climate change alone averaged 63% by 2080, but 74% when also considering land-cover change. Refuge areas amounted to 2000-42,000 km(2) depending on thresholds used, with 900-17,000 km(2) outside the current species range. We demonstrate that efforts to halt deforestation could mediate some orang-utan habitat loss, but further decline of the most suitable areas is to be expected given projected changes to climate. Protected refuge areas could therefore become increasingly important for ongoing translocation efforts. We present an approach to help identify such areas for highly threatened species given environmental changes expected this century.

Targeted conservation to safeguard a biodiversity hotspot from climate and land-cover change.

Responses of biodiversity to changes in both land cover and climate are recognized [1] but still poorly understood [2]. This poses significant challenges for spatial planning as species could shift, contract, expand, or maintain their range inside or outside protected areas [2-4]. We examine this problem in Borneo, a global biodiversity hotspot [5], using spatial prioritization analyses that maximize species conservation under multiple environmental-change forecasts. Climate projections indicate that 11%-36% of Bornean mammal species will lose ≥ 30% of their habitat by 2080, and suitable ecological conditions will shift upslope for 23%-46%. Deforestation exacerbates this process, increasing the proportion of species facing comparable habitat loss to 30%-49%, a 2-fold increase on historical trends. Accommodating these distributional changes will require conserving land outside existing protected areas, but this may be less than anticipated from models incorporating deforestation alone because some species will colonize high-elevation reserves. Our results demonstrate the increasing importance of upland reserves and that relatively small additions (16,000-28,000 km(2)) to the current conservation estate could provide substantial benefits to biodiversity facing changes to land cover and climate. On Borneo, much of this land is under forestry jurisdiction, warranting targeted conservation partnerships to safeguard biodiversity in an era of global change.

Coming down from the trees: is terrestrial activity in Bornean orangutans natural or disturbance driven?

The orangutan is the world's largest arboreal mammal, and images of the red ape moving through the tropical forest canopy symbolise its typical arboreal behaviour. Records of terrestrial behaviour are scarce and often associated with habitat disturbance. We conducted a large-scale species-level analysis of ground-based camera-trapping data to evaluate the extent to which Bornean orangutans Pongo pygmaeus come down from the trees to travel terrestrially, and whether they are indeed forced to the ground primarily by anthropogenic forest disturbances. Although the degree of forest disturbance and canopy gap size influenced terrestriality, orangutans were recorded on the ground as frequently in heavily degraded habitats as in primary forests. Furthermore, all age-sex classes were recorded on the ground (flanged males more often). This suggests that terrestrial locomotion is part of the Bornean orangutan's natural behavioural repertoire to a much greater extent than previously thought, and is only modified by habitat disturbance. The capacity of orangutans to come down from the trees may increase their ability to cope with at least smaller-scale forest fragmentation, and to cross moderately open spaces in mosaic landscapes, although the extent of this versatility remains to be investigated.

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.

Recent morphological and molecular studies led to the recognition of two extant species of clouded leopards; Neofelis nebulosa from mainland southeast Asia and Neofelis diardi from the Sunda Islands of Borneo and Sumatra, including the Batu Islands. In addition to these new species-level distinctions, preliminary molecular data suggested a genetic substructure that separates Bornean and Sumatran clouded leopards, 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 subspecies differentiation using additional molecular (mainly historical) samples of eight Bornean and 13 Sumatran clouded leopards; a craniometric analysis of 28 specimens; and examination of pelage morphology of 20 museum specimens and of photographs of 12 wild camera-trapped animals. Molecular (mtDNA and microsatellite loci), craniomandibular and dental analyses strongly support the differentiation of Bornean and Sumatran clouded leopards, but pelage characteristics fail to separate them completely, 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 International Code of Zoological Nomenclature.