Phylogeographie des Nebelparders (Neofelis nebulosa, Griffith 1821) und seine Ökologie und Verbreitung in Sabah, Malaysia.

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1 Universität Würzburg Biologische Fakultät Lehrstuhl für Tierökologie & Tropenbiologie Phylogeographie des Nebelparders (Neofelis nebulosa, Griffith 1821) und seine Ökologie und Verbreitung in Sabah, Malaysia. Phylogeography of clouded leopards (Neofelis nebulosa, Griffith 1821) and their ecology and distribution in Sabah, Malaysia. Erstgutachter: Prof. Dr. K. E. Linsenmair Zweitgutachter: Prof. Dr. H. Hofer Diplomarbeit Mai 2007

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3 Diplomarbeit Index Index Abbrevations... IV Zusammenfassung... 1 Abstract... 3 General Introduction... 5 Large carnivores as vulnerable and indicator species... 6 Large carnivores as umbrella species... 6 Large carnivores as keystone species... 7 Large carnivores as flagship species... 7 The Clouded Leopard: a biological review... 7 Distribution... 7 Description... 8 Habitat... 9 Behaviour... 9 Diet Population and protection status General Objectives Chapter 1 Clouded leopard phylogeny Introduction Materials and Methods Samples and DNA extraction Mitochondrial DNA analysis Microsatellite markers Population structure analysis Results Mitochondrial DNA analysis Microsatellite analysis Population substructures Estimation of the coalescence time of genetic variations in clouded leopards Discussion Management implications I

4 Index Diplomarbeit Chapter 2 Clouded leopard ecology Introduction Non-invasive methods Scent marking Status and behaviour of Sundaland clouded leopards Main goals Methods Study area Topography and soils Climate Flora Fauna Main Research Area Determining the size of the area surveyed Data collection Transect surveys Scent stations Night surveys Track measurement Laboratory analysis Statistical and analytical analysis Application of the results on landscape level Results Recorded mammal species Scent stations Faecal analysis Scent marking behaviour of Sundaland clouded leopards Individual identification by photographs Tracking Individual identification by tracks Population size and density Distribution in Sabah II

5 Diplomarbeit Index 4. Discussion Mammal species in Tabin Night surveys Scent stations Molecular scatology Scent marking in clouded leopards Clouded leopard abundance Rigorous track classification method Clouded leopard distribution in Sabah General conclusion Acknowledgment References Appendix Erklärung III

6 Abbreviations Diplomarbeit Abbreviations BS CI CITES CFR Dps / Dkf DVCA ESU IUCN LD ME / ML / MP mya NJ PC / PCA SWD TBR TPR TS TWR VJR W bootstrap support confidence interval Convention of International Trade in Endangered Species commercial forest reserve genetic distance estimators: proportion of shared alleles / kinship coefficient Danum Valley Conservation Area evolutionary significant unit World Conservation Union (International Union for the Conservation of Nature and Natural Resources) linkage disequilibrium minimum evolution / maximum likelihood / maximum parsimony million years ago neighbor-joining principal component / principal component analysis Sabah Wildlife Department tree-bisection reconnection totally protected reserve track-set Tabin Wildlife Reserve virgin jungle reserve buffer width IV

7 Diplomarbeit Zusammenfassung Zusammenfassung Im Zuge des kontinuierlichen Rückgangs der tropischen Regenwälder werden die verbleibenden ungestörten Habitate immer kleiner und fragmentierter. Diese ökologischen Veränderungen bedeuten besonders für viele große Raubtiere eine starke Gefährdung. Auch der Nebelparder (Neofelis nebulosa, Griffith 1821), der ausschließlich in tropischen und subtropischen Regenwäldern in Südostasien vorkommt und zu den bisher am wenigsten untersuchten Katzenarten gehört, ist dieser Gefahr ausgesetzt. Genetische und morphologische Untersuchungen führten kürzlich dazu, dass eine Reklassifizierung der Nebelparder auf Borneo (N. nebulosa diardi) zu einer eigenen Art (N. diardi) vorgeschlagen wurde. Da die genetische Studie jedoch nur auf drei Individuen von Borneo basierte, habe ich in meiner Diplomarbeit die Neueinteilung mit zusätzlichen Proben von Borneo (N = 4) überprüft. Ich konnte darüber hinaus Museumsmaterial von Tieren von Sumatra (N = 3), die bisher nicht genetisch untersucht wurden, sammeln. Meine Ergebnisse, die auf Sequenzen von drei mitochondrialen Genfragmenten (zusammen 900 Bp) und 18 Mikrosatelliten basieren, unterstützen die Unterscheidung in N. nebulosa und N. diardi. Die beiden Arten wiesen 41 fixierte Nukleotidunterschiede auf und bei acht der analysierten Mikrosatelliten gab es keine Überlappungen der Allele. Diese genetischen Differenzen sind vergleichbar mit Unterschieden zwischen anerkannten Arten in der Schwestergattung Panthera. Ferner konnte ich zeigen, dass auch die Tiere auf Sumatra zu der neu eingeteilten Art N. diardi gehören, da die analysierten Individuen von Sumatra den Proben von Borneo genetisch ähnelten. Aufgrund des Ursprungs von N. diardi auf zwei Inseln im Sundaschelf schlage ich den deutschen Namen Sundaland Nebelparder vor. Außerdem habe ich sowohl in der mtdns Analyse als auch in der Mikrosatellitenuntersuchung einen genetischen Unterschied zwischen den Populationen auf Borneo und Sumatra festgestellt. Die Ursache dafür ist ein reduzierter Genfluss zwischen den beiden Populationen, und deshalb empfehle ich die Unterteilung von N. diardi in zwei Unterarten. Aufgrund dieser Reklassifizierung sollte man nicht nur die beiden Arten N. nebulosa und N. diardi, sondern auch die verschiedenen Populationen auf Borneo und Sumatra getrennt voneinander behandeln. Eine Unterteilung der Nebelparder in zwei Arten und Unterarten hat zur Folge, dass sich ihre Verbreitungsgebiete reduzieren und sich dadurch der Grad ihrer Bedrohung erhöht. Umso wichtiger ist deshalb ein verstärkter Schutz der unterschiedlichen Populationen. Diese Aufgabe gestaltet sich jedoch aufgrund des teilweise nachtaktiven und scheuen Verhaltens der Tiere und ihres Vorkommens in geringen Populationsdichten in schwer zugänglichen Gebieten als äußerst schwierig. 1

8 Zusammenfassung Diplomarbeit Bisher wurden meist sehr kostspielige und zeitaufwändige Methoden angewendet um die Ökologie großer Raubtiere in Regenwäldern zu erforschen. In dieser Arbeit habe ich verschiedene nicht invasive Methoden im Tabin Wildlife Reserve im Nordosten von Borneo (Sabah) getestet. Mit Lockstoffen beköderte Haarfallen und molekulare Kotanalyse konnten nicht erfolgreich angewendet werden. Im Gegensatz dazu stellten sich Nachtfahrten mit Scheinwerfern und Spurenanalysen als kostengünstige und relativ einfach anzuwendende Methoden dar. Auf vier Nachtfahrten wurden Sundaland Nebelparder gesichtet. Mit Hilfe von Fotos gelang es mir zwei Individuen im südlichen Teil des Untersuchungsgebietes anhand ihrer Fellzeichnung zu identifizieren. Des Weiteren konnte ich in der Feldarbeit zwei, möglicherweise drei, verschiedene Formen eines Markierungsverhaltens zeigen. Ein solches Verhalten war zwar von anderen Katzenarten bereits bekannt, ist jedoch bei Nebelpardern noch nie beschrieben worden. Während meiner täglichen Untersuchungen auf Transekten, entlang von Schotterstraßen, Bachläufen und Waldwegen habe ich sechs Spurenfolgen von Nebelpardern aufgenommen. Diese konnten mit Hilfe von multivariater Statistik in vier Gruppen eingeteilt werden, wobei anzunehmen ist, dass jede Gruppierung einen Nebelparder repräsentiert. Die Anwendung eines Fang-Wiederfang Modells erlaubte eine ungefähre Abschätzung der Nebelparderdichte in meinem Untersuchungsgebiet. In dem 56 km² großen Areal habe ich mittels der Spurenanalyse fünf Individuen ( ± 2,26 SE) und im 19 km² großen südlichen Teil des Untersuchungsgebietes mittels Fotoanalyse zwei Nebelparder ( ± 0,59 SE) errechnet. Auf Grundlage dieser Abschätzungen ergab sich eine Dichte von neun ( ± 4,36 SE für die Spuren) und 10,5 ( ± 3,1 SE für die Fotos) Nebelpardern auf 100 km² im Tabin Wildlife Reserve. Die ähnlichen Ergebnisse für die zwei unabhängig voneinander kalkulierten Dichten unterstützen die Annahme, dass die wirkliche Dichte im 95 % Konfidenzintervall von acht bis 17 Individuen auf 100 km² liegt. Trotzdem möchte ich aufgrund der geringen Anzahl an Fängen und Wiederfängen in beiden Ansätzen betonen, dass diese Dichteangabe als eine erste grobe Abschätzung und Arbeitshypothese für weitere Forschungen anzusehen sein sollte. Des Weiteren habe ich unter Berücksichtigung des Schutzstatus der formal geschützten Gebiete (kommerziell genutzt oder reines Schutzgebiet), ihrer Größe und des bestätigten Vorkommens an Nebelpardern versucht, seine Verbreitung über ganz Sabah zu bestimmen. Basierend auf Bestandsaufnahmen vom Sabah Wildlife Department konnte ich zeigen, dass zurzeit Nebelparder in 25 % der Landfläche Sabahs vorkommen, aber nur wenige dieser Gebiete unterliegen einem totalen Schutzstatus. Daher schlage ich vor, einen Schwerpunkt auf das nachhaltige Management der kommerziell genutzten Waldgebiete zu legen, um das langzeitige Überleben der Nebelparder in Sabah zu garantieren. 2

9 Diplomarbeit Abstract Abstract Associated with the continuous loss of tropical rainforests, natural habitats become more and more fragmented leading to rampant ecological changes which place most top carnivores under heavy pressure. The clouded leopard (Neofelis nebulosa, Griffith 1821) is one of the least studied cat species and occurs exclusively in subtropical and tropical rainforests in south-east Asia. Recently, reclassification of Bornean clouded leopards (N. nebulosa diardi) to species level (N. diardi) was suggested based on molecular and morphological evidence. Since the genetic results were based on only three Bornean samples I re-evaluated this partition using additional samples of Bornean clouded leopards (N = 4). I was also able to include specimens from Sumatra (N = 3), which were lacking in the previous analysis. I found strong support for the distinction between N. nebulosa and N. diardi based on three fragments of mtdna (900 bp) and 18 microsatellites. Forty-one fixed mitochondrial nucleotide differences and non-overlapping allele sizes in eight of 18 microsatellite loci distinguished N. nebulosa and N. diardi. This is equivalent to the genetic divergence among recognized species in the genus Panthera. Sumatran clouded leopards clustered with specimens from Borneo, suggesting that Sumatran individuals also belong to N. diardi. Referring to their origin on two Sunda Islands I propose to give N. diardi the common name Sundaland clouded leopard. Additionally, a significant population subdivision was apparent among N. diardi from Sumatra and Borneo based on mtdna and microsatellite data. The reduced gene flow between these islands suggests the recognition of two subspecies of N. diardi. Based on this reclassification of clouded leopards not only the two species N. nebulosa and N. diardi, but also the populations of N. diardi on Borneo and Sumatra should be managed separately. This research will give a good example for the importance of taxonomic splitting for conservation. The two species and the distinct populations on Borneo and Sumatra face a much greater risk of extinction due to smaller distribution ranges, than previously assessed based on the former classification. Therefore more effort is needed to protect the different populations from extinction. However, censussing and monitoring of these species is extremely difficult due to their partly nocturnal and far-ranging behaviour as well as their low densities in densely vegetated and remote areas. Consequently little is known about their behaviour and status. So far various methods have been used to determine the status of top carnivore populations in rainforest habitats, most of them costly in terms of equipment and time. In this study, performed in Tabin Wildlife Reserve in north-eastern Borneo (Sabah) I evaluated different non-invasive methods for investigating secretive carnivores occurring in 3

10 Abstract Diplomarbeit tropical rainforests. Scent stations as hair-traps as well as the application of molecular scatology were used unsuccessfully. In contrast, I could show that night surveys and rigorous track classification are useful, cheap and easy-applied methods for research on elusive carnivores. During four night spotlight surveys clouded leopards were observed. I detected two different forms of scent marking for Sundaland clouded leopards ; micturition, and cheek rubbing. In contrast to other larger cats territorial marking was virtually unknown for clouded leopards before. During daily transect surveys along roadways, streams, and jungle trails six track-sets were recorded. Multivariate analysis of those track-sets grouped the tracks in four clusters, suggesting that four different individuals left the tracks. On the basis of their distinctive coat pattern two clouded leopards could be individually identified by the analysis of photographs, obtained during night drives in the southern part of the research area. I used these data to apply a capture-recapture analysis to roughly estimate the abundance of clouded leopards within my study site. The population size in the 56 km² research area was estimated to be five individuals ( ± 2.26 SE) for the track analysis and two animals ( ± 0.59 SE) in a 19 km² fragment of the research area used for photograph analysis. I obtained densities of clouded leopards based on the population estimates of nine ( ± 4.36 SE for tracks) and 10.5 ( ± 3.1 SE for photographs) per 100 km² in Tabin Wildlife Reserve. The consistent population estimates from two independently applied methods support that the density lies most likely between the approximately 95 % confidence interval of eight to 17 individuals per 100 km². However, due to the low number of captures and recaptures in my study I would like to emphasise that my calculated density should rather be taken as rough estimates and first working hypothesis than a true number. I extrapolated my local-scale results to regional landscape level, taking into account the conservation status of all reserves (totally protected or commercial forest reserves) in Sabah and their size and presence of clouded leopards. I showed that to date clouded leopards are still confirmed in approximately 25 % of Sabah, but that only a few reserves are totally protected and these areas are inhabited by just a few hundred individuals. The remaining reserves are classified as commercial forest reserves. Therefore, I suggest placing a higher priority on sustainable management of these commercial forest reserves to ensure the long term persistence of viable clouded leopard populations. 4

11 Diplomarbeit General Introduction General Introduction At present, species are becoming extinct at a rate between 100 and 1000 times of natural background rates (Balmford 1996). Only around 5 % of the planet s surface is protected in some form from exploitation (Gittleman et al. 2001). The ongoing habitat loss will produce much higher extinction rates with potential disappearance of up to half of the world s species (Pimm et al. 1995). Of 11 mammal orders, five have a significantly higher number of threatened species than expected (artiodactyls, insectivores, primates, perrissodactyls, sirenians) (Mace & Balmford 2000). However, carnivores are not one of them nor does any carnivore family have an unusually high level of threatened species (Mace & Balmford 2000). Even though carnivores may fair relatively well in general, historical and current patterns of extinction clearly indicate that large carnivorous species with restricted ranges are highly threatened (Gittleman et al. 2001). The extinction vulnerability among species is frequently caused by particular biological traits (Terborgh 1974; Purvis et al. 2000), e. g. small or declining population sizes, low population density, large home ranges, large body size, little genetic variability or species which are hunted by humans. In many ways these summed-up characteristics reflect exactly the biology of large carnivores (Gittleman et al. 2001). In conservation biology species are often classified into the following categories (Gittleman et al. 2001): vulnerable species (species most likely to become extinct), indicator species (reflect critical environmental damage), umbrella species (species requiring large areas and thus, if protected, will in turn protect other species), keystone species (play a pivotal role in ecosystems) and flagship species (popular species, which attract much attention). Each classification informs whether a species deserves particular conservation and protection efforts. Remarkably, all of these labels fit on most of the large carnivorous species. The following examples emphasise the specialty of carnivores, and give reasons why carnivores should receive closer attention and disproportionate resources. 5

12 General Introduction Diplomarbeit Large carnivores as vulnerable and indicator species Fundamentally, the biological niche of large carnivores at the top of the food chain means that they will always be less abundant than their prey and require relatively large home ranges. As a consequence these species are the first to suffer when human populations expand and cultivate previously untouched habitats (Sillero-Zubiri & Laurenson 2001). Therefore top predators are more vulnerable to habitat destruction and fragmentation than many other species. Consequently it is believed that the presence and fluctuations of top predators reflect the status of other species in the community as well as the chemical and/or physical changes in the environment (Landres et al. 1988). This makes many larger carnivores good indicator species. Today, many carnivores are confined to protected areas with patchy distributions, due to dramatic declines in suitable habitats over the past few hundred years. As a result of isolation of the remaining populations, recolonisation of vacant areas becomes less likely, as well as the maintenance of sink populations by immigration. The enduring populations, often less than 100 individuals in size, are expected to be more prone to extinction than larger populations, partly because deterministic declines can drive them to extinction more rapidly (Fahrig 1997). Furthermore demographic bottlenecks resulting in a reduction of the molecular genetic variation can impair the reproductive potential of a population and thus its long term health and viability (Wildt et al. 1987). The Florida panther (Puma concolor coryi) is a well documented example of inbreeding consequences in which lower heterozygosity is associated with a suite of physiological problems. For example sperm viability is times lower than in other panther subspecies and 58 % of the males are cryptorchids (absence of one or both testes from the scrotum) (Roelke et al. 1993). In addition cardiac defects and disease incidences are high and growth arrest is indicated by high incidences of stress lines in the cortical bones (Roelke et al. 1993). Large carnivores as umbrella species The protection of large carnivores requires not only huge reserves, but also vital prey populations. For many carnivores the prey abundance and availability, as well as geographic variation in food resources, influence the population viability and density (Fuller & Murray. 1998). As a consequence planning assessments and protection of carnivores prey are also important for carnivore conservation. Thus, many other species will fall under that umbrella, if large tracts of land get managed with the intention to protect large predators. Nepal s Chitwan National Park is a well known case study of single species conservation. The unusual 6

13 Diplomarbeit General Introduction forceful blend of protection in the park leads to the highest tiger (Panthera tigris) density in the world (Dinerstein et al. 1999), but this protection also has a lasting positive effect on the population density of other species (e. g. one-horned rhinoceros Rhinocerus unicornis, gaur Bos gaurus). Large carnivores as keystone species Large carnivores, as predators, limit the number of herbivores and smaller carnivores (Wright et al. 1994), a so called top-down regulation. Subsequently due to removal of dominant carnivores smaller carnivores undergo population increases, a phenomenon called mesopredator release (Soulé et al. 1988). For example densities of predators of invertebrates, seed predators and herbivores are 10 to 100 times higher on Barro-Colorado Island compared to similar habitats on the mainland where top carnivores are present (Terborgh et al and 2006). Hence, there can be no doubt that carnivores, as predators, play a vital role in the maintenance of biodiversity, stability and integrity of various communities (Paine 1966; Berger 1999; Crooks & Soulé. 1999; Terborgh et al and 2006; Sergio et al. 2005; Johnson et al. 2007). Large carnivores as flagship species Many large carnivores can serve as flagship species due to their charismatic nature. These flagships can be used to anchor a conservation campaign, because they raise public interest and sympathy. The Florida panther is one of these flagship species. Used as a poster-animal it became a symbol and leading element of the entire conservation campaign (Simberloff 1998). Especially large cats galvanize public interest towards a greater goal of habitat conservation. The Clouded Leopard: a biological review Distribution In the past, clouded leopards were distributed in most parts of south-east Asia, ranging from Nepal, Assam to mainland south-east Asia and southern China. Furthermore clouded leopards were distributed on the Islands of Borneo, Sumatra and historically they were found on Taiwan and Java. However, on Java clouded leopards became extinct in the Holocene (Meijaard 2004). On Taiwan the Formosan clouded leopard is thought to be extinct in the wild: the last confirmed sighting of a clouded leopard was in 1983 (Rabinowitz 1988) and in 1989 the skin of a small clouded leopard was found in the Tayloko region (Wang et al. 1995). 7

14 General Introduction Diplomarbeit Compared to distribution ranges of other Pantherinae like leopards and lions (Panthera leo), the extension of clouded leopards is very restricted. Description Clouded leopards are medium sized cats, weighing between kg, even though on Borneo males were recorded with a body weight over 30 kg (Sabah Wildlife Department pers. comm.). The clouded leopard is named after the distinctive cloud-shaped blotches on its fur. The base colour of the fur is pale yellow to rich brown. The blotches are edged with dark brown or black and become brighter towards the middle. The animal s underside is pale or white with few spots. Neck and back are streaked dark brown or black, whereas the head and legs are usually spotted. The heavily furred tail is marked with broken black rings. Completely black specimens, similar to the black panther (Panthera pardus) (Banks 1931; Davies & Payne 1982; Rabinowitz et al. 1987), or pale, whitish individuals have been reported anecdotally on Borneo (Davies & Payne 1982). Several adaptations like flexible joints in their hind feet, which allow them to rotate their feet more than any other felid, give clouded leopards amazing arboreal skills. Like the margay a) b) Figure 1 Male clouded leopard (Neofelis nebulosa) photographed at Duisburg zoo. (a) shows the proportionately short legs with large broad paws. (b) shows the exceptionally long and plushy tail. 8

15 Diplomarbeit General Introduction (Leopardus wiedii) of South America, clouded leopards can climb down tree trunks, squirrellike headfirst (Hemmer 1968). Proportionately short legs (Figure 1 a Page 8) provide excellent leverage and a low centre of gravity for climbing while large broad paws with sharp claws allow clouded leopards a good grip on tree branches (Figure 1 a). An exceptionally long and plushy tail, which usually measures between 75 and 90 cm and is almost equivalent to their head-body length (Mehta & Dhewaju 1990), is extremely important as a balancing aid (Figure 1 b Page 8). These arboreal skills of the clouded leopard gave rise to the Malaysian name harimau-dahan meaning branch-tiger. Recent studies on skull morphology revealed some saber-tooth characters in clouded leopards (Christiansen 2006). Christiansen & Adolfssen (2005) measured various typical skull characteristics and found that Neofelis has the largest gape of any extant carnivores. Gapes approaching an angle of 90 are similar only to extinct saber-tooth cats whereas all other extant felids can only open their mouth up to an angle of (Christiansen 2006). This huge gape in clouded leopards is linked to the longest canines in proportion to body weight of all extant felids (Guggisberg 1975). Habitat Based on anecdotal observations clouded leopards have been described as inhabiting principally evergreen tropical rainforest (Pocock 1939; Wood 1949; Prater 1971), but more recently clouded leopards have also been reported in other types of forests such as secondary and logged forest (Davies & Payne 1982; Rabinowitz et al. 1987; Santiapillai & Ashby 1988). In subtropical Nepal the clouded leopard occurs in marginal dry woodland, but it has also been recorded in shrub forest and tall grasslands (Dinerstein & Mehta 1989). Davies and Payne (1982) confirmed the presence of clouded leopards in mangrove swamps on Borneo. In the Himalayan foothills clouded leopards have been recorded up to an elevation of 2,600 m (Choudhury 1997), possibly as high as 3,000 m (Jerdon 1874). All these sightings suggest that the species might be more flexible in its habitat needs than previously thought. Behaviour Compared to other big cats very little is known about the natural history of clouded leopards. Most available information is anecdotal, based on local surveys, sightings and interviews (Rabinowitz et al. 1987; Davies 1990) or stems from captive observations (Hemmer 1968; Nowell & Jackson 1996; Law & Tatner 1998; Wielebnowski et al. 2002). A total of only 9

16 General Introduction Diplomarbeit seven clouded leopards have ever been radio-collared (Dinerstein & Mehta 1989; Austin & Tewes 1999; Grassman et al. 2005). Many early accounts depict this carnivore as an arboreal and nocturnal denizen of dense primary forest (Pocock 1939; Wood 1949). Despite their arboreal talents there is no field evidence to support this assumption. Recent studies showed that clouded leopard movements are usually terrestrial (Rabinowitz et al. 1987; Dinerstein & Mehta 1989; Austin & Tewes 1999; Grassman et al. 2005) and that clouded leopards use trees mainly for resting (Rabinowitz et al. 1987; Davies 1990). According to Davies and Payne (1982) and Rabinowitz et al. (1987), clouded leopards were seen following jungle trails and roads in Borneo. Daily distances of 1,000-2,700 meters and home ranges between 23 and 45 km² (Grassman et al. 2005; Austin & Tewes 1999) cannot be covered without spending much time on the ground. These home-ranges exceed home-range sizes of male leopards in Thailand (Rabinowitz 1989; Grassman 1999). Early accounts which classified the clouded leopard as strictly nocturnal got temporary confirmed by photographs obtained by camera trapping in Sumatras Gunung Leuser National Park at night (Griffiths 1993). There were speculations that clouded leopards are more strictly nocturnal in areas where they occur sympatrically with tigers like in Gunung Leuser National Park (NP) and that they are less nocturnal on Borneo in absence of other large carnivores (Selous & Banks 1935; Rabinowitz et al. 1987). However recent data from Thailand, where the clouded leopard occurs sympatrically with superior carnivores, indicate that the clouded leopard is not as strictly nocturnal as previously thought and is more crepuscular (Grassman et al. 2005). Due to their extremely secretive nature, virtually nothing is known of their social behaviour in the wild. Most likely they live solitary, similar to most other big cats, unless associated with a mate for breeding or females accompanied by cubs. Diet Pocock (1939) surmised from the deep penetration of its bite, attested by the long canines and from its powerful build that clouded leopards are obviously adopted for preying relatively large ungulate prey. However, their chief prey has been reported to consist of birds, primates and small mammals, as well as larger prey, such as porcupines, deer and pigs (Sus sp.) (Banks 1931; Selous & Banks 1935; Prater 1971; Griffiths 1993; Grassman et al. 2005). In a riverine forest in Sabah, a clouded leopard was observed feeding on a proboscis monkey (Nasalis larvatus) in the branches of a small tree (C. Prudente, pers. com.) and in Thailand clouded leopards were sighted hunting pig-tailed macaques (Macaca nemestrina) (Davies 1990). 10

17 Diplomarbeit General Introduction Interestingly Grassman et al. (2005) found a pangolin (Manis javanica) which was killed by a clouded leopard. Population and protection status The clouded leopard is listed, at present, by IUCN as vulnerable (IUCN 2006), as endangered under the United States Endangered Species Act and it is listed on CITES Appendix I, which bans international commerce. Due to a lack of research information, population estimates do not exist, and still little known is about the actual status in any part of its geographic range (Sunquist & Sunquist 2002). However, it is thought that the number of wild clouded leopards is declining throughout its range (Sunquist & Sunquist 2002). The foremost threat is deforestation and cultivation of previously untouched habitats (Nowell & Jachson 1996). Secondly, in some areas clouded leopards are widely hunted for their teeth and decorative pelt or for bones for traditional Asian medicine (Tan Bangjie 1984; Nowell & Jackson 1995). In surveys of south-east Asian black markets clouded leopard pelts were traded openly on a regular base (Tan Bangjie 1984; Nowell & Jackson 1995; Sunquist & Sunquist 2002). General Objectives In one part of this study I tried to collect as many genetic samples of Bornean and Sumatran clouded leopards as possible to contribute to the taxonomic designation of clouded leopards. Recently reclassification of Bornean clouded leopards (N. nebulosa diardi) to species level (N. diardi) was suggested based on molecular and morphological evidence (Buckley-Beason et al. 2006; Kitchener et al. 2006). Since the genetic results were based solely on three Bornean samples I re-evaluated this partition utilising different molecular markers. In a second part of this study I investigated the status of clouded leopards in a part of its natural habitat. The field work was conducted in Tabin Wildlife Reserve which forms a totally protected area in north-eastern Borneo. During this field study I examined different noninvasive methods to study clouded leopards in a tropical rainforest. Main goals included estimation of population size and of density of clouded leopards in my study area. An extrapolation of this information to other forested areas in Sabah was performed to provide a first rough assessment of the status of clouded leopards region wide. The aim of this study was to contribute to effective conservation of one of the most threatened cat species in Asia. 11

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19 Diplomarbeit Chapter 1: Clouded leopard phylogeny Introduction Chapter 1 Clouded leopard phylogeny revisited: Support for species recognition and population division between Borneo and Sumatra 1. Introduction The taxonomic history of clouded leopards is complex and their systematic classification and phylogenetic status remained obscure for a long time (Pocock 1917; Haltenorth 1936; Hemmer 1964; Hemmer 1968). The clouded leopard was first described by Griffith (Griffith 1821) as Felis nebulosa, based on a captive individual most probably from southern China. Later the genus name was changed to Neofelis (Gray 1867). After this description a controversial discussion on the systematic position of clouded leopards within the family Felidae started. For example Leyhausen (1990) listed Neofelis as a full genus, but also included the tiger within that genus. Due to a resembling coat pattern Corbet and Hill (1992) placed clouded leopards and marble cats (Pardofelis marmorata) in the genus Pardofelis. Despite this past disputes Neofelis morphological characteristics and vocalizations finally led to the conclusion that the Neofelis and the Panthera genus form one monophyletic group named Pantherinae (Werdelin 1983; Peters & Hast 1994). Later phylogenetic studies confirmed that clouded leopards belong to the Panthera lineage but separated first from their common ancestor approximately 6 million years ago (mya) (Johnson & O Brien 1997; Mattern & McLennan 2000; Yu & Zhang 2005; Johnson et al. 2006). However, based on a broad analysis of felid skull morphologies Christiansen (2006) showed, that clouded leopards are standing out from the genus Panthera as well as from all other extant felids. Two years after Griffith described Felis nebulosa (Griffith 1821), Cuvier (1823) depicted an individual from Sumatra and due to morphologically differences he classified it as an additional species Felis diardi. Hodgson (1853) characterised a specimen from Nepal as a third species Felis macrosceloides and Swinhoe (1862) described another species Leopardus brachyurus based on a specimen from Formosa (Taiwan). Those regional species have subsequently been regarded as subspecies of Felis nebulosa, whereas the genus name was changed to Neofelis (Gray 1867) (Table 1). Figure 2 shows the geographical range of the four 12

20 Chapter 1: Clouded leopard phylogeny Introduction Diplomarbeit putative subspecies. However, this old classification was only based on a single or few specimens, but modern taxonomic classifications require extensive sampling from throughout the species range (Corbet 1970; Groves 2001). Recently Buckley-Beason et al. (2006) and Kitchener et al. (2006) reanalysed the phylogenetic relationship among clouded leopards due to a lack of molecular and Figure 2 shows the geographical distribution of clouded leopards in south-east Asia. Different colours stand for broadly based morphological the four putative clouded leopard subspecies based on comparisons. A reclassification of historical descriptions. Bornean and Sumatran clouded leopards N. nebulosa diardi to species level N. diardi was suggested (Kitchener et al. 2006). Morpholometric analysis of pelages by Kitchener et al. (2006) showed that Sumatran clouded leopards as well as Bornean individuals differ in a similar manner to individuals from mainland south-east Asia, with the size of the cloud markings being the primary factor for distinction. Kitchener et al. (2006) did not find any evidence for recognition of the subspecies N. nebulosa macrosceloides and N. nebulosa brachyurus. Buckley-Beason et al supported this reclassification based on mtdna, nuclear DNA sequences, microsatellite analysis, and fixed chromosomal differences for Table 1 Recognised subspecies of the clouded leopard (Neofelis nebulosa) by the IUCN Cat Specialist Group (Nowell & Jackson 1996).* Subspecies Geographical distribution N. nebulosa macrosceloides Nepal, India, Bhutan, Bangladesh, Myanmar N. nebulosa nebulosa Myanmar, Thailand, Cambodia, Vietnam, southern China, Peninsula Malaysia N. nebulosa brachyurus Taiwan N. nebulosa diardi Sumatra, Borneo * recent reclassification suggestions by Buckley-Beason et al. (2006) and Kitchener et al. (2006) are not considered 13