Andrew C. Kitchener

Spatial and temporal analysis of second-generation anticoagulant rodenticide residues in polecats (Mustela putorius) from throughout their range in Britain, 1992–1999

Polecats (Mustela putorius) in Britain are currently expanding their range eastwards from Wales to reoccupy central and eastern areas of England. Second-generation anticoagulant rodenticides (SGARs), to which polecats are exposed by eating contaminated prey, are used more extensively in these central and eastern regions, leading to fears of increased exposure, and possible resultant mortality. We measured bromadiolone, difenacoum, flocoumafen and brodifacoum concentrations in the livers of 50 polecats from areas that included newly recolonised habitats and found that at least one SGAR was detected in the livers of 13 out of 37 (35.1%) male and 5 out of 13 (38.5%) female polecats. Difenacoum and bromadiolone were detected most frequently. We then combined these data with measurements on another 50 individuals from earlier studies to create a dataset for 100 polecats collected throughout the 1990s from across the whole of their current range. Using this dataset, we determined if there was any evidence that contamination in polecats had increased during the 1990s and whether animals from England were more contaminated than those from Wales, as might be expected given regional differences in the patterns of SGAR use. Overall, 31 of the 100 polecats analysed to date contained SGAR residues. The incidence was a little higher (40%) in animals that died between January and June and this probably better reflects the overall proportion of animals that are sub-lethally exposed. There was no statistically significant change during the 1990s in the proportion of polecats exposed to SGARs nor any evidence that greater use of SGARs in England resulted in more contamination of polecats. Contrary to expectation, the proportion of animals that contained difenacoum was marginally higher in Wales than elsewhere.

Hybridization and the phylogenetic relationship between polecats and domestic ferrets in Britain

Ferrets (Mustela furo) were domesticated from polecats (M. putorius, M. eversmannii) over 2000 years ago. Following their introduction to Britain, they escaped and hybridized with native European polecats (M. putorius). Native polecats declined to the point of near extinction prior to World War I, but have recently begun to expand from a Welsh refugium. Concern has arisen as to the extent of polecat/ferret introgression, and in particular, whether the expanding population is of mainly hybrid origin. Therefore, mitochondrial DNA sequencing was used to investigate polecat genetic diversity in Britain. Two geographically distinct lineages were found, where one may be ancestral to the British polecat, and the other to the domestic ferret. The ancestral distribution of each lineage, or assortative mating is sufficient to explain the observed pattern. A further comparison between the distribution of the polecat phenotype and mitochondrial haplotype implies that the current population expansion may be mediated by dispersing male polecats hybridizing with female feral ferrets. However, the wild source of the ferret remains obscure. Relatively recent speciation from European mink (M. lutreola) and black-footed ferrets (M. nigripes), and/or the effects of hybridization result in an unresolved molecular phylogeny.

Reconstructing Mammalian Phylogenies: A Detailed Comparison of the Cytochrome b and Cytochrome Oxidase Subunit I Mitochondrial Genes

The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cyt b more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cyt b correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cyt b also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5–2.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.

A diagnosis for the Scottish wildcat (Felis silvestris): a tool for conservation action for a critically‐endangered felid

A recent estimate suggests that the Scottish wildcat may be critically endangered. Nevertheless, there is still no uncontroversial method for diagnosing the Scottish wildcat. We analysed morphological differences between wild-living cats in Scotland on the basis of 20 pelage characters, scoring from 1 (domestic cat) to 3 (wildcat), in combination with 40 skull parameters and intestinal length. A cluster analysis, based on Principal Components derived from the scores for pelage characters, showed that the wild-living cats fell into three main groups without any ap rioriclassification. Each group corresponds well to the traditional characteristics of wildcats, hybrids and domestic cats, respectively, and the former two each show higher levels of morphological homogeneity compared with the third group. The three groups are most significantly differentiated by seven pelage characters: (1) extent of dorsal stripe, (2) shape of tail tip, (3) distinctness of tail bands, (4) presence/absence of broken stripes and (5) spots, on flanks and hindquarters, (6) shape and number of stripes on nape and (7) on the shoulders. Most Group-1 cats (75.6%, n = 74), but none of the other two groups, score more than 2 for all seven characters. All Group-3 cats (n = 35) and some Group-2 cats (19.2%, n = 26), but no Group-1 cats, scored 1 for one or more of the seven characters. We propose that Group-1, which is the furthest from the domestic cat in all criteria, should be used to define the Scottish wildcat. However, in practice, if a wild-living cat does not score 1 for any of the seven characters it should be treated as a wildcat in the field. These definitions provide a simple way of diagnosing a Scottish wildcat scientifically, as well as practically, which will effectively facilitate conservation action and the enforcement of protective legislation.

Second-generation rodenticides and polecats (Mustela putorius) in Britain.

In Britain, polecats Mustela putorius hunt around farm buildings, especially in winter, and, as a result, may be secondarily exposed to rodenticides by eating contaminated prey. This paper reports the first survey of second-generation rodenticides in polecats. Twenty-nine adult polecats which had been killed either accidentally on roads (24) and in traps (4), or had died of an unknown cause (1) were collected during 1992-1994. The livers of 24 animals and the stomach walls of the remaining five, for which the livers were not available, were analysed for difenacoum, bromadiolone, brodifacoum and flocoumafen. In total, rodenticide residues were detected in 31% of the polecats analysed. Residues were found in seven of the 24 livers (29%) and in two of the five stomachs analysed (40%). Difenacoum was detected most frequently (28% of animals), and was the only rodenticide in the stomach, while bromadiolone and brodifacoum were detected in only 10% and 3% of polecats, respectively. Flocoumafen was not detected in any animals. More than one rodenticide occurred in the livers of two animals; one contained difenacoum and bromadiolone, the other also contained brodifacoum. There was no sex bias in the proportion of animals containing rodenticides. Animals with detectable residues came from more than one county and were collected only during January-April in each year.

An archaeological and historical review of the relationships between felids and people.

ABSTRACT A review of the archaeological and historical records reveals several lines of evidence that people have had close relationships with felids. Almost 40% of felid species have been tamed on all continents, excluding Europe and Oceania, but only one species was domesticated. However, taming occurred mostly in five felid lineages, mostly in South and Central America, and Southwest Asia and North Africa, which is consistent with the early development of permanent human settlements and agriculture in these regions. In the Old World, probably since the beginning of the Neolithic, the first farmers encouraged commensal small carnivorans, which had been attracted either by rodent pests or scavenging opportunities. Recent genetic evidence supports archaeological evidence for the domestic cats origin in the Fertile Crescent of Mesopotamia. However, full domestication may have occurred only in ancient Egypt, where breeding of imported Mesopotamian wildcats may have been controlled, thereby allowing artificial selection, and suggesting that putative early domestic cats were most probably tamed wildcats. As there was no tameable, small felid in Europe, other indigenous carnivorans, such as mustelids, viverrids, and herpestids, were tamed instead. They were slowly replaced as the domestic cat spread gradually throughout Europe, principally with the Romans. The wildcat was fully domesticated, owing probably to a specific set of human cultural events and requirements, rather than as a consequence of a unique tendency to tameness in some populations of Felis silvestris. The global spread of the domestic cat probably obviated the need for domestication of other small felids elsewhere.

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.

Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK

Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNA was detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR-RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6·0 to 44·4% depending on the host. PCR-RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Planning tiger recovery: Understanding intraspecific variation for effective conservation

Radical revision of tiger taxonomy for a pragmatic and scientifically sound approach to tiger conservation management. 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.

The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification—Implications for forensic purposes

The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection. To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular approaches have been developed to identify biological material as being of tiger in origin. Some countries also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify tiger species and subspecies simultaneously; this test is based on identification of single nucleotide polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from four of the five extant putative tiger subspecies that currently exist in the wild were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species available on GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses. Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three of which were specific to P. t. sumatrae and the other three were specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies (approximately 0.26 pg), indicating that it will work on trace amounts of tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to identify the presence of tiger within mixed samples.