Skull morphology diverges between urban and rural populations of red foxes mirroring patterns of domestication and macroevolution.

Human activity is drastically altering the habitat use of natural populations. This has been documented as a driver of phenotypic divergence in a number of wild animal populations. Here, we show that urban and rural populations of red foxes (Vulpes vulpes) from London and surrounding boroughs are divergent in skull traits. These changes are primarily found to be involved with snout length, with urban individuals tending to have shorter and wider muzzles relative to rural individuals, smaller braincases and reduced sexual dimorphism. Changes were widespread and related to muscle attachment sites and thus are likely driven by differing biomechanical demands of feeding or cognition between habitats. Through extensive sampling of the genus Vulpes, we found no support for phylogenetic effects on skull morphology, but patterns of divergence found between urban and rural habitats in V. vulpes quantitatively aligned with macroevolutionary divergence between species. The patterns of skull divergence between urban and rural habitats matched the description of morphological changes that can occur during domestication. Specifically, urban populations of foxes show variation consistent with 'domestication syndrome'. Therefore, we suggest that occurrences of phenotypic divergence in relation to human activity, while interesting themselves, also have the potential to inform us of the conditions and mechanisms that could initiate domestication. Finally, this also suggests that patterns of domestication may be developmentally biased towards larger patterns of interspecific divergence.

Radiographic assessment of the skeletons of Dolly and other clones finds no abnormal osteoarthritis.

Our recent report detailing the health status of cloned sheep concluded that the animals had aged normally. This is in stark contrast to reports on Dolly (first animal cloned from adult cells) whose diagnoses of osteoarthritis (OA) at 5½ years of age led to considerable scientific concern and media debate over the possibility of early-onset age-related diseases in cloned animals. Our study included four 8-year old ewes derived from the cell line that gave rise to Dolly, yet none of our aged sheep showed clinical signs of OA, and they had radiographic evidence of only mild or, in one case, moderate OA. Given that the only formal record of OA in Dolly is a brief mention of a single joint in a conference abstract, this led us to question whether the original concerns about Dolly's OA were justified. As none of the original clinical or radiographic records were preserved, we undertook radiographic examination of the skeletons of Dolly and her contemporary clones. We report a prevalence and distribution of radiographic-OA similar to that observed in naturally conceived sheep, and our healthy aged cloned sheep. We conclude that the original concerns that cloning had caused early-onset OA in Dolly were unfounded.

Rensching cats and dogs: feeding ecology and fecundity trends explain variation in the allometry of sexual size dimorphism.

The tendency for sexual size dimorphism (SSD) to increase with body mass in taxa where males are larger, and to decrease when females are larger, is known as Rensch's rule. In mammals, where the trend occurs, it is believed to be the result of a competitive advantage for larger males, while female mass is constrained by the energetics of reproduction. Here, we examine the allometry of SSD within the Felidae and Canidae, demonstrating distinctly different patterns: in felids, there is positive allometric scaling, while there is no trend in canids. We hypothesize that feeding ecology, via its effect on female spacing patterns, is responsible for the difference; larger male mass may be advantageous only where females are dispersed such that males can defend access to them. This is supported by the observation that felids are predominately solitary, and all are obligate carnivores. Similarly, carnivorous canids are more sexually dimorphic than insectivores and omnivores, but carnivory does not contribute to a Rensch effect as dietary variation occurs across the mass spectrum. The observed inter-familial differences are also consistent with reduced constraints on female mass in the canids, where litter size increases with body mass, versus no observable allometry in the felids.

The genetic legacy of the 19th-century decline of the British polecat: evidence for extensive introgression from feral ferrets.

In the 19th century, the British polecat suffered a demographic contraction, as a consequence of direct persecution, reaching its lowest population in the years that preceded the First World War. The polecat is now recovering and expanding throughout Britain, but introgressive hybridization with feral ferrets has been reported, which could be masking the true range of the polecat and introducing domestic genes into the species. We used a fragment of the mitochondrial DNA control region and 11 microsatellite loci to characterize the frequency and extent of hybridization and introgression between the two species and assess whether the 19th-century decline corresponded to a genetic bottleneck in the polecat. The proportion of admixture detected in the wild was high (31%) and hybrids were more frequently found outside Wales, suggesting that hybridization is more likely to occur along the eastern edge of the polecat's range expansion. The patterns observed in the mitochondrial and nuclear DNA data show that introgression was mediated by crosses between male polecats and female ferrets, whose offspring backcrossed with polecats. No first-generation (F1 ) hybrids were identified, and the broad range of observed admixture proportions agrees with a scenario of past extensive hybridization between the two species. Using several different methods to investigate demographic history, we did not find consistent evidence for a genetic bottleneck in the British polecat, a result that could be interpreted as a consequence of hybridization with ferrets. Our results highlight the importance of the Welsh polecat population for the conservation and restoration of the genetic identity of the British polecat.

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.

Detection of Neospora caninum in wild carnivorans in Great Britain.

Samples of brain and other tissues were collected from 99 ferrets (Mustela furo), 83 red foxes (Vulpes vulpes), 70 European polecats (Mustela putorius), 65 American mink (Neovison vison), 64 Eurasian badgers (Meles meles) and 9 stoats (Mustela erminea), from around Great Britain. DNA was extracted from approximately 1g of tissue and tested by specific nested ITS1 PCR for Neospora caninum. The results from the PCR demonstrated that Neospora specific DNA was detected in all species of wild carnivorans with the exception of the stoats (0/9). Neospora DNA positive samples were detected in: polecats 18.6% (13/70), badgers 10.9% (7/64), ferrets 10.1% (10/99), foxes 4.8% (4/83) and mink 4.6% (3/65). In the badgers N. caninum DNA positive samples were found in brain (n=2), liver (n=2) and neck muscle (n=3). Selected positive ITS1 DNA sequences were submitted to Genbank. Sequence UKwildlife1 (accession number JX857862) was found in two badgers, whilst UKwildlife2 and UKwildlife3 (accession numbers JX857863 and JX857864 respectively) were found in ferrets, all three sequences demonstrated point mutations at a single base, while sequence UKwildlife4 (accession number JX857865) was found in all the species that tested positive and showed complete identity when compared against published reference sequences for: N. caninum (Nc Liverpool isolate, EU564166). Our data shows that almost all the wild carnivoran mammal species tested are intermediate hosts for N. caninum and are therefore capable of acting as reservoirs of infection for other species. These species could also act as useful sentinel species, demonstrating the presence of the parasite in particular geographical and environmental locations.

The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms.

There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.

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.

Genetic diversity and introgression in the Scottish wildcat.

This paper describes a genetic analysis of wild-living cats in Scotland. Samples from 230 wild-living Scottish cats (including 13 museum skins) and 74 house cats from England and Scotland were surveyed for nine microsatellite loci. Pelage characteristics of the wild-living cats were recorded, and the cats were then grouped into five separate categories depending on the degree to which they conformed to the characteristics attributed to Felis silvestris Schreber, 1775. Allele frequency differences between the morphological groups are greater than those among the three house cat samples. Analysis of genetic distances suggests that more of the differences between individuals can be explained by pelage than geographical proximity, and that pelage and geographical location are not confounded. Ordination of the genetic distances suggests two main groups of wild-living cats, with intermediates, and one group is genetically very similar to the house cats, while the other group contains all cats taxonomically identified as wildcat based on morphology. A genetic mixture analysis gives similar results to the ordination, but also suggests that the genotypes of a substantial number of cats in the wildcat group are drawn from a gene pool with genotypes in approximately equilibrium proportions. We argue that this is evidence that these cats do not have very recent domestic ancestry. However, from the morphological data it is highly likely that this gene pool also contains a contribution from earlier introgression of domestic cat genes.

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.