Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.     

Genetics and evolution of the mink Lpm-system. VIII. Characteristic changes in the antigenic structure of Lpm-protein and alpha2- macroglobulin in Mustelidae family

Interspecific variability of the isotypic structure of two macroglobulins (Lpm and alpha 2M) in Mustelidae family was investigated using monospecific rabbit antisera against Lpm and alpha 2M Mustela vison. It was established that all the species studied may be divided into four groups, according to the degree of their homology with Lpm: 1) M. vison; 2) M. altaica, M. erminea, Kolonocus sibirica, Putorius eversmany, P. putorius, Luteola lutreola; 3) Vormela peregusna; 4) Martes martes, M. foina, M. zibellina. The greatest divergence of Martes gene species from M. vison and other species studied is determined by two different immunogenetic approaches, the first based on the whole isotype of Lpm and the other using allotypic markers of individual Lpm genes. The variability of the isotypic structure of alpha 2M was lower than that revealed in Lpm. V. peregusna is the only species which significantly differs from all above-mentioned species, including M. vison. The differences established for degree of variability between Lpm and alpha 2M in Mustelidae family may be connected with the evolutionary peculiarities of these two genetic system of serum alpha-macroglobulins, which being phylogenetically related, are still structurally divergent.     

First U.S. record of the hard tick Ixodes (Pholeoixodes) gregsoni Lindquist, Wu, and Redner

Ixodes (Pholeoixodes) gregsoni Lindquist, Wu, and Redner, a species of hard tick described in 1999 in Canada, was recovered from a harvested fisher (Martes pennanti Erxleben) and a domestic cat (Felis silvestris catus Ragni and Randi) in Vermont in 2001 and from harvested mink (Mustela vison Schreber) in Maine in 2003. These samples are the first records of this species within the United States. Although knowledge of this tick's natural history and distribution are still preliminary, these records indicate a possible greater distribution for I. gregsoni than initially perceived. Although its status as a disease vector is presently unknown, natural resource professionals should be aware of the possibility of this tick's occurrence in the northeastern United States.     

Geographic and host range of the nematode Soboliphyme baturini across Beringia

The nematode Soboliphyme baturini Petrov, 1930, was found to represent a single species with a relatively broad geographic range across Beringia and northwestern North America on the basis of the assessment of molecular sequence data for adult and juvenile parasites. Refuted are hypotheses suggesting that several cryptic species could be partitioned either among an array of mustelid definitive hosts or across the vast region that links North America and Eurasia. Host specificity for this species is examined on the basis of a comprehensive list for definitive hosts, derived from new field surveys and existing literature for S. baturini. Only 5 mustelids (Gulo gulo, Martes americana, M. caurina, M. zibellina, and Neovison vison) appear to have significant roles in the life history, persistence, and transmission of this nematode. Soboliphyme baturini readily switches among M. americana, M. caurina, Mustela erminea, or N. vison at any particular locality throughout its geographic range in North America, although Martes spp. could represent the source for nematodes in a broader array of mustelids. Molecular analyses (243 base pairs of mitochondrial gene nicotinamide dehydrogenase [ND4]) suggest that hypotheses for host specificity across an array of mustelid definitive hosts are not supported. The life cycle of S. baturini is explored through a review of diet literature for 2 marten species, M. americana and M. caurina, and other mustelids across the Holarctic. Shrews (Soricomorpha: Soricidae) comprise >8% of prey for these species of Martes, suggesting their putative role as paratenic hosts. Juvenile nematodes found in the diaphragms of soricids are genetically identical to adult S. baturini found in the stomachs of mustelids at the same locations in both Asia and North America, corroborating a role in transmission for species of Sorex.     

Echinococcus multilocularis in carnivores from the Klatovy district of the Czech Republic

A unique human case of alveolar echinococcosis was described in 1979 from the Klatovy district of the Czech Republic. However, there were no previous epidemiological studies in this area focusing on detection of the source of infection--Echinococcus multilocularis adults producing eggs. During the period June 1997 to April 1999, 29 out of a total of 46 (63.3%) red foxes (Vulpes vulpes) in the Klatovy district and one of four foxes (25.0%) in the Pilsen South district were found to be infected with adult worms of E. multilocularis. No E. multilocularis adults were found in other animals from the Klatovy district (i.e. three specimens of Martes martes, two Martes foina, one Mustela erminea, two Meles meles and one Felis catus f. domestica). An examination of faecal samples from 55 dogs (Canis familiaris) from the Klatovy district resulted in the detection of E. multilocularis DNA in one (1.8%) sample. The present results support the possibility that human alveolar echinococcosis previously described in the Czech Republic had the character of an autochthonous infection. There are also indications of a potential risk of infection to humans.     

Evolutionary trends of the mitochondrial lineage differentiation in species of genera Martes and Mustela

We compared partial sequences (402 bp) of the mitochondrial cytochrome b gene in 68 individuals of martens (Martes), weasels (Mustela) and their relatives from the Northern Hemisphere to identify the modes of geographic differentiation in each species. We then compared complete sequences (1140 bp) of the gene in 17 species of the family Mustelidae to know the spatial and temporal modes of speciation, constructing linearized trees with transversional substitutions for deeper lineage divergences and with transversions and transitions for younger lineages. Our data suggested that these lineages of Martes and Mustela differentiated in a stepwise fashion with five radiation stages from the generic divergences (stage I) to the intraspecific divergences (stage V), during the last 10 or 20 million years as the fossil evidence suggests. In the lineage of Martes, the first offshoots are of Martes flavigula, M. pennanti, and Gulo gulo (stage II), the second is M. foina (stage III), and the third are M. americana, M. martes, M. melampus, and M. zibellina (stage IV). The divergence of the lineages of Mustela is likely to have taken place concurrently with the radiations of the Martes. These divergence processes are attributable in part to the geographic allocation along the two continents, North America and Eurasia, as well as among peripheral insular domains, such as Taiwan and the Japanese Islands. In addition, the Eurasian continent itself was shown to have been involved in the species diversification in the martens and weasels.     

Independent nonframeshift deletions in the MC1R gene are not associated with melanistic coat coloration in three mustelid lineages

Sequence variation within the 5' flanking (about 240 bp) and exon regions (426 bp) of the melanocortin-1 receptor (MC1R) gene was examined to determine the potential role of this protein in the melanistic coat coloration of 17 mustelid species in four genera: Gulo (wolverines), Martes (martens), Mustela (weasels), and Meles (badgers). Members of the genera Mustela and Meles, together with Martes flavigula and Martes pennanti, were shown to have intact gene sequences. However, several "in frame" deletions of the MC1R gene region implicated in melanism of other species were detected within members of the genera Martes and Gulo. For instance, Gulo gulo possessed a 15 bp deletion in the second transmembrane domain coding region, while Martes americana, Martes melampus, Martes zibellina, and Martes martes shared a 45 bp deletion overlapping this area. In addition, Martes foina was found to possess a 10 bp insertion followed closely by a 28 bp deletion immediately downstream of the deletion found in other martens. Notably, none of these indels was associated with a melanistic phenotype. Phylogenetic analysis revealed that each of these nonrandomly distributed deletions arose independently during the evolution of this family. Specific indel-neighboring motifs appear to largely account for the biased and repeated occurrence of deletion events in the Martes/Gulo clade.     

Mitochondrial phylogeography and population history of pine martens Martes martes compared with polecats Mustela putorius

The flora and fauna of Europe are linked by a common biogeographic history, most recently the Pleistocene glaciations that restricted the range of most species to southern refugial populations. Changes in population size and migration, as well as selection, have all left a signature on the genetic differentiation. Thus, three paradigms of postglacial recolonization have been described, inferred from the patterns of DNA differentiation. Yet some species, especially wide-ranging carnivores, exhibit little population structuring between the proposed refugia, although relatively few have been studied due to the difficulty of obtaining samples. Therefore, we investigated mitochondrial variation in pine martens, Martes martes, in order to understand the extent to which they were affected by glacial cycles, and compared the results with an analysis of sequences from polecats, Mustela putorius. A general lack of ancient lineages, and a mismatch distribution that is consistent with an expanding population, is evidence that the present-day M. martes and Mu. putorius in central and northern Europe colonized from a single European refugium following a recent glaciation. There has also been interspecific mitochondrial introgression between M. martes and the sable M. zibellina in Fennoscandia.     

Phylogenetic relationships and divergence times among mustelids (Mammalia: Carnivora) based on nucleotide sequences of the nuclear interphotoreceptor retinoid binding protein and mitochondrial cytochrome b genes

Phylogenetic relationships among 20 species-group taxa of Mustelidae, representing Mustelinae (Mustela, Martes, Gulo), Lutrinae (Enhydra), and Melinae (Meles), were examined using nucleotide sequences of the nuclear interphotoreceptor retinoid binding protein (IRBP) and mitochondrial cytochrome b genes. Neighbor-joining and maximum-parsimony phylogenetic analyses on these genes separately and combined were conducted. While IRBP performed better than cytochrome b in recovering more-inclusive clades, cytochrome b demonstrated more resolving power in recovering less-inclusive clades. Strong support was found for a close affinity of Enhydra with Mustela to the exclusion of Martes and Gulo (causing Mustelinae to be paraphyletic); the most-basal position of Mustela vison within Mustela, followed by Mustela erminea; an association of Mustela lutreola, Mustela itatsi, Mustela sibirica, and the subgenus Putorius (including Mustela putorius and Mustela eversmanii), to the exclusion of Mustela nivalis and Mustela altaica; and a basal position of Mustela itatsi to a clade containing Mustela sibirica and Putorius. Whereas cytochrome b strongly supported Mustela lutreola as the sister species to Putorius, IRBP strongly supported its basal placement to the Mustela itatsi-Mustela sibirica-Putorius clade. The low level of sequence divergence in cytochrome b between Mustela lutreola and Putorius is therefore a result of interspecific mitochondrial introgression between these taxa, rather than a recent origin of Mustela lutreola in a close relationship to Putorius. Time estimates inferred from IRBP and cytochrome b for mustelid divergence events are mostly in agreement with the fossil record.     

Serologic survey for selected viral pathogens in free-ranging endangered European mink (Mustela lutreola) and other mustelids from south-western France

To investigate the possible role of selected pathogens in the decline of endangered European mink (Mustela lutreola) populations and the potential for these pathogens to affect mink survival, a serologic survey was conducted using serum samples collected from March 1996 to March 2003 in eight departments of south-western France. In total, 481 free-ranging individuals of five mustelid species (including the European mink) were tested. Sympatric mustelids can serve as sentinels to determine the presence of antibodies to viruses in the study area that could potentially infect mink. Antibodies to Canine distemper virus (CDV) were detected in all species; 9% of 127 European mink, 20% of 210 polecats (Mustela putorius), 5% of 112 American mink (Mustela vison), 33% of 21 stone marten (Martes foina) and 5% of 20 pine marten (Martes martes). Antibody prevalence was significantly higher in stone marten and polecats, possibly because their ranges overlap more closely with that of domestic species than that of the other species tested. Antibodies to Canine adenovirus were detected in all species but the pine marten; antibody prevalence estimates ranging from 2% to 10%. Antibodies to canine parainfluenza virus were detected in 1% of European mink, 1% of American mink and 5% of tested polecats but were not detected in Martes species. Antibodies to Rabies virus (RV) were detected in three animals, possibly because of interspecies transmission of bat lyssaviruses as the sampling area is considered to be free of RV, or to a lack of test specificity, as antibody titers were low. The high antibody prevalence to potentially lethal CDV suggests that this pathogen could have significant effects on the free-ranging populations and has implications for the conservation efforts for the endangered European mink.     

Sylvatic trichinosis in Ontario, Canada

Samples of muscle from 4,773 specimens of 18 species of wild mammals from Ontario were examined for Trichinella. One of 12 mink (Mustela vison), 83 of 1,821 fisher (Martes pennanti) and 68 of 1,980 marten (Martes americana) had T. spiralis. Prevalences of infections by Trichinella were determined for fisher and marten from the Algonquin region, over a 10-yr period. Prevalences ranged from 0.9-9.2% in fisher and 1.3-8.7% in marten indicating that the parasite is well-established in the region. Prevalences of Trichinella increased with age of both fisher and marten. Intensities determined for the 1981-1982 sample ranged from 0.4-15.8 larvae/g for fisher and 22.4-159.7 larvae/g for marten. Higher intensities were not correlated with older hosts. Fisher and marten appeared to be the key hosts maintaining Trichinella in the Algonquin region, but transmission dynamics were unclear. Transmission may include an unidentified small rodent or other host and natural carrion-feeding.     

Leptospirosis in free-ranging endangered European mink (Mustela lutreola) and other small carnivores (Mustelidae, Viverridae) from southwestern France

To study the possible role of disease in the decline of endangered European mink (Mustela lutreola), we conducted a survey of antibody prevalence and renal carriage of pathogenic leptospira (Leptospira interrogans sensu lato) using serum and kidney samples collected from 1990 to 2007 from several free-ranging small carnivores and farmed American mink (Mustela vison) in southwestern France. An indirect microscopic agglutination test using a panel of 16 serovars belonging to 6 serogroups (Australis, Autumnalis, Icteroh?morrhagi?, Grippotyphosa, Panama, Sejroe) revealed antibodies in all species, with significant differences in antibody prevalences: 74% in European mink (n=99), 65.4% in European polecats (Mustela putorius, n=133), 86% in American mink (n=74), 89% in stone martens (Martes foina, n=19), 74% in pine martens (Martes martes, n=19), 35% in common genets (Genetta genetta, n=79), and 31% in farmed American mink (n=51). Serogroups Australis and Icteroh?morragi? were dominant in most free-ranging species; serogroup Grippotyphosa had high prevalences in European mink. Such high antibody prevalences have never been reported. They are probably related to the large number of known reservoirs, rats (Rattus spp.), muskrat (Ondatra zibethicus), and coypu (Myocastor coypu), in the study area. The polymerase chain reaction test specific for pathogenic leptospiral DNA detected renal carriage in 23% of 34 European mink, 22% of 18 polecats, and 15% of 33 free-ranging American mink, with no significant differences. Renal carriage shows that mustelids may shed leptospira for short periods, but their epidemiologic role is probably limited. High antibody prevalences suggest that the disease is unlikely to be highly pathogenic for these species; however, chronic forms of the disease (abortions, renal lesions) could reduce the reproductive success or life span of infected animals. Further studies on the pathogenicity of leptospirosis in these populations are needed to measure its impact on the population dynamics of these rodent predators.     

Antibodies to Aleutian mink disease parvovirus in free-ranging European mink (Mustela lutreola) and other small carnivores from southwestern France

Owing to the rapid decline of the European mink (Mustela lutreola) in France, a national conservation action plan has been initiated, in which scientific research to improve understanding of the causes of the decline is one of the primary objectives. In order to investigate the possible role of Aleutian disease parvovirus (ADV) in decline of the species, a serologic survey was conducted from March 1996 to March 2002 in 420 free-ranging individuals of six species of small carnivores distributed in eight departments of southwestern France. Antibodies to ADV were detected in 17 of 75 American mink (Mustela vison), 12 of 99 European mink, 16 of 145 polecats (Mustela putorius), four of 17 stone martens (Martes foina), one of 16 pine martens (Martes martes), and three of 68 common genets (Genetta genetta). Seroprevalence was significantly higher in American mink than in other species. Seropositive individuals with gamma globulin levels >20% were observed in four European mink, four American mink, two stone martens, and one pine marten. Geographic distribution of positive animals indicates the virus has spread to all areas where European mink are found. Furthermore, a trend of increasing prevalence seems to appear in Mustela sp. sympatric with American mink. Although further investigations are necessary to evaluate the role of ADV in decline of European mink, evidence of the virus in the wild at the levels found in our study has implications for conservation of this species.     

Does removal of an alien predator from small islands in the Baltic Sea induce a trophic cascade?

Changes in carnivore abundance can alter the distribution and abundance of plants on a community wide basis, an effect known as a trophic cascade. Because alien predators can have a disproportionate impact, compared to native predators, on herbivore populations, they may induce stronger trophic cascades in plant communities than native predators. We studied the indirect effects of the removal of an alien predator, the American mink Mustela vison on plant communities on small islands in the Baltic Sea, SW Finland. Mink had been removed from a group of islands for 12 yr, while another group of islands with mink presence served as a control area. Field voles Microtus agrestis and bank voles Myodes glareolus exert strong grazing pressure on the island vegetation and are an important part of mink diet. On nine islands of the mink removal area and five islands of the control area we studied the vegetation in ten randomly chosen plots; five in herbaceous and five in woody (i.e. dwarf shrub) vegetation. We studied the cascading impacts of mink predation on grassy and woody vegetation using the Shannon diversity and equitability indices and comparing abundances of different species between mink removal and control islands. Diversity and equitability of plant communities were higher on mink removal islands. In grassy patches, abundances of several species differed between mink removal and control islands. Our results demonstrate, for the first time, that alien predator removal may induce a trophic cascade on small islands.     

Population genetic structure and distribution of introduced American mink (<Emphasis Type="Italic">Mustela vison</Emphasis>) in Spain,based on microsatellite variation

The population genetic structure of an invasive species in Spain, the American mink (Mustela vison), was investigated using microsatellite DNA markers. This semi-aquatic carnivore, originating from North America, was imported into Europe for fur farming since the beginning of the 20th century. Due to massive escapes, farm damages, deliberate releases and/or accidents, feral mink populations were established in the aquatic ecosystems of many European countries, including Spain. We genotyped 155 American mink originating from the Spanish regions Basque Country, Catalonia, Castilla-Leon, Aragon, Valencia and Galicia using 10 polymorphic microsatellite loci to highlight population genetic structure, distribution and dispersal. M. vison populations in Spain appear differentiated and not yet connected by gene flow. Bayesian clustering analyses and spatial analyses of molecular variance detected four inferred clusters, overall coinciding with the sampled geographical localities. Preliminary testing shows moderate to large estimated effective population sizes. Molecular analyses result useful to provide baseline data for further research on the evolution of invasive mink populations, as well as support local management strategies and indirectly benefit the conservation of threatened species in Spain, such as the endangered European mink (Mustela lutreola), and the polecat (Mustela putorius), which share the habitat with the American mink. This paper is dedicated to the memory of Xavier Domingo-Roura.     

Mustelids from Sackdilling Cave (Bavaria,Germany) and their biostratigraphic significance

Sackdilling Cave is a karst fissure filled with a fossiliferous breccia including abundant fossil remains, mostly snails and small mammals. The taxonomic status of mustelid remains from this cave was previously regarded as controversial and, consequently, different authors listed different species from the locality. A detailed revision of the material shows the presence of five mustelid species: Meles sp., Martes vetus, Mustela strandi, Mustela palerminea, and Mustela praenivalis. Among them, especially noteworthy is the presence of the type specimen of Martes vetus. The Sackdilling holotype resembles Martes foina and differs from that of Martes martes in a short and broad viscerocranium; wide and less extended forward incisor row; compressed, short and wide temporal region; large, strongly inflated and convex tympanic bullae; and narrow P3 with weak lingual bulge. Some dental characters are specific for Martes vetus and show some intermediate values between Martes foina than Martes martes. Among them are the P4 protocone length and the M1 trigon length. The revised material from Sackdilling Cave of Mustela palerminea and Mustela praenivalis was compared with that from other Early and early Middle Pleistocene sites of Europe and showed the presence of intermediate characters. Subsequently, based on the entire mustelid assemblage and other faunal elements (mainly rodents), the age of this fauna was estimated as ca. 0.9–0.7 Ma.     

辽宁朝阳马山洞的食肉类化石

本文主要记述了辽宁省朝阳市龙城区马山洞发现的食肉类化石。堆积物的上部主要为颗粒细小的砂质黏土, 而下部主要为直径较大的角砾。2007年出土的食肉目化石有: 似浣熊貉(Nyctereutes procyonoides)、赤狐(Vulpes vulpes)、狗獾(Meles meles)、黄鼬(Mustela sibirica)、中华猫(Felis chinensis)和疑似虎(Panthera tigris)?。其中前5个种在形态上与现生种类非常接近; 而最后一个种除与现生虎比较接近外, 与现生狮子也有一定的相似性。     

Fruit consumption by carnivores in Mediterranean Europe

• 1 We reviewed patterns of fruit consumption amongst 10 species of mesocarnivores: red fox Vulpes vulpes, weasel Mustela nivalis, stoat Mustela erminea, polecat Mustela putorius, stone marten Martes foina, pine marten Martes martes, Eurasian badger Meles meles, common genet Genetta genetta, Egyptian mongoose Herpestes ichneumon and wildcat Felis silvestris in Mediterranean Europe.
• 2 The 65 reviewed studies recorded 79 different fruits eaten by carnivores, 58 of which were identified to species. Most records (63%) were of fleshy fruits with high pulp content. The frequency of occurrence of fruit items varied widely amongst species and regions. Four of the carnivore species (red fox, stone marten, badger and common genet) included more than 30 fruit species in their diet.
• 3 A longitudinal pattern was detected in the consumption of fruit in the Mediterranean region, with the frequency of occurrence of fruit consumption increasing towards the east.

     

Notes on the food of feral mink Mustela vison in England and Wales

There is some apprehension about the possible effect of the introduced American mink Mustela vison upon the indigenous British fauna, either as a predator exerting new and excessive pressure upon native prey species, or as a carnivore competing for prey resources against native carnivores. Concern is also felt by preservers of game, and by keepers of small domestic animals (poultry and rabbits) about the mink's potential as a pest. To investigate the food habits of the mink in England and Wales the alimentary canals of 1165 trapped animals were examined, yielding 204 samples for analysis. The resulting information provides a general picture of the mink's diet, including seasonal variations. Comparisons were possible with similar work carried out in other countries, and with the diets of other British carnivores. Tentative conclusions were reached regarding the broad pattern of food relationships between the mink and other British animals, wild and domestic.     

鼬科动物线粒体DNA控制区结构分析

利用PCR技术获得紫貂(Martes zibellina)和黄喉貂(Martes flavigula)线粒体DNA控制区全序列,并结合从GenBank中下载的9种鼬科动物相应序列,用ClustalX排序后对控制区结构进行分析,识别出延长终止序列区、中央区和保守序列区3个区域,指出了一个终止相关序列ETAS1及8个保守序列(CSB-F、E、D、C、B、1、2和3),并给出了序列通式,在CSB1和CSB2之间发现不同形式的短重复序列.此外,以狼为外类群,应用邻接法构建鼬科线粒体控制区全序列的系统进化树,结果表明:臭鼬亚科最先从鼬科中分化出来,随后剩余类群分为两大支系,即貂属种类与貂熊聚为一支,并与獾亚科的狗獾形成姐妹群;另一支为水獭亚科的物种与鼬属的林鼬形成姐妹群,再与虎鼬聚在一起,狗獾与貂属的紫貂亲缘关系最近,水獭亚科与鼬属亲缘关系最近.