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We examined the nucleotide sequences of the mitochondrial NADH dehydrogenase subunit 2 gene (976 base pairs) for 279 individuals of the sable Martes zibellina (Carnivora, Mustelidae), derived from diverse areas throughout the regions of the Ural Mountains to the Russian Far East on the Eurasian continent and the peripheral peninsula (Kamchatka) and islands (Sakhalin, Hokkaido, and southern Kurils). The demographic history of the sable and its migration history to the eastern peripheral peninsula and islands were inferred using phylogeographic approaches. The analyses confirmed the previously found major lineages for the examined sables and further identified novel sublineages. Our data also support that a lineage, which is endemic to the eastern marginal islands (Sakhalin, Hokkaido, and southern Kurils), was produced by the demographic expansion of an ancestral lineage in the Eurasian continent. The most recent common ancestor of the Sakhalin, Hokkaido, and southern Kuril sables was estimated to exist during the Late Pleistocene. We also determined that another lineage exists on Sakhalin and is shared by the Far East Primorsky population. Our results indicate multiple migration events onto Sakhalin from the continent and suggest the importance of the formation of several straits to the distribution of sable lineages. Meanwhile, Kamchatka is dominated by a sole lineage which would also have followed the demographic expansion on the Eurasian continent. The Russian Far East was indicated as the source area for lineage diversifications; in this region, genetic diversity was relatively high, which is consistent with previous studies.

The vertical distribution of introduced Siberian weasels Mustela sibirica and endemic Japanese weasels M. itatsi in the Seburi Mountains in Kyushu, Japan, was examined from October 1996 to February 1998. Siberian weasels occurred near villages with paddy and cultivated fields, whereas Japanese weasels occurred in grasslands and plantations. The dispersion of yearlings destabilized the distributions of both weasel species. The horizontal distribution of both species throughout Japan was examined by means of collection of dead specimens and by trapping from March 1998 to March 2002. The eastern boundary of the distribution of the Siberian weasel was Fukui, Nagano, and Aichi prefectures; however, the distribution is expanding slowly eastward. The Siberian weasel cannot invade new habitats that lack nearby villages in Seburi, and cannot expand its range in the eastern area of Aichi, where Japanese weasels are dominant. The presence of the Japanese weasel likely prevents expansion of the distribution of the Siberian weasel.

We examined sequence variation in mitochondrial and nuclear genes of seven species of the genus Martes (Mustelidae, Carnivora): Martes americana (American marten), Martes flavigula (yellow-throated marten), Martes foina (beech marten), Martes martes (pine marten), Martes melampus (Japanese marten), Martes pennanti (fisher) and Martes zibellina (sable), focusing on the phylogenetic history of the Hokkaido subspecies of the sable, Martes zibellina brachyura. Nucleotide sequence analysis of the mitochondrial cytochrome b gene confirmed the view that the Hokkaido sable population has lower genetic diversity. In contrast, network analysis of a nuclear gene related to coat colour, melanocortin-1 receptor (Mc1r), revealed two different haplogroups for this population: one shared with that of Russian sables and the other specific to this population but with a close relationship with the American and Japanese martens, implying that these endemic haplotypes are composed of uncharacterised ancestral lineages of a past population. We also examined the sequence variation in a neighbouring nuclear gene, transcription factor 25 (Tcf25), located about 5 kb upstream from the Mc1r gene, and found similar trends. The sable genome leaves the impression that Hokkaido hosted ancient marten lineages, with subsequent recent migrations from the continent. The validity of a candidate Mc1r mutation for the entirely yellow coat observed on Hokkaido sables was also discussed.

We analyzed a concatenated (8492. bp) nuclear-mitochondrial DNA data set from 44 musteloids (including the first genetic data for Lyncodon patagonicus) with parsimony, maximum likelihood, and Bayesian methods of phylogenetic and biogeographic inference and two Bayesian methods of chronological inference. Here we show that Musteloidea emerged approximately 32.4-30.9 million years ago (MYA) in Asia, shortly after the greenhouse-icehouse global climate shift at the Eocene-Oligocene transition. During their Oligocene radiation, which proceeded wholly or mostly in Asia, musteloids diversified into four primary divisions: the Mephitidae lineage separated first, succeeded by Ailuridae and the divergence of the Procyonidae and Mustelidae lineages. Mustelidae arose approximately 16.1 MYA within the Mid-Miocene Climatic Optimum, and extensively diversified in the Miocene, mostly in Asia. The early offshoots of this radiation largely evolved into badger and marten ecological niches (Taxidiinae, Melinae, Mellivorinae, Guloninae, and Helictidinae), whereas the later divergences have adapted to other niches including those of weasels, polecats, minks, and otters (Mustelinae, Ictonychinae, and Lutrinae). Notably, and contrary to traditional beliefs, the morphological adaptations of badgers, martens, weasels, polecats, and minks each evolved independently more than once within Mustelidae. Ictonychinae (which is most closely related to Lutrinae) arose approximately 9.5-8.9 MYA, most likely in Asia, where it diverged into the Old World Ictonychini (Vormela, Poecilictis, Ictonyx, and Poecilogale) and New World Lyncodontini (Lyncodon and Galictis) lineages. Ictonychini presumably entered Africa during the Messinian Salinity Crisis (at the Miocene-Pliocene transition), which interposed the origins of this clade (approximately 6.5-6.0 MYA) and its African Poecilictis- Ictonyx- Poecilogale subclade (approximately 4.8-4.5 MYA). Lyncodontini originated approximately 2.9-2.6 MYA at the Pliocene-Pleistocene transition in South America, slightly after the emergence of the Panamanian land bridge that provided for the Great American Biotic Interchange. As the genera Martes and Ictonyx (as currently circumscribed) are paraphyletic with respect to the genera Gulo and Poecilogale, respectively, we propose that Pekaniaand Poecilictis be treated as valid genera and that Martes pennanti and Ictonyx libyca, respectively, be assigned to these genera. Fil: Sato, Jun J.. Fukuyama University; Japón Fil: Wolsan, Mieczyslaw. Polish Academy of Sciences; Argentina Fil: Prevosti, Francisco Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”; Argentina Fil: D'Elía, Guillermo. Universidad Austral de Chile; Chile Fil: Begg, Colleen. No especifica; Fil: Begg, Keith. No especifica; Fil: Hosoda, Tetsuji. Taikyu High School; Japón Fil: Campbell, Kevin L.. University of Manitoba; Canadá Fil: Suzuki, Hitoshi. Hokkaido University; Argentina

Intra-species genetic variations of the sable Martes zibellina (Carnivora, Mustelidae), originating from Russian Far East and Hokkadio, were assessed by using nucleotide sequences of the mitochondrial NADH dehydrogenase subunit 2 gene (976 base pairs). Evaluation of the genetic diversity of the sables demonstrated that populations in the southern Primorsky territory in Russian Far East harbors high genetic diversity. We assumed that the high genetic variations might have been due to effects of refugia, secondary admixture of allopatrically differentiated lineages, or massive anthropogenic introductions. Molecular phylogenetic (maximum likelihood and Bayesian inference approaches) and network (median joining method) analyses clarified that sables in Hokkaido was monophyletic. Bayesian-relaxed molecular dating approach estimated the date for migration of sables into Hokkaido to lie between 0.10–0.27 Myr BP. Considering the geological evidence, the Late Pleistocene was presumed to be the plausible ...

Phylogenetic relationships among species of the family Mustelidae were examined using the combined nucleotide sequences of the three mitochondrial genetic loci (cytochrome b (MT-CYB; 1140 bp), NADH dehydrogenase subunit 2 (MT-ND2; 1044 bp), and displacement loop (MT-DLOOP; 540 bp)), with special emphasis on the phylogenetic history of four Taiwanese mustelid species: Martes flavigula (Boddaert, 1785), Melogale moschata (Gray, 1831), Mustela nivalis L., 1766, and Mustela sibirica Pallas, 1773. Maximum likelihood phylogenetic analysis of the combined sequences of the mitochondrial genetic loci produced a topology largely congruent with that of previous studies at the species level. Analyses of intraspecific genetic variations revealed two Melogale moschata individuals from Taiwan and Vietnam that showed genetic distances comparable with interspecific variations within the mustelid lineages. Furthermore, Mustela nivalis, recently discovered in Taiwan, was not as genetically differentiated from other continental conspecific individuals as a previous morphological survey suggested. Divergence time estimations for the mustelid lineages of Taiwan and the Eurasian continent by the Bayesian relaxed molecular clock approach suggested multiple colonization of Taiwan by mustelids from the continent during the Pleistocene, creating a hierarchical pattern of endemism based on the differential isolation history of the mustelid species in Taiwan.

Considerable long-standing controversy and confusion surround the phylogenetic affinities of pinnipeds, the largely marine group of "fin-footed" members of the placental mammalian order Carnivora. Until most recently, the two major competing hypotheses were that the pinnipeds have a single (monophyletic) origin from a bear-like ancestor, or that they have a dual (diphyletic) origin, with sea lions (Otariidae) derived from a bear-like ancestor, and seals (Phocidae) derived from an otter-, mustelid-, or musteloid-like ancestor. We examined phylogenetic relationships among 29 species of arctoid carnivorans using a concatenated sequence of 3228 bp from three nuclear loci (apolipoprotein B, APOB; interphotoreceptor retinoid-binding protein, IRBP; recombination-activating gene 1, RAG1). The species represented Pinnipedia (Otariidae: Callorhinus, Eumetopias; Phocidae: Phoca), bears (Ursidae: Ursus, Melursus), and Musteloidea (Mustelidae: Mustela, Enhydra, Melogale, Martes, Gulo, Meles; Procyonidae: Procyon; Ailuridae: Ailurus; Mephitidae: Mephitis). Maximum parsimony, maximum likelihood, and Bayesian inference phylogenetic analyses of separate and combined datasets produced trees with largely congruent topologies. The analyses of the combined dataset resulted in well-resolved and well-supported phylogeny reconstructions. Evidence from nuclear DNA evolution presented here contradicts the two major hypotheses of pinniped relationships and strongly suggests a single origin of the pinnipeds from an arctoid ancestor shared with Musteloidea to the exclusion of Ursidae.

Phylogenetic relationships among the ferret-badger Melogale moschata, the skunk Mephitis mephitis, and 21 other arctoid carnivorans, representing Mustelidae (Mustelinae: Mustela, Martes, Gulo; Lutrinae: Enhydra; Melinae: Meles), Procyonidae (Procyon), and Ursidae (Ursus, Melursus), were evaluated through maximum-parsimony phylogenetic analysis of concatenated partial nucleotide sequences of the nuclear recombination-activating gene 1 (RAG1) and gene encoding interphotoreceptor retinoid-binding protein (IRBP). The analysis strongly supports Melogale as more closely related to a musteline-lutrine clade (containing Mustela and Enhydra) than to Meles or another musteline clade containing Martes and Gulo (causing Melinae and Mustelinae, as traditionally circumscribed, to be nonmonophyletic). This, together with known morphological and karyological evidence for nonmeline affinities of Melogale, justify the exclusion of the ferret-badgers from the monophyletic Melinae. Therefore, we recommend that Melogale be classified in a distinct mustelid subfamily, the monotypic Helictidinae. Our analysis also strongly supports an outgroup position of the skunks to a clade containing Procyonidae and the nonmephitine Mustelidae (causing Mustelidae, as traditionally circumscribed, to be paraphyletic). This position of the skunks agrees with results of most previous genetic studies. However, it is contradicted by known morphological evidence from both living and fossil taxa, as well as genetic evidence from protein electrophoresis. These consistently support the traditional placement of the skunks within the monophyletic Mustelidae (recently in a close relationship to Lutrinae). Therefore, we consider the recent elevation of the skunks to the level of family as premature, and recommend that this clade be left at the subfamily level (Mephitinae) within the family Mustelidae, pending further evidence.

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.

Gobo Shoko High School, Gobo, Wakayama 644-0012, JapanGenetic contamination resulting from the introduction ofnon-native animals has become a hot issue in conserva-tion biology. The Japanese marten Martes melampusmelampus, which is endemic to Honshu, Shikoku, andKyushu, Japan (Mammalogical Society of Japan 1997),was introduced to Hokkaido, northern Japan, where thesable, M. zibellina brachyura, is indigenous and wherehybridisation between the two species has been antici-pated. The Hokkaido population of M. m. melampus isthought to be descended from individuals that were in-troduced artificially into Hokkaido from Honshu duringthe 1940s/1950s (e.g. Inukai 1975; Hosoda et al. 1999).Over the 60 years since introduction, the distribution ofM. m. melampus has expanded to include a large area ofsouthern and central Hokkaido (Murakami and Ohtaishi2000). Martes zibellina and M. melampus are known to besufficiently closely related, phylogenetically (Hosodaet al. 1997, 1999, 2000; Kurose et al. 1999), to yieldhybrids, although a preliminary genetic survey ofHokkaido’s sable and marten did not show such signs(Hosoda et al. 1999, 2000). In Europe, introgressionof mitochondrial DNA from M. z. to M. martes hasbeen documented (Davison et al. 2001). In order to assess the likelihood of interspecificgenetic crosses occurring, karyological comparisons arenecessary. Differentially stained karyotypes are reportedamong individuals of M. zibellina ssp. from Russia (Gra-phodatsky et al. 1977; Graphodatsky & Radzabli 1988)and of M. m. melampus from Honshu (Obara 1982, 1987,1991), but not from M. z. brachyura from Hokkaido. Inthis study, we analyzed the karyotype of M. z. brachyurain Hokkaido using conventional and differential stainingmethods.

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.

We examined the intra- and inter-specific genetic relationships of the Japanese marten Martes melampus and the sable M. zibellina using cytoplasmic and nuclear DNA markers. The interspecific sequence divergences in the 402 base pairs of the mitochondrial cytochrome b gene averaged 3.3%. The extent of the divergences among thirteen individuals of M. melampus collected from Honshu, Shikoku, Kyushu and Tsushima was small (≤0.5%), irrespective of their fur color variation. A somewhat higher degree of intra-specific variation (up to 1.3%) was observed among M. zibellina specimens, but the extent of inter-populational variation between Primorye, Russia, and Hokkaido, Japan, was not so high (minimum 0.2%), suggesting that there has been recent genetic communication between Hokkaido and the continent. Among the 24 restriction sites of the nuclear ribosomal DNA spacer, there was no difference within either species, however one site differed between the two species. Using these molecular markers we confirmed that an amimal from Hokkaido, showing the typical morphological characteristics of M. melampus, possessed the same genotype as M. melampus from Honshu. From these results and descriptions in the literature, we presumed that the animal in question could be a descendent of M. melampus introduced to Hokkaido from Honshu by fur farmers about 50 years earlier. Eight animals examined from Hokkaido showed no indication of hybridization between the two species.

We analyzed the restriction fragment length polymorphisms in the spacer regions of ribosomal DNA (rDNA), using twelve restriction enzymes, to examine whether the Iriomote cat is related to the leopard cat (Felis bengalensis). A restriction map for each taxon was constructed and the major taxon-specific types of repeating unit (repetypes) were characterized on the basis of the arrangements of restriction sites. The Iriomote cat and the leopard cat share a common repetype but this repetype is different from that of the domestic cat (F. catus) with an estimated sequence divergence of 1.5% and from that of the ocelot (F. paradalis) with an estimated sequence divergence of 2.5%. These results indicate that, phylogenetically, the Iriomote cat is closely related to the leopard cat and that the ancestral population moved from the continent to Iriomote Island quite recently. The rDNA arrays of the leopard cat exhibit considerable intragenomic size-variation, which is thought to have emerged as a result of differences in numbers of repeated DNA segments, whereas the extent of such size-variation is much lower in the rDNA of the Iriomote cat. It appears that, even though migration of the Iriomote cat occurred relatively recently, the population has diverged to some extent from its continental counterpart, perhaps via fixation of preexistent intraspecific variations rather than by generation of new variations.

Heterogeneity in the external spacers of the ribosomal DNA (rDNA) from eight species of terrestrial carnivores (Canidae, Mustelidae) was analyzed using twelve different restriction enzymes and cloned mouse rDNA probes. We constructed species-specific maps of restriction sites on rDNA. Based on differences in the arrangements of the restriction sites, sequence divergence among the repeating units of rDNA was estimated in order to construct a molecular-phylogenetic tree and to estimate the timing of the divergence of species. The divergence between Nyctereutes procyonoides viverrinus and Vulpes vulpes japonica was 8.4%. The sequence divergence between Mustela putorius furo versus Mustela sibirica itatsi, Mustela nivalis nivalis and Martes melampus melampus was 0.65%, 1.59% and 7.27%, respectively.

Molecular phylogenetic analyses of combined mitochondrial DNA sequences (2814 bp; cytochrome b gene, displacement loop region, and NADH dehydrogenase subunit 2 gene) identified nine groups among 49 individual Japanese martens, Martes melampus, collected from several areas in Japan. The grouping was not correlated with winter coat color, but was consistent with geography. In particular, the monophyly of 29 Tsushima martens, M. m. tsuensis, was supported by strong clade support and topological tests. Haplotype and nucleotide diversities were much lower for the Tsushima population than for any population on the Japanese main islands. In addition, analyses of heterozygosity in nuclear growth hormone receptor gene sequences (654 bp) showed genetic homogeneity for the Tsushima population. This evidence supports the view that the Tsushima marten's long history of isolation on small islands is responsible for its genetic distinctiveness and uniformity, validating the Tsushima population as an evolutionarily significant unit.

Sequence variation within the 5' flanking (about 240 bp) and exon regions (426 bp) of the melanocortin- I receptor (MCIR) gene was examined to determine the potential role of this protein in the melanistic coat colorationof 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 pennonti, were shown to have intact gene sequences. However, several "in frame'' deletions of the MCIR 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.

In a survey of 228 tuberculous patients who had received kanamycin (KM) treatment, KM-resistant Escherichia coli or Escherichia freundii were isolated from 29. From the patients who had not received KM-therapy, KM-resistant strains were not isolated. Of the 29 KM-resistant strains isolated, 5 E. coli strains were also resistant to other aminoglycoside antibiotics, i.e., paromomycin, fradiomycin and streptomycin. They were also resistant to chloramphenicol and sulfanilamide and all of these resistances could be transferred to E. coli strains by conjugation. The resistance was considered to be conferred by an R factor. The remaining 24 KM-resistant strains were identified as E. freundii. In these level of KM-resistance was rather low and conjugal transfer was not observed. All were resistant to paromomycin but sensitive to fradiomycin. When 381 KM-sensitive strains were plated on an agar plate containing 25 μg of KM/ml, 23 KM-resistant strains were isolated. The mutants thus obtained were similar to the second type of 24 KM-resistant strains isolated from patients. Thus it was strongly suggested that the 24 strains carrying low levels of KM- and paromomycin-resistance, that is, conjugally nontransmissible, appeared by spontaneous mutation in the intestines of patients who had received KM-treatment.