Masahiro A. Iwasa

Complex structure of B-chromosomes in two mammalian species: Apodemus peninsulae (Rodentia) and Nyctereutes procyonoides (Carnivora)

B-chromosomes (Bs) of two mammalian species, raccoon dog (Nyctereutes procyonoides, Carnivora) and Asian wood mouse (Apodemus peninsulae, Rodentia) were investigated using chromosome segment microdissection and double-colour FISH. In the raccoon dog, all B-chromosomes showed homology with each other but not with the A-chromosomes. Two segment-specific probes (from proximal and distal parts of B) have been localized in corresponding chromosome parts, with significant variation in their sizes. In Asian wood mice, two types of B-specific chromatin were revealed – B1 and B2. Most Bs were either B1 or B2 specific; furthermore, some Bs were found to be composed of both types of chromatin. B-chromosome-specific libraries of A. peninsulae contain sequences homologous to the heterochromatic regions of sex and some A-chromosomes and dispersed repeated sequences. B1-specific probes gave signals on sex chromosomes of Apodemus speciosus and Apodemus agrarius. The origin and evolution of B-chromosomes in mammals are discussed.

A spatial aspect on mitochondrial DNA genealogy in Apodemus peninsulae from East Asia.

Apodemus peninsulae is a field mouse that inhabits the broad-leafed forests of temperate Eurasia. We examined the mitochondrial cytochrome b gene in 57 individuals of A. peninsulae from northeastern Asia, including Siberia, Primorye, Magadan region, Sakhalin, Hokkaido, and the Korean Peninsula. The genealogy of the mitochondrial DNA (mtDNA) in A. peninsulae was shown to have substantial geographic affinity, suggesting geographic architecture of northeastern Asia, including the islands of Sakhalin and Hokkaido, played important roles on the cladogenesis. Taking into account the presence of region-specific anciently divergent mtDNA types, three parts of the regions of Primorye, Siberia, and the Korean Peninsula can be denoted as refugia for A. peninsulae during the substantial period of the Quaternary glacial ages. Among the geographic regions examined, Primorye is likely to be the most influential one, from which the mtDNA is thought to have migrated to the neighboring regions of Sakhalin, Hokkaido, the Magadan region, and Siberia during the evolution of this species.

EVOLUTIONARY NETWORKS OF MATERNAL AND PATERNAL GENE LINEAGES IN VOLES (EOTHENOMYS) ENDEMIC TO JAPAN

Abstract Sequence variations in the mitochondrial cytochrome-b (Cytb) gene and the sex-determining gene of Y chromosome (Sry), which show maternal and paternal inheritance, respectively, were examined in 2 species of Japanese voles of the genus Eothenomys (Arvicolinae), E. andersoni and E. smithii, to elucidate phylogenies within and between species. Considerable intraspecific variation in Cytb was observed in both species, with clear geographic affinities, northeastern and central Honshu, Kii Peninsula of Honshu, Shikoku, and Kyushu (Japan), suggesting fairly ancient colonization in geographic areas. Genealogy of Cytb, however, was complicated and clearly discordant with current taxonomy. For example, the Kii Peninsula population of E. andersoni and the Shikoku population of E. smithii had unique haplotypes that differed from those of the remaining populations of both species. Sry gene variation, in contrast, was species specific and was polymorphic in E. andersoni with substantial geographic distribution, but it was monomorphic in E. smithii, suggesting a recent geographic expansion of E. smithii eastward. We attribute the complicated population genetics to topographic complexity of the Japanese islands and suggest that rearrangement of species was associated with climatic changes during the Quaternary glaciation on the basis of an assumption of divergence time among several murine rodents.

Geographic Patterns of Cytochrome b and Sry Gene Lineages in the Gray Red-Backed Vole Clethrionomys rufocanus from Far East Asia Including Sakhalin and Hokkaido

Abstract The gray red-backed vole, Clethrionomys rufocanus, from Far East Asia including Sakhalin and Hokkaido is known to harbor intraspecific morphological and cytogenetic variations. Here we analyzed geographic variation in the mitochondrial cytochrome b (Cytb) gene and Y chromosome specific Sry gene by a PCR direct sequencing technique. Determination of sequences in the Cytb gene (1140 bp) in 29 individuals provided 28 haplotypes and construction of a neighbor joining tree clearly indicated that they were grouped into four major lineages, which predominated in Primorskyi territory, Kamchatska-Magadan, Sakhalin, and Hokkaido-Kuril, with 0.02–0.04 sequence divergence (Kimuras distance, all substitutions, all codon positions). The sequences for the Sry region (336–366 bp) included a variable repetitious region of microsatellites such as TG(TC)2. In total five sequences were recognized which can be grouped into two forms, continental and insular (Sakhalin and Hokkaido) based on the presence or absence of a segment of TG(TC)3TG(TC)4. It was revealed that, therefore, the population of Sakhalin possessed their own type of mitochondrial DNA but the same Sry gene as Hokkaido. A similar trend can be seen in the continental populations. Our results suggested that the population of C. rufocanus in each of the geographic domains accumulated own genetic elements in part but genetic exchanges between neighboring populations occurred during the course of evolution. It is noteworthy that the insular domains, Sakhalin and Hokkaido, have played an important role in raising the amount of genetic diversity in small rodent species.

Local differentiation of Clethrionomys rutilus in northeastern Asia inferred from mitochondrial gene sequences

Summary Mitochondrial DNA cytochrome b (Cytb) gene sequences were used to assess the evolutionary history of the northern red-backed vole, Clethrionomys rutilus , in northeastern Asia. Neighbor-joining and maximum likelihood trees constructed with the Cytb gene sequences (1140 bp) of 27 samples revealed four major local lineages; those represented by haplotypes from central Siberia, far eastern Siberia, Alaska-Kamchatskasol;Sakhalin, and Hokkaido. These lineages differed from one another with sequence divergences ranging from 0.0160 to 0.0298 (Kimuras distance, all substitutions at all codon positions). These findings imply that C. rutilus has inhabited the local areas during a longterm period of the evolutionary time, such as the last one or two million years, as observed in another common species of red-backed vole C. rufocanus with similar geographic distribution. The intraspecies geographic partition, however, differs between the species, implying that these two species have experienced different evolutionary histories in geographic expansion and genetic exchanges among local populations.

MOLECULAR PHYLOGENY OF RED-BACKED VOLES IN FAR EAST ASIA BASED ON VARIATION IN RIBOSOMAL AND MITOCHONDRIAL DNA

Phylogenetic relationships among various species of red-backed voles (Clethrionomys and Eothenomys) from areas surrounding the Sea of Japan were assessed by examining variation in the nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA). The rDNA data indicated at least six phylogenetically distinct lineages represented by C. rufocanus, C. rex, C. rutilus, E. andersoni, E. smithii, and E. melanogaster. Lineages of rufocanus-rex and andersoni-smithii can be regarded as sister species, respectively. The mtDNA data generally were congruent but also suggested the splitting of two additional species lineages, C. regulus from C. rufocanus and E. imaizumii from E. andersoni-smithii. Our data revealed that these species have a complex evolutionary history, which includes interspecific gene flow.

Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype

Summary. Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (>96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest that our ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.

Synthesis of Seoul virus RNA and structural proteins in cultured cells

Summary.  Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i. = 0.25, the plus-strand RNA was detected within 1 h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2 hpi, and accumulated as scattered granules in the cytoplasm until 24 hpi. In contrast, the G2 protein first appeared at 8 hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24 hpi. Infectious virus particles were released into the medium at 24 h hpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2 hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.

Evolutionary significance of chromosome changes in northeastern Asiatic red-backed voles inferred with the aid of intron 1 sequences of the G6pd gene

To evaluate the evolutionary significance of karyotypic characters in red-backed voles (Rodentia, Arvicolinae, genera Clethrionomys and Eothenomys) from northeastern Asia, we constructed phylogenetic trees based on intron 1 sequences (ca. 580 bp) of the X-linked gene G6pd. Phylogenetic trees, constructed using neighbor-joining and maximum-likelihood methods, revealed two major clades in red-backed voles corresponding to the glareolus- and the rufocanus-cytotypes defined by a reciprocal translocation between chromosomes 1 and 9 based on G-band patterns. If confirmed, two types of X chromosome, acrocentric X and subtelocentric X, which have often been claimed to be traits of evolutionary and taxonomic significance, should be considered as examples of homoplasy because they occur sporadically in members of both rufocanus- and glareolus-cytotypes in red-backed voles.

Colonization history of the Japanese water shrewChimarrogale platycephala, in the Japanese Islands

To evaluate the intraspecific evolutionary history and local differentiation of the Japanese water shrewChimarrogale platycephala (Temminck, 1842), we an a lyzed the mitochondrial cytochromeb (Cytb) sequence divergence for samples from 55 localities in the Japanese is lands of Honshu and Kyushu. According to phylogenetic trees based on theCytb data, there were fourCytb haplotype lineages, which showed rough affinities with geographic areas, namely, Eastern/Central Honshu, the Kinki District of Western Honshu, the Chugoku District of Western Honshu, and Kyushu. However, in the alpine areas of the boundary between the Kinki and Chugoku Districts, complicated distribution patterns of theCytb haplotypes were revealed. Considering the present data and geological history in the Quaternary, we hypothesized the following evolutionary scenario. First, differ entiation and division into four primary ancestral geographic colonies of the shrews occurred in hypothetical refugia in the mid — late Pleistocene. Subsequently, rapid expansion occurred and caused the complicated distribution patterns of theCytb haplotypes in the boundary areas owing to the complex topography during the late stage of the Quaternary.