Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

Occurrence of two types of Hynobius naevius in Northern Kyushu, Japan (Amphibia: Urodela)

A survey to examine genetic variation among Hynobius naevius from four localities of Fukuoka Pref., northern Kyushu, Japan, resulted in the detection of two, sympatric, genetic types (A and B) that are clearly different in the allelic frequencies of four loci (ACOH-A, ACOH-B, ADH-A, and SOD-A) in each locality. Morphological investigations between the two genetic types also proved that they are clearly discriminated; the type A is about 75 mm in SVL, lacks mottling pattern on bluish purple dorsum, and possesses relatively short vomerine teeth series, while the type B is about 60 mm in SVL, and has light mottling on reddish purple ground color. These results strongly suggest that reproductive isolation occurs between these two types, and that they could be regarded as separate species. Populations from Toyota-cho, western Honshu, and Yabe-machi, central Kyushu, both close to Fukuoka Pref., were very similar to the types A and B, respectively. From these results, we consider that two evolutionary lineages that first evolved allopatrically in western Honshu and southern Kyushu secondarily contacted and became sympatric in the region of northern Kyushu.     

Phylogenetic relationships of Hynobius naevius (Amphibia: Caudata) as revealed by mitochondrial 12S and 16S rRNA genes

Using mitochondrial 12S and 16S rRNA sequences, we investigated phylogenetic relationships among populations of the endemic Japanese salamander Hynobius naevius. Monophyly of this species was recovered only in the maximum parsimony tree and was unresolved in maximum likelihood and Bayesian trees. Instead the following four haplotype clades consistently emerged clearly: Clade 1 from northwestern Kyushu, Clade 2 from Chugoku and northeastern Kyushu, Clade 3 from western Shikoku and Kyushu, and Clade 4 from Chubu-Kinki and central-eastern Shikoku. Of these, Clades 1 and 2, and Clades 3 and 4, respectively, correspond to Groups A and B previously recognized from the analyses of allozyme data in this species, but monophyly of these groups was not strongly supported. Unlike the previous results, the western and eastern samples from Shikoku did not form a clade, and were grouped with Kyushu-B in Clade 3 and Chubu-Kinki in Clade 4, respectively. The reason for this conflict between mtDNA and allozyme results is unknown, but might be related to retention of ancestral mtDNA polymorphism in Shikoku populations. Nearly simultaneous divergence of as many as four lineages in wide-ranging H. naevius is inferred for the late Miocene-Pliocene history of this taxon.     

Allozymic variation of Hynobius kimurae Dunn (Amphibia, Caudata)

An electrophoretic survey was conducted to examine genetic divergence among 21 populations of a lotic-breeding salamander Hynobius kimurae from Honshu, Japan. Genetically H. kimurae proved to be specifically distinct from H. naevius. Hynobius kimurae is divided genetically into two groups of local populations, and the five populations from the eastern area are genetically distinct from the remaining populations of the central and western areas. Less prominent genetic differentiation was revealed between the western and central populations. Separation of the eastern and central-western groups are discussed in relation to the formation of the Japanese mainland.     

Systematic Relationships of Hynobius okiensis among Japanese Salamanders (Amphibia: Caudata)

We conducted an electrophoretic survey to examine systematic relationships of a lotic-breeding salamander Hynobius okiensis endemic to Dogo Island of the Oki Islands, Japan, with several lentic and lotic-breeding Japanese species. Genetically H. okiensis with 2n=56 chromosomes was closer to the lentic-breeding H. nebulosus group (H. nebulosus and H. dunni) with the same chromosome number than to the lotic-breeding H. naevius group (H. naevius and H. kimurae) and H. boulengeri with 58 chromosomes. Chromosome number reduction from 58 to 56, possibly accompanied with a change in breeding environment from streams to still waters, is estimated to have first occurred in the nebulosus group of Hynobius. A reversal only in breeding habits then seems to have followed in steep, montane environments of the small island of Dogo, resulting in the speciation of H. okiensis.     

A new species of salamander of the genus Hynobius from Central Honshu, Japan (Amphibia, Urodela)

We describe a small salamander from south Central Honshu, Japan, as a new species, Hynobius katoi. The genetic distances between this species and several named species, including sympatric H. kimurae, derived from allozyme data from a starch gel electrophoresis, proved to be sufficiently large to differentiate it at a specific rank. Distribution of this species is confined to the montane regions of Shizuoka and Nagano Prefectures, on the Akaishi Mountains of the Chubu District, central Japan. It is regarded as a member of the naevius group of Hynobius, characterized by small number of large, pigmentless ova. The species differs from the other species of the naevius group by the combination of relatively small body size, nearly spotless body, relatively few vomerine teeth forming moderately shallow series, and unique electrophoretic pattern of isozymes.     

Genetic relationships among salamanders of the genus Hynobius (Amphibia, Caudata) from Korea and southwestern Japan

We performed allozyme analysis for three Korean (Hynobius leechii, H. quelpaertensis, and H. yangi) and three Japanese (H. nebulosus, H. tsuensis, and H. dunni) salamanders to clarify their interspecific relationships using H. naevius as an outgroup. The genetic distances (Nei's D) within ingroup species ranged from 0.11 to 0.78 with a mean of 0.33. In the NJ and CONTML trees, monophyly of the ingroup was not supported and Korean H. quelpaertensis and H. leechii diverged first from the remaining species, which together formed a weakly supported clade. Korean H. yangi, long identified as H. leechii, was closer to Japanese H. nebulosus (D=0.108) and H. tsuensis (D=0.138) than to Korean H. leechii (D=0.197) and H. quelpaertensis (D=0.305). Hynobius tsuensis and H. nebulosus were very close (D=0.108) despite their different breeding habits. A geohistorical hypothesis is proposed to explain the divergence of the six species.     

三种小鲵和极北鲵的核型

本文报道了安吉小鲵（Ｈｙｎｏｂｉｕｓａｍｊｉｅｎｓｉｓ）、中国小鲵（Ｈ．ｃｈｉｎｅｎｓｉｓ）、东北小鲵（Ｈ．ｌｅｅｃｈｉ）（辽宁桓仁）和极北鲵（Ｓａｌａｍａｎｄｒｅｌａｋｅｙｓｅｒｌｉｎｇｉ）（吉林白河）的核型。３种小鲵２ｎ＝５６,染色体按大小和形状分为大、中、小型双臂和小型单臂４组。极北鲵２ｎ＝６２,染色体按大小和形状可分为大、中、小型３组。对东北小鲵辽宁桓仁与辽宁千山标本核型的显著差异结合分类学问题进行了讨论。对３种小鲵核型进行了比较分析,并讨论了中国的小鲵属核型特点。比较了不同地区极北鲵核型的异同,并将其与小鲵属核型进行了比较。     

Cytogenetic studies of Hynobiidae (Urodela)XVIII. A ZZ/ZW sex-determining mechanism in a hynobiid salamander species,Hynobius tokyoensis Tago

The karyotype of Hynobius tokyoensis (2n = 56) was analyzed using three kinds of banding methods to determine the morphological differentiation of the sex chromosomes of this species. Salamanders and egg sacs were collected from seven localities around Tokyo, Japan. Of 28 chromosome pairs, microchromosome No. 21 was identified as a ZZ/ZW-type sex chromosome. The Z chromosome was acrocentric, whereas the W chromosome was submetacentric, with a heterochromatic, elongated short arm. Interestingly, the W chromosome is of three distinct types, W(A), W(B), and W(C), based on R-banding and Ag-NOR patterns. W(A) was detected in five populations from southern habitats, whereas W(B) and W(C) were detected in one population each from northern habitats. W(A), W(B), and W(C) were all found to carry Ag-NORs on their heterochromatic short arms. Considering the karyotypes of other species belonging to the same genus, we discuss the evolution of the sex chromosomes of H. tokyoensis.     

中国小鲵的核型

中国小鲵(Hynobius chinensis)被描述于116年前,我国学者于2005年在模式产地湖北宜昌再次采到标本,至此该物种的有效性得以确认。以2007年采于模式产地的中国小鲵胚胎为材料,采用Giemsa染色方法,对中国小鲵的核型进行了分析。结果表明,中国小鲵的二倍体染色体数为56,28对染色体按大小和形状可以分为4组:第一组,包括Nos.1～9,由大型m,sm,st染色体组成;第二组为中型双臂染色体组,由Nos.10～13组成,包含m,sm染色体;第三组,包括Nos.14～18,由小型m染色体组成;第四组,包括Nos.19～28,由小型t染色体组成。与小鲵属其他物种相比,中国小鲵核型与具染色体数为2n=56的类群核型大致相同,差异出现在染色体分组和相应染色体着丝点类型上。     

A new species of lentic breeding Korean salamander of the genus Hynobius (Amphibia, Urodela)

A new species of salamander, Hynobius yangi, is described from Kori (=Hyoam-ri), Busan-shi, southeastern Korea. It is a lentic breeder belonging to the H. nebulosus species group, and has long been confused with another Korean species H. leechii. The new species, however, is genetically substantially differentiated from Korean and Japanese relatives. Hynobius yangi is morphologically very similar to H. leechii, but could be differentiated from it by the tail shape, degree of limb separation, shape of vomerine teeth series, shape of egg sac, and dorsal coloration.     

中国西南部小鲵属的遗传分化

运用同工酶电泳和DNA测序技术,我们研究了中国东南部两种小鲵的遗传多样性。同工酶数据显示义乌小鲵和安吉小鲵之间存在中等水平的种间差异。而线粒体DNA序列数据则显示两种之间存在较高水平的种间差异。安吉小鲵种群存在明显的近亲繁殖现象;义乌小鲵种群间存在明显的分化。舟山群岛上义乌小鲵种群明显有别于大陆种群。     

Sperm motility-initiating activity in the egg jelly of the externally-fertilizing urodele amphibian, Hynobius lichenatus

Low osmolality initiates sperm motility during the external fertilization of aquatic anuran amphibians. It is thought that this process occurs also in urodeles, but this has not been fully examined in these species. We report here that fertilization was achieved in the externally fertilizing hynobiid, Hynobius lichenatus, by direct insemination onto the egg jelly surface without initial exposure of the sperm to a hypoosmotic solution. To identify the factors in addition to low osmolality that initiate sperm motility in Hynobius, we suspended the sperm of this amphibian in egg jelly extract (JE), and about 90% began to move within 1 min. This indicated the presence of a substance in JE that promotes motility initiation, as is also the case in the newt, Cynops pyrrhogaster. To examine whether this JE factor is homologous to the sperm motility-initiating substance (SMIS) in the newt, we tested for possible inter-species cross-reactivity of the JE. The percentage of moving Cynops sperm was increased to 67% in Hynobius JE at 5 min, and 65% of the Hynobius sperm began to move in Cynops JE within 1 min, indicating that JE is indeed cross-reactive between these species of salamander and newt. Concomitantly, pretreatment of Hynobius JE with Fab fragments of a Cynops SMIS monoclonal antibody resulted in a decreased number of moving Hynobius sperm. Immunoblotting further suggested that the substance in Hynobius JE responsible for motility initiation has an 18 kDa molecular mass, with an isoelectric point at 7.5.     

Karyotypes of two rare species of hynobiid salamanders from Taiwan,Hynobius sonani (Maki) and Hynobius formosanus Maki (Urodela)

Two salamanders endemic to Taiwan, Hynobius sonani and Hynobius formosanus, are similar to Japanese hynobiids in having 2n = 58 chromosomes and by the absence of a medium-sized telocentric pair. H. sonani has 6 metacentric and 10 telocentric microchromosome pairs, while H. formosanus has 5 metacentric and 11 telocentric microchromosome pairs. The karyotype of H. sonani is nearly identical to that of the Japanese hynobiid salamander H. boulengeri. Karyophylogenetic relationships among forms of mountain-stream type hynobiids of Taiwan and Japan are discussed.This paper is dedicated to late Mr. Tadao Yamamoto who died on November 2, 1985.     

挂榜山小鲵的核型(有尾目:小鲵科)

本文首次报道了挂榜山小鲵Hynobius guabangshanensis 的核型.其2n=56,分为4组,其中9对大型染色体,4对双臂中型染色体,5对双臂小型染色体和10对单臂小型染色体.挂榜山小鲵具有池塘类型小鲵的特征,即2n=56以及缺少中端着丝粒染色体,这与挂榜山小鲵的生态和形态特征也属池塘类型相符.     

Biogeographical consequences of Cenozoic tectonic events within East Asian margins: a case study of Hynobius biogeography

Few studies have explored the role of Cenozoic tectonic evolution in shaping patterns and processes of extant animal distributions within East Asian margins. We select Hynobius salamanders (Amphibia: Hynobiidae) as a model to examine biogeographical consequences of Cenozoic tectonic events within East Asian margins. First, we use GenBank molecular data to reconstruct phylogenetic interrelationships of Hynobius by bayesian and maximum likelihood analyses. Second, we estimate the divergence time using the bayesian relaxed clock approach and infer dispersal/vicariance histories under the 'dispersal-extinction-cladogenesis' model. Finally, we test whether evolutionary history and biogeographical processes of Hynobius should coincide with the predictions of two major hypotheses (the 'vicariance'/'out of southwestern Japan' hypothesis). The resulting phylogeny confirmed Hynobius as a monophyletic group, which could be divided into nine major clades associated with six geographical areas. Our results show that: (1) the most recent common ancestor of Hynobius was distributed in southwestern Japan and Hokkaido Island, (2) a sister taxon relationship between Hynobius retardatus and all remaining species was the results of a vicariance event between Hokkaido Island and southwestern Japan in the Middle Eocene, (3) ancestral Hynobius in southwestern Japan dispersed into the Taiwan Island, central China, 'Korean Peninsula and northeastern China' as well as northeastern Honshu during the Late Eocene-Late Miocene. Our findings suggest that Cenozoic tectonic evolution plays an important role in shaping disjunctive distributions of extant Hynobius within East Asian margins.     

Phylogenetic relationships and phylogeography of Hynobius tokyoensis (Amphibia: Caudata) using complete sequences of cytochrome b and control region genes of mitochondrial DNA

Using complete sequences of cytochrome b (cyt b) and control region (CR; D-loop) genes of mitochondrial DNA, we investigated the phylogenetic relationships and phylogeography among populations of the salamander Hynobius tokyoensis in northern Japan. Inclusion of populations from Aichi, Chubu region within Hynobius nebulosus by previous results was confirmed because they formed a clade with H. nebulosus. Monophyly of H. tokyoensis was supported in all trees analyzed, and two clades consistently emerged: clade A from northeastern Kanto (Ibaraki) to southern Tohoku (Fukushima), and clade B from the remaining area south of northern Kanto (Tochigi). These two clades are estimated to have an old history of divergence during the late Pliocene, when the present-day Kanto Plain was under the sea. Within clade B, three haplotypes groups (I-III) are hypothesized to have diverged more recently, i.e., between the early and mid-Pleistocene, probably in relation to glacial events. The ancestral groups I and II were separated at the present-day western Kanto and Boso Peninsula areas, respectively, and subsequently, group II invaded northward to the present-day northern Kanto area and gave rise to group III. Populations of the Miura Peninsula are thought to have multiple origins, one directly from Boso and another from a more recent southward invasion from the northern Kanto area. Cytochrome b appears to have evolved more quickly than CR genes in H. tokyoensis, and thus will be more useful for phylogenetic analyses and formulating conservation measures.     

Estimation of age structure by skeletochronology of a population of Hynobius nebulosus in a breeding season (Amphibia,Urodela)

Using skeletochronology, we determined the age structure of adult Hynobius nebulosus from Kyoto in the breeding season of 1998. From previously marked individuals, the lines of arrested growth proved to be formed once per year, indicating the number of winters each salamander experienced. The age at first reproduction was estimated to be 2.8-2.9 yrs of age in males and 3.8-3.9 yrs in females. The oldest males and females were 9.8-9.9 and 5.8-5.9 yrs of age, respectively, and, therefore the longevity in this species was estimated to be more than 9 yrs for males and 5 yrs for females. The growth curve of male's body size estimated indicated that the growth rate much decreases after males attained sexual maturity. Because body sizes of adults greatly vary even within an age class, it is dangerous to estimate individual age from the size frequency data at least in adults. We discussed age properties in Hynobius by comparing lentic and lotic breeders.     

Cytological evidence for a complex of species within the taxon Bactrocera tau (Diptera: Tephritidae) in Thailand

Analysis of mitotic karyotypes of wild specimens of larvae of the Bactrocera tea-like fruit flies (Diptera: Tephritidae) in Thailand has revealed seven distinct chromosome forms, based on the amount and distribution of heterochromatin in sex chromosomes and autosomes. Such cytological differences are perfecdy correlated with morphological observations and molecular genetics data. These findings clearly suggest that B. tau is a cluster of at least seven closely related species temporarily designated as species A (= B. tau) , B, C, D, E, F and G. On die basis of the gross quantity of heterochromatin accumulation in the genome, three groups of mitotic karyotypes can be recognized. Group 1 comprises species A and E. Species E specifically occurs only in fruits of Strychnos thorelii while species A attacks many kinds of host plants. Group 2 contains species B, F and G. Species B has been found only in Siphonodon celastrineus fruit, whereas species F and G attack die same host species, the medically important plant, Hydnocarpus anthelminthicus , albeit in different localities. Group 3 includes species C and D, each of which comprises larger amounts of pericentric heterochromatin in all chromosomes than die other two groups. Hence, diese two species are cytologically remote from those of groups 1 and 2. Species C and D occur allopatrically but they attack the same host plant species, Momordica cochinchinensis. Thus, genetic differentiation at the chromosomal level to specific host plant species and geographic isolation seem to play an important role in speciation of members of the B. tau complex.