New data on the distribution of the Volturno spined loach Cobitis zanandreai (Teleostei: Cobitidae)

The Volturno Spined Loach Cobitis zanandreai is thought to be restricted to only two places in Italy, the Fondi Lake (Latium) and the Volturno river basin (Campania). In this study we carried out a review of the available literature and surveyed 37 sites of potential occurrence. We discovered seven previously unknown populations of C. zanandreai in the Liri-Garigliano and the Pontine plain watersheds, significantly expanding the known limits of its distribution. We found the invasive C. bilineata only in the Amaseno River. Our results agree with records from the 19th and 20th centuries, highlighting the importance of historical data in understanding current species distribution. Our findings provided a substantial distribution update that allow to assess the species conservation status more objectively.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae: Cobitis) and their phylogenetic positions

The striata complex, a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.     

Comparative histology of the testes of the spined loach Cobitis taenia L. and natural allotetraploids of Cobitis (Pisces, Cobitidae)

The spined loach Cobitis taenia L. creates exclusively diploid and mixed diploid–polyploid populations. Allotriploid females, which co-exist with C. taenia or C. elongatoides and a few tetraploid males and females dominate in most Cobitis mixed populations. They reproduce gynogenetically and produce triploid eggs that are stimulated to development by sperm from Cobitis males. Some of these eggs are fertilized, which leads to the production of bisexual tetraploids. Males of C. taenia (2n = 48) from a diploid population in Lake Klawój, Northern Poland (46 individuals) and from a mixed Cobitis population in the Bug River, Eastern Poland (7 individuals), and three tetraploid males (4n = 98) from the same mixed population were examined. All the fish were analyzed karyologically and histologically. Tubules with cysts of the testes of C. taenia from both populations were filled with germ cells at various developmental stages. Among fishes from Lake Klawój sperm maturation in batches simultaneous with the batch spawning of C. taenia females was found. The testes of the loach C. taenia, from a mixed population in the Bug River, were filled with spermatozoa over the entire reproductive season. Sperm maturation in batches was not observed. Sperm maturation in batches seems to be only connected with a few diploid males in this population. So, a continuous process of spermatogenesis in their testes is required. Only in the testes of all tetraploid Cobitis males were cells characteristic of the early stages of spermatogenesis observed, i.e. without spermatids and spermatozoa. Furthermore, the histological sections of the testis of a male captured in August, revealed fragments with connective tissue between the germ cells. However the participation of tetraploid, infertile Cobitis males in the process of reproduction in the investigated mixed population remains controversial. The results obtained so far, reveal that even the infertile sperm of tetraploid males may induce gynogenesis in Cobitis triploid females.     

Comparative morphology and histology of the ovaries of the spined loach Cobitis taenia L. and natural allopolyploids of Cobitis (Cobitidae)

The maturity of ovaries, gonado-somatic index and size of oocytes in particular developmental stages during the reproductive period of spined loach Cobitis taenia and allotriploid and allotetraploid Cobitis were examined. The ploidy levels of all the loaches were determined according to karyotypes. All the investigated females spawned multiple times and the spawning period was from the end of May (water temperature c. 18·5° C) to mid- or the end of July. Some of the triploids had a longer spawning period which lasted until the end of August. Each female could lay several portions of eggs which differed in size and number during the spawning season. The relative proportion of oocytes of each stage in the ovaries during the reproductive period changed. The sizes of oocytes at the particular stages of triploids were statistically significantly larger than oocytes in the respective stages in C. taenia (diploid). The sizes of the oocytes (each stage) of C. taenia from both populations were not significantly different.     

Biochemical genetic evidence for a hybrid origin of spined loach, Cobitis taenia taenia, in Japan

Starch gel electrophoresis and isoelectric focusing were performed on sarcoplasmic proteins from three taxa of cobitid fish in Japan, Cobitis biwae , C. taenia striata and C. l. taenia . These taxa are hardly distinguishable from each other by external appearance or morphological characters. Electrophoretic patterns of sarcoplasmic proteins from white muscle suggested fixed allelic differences between C. biwae and C. t. striata at two protein-coding loci (parvalbumin and creatine kinase). The third taxon, C. t. taenia , showed composite banding patterns with respect to the above two proteins, suggesting C. t, taenia originated from hybridization between C. biwae and C, t. striata . Previously, C. t, taenia and C. t. striata were believed to have shared a common ancestor subsequent to their divergence from C. biwae .     

Diverse processes shape deep phylogeographical divergence in Cobitis sinensis (Teleostei: Cobitidae) in East Asia

Little has been known about the impacts of past vicariance events on the phylogeography and population structure of freshwater fishes in East Asia. The aims of this study are to assess the genetic variability with extensive sampling throughout the range of Chinese spiny loach, Cobitis sinensis, and to infer the genetic structure and evolutionary history of populations. Cobitis sinensis in China may have initiated from two ancestral populations, namely Yangtze and Pearl Rivers, which diverged about 7.24 MYA likely due to drainage systems alteration. In the phylogroup I, a southward dispersal event occurred from East China (Yangtze River) to south ZheMin and Hainan subregions, followed by eastward dispersal from ZheMin to south Taiwan. In the phylogroup II, eastward colonization took place from Pearl River to north Taiwan in the late Pliocene, coupled with loss of genetic diversity in the island populations. This study showed that Cenozoic tectonic movements and climatic and sea‐level fluctuations may have shaped the genetic structure of C. sinensis in concert. Highly diverged mtDNA sequences suggest existence of cryptic species in morphospecies C. sinensis.     

Iksookimia yongdokensis, a new cobitid fish (Pisces: Cobitidae) from Korea with a key to the species ofIksookimia

A new cobitid fish,Iksookimia yongdokensis, is described from rivers flowing to the southeastern coast of Korea. The species is distinguishable from its congeners by the following characteristics: reduced laminar circularis at the base of the pectoral fins in males; color pattern including 9–13 vertical bars on the body; 13–14 gill rakers; 17–20 caudal vertebrae; and small scales with a large focal area. Its unique discontinuous distribution on the Korean Peninsula is discussed and the biogeographic implications considered.     

Antennatus flagellatus (Teleostei: Antennariidae), a new species of frogfish from southern Japan

A new species of frogfish,Antennatus flagellatus, is described on the basis of two specimens from Kashiwajima Island, Kochi Pref., southern Japan. It is distinguished from congeneric species as follows: illicium long, more than 40% SL and about 3.6 times length of second dorsal spine; caudal peduncle present.     

Evidence of a secondary interspecific mitochondrial DNA introgression in the pond loach Misgurnus dabryanus (Teleostei: Cobitidae) population introduced in Japan

The pond loach Misgurnus dabryanus is a freshwater fish with a distribution range spanning the eastern part of the Asian continent, the Korean Peninsula, and Taiwan. The pond loach was transplanted to the Japanese archipelago through the co-inclusion with dojo loach (Misgurnus anguillicaudatus species complex) populations, which were imported live from China for food materials, and it is currently distributed widely across Japan. A previous mitochondrial DNA (mtDNA) analysis revealed that a pond loach population in Ehime Prefecture (Shikoku Island, Japan) included two highly diverged mtDNA groups (Groups I and II). To examine the origin of these two distinct forms of mtDNA within the Japanese pond loach population, we performed phylogenetic analyses using sequences based on the mtDNA of cytochrome oxidase b (cyt b) and the nuclear DNA recombination activating gene 1 (RAG-1). We also conducted a random amplified polymorphic DNA (RAPD) analysis to examine the establishment of reproductive isolation between sympatric pond loaches with two different mtDNA groups. Our mtDNA phylogenetic results indicated that the two diverged pond loach mtDNA sequences showed polyphyletic relationships among Misgurnus species and its related genus Cobitis. In contrast, there were no clear divergence in nuclear DNA among the pond loaches irrespective of their mtDNA groups, and they all formed monomorphic clades in the phylogenetic relationships among the species. The discrepancy between the mtDNA and nuclear DNA genes support that the existence of two diverged forms of DNA within the pond loach population could be attributed to past mtDNA introgressions from other species rather than convergent evolution. Previous mtDNA phylogenetic studies among Cobitidae revealed that the dojo loach also consisted of two genetically diverged polyphyletic clades: an original Misgurnus mtDNA and an introgressed mtDNA from Cobitis species. In our mtDNA result, the Group II haplotype of the pond loach was included in the mtDNA from the introgressed dojo loach. This suggested that the Group II haplotype was derived from introgressed dojo loach mtDNA. The close relationships between the introgressed dojo loach and the pond loach mtDNA indicated that this secondary introgression had recently occurred via hybridization in a recent artificial aquaculture or transportation process. Common RAG-1 alleles and RAPD bands were shared between the sympatric pond loaches with original and introgressed mtDNAs. This indicates that the introgressed mtDNA haplotype is included as one of the polymorphic genotypes within the pond loach populations, and does not represent existence of different cryptic species.     

Chromosomal inheritance of parental rDNAs distribution pattern detected by FISH in diploid F1 hybrid progeny of Cobitis (Teleostei,Cobitidae) species has non-Mendelian character

This study was conducted to describe the major and the minor rDNA chromosome distribution in the spined loach Cobitis taenia (2n = 48) and the Danubian loach Cobitis elongatoides (2n = 50), and their laboratory-produced diploid reciprocal F₁ hybrid progeny. It was tested by fluorescence in situ hybridisation (FISH) whether the number of 28s and 5s rDNA sites in the karyotypes of diploid hybrids corresponds to the expectations resulting from Mendelian ratio and if nucleolar organiser regions (NOR)were inherited from both parents or nucleolar dominance can be observed in the induced F₁ hybrid progeny. Ten (females) or twelve (males) 28s rDNA loci were located in nine uniarm chromosomes of C. taenia. Two of such loci terminally bounded on one acrocentric chromosome were unique and indicated as specific for this species. Large 5s rDNA clusters were located on two acrocentric chromosomes. In C. elongatoides of both sexes, six NOR sites in terminal regions on six meta-submetacentric chromosomes and two 5s rDNA sites on large submetacentrics were detected. The F₁ hybrid progeny (2n = 49) was characterised by the intermediate karyotype with the sites of ribosome synthesis on chromosomes inherited from both parents without showing nucleolar dominance. 5s rDNA sites were detected on large submetacentric and two acrocentric chromosomes. The observed number of both 28s and 5s rDNAs signals in F₁ diploid Cobitis hybrids was disproportionally inherited from the two parental species, showing inconsistency with the Mendelian ratios. The presented rDNA patterns indicate some marker chromosomes that allow the species of the parental male and female to be recognised in hybrid progeny. The 5s rDNA was found to be a particularly effective diagnostic marker of C. elongatoides to partially discern genomic composition of diploid Cobitis hybrids and presumably allopolyploids resulting from their backcrossing with one of the parental species. Thus, the current study provides insight into the extent of rDNA heredity in Cobitis chromosomes and their cytotaxonomic character.     

Spawning habitat in the spined loach, <Emphasis Type="Italic">Cobitis taenia</Emphasis> (Cypriniformes: Cobitidae)

Spawning habitat of the endangered spined loach Cobitis taenia was studied in northwestern Germany. The distribution of eggs of the loach in the field was best correlated with the density of vegetation but had little correlation with current velocity, water depth, or bottom substrate. In the aquarium, the loach chose dense vegetation for an oviposition site, as inferred by the direct observations of spawning and the location of spawned eggs. It is concluded that such habitat specialization is an important element in the autecology of the endangered fish and a critical stage in the habitat requirements of its populations.     

A new cottid species, Icelus sekii (Perciformes: Cottoidei), from Hokkaido, Japan

A new cottid species, Icelus sekii, is described on the basis of six specimens collected from off Rausu and Urakawa, Hokkaido Island, Japan. This species is distinguished from its congeners by the following combination of characters: supraocular and parietal spines absent; nuchal spine obscure; uppermost preopercular spine unbranched; no scales between dorsal scale row and lateral line scale row, and no scales below lateral line scale row; supraocular, parietal, and nuchal cirri present; five dark brown saddles dorsolaterally; anal fin rays 13; pectoral fin rays 15; vertebrae 12 + 24–25 = 36–37. Icelus sekii can be mature at the smallest size among the species of Icelus. As a secondary sexual character, the male holotype has unique ensiform flaps on the distal tips of the first dorsal fin.     

A new species of armored sea robin, Paraheminodus kamoharai (Teleostei: Peristediidae), from the Philippines

A new peristediid, Paraheminodus kamoharai, is described from three specimens (106.9–114.8mm SL) collected from the Sulu Sea, the Philippines. The new species is easily distinguished from its congeners (Paraheminodus laticephalus and P. murrayi) in having 33 bony plates in the upper lateral row, the 24–25th to 30th bony plate each in the upper lateral row with a forwardly directed spine, 17–19 gill rakers on the lower limb, a long slender rostral projection (43.4–47.0% of head length), and short upper (41.1–42.3%) and lower jaws (36.5–37.2%).     

A new species of scabbardfish, Evoxymetopon macrophthalmus (Scombroidei: Trichiuridae), from Okinawa, Japan

Evoxymetopon macrophthalmus sp. nov. is described on the basis of a single specimen collected off Okinawa Island, Ryukyu Islands, Japan. Among the two valid species of Evoxymetopon, the new species is more similar to E. poeyi in having the first dorsal-fin spine enormously large, compressed, and sword shaped. It differs from E. poeyi in having the upper profile of head uniformly convex without a prominent sagittal crest (vs. with a prominent sagittal crest), tip of first anal pterygiophore connected to the last rib in the anterior part, while the first hemal spine is long and connected to the posterior margin of pterygiophore bone plate (vs. connected to the first hemal spine only), a larger eye, 4.5 times in head length (vs. 5.5–5.9 times), 15 externally visible anal-fin soft rays (vs. 20), 9 upper gill rakers of the first arch (vs. 4–6), and 60 caudal vertebrae (vs. 64).     

Cryptic clonal lineages and genetic diversity in the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from nuclear and mitochondrial DNA analyses

In the loach Misgurnus anguillicaudatue, the asexual lineage, which produces unreduced clonal diploid eggs, has been identified. Among 833 specimens collected from 54 localities in Japan and two localities in China, 82 candidates of other lineage(s) of cryptic clones were screened by examining RFLP (restriction fragment length polymorphism)-PCR haplotypes in the control region of mtDNA. This analysis was performed because triploid loaches arise from the accidental incorporation of the sperm nucleus into unreduced diploid eggs of a clone. The categorization of members belonging to three newly identified lineages (clones 2–4) and the previously identified clonal lineage (clone 1) was verified by evaluating the genetic identity between two or more individuals from each clonal lineage based on RAPD (random amplified polymorphic DNA)-PCR and multilocus DNA fingerprints. We detected 75 haplotypes by observing the nucleotide status at variable sites from the control region of mtDNA. Phylogenic trees constructed from such sequences showed two highly diversified clades, A and B, that were beyond the level common for interspecific genetic differentiation. That result suggests that M. anguillicaudatus in Japan is not a single species entity. Two clone-specific mtDNA sequences were included in clade A, and the loaches with such sequences may be the maternal origin of the clones. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

中国广西盲鳅一新种——环江高原鳅(英文)

2007年4月,在广西壮族自治区环江县川山镇乐衣村附近一洞穴采集到一批盲鳅类标本。经鉴定,为高原鳅属(Triplophysa)鱼类一新种,被命名为环江高原鳅(Triplophysa huangjiangensis)。该新种以以下组合特征与高原鳅属的其他已知种类相区别:无眼;通体无鳞;尾鳍分叉;尾柄上、下叶具发达的软鳍褶;背鳍后缘平截;背鳍起点距尾鳍基的距离短于距吻端的距离;背鳍分枝鳍条数为8~9;胸鳍分枝鳍条数为10~11;腹鳍分枝鳍条数为6~7;臀鳍分枝鳍条数为6;尾鳍分枝数为13~14;胸鳍长为胸鳍起点至腹鳍起点间距的52.3%~70.7%;腹鳍起点位于背鳍起点之前下方;外侧吻须最长,为侧面头长的47.0%~73.8%;前鼻孔位于一短管中,末端延长呈须状;鳔后室发达,末端可伸达腹鳍起点。     

Phylogeography and the maternal origin of the tetraploid form of the Japanese spined loach, <Emphasis Type="Italic">Cobitis biwae</Emphasis>, revealed by mitochondrial DNA analysis

The Japanese spined loach Cobitis biwae includes the tetraploid form, which has 96 chromosomes, as well as the diploid form, which has 48 chromosomes. In the present study, we analyzed the mitochondrial DNA (mtDNA) to examine the genetic relationships among 82 populations of diploid–tetraploid complexes of C. biwae. Restriction fragment length polymorphism (RFLP) analysis of the ND1 region on 202 individuals revealed that C. biwae contains three genetically divergent major groups corresponding to geographical proximities (the Eastern, Western, and Kochi groups). Phylogenetic analyses (neighbor-joining, NJ and maximum-parsimony, MP) of a part of the cytochrome b gene sequence (748bp) in 31 individuals supported the three major groups recognized by RFLP, and indicated considerable genetic differentiation between the Western group and the other two groups (average, 15.2%). The genetic relationship and distribution pattern of the three major groups hypothesized two major dispersions of C. biwae during the middle Miocene: first, the ancestor of the Eastern and Kochi groups had spread from the West through wide regions of present-day Honshu and Shikoku Islands, and following that period, the ancestor of the Western group with a different mtDNA composition, probably resulting from the mtDNA introgression from C. takatsuensis, moved into western Japan and pushed the former north eastward. All tetraploid form populations were included in the Western group and treated as a monophyletic cluster with low genetic divergence. It is notable that two diploid populations geographically adjacent to the tetraploid range were genetically closely related to the tetraploid forms. This result suggests that these diploid populations were directly related to the maternal origin of the tetraploid form.