Spined Loach Research Articles

Threatened Fishes of the World, Cobitis puncticulata (Erk’akan, Atalay-Ekmekçi & Nalbant, 1998) (Cobitidae)

Keywords Cobitis puncticulata.Brownspinedloach.Distribution.WesternAnatoliaCommon name: Brown spined loach (English) Kah-verengi copcu baligi (Turkish). Conservation status:Critically Endangered (IUCN 2009). Identification:Brownish body, consisting only of small dark-greydots through the body and head and blotches of Z2and Z4 (Gambetta 1934) are very small, indistinct orconfluent (Kottelat and Freyhof 2007). A strong keelis present above and below the caudal peduncle.Sexually dimorphic; males have one lamina circularisor scale of Canaestrini in the second pectoral fin ray(Erkakan et al. 1998). Females up to78 mm SL, malesare smaller. Distribution: Apart from the Kocacay andthe Karadere from the Manyas (Kus) Lake drainage(Erk’akan and Ekmekci 2000), recorded from theRiver Evros in Greece (Thrace) and in the UluabatLake catchment in the east of Manyas Lake (Freyhofet al. 2008). In 2005, we found this species inBakacak Stream a tributary of the Kocabas drainage(Marmara Sea) in Canakkale (Ekmekci et al. 2008).Abundance: Recorded as rare by IUCN (Crivelli 2006).Habitat and ecology: Still to moderate flowingstreams, with muddy or sand substrate. Reproduction:No data, only known to spawn in spring. Threats:InTurkey, pollution and flow regulation are the mostimportant threats, with decline of the habitat qualityand further loss of habitat. No threats were recorded inthe lower Evros. Conservation recommendations:Water level change causing habitat loss should becontrolled. Detailed information is required on thepopulation size and biology. Remarks: After recordingC. puncticulata from two new localities, Freyhof et al.(2008) suggested changing the status to “vulnerable”.We have recently extend the range of this species to a

Ichthyofauna of the rivers in the Romincka Forest (Pregola River basin, northeastern Poland)

The composition and structure of the ichthyofauna of the rivers of the Romincka Forest (Pregola River basin, northeastern Poland) was investigated. Twenty-two fish species were confirmed to occur, four of which are protected: bullhead, Cottus gobio L.; spined loach, Cobitis taenia L.; bitterling, Rhodeus sericeus (Pall.); stone loach, Barbatula barbatula (L.). Species richness was the highest in the Bludzia River, in which 17 fish species belonging to 6 ecological reproductive guilds were confirmed. The ichthyofauna of the other rivers in the forest generally comprised 6-10 fish species belonging to 4-5 ecological reproductive guilds. The number of fish occurring at sampling sites decreased with increased river gradient, while the number of rheophilic species increased.

Characterization of polymorphic microsatellite loci in the endangered Miho spine loach (Iksookimia choii) and cross‐species amplification within the Cobitidae family

The Miho spine loach (Iksookimia choii) is an endangered freshwater fish endemic to Korea. To aid a conservation programme for this species, 20 polymorphic microsatellite loci were isolated and characterized in 67 individuals from three populations. The number of alleles per locus ranged from 8 to 33. Within populations, the observed and expected heterozygosities ranged from 0.59 to 1.00 and from 0.56 to 0.96, respectively, indicating its usefulness in population genetics studies. Cross-species amplification of the loci was tested in six species of the family Cobitidae, and 18 loci were amplified in two or more related species.

Study of Fish Fauna in Tamjin River on Jangheung Dam Construction

In order to perform environmental impact assessment on Jangheung Dam construction, we investigated fish fauna and community from February to November 2005. Fishes were collected seasonally in 3 sites at upstream and 5 sites at downstream of the dam using cast net and kick net. A total number of fish caught from the study area was 8,733 fish representing 15 families and 45 species, and Cyprinidae and Gobiidae Families had twenty-four (53.3%) and four (8.9%) species, respectively. However, rest of families composed of one to three species. There was the 2nd-class endangered species, Sand lamprey (Lampetra reissneri), found in the upstream (Site 3) of the dam, and fourteen Korean endemic species (31.1%) including southern king spine loach (Iksookimia hugowolfeldi). In the mean time, one foreign introduced species, Crucian carp. (Carassius cuvieri) was mostly found in upstream of the dam among the 45 species. The number of species in each site ranged 20 to 34 species, and it tended to increase toward to downstream. The species of pale chub (Zacco platypus, 43.5% relative abundance) and dark chub (Zacco temminckii, 14.7%) were dominant and subdominant species, and 14 species including eel (Anguilla japonica) comprised less than 0.1% relative abundance (rare species) of the total catch. Compared to assessments in previous years (1998~2004), the number of species found in 2005 was the maximum. Fifteen species were not confirmed at this year even if they were found in previous years, and some of them including hickory shad (Konosirus punctatus) were peripheral species of fish collected in only single year. Whereas three species (Lampetra reissneri, Carassius cuvieri, Hemiculter eigenmanni) was confirmed for the first time in 2005. Based on the results, modification of fish fauna and community in Tamjin River watershed should be monitored in detail in order to assess any negative effect due to dam construction, and additional investigation for the endangered species needs to be performed to establish conservation of their habitat and population.

Development of nine polymorphic microsatellite loci in the spined loach, Cobitis taenia, and cross‐species amplification in the related species C. elongatoides, C. taurica and C. tanaitica

Nine polymorphic microsatellite loci were developed for the spined loach, Cobitis taenia (Teleostei: Cobitidae). The loci were validated using 50 individuals from a population in Belgium. Moderate to high levels of polymorphism were detected (two to 11 alleles). In addition, most markers amplified successfully in three closely related taxa that are known to hybridize with C. taenia: C. elongatoides, C. taurica and C. tanaitica. Some of the loci are most likely diagnostic among species. These markers will be valuable for the study of the historical and contemporary interactions within C. taenia and the Cobitis species complex.

Making it on their own: sperm-dependent hybrid fishes (Cobitis) switch the sexual hosts and expand beyond the ranges of their original sperm donors

Interspecific hybridization may result in asexual hybrid lineages that reproduce via parthenogenesis. Contrary to true parthenogens, sperm-dependent asexuals (gynogens and hybridogens) are restricted to the range of bisexual species, generally the parental taxa, by their need for a sperm donor. It has been documented that asexual lineages may rarely use sperm from a non-parental species or even switch a host. The available literature reports do not allow distinguishing, between whether such host switches arise by the expansion of asexuals out of their parental's range (and into that of another's) or by the local extinction of a parental population followed by a host switch. The present study combines new and previously collected data on the distribution and history of gynogenetic spined loaches (Cobitis) of hybrid origin. We identified at least three clonal lineages that have independently switched their sperm dependency to different non-parental Cobitis species, and in cases incorporated their genomes. Our current knowledge of European Cobitis species and their hybrids suggests that this pattern most probably results from the expansion of gynogenetic lineages into new areas. Such expansion was independent of the original parental species. This suggests that sperm dependence is not as restrictive to geographical expansion when compared with true parthenogenesis as previously thought.

Geographic differentiation of Cobitis shikokuensis inferred from mtDNA RFLP analysis

Mitochondrial DNA divergence among populations of the Japanese spinous loach Cobitis shikokuensis, endemic to Shikoku Island, was investigated by restricted fragment length polymorphism analysis. A total of 68 restriction sites on DNA fragments from the cytochrome b to D-loop regions and from the 12S rRNA to 16S rRNA regions, amplified by PCR, were analyzed. A total of 12 haplotypes (plus 6 in outgroups) were detected in 268 specimens collected from 19 localities in seven rivers (and 41 specimens from four localities in three rivers in outgroups). Three of the seven river populations of C. shikokuensis were shown to have unique haplotypes, and four of the seven river populations were monomorphic. The nested structure of the haplotype network for populations of C. shikokuensis exhibited two large clades corresponding to (1) populations from the Shimanto River and its neighbors and (2) two genetically divergent populations in the Shigenobu and Iwamatsu Rivers. The population from the Shimanto River, the largest river inhabited by C. shikokuensis, maintains great haplotype diversity as well as the allozyme diversity previously reported. On the other hand, populations from the Hiji River, the second largest river, which exhibited the highest allozyme diversity, were monomorphic in their mtDNA. The nested clade analysis (NCA) revealed that past fragmentation between the above two clades could occur in the initial distribution process of C. shikokuensis. The large genetic divergence of two river populations from the Shigenobu and Iwamatsu Rivers was inferred to be caused by a process of long distance colonization and fragmentation. MtDNA introgression into the Hiji River population from southern river populations was suggested. Taking genetic divergence into consideration, each river population of C. shikokuensis should be conserved separately as like a distinct species, and conservation programs for the small populations showing less genetic variability should be invoked as soon as possible.

Diversity of European spined loaches (genus Cobitis L.): an update of the geographic distribution of the Cobitis taenia hybrid complex with a description of new molecular tools for species and hybrid determination

Although the unique features of asexual reproduction and hybridization among European spined loaches (genus Cobitis) have recently attracted the attention of conservation biologists, faunists and evolutionary biologists, the research has suffered from uncertain identification of specimens and their genomes because of the extreme morphological similarity of all the species within the hybrid complex. In this article, a Europe‐wide study is reported, which was performed on samples collected by several research teams. Several complementary methodologies, such as allozyme analysis, karyotyping, flow cytometry and DNA sequencing allowed us to confirm or reject the existence of all previously reported species and their hybrids as well as to uncover several new hybrid biotypes. The biogeography of all the known biotypes, that is, parental species and hybrid biotypes, has been summarized here and the taxonomic position of two undescribed putative species mentioned in previous publications has been established. New polymerase chain reaction restriction fragment length polymorphism markers for species determination have further been developed and applied, which would allow the unambiguous identification of parental species and their genomes in the known hybrid biotypes within the complex.

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.

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (Cobitis: Teleostei), and their ability to establish successful clonal lineages—on the evolution of polyploidy in asexual vertebrates

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides x C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.

Performance of a pool-and-weir fish pass for small bottom-dwelling freshwater fish species in a regulated lowland river

Abstract Although small benthic freshwater fish species are an important biological component of fish assemblages and free instream movement is indispensable for their survival, they are often neglected in fish pass performance studies. In this study, a capture-mark-recapture approach was used to assess whether small bottom-dwelling species, including gudgeon (Gobio gobio), stone loach (Barbatula barbatula), spined loach (Cobitis taenia) and bullhead (Cottus gobio), were able to cross a pool-and-weir fish pass in a regulated lowland river. Some tagged individuals of stone loach (18%), gudgeon (7%) and spined loach (2%) managed to successfully ascend the fish pass under study, despite the fact that water velocity levels in the different overflows of the facility (between 0.55-1.22 m/s) exceeded the critical swimming speed of all three species. Although this suggests that a pool-and-weir fish pass is a able to facilitate upstream movement of some small benthic species in a regulated river, more detailed research incorporating advanced tagging and retrieving techniques is necessary.

Cobitis pontica sp. nova—a new spined loach species (Cobitidae) from the Bulgarian waters

Spined loaches from the Veleka River (Black Sea coast of Bulgaria), earlier confirmed to represent a separate evolution lineage within a 50-chromosome morph occurring in the Black Sea basin, are described as a new species. This species has the karyotype (2n = 50, NF = 90) similar to one from earlier described C. taurica occurring in the Crimean Peninsula, but differs from it by larger and less numerous spots in the fourth Gambetta’s zone of pigmentation and more anterior position of suborbital spine.

A new loach, Cobitis shikokuensis (Teleostei: Cobitidae), from Shikoku Island, Japan

A new species of spinous loach, Cobitis shikokuensis, is described based on 297 specimens from Shikoku Island, Japan. The new species was formerly known as the Shikoku group of Cobitis takatsuensis. It can be distinguished from other species of Cobitis and closely related genera by a combination of the following characters: dorsal fin with 6 branched soft rays; anal fin with 5 branched soft rays; one brownish streak across eye from the tip of nose, no streak on cheek; a black spot smaller than eye diameter near the dorsal corner of the caudal fin base; 3–5 small brownish speckles on ventral side of caudal peduncle; high caudal peduncle with well-developed fleshy keels on dorsal and ventral side; a lamina circularis at base of dorsal part of pectoral fin absent; first branched soft ray of pectoral fin broad in males; pectoral soft rays widely branched from the approximate midpoint; last anal fin ray with 2 elements; interorbital width 11.2–17.1% of head length.

Dispersal of invading trematodes Nicolla skrjabini (Iwanitzky, 1928) and Plagioporus skrjabini Kowal, 1951 (Trematoda: Opecoelidae) in the Volga

The species compositions of fish trematodes in the neighboring Volga and Don basins are similar in many ways, however, there are some differences. One difference is that trematodes of the genera Nicolla and Plagioporus were not previously recorded in the Volga basin. Their absence was accounted for by the fact that their initial hosts, snails of the genera Lithoglyphus (Gastropoda: Lithoglyphidae) and Theodoxus (Gastropoda: Neritidae), did not occur in the Volga until recently. At present, these snails, trematodes, and some other parasites have successfully naturalized in the Volga and actively expand their ranges in its basin (Zhokhov and Pugacheva, 2001). Here, we present new data on the dispersal of these invaders in the Volga basin. The distribution of the trematode Nicolla skrjabini Iwanitzki, 1928 is limited to the rivers of the Azov‐ Black Sea and Baltic basins. That of the trematode Plagioporus skrjabini Kowal, 1951 is confined to rivers of the Azov‐Black Sea basin alone. The life cycles of these parasites are known. For N. skrjabini , the first intermediate host is the snail Lithoglyphus natocoides Pfeiffer, and the second intermediate host (according to experimental data) is Gammarus balcanicus Schaferna (Sten’ko, 1976). This trematode has a wide range of definitive hosts that comprises 27 fish species. In P. skrjabini , the snail Theodoxus fluviatilis and six gammarid species are involved in the life cycle (Chernogorenko et al., 1978). The definitive hosts of P. skrjabini are Cobitis taenia L., Neogobius fluviatilis Pall., and Mesogobius batrachocephalus Pall. In 1952, the Volga‐Don connected the two river basins, and the snails with associated trematodes found an opportunity to invade the Volga. The snails of the genus Lithoglyphus appeared in the lower Volga in the late 1960s (Belyavslaya and Vyushkova, 1971). In 1971, they were already numerous in the Volga delta (Pirogov, 1972). At present, these snails occur almost all over the Volga delta and form very dense aggregations (up to 11 180 ind./m 2 ) in the upper part of the delta and in arms of its western part (Biserova, 1990, 1996;

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid-polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid-polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3-6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.

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 Klawoj, 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 Klawoj 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.

European colonization by the spined loach (Cobitis taenia) from Ponto‐Caspian refugia based on mitochondrial DNA variation

In the last 20 years, new species, asexual reproduction, polyploidy and hybridization have all been reported within the genus Cobitis. An understanding of the current distribution and baseline phylogeographical history of 'true' nonhybrid Cobitis species is crucial in order to unravel these discoveries. In the present work, we investigated the phylogeography of the spined loach, Cobitis taenia, using 1126 bp of the mitochondrial cytochrome b gene from 174 individuals collected at 47 sites. In total, 51 haplotypes that differed at 49 positions (4.35%) were detected. We deduce that C. taenia survived European glaciations in at least three refugees in the Ponto-Caspian area. Two of these refugees each provided a major lineage that recolonized Europe in separate directions: one westward to England and the other spreading north into Russia before moving west. A third (minor) lineage that contributed little to the recolonization of Europe was also revealed--remaining near its Black Sea refuge. However, more recent history was difficult to resolve with colonization from a more western refugium during the last glacial maximum (LGM) a distinct possibility. Nested clade analysis indicates a pattern of restricted gene flow with isolation by distance at the first two levels and overall. Unlike many other European freshwater fish species, the Danube is not part of the current distribution of C. taenia, nor was it used as either a refuge or a source of colonization of Europe. Low genetic diversity within C. taenia suggests that its colonization of Europe is relatively recent. Demographic analyses revealed a history of recent expansion and isolation by distance.

Phylogenetic relationships among the Ibero-African cobitids ( Cobitis, cobitidae) based on cytochrome b sequence data

We estimated the phylogenetic relationships of all Ibero-African spined loaches of the genus Cobitis using the complete mitochondrial cytochrome b gene (1140 bp). We analysed 93 individuals of seven cobitid species found in all the principal drainages of the Iberian Peninsula and North Africa. A molecular phylogeny was used to revise current systematics of the Ibero-African Cobitis species and to infer a biogeographical model for Cobitis in the Western Mediterranean area during the Cenozoic period. Phylogenetic analysis provided support for the monophyly of two mtDNA clades: Clade A or Italian Clade with the Italian species ( C. bilineata, C. zanandreai), and Clade B or the Ibero-African Clade. The Ibero-African Clade included all species endemic for the Iberian Peninsula ( C. vettonica, C. calderoni, and C. paludica) and North Africa ( C. maroccana). The species C. paludica does not constitute a natural group, and could be divided into at least four monophyletic mtDNA lineages with moderate to high bootstrap values and posterior probability support. Phylogenetic relationships of the Ibero-African species were not resolved satisfactorily, but in all analyses C. calderoni from Northern Iberian Peninsula was basal. We have re-calibrated a molecular clock for the genus Cobitis (0.68% per million year by pairwise) using populations inhabiting both sides of the Gibraltar Strait. Application of this Cobitis mtDNA clock provides evidence that the Messinian salinity crisis played a primary role in the diversification of some Ibero-African cobitid species. The basal polytomies of the Ibero-African Clade might suggest that all mtDNA lineages diversified rapidly.

Ice age cloning – comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation

Recent advances in population history reconstruction offered a powerful tool for comparisons of the abilities of sexual and clonal forms to respond to Quaternary climatic oscillations, ultimately leading to inferences about the advantages and disadvantages of a given mode of reproduction. We reconstructed the Quaternary historical biogeography of the sexual parental species and clonal hybrid lineages within the Europe-wide hybrid complex of Cobitis spiny loaches. Cobitis elongatoides and Cobitis taenia recolonizing Europe from separated refuges met in central Europe and the Pontic region giving rise to hybrid lineages during the Holocene. Cobitis elongatoides due to its long-term reproductive contact with the remaining parental species of the complex--C. tanaitica and C. spec.--gave rise to two clonal hybrid lineages probably during the last interglacial or even earlier, which survived the Würmian glaciation with C. elongatoides. These lineages followed C. elongatoides postglacial expansion and probably decreased its dispersal rate. Our data indicate the frequent origins of asexuality irrespective of the parental populations involved and the comparable dispersal potential of diploid and triploid lineages.

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.