Examination of unidentifiable spined loach individuals found in the overlap zone of two tetraploid species within a single river in Japan

An endangered tetraploid spined loach species, Cobitis takenoi (Cypriniformes: Cobitidae; hereafter called Tango loach) is known to inhabit only a single river in Kyoto Prefecture, Japan. Since Tango loach was discovered recently, in 2010, and only described in 2016, its morphology, ecology, and genetics are not well studied. Another tetraploid spined loach species Cobitis sp. BIWAE type A (hereafter, called Ohshima loach) inhabits the same river. The two loaches are reported as morphologically distinguishable from each other. Although the habitats of the two species in the river are segregated (Ohshima loach and Tango loach inhabit the upper and lower reaches, respectively), they overlap to a small degree in the boundary area. Recently, some individuals with morphological characteristics that are intermediate between the two species were found in the overlap zone. It was suspected that hybrids between the two species were produced since breeding seasons of the two species overlapped. To investigate whether the two species produce hybrids, we performed mitochondrial and nuclear DNA analyses on the unidentifiable individuals. Eight individuals unidentifiable to the species level collected in the river between 2017 and 2018 were examined and compared with the Tango and Ohshima loach species. Using mitochondrial DNA (mtDNA) cytochrome b analysis, we found that six individuals had mtDNA types identical to Tango loach and two individuals had mtDNA types identical to Ohshima loach. Furthermore, sequencing analysis of nuclear recombination activating gene 1 (RAG-1) revealed that each species had species-specific alleles. The phylogenetic analysis indicated that alleles in Tango loach were divided into two clusters and those from Ohshima loach formed a single cluster. There were no discrepancies in the combination between mtDNA and nuclear DNA species types within each specimen. DNA fingerprinting analysis (AFLP) showed that the species-unidentifiable individuals exhibited distinctly segregated genetic groups corresponding with Tango and Ohshima loaches. In summary, no hybrids were detected from among any unidentifiable individual examined in this study. New conventional genetic method for discriminating the two sympatric loach species developed here can be effective tool for the conservation of the Tango loach since there was no strict diagnostic morphological character between them.     

Molecular and morphological analyses revealed a cryptic species of dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) in Japan

Although it has been reported that populations of the Japanese dojo loach Misgurnus anguillicaudatus (Cypriniformes: Cobitidae) belong to two distinct mitochondrial (mt)DNA (Type I and Type II), the taxonomic status of the species remains unresolved. To address this question, nuclear DNA and morphological analyses were performed on M. anguillicaudatus population in the Nakaikemi Wetland, where Type I and Type II lineages are sympatric. Results suggest the existence of a cryptic species (Type I) within the Japanese dojo loach.     

Molecular phylogeny and intraspecific structure of loaches (genera <Emphasis Type="Italic">Cobitis</Emphasis> and <Emphasis Type="Italic">Misgurnus</Emphasis>) from the Far East region of Russia and some conclusions on their systematics

Russian Far East loaches of the genera Cobitis and Misgurnus are among members of the family Cobitidae with poorly understood systematics. In this study we present phylogenetic hypotheses based on mitochondrial (cytochrome b) and nuclear (RAG-1) sequences. All analyses recovered comparable topological phylogenies, and all data sets supported the non-monophyly of the genera Cobitis and Misgurnus. Both genera are represented by multiple lineages that in some cases do not correspond to the species described. We found some phylogenetic incongruities for the genus Misgurnus (M. mohoity and M. anguillicaudatus) that are explained by ancient hybridization, as was suggested previously for M. anguillicaudatus. The revealed phylogenetic relationships suggest that Paramisgurnus should be treated as a synonym of Misgurnus and M. bipartitus as a synonym of M. mohoity. All analyses recovered C. choii as a member of the genus Cobitis, confirming previous taxonomic conclusions. Most of the molecular lineages found follow currently recognized taxa with some exceptions, such as M. anguillicaudatus and C. lutheri. Phylogenetic relationships recover several unrelated lineages of M. anguillicaudatus and suggest additional studies to solve current taxonomic uncertainty. We found that C. lutheri is a non-natural group that contains two unrelated lineages: specimens of C. lutheri from the Far East of Russia collected close to the type locality and a second lineage with specimens of C. lutheri from Korea, the identification of which must be revised. The study provides evidence of the presence of the Far East species M. nikolskyi in Sakhalin Island, but simultaneously shows conspicuous genetic distinctiveness between the island and the mainland populations.     

Comparative and evolutionary analysis in natural diploid and tetraploid weather loach Misgurnus anguillicaudatus based on cytochrome b sequence data in central China

To obtain the phylogenetic relationship between diploid and tetraploid Misgurnus anguillicaudatus, the mitochondrial cyt b gene in the diploid and tetraploid weather loach were isolated and sequenced. The DNA sequences were analyzed using MEGA 3.0 software to determine the phylogenetic relationship. Forty-five variable sites among cyt b gene sequences and 18 amino acid substitutions occurred within the diploid and tetraploid loaches as deduced from the nucleotide sequences analysis of the cyt b gene. The nucleotide pairwise distance between diploid and tetraploid loach ranged from 0.001 to 0.025. Phylogenetic analysis revealed evolutionary relationships between diploid and tetraploid loach. Our results indicated a significant difference between diploid and tetraploid loach about the cyt b gene. AMOVA analysis indicated that there were no significant genetic variations within diploid loaches (Fst = 0.2529, P > 0.05) and within tetraploid loaches (Fst = 0.0564, P > 0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst = 0.7634, P < 0.05). Thus, it is concluded that no reproductive isolation was found within the same cytotypes of different localities, but there was reproductive isolation between these two cytotypes. The diploid loach existed before the tetraploid loach in nature. The present study is the first to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.     

Molecular Evidence for Multiple Origins of the European Spined Loaches (Teleostei,Cobitidae)

We present a phylogenetic investigation of the Northern Clade, the major monophyletic clade within the freshwater fish family Cobitidae, one of the most prominent families of freshwater fishes found in Asian and European waters. Phylogenetic reconstructions based on the cytochrome b and RAG-1 genes show the genera Microcobitis, Sabanejewia, Koreocobitis and Kichulchoia as monophyletic groups. These reconstructions also show a Cobitis sensu lato and a Misgurnus sensu lato group. The Cobitis sensu lato group includes all species of Cobitis, Iksookimia, Niwaella and Kichulchoia, while the Misgurnus sensu lato group includes Misgurnus, Paramisgurnus and Koreocobitis. Although the monophyly of both the Cobitis sensu lato and Misgurnus sensu lato groups is supported, relationships within the groups are incongruent with current generic definitions. The absence of monophyly of most genera included in the Cobitis sensu lato group (Cobitis, Iksookimia and Niwaella) or their low genetic differentiation (Kichuchoia) supports their consideration as synonyms of Cobitis. Molecular phylogenies indicate that the Asian species of Misgurnus experienced a mitochondrial introgression from a lineage of Cobitis. We also find two nuclear haplotypes in the same Cobitis species from the Adriatic area that, in the absence of morphological differentiation, may indicate molecular introgression. Most lineages within the Northern Clade consist of species found in East Asia. However, some lineages also contain species from Europe and Asia Minor. The phylogenetic relationships presented here are consistent with previous studies suggesting an East Asian origin of the Northern Clade. According to the current distributions and phylogenetic relationships of the Misgurnus sensu lato and Cobitis clade lineages, particularly of M. fossilis and C. melanoleuca, the range expansion of East Asian species into Europe was most likely via Siberia into Northern and Central Europe. Phylogenetic analyses also show that the Cobitis sensu lato group consists of two clear subgroups (I and II), each presenting geographical differences. Subgroup I is distributed exclusively in East Asian drainages with an Eastern European offshoot (C. melanoleuca), whereas Subgroup II includes species widespread throughout Europe (including the Mediterranean), Asia Minor, the Black Sea and the Caucasus, with some lineages related to species restricted to East Asia.     

Molecular characteristics of mitochondrial DNA and phylogenetic analysis of the loach (Misgurnus anguillicaudatus) from the Poyang Lake

The goal of our study was to investigate the molecular characteristics of mitochondrial DNA (mtDNA) and phylogenetic construction of the weather loach (Misgurnus anguillicaudatus) in Poyang Lake. The complete mitochondrial genome was 16,634?bp, and the gene order was identical to that of teleost fishes. Compared with the previous reported weather loach in China, there were numerous nucleotide substitutions and length polymorphisms on the structural genes of mitochondrial DNA in the loach from the Poyang Lake. The Phylogenetic tree indicated that the loach had its own molecular characteristics and was somewhat different from those in other regions of China. Fourteen unique haplotypes of the cytochrome b (cyt b) gene were obtained from 300 weather loaches. The Phylogenetic tree based on the cyt b gene showed that the loaches were substructured into two different populations in The Poyang Lake. Results indicated that the loaches in Poyang Lake not only showed the same phylogeny as the loaches in other areas of China, but also generated its own unique phylogenetic relationships.     

Comparative anatomy of the cheek muscles of Korean cobitid fishes (Ostariophysi: Cypriniformes), with the comments on their phylogenetic relationships

Cheek muscles of 16 species of all Korean cobitid species belong to six genera were examined and described in detail, with comments on their phylogenetic relationships made on the basis of characteristics of cheek muscles. Korean cobitids were divided into two groups, the Cobitis and Misgurnus groups, by five characters related to three cheek muscles (adductor mandibulae, preorbitalis, and retractor arcus palatine). The former group, comprising Cobitis, Iksookimia, Kichulchoia, Koreocobitis, and Niwaella, is defined by four apomorphies, and the latter, including Misgurnus, by a single apomorphy.     

鄱阳湖区泥鳅遗传多样性研究

采用同工酶电泳、RAPD引物PCR扩增和线粒体DNA细胞色素b基因全序列3种方法,对鄱阳湖3种斑纹泥鳅的遗传结构进行分析。同工酶电泳显示3种斑纹泥鳅在LDH、MDH和EST这3种同工酶之间存在群体和组织差异;RAPD试验结果显示6条RAPD引物在18尾鄱阳湖泥鳅个体中共扩增出了169条不同分子量的RAPD标记,其中多态性带101条,多态性带的比例为59.56%,平均每个引物产生9.39条带和5.61条多态性带,不同斑纹泥鳅的遗传多样性指数显示3种斑纹泥鳅的遗传水平处于较高水平。以特异性引物进行PCR扩增,获得了1140bp的细胞色素b基因全序列。结果显示,3种不同斑纹泥鳅中检测出了1个共有单倍型和3个特有单倍型。与共有单倍型相比,大花斑泥鳅特有单倍型有91个变异位点,而小花斑泥鳅与无花斑泥鳅特有单倍型的变异位点为20和24个。基于单倍型构建的NJ树和MP树,显示大花斑泥鳅的特有单倍型EU145023处于独立的分支上,这反映出鄱阳湖地区不同斑纹的泥鳅之间在线粒体DNACytb基因中出现了一定距离的分化。       

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.     

Long-term stability of the seasonal succession of different zooplankton species in a brackish water lagoon (southern Baltic Sea)

While it has been widely suggested that freshwater fishes from East Asia invaded the western Palaearctic, details about this process are largely unknown. Here, using the cytochrome b gene, we evaluated the phylogenetic relationships of a small group of Eurasian primary freshwater fishes (Cobitidae), which are widely distributed and species rich in East Asia and Europe, with the purpose of inferring their invasion process of Europe from East Asia. Though phylogenetic relationships of cobitids were not well resolved, our analysis could identify three sister groups formed by the European and East Asian cobitids, which brought new insights into the biogeography of the genera Cobitis, Misgurnus, and Sabanejewia. The present results support the view that Asian cobitid fishes may have invaded Europe at least five times independently, and once reverse colonization of European cobitids to East Asia could also be found. Ancestral Sabanejewia might have been the first cobitids to cross Siberia and invade the EMZS (Euro-Mediterranean zoogeographic subregion) about 33.54 million years ago (MYA). One lineage of Cobitis and the ancestor of Misgurnus fossilis (Linnaeus) almost in the same time invaded the Europe, responding to 16.71 MYA and 16.59 MYA, respectively. Three different lineages of Cobitis were found to have invaded the EMZS from East Asia, and once reverse invasion to East Asia occurred to one subclade of European Cobitis. And our data also suggest that the diversity of East Asian cobitid fishes, especially of the genus Cobitis, is greatly underestimated. Handling editor: C. Sturmbauer     

Detection of hybridization between two loach species (<Emphasis Type="Italic">Paramisgurnus dabryanus</Emphasis> and <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and “supposed hybrids” with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 “supposed hybrids” was identified to be F₁ hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.     

Environmental DNA analysis provides an overview of distribution patterns of two dojo loach species within the Naka-ikemi Wetland,Fukui Prefecture,Japan

Ichthyological Research - In the Naka-ikemi Wetland, a cryptic species of the dojo loach, Misgurnus sp. Type I (called Type I loach herein), inhabits along with the common nominal species,...     

Indirect positive effects of agricultural modernization on the abundance of Japanese tree frog tadpoles in rice fields through the release from predators

In Japan, the density of Japanese tree frogs (Hyla japonica) may not be necessary high in traditional rice fields compared to modern fields because the modernization of drainage systems from shallow earthen ditches to deep concrete-line ones reduces the abundance of predators/competitors for tadpoles, such as dojo loach (Misgurnus anguillicaudatus). This hypothesis was tested using two approaches. First, laboratory experiments revealed that the presence of the loach significantly reduced the survival of tree frog eggs and tadpoles. Second, observational studies in rice fields showed that the direct effect of modernization on tadpoles was not significant but the indirect (and thus total) effects were weakly positive due to the reduction in the density of dojo loach. Our study suggests that recent agricultural intensification in rice fields does not negatively impact all aquatic species but that some species may not be affected or can even flourish by having tolerance for the changes and release from potential predators or competitors.     

Evidence for selective sweeps by Wolbachia infections: phylogeny of Altica leaf beetles and their reproductive parasites

Infections with maternally inherited Wolbachia bacteria may have dramatic influences on reproductive traits and speciation patterns of their hosts. We here show that in the beetle genus Altica, infection has influenced phylogenetic patterns of the host's mtDNA and different strains led to repeated selective sweeps. By comparing a COI/II‐based phylogeny of the hosts with a phylogeny of the bacteria based on ftsZ, we show that cospeciation is rare and restricted to few recently diverged species. While in general each species apparently harbours a single Wolbachia strain, Altica lythri presents a strikingly different pattern: in the polyphyletic species, three highly divergent mtDNA haplotypes (2.1–4.6% p‐distance) are coupled with three different Wolbachia strains (wLytA1, wLytA2 and wLytB). These haplotypes and Wolbachia strains are widely distributed and mostly found in sympatry. A phylogeny based on microsatellite data supports the monophyly of A. lythri. The discrepancy between mtDNA and nuclear phylogeny may best be explained by interspecific hybridization that led to introgression of mtDNA coupled with a different Wolbachia strain. Selective sweeps apparently drove the introgressed haplotypes to widespread distribution. As for effects of Wolbachia on reproduction, infection with wLytA1 appears to be correlated with a substantial sex ratio distortion, which was most prominent in A. lythri.     

Circannual vitellogenin levels in Chinese loach <Emphasis Type="Italic">(Misgurnus anguillicaudatus)</Emphasis>

To improve the use of the Chinese loach, Misgurnus anguillicaudatus, as a sentinel species for environmental investigations, normal ranges of plasma vitellogenin (Vtg) was studied. Male and female loaches were collected every 2 months from November 2004 to September 2005, and a specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) was used to determine plasma Vtg levels. Our data indicated that the average maximum Vtg level of female loaches (757 ± 356 μg mL⁻¹) appeared in March, approximately 1 month before spawning. Female plasma Vtg significantly correlated with gonadosomatic index (GSI) which indicated that female plasma Vtg can be used as an indicator of the reproductive stages of the ovary. Low detectable plasma Vtg levels was detected in a number of male loaches, and the highest average plasma Vtg concentration attained to the level of μg mL⁻¹; after exposure to estrogenic compounds by waterborne, injection or oral food, Vtg can be induced in male fish, so the presence of Vtg in male loaches might be attributed to the ingestion of estrogenic substances in food, the contamination of estrogenic compounds from the living environment, or the estrogen used by the local fish breeders to accelerate the rate of growth during artificial culture. The results indicate that Vtg levels in male and female Chinese loaches will be very helpful in field studies which use Chinese loach as a sentinel species.     

A preliminary investigation into the use of the invasive golden apple snail,Pomacea canaliculata (Lamarck, 1822), as a water purifier and food source in the breeding ponds of the oriental weatherloach Misgurnus anguillicaudatus (Cantor, 1842)

This study investigated the feasibility of using an invasive snail, Pomacea canaliculata, as a food source and water purifier for the commercial breeding of the loach Misgurnus anguillicaudatus. The predatory potential of M. anguillicaudatus (3.5–5.5?g) against hatchling snails was evaluated in aquaria and simulated paddy fields. Some hatchling snails left the water to avoid being preyed upon by the loaches, and approximately 10 hatchlings died per day in the presence of five loaches in aquaria, whereas a weaker snail control effect was observed in the simulated paddy fields. The growth of rice seedlings (Oryza sativa) was not reduced by the presence of hatchling snails alone, but the shoot biomass of seedlings coexisting with snails was promoted after introducing the loaches. Additionally, the presence of P. canaliculata adults improved the aquatic environment in the short term for loach breeding by decreasing the turbidity of the water. Importantly, M. anguillicaudatus (12–18?g) mortality decreased and its weight increased in the presence of adult snails.     

Genetic and environmental variations in quantitative characters in fishes: a comparative analysis of monoclonal triploid and bisexual tetraploid spined loaches (<Emphasis Type="Italic">Cobitis</Emphasis>, Cobitidae)

The heritability estimates of 25 external morphometric characters and 23 craniometric indices are obtained by use of variances in monoclonal all-female triploids and bisexual tetraploids of spined loaches (genus Cobitis, Cobitidae) collected from the same breeding biotope. Most of studied traits demonstrate low heritability confirming previous conclusion on the similarity between external morphometric characters and craniological indices in relative effects of genetic and environmental components in their total phenotypic variation. Low heritability estimates in most of external morphological traits correspond to their low diagnostic value in Cobitis species. As a whole, in spite of certain deviations, studies on clonal forms do not refute the concept on higher heritability estimates in diagnostically significant traits in comparison with traits without diagnostic values in the same taxonomic group. Low heritability in most morphometric traits more probably is resulted from their low additive genetic variation caused by strong selection of evolutionary developed specific body shape in spined loaches, because strong selection should reduce the genetic variance in body proportions to minimal size. Sex differences observed in heritability estimates should be interpreted as a result of linkage of several additive genes controlling these traits to sex chromosomes. A few characters demonstrating high heritability estimates up to 0.492–0.580 are of great interest for taxonomic and phylogenetic studies in genus Cobitis and related taxa.     

Morphological comparison and DNA barcoding of four closely related species in the genera Misgurnus and Paramisgurnus (Cypriniformes: Cobitidae)

To evaluate the feasibility of morphological and genetic identification of the closely related species in the genera Misgurnus and Paramisgurnus, the morphological characters of four species in these genera and DNA barcoding of five loaches (P. dabryanus, M. anguillicaudatus, M. bipartitus, M. mohoity, and Barbatula toni) were investigated. Twelve morphological characters were measured in 542 individuals to perform the comparative analysis. Among these characters, only the caudal peduncle length (L_CP) revealed significant difference (P < 0.05) among these four species. The clustering based on morphological characters formed two clusters (P. dabryanus and M. anguillicaudatus; M. bipartitus and M. mohoity). A total of 186 COI fragments for the five loaches investigated were sequenced and analyzed. The results showed that interspecific K2P distance was much higher than intraspecific distance within the five species. Bayesian inference of phylogeny showed that individuals of these species were divided into five specific clades. Meanwhile, the COI fragments exhibited 22 character attributes for the differentiation of the five loach species based on character-based method. Our results suggested that DNA barcoding based on COI can be used as an efficient identifier of these five loach species; the combination of distance-based method, Bayesian inference and character-based approach provides higher resolution of identification at species level.     

Phosphoglucomutase electrophoretic patterns of the loach Cobitis biwae

Phosphoglucomutase of two groups of the loach Cobitis biwae , a fresh-water teleost, was examined by starch gel electrophoresis. Some of the fishes had six-banded electrophoretic patterns not observed in other loach species (e.g. Misgurnus anguillicaudatus and Cobitis delicata ). The complexity of the pattern is explainable by an assumption that the loaches are tetraploid fishes. The result suggests that a diploid-tetraploid relationship exists within the species Cobitis biwae .     

New haplotype of the complete mitochondrial genome of <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> and its species-specific DNA markers: distinguishing <Emphasis Type="Italic">C. siamensis</Emphasis> from <Emphasis Type="Italic">C. porosus</Emphasis> in Thailand

Based on molecular phylogeny of available complete mitochondrial DNA (mtDNA) genome sequences reveals that Crocodylus siamensis and C. porosus are closely related species. Yet, the sequence divergence of their mtDNA showed only a few values under conspecific level. In this study, a new haplotype (haplotype2, EF581859) of the complete mtDNA genome of Siamese crocodile (C. siamensis) was determined. The genome organization, which appeared to be highly similar to haplotype1 (DQ353946) mtDNA genome of C. siamensis, was 16,814 bp in length. However, the sequence divergence between the two genomes differed by around 7–10 and 0.7–2.1% for the haplotype1 between C. siamensis and C. porosus (AJ810453). These results were consistent with the phylogenetic relationship among the three genomes, suggesting that C. siamensis haplotype1 mtDNA genome might be the hybrid or the intraspecific variation of C. porosus. On the other hand, our specimen was found to be a true C. siamensis. Simultaneously, the seven species-specific DNA markers designed based on the distinctive site between haplotype2 mtDNA sequences of C. siamensis and haplotype1 mtDNA sequence of C. siamensis–C. porosus were successfully used to distinguish C. siamensis from C. porosus. These effective markers could be used primarily for rapid and accurate species identification in population, ecology and conservation studies.