Selection shapes the patterns of codon usage in three closely related species of genus Misgurnus

Neutrality plots revealed that selection probably dominates codon bias, whereas mutation plays only a minor role, in shaping the codon bias in three loaches, Misgurnus anguillicaudatus, M. mohoity, and M. bipartitus. These three species also clearly showed similar tendencies in the preferential usage of codons. Nineteen, nine, and 14 preferred codon pairs and 179, 182, and 173 avoided codon pairs were also detected in M. anguillicaudatus, M. bipartitus, and M. mohoity, respectively, and the most frequently avoided type of cP3-cA1 dinucleotide in these species was nnUAnn. The expression-linked patterns of codon usage revealed that higher expression was associated with higher GC₃, lower ENC, and a smaller proportion of amino acids with high size/complexity (S/C) scores in these three species. These results elucidate selectively driven codon bias in Misgurnus species, and reveal the potential importance of expression-mediated selection in shaping the genome evolution of fish.     

Effect of loach consumption on the reproduction of giant water bug Kirkaldyia deyrolli: dietary selection, reproductive performance, and nutritional evaluation

Rice fields provide major habitats for lentic aquatic insects including the giant water bug Kirkaldyia (=Lethocerus) deyrolli (Heteroptera: Belostomatidae) in Japan. Previous researchers have emphasized that conserving populations of the frogs, Hyla japonicus and Rana nigromaculata, is very important for preserving K. deyrolli because these frogs were found to be a major component of the diet of K. deyrolli adults. However, these previous studies were carried out in rice fields with no loaches, Misgurnus anguillicaudatus, which were probably been inhabited by loaches in the past. A series of field surveys and laboratory experiments were conducted to determine the dietary preference of K. deyrolli adults for loaches, frogs, and aquatic insects; the reproductive effects of consuming these foods; and their protein content. In the rice fields, K. deyrolli adults ate loaches when they were available. When the three prey species (frog: R. nigromaculata, loach: M. anguillicaudatus, and dragonfly: Orthetrum albistylum speciosum) were supplied in laboratory conditions, K. deyrolli adults ate more loaches than frogs or dragonflies. In addition, K. deyrolli adults provided with loaches or frogs laid more egg masses within the limited breeding season than when provided with dragonflies. The edible parts of the loach bodies were the largest of the three prey types. It is possible that K. deyrolli adults have been forced to eat frogs due to reductions in the population density of loaches because modern rice cultivation obstructs loach migration routes and reduces their spawning grounds.     

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.     

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.     

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.     

Movement and assemblage of fish in an artificial wetland and canal in a paddy fields area,in eastern Japan

This study, carried out in a paddy fields area in eastern Japan, investigated the timing and patterns of fish movement and assemblage in an artificial wetland and canal. The number of Misgurnus anguillicaudatus and Lefua echigonia immigrating and emigrating between the wetland and the canal accounted for 80–90 % of all the sampled fish. M. anguillicaudatus, L. echigonia, Pseudorasbora parva and Rhynchocypris lagowskii were dominant in the wetland. Immigration of mature M. anguillicaudatus and L. echigonia was detected between late winter and spring. The standard length of the two loach species in the wetland was smaller than that in the canal. These results confirm that wetlands play a role in spawning and nursery for the two species of loaches. The standard length of P. parva in the wetland was smaller than in the canal. This suggests that the wetland was a more suitable spawning and nursery area for this fish species than the canal. L. echigonia used the wetland as a spawning and nursery area, but previous studies reported that the loach did not use paddy fields near the wetland. This could be because the paddy fields were irrigated between June and September and this period did not largely overlap with the fish spawning season. Therefore, we conclude that the conservation and restoration of wetlands, where water is present throughout the year, will contribute toward the preservation of the fish population in a paddy fields area.     

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.     

Species diversity of Onchidiidae (Eupulmonata: Heterobranchia) on the mainland of China based on molecular data

The identification of members of the Onchidiidae is based on morphological characters; this is often time-consuming and can be inconclusive. In order to explore the species diversity of onchidiids in China, we provide a phylogeny constructed using partial sequences of two mitochondrial genes (16S rRNA and COI) and one nuclear ribosomal RNA gene (28S rRNA) from 32 samples comprising five genera. The topology, using both Bayesian and Maximum Likelihood inference methods, showed that the taxa clustered in two main groups of six species, one of which included Platevindex mortoni, Platevindex sp. and Onchidium ‘struma’; the other included Paraoncidium reevesii, Onchidella sp. and Peronia verruculata. It is clear that COI will be useful in discriminating onchidiid species-group taxa.     

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.     

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 and morphological characterization of two aphid genera,Acyrthosiphon and Aulacorthum (Hemiptera: Aphididae)

The taxonomic histories of two genera, Acyrthosiphon and Aulacorthum, have been controversial because of the ambiguous morphology used as criteria to distinguish these genera. In this study, molecular and morphological characters were analyzed to determine whether the current classification of these genera is reasonable. Two phylogenetic trees based on five genes (COI, COII, srRNA, lrRNA, and EF1a) supported the distinctiveness of Acyrthosiphon and Aulacorthum, and a canonical discriminant analysis using 19 morphological characters showed that the two genera were separated by five morphological characters (HTB, C, Ant.III, HFM, and SIPH). Based on these results, we confirm that the current classification of Acyrthosiphon and Aulacorthum is supported by molecular and morphological characters.     

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.     

Molecular Analysis of the Freshwater Prawn Macrobrachium olfersii (Decapoda,Palaemonidae) Supports the Existence of a Single Species throughout Its Distribution

Macrobrachium olfersii is an amphidromous freshwater prawn, widespread along the eastern coasts of the Americas. This species shows great morphological modifications during ontogenesis, and several studies have verified the existence of a wide intraspecific variation. Because of this condition, the species is often misidentified, and several synonyms have been documented. To elucidate these aspects, individuals of M. olfersii from different populations along its range of distribution were investigated. The taxonomic limit was established, and the degree of genetic variability of this species was described. We extracted DNA from 53 specimens of M. olfersii, M. americanum, M. digueti and M. faustinum, which resulted in 84 new sequences (22 of 16S mtDNA, 45 of Cythocrome Oxidase I (COI) mtDNA, and 17 of Histone (H3) nDNA). Sequences of three genes (single and concatenated) from these species were used in the Maximum Likelihood and Bayesian Inference phylogenetic analyses and COI sequences from M. olfersii were used in population analysis. The genetic variation was evaluated through the alignment of 554 bp from the 16S, 638 bp from the COI, and 338 bp from the H3. The rates of genetic divergence among populations were lower at the intraspecific level. This was confirmed by the haplotype net, which showed a continuous gene flow among populations. Although a wide distribution and high morphological intraspecific variation often suggest the existence of more than one species, genetic similarity of Caribbean and Brazilian populations of M. olfersii supported them as a single species.     

Morphometrics of the final instar exuviae of five cicada species occurring in urban areas of central Korea

Cicadas produce loud calling songs for mate attraction that can be a nuisance to city dwellers in Korea. Exuviae of final instars can be used to estimate population density. We investigated the morphological characteristics of final instar exuviae for five cicada species that are abundant in central Korea: Cryptotympana atrata, Hyalessa fuscata, Graptopsaltria nigrofuscata, Meimuna opalifera, and Meimuna mongolica. The characters analyzed were pro-mesonotum length, pronotum width, pronotum length, head width, abdominal circumference, body length, wing length, and femoral tooth angle. The results of a general linear model showed that species and sex were significant for morphological characters, and the result of post hoc analyses revealed that the mean values of most of the morphological characters measured were significantly different among the five cicada species. Three groups, Cryptotympana atrata, H. fuscata/G. nigrofuscata, and M. opalifera/M. mongolica, were distinctively identified based on most morphological characters. The distributions of femoral tooth angles did not overlap between H. fuscata and G. nigrofuscata, and the distributions of abdominal circumference did not overlap between M. opalifera and M. mongolica. Thus, the exuviae of all five cicada species could be easily distinguished based on morphological characters. We also provide a morphological key for the exuviae of all five cicada species in Korea.     

Morphological and molecular characterization of two Trichodina (Ciliophora,Peritrichia) species from freshwater fishes in China

In the present study, we provide morphological and molecular characterization of two Trichodina species, T. acuta Lom, 1970 and T. funduli Wellborn, 1967, isolated from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. Morphological characters of the two Trichodina species were mainly investigated on the basis of dry silver nitrate-impregnated specimens. Both species are medium-sized and possess well-developed denticles comprising strongly sickle-shaped blades, well-developed central parts, and straight rays. Trichodina acuta can be easily distinguished from the other Trichodina species that possess a clear central circle by the well-developed sharp blade apophysis, and the gap between ray tip and central circle. Trichodina funduli is a poorly known species that is easily confused with T. heterodentata Duncan, 1977, however the latter species has thinner denticles. The small subunit ribosomal RNA gene sequences of Trichodina acuta and T. funduli were incorporated into phylogenetic analyses. Our findings suggest that the phylogenetic lineage of trichodinids might not correspond with their living environments, host species or even some morphological characteristics.     

Morphology and ecology of Pacific sturgeons

The six North Pacific members of the genus Acipenser have generally similar environmental requirements and share several specific morphological features. This Pacific group consists of green sturgeon, Acipenser medirostris, Sakhalin sturgeon, A. mikadoi, white sturgeon, A. transmontanus, Chinese sturgeon, A. sinensis, Dabry’s sturgeon, A. dabryanus and Amur sturgeon, A. schrenkii. A qualitative morphological examination of these species revealed synapomorphic characters that suggest close phyolgenetic relationships resulting from common ancestry of eastern and western species within the Pacific Rim. An evolutionary taxonomic approach, considering a reduction of characters from east to west, would suggest an Asian ancestry for all Pacific sturgeons. In contrast however, a phylogenetic approach using derived character states suggests a North American ancestry. Besides a common ancestry for Chinese and American sturgeons, the taxonomic status of A. mikadoi is discussed.     

Mitochondrial COI and morphological evidence for host specificity of the black cherry aphids Myzus cerasi (Fabricius, 1775) collected from different cherry tree species in Europe (Hemiptera,Aphididae)

Partial sequences of the mitochondrial COI gene of forty eight European and two Turkish population samples of Myzus cerasi from different winter hosts (Prunus spp.) were subjected to phylogenetic analyses. The analysed M. cerasi samples emerged as paraphyletic relative to a Myzus borealis sample used as an out-group, and formed two major clades in neighbor joining, maximum parsimony, maximum likelihood and Bayesian inference trees, corresponding to subspecies living specifically on Prunus avium and P. cerasus. Multivariate discriminant analysis (method of canonical variates) was applied to find out if morphological variation of samples correlated with mitochondrial COI and host plant information. Mean scores on the first two canonical variables clustered samples fully in accordance with their COI haplotypes and host plants confirming the existence of two morphologically similar winter host - specific subspecies of M. cerasi in Europe. No single morphological character enabled satisfactory discrimination between apterous viviparous females of the two subspecies. A three-character linear discriminant function enabled 92.37% correct identification of apterous viviparous females of M. cerasi cerasi (n = 118) and 93.64% of M. cerasi pruniavium (n = 110). A key for the morphological identification of the two subspecies is presented and their taxonomic status is discussed.     

Phylogeny of the dragonfly genus Sympetrum (Odonata: Libellulidae)

The libellulid dragonfly genus Sympetrum has been recognized since 1833, but lacks any morphological synapomorphies to unite the taxon. Previous researchers have disagreed over which species belong in Sympetrum, bringing the monophyly of the genus into question. We use DNA sequence data from 6 genetic loci (16S, tRNA-valine, 12S, elongation factor 1 alpha, cytochrome oxidase subunit I, and the second internal transcribed spacer region) and 25 morphological characters (mainly genitalic) to test the monophyly of Sympetrum with Bayesian inference and maximum likelihood analyses. Under Bayesian inference, all Sympetrum species included in this study form a clade, which also contains the Hawaiian monotypic genus Nesogonia, often considered a close relative of Sympetrum. Phylogenetic analyses also reveal at least six strongly supported clades (treated as species groups) within Sympetrum, but relationships between these species groups remain unresolved or unsupported. Although the relationships between Sympetrum species groups remain unresolved, several species groups include taxa from multiple biogeographic regions/continents, and the species group sister to the rest of Sympetrum contains migratory species from the New World and Africa. This pattern suggests a complex biogeographic history in Sympetrum shaped by vicariance and dispersal. Preliminary estimates of the divergence dates of Sympetrum species groups outline a rapid radiation of the groups approximately 32-38 million years ago, possibly influenced by cooling and drying climates of the late Eocene and early Oligocene.     

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.