Phylogeography of loaches of the genus lefua (balitoridae, cypriniformes) inferred from mitochondrial DNA sequences

In order to elucidate phylogenetic relationships and intraspecific variations and to infer the evolutionary process of loaches of the genus Lefua, we analyzed nucleotide sequences of the mitochondrial D-loop region of 100 specimens obtained from 97 localities in Japan and Korea. The genus Lefua includes three described species, L. nikkonis, L. echigonia, and L. costata and an undescribed species, Lefua sp. Our results showed that each species of Lefua formed a monophyletic group, indicating clearly that Lefua species can be genetically distinguished from one another. Lefua nikkonis was the most closely related to L. costata, while L. sp. was the most closely related to L. echigonia. Specimens of L. sp. were grouped into two intraspecific populations and specimens of L. echigonia were grouped into six populations. These populations were well separated geographically from one another by mountain ranges and highlands. We estimated the evolutionary time for splitting of the species and intraspecific populations, and speculated on the evolutionary process of the genus Lefua. Species of Lefua are severely threatened. Fundamental genetic information is indispensable for conservation. We presented genetic background in order to protect these threatened loaches.     

Parallel evolution in eight-barbel loaches of the genus Lefua (Balitoridae, Cypriniformes) revealed by mitochondrial and nuclear DNA phylogenies

The evolutionary history of eight-barbel loaches of the genus Lefua contains important phylogenetic information that will aid in resolution of the faunal formations and evolutionary histories of Japanese and East Asian freshwater fishes. Our sequencing of the mitochondrial D-loop region in a large number of samples allowed construction of the most comprehensive phylogeny of these loaches to date; we demonstrated monophyly of five Lefua species and identified populations of Lufua. sp. and Lefua echigonia. Loaches inhabiting the Tokai region in Japan were morphologically and ecologically indistinguishable from Lefua sp. However, they were included in the L. echigonia lineage. We determined a novel phylogeny by sequencing the nuclear ribosomal S7 subunit and showed that nuclear DNA phylogeny essentially matched the mitochondrial DNA phylogeny. Loaches from the Tokai region were part of the L. echigonia lineage, indicating parallel evolution between Tokai loaches and Lefua sp. in western Japan. We presented the most robust phylogeny to date using concatenated mitochondrial and nuclear sequences. The wealth of molecular information allowed us to speculate on evolutionary processes in the genus Lefua.     

Incongruence between mtDNA phylogeny and morphologial and ecological characters in loaches of the genus Lefua (Balitoridae, Cypriniformes)

To elucidate the phylogenetic position of Lefua loaches from Aichi and Shizuoka Prefectures of Honshu Island, Japan, we determined their nucleotide sequences for the mitochondrial D-loop region and compared these to sequences from four other Lefua species: L. costata, L. nikkonis, L. echigonia, and L. sp. Loaches identified as L. sp. on the basis of morphology comprised a clade (the L. sp. Tokai population) that grouped together with L. echigonia; hence, the processes involved in evolution within the genus Lefua were unclear. We performed randomly amplified polymorphic DNA (RAPD) analyses to obtain genetic information on nuclear DNA. The RAPD patterns of the L. sp. Tokai population differed from those of the local L. echigonia and L. sp. populations. The L. sp. Tokai population was similar to L. echigonia with regard to mitochondrial DNA but differed from L. echigonia and L. sp. with respect to nuclear DNA; this indicated that the evolutionary background of the L. sp. Tokai population was unique. We suggest that introgression of mitochondria occurred from L. echigonia to the L. sp. Tokai population, and speculate on the process of evolution of the latter population of Lefua. As with six L. echigonia populations and two L. sp. populations, we regard the L. sp. Tokai population as an evolutionary significant unit (ESU) that qualifies for protection as an endangered loach.     

Genetic population structure of the eight-barbel loach Lefua echigonia in the Ise Bay region,a single paleo-river basin in central Honshu,Japan

Ichthyological Research - We examined the genetic population structure of Lefua echigonia in the Ise Bay region based on the nucleotide sequences of the mitochondrial DNA cytochrome b region....     

Morphologies and population genetic structures of the eight-barbel loach of the genus Lefua on southern Sakhalin

Ichthyological Research - Coldwater, primary freshwater, fish such as the eight-barbel loach, Lefua nikkonis, are thought to have colonised Hokkaido from the continental Far East via Sakhalin...     

Phylogenetic relationships and intraspecific variations of loaches of the genus Lefua (Balitoridae,Cypriniformes)

Three nominal species are known in East Asian balitorid loaches of the genus Lefua, i.e. L. echigonia, L. nikkonis, and L. costata. Lefua echigonia, with large morphological variations was recently separated into two groups, L. echigonia including the holotype and L. sp., based on morphological and ecological traits. We performed protein and DNA analyses to elucidate phylogenetic relationships among loaches of the genus Lefua and to settle the taxonomic status of L. sp. We also investigated intraspecific variations in L. echigonia s. str. to shed light on the process of formation of freshwater fish fauna in Japan. Protein analyses using two-dimensional gel electrophoresis showed that genetic distances between L. sp. and L. echigonia s. str. and between L. sp. and L. nikkonis were as large as that between L. echigonia s. str. and L. nikkonis. DNA analyses of the mitochondrial D-loop region showed that L. sp. and L. echigonia s. str. were monophyletic, respectively, while neither L. nikkonis nor L. costata was monophyletic and these species formed together a clade. The results supported the specific status of L. sp. and proposed reevaluation of the taxonomic status of L. nikkonis and L. costata. DNA analyses also showed that L. sp. was more closely related to L. echigonia s. str. than to the L. nikkonis-L. costata complex, and four local populations were distinguished in L. echigonia s. str. Distribution patterns of the four local populations of L. echigonia s. str. in Japan were approximately congruent with those of the medaka, Oryzias latipes, suggesting that differentiation in the two distantly related fishes have a common historical background.     

Length‐weight relationships of seven fish species from the Wutong River,northeast China

Length‐weight relationships (LWR) were calculated for seven fish species from the Wutong River, northeast China(47°58'‐47°10'N; 130°07'‐130°48'E), including Ladislavia taczanowskii Dybowski, 1869; Gobio cynocephalus Dybowski, 1869; Rhynchocypris lagowskii (Dybowski, 1869); Lefua costate (Kessler, 1876); Cobitis lutheri Rendahl, 1935; Cobitis sibirica Gladkov, 1935; Perccottus glenii Dybowski, 1877. All fish specimens were collected by electric fishing gear (voltage, 12 V) and gillnets (mesh size: 1.1 cm × 1.1 cm, 1.4 cm × 1.4 cm, 1.8 cm × 1.8 cm, 2.5 cm × 2.5 cm) from May to July 2017. The a values ranged from 0.0038 (Cobitis lutheri) to 0.0084 (Lefua costata), and b values from 2.716 (Lefua costata) to 3.450 (Perccottus glenii) and r² values from 0.973 (Cobitis lutheri) to 0.995 (ladislavia taczanowskii). This study provides the first published the LWRs for the five species, and new maximum total lengths were recorded for two species, including Gobio cynocephalus and Lefua costata.     

Isolation and characterization of polymorphic microsatellite DNA markers in the Japanese eight‐barbel loach,Lefua echigonia

We isolated and characterized 17 polymorphic microsatellite loci for the Japanese eight‐barbel loach, Lefua echigonia, an endangered freshwater species in streams including agricultural canals in Japan. The number of observed alleles for each locus ranged from two to nine, and the values of observed and expected heterozygosities ranged from 0.125 to 0.844 and from 0.148 to 0.876, respectively. All loci conformed to Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

On the systematics and phylogeography of eight-barbel loaches the genus Lefua (Cobitoidea: Nemacheilidae): mtDNA typing of L. pleskei

Comparative analysis of personal sequence data for the mtDNA control region (926 to 928 bp) from eight-barbel loaches inhabiting eight localities in the Amur River basin (4) and the Sea of Japan (4) and the GeneBank/NCBI data for the Lefua individuals from the other regions of the world showed that eight-barbel loaches from Primorskii krai water basins were marked by a specific group of mtDNA haplotypes. This finding is considered as supporting the species status of L. pleskei. Genetic distances within L. pleskei are small (on average 0.355) and close to those within L. nikkonis (on average 0.48%). The distances between this species pair are the least (on average 2.15%) among all other pairs compared. In MP, ML, and Bayesian trees, L. pleskei and L. nikkonis haplotypes formed a common clade with high statistically significant support. In all tree variants, L. costata mtDNA haplotypes were located out of the group of interest. A clade consisting of highly diverged lineages of Lefua sp. and L. echigonia haplotypes occupied even more independent position. The mtDNA haplotypes of L. pleskei and L. costata from the Amur River basin were evolutionary young and derived from the haplotypes found in these species from the Sea of Japan (L. pleskei) or the Yellow Sea (L. costata) basins. It is thereby suggested that both species rather recently migrated into the Amur River system. According to the molecular clock data, basal diversification of the eight-barbel loach lineages took place at the end of middle Miocene (about 11 to 12 Myr ago), while divergence of L. pleskei and L. costata ancestral forms probably occurred approximately, 5 Myr ago. Since all main lineages of eight-barbel loaches were found in the Sea of Japan basin (continental coastline and the islands), the divergence order and dispersal patterns of the Lafua species might have been largely determined by the geological development pattern of this water body and the adjacent territories.     

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.     

A laboratory study of the effects of shelter availability and invasive crayfish on the growth of native stream fish

1. The alteration of stream habitats by urbanisation reduces the availability of shelter. Reduced shelter availability may increase both predation risks and metabolic costs, negatively affecting the growth performance of stream fish. Although urbanised streams often allow the establishment of invasive species, the additive or interactive effects of shelter availability and invasive species are rarely explored. The invasive red swamp crayfish (Procambarus clarkii) is a strong shelter competitor and predator in streams. 2. We conducted a stream‐channel experiment to investigate how shelter availability and this invasive crayfish species affect the growth performance (measured as the loss in body mass under starved conditions) of two native benthic species, Japanese crucian carp (Carassius auratus complex) and an endangered cobitid fish (Lefua echigonia). 3. We found that both the absence of shelter and the presence of crayfish increased body mass loss in the crucian carp. However, the growth performance of the crucian carp was improved by the presence of shelter except in the presence of crayfish, in which case the shelter available did not ameliorate the negative effect of the crayfish on growth performance (i.e. an interactive effect). This result suggests that crayfish may affect the growth performance of the crucian carp through chemical cues as well as through shelter occupancy. 4. In contrast, shelter availability is the primary factor affecting the body mass loss of the cobitid fish. However, the growth performance of these fish was unaffected by the presence of crayfish. The change in the growth performance of the cobitid fish in response to the absence of shelter was greater than that of the crucian carp. This finding suggests that the cobitid fish may be more vulnerable than the crucian carp to a reduction in shelter availability. 5. Our study demonstrates that reduced shelter availability and/or crayfish invasion can have significant, indirect negative effects on the growth performance of native fish, but whether those effects are interactive might vary depending on the species. Our findings have clear implications for stream restoration and habitat assessment.     

Phylogeography of the eight-barbel loach <Emphasis Type="Italic">Lefua nikkonis</Emphasis> (Cypriniformes: Nemacheilidae): how important were straits in northern Japan as biogeographical barriers?

Many straits in the Japanese archipelago have been proposed as biogeographical boundaries, but there is disagreement regarding their importance as historic barriers against dispersal of terrestrial and freshwater taxa. Mitochondrial DNA haplotype and phylogenetic analyses of Lefua nikkonis, a primary freshwater fish inhabiting northern Japan and descendent from Siberia, revealed that the species is genetically structured within its geographic range, but that two major haplotypes are widely distributed across the Ishikari Lowland of Hokkaido Island as well as across the Tsugaru Strait between Hokkaido and Honshu Islands, two well-known biogeographical boundaries of northern Japan. The two major haplotypes were separated from each other by only one mutational step, and many other haplotypes, including those endemic to the region south of these barriers, have diverged from the major haplotypes, suggesting rapid range expansion and local differentiation. Divergence-time estimates, based on vicariance of the Honshu endemic congener L. echigonia via uplift of the Central Highlands, demonstrated that the southward dispersal of L. nikkonis from Hokkaido Island to Honshu Island occurred less than 0.08–0.19 Mya, suggesting that a land bridge emerged at the Tsugaru Strait during the Riss glaciation. Given that other freshwater taxa crossed the strait earlier (during the Middle Pleistocene), it is likely that land bridges in the strait have repeatedly emerged. The fact that L. nikkonis invaded only the northern part of Honshu, and that many other freshwater species also have the limit of their distribution ranges in this area as well, indicates that a faunal transition zone might persist even without the Tsugaru Strait. Thus, straits and lowlands in northern Japan are likely to have been less important as dispersal barriers to freshwater taxa than is currently thought.     

中国北鳅谱系地理学分析

北鳅(Lefua costata)为冷水性鱼类, 分布于淮河以北, 分析遗传结构能够反映其适应环境变迁的响应。基于线粒体D-loop区211条序列分析了我国北鳅的谱系地理学和遗传多样性, 样本采自9条水系共18个样点。单倍型分析显示共计55个单倍型, 呈高单倍型多样性(h=0.9304)和高核苷酸多样性(π=0.0087)。单倍型多样性和核苷酸多样性最高的为辽东半岛种群(LD) (h=0.8920, π=0.0074), 其他地理种群距辽东半岛越远, 单倍型多样性越低。遗传距离(K2P)、群体间的遗传分化指数(F_ST)及AMOVA分子方差分析均显示LD种群与其他地理种群存在显著分化。基于单倍型构建的分子系统发育树和单倍型网络关系图均显示9条水系的单倍型不能各自聚类, 形成3大分支: 分支A以LD种群为主; 分支B含所有地理种群; 分支C以淮黄河种群(HH)和海滦河种群(HL)为主。中性检验和错配分析显示不同地理种群有扩张现象。基于化石校准点和北鳅鱼类D-loop序列1.00%Ma的平均进化速率评估各地理种群的分歧时间, 为2.3784— 0.0477Ma前, LD种群与其他地理种群分化时间最早(2.3784 Ma前), 推测LD种群受第四纪冰期影响较小, 辽东半岛为我国北鳅起源地之一, 其他地理种群以辽东半岛为中心借助松辽古大湖和黄渤海平原河口等发生多次迁移。     

A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species

The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 °C for 10 s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG), and methanol (MeOH) at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%; glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25 mM NaCl and 50 mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 °C for 10 s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50 mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg⁻¹ mL⁻¹). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully.     

Genetic population structure of <Emphasis Type="Italic">Lefua echigonia</Emphasis> inferred from allozymic and mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> variations

We examined the genetic population structure of Lefua echigonia (Japanese name, hotoke-dojo) using polymerase chain reaction restriction-fragment length polymorphisms (PCR-RFLPs) of the mitochondrial cytochrome b gene and two allozymic loci. The phylogenetic relationships of L. echigonia and those among L. echigonia, Lefua sp. (nagare hotoke-dojo), and L. nikkonis (ezo hotoke-dojo) were also investigated based on the nucleotide sequences of the cytochrome b gene. PCR-RFLP analysis revealed 18 mitotypes in L. echigonia, 2 in Lefua sp., and 1 in L. nikkonis. Phylogenetic trees based on the cytochrome b sequences indicated that the 18 mitotypes in L. echigonia were divided into five distinct groups (South-Kanto, North-Kanto, Tohoku, Echigo, and Tokai-Kinki clades) that differed by 8.5–15.3%, reflecting region-specific geographic distributions. The distributions of alleles in two allozymic loci roughly corresponded to those of the mitotype groups. The divergence times of the five groups were estimated to be about 3.4–7.7 million years ago by applying a general rate for mitochondrial DNA, suggesting that the divergence among them might have occurred in the late Tertiary. It can be inferred that the regional differentiation of each group was mainly due to geographic isolation and that this has been maintained, because the boundaries among the groups corresponded to geological features. The trees also supported the existence of three taxa, L. echigonia, Lefua sp., and L. nikkonis. We concluded that Lefua sp. was distinguished from other species in Lefua by morphological and ecological characters and also by genetic divergences of the cytochrome b gene. Our study also demonstrated the superior efficacy and simplicity of PCR-RFLP analysis as a method for detecting genetic variation in L. echigonia.     

Movement patterns of small benthic fish in lowland headwater streams

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