Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

Nemacheilus pezidion, a new species of loach from southern Laos (Teleostei: Nemacheilidae).

Nemacheilus pezidion, new species, is described from the Xe Kong watershed, Mekong drainage, in Attapeu province, southern Laos. It is distinguished from all other Nemacheilidae in Southeast Asia by its unique colour pattern consisting of a black midlateral stripe and a middorsal row of 1115 narrow saddles. Besides, the male has a globulous suborbital flap with tubercles along its posterior edge, and the pectoral fin with thickened anterior rays, and branched rays 14 and unculiferous pads behind them covered by small tubercles. It was found in habitats with moderate flow, on mud to gravel bottom. Nemacheilus pezidion was earlier misidentified as N. longistriatus.

Molecular characterization and nutritional regulation of sodium-dependent glucose cotransporter 1 (Sglt1) in blunt snout bream (Megalobrama amblycephala).

Fish has poor utilization capacity for glucose metabolism. The possible reasons are related to the core regulatory elements of glucose metabolism: transport proteins. Studies on the species and functions of Sglt1 in aquatic animals are scarce, therefore further studies are needed. In this study, the full length of blunt snout bream (Megalobrama amblycephala) sglt1 (Masglt1) was 2965 bp including 5'-UTR region of 168 bp and a 3'-UTR region of 820 bp. Masglt1 have a highest sequence homology in Cypriniformes fish. MaSglt1 protein was identified as a transmembrane protein with 14 α-helix structures locating plasma membrane by the methods of predicted tertiary structure and immunohistochemical staining. MaSglt1 protein has a hollow channel forms which could be specifically coupled with two Na+ ions to recognize glucose and carry out transmembrane transport. High sglt1 mRNA was found in the intestine and kidney. The mRNA levels of intestinal sglt1 had a positive correlation with dietary starch levels at 3 h after feeding, and the mRNA was significantly higher than that at 24 h, however, the mRNA levels of renal sglt1 presented results opposite to those of intestinal sglt1. The mRNA levels of renal sglt1 had a positive correlation with dietary starch levels at 24 h after feeding, and the expression was significantly higher than that at 3 h. These results confirmed that Masglt11 was mainly found in the intestine and kidney and was located in the cell membrane, playing a role in glucose homeostasis.

Oxynoemacheilus isauricus, a new nemacheilid loach from Central Anatolia (Teleostei: Nemacheilidae).

Oxynoemacheilus isauricus, new species, from the Lakes Beysehir and Sugla basins in Central Anatolia is distinguished from all other species of the O. angorae group by having a very slender caudal peduncle (its depth 2.22.6 times in its length). The new species is further distinguished by having a short head (head length 2124% SL), and a midlateral series of irregularly shaped blotches on the flank. Oxynoemacheilus isauricus is also distinguished by a minimum K2P sequence divergence of 7.5% and 8.0% in the mtDNA-COI barcode region from O. eregliensis and O. atili, its closest relatives.

Oxynoemacheilus nasreddini, a new nemacheilid loach from Central Anatolia (Teleostei: Nemacheilidae).

Oxynoemacheilus nasreddini, new species, from Lake Aksehir, Eber, Egirdir, and Ilgin basins in Central Anatolia is distinguished from other species of Oxynoemacheilus in Central Anatolia by having irregularly shaped blotches on flank; 24 dark-brown bands on caudal fin; a slender body, and an emarginate caudal fin in which the shortest middle caudal-fin ray is 7691% of the longest ray of the upper caudal-fin lobe. Molecular data suggest that the new species is closely related to O. mediterraneus from the Aksu and Köprüçay drainages. Although the two species are separated by only a minimum K2P distance of 1.2% in the mtDNA COI barcode region, they are well distinguished morphologically. The situation is complicated by loaches from Lake Egirdir basin that are identified as O. nasreddini by their morphological character states but are only distinguished from O. mediterraneus by a minimum K2P distance of 0.2% in the mtDNA COI barcode region.

A new loach species of Troglonectes (Teleostei: Nemacheilidae) from Guangxi, China.

A new species of the genus Troglonectes is described from Guangxi Zhuang Autonomous Region, China. Troglonectes hechiensis sp. nov. can be easily distinguished from its congeners based on the following characters: eyes normal; whole body covered by scales except head, throat, and abdomen; lateral line incomplete; caudal fin concave; and color pattern present on body.

FISH Identifies Chromosome Differentiation Between Contemporary Genomes of Wild Types and the Ancestral Genome of Unisexual Clones of Dojo Loach, Misgurnus anguillicaudatus.

In dojo loach (Misgurnus anguillicaudatus), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.

Oxynoemacheilus sarus/, a new nemacheilid loach from the lower Ceyhan and Seyhan in southern Anatolia (Teleostei: Nemacheilidae).

Oxynoemacheilus sarus, new species, is described from the lower Ceyhan and Seyhan drainages in southern Anatolia. It is distinguished from other Oxynoemacheilus species in Cilicia (including the Göksu, Seyhan and Ceyhan drainages) by possession of a series of irregularly shaped midlateral blotches, 3-5 dark-brown bands on the caudal fin, a complete lateral line, a forked caudal fin (shortest middle caudal-fin ray is 56-70% of longest ray of the upper caudal-fin lobe), the caudal peduncle depth 1.4-1.8 times in its length, and a suborbital groove in male individuals. The new species occurs in sympatry with superficially similar O. seyhanicola and O. evreni, and is distinguished by colour pattern as well as morphometric and molecular characters. Molecular data suggest that the closest relatives to the new species in our dataset are O. euphraticus and O. shehabi, which is characterised by a minimum K2P distance of 3.6% and 3.8%, respectively, in the COI mtDNA barcode region.

A review of the Cypriniform tribe Yunnanilini Prokofiev, 2010 from China, with an emphasis on five genera based on morphologies and complete mitochondrial genomes of some species.

The loach tribe Yunnanilini from China is reviewed here using morphological characters and complete mitochondrial genomes of select species. Molecular data suggest that the tribe Yunnanilini is not monophyletic and can be divided into three clades. Species of the Yunnanilus nigromaculatus group form an independent genus and are placed in Eonemachilus. In the phylogenetic tree, Y. jinxiensis clusters with Paranemachilus genilepis, and Y. pulcherrimus clusters with Micronemacheilus cruciatus, indicating that Y. jinxiensis and Y. pulcherrimus belong to Paranemachilus and Micronemacheilus, respectively. Based on morphological data, Y. bailianensis and Y. longibarbatus are placed in Heminoemacheilus, while Y. jinxiensis and Y. pulcherrimus, are placed in Paranemachilus and Micronemacheilus, respectively. Yunnanilus niulanensis and Y. qujinensis are treated as junior synonyms of Eonemachilus caohaiensis. Eonemachilus, Micronemacheilus, and Yunnanilus are show short separation between anterior and posterior nostrils. The genera can be distinguished from each other by mouth structure, lateral line and cephalic lateral-line canals, and papillae on median part of both lips. The anterior and posterior nostrils of Heminoemacheilus and Paranemachilus are closely set. Paranemachilus and Micronemacheilus are distinguished by cheeks covered with scales and lips with papillae, respectively. Our phylogenetic tree and morphological characters support Traccatichthys as a valid genus, which can be distinguished from Micronemacheilus by anterior and posterior nostrils closely set (vs. clearly separated). Four species are placed in Traccatichthys.

emOxynoemacheilus/em emphasicus/em, a new nemacheilid loach from the eastern Black Sea basin with some remarks on other Caucasian emOxynoemacheilus/em (Teleostei: Nemacheilidae).

Oxynoemacheilus phasicus, new species, is described from the Rioni and Enguri River drainages in Georgia. It is distinguished from other Oxynoemacheilus species in the O. brandtii group by a mottled or marbled flank pattern in adults, a slightly emarginate caudal fin, and a deep caudal peduncle. Molecular data suggest that the new species is characterized by a minimum K2P distance of 7.5% from O. brandtii from the Kura drainage in the mtDNA COI barcode region. Oxynoemacheilus brandtii and O. elsae are re-diagnosed. A very slender Oxynoemacheilus from the Aras drainage clusters as sister to O. elsae in our molecular analysis and not with O. brandtii from the Kura River. However, it is identified as O. brandtii as it is indistinguishable from this species in morphological characters.

One northward, one southward: Contrasting biogeographical history in two benthic freshwater fish genera across Southeast Asia (Teleostei: Cobitoidea: Nemacheilus, Pangio).

Southeast Asia is one of the world's biodiversity hotspots, and the high level of diversity and endemism was reached by colonisation events as well as internal diversification. We investigate the phylogenetic relationships and biogeographic history of the loach genus Nemacheilus, which is widely distributed and common across freshwaters of Southeast Asia. In addition we present the ancestral range reconstruction of the related loach genus Pangio that commonly occurs in the same region as Nemacheilus. Our results reveal that the species currently classified as Nemacheilus in fact are a polyphyletic assemblage; most species are now retaining in a monophyletic Nemacheilus sensu stricto and five species belong to different lineages. We further indicate the existence of hidden diversity within Nemacheilus in the form of several undescribed species. Three major clades (Selangoricus, Masyae and Ornatus) are found within the genus Nemacheilus sensu stricto. These clades generally correspond to the species groups formerly defined on the basis of their pigmentation pattern. The biogeographic analyses show that Nemacheilus most likely originated in mainland Southeast Asia and subsequently expanded in a southward direction to Borneo, Sumatra and Java and the southern Malay Peninsula. In contrast, the genus Pangio originated in Sundaland, from where it extended several times northwards into Indochina and to northern India. Our results demonstrate that small freshwater fishes with restricted dispersal ability are very helpful for the reconstruction of biogeographic history. The contrasting biogeographic history of these two groups of small, benthic and related fish show how complex and case-specific the processes that lead to the biodiversity richness of Southeast Asia are.

Yunnanilus chuanheensis, a new loach species (Cypriniformes: Nemacheilidae) from the upper Lixianjiang River in Yunnan, China.

A new loach species (Cypriniformes: Nemacheilidae: Yunnanilini), Yunnanilus chuanheensis sp. nov., was caught from Chuanhe in the upper reaches of the Lixianjiang River, a tributary of the Red River in Nanjian County, Yunnan Province, China. This species is a member of the traditional Y. pleurotaenia species group based on the presence of a lateral line and cephalic lateral-line canals. It can be distinguished from other species in the Y. pleurotaenia species group by the following characters: lips without papillae, anterior and posterior nostrils separated, whole body scaled, eye diameter smaller than interorbital width, outer gill raker absent on first gill arch, eye diameter greater than 18% of head length, 10-11 inner gill rakers on first gill arch, and lateral line not extending to vertical through dorsal fin insertion. To the best of our knowledge, this is the first Yunnanilus species recorded from the Red River drainage.

Skeletal and muscular pelvic morphology of hillstream loaches (Cypriniformes: Balitoridae).

The rheophilic hillstream loaches (Balitoridae) of South and Southeast Asia possess a range of pelvic girdle morphologies, which may be attributed to adaptations for locomotion against rapidly flowing water. Specifically, the connectivity of the pelvic plate (basipterygium) to the vertebral column via a sacral rib, and the relative size and shape of the sacral rib, fall within a spectrum of three discrete morphotypes: long, narrow rib that meets the basipterygium; thicker, slightly curved rib meeting the basipterygium; and robust crested rib interlocking with the basipterygium. Species in this third category with more robust sacral rib connections between the basipterygium and vertebral column are capable of walking out of water with a tetrapod-like lateral-sequence, diagonal-couplet gait. This behavior has not been observed in species lacking direct skeletal connection between the vertebrae and the pelvis. The phylogenetic positions of the morphotypes were visualized by matching the morphological features onto a novel hypothesis of relationships for the family Balitoridae. The morphotypes determined through skeletal morphology were correlated with patterns observed in the pelvic muscle morphology of these fishes. Transitions towards increasingly robust pelvic girdle attachment were coincident with a more anterior origin on the basipterygium and more lateral insertion of the muscles on the fin rays, along with a reduction of the superficial abductors and adductors with more posterior insertions. These modifications are expected to provide a mechanical advantage for generating force against the ground. Inclusion of the enigmatic cave-adapted balitorid Cryptotora thamicola into the most data-rich balitorid phylogeny reveals its closest relatives, providing insight into the origin of the skeletal connection between the axial skeleton and basipterygium.

A new species of Triplophysa (Cypriniformes, Nemacheilidae) from Weihe River in Gansu Province, China.

A new species of Tibetan loach, Triplophysa weiheensis sp. nov., is described from the Weihe River in Gansu Province, China, based on morphological and molecular analyses. The new species can be distinguished from all known congeners by a unique combination of the following characters: scaleless; snout abruptly sloping downward, anterior to anterior nostril; lower jaw crescentic, not sharp; body without obvious mottling; lateral line interrupted on posterior trunk at pelvic-fin distal extremity; caudal-peduncle length 2.0-2.7 times its depth; branched rays of pectoral fin 10-11; branched rays of pelvic fin 5-6; inner gill rakers on 1 st gill arch 14-16; vertebrae 4+34-36; intestine with 6-7 loops, length ca. 1.8 times SL ( n=3); bony capsule of air bladder small and thin; posterior chamber of air bladder absent.

Resolving an unnoticed diversity within the Schistura robertsi species complex (Teleostei: Nemacheilidae) using molecules and morphology.

The Schistura robertsi species complex is a group of freshwater fish inhabiting streams in southeast Myanmar as well as in western and southern Thailand. In southern Thailand, the distribution exceeds the biogeographically important 'Surat Thani - Krabi line'. The complex is believed to include five described and one undescribed species, but monophyly and systematics of the group have never been studied explicitly. The present study aims to resolve the number of species within the Schistura robertsi group as well as their distribution areas and phylogenetic relations. We analysed mitochondrial and nuclear sequence data of 86 specimens from 47 localities and 18 morphological characters of 193 specimens. The phylogenetic analyses revealed the S. robertsi complex to be monophyletic and to be composed of ten major lineages. Six of them correspond to the known described or undescribed species, but another four newly identified clades reveal the existence of an overlooked diversity within the group. All genetic lineages are statistically highly supported and all are morphologically diagnosable, suggesting that they represent distinct species. The distribution areas of several clades overlap, the cases of direct co-occurrence show no sign of hybridisation.

Mitogenomic characterization and systematic placement of the Congo blind barb Caecobarbus geertsii (Cypriniformes: Cyprinidae).

This study presents the first complete mitochondrial genome (mitogenome) of Caecobarbus geertsii, the Congo blind barb, a cave-dwelling, CITES-protected, cyprinid fish endemic to the Lower Congo basin (DRC). The length of the circular mitogenome is 16,565 base pairs. The 13 protein coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes are similar in position and direction to those of other members of the family Cyprinidae. Phylogenetic analyses including 28 complete mitogenomes from representatives of the subfamily Smiliogastrinae (Cyprinidae), showed that Caecobarbus was nested within a clade including representatives of the genus Enteromius. The data presented in this study provide information on the molecular identification and classification of this threatened species. The results further suggest the need for a taxonomic revision of the genus Enteromius.

Disentangling the taxonomy of the subfamily Rasborinae (Cypriniformes, Danionidae) in Sundaland using DNA barcodes.

Sundaland constitutes one of the largest and most threatened biodiversity hotspots; however, our understanding of its biodiversity is afflicted by knowledge gaps in taxonomy and distribution patterns. The subfamily Rasborinae is the most diversified group of freshwater fishes in Sundaland. Uncertainties in their taxonomy and systematics have constrained its use as a model in evolutionary studies. Here, we established a DNA barcode reference library of the Rasborinae in Sundaland to examine species boundaries and range distributions through DNA-based species delimitation methods. A checklist of the Rasborinae of Sundaland was compiled based on online catalogs and used to estimate the taxonomic coverage of the present study. We generated a total of 991 DNA barcodes from 189 sampling sites in Sundaland. Together with 106 previously published sequences, we subsequently assembled a reference library of 1097 sequences that covers 65 taxa, including 61 of the 79 known Rasborinae species of Sundaland. Our library indicates that Rasborinae species are defined by distinct molecular lineages that are captured by species delimitation methods. A large overlap between intraspecific and interspecific genetic distance is observed that can be explained by the large amounts of cryptic diversity as evidenced by the 166 Operational Taxonomic Units detected. Implications for the evolutionary dynamics of species diversification are discussed.

Psilorhynchus nahlongthai, a new fish species (Teleostei: Psilorhynchidae) from the Brahmaputra drainage, northeast India.

Psilorhynchus nahlongthai, a new psilorhynchid fish, is described from the Diyung River, a tributary to the Kopili River (itself a southern tributary of the Brahmaputra drainage) in Assam, northeast India. It is placed in the Psilorhynchus balitora species group and can be easily distinguished from all other members of this group by a combination of the following characters: dense and prominent tuberculation on the head region; thick and long pre- and post-epiphyseal fontanelles on the neurocranium; 9 + 8 caudal-fin rays; and 34 (24 + 10) vertebrae. Genetic divergence between P. nahlongthai and members of the P. balitora species group from the Brahmaputra and neighbouring drainages, with K2P distances ranging 3.7%-14.7% and 7.4%-20.7% in the mitochondrial COI and cyt b gene datasets respectively, support its report as a new species.

Characterization of the complete mitochondrial genome of Barilius malabaricus and its phylogenetic implications.

Bariliine fishes are important components of the ornamental as well as subsistence fishery sectors in India. Many of the species in the genus Barilius are threatened by habitat loss and therefore need to be met with conservation initiatives. Effective conservation measures, however, require clarification of species identities and resolution of the validity of many species currently treated as synonymous or sub-species. The complete mitochondrial genome data provide better insight into phylogenetic information than the short fragment or single gene based analysis. Thus, we have sequenced the complete mitochondrial genome of Barilius malabaricus, one of the important fish species in the fresh water ornamental sector, for better understanding its phylogenetic status. The 16,519 bp mitochondrial genome consists of 37 genes which classifies as 22 tRNA, 13 protein coding and 2 ribosomal RNA genes and a control region. Overall, the mitochondrial genome bears the typical gene order and composition as in other fishes. Further, the COI, cytochrome b and 16S rRNA gene sequences revealed that, B. malabaricus is genetically closer to B. canarensis and B. bakeri compared to other Barilius species. Also, the Barilius species of west flowing rivers in Western Ghats were consistently recovered as a clade distinct from other species. We therefore suggest to retain the genus name Barilius for the species from the Western Ghats until a comprehensive analysis based on both morphological and molecular markers reveals the relationship between species now variously placed in the genera Barilius and Opsarius in greater detail.

Oreonectes guilinensis (Teleostei, Cypriniformes, Nemacheilidae), a new loach species from Guangxi, China.

A new loach Oreonectes guilinensis sp. nov. is described from Guangxi Zhuang Autonomous Region, China. It is the second surface-dwelling fish identified in the genus Oreonectes after the type species Oreonectes platycephalus. This new species can be distinguished from other species within the genus by the combination of the following features: a round caudal fin, 13-14 branched rays, an incomplete lateral line with 4-6 pores, a short pelvic fin located some distance from the anus, a scale-covered body, a stout build (body width 14.0%-16.7% of standard length), a posterior chamber of well-developed airbladder and a yellowish-brown body with a lateral dark brown strip. The maximum likelihood phylogenetic analysis based on cytb gene recovered this new species in a well-supported clade with the type species O. platycephalus, sister to the Lefua clade. Morphological comparisons as well as our phylogenetic tree support Oreonectes shuilongensis, Oreonectes daqikongensis, Oreonectes jiarongensis as species of Troglonectes. Based on our results there are 6 valid species in the genus Oreonectes.