Taxonomic revision of the Southeast Asian brook barb genus Poropuntius Smith, 1931 (Teleostei, Cyprinidae) with description of a new species from Vietnam.

Molecular data from samples encompassing 22 nominal species of Poropuntius indicate that the species-level diversity in the genus has been vastly overestimated, likely due to inadequate taxon and geographic sampling and reliance on morphological characters that vary intra-specifically. The latter includes discrete mouth morphologies related to alternate feeding strategies (ecomorphs) within populations. One new species is described, Poropuntiusanlaoensis Hoàng, Pham & Tran, sp. nov., and 17 synonyms of six valid species names of Poropuntius, P.krempfi, P.alloiopleurus, P.huangchuchieni, P.laoensis, P.kontumensis, and P.deauratus, are recognised. Additional taxonomic changes in this widespread and generally poorly known genus are likely as more molecular and morphological data become available.

Genome-wide analysis reveals the contributors to fast molecular evolution of the Chinese hook snout carp (Opsariichthys bidens).

Variations in molecular evolutionary rate have been widely investigated among lineages and genes. However, it remains an open question whether fast rate of molecular evolution is driven by natural selection or random drift, and how the fast rate is linked to metabolic rate. Additionally, previous studies on fast molecular evolution have been largely restricted to concatenated matrix of genes or a few specifically selected genes, but less is known for individual genes at the genome-wide level. Here we addressed these questions using more than 5000 single-copy orthologous (SCO) genes through comparative genomic and phylogenetic analyses among fishes, with a special focus on a newly-sequenced clupeocephalan fish the Chinese hook snout carp Opsariichthys bidens. We showed O. bidens displays significantly higher mean substitution rate and more fast-evolving SCO genes (2172 genes) than most fishes studied here. The rapidly evolving genes are enriched in highly conserved and very basic functions such as translation and ribosome that are critical for biological fitness. We further revealed that ∼25 % of these fast-evolving genes exhibit a constant increase of substitution rate from the common ancestor down to the present, suggesting a neglected but important contribution from ancestral states. Model fitting showed that ∼85 % of fast-evolving genes exclusive to O. bidens and related species follow the adaptive evolutionary model rather than random-drift model, and 7.6 % of fast-evolving genes identified in O. bidens have experienced positive selection, both indicating the reflection of adaptive selection. Finally, metabolic rate was observed to be linked with substitution rate in a gene-specific manner. Overall, our findings reveal fast molecular evolution of SCO genes at genome-wide level in O. bidens, and uncover the evolutionary and ecological contributors to it.

Contrasting genetic and morphological differentiation among geographical lineages of a stenotopic miniature rasborine, Boraras maculatus, in Peninsular Malaysia.

The variability in the stenotopic miniature rasborine Boraras maculatus (Cypriniformes: Danionidae: Rasborinae) across acidic-water habitats of Peninsular Malaysia (PM) was investigated using two molecular markers (the mitochondrial cytochrome c oxidase subunit I [COI] gene and the nuclear rhodopsin gene), as well as morphological evidence. Molecular phylogenetic analyses revealed differentiation among populations of B. maculatus in PM with the distinction of four allopatric lineages. Each of them was recognized as a putative species by automatic species delimitation methods. These lineages diverged from each other between 7.4 and 1.9 million years ago. A principal component analysis (PCA) was conducted to examine the multivariate variation in 11 morphometric measurements among three of these lineages. PCA results showed a significant overlap in morphological characteristics among these lineages. Additionally, a photograph-based machine learning approach failed to fully differentiate these lineages, suggesting limited morphological differentiation. B. maculatus represents a case of morphological stasis in a stenotopic miniature species. Strong habitat preference, coupled with long-term habitat fragmentation, may explain why each lineage of B. maculatus has a restricted distribution and did not disperse to other regions within and outside of PM, despite ample possibilities when the Sunda shelf was emerged and drained by large paleodrainages for most of the past 7 million years. The conservation status of B. maculatus and its peat swamp habitats are discussed, and it is concluded that peat swamps comprise several evolutionary units. Each of these units is considered a conservation unit and deserves appropriate protection.

Chromosome-level reference genomes of two imperiled desert fishes: spikedace (Meda fulgida) and loach minnow (Tiaroga cobitis).

North American minnows (Cypriniformes: Leuciscidae) comprise a diverse taxonomic group, but many members, particularly those inhabiting deserts, face elevated extinction risks. Despite conservation concerns, leuciscids remain under sampled for reference assemblies relative to other groups of freshwater fishes. Here, we present 2 chromosome-scale reference genome assemblies spikedace (Meda fulgida) and loach minnow (Tiaroga cobitis) using PacBio, Illumina and Omni-C technologies. The complete assembly for spikedace was 882.1 Mb in total length comprised of 83 scaffolds with N50 = 34.8 Mb, L50 = 11, N75 = 32.3 Mb, and L75 = 18. The complete assembly for loach minnow was 1.3 Gb in total length comprised of 550 scaffolds with N50 = 48.6 Mb, L50 = 13, N75 = 42.3 Mb, and L75 = 20. Completeness assessed via Benchmarking Universal Single-Copy Orthologues (BUSCO) metrics using the Actinopterygii BUSCO database showed ∼97% for spikedace and ∼98% for loach minnow of complete BUSCO proportions. Annotation revealed approximately 32.58 and 29.04% of spikedace and loach minnow total genome lengths to be comprised of protein-coding genes, respectively. Comparative genomic analyses of these endangered and co-distributed fishes revealed widespread structural variants, gene family expansions, and evidence of positive selection in both genomes.

Social behaviors as welfare indicators in teleost fish.

Animal welfare is a key issue not only for aquaculture industry and food production, but also for daily husbandry practices in research topics related to physiology in wild and farmed animals. In this context, teleost fish constitute interesting models to assess alternative welfare indicators because of their wide diversity in reproductive and social structures. Any framework for assessing teleost fish welfare needs to account for the physiological mechanisms involved in each species as a first step. A comprehensive approach should also take into account how these physiological and behavioral parameters can be altered by environmental enrichment considering the specific requirements in each case and identifying intrinsic biological characteristics of individual species. This review will show how cortisol and sex steroids regulate social behavior in teleost fish, and how different aspects of social behavior can be employed as welfare indicators according to specific characteristics in each case. This article will consider evidence in teleost fish, including cichlids, characids and cyprinids with different reproductive strategies and social structures (e.g., territorial social hierarchies or shoaling behavior). Neotropical species will be particularly emphasized. The main laboratory-based animal welfare indicators are cortisol, a classical stress hormone, together with sex steroids. Considering that the endocrine landscape is intrinsically related to social behavior, reproductive and agonistic behavioral traits such as aggression, anxiety and courtship are key elements to assess welfare under housing and culture conditions. This review highlights the importance of assessing physiological mechanisms and identifying behavioral characteristics in teleost fish, especially in Neotropical species, as a baseline to understand which environmental enrichment can improve animal welfare in each individual species.

Complete mitochondrial genome of Belligobio pengxianensis (Cypriniformes: Gobionidae).

Belligobio pengxianensis is a small fish endemic to the upper Yangtze River of China. In this study, the complete mitochondrial genome of B. pengxianensis is determined for the first time, and it should become a reference sequence to aid in species identification, biodiversity monitoring and conservation. The mitogenome has overall length of 16,610 bp and AT content of 55.23%, including 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one non-coding control region. The results of phylogenetic analyses show that B. pengxianensis is nested within the genus Hemibarbus.

The complete mitochondrial genome of Pethia padamya (Actinopteri, Cyprinidae).

Pethia padamya (Kullander and Britz, 2008) is a freshwater fish distributed in the Mekong River basin of Thailand. It has beautiful colors and can be used as an ornamental fish. The complete mitochondrial genome of P. padamya was determined using next-generation sequencing technology and its characteristics were analyzed. The mitochondrial genome is a closed circular molecule comprising 16,792 bp, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a major non-coding region. The overall base composition of the mitochondrial genome is 32.47% A, 25.39% C, 26.08% T, and 16.06% G, with a high A + T bias of 58.55%. Phylogenetic analysis revealed P. padamya as a sister group of Pethia conchonius+(Pethia ticto+Pethia cumingii) and Pethia gelius with maximal support, providing support for the monophyly of the genus Pethia based on concatenated nucleotide sequences. The results of this study proved the monophyly of the genus Pethia. These data for the first time provide information on the complete mitochondrial genome of P. padamya and can contribute to further studies on the biodiversity and management of P. padamya.

Deep genome-wide divergences among species in White Cloud Mountain minnow Tanichthys albonubes (Cypriniformes: Tanichthyidae) complex: Conservation and species management implications.

Identifying cryptic species is important for the assessments of biodiversity. Further, untangling mechanisms underlying the origins of cryptic species can facilitate our understanding of evolutionary processes. Advancements in genomic approaches for non-model systems have offered unprecedented opportunities to investigate these areas. The White Cloud Mountain minnow (Tanichthys albonubes) is a popular freshwater pet fish worldwide but its wild populations in China are critically endangered. Recent research based on a few molecular markers suggested that this species in fact comprised seven cryptic species, of which six were previously unknown. Here, we tested six of these cryptic species and quantified genomic interspecific divergences between species in the T. albonubes complex by analyzing genome-wide restriction site-associated DNA sequencing (RADseq) data generated from 189 individuals sampled from seven populations (including an outgroup congeneric species, T. micagemmae). We found that six cryptic species previously suggested were well supported by RADseq data. The genetic diversity of each species in the T. albonubes complex was low compared with T. micagemmae and the contemporary effective population sizes (Ne) of each cryptic species were small. Phylogenetic analysis showed seven clades with high support values confirmed with Neighbor-Net trees. The pairwise divergences between species in T. albonubes complex were deep and the highly differentiated loci were evenly distributed across the genome. We proposed that the divergence level of T. albonubes complex is at a late stage of cryptic speciation and lacking gene flow. Our findings provide new insights into cryptic speciation and have important implications for conservation and species management of T. albonubes complex.

Phylogenomics and classification of Notropis and related shiners (Cypriniformes: Leuciscidae) and the utility of exon capture on lower taxonomic groups.

North American minnows of the Shiner Clade, within the family Leuciscidae, represent one of the most taxonomically complex clades of the order Cypriniformes due to the large number of taxa coupled with conserved morphologies. Species within this clade were moved between genera and subgenera until the community decided to lump many of the unclassified taxa with similar morphologies into one genus, Notropis, which has held up to 325 species. Despite phylogentic studies that began to re-elevate some genera merged into Notropis, such as Cyprinella, Luxilus, Lythrurus, and Pteronotropis, the large genus Notropis remained as a taxonomic repository for many shiners of uncertain placement. Recent molecular advances in sequencing technologies have provided the opportunity to re-examine the Shiner Clade using phylogenomic markers. Using a fish probe kit, we sequenced 90 specimens in 87 species representing 16 genera included in the Shiner Clade, with a resulting dataset of 1,004 loci and 286,455 base pairs. Despite the large dataset, only 32,349 bp (11.29%) were phylogenetically informative. In our maximum likelihood tree, 78% of nodes are 100% bootstrap supported demonstrating the utility of the phylogenomic markers at lower taxonomic levels. Unsurprisingly, species within Notropis as well as Hudsonius, Luxilus, and Alburnops are not resolved as monophyletic groups. Cyprinella is monophyletic if Cyprinella callistia is excluded, and Pteronotropis is monophyletic if it includes Hudsonius cummingsae. Taxonomic changes we propose are: restriction of species included in Alburnops and Notropis, elevation of the subgenus Hydrophlox, expansion of species included in Miniellus, movement of Hudsonius cummingsae to Pteronotropis, and resurrection of the genera Coccotis and Paranotropis. We additionally had two specimens of three species, Notropis atherinoides, Ericymba amplamala, and Pimephales vigilax and found signficant differences between the localities (1,086, 1,424, and 845 nucleotides respectively).

The complete mitochondrial genome of Mesogobio lachneri (Cypriniformes: Gobionidae) from Northeast Asia.

Although Mesogobio lachneri is the type species of the genus Mesogobio, its systematic position and status have remained unresolved to date. In this study, for the first time, we report the complete mitochondrial genome of M. lachneri using Sanger sequencing. It is a circular genome with a length of 16,602 bp, comprising 22 tRNAs, 13 protein-coding genes (PCGs), two rRNAs, and one non-coding control region. Our phylogenetic analysis reveals that M. lachneri is the close relative of the genus Gobio, indicating that Mesogobio may be a valid genus.

Towards the phylogenetic placement of the enigmatic African genus Prolabeops Schultz, 1941.

The small cyprinid genus Prolabeops Schultz, 1941 is restricted to the Nyong and Sanaga River systems in Cameroon. In the past, the genus had been suggested to be either a member of the Labeoninae, Torinae or the Smiliogastrinae mainly on the basis of morphological similarities, and it is nowadays considered as incertae sedis within the Cypriniformes. This study provides the first attempt to reveal the phylogenetic position of Prolabeops using molecular data. For this purpose, the authors sequenced a large fraction of the mitochondrial genome (c. 13,600 bp), including all mitochondrial protein coding genes, of two Prolabeops melanhypopterus specimens and an additional four Enteromius specimens. The large-scale phylogenetic analysis was based on an alignment including all mitochondrial protein coding genes of 902 specimens representing c. 899 cypriniform species. Prolabeops was clearly recovered within the African Smiliogastrinae, forming a weakly supported clade together with Enteromius jae, Enteromius hulstaerti and Barboides gracilis. The study data underline the urgent need of a thorough taxonomic revision of the small African barbs collectively placed in the genus Enteromius.

Phylogeographical Analysis of the Freshwater Gudgeon Huigobio chenhsienensis (Cypriniformes: Gobionidae) in Southern China.

The freshwater gudgeon Huigobio chenhsienensis (Cypriniformes: Gobionidae) is a small fish endemic to southern China. In this study, we used mitochondrial cytochrome b gene (Cytb), from wide-ranging samplings of H. chenhsienensis from the Ou River (the central of southern China) to the Yangtze River Basin (the northernmost part of southern China) to explore genetic variations and the evolutionary history of H. chenhsienensis in southern China. In total, 66 haplotypes were identified from Cytb sequences of 142 H. chenhsienensis individuals, which could be divided into lineages A, B, and C with divergence times of ~4.24 Ma and ~3.03 Ma. Lineage A was distributed in the lower reaches of the Yangtze River, the Oujiang River, and the Jiao River, lineage B was distributed in the Qiantang River and the Cao'e River, whereas lineage C was restricted to the Poyang Lake drainage from the middle reaches of the Yangtze River. Lineage A could be subdivided into sub-lineages A-I, A-II, A-III, and A-IV, with divergence times of 1.30, 0.97, and 0.44 Ma. Lineage C could be subdivided into sub-lineages C-I and C-II, with a divergence time of 0.85 Ma. Our findings indicate that climate change during the Pliocene and Pleistocene eras, as well as the limited dispersal ability of H. chenhsienensis, have been major drivers for shaping the phylogeographical patterns of H. chenhsienensis.

Common carp (Cyprinus carpio) sperm reduction during short-term in vitro storage at 4 °C.

This study was designed to optimize a short-term storage protocol for common carp sperm at 4 °C under aerobic condition. Sperm from individual males were collected directly with or without extenders. The results demonstrated that in general, it was similar effect to collect sperm directly in extenders and keeping sperm for 0.5 h after collection without extenders. Sperm was diluted with eight selected extenders (sperm: extender = 2:1, 1:1 and 1:9) and undiluted sperm was used as a control. Sperm and seminal plasma parameters (sperm motility, velocity, membrane integrity, sperm concentration, osmolality and pH in seminal plasma) were evaluated in sperm stored on ice under aerobic conditions at 0, 2, 4, 6 and 8 days post stripping (DPS) using the computer- assisted sperm analysis system. Results showed that 1:1 and 2:1 dilution maintained higher sperm function and more sperm for a longer period. After 8 DPS, the best sperm quality and quantity was recorded in the common carp seminal plasma supplemented with 50 mM NaCl, Cejko extender (2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl, pH 7.5 and 310 mOsm/kg) supplemented with/without 25 mM KCl/NaCl. The reduction of spermatozoa number with time during short-term storage but varied according to different dilution ratios and extenders. At 8 DPS, sperm count has dropped by 22.9 % in a dilution of 1:1 compared to 50.3 % in sperm without dilution. Extenders with diluted 1:1, especially Cejko solution, largely postponed sperm reduction, 21.3 % compared to 55.5 % for sperm stored without extenders.

Descriptions of six new species in White Cloud Mountain minnow Tanichthys albonubes complex (Cypriniformes: Tanichthyidae) in southern China.

Tanichthys albonubes is a critically endangered freshwater fish in South China and is classified as a second-class state-protected animal in China. It is also a colourful ornamental fish and has been introduced into many countries and regions around the world. Previous studies have revealed that there are seven cryptic species in the T. albonubes complex. Here, six new species of these cryptic species were diagnosed and described based on external morphology, colour patterns and osteological characteristics: Tanichthys shenzhenensis sp. nov., Tanichthys huidongensis sp. nov., Tanichthys luheensis sp. nov., Tanichthys dongxingensis sp. nov., Tanichthys guipingensis sp. nov. and Tanichthys hainanensis sp. nov. In addition, an identification key to the genus Tanichthys was provided. The key diagnosable characteristics of six new species in T. albonubes complex include the margin colour of dorsal and anal fins, the number of branched dorsal and anal fins, the relative position of the orbitosphenoid to the parasphenoid, the states of the neural complex, the states of the neural spine of the fourth vertebra and the anterior process of the innominatum. These six new species were also well supported by the phylogenetic tree and uncorrected p-distances based on Cyt b sequences. The distribution pattern of the genus Tanichthys is characterized by point-like, discontinuous distribution and geographically isolated on a large scale with narrow distribution ranges of each species. This study provides scientific data for the taxonomy, conservation status assessment and evolution of Tanichthys.

História do Zebrafish na pesquisa científica: uma breve revisão

O zebrafish (Danio rerio) foi descrito pela primeira vez pelo escocês e zoólogo Francis Hamilton, sendo uma das mais de 20 espécies do gênero Danio e uma das mais de 4.000 espécies dentro da ordem Cypriniformes. Na década de 80, o biólogo George Streisinger, professor do Instituto de Biologia Molecular da Universidade de Oregon - Estados Unidos é reconhecido como o fundador do modelo animal zebrafish, ele utilizou-o para explorar a base genética do desenvolvimento neural dos vertebrados, estudo esse publicado no periódico Nature. O zebrafish tem uma série de atributos que o torna um excelente modelo de pesquisa, cerca de 70% de seus genes são ortólogos aos genes humanos, sendo que destes, 82% possuem relevância a doenças humanas. Trabalhar com o zebrafish possibilita mais praticidade por ser eficiente e com baixo custo, vem substituindo cada vez mais as pesquisas realizadas com roedores. Inicialmente tornou-se popular como modelo de desenvolvimento de vertebrados, pelos embriões serem transparentes e por desenvolverem rapidamente. Na atualidade, a pesquisa está se expandindo para áreas, como farmacologia, pesquisa clínica como modelo de diversas doenças, descoberta de medicamentos e estudos toxicológicos, sendo esse último um dos campos onde se concentra uma parte significativa da produção científica brasileira. As pesquisas crescem para o aperfeiçoamento dos dados já existentes, reiterar as semelhanças com a biologia humana e para a descoberta de novos conhecimentos, que expandirão de forma positiva nosso desenvolvimento na saúde animal e humana.

Evolutionary history of the Pelasgus minnows (Teleostei: Leuciscidae), an ancient endemic genus from the Balkan Peninsula.

The Balkans are one of the European biodiversity hotspots, hosting outstandingly rich, yet threatened, flora and fauna. This region hosts one of the richest endemic freshwater ichthyofauna in Europe, including several genera occurring exclusively here. One of these is the genus of the primary freshwater minnows Pelasgus, which was designated only in 2007. The genus is one of the most ancient genera of the family Leuciscidae and comprises seven small-bodied species, inhabiting slower, well-vegetated courses of rivers. This work is the first molecular multilocus phylogeny of the genus, based on one mitochondrial and three nuclear markers. In total, 305 individuals across almost entire distribution range of the genus were analysed. We inferred the evolutionary history of the species by comparing the results of our calibrated multilocus coalescent species-tree to palaeogeological events. The diversification of the genus started in the early Miocene and continued through to the beginning of Pleistocene. We identified the regions of the oldest colonization by Pelasgus, the drainages of the ancient lakes Ohrid and Prespa, and the southernmost part of the Peloponnese, hosting Pelasgus minutus, P. prespensis and P. laconicus, respectively. We showed that P. prespensis is not endemic to Lake Prespa, as previously thought; it occurs also in the Albanian River Devoll. This corroborates the emerging opinion that the endemic taxa of ancient lakes evolved within larger-scale historic drainages and not only within the lakes. Our results showed that the species with the most recent common ancestor of the early Pliocene origin, P. thesproticus, P. epiroticus, P. stymphalicus and P. marathonicus, have neighbouring distribution ranges. Pelasgus epiroticus is especially interesting, not only for its pronounced genetic diversity with a geographic pattern, but also for being found at three localities within the native distribution range of P. stymphalicus as a result of a translocation. At two of these localities, we identified hybrids between the two species, and at one of them, the genetically pure native species was not found at all. This points to a threat of the loss of the native ichthyofauna due to unintentional translocations.

Complete mitochondrial genome of Ladislavia taczanowskii (Cypriniformes: Gobionidae).

Mitochondrial genomes of two individuals of Tachanovsky's gudgeon Ladislavia taczanowskii have been determined on the basis of Sanger dideoxy sequencing. The gene compositions of two genomes contain 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region with the same length 16,614 bp. The phylogenetic tree reveals that the monotypic genus Ladislavia is a sister group of the subfamily Gobioninae within the family Gobionidae.

Characterization of the complete mitochondrial genome of Aphyocypris chinensis (Cypriniformes, Xenocyprididae), and its phylogenetic position in Cypriniformes.

Aphyocypris chinensis Günther, 1868 is a small freshwater fish of the family Xenocyprididae (Cypriniformes). In this study, we determined its complete mitochondrial genome and phylogenetic position in Cypriniformes. The complete mitochondrial genome is 16,608 bp in size, containing 13 protein-coding genes, two RNAs, 22 tRNAs, and a control region. It has the typical vertebrate mitochondrial gene arrangement. Our mitogenomic phylogeny revealed that A. chinensis belongs to Xenocyprididae, rather than Danionidae. This mitogenome information could play an essential role in resolving the conflict over its current taxonomic status in Cypriniformes.

To lump, to split or to maintain? Molecular taxonomy of the spotted barb Barbodes binotatus (Cyprinidae) and closely related species in Peninsular Malaysia.

The taxonomic status of the Southeast Asian spotted barb, Barbodes binotatus (Teleostei: Cyprinidae), has puzzled researchers because of large but inconsistent geographic variation of its body melanin marking pattern. In this study, the authors appraise the differentiation of B. binotatus and two closely related species, Barbodes rhombeus and saddle barb, Barbodes banksi, in Peninsular Malaysia using mitochondrial and nuclear markers. The results of this study reveal that the Peninsular Malaysia populations of each of the three species form largely reciprocal monophyletic lineages that differ from each other by a minimum of 2.3% p-genetic distance using COI gene. Nonetheless, specimens of B. binotatus in Peninsular Malaysia are only distantly related to specimens of B. binotatus in Java (type locality). The monophyly of B. banksi is not refuted although specimens of Peninsular Malaysia are genetically distinct from those of Sarawak (type locality). The authors discuss alternative hypotheses whether each of these three valid species is a single species or each of the main five genetic lineages revealed in this study represents a distinct species. Preliminary investigations reveal a mito-nuclear discordance at one locality in Peninsular Malaysia where B. binotatus and B. banksi co-occur. Further studies should inform on the extent of reproductive porousness between these two lineages and others.

Re-description of the loach species Leptobotia citrauratea (Teleostei, Botiidae), with the description of L. brachycephala from southern Zhejiang Province, China.

Leptobotia citrauratea (Nichols, 1925), a loach species, originally described from Dongting Lake, was recently rehabilitated, based on the examination of the holotype and non-topotypical specimens. Several field surveys conducted from 2016 to 2019 in Zhejiang, Jiangxi and Hunan Provinces, P.R. China, yielded many specimens of Leptobotia which were initially identified as L. citrauratea. Molecular and morphological analyses of these specimens demonstrated that two distinct species are involved. One was identified as L. citrauratea, represented by specimens from both the Poyang and Dongting Lake (type locality) systems in Jiangxi and Hunan Provinces, and the other species is described as L. brachycephala, represented by specimens from the Ou-Jiang and Qu-Jiang, two coastal rivers of Zhejiang Province, China. Leptobotia brachycephala resembles L. citrauratea and L. micra in having a row of orange dots or an orange stripe along the dorsal mid-line of the body, extending from the nape to the caudal-fin base - a unique character in Leptobotia. Leptobotia brachycephala differs from L. citrauratea and L. micra Bohlen & Slechtová, 2017, in caudal-fin shape and pelvic-fin insertion and proportional measurements including caudal-fin length, head length, predorsal length and anal-fin length. Its species status was further corroborated by position in a molecular phylogenetic analysis, based on the mitochondrial cyt b gene and its minimum uncorrected p-distance (2.9%) from congeneric species.