Molecular Identification of Acrossocheilus jishouensis (Teleostei: Cyprinidae) and Its Complete Mitochondrial Genome.

Molecular identification, such as DNA barcoding, is a useful tool that is widely applied in distinguishing species. To identify the cyprinid Acrossocheilus jishouensis, which was previously known to be restricted to only its type locality, we conducted molecular identification of this species based on 23 samples in five localities. Molecular identification based on the mitochondrial COI gene sequence showed that the morphologically similar samples from the five populations were all A. jishouensis, as the mean genetic distances between populations were very small (0.1-1.6%); thus, the distribution of this species was substantially expanded. The whole mitochondrial genome of one sample was also assembled, which was 16,594 bp in length and consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and one control region. All PCGs began with ATG except the COI gene, which started with GTG; seven PCGs used the complete stop codon TAA, while four terminated in T(AA) and two ended with TAG. The overall base composition reflected a higher proportion of A+T than G+C and a positive AT-skew and negative GC-skew pattern except for the opposite in ND6. Phylogenetic relationships inferred using BI and ML methods revealed that both Acrossocheilus and Onychostoma were nonmonophyletic, which indicated that the traditional diagnoses between these two genera need to be assessed further. The results of this study not only expanded the known distribution ranges of A. jishouensis, but also provided a valuable data resource for future molecular and evolutionary studies of Acrossocheilus and other cyprinids in Barbinae.

Two Complete Mitochondrial Genomes of Leptobrachium (Anura: Megophryidae: Leptobrachiinae): Characteristics, Population Divergences, and Phylogenetic Implications.

The mustache toads Leptobrachium boringii and Leptobrachium liui are two attractive species in Megophryidae, in which adult males have mustache-like keratinized nuptial spines on their upper lip. However, both are under threat due to multiple factors, of which scientific studies are still very limited. In this study, two new complete mitochondrial genomes of L. boringii and L. liui were sequenced, assembled, and annotated based on next-generation sequencing. The mitogenome lengths of L. boringii and L. liui were found to be 17,100 and 17,501 bp, respectively, with both containing 13 protein coding genes, 23 tRNAs, 2 rRNAs, and 1 non-coding control region. Nucleotide diversity analyses indicate that atp8, atp6, and nad2 showed higher nucleotide diversity than cox1, cox3, and cytb. The intraspecific genetic distances among three different populations of L. boringii exceed 4%, and those between two populations of L. liui reach 7%. Phylogenetic relationships support their division into two subfamilies of Megophryidae (Leptobrachiinae and Megophryinae) as well as two species groups within Leptobrachium, corresponding to the number of keratinized nuptial spines (10-48 in the L. boringii species group vs. 2-6 in the L. liui species group). The two new mitogenomes reported in this study provide valuable data for future molecular evolutionary and conservation studies of the genus Leptobrachium and other Megophryidae toads.

Complete mitochondrial genome of Rectorisluxiensis (Teleostei, Cyprinidae): characterisation and phylogenetic implications.

Mitochondrial genomes (mitogenomes) are widely used in scientific studies on phylogenetic relationships, molecular evolution and population genetics. Here, we sequenced and analysed the mitogenome of Rectorisluxiensis, a Yangtze River drainage endemic, but threatened cyprinid fish of Labeoninae. The complete mitogenome of R.luxiensis was 16,592 bp in length, encoding 13 protein coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a control region. The mitogenome showed a high A+T content (58.2%) and a positive AT-skew (0.10) and negative GC-skew (-0.25) base composition pattern. All the 13 PCGs were found to start with ATG codons, except for the COXI, in which GTG was the start codon. The ratio of non-synonymous and synonymous substitutions (Ka/Ks) of all the 13 PCGs were less than 1, indicating negative or purifying selection evolved in these genes. Comparatively speaking, the evolutionary rate of ATP8 was the fastest and ND4L was the slowest. All tRNAs could fold into a typical cloverleaf secondary structure, except tRNASer1 that lacked a dihydrouridine arm. Phylogenetic relationships, based on the PCGs dataset of 91 mitogenomes of Labeoninae, showed that R.luxiensis grouped with Rectorisposehensis and they formed a monophyletic Rectoris. However, many non-monophyletic genera were revealed in labeoninae fishes, such as Cirrhinus, Decorus, Garra, Labeo and Pseudocrossocheilus, which indicated that the validities of some traditional genera required a further check. This study reported the complete mitogenome of R.luxiensis for the first time, which provided valuable data for future molecular evolution and conservation related studies of Rectoris and other species in Labeoninae.

Husbandry, Captive Breeding, and Field Survey of Chinese Giant Salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus) is the largest extant amphibian species in the world with adults capable of reaching 2 m in length. Wild populations of A. davidianus have declined dramatically during the last century, making it also one of the top threatened species globally. Fortunately, aquaculture for this species developed in China during the 1970s has been extremely successful. Many relevant commercial products of A. davidianus have been produced in recent years on account of its nutritional and medicinal values. Balancing conservation and utilization will be key to the future destiny of A. davidianus. In this chapter, we describe detailed protocols for husbandry in indoor and outdoor facilities, captive breeding under natural-imitative conditions and using artificial insemination, and surveying and monitoring A. davidianus in the field. The protocols presented here aim to make the practices of A. davidianus operative and increase public awareness of this mystical and precious species.

Reintroduction modifies the intraspecific variations of symbiotic microbes in captive bred Chinese giant salamander.

Microorganisms play as fundamental contributors to maintain hosts' fitness, which can be shaped by external environment. Moreover, symbiotic microbiome also varied within species (e.g., between sexes and developmental stages). However, we still need more studies to quantify whether the intraspecific variation patterns of symbiotic microbes can be modified with the change of environment. The Chinese giant salamander (CGS; Andrias davidianus) is a Critically Endangered species. Despite quantitative captive bred individuals were released to rebuild wild populations, the effectiveness is limited. More importantly, no studies have revealed the adaptation of released CGSs to the complex field conditions. In the present study, we explored whether reintroduction can reshape the intraspecific variations of symbiotic microbiota in captive bred CGSs using high-throughput amplicon sequencing of the16S rRNA gene. We found no significant difference of symbiotic microbiome in captive bred males and females, but released males and females differed significantly in skin microbiome. Juveniles had higher diversity of microbial symbiont than adults in hatchery, but lower diversity in field. Moreover, dominant bacterial taxa differed between juveniles and adults in both hatchery and field. Importantly, this symbiotic microbiome variations within species can be modified (alpha and beta diversity, and community composition) when captive bred individuals were released to the field. Overall, we observed a lower alpha diversity and higher relative abundance of Chryseobacterium, Plesiomonas, and Acinetobacter in the bacterial community of captive bred individuals. Instead, higher alpha diversity of symbiotic microbiota and higher relative abundance of S24-7 and Lactobacillus was detected in released individuals. These modifications may associate with the change of living environment, as well as the specific behavior within CGSs (e.g., movement patterns and foraging activities). Future studies can incorporate other approaches (e.g., blood physiology) to better evaluate the growth and health of reintroduced CGSs.

Characterization, Comparison of Two New Mitogenomes of Crocodile Newts Tylototriton (Caudata: Salamandridae), and Phylogenetic Implications.

Mitochondrial genomes (mitogenomes) are valuable resources in molecular and evolutionary studies, such as phylogeny and population genetics. The complete mitogenomes of two crocodile newts, Tylototriton broadoridgus and Tylototriton gaowangjienensis, were sequenced, assembled, and annotated for the first time using next-generation sequencing. The complete mitogenomes of T. broadoridgus and T. gaowangjienensis were 16,265 bp and 16,259 bp in lengths, which both composed of 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and 1 control region. The two mitogenomes had high A + T content with positive AT-skew and negative GC-skew patterns. The ratio of non-synonymous and synonymous substitutions showed that, relatively, the ATP8 gene evolved the fastest and COI evolved the slowest among the 13 PCGs. Phylogenetic trees from BI and ML analyses resulted in identical topologies, where the Tylototriton split into two groups corresponding to two subgenera. Both T. broadoridgus and T. gaowangjienensis sequenced here belonged to the subgenus Yaotriton, and these two species shared a tentative sister group relationship. The two mitogenomes reported in this study provided valuable data for future molecular and evolutionary studies of the genus Tylotoriton and other salamanders.

Allelically and Differentially Expressed Genes After Infection of Edwardsiella ictaluri in Channel Catfish as Determined by Bulk Segregant RNA-Seq.

Identification of genetic markers associated with resistance against enteric septicemia of catfish (ESC) is of great interest for genetic enhancement programs of catfish. In the present study, bulk segregant RNA-Seq analysis was applied to determine differentially expressed genes and alleles after ESC infection. Here we report three genomic regions on LG1, LG12, and LG26, containing significant single-nucleotide polymorphisms (SNPs). These genomic regions aligned well with quantitative trait loci (QTL) previously identified. Within the QTL regions, eleven genes were found to be differentially regulated between phenotypic bulks. Importantly, the QTL on linkage group 1 (LG1) were found to be expressed in the liver, whereas the QTL on LG12 and LG26 were expressed in the intestine, suggesting multiple mechanisms of ESC resistance. It is apparent that apolipoproteins may be important for ESC resistance as the QTL on LG1 included the 14-kDa apolipoprotein genes that are both allelically expressed and differentially expressed between the resistant and susceptible bulks. Traf2 and NCK-interacting protein kinase (TNIK) were found in the QTL on LG12, and it was downregulated in resistant fish, suggesting the importance of NCK downregulation in ESC resistance, as previously reported. In addition, we observed divergent gene expression patterns between the liver and intestine after infection. Immune/inflammatory-related processes were overrepresented from liver DEGs, while those DEGs identified from intestine were enriched for proteolysis and wounding processes. Taken together, the BSR-Seq analysis presented here advanced the knowledge of ESC resistance, providing information of not only positions of QTL but also genes and their differential expression between resistant and susceptible fish, making it one step closer to the identification of the causal genes for ESC resistance.

Whole-Genome Sequencing of Sinocyclocheilus maitianheensis Reveals Phylogenetic Evolution and Immunological Variances in Various Sinocyclocheilus Fishes.

An adult Sinocyclocheilus maitianheensis, a surface-dwelling golden-line barbel fish, was collected from Maitian river (Kunming City, Yunnan Province, China) for whole-genome sequencing, assembly, and annotation. We obtained a genome assembly of 1.7 Gb with a scaffold N50 of 1.4 Mb and a contig N50 of 24.7 kb. A total of 39,977 protein-coding genes were annotated. Based on a comparative phylogenetic analysis of five Sinocyclocheilus species and other five representative vertebrates with published genome sequences, we found that S. maitianheensis is close to Sinocyclocheilus anophthalmus (a cave-restricted species with similar locality). Moreover, the assembled genomes of S. maitianheensis and other four Sinocyclocheilus counterparts were used for a fourfold degenerative third-codon transversion (4dTv) analysis. The recent whole-genome duplication (WGD) event was therefore estimated to occur about 18.1 million years ago. Our results also revealed a decreased tendency of copy number in many important genes related to immunity and apoptosis in cave-restricted Sinocyclocheilus species. In summary, we report the first genome assembly of S. maitianheensis, which provides a valuable genetic resource for comparative studies on cavefish biology, species protection, and practical aquaculture of this potentially economical fish.

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Water quality under tourism disturbance was simulated through controlling the water intake of the ecological breeding ponds of Chinese giant salamander (Andrias davidianus, CGS). Both the reproductive behavior (oviposition and parental care) and capacity (relative egg production, fertilizing rate of eggs, and hatching rate of fertilized eggs) of CGS were examined using a real-time infrared digital monitoring system. The relationships among reproductive behavior, capacity, and the corresponding parameters of water quality were analyzed, to understand how water quality under tourism disturbance would affect the reproductive behavior and capacity of CGS. The examined oviposition behavior and capacity of CGS showed no variation in general, but the parental care behavior such as tail fanning and agitation time of the male CGS were prolonged significantly in the groups under tourism disturbance. Such prolonged behaviors would help increase the content of dissolved oxygen (DO) to meet the high demands of DO during embryonic development of CGS. In addition, the overall hatching time of fertilized eggs was increased significantly under disturbance conditions when it compared with the control, which would ensure the overall hatching rate among these comparative groups unaffected. In summary, the prolongations of some reproductive behavior (tail fanning and agitation of the male CGS and the development time of fertilized egg) would be a kind of positive actions of CGS in response to the changes of water quality resulted from tourism disturbance.

Construction of a chromosome-level genome assembly for genome-wide identification of growth-related quantitative trait loci in Sinocyclocheilus grahami (Cypriniformes, Cyprinidae).

The Dianchi golden-line barbel, Sinocyclocheilus grahami (Regan, 1904), is one of the "Four Famous Fishes" of Yunnan Province, China. Given its economic value, this species has been artificially bred successfully since 2007, with a nationally selected breed (" S. grahami, Bayou No. 1") certified in 2018. For the future utilization of this species, its growth rate, disease resistance, and wild adaptability need to be improved, which could be achieved with the help of molecular marker-assisted selection (MAS). In the current study, we constructed the first chromosome-level genome of S. grahami, assembled 48 pseudo-chromosomes, and obtained a genome assembly of 1.49 Gb. We also performed QTL-seq analysis of S. grahami using the highest and lowest bulks (i.e., largest and smallest size) in both a sibling and random population. We screened two quantitative trait loci (QTLs) (Chr3, 14.9-39.1 Mb and Chr17, 4.1-27.4 Mb) as the major growth-related locations. Several candidate genes (e.g., map2k5, stat1, phf21a, sox6, and smad6) were also identified, with functions related to growth, such as cell differentiation, neuronal development, skeletal muscle development, chondrogenesis, and immunity. These results built a solid foundation for in-depth MAS studies on the growth traits of S. grahami.

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.

Comprehensive transcriptional changes in the liver of Kanglang white minnow (Anabarilius grahami) in response to the infection of parasite Ichthyophthirius multifiliis.

Abstract: The notorious parasite Ichthyophthirius multifiliis (Ich) has been recorded worldwide in fish species and causes white spot disease, posing major threats and resulting in severe losses to international fish production. Extensively effective strategies for treating Ich are not available yet, and genetic mechanisms of hosts in response to the parasite are still largely unknown. In this study, we selected Kanglang white minnow (KWM, Anabarilius grahami) to examine its liver transcriptional changes after Ich infection, as white spot disease is one bottleneck problem in exploring this economically important species. We divided the experimental fishes into three groups (control, early-infected, and late-infected) to examine differentially expressed genes (DEGs). A total of 831 DEGs were identified and classified into 128 significantly enriched GO (Gene Ontology) terms and 71 significantly enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Most of these terms or pathways were functionally enriched in immunity, inflammatory response, and apoptosis, such as nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling, tumor necrosis factor (TNF) signaling, interleukin-17 (IL-17) signaling, and apoptosis pathways. We also identified 178 putative antimicrobial peptides (AMPs) and AMP precursors based on our previously reported genome assembly of KWM, and revealed that the expressional patterns varied according to different types. In summary, our work reported the first comprehensive transcriptional changes in KWM in response to the exogenous infection of Ich, which would lay a solid foundation for in-depth studies on disease defense or resistant strains selection in this valuable fish.

Sinocyclocheilus sanxiaensis, a new blind fish from the Three Gorges of Yangtze River provides insights into speciation of Chinese cavefish.

A blind fish of Sinocyclocheilus (Cypriniformes: Cyprinidae) was caught in open water in the Three Gorges (Sanxia) reservoir, at a depth of 20 m in the mainstream of Yangtze River in Zigui County, Hubei Province, China. This fish can be easily distinguished from all other congeners by external morphological characteristics, and is estimated to have diverged from its sister group about 0.55 million years ago (Ma). The geologically well separated locality of this species has expanded the distribution of Sinocyclocheilus cavefish from around N25°(latitude) to above N30°. Herein, we describe this new species as Sinocyclocheilus sanxiaensis sp. nov., and discuss the possible reasons why the species appears, surprisingly, in the Three Gorges reservoir.

Evolution of the intermuscular bones in the Cyprinidae (Pisces) from a phylogenetic perspective.

Intermuscular bones (IBs) are widely present in morphologically generalized teleost fishes and are commonly found in the Cyprinidae. Intermuscular bones are small, hard spicules of bone that are formed by ossification in the myosepta between neighboring myomeres. Why fish have IBs, and whether there is any evolutionary pattern to their occurrence, has been poorly understood. However, the presence of IBs does substantially affect the meat quality and commercial values of many cyprinid fishes in aquaculture. In this study, we sampled 592 individuals of cyprinid fishes to systematically investigate the evolution of IBs from a phylogenetic point of view. We found that the total number of IBs in the Cyprinidae ranged from 73 to 169, and we clarified that only two categories of IBs (epineural and epipleural) were present in all examined cyprinids. Most of the IBs were distributed in the posterior region of the fish, which might be an optimal target for selecting fewer IB strains in aquaculture. There was a positive correlation between IBs and the number of vertebrae, thus making it possible to predict the approximate number of IBs by counting the number of vertebrae. Although the IBs displayed some correlation with phylogenetic relationships in some lineages and to ecological factors such as diet (especially carnivore), in an overall view the variations of IBs in cyprinids were extremely diverse. The number and patterns of IBs in these fishes may reflect their phylogenetic history, but have been shaped by multiple environment factors. In this study, we also confirmed that X-ray photography remains an optimal and reliable method for the study of IBs.

Whole-Genome Sequencing of the Giant Devil Catfish, Bagarius yarrelli.

As one economically important fish in the southeastern Himalayas, the giant devil catfish (Bagarius yarrelli) has been known for its extraordinarily large body size. It can grow up to 2 m, whereas the non-Bagarius sisorids only reach 10-30 cm. Another outstanding characteristic of Bagarius species is the salmonids-like reddish flesh color. Both body size and flesh color are interesting questions in science and also valuable features in aquaculture that worth of deep investigations. Bagarius species therefore are ideal materials for studying body size evolution and color depositions in fish muscles, and also potential organisms for extensive utilization in Asian freshwater aquaculture. In a combination of Illumina and PacBio sequencing technologies, we de novo assembled a 571-Mb genome for the giant devil catfish from a total of 153.4-Gb clean reads. The scaffold and contig N50 values are 3.1 and 1.6 Mb, respectively. This genome assembly was evaluated with 93.4% of Benchmarking Universal Single-Copy Orthologs completeness, 98% of transcripts coverage, and highly homologous with a chromosome-level-based genome of channel catfish (Ictalurus punctatus). We detected that 35.26% of the genome assembly is composed of repetitive elements. Employing homology, de novo, and transcriptome-based annotations, we annotated a total of 19,027 protein-coding genes for further use. In summary, we generated the first high-quality genome assembly of the giant devil catfish, which provides an important genomic resource for its future studies such as the body size and flesh color issues, and also for facilitating the conservation and utilization of this valuable catfish.

Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish.

Body size is one of the most important attributes of a species, but the basic question of why and how each species reaches a different "right size" is still largely unknown. Herein, three phylogenetically closely related catfishes from Sisoridae, including one extraordinarily large-sized Bagarius yarrelli and two average-sized Glyptothorax macromaculatus and Oreoglanis setiger, were comparatively studied using RNA-Seq. Approximately 17,000 protein-coding genes were annotated for each of the three fishes, and 9509 genes were identified as high-confidence orthologous gene pairs. Comparative expressions uncovered a similar functional cluster about ribosome biogenesis was enriched in different tissues of the upregulated genes of Bagarius yarrelli. Moreover, differentially expressed genes and positively selected genes revealed that the glycolysis/pyruvate metabolism and cell cycle pathways have also greatly enhanced in this large-sized species. In total, 20 size-related candidate genes (including two growth modulators: the serine/threonine-protein kinases 3 (AKT3) and adaptor protein 1 (SH2B1), and a crucial pyruvate kinase (PKM2A)) were identified by multiplying comparative analyses along with gene functional screening, which would play major roles in enabling the large body size associated with Bagarius yarrelli and provide new insights into body size evolution. In conjunction with field observations and morphological comparisons, we hypothesize that habitat preferences promote size divergence of sisorids.

Genome Assembly for a Yunnan-Guizhou Plateau "3E" Fish, Anabarilius grahami (Regan), and Its Evolutionary and Genetic Applications.

A Yunnan-Guizhou Plateau fish, the Kanglang white minnow (Anabarilius grahami), is a typical "3E" (Endangered, Endemic, and Economic) species in China. Its distribution is limited to Fuxian Lake, the nation's second deepest lake, with a significant local economic value but a drastically declining wild population. This species has been evaluated as VU (Vulnerable) in the China Species Red List. As one of the "Four Famous Fish" in Yunnan province, the artificial breeding has been achieved since 2003. It has not only re-established its wild natural populations by reintroduction of the artificial breeding stocks, but also brought a wide and popular utilization of this species to the local fish farms. A. grahami has become one of the main native aquaculture species in Yunnan province, and the artificial production has been emerging in steady growth each year. To promote the conservation and sustainable utilization of this fish, we initiated its whole genome sequencing project using an Illumina Hiseq2500 platform. The assembled genome size of A. grahami is 1.006 Gb, accounting for 98.63% of the estimated genome size (1.020 Gb), with contig N50 and scaffold N50 values of 26.4 kb and 4.41 Mb, respectively. Approximately about 50.38% of the genome was repetitive. A total of 25,520 protein-coding genes were subsequently predicted. A phylogenetic tree based on 4,580 single-copy genes from A. grahami and 18 other cyprinids revealed three well-supported subclades within the Cyprinidae. This is the first inter-subfamily relationship of cyprinids at genome level, providing a simple yet useful framework for understanding the traditional but popular subfamily classification systems. Interestingly, a further population demography of A. grahami uncovered a historical relationship between this fish and Fuxian Lake, suggesting that range expansion or shrinkage of the habitat has had a remarkable impact on the population size of endemic plateau fishes. Additionally, a total of 33,836 simple sequence repeats (SSR) markers were identified, and 11 loci were evaluated for a preliminary genetic diversity analysis in this study, thus providing another useful genetic resource for studying this "3E" species.

A Review of Molecular Responses of Catfish to Bacterial Diseases and Abiotic Stresses.

Catfish is one of the major aquaculture species in the United States. However, the catfish industry is threatened by several bacterial diseases such as enteric septicemia of catfish (ESC), columnaris disease and Aeromonas disease, as well as by abiotic stresses such as high temperature and low oxygen. Research has been conducted for several decades to understand the host responses to these diseases and abiotic stresses. With the development of sequencing technologies, and the application of genome-wide association studies in aquaculture species, significant progress has been made. This review article summarizes recent progress in understanding the molecular responses of catfish after bacterial infection and stress challenges, and in understanding of genomic and genetic basis for disease resistance and stress tolerance.

The Complete Mitochondrial Genome of Glyptothorax macromaculatus Provides a Well-Resolved Molecular Phylogeny of the Chinese Sisorid Catfishes.

Previous phylogenetic analyses of the Chinese sisorid catfishes have either been poorly resolved or have not included all the 12 sisorid genera. Here, we successfully assembled the first complete mitochondrial genome of the sisorid fish Glyptothorax macromaculatus. Based on this novel mitochondrial genome and previously published mitochondrial genomes in the Sisoridae, we generated maximum likelihood and Bayesian phylogenies. We dated our preferred topology using fossil calibration points. We also tested the protein-coding genes in the mitochondrial genomes of the glyptosternoid fishes for signals of natural selection by comparing the nucleotide substitution rate along the branch ancestral to the glyptosternoid fishes to other branches in our topology. The mitochondrial sequence structure of G. macromaculatus was similar to those known from other vertebrates, with some slight differences. Our sisorid phylogenies were well-resolved and well-supported, with exact congruence between the different phylogenetic methods. This robust phylogeny clarified the relationships among the Chinese sisorid genera and strongly supported the division of the family into three main clades. Interestingly, the glyptosternoid divergence time predicted by our molecular dating analysis coincided with the uplift of the Tibetan Plateau, suggesting that geology may have influenced speciation in the Sisoridae. Among the mitochondrial protein-coding genes, atp8 may have most rapidly evolved, and atp6 may have been subjected to positive selection pressure to adapt to high elevations. In summary, this study provided novel insights into the phylogeny, evolution and high-altitude adaptions of the Chinese sisorid fishes.