Discovery of a wild, genetically pure Chinese giant salamander creates new conservation opportunities.

Effective conservation of threatened biota relies on accurate assessments and scientific guidance. As an unfortunate example, Chinese giant salamanders ( Andrias, CGS) remain critically endangered in nature. Misguided conservation efforts, e.g., commercial propagation and releasing of millions of likely non-indigenous or interspecific hybrids, have further compromised conservation initiatives. Limited information on wild populations of CGS poses a significant conservation challenge. Following 18-month long field monitoring, we now report the discovery of a wild population of CGS in a closed nature reserve in Jiangxi Province, China. Genomic assessments reveal its genetic distinctiveness and do not detect genetic admixture with other species. Based on morphological and molecular evidences, we describe this CGS as a new species Andrias jiangxiensis sp. nov. This is the only known species of CGS today with a genetically pure, reproducing, in situ population. This discovery emphasizes the important role that closed nature reserves play in protecting species, and the necessity of integrating long-term field monitoring and genetic assessments. It sets a new pathway for discovering and conserving endangered species, especially for those biotas that are similarly being extirpated by anthropogenic translocations and overexploitation.

Genetic diversity and reproductive success of a wild population of Chinese sturgeon ( Acipenser sinensis) from the Yangtze River inferred from juveniles born in 2014.

The Chinese sturgeon ( Acipenser sinensis Gray, 1835) is a large anadromous fish species, which is under considerable threat due to dramatic declines in population numbers. In the current study, population genetic diversity and individual reproductive success were assessed using nuclear microsatellite markers (simple sequence repeat, SSR) and complete mitochondrial (mtDNA) genome analysis of juveniles born in 2014. Results showed the existence of size polymorphism in the mtDNA genome of Chinese sturgeon, which was caused by a repeat motif. Population genetic diversity was high based on both SSR ( Ho: 0.728±0.211; He: 0.779±0.122) and mtDNA genome analyses ( H: 0.876±0.0035; Pi: 0.0011±0.0010). A positive inbreeding coefficient ( FIS: 0.066±0.143) was also found, indicating the occurrence of inbreeding. Reconstruction of sibling groups identified 11 mothers and 11 fathers involved in reproduction of Chinese sturgeons in 2014. Variance in individual reproductive success was not significant, with reproductive success of parent fish instead shown to be relatively even ( P=0.997>0.05), thus suggesting the absence of sweepstakes reproductive success (SRS). These results indicate that, in regard to conservation, loss of genetic diversity due to the effects of SRS is not of particular concern. However, we must focus on having an adequate number of adults and suitable environmental conditions to ensure that fish can reproduce.

Genetic diversity and temporal changes of an endemic cyprinid fish species, Ancherythroculter nigrocauda, from the upper reaches of Yangtze River.

Small populations with low genetic diversity are prone to extinction. Knowledge on the genetic diversity and structure of small populations and their genetic response to anthropogenic effects are of critical importance for conservation management. In this study, samples of Ancherythroculter nigrocauda, an endemic cyprinid fish from the upper reaches of Yangtze River, were collected from five sites to analyze their genetic diversity and population structure using mitochondrial cytochrome b gene and 14 microsatellite loci. Haplotype diversity, nucleotide diversity, and expected heterozygosity indicated that the A. nigrocauda populations had low genetic diversity, and decreased heavily from 2001 to 2016. Significant genetic differentiation was found among different populations in the cyt b gene and SSR markers based on the genetic differentiation index (F ST), whereas no differentiation was found in 2001. Haplotype genealogy showed that eight out of 15 haplotypes were private to one population. The SSR STRUCTURE analysis showed that there were four genetic clusters in the A. nigrocauda samples, with each population forming a single cluster, except for the Chishui River (CSR) and Mudong River (MDR) populations, which formed a common cluster. Therefore, loss of genetic diversity and increased genetic differentiation were found in the A. nigrocauda populations, which could be attributed to dam construction, overfishing, and water pollution in the upper Yangtze River. It is therefore recommended that the government should ban fishing, control water pollution, increase river connectivity, and establish artificial breeding and stocking.

Evolution and phylogenetic application of the MC1R gene in the Cobitoidea (Teleostei: Cypriniformes).

Fish of the superfamily Cobitoidea sensu stricto (namely loaches) exhibit extremely high diversity of color patterns, but so far little is known about their evolutionary mechanism. Melanocortin 1 receptor gene (MC1R) plays an important role during the synthesis of melanin and formation of animal body color patterns. In this study, we amplified and sequenced the partial MC1R gene for 44 loach individuals representing 31 species of four families. Phylogenetic analyses yielded a topology congruent with previous studies using multiple nuclear loci, showing that each of the four families was monophyletic with sister relationships of Botiidae+ (Cobitidae+(Balitoridae+Nemacheilidae)). Gene evolutionary analyses indicated that MC1R in loaches was under purifying selection pressure, with various sites having different dN/dS values. Both Botiidae and Cobitidae had lower dN/dS values than those of background lineages, suggesting their evolution might be strongly affected by purifying selection pressure. For Balitoridae and Nemacheilidae, both had larger dN/dS values than those of background lineages, suggesting they had a faster evolutionary rate under more relaxed selection pressure. Consequently, we inferred that the relatively stable color patterns in Botiidae and Cobitidae might result from the strong purifying selection pressure on the MC1R gene, whereas the complicated and diverse color patterns in Balitoridae and Nemacheilidae might be associated with the relaxed selection pressure. Given the easy experimental procedure for the partial MC1R gene and its excellent performance in reconstructing phylogeny, we suggest this gene could be used as a good molecular marker for the phylogenetic study of fish species.

[Phylogeographic analyses of Sarcocheilichthys nigripinnis(Teleostei: Cyprinidae)based on mitochondrial DNA Cyt b gene sequences].

Sarcocheilichthys nigripinnis is a small cyprinid fish widely distributed in East Asia, and it has been widely used in biogeographic analyses of freshwater fishes in China. In the present study, 142 S. nigripinnis individuals from 20 sampling sites in eight river systems were collected to investigate its phylogeography and genetic variations.. Populations from the Yellow River represent northern clade and all others represent southern clade. The results showed that 56 haplotypes were identified as mitochondrial DNA (mtDNA) Cyt b gene of 1 140 bp length. Relatively high haplotype diversity (h=0.971) and low nucleotide diversity (π=0.0212) were detected, and the estimated average genetic distance was 2.2%. Moreover, a neighbor-joining(NJ)tree revealed seven strongly supported lineages. Populations from the Yellow River were located at the basal position, whereas the remaining populations were more derived. Our results indicate that S. czerskii might have been evolved from S. nigripinnis in the Yellow River ~1.03 Ma. Based on the phylogeographical analysis of S. nigripinnis, we also suggest that vicariance, following mountain uplift and drainage isolation, plays an important role in producing evolutionary lineage differentiations. Moreover, molecular dating estimated that the divergence time of S. czerskii could be dated back to 0.95~3.92 Ma. Mismatch distribution analysis and neutrality tests also suggested the recent demographic expansions of S. nigripinnis populations, and that the effects of Pleistocene climatic changes could be a vital factor of the population dynamics of S. nigripinnis.

[Skeletal anatomy and phylogenetic position analysis of Gobiocypris rarus].

The phylogenetic position of Gobiocypris rarus, a small cyprinid fish of interest in many biological areas due to its unique characteristics, is still under debate. At the morphological view, it belongs to the Danioninae subfamily of Cyprinidae; however, recent molecular research recognizes it as a member of the Gobioninae subfamily. To investigate the phylogenetic position of Gobiocypris rarus, we prepared transparent skeleton specimens, selected 47 characteristics and reconstructed the phylogenetic tree using PAUP. The results indicated that Gobiocypris rarus was clustered with Gobioninae, which was in agreement with recent molecular phylogenetic conclusions.

Phylogenetic relationships of the Cobitoidea (Teleostei: Cypriniformes) inferred from mitochondrial and nuclear genes with analyses of gene evolution.

The superfamily Cobitoidea of the order Cypriniformes is a diverse group of fishes, inhabiting freshwater ecosystems across Eurasia and North Africa. The phylogenetic relationships of this well-corroborated natural group and diverse clade are critical to not only informing scientific communities of the phylogeny of the order Cypriniformes, the world's largest freshwater fish order, but are key to every area of comparative biology examining the evolution of traits, functional structures, and breeding behaviors to their biogeographic histories, speciation, anagenetic divergence, and divergence time estimates. In the present study, two mitochondrial gene sequences (COI, ND4+5) and four single-copy nuclear gene segments (RH1, RAG1, EGR2B, IRBP) were used to infer the phylogenetic relationships of the Cobitoidea as reconstructed from maximum likelihood (ML) and partitioned Bayesian Analysis (BA). Analyses of the combined mitochondrial/nuclear gene datasets revealed five strongly supported monophyletic Cobitoidea families and their sister-group relationships: Botiidae+(Vaillantellidae+(Cobitidae+(Nemacheilidae+Balitoridae))). These recovered relationships are in agreement with previous systematic studies on the order Cypriniformes and/or those focusing on the superfamily Cobitoidea. Using these relationships, our analyses revealed pattern lineage- or ecological-group-specific evolution of these genes for the Cobitoidea. These observations and results corroborate the hypothesis that these group-specific-ancestral ecological characters have contributed in the diversification and/or adaptations within these groups. Positive selections were detected in RH1 of nemacheilids and in RAG1 of nemacheilids and genus Vaillantella, which indicated that evolution of RH1 (related to eye's optic sense) and RAG1 (related to immunity) genes appeared to be important for the diversification of these groups. The balitorid lineage (those species inhabiting fast-flowing riverine habitats) had, as compared with other cobitoid lineages, significantly different dN/dS, dN and dS values for ND4 and IRBP genes. These significant differences are usually indicative of weaker selection pressure, and lineage-specific evolution on genes along the balitorid lineage. Furthermore, within Cobitoidea, excluding balitorids, species living in subtropics had significantly higher dN/dS values in RAG1 and IRBP genes than those living in temperate and tropical zones. Among tropical cobitoids, genes COI, ND5, EGR2B, IRBP and RH1, had a significantly higher mean dS value than those species in subtropical and temperate groups. These findings suggest that the evolution of these genes could also be ecological-group-specific and may have played an important role in the adaptive evolution and diversification of these groups. Thus, we hypothesize that the genes included in the present study were actively involved in lineage- and/or ecological-group-specific evolutionary processes of the highly diverse Cobitoidea. These two evolutionary patterns, both subject to further testing, are hypothesized as integral in the diversification with this major clade of the world's most diverse group of freshwater fishes.

[Phylogenetic relationships among Cobitoidea based on mitochondrial ND4 and ND5 gene sequences].

In the present study, we cloned and sequenced 10 new ND4 and ND5 gene sequences of Cobitoidea. These sequences were used to reconstruct phylogenetic relationships together with those of 15 other species downloaded from GenBank. The results showed that the length of ND4 gene sequence was 1 380-1 387 bp with ATG as starting codon and incomplete termination signal as terminated codon; the length of ND5 gene sequence was 1 821-1 839 bp with ATG as starting codon and TAA or TAG as terminated codon; three tRNA genes coding tRNAs that carry hisidine, serine and leucine respectively, were inserted between ND4 and ND5 genes. A, T, C and G accounted for 30.4%, 27.3%, 14.2% and 28.1% in ND4/ND5 gene (including intervening three tRNA genes). The content of A+T (57.7%) is higher than that of G+C (42.3%). The estimated Ti/Tv ratio was 1.586. With Danio rerio and Cyprinus carpio as outgroups, the phylogenetic relationships of Cobitoidea were analyzed using maximum parsimony (MP) method, maximum likelihood (ML) method and Bayesian analyses (BI). Results of all the three methods indicated that Cobitinae, Nemacheilinae, Botiinae, Balitoridae and Vaillantellidae were all monophyletic respectively, and their interrelationships were: (Vaillantellidae + (Botiinae + (Cobitinae + (Nemacheilinae + Balitoridae)))), which was consistent with the previous studies based on the whole mitogenome and some nuclear genes sequences (eg. RAG1 gene). However, the present results differ from those based upon other mtDNA genes sequences (e.g. the cyt b and D-loop showed that Nemacheilinae grouped with Cobitinae forming sister-group, then they clustered with Balitoridae). The difference could be due to the phylogenetic information carried by different length sequence: Longer sequences carry more information and result in more reliable phylogenetic trees.

[Geographical speciation process of the genus Lepturichthys (Cypriniformes: Balitoridae)].

The genus Lepturichthys is endemic in China, including two valid species, L. fimbriata and L. dolichopterus. The former is widely distributed in the Upper and Middle Yangtze River, while the latter only in Minjiang River. In the present study, the mitochondrial cytochrome b gene sequences were analyzed for 20 individuals of L. fimbriata collected from the Upper and Middle Yangtze River and eight individuals of L. dolichopterus collected from Minjiang River. This paper aimed to investigate the geographical speciation process of the genus Lepturichthys. The results showed that the genetic variations among different haplotypes of L. fimbriata ranged from 0.1% to 1.8%, with an average of 0.7%, and from 0.6% to 1.3%, with an average of 0.7% among different haplotypes of L. dolichopterus. The variations between L. fimbriata and L. dolichopterus were also very low, ranging from 0.2% to 1.8%, with an average of 0.9%. Two methods, Neighbor Joining (NJ) and Bayesian analysis (BI), were used for constructing the molecular phylogenetic trees. Both NJ and BI trees indicated that all Lepturichthys specimens formed as a monophyletic group; the L. dolichopterus samples also formed as monophyletic group, having a derived position in the phylogenetic trees, while the L. fimbriata samples formed as a paraphyletic group. Both L. fimbriata and L. dolichopterus are valid species in morphology. Based on molecular and morphological data, we presumed that the geographical speciation process of the genus Lepturichthys could be as following: during the Quaternary Glacial period, L. fimbriata in the Middle and Lower Yangtze River dispersed to the drainages of Southeast China along the Continental Shelf of East China Sea, one population spread to the Minjiang River and envolved as a new species (L. dolichopterus) because of considerable environmental difference, while the remaining L. fimbriata populations in the Yangtze River formed as a paraphyly. The other similar geographical speciation patterns are discussed in the present study as well.

Seasonal variation in drifting eggs and larvae in the upper Yangtze, China.

From 5 March to 25 July 2008, ichthyoplankton drifting into the Three Gorges Reservoir from the upper reaches of the Yangtze River were sampled daily to investigate the species composition, abundance, and seasonal variation in early-stage fishes in this area. Twenty-eight species belonging to five orders and 17 families or subfamilies were identified by analyzing fish eggs and larvae, and a total of 14.16 billion individuals were estimated drifting through the sampling section during the investigation. Among the ichthyoplankton sampled, species in Cultrinae, Cobitidae, Gobioninae and Gobiidae, along with the common carp (Cyprinus carpio Linnaeus), comprised 89.6% of the total amount. Six peaks of drift density were identified during the sampling period, and a significant correlation was found between drift density with water discharge. The dominant species were different in each drift peak, indicating different spawning times for the major species. The total amount of the four major Chinese carps that drifted through the sampling section was estimated as 0.88 billion, indicating an increase in the population sizes of these species in the upper reaches of the Yangtze River after construction of the Three Gorges Dam. Actually, these reaches have become the largest spawning area for the four major Chinese carps in the Yangtze River. The large total amount of eggs and larvae drifting through this section demonstrated that the upper reaches of the Yangtze River provided important spawning sites for many fish species, and that conservation of this area should be of great concern.

Phylogenetic relationships of the cypriniformes tested by mtDNA 12S rRNA sequence variations.

Cypriniformes is the largest order of freshwater fishes. The phylogenetic relationships of the Cypriniformes have been studied by many investigators based on morphological characters, but no agreement has been reached. In the present paper, complete mitochondrial 12S rRNA sequences of five families of the Cypriniformes were determined and analyzed to test the present morphological hypotheses. After alignment, there are 1000 sites, among which 467 sites are in stems and 533 sites in loops. 395 sites are variable, and of which 267 are informative. Neighbor-joining and Maximum Parsimony methods were employed for phylogenetic analysis. The results indicate that the cyprinids form a monophyletic group while the non-cyprinid cypriniforms form another one, which is in accordance with the hypothesis proposed by Siebert. The Cyprinidae consists of three main clades, the Danionini, the Cyprinini, and the Leuciscini. However, in the non-cyprinid cypriniforms, the relationships could not be resolved clearly. The Cobitidae is polyphyletic. Homalopteridae, Noemacheilinae and Cobitinae may have closer relationships.