| Misgurnus species(Cypriniformes: Cobitidae),commonly known as Chinese loaches,widely occurs in rivers,lakes,ponds and ricefields.Given their high nutritional value and delicacy,the loach species are widely appreciated and consumed in the local markets,and Misgurnus species are considered as several of most valuable fisheries resources and have become potentially important targets for aquaculture,domestication,and stock enhancement.However,in recent years,the catching production and the wild resources of these two species have been declining each year due to over-fishing and environmental deterioration.Meanwhile,due to the artificial releasing of cultured loaches in the local religious activities without scientific guidance,the aquatic biodiversity of wild resources is severely impacted.Despite their economic and ecological importance,little information could be available about the germplasm resource,evolutionary status,and adaptive evolution of these species.Therefore,the survey of the Misgurnus species resources was undertaken by this study in the past several years.To evaluate the feasibility of morphological and genetic identification of three Misgurnus species and closely related loach species,the morphological characters and DNA barcoding were investigated.Subsequently,the phylogenetic relationship of genus Misgurnus in Cobitidae and the evolutionary status of species in genus Misgurnus were investigated based on the mitochondrial genomes.Meanwhile,the population structure analyzes of these Misgurnus species in their mainly distributed areas were performed.Also,a comprehensive transcriptome analysis of these three species was undertaken to investigate their evolutionary rates and to identify the genes that may have facilitated lineage-specific thermal adaptations of these three species.We examined optimal codon and amino acid usage based on the recently available large-scale RNA-seq data for three Misgurnus species.Using these data,we investigated the codon usage patterns and whether codon usage and amino acid frequencies have been optimized in the highly expressed genes of these three closely related organisms.The main results of this study are as following: 1.Species distribution,morphylogy and DNA barcoding of Misgurnus speicesThree species,M.anguillicaudatus,M.mohoity,and M.bipartitus,are distributed throughout China,except on the Tibetan plateau.M.anguillicaudatus is widespread found in most areas,and it mainly distributes in the region of middle and lower reaches of the Yangtze River Basin.M.bipartitus is restricted to north of the Yellow River in China,and the major distribution area is limited in Songhua River and Liao River basin.M.mohoity only occurs in the Amur River Basin in northeast China.Twelve morphological characters of four loach species(three Misgurnus species above,P.dabryanus)in Misgurnus and Paramisgurnus and DNA barcoding of five loaches(including Barbatula toni)were investigated.Twelve morphological characters were measured in 542 individuals to perform the comparative analysis.Among these characters,only the caudal peduncle length(LCP)revealed significant difference(P < 0.05)among these four species.Notably,the predorsal distance(DPR)of M.mohoity is the highest among the four species,and the caudal peduncle height(HCP)and body width(WB)are sighnificant difference in three Misgurnus species.The clustering based on morphological characters formed two clusters(P.dabryanus and M.anguillicaudatus;M.bipartitus and M.mohoity).Meanwhile,a total of 186 cox1 fragments for the five loaches investigated were sequenced and analyzed.The results showed that interspecific K2 P distance was much higher than intraspecific distance within the five species.Bayesian inference of phylogeny showed that individuals of these species were divided into five specific clades.Meanwhile,the cox1 fragments exhibited 22 character attributes for the differentiation of the five loach species based on character-based method.These results suggested that DNA barcoding based on cox1 can be used as an efficient identifier of these five loach species.2.The phygenetic relationship and adaptive evolution of Misgurnus speciesThe phylogenetic relationships of 59 Cobitoidea species were reconducted based on the mitochondrial genomes.The phylogenetic trees constructed by Bayesian inference and maximum likelihood methods revealed a congruent topological structure.Cobitoidea species were divided into four major clusters,and the evolutionary relationship is as following:(Botiinae +(Balitoridae +(Cobitinae + Nemacheilinae))).It is noteworthy that the species in genus Misgurnus are not in a monophyletic group and divided into two divergent clades.M.bipartitus and M.anguillicaudatus were clustered into Cobitis group;M.mohoity and M.nikolskyi was embedded in the P.dabryanus and Koreocobitis lineages.The codon usage bias and evolutionary ratios of 13 PCGs were investigated in seven closely related loach species.The results of calculation using free-ratio model in PAML revealed that Ka/Ks ratios of Misgurnus species were much lower than the species in genera Paramisgurnus and Cobitis.The analysis of branch-site model showed that cox2 was positively selected in M.bipartitus and M.anguillicaudatus lineages(? = 10.92,P < 0.01),and the positively selected sites occured in the positions of 137 bp and 217 bp based on the BEB analysis.The clustering analysis based on the RSCU values in 21 Cobitinae species revealed that the clustering relationships of Misgurnus coincided with the phylogenetic relationships.3.The population structure of three Misgurnus speciesThe population structure of M.anguillicaudatus in Yangtze River basin was investigated based on genotyping by sequencing.The results revealed that the main genotypes are dominant in the populations,and the heterozygosity ranged from 0.031 to 0.097.Population structure analysis indicated that the populations in Yangtze River basin were divided into two subgroups,and the significant genetic difference between upper reaches with middle and lower reaches was observed.The FST indicated the high genetic differentiation among the populations,especially between populations in upper reaches and other populations.The neighbor joining tree revealed that the individuals were grouped into two major clusters,including populations in upper reaches and populations in middle and lower reaches.The results of the scatter plot based on PCA also revealed the genetic differentiation.To provide valuable insights into the germplasm resource of M.bipartitus and M.mohoity,population structure analysis were performed based on mitochondrial DNA.Totally,19 and 21 haplotypes were identified from the individuals of M.bipartitus and M.mohoity based on cox1 gene,respectively.High haplotype diversity and low nucleotide diversity were found within the populations of M.bipartitus,and the populations of M.mohoity exhibited both high levels of haplotype diversity and nucleotide diversity.Meanwhile,high levels of genetic differentiation and genetic variation between populations in Liao River basin(PAJ,FUS and HUR)and populations in Songhua River basin(MDJ,FUY and FUJ)were observed in M.bipartitus;the populations of M.mohoity also revealed high genetic differentiation and high among-population genetic variations.4.The adaptive evolution of three Misgurnus speciesThe geographic distribution of three Misgurnus species,M.anguillicaudatus,M.bipartitus,and M.mohoity,displays a specific pattern in China,coincident with temperature zones.In this study,we sequenced the transcriptomes of these three species and used the sequences to investigate the lineage-specific adaptations within the genus Misgurnus.In total,51 orphan genes(19 in M.anguillicaudatus,18 in M.bipartitus,and 14 in M.mohoity)that may contribute to the species-specific adaptations,were identified.An analysis of 1392 one-to-one orthologous genes revealed significantly higher ratios of nonsynonymous to synonymous substitutions in the M.mohoity lineage than in M.anguillicaudatus.The genes displaying signatures of positive selection and rapid evolution in Misgurnus were involved in four function categories,(1)energy metabolism,(2)signal transduction,(3)membrane,and(4)cell proliferation or apoptosis,implying that these candidate genes play critical roles in the thermal adaptation of the fish to their living environments.We also detected more than five positively selected sites in cldn15 lb and isca1,which function as important factors in paracellular Na+ transport and Fe/S cluster assembly,respectively.Overall,our study provides valuable insights into the adaptive evolution of loaches from different temperature zones in China and is the foundation for future studies to clarify the genetic basis of temperature adaptation in fishes.5.Codon usage in three closely related species of genus MisgurnusCodon usage bias,explained as the balance between translational selection and natural mutation,is a widespread phenomenon,extending from prokaryotes to eukaryotes.Selection favors specific codons that promote the efficient and accurate translation of genes.The neutrality plots revealed that selection probably dominates codon bias,whereas mutation plays only a minor role in shaping the codon bias in three loaches,M.anguillicaudatus,M.mohoity,and M.bipartitus.In all three loaches,synonymous codon usage order(SCUO)showed a strong “U” nonlinear correlation with GC3,and ENC values correlated inversely with the GC3.Genes with higher GC3 in the three loaches were preferentially involved in “binding”,“metabolic process”,and “cellular process”.These three species also clearly show similar tendencies in the preferential usage of codons,and CUG(Leu),AGA(Arg),AGG(Arg),and GUG(Val)had the highest RSCU values.Nineteen,nine,and 14 preferred codon pairs and 179,182,and 173 avoided codon pairs were also detected in M.anguillicaudatus,M.bipartitus,and M.mohoity,respectively,and the most frequently avoided type of cP3-cA1 dinucleotide in these species was nnUAnn.The expression-linked patterns of codon usage revealed that the high expression was associated with higher GC3,lower ENC,and a smaller proportion of amino acids with high size/complexity(S/C)scores in these three species.The codons and amino acids in the highly expressed genes in these loaches were also biased.A comparative analysis of the codon usage in 27 vertebrates divided fishes into two major clusters,and these loaches were grouped with Danio rerio,coinciding exactly with their evolutionary status. |
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