| Misgurnus species(Cypriniformes: Cobitidae),with high protein and low fat,are nutrient-rich aquatic economic animals,which are widely appreciated and consumed in domestic and foreign markes.Three species,Misgurnus mohoity,Misgurnus bipartitus and Misgurnus anguillicaudatus,are native loaches in China.M.anguillicaudatus has been widespread found in most river basins in China,except Qinghai-Tibet Plateau.In contrast,M.bipartitus is confined to the north of the Yellow River,and M.mohoity only occurs in Songhua River basin.Significant scientific values are found in the three species due to their specific distribution ranges.M.anguillicaudatus is also interesting from the scientific perspective for its natural variability in ploidy levels,which is rare in other animals.M.anguillicaudatus have been an ideal raw material for the studies of cytogeography,hylogeography and polyploid evolution owing to its abundant polyploidy levels.However,in recent years,the amount of wild loach resources has been repidly reduced due to overfishing and deterioration of ecological conditions.Therefore,the survey of the Misgurnus species and polyploid M.anguillicaudatus resources across China was undertaken by this study.The genetic diversity,population genetic structure,demgraphic and migration history of three Misgurnus species were investigated using cytochrome b(Cyt b),control region(CR)sequences and polymorphic nuclear microsatellite markers(SSR),expecting to provide scientific evidences for the genetic evolution and germplasm resources of Misgurnus and polyploid M.anguillicaudatus.The main results are as follows:1.Development of microsatellite markers in MisgurnusIn this study,there were 38 polymorphic SSR markers with expected fragment length separated from three species of Misgurnus through transcriptome data.Finally,12 SSR primers were selected with high polymorphism and achieving cross-species amplification in three species of Misgurnus.The genetic diversity detected by 12 SSR primers was M.anguillicaudatus > M.mohoity > M.bipartitus,and there were differences in polymorphisms of different SSR loci in different species.All the 12 polymorphic microsatellite markers will be valuable for further population evolution research of Misgurnus and related species.2.Population genetics of M.mohoityGenetic variation in the 60 individuals of M.mohoity from 6 geographic populations were analyzed using concatenated mt DNA Cyt b+CR(1211 bp)sequence.High mt DNA genetic diversity was observed in M.mohoity at the species level.The lowest genetic diversity was detected in western Qiqihar(QQ)population,whereas northern Tongjiang(TJ)population was characterized by highest genetic diversity.Intraspecific phylogeography and population genetic structure revealed that no monophyletic group or distinct geographical genetic lineages was formed from six populations.Tongjiang(TJ)and Qiqihar(QQ)populations were observed high genetic differentiation with other populations.Mismatch distribution and neutrality test showed that six populations of M.mohoity were relatively stable and did not experience population expansion events.Genetic variation in the 97 individuals of M.mohoity from 6 geographic populations were analyzed using 12 microsatellite markers.A total of 58 alleles were detected.Genetic diversity analysis revealed that obvious lower genetic index was found in western Qiqihar(QQ)population,compared to higher genetic index in northern Tongjiang(TJ)and Fuyuan(FY)populations.The results of population genetic structure analysis showed that there was a greater degree of genetic differentiation between Tongjiang(TJ)/ Qiqihar(QQ)and other populations,while the genetic variation among the southeastern populations was lower.NJ phylogenetic,PCo A and STRUCTURE analysis showed that Tongjiang(TJ)population and Qiqihar(QQ)population could form monophyletic branch,respectively,which were significantly separated from the other four populations and had high genetic specificity.3.Population genetics of M.bipartitusGenetic variation in the 133 individuals of M.bipartitus from 14 geographic populations were analyzed using concatenated mt DNA Cyt b+CR(1211 bp)sequence.Low mt DNA genetic diversity was observed in M.bipartitus at the species level,and slightly higher genetic diversity was observed in Liao River basin compared to Songhua River basin.Intraspecific phylogeography and population genetic structure revealed that no monophyletic group or distinct geographical genetic lineages was formed from 14 populations,which indicating the overall high genetic similarity in M.bipartitus.Historical population dynamics analysis indicated that M.bipartitus,especially populations in Songhua River basin,may have experienced population expansion events during the Last Interglacial Period.Genetic variation in the 324 individuals of M.bipartitus from 17 geographic populations were analyzed using 12 microsatellite markers.A total of 65 alleles were detected.Population genetic diversity analysis showed that there was high genetic consistency in M.bipartitus with no significant differences among geographical populations.Population genetic structure analysis indicated that genetic distances among M.bipartitus populations were at the similar level.NJ phylogenetic,PCo A and STRUCTURE analysis revealed that there was low genetic variation in M.bipartitus and no obvious genetic clades were found among populations.4.Population genetics and phylogeography of polyploid M.anguillicaudatusIn this study,a total of 7479 M.anguillicaudatus specimens from 168 populations were genotyped and analysed throughout its distribution range in China,except for the western plateau.Three ploidy levels were detected: diploid,triploid and tetraploid.In contrast to the widely distributed diploids,polyploid populations were primarily found at locations in the central Yangtze River basin,namely Han River,Dongting Lake and Poyang Lake,but some were also found in the Haihe and Pearl River basins.The survey found that there was no apparent pattern to the ploidy composition in populations where polyploid specimens appeared;distribution of polyploid specimens was limited to some regions,but there were no clearly allopatric geographical ranges or boundaries dividing diploid and polyploid populations of M.anguillicaudatus in China.Partial CR(605 bp)and Cyt b(606 bp)sequences were successfully amplified from 351 samples of M.anguillicaudatus,among which 216 were diploids and 135 were tetraploids.Phylogenetic analysis found that phylogenetic division did not fully correspond to the ploidy structure across the distribution range.Instead,sympatric diploids and tetraploids generally clustered within the same lineage,congruent with the geographic distribution splitting into five lineages.The lowest haplotype and nucleotide diversities were detected in the populations of Haihe River,whereas the Pearl River populations were characterized by highest genetic diversity.Haplotype and nucleotide diversities of the three rivers in the Yangtze River basin exhibited equally high values.Estimates of directional gene flow between regions indicated that the migration of tetraploid populations was lower than diploid populations.The migration among the tetraploid populations was predominantly symmetrical with no obvious directionality.In contrast,the migration between diploid populations was asymmetrical,exhibiting a general trend of migration towards the localities in the Yangtze basin,with north / south to center and west to east as dominant migratory directions.5.Population genetics and phylogeography of MisgurnusGenetic variation in the 635 individuals of Misgurnus from 76 geographic populations were analyzed using concatenated mt DNA Cyt b+CR(1211 bp)sequence.Lower mt DNA genetic diversity was observed in M.bipartitus at the species level,compared to higher genetic diversity in M.mohoity and M.anguillicaudatus.Among different basins of M.anguillicaudatus populations,the lowest genetic diversity was detected in the populations of Songliao River,whereas the Pearl River populations were characterized by highest genetic diversity.Haplotype and nucleotide diversities in other river basins exhibited equally high values.Phylogeography and population genetic structure revealed that M.mohoity and M.bipartitus could form monophyletic lineages,meanwhile the populations of M.anguillicaudatus splitted into five lineages congruent with the geographic distribution.Historical population dynamics analysis indicated that M.bipartitus and Yangtze River populations of M.anguillicaudatus may have experienced population expansion events during the Quaternary Pleistocene.Genetic variation in the 1352 individuals of Misgurnus from 72 geographic populations were analyzed using 12 microsatellite markers.A total of 97 alleles were detected.Population genetic diversity analysis showed that there was no significant differences among species of Misgurnus and geographical populations.Population genetic structure analysis indicated significant difference among species of Misgurnus as well as geographical regions.NJ phylogenetic and PCo A analysis identified two genetic clades in Misgurnus populations,of which Yangtze River,Pearl River and Hainan Island populations of M.anguillicaudatus claded into a lineage,the other river populations of M.anguillicaudatus together with M.bipartitus and M.mohoity claded into another lineage.STRUCTURE analysis revealed that three species of Misgurnus could be distinguished obviously. |
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