| Identification of conservation units is crucial for the monitoring and management of endangered species.The genetic characteristics of populations,including population structure,genetic diversity,gene flow and local adaptation etc.,are the fundamentals of fashioning conservation units.The extent of genetic variability of a population determines the ability of that population to adapt to the environment through natural selection.The roughskin sculpin,Trachidermus fasciatus Heckel(Scorpaeniformes: Cottidae),is a small,benthic,carnivorous,and catadromous fish species.It was widely distributed along the east coast of China,west and south coast of the Korean Peninsula,and Kyushu coast of Japan.However,due to anthropogenic activities such as overfishing,environmental pollution,construction of dams,and deterioration of spawning grounds,T.fasciatus has declined in abundance and even gone extinct across most of its historical range in China and its current distribution has become highly fragmented in the past decades.Considering its biological,ecological,and endangered status in China,T.fasciatus has been included in the List of the Wildlife under Special State Protection as a second class state protected aquatic animal by the Chinese government in 1988.A better understanding of genetic diversity and population genetic status of T.fasciatus are crucial for designing suitable management guidelines.However recent studies on population structure of T.fasciatus along the coast of China have produced conflicting results.The genome research is still lacking,which will further restrict the management and protection for T.fasciatus.This study focused on the conservation genetics of T.fasciatus by using microsatellites and population genomics techniques,which would be crucial for designing management guidelines.Main results are presented as follows:1.We developed 25 highly polymorphic microsatellites by using magnetic beads enrichment procedure.The genotyping results based on 24 individuals from Fuyang suggested high levels of genetic diversity: the number of alleles per locus ranged from 6 to 28,polymorphic information content ranged from 0.50 to 0.95,and the expected and observed heterozygosities ranged from 0.54 to 0.97 and 0.42 to 1.00 respectively.2.We developed genome-wide SNPs for 27 individuals in two populations of T.fasciatus based on RAD-seq.An optimized high-efficient assembly method for multiindividuals was developed.And the results suggested its high accuracy and efficiency,a total of 176,619 contigs were generated with mean length of 572 bp and N50 of 603 bp,and the GC content was 41.6%.Most of the loci(96.7%)were each assembled into one contig,98.0% of the input reads were properly mapped to the generated contigs.174,017 contigs were retained for downstream SNP discovery and population analysis.A total of 1,612,103 SNPs were detected based on the above RAD contigs,of these 44,066 SNPs were retained after quality filtering.The genetic divergence between the two populations was high and significant(FST = 0.0705,P < 0.01),suggesting the efficiency of RAD contigs.Moreover,in order to overcome the limitation of utilization of paired reads in STACKS,the bounded error SNP calling model in STACKS was also modified to call SNPs from paired reads.3.Genetic variation for 232 individuals in 10 population of T.fasciatus were assessed by 16 polymorphic microsatellites.Genetic diversity were high within populations,polymorphic information content ranged from 0.793 to 0.924,and the observed and expected heterozygosities ranged from 0.734 to 0.872 and 0.835 to 0.847 respectively.The Qingdao population showed both the lowest levels of genetic diversity and effective population size,suggesting its priority of protection.Results of population structure analysis indicated that there were seven genetic clusters among the sampled populations,with “Dandong”,“Dalian” and “Qinhuangdao” forming into one group,“Dongying” and “Weifang” forming into one group,and the rest five populations(“Wendeng”,“Rongcheng”,“Qingdao”,“Fuyang”,“Ariake Sea”)grouping into distinct clusters respectively.AMOVA suggested significant divergence among the seven groups,and all the pairwise FST between populations were significant(P < 0.01)except for the pair between Dongying and Weifang,the global FST was 0.048.Recent migration rates suggested low levels of gene flow among nine populations of China,and there was no pattern of isolation by distance among populations.These results suggested habitat fragmentation and genetic drift were the main causes for the significant genetic divergence among populations.4.Genetic structure and local adaptation for 180 individuals in eight population of T.fasciatus were assessed by population genomic techniques.32 individuals were selected for RAD contigs assembly based on our optimized assembly method,which yielded a total of 262,533 contigs with mean length of 517 bp and N50 of 566 bp,GC content of 41.6%.A total of 237,947 contigs containing the enzyme cutting sites were retained for downstream analysis,which corresponded to 237,828 RAD loci.A total of 3,436,092 SNPs were detected based on 180 individuals in eight populations of T.fasciatus,of these 10,153 SNPs were retained after quality filtering.Results of population structure analysis indicated that there were six genetic clusters among eight populations,with “Dandong” and “Dalian” forming into one cluster,“Dongying” and “Weifang” forming into one cluster,and the rest four populations(“Wendeng”,“Rongcheng”,“Qingdao”,“Fuyang”)forming into distinct clusters respectively.AMOVA suggested the significant genetic divergence among the six genetic clusters(FCT = 0.081,P < 0.01).Pairwise FST were similar to the results based on microsatellites but with high values,overall FST was 0.103.The Qingdao population owned the lowest genetic diversity and highest genetic differentiation,suggesting the priority of protection.A total of 414 outliers related either to FST or to environmental factors were detected,of these 18 outliers were both FST-outliers and environmental related.GO characterization of 414 Outliers were classified into biological process,cellular component and molecular function,such as metabolism,response,regulation and catalytic activity etc..Furthermore,we found several genes and pathways related to metabolism or transport,especially the synthesis and metabolism of fatty acids.The results suggested the relevance of ocean temperature with local adaptation of T.fasciatus.Genetic structure analysis based on adaptive loci suggested populations with same ocean temperature grouped together,with “Dandong”,“Dalian”,“Dongying”,“Weifang” forming into one group,“Wendeng” and “Rongcheng” forming into one group,“Qingdao” forming into one group,“Fuyang” forming into one group.The genetic structure based on neutral loci was similar to that based on all loci,suggesting the main causes of genetic structure of T.fasciatus were neutral forces,which implied the strong effects of genetic drift.Overall,we have developed two kinds of genetic markers,microsatellites and genome-wide SNPs,both the results based on the above makers indicated the significant genetic structure among populations and local adaptations within populations of T.fasciatus.Our results suggested that the eight populations(“Dandong”,“Dalian”,“Dongying”,“Weifang”,“Rongcheng”,“Wendeng”,“Qingdao”,“Fuyang”)of T.fasciatus in China should be defined into seven MUs(“Dongying” and “Weifang” could be defined as one MUs,and the rest six populations as distinct MUs)and four adaptive groups(“Dandong”,“Dalian”,“Dongying” and “Weifang” should be defined as one adaptive group,“Rongcheng” and “Wendeng” should be defined as one adaptive group,“Qingdao” and “Fuyang” are distinct adaptive groups respectively).Habitat fragmentation and genetic drift due to anthropogenic activities were responsible for the significant genetic differentiation among populations.We highlight the need for in situ conservation efforts for T.fasciatus across its entire distribution range to preserve overall evolutionary potential and to avoid germplasm confusion.The generated genetic markers are valuable genetic resources,and our results have filled the gap of genome research and clarified the genetic structure and the mechanism of local adaptation for T.fasciatus,which are crucial for designing guidelines of conservation units. |
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