| Boleophthalmus pectinirostris is a very unique amphibious fish.It is listed as one of the"Ten Marine Treasures"by the World Wide Fund for Nature or World Wildlife Fund(WWF).It is widely distributed in the Yellow Sea,East China Sea and South China Sea.In recent years,B.pectinirostris has been threatened by environmental pollution and overfishing.In order to effectively protect the germplasm resources of B.pectinirostris in China,it is necessary to study the genealogical geographical distribution pattern and evolutionary mechanism of its existing populations,and explore its causes,so as to provide a data basis for the effective protection,rational development and scientific management of B.pectinirostris resources in China.Therefore,this study used mitochondrial D-loop region,mitochondrial whole genome and nuclear gene transcriptome as molecular markers to analyze the geographic pattern and historical dynamics of B.pectinirostris populations in China,discussed the driving force of paleogeographic history,current ecological isolation and human activities on its population differentiation,and revealed the relative importance of neutral and adaptive evolution in its population evolution.The main results are as follows:1.A total of 185 D-loop sequences with lengths of 696 bp were determined for the analysis of population geographic patterns and differentiation using mitochondrial control region fragments in nine B.pectinirostris populations(Rushan;Chengyang;Ganyu;Daishan;Fenghua;Longhai;Zhujiangkou;Hepu;and Longhua)along the Chinese coast.The results showed that there was a high genetic diversity in Chinese coastal marsupials.A total of 6insertion or deletion sites and 112 polymorphic sites were detected,including 62 single mutant sites and 50 parsimony informative sites.The overall haplotype diversity(H_d)was0.985,the nucleotide diversityπwas 0.012,and the average nucleotide difference number(k)was 8.371.Compared with various populations,the Shandong Rushan population(H_d=0.810,π=0.00576)and Shandong Chengyang population(H_d=0.747,π=0.0037)had relatively low genetic diversity,other populations are relatively high.Analysis of population genetic structure showed that the nine B.pectinirostris populations in China could be dividedinto two clades:Clade A is Rushan population in Shandong;Clade B was composed of all populations except Rushan population,and Subclade BI(Chengyang population in Shandong)and Subclade BII(all populations in the south of Shandong)were differentiated in Clade B.Both neutral test and nucleotide mismatch analysis showed that the Chinese B.pectinirostris populations has experienced population expansion events.The expansion times of Clade A,Subclade BI and Subclade BII are 0.456～0.911,0.513～1.026 and2599～5197 years ago,respectively.2.A total of 61 full-length mitochondrial sequences with lengths of 16,980～17,243 bp were determined using the mitochondrial whole genome to analyze the population geographic patterns and adaptive evolution of nine B.pectinirostris populations(Rushan;Chengyang;Ganyu;Daishan;Fenghua;Longhai;Zhujiangkou;Hepu;and Longhua)along the Chinese coast.The results supported the above research results of mitochondrial D-loop region:279 insertion or deletion sites were detected,most of which were caused by a 130bp tandem repeat sequence in the D-loop region repeated at different times;670 polymorphic sites were detected,including 444 single mutation sites and 226 parsimony information sites.The overall haplotype number(N_h)was 61,haplotype diversity(H_d)was 1,nucleotide diversity(π)was 0.0033,and the average nucleotide difference number(k)was 55.972,indicating that the overall genetic diversity level of Chinese B.pectinirostris was high.Compared with various populations,the genetic diversity of Rushan population(H_d=1,π=0.0004)and Chengyang population(H_d=1,π=0.0005)were lower than that of other populations.The nine populations of B.pectinirostris in China could be divided into two major clades:Clade A was the Shandong Rushan population,Clade B was composed of all populations except Rushan population,and Subclade BI(Chengyang population)and Subclade BII(all populations in the south of Shandong)were differentiated in Clade B.The genetic distance between Clade A and Clade B is relatively far(0.0052～0.0057);the genetic differentiation index reached 0.652～0.920.Adaptive evolutionary analysis did not screen for genes and site to positive selection among the 13 protein-coding genes in the mitochondria of Chinese B.pectinirostris,but we detected 4 and 1 fixed amino acid changes in Rushan population and Chengyang respectively.After detection,these changes had a partial impact on the physical and chemical properties of mitochondrial protein,and its exact biological significance needs to be further studied in the future.3.We analyzed the geographical pattern and adaptive evolution of five B.pectinirostris populations(Rushan,Chengyang,Pingyang,Longhai,And Hepu)in coastal China by using nuclear gene transcriptomic analysis.A total of 1210408 SNPs of high quality were obtained from 24 individuals through filter and comparison of original data.Genetic diversity analysis showed that the average nucleotide diversity index(Pi)of the five populations was 0.243,the observed value of average heterozygosity(H_o)was 0.089,and the average heterozygosity(H_e)was 0.215.The heterozygosity of Rushan(H_o=0.039,H_e=0.054)and Chengyang population(H_o=0.076,H_e=0.106)in Shandong was relatively lower than that of other populations.The results of principal component analysis(PCA)and NJ clustering showed that the five B.pectinirostris populations in China could be divided into three Clades,Rushan population and Chengyang population were one Clades respectively,and three populations in the south of Shandong constituted the third Clades.The genetic distance between populations ranged from 0.0336 to 0.0620,among which Rushan and Chengyang populations(0.0530),Rushan and the three southern populations(0.0503～0.0620)and Chengyang and the three southern populations were far away(0.0494～0.0542),while the genetic distance within the three southern populations was close.Adaptive evolution analysis showed that Rushan and Chengyang populations had 41 and 182 positive selection sites respectively compared with the southern population.Functional annotation and enrichment analysis showed that the candidate genes corresponding to these positive selection sites were mostly related to energy generation,fat metabolism,carbohydrate metabolism,biofilm function,synaptic development and immunity,which may be related to the adaptive evolution of B.pectinirostris in the two places due to their adaptation to high latitude and low temperature conditions.The results suggest that adaptive evolution may also play an important role in the population evolution of B.pectinirostris along the coast of China. |
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