Study On Phylogeography And Adaptive Differentiation Of Octopus Ocellatus Along The Coast Of China

Octopus ocellatus,also known as rice octopus(fangsiao),small octopus(Taiwan),and Short-lega octopus.Belonging to the cephalopod(Cephalopoda)Octopoda(Octopodidae)Octopus,a soft animal cephalopods.Widely distributed in the Yellow Sea,Bohai,East China Sea and South China Sea.In recent years,with the vigorous development of China's marine cephalopods resource,octopus resources in China have been widely developed.Development of resources inevitably requires in-depth understanding of their population structure and genetic variation.Based on it,the paper uses wild populations in different sea areas in China as experimental materials.Using morphological multivariate analysis combined with mitochondrial DNA molecular marker technology and simplified genomics sequencing technology,the geographic pattern and adaptive differentiation of the population of Chinese coastal O.ocellatus were studied from the perspective of appearance to molecular to genomics level,provide theoretical basis for management and conservation of O.ocellatus germplasm resources.The main results of this study are summarized follows:(1)A total of 12 morphological variable indicators of 6 O.ocellatus geography groups were analyzed using morphometric methods.The results show: The ratio of the brachial length to the body length and the length of the funnel to the body length have reached significant levels.Analysis of differences in morphological traits among populations based on Multivariate analysis methods.Based on the principal component analysis,a total of three principal components were constructed.Principal component one contributed 47.549 %,principal component two contributed 13.679 %,principal component three contributed 8.497 %,and cumulative contribution rate reached 69.743 %.Scatterplots constructed from principal component one and principal component two clearly show that there are more obvious boundaries between geographical groups in the north and the south.Cluster analysis clustered the six groups into three groups,of which Dalian and Nantong were clustered together in the north of the Yangtze River and three groups in the south of the Yangtze River,such as Shanghai,Zhoushan,and Zhanjiang.The Dongshan group gathered in a separate branch.(2)The genetic geography patterns of 14 individuals of the coastal O.ocellatus geography: Dalian,Yantai,Qingdao,Nantong,Shanghai,Xiamen,Dongshan and Zhanjiang,were analyzed using total mitochondrial genome sequencing methods.O.ocellatus resources are rich in genetic diversity.The O.ocellatus mitochondrial genome sequence length 15977 bp-15990 bp,GC content between 22.40 %-22.93 %.It encodes 13 genes(COI,COII,COIII,ATP6,ATP8,ND1–6,ND4L),22 t RNAs,2 r RNAs,and a D-loop region.The 14 mitochondrial genomes contain 1540 variable information sites,of which 984 are simple information sites.The nucleotide diversity index of the 13 encoded protein genes in the mitochondrial genome of O.ocellatus was between 0.016-0.06513.There were 14 haplotypes in 14 sequences,with a haplotype diversity index(Hd)of 1,a nucleotide diversity index(pi)of 0.03224,and an average number of nucleotide differences(K)of 514.57.Cluster analysis showed that 8 groups clearly differentiated into two groups,one consisting of Dalian,Qingdao,Yantai and Nantong groups north of the Yangtze River,and the other consisted of Shanghai,Dongshan,Xiamen and Zhanjiang south of the Yangtze River.The genetic distance of the two groups was 0.048;the differentiation coefficient was 0.63544(P<0.01).The ATPase 6 and ND2 gene sequencing techniques with higher genetic variation in the mitochondrial genome were selected to further analyze the genetic structure and variation of the 8 O.ocellatus geographical populations.The results of population differentiation and mitochondrial whole genome differentiation were the same.A total of 46 polymorphic loci were detected in the 575 bp ATPase 6 gene fragment.There were a total of 14 haplotypes in 8 individuals and 163 individuals with a haplotype diversity index(Hd)of 0.670.The index(Pi)is 0.02833 and the average number of nucleotide differences is(K)16.434;A total of 69 polymorphic loci were detected in the 639 bp fragment of ND2 gene.The proportion of polymorphic loci was 11.52 %.There were a total of 28 haplotypes in 152 individuals in 8 populations.Haplotype diversity index(Hd))was 0.817,the nucleotide diversity index(Pi)was 0.03327,and the average nucleotide difference(K)was 20.892.This species has higher levels of genetic variation than most other marine cephalopods.The clustering results showed that there was a significant genetic differentiation among O.ocellatus populations in China(P<0.05).The eight populations have clearly differentiated into a group consisting of northern groups such as Dalian,Qingdao,Yantai and Nantong,and a group consisting of southern groups such as Shanghai,Dongshan,Xiamen and Zhanjiang.The differentiation coefficient of the two groups in the ATPase 6 gene was 0.98471(P<0.01),the gene flow was 0.0078(Nm<1).The AMOVA test showed that 98.47% of the genetic differences existed between groups,and 1.53% of the genetic differences existed within the population.The differentiation coefficient of the two groups showed 0.91252(P<0.01)in the ND2 gene and 0.048(Nm<1)in the gene flow.The AMOVA test showed that 96.05% of the genetic differences existed between the groups.There was a 3.95 % genetic difference within the population.(3)Based on RADseq(Restriction-site associated DNA sequencing)technology,the whole genome of O.ocellatus wild populations was scanned.This study collected a total of 72 samples from 6 populations in Yellow Sea and East China Sea and Bohai Sea.A total of 1821 M tag sequences were obtained after the data was filtered.A total of 9009 single nucleotide polymorphism(SNP)sites were obtained by splitting approximately 10 M resds per individual.The analysis of genetic differentiation FST and STRUCTURE further confirmed the results of morphological and mitochondrial genome analysis.The two major groups of the Yangtze River differentiated significantly.Adaptive evolution analysis shows that except neutral evolution,adaptive evolution may play an important role in population differentiation.Using BAYESCENV and LFMM methods to screen loci,1817 sites that may be affected by environmental selection were found.SEED2 software was used to correlate the reference two-spotted quail genomes and 22 sites were screened out by both methods.By blast alignment on NCBI,these selected genes are mainly related to energy metabolism,information transmission,and formation of some signaling pathways.Finally,it is speculated that the causes of these differences may be related to the local seawater temperature,salinity,dissolved oxygen and climate and other factors.This study provides a scientific basis for enriching the genetic diversity of the conservation population of O.ocellatus.