Genetic Structure And Connectivity Differentiation Of Two Morphologic Populations Of Galaxea Fascicularis

As the environmental pressures（rising surface seawater temperature,ocean acidification,seawater pollution and human exploitation）increased severely,the frequency and scale of coral bleaching events are unprecedented.In essence,the genetic diversity of reef coral determines its environmental adaptability and resilience.Therefore,accurate assessment of genetic diversity and maintenance of dynamic gene flow is the prerequisite for the development of accurate coral reef protection strategies.However,reef-building corals have extremely complex life history,complex and diverse asexual and sexual reproduction systems,different levels of gene flow and serious generational overlap.These characteristics determine the genetic structure of the subtle and complex populations and aggregates of reef-building corals.Therefore,it is necessary to develop and identify stable amplified and highly polymorphic microsatellite markers,and explore the population genetics of reef corals.In this study,we developed and identified transcriptome-based microsatellite markers for the representative species Galaxea fascicularis（Gf）corals in Hainan-Xisha Islands,and analyzed their genetic structure and inter-regional connectivity patterns.To further explore the relationship between the two subgroups,the mitochondrial genome of 18 Gf individuals was sequenced and compared,The main findings are as follows:（1）Development and analysis of microsatellite markers58 microsatellite sequences were screened from Gf assembled transcriptome data.Using Gen Bank BLAST to align the sequences,Potential homologous genes were found in17 sequences,of which,13 have the E-values<1.0E^-10.The microsatellite locus Gf-E21and Gf-E50 of two sequences were used in the subsequent population genetics studies.DNA of 4 Gf coral individuals from different sources was used to test the amplification of58 microsatellite locus,16 loci that can be specifically amplified in both s L and h S type and have amplification efficiency more than 75%,were selected and used to scan 7 HIFR geographic populations（Mlt,Wt,Cq,Fjz,Hh,Yl B,Lht）and 3 Xs R geographical populations（Qly,Hg R,Yz R）,In total 245 individuals.After Ewens-Watterson tests,12neutral markers were identified and used for the subsequent population genetic analysis of populations.（2）Microsatellite genetic structure of 10 geographical populationsBefore population genetic analysis,the redundant genetic homogeneity individuals（10/12 loci’s genotypes were the same）were determined and eliminated.30（12.2%）genetic homogeneity individuals were found in 4 geographical populations（Hg R、Wt、Fjz、Qly）.There are 4（Gf-E51、36、48 and 50）and 7（Gf-28、37、3641、48、15 and 50）loci in HIFR and Xs R populations,respectively,consistent with Hardy-Weinberg equilibrium（HWE,after Bonferroni correction）..The average allele richness was 4.7±0.32（5.1,Mlt-4.2,Wt）and 4.6±0.34（5.1,Hg R-4.3,Yz R）,in HIFR and Xs R populations,respectively,The average observed heterozygosity of Xs R was higher than that of HIFR,they are 0.73±0.02（0.76,Yz R-0.70,Hg R）and 0.60±0.04（0.66,Mlt-0.54,Hh）,in HIFR and Xs R populations,respectively.The inbreeding coefficient F_ISof HIFR is 0.19±0.05,significantly higher that that of Xs R（-0.03±0.08）.The AMOVA of population genetic differentiation among HIFR populations was 0.032,while that of Xs R is 0.065.The cluster analysis showed that HIFR groups and Xs R groups were clustered together respectively,indicating that there is geographic isolation.（3）Genetic structure and connectivity differentiation of geographic subpopulations of s L and h S coralsOur previous study found that Gf can be divided into two morphologies,s L and h S,based on the differences in their punctured cyst morphology and mitochondrial genotype.There are significant differences in heat resistance and stress resistance between them.According to these two morphological types,each geographic population was divided into geographic subgroups for population genetic analysis,and the results were as follows:the average allele richness of 12 loci was 3.5±0.34（4.2%）in s L and h S subpopulations is3.5±0.34（4.2,HGR/s L-2.9,Cq/s L）and 3.4±0.15（3.6,Yl B/h S-3.2,Hh/h S）,respectively.Average observed heterozygosity is 0.67±0.07（0.75,Yz R/s L-0.54,Cq/s L）,0.63±0.08（0.77,Qly/h S-0.51,Fjz/h S）,respectively.Overall,the genetic diversity of s L subpopulations was higher than that of h S.The loci consistent with HWE in s L subgroup and h S subgroup were 100%and 83.3%,respectively,which is significantly higher than that of geographical groups.The inbreeding coefficient F_ISof s L subgroup 0.01±0.13,while that of h S is-0.02±0.10.The genetic differentiation F_STof s L and h S in the HIFR and Xs R regions were 0.075/0.077 and 0.075/0.084.No significant differentiation was found.And between the two regions,the genetic differentiation of s L subgroups is 0.117,the percentage of molecular variance between regions is as high as 9%,while the AMOVA of h S is only 0.083.The cluster analysis showed that the s L subpopulations of HIFR and Xs R were divided into two branches,while hS subpopulations gather together.The gene flow of s L subsets was significantly smaller than that of h S at the HIFR-Xs R spatial scale.The results suggest that the geographical isolation in s L is significant while the gene flow in h S is smooth.（4）Comparative analysis of mitochondrial genomes of two morphological types of Galaxea fascicularisComparative analysis of the mitochondrial genome of 18 Galaxea fascicularis individuals（8 s L,10 h S）.Comparative analysis revealed 10 SNPs（single nucleotide polymorphism）in the mitochondrial genome genes,of which 6 were type-specific SNPs.There are 6 SNPs on the 4 protein-coding genes,4 of which are type-specific SNPs.The two SNPs with non-synonymous mutations are located on COI and COII,respectively.There are 2 SNPs on each of the 2 r RNA genes,and each has a type-specific SNP.There are 12 SNPs in the intergenic region,9 of which are type-specific,and there are 5type-specific SNPs in the putative control region（between the COII gene and 12s r RNA）.The 22 SNPs in the entire genome are all base transition SNPs without base transversion.8non-type-specific SNPs,only one base transition is in the s L type,and the remaining 7 base transition are in the h S type,indicating that the overall diversity of the h S type is higher than that of the s L type in the mitochondrial genome.This was contrary to the level of nuclear microsatellite DNA diversity（s L>h S）.Compared with s L type mitochondrial genome,h S lose 290 bp between Cytb and ND2 due to reverse transcriptional transposition,and there is 20 bp insertion between COXI and t RNA^met.Phylogeny analysis of s L/h S and other 57（complex clade、Robust clade、Corallimorpharia、Antipatharia、Hydrozoa、Alcyonacea、Actiniaria）cnidarians showed that,s L and h S still belong to the same species,they clustered with the complex clade of stony coral.