Population Genetics Of Megalobrama Species Based On The Whole Genome Re-sequencing

The genus Megalobrama belongs to the cyprinid subfamily Cultrinae,which is one of the most economically important freshwater fish genera in China.In recent years,the wild resources of Megalobrama have declined seriously as a result of deteriorating environmental conditions and anthropogenic factors.However,there are few reports on the conservation of germplasm resources and population genetics of Megalobrama.With the development of high-throughput sequencing technology,combinations of genomics and population genetics not only obtain the genetic information but also indicate the formation of species diversity and their environmental adaptability.This study consists of the following contents:(1)explore the morphological variation among the four Megalobrama species by X-ray photography;(2)investigate the genetic diversity and genetic differentiation of Megalobrama populations by mitochondrial markers;(3)analyze the genetic variation and genetic structure of Megalobrama populations inferred by whole genome resequencing;(4)determine the population history and feeding habits of Megalobrama,and reveal the molecular mechanisms of environmental adaptability of Megalobrama populations.Our findings will ascertain the population history and genetic diversity,and illuminate the understanding of the evolution of Megalobrama populations and molecular mechanisms of their environmental adaptability.These findings will represent a significant contribution to conversation and utilization of germplasm resources in Megalobrama.The results are listed as follows:1.Morphological variation among the four Megalobrama species inferred by X-ray photographyA total of 34 morphometric traits and 12 meristic traits of the four Megalobrama species were detected and analyzed by X-ray photography.We found significant differences among the four species in some of the morphometric traits,including head length/body length,caudal peduncle length/body length,2-4/body length,2-5/body length,4-5/body length and 6-9/body length ratios,which could therefore be used to distinguish among these species.The first three principal components could explain66.61% of the total variation in the morphology of four species.The first principal component(PC1)accounted for 39.78% of the variation among the samples and reflected differences in morphological traits associated with dorso-ventral axis.PC2 accounted for 17.25% of the variation among the samples,and it was strongly influenced by traits involved with head–tail axis.PC3 accounted for 9.58% of the morphological variation and was mostly associated with the traits that directly affect the locomotion.The results indicated that M.hoffmanni had the farthest genetic relationship with the other three species,while M.skolkovii and M.pellegrini had the closest genetic relationship.M.pellegrini was used as a model to investigate differences between the X-ray photographic method and traditional manual measurement method.The independent-samples T test result showed that 17 morphological traits were significantly different between the two methods;among these,the manual method produced larger values than the X-ray method for 11 traits.We assume that it may have been caused by the curvature of the fish body surface.2.Genetic diversity and genetic differentiation of Megalobrama populations inferred by mitochondrial markers.The concatenated sequences of mitochondrial cytochrome b(Cytb)and control region(CR)were used to analyze the genetic variation of 15 Megalobrama populations.We identified 47 Cytb+CR haplotypes.Haplotype network showed that all of the sampled populations clustered into three branches: M.hoffmanni,M.amblycephala,and a third branch comprised M.skolkovii and M.pellegrini.The result showed that haplotype diversity and nucleotide diversity of M.hoffmanni and M.skolkovii were high,and that M.hoffmanni was genetically the most divergent of the four species.There was a high genetic differentiation among the four Megalobrama species,and genetic distance among populations was not affected by geographical distance.All four Megalobrama species mostly exhibited unique Cytb+CR haplotypes,but Hap1 and Hap25 were shared by M.amblycephala and M.skolkovii.Our study showed that there was a high level of gene flow between the Liangzi Lake(LZL)population of M.amblycephala and Jinsha River Reservoir(JS)population of M.skolkovii.It indicates that there may be mitochondrial introgression between the two species.Interestingly,the number of variable sites was the highest in the M.skolkovii populations.This suggests that gene flow,as one of the sources of genetic variation,may be contributing to the relatively high genetic diversity of M.skolkovii.Mismatch analysis revealed that many Megalobrama populations accumulated low frequency alleles,while a few populations accumulated many moderate frequency alleles.3.Genetic variation and genetic structure of Megalobrama populations inferred by whole genome resequencingWe used the M.amblycephala genome as reference genome,and investigated the variation information of different Megalobrama populations.The results showed that the number of SNPs in the M.amblycephala,M.hoffmanni,M.pellegrini,and M.skolkovii were 16,235,392,16,133,317,6,825,670,17,179,046,respectively.These variation sites mainly distributed in the intergenic regions,intron regions,3'untranslated regions,5'untranslated regions,non-synonymous mutations,and synonymous mutations.The number of In Del sites contained 3,737,686 in M.amblycephala,4,097,403 in M.hoffmanni,1,649,876 in M.skolkovii,and 4,006,423 in M.pellegrini.The number of SVs consisted of 108,169 in M.amblycephala,95,589 in M.hoffmanni,59,356 in M.pellegrini,and 102,721 in M.skolkovii.The number of CNVs contained 39,777 in M.amblycephala,27,854 in M.hoffmanni,20,665 in M.pellegrini,and 35,930 in M.skolkovii.The phylogenetic tree,population genetics structure and principal component analysis revealed that all Megalobrama samples could be divided into six subgroups,including M.amblycephala,M.pellegrini,Hainan M.hoffmanni,Pearl River M.hoffmanni,Fuyuan M.skolkovii and other M.skolkovii populations.The differentiation index(Fst)showed that there was high genetic differentiation between M.hoffmanni and other Megalobrama species.Moreover,the introgression from M.amblycephala promoted the increase of the nucleotide polymorphism of M.skolkovii,and further accelerated its LD decay rate.4.Demographic history and feeding habits of Megalobarma speciesBy combining divergence time and ancestral distribution geography,it is inferred that the ancestors of genus Megalobrama may have been initially distributed in the Pearl River,some spread to Hainan Island due to sea-level fall during the glacial period and others spread to northern China.The decline in sea level during the glacial period created a broad continental shelf and land bridges in the South China Sea,which increased the suitable habitat of M.hoffmanni and caused the population expansion.The effective populations size of M.amblycephala,M.skolkovii and M.pellegrini in the Yangtze River showed decrease trend during the Quaternary glacial period,and did not increase until the climate warming in the late pleistocene.M.amblycephala mainly feeds on high-fiber,low-energy aquatic plants,it enhanced energy metabolism through the oxidation of fatty acids and adaptive changes of lipid metabolism.M.hoffmanni is omnivorous fish that mainly promotes the intestinal absorption of cholesterol,fat and lipophilic vitamins through cholesterol metabolism.