Research On The Morphological Variation And Genetic Relationship Of Chinese Crocodile Lizard(Shinisaurus Crocodilurus) Amomg Different Geographical Populations

Shinisaurus crocodilurus,which belongs to the family Shinisauridae and the genus Shinisaurus,is the ancient reptile residued in South China after Quaternary glacial.It is a first class protected animal in China,and listed in CITES-appendix II in 1989.As a result of habitat destruction and Human hunting,this species declines sharply in quantity and is on verge of extinction.Therefore,understanding the genetic variation and geographical relationship of this species is of great significance for the protection and management of the crocodile lizard.This research used two molecular markers of microsatellites DNA and mitochondria DNA,and associated with the morphological differences between different regions,to study genetic diversity and geographical relationship of 16 different geographic populations.(1)S.crocodilurus populations have some differences in the morphology among four distribution areas(Guangxi Daguishan,Guangdong Luokeng,Guangdong Maoming and Vietnam),Daguishan's population is significantly(P<0.05)larger than Luokeng's and Maoming's in SVL and HW(Head Width),slightly larger than vietnam's but not significant.Female S.crocodilurus in Maoming's populations have more lamellae beneath fourth toe(L~4)than that in Luokeng's populations,and males have more L~4 than that in Luokeng's and Daguishan's populations(P<0.05).(2)The results of microsatellites DNA showed that 107 alleles was found by using10 microsatellite loci.The average number of alleles,effective allele,observed heterozygosity and expected heterozygosity among the 16 crocodile lizards populations was 4.975,3.159,0.465 and 0.624,respectively.The results of mitochondria DNA showed that 10 haplotypes were detected from 98 individuals.Haplotype diversity and nucleotide polymorphism were 0.195246 and 0.000303,respectively.Both of them were very low.Both microsatellites DNA and mitochondria DNA results showed low genetic diversity of the species.(4)We discuss genetic structure and phylogeny of S.crocodilurus based on microsatellites markers and mitochondria markers.The results of microsatellites markers showed that the pairwise population Fst values were at the range from 0.032 to 0.267,and most of them were at the range from 0.05 to 0.25.Genetic differentiation between populations was above medium level.The result of Mantel Test showed that 15 natural populations(apart from the Guiping raising population)existed significant isolation-by-distance model(r=0.67,p=0.01).There was a very significant positive correlation between geographical distance and genetic distance.The results of Structure clustering analysis using microsatellite markers showed that 16 populations divided into4 clusters including GuangxiDaguishan(DCC,CSC,DSC,YSC),Guangdong Luokeng(MBC,CSK,DBT,SK),Guangdong Maoming(LZD,LHD),and Vietnam(HH,TYT,DS,YT).A Neighbour-Joining phylogenetic tree was built based on the Nei's genetic distance of microsatellites markers.The NJ phylogenetic tree showed 16 populations were divided into two major branches of China and Vietnam.Guangxi Daguishan and Guangdong Luokeng's population were assigned to two branchlets after separated from Guangdong Maoming's population.Three phylogenetic trees of NJ,ML and UPMGA based on mitochondria markers showed that 16 populations of S.crocodilurus were divided into two major branches of China and Vietnam.Median-Joining network relationship showed that 10 haplotypes were clustered into three branches including Guangxi and Guangdong Luokeng populations,Guangdong Maoming populations and Vietnam populations.But no centric haplotype was found.Analysis of molecular variation(AMOVA)based on microsatellite markers was showed that variations within population were the major variations source.However,AMOVA based on mitochondria markers showed that most variations were among populations.(5)Based on microsatellite markers,the results of bottleneck effect showed that 12populations of 4 clusters under 3 models(I.A.M,T.P.M,and S.M.M)and Mode-shift test have undergone significant Bottleneck effect excepting 4 populations of Guangdong Luokeng.(6)The population history of S.crocodilurus was discussed based on mitochondria DNA.Values of Tajima's D,Fu and Li's D,Fu and Li's F were significantly greater than zero(P<0.05).It suggested that populations deviated from neutrality selection.The populations may undergo Bottleneck effect or equilibrium selection.