Molecular Systematic Study On Some Species Of Triculine

Triculinae is a subfamily of Pomatiopsidae(Gastropoda).It mainly distributes insoutheast Asia and southern China.Over 50 species were found in China.It is known that,some species of generaTricula,Neotricula and Robertsiella can act as intermediate hosts ofSchistosoma sinensium,S.mekongi,S.malayensis and Paragonimus.In the past,theTriculinae were identified based on the shell morphology and formula of radula.More andmore studies used molecular teclmiques recently,however,the molecular data on Triculinaewas still very limited.The present study was implemented to reconstruct the phylogeneticrelationship among some species of Triculinae,using the molecular markers of cytocrome coxydase subunit 1(COI),large subunit RNA(16S)in mitochondrial genome,and nuclearlarge subunit RNA(28S)and the first internal transcribed spacer(ITS 1).Partial fragments of COI,16S rDNA,28S rDNA and ITS 1 were amplified by PCR and thensequenced for samples from 11 localities of six provinces in China.The sequences of otherspecies of Triculinae and two outgroups were retrieved from GenBank and added to thefour datasets respectively.The base composition,substitution saturation and phylogeneticsignal within each gene fragment were analyzed by sofiwares.The sequences were alignedglobally by CLUSTAL X and the ITS1 sequences were folded by Mfold with defaultparameters.Then the phylogenetic relationships were reconstructed using MP,ML,and NJcriteria for individual and combined datasets by PAUP ver.4.0b10.We compared thetopologies of those trees and made conclusions as follows: 1.After alignment we obtained a 598 bp fragment of COI,513 bp of 16S and 760 bp of 28S.The nucleotide divergences among the three geneswere 35.6%,31% and 14%,which was25.6% for the combined dataset;the base contents of A and T were 70%,66% and 41%,which was 52% for the combined dataset.The frequency of transition was more than that oftransversion for all three sequences.The ITS1 sequence showed great divergence,thesequence variation ranged from 1.2% to 56.8%,and the average A and T content was 51%.The sequences of T.pingi were different from T.hsiangi at only 3 bp in COI,1 bp in 16Sand 28S,and 5 bp in ITS1 respectively,the identity was over 99%.Among those samples inHubei Province,except T.sp.H-SHY and T.hongshanensis,the average similarity was over95%.2.The result of base substitution saturation analysis for the three sequences showed nosignificant saturation for COI and 28S,but the transition in 16S rDNA has reachedsaturation.ILD test indicated no statistically significant incongruence among the two,threeand four combined dataset,excluding a little incongruence between 16S and 28S.Thisincongruence was not induced by the resolution of the 16S sequence,maybe owing to thesignal noise.So the combined sequences were analyzed.3.According to the folding results of all 12 samples,a general model of ITS 1 secondarystructure was concluded,namely,an open multi-branch loop with four helices.Itdemonstrated that the secondary structure was more conserved than primary structure,additionally,the 3' end was more conserved than 5' end.4.The topology of phylogenetic trees with individual gene fragment was a bit differentfrom that with combined dataset.However,there was congruence in some general clades.The topology of phylogenetic trees with combined dataset(COI,16S and 28S)wasconsistent with that of ITS1,irrespective of different methods of ML,MP and NJ,and alltrees had high bootstrap values.Those demonstrated that combined analysis could increasethe phylogenetic signal and decrease noise;secondary structure does have significantimplication to sequence alignment and also phylogenetic analysis. 5.The result indicated that Triculinae may be a monophyletic group.The taxa in Fujian,Guangxi and Zhejiang provinces were in one clade and those from Hunan,Sichuan andHubei provinces were in another clade.The subclades of(T.sp.H-SNJ,T.sp.H-XSH,T.sp.H-YX,T.pingi and T.hsiangi)and(T.sp.H-SHY-T.hortensis)had high supports allthrough.It can be presumably concluded that the phylogenetic relationships among the Triculinaespecies in China are partly related with the geographical and ecological characters.Thespecies among Hubei Province showed extreme affinity with each other except T.hongshanensis and T.sp.H-SHY,they may be a species complex.T.pingi and T.hsiangishould be congeneric species.