Studies On Molecular Taxonomy, Phylogeny And Genetic Diversity Of Chondrus Stackhouse (Florideophyceae, Rhodophyta)

Nuclear ribosomal 18S rRNA gene,internal transcribed spacer(ITS),mitochondrial cox2-3 spacer sequences,and inter simple sequence repeat(ISSR) marker technique were applied to study the molecular taxonomy,phylogeny and evolution,population genetics of red algae Chondrus Stackhouse,aiming to understand the origin and mechanism of molecular evolution of this red algae.(1) The nuclear-encoded small-subunit ribosomal RNA gene(18S rDNA) and internal transcripbed spacer(ITS) of 16 isolates of Chondrus from 8 countries were sequenced.A total of 1796 nucleotides and 710 nucleotide were obtained and aligned with phylogenetic analysis conducted for 18S rRNA and nrDNA ITS,respectively. NrDNA ITS data indicate that the entity from Dalian,China,regarded as C.spl is C. nipponicus,although which is not consistent with the result from 18S rRNA gene analysis(C.sp1 may be C.pinnulatus).The C.sp2 previously depicted as C.yendoi or Mazzaella japonica may belong to genus Chondrus.So,at least three Chondrus species,C.ocellatus,C.nipponicus,C.armatus and C.pinnulatus are distributed in China.However,the entity from Connemara,Ireland,named C.crispus,is not a Chondrus species but that ofMastocarpus stellatus,although it is similar to C.crispus morphologically.Phylogenetic analysis based on completel8S rDNA sequence data show that genus Chondrus includes 3 main lineages:the Northem Pacific lineage containing C.ocellatus,C.yendoi,and C.nipponicus;C.armatus and C.pinnulatus formed the sub- North Pacific lineage;and Northern Atlantic Ocean lineage comprises samples of C.crispus from Canada,Portugal,Ireland,Germany and France.The phylogenetic relationships indicate that genus Chondrus might have a North Pacific ancestral origin,and radiated to North Atlantic area and formed species C crispus.(2) Eighteen isolates of the-red algae Chondrus crispus were collected from Northern Atlantic sites,togethor with C.ocellatus,C.yendoi and C.pinnulatus from the North Pacific.The nuclear rDNA internal transcribed spacer(ITS) was sequenced and compared,spanning both the ITS regions and the 5.8S rRNA gene.Percentage of nucleotide variation for C.crispus ranged from 0.3%to 4.0%.Phylogenetic analyses were performed using maximum parsimony,neighbor-joining and minimum evolution methods.They showed that two main clades existed within the C.crispus samples examined and that suggested C.crispus had a single Atlantic origin.The clustering however did not follow the geographic origin.We hypothesized that the current distribution of C.crispus populations might be a result of three main factors: temperature boundaries,paleoclimate and paleoceanography.ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.(3) Genetic diversity and population structure analysis were conducted on 7 wild populations(139 individuals) of C.crispus with ISSR markers.Based on the results of PCR amplifying optimization and primers screening,eighteen primers were totally yielded 184 loci,of which 174 were polymorphic.Statistic analysis showed that Ne's gene diversity(He) and Shannon's information index(I) were 0.1228 and 0.2127, respectively.Gene diversity(H_t) among 7 populations of C.crispus was 0.2922,and gene differentiation index(G_st) was 0.7463,which indicated that 74.63 percent of differentiation occurred among populations,the rest(25.37%) was in populations.All these data suggested that extinct genetic differentiation existed in Northern Atlantic C. crispus populations.(4) Phylogenetic analyses using minimum -evolution and maximum-parsimony methods from ITS data show that Rhodomelaceae in Florideophyceae is closely related to Bangiophyceae evolutionally.In Florideophyceae,Gigartinales has the highest evolutional status,then Halymeniaceae,Gelidiaceae,Delesseriaceae and Liagoraceae,and Rhodomelaceae the lowest.In Gigartinales,Gigartinaceae and Dumontiaceae have close relationship,while Chondrus and Mazzaella,which are difficult to discriminate from morphology,formed a fairly compact clade.The low divergence among Chondrus species is justified considering its recent origin,and suggests that ITS sequence would provide a valid method for molecular phylogenetic analysis,and to reconstruct the phylogeny within the Rhodophyta on order and family levels.The intergenic region cox2-3 ranged from 134 to 400 bp in length,with great variation in the 13 genus of Rhodophyta,and A+T content varied from 49.1%to 61.4%.The average intergeneric variant nucleotides number is 34,and if genus Chondrus excluded,the interspecific nucleotide variant rate is about from 9.45%to 17.78%.The NJ and MP phylogenetie trees show that in the 13 red algal genus,genus Hypnea in Gelidiales is close to genus Catenella in Gigartinales in evolutionary relationships.In order Ceramiales,genus Dasya and genus Heterosiphonia in family Dasyaceae have close relationships to genus Bostrychia in family Rhodomelaceae and genus Caloglossa in family Delesseriaceae,and genus Asparagopsis in order Bonnemaisoniales is close to genus Batrachospermum in family Batrachospermaceae. Our results suggest that mitochondrial intergenic region(cox2-3) sequence has great practicality in algal phylogenetic and systematic studies,especially to Rhodophyta at intergeneric or interspecific level,it could be used for reconstructing the red algal intergeneric or intrageneric phylogenetic relationships.