Researches Of The Phylogenetic Relationships Among 13 Ardeids And Genetic Diversity Of The Chinese Egret

The present study studied the phylogenetic relationships among 13 ardeids (Little Egret E. garzetta, Chinese Egret E. eulophotes, Eastern Reef Egret E. sacra, White-faced Egret E. novaehollandiae, Grey Heron A. cinerea, Great Egret A. alba, Cattle Egret B. ibis, Chinese Pond-Heron A. bacchus, Black-crowned Night-Heron N. nycticorax, White-eared Night-Heron G. magnificus, Yellow Bittern I. sinensis,BlackBittern I. flavicollis and Great Bittern B. stellaris) and the genetic diversity of the E. eulophotes, based on the 14 mitochondrial genes and 5'-end of the mitochondrial control region, respectively.Mitochondrial fragments from 12S rRNA to Cytb of each ardeids were amplified directly from the total DNA using 13 pairs of universal primers designed by present study. The obtained mtDNA sequences of the 12 ardeids were ranged from 14737 to 14764 bp in length and had gene order consistent with those of other avian. After downloaded sequences of White-faced Egret E. novaehollandiae, Black-faced Spoonbill P. minor and Crested Ibis N. nippon from Genbank, the sequences of two rRNA genes and 12 protein coding genes were aligned individual before used in phylogenetic analysis. The results of the alignment of 13325 bp sequences of all the 15 species indicated that there were 4875 variable sites, of which 3384 were parsimony-informative sites. The average transition (Ts) and transversion (Tv) were 1186 and 463, respectively, with value of R (Ts/Tv) being 2.6. The uncorrected p distances ranged from 0.153 to 0.166 between outgroup and ingroup, and from 0.018 to 0.139 among the ingroup species.In the phylogenetic trees of 13 ardeids, the NJ, ML and BI tree were identical in tree topologies, which all indicated that 13 ardeids first spilt into two clades, one was formed by B. stellaris, I. Jlavicollis and I. sinensis and the later two grouped firstly. The remaining ten ardeids formed anther clade and A. bacchus spilt at the base. G. magnificus, E. novaehollandiae, E. garzetta, E. eulophotes, and E. sacra formed a cluster and the later two grouped firstly and then the E. garzetta, E. novaehollandiae, G .magnificus in turn. N. nycticorax, B. ibis, A. alba and A. cinerea formed another cluster and the later two grouped firstly and then B. ibis, A. cinerea in turn.In the three MP trees, the tree with equally weighted and the tree weighted 0.5 of third position were identical in tree topologies, which different to NJ, ML and BI trees in A. alba grouping with B. ibis, G magnificus grouping with A. bacchus firstly.The MP tree with Ts:Tv in the third position weighted 1:2.2 also indicated that support A. alba grouped with B. ibis firstly, but A. bacchus and the other two clusters were parallel relationships.In genetic diversity of E eulophotes, a 433 bp DNA fragment at 5' end of the mitochondrial control region was surveyed. Among 90 individual samples, 31 haplotypes were defined by 30 polymorphic sites, of which three were found across all populations, eleven were shared by two populations and seventeen were unique for single population. The p-distances among haplotypes ranged from 0.0023 to 0.0208, with average of 0.0111. The parsimony network indicated the relationships among haplotypes were not apparent correlated to their populations.Haplotype diversity (h) of populations in Xiaocaiyu, Riyu and Xingrentuo were 0.860±0.057,0.878±0.036 and 0.961±0.022, respectively, while nucleotide diversity (π) were 0.0076±0.0010,0.0096±0.0008 and 0.0086±0.0006, respectively. Average number of nucleotide differences (k) of populations in Xiaocaiyu, Riyu and Xingrentuo were 3.27,4.138 and 3.720, respectively. With respect to those in E. garzetta population (h = 0.954±0.022,π= 0.0145±0.0014 and k = 6.297), haplotype diversity was no significant different between them but theπand k were related low in E. eulophotes, the lowest n and k in E. eulophotes (Xiaocaiyu) were both only about 52% of that in E. garzetta and the highst n and k in E. eulophotes (Xiaocaiyu) were both only about 66% of that in E. garzetta. The result of neutrality test and mismatch distribution analysis (SSD = 0.0081, P = 0.12) both revealed population expansion in E. eulophotes, which happened in about 13000 -64000 year ago.Analysis of molecular variance (AMOVA) revealed that more than 95% variation was within populations, and differentiation among populations was less than 5% but statistic significant (distance-basedΦ_st = 0.0419, P < 0.05; haplotype-basedΦ_st = 0.03267, P < 0.01). Pairwise comparisons indicated that significant differentiation was between populations of Xiaocaiyu and Xingrentuo (distance-based F_st = 0.0743, P<0.01; haplotype-based Fst = 0.0316, P < 0.05), but no between Xiaocaiyu and Riyu (distance-based Fst = 0.0148, P > 0.05; haplotype-based Fst = 0.0212, P > 0.05). Significant differentiation was found between populations of Riyu and Xingrentuo only based on haplotype frequence estimated (Fst = 0.0444, P < 0.01) but no based on distance estimated (Fst = 0.0397, P > 0.05).In conclusion, (1) the phylogenetic relationships among 13 ardeids in present study were: bitterns were monophyletic and first divergen; night herons were not monophyletic, of which G. magnificus had close related to the genus of Egretta and N. nycticorax had close related to the genus of Ardea; E. novaehollandiae belonging to the genus of Egretta was supported; among the four egrets, E. sacra had close related to E. eulophotes and E. novaehollandiae was more diverge; A. alba and B. ibis had close related to the genus of Ardea and they belonged to the genus of Ardea was supported; A. bacchus was the first divergen from the ten ardeinae but needed further researches, because of low support values. (2) The genetic divergences of E. eulophotes were relatively low; population expansion ever happened in demographic history of E. eulophotes; E. eulophotes had a significant population genetic structure but limited geographic structure; implications for conservation strategies were that the populations of Xiaocaiyu and Riyu formed a management unit and the population of Xingrentuo formed another management unit.