Phylogenetic Relationships In Zosteraceae And Population Genetics Of Zostera Marina Linnaeus

Zostera marina Linnaeus, a vital member of Zostera (Zosteraceae, Helobiae), is a dominant seagrass species and a monoecious marine angiosperm distributed in the northern hemisphere. Because of the different classification results of morpholgy and molecule, we analysed phylogenetic relationships in Zosteraceae and five Zostera in China based on partial nucleotide sequences of matK,rbcL and ITS. Population genetics of Z. marina based on AFLP and SSR also should be discussed.1. Partial nucleotide sequences of matK and ITS gene were sequenced and analyzed for Zosteraceae, and nucleotide composition analysis indicated a strong bias against cytimidine (C) in both fragments. 228 nucleotide substitutions were found in ITS gene, showing the high genetic polymorphism. 249 nucleotide substitutions were checked in matK gene, and most of them were synonymous transitions at the third codon positions. The Zosteraceae species had a certain degree of differentiation at the amino acid level. Based on partial sequences of matK and ITS gene, phylogenetic trees were constructed by NJ, MP, ML and Bayes methods and the results were consistent. The phylogenetic trees showed four separate lineages: (1) subgenus Zostera, (2) Heterozostera, (3) subgenus Zosterella and (4) Phyllospadix. The pairwise percentage divergences between the samples of subgenus Zostera and subgenus Zosterella were from 29.09% to 35.51% and were much higher than the standard divergence among genera of most angiosperm, which were from 9.60% to 28.80%. Both of them would be the generic rank. We agreed with the notion of Tomlinson and Posluszny, who recommended taxonomic delimition of Zosteraceae as four genera.2. Fragments of the chloroplast (matK and rbcL) and the nuclear ribosome (ITS) regions were successfully suitable for phylogenetic relationship analysis. With sequencing by PCR amplification, the paper analyzed the phylogenetic relationship of 5 Zosteraceae species (Zostera marina, Z. japonica, Z. caespitosa, Z. asiatica and P. iwatensis), which were collected from Ireland, Japan, Korea, China and GenBank. Partial nucleotide sequences of matK, rbcL gene and ITS were analyzed for five Zosteraceae species, and nucleotide composition analysis indicated a strong bias against cytimidine (C) in three fragments. 172 nucleotide substitutions were found in ITS fragment, showing the high genetic polymorphism. 52 nucleotide substitutions were checked in matK gene and 20 nucleotide substitutions were checked in rbcL gene, most of them were synonymous transitions at the third codon positions. The five species had a certain degree of differentiation at the amino acid level. Based on partial sequences of ITS, matK and rbcL gene, phylogenetic trees were constructed by NJ, MP, ML and Bayes methods, the results were consistent and the phylogenetic trees showed three separate lineages. The minimum pairwise percentage divergences in the samples of Z. japonica and other Zostera species was 19.33% and much higher than the standard (9.60%-28.80%) among genera of most angiosperm and it would be the generic rank from the molecular data. Based on the synonymous nucleotide substitution rate of the rice chloroplast genome matK combined with rbcL gene, we estimated the divergence time between five Zosteraceae species was approximately ranged from Eocene to Pliocene. The ITS sequences of all Z. marina samples were identical, which fell into the same cluster. It was concluded that variation within ITS region of Z. marina was not correlated to their geographical distance, and ITS region was unsuitable for identification of population groups on a regional or oceanic scale. The research further elaborated phylogenetic relationship of five Zosteraceae species, also provided theoretic basis for seagrass phylogeny in China.3. Genetic relationships of Z. marina were analyzed using AFLP analysis. A total of 235 bands were generated from 63 individuals by 6 primers combinations; of these, 160 (68.09%) were polymorphic. The population with the highest Nei's gene diversity value (H) and Shannon's information index value (I) was detected in the population Finavarra (0.072 and 0.115), while the lowest was population Jiaonan (0.007 and 0.011). Genetic similarity values among 6 populations ranged from 0.660 to 0.924 with the corresponding distance values ranging from 0.076 to 0.340. The results of AMOVA analysis indicated that 83.29% of the species'total genetic variation was due to differences among populations and 16.71% occurred within populations. By screening the UPGMA tree, population Tian'ehu was genetically closer to population Jiaonan than population Lidao, which may be attributable to eelgrass meadow fragments and some marine gyres/currents. The geographic distance was responsible for the genetic differentiation among populations in China (Lidao, Tian'ehu and Jiaonan), Japan (Tokyo Bay), Korea (Naepo) and Ireland (Finavarra). The results of the present study highlight the utility of DNA-based markers for conservation in genetically depauperate species and any future restoration and conservation projects use only locally eco-sourced materials for population augmentations.4. Genetic diversity and population structure of seven natural populations of Z. marina were investigated by using microsatellite markers (SSR). A total of 57 alleles were identified in 148 individuals across the four microsatellite primers analysed, with a mean value of 14 alleles per locus. The mean expected heterozygosity (He) and observed heterozygosity (Ho) across all populations were 0.687 and 0.417, respectively, and a higher level of diversity was found in population Qingdao Bay (A=7.750, AR=7.043) than other populations. FST values suggested moderate genetic differentiation within most of the Z. marina populations. From the UPGMA tree, four populations in China clustered together, and the genetic relationships may be attributable to eelgrass meadow fragments. The geographic distance was responsible for the genetic differentiation among populations in China (Lidao, Tian'ehu, Qingdao Bay and Dalian), Korea (Naepo), Japan (Tokyo Bay) and Ireland (Finavarra). Results of possible number of clusters agreed with the seagrass origin of East Asia. Population Qingdao Bay of the species has higher genetic diversity. Thus, populations of the region deserve prior conservation and utilization for breeding programmes.