Analyses Of Chloroplast Genome Of Smilax China L.and Comparative Studies Of CpDNA Genomes Of Monocots

The monocot clade is a monophyly lineage that diverged early during angiosperm evolution. Relationships within the monocot clade have been addressed in a number of phylogenetic studies and major clades have been identified. The placement of Liliales, however, has strikingly varied among previous studies. The rapid growth of complete chloroplast genome sequence data provides a wealth of new information for studying the evolution of chloroplast genomes and the phylogeny of angiosperms. As a result,212 plastid genomes of green plants have been sequenced. The lack of cpDNA genome sequence data for Liliales has hampered an evaluation of the phylogeny relationships of monocots from a phylogenomic perspective. Smilacaceae is a monogeneric family of Liliales consisting of about 200～250 species widely distributed in tropical, subtropical, and temperate regions. In the present study, we report the sequence and characterization of the complete cpDNA genome from the Liliales species, Smilax china L. Then, comparative analyses have been conducted among the known monocot chloroplast genomes by the means of bioinformatics. Furthermore, we explored the effects of gene functions, natural selection and nucleotide substitution rate on phylogenetic inference of monocot clades. We further conducted phylogenetic analyses of monocot based on the combined 63 common protein genes in order to evaluate the phylogenetic position of Liliales from a phylogenomic perspective. The main results of our research are as follows:1. Sequencing and analysis of the chloroplast genome of Smilax china L.The chloroplast genome of Smilax china was determined to be a circular DNA molecule of 157,878bp with a quadripartite structure typical of the majority of land plant chloroplast chromosomes. It includes two inverted repeat regions (IRa and IRb) of 27,367bp separated by a large single copy region (LSC) and a small single copy region (SSC) of 84,608bp and 18,536bp, respectively. A total of 135 genes were identified in the Smilax cpDNA genome. Of the 135 genes,21 are duplicated in the IRs. Thus there are 114 unique genes in the cpDNA genome of Smilax china. Seventy three of the 114 genes are polypeptide genes,30 are distinct tRNA genes, four are rRNA genes, six are conserved reading frames (ycfs), and three are pseudogenes.2. Comparison of chloroplast genome within monocotsComparision of cp genome showed that all the monocot cp genomes contain four parts (LSC-IRa-SSC-IRb). The lengths of monocot cp genomes are between 135kb and 170kb except that of Rhizanthella gardneri (Orchidaceae), which is only 59,190bp resulting from a major genome contraction. The cp genome length of Poaceae is between 135kb and 141kb, which is much less than that of other monocot lineages (146-170kb). On the other hands, the cp genome length of Alismataceae (165-170kb) is much longer than those of other monocot lineages (<161kb). Comparision of monocot chloroplast genomes indicated that three inversions (Ⅰ,Ⅱ,Ⅲ) were identified in cp genomes of Poaceae. Analysis of "genome contents" study identified eight divergent hotspots in the monocot chloroplast genome.3. Heterogeneity of substitution rate and natural selection force among genes and among the four functional gene groupsThere are 63 protein-coding genes identified as common to the monocot cpDNA genomes. The 63 protein-coding genes were partitioned into four functional groups: (1) gene expression; (2) photosynthetic apparatus; (3) photosynthetic metabolism; and (4) a miscellaneous group. Nonsynonymous (dN), synonymous (dS), total nucleotide substitution rate (Ts+Tv) and natural selection force (dN/dS) are heterogeneous among genes and lineages.4. Effects of gene function, selection force, and substitution rate on inference of monocot phylogenyPhylogenetic analyses of the 2-codon and 3-codon data from the four functional gene groups resulted in different topologies, and trees resulting from the 2-codon (1st+2nd) and 3-codon (1st+2nd+3rd) data of the same group genes are different in some lineages. The two trees (2-codon data and 3-codon data) of the same selection force group have the same topology, only different in the bootstrap values at some nodes. All the trees basically support the commonly recognized phylogeny of monocots except the variable topologies and unreliable bootstrap values in some special lineages such as Liliales, Asparagales and Dioscoreales. Seletion force exerts a strong effect on inference of monocot phylogeny.5. Phylogenetic analyses of the monocots using combined 63 gene sequence dataCompared with trees from gene groups, trees from 2-codon,3-codon,3rd-codon and amino acids data of the 63 protein-coding genes are more consistent in topology and have higher bootstrap values. However, incongruence still exists regarding the placements of Liliales, Asparagales and Dioscoreales. The 2-codon tree supports a paraphyletic relationship between Liliales (Smilax) and Dioscoreales (Dioscorea), consistent with previous phylogenetic studies such as APGⅢ(2009). However, the 3-codon tree supports the monophyly of Liliales(Smilax) and Dioscoreales (Dioscorea). Topology test using SH test with the 3-codon data indicates that the 3-codon tree that support the sisiter relationship between Liliales(Smilax) and Dioscoreales(Dioscorea) ia the best, suggesting this relationship is better supported by the total cp genome data.