The Complete Chloroplast Genomes of Gynostemma Reveal the Phylogenetic Relationships of Species within the Genus

Abstract

Gynostemma is an important medicinal and food plant of the Cucurbitaceae family. The phylogenetic position of the genus Gynostemma in the Cucurbitaceae family has been determined by morphology and phylogenetics, but the evolutionary relationships within the genus Gynostemma remain to be explored. The chloroplast genomes of seven species of the genus Gynostemma were sequenced and annotated, of which the genomes of Gynostemma simplicifolium, Gynostemma guangxiense and Gynostemma laxum were sequenced and annotated for the first time. The chloroplast genomes ranged from 157,419 bp (Gynostemma compressum) to 157,840 bp (G. simplicifolium) in length, including 133 identical genes: 87 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene. Phylogenetic analysis showed that the genus Gynostemma is divided into three primary taxonomic clusters, which differs from the traditional morphological classification of the genus Gynostemma into the subgenus Gynostemma and Trirostellum. The highly variable regions of atpH-atpL, rpl32-trnL, and ccsA-ndhD, the repeat unilts of AAG/CTT and ATC/ATG in simple sequence repeats (SSRs) and the length of overlapping regions between rps19 and inverted repeats(IRb) and between ycf1 and small single-copy (SSC) were found to be consistent with the phylogeny. Observations of fruit morphology of the genus Gynostemma revealed that transitional state species have independent morphological characteristics, such as oblate fruit and inferior ovaries. In conclusion, both molecular and morphological results showed consistency with those of phylogenetic analysis.

Keywords: Gynostemma; chloroplast genome; phylogeny; taxonomic.

Figure 8

Fruit morphology of some Gynostemma species. (A): G. microspermum (capsule with a persistent perianth and style); (B): G. compressum, (C): G. caulopterum, (D): G. guangxiense and (E): G. pentagynum (oblate fruit with an inferior ovary and a persistent perianth and style); (F): G.longipes (berry without a persistent perianth and style).

Figure 1

Chloroplast genome maps of Gynostemma. Genes transcribed counterclockwise are present inside of the circle. Genes transcribed clockwise are present outside of the circle. The colour of the genes corresponds to the function of the genes. The dashed area of the inner circle indicates the GC content of the genome. * Genes with intron.

Figure 2

Comparative analysis plots of RSCU values of seven species in the genus Gynostemma. The bar chart above each amino acid shows RSCU values within Gynostemma species. Each bar represents a species, from left to right: G. burmanicum, G. caulopterum, G. compressum, G. guangxiense, G. laxum, G. longipes and G. simplicifolium.

Figure 3

Repeat sequence signature map. (A): SSRs for repeated unit classification. (B): comparison of mononucleotide to tetranucleotide repeat sequence types. (C): palindromic, forward, reverse, and complementary repeat sequence types; (D): Repeat length classification of long repeat sequences. * Represents a newly sequenced species of the genus Gynostemma.

Figure 4

Comparison of the chloroplast genomes of Gynostemma. Global Shuffle-LAGAN alignment was performed on the mVISTA website. Grey arrows above the alignment represent the genes. In each plot, the vertical scale represents the percent identity (50 to 100%). Genome regions are colour-coded as protein-coding exon (blue), rRNA or tRNA (sky blue), and conserved noncoding sequences (CNS, red). * Represents a newly sequenced chloroplast genome of Gynostemma species in this study.

Figure 5

Comparative analysis of nucleotide diversity (Pi) values among chloroplast genomes of Gynostemma species. The pink line is twice the median Pi value (Pi = 0.0124).

Figure 6

Comparison of SC/IR junctions among the chloroplast genomes of Gynostemma species. JLB (junction of LSC/IRb), JSB (junction of SSC/IRb), JSA (junction of SSC/IRa), JLA (junction of LSC/IRa). * Represents newly sequenced chloroplast genomes of Gynostemma species in this study.

Figure 7

Phylogenetic tree of complete chloroplast genomes of the genus Gynostemma based on maximum likelihood (ML) and Bayesian inference (BI) methods. The Bayesian posterior probabilities/ML bootstrap support values are displayed at the nodes. The colour of the species name indicates the subgenus: blue—subgenus Gynostemma, yellow—subgenus Trirostellum. branch length representative substitutions per site. * Represents newly sequenced chloroplast genomes of Gynostemma species in this study.