Comparative and phylogenetic analysis of the complete chloroplast genomes of six Polygonatum species (Asparagaceae)

Abstract

Polygonatum Miller belongs to the tribe Polygonateae of Asparagaceae. The horizontal creeping fleshy roots of several species in this genus serve as traditional Chinese medicine. Previous studies have mainly reported the size and gene contents of the plastomes, with little information on the comparative analysis of the plastid genomes of this genus. Additionally, there are still some species whose chloroplast genome information has not been reported. In this study, the complete plastomes of six Polygonatum were sequenced and assembled, among them, the chloroplast genome of P. campanulatum was reported for the first time. Comparative and phylogenetic analyses were then conducted with the published plastomes of three related species. Results indicated that the whole plastome length of the Polygonatum species ranged from 154,564 bp (P. multiflorum) to 156,028 bp (P. stenophyllum) having a quadripartite structure of LSC and SSC separated by two IR regions. A total of 113 unique genes were detected in each of the species. Comparative analysis revealed that gene content and total GC content in these species were highly identical. No significant contraction or expansion was observed in the IR boundaries among all the species except P. sibiricum1, in which the rps19 gene was pseudogenized owing to incomplete duplication. Abundant long dispersed repeats and SSRs were detected in each genome. There were five remarkably variable regions and 14 positively selected genes were identified among Polygonatum and Heteropolygonatum. Phylogenetic results based on chloroplast genome strongly supported the placement of P. campanulatum with alternate leaves in sect. Verticillata, a group characterized by whorled leaves. Moreover, P. verticillatum and P. cyrtonema were displayed as paraphyletic. This study revealed that the characters of plastomes in Polygonatum and Heteropolygonatum maintained a high degree of similarity. Five highly variable regions were found to be potential specific DNA barcodes in Polygonatum. Phylogenetic results suggested that leaf arrangement was not suitable as a basis for delimitation of subgeneric groups in Polygonatum and the definitions of P. cyrtonema and P. verticillatum require further study.

Figure 1

Gene map of the chloroplast genome among the Polygonatum species. Genes inside and outside the circle transcribed in counter-clockwise and clockwise respectively. The dark gray and light gray areas inside the inner circle indicate GC content and AT content respectively. LSC (Large single-copy), SSC (Small single-copy) and the inverted repeats (IRa, IRb) were denoted inner the circle.

Figure 2

Relative synonymous codon usage (RSCU) value of 20 amino acids and stop codons of seven Polygonatum and two Heteropolygonatum species based on protein-coding sequences in chloroplast genomes. The colors of the bar correspond to the colors of codons. Each amino acid corresponds to nine histograms, and y-axis represents the RSCU value. The order of each six columns from left to right is P. campanulatum, P. filipes1, P. franchetii, P. zanlanscianense1, P. cyrtonema1, P. sibiricum1, P. kingianum 2, H. alternicirrhosum and H. ginfushanicum.

Figure 3

Analysis of long dispersed repeats in the cp genomes of seven Polygonatum and two Heteropolygonatum species. (A) The number of the four types of long repeats. (B) Distribution ratio of repeats in regions of the cp genome. (C) Distribution ratio of repetitive sequences in functional regions. (D) Proportion of repeats in different length intervals of the chloroplast genome.

Figure 4

Simple sequence repeats (SSRs) analysis of the complete chloroplast genomes of the seven Polygonatum and two Heteropolygonatum species. (a) Numbers of mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide repeats. (b–j). Frequencies of SSRs motifs in different repeat class types.

Figure 5

Alignment of chloroplast genomes of Heteropolygonatum alternicirrhosum, H. ginfushanicum, Polygonatum campanulatum, P. filipes1, P. franchetii, P. zanlanscianense1, P. cyrtonema1, P. sibiricum1, P. kingianum2. The grey arrows at the top represent the direction of gene translation, and the y-axis indicates the percentage identity between 50 and 100%. (Exon: protein codes; UTR: tRNAs and rRNAs; CNS: conserved noncoding sequences).

Figure 6

Genomic rearrangement of the seven Polygonatum and two Heteropolygonatum. Blocks in different colors correspond to different gene types. Black: transfer RNA (tRNA); green: intron-containing Trna; Red: ribosomal RNA; White: protein-coding genes (PCGs).

Figure 7

Comparative analysis of the LSC, IR and SSC boundary regions in the nine chloroplast genomes.

Figure 8

Nucleotide diversity analysis of the complete chloroplast genomes of the seven Polygonatum and two Heteropolygonatum (window length: 600 bp; step size: 200 bp).

Figure 9

Phylogenetic relationships of the 57 cp sequences of Polygonatum and 4 of Heteropolygonatum, with Maianthemum henryi set as the outgroup. Maximum likelihood (ML) and Bayesian inference (BI) methods were used to reconstruct the tree. Only ML tree was shown, because of the highly identified topologies of ML tree and BI tree. The value of ML supports and Bayesian posterior probabilities were shown above the branches. The cp genomes newly sequenced in this study are highlighted with red triangle marks.