Comparative analysis of plastid genomes within the Campanulaceae and phylogenetic implications

Abstract

The conflicts exist between the phylogeny of Campanulaceae based on nuclear ITS sequence and plastid markers, particularly in the subdivision of Cyanantheae (Campanulaceae). Besides, various and complicated plastid genome structures can be found in species of the Campanulaceae. However, limited availability of genomic information largely hinders the studies of molecular evolution and phylogeny of Campanulaceae. We reported the complete plastid genomes of three Cyanantheae species, compared them to eight published Campanulaceae plastomes, and shed light on a deeper understanding of the applicability of plastomes. We found that there were obvious differences among gene order, GC content, gene compositions and IR junctions of LSC/IRa. Almost all protein-coding genes and amino acid sequences showed obvious codon preferences. We identified 14 genes with highly positively selected sites and branch-site model displayed 96 sites under potentially positive selection on the three lineages of phylogenetic tree. Phylogenetic analyses showed that Cyananthus was more closely related to Codonopsis compared with Cyclocodon and also clearly illustrated the relationship among the Cyanantheae species. We also found six coding regions having high nucleotide divergence value. Hotpot regions were considered to be useful molecular markers for resolving phylogenetic relationships and species authentication of Campanulaceae.

Fig 1

Plastid genomes of Cyananthus flavus (A), Cyclocodon parviflorus (B), and Codonopsis hongii (C). Genes inside the circle are transcribed clockwise, and genes outside the circle are transcribed counter-clockwise. The dark-gray inner circle corresponds to the GC content, and the light-gray represents the AT content.

Fig 2. Percentages of variable sites in protein-coding regions.

The blue line indicates the comparison of eleven species among the family Campanulaceae; the gray line indicates the comparison of six Cyanantheae species; the orange line indicates the Campanuleae species; the yellow line indicates the out-group. X axis: position of the midpoint of a window. Y axis: nucleotide diversity of each window.

Fig 3. Comparison of the borders between IR and LSC/SSC regions and the gene composition of IR regions.

Fig 4. Codon contents of 20 amino acids and stop codons in all protein-coding genes of the Campanulaceae plastomes.

The color of the histogram corresponds to the color of codons.

Fig 5. Heatmap analysis for codon distribution of all protein-coding genes of all considered species.

Colour key: Higher red values indicate higher RSCU values, and lower blue values indicate lower RSCU values.

Fig 6. Frequency of three types of dispersed repeats by length.

(F: forward, P: palindrome, R: reverse).

Fig 7. The distribution maps of simple sequence repeats (SSR).

Classification of SSRs by repeat types. p1, mononucleotides (mono-); p2, dinucleotides (di-); p3, trinucleotides (tri-); p4, tetranucleotides (tetra-); p5, pentanucleotides (penta-); p6, hexanucleotides (hex-); c, compound.

Fig 8. Repeat number in the different regions of Campanulaceae plastomes, including Tandem repeats, Dispersed repeats and SSR repeats.

The yellow line refers to SSC regions, the gray lines refers to IRa regions, the orange line refers to LSC regions, and the blue line refers to the total plastome.

Fig 9. Phylogenetic relationship of all Campanulaceae species by using the 76 protein-coding genes, based on the Maximum likelihood (ML) analysis and Bayesian inference (BI) analysis.