Evolutionary Analysis of Plastid Genomes of Seven Lonicera L. Species: Implications for Sequence Divergence and Phylogenetic Relationships

Abstract

Plant plastomes play crucial roles in species evolution and phylogenetic reconstruction studies due to being maternally inherited and due to the moderate evolutionary rate of genomes. However, patterns of sequence divergence and molecular evolution of the plastid genomes in the horticulturally- and economically-important Lonicera L. species are poorly understood. In this study, we collected the complete plastomes of seven Lonicera species and determined the various repeat sequence variations and protein sequence evolution by comparative genomic analysis. A total of 498 repeats were identified in plastid genomes, which included tandem (130), dispersed (277), and palindromic (91) types of repeat variations. Simple sequence repeat (SSR) elements analysis indicated the enriched SSRs in seven genomes to be mononucleotides, followed by tetra-nucleotides, dinucleotides, tri-nucleotides, hex-nucleotides, and penta-nucleotides. We identified 18 divergence hotspot regions (rps15, rps16, rps18, rpl23, psaJ, infA, ycf1, trnN-GUU-ndhF, rpoC2-rpoC1, rbcL-psaI, trnI-CAU-ycf2, psbZ-trnG-UCC, trnK-UUU-rps16, infA-rps8, rpl14-rpl16, trnV-GAC-rrn16, trnL-UAA intron, and rps12-clpP) that could be used as the potential molecular genetic markers for the further study of population genetics and phylogenetic evolution of Lonicera species. We found that a large number of repeat sequences were distributed in the divergence hotspots of plastid genomes. Interestingly, 16 genes were determined under positive selection, which included four genes for the subunits of ribosome proteins (rps7, rpl2, rpl16, and rpl22), three genes for the subunits of photosystem proteins (psaJ, psbC, and ycf4), three NADH oxidoreductase genes (ndhB, ndhH, and ndhK), two subunits of ATP genes (atpA and atpB), and four other genes (infA, rbcL, ycf1, and ycf2). Phylogenetic analysis based on the whole plastome demonstrated that the seven Lonicera species form a highly-supported monophyletic clade. The availability of these plastid genomes provides important genetic information for further species identification and biological research on Lonicera.

Keywords: Lonicera; phylogenetic relationship; plastid genome; positive selection; repeat sequences.

Figure 1

Chloroplast genome map for three Lonicera species. Genes located outside the outer rim are transcribed in a counterclockwise direction, whereas genes inside the outer rim are transcribed in a clockwise direction (as indicated by grey arrows). The colored bars indicate known different functional groups. The dashed gray area in the inner circle shows the percentage GC content of the corresponding genes. LSC, SSC, and IR denote large single copy, small single copy, and inverted repeat, respectively.

Figure 2

Amino acid (a) and codon usage (b) frequencies of the protein-coding sequences in the seven Lonicera chloroplast genomes. *: Termination codon.

Figure 3

Analysis of repeated sequences in the seven Lonicera chloroplast genomes. (a) Frequency of selected motifs of simple sequence repeats (SSRs); (b) Frequency of repeat sequences of length >30 bp; (c) Composition of the repeats in seven Lonicera species.

Figure 4

Percentages of variable characters in homologous regions among the chloroplast genomes of seven Lonicera species: (a) coding region and (b) noncoding region. The homologous regions are oriented according to their locations in the chloroplast genome.

Figure 5

Sequence alignment of chloroplast genomes from seven Lonicera species. Sequences of chloroplast genomes were aligned and compared using the mVISTA program. The horizontal axis (x) indicates the coordinates within the chloroplast genome. The vertical scale (y axis) indicates the percentage identity, ranging from 50 to 100%. The grey arrows indicates the direction of each gene. Purple bars represent exons, orange bars show conserved non-coding sequences.

Figure 6

Comparison of the border positions of large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions in the chloroplast genomes in seven Lonicera species.

Figure 7

Phylogenetic tree obtained for seven Lonicera species based on the complete chloroplast genomes. The first number of the slashes on the branches shows the posterior probabilities according to Bayesian inference, the second number shows the bootstrap values obtained by maximum likelihood analyses, and the third number shows the bootstrap values obtained by maximum parsimony analyses.