Comparative Analyses of Euonymus Chloroplast Genomes: Genetic Structure, Screening for Loci With Suitable Polymorphism, Positive Selection Genes, and Phylogenetic Relationships Within Celastrineae

Abstract

In this study, we assembled and annotated the chloroplast (cp) genome of the Euonymus species Euonymus fortunei, Euonymus phellomanus, and Euonymus maackii, and performed a series of analyses to investigate gene structure, GC content, sequence alignment, and nucleic acid diversity, with the objectives of identifying positive selection genes and understanding evolutionary relationships. The results indicated that the Euonymus cp genome was 156,860-157,611bp in length and exhibited a typical circular tetrad structure. Similar to the majority of angiosperm chloroplast genomes, the results yielded a large single-copy region (LSC) (85,826-86,299bp) and a small single-copy region (SSC) (18,319-18,536bp), separated by a pair of sequences (IRA and IRB; 26,341-26,700bp) with the same encoding but in opposite directions. The chloroplast genome was annotated to 130-131 genes, including 85-86 protein coding genes, 37 tRNA genes, and eight rRNA genes, with GC contents of 37.26-37.31%. The GC content was variable among regions and was highest in the inverted repeat (IR) region. The IR boundary of Euonymus happened expanding resulting that the rps19 entered into IR region and doubled completely. Such fluctuations at the border positions might be helpful in determining evolutionary relationships among Euonymus. The simple-sequence repeats (SSRs) of Euonymus species were composed primarily of single nucleotides (A)n and (T)n, and were mostly 10-12bp in length, with an obvious A/T bias. We identified several loci with suitable polymorphism with the potential use as molecular markers for inferring the phylogeny within the genus Euonymus. Signatures of positive selection were seen in rpoB protein encoding genes. Based on data from the whole chloroplast genome, common single copy genes, and the LSC, SSC, and IR regions, we constructed an evolutionary tree of Euonymus and related species, the results of which were consistent with traditional taxonomic classifications. It showed that E. fortunei sister to the Euonymus japonicus, whereby E. maackii appeared as sister to Euonymus hamiltonianus. Our study provides important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Euonymus species.

Keywords: Euonymus; adaptive evolution; chloroplast genome; molecular marker; phylograms.

Figure 1

Chloroplast genome maps of Euonymus species. The species name and specific information regarding the genome (length, GC content, and the number of genes) are depicted in the center of the plot. Extending outward, the middle two layers are the nucleotide diversity of E.fortunei (inner) and E. maackii (Outer) compared with E. phellomanus, respectively.

Figure 2

The GC (%) composition in different positions of coding sequence (CDS) region of species within Celastraceae.

Figure 3

Comparison of the borders of large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions among seven Celastraceae cp genome.

Figure 4

The comparative analysis of codon usage bias in 12 Celastrineae species. (A) Codon adaptation index (CAI), (B) Codon bias index (CBI), (C) Frequency of optimal codons index (Fop), (D) Effective number of codons (ENc). (E) GC of synonymous codons in third position (GC3s).

Figure 5

(A) Analysis of simple-sequence repeats (SSRs) in the chloroplast genomes of seven Celastraceae species; (B) Frequency of SSRs in the LSC, IR, SSC region; (C) Frequency of SSRs in the intergenic regions, protein-coding genes, and introns; (D) Number of Palindromic repeat, Direct repeat, Reverse repeat, Complement repeat; (E) Distribution of tandem repeats in genomic regions and exon, intergenic spacer (IGS), and intron regions.

Figure 6

Comparison of six Euonymus cp genomes using mVISTA, with the E. phellomanus genome as the reference. The y-axis represents the percent identity within 50–100%. Gray arrows indicate the direction of gene transcription. Blue blocks indicate conserved genes, while red blocks indicate conserved noncoding sequences (CNS).

Figure 7

Comparison of nucleotide diversity (Pi) values among the six Euonymus species (window length: 300bp, step size: 200bp). X-axis, position of the midpoint of each window; Y-axis, nucleotide diversity (pi) of each window.

Figure 8

Pairwise Ka/Ks ratios 12 Celastrineae species. This heatmap shows pairwise Ka/Ks ratios between every sequence in the multigene nucleotide alignment.

Figure 9

Phylogenetic trees of the Euonymus species based on the chloroplast genome by MP. (A) Phylogenetic tree constructed using the complete chloroplast genome data. (B) Phylogenetic tree constructed using single copy genes; (C) Phylogenetic tree constructed using LSC data; (D) Phylogenetic tree constructed using SSC data; and (E) Phylogenetic tree constructed using IR data.