Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution

Abstract

Euonymus hamiltonianus and its relatives (Celastraceae family) are used for ornamental and medicinal purposes. However, species identification in Euonymus is difficult due to their morphological diversity. Using plastid genome (plastome) data, we attempt to reveal phylogenetic relationship among Euonymus species and develop useful markers for molecular identification. We assembled the plastome and nuclear ribosomal DNA (nrDNA) sequences from five Euonymus lines collected from South Korea: three Euonymus hamiltonianus accessions, E. europaeus, and E. japonicus. We conducted an in-depth comparative analysis using ten plastomes, including other publicly available plastome data for this genus. The genome structures, gene contents, and gene orders were similar in all Euonymus plastomes in this study. Analysis of nucleotide diversity revealed six divergence hotspots in their plastomes. We identified 339 single nucleotide polymorphisms and 293 insertion or deletions among the four E. hamiltonianus plastomes, pointing to abundant diversity even within the same species. Among 77 commonly shared genes, 9 and 33 were identified as conserved genes in the genus Euonymus and E. hamiltonianus, respectively. Phylogenetic analysis based on plastome and nrDNA sequences revealed the overall consensus and relationships between plastomes and nrDNAs. Finally, we developed six barcoding markers and successfully applied them to 31 E. hamiltonianus lines collected from South Korea. Our findings provide the molecular basis for the classification and molecular taxonomic criteria for the genus Euonymus (at least in Korea), which should aid in more objective classification within this genus. Moreover, the newly developed markers will be useful for understanding the species delimitation of E. hamiltonianus and closely related species.

Fig 1. Gene map and plastome variations.

SNPs were measured in every 2 kb sliding window with a 500 bp sliding length. 1–4: Maximum variant number scale in each map is 6; 1. E. hamiltonianus (Hantaek)- specific SNP density map; 2. E. hamiltonianus (Hongcheon)-specific SNP density map; 3. E. hamiltonianus (Jeju)-specific SNP density map; 4. E. hamiltonianus (‘Snow’)-specific SNP density map.

Fig 2. Genomic structures and diversity of rDNAs in Euonymus.

A and B show the structures of 45S rDNA and 5S rDNA, respectively. Variations are marked based on E. hamiltonianus (Hantaek) as a reference and are highlighted by red and black lines. Red and black lines indicate SNPs and InDels, respectively.

Fig 3. IR junctions in ten Euonymus plastomes.

A) In every species, rps19 is 279 bp long and is present at the IR junction. rps19 is present in the IR in four Euonymus individuals and in the LSC in six Euonymus individuals. The right IR junctions on the ycf1b genes are marked. B) and C) Nucleotide diversity in the left and right junctions, respectively. SNP and InDels are indicated by red and black lines, respectively. All variants are marked based on E. hamiltonianus (Hantaek) as a reference. The regions upstream of 735 bp in C) have no variants.

Fig 4. Pi value for each plastome region among the ten Euonymus lines.

The total pi value among whole plastomes was calculated as 0.00695 using DnaSP with the sliding window method. The sliding window size was 600 bp with a 200 bp sliding length. Six regions were estimated as divergence hotspots among Euonymus, as they had higher pi values (> 0.02). IR regions had lower pi values than SC regions (LSC and SSC).

Fig 5. Phylogenetic tree of the ten Euonymus accessions based on plastome and 45S rDNA sequences.

The tree on the left was drawn using common plastome coding sequences, and the tree on the right was drawn using common 45S rDNA sequences. The phylogenetic trees were constructed using the Bayesian Inference method. Bootstrap (ML method) and posterior probability values greater than 50% were shown. The supporting values separated by slash are bootstrap values and posterior probability, respectively.

Fig 6. Representative genotypes and scheme used to develop barcoding markers.

EHV: E. hamiltonianus (Hantaek), EHH: E. hamiltonianus (Hongcheon), EHJ: E. hamiltonianus (Jeju), EHS: E. hamiltonianus (‘Snow’), EE: E. europaeus.