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

PLOS ONE RESEARCH ARTICLE Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution Young Sang Park1, Jong-Soo Kang ID1, Jee Young Park1, Hyeonah Shim1, Hyun Ok Yang2, Jung Hwa Kang3, Tae-Jin Yang ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Agriculture, Forestry and Bioresources, Plant Genomics & Breeding Institute, College of Agriculture & Life Sciences, Seoul National University, Seoul, Korea, 2 Department of Integrative Biological Sciences and Industry, Sejong University, Seoul, Korea, 3 Hantaek Botanical Garden, Yongin, Korea * Abstract OPEN ACCESS Citation: Park YS, Kang J-S, Park JY, Shim H, Yang HO, Kang JH, et al. (2022) Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution. PLoS ONE 17(10): e0275590. https://doi.org/10.1371/ journal.pone.0275590 Editor: Sudhindra R. Gadagkar, Midwestern University, UNITED STATES Received: December 20, 2021 Accepted: September 20, 2022 Published: October 5, 2022 Copyright: © 2022 Park et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All the data in this study were deposited in NCBI GenBank. The accession numbers of plastomes and rDNAs are listed in Tables 1 and 2, respectively. 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. Funding: This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9A5030733). The funders had no role in study design, data collection and analysis, PLOS ONE | https://doi.org/10.1371/journal.pone.0275590 October 5, 2022 1 / 19 PLOS ONE decision to publish, or preparation of the manuscript. Competing interests: The authors declare that there are no competing interests. Complete plastomes and nrDNAs of Euonymus Introduction Euonymus hamiltonianus Wall., a plant belonging to the Celastraceae family, is widely distributed from Northern India to Far East Asia (http://www.plantsoftheworldonline.org). E. hamiltonianus are valuable ornamental plants due to their beautiful shapes and colors. However, the morphological variations lead to ambiguous delimitation of this species. The uncertainty of species delimitation is not only the case for E. hamiltonianus but also for other closely related species. Fruit morphology of Euonymus was used to divide them into five sections, but molecular phylogeny of Euonymus was poorly supported in the previous study [1]. Although Celastraceae is a large family containing 96 genera and 1,350 species, few studies have been conducted for the genus Euonymus [2–4]. Universal DNA barcoding regions such as plastid matK, rbcL, and nuclear ribosomal internal transcribed spacer (nrITS) regions have been utilized to construct the phylogenies of Celastraceae [5–8]. Nevertheless, the intrageneric boundaries are still unclear due to the limitations of using a few short barcoding regions. To date, most studies in Euonymus have been conducted on individual species [9–12]. Even though an interspecies comparison of the Euonymus plastomes was recently conducted, no indepth analysis of one species with its relatives has been performed [3]. Therefore, to address the plastome diversity and evolution of E. hamiltonianus and its relatives, it is important to conduct in-depth comparative analysis using various resources. Plastomes have been widely employed for phylogenetic studies and DNA barcoding in various plants due to their uniparental inheritance (usually maternal inheritance) in most land plants [13]. Uniparental inheritance could reduce genetic diversity, but also provide simplicities in tracking ancestors (usually maternal) and obtaining genetic information with less heterogeneity [14]. In most land plants, plastid and mitochondrial genomes exhibit contrasting patterns in genomic features such as genome size, genome structure, gene content, and nucleotide substitution rates. Although plastomes have conserved genome structure and gene content, nucleotide substitution rates of plastid genes are generally faster than those of mitochondrial genes. In contrast, mitochondrial genomes display complicated genome structure and variable gene content, but substitution rates of mitochondrial genes are slower than those of plastid genes [15–18]. These genomic features have allowed plastomes to accumulate variations among species at a moderate rate [19,20]. Nuclear ribosomal DNAs such as 45S rDNA and 5S rDNA exist in a tandem repeat array of hundreds to thousands of copies and are genetically conserved due to concerted evolution [21,22]. However, the ITS regions, ITS1 between 18S and 5.8S and ITS2 between 5.8S and 26S, of the 45S rDNA are relatively variable at the sequence level. These characteristics of the nrDNA region provide useful genetic information for phylogenetic studies and DNA barcoding [23]. In addition (...truncated)