The complete chloroplast genome sequence of Viola labradorica (Violaceae)
Article information
Abstract
Viola labradorica is commonly known as Alpine violet, American dog violet, Dog violet, and Labrador violet. This species is distributed throughout eastern Canada and North America. In the present study, we sequenced the chloroplast genome of V. labradorica for the first time and performed a phylogenetic analysis of the genus Viola. The length of the chloroplast genome of V. labradorica was 158,751 bp. A large single-copy region (87,211 bp), a small single-copy region (17,344 bp), and two inverted repeat regions (27,098 bp each) were identified. A phylogenetic analysis was conducted using 77 protein-coding genes from the chloroplast genomes of 34 Viola. Salix koriyanagi (Salicaceae) was used as an outgroup. The genus Viola forms a monophyletic clade. Among the clades for the genus Viola, the sect. Viola formed a clade and was divided into two subclades: subsect. Viola and subsect. Rostratae. Within the subsect. Rostratae, V. labradorica was placed in a basal position. These results contribute to a clear identification of the phylogenetic position of V. labradorica in the subsect. Rostratae.
INTRODUCTION
Viola L. is the largest genus of Violaceae Batsch, and the genus Viola contains 658 species (Marcussen et al., 2022). Viola labradorica Schrank has several common names, such as Alpine violet, American dog violet, Dog violet, and Labrador violet. V. labradorica is distributed throughout eastern Canada and North America (GBIF Secretariat, 2022). According to Platt (1950), V. labradorica and V. walteri are morphologically similar in the Appalachians; they present stipular teeth, pubescent adaxial leaves, petioles, and peduncles. However, V. labradorica has small spar stipular teeth and scattered hairs on its upper leaves. Therefore, this species differs from V. walteri (Platt, 1950). The colors of the petals are lavender violet to violet, and the two lateral petals are sparsely bearded (Little and McKinney, 2015). Previous studies of this species included morphology, scanning electron microscope observations of the stigmas, and a flavonoid analysis (Platt, 1950; Ballard, 1992). Systematic studies of chloroplast DNA sequences were conducted by Shooner et al. (2015). However, V. labradorica has never been compared within the section or genus categories. Here, we report the chloroplast genome of V. labradorica for the first time and reconstruct the phylogeny of the genus Viola. These results will serve as a valuable resource for comprehensive taxonomic studies of the subsection Rostratae.
MATERIALS AND METHODS
Fresh leaves of V. labradorica were obtained from the greenhouse of Kangwon National University in Korea, and DNA was extracted. The seeds were purchased from Rareplants.eu (http://www.rareplants.de), and seed-germinating individuals were identified by referring to the original description (Schrank, 1818), lectotype specimen (K000327814 [digital image]), and a description by Little and McKinney (2015). A voucher specimen (KWNU 100770) was deposited into the Kangwon National University Herbarium. DNA was extracted using the DNA Plant Mini Kit (Qiagen Inc., Valencia, CA, USA), with sequencing done on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). We obtained paired-end reads with an average read length of 301 bp from 5,493,560 raw reads; de novo assembly was conducted using Geneious 7.1.9 (Biomatters Ltd., Auckland, New Zealand), and 628,897 contigs were aligned. The complete chloroplast sequence was annotated based on the online program GeSeq (Tillich et al., 2017) and was manually edited through a comparison with the caulescent plants Viola: V. acuminata, V. collina, and V. grypoceras (GenBank accession Nos. MW802528, OM177181, and OM055663, respectively). A circular genome map of V. labradorica was drawn using CPGView (Liu et al., 2023). The cis-splicing genes and the trans-splicing gene of rps12 were drawn using CPGView (Liu et al., 2023). For a phylogenetic analysis of V. labradorica, 77 protein-coding genes from 34 Viola species (Go and Yoo, 2023) and one outgroup (Salix koriyanagi; GenBank accession No. MK120982) were aligned using MAFFT v7.017 (Katoh et al., 2002). Phylogenetic analyses were conducted by means of the maximum likelihood method using RAxML v8.2.12 (Stamatakis, 2014) under the GTR + I model with 1,000 bootstrap replicates and with Bayesian inference using MrBayes 3.2.2 (Ronquist et al., 2012) on the CIPRES Science Gateway (http://www.phylo.org) (Miller et al., 2010). We used the intraspecific classification system of Marcussen et al. (2022).
RESULTS AND DISCUSSION
The complete chloroplast genome of V. labradorica (GenBank accession No. OR003906) has a total length of 158,751 bp (GC content: 36.1%). This genome exhibits a quadripartite structure, consisting of a large single-copy region with a length of 87,211 bp, a small single-copy region with a length of 17,344 bp, and two inverted repeats (IRs) of 27,098 bp each (Fig. 1). The chloroplast genome of V. labradorica contains 111 genes, with 77 proteincoding genes, 29 tRNA genes, four rRNA genes, and one pseudogene. In total, the IR regions contained the following 19 duplicated genes: ndhB, rpl2, rpl23, rps7, rps12, ycf1, ycf2, ycf15, rrn16, rrn23, rrn4.5, rrn5, trnA-UGC, trnICAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, and trnV-GAC (Table 1). In the chloroplast genome of V. labradorica, eight protein-coding genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, and rpoC1) and six tRNA genes (trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) contained one intron, and three genes (clpP, rps12, and ycf3) contained two introns. The protein-coding genes with introns had the structures of 13 genes, including 12 cis-splicing genes (atpF, rpoC1, ycf3, clpP, petB, petD, rpl16, and ndhA, with reverse duplications for rpl2 and ndhB), and one trans-splicing gene (rps12).
Chloroplast genomic map of the V. labradorica chloroplast genome. This circular map was drawn using CPGView (http://www.1kmpg.cn/cpgview/) and contains seven tracks. From the center, the first track shows dispersed repeats, with direct repeats in red and palindromic repeats in green. The second track presents long tandem repeats as short blue bars. Short tandem repeats or microsatellite sequences are depicted in the third track as short bars in various colors. The fourth track delineates regions in different colors, showing small single-copy (light blue), inverted repeats (grey), and large single-copy (light green) regions. The GC content of the genome is depicted in the fifth track. Genes are shown in the sixth track, with their corresponding functional classifications indicated. The transcription directions for the inner and outer genes are clockwise and counterclockwise, respectively. The outermost track shows quadripartite structures in different line patterns, with small single-copy (thick dotted line), inverted repeats (solid lines), and large single-copy (dotted line) cases shown. Functional gene classifications are displayed in the bottom left corner.
Genes Contained in the Chloroplast Genome of Viola labradorica (111 gene species)
The gene order and gene direction of V. labradorica were very similar to another Viola, allowing us to confirm that the chloroplast genome of Viola is highly conserved, as found in previous reports (Cheon et al., 2019; Cao et al., 2022). We conducted a phylogenetic analysis, with the results showing that Viola strongly indicated monophyly (BS = 100). A phylogenetic tree was distinguished for each section according to the classification system (Marcussen et al., 2022), except for the section Chamaemelanium, for which conflicting opinions exist in the literature (Becker, 1925; Clausen, 1929, 1964). Specifically, the section Viola formed a monophyletic clade and then was divided into two well-supported subclades, denoted as subsection Viola and subsection Rostratae (Fig. 2). In these two subsections, V. labradorica belongs to the subsection Rostratae and forms a sister group with five other species. The subsection Rostratae subclade supports the classification of Viola (Marcussen et al., 2022). Here, we report the first complete chloroplast genome sequence of V. labradorica and a phylogenetic analysis of Viola. Our results will assist in the effort to construct the comprehensive classification system of the Viola subsection Rostratae.
Maximum likelihood tree based on 77 protein-coding genes from the complete chloroplast genomes of 34 Viola and one Salix as an outgroup (MK120982). The position of V. labradorica is highlighted in bold. Bootstrap (BP) values and Bayesian Posterior probabilities (PP) values are displayed above the branch nodes, with asterisks indicating node BS = 100% and PP = 1.00.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MIST) (NRF-2022R1F1A1067342). The authors are grateful to anonymous reviewers who provided invaluable comments on the early version of the manuscript.
Notes
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.