Europe PMC

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): Comparative analysis with congeneric species.

Author information

Affiliations

  • 1. Department of Biological Sciences, Kangwon National University, Chuncheon, South Korea.
      Authors
    • Kim KA 1
    (1 author)
  • 2. Department of Biological Science, Sangji University, Wonju, South Korea.
      Authors
    • Cheon KS 2
    (1 author)

ORCIDs linked to this article

Plos one, 18 Mar 2021, 16(3):e0248788
https://doi.org/10.1371/journal.pone.0248788 PMID: 33735287 PMCID: PMC7971521

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
Free full text in Europe PMC

Abstract 

Adenophora racemosa, belonging to the Campanulaceae, is an important species because it is endemic to Korea. The goal of this study was to assemble and annotate the chloroplast genome of A. racemosa and compare it with published chloroplast genomes of congeneric species. The chloroplast genome was reconstructed using de novo assembly of paired-end reads generated by the Illumina MiSeq platform. The chloroplast genome size of A. racemosa was 169,344 bp. In total, 112 unique genes (78 protein-coding genes, 30 tRNAs, and 4 rRNAs) were identified. A Maximum likelihood (ML) tree based on 76 protein-coding genes divided the five Adenophora species into two clades, showing that A. racemosa is more closely related to Adenophora stricta than to Adenophora divaricata. The gene order and contents of the LSC region of A. racemosa were identical to those of A. divaricata and A. stricta, but the structure of the SSC and IRs was unique due to IR contraction. Nucleotide diversity (Pi) >0.05 was found in eleven regions among the three Adenophora species not included in sect. Remotiflorae and in six regions between two species (A. racemosa and A. stricta).

Free full text 

Logo of plosoneLink to Publisher's site
PLoS One. 2021; 16(3): e0248788.
Published online 2021 Mar 18. https://doi.org/10.1371/journal.pone.0248788
PMCID: PMC7971521
PMID: 33735287

Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): Comparative analysis with congeneric species

Kyung-Ah Kim, Data curation, Funding acquisition, Investigation, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing1,2 and Kyeong-Sik Cheon, Conceptualization, Data curation, Formal analysis, Investigation, Resources, Software, Supervision, Visualization, Writing – original draft, Writing – review & editing3,*
Tzen-Yuh Chiang, Editor
Author information Article notes Copyright and License information Disclaimer

Associated Data

Supplementary Materials
Data Availability Statement

Abstract

Adenophora racemosa, belonging to the Campanulaceae, is an important species because it is endemic to Korea. The goal of this study was to assemble and annotate the chloroplast genome of A. racemosa and compare it with published chloroplast genomes of congeneric species. The chloroplast genome was reconstructed using de novo assembly of paired-end reads generated by the Illumina MiSeq platform. The chloroplast genome size of A. racemosa was 169,344 bp. In total, 112 unique genes (78 protein-coding genes, 30 tRNAs, and 4 rRNAs) were identified. A Maximum likelihood (ML) tree based on 76 protein-coding genes divided the five Adenophora species into two clades, showing that A. racemosa is more closely related to Adenophora stricta than to Adenophora divaricata. The gene order and contents of the LSC region of A. racemosa were identical to those of A. divaricata and A. stricta, but the structure of the SSC and IRs was unique due to IR contraction. Nucleotide diversity (Pi) >0.05 was found in eleven regions among the three Adenophora species not included in sect. Remotiflorae and in six regions between two species (A. racemosa and A. stricta).

Introduction

Among the angiosperms, Campanulaceae are known to have the chloroplast genomes with the most structural changes, along with Geraniaceae and Fabaceae [1–11]. Among the Campanulaceae, Adenophora species in particular have very different chloroplast genome structures due to many rearrangements [12,13]. Although many studies have been carried out on the genus Adenophora, its accurate phylogenetic relationships and taxonomic position are not clear [12–19]. Therefore, it is expected that the difference in chloroplast genome structure among Adenophora species may be used as important information to solve the phylogenetic relationships and taxonomic positions of various species that are currently unclear.

The genus Adenophora, which belongs to Campanulaceae, is a perennial herbaceous plant genus with ca. 50–100 species that are distributed in temperate regions in Eurasia [12,13]. This genus is commonly called “Adenophora Radix” and is an important plant resource used as an herbal medicine [20,21].

Among Adenophora species, Adenophora racemosa J. Lee & S. Lee, discussed in this study, is endemic to Korea and was first described by Lee and Lee [22] after collection from Mt. Odae National Park in Korea. This species is considered closely related to Adenophora divaricata Franch. & Sav., Adenophora tyosenensis Nakai ex T.H. Chung and Adenophora pulcher Kitam. owing to morphological characteristics such as four-leaf verticillation, regular teeth on the leaf margins, and a pale green colour of the adaxial surface of the leaf basin. However, A. racemosa is distinguished from A. divaricata in that the inflorescence is a panicle, and it is distinguished from A. tyosenensis and A. pulcher by an urceolate corolla reminiscent of that of lily of the valley (Convallaria keiskei Miq.) [22].

In relatively recent molecular phylogenetic studies, however, the phylogenetic relationships and taxonomic position of A. racemosa were not clear because it exhibited unresolved paraphyly with related taxa [13–15]. Furthermore, the phylogenetic relationships and taxonomic position of many Adenophora species are currently ambiguous.

In this study, therefore, we reported the complete chloroplast genome sequence of A. racemosa, an endemic of Korea, and compared the sequence to those of four published congeneric chloroplast genomes, i.e., those from Adenophora divaricata, Adenophora erecta S.T. Lee, J.K. Lee & S.T. Kim, Adenophora remotiflora (Siebold & Zucc.) Miq., and Adenophora stricta Miq. We found that A. racemosa has a previously unreported unique chloroplast genome structure caused by IR contraction, important evidence supporting its recognition as an independent species. We believe that the results of this study can be used as important information for obtaining new insights into the evolutionary history of the genus Adenophora. Additionally, the marker information presented in this study is considered to be very useful information for further studies aiming to determine the exact phylogenetic relationships of Adenophora species.

Materials and methods

Sample collection, DNA extraction and chloroplast genome sequencing

Since A. racemosa is not endangered and protected species, plant materials were collected without permission. The plant material of A. racemosa was collected from Mt. Gaya (35° 49’ 21.5” N, 128° 07’ 18.3” E) in Gyeongsangnam-do Province of South Korea, and a voucher specimen (voucher no. KWNU93473) was deposited in Kangwon National University Herbarium (KWNU).

Total DNA was extracted from approximately 100 mg of fresh leaves using a DNA Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). Genomic DNA was used for sequencing on the Illumina MiSeq (Illumina Inc., San Diego, CA, USA) platform.

Assembly and genome mapping

Chloroplast genome assembly was conducted by the de novo assembly protocol [23] via the Phyzen bioinformatics pipeline (http://phyzen.com). The DNA of A. divaricata was sequenced to produce 8,361,496 raw reads with a length of 301 bp. Low-quality sequences (Phred score < 20) were trimmed using CLC Genomics Workbench (version 6.04; CLC Inc., Arhus, Denmark). After trimming, the library for A. racemosa included 6,991,585 reads. Then, de novo assembly was implemented using the CLC Genome Assembler (http://www.clcbio.com/products/clc-assembly-cell). A total of 107,248 reads were aligned and selected form chloroplast contigs using the nucmer tool in MUMmer [24]. The draft genome contigs were merged into a single contig by joining overlapping terminal sequences of each contig. Additionally, the chloroplast genome coverage was estimated using CLC Genomics Workbench (version 6.04; CLC Inc.).

The protein-coding genes, transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) in the chloroplast genome were predicted and annotated using Dual Organellar GenoMe Annotator (DOGMA) with the default parameters [25] and manually edited by comparison with the published chloroplast genome sequences of Campanulaceae. tRNAs were confirmed using tRNAscan-SE [26]. A circular chloroplast genome map was drawn using the OGDRAW program [27].

Phylogenetic analyses

Two genes (rpl23 and clpP) among the total 78 PCGs were excluded from the phylogenetic analysis data matrix, since most of these gene regions were deleted, and only a few regions existed in the chloroplast genomes of Adenophora species. A total of 76 protein-coding genes from 13 species (see S1 Table for accession numbers) were compiled into a single file of 83,906 bp (S2 Table) and aligned with MAFFT [28]. Twelve Campanulaceae s. str. species were selected as the ingroups, and one species (Lobelia chinensis Lour.) was chosen as the outgroup. Maximum likelihood (ML) analyses were performed using RAxML v7.4.2 with 1000 bootstrap replicates and the GTR+I model [29]. Bayesian inference (ngen = 1,000,000, samplefreq = 200, and burninfrac = 0.25) was carried out using MrBayes v3.0b3 [30], and the best substitution model (GTR+I) was determined by the Akaike information criterion (AIC) in jModelTest version 2.1.10 [31].

Comparative analysis of genome structure

mVISTA was used to compare similarities among the five Adenophora species using shuffle-LAGAN mode [32]. The annotated A. racemosa chloroplast genome was used as a reference. Additionally, the genome structures of the five Adenophora species were compared using MAUVE [33].

Nucleotide diversity and Ka/Ks ratio analysis

To assess complete nucleotide diversity (Pi) among the five Adenophora chloroplast genomes, the complete chloroplast genome sequences were aligned using the MAFFT [28] aligner tool and manually adjusted with BioEdit [34]. We then performed sliding window analysis to calculate the nucleotide variability (Pi) values using DnaSP 6 [35] with a window length of 600 bp and a step size of 200 bp [36]. The 75 protein-coding genes were extracted and aligned separately using MAFFT [28] to estimate the synonymous (Ks) and nonsynonymous (Ka) substitution rates. The Ka/Ks for each gene was estimated in DnaSP [35].

Results

Feature of the Adenophora chloroplast genomes

The chloroplast genome of Adenophora racemosa (GenBank accession no. MT012303) has been submitted to GenBank of the National Center for Biotechnology Information (NCBI). The complete chloroplast genome of A. racemosa is 169,344 bp in length, with an average mean coverage depth of 159-fold (S1 Fig). It exhibits a typical quadripartite architecture, with an LSC (large single copy), an SSC (small single copy) and a pair of IRs (inverted repeats) of 122,518 bp, 29,588 bp and 8619 bp, respectively (Fig 1; Table 1).

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g001.jpg
Gene map of the Adenophora racemosa chloroplast genome.

Genes inside the circle are transcribed clockwise, and genes outside are transcribed counterclockwise. The dark grey inner circle corresponds to the GC content, and the light-grey circle corresponds to the AT content.

Table 1

Comparison of chloroplast genome features of five Adenophora species.
FeatureA. racemosaA. strictaA. divaricataA. erectaA. remotiflora
GenBank accession No.MT012303KX462131KX462129KX462130KP889213
Genome size169,344159,759176,331173,324171,724
Large single copy (LSC)122,518112,321113,353105,861105,555
Small single copy (SSC)29,58827,238864811,26711,295
Inverted repeat (IR)861910,10027,16528,09827,437
Number of unique protein-coding genes7878787878
Number of tRNAs3030303030
Number of rRNAs44444
G+C (%)
    Large single copy (LSC)36.137.137.137.537.5
    Small single copy (SSC)35.635.433.035.034.9
    Inverted repeat (IR)52.351.042.241.842.0
    Total genome37.738.538.538.738.8

The total length of the chloroplast genomes of five Adenophora species, i.e., A. racemosa and four species analysed in a previous study (A. divaricata, A. erecta, A. remotiflora, and A. stricta), ranged from 159,759 to 176,331 bp (Table 1). The length of the LSC regions in the five chloroplast genomes anged from 105,555 to 122,518 bp, and the SSC and IR were 8648 to 29,588 bp and 8619 to 28,098 bp in length, respectively. In the chloroplast genome of A. racemosa, very long sequences were inserted into two IGSs (intergenic spacers) of psbB-rpl20 and ψpsbJ-ycf3, resulting in an extended LSC region (Fig 2; Table 1).

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g002.jpg
Visualization of alignment of five Adenophora chloroplast genomes using A. racemosa as a reference.

The vertical scale indicates the percent identity, ranging from 50% to 100%. Coding regions, RNAs, and non-coding regions are marked in purple, sky blue, and red, respectively.

Additionally, each of the five chloroplast genomes contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs (tRNA), and 4 ribosomal RNAs (rRNA). The G+C contents in the five chloroplast genomes ranged from 37.7 to 38.8%.

Cheon et al. [12] reported that three genes (rpl23, infA, and clpP) in Adenophora chloroplast genomes were pseudogenized, two tRNAs (trnI-CAU and trnV-GAC) and one gene (psbJ) had one additional copy and two additional copies, respectively, and part of three genes (psbB, ycf3, and rrn23) was duplicated. The A. racemosa chloroplast genome analysed in this study had the same characteristics. The 5’ exon of the rps12 gene in the A. racemosa chloroplast genome was located in the SSC region due to IR contraction, making it identical to the chloroplast genome of A. stricta. Meanwhile, trnQ-UUG in the chloroplast genome of A. racemosa had an additional copy in the LSC region.

Phylogenetic analyses of Campanulaceae

The ML (maximum likelihood) tree formed the following two clades: platycodonoids and campanuloids. The campanuloids formed two subclades: the Campanula s. str. clade and Rapunculus clade. All nodes in the ML tree were strongly supported, with 100% BP (bootstrap) and 1.00 PP (Bayesian posterior probability) values (Fig 3).

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g003.jpg
The ML tree based on 76 protein coding genes from 13 chloroplast genomes.

The 100% bootstrap (BP) value and 1.00 Posterior probability (PP) value are marked with *.

In the Campanula s. str. clade, Trachelium caeruleum L. formed a basal branch, and Campanula zangezura (Lipsky) Kolak. et Serdjukova was sister to Campanula punctata Lam. and the Campanula takesimana Nakai clade. Within the Rapunculus clade, Hanabusaya asiatica (Nakai) Nakai was the earliest-diverging lineage and was sister to all other species in the clade. Additionally, five Adenophora species were divided into two subclades: a clade containing the sect. Remotiflorae species (A. remotiflora and A. erecta) and a clade containing the remaining three Adenophora species. Furthermore, A. divaricata was sister to the A. stricta and A. racemosa clade.

The structural changes of Adenophora chloroplast genomes

The gene order and contents of the LSC region of A. racemosa were identical to those of A. divaricata and A. stricta. In the results of previous study [12], the LSC of A. divaricata and A. stricta were confirmed that inversion of two large gene blocks (trnT-UGU-ndhC, and psbJ-ψpsbJ) were occurred when compared to LSC of sect. Remotiflorae speices, A. erecta and A. remotiflora. Cheon et al [12] also reported that the gene order and contents of the IR and SSC in two sect. Remotiflorae species and A. divaricata were the same, but the IR of A. stricta was identified as being much shorter than that of other Adenophora species due to IR contraction. Meanwhile, the IR of A. racemosa was identified as the shortest among the five studied Adenophora species because IR contraction, including partial contraction of psbB, trnN-GUU, and trnR-AGC, contraction further occurred in the A. racemosa chloroplast genome than in the A. stricta chloroplast genome (Fig 4; Table 1).

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g004.jpg
IR contraction in the Adenophora racemosa chloroplast genome.

Nucleotide diversity and Ka/Ks ratio

The average nucleotide diversity (Pi) among the five Adenophora chloroplast genomes and all chloroplast genomes except those of the two sect. Remotiflorae species were estimated to be 0.087 and 0.010, respectively. Additionally, the Pi between the two chloroplast genomes of A. racemosa and A. stricta, the species with the closest phylogenetic relationship with A. racemosa, was estimated to be 0.009, ranging from 0 to 0.383 (Fig 5). In the five chloroplast genomes, seven regions (rpoA-petD, psbB-rpl20, ycf3-ropB, ndhD-trnI, ndhF-rpl32, and two ycf1 regions) showed high values of Pi (> 0.05). In the results for the groups of three species and two species, 11 (rpoA-petD, trnL-rpl20, psbJ-ndhC, trnT-psbJ, trnC-petN, psbJ-ycf3, ycf3-rpoB, rpoC2, ndhF-rpl32, and two ycf1 regions) and seven regions (trnL-rpl20, trnT-psbJ, trnC-petN, psbJ-ycf3, ndhF-rpl32, and ycf1) showed a high value of Pi (> 0.05), respectively.

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g005.jpg
Sliding window analysis of Adenophora chloroplast genomes.

A; Pi values of five Adenophora species, B; Pi values of three Adenophora species, excluding the two sect. Remotiflorae species, C; Pi values of A. stricta and A. racemosa.

The Ka (non-synonymous)/Ks (synonymous) ratio was calculated for the 75 protein-coding genes of three Adenophora species, namely, A. divaricata, A. stricta, and A. racemosa (Fig 6; S3 Table). Comparison between A. divaricata and A. stricta revealed high values of 1 or more in seven gene regions (matK, rpoB, rpoC1, rpoC2, ycf2, ndhF, and ycf1), and that between A. divaricata and A. racemosa showed that 5 gene regions (matK, rpoB, rpoC1, rpoC2, and ycf1) had a value of 1 or more. Furthermore, only one region showed a high value of more than 1 between A. stricta and A. racemosa, which showed the closest phylogenetic relationship.

An external file that holds a picture, illustration, etc. Object name is pone.0248788.g006.jpg
The Ka/Ks ratio of Adenophora chloroplast genomes for individual genes.

Discussion

Chloroplast genome organization in Adenophora

The lengths of the LSC of A. divaricata, A. stricta, and A. racemosa were longer than those of the two sect. Remotiflorae species (A. erecta and A. remotiflora). Additionally, A. racemosa had the longest LSC among the five Adenophora species. The difference in the lengths of LSC regions between sect. Remotiflora and the remaining three species is judged to be due to sequence mutations of the inversion end point of two large gene blocks. Also, we confirmed that the difference lengths of IRs and SSC regions among the three Adenophora species except two sect. Remotiflorae species were attributed to IR contraction (Fig 4).

Adenophora species are known to be difficult to distinguish because of their overlapping morphological characters [13]. In particular, A. racemosa, discussed in this study, has morphological characteristics that are very similar to those of A. divaricata, which makes it very difficult to distinguish the two species. Therefore, the difference in chloroplast genome structure between the two species identified in this study is considered to be very useful information for distinguishing between the two species.

Suggestions for classification system of genus Adenophora

The ML tree in this study showed that Adenophora forms a monophyletic clade divided into two subclades, one containing the two sect. Remotiflorae species and another containing the remaining three species. In the clade containing the remaining three species, A. racemosa has a closer relationship with A. stricta than with A. divaricata. We think that these relationships have important implications because they are different from the relationships in the recent classification system.

The classification system of Adenophora has been established by many studies [37–44], and the species in this genus are divided into sections mainly by leaf arrangement and disk shape. Among the five Adenophora species discussed in this study, accordingly, it is common to treat A. erecta and A. remotiflora as belonging to sect. Remotiflorae, A. divaricata and A. racemosa as belonging to sect. Platyphyllae, and A. stricta as belonging to sect. Gmelinianae. However, the two species belonging to sect. Platyphyllae exhibited paraphyly, and these phylogenetic relationships were different from the relationships in the current classification system. Of course, this study was carried out with only a few taxa, which makes it difficult to discuss the complete phylogenetic relationships of Adenophora. However, paraphyletic relationships have been confirmed in this study, and we think that in-depth studies are necessary to delimit the sections of Adenophora, except sect. Remotiflorae.

Evolution of protein-coding genes in Adenophora species

The Ka/Ks ratio may indicate which selection pressure is acting on a particular PCGs. Ka/Ks > 1 and Ka/Ks < 1 indicate that the gene is affected by positive selection and negative selection, respectively, and a value of 0 indicates neutral selection [36,45].

The Ka/Ks ratio of Adenophora species was calculated for the first time in this study. As a result, between A. divaricata and A. stricta, there were two more positively selected genes (ycf2 and ndhF) than between A. divaricata and A. racemosa. Additionally, between A. racemosa and A. stricta, 62 and 12 genes were calculated to be under neutral selection and negative selection, respectively, and only 1 gene (ycf1) was identified as being under positive selection (Fig 4; S3 Table).

In the Caesalpinioideae of Leguminosae, known as one of the groups with the most structural changes in the chloroplast genome, four genes (ndhD, ycf1, infA and rpl23) and three genes (psbH, clpP, and rps16) were identified as being under positive selection [36,46], respectively. In the Convolvulaceae and Araceae, three genes (accD, cemA, and ycf2) and only one gene (rps12) were positively selected, respectively. Moreover, ycf1 was identified as the gene with the most accelerated mutation rates among the species in this study, and ycf1 was found to have the highest sequence mutation rates among the protein-coding genes in a previous study including sect. Remotiflorae species [12].

Useful molecular marker information for Adenophora phylogenetics

We think that marker information that can best describe the phylogenetic tendencies of the remaining sections (except sect. Remotiflorae) is most needed at this point. In a previous study [13], because sect. Remotiflorae formed a monophyletic group, there was no issue in classifying it as a section.

The results of this study using the sliding window method among the three Adenophora species (Fig 3B) showed that the nucleotide diversity in eleven regions, including three gene regions and eight IGS (intergenic spacer) regions, had high calculated values (> 0.05). We think that six regions (Fig 3C), namely, trnL-rpl20, trnT-psbJ, trnC-petN, psbJ-ycf3, ndhF-rpl32, and ycf1, among the eleven regions have particularly high phylogenetic resolution because their nucleotide diversity values were high in two species that showed a close phylogenetic relationship in the ML tree (Fig 2).

Conclusion

In this study, we assembled the chloroplast genome of A. racemosa, which had a total length of 169,344 bp. The IR of A. racemosa was identified as the shortest among the Adenophora species because of IR contraction. A. racemosa is not easy to distinguish because its morphological characteristics are very similar to those of A. divaricata. Therefore, the different structures of the chloroplast genomes are considered to be very useful information for distinguishing between the two species. The ML tree results showed that A. racemosa is more closely related to A. stricta than to A. divaricata, indicating a clear problem with the current classification system for Adenophora. Therefore, we think that further in-depth phylogenetic studies of Adenophora are needed, and the molecular marker information presented in this study is expected to be very useful for such studies.

Supporting information

S1 Fig

The mapped read depth of A. racemosa chloroplast genome.

(TIFF)

S1 Table

The GenBank accession numbers of all the 13 chloroplast genomes used for phylogenetic analysis.

(DOCX)

S2 Table

The length and aligned length of each gene used for phylogenetic analysis.

(XLSX)

S3 Table

Ka/Ks ratio of three Adenophora species, A. divaricata, A, stricta, and A. racemosa.

(XLSX)

Funding Statement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1A6A3A11029236).

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

References

1. Cosner ME, Raubeson LA, Jansen RK. Chloroplast DNA rearrangements in Campanulaceae: phylogenetic utility of highly rearranged genomes. BMC Evolutionary Biology. 2004; 4:27. 10.1186/1471-2148-4-27 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
2. Cosner ME, Jansen RK, Palmer JD, Downie SE. The highly rearranged chloroplast genome of Trachelium caeruleum (Campanulaceae): multiple inversions, inverted repeat expansion and contraction, transposition, insertions/deletions, and several repeat families. Current Genetics. 1997; 31:419–429. 10.1007/s002940050225 [Abstract] [CrossRef] [Google Scholar]
3. Cheon KS, Yoo KO. Complete chloroplast genome sequence of Hanabusaya asiatica (Campanulaceae), an endemic genus to Korea. Mitochondrial DNA Part A. 2016; 27: 1629–1631. 10.3109/19401736.2014.958702 [Abstract] [CrossRef] [Google Scholar]
4. Cheon KS, Kim KA, Jang SK, Yoo KO. Complete chloroplast genome sequence of Campanula takesimana (Campanulaceae), an endemic to Korea. Mitochondrial DNA Part A. 2016; 27:2169–2171. 10.3109/19401736.2014.982610 [Abstract] [CrossRef] [Google Scholar]
5. Guisinger MM, Kuehl JNV, Boore JL, Jansen RK. Genome-wide analyses of Geraniaceae plastid DNA reveal unprecedented patterns of increased nucleotide substitutions. PNAS. 2008; 105:18424–18429. 10.1073/pnas.0806759105 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
6. Haberle RC, Fourcade HM, Boore JL, Jansen RK. Extensive rearrangements in the chloroplast genome of Trachelium caeruleum are associated with repeats and tRNA genes. Journal of Molecular Evolution. 2008; 66:350–361. 10.1007/s00239-008-9086-4 [Abstract] [CrossRef] [Google Scholar]
7. Hong CP, Park J, Lee Y, Lee M, Park SG, Uhm Y, et al.. accD nuclear transfer of Plantycodon grandiflorum and the plastid of early Campanulaceae. BMC Genomics, 2017; 18:607. 10.1186/s12864-017-4014-x [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
8. Marcussen T, Meseguer AS. Species-level phylogeny, fruit evolution and diversification history of Geranium (Geraniaceae). Molecular Phylogenetics and Evolution. 2017; 110:134–149. 10.1016/j.ympev.2017.03.012 [Abstract] [CrossRef] [Google Scholar]
9. Schwarz EN, Ruhlman TA, Sabir JSM, Hajrah NH, Alharbi NS, Al-Malki AL, et al.. Plastid genome sequences of legumes reveal parallel inversions and multiple losses of rps16 in papilionoids. Journal of Systematics and Evolution. 2015; 53:458–468. 10.1111/jse.12179 [CrossRef] [Google Scholar]
10. Weng ML, Blazier JC, Govindu M, Jansen RK. Reconstruction of the Ancestral Plastid Genome in Geraniaceae Reveals a Correlation between Genome Rearrangements, Repeats, and Nucleotide Substitution Rates. Molecular Biology and Evolution. 2014; 31:645–659. 10.1093/molbev/mst257 [Abstract] [CrossRef] [Google Scholar]
11. Yoo KO, Cheon KS, Kim KA. Complete chloroplast genome sequence of Campanula punctata Lam. (Campanulaceae). Mitochondrial DNA Part B. 2016; 1:192–193. 10.1080/23802359.2016.1149791 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
12. Cheon KS, Kim KA, Yoo KO. The complete chloroplast genome sequences of three Adenophora species and comparative analysis with Campanuloids species (Campanulaceae). Plos ONE. 2017; 12: e0183652. 10.1371/journal.pone.0183652 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
13. Kim KA. Phylogenetic study of the genus Adenophora (Campanulaceae). Ph.D. Thesis, Kangwon National University. 2016.
14. Kim KA, Yoo KO. Phylogenetic relationships of Korean Campanulaceae based on PCR-RFLP and ITS sequences. Korean Journal of Plant Taxonomy. 2011; 41:119–129. 10.11110/kjpt.2011.14.2.119 [CrossRef] [Google Scholar]
15. Kim KA, Yoo KO. Phylogenetic relationships of Korean Campanulaceae based on chloroplast DNA sequences. Korean Journal of Plant Taxonomy. 2012; 42:282–293. 10.11110/kjpt.20125.42.4.282 [CrossRef] [Google Scholar]
16. Kim KA, Cheon KS, Jang SK, Yoo KO. Complete chloroplast genome sequence of Adenophora remotiflora (Campanulaceae). Mitochondrial DNA Part A. 2016; 27:2963–2964. 10.3109/19401736.2015.1060461 [Abstract] [CrossRef] [Google Scholar]
17. Eddie WMM, Shulkina T, Gaskin J, Haberle RC, Jansen RK. Phylogeny of Campanulaceae s. str. inferred from ITS sequences of nuclear ribosomal DNA. Annals of the Missouri Botanical Garden. 2003; 90:554–575. 10.2307/3298542 [CrossRef] [Google Scholar]
18. Tu P, Niu Y, Xu L, Xu G. Microscopic identification of the powder of roots of genus Adenophora. I. The roots of sect. Basiphyllae and sect. Pachydiscus. China journal of Chinese Materia Medica. 1996; 21:581–585. [Abstract] [Google Scholar]
19. Tu P, Niu Y, Xu L, Xu G. Microscopic identification of the powder of roots of genus Adenophora: II. The roots of sect. Remotiflorae and sect. Adenophora. China journal of Chinese Materia Medica. 1997; 22:67–72. [Abstract] [Google Scholar]
20. Cooperation teaching materials compilation committee of oriental medicine college in Korea. Herbal medicine. Seoul: Younglimsa Press; 2005. 10.1097/01.md.0000172299.72364.95 [CrossRef] [Google Scholar]
21. Ji YJ, Moon BC, Lee AY, Chun JM, Choo BK, Kim HK. Molecular phylogenetic position of Adenophora racemosa, an endemic species in Korea. Korean Journal of Crop Science. 2010; 18:379–388. [Google Scholar]
22. Lee J, Lee S. Adenophora racemosa (Campanulaceae), a new species from Korea. Korean Journal of Plant Taxonomy. 1990; 20:121–126. 10.11110/kjpt.1990.20.2.121 [CrossRef] [Google Scholar]
23. Cho KS, Yun BK, Yoon YH, Hong SY, Mekapogu M, Kim KH, et al.. Complete chloroplast genome sequence of tartary buckwheat (Fagopyrum tataricum) and comparative analysis with common buckwheat (F. esculentum). Plos ONE. 2015; 10:e0125332. 10.1371/journal.pone.0125332 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
24. Delcher AL, Salzberg SL, Phillippy AM. Using MUMmer to identify similar regions in large sequence sets. Current Protocols in Bioinformatics. 2003; 10:3. 10.1002/0471250953.bi1003s00 [Abstract] [CrossRef] [Google Scholar]
25. Wyman SK, Jansen RK, Boore JL. Automatic annotation of organellar genomes with DOGMA. Bioinformatics. 2004; 20:3252–3255. 10.1093/bioinformatics/bth352 [Abstract] [CrossRef] [Google Scholar]
26. Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Research. 2005; 33:W686–W689. 10.1093/nar/gki366 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
27. Lohse M, Drechsel O, Bock R. OrganellarGenomeDRAW (OGDRAW): A tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Current Genetics. 2007; 52:267–274. 10.1007/s00294-007-0161-y [Abstract] [CrossRef] [Google Scholar]
28. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research. 2002; 30:3059–3066. 10.1093/nar/gkf436 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
29. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006; 22:2688–2690. 10.1093/bioinformatics/btl446 [Abstract] [CrossRef] [Google Scholar]
30. Huelsenbeck JP, Ronquist R. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics. 2001; 17:754–755. 10.1093/bioinformatics/17.8.754 [Abstract] [CrossRef] [Google Scholar]
31. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods. 2012; 9:772. 10.1038/nmeth.2109 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
32. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Research. 2004; 32:W273–W279. 10.1093/nar/gkh458 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
33. Darling ACE, Mau B, Blattner FR, Perna NT. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Research. 2004; 14:1394–1403. 10.1101/gr.2289704 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
34. Hall TH. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleric Acids Symposium Series. 1999; 41:95–98. 10.14601/phytopathol_mediterr-14998u1.29 [CrossRef] [Google Scholar]
35. Rozas J. Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al.. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution. 2017; 34:3299–3302. 10.1093/molbev/msx248 [Abstract] [CrossRef] [Google Scholar]
36. de Souza UJB, Nunes R, Targueta CP, Diniz-Filho JA, Telles MPC. The complete chloroplast genome of Stryphnodendron adstringens (Leguminosae–Caesalpihioideae): comparative analysis with related Mimosoid species. Scientific reports. 2019; 9:14206. 10.1038/s41598-019-50620-3 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]
37. Korshinsky S. Untersuchungen uber die Russischen Adenophora-Arten. Saint Petersburg: Mémoires de l'Academie Imperiale des Sciences de Saint Petersbourg; 1894. [Google Scholar]
38. Fedorov AA. Adenophora. In: Shishkia BK editor. Flora of the U.S.S.R. vol 24. Moskva: Academii Nauk SSSR; 1957. pp. 246–267. [Google Scholar]
39. Hong DY. Adenophora Fisch. In: Lu a, Chen S. editors. Flora reipublicae popularis sinicae vol 73. Beijing: Science Press; 1983. pp. 92–139. [Google Scholar]
40. Fu CX, Liu MY. Study on the taxonomy of Adenophora Fischer in Heilongjiang Province. Natural Science Journal of Harbin Normal University. 1986; 2:41–52. [Google Scholar]
41. Okazaki J. Adenophora Fisch. In: Iwatsuki K, Yamazaki T, Boufford DE, Ohba H. editors. Flora of Japan. 3a. Tokyo: Kodansha Ltd.; 1993. pp. 406–401. [Google Scholar]
42. Lee JK, Lee ST. A taxonomic study of the genus Adenophora in Korea. J. Nat. Sci. Sungkyunkwan Univ. 1994; 45:15–34 (1994). [Google Scholar]
43. Yoo, K. O. Taxonomic studies on the Korean Campanulaceae. PhD Thesis. Kangwon National University. 1995.
44. Tu P. E., Chen H. B., Xu G. J. & Xu L. S. Classification and evolution of the genus Adenophora Fischer in Cheina. Acta Botanica Boreali-Occidentalia Sinica. 1998; 18: 613–621. [Google Scholar]
45. Nei M. & Kumar S. Molecular evolution and phylogenetics. Oxford university press; 2000. [Google Scholar]
46. Liu W. et al.. Complete chloroplast genome of Cercis chiniana (Fabaceae) with structural and genetic comparison to six species in Caesalpinioideae. International Journal of Molecular Sciences. 2018; 19:1286. [Europe PMC free article] [Abstract] [Google Scholar]
2021; 16(3): e0248788.
Published online 2021 Mar 18. 10.1371/journal.pone.0248788.r001

Decision Letter 0

Tzen-Yuh Chiang, Academic Editor
Copyright and License information Disclaimer

3 Feb 2021

PONE-D-21-00944

Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): comparative analysis with congeneric species

PLOS ONE

Dear Dr.Cheon,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at gro.solp@enosolp. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dr. Cheon et al’s MS entitled “Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): comparative analysis with congeneric species” was to assemble and annotate the chloroplast genome sequence of Adenophora racemosa, an endemic of Korea, and compare the sequence with four published chloroplast genomes of congeneric species. The MS is basically written clearly. The results are clearly presented and the discussions are basically reasonable and sound, but there are some issues need to be clarified. In addition, I do not see any significant novelties of the MS in the data analysis approach and conclusion found in this MS.

1. The genus Adenophora contains many species. Why did you select only five species from this MS for analysis? What are the selection criteria?

2. Please also use the regions with high values of Pi (> 0.05) for phylogenetic analysis, and compare with the results of the 76 protein-coding genes.

3. L21-L22: “genome” should be changed to “chloroplast genome”.

4. L22-L33: “Adenophora racemosa” should be changed to “A. racemosa”.

5. L102： In this study, 78 protein-coding genes were identified, but why 76 protein-coding genes were used for phylogenetic analyses?

6. L118: “Adenophora” should be rendered in italic font.

7. L130: “169,344” should be changed to “169,344 bp”.

8. L132: “Small single copy” should be changed to “small single copy”.

9. L165-L166: “13 Campanulaceae and one outgroup” is inaccurate, please rephrase it.

10. L198: Check if “seven regions” is correct, I’m counting “six regions”.

11. L205: Please check the “75 protein-coding genes”. The L122 in Materials and Methods described “76 protein-coding genes”.

12. L261-L264: Where possible, please rewrite these sentences to avoid high similarity to ref. [36].

13. The latitude and longitude of the sampling location is missing.

14. Line 118, italic for the genus name.

15. Line 130, change '169,344 in' to '169,344 bp in'.

16. The method in the article shows that ML and BI (Bayesian Inferce) are used to construct a phylogenetic tree (line 105-108). The result refers to the ML tree and the branch support of the two methods (BP and PP), and fig2 is ML tree. The description of the BI tree is missing here (line 159-163).

17. Line 191, change 'two Remotiflorae' to 'two sect. Remotiflorae'.

18. The comparison of the Ka/Ks ratio is only made among three species. I think we should compare all the five species in this genus, and there may be different findings, which will help understand the phylogenetic relationship between these five species.

19. Ka/Ks is the result of pairwise comparison in DnaSP. By counting and comparing the number of regions with a ratio greater than 1, it is helpful to judge the distance of relationship. But for whether a gene has undergone positive selection, a more conventional method needs to be carried out under the framework of phylogeny.

20. Line 225, change 'occurred one more time in' to 'contraction further occurred in'.

21. Line 269, the ycf1 gene should be under positive selection.

22. The formats of the cited documents are not uniform. For example, line 377 and 380, lack year information. Line 380, change '20; 3252-3255 ' to '20:3252-3255 '. And line 404, line 411, line 416, line 418, 422 Line, line 424, line 428, line 432 and so on.

Reviewer #2: Major points

1) Professional English proof-reading is recommended.

2) Re-writing of Discussion is recommended. For example, Figs 5 and 6 should be first presented and explained in Result but not in Discussion (Lines 221-226).

3) GenBank Acc. No. MT012303 is not available now.

4) It is needed to present an evidence to confirm IR contraction. For example, NGS-read mapping coverage graph on chloroplast genome or PCR validation results.

Minor points

1) Plastome, plastid genome, chloroplast genome, and cp genome; these terms are needed to be unified into a single term, such as chloroplast genome.

2) Lines 89-90: More detailed explanation is needed. Do “107,248 reads” mean the reads from chloroplast genome or chloroplast contigs? It is needed to present a reference chloroplast genome sequence used for nucmer tool-based selection.

3) Lines 269-270: “negative selection” is thought to be “positive selection”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at gro.solp@serugif. Please note that Supporting Information files do not need this step.

    2021; 16(3): e0248788.
    Published online 2021 Mar 18. 10.1371/journal.pone.0248788.r002

    Author response to Decision Letter 0

    Copyright and License information Disclaimer

    22 Feb 2021

    Response to Reviewers

    We are pleased to resubmit for publication the revised version of PONE-D-21-00944 “Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): comparative analysis with congeneric species” We appreciated the constructive criticisms of the reviewers. We have addressed each of their concerns as outlined below.

    Journal requirements:

    1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

    https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

    https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

    → We checked and reflected the template style of PLOS ONE at the request of the journal.

    2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

    → We have added to the manuscript what you pointed out.

    Reviewer 1.

    1. The genus Adenophora contains many species. Why did you select only five species from this MS for analysis? What are the selection criteria?

    → As you mentioned, genus Adenophora consist of many species (approximately 50-100 species). However, the studies about cp genome of this genus were not conducted except for only two studies (Cheon et al. 2016, 2017), and accordingly, the number of taxa we could use for comparative analyses were very limited to 5 taxa including the one species (Adenophora recemosa) discussed in this study.

    2. Please also use the regions with high values of Pi (> 0.05) for phylogenetic analysis, and compare with the results of the 76 protein-coding genes.

    → As you suggested, we performed a phylogenetic analysis using genetic markers with high Pi values. However, the tree was exactly the same as the phylogenetic tree based on 76 PCGs. This result is thought to be because the molecular markers proposed by us is a genetic regions suitable for phylogeny of genus Adenophora. Therefore, we have not added any relevant information to the manuscript. Additionally, we are preparing to conduct extensive study on the genus Adenophora using the molecular marker regions devised by us. Upon completion of this study, we promise to publish in the PLOS ONE.

    3. L21-L22: “genome” should be changed to “chloroplast genome”.

    → We checked it and changed it as you suggested.

    4. L22-L33: “Adenophora racemosa” should be changed to “A. racemosa”.

    → We checked it and changed it as you suggested.

    5. L102： In this study, 78 protein-coding genes were identified, but why 76 protein-coding genes were used for phylogenetic analyses?

    → Because two genes (rpl23 and clpP) were existed only few gene regions in the Adenophora plastomes due to sequence deletion, we were conducted the phylogeny exclude these genes. Also, we added the information of these in the Materials and Methods section.

    6. L118: “Adenophora” should be rendered in italic font.

    → We checked it and changed it as you suggested.

    7. L130: “169,344” should be changed to “169,344 bp”.

    → We checked it and changed it as you suggested.

    8. L132: “Small single copy” should be changed to “small single copy”.

    → We checked it and changed it as you suggested.

    9. L165-L166: “13 Campanulaceae and one outgroup” is inaccurate, please rephrase it.

    → We have modified the Fig. 3. legend you pointed out as follows: The ML tree based on 76 protein coding genes from 13 plastomes.

    10. L198: Check if “seven regions” is correct, I’m counting “six regions”.

    → For what you pointed out, we double-checked it, and as a result, two regions of the ycf1 gene show high value (>0.05), so a total of seven regions are correct.

    11. L205: Please check the “75 protein-coding genes”. The L122 in Materials and Methods described “76 protein-coding genes”.

    → We double-check and ‘75 protein coding gene’ is correct because it was confirmed that three genes (rpl23, infA, and clpP) were pseudogenized. It is noted in the ‘Feature of the Adenophora plastomes’ in the ‘Results’ section. We also modified ‘76 protein coding genes’ to ‘75 protein coding genes’ in the Materials and Methods section.

    12. L261-L264: Where possible, please rewrite these sentences to avoid high similarity to ref. [36].

    → As you pointed out, the sentences are written very similar to reference 36. Therefore, we have modified the sentences as follows: The Ka/Ks ratio may indicate which selection pressure is acting on a particular PCGs. Ka/Ks > 1 and Ka/Ks < 1 indicate that the gene is affected by positive selection and negative selection, respectively, and a value of 0 indicates neutral selection [36, 45].

    13. The latitude and longitude of the sampling location is missing.

    → We added the GPS information of sampling location.

    14. Line 118, italic for the genus name

    → We modified it as italic.

    15. Line 130, change '169,344 in' to '169,344 bp in'.

    → We modified it.

    16. The method in the article shows that ML and BI (Bayesian Inferce) are used to construct a phylogenetic tree (line 105-108). The result refers to the ML tree and the branch support of the two methods (BP and PP), and fig2 is ML tree. The description of the BI tree is missing here (line 159-163).

    → We reconstructed the phylogenetic tree using Maximum likelihood method. Also, the BP value based on bootstrap method and PP value based on Bayesian inference were performed to confirm the support values of each node in ML tree.

    17. Line 191, change 'two Remotiflorae' to 'two sect. Remotiflorae'.

    → We modified it.

    18. The comparison of the Ka/Ks ratio is only made among three species. I think we should compare all the five species in this genus, and there may be different findings, which will help understand the phylogenetic relationship between these five species.

    → Since the three species except two sect. Remotiflorae species are morphologically very similar, we conducted an analysis that included only three species (A. divaricate, A. stricta, and A. racemosa). We understand that including all five species as suggested by you may result in different. However, we think it is better to present more concise and intensive analysis results. Please understand with your broad generosity.

    19. Ka/Ks is the result of pairwise comparison in DnaSP. By counting and comparing the number of regions with a ratio greater than 1, it is helpful to judge the distance of relationship. But for whether a gene has undergone positive selection, a more conventional method needs to be carried out under the framework of phylogeny.

    → Thank you for your valuable opinion. But we do not understand what the ‘more conventional method’ you mentioned means. If you can tell us how, we will add it in the next review.

    20. Line 225, change 'occurred one more time in' to 'contraction further occurred in'.

    → We modified it.

    21. Line 269, the ycf1 gene should be under positive selection.

    → We modified it.

    22. The formats of the cited documents are not uniform. For example, line 377 and 380, lack year information. Line 380, change '20; 3252-3255 ' to '20:3252-3255 '. And line 404, line 411, line 416, line 418, 422 Line, line 424, line 428, line 432 and so on.

    → We modified it.

    Reviewer 2.

    Major points

    1) Professional English proof-reading is recommended.

    → Manuscript has been completely revised by a professional English translation agency.

    2) Re-writing of Discussion is recommended. For example, Figs 5 and 6 should be first presented and explained in Result but not in Discussion (Lines 221-226)

    → We have re-written the discussion as your suggestion.

    3) GenBank Acc. No. MT012303 is not available now.

    We think that GenBank accession number will be released soon. We submitted a sequin file on the chloroplast genome of Adenophora racemosa to NCBI in Feb. 2020. At the time of submission, we had requested to NCBI that genome information of A. racemosa be not released for a year because of a group doing study very similar to ours.

    4) It is needed to present an evidence to confirm IR contraction. For example, NGS-read mapping coverage graph on chloroplast genome or PCR validation results.

    → We added the NGS-read mapping coverage graph as a supplementary Figure 1.

    Minor points

    1) Plastome, plastid genome, chloroplast genome, and cp genome; these terms are needed to be unified into a single term, such as chloroplast genome.

    → We have unified the words you pointed out as the ‘chloroplast genome’.

    2) Lines 89-90: More detailed explanation is needed. Do “107,248 reads” mean the reads from chloroplast genome or chloroplast contigs? It is needed to present a reference chloroplast genome sequence used for nucmer tool-based selection.

    → ‘107,248 reads’ mean the reads from chloroplast contigs. We added this on the Materials and Methods section.

    3) Lines 269-270: “negative selection” is thought to be “positive selection”.

    → We modified it.

    Attachment

    Submitted filename: Response to Reviewers.docx

      2021; 16(3): e0248788.
      Published online 2021 Mar 18. 10.1371/journal.pone.0248788.r003

      Decision Letter 1

      Tzen-Yuh Chiang, Academic Editor
      Copyright and License information Disclaimer

      5 Mar 2021

      Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): comparative analysis with congeneric species

      PONE-D-21-00944R1

      Dear Dr. Cheon,

      We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

      Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

      An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at gro.solp@gnillibrohtua.

      If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact gro.solp@sserpeno.

      Kind regards,

      Tzen-Yuh Chiang

      Academic Editor

      PLOS ONE

      Additional Editor Comments (optional):

      Reviewers' comments:

      Reviewer's Responses to Questions

      Comments to the Author

      1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

      Reviewer #1: All comments have been addressed

      Reviewer #2: All comments have been addressed

      **********

      2. Is the manuscript technically sound, and do the data support the conclusions?

      The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

      Reviewer #1: Yes

      Reviewer #2: Yes

      **********

      3. Has the statistical analysis been performed appropriately and rigorously?

      Reviewer #1: Yes

      Reviewer #2: Yes

      **********

      4. Have the authors made all data underlying the findings in their manuscript fully available?

      The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

      Reviewer #1: Yes

      Reviewer #2: No

      **********

      5. Is the manuscript presented in an intelligible fashion and written in standard English?

      PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

      Reviewer #1: Yes

      Reviewer #2: Yes

      **********

      6. Review Comments to the Author

      Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

      Reviewer #1: I have no additional comments for this manuscript, including concerns about dual publication, research ethics, or publication ethics.

      Reviewer #2: GenBank Acc. No. MT012303 is not available now. Authors should request immediately open of the sequence information to NCBI GenBank via e-mail.

      **********

      7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

      If you choose “no”, your identity will remain anonymous but your review may still be made public.

      Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

      Reviewer #1: No

      Reviewer #2: No

        2021; 16(3): e0248788.
        Published online 2021 Mar 18. 10.1371/journal.pone.0248788.r004

        Acceptance letter

        Tzen-Yuh Chiang, Academic Editor
        Copyright and License information Disclaimer

        9 Mar 2021

        PONE-D-21-00944R1

        Complete chloroplast genome sequence of Adenophora racemosa (Campanulaceae): comparative analysis with congeneric species

        Dear Dr. Cheon:

        I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

        If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact gro.solp@sserpeno.

        If we can help with anything else, please email us at gro.solp@enosolp.

        Thank you for submitting your work to PLOS ONE and supporting open access.

        Kind regards,

        PLOS ONE Editorial Office Staff

        on behalf of

        Dr. Tzen-Yuh Chiang

        Academic Editor

        PLOS ONE

          Articles from PLOS ONE are provided here courtesy of PLOS

          Full text links 

          Read article at publisher's site: https://doi.org/10.1371/journal.pone.0248788

          Read article for free, from open access legal sources, via Unpaywall: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0248788&type=printableUnpaywall

          Citations & impact 

          Impact metrics

          7
          Citations
          Jump to Citations
          20
          Data citations
          Jump to Data

          Citations of article over time

          Smart citations by scite.ai
          Smart citations by scite.ai include citation statements extracted from the full text of the citing article. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
          Explore citation contexts and check if this article has been supported or disputed.
          https://scite.ai/reports/10.1371/journal.pone.0248788

          Supporting
          Mentioning
          Contrasting
          1
          4
          0

          Article citations

          Go to all (7) article citations

          Data 

          Data behind the article

          This data has been text mined from the article, or deposited into data resources.

          BioStudies: supplemental material and supporting data

          Nucleotide Sequences (5)

          Similar Articles 

          To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

          Funding 

          Funders who supported this work.

          Ministry of Education (1)