The Complete Chloroplast Genome Sequence of Pseudolysimachion pyrethrinum var. gasanensis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors describe the new plastome of a variety they alone recognize. The manuscript is well-written as a typical plastome description. However, I have identified several significant issues the authors need to address.

The phylogenetic hypothesis published by Albach et al. (2004), which the authors used, suggests they may not know how to interpret a phylogeny or that they deliberately do not accept the position of their study species within the genus Veronica. In that work, Veronica subg. Pseudolysimachion is shown to be monophyletic. The authors fail to mention or discuss the recognition of Pseudolysimachion as a separate genus in the current taxonomic context.

Plants of the World Online (POWO) recognizes Pseudolysimachion pyrethrinum var. gasanensis M.Kim & H.Jo (2017) as a heterotypic synonym of Veronica pyrethrina Nakai (see link: https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:812548-1#synonyms). This directly contradicts the taxonomic premise of the manuscript.

There is an inconsistency in the GenBank submission. The uploaded sequence (accession PX593256) is named Veronica pyrethrina, while the manuscript argues for recognition at the different species and variety. Furthermore, why was the first plastome uploaded in this study (accession PX593255) not used in the comparative analysis?

The authors state: “Our phylogenetic analysis (Figure 5) provides molecular clarity to previous taxonomic ambiguities, strongly supporting the monophyly of Pseudolysimachion.” This claim is inaccurate. Their own results show the genus Veronica as polyphyletic, a finding which contradicts the monophyly supported by Albach et al. (2004). Therefore, the analysis does not resolve ambiguity but rather introduces a new conflict that requires explanation.

Major Concerns:

Taxonomic Consistency: The manuscript's argument for recognizing Pseudolysimachion is not aligned with current authoritative sources (POWO) or its own data labels (GenBank).

Interpretation of Results: The conclusion about the monophyly of Pseudolysimachion appears to be a misinterpretation of the presented phylogenetic tree, which actually shows Veronica as polyphyletic.

These issues must be thoroughly revised and clarified before the manuscript can be considered for publication.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

We thank the reviewer for the critical and constructive comments on our manuscript. We have carefully addressed the taxonomic concerns and clarified the phylogenetic interpretations as suggested.

Comment 1: Concerning the recognition of Pseudolysimachion as a separate genus vs. Veronica subg. Pseudolysimachium.

• Response: We acknowledge the reviewer’s perspective regarding the classification of Pseudolysimachion as a subgenus within Veronica, as suggested by Albach et al. (2004, 2005). However, the taxonomic rank of this group remains a subject of ongoing discussion in the botanical community. In East Asian botany, particularly in the Flora of Korea, Pseudolysimachion has been consistently recognized as a distinct genus based on morphological characters such as the long corolla tube and specific capsule structure [4, 5, 15]. Our study follows this established regional taxonomic framework. We have revised the Discussion to acknowledge both taxonomic viewpoints, providing a more balanced context for the readers.

Comment 2: Concerning the POWO database treating P. pyrethrinum var. gasanensis as a synonym of Veronica pyrethrina.

• Response: While we respect the global "lumping" approach often reflected in the POWO database, Pseudolysimachion pyrethrinum var. gasanensis was described as a new variety based on its distinct diminutive stature and specific habitat (limestone soil conditions), which differentiate it from the typical P. pyrethrinum [5]. One of the primary goals of our study was to provide molecular evidence (complete plastome and SSR markers) to re-evaluate these regional taxonomic ambiguities. Our results show clear genetic divergence that supports its recognition at the varietal level within the Korean flora. We have added a section in the Discussion highlighting the importance of using high-resolution genomic data to supplement morphological classifications in regional endemics.

Comment 3: Inconsistency in GenBank labels (PX593256) and the unused sequence (PX593255).

• Response: We apologize for the confusion regarding the GenBank labels. The use of Veronica pyrethrina for the accession PX593256 was a reflection of the GenBank database's default nomenclature. We will update the metadata to align with the taxonomic names used in this manuscript.
• Regarding PX593255: Although we initially registered the sequence of the typical P. pyrethrinum (PX593255) in GenBank, a subsequent quality control analysis revealed that its sequencing depth was insufficient for the high-resolution comparative analysis required for this study. To ensure the scientific integrity of our findings, we decided to exclude it from the current manuscript. We are currently re-sequencing this sample to achieve higher coverage and plan to present these results in a future publication focused on organelle genomics (e.g., Mitochondrial DNA). For this study in Horticulturae, we prioritized the high-quality plastome of P. pyrethrinum var. gasanensis (PX593256) because of its unique compact growth habit, which holds significant potential for the development of new potted plant cultivars.

Comment 4: Concerning the interpretation of monophyly and the polyphyly of Veronica.

• Response: We agree that our initial statement regarding the monophyly of Pseudolysimachion required more precise phrasing. In our phylogenetic tree (Figure 5), Pseudolysimachion forms a well-supported monophyletic clade, separate from the other Veronica species included. We recognize that this makes the genus Veronica (as traditionally defined) appear polyphyletic in our tree. This result actually highlights the evolutionary distinctness of the Pseudolysimachion lineage, further supporting our argument for its recognition as a separate genus. We have revised the Discussion to clarify that our results support the monophyly of the Pseudolysimachion clade and discussed how this relates to the broader phylogenetic hypotheses of the tribe Veroniceae.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

There are several questions/issues that must be resolved before the paper can be published.

In the SSR analysis, only the SSR type and distribution of P. pyrethrinum var. gasanensis were reported. Comparisons must be made with some closely related species in the discussion, such as SSR quantity, density, type distribution, and location preference (LSC/SSC/IR).

In the third paragraph of the introduction, it was mentioned that P. pyrethrinum var. gasanensis adapt to specific habitats (limestone habitats). So it should be combined with Ka/Ks analysis to conduct evolutionary correlation analysis in the discussion.

Figure 4 presents a lot of relevant information, but the description is too simplistic. The authors should quantitatively compare the length of the IR region, the precise distance (bp) of genes (such as rps19, ycf1) crossing the boundary, and discuss the similarities and differences with closely related species.

In Figure 5, 17 complete chloroplast genomes of related taxa need to list their references.

Author Response

Response to Reviewer 2 Comments

We sincerely appreciate the reviewer’s positive feedback and the insightful suggestions to improve the comparative aspects of our manuscript. We have performed additional analyses to address each point.

Comment 1: SSR analysis comparison with closely related species.

• Response: As suggested, we have expanded the discussion on SSRs by comparing our findings with closely related taxa within the Veroniceae (e.g., nakaianum and P. kiusianum var. diamantiacum). We found that while the total number of SSRs is relatively conserved (ranging from 38 to 45), the distribution of compound SSRs and specific loci in the intergenic spacers (IGS) such as trnS-trnG provides unique footprints for var. gasanensis. We have added a comparative summary of SSR quantity, density, and location preferences (LSC/SSC/IR) in the Discussion.

Comment 2: Evolutionary correlation analysis (Ka/Ks) and limestone habitat adaptation.

• Response: This is a very insightful suggestion. We have integrated the Ka/Ks analysis results with the ecological characteristics of pyrethrinum var. gasanensis. Specifically, we discussed how the strong purifying selection (Ka/Ks < 1.0) in core photosynthetic and metabolic genes may reflect the evolutionary pressure to maintain functional efficiency in the specific nutrient-limited and alkaline conditions of limestone habitats. We have enhanced the Discussion to correlate these molecular signatures with the variety’s adaptive strategies.

Comment 3: Quantitative comparison of IR boundaries (Figure 4).

• Response: We have revised the description of Figure 4 to be more detailed and quantitative. We now provide the precise distances (bp) of key genes such as rps19, ndhF, and ycf1 from the JLB (LSC/IRb), JSB (IRb/SSC), JSA (SSC/IRa), and JLA (IRa/LSC) junctions. Furthermore, we have compared these boundary positions with those of closely related Pseudolysimachion species, noting the slight expansion of the IR region in pyrethrinum var. gasanensis compared to its relatives, which contributes to its unique plastome length.

Comment 4: References for the 17 taxa used in Figure 5.

• Response: We have updated the caption of Figure 5 and the corresponding section in the Materials and Methods to include a comprehensive list of references for all 17 complete chloroplast genomes used in the phylogenetic analysis. These references have also been added to the main reference list (Refs. [12, 13, 28, 29, 31, 32]).