Review Reports - First Plastome Sequences of Two Endemic Taxa of Orbea Haw. from the Arabian Peninsula: Comparative Genomics and Phylogenetic Relationships Within the Tribe Ceropegieae (Asclepiadoideae, Apocynaceae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study sequenced the chloroplast genomes of Orbea sprengeri subsp. commutata and Orbea wissmannii var. eremastrum, providing important references for the phylogenetic relationships of Ceropegieae plants. My comments are as follows:

L94–L111: The description of the roles and effects of Orbea species appears overly detailed and could be streamlined. It might also be considered whether this part could be merged with the following paragraph.

When Orbea sprengeri subsp. commutata and Orbea wissmannii var. eremastrum first appear in the text, they may be presented in full. Subsequently, abbreviations (e.g., in sections 2.1, 2.3, etc.) could be used for brevity. The same principle should be applied to other species mentioned, and a thorough check throughout the manuscript is recommended.

L133–L135: The formatting here seems inconsistent with the following sections.

In Table 5, could the column “Species” be presented using standard species abbreviations? This would likely improve readability for readers.

Additionally, the formatting of the References section could be checked to ensure consistency throughout.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript and for your valuable comments and professional advice. In response to your suggestions, I have revised the relevant sections of the manuscript accordingly. Detailed responses to each comment are provided in the cover letter, and all corresponding revisions and corrections have been highlighted in red in the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study reports for the first time the complete chloroplast genomes of two Orbea species endemic to the Arabian Peninsula and conducts systematic comparative genomic and phylogenetic analyses. The work fills a gap in plastid genomic data for this group in the region and provides valuable insights into plastid evolution within the tribe Ceropegieae. The experimental design is sound and the data foundation is solid; however, shortcomings are present in the completeness of methodological details, standard of data presentation, depth of results interpretation, and clarity of figures.

For the meaning of using the cp genome, the introduction needs to improve, such as why cp genome is good for using as phylogeny studies. Please read the recent papers as: Chloroplast phylogenomics reveals the maternal ancestry of cultivated chrysanthemums (10.48130/gcomm-0025-0019) and The Mendelian pea pan-plastome: insights into genomic structure, evolutionary history, and genetic diversity of an essential food crop （10.48130/gcomm-0024-0004）for their application in phylogeny and population study. It will increase the meaning of cp background research.

In Materials and Methods，this section lacks critical details. For example, in section 2.3, it is mentioned that de novo assemblies were independently performed using GetOrganelle to validate the results, but the specific runtime parameters are not provided. Please supplement the key parameters used for GetOrganelle.
The specific gene information at the IR boundaries for each species in Figure 5 is not presented intuitively. It is recommended to directly annotate the names of genes spanning or adjacent to the four junctions—LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC—on the map for each species.
Figure 7, the mVISTA plot, illustrates the sequence conservation patterns, but the key hypervariable regions identified in the main text are not distinctly marked in the figure. It is suggested to add labeled arrows or shaded boxes to highlight these high-Pi regions, thereby visually linking the results from Figure 6 with this figure.
The layout of Table 5 is relatively dense. Please reformat Table 5, supplement it with key parameters such as the lengths of the LSC, SSC, and IR regions, GC content, and gene counts for each species, and ensure accurate alignment of the data.
In Figure 12, genes with Ka/Ks values > 1 are only labeled as "adaptive." It is recommended to supplement the main text with a discussion on their potential functional adaptive context.
In the Discussion, line 673 proposes six hypervariable regions as potential DNA barcodes, but the description is limited to comparisons with other studies. It is suggested to briefly expand the discussion by stating that the candidate loci identified in this study lay the groundwork for future targeted DNA barcode validation research within the tribe Ceropegieae, particularly among morphologically challenging closely related species. This would direct the findings of this study toward a clear downstream application. And also pointing out the meaning of mutation for organellar genomes as paper like Rethinking the mutation hypotheses of plant organellar DNA.

Author Response

Dear Reviewer,

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study presents the assembly, annotation, and comparative analysis of chloroplast genomes from two Orbea taxa native to southwestern Saudi Arabia, followed by phylogenomic reconstruction within the tribe Ceropegieae. By generating and analyzing novel plastome data, the authors address a clear gap in genomic resources for Arabian Orbea species and provide a valuable foundation for future evolutionary and conservation studies. Overall, the manuscript is well organized, methodologically sound, and clearly written. The use of established bioinformatic pipelines and comparative plastome analyses is appropriate, and the results are consistent with previous studies on plastid genome evolution in Apocynaceae. Howver, I believe the manuscript would benefit from several revisions, primarily focused on the Discussion, to improve conceptual clarity and strengthen the biological interpretation of the results.

Major comments

In the Discussion, the plastome-based phylogenetic results are presented in a way that may overstate their ability to fully resolve evolutionary relationships and species boundaries. While plastid genomes are powerful tools for phylogenomic inference, they represent a single, maternally inherited locus and may be affected by processes such as chloroplast capture, introgression, or lineage sorting. I suggest rephrasing these statements from a plastid-based evolutionary hypothesis point of view and discussing how future integration of nuclear genomic data could complement and refine the current findings.
Interpretations linking plastome features or phylogenetic patterns to environmental adaptation (e.g., montane or arid habitats) are presented without direct tests of selection or functional validation. Thus, use more cautious language (e.g., “may reflect,” “is consistent with,” “could be associated with”) or rephrase it.
The Discussion also highlights the importance of the results for conservation, which is appropriate, but the practical implications remain somewhat general. Please, promote a clearer explanation of how plastome data can be applied in conservation contexts and distinguish between immediate applications of the present data and longer-term perspectives requiring additional datasets.

Minor comments

I believe that some analytical choices would benefit from additional justification, particularly those associated with the thresholds and parameters used for repeat and SSR detection and the criteria for identifying highly variable regions in comparative analyses.
Double-check the Methods and Results sections to ensure consistent terminology when referring to plastome regions (LSC, SSC, IRs) and comparative datasets.
Figures 4, 5, 6, 9, 10, and 12 contain complementary information. The authors may consider moving some of these figures to the Supplementary Material or combining selected panels into a single composite figure to improve conciseness and readability of the main text.

Author Response

Dear Reviewer,

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I have reviewed the revised manuscript and the authors’ responses and am satisfied that all major points raised in my previous review have been fully and appropriately addressed. The revisions have improved the conceptual clarity of the Discussion, strengthened the justification of methodological choices, and resulted in a more balanced and cautious biological interpretation. The manuscript is now clear, coherent, and technically sound.

I have no further major concerns and support acceptance of the revised version. I would like to offer three minor, optional comments that may further strengthen the manuscript:

In a few places, interpretations of Ka/Ks > 1 could be phrased more cautiously (e.g., “signals consistent with positive selection”), given the plastome-based and pairwise nature of the analysis.
The discussion of possible plastome heteroplasmy in Orbea wissmannii var. eremastrum is interesting but could more explicitly emphasize its speculative nature and the need for further validation.
Some statements could be slightly refined to stress that conclusions mainly apply to the Arabian Orbea taxa analyzed here, rather than to the genus as a whole.

These are minorsuggestions only and do not affect the overall conclusions or quality of the study.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review the manuscript again and for your valuable comments, which have helped to improve the quality of the article. In response to your suggestions, I have revised the relevant sections of the manuscript accordingly. Detailed responses to each comment are provided in the attached file, and all corresponding revisions and corrections have been highlighted in yellow in the revised manuscript.

Author Response File: Author Response.pdf