Review Reports - Phylogeography of Halophytes Across Saline Landscapes: A Systematic Review of Coastal–Inland Connectivity and Genetic Differentiation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant topic, and the idea of synthesizing phylogeographic patterns across coastal and inland halophytic systems is interesting. However, in its current form it requires MAJOR and substantial revision. The central limitation is the restricted dataset (20 studies), which does not allow the authors to robustly address global patterns or geographic bias. Many of the conclusions are therefore overstated relative to the available evidence. In the Introduction (p. 2), the conceptual framing of halophytes needs clarification. Halophytes should be presented as multiple independent evolutionary lineages that have converged on salt tolerance, rather than as a single group repeatedly adapting to salinity. The discussion of classifications (glycophytes, obligate and facultative halophytes) should not be framed as opposing systems, but as complementary frameworks. The concept of “strong genetic structure” is unclear and should be defined. In addition, the comparison between inland and coastal adaptation is oversimplified, as coastal systems also present strong environmental gradients (see for example the halophytes that thrive closer to the see with tidal inundation, anoxia, salinity variation) that require highly localized adaptation (For example, Salicornia perennis in Atlantic Europe). In the early framing of results (p. 3), statements such as “by far” are not supported analytically and should be justified quantitatively or removed. More importantly, the small number of studies considered for the final outcomes creates bias itself. There is no papers on Australia, New Zeland, South America, etc. See for example new phylogenomic studies in Tecticornia: Žerdoner Čalasan A, Morales-Briones DF, Kadereit G, Shepherd KA. Evolutionary history of Australian samphires (Salicornieae, Amaranthaceae). Ann Bot. 2026 Jan 9;137(1):247-266. doi: 10.1093/aob/mcaf232. PMID: 40997136; PMCID: PMC12784080. Regarding Figure 1 (p. 4), the conceptual model is unclear in its treatment of gene flow, particularly the coexistence of strong and weak gene flow between systems. I cannot understand that chart. More importantly, the manuscript lacks a clear operational definition of “halophyte”, which is essential for study selection. This becomes critical when taxa such as Arabidopsis are included: is it a halophyte? In the Methods (p. 5), the inclusion of only 20 studies is a major limitation. The search strategy appears too restrictive and should be expanded to include additional markers (e.g. ETS and other commonly used plastid regions (atpB-rbcL, matK-trnK) and a broader taxonomic scope. A supplementary table listing all screened studies and reasons for exclusion would improve transparency. Figure 2 (p. 6) appears unnecessary, as it does not add information beyond the text and is based on a very limited dataset. One of the banners state n = 0 which I think does not provide any futher information. In the Results, the issue of dataset size becomes critical. In the section on geographic bias (p. 7,,,), it is not appropriate to infer bias from only 20 studies. The authors should expand the dataset substantially and explicitly include key halophytic genera that are currently missing from the synthesis. These include, among others, Suaeda, Salicornia and the broader Salicornioideae (e.g. Arthrocaulon, Halopeplis, Halocnemum, Kalidium, Halostachys, Tecticornia…), as well as Spartina (now Sporobolus; e.g. S. maritima, S. alterniflora, S. densiflora, S. patens), Distichlis, Batis, Limonium, and halophytic species within Juncus and Atriplex to name a few of them. These groups are well represented in the literature, including taxonomic, systematic and phylogeographic studies, and their absence suggests that the search strategy is incomplete. As noted (p. 8), the lack of studies from South America and Australia further weakens any global interpretation. Authors mentioned lack of studies in Tunisia. Please see some of them where Tunisian plants are considered (for example): Ramírez, E., Sánchez-Gavilán, I., Rufo, L., Sánchez-Mata, D., & de la Fuente, V. (2022). Morphology, anatomy and phylogeny of the two sister halophytic genera Microcnemum and Arthrocnemum (Salicornioideae/Amaranthaceae). Plant Biosystems - An International Journal Dealing with All Aspects of Plant Biology, 156(6), 1422–1437. https://doi.org/10.1080/11263504.2022.2056649 Hayder, Z., Gaied, R.B., Tlili, A. et al. Phylogenetic and morphological studies of Sarcocornia (L.) A.J. Scott and Salicornia L. (Chenopodiaceae) and insights into plant diversity with first record of two species new for Tunisia. Genet Resour Crop Evol 70, 717–729 (2023). https://doi.org/10.1007/s10722-022-01454-y There are also taxonomic inconsistencies (p. 8), such as the treatment of Sarcocornia as separate from Salicornia, despite being phylogenetically embedded within it (Piirainen et al. 2017, cited in the text). Throughout the Results, the inclusion of Arabidopsis as a halophyte remains questionable (p. 9) and should be justified or removed. Tables are a major weakness. Table 1 (p. 10) is incomplete, includes taxa that are not strict halophytes (e.g. Phragmites, which is more typical of brackish environments), and omits many key halophytic genera listed above. This results in a biased and unrepresentative dataset. Table 2 (p. 12–13) contains entries that are not clearly defined at genus or species level and includes statements without proper citations. Salicornioideae is not a genus or mixed halophytes. Overall, the dataset is too limited in terms of genera, species and studies. In the Discussion (p. 15), the text is overly long and largely repetitive of the Results. Both sections state essentially the same conclusions and in both sections are citatitions. The authors should reduce redundancy and focus on interpretation. Claims regarding global patterns and bias should not be made without a substantially expanded dataset. The reference list appears many studies toward South African literature, at least 8 or 9 (please, check). Are all of them included in the 20 studies analyzed? In that case, wouldn’t Africa be underrepresented? I think the paper needs also an Appendix table with the studies selected and more related information. But from my point of view, this would be only possible after a strong search with the main plants that thrive in saline ecosystems both inland and coastal worldwide. That requires a gigantic new effort.... Overall, I recommend a very substantial major revision. The manuscript might have potential and being of interest if authors address greater expansion of the dataset, clearer taxonomic and conceptual criteria, and a more rigorous and cautious synthesis before it can be considered for publication.

Author Response

Comment:

Response:

We thank the reviewer for this valuable comment. We agree that a substantially larger number of studies are available on the phylogeny, systematics, taxonomy, and population genetics of halophytes globally. However, the objective of this review was not to provide a comprehensive synthesis of all genetic studies conducted on halophytes. Rather, the review specifically focused on studies that directly examined phylogeographic patterns, genetic diversity, population structure, or connectivity across both coastal and inland saline environments, thereby allowing explicit comparisons between these habitat types.

To ensure that the review remained aligned with its objectives, studies were only included if they investigated halophytic taxa occurring in both coastal and inland saline habitats or explicitly compared populations across these contrasting environments. Studies focusing exclusively on coastal salt marshes or exclusively on inland saline habitats were excluded, even when they involved genera such as Salicornia, Suaeda, Atriplex, Spartina (Sporobolus), Limonium, Distichlis, or members of the Salicornioideae. This criterion was stated explicitly in the inclusion and exclusion criteria within the Materials and Methods section.

Consequently, the final dataset represents the subset of studies that directly address the review's central research questions regarding habitat connectivity, genetic differentiation, and phylogeographic divergence between coastal and inland saline environments. We have revised the manuscript to clarify this rationale and to emphasise that the identified geographic patterns and research gaps apply specifically to studies comparing coastal and inland saline systems, rather than to the broader body of halophyte genetic research.

Comment:

In the Introduction (p. 2), the conceptual framing of halophytes needs clarification. Halophytes should be presented as multiple independent evolutionary lineages that have converged on salt tolerance, rather than as a single group repeatedly adapting to salinity.

The discussion of classifications (glycophytes, obligate and facultative halophytes) should not be framed as opposing systems, but as complementary frameworks.

Response:
We thank the reviewer for this helpful comment. To clarify the definition and conceptual framing of halophytes, we have incorporated the widely accepted definition of Flowers and Yeo (1986), which defines halophytes as salt-tolerant plants capable of completing their life cycle at salinity levels exceeding 200 mM. We also expanded the text to highlight the taxonomic diversity of halophytes by referencing the eHALOPH database, which currently recognises more than 2,500 halophytic species worldwide. The revised text emphasises that halophytes represent a diverse assemblage of salt-tolerant species occurring across numerous plant lineages rather than a single taxonomic or evolutionary group.

In addition, we revised the discussion of halophyte classification to reflect the evolution of the concept from a simple dichotomy between halophytes and glycophytes to a continuum of salinity tolerance. Earlier classifications distinguished salt-tolerant and salt-sensitive species as discrete groups, whereas later frameworks recognised obligate, facultative, and other functional categories of halophytes. More recent perspectives view salinity tolerance as a gradient ranging from highly sensitive glycophytes to euhalophytes capable of tolerating repeated exposure to seawater. This revision better reflects the diversity of physiological responses to salinity observed across plant lineages and aligns with the reviewer's suggestion that halophytes should not be considered a single evolutionary entity. Given that the focus of this review is on phylogeographic patterns and genetic connectivity between coastal and inland saline habitats, we elected not to include a detailed discussion of the multiple independent evolutionary origins of salt tolerance, but we agree that halophytes comprise numerous lineages that have evolved adaptations to saline environments independently.

Comment:

The concept of “strong genetic structure” is unclear and should be defined.

Response: We thank the reviewer for this comment. We agree that the term "strong genetic structure" may be ambiguous when used without definition. To improve clarity, we revised the text to refer more explicitly to increased genetic differentiation among populations resulting from reduced gene flow, habitat fragmentation, and isolation. Because this statement occurs in the Introduction, where the objective is to provide context rather than present results, we avoided using technical population genetic metrics and instead described the expected patterns in terms of population differentiation and localised adaptation.

In addition, the comparison between inland and coastal adaptation is oversimplified, as coastal systems also present strong environmental gradients (see for example the halophytes that thrive closer to the see with tidal inundation, anoxia, salinity variation) that require highly localized adaptation (For example, Salicornia perennis in Atlantic Europe).

Response: We thank the reviewer for highlighting this important point. We agree that the original text may have overstated the contrast between coastal and inland systems. Coastal salt marshes are characterised by strong environmental gradients, including variation in tidal inundation, salinity, sediment dynamics, and oxygen availability, which can promote localised adaptation despite ongoing connectivity. To address this, we have revised the Introduction to acknowledge that adaptive divergence can occur in both coastal and inland saline environments. We also incorporated relevant literature, including Foust et al. (2016), which demonstrated genetic and epigenetic differentiation associated with environmental gradients in coastal salt marsh species. The revised text therefore emphasises that differences between coastal and inland systems relate primarily to the relative importance of connectivity versus habitat isolation, rather than the presence or absence of local adaptation.

Comment:

In the early framing of results (p. 3), statements such as “by far” are not supported analytically and should be justified quantitatively or removed. More importantly, the small number of studies considered for the final outcomes creates bias itself.

Response:

We thank the reviewer for this observation. We have carefully reviewed the manuscript and removed or revised any wording that could imply unsupported quantitative differences. Where appropriate, descriptive statements have been replaced with explicit frequencies and percentages derived from the dataset. We agree that the relatively small number of included studies limits the extent to which broad generalisations can be made. However, the final dataset reflects the predefined eligibility criteria of this review, which focused specifically on studies examining phylogeographic patterns, genetic diversity, and connectivity across both coastal and inland saline environments. As a result, the dataset represents a specialised subset of the broader halophyte literature rather than all available genetic studies on halophytes. To address this concern, we have clarified in the Results and Discussion that the observed geographic and methodological patterns apply only to this subset of studies and should not be interpreted as representative of the entire field of halophyte phylogeography.

Comment:

There is no papers on Australia, New Zeland, South America, etc. See for example new phylogenomic studies in Tecticornia: Žerdoner Čalasan A, Morales-Briones DF, Kadereit G, Shepherd KA. Evolutionary history of Australian samphires (Salicornieae, Amaranthaceae). Ann Bot. 2026 Jan 9;137(1):247-266. doi: 10.1093/aob/mcaf232. PMID: 40997136; PMCID: PMC12784080.

Response:
We thank the reviewer for highlighting this recent study and for drawing our attention to the expanding literature on halophyte evolution in Australia. We have examined the paper by Žerdoner Čalasan et al. (2026), which provides valuable insights into the evolutionary history and phylogenomic relationships of Australian samphires (Tecticornia and related Salicornieae taxa). We agree that this study contributes substantially to our understanding of halophyte diversification and biogeographic history.

However, our review was specifically designed to synthesise studies investigating phylogeographic patterns, genetic diversity, population structure, and connectivity across both coastal and inland saline environments. Although the study includes taxa occurring in coastal and inland habitats, its primary focus is on phylogenomic reconstruction and evolutionary relationships among species rather than population-level genetic differentiation, gene flow, or phylogeographic connectivity between coastal and inland populations. Consequently, it did not meet our predefined inclusion criteria.

We have nevertheless acknowledged in the Discussion that important phylogenetic and phylogenomic studies have been conducted in regions such as Australia and that these studies provide valuable insights into the evolutionary history of halophytes. We further note that the limited representation of Australia, New Zealand, and South America in our final dataset reflects the specific scope and eligibility criteria of this review rather than the absence of halophyte genetic research in these regions.

Comment:

Regarding Figure 1 (p. 4), the conceptual model is unclear in its treatment of gene flow, particularly the coexistence of strong and weak gene flow between systems. I cannot understand that chart.

Response:

We thank the reviewer for this comment. We agree that the original figure may have implied multiple levels of contemporary gene flow between coastal and inland populations, which could be interpreted as contradictory. The figure has been revised to distinguish clearly between (i) high contemporary connectivity among coastal populations, (ii) reduced connectivity among inland populations, and (iii) rare or historical dispersal events linking coastal and inland systems. The revised figure therefore focuses on contrasting patterns of connectivity and isolation rather than suggesting simultaneous strong and weak gene flow between the same populations.

Comment:

More importantly, the manuscript lacks a clear operational definition of “halophyte”, which is essential for study selection. This becomes critical when taxa such as Arabidopsis are included: is it a halophyte?

Response:

We thank the reviewer for highlighting this important issue. To improve clarity and consistency, we have strengthened the operational definition of halophytes in both the Introduction and Methods, adopting the widely accepted definition of Flowers and Yeo (1986), which defines halophytes as plants capable of completing their life cycle at salinity levels exceeding 200 mM NaCl. Following this clarification, we re-evaluated all included studies and agree that Arabidopsis thaliana is generally regarded as a salt-tolerant glycophyte rather than a true halophyte. Although the study by Busoms et al. (2015) provides valuable insights into local adaptation to coastal salinity, it does not focus on a halophytic species and therefore falls outside the scope of this review. The study has consequently been removed from the final synthesis and associated analyses.

Comment:

In the Methods (p. 5), the inclusion of only 20 studies is a major limitation. The search strategy appears too restrictive and should be expanded to include additional markers (e.g. ETS and other commonly used plastid regions (atpB-rbcL, matK-trnK) and a broader taxonomic scope.

Response:

We thank the reviewer for this suggestion. Following this recommendation, we conducted additional searches incorporating the suggested molecular markers (ETS, atpB-rbcL, matK-trnK, and related plastid regions). However, these searches did not identify additional studies that met our predefined inclusion criteria.

Importantly, the objective of this review was not to provide a comprehensive synthesis of all phylogenetic or phylogeographic studies on halophytes globally. Rather, the review specifically focused on studies investigating genetic diversity, population genetics, phylogeography, or evolutionary connectivity between coastal salt marsh and inland saline populations, or studies explicitly including both habitat types within their analyses. Consequently, many phylogenetic, taxonomic, and genomic studies on halophytes were excluded because they focused exclusively on coastal or inland systems without examining connectivity or comparisons between these environments.

To further ensure that relevant taxa were not overlooked, we conducted an independent search using the eHALOPH database. Using the keyword "DNA", 136 halophytic species were identified. The reference lists associated with each species profile were subsequently examined individually for evidence of phylogenetic, population genetic, or phylogeographic studies involving both coastal and inland saline habitats. This additional screening did not yield further studies that satisfied the inclusion criteria.

We have clarified these points in the Methods section to better distinguish between the scope of this review and broader reviews of halophyte systematics or phylogenomics.

Response:

A supplementary table listing all screened studies and reasons for exclusion has been provided as supplementary tables S1 and S2, see manuscript.

To improve transparency, we have added Supplementary Table S1 listing all studies assessed at the full-text stage together with reasons for exclusion, and Supplementary Table S2 summarising the characteristics of all studies included in the final synthesis. The relatively small number of eligible studies reflects the scarcity of phylogeographic investigations explicitly examining halophyte populations across both coastal and inland saline habitats, which itself represents an important knowledge gap identified by this review. See the manuscript edits.

Comment:

Figure 2 (p. 6) appears unnecessary, as it does not add information beyond the text and is based on a very limited dataset. One of the banners state n = 0 which I think does not provide any futher information.

Response: We thank the reviewer for this comment. Figure 2 has been retained because it represents the PRISMA 2020 study selection workflow, which is recommended for systematic reviews and provides transparency regarding the identification, screening, eligibility assessment, and inclusion of studies. Although the final dataset is relatively small, the figure allows readers to track the application of the inclusion and exclusion criteria and therefore improves reproducibility. We agree that some elements of the figure can be simplified, and we have revised the figure to improve clarity. The "Reports not retrieved (n = 0)" box was retained to remain consistent with the PRISMA 2020 reporting framework and to explicitly indicate that all potentially eligible full-text articles were successfully obtained and assessed.

Comment:

In the Results, the issue of dataset size becomes critical. In the section on geographic bias (p. 7,,,), it is not appropriate to infer bias from only 20 studies. The authors should expand the dataset substantially and explicitly include key halophytic genera that are currently missing from the synthesis. These include, among others, Suaeda, Salicornia and the broader Salicornioideae (e.g. Arthrocaulon, Halopeplis, Halocnemum, Kalidium, Halostachys, Tecticornia…), as well as Spartina (now Sporobolus; e.g. S. maritima, S. alterniflora, S. densiflora, S. patens), Distichlis, Batis, Limonium, and halophytic species within Juncus and Atriplex to name a few of them. These groups are well represented in the literature, including taxonomic, systematic and phylogeographic studies, and their absence suggests that the search strategy is incomplete.

Response: We thank the reviewer for this valuable comment. We agree that a substantially larger number of studies are available on the phylogeny, systematics, taxonomy, and population genetics of halophytes globally. However, the objective of this review was not to provide a comprehensive synthesis of all genetic studies conducted on halophytes. Rather, the review specifically focused on studies that directly examined phylogeographic patterns, genetic diversity, population structure, or connectivity across both coastal and inland saline environments, thereby allowing explicit comparisons between these habitat types.

Comment:

As noted (p. 8), the lack of studies from South America and Australia further weakens any global interpretation. Authors mentioned lack of studies in Tunisia. Please see some of them where Tunisian plants are considered (for example): Ramírez, E., Sánchez-Gavilán, I., Rufo, L., Sánchez-Mata, D., & de la Fuente, V. (2022). Morphology, anatomy and phylogeny of the two sister halophytic genera Microcnemum and Arthrocnemum (Salicornioideae/Amaranthaceae). Plant Biosystems - An International Journal Dealing with All Aspects of Plant Biology, 156(6), 1422–1437. https://doi.org/10.1080/11263504.2022.2056649 Hayder, Z., Gaied, R.B., Tlili, A. et al. Phylogenetic and morphological studies of Sarcocornia (L.) A.J. Scott and Salicornia L. (Chenopodiaceae) and insights into plant diversity with first record of two species new for Tunisia. Genet Resour Crop Evol 70, 717–729 (2023). https://doi.org/10.1007/s10722-022-01454-y

Response:
We thank the reviewer for these suggestions and for drawing our attention to additional studies from Tunisia. We note that the study by Hayder et al. (2023) was already included in our final synthesis and therefore Tunisia is represented within the review dataset. We have revised the text to clarify that our concern relates to the limited overall representation of African studies rather than the complete absence of studies from Tunisia.

We also examined the study by Ramírez et al. (2022). While this work provides valuable insights into the morphology, anatomy, and phylogenetic relationships of halophytic taxa, it does not investigate population genetic structure, phylogeography, genetic diversity, or connectivity between coastal and inland saline populations and therefore does not meet the predefined inclusion criteria of this review.

More broadly, we acknowledge that substantial halophyte research has been conducted in regions such as Australia, South America, and North Africa. However, our review specifically targeted studies examining phylogeographic patterns, population genetic structure, and connectivity across coastal and inland saline environments. Consequently, the limited representation of these regions in the final dataset reflects the scarcity of studies meeting these specific criteria rather than an absence of halophyte research more generally.

Comment:

There are also taxonomic inconsistencies (p. 8), such as the treatment of Sarcocornia as separate from Salicornia, despite being phylogenetically embedded within it (Piirainen et al. 2017, cited in the text).

Response:
We thank the reviewer for highlighting this taxonomic issue. We agree that Sarcocornia is now generally regarded as phylogenetically embedded within Salicornia following the taxonomic revision proposed by Piirainen et al. (2017). In the manuscript text, we have adopted the currently accepted taxonomy and refer to these taxa as Salicornia. However, several studies included in the review were published prior to this revision and originally reported their results under the genus Sarcocornia. To maintain consistency with the source literature while acknowledging current taxonomy, we have revised the relevant tables to present these taxa as Sarcocornia (≡ Salicornia). A note has also been added to clarify that historical species names were retained where necessary to reflect the terminology used in the original publications.

Comment:

Throughout the Results, the inclusion of Arabidopsis as a halophyte remains questionable (p. 9) and should be justified or removed. Tables are a major weakness. Table 1 (p. 10) is incomplete, includes taxa that are not strict halophytes (e.g. Phragmites, which is more typical of brackish environments), and omits many key halophytic genera listed above. This results in a biased and unrepresentative dataset.

Response:
We thank the reviewer for these comments. Following the reviewer's earlier recommendation, the study involving Arabidopsis thaliana has been removed from the final synthesis because A. thaliana is generally regarded as a salt-tolerant glycophyte rather than a halophyte under the definition adopted in this review.

Regarding Phragmites australis, we acknowledge that this species is not a strict or obligate halophyte. However, it is widely recognised as a facultative halophyte, occurring across freshwater, brackish, and saline environments, and is included in major halophyte databases such as eHALOPH. The study was therefore retained because it met the predefined inclusion criteria and explicitly examined genetic patterns across coastal and inland saline habitats.

We respectfully suggest that the apparent omission of several major halophytic genera reflects the specific scope of this review rather than a deficiency in the dataset. The objective of this study was not to compile all phylogenetic, systematic, or genetic studies on halophytes globally, but rather to synthesise studies investigating phylogeographic patterns, population genetic structure, genetic diversity, and connectivity between coastal and inland saline populations. Many of the genera identified by the reviewer (e.g., Tecticornia, Microcnemum, Arthrocnemum, and others) are represented by important phylogenetic or taxonomic studies, but these did not satisfy the inclusion criteria because they lacked population-level analyses, phylogeographic inference, or explicit comparisons between coastal and inland habitats. We have clarified this distinction in both the Methods and Discussion sections.

Comment:

Table 2 (p. 12–13) contains entries that are not clearly defined at genus or species level and includes statements without proper citations. Salicornioideae is not a genus or mixed halophytes. Overall, the dataset is too limited in terms of genera, species and studies.

Response:
We thank the reviewer for this observation. We agree that the original heading may have implied a taxonomic rank that was not consistently represented across all entries. To address this issue, the column heading has been revised from "Genus" to "Taxon", which more accurately accommodates the range of taxonomic levels represented in the included studies, including species (Triglochin maritima), genera (Salicornia), subfamilies (Salicornioideae), species complexes (Ruppia spp.), and multi-species assemblages. We also removed Arabidopsis following the reviewer's earlier recommendation and revised several category labels to improve clarity. Furthermore, citations have been checked and added where necessary to ensure that all phylogenetic and phylogeographic patterns presented in the table are appropriately referenced.

Regarding the limited taxonomic representation, we respectfully note that the taxa included in Table 2 reflect only those studies meeting the predefined inclusion criteria, namely investigations of phylogeographic patterns, genetic diversity, population structure, or connectivity involving both coastal and inland saline environments. Consequently, the table should not be interpreted as a comprehensive summary of all halophyte taxa studied globally, but rather as a synthesis of the taxa represented within the final review dataset.

Comment:

In the Discussion (p. 15), the text is overly long and largely repetitive of the Results. Both sections state essentially the same conclusions and in both sections are citatitions. The authors should reduce redundancy and focus on interpretation.

Response:
We thank the reviewer for this constructive comment and agree that the distinction between the Results and Discussion sections could be improved. In response, we substantially revised both sections to reduce redundancy and ensure a clearer separation between the presentation of findings and their interpretation.

The Results section was rewritten to focus primarily on reporting the patterns identified across the included studies, including phylogeographic structure, genetic diversity patterns, molecular markers, and the frequency of reported drivers of differentiation. Several descriptive and interpretative statements were removed from the Results and reserved for the Discussion.

The Discussion was subsequently streamlined to emphasise the broader implications of the findings, including the roles of habitat connectivity, fragmentation, historical biogeography, environmental selection, and methodological limitations. Repetitive descriptions of individual study outcomes were removed, and citations were retained only where necessary to support interpretation or place the findings within a broader evolutionary context. These revisions have substantially reduced overlap between the two sections and strengthened the Discussion's focus on synthesis, interpretation, and future research directions.

Comment:

Claims regarding global patterns and bias should not be made without a substantially expanded dataset. The reference list appears many studies toward South African literature, at least 8 or 9 (please, check). Are all of them included in the 20 studies analyzed? In that case, wouldn’t Africa be underrepresented?

Response:
We thank the reviewer for this observation. We agree that the relatively small number of eligible studies limits the extent to which broad global generalisations can be made. To address this concern, we have revised the manuscript to clarify that the observed geographic and methodological patterns apply specifically to the subset of studies meeting our predefined inclusion criteria rather than to the entirety of halophyte genetic research worldwide. We have also moderated several statements referring to "global patterns" and "geographic bias" to better reflect the scope of the dataset.

Regarding the South African references, not all references cited in the manuscript formed part of the final systematic review dataset. Several South African studies were cited in the Introduction and Discussion to provide ecological, biogeographic, and regional context for saline habitats and halophyte diversity. However, only studies meeting the predefined inclusion criteria—namely those investigating phylogeography, population genetics, genetic diversity, or connectivity between coastal and inland saline environments were included in the final synthesis. Consequently, the number of South African references cited in the manuscript exceeds the number of South African studies included in the review dataset.

We have clarified this distinction in the Methods and Discussion sections to avoid confusion between references cited for contextual background and studies included in the systematic review.

Comment:

I think the paper needs also an Appendix table with the studies selected and more related information. But from my point of view, this would be only possible after a strong search with the main plants that thrive in saline ecosystems both inland and coastal worldwide. That requires a gigantic new effort.... Overall, I recommend a very substantial major revision. The manuscript might have potential and being of interest if authors address greater expansion of the dataset, clearer taxonomic and conceptual criteria, and a more rigorous and cautious synthesis before it can be considered for publication.

Response:
We thank the reviewer for the detailed assessment and constructive suggestions. We agree that transparency regarding the studies included in the review is important. In response, we have added a supplementary table providing detailed information on all studies included in the final synthesis, including taxon, geographic region, habitat type, molecular markers, study objectives, and key findings. We have also provided a supplementary table documenting studies assessed for eligibility and the reasons for exclusion, thereby improving the transparency and reproducibility of the review process.

We further clarified the operational definition of halophytes, revised the taxonomic treatment of several groups, removed Arabidopsis from the final synthesis, expanded the search methodology to include screening of the eHALOPH database (version 7.10; 136 species screened), and strengthened the description of the inclusion and exclusion criteria. The Results and Discussion sections were substantially revised to reduce redundancy and improve the distinction between reporting and interpretation.

With respect to expansion of the dataset, we respectfully note that the objective of this review was not to synthesise all genetic, phylogenetic, or phylogenomic studies of halophytes worldwide. Rather, the review specifically focused on studies examining phylogeography, population genetic structure, genetic diversity, and connectivity across both coastal and inland saline environments. Many studies involving major halophytic genera were identified during the search process but did not meet the predefined eligibility criteria because they focused exclusively on coastal or inland populations, taxonomy, phylogenomics, or species-level evolutionary relationships. We have clarified this distinction throughout the manuscript and have moderated statements regarding global patterns to ensure that conclusions are framed within the scope of the available evidence.

We appreciate the reviewer's recognition of the potential value of the manuscript and believe that the revisions undertaken have substantially strengthened the methodological transparency, taxonomic consistency, and interpretation of the findings.

Reviewer 2 Report

Comments and Suggestions for Authors This manuscript is an abstract of a systematic review. The content is clear, logically rigorous, scientifically sound, and written in a formal academic style. The topic focuses on genetic structure differences in halophytic plants between coastal and inland saline habitats, which holds clear scientific significance and conservation application value. However, as a global systematic review, the sample size of only 20 studies is relatively small. Additionally, directly comparing genetic diversity indices across studies that use different molecular markers is challenging. It is recommended to reference comparison methods and conclusions from similar reviews on non-halophytic plants. Besides the driving factors listed in the text, multiple other factors can influence the genetic diversity patterns of species, such as the timing of speciation, species distribution range, geographic scope and sampling coverage of the studies, population size and population dynamics, and the direction of dispersal and migration between coastal and inland areas, among other complex factors. Overall, this is a scientifically valuable review paper. With appropriate revisions and improvements, it is recommended for publication.

Author Response

Comment:

This manuscript is an abstract of a systematic review. The content is clear, logically rigorous, scientifically sound, and written in a formal academic style. The topic focuses on genetic structure differences in halophytic plants between coastal and inland saline habitats, which holds clear scientific significance and conservation application value.

Response:
We thank the reviewer for their positive assessment of the manuscript and for recognising the scientific significance and potential conservation value of this review. We appreciate the constructive feedback provided and have addressed each comment below to further strengthen the manuscript.

Comment:

However, as a global systematic review, the sample size of only 20 studies is relatively small.

Response:
We agree that the final dataset is relatively small. However, this reflects the scarcity of studies explicitly examining phylogeographic patterns, genetic diversity, population structure, or connectivity across both coastal and inland saline environments. To address this concern, we expanded the search strategy, incorporated additional search terms and molecular markers, screened the eHALOPH database (version 7.10; 136 species screened), and clarified the inclusion criteria. We have also moderated statements referring to global patterns and emphasised throughout the manuscript that the findings apply specifically to the subset of studies meeting the predefined eligibility criteria. We further highlight this limitation as an important knowledge gap and future research priority. Additionally, directly comparing genetic diversity indices across studies that use different molecular markers is challenging. It is recommended to reference comparison methods and conclusions from similar reviews on non-halophytic plants.

Comment:

Additionally, directly comparing genetic diversity indices across studies that use different molecular markers is challenging. It is recommended to reference comparison methods and conclusions from similar reviews on non-halophytic plants.

Response:
We thank the reviewer for this observation and agree that direct comparisons among studies employing different molecular markers should be interpreted cautiously. The objective of this review was not to perform a quantitative meta-analysis of genetic diversity metrics, but rather to identify broad phylogeographic patterns and methodological trends across studies. As noted in the Discussion, the included studies used a variety of marker systems (e.g., cpDNA, ITS, AFLPs, microsatellites, and SNPs), each differing in inheritance patterns, mutation rates, and analytical resolution. Consequently, comparisons of genetic diversity indices among studies were interpreted qualitatively rather than quantitatively. We have further clarified this limitation in the Discussion and emphasise that the diversity of marker systems highlights the need for greater methodological standardisation and wider adoption of genomic approaches in future halophyte phylogeographic research.

Comment:

Besides the driving factors listed in the text, multiple other factors can influence the genetic diversity patterns of species, such as the timing of speciation, species distribution range, geographic scope and sampling coverage of the studies, population size and population dynamics, and the direction of dispersal and migration between coastal and inland areas, among other complex factors.

Response:
We agree with the reviewer that genetic diversity patterns are influenced by numerous interacting ecological, demographic, historical, and evolutionary processes. In response, we have expanded the Discussion to acknowledge additional factors that may influence genetic diversity and phylogeographic structure, including species age and evolutionary history, geographic range size, sampling extent, population size and demographic dynamics, and dispersal directionality between coastal and inland habitats. We also emphasise that the drivers identified in this review represent those most frequently reported in the included studies rather than an exhaustive list of mechanisms influencing halophyte evolution.

Comment:

Overall, this is a scientifically valuable review paper. With appropriate revisions and improvements, it is recommended for publication.

Response:

We sincerely thank the reviewer for their positive evaluation and constructive recommendations.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely topic by synthesizing phylogeographic patterns of halophytes across coastal and inland saline ecosystems. It provides a comprehensive overview of molecular approaches, genetic differentiation, and regional research biases, with particular emphasis on the underrepresentation of African inland saline systems. The review is generally well organized and scientifically relevant; however, following suggestions needs to be incorporated before publication.

The title is slightly long and may be condensed for clarity and readability.
In abstract, mention methodological limitation or research gap.

additional mention of the main methodological limitation or research gap would strengthen the concluding statement.
Minor grammatical corrections are needed, e.g., “hosts specialized halophytic communities” should be “host specialized halophytic communities.”
In Introduction, add recent literature (2024-26) to strengthen the novelty and contemporary relevance.
In Figure 1, mention the version of ChatGPT used.
In Materials and Methods: add following details: How studies were assessed for quality or bias; Whether duplicate screening was performed by multiple reviewers. How disagreements between reviewers were resolved.
The search strategy would benefit from including exact search dates.
Google Scholar screening criteria should be described more rigorously because only the first 200 results were screened.
The authors mention quantitative synthesis, but no meta-analytical framework or statistical synthesis is presented.
Ensure that the PRISMA diagram follows the latest PRISMA 2020 formatting recommendations.
The manuscript should specify whether citation tracking and manual searches contributed additional records.

In Molecular Markers and Diversity Metrics: percentages should be reported consistently with sample sizes for clarity. A supplementary table summarizing all included studies and markers may be used for better understanding.
In Regional Research Effort: Add discussion on why African systems remain understudied would strengthen the interpretation

Genus and species names should be in italics.
In Drivers of Genetic Differentiation: Quantitative support for the percentage-based claims (e.g., “~17%”, “~50%”) should be clarified with exact study counts.
In Table 3: separate groping of ecological, historical, and evolutionary drivers may be considered.
In case study section: add one more comparative example from Asia or Africa.
In discussion: avoid repetition of results.
In Conclusion: At the end add a statement emphasizing the global conservation importance of inland saline ecosystems.
In references: check all references for completeness and consistent formatting. Check if all in-text references are included in the reference list and vice versa.
A thorough proofreading of the MS for language and grammatical errors is essential.

Comments on the Quality of English Language

A thorough proofreading of the MS for language and grammatical errors is essential.

Author Response

Comment:

The title is slightly long and may be condensed for clarity and readability.

Phylogeography of Halophytes Across Saline Landscapes: A Systematic Review of Coastal–Inland Connectivity and Genetic Differentiation

Comment:

In abstract, mention methodological limitation or research gap.

additional mention of the main methodological limitation or research gap would strengthen the concluding statement.

Thank you for the suggestion. The authors have revised the abstract and added the limitation of this review.

Comment:

Minor grammatical corrections are needed, e.g., “hosts specialized halophytic communities” should be “host specialized halophytic communities.”

Thank you for the suggestion; see manuscript for revised text and grammar.

Comment:

In Introduction, add recent literature (2024-26) to strengthen the novelty and contemporary relevance.

Thank you for the suggestion; see manuscript for revised Introduction with recent literature added.

Comment:

In Figure 1, mention the version of ChatGPT used.

Fixed in the manuscript.

Comment:

In Materials and Methods: add following details: How studies were assessed for quality or bias; Whether duplicate screening was performed by multiple reviewers. How disagreements between reviewers were resolved.

Thank you for highlighting this issue; the authors have revised the materials and methods section, and a supplementary table showing how the studies were screened, included or excluded for the purpose of this review was also added. See manuscript and response to Reviewer 1’s comments.

Comment:

The search strategy would benefit from including exact search dates.

Thank you for this; an Excel sheet with the exact search dates for the search strategy was added as a supplementary table to show no bias was done in this study.

Comment:

Google Scholar screening criteria should be described more rigorously because only the first 200 results were screened.

We now specify that only the first 200 results sorted by relevance were screened, and we justify this choice as a reproducible and pragmatic approach. The eHALOPH database was also used to assist with the screening and search strategy of the paper for further descriptions; see the response above from Reviewer 1.

Comment:

The authors mention quantitative synthesis, but no meta-analytical framework or statistical synthesis is presented.

We have corrected and revised the results description section to avoid implying a meta‑analytical framework.

Comment:

Ensure that the PRISMA diagram follows the latest PRISMA 2020 formatting recommendations.

Updated to conform with PRISMA 2020 formatting recommendations.

Comment:

The manuscript should specify whether citation tracking and manual searches contributed additional records.

Clarified that citation tracking contributed additional records.

Comment:

In Molecular Markers and Diversity Metrics: percentages should be reported consistently with sample sizes for clarity. A supplementary table summarizing all included studies and markers may be used for better understanding.

Percentages are now reported consistently with sample sizes. A supplementary Table 2 summarising all included studies, markers, and metrics has been added.

Comment:

In Regional Research Effort: Add discussion on why African systems remain understudied would strengthen the interpretation

Thank you for the comment; this was discussed in the Discussion section to state that the understudy of African systems could be attributed to the GDP, as highlighted by Beheregaray (2008).

Comment:

Expanded discussion on why African systems remain understudied, highlighting limited funding, logistical challenges, and fewer genomic resources.

Comment:

Genus and species names should be in italics.

Corrected to italics throughout the manuscript.

Comment:

In Drivers of Genetic Differentiation: Quantitative support for the percentage-based claims (e.g., “~17%”, “~50%”) should be clarified with exact study counts.

The authors have revised this to state the exact amount instead of percentages throughout the results section.

Comment:

In Table 3: separate groping of ecological, historical, and evolutionary drivers may be considered.

Reorganised to separate ecological, historical, and evolutionary drivers.

Comment:

In case study section: add one more comparative example from Asia or Africa.

Added a comparative example from Asia to balance geographic coverage.

Comment:

In discussion: avoid repetition of results.

Revised to reduce repetition of results.

Comment:

In Conclusion: At the end add a statement emphasizing the global conservation importance of inland saline ecosystems.

Added a statement emphasising the global conservation importance of inland saline ecosystems.

Comment:

In references: check all references for completeness and consistent formatting. Check if all in-text references are included in the reference list and vice versa.

Checked for completeness and consistency; ensured all in‑text citations match the reference list.

Comment:

A thorough proofreading of the MS for language and grammatical errors is essential.