Genomic Diversity and Species Boundaries of the Chilean Silversides Fishes (Atheriniformes, Atherinopsidae)

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

In this study, the genomic diversity of Chilean silversides (Odontesthes and Basilichthys) and their species boundaries were analysed using RADseq. The study included 78 Odontesthes and 60 Basilichthys individuals and used phylogeny, structural analyses (structure, DAPC, PCA), genetic distances (Mantel's test), and Bayesian species delimitation (BFD) to investigate the phylogenetic relationships and population differentiation of the species of the two genera. The results showed that 1) the freshwater Odontesthes species, O. mauleanum, and the estuarine species, O. brevianalis, are not significantly differentiated at the genetic level, and that morphological differences may be related to phenotypic plasticity or local adaptation; 2) the traditionally taxonomically placed B. microlepidotus in the genus Basilichthys is similarly differentiated from the formerly synonymous species, B. australis, which share a lack of species-level differentiation but show geographic isolation by distance from south to north, while the arid Atacama Desert separates Chilean and Peruvian populations in terms of gene exchange; 3) differences in the genetic structure of the two genera may be related to the potential for oceanic exchange: Odontesthes has gene flow with oceanic populations via estuaries, whereas Basilichthys has gene flow as Basilichthys evolved in freshwater isolation.

The problems with the MS are as follows:

Comments 1:、The relationship between taxonomic controversies and conservation needs is not sufficiently emphasized in the study. The silverside is a threatened species in Chile, but the study did not explore in depth the impact of ambiguous species boundaries on conservation strategies (e.g. population management, habitat restoration). It is recommended that the barriers to conservation action caused by confusion in species classification be included in the introduction to increase the relevance of the study to conservation biology.

Response 1: We thank the referee for his/her careful reading of the manuscript, his positive comments on our work, and recommending publication in Diversity. We included a paragraph on the relevance of the confusion in species classification to conservation and management actions for commercially valuable fish such as silversides in the introduction section:

Line 118: “Defining the taxonomic boundaries of species can have significant repercussions not only for science but also for conservation strategies, especially for threatened species, as demonstrated by these silversides in Chile. Taxonomic controversies hamper the effective implementation of conservation actions, such as population management and habitat restoration. This confusion can hinder the accurate identification of at-risk populations and the assessment of their conservation status. Therefore, addressing these taxonomic barriers is crucial to strengthen the relevance of conservation biology studies, thereby ensuring appropriate and effective management strategies for the protection of vulnerable species of commercial interest. The silverside, in particular, has been studied to be affected by pollution, driven by land use, industrial operations, and urban growth in Chilean freshwater ecosystems (Cortés-Miranda et al., 2025).”

Comments 2: The study did not integrate morphological data to explain the conflict between genetic and phenotypic differentiation (e.g. the mismatch between scale characters and genotypes of O. mauleanum); moreover, the study did not adequately discuss the mechanisms driving population structure, e.g. the marine gene penetration event (the) hybridisation of O. regia with freshwater species is only based on speculation from previous literature and lacks targeted validation. It is recommended to complement morpho-genomic association analysis with gene flow simulation or ABBA-BABA testing for suspected hybridisation events (e.g. Lake LLAN populations).

Response 2: We thank the Referee for pointing out this remark. The study is based on genomic data and does not include morphological data. For the genus Basilishthys, the morphological data analyzed are part of the doctoral thesis of one of the authors (Roberto Cifuentes) but have already been published by Veliz et al. (2012). For the genus Odontesthes, the mechanisms that determine the hybridization of O. regia with freshwater species were not analyzed in the present study because this analysis has already been performed by the authors in another work (Hugues et al. 2020). Four of the five authors of the present work are part of the aforementioned in 2020 study where ABBA-BABA were performed to detect possible hybridization events (e.g., populations from Lake LLAN). This is in no way a reflection of previous speculations from the literature, on the contrary, we base our discussion on our own results and scientific publications. Unfortunately, we currently lack reference material for morphological analyses of the genus Odontesthes. Furthermore, it is important to clarify that Odontesthes brevianalis and O. mauleanum are difficult to distinguish and are commonly named based on their typical habitat: estuarine/fluvial in the case of O. brevianalis and primarily lacustrine in the case of O. mauleanum. These two species are distinguished by a single morphological feature: the presence of markedly crenate scales along the flank of O. mauleanum, or restricted only to the caudal peduncle in the case of O. brevianalis (Dyer, 2006). However, this feature mentioned in the literature does not appear to be robust to observations of collected individuals (personal communication).

Dyer, B. S. (2006). Systematic revision of the South American silversides (Teleostei, Atheriniformes). Biocell, 30, 69–88.

Comments 3: The effect of glaciation on genetic structure is too general, and not modelled with specific glacial events and population dispersal thresholds; adaptive evolution at the genome level (e.g. salinity responsive genes) is not addressed. It is suggested that ecological niche modelling (ENM) be introduced to quantify glacial refugia and dispersal pathways.

Response 3: Thanks for the feedback. As another reviewer suggested, the final discussion section, titled "The Role of Glaciations and Geological Events in Shaping Genetic Patterns," was removed from the manuscript. Since the present study does not perform a time-calibrated analysis, it provides no evidence to support or refute the influence of specific geological events on the distribution of the populations analyzed.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. 

2. Questions for General Evaluation

Reviewer’s Evaluation

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Dear Editor,

The current manuscript presents an interesting study on the genetic diversity of two genera of Atheriniformes found in Chilean hydrographic basins. The study provides a detailed methodology, and most of the results discussed are well supported. However, there are some issues that need to be addressed before the manuscript can be accepted for publication. The main concerns requiring revision are as follows:

Comments 1: - The first issue that caught my attention is the use of terms such as “phylogenetic analysis” or “phylogenetic reconstruction” when referring to analyses in which the terminals are individuals of the same species. Although this is a common mistake, it should be corrected. An analysis can only be accurately described as a “phylogenetic analysis” when it investigates phylogenetic relationships, that is, relationships among species or higher taxonomic levels. Intraspecific relationships, by contrast, are genealogical or tokogenetic in nature, not phylogenetic. Therefore, only analyses with species or higher taxonomic units as terminals should be referred to as phylogenetic. Similarly, it is incorrect to describe a species as “monophyletic.” Please revise these terms accordingly throughout the manuscript.

Response 1:  We thank Referee 2 for bringing this important point to our attention. In fact, we did not use "phylogenetic analysis" or "phylogenetic reconstruction" with care. As referee 2 mentions, in all our analyses the terminals are individuals, however, in the maximum likelihood analysis using IQ-Tree (Nguyen et al. 2015) several representatives of different species were used: the Odontesthes tree comprises O. regia , O. brevianalis , and O. mauleanum; and the Basilichthys tree comprises B. semotilus, B. australis (supposedly junior synonymous species), and B. microlepidotus. Only for this analysis we keep the term “phylogenetic”. This is consistent with our study's objective: "to analyze the diversity within and among species of Chilean silversides using genomic data (RADseq) to better characterize the genetic boundaries between species". Initially, these analyses were intended to represent phylogenetic relationships among species; however, the results revealed otherwise.

Once the synonymy of certain species has been confirmed or suggested (e.g., B. australis and B. microlepidotus), in all other analyses we changed the term “phylogenetic” to “genealogical or tokogenetic”. This ensures that the terminology accurately reflects the nature of the relationships being studied.

Nguyen, L.T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2015, 32, 268–274, doi:10.1093/molbev/msu300.

Comments 2: - The final section of the discussion, titled “The role of glaciations and geological events in shaping genetic patterns,” should be removed from the manuscript. Since the current study does not perform a time-calibrated analysis, it does not provide evidence to support or refute the influence of specific geological events on the distribution of the analysed populations. As a result, this section is not supported by the data presented. Please exclude it from the manuscript.

Response 2: Thanks for the comment. We agree. We removed the section from the manuscript.

Comments 3: - Lines 131-133: “Initial assignment of individuals of Odontesthes to species was based on locality: estuarine or coastal fishes were assigned to O. brevianalis, while individuals from lakes and deep rivers to O. mauleanum”. Since the authors indicate that species within the same genus are very similar and require close examination of specific characteristics for accurate identification, the manuscript must clearly explain how the individuals were identified. As currently written, the text gives the impression that specimens were identified solely based on the locality where they were collected. If this is the case, it raises serious concerns about the reliability of the data, as such an approach could lead to misidentifications. This, in turn, might explain the placement of certain terminals in Figure 2 and could potentially affect the overall results of the study. Please clarify the identification procedures used and verify whether the results need to be re-evaluated accordingly.

Response 3: Thank you for pointing this out. We agree with your comment and have carefully revised the terminology throughout the manuscript to ensure accuracy. Odontesthes brevianalis and O. mauleanum were difficult to distinguish in the field, and are named here based on typical habitat, which is estuarine/riverine for O. brevianalis, and primarily lacustrine for O. mauleanum. These two species are separated by a single morphological character, the presence of “noticeably crenate scales” along the side of O. mauleanum, or restricted only to the caudal peduncle for O. brevianalis (Dyer, 2006). However, this feature mentioned in the literature does not seem to be robust to observations of individuals (personal communication).

Dyer, B. S. (2006). Systematic revision of the South American silversides (Teleostei, Atheriniformes). Biocell, 30, 69–88.

As minor issues:

Comments 4: - Line 66: “Odontesthes” The name of the genus should be italicized.

Response 4: Done.

Comments 5: - Lines 102-103: “Veliz et al. [21] showed that this species and B. microlepidotus form (sic) a monophyletic group and share the most common mitochondrial haplotypes”. Please revise this sentence. The fact that two species form a monophyletic group only indicates that they are sister species. It should be made clear that, in this case, they were considered synonyms because neither represents a genetically exclusive lineage.

Response 5:We agree. We've modified the sentence to make sense.

Comments 6:  - Lines 412-413: “Previous studies suggest that individuals may use marine routes for such movements”. Could headwater capture events be related to the current distribution of these groups?

Response 6: Certainly, headwater capture events could be related to the dispersal of these groups. In fact, the two processes are not mutually exclusive. However, there is no evidence yet to support the occurrence of headwater capture events in this group. Given that silversides have a broad capacity to survive in different salinity ranges (Hughes et al 2017; Hughes et al 2020) and that the habitat reconstruction of their most recent ancestor appears to be marine, previous studies suggest that individuals could use marine routes for such movements.

Hughes, L.C.; Somoza, G.M.; Nguyen, B.N.; Bernot, J.P.; González-Castro, M.; Díaz de Astarloa, J.M.; Ortí, G. Transcriptomic Differentiation Underlying Marine-to-Freshwater Transitions in the South American Silversides Odontesthes aArgentinensis and O. Bbonariensis (Atheriniformes). Ecol. Evol. 2017, 7, 5258–5268, doi:10.1002/ece3.3133.

Hughes, L.C.; Cardoso, Y.P.; Sommer, J.A.; Cifuentes, R.; Cuello, M.; Somoza, G.M.; González-Castro, M.; Malabarba, L.R.; Cussac, V.; Habit, E.M.; et al. Biogeography, Habitat Transitions and Hybridization in a Radiation of South American Silverside Fishes Revealed by Mitochondrial and Genomic RAD Data. Mol. Ecol. 2020, 29, 738–751, doi:10.1111/mec.15350.

Comments 7: - Line 451: “which also gives it a high degree of endemism”. This sentence seems inconsistent with the context of the current study, as both analysed genera exhibit broad geographic distributions.

Response 7: We thank the reviewer for their comment. However, we do not consider the term to be inconsistent, as the phrase refers to the high degree of endemism characteristic of Chilean ichthyofauna in general, a concept well documented in previous studies (Habit et al., 2006; Vargas et al., 2015). Regarding the species analyzed in this study (O. brevianalis, O. mauleanum, and B. microlepidotus), they have geographic distributions restricted to Chile, only in the southernmost trans-Andean sectors of Patagonia, which reinforces their relevance as endemism for the described region.

Habit, Evelyn, Dyer, Brian, & Vila, Irma. (2006). Estado de conocimiento de los peces dulceacuícolas de Chile. Gayana (Concepción), 70(1), 100-113. https://dx.doi.org/10.4067/S0717-65382006000100016

Vargas, P. V., Arismendi, I., & Gomez-Uchida, D. (2015). Evaluating taxonomic homogenization of freshwater fish assemblages in Chile. Revista chilena de historia natural, 88, 1-10.

Comments 8: - Line 454: “We have confirmed the synonymy of B. microlepidotus …” As scientists we may corroborate of contest a hypothesis, but we will never confirm, or prove it definitively.

Response 8: Thanks for the comment. We agree. We changed the word "confirm" to "corroborate".

Comments 9: - Lines 464-466: “However, more research is needed to better understand the factors driving genetic differentiation of silverside populations”. This type of statement is commonly found at the end of scientific papers, but it can become vague or uninformative. Please specify what kind of future research is needed to improve our understanding of the genetic differentiation among these populations.

Response 9: Thank you, referee 2, for the comment. We agree that this sentence may be vague or uninformative. We've added a few final lines to the text indicating what kind of future research is needed to improve our understanding of the genetic differentiation between these populations.

Lines 494-500: ”However, further research is needed to better understand the factors driving genetic differentiation in silverside populations. Future research using more integrative approaches could be very useful for decision-making, such as: (i) comparing geographically separated populations from different habitat types (fjords, rivers, lakes, ocean); (ii) combining genetics with oceanographic/hydrological models that simulate currents and larval dispersal; (iii) and relating genetic differences to environmental variables (salinity, temperature, oxygen, pH, pollution) using genotype-environment association models (Redundancy Analysis-RDA , Latent Factor Mixed Models-LFMM).”