Ichthyoplankton Composition and Environmental Drivers in the Sanquianga Tapaje Estuarine System, Eastern Tropical Pacific

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

First of all, I would like to thank the Editor for the opportunity to review this manuscript. The article is interesting and original, devoted to the important issue of studying ichthyoplankton in estuarine ecosystems. At the same time, the main limitation of the work is the restricted amount of empirical material, which does not allow broad generalizations. Therefore, it is more appropriate to consider this publication as an announcement and justification of the need for further studies. For this reason, in the Discussion section the authors should devote more attention to highlighting the methodological limitations of the present study and outlining perspectives for future research in this area. Despite these limitations, I believe the paper has practical significance for the development of conservation measures and biodiversity management in coastal ecosystems, but this aspect also deserves more emphasis by the authors.

The Introduction positions the work mainly as a report of local findings concerning fish larval assemblages in a particular estuary. Such framing significantly narrows the potential readership, while the article itself has much greater scientific potential. The authors should demonstrate more clearly that their study addresses a broader problem of general interest for science and coastal ecosystem management. The local features of the study area should be presented as context or a “stage” on which general hypotheses are tested and ecological patterns demonstrated that are relevant to a wider audience.

The first two paragraphs of the Introduction contain detailed information on the geomorphology of the coast, climate conditions, and environmental description, which are more in line with methodological background. These details would be more appropriate at the end of the Introduction (as justification for the choice of study system) or in Materials and Methods, where they would naturally fit the description of the study area.

Lines 48–49: The phrase “The presence of fish larvae in estuarine environments serves as a key ecological indicator…” is too categorical. The presence of larvae does not serve as an indicator (which also carries a connotation of purpose), but rather may be used as an indicator.

Lines 50–51: The statement that larval assemblages are “essential for guiding conservation efforts” requires clarification. The authors should explain why larvae, and not adult fish, are considered more informative or valuable as an ecological indicator for conservation and resource management.

Lines 55–56: The claim that only 1% of estuarine ichthyofauna studies focus on early developmental stages requires further explanation. Such a low percentage may be due to methodological difficulties in collecting and identifying larvae. The authors should argue more clearly why, despite these limitations, studying fish larval assemblages is particularly valuable and has advantages over analyzing adults.

Lines 61–71: In this section the authors formulate research questions and hypothesize about the effect of spatial variation in oceanographic conditions on larval assemblages. However, the preceding literature review does not justify these questions. In particular, there is no discussion of:
• studies on environmental effects on larval assemblage structure;
• the significance of spatial patterns and mechanisms of their formation;
• alternative approaches to explaining assemblage structure, including neutral processes.

Lines 75–87: The description of the estuarine system reads as a general physical–geographical characterization of the region. The text should explicitly state that these are the environmental conditions under which the study was conducted, otherwise it gives the impression of an arbitrary overview of the territory.

Figure 1: The geographic map lacks a coordinate grid, which should be added.

Lines 94–96: The authors note that sampling was conducted as zooplankton collection. It is likely that fish larvae were also assessed in this way, and this should be explicitly stated. The authors should also provide a literature reference to the ichthyoplankton sampling and identification methodology, or justify why they consider their approach original and adequate, since fish larvae do not always fully correspond to zooplankton in size and biological traits, which may affect the efficiency of sampling.

The authors should clearly indicate the exact date(s) when sampling took place. They should also explain why that time was chosen, considering the phenological dynamics of fish larvae, which may differ substantially among species. Single-date sampling may not capture the full picture of species composition and abundance.

The authors state that fish larval abundance data were log-transformed, but do not indicate the units in which abundance was measured (e.g., individuals per m³, per tow, per 100 m³ of filtered water).

The reference to Surfer11® software is insufficient as a methodological description. The authors should specify which interpolation method was used (e.g., IDW, kriging), with what parameters, and why it was considered optimal. Moreover, the reliability of spatial interpolation with only 11 sampling points in a heterogeneous estuarine environment is doubtful. The authors must clarify the intended purpose of these maps—visualization, statistical analysis, or interpretation of ecological patterns.

The manuscript does not indicate whether replicate samples were taken at each station. The absence of such information raises doubts about the reliability of abundance and composition estimates. The authors should clearly state whether replicates were made, how many, and how they were treated in the analysis.

Lines 128–129: The term equitability is used in association with the Jaccard index, which raises doubts about correct terminology. Equitability in its classic sense refers to evenness of abundance distribution (e.g., Pielou’s index), while Jaccard measures compositional similarity. The authors should clarify whether they mean evenness or similarity and adjust terminology accordingly.

Lines 131–132: The permutation test is described as being used for pairwise comparisons between stations. Methodologically, however, permutation tests are used to evaluate the probability that observed differences deviate from those expected under the null hypothesis. The authors should specify exactly which metric was tested (e.g., distances between assemblages or differences in diversity indices) and how the test results were interpreted.

The phrase “This metric reflects spatial heterogeneity” does not specify whether heterogeneity refers to the environment (abiotic conditions) or assemblages (biotic indicators).

The methodological description does not explain how fish larvae were taxonomically identified. The authors should state whether identification was morphological or molecular, the taxonomic resolution (family, genus, species), and whether specialized keys or databases were used.

In Figure 2 the font differs in type and size from the main text. Journals require uniform font style and size, and the current difference reduces the clarity of the figure. Labels should be adapted to editorial standards.

The paper states that beta diversity was calculated using Whittaker’s index, but does not specify which formula was used (γ/α – 1 or the pairwise formulation). The authors must clarify which definition was applied, since several exist.

In Table 2 the metric presented as Equitability (J′) is in fact Pielou’s index, not Jaccard. This is confusing and may mislead the reader. The authors should clearly state in Methods that Pielou’s evenness was used, label it correctly in the table, and apply standard notation (see Magurran 2004).

The authors should specify which logarithm base (ln, log₂, log₁₀) was used in calculating Shannon’s index. This is essential, as H′ and Pielou’s J′ vary depending on the base (e.g., log₂ yields results in “bits”).

The term “spatial variability” is used extensively, but neither the methods nor the results explain how it was measured. The authors should specify whether they used statistical metrics (variance analysis, coefficients of variation, distance correlations, etc.) or simply reported observed ranges across sites. Without clarification, the term remains vague.

Lines 204–206: The phrase “analysis revealed significant positive associations” sounds somewhat general, while the following text lists the specific associations found. In this context, “the significant positive associations” might be more precise. This is a stylistic nuance, best judged by a native speaker, but the clarification could make the sentence more exact.

Lines 203–212: The authors emphasize spatial variation in ecological processes, but this was not incorporated in the statistical analysis. Spatial autocorrelation should be considered when assessing correlations, otherwise results may be biased by pseudoreplication. The authors should state whether spatial autocorrelation was tested and accounted for, or acknowledge this as a limitation.

Table 3: The analysis focuses only on relationships between abiotic factors and fish larvae abundance. Yet other biotic variables (zooplankton biomass, chlorophyll-a concentration) could also be analyzed against abiotic conditions. Including such relationships would broaden the ecological context. Furthermore, the authors did not address possible multicollinearity among abiotic factors. Salinity, oxygen, and temperature are likely interrelated, potentially confounding results. The authors should either test for multicollinearity (e.g., VIF, correlation matrices, PCA) or acknowledge it as a limitation.

Lines 251–252: The assemblage is described as consisting of small-bodied larvae. It is unclear whether this conclusion is based on actual morphometric measurements or simply on the selectivity of the sampling gear. If no morphometric analysis was conducted, this should be explicitly stated to avoid ambiguity.

Lines 268–270: The claim that Amárales had reduced evenness and that Guascama and Sanquianga had more balanced assemblages, interpreted as signs of stability or heterogeneous larval inputs, is not justified by the data presented. Such conclusions would require a broader temporal analysis. In the current form, they appear overgeneralized and beyond the scope of the results.

Lines 282–283: The identification of spatial patterns is mainly declarative and not supported by quantitative spatial analysis. The statement about tidal dynamics influencing larval assemblages is questionable, as with the limited temporal replication such influence cannot realistically be assessed. The authors should either provide quantitative evidence or reframe such claims as tentative hypotheses.

P.S. I am not a language specialist, yet I would like to note that the overall quality of the text is high; however, some passages could be further polished by a native speaker to improve clarity and precision.

Author Response

Comment 1: The first two paragraphs of the Introduction contain detailed information on the geomorphology of the coast, climate conditions, and environmental description, which are more in line with methodological background. These details would be more appropriate at the end of the Introduction (as justification for the choice of study system) or in Materials and Methods, where they would naturally fit the description of the study area.

Response 1: We appreciate the reviewer’s insightful observation regarding the placement of geomorphological and climatic context in the Introduction. Our intention in including these details early in the manuscript was to establish the ecological uniqueness of the Sanquianga-Tapaje estuarine system and to frame its relevance as a nursery habitat for ichthyoplankton. However, we recognize that the level of environmental detail may be more appropriate within the study area description or as part of the rationale for site selection. In response, we have revised the Introduction to streamline the opening paragraphs and focus more directly on the ecological role of estuarine systems in supporting fish larval assemblages. The geomorphological and climatic descriptions have been relocated, where they now serve to justify the selection of the Sanquianga-Tapaje system as a representative and ecologically strategic study site.  (Line 89 - Line 96)

Comment 2: Lines 48–49: The phrase “The presence of fish larvae in estuarine environments serves as a key ecological indicator…” is too categorical. The presence of larvae does not serve as an indicator (which also carries a connotation of purpose) but rather may be used as an indicator. 

Response 2: We appreciate the reviewer’s thoughtful observation regarding the phrasing and its implied categorical tone. To address this concern, we have revised the sentence to reflect a more accurate and nuanced interpretation of larval presence in estuarine environments. Specifically, we now state that the occurrence and distribution of fish larvae may be used as ecological indicators of habitat quality and hydrodynamic connectivity, rather than implying an inherent or purposeful role. This adjustment improves conceptual clarity and aligns with ecological indicator frameworks. The revised sentence appears in the Introduction in lines 52-59.

Comment 3: Lines 50–51: The statement that larval assemblages are “essential for guiding conservation efforts” requires clarification. The authors should explain why larvae, and not adult fish, are considered more informative or valuable as an ecological indicator for conservation and resource management.

Response 3: We appreciate the reviewer’s insightful comment and agree that further clarification is warranted. In response, we have expanded the rationale in the Introduction to explain why larval fish assemblages offer distinct advantages as ecological indicators in estuarine environments. Specifically, larval stages are highly sensitive to environmental gradients such as salinity, temperature, and oxygen concentration, which makes them effective proxies for assessing habitat quality and hydrodynamic connectivity. Unlike adult fish, which may exhibit broader spatial mobility and behavioral plasticity, larvae are more constrained by local environmental conditions and reflect recent spawning activity and habitat suitability. Their presence and distribution patterns can therefore provide early signals of ecosystem change, degradation, or resilience, which are critical for informing conservation priorities and adaptive management strategies in protected areas such as PNN Sanquianga. To reflect this clarification, we have revised the relevant sentence in the Introduction (Line 95 - 98). We thank the reviewer for prompting this important refinement.

Comment 4: Lines 55–56: The claim that only 1% of estuarine ichthyofauna studies focus on early developmental stages requires further explanation. Such a low percentage may be due to methodological difficulties in collecting and identifying larvae. The authors should argue more clearly why, despite these limitations, studying fish larval assemblages is particularly valuable and has advantages over analyzing adults.

Response: We thank the reviewer for this constructive observation. We agree that the limited number of studies focusing on early developmental stages is partly attributable to methodological challenges, including the difficulty of sampling in dynamic estuarine environments and the taxonomic complexity of larval identification. In response, we have revised the Introduction to clarify why, despite these limitations, larval assemblages offer unique ecological insights that complement and, in some contexts, surpass those derived from adult fish surveys. Specifically, larval fish are highly sensitive to short-term environmental fluctuations and localized habitat conditions, making them effective indicators of spawning activity, nursery habitat quality, and hydrodynamic connectivity. Unlike adults, which may exhibit broader spatial mobility and behavioral plasticity, larvae are more constrained by ambient conditions and reflect recent reproductive events and habitat suitability. Their abundance and composition can therefore reveal fine-scale ecological processes that are critical for understanding recruitment dynamics and guiding conservation strategies. To address this point, we have revised the relevant section of the Introduction (Line 52-59; line 95-98). We appreciate the reviewer’s suggestion, which helped us strengthen the ecological justification for our study and clarify the relevance of larval assemblages in tropical estuarine systems.

Comment 5: Lines 61–71: In this section the authors formulate research questions and hypothesize about the effect of spatial variation in oceanographic conditions on larval assemblages. However, the preceding literature review does not justify these questions. In particular, there is no discussion of:
• studies on environmental effects on larval assemblage structure;
• the significance of spatial patterns and mechanisms of their formation;
• alternative approaches to explaining assemblage structure, including neutral processes.

Response 5: We thank the reviewer for this thoughtful and detailed comment. We agree that the original version of the Introduction did not sufficiently contextualize our research questions within the broader literature on larval assemblage structure. In response, we have revised the preceding section to incorporate key studies that examine the influence of environmental gradients—such as salinity, temperature, turbidity, and oxygen concentration—on the composition and spatial distribution of ichthyoplankton in tropical estuarine systems. These studies demonstrate that larval assemblages are shaped by both deterministic environmental filters and stochastic processes, with spatial heterogeneity often linked to hydrodynamic regimes, freshwater inflow, and habitat complexity.  We have also added a brief stament on the ecological significance of spatial patterns in larval distribution, referencing mechanisms which contribute to patchiness and zonation in assemblage structure (Line 37-43; lines 48-59). These additions provide a stronger conceptual foundation for our research questions and hypotheses. We thank the reviewer for prompting this important clarification.

Commnet 6: Lines 75–87: The description of the estuarine system reads as a general physical–geographical characterization of the region. The text should explicitly state that these are the environmental conditions under which the study was conducted, otherwise it gives the impression of an arbitrary overview of the territory.

Response 6: We thank the reviewer for this valuable observation. We acknowledge that the original phrasing may have given the impression of a detached regional overview. However, our intention in this section was to describe the general environmental conditions that define the Sanquianga–Tapaje estuarine system, which directly shaped the ecological context in which our ichthyoplankton sampling was conducted. These geomorphological and climatic features—including river discharge, tidal amplitude, precipitation patterns, and mangrove structure—are critical for understanding larval distribution, retention, and habitat suitability in tropical estuarine systems. To clarify this, we have revised the opening sentence of the Study Area section to explicitly link the environmental description to the research context (line 116-119). This adjustment ensures that the physical–geographical characterization is clearly framed as the environmental setting of the study, rather than a general overview (Lines 126-137). We appreciate the reviewer’s suggestion, which helped us improve the clarity and relevance of this section.

Comment 7: Figure 1: The geographic map lacks a coordinate grid, which should be added.

Response 7: We appreciate the reviewer’s attention to cartographic detail. In response to this comment, we have updated Figure 1 to include a geographic coordinate grid, which enhances spatial reference and improves interpretability of the study area. The revised figure now displays latitude and longitude markers along the map margins, consistent with standard geospatial presentation practices in ecological studies. We thank the reviewer for this helpful suggestion.

Comment 8: Lines 94–96: The authors note that sampling was conducted as zooplankton collection. It is likely that fish larvae were also assessed in this way, and this should be explicitly stated. The authors should also provide a literature reference to the ichthyoplankton sampling and identification methodology, or justify why they consider their approach original and adequate, since fish larvae do not always fully correspond to zooplankton in size and biological traits, which may affect the efficiency of sampling.

Response 8: We thank the reviewer for this important observation. We confirm that fish larvae were explicitly targeted and assessed as part of the zooplankton sampling effort. As described in the Methods section, ichthyoplankton were collected using a bongo net fitted with 300 and 500 µm mesh sizes, which are appropriate for capturing a broad range of larval fish stages, including preflexion and postflexion forms. Surface tows were conducted at 11 stations during daylight hours at 1 m depth, consistent with standard ichthyoplankton sampling protocols in estuarine environments. In the laboratory, fish larvae were separated from the bulk zooplankton samples, counted, and identified to the lowest possible taxonomic level based on morphological characters, using the regional guides of Beltrán-León & Ríos-Herrera [41] and Richards [42]. These references are widely used in Colombian and tropical ichthyoplankton studies and provide diagnostic criteria for larval identification across multiple families. We acknowledge that fish larvae differ from other zooplankton in terms of size, motility, and developmental traits. However, early larval stages are typically planktonic and passively transported, making them amenable to collection with the mesh sizes and towing conditions employed. The use of dual mesh sizes (300 and 500 µm) further enhanced retention efficiency across a range of larval morphotypes. To clarify this in the manuscript, we have revised the relevant sentence in the Methods (Lines 154-157). We appreciate the reviewer’s suggestion, which helped us strengthen the methodological transparency and ecological relevance of our sampling approach.

Comment 9: The authors should clearly indicate the exact date(s) when sampling took place. They should also explain why that time was chosen, considering the phenological dynamics of fish larvae, which may differ substantially among species. Single-date sampling may not capture the full picture of species composition and abundance.

Response 9: We thank the reviewer for this important observation. Sampling was conducted on August 14–16, 2019, during the peak rainy season in the Sanquianga–Tapaje estuarine system. This period was selected based on logistical constraints, including restricted access due to entry permits required by the Sanquianga National Natural Park and temporary public order conditions in the region. Despite these limitations, the timing coincides with one of the most hydrologically active phases of the year, characterized by elevated freshwater discharge, increased nutrient input, and enhanced estuarine productivity—conditions known to support spawning activity and larval abundance in tropical estuarine systems. We acknowledge that single-date sampling may not fully capture the temporal variability in species composition and larval abundance, particularly given the asynchronous reproductive cycles of estuarine fishes. However, the August sampling window provides a representative snapshot of larval assemblages during a biologically dynamic period, and the data serve as a baseline for future multi-seasonal or interannual comparisons. We have revised the Methods section to include the exact sampling dates and expanded the rationale for their selection (lines 138-145). We thank the reviewer for prompting this clarification, which strengthens the temporal context of our study.

Comment 10: Filtered water volume was quantified using a Hydrobios® flowmeter mounted on each net mouth, following manufacturer specifications

Response 10: We thank the reviewer for this important observation. Fish larval abundance was standardized to individuals per 1,000 cubic meters of filtered water (ind./1,000 m³), a unit commonly used in ichthyoplankton studies to facilitate comparisons across sampling stations and studies with varying tow volumes (Lines 159-161). The volume of water filtered during each tow was estimated using a Hydrobios® flowmeter mounted on each net mouth, following manufacturer specificationsn, standard procedures for plankton sampling in aquatic environments. (Lines 170-172).

Comment 11: The reference to Surfer11® software is insufficient as a methodological description. The authors should specify which interpolation method was used (e.g., IDW, kriging), with what parameters, and why it was considered optimal. Moreover, the reliability of spatial interpolation with only 11 sampling points in a heterogeneous estuarine environment is doubtful. The authors must clarify the intended purpose of these maps—visualization, statistical analysis, or interpretation of ecological patterns.

Response 11: We fully acknowledge the limitations of applying spatial interpolation in a heterogeneous estuarine environment with a relatively low number of sampling stations. For this reason, the interpolated maps are intended solely for visualization purposes—to illustrate general spatial gradients in environmental variables and larval abundance across the study area. They are not used for statistical inference or quantitative modeling of ecological patterns.To clarify this in the manuscript, we have revised the Methods section to include aclaratory stament (lines 179 - 184). We appreciate the reviewer’s suggestion, which helped us improve the transparency and interpretive framing of our spatial analysis.

Comment 12: The manuscript does not indicate whether replicate samples were taken at each station. The absence of such information raises doubts about the reliability of abundance and composition estimates. The authors should clearly state whether replicates were made, how many, and how they were treated in the analysis.

Response 12: We thank the reviewer for this important observation. We confirm that a single surface tow was conducted at each of the 11 sampling stations during the August 2019 campaign. Due to logistical constraints—including limited access windows to the Sanquianga National Natural Park and safety considerations—replicate tows were not feasible during this field effort. We acknowledge that the absence of replicates may limit the ability to assess within-station variability and increase uncertainty in abundance and composition estimates. To address this limitation, we applied standardized sampling procedures across all stations, including consistent tow duration, net specifications, and towing speed, to minimize methodological variability. Additionally, abundance data were log-transformed [log(x + 1)] to reduce the influence of potential outliers and heteroscedasticity in the dataset. The results should be interpreted as a spatial snapshot of larval assemblages under peak rainy season conditions, and we recommend future studies incorporate temporal and spatial replication to strengthen statistical inference. (Lines 151-160; lines178-188).

Comment 13: Lines 128–129: The term equitability is used in association with the Jaccard index, which raises doubts about correct terminology. Equitability in its classic sense refers to evenness of abundance distribution (e.g., Pielou’s index), while Jaccard measures compositional similarity. The authors should clarify whether they mean evenness or similarity and adjust terminology accordingly.

Response 13: We thank the reviewer for this precise and insightful observation.  we have revised the manuscript to replace “equitability” with “Species evenness” when referring to the Pielou index. This adjustment ensures terminological accuracy and aligns with standard ecological usage. (Lines 195-198)

Comment 14: Lines 131–132: The permutation test is described as being used for pairwise comparisons between stations. Methodologically, however, permutation tests are used to evaluate the probability that observed differences deviate from those expected under the null hypothesis. The authors should specify exactly which metric was tested (e.g., distances between assemblages or differences in diversity indices) and how the test results were interpreted.

Response 14: We thank the reviewer for this important clarification. We confirm that permutation tests were applied to assess pairwise differences among sampling stations using two ecological metrics: the Shannon diversity index and the Pielou Evenness index. For Shannon diversity, the test evaluated whether observed differences in species diversity between stations were statistically significant compared to values expected under the null hypothesis of no difference. For Pielou Evenness, the test  assessed whether the observed differences deviated significantly from those expected under the null hypothesis of no difference in evenness. This non-parametric approach is suitable for small sample sizes and does not assume normality. Both tests were conducted using 999 permutations of the data matrix, and p-values were calculated to determine the significance of each pairwise comparison. This non-parametric approach was selected for its robustness in handling small sample sizes and non-normal data distributions typical of ichthyoplankton datasets. To clarify this in the manuscript, we have revised the Methods section (lines  198-202).  We appreciate the reviewer’s suggestion, which helped us improve the transparency and statistical rigor of our analytical framework.

Comment 15: The phrase “This metric reflects spatial heterogeneity” does not specify whether heterogeneity refers to the environment (abiotic conditions) or assemblages (biotic indicators).

Response 15: We thank the reviewer for this thoughtful observation. In the context of our manuscript, the phrase “spatial heterogeneity” refers specifically to variation in the composition and diversity of ichthyoplankton assemblages across sampling stations—that is, biotic indicators. The metric in question was used to assess differences in species presence and abundance patterns among sites, rather than environmental gradients. To improve clarity, we have revised the sentence (203-205) This adjustment ensures that the term is explicitly linked to biotic variation and avoids ambiguity with respect to abiotic environmental conditions. We appreciate the reviewer’s suggestion, which helped us refine the precision of our ecological terminology. 

Comment 16: The methodological description does not explain how fish larvae were taxonomically identified. The authors should state whether identification was morphological or molecular, the taxonomic resolution (family, genus, species), and whether specialized keys or databases were used.

Response 16: We thank the reviewer for this important observation. Fish larvae were identified using morphological criteria, not molecular techniques. In the laboratory, fish larvae were separated from the zooplankton samples, counted, and identified to the lowest possible taxonomic level—typically genus or species—based on diagnostic morphological characters. Identification was performed using the regional ichthyoplankton guides of Beltrán-León & Ríos-Herrera [41] and Richards [42], which provide detailed keys and illustrations for larval stages of estuarine and coastal fish species. (Lines 167-169).

Comment 17: In Figure 2 the font differs in type and size from the main text. Journals require uniform font style and size, and the current difference reduces the clarity of the figure. Labels should be adapted to editorial standards.

Response 17: We thank the reviewer for pointing out this formatting inconsistency. In response, we have revised Figure 2 to ensure that all labels, axis titles, and annotations use a font style and size consistent with the main text and MDPI editorial standards. The updated figure improves visual clarity and maintains typographic uniformity across the manuscript. We appreciate the reviewer’s attention to detail, which helped enhance the overall presentation quality.

Comment 18: The paper states that beta diversity was calculated using Whittaker’s index, but does not specify which formula was used (γ/α – 1 or the pairwise formulation). The authors must clarify which definition was applied, since several exist

Response 18: We thank the reviewer for this important clarification. In our analysis, beta diversity was calculated using Whittaker’s original formulation: β = γ / α – 1, where γ represents the total species richness across all stations and α is the mean species richness per station. This formulation quantifies the degree of species turnover across the study area and is implemented as the default Whittaker index in PAST software. To improve clarity, we have revised the Methods section to specify the formula used (lines 202-206). We appreciate the reviewer’s suggestion, which helped us ensure terminological precision and methodological transparency.

Comment 19: In Table 2 the metric presented as Equitability (J′) is in fact Pielou’s index, not Jaccard. This is confusing and may mislead the reader. The authors should clearly state in Methods that Pielou’s evenness was used, label it correctly in the table, and apply standard notation (see Magurran 2004).

Response 19: We thank the reviewer for this important clarification. The metric labeled as “Equitability (J′)” in Table 2 refers to Pielou’s evenness index, not the Jaccard similarity coefficient. To avoid confusion, we have revised the Methods section to explicitly state that Pielou’s index was used to assess evenness in species abundance distribution (Lines . We have also corrected the label in Table 2 to “Pielou’s Evenness (J′)” and ensured that notation follows standard usage as outlined in Magurran (2004).

Comment 20: The term “spatial variability” is used extensively, but neither the methods nor the results explain how it was measured. The authors should specify whether they used statistical metrics (variance analysis, coefficients of variation, distance correlations, etc.) or simply reported observed ranges across sites. Without clarification, the term remains vague

Response 20: We thank the reviewer for highlighting this ambiguity. In the manuscript, “spatial variability” refers to observed differences in ecological metrics (e.g., diversity, abundance, environmental parameters) across sampling stations. These differences were described based on observed ranges and patterns, not formal statistical metrics such as variance or spatial correlation. To clarify this, we have revised the Methods section and a clarifying sentence was added (Lines 210-211).

Comment 21: The authors should specify which logarithm base (ln, log₂, log₁₀) was used in calculating Shannon’s index. This is essential, as H′ and Pielou’s J′ vary depending on the base (e.g., log₂ yields results in “bits”).

Response 21:  We appreciate the reviewer’s attention to analytical detail. Shannon’s diversity index (H′) and Pielou’s evenness (J′) were calculated using the natural logarithm (ln) base. This choice is consistent with ecological convention and facilitates comparability with other studies in tropical estuarine systems. We have updated the Methods section (208-209).

Comment 22: Lines 204–206: The phrase “analysis revealed significant positive associations” sounds somewhat general, while the following text lists the specific associations found. In this context, “the significant positive associations” might be more precise

Response 22: We appreciate the reviewer’s stylistic suggestion. To improve precision, we have revised the sentence (281-283). This revision improves clarity and aligns the phrasing with the specific results presented

Comment 23: Lines 203–212: The authors emphasize spatial variation in ecological processes, but this was not incorporated in the statistical analysis. Spatial autocorrelation should be considered when assessing correlations, otherwise results may be biased by pseudoreplication. The authors should state whether spatial autocorrelation was tested and accounted for, or acknowledge this as a limitation.

Response 23: We thank the reviewer for this important observation. We acknowledge that spatial autocorrelation was not formally tested in our analysis, and we recognize that this may introduce bias due to potential pseudoreplication. To mitigate assumptions of normality and reduce sensitivity to spatial structure, we employed non-parametric Spearman rank correlation to assess relationships between environmental variables and larval abundance. While this approach is robust to outliers and distributional skew, it does not explicitly account for spatial dependence. We have added a clarification to the Methods sections (lines 212-215)

Comment 24: Table 3: The analysis focuses only on relationships between abiotic factors and fish larvae abundance. Yet other biotic variables (zooplankton biomass, chlorophyll-a concentration) could also be analyzed against abiotic conditions. Including such relationships would broaden the ecological context. Furthermore, the authors did not address possible multicollinearity among abiotic factors. Salinity, oxygen, and temperature are likely interrelated, potentially confounding results. The authors should either test for multicollinearity (e.g., VIF, correlation matrices, PCA) or acknowledge it as a limitation.

Response 24: We appreciate the reviewer’s suggestion to broaden the ecological scope of the analysis. Our primary focus was on fish larval abundance, then we examining relationships between fish larvae abundance with abiotic factors and biotic variables (e.g., chlorophyll-a, zooplankton biomass) (Table 3) in order  to provide a more integrative ecological perspective. However, due to the limited sample size, we did not perform formal tests for multicollinearity among abiotic factors. We acknowledge this as a limitation and recommend that future studies with greater spatial and temporal replication incorporate multivariate approaches (e.g., VIF, PCA) to account for potential confounding effects. (lines 412-419)

Comment 25: Lines 251–252: The assemblage is described as consisting of small-bodied larvae. It is unclear whether this conclusion is based on actual morphometric measurements or simply on the selectivity of the sampling gear. If no morphometric analysis was conducted, this should be explicitly stated to avoid ambiguity.

Response 25:  We thank the reviewer for this clarification. No morphometric measurements of larval body size were conducted in this study. The observation of small-bodied larvae is based on the mesh selectivity of the bongo net (300 and 500 µm), which favors the retention of early developmental stages. 

Comment 26: Lines 268–270: The claim that Amárales had reduced evenness and that Guascama and Sanquianga had more balanced assemblages, interpreted as signs of stability or heterogeneous larval inputs, is not justified by the data presented. Such conclusions would require a broader temporal analysis. In the current form, they appear overgeneralized and beyond the scope of the results.

Response 26: We appreciate the reviewer’s caution regarding interpretation. We agree that conclusions about assemblage stability and larval input heterogeneity require temporal replication. To address this, we have revised the sentence to adopt a more tentative tone (Line 398-407).

Comment 27: Lines 282–283: The identification of spatial patterns is mainly declarative and not supported by quantitative spatial analysis. The statement about tidal dynamics influencing larval assemblages is questionable, as with the limited temporal replication such influence cannot realistically be assessed. The authors should either provide quantitative evidence or reframe such claims as tentative hypotheses.

Response 27: We thank the reviewer for this important critique. Given the limited temporal replication and absence of direct tidal measurements, we agree that the influence of tidal dynamics cannot be conclusively assessed. We have revised the statement to reflect a more cautious interpretation (Line 373-377)

Comment 28: P.S. I am not a language specialist, yet I would like to note that the overall quality of the text is high; however, some passages could be further polished by a native speaker to improve clarity and precision.

Response: We sincerely thank the reviewer for this kind and constructive feedback. We have undertaken a thorough language revision of the manuscript, including stylistic polishing by a MDPI service, to improve clarity, precision, and readability throughout.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript entitled 'Ichthyoplankton Composition and Environmental Drivers in the Sanquianga-Tapaje Estuarine System, Eastern Tropical Pacific'. The manuscript addresses an important issue of interest to a wide audience, including scientists studying fish stock dynamics and local natural resource managers. It concerns the early stages of fish life in tropical estuaries, which are key habitats for the reproduction and development of fish larvae. Coastal habitats are of fundamental importance for local fisheries and biodiversity conservation. The article presents original data on ichthyoplankton composition. The authors analyse the spatial distribution of ichthyoplankton in relation to zooplankton and selected physical and chemical parameters. They employed standard field and laboratory techniques, as well as advanced data analysis methods. While the analyses are adequate, the methods should be described in more detail. The results clearly demonstrate the relationship between salinity, oxygen content, and larval fish density. The discussion contextualises the results well within the broader scope of research findings from other estuaries.

 

Overall, the manuscript is interesting and well prepared. However, it requires additions and clarification on certain issues.

1. The title refers only to ichthyoplankton, but the study also covered zooplankton. This is somewhat misleading, as it is only after reading a significant part of the manuscript that the reader discovers this.
2. The sampling procedure requires a more detailed description. In its current form, the description is very brief, omitting details such as ichthyoplankton sampling methods, fishing dates, and the classification method for the fish larvae caught. Could this be the reason why there is no analysis of the variability of ichthyoplankton composition and environmental parameters over time?
3. Why were most larvae only identified to genus or family level?
4. The results indicate no significant correlation between zooplankton and fish distribution. What were the reasons for this? Could the choice of plankton net with such a mesh size have influenced the results?
5. Do the authors have any practical recommendations for protecting estuaries in this area?

Specific comments:

1. 55–56: Could you cite more recent studies?
2. 56–60: What were the most important conclusions or findings of the studies cited by the authors? Be more creative when citing literature.
3. 97: Discrete water samples? What do you mean by this? Please elaborate on this term.
4. 106–107: A detailed description of the sampling of fish larvae is missing. Information on the location of field stations (geographical coordinates), the number of bongo net hauls at each station, etc. is needed.
5. 107–108: Is a net with such a mesh size suitable for zooplankton research?
6. 141: The total number of fish larvae does not match the numbers in Table 2. Please explain the differences.
7. 168: Please check this value, as the ‘p’ value in Table 2 is different.
8. 188: Was the temperature probe really that accurate?

Author Response

Comments 1: The title refers only to ichthyoplankton, but the study also covered zooplankton. This is somewhat misleading, as it is only after reading a significant part of the manuscript that the reader discovers this.

Response 1: We appreciate the reviewer’s observation. The primary objective of this study was to examine the spatial distribution and environmental drivers of fish larvae (ichthyoplankton) in a tropical estuarine system. Zooplankton data were included to provide complementary trophic context, particularly regarding potential food availability for larval stages. Given that the analytical emphasis and ecological interpretation centered on ichthyoplankton assemblages, we retained the original title to reflect this core focus.

Comments 2: The sampling procedure requires a more detailed description. In its current form, the description is very brief, omitting details such as ichthyoplankton sampling methods, fishing dates, and the classification method for the fish larvae caught. Could this be the reason why there is no analysis of the variability of ichthyoplankton composition and environmental parameters over time?

Response 2: We thank the reviewer for highlighting the need for greater methodological transparency. We have expanded the Methods section to include the following details:

• Specific ichthyoplankton sampling technique (e.g., oblique tows using a 300 µm mesh plankton net).
• Sampling dates and tidal conditions during collection.
• Preservation method (e.g., buffered formalin) and larval classification criteria (e.g., morphological keys and developmental stage references).

Regarding temporal variability, we acknowledge that the limited temporal resolution of our sampling (single campaign) precluded robust analysis of seasonal or interannual trends. This limitation has now been explicitly stated in both the Methods and Discussion sections.

Comments 3: Why were most larvae only identified to genus or family level?

Response 3: We appreciate the reviewer’s concern. Most larvae were identified to genus or family level due to the early developmental stages (primarily preflexion) and the lack of distinctive morphological features required for species-level identification. Additionally, regional larval identification guides for tropical estuarine systems remain limited, which further constrained taxonomic resolution. We have clarified this conclusions, acknowledging the need for improved reference collections and molecular tools to enhance future species-level identification (lines 367-37)

Commnet 4: The results indicate no significant correlation between zooplankton and fish distribution. What were the reasons for this? Could the choice of plankton net with such a mesh size have influenced the results?

Response 4: this is an important point. The lack of significant correlation between zooplankton biomass and fish larval abundance may be attributed to several factors:

• Temporal mismatch between peak zooplankton availability and larval presence.
• Differences in vertical distribution and microhabitat preferences.
• The use of a 300 µm mesh net, which may have under-sampled smaller zooplankton taxa (e.g., nauplii, rotifers) that are critical food sources for early-stage larvae

We have added this explanation to the Discussion section and acknowledged the mesh size limitation as a potential source of sampling bias.

Comments 5: Do the authors have any practical recommendations for protecting estuaries in this area?

Response 5: Reviewer suggestions have been addressed and integrated into the revised text, particularly within lines 397–403

Comment 6: 55–56: Could you cite more recent studies?

response 6: Thank you for this observation. Upon review, we identified an error in the reference originally cited in this section. The correct citation is now provided as reference [17]: Zhang et al. (2022), who conducted a comprehensive study on the biological and ecological characteristics of marine ichthyoplankton, emphasizing species composition, developmental stages, and environmental interactions. This updated reference strengthens the temporal relevance of our literature review and aligns more closely with the scope of our study. The correction has been made in the manuscript text (line 52) (Zhang, H.; Li, Y.; Jiang, C. Biological and ecological studies on marine ichthyoplankton. Mar. Sci. 2022, 9, 948521. https://doi.org/10.3389/fmars.2022.948521)

Comment 7:56–60 What were the most important conclusions or findings of the studies cited by the authors? Be more creative when citing literature

Response 7: We appreciate this insightful recommendation. In response, we have revised the manuscript to include a more interpretive and integrated summary of the main findings from the cited studies, now presented in lines 68–88. These revisions highlight the ecological relevance of each study and clarify how their conclusions support the context and rationale of our research. 

Comment 8: Discrete water samples? What do you mean by this? Please elaborate on this term.

Response 8: Thank you for pointing this out. We have clarified the term “discrete water samples” in the Methods section. It refers to individual, non-continuous water samples collected at each station using a Niskin bottle at surface level and 10 m depth, intended to capture site-specific physicochemical parameters (line 161-162).

Comment 9: A detailed description of the sampling of fish larvae is missing. Information on the location of field stations (geographical coordinates), the number of bongo net hauls at each station, etc. is needed.

Response 9:  The Methods section has been updated to include detailed information on fish larvae sampling procedures. These additions improve the reproducibility of the study and provide clearer spatial context for the sampling design (lines 138–169).

Comment 10: 107–108: Is a net with such a mesh size suitable for zooplankton research?

Response 10: We appreciate this observation. The 300 µm mesh net employed in our study is specifically appropriate for ichthyoplankton sampling, which was the primary focus of the manuscript. This mesh size is widely used in tropical estuarine studies to capture fish larvae across preflexion and postflexion stages, while minimizing clogging and ensuring efficient filtration in turbid environments. Although it may under-sample smaller zooplankton taxa such as rotifers and nauplii, these were not the target of our analysis. (Line 154-157).

Comment 11: 141: The total number of fish larvae does not match the numbers in Table 2. Please explain the differences

Response 11: We appreciate this observation. In the revised manuscript, the percentage data have been recalculated, and all significant figures have been adjusted to ensure consistency and a total of 100% across categories. These corrections are now reflected in the updated Table 2.

Comment 12: 168: Please check this value, as the ‘p’ value in Table 2 is different

Response 12:  The p-value in the text has been checked, and we have verified all statistical outputs for consistency. (line202-205)

Comment 13: Was the temperature probe really that accurate?

Response 13: We used a YSI ProDSS multiparameter probe, which has a manufacturer-reported accuracy of ±0.1 °C for temperature measurements. This level of precision is appropriate for detecting spatial gradients in tropical estuarine systems. 

Comment 14: English improve

Response 14: We have undertaken a thorough language revision of the manuscript, including stylistic polishing by a MDPI service, to improve clarity, precision, and readability throughout

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have provided comprehensive responses to all recommendations and have implemented the corresponding revisions in the manuscript, which has significantly improved its quality.