Microsatellite-Based Evaluation of Genetic-Distance-Driven Crossbreeding in the Endangered Freshwater Fish Pseudopungtungia nigra

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a highly relevant topic in conservation genetics and artificial breeding of endangered freshwater fishes, using Pseudopungtungia nigra as a model species. The study contributes valuable empirical evidence on how genetic-distance-based crossbreeding can influence genetic diversity and early developmental performance, supported by microsatellite marker analysis. The methodology and objectives are generally well-aligned, and the results have potential application in conservation breeding and population restoration programs.

However, the manuscript requires major language revision, improved figure descriptions, clarification of experimental design, and deeper biological interpretation of statistical findings. While the technical foundation is sound, the presentation and discussion need refinement to ensure scientific clarity and publication readiness. All comments are organized from Title to References, including line-specific notes and overall evaluation.

1) Title and Abstract

Lines 1–27

• Comment (L1–3): Title is overly long and slightly awkward. Suggest revision to:
  “Microsatellite-Based Evaluation of Genetic-Distance-Driven Crossbreeding in the Endangered Freshwater Fish Pseudopungtungia nigra.”
  This reads more smoothly and reflects the study focus.
• L9–27: Abstract is comprehensive but wordy. Condense repetitive expressions like “genetic and fitness outcomes” → “genetic diversity and fitness traits.”
• L17–24: Avoid overinterpretation—“infinite effective population size” is misleading; clarify as “undeterminable due to small sample size.”
• L25–27: The keyword list is acceptable, but “genetic distance” and “genetic diversity” overlap; replace one with “microsatellite analysis.”

Key issues:

• No quantitative p-values or sample sizes in the abstract.
• The “Key Contribution” section is strong but somewhat self-promotional—simplify language.

2) Introduction

Lines 35–87

• L36–41: Nicely sets context but too general; cite recent global reviews on fish restoration genetics (e.g., Lutz et al., 2021).
• L46–54: Needs smoother flow—combine into one paragraph. Avoid repeating “endangered” too many times.
• L65–74: Well-written section on balancing diversity and local adaptation. Could add brief mention of outbreeding depression thresholds (Frankham 2011).
• L76–82: Claim that “few studies have been reported” needs supporting references—add 2–3 more relevant freshwater fish studies.
• L83–87: The last paragraph is repetitive and lacks a clear hypothesis. Suggest rewriting:

“This study tests whether crossbreeding between genetically distant pairs enhances genetic diversity and early survival in P. nigra without causing divergence from the broodstock population.”

3) Materials and Methods

Lines 88–198

• L90–98: Include GPS coordinates in Figure 1 legend for reproducibility.
• L105–117: Good explanation of VIE tagging, but could add information about tag retention validation or error rate.
• L118–136: DNA extraction description is detailed but too procedural—shorten brand names and catalog numbers.
• L127–137 (Table 1): Marker information table is fine but references (e.g., “Bang et al. 2023, Kim et al. 2025”) should be formatted consistently.
• L139–153: Table 2 on genetic distance lacks standard deviation or measure of genetic similarity; clarify if values are Nei’s or Cavalli-Sforza distance.
• L157–198: Statistical methods are described clearly, but details like “PHWE analyses” (L164) should be fully written as “Probability of Hardy–Weinberg Equilibrium.”
• Add a flow diagram summarizing sample collection, genotyping, and analysis—currently text-heavy.

Major Issue:

• Ethical approval is mentioned later but not here. Ethical statement should be placed at the end of “Materials and Methods.”
• Replication: Crossing appears to be single pair per genetic-distance category. Clarify if replicates (n>1) were performed or pooled.

4) Results

Lines 199–264

• L201–213 (Table 3): Provide standard errors for diversity estimates.
• L214–222 (Table 4): Interpretation of “infinite Ne” must be cautious—add a note in the Results explaining estimation uncertainty.
• L223–232 (Table 5): Statistical significance (p-values) should accompany FST results.
• L233–249 (Figures 3–4): Figures should be better annotated—currently unclear what colors correspond to which groups. Add clear legends.
• L250–257 (Figure 5): Include n-values and error bars with SD or SE. Use same scale for all groups for visual clarity.

Comment: Results are descriptive but lack biological linkage—authors should highlight what levels of heterozygosity imply for restoration programs.

5) Discussion

Lines 265–350

• L266–277: Good interpretation of heterozygosity, but very repetitive—merge redundant sentences.
• L279–283: Strong observation linking outcrossing and adaptability; however, cite at least one empirical freshwater fish example.
• L286–299: The bottleneck discussion is insightful but needs numerical linkage to Table 4.
• L300–308: The explanation for “infinite Ne” is excellent and shows understanding of statistical bias.
• L309–334: Discussion of genetic structure is valid but can be shortened; STRUCTURE vs. DAPC comparison is overly detailed for Discussion.
• L335–350: Hatching and survival rate explanation lacks mechanistic reasoning—should mention maternal egg quality or gamete compatibility.
• Add a final paragraph clearly stating management implications: how findings can be used for P. nigra conservation programs.

6) Conclusion

Lines 351–363

• Good summary but repetitive. Simplify to 4–5 sentences focusing on:
• Key result: higher hatching rate in genetically distant pairs.
• Conservation implication: use moderate genetic distance for restoration breeding.
• Future work: larger broodstock sample and genomic markers.

7) Author Contributions, Funding, and Ethics

Lines 364–378

• L364–369: Author contribution statement has redundancy; ensure compliance with MDPI format.
• L370–377: “Ethical review waived due to reason” is incomplete and unclear—must specify institutional policy and justification.
• L378: Typo: “There authors” → “The authors.”

8) References

Lines 379–527

• Citation style inconsistent—some years italicized, some not.
• Missing DOIs in several places (e.g., refs 8–9).
• Ref 31: “Microsatellite records for volume 12, issue 2. 2020.” is incomplete; must provide journal name.
• Ref 37–38: Update titles for clarity.
• Remove unnecessary details like “doi:” repetition.

9) Language and Technical Quality

• Numerous grammatical errors:
• “crossing group based on genetic distance” → “crossbred groups based on genetic distance.”
• “are separated and management in a 100 L tank” → “were separated and maintained in 100 L tanks.”
• Use consistent tense—past for Methods and Results, present for Discussion.
• Figures require higher resolution and consistent font style.

 

Recommendation

The manuscript presents important findings but requires major revision before it can be considered for publication. I recommend substantial language editing, figure and reference correction, and clearer justification and interpretation of experimental results. With these revisions, the paper could make a meaningful contribution to conservation genetics and artificial breeding management of endangered freshwater fish species.

 

Comments on the Quality of English Language

• Numerous grammatical errors found. Edit by Native language speaker

• Use consistent tense—past for Methods and Results, present for Discussion.

Author Response

Comment . Comment (L1–3): Title is overly long and slightly awkward. Suggest revision to:

“Microsatellite-Based Evaluation of Genetic-Distance-Driven Crossbreeding in the Endangered Freshwater Fish Pseudopungtungia nigra.”

This reads more smoothly and reflects the study focus.

1. Thanks for the review. Edited according to comments.

[Microsatellite-Based Evaluation of Genetic-Distance-Driven Crossbreeding in the Endangered Freshwater Fish Pseudopungtungia nigra]

 

Comment . L9–27: Abstract is comprehensive but wordy. Condense repetitive expressions like “genetic and fitness outcomes” → “genetic diversity and fitness traits.”

L17–24: Avoid overinterpretation—“infinite effective population size” is misleading; clarify as “undeterminable due to small sample size.”

L25–27: The keyword list is acceptable, but “genetic distance” and “genetic diversity” overlap; replace one with “microsatellite analysis.”

1. Thanks for the review. Edited according to comments.

[This study examined the genetic diversity and fitness traits of crosses between genetically distant (HGD) and closely related (LGD) broodstock individuals of Pseudopungtungia nigra, an endangered Korean freshwater fish. Using ten microsatellite loci, we evaluated genetic diversity, population structure, and early survival performance among crossbreeds and their broodstock.]

[The broodstock and both crossbred groups displayed bottleneck signals, while LD-based effective population size was infinite for the broodstock and HGD but finite for LGD, suggesting estimation bias because the parameter was undeterminable due to small sample size.]

 

Comment . No quantitative p-values or sample sizes in the abstract.

The “Key Contribution” section is strong but somewhat self-promotional—simplify language.

1. Thanks for the review. Edited according to comments.

[Both HGD and LGD progenies showed deviations from Hardy–Weinberg equilibrium and exhibited higher observed heterozygosity than expected, indicating the influence of artificial selection. The broodstock and both crossbred groups displayed bottleneck signals, while LD-based effective population size was infinite for the broodstock and HGD but finite for LGD, suggesting estimation bias because the parameter was undeterminable due to small sample size (each group, n=28-30). STRUCTURE and DAPC analyses revealed that HGD_20 was most genetically similar to the broodstock population, while LGD and HGD_19 formed distinct clusters. Hatching rate was 1.5-fold higher in HGD compared with LGD (P<0.05), although survival did not differ significantly (P>0.05). These results highlight that crossbreeding based on genetic distance can enhance genetic diversity and hatching performance without causing excessive genetic divergence from the parental population, offering a practical model for the genetic management of endangered fish restoration.]

[This study shows that genetic-distance based artificial crossing in the endangered freshwater fish Pseudopungtungia nigra can improve hatching performance while preserving genetic similarity to the broodstock, providing a practical approach for balancing genetic diversity and fidelity in conservation breeding programs.]

 

Comment . L36–41: Nicely sets context but too general; cite recent global reviews on fish restoration genetics (e.g., Lutz et al., 2021).

1. Thanks for the review. Edited according to comments.

[Consistent with this logic, Lutz et al. [8] demonstrated that reintroducing the endangered freshwater fish Macquaria australasica from multiple, genetically differentiated source populations produced a genetically diverse, self‐recruiting population, reducing the risk of harmful inbreeding and enhancing survival and growth.]

 

Comment . L46–54: Needs smoother flow—combine into one paragraph. Avoid repeating “endangered” too many times.

1. Thanks for the review. Edited according to comments.

[Pseudopungtungia nigra is an endangered fish found only in the Geumgang and Mangyeonggang Rivers of the Korean Peninsula [11,12]. Although previous population genetic and ecological studies provide a favourable basis for restoration planning [11,12], the broodstock available for release are constrained by collection limits, as the Korean government restricts the number of captive broodstock to fewer than 30 individuals, thereby limiting the effectiveness of artificial insemination–based programs [12]. Under such conditions, where only small broodstock pools can be maintained, hybridization strategies that increase or at least maintain genetic diversity become essential for successful restoration [7,13,14].]

 

Comment . L65–74: Well-written section on balancing diversity and local adaptation. Could add brief mention of outbreeding depression thresholds (Frankham 2011).

1. Thanks for the review. Edited according to comments.

[Empirical guidelines indicate that the risk of outbreeding depression increases when crosses involve populations that differ in karyotype, occupy markedly different environments, or have been isolated for long periods, whereas it is expected to be low when populations of the same species share karyotypes, inhabit similar environments, and have been separated for less than a few hundred years [21,22].]

 

Comment . L76–82: Claim that “few studies have been reported” needs supporting references—add 2–3 more relevant freshwater fish studies.

1. Thanks for the review.

 [but few studies have been reported on genetic distance-based crossbreeding of endangered freshwater fishes [8,23,28-34].]

[32. del Mar Ortega-Villaizan, M.; Noguchi, D.; Taniguchi, N. Minimization of genetic diversity loss of endangered fish species captive broodstocks by means of minimal kinship selective crossbreeding. Aquaculture 2011, 318, 239–243.

33. Sriphairoj, K.; Kamonrat, W.; Na-Nakorn, U. Genetic aspect in broodstock management of the critically endangered Mekong giant catfish, Pangasianodon gigas in Thailand. Aquaculture 2007, 264, 36–46.
34. Pavlova, A.; Beheregaray, L.B.; Coleman, R.; Gilligan, D.; Harrisson, K.A.; Ingram, B.A.; Kearns, J.; Lamb, A.M.; Lintermans, M.; Lyon, J. Severe consequences of habitat fragmentation on genetic diversity of an endangered Australian freshwater fish: A call for assisted gene flow. Evolutionary Applications 2017, 10, 531–550.]

 

Comment . L83–87: The last paragraph is repetitive and lacks a clear hypothesis. Suggest rewriting:

“This study tests whether crossbreeding between genetically distant pairs enhances genetic diversity and early survival in P. nigra without causing divergence from the broodstock population.”

1. Thanks for the review. Edited according to comments.

[This study tests whether crossbreeding between genetically distant pairs enhances genetic diversity and early survival in P. nigra without causing divergence from the broodstock population.]

 

Comment . L90–98: Include GPS coordinates in Figure 1 legend for reproducibility.

1. Thanks for the review. Edited according to comments.

 

Comment . L105–117: Good explanation of VIE tagging, but could add information about tag retention validation or error rate.

1. Thanks for the review. Edited according to comments.

[Previous studies on a variety of freshwater and marine fishes have reported high retention of visible implant elastomer (VIE) tags (typically >90–100%) over periods of several months to at least six months, with negligible effects on growth or survival [35]. In this study, fluorescent VIE tags were therefore used to pre-identify broodstock individuals for subsequent genotype verification.]

 

Comment . L118–136: DNA extraction description is detailed but too procedural—shorten brand names and catalog numbers.

1. Thanks for the review. Edited according to comments.

 [Genomic DNA was extracted from ethanol-preserved fins using a commercial kit (Genomic DNA Prep Kit, BioFact, Seoul, Korea) following the manufacturer’s instructions, quantified with a NanoDrop ND-1000 (Thermo Scientific), diluted to working concentration (50 ng), and stored at 4 °C.]

 

Comment . L127–137 (Table 1): Marker information table is fine but references (e.g., “Bang et al. 2023, Kim et al. 2025”) should be formatted consistently.

1. Thanks for the review. Edited according to comments.

[Bang et al. [36], Kim et al. [12]]

 

Comment . L139–153: Table 2 on genetic distance lacks standard deviation or measure of genetic similarity; clarify if values are Nei’s or Cavalli-Sforza distance.

1. Thanks for the review. Nei's genetic distance D is an “index” calculated by combining multiple markers for a pair of individuals (or populations), so it is not usually reported as mean ± SD.

[Pairwise Nei’s genetic distance between female and male broodstock]

 

Comment . L157–198: Statistical methods are described clearly, but details like “PHWE analyses” (L164) should be fully written as “Probability of Hardy–Weinberg Equilibrium.”

1. Thanks for the review. Edited according to comments.

[The P_HWE (Probability of Hardy–Weinberg Equilibrium) analyses were performed using GENEPOP v4.2 [41]. Two methods were used to estimate bottlenecks.]

 

Comment . Add a flow diagram summarizing sample collection, genotyping, and analysis—currently text-heavy.

1. Thanks for the review. Edited according to comments.

 

Figure 3. Workflow summarizing sampling, VIE tagging, DNA extraction and genotyping, analysis of genetic distance between individuals, rearing and induced maturation, genetic-distance based crosses (HGD and LGD groups), and early survival rate analysis in P. nigra. (a) Collection of 30 individuals from the Yudeungcheon Stream in the Geumgang River basin; (b) Individual identification using VIE tags; (c) DNA extraction from fin tissue and genotyping at ten microsatellite loci; (d) Estimation of genetic distance between female and male broodstock based on Nei’s distance; (e) Rearing and induced maturation of broodstock in 100-L tanks with artificial feeding; (f) Artificial crosses between individuals assigned to HGD and LGD groups; adhesive eggs were attached to plastic mesh for incubation (photo); (g) Measurement of final hatching rate and early larval survival.

 

Comment . Ethical approval is mentioned later but not here. Ethical statement should be placed at the end of “Materials and Methods.”

1. Thanks for the review. Edited according to comments.

[2.5. Ethical approval

All procedures involving live P. nigra followed the guidelines for the care and use of experimental animals of Soonchunhyang University (2018-35). Collection, handling, and release of P. nigra were authorized by the Geumgang River Basin Environmental Office and the Jeonbuk Regional Environmental Office of the Ministry of Environment (permit nos. 2018-35, 2019-26, 2018-16, and 2019-15).]

 

Comment . Replication: Crossing appears to be single pair per genetic-distance category. Clarify if replicates (n>1) were performed or pooled.

1. Thanks for the review. Each pair of mating groups was divided into three replicates.

 [Each cross group (HGD_20, HGD_19, LGD_11, and LGD_9) was maintained in a single incubation tank to minimize environmental variation, and eggs were divided into three well-aerated plastic mesh hatching nets so that hatching and survival rates could be estimated separately for three replicates under identical tank conditions.

 

Comment . L201–213 (Table 3): Provide standard errors for diversity estimates.

1. Thanks for the review. Edited according to comments.

Table 3. Expected and observed heterozygosity of broodstock and F1 progenies from cross groups on the basis of genetic distance

Group name	Genetic distance index	No. of individuals	NA	HE	HO	FIS	PHWE
Broodstock	-	28	5.8±1.8	0.739±0.120	0.697±0.158	0.058	0.221
HGD	19	30	2.7±0.3	0.549±0.050	0.787±0.082	−0.445	0.000***
	20	30	2.9±0.3	0.547±0.071	0.800±0.108	−0.448	0.000***
LGD	11	29	2.9±0.3	0.538±0.035	0.638±0.060	−0.227	0.000***
	9	30	2.6±0.4	0.469±0.071	0.573±0.094	−0.190	0.000***

N_A: Average number of alleles, H_E: . Expected heterozygosity, H_O: observed heterozygosity, F_IS: Inbreeding coefficient, P_HWE: Hardy-Weinberg equilibrium, ^***P < 0.001.

 

Comment . L214–222 (Table 4): Interpretation of “infinite Ne” must be cautious—add a note in the Results explaining estimation uncertainty.

1. Thanks for the review. Edited according to comments.

 [In the LD-based estimates, values reported as “infinite” for the broodstock and HGD cross groups should be interpreted as indicating that N_e could not be reliably bounded from above rather than as evidence of an extremely large population size, reflecting estimation uncertainty associated with limited sample size and low levels of linkage disequilibrium. We therefore treat these estimates qualitatively (very large or undeterminable N_e) and focus on contrasts with the finite Ne obtained for the LGD cross group.]

 

Comment . L223–232 (Table 5): Statistical significance (p-values) should accompany FST results.

1. Thanks for the review. Edited according to comments.

Table 5. F_ST among populations according to microsatellite of cross group and broodstock of P. nigra

	Broodstock	HGD_20	HGD_19	LGD_11	LGD_9
Broodstock	-	0.000	0.000	0.000	0.000
HGD_20	0.105	-	0.000	0.000	0.000
HGD_19	0.152	0.224	-	0.000	0.000
LGD_11	0.201	0.316	0.321	-	0.000
LGD_9	0.170	0.288	0.298	0.371	-

Pairwise genetic differentiation significant level P-values (above), F_ST: Pairwise genetic differentiation of microsatellite (below).

 

Comment . L233–249 (Figures 3–4): Figures should be better annotated—currently unclear what colors correspond to which groups. Add clear legends.

1. Thanks for the review. Edited according to comments.

 [Analysis of the genetic structure of broodstock and cross groups of P. nigra using STRUCTURE. The curve shows ΔK as a function of the number of clusters (K), with a clear maximum at K = 3. The bar plots display individual cluster membership coefficients for K = 3–5; each vertical bar represents one individual, and individuals are grouped along the x-axis by sampling group (Broodstock, HGD_20, HGD_19, LGD_11, LGD_9). Colors indicate proportional membership in each inferred genetic cluster (Cluster 1 = red, Cluster 2 = yellow, Cluster 3 = green, Cluster 4 = blue, Cluster 5 = magenta), as specified in the legend.]

Comment . L250–257 (Figure 5): Include n-values and error bars with SD or SE. Use same scale for all groups for visual clarity. Comment: Results are descriptive but lack biological linkage—authors should highlight what levels of heterozygosity imply for restoration programs.

1. Thanks for the review. Edited according to comments.

Figure 6. Survival and hatching rates of fertilized P. nigra eggs following crossbreeding of genetic distance. Significant differences are indicated as different letters above the error bars within each variable (P<0.05). Same capital and small letters mean no significant difference among survival rate (P>0.05). The hatching and survival rates of HGD were combined as the average of HGD_20 and HGD_19, and the LGD values ​​were also the same as the combined values ​​of LGD_11 and LGD_9. Values are means ± SE based on HGD and LGD replicates per genetic-distance group.

[Taken together with the higher observed heterozygosity of HGD cross groups compared with LGD (Table 3), these patterns indicate that crosses between more genetically distant parents can increase heterozygosity and improve hatching performance without compromising early survival, which is advantageous for restoration programs that aim to release genetically diverse but viable offspring.]

 

Comment . L266–277: Good interpretation of heterozygosity, but very repetitive—merge redundant sentences.

1. Thanks for the review. Edited according to comments.

[Because cross groups were generated by 1:1 matings between selected broodstock pairs, they carried fewer alleles on average than the broodstock population, presumably because not all rare parental alleles could be transmitted under this design [19]. Consistent with this, both the mean number of alleles and HE were lower in HGD and LGD than in the broodstock, whereas NA and HE were similar between the two cross groups [47]. In both HGD and LGD, H_O exceeded H_E, a pattern typical of artificially selected or managed crosses [28]. HGD showed higher H_O than LGD, indicating greater heterozygosity and indirectly suggesting that the HGD cross group retained relatively more genetic diversity than the LGD cross group [17,42].]

 

Comment . L279–283: Strong observation linking outcrossing and adaptability; however, cite at least one empirical freshwater fish example.

1. Thanks for the review. Edited according to comments.

 [It is primarily associated with environmental adaptability, with reports suggesting that outcrossing enhances adaptability [8,26,48,49].]

Lutz, M. L., Tonkin, Z., Yen, J. D. L., Johnson, G., Ingram, B. A., Sharley, J., Lyon, J., Chapple, D. G., Sunnucks, P., & Pavlova, A. (2021). Using multiple sources during reintroduction of a locally extinct population benefits survival and reproduction of an endangered freshwater fish. Evolutionary Applications, 14(4), 950–964. https://doi.org/10.1111/eva.13173

Robinson, Z. L., Coombs, J. A., Hudy, M., Nislow, K. H., Letcher, B. H., & Whiteley, A. R. (2017). Experimental test of genetic rescue in isolated populations of brook trout. Molecular Ecology, 26(17), 4418–4433. https://doi.org/10.1111/mec.14225

 

Comment . L286–299: The bottleneck discussion is insightful but needs numerical linkage to Table 4.

1. Thanks for the review. Edited according to comments.

[In the broodstock population, HWE did not deviate, indicating that the current population is approximately in genetic equilibrium and that allele frequencies are expected to remain stable across generations under random mating [50,51]. However, BOTTLENECK analyses showed significant heterozygosity excess under all three mutation models (IAM, TPM, SMM; all Wilcoxon P<0.001) and a shifted allele frequency mode not only in the broodstock but also in all cross groups (Table 4), indicating a recent reduction in effective size in which rare alleles are lost more rapidly than H_E declines [18,42]. This suggests that the bottleneck phenomenon in the broodstock population has also spread to the HGD and LGD groups [47,52,53].]

 

Comment . L300–308: The explanation for “infinite Ne” is excellent and shows understanding of statistical bias.

1. Thanks for the review. Added content.

 [Such effects are not merely theoretical: in supplemented salmonid populations, supportive breeding has been shown to double the census number of spawners while reducing the overall effective population size of the wild-plus-hatchery system by about two-thirds, illustrating how variance in family size and LD inflation can severely depress N_e despite apparent demographic gains [52,57].]

 

Comment . L309–334: Discussion of genetic structure is valid but can be shortened; STRUCTURE vs. DAPC comparison is overly detailed for Discussion.

1. Thanks for the review. Edited according to comments.

[Cross groups produced on the basis of genetic distance showed the smallest genetic differences from the broodstock, indicating that HGD and LGD progenies are largely derived from the existing broodstock gene pool. Among them, HGD_20 exhibited the lowest F_ST and cluster assignments most similar to the broodstock, suggesting that this cross group is genetically closest to the parental population [58]. Taken together, the F_ST, STRUCTURE (K = 3), and DAPC results consistently indicate that the broodstock and HGD_20 form a genetically similar cluster, whereas HGD_19, LGD_11, and LGD_9 are more differentiated.

]

Comment . L335–350: Hatching and survival rate explanation lacks mechanistic reasoning—should mention maternal egg quality or gamete compatibility.

Add a final paragraph clearly stating management implications: how findings can be used for P. nigra conservation programs.

1. Thanks for the review. Edited according to comments.

[From a management perspective, our results indicate that crosses between high genetically distant parents (HGD) can increase heterozygosity and hatching performance while maintaining close genetic similarity to the broodstock, making them suitable candidates for producing release in P. nigra restoration programs. In practical terms, broodstock management could prioritize pairing schemes that avoid very closely related individuals (LGD-type crosses) and instead target an high range of genetic distances, while routinely monitoring heterozygosity and allelic richness to prevent loss of diversity across generations. These strategies are consistent with conservation, breeding, and management practices for other endangered fish species. Carefully designed crossbreeding has been shown to improve the fitness of reintroduced or supplemented populations and reduce genetic risk, and can be directly integrated into ongoing P. nigra release and habitat restoration efforts. Future studies suggest the need to include more variables, such as fertilization rate, hatching rate, survival rate, and 7 day survival rate.]

 

Comment . Lines 351–363 Good summary but repetitive. Simplify to 4–5 sentences focusing on:

Key result: higher hatching rate in genetically distant pairs.

Conservation implication: use moderate genetic distance for restoration breeding.

Future work: larger broodstock sample and genomic markers.

1. Thanks for the review. Edited according to comments.

 [This study shows that crosses between genetically distant broodstock pairs (HGD) achieved markedly higher hatching rates than crosses between closely related pairs (LGD), while retaining genetic compositions broadly similar to the broodstock population. These results indicate that using an appropriate intermediate range of genetic distances among parents can serve as a practical guideline for designing artificial crosses in Pseudopungtungia nigra restoration programs, enhancing heterosis and hatching success without causing excessive genetic divergence from the source population. Future work should increase the number of maternal families and apply genome-wide markers (e.g. SNP panels) to refine the optimal genetic distance for crossing and to track how genetic diversity and fitness respond over time in restored populations.]

 

Comment . L364–369: Author contribution statement has redundancy; ensure compliance with MDPI format.

L370–377: “Ethical review waived due to reason” is incomplete and unclear—must specify institutional policy and justification.

L378: Typo: “There authors” → “The authors.”

1. Thanks for the review. Errors in the manuscript have been corrected.

[All procedures involving live P. nigra followed the guidelines for the care and use of experimental animals of Soonchunhyang University (2018-35). Additionally, Geumgang River Basin Environmental Office (permit nos.: 2018-35, 2019-26) and Jeonbuk Regional Environmental Office of the Ministry of Environment (permit nos.: 2018-16, 2019-15).]

 

Comment . Citation style inconsistent—some years italicized, some not.

Missing DOIs in several places (e.g., refs 8–9).

Ref 31: “Microsatellite records for volume 12, issue 2. 2020.” is incomplete; must provide journal name.

Ref 37–38: Update titles for clarity.

Remove unnecessary details like “doi:” repetition.

1. Thanks for the review. Edited according to comments.

 

Comment . Numerous grammatical errors:

“crossing group based on genetic distance” → “crossbred groups based on genetic distance.”

“are separated and management in a 100 L tank” → “were separated and maintained in 100 L tanks.”

Use consistent tense—past for Methods and Results, present for Discussion.

Figures require higher resolution and consistent font style.

1. Thanks for the review. Edited according to comments.

 

Comment . The manuscript presents important findings but requires major revision before it can be considered for publication. I recommend substantial language editing, figure and reference correction, and clearer justification and interpretation of experimental results. With these revisions, the paper could make a meaningful contribution to conservation genetics and artificial breeding management of endangered freshwater fish species.

1. Thanks for the review. Based on comments, we have revised the language, revised the figures and references, and revised the discussion.

 

 

Comment . Numerous grammatical errors found. Edit by Native language speaker

Use consistent tense—past for Methods and Results, present for Discussion.

1. Thanks for the review. English proofreading and consistent tense usage were used.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Overall I like the paper and it is informative.  I would like to see a little additional information in the methods to clarify some details, please see my comments.  I think there might be a discrepancy between the words and what is in table 2 (M9-F8)?

Please see my attached comments for questions and suggestions.

Comments for author File: Comments.pdf

Author Response

Comment . Line 93: “Thirty adults” is mentioned but there are only 28 individuals in table 2?

1. Thank you for review. Thirty individuals were collected, but two died during rearing, so only 28 individuals were used.

[Thirty individuals were collected, but two died during rearing, so only 28 individuals were used.]

Comment . Figure 1. The large red arrow is confusing. I suggest removing it.

1. Thank you for review. Edited according to comments.

 

Comment . Line 98. Figure 1 caption: “red circles”? There is only one red dot. Suggest changing to “red dot” or “red circle” to indicate single red circle.

1. Thank you for review. Edited according to comments.

[red circle]

Comment . Lines 127–128: Why were only ten chosen and why did you choose the specific ten microsatellites?

Were default values used for your analysis used across the methods? Please specify if default parameters are used for each one of your programs and if not, please specify which parameter values were used and why.

1. Thank you for review. Edited according to comments. Parameters have also been added.

[We selected 10 markers because they showed moderate to high polymorphism (PIC value), which is suitable for estimating genetic diversity and genetic distance between individuals.]

[The Ne was estimated in NeEstimator v2 [43] using the linkage-disequilibrium method under a random-mating model, with a critical allele frequency threshold of Pcrit = 0.02.]

[Tests for departure from PHWE (Probability of Hardy–Weinberg Equilibrium) in GENEPOP v4.2 were conducted using the default Markov-chain parameters.]

[We performed statistical and visualization analyses to determine the genetic diversity information and structure of the crossbred group and broodstock. Expected (HE) and observed heterozygosity (HO) were calculated in Cervus v3.0 using default options]

[The number of principal components to retain was chosen PCs was 80.]

 

Comment . Lines 135: Why 28 genotypes and not all 30 individuals captured?

1. Thank you for review. Edited according to comments.

[Thirty fish were collected, but two died during rearing, so only 28 were used for genetic distance analysis.]

 

Comment . Lines 142–143: “distances 9 and 11” are specified for the LGD group. However, when looking at table 2. It has “7” highlighted for M9-F8. There must be a mistake in the writing, please fix or in the table. Table 3 also shows a genetic distance of 9? Why were the genetic distances of 9, 11, 19, and 20 chosen from the specific pairings when there are other distances like 7 and 8 and 24 and 22? Why only two pairings for each group? Please specify what a long genetic distance is and provide references when needed.

10. Thank you for review. Fixed the error with M12-F10. Because the table only selects mating groups with a long genetic distance among mature individuals, not all individuals that are mature, it was not possible to select only mating groups with a very high genetic distance value, such as 22.

[Because it was not feasible to perform artificial crosses for all possible female–male combinations, we restricted the experiment to a limited number of mating groups. Among broodstock pairs in which both sexes were simultaneously sexually mature and in good condition and could produce sufficient eggs and milt for artificial spawning, we selected the pairs with genetic distances of 19 and 20, as well as those with the minimum distances of 9 and 11.]

 

Comment .

Line 149: is there a citation for the “dry method”?

Line 161: first time F1 is used. Please state what F1 is.

Line 164: first time using PHWE . Please state what PHWE is.

Line 166: What is IAM?

Line 167: What is TPM?

Line 167: stepwise mutation model is specified as SMM, thank you!

Line 169: First time Ne is used. Please state what Ne is.

Line 175: First time DAPC is used. Please state what DAPC is.

1. Thank you for review. Edited according to comments.

[F₁ progeny (first-generation progeny)]

[P_HWE (Probability of Hardy–Weinberg Equilibrium)]

[recent bottlenecks were tested under the Infinite Allele Model (IAM). A Two-Phase Model (TPM) and stepwise mutation model (SMM) were used to estimate]

[The effective population size (N_e) was estimated in NeEstimator v2 [44] using the linkage-disequilibrium method under a random-mating model, with a critical allele frequency threshold of Pcrit = 0.02.]

[Discriminant Analysis of Principal Components (DAPC)]

 

Comment . Line 178–180: The “Hatching rate” definition is a little confusing. Do you mean: Hatching Rate was defined as the time point when 100% hatching was complete at a water temperature at 20.0±0.5 °C

1. Thank you for review. Edited according to comments.

[The hatching time was defined as the point at which all eggs (100%) had hatched at 20.0 ± 0.5 °C, and the hatching rate was calculated as the percentage of hatched larvae relative to the total number of eggs.]

 

Comment . Line 187: is P < 0.05 correct to use or should it be an a < 0.05 because and ANOVA was used? Why was a Duncan’s test used instead of a Tukey adjustment?

1. Thank you for review. Regarding the choice of post hoc test, we first examined normality and homogeneity of variances. When the assumptions of normality and homoscedasticity were satisfied, we performed one-way ANOVA followed by Duncan’s multiple range test at α = 0.05. When the homogeneity of variances assumption was not met, we applied alternative tests that do not assume equal variances, and used post hoc procedures appropriate for unequal variances instead of Tukey.

[Statistical analyses were performed in SPSS v12.0 (Chicago, Illinois, SPSS Inc.) using one-way ANOVA. Because homogeneity of variance was not met for survival rate, post hoc comparisons used Dunnett T3; because homogeneity was met for hatching rate, Duncan’s multiple range test was applied (P < 0.05).]

 

Comment . Line 189: Why was there a range of 29 to 30 larvae selected?

30. Thank you for review. We thank the reviewer for this comment. For each cross, we aimed to genotype approximately 30 F1 larvae because a sample size of about 30 individuals per family is generally considered sufficient to characterize microsatellite loci genotypes and to detect segregation patterns within a cross. Accordingly, we randomly selected 30 larvae per cross for microsatellite genotyping. In one cross, however, one larva was inadvertently lost (insufficient tissue for DNA extraction), so the final sample size for that group was 29 instead of 30.

 

Comment . Line 194 and Line 188: Usually a sentence does not start with an abbreviation. “First generation progeny…”?

1. Thank you for review. Edited according to comments.

[First-generation progeny (F₁) larvae produced from each crossbred group were sampled at the time survival was assessed. For each crossbred group, 29 to 30 larvae were randomly selected, preserved whole in 99.9% ethanol, and stored at 4 °C. Genomic DNA was extracted from whole larvae in 1.5 ml microtubes using the PCRBIO Rapid Extract Lysis Kit (PCR Biosystems, London) following the manufacturer’s protocol and used for genetic diversity analyses.

First-generation progeny (F₁) genotypes were determined with the same ten microsatellite markers used for broodstock genotyping (Table 2). PCR conditions were identical to those in Section 2.3. Genotyping analyzed on an ABI 3730xl DNA Analyzer (Applied Biosystems, USA). Allele sizes were scored with Peak Scanner software v1.0 (Applied Biosystems, Foster City, CA, USA).

]

Comment . Line 216: Eeective population size (Ne) is being stated here but should be stated above when it is first used.

1. Thank you for review. Edited according to comments.

 

Comment . Line 217: The Ne isn’t a range of 65–114, there are only two numbers, correct? Suggest changing to “LGD had an Ne of 65 or 114.

1. Thank you for review.

[LGD had an N_e of 65 or 114.]

 

Comment . Line 253 and 256: what was the actual p-value? I believe that p-values should be reported.

1. Thank you for review.

[HGD than in LGD (P < 0.05; P-value: 0.001)]

[Survival rate did not differ significantly among groups (P > 0.05; P-value: 0.166)]

 

Comment . Lines 293–294: The broodstock only came from 30 sampled individuals taken from the wild, correct? Is the source population bottlenecked or was the act of sampling and taking 30 individuals creating the bottleneck? The study species is endangered so it would be reasonable that there was a recent bottleneck. Has there been any other research done on the source population? This concept should be expanded on within the paragraph. Sure the data shows a bottleneck but acknowledge the dieerent reasons to why this could be possible.

1. Thank you for review. The original population was tested for a bottleneck and found to be bottlenecked. YD (2019) is the original population.

[The broodstock used in this study were derived from the YD (2019) population sampled in 2019, which has previously been shown to exhibit pronounced historical and recent bottlenecks and elevated inbreeding based on microsatellite loci analyses [12]. Thus, the reduced genetic diversity and strong bottleneck signals detected in our broodstock and crossbred groups are more likely to reflect the demographic history of this source population than a novel bottleneck created by sampling 30 individuals for artificial propagation [12]. Given that P. nigra is an endangered species with small and fragmented populations, long-term reductions in effective population size at YD may constrain the genetic variation available for broodstock formation and subsequent crosses [12].]

[Kim, K. R., Kwak, Y. H., Sung, M. S., Cho, S. J., & Bang, I. C. (2023). Population structure and genetic diversity of the endangered fish black shinner Pseudopungtungia nigra (Cyprinidae) in Korea: A wild and restoration population. Scientific Reports, 13(1), 9692.]

 

Comment . Line 302: What factors in your procedures could have led to the Ne of infinity? You have a small sample size correct?

1. Thank you for review.

We thank the reviewer for this important question. In our study, contemporary effective population size (Ne) was estimated using the linkage-disequilibrium (LD) method implemented in NeEstimator. In this framework, “infinite” Ne does not indicate that the population is truly infinite, but rather that the LD signal is too weak to provide an upper bound for Ne under the given data conditions.

In our analyses, each group consisted of only 29–30 individuals genotyped at 10 microsatellite loci. This relatively small sample size per group, combined with the limited number of loci (and the exclusion of rare alleles by applying a minor allele-frequency threshold), reduces the amount of LD information available for Ne estimation. As a result, for some groups the confidence intervals became extremely wide and the upper confidence limit did not converge, so NeEstimator returned “infinite” Ne. In other words, these values should be interpreted as “very large or not estimable with our data” rather than as evidence of a biologically infinite population size.

The small sample sizes per group in this study reflect practical and ethical constraints on removing individuals of an endangered species from the wild for broodstock and experimental crosses. We have now clarified in the revised manuscript that “infinite” Ne values arise from the limited sample size and marker set in the LD-based method, and that these estimates are interpreted cautiously as indicating that Ne is too large to be reliably bounded with the available data.

 

Comment . Lines 323–334: The first two sentences are a little confusing. “were the most genetically similar” kind of leads the readers to believe that the Fst values was relatively low, indicating the similar genetics. In the results section it is mentioned that there is an “intermediate levels of genetic dieerentiation”. I suggest restating the primary objective

1. Thank you for review. Our intention was not to imply that the FST values indicate low differentiation in an absolute sense, but rather that some crossbred groups show relatively lower differentiation from the broodstock compared with other crosses. As reported in the Results, the broodstock shows the lowest pairwise differentiation with HGD_20 (FST = 0.105), followed by HGD_19, LGD_11, and LGD_9 (FST = 0.152–0.201), whereas FST among the crossbred groups themselves is higher (0.224–0.371), indicating intermediate levels of genetic differentiation.

[The primary objective of this study is to generate crossbred groups that differ in genetic distance while remaining genetically comparable to the broodstock population. Consistent with this objective, the broodstock shows the lowest pairwise differentiation with HGD_20 and slightly higher differentiation with HGD_19 (F_ST = 0.105–0.152), indicating that these high-distance crosses retain relatively greater similarity to the source population than the LGD groups, even though all pairwise FST values still fall within the range of moderate genetic differentiation among groups. Thus, our genetic-distance-based crossing scheme produces crossbred groups that differ in genetic distance while maintaining a moderate level of genetic similarity to the broodstock. Although this study achieves its primary objective, future genetic-distance-based crossing schemes could produce crossbred groups that differ more markedly in their genetic similarity to the source population [13,60,61]. This possibility is a critical consideration for restoration programs targeting endangered species. In such species, where artificial crossing is unavoidable because of limited population size, the practical goal is to generate crossbred populations that maximize genetic diversity while avoiding excessive genetic divergence among crossbred groups and between crossbred groups and the source population. Future work therefore needs to integrate both genetic distance and similarity to the maternal population when selecting broodstock, and to evaluate the performance of such schemes across multiple endangered populations [62,63].]

 

Comment . Lines 327–330, then talk about HGD_20 and HGD_19.

Line 336: Here you mention long genetic distances but have not described what is long and why you chose the distances. Please add this type of information into the methods and then discuss in the discussion when needed.

1. Thank you for review.

 

[The primary objective of this study is to generate crossbred groups that differ in genetic distance while remaining genetically comparable to the broodstock population. Consistent with this objective, the broodstock shows the lowest pairwise differentiation with HGD_20 and slightly higher differentiation with HGD_19 (F_ST = 0.105–0.152), indicating that these high-distance crosses retain relatively greater similarity to the source population than the LGD groups, even though all pairwise F_ST values still fall within the range of moderate genetic differentiation among groups. Thus, our genetic-distance based crossing scheme produces crossbred groups that differ in genetic distance while maintaining a moderate level of genetic similarity to the broodstock. Although this study achieves its primary objective, future genetic-distance based crossing schemes could produce crossbred groups that differ more markedly in their genetic similarity to the source population [13,60,61]. This possibility is a critical consideration for restoration programs targeting endangered species. In such species, where artificial crossing is unavoidable because of limited population size, the practical goal is to generate crossbred populations that maximize genetic diversity while avoiding excessive genetic divergence among crossbred groups and between crossbred groups and the source population. Future work therefore needs to integrate both genetic distance and similarity to the maternal population when selecting broodstock, and to evaluate the performance of such schemes across multiple endangered populations [62,63].]

[For the purposes of this study, we defined “high genetic distance (HGD)” as parental pairs located in the upper range of the Nei’s D distribution among broodstock, and “low genetic distance (LGD)” as parental pairs located in the lower range.]

[To evaluate the effect of increased parental genetic distance on hybrid vigor, we compared hatchability and survival between crossbred groups generated from high (HGD; Nei’s distance = 19–20) and low (LGD; Nei’s distance = 9–11) genetic-distance pairs.]

 

Comment . Overall I like the paper and it is informative.  I would like to see a little additional information in the methods to clarify some details, please see my comments.  I think there might be a discrepancy between the words and what is in table 2 (M9-F8)?

1. Thank you for review. Edited according to comments.

[Accordingly, the pairs with distances of 19 and 20 (M14–F12, M8–F9) were designated as high genetic distance (HGD), whereas the pairs with distances of 9 and 11 (M6–F6, M12–F10) were designated as low genetic distance (LGD) (Table 2).]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

It may be accepted for publication.