Spatial Patterns of Neutral and Functional Genetic Variations along Dendritic Networks of Riverscape in Brown Trout Populations

Round 1

Reviewer 1 Report

This paper reports the spatial patterns of genetic variation among neutral and functional genetic markers of brown trout populations distributed along the Garonne and Ariege sub-river basins interconnected by dendritic networks structured by elevation.  A total of 318 individuals were collected among 16 localities, with about 20 individuals sampled per locality. The manuscript is well-written with only a few grammatical improvements necessary. Three hypotheses that may account for differences in genetic diversity among localities, and also relative to the marker type (neutral vs functional). 1) Unidirectional water flow may result in downstream-biased dispersal and asymmetric gene flow, 2) Due to increased river width downstream the larger habitat would result in larger effective population sizes, and 3) Upstream-directed colonization after glacial events may lead to loss of genetic variation upstream. While the first two hypotheses are tested in this study, the third is not mentioned in the discussion. Further, the authors incorrectly cite in support of this third hypothesis the work of Taberlet et al. (1988). In that paper, the authors could not find any correlation between genetic data and Quaternary glacial events. Further, in the ten taxonomic groups compared in that study, no aquatic species were included, and only four members of Triturus spp. (an amphibian) was included, which would not match the dispersal potential of brown trout. I suggest that the authors remove that third hypothesis from their introduction, as it is incorrectly cited and not explored further in this manuscript.

Grammatical suggestions

Page 4,

Line 118. Change 'supposedly' with 'putative'

Page 5,

Line 155. Remove 'First,'. Start the sentence with "We tested..."

Line 165. Change 'Based on' with 'We used..."

Line 171. Change 'few' with 'least'

Line 172.  'parameter' should be possessive, i.e., parameter's

Line 182. Change the first sentence to: "The technical replicates for 54 individuals were identical."  

Line 210 and Table 2. The significant figures for the Fst values should be the same (three figures) for all calculations. Accordingly, change 0.06 to 0.060 (two instances), and 0.04 to 0.040, in both the text and Table 2.

Page 8

Line 250. Change 'less' for 'least'

Page 9

Line 261. Add 'as follows.' after '...neutral markers'

Lines 283-283. I suggest rewriting this sentence as follows: Furthermore, we noticed that populations considered as "isolated' were actually those located upstream relative to all other populations (but not strictly speaking upstream the river, Table 1, Fig. 1). 

see grammatical suggestions/comments above

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript aims to evaluate hypothesis on genetic variation in neutral and putative functional markers among brown trout populations in the Garonne-Ariège basin. These hypotheses are summarized at lines 81-93 and included higher diversity at downstream locations for any studied marker (neutral/functional) and higher differentiation among upstream locations at neutral loci (by isolation and drift expected in smaller populations) but not at functional loci that are subject “to convergent selective pressures in all upstream sites”. This later could be not right as upstream areas likely preserve better habitat quality for brown trout (see Tisseuil et al 2012a for the study region) and therefore selective pressures are relaxed there, being drift and isolation the main evolutionary factors for these upstream populations even at functional markers.

As the authors recognizes (lines 278-280, and 301-302) their analyses suffer from an unbalanced sampling strategy, with rivers sampled in a unique location (ARZDur, TOUlar, or HERPey) whereas other were sampled at several locations (Ariège river). Thus, the mean distance from other locations is not a proper variable to assess the hypothesis presented in the manuscript based on upstream-downstream divergences. Results using the distance to the Garonne -Ariège confluence (Figure S1) do not fit with the hypothesis on local diversity. I suggest, the authors declare “a priori” upstream and downstream locations at each sub-basin. For instance, ARABas and BOUArg are “upstream” and LEZAUB, SALGir and SALTau are “downstream” in the “Garonne” subbasin, meanwhile ARISav and ORIAx are “upstream” and ARIPam and ARIVar downstream in the Ariège one (or use information in Tissueil et al 2012b). Then FSTAT analyses could be used to compare AR (and why not observed and expected heterozygosity?) and FST between the upstream and downstream group of populations.

I also suggest a more classical Mantel test to correlate genetic differentiation (FST) with hydrological distances between pairs of populations. In addition, we should be aware of the effect of anthropogenic barriers (dams, weirs...) on the population structure of brown trout (see for example González-Ferreras et al 2022), and particularly if anadromous trout (sea trout) are present in the studied area, as hybridization between resident and anadromous fish may increase diversity downstream of the barriers (Bernas et al 2021). The main hydrological barriers in the studied region should be included in Figure 1, particularly those that impede the migration of brown trout.

The paper should also consider that brown trout have been object of fishery management for more than a century. Releases from domestic hatchery stocks of non-native origin have been a common practice around Europe, including France (see Berrebi et al 2021 and Bohling et al 2016). As a result, stocking have introduced non-native alleles increasing local diversity but reducing population differentiation (e.g., Vera et al 2023). It should be nice if the authors were able to estimate the genetic impact of hatchery releases in the studied populations (see Saint-Pé et al 2019 ref #34 in the manuscript or) and use this variable in their analyses. Prunier et al 2022 reported hatchery releases at l’One river from the Garonne basin and they observed that the classical pattern of downstream increase in genetic diversity may be reversed when considering highly introgressed populations.

Cited references:

Bernas R, Was-Barcz A, Arnyasi M, et al. Evidence of unidirectional gene flow in a fragmented population of Salmo trutta L. Scientific Reports 2021;11(1), doi:10.1038/s41598-021-02975-9

Berrebi P, Horvath Á, Splendiani A, et al. Genetic diversity of domestic brown trout stocks in Europe. Aquaculture 2021;544(June), doi:10.1016/j.aquaculture.2021.737043

Bohling J, Haffray P, Berrebi P. Genetic diversity and population structure of domestic brown trout (Salmo trutta) in France. Aquaculture 2016;462(1-9, doi:https://0-doi-org.brum.beds.ac.uk/10.1016/j.aquaculture.2016.04.013

González-Ferreras AM, Leal S, Barquín J, et al. Patterns of genetic diversity of brown trout in a northern Spanish catchment linked to structural connectivity. Aquatic Sciences 2022;84(4), doi:10.1007/s00027-022-00877-0

Prunier JG, Saint‐Pé K, Tissot L, et al. Captive‐bred ancestry affects spatial patterns of genetic diversity and differentiation in brown trout (Salmo trutta) populations. Aquatic Conservation: Marine and Freshwater Ecosystems 2022;32(9):1529-1543, doi:10.1002/aqc.3826

Tisseuil C, Vrac M, Grenouillet G, et al. Science of the Total Environment Strengthening the link between climate, hydrological and species distribution modeling to assess the impacts of climate change on freshwater biodiversity. Science of the Total Environment, The 2012a;424(193-201, doi:10.1016/j.scitotenv.2012.02.035

Tisseuil C, Leprieur F, Grenouillet G, et al. Projected impacts of climate change on spatio-temporal patterns of freshwater fish beta diversity: A deconstructing approach. Global Ecology and Biogeography 2012b;21(12):1213-1222, doi:10.1111/j.1466-8238.2012.00773.x

Vera M, Aparicio E, Heras S, et al. Regional environmental and climatic concerns on preserving native gene pools of a least concern species: Brown trout lineages in Mediterranean streams. Science of The Total Environment 2023;862(160739, doi:https://0-doi-org.brum.beds.ac.uk/10.1016/j.scitotenv.2022.160739

Other comments related with Methods:

1- Figure 2. As commented HO and HE estimates on local diversity can be included in this analyses (and preferably using distance to the Garonne-Ariège confluence (as in Figure S1)).

2- Figure 3. As indicated a classical Mantel test determining relationships between hydrological (Km) and genetic (FST) distance matrices should be better to evaluate isolation by distance among study locations. Note that log transformations for hydrological/geographical distances, and modifications of FST (FST/(1-FST)) have been proposed to improve this test.

3- In addition to the analyses performed in the manuscript (and suggestions indicated above) the authors can do analyses on the Genetic Population Structure for each kin of marker to check for the conformance among their results (e.g., STRUCTURE software for neutral SNPs and for functional SNPs and MHC gene).

4- To identify selective processes, the authors could do an FST-outlier analysis for the full set of markers (Arlequin software can do this analysis).

Minor comments:

1- Figure 1, Le Sios location code (SIOTra in Table 1) is not in this Figure.

2- As commented before, local diversity estimates based on expected and observed heterozygosity at the studied locations could be also compared in addition to Allele richness (values are already available in Table 2)

3-Sentences in lines 183 to 192 is confusing. Only a single locus showed HWE deviations and was removed from the study, but several populations showed significant deviations at neutral SNPs and MHC classIIB gene.

4- Table 3 is not cited in the text.

5- Sentence in lines 196-197 is duplicated in lines 212-213.

6-Appendix S1.4 sheet was not available in the excel file i received

English looks OK, at least for non-native speakers

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

This revised version of the manuscript has addressed all my concerns on the previous one. I specifically appreciate using the distance to the confluence and centrality instead of mean hydrological distance of each sample site to the others. Despite the manuscript may be suffering from unbalanced sampling in some subbasins (large centrality scores are received by the downstream locations -SALtau, ARIpam and ARIVar)- at the Garone and Ariège rivers where several sites were sampled in upstream waters), results are clearer now.  I just have some minors comments detailed below:

1-Supplementary material: in the final version place Figure 1 just before Appendix 5.

2- Similarly, in the manuscript place Figure 1 close to the Figure 1’s caption.

3- Appendix 4 on introgressive hybridization with domestic strains, clarify that these results refer to neutral SNPs markers. Note that at location TOULar all sampled fish looks like admixed fish. This fact could explain why this location presents a singular genetic unit in Figure 5A of the text.  I suggest modification in lines 184-186, and 322-325. For instance:

In line 184: A preliminary analysis using neutral SNPs,…, allowed us to confirm that only the Touyre River sampled was impacted from captive-bred strains (Appendix 4).

In line 324 : … and the Touyre River impacted by hatchery strains (Fig. 5A1, Fig 1).

4- Sentence in line 240-241 looks as reiterative with that in lines 234-236, and can be removed from the manuscript

5-Sentence in lines 482-484 is awkward: I suggest modifying to: Patterns of population structure and local diversity observed for neutral SNPs reflected genetic drift and limited gene flow among populations.

6- In line 507 I suggest replacing “, but” by “despite” and then in line 508 replacing “evolutionary forces” by “drift and isolation” and later in line adding “adaptive response to” before “environmental pressures”.

Author Response

Please see the attachment

Author Response File: Author Response.docx