Genetic Diversity and Population Structure of Shoshone Sculpin Cottus greenei in the Hagerman Valley of South-Central Idaho

Round 1

Reviewer 1 Report

In the manuscript "Genetic diversity and population structure of Shoshone sculpin Cottus greenei in the Hagerman Valley of south-central Idaho", the authors identified a suite of microsatellite loci that amplify well and exhibit variation within and between Shoshone sculpin populations. Additionally, They analyzed genetic diversity, structure, and effective population size of the micro-endemic Shoshone sculpin, using range-wide sampling and microsatellite DNA analyses. The present study confirmed that Shoshone sculpin are a highly genetically structured with very high levels of genetic differentiation among most populations.

This authors reveal the genetic diversity and structure across the distribution range of Shoshone sculpin, which was valuable for preservation of this species.

Minor points:
-In Table 1 and table 4, the authors should indicate the meaning of each indicator, put it in the title or add a note.

-In Table 1, the value “N” of Fisher Lake is “50*”, the meaning of the asterisk needs to be clarified here.

-Line 105, the “Table 1” should be “Table 2”.

-In Table 5 title, there is “P-values < 0.05 are considered significant”. But in results, there is “No populations showed evidence of a recent bottleneck under any of the three tests for both mutational models (Table 5)”. The authors should explain the relationship between the significant P-values and bottlenecks in populations in table or in Methods.

-The format of references is not uniform. For example in line 413.

Author Response

Dear Editors,

Please find attached our revisions for the manuscript “Genetic diversity and population structure of Shoshone sculpin Cottus greenei in the Hagerman Valley of south central Idaho”.  We thank the reviewers for their time and effort and believe their comments have improved the manuscript. We have done our best to address their suggested edits and comments. Responses to their primary suggestions can be found below highlighted in bold.

Reviewer 1:

Minor points:
-In Table 1 and table 4, the authors should indicate the meaning of each indicator, put it in the title or add a note.

MRC: Changed as suggested

-In Table 1, the value “N” of Fisher Lake is “50*”, the meaning of the asterisk needs to be clarified here.

MRC: Fixed

-Line 105, the “Table 1” should be “Table 2”.

MRC: Fixed

-In Table 5 title, there is “P-values < 0.05 are considered significant”. But in results, there is “No populations showed evidence of a recent bottleneck under any of the three tests for both mutational models (Table 5)”. The authors should explain the relationship between the significant P-values and bottlenecks in populations in table or in Methods.

MRC: We added explanations in both the table and the methods.

-The format of references is not uniform. For example in line 413.

MRC: We have update references that were cited in the paper, but not included in the references section.  We removed references in the references section that were not cited in the paper.  We corrected the citation on line 413.

Reviewer 2 Report

This manuscript by Campbell et al. uses microsatellite genotypes, from markers developed in Cottus spp., to elucidate the population genetic structure of a Snake River, Idaho, USA microendemic sculpin fish species, Cottus greenei, which is a spring-fed stream specialist. They examine data from over a thousand individuals, screened for sympatric Cottus species and hybrids, and discover that, not unlike other Cottus species, there is significant population structure both in the degree of divergence between locales, reflecting little to no contemporary gene flow between many locations, as well as significant differences in genetic diversity among a number of locales.

I find that the data are sufficient to reveal what the authors aim to do, that the methods, while not cutting edge, are robust and adequate to clarify the patterns described in the objectives, and that the inferences drawn are in keeping with the compendium of results. I would not object to the manuscript being published in Fishes, and though I believe the following minor revisions would improve the contextualization and impact of the manuscript for readership, I do not believe they are absolutely necessary for publication.

First, I thought that the estimates of effective population size, which reasonable and important to make, did not end up revealing anything because of limitations outside the authors’ control (ratio of sample size to population size, overlapping generations, etc.). These constraints are well known, and while I appreciate the authors’ being explicit about them, I think their discussion could be much abbreviated.

Indeed, it surprised me that the authors went to such great extent to discuss those limitations on estimating Ne but said precious little about the conservation implications of their results. What the authors essentially have discovered is that many springs hold a considerable and unique portion of the genetic diversity of the species as a whole, which, again, is a microendemic. That implies that degradation of any spring segment, through targeted modification or surrounding land use changes (e.g. irrigation), may irreparably deprive the species of evolutionary potential. Moreover, it implies that previous modifications to the spring/seepage system likely already extirpated numerous unique sculpin populations. I appreciate that the authors, whose instincts are likely to be to provide objective results without prognosticating what they imply about human actions, but in this case I think some prognostication is warranted.

Finally, I think it is important to put these results in more biogeographic and evolutionary context. Recently, Young et al. (Western North American Naturalist 82(2)), which the authors fail to cite, discovered that Cottus greenei is the most divergent member of a species complex of Cottus from the American West (C. beldingii). Several of the other members of this species complex show limited distributions (endemism) making C. greenei not unlike its relatives, though I did not check about ecological similarities (e.g. cold-water specialization). Moreover, regardless of how much stock one puts in their topologies, these authors also note that the distribution of C. greenei in the Snake River Valley can only have occurred through range modification or dispersal from areas not inundated by the Lake Bonneville flood, circa 17.4 kya, since the divergence of this species from the rest of the complex appears to precede that geological event. In summary, I think more context as to how C. greenei came to be isolated and inhabit this particular spring system (e.g. was the range likely much larger historically and reduced through western aridification following deglaciation…) would help to contextualize the present results.

Minor concerns:

The heterozygote deficiency (i.e. ratio of He to Ho) noted in the results, while perhaps non-zero, seems minor and within the range of most “healthy” populations. I would also suspect that limited population size (drift and minor inbreeding) and population structure could also erase the effects of past population bottlenecks. Without greater resolution and management of potential linkage and null allele effects in these microsatellite loci, I would be very cautious of even reporting evidence of population expansion based on heterozygosity tests, considering it could be used to undermine efforts to conserve this vulnerable species.

There were quite a few cited references missing from the bibliography.

Author Response

Dear Editors,

Please find attached our revisions for the manuscript “Genetic diversity and population structure of Shoshone sculpin Cottus greenei in the Hagerman Valley of south central Idaho”.  We thank the reviewers for their time and effort and believe their comments have improved the manuscript. We have done our best to address their suggested edits and comments. Responses to their primary suggestions can be found below highlighted in bold.

Reviewer 2:

-Abbreviated: Consider abbreviating discussion on effective population size.

MRC: We felt that given the importance that effective population size is given in conservation and management it was important to provide a detailed discussion on the limitations of estimating this parameter in general and specifically within the context of our study.  Sample sizes of 50 per population are often recommended for studies examining the genetic diversity and structure of a species.  However, we wanted to emphasize the limitations of these sizes when the true NE is likely significantly higher than 50.  Future work with this species will focus on increased samples sizes and temporal sampling.

- I think some prognostication is warranted.

MRC: We feel this manuscript provides a needed introduction in explaining the genetic structure of a previously unstudied species. We emphasize the species’ uniqueness, extreme habitat requirements, it's endemism and its conservation status.  We state that the preservation of the genetic diversity of this species will require the protection and preservation of multiple isolated populations.  We are hesitant to expand a discussion on conservation implications given that we did not conduct a detailed assessment of population abundance and habitat quality/stability throughout the species’ range.

- Its important to put these results in more biogeographic and evolutionary context.

MRC:  We agree that it is very appropriate to cite Young et al’s (2022) work indicating that Shoshone sculpin are the most divergent member within the C. beldingii complex.  We have included that information in the introduction.  We were really interested in the statement in Young et al’s paper that Shoshone sculpin may have “persisted in refugia above the floodwaters that it no longer occupies” due to the Bonneville flood.  However, it is very difficult to speculate on the species’ distribution before, during, and after the Bonneville flood.  

- There were quite a few cited references missing from the bibliography.

MRC: We have update references that were cited in the paper, but not included in the references section.  We removed references in the references section that were not cited in the paper.