Depth-Dependent Phenotypic Plasticity Differs Between Two Deep-Freshwater Amphipod Scavengers of the Genus Ommatogammarus Despite Similarly Low Genetic Diversity in Ancient Lake Baikal

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article is about two genetically similar species of endemic amphipods that are adapted to life at great depths in an ancient lake, their phenotypic differences associated with the peculiarities of their deep and dark habitat. I find it interesting in terms of understanding the evolutionary transformations of the biota in ancient unique lakes and determining the limits of the adaptive capabilities of relict species. It is written using modern methods and molecular analysis. I support its publication, but the text needs stylistic editing and some important clarifications.

I recommend replace the Title «Differences in Depth-Related Phenotypic Plasticity between Two Deep-Freshwater Ommatogаmmarus Amphipod Scavengers Over Similarly Low Genetic Diversity Throughout Ancient Lake Baikal»

by more clear for readers:

“Depth-dependent phenotypic plasticity differs in two amphipod scavengers (genus Ommatogammarus) despite similarly low genetic diversity in ancient Lake Baikal”

I propose some Minor changes.

Lines 18-19. Deep-water ecosystems make up the world's largest ecosystem, although they are poorly understood.

Lines 22-25. This study examines the genetic diversity and depth-related traits of two deep-water amphipod scavengers, Ommatogammarus flavus and O. albinus, which are dominant in Baikal's deepwater community.

Lines 26-28. Our results revealed low intraspecific diversity of the cytochrome c oxidase subunit I gene marker fragment both vertically and horizontally, indicating the absence of significant barriers in the distribution of both species and the existence of a “bottleneck” event in their evolutionary history.

Lines 29-30 … O. flavus, as well as eye colour controlling stable opsin expression in both species

Line 32 … and help to characterize the ecological differences between

Everywhere change “deep-sea habitats” by deep-water habitats or environments… since you are talking not only about the sea but  especially about a freshwater lake.

Lines 46-47. … and related evolutionary processes remain to be understood.

Lines 97-98. Comparative roles of horizontal and vertical diversification of deep-water amphipod fauna. Role in what? It is unclear sentence.

Lines 102-103 ... At the same time, at least one species presents the same haplotypes for samples collected at several hundred kilometers
[31]. Please add name of species.

The introduction seems too broad. I would suggest staying closer to the topic of adaptations and genetic and phenotypic diversity of deep-water species, and it would be better to omit the information that will not be discussed further in the article and within the framework of the stated topic (for example about shallow inhabitants) .

Lines 149-152 … Apart from horizontal geographic diversity…… to study vertical diversity ? It is not clear what vertical diversity and horizontal diversity are. Do you mean genetic diversity depending on the spatial location (Lake region), and depth on the place?  Why in the "deep ocean" and not a lake?Rewrite the sentence for clarity. 

Phenotypic differences were only observed at different depths in one area (Bolshie Koty), and not in different areas of the lake (9 stations in total). Were they not noted? It is necessary to clarify why such a design of the work - genetic diversity in different regions, phenotypics in one, is this a unique case of the place, or will it be the same in other areas of the lake?

Lines 358-359 and 380-387 should be in Methods.

Discussion is acceptable.

The conclusions are fully confirmed by the results.

Overall, I like the work. Consider the comments, we will rearrange the text a little and improve English. 

 

Comments on the Quality of English Language

English is difficult to understand in places. Read and simplify sentences for clarity, then review with a professional editor.

Author Response

Comment 1: This article is about two genetically similar species of endemic amphipods that are adapted to life at great depths in an ancient lake, their phenotypic differences associated with the peculiarities of their deep and dark habitat. I find it interesting in terms of understanding the evolutionary transformations of the biota in ancient unique lakes and determining the limits of the adaptive capabilities of relict species. It is written using modern methods and molecular analysis. I support its publication, but the text needs stylistic editing and some important clarifications.

Response 1: We would like to thank the Reviewer for taking the time and effort to evaluate our work and for their kind words. We appreciate and share the concerns raised by the Reviewer and updated the text accordingly; we also had it edited with an emphasis on academic style and concise sentences. Please find detailed responses below.

 

Comment 2: I recommend replace the Title «Differences in Depth-Related Phenotypic Plasticity between Two Deep-Freshwater Ommatogаmmarus Amphipod Scavengers Over Similarly Low Genetic Diversity Throughout Ancient Lake Baikal» by more clear for readers:

“Depth-dependent phenotypic plasticity differs in two amphipod scavengers (genus Ommatogammarus) despite similarly low genetic diversity in ancient Lake Baikal”

Response 2: Thank you for this idea! Indeed, the proposed title is more clear and concise. We adopted it almost fully but find it important to mention that this work is performed in a freshwater system. The title now reads:

Depth-Dependent Phenotypic Plasticity Differs between Two Deep-Freshwater Amphipod Scavengers of the Genus Ommatogаmmarus despite Similarly Low Genetic Diversity in Ancient Lake Baikal

 

Comment 3:

I propose some Minor changes.

Lines 18-19. Deep-water ecosystems make up the world's largest ecosystem, although they are poorly understood.

Response 3: Thank you for this suggestion. We used it to update the sentence but left the word “environment” in place to avoid duplication of the word “ecosystem”. The sentence now reads (lines 17-18 in the updated manuscript without tracked changes):

“Although deep-water environments make up the world’s largest ecosystem, they are poorly characterized.”

 

Comment 4: Lines 22-25. This study examines the genetic diversity and depth-related traits of two deep-water amphipod scavengers, Ommatogammarus flavus and O. albinus, which are dominant in Baikal's deepwater community.

Response 4: Thank you for this suggestion. We agree that the sentence could be hard to read, so we split it following the advice. The sentence now reads (lines 21-24):

“This study examines the genetic diversity across broad vertical (~1 km) and horizontal (~500 km) ranges, as well as depth-related traits in two deep-water scavengers Ommatogammarus flavus (Dybowsky, 1874) and Ommatogammarus albinus (Dybowsky, 1874).”

The full names of the species and their author description are provided in full at first mention as per the International Code for Zoological Nomenclature.

 

Comment 5: Lines 26-28. Our results revealed low intraspecific diversity of the cytochrome c oxidase subunit I gene marker fragment both vertically and horizontally, indicating the absence of significant barriers in the distribution of both species and the existence of a “bottleneck” event in their evolutionary history.

Response 5: Thank you. We updated the sentence, and it now reads (lines 24-27):

“Our results revealed low intraspecific diversity of the cytochrome c oxidase subunit I gene marker fragment across locations and depths, indicating the absence of significant barriers in the distribution of either species and a bottleneck event in their evolutionary histories.”

 

Comment 6: Lines 29-30 … O. flavus, as well as eye colour controlling stable opsin expression in both species

Response 6: We understand now that the sentence may have been confusing and are grateful to the Reviewer for helping us realize this issue. We have rewritten the sentence, and the phrase now reads (line 28):

“O. flavus, as well as in eye color for both species.”

 

Comment 7: Line 32 … and help to characterize the ecological differences between

Response 7: Implemented (lines 29-30). Thank you!

 

Comment 8: Everywhere change “deep-sea habitats” by deep-water habitats or environments… since you are talking not only about the sea but especially about a freshwater lake.

Response 8: Thank you for the suggestion. We double-checked the usage of “deep-sea” and “deep-water”. There are 22 entries of “deep-water” and three of “deep-sea” in the text. In those three cases, we specifically discuss literature on deep-sea adaptation (line 40), compare deep-sea to deep-freshwater conditions (line 52) or speak specifically about deep-sea research (line 73). Whenever we speak about more general information, we use deep-water.

 

Comment 9: Lines 46-47. … and related evolutionary processes remain to be understood.

Response 9: Implemented (line 47). Thank you!

 

Comment 10: Lines 97-98. Comparative roles of horizontal and vertical diversification of deep-water amphipod fauna. Role in what? It is unclear sentence.

Response 10: Indeed, the sentence was unclear. Thank you for noticing it. The sentence now reads (lines 94-96):

“In this study, our objective was to provide initial insights into the comparative roles of the horizontal and vertical distances in the diversification of deep-water amphipod fauna in Lake Baikal.”

 

Comment 11: Lines 102-103 ... At the same time, at least one species presents the same haplotypes for samples collected at several hundred kilometers [31]. Please add name of species.

Response 11: indeed, this information was missing. The species in question is a shallow-water species Eulimnogammarus cyaneus (Dybowsky, 1874). The sentence containing this information was removed from the text according to the suggestion in the comment directly below.

 

Comment 12: The introduction seems too broad. I would suggest staying closer to the topic of adaptations and genetic and phenotypic diversity of deep-water species, and it would be better to omit the information that will not be discussed further in the article and within the framework of the stated topic (for example about shallow inhabitants) .

Response 12: Thank you for this suggestion. The information about shallow-water species is now condensed into one sentence (lines 97-99). We could not omit it completely, as it explains our choice of genetic markers and also is further brought up in the discussion (lines 403-406).

We totally agree that the Introduction may seem long, but it is also very important to note that almost half of it comprises the description of the species. This text could theoretically be replaced by several literature references, but the access to these sources is fairly limited (many of them are either in Russian or in German and/or unavailable online), so we feel that it is important to briefly summarize the relevant knowledge accumulated during the last 150 years.

In addition, we went through the Introduction and shortened the text wherever it was possible.

 

Comment 13: Lines 149-152 … Apart from horizontal geographic diversity…… to study vertical diversity ? It is not clear what vertical diversity and horizontal diversity are. Do you mean genetic diversity depending on the spatial location (Lake region), and depth on the place? Why in the "deep ocean" and not a lake?Rewrite the sentence for clarity.

Response 13: Thank you for your question. Yes, exactly, horizontal (also sometimes called geographic) diversity is diversity across spatial locations, and vertical (or bathymetric) diversity is diversity across depths. We first use these terms above, when describing some relevant studies in deep-sea amphipods, and have now added more clarifications to it (lines 70-72). Throughout the rest of the text, we decided to use horizontal/vertical as the most clear pair.

By the part of the sentence that reads “even though to a smaller scale than in the deep ocean”, we meant that the ranges of depths in Baikal (maximum depth 1.6 km) are much smaller than in the ocean (maximum depth 11 km).

The sentence has been rewritten to read more easily:

“We sampled a range of geographic locations to study horizontal diversity (>500-km distance between the furthest points) and a range of depths at a small distance (1-km depth range at 2-km horizontal distance) to study vertical diversity.”

 

Comment 14: Phenotypic differences were only observed at different depths in one area (Bolshie Koty), and not in different areas of the lake (9 stations in total). Were they not noted? It is necessary to clarify why such a design of the work - genetic diversity in different regions, phenotypics in one, is this a unique case of the place, or will it be the same in other areas of the lake?

Response 14: Thank you for this interesting question! Bolshie Koty is located in the part of Baikal characterized by very steep slopes and great depth, which allowed us to sample at five depths, from 25 to 1000 meters, at very small distances and within one sampling campaign. We mention this information in the Materials and Methods section now (lines 169-173). Such a range of depths would be unrealistic at many other places. Thus, most other sampling places (six out of nine) were analyzed at only one or two very close depths, and samples were taken only in ethanol, so measuring carotenoid concentration was not possible. However, in Baikalsk we did sample depths from 150 to 1000 meters and saw the same tendency regarding body and eye color, even though it could not be analyzed quantitatively due to much smaller sample. We added this information to the manuscript (lines 326-327).

 

Comment 15: Lines 358-359 and 380-387 should be in Methods.

Response 15: Thank you for this suggestion. Indeed, we removed methodological information or moved it to the Methods.

 

Comments 16-18:

Discussion is acceptable.

The conclusions are fully confirmed by the results.

Overall, I like the work. Consider the comments, we will rearrange the text a little and improve English.

 

Responses 16-18:

Thank you for your kind words! We did our best to improve the readability and are grateful for the suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting and important manuscript well worth publishing. My fairly minor concerns are two-fold. First, some of the expressions used by the authors are confusing, so rephrasing is required (see the text with my comments). The writing style is general can be more concise. Second, the structure of the manuscript can be improved. Currently the results section is a curious mix of results, material & methods and some elements of discussion. This issue needs to be addressed before the ms can be accepted for publication.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

As above

Author Response

Comment 1:

This is an interesting and important manuscript well worth publishing. My fairly minor concerns are two-fold. First, some of the expressions used by the authors are confusing, so rephrasing is required (see the text with my comments). The writing style is general can be more concise. Second, the structure of the manuscript can be improved. Currently the results section is a curious mix of results, material & methods and some elements of discussion. This issue needs to be addressed before the ms can be accepted for publication.

 

Response 1:

We would like to thank the Reviewer for their kind words and for the time invested in helping us improve the manuscript. We have followed the suggestions and updated the text accordingly. Below are the comments from the file in the form of “highlighted text / comment” and our responses.

 

Comment 2:

L48 “Ancient / how ancient?”

Response 2: Thank you for your question. Ancient lake is an accepted term meaning that a lake is at least several million years old, as opposed to the majority of lakes that exist for no longer than 20,000 years. We refer to a review on ancient lakes [6] in the sentence below.

Moreover, the question of Lake Baikal age is not something that can be described in a simple sentence. It formed when multiple small water bodies merged. Thus, depending on what we describe as the lake, its age can be estimated as 25–30 or even 70 million years. We speak about relevant information related to the formation of the deep-water zone in the Discussion section (lines 393-394 according to the updated manuscript files without tracked changes), but adding all this information to the Introduction does not seem relevant.

 

Comment 3:

L49 “high depths-shaped / meaning?”

Response 3: meaning that these species evolved in the conditions of adaptation to high depths. This sentence was rewritten in response to another comment, and this awkward expression has been removed.

 

Comment 4:

L100 “deep / how deep is deep?”

Response 4: Formally, deep-water is usually defined as at least 200 meters (mentioned in the Introduction, line 37). In this particular case, the studied species dwell to the depths of ~1300 or 1642 meters and prevail at species-specific depths. This information is detailed in the next paragraph (lines 129-135). Unfortunately, incorporating it to the same sentence makes it harder to read.

 

Comment 5:

L120 “form stable phylogenetic group / ?”

Response 5: indeed, it would be more correct to speak about a well-supported phylogenetic cluster (corrected now). Thank you for your question.

 

Comment 6:

L121 “than within / those”

Response 6: corrected. Thank you!

 

Comment 7:

L123 “O. flavus and O. albinus / “

Response 7: There was no textual comment there but we suppose that the concern may have been connected to the words “we will name the species O. flavus and O. albinus”, which is indeed awkward. It is now corrected and reads “we will use the names O. flavus and O. albinus”.

 

Comment 8:

L137 “intersect / overlap”

Response 8: implemented. Thank you!

 

Comment 9:

L158 “different / delete”

Response 9: implemented. Thank you!

 

Comment 10:

L305-313 “We started by exploring the genetic diversity of O. flavus and O. albinus from differ306 ent depths in Bolshie Koty, near the Biological Station of the ISU. This station is located in

307 the southwestern part of the lake, which is characterized by steep declining slopes ([71];

308 http://bic.iwlearn.org/en/atlas/atlas/127-angles-of-inclination-of-map)), thus providing us

309 with a rare opportunity to sample at different depths (25 to 1000 m) at relatively short

310 horizontal distances (around 2-km distance from the shore to 1000-m point). We used the

311 Folmer COI fragment as the marker sequence, as this approach has been successfully ap312 plied to study genetic diversity in other Baikal amphipods before and other deep-water

313 amphipods (see the Introduction section). / Meterials and Methods, not Results”

Response 10: Thank you for this suggestion! We moved methodological information to the corresponding section. This text now reads (lines 301-302):

“We started by exploring the genetic diversity of O. flavus and O. albinus from 25 to 1000 m in Bolshie Koty, where the steep slopes of the lake allowed for such analysis. ”

The explanation of the choice of the sampling point is important for the logical flow (the other reviewer had a question on it), so it was important to retain it. All the other information is now in the Materials & Methods section.

 

Comment 11:

L316 “morphologically united sp / what does it men? rephrase”

Response 11: it means the same morphological species. Corrected now. Thank you for your suggestion!

 

Comment 12:

L323-324 “Then, we went on to sample O. flavus and O. albinus from nine points in all three basins of the lake to analyze their horizontal diversity (Figure 2). Even though a few unique / Also not results”

Response 12: Thank you for your suggestion. We changed the wording to shift the focus from conducted work to the obtained result. The sentence now reads (lines ...):

“Then, we analyzed horizontal diversity of O. flavus and O. albinus from nine points in all three basins of the lake (Figure 2).”

 

Comment 13:

L327-329 “Thus, either these

328 species do not form isolated groups of populations in the basins or formed such groups

329 too recently to be distinguishable by mitochondrial sequences. The d/ This is already discussion”

Response 13: Thank you! The sentence has been removed.

 

Comment 14:

L337 “ O. flavus differed in their visual appearance, m / differed from what? Confusing, please re-formulate”

Response 14: we meant that individuals from different depths differed in their appearance but shortened this phrase to avoid word repetition.

This part of the sentence has been rewritten and now reads (lines 322-323):

“we found that O. flavus from different depths varied in their appearance, most importantly and most clearly in coloration.”

 

Comment 15:

L363 “the decision to revisit the characterize / meaning”

Response 15: yes, we vehemently agree that the sentence was awkward and grammatically incorrect. We meant that first we made the observation and then checked it with additional data. The sentence has been rewritten and now reads (lines 346-348):

“This observation, in conjunction with the unusual appearance of O. flavus from this lowest depth, prompted us to examine the color of individuals of different sizes specifically from this depth.”

 

Comment 16:

L385 “opsins sequences between depths, at which eyes looked different, we compared the sequences of opsins in animals from different depths. The same animals that were used for / not Results”

Response 16: The first half of this phrase was already removed due to suggestion from the other Reviewer. Thank you for this suggestion.

 

Comment 17:

L411-412 “formed to occupy deep-water niches in two different basins of the lake and then

412 contacted when the lake progressively deepened / re-phrase please”

Response 17: Thank you for this suggestion. We rewrote this sentence to make it clearer. It is now divided into two and reads (lines 388-391):

“A plausible hypothesis could be that these species originally formed and occupied deep-water niches in two different basins of the lake. Then, when the lake progressively deepened, the species came into secondary contact.”

 

Comment 18:

L416 “much higher / Details? What was the water temparuture?”

Response 18: Thank you for this question! Unfortunately, it is impossible to determine the temperature several million years ago with any reasonable precision. This thesis was derived from the known information on historical climate changes and projected air temperatures. However, we totally agree that our sentence sounded more confident than it should have, so it was reformulated to correctly reflect the available information (lines 394-395):

“However, before approximately 6 million years ago the deep-water zone was probably anoxic and uninhabitable for multicellular life due to a warmer climate [78,79].”

 

Comment 19:

L430 “should mean / suggests”

Response 19: Implemented, thank you!

 

Comment 20:

L433 “ for a long time / How long? Be precise please“

Response 20: for at least 10 million years. This information with a reference has been added in parentheses.

 

Comment 21:

L450 “subwater / underwater?”

Response 21: indeed, it should have been “underwater”. Changed as suggested, thank you!

 

Comment 22:

L451 “bringing the bottom to shallower depths than / re-phrase please, unclear”

Response 22: Thank you. The wording is now changed to:

“making depths shallower than those preferred by O. albinus”