Status and Life History Traits of Simpsonaias ambigua (Salamander Mussel) in Ontario, Canada

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I enjoyed reading this paper. It is well researched, very clear, and concise. A few minor questions/additions.

Line 80. A question comes to mind while reading about the search techniques, why was snorkeling or scuba diving not employed in deeper waters? It has been effective in Simpsonaias surveys elsewhere? My understanding is that Necturus live not just in shallow water and Simpsonaias are also not restricted to wadable waters.

Line 380 Should the verb was be changed to were?

Line 481. Add the word “to” to “entrance the bait”.

Line 586 the name Simpsonaias ambigua should be in italics.

Author Response

Thank you for taking the time to review our manuscript. Below are the responses to your comments.

Comment 1: I enjoyed reading this paper. It is well researched, very clear, and concise. A few minor questions/additions.

Response 1: Thank you very much for your kind comment.

Comment 2. Line 80. A question comes to mind while reading about the search techniques, why was snorkeling or scuba diving not employed in deeper waters? It has been effective in Simpsonaias surveys elsewhere? My understanding is that Necturus live not just in shallow water and Simpsonaias are also not restricted to wadable waters.

Response 2: Thank you for your comment. You’re absolutely right, these species aren’t limited to wadeable waters. We didn’t use scuba or snorkel gear in the deeper areas of the Sydenham River due to its poor water clarity. With visibility being less than a foot, diving or snorkeling simply isn’t feasible. That’s why we developed alternative methods, to find these species in water bodies with poor visibility. This point was stated in lines 475-476: “Trapping of the host species is particularly useful when the population of N. maculosus has a high density and when water visibility or depth are limiting factors”. We added “The other two methods are suitable when exploring large areas for the first time; as they help narrow the search area using traditional surveys, when water visibility or depth are limiting factors to conduct flipping” (lines 526-529).

Bogan and Loci 2009, collected S. ambigua in less than 30 feet of water in Allegheny River Pools. Even though they don’t specify that they used scuba, given the depth they found individuals is fair to speculate they did. Therefore, scuba and snorkeling are appropriate if conditions allow.

Comment 3: Line 481. Add the word “to” to “entrance the bait”.

Response 3: Thank you for your suggestion. We added “to” to that line.

Comment 4: Line 586 the name Simpsonaias ambigua should be in italics.

Response 4: Thanks for pointing this out, we updated the reference so Simpsonaias ambigua is in italics.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear colleagues,

This is nice paper, focusing on a rare freshwater mussel species. It thoroughly describes the reproductive biology, life history, ecological preferences, and age of the Salamander Mussel (Simpsonaias ambigua) in Ontario. This work is based on a long-term field survey. The manuscript is well-written and concise. I don't have any major comments but a few small typos are highlighted below.

Line 30: It is small (maximum 50 mm long and 28 mm height, ... => It is small (maximum 50 mm long and 28 mm height), ...

Line 39: Simpsonaias ambigua is endemic to North American ... => Simpsonaias ambigua is endemic to North America ...

Line 416: were founds => were found

Lines 512-513: "that species of the Simpsonsais genera do have characteristics that indicate a periodic life history strategy...". First, the name of this genus is Simpsonaias, not Simpsonsais. Second, this sentence need to be revised. "species of the Simpsonsais genera" - it is a single genus, Simpsonaias. Why genera?! Moreover, this genus is currently monotypic - it contains the only species, Simpsonaias ambigua. So, something like: "that the genus Simpsonaias does have characteristics that indicate a periodic life history strategy..." or "that Simpsonaias mussels do have characteristics that indicate a periodic life history strategy...".

 

Kind regards,

 

Author Response

Comment 1: This is nice paper, focusing on a rare freshwater mussel species. It thoroughly describes the reproductive biology, life history, ecological preferences, and age of the Salamander Mussel (Simpsonaias ambigua) in Ontario. This work is based on a long-term field survey. The manuscript is well-written and concise. I don't have any major comments but a few small typos are highlighted below.

Response 1: Thank you very much for your kind comment.

Comment 2: Line 30: It is small (maximum 50 mm long and 28 mm height, ... => It is small (maximum 50 mm long and 28 mm height), ...

Response 2: Thank you for this observation, we added the “)”.

Comment 3: Line 39: Simpsonaias ambigua is endemic to North American ... => Simpsonaias ambigua is endemic to North America

Response 3: Thank you for noting this, we eliminated the “n” in American.

Comment 4: Line 416: were founds => were found

Response 4: Thank you for noting this, we eliminated the additional “s”.

Comment 5: Lines 512-513: "that species of the Simpsonsais genera do have characteristics that indicate a periodic life history strategy...". First, the name of this genus is Simpsonaias, not Simpsonsais. Second, this sentence need to be revised. "species of the Simpsonsais genera" - it is a single genus, Simpsonaias. Why genera?! Moreover, this genus is currently monotypic - it contains the only species, Simpsonaias ambigua. So, something like: "that the genus Simpsonaias does have characteristics that indicate a periodic life history strategy..." or "that Simpsonaias mussels do have characteristics that indicate a periodic life history strategy...".

Response 5: Thank you for your suggestion. We incorporated these changes to the manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

General comment

I liked the MS and there are some sound novel interesting findings.

Major comment

My problem with the MS are the eDNA sections. From what is written it doesn’t appear that you took and tested water samples for sites/systems where you found the mussels? Hence although you did run + procedural controls, because you cannot verify for this study that the eDNA sampling method works when mussels were present, you cannot ascribe what a failure to detect mussels arises from, i.e. no mussels or an inadequate sampling method. Although the method has worked elsewhere, you acknowledge yourself that the sampling method you used for this study might be flawed. My recommendation is to remove all of the eDNA sections and just make it a paper about a survey of and ecology/biology of Simpsonaias ambigua. There is more than enough interesting and novel stuff in that.

Minor comment

I think you could condense the MS by at least 25%. I have attached a Word doc. version of the MS where I started to that.

Comments for author File: Comments.pdf

Author Response

Comment 1:

General comment

I liked the MS and there are some sound novel interesting findings.

Response 1: Thank you very much for your kind comment.

Comment 2:

Major comment

My problem with the MS are the eDNA sections. From what is written it doesn’t appear that you took and tested water samples for sites/systems where you found the mussels? Hence although you did run + procedural controls, because you cannot verify for this study that the eDNA sampling method works when mussels were present, you cannot ascribe what a failure to detect mussels arises from, i.e. no mussels or an inadequate sampling method. Although the method has worked elsewhere, you acknowledge yourself that the sampling method you used for this study might be flawed. My recommendation is to remove all of the eDNA sections and just make it a paper about a survey of and ecology/biology of Simpsonaias ambigua. There is more than enough interesting and novel stuff in that.

Response 2: Thank you for your thoughtful comment; you’ve raised an important point. While we didn’t remove the eDNA sections as you suggested, we added some clarifying text. In 2019, we tested the primers in the Sydenham River at site SR-05, where S. ambigua is present and was surveyed for this manuscript. We had positive detections there, which serve as positive controls. However, since this data has been published elsewhere, we can’t include it in the methods or results of this manuscript.

This is the text added to the manuscript: Lines 161-164: ”Porto-Hannes et al. [18] previously developed a quantitative PCR assay and optimized methods to detect S. ambigua eDNA in the laboratory (e.g., holding tank water) and in the field at sites in Ontario where this species is known to occur (East Sydenham River, site SR-05; Figure 1a)”.

Comment 3:

Minor comment

I think you could condense the MS by at least 25%. I have attached a Word doc. version of the MS where I started to that.

Response 3:

Thank you for taking the time to review the manuscript and provide valuable suggestions for condensing the text. We have implemented many of your recommendations, except for those regarding the eDNA sections, which we addressed in our response to comment 2.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

                             The additional text clarifying that you had sampled a site for eDNA where the mussels were present and did detect them there is perfect. However, this has opened a fresh can of worms, again relating to the reliability of the eDNA methodology/sensitivity.

In your 2023 paper, I liked it a lot – nice work, where you develop the markers you failed to detect the mussels consistently unless they were a large population. From the paper:

“Most detections of S. ambigua eDNA were observed at siteLSC-SYR-05,which is downstream of the reach of the Sydenham River that appears to support the largest populations of the species. However, we detected eDNA in only two of three field replicates and two to three qPCR replicates at this site. At sites LSC-SYR-29 andLSC-SYR-44, we detected eDNA in only one of three field replicates and one qPCR replicate. We did not detect eDNAatsiteLSC-SYR33, which appears to support only small populations of S. ambigua and maybe near the upstream limit of the species in the river (see previous text).”

In essence the method applied is only reliable (ignoring other stochastic & environmental considerations) if “large” populations exist. Hence a lack of detection doesn’t necessarily mean a lack of mussels, just that the method isn’t sensitive enough as determined by your 2023 publication. Yes following the rule of parsimony given the that only dead shells were present it is likely there are no mussels in the rivers you tested but we can’t use your eDNA work to state that categorically.

I going to suggest again that you cut the eDNA sections from the MS – as said previously because you can’t use it to confirm mussels weren’t present, it doesn’t in my opinion add anything to the MS. Alternatively, you need to point out that the eDNA methodology utilised is limited in resolution and  a negative finding cannot be regarded as definitive, i.e. that there aren’t any mussels there (sorry).

Author Response

Comment 1: The additional text clarifying that you had sampled a site for eDNA where the mussels were present and did detect them there is perfect. However, this has opened a fresh can of worms, again relating to the reliability of the eDNA methodology/sensitivity.

In your 2023 paper, I liked it a lot – nice work, where you develop the markers you failed to detect the mussels consistently unless they were a large population. From the paper:

“Most detections of S. ambigua eDNA were observed at siteLSC-SYR-05,which is downstream of the reach of the Sydenham River that appears to support the largest populations of the species. However, we detected eDNA in only two of three field replicates and two to three qPCR replicates at this site. At sites LSC-SYR-29 andLSC-SYR-44, we detected eDNA in only one of three field replicates and one qPCR replicate. We did not detect eDNAatsiteLSC-SYR33, which appears to support only small populations of S. ambigua and maybe near the upstream limit of the species in the river (see previous text).”

In essence the method applied is only reliable (ignoring other stochastic & environmental considerations) if “large” populations exist. Hence a lack of detection doesn’t necessarily mean a lack of mussels, just that the method isn’t sensitive enough as determined by your 2023 publication. Yes following the rule of parsimony given the that only dead shells were present it is likely there are no mussels in the rivers you tested but we can’t use your eDNA work to state that categorically.

I going to suggest again that you cut the eDNA sections from the MS – as said previously because you can’t use it to confirm mussels weren’t present, it doesn’t in my opinion add anything to the MS. Alternatively, you need to point out that the eDNA methodology utilized is limited in resolution and a negative finding cannot be regarded as definitive, i.e. that there aren’t any mussels there (sorry).

Response 1: Thank you one more time for your kind and encouraging words. Also, for taking the time to read our 2023 manuscript and for your comments. We have decided to make the changes you suggested. We eliminated most of the text you indicated expect for lines 257-272 (from the latest manuscript), because these are methods to confirm that the glochidia-like structures found in the Mudpuppy’s gills are Simpsonaias ambigua and not other gill parasites. We also updated Figure 2, eliminating the sites where we conducted eDNA sampling.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Nicely done.