Evaluation of the “Bottleneck” Effect in an Isolated Population of Microtus hartingi (Rodentia, Arvicolinae) from the Eastern Rhodopes (Bulgaria) by Methods of Integrative Analysis

Round 1

Reviewer 1 Report

This is interesting paper, presenting new data and analyses that have to be published. However, there is one significant problem that need to be solved before publication. This problem is that authors presented isolated position of population of M. hartingi from the Eastern Rhodopes and it’s passing thorough “bottleneck” stage of the population dynamics as established known facts, whereas these are only author’s hypotheses not verified with available data.

Authors demonstrated the absence of connection of the population of Eastern Rhodopes and M. h. strandzensis in Bulgaria, but such connection can occur in NE Greece and N Turkey. To reject (or support) this connection, it is necessary to undertake extensive field survey and/or species distribution modeling coupled with population connectivity analysis. In the absence of survey data and results of distribution analyses, isolated position of the population of Eastern Rhodopes can be accepted as hypothesis only. Relatively high genetic distance of Eastern Rhodopes population and M. h. strandzensis can be considered as an indirect support of this hypothesis, but also may be the track of the past isolation, the result of isolation by distance, or just a sampling error (only two specimens from E Rhodopes are studied genetically).

Concerning the “bottleneck” stage of the population dynamics, there are several genetic tests that can support this hypothesis, but these tests can’t be applied in author’s case due to too small sample size. Developmental aberrations found in the population of Eastern Rhodopes are (most probably) indications of the high inbreeding rate, but it is only indirect support of the “bottleneck” hypothesis.

I would recommend to re-write some parts of Abstract, Introduction and Discussion to divide unambiguously well-established facts and author’s hypotheses. From this point of view, the Title also should be changed to avoid reader’s misunderstanding.

To make understanding the spatial relations of studied populations of M. hartingi easier for readers, I would recommend to add the map showing positions of these populations to the manuscript.

One more minor note: when you write “Macedonia” (page 2, line 46; page 4, Table 1; page 11, Fig. 6; page 13, line 384), please be more precise and indicate what do you mean, one of the Greece provinces (Eastern, Western or Central Macedonia) or Republic of North Macedonia (FYROM).

Author Response

I am highly obliged to the revier 1 for his very useful corrections, which helped us to improve the manuscript.

This is interesting paper, presenting new data and analyses that have to be published. However, there is one significant problem that need to be solved before publication. This problem is that authors presented isolated position of population of M. hartingi from the Eastern Rhodopes and it’s passing thorough “bottleneck” stage of the population dynamics as established known facts, whereas these are only author’s hypotheses not verified with available data.

1. Authors demonstrated the absence of connection of the population of Eastern Rhodopes and M. h. strandzensisin Bulgaria, but such connection can occur in NE Greece and N Turkey. To reject (or support) this connection, it is necessary to undertake extensive field survey and/or species distribution modeling coupled with population connectivity analysis. In the absence of survey data and results of distribution analyses, isolated position of the population of Eastern Rhodopes can be accepted as hypothesis only. Relatively high genetic distance of Eastern Rhodopes population and M. h. strandzensiscan be considered as an indirect support of this hypothesis, but also may be the track of the past isolation, the result of isolation by distance, or just a sampling error (only two specimens from E Rhodopes are studied genetically).

We agree that complete isolation of the population of Eastern Rhodopes can be accepted as hypothesis only. Nevertheless, in confirming this hypothesis, we received a lot of facts. We believe that the identified specificity in the morphological structure of the population of Eastern Rhodopes, as well as its isolation from other forms of M. hartingi, according to Cytb analysis, most likely indicate the ancient isolation of this form. This is confirmed both by comparisons with our data and with data from literature sources. We certainly agree that it is necessary to undertake extensive field survey and/or species distribution modeling coupled with population connectivity analysis. In addition, T. Zorenko and N. Atanasov explored a significant part of Bulgaria towards the Mariza River and did not find suitable habitats for M. hartingi (for more details, see Kryštufek, et al, 2018. The Mariza River probably also contributed to the isolation of M. hartingi from E. Rhodopes See figure

It is also known that this vole avoids agricultural land and lives mainly on land that is not cultivated (Bontzorlos V.A., 2009. The trophic ecology of barn owl in the agricultural ecosystems of central Greece: its application in the distribution and abundance of its prey Doctoral dissertation, Universidad de Salamanca). Thus, these voles have high requirements for habitats, which are few and only fragments. Voles of the subgenus Sumeriomys are also quite spatially conservative. When settling in a new place, these voles first organize a social structure in the population and only then proceed to reproduction (Kasatkin, M.V., Isaev, S.I., and Savitskaya, L.E., 1998. Some features of the ecology of the social vole (Microtus socialis) in the black lands of Kalmykia during the period of population growth, Zool. Journal. 77(5) 582-592).

2. Concerning the “bottleneck” stage of the population dynamics, there are several genetic tests that can support this hypothesis, but these tests can’t be applied in author’s case due to too small sample size. Developmental aberrations found in the population of Eastern Rhodopes are (most probably) indications of the high inbreeding rate, but it is only indirect support of the “bottleneck” hypothesis.

The supposed existence of the population of Eastern Rhodopes in the “bottleneck” mode is confirmed by the detected genetically determined disorders in the calcification of the skull in all examined individuals, which could have arisen only as a result of inbreeding. Similar disturbances have not been found in any other M. hartingi population.

3. I would recommend to re-write some parts of Abstract, Introduction and Discussion to divide unambiguously well-established facts and author’s hypotheses. From this point of view, the Title also should be changed to avoid reader’s misunderstanding.

The Abstract presents only the methods and results. In the Introduction our former results as well as the data from literature are presented. The main objection of the reviewer concerns our consideration of the "bottleneck". A detailed answer to this remark is in our 1st and 2nd answers. We tried to make a distinction in the Discussion in order to separate the unambiguously established facts and our hypotheses. In addition, we reduced some of the text in the section that is too speculative.

4. To make understanding the spatial relations of studied populations of M. hartingieasier for readers, I would recommend to add the map showing positions of these populations to the manuscript.

Region map is added – Figure 1.

5. One more minor note: when you write “Macedonia” (page 2, line 46; page 4, Table 1; page 11, Fig. 6; page 13, line 384), please be more precise and indicate what do you mean, one of the Greece provinces (Eastern, Western or Central Macedonia) or Republic of North Macedonia (FYROM).

We've made a clarification. In all cases, we mean North Macedonia.

 

Author Response File: Author Response.docx

Reviewer 2 Report

- you can find the article I reviewed in the attached file. - I marked the necessary corrections on the text (especially Turkish location names are wrong and should be corrected). The article is original, it makes an important contribution to the taxonomy of the genus and to the zoogeography of Europe. - It is mentioned in the article that 2 gene regions (mitochondrial Cytb and nuclear GHR) were studied. - However, in the article it seems as if the GHR gene has been forgotten. There is no methodical information with GHR and it has not been evaluated in the results either. - Apart from this, providing more information about Cytb findings (such as haplotype diversity, nucleotide diversity, number of polymorphic sites, number of mutations..so on) will make the article more meaningful. In this way, the genetic bottleneck that the species is likely exposed to can be better understood.

Comments for author File: Comments.pdf

Author Response

We are very pleased to the revier 2 for his corrections, which were very important. All the corrections were put in the text.

 you can find the article I reviewed in the attached file. - I marked the necessary corrections on the text (especially Turkish location names are wrong and should be corrected). The article is original, it makes an important contribution to the taxonomy of the genus and to the zoogeography of Europe. - It is mentioned in the article that 2 gene regions (mitochondrial Cytb and nuclear GHR) were studied. - However, in the article it seems as if the GHR gene has been forgotten. There is no methodical information with GHR and it has not been evaluated in the results either. - Apart from this, providing more information about Cytb findings (such as haplotype diversity, nucleotide diversity, number of polymorphic sites, number of mutations..so on) will make the article more meaningful. In this way, the genetic bottleneck that the species is likely exposed to can be better understood.

We have made all the corrections to the text of the manuscript. As for “more information about Cytb findings (such as haplotype diversity, nucleotide diversity, number of polymorphic sites, number of mutations..so on)”. The number of variable and parsimony informative sites is indicated in the results section (paragraph 3.2), and the nucleotide and haplotype diversity for the sample formed with two individuals are not informative.

Author Response File: Author Response.docx