Old lake versus young taxa: a comparative phylogeographic perspective on the evolution of Caspian Sea gastropods (Neritidae: Theodoxus)

The Caspian Sea has been a highly dynamic environment throughout the Quaternary and witnessed major oscillations in lake level, which were associated with changes in salinity and habitat availability. Such environmental pressures are considered to drive strong phylogeographic structures in species by forcing populations into suitable refugia. However, little is actually known on the effect of lake-level fluctuations in the Caspian Sea on its aquatic biota. We compared the phylogeographic patterns of the aquatic Neritidae snail genus Theodoxus across the Pontocaspian region with refugial populations in southern Iran. Three gene fragments were used to determine relationships and divergence times between the sampled populations in both groups. A dated phylogeny and statistical haplotype networks were generated in conjunction with the analyses of molecular variance and calculations of isolation by distance using distance-based redundancy analyses. Extended Bayesian skyline plots were constructed to assess demographic history. Compared with the southern Iranian populations, we found little phylogeographic structure for the Pontocaspian Theodoxus group, with more recent diversification, homogeneity of haplotypes across the Pontocaspian region and a relatively stable demographic history since the Middle Pleistocene. Our results argue against a strong influence of Caspian Sea low stands on the population structure post the early Pleistocene, whereas high stands may have increased the dispersal possibilities and homogenization of haplotypes across the Pontocaspian region during this time. However, during the early Pleistocene, a more dramatic low stand in the Caspian Sea, around a million years ago, may have caused the reduction in Theodoxus diversity to a single lineage in the region. In addition, our results provide new insights into Theodoxus taxonomy and outlooks for regional conservation.

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We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Thanks for your submission. We hope you find the reviewers' comments constructive as you revise, and we look forward to the next version. Best wishes.

Associate Editor's comments (Dr Kristina Sefc):
The reviewers of the manuscript are both impressed by the remarkable phylogeographic sampling underlying the study, and are mostly in agreement with the presented work. Please respond to the concerns (as raised by reviewer 1) regarding the use of lineage-through-time plots and Bayesian skyline plots in your analysis. I agree with the reviewer that it is preferable to omit analyses if assumptions are violated or if there's a risk to arrive at spurious results. Regarding the BSP, if a literature survey (e.g. simulation studies testing effects of population structure on BSP analyses) suggests that the structure in your data might pose a problem, you could consider to run analyses for the individual haplogroups separately and omit divergent lineages that include only few haplotypes. Please also provide the additional information and discussions as suggested by reviewer 1, and follow the comments of reviewer 2.
Reviewers' Comments to Author: This study provides a comparative phylogeographic analyses of Theodoxus gastropods from the Pontocaspian region and Southern Iran. The study is based on a good dataset with samples from quite a few distinct localities well spread across the study area. Although the manuscript is generally well written and most of the analyses/conclusions drawn from the analyses are ok, I do have some comments that need to be considered before this ms is ready for publication: Major comments on analyses: 1. General comment on LTT-plots: LTT plots are typically NOT used for assessing demographic histories, but rather to infer/visualise the accumulation of distinct species/phylogenetic lineages through time. LTTs are not very useful for inferring intraspecific patterns of diversification as here LTTs would only make sense if you had all (or nearly all) the species' haplotypes included. As this is something you cannot be sure of, I strongly recommend removing LTTs from your ms.
2. Bayesian Skyline Plots: You have lots of phylogeographic structure in your data, in particular in the Iranian dataset, but also in the Pontocaspian data (even if you state that there is hardly and population genetic structuring). It is well known that BSPs might be heavily affected by population structure and there are already some studies available that tested the effect of population structure and sampling design on the performance of BSPs. I strongly recommend to check the relevant literature and adapt your analysis strategy and/or interpretation of the BSP patterns accordingly.
Minor comments: 1. Summary & Materials and methods: You write that your study is baed on three amplified gene fragments. It's never mentioned you also sequenced them ...
2. I strongly suggest to provide some more information on the biology/ecology of the study species (habitat preference, specialist or generslist, ...) and the hydrological systems(s) (potential habitat discontinuities in the Caspian Sea that are potential parriers to dispersal, hydrological connection to other systems, ...). This info is important for interpreting the patterns you find.
3. Summary, Results & Discussion: Is the resolution (mutation rate) of your markers good enough to test for a potential influence of Pleistocene lake level fluctuations on population structure? 4. Introduction, line 47: How old is the Caspian Sea? 5. Introduction, line 52: In what sense is the Caspian Sea a unique model system to study the effect of palaeo-environmental changes on the phylogeographic struture in long-lived lake biota? It's certainly an excellent model system, but I can think of quite a few other systems that are equally well (or maybe even better) suited -e.g., just think of the East African Great Lakes. 6. M&M, page 2, line 48: Why did you use the BD tree prior? Any specific reasons for doing so? I'm asking because you are looking at a mixture of inter-and (mainly) intraspecific data and and tree prior choice might not be a trivial issue here.  Table S1, there's quite some structure, within the three main clades/refugial lineages. I think you can and should put much more emphasis on the phylogeographic patterns in the discussion, e.g. how haplogroup V got distributed over such a large area, from the Black Sea over rivers northwest of the Caspian Sea and the Caspian Sea to rivers southeast of the Caspian Sea, whereas the two subclades of haplogroup VI have very narrow distribtutions. By the way, why don't you consider these two subclades different haplogroups? This would make much more sense.
13. Discussion: Please discuss your findings also in light of what is known on habitat preferences of the species. E.g., are Theodoxus habitat specialists or rather generalists? And are there any obvious habitat discontinuities that might act as dispersal barriers?
Reviewer: 2 The manuscript reports on a phylogenetic reconstruction of the neritid genus Theodoxus from the Caspian Sea region. The authors have a range of samples from across the region, and used three genes (two mitochondrial, one nuclear) for their inferences. There was evidence of a general lack of reciprocal monophyly among species, but instead isolation by distance was present. The authors also undertook reconstructions of historic population sizes using their data, and speculate on potential links between climate and present day diversity. Overall this is a nice paper based on a hard-won dataset. The analyses appear appropriate to the questions being asked, and the results are fairly clear. Most of my comments relate to relatively minor issues of presentation. P2, L40-43. I'm not convinced there is any evidence here of effects of low lake levels on population genetic structure. At best the evidence is that high stands may have provided opportunities for genetic homogenisation.

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Most of my previous concerns have been addressed satisfactorily. Two issues, however, remain: 1. lines 175-177 (and comment 10 of the original review): ... no, I didn't misread the sentence. Note that each node in the tree represents a divergence event. In fact, you can delete this entire sentence as it essentially conveys the same information as the previous sentence (lines 174-175).
2. EBSPs: Yes, doing separate analyses for the two Pontocaspian phylogroups (now presented as Supplementary info) is the right way to deal with these data. And yes, for most of the Iranian phylogroups sample size/diversity is too small to allow for doing EBSP analyses. This, however, is no justification for pooling all these four pretty divergent phylogroups (cryptic species?) for a single EBSP analysis. You have virtually no intraphylogroup diversity in the Iranian samples and with the large interphylogroup divergence you inevitably get a signature of a drastic recent population decline in your EBSP. It's simply not possible to do reliable EBSP analyses with these Iranian samples. I suggest you have a look at pevious studies that evaluated the performance of (E)BSPs in the presence of population structure. Specifically, in highly structured populations, drastic recent population size declines were observed (similar to what you found with your data) even though the sequences were simulated under a constant population size scenario. Two relevant studies that come to my mind are Heller et al. 2013 PLoS One & Grant 2015 J Hered. I think you'll have to have a more critical look at your EBSPs, especially since these data are essential for your discussion/conclusions on conservation aspects in your gastropods. Manuscript ID RSOS-190965.R1 entitled "Old lake vs. young taxa: a comparative phylogeographic perspective on the evolution of Caspian Sea gastropods (Neritidae: Theodoxus)" which you submitted to Royal Society Open Science, has been reviewed. The comments of the reviewer(s) are included at the bottom of this letter.
Please submit a copy of your revised paper before 19-Sep-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers.
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In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections before the reference list: • Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
• Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Dear authors, I'd like to thank you for addressing most of the concerns raised in the first round of review. The issue of pooling divergent lineages for the EBSPs still remains and actually represents a rather serious one since an important conclusion -regarding the decline in population size -may be based on an artifact in the analysis. Please see the reviewer's comments for details. If the sampling / the data don't allow to analyse the demographic history of the Iranian populations, then you'll have to consider dropping this part from the manuscript (or interpret the network structures of the mtDNA and ncDNA sequences verbally -less diversity in the mt than nc genomes within each phylogroup may indeed point to a recent bottleneck); or at least discuss the problem associated with pooling the divergent lineages and make clear that there's a risk of a spurious result. Sincerely, Kristina Sefc Subject Editor Comments to Authors: Thank you for addressing previous comments. Please note that the reviewer and the AE still feel there is a major issue to address. In your resubmission please make the necessary edits and address the comments. If the AE does not feel these are sufficiently addressed we will not be able to consider the manuscript further. Best wishes.
Reviewer comments to Author: Reviewer: 1 Most of my previous concerns have been addressed satisfactorily. Two issues, however, remain: 1. lines 175-177 (and comment 10 of the original review): ... no, I didn't misread the sentence. Note that each node in the tree represents a divergence event. In fact, you can delete this entire sentence as it essentially conveys the same information as the previous sentence (lines 174-175).
2. EBSPs: Yes, doing separate analyses for the two Pontocaspian phylogroups (now presented as Supplementary info) is the right way to deal with these data. And yes, for most of the Iranian phylogroups sample size/diversity is too small to allow for doing EBSP analyses. This, however, is no justification for pooling all these four pretty divergent phylogroups (cryptic species?) for a single EBSP analysis. You have virtually no intraphylogroup diversity in the Iranian samples and with the large interphylogroup divergence you inevitably get a signature of a drastic recent population decline in your EBSP. It's simply not possible to do reliable EBSP analyses with these Iranian samples. I suggest you have a look at pevious studies that evaluated the performance of (E)BSPs in the presence of population structure. Specifically, in highly structured populations, drastic recent population size declines were observed (similar to what you found with your data) even though the sequences were simulated under a constant population size scenario. Two relevant studies that come to my mind are Heller et al. 2013 PLoS One & Grant 2015 J Hered. I think you'll have to have a more critical look at your EBSPs, especially since these data are essential for your discussion/conclusions on conservation aspects in your gastropods. I am pleased to inform you that your manuscript entitled "Old lake vs. young taxa: a comparative phylogeographic perspective on the evolution of Caspian Sea gastropods (Neritidae: Theodoxus)" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact --if you are going to be away, please nominate a coauthor (if available) to manage the proofing process, and ensure they are copied into your email to the journal. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published.

Reference Number: RSOS-190965
We very much appreciate the time and effort of the reviewers and the editors in assessing our paper and we are grateful for their useful comments. Below we list all critical comments and suggestions of the reviewers (in italics), together with our point-by-point replies (in blue). Attached at the very end is the revised version of the research paper with the 'Track Changes -All Markup' enabled, for easy reference of where changes have been made.

Please respond to the concerns (as raised by reviewer 1) regarding the use of lineage-through-time plots and Bayesian skyline plots in your analysis. I agree with the reviewer that it is preferable to omit analyses if assumptions are violated or if there's a risk to arrive at spurious results. Regarding the BSP, if a literature survey (e.g. simulation studies testing effects of population structure on BSP analyses) suggests that the structure in your data might pose a problem, you could consider to run analyses for the individual haplogroups separately and omit divergent lineages that include only few haplotypes.
We have removed the LTT plots and improved our Bayesian skyline analyses as suggested. Please see replies to reviewer 1: major comments 1 & 2 and minor comment 8.

1) General comment on LTT-plots: LTT plots are typically NOT used for assessing demographic histories, but rather to infer/visualise the accumulation of distinct species/phylogenetic lineages through time. LTTs are not very useful for inferring intraspecific patterns of diversification as here LTTs would only make sense if you had all (or nearly all) the species' haplotypes included. As this is something you cannot be sure of, I strongly recommend removing LTTs from your ms.
We agree with the suggestion and have remove the LTT plots and all content related to them.

2) Bayesian Skyline Plots: You have lots of phylogeographic structure in your data, in particular in the Iranian dataset, but also in the Pontocaspian data (even if you state that there is hardly and population genetic structuring). It is
Appendix A well known that BSPs might be heavily affected by population structure and there are already some studies available that tested the effect of population structure and sampling design on the performance of BSPs. I strongly recommend to check the relevant literature and adapt your analysis strategy and/or interpretation of the BSP patterns accordingly.
We are interested in the overall trends in both groups rather than the phylogroup specific trends. As most phylogroups have overlapping ranges (and thus likely have experienced similar environmental histories), the impact bias of a specific phylogroup on the overall pattern should be limited. Moreover, some phylogroups (specifically those in Iran) are too small to construct reliable skyline plots individually. Nevertheless we note the reviewer's concern and the associate editor's suggestions and have constructed EBS plots (also see minor comment 8 of reviewer 1) for all phylogroups where ESS values < 200 could be established (specifically phylogroups V and VI). These show little deviation from the overall trend from the Pontocaspian group. We have added the new additional results to the supplementary information and made alterations to the methods and results chapters. See: L150-168 & L199-208 and Appendix A; figure S1.

1) Summary & Materials and methods: You write that your study is based on three amplified gene fragments. It's never mentioned you also sequenced them ...
We have now added sequencing information in the 'materials and methods' section. See: L100-101.
2) I strongly suggest to provide some more information on the biology/ecology of the study species (habitat preference, specialist or generalist, ...) and the hydrological systems(s) (potential habitat discontinuities in the Caspian Sea that are potential barriers to dispersal, hydrological connection to other systems, ...). This info is important for interpreting the patterns you find.

3) Summary, Results & Discussion: Is the resolution (mutation rate) of your markers good enough to test for a potential influence of Pleistocene lake level fluctuations on population structure?
This largely depends on the duration of the lake level fluctuation and is critically discussed (see L222-224 & L237-239). Moreover, there are a number of recently published studies on Theodoxus, using these three genes, to assess phylogeographic patterns and evolutionary history on the intraspecific level with success (i.e. showed to have sufficient resolution to detect a variety of major influences) [1-5]. As most Theodoxus intraspecific diversification occurs after the Pliocene [6], this suggest they variable enough to detect the effect of major lake level changes during this period.

4) Introduction, line 47: How old is the Caspian Sea?
Added the age of the Caspian Sea as suggested. See: L37-38.

5) Introduction, line 52:
In what sense is the Caspian Sea a unique model system to study the effect of palaeo-environmental changes on the phylogeographic structure in long-lived lake biota? It's certainly an excellent model system, but I can think of quite a few other systems that are equally well (or maybe even better) suited -e.g., just think of the East African Great Lakes.

6) M&M, page 2, line 48: Why did you use the BD tree prior? Any specific reasons for doing so? I'm asking because you are looking at a mixture of inter-and (mainly) intraspecific data and tree prior choice might not be a trivial issue here.
While the 'coalescent population (constant and exponential) tree priors' are more orientated towards population level studies, we selected the birth death-prior due to having more than one species and not wanting to assume all Caspian Sea morphospecies belonged to the same phylogenetic species. Moreover, according to the latest published research [7], the choice of tree prior and molecular clock does not substantially affect phylogenetic patterns or diversification rates.

8) M&M, page 3, line 16+: Did you do a BSP (as stated in the ms) or an EBSP analysis? I'm asking because as far as I know the standard BSP analysis does not allow for multi locus data. Please clarify.
Changed as suggested. All BS plots have now been reconstructed using the EBSP method and content/references adapted. See: L26, L150-168, L199-208 & L298-307, figure 5 + caption and Appendix A; figure S1.

9) M&M, page 3, line 25: Did you really use the 95% confidence intervals or rather the 95% HPD intervals (which would be the standard way).
This should have been 'HPD'. Changed as suggested. See: L120. We feel the reviewer may have misread the sentence (See L174-176). We specifically state 'between phylogroups' and not 'within and between phylogroups'. We are specifically describing the divergence events of I, II, III and IV from each other; and V and VI from each other.

11) Results, page 3, lines 38+: It would be good to indicate the different morphospecies you refer to here also in the tree.
Changed as suggested. We have adapted figure 3 to also indicate species names. See: figure 3 + caption.

12) Discussion, page 4, line 1: Where is the evidence for the proposed catastrophic bottleneck? This is not based on your data, or is it? Please clarify.
We partly agree with the reviewer, in that the early bottleneck discussed is somewhat speculative as the extended Bayesian skyline plots cannot cover this period of time to adequately test it. The reasoning is based on the presence of diverse fossil data in the absence of early molecular diversification in the phylogeny (all diversity stems from a single lineage over this period) and low nucleotide diversity (indicating a young species flock). This is a reasonable explanation given the data available and the results we have. However we do critically discuss our interpretation and note the speculativeness thereof. We agree with the reviewer that more discussion needs to be included regarding the broad distribution of phylogroups V and VI. As such we have added some discussion about the effects of high stands and how they may have driven homogeneity of haplotypes across the system (also see response to reviewer 2, minor comment 1). However the two 'subclades' in phylogroup VI are not supported by posterior probabilities in the phylogeny and nor are the differences conserved across the AMOVAs of all genes. As such, discussion of 'narrow ranges' or the general patterns within subclades of phylogroup V or VI would be very dubious. We agree with the reviewer that there is little evidence to support low stands having any effect on population structure; indeed that is primarily our outcome. However, following the suggestion, we have adapted our text to put more focus on the homogenising effects of high stands. See: L30-31, L240-247 & L322-324.

3) P3, L23. Use "should possess similar"
Changed 'mimic' to 'possess' as suggested. See: L85. Parallel evolution is unlikely given its rarity, especially considering that the amount of mutations that have occurred and the extended geological time over which these took place. Moreover, although not discussed or shown, the majority of key morphological characters are conserved, such as those related to the operculum (which is often used to distinguish species). As this is not really in the scope of this study, it will be addressed in a follow-up article currently in preparation by the authors reviewing the taxonomy and morphology of all extant Theodoxus spp.
Volume number now added. See: reference 70.

16
The Caspian Sea has been a highly dynamic environment throughout the Quaternary and witnessed major oscillations 17 in lake level, which were associated with changes in salinity and habitat availability. Such environmental pressures 18 are considered to drive strong phylogeographic structure in species by forcing populations into suitable refugia.

19
However, little is actually known on the effect of lake level fluctuations in the Caspian Sea on its aquatic biota. We  Theodoxus taxonomy and outlooks for regional conservation.

36
The Caspian Sea is one of the largest and oldest lakes on the planet. Barring episodic overflow events, the Caspian 37 Sea has been an isolated endorheic basin since at least the early Pliocene, around 5.3 million years ago (Ma) [8].

38
Today, the Caspian Sea and its catchment cover an area of approximately 3,500,000 km 2 [9,10]. It is renowned for 39 dramatic historical lake level fluctuations and salinity shifts, primarily as a consequence of glacial cycles during the 40 Quaternary [8,11]. Moreover, temporary connections occurred between the Caspian Sea and other Pontocaspian 41 basins during high-stands (i.e. the Aral, Azov and Black seas [8]).) during high stands. These changes are 42 hypothesized to have had a major effect on the evolutionary history of its aquatic biota, such as the highly endemic 43 gastropod fauna [12]. Such fluctuations make the Caspian Sea a uniquesuitable model system to study the effect of 44 palaeo-environmental change on phylogeographic structure in long-lived lake biota.

46
The recurrent fluctuations in salinity and lake level during the Quaternary may have created periodic temporal refugia. Isolated Caspian sub-basins during low stands or spring sources and riverine systems during periods of 48 increased salinity are potential candidate refugia. The restricted gene flow across such refugial populations are 49 expected to generate strong phylogeographic structure within the species as a consequence of isolated evolutionary 50 histories. Compared to the large extent of the Caspian drainage basin, surprisingly only a lowsmall number of in the Caspian drainage basin [14,[16][17][18]21]. Although this may be a genuine evolutionary pattern (e.g. due to a lack 55 of genetic breaks), there may be a bias. Often, taxa were selected whichthat are easily dispersed, or the sampling design was rather narrow across the Caspian basin itself.

58
Gastropods can make good candidates for documenting responses to environmental changes as they often have understanding of the effects of major salinity and lake level changes on the evolutionary history of Pontocaspian gastropods, but also improveenhance our understanding of the phylogeography and the existence of refugia in the although they depend on well oxygenated water and environments with hard substrate, where they can graze on algae 66 (i.e. rocks and stones, shell beds or aquatic plants) [27]. Due to the abundance of these environments in the

71
[29] and molecular analyses [6] have led to speculations that these four may represent a single species [12]. Here, we

81
We hypothesise that repeated fluctuations in lake level andCaspian Sea low stands with increased salinity (perhaps

Sample collection and laboratory protocols 93
Theodoxus specimens were collected and stored following Sands et al. [6]. We included the genetic data of either five 94 or ten specimens per location to allow for robust analyses (table 1;

165
To construct the EBS plots, the output EBSP log files were imported into the BEAST package tool EBSPAnalyzer

178
However, there is both polyphyly and some paraphyly between the morphospecies T. pallidus (Dunker, 1861) and T.
individuals from various locations and similarly suggest paraphyly and polyphyly among morphospecies commonly figure 3).

Phylogeographic structure
diversity is far lower in the Caspian group as compared to southern Iran, indicative of a younger flock (table 2).
distantly connected Pontocaspian localities (Appendix A, table S1). Similar AMOVA results were also found for Appendix A, figures S1 and S2B, supporting information). 217 tables S1 and S2; figures S1 and S2).). Therefore, our hypothesis that major low stands drove strong phylogeographic

234
The weak phylogeographic structure observed in the Pontocaspian Theodoxus group is also found among other

263
Furthermore, tectonic activity in the Caucasus and Alborz mountains may have affected the lifespan and suitability of 264 springs as refugia [68][69][70][71]. Comparable examples of dramatic and sustained low stands have been shown for some 265 ancient lakes to reduce diversity in groups to a single lineage [72]. A well-documented similar case may be Lake

266
Malawi where two recent gastropod species flocks have arisen from a single surviving lineage [73,74]. species studied by Sands et al. [6]. As such, it is highly likely that these Theodoxus groups share a sister relationship.

272
On the intraspecific level however, strong paraphyly can be detected within each group when comparing the 273 phylogroups and morphospecies included (table 1; figure 3).

275
The southern Iranian phylogroups II and III contain specimens attributed to T. pallidus, while phylogroups I, III and

276
IV contain specimens attributed to T. doriae. This not only shows polyphyly between species on a phylogroup level

277
(phylogroups I, II and IV; table 1; figure 3), but additionally that there is paraphyly within phylogroups (phylogroup 278 III; table 1; figure 3). Both species were originally described from Iran and already considered synonymous by

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Starmühlner and Edlauer [75]. Glöer and Pešić [76] recently reviewed the material of T. pallidus studied by already been synonymised based on morphological similarities [12,29], which was already supported through 291 previous phylogenetic analyses [6], the inclusion of T. schultzii and T. major has only been speculated [12]. Our study 292 reaffirms these earlier suggestions and warrants the synonymy of all four morphospecies based on molecular data,

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with the name Theodoxus major Issel, 1865 having priority.

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While early humans probably had little environmental footprints as compared to today, an ever growing pressure for 301 water resources in conjunction with these climatic changes [80][81][82] may have posed increasing threats for T. pallidus.

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As spring systems often contain unique and endemic haplotypes, dedicated conservation efforts of a variety of spring 303 systems are critical to protect this species (and potentially other aquatic species in southern Iran).

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On the other hand, our analysis suggests that T. major has expanded over the last 105 kayr (figure 5a5). Considering 306 the entire range of the 95% confidence interval, however, both a population decline and increase is feasible. Already 307 for a number of Pontocaspian species population declines are evident [12]. Several species of Pontocaspian molluscs 308 are even thought to have gone extinct during the past century [12]. The causes for these declines are not well 309 established given the general lack of data for most species [12], but may be linked to anthropogenic activity [83].     , with the 95% credibility interval given in parentheses and as grey bars for in--group taxa. Small red squares at nodes (with darkened node bars and, in some instances, dates) indicate significant posterior probabilities of divergence events. Parallel to each supported phylogroup, coloured bars indicate the localities and respective morphospecies of the included specimens as defined in the key on the left (also see figure 2).. Caspian Sea lake level variations over the last 1.5 Ma (relative to absolute sea level) and regional stratigraphy (following the "short--Akchagylian" option) are adapted from Krijgsman et al. [8] (Khv. = Khvalynian).  In the BS plots, the The central line of each plot represents the median value, while in the LTT plots the line rather constitutes the mean. In both plots and the shaded area indicates the 95% confidence interval and floating grey bars indicate the 95% confidence interval associated with the onset of intraspecific diversification. The LTT and BS plots indicaterespectively. Note the EBS plots depict marginally younger intraspecific diversitydifferent starting dates for each group when compared withto the phylogeny. Importantly however, there is strong overlap inof EBS plot starting dates with the 95% confidence intervalsHPDs established among the analysesfor the onset of intraspecific diversification in each group as shown in the phylogeny (figure 3).

Reference Number: RSOS-190965
We very much appreciate the time and effort of the reviewer and the editors in assessing our revised paper and we are grateful for their additional comments. Below we list all critical comments and suggestions of the associate editor and reviewer (in italics), together with our point-by-point replies (in blue).

ASSOCIATE EDITOR (Dr Kristina Sefc):
I'd like to thank you for addressing most of the concerns raised in the first round of review. The issue of pooling divergent lineages for the EBSPs still remains and actually represents a rather serious one since an important conclusion -regarding the decline in population size -may be based on an artefact in the analysis. Please see the reviewer's comments for details. If the sampling / the data don't allow to analyse the demographic history of the Iranian populations, then you'll have to consider dropping this part from the manuscript (or interpret the network structures of the mtDNA and ncDNA sequences verbally -less diversity in the mt than nc genomes within each phylogroup may indeed point to a recent bottleneck); or at least discuss the problem associated with pooling the divergent lineages and make clear that there's a risk of a spurious result.
We note the feedback of the reviewer and the associate editor regarding the EBSPs. We understand there may still be a potential bias from pooling the Iranian data without being able to assess the EBSP trends in individual Iranian phylogroups due to the ESS values lacking support (potentially as a result of small sample sizes of the phylogroups). We have thus followed the associate editor's suggestions: 1) We have added further interpretation of our networks and haplotypic diversity to add support for a bottleneck within the Iranian species as observed in the EBSP (see L171-174, L190-191 & L284-286).
2) We now note that "caution" should be taken with the Iranian group's EBSP when interpreting the trend of the pooled data (figure 5) given the bias that may be caused (L190-192, L282-284 & L289-290). We reference the papers noted by the reviewer in this regard (L135 & L283-284). However, we also note that Heller et al. (2013) indicates that at least in part some of these concerns may be mitigated by a balanced sampling strategy such as our own (L284). Moreover we note that even neglecting the EBSP (from a conservation perspective), given current climate shifts and anthropogenic threats to spring habitats in the region, conservation efforts may still be needed to protect this species (L290-293).