Phyllopteryx taeniolatus, is an iconic and endemic fish found across temperate reefs of southern Australia. Despite its charismatic nature, few studies have been published, and the extent of population sub-structuring remains poorly resolved. Here we used 7462 single nucleotide polymorphisms (SNPs) to identify the extent of population structure in the weedy seadragon along the temperate southeast coast of Australia. We identified four populations, with strong genetic structure (FST = 0.562) between them. Both Discriminant Analysis of Principle Components (DAPC) and Bayesian clustering analyses support four distinct genetic clusters . In addition to these genetic differences, geographical variation in external morphology was recorded, with individuals from New South Wales shaped differently for a few measurements to those from the Mornington Peninsula (Victoria). We posit that these genetic and morphological differences suggest that the Victorian population of P. taeniolatus was historically isolated by the Bassian Isthmus during the last glacial maximum and should now be considered at least a distinct population. We also recorded high levels of genetic structure among the other locations. Based on the genomic and to a degree morphological evidence presented in this study, we recommend that the Victorian population be managed separately from the eastern populations .The common or weedy seadragon,  Marine fishes often inhabit wide geographic ranges, have large populations with high genetic diversity, and display low levels of genetic structuring due to high larval dispersal capacity –5. ConsePhycodurus eques, the weedy seadragon Phyllopteryx taeniolatus , and the recently discovered ruby seadragon Phyllopteryx dewysea Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Klanten et al. “Genomic and morphological evidence of distinct subpopulations in the endemic weedy seadragon Phyllopteryx taeniolatus (Syngnathidae) along the east coast of Australia”.SummaryKlanten et al., present a population genetics study on the weedy seadragon (Phyllopteryx taeniolatus) using SNP sequence data generated from ddRADseq. This work builds on previous studies that used mitochondrial and microsatellite sequence data. In this study the authors have contributed further evidence for significant population structuring at a regional scale that has implications for the management of this species. They have also incorporated morphological measurements as further evidence of population isolation and interpret these results along with the genetic data with reference to the historical Bassian Isthmus as a barrier to dispersal.The SNP data generated in this study have been assembled and filtered appropriately to produce a high-quality dataset for which to analyse genetic diversity and patterns among the populations. The authors have used multiple different analyses to test for genetic population structure, and these have been carried out appropriately for the dataset. The phylogenetic analyses are not suitable for this data set however, and should be removed (comments below). There is also widespread unjustifiable use of the word hybridisation, which needs to be removed. Otherwise, this is a robust contribution to understanding these iconic fish.1. Line 59: “…” isn’t correct as common names are not controlled by the ICZN. Suggest change to: .2. Line 62: Ruby seadragons are thought to have prehensile tails .3. Lines 77-88: You state that mtDNA showed strong population structure for weedy seadragons, and lowered genetic diversity on the east coast. Yet at the end of this paragraph you state that leafy seadragon genetic diversity was moderate to low, a similar pattern to the weedy. This last statement is in contrast with [28] which actually reports some areas with quite high diversity, but showed low diversity for the east coast. You either need to elaborate further on this complexity, or just shorten the final sentence of the paragraph so it does not include a species comparison.4. Line 149: Were pairwise Fst values also calculated in Arlequin or using R? This is not explicitly stated in the Methods section but is stated in the caption to Table 1.5. Line 155: EAC has not been mentioned in text before here, though it is explained in the Discussion (line 312), I would write it out in full here as well.6. Have you corrected for different sample sizes among populations when calculating expected Heterozygosity?7. It is not clear exactly how the geographic distances were obtained, using the Ruler tool, by hand in Google Earth, or calculating the edge length of a shapefile? Would this be easy to replicate if it has been measured by hand?8. Can you display the Evanno delta-K plot from STRUCTURE, and the BIC plot from DAPC that you used to determine the number of appropriate population clusters (K=4). These plots could be added to the Supplementary Material.9. Line 238: “Our unrooted phylogeny based on Maximum likelihood inferences supports the structure analyses (K = 4) distinguishing populations between CNSW, SNSW, VIC and TAS .” Actually, your unrooted tree shows three clusters and a grade. You may wish to use midpoint rooting to assist with interpretation here. It’s hard to see what is going on with this figure. Having said that, I do not think that its valid to apply this evolutionary model to a SNP data set. RAxML is definitely not designed with SNP data in mind, and you would be better off using software that deals with the assumptions created by this type of data set. If you can’t do this, I would suggest removing this part altogether. It doesn’t really add anything unique to your conclusions.10. Line 238: You state that the “Eden population in southern NSW is a monophyletic clade within the broader NSW group, instead of its own lineage .” This does not make grammatical sense with regard to monophyly, and needs clarification.11. Line 245: “These results would indicate possible hybridisation between CNSW, SNSW and TAS but not VIC .” Firstly, this seems more like Discussion than results, and needs to move to that section. Hybridisation is not the only source of genetic similarity, so do check your assumptions here, and expand on all possibilities unless you can test amongst them. Having said that, hybridisation is not the right term to apply to interbreeding among populations.12. Line 271: “Furthermore, the smaller-sized seadragons in VIC also appear to have a darker colour pattern compared to NSW and TAS seadragons .” I don’t think you should refer to colour based on the single images displayed, and those that lack a colour standard. There are so many uncontrolled variables here.13. Lines 278-280: Think about removing phylogenetic inference, as suggested above.14. Lines 286-288: You may wish to also cite [28] here, as it also showed that the Portsea/Flinders animals were distinct from NSW and TAS.15. Line 288-290: “The Bassian Isthmus [29] which has acted as a barrier to marine dispersal for 20-25 kya during the last glacial maximum may have prevented mixing between these populations, during which Victorian weedy seadragons diverged in isolation.” I would acknowledge that this has already been suggested, which you state in your introduction, but do not link to here.16. Line 292: “been ample opportunity for hybridisation”. Do you mean ample opportunity for secondary contact?You don’t usually here about populations hybridising, rather different species hybridising.17. Line 297-298: “we suggest the Victorian P. taeniolatus to be a subpopulation, alternatively weedy seadragons along southeast coast of Australia are forming a species complex .” It has already been demonstrated that, mitochondrially, the VIC animals are not the closest relatives of the east coast populations, so your interpretations here are somewhat skewed by the fact your sampling gaps increase the differences you see. You really have very little evidence for suggesting a species complex, and I suggest you remove this speculation from here and line 316-320. You may say it’s possible, but you have no evidence for this at present. Also, it would be prudent to refer to the study which has sampled them across the distributional range and did not find evidence for this!18. Line 303: you should remove discussion about colour differences as it was not tested here.19. Line 309: issues with ‘hybridisation’ as discussed above. You just mean interbreeding among populations, which is the foundation of the biological species concept.20. Lines 321 – 333: In the first sentence of this paragraph you state the difficulty in comparing the genetic diversity of the SNP dataset to other studies on syngnathids which have used microsatellite sequence data. As the number of alleles does have an effect on expected Heterozygosity values I would urge caution in directly comparing He values between different species and studies where the number of alleles may differ, particularly when the total number of alleles for this study is likely to be much higher given that thousands of SNP loci were used.21. Line 358: Again, remove terminology ‘hybrids’22. Line 367: make ‘scientist’ plural23. References- many do not italicize species names. this needs to be fixed.Reviewer #2: Comments to authorsThis manuscript reports RAD sequencing of individuals of common seadragons (Phyllopteryx taeniolatus) in the eastern part of their range from New South Wales, Tasmania and Victoria. The authors use the data to investigate population structuring and genetic diversity. Morphological measurements are taken to investigate morphological differentiation. The study contributes important data for this charismatic species and uses the usual population genetic tools to find substantial population structure and some evidence for morphological differences. I overall find the paper interesting but suggest to tone down some of the conclusions to better fit the data.Main pointsSpecies complexThe authors suggest in the discussion that Victorian and New South Wales populations may be reproductively isolated to form a species complex. This conclusion is in my opinion not supported by the data. As I outline below, the morphological differences need further investigation. The genetic differentiation between those populations is large as per FST but that per se does not imply complete reproductive isolation. The Structure analyses at K=2 indicate some potential shared ancestry components. For such a far reaching claim, one would need additional analyses explicitly showing the absence of gene flow and, as the authors acknowledge, ideally more than one sampling site in Victoria and from the remaining distribution.The conclusion that Victorian populations should be separately managed should therefore be carefully considered. I also wonder what the recommendation would be about Tasmanian and New South Wales populations. According to the given scenario. they were possibly historically connected when the Bassian Strait was closed but now they are separated. What would be the recommendation for their management given that their differentiation towards New South Wales is smaller?Morphological analysisI find the conclusion that the New South Wales and Victorian populations are morphologically distinct premature. The main difference is a difference in body size. A difference in body size could reflect temperature differences or differences in demographics or age structure of the population or even just sampling younger individuals in Victoria. These possibilities should be at least carefully discussed.We can expect that all other metrics scale allometrically with body size, hence it is not surprising to find further differences between the two populations. The analyses should be performed with measurements that were scaled by the body size.It is further not clear if the study separated males and females, which do show sexual dimorphism at least in the body depth. It would be great to test here if there are other differences between males and females.The discussion compares Tasmanian to New South Wales individuals to support evolutionary scenarios but this data was not included in the main analysis. The Tasmanian individuals either need to be included in the main analysis or this discussion needs to be explicit that it is speculative.Lastly, comparing only New South Wales and Victoria may miss variation that is being seen in the genetics. I would be interested to see a PCA of the morphological measurements of all individuals including Tasmania. It would be interesting to see if morphological structure follows genetic structure.Data availabilityInformation on sequencing statistics and genotyping metrics for each sample need to be given.Please give the vcf or genepop files and fasta files of loci should be deposited in Data Dryad or uploaded as Supplementary Material if file size allows.Minor commentshttps://www.fishnames.com.au/.L.1 Title: I personally also like “weedy” seadragon too but common seadragon should be used to support the attempt to standardize names L.4 Rephrase to “Population genomics of the weedy seadragon” would be more appropriate given the titleL.23 The IUCN citation (?) seems misplaced here.L.23 Unclear, range wide genetic population data is available in Wilson, Stiller & Rouse 2017.L.28 replace  with L.29 It is unclear how IBD can support the existence of 4 distinct genetic clusters, because IBD measures the degree of increasing genetic differentiation with geographic distance and does not delineate clustersL.54 Spell out scientific name Syngnathidae first and then refer with the common name in the second sentenceL.65 missing )L.74 Please cite the version of the IUCN assessment because the status of common seadragons has changed throughout different assessments. The IUCN status (now reversed to Least Concern) should also be given.L.84 There is controversy in the exact age of the Last Glacial Maximum but 20-25 KY seems to old. Lewis, S. E., Sloss, C. R., Murray-Wallace, C. V., Woodroffe, C. D., & Smithers, S. G. (2013). Post-glacial sea-level changes around the Australian margin: a review. Quaternary Science Reviews, 74, 115–138.L.100 Reference to Supp Table 2 with collection neededL.107 Please specify how many individuals were measured from each locality.L.123 What barcodes were used? Were they single or dual barcodes?L.140 Please provide some detail of how STACKS calls SNPs as this is an important factor determining the number and quality of the raw SNPs. How was the single SNP chosen, randomly or according to best depth of coverage?L.142 What was the requirement for depth of coverage?L.144 Abbreviations should be defined as observed heterozygosity (Ho) etc.L.152 How many replicates to assess significance?L.159 The sentence seems incomplete.L.165 Please also include a simple PCA that does not require setting a value of K. DAPC aims to maximize the distance between K groups and can often appear like heavier structuring than in the data. A simple PCA allows to look at clustering with very little assumptions.L.165 I may be wrong here but DAPC is PCA based which means that it cannot handle missing data. How were missing data points replaced? What K was chosen for DAPC?L.174 What dataset was this done on? SNPs only or RAD loci, were invariant RAD loci included?L.176 How was the substitution model chosen? I assume the analysis was done on the concatenation of all RAD loci without partitioning?L.185 What was the sample size for VIC and NSW? Common seadragons are sexually dimorphic with deeper bodies in the females - was this accounted for in the analyses?L.196 Please provide a table of sequencing and SNP statistics for each individual. The main text also needs statistics on average missing proportionsL.197 Unclear which groups this FST value is comparing.L.206 Please add the population description as in Table 3 to Table 2.L.223 Is allelic richness useful for binary SNP markers?L.233 I think it would be more logical to place the Mantel test directly after the FST.L.233 and following Please use past tense to describe results.L.237 DAPC typoL.241 The interpretation of a monophyletic Eden population and its relationship to the NSW populations depends on the rooting. This needs to be reformulated. It is also not clear what “instead of its own lineage” means. Eden is its own lineage.L.245 Given the concerns regarding the proposed species complex, “gene flow” would be the better term to use over “hybridisation”.L.260 Figure legend should mention that only branches with 100 bootstrap support are annotated.L.265 We would expect allometric scaling of most of these measurements. Where the measurements scaled by the total TL?L.291 The isthmus opened later, detailed reconstructions and dates are in Lambeck, K., & Chappell, J. (2001). Sea level change through the last glacial cycle. Science, 292(5517), 679–686.L.292 Gene flow is more appropriate in this context.L.293 The FST values are large but they do not indicate complete isolation.L.303 Please give the source of this information.L.313 distant?L.313 is almost identical to L296 and following without providing new evidenceL.329 write out microsatellitesL.328 This sentence is hard to follow.Figure 1 Sydney is all CAPS, other localities not. Would be helpful to add sample size to the map.Suppl Table 3 Some measurements have more then 10 decimal places which cannot possibly be the accuracy of the ImageJ measurements**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  4 Sep 2020Response to Reviewers:Manuscript: PONE-D-20-17523Genomic and morphological evidence of distinct subpopulations in the endemic common (weedy) seadragon Phyllopteryx taeniolatus (Syngnathidae) along the east coast of AustraliaReviewer #1:SummaryKlanten et al., present a population genetics study on the weedy seadragon (Phyllopteryx taeniolatus) using SNP sequence data generated from ddRADseq. This work builds on previous studies that used mitochondrial and microsatellite sequence data. In this study the authors have contributed further evidence for significant population structuring at a regional scale that has implications for the management of this species. They have also incorporated morphological measurements as further evidence of population isolation and interpret these results along with the genetic data with reference to the historical Bassian Isthmus as a barrier to dispersal. The SNP data generated in this study have been assembled and filtered appropriately to produce a high-quality dataset for which to analyse genetic diversity and patterns among the populations. The authors have used multiple different analyses to test for genetic population structure, and these have been carried out appropriately for the dataset. The phylogenetic analyses are not suitable for this data set however, and should be removed (comments below). There is also widespread unjustifiable use of the word hybridisation, which needs to be removed. Otherwise, this is a robust contribution to understanding these iconic fish.Response: We thank Reviewer 1 for their constructive comments. We accept the reviewers point about the phylogenetic tree  and have opted to remove this from our manuscript. It is unnecessary as our conclusions are well supported by the remaining analyses. We also edited the word hybridisation using terms such as ‘interbreeding’ Note: We added additional citations (suggested by reviewers) so the citation # in our manuscript have changed. Specific comments:1. Line 59: “…” isn’t correct as common names are not controlled by the ICZN. Suggest change to: .Response: Edited as suggested by reviewer. We also mention (Title and Abstract) both “common or weedy”.2. Line 62: Ruby seadragons are thought to have prehensile tails .Response: Thank you for pointing this out, we changed the sentence to make sure it’s only weedy seadragons that have no prehensile tails. “Phyllopteryx taeniolatus are weak swimmers and lack the prehensile tail of seahorses ...”We also added Rouse et al. 2007 [22] as a reference. 3. Lines 77-88: You state that mtDNA showed strong population structure for weedy seadragons, and lowered genetic diversity on the east coast. Yet at the end of this paragraph you state that leafy seadragon genetic diversity was moderate to low, a similar pattern to the weedy. This last statement is in contrast with [28] which actually reports some areas with quite high diversity, but showed low diversity for the east coast. You either need to elaborate further on this complexity, or just shorten the final sentence of the paragraph so it does not include a species comparison.Response: We edited the sentence so that it does not include a comparison. “Another study [34] on the leafy seadragon (Phycodurus eques), using both mtDNA and microsatellites, also found strong population structure especially between western and southern Australian populations, with high to low genetic diversity.”4. Line 149: Were pairwise Fst values also calculated in Arlequin or using R? This is not explicitly stated in the Methods section but is stated in the caption to Table 1.Response: We added “… in Arlequin ..” to clarify sentence.5. Line 155: EAC has not been mentioned in text before here, though it is explained in the Discussion (line 312), I would write it out in full here as well.Response: Eastern Australian Current (EAC) now written in full.6. Have you corrected for different sample sizes among populations when calculating expected Heterozygosity?Response: We used Nei’s calculation of heterozygosity using the R package pegas. Nei’s equation is as follows: H ^=n/(n-1)(1-∑_(i=1)^k▒p_i^2 )This calculation does not correct for different sample sizes. Without this correction values calculated from small samples sizes are likely to be biased upward. More to the point we don’t emphasise heterozygosity comparisons between populations and only consider the global  values as they relate to other species. 7. It is not clear exactly how the geographic distances were obtained, using the Ruler tool, by hand in Google Earth, or calculating the edge length of a shapefile? Would this be easy to replicate if it has been measured by hand?Response: we measured by hand using Ruler tool ‘Path’ in Google Earth. “… using Google Earth following the Eastern Australian Current (EAC) along the east coast to Tasmania using the Ruler tool Path by hand.”8. Can you display the Evanno delta-K plot from STRUCTURE, and the BIC plot from DAPC that you used to determine the number of appropriate population clusters (K=4). These plots could be added to the Supplementary Material.Response: Plots have now been added to Supporting information .9. Line 238: “Our unrooted phylogeny based on Maximum likelihood inferences supports the structure analyses (K= 4) distinguishing populations between CNSW, SNSW, VIC and TAS .” Actually, your unrooted tree shows three clusters and a grade. You may wish to use midpoint rooting to assist with interpretation here. It’s hard to see what is going on with this figure. Having said that, I do not think that its valid to apply this evolutionary model to a SNP data set. RAxML is definitely not designed with SNP data in mind, and you would be better off using software that deals with the assumptions created by this type of data set. If you can’t do this, I would suggest removing this part altogether. It doesn’t really add anything unique to your conclusions.Response: We removed any text referring to phylogenetic tree and Figure 5 as suggested above from our manuscript.10. Line 238: You state that the “Eden population in southern NSW is a monophyletic clade within the broader NSW group, instead of its own lineage .” This does not make grammatical sense with regard to monophyly, and needs clarification.Response: This sentence has been removed, as we removed all text relating to phylogenetic tree and Figure 5.11. Line 245: “These results would indicate possible hybridisation between CNSW, SNSW and TAS but not VIC .” Firstly, this seems more like Discussion than results, and needs to move to that section. Hybridisation is not the only source of genetic similarity, so do check your assumptions here, and expand on all possibilities unless you can test amongst them. Having said that, hybridisation is not the right term to apply to interbreeding among populations.Response: Removed this sentence from results section.12. Line 271: “Furthermore, the smaller-sized seadragons in VIC also appear to have a darker colour pattern compared to NSW and TAS seadragons .” I don’t think you should refer to colour based on the single images displayed, and those that lack a colour standard. There are so many uncontrolled variables here.Response: We removed this sentence. Furthermore, the whole section on morphology has changed as per Reviewer 2.13. Lines 278-280: Think about removing phylogenetic inference, as suggested above.Response: Removed from manuscript.14. Lines 286-288: You may wish to also cite [28] here, as it also showed that the Portsea/Flinders animals were distinct from NSW and TAS.Response: We added citation Wilson et al. 2017 [30]. “Of note is that all individuals from two sites in Victoria (Flinders Bay and Portsea) are highly distinct, genetically and to a degree morphologically, to the main populations of central and southern NSW, as well as Tasmania [see also 30].”15. Line 288-290: “The Bassian Isthmus [29] which has acted as a barrier to marine dispersal for 20-25 kya during the last glacial maximum may have prevented mixing between these populations, during which Victorian weedy seadragons diverged in isolation.” I would acknowledge that this has already been suggested, which you state in your introduction, but do not link to here.Response: We added Wilson et al 2017 [30] in citation to this sentence.“The Bassian Isthmus [30–32] which has acted as a barrier to marine dispersal …”16. Line 292: “been ample opportunity for hybridisation”. Do you mean ample opportunity for secondary contact? You don’t usually here about populations hybridising, rather different species hybridising.Response: changed and used gene flow“Since the opening of the Bassian Isthmus, approximately 14-25 kya, there has been ample opportunity for gene flow, therefore the question arises …”17. Line 297-298: “we suggest the Victorian P. taeniolatus to be a subpopulation, alternatively weedy seadragons along southeast coast of Australia are forming a species complex .” It has already been demonstrated that, mitochondrially, the VIC animals are not the closest relatives of the east coast populations, so your interpretations here are somewhat skewed by the fact your sampling gaps increase the differences you see. You really have very little evidence for suggesting a species complex, and I suggest you remove this speculation from here and line 316-320. You may say it’s possible, but you have no evidence for this at present. Also, it would be prudent to refer to the study which has sampled them across the distributional range and did not find evidence for this!Response: We have changed this section and the next and hypothesis that by extending the study in future to include individuals from the entire distribution range and using genome-wide SNPs, this study has increased the power of analysis both by including a large number of markers, and by having markers in more biologically relevant regions of the genome. We would like to point out that mitochondrial data frequently has a different gene tree from nuclear data and is a tiny subset of the genes available for divergence. In addition, to having many more loci (both mt & nDNA), nuclear markers provide a better picture of divergence because they both code for more proteins, and are not only matrilinear, but are less affected by introgression and diverge faster.We agree that this study alone does not provide sufficient evidence that they are different species, but our populations (and particularly Victoria) are quite divergent with FST values well into what separates species in many other organisms. This in fact is important for future management and conservation.Please see page 14 in MS where we elaborate on this issue.18. Line 303: you should remove discussion about colour differences as it was not tested here.Response: We removed this section.19. Line 309: issues with ‘hybridisation’ as discussed above. You just mean interbreeding among populations, which is the foundation of the biological species concept.Response: Changed to ‘.. interbreeding .. ‘ 20. Lines 321 – 333: In the first sentence of this paragraph you state the difficulty in comparing the genetic diversity of the SNP dataset to other studies on syngnathids which have used microsatellite sequence data. As the number of alleles does have an effect on expected Heterozygosity values I would urge caution in directly comparing He values between different species and studies where the number of alleles may differ, particularlywhen the total number of alleles for this study is likely to be much higher given that thousands of SNP loci were used.Response: We agree and have now added this statement to the end of that paragraph“However, these data should be interpreted with caution. Theoretically, the maximum heterozygosity derived from biallelic loci (such as the SNPs employed here) is 0.5. On the other hand, heterozygosity at microsatellite loci can theoretically reach 1.0.”21. Line 358: Again, remove terminology ‘hybrids’Response: Removed and replaced with “interbreeding”22. Line 367: make ‘scientist’ pluralResponse: Corrected23. References- many do not italicize species names. this needs to be fixed.Response: Fixed all references, thank you for pointing out.Reviewer #2Main pointsSpecies complex:The authors suggest in the discussion that Victorian and New South Wales populations may be reproductively isolated to form a species complex. This conclusion is in my opinion not supported by the data. As I outline below, the morphological differences need further investigation. The genetic differentiation between those populations is large as per FST but that per se does not imply complete reproductive isolation. The Structure analyses at K=2 indicate some potential shared ancestry components. For such a far reaching claim, one would need additional analyses explicitly showing the absence of gene flow and, as the authors acknowledge, ideally more than one sampling site in Victoria and from the remaining distribution. The conclusion that Victorian populations should be separately managed should therefore be carefully considered. I also wonder what the recommendation would be about Tasmanian and New South Wales populations. According to the given scenario. they were possibly historically connected when the Bassian Strait was closed but now they are separated. What would be the recommendation for their management given that their differentiation towards New South Wales is smaller?Response: We thank Reviewer 2 for their constructive comments. As Reviewer 1 (point 17 above) also pointed to this issue, we clarified that our data has limited evidence, for the moment, for a species complex, and we suggest that much more sampling (across its entire range) using genome-wide SNPs will be needed. We would like to leave this hypothesis in our manuscript as a possibility (assumption) that it may occur. It will open dialogue and direct to more studies into this species. We have elaborated on this extensively in our discussion see p.14 in MS .Regarding management we are suggesting that each state should manage their populations in particular VIC.Morphological analysis:I find the conclusion that the New South Wales and Victorian populations are morphologically distinct premature. The main difference is a difference in body size. A difference in body size could reflect temperature differences or differences in demographics or age structure of the population or even just sampling younger individuals in Victoria. These possibilities should be at least carefully discussed. We can expect that all other metrics scale allometrically with body size, hence it is not surprising to find further differences between the two populations. The analyses should be performed with measurements that were scaled by the body size.It is further not clear if the study separated males and females, which do show sexual dimorphism at least in the body depth. It would be great to test here if there are other differences between males and females.The discussion compares Tasmanian to New South Wales individuals to support evolutionary scenarios but this data was not included in the main analysis. The Tasmanian individuals either need to be included in the main analysis or this discussion needs to be explicit that it is speculative.Lastly, comparing only New South Wales and Victoria may miss variation that is being seen in the genetics. I would be interested to see a PCA of the morphological measurements of all individuals including Tasmania. It would be interesting to see if morphological structure follows genetic structure.Response: We have now analysed morphological differences via examination of external metrics vs total length  and via ordination LDA and PCA . We removed Table 3 and re-wrote the morphology sections in Methods and Results.The biplots suggest that several metrics differ between Victorian individuals and both NSW and Tasmania, supporting our statement that some morphological differences were apparent, by removing ontogenetic confounding and suggesting overall shape differences. We now note this and that all measured individuals were adults. Unfortunately, not all specimens were sexed so we comment on this too. We were able to determine sex for over half the specimens , so could not fully analyse sex differences in morphology, but argue against that different sex ratios at each location are driving differences in shape. We included Tasmanian samples (n=5 only) separately in biplots and both LDA and PCA plots (see above). Data availability:Information on sequencing statistics and genotyping metrics for each sample need to be given. Please give the vcf or genepop files and fasta files of loci should be deposited in Data Dryad or uploaded as Supplementary Material if file size allows.Response: We have added # of reads and % missing loci for each individual to S1 Table. Also, we have now uploaded a vcf and genepop file as S1 Data and S2 Data as supporting data files. We added following sentence:“Fastq sequence files were deposited in NCBI’s Sequence Read Archive (accession numbers: SRR12395874-12395945) and the associated metadata is available at GEOME [36] and is provided in S1 Table. Data generated as vcf file (S1 Data) and as genepop file (S2 Data) are available as supporting information.”Minor comments:https://www.fishnames.com.au/.L.1 Title: I personally also like “weedy” seadragon too but common seadragon should be used to support the attempt to standardize names Response: We agree that common seadragon is proper, however these animals in particular are known widely as ‘weedy’ and is used generally in the literature , as well as the IUCN Red List. We have added “common” to the title as well as at the start of the Abstract, and do say that the proper name is common, see also our response to Reviewer 1.L.4 Rephrase to “Population genomics of the weedy seadragon” would be more appropriate given the titleResponse: Changed as suggested.L.23 The IUCN citation (?) seems misplaced here.Response: Removed whole sentence.L.23 Unclear, range wide genetic population data is available in Wilson, Stiller & Rouse 2017.Response: Removed that sentence see above.L.28 replace  with Response: CorrectedL.29 It is unclear how IBD can support the existence of 4 distinct genetic clusters, because IBD measures the degree of increasing genetic differentiation with geographic distance and does not delineate clustersResponse: We removed sentence. Instead related our reults of genetic clusters to DAPC and Bayesian clustering analysis.L.54 Spell out scientific name Syngnathidae first and then refer with the common name in the second sentenceResponse: CorrectedL.65 missing )Response: CorrectedL.74 Please cite the version of the IUCN assessment because the status of common seadragons has changed throughout different assessments. The IUCN status (now reversed to Least Concern) should also be given.Response: Added sentence: “The weedy seadragon’s status on the IUCN Red List was changed in 2017 from “Near Threatened” (NT) to “Least Concern” (LC) but their population trend is still considered as “Decreasing”https://dx.doi.org/10.2305/IUCN.UK.2017-2.RLTS.T17177A67624517.en).” . Post-glacial sea-level changes around the Australian margin: a review. Quaternary Science Reviews, 74, 115–138.Response: Thank you for the REF which we added now. We would like to add that this particular paper concentrated on the post-glacial sea level fluctuations and does mention several times that the exact LGM times for Tasmania/Vic are controversial. According to Lambeck and Chappell 2001 as you also mentioned below (added this REF as well), the peak of LGM was around 25kya.We based our dates on the isthmus (land bridge) that existed when VIC and TAS were connected (thus separating marine organisms between east and west coast) see also Lambeck and Chappell 2001. The opening of land bridge to the east was about 14 kya suggested by Lambeck and Chapell’s 2001 Fig 4. L.100 Reference to Supp Table 2 with collection neededResponse: Added. “In total 82 P. taeniolatus were sampled for genetic analysis between astral summer 2014/15 – 2018 (S1 Table).”L.107 Please specify how many individuals were measured from each locality.Response: Added sentence and provide full data now in S2 Table (morphology).“In total we measured 45 individuals, 20 each from NSW and VIC and 5 from TAS (S2 Table).”L.123 What barcodes were used? Were they single or dual barcodes?Response: Sentence added. “The RADSeq data generated by AGRF used single barcodes that are incorporated into the names of the Fastq files for each individual.” L.140 Please provide some detail of how STACKS calls SNPs as this is an important factor determining the number and quality of the raw SNPs. How was the single SNP chosen, randomly or according to best depth of coverage?Response: We have added a reference to the manuscript Rochette et al. (2019) that explains in section 2.8 the Bayesian genotype caller employed by STACKs V2 to call SNPs. We used the ‘write_single_snp’ option which outputs the first SNP per locus and the default setting which for min coverage with is 3x per individual which resulted in a mean coverage of 21.1 x per individual per locus. See text below. The manuscript now says:“Demultiplexing of the sequence data and initial quality control was conducted at AGRF with STACKS v.1.41  using default settings. Only sequences with exact barcodes were retained and sequences were trimmed to 140 bp. All other data processing took place at the Genomics and Bioinformatics Cluster at the University of Central Florida using STACKS v.2.0b. Loci were assembled using the denovo pipeline using default settings except we allowed for four mismatches between stacks within individuals (-M) and four mismatches between sample loci when building the catalog (-n). See Rochette et al. [39] for a detailed explanation of the Bayesian genotype caller employed in STACKS.Stacks in which the numbers of reads were more than two standard deviations above the mean were assumed to be repetitive elements and removed from the data set. To eliminate poor quality samples, those individuals with fewer than 200,000 reads or less than 60% of loci were excluded from further analysis, resulting in a final sample size of 72 individuals. Using the ‘populations’ function in STACKS, we eliminated those loci with observed heterozygosity greater than 0.7 (--max-obs-het) and applied a minor allele frequency cut-off of 0.05 (--min-maf). We retained only those loci that amplified in ≥ 80% of individuals (-r). Output files were created by implementing the ‘write_single_snp’ option which outputs the first SNP per locus.”And the results read:“A total of 19,608 loci were resolved across 72 individuals of which 7462 were variable and passed filtering. Samples had an average coverage 21 reads per SNP with a data matrix that was 92.6% complete .”Please see comments below for more information by author of STACKs.L.142 What was the requirement for depth of coverage?Response: The default settings for STACKs is 3x. We used the default which results in an average coverage of 21x per individual per locus. This decision was made after a conversation with the authors of STACKs Julian Catchen.From the Author of Stacks: “There is no minimum depth to call SNPs. This has always been handled by the SNP model, which calculates the likelihood of each position being a heterozygote and a homozygote . The model then weighs the evidence for a heterozygous versus a homozygous position using a likelihood ratio test resulting in a p-value. If you want to require more evidence, that is more reads, to support a particular SNP call, you can increase the required p-value to consider the likelihood ratio test statistically significant . The default p-value is (of course) 0.05, but you can lower this, which will require more evidence for a call to be declared statistically significant. Using -m at the population-program stage to randomly drop alleles that have already proven to be statistically significant is a bad idea and will result in random allele drop out. I regret ever making this option available and removed it when given the chance with the 2.0 release.”https://groups.google.com/forum/#!topic/stacks-users/IzeSy3nw5Bg L.144 Abbreviations should be defined as observed heterozygosity (Ho) etc.Response: AddedL.152 How many replicates to assess significance?Response: We are unsure where this ‘replicates to assess significance’ is? Line 151-153 reads: “…. comparisons according to Benjamini and Yekutieli [37] by controlling false discovery rates using the p.adjust function in R. In addition, we tested for Isolation by Distance [38] using a Mantel test and 100,000 permutations, as implemented in the R package ade4 v.1.7-15 [39].”As far as we can tell there are no missing # of replicates in this section. L.159 The sentence seems incomplete.Response: Edited: “To explore the possibility of population structuring, we used two methods, Bayesian clustering algorithms of STRUCTURE and Discriminant Analysis of Principle Components (DAPC).”L.165 Please also include a simple PCA that does not require setting a value of K. DAPC aims to maximize the distance between K groups and can often appear like heavier structuring than in the data. A simple PCA allows to look at clustering with very little assumptions.Response: We have now included PCAs for our major groups in supplemental materials . These do indeed show less clustering compared to the DAPC plots. However, multiple lines of evidence support the DAPC clusters including F-statistics and STRUCTURE analyses so the major conclusions of our study have not changed. L.165 I may be wrong here but DAPC is PCA based which means that it cannot handle missing data. How were missing data points replaced? What K was chosen for DAPC?Response: Agreed. We’ve added the following text. “Because PCA analyses require no missing data (and DAPC uses PCA) we replaced missing data with the mean allele frequency for each locus using the method scaleGen in ADEGENET.”A k of 4 was chosen for DAPC, see the S1 Fig for the BIC graph used to determine this. L.174 What dataset was this done on? SNPs only or RAD loci, were invariant RAD loci included?Response: The phylogenetic tree has been removed from the paper as per Reviewer 1.L.176 How was the substitution model chosen? I assume the analysis was done on the concatenation of all RAD loci without partitioning?Response: The phylogenetic tree has been removed from the paper (see above).L.185 What was the sample size for VIC and NSW? Common seadragons are sexually dimorphic with deeper bodies in the females - was this accounted for in the analyses?Response: See also Reviewer 1 above, we changed the morphology section. We added sex where it was possible.L.196 Please provide a table of sequencing and SNP statistics for each individual. The main text also needs statistics on average missing proportionsResponse: These data have been added to S1 Table and a line in the text has been added, only 7.4% missing. “Samples had an average coverage of 21 reads per SNP with a data matrix that was 92.6% complete .”L.197 Unclear which groups this FST value is comparing. Response: Added to new S3 Table A (AMOVA results).L.206 Please add the population description as in Table 3 to Table 2.Response: Table 3 (morphology students t-test) has been removed and instead we have plotted all measurements against total length to eliminate allopatric patterns. L.223 Is allelic richness useful for binary SNP markers?Response: We used this so other studies can compare. Some studies use Ho and/or He but a few terrestrial studies use allelic richness. During the course of writing the MS and comparing to other studies using SNPs  most authors used allelic richness more often than He or Ho. We attempted to show allelic richness of seadragons for future comparisons to other organisms. L.233 I think it would be more logical to place the Mantel test directly after the FST.Response: Edited result section moved Mantel test after pairwise Fst.L.233 and following Please use past tense to describe results.Response: correctedL.237 DAPC typoResponse: correctedL.241 The interpretation of a monophyletic Eden population and its relationship to the NSW populations depends on the rooting. This needs to be reformulated. It is also not clear what “instead of its own lineage” means. Eden is its own lineage.Response: Removed this sentence as we removed the phylogenetic tree as per Reviewer 1. L.245 Given the concerns regarding the proposed species complex, “gene flow” would be the better term to use over “hybridisation”.Response: Removed this sentence as per Reviewer 1 and in discussion we edited to ‘interbreeding’.L.260 Figure legend should mention that only branches with 100 bootstrap support are annotated.Response: Figure 5 was removed as per Reviewer 1L.265 We would expect allometric scaling of most of these measurements. Where the measurements scaled by the total TL?Response: Added S4 Fig plotting all 15 measurements against total length. In discussion:“While individuals from Victoria were somewhat smaller, biplots  indicate that shaped differences summarised in Fig 5A were not simply length-related. Most NSW individuals were not sexed but it is unlikely skewed sex ratios of samples by location can account for shape differences.”L.291 The isthmus opened later, detailed reconstructions and dates are in Lambeck, K., & Chappell, J. (2001). Sea level change through the last glacial cycle. Science, 292(5517), 679–686.Response: Added citation Lambeck & Chappel clearly state that the land bridge was closed effectively until ca. 14kya but started to break up from 14kya albeit fluctuating.L.292 Gene flow is more appropriate in this context.Response: CorrectedL.293 The FST values are large but they do not indicate complete isolation.Response: For SNP data there are indeed very large. See more of our explanation above for Reviewer 1. We have edited the sentence and also added Wilson et al 2017.L.303 Please give the source of this information.Response: we removed colour patterns as suggested by Reviewer 1.L.313 distant?Response: Corrected L.313 is almost identical to L296 and following without providing new evidenceResponse: Edited, this whole section has been re written see p.14 in our MS. L.329 write out microsatellitesResponse: CorrectedL.328 This sentence is hard to follow.Response: Edited: “This low genetic diversity in both the endemic weedy (this study) and leafy [34] seadragons stands in comparison with other members of the family Sygnathidae, which have moderate to high heterozygosity. Specifically, He ranged from 0.452 – 0.949 in five studies of widespread seahorses  and from 0.752 – 0.958 in four widespread pipefishes .”Figure 1 Sydney is all CAPS, other localities not. Would be helpful to add sample size to the map.Response: Corrected, and sample sizes for each population have been added to legend of Fig 1.Suppl Table 3 Some measurements have more then 10 decimal places which cannot possibly be the accuracy of the ImageJ measurementsResponse: Corrected this table is now S2 Table.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 22 Sep 2020PONE-D-20-17523R1Genomic and morphological evidence of distinct populations in the endemic common (weedy) seadragon Phyllopteryx taeniolatus (Syngnathidae) along the east coast of AustraliaPLOS ONEDear Dr. Klanten,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2: Klanten et al. have done a careful job at addressing the reviewers’ concerns and have added useful figures and additional material that improve the manuscript. My remaining concerns are regarding the still strong emphasis on a species complex, for which I do not see sufficient evidence. There are also questions regarding the PCA, which I think is a simple plotting error.1) Both reviewer 1 and myself have raised concerns about the suggestion of a species complex in Victoria and New South Wales. The authors have made modifications in calling for extended sampling and the improved resolution that nuclear markers can give, which are useful additions. However, the text still strongly implies that there is a species complex but that separate species are “not yet advocated”. The presented evidence based on allele frequency differences analyzed in a number of ways is not strong enough to draw that conclusion. This needs justification from the literature. As I have pointed out in my last review, this view is omitting the results from Structure at K=2 that does show shared ancestry components between Victoria and the eastern sites. If there was reproductive isolation, one would expect completely separate ancestry proportions. The Structure results need to be mentioned.2) Along the same vein, there are also multiple mentions of “very restricted to no gene flow” or similar. This lies on the assumption that FST values reflect migration rates. In practice, this relationship is not well established because there are also other factors which impact allele frequency differences ” Heredity. 1999;82:117–125). It would be more appropriate to refer to “significant allele frequency differences” or “significant genetic differentiation”, because this is what has actually been measured with FST.3) I thank the authors for including the PCA analysis. I think there may have been a mistake in the PCA plotting. I wondered how such clear clustering in DAPC can result in the PCA plot as shown in Figure S3, where several individuals were not clustering with their parent population. If that was true this would be concerning because PCA and DAPC are conceptually very similar. I repeated the analysis (on genepop file with four population groups separated) and found a clear assignment of individuals to populations (see code and plot below). The structure of the PCA looks similar to Figure S3 but it looks like the coloring of the individuals according to their populations is off in Figure S3. There must be a glitch in the R script that the authors used. Good news is that the PCA will support the DAPC results much better and the authors could consider to include it in the main text (up to the authors).Minor commentsL.67 Not sure if reference 19 is appropriate for the number of Syngnathidae species; Reference 18 Eschmeyer actually lists 321 valid speciesL.129ff. May be more readable as complete sentences?L.219 Typo in “Principal components”L.230 Typo in “patterns”L.231 Population code “SYD” has not been defined before.L.258 Unclear what the difference between FST = 0.561 and FST = 0.630 (L.262) is.L.289 Move “(FST ranging 0.636– 0.703)” to the end of the sentence to include TASL.313 Reference to Supplementary Figure S1 needed.L.370 Past tense? “were highly distinct”L.373 Move references to after “as a barrier to marine dispersal for 20-25 kya374 during the last glacial maximum (LGM)”L.376 The strait opened 14 ka ago according to Lambeck & Chappell, so gene flow may have been reinstated between 14 kya and today, not 14-25 kya.L.379 Type in “shape differences”L.380 It does not seem obvious why sex differences would not affect the observed differences.L.383 It is more appropriate to refer to allele frequency differences than gene flow (see above).L.386 Need a reference that the found differentiation is extraordinarily high.L.388 This indicates that some of the RAD loci obtained the mitochondrial genome. Is this likely? Intuitively, I would think that is unlikely to obtain mitochondrial RAD loci but maybe this is different in this case. If there were mitochondrial loci in the dataset, these would have to get filtered or modeled separately from nuclear loci because they are haploid and have a different effective population size.L.398 This paragraph seems isolated and the point is not clear.L.405 Unclear which populations “this division” refers to. The previous sentence states that TAS and NSW populations were historically connected, not divided. The following sentence also talks about similarities, rather than divisions.L.415 change to “”?L.435 Reference needed: “less resilient under normal circumstances, but instead have a smaller gene pool due to smaller population sizes and restricted distribution ranges”L.436 It is more appropriate to refer to allele frequency differences than gene flow (see above).L.440 Reference needed: “increase in anthropogenic stressors resulting in habitat loss (kelp and seagrass) and an increase of sea surface temperature”L.458 It is more appropriate to refer to allele frequency differences than gene flow (see above).**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentCommon_seadragon review.pdfSubmitted filename: Click here for additional data file. 5 Nov 2020Response to Reviewer Comments:Klanten et al. have done a careful job at addressing the reviewers’ concerns and have addeduseful figures and additional material that improve the manuscript. My remaining concerns areregarding the still strong emphasis on a species complex, for which I do not see sufficientevidence. There are also questions regarding the PCA, which I think is a simple plotting error.1) Both reviewer 1 and myself have raised concerns about the suggestion of a speciescomplex in Victoria and New South Wales. The authors have made modifications incalling for extended sampling and the improved resolution that nuclear markers can give,which are useful additions. However, the text still strongly implies that there is a speciescomplex but that separate species are “not yet advocated”. The presented evidencebased on allele frequency differences analyzed in a number of ways is not strongenough to draw that conclusion. This needs justification from the literature. As I havepointed out in my last review, this view is omitting the results from Structure at K=2 thatdoes show shared ancestry components between Victoria and the eastern sites. If therewas reproductive isolation, one would expect completely separate ancestry proportions.The Structure results need to be mentioned.Response: Structure Harvester analyses indicate the most likely K is four. This is supported by DAPC and in part by AMOVA. We added the following text “Each population harboured private alleles including CNSW (1721), SNSW (348), TAS (563), and VIC (3918). While none of these were fixed a substantial proportion reached an allele frequency of 0.9 meaning they were nearly fixed .” The pattern presented in the Structure plots when looking at all values of K can be explained by incomplete lineage sorting within recently diverged lineages. Further, we would not expect completely separate ancestry proportions, as it takes time for such differences to fix, and these were likely in full contact during the last interglacial period (about 120 kya), and intermittently until the glacial maximum about 40 kya. Finally, due to our results and west to east current of the Bass Strait, there may be occasional unidirectional introgression of Victorian genes to the eastern populations, but the reverse is highly unlikely, which is why we specifically noted this population even though the others also demonstrate high differentiation.We agree with the Reviewer that more evidence is still needed to confirm distinct species but note that the genetic data, backed up by morphological, are entirely consistent, with the hypothesis that these are diverging populations that are not randomly mating and possibly not mating at all. These, we believe are important considerations for conservation.  We feel that it would be remiss to ignore a reasonable scientific discussion of our results and their interpretation, which we strongly feel should include a hypothesis of a subspecies and how we might further test this. Apart from the evolutionary and ecological implications, conservation management of this iconic species will depend on species population structure across its range. Therefore, our Discussion includes such speculation and a way forward. However, note that we do not use these terms (subspecies) in our Title, Abstract, and Results.We would also like to draw the Reviewers attention to a few recent articles that support genome level studies to delineate species-level structure  examples are: https://doi.org/10.1371/journal.pone.0189417. 1. Baumsteiger et al. 2017, Genomics clarifies taxonomic boundaries in a difficult species complex. PLoS ONE 12(12): e0189417. 2. Roux et al. 2016, Shedding Light on the Grey Zone of Speciation along a Continuum of Genomic Divergence. PLoS Biol 14(12): e2000234. doi:10.1371/journal.pbio.2000234.3. Beheregaray et al. 2017, Genome-wide data delimits multiple climate-determined species rangesin a widespread Australian fish, the golden perch (Macquaria ambigua) Mol. Phylog. Evol 111, 65-75.2) Along the same vein, there are also multiple mentions of “very restricted to no gene flow”or similar. This lies on the assumption that FST values reflect migration rates. Inpractice, this relationship is not well established because there are also other factorswhich impact allele frequency differences ” Heredity .1999;82:117–125). It would be more appropriate to refer to “significant allele frequencydifferences” or “significant genetic differentiation”, because this is what has actually beenmeasured with FST.Response: McCauley and Whitlock’s argument is that one should not directly measure gene flow using Wright’s F-statistics, and we do not do so in this paper. We accept that such a calculation would be inaccurate, however, references to the direction and loose magnitude of link between gene flow and genetic differentiation are still valid. We have altered much of the wording to reduce confusion but have retained it where appropriate. 3) I thank the authors for including the PCA analysis. I think there may have been a mistakein the PCA plotting. I wondered how such clear clustering in DAPC can result in the PCAplot as shown in Figure S3, where several individuals were not clustering with theirparent population. If that was true this would be concerning because PCA and DAPC areconceptually very similar. I repeated the analysis (on genepop file with four populationgroups separated) and found a clear assignment of individuals to populations (see codeand plot below). The structure of the PCA looks similar to Figure S3 but it looks like thecoloring of the individuals according to their populations is off in Figure S3. There mustbe a glitch in the R script that the authors used. Good news is that the PCA will supportthe DAPC results much better and the authors could consider to include it in the maintext (up to the authors).Response: We thank Reviewer 2 for catching this! We corrected our code (apparently an older table was used where some of the samples were incorrectly coded). Minor commentsL.67 Not sure if reference 19 is appropriate for the number of Syngnathidae species;Reference 18 Eschmeyer actually lists 321 valid speciesResponse: Corrected and removed REF 19L.129ff. May be more readable as complete sentences?Response: EditedL.219 Typo in “Principal components”Response: Corrected L.230 Typo in “patterns”Response: Corrected L.231 Population code “SYD” has not been defined before.Response: Corrected to NSW (typo as many samples measured were from Sydney area)L.258 Unclear what the difference between FST = 0.561 and FST = 0.630 (L.262) is.Response: We clarified the difference between the two Fst values. One was for all 9 pops , the second Fst was for grouping the 9 pop’s into 4 areas.L.289 Move “(FST ranging 0.636– 0.703)” to the end of the sentence to include TASRespose: Moved to the end of sentenceL.313 Reference to Supplementary Figure S1 needed.Response: AddedL.370 Past tense? “were highly distinct”Response: CorrectedL.373 Move references to after “as a barrier to marine dispersal for 20-25 kya 374 during the last glacial maximum (LGM)”Response: Moved referenceL.376 The strait opened 14 ka ago according to Lambeck & Chappell, so gene flow mayhave been reinstated between 14 kya and today, not 14-25 kya.Response: CorrectedL.379 Type in “shape differences”Response: CorrectedL.380 It does not seem obvious why sex differences would not affect the observeddifferences.Response: We clarified this by adding a sentence in the Results  and explained why we cannot report a skewed sex ratio in the discussion . They did not influence our linear trendline for body depth, however to resolve this we would need to measure more females especially in NSW where females can be easily distinguished in situ by body depth.L.383 It is more appropriate to refer to allele frequency differences than gene flow (seeabove).Response: We thank the reviewer but can’t find a reference to gene flow on line 383. L.386 Need a reference that the found differentiation is extraordinarily high.Response: Added Ref 54.L.388 This indicates that some of the RAD loci obtained the mitochondrial genome. Isthis likely? Intuitively, I would think that is unlikely to obtain mitochondrial RAD loci but maybethis is different in this case. If there were mitochondrial loci in the dataset, these would have toget filtered or modeled separately from nuclear loci because they are haploid and have adifferent effective population size.Response: We have removed the references to this. We do believe some mitochondrial loci were sequenced, but haploid loci would have been filtered out when we only retained polymorphic sites.L.398 This paragraph seems isolated and the point is not clear.Response: We moved this sentence up, to where we talk about isolation distinguishing populationsL.405 Unclear which populations “this division” refers to. The previous sentence statesthat TAS and NSW populations were historically connected, not divided. The following sentencealso talks about similarities, rather than divisions.Response: We edited and moved this sentence up where we talk about Vic pop managed separately to NSW, TAS pop’s.L.415 change to “”?Response: Added now it’s REF 29L.435 Reference needed: “less resilient under normal circumstances, but instead havea smaller gene pool due to smaller population sizes and restricted distribution ranges”Response: Added references L.436 It is more appropriate to refer to allele frequency differences than gene flow (seeabove).Response: Edited to genetic differentiation L.440 Reference needed: “increase in anthropogenic stressors resulting inhabitat loss (kelp and seagrass) and an increase of sea surface temperature”Response: Added referencesL.458 It is more appropriate to refer to allele frequency differences than gene flow (seeabove).Response: We would like to keep ‘restricted gene flow’ as this section is in general terms and appropriate as it concerns management perspectives.Attachment2nd Response to Reviewer.docxSubmitted filename: Click here for additional data file. 23 Nov 2020Genomic and morphological evidence of distinct populations in the endemic common (weedy) seadragon Phyllopteryx taeniolatus (Syngnathidae) along the east coast of AustraliaPONE-D-20-17523R2Dear Dr. Klanten,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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Krascheninnikovia ceratoides (Chenopodiaceae) is a plant characteristic of dry steppe and semi-desert formations. Earlier studies showed that the ancestor of this autochthonous steppe element originated in Central Asia during the Miocene/Pliocene, i.e., in the same region and at the same time as the first appearance of steppe vegetation. However, as the extant lineages of Krascheninnikovia ceratoides diversified only 2.2 ± 0.9 Mya, it may represent a modern element of current dry steppe and semi-desert formations, rather than a component of the first steppe precursors of the Miocene. As such, it may have capitalised on the climatic conditions of the cold stages of the Quaternary to expand its range and colonise suitable habitats outside of its area of origin. To test this hypothesis, phylogeographic methods were applied to high-resolution genotyping-by-sequencing data. Our results indicate that Krascheninnikovia originated in western Central Asia and the Russian Altai, then spread to Europe in the West, and reached North America in the East. The populations of eastern Central Asia and North America belong to the same clade and are genetically clearly distinct from the Euro-Siberian populations. Among the populations west of the Altai Mountains, the European populations are genetically distinct from all others, which could be the result of the separation of populations east and west of the Urals caused by the Pleistocene transgressions of the Caspian Sea.Constituting one of Earth’s major biomes, steppes are characterised by naturally treeless extra-tropical vegetation. The formation of the Eurasian steppe belt, the largest steppe region in the world, began in Central Asia during the Neogene. In the glacial stages of the Pleistocene, steppe displaced forest vegetation, which in turn recolonised the area during the warmer interglacial periods, thus affecting the distribution of plants adapted to these habitats.  The Eurasian steppe belt is the largest steppe region in the world, stretching from the Hungarian basin in the West to the Amur River in the East. Its formation presumably started in Central Asia in the Neogene1 [and literature cited therein]. Significant Northern Hemispheric glaciation started at the beginning of the Pleistocene. In the last 2.6 Million years (My), numerous cold-warm cycles have occurred, as indicated by oxygen isotope studies of marine sediments4. During the cool glacial periods, steppes spread at the expense of forests, while forests recolonised the same areas in the warmer interglacial periods5. The distribution range of plants growing in steppe and semi-desert formations has expanded and contracted accordingly6. Some important landscape structures that may have affected the ability of steppe plants to migrate within their appropriate habitat during the ice ages were permanent, such as the Altai Mountains in Russia and Mongolia, or the Khangai Mountains of Central Mongolia7. On the other hand, temporarily large bodies of water in the area south of the Ural Mountains, caused by transgressions of the Caspian Sea during the ice ages, may have inhibited the migration of steppe plants for only a limited time10.Steppes are characterised as naturally treeless extra-tropical vegetation dominated by drought-resistant xeromorphic grasses, perennial herbs, and low shrubs, geophytes and therophytesArtemisia-Chenopodiaceae cold steppe (tundra-steppe) belt5 can be found nowadays only in azonal steppe islands and in artificial, man-made steppe-like habitats12. In recent years, steppe plants and their shared history with the Eurasian steppe belt have come under the focus of much research21.In Central Europe, the remains of the more or less continuous Pleistocene Krascheninnikovia ceratoides (L.) Gueldenst. (Chenopodiaceae) is a particularly suitable subject for reconstructing the evolutionary history of dry steppe and semi-desert formations. It is widespread throughout the Northern Hemisphere, growing on shortgrass steppes, desert steppes, and in semi-deserts23. Although its high morphological variation has led to the recognition of a variety of taxonomical names, Krascheninnikovia is now thought to constitute a single species with two subspecies, the Eurasian K. ceratoides subsp. ceratoides and the North American K. ceratoides subsp. lanata23. The distribution of K. ceratoides subsp. ceratoides is broad and continuous, extending from Mongolia and northern China in the East to the southern Volga and Don regions (European Russia) in the West  to high elevations of 4500–4800 m in the southern part of its range, such as in the Pamir and Ladakh mountain ranges27. In Asia, the plant is used for firewood and fodder, with its overuse leading to shrinking population sizes and concomitant possible land degradation as a consequence of increased erosion28. This phenomenon is called the Teresken syndrome for Teresken, the common name for K. ceratoides in Russian29. In North America it is used as fodder for livestock in winter (‘winterfat’)31. Krascheninnikovia ceratoides plants can reach an age of 300 years32, and first reproduction only occurs in plants older than 25 years33. The species is believed to be outcrossing34. The bractlets surrounding its fruit and utricle are densely covered with long hairs, which may facilitate dispersal by wind.K. ceratoides throughout its distribution range. Diploids with 2n = 18 chromosomes have been reported from the Ural Mountains of western Russia, across Kazakhstan and Kyrgyzstan to Mongolia and China40. Tetra- and/or hexaploids (2n = 36 and 54) have been reported from Europe to the Russian Altai Mountains46, and south of this range in Armenia47, China39, Iran48, Kyrgyzstan37, and Tajikistan40. A mixed population of di-, tri-, tetra-, penta-, and hexaploids  has been reported from Kyrgyzstan37. Diploid and tetraploid populations occur in North America49.Various ploidy levels exist in Krascheninnikovia originated c. 5 to 23 Million years ago (Mya) 51; 17.3 Mya (12.0‒23.5 Mya)35), while the crown group diversified 2.2 ± 0.9 Mya35. With a minimum age of more than 5 My, K. ceratoides has persisted throughout several global climatic changes including the glacial and interglacial phases of the Quaternary. Its diversification into extant lineages took place during the Gelasian , and seemingly correlates with the appearance of modern types of desert and semi-desert. These are, together with semi-arid temperate grasslands such as prairie, steppe, and pampas, a Plio-/Pleistocene phenomenon52. Therefore, K. ceratoides could have been among the first plants that inhabited modern-type dry steppe areas.According to previous studies, the stem group of 35 reconstructed the biogeographical history of Krascheninnikovia using a few molecular markers from its nuclear and chloroplast genomes . Genetic analyses relying on single loci may reflect only the history of these particular loci, rather than the history of the species studied. Therefore, a whole-genome approach is favourable, where single loci have less weight and cannot influence the result as strongly. To investigate the spatio-temporal diversification of K. ceratoides in more detail, we sequenced populations of the species from throughout most of its distribution range employing genotyping-by-sequencing (GBS). We then inferred phylogenetic trees and networks from the GBS data, conducted ancestral range reconstructions, and measured genome size. We specifically addressed the following questions: (1) Where did Krascheninnikovia originate? (2) Can GBS provide the appropriate resolution to retrace its colonising history and migration routes? (3) Can the retrieved phylogeographic and population genetic patterns be related to palaeogeographical and palaeoenvironmental events during the evolutionary history of the Eurasian steppe? With this, our overall aim was to contribute to the knowledge of the florogenesis of the Eurasian steppe belt.Seidl et al.Krascheninnikovia ceratoides leaves were collected in the field and dried with silica gel or taken from herbarium material. Leaves from up to five individuals per population were collected. The five individual specimens from Kyrgyzstan combined into population P025 came from two different localities about 80 km apart. In total, 183 accessions from 43 different populations of K. ceratoides were used for GBS analyses  were included, as well as two eastern Middle Asian populations of K. ceratoides subsp. ceratoides, which were originally determined as K. ewersmanniana . Axyris hybrida and Ceratocarpus arenarius were used as outgroups. Further details to the collected samples can be found in Supplementary Table ses Fig. . Most ofThe present study complies with relevant institutional, national, and international guidelines and legislation.K. ceratoides leaf tissue by flow cytometry, using an internal standard (either Pisum sativum L. ‘Kleine Rheinländerin’ (2C = 8.8 pg53) or Petroselinum crispum (Mill.) Fuss (2C = 4.5 pg54)) and the CyStain PI Absolute P kit  according to the manufacturer’s instructions. The relative fluorescence of at least 5000 particles per sample was recorded with a CyFlow Space .We determined the genome size and ploidy level of 99 samples of 35 were complemented with additional individuals from both the same and new populations  (single-end sequencing of 100 bp fragments). To achieve the same genomic coverage for individuals with different ploidies, twice the amount of DNA was used for tetraploids as for diploids. Two individuals (one from population P029 and one from population P072) were sequenced twice to assess the reproducibility of the method.Genotyping-by-sequencing library preparation followed Poland et al.56 for the sorting, filtering and de novo assembly of raw read data. To find the optimal parameter settings, we ran STACKS ‘denovo_map’ on a reduced data set 59 with the concatenated consensus sequence of the respective individuals serving as a reference. BWA settings were the following: maximum gap length of twelve, maximum of one gap per read, and a mismatch parameter of 0.0160. These bam-files were used as input for nQuire using the denoising option and for ploidyNGS using the “guess ploidy” option.To estimate ploidy levels from GBS data, we used nQuireKrascheninnikovia, an analysis was performed in R61 using the LEA62 package, using the data set of unlinked SNPs in Variant Call Format (VCF) of ingroup samples only. The ploidy was set to tetraploid. One to fifteen hypothetical ancestral populations were tested with 100 repetitions each. The number of clusters was chosen based on minimal cross-entropy. To show the result per population, the affiliation to each hypothetical ancestral population was calculated per population as the mean over its individuals and shown as pie charts on a map. To validate the results using a different approach, both Discriminant Analysis of Principal Components (DAPC) and Principle Coordinate Analysis (PCoA) were performed using the R packages64 adegenet and dartR, respectively, based on the ingroup data set of unlinked SNPs.To infer the population genetic structure of Krascheninnikovia as consensus sequences per population, after removing all uninformative sites65. Phylogenetic analyses were conducted by applying the maximum likelihood (ML) criterion using66 IQ-TREE v.2.0.367 with the GTR+G model and correction for ascertainment bias68. SH-like approximate likelihood ratio test69 and ultrafast bootstrapping (UFBoot)70 were performed with 1,000 repetitions each to obtain bootstrap support (aLRT/UFBoot BS) for nodes. The outgroups Axyris and Ceratocarpus are genetically too distant to assess the correct rooting, so midpoint rooting was used, which resulted in a tree with basal positions for populations of the Russian Altai. The basal population of the IQ-TREE analysis in Seidl et al.35, P242, originated in the Russian Altai as well. The splits data was visualised in SplitsTree v4.14.871 and the consensus tree in FigTree v1.4.372.Unlinked SNPs of 6,029 loci were retained in the PHYLIP file of 73. Populations were assigned to biogeographic regions according to their origin based on Brummit74: Central Europe, eastern Europe, western Middle Asia, central Middle Asia, eastern Middle Asia, southern Middle Asia, the Russian Altai Mountains, western Central Asia, southern Central Asia, eastern Central Asia, and North America. Only loci with called bases present in all populations were used. Rarefaction sample sizes for each ‘SNP locus’ were two populations from each region, and two alleles (‘genes’ in HP-Rare) from each population. Mean rarefaction and standard deviation were then calculated per region.To calculate private allelic richness per region, a rarefaction analysis was performed in HP-Rare75 as input tree for the BioGeoBears v. 1.1.178 R package for R ver. 3.5.078. The assignment of populations to regions was described above. The analysis was performed based on two different migration hypotheses: free migration between all areas, and restricted migration due to the distance between areas (0.8 for regions with a common land border and 0.2 for areas without a common land border). Three different biogeographic models were tested: DEC, DIVALIKE, and BAYAREALIKE, all both with and without the jumping parameter “j”, which determines the probability of jump dispersals. Based on AICc values, DIVALIKE+J with restricted migration was chosen as the most likely model. DIVALIKE is a likelihood interpretation of parsimony-based DIVA and allows the same processes79 such as dispersal, extinction, narrow sympatry, narrow and widespread vicariance, but not, e.g., subset sympatry.An ultrametric tree was built based on the ML tree using PATHd8The mean 2C genome sizes (± standard deviation) of diploid (N = 38) and tetraploid N = 61) individuals were 2.8 ± 0.3 pg and 5.8 ± 0.2 pg, respectively. The populations measured were uniformly di- or tetraploid. The populations from Central Europe, western Middle Asia, and the Russian Altai Mountains were all tetraploid, but in most regions both diploid and tetraploid populations were found , western Central Asia , and eastern Central Asia (P210) were found to be tetraploid; of P025, all individuals were found to be tetraploid except P025-I0223, which was diploid in both analyses, by GBS and flow-cytometry data. Consequently, we consider it very likely that P025-I0223 came from one of the two Kyrgyz localities, and the other four P025 individuals from the other locality, which is ~ 80 km away. P025-I0223 (hereinafter P025_D) and the other four individuals (hereinafter P025_T) were therefore treated as two different populations in all analyses. Of population P034, one individual (P034-I0317) was tetraploid, while all others were diploid. In all populations except for P025 and P034, the GBS-estimated ploidy of unmeasured samples corresponded to the ploidy of measured individuals from the same population.Krascheninnikovia. Of those loci, 6029 were kept after removing all uninformative sites. In the PHYLIP file including the outgroups, only 500 SNPs of Axyris and 525 SNPs of Ceratocarpus were retained, while the populations of Krascheninnikovia comprised on average 3731 (± 883) SNPs. Due to this huge difference of the amount of recovered data, the analyses including the outgroups may not be meaningful and were dismissed.Of 24,249 loci, composed of 2,625,739 sites, 21,254 variant and unlinked sites remained in the STACKS VCF file of LEA assigned all individuals to hypothetical ancestral populations based on their genetic composition. After 100 repetitions, eight hypothetical ancestral populations (K) were chosen based on the cross-entropy criterion , was inferred between the populations east of the Khangai Mountains in Central Mongolia (North America and eastern Mongolia), and the populations west of the Khangai Mountains. The second deepest split (K = 3) is in the area of the Ural Mountains, dividing the populations west of the Khangai Mountains. With K = 4, the Mongolian populations east of the Khangai Mountains and the North American populations each form a group of their own. The populations between the Ural and Khangai Mountains split in two groups according to their ploidy with K = 5: diploid populations are assigned to the light green cluster, while tetraploid populations are assigned to the dark green cluster. With K = 6, the populations of the Russian Altai and western Central Asia form their own group (dark blue). With K = 7, population P238 from southern Central Asia forms its own cluster (light blue), as does P081 with K=8 (black).To illustrate the consecutive splits between the hypothetical ancestral populations, the cluster membership was depicted as pie charts on a map for all K values from two to eight Fig. . The deeSimilarly to LEA, DAPC analysis clusters individuals according to their genetic composition Fig. . Four clSamples from Europe, Middle Asia, southern Central Asia, the Russian Altai and western Central Asia clustered together in the Principal Coordinate Analysis Fig. . Apart fK. ceratoides comprises two fully supported major genetic groups: populations from the Russian Altai, eastern Central Asia, and North America form one clade, which is a sister to a clade comprising the remaining accessions from the Russian Altai and from southern Central Asia to Central Europe  and the Russian Altai Mountains (dark blue)  and from there to North America (lilac). From the Russian Altai Mountains, it migrated to southern Central Asia and to eastern Middle Asia (light green). From eastern Middle Asia, it then migrated again to southern Central Asia (light blue) and to southern Middle Asia (brown). It migrated further west to central Middle Asia (pink) and to western Middle Asia (dark green), and on to eastern Europe (red) and Central Europe (blue).Based on our BioGeoBEARS analysis, the ancestral range of ue) Fig. . From weThe highest private allelic richness was found in western Central Asia (0.014 ± 0.002), followed by eastern Central Asia (0.013 ± 0.004) and the Russian Altai Mountains  have divided the populations east and west of the mountains and prevented gene flow for a long time. This split occurred in the early stages of diversification, after Krascheninnikovia migrated from the Altai Mountains to eastern Central Asia  on the one hand and eastern Central Asia (East Mongolia) on the other was found in all phylogenetic inferences Figs. , 4 as wees Figs. , which hKrascheninnikovia migrated west to Middle Asia.Starting from its likely area of origin in western Central Asia and the adjacent Altai mountains, 85. According to our biogeographic analysis, this probably also affected the phylogeographic patterns of K. ceratoides, which was probably present on the Kazakhstan plain and even further west at this time. This damming would have caused range splits and contractions, followed by isolation and subsequent expansion. Repeated secondary contact may be the explanation for the absence of monophyly for individuals from eastern Middle Asia in our IQ-TREE tree  Korn.-Trotzky12, Goniolimon speciosum (L.) Boiss.18, Capsella Medik.13, Allium cretaceum N.Friesen & Seregin, and A. montanostepposum N.Friesen & Seregin14 were affected by these backwater events as well.During the Middle Pleistocene, glaciers dammed the Siberian Ob-Irtysh-Tobol river system, leading to backwaters that reached the foothills of the Kazakhstan Highlands and the Altai Mountains. The resulting landscape of lakes and swampy areas was not a suitable habitat for steppe plantsKrascheninnikovia migrated stepwise from Central Asia to Middle Asia and to Europe. Then, the European populations were separated from populations east of the Urals  and southern Central Asia  belong to the light green cluster in our LEA analysis, suggesting a common ancestry. The same cluster membership is shared by the diploid individuals from central Middle Asia (P034) and eastern Europe , which are admixed. The westward migration of K. ceratoides was possibly accomplished by diploid individuals, followed by (auto-)polyploidization in the newly colonised areas, a process which occurred several times independently as a response to climatic fluctuations in the Pleistocene104.Diploid and tetraploid populations of sia Fig. 34, 47. MKrascheninnikovia. While some argue that Krascheninnikovia is monospecific with only two subspecies ceratoides and lanata22, others distinguish several entities at the specific level, such as K. arborescens, K. ewersmanniana, and K. compacta from Middle and Central Asia90. Our data did not include samples representing these potential taxa, except for two populations identified as K. ewersmanniana (P069 and P070). Therefore, this study is not suitable to conclude on this topic. The results of our analyses support however the recognition of subspecies lanata as defined by Heklau and Röser23, as the North American individuals form a clearly distinguishable genetic lineage. Our data did not support the samples identified as K. ewersmanniana to belong to a distinct clade, as they are nested at different positions among morphologically typical subsp. ceratoides specimens from eastern Middle Asia. However, to confirm this hypothesis thorough morphological studies are necessary. Thus, we concur with the conclusions of Heklau and Röser23, recognizing (at the moment) two subspecific entities, i.e. subsp. ceratoides and subsp. lanata within monotypic Krascheninnikovia.There is an ongoing discussion about potential subspecies and species in the genus Krascheninnikovia and its connection to the history of the Eurasian steppe belt, confirming and refining previous findings using GBS. The observed phylogeographic patterns reveal the common history of Krascheninnikovia and the steppe belt: The Krascheninnikovia lineage represents an autochthonous steppe element that evolved in situ during the Miocene when steppe precursors made their first appearance in Central Asia. Additionally, the Caspian Sea transgressions and the ice-dammed backwaters of the Ob-Irtysh-Tobol river system during the Pleistocene left clear imprints on the population genetic structure that persist until today. Nevertheless, there is still much to be investigated: The inclusion of further samples from the southernmost part of the range in Eurasia and from North America in future GBS analyses of Krascheninnikovia could allow the narrowing down of the area of origin and conclusions to be drawn about the number of migration events to America that have taken place.Our analyses provide insights into the history of Supplementary Information