Gene Flow and Recruitment Patterns among Disjunct Populations of Allocasuarina verticillata (Lam.) L.A.S. Johnson
, Nigel England 2, Linda Broadhurst 3, Lan Li 3, Chonglu Zhong 1 and David Bush 2Round 1
Reviewer 1 Report
I would like to thank to the authors of this study all the clarifications and improvements made on the manuscript. I believe that the manuscript has been significantly improved and now warrants publication in forests after, maybe, minor English spell check that I do not feel qualified to judge.
Author Response
We gratefully acknowledge the support for the manuscript improvement. The manuscript have been carefully checked by a native-English-speaking co-auther.
Reviewer 2 Report
Dear Editor and Authors,
my comments to the improved manuscript are included in the attached file.
Comments for author File: 
Comments.pdf
Author Response
We really appreciate the reviewers’ efforts and attentions to detail in improving this manuscript. Detailed changes of the manuscript are provided in attached cover letter.
Author Response File: 
Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript entitled “Gene flow and recruitment patterns among disjunct populations of Allocasuarina verticillate” by Zhang et al. explores the effects of population size, reproductive patterns and pollen and seed dispersal on among-population genetic diversity, genetic differentiation and structure using field survey and microsatellite marker techniques. The manuscript is generally well-written, with the introduction providing sufficient background and including all relevant references. The research design is appropriate and the methods adequately described to ensure the reproducibility of the study. The results are also adequately presented. I consider that the questions addressed and the results obtained from this study are of interest for the readers of forests journal. However, I have identified some weaknesses that should be addressed, and that are explained in further detail in the following lines. Among them, the most concerning to me is that some of the conclusions drawn by the authors in the discussion and conclusions are not supported by the results and/or they need clarification.
Introduction:
Page 2, 1st paragraph: “For example, some plants can regenerate asexually to ensure population survival and persistence in the face of unfavorable environmental conditions, for example if mate-availability is limited or if pollen vectors are absent.”.
- Comment: Style error, “for example” is written twice. Maybe remove the first “for example”.
Page 2, 2nd paragraph: “Importantly, however, recruitment, sustainability and extension of fragmented populations rely on seed-dispersal and seedling-establishment (Howe and Miriti 2004; Hampe et al. 2008). “
- Comment: Could you explain briefly why is that?
Page 2, 4th paragraph: “Allocasuarina verticillata (Lam.) L.A.S.Johnson is a small, tree (5-9 m tall), widely distributed across southeastern mainland Australia and Tasmania. “
- Comment: Do you mean “Allocasuarina verticillata (Lam.) L.A.S.Johnson is a small tree (5-9 m tall), widely distributed across southeastern mainlands in Australia and Tasmania. “?
Page 3, 1st paragraph: “mechanical dis-turbances such as track building associated with infrastructure development such as radio mast installations on the hills “
- Comment: You have repeated “such as” twice in the same sentence
Page 3, 1st paragraph: “An intense fire in 1952 on the slopes of Mt Stromlo resulted in very dense stands of A. verticillata that are still extant in 2020, following another major fire in 2003. “
- Comment: I do not understand this sentence: How “an intense fire” can result in “very dense stands” of a tree.
Material and methods
Page 3, last paragraph: “When sampling, individuals were categorized into five groups: field-grown seedlings i.e. young trees lacking reproductive structures and less than 3 m in height; females, (tree bearing cones); males, trees bearing male inflorescences; monoecious, trees bearing both cones and male inflorescences; gender unknown, (trees >3 m tall but bearing no cone or male inflo- rescence). Female, male, monoecious and gender unknown trees were regarded as adults. “
- Comment: Could you explain briefly why this classification was chosen (what questions would you allow to answer this sampling design? It is unclear to me (one line or two at the most)
Page 4, 1st paragraph: “Other tree and shrub species co-occurring in the stands of A. verticillata included Eucalyptus mannifera, E. melliodora, E. rossii, E. blakelyi, E. macrorhyncha and Exocarpus cupressiformis. “
- Comment: What is the purpose of including this sentence in the material and methods section? Are these species used in the sampling? If not, I consider that it fits better in the introduction (or in the discussion only if it provides useful information for the understanding of the study and/or the interpretation of the results).
Page 6, 2nd paragraph: “Forty Mt. Painter and 48 Cooleman Ridge seedlings”
- Comment: Change “Forty” by “40”.
Page 7, paragraph 3: “Twelve SSR makers were selected by means of screening and optimization of 48 EST-SSR markers that had previously been developed for Casuarina equisetifolia subsp. equisetifolia (Zhang et al., 2016; Xu et al. 2018) and six genomic-derived SSR markers developed for A. verticillata by Broadhurst (2011). “
- Comment: Why did you use these two types of SSR markers (EST-SSR and genomic-derived SSR)?
Page 10, paragraph 2: “All mature male trees across the three isolated populations were considered candidate fathers. Similarly, maternity analyses of seedlings (47 from Coole- man Ridge, 55 from Mt. Painter) were performed and all the mature female trees of the four isolated populations were considered candidate mothers. “
- Comment: Are there four or three isolated populations?
Results
Page 10, paragraph 3: “There were positive correlations between effective population size (number of parental trees ) and percentage of viable seed in the three fragmented populations, rather than between actual population size and the percentage of viable seed, suggesting that it is effective population size instead of ac- tual population size that plays a key role in influencing reproductive success of A. verticillata. “
- Comment: Where are the table containing the results of these analyses (R, p-value)? What statistical method was used to perform the correlation (e.g. Pearson)?
Page 11, paragraph 1: “A negative correlation between effective population size (number of trees possessing reproductive structures) and identified clonal individuals; 90 vs. 3, 43 vs. 8 and 26 vs. 12 at Cooleman Ridge, Mt. Painter and Isaacs Ridge populations respectively, was noted. “
- Comment: Again, what statistical method was used to calculate this correlation? Where are the results of this analysis? (the Pearson´s R and the p-value)?
Page 16, paragraph 1: “The AMOVA revealed that only a small proportion of the genetic variation (8 %) was partitioned among the four populations and most genetic variation (92 %) was generated within the populations. “
- Comment: Where are the results for the AMOVA?
Discussion
Page 18, last paragraph: “This study of A. verticillata on geographically isolated hills in the ACT provides evidence of limited wind-mediated gene flow, presumably via long-distance pollen dispersal, among subpopulations; predominantly short-distance seed dispersal, despite the species’ winged seed suggesting adaptation to anemochorous dispersal; and, strong evidence that asexual reproduction plays an important part in the species’ strategy for adaptation to challenging sites. “
- Comment: I do not see the “predominantly short-distance seed dispersal”. 24 out of 47 offspring seedlings at Cooleman Ridge were not identified from within- and among-population female trees. 23 out of 55 offspring seedlings at Mt. Painter, were not identified from within- and among-population female trees. This is almost 50% of the individuals coming from seeds from longer distances. This contrasts with the results of pollen dispersal, where the 48 open-pollinated seedlings at Cooleman and 34 of 40 seedlings at Mt. Painter were assigned at 6 male trees belonging to the four studied populations. Thus, it seems like the seed dispersal plays a major role in longer distance gene flow rather than pollen. However, you keep claiming that pollen is the main mode to long-distance gene flow for this species throughout the discussion, e.g. Page 20, paragraph 3: “We presume that the main mode of genetic exchange among isolated stands of A. verticillata in the ACT is via wind-transported pollen.” And Page 20 paragraph 5 “Anemochoric long distance seed dispersal is far less likely to be a significant driver of gene flow among localities in this study. Though the seed is winged, it would only be likely to travel comparatively short distances, even under strong wind conditions.”. You provide a clarification later on in the discussion (page 21, the entire first paragraph) but I would suggest to organize the discussion differently to make it easier for the reader to understand your claims.
Page 20, paragraph 2: “Statistically significant among-popula- tion gene flow (Tm) was also found (Table 7), suggesting that hard barriers to gene flow do not exist among the four populations, even though they are relatively spatially distant (pairwise distance range from 7.6 to 14.2 km) “
- Comment: Where is table 7?
Conclusion
“The species has several adaptations that explain previous observations that it appears to thrive under the influence of these environmental conditions and anthropogenic disturbance. “
- Comment: I suggest to rewrite this sentence. It is difficult to read and understand
“while seed dispersal appears to be predominantly over shorter distances within populations. “
- Comment: Again, in page 21, 1st paragraph you claim that you do not have enough information to draw conclusions about long-distance maternal seed dispersal. If you do not have enough information, you cannot draw a conclusion. Why did you keep claiming that seed dispersal appears to be predominantly over shorter distances”? Actually, your results point to a long-distance dispersion, although you need more data to determine kinship among individuals for which no pedigree exists, as you claim in page 21, paragraph 1. I would suggest to remove this claim about a likely short-distance seed dispersal from all the discussion (it appears several times as I have previously pointed out) unless you have data to support this claim.
Author Response
Please see the attachment.
Reviewer 2 Report
The reviewed paper reports the results of analyses in which 12 microsatellite loci were used to study the genetic diversity and differentiation as well as the spatial genetic structure among four populations of Allocasuarina verticillata from south eastern Australia. Based on the obtained results authors discuss the revealed geographical pattern of genetic differentiation of studied species and make hypotheses that explain the observed pattern of variation.
The reviewed paper is an example of an interesting study with an appropriate methodological side and clearly delineated objectives of the work. The applied methodology is correct and suitable for the realization of the paper's objectives. The comprehensive statistical analysis of the obtained results is a strong point of the study. The results description is adequate and the conclusions are well supported. English language and style in the reviewed manuscript are fine.
As there is no line numbering in reviewed manuscript my comments (minor flaws) were incorporated into the manuscript file which was attached to this Review report.
Comments for author File: Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
