DNA Markers Reveal Genetic Associations among 11,000-Year-Old Scots Pine (Pinus sylvestris L.) Found in the Baltic Sea with the Present-Day Gene Pools in Lithuania

Round 1

Reviewer 1 Report

This study makes a significant contribution to the understanding of evolutionary history of Scots pine in the Baltic region.

Minor comments:

Ln. 30  ”as Scots pine is of major fundamental interest in revealing the evolution” Either ”major” or ”fundamental”.

Ln. 32  ”are the first waves of post-glacial migrants” Reference needed.

Ln. 45-46   ” Like in our material, the Scots pines were already present in the eastern Baltic seacoast ca. 12 000 years BP.”  Avoid anticipating your own results in the Introduction.

Ln. 57  ”starching over whole Europe” probably ”stretching”

Ln. 67  ”from variable refugia leading to a” Perhaps ”distinct refugia”

Ln. 68-71: I suggest mentioning the research questions in the same paragraph with the objectives

Figure 2 with morphotypes is not necessary. It might be included as supplementary material.

Figure 3: Please explain the values in the legend. Is it really necessary to have three digits after comma (dot?) (e.g., 20,886)?

Ln 220 ”we successfully amplified acceptable quality alleles in the 11 nuclear SSR loci in seven out of 7 sea pine individuals (Table 2).” In 7 out of 9?

Figure 5 Please explain the abbreviation BOG and NORM in the Figure caption.

Ln. 278  ”each single pollen grain contains different genotype” genotypes?

Ln. 285 ”plant materials and is its yield is markedly higher” Perhaps ”and its yield is markedly …”

Ln. 289 ”vegetation 33-55 m. below” delete the dot.

Ln. 326 ” 133000 BP up to the present-day.” 13300 BP?

Ln. 358 ”indicating their origin from the southern glacial refugia in Balkans.” Mitotype A is found in Southern Europe (i.e., including Italian peninsula) not only in the Balkans. Please rephrase here and in the abstract.

Author Response

Thanks for the comments improving our manuscript. The comments are addressed in the attached file.

Author Response File: Author Response.doc

Reviewer 2 Report

This is an interesting work with reliable methodology, as it refers to laboratory work and protocols designed to avoid genetic contamination. The overall problem is interesting, as the authors intend to verify the lineage of Scots pine that migrated to Lithuanian forests, and the possibility to test it based on ancient wood is particularly appealing because it may inform about one of the lineage which migrated first.

However, the manuscript is inconsistent in several parts. Furthermore, this needs clarifications.

The authors genotyped some ancient trees for microsatellites to find the highest probability of assignment of these trees to present in Lithuania. I would like to see the rationale for such comparisons. Nowadays, forests represent populations that could be descendants of first migrants, however, there were so many generations since the first migrations, and also, gene flow in scots pine is so extensive that I doubt that there is any significant differentiation among nowadays populations over such a limited area such as Lithuanian country. The authors could analyze genetic diversity and differentiation levels among the sampled ‘autochthonous’ populations, and this information is missing.

Also, I do not see the rationale for involving morphotypes of particular populations. This issue seems to be out of the paper's main course and is artificially included in this manuscript. The extent of association of ancient pines to wetland or drysite populations is unclear, and the data do not support conclusions.

I suggest making the manuscript more focused on the genetic lineage analyses and making a more in-depth analysis of population genetic diversity, including population differentiation levels. With such a large number of populations and trees, I would even suggest making the inference about the genetic clusters (Bayesian methods such as Structure could be useful), which could provide some more insights into the history of Scots pine in these areas.

Below are some minor comments:

Line 60-62, please provide the exact reference of the study of 54 populations, because there is statement ‘our earlier study…’

Line 75 change ‘We also in tested’ to ‘We also tested’

Line 109, the authors genotyped 27 autochthonous populations, but there is no analysis about these population, for example, what was the diversity, or what was the level of population differentiation, We have no information about even basic parameters of these populations,

Line 172-181 – while for mtDNA marker, there is a detailed description of how the fragment size was measured (ABI system and GeneMapper), there is no information on how the genotypes of 27 autochthonous populations were genotyped. Only PCR amplification is described.

Line 183, 186 write whole word ‘software’ not ‘soft’

Line 220 – “in seven out of 7 sea pine individuals”, shouldn’t it be ‘in 7 out of 9’ ??

Line 231 – change ‘fir’ to for

Author Response

Dear Reviewer,

We thank the reviews for the comments and efforts in improving our paper. Our study brings 3 main messages to the scientific community: (a) salty seawater well conserves DNA in wood, this may promote ore findings for the underwater artefacts (b) shed light on postglacial migration of Scots pine in the Baltic region (c) investigates the long-term temporal genetic associations.

The later point received remarks, that these associations are likely to be random over such a long period as 10 000 years. The fact we have proven is that there are long-term temporal genetic associations in Scots pine. Yes, it is a possibility that these are random associations. However, gene pools may be locally conserved over a long time. No one has studied for how long. Why not for 10 000 years? It is the neutral part of the genome, not markedly affected by the changes in the environment over years. Gene frequencies in the neutral part of the genome in a widespread wind-pollinated species with a large population size likely to be free of strong defects of mutations, drift and natural selection and temporally stay at HW equilibrium over many generations. Why not 10000 years (ca. 30 non-overlapping pine generations of 300 years each). In support of these non-random associations, we found that the temporal associations were consistently strong with all present-day southern populations genotyped in our study. Also, in the Discussion, we noted that the fossil pollen data shows that Scots pine in southern Lithuania has been present all the time over the last 10 000 years. While in the other parts of Lithuania Scots pine presence was not permanent.

There were suggestions to expand the study by presenting genetic comparisons among the present-day populations. However, we intend to do this in a separate study. The present study is already long.

Darius

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

The manuscript was amended mostly according to reviewer suggestions.

However, line 327, 

300 years as a generation time is much overestimated. Scots pine reaches the highest fecundity when it is about 100 years old, therefore generation should be counted as 100 years and not 300.

Author Response

OK thanks for remarking we changed the generation time to 100 years and revised the sentence accordingly. We marked it in green.

Author Response File: Author Response.docx