PONE-D-22-27124Barley systematics and taxonomy foreseen by seed morphometric variationPLOS ONE

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Please clarify the problem statement in introduction section clearly and justify it in discussion section. Its needed to answer the reviewer comments before further process.

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Jeanty et al. brings a rather important contribution to the field of archaeobotany that will be of relevance for researchers in other disciplines, such as agronomy and plant physiology. The demonstration that barley types (2 row/6row, grain nakedness, growth habit) can be discriminated based on grain morphology is immensely relevant and exciting. It also dispels the fears that interannual or environmental variation might render comparisons based on grain morphology ineffective.

I therefore recommend its publication in PLoS One with only some suggestions for the authors’ consideration.

Line 20 – no need to have a comma between “variation” and “on the grains”.

Line 68 – In fact, several recent genomic studies have highlighted the polyphyletic and multiregional origin of barley domestication. See, for example,

Allaby, R. G. (2015). Barley domestication: the end of a central dogma?. Genome Biology, 16(1), 1-3.

Poets, A. M., Fang, Z., Clegg, M. T., & Morrell, P. L. (2015). Barley landraces are characterized by geographically heterogeneous genomic origins. Genome biology, 16(1), 1-11.

Pankin, A., Altmüller, J., Becker, C., & von Korff, M. (2018). Targeted resequencing reveals genomic signatures of barley domestication. New Phytologist, 218(3), 1247-1259.

Line 77 – There are other ancient DNA studies from barley grains that have been useful to study its origins and evolution. See, for example,

Palmer, S. A., Moore, J. D., Clapham, A. J., Rose, P., & Allaby, R. G. (2009). Archaeogenetic evidence of ancient Nubian barley evolution from six to two-row indicates local adaptation. PLoS One, 4(7), e6301.

Hagenblad, J., Morales, J., Leino, M. W., & Rodríguez-Rodríguez, A. C. (2017). Farmer fidelity in the Canary Islands revealed by ancient DNA from prehistoric seeds. Journal of Archaeological Science, 78, 78-87.

Line 112 – For the sake of readability, please make the numbering of aims here the same as in the Abstract.

Line 127 – Why 66 instead of 58 accessions? What is the difference between a “variety” and an “accession” in this context? Sounds confusing.

Line 158 – why only 784 SNPs when previously (line 60) there were 1056 SNPs? I presume this is the number of SNPs used for these particular accessions, but please clarify.

Line 158 – Also, as a suggestion, it would be nice to do a PCA for the accessions used, based on the genetic data, colouring the points with the same colour-scheme used in Figure 1 for 2-rowed hulled, 2-rowed naked, etc. It is not necessary for the overall story but it would be an interesting addition.

Also, please make the SNP data available as an Excel Spreadsheet as a supplementary file. This open-data practice is standard in the biological sciences.

Line 188 – Can we have a value for the “within”-variety variations or does the study described in Lines 138-142 make this unnecessary?

Line 234 – when discussing the results for genetic information and grain morphometrics, please address the issue that the number of SNPs used is to low for finding correlations between genetics and type. Intuitively, population structure will be more relevant than phenotype. For finding grain measurements causative SNPs a Genome-Wide Association Study (GWAS) study would have to be performed with hundreds of thousands of SNPs. You do mention in lines 357-359 studies looking at QTLs but perhaps refer to GWAS studies, for example:

Sharma, R., Draicchio, F., Bull, H., Herzig, P., Maurer, A., Pillen, K., ... & Flavell, A. J. (2018). Genome-wide association of yield traits in a nested association mapping population of barley reveals new gene diversity for future breeding. Journal of Experimental Botany, 69(16), 3811-3822.

Reviewer #2: In this paper, the authors had used a dataset of 58 varieties of French-origin barley to assess the taxonomic signal in grain measurements and test the impact of various factors on grain size and shape. They had also compared the relationship between morphometric and genetic proximity. I did not see any novelty in this manuscript. The work have not designed well. The methods of data collections and presentation of the results were not appropriate.

Please see my comments below:

- A small sample (58 French barley genotypes) size is a limitation of this study, as it may not provide enough power to detect differences or relationships that may exist.

- 54 barley genotypes grown in a field for only one year may not provide enough data to draw meaningful conclusions about their long-term performance or stability. If the goal of the study is to understand the characteristics and behavior of grain measurements, it may be necessary to grow them for multiple years or in different environments to get a more complete understanding of their characteristics.

Reviewer #3: The authors describe a new method to cluster barley seeds from their morphology e.g. into 2-row and 6-row types. This may be useful in many respects, and they claim that the method is relevant for analysis and classification of archaeological grains. However, this remains very speculative for a number of reasons: First of all, the analysis presented in this manuscript are all conducted on non-charred grains. This is contradictory to the idea, that the method is developed to be used for archeological grains, since the absolute majority of archaeological grains are charred. Other studies have shown that the morphology of grains changes when they are charred. We can therefore not be sure, if the method is applicable for charred grains. Secondly, the authors do not demonstrate an analysis of any archaeological grains and how well the method can cluster them into subgroups. This is essential for the conclusion that the method is relevant for archaeobotany. Another important discussion point is that (pre-historic) archaeological barley grains are significantly smaller than extant varieties. This suggests that extensive breeding has increased the size of barley grains over the last millennia. Therefore it is unclear if the method is applicable to pre-historic barley grains.

The method developed may be very useful in many respects. However, for the above reasons the conclusion drawn by the authors, that the method can be sued to analyze pre-historic grains is not documented by the presented data. Interpreted in another context the method could certainly be acceptable for publication.

Reviewer #4: This is a timely paper with potentially significant impacts on a number of fields, including archaeology, as the authors rightly suggested. The authors conducted vigorous analysis on a large dataset (58 varieties) of barley caryopses. The results suggest that caryopses metrics correspond to ear morphotypes (2/6-row; hulled/naked), as previously speculated by scholars but now approved with robust quantified evidence. The new data concerning sowing times and grain metric variations is excellent and constitute a significant contribution. I have a couple of referencing comments. The conclusion on that growing condition does not impose major effects on grain morphology, in relative terms, resonates with Ritchey and colleagues' work (2022), suggesting human choice and varietal difference played a key role in regional variations of barley grain morphotypes in prehistory, not the environment. Additional reference to Liu et al. 2017 on the eastern dispersal of barley will be helpful in covering a global perspective of early barley cultivation. Otherwise, this should be published, and I have no qualms with the manuscript.

Liu, X., D.L. Lister, Z. Zhao, C.A. Petrie, X. Zeng, P.J. Jones, R. Staff, A.K. Pokharia, J. Bates, R.N. Singh, S.A. Weber, G. Motuzaite Matuzeviviute, G. Dong, H. Li, H. Lü, H. Jiang, J. Wang, J. Ma, D. Tian, G. Jin, L. Zhou, X. Wu & M.K. Jones, 2017. Journey to the East: diverse routes and variable flowering times for wheat and barley en route to prehistoric China. PLOS ONE, 12(11), e0209518.

Ritchey, M. M., Y. Sun, G. Motuzaite Matuzeviciute, S. Shaoda, A. K. Pokharia, M. Spate, L. Tang, J. Song, H. Li, G. Dong, P. Vaiglova, M. Frachetti and X. Liu, 2022. The Wind that Shakes the Barley: the role of eastern Eurasian cuisines and environments on barley grain size. World Archaeology, 53(1): 1-18.

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Reviewer #1: Yes: Hugo R. Oliveira

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Barley systematics and taxonomy foreseen by seed morphometric variation

PONE-D-22-27124R1

Dear Dr. Jeanty,

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Kind regards,

Muhammad Abdul Rehman Rashid, PhD

Academic Editor

PLOS ONE

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