PONE-D-22-34359Interactions between Trypillian farmers and North Pontic forager-pastoralists in Eneolithic central UkrainePLOS ONE

Dear Dr. Nikitin,

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please 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. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The relation between Tripolje complex farming communities and advanced foragers belonging to Stredny Stog has been contested. The archaeology speaks of cultural exchange in pottery as well as certain farming practices. Since we lack human bones, a single bone is here undergoing a full interdisciplinary treatment to illuminate its origin, which points to Stregny Stog thus confirming archaeological evidence of contacts.

The paper provides a full contextual background to the archaeological problem, and provides as well a full methodological presentation of all analyses. AN exemplary presentation.

The conclusion could have been more bold, providing some tentative hypotheses as to the nature of this interaction, whether peaceful e.g. trade or rather captives. Or something in between.

Reviewer #2: Nikitin and colleagues present a joint analysis of isotopic and genetic data obtained from one individual from the Kolomiytsiv Yar Tract (KYT) in central Ukraine, which belongs to the Trypillian culture (the eastern part of the Cucuteni-Trypillia complex). During the late Neolithic, the Cucuteni-Trypillia complex occupied a large geographic area, encompassing major parts of present-day eastern Romania, Moldova, and Ukraine, and is characterised by the emergence of settlement mega-sites with several hundreds of inhabitants. Yet, skeletal material from the Trypillian culture is rare and thus only limited DNA data was so far produced. To my knowledge, genome-wide information of only 20 individuals from Ukraine, and four individuals from Moldova are currently available. Consequently, this study provides a relevant addition to the current archaeogenetic record. According to the novel radiocarbon date reported in this study, the analysed individual belongs to the Trypillia BI-II stage, which is associated in the east with the introduction of pottery traditions potentially adopted from neighbouring steppe pastoralist groups, especially the so-called “Cucuteni C ceramics”. Since this kind of pottery is also found at the KYT, indicating some cultural exchange between the local Trypillian population and the adjacent steppe pastoralists, the authors investigate whether this cultural signal is also reflected in genome (and isotopic signature) of the reported individual. For this purpose, the authors report novel carbon and nitrogen data to reconstruct the diet of the individual as well as strontium and genome-wide DNA data to trace the origin and ancestry of the specimen. Indeed, the nitrogen and carbon isotope values indicate that the individual’s diet was different to the diet of the local Eneolithic farmers but is shifted in the direction of the fisher-forager-pastoralists of the North Pontic steppe. The authors highlight that the diet of the individual could also reflect “a mixture of farming, pastoralist and foraging subsistence practice”. Genetically, the individual is remarkable since it clusters outside the genetic variation of previously published Trypillia samples, which carry mainly Early European Farmer ancestry, and clusters closely with early Bronze Age Yamnaya individuals, proving gene low from the steppe pastoralists into Trypillia farmer communities. Consistently, the strontium isotope values of the bone sepcimens fall in the range of previously published Yamnaya individuals. Yet, the individual is genetically not cladal with Yamanya individuals (as shown using F4 statistics), suggesting that the individual is ultimately admixed between a major Steppe source and a minor WHG enriched source.

In my opinion, the paper combines comprehensibly different lines of evidence to present a convincing story. The authors provide the first individual with Trypillia cultural affiliation that carries a majority of Steppe-derived ancestry, which is a great addition to the archaeogenetic record (especially for a geographic region in a period that is so far understudied) and service to the aDNA community. As far as I can assess the methodology, the analyses were conducted according to the scientific standard. The laboratory protocols for DNA and isotope extraction and processing are described in detail and cite the relevant references. Before population genetic analysis, data quality was ensured by measuring contamination on the mtDNA level. The authors applied well-established population genetic methods to the genome-wide data, namely PCA and F4-statistics. For both the isotopic measurements and population genetic methods, all relevant results were reported (Standard deviations for isotope values, Z-values for F4-statistics, etc). Therefore, I consider this paper to be technically sound.

My comments regarding the scientific quality are as follows:

The paragraph “Ceramic assemblage at Kolomiytsiv Yar Tract” (lines 257 to 270) in the Results and Discussion section does not report new results, so maybe it would be more appropriate to move its content to the introduction.

In the lines 306 to 307, the authors write “Overall, the KYT diet appears comparable to the diet of fisher-forager-pastoralists of the North Pontic steppe, but not to the diet of farmers of Eneolithic Pontic coast and adjacent forest steppe areas (Figure 3)”. In my opinion, this is a contestable statement as the sample plots between all six reference groups and does not cluster with any of them specifically. While the authors also state that “the diet of the KYT individual could also be reflecting a mixture of farming, pastoralist and foraging subsistence practices”, which mitigates the previous assertion, I would advocate for a more cautious conclusion.

In the segment “Strontium isotope analysis” in the Results and Discussion section (lines 331 to 397), the authors reference several previously published 87Sr/86Sr ratios for Eneolithic and Yamnaya populations from Ukraine. Without knowledge of these publications, it was a difficult for me to follow, thus I wonder if it would be possible to summarise the strontium values in a similar form as it was done for the nitrogen and carbon values in Figure 3.

In line 416 the authors write “Whole genome analysis of the KYT specimen produced 76550 Singe Nucleotide Polymorphism “hits” (…)”. I would suggest to rephrase that since it is not clear to me what “whole genome analysis” means in this context. I guess this describes “sequencing of the aDNA libraries after 1240k target enrichment”. In general, since the data is SNP captured and not shotgun sequenced, I would advocate for the use of the term “genome-wide data” instead of “whole genome data”. Also, I cannot not reconstruct why only 76550 SNPs were genotyped, when (according to lines 247 to 248) “a total of 357,145 targeted SNPs on chromosomes [were] covered by at least one sequence”. Additionally, I would like to know the number of SNPs available for PCA. In Table S2, only the 1240k SNPs are reported, yet the PCA was run on Human Origins data, which is only a subset of the 1240k SNP set.

In lines 432 to 436, the authors write “A plausible scenario is that the KYT individual has much of their ancestry from a Proto-Yamna population (Chintalapati et al. 2022), while also being admixed with Serbia/Iron Gates Mesolithic, the latter possibly coming from the Neolithic populations of the Dnipro Valley that have been shown to carry WHG admixture (Mathieson et al. 2018)”. The paper could highly profit from formally testing this hypothesis, e.g. using qpAdm. While PCA and F4-statistics clearly suggest a major proportion of Steppe ancestry in the KYT-SSX individual, qpAdm testing would provide a (citable) percentage and could be furthermore applied to identify the source population that provided the smaller ancestry proportion (which causes the shift away from Yamnaya in the PCA).

I have also noticed some smaller typos which should be fixed after another round of proof-reading, e.g.:

Line 56 „The Yamma cultural (…)“

Line 237 „We took a sample of xx”

In Figure 3 “Dnipro Valley Fihsers” and “Dniester Delta Eneolithic Fiher”

Summarising, I would like to see this study published and think that after some small adjustments the manuscript should be ready for publication.

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Reviewer #1: Yes: kristian kristiansen

Reviewer #2: No

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Interactions between Trypillian farmers and North Pontic forager-pastoralists in Eneolithic central Ukraine

PONE-D-22-34359R1

Dear Dr. Nikitin,

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. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mateusz Baca

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors satisfactory addressed the main issues raised by the Reviewer 2. They provided the correct number of SNPs from the 1230k dataset and the number of SNPs used for PCA. They also provided a more cautious assessment of the analysis of dietary isotopes. In my opinion, the paper is now ready for publication in PlosONE.

Reviewers' comments: