Reconstruction of the lifeways of Central European Late Bronze Age communities using ancient DNA, isotope and osteoarchaeological analyses

Domain: chemistry
Published: 2026-02-24
Editorial outcome: minor_revision

Abstract
The Late Bronze Age (ca. 1300–800 BCE) of Central Europe is often characterised as a period of increasing mobility, socioeconomic transformation, environmental fluctuations, and expanding cultural networks. However, reconstructing the demographic aspects of these changes has been hindered by cremation being the dominant mortuary practice, limiting biomolecular approaches. Here, we integrate ancient DNA, oxygen and strontium isotope analyses, and osteoarchaeology to examine rare inhumation burials from Kuckenburg and Esperstedt in Central Germany (n = 36) and compare them to contemporaneous inhumations from the neighbouring regions of South Germany, Bohemia (Czechia) and Southwest/Central Poland (n = 33). Genome-wide data show genetic continuity with preceding Early Bronze Age populations, alongside gradual increases in Early European Farmer-related ancestry, albeit with regionally different timing and extent, reflecting a nuanced pattern of mobility and admixture. Oxygen and strontium isotope data from Central Germany indicate that most individuals match the local isotope signal, including those who were cremated or had a different diet, and with only a few isotopic outliers, suggesting that mobility was present but not extensive. Overall, our findings suggest that the diverse inhumation practices at Kuckenburg and Esperstedt were culturally motivated, reflecting local traditions and ongoing regional interconnectedness rather than the influx of new genetic groups or non-local individuals.

Introduction
The Late Bronze Age (LBA, ca. 1300–800 BCE) in Central Europe was marked by profound cultural and social transformations, including long-distance trade and increasing interconnectedness, climatic and ideological shifts, and mobility1–4. A change in mortuary practices was also observed, with a transition from inhumation to cremation becoming the predominant practice. This transformation was so pronounced and widespread that it defined the period through its naming as the so-called Urnfield culture5 when cremated remains were placed in urns and buried in flat cemeteries. Based on recent archaeological findings, this practice is proposed to have originated in the middle Danube region, including areas of modern-day Lower Austria, Moravia, southwest Slovakia, and parts of Hungary, in the period between ca. 2000 and 1600 BCE, before spreading more widely throughout Europe and becoming consolidated in the classic Urnfield horizon after 1300 BCE6. This mortuary tradition significantly limits our ability to reconstruct the lifeways of people, such as their mobility patterns, diet, genetic ancestry and biological relatedness during this period, mainly due to the limitations of applying biomolecular methods to cremated remains7–10.

Ancient DNA (aDNA) has emerged as a critical tool for reconstructing human population histories and their relationship with cultural, economic, and linguistic changes in the past11–13. From an archaeogenetic perspective, the genomic profile of Bronze Age (BA) Central Europeans was largely established in the preceding 3rd millennium BCE, as the result of genetic mixing of Middle/Late Neolithic farming groups (carrying so-called Early European Farmer (EEF)-, and Western Hunter-Gatherer (WHG)-related ancestries) and pastoralist groups from Eastern Europe carrying steppe-related ancestry14–19, resulting in largely similar but regionally slightly different ancestry profiles. Specifically, for regions in today’s southern Germany, Switzerland, Austria and Hungary, a higher amount of EEF-related ancestry (and conversely a lower amount of steppe-related ancestry) has been documented relative to more northern regions such as Central Germany, Bohemia and Poland, already since the Bell Beaker and Early Bronze Age (EBA) periods16–18,20–22. In Bohemia, the shift from the EBA/Únětice to the Middle BA/Tumulus and LBA/Knovíz culture is also associated with an increase in EEF ancestry18, whereas such a shift was not observed in data from Poland associated with the Trzciniec culture21, whose roots lie largely in the forest zone of Europe.

Meanwhile, oxygen and strontium isotope analyses have been used to investigate mobility patterns23–25. For example, strontium evidence from Central Germany indicates that mobility was present in the EBA, with Únětice-period sites showing a mix of local and a smaller number of non-local individuals and indications of female exogamy26,27. While in southern Germany, high numbers of non-local individuals are reported, especially among female individuals, consistent with pronounced mobility and female exogamy20,28. The lack of such datasets for the LBA of much of Central Europe remains a temporal gap. Yet, despite the extent of cremation during the LBA, this practice was not universally adopted. For instance, the Unstrut group (ca. 1325-750 BCE), located between the Thuringian Forest and the Saale River in Central Germany and the focus of this study, continued to bury their dead for nearly 500 years, even as neighbouring groups, such as the Saalemündungs Group, transitioned to cremation29–31. Specifically, the mortuary practices of the Unstrut group are characterised by inhumation burials, where bodies are placed in a stretched supine position, oriented NW–SE, sometimes accompanied by stone packing, but also include a variety of other ways of burial, such as settlement or skull-only depositions31. Such practices align with broader patterns of LBA ‘special’ or ‘atypical’ burials (German: Sonderbestattungen) found across Central Europe, including Central Germany32, the Upper Tisza region33, Transylvania34, Moldova34, settlement burials of the Knovíz group in Czechia35, child burials within settlements at Şoimuş–Teleghi in southwestern Transylvania36, and settlement burials in Moravia, Austria, and South Germany37 among other examples38.

This study’s main focus lies on two archaeological sites in Saxony-Anhalt, Central Germany, associated with the Unstrut group: Esperstedt and Kuckenburg (Supplementary Note 1, Supplementary Fig. 1 and 12). Together, these sites form an extensive complex comprising a fortified settlement on a spur overlooking the Weida river (Kuckenburg)39–43, a second possibly fortified settlement (Esperstedt) at the opposite side of the Weida valley44, and a large bi-ritual graveyard45 situated just outside the ditch bordering the settlement to the west (Fig. 1a). For this study, we use a local, project-specific division of the LBA into two main phases: 1 (early) ca. 1300–1050 BCE and 2 (late) ca. 1050–800 BCE. In relative-chronological terms, phase 1 (early LBA) corresponds to the Central European phases BzD and HaA, whereas phase 2 (late LBA) corresponds to the Central European phase HaB46. This division is based mainly on the settlement and dietary history of the Esperstedt/Kuckenburg micro-region in the LBA47, which show, at the current state of research, marked changes at the turn from the 2nd to the 1st millennium BCE.

Mortuary practices at these two sites reflect the characteristic diversity of the northern Unstrut group, including burials in settlement pits and ditches, and the graveyard (Supplementary Figs. 1–28). While inhumation was the predominant practice, a few cremation burials have also been identified44. Additionally, the settlement of Kuckenburg includes skull-only depositions and multi-stage funerary rites39,40 (Supplementary Figs. 13–28). Both the way that individuals were laid to rest and the bioarchaeological and biomolecular study of their remains can inform us about important aspects of their lived and embodied experience, such as their diet, origins, mobility patterns, social organisation, socioeconomic status, kinship practices (biological or social), degree of social integration within the community, and even societal perceptions of identity, ideas of personhood, and construction and negotiation of social memory48–51. Hence, by analysing the individuals buried at Kuckenburg and Esperstedt, we aim to address gaps in our understanding of Central European LBA communities on site-specific and regional scales, especially as forms of settlement burials are widespread across Central Germany, Bohemia, and Moravia and therefore provide a broader comparative framework.

Given the general scarcity of inhumations during the Urnfield period, we generated, co-analysed and report new data from eleven additional LBA sites in today’s Bohemia/Czechia (n = 6), Southwest/Central Poland (n = 3), Thuringia/Germany (n = 1) and South Germany (n = 1) (Fig. 1b, Supplementary Notes 1.3 to 1.13, Supplementary Figs. 29-31). Leubingen in Thuringia is part of the main distribution area of the Unstrut group, whereas the sites in Bohemia and Southwest/Central Poland are attributed to the respective regional variants of the Urnfield cultural complex, such as the Knoviz and Štitary cultures in Bohemia or the Lusatian culture in Poland. Within the Urnfield complex, these represent a mosaic of regional groups differentiated by variations in burial practices, ceramic traditions, and material culture, but unified by parallel developmental trajectories and cultural traditions, including the predominance of cremation. The majority of the less common inhumations were found in settlement pits with very few or no grave goods. An exception is the LBA cemetery of Neckarsulm in South Germany, which has been assigned to the untermainisch-schwäbische Gruppe (German term), a local form of the Urnfield culture. This site is unique in that it represents a large necropolis of 32 inhumation graves, and in that it was interpreted archaeologically and anthropologically as the burial place of an all-male group. Previous interpretations have discussed whether these individuals represent retinues with a flat hierarchy or so-called warrior bands within a broadly segmentary social structure, while explicitly rejecting the concept of a hereditary warrior elite (Supplementary Note 1.4)1,52–54.

Taken together, adopting a primarily site-specific perspective (i.e., focusing on the sites of Esperstedt and Kuckenburg), we aim to characterise the genetic ancestry and mobility patterns of LBA individuals in Central Germany and assess their degree of continuity or change since the EBA in comparison with new and published data from contemporaneous groups from Central and South Germany, Czechia, and Poland (Fig. 1b). Specifically, we examine how factors such as age at death, genetic sex, socioeconomic status (using grave goods as a proxy), ancestry, mobility, cultural influences, and ideological shifts influenced mortuary choices in this region. Finally, we assess whether inhumation and cremation represented parallel burial traditions within the same community. We employ an interdisciplinary approach to address these questions by integrating aDNA, isotopic, and osteoarchaeological analyses. Through this approach, we aim to reach a more nuanced understanding of the lifeways of LBA communities and the motivation behind their choice of diverse mortuary practices.

Results
Genetic ancestry in the Late Bronze Age
We successfully retrieved aDNA from 75 LBA inhumations from Central Europe. All samples passed quality control, meeting criteria such as endogenous DNA content > 0.1% and characteristic aDNA damage patterns at the 5’ and 3’ ends of fragments55 in initial shotgun sequencing. After enrichment for 1.24 million single-nucleotide polymorphisms (SNPs)56, individuals (n = 69) with nuclear DNA contamination < 5%, mitochondrial DNA contamination < 10% and > 30,000 SNPs covered on the 1240k panel, were retained for downstream analysis. Genetic sex determination identified 32 female and 37 male individuals (see Methods section for details). For individuals with both genetic and osteoarchaeological sex assessments (n = 39), the results agreed in 85% of cases (Supplementary Data 1). We also called mitochondrial (mt) haplotypes (> 3000 mt reads) and Y-chromosome haplogroups for the individuals with sufficient coverage. We observed 51 different mt haplotypes in 64 individuals with enough mt reads for reliable mt haplogroup calling, broadly resembling the mt haplogroup distribution in Bronze Age Central Europe (ref. 17, 57). The majority of male individuals carried either Y haplogroup I2a1 (n = 11) or R1b1 (n = 21), with two individuals carrying R1a (LBP001, NCE005), one individual (KUC023, Supplementary Note 2.4) carrying I1a3b2 and another (LEU020) I2a2a2 (Supplementary Data 1).

We conducted a Principal Component Analysis (PCA) on modern West Eurasian populations, on which we projected the newly analysed and published ancient individuals of interest (Fig. 2a), as well as ADMIXTURE analysis (Supplementary Note 2.1, Supplementary Fig. 32) and a series of f4-statistics (Supplementary Data 3), to genetically characterise the new LBA individuals and to assess their affinity to preceding EBA/Middle Bronze Age (MBA) populations (see Supplementary Data 2a and 2b for a list of populations used in this study), to nearby regional populations, and potential genetic variation linked to different mortuary practices (excluding cremated individuals). Specifically, the f4-statistics of the form f4(outgroup, X; Y, Z) use Old Africa as an outgroup, while X represents Early European Farmer (EEF-Balkan_N, Turkey_N), Western European hunter-gatherers (WHG-WHGA and WHGB), or steppe-related ancestry (OldSteppe) (as defined in Patterson et al. 18 and shown in Supplementary Data 2b, column Group ID_published), and Y and Z are the test populations of interest to assess changes in allele sharing in regard to the ancestry source X over time (see Methods for details). Positive f4-values indicate that population Z shares more alleles with X than population Y, while negative values indicate that population Y shares more alleles with X than population Z. Values near zero suggest no significant difference in allele sharing with X between Y and Z.

In PCA space, early LBA individuals from Central Germany plot within the genetic variation of published EBA individuals associated with the Únětice culture, suggesting regional genetic continuity27,56. This observation is supported by f4-statistics values close to zero (f4(Old Africa, EEF; Germany_EBA_Unetice, Central_Germany_early_LBA) = 0.000345, Z = 2.659, n = 1,072,545 SNPs), which indicates no significant difference in allele sharing between the EBA individuals and the early LBA individuals from Central Germany (Supplementary Data 3, Test 1). These individuals also overlap with the only published LBA genome from Central Germany, from the archaeological site of Halberstadt56. However, most late LBA individuals from Central Germany are shifted slightly to a lower position on PC2, suggesting an increase in EEF-related ancestry and a reduction in steppe-related ancestry, which is confirmed by positive f4-statistics of the form f4(Old Africa, EEF; Germany_EBA_Unetice, Central_Germany_late_LBA) = 0.000677, Z = 7.174, n = 1,106,172 SNPs (Supplementary Data 3, Test 2). A few of these individuals (e.g., KUC022) also overlap with individuals from the Tollense battlefield, in northern Germany58, as well as published and newly generated individuals from the LBA Knovíz culture in Czechia18 (Fig. 2a). KUC022 is a genetic outlier according to f4(Old Africa, EEF; KUC022, Central_Germany_late_LBA) = -0.000829, Z = -3.078, n = 624,616 SNPs (Supplementary Data 3, Test 3). Due to the absence of aDNA data from the MBA in Central Germany, direct comparisons with the immediately preceding centuries are not possible.

For comparison, we also analysed 12 individuals from the unique LBA cemetery of Neckarsulm (NES) in South Germany (Supplementary Note 1.4)54,59. Genetic sex determination confirmed osteoarchaeological assignments, supporting the initial assessment that all 12 individuals were male59. A comparison of the South and Central Germany individuals in PCA space showed distinct genetic profiles: NES individuals occupy a lower position on PC2 (Fig. 2a), suggesting higher EEF-related ancestry and a difference in ancestry between the two regions, confirmed by f4(Old Africa, EEF; Central_Germany_LBA, South_Germany_LBA) = 0.000887, Z = 6.638, n = 1,040,306 SNPs (Supplementary Data 3, Test 4). NES individuals also appear to carry more EEF-related ancestry than the local EBA populations in South Germany16,20, which is confirmed by positive f4-statistics of the form f4(Old Africa, EEF; South_Germany_EBA, South_Germany_LBA) = 0.000677, Z = 4.562, n = 1,054,321 SNPs (Supplementary Data 3, Test 5).

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript offers new genetic and isotopic analyses of ca. 70 LBA Central European inhumations in order to reconstruct population and mobility at the end of the second and start of the first millennia BC – a period when the dominant funerary rite was cremation, limiting our ability to conduct some biomolecular research. As such, it fills a major knowledge gap in our understanding of the population history of later prehistoric Europe. It is also innovative in its extremely thoughtful integration of genetics, isotopic data, and mortuary archaeology. I also want to note that the entire text is exceptionally well written with a crystal clear methods section and a really thoughtful discussion. I commend the authors on work of this quality. The figures are generally clear though sometimes difficult to read due to the lower resolution of reviewer pdfs. The supplementary material are complete, easily navigable and largely helpful. I was surprised not to see a discussion of the isotopic work in the supplementary material – I think this would be welcome.\n\nAll my specific comments are minor and just to help tweak and clarify the text\nAbstract\nLn 54-56 “However, reconstructing the demographic and social impacts of these changes has been hindered by cremation being the dominant mortuary practice, limiting biomolecular approaches.” – this is a nitpick, but the dominant rite being cremation does not hinder our reconstruction of the *social impacts* of mobility and transformation – we can do that just fine without biomolecular data\nManuscript\nLn 81 “buried in fields” – change fields to “flat cemeteries”\nLn 99 “accompanied by stone packs” – unclear phrasing. Does this mean “stone packing”?\nLn 122 “outside the second settlement's “gates”” – perhaps “outside the entry to the second settlement”? ‘Gates’ in scare-quotes here is confusing, especially as the figure referenced does not show much detail of either enclosure\nLn 304-05 “without being able to locate the allochthonous source concretely” – this seems in fact entirely expected within the scenario outlined by the authors of regular small-medium scale mobility and more intensive connectivity. Instead of a single or major pt A   pt B geneflow event, people intermingle and become mixed with shifting patterns of ancestry resulting rather than one or more ‘sources’ of this ancestry. I’d encourage the authors to trust their data and perhaps think away from ‘origins’ to the social practices and relationships underlying their data (remembering too that all of the ‘populations’ that are being modelled in these admixture models are statistical entities not bounded, isolated groups of self-identifying people).\nLn 525 “those receiving formal burial” – are you including inhumed and cremated interments here or just inhumations? There’s a bit of slippage in this section where it’s not always clear whether you’re addressing the totality of the burial community, or the group of inhumations assessed in this manuscript.\nLn 534-36 “Despite the local adherence to inhumation practices, which might initially suggest a conservative cultural stance, the Unstrut group was connected to neighbouring, predominantly southern LBA groups” – the sentence implies that (a) retention of an older burial rite is conservative (a word with strong implications of unchanging), (b) that conservative people are not in contact with other communities – neither of these is true. Traditions and old ways are enacted because they are valuable in the present – many traditions are themselves invented or reinvented or reinvested with new meaning to address dynamic presents. Moreover, there is no reason to assume that a group with deeper rooted or intensely local funerary rites is not in touch with neighbours or that being in touch with neighbours (even having children with them) will necessarily lead to changes in burial rite or the adoption of the outside burial rite. Underlying this sentence is an unwarranted (and probably unconscious) evolutionary logic that does not really survive being closely examined. I’d suggest a rethink or a rephrase. Maybe to something like “Even as the Unstrut group developed and maintained connections to neighbouring, predominantly southern LBA groups, they continued to practice inhumation burial, a rite with a deep history in this part of central Germany”\nThe authors might find the discussion of parallel, distinct Neolithic Irish mortuary rites in Smythe et al (2025 - https://doi.org/10.1017/S0959774325000058) of interest.\nLn 549 – typo (two commas)\nReferences\n#34: this is a chapter in The Oxford Handbook of the European Bronze Age, not a book or report as it’s presently cited\nFigures\nFig 3b: I think the authors might experiment a bit with this image – at present, though they note in the text that data are missing from some periods, it’s not visually obvious that this is the case. To make the four graphs more coherent amongst each other, I think it might be useful for them to have all the same X axis, even if some categorise (e.g. MBA Central Europe) n=z and the space is empty.\nThere is I think a missing figures relating to the Sr data: A broader Sr basemap so that your local values can be visually put in context with the wider region what proximity to these sites falls within the local range (e.g. if most people are travelling and living their lives on the local geology, is that within a 15 km range, a 50km range, a 200 km range?). It would be useful to indicate as well where you collected each of your baseline samples for your tightly defined local area.\nSupplementary material\nSM part 1: I would have liked a couple of maps to locate the cemeteries more precisely and then locate the graves within them. The photographs are extremely welcome, but a diagram of their placement would be very helpful for future researchers.\nSupplementary tables: a contents tab that summarises what is in each of the sheets would be welcome but is optional\nOverall, I very much enjoyed reading this manuscript! Catherine Frieman", "reviewer_2": "The attachment contains a formatted version of these comments.\nReviewer’s comments on “Ancient DNA, isotope and osteoarchaeological 1 analyses inform on lifeways of Late Bronze Age communities in Central Europe”\nJournal questions\nWhat are the noteworthy results?\nThe paper presents results from period and location that is otherwise poorly represented because of the destruction of genetic material by the dominant cremation rite during the Urnfield culture. It demonstrates that cultural changes were accompanied by gradual changes in ancestry.\nWill the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nThis is a significant addition to the literature on the genetics of late bronze age Europe. It also integrates isotopic and cultural data to an extent that has previously been unusual in the ancient DNA literature.\nDoes the work support the conclusions and claims, or is additional evidence needed?\nI consider that the data and analysis supports the conclusions, though some minor aspects of methods and results need improved presentation.\nAre there any flaws in the data analysis, interpretation and conclusions? - Do these prohibit publication or require revision?\nThere is nothing here that I consider prohibits publication, but see the detailed comments below on some deficiencies in the analysis, and caveats on interpretation.\nIs the methodology sound? Does the work meet the expected standards in your field?\nYes, the overall approach is sound and uses established methods. The methods meet expected standards, as does most of the data reporting, but see below for some omissions.\nIs there enough detail provided in the methods for the work to be reproduced?\nFor the most part. I am not expert in the genetic methods but they are presented at the same level of detail I have seen in other papers and I am able to follow how the results were produced. Most of the isotopic and dating methods are presented fully enough, but there are some missing (see below). Statistical methods are adequately presented.\nDetailed comments\nLines 200-238 use f4 statistics of the form f4(OldAfrica, EEF, X, Y) and cite supplementary table 3, but comparisons with EEF as population2 do not appear in that table.\nThe tests used are “either a non-parametric Kruskal-Wallis test or a parametric ANOVA, depending on the data distribution” (lines 275-276 and 1013-1014). This may lead to incoherent results as K-W comparisons are based on medians and ANOVA on means. It would be better to use the same test throughout. Figure 3b shows one sample size is small (n=11) for the ANOVA, which makes deviations from normality hard to detect and suggests the use of K-W. However, the p-values here are so low this is unlikely to change the conclusions. What is the meaning of one, two and three asterisks?\nLines 365-374: When reporting ROH the units should be given, presumably cM. Supp Table 7 shows that KNE028 and KUC027 also have sum_roh>4 over 50, and the former has sum_roh greater than KUC010 in all ranges, but they are not discussed here. Why?\nMissing data and/or methods. The methods indicate carbon and oxygen isotope analyses were performed using enamel carbonate (lines 992-1009), but Supp Table 8 only reports δ18O and not δ¹³C. Figure 6d shows δ¹³C collagen results, but stable isotope measurements on collagen are not described in the methods, which only include collagen preparation and measurement for radiocarbon. I could not find the δ¹³C data plotted in figure 6d in the supplementary information (“13C” appears in the file only in Supp Table 12), though millet consumption yes/no is in Supp Table 1. Statistical analysis of both collagen and enamel δ¹³C data is reported (lines 480-481 and Supp Table 12). Does some of this data come from Wahl & Price (2013)? Please could δ¹³C carbonate, δ¹³C collagen and δ¹⁵N collagen be added to supp Table 8?\nLocal oxygen isotope range. Lines 384-385: one cannot define a human local δ18O baseline from animals as there is “marked species specificity in the relationship between drinking water and body water” (ref. 16) with a strong effect of body mass, making hamsters particularly unsuitable. The construction of this baseline is not described in the methods. The range of faunal values in Supp Table 8 is -8.3 to -5.6 ‰, but the range in Figure 6c is greater than this, from below -9 to above -5 ‰. Without data to indicate the local range, I think the best approach is to look for outliers. Here none of the values are more than 1.5xIQR from the quartiles, so no outliers are detected.\nLine 418: C3 plants would be ubiquitous on all local geological formations, not found on different ones to millet, but millet may have been restricted to certain geological formations.\nLines 688-689: It would be helpful if Supp Table 1 indicated which are the new dates and which are from Orfanou et al (2024).\nLine 925 “when possible, we sampled the enamel from both first and third molars” This does not seem to have been done. Supp Table 8 shows only one individual with two teeth sampled, and those were M2 and M3. Overall M2 is the most frequently sampled tooth.\nLine 927 “δ18O values from M1 should be interpreted with caution as they can be influenced by breastfeeding” This is true. These samples need to be marked when plotted in figure 6, and perhaps omitted from the outlier analysis I suggested above.\nLines 990-1009: the reference points (VPDB or VSMOW) for the δ values should be stated at least once.\nSupp Table 6 IBD: the units, presumably cM, should be given.\nSupplementary Text\nLine 83 “Dating: LBA (MAMS-57256: 941 ± 41 BC cal (Cal 1-sigma))”\nLine 241 “Dating: 14C: cal BC 894–830 (Cal 1-sigma), cal BC 902–819 (Cal 2-sigma).”\nLine 921 “Individual A - MAMS-52806: 3044±25 BP”\nThe first two (and many others) are not properly reported radiocarbon dates. The third (like others in pages 47 to 49 of the supplement) is inconsistent in not supplying calendar dates. The calibrated result should be expressed as a range with a probability; following Supp Table 1 for line 83 this would be 1006-840 BC (95.4%). According to the methods “All calibrated dates are reported in the text as 95.4% probability ranges” and this should also be the convention for every date in the supplementary text. The laboratory code for the date should always be included.\nWhere there are multiple individuals in a grave it would be useful to know which of them the radiocarbon date comes from. Where there are multiple dates, the individuals should be identified with the relevant date (e.g. lines 498-504)\nLine 113: “1173,5 ± 47,5” this is spurious precision – the calibration data is at 5 year intervals and dates cannot be determined to a fraction of a year. Round to the whole year.\nMany descriptions contain mentions of profiles A-B, C-D and E-F, but these are not marked on the images and are therefore not meaningful to the reader.\nSupplementary Figure 3: what do the colours in this figure indicate?\nMinor changes\nFigure 4 I can’t see any greyed out lines here. Are these non-working models the lighter colours?\nLine 549 delete extra comma\nLine 687 “95.4% (2σ) probability ranges” – delete “2σ” these are not standard deviations.\nLines 980: ±13 should be ±0.000013\nLine 995: NaCIO should be NaClO (lower case l not uppercase I)\nLine 1000: CO2 should be CO2\nLine 997: liophilized should be lyophilized\nSupplementary throughout:\nuse point not comma for a decimal;\nuse raised “” not lowered „“\nStandardise age group names: choose one of juvenil, juvenile or juvenis; matur, mature or maturus; adult or adultus.\nSupp Line 355: “right stool” – I don’t know what this means\nSupp Lines 498-504: remove ‘mit’", "reviewer_3": "The paper by Eleftheria Orfanou and contributors aimed to address aDNA, provenance isotope analysis, combined with osteoarchaeological analysis, to understand the lifeways of populations from central Europe during the Late Bronze Age. Unfortunately, as I am unable to comment on some of the core aspects of the paper, such as genetic and isotope analysis, this review will focus on the osteoarchaeology/ palaeopathology/ funerary aspects of the paper.\nContent and interpretation\n-Lines 455-457: the authors interpret the lack of biological relationships between the individuals buried within a complex mortuary feature (39/2018) as resulting from cultural or ritual criteria instead of biological relatedness. Now, can some of the explanations be attributed to ‘opportunistic or convenient burial reasons’? For instance, it would not be irrational to think that if both individuals displayed perimortem trauma that resulted from a fatal event occurring in close temporality to one another, wouldn’t it be convenient to bury both (genetically unrelated) individuals within the same grave? For this reason, I suggest that, in addition to ‘cultural or ritual criteria’, the idea of logically opportunistic or convenient burial should be considered as an explanation.\n-Lines 497-498: stated that ‘these inhumations also provided us with a rare opportunity to assess the physiological stress and trauma of the people in LBA central Germany. You commence the descriptions, indicating that ‘degenerative diseases were most common in older individuals’ now, do you consider joint diseases to be physiologically related or trauma-related? Unless you believe that joint diseases represent ‘trauma to the joints’, I think that instead of referring to ‘physiological stress and trauma’, it would be better suited to inform the audience that you looked at palaeopathological lesions/conditions (or just the palaeopathology of the populations). These will include joint diseases, trauma, and metabolic conditions (markers of non-specific physiological stress). Also, in Line 505, you mentioned tuberculosis, which is neither a form of trauma nor a stress marker; therefore, be careful when stating that you observed the evidence for trauma and physiological stress because your analysis went beyond that and included more types of pathologies. For this reason, I suggest you use the term or talk about ‘pathologies or palaeopathologies in general’\n-Line 503: replace ‘stress markers’, which is a broad term, with ‘Non-specific marker of physiological stress’, which is the common term used to describe cribra orbitalia, porotic hyperostosis and dental enamel hypoplasia. Replace ‘stress markers’ everywhere else in the paper when you are referring to these markers and use the one suggested above.\n-Line 505: what do you mean by ‘spinal tuberculosis’? If you only found periosteal reactions (new bone formation on visceral aspects of ribs) without the pathognomonic signs of destructive lesions in the spine, then you cannot be certain it was TB, just a non-specific infection.\n-Lines 521-525: the summary of the palaeopathological evidence (or the discussion, Lines 610-649) could have included some interpretations on the dental disease. If you were interested in the lifeways of these populations… What were the health implications associated with the findings of Streptococcus mutans and Parvimonas?\nFinally, was the interpretation of body positions out of the scope of analysis? Some individuals were buried prone, which, if analysed in conjunction with isotope analysis and grave goods, could have funerary implications that could contribute to addressing the lifeways of these populations.\nTypos and grammar:\n-Line 549: delete one comma\n-Line 676: change ‘was’ with ‘were’\n-Lines 1035-1036: accession number and URL link not provided", "reviewer_4": "Orfanou and colleagues present a genomic, isotopic, and osteoarchaeological analysis of rare inhumation burials from populations in Central Europe associated with the Urnfield culture, during the Late Bronze Age (LBA). The focus of the manuscript is site-specific and regional, targeting a local population present at the archaeological sites of Kuckenburg and Esperstedt in Central Germany, but they supplement with novel data derived from contemporaneous ancient individuals from South Germany, Bohemia (Czechia) and southeastern Poland. They expand the genomic analyses further with previously published contemporary and ancient human datasets. They conclude for the local Kuckenburg and Esperstedt population that there were no associations between mortuary practice and genetic sex, relatedness, geographic origin, or inferred socioeconomic status. The community showed cultural flexibility, with inhumation vs cremation behavior possibly negotiated with fluctuating ideas of identity based on external influences, as regional interconnectedness grew. This local and more nuanced view of an ancient community is commendable, the paper is generally well written and clear, and I see no deep methodological faults.\nThere are improvements that could be made to strengthen the manuscript. I will discuss these generally, and then provide more detailed line edits below.\nThe first concerns the incorporation of the new genomic data from South Germany, Czechia, and Poland. There is no background information provided for these individuals in the Introduction, and they are skipped completely some section (e.g. Mortuary practices and physiological stress), which makes them feel ‘tacked-on’ to the project (I am aware the burial details are in the supplemental). Apart from these individuals being contemporaneous in age and (apparently) associated with the Urnfield Culture, the reader is left unclear why these are being compared to the Unstrut group; are they the only other Urnfield populations sampled (i.e. the AADR lacks representatives for this culture, given the prevalence of cremation)? Where do these populations fit into the question presented in the introduction, of cultural and biological continuity vs change, in Central Europe, during the Bronze Age? As a more detailed example, should we have expected different ratios of EEF, Steppe, and WHG than what is estimated with the qpAdm results in Figure 2b? Are those results very surprising, from an archaeological/cultural standpoint?\nThe second concerns the continued theme throughout the manuscript of fluctuating Early European Farmer (EEF) related ancestry. There doesn’t seem to be a consistent story told about population continuity vs admixture, but I’m sure a few sentences would clear it up. Some examples: The paper highlights a fairly consistent increase in EEF ancestry over time in all regions (Figure 3), but the qpAdm modeling suggests population continuity from EBA to early LBA (Central Germany) or continuity from MBA to LBA (Bohemia and Poland). In the strontium analyses, everyone seems local, regardless of time depth, genetic sex, or mortuary practice – where in the increase in EEF ancestry coming from? In the dietary analyses, there does seem to be a correlation with a return to C3 plants and EEF-related ancestry, in the late LBA, but not with the earlier switch to millet. The authors then bounce between suggesting a change in ancestry with the former, but not one with the latter. Settlement contexts is another example.\nThe authors could expand the mtDNA and NRY analyses and interpretations. The continental spread and phylogenetic history of the haplotypes typed are not discussed, even in passing. Are any of them surprising (the two CT individuals?)? All expected? How about the drastically increased mitochondrial diversity? KUC023 has a unique NRY haplotype and an elevated Sr ratio, but is not apparently a genetic outlier. What might be going on there?\nSome quick comments on the methods:\nWas aDNA extraction attempted on any of the bones from the cremated remains? Are the authors just not reporting libraries at all that didn’t pass internal endogenous DNA scan requirements? They had the bones for the isotopic work.\nIn your biological relatedness analyses, KIN (https://doi.org/10.1186/s13059-023-02847-7) seems to be growing in popularity and perhaps should be attempted here? Furthermore, is the limited relatedness detected at Kuckenburg and Esperstedt to be expected? The authors could expound on this more.\nSpecific Comments:\nPage 3, lines 81-83: Give a date range here?\nPage 5, lines 139-141: Are the \"Sonderbestattungen\" burials the only unique archaeological/cultural aspect of the Kuckenburg and Esperstedt settlements? Would the results presented here be applicable to the other non-cremation sites mentioned in Paragraph 2 of the introduction? How broadly applicable would these analyses be to Bronze Age central European communities, or do the authors want the takeaway to be a hyper site-specific perspective?\nPage 5, lines 162-164: I can follow why NES005 was thrown away, but I'm not following why NES006 wasn't - high contamination with angsd and hapcon, but low with ContamMix. Have you considered you might have sex chromosomal aneuploidies? Could check with karyo_RxRy? Also, if I'm following your column BW in Sup Table 1, you only threw out 2 individuals (though NES006 is in red), which seems to leave 72 individuals for other analyses; not 70. Are there duplicates? Column H has 4 \"same\" values and maybe that is where the difference is. If so, mention 2 duplicates were found in 4 analyses.\nPage 7, lines 181-182: Which individuals (genomic data) are the authors using to define these three groups in their analyses? I can’t find those group IDs in any of the supplementary tables. If they are consistent samples that everyone uses, define and give appropriate references.\nPage 7, lines 183-187: This belongs more in the Methods section, than in Results\nPage 7, lines 194-196 (Figure 2b Legend): An expanded introduction that includes the S. Germany, Czechia, and Poland individuals would help give us more perspective of how we should be interpreting the qpAdm models in comparison to the Unstrut population.\nPage 8, lines 219-220: A description of what makes this cemetery unique likely belongs in the introduction.\nPage 8, line 240: Edit to “…generated, as well as published, LBA…”\nPage 9, lines 256-258: I don't understand where the support for this sentence comes from in the preceding paragraph, as I don't see any graphics or data in Supp Table 3 showing any early bronze age comparisons?\nPage 9, lines 260-269: Why are the individuals from Poland, who seem to have the lowest proportion of EEF-related ancestry, not discussed in this paragraph?\nPage 9, lines 271-274: Can you be more clear where you are getting the proportions used for Figure 3a/b, because they don't seem to exactly match the values from qpAdm listed in Figure 2b (for the LBA individuals, at least). This can be put in the text or in the figure legend.\nPages 12-13, lines 333-342: Also discuss LNV001 and SNY001, who are highlighted as outliers in this manuscript, in the PCA, and in Figure 5 below.\nPage 14, lines 365-374: Why are KNE028, KUC027 not being discussed in the hapROH analysis? Do they not also have elevated levels? See Supplemental Figure 3.\nPage 14, lines 386-396: None of the genetic outliers appear non-local in the isotopic analyses. Can the authors discuss/highlight this?\nPage 15, line 406: How as millet consumption determined? I see the column in Sup Table 1, but not a description.\nPage 21, line 571: By “this dietary shift” I assume the authors are referring to the return C3 plants, but please clarify.\nPage 27, line 730: The 1240k capture needs references; I assume you aren’t using the new Twist kit.\nPage 27, lines 733-736: Both the mtDNA and NRY capture protocols/kits need references.\nPage 30, line 804: I assume these genotypes are from the preceding ATLAS calls? Please be more specific in the \"Population Genetic Analyses\" when you used the pseudo-haploid calls and when you used the ATLAS genotypes.\nPage 30, line 807: spelling “pubblication”\nPage 31, lines 831-833: Was the HO dataset *only* used for the PCA and Admixture analyses? If nothing else, say that. I would also state the limited HO dataset was necessary to include many of the contemporary populations.\nPage 31, lines 853: Here and elsewhere, when you bring up population group labels like this, reference your Supplementary Table 2b.\nPage 31, lines 856-857: As already previously mentioned, we are never given who are included in these groups.\nPage 32, lines 867-872: Are these labels from the AADR or from labels in Sup Table 2b? I don’t see some of them in the supplementary tables.\nPage 37, lines 1031-1032: I applaud the authors for including this level of detail for their statistical tests."}, "author_response": "Point-by-point response to the and Reviewers\nReviewer 1\nThis manuscript offers new genetic and isotopic analyses of ca. 70 LBA\nCentral European inhumations in order to reconstruct population and mobility at the end\nof the second and start of the first millennia BC – a period when the dominant\nfunerary rite was cremation, limiting our ability to conduct some biomolecular\nresearch. As such, it fills a major knowledge gap in our understanding of the\npopulation history of later prehistoric Europe. It is also innovative in its extremely\nthoughtful integration of genetics, isotopic data, and mortuary archaeology. I also\nwant to note that the entire text is exceptionally well written with a crystal clear\nmethods section and a really thoughtful discussion. I commend the authors on\nwork of this quality. The figures are generally clear though sometimes difficult to\nread due to the lower resolution of reviewer pdfs. The supplementary material are\ncomplete, easily navigable and largely helpful.\nWe sincerely thank Reviewer 1 for the positive and constructive evaluation of our\nmanuscript. Regarding the embedded figures, we have ensured that the final figures are\nprovided at a higher level of resolution.\nI was surprised not to see a discussion of the isotopic work in the supplementary\nmaterial – I think this would be welcome.\nSupplementary Note 3 should have been named isotope analyses instead of statistical\nanalyses, since it contains statistical analyses of isotope data. This has now been\ncorrected. Furthermore, we have now expanded this section with a paragraph regarding\n1\nδ18O outlier detection (1.5×IQR method) in human enamel (Supplementary Note 3.3)\nand a section on the regional 87Sr/86Sr isoscape context (Supplementary Note 3.2).\nAbstract\nLn 54-56 “However, reconstructing the demographic and social impacts of these\nchanges has been hindered by cremation being the dominant mortuary practice,\nlimiting biomolecular approaches.” – this is a nitpick, but the dominant rite being\ncremation does not hinder our reconstruction of the *social impacts* of mobility\nand transformation – we can do that just fine without biomolecular data.\nWe agree that the dominance of cremation does not hinder archaeological\nreconstructions of social impact. We have therefore revised the sentence to refer\nspecifically to the demographic aspects accessible through biomolecular methods. The\nsentence now reads: “However, reconstructing the demographic aspects of these\nchanges has been hindered by cremation being the dominant mortuary practice,\nlimiting biomolecular approaches.”\nManuscript\nLn 81 “buried in fields” – change fields to “flat cemeteries”\nWe have changed this in the text.\nLn 99 “accompanied by stone packs” – unclear phrasing. Does this mean “stone\npacking”?\nWe have changed this in the text.\nLn 122 “outside the second settlement's 'gates'” – perhaps “outside the entry to the\nsecond settlement”? ‘Gates’ in scare-quotes here is confusing, especially as the\nfigure referenced does not show much detail of either enclosure.\nWe have replaced “outside the second settlement’s ‘gates’” with “outside the ditch\nbordering the settlement to the west” to improve clarity and avoid potential confusion.\nLn 304-05 “without being able to locate the allochthonous source concretely” – this\nseems in fact entirely expected within the scenario outlined by the authors of\nregular small-medium scale mobility and more intensive connectivity. Instead of a\nsingle or major pt A pt B geneflow event, people intermingle and become mixed\nwith shifting patterns of ancestry resulting rather than one or more ‘sources’ of\nthis ancestry. I’d encourage the authors to trust their data and perhaps think away\nfrom ‘origins’ to the social practices and relationships underlying their data\n(remembering too that all of the ‘populations’ that are being modelled in these\nadmixture models are statistical entities not bounded, isolated groups of self-\nidentifying people).\nand connectivity rather than discrete population movements. We have revised the\nphrasing to avoid implying a single allochthonous source, replacing “without being able\nto locate the allochthonous source concretely” with “without being able to be more\nspecific”.\nLn 525 “those receiving formal burial” – are you including inhumed and cremated\ninterments here or just inhumations? There’s a bit of slippage in this section where\nit’s not always clear whether you’re addressing the totality of the burial\ncommunity, or the group of inhumations assessed in this manuscript.\nIn this section, we are indeed referring to the group of inhumations assessed in this\nstudy. We have revised the sentence to read: “at least for the inhumed individuals\nreceiving formal burial” to make this distinction clearer.\nLn 534-36 “Despite the local adherence to inhumation practices, which might\ninitially suggest a conservative cultural stance, the Unstrut group was connected to\nneighbouring, predominantly southern LBA groups” – the sentence implies that\n(a) retention of an older burial rite is conservative (a word with strong implications\nof unchanging), (b) that conservative people are not in contact with other\ncommunities – neither of these is true. Traditions and old ways are enacted\nbecause they are valuable in the present – many traditions are themselves invented\nor reinvented or reinvested with new meaning to address dynamic presents.\nMoreover, there is no reason to assume that a group with deeper rooted or\nintensely local funerary rites is not in touch with neighbours or that being in touch\nwith neighbours (even having children with them) will necessarily lead to changes\nin burial rite or the adoption of the outside burial rite. Underlying this sentence is\nan unwarranted (and probably unconscious) evolutionary logic that does not really\nsurvive being closely examined. I’d suggest a rethink or a rephrase. Maybe to\nsomething like “Even as the Unstrut group developed and maintained connections\nto neighbouring, predominantly southern LBA groups, they continued to practice\ninhumation burial, a rite with a deep history in this part of central Germany” The\nauthors might find the discussion of parallel, distinct Neolithic Irish mortuary rites\nin Smythe et al (2025 - https://doi.org/10.1017/S0959774325000058) of interest.\nThank you for this comment. We agree that our original phrasing unintentionally implied\nan evolutionary logic that is neither accurate nor theoretically appropriate. We have now\nrevised the sentence, as suggested, to avoid these implications. The text now reads: “Even\nas the Unstrut group developed and maintained connections to neighbouring,\npredominantly southern LBA groups, as evidenced also by the continuous increase in EEF-\nlike ancestry, they continued to practice inhumation burial, a rite with a deep history in\nthis part of Central Germany.” Thank you as well for directing us to Carlin N, Smyth J,\nFrieman CJ, et al (2025). We found their interpretive framework very helpful and\n3\ninteresting, and we now cite this work in our discussion when addressing limited biological\nrelatedness in prehistoric mortuary contexts.\nLn 549 – typo (two commas) Done.\nReferences\n#34: This is a chapter in The Oxford Handbook of the European Bronze Age, not a\nbook or report as it’s presently cited\nThe reference has been corrected to indicate that this work is a chapter within The\nOxford Handbook of the European Bronze Age.\nFigures\nFig 3b: I think the authors might experiment a bit with this image – at present,\nthough they note in the text that data are missing from some periods, it’s not\nvisually obvious that this is the case. To make the four graphs more coherent\namongst each other, I think it might be useful for them to have all the same X axis,\neven if some categories (e.g. MBA Central Europe) n=z and the space is empty.\nWe have updated Figure 3b accordingly.\nThere is I think a missing figures relating to the Sr data: A broader Sr basemap so\nthat your local values can be visually put in context with the wider region what\nproximity to these sites falls within the local range (e.g. if most people are\ntravelling and living their lives on the local geology, is that within a 15 km range, a\n50km range, a 200 km range?). It would be useful to indicate as well where you\ncollected each of your baseline samples for your tightly defined local area.\nWe agree that it is very useful to visualise where our sites are located in relation to the\nsurrounding strontium isotope values. For this reason, we have now included, in the\nSupplementary Text 3.2, a map showing the modelled bioavailable 87Sr/86Sr isoscape for\nCentral Europe, adapted from Bataille et al. (2020). On this map, we indicate the\nlocations of our sites (Kuckenburg and Esperstedt), from which the human and faunal\nindividuals used for the analyses were collected, as well as the site of Neckarsulm, for\nwhich published strontium isotope data are available. This addition will allow readers to\nbroadly situate our measured values within the wider regional context. We also note, as\ndiscussed in the Supplementary Text 3.2, that equifinality (i.e., the occurrence of similar\n87Sr/86Sr ratios in geologically distinct regions) must be considered when interpreting\nsuch data. A new figure illustrating this information has been added as Supplementary\nFigure 7a and b. We have also added a sentence in the main text directing the reader to\nthe Supplementary Text, lines that reads as “We additionally provide a modelled\nbioavailable 87Sr/86Sr isoscape for Central Europe (Supplementary Text 3.2,\nSupplementary Fig. 7a,b), adapted from Bataille et al. (2020), to place these values\nwithin a broader regional context.”\nSM part 1: I would have liked a couple of maps to locate the cemeteries more\nprecisely and then locate the graves within them. The photographs are extremely\nwelcome, but a diagram of their placement would be very helpful for future\nresearchers.\nAn overview of all archaeological sites, including Kuckenburg and Esperstedt, is\nalready provided in Figure 1. However, we agree that a more detailed map showing the\nprecise locations of the burials is necessary. For this reason, we have now added\ndetailed maps of the location of the Kuckenburg (Supplementary Fig.2) and Esperstedt\n(Supplementary Fig.1) burials in Supplementary Note 1, under the respective site\ndescriptions, as these two sites form the core of our study.\nSupplementary tables: a contents tab that summarises what is in each of the sheets\nwould be welcome but is optional.\nWe have now added a contents tab at the beginning of the Supplementary Tables file,\nsummarising the content of each sheet.\nOverall, I very much enjoyed reading this manuscript!\nThank you!\nThe paper presents results from period and location that is otherwise poorly\nrepresented because of the destruction of genetic material by the dominant\ncremation rite during the Urnfield culture. It demonstrates that cultural changes\nwere accompanied by gradual changes in ancestry. This is a significant addition to\nthe literature on the genetics of late bronze age Europe. It also integrates isotopic\nand cultural data to an extent that has previously been unusual in the ancient DNA\nliterature. I consider that the data and analysis supports the conclusions, though\nsome minor aspects of methods and results need improved presentation. There is\nnothing here that I consider prohibits publication, but see the detailed comments\nbelow on some deficiencies in the analysis, and caveats on interpretation. Yes, the\noverall approach is sound and uses established methods. The methods meet\nexpected standards, as does most of the data reporting, but see below for some\nomissions. For the most part. I am not expert in the genetic methods but they are\npresented at the same level of detail I have seen in other papers and I am able to\nfollow how the results were produced. Most of the isotopic and dating methods are\npresented fully enough, but there are some missing (see below). Statistical methods\nare adequately presented.\nWe sincerely thank Reviewer 2 for their positive assessment of our study.\nDetailed comments\nLines 200-238 use f4 statistics of the form f4(OldAfrica, EEF, X, Y) and cite\nsupplementary table 3, but comparisons with EEF as population2 do not appear in\nthat table.\nWe thank the reviewer for pointing out this omission. The comparisons with EEF were\nindeed included in the analyses, but the label was missing from Supplementary Table 3.\nWe have now added the EEF label to the table to clarify this.\nThe tests used are “either a non-parametric Kruskal-Wallis test or a parametric\nANOVA, depending on the data distribution” (lines 275-276 and 1013-1014). This\nmay lead to incoherent results as K-W comparisons are based on medians and\nANOVA on means. It would be better to use the same test throughout. Figure 3b\nshows one sample size is small (n=11) for the ANOVA, which makes deviations\nfrom normality hard to detect and suggests the use of K-W. However, the p-values\nhere are so low this is unlikely to change the conclusions. What is the meaning of\none, two and three asterisks?\nThank you for your comment.\nWe fully agree. In fact, while double-checking the data we noticed data from 7\nunpublished EBA individuals from Central Germany which are reserved for a separate\nstudy. After removing them we applied the same Kruskal-Wallis test to all regions. We\nhave also clarified in the Figure 3b caption that one, two, three, and four asterisks\n6\nindicate significance levels of p < 0.05, p < 0.01, p < 0.001, and p < 0.0001,\nrespectively.\nLines 365-374: When reporting ROH, the units should be given, presumably cM.\nSupp Table 7 shows that KNE028 and KUC027 also have sum_roh>4 over 50, and\nthe former has sum_roh greater than KUC010 in all ranges, but they are not\ndiscussed here. Why?\nWe have now included KUC027 and KNE028 in the discussion of individuals with\nelevated ROH values. Both display moderate ROH levels (sum_roh > 4 = 83.72 cM and\n83.89 cM, respectively), comparable to KUC010 (72.68 cM). In addition, we have\nspecified the units (cM) for all ROH values in the text and added this information to the\ncaption of Supplementary Table 7.\nMissing data and/or methods\nThe methods indicate carbon and oxygen isotope analyses were performed using\nenamel carbonate (lines 992-1009), but Supp Table 8 only reports δ18O and not\nδ¹³C.\nThe δ¹³C results were not originally included because they were published in Orfanou et\nal. (2024). We agree that presenting them here improves completeness and\ncomparability. We have therefore added the δ¹³C values as an additional column next to\nthe δ18C results in Supplementary Table 8.\nFigure 6d shows δ¹³C collagen results, but stable isotope measurements on collagen\nare not described in the methods, which only include collagen preparation and\nmeasurement for radiocarbon. I could not find the δ¹³C data plotted in Figure 6d\nin the supplementary information (“13C” appears in the file only in Supp Table\n12), though millet consumption yes/no is in Supp Table 1.\nStatistical analysis of both collagen and enamel δ¹³C data is reported (lines 480-481\nand Supp Table 12). Does some of this data come from Wahl & Price (2013)?\nPlease could δ¹³C carbonate, δ¹³C collagen and δ¹⁵N collagen be added to supp\nTable 8?\nThe δ13C_collagen results plotted in Figure 6d were published in Orfanou et al. (2024),\nwhere all isotopic measurements are reported in Supplementary Table S2\n(Isotopes_Overview). The methods for collagen extraction and stable isotope\nmeasurement are also described in detail in that publication. To facilitate cross-\nreferencing and improve accessibility, we have now added the δ13C and δ15N collagen\nvalues as new columns in Supplementary Table 8 of the present study. All requested\ndata (δ13C carbonate, δ13C collagen, and δ15N collagen) are now summarised in\nSupplementary Table 8.\nbaseline from animals as there is “marked species specificity in the relationship\nbetween drinking water and body water” (ref. 16) with a strong effect of body\nmass, making hamsters particularly unsuitable. The construction of this baseline is\nnot described in the methods. The range of faunal values in Supp Table 8 is -8.3 to\n-5.6 ‰, but the range in Figure 6c is greater than this, from below -9 to above -5\n‰. Without data to indicate the local range, I think the best approach is to look for\noutliers. Here, none of the values are more than 1.5xIQR from the quartiles, so no\noutliers are detected.\nWe agree that a human δ18O baseline should not be derived from faunal values for the\nreasons explained above. Following the reviewer’s recommendation, we applied a non-\nparametric 1.5×IQR approach to the human δ18O dataset to identify potential non-local\nindividuals. In this test, we excluded individuals whose sampled tooth was an M1, as\ntheir values may be affected by breastfeeding. These individuals are still shown in\nFigure 6c, where they are labelled with “M1”. No δ18O outliers were detected based on\nthis approach. The results of the test have been added to Supplementary Table 12 under\n“4. δ18O IQR summary (humans, excluding M1)”, and a paragraph explaining the\nprocess has been added in Supplementary Text under section “3.3 δ18O outlier detection\n(1.5×IQR method) in human enamel”. We have also removed the oxygen baseline from\nFigure 6c and corrected the figure and caption. Finally, we removed the corresponding\nsentence referring to the oxygen baseline from lines 384 - 385.\nLine 418: C3 plants would be ubiquitous on all local geological formations, not\nfound on different ones to millet, but millet may have been restricted to certain\ngeological formations.\nWe agree with this clarification, and we have now revised the sentence to read “Millet\nconsumers displayed higher 87Sr/86Sr values, which may suggest that millet was grown in\nareas with different underlying geology, whereas C plants would have been ubiquitous\n3\nacross the local landscape”.\nLines 688-689: It would be helpful if Supp Table 1 indicated which are the new\ndates and which are from Orfanou et al (2024).\nWe have now added an additional column, Date_Source, after the Date_Note column in\nSupplementary Table 1, indicating which radiocarbon dates were produced in our\nprevious study (Orfanou et al., 2024) and which were generated for the present study.\nLine 925 “when possible, we sampled the enamel from both first and third molars”\nThis does not seem to have been done. Supp Table 8 shows only one individual\nwith two teeth sampled, and those were M2 and M3. Overall M2 is the most\nfrequently sampled tooth.\npotential movement across different life stages. However, due to the limited\npreservation and availability of these teeth, we predominantly sampled second molars\n(M2). We have clarified this in the text and revised the sentence accordingly, which\nnow reads “For the inhumations, our initial aim was to sample enamel from both first\nand third molars to assess potential movement across different life stages (childhood vs.\nadolescence). However, due to the limited preservation and availability of these teeth,\nwe predominantly sampled second molars (M2), which provide information about early\nto mid-childhood”.\nLine 927 “δ18O values from M1 should be interpreted with caution as they can be\ninfluenced by breastfeeding” This is true. These samples need to be marked when\nplotted in figure 6, and perhaps omitted from the outlier analysis I suggested\nabove.\nWe have marked the samples in Figure 6c as “M1”, and we have excluded them from\nthe outlier analysis.\nLines 990-1009: the reference points (VPDB or VSMOW) for the δ values should\nbe stated at least once.\nThey are now stated in the text “ Final stable carbon (δ13C, reported relative to VPDB)\nand oxygen (δ18O, reported relative to VSMOW) isotope values …”\nSupp Table 6 IBD: the units, presumably cM, should be given.\nWe have now specified in the caption of Supplementary Table 6 that all IBD segment\nlengths are reported in centimorgans (cM).\nSupplementary Text\nLine 83 “Dating: LBA (MAMS-57256: 941 ± 41 BC cal (Cal 1-sigma))”\nCorrected.\nLine 241 “Dating: 14C: cal BC 894–830 (Cal 1-sigma), cal BC 902–819 (Cal 2-\nsigma).”\nCorrected.\nLine 921 “Individual A - MAMS-52806: 3044±25 BP”\nThe first two (and many others) are not properly reported radiocarbon dates. The\nthird (like others in pages 47 to 49 of the supplement) is inconsistent in not\nsupplying calendar dates. The calibrated result should be expressed as a range\n9\nwith a probability; following Supp Table 1 for line 83, this would be 1006-840 BC\n(95.4%). According to the methods, “All calibrated dates are reported in the text\nas 95.4% probability ranges”, and this should also be the convention for every date\nin the supplementary text. The laboratory code for the date should always be\nincluded.\nCorrected.\nWhere there are multiple individuals in a grave, it would be useful to know which\nof them the radiocarbon date comes from. Where there are multiple dates, the\nindividuals should be identified with the relevant date (e.g. lines 498-504)\nWe specified this in the Supplementary Text.\nLine 113: “1173,5 ± 47,5” this is spurious precision – the calibration data is at 5\nyear intervals, and dates cannot be determined to a fraction of a year. Round to\nthe whole year.\nCorrected.\nMany descriptions contain mentions of profiles A-B, C-D and E-F, but these are\nnot marked on the images and are therefore not meaningful to the reader.\nRemoved from the text since they are not informative.\nSupplementary Figure 3: What do the colours in this figure indicate?\nThe coloured bars represent different ROH length bins: dark blue = 4 - 8 cM, light blue\n= 8 - 12 cM, yellow = 12 - 20 cM, and red = > 20 cM. We have now included this\ninformation in the figure caption.\nMinor changes\nFigure 4: I can’t see any greyed out lines here. Are these non-working models the\nlighter colours?\nThe non-working models are indeed indicated by lighter-coloured bars rather than\ngreyed-out ones. We have updated the figure caption accordingly to clarify this.\nLine 549: delete extra comma\nDeleted.\nLine 687: “95.4% (2σ) probability ranges” – delete “2σ” these are not standard\ndeviations.\nLines 980: ±13 should be ±0.000013\nCorrected.\nLine 995: NaCIO should be NaClO (lower case l not uppercase I)\nThanks. Done.\nLine 1000: CO2 should be CO2\nWe have corrected the chemical formula to display CO with a subscript in the revised\n2\nmanuscript.\nLine 997: liophilized should be lyophilized\nDone.\nSupplementary throughout:\nuse point not comma for a decimal;\nWe have replaced commas with points as decimal separators throughout the manuscript.\nuse raised “” not lowered „“\nWe have replaced the German-style quotation marks („ “) with English-style raised\nquotation marks (“ ”) throughout the manuscript.\nStandardise age group names: choose one of juvenil, juvenile or juvenis; matur,\nmature or maturus; adult or adultus.\nWe have standardised the age group terminology throughout the main text,\nsupplementary text, and supplementary tables, using the categories infant, juvenile,\nadult, mature, and senile.\nSupp Line 355: “right stool” – I don’t know what this means\nThe term “right stool” was a mistranslation of the German “rechte Hockerbestattung”,\nmeaning an individual buried in a right-crouched (flexed) position. We have now\ncorrected the text accordingly.\nSupp Lines 498-504: remove ‘mit’\nThe paper by Eleftheria Orfanou and contributors aimed to address aDNA,\nprovenance isotope analysis, combined with osteoarchaeological analysis, to\nunderstand the lifeways of populations from central Europe during the Late\nBronze Age. Unfortunately, as I am unable to comment on some of the core aspects\nof the paper, such as genetic and isotope analysis, this review will focus on the\nosteoarchaeology/palaeopathology/funerary aspects of the paper.\nWe thank Reviewer 3 for their expertise on the osteoarchaeological, palaeopathological,\nand funerary aspects of our study.\nContent and interpretation\n-Lines 455-457: the authors interpret the lack of biological relationships between\nthe individuals buried within a complex mortuary feature (39/2018) as resulting\nfrom cultural or ritual criteria instead of biological relatedness. Now, can some of\nthe explanations be attributed to ‘opportunistic or convenient burial reasons’? For\ninstance, it would not be irrational to think that if both individuals displayed\nperimortem trauma that resulted from a fatal event occurring in close temporality\nto one another, wouldn’t it be convenient to bury both (genetically unrelated)\nindividuals within the same grave? For this reason, I suggest that, in addition to\n‘cultural or ritual criteria’, the idea of logically opportunistic or convenient burial\nshould be considered as an explanation.\nWe agree that opportunistic practices should also be considered as a possible\nexplanation. For this reason, the text now reads “cultural, ritual, or opportunistic\ncriteria” to reflect that such practical considerations may have influenced the joint\ndeposition of these individuals.\n-Lines 497-498: stated that ‘these inhumations also provided us with a rare\nopportunity to assess the physiological stress and trauma of the people in LBA\ncentral Germany. You commence the descriptions, indicating that ‘degenerative\ndiseases were most common in older individuals’ now, do you consider joint\ndiseases to be physiologically related or trauma-related? Unless you believe that\njoint diseases represent ‘trauma to the joints’, I think that instead of referring to\n‘physiological stress and trauma’, it would be better suited to inform the audience\nthat you looked at palaeopathological lesions/conditions (or just the\npalaeopathology of the populations). These will include joint diseases, trauma, and\nmetabolic conditions (markers of non-specific physiological stress). Also, in Line\n505, you mentioned tuberculosis, which is neither a form of trauma nor a stress\nmarker; therefore, be careful when stating that you observed the evidence for\ntrauma and physiological stress because your analysis went beyond that and\nincluded more types of pathologies. For this reason, I suggest you use the term\nor talk about ‘pathologies or palaeopathologies in general’\nreflect the scope of our analysis. We have now changed the section title to “Mortuary\npractices and palaeopathology in LBA Central Germany.” We have also replaced\n“physiological stress and trauma” with “palaeopathological lesions/conditions,” and the\nphrase now reads as: “These inhumation burials also provided us with a rare\nopportunity to assess the palaeopathological lesions/conditions of the people in LBA\nCentral Germany.” This revised version reflects more accurately the different\ncategories of pathological evidence considered in our study.\n-Line 503: replace ‘stress markers’, which is a broad term, with ‘Non-specific\nmarker of physiological stress’, which is the common term used to describe cribra\norbitalia, porotic hyperostosis and dental enamel hypoplasia. Replace ‘stress\nmarkers’ everywhere else in the paper when you are referring to these markers\nand use the one suggested above.\nWe have replaced the term “stress markers” with “non-specific markers of physiological\nstress” throughout the manuscript.\n-Line 505: what do you mean by ‘spinal tuberculosis’? If you only found periosteal\nreactions (new bone formation on visceral aspects of ribs) without the\npathognomonic signs of destructive lesions in the spine, then you cannot be certain\nit was TB, just a non-specific infection.\nWe identified several individuals showing lesions consistent with possible or early-\nstage spinal tuberculosis, manifesting as intraosseous, round or oval cavities within the\nvertebral bodies. Definite evidence of tuberculosis is observed in individual KUC012\n(16_2009), where similar lesions also occur in other skeletal elements. This evidence is\npresented in the Supplementary Table 13 and includes mild spondylitis of the vertebral\nbodies T4-L2, osteitis of the tibia, periostitis of the tibiae, among other changes. We\nnow direct the reader to the Supplementary Table 13 by adding the following in the\nmain text: “ Non-adults have also a higher incidence of spinal tuberculosis (e.g.,\nindividual KUC012, Supplementary Table 13)”.\n-Lines 521-525: the summary of the palaeopathological evidence (or the discussion,\nLines 610-649) could have included some interpretations on the dental disease. If\nyou were interested in the lifeways of these populations… What were the health\nimplications associated with the findings of Streptococcus mutans and\nParvimonas?\nWe thank the reviewer for this suggestion. We did not analyse in depth the dental status\nand disease-related changes to the teeth and gums of the individual skeletal remains in\nthis study. As part of the investigation, however, we did document the dental status and\nrecorded notable pathologies for completeness. A more detailed assessment of dental\n14\nhealth lies beyond the scope of the present study but will be addressed in future work,\nonce more individuals become available. What we can say from the data available to\ndate is that, for the burials where dental status could be assessed, the individuals\ngenerally show good dental health. Calculus and mild caries are commonly observed,\nwhile isolated cases of severe caries also occur, ranging from pulpa aperta to\npathological changes in the jawbone (e.g., KUC004). However, such severe cases are\nrare within the examined population. We added this observation to the text, and now it\nreads as “In addition, for the burials where dental status could be assessed, the\nindividuals generally showed good dental health. Calculus and mild caries were\ncommonly observed, while isolated cases of severe caries also occurred, ranging from\npulpa aperta to pathological changes in the jawbone (e.g., individual KUC004,\nSupplementary Table 13). However, such severe cases were rare within the examined\npopulation”.\nRegarding the health implications associated with the findings of S. mutans and P. micra it is\ndifficult to draw direct conclusions. Both pathobionts are commonly found in the oral\nmicrobiome where they can cause caries and periodontitis. However, this can be prevented by\ngood hygiene. On the other hand, it can be favoured by a carbohydrate rich diet. Detecting\nboth pathogens, in particular S. mutans, is in so far no surprise, as caries has been observed in\nsome individuals. We therefore added: “These pathobionts are commonly involved in the\ndevelopment of dental caries and periodontitis, respectively, often favoured by a\ncarbohydrate rich diet. Their detection is in line with the paleopathological observations”.\nFinally, was the interpretation of body positions out of the scope of analysis?\nSome individuals were buried prone, which, if analysed in conjunction with isotope\nanalysis and grave goods, could have funerary implications that could contribute\nto addressing the lifeways of these populations.\nWe agree with the reviewer that the body positions of the individuals could provide\nvaluable insights when considered alongside isotopic data and grave goods. However, in\nour current dataset, the variation in body positions is high, preventing the formation of\nstatistically meaningful groups for analysis. Moreover, at this stage, only a few\nindividuals are associated with grave goods. For these reasons, we did not pursue an in-\ndepth interpretation of body positions in this study. We recognise the potential\nsignificance of this aspect and plan to explore it further in future research, when more\nindividuals become available.\nTypos and grammar:\n-Line 549: delete one comma\nDone.\n-Line 676: change ‘was’ with ‘were’\nDone.\n-Lines 1035-1036: accession number and URL link not provided\nWe added the accession number: PRJEB98508 and the link will go live upon\nacceptance.\nOrfanou and colleagues present a genomic, isotopic, and osteoarchaeological\nanalysis of rare inhumation burials from populations in Central Europe associated\nwith the Urnfield culture, during the Late Bronze Age (LBA). The focus of the\nmanuscript is site-specific and regional, targeting a local population present at the\narchaeological sites of Kuckenburg and Esperstedt in Central Germany, but they\nsupplement with novel data derived from contemporaneous ancient individuals\nfrom South Germany, Bohemia (Czechia) and southeastern Poland. They expand\nthe genomic analyses further with previously published contemporary and ancient\nhuman datasets. They conclude for the local Kuckenburg and Esperstedt\npopulation that there were no associations between mortuary practice and genetic\nsex, relatedness, geographic origin, or inferred socioeconomic status. The\ncommunity showed cultural flexibility, with inhumation vs cremation behavior\npossibly negotiated with fluctuating ideas of identity based on external influences,\nas regional interconnectedness grew. This local and more nuanced view of an\nancient community is commendable, the paper is generally well written and clear,\nand I see no deep methodological faults. There are improvements that could be\nmade to strengthen the manuscript. I will discuss these generally, and then provide\nmore detailed line edits below.\nWe sincerely thank Reviewer 4 for their positive and thoughtful evaluation of our\nmanuscript.\nThe first concerns the incorporation of the new genomic data from South\nGermany, Czechia, and Poland. There is no background information provided for\nthese individuals in the Introduction, and they are skipped completely some section\n(e.g. Mortuary practices and physiological stress), which makes them feel ‘tacked-\non’ to the project (I am aware the burial details are in the supplemental).\nApart from these individuals being contemporaneous in age and (apparently)\nassociated with the Urnfield Culture, the reader is left unclear why these are being\ncompared to the Unstrut group; are they the only other Urnfield populations\nsampled (i.e. the AADR lacks representatives for this culture, given the prevalence\nof cremation)?\nOur study centres on regions in today’s Central Germany, where inhumations are\npresent during the LBA, while cremation was the predominant mortuary practice.\nBecause of this mortuary practice, genetic data from South Germany, Czechia, and\nPoland are also scarce. Due to this scarcity of LBA individuals from Central Europe in\npublic repositories (e.g. POSEIDON or AADR), aside from the few published we rely\nheavily on the newly generated genetic data from these neighbouring regions to situate\nthe LBA Unstrut group within a wider Central European chronological context. The\nindividuals associated with the Knovíz culture in Czechia date to the LBA/Urnfield\nperiod and share broadly comparable mortuary practices with Central Germany, as we\nmention in the Introduction. Even though published Knovíz data exist, our new\n17\nindividuals provide additional, regionally relevant genetic data from the LBA. The four\nLBA/Lusatian/Urnfield individuals from Poland are also used for genetic comparison\nonly. They are included because they are contemporaneous, and reasonable\ngeographical proxies given the cultural and demographic interconnectedness of the\nperiod. As such, the combined genomic data from today’s Germany, Czechia and\nPoland presents the first solid LBA genetic record for Central Europe and thus a useful\nbaseline for future studies that wish to integrate multi-proxy analyses across wider\nregions in Europe. We have added clarifying sentences in the main text, emphasising\nthat the southern German, Czech, and Polish individuals are used exclusively as a\ngenetic comparative dataset. The text now reads as “ We adopt a site-specific\nperspective (i.e., focusing on the sites of Esperstedt and Kuckenburg), where we\nreconstruct individual bioarchaeological profiles, and we contextualise their genetic\nancestry through comparison of the newly analysed individuals with published\ncontemporaneous groups from Central and South Germany, Czechia, and Poland (Fig.\n1b)”.\nWhere do these populations fit into the question presented in the introduction, of\ncultural and biological continuity vs change, in Central Europe, during the Bronze\nAge? As a more detailed example, should we have expected different ratios of EEF,\nSteppe, and WHG than what is estimated with the qpAdm results in Figure 2b?\nAre those results very surprising, from an archaeological/cultural standpoint?\nFrom an archaeogenetic perspective, the proportions of EEF-, Steppe- and WHG-related\nancestry observed in our LBA individuals are broadly consistent with expectations, i.e. what\nis known from the reported genomic ancestry profiles of individuals dating to the Bronze Age\nperiod in Central Europe, and in fact across most regions of Europe more generally\n(Mathieson et al. 2018, Olalde et al. 2019, Furtwängler et al. 2020, Papac et al. 2021,\nPatterson et al. 2021, Lazaridis et al. 2024, to name but a few).\nSpecifically, for South Germany, elevated EEF ancestry relative to Central Germany has been\ndocumented since the Early Bronze Age (Mittnik et al. 2019; Furtwängler et al. 2020,\nGretzinger et al. 2024), and our results further confirm this observation, as mentioned in lines\n292 – 305 in the main text. In Bohemia, the shift from the EBA/Únětice to the MBA/Tumulus\nand LBA/Knovíz cultures is also associated with higher EEF ancestry, in line with previous\nstudies (Patterson et al. 2021), as mentioned in lines 306-316 in the main text. Lastly, in\nPoland, Chyleński et al. (2023) did not detect an increase in EEF-related ancestry from the\nEBA to the MBA. However, this results from the fact that the analysed material comes from\nthe Trzciniec culture, whose roots lie largely in the forest zone of Europe. Samples from\nPoland — and in fact from Silesia (south-western Poland) — may differ from what Chyleński\nobserved. Silesia is much closer, in many respects, to the regions of Bohemia and Moravia,\nso this should not be surprising. We have added a few sentences presenting the genetic\nbackground for the Early Bronze Age in Central Europe to the Introduction, lines 90 to 110.\nFor Central Germany, however, the increase in EEF ancestry from the early to the late phase\nof the LBA represents a new finding based on the newly reported data. From an\n18\narchaeological perspective, it does not come as a complete surprise given the degree of\ninterconnectedness and long-term interaction among neighbouring LBA groups\nvisible/attested in the archaeological record, as discussed specifically in lines 585-590 of the\ndiscussion for the southern parts of Germany and Bohemia. Hence, a delayed increase of EEF\nancestry in Central Germany could simply be the result of geographic distance. Finally, while\npublished LBA genomic data from Poland remain limited, the higher EEF ancestry observed\nin our four individuals follows the broader trend of increasing EEF ancestry in the LBA in\nCentral Europe. Therefore, our qpAdm results agree, for most regions, with established\npatterns.\nMore generally, the observed genetic shifts during the LBA are detectable but subtle in\ncomparison to major turnover events in preceding millenia such as the Neolithic transition or\nthe arrival of steppe-related ancestry. To our knowledge, there is no strong prediction from\nthe archaeological record as to which degree a particular cultural influence should be\nreflected in the genetic ancestry profiles of the associated LBA individuals.\nThe second concerns the continued theme throughout the manuscript of\nfluctuating Early European Farmer (EEF) related ancestry. There doesn’t seem to\nbe a consistent story told about population continuity vs admixture, but I’m sure a\nfew sentences would clear it up. Some examples: The paper highlights a fairly\nconsistent increase in EEF ancestry over time in all regions (Figure 3), but the\nqpAdm modeling suggests population continuity from EBA to early LBA (Central\nGermany) or continuity from MBA to LBA (Bohemia and Poland).\nAlthough EEF ancestry increases overall across the entire Bronze Age period, the\ntiming, extent, and mechanisms differ by region. We have clarified this in the relevant\nresults and discussion sections to avoid implying a uniform demographic process and to\nemphasise the nuances the new data offers with regards to periods of stasis vs. periods\nof change (Fig. 3b), and in particular in light of contextual evidence from isotopic and\narchaeological data.\nIn the strontium analyses, everyone seems local, regardless of time depth, genetic\nsex, or mortuary practice – where is the increase in EEF ancestry coming from?\nStrontium and ancient DNA analyses capture mobility at different temporal levels.\nSpecifically, strontium isotope ratios reflect childhood residence since tooth enamel\nforms early in life and does not remodel. Hence, strontium isotope analysis can detect\nonly first-generation migrants. Genetic ancestry, on the other hand, records population\ninteractions over many generations. As a result, individuals can appear isotopically local\nwhile still carrying ancestry components introduced into the region by earlier gene flow\nand interaction with neighbouring groups in the interconnected Late Bronze Age world.\nConcerning the potential sources of the increased EEF ancestry, we refer to this in the\nresults section of the main text, e.g. in lines 330-343 and discuss this in lines 583-590.\nplants and EEF-related ancestry, in the late LBA, but not with the earlier switch to\nmillet. The authors then bounce between suggesting a change in ancestry with the\nformer, but not one with the latter. Settlement contexts is another example.\nConcerning the diet of the individuals, the association between higher EEF ancestry and\nthe late LBA return to C plants does not necessarily imply causation. This pattern may\n3\nreflect changes in agricultural practices, environment, culinary preferences or cultural\ninfluence rather than population movement. Indeed, we did not observe a change in the\nancestry of the individuals when millet became part of the diet in the early phase of the\nLBA. The same can be applied to the settlement contexts, that is, an increase in\nsettlement contexts is not necessarily caused by a change in the ancestry of individuals.\nWe have rearranged the relevant parts of the discussion and have now emphasised this\naspect in the main text.\nThe authors could expand the mtDNA and NRY analyses and interpretations. The\ncontinental spread and phylogenetic history of the haplotypes typed are not\ndiscussed, even in passing. Are any of them surprising (the two CT individuals?)?\nAll expected? How about the drastically increased mitochondrial diversity?\nKUC023 has a unique NRY haplotype and an elevated Sr ratio, but is not\napparently a genetic outlier. What might be going on there?\nCT is a very basal Y haplogroup, and we could only resolve these individuals to this\nresolution due to poor coverage. CT simply indicates that these individuals do not carry\nY haplogroups from the A and B branches (which is not surprising, as these are\nconstrained to Africa in the human past). Aside from these calls, and aside from\nKUC023, we observe only common haplogroups from I1, I2, R1a, and R1b, which are\nexpected at this time.\nIndividual KUC023 carries Y-haplogroup I-Z63, which is rare and, as a result, poorly\nunderstood. The earliest find of a derived haplogroup is in Denmark (McColl et al.\n2024), dated to 400-220 BC. We find mostly evidence for individuals carrying similar\nZ63-derived sub-lineages in both Northern Europe and Austria/Hungary in the later Iron\nAge and Medieval periods (or even later), but this tells us little about their origin or\ndistribution during the LBA. There is a general hypothesis that it originated in\nScandinavia, as it is still found there and in the British Isles today. Hence, this may\npoint to a more northern ancestry source in this individual’s past, which may not be\nreadily obvious genetically due to overall heterogeneity in the region at the time. This is\nnow mentioned in the Supplementary Note 2.4.\nRegarding the mitochondrial diversity, we observed 24 different mtDNA haplogroups in\n28 female individuals, broadly resembling the mt haplogroup distribution in Bronze\nAge Central Europe (e.g., Brandt et al. 2013, Science; or Papac et al. 2021 (Unetice).\nThis is now mentioned in the main text.\nWas aDNA extraction attempted on any of the bones from the cremated remains?\nAre the authors just not reporting libraries at all that didn’t pass internal\nendogenous DNA scan requirements? They had the bones for the isotopic work.\nWe did not attempt DNA extraction from the cremated bones used for strontium isotope\nanalysis, as these samples were fully calcined, therefore lacked preserved organic\nmaterial. Moreover, the samples selected for isotopic work were mostly fragmented\nlong bone fragments, which are generally less suitable for aDNA analysis. We did,\nhowever, attempt DNA extraction from one cremated petrous bone from Kuckenburg\n(KUC029), which did not yield usable results (very low endogenous DNA and no\ndamage profiles). We agree that this information should be reported, and have now\nincluded the individual KUC029 Supplementary Table 1.\nIn your biological relatedness analyses, KIN (https://doi.org/10.1186/s13059-023-\n02847-7) seems to be growing in popularity and perhaps should be attempted\nhere?\nWe have now included KIN (Popli et al. 2023) in our biological relatedness analyses\nunder Supplementary Table 5c. The results are consistent with those obtained using\nREADv2 and BREADR, confirming the inferred degrees of relatedness among the\nindividuals. Details of this analysis have been added to the Methods (Biological\nrelatedness) section.\nFurthermore, is the limited relatedness detected at Kuckenburg and Esperstedt to\nbe expected? The authors could expound on this more.\nLimited close biological relatedness within cemeteries and mortuary contexts is\nincreasingly recognised and described in prehistoric Europe. Although these examples\ncome from different periods and regions, many studies now report low levels of close\nbiological relatedness within mortuary groups. A recent study from Neolithic Ireland\n(Carlin et al. 2025), for example, shows that even long-used, well-sampled mortuary\ncontexts often contain individuals who are only distantly related or unrelated, reflecting\nsocially defined mortuary practices rather than biological kin groups. Similar patterns\nhave been identified in Early Bronze Age Central Europe (Knipper et al. 2017; Mittnik\net al. 2019), where cemeteries often also contain individuals of diverse origins with few\nclose biological relatives. Another example comes from Knipper et al. 2020, which\nexamines a 5th-century AD cemetery at Mözs in Pannonia. Isotopic analyses revealed a\npopulation of diverse geographical origins that formed a socially constituted community\nrather than a biologically related one. These examples, which should be used with\nappropriate caution, suggest that the limited relatedness observed at Kuckenburg and\nEsperstedt is not that unexpected and may reflect selective mortuary traditions and\ncommunity belonging. The inclusion and analysis of more individuals in future studies\nwill refine this picture further. We have now added a paragraph to the Discussion, lines\n694 - 709, that expands on this.\nPage 3, lines 81-83: Give a date range here?\nWe have now adjusted the text to read as “Based on recent archaeological findings, this\npractice is proposed to have originated in the middle Danube region, including areas of\nmodern-day Lower Austria, Moravia, southwest Slovakia, and parts of Hungary6, in the\nperiod between ca. 2000 and 1600 BCE, before spreading more widely throughout\nEurope and becoming consolidated in the classic Urnfield horizon after 1300 BCE”.\nPage 5, lines 139-141: Are the \"Sonderbestattungen\" burials the only unique\narchaeological/cultural aspect of the Kuckenburg and Esperstedt settlements?\nWould the results presented here be applicable to the other non-cremation sites\nmentioned in Paragraph 2 of the introduction? How broadly applicable would\nthese analyses be to Bronze Age central European communities, or do the authors\nwant the takeaway to be a hyper site-specific perspective?\nAs the phenomenon of “settlement burials” (in all its varied definitions) is quite common not\nonly in the southern parts of present-day Saxony-Anhalt and Bohemia but also across a wide\nrange from Central Germany to present-day Moravia, the results of our analysis are not\nlimited to a hyper–site-specific perspective. Rather, they provide a model for ritual and social\nactivities in these regions. We have clarified this point in the text, and now it reads as\n“Hence, by analysing the individuals buried at Kuckenburg and Esperstedt, we aim to\naddress gaps in our understanding of Central European LBA communities on site-specific\nand regional scales, especially as forms of settlement burials are widespread across Central\nGermany, Bohemia, and Moravia and therefore provide a broader comparative framework”.\nPage 5, lines 162-164: I can follow why NES005 was thrown away, but I'm not\nfollowing why NES006 wasn't - high contamination with angsd and hapcon, but\nlow with ContamMix. Have you considered you might have sex chromosomal\naneuploidies? Could check with karyo_RxRy? Also, if I'm following your column\nBW in Sup Table 1, you only threw out 2 individuals (though NES006 is in red),\nwhich seems to leave 72 individuals for other analyses; not 70. Are there\nduplicates? Column H has 4 \"same\" values, and maybe that is where the\ndifference is. If so, mention 2 duplicates were found in 4 analyses.\nThank you for pointing this out. NES006 should indeed have been excluded. Although\nContamMix produced a lower contamination estimate, both ANGSD and HapCon indicated\nhigh contamination levels. We also re-examined NES006 regarding potential sex-\nchromosomal aneuploidies, and the individual is confidently classified as XY with posterior\nprobability ~1. No NES individuals were flagged as potentially carrying sex chromosomal\naneuploidies. Regarding the sample numbers, four samples represent two biological\nindividuals (as noted in Supplementary Table 1, column H “same”, and now specified in the\nNotes of the same table). After removing these duplicates, the dataset contains 69 unique\n22\nindividuals. We have now clarified this in the text and corrected the numbers accordingly.\nWe have also updated all relevant tests and figures to exclude NES006.\nPage 7, lines 181-182: Which individuals (genomic data) are the authors using to\ndefine these three groups in their analyses? I can’t find those group IDs in any of\nthe supplementary tables. If they are consistent samples that everyone uses, define\nand give appropriate references.\nThank you for highlighting this issue. The three main ancestry groups used in our f -4\nstatistics follow the publication of Patterson et al. (2022). We have now specified in the\ntext how EEF, WHG, and Steppe correspond to the entries in Supplementary Table 2b,\nand indicated the column (“Group ID_published”) where they can be found.\nPage 7, lines 183-187: This belongs more in the Methods section than in Results\nWe agree that these sentences read more like a methods section. However, we chose to\ninclude this brief explanation in the Results to help readers interpret the described tests\nwithout needing to navigate back and forth between sections.\nPage 7, lines 194-196 (Figure 2b Legend): An expanded introduction that includes\nthe S. Germany, Czechia, and Poland individuals would help give us more\nperspective of how we should be interpreting the qpAdm models in comparison to\nthe Unstrut population.\nWe agree that a brief review of the general genetic background of Bronze Age Central\nEurope was missing from the Introduction. We have added a full paragraph (lines 90-\n103) including the relevant literature to set the scene for the interpretation of the newly\nreported results of this study.\nPage 8, lines 219-220: A description of what makes this cemetery unique likely\nbelongs in the introduction.\nThank you for this suggestion. However, we believe that a short sentence explaining why the\nNeckarsulm cemetery is considered unique fits best at the beginning of the Results section, as\nit helps situate the reader for the upcoming discussion of results. We have now expanded this\nsentence and direct the reader to the more detailed archaeological description provided in\nSupplementary Note 1.4, where the site and the burials are discussed. The text now reads:\n“For comparison, we also analysed 12 individuals from the unique LBA cemetery of\nNeckarsulm (NES) in South Germany, which has been interpreted archaeologically as the\nburial place of an all-male “warrior community” (Supplementary Note 1.4)”. We also\nreference the publications by Knöpke (2009) and Wahl & Price (2013), which offer\ncomprehensive archaeological and isotopic interpretations of the site. In our study, we only\nprovide new genetic data for these individuals.\nWe have revised the sentence.\nPage 9, lines 256-258: I don't understand where the support for this sentence\ncomes from in the preceding paragraph, as I don't see any graphics or data in\nSupp Table 3 showing any early Bronze Age comparisons?\nWe have now added all supporting f -statistics for this sentence to the text (lines 268–287)\n4\nand added all the corresponding tests in Supplementary Table 3. We have also adjusted the\nsentence in the main text for clarity, which now reads: “Together, the observed patterns attest\nto similar shifts of genetic ancestry in all regions, characterised by increasing proportions of\nEEF-related ancestry between the Early and the Late Bronze Age, although the timing,\nextent, and mechanisms of this shift differ between regions”.\nPage 9, lines 260-269: Why are the individuals from Poland, who seem to have the\nlowest proportion of EEF-related ancestry, not discussed in this paragraph?\nWe have now included the Polish individuals in this paragraph, which now reads as “The\nfour LBA individuals from SW/Central Poland also show a lower proportion of EEF-related\nancestry (37.4 ± 2.2 %) similar to those from Central Germany. However, given the very\nsmall sample size, it is unclear whether these individuals are representative of Polish LBA\ngroups more broadly, and therefore these results should be interpreted with caution”.\nPage 9, lines 271-274: Can you be more clear where you are getting the proportions\nused for Figure 3a/b, because they don't seem to exactly match the values from qpAdm\nlisted in Figure 2b (for the LBA individuals, at least). This can be put in the text or in\nthe figure legend.\nThe proportions used for Figure 3a/b are the qpAdm distal ancestry estimates presented in\nSupplementary Table 4a (qpADM_distal). These are the same proportions as in Figure 2b.\nWe have clarified this in both figure captions. We have now added the EEF proportions that\nare used in Figure 3a/b for the published individuals in Supplementary Table 4a as well.\nPages 12-13, lines 333-342: Also discuss LNV001 and SNY001, who are highlighted\nas outliers in this manuscript, in the PCA, and in Figure 5 below.\nThey are now mentioned in this section, and the text reads as “LNV001 and SNY001\nfrom Bohemia show highest genetic similarity to individuals from Central Germany”.\nPage 14, lines 365-374: Why are KNE028, KUC027 not being discussed in the\nhapROH analysis? Do they not also have elevated levels? See Supplemental Figure\n3.\nelevated ROH values. Both display moderate ROH levels (sum_roh > 4 = 83.72 cM and\n83.89 cM, respectively), comparable to KUC010 (72.68 cM). In addition, we have\nspecified the units (cM) for all ROH values in the text and added this information to the\ncaption of Supplementary Table 7.\nPage 14, lines 386-396: None of the genetic outliers appear non-local in the isotopic\nanalyses. Can the authors discuss/highlight this?\nWe copy here our answer from above:\nStrontium and ancient DNA analyses capture mobility at different temporal levels.\nSpecifically, strontium isotope ratios reflect childhood residence since tooth enamel\nforms early in life and does not remodel. Hence, strontium isotope analysis can detect\nonly first-generation migrants. Genetic ancestry, on the other hand, records population\ninteractions over many generations. As a result, individuals can appear isotopically local\nwhile still carrying ancestry components introduced into the region by earlier gene flow\nand interaction with neighbouring groups in the interconnected Late Bronze Age world.\nWe have added this note in Supplementary Note 3.2.\nPage 15, line 406: How is millet consumption determined? I see the column in Sup\nTable 1, but not a description.\nMillet consumption was inferred from δ13C (and δ15N) values measured on bone\ncollagen, following established interpretative thresholds from previous isotope studies\nin the region (e.g., Filipović et al., 2020; Le Huray, 2005, also see Orfanou et al., 2024,\nfor a full explanation of the method). We set a δ13C collagen value of –18‰ as the\nthreshold for increased consumption of C plants. Values higher than –18‰ indicate the\n4\nconsumption of a mixed C /C diet and moderate millet intake, while values higher than\n3 4\n–12‰ correspond to a diet predominantly based on C plants (Filipović et al., 2020;\nmillet consumption was determined. The threshold note has also been added in Figure\n6d above the dotted line of –18‰.\nPage 21, line 571: By “this dietary shift”, I assume the authors are referring to the\nreturn C3 plants, but please clarify.\nIndeed, this is what we mean by “this dietary shift”. We have clarified this in the text by\nreplacing “this dietary shift” with “the return to C -plant-based diets”.\nthe new Twist kit.\nWe have now added the appropriate reference for the 1240k capture method.\nreferences.\nWe have now included the relevant references for the mitochondrial and Y\nchromosomes capture protocols.\nPage 30, line 804: I assume these genotypes are from the preceding ATLAS calls?\nPlease be more specific in the \"Population Genetic Analyses\" when you used the\npseudo-haploid calls and when you used the ATLAS genotypes.\nWe thank the reviewer for addressing this ambiguity. A specification was added in the\n“Imputation” and “Population Genetic Analyses” sections. We specified that “Diploid\nautosomal genotypes produced with ATLAS” were used for imputation with GLIMPSE\n(“Imputation” section); we also now specify that “All the population genetic analyses\nbesides IBD analyses were conducted with pseudo-haploid genotypes produced with\npileupCaller” in the “Reference dataset” section, and then that regarding the dataset for\nIBD analyses “Diploid genotypes imputed with GLIMPSE and with >500,000 SNPs and\ngenotype probabilities >0.7 were included in the analysis.” in the “IBDs” section.\nPage 30, line 807: spelling “pubblication”\nCorrected.\nPage 31, lines 831-833: Was the HO dataset *only* used for the PCA and\nAdmixture analyses? If nothing else, say that. I would also state the limited HO\ndataset was necessary to include many of the contemporary populations.\nYes, the Human Origins (HO) dataset was only used for PCA and ADMIXTURE\nanalyses. We have clarified this in the revised text and noted that the HO dataset was\nchosen to enable inclusion of a larger number of modern comparative populations.\nPage 31, line 853: Here and elsewhere, when you bring up population group labels\nlike this, reference your Supplementary Table 2b.\nDone.\nPage 31, lines 856-857: As already previously mentioned, we are never given who is\nincluded in these groups.\nThe three main ancestry groups used in our f -statistics follow the publication of\ncorrespond to the entries in Supplementary Table 2b, and indicated the column (“Group\nID_published”) where they can be found.\nTable 2b? I don’t see some of them in the supplementary tables.\nThe labels correspond to the AADR v54.1.p1 group designations and are also provided\nin Supplementary Table 2b under the column “Group ID.” We have now clarified this in\nthe text by explicitly referencing Supplementary Table 2b alongside the labels.\nPage 37, lines 1031-1032: I applaud the authors for including this level of detail for\ntheir statistical tests.\nThank you.\nReviewer #1 (Remarks to the Author):\nI was really pleased to see the careful and thoughtful revisions and responses to the reviewers.\nThe authors have made an already strong paper excellent. I have no further comments - well\ndone!\n-Catherine Frieman\nThank you. Much appreciated.\nReviewer #2 (Remarks to the Author):\nThe authors have provided a comprehensive reply to the comments and have made\nimprovements to their manuscript. I have only one further comment: the reply makes clear the\nsource of the collagen isotopic data. For complete clarity I think they ought to cite Orfanou et\nal. (2024) as the source in the caption for Figure 6d.\nThank you very much. A reference to our dietary isotope study is now included in the caption of\nFigure 6.\nReviewer #3 (Remarks to the Author):\nI have reviewed the manuscript for the second time, and in my opinion, the authors satisfactorily\nmodified the text in accordance with my former suggestions. I appreciate the time and effort\nthey have put into acknowledging these. Thus, I see no reason why the manuscript could be\npublished.\nThank you. Much appreciated.\nReviewer #4 (Remarks to the Author):\nI appreciate the authors for responding positively and meticulously to most of my suggestions. I\nnow read a more nuanced and regional take on the EEF ancestry changes, more highlights of\nwhen results matched or didn’t match expectations, and expanded discussions of the diet\ncorrelations and relatedness measurements. More specifically, thanks to the authors for running\nKIN, for reporting on the single cremation they attempted aDNA analysis on, for the new\nsupplemental note on KUC023, for clarifying which genotype sets were used when, and for\nproviding much more detail on ancestry group names and membership, both in the text and in\nsupplemental tables – this makes future replication easier and our field better.\nWe agree and highly appreciate the thorough review. Many thanks!\n(All subsequent line references are to the tracked changes pdf)\nIn regards to the individuals genotyped from South Germany, Bohemia, and SW/Central Poland,\nI acknowledge the broader genetic background of Bronze Age Central Europeans given in the\nIntroduction and I acknowledge that the authors more consistently highlight these individuals in\nthe Results. I am still unclear why, however, these individuals are only being introduced to us\nhalfway through the Results, particularly when most of the first few paragraphs of that section\nincorporate them as part of a larger Central European LBA gene pool and they are identified in\nFigure 1. While I understand the focus is on the Esperstedt and Kuckenburg Unstrut group,\nwould it truly disorganize the manuscript to summarize those 3 additional populations in a\nminimized version of what you do in lines 132-159 for your main site(s)? You describe as much\nin your peer review rebuttal notes. Instead, we still only get a single sentence heads up, in the\nfinal paragraph of the introduction, of their existence, even though they represent almost half of\nyour (newly contributed) dataset. It is the authors’ prerogative to bury (ha) or to highlight sites,\nbut I do think it is a missed opportunity. If LBA individuals from Central Europe are universally\nscarce, shouldn’t any and all be meaningful?\nWe fully agree. It was and is clearly not our intention to place some sites over others. The\nreason why our main focus lies on Esperstedt and Kuckenburg is because the locally funded\nproject was built around these two sites associated with the Unstrut group, including the\ngeneration and evaluation of all contextual data, such as mortuary practices, dietary and\nmobility isotopes, anthropology and other archaeological meta-information.\nWe now have introduced the co-analysed comparative sites/regions in the introduction and refer\nto the respective Supplementary Notes for more specific details on the sites and the\ninhumations studied.\nSome new small edits:\nIn regards to KUC023, fix the reference at line 190 to say ‘Supplementary Note 2.4’ instead of\n‘Supplementary Table 2.4’, and add a reference to Supplemental Note 2.4 somewhere in lines\n433-436.\nThanks. This is done.\nAdditionally include a short note of KUC027 and KUC010 in Supplementary Note 2.3, as you\nhave already done in the main body of the text. That is where the reader will see the actual\nresults.\nThank you. A short note is now added to the Supplementary Note 2.3.\nThank you for clarifying on final sample counts (69 passing quality control), though your edits on\nlines 186-187 now says “51 different mtDNA haplotypes in 64 individuals”, and I’m unclear why.\nThank you for this comment. The reason why mitochondrial haplogroups could be assigned to\nonly 64 of the 69 individuals passing quality control is that five individuals did not have enough\nmitochondrial reads for reliable mtDNA haplogroup calling. This clarification has now been\nadded to the main text, and it now reads as “We observed 51 different mt haplotypes in 64\nindividuals, with enough mt reads for reliable mtDNA haplogroup calling, broadly resembling the\nmt haplogroup distribution in Bronze Age Central Europe.”\nThere are some changes in font style at lines 944-946 and 1084-1086.\nThanks for spotting. The font style in these lines was already fixed in the clean version, as is in\nthe resubmitted 2nd revision."}]

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