Two longobard-associated cemeteries

We performed a deep genomic characterization of individuals buried in two cemeteries of the sixth to seventh centuries CE that have material culture associated with the Longobards. Both are considered key sites with regard to the proposed migration from Pannonia to Italy. The first cemetery, Szólád, is located in present-day Hungary (Supplementary Figure 1). There are 45 graves (Fig. 1b), all of which are dated to the middle third of the sixth century based on a combination of stylistic elements of the grave goods and radiocarbon (2-sigma range of 412–604 CE, Supplementary Table 1) analysis14. Archeological, stable isotope, and mtDNA (HVS-1) analyses suggested that Szólád was occupied for only ~20/30 years by a mobile group of Longobard-era settlers14. The female to male ratio (sexing being based primarily on genetic data, or in its absence, anatomy) is 0.65. Graves in this cemetery are organized such that a core group (N = 18), mostly of male individuals, is surrounded by a half-ring of females (N = 11) (Supplementary Figure 2). Most of these individuals lie in elaborate graves with ledge walls and wooden chambers all in the same orientation, furnished with numerous artifacts such as beads, pottery, swords, and shields. The remaining 16 Longobard-period graves are more diverse in relation to the sex of the individuals, as well as to the quality of grave construction and richness of artifacts. Archeologists also recovered two bodies (AV1, AV2) that derive from a later occupation of the region by the Avars in the fill of the Longobard-period grave (SZ27)15, as well the skeletal remains from an individual dating to the Bronze Age 10 m north of the cemetery (SZ1). See Supplementary Note 1, Supplementary Figures 1–11, and Supplementary Table 1 for the archeological context of Szólád.

The other cemetery, Collegno, is near Turin (Supplementary Figure 12), northern Italy, and was in use from the late sixth through the eighth centuries, the earliest period of the Longobard kingdom in Italy16. We studied the 57 graves that date between 580 and 630 CE based on artifact typologies (Fig. 1c) and that represent the first of three major periods of occupation. The types and range of grave goods in these 57 interments are comparable to those recovered at Szólád. However there is also evidence for a gradual cultural and religious evolution, with some practices disappearing in later decades. While there are no ledged graves, some are constructed via a wooden chamber structure, and there is the skeleton of a horse (devoid of head) in both cemeteries. See Supplementary Note 3 and Supplementary Figures 12–22 for the archeological context of Collegno.

Illumina sequencing of DNA extracts from petrous bone17 and teeth identified 39 and 24 samples from Szólád and Collegno, respectively, for which there was high endogenous content, high library complexity, and patterns of postmortem damage (PMD) characteristic of ancient DNA (Supplementary Note 4, Supplementary Data 1). Analysis of X-chromosome-mapped reads in males and mtDNA in both sexes revealed low levels of estimated contamination in almost all samples (mean ~1%), although one, CL31, had a value of 27% and 7% using the X and mtDNA, respectively. While we include this sample in certain individual-based analyses, its results should be treated with caution. Genomic libraries for the majority of samples (60 out of 63) underwent partial UDG treatment18. Endogenous DNA content was sufficient (33–67%, mean 57%) for 10 male samples from Szólád to undergo whole-genome sequencing (WGS), with a mean genome-wide coverage across samples of 11.3×. The remaining 53 samples underwent an in-solution capture targeting 1.2 M single nucleotide polymorphisms (SNPs) (henceforth 1240 K capture)19,20. The average coverage at these SNPs (excluding the whole genomes) was ~1.5x and the mean number of genotyped SNPs per sample was ~522 K (Supplementary Note 5). Unless noted, we considered 33 and 22 samples from Szólád and Collegno for downstream analysis, respectively (three and one samples from Szólád and Collegno had fewer than 30,000 usable SNPs, while samples SZ1, AV1, AV2, and CL36 were found not to belong to the same occupation period as the other samples).

In addition, we assembled comparative SNP data for different modern reference Eurasian samples, using directly genotyped SNPs from WGS21,22 or by imputation23,24, as well as 435 ancient West Eurasians from 6300 to 300 BC20,25,26 (Supplementary Note 6 and Supplementary Data 2). We also included comparative genomic data from 18 Eurasian genomes27,28,29 that are from similar time periods as the two cemeteries focused on here.

Two primary central/northern and southern groups

Principal component analysis (PCA) of samples from Szólád and Collegno against modern reference sets infers that our ancient samples possess genetic ancestry that overlaps overwhelmingly with modern Europeans (Fig. 2a, Supplementary Note 7, Supplementary Figures 23–25). However, they do not cluster with individuals from their respective modern countries of origin. Instead, samples from both cemeteries demonstrate a diverse distribution, with two broad clusters around modern northern and southern individuals, as well as individuals of intermediate ancestry. This north/south axis of genetic variation is also observed when examining only our ancient samples, demonstrating that our results are not a bias introduced due to the reliance on modern reference populations or close kinship (Supplementary Figures 26–28).

Fig. 2 Principal component analysis of Szólád and Collegno. a Procrustes PCA of modern Europeans (faded small dots are individuals, larger circle is median of individuals) along with samples from Szólád (filled circles), Collegno (filled triangles) and other Migration Period samples. Szólád and Collegno samples are filled with colors based on estimated ancestry from ADMIXTURE. NWE, modern northwest Europe; NE, modern northern Europe; NEE, modern northeast Europe; CE, modern central Europe; EE, modern eastern Europe; WE, modern western Europe; SE, modern southern Europe; SEE, modern southeast Europe; HUN, modern Hungarian. b Procrustes PCA of modern and Bronze Age Europeans along with samples from Szólád and Collegno and other Migration Period samples. Gray dots are modern Europeans as shown in a. NWb, Bronze Age northwest Europe; Nb, Bronze Age northern Europe; NEb, Bronze Age northeast Europe; Cb, Bronze Age Europe; Eb, Bronze Age Europe; Wb, Bronze Age western Europe; SEb, Bronze Age southeast Europe; ITb, Bronze Age Italy; ITn, Neolithic Italy Full size image

We next analyzed our ancient samples using supervised model-based clustering analysis implemented in ADMIXTURE30 against a worldwide panel of modern reference samples (Supplementary Note 8, Supplementary Figures 29–43). The major genetic component in both Szólád and Collegno is CEU + GBR (it was difficult to consistently distinguish the ancestry coefficients for these two populations), with a mean of 64% and 57% across samples, respectively. TSI is the second most prominent component (mean of 25% and 33% across samples, respectively) (Fig. 3). An unsupervised analysis on a set of unrelated samples from Szólád and Collegno demonstrated a similar CEU + GBR vs. TSI- like structure (Supplementary Figures 44–46). By crudely assigning individuals to five color-coded groups based on relative ancestry components, a clear correspondence can be observed between our ADMIXTURE and PCA analysis. Analysis of Y-chromosomes in males generally reveals a highly concordant pattern to the autosomes with regard to haplogroups that are most predominant in modern central/northern and southern Europeans (Supplementary Note 9, Supplementary Figures 47–49, Supplementary Data 3, Supplementary Table 2).

Fig. 3 Genetic structure of Szólád and Collegno. Model-based ancestry estimates from ADMIXTURE for Szólád (a) and Collegno (b) using 1000 Genomes Project Eurasian and YRI populations to supervise analysis. Note that high contamination was identified in CL31 and overlaid with a pink hue in b Full size image

A population assignment analysis (PAA) that estimates uncertainty in genetic ancestry assignment finds that individuals with high CEU + GBR ancestry are assigned to countries from all over modern central, northern, and northwest Europe (Supplementary Note 10, Supplementary Figure 50, Supplementary Data 4). We refer to this as central/northern ancestry as it is generally difficult to distinguish this with more precision given the resolution of our data. A series of D-statistics analyses of the form D (ancient, ancient, modern_reference, outgroup) confirms the close relationship amongst our ancient samples assigned as being of primarily central/northern ancestry from both Szólád and Collegno (there are even four Szólád-Collegno pairs that appear to form significant clade compared to all other modern populations), while those of southern ancestry show more potential structure (Supplementary Note 11, Supplementary Figures 51–60, Supplementary Table 3). An analysis of rare variants27 in our nine medieval whole genomes from Szólád is consistent in terms of relative amounts of central/northern and southern ancestry, demonstrating that our results are not the result of any SNP ascertainment bias (Supplementary Note 12, Supplementary Figures 61–64, Supplementary Table 4).

We also examined our ancient samples within the context of the prehistoric groups that were the major contributors to modern European genetic variation: Paleolithic hunter-gatherers (WHG), Neolithic farmers (EEF), and Bronze Age Steppe herders (SA). Both PCA and supervised and unsupervised ADMIXTURE analyses (Supplementary Figures 65-72) essentially reiterate the same structure amongst our ancient medieval samples, with greatest EEF ancestry in those individuals demonstrating similarities to modern southern Europeans and greater WHG + SA ancestry in those that resembled modern northern Europeans (with WHG being predominant in northwest Europe and SA in northeast Europe).

Relating our results to questions of migration requires us to understand to what extent the geospatial distribution of genetic diversity today reflects that of the Migration Period ~50–60 generations ago. While previous sampling from the era has been limited, we note that published fourth- to seventh-century genomes from Britain, Bavaria, Lithuania, and the Caucasus, analyzed alongside our own ancient samples, cluster close to their modern counterparts. The next temporally closest major European sample to the Migration Period involves a large number of recently characterized Bronze Age individuals that are ~100 generations separated from the Migration Period20,25,26. Though there are discrepancies, we find a general genetic similarity between individuals sampled from the same location today and in the Bronze Age at a continent-wide scale when considering northern and southern ancestry (Fig. 2, Supplementary Figures 73, 74), suggesting that the strong isolation-by-distance pattern observed in modern day Europeans was emerging ~4000 years ago (and presumably would have been even more similar ~1500 years ago). Based on PCA and D-statistic analyses (Supplementary Figures 75–84), individuals from Szólád and Collegno with high CEU + GBR ancestry that make up the majority of our sample are significantly closer to Bronze Age central, northwestern, eastern (Polish), and (at least using PCA) northern Europeans than Bronze Age Hungarians. We found no evidence that such ancestry was present in northern Italy during this time (who instead resemble modern southern and Iberian Europeans), which would be consistent with inferred long term barriers to gene flow in Europe across the Alps31. As noted previously26, Bronze Age populations and Hungarian Scythians from the third to sixth centuries BCE are diverse, with most sharing similarity with modern southern Europeans, though a minority are found in close proximity to the central/northern samples from Szólád and Collegno in the PCA. Overall, we suggest that based on modern and Bronze Age data, the high CEU + GBR ancestry observed in both Szólád and in particular Collegno is unusual.

Both cemeteries are organized around biological kinship

We utilized lcMLkin32 to infer pairwise biological relatedness within ancient cemeteries at an unprecedented level compared to previous studies33,34 (Supplementary Note 13, Supplementary Figures 85–87, Supplementary Data 5). We hereafter refer to groups of biologically related individuals as kindred as a shorthand, though we recognize that kinship in the traditional archeological/anthropological sense encompasses a much broader range of social relationships35.

Within Szólád we identified four kindreds among the Longobard era burials (gray shadings in Fig. 1b), with one particularly large one (Kindred SZ1, Fig. 1b). Kindred SZ1 (Fig. 4a) spans three generations and consists of ten individuals in close spatial proximity. Seven individuals all share recent identity-by-descent (IBD) from SZ24 (one of the oldest individuals in the cemetery, between ~45 and 65 years old (yo.)), while another two individuals, SZ15 and SZ19, are more remotely connected genealogically to the kindred via SZ6, a young male aged 8–12 yo. at the time of death. While SZ6 is related to all other individuals in this kindred, sharing the greatest IBD with siblings SZ8 and SZ14, we are unable to determine the exact genealogical relationships involved, likely because of its low SNP coverage (0.048×).

Fig. 4 Major kindreds in Szólád and Collegno. Kindreds SZ1 (a), CL1 (b), and CL2 (c), with colors corresponding to criteria and labeling in Fig. 2. ADMIXTURE coefficients (vertical bars on side of each individual) estimated using 1000 Genomes21 European populations only. Dashed lines indicate relationships of unknown degree (including past inbreeding in the parents of CL97). d Displays strontium isotope ratios for Kindreds CL1 and CL2 where available. Estimate of local range at Collegno is shown using black shading, as detailed in Fig. 4. Numbers indicate generations (1 being the oldest, 3 being the youngest) Full size image

Individuals in this kindred were buried with a rich diversity of grave goods, and all but one was buried in elaborate ledge graves. Only two members of this kindred are female, SZ8 and SZ19, who are estimated to be 3–5 and 17–25 yo. respectively at death. The rest are males, aged from ~1 to ~65 yo. These graves occupy a prominent position in the northwest of the cemetery, with all but SZ19 found amongst the core group (she is instead found in the external half-ring of women; Supplementary Figure 2). Six male individuals in this kindred were buried with weapons, despite three (SZ7, SZ14, and SZ15) being teenagers at the time of death (12–17 yo.). The adult males in this kindred (SZ24, SZ13, and SZ22) appear to have had access to a diet particularly high in animal protein, as inferred from nitrogen isotope analysis14. Individual SZ13 has the deepest grave and is the only individual in the whole cemetery whose burial includes a weighing scale and a horse, which may be an indication of his differentiated status in that society. We note that this kindred lacks adult female descendants of SZ24, though we were unable to sample some of the female graves in the half-ring structure (unsampled graves SZ21 and SZ29 could still be potential mothers of the third generation; individuals SZ17 and SZ26 can be excluded based on mtDNA analysis by Vai et al.36).

While individuals in this kindred are predominantly of a central/northern European genetic ancestry, they are not genetically homogenous. Again, SZ19 is an outlier, strikingly possessing 100% TSI ancestry. In addition SZ6 and the two third-generation siblings SZ8 and SZ14 also possess a small but noticeable TSI ancestry component. Assuming that one of the siblings’ parents represented the central European ancestry seen in their two uncles (SZ13 and SZ22), we inferred (using an adapted version of spatial ancestry analysis (SPA) Supplementary Note 14, Supplementary Figures 88–96) that the other parent likely possessed an ancestry that most resembled modern day French individuals (Supplementary Figures 92–93). This latter individual would probably have been female, as while SZ14 has a similar Y chromosome to SZ13 and SZ22, both siblings possess a different mtDNA type to their uncle's (I3 vs. N1b2).

In Collegno, we identified three kindreds, with one particularly extensive one. Nine of the ten individuals from the largest kindred (Kindred CL1; Figs. 1c and 4b) were buried in elaborate graves and/or with artifacts. In contrast to Szólád, individuals with close biological relationships occupied spatially distant graves. Interestingly, the spatial cluster with six individuals is chronologically older (570/590-610 CE) than the more westerly trio (Supplementary Figure 19). Kindred CL2 (Fig. 4c) is found in the east part of Collegno, with graves positioned in a row running north to south (Fig. 1c).

Similar to Kindred SZ1, Kindred CL1 is predominantly of central/northern European ancestry. However, while genetically quite similar, on average members of this kindred possess slightly less FIN ancestry and are thus more shifted towards northwestern Europe in the PCA, SPA, and PAA. In addition, this group is again not genetically homogenous, with the unsampled father of CL87 being of much greater central/northern European ancestry than the mother, CL102, who has an ancestry profile again most consistent with modern day France (Supplementary Figure 95). Kindred CL2 also has a more mixed genetic ancestry based on the ADMIXTURE analysis that would generally be associated with a more modern central European ancestry than Kindreds SZ1 and CL1. Interestingly the grave goods of the daughter, CL47, and an unsampled adjacent female, CL48, resemble burials from this time in southern France and Switzerland (Supplementary Note 3). Members of these two large kindred groups also appear to have generally consumed more animal protein than other individuals in the cemetery (Supplementary Figure 97).

Ancestry is associated with elements of material culture

In both cemeteries individuals with predominantly central/northern and southern European ancestry possess very distinctive grave furnishings. In order to quantify this relationship, we classified individuals into either Northern (N) or Southern (S) groups based on their proportion of CEU + GBR + FIN ancestry versus TSI + IBS ancestry, and used these to conduct a series of Fisher’s exact tests for their association with material culture (we note that our results were robust to our specific ancestry cutoffs; see Supplementary Note 15, Supplementary Table 5). We focused on artifacts potentially associated with either specific cultural traditions (e.g., S-brooches and stamped pottery) or individual profession or status (e.g., war weapons). In both Szólád and Collegno individuals with N ancestry were significantly more often buried with grave goods (p-value < 0.0071, Fisher exact test, Supplementary Table 6). In contrast, no S individual was buried with such artifacts, with only two exceptions (females SZ19 and SZ31). This association between genetic ancestry and material culture is particularly significant for beads (from necklaces and pendants) and food offerings in Szólád, as well as weapons in both Collegno and Szólád (Supplementary Table 7). We note that one artifact in grave SZ19 is stylistically distinct (possibly Roman) from the artifacts found in other graves in the same cemetery. Grave type (p-value < 0.02, Fisher exact test, Supplementary Table 8) also significantly differs between groups in both cemeteries, with N individuals presenting graves with wooden elements, as opposed to simple pits (more common amongst graves with S individuals).

Comparison of genetic and strontium isotope data

We also generated new strontium isotope data (87Sr/86Sr) for Collegno to complement the existing data from Szólád14 and analyzed them within the context of our genomic data in order to better understand patterns of immigration to these two sites (Supplementary Note 16, Supplementary Figures 97–104, Supplementary Tables 9-13). Within Szólád we find that adult individuals with both predominantly central/northern and southern genomic ancestry possess similar non-local signatures (Alt et al.14 described this as Range I) (Fig. 5). This might suggest that individuals from both ancestry groups immigrated into Szólád together despite the differences in material culture. However, we also note generally a quite diverse non-local range amongst adults with central/northern ancestry (for example SZ4 and SZ16 are extreme outliers), suggesting that not all individuals originated from the same location prior to settling in Szólád.

Fig. 5 Strontium isotope ratios in Szólád and Collegno. Evidence for migrants at Collegno and Szólád, as suggested by 87Sr/86Sr values of human tooth enamel and environmental reference samples. Isotopic data from Szólád were taken from the ref.14. The vertical lines indicate where multiple teeth were sampled from the same individual. The gray band denotes the local bioavailable strontium isotope range. Individuals falling outside of this are considered non-local. A: Collino di Superga (clays and marls); B: Lago Piccolo di Avigliana (glacial deposits); C: Castello di Avigliana (serpentinite, green schist); D: Collegno site (Pleistocene gravels); E: Bakony Mountains; F: vegetation south of Lake Balaton; G: water south of Lake Balaton; H: soil near Szólád (Szólád data from the ref.14). Color coding scheme corresponds to ancestry criteria and labeling in Fig. 2, though samples represented in black are those lacking genomic data. To ease readability of the figure, the 87Sr/86Sr values of each ancestry group are arranged in ascending order Full size image

In contrast, in Collegno it was notable that the five individuals with major southern ancestry primarily assigned to Italy using PAA exhibited local strontium isotope signatures. When examining the two major kindred, we observe the striking general pattern that earlier generations had strontium isotope values that diverged from the local range more than later generations (Fig. 4d, Supplementary Figure 100). This appears to fit a model of individuals of central/northern European ancestry migrating and settling in Collegno amongst a set of local individuals of primarily Italian origin.