Most Phoenician trading ports were initially founded in locations where there was already an established indigenous community, resulting in integration of locals into the growing Phoenician settlement. The settlement history of Ibiza offers a valuable opportunity to investigate the genetic makeup of a Phoenician population where there was a relatively very small indigenous population at the time of Phoenician arrival. Being a likely secondary Phoenician settlement from the city of Gadir, the early population of Ibiza also potentially provides information about Phoenician settlements on the Iberian mainland. The data presented here adds to our previous research investigating admixture in Phoenician settlements across the Western Mediterranean3,23. It also helps dissect and possibly identify the origins of the various populations that ultimately contributed to the genetic makeup of the modern Ibizans.

Our analyses of the mitogenomes of the ancient samples from Ibiza indicate a predominantly European maternal ancestry for the population (Fig. 1). Haplogroups H1 and H3, which either emerged from the Franco-Iberian refugia of the LGM50,51, or arrived with early Neolithic expansions, account for 50% of the Ibizan ancient samples (see Table 1). Szecseny-Nagy et al.38 also found similarly high rates of haplogroup H in their analyses of the Hyper-Variable Region (HVR) of Neolithic and Early Bronze Age Iberian samples. Investigating coding region SNPs, they showed that among haplogroup H, 65% belonged to hg H1 and 14% to hg H3. Particularly high frequencies of H1 and H3 are also seen in ancient and modern Sardinians3,34. The majority of the remaining haplogroups present in the Phoenician population of Ibiza, U4a, U5b, and T2b have all been identified in Mesolithic or Neolithic populations in Western Europe including Iberia38,52,53. Haplogroup U5b3 frequencies, though generally low across Europe, are the highest today in Sardinia and Spanish Andalusia54 and are thus also consistent with an Iberian origin or possible contacts with Phoenician Sardinia. Haplogroup J1c is thought to have an eastern Mediterranean origin and has been identified in several Neolithic samples in Eastern Europe and in a Chalcolithic sample (2880–2630 BCE) from the El Mirador Cave site, Atapuerca, Spain55. Haplogroup J1c has not been found previously in any ancient samples in the Levant and appears to have been distributed across Europe with the Neolithic expansion, suggesting that it may have been picked up in Northern Anatolia30. Olivieri et al.34 found a J1c3 haplotype in one of their ancient samples from Sardinia (MA74), dated to 6190–6000 calBP and thus its presence in our Puig des Molins samples is not unexpected, as this lineage was clearly present in the Western Mediterranean region at the time that Ibiza was settled. The primary Phoenician settlers of Ibiza are likely to have be males who sailed from Gadir and the presence of these haplogroups provides additional support to our previous work suggesting that local female integration was a hallmark of Phoenician settlement history across the Mediterranean basin3.

Our previous analyses of Phoenician mtDNA from Monte Sirai, Sardinia, another Western Mediterranean Phoenician and Punic settlement, indicated significant integration of indigenous women among the predominantly male Phoenician settlers3. We suggest a similar pattern is indicated for the Phoenician settlement of Ibiza. When we investigated the relationships of mitogenomes of Phoenician populations from Lebanon, Sardinia and Ibiza (Fig. 5) we found that there was no particular affinity amongst Phoenician populations (circles) when compared to pre-Phoenician or modern populations from those locations. This may indicate that local female integration was indeed a common strategy of Phoenician settlements. We expect that the integration of indigenous women from south central coastal Iberia into the Phoenician settlement of Gadir must have also occurred and that the ancient mitochondrial lineages observed Ibiza were brought along from Gadir or, alternatively, from some other Phoenician settlement in the Central or Western Mediterranean. Analyses of ancient Phoenician samples from Gadir and complete mitogenomic sequencing of ancient samples from across the Iberian Peninsula are needed to fully test this explanation. Analyses of HVR sequences from Neolithic and Early Bronze Age samples from the Iberian peninsula38 and modern southern Iberian populations56 indicate that there may be differences between the southwest and southeast Iberian populations, and it may be possible to locate particular regions of the Iberian coast from which these mtDNA lineages originated.

There was a sizable increase in the number of the Punic burials at Puig des Molins during the 5th and 4th centuries BCE, indicating a surge in the population size of Ibiza10. This increase is largely attributed by historians to the movement of people from other prospering Phoenician/Punic settlements in nearby Sardinia and Carthage10. Despite anthropological evidence supporting the presence of North and sub-Saharan African ancestry in Punic individuals from the Island of Ibiza57, mitochondrial lineages exclusively associated with North Africa or the Near East were not observed in our ancient Ibizan samples, although several of these lineages were previously identified in Phoenician samples from Sardinia, i.e. haplogroup N1b1a53. Our results cannot, however, rule out the arrival of admixed individuals with European maternal ancestry from Phoenician sites in Sardinia, North Africa or the Iberian mainland. In fact, we previously identified a likely Iberian mtDNA haplotype, U5b2c1, in a young Carthaginian23, suggesting the presence of an admixed population there by late 6th century BCE. We also note that haplogroups H1 and H3, while European in origin, are also found at high frequency in North African populations, particularly H1, which is today found at levels of 40–45% in northwest Africa56,58. Haplogroup H1bc was identified in a Bronze Age sample from Sidon29 and H1 is present in modern Lebanese3. It is therefore possible, that these H lineages could have been brought to Ibiza, with Phoenicians from either the Levantine homeland or North Africa. It is a more parsimonious suggestion, however, that their presence in Ibiza is due to a more direct European and probably and Iberian source, particularly given the lack of any mitogenomes of clear North African or Near Eastern origin identified in the ancient Ibizan population sampled thus far. The morphological characteristics indicating African ancestry in crania from Punic contexts in Ibiza identified by Márquez-Grant57 could be consistent with our aDNA results if the African admixture in Ibiza was male dominated. Alternatively, aDNA analyses of additional Phoenician samples from Ibiza may indeed find non-European mtDNA lineages, but to date, we have only observed what appears to be a typical European, and primarily Western European signature.

Unfortunately, to really dissect population mtDNA histories generally, and those of southwest Europe or any region with high rates of haplogroup H in particular, complete mitogenome sequencing is necessary. Despite the large sample sizes for studies of HVR sequences for the Iberian Peninsula38,59, there are few complete ancient mitogenome sequences publicly available particularly beyond the Basque region. As Hernandez et al.56 point out, secure assignment to haplogroup H is not possible based on control region sequencing alone, as it requires identification of T7028C. Further sub-typing of haplogroup H also requires the identification of key coding region SNPs (for example, G3010A for H1, T6776C for H3).

In addition to the ancient Ibiza samples, we sequenced a complete mitogenome of a Late Chalcolithic/Early-Bronze Age sample (MS10589) from the Ca na Costa site on the island of Formentera which was determined to belong to the K1a1b haplogroup. Mathieson et al.55 identified K1a1b1 in a Middle Neolithic Iberian sample (Mina3) with an archaeological context dated to 3900–3600 BCE. In their analyses of mtDNA hypervariable region sequences obtained from ancient samples from Neolithic and Iron-Age necropoli from Mallorca and Menorca, Simón et al.39 found that 4.34% belonged to Haplogroup K, with the highest frequency (10%) in the oldest (Neolithic) necropolis, Cova des Pas, on Minorca. Our K1a1b result may indicate that the initial settlement of Formentera, prior to the arrival of the Phoenicians, also likely came from the mainland or via Mallorca, an observation presented by Bellard4. Olivieri et al.34 found K1a and K1b1b1 in pre-Phoenician Sardinians, but we have yet to find haplogroup K in any of our Phoenician samples.

Ibiza has had a complex history of resettlement periods since the time of the Phoenicians. In addition to a Phoenician founding population, modern Ibizans would have had potential genetic influences from later Punic settlements, like Carthage and our ancient DNA data are consistent with this scenario. Historic records would suggest that the Ibizan genetic signature was also likely influenced by the arrival of Arab and Berber populations during the first millennium CE and finally, from 1200 CE, it was said to be overwhelmed by mainland colonists. This might explain the discontinuity between the ancient and modern DNA results we observed, shown in Fig. 3 and is consistent with the results of Ramon and colleagues13, based on HLA markers and blood groups in Ibizans, and earlier mtDNA14 and Y-chromosome15 studies which show affinities to North African and Near Eastern populations.

Our TempNet results showed little continuity between the Phoenician inhabitants of the island and the modern Ibizan population. No shared haplotypes were found and only one haplogroup, T2b, was found in both groups. Perhaps most surprising was that neither haplogroups H1 or H3 were found in the modern Ibizans. These two haplogroups were not only the most common in our ancient population but are found at high frequencies in both modern North African and Western European populations generally56. The modern Ibizan population is singularly unusual in the entire Western Mediterranean region for their apparent lack of these two common haplogroups. It is possible that the mtDNA frequencies are so different from the ancient samples due to random genetic drift, which is common in small island populations. The small sample size (n = 18) could also be a factor, though our result indicating that the modern Ibizans are genetically quite different to mainland populations is consistent with previous studies13,14,16,39.

Haplogroups T1 and V, found in the modern Ibizans, are lineages found at reasonably high frequencies in the Near East. Haplogroup V is also found in both ancient and modern populations from the Iberian Peninsula and in ancient samples from Mallorca and Minorca, and T1 is also found at low frequency in mainland Spain today14,32,55. These lineages are also found in Berbers which could explain their presence in the modern Ibizans. Haplogroup L2c however, which was identified in two unrelated, modern Ibizans, has not been found in Berbers60. It is typically a West African, sub-Saharan lineage and it may have been introduced during the Islamic expansion which had significant exchange with the entire African continent. Alternatively, it could have been a more recent arrival, perhaps the result of the transatlantic slave trade59. Botigué et al.61, showed in their whole genome analyses that sub-Saharan ancestry was less than 1% in Europe, except for the Canary Islands, where Maca-Meyer et al.62 found L2c in both 17th and 18th century historic and modern individuals. Another sub-Saharan lineage, L1b has also been identified in a Late Chalcolithic population in central Iberia38 and in one 7th century CE sample from Mallorca39.

The remaining lineages identified in modern Ibizans are all either exclusively European markers (e.g. J2b1a63) or are widely distributed across Europe and the Near East and identifying the specific origin of the population influx that causes the discontinuity between ancient and modern Ibizans is difficult. The F ST results for the complete mitogenomes shown in Fig. 4 do not indicate a single likely source, though the closest population to modern Ibizans appears to be France. France may be acting as a proxy for a more likely source of settlers to Ibiza, namely Catalonia, since it has been shown to be genetically closely related64. Further whole genome analyses of the modern indigenous Ibizans would provide the necessary data to fully assess the origins of this population, but from a mitochondrial perspective, it appears that they are not directly related to the Phoenician and Punic founding populations and thus this is not the explanation for the unusual genetic signature.

While the mitochondrial evidence suggests that the founding female population of Ibiza was primarily derived from a mainland Iberian source, the results of the whole genome analyses of sample MS10614 indicate a significant Eastern Mediterranean/Levantine component. Sample MS10614 comes from an individual that was part of a collective burial inside a Punic hypogeum at Can Portes des Jurat, Cas Molí20. The sample was directly radiocarbon dated to 361–178 cal BCE. The archaeological context of the burial also suggests a 3rd century BCE date and thus this individual lived during the period of greater Punic influence of Ibiza or just after this period of population growth.

As can be seen in Fig. 6a,b, the Ibizan Phoenician sample, MS10614, plots in between modern Levantine and Iberian populations but closer to both of these than to modern North African populations. In Fig. 6c, it is positioned most closely to a Levantine Neolithic sample, but in between a Sidon Bronze Age and European/Iberian Bronze Age samples. The ADMIXTURE result (k = 2) in Fig. 7 also indicates that the genetic ancestry of this individual was intermediate between an Iberian Bronze Age and Levantine Bronze Age individuals. The result of k = 3, however, shows a third component (dark blue) found in Anatolian and Levantine Neolithic and Bronze Age individuals. This component is present in the ancient Ibizan and also present in modern Spanish. We suggest that this third component represents the Western Iranian Neolithic farmers that had admixed with the Levantine and Anatolian Neolithic populations30. Levantine Bronze Age populations were further admixed with this component from both Anatolian Chalcolithic and Levantine Neolithic ancestry. This component is not present in ancient Iberians, supporting the notion that the main ancestral population of early European farmers were the Neolithic North-Western Anatolians before they were admixed with the Iranians30. The presence of this component in modern Iberians is likely the result of historic influences.

The mtDNA haplogroup of this individual, T2b, indicates that the direct maternal ancestry is likely local and the Levantine contribution may therefore represent paternal ancestry. This finding is in line with previous work by our group using Y-chromosome data showing East Mediterranean Y signatures across the Phoenician settlements in the West, including Ibiza. These signatures, attributed to the Phoenicians, were found to be present at the frequency of 6% among the modern male populations studied, indicating a substantial Phoenician male presence in settlements across the Western Mediterranean65. The Near Eastern component to the genome of this individual may represent that of the founding Phoenician population, though the possibility that this individual or a recent ancestor may have arrived directly from the Levantine homeland or from Punic settlements in North Africa or elsewhere in the Phoenician interaction network cannot be ruled out, though the sample does not appear to be closely related to modern North African populations as shown in Fig. 6b.

In conclusion, our results show a complex pattern of settlement of the island of Ibiza. We demonstrate a clear genetic discontinuity between the early Phoenician settlers and the modern inhabitants of the island based on the mtDNA results. Thus, the unusual genetic signature of modern Ibizans is not likely to be the result of their Phoenician ancestry, at least from a mitochondrial perspective. It appears that multiple population arrivals through invasions or other movements combined with periods of population instability since the early Phoenician settlement may have led to a total reshuffling of the genetic makeup of this island. Over the last several centuries Ibiza witnessed population growth supported by the arrival of mainland populations to the island, followed by significant population reduction resulting from the bubonic plague and malaria13. These events, combined with a founder effect, and inbreeding common in islands with a relatively small population such as Ibiza, could have resulted in the loss of the indigenous mtDNA signatures observed in the ancient samples we analysed. It appears that the lineages present in the Phoenicians of Ibiza were replaced by different European mtDNA haplogroups (which appear to be most closely related to those of modern French but likely also similar to Catalonians). Despite this lack of continuity observed in the mitochondrial genomes, previous Y chromosome analyses suggest that there is still some Phoenician signature in the modern Ibizan population. This is consistent with historical evidence suggesting that Phoenician influence in the West was male dominated and indicates that there was not a total replacement of the Ibizan founding population. Comparisons of the ancient Phoenician whole genome data with whole genome data from modern Ibizans will help clarify this point further. Finally, the whole genome data obtained from the ancient Ibizan sample belonged to an individual with a significant Eastern Mediterranean component, suggesting an admixed Phoenician community in Ibiza during the 3rd century BCE. While this result is consistent with the archaeological evidence and further indicates that diversity was a hallmark of Phoenician societies, it also highlights the complexity of island population settlements and underscores the importance of the inclusion of ancient DNA analysis in population genetics.