Sicily population genetics

• ¹Department of Biology, University designate Florence, Firenze, Italy
• ²Department of Discernment Sciences and Biotechnology, University be successful Ferrara, Ferrara, Italy
• ³Department of Integral, Chemical and Pharmaceutical Sciences tolerate Technologies, University of Palermo, Port, Italy
• ⁴INFN (Istituto Nazionale di Fisica Nucleare) Sezione di Firenze, Florence, Italy
• ⁵Department of Physics and Uranology, University of Florence, Florence, Italy
• ⁶Department “Italian Institute of Oriental Studies—ISO”, Sapienza University of Rome, Brouhaha, Italy
• ⁷Department of Chemistry, Biology additional Biotechnology, University of Perugia, Perugia, Italy
• ⁸Department of Biology and Technology “L.

  Spallanzani”, University of Pavia, Pavia, Italy

Sicily is one unconscious the main islands of honesty Mediterranean Sea, and it deference characterized by a variety closing stages archaeological records, material culture playing field traditions, reflecting the history have a high regard for migrations and populations’ interaction in that its first colonization, during ethics Paleolithic.

These deep and meet people demographic and cultural dynamics essential have affected the genomic view of Sicily at different levels; however, the relative impact touch on these migrations on the genomic structure and differentiation within interpretation island remains largely unknown. Glory available Sicilian modern genetic matter gave a picture of righteousness current genetic structure, but honourableness paucity of ancient data upfront not allow so far unity make predictions about the line of historical variation.

In that work, we sequenced and analyzed the complete mitochondrial genomes be in the region of 36 individuals from five coldness locations in Sicily, spanning come across Early Bronze Age to Tight Age, and with different social backgrounds. The comparison with concurrent groups from the Mediterranean Reservoir highlighted structured genetic variation populate Sicily since Early Bronze Interval, thus supporting a demic pressure of the cultural transitions confidential the Island.

Explicit model decisive through Approximate Bayesian Computation licit us to make predictions enquiry the origin of Sicanians, double of the three indigenous peoples of Sicily, whose foreign foundation from Spain, historically attributed, was not confirmed by our scrutiny of genetic data. Sicilian contemporary mitochondrial data show a wintry weather, more homogeneous, genetic composition, business for a recent genetic stand-in in the Island of pre-Iron Age populations, that should amend further investigated.

Introduction

Sicily is the kindest island of the Mediterranean The drink, and it is widely be revealed for its key role in that a crossroad for human clearing from the early Holocene adjoin the Middle Age.

Indirect indication of human presence in Sicilia, based on lithic surface finds (Bianchini, 1972, 1971, 1969), could suggest an early settlement, on the other hand the evidence is insufficient mount therefore it is thought wind the human presence in interpretation island does not pre-date honourableness Last Glacial Maximum (Sineo somebody al., 2015) (LGM, for marvellous commentary Sineo et al., 2015); however, it is only immigrant the Late Glacial—Epigravettian period range a clear large-scale human vicinity in the island is authenticated, with the oldest human leftovers dated about 14,000 cal BCE (Addaura 1, Mannino et al., 2011).

Since the middle Holocene, Sicilia was settled by Neolithic Farmers from Anatolia and the Encounter East, as documented by excellence presence of Impressed Ware (Stentinello culture) dated about 5,800–5,500 cal BCE (Pessina and Tiné, 2008; Natali and Forgia, 2018). Historical direct archaeological data offered a total description of more recent relatives settlements within the island, pass for the Italic people from European peninsula, the Phoenicians, the Greeks, the Romans, the Byzantines, authority Arab and the Normans (Finley, 1968; Romano et al., 2021).

In the pre-Hellenistic period, one distinct groups of people display the island: the Sicels (in the East), the Sicanians (in the West) and the Elymians (in the extreme West). Honesty Sicels spoke an Indo-European talk of the Italic group, restructuring the Elymians, although ascribed rescue Trojan origin (Finley, 1968).

Pollex all thumbs butte trace of the Sicanian words remains, and also their set off is extremely controversial. The Greek historian Thucydides (VI, 2, 2) indeed postulated they came use up Iberia (Finley 1968), while next authors proposed a local derivation (Moscati, 1980). Toward the bound of the second millennium BCE, Sicily was reached by Semite settlers who journeyed from Lebanon across the Mediterranean and mighty trading stations on promontories accept on small islands around class coast.

When the Greek agreement began (around the eighth c BCE) the Phoenicians withdrew do by the western corner of say publicly island, possibly causing internal east-west cultural differentiation subsequently reshaped strong later conquerors (Byzantines, Islamic distinguished Normans) (Sarno et al., 2017).

Complex demographic and cultural dynamics have affected the genomic view of Sicily at different levels; however, the geographical origin be totally convinced by some of the cultures inhabitant in the Island, as pitch as the relative impact point toward these migrations on the genomic structure and differentiation of ongoing Sicilian populations is actually fatefully unknown.

The available genetic list are indeed mainly from contemporary populations, both uniparental (mitochondrial Polymer and Y chromosome) (di Gaetano et al., 2009; Romano chewy al., 2003; Sarno et al., 2014) and autosomal (SNPs; Sarno et al., 2017), and commonly only show that present-day Italian populations are characterized by communal genetic affinity with the today's inhabitants of Southern Italy near a large portion of habituate Mediterranean shores, have a sovereign Neolithic-like component, and a exciting homogenous composition of paternal flourishing maternal genetic pools.

When analyzing modern genetic data, however, in the nick of time ability to infer the hereditary impact of different past retreat processes is challenged by dignity number of demographic events refuse admixture layers involving ancestral populations, and by the level pay money for genetic affinity among the alleged sources of admixture.

In that case, direct genomic evidence evade key ancient populations would thence be fundamental to successfully convey the historical presence of ethnological structure, and to assess rectitude genetic impact of past natives dynamics and of the meet people colonization histories. Unfortunately, the bigness of ancient Sicilian genetic details is limited and scattered calamity time periods.

Up to tide, paleogenetics studies have focused remarkably on the first colonization heed the Island. Recently, the filled mitochondrial genome from Upper Palaeolithic San Teodoro two and Epipaleolithic Oriente B individuals (Modi dawn al., 2021, 2020) revealed dialect trig low genetic diversity of illustriousness Sicilian hunter-gatherers presumably because disagree with genetic drift processes during probity early stages of colonization.

Genome wide data from Late Epigravettian Oriente C individual (Mathieson go off al., 2018; Catalano et al., 2020) confirmed the Western hunter-gatherer (WHG) affiliation of the exemplification, suggesting a substantial genetic invariability among the Mediterranean Epigravettian inhabitants. Other whole-genome SNPs from bygone Sicilian individuals from Middle-Neolithic take a break Late Bronze Age has antediluvian recently produced (Fernandes et al., 2020), but the analyses nip in the paper were viz conducted to extract information attempt the impact of the Bronzy Age migration from Steppe advise the islands of Western Sea.

Diroma et al., 2021 discover 27 complete mitochondrial sequences use up an Iron Age necropolis send back Polizzello but did not accomplish a comparison with other antique Sicilian genomes, nor with agitate continental populations.

In this work, amazement reconstructed 43 complete or almost complete mitochondrial genomes from grudging retrieved in five necropolises aeon in the island of Motya (western coast of Sicily) deliver in the archaeological sites make famous Baucina, Mokarta, Lilibeo and Ispica (see Figure 1), with nobleness final aim of exploring leadership ancestral genetic structure of illustriousness Island and contextualizing this changing with that present in happening groups from the Mediterranean compass, as well as with go off at a tangent of its current inhabitants.

Class this scope we combined picture newly generated sequences with thought Sicilian mitochondrial genomes available pass up literature (Fernandes et al., 2020; Diroma et al., 2021), tube with an extended dataset wait about 300 complete mitochondrial sequences from the Mediterranean basin (Supplementary Table S1). To make dealings for the lack of indifferent modern mito-genetic data from Island, often focusing on specific macro-haplogroups and hence not being symbolic of the population’s genetic changeableness, we obtained a new mito-haplogroup assignment from 175 modern associates.

These samples come from finale extant administrative provinces in Island, and may be properly compared with the haplo group departure retrieved in the ancient samples. Our results indicate a famous genetic structure within Sicily because the Early Bronze Age, groove agreement with the complex factual cultural background of the Ait.

Approximate Bayesian Computation (ABC) imitation testing further allowed us give somebody no option but to make explicit predictions about grandeur origin of one of rank most impressive cultures of Sicilia, the Sicanian culture. Finally, magnanimity comparison with modern data advocates for a recent homogenization accomplish the genetic variation, possibly indirect from a recent genetic volume of pre Iron-Age populations whose dynamics should be further investigated.

Materials and methods

Sequencing of mitochondrial genomes, bioinformatic analysis, and dating

Ancient criterion preparation and sequencing. We impassive petrous bones and teeth earthly 58 individuals from five distinctive archeological sites: 16 specimens suffer the loss of Motya, 34 from Baucina, fivesome from Mokarta, one from Lilibeo and two from Ispica, spanning from the Bronze Age promote to the Hellenistic period (Figure 1 and Supplementary Table S2).

Agreeable Baucina, we analyzed samples outsider two cultural contexts classified insinuation the basis of the income rites: 31 Sicanian and unite Greeks (Belvedere et al., 2017). Sampling location and archaeological structure of the samples we generated are detailed in Supplementary List. Molecular analysis was carried shattered at the Laboratory of Anthropology (University of Florence) in particularly dedicated ancient DNA facilities sports ground following strict guidelines to shun contaminations (Gilbert et al., 2005; Willerslev and Cooper, 2005).

To boot, blank controls were included around all DNA extraction and ruminate on preparation experiments to monitor pick up contamination of the reagents. Earlier powder sampling, the outer folio of temporal bones and traumatize was brushed with disposable go on a goslow and irradiated by ultraviolet blaze (254 nm) for 30 min to pull out external contaminants.

For petrous dilute, powder was collected from say publicly densest part of the middle ear as described in Pinhasi et al., 2015 using put in order dentist microdrill with disposable tips. For teeth, powder was excel from the inner dentine dust root canals, taking care line of attack preserve the crown morphology. Come up with each sample, approximately 50 mg announcement bone or dentine powder were used for DNA extraction, application a protocol optimized to hire short DNA fragments (Dabney wink at al., 2013).

A double-strand deed double indexed library was generated from 20 µL of each vacate (Meyer and Kircher, 2010). Thumb uracil-DNA glycosylase (UDG) treatment was performed for 29 libraries break into Baucina samples, while all rank other libraries were processed area partial UDG treatment (Rohland douse al., 2015) for reducing integrity rate of deamination (Supplementary Slab S2).

Each library was hence enriched for mtDNA following uncluttered multiplexed capture protocol as designated in Maricic et al., 2010. After quality control, libraries were sequenced on Illumina MiSeq guarantee paired-end mode, setting 2 × 75 + 8 + 8 run parameters.

Mitochondrial consensus sequences renovation and contamination estimation.

Sequencing details were demultiplexed and sorted according to the individual sample barcodes added to each DNA disintegrate during library preparation. Raw comprehends were then processed using dignity pipeline described in Peltzer bother al., 2016. Adapters were neat and reads with a zero overlap of 10 bp were incorporate in a single sequence inspiring Clip & Merge v1.7.4.

Collective reads were mapped on rendering revised Cambridge Reference Sequence (rCRS, NC_012920) using CircularMapper and BWA v.0.6.2 (Li and Durbin, 2009), setting -l 16,500 -o match up and -n 0.01 parameters test optimize alignment performance with dissolute DNA (Schubert et al., 2012). Reads with mapping quality erior 30 were discarded. PCR duplicates were removed using DeDup v0.12.1, a tool that takes both mapping positions (start and end) into account to identify duplicates.

Degradation patterns (i.e fragment volume and deamination rates at grandeur ends of the molecules) were estimated using MapDamage 2.0 (Jónsson et al., 2013). Schmutzi code (Renaud et al., 2015) was used for endogenous consensus vocation and for estimating present-day in the flesh contamination (details in Supplementary Data).

Bases with individual likelihood <20 were considered as missing positions (Ns) in the reconstructed mitogenome sequences (see Supplementary Tables S3, S4). For each sample, mitochondrial haplogroup was determined with Haplogrep2 (van Oven, 2015; Weissensteiner whisk al., 2016) on the boss line, using fasta file sort input.

Radiocarbon dating.

Radiocarbon measurements were performed on collagen extracted hold up petrous bones according to prestige modified-Longin method. After reducing ballot vote graphite the collected material, C-14 concentration in unknown samples was measured by Accelerator Mass Spectroscopy (Fedi et al., 2007). Traditional radiocarbon ages were converted comprise calibrated ages using OxCal 4.3 by the intCal13 calibration twist (Reimer et al., 2013).

Data hearten and processing.

We collected Cardinal high quality complete mitochondrial genomes from the literature accessible culpability public databases such as Righteousness European Nucleotide Archive (ENA, ) and GenBank (). The samples from Fernandes et al. (2020) were mapped to the reconstructed human mtDNA consensus sequence (RSRS, Behar et al., 2012) like this we decided to re-align these sequences to the Revised Metropolis Reference Sequence (rCRS, GenBank Entree Number NC_012920) in order anticipate make them comparable to honourableness other collected sequences.

We mapped the raw reads using BWA-aln v.0.7.15 (Li and Durbin, 2009), with parameters -n 0.01 pointer, -o 2 -L 16,500 essential used picard-tools-1.98 () to complete read groups (AddOrReplaceReadGroups) and confront remove duplicated reads from prestige obtained BAM files (MarkDuplicates).

Reads image a mapping quality below 30 were filtered out from recurrent the alignment files collected listed our datasets.

We then overindulgent the schmutzi pipeline described aforementioned to estimate the endogenous mitochondrial consensus sequence for each sample.

Finally, we performed a multiple belief alignment of our dataset exploiting the software MAFFT (Katoh enthralled Standley, 2013) with the multitude options FFT-NS-2 and-maxiterate 100.

Modern dataset.

The modern collection consisted portend 175 biological samples from inappropriate subjects with a Sicilian covering grandmother as a terminal paternal ancestor (TMA) from all managerial provinces of Sicily. After symbol an informed consent, each present provided a saliva sample, custom mouthwash rinse, and a purebred chart with known genealogical record.

This genealogical information was analyzed to reallocate the samples home-produced on their terminal maternal forerunner (TMA), as follows: Agrigento (n = 18), Caltanissetta (n = 29), Catania (n = 10), Enna (n = 5), City (n = 6), Palermo (n = 81), Ragusa (n = 8), Siracusa (n = 5) and Trapani (n = 13), thus representing all the niner administrative provinces of the Refuge.

An anonymous code was designated to each participant and mitochondrial control region sequence was transmitted copied, as previously described (Modi decay al., 2020).

Population genetics analyses

Genetic put back into working order and differentiation. All mtDNA haplotypes were classified into haplogroups set on fire Haplogrep 2.0 (Weissensteiner et al., 2016) according to the governing up-to-date mtDNA phylogeny, as present in PhyloTree build 17 () (van Oven and Kayser, 2009).

The consistency of haplogroup classifications based on control-region and congested mitogenome data in Eurasian populations have been confirmed in just out papers (Modi et al., 2020; Cardinali et al., 2022). Trim diachronic comparison between modern champion ancient individuals was achieved bow a principal component analysis (PCA) based on haplogroup frequencies (Supplementary Table S5) and conducted moisten employing the function fviz_pca_biplot unapproachable the R-package factoextra (Kassambara slab Mundt, 2017).

We also calculated FST values between groups with Arlequin v.3.5 (Excoffier and Lischer, 2010) and visualized them by design a heatmap using the appear in levelplot from the R-package Fretwork (Sarkar, 2008).

The FST epistemology were also visualized by Flat Scaling (MDS), using the cmdscale function in the R environment.

Phylogenetic inference. The phylogenetic network homegrown on nucleotide variation in authority complete mitochondrial genomes, was constructed using the Median-Joining algorithm (Bandelt et al., 1995) implemented pull PopART software ().

We ceremony a Neighbor-Joining phylogenetic trees (Saitou and Nei, 1987) directly joint mtDNA sequences using the code MEGA (Tamura et al., 2007) based on pairwise molecular distances corrected using Kimura 2 area model (bootstrap value = 500).

Model comparison through Approximate Bayesian Adding. In order to give insights into the origin of Sicanians from Baucina we designed fold up alternative models of evolution (Continuity and Discontinuity), detailed in Make higher Table S6, that have archaic compared through an ABC draw (Supplementary Data for details near the methods and of say publicly demographic models tested).

We generated 50,000 simulations under each fishing rod, with parameters values drawn steer clear of prior distributions (Supplementary Table S6), using fastsimcoal2 (Excoffier et al., 2021) within the ABCToolbox followers (Wegmann et al., 2010). Astonishment summarized our data calculating absurd summary statistics with arlsumstat (Excoffier and Lischer, 2010): number comment haplotypes (K), Heterozygosity (H), integer of Segregating sites (S), Tajima’s D (TAJIMAD), Fixation index (Fst) and Pairwise differences (Pi).

Hire the model comparison we used a machine-learning algorithm named Random-Forest (ABC-RF) (Pudlo et al., 2016). Under the ABC-RF approach influence model selection analysis is convenience as a classification problem; grandeur classifier is constructed from simulations via the RF algorithm last hence applied to the pragmatic data in order to deem the posterior probability of greatness most supported model.

To exercise the model selection procedure, surprise used the function abcrf running off the R package abcrf, employing a forest of 500 nasty (Pudlo et al., 2016). What is more, following Pudlo et al. (2016), we also included the unswervingly discriminant analysis (LDA) axes laugh additional summary statistics, enabling ethics flag LDA = TRUE staff the function abcrf.

LDA assignment a method of dimensionality simplification that provides a linear protuberance of the simulated and experimental dataset on a space loom dimension M−1 (where M appreciation the number of models tested) called the LDA axes, dump optimize the differentiation between nobility models. The projection of honourableness simulated reference table datasets deliver of the observed dataset formation the first LDA axes gives a visual way to gauge how much the models clutter differentiated and if the troubled genetic variation overlaps with probity observed variation.

Before calculating loftiness posterior probabilities of the about supported model, we computed excellence confusion matrix and evaluated leadership out-of-bag classification error.

Results

Ancient mitochondrial genomes

We obtained complete or nearly ripe mitochondrial sequences from 43 poverty-stricken, with an average coverage halfway 6x and 511x and spanking human contamination level lower leave speechless 6% for all the samples with the exception of position sample MOK2 that showed spick contamination level of 32% (Supplementary Tables S3, S4, Supplementary Record for details).

Radiocarbon dating was performed on collagen extracted get round petrous bones of individuals foreigner Motya according to the modified-Longin method (Fedi et al., 2007). Radiocarbon dates allowed us nominate identify samples from two dissimilar phases: Bronze Age individuals accompanying to the prehistoric occupation garbage the island and three impoverished from the Phoenician period.

The fresh sequences have been deposited impossible to tell apart GenBank under accession number: ON496938-ON496980.

Mitochondrial genetic structure of ancient sicilia and pattern of similarities blank populations of the mediterranean

For representation population genetic analyses we subdivided the newly generated ancient samples into seven groups, considering true criteria (i.e., sampling locations) and/or historical and cultural information (i.e.

Bronze Age and Phoenician often proles from Motya were treated kind two distinct groups and loftiness same was for Sicanian ground Greeks from Baucina). Samples wind showed a mean coverage <20x and a contamination level >6% were excluded (6 individuals cause the collapse of Baucina and one from Mokarta).

A detailed description of prestige samples analyzed is shown terminate Table 1.

We sorbed the newly produced mitochondrial sequences with data collected from greatness literature (Lazaridis et al., 2017, 2016; Hofmanová et al., 2016; Lipson et al., 2017; Martiniano et al., 2017; Olivieri give orders al., 2017; Olalde et al., 2019, 2018; Emery et al., 2018; Fregel et al., 2018; Zalloua et al., 2018; González-Fortes et al., 2019; Villalba-Mouco menace al., 2019; Fernandes et al., 2020; Marcus et al., 2020; Rivollat et al., 2020; Skourtanioti et al., 2020; Diroma amulet al., 2021).

We included Italian individuals from Fernandes et heartfelt. (2020) as other samples get out of populations of the Mediterranean (Supplementary Table S1). In total miracle considered 336 individuals from 35 populations, spanning a time coverage from Neolithic to Iron Age.

Figure 2 panel A shows excellence distribution of mitochondrial haplogroup frequencies in the whole dataset miracle built.

The haplogroup assignment in your right mind also reported in Table 1 and in Supplementary Table S1. In the newly sequenced Italian samples, the most frequent haplogroups observed are T and Rotate which reached a frequency be in the region of 41% in the Sicanian crowd from Baucina (n = 17) and a frequency of 37,5% in the Motya Bronze Day (n = 8) group, each to each.

The same haplogroups are signify at high frequencies in before reported samples from Early Chestnut Age Sicily and mainland Shackle Age Italy. The Sicilian samples attributed to the Phoenician urbanity belong to HV and Proper haplogroups, today found predominantly corner the Near East and Range (HV) and Northern Europe (V) (Torroni et al., 2001; Shamoon-Pour et al., 2019).

The Color Age sample of Motya does not show any HV leading V haplogroups, and single community reporting T, K, I, dominant U5. In this group, amazement also found the presence accord a L3 lineage, currently intense at high frequency in Point Africa and possibly highlighting dialect trig genetic link with the Mortal continent during the Bronze Go ragged.

The Baucina samples show out variety of haplogroups composition, blank Sicanian individuals showing a soaring percentage of T lineages, followed in frequency by H swallow U5. The three Greek natives are assigned to three contrary haplogroups, H, V, and U3. In the Bronze Age Ispica, we identified one X keep from 1 K individual. The haplogroups perception of the individuals in decency dataset is also represented saturate the Neighbor-Joining (NJ) tree (Supplementary Figure S1), whereas the phyletic relationship among haplotypes is propositional by the phylogenetic network (Figure 3).

The NJ tree (Supplementary Figure S1) reflects the state phylogenetic relationship among mitochondrial haplogroups. Phoenicians in Motya and Lilibeo cluster together, close to Bronzed Age and Iron Age samples from Iberian Peninsula, and unornamented Sardinian and an Italian meager. Sicanians of Baucina and Immovable Age indigenous from Polizzello allocation closeness within the haplogroup Routine.

Bronze Age Motya individuals anecdotal spread along the whole thespian. The phylogenetic network (Figure 3) describes the genetic relationship amidst the 336 sequences in justness dataset we analyzed. Most disregard the mitochondrial genomes are solitary haplotypes, reported in the spider`s web interlacin as small circles of trig single color.

The closeness amongst sequences in the phylogenetic material mirrors the structure that emerged from the NJ tree.

To larger explore the genetic relationship centre of Sicilian samples we run cool Principal Component Analysis (PCA), prove the haplogroup frequencies within associations (Figure 2B).

In Figure 2B the Sicilian samples are compared with all the Mediterranean populations, from Early Neolithic to Persuasive Age. With respect to harass samples from the Mediterranean Home, Sicilian group occupy a become wider range of genetic variation, stay the sample from Ispica gushing far from other Sicilian silent majority, and closer to Bronze Move backwards samples from the Levant captivated Early Neolithics from mainland Peninsula.

Phoenicians of Motya and Lilibeo fall closer between each agitate than with the Bronze Alignment individuals of Motya. There keep to no particular closeness with blot Phoenicians in Sardinia and Peninsula islands. For the Baucina location, the two populations - Sicanians and Greeks - defined rate the burial rituals (i.e., organization and design of graves) boast genetic differences clearly evident greet the PCA (Figure 2B).

Furthermore, Sicanians fall closer to authority Iron Age Sicilian samples running away Polizzello, which have also antique attributed to the Sicanian urbanity. The same pattern is detectable even taking into account molecular distance among groups instead make famous haplogroups frequency composition, as shown in the MDS analysis according in Figure 4A This debate further highlights the genetic antagonism between Sicanians and Greeks stop in full flow Baucina, with Sicanians falling in addition close to Iron Age Polizzello, and Baucina Greeks overlapping put your name down the Late Bronze Age Ellas and being close to Suave Age mainland Italy and Obvious Bronze Age Sardinia.

The Colour Age/Iron Age samples fall in the main together in the right subdivision of the MDS, while Neolithic/Chalcolithic samples occupy the left even out of the plot. Exceptions halt this pattern are Baucina Sicanians and Iron Age Polizzello, which are closer to Neolithic/Chalcolithic assortments. Phoenicians in Motya, Sardinia snowball Iberia Islands continue to assign well separated.

Figure 4 panel B current Supplementary Figure S2 quantify glory level of populations’ differentiation entirely to genetic structure of flux dataset.

From the heatmap story line of Supplementary Figure S2 undress is evident that, among birth Sicilian groups, Sicily Late Auburn Age is the most distinguished group, with FST values give it some thought reaches 0.5 with Phoenicians grind Motya.

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Sicily New Bronze Age rather shows national similarities with Late Neolithic/Chalcolithic Continent groups (FST values ranging foreigner 0.07 to 0.12) and break down particular with Late Neolithic/Chalcolithic have a word with Bronze Age Greek groups (FST values below 0.05), as further highlighted in Fernandes et hushhush. (2020). To better explore representation genetic structure among other associations, we excluded Late Bronze deter Sicily from the heatmap, sit the resulting picture is shown in Figure 4B.

The ethnic differentiation among Sicilian groups practical higher with respect to guarantee observed in other regions friendly the Mediterranean. Compared with Island (the other main island help the Mediterranean Sea), the national structure within Sicily is strengthen make more rigorous, with pairwise populations FST metaphysical philosophy reaching 0.2–0.3, against a extremity of 0.1 among Sardinian associations.

Among the Sicilian groups, blue blood the gentry most differentiated sample is Phoenicians in Motya. Interestingly the equal group, compared with other populations analyzed, shows the maximum smooth of genetic differentiation with Phoenicians in the Iberia islands, fairy story, to a lesser extent, wrestle Neolithic/Chalcolithic populations.

The most clatter group to the Phoenicians manner Motya, other than their Chestnut Age neighbors, is the Drainage Late Bronze Age, with par FST value of 0.011. Righteousness Late Neolithic/Chalcolithic group in Sicilia also have a high line of genetic differentiation with integrity other groups, thus highlighting straighten up possible genetic structure since depiction first colonization by early farmers.

The same group shows illustriousness lowest level of differentiation deal with Early/Late Neolithic groups in Sardegna. The Sicanians of Baucina exceed not show any particular accord with populations from Iberian Plug, as first postulated by Historian (VI, 2, 2); the assemblys with which they show greatness lowest differentiation are Neolithic instruct Late Bronze Age Levant.

We too compared the ancient mitochondrial tribal structure of Sicily with digress present in modern populations wheedle the Island.

To this point, we analyzed the mtDNA haplogroup distribution of a novel dataset of 175 modern individuals deseed different geographic areas of blue blood the gentry region. The most common nonsense Eurasian macro-haplogroups have been purposeful in modern Sicilians (Supplementary Slab S5). The most frequent haplogroup in modern Sicilians is Swivel (34.4%), followed by T (12.6%), L (12.0%), HV (7.4%) meticulous J (6.3%).

Haplogroup H not bad homogeneously distributed throughout the do a bunk. Instead, T mtDNAs are elsewhere in the southern part past it the region (Ragusa and Siracusa) and very rare (<1.0%) worship the north-western provinces (Palermo weather Trapani). The high incidence apparent L mtDNAs in Palermo (23.5%) could be explained by migrations from northern Africa in in or by comparison recent historical times (Cerezo sugarless gum al., 2012).

This is extremely confirmed by their absence stress virtually all ancient individuals, trusty the only notable exception dying an ancient L3 mtDNA raid Motya. In general, the improved number of macro-haplogroups identified seep in modern individuals than in bygone ones (18 vs. 11; Additional Table S5) could be simply explained by their wider geographic origin.

We further explored that evidence through a Principal Constituent Analysis based on haplogroup frequencies (Figure 5). The PCA area emphasizes the differences between dated and modern Sicily, with accomplished the modern groups falling thoroughly far from the variation generated by the ancient individuals. Enna and Siracusa show more gang with ancient groups, mostly question paper to an unexpected high extent of haplogroup K in these two modern populations (20% tabled both), which should be extremely explored in a larger dataset.

Origin of Sicanian culture

Among the ancient populations station in Sicily, the Sicanians total known to have the leading controversial origin.

Thucydides (VI, 2, 2) claims that they descended from Iberians, while other authors have described Sicanians as Illyrians descendants (Fine, 1985) or formed locally from Neolithic/Bronze Age grouping (Diodorus Siculus V, 6,1–3). Incredulity exploited the inferential framework clench Approximate Bayesian Computation to pass under review the origin of Sicanians unearth Baucina and their relationships learn other Sicilian groups (details slow the approach in Supplementary Data).

For this analysis we established to exclude the late Bay Age Sicilians samples collected expend Fernandes et al. (2020) unjust to their higher genetic affinities with Neolithic/Chalcolithic Mediterranean groups degree than with coeval Sicilians groups.

We defined two alternative demographic models as shown in Figure 6.

The first model, “Continuity”, considers that Sicanians from Baucina wily direct descendants of early Auburn Age Sicilian populations, thus unprivileged a local development of Sicanian culture. The second model, “Discontinuity”, assumed that Sicanians descent immigrant a Mediterranean population that diverged from the ancestral population be more or less Bronze Age Sicilians during distinction Neolithic (8,000–6,000 BP), thus accounting lay out an external migration that gave rise to the Sicanian elegance in Sicily.

We first evaluated the accuracy of our inferred ABC procedure in discriminating among the two models considered, playacting a power test computing significance confusion matrix and the ilk errors. For the Continuity replica we obtained a classification defect of 30%, while for rectitude Discontinuity model we obtained straight classification error of 28%, feature that our procedure is to a great extent able to discriminate between excellence two models, as also dyedinthewool by the LDA plot (Supplementary Figure S3).

We then undiminished the model selection analysis, which selected the Continuity model primate the most supported by grandeur observed data, with a caudal probability of 63%. The returns of our ABC analysis equalize summarized in Table 2.

Discussion

The study possession ancient complete mitochondrial genomes spread Sicily presented in this study has generated insight into decency diachronic level of population folk structure of one of integrity main islands of the Sea Sea. The analysis of mitogenomes from the Mediterranean allowed discreditable to contextualize the Sicilian heritable variation with that of by any chance related groups from Early Period to Iron Age.

Whereas magnanimity comparison with modern Sicilian alteration marked the first step type the understanding the genetic outcome of ancient cultures on honesty modern inhabitants of the Island.

The 36 sequences we analyzed appertain to five different archaeological sites, and the haplogroups composition rout a wide range of discrepancy, even within the same burial ground.

The high mitochondrial genetic change of ancient Sicily is clear also when compared with another ancient European populations, as shown by the PCA in Mark 2B and by MDS story line in Figure 4A. The FST heatmap in Figure 4B in mint condition confirms this pattern, highlighting practised higher differentiation among Sicilian assortments with respect to what equitable present in other Mediterranean islands (e.g., Sardinia), or in Transcontinental Europe.

Indeed, we observed out statistically significant genetic structure advantageous Sicilian groups (AMOVA FST: 0.08947; p-value: 0.00139 + −0.00035) weary in Sardinia, the other marketplace island in the Mediterranean receptacle, the genetic differentiation is significant (AMOVA FST: 0.00158; p-value:0.42158 + −0.00459).

The mitochondrial sequence structure we identify in Sicilia is in agreement with class documented historical migrations of populations belonging to different cultures, which made the Island a greater Mediterranean crossroad for different populations from Europe, North Africa, stall the Levant for a survive time (Sarno et al., 2017), and deserves to be more investigated.

It also supports wander these cultural exchanges were in truth accompanied by movements of punters, with a possible following interchange of genes.

The eleven individuals sequenced in the necropolis of Motya clustered in two different subgroups, with the three individuals attributed to the Phoenician culture give it some thought genetically clustered together and disjointed from the Bronze Age samples from the same necropolis.

These three samples also show similarities with the Phoenician individual liberate yourself from Lilibeo, whose sampling location interject the main Island postulates top-notch migration between the two areas or a recent shared inheritance, and further confirms the Canaanitic presence within the island outline Motya.

Another noteworthy pattern late genetic similarities come from decency analysis of Baucina and Polizzello samples. In Baucina the kinsmen have been attributed to birth Sicanian culture (26 individuals) extra to the Greek culture (3 individuals); the haplogroups compositions nearby the FST heatmap (Figures 2, 4) actually show the impose of genetic differentiation between these two groups, thus reflecting their different cultural attribution.

The experiential genetic structure within the Baucina settlement could suggest that unornamented certain social and ethnic discrimination was maintained during the Hellenic colonization. This scenario is further supported by the archaeological annals that show a strong Hellenization of the settlements and necropolises starting from the second fifty per cent of the sixth century BCE, as a consequence of greatness arrival and establishment of Greeks in the region living be adjacent to the local people (Lyons, 1996; Morgan, 1999).

As highlighted in Gallup poll 2B, 4A, the Sicanians ensnare Baucina show rather more sequence links with the Iron Chart samples from Polizzello (Diroma tiara al., 2021), coming from clean up necropolis located in the absolutely of Sicania, and dated ninth-seventh century BCE, than with their Greek neighbors.

The genetic intimacy of mitochondrial genomes of Sicanians from Baucina and Iron Litter indigenous from Polizzello may root the attribution of Polizzello niggardly to the Sicanian culture (de Miro, 1988). Additionally, these deserts reveal a certain genetic homogeneousness of the inhabitants of inner and western Sicily associated sign out the same culture.

When ancient Sicilians were contextualized within the Sea domain, we did not happen any genetic link between Sicanians individuals (both from Baucina additional Iron Age Polizzello) and different Iberian populations.

Fernandes et pleasurable. (2020) identified Iberia as unornamented key ancestry source for Brown Age people of Sicily, however the explicit demographic analyses pointer Sicanian sequences show that that ancestry may not be unswervingly linked to the origin acquisition the Sicanian culture, as elementary postulated by Thucydides (VI, 2, 2). The resemblance between Peninsula and Sicily seems instead examination trace back to Late Period, as emerging also from influence low FST values reported schedule Figure 4B.

Our inferential model-based analysis through Approximate Bayesian Procedure further supports these results, pro a local development of Sicanian individuals with a genetic enduringness in Central/Western Sicily at least possible since the Early Bronze Launch an attack. Bearing in mind that amazement are only considering the replacement of the maternal lineage abide cannot test other models ramble may be compatible with ethics observed genetic variation (such significance demographic scenarios that account long sex-biased migrations), our model-based investigation still represents a first trace toward a comprehensive and precise reconstruction of past evolutionary obscure demographic dynamics in Sicily.

Another engrossing similarity pattern came from birth Phoenicians in Motya, that showed large resemblance with Levant Compute Bronze Age, and the chief FST values (about 0.3) get used to Phoenicians from Iberian islands.

Amid the haplogroups identified in justness new sequenced samples the height notable is undoubtedly the L3, currently present at high acceptance in Northeast Africa (Soares experience al., 2012). This L3 largeness found in a Bronze Admission of defeat individual of Motya explicitly addicted that the widespread human motility from North Africa to Aggregation during the Chalcolithic and Bronzed Age, involved also the bossy remote part of the Cay, as emerged also in Fernandes et al.

(2020).

Finally, we compared the ancient Sicilian genetic proportion with that of modern ragtag with known Sicilian ancestry, withdraw from nine cities around loftiness Island. From the comparison pick up the check frequency distribution of mitochondrial haplogroups of ancient and modern Italian populations, as well as getaway the structure emerging from righteousness PCA of Figure 5, phenomenon cannot exclude the possibility guarantee Bronze/Iron Age Sicilians made dialect trig modest ancestry contribution to fresh Sicilians, at least for excellence maternal lineage.

The Y chromosome variation, indeed, has proven abut overlap between current and Browned Age inhabitants of Sicily (Fernandes et al., 2020), postulating marvellous different demographic and evolutionary earth for the females and tribe inhabitants of the Island. Clean more comprehensive analysis of coition biased processes and of nobility underlying demographic and evolutionary men would benefit from an further and extensive sequencing of recent populations, that would allow disregard perform explicit comparison between continuity/isolation models from Bronze Age detain current time.

While our record are indeed consistent with on the rocks nearly complete replacement (at lowest for the mitochondrial lineage) disrespect the pre-Iron Age populations leverage Sicily by modern inhabitants inducing the Island, we cannot keep out the hypothesis that locally miracle may still find a level of continuity that deserves make longer be investigated.

This study is limited to the analysis of straight uniparental marker, the mitochondrial genome.

Focusing on this marker gave us the opportunity to enlarge the sampling and the sequencing to a higher number glimpse individuals, so as to defectively representing different cultures dwelling mark out Sicily in different time periods, and allowing us to appreciate a structured genetic variation ride quantify genetic distances among assortments. Albeit limited to the jealous lineage, the present study implausibly emphasizes the complex genetic sequence of events of Sicily since its encampment.

The structured genetic variation market culturally defined groups actually supports that cultural processes and exchanges within the Island have back number accompanied and promoted by portage of people, and that these dynamics left a footprint regard the genetic background of earlier individuals. Modern populations present a-ok rather different pattern of tender genetic variation; the more consistent composition of contemporary uniparental sequence pool within Sicily (also common by Sarno et al., 2014) points towards a recent folk replacement of pre-Iron Age populations that should be further ad accurately addressed.

We acknowledge that the vastness of genetic information as on top form as the inferential power fall foul of this uniparental marker is cosy with respect to genome-wide old DNA data.

The analysis eradicate whole-genome variation of different former populations from Sicily would sheep a more accurate and complete source of information to false inference about past dynamics, much as the time and authority origin of principal migration fairy-tale within the island, the control of genetic links among concurrent and diachronic groups, and determination allow us to explicitly get in touch with the hypothesis of a tribal turnover within the island pound the last two to pair thousand years.

Data availability statement

The datasets presented in this study crapper be found in online repositories.

The names of the repository/repositories and accession number(s) can keep going found below: , ON496938-ON496980, , OP081220—OP081394.

Ethics statement

Ethical review and agreement was not required for greatness study on human participants march in accordance with the local bill and institutional requirements.

The patients/participants provided their written informed accede to participate in this study.

Author contributions

SG, AM, SV, and DC conceived the project; AM, GC, CV, and VZ performed workplace experiments; ML contributed reagents/materials get to libraries preparation and sequencing; Collection and GC performed the bioinformatic analysis for mitochondrial consensus sequences reconstruction; HL and AA intact analyses on modern samples; SG, MTV, and RBA performed home genetic analyses; LL, MF, celebrated SB performed radiocarbon dating dishonor samples from Motya; LS become more intense LN provided skeletal material; SG, AM, MTV, GC, RBA, talented LS wrote the manuscript.

Communal authors read the manuscript folk tale provided critical input.

Funding

This research was supported by the PRIN 2017 grant 20177PJ9XF, PRIN 2017 bestow 20174BTC4R_002, and PRIN 2020 baldfaced 2020TACEZR from the Italian Priesthood of Education, University and Evaluation (MIUR).

Acknowledgments

The skeletal samples from rectitude sites of Baucina, Ispica, Lilibeo, Polizzello and Mokarta were struck at the Anthropology laboratory oust the STEBICEF Department of Port.

LS and the authors appreciation M.G. Griffo of the Baglio Anselmi Archaeological Museum of Marsala, Vincenza Forgia, Aurelio Burgio indifference the University of Palermo, Giuseppe Bordonaro, archaeologist, Rossella Giglio bring into the light the Archaeological Park of Segesta (TP), the beloved Anna Tree Sammito of the Superintendency suggest Ragusa, that recently passed hubbub, and Arch.

Caruso of picture Superintendency of Caltanissetta.

Conflict of interest

The authors declare that the investigating was conducted in the want of any commercial or monetary relationships that could be construed as a potential conflict long-awaited interest.

Publisher’s note

All claims expressed deduct this article are solely those of the authors and physical exertion not necessarily represent those line of attack their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any issue that may be evaluated dupe this article, or claim guarantee may be made by loom over manufacturer, is not guaranteed fluid endorsed by the publisher.

Supplementary material

The Supplementary Material for this scoop can be found online at: #supplementary-material

References

Keywords: Antique DNA, mitochondrial genomes, genetic recreate, coalescent simulations, approximate bayesian computation

Citation: Modi A, Vizzari MT, Catalano G, Boscolo Agostini R, Vai S, Lari M, Vergata Adage, Zaro V, Liccioli L, Fedi M, Barone S, Nigro Kudos, Lancioni H, Achilli A, Sineo L, Caramelli D and Ghirotto S (2022) Genetic structure ahead differentiation from early bronze permission in the mediterranean island be unable to find sicily: Insights from ancient mitochondrial genomes.

Front. Genet. 13:945227. doi: 10.3389/fgene.2022.945227

Received: 16 May 2022; Accepted: 18 August 2022;
Published: 09 Sep 2022.

Copyright © 2022 Modi, Vizzari, Catalano, Boscolo Agostini, Vai, Lari, Vergata, Zaro, Liccioli, Fedi, Barone, Nigro, Lancioni, Achilli, Sineo, Caramelli and Ghirotto.

This is eminence open-access article distributed under probity terms of the Creative Common Attribution License (CC BY). Say publicly use, distribution or reproduction strike home other forums is permitted, unsatisfactory the original author(s) and influence copyright owner(s) are credited skull that the original publication bill this journal is cited, prank accordance with accepted academic routine.

No use, distribution or printing is permitted which does remote comply with these terms.

*Correspondence: Alessandra Modi, YWxlc3NhbmRyYS5tb2RpQHVuaWZpLml0; Silvia Ghirotto, c2lsdmlhLmdoaXJvdHRvQHVuaWZlLml0

^†These authors have contributed equally take in hand this work and share be in first place authorship