Main roads and land cover shaped the genetic structure of a Mediterranean island wild boar population

Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.     

Genomic diversity and differentiation of a managed island wild boar population

The evolution of island populations in natural systems is driven by local adaptation and genetic drift. However, evolutionary pathways may be altered by humans in several ways. The wild boar (WB) (Sus scrofa) is an iconic game species occurring in several islands, where it has been strongly managed since prehistoric times. We examined genomic diversity at 49 803 single-nucleotide polymorphisms in 99 Sardinian WBs and compared them with 196 wild specimens from mainland Europe and 105 domestic pigs (DP; 11 breeds). High levels of genetic variation were observed in Sardinia (80.9% of the total number of polymorphisms), which can be only in part associated to recent genetic introgression. Both Principal Component Analysis and Bayesian clustering approach revealed that the Sardinian WB population is highly differentiated from the other European populations (F_ST=0.126–0.138), and from DP (F_ST=0.169). Such evidences were mostly unaffected by an uneven sample size, although clustering results in reference populations changed when the number of individuals was standardized. Runs of homozygosity (ROHs) pattern and distribution in Sardinian WB are consistent with a past expansion following a bottleneck (small ROHs) and recent population substructuring (highly homozygous individuals). The observed effect of a non-random selection of Sardinian individuals on diversity, F_ST and ROH estimates, stressed the importance of sampling design in the study of structured or introgressed populations. Our results support the heterogeneity and distinctiveness of the Sardinian population and prompt further investigations on its origins and conservation status.     

Introgressive hybridisation between domestic pigs (Sus scrofa domesticus) and endemic Corsican wild boars (S. s. meridionalis): effects of human-mediated interventions

Owing to the intensified domestication process with artificial trait selection, introgressive hybridisation between domestic and wild species poses a management problem. Traditional free-range livestock husbandry, as practiced in Corsica and Sardinia, is known to facilitate hybridisation between wild boars and domestic pigs (Sus scrofa). Here, we assessed the genetic distinctness and genome-wide domestic pig ancestry levels of the Corsican wild boar subspecies S. s. meridionalis, with reference to its Sardinian conspecifics, employing a genome-wide single nucleotide polymorphism (SNP) assay and mitochondrial control region (mtCR) haplotypes. We also assessed the reliance of morphological criteria and the melanocortin-1 receptor (MC1R) coat colour gene to identify individuals with domestic introgression. While Corsican wild boars showed closest affinity to Sardinian and Italian wild boars compared to other European populations based on principal component analysis, the observation of previously undescribed mtCR haplotypes and high levels of nuclear divergence (Weir’s θ > 0.14) highlighted the genetic distinctness of Corsican S. s. meridionalis. Across three complementary analyses of mixed ancestry (i.e., STRUCTURE, PCADMIX, and ELAI), proportions of domestic pig ancestry were estimated at 9.5% in Corsican wild boars, which was significantly higher than in wild boars in Sardinia, where free-range pig keeping was banned in 2012. Comparison of morphologically pure- and hybrid-looking Corsican wild boars suggested a weak correlation between morphological criteria and genome-wide domestic pig ancestry. The study highlights the usefulness of molecular markers to assess the direct impacts of management practices on gene flow between domestic and wild species.Subject terms: Conservation genomics, Genetic hybridization, Structural variation     

Mitochondrial haplotypes of European wild boars with 2n = 36 are closely related to those of European domestic pigs with 2n = 38

Wild boars from Western Europe have a 2n = 36 karyotype, in contrast to a karyotype of 2n = 38 in wild boars from Central Europe and Asia and in all domestic pigs. The phylogenetic status of this wild boar population is unclear, and it is not known if it has contributed to pig domestication. We have now sequenced the mtDNA control region from 30 European wild boars (22 with a confirmed 2n = 36 karyotype) and six Asian wild boars (two Hainan and four Dongbei wild boars) to address this question. The results revealed a close genetic relationship between mtDNA haplotypes from wild boars with 2n = 36 to those from domestic pigs with 2n = 38. Thus, we cannot exclude the possibility that wild boars with 2n = 36 may have contributed to pig domestication despite the karyotype difference. One of the European wild boars carried an Asian mtDNA haplotype, and this most likely reflects gene flow from domestic pigs to European wild boars. However, this gene flow does not appear to be extensive because the frequency of Asian haplotypes detected among European wild boars (c. 3%) were 10-fold lower than among European domestic pigs (c. 30%). Previous studies of mtDNA haplotypes have indicated that pig populations in Europe and Asia have experienced a population expansion, but it is not clear if the expansion occurred before or after domestication. The results of the present study are consistent with an expansion that primarily occurred prior to domestication because the mtDNA haplotypes found in European and Asian wild boars did not form their own clusters but were intermingled with haplotypes found in domestic pigs, indicating that they originated from the same population expansion.     

Are captive wild boar more introgressed than free-ranging wild boar? Two case studies in Italy

Hybridization between wild boar (Sus scrofa) and domestic pig occurred in the past and still occurs today, having great evolutionary and management implications. In fact, genetic introgression from the domestic form may alter traits like behavior, reproduction rate, and immunology in wild populations, with likely demographic impacts. Thus, it is crucial to understand under what conditions hybridization occurs in S. scrofa. Captive crosses with domestic pigs (released or escaped) have been suggested to constitute the major source of the spread of domestic genes into wild boar populations. However, to date, few studies have assessed the degree of admixture in farmed animals in comparison to the surrounding wild populations. With this purpose, we analyzed microsatellite loci in wild boar sampled in breeding stations and in the local wild population in two Italian regions (Sardinia and Piedmont). Both captive populations had lower allelic richness than the corresponding wild population, but a similar expected heterozygosity. In Piedmont, introgression from the domestic form into the wild population seems to be extremely low, while there are significant signs of admixture in the sampled breeding stations. In Sardinia, instead, the captive sample did not differ significantly from the wild population, which showed moderate signs of introgression. We conclude that hybridization in nature seems to play the key role in Sardinia, while intentional hybridization in captivity is the major source of introgression in Piedmont. Our findings emphasize the need for a routine genetic monitoring of wild boar captive populations, coupled with reference data on the neighboring wild populations.     

Spatial genetic structure of Aquilegia taxa endemic to the island of Sardinia

Background and Aims

The Mediterranean Basin is one of the most important regions for the Earth''s plant biodiversity; however, the scarcity of studies on fine scale patterns of genetic variation in this region is striking. Here, an assessment is made of the spatial genetic structure of all known locations of the three Sardinian endemic species of Aquilegia in order to determine the relative roles of gene flow and genetic drift as underlying evolutionary forces canalizing the divergence of Sardinian Aquilegia taxa, and to see if the spatial genetic structure found fits the current taxonomic differentiation of these taxa.

Methods

DNA from 89 individuals from all known locations of Aquilegia across Sardinia was analysed by means of amplified fragment length polymorphism (AFLP) markers. Both principal co-ordinates analysis (PCoA) and Bayesian clustering analyses were used to determine the spatial genetic structure irrespective of any taxonomic affiliation. Historical effects of gene flow and genetic drift were assessed by checking for the existence of isolation-by-distance patterns.

Key Results

STRUCTURE and PCoA analyses revealed a pattern of genetic variation geographically structured into four spatial genetic groups. No migration–drift equilibrium was detected for Aquilegia in Sardinia, when analysed either as a whole or in individual groups. The scenario approached a Case III pattern sensu Hutchinson and Templeton, which is associated with extreme isolation conditions where genetic drift has historically played a dominant role over gene flow.

Conclusions

The pattern of genetic variation of Sardinian taxa of Aquilegia indicates that genetic drift has been historically more influential than gene flow on population structure of Sardinian species of Aquilegia. Limited seed dispersal and divergent selection imposed by habitat conditions have been probably the main causes reinforcing post-Pleistocene geographical isolation of Aquilegia populations. The spatial genetic structure found here is not fully compatible with current taxonomic affiliations of Sardinian Aquilegia taxa. This is probably a consequence of the uncoupling between morphological and genetic patterns of differentiation frequently found in recently radiated taxa.     

Genetic evidence for introgression between domestic pigs and wild boars (Sus scrofa) in Belgium and Luxembourg: a comparative approach with multiple marker systems

Hybridization between wild species and their domestic relatives can be an important conservation and management problem. Genetic purity of the wild species is desirable per se and the phenomenon can have unpredictable evolutionary consequences. Declining European wild boar populations were frequently restocked with farmed wild boars that sometimes had been crossed with domestic pigs. We used simple polymerase chain reaction‐based diagnostic tests to detect the presence of mitochondrial DNA and coat colour alleles of domestic origin in wild boars from Belgium, Luxembourg, and western Germany. Microsatellite genotypes were used to test for genetic admixture between the wild boars and domestic pigs. Although almost one‐third of all Luxembourg wild boars carried Asian mitochondrial DNA haplotypes originating from domestic pigs, microsatellite‐based clustering only identified four putatively admixed individuals in Luxembourg. By contrast, clustering identified wild boar × domestic hybrids in most sampling locations in Belgium. We interpret these results as evidence of releases of hybrid captive‐reared wild boars. Our results emphasize the need (if working with classical markers) to use different systems to obtain an understanding as to whether hybridization between wild and domestic relatives might have affected the genetic make‐up of a local population. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 104–115.     

Inferring the origin and genetic diversity of the introduced wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) populations in Argentina: an approach from mitochondrial markers

The Eurasian wild boar (Sus scrofa Linnaeus, 1758) was introduced into Argentina at the beginning of the twentieth century when individuals from Europe were taken to La Pampa province for hunting purposes. Starting from there, a dispersal process began due to the invasive characteristics of the species and to human-mediated translocations. The main objective of this study was to characterize for the first time, the phylogenetic relationships among wild boars from Argentina with those from Uruguay, Europe, Asia, and the Near East, along with diverse domestic pig breeds in order to corroborate the historical information about the origin of the local populations. To this end, we used mitochondrial Control Region and Cytochrome b sequences from sampled Argentinian wild boars and retrieved from GenBank. The results showed that the majority of the Argentinian wild boar populations descend from European lineages, in particular of the E1 clade, according to the historical records. Remarkably, the population of El Palmar National Park had Asian origin that could be attributed to hybridization with local domestic pigs or to unrecorded translocations. Finally, genetic diversity in Argentinian populations was lower than in Europe and Uruguay meaning that wild boar in Argentina is still under the influence of founder effect and has experienced minor genetic introgression from domestic pigs, representing in this sense a reservoir of the original wild boar genetic variability.     

Population structure in Sardinia on the basis of quantitative palmar dermatoglyphic traits

We describe the genetic structure of twenty Sardinian subpopulations using quantitative palmar dermatoglyphic traits (a-b, and A-d counts, atd angle value, coefficient of Turpin and Lejeune, main line index, mean of A-, B-, C, and D-line terminations) of 3777 subjects (2043 males and 1734 females). The twenty subdivisions represent sixteen historical-geographic areas of the island in which people speaks Sardinian language, Sassarese and Gallura areas in which people speaks two Italian dialects, and the two communities of Alghero (Catalan speaking) and Carloforte (Ligurian speaking). Analysis was carried out for both hands and both sexes combined and using R-matrix technique and the extension of the Harpending-Ward model to quantitative traits according to Relethford & Blangero (1990). Multivariate minimum F_st value (0.0127) is higher than that of most. Mediterranean populations and shows the importance of isolation and genetic drift as evolutive forces at the basis of microdifferentiation among the Sardinian subpopulations considered. However, when the four populations not speaking Sardinian language are removed from the analysis, the value of F_st decreases to 0.008. The regression of mean genetic variance on distances from the centroid (r_ii values) states the marked effect of the genetic drift for Nuorese and Barbagia di Ollolai subdivisions (placed in the inner and mountainous areas of Sardinia) and reveals considerable levels of admixture for Carloforte subdivision. The contemporary genetic structure of these groups reflects their historical, linguistic and geographic characteristics. On the whole, our analysis confirms the usefulness of quantitative dermatoglyphic traits in studying genetic population structure.     

TYR基因外显子1的序列变异

为了分析家猪与野猪的遗传多样性及起源,测定了来自１２个中国地方家猪品种、３个欧洲引进猪品种以及8个中国野猪和2个越南野猪共36个个体的酪氨酸酶基因（TYR）外显子1的序列,共检出６个单核苷酸多态性位点（SNPs）,且这6个位点的变异均为同义突变,根据这些变异可将酪氨酸酶基因DNA序列归结为４种单倍型。结合已发表的数据,构建了简约中介网络图。 在网络图中,单倍型TYR*2主要为欧洲家猪与欧洲野猪和三条亚洲家猪染色体。大部分亚洲家猪和野猪共享单倍型TYR*1,表明这是一个亚洲类型的单倍型;同时也有部分欧洲家猪与野猪携带这一单倍型。 而单倍型TYR*3和TYR*4为本研究检测到的稀有单倍型,这两种单倍型主要由中国家猪与亚洲野猪组成。这种网络图结构支持家猪的欧洲和亚洲独立起源学说,同时也表明相当部分的欧洲家猪品种受到亚洲猪的基因渗透,而少量中国家猪和日本野猪也受到了欧洲猪的基因渗透。     

A genetic method to distinguish crossbred Inobuta from Japanese wild boars

To distinguish pig-wild boar crossbred Inobuta from Japanese wild boar populations, a genetic method by using mitochondrial DNA (mtDNA) haplotypes and the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) was developed. Sixteen mtDNA haplotypes from 152 wild boars from Kyushu, Shikoku and Honshu islands of Japan were distinct from those from Asian and European domestic pigs. Five alleles of GPIP were classified into two groups: 1). alleles GPIP*1, GPIP*3 and GPIP*3a from Japanese wild boars, Asian wild boars and domestic pigs; 2). alleles GPIP*4 and GPIP*4a from European wild boars and domestic pigs. An extensive genetic survey was done to distinguish the crossbred Inobuta from 60 wild boars hunted on Tsushima Island, Goto Islands, and Nagasaki and Ooita Prefectures. The mtDNA haplotypes from the 60 samples showed Japanese wild boars, but four wild boar samples from Nagasaki Prefecture had the European GPIP allele, GPIP*4. These results showed that nuclear DNA polymorphism analysis is useful, in addition to mtDNA haplotype assay, to detect "Inobuta" having the European genotype from Japanese wild boar populations.     

Diversity of wild and domestic pig populations estimated by a set of serum allotypes

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.     

Population structure of the wild soybean (Glycine soja) in China: implications from microsatellite analyses

Background and Aims Wild soybean (Glycine soja), a native species of East Asia, is the closest wild relative of the cultivated soybean (G. max) and supplies valuable genetic resources for cultivar breeding. Analyses of the genetic variation and population structure of wild soybean are fundamental for effective conservation studies and utilization of this valuable genetic resource. Methods In this study, 40 wild soybean populations from China were genotyped with 20 microsatellites to investigate the natural population structure and genetic diversity. These results were integrated with previous microsatellite analyses for 231 representative individuals from East Asia to investigate the genetic relationships of wild soybeans from China. Key Results Analysis of molecular variance (AMOVA) revealed that 43·92 % of the molecular variance occurred within populations, although relatively low genetic diversity was detected for natural wild soybean populations. Most of the populations exhibited significant effects of a genetic bottleneck. Principal co-ordinate analysis, construction of a Neighbor-Joining tree and Bayesian clustering indicated two main genotypic clusters of wild soybean from China. The wild soybean populations, which are distributed in north-east and south China, separated by the Huang-Huai Valley, displayed similar genotypes, whereas those populations from the Huang-Huai Valley were different. Conclusions The previously unknown population structure of the natural populations of wild soybean distributed throughout China was determined. Two evolutionarily significant units were defined and further analysed by combining genetic diversity and structure analyses from Chinese populations with representative samples from Eastern Asia. The study suggests that during the glacial period there may have been an expansion route between south-east and north-east China, via the temperate forests in the East China Sea Land Bridge, which resulted in similar genotypes of wild soybean populations from these regions. Genetic diversity and bottleneck analysis supports that both extensive collection of germplasm resources and habitat management strategies should be undertaken for effective conservation studies of these important wild soybean resources.     

Diversity of wild and domestic pig populations estimated by a set of serum allotypes

Variation of serum protein allotypes serving as genetic markers of the blood has been analyzed in 29 populations of the domestic pig and subspecies of the wild boar. The population biodiversity and genetic structure have been estimated by two methods: by the frequencies of allotype combinations and with the use of a map constructed in the space of two principal components. The results obtained are the basis for determining the characteristics of the microevolution of wild boars and formation of the breeds of domestic pigs.     

Genetic structure of the wild boar population in Portugal: Evidence of a recent bottleneck

The present study assesses the degree of genetic structure and the presence of recent genetic bottlenecks in the wild boar population in Portugal. One hundred and ten individuals were sampled after capture during organised legal drive hunts, conducted in 58 municipalities across the continental territory, during the game seasons of 2002/2003 and 2003/2004. Individuals were genetically typed at six microsatellite loci using multiplex PCR amplification. Significant deviations from Hardy–Weinberg equilibrium were found for the total population of wild boar in Portugal. Wild boar population genetic structure was assessed using Bayesian methods, suggesting the existence of three subpopulations (North, Centre and South). Tests were conducted to detect the presence of potential migrants and hybrids between subpopulations. After exclusion of these individuals, three sets of wild boars representative of respective subpopulations were distinguished and tested for the effects of recent bottlenecks. Genetic distances between pairs of subpopulations were quantified using F_ST and R_ST estimators, revealing a variation of 0.138–0.178 and 0.107–0.198, respectively. On the basis of genetic and distribution data for Portuguese wild boar from the beginning of the 20th century, a model of strong demographic decline and contraction to isolated refuge areas at the national level, followed by a recovery and expansion towards former distribution limits is suggested. Some evidence points to present admixture among subpopulations in contact areas.     

Comparative landscape genetic analyses show a Belgian motorway to be a gene flow barrier for red deer (Cervus elaphus), but not wild boars (Sus scrofa)

While motorways are often assumed to influence the movement behaviour of large mammals, there are surprisingly few studies that show an influence of these linear structures on the genetic make-up of wild ungulate populations. Here, we analyse the spatial genetic structure of red deer (Cervus elaphus) and wild boars (Sus scrofa) along a stretch of motorway in the Walloon part of Belgium. Altogether, 876 red deer were genotyped at 13 microsatellite loci, and 325 wild boars at 14 loci. In the case of the red deer, different genetic clustering tools identified two genetic subpopulations whose borders matched the motorway well. Conversely, no genetic structure was identified in the case of the wild boar. Analysis of isolation-by-distance patterns of pairs of individuals on the same side and on different sides of the motorway also suggested that the road was a barrier to red deer, but not to wild boar movement. While telemetry studies seem to confirm that red deer are more affected by motorways than wild boar, the red deer sample size was also much larger than that of the wild boars. We therefore repeated the analysis of genetic structure in the red deer with randomly sub-sampled data sets of decreasing size. The power to detect the genetic structure using clustering methods decreased with decreasing sample size.     

The robust phylogeny of Korean wild boar (<Emphasis Type="Italic">Sus scrofa coreanus</Emphasis>) using partial D-loop sequence of mtDNA

In order to elucidate the precise phylogenetic relationships of Korean wild boar (Sus scrofa coreanus), a partial mtDNA D-loop region (1,274 bp, NC_000845 nucleotide positions 16576-1236) was sequenced among 56 Korean wild boars. In total, 25 haplotypes were identified and classified into four distinct subgroups (K1 to K4) based on Bayesian phylogenetic analysis using Markov chain Monte Carlo methods. An extended analysis, adding 139 wild boars sampled worldwide, confirmed that Korean wild boars clearly belong to the Asian wild boar cluster. Unexpectedly, the Myanmarese/Thai wild boar population was detected on the same branch as Korean wild boar subgroups K3 and K4. A parsimonious median-joining network analysis including all Asian wild boar haplotypes again revealed four maternal lineages of Korean wild boars, which corresponded to the four Korean wild boar subgroups identified previously. In an additional analysis, we supplemented the Asian wild boar network with 34 Korean and Chinese domestic pig haplotypes. We found only one haplotype, C31, that was shared by Chinese wild, Chinese domestic and Korean domestic pigs. In contrast to our expectation that Korean wild boars contributed to the gene pool of Korean native pigs, these data clearly suggest that Korean native pigs would be introduced from China after domestication from Chinese wild boars.     

东北亚地区野猪种群mtDNA遗传结构及系统地理发生

研究测定了中国东北、华北及四川西部72个野猪(Susscrofa)个体线粒体控制区全序列,并结合GenBank报道的日本野猪(S.s.leucomystax)、琉球野猪(S.s.riukiuanus)72个同源区序列,分析了东北亚地区野猪线粒体DNA的变异及系统地理格局。在东北亚地区野猪的线粒体控制区共发现42个变异位点,均为转换,共定义了34个单元型。单元型之间的系统发生分析表明,东北亚地区野猪来自同一祖先。东北亚地区野猪现生种群具有显著的种群遗传结构,其中日本野猪与分布于中国东北地区的东北野猪之间亲缘关系较近;而琉球野猪则与华北野猪间亲缘关系较近,与日本野猪和东北野猪间的关系相对较远。嵌套进化枝系统地理分析(Nestedcladephylogeographicalanalysis,NCPA)表明:东北亚地区野猪由同一祖先经过长距离的迁徙而形成现生各种群(或亚种);琉球野猪应起源于大陆野猪,其种群演化可能经历了片断化事件;华北野猪呈现南部种群遗传多样性高的特点,其种群内部曾经历了一次分布区由南向北的扩张     

High domestic pig contribution to the local gene pool of free-living European wild boar: a case study in Poland

Rates of hybridization between wild and domesticated animals appear to be increasing worldwide. Recent results suggest that genetic introgression from domestic swine into European wild boar is much more common in local populations than expected, based on pan-European studies. Thus, we screened the genetic purity of 265 free-living wild boars from two hunting areas in Poland by genotyping the melanocortin receptor 1 gene (MC1R) for polymorphism. Unexpectedly, high numbers of individuals with domestic genes (24%) were identified. This suggests that mixed ancestry may be common in Polish wild boar. Among admixed individuals, backcrosses with domestic pig and/or introgressed wild boars were detected (2%). Multiple commercial domestic pig breeds are possibly involved in the introgression observed in the study populations. In addition, the absence of significant differences in the frequency of wild-type allele among two hunting areas suggests high dispersal of individuals and gene flow among populations. We conclude that further study is needed to better understand the mechanisms and sources of introgression in wild boars in Poland.     

Craniofacial morphometric variation and the biological history of the peopling of Sardinia

The aim of this work is to explore the pattern of craniofacial morphometric variation and the relationships among five prehistoric Sardinian groups dated from Late Neolithic to the Nuragic Period (Middle and Late Bronze Age), in order to formulate hypotheses on the peopling history of Sardinia. Biological relationships with coeval populations of central peninsular Italy were also analysed to detect influences from and towards extra-Sardinian sources. Furthermore, comparison with samples of contemporary populations from Sardinia and from continental Italy provided an indication of the trend leading to the final part of the peopling history. Finally, Upper Palaeolithic and Mesolithic samples were included in the analyses to compare the prehistoric Sardinians with some of their potential continental ancestors. The analysis is based on multivariate techniques including Mahalanobis D² distance, non-parametric multidimensional scaling (MDS) and principal component analysis (PCA). The results showed the tendency to progressive differentiation between Sardinian groups and peninsular Italian groups, with the possible exception of a discontinuity showed by the Bonnànaro (Early Bronze Age) Sardinian sample. Several aspects of the morphological results were found to agree with the current genetic evidence available for the present-day Sardinian population and a Nuragic sample: (1) biological divergence between the Sardinian and peninsular Italian populations; (2) similarity/continuity among Neolithic, Bronze Age and recent Sardinians; (3) biological separation between the Nuragic and Etruscan populations; (4) contribution of a Palaeo-Mesolithic gene pool to the genetic structure of current Sardinians.