Identifying native animals in crossbred populations: the case of the Sardinian goat population

The aim of this work was to develop a strategy for using a genetic analysis for identifying native animals in regions where local breeds have been crossed with improved breeds and then compare that strategy to the overall morphology and breeding histories of the herds for identifying these animals. The experiment included the Sardinian goat population, which is a crossbred of native animals with the Maltese breed. Whole herds were assigned to Maltese (five herds; 49 animals), crossbred (18 herds; 117 animals) or Sardinian (12 herds; 164 animals) groups. For the genetic analysis, genotypes of 22 microsatellites were determined on 330 animals, and basic measurements of genetic diversity were calculated. Genetic variability in the microsatellites was different in the three groups. High positive F _IS showed that inbreeding existed in the subpopulations. The index of genetic differentiation, Nei's standard genetic distance and Reynolds' genetic distance were calculated and found to be significantly different between the three groups. The Sardinian and Maltese groups were the most distant whereas the crossbred group was closer to the Sardinian group. The proportion of the genome derived from two ancestral populations (native Sardinian and Maltese) was assessed using the structure software. Animals were assigned to three clusters on the basis of native Sardinian thresholds. A good correspondence between the empirical (morphology and breeding histories) and the objective genetic analysis was found. Both approaches indicate the presence of three different subpopulations in the Sardinian goat population.     

Endemic Cyprus Warbler Sylvia melanothorax and colonizing Sardinian Warbler Sylvia melanocephala show different habitat associations

Anthropogenic habitat change and assisted colonization are promoting range expansions of some widespread species with potential consequences for endemic fauna. The recent colonization of Cyprus by breeding Sardinian Warblers Sylvia melanocephala has raised concerns that it might be displacing the closely related and endemic Cyprus Warbler Sylvia melanothorax. Habitat associations of both species were examined using models of abundance within the 95% density kernel of the Sardinian Warbler’s range and also outside this range for Cyprus Warbler. Within the Sardinian Warbler’s range, the two species were associated with subtly different scrub habitats. Outside the Sardinian Warbler’s range, the Cyprus Warbler differed again in its habitat association, but this probably resulted from marked differences in habitat extent and availability in different parts of the island rather than from competitive displacement, as none of the habitat or land‐use elements differentially associated with Cyprus Warblers was positively associated with Sardinian Warbler occurrence. This suggests that the Sardinian Warbler has exploited a different niche, rather than displacing the endemic species, and has perhaps benefitted from changing land‐use patterns, particularly recent fallows and abandoned agriculture, in contrast to the stronger association of Cyprus Warblers with semi‐natural scrub.     

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.     

Growth charts of head length and breadth for regional areas? A study in Sardinia (Italy)

There exist few standards of head length and breadth from childhood to adulthood in Europoid populations. Moreover, such standards are based on samples that cannot be used as references for all populations since they were taken from different ethnic groups and from different periods. The aims of this study were: (1) to test whether standards derived from North Americans of European extraction can be used to assess the Sardinian population; and (2) to produce growth charts for head length and breadth for Sardinian males and females from 3 to 22 years of age.The cross sectional sample consisted of 9,721 subjects of Sardinian origin (4,884 males and 4,837 females), aged 3–22 years, measured from 1998 to 2008. Growth percentiles were produced with the LMS method. The mean values for each sex in each age class (3–18 years) are almost always significantly lower for both head length and breadth than the corresponding North American values. The exceptions are the head length of boys of 14 years and girls of 16–18 years where values for Sardinians are lower, but not significantly so. The results show that the North American standards are not appropriate for the assessment of Sardinian children. For the Sardinian population, specific regional growth charts should be used to correctly evaluate the normal range and the cut-off points of the extreme percentiles.     

Mitochondrial CR-1 Variation in Sardinian Hares and Its Relationships with Other Old World Hares (Genus Lepus)

Among the European fauna, the Sardinian hare (Lepus sp.) is peculiar in that it differs from all other hares inhabiting the continent. Here, we report on the variation of a 461 bp sequence of hypervariable domain 1 of the mitochondrial control region, examined in 42 hares collected throughout Sardinia and compared to the corresponding sequences of different Lepus taxa. Seventeen novel haplotypes were found in the Sardinian population, resulting in a haplotype diversity of 0.840 and a nucleotide diversity of 0.012. As a result of Bayesian and principal coordinates analyses, Sardinian hares were grouped with North African hares, constituting a monophyletic clade that diverges from all other Old World hares, including Cape hares from South Africa and East Asia. Hence, our data agree that populations inhabiting North Africa and Sardinia form a distinct taxon, which could possibly be included in the L. capensis superspecies. Moreover, two corresponding lineages can be found in Sardinia and Tunisia, providing evidence of a common origin of the two populations and thus supporting the hypothesis that North African hares were introduced into the island in historical times. Our data show that the two lineages differ in their geographic distribution throughout the island and that the wild Sardinian population also shows the signature of a postintroduction demographic expansion.     

Differentiation under isolation and genetic structure of Sardinian hares as revealed by craniometric analysis,mitochondrial DNA and microsatellites

Hares (Lepus capensis Linnaeus 1758) were probably introduced into Sardinia in historical times. Previous studies indicated North Africa as the most likely source area but did not exclude the occurrence of hybridization events with continental brown hares (L. europaeus Pallas 1778) perhaps introduced for hunting purposes. We implemented both morphometric and genetic approaches to verify the genetic isolation of the Sardinian population. Specifically, we conducted a multivariate analysis of craniometric data and analysed 461 bp of the mitochondrial control region and 12 autosomal microsatellites in Sardinian hares, using North African cape hares and European brown hares as reference populations. Sardinian hares displayed a peculiar skull shape. In agreement, both nuclear and mitochondrial markers remarked the distinctiveness of this population. Observed and expected heterozygosity were 0.52 and 0.61, while haplotype and nucleotide diversity were 0.822 and 0.0129. Self‐assignment based on Bayesian cluster analysis was high (average membership 0.98), and no evident signs of introgression from continental brown hares were found. Our results support the hypothesis that the Sardinian hares have been introduced from North Africa, remained genetically isolated since the founding event and evolved independently from the source population. This long‐lasting isolation and the consequent genetic drift resulted in a differentiation, perhaps accompanied by an adaptation to local environmental conditions.     

Genetic variation in prehistoric Sardinia

We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age and Iron Age, between 3,430 and 2,700 years ago. After eliminating the samples that, in preliminary biochemical tests, did not show a high probability to yield reproducible results, we obtained 23 sequences of the mitochondrial DNA control region, which were associated to haplogroups by comparison with a dataset of modern sequences. The Nuragic samples show a remarkably low genetic diversity, comparable to that observed in ancient Iberians, but much lower than among the Etruscans. Most of these sequences have exact matches in two modern Sardinian populations, supporting a clear genealogical continuity from the Late Bronze Age up to current times. The Nuragic populations appear to be part of a large and geographically unstructured cluster of modern European populations, thus making it difficult to infer their evolutionary relationships. However, the low levels of genetic diversity, both within and among ancient samples, as opposed to the sharp differences among modern Sardinian samples, support the hypothesis of the expansion of a small group of maternally related individuals, and of comparatively recent differentiation of the Sardinian gene pools. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nuclear and chloroplast microsatellite diversity in Phaseolus vulgaris L. from Sardinia (Italy)

Studies of the level and the structure of the genetic diversity of local varieties of Phaseolus vulgaris are of fundamental importance, both for the management of genetic resources and to improve our understanding of the pathways of dissemination and the evolution of this species in Europe. We have here characterized 73 local bean populations from Sardinia (Italy) using seed traits and molecular markers (phaseolins, nuSSRs and cpSSRs). American landraces and commercial varieties were also included for comparison. We see that: (a) the Sardinian material is distinct from the commercial varieties considered; (b) the variation in the seed traits is high and it mostly occurs among populations (95%); (c) compared to the American sample and the commercial varieties, the Sardinian collection has a low level of diversity; (d) the majority (>95%) of the Sardinian individuals belong to the Andean gene pool; (e) the Sardinian material shows a strong genetic structure, both for cpSSRs and nuSSRs; (f) the nuSSRs and cpSSRs concur in differentiating between gene pools, but a lack of congruence between nuclear and chloroplast has been observed within gene pools; and (g) there are three putative hybrids between the Andean and Mesoamerican gene pools. Despite the relatively low level of diversity, which is probably due to a strong founder effect, the Sardinian landraces are worth being conserved and studied further because of their distinctiveness and because hybridization within and between the gene pools could generate variation that will be useful for breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Properdin factor B polymorphism in four Sardinian villages

A sample of healthy unrelated individuals was typed for properdin factor B (Bf) polymorphism in four Sardinian villages. Two villages, Desulo and Tonara, are located in the highlands; the other two, Orosei and Galtellì, are located in the lowlands. No heterogeneity was found between the highland and the lowland villages, whereas a significant difference was found between the Sardinian villages and continental Italy. The allele Bf-F1 shows the highest gene frequency so far observed. Typically Sardinian is the gametic association (haplotype) HLA-A30, Cw5, B18, BfF1, DR3.     

Quantitative dermatoglyphics in males and females of five Sardinian linguistic groups

Discriminant analysis of 23 digital and 15 palmar quantitative dermatoglyphic variables of 1364 Sardinians, 689 males and 675 females, were performed to identify biological relationships among five Sardinian linguistic groups of both sexes. By ANOVA 17 of the 23 digital variables and 4 of the 15 palmar variables were significantly different among males and females of the Sardinian linguistic groups. By MANOVA the results indicate high intergroup heterogeneity also within each sex. By stepwise discriminant analysis with p≤0.01 as F-to-enter and p≤0.05 as F-to-remove, only 4 of the 38 digital and palmar variables were in the model (URC R5, RRC L5, RRC R5, URC R4). The pattern of interpopulation biological relationships shows a clearly distinct position of the Gallurian group (both males and females) which speaks an Italian dialect. The properly Sardinian linguistic groups (Campidanian and Logudorian), the Sassarian group (which speaks and Italian dialect) and the Alghero group (which speaks Catalan) were close to one another. This pattern agrees more with the ethno-historical background than with the linguistic one.     

In vitro dissection and development of sheep embryos after preservation at very low temperature (-196 degrees C)

Embryos obtained from Sardinian breed ewes superovulated with FSH-p (Sigma) were frozen at -196 degrees C in liquid nitrogen. After 6 months storage, the embryos (12, all at the compact morula stage), were thawed in a water bath at 39 degrees C for 30 minutes. Six embryos were dissected with a Leitz micromanipulator using a simplified technique. Both demi and intact embryos, were cultured in medium TCM 199 + 10% FCS at 38 degrees C in 5% CO2 for 24 hours. Only five demi-embryos (41.6%) became blastocysts versus 4 whole embryos (66.6%) after the culture period. Splitting as our results show, lowers the embryo viability after freezing-thawing, but it can be used in certain instances to obtain genetic improvement in the Sardinian breed.     

Genetic structure of the Sardinian population at the D1S80 VNTR locus and population diversity

We typed the Sardinian population at the D1S80 VNTR locus. Nineteen alleles were detected in a sample of 92 unrelated individuals, allele frequency distribution showing a modal pattern mostly in agreement with other Caucasoid populations. A high degree of heterozygosity (observed value=80.4%) was present. Goodness-of-fit tests demonstrated no departure from Hardy-Weinberg expectations. Data regarding heterozygosity, number of alleles and singletons appeared in accordance with the IAM mutation-drift equilibrium model and showed no evidence of hidden substructuring. Allele 34 exhibited in Sardinians the highest frequency never observed in Caucasians. Nonetheless, the comparison with other European populations did not disclose Sardinian genetic peculiarity. Indeed, measures of genetic divergence among Europeans demonstrated definitely smaller values at the D1S80 locus in comparison with those calculated over a high number of (pre-DNA) polymorphic loci. High mutation rate and selective neutrality typical of VNTRs could account for the observed moderate genetic divergence. Isolation and genetic drift, on the other hand, may have determined certain deviations in allele frequency distribution, as occurred to allele 34 in the Sardinian population.     

Directional asymmetry of finger ridge counts in females of Sardinian linguistic groups

The data used in this study are the fingerprints of 744 females from 6 Sardinian linguistic groups. We analyzed 15 dermatoglyphic variables of directional asymmetry, namely the right vs left signed differences between i-th homologous fingers for larger ridge counts, radial counts and ulnar counts. Principal components analysis of the dermatoglyphic variables in the females of Sardinian linguistic groups revealed a pattern of biological relations among the groups which is consistent with their linguistic, but especially historical and demographic, backgrounds.     

Conservation genetics of the endangered red deer from Sardinia and Mesola with further remarks on the phylogeography of Cervus elaphus corsicanus

Red deer Cervus elaphus of the endangered populations from Sardinia and Mesola Wood, northern Italy, were analysed for genetic variation at 531 bp of the mitochondrial control region and 12 polymorphic nuclear microsatellite loci. A phylogenetic analysis was conducted including additional data from the literature to gain insight into the phylogeographical origin of the Sardinian subspecies C. e. corsicanus . Microsatellite variation was low in both populations but Sardinia showed comparatively high variability at the control region. Management recommendations are discussed. In particular, the Mesola red deer, the only remaining indigenous Italian population, ought to be managed to increase the effective population size and should be subdivided into two or more populations. As to the phylogeography of the Sardinian population, microsatellite data favoured mainland Italy as the place of origin in that Sardinia and Mesola showed the smallest distance values and were paired together in trees with high bootstrap support. However, the mitochondrial data only partially confirmed this conclusion but showed great similarity between Sardinian and Spanish red deer. Possible explanations for this discrepancy and general limits of mitochondrial sequences in resolving demographic and biogeographical processes of the recent past are discussed.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 691–701.     

Morphological variation among populations of Aphanius fasciatus Nardo, 1827 (Teleostei, Cyprinodontidae) from the Mediterranean

The amount of osteological variation among 11 Italian killifish Aphanius fasciatus populations was examined by the univariate and multivariate analysis of 40 morphometric and meristic variables of the skull and vertebral column. Populations were sampled in three geographically distinct areas (the Adriatic, Sardinia and Sicily). The statistical analysis confirmed that several populations were well differentiated. In particular, discriminant analysis revealed a strong discriminating power of the morphometric variables. Morphometrics of the vertebrae, bony elements of the pharyngeal jaws, supraoccipital and parasphenoid were the most important in discriminating populations. The dendrogram obtained by UPGMA cluster analysis shows the separation of the south-eastern Sicilian populations, that of the Sardinian populations and that of the central-northern Sicilian plus Adriatic populations, as well as the isolation of the Sicilian population from Pantano Viruca and of the Sardinian populations from Pauli Figu from all the others. The significance of the observed differentiation pattern is discussed.     

Initiation codon mutation as a cause of alpha thalassemia

Cloning and sequence analysis of the alpha-globin genes from a Sardinian patient with the nondeletion type of hemoglobin-H disease revealed a new type of thalassemia lesion. A mutation in the alpha 2-globin gene changes the initiation codon ATG to ACG and abolishes the function of this gene. Globin mRNA output from the affected alpha 2 locus is decreased relative to the alpha 1 locus. The mutation is detectable in genomic DNA by restriction analysis with the enzyme NcoI. Of the seven Sardinian patients with nondeletion alpha thalassemia screened with this enzyme, six had the initiation codon lesion.     

A systematic and biogeographical study of epi- and hypogean populations of the Proasellus species group from Sardinia, central Italy and Jordan: allozyme insights

Genetic variation at 18 enzymatic loci was investigated in 10 epigean and three hypogean populations belonging to the Proasellus (Asellidae, Crustacea) species group, from Sardinia, central Italy, and Jordan, in order to clarify their systematics and evolutionary relationships. We found unexpectedly high levels of genetic divergence, which allowed us to recognize a number of genetically distinct species presently hidden under the name Proasellus coxalis . The group of Sardinian populations is the most genetically differentiated. Among the central Italian populations, the northernmost population is the earliest branch in the proposed phyletic scheme, whereas the remaining populations are genetically homogeneous and clustered as the sister taxon of the Sardinian populations. The Jordanian population is genetically differentiated versus both the central Italian and Sardinian groups. The evolutionary relationships seem to indicate that several waves of colonization by this genus occurred in Europe. The evolutionary times inferred from genetic distance data place the main splitting events at the end of the Miocene, in agreement with the paleogeography of the study areas.     

Effects of human perturbation on the genetic make-up of an island population: the case of the Sardinian wild boar

Game species are often manipulated by human beings, whose activities can deeply affect their genetic make-up and population structure. We focused on a geographically isolated wild boar population (Sardinia, Italy), which is classified, together with the Corsican population, as a separate subspecies (Sus scrofa meridionalis). Two hundred and ten wild boars collected across Sardinia were analysed with a set of 10 microsatellites and compared with 296 reference genotypes from continental wild populations and to a sample of domestic pigs. The Sardinian population showed remarkable diversity and a high proportion of private alleles, and strongly deviated from the equilibrium. A Bayesian cluster analysis of only the Sardinian sample revealed a partition into five subpopulations. However, two different Bayesian approaches to the assignment of individuals, accounting for different possible source populations, produced consistent results and proved the admixed nature of the Sardinian population. Indeed, introgressive hybridization with boars from multiple sources (Italian peninsula, central Europe, domestic stocks) was detected, although poor evidence of crossbreeding with free-ranging domestic pigs was unexpectedly found. After excluding individuals who carried exotic genes, the population re-entered Hardy-Weinberg proportions and a clear population structure with three subpopulations emerged. Therefore, the inclusion of introgressed animals in the Bayesian analysis implied an overestimation of the number of clusters. Nonetheless, two of them were consistent between analyses and corresponded to highly pure stocks, located, respectively, in north-west and south-west Sardinia. This work shows the critical importance of including adequate reference samples when studying the genetic structure of managed wild populations.