Genetic diversity of the sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) in Central Europe and the western part of the Balkan Peninsula and evidence of marron genotype introgression into wild populations

The sweet chestnut (Castanea sativa Mill.) is a widely spread and important multipurpose tree species in the Mediterranean area, which has played an important role in human history. Natural events, such as glaciations, and human influence played significant roles in the distribution and genetic makeup of the sweet chestnut. In order to better understand how natural and human-mediated past events affected the current genetic diversity and structure of the sweet chestnut, we analysed populations from Central Europe and the western part of the Balkan Peninsula, utilizing ten polymorphic nuclear microsatellite markers. The study revealed the existence of three genetically and, to a large extent, geographically distinct and well-defined groups of sweet chestnut populations. Two not entirely separated groups of populations were detected in the northern part of the studied area and one in the southern. Our results indicate that the genetic structure of sweet chestnut populations in Central Europe and the western part of the Balkan Peninsula is the result of both natural colonization events and significant and lengthy human impact. Furthermore, it has been proven that the gene flow between cultivated/grafted trees’ and wild chestnut stands can influence their genetic structure. However, our results reveal that cultivated-to-wild introgression in the sweet chestnut is dependent on the close proximity of chestnut orchards and naturally occurring populations.     

Unraveling the naturalness of sweet chestnut forests (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) in central Spain

This paper describes the patterns and processes of vegetation change and fire history in the Late Holocene (c. 3,140 cal bp) palaeoecological sequence of El Tiemblo, in a mountainous area in central Spain (Gredos range, Spanish Central System), and provides the first Iberian pollen sequence undertaken within a Castanea sativa-dominated woodland. These new data reassess not only the autochthonous nature of the species in the region and in the Iberian Peninsula, but also the naturalness of well-developed sweet chestnut forests. The study focuses on anthropogenic dynamics linked both to the effects of livestock husbandry and the use of fire for forest clearance. With this aim, non-pollen palynomorphs (coprophilous fungi ascospores) and charcoal accumulation rate are useful indicators for assessing the increasing role of human influence on vegetation.     

Phylogeography of the invasive weed Hypochaeris radicata (Asteraceae): from Moroccan origin to worldwide introduced populations

In an attempt to delineate the area of origin and migratory expansion of the highly successful invasive weedy species Hypochaeris radicata, we analysed amplified fragment length polymorphisms from samples taken from 44 populations. Population sampling focused on the central and western Mediterranean area, but also included sites from Northern Spain, Western and Central Europe, Southeast Asia and South America. The six primer combinations applied to 213 individuals generated a total of 517 fragments of which 513 (99.2%) were polymorphic. The neighbour-joining tree presented five clusters and these divisions were supported by the results of Bayesian analyses: plants in the Moroccan, Betic Sierras (Southern Spain), and central Mediterranean clusters are all heterocarpic. The north and central Spanish, southwestern Sierra Morena, and Central European, Asian and South American cluster contain both heterocarpic (southwestern Sierra Morena) and homocarpic populations (all other populations). The Doñana cluster includes two homocarpic populations. Analyses of fragment parameters indicate that the oldest populations of H. radicata are located in Morocco and that the species expanded from this area in the Late Quaternary via at least three migratory routes, the earliest of which seems to have been to the southwestern Iberian Peninsula, with subsequent colonizations to the central Mediterranean area and the Betic Sierras. Homocarpic populations originated in the southwestern Iberian Peninsula and subsequently spread across north and central Spain, Central Europe and worldwide, where they became a highly successful weed.     

Role of domestication in shaping <Emphasis Type="Italic">Castanea sativa</Emphasis> genetic variation in Europe

The genetic structure of sweet chestnut (Castanea sativa Mill.) across Europe was assessed using 73 inter-simple sequence repeat markers to screen 1,768 individuals from 68 stands distributed across 29 sites in five European countries (Italy, France, Spain, Greece, and UK). At each site, trees were sampled from three distinct management types (domestication levels): naturalized stands, managed coppice, and grafted fruit orchards. In more than a third of the orchards, nonlocal genetic material (grafted clones) were evident, showing (as predicted) large differences from the other two domestication levels for most of the within-population genetic diversity parameters estimated. Randomly generated linkage disequilibrium analysis revealed weak though significant differences in two-locus allelic correlations between naturalized stands and coppice, suggesting that long-term management techniques may influence the genetic makeup of the populations. Multivariate analysis revealed the existence of five distinct gene pools across the study area; three were located in Greece, one on the northwestern coast of the Iberian peninsula and a large gene pool covering the rest of the Mediterranean basin. The implications of the results are discussed in relation to developing conservation strategies for chestnut genetic resources in Europe.     

Landscape genetic structure of chestnut (Castanea sativa Mill.) in Spain

The current need for forest conservation and management has driven a rapid expansion of landscape genetics approach. This discipline combines tools from molecular genetics, landscape ecology and spatial statistics and is decisive for improving not only ecological knowledge but also for properly managing population genetic resources. This approach could be appropriate to sweet chestnut (Castanea sativa Mill.), a multipurpose species of great economic importance in the Mediterranean basin and a species considered to be a good model of integration between natural and human-driven distribution of diversity. Sixteen chestnut populations, covering the distribution range of the species in Spain, were analysed using seven microsatellite markers. Results revealed a high level of genetic diversity in Spanish chestnut populations, which in part followed a geographical pattern, although distribution was not homogeneous. Likewise, areas particularly rich in diversity were detected, facilitating the development of a hypothesis about the history of chestnut in Spain. In conclusion, these results provide valuable baseline data for more in-depth studies on chestnut landscape genetics that can contribute to its conservation.     

Chestnut cultivar diversification process in the Iberian Peninsula, Canary Islands, and Azores

This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.     

Variation in grafted European chestnut and hybrids by microsatellites reveals two main origins in the Iberian Peninsula

This is the first known large-scale molecular study of simple sequence repeats (SSR) loci based on samples from grafted trees found in the Iberian Peninsula and Canary Islands and hybrids. Interspecific hybrids resistant to ‘ink disease’ (Phytophthora spp.) were obtained in France, Portugal and Spain, although difficult to distinguish by morphology. This study focuses on genetic variation using 10 SSRs (11 unlinked loci) from clonally propagated cultivars (574 accessions) of European chestnut (Castanea sativa) and hybrids (71 accessions). They were compared with a representative sample of exotic chestnut species present in the Atlantic area, 47 accessions of Castanea crenata, 37 of Castanea mollissima and 33 of Castanea henryi. Accessions were analysed using a model-based Bayesian procedure (Structure), factorial correspondence analysis and analysis of molecular variance. The main chestnut species, hybrids and alien introgressions were differentiated. Two main origins of variability in European cultivated chestnut were found in the Iberian Peninsula, one in the North and a second in the Centre. Andalusian and Canary Island accessions could be assigned to both of these zones, which indicate that they could have been colonised with cultivars originating from either zone, in the case of the Canary Islands from the sixteenth century on.     

Genetic structure,relationships and admixture with wild relatives in native pig breeds from Iberia and its islands

Background

Native pig breeds in the Iberian Peninsula are broadly classified as belonging to either the Celtic or the Mediterranean breed groups, but there are other local populations that do not fit into any of these groups. Most of the native pig breeds in Iberia are in danger of extinction, and the assessment of their genetic diversity and population structure, relationships and possible admixture between breeds, and the appraisal of conservation alternatives are crucial to adopt appropriate management strategies.

Methods

A panel of 24 microsatellite markers was used to genotype 844 animals representing the 17 most important native swine breeds and wild populations existing in Portugal and Spain and various statistical tools were applied to analyze the results.

Results

Genetic diversity was high in the breeds studied, with an overall mean of 13.6 alleles per locus and an average expected heterozygosity of 0.80. Signs of genetic bottlenecks were observed in breeds with a small census size, and population substructure was present in some of the breeds with larger census sizes. Variability among breeds accounted for about 20% of the total genetic diversity, and was explained mostly by differences among the Celtic, Mediterranean and Basque breed groups, rather than by differences between domestic and wild pigs. Breeds clustered closely according to group, and proximity was detected between wild pigs and the Mediterranean cluster of breeds. Most breeds had their own structure and identity, with very little evidence of admixture, except for the Retinto and Entrepelado varieties of the Mediterranean group, which are very similar. Genetic influence of the identified breed clusters extends beyond the specific geographical areas across borders throughout the Iberian Peninsula, with a very sharp transition from one breed group to another. Analysis of conservation priorities confirms that the ranking of a breed for conservation depends on the emphasis placed on its contribution to the between- and within-breed components of genetic diversity.

Conclusions

Native pig breeds in Iberia reveal high levels of genetic diversity, a solid breed structure and a clear organization in well-defined clusters.     

Non-congruent geographic patterns of genetic divergence across European species of <Emphasis Type="Italic">Branchinecta</Emphasis> (Anostraca: Branchinectidae)

Three of the five European species of Branchinecta have a disjunct distribution. In this study, we analyze populations of B. ferox and B. orientalis for mitochondrial (cox1) and nuclear (ITS2) molecular markers. We compare intraspecific genetic divergences between geographically distant populations of B. orientalis, from its only known Spanish population (originally described as B. cervantesi) and from a Hungarian population (assigned to B. orientalis since its discovery), with data from two relatively close Iberian populations of B. ferox. Results indicate that isolation between B. ferox and B. orientalis clades is ancient, and that the clade including the two Iberian populations of B. ferox is geographically structured. Conversely, Iberian and Hungarian populations of B. orientalis do not show geographical structure for the mitochondrial fragment. Lack of geographic structure coupled with very low genetic distances indicates that current Iberian and Hungarian populations of B. orientalis originated from a common population stock, and that the time elapsed since their separation has not been long enough to render the clades reciprocally monophyletic. We hypothesize that colonization of the Iberian Peninsula by B. orientalis is probably the consequence of a single recent dispersal event, and consequently we confirm the synonymy between B. cervantesi and B. orientalis.     

Sex-biased initial eggs favours sons in the slightly size-dimorphic Scops owl (Otus scops)

There is considerable controversy concerning the origin of Iberian populations of the Mediterranean chameleon, Chamaeleo chamaeleon . Current opinion dictates that Spanish populations result from introductions during the 18^th and 19^th centuries, with subsequent translocations from the original populations to other parts of Spain. The Portugese population in the Algarve is believed to have been introduced from Africa or Spain during the 1920s. However, Holocene remains of chameleons suggest that the Malaga population at least could have a much older origin. Analysis of sequences from the mitochondrial 16S ribosomal RNA gene of samples from the Iberian Peninsula and North Africa revealed a double origin for the Iberian population. The Mediterranean Iberian (Malaga) population is closely related to Mediterranean North African populations, with Atlantic Iberian populations more closely related to populations of the Atlantic coast of North Africa. The overall genetic differentiation and diversity observed was very low, preventing precise dating of the colonization events. However this low level of differentiation is not consistent with Plio-Pleistocene colonization, the assumed timing for a natural colonization event and suggests that chameleons were probably introduced twice by man in the recent past. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 1–7.     

On the Iberian endemism <Emphasis Type="Italic">Eurylophella iberica</Emphasis> Keffermuller and Da Terra 1978 (Ephemeroptera,Ephemerellidae): current and future potential distributions,and assessment of the effectiveness of the Natura 2000 network on its protection

Eurylophella iberica Keffermüller and Da Terra, 1978 is an endemic insect species of the Iberian Peninsula whose distribution has been poorly studied to date with rather old and scattered records. Here we compiled all existing distribution records and add new records from recent sampling activities. We also used this updated distributional information and environmental data (climate and geology) to estimate both current and future potential distributions in different climate change scenarios. We found that currently ca. 50% of the total Iberian region could present suitable environmental conditions for E. iberica (all the Iberian Peninsula, save the most eastern and Mediterranean areas). However, the potential distributions estimated when considering future climate change scenarios showed a marked reduction in the areas with suitable environmental conditions for the species, especially in the south. The northwest part of the Iberian Peninsula is a crucial zone for the future survival of this endemic species. We also found that most populations that occur in areas with suitable (both current and future) environmental conditions fall outside the Natura 2000 network of protected areas. Our results represent the first attempt to estimate the potential distribution of this endemic species providing important insights for its conservation.     

The Consumption of Acorns (from <Emphasis Type="Italic">Quercus</Emphasis> spp.) in the Central West of the Iberian Peninsula in the 20th Century

There is evidence of the consumption of acorns from Quercus species in the Iberian Peninsula from prehistory through the 20th century up until the 1960s. Acorns were used primarily for human consumption, mainly during food shortages. The high abundance and even distribution of Quercus tree species made it possible for acorn consumption to be widespread across the Iberian Peninsula. The favored species was the holm oak (Quercus ilex subsp. ballota), because a large part of its harvest consists of sweet acorns, while in other species the acorns are almost always bitter. People developed a substantial knowledge base underpinning a great variety of uses of acorns, from eating them directly from the tree to preparation with very simple treatments, such as drying, roasting, or boiling. By manipulating levels of bitterness in a number of species, cooks were able to prepare dishes that ranged from salty to sweet.Based on interviews with knowledgeable people and a review of ethnobotanical papers, this article describes the forms of consumption, the processed products, and the other uses of acorns of the species of the genus Quercus in the central west of the Iberian Peninsula. We also suggest why acorns lost their prominence in the late 1960s. At present, the main use of the acorn is as food for Iberian black pigs to obtain quality sausages. In addition, new products such as acorn liquor, caramels, and other items have recently appeared, marketed as distinctive products on a small scale.     

Genetic variability and diversification process in local pear cultivars from northwestern Spain using microsatellites

This is the first known large-scale molecular study of simple sequence repeats loci based on pear cultivars from the northwestern Iberian Peninsula. Most of the Spanish pear crop (one of the largest in Europe) is based on the Spanish cultivar Blanquilla and various other foreign cultivars. However, local cultivars can still be found in old orchards in northwestern Spain. Between 1978 and 1981, the Centro de Investigaciones Agrarias de Mabegondo (Xunta de Galicia) established a Germplasm Bank of local pear cultivars containing 221 accessions. In the current study, these were analysed and compared with 20 commercial cultivars of Pyrus spp. using 19 microsatellites. We identified 127 genotypes out of 221 accessions with an average of 43% clonality in this collection. Genotypes were analysed using a model-based Bayesian procedure (Structure), factorial correspondence analysis and molecular variance analysis, and Jaccard coefficients were estimated. Four reconstructed populations were identified by Structure, one related to Asian cultivars, two to French and English cultivars and one to Galician cultivars. The four identified groups of pears had evolved independently. This study explains the diversification process in pear cultivars from northwestern cultivars based on hybridization (16%), selection of triploids (38%) and the introgression of commercial cultivars in the collection (4%).     

Distribution patterns of the peracarid crustaceans associated with the alga <Emphasis Type="Italic">Corallina elongata</Emphasis> along the intertidal rocky shores of the Iberian Peninsula

Spatial patterns of intertidal peracarids, associated with the alga Corallina elongata, were studied along the whole Iberian Peninsula. A total of 28,215 specimens were collected, comprising 78 different species (57 amphipods, 16 isopods, 4 tanaids and 1 cumacean), most of them with Atlantic-Mediterranean distribution (60%) and only 9% of Mediterranean endemics. Gammarids were dominant in abundance and number of species, representing more than 70% of the total peracarids. The most common species collected during the present study were the caprellid Caprella penantis, the gammarids Hyale schmidti, Hyale stebbingi, Jassa cf. falcata and Stenothoe monoculoides, the isopod Ischyromene lacazei and the tanaid Tanais dulongii. Caprellids and tanaidaceans presented their highest populations in the stations of the Strait of Gibraltar, whereas isopods were more abundant in Atlantic stations. Univariate analyses did not reflected differences in number of species, abundance and Shannon-Weaver diversity between Mediterranean and Atlantic. However, cluster analyses and Whittaker index, as measure of ß-diversity, showed a different species composition between Mediterranean and Atlantic and a replacement of species along the coast, especially at the Strait of Gibraltar. The turnover mainly affected species of the same genera, probably related with sympatric speciation. CCA and BIO-ENV analyses showed high correlations between environmental measures (especially conductivity) and peracarid distribution. Mediterranean species tolerated higher values of conductivity and temperature, while Atlantic species were associated with stations characterized by higher oxygen concentrations.     

Genome data from a sixteenth century pig illuminate modern breed relationships

Ancient DNA (aDNA) provides direct evidence of historical events that have modeled the genome of modern individuals. In livestock, resolving the differences between the effects of initial domestication and of subsequent modern breeding is not straight forward without aDNA data. Here, we have obtained shotgun genome sequence data from a sixteenth century pig from Northeastern Spain (Montsoriu castle), the ancient pig was obtained from an extremely well-preserved and diverse assemblage. In addition, we provide the sequence of three new modern genomes from an Iberian pig, Spanish wild boar and a Guatemalan Creole pig. Comparison with both mitochondrial and autosomal genome data shows that the ancient pig is closely related to extant Iberian pigs and to European wild boar. Although the ancient sample was clearly domestic, admixture with wild boar also occurred, according to the D-statistics. The close relationship between Iberian, European wild boar and the ancient pig confirms that Asian introgression in modern Iberian pigs has not existed or has been negligible. In contrast, the Guatemalan Creole pig clusters apart from the Iberian pig genome, likely due to introgression from international breeds.     

MSP-PCR and RAPD molecular biomarkers to characterize<Emphasis Type="Italic">Amanita ponderosa</Emphasis> mushrooms

Amanita ponderosa is a specie of wild edible mushrooms growing spontaneously in some Mediterranean microclimates, namely in Alentejo and Andaluzia, in the Iberian Peninsula. The nutritional values of these fungi make them highly exportable. Due to the wide diversity of mushrooms in nature, it is essential to differentiate and to identify the various edible species. RAPD markers have been used as a valuable tool to distinguish the different genotypes, although this method has not yet been used toAmanita ponderosa. Two methods were used to establish different genetic fingerprinting patterns of edible mushrooms. Samples ofAmanita ponderosa were collected in six different regions of the southwest of the Iberian Peninsula and compared by RAPD-PCR and MSP-PCR. Additionally, to compare molecular profiles with others genera of edible mushrooms, three species of Basidiomycetes (Pleurotus ostreatus, Lactarius deliciosus andCoriolus versicolor) and an Ascomycete were used. Results showed that some molecular markers discriminate among an Ascomycete from Basidiomycetes (Amanita ponderosa, Pleurotus ostreatus, Lactarius deliciosus andCoriolus versicolor) and discriminate among the different genera within basidiomycetes, as it is expected. Moreover, OPF-6, OPG-2, OPG3 and M13 primes allowed to unravel a level of genetic polymorphism withinAmanita ponderosa mushrooms collected from different geographic origin.     

Mitochondrial haplotype variation and phylogeography of Iberian brown trout populations

The biogeographical distribution of brown trout mitochondrial DNA haplotypes throughout the Iberian Peninsula was established by polymerase chain reaction-restriction fragment polymorphism analysis. The study of 507 specimens from 58 localities representing eight widely separated Atlantic-slope (north and west Iberian coasts) and six Mediterranean drainage systems served to identify five main groups of mitochondrial haplotypes: (i) haplotypes corresponding to non-native, hatchery-reared brown trout that were widely distributed but also found in wild populations of northern Spain (Cantabrian slope); (ii) a widespread Atlantic haplotype group; (iii) a haplotype restricted to the Duero Basin; (iv) a haplotype shown by southern Iberian populations; and (v) a Mediterranean haplotype. The Iberian distribution of these haplotypes reflects both the current fishery management policy of introducing non-native brown trout, and Messinian palaeobiogeography. Our findings complement and extend previous allozyme studies on Iberian brown trout and improve present knowledge of glacial refugia and postglacial movement of brown trout lineages.     

Instant domestication process of European chestnut cultivars

This study presents the results of the first genetic analysis of ancient chestnut trees (Castanea sativa Mill.) in Italy and in the Iberian Peninsula to better understand the effect of grafting on the domestication process of chestnut and to investigate the impacts of early selection and improvement on the genetic diversity retained. We evaluated 105 giant ancient trees from Italy, Spain and Portugal and compared them with the European Union (EU) database of chestnut cultivars by using a set of 24 simple sequence repeats (SSRs; microsatellite markers). We measured the perimeter (girth) at the diameter at breast height (DBH). Samples from both the canopy and the roots of each tree were analysed to distinguish which trees were self‐rooted and which were grafted. Diversity was compared using standard metrics and model‐based approaches based on the expected heterozygosity (He) at equilibrium. We could differentiate 91 new genotypes; 9.6% matched known chestnut cultivars. We found the first evidences of cultivation, that is, grafting to produce “instant domestication” in Galicia and in the Douro Valley in trees of 14‐m perimeter (15th century) and in the Basque Country (first report in that area) in a tree of 11.5‐m perimeter (16th century). In Italy, the cultivar “Marrone Fiorentino” was found in some giant trees with perimeters of 8 and 9 m (17th‐18th centuries) in the Toscana and Umbria. Those findings matched with written references in Portugal from the 16th century and from the 18th century in Spain. “Instant domestication” could be dated back to the 15th century and was related to the wild populations existing in the same areas where cultivars are being propagated, without a different genetic structure for wild chestnut trees and with a high diversity maintained through the initiation of domestication.     

Genetic introgression on freshwater fish populations caused by restocking programmes

The brown trout (Salmo trutta L.) is one of the best studied native salmonids of Europe. Genetic studies on this species suggest that a large proportion of the evolutionary diversity corresponds to southern European countries, including the Iberian Peninsula, where this study is focused. Stocking activities employing non-indigenous hatchery specimens together with the destruction and fragmentation of natural habitats are major factors causing a decrease of native brown trout populations, mostly in the Mediterranean basins of the Iberian Peninsula. The main aim of the present work is to examine the genetic structure of the brown trout populations of the East Cantabrian region, studying the consequences of the restocking activities with foreign hatchery brown trout specimens into the wild trout populations. We have based our study on the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism technique conducted on a mitochondrial fragment of 2700 base pairs and on the lactate dehydrogenase locus of the nuclear DNA. Our results show higher introgression rates in the Ebro (Mediterranean) basin than in the Cantabrian rivers. This revised version was published online in July 2006 with corrections to the Cover Date.     

New large threatened populations of <Emphasis Type="Italic">Phengaris nausithous</Emphasis> discovered in the SW of Europe

The near threatened Phengaris nausithous is distributed in two large mountainous areas in the northern part of the Iberian Peninsula, living in small dispersed populations. During July and August of 2012–2015 we systematically searched for the species in the southeastern part of the Cordillera Cantábrica. We found 38 new populations grouped into seven metapopulations. Two of the latter are the largest known, representing 34 % of the species’ occurrence range in Spain. The discovery of these new metapopulations does not improve the conservation status of P. nausithous in the SW of Europe because a land consolidation project currently underway and an established peat extraction plant could lead to the imminent extinction of the largest metapopulations. However, there is still time to reverse the present course of change. Knowledge of these recently discovered populations allows the establishment of management measures to protect them over the long term.