Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999     

Mitochondrial DNA variation and RAPD mark oleasters,olive and feral olive from Western and Eastern Mediterranean

The study of genetic diversity within the olive-tree (cultivated and wild forms) may be useful to reveal agronomic traits in the wild germplasm and to try to understand the history of the olive-tree domestication. In this way, a study of nuclear and mitochondrial DNAs of cultivated and wild olives from two Corsican and Sardinian Mediterranean islands was performed using RAPD and RFLP markers. Our results show that most of the varieties and most of the oleasters were separated using the UPGMA dendrogram based on the Nei and Li similarity index. Most of oleasters carried either the MOM or MCK mitotype, characteristic of olives in the Western Mediterranean, whereas most of the varieties carried the ME1 mitotype, characteristic of olives in the East Mediterranean. The results indicate that the combination of mitotype and RAPD markers can be used as a powerful tool for differentiating two groups in the wild forms: the Western true oleasters and the feral forms. The true oleasters are characterized by a Western mitotype and a Western RAPD pattern. Feral forms originate either from varieties or from hybridisation between a variety and an oleaster. Consequently, as expected, some of them aggregated with the varieties from which they were derived. The other feral forms are clustered with the oleasters and were detected only by their mitotype determination. This study has also permitted us to differentiate two populations of cultivated olives in Corsica: one with close relationships with Italian varieties (influenced by the East) and one selected from local oleasters probably due to a better local adaptation than foreign varieties.     

The use of molecular markers for germplasm management in a French olive collection

With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rh?ne Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.     

Genetic diversity and gene flow between the wild olive (oleaster, Olea europaea L.) and the olive: several Plio-Pleistocene refuge zones in the Mediterranean basin suggested by simple sequence repeats analysis

Aim The oleaster is believed to have originated in the eastern Mediterranean, implying that those in the western Mediterranean basin could be feral. Several studies with different molecular markers (isozymes, random amplified polymorphic DNA, amplified fragment length polymorphism) have shown a cline between the eastern and the western populations, which supports this hypothesis. To reconstruct the post‐glacial colonization history and establish a relationship between olive and oleaster populations in the Mediterranean basin, analyses were carried out on the genetic variation of chloroplast DNA (chlorotype) and at 12 unlinked simple sequence repeat (SSR) loci, sampling a total of 20 oleaster groves. Location This is the first known large‐scale molecular study of SSR loci based on samples of both oleasters and cultivars from the entire Mediterranean basin. Methods Samples were taken from 166 oleasters in 20 groves of modern populations, and 40 cultivars to represent molecular diversity in the cultivated olive. The Bayesian method and admixture analysis were used to construct the ancestral populations (RPOP; reconstructed panmictic oleaster populations) and to estimate the proportion of each RPOP in each tree. If one tree can be assigned to two or more RPOPs, it can be regarded as a product of hybridization between trees from different populations (i.e. admix origin). Results On this first examination of the SSR genetic diversity in the olive and oleaster, it was found to be structured in seven RPOPs in both eastern and western populations. Based on different population genetic methods, it was shown that: (1) oleasters are equally present in the eastern and the western Mediterranean, (2) are native, and (3) are not derived from cultivars. Chlorotypes (one and three in the eastern and western Mediterranean, respectively) revealed fruit displacement for the oleasters. Main conclusions Oleaster genetic diversity is divided into seven regions that could overlay glacial refuges. The gradient, or cline, of genetic diversity revealed by chloroplast and SSR molecular markers was explained by oleaster recolonization of the Mediterranean basin from refuges after the last glacial event, located in both eastern and western regions. It is likely that gene flow has occurred in oleasters mediated by cultivars spread by human migration or through trade. Animals may have helped spread oleasters locally, but humans have probably transported olives but not oleaster fruits over long distances. We found that cultivars may have originated in several RPOPs, and thus, some may have a more complex origin than expected initially.     

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which differed in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to five distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, confirming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same specific pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones efficiently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas. Received: 25 November 1998 / Accepted: 19 December 1998     

Allozyme variation of oleaster populations (wild olive tree) (Olea europaea L.) in the Mediterranean Basin

As a result of the early domestication and extensive cultivation of the olive tree throughout the Mediterranean Basin, the wild-looking forms of olive (oleasters) presently observed constitute a complex, potentially ranging from wild to feral forms. Allozyme variation was analysed at 10 loci in 31 large and 44 small oleaster populations distributed in various habitats of the Mediterranean Basin and in two populations of the wild subspecies Olea europaea subsp (ssp) guanchica, endemic to the Canary islands and closely related to oleasters. At eight polymorphic loci, 25 alleles were identified. Genetic evidence that nondomesticated oleasters still survive locally was provided by the occurrence of four and one alleles shared exclusively by the eight western and two eastern oleaster populations, respectively, which were collected in forests potentially containing genuinely wild forms according to environmental, historical and demographic criteria. As reported previously from cytoplasmic and RAPDs analysis, substantial genetic differentiation was observed between the eastern oleaster populations genetically close to most olive clones cultivated in the Mediterranean Basin, and the western populations that are related to the wild Canarian populations. In addition, the occurrence of significantly lower heterozygosity in cultivated olive than in oleasters, whatever their origin, suggests that intensive selection involving inbreeding has taken place under cultivation to obtain particular characteristics in the olive cultivars.     

Genetic diversity and relationships of wild and cultivated olives in Turkey

In the present study, we proposed to determine the genetic diversity and relationships between local cultivars and wild olive trees from three important olive-growing regions, i.e., Marmara, Aegean, and Mediterranean, of Turkey. This is the first known large-scale molecular study to investigate the relationships between local cultivars and wild olives from the eastern Mediterranean basin. Two hundred and four oleaster trees and 27 cultivars were sampled to represent molecular diversity. We used 11 simple sequence repeat and 13 sequence-related amplified polymorphism markers to assess genetic variations and inter-relationships among the samples. The results of the analysis showed differences in the levels of allelic composition and heterozygosity between cultivated and wild olive trees. The observation of a high proportion of a certain wild-type genetic background in the cultivars may indicate the former use of local wild trees in olive domestication in Turkey, a possible autochthonal origin of cultivars. “Gemlik” was found to be the most common olive cultivar in the Marmara region and most of the wild olive samples from this region may be feral forms derived from cultivar seed spreading. The information obtained from this study can help to assist the management of an olive collection and sheds some light on the origin of Turkish olive cultivars.     

Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars

One hundred and two olive RAPD profiles were sampled from all around the Mediterranean Basin. Twenty four clusters of RAPD profiles were shown in the dendrogram based on the Ward’s minimum variance algorithm using chi-square distances. Factorial discriminant analyses showed that RAPD profiles were correlated with the use of the fruits and the country or region of origin of the cultivars. This suggests that cultivar selection has occurred in different genetic pools and in different areas. Mitochondrial DNA RFLP analyses were also performed. These mitotypes supported the conclusion also that multilocal olive selection has occurred. This prediction for the use of cultivars will help olive growers to choose new foreign cultivars for testing them before an eventual introduction if they are well adapted to local conditions. Received: 10 April 2000 / Accepted: 15 May 2000     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Microbial communities associated with the root system of wild olives (Olea europaea L. subsp. europaea var. sylvestris) are good reservoirs of bacteria with antagonistic potential against Verticillium dahliae

Wild olive trees, namely oleaster, are considered the ancestor of cultivated olive and a unexplored source of genetic variability that might contain important traits of agronomic and biotechnological interest. The longevity and genetic diversity of oleasters may have favoured selection of specific and well adapted rhizosphere microbial populations that can constitute unique reservoirs of microbial antagonists of Verticillium dahliae, the main soilborne fungal pathogen of olive worldwide. The objective of this present study was to determine the structure and diversity of bacterial communities in the rhizosphere and endosphere of oleaster from 11 havens in Cádiz and Córdoba provinces of Andalusia, southern Spain. To carry out the study we used a multiphasic approach. First, the occurrence and diversity of rhizosphere bacteria was monitored by a cultivation-independent-approach, using fluorescent terminal restriction fragment length polymorphism (FT-RFLP) analyses of amplified 16S rDNA sequences. FT-RFLP patterns revealed a high heterogeneity in the composition of the sampled rhizosphere bacterial communities and suggested the existence of plant genotype-site-specific communities, with each oleaster haven being a unique reservoir of bacterial diversity. Secondly, to investigate the antagonistic potential of these root-associated bacterial populations, a total of 675 bacterial isolates obtained from oleaster rhizosphere and endosphere were screened by dual testing for inhibition of in vitro growth of the highly virulent, olive defoliating pathotype of V. dahliae. Out of 675 tested bacterial isolates, 94 (14%) showed a strong antagonistic activity against a defoliating V. dahliae pathotype. Of the antagonistic bacteria, a slightly lower proportion (12.9% of total bacteria) were inhabitant of the oleaster rhizosphere compared to that in the endosphere (16.5%). The biotechnological potential of those isolates was assessed by in vitro production of different hydrolytic enzymes, indole-1.3-acetic acid (IAA), siderophores, and antimicrobial compounds. Overall, most of bacterial antagonists (58.5 to 78.3%) showed proteolytic, lipolytic, and chitinolytic activity, and produced IAA and siderophores. Finally, analysis of the 16S rDNA gene sequence indicated that most of the 94 bacterial antagonists belong to genera Bacillus (56.4%), Pseudomonas (27.7%), and Paenibacillus (7.4%). Overall, the rhizosphere and endosphere of wild olives were proved as a good reservoir of bacteria antagonists against V. dahliae. Several of those bacteria showing high and broad antagonism potential may therefore be considered for further analyses as promising biocontrol agents against V. dahliae in olive.     

On the origin of the invasive olives (Olea europaea L., Oleaceae)

The olive tree (Olea europaea) has successfully invaded several regions in Australia and Pacific islands. Two olive subspecies (subspp. europaea and cuspidata) were first introduced in these areas during the nineteenth century. In the present study, we determine the origin of invasive olives and investigate the importance of historical effects on the genetic diversity of populations. Four invasive populations from Australia and Hawaii were characterized using eight nuclear DNA microsatellites, plastid DNA markers as well as ITS-1 sequences. Based on these data, their genetic similarity with native populations was investigated, and it was determined that East Australian and Hawaiian populations (subsp. cuspidata) have originated from southern Africa while South Australian populations (subsp. europaea) have mostly derived from western or central Mediterranean cultivars. Invasive populations of subsp. cuspidata showed significant loss of genetic diversity in comparison to a putative source population, and a recent bottleneck was evidenced in Hawaii. Conversely, invasive populations of subsp. europaea did not display significant loss of genetic diversity in comparison to a native Mediterranean population. Different histories of invasion were inferred for these two taxa with multiple cultivars introduced restoring gene diversity for europaea and a single successful founder event and sequential introductions to East Australia and then Hawaii for cuspidata. Furthermore, one hybrid (cuspidata x europaea) was identified in East Australia. The importance of hybridizations in the future evolution of the olive invasiveness remains to be investigated.     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

Substantial genetic diversity in cultivated Moroccan olive despite a single major cultivar: a paradoxical situation evidenced by the use of SSR loci

To assess the genetic diversity in Moroccan cultivated olive, Olea europaea L. subsp. europaea, we performed molecular analysis of olive trees sampled in four geographic zones representing all areas of traditional olive culture. The analysis of 215 trees using 15 simple sequence repeat (SSR) loci revealed 105 alleles distributed among 60 SSR profiles. The analysis of chloroplast deoxyribonucleic acid polymorphism for these 60 olive genotypes allowed to identify four chlorotypes: 42 CE1, one CE2, nine COM1 and eight CCK. Among the 60 SSR profiles, 52 corresponded to cultivated olive trees for which neither denomination nor characterisation is available. These local olive genotypes displayed a spatial genetic structuring over the four Moroccan geographic zones (northwest, north centre, Atlas and southwest), as pairwise Fst values ranged from 0.0394 to 0.1383 and varied according to geographic distance. As single alleles detected in local olive were also observed in Moroccan oleaster populations, results suggest that plant material was mainly selected from indigenous populations. The assumption that Picholine marocaine cultivar is a multi-clonal cultivar was not supported by our data because we found a single genotype for 112 olive trees representing 31 to 93% of the olives sampled locally in the 14 different areas. Picholine marocaine and the few other named cultivars do not seem to belong to the same gene pools as the unnamed genotypes cultivated only locally. The situation is paradoxical: a substantial genetic diversity in Moroccan olive germplasm, probably resulting from much local domestication, but a single cultivar is predominant.     

Range and Frequency of Africanized Honey Bees in California (USA)

Africanized honey bees entered California in 1994 but few accounts of their northward expansion or their frequency relative to European honey bees have been published. We used mitochondrial markers and morphometric analyses to determine the prevalence of Africanized honeybees in San Diego County and their current northward progress in California west of the Sierra Nevada crest. The northernmost African mitotypes detected were approximately 40 km south of Sacramento in California’s central valley. In San Diego County, 65% of foraging honey bee workers carry African mitochondria and the estimated percentage of Africanized workers using morphological measurements is similar (61%). There was no correlation between mitotype and morphology in San Diego County suggesting Africanized bees result from bidirectional hybridization. Seventy percent of feral hives, but only 13% of managed hives, sampled in San Diego County carried the African mitotype indicating that a large fraction of foraging workers in both urban and rural San Diego County are feral. We also found a single nucleotide polymorphism at the DNA barcode locus COI that distinguishes European and African mitotypes. The utility of this marker was confirmed using 401 georeferenced honey bee sequences from the worldwide Barcode of Life Database. Future censuses can determine whether the current range of the Africanized form is stable, patterns of introgression at nuclear loci, and the environmental factors that may limit the northern range of the Africanized honey bee.     

Substoichiometrically different mitotypes coexist in mitochondrial genomes of Brassica napus L

Cytoplasmic male sterility (CMS) has been identified in numerous plant species. Brassica napus CMS plants, such as Polima (pol), MI, and Shaan 2A, have been identified independently by different researchers with different materials in conventional breeding processes. How this kind of CMS emerges is unclear. Here, we report the mitochondrial genome sequence of the prevalent mitotype in the most widely used pol-CMS line, which has a length of 223,412 bp and encodes 34 proteins, 3 ribosomal RNAs, and 18 tRNAs, including two near identical copies of trnH. Of these 55 genes, 48 were found to be identical to their equivalents in the "nap" cytoplasm. The nap mitotype carries only one copy of trnH, and the sequences of five of the six remaining genes are highly similar to their equivalents in the pol mitotype. Forty-four open reading frames (ORFs) with unknown function were detected, including two unique to the pol mitotype (orf122 and orf132). At least five rearrangement events are required to account for the structural differences between the pol and nap sequences. The CMS-related orf224 neighboring region (～5 kb) rearranged twice. PCR profiling based on mitotype-specific primer pairs showed that both mitotypes are present in B. napus cultivars. Quantitative PCR showed that the pol cytoplasm consists mainly of the pol mitotype, and the nap mitotype is the main genome of nap cytoplasm. Large variation in the copy number ratio of mitotypes was found, even among cultivars sharing the same cytoplasm. The coexistence of mitochondrial mitotypes and substoichiometric shifting can explain the emergence of CMS in B. napus.     

Genetic structure and core collection of the World Olive Germplasm Bank of Marrakech: towards the optimised management and use of Mediterranean olive genetic resources

The conservation of cultivated plants in ex-situ collections is essential for the optimal management and use of their genetic resources. For the olive tree, two world germplasm banks (OWGB) are presently established, in Córdoba (Spain) and Marrakech (Morocco). This latter was recently founded and includes 561 accessions from 14 Mediterranean countries. Using 12 nuclear microsatellites (SSRs) and three chloroplast DNA markers, this collection was characterised to examine the structure of the genetic diversity and propose a set of olive accessions encompassing the whole Mediterranean allelic diversity range. We identified 505 SSR profiles based on a total of 210 alleles. Based on these markers, the genetic diversity was similar to that of cultivars and wild olives which were previously characterised in another study indicating that OWGB Marrakech is representative of Mediterranean olive germplasm. Using a model-based Bayesian clustering method and principal components analysis, this OWGB was structured into three main gene pools corresponding to eastern, central and western parts of the Mediterranean Basin. We proposed 10 cores of 67 accessions capturing all detected alleles and 10 cores of 58 accessions capturing the 186 alleles observed more than once. In each of the 10 cores, a set of 40 accessions was identical, whereas the remaining accessions were different, indicating the need to include complementary criteria such as phenotypic adaptive and agronomic traits. Our study generated a molecular database for the entire OWGB Marrakech that may be used to optimise a strategy for the management of olive genetic resources and their use for subsequent genetic and genomic olive breeding.     

Historical biogeography of olive domestication (Olea europaea L.) as revealed by geometrical morphometry applied to biological and archaeological material

Aim This study intends to improve our understanding of historical biogeography of olive domestication in the Mediterranean Basin, particularly in the north-western area. Location Investigations were performed simultaneously on olive stones from extant wild populations, extant cultivated varieties from various Mediterranean countries, and archaeological assemblages of Spanish, French and Italian settlements. Methods A combination of morphometrics (traditional and geometrical) allowed us to study both the size and shape of endocarp structure. Concerning shape, a size-standardized method coupled with fitted polynomial regression analysis was performed. Results We found morphological criteria for discriminating between wild and cultivated olive cultivars, and established patterns of morphological variation of olive material according to the geographical origin (for extant material) and to the age of the olive forms (for archaeological material). Levels of morphological convergences and divergences between wild olive populations and cultivated varieties are presented as evidence. Main conclusions Morphological changes of endocarps of olive under domestication at both geographical and chronological scales provide new criteria for the identification of olive cultivars. They allow to determine the origins of cultivated forms created and/or introduced in the north-western Mediterranean regions and to understand how human migrations affected the rest of the Western Mediterranean regions. A model of diffusion of olive cultivation is proposed. It shows evidence of an indigenous origin of the domestication process, which is currently recognized in the north-western area since the Bronze Age.     

Plastid and nuclear DNA polymorphism reveals historical processes of isolation and reticulation in the olive tree complex (Olea europaea)

Aim The olive tree is considered one of the best indicators of the Mediterranean climate. The species’ distribution is associated with geographical and bioclimatic factors, as well as being influenced by a long period of cultivation. Despite concerted efforts of different research groups, the origin of the Mediterranean olive tree still remains elusive. In the present study, relationships between taxa and populations covering the entire range of Olea europaea were investigated using both maternal (plastid genome) and biparental (nuclear genome) markers to disclose evolutionary patterns in the olive complex. Phylogenetic and phylogeographical results of the two‐genome analyses were interpreted in a biogeographical context. Location Mediterranean, temperate and subtropical floristic regions of the Old World. Methods Phylogeographical reconstructions of plastid DNA polymorphism were performed using microsatellites, restriction sites and indels on a wide sample of 185 representative trees across the Old World, including 28 herbarium specimens from remote areas. Additionally, the potential utility of one ITS‐1 pseudogene for phylogenetic analyses was explored using Bayesian and maximum parsimony approaches on a subsample of 38 olive trees. Results Forty plastid haplotypes were recognized and split into two lineages and seven sublineages. The analysis of ITS‐1 sequences also allowed the identification of seven well differentiated groups. Distribution of plastid and ribosomal DNA lineages was congruent, but particular cases of phylogenetic incongruence were disclosed (particularly in the Sahara and Madeira). Lastly, two divergent ITS‐1 copies were isolated from the same sample of four individuals of different subspecies. Main conclusions Phylogenetic congruence of both ITS‐1 and plastid lineages suggested an evolutionary scenario of predominant isolation during the Plio‐Pleistocene in Macaronesia, the Mediterranean, southern Africa, eastern Africa and Asia. The Saharan desert appeared to have played an important role of vicariant barrier between southern and northern African populations in early times. Incongruence of some plastid and nuclear results, as well as intermingled ITS‐1 copies of different lineages in single individuals, was interpreted as a result of recurrent reticulation events in the olive complex. We identified an ancient hybrid zone from the Sahara to north‐eastern African mountains, where divergent plastid and nuclear lineages still co‐exist. Results of this paper, and previous studies, suggest that the cultivated olive originated from a pre‐Quaternary Mediterranean ancestor, with no evidence for a recent hybrid origin. In contrast, a continuous process of olive domestication through local hybridization events of cultivated trees with natural populations may have brought about a remarkably high genomic diversity among cultivated trees across the Mediterranean.     

Mitochondrial DNA phylogeny and the reconstruction of the population history of a species: the case of the European anchovy (Engraulis encrasicolus)

Analysis of mitochondrial DNA restriction fragment length polymorphism in European anchovy (Engraulis encrasicolus) revealed a large number of mitotypes that form two distinct clusters (phylads). Phylad A consists of one common mitotype and many rare secondary mitotypes that are one mutational step removed from the main type. Nucleotide diversity and number of homoplasious changes are low. Phylad B has a complex pattern of mitotype connectedness, high nucleotide diversity, and a large number of homoplasious changes. It is suggested that the two phylads evolved in isolation from each other and that present coexistence is the result of a secondary contact. Moreover, phylad A has a "star" phylogeny, which suggests that it has evolved in a population that experienced a drastic bottleneck followed by an explosion of size. Phylad A is practically the only phylad present in the Black Sea, with its frequency dropping to 85% in the northern Aegean, and to 40% in the rest of Mediterranean and the Bay of Biscay. The Black Sea is, therefore, the most likely place of origin of phylad A. Molecular data are consistent with a population bottleneck in the Black Sea during the last glaciation event and a subsequent exit of phylad A with the outflow into the Aegean following the ice melting. Phylogenetic analysis of anchovy mtDNA provides a reconstruction of population history in the Mediterranean, which is consistent with the geological information.      

Combination of chloroplast and mitochondrial DNA polymorphisms to study cytoplasm genetic differentiation in the olive complex ( Olea europaea L.)

Four hundred and four individuals belonging to the species Olea europaea were characterised using mitochondrial DNA (mtDNA) RFLPs. Twelve mitotypes were distinguished. The combination of mtDNA information with cpDNA polymorphism (characterised in a previous study) led us to recognise 20 cytoplasmic lineages of which seven were found in the Mediterranean area (oleasters, cultivars and O. e. subsp. maroccana). In the olive complex, strong cytoplasm genetic differentiation was revealed ( F(st) = 0.73). Very strong linkage disequilibrium between cpDNA and mtDNA polymorphisms was observed, particularly in the Mediterranean subspecies europaea. This high congruence between genetic structure based on cpDNA or mtDNA sustains a low level of recurrent mutation in both organelle DNAs and, thus, the polymorphisms used in this study were pertinent to reconstruct olive phylogeography. In the Mediterranean area, genetic drift due to population regression during Quaternary glaciations, and founder effects associated with the postglacial seed dissemination, have probably contributed to the existence of a high genetic linkage disequilibrium between cpDNA and mtDNA polymorphisms. Thus, four Mediterranean cytoplasmic lineages, clearly distinguished both by cpDNA and mtDNA polymorphisms, most likely reflect four distinct relic populations during Quaternary glaciations. Finally, O. e. subsp. maroccana from South Morocco, which also displayed specific cytoplasmic lineages, should be considered as another relic Mediterranean population.