Allozyme variation in cork oak (Quercus suber L.): the role of phylogeography and genetic introgression by other Mediterranean oak species and human activities

 Genetic variation in the cork oak (Quercus suber L.) was investigated using 11 loci from seven enzyme systems in 40 populations sampled over the entire distribution of this species in the western Mediterranean Basin. Mean heterozygosity values over the polymorphic loci (Ho=0.283), the percentage of polymorphic populations (M=0.76), and the total genetic diversity (Ht=0.31) from which 11% was accounted for among-population variation, are among the highest recorded in oak species. In contrast to previous results in holm oak (Q. ilex L.), another evergreen species in the same area, cork oak possessed a smaller allele pool and a lower average number of alleles per locus and per population (A=2.0). More particularly, very few low-frequency alleles were observed in cork oak except for eight populations in which allozyme polymorphism at locus Pgi 1, diagnostic between both species, indicates that these low-frequency alleles are introgressed from holm oak. On the basis of the genetic distance estimated from allozyme frequencies, 32 of the 40 cork oak populations studied were classified into two very distinct sets which also corresponded to distinct geographic areas. One set gathered together the 18 populations from the Iberian peninsula and two adjacent areas in France, i.e. the centre of origin of cork oak, according to paleobotanical data. This set was characterized by a larger allele pool, a higher within-population genetic diversity and a lower differentiation between populations than was observed in the other set, which comprised the populations from North Africa, Sicily, Sardinia, Corsica, continental Italy and the region of Provence (southeastern France). In these more southern and eastern disjunct areas, cork oak migration from Iberia may have occurred at different periods since the end of the Tertiary. The possible effect of human activity on cork oak genetic structure, i.e. the selection of good-quality cork, acorn over-use for animal food, and even human nutrition, is discussed. Received: 3 March 1998 / Accepted: 19 March 1998     

Multiple origins of polyploidy and the geographic structure of Heuchera grossulariifolia

Multiple origins of polyploidy from an ancestral diploid plant species were investigated using restriction site polymorphism and sequence variation in the chloroplast DNA (cpDNA) of Heuchera grossulariifolia (Saxifragaceae). Phylogenetic analysis indicated that autopolyploidy has arisen at least twice in the evolutionary history of this species and potentially up to as many as seven times. These results suggest a greater range of independent polyploid origins as compared to a previous study of H. grossulariifolia using cpDNA restriction sites that indicated a minimum of three independent origins. Moreover, most polyploid populations did not contain cpDNA haplotypes from a single origin, but rather combined haplotypes from at least two polyploid origins. Past migration among polyploid populations of independent origin or localized polyploid formation may explain the distribution of polyploid haplotypes within and among populations. The analysis also revealed a discrepancy between relatedness and geographical location. In nearly all sympatric populations of diploids and polyploids, polyploids had the same cpDNA haplotypes as diploids from a geographically remote population. This geographical discordance has several possible explanations, including small sample sizes, extinction of parental diploid haplotypes, chloroplast introgression, and homoplasy in the cpDNA sequence data. We conclude that the recurrent formation of polyploids is an important evolutionary mechanism in the diversification of H. grossulariifolia .     

Landscape dynamics in Mediterranean oak forests under global change: understanding the role of anthropogenic and environmental drivers across forest types

The Mediterranean region is projected to be extremely vulnerable to global change, which will affect the distribution of typical forest types such as native oak forests. However, our understanding of Mediterranean oak forest responses to future conditions is still very limited by the lack of knowledge on oak forest dynamics and species‐specific responses to multiple drivers. We compared the long‐term (1966–2006) forest persistence and land cover change among evergreen (cork oak and holm oak) and deciduous oak forests and evaluated the importance of anthropogenic and environmental drivers on observed changes for Portugal. We used National Forest Inventories to quantify the changes in oak forests and explored the drivers of change using multinomial logistic regression analysis and an information theoretical approach. We found distinct trends among oak forest types, reflecting the differences in oak economic value, protection status and management schemes: cork oak forests were the most persistent (62%), changing mostly to pines and eucalypt; holm oak forests were less persistent (53.2%), changing mostly to agriculture; and deciduous oak forests were the least persistent (45.7%), changing mostly to shrublands. Drivers of change had distinct importance across oak forest types, but drivers from anthropogenic origin (wildfires, population density, and land accessibility) were always among the most important. Climatic extremes were also important predictors of oak forest changes, namely extreme temperatures for evergreen oak forests and deficit of precipitation for deciduous oak forests. Our results indicate that under increasing human pressure and forecasted climate change, evergreen oak forests will continue declining and deciduous oak forests will be replaced by forests dominated by more xeric species. In the long run, multiple disturbances may change competitive dominance from oak forests to pyrophytic shrublands. A better understanding of forest dynamics and the inclusion of anthropogenic drivers on models of vegetation change will improve predicting the future of Mediterranean oak forests.     

Combined ecological niche modelling and molecular phylogeography revealed the evolutionary history of Hordeum marinum (Poaceae)--niche differentiation, loss of genetic diversity, and speciation in Mediterranean Quaternary refugia

The Hordeum marinum species group consists of two annual grasses of western Eurasian saline meadows or marshes. The two grasses split in the Quaternary about two million years ago. Hordeum marinum and the diploid of Hordeum gussoneanum (2x) co-occur throughout the Mediterranean basin, while the autotetraploid cytotype of H. gussoneanum (4x) overlaps with its diploid progenitor geographically only in the utmost Eastern Mediterranean, extending from there eastwards into Asia. Using chloroplast sequences of the trnL-F region, six newly developed chloroplast microsatellite loci, ecological predictive models based on climate data, and the present geographical distribution of the two species we analysed differentiation processes in the H. marinum group. The chloroplast data indicated clear differences in the history of both species. For H. marinum we found a subdivision between genetically variable populations from the Iberian Peninsula and the more uniform populations from the remaining Mediterranean. As an explanation, we assume Pleistocene fragmentation of an earlier widespread population and survival in an Iberian and a Central Mediterranean glacial refuge. Chloroplast variation was completely absent within the cytotypes of H. gussoneanum, indicating a severe and recent genetic bottleneck. Due to this lack of chloroplast variation only the combination of ecological habitat modelling with molecular data analyses allowed conclusions about the history of this taxon. The distribution areas of the two cytotypes of H. gussoneanum overlap today in parts of Turkey, indicating an area with similar climate conditions during polyploid formation. However, after its origin the polyploid cytotype underwent a pronounced ecological shift, compared to its diploid progenitor, allowing it to colonize mountainous inland habitats between the Mediterranean basin and Afghanistan. The extant sympatric occurrence of H. marinum and H. gussoneanum 2x in the Mediterranean region is interpreted as a result of secondary contact after fast Holocene range expansion out of different ice age refugia.     

Climate response of cork growth in the Mediterranean oak (Quercus suber L.) woodlands of southwestern Portugal

In the Mediterranean climate regions, drought events are expected to affect the growth of forests ecosystems by changing trees growth rates and eventually inducing shifts in their growth patterns. Cork oak (Quercus suber L.) is a strictly western Mediterranean tree species periodically harvested for its bark, the cork. So far, cork oak has received limited attention for dendroclimatological studies due to its typical faint and erratic tree wood rings. Moreover, its distinct cork rings chronologies have been completely neglected. In this study we introduce an approach using cork ring chronologies dated back 9–10 years for climate response. Despite enhancing interannual variability and increasing statistical response to short-term climatic variability, still poorly understood, this study will possibly allow infer long-term climate response. We analyzed the cork ring chronologies of 55 cork samples collected in mature (under exploitation) trees in three distinct locations in southwestern Portugal. Cork growth recorded a high climate signal, with highly significant and coherent responses to the yearly climate-related sources of variation. We successfully assessed trends of cork growth via correlation analysis including selected climate variables among mean monthly temperature, monthly precipitation and, on an annual basis, eight precipitation indices. The high mean sensitivities and inter-series correlations found for cork ring chronologies combined with the significant variance explained by climate variables suggest that climate is likely one dominant signal that affects cork growth, but local environmental stresses can decisively affect this (climate) signal. Assuming cork growth as a proxy for cork oak growth, it seems conceivable that despite the trees being highly resistant to drought stress, cork oak woodlands in southwestern Portugal would have to face lesser growth in a global warming scenario.     

The Balearic Islands: a reservoir of cpDNA genetic variation for evergreen oaks

Aim To analyse the role of the Balearic Islands as a refuge area for evergreen Quercus (cork oak: Quercus suber L., holm oak: Q. ilex L., kermes oak: Q. coccifera L.), by using molecular, historical and palaeobotanical data. Location The Western Mediterranean Basin (Balearic Islands, eastern Iberia, Provence, Sardinia, Corsica, Sicily, Malta, Italy, Northern Africa). Methods We sampled 108 populations and used the PCR‐RFLP technique with five universal cpDNA primers to define haplotypes in the sampled populations. Diversity, differentiation parameters and spatial analysis of the populations, using a spatial version of amova , were linked to the geological history of the Western Mediterranean Basin in order to explain the present spatial pattern of the evergreen Quercus populations in the Balearics. Results Evergreen Quercus cpDNA shows a complex structure, with remnants of ancient diversity in the Balearics. Balearic populations of holm oak are related to Iberian populations, while for cork and kermes oaks, we found both Tyrrhenian and Iberian haplotypes. Main conclusions The complex spatial patterns of cpDNA in Balearic evergreen Quercus appears explicable in terms of a combination of physical (vicariance and long distance dispersal) and biological (introgressive hybridization) factors. The Balearics constitute a glacial refuge area and a reservoir of genetic variation with traces of ancient diversity from Messinian–Pliocene stages.     

Chloroplast DNA variation in the Quercus affinis-Q. laurina complex in Mexico: geographical structure and associations with nuclear and morphological variation

The geographical distribution of chloroplast DNA (cpDNA) variation in 39 populations of two hybridizing Mexican red oaks, Quercus affinis and Q. laurina, was investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Six haplotypes were identified. Of these, two (H1 and H4), separated by four mutations, had high frequencies (58 and 23% of the individuals, respectively) and were present across the whole geographical range of both species, often co occurring in the same populations. The other four haplotypes were rare, geographically restricted, and are probably derived from the two frequent haplotypes. Latitudinal or other clinal patterns in diversity levels or haplotype composition of populations were not apparent. The pattern of haplotype distribution was characterized by some mosaicism, with contrasting populations often situated in proximity. Average within-population diversity (hS=0.299) and population differentiation (GST=0.499) were, respectively, higher and lower than values reported in previous studies of oak species. There was evidence for phylogeographical structure, as indicated by NST (0.566) being significantly higher than GST. Haplotypic variation was largely species-independent, although some very weak associations were detected between haplotypes H1 and H4 and morphological and nuclear molecular variation correspondingly characterizing Q. affinis and Q. laurina. These oaks probably did not experience a marked restriction to one or a few particular subregions of their present range during the last glacial cycle. It is more likely that substantial populations persisted throughout several episodes of climatic change, but experienced recurrent latitudinal and altitudinal migrations which may have caused the widespread distribution of haplotypes H1 and H4 and frequent intermixing of populations.     

Contrasting patterns of historical colonization in white oaks (Quercus spp.) in California and Europe

Phylogeography allows the inference of evolutionary processes that have shaped the current distribution of genealogical lineages across a landscape. In this perspective, comparative phylogeographical analyses are useful in detecting common historical patterns by either comparing different species within the same area within a continent or by comparing similar species in different areas. Here, we analyse one taxon (the white oak, genus Quercus, subgenus Quercus, section Quercus) that is widespread worldwide, and we evaluate its phylogeographical pattern on two different continents: western North America and Western Europe. The goals of the present study are: (i) to compare the chloroplast genetic diversity found in one California oak species vs. that found in the extensively studied European oak species (in France and the Iberian Peninsula); (ii) to contrast the geographical structure of haplotypes between these two taxa and test for a phylogeographical structure for the California species. For this purpose, we used the same six maternally inherited chloroplast microsatellite markers and a similar sampling strategy. The haplotype diversity within site as well as the differentiation among sites was alike in both taxa, but the Californian species has higher allelic richness with a greater number of haplotypes (39 vs. 11 in the European white oak complex). Furthermore, in California these 39 haplotypes are distributed locally in patches while in the European oaks haplotypes are distributed into lineages partitioned longitudinally. These contrasted patterns could indicate that gene movement in California oak populations have been more stable in response to past climatic and geological events, in contrast to their European counterparts.     

Genetic variation at allozyme and RAPD loci in sessile oak Quercus petraea (Matt.) Liebl.: the role of history and geography

The nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at eight allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and postglacial history on patterns of nuclear variation. Because of its small effective population size and maternal transmission, the chloroplast genome is a good marker of population history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations and, secondly, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation ( G _ST) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. Possibly, divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci assayed, RAPDs probably provided a less biased picture of the relative role of geography and history.     

Identifying signatures of natural selection in cork oak (Quercus suber L.) genes through SNP analysis

Cork oak (Quercus suber L.) is an evergreen tree species endemic to the western Mediterranean Basin with a major economical, social and ecological relevance, associated with cork extraction and exploitation. In the last years, cork oak stands have been facing a significant decline, which may be aggravated by the climate changes that are predicted to occur within cork oak distribution range during this century. Under this scenario, the assessment of adaptive genetic variation is essential to understand how cork oak may cope with these threats and to delineate strategies for the management of its genetic resources. In this study, six candidate genes possibly significant for environmental adaptation were analysed in cork oak populations from its entire distribution range. Signatures of natural selection were investigated using population genetic statistics and environmental association tests under alternative scenarios of population genetic structure. Signals of balancing selection were detected in the putative non-expressor of pathogenesis-related gene 1 (NPR1), involved in plant defence response against pathogens, in auxin response factor 16 (ARF16), a gene implicated in root development, in RAN3, also involved in developmental processes, and in glutamine synthetase nodule isozyme (GS), involved in nitrogen fixation. Furthermore, for ARF16, a class I heat shock protein (sHSP) and GS, associations were found between SNP allele and haplotype frequencies and several spatial and climatic variables, suggesting that these genes may have a role on cork oak local adaptation. In this study, the first steps were taken into gathering information on cork oak adaptation to environmental conditions.     

Multiple Recruitment Limitation Causes Arrested Succession in Mediterranean Cork Oak Systems

Lack of tree regeneration and persistency of species-poor shrublands represent a growing problem across Mediterranean evergreen oak forests. What constrains forest regeneration is poorly understood, and restoration attempts have been largely unsuccessful. We assessed the contribution of four different mechanisms of tree recruitment limitation (that is, source, dispersal, germination, and establishment) in a cork oak (Quercus suber) system in southern Portugal. Using a combination of field studies and experiments, we quantified seed production, seed removal and dispersal, seed survival and germination, seedling establishment and survival, as well as cork oak natural regeneration for the three dominant vegetation types in this system (Cistus ladanifer shrubland, oak forest, and oak savanna). We found that all four forms of cork oak recruitment limitation were significantly more severe in shrublands than in oak forests and savannas, so that oak seedling recruitment in shrubland was impeded in multiple ways. Our results explain why transitions from shrublands to oak savannas and forests are extremely difficult, and that the release from arrested succession in this system requires the simultaneous relief of multiple constraints on recruitment limitation in the early life history of oaks. These results have important implications for the restoration and conservation of Mediterranean oak systems.     

Chloroplast DNA variation and population structure in the widespread forest tree, Eucalyptus grandis

Recognition of genetic structure of populations and the ability to identify vulnerable populations is useful for the formation of conservation management strategies for plants. Eucalyptus grandis is a tall forest tree that has a major area of occurrence in subtropical eastern Australia, with smaller populations located in the east coast tropics. Many widespread forest species exhibit population differentiation that corresponds to geographic regions. However, Eucalyptus grandis appears to be an exception based on isozyme and morphological data. This is intriguing given a large discontinuity between northern populations and those in the southern part of the species range. In this study, the distribution of a maternally inherited chloroplast locus was examined because it was more likely to reveal genetic structure due to the slower evolution of the chloroplast genome and limited dispersal of seed in eucalypts. As expected, the G _ST for chloroplast DNA was higher than that for nuclear DNA but indicated low population differentiation for a forest tree species. Phylogeographic analysis indicated that the 15 populations grouped into three broad geographical regions; however, overall population structure was weak suggesting that the large geographical disjunction in the distribution of E. grandis may be relatively recent. A paradigm for conservation management of E. grandis based on chloroplast DNA haplotype distribution would take into account the low differentiation among populations.     

Geographical diversification of the genus Cicer (Leguminosae: Papilionoideae) inferred from molecular phylogenetic analyses of chloroplast and nuclear DNA sequences

Cicer L. (Leguminosae: Papilionoideae) consists of 42 species of herbaceous or semi-shrubby annuals and perennials distributed throughout the temperate zones of the Northern Hemisphere. The origin and geographical relationships of the genus are poorly understood. We studied the geographical diversification and phylogenetic relationships of Cicer using DNA sequence data sampled from two plastid regions, trnK / matK and trnS - trnG , and two nuclear regions, the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA, from 30 species. The results from the phylogenetic analyses of combined nuclear and chloroplast sequence data revealed four well-supported geographical groups: a Middle Eastern group, a West-Central Asian group, an Aegean–Mediterranean group, and an African group. Age estimates for Cicer based on methods that do not assume a molecular clock (for example, penalized likelihood) demonstrate that the genus has a Mediterranean origin with considerable diversification in the Miocene/Pliocene epochs. Geological events, such as mountain orogenesis and environmental changes, are major factors for the dispersal of Cicer species. The early divergence of African species and their geographically distinct region in the genus suggest a broader distribution pattern of the genus in the past than at present.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154 , 175–186.     

Regional-scale differentiation and phylogeography of a desert plant <Emphasis Type="Italic">Allium mongolicum</Emphasis> (Liliaceae) inferred from chloroplast DNA sequence variation

Past geological and climatic changes have promoted regional-scale intraspecific differentiation and range contraction/expansion in many temperate plants. However, little is known about how the desert species in central Asia responded to past geological and climatic changes, especially for a few widespread desert plants. In the present study, we aimed to survey the population structure and phylogeographical history of Allium mongolicum, which is widely distributed in the deserts of northwestern China. We sequenced two chloroplast DNA fragments (accD-psaI and psbA-trnH) for 418 individuals from 38 populations across the whole range of the species. Fourteen chlorotypes were identified, and three out of them were dominant. All populations were divided into three larger distinct groups by SAMOVA, which was largely congruent with the geographical division based on the Monmonier’s maximum-difference algorithm. Each of the groups occupied a distinct geographical region with a specific dominant chlorotype. Analysis of molecular variance showed that a high proportion of the total genetic variation (70.05%) existed among the three regions. The demographic dynamic tests indicated that the desert species had experienced a sudden regional-scale range expansion/recolonization in the Quaternary glaciers, which was further identified by the ecological niche modeling. These results suggest that the species has a distinct regional-scale differentiation as well as multiple geographically isolated refugia. Our results further enforce the idea that the environmental changes since the late Miocene greatly promoted differentiation of desert plants in northwestern China, and the Quaternary climatic oscillations played an important role in structuring the current populations of these species.     

Microsatellite analysis of the phylogeography, Pleistocene history and secondary contact hypotheses for the killifish, Fundulus heteroclitus

The mummichog, Fundulus heteroclitus, exhibits extensive latitudinal clinal variation in a number of physiological and biochemical traits, coupled with phylogeographical patterns at mitochondrial and nuclear DNA loci that suggest a complicated history of spatially variable selection and secondary intergradation. This species continues to serve as a model for understanding local and regional adaptation to variable environments. Resolving the influences of historical processes on the distribution of genetic variation within and among extant populations of F. heteroclitus is crucial to a better understanding of how populations evolve in the context of contemporary environments. In this study, we analysed geographical patterns of genetic variation at eight microsatellite loci among 15 populations of F. heteroclitus distributed throughout the North American range of the species from Nova Scotia to Georgia. Genetic variation in Northern populations was lower than in Southern populations and was strongly correlated with latitude throughout the species range. The most common Northern alleles at all eight loci exhibited concordant latitudinal clinal patterns, and the existence of an abrupt transition zone in allele frequencies between Northern and Southern populations was similar to that observed for mitochondrial DNA and allozyme loci. A significant pattern of isolation by distance was observed both within and between northern and southern regions. This pattern was unexpected, particularly for northern populations, given the recent colonization history of post-Pleistocene habitats, and was inconsistent with either a recent northward population expansion or a geographically restricted northern Pleistocene refugium. The data provided no evidence for recent population bottlenecks, and estimates of historical effective population sizes suggest that post-Pleistocene populations have been large throughout the species distribution. These results suggest that F. heteroclitus was broadly distributed throughout most of its current range during the last glacial event and that the abrupt transition in allele frequencies that separate Northern and Southern populations may reflect regional disequilibrium conditions associated with the post-Pleistocene colonization history of habitats in that region.     

Complex phylogeographic patterns indicate Central American origin of two widespread Mesoamerican <Emphasis Type="Italic">Quercus</Emphasis> (Fagaceae) species

The northern Neotropical region is characterized by a heterogeneous geological and climatic history. Recent studies have shown contrasting patterns regarding the role of geographic elements as barriers that could have determined phylogeographic structure in various species. Recently, the phylogeography and biogeography of Quercus species have been studied intensively, and the patterns observed so far suggest contrasting evolutionary histories for Neotropical species in comparison with their Holarctic relatives. The goal of this study was to describe the phylogeographic structure of two Neotropical oak species (Quercus insignis and Quercus sapotifolia) in the context of the geological and palaeoclimatic history of the northern Neotropics. Populations through the distribution range of both species were collected and characterized using nine chloroplast DNA microsatellite loci. Both oak species showed high levels of genetic diversity and strong phylogeographic structure. The distribution of genetic variation in Q. insignis suggested an influence of two major barriers, the Isthmus of Tehuantepec and the Nicaraguan Depression, while Q. sapotifolia exhibited a genetic structure defined by the heterogeneity of the Chortis highlands. The haplotype networks of both species indicated complex histories, suggesting that colonization from the Sierra Madre de Chiapas to central Mexico and from the north of the Nicaraguan Depression to the Costa Rican mountains may have occurred during different stages, and apparently more than one time. In conclusion, the phylogeographic structure of Neotropical oak species seems to be defined by a combination of geological and climatic events.     

Phylogeography of the planktonic chaetognath Sagitta setosa reveals isolation in European seas

Numerous planktonic species have disjunct distribution patterns in the world's oceans. However, it is unclear whether these are truly unconnected by gene flow, or whether they are composed of morphologically cryptic species. The marine planktonic chaetognath Sagitta setosa Müller has a discontinuous geographic distribution over the continental shelf in the northeastern Atlantic, Mediterranean Sea, and Black Sea. Morphological variation between these populations has been described, but overlaps and is therefore unsuitable to determine the degree of isolation between populations. To test whether disjunct populations are also genetically disjunct, we sequenced a 504-bp fragment of mitochondrial DNA comprising the cytochrome oxidase II region of 86 individuals. Sequences were highly variable; each represented a different haplotype. Within S. setosa, sequence divergence ranged from 0.2 to 8.1% and strong phylogeographic structure was found, with four main groups corresponding to the northeastern Atlantic, Mediterranean Sea (including Ligurian Sea, Tyrrhenian Sea and Gulf of Gabes), Adriatic Sea, and Black Sea. Two of these (Atlantic and Black Sea) were resolved as monophyletic clades, thus gene flow between disjunct populations of S. setosa has been extremely limited and lineage sorting has taken place. The deepest divergence was between Atlantic and Mediterranean/Black Sea populations followed by a split between Mediterranean and Black Sea populations. The Mediterranean/Black Sea clade comprised three groups, with the Adriatic Sea as the most likely sister clade of the Black Sea. These data are consistent with a colonization of the Black Sea from the Mediterranean. Furthermore, a possible cryptic species was found in the Black Sea with 23.1% sequence divergence from S. setosa. Two possibilities for the evolutionary origin of this species are proposed, namely, that it represents a relict species from the ancient Paratethys, or that it represents another chaetognath species that colonized the Black Sea more recently. Even though the exact timing of disjunction of S. setosa populations remains unclear, on the basis of the geological and paleoclimatic history of the European basins and our estimates of net nucleotide divergence, we suggest that disjunct populations arose through vicariance resulting from the cyclical changes in temperature and sea levels during the Pleistocene. We conclude that these populations have remained disjunct, not because of limited dispersal ability, but because of the inability to maintain viable populations in suboptimal, geographically intermediate areas.     

A set of primers for the amplification of chloroplast microsatellites in Quercus

The increase in demand for the certification of oak seed lots, as well as control of the geographical origin of oak wood, has led us to develop powerful genetic markers permitting us to discriminate among provenance regions. With the aim of detecting new chloroplast variants, we have identified 17 potential cpSSRs motifs from available oak sequences and tested their variability among French oak populations. Six loci were polymorphic at the intraspecific level in Quercus petraea and Q. robur. Moreover, conservation of the primer pairs was checked on a set of 21 forest tree species and they were all shown to work well on several Quercus species, and even within Fagacaea.