Marie-Hélène Pemonge

Chloroplast DNA variation in European white oaks: Phylogeography and patterns of diversity based on data from over 2600 populations

A consortium of 16 laboratories have studied chloroplast DNA (cpDNA) variation in European white oaks. A common strategy for molecular screening, based on restriction analysis of four PCR-amplified cpDNA fragments, was used to allow comparison among the different laboratories. A total of 2613 oak populations (12,214 individual trees from eight species) were sampled from 37 countries, and analysed with the four fragments. They belong to eight related oak species: Quercus robur, Q. petraea, Q. pubescens, Q. frainetto, Q. faginea, Q. pyrenaica, Q. canariensis and Q. macranthera. During this survey, 45 chloroplast variants were detected and are described together with their phylogenetic relationships, but several of these haplotypes were pooled when there were some risks of confusion across laboratories during the survey, and finally 32 remained that were mapped and used in diversity analyses. A strong phylogeographic structure is apparent from the data, where related haplotypes have broadly similar geographic distributions. In total, six cpDNA lineages are identified, which have distinct geographic distributions, mainly along a longitudinal gradient. Most haplotypes found in northern Europe are also present in the south, whereas the converse is not true, suggesting that the majority of mutations observed were generated prior to postglacial recolonisation, corroborating the conclusions of earlier studies. The description of a new western European lineage constitutes a major finding, compared to earlier phylogenetic treatments. Although the eight oak species studied systematically share cpDNA variants when in sympatry, they partition cpDNA diversity differently, as a consequence of their different ecology and life history attributes. Regional differences in levels of differentiation also exist (either species-specific or general); these seem to be related to the intensity of past and present management of the forests across Europe but also to the level of fragmentation of the range within these regions.

An enlarged set of consensus primers for the study of organelle DNA in plants

the chloroplast genome (cpDNA) in population genetics studies of plants. Indeed, despite its low substitution rate (Wolfe et al. 1987), its typically uniparental mode of transmission, and hence clonal mode of evolution is a unique feature of cytoplasmic genomes of great interest for evolution studies. The plant mitochondrial genome (mtDNA) shares these characteristics, with however, an even slower substitution rate, but is much less studied because it varies enormously in size and gene arrangement (Palmer 1992). Consensus primers which are homologous to the most conserved coding regions of cpDNA or mtDNA but amplify the more variable noncoding regions are likely therefore to be very useful. Here, we greatly enlarge a previous list of primers published in this journal (Demesure et al. 1995). A total of seven pairs of cpDNA primers and nine pairs of mtDNA primers are described. Moreover, the degree of conservation of the whole set (16 and 12 pairs of cpDNA and mtDNA primers, respectively) is tested by amplification on five species, three angiosperms (two dicotyledons and one monocotyledon), a pine (gymnosperm) and a fern (pteridophyte), i.e. on a broader taxonomic range than in the former study. The primers are also evaluated by comparison with sequences available in the molecular databases. The primers were designed in order to be as consensual as possible, although the chloroplast genome of tobacco (i.e. a dicotyledon) was used as reference, and full homology with dicotyledon sequences was also given priority in the case of the mtDNA primers. Their design was facilitated by the availability of an increasing number of complete sequences in the molecular databases. Conserved coding sequences flanking the regions to be amplified were identified with the computer software B L A S T (Altschul et al. 1990). The cpDNA primers are all located in the relatively variable large single-copy region (Palmer 1985). For the mtDNA primers, introns of genes available in the molecular databases (e.g. subunits 1, 4, 5 and 7 of NADH dehydrogenase) were selected. Finally, the exact position and length of the primers were chosen according to their thermodynamic parameters using the Oligo Primer Analysis Software version 4.1 (Rychlik et al. 1990). The annealing temperatures were then manually optimized with a PHC3 (Techne) apparatus and then more rapidly with a Robocycler Gradient 96 (Stratagene). The list of the new pairs of primers is given in Table 1 along with their sequence (from 5′ to 3′) and annealing temperature. Because optimal PCR conditions can vary according to the thermocycler, the temperatures given here are only indicative. The degree of conservation of the whole set is described in Table 2. First, the exact position of the primers and the expected length of the fragment is provided for a reference sequence. Then, the list of species that were successfully amplified and the list of the species retrieved from databases that are likely to amplify, as judged from the orientation and homology of their sequences compared with those of the primers, are given. The 28 pairs of primers were directly tested by PCR on five species: Quercus robur L. (pedunculate oak, Fagaceae, Dicotyledons, Angiosperms), Ilex aquifolium L. (holly, Aquifoliaceae, Dicotyledons, Angiosperms), Arundinaria japonica (bamboo, Poaceae, Monocotyledons, Angiosperms), Pinus pinaster Ait. (maritime pine, Pinaceae, Gymnosperms) and Polystichum filix-mas (L.) Roth (male fern, Aspidiaceae, Pteridophytes). The cpDNA primers were also checked against the complete chloroplast sequences of two cereals, Zea mays (X86563) and Oryza sativa (X15901), of a gymnosperm, Pinus thunbergii (D17510), and of a bryophyte, Marchantia polymorpha (X04465). The mtDNA primers were checked against the only complete mtDNA sequence available to date, i.e. that of Marchantia polymorpha (M68929). This was completed by making comparisons with mtDNA genomic sequences of variable sizes, as far as these sequences encompassed the positions of both primers from a pair. Total DNA was extracted following the procedure described in Dumolin et al. (1995). The amplifications were performed as detailed in Demesure et al. (1995). The fragments were separated on 1% agarose gels and stained with ethidium bromide. PRIMER NOTE

Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed‐species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) × Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.

Novel perspectives in wood certification and forensics: dry wood as a source of DNA.

The importance of wood for human societies can hardly be understated. If dry wood were amenable to molecular genetic investigations, this could lead to major applications in wood forensics, certification, archaeology and palaeobotany. To evaluate the potential of wood for molecular genetic investigations, we have attempted to isolate and amplify, by PCR, DNA fragments of increasing size corresponding to all three plant genomes from different regions of 10 oak logs. Stringent procedures to avoid contamination with external DNA were used in order to demonstrate the authenticity of the fragments amplified. This authenticity was further confirmed by demonstrating genetic uniformity within each log using both nuclear and chloroplast microsatellites. For most wood samples DNA was degraded, and the sequences that gave the best results were those of small size and present in high copy number (chloroplast, mitochondrial, or repeated nuclear sequences). Both storage conditions and storage duration play a role in DNA conservation. Overall, this work demonstrates that molecular markers from all three plant genomes can be used for genetic analysis on dry oak wood, but outlines some limitations and the need for further evaluation of the potential of wood for DNA analysis.

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600‐years‐old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite‐type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.

Chloroplast DNA variation of oaks in France and the influence of forest fragmentation on genetic diversity

Chloroplast DNA variation was studied in a total of 878 French oak populations from four different species. Three main cpDNA lineages were found, which have well-demarcated distributions in the country. The study of the distribution of haplotypes in each species supports the view that the four species were restricted to different refugia during the last ice-age. This is evident despite the fact that extensive cpDNA introgression occurred during and after postglacial recolonisation. Nevertheless, the individual species have different ecological requirements and also differ in their ability to hybridise, resulting in heterogeneous levels of partitioning of cpDNA diversity and incomplete cpDNA introgression. The first analysis of the effect of the landscape structure on genetic diversity in these oak species is presented here. The only discernible effect of landscape structure on cpDNA diversity was found in Quercus robur, and is very weak and rather counterintuitive. The biology and abundance of these oak species may make them particularly resistant to fragmentation; in addition, artificial seed flow may complicate the picture, and will require more direct investigations.

Checking the geographical origin of oak wood: molecular and statistical tools

New methods for better identification of timber geographical origin would constitute an important technical element in the forest industry, for phytosanitary certification procedures or in the chain of custody developed for the certification of timber from sustainably managed forests. In the case of the European white oaks, a detailed reference map of chloroplast (cp) DNA variation across the range exists, and we propose here to use the strong geographical structure, characterized by a differentiation of western vs. eastern populations, for the purpose of oak wood traceability. We first developed cpDNA markers permitting the characterization of haplotype on degraded DNA obtained from wood samples. The techniques were subsequently validated by confirming the full correspondence between genotypes obtained from living tissues (buds) and from wood collected from the same individual oak. Finally, a statistical procedure was used to test if the haplotype composition of a lot of wood samples is consistent with its presumed geographical origin. Clearly, the technique cannot permit the unambiguous identification of wood products of unknown origin but can be used to check the conformity of genetic composition of wood samples with the region of alleged origin. This could lead to major applications not only in the forest industry but also in archaeology or in palaeobotany.

Efficient mitigation of founder effects during the establishment of a leading-edge oak population.

Numerous plant species are shifting their range polewards in response to ongoing climate change. Range shifts typically involve the repeated establishment and growth of leading-edge populations well ahead of the main species range. How these populations recover from founder events and associated diversity loss remains poorly understood. To help fill this gap, we exhaustively investigated a newly established population of holm oak (Quercus ilex) growing more than 30 km ahead of the nearest larger stands. Pedigree reconstructions showed that plants belong to two non-overlapping generations and that the whole population originates from only two founder trees. The four first-generation trees that have reached maturity showed disparate mating patterns despite being full-sibs. Long-distance pollen immigration was notable despite the strong isolation of the stand: 6 per cent gene flow events in acorns collected on the trees (n = 255), and as much as 27 per cent among their established offspring (n = 33). Our results show that isolated leading-edge populations of wind-pollinated forest trees can rapidly restore their genetic diversity through the interacting effects of efficient long-distance pollen flow and purging of inbred individuals during recruitment. They imply that range expansions of these species are primarily constrained by initial propagule arrival rather than by subsequent gene flow.

European Neolithization and Ancient DNA: An Assessment

Neolithic processes underlying the distribution of genetic diversity among European populations have been the subject of intense debate since the first genetic data became available. However, patterns observed in the current European gene pool are the outcome of Paleolithic and Neolithic processes, overlaid with four millennia of further developments. This observation encouraged paleogeneticists to contribute to the debate by directly comparing genetic variation from the ancient inhabitants of Europe to their contemporary counterparts. Pre‐Neolithic and Neolithic paleogenetic data are becoming increasingly available for north and northwest European populations. Despite the numerous problems inherent in the paleogenetic approach, the accumulation of ancient DNA datasets offers new perspectives from which to interpret the interactions between hunter‐gatherer and farming communities. In light of information emerging from diverse disciplines, including recent paleogenetic studies, the most plausible model explaining the movement of Neolithic pioneer groups in central Europe is that of leapfrog migration.