Rémy J. Petit

High level of genetic differentiation for allelic richness among populations of the argan tree (Argania spinosa (L.) Skeels) endemic to Morocco.

Genetic diversity at nine isozyme loci was surveyed in an endangered tree species, the argan tree, endemic to south-western Morocco. The species is highly diverse (3.6 alleles/locus) with populations strongly differentiated from each other (FST=0.25). This example is used to illustrate a method for standardizing measures of allelic richness in samples of unequal sample sizes, which was developed for the estimation of the number of species and relies on the technique of rarefaction. In addition, it is shown that the measure of subdivision, θST, obtained when allelic richness is used in place ofh (Neis index of diversity), is much larger than the FST [e.g. θST(40)=0.52, where (40) indicates the specified sample used to estimate the allelic richness]. This suggests that rare alleles (which strongly influence measures of allelic richness) have a more scattered distribution than more frequent ones, a result which raises special conservation issues for the argan tree.

Comparative organization of chloroplast, mitochondrial and nuclear diversity in plant populations

Plants offer excellent models to investigate how gene flow shapes the organization of genetic diversity. Their three genomes can have different modes of transmission and will hence experience varying levels of gene flow. We have compiled studies of genetic structure based on chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear markers in seed plants. Based on a data set of 183 species belonging to 103 genera and 52 families, we show that the precision of estimates of genetic differentiation (GST) used to infer gene flow is mostly constrained by the sampling of populations. Mode of inheritance appears to have a major effect on GST. Maternally inherited genomes experience considerably more subdivision (median value of 0.67) than paternally or biparentally inherited genomes (∼0.10). GST at cpDNA and mtDNA markers covary narrowly when both genomes are maternally inherited, whereas GST at paternally and biparentally inherited markers also covary positively but more loosely and GST at maternally inherited markers are largely independent of values based on nuclear markers. A model‐based gross estimate suggests that, at the rangewide scale, historical levels of pollen flow are generally at least an order of magnitude larger than levels of seed flow (median of the pollen‐to‐seed migration ratio: 17) and that pollen and seed gene flow vary independently across species. Finally, we show that measures of subdivision that take into account the degree of similarity between haplotypes (NST or RST) make better use of the information inherent in haplotype data than standard measures based on allele frequencies only.

INVITED REVIEW: Comparative organization of chloroplast, mitochondrial and nuclear diversity in plant populations

Plants offer excellent models to investigate how gene flow shapes the organization of genetic diversity. Their three genomes can have different modes of transmission and will hence experience varying levels of gene flow. We have compiled studies of genetic structure based on chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear markers in seed plants. Based on a data set of 183 species belonging to 103 genera and 52 families, we show that the precision of estimates of genetic differentiation (GST) used to infer gene flow is mostly constrained by the sampling of populations. Mode of inheritance appears to have a major effect on GST. Maternally inherited genomes experience considerably more subdivision (median value of 0.67) than paternally or biparentally inherited genomes (∼0.10). GST at cpDNA and mtDNA markers covary narrowly when both genomes are maternally inherited, whereas GST at paternally and biparentally inherited markers also covary positively but more loosely and GST at maternally inherited markers are largely independent of values based on nuclear markers. A model‐based gross estimate suggests that, at the rangewide scale, historical levels of pollen flow are generally at least an order of magnitude larger than levels of seed flow (median of the pollen‐to‐seed migration ratio: 17) and that pollen and seed gene flow vary independently across species. Finally, we show that measures of subdivision that take into account the degree of similarity between haplotypes (NST or RST) make better use of the information inherent in haplotype data than standard measures based on allele frequencies only.

Gene flow and species delimitation

A defining feature of species is that their constituting populations are connected by gene flow. However, interspecific gene flow (introgression) can affect species integrity. If some genome components were less prone to introgression than others, they should be particularly suitable to delimitate species. Recent simulation studies have predicted a negative correlation between intra- and interspecific gene flow, suggesting that markers associated with the most dispersing sex should better delimitate species. A review of studies of introgression in species with sex-biased dispersal largely confirms this prediction. Hence, species delimitation should be more effective with markers experiencing high levels of gene flow, a simple but not widely appreciated prediction.

THE HIDDEN SIDE OF INVASIONS: MASSIVE INTROGRESSION BY LOCAL GENES

Abstract Despite hundreds of reports involving both plants and animals, the mechanisms underlying introgression remain obscure, even if some form of selection is frequently invoked. Introgression has repeatedly been reported in species that have recently colonized a new habitat, suggesting that demographic processes should be given more attention for understanding the mechanisms of introgression. Here we show by spatially explicit simulations that massive introgression of neutral genes takes place during the invasion of an occupied territory if interbreeding is not severely prevented between the invading and the local species. We also demonstrate that introgression occurs almost exclusively from the local to the invading species, especially for populations located far away from the source of the invasion, and this irrespective of the relative densities of the two species. This pattern is strongest at markers experiencing reduced gene flow, in keeping with the observation that organelle genes are often preferentially introgressed across species boundaries. A survey of the literature shows that a majority of published empirical studies of introgression during range expansions, in animals and in plants, follow the predictions of our model. Our results imply that speciation genes can be identified by comparing genomes of interfertile native and invading species pairs.

Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence

Abstract The geographic distribution throughout Europe of each of 32 chloroplast DNA variants belonging to eight white oak species sampled from 2613 populations is presented. Clear-cut geographic patterns were revealed by the survey. These distributions, together with the available palynological information, were used to infer colonisation routes out of the glacial period refugia. In western Europe in particular, movements out of the Iberian and the Italian Peninsulas can be clearly identified. Separate refugia are also present in eastern Balkans, whereas further west in this peninsula similarities with Italy were evident. Movements resulting in the exchange of haplotypes between refugia both during the present interglacial and probably also during earlier glacial cycles were therefore inferred. The consequences of these past exchanges is that phylogenetically divergent haplotypes have sometimes followed very similar colonisation routes, limiting somewhat the phylogeographic structure. Cases of geographic disjunction in the present-day distribution of haplotypes are also apparent and could have been induced by the existence of rapid climatic changes at the end of the glacial period (specifically the Younger Dryas cold period), which resulted in range restriction following an early warm period during which oak first expanded from its primary refugia. This cold phase was followed by a new period of expansion at the outset of the Holocene, involving in some cases ‘secondary’ refugia. It is expected that these short climate oscillations would have led to a partial reshuffling of haplotype distribution. Early association between haplotypes and oak species are also suggested by the data, although extensive introgression among species has ultimately largely blurred the pattern. This implies that colonisation routes may have been initially constrained by the ecological characteristics of the species hosting each chloroplast variant. We suggest for instance that two oak species distributed in the north of the Iberian Peninsula ( Quercus petraea and Q. pubescens ) are recent post-glacial immigrants there. When considered together, conclusions on the location of glacial period refugia and the colonisation routes derived from molecular information and fossil pollen data appear to be both largely compatible and complementary.

Current trends in microsatellite genotyping.

Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost‐effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next‐generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non‐model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well‐established strategy including standardized high‐throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

CHLOROPLAST DNA PHYLOGEOGRAPHY OF THE COMMON BEECH (FAGUS SYLVATICA L.) IN EUROPE

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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