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.

Loci considered to be under selection are generally avoided in attempts to infer past demographic processes as they do not fit neutral model assumptions. However, opportunities to better reconstruct some aspects of past demography might thus be missed. Here we examined genetic differentiation between two sympatric European oak species with contrasting ecological dynamics (Quercus robur and Quercus petraea) with both outlier (i.e. loci possibly affected by divergent selection between species or by hitchhiking effects with genomic regions under selection) and nonoutlier loci. We sampled 855 individuals in six mixed forests in France and genotyped them with a set of 262 SNPs enriched with markers showing high interspecific differentiation, resulting in accurate species delimitation. We identified between 13 and 74 interspecific outlier loci, depending on the coalescent simulation models and parameters used. Greater genetic diversity was predicted in Q. petraea (a late-successional species) than in Q. robur (an early successional species) as introgression should theoretically occur predominantly from the resident species to the invading species. Remarkably, this prediction was verified with outlier loci but not with nonoutlier loci. We suggest that the lower effective interspecific gene flow at loci showing high interspecific divergence has better preserved the signal of past asymmetric introgression towards Q. petraea caused by the species' contrasting dynamics. Using markers under selection to reconstruct past demographic processes could therefore have broader potential than generally recognized.

Multiplex PCR is a fast and cost-effective technique allowing increased genotyping throughput of microsatellites. We developed two multiplexes for Quercus petraea and Q. robur, a 12-plex of EST-SSRs (eSSRs) and an 8-plex of genomic SSRs (gSSRs). We studied the origin of allele calling errors at the human reader and software levels. We showed that the robustness of allele identification can be improved by binning on raw peak sizes prior to genetic data analysis. We checked through simulation the power of these markers for species delimitation and hybrid detection. The resolution achieved with all 20 markers was greatly improved compared to that of previous studies based on a subset of the markers. Preliminary PCR tests suggest that these multiplexes might be useful to study other oak species as well. The strategy used for multiplex microsatellite development (from PCR conditions to the definition of allele calling rules) should be broadly applicable.

Natural hybridization is attracting much interest in modern speciation and conservation biology studies, but the underlying mechanisms remain poorly understood. In particular, it is unclear why environmental changes often increase hybridization rates. To study this question, we surveyed mating events in a mixed oak stand and developed a spatially explicit individual-based hybridization model. This model, where hybridization is frequency-dependent, pollen is nonlimiting and which allows immigrant pollen to compete with local pollen, takes into account species-specific pollen dispersal and sexual barriers to hybridization. The consequences of pollen limitation on hybridization were studied using another simple model. The results indicate that environmental changes could increase hybridization rates through two distinct mechanisms. First, by disrupting the spatial organization of communities, they should decrease the proportion of conspecific pollen available for mating, thus increasing hybridization rates. Second, by decreasing the density of conspecifics, they should increase pollen limitation and thus hybridization rates, as a consequence of chance pollination predominating over deterministic pollen competition. Altogether, our results point to a need for considering hybridization events at the appropriate level of organization and provide new insights into why hybridization rates generally increase in disturbed environments.

A growing body of evidence from community genetics studies suggests that ecosystem functions supported by plant species richness can also be provided by genetic diversity within plant species. This is not yet true for the diversity-resistance relationship as it is still unclear whether damage by insect herbivores responds to genetic diversity in host plant populations. We developed a manipulative field experiment based on a synthetic community approach, with 15 mixtures of one to four oak (Quercus robur) half-sib families. We quantified genetic diversity at the plot level by genotyping all oak saplings and assessed overall damage caused by ectophagous and endophagous herbivores along a gradient of increasing genetic diversity. Damage due to ectophagous herbivores increased with the genetic diversity in oak sapling populations as a result of higher levels of damage in mixtures than in monocultures for all families (complementarity effect) rather than because of the presence of more susceptible oak genotypes in mixtures (selection effect). Assemblages of different oak genotypes would benefit polyphagous herbivores via improved host patch location, spill over among neighbouring saplings and diet mixing. By contrast, genetic diversity was a poor predictor of the abundance of endophagous herbivores, which increased with individual sapling apparency. Plant genetic diversity may not provide sufficient functional contrast to prevent tree sapling colonization by specialist herbivores while enhancing the foraging of generalist herbivores. Long term studies are nevertheless required to test whether the effect of genetic diversity on herbivory change with the ontogeny of trees and local adaptation of specialist herbivores.

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade-off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even-aged stand, trees of the late-successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early-successional species (Q. robur). A large-scale paternity study and a spatially explicit individual-based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition-colonization trade-off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.

Although interfertility is the key criterion upon which Mayr’s biological species concept is based, it has never been applied directly to delimit species under natural conditions. Our study fills this gap. We used the interfertility criterion to delimit two closely related oak species in a forest stand by analyzing the network of natural mating events between individuals. The results reveal two groups of interfertile individuals connected by only few mating events. These two groups were largely congruent with those determined using other criteria (morphological similarity, genotypic similarity and individual relatedness). Our study, therefore, shows that the analysis of mating networks is an effective method to delimit species based on the interfertility criterion, provided that adequate network data can be assembled. Our study also shows that although species boundaries are highly congruent across methods of species delimitation, they are not exactly the same. Most of the differences stem from assignment of individuals to an intermediate category. The discrepancies between methods may reflect a biological reality. Indeed, the interfertility criterion is an environment-dependant criterion as species abundances typically affect rates of hybridization under natural conditions. Thus, the methods of species delimitation based on the interfertility criterion are expected to give results slightly different from those based on environment-independent criteria (such as the genotypic similarity criteria). However, whatever the criterion chosen, the challenge we face when delimiting species is to summarize continuous but non-uniform variations in biological diversity. The grade of membership model that we use in this study appears as an appropriate tool.