Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.

Times Cited: 76International audienceIn the European Alps, Rhonodendron ferrugineum can constitute dense populations with almost 100% of cover. The developmental pattern by layering and the resulting complexity of population structure make it difficult to identify distinct clones even by excavation. Therefore genotypic structure of a R. ferrugineum population, in the French Alps, was inferred from AFLP markers. In a first step, we analysed 400 samples using AFLP profiles generated by one selective primer pair. Seventeen bands out of 25 were polymorphic (68%). We identified a total of 32 multilocus genotypes. In a second step, the 32 genotypes were verified by applying two additional primer pairs to the two most distant samples from each genotype. The mean similarity (proportion of band sharing) between pairs of clones was 0.85 (range from 0.52 to 0.94). The spatial distribution of clones showed that vegetative spreading mainly occurred down a slope. Based on an annual shoot mean growth of 2.6 cm/year and the size of the widest clone, we estimated the age of the oldest individual to be at least 300 years. A single genotype can occupy a large surface and sometimes form a dense patch, suggesting that this species adopts a phalanx growth form with limited intermingling of some genets

Subspecies complexes may provide valuable insights into the early stages of the speciation process. The bluethroat (Luscinia svecica) consists of many morphologically distinct subspecies that differ most strikingly in the ornamental colour pattern of the male throat. We investigated the genetic and phenotypic differentiation in this subspecies complex, using (i) microsatellite genotyping (11 loci) of a sample of 364 individuals from bluethroat populations in Europe and Asia, and (ii) spectrometric and morphological measurements of a sample of 131 museum skin specimens. Population genetic analyses, based on microsatellite allele frequency variation, revealed a slight but significant overall population differentiation (F(ST) = 0.042). There was a well-differentiated southern group of subspecies with white or no throat spots and a less-differentiated northern group of chestnut-spotted populations. Phylogenetic analyses indicated that the southern all-blue and white-spotted forms are ancestral to the chestnut-spotted subspecies. In addition to the qualitative variation in throat plumage pattern already described in the literature, we found significant quantitative variation among subspecies in hue, chroma and brightness of the ultraviolet (UV)/blue throat coloration, and this variation seemed to be unrelated to the phylogenetic distance between subspecies.

We assessed the mitochondrial DNA sequence divergence of a 718 bp fragment of the control region and 1007 bp of the cytochrome b gene between two allopatric morphologically different subspecies of bluethroat (Luscinia svecica). None of the 17 total haplotypes was shared between L. s. namnetum and L. s. svecica. However, the mean distances between subspecies were very low for both fragments (0.00168 +/- 0.00001 (mean +/- SE) for the control region; 0.00306 +/- 0.00016 for the cytochrome b gene). Only one substitution made the two subspecies genetically differentiated, highlighting their recent divergence. Interestingly, the control region was not more variable than the cytochrome b gene.

We analysed mitochondrial DNA (mtDNA) sequences from 154 bluethroats (Luscinia svecica) sampled at 21 sites throughout much of their Eurasian range. A previously reported, single base-pair mtDNA difference between L. s. svecica and L. s. namnetum was inconsistent upon expanded geographical sampling. A significant FST value (0.29) and an isolation-by-distance effect show the existence of geographical differentiation. Phylogenetic analysis of haplotypes revealed northern and southern groups, although lineage sorting is incomplete. There was no geographical structure to the haplotype tree within groups, and currently recognized subspecies were not supported. A minimum evolution tree based on pairwise mtDNA genetic distances among average samples showed the same two broadly distributed northern and southern groups. These groups abut in the centre of the latitudinal range, and were possibly isolated by forest that developed and spread westward over the last 15 000 years. Pairwise FST values averaged 0.16 in the southern group, 0.04 in the northern group, and 0.42 between groups. Mismatch distributions suggested population growth in each group, with that in the south being more recent. In the northern group, the geographical pattern in tau suggested northward and eastward expansion. Analysis of nucleotide diversity suggested westward expansion in the southern group. The northern group had higher nucleotide diversity than the southern group, consistent with a larger current population size in the north. Given the significant FST, incompletely sorted haplotype tree, and broadly patterned minimum evolution tree, L. svevica appears to represent a species at an intermediate stage of differentiation between panmixia and reciprocal monophyly.

Common Crossbill subspecies have been described according to morphological traits, vocalizations and geographical distribution. In this study, we have tried to determine whether the subspecies correspond to clear-cut mitochondrial DNA lineages, by sequencing 717 bp of the control region from individuals taken at several sampling locations in North America and the Western Palaearctic. We find 22 haplotypes from the 37 sampled individuals with a mean divergence of 0.0118 +/- 0.0069 (mean +/- SD). We find a mixing of the mitochondrial haplotypes at the continental level among the different types or subspecies previously described. Morphological differentiation (in bill size and shape essentially) shows the possibility of rapid local adaptation to fluctuating resources (coniferous seeds), without necessarily promoting the development of reproductive barriers between morphs.

We tested the use of amplified fragment length polymorphism (AFLP) to assess the frequency of extra-pair parentage in a bluethroat (Luscinia svecica namnetum) population. Thirty-six families totalling 162 nestlings were analysed. Using a combination of three primer pairs, we reached an exclusion probability of 93% for the population. This probability can reach 99% considering families independently. We revealed that extra-pair fertilizations are very common: 63.8% of all broods contain at least one extra-pair young, totalling 41.9% of all young analysed. However, with the technique and the three primer pairs used it was not possible to attribute the parentage exclusions to extra-pair paternity, maternity or both. As brood parasitism has never been reported in this species, it seems likely that the exclusions are due to extra-pair males. This study shows that dominant AFLP markers can be useful for studying the mating system of taxa for which no microsatellite primers are available. This technique allows the approximate estimation of parentage exclusions despite the fact that it is not possible to know which parent has to be excluded.

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