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International audience; Because of the demand for controlling livestock diets, two methods that characterize the DNA of plants present in feces were developed. After DNA extraction from fecal samples, a short fragment of the chloroplastic trnL intron was amplified by PCR using a universal primer pair for plants. The first method generates a signature that is the electrophoretic migration pattern of the PCR product. The second method consists of sequencing several hundred DNA fragments from the PCR product through pyrosequencing. These methods were validated with a blind analysis of feces from concentrate- and pasture-fed lambs. The signature method allowed differentiation of the two diets and confirmed the presence of concentrate in one of them. The pyrosequencing method allowed the identification of up to 25 taxa in a diet. These methods are complementary to the chemical methods already used. They could be applied to the control of diets and the study of food preferences.

International audience; Knowledge about the phylogenetic history, genetic variation and ecological requirements of a species is important for its conservation and management. Unfortunately, for many species this information is lacking. Here we use multiple approaches (phylogenetics, population genetics and ecological modelling) to evaluate the evolutionary history and conservation status of Capra walie, an endangered flagship species of wild goat endemic to Ethiopia. The analysis of mitochondrial cytochrome b and Y-chromosome DNA sequences suggests that C. walie forms a monophyletic clade with Capra nubiana, but potentially has been isolated for up to 0.8 million years from this closely related species. Microsatellite DNA analyses show that C. walie has very low genetic variation (mean heterozygosity=0.35) compared with other endangered mammals. This reduced variation likely derives from a prolonged demographic decline and small effective population size. Ecological niche modelling using the bioclimatic features of habitats occupied by C. walie, suggests ecological differences between C. walie and C. nubiana, and identifies the areas most suitable for future reintroductions of C. walie. The genetic and bioclimatic data suggest that C. walie is distinct and requires immediate conservation actions including genetic monitoring and reintroductions to establish independent populations. This study illustrates how combining noninvasive sampling along with genetic and ecological (bioclimatic) approaches can help assess conservation status of poorly known species.

Accurate genotyping of complex systems, such as the major histocompatibility complex (MHC) often requires simultaneous analysis of multiple co-amplifying loci. Here we explore the utility of the massively parallel 454 sequencing method as a universal tool for genotyping complex MHC systems in nonmodel vertebrates. The power of this approach stems from the use of tagged polymerase chain reaction (PCR) primers to identify individual amplicons which can be simultaneously sequenced to the arbitrarily chosen coverage. However, the error-prone sequencing technology poses considerable challenges as it may be difficult to discriminate between sequencing errors and true rare alleles; due to complex nature of artefacts and errors, efficient quality control is required. Nevertheless, our study demonstrates the parallel 454 sequencing can be an efficient genotyping platform for MHC and provides an alternative to classical genotyping methods. We introduced procedures to identify the threshold that can be used to reduce number of genotyping errors by eliminating most of artefactual alleles (AA) representing PCR or sequencing errors. Our procedures are based on two expectations: first, that AA should be relatively rare, both overall and on per-individual basis, and second, that most AA result from errors introduced to sequences of true alleles. In our data set, alleles with an average per-individual frequency below 3% most likely represented artefacts. This threshold will vary in other applications according to the complexity of the genotyped system. We strongly suggest direct assessment of genotyping error in every experiment by running a fraction of duplicates: individuals amplified in independent PCRs.

International audience; To test the influence of various species traits, elevation and phylogeographical history on the genetic diversity of high-mountain plants in the Alps and Carpathians. The regular sampling grid comprised the whole range of the European Alps and the Carpathians. Twenty-two high-mountain plant species were exhaustively sampled and their genetic diversity was assessed with amplified fragment length polymorphisms (AFLPs). ANOVAs were used to check for relationships between species traits and species genetic diversity, and to test whether genetic diversity was influenced by altitude and phylogeographical history (i.e. Alps versus Carpathians). In both mountain systems, species dispersed and pollinated by wind showed higher genetic diversity than species with self or insect pollination, and with animal- or gravity-dispersed seeds. Only in the Alps did altitudinal range size affect species genetic diversity significantly: species with narrow altitudinal ranges in the highest vegetation belts had significantly higher genetic diversity than those expanding over wide altitudinal ranges. Genetic diversity was species specific and significantly higher in the Alps than in the Carpathians, but it was not influenced by elevation. Wind pollination and wind dispersal seem to foster high genetic diversity. However, species traits are often associated and their effects on genetic diversity cannot be clearly disentangled. As genetic diversity is species specific, comparisons across species need to be interpreted with care. Genetic diversity was generally lower in the Carpathians than in the Alps, due to higher topographical isolation of alpine habitats in the Carpathians and this mountain massif's divergent phylogeographical history. Elevation did not influence genetic diversity, challenging the long-held view of decreasing genetic diversity with increasing elevation in mountain plants.

International audience; DNA barcoding - taxon identification using a standardized DNA region - has received much attention recently, and is being further developed through an international initiative. We anticipate that DNA barcoding techniques will be increasingly used by ecologists. They will be able to not only identify a single species from a specimen or an organism's remains but also determine the species composition of environmental samples. Short DNA fragments persist in the environment and might allow an assessment of local biodiversity from soil or water. Even DNA-based diet composition can be estimated using fecal samples. Here we review the new avenues offered to ecologists by DNA barcoding, particularly in the context of new sequencing technologies.

Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.

The emergence of farming during the Neolithic transition, including the domestication of livestock, was a critical point in the evolution of human kind. The goat ( Capra hircus ) was one of the first domesticated ungulates. In this study, we compared the genetic diversity of domestic goats to that of the modern representatives of their wild ancestor, the bezoar, by analyzing 473 samples collected over the whole distribution range of the latter species. This partly confirms and significantly clarifies the goat domestication scenario already proposed by archaeological evidence. All of the mitochondrial DNA haplogroups found in current domestic goats have also been found in the bezoar. The geographic distribution of these haplogroups in the wild ancestor allowed the localization of the main domestication centers. We found no haplotype that could have been domesticated in the eastern half of the Iranian Plateau, nor further to the east. A signature of population expansion in bezoars of the C haplogroup suggests an early domestication center on the Central Iranian Plateau (Yazd and Kerman Provinces) and in the Southern Zagros (Fars Province), possibly corresponding to the management of wild flocks. However, the contribution of this center to the current domestic goat population is rather low (1.4%). We also found a second domestication center covering a large area in Eastern Anatolia, and possibly in Northern and Central Zagros. This last domestication center is the likely origin of almost all domestic goats today. This finding is consistent with archaeological data identifying Eastern Anatolia as an important domestication center.

International audience; CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum (R) Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog ( Rana catesbeiana ) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.

International audience; The distributional range of alpine plants experienced dramatic changes during the Quaternary ice ages. These changes offer many opportunities for studying the impact of habitat contraction and fragmentation on both species and genetic diversity. Here, we examined the amplified fragment length polymorphism-based genetic diversity in the sedge Carex curvula All. in relation to the species diversity of siliceous European alpine grasslands in which the sedge is dominant. We found no relationship or even a negative relationship between genetic and species diversity indices. Local species richness was associated with the regional pool size of siliceous alpine species, which was itself dependent on the extant area of suitable habitats for these species. Genetic diversity of C. curvula was primarily shaped by the presumed location of glacial refugia and the routes of post-glacial colonization. We conclude that the two levels of diversity are not positively correlated because genotypes and species do not respond similarly to the spatial dynamics of suitable habitats induced by Quaternary temperature changes.

International audience; We introduce a new method to detect signatures of natural selection in the genome based on the application of spatial analysis, with the contribution of Geographical Information Systems, environmental variables, molecular data, and multiple univariate logistic regressions. Its use allows the identification of the same genomic regions as revealed by a theoretical approach in population genomics, but also the uncovering of environmental parameters responsible for selection which was so far difficult. This research also opens up avenues for GISciences research in molecular ecology.

International audience; Noninvasive DNA sampling allows studies of natural populations without disturbing the target animals. Unfortunately, high genotyping error rates often make noninvasive studies difficult. We report low error rates (0.0-7.5%/locus) when genotyping 18 microsatellite loci in only 4 multiplex polymerase chain reaction amplifications using fecal DNA from bighorn sheep (Ovis canadensis). The average locus-specific error rates varied significantly between the 2 populations (0.13% vs. 1.6%; P < 0.001), as did multi-locus genotype error rates (2.3% vs. 14.1%; P < 0.007). This illustrates the importance of quantifying error rates in each study population (and for each season and sample preservation method) before initiating a noninvasive study. Our error rates are among the lowest reported for fecal samples collected noninvasively in the field. This and other recent studies suggest that noninvasive fecal samples can be used in species with pellet-form feces for nearly any study (e.g., of population structure, gene flow, dispersal, parentage, and even genome-wide studies to detect local adaptation) that previously required high-quality blood or tissue samples.

Les comptages de crottes sont largement utilisés pour estimer l'abondance et la densité des céphalophes dans les forêts tropicales africaines. Cependant, une des limites importantes de cette méthode est que l'identification de l'espèce basée sur les caractéristiques morphologiques des excréments est extrêmement difficile dans la plupart des cas. Certains auteurs émettent l'hypothèse qu'il est au moins possible de distinguer entre les crottes de céphalophes bleus, du Natal et à dos jaune. Cependant, cette étude est la première tentative de tester la fiabilité de l'identification sur le terrain. Dans cette étude, nous suggérons une méthode basée sur un protocole génétique non invasif pour identifier l'espèce correspondant aux crottes. La comparaison entre identifications de terrain et génétiques montre que seules les crottes de Cephalophus silvicultor ont identifiées sans erreur sur le terrain. Le taux d'erreurs est élevé pour le céphalophe du Natal et le céphalophe bleu, et toute tentative pour faire la distinction entre ces espèces aboutirait à de fausses estimations pour les deux espèces. Nous recommandons de faire des analyses d'ADN pour s'assurer que l'identification des espèces est fiable lorsque l'on a recours aux comptages de crottes pour le recensement des céphalophes. Cet input méthodologique va probablement consolider la technique de comptage des crottes et augmenter sa fiabilité dans les estimations de l'abondance des espèces de céphalophes et dans les études de leur distribution spatiale.

International audience; The importance of the conservation of all three fundamental levels of biodiversity (ecosystems, species and genes) has been widely acknowledged, but only in recent years it has become technically feasible to consider intraspecific diversity, i.e. the genetic component to biodiversity. In order to facilitate the assessment of biodiversity, considerable efforts have been made towards identifying surrogates because the efficient evaluation of regional biodiversity would help in designating important areas for nature conservation at larger spatial scales. However, we know little about the fundamental relationships among the three levels of biodiversity, which impedes the formulation of a general, widely applicable concept of biodiversity conservation through surrogates. Here, we present the set-up of an international, interdisciplinary project, INTRABioDiv (http://www.intrabiodiv.eu), which studied vascular plant biodiversity at a large scale, i.e. across the European Alps and the Carpathians. Our assessment comprises species richness (high-mountain flora), genetic variation (amplified fragment length polymorphisms, AFLPs) and environmental diversity (modelled potential habitat diversity). Our primary aims were to test for correlations between intra- and interspecific diversity and to identify possible environmental surrogates to describe biodiversity in the two study regions. To the best of our knowledge, INTRABioDiv represents the first multispecies study on intraspecific, molecular-genetic variation in relation with species and habitat diversity. Here, we outline the theoretical background, our sampling scheme, the technical approaches and the feasibility of a concentrated and standardized sampling effort. We further show exemplary results. Our three data sets will be made freely available and will provide a playground for further hypothesis testing in conservation, ecology or evolution open to the scientific community. (C) 2008 Rubel Foundation, ETH Zurich. Published by Elsevier GmbH. All rights reserved.

Times Cited: 4; International audience; DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.

Times Cited: 1; International audience; Asian bears face major threats due to the impact of human activities as well as a critical lack of knowledge about their status, distribution and needs for survival. Once abundant in northern Pakistan, the Himalayan brown bear (Ursus arctos isabellinus) has been exterminated in most of its former distribution range. It presently occurs sparsely, in small populations, the Deosai National Park supporting the largest isolate. This decline might imply a reduction in genetic diversity, compromising the survival of the population. Using a combination of fecal DNA analysis and field data, our study aimed at assessing the size and genetic status of the Deosai population and give guidelines for its conservation and management. Using fecal genetic analysis, we estimated the population to be 40-50 bears, which compares well with the field census of 38 bears. The northern Pakistani brown bear population may have undergone an approximate 200-300-fold decrease during the last thousand years, probably due to glaciations and the influence of growing human population. However, in spite of the presence of a bottleneck genetic signature, the Deosai population has a moderate level of genetic diversity and is not at immediate risk of inbreeding depression. Gene flow might exist with adjacent populations. We recommend careful monitoring of this population in the future both with field observations and genetic analyses, including sampling of adjacent populations to assess incoming gene flow. The connectivity with adjacent populations in Pakistan and India will be of prime importance for the longterm survival of Deosai bears. (c) 2006 Elsevier Ltd. All rights reserved.

Times Cited: 1; International audience; The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.

Times Cited: 2; International audience; 1. We studied male yearly reproductive success (YRS) and its determinants (phenotypic characteristics, age, population density) in two Scandinavian brown bear populations, using molecular techniques to determine paternity. 2. We found a significant difference in male YRS between the study areas, with lower YRS in the south than in the north. 3. In general, older and larger males had higher YRS. Older males may be more experienced in competition for reproduction (male dominance). Large body size is of direct benefit in male-male competition and of advantage in endurance competition for the access to females. 4. Age was relatively more important for YRS in the north and body size was more important in the south, due perhaps to differences in male age structure due to illegal killing. A single old male dominated the reproduction in the north during the study, which resulted most probably in the relatively higher importance of age in the north. In the south, with a more even male age structure, no single male was able to dominate, probably resulting in a more intense competition among males, with body size as the deciding factor. 5. Male YRS was correlated positively with population density. This may be related to the structure of the expanding bear population, with female densities declining towards the population edge. 6. Internal relatedness, a measure of genetic heterozygosity, was correlated negatively with YRS, suggesting that outbred individuals have a higher YRS. Individual heterozygosity at key or many loci may reflect male physical qualities and condition-sensitive traits, which may benefit males directly in contest or in sperm competition.

Times Cited: 2; International audience; The ability of species to track their ecological niche after climate change is a major source of uncertainty in predicting their future distribution. By analyzing DNA fingerprinting ( amplified fragment-length polymorphism) of nine plant species, we show that long-distance colonization of a remote arctic archipelago, Svalbard, has occurred repeatedly and from several source regions. Propagules are likely carried by wind and drifting sea ice. The genetic effect of restricted colonization was strongly correlated with the temperature requirements of the species, indicating that establishment limits distribution more than dispersal. Thus, it may be appropriate to assume unlimited dispersal when predicting long-term range shifts in the Arctic.

International audience; The population concept is central in evolutionary and conservation biology, but identifying the boundaries of natural populations is often challenging. Here, we present a new approach for assessing spatial genetic structure without the a priori assumptions on the locations of populations made by adopting an individual-centred approach. Our method is based on assignment tests applied in a moving window over an extensively sampled study area. For each individual, a spatially explicit probability surface is constructed, showing the estimated probability of finding its multilocus genotype across the landscape, and identifying putative migrants. Population boundaries are localized by estimating the mean slope of these probability surfaces over all individuals to identify areas with genetic discontinuities. The significance of the genetic discontinuities is assessed by permutation tests. This new approach has the potential to reveal cryptic population structure and to improve our ability to understand gene flow dynamics across landscapes. We illustrate our approach by simulations and by analysing two empirical datasets: microsatellite data of Ursus arctos in Scandinavia, and amplified fragment length polymorphism (AFLP) data of Rhododendron ferrugineum in the Alps.

Times Cited: 1; International audience; It is difficult to obtain fossil data from the 10% of Earth's terrestrial surface that is covered by thick glaciers and ice sheets, and hence, knowledge of the paleoenvironments of these regions has remained limited. We show that DNA and amino acids from buried organisms can be recovered from the basal sections of deep ice cores, enabling reconstructions of past flora and fauna. We show that high-altitude southern Greenland, currently lying below more than 2 kilometers of ice, was inhabited by a diverse array of conifer trees and insects within the past million years. The results provide direct evidence in support of a forested southern Greenland and suggest that many deep ice cores may contain genetic records of paleoenvironments in their basal sections.

Goats were among the first farm animals domesticated, ≈10,500 years ago, contributing to the rise of the “Neolithic revolution.” Previous genetic studies have revealed that contemporary domestic goats ( Capra hircus ) show far weaker intercontinental population structuring than other livestock species, suggesting that goats have been transported more extensively. However, the timing of these extensive movements in goats remains unknown. To address this question, we analyzed mtDNA sequences from 19 ancient goat bones (7,300–6,900 years old) from one of the earliest Neolithic sites in southwestern Europe. Phylogenetic analysis revealed that two highly divergent goat lineages coexisted in each of the two Early Neolithic layers of this site. This finding indicates that high mtDNA diversity was already present >7,000 years ago in European goats, far from their areas of initial domestication in the Near East. These results argue for substantial gene flow among goat populations dating back to the early neolithisation of Europe and for a dual domestication scenario in the Near East, with two independent but essentially contemporary origins (of both A and C domestic lineages) and several more remote and/or later origins.

International audience; Based on the sexually selected infanticide (SSI) hypothesis, infanticide can be an adaptive mating strategy for males, but this is has rarely been documented in non-social mammals. This phenomenon should not benefit females, so one would expect females to evolve mating counter strategies in order to protect their infants from infanticidal males. Cases of SSI are extremely difficult to document in the field, especially for non-social species. Using field observations and genetic methods, we describe mating strategies employed by both sexes of brown bears (Ursus arctos) in relation to SSI. We present evidence for the first time suggesting that infanticide is an adaptive male mating strategy in this non-social carnivore, as all requirements for SSI are fulfilled (1) infanticide shortens the time to the mother's next estrus, (2) the perpetrator is not the father of the killed infants, and (3) putative perpetrators sire the next litter. Moreover, all infanticide cases occurred during the mating season. We expected that primarily immigrant males were infanticidal, as in social species. However, we found that resident adult males commonly committed infanticide. Perhaps they recognize females they have mated with previously. Moreover, we used DNA-based parentage testing to demonstrate a minimum of 14.5% of multiple paternities (up to 28% for litters with at least three young). Female promiscuity to confuse paternity may be an adaptive counter strategy to avoid infanticide.

Rapidly evolving genes (e.g. candidate selected loci) are of increasing interest to molecular ecologists and conservation geneticists. Here, we report primers for five regions from three independent nuclear reproductive genes that reliably generate polymorphic sequences across the widespread wild goats of the Capra ibex species group and likely many other species of bovids. From three to nine single-nucleotide polymorphisms (SNPs) were identified in each gene region among C. ibex subspecies. Average numbers of SNPs per 1000 bp across all five gene regions was 15.0, with a high of 25.3 in the ZP3 exons 3 and 4 sequence and a low of 6.1 in the TNP1 sequence.

International audience; Background The reconstruction of biological processes and human activities during the last glacial cycle relies mainly on data from biological remains. Highly abundant tissues, such as wood, are candidates for a genetic analysis of past populations. While well-authenticated DNA has now been recovered from various fossil remains, the final ‘proof' is still missing for wood, despite some promising studies. Scope The goal of this study was to determine if ancient wood can be analysed routinely in studies of archaeology and palaeogenetics. An experiment was designed which included blind testing, independent replicates, extensive contamination controls and rigorous statistical tests. Ten samples of ancient wood from major European forest tree genera were analysed with plastid DNA markers. Conclusions Authentic DNA was retrieved from wood samples up to 1000 years of age. A new tool for real-time vegetation history and archaeology is ready to use.

Times Cited: 24; International audience; Today, with the rapid development of population genomics, the genetic basis of adaptation can be unraveled directly at the genome level, without any prerequisites about the selectively advantageous genes or traits. For nonmodel species, it is now possible to screen many markers randomly scattered across the genome and to distinguish between the neutral genetic background and outlier loci displaying an atypical behavior (e.g., a higher differentiation between populations). This study investigated the genetic frame of adaptation to a gradient of altitude in the common frog (Rana temporaria) by means of a genome scan based on 392 amplified fragment length polymorphism markers. Using two outlier detection methods never applied to dominant data so far, we sought for loci with a genetic differentiation diverging from neutral expectations when comparing populations from different altitudes. All the detected loci were sorted out according to their most probable cause for outlier behavior and classified as false positives, outliers due to local effects, or outliers associated with altitude. Altogether, eight good candidate loci were identified as potentially involved in adaptation to altitude because they were picked out in several independent interaltitude comparisons. This result illustrated the potential of genome-wide surveys to reveal selection signatures along selection gradients, where the association between environmental variables and fitness-related traits may be complex and/or cryptic. In this article, we also underlined the need for confirmation of the selection footprints for the outlier loci. Finally, we provided some preliminary insights into the genetic basis of adaptation along an altitudinal cline in the common frog.

International audience; Populations of the European Spadefoot toad (Pelobates fuscus) have experienced recent declines all over Europe, but these appear to be more intense in north and western Europe. Due to the toad's fossorial nature and specific habitat requirements, environmental conditions have played a major role in structuring current populations. We examined the phylogeographic structure in P. fuscus from 16 localities throughout Europe using mitochondrial cytochrome b gene sequence analysis. Sequence divergence among haplotypes was low (0.54 +/- 0.15%). Three very closely related haplotypes occupy northern and western parts of Europe whereas 12 others were observed among samples from south-eastern Europe, including the Balkans. Our results suggest that toads only recently colonized the northern and western parts of Europe following glacial retreat. This expansion probably took place in steppic-like areas during the younger Dryas cold interval, about 12,900-11,500 years ago. Restricted gene flow with an isolation-by-distance population structure characterises a major part of its distribution range. Based on our results we suggest that the northern and western lineages should be considered as distinct conservation units, while the south-eastern populations from the refugial areas, where nearly all genetic polymorphism occurs and populations appear less vulnerable, should receive special attention.

Times Cited: 2; International audience; The distribution of genetic variation within and among 20 European sites infested by the large pine weevil, Hylobius abietis, was analysed using dominant amplified fragment length polymorphism markers. Analysis of molecular variance was performed at the European, regional and local scales. Most of the genetic variability was found within rather than among populations and the global fixation index averaged over loci was low (0.07). We found no evidence of genetic drift, even in relatively isolated sites. This genetic pattern tends to confirm the high dispersal ability of the weevil and the influence of human-mediated expansion of its range through conifer plantations across Europe since the 19th century. Assignment tests demonstrated that the regional forest is a pertinent geographic scale for defining populations in the large pine weevil. Testing the potential influence of the larval host-plant identity (Scot Pine vs Norway Spruce) on the genetic structure revealed a weak but significant effect in two of the three regions tested (in Ardeche and in Limousin but not in Finland). One locus varied with host-plant use in the two French regions, indicating a potential role in host-plant adaptation. However, host-race formation is not observed in H. abietis; we discuss this result in the light of our current knowledge of this insect's biology. Altogether, this study shows that the use of different host plants for development does not constitute a strong barrier to gene flow for H. abietis and confirms the high dispersal ability of this forest pest.

Times Cited: 15; International audience

Times Cited: 66; International audience; Although genotyping errors affect most data and can markedly influence the biological conclusions of a study, they are too often neglected. Errors have various causes, but their occurrence and effect can be limited by considering these causes in the production and analysis of the data. Procedures that have been developed for dealing with errors in linkage studies, forensic analyses and non-invasive genotyping should be applied more broadly to any genetic study. We propose a protocol for estimating error rates and recommend that these measures be systemically reported to attest the reliability of published genotyping studies.

International audience; The systematics of the genus Capra remain controversial in spite of studies conducted using morphology, mtDNA, and allozymes. Here, we assess the evolutionary history of Capra (i) using phylogenetic analysis of two nuclear genes located on the Y-chromosome and (ii) previously published and new cytochrome b sequences. For the Y-chromosome phylogeny, we sequenced segments from the amelogenin (AMELY) and zinc finger (ZFY) genes from all of the eight wild taxa and from domestic goats (Capra hircus). Phylogenetic analysis of the Y-chromosome data revealed two well-defined clades. The domestic goat (C. hircus), the bezoar (Capra aegagrus), and the markhor (C. falconeri) belong to one clade (ML bootstrap value [BP]: 98%), suggesting that domestic goats originated from one or both of these wild species. The second clade (ML BP: 92%) is comprised of all the other wild species. Horn morphology is generally concordant with the Y-chromosome phylogeny. The mtDNA data also revealed two well-defined clades. However, the species in each clade are different from those inferred from the Y-chromosome data. To explain the discordance between Y-chromosome and mtDNA phylogenies, several hypotheses are considered. We suggest that a plausible scenario involves mtDNA introgression between ancestral taxa before the relatively recent colonization of Western Europe, the Caucasus Mountains, and East Africa by Capra populations. (c) 2006 Elsevier Inc. All rights reserved.

The amplified fragment length polymorphism (AFLP) technique is one of the most informative and cost-effective fingerprinting methods. It produces polymerase chain reaction (PCR)-based multi-locus genotypes helpful in many areas of population genetics. This chapter focuses on technical laboratory information to successfully develop the AFLP technique for vertebrates. Several AFLP protocols are described, as well as recommendations about important factors of the procedure such as the choice of enzyme and primer combinations, the choice and scoring of markers, the influence of the genome size on the AFLP procedure, and the control and estimation of genotyping errors. Finally, this chapter proposes a troubleshooting guide to help resolve the main technical difficulties encountered during the AFLP procedure.

International audience

International audience; We address the impact of the ice age cycles on intraspecific cpDNA diversity, for the first time on the full circumboreal-circumarctic scale. The bird-dispersed bog bilberry (or arctic blueberry, Vaccinium uliginosum) is a key component of northern ecosystems and is here used to assess diversity in previously glaciated vs. unglaciated areas and the importance of Beringia as a refugium and source for interglacial expansion. Eighteen chloroplast DNA haplotypes were observed in and among 122 populations, grouping into three main lineages which probably diverged before, and thus were affected more or less independently by, all major glaciations. The boreal 'Amphi-Atlantic lineage' included one haplotype occurring throughout northern Europe and one occurring in eastern North America, suggesting expansion from at least two bottlenecked, glacial refugium populations. The boreal 'Beringian lineage' included seven haplotypes restricted to Beringia and the Pacific coast of USA. The 'Arctic-Alpine lineage' included nine haplotypes, one of them fully circumpolar. This lineage was unexpectedly diverse, also in previously glaciated areas, suggesting that it thrived on the vast tundras during the ice ages and recolonized deglaciated terrain over long distances. Its largest area of persistence during glaciations was probably situated in the north, stretching from Beringia and far into Eurasia, and it probably also survived the last glaciation in southern mountain ranges. Although Beringia apparently was important for the initial divergence and expansion of V. uliginosum as well as for continuous survival of both the Beringian and Arctic-Alpine lineages during all ice ages, this region played a minor role as a source for later interglacial expansions.

International audience

Times Cited: 4; International audience; The amplified fragment length polymorphism (AFLP) technique is one of the most informative and cost-effective fingerprinting methods. It produces polymerase chain reaction (PCR)-based multi-locus genotypes helpful in many areas of population genetics. This chapter focuses on technical laboratory information to successfully develop the AFLP technique for vertebrates. Several AFLP protocols are described, as well as recommendations about important factors of the procedure such as the choice of enzyme and primer combinations, the choice and scoring of markers, the influence of the genome size on the AFLP procedure, and the control and estimation of genotyping errors. Finally, this chapter proposes a troubleshooting guide to help resolve the main technical difficulties encountered during the AFLP procedure.

International audience; Noninvasive genetic methods can be used to estimate animal abundances and offer several advantages over conventional methods. Few attempts have been made, however, to evaluate the accuracy and precision of the estimates. We compared four methods of estimating population size based on fecal sampling. Two methods used rarefaction indices and two were based on capture-mark-recapture (CMR) estimators, one combining genetic and field data. Volunteer hunters and others collected 1904 fecal samples over 2 consecutive years in a large area containing a well-studied population of brown bears (Ursus arctos). On our 49,000-km2 study area in south-central Sweden, population size estimates ranged from 378 to 572 bears in 2001 and 273 to 433 bears in 2002, depending on the method of estimation used. The estimates from the best model in the program MARK appeared to be the most accurate, based on the minimum population size estimate from radio-marked bears in a subsection of our sampling area. In addition, MARK models included heterogeneity and temporal variation in detection probabilities, which appeared to be present in our samples. All methods, though, incorrectly suggested a biased sex ratio, probably because of sex differences in detection probabilities and low overall detection probabilities. The population size of elusive animals can be estimated reliably over large areas with noninvasive genetic methods, but we stress the importance of an adequate and well-distributed sampling effort. In cases of biased sampling, calibration with independent estimates may be necessary. We recommend that this noninvasive genetic approach, using the MARK models, be used in the future in areas where sufficient numbers of volunteers can be mobilized.

Times Cited: 13; International audience; We show that Alpine ibex (Capra ibex) and Corsican mouflon (Ovis musimon) faeces yield useful DNA for microsatellite analysis, however, we detected higher genotyping error rates for spring faeces than for winter faeces. We quantified the genotyping error rate by repeatedly genotyping four microsatellites. Respectively, 99 and 95% of mouflon and ibex genotyping repetitions provided a correct genotype using winter samples, whereas spring samples provided only 52 and 59% correct genotypes. Thus, before starting a noninvasive study, we recommend that researchers conduct a pilot study to quantify genotyping error rates for each season, population and species to be studied.

The history and origins of the donkey are particularly interesting because, together with the horse, the donkey ( Equus asinus ) has been used for the transportation of people, possessions, and produce in many cultures. This domestication marks a major cultural shift away from sedentary, agrarian

Times Cited: 2; International audience; During the last century, North African ungulate species have suffered from habitat loss and over-hunting. Gazella dorcas (Antilopidae subfamily) and Ammotragus lervia (Caprinae subfamily) are among the ungulates that have suffered most. To help to protect these species, conservation programs and population genetics studies are being implemented. Here, we tested 30 published microsatellite primer-pairs from Bovids (cattle, sheep and goat) on eight individuals from each species. From the 30 loci tested, 20 amplified well and showed moderate allelic richness (3.75 and 4.65 mean number of alleles per species, respectively, for G. dorcas and A. lervia), and moderatly high heterozygosity (0.53 and 0.63 per species, respectively). These 20 polymorphic markers will facilitate conservation and genetic studies in these two species, and promise to be widely useful across divergent ungulate taxa.

Times Cited: 22; International audience

Times Cited: 38; International audience; Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.

Times Cited: 3; International audience; The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification. (C) 2003 Elsevier Inc. All rights reserved.

International audience; Inferring phylogenetic relationships among closely related plant species is often difficult due to the lack of molecular markers exhibiting enough nucleotide variability at this taxonomic level. Moreover, gene tree does not necessary represent the true species tree because of random sorting of polymorphic alleles in different lineages. A solution to these problems is to use many amplified fragment length polymorphisms (AFLP) distributed throughout the whole genome, to infer cladistic and phenetic among-species relationships. Phylogenetic relationships among interfertile species of Trollius L. (Ranunculaceae) were investigated using nuclear DNA (ITS1 + 5.8S rRNA + ITS2) and chloroplast DNA (trnL intron and trnL–trnF intergene spacer) sequences, and AFLP markers. ITS sequences were not informative at the intrageneric level, but confirmed the sister relationship between Trollius and Adonis genera, and provided new information on the phylogenetic relationships among five Ranunculaceae genera. Chloroplast DNA was more informative among Trollius species, but not consistent with the sections previously described. AFLP proved to be a powerful tool to resolve the complex genetic relationships between the morphological entities constituting the genus Trollius. Although as much as 76.1% of the total AFLP variability was found within a priori defined morphological groups, the remaining 23.9% variability differentiating groups was sufficient to generate congruent and robust cladistic and phenetic trees. Several morphological traits, independent from those used to define groups, were mapped onto the molecular phylogeny, and their evolution discussed in relation to the absence/presence of pollinator-seed parasite Chiastocheta flies.

Times Cited: 121; International audience; Population genomics has the potential to improve studies of evolutionary genetics, molecular ecology and conservation biology, by facilitating the identification of adaptive molecular variation and by improving the estimation of important parameters such as population size, migration rates and phylogenetic relationships. There has been much excitement in the recent literature about the identification of adaptive molecular variation using the population-genomic approach. However, the most useful contribution of the genomics model to population genetics will be improving inferences about population demography and evolutionary history.

Times Cited: 3; International audience; For future investigations of the mating system of a highly social mammal (Marmota marmota), we identified 16 new microsatellites using an enrichment protocol. Five loci were revealed to be polymorphic. The polymorphism was rather low (two to six alleles among 24 individuals). However, these markers, added to the other six published microsatellites for M. marmota and Spermophilus citellus, will help to assess dispersal patterns and test for genetic monogamy in alpine marmots from the European Alps.

Times Cited: 17; International audience; For some French Registered Designation of Origin (RDO) cheeses Prim'Holstein's milk is not allowed for cheese making (e.g., Reblochon, Abondance, and Beaufort cheeses). To find molecular markers for Prim'Holstein's milk detection in RDO cheese, four genes affecting coat color in cattle (c-kit, MGH, TYRP1, and MC1R) have been sequenced for three mountain breeds and the Prim'Holstein breed. Only the MC1R gene (E-locus) has shown variation between the four breeds. Among the 25 French and Italian breeds sequenced for the MC1R gene, only the Vosgienne breed has presented the same allele as the black Prim'Holstein breed (E-D). A quick and easy DNA-based method to detect Holstein's milk in RDO cheese is proposed based on E-D allele detection. A DNA extraction from cheese, a preamplification of the gene and a competitive oligonuleotide priming PCR on MC1R mutations were performed. Using an automated sequencer, differences in fluorescence and fragment size reveal the allele type. This simple approach provides good reproducibility and is shown to be relatively sensitive, with a detection limit of about 1 % of Holstein's milk in milk curd.