Publications

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    ABSTRACT: Environmental DNA (eDNA) metabarcoding is increasingly used to study present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of presence / absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts, and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in presence of observational errors can successfully estimate true prevalence, detection probability, and false positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability, and false positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies, and could be explicitly estimated to improve the reliability of results.This article is protected by copyright. All rights reserved.
    Molecular Ecology Resources 10/2014; · 7.43 Impact Factor
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    ABSTRACT: DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.
    Biology letters. 09/2014; 10(9).
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    ABSTRACT: The interactions between botanical composition of pasture, quality of herbage grazed, performances of dairy cows and sensory and nutritional properties of dairy products were investigated using an integrated system approach. Two contrasting grazing systems were evaluated from May to September in two years. The treatments included a continuous grazing system (DIV) managed at a lenient stocking rate (1.0 LU ha−1) on a botanically-rich permanent pasture, and a rotational grazing system (PROD) set up at a higher stocking rate (1.7 LU ha−1) on a former temporary grassland presenting moderate biodiversity. DIV aimed to maximize biodiversity and obtain high sensory and nutritional quality cheese, whereas PROD was oriented towards milk production and herbage quality. In each system, 12 non-feed-supplemented Montbéliarde cows were used. The DIV system led to higher milk production per cow in the early grazing season than the PROD system (22.2 vs. 19.9 kg d−1). At the beginning of summer, this milk production pattern was inverted following a decrease in grass nutritive value in the DIV system. In parallel, DIV cows showed a more marked loss of body condition than PROD cows over the season. In terms of milk fatty acid profile, the DIV system proved very interesting early in the grazing season but lost its value over time as the herbage matured. Cheese sensory properties differed between systems only after a long ripening period (6 months). Regarding the ecological performances, the DIV plot showed greater botanical and entomological biodiversity than the PROD plot. This study provides evidence that the balance between animal performances, dairy product quality and biodiversity in dairy systems is more complex than previously thought, since the expected benefits of each system vary markedly over periods. The evolution of herbage vegetation stage during the grazing season combined with the botanical composition of the pasture is a key component for understanding these variations.
    Agriculture Ecosystems & Environment 03/2014; 185:231–244. · 2.86 Impact Factor
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    ABSTRACT: Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25-15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.
    Nature 02/2014; 506(7486):47-51. · 38.60 Impact Factor
  • François Pompanon
    01/2014: pages 3061-3064;
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    ABSTRACT: Roads are of major concern in conservation biology, as they are known to restrict animal movements through landscape fragmentation, and may therefore impact genetic patterns in native terrestrial organisms. We assessed the effect of two large-scale transportation infrastructures (LTIs), a 40-year-old highway and a 30 year-old high-speed railway, on the spatial genetic structure of the alpine newt Ichthyosaura alpestris, a highly nomadic amphibian. Genetic data were gathered following a targeted individual-based sampling scheme and analysed using both overlay and correlative methods. While simulations suggested that the highway may be old enough for a significant barrier effect to be detected, LTIs were never detected as barriers to gene flow: inferred genetic boundaries rather coincided with transition zones between major landscape entities. Further-more, spatial principal component analysis, a method designed to reveal cryptic genetic spatial patterns in high gene flow species, counter-intuitively suggested that the highway may act as a potential dispersal corridor in low-quality habitats, thus challenging traditional hypotheses on road impacts in amphibians. Our study showed that con-sidering local interactions between species, infrastructures and landscape-specific characteristics is essential for better understanding the potential impacts of roads on movement patterns in terrestrial organisms.
    Conservation Genetics 11/2013; · 2.18 Impact Factor
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    ABSTRACT: The Podismini are melanopline grasshoppers with a Holarctic distribution and well represented in the Eurasian fauna. To investigate their controversial taxonomy and evolutionary history, we studied 86%, 78% and 33% respectively of the Eurasian, European and Asian Palaearctic genera (Otte, 1995; Eades et al., 2013). We reconstructed parsimony, maximum likelihood and Bayesian phylogenies using fragments of four genes (ITS1, 16S, 12S, CO2). We applied a Bayesian molecular clock to estimate the times of species divergence, and the event-based parsimony method to depict the biogeographic framework of the diversification. Our results suggest that the selected Eurasian Podismini constitute a monophyletic group inside the Melanoplinae, provided it includes the North American genus Phaulotettix. The clades proposed by the present study inside the Podismini do not fit the older morphological or cytological classifications, but are in agreement with more recent proposals. Furthermore, our results can be explained by a plausible biogeographic history in which the present geographical distribution of the Eurasian Podismini resulted from known changes, to the Cenozoic climate and vegetation, induced by major geological events including the genesis of high mountain chains (e.g., Himalayas, Altay, Alps) and large deserts (e.g., Gobi, Karakoum, Taklamakan), and the opening of marginal seas (e.g., Bering, Japanese and Yellow Seas).
    Molecular Phylogenetics and Evolution 09/2013; · 4.07 Impact Factor
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    ABSTRACT: Genetic data are increasingly used in landscape ecology for the indirect assessment of functional connectivity, that is, the permeability of landscape to movements of organisms. Among available tools, matrix correlation analyses (e.g. Mantel tests or mixed models) are commonly used to test for the relationship between pairwise genetic distances and movement costs incurred by dispersing individuals. When organisms are spatially clustered, a population-based sampling scheme (PSS) is usually performed, so that a large number of genotypes can be used to compute pairwise genetic distances on the basis of allelic frequencies. Because of financial constraints, this kind of sampling scheme implies a drastic reduction in the number of sampled aggregates, thereby reducing sampling coverage at the landscape level. We used matrix correlation analyses on simulated and empirical genetic data sets to investigate the efficiency of an individual-based sampling scheme (ISS) in detecting isolation-by-distance and isolation-by-barrier patterns. Provided that pseudo-replication issues are taken into account (e.g. through restricted permutations in Mantel tests), we showed that the use of interindividual measures of genotypic dissimilarity may efficiently replace interpopulation measures of genetic differentiation: the sampling of only three or four individuals per aggregate may be sufficient to efficiently detect specific genetic patterns in most situations. The ISS proved to be a promising methodological alternative to the more conventional PSS, offering much flexibility in the spatial design of sampling schemes and ensuring an optimal representativeness of landscape heterogeneity in data, with few aggregates left unsampled. Each strategy offering specific advantages, a combined use of both sampling schemes is discussed.
    Molecular Ecology 08/2013; · 6.28 Impact Factor
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    François Pompanon, Emeline Pettex, Laurence Després
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    ABSTRACT: Life history and spatio-temporal patterns of resource utilisation were characterised in four Chiastocheta (Diptera: Anthomyiidae) species, whose larvae compete as seed predators on Trollius europaeus fruits. Interspecific co-occurrence was observed in 80% of the resource patches (= Trollius fruits) in the two communities studied. Isolated larvae from all species had a similar food intake, but differed in development time and size at emergence. Different species exhibit contrasting resource exploitation strategies with specific mining patterns and a partial temporal shift. Two species exhibited particularly singular strategies. C. rotundiventris escaped from strong interactions with other species because it was the first species to develop and the only one to exploit the central pith of Trollius fruits. The key role of this species as the main pollinator of the host-plant appears to be a by-product of constraints imposed by occupying a restricted niche. Although the resource is ephemeral due to seed dispersal, C. dentifera, the last species to oviposit, is not disadvantaged because it has a short development time and rapid food intake. The different patterns can partly explain the stability of Chiastocheta communities, but do not prevent competition to occur at high larval densities.
    Acta Oecologica 06/2013; · 1.62 Impact Factor
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    ABSTRACT: Wildlife is considered to be mainly exposed to environmental contaminants via oral route. Food web analysis is therefore crucial in environmental risk assessment and management. In this context, diet information is commonly obtained from literature and little attention has been given to site-specific considerations (habitat, season…), which might considerably affect diet composition and thus wildlife exposure assessment. By associating next-generation sequencing and DNA barcoding techniques, the metabarcoding molecular approach of diet analysis could achieve a better taxonomic identification of food items with lower time investment than traditional micro- and macro-histological observations of food remains. In this context, the aim of this study is to evaluate the use of this new molecular method to investigate the diet of wild small mammals exposed to arsenic (As) and eventually to better understand and model its transfers. On a partially remediated former gold mine in southern France, small mammals were captured in spring and autumn in zones differing by their remediation treatment and in a control site. Botanical surveys were also performed on the polluted site. DNA was extracted from stomach content (n=96) and faeces (n=19) and then amplified with 3 primers sets, allowing to get plants and invertebrates (molluscs, arthropods and earthworms) DNA. Finally, amplified DNA was sequenced on next-generation sequencer. Preliminary results on plant DNA showed that 95% of the sequences were at least identified to family level and among them 13% were identified up to species level. This attests the possibility to reach a precise taxonomic level with this molecular method. Stomach contents and faeces from a given small mammal specimen gave complementary information, probably because they correspond to different meals. The results also suggested different food patterns among small mammal species, probably related to their foraging ecology. Comparisons between plant taxa identified in the diet and local botanical survey showed some mismatches, which may be related to small mammal mobility during their foraging activity. As a conclusion, the recent DNA metabarcoding is a promising approach to relate local food web structure to resource availability and thus to better understand pollutant trophic transfer in ecosystems.
    SETAC Europe, Glasgow, UK; 05/2013
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    Oryx 01/2013; · 1.62 Impact Factor
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    ABSTRACT: Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches.
    Molecular Ecology 04/2012; 21(15):3647-55. · 6.28 Impact Factor
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    ABSTRACT: Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.
    Molecular Ecology 04/2012; 21(8):2045-50. · 6.28 Impact Factor
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    ABSTRACT: DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling/extraction procedure and opens the possibility to fully standardize biodiversity surveys.
    Molecular Ecology 02/2012; 21(8):1816-20. · 6.28 Impact Factor
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    ABSTRACT: Diet analysis is a prerequisite to fully understand the biology of a species and the functioning of ecosystems. For carnivores, traditional diet analyses mostly rely upon the morphological identification of undigested remains in the faeces. Here, we developed a methodology for carnivore diet analyses based on the next-generation sequencing. We applied this approach to the analysis of the vertebrate component of leopard cat diet in two ecologically distinct regions in northern Pakistan. Despite being a relatively common species with a wide distribution in Asia, little is known about this elusive predator. We analysed a total of 38 leopard cat faeces. After a classical DNA extraction, the DNA extracts were amplified using primers for vertebrates targeting about 100 bp of the mitochondrial 12S rRNA gene, with and without a blocking oligonucleotide specific to the predator sequence. The amplification products were then sequenced on a next-generation sequencer. We identified a total of 18 prey taxa, including eight mammals, eight birds, one amphibian and one fish. In general, our results confirmed that the leopard cat has a very eclectic diet and feeds mainly on rodents and particularly on the Muridae family. The DNA-based approach we propose here represents a valuable complement to current conventional methods. It can be applied to other carnivore species with only a slight adjustment relating to the design of the blocking oligonucleotide. It is robust and simple to implement and allows the possibility of very large-scale analyses.
    Molecular Ecology 01/2012; 21(8):1951-65. · 6.28 Impact Factor
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    ABSTRACT: This study introduces a novel DNA sampling method in amphibians using skin swabs. We assessed the relevancy of skin swabs relevancy for genetic studies by amplifying a set of 17 microsatellite markers in the alpine newt Ichthyosaura alpestris, including 14 new polymorphic loci, and a set of 11 microsatellite markers in Hyla arborea, from DNA collected with buccal swabs (the standard swab method), dorsal skin swabs and ventral skin swabs. We tested for quality and quantity of collected DNA with each method by comparing electrophoresis migration patterns. The consistency between genotypes obtained from skin swabs and buccal swabs was assessed. Dorsal swabs performed better than ventral swabs in both species, possibly due to differences in skin structure. Skin swabbing proved to be a useful alternative to buccal swabbing for small or vulnerable animals: by drastically limiting handling, this method may improve the trade-off between the scientific value of collected data, individual welfare and species conservation. In addition, the 14 new polymorphic microsatellites for the alpine newt will increase the power of genetic studies in this species. In four populations from France (n=19-25), the number of alleles per locus varied from 2 to 16 and expected heterozygosities ranged from 0.04 to 0.91. Presence of null alleles was detected in two markers and two pairs displayed gametic disequilibrium. No locus appeared to be sex-linked.
    Molecular Ecology Resources 01/2012; 12(3):524-31. · 7.43 Impact Factor
  • Pompanon F, Bonin A
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    ABSTRACT: Population genomics is a recently emerged discipline, which aims at understanding how evolutionary processes influence genetic variation across genomes. Today, in the era of cheaper next-generation sequencing, it is no longer as daunting to obtain whole genome data for any species of interest and population genomics is now conceivable in a wide range of fields, from medicine and pharmacology to ecology and evolutionary biology. However, because of the lack of reference genome and of enough a priori data on the polymorphism, population genomics analyses will still involve higher constraints for researchers working on non-model organisms, as regards the choice of the genotyping/sequencing technique or that of the analysis methods. Therefore, Data Production and Analysis in Population Genomics purposely puts emphasis on protocols and methods that are applicable to species where genomic resources are still scarce. It is divided into three convenient sections, each one tackling one of the main challenges facing scientists setting up a population genomics study. The first section helps devising a sampling and/or experimental design suitable to address the biological question of interest. The second section addresses how to implement the best genotyping or sequencing method to obtain the required data given the time and cost constraints as well as the other genetic resources already available. Finally, the last section is about making the most of the (generally huge) dataset produced by using appropriate analysis methods in order to reach a biologically relevant conclusion. Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, advice on methodology and implementation, and notes on troubleshooting and avoiding known pitfalls.
    Methods in Molecular Biology edited by Walker JM, 01/2012; Humana Press., ISBN: 978-1-61779-869-6
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    ABSTRACT: The goat (Capra hircus) is one of the earliest domesticated species ca. 10,500 years ago in the Middle-East where its wild ancestor, the bezoar (Capra aegagrus), still occurs. During the Neolithic dispersal, the domestic goat was then introduced in Europe, including the main Mediterranean islands. Islands are interesting models as they maintain traces of ancient colonization, historical exchanges or of peculiar systems of husbandry. Here, we compare the mitochondrial genetic diversity of both medieval and extant goats in the Island of Corsica that presents an original and ancient model of breeding with free-ranging animals. We amplified a fragment of the Control Region for 21 medieval and 28 current goats. Most of them belonged to the A haplogroup, the most worldwide spread and frequent today, but the C haplogroup is also detected at low frequency in the current population. Present Corsican goats appeared more similar to medieval goats than to other European goat populations. Moreover, 16 out of the 26 haplotypes observed were endemic to Corsica and the inferred demographic history suggests that the population has remained constant since the Middle Ages. Implications of these results on management and conservation of endangered Corsican goats currently decimated by a disease are addressed.
    PLoS ONE 01/2012; 7(1):e30272. · 3.53 Impact Factor
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    ABSTRACT: Accurate information about the diet of large carnivores that are elusive and inhabit inaccessible terrain, is required to properly design conservation strategies. Predation on livestock and retaliatory killing of predators have become serious issues throughout the range of the snow leopard. Several feeding ecology studies of snow leopards have been conducted using classical approaches. These techniques have inherent limitations in their ability to properly identify both snow leopard feces and prey taxa. To examine the frequency of livestock prey and nearly-threatened argali in the diet of the snow leopard, we employed the recently developed DNA-based diet approach to study a snow leopard population located in the Tost Mountains, South Gobi, Mongolia. After DNA was extracted from the feces, a region of ∼100 bp long from mitochondrial 12S rRNA gene was amplified, making use of universal primers for vertebrates and a blocking oligonucleotide specific to snow leopard DNA. The amplicons were then sequenced using a next-generation sequencing platform. We observed a total of five different prey items from 81 fecal samples. Siberian ibex predominated the diet (in 70.4% of the feces), followed by domestic goat (17.3%) and argali sheep (8.6%). The major part of the diet was comprised of large ungulates (in 98.8% of the feces) including wild ungulates (79%) and domestic livestock (19.7%). The findings of the present study will help to understand the feeding ecology of the snow leopard, as well as to address the conservation and management issues pertaining to this wild cat.
    PLoS ONE 01/2012; 7(2):e32104. · 3.53 Impact Factor
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    ABSTRACT: L orsque les herbivores domestiques exploitent des couverts prairiaux complexes, caractérisés par une hétérogénéité structurelle et une diversité spécifique de la végétation importantes, ils sont confrontés à une multitude d'options de sélection alimentaire. Connaître les espèces végétales qu'ils sélectionnent est un élément important pour comprendre les interactions entre l'herbe, les animaux et les produits qui en sont issus. Ces nouveaux éléments de connaissance pourraient également intéresser fortement les filières de qualité qui s'attachent à démontrer les liens au terroir pour consolider leur spécificité. Une part importante du lien entre le produit et son terroir s'établit en effet à travers l'alimentation du troupeau et les caractéristiques du régime alimentaire (COULON et al., 2003 ; MARTIN et al., 2005 ; LUCAS et al., 2006 ; FARRUGGIA et al., 2009). Accéder à ce niveau de finesse dans la connaissance des espèces ingérées n'est cependant pas aisé. La prairie permanente est en effet constituée d'un mélange intime d'espèces végétales qui rend complexe la détermination de la sélection de l'animal au niveau de l'espèce. 43 Fourrages (2012) 209, 43-51
    Fourrages 01/2012; 209:43-51. · 0.39 Impact Factor

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