En six annees de travaux de recherche, le Laboratoire de Biologie des Populations d'altitude a mis au point des techniques de la biologie moleculaire qui ont permis de developper une biologie des populations basee sur la genetique moleculaire et cela pour une petite population en danger d'extinction, les Ours des Pyrenees. A partir de sequences d'ADN (zone de controle de l'ADN mitochondrial) extraites, en tres faibles quantites, de poils ou de crottes recueillis sur le terrain, il a ete possible (i) d'evaluer les degres de parente entre differentes populations europeennes, (ii) de connaitre le sexe des individus, (iii) d'elaborer une sorte de carte d'identite (a l'aide de six sequences microsatellites) pour chaque individu d'ou une connaissance precise de l'effectif et cela sans avoir a capturer des ours (methodes dites non-invasives). Les resultats obtenus ouvrent de nouvelles perspectives en biologie de la conservation.

The remnant Brown Bear (Ursus arctos) population in the Pyrenees mountains on the border between France and Spain is among the most threatened in Europe. To obtain critical information for the management of this Brown Bear population, the French Ministry of the Environment initiated a research programme in 1991 with three major goals : (i) identify the potential conservation units at the European level, (ii) develop a method to determine the sex of free-ranging bears, (iii) establish unique genetic identification of all remaining individuals using noninvasive sampling techniques. Previous studies have demonstrated the potential for hair or faeces collected in the field to provide a suitable source of DNA for genotyping and sexing free-ranging animals. Despite recent advances, noninvasive genetic sampling represents a difficult challenge as hair and faeces provide only picogram quantities of degraded template DNA. Under these limiting conditions, there are two major genotyping errors which may lead to inaccurate results : an allele of a heterozygous individual may not be detected, or PCR-generated alleles or false alleles may arise. To overcome these difficulties, a multiple-tubes approach ha been recently developed, Using this method, the DNA extract is distributed among seven tubes and amplified separately to determine the genotype with a 99% accuracy. Before the beginning of this genetic study, field data based mainly on the recording of track sizes suggested the presence of only several animals. Five unique genotypes were identified from the six polymorphic microsatellite loci The genotypic data for each sample were compared with the corresponding track size in order to obtain a minimum estimate of the population size. Because one genotype was found with two nonoverlapping track sizes, we were able to detect six individual bears. The g genotype of one bear was not observed after 1993. The population is composed of one yearling, three adult males and one adult female, This work extends the application of molecular methods in conservation biology by demonstrating that not only is population size accessible, but also sex ratios and individual home ranges can be established.

Domestic animals have played a key role in human history. Despite their importance, however, the origins of most domestic species remain poorly understood. We assessed the phylogenetic history and population structure of domestic goats by sequencing a hypervariable segment (481 bp) of the mtDNA control region from 406 goats representing 88 breeds distributed across the Old World. Phylogeographic analysis revealed three highly divergent goat lineages (estimated divergence >200,000 years ago), with one lineage occurring only in eastern and southern Asia. A remarkably similar pattern exists in cattle, sheep, and pigs. These results, combined with recent archaeological findings, suggest that goats and other farm animals have multiple maternal origins with a possible center of origin in Asia, as well as in the Fertile Crescent. The pattern of goat mtDNA diversity suggests that all three lineages have undergone population expansions, but that the expansion was relatively recent for two of the lineages (including the Asian lineage). Goat populations are surprisingly less genetically structured than cattle populations. In goats only ≈10% of the mtDNA variation is partitioned among continents. In cattle the amount is ≥50%. This weak structuring suggests extensive intercontinental transportation of goats and has intriguing implications about the importance of goats in historical human migrations and commerce.

Traditional classification in the genus Capra is based mainly on horn morphology. However, previous investigations based on allozyme data are not consistent with this classification. We thus reexamined the evolutionary history of the genus by analyzing mitochondrial DNA (mtDNA) sequence variation. We collected bone samples from museums or dead animals found in the field. Thirty-four individuals were successfully sequenced for a portion of the mtDNA cytochrome b gene and control region (500 bp in total). We obtained a star-like phylogeny supporting a rapid radiation of the genus. In accordance with traditional classification, mtDNA data support the presence of two clades in the Caucasus and the hypothesis of a domestication event in the Fertile Crescent. However, in conflict with morphology, we found that C. aegagrus and C. ibex are polyphyletic species, and we propose a new scenario for Capra immigration into Europe.

Pyrenean brown bears Ursus arctos are threatened with extinction. Management efforts to preserve this population require a comprehensive knowledge of the number and sex of the remaining individuals and their respective home ranges. This goal has been achieved using a combination of noninvasive genetic sampling of hair and faeces collected in the field and corresponding track size data. Genotypic data were collected at 24 microsatellite loci using a rigorous multiple‐tubes approach to avoid genotyping errors associated with low quantities of DNA. Based on field and genetic data, the Pyrenean population was shown to be composed at least of one yearling, three adult males, and one adult female. These data indicate that extinction of the Pyrenean brown bear population is imminent without population augmentation. To preserve the remaining Pyrenean gene pool and increase genetic diversity, we suggest that managers consider population augmentation using only females. This study demonstrates that comprehensive knowledge of endangered small populations of mammals can be obtained using noninvasive genetic sampling.

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.

Some small European populations of the brown bear (Ursus arctos) are threatened by the risk of extinction in the near future. The reinforcement of these populations with bears from other regions might provide a solution to their future survival. However, before any population transfer, the different conservation units must be identified. The phylogeographic approach has been advocated for this purpose. The different European populations were assayed for mitochondrial (mt) DNA polymorphism. A remarkable degree of concordance was found between the geographic distribution and the mtDNA haplotypes. Two clearly distinct lineages differing by more than 7% in mtDNA control region sequences were found and, furthermore, the western lineage appears to be organized into two clades which correspond to two different ancestral refugia. The potential conservation units can be deduced from these results, and a management policy can consequently be inferred. This study clearly demonstrates the relevance of the molecular phylogeographic approach to the identification of conservation units.

As an aid to the management of the Pyrenean population of the brown bear Ursus arctos, a sexing method based on the amplification of a Y chromosome specific sequence has been developed, and tested using hairs found in the field as a source of DNA. This method involves a two-step polymerase chain reaction (PCR) which allows the detection of a very small amount of DNA, probably a single SRY gene molecule. The sex can reliably be identified using about 50pg of DNA extract as template. It is possible that this approach could, with adjustments, be used to identify the sex of other species of eutherian mammals.

Total DNA has been extracted from cells found at the base of a single hair plucked from a captive Pyrenean brown bear (U. arctos). A 307-base pair portion of the cytochrome b gene (encoded by mitochondrial DNA) has been sequenced using DNA amplified by the polymerase chain reaction (PCR). A phylogenetic tree has been constructed, based on this and three other homologous sequences recently published by Shields and Kocher [4]. It suggests that the Pyrenean brown bear was already separated well before the recent split between the Alaskan brown bear (U. arctos) and the polar bear (U. maritimus).

Mitochondrial DNA (mtDNA) from 25 blue tits Parus caeruleus sampled from two populations of the Grenoble region (France) was assayed for polymorphism with 17 restriction endonucleases. Nine genotypes were found. Several mtDNA genotypes were also analysed by amplification via the polymerase chain reaction (PCR) and direct sequencing of 903 bp of the cytochrome b gene. The mtDNA polymorphism is greater in P. caeruleus than in other comparable bird species and results from the presence of two clearly differentiated mitochondrial lineages. Using the data of restriction polymorphism, the mean sequence divergence between individuals of the two lineages is 1.23%. Therefore, P. caeruleus should fall into the category II of phylogeographic pattern sensu Avise et al. (1987): discontinuous mtDNA genotypes which co-occur in the same region. P. caeruleus, like humans and other mobile species with high gene flow, seems to have lost its geographic structure in terms of mtDNA phylogeny. This unusual mitochondrial polymorphism can be explained by the recent admixture of two long-term isolated populations. This could be accounted for by two different scenarios. One assumes a simultaneous post-glacial colonization of the Grenoble region by two isolated European populations of P. caeruleus. Alternatively, hybridization between P. caeruleus and P. cyanus could have caused the observed pattern of mtDNA variation.

A morphometric analysis of Purkinje cells in the developing cerebellar cortex of the chick was performed in normal animals and embryos made hypothyroid by one or two spaced injections of tetramethylthiourea. Profiles of 162 Purkinje cells, from Golgi-Cox treated sections were analysed. Soma area, perimeter and circularity index, cumulative length of the dendrites and number of dendritic bifurcations were studied. The results showed significant differences between control and hypothyroid animals. There were no important differences between birds rendered transiently hypothyroid with a single injection and those made chronically hypothyroid with dual injections. This confirms that the Purkinje cell is very dependent on thyroid hormone especially during the early phases of its morphogenesis. The development of the Purkinje cell was the most affected process of cerebellar cortex maturation in the thyroid-deficient chick. The dendritic arborization was particularly hypoplastic. Moreover, a dynamic balance appeared to exist between the development of the dendritic arborization and that of the perikaryon.