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Theses

"Genetics of the Scandinavian brown bear (Ursus arctos): implication for biology and conservation"

Abstract : This thesis deals with the application of molecular tools, combined with field data, in wildlife management, in conservation and in understanding species' biology and behavior. We used the brown bear (Ursus arctos) as a model species and the Scandinavian bear population as a case study. The first part of this thesis is a methodological part, in which we developed or reviewed technical aspects in molecular biology and parentage analysis; the second part is devoted to the application of molecular genetics to estimate population sizes and to understand mating systems.
Noninvasive methods are gaining widespread use in genetic studies as they do not require the handling or disturbance of the study animal. However, DNA recovered from noninvasive samples, such as hairs or feces, is usually degraded and/or in small quantities, leading to genotyping errors and resulting in the identification of incorrect genotypes. This is a major concern, especially for small or endangered populations, as it can lead to biases in population size estimates. With the aim of increasing the quality and quantity of the desired DNA template, and to avoid the need for numerous replicates, we devised a two-step polymerase chain reaction (PCR) method. This “multiplex pre-amplification method” was tested on different species and compared with a conventional PCR approach. It significantly improved microsatellite amplification and decreased error rates for fecal DNA in limiting conditions. To more specifically amplify DNA from noninvasive samples of brown bears, we also redesigned microsatellite primers and one sex-specific primer and combined a semi-nested PCR with the multiplex pre-amplification method. These new approaches could be transposed to other species where conventional PCR methods experience low success due to limiting DNA concentration and/or quality.
Genotyping errors remain a taboo subject in population genetics studies, in spite of their occurrence in most datasets and the negative consequences they may cause in the interpretation of the results. We considered four case studies representing a large variety of population genetics investigations, to track genotyping errors and identify their causes. In these datasets the estimated genotyping error rate ranged from 0.8% to 2.6%, depending on the study organism and the marker used. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs (Amplified Fragment Length Polymorphism), but in both cases, human factors were non-negligible error generators. We present suggestions to limit and quantify genotyping errors at each step of the genotyping process and recommend the systematic reporting of the error rate in population genetics studies.
Parentage analyses using multilocus genotypes are widely used to assess reproductive success, mating patterns, kinship and fitness in natural populations. Several approaches, based on maximum likelihood estimations and /or Bayesian inference, have been recently developed, but they often remain theoretical and difficult for biologists to apply. However, there is a clear lack of parentage assignment softwares that are able to consider several generations of individuals and that allow the determination of both parents without any prior assumptions. We developed the software PARENTE to conduct parentage inference using molecular data from diploid codominant markers. Based on the principle of genetic compatibility, PARENTE looks for maternity, paternity or simultaneously for both potential parents, using multilocus genotypes and birth and death dates of individuals (if available). It also calculates the probability of successfully allocating an individual offspring to its parents.
Estimates of population size and population density are essential for successful management and conservation of species. However, few attempts have been made to evaluate the accuracy of the estimates obtained. Using the protocols developed for amplifying fecal DNA, we first compared four census methods based on noninvasive genetic methods. Two methods used rarefaction indices and two were based on capture-mark-recapture (CMR) estimators. A total of 1904 fecal samples were collected over 2 consecutive years in a 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 used. Based on a calculated minimum population size from radio-telemetry data, we concluded that the estimate from the best model in program MARK, a CMR estimator, was the most accurate. This model included heterogeneity and temporal variation in detection probabilities, which appeared to be present in our samples. Second, we evaluated the reliability of three traditional field methods in comparison with the best performing noninvasive genetic method in a smaller study area (7,328 km²). All three field methods tended to underestimate population size; the genetic method using the MARK estimator seemed to perform the best. We concluded that approximately 550 (482-648) bears were present in the 49,000-km² study area and 223 (188-282) bears were present in the 7,328-km² study area during 2001 and 2002. We suggest that the brown bear has reached a threshold density in the core area and currently expands on the edge of this area. A cost/benefit analysis showed that the noninvasive genetic method was less expensive than the most reliable field method and it is preferable from an ethical point of view. In conclusion, we recommend the use of noninvasive genetic methods, using the MARK estimator, to estimate population size over large areas. We also point out the importance of an adequate and well-distributed sampling effort and advise calibration with independent estimates in case of biased sampling, if possible. Future studies should aim at collecting 2.5 to 3 times the number of fecal samples as the “assumed” number of animals. These studies also confirmed that the present management of the Scandinavian bears has been successful and that this population is in a good conservation status.
The knowledge of mating systems is important for understanding the evolution of sexual selection. We studied two major aspects of the brown bear mating system, namely the mating strategies employed by both sexes in relation to sexually selected infanticide (SSI) and female mate selection. Infanticide, the killing of dependent young, can be considered as sexually selected and adaptive for males, if the following three requirements are fulfilled: i) infanticide shortens the time to the mother's next estrus, ii) the perpetrator is not the father of the killed infants, and iii) perpetrators sire the female's next litter. However, this is not of benefit for females and they may have evolved counterstrategies in order to defend their infants against infanticidal males. We documented eight cases of infanticide in the field. From genetic samples collected at the sites and from observations, we verified that all requirements for SSI were fulfilled, suggesting that SSI may be an adaptive male mating strategy in this nonsocial carnivore. Contrary to social species, where mostly immigrant males kill young, mainly resident adult males were infanticidal in Scandinavian brown bears. This implies that they are able to differentiate their own progeny from unrelated cubs, perhaps by recognizing the females they mated with. Moreover, we genetically documented a minimum of 14.5% multiple paternities (28% for litters with 3 young or more). Female promiscuity to confuse paternity may therefore be an adaptive counterstrategy to avoid SSI. Further, we assessed on which criteria female brown bears chose their reproductive partner(s). We hypothesized that females may be faced with a dilemma: either select a high quality partner based on morphological or genetic criteria, as suggested by theories of mate choice, or rather mate with future potentially infanticidal males, i.e. the geographically closest males. We tested whether different male traits influenced paternity determination and found that females significantly selected the geographically closest males, but also the more heterozygous, largest and oldest males. We suggest that female brown bears might mate with the closest males as a counter-strategy to infanticide and exercise a post-copulatory cryptic choice, based on morphological traits such as body size or dominance, reflecting male genetic quality.
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Eva Bellemain. "Genetics of the Scandinavian brown bear (Ursus arctos): implication for biology and conservation". Ecology, environment. Université Joseph-Fourier - Grenoble I, 2004. English. ⟨tel-00122944⟩

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