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

机译：“斯堪的纳维亚棕熊（Ursus arctos）的遗传学：对生物学和保护的影响”

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摘要

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.
机译：本文研究了分子工具与野外数据相结合在野生动植物管理，保护以及了解物种的生物学和行为方面的应用。我们以棕熊（Ursus arctos）为模型物种，以斯堪的纳维亚熊种群为案例研究。本文的第一部分是方法论部分，其中我们开发或回顾了分子生物学和亲本分析的技术方面。第二部分专门介绍分子遗传学在估计种群数量和了解交配系统方面的应用。由于无创方法不需要处理或干扰研究动物，因此已在基因研究中得到广泛使用。但是，从非侵入性样本（例如头发或粪便）中回收的DNA通常会被降解和/或少量降解，从而导致基因分型错误并导致对不正确的基因型的鉴定。这是一个主要问题，尤其是对于小规模或濒临灭绝的人口而言，因为这可能导致人口规模估计出现偏差。为了提高所需DNA模板的质量和数量，并避免大量重复的需求，我们设计了一种两步聚合酶链反应（PCR）方法。在不同物种上测试了这种“多重预扩增方法”，并将其与常规PCR方法进行了比较。它在有限条件下显着改善了微卫星扩增，并降低了粪便DNA的错误率。为了更具体地从棕熊的非侵入性样品中扩增DNA，我们还重新设计了微卫星引物和一种性别特异性引物，并将半巢式PCR与多重预扩增方法相结合。这些新方法可能会转移到其他常规PCR方法由于限制DNA浓度和/或质量而导致成功率很低的物种中。尽管大多数数据集中存在基因分型错误，但遗传错误仍然是禁忌话题。可能导致结果解释。我们考虑了四个代表各种人口遗传学研究的案例研究，以追踪基因分型错误并确定其原因。在这些数据集中，估计的基因分型错误率介于0.8％至2.6％之间，具体取决于研究生物和所使用的标记。错误的主要来源是微卫星的等位基因缺失和AFLP（扩增片段长度多态性）的峰强度差异，但在两种情况下，人为因素都是不可忽略的错误产生器。我们提出了限制和量化基因分型过程中每个步骤的建议，并建议系统报告群体遗传学研究中的错误率。使用多基因座基因型的亲本分析被广泛用于评估生殖成功，交配方式，亲属关系和适应性自然人口。最近已经开发了几种基于最大似然估计和/或贝叶斯推论的方法，但是它们通常仍然是理论上的，生物学家很难应用。但是，显然缺乏能够考虑几代人的父母身份分配软件，并且无需任何先决条件就可以确定父母双方。我们开发了软件PARENTE，使用来自二倍体共性标记的分子数据进行亲缘推断。根据遗传相容性原则，PARENTE使用多基因座基因型和个体的出生和死亡日期（如果可用）寻找潜在的父母的生育，陪产或同时生育。它还计算成功将单个后代分配给其父母的概率。种群规模和种群密度的估算对于成功管理和保护物种至关重要。但是，很少有人尝试评估所获得估计的准确性。使用开发用于扩增粪便DNA的方案，我们首先比较了基于非侵入性遗传方法的四种普查方法。两种方法使用稀疏指数，两种方法基于捕获标记捕获（CMR）估计量。连续2年在瑞典中南部的49,000平方公里研究区总共收集了1904份粪便样本。根据使用的方法，估计的人口规模在2001年为378至572头熊，在2002年为273至433头熊。根据从无线电遥测数据计算出的最小人口规模，我们得出结论，根据MARK程序中的最佳模型（CMR估计器）进行的估计是最准确的。该模型包括检测概率中的异质性和时间变化，这似乎存在于我们的样本中。其次，我们在较小的研究区域中评估了三种传统现场方法与性能最佳的非侵入式遗传方法的可靠性（7，328平方公里）。这三种实地方法都倾向于低估人口规模。使用MARK估算器的遗传方法似乎表现最好。我们得出的结论是，在2001年和2002年之间，在49,000平方公里的研究区域中约有550（482-648）头熊，在7,328km²的研究区域中有223（188-282）头熊。核心区域的阈值密度，目前在该区域的边缘扩展。成本/收益分析表明，非侵入性遗传方法比最可靠的现场方法便宜，并且从伦理角度来看，它是可取的。总而言之，我们建议使用无创遗传方法和MARK估算器来估算大面积人群的数量。我们还指出了进行充分且分布合理的采样工作的重要性，并在可能的情况下建议在有偏见的情况下使用独立的估计值进行校准。未来的研究目标应是收集粪便样本数量是“假定”动物数量的2.5到3倍。这些研究还证实，目前对斯堪的纳维亚熊的管理是成功的，该种群处于良好的保护状态。交配系统的知识对于理解性别选择的演变非常重要。我们研究了棕熊交配系统的两个主要方面，即性别选择的杀婴剂（SSI）和雌性配偶的选择。如果满足以下三个条件，则杀婴即杀害未成年的年轻人，可以认为是按性别选择的，并且适合于男性：i）杀婴缩短了母亲下一次发情的时间； ii）犯罪者不是被杀者的父亲婴儿，以及iii）肇事者为雌性的下一胎产父。但是，这对女性没有好处，为了保护自己的婴儿免遭杀婴性男性的侵害，他们可能已经制定了应对策略。我们在现场记录了8例杀婴事件。从现场收集的遗传样本和观察结果中，我们验证了对SSI的所有要求均已满足，这表明SSI可能是这种非社会食肉动物的一种适应性雄性交配策略。与社会物种相反，那里的大多数移民男性杀死年轻人，而成年男性主要在斯堪的纳维亚棕熊中杀婴。这意味着它们能够通过识别与之交配的雌性，使其自己的后代与无关的幼崽区分开。此外，我们通过遗传学方法记录了至少14.5％的多重花粉（对于3个或更多年幼的猫砂，则为28％）。因此，女性滥交父母身份可能是避免SSI的适应性对策。此外，我们评估了雌性棕熊选择其生殖伴侣的标准。我们假设雌性可能面临两难选择：要么根据配偶选择理论建议，要么根据形态或遗传标准选择高质量的伴侣，要么宁愿与未来可能杀婴儿的雄性交配，即地理位置最接近的雄性。我们测试了不同的男性特征是否会影响亲子鉴定，并发现女性明显选择了地理上最接近的男性，而且选择了杂合度更高，最大和最老的男性。我们建议雌性棕熊可能与最接近的雄性棕熊交配，以作为对付婴儿杀手的对策，并根据体型或优势地位等形态特征，行使交配后的隐秘选择，以反映雄性遗传品质。

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    Bellemain Eva;

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