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Cercueil, A., Bellemain, E., Manel, S. 2002. PARENTE: A software package for parentage analysis. Journal of Heredity. 93(6), 458-459. — by Eva Bellemain — last modified 2011-01-30 20:25

PARENTE is a user-friendly software package that conducts 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. PARENTE is free and can be downloaded from : http://www2.ujf-grenoble.fr/leca/membres/manel.html.

Piggott, M.P., Bellemain, E. Taberlet, P., Taylor, A. 2004. A multiplex pre-amplification method that significantly improves microsatellite amplification and error rates for faecal DNA in limiting conditions. Conservation Genetics. 5(2), 417-420. — by Eva Bellemain — last modified 2011-01-30 21:32

 

Bellemain, E., Taberlet, P. 2004. Improved noninvasive genotyping method: application to brown bear (Ursus arctos) feces. Molecular Ecology Notes. 4, 519-522 — by Eva Bellemain — last modified 2011-01-30 20:27

We redesigned new microsatellite primers and one sex-specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi-nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.

Bonin, A., Bellemain, E., Bronken Eidesen, P., Pompanon, F., Brochmann, C., Taberlet, P. 2004. How to track and assess genotyping errors in population genenics studies Molecular Ecology (Invited Review) 13, 3261-3273. — by Eva Bellemain — last modified 2011-01-30 20:29

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.

Manel, S., Bellemain, E., Swenson, J.E., Francois, O. 2004. Assumed and inferred spatial structure of populations: the Scandinavian brown bears revisited. Molecular Ecology. 13, 1327-1331 — by Eva Bellemain — last modified 2011-01-30 20:31

We reanalysed the spatial structure of the Scandinavian brown bear ( Ursus arctos) population based on multilocus genotypes. We used data from a former study that had presumed a priori a specific population subdivision based on four subpopulations. Using two independent methods (neighbour-joining trees and Bayesian assignment tests), we analysed the data without any prior presumption about the spatial structure. A subdivision of thepopulation into three subpopulations emerged from our study. The genetic pattern of these subpopulations matched the three geographical clusters of individuals present in the population. We recommend considering the Scandinavian brown bear population as consisting of three (instead of four) subpopulations. Our results underline the importance of determining genetic structure from the data, without presupposing a structure, even when there seems to be good reason to do so.

Tallmon, D., Bellemain, E., Swenson, J.E., Taberlet, P. 2004. Genetic monitoring of Scandinavian brown bear effective population size and immigration Journal of Wildlife Management 68(4), 960-965 — by Eva Bellemain — last modified 2011-01-30 20:33

We used microsatellite marker data taken from Scandinavian brown bear (Ursus arctos) tissue samples collected by hunters and biologists to estimate population genetic parameters important for bear management. Specifically, we show evidence of a small effective population size (Ne = 44.8; 95% CI: 30.9 to 73.2) and low rates of immigration (m^= 0.01; 95% CI: 0.00 to 0.05) into the brown bear population along the southern edge of their range in Scandinavia. The ratio of genetic effective size to population size is approximately 0.07–0.17, which falls within the range of values found in previous studies of brown bears. The large confidence intervals around the immigration estimate reflect considerable uncertainty. Nonetheless, these values deserve attention because they are near thresholds of short-term management concern and worthy of long-term monitoring. If the genetic effective size remains this small and immigration remains low, then this population could be subject to the loss of fitness as a consequence of inbreeding effects.

Bellemain, E., Swenson, J.E., Tallmom, D.A., Brunberg, S., Taberlet, P. 2005. Estimating population size of elusive animals using DNA from hunter-collected feces: comparing four methods for brown bears. Conservation Biology, 19(1), 150-161 — by Eva Bellemain — last modified 2011-01-30 20:34

Noninvasive genetic methods can be used to estimate animal abundances and offer several advantages over conventional methods. However few attempts have been made 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 a total of 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-km_ 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 program MARK appeared to be the most accurate, based on the minimum population size estimate from radiomarked 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. However, all methods 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 case of biased sampling, calibration with independent estimates may be necessary. We recommend this noninvasive genetic approach, using the MARK models, to be used in the future in areas where sufficient numbers of volunteers can be mobilized.

Bellemain, E., Swenson, J.E., Taberlet, P. 2006. Mating strategies in relation to sexually selected infanticide in a nonsocial carnivore: the brown bear. Ethology, 111, 1-14 — by Eva Bellemain — last modified 2011-01-30 20:50

Based on the sexually selected infanticide (SSI) hypothesis, infanticide can be an adaptive mating strategy for males, but this is controversial. This phenomenon should not benefit females, so one would expect females to evolve mating counter strategies in order to defend their infants against infanticidal males. Cases of SSI are extremely difficult to document in the field, especially for nonsocial 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 that infanticide might be an adaptive male mating strategy in this non social carnivore, as all requirements for SSI 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) putative perpetrators sire the next litter. One would expect that infanticide in nonsocial species should be ascribed primarily to immigrant males, as in social species. However, our results indicate that SSI by resident adult males can also be common. Perhaps they recognize females they have mated with previously. Moreover, we use DNA-based parentage testing to demonstrate a minimum of 14.5% of multiple paternities (up to 28% for litters with 3 young or more). Female promiscuity to confuse paternity may therefore be an adaptive counter strategy to avoid infanticide.

Støen, O-G., Bellemain, E., Sæbø, S., Swenson, J.E. 2006. Kin-related spatial structure in brown bears Ursus arctos. Behavioural Ecology and Sociobiology. 59, 191-197 — by Eva Bellemain — last modified 2011-01-30 20:52

Kin-related social structure may influence reproductive success and survival and, hence, the dynamics of populations. It has been documented in many gregarious animal populations, but few solitary species. Using molecular methods and field data we tested: (1) whether kin-related spatial structure exists in the brown bear (Ursus arctos), which is a solitary carnivore, (2) whether home ranges of adult female kin overlap more than those of non-kin, and (3) whether multigenerational matrilinear assemblages, i.e. aggregated related females, are formed. Pairwise genetic relatedness between adult (5 years and older) female dyads declined significantly with geographic distance, whereas this was not the case for male-male dyads or opposite sex dyads. The amount of overlap of multiannual home ranges was positively associated with relatedness among adult females. This structure within matrilines is probably due to kin recognition. Plotting of multiannual home range centers of adult females revealed formation of two types of matrilines, matrilinear assemblages exclusively using an area and dispersed matrilines spread over larger geographic areas. The variation in matrilinear structure might be due to differences in competitive abilities among females and habitat limitations. The influence of kin-related spatial structure on inclusive fitness needs to be clarified in solitary mammals.

Pompanon, F., Bonin, A., Bellemain, E., Taberlet, P. 2005 Genotyping errors: causes, consequences and solutions. Nature Reviews Genetics 6, 847-856. — by Eva Bellemain — last modified 2008-01-05 09:49

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.

Bellemain, E., Zedrosser, A., Manel, S., Taberlet, P., Waits, L.P., Swenson, J.E. 2006. The dilemma of female mate selection in the brown bear, a species with sexually selected infanticide. Proceedings of the Royal Society of London, Series B 273, 283-291 — by Eva Bellemain — last modified 2008-01-05 09:49

Because of differential investment in gametes between sexes, females tend to be the more selective sex. Based on this concept, we investigate mate selection in a large carnivore: the brown bear (Ursus arctos). We hypothesise that, in this species with sexually selected infanticide (SSI), females may be faced with a dilemma: either select a high-quality partner based on phenotypic criteria, as suggested by theories of mate choice, or rather mate with future potentially infanticidal males as a counter strategy to SSI. We evaluated which male characteristics were important in paternity assignment. Among males available in the vicinity of the females, the largest, most heterozygous and less imbred and also the geographically closest males were more often the fathers of the female’s next litter. We suggest that female brown bears may select the closest males as a counter-strategy to infanticide and exercise a post-copulatory cryptic choice, based on physical attributes, such as a large body size, reflecting male genetic quality. However, male-male competition either in the form of fighting before copulation or during the postcopulatory phase, in the form of sperm competition, cannot entirely be ruled out.

Solberg, H., Bellemain, E., Drageset, O.M., Taberlet, P., Swenson, J.E. 2006. An evaluation of field and genetic methods to estimate brown bear (Ursus arctos) population size. Biological conservation 128, 158-168. — by Eva Bellemain — last modified 2008-01-05 09:49

Estimates of population size and density are essential for successful management and conservation of any species. Although there are a variety of methods available for estimating abundance and density of populations, most studies rely on only one estimator and very few studies have compared and critically evaluated the adequacy and the cost of these methods. We used the brown bear (Ursus arctos) in south-central Sweden to compare the performance of three different methods of estimating population size, including methods based on conventional field data as well as on non-invasive genetic data. The method based on observations of females with cubs underestimated the true population size, as the estimates were below the number of unique genotypes determined from fecal data inside the study area. The genetic method using the closed population MARK estimator, as recommended in a previous study, seemed to perform the best. We conclude that approximately 223 (188-282) bears were present in our 7,328 km_ study area during 2001 and 2002 and suggest that this hunted brown bear population has been relatively stable for about ten years. The non-invasive genetic method was less expensive than the most reliable traditional field method, and preferable from an ethical point of view. We recommend that future studies should aim at collecting 2.5 to 3 times the number of faecal samples as the “assumed” number of animals.

Miquel, C., Bellemain, E., Poillot, C., Taberlet, P. 2006. Quality indexes to assess genotypes reliability for studies using non-invasive sampling and multiple-tube approach. Molecular Ecology Notes 6, 985-988. — by Eva Bellemain — last modified 2008-01-05 09:50

In noninvasive studies, the intersample variance in DNA quality and quantity is large, and produces multilocus genotypes of highly variable quality. Here we propose a standardized method for testing the reliability of the genotyping procedure when using the multiple-tube approach. The quality indexes generated will allow reliable comparisons among samples, loci, studies, and field and/or laboratory protocols. These indexes represent a powerful tool for the quality management of noninvasive studies.

Bellemain, E., Nawaz, A., Valentini, A., Swenson, J.E, Taberlet, P. 2007. Genetic tracking of the brown bear in northern Pakistan and implications for conservation. Biological Conservation 134, 537-547. — by Eva Bellemain — last modified 2008-01-05 09:50

Asian bears face major threats due to the impact of human activity 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 long-term survival of Deosai bears.

Zedrosser, A., Bellemain, E., Taberlet, P., Swenson, J.E. 2007. Genetic estimates of annual reproductive success in male brown bears: the effects of body size, age, heterozygosity and population density. Journal of Animal Ecology 76(2):368-375. — by Eva Bellemain — last modified 2008-01-05 09:50

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. We found a significant difference in male YRS between the study areas, with lower YRS in the south than in the north. 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. 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. 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. 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.

Manel, S., Berthoud, F., Bellemain, E. Gaudeul, M., Swenson, J.E., Luikart, G., Waits, L.P. Intrabiodiv consortium, Taberlet, P. 2007. A new individual-based geographic approach for identifying genetic discontinuities. Molecular Ecology 16(10): 2031-2043. — by Eva Bellemain — last modified 2008-01-05 13:09

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.

Valentini, A., Miquel, C., Nawaz, M.A., Bellemain, E., Coissac, E., Pompanon, F., Nascetti, G. et al. 2008. New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach. Molecular Ecology, 2009, 9, 51-60 — by Eva Bellemain — last modified 2011-01-30 20:56

The development of DNA barcoding (species identification using a standardized DNA sequence), and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis. DNA fragments shorter than 100-150 base pairs remain in a much higher proportion in degraded DNA samples and can be recovered from faeces. As a consequence, by using universal primers that amplify a very short but informative DNA fragment, it is possible to reliably identify the plant taxon that has been eaten. According to our experience and using this identification system, about 50% of the taxa can be identified to species using the trnL approach, i.e. using the P6 loop of the chloroplast trnL (UAA) intron. We demonstrated that this new method is fast, simple to implement, and very robust. It can be applied for diet analyses of a wide range of phytophagous species at large scales. We also demonstrated that our approach is efficient for mammals, birds, insects, and molluscs. This method opens new perspectives in ecology, not only by allowing large-scale studies on diet, but also by enhancing studies on resource partitioning among competing species, and describing food webs in ecosystems.

Bellemain, E. and Ricklefs, R.E. 2008. Are islands the end of the colonisation road? Trends in Ecology and Evolution, 23 (8), 461-468 — by Eva Bellemain — last modified 2011-01-30 21:18

Ecologists have, up to now, widely regarded colonization of islands from continents as a one-way journey, mainly because of widely accepted assertions that less diverse island communities are easier to invade. However, continents present large targets and island species should be capable of making the reverse journey, considering they are the direct descendants of successful colonists and provided that they have not lost their dispersal abilities. Recent mapping of geography onto molecular phylogenies has revealed several cases of ‘reverse colonization’ (from islands to continents). We suggest this phenomenon warrants closer attention in ecology and biogeography. Assessing its significance will contribute to understanding the role of dispersal and establishment in biogeographic distributions and the assembly of natural biotas.

Bellemain, E., Bermingham, E., Ricklefs, R.E. 2008. The dynamic evolutionary history of the bananaquit (Coereba flaveola) in the Caribbean revealed by a multigene analysis. BMC Evolutionary Biology, 8:240 doi:10.1186/1471-2148-8-240 — by Eva Bellemain — last modified 2008-09-10 10:57

Background: The bananaquit (Coereba flaveola) is a small nectivorous and frugivorous emberizine bird (order Passeriformes) that is an abundant resident throughout the Caribbean region. We used multi-gene analyses to investigate the evolutionary history of this species throughout its distribution in the West Indies and in South and Middle America. We sequenced six mitochondrial genes (3744 base pairs) and three nuclear genes (2049 base pairs) for forty-four bananaquits and three outgroup species. We infer the ancestral area of the present-day bananaquit populations, report on the species' phylogenetic, biogeographic and evolutionary history, and propose scenarios for its diversification and range expansion. Results: Phylogenetic concordance between mitochondrial and nuclear genes at the base of the bananaquit phylogeny supported a West Indian origin for continental populations. Multi-gene analysis showing genetic remnants of successive colonization events in the Lesser Antilles reinforced earlier research demonstrating that bananaquits alternate periods of invasiveness and colonization with biogeographic quiescence. Although nuclear genes provided insufficient information at the tips of the tree to further evaluate relationships of closely allied but strongly supported mitochondrial DNA clades, the discrepancy between mitochondrial and nuclear data in the population of Dominican Republic suggested that the mitochondrial genome was recently acquired by introgression from Jamaica. Conclusion: This study represents one of the most complete phylogeographic analyses of its kind and reveals three patterns that are not commonly appreciated in birds: (1) island to mainland colonization, (2) multiple expansion phases, and (3) mitochondrial genome replacement. The detail revealed by this analysis will guide evolutionary analyses of populations in archipelagos such as the West Indies, which include islands varying in size, age, and geological history. Our results suggest that multi-gene phylogenies will permit improved comparative analysis of the evolutionary histories of different lineages in the same geographical setting, which provide replicated "natural experiments" for testing evolutionary hypotheses.

Pages, M., Maudet, C., Bellemain, E., Taberlet, P., Hugues, S., Hänni, C. 2009. Sex your Ursid free. Conservation Genetics. DOI 10.1007/s10592-008-9650-x — by Eva Bellemain — last modified 2011-01-30 21:27

In this paper, we characterise three sex-specific genes (ZFX/Y, SRY, AMLX/Y) for all eight extant bear species and propose a new, robust and accurate molecular procedure to identify the sex of bears from non-invasive samples and fossil remains. These materials contain tiny amounts of poorly preserved deoxyribonucleic acid (DNA), leaving Polymerase Chain Reaction (PCR) amplification very prone to contamination and difficult to analyse. By taking into account the ancient DNA requirements, the duplex procedures that we developed are efficient not only on DNA extracted from bear faeces but also on ancient DNA extracted from a brown bear fossil 7,500 years old. Defined specifically for ursids, the procedure for faecal samples (co-amplification of ZFX/Y and SRY markers) appears more accurate than other published procedures, as it prevents cross-amplification of potential ingested prey and contamination (19 non-ursid species tested). This system can be applied to threatened bear populations to improve the reliability of sex-ratio and population-size estimates based on non-invasive samples.

Aarnes, S.G., Bellemain, E., Eiken, H.G. & Wartiainen, I. 2009. Interlaboratory comparison of genetic profiles of brown bears from Sweden (Laboratoire d’Ecologie Alpine) and Norway (Bioforsk Svanhovd). Bioforsk Report Vol. 4 No. 133. Bioforsk, Svanhovd — by Eva Bellemain — last modified 2011-01-30 21:27

Comparisons of individual DNA-profiles between different laboratories require that the data can be standardized. In this study, we compared DNA profiles of brown bears (Ursus arctos) from Sweden with DNA profiles of Norwegian brown bears. Brown bear samples from Sweden were analyzed at Laboratoire d’Ecologie Alpine (LECA) in France, while the samples collected in Norway were analyzed in the DNA laboratory at Bioforsk Svanhovd. In April 2008, DNA from 38 different bears were analyzed both at LECA in France and at Bioforsk Svanhovd in Norway, which allowed to estimate a first calibrations keys and normalise the data. Calibrations key calculated from this study were used, in 2009, on genotypes from 79 individuals from south-Norway to compare with the genotypes in the database for Swedish brown bears. The initial comparison gave no match. In this study, new calibration keys were determined in order to make the genotypes from Norwegian bears comparable with the whole Swedish bear genetic database. The comparison based on the new calibration key included 165 individuals from Norway (time period 2005-2009) and gave 42 matches with individuals from the database for Swedish brown bears (time period 2001-2009). Marker MU59 did not function well in this calibration and additional analyses are needed to sort out the problems with this marker.

Bellemain, E., Carlsen, T., Coissac, E., Taberlet, P., Brochmann, C., Kauserud, H. 2010. ITS as a DNA barcode in fungi: An in silico approach reveals potential PCR biases. BMC Microbiology. 10:189 — by Eva Bellemain — last modified 2011-01-30 21:27

Background: During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates. Results: Some of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi. Conclusions: We conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.

Swenson, J.E., Taberlet, P., Bellemain, E. Genetics and conservation of European brown bears. 2011. In press. Mammal Review. — by Eva Bellemain — last modified 2011-01-30 21:23

1. We review the genetics research that has been conducted on the European brown bear Ursus arctos, one of the genetically best-studied mammalian species. 2. The first genetics studies on European brown bears were on phylogeography, as a basis for proposed population augmentations. Two major mitochondrial DNA lineages, western and eastern, and two clades within the western lineage were found. This led to a hypothesis that brown bears had contracted to southern refugia during the last glacial maximum. More recent results suggest that gene flow among brown bears blurred this structure and they survived north of these putative refugia. Thus, today’s structure might be a result of population fragmentation caused by humans. 3. The nuclear diversity of European brown bears is similar in range to that in North American bears: low levels occur in the small populations and high levels in the large populations. 4. Many non-invasive genetic methods, developed during research on brown bears, have been used for individual identification, censusing populations, monitoring migration and gene flow, and testing methods that are easier to use in endangered populations and over large areas. 5. Genetics has been used to study many behavioural and population ecological questions that have relevance for the conservation and management of brown bears. 6. The European brown bear has served, and will continue to serve, as a model for the development of methods, analyses and hypotheses in conservation genetics.