Olivier Lepais

Current trends in microsatellite genotyping.

Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost‐effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next‐generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non‐model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well‐established strategy including standardized high‐throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

Estimation of bumblebee queen dispersal distances using sibship reconstruction method

Dispersal ability is a key determinant of the propensity of an organism to cope with habitat fragmentation and climate change. Here we quantify queen dispersal in two common bumblebee species in an arable landscape. Dispersal was measured by taking DNA samples from workers in the spring and summer, and from queens in the following spring, at 14 sites across a landscape. The queens captured in the spring must be full sisters of workers that were foraging in the previous year. A range of sibship reconstruction methods were compared using simulated data sets including or no genotyping errors. The program Colony gave the most accurate reconstruction and was used for our analysis of queen dispersal. Comparison of queen dispersion with worker foraging distances was used to take into account an expected low level of false identification of sister pairs which might otherwise lead to overestimates of dispersal. Our data show that Bombus pascuorum and B. lapidarius queens can disperse by at least 3 and 5 km, respectively. These estimates are consistent with inferences drawn from studies of population structuring in common and rare bumblebee species, and suggest that regular gene flow over several kilometres due to queen dispersal are likely to be sufficient to maintain genetic cohesion of ubiquitous species over large spatial scales whereas rare bumblebee species appear unable to regularly disperse over distances greater than 10 km. Our results have clear implications for conservation strategies for this important pollinator group, particularly when attempting to conserve fragmented populations.

Cryptic differences in dispersal lead to differential sensitivity to habitat fragmentation in two bumblebee species

Habitat loss has led to fragmentation of populations of many invertebrates, but social hymenopterans may be particularly sensitive to habitat fragmentation due to their low effective population sizes. The impacts of fragmentation depend strongly on dispersal abilities, but these are difficult to quantify. Here, we quantify and compare dispersal abilities of two bumblebee species, Bombus muscorum and Bombus jonellus, in a model island system. We use microsatellites to investigate population genetic structuring, dispersal and spatial patterns in genetic diversity. Populations of both species showed significant structuring, and isolation by distance, but this was markedly greater in B. muscorum (θ = 0.13) than in B. jonellus (θ = 0.034). This difference could reflect a higher effective population size in B. jonellus compared to B. muscorum, but this is not consistent with the observed abundance of the two species. We argue that it is more likely that B. jonellus has a higher propensity to disperse than B. muscorum. This will influence their relative susceptibility to habitat fragmentation and may in part explain differential declines of mainland populations of these and other bumblebee species.

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state‐of‐the‐art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north‐eastern Australia, we show that high‐throughput shotgun pyrosequencing technology, so‐called second‐generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large‐scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200 908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost‐effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.

Reconstructing demographic events from population genetic data: the introduction of bumblebees to New Zealand

Four British bumblebee species (Bombus terrestris, Bombus hortorum, Bombus ruderatus and Bombus subterraneus) became established in New Zealand following their introduction at the turn of the last century. Of these, two remain common in the United Kingdom (B. terrestris and B. hortorum), whilst two (B. ruderatus and B. subterraneus) have undergone marked declines, the latter being declared extinct in 2000. The presence of these bumblebees in New Zealand provides an unique system in which four related species have been isolated from their source population for over 100 years, providing a rare opportunity to examine the impacts of an initial bottleneck and introduction to a novel environment on their population genetics. We used microsatellite markers to compare modern populations of B. terrestris, B. hortorum and B. ruderatus in the United Kingdom and New Zealand and to compare museum specimens of British B. subterraneus with the current New Zealand population. We used approximate Bayesian computation to estimate demographic parameters of the introduction history, notably to estimate the number of founders involved in the initial introduction. Species‐specific patterns derived from genetic analysis were consistent with the predictions based on the presumed history of these populations; demographic events have left a marked genetic signature on all four species. Approximate Bayesian analyses suggest that the New Zealand population of B. subterraneus may have been founded by as few as two individuals, giving rise to low genetic diversity and marked genetic divergence from the (now extinct) UK population.

High Genetic Diversity and Distinctiveness of Rear-Edge Climate Relicts Maintained by Ancient Tetraploidisation for Alnus glutinosa

Populations located at the rear-edge of a species’ distribution may have disproportionate ecological and evolutionary importance for biodiversity conservation in a changing global environment. Yet genetic studies of such populations remain rare. This study investigates the evolutionary history of North-African low latitude marginal populations of Alnus glutinosa Gaertn., a European tree species that plays a significant ecological role as a keystone of riparian ecosystems. We genotyped 551 adults from 19 populations located across North Africa at 12 microsatellite loci and applied a coalescent-based simulation approach to reconstruct the demographic and evolutionary history of these populations. Surprisingly, Moroccan trees were tetraploids demonstrating a strong distinctiveness of these populations within a species otherwise known as diploid. Best-fitting models of demographic reconstruction revealed the relict nature of Moroccan populations that were found to have withstood past climate change events and to be much older than Algerian and Tunisian populations. This study highlights the complex demographic history that can be encountered in rear-edge distribution margins that here consist of both old stable climate relict and more recent populations, distinctively diverse genetically both quantitatively and qualitatively. We emphasize the high evolutionary and conservation value of marginal rear-edge populations of a keystone riparian species in the context of on-going climate change in the Mediterranean region.

Triploid bumblebees indicate a direct cost of inbreeding in fragmented populations

Hymenopteran species with single‐locus complimentary sex‐determination (sl‐CSD) face an additional cost of inbreeding because of a loss of diversity at the sex‐determining locus. Laboratory studies of a range of Hymenoptera have found that a small percentage of diploid males produce viable diploid sperm, and that if these males mate, then the resultant females produce triploid offspring that are sterile. Here, we use microsatellite markers to determine the frequency of triploid individuals of Bombus muscorum and B. jonellus in a model island system. Triploids were found in populations of both species. Observed triploid frequencies of up to 8% were detected, and estimated total frequencies peaked at 20% with respect to normal diploid workers. For both species, triploid frequency was negatively correlated with surrogates of population size, providing direct evidence for inbreeding in small populations. Populations limited to <∼15 km2 of suitable habitat were particularly likely to harbour triploids. Estimated total triploid frequencies were higher in B. muscorum than in B. jonellus, perhaps due to the greater dispersal range of the latter species. Implications for the conservation of rare social hymenopterans are discussed.

De Novo Discovery and Multiplexed Amplification of Microsatellite Markers for Black Alder (Alnus glutinosa) and Related Species Using SSR-Enriched Shotgun Pyrosequencing

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17 855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.

Thirteen microsatellites developed by SSR-enriched pyrosequencing for Solanum rostratum (Solanaceae) and related species

PREMISE OF THE STUDY Microsatellite markers were developed using second-generation sequencing in Solanum rostratum as a tool to study the reproductive biology and genetic structure of this invasive species. METHODS AND RESULTS Thirteen microsatellites were successfully discovered and amplified in a single multiplexed PCR. All loci showed genetic variation in S. rostratum. Cross-amplification in five closely related taxa was successful for a subset of loci. CONCLUSIONS The set of 13 microsatellite markers developed here provides a time-effective and cost-effective genetic tool to study the reproductive biology of S. rostratum. The demonstrated transferability of the PCR multiplex to related taxa also highlights its usefulness for evolutionary studies across Solanum sect. Androceras.