Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

Whole genome sequencing is helping generate robust phylogenetic hypotheses for a range of taxonomic groups that were previously recalcitrant to classical molecular phylogenetic approaches. As a case study, we performed a shallow shotgun sequencing of eight species in the tropical tree family Chrysobalanaceae to retrieve large fragments of high-copy number DNA regions and test the potential of these regions for phylogeny reconstruction. We were able to assemble the nuclear ribosomal cluster (nrDNA), the complete plastid genome (ptDNA) and a large fraction of the mitochondrial genome (mtDNA) with approximately 1000×, 450× and 120× sequencing depth respectively. The phylogenetic tree obtained with ptDNA resolved five of the seven internal nodes. In contrast, the tree obtained with mtDNA and nrDNA data were largely unresolved. This study demonstrates that genome skimming is a cost-effective approach and shows potential in plant molecular systematics within Chrysobalanaceae and other under-studied groups.

Collections of specimens held by natural history museums are invaluable material for biodiversity inventory and evolutionary studies, with specimens accumulated over 300 years readily available for sampling. Unfortunately, most museum specimens yield low-quality DNA. Recent advances in sequencing technologies, so called next-generation sequencing, are revolutionizing phylogenetic investigations at a deep level. Here, the Illumina technology (HiSeq) was used on herbarium specimens of Sartidia (subfamily Aristidoideae, Poaceae), a small African-Malagasy grass lineage (six species) characteristic of wooded savannas, which is the C3 sister group of Stipagrostis, an important C4 genus from Africa and SW Asia. Complete chloroplast and nuclear ribosomal sequences were assembled for two Sartidia species, one of which (S. perrieri) is only known from a single specimen collected in Madagascar 100 years ago. Partial sequences of a few single-copy genes encoding phosphoenolpyruvate carboxylases (ppc) and malic enzymes (nadpme) were also assembled. Based on these data, the phylogenetic position of Malagasy Sartidia in the subfamily Aristidoideae was investigated and the biogeographical history of this genus was analysed with full species sampling. The evolutionary history of two genes for C4 photosynthesis (ppc-aL1b and nadpme-IV) in the group was also investigated. The gene encoding the C4 phosphoenolpyruvate caroxylase of Stipagrostis is absent from S. dewinteri suggesting that it is not essential in C3 members of the group, which might have favoured its recruitment into a new metabolic pathway. Altogether, the inclusion of historical museum specimens in phylogenomic analyses of biodiversity opens new avenues for evolutionary studies.

Ant -fungus associations are well known from attine ants, whose nutrition is based on a symbiosis with basidiomycete fungi. Otherwise, only a few non-nutritional ant -fungus associations have been recorded to date. Here we focus on one of these associations involving Allomerus plant-ants that build galleried structures on their myrmecophytic hosts in order to ambush prey. We show that this association is not opportunistic because the ants select from a monophyletic group of closely related fungal haplotypes of an ascomycete species from the order Chaetothyriales that consistently grows on and has been isolated from the galleries. Both the ants' behaviour and an analysis of the genetic population structure of the ants and the fungus argue for host specificity in this interaction. The ants' behaviour reveals a major investment in manipulating, growing and cleaning the fungus. A molecular analysis of the fungus demonstrates the widespread occurrence of one haplotype and many other haplotypes with a lower occurrence, as well as significant variation in the presence of these fungal haplotypes between areas and ant species. Altogether, these results suggest that such an interaction might represent an as-yet undescribed type of specific association between ants and fungus in which the ants cultivate fungal mycelia to strengthen their hunting galleries.

Understanding the mechanisms responsible for phenotypic diversification within and among species ultimately rests with linking naturally occurring mutations to functionally and ecologically significant traits. Colour polymorphisms are of great interest in this context because discrete colour patterns within a population are often controlled by just a few genes in a common environment. We investigated how and why phenotypic diversity arose and persists in the Zosterops borbonicus white-eye of Reunion (Mascarene archipelago), a colour polymorphic songbird in which all highland populations contain individuals belonging to either a brown or a grey plumage morph. Using extensive phenotypic and genomic data, we demonstrate that this melanin-based colour polymorphism is controlled by a single locus on chromosome 1 with two large-effect alleles, which was not previously described as affecting hair or feather colour. Differences between colour morphs appear to rely upon complex cis-regulatory variation that either prevents the synthesis of pheomelanin in grey feathers, or increases its production in brown ones. We used coalescent analyses to show that, from a ‘brown’ ancestral population, the dominant ‘grey’ allele spread quickly once it arose from a new mutation. Since colour morphs are always found in mixture, this implies that the selected allele does not go to fixation, but instead reaches an intermediate frequency, as would be expected under balancing selection.

The study showed that NGS can be used to generate abundant phylogenetic information rapidly, opening new avenues for grass phylogenetics. These data clearly showed that Lecomtella forms an isolated lineage, which, in combination with its morphological peculiarities, justifies its treatment as a separate tribe: Lecomtelleae. New descriptions of the tribe, genus and species are presented with a typification, a distribution map and an IUCN conservation assessment.

In theory, mutualisms are intrinsically unstable, and the search for the maximum profit at the minimum cost should lead every mutualist to become a parasite. From an empirical point of view, mutualisms are ubiquitous and of major importance to ecosystems, suggesting the existence of mechanisms that enhance the maintenance of such relationships. We focused on the obligatory myrmecophytic association between the Neotropical plant Hirtella physophora (Chrysobalanaceae) and the ant Allomerus decemarticulatus (Myrmicinae). The plant shelters the ants in leaf pouches in exchange for protection from phytophagous insects. We experimentally demonstrated that the ants partially castrate their host plant by destroying almost two-thirds of its floral buds. The ants also impede pollination through their presence and interactions with pollinators. These results reveal that ant activity negatively affects the plant's reproduction both directly and indirectly. This dual negative effect does not result in the complete castration of the plant. We also highlight major limitations to plant reproduction due to the spontaneous abscission of flowers and to the limited quantity and/or poor quality of the pollen. These limitations must not be overlooked since they can alter the outcome of the association of H. physophora with its ant partner. We therefore conclude that the evolutionary fate of the relationship depends on both ant castration intensity and obstacles to plant fertilization not related to the presence of ants.

Although associations between myrmecophytes and their plant ants are recognized as a particularly effective form of protective mutualism, their functioning remains incompletely understood. This field study examined the ant-plant Hirtella physophora and its obligate ant associate Allomerus decemarticulatus. We formulated two hypotheses on the highly specific nature of this association: (1) Ant presence should be correlated with a marked reduction in the amount of herbivory on the plant foliage; (2) ant activity should be consistent with the "optimal defense" theory predicting that the most vulnerable and valuable parts of the plant are the best defended. We validated the first hypothesis by demonstrating that for ant-excluded plants, expanding leaves, but also newly matured ones in the long term, suffered significantly more herbivore damage than ant-inhabited plants. We showed that A. decemarticulatus workers represent both constitutive and inducible defenses for their host, by patrolling its foliage and rapidly recruiting nestmates to foliar wounds. On examining how these activities change according to the leaves' developmental stage, we found that the number of patrolling ants dramatically decreased as the leaves matured, while leaf wounds induced ant recruitment regardless of the leaf's age. The resulting level of these indirect defenses was roughly proportional to leaf vulnerability and value during its development, thus validating our second hypothesis predicting optimal protection. This led us to discuss the factors influencing ant activity on the plant's surface. Our study emphasizes the importance of studying both the constitutive and inducible components of indirect defense when evaluating its efficacy and optimality.