Pierre-Jean G. Malé

High‐throughput microsatellite isolation through 454 GS‐FLX Titanium pyrosequencing of enriched DNA libraries

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.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross‐tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family.

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.

From museums to genomics: old herbarium specimens shed light on a C3 to C4 transition

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.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2010 - 31 July 2010.

This article documents the addition of 205 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Bagassa guianensis, Bulweria bulwerii, Camelus bactrianus, Chaenogobius annularis, Creontiades dilutus, Diachasmimorpha tryoni, Dioscorea alata, Euhrychiopsis lecontei, Gmelina arborea, Haliotis discus hannai, Hirtella physophora, Melanaphis sacchari, Munida isos, Thaumastocoris peregrinus and Tuberolachnus salignus. These loci were cross‐tested on the following species: Halobaena caerulea, Procellaria aequinoctialis, Oceanodroma monteiroi, Camelus ferus, Creontiades pacificus, Dioscorea rotundata, Dioscorea praehensilis, Dioscorea abyssinica, Dioscorea nummularia, Dioscorea transversa, Dioscorea esculenta, Dioscorea pentaphylla, Dioscorea trifida, Hirtella bicornis, Hirtella glandulosa, Licania alba, Licania canescens, Licania membranaceae, Couepia guianensis and 7 undescribed Thaumastocoris species.

Phylogenomics and taxonomy of Lecomtelleae (Poaceae), an isolated panicoid lineage from Madagascar

BACKGROUND AND AIMS An accurate characterization of biodiversity requires analyses of DNA sequences in addition to classical morphological descriptions. New methods based on high-throughput sequencing may allow investigation of specimens with a large set of genetic markers to infer their evolutionary history. In the grass family, the phylogenetic position of the monotypic genus Lecomtella, a rare bamboo-like endemic from Madagascar, has never been appropriately evaluated. Until now its taxonomic treatment has remained controversial, indicating the need for re-evaluation based on a combination of molecular and morphological data. METHODS The phylogenetic position of Lecomtella in Poaceae was evaluated based on sequences from the nuclear and plastid genomes generated by next-generation sequencing (NGS). In addition, a detailed morphological description of L. madagascariensis was produced, and its distribution and habit were investigated in order to assess its conservation status. KEY RESULTS The complete plastid sequence, a ribosomal DNA unit and fragments of low-copy nuclear genes (phyB and ppc) were obtained. All phylogenetic analyses place Lecomtella as an isolated member of the core panicoids, which last shared a common ancestor with other species >20 million years ago. Although Lecomtella exhibits morphological characters typical of Panicoideae, an unusual combination of traits supports its treatment as a separate group. CONCLUSIONS 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.

Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data.

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.

An ant symbiont directly and indirectly limits its host plant’s reproductive success

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.

Predation success by a plant-ant indirectly favours the growth and fitness of its host myrmecophyte

Mutualisms, or interactions between species that lead to net fitness benefits for each species involved, are stable and ubiquitous in nature mostly due to “byproduct benefits” stemming from the intrinsic traits of one partner that generate an indirect and positive outcome for the other. Here we verify if myrmecotrophy (where plants obtain nutrients from the refuse of their associated ants) can explain the stability of the tripartite association between the myrmecophyte Hirtella physophora, the ant Allomerus decemarticulatus and an Ascomycota fungus. The plant shelters and provides the ants with extrafloral nectar. The ants protect the plant from herbivores and integrate the fungus into the construction of a trap that they use to capture prey; they also provide the fungus and their host plant with nutrients. During a 9-month field study, we over-provisioned experimental ant colonies with insects, enhancing colony fitness (i.e., more winged females were produced). The rate of partial castration of the host plant, previously demonstrated, was not influenced by the experiment. Experimental plants showed higher δ15N values (confirming myrmecotrophy), plus enhanced vegetative growth (e.g., more leaves produced increased the possibility of lodging ants in leaf pouches) and fitness (i.e., more fruits produced and more flowers that matured into fruit). This study highlights the importance of myrmecotrophy on host plant fitness and the stability of ant-myrmecophyte mutualisms.

The karyotypes of Gigantiops destructor (Fabricius) and other ants from French Guiana (Formicidae)

Abstract The aim of this study, which was conducted in French Guiana, was to characterize the karyotypes of nine ant species belonging to the genera Anochetus, Apterostigma, Cyphomyrmex, Camponotus, Gigantiops, Myrmicocrypta, Odontomachus and Pseudomyrmex, and to compare them with published data. We present the first descriptions of the karyotypes of Gigantiops destructor (Fabricius), an endemic Formicinae of the Amazonian region, which is the only living species in the tribe Gigantiopini, and of a species from the poorly-known cryptic genus Myrmicocrypta, which belongs to the Myrmicinae tribe Attini.