Publications

Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.

A new species of hymenolepidid cestodes from Sephanoides sephaniodes (Trochilidae) found in Chile is described. The most characteristic features of Colibrilepis pusilla gen. nov., sp. nov. are the lack of rostellum, a cirrus sac with a thick-walled distal end (separated by a constriction) and protruding into genital atrium, a thick-walled saccular uterus filling entire median field of the gravid proglottis and the small number of eggs containing thick walled embryophores with polar swellings. Staphylepis is the most similar genus but differs in its apical structure because of the presence of a rudimentary rostellum. Moreover, molecular phylogenetic analyses show that Staphylepis and Colibrilepis are not sister taxa.

Metagenetics represents an efficient and rapid tool to describe environmental diversity patterns of microbial eukaryotes based on ribosomal DNA sequences. However, the results of metagenetic studies are often biased by the presence of extracellular DNA molecules that are persistent in the environment, especially in deep-sea sediment. As an alternative, short-lived RNA molecules constitute a good proxy for the detection of active species. Here, we used a metatranscriptomic approach based on RNA-derived (cDNA) sequences to study the diversity of the deep-sea benthic foraminifera and compared it to the metagenetic approach. We analyzed 257 ribosomal DNA and cDNA sequences obtained from seven sediments samples collected in the Sea of Japan at depths ranging from 486 to 3665 m. The DNA and RNA-based approaches gave a similar view of the taxonomic composition of foraminiferal assemblage, but differed in some important points. First, the cDNA dataset was dominated by sequences of rotaliids and robertiniids, suggesting that these calcareous species, some of which have been observed in Rose Bengal stained samples, are the most active component of foraminiferal community. Second, the richness of monothalamous (single-chambered) foraminifera was particularly high in DNA extracts from the deepest samples, confirming that this group of foraminifera is abundant but not necessarily very active in the deep-sea sediments. Finally, the high divergence of undetermined sequences in cDNA dataset indicate the limits of our database and lack of knowledge about some active but possibly rare species. Our study demonstrates the capability of the metatranscriptomic approach to detect active foraminiferal species and prompt its use in future high-throughput sequencing-based environmental surveys.

The vomeronasal organ (VNO) is an olfactory structure that detects pheromones and environmental cues. It consists of sensory neurons that express evolutionary unrelated groups of transmembrane chemoreceptors. The predominant V1R and V2R receptor repertoires are believed to detect airborne and water-soluble molecules, respectively. It has been suggested that the shift in habitat of early tetrapods from water to land is reflected by an increase in the ratio of V1R/V2R genes. Snakes, which have a very large VNO associated with a sophisticated tongue delivery system, are missing from this analysis. Here, we use RNA-seq and RNA in situ hybridization to study the diversity, evolution, and expression pattern of the corn snake vomeronasal receptor repertoires. Our analyses indicate that snakes and lizards retain an extremely limited number of V1R genes but exhibit a large number of V2R genes, including multiple lineages of reptile-specific and snake-specific expansions. We finally show that the peculiar bigenic pattern of V2R vomeronasal receptor gene transcription observed in mammals is conserved in squamate reptiles, hinting at an important but unknown functional role played by this expression strategy. Our results do not support the hypothesis that the shift to a vomeronasal receptor repertoire dominated by V1Rs in mammals reflects the evolutionary transition of early tetrapods from water to land. This study sheds light on the evolutionary dynamics of the vomeronasal receptor families in vertebrates and reveals how mammals and squamates differentially adapted the same ancestral vomeronasal repertoire to succeed in a terrestrial environment.

The European genus Ophrys (Orchidaceae) is famous for its insect-like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives.

Cucolepis gen. n. is erected as monotypic for Cucolepis cincta sp. n., a new species of cyclophyllidean cestode of the family Paruterinidae. The new species is described from the squirrel cuckoo, Piaya cayana Lesson (Aves: Cuculiformes), taken from two localities in Paraguay in 1984 and 1985. This new genus is most similar to the genus Triaenorhina Spasskii et Shumilo, 1965 in terms of the hook morphology and large epiphyseal structures extending from both the handle and guard, but differs in several aspects of the strobilar morphology, such as the shape of the cirrus sac, genital atrium, uterus and paruterine organ. The strobilar morphology of the new genus strongly resembles that of the genus Francobona Georgiev et Kornyushin, 1994, especially the shape of the cirrus sac and genital atrium, yet Francobona spp. lack, the developed epiphyseal structures observed in species of Cucolepis and Triaenorhina. Previous records and the nature of parasite-host associations between cuculiform birds and their cestode parasites are discussed.

The osteology of Thaiichthys buddhabutrensis, nov. gen., from the Late Jurassic – Early Cretaceous of Thailand is described on the basis of a collection of well-preserved specimens. The mode of preservation of the material allows describing the external anatomy, as well as some elements of the internal anatomy (braincase, elements of the vertebral column). Most of the cranial and postcranial skeleton shows a rather conservative anatomy for ‘semionotiformes’, but the jaw apparatus displays specializations. Variations observed in the ossification pattern of the skull roof and of the cheek, in the morphology of the median dorsal scales and in fin rays’ count indicate that caution should be applied when these characters are used in diagnoses and in phylogenetic analyses. A phylogenetic analysis including a set of gars, of ‘semionotiformes’, of Macrosemiiformes and of Halecomorphi shows the following features: (1) the monophyly of Holostei; (2) sister-pair relationships between Tlayuamichin/Semiolepis, Isanichthys/’Lepidotes’ latifrons and Araripelepidotes/Pliodetes; (3) the latter pair, together with Thaiichthys and possibly ‘Lepidotes’ mantelli, are resolved as stem Lepisosteiformes; and (4) the ‘semionotiformes’ (a group gathering species of Semionotus and Lepidotes) do not form a clade.

Rhizaria is one of the six supergroups of eukaryotes, which comprise the majority of amoeboid and skeleton-building protists living in freshwater and marine ecosystems. There is an overall lack of molecular data for the group and therefore the deep phylogeny of rhizarians is unresolved. Molecular data are particularly scarce for the clade of Retaria, which include two prominent groups of microfossils: foraminiferans and radiolarians. To fill this gap, we have produced and sequenced EST libraries for 14 rhizarian species including seven foraminiferans, Gromia and six taxa belonging to traditional Haeckel's Radiolaria: Acantharea, Polycystinea, and Phaeodarea. A matrix was constructed for phylogenetic analysis based on 109 genes and a total of 56 species, of which 22 are rhizarians. Our analyses provide the first multigene evidence for branching of Phaeodarea within Cercozoa, confirming the polyphyly of Haeckel's Radiolaria. It confirms the monophyly of Retaria, a clade grouping Foraminifera with other lineages of Radiolaria. However, contrary to what could be expected from morphological observations, Foraminifera do not form a sister group to radiolarians, but branch within them as sister to either Acantharea or Polycystinea depending on the multigene data set. While the monophyly of Foraminifera and Acantharea is well supported, that of Polycystinea, represented in our data by Spumellaria and Collodaria is questionable. In view of our study, Haeckel's Radiolaria appears as both, a polyphyletic and paraphyletic assemblage of independent groups that should be considered as separate lineages in protist classification.

We present here the results of the Analysis of HLA Population Data (AHPD) project of the 16th International HLA and Immunogenetics Workshop (16IHIW) held in Liverpool in May-June 2012. Thanks to the collaboration of 25 laboratories from 18 different countries, HLA genotypic data for 59 new population samples (either well-defined populations or donor registry samples) were gathered and 55 were analysed statistically following HLA-NET recommendations. The new data included, among others, large sets of well-defined populations from north-east Europe and West Asia, as well as many donor registry data from European countries. The Gene[rate] computer tools were combined to create a Gene[rate] computer pipeline to automatically (i) estimate allele frequencies by an expectation-maximization algorithm accommodating ambiguities, (ii) estimate heterozygosity, (iii) test for Hardy-Weinberg equilibrium (HWE), (iv) test for selective neutrality, (v) generate frequency graphs and summary statistics for each sample at each locus and (vi) plot multidimensional scaling (MDS) analyses comparing the new samples with previous IHIW data. Intrapopulation analyses show that HWE is rarely rejected, while neutrality tests often indicate a significant excess of heterozygotes compared with neutral expectations. The comparison of the 16IHIW AHPD data with data collected during previous workshops (12th-15th) shows that geography is an excellent predictor of HLA genetic differentiations for HLA-A, -B and -DRB1 loci but not for HLA-DQ, whose patterns are probably more influenced by natural selection. In Europe, HLA genetic variation clearly follows a north to south-east axis despite a low level of differentiation between European, North African and West Asian populations. Pacific populations are genetically close to Austronesian-speaking South-East Asian and Taiwanese populations, in agreement with current theories on the peopling of Oceania. Thanks to this project, HLA genetic variation is more clearly defined worldwide and better interpreted in relation to human peopling history and HLA molecular evolution.

The genetic diversity of Europeans has been shaped by various evolutionary forces including their demographic history. Genetic data can thus be used to draw inferences on the population history of Europe using appropriate statistical methods such as computer simulation, which constitutes a powerful tool to study complex models.