Improving soil bacterial taxa-area relationships assessment using DNA meta-barcoding.

The evaluation of the taxa-area relationship (TAR) with molecular fingerprinting data demonstrated the spatial structuration of soil microorganisms and provided insights into the processes shaping their diversity. The increasing use of massive sequencing technologies in biodiversity investigations has now raised the question of the advantages of such technologies over the fingerprinting approach for elucidation of the determinism of soil microbial community assembly in broad-scale biogeographic studies. Our objectives in this study were to compare DNA fingerprinting and meta-barcoding approaches for evaluating soil bacterial TAR and the determinism of soil bacterial community assembly on a broad scale. This comparison was performed on 392 soil samples from four French geographic regions with different levels of environmental heterogeneity. Both molecular approaches demonstrated a TAR with a significant slope but, because of its more sensitive description of soil bacterial community richness, meta-barcoding provided significantly higher and more accurate estimates of turnover rates. Both approaches were useful in evidencing the processes shaping bacterial diversity variations on a broad scale. When different taxonomic resolutions were considered for meta-barcoding data, they significantly influenced the estimation of turnover rates but not the relative importance of each component process. Altogether, DNA meta-barcoding provides a more accurate evaluation of the TAR and may lead to re-examination of the processes shaping soil bacterial community assembly. This should provide new insights into soil microbial ecology in the context of sustainable use of soil resources.

New nuclear markers and exploration of the relationships among Serraniformes (Acanthomorpha, Teleostei): the importance of working at multiple scales.

We explore the relationships within Serraniformes (Li et al., 2009) using a dense taxon sampling and seven nuclear markers. Six had already used been for teleost phylogeny (IRBP, MC1R, MLL4, Pkd1, Rhodopsin, and RNF213) at other scales, and one (MLL2) is new. The results corroborate the composition of Serraniformes described in previous publications (some Gasterosteiformes, Perciformes and Scorpaeniformes). Within the clade, Notothenioidei and Zoarcoidei are each monophyletic. Cottoidei was not monophyletic due to placement of the genus Ebinania (Psychrolutidae). Our independent data confirm the sister-group relationship of Percophidae and Notothenioidei as well as the division of Platycephaloidei in four different groups (Bembridae, Platycephalidae, Hoplichthyidae and Peristediidae with Triglidae). Within Cottoidei, Liparidae and Cyclopteridae formed a clade associated with Cottidae, the genus Cottunculus (Psychrolutidae), and Agonidae. Serranidae and Scorpaenidae are not monophyletic, with the Serranidae divided in two clades (Serraninae and Epinephelinae/Anthiinae) and Scorpaenidae including Caracanthidae and the genus Ebinania (Psychrolutidae). We discuss some morphological characters supporting clades within the Scorpaenidae.

A study of a population of Nyssomyia trapidoi (Diptera: Psychodidae) caught on the Pacific coast of Ecuador.

BACKGROUND: Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species. METHODS: Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed. RESULTS: Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA. CONCLUSION: Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.

Phylogenetic footprints of an Antarctic radiation: the Trematominae (Notothenioidei, Teleostei).

The teleost suborder Notothenioidei is restricted to the Southern Ocean and has been described as a species flock spanning the whole of it. Within the suborder, the subfamily Trematominae is important for coastal Antarctic ecosystems. The eleven Trematomus species occupy a large range of ecological niches. The genus is monophyletic if the genus Pagothenia (two additional species) and Cryothenia amphitreta, also nested within it, are included. Although the Trematominae have received much interest, the relationships among these fourteen species are still unclear. Several recent studies have tried to resolve these interrelationships; however no complete and clear picture has emerged, probably because of the use of a low number of insufficiently variable markers. The only common results places T. scotti as the sister-group of the rest of the subfamily and T. loennbergi close to T. lepidorhinus. We use here more variable markers. Four nuclear markers, two of which are new, and a mitochondrial marker for the biggest trematomine sampling ever gathered (14 species, 78 specimens). We found that several nuclear haplotypes are shared by several species (mostly in very closely related species). The haplotype patterns coupled with the cytogenetics of the subfamily suggest that a phenomenon of incomplete lineage sorting (ILS) is likely to be at play. Using a calibration linked to fossil evidence, we evaluate the relative ages of each clade within the Trematominae to assess the proximity of the speciation events to one another. The main trematomine diversification was recent and sudden.

DNA from soil mirrors plant taxonomic and growth form diversity.

Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches.

Large-scale species delimitation method for hyperdiverse groups.

Accelerating the description of biodiversity is a major challenge as extinction rates increase. Integrative taxonomy combining molecular, morphological, ecological and geographical data is seen as the best route to reliably identify species. Classic molluscan taxonomic methodology proposes primary species hypotheses (PSHs) based on shell morphology. However, in hyperdiverse groups, such as the molluscan family Turridae, where most of the species remain unknown and for which homoplasy and plasticity of morphological characters is common, shell-based PSHs can be arduous. A four-pronged approach was employed to generate robust species hypotheses of a 1000 specimen South-West Pacific Turridae data set in which: (i) analysis of COI DNA Barcode gene is coupled with (ii) species delimitation tools GMYC (General Mixed Yule Coalescence Method) and ABGD (Automatic Barcode Gap Discovery) to propose PSHs that are then (iii) visualized using Klee diagrams and (iv) evaluated with additional evidence, such as nuclear gene rRNA 28S, morphological characters, geographical and bathymetrical distribution to determine conclusive secondary species hypotheses (SSHs). The integrative taxonomy approach applied identified 87 Turridae species, more than doubling the amount previously known in the Gemmula genus. In contrast to a predominantly shell-based morphological approach, which over the last 30 years proposed only 13 new species names for the Turridae genus Gemmula, the integrative approach described here identified 27 novel species hypotheses not linked to available species names in the literature. The formalized strategy applied here outlines an effective and reproducible protocol for large-scale species delimitation of hyperdiverse groups.

Comprehensive sampling reveals circumpolarity and sympatry in seven mitochondrial lineages of the Southern Ocean crinoid species Promachocrinus kerguelensis (Echinodermata).

Sampling at appropriate spatial scales in the Southern Ocean is logistically challenging and may influence estimates of diversity by missing intermediate representatives. With the assistance of sampling efforts especially influenced by the International Polar Year 2007-2008, we gathered nearly 1500 specimens of the crinoid species Promachocrinus kerguelensis from around Antarctica. We used phylogeographic and phylogenetic tools to assess its genetic diversity, demographic history and evolutionary relationships. Six phylogroups (A-F) identified in an earlier study are corroborated here, with the addition of one new phylogroup (E2). All phylogroups are circumpolar, sympatric and eurybathic. The phylogeny of Promachocrinus phylogroups reveals two principal clades that may represent two different cryptic species with contrasting demographic histories. Genetic diversity indices vary dramatically within phylogroups, and within populations, suggesting multiple glacial refugia in the Southern Ocean: on the Kerguelen Plateau, in the East Weddell Sea and the South Shetland Islands (Atlantic sector), and on the East Antarctic continental shelf in the Dumont d'Urville Sea and Ross Sea. The inferences of gene flow vary among the phylogroups, showing discordant spatial patterns. Phylogroup A is the only one found in the Sub-Antarctic region, although without evident connectivity between Bouvet and Kerguelen populations. The Scotia Arc region shows high levels of connectivity between populations in most of the phylogroups, and barriers to gene flow are evident in East Antarctica.

Macroevolution of venom apparatus innovations in auger snails (Gastropoda; Conoidea; Terebridae).

The Terebridae are a diverse family of tropical and subtropical marine gastropods that use a complex and modular venom apparatus to produce toxins that capture polychaete and enteropneust preys. The complexity of the terebrid venom apparatus suggests that venom apparatus development in the Terebridae could be linked to the diversification of the group and can be analyzed within a molecular phylogenetic scaffold to better understand terebrid evolution. Presented here is a molecular phylogeny of 89 terebrid species belonging to 12 of the 15 currently accepted genera, based on Bayesian inference and Maximum Likelihood analyses of amplicons of 3 mitochondrial (COI, 16S and 12S) and one nuclear (28S) genes. The evolution of the anatomy of the terebrid venom apparatus was assessed by mapping traits of six related characters: proboscis, venom gland, odontophore, accessory proboscis structure, radula, and salivary glands. A novel result concerning terebrid phylogeny was the discovery of a previously unrecognized lineage, which includes species of Euterebra and Duplicaria. The non-monophyly of most terebrid genera analyzed indicates that the current genus-level classification of the group is plagued with homoplasy and requires further taxonomic investigations. Foregut anatomy in the family Terebridae reveals an inordinate diversity of features that covers the range of variability within the entire superfamily Conoidea, and that hypodermic radulae have likely evolved independently on at least three occasions. These findings illustrate that terebrid venom apparatus evolution is not perfunctory, and involves independent and numerous changes of central features in the foregut anatomy. The multiple emergence of hypodermic marginal radular teeth in terebrids are presumably associated with variable functionalities, suggesting that terebrids have adapted to dietary changes that may have resulted from predator-prey relationships. The anatomical and phylogenetic results presented serve as a starting point to advance investigations about the role of predator-prey interactions in the diversification of the Terebridae and the impact on their peptide toxins, which are promising bioactive compounds for biomedical research and therapeutic drug development.

Morphometric and molecular characterization of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) from the Ecuadorian Amazon Basin with description of a new species.

Several populations of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) were collected from the Yasuní National Park in the Ecuadorian Amazon region. The specimens comprised the species Psychodopygus geniculatus, Psychodopygus luisleoni and Psychodopygus corossoniensis. Within Ps. geniculatus, we observed two populations, one with a narrow paramere and relatively short genital filaments and the other characterized by a wider coxite and longer genital filaments. A multiple approach combining morphology, morphometry and DNA sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and the mitochondrial cytochrome b gene was carried out. Morphological, morphometric and molecular data strongly suggested the presence of two populations within Ps. geniculatus. The lack of intermediate forms within these populations supported the proposal of two sympatric species. This report describes Psychodopygus francoisleponti n. sp.

Cantharellus texensis sp. nov. from Texas, a southern lookalike of C. cinnabarinus revealed by tef-1 sequence data.

This contribution to the genus Cantharellus in North America deals with the smaller, reddish pink species from the Gulf of Mexico states and eastern United States. C. texensis sp. nov. is presented as a new southern lookalike of C. cinnabarinus. The morphological species concepts are supported by newly generated molecular sequence data from the protein coding gene tef1. Similarities to C. persicinus, a third pinkish taxon, are discussed. The very different microscopic features for the three taxa are illustrated in detail. C. cinnabarinus is neotypified. C. minor forma intensissima is considered to be possibly unrelated not only to the discussed taxa in this paper but also to typical C. minor.

CpDNA-based species identification and phylogeography: application to African tropical tree species.

Despite the importance of the African tropical rainforests as a hotspot of biodiversity, their history and the processes that have structured their biodiversity are understood poorly. With respect to past demographic processes, new insights can be gained through characterizing the distribution of genetic diversity. However, few studies of this type have been conducted in Central Africa, where the identification of species in the field can be difficult. We examine here the distribution of chloroplast DNA (cpDNA) diversity in Lower Guinea in two tree species that are difficult to distinguish, Erythrophleum ivorense and Erythrophleum suaveolens (Fabaceae). By using a blind-sampling approach and comparing molecular and morphological markers, we first identified retrospectively all sampled individuals and determined the limits of the distribution of each species. We then performed a phylogeographic study using the same genetic data set. The two species displayed essentially parapatric distributions that were correlated well with the rainfall gradient, which indicated different ecological requirements. In addition, a phylogeographic structure was found for E. suaveolens and, for both species, substantially higher levels of diversity and allelic endemism were observed in the south (Gabon) than in the north (Cameroon) of the Lower Guinea region. This finding indicated different histories of population demographics for the two species, which might reflect different responses to Quaternary climate changes. We suggest that a recent period of forest perturbation, which might have been caused by humans, favoured the spread of these two species and that their poor recruitment at present results from natural succession in their forest formations.

A molecular phylogenetic framework for the Muricidae, a diverse family of carnivorous gastropods.

With over 1600 extant described species, the Muricidae are one of the most species-rich and morphologically diverse families of molluscs. As predators of molluscs, polychaetes, anthozoans barnacles and other invertebrates, they form an important component of many benthic communities. Traditionally, the classification of muricids at specific and generic levels has been based primarily on shells, while subfamilies have been defined largely by radular morphology, although the composition and relationships of suprageneric groups have never been studied exhaustively. Here we present the phylogenetic relationships of 77 muricid species belonging to nine of the ten currently recognized subfamilies, based on Bayesian inference and Maximum Likelihood analyses of partial sequences of three mitochondrial (12S, 16S and COI) and one nuclear (28S) genes. The resulting topologies are discussed with respect to traditional subfamilial arrangements, and previous anatomical and molecular findings. We confirm monophyly of each of the subfamilies Ergalataxinae, Rapaninae, Coralliophilinae, Haustrinae, Ocenebrinae and Typhinae as previously defined, but earlier concepts of Muricinae, Trophoninae and Muricopsinae are shown to be polyphyletic. Based on our phylogenetic hypothesis, a new arrangement of these subfamilies is proposed.

Finding candidate genes under positive selection in Non-model species: examples of genes involved in host specialization in pathogens.

Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host-pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17,000-32,000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty-two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non-model organisms for which candidate genes for diversifying selection are needed.

Low genetic diversity in the masked palm civet Paguma larvata (Viverridae).

The masked palm civet is distributed through south-east Asia, China and the Himalayas. Because of its potential role in the severe acute respiratory syndrome (SARS) epidemic, it has become important to gather information on this species, and notably to provide a tool to determine the origin of farm and market animals. For this purpose, we studied the genetic variability and the phylogeographic pattern of the masked palm civet Paguma larvata. First, two portions of mitochondrial genes, cytochrome b and the control region, were sequenced for a total of 76 individuals sampled from China, the Indochinese region and the Sundaic region. Results indicated a low genetic variability and suggested a lack of a phylogeographic structure in this species, which do not allow inferring the geographic origin of samples of unknown origin, although it is possible to distinguish individuals from China and the Sundaic region. This low variation is in contrast to the well-marked morphological differentiation between the populations in the Sundaic and Chinese-Indochinese regions. We also used five microsatellite loci to genotype 149 samples from two wild and four farmed populations in China, where the masked palm civet is farmed and where the SARS coronavirus was isolated. These analyses also showed a reduced variability in Chinese civets and showed that farmed populations did not exhibit a lower genetic diversity than wild populations, suggesting frequent introductions of wild individuals into farms.

Molecular phylogeny of the Dictyotales and their position within the Phaeophyceae, based on nuclear, plastid and mitochondrial DNA sequence data.

For the first time, on the basis of nuclear, plastid and mitochondrial sequence data, the most comprehensive molecular phylogeny of the Dictyotales to date is presented, in a broad context where all brown algal orders are included (except Discosporangiales, Ascoseirales and Nemodermatales). A veto supertree approach was used here to evaluate congruency and conflicts between genes: phylogenetic signal was congruent and mainly carried by chloroplastic information. Supermatrix analyses (BI, ML and MP) revealed that Dictyotales is sister to Onslowiales, this ensemble being sister of a clade also encompassing Sphacelariales and Syringodermatales. The family Scoresbyellaceae is merged into the family Dictyotaceae. Furthermore, the current subdivision of the Dictyotaceae into two tribes was not supported. The enigmatic genus Stoechospermum was shown to belong to the same clade as Dictyota, Rugulopteryx, Scoresbyella and Canistrocarpus. Homoeostrichus and Dictyopteris did not appear monophyletic. Zonaria stipitata clustered with the Spatoglossum species; since this is consistent with its morphological features, the new combination Spatoglossum stipitatum is proposed accordingly.

Starting to unravel the toxoglossan knot: molecular phylogeny of the "turrids" (Neogastropoda: Conoidea).

The superfamily Conoidea is one of the most speciose groups of marine mollusks, with estimates of about 340 recent valid genera and subgenera, and 4000 named living species. Previous classifications were based on shell and anatomical characters, and clades and phylogenetic relationships are far from well assessed. Based on a dataset of ca. 100 terminal taxa belonging to 57 genera, information provided by fragments of one mitochondrial (COI) and three nuclear (28S, 18S and H3) genes is used to infer the first molecular phylogeny of this group. Analyses are performed on each gene independently as well as for a data matrix where all genes are concatenated, using Maximum Likelihood, Maximum Parsimony and Bayesian approaches. Several well-supported clades are defined and are only partly identifiable to currently recognized families and subfamilies. The nested sampling used in our study allows a discussion of the classification at various taxonomical levels, and several genera, subfamilies and families are found polyphyletic.

Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development.

BACKGROUND: The acronym CHARGE refers to a non-random cluster of malformations including coloboma, heart malformation, choanal atresia, retardation of growth and/or development, genital anomalies, and ear anomalies. This set of multiple congenital anomalies is frequent, despite rare patients with normal intelligence, and prognosis remains poor. Recently, CHD7 gene mutations have been identified in CHARGE patients; however, the function of CHD7 during development remains unknown. METHODS: We studied a series of 10 antenatal cases in whom the diagnosis of CHARGE syndrome was suspected, considering that a careful pathological description would shed light on the CHD7 function during development. CHD7 sequence analysis and in situ hybridisation were employed. RESULTS: The diagnosis of CHARGE syndrome was confirmed in all 10 fetuses by the identification of a CHD7 heterozygous truncating mutation. Interestingly, arhinencephaly and semi-circular canal agenesis were two constant features which are not included in formal diagnostic criteria so far. In situ hybridisation analysis of the CHD7 gene during early human development emphasised the role of CHD7 in the development of the central nervous system, internal ear, and neural crest of pharyngeal arches, and more generally showed a good correlation between specific CHD7 expression pattern and the developmental anomalies observed in CHARGE syndrome. CONCLUSIONS: These results allowed us to further refine the phenotypic spectrum of developmental anomalies resulting from CHD7 dysfunction.