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.

Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophies (CMDs), are heterogeneous autosomal recessive disorders. Their severe manifestations consist of early hypotonia and weakness, markedly delayed motor milestones and contractures, often associated with joint deformities. Histological changes seen in muscle biopsies consist of large variations in muscle fibre size, a few necrotic and regenerating fibres and a marked increase in endomysial collagen tissue. Diagnosis is based on clinical features and on morphological changes. In several CMD cases, we have demonstrated an absence of one of the components of the extracellular matrix around muscle fibres, the merosin M chain, now referred to as the alpha 2 chain of laminin-2 (ref.3). We localized this CMD locus to chromosome 6q2 by homozygosity mapping and linkage analysis. The laminin alpha 2 chain gene (LAMA2) maps to the same region on chromosome 6q22-23 (ref. 5). We therefore investigated LAMA2 for the presence of disease-causing mutations in laminin alpha 2 chain-deficient CMD families and now report splice site and nonsense mutations in two families leading presumably to a truncated laminin alpha 2 protein.

A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease characterized by degeneration of the motor neurons of the spinal cord causing proximal paralysis with muscle atrophy. The region on chromosome 5q13 encompassing the disease gene is particularly unstable and prone to large-scale deletions whose characterization recently led to the identification of the survival motor neuron (SMN) gene. We now present a genetic analysis of 54 unrelated Spanish SMA families that has revealed a 4-basepair (bp) deletion (AGAG) in exon 3 of SMN in four unrelated patients. This deletion, which results in a frameshift and a premature stop codon, occurs on the same haplotype background, suggesting that a single mutational event is involved in the four families. The other patients showed either deletions of the SMN gene (49/54) or a gene conversion event changing SMN exon 7 into its highly homologous copy (cBCD541, 1/54). This observation gives strong support to the view that mutations of the SMN gene are responsible for the SMA phenotype as it is the first frameshift mutation reported in SMA.

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia).

Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. We previously localized the SJS1 locus to chromosome 1p34-p36.1 and found no evidence of genetic heterogeneity. Here we describe mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) in three SJS1 families. In so doing, we have identified the first human mutations in HSPG2, which underscore the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability.

A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family.

A candidate gene for Branchio-Oto-Renal (BOR) syndrome was identified at chromosome 8q13.3 by positional cloning and shown to underlie the disease. This gene is a human homologue of the Drosophila eyes absent gene (eya), and was therefore called EYA1. A highly conserved 271-amino acid C-terminal region was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. The expression pattern of the murine EYA1 orthologue, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. In the developing kidney, the expression pattern is indicative of a role for Eya1 in the metanephric cells surrounding the 'just-divided' ureteric branches.

Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Among the four loci causing AD-HSP identified so far, the SPG4 locus at chromosome 2p2-1p22 has been shown to account for 40-50% of all AD-HSP families. Using a positional cloning strategy based on obtaining sequence of the entire SPG4 interval, we identified a candidate gene encoding a new member of the AAA protein family, which we named spastin. Sequence analysis of this gene in seven SPG4-linked pedigrees revealed several DNA modifications, including missense, nonsense and splice-site mutations. Both SPG4 and its mouse orthologue were shown to be expressed early and ubiquitously in fetal and adult tissues. The sequence homologies and putative subcellular localization of spastin suggest that this ATPase is involved in the assembly or function of nuclear protein complexes.

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.

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.

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.

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.

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.

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.

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.

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.