Julie Reveillaud

Minimum entropy decomposition: Unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences

Molecular microbial ecology investigations often employ large marker gene datasets, for example, ribosomal RNAs, to represent the occurrence of single-cell genomes in microbial communities. Massively parallel DNA sequencing technologies enable extensive surveys of marker gene libraries that sometimes include nearly identical sequences. Computational approaches that rely on pairwise sequence alignments for similarity assessment and de novo clustering with de facto similarity thresholds to partition high-throughput sequencing datasets constrain fine-scale resolution descriptions of microbial communities. Minimum Entropy Decomposition (MED) provides a computationally efficient means to partition marker gene datasets into ‘MED nodes’, which represent homogeneous operational taxonomic units. By employing Shannon entropy, MED uses only the information-rich nucleotide positions across reads and iteratively partitions large datasets while omitting stochastic variation. When applied to analyses of microbiomes from two deep-sea cryptic sponges Hexadella dedritifera and Hexadella cf. dedritifera, MED resolved a key Gammaproteobacteria cluster into multiple MED nodes that are specific to different sponges, and revealed that these closely related sympatric sponge species maintain distinct microbial communities. MED analysis of a previously published human oral microbiome dataset also revealed that taxa separated by less than 1% sequence variation distributed to distinct niches in the oral cavity. The information theory-guided decomposition process behind the MED algorithm enables sensitive discrimination of closely related organisms in marker gene amplicon datasets without relying on extensive computational heuristics and user supervision.

Host-specificity among abundant and rare taxa in the sponge microbiome.

Microbial communities have a key role in the physiology of the sponge host, and it is therefore essential to understand the stability and specificity of sponge–symbiont associations. Host-specific bacterial associations spanning large geographic distance are widely acknowledged in sponges. However, the full spectrum of specificity remains unclear. In particular, it is not known whether closely related sponges host similar or very different microbiota over wide bathymetric and geographic gradients, and whether specific associations extend to the rare members of the sponge microbiome. Using the ultra-deep Illumina sequencing technology, we conducted a comparison of sponge bacterial communities in seven closely related Hexadella species with a well-resolved host phylogeny, as well as of a distantly related sponge Mycale. These samples spanned unprecedentedly large bathymetric (15–960 m) gradients and varying European locations. In addition, this study included a bacterial community analysis of the local background seawater for both Mycale and the widespread deep-sea taxa Hexadella cf. dedritifera. We observed a striking diversity of microbes associated with the sponges, spanning 47 bacterial phyla. The data did not reveal any Hexadella microbiota co-speciation pattern, but confirmed sponge-specific and species-specific host–bacteria associations, even within extremely low abundant taxa. Oligotyping analysis also revealed differential enrichment preferences of closely related Nitrospira members in closely related sponges species. Overall, these results demonstrate highly diverse, remarkably specific and stable sponge–bacteria associations that extend to members of the rare biosphere at a very fine phylogenetic scale, over significant geographic and bathymetric gradients.

Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy

Background The Astrophorida (Porifera, Demospongiae p) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution. Methodology/Principal Findings With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deep- and shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 5′ end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella). Conclusion The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can be explained by the banality of convergent evolution and secondary loss in spicule evolution. These processes have taken place many times, in all the major clades, for megascleres and microscleres.

Species boundaries and phylogenetic relationships between Atlanto-Mediterranean shallow-water and deep-sea coral associated Hexadella species (Porifera, Ianthellidae)

Coral reefs constitute the most diverse ecosystem of the marine realm and an increasing number of studies are focusing on coral species boundaries, distribution, and on processes that control species ranges. However, less attention has been paid to coral associated species. Deep-sea sponges dominate cold-water coral ecosystems, but virtually nothing is known about their molecular diversity. Moreover, species boundaries based on morphology may sometimes be inadequate, since sponges have few diagnostic characters. In this study, we investigated the molecular diversity within the genus Hexadella (Porifera, Demospongiae, Verongida, Ianthellidae) from the European shallow-water environment to the deep-sea coral ecosystems. Three molecular markers were used: one mitochondrial (COI) and two nuclear gene fragments (28S rDNA and the ATPS intron). Phylogenetic analyses revealed deeply divergent deep-sea clades congruent across the mitochondrial and nuclear markers. One clade contained specimens from the Irish, the Scottish, and the Norwegian margins and the Greenland Sea (Hexadella dedritifera) while another clade contained specimens from the Ionian Sea, the Bay of Biscay, and the Irish margin (H. cf. dedritifera). Moreover, these deeply divergent deep-sea clades showed a wide distribution suggesting a connection between the reefs. The results also point to the existence of a new deep-sea species (Hexadella sp.) in the Mediterranean Sea and of a cryptic shallow-water species (Hexadella cf. pruvoti) in the Gorringe Bank. In contrast, low genetic differentiation between H. cf. dedritifera and H. pruvoti from the Mediterranean Sea was observed. All Hexadella racovitzai specimens from the Mediterranean Sea (shallow and deep) to the Atlantic formed a monophyletic group.

Phylogenetic Relationships among NE Atlantic Plocamionida Topsent (1927) (Porifera, Poecilosclerida): Under-Estimated Diversity in Reef Ecosystems

Background Small and cryptic sponges associated with cold-water coral reefs are particularly numerous and challenging to identify, but their ecological and biochemical importance is likely to compete with megabenthic specimens. Methodology/Principal Findings Here we use a combination of the standard M1M6 and I3M11 partitions of the COI fragment, partial rDNA 28S sequences and morphology to delineate small encrusting Plocamionida species. In total, 46 specimens were retrieved from seven shallow to deep-water coral locations, crossing 3,000 km along the European margins. Our work provides evidence that the Plocamionida ambigua f. tylotata and f. grandichelata can be considered valid species, whereas Plocamionida ambigua f. tornata corresponds to the species P. ambigua. Within the monophyletic group of Plocamionida, P. microcionides is shown as really divergent from the other taxa, and four putative new Plocamionida species are suggested. Conclusions/Significance This study shows that the use of molecular and morphological information in an integrative approach is a powerful tool for the identification of sponge species, and suggests that an under-estimated biodiversity of sponges occurs in cold-water coral reefs.

First sighting of active fluid venting in the Gulf of Cadiz

The Mercator mud volcano, located in the Gulf of Cadiz off the coasts of Spain, Portugal, and Morocco (Figure 1), may provide an accessible field laboratory for studying local active venting and its possible internal and external controls. The recent discovery of the first active deep ocean ‘brown smoker’ chimney in this area can possibly be linked with the disintegration of a gas hydrate layer between the seafloor and a subsurface level that is dependent on pressure and temperature. For more than a decade, the international marine scientific community has deployed considerable efforts in exploring the Gulf of Cadiz. Since the discovery of the Gulfs first mud volcano in 1999, research cruises have steadily unveiled one mud volcano after another [Gardner, 2001; Pinheiro et al., 2003; Somoza et al., 2003]. These mud volcanoes are clustered in several fields on the Portuguese, Spanish, and Moroccan margins (Figure 1). Extensive geophysical evidence of shallow gas and subsurface fluid flow has been reported in the Gulf of Cadiz [Baraza and Ercilla 1996; Pinheiro et al., 2003].

Relevance of an integrative approach for taxonomic revision in sponge taxa: case study of the shallow-water Atlanto-Mediterranean Hexadella species (Porifera : Ianthellidae : Verongida)

Abstract. The identification of sponges that lack a mineral skeleton is always highly challenging, especially for Hexadella species, which are also fibreless. Recently, the yellow species Hexadella pruvoti Topsent was identified as a cryptic species complex while the pink coloured Hexadella racovitzai Topsent showed two highly divergent lineages. We performed a COI phylogenetic reconstruction using 27 new Mediterranean Hexadella samples in order to confirm the presence of divergent lineages within both shallow-water species. Specimens were described with an integrative approach combining morphological and cytological investigations, biochemical profiling and assessment of natural toxicity in order to identify diagnostic characters for each taxon. H. topsenti, sp. nov. is distinguished from H. racovitzai by its colour, its surface network shape, divergent secondary metabolite patterns and toxicity values. H. crypta, sp. nov. differs from H. pruvoti by a different encrusting growth form when alive, and by distinctively colouring the ethanol fixative solution. In addition, H. pruvoti and H. crypta show different types of cells with inclusions as well as distinct metabolic fingerprints. Natural toxicity values, however, do not permit the separation of H. pruvoti and H. crypta. Our work shows that only the use of a combination of complementary tools can provide relevant descriptions for some problematic taxa.