Taxonomic and Functional Microbial Signatures of the Endemic Marine Sponge Arenosclera brasiliensis

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (similar to 150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.

Novel organization of the mitochondrial genome in the deep-sea coral, Madrepora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications

Madrepora is one of the most ecologically important genera of reef-building scleractinians in the deep sea, occurring from tropical to high-latitude regions. Despite this, the taxonomic affinities and relationships within the genus Madrepora remain unclear. To clarify these issues, we sequenced the mitochondrial (mt) genome of the most widespread Madrepora species, M. oculata, and compared this with data for other scleractinians. The architecture of the M. oculara mt genome was very similar to that of other scleractinians, except for a novel gene rearrangement affecting only cox2 and cox3. This pattern of gene organization was common to four geographically distinct M. oculata individuals as well as the congeneric species M. minutiseptum, but was not shared by other genera that are closely related on the basis of cox1 sequence analysis nor other oculinids, suggesting that it might be unique to Madrepora. (C) 2012 Elsevier Inc. All rights reserved.

Taxonomic sufficiency of polychaete taxocenes for estuary monitoring

The polychaetes assemblage structure was used in order to investigate taxonomic sufficiency in a heavily polluted tropical bay. Species abundance was aggregated into progressively higher taxa matrices (genus, family, order) and was analyzed using univariate and multivariate techniques. Polychaetes distribution in Guanabara Bay (GB) was in accordance with a pollution gradient, probably ruled by the organic enrichment, consequent effects of hypoxia and altered redox conditions coupled with prevailing patterns of circulation. Within the sectors of GB, an increasing gradient in species richness and occurrence was observed, ranging from the azoic and impoverished stations in the inner sector to a well-structured community in terms of species composition and abundance inhabiting the outer sector. Multivariate statistical analysis showed similar results when species were aggregated into genera and families, while greater difference occurred at coarser taxonomic identification (order). The literature about taxonomic sufficiency has demonstrated that faunal patterns at different taxonomic levels tend to become similar with increased pollution. In GB, an analysis carried out solely at family level is perfectly adequate to describe the environmental gradient, considered a useful tool for a quick environmental assessment. (C) 2011 Elsevier Ltd. All rights reserved.

Phylogenetic analysis of Codium species from Brazil, with the description of the new species C. pernambucensis (Bryopsidales, Chlorophyta)

The genus Codium comprises c. 125 species widely distributed in marine coastal environments throughout the world. Due to morphological plasticity, the taxonomic delimitation of Codium species can be difficult. Sequences of the first exon of the large subunit of RUBISCO (rbcL) have been used in the molecular delimitation of species and for phylogenetic purposes. In the present study, we complement previous morphological work on Brazilian Codium species with molecular systematics. Based on the partial rbcL sequences, seven species are recognized along the Brazilian coast: C. decorticatum, C. intertextum, C. isthmocladum, C. profundum, C. spongiosum, C. taylorii and the new species Codium pernambucensis. Ten unique sequences were obtained among the samples examined, which we used in combination with previously published sequences to infer molecular phylogenies using various methods. The resulting trees showed three principal monophyletic groupings: Clade A with species having a prostrate habit, not branched, and mostly with small, grouped utricles; Clade B primarily consisting of upright species with cylindrical branches and large individual utricles; and Clade C composed of upright species with cylindrical branches that are slightly flattened, and have intermediate-sized individual utricles. The Brazilian species grouped with morphologically similar taxa from other geographic localities, and are present in all three main clades. A new sprawling species, Codium pernambucensis is described based on morphology and molecular analyses.

The use of image analysis systems for the assesment of zooplankton physiological rates and carbon fluxes

Trabajo realizado por: Garijo, J. C., Hernández León, S.

Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Emended description of Actinobacillus capsulatus Arseculeratne 1962, 38AL

The taxonomic position of Actinobacillus capsulatus, a member of the family Pasteurellaceae found in rabbits, hares and hamsters, has been challenged. 16S rRNA gene (rrs) sequence data show the species to be heterogeneous. Using a polyphasic approach, 23 strains that were identified previously as belonging, or closely related, to A. capsulatus were analysed. Eighty characters were included in the phenotypic analysis. Phylogenetic analysis was done based on rrs, rpoB, infB and recN sequences. In addition, the recN sequence similarities were used to calculate the whole-genome sequence relatedness of all strains investigated as well as that with other members of the family Pasteurellaceae. The phenotypic analysis allowed identification of five groups. The major group of 17 strains could be classified as A. capsulatus. Two hamster isolates were closely related to A. capsulatus but differed in a few characters. Single isolates from a rabbit and snowshoe-hare were phenotypically related to Actinobacillus suis. One rabbit isolate was related to the genus Mannheimia, while another isolate could not be classified phenotypically with known taxa. The phylogenetic analysis confirmed the phenotypic grouping. In contrast to the rrs-based tree, the A. capsulatus strains clustered unambiguously with the type species and related species of the genus Actinobacillus in the rpoB-, infB- and recN-based trees. Genome similarity comparison using recN finally confirmed the high genomic relationship of the A. capsulatus strains with the type species and related species of the genus Actinobacillus and allowed a clear assignment of the other unrelated strains to the phenotypic and phylogenetic clusters outlined. The present findings allow the description of A. capsulatus to be emended and separate it more clearly from other species, both phenotypically and genotypically. The type strain of A. capsulatus is CCUG 12396(T) (=Frederiksen 243(T)=ATCC 51571(T)=NCTC 11408(T)=CIP 103283(T)).

Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA)

Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.

How does the taxonomic status of allopatric populations influence species richness within African cichlid fish assemblages?

Aim  Current estimates of species richness within rapidly evolving species flocks are often highly dependent on the species status of allopatric populations that differ in phenotypic traits. These traits may be unreliable indicators of biological species status and systematists may have inconsistently assigned species among lineages or locations on the basis of these traits, thus hampering comparative studies of regional species richness and speciation rates. Our aim was to develop a method of generating standardized estimates of regional species richness suitable for comparative analysis, and to use these estimates to examine the extent and consistency of species assignment of allopatric populations within rapidly evolving cichlid fish flocks present in three east African lakes. Location  Lakes Malawi, Victoria and Tanganyika. Methods  Using published taxon co-occurrence data, a novel approach was employed to calculate standardized ‘minimum’ estimates of regional species richness for hard substrate associated complexes of cichlids within each of the lakes. Minimum estimates were based on an explicit assumption that if taxa present on equivalent habitats have disjunct distributions, then they are allopatric forms of the same species. These estimates were compared with current observed ‘high-end’ regional species richness estimates for those complexes to determine the consistency of species assignment of allopatric populations between lineages within a lake. A ‘sympatry’ index was developed to enable comparisons of levels of species assignment of allopatric populations between-lakes to be made. Results  Within each lake, the minimum and high-end estimates for species richness were significantly correlated across complexes, indicating that the complexes that contain more recognized species contain the most genuine biological species. However, comparisons of complexes among lakes revealed considerable differences. For equivalent geographical areas, substantially higher proportions of recognized species were totally allopatric within the studied Lake Malawi and Lake Victoria complexes, than those of Lake Tanganyika. Main Conclusions  Among African lakes, levels of assignment to species status of allopatric populations were found to be distinctly different. It is unclear whether the discrepancies are a consequence of differences between the lake faunas in degrees of phenotypic divergence among allopatric populations, or are simply the result of inconsistent taxonomic practices. In either case, these results have considerable wider relevance for they emphasize that quantitative measures of regional and beta diversity are critically dependent on the species status of allopatric populations, an issue usually neglected in comparative studies of species richness. The technique introduced here can be used to standardize measures of regional diversity of lineages for comparative analyses, potentially enabling more accurate identification of processes influencing rates of speciation.

A global seasonal surface ocean climatology of phytoplankton types based on CHEMTAX analysis of HPLC pigments

Much advancement has been made in recent years in field data assimilation, remote sensing and ecosystem modeling, yet our global view of phytoplankton biogeography beyond chlorophyll biomass is still a cursory taxonomic picture with vast areas of the open ocean requiring field validations. High performance liquid chromatography (HPLC) pigment data combined with inverse methods offer an advantage over many other phytoplankton quantification measures by way of providing an immediate perspective of the whole phytoplankton community in a sample as a function of chlorophyll biomass. Historically, such chemotaxonomic analysis has been conducted mainly at local spatial and temporal scales in the ocean. Here, we apply a widely tested inverse approach, CHEMTAX, to a global climatology of pigment observations from HPLC. This study marks the first systematic and objective global application of CHEMTAX, yielding a seasonal climatology comprised of ~1500 1°x1° global grid points of the major phytoplankton pigment types in the ocean characterizing cyanobacteria, haptophytes, chlorophytes, cryptophytes, dinoflagellates, and diatoms, with results validated against prior regional studies where possible. Key findings from this new global view of specific phytoplankton abundances from pigments are a) the large global proportion of marine haptophytes (comprising 32 ± 5% of total chlorophyll), whose biogeochemical functional roles are relatively unknown, and b) the contrasting spatial scales of complexity in global community structure that can be explained in part by regional oceanographic conditions. These publicly accessible results will guide future parameterizations of marine ecosystem models exploring the link between phytoplankton community structure and marine biogeochemical cycles.

(Appendix) Analysis of benthic foraminifera from ODP Leg 181 and DSDP Leg 90

Canonical correspondence analysis indicates that the distribution of Neogene benthic foraminiferal faunas (>63 µm) in seven DSDP and ODP sites (500-4500 m water depth) east of New Zealand (38-51°S, 170°E-170°W) is most strongly influenced by depth (water mass stratification), and secondly by age (palaeoceanographic changes influencing faunal composition and biotic evolution). Stratigraphic faunal changes are interpretted in terms of the pulsed sequential development of southern, and later northern, polar glaciation and consequent cooling of bottom waters, increased vertical and lateral stratification of ocean water masses, and increased overall and seasonal surface water productivity. Oligocene initiation of the Antarctic Circumpolar Current and Deep Western Boundary Current (DWBC), flowing northwards past New Zealand, resulted in extensive hiatuses throughout the Southwest Pacific, some extending through into the Miocene. Planktic foraminiferal fragmentation index values indicate that carbonate dissolution was significant at abyssal depths throughout most of the Neogene, peaking at upper abyssal depths in the late Miocene (11-7 Ma), with the lysocline progressively deepened thereafter. Miocene abyssal faunas are dominated by Globocassidulina subglobosa and Oridorsalis umbonatus, with increasing Epistominella exigua after 16 Ma at upper abyssal depths. Peak abundances of Epistominella umbonifera indicate increased input of cold Southern Component Water to the DWBC at 7-6 Ma. Faunal association changes imply establishment of the modern Oxygen Minimum Zone (upper Circumpolar Deep Water) in the latest Miocene. Significant latitudinal differences between the benthic foraminiferal faunas at lower bathyal depths indicate the existence of an oceanic front along the Chatham Rise (location of present Subtropical Front), since the early late Miocene at least, with more pulsed productivity (higher E. exigua) along the south side. Modern Antarctic Intermediate Water faunal associations were established north of the Chatham Rise at 10-9 Ma, and south of it at 3-1.5 Ma. Middle-upper bathyal faunas on the Campbell Plateau are dominated by reticulate bolivinids during the early and middle Miocene, indicative of sustained productivity above relatively sluggish, suboxic bottom waters. Faunal changes and hiatuses indicate increased current vigour over the Campbell Plateau from the latest Miocene on. Surface water productivity (food supply) appears to have increased in three steps (at times of enhanced global cooling) marked by substantially increased relative abundance of: (1) Abditodentrix pseudothalmanni, Alabaminella weddellensis, Cassidulina norvangi (16-15 Ma, increased pulsed productivity); (2) Bulimina marginata f. aculeata, Nonionella auris, Trifarina angulosa, Uvigerina peregrina (3-1.5 Ma, increased overall productivity); and (3) Cassidulina carinata (1-0.5 Ma, increased overall productivity). Three intervals of deep-sea benthic foraminiferal taxonomic turnover are recognised (16-15, 11.5-10, 2-0.5 Ma) corresponding to intervals of enhanced global cooling and possible productivity changes. The late Pliocene-middle Pleistocene extinction, associated with increasing Northern Hemisphere glaciation, culminating in the middle Pleistocene climatic transition, was more significant in the study area than the earlier Neogene turnovers.

Metadata und statistic analysis of archael and bacterial sequences originating from sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

DNA extraction was carried out as described on the MICROBIS project pages (http://icomm.mbl.edu/microbis ) using a commercially available extraction kit. We amplified the hypervariable regions V4-V6 of archaeal and bacterial 16S rRNA genes using PCR and several sets of forward and reverse primers (http://vamps.mbl.edu/resources/primers.php). Massively parallel tag sequencing of the PCR products was carried out on a 454 Life Sciences GS FLX sequencer at Marine Biological Laboratory, Woods Hole, MA, following the same experimental conditions for all samples. Sequence reads were submitted to a rigorous quality control procedure based on mothur v30 (doi:10.1128/AEM.01541-09) including denoising of the flow grams using an algorithm based on PyroNoise (doi:10.1038/nmeth.1361), removal of PCR errors and a chimera check using uchime (doi:10.1093/bioinformatics/btr381). The reads were taxonomically assigned according to the SILVA taxonomy (SSURef v119, 07-2014; doi:10.1093/nar/gks1219) implemented in mothur and clustered at 98% ribosomal RNA gene V4-V6 sequence identity. V4-V6 amplicon sequence abundance tables were standardized to account for unequal sampling effort using 1000 (Archaea) and 2300 (Bacteria) randomly chosen sequences without replacement using mothur and then used to calculate inverse Simpson diversity indices and Chao1 richness (doi:10.2307/4615964). Bray-Curtis dissimilarities (doi:10.2307/1942268) between all samples were calculated and used for 2-dimensional non metric multidimensional scaling (NMDS) ordinations with 20 random starts (doi:10.1007/BF02289694). Stress values below 0.2 indicated that the multidimensional dataset was well represented by the 2D ordination. NMDS ordinations were compared and tested using Procrustes correlation analysis (doi:10.1007/BF02291478). All analyses were carried out with the R statistical environment and the packages vegan (available at: http://cran.r-project.org/package=vegan), labdsv (available at: http://cran.r-project.org/package=labdsv), as well as with custom R scripts. Operational taxonomic units at 98% sequence identity (OTU0.03) that occurred only once in the whole dataset were termed absolute single sequence OTUs (SSOabs; doi:10.1038/ismej.2011.132). OTU0.03 sequences that occurred only once in at least one sample, but may occur more often in other samples were termed relative single sequence OTUs (SSOrel). SSOrel are particularly interesting for community ecology, since they comprise rare organisms that might become abundant when conditions change.16S rRNA amplicons and metagenomic reads have been stored in the sequence read archive under SRA project accession number SRP042162.

A method to incorporate the effect of taxonomic uncertainty on multivariate analyses of ecological data

Researchers in ecology commonly use multivariate analyses (e.g. redundancy analysis, canonical correspondence analysis, Mantel correlation, multivariate analysis of variance) to interpret patterns in biological data and relate these patterns to environmental predictors. There has been, however, little recognition of the errors associated with biological data and the influence that these may have on predictions derived from ecological hypotheses. We present a permutational method that assesses the effects of taxonomic uncertainty on the multivariate analyses typically used in the analysis of ecological data. The procedure is based on iterative randomizations that randomly re-assign non identified species in each site to any of the other species found in the remaining sites. After each re-assignment of species identities, the multivariate method at stake is run and a parameter of interest is calculated. Consequently, one can estimate a range of plausible values for the parameter of interest under different scenarios of re-assigned species identities. We demonstrate the use of our approach in the calculation of two parameters with an example involving tropical tree species from western Amazonia: 1) the Mantel correlation between compositional similarity and environmental distances between pairs of sites, and; 2) the variance explained by environmental predictors in redundancy analysis (RDA). We also investigated the effects of increasing taxonomic uncertainty (i.e. number of unidentified species), and the taxonomic resolution at which morphospecies are determined (genus-resolution, family-resolution, or fully undetermined species) on the uncertainty range of these parameters. To achieve this, we performed simulations on a tree dataset from southern Mexico by randomly selecting a portion of the species contained in the dataset and classifying them as unidentified at each level of decreasing taxonomic resolution. An analysis of covariance showed that both taxonomic uncertainty and resolution significantly influence the uncertainty range of the resulting parameters. Increasing taxonomic uncertainty expands our uncertainty of the parameters estimated both in the Mantel test and RDA. The effects of increasing taxonomic resolution, however, are not as evident. The method presented in this study improves the traditional approaches to study compositional change in ecological communities by accounting for some of the uncertainty inherent to biological data. We hope that this approach can be routinely used to estimate any parameter of interest obtained from compositional data tables when faced with taxonomic uncertainty.