public read Prof. Fabiano Thompson Federal University of Rio de Janeiro Assistent Professor

Av. Carlos Chagas Fo. S/N CCS - IB - Laboratory of Microbiology e SAGE-COPPE - BLOCO A (Anexo) A3 - sl 102 RIo de Janeiro RJ 21941-599 Brazil

+55 21 3938-6567 +55 21 3938-6567 CCS - IB - Laboratory of Microbiology e SAGE-COPPE - BLOCO A (Anexo) A3 - sl 102 http://www.microbiologia.biologia.ufrj.br 1414421565596 Principal Investigator Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world’s largest continuous rhodolith bed (of B21000km2) and has one of the largest marine CaCO3 deposits (producing 25 megatons of CaCO3 per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia- oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16lmol carbonm Rhodoliths holobionts carbon cycle biomineralization Abrolhos Bank Abrolhos Bank -38.625 -36.5 -17.0 -19.0 1414421565596

This study was carried out in the Abrolhos Shelf off eastern Brazil. Rhodoliths were collected by scuba diving in December 2010 from three different sites near two recently described sinkhole-like structures called Buracas (Bastos et al., 2013). Buracas are cup- shaped depressions on the seafloor and their suggested function is to trap and accumulate organic matter, thus functioning as productivity hotspots in the mid- and outer shelf of the central portion of the Abrolhos Bank (Cavalcanti et al., 2013). Seven rhodoliths were sampled as follows: two individual rhodoliths from the shallower portion (27 m) (17.813301 S/38.237441 W) outside the first Buraca; three from the inner region of the same Buraca (43 m deep) (17.813991 S/38.243061 W) and two from a deeper point (51 m) (17.913611 S/37.909361 W) near another sinkhole-like structure (Supplementary Table S1) away from the first (Figure 1). Mono- specific rhodolith-forming CCA were selected by visual inspection. Immediately after collection, the specimens were frozen in liquid nitrogen in the field. For comparison purposes, surrounding water samples (8l) collected using sterivex 0.2-mm filters at exactly the same points over the rhodolith beds at the first Buraca (inside—43 m; outside—27 m) and outside the deeper Buraca (51m) were used. A detailed description of the region of the Buracas, water sampling and metagenomic characterization of the planktonic microbial community is available in the work by Cavalcanti et al. (2013).

In the laboratory, a small fragment of each rhodolith sample (B1 cm2) was sterilely macerated with liquid nitrogen without the separation of epibionts to provide a representation of the entire rhodolith holobiont as previously defined (calcifying algae plus associated microbes, fauna and flora). A subsequent step using CTAB buffer with 100 mM of EDTA and a PowerSoil purification column was used to gather the DNA of high-molecular-weight rhodolith holobionts as described by Garcia et al. (2013). High-quality total DNA extracted from rhodoliths was then sequenced using a 454 GS Jr machine (454 Life Sciences, Branford, CT, USA) and the GS Jr Titanium-sequencing process. Sequences were submitted to the MG-RAST 3.1 server (Metagenomics-Rapid Annotation Using Subsystems Technology) (Meyer et al., 2008) and quality filtered. Post-quality-control (QC) sequences were annotated using the (SEED) Subsystems Technology for functional classification (Overbeek et al., 2005) and the GenBank database for phyloge- netic analyses. All BLAST queries were performed with a maximum expected cutoff value of 10e–5.

The Statistical Analysis of Metagenomic Profiles (STAMP v.2.0.0) software was used for statistical analysis (Parks and Beiko, 2010). For comparison purposes, rhodolith metagenomes were compared with water metagenomes from the same site (Cavalcanti et al., 2013). The water and rhodolith samples were compared by using a two-sided Welch’s t-test with 95% confidence intervals calcu- lated by inverting the Welch’s test and by using the Benjamin–Hochberg FDR multiple test correction. A principal component analysis was conducted using the STAMP software package to compare the taxonomic grouping based on the taxonomic class contributions of each metagenome from both the rhodolith and the surrounding seawater. To isolate the relative contributions of taxonomic groups within the rhodolith metagenomes, the most abundant groups (Alphaproteobacteria, Gammapro- teobacteria, Eukarya, Betaproteobacteria, Deltapro- teobacteria, Actinobacteria, Firmicutes, Bacteroidetes, Cyanobacteria, Planctomycetes, Archaea and Others) were functionally re-annotated using the Work- bench tool, and their functions were compared with an analysis of variance using a Tukey–Kramer post- hoc test, an eta-squared effect size and a Benjamin– Hochberg multiple test correction. In all these cases, P-values of o5% were considered statistically significant.