The ISME Journal (2014) 8, 52–62 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean Giselle S Cavalcanti1, Gustavo B Gregoracci1, Eidy O dos Santos1, Cynthia B Silveira1, Pedro M Meirelles1, Leila Longo2, Kazuyoshi Gotoh3, Shota Nakamura3, Tetsuya Iida3, Tomoo Sawabe4, Carlos E Rezende5, Ronaldo B Francini-Filho6, Rodrigo L Moura1, Gilberto M Amado-Filho2 and Fabiano L Thompson1 1Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil; 2Instituto de Pesquisas Jardim Botaˆnico do Rio de Janeiro, Rio de Janeiro, Brazil; 3Laboratory of Genomic Research on Pathogenic Bacteria, International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 4Laboratory of Microbiology, Hokkaido University, Sapporo, Japan; 5Environmental Science Laboratory, Campos dos Goytacazes, UENF, Rio de Janeiro, Brazil and 6Department of Engineering and Environment, Federal University of Paraı´ba, Paraı´ba, Brazil 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 2 continuous rhodolith bed (of B21 000 km ) 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 (52.16 lmol carbon m À 2 s À 1), allowing the entire Abrolhos rhodolith bed to produce 5.65 Â 105 tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean. The ISME Journal (2014) 8, 52–62; doi:10.1038/ismej.2013.133; published online 29 August 2013 Subject Category: Microbe-microbe and microbe-host interactions Keywords: rhodoliths; holobionts; carbon cycle; biomineralization; Abrolhos Bank Introduction The term holobiont refers to any organism and all of its associated symbiotic microbes (parasites, mutu- Eukaryote–microbe associations are increasingly alists, synergists and amensals) (Rosenberg and recognized as being essential for eukaryotic host Zilber-Rosenberg, 2011), including endobionts and health and metabolism, enabling the expansion of epibionts that perform diverse ecological roles their physiologic capacities (Rosenberg et al., 2007). (Wahl et al., 2012). A holobiont occupies and adapts to an ecological niche, and is able to employ Correspondence: FL Thompson, Av. Carlos Chagas Fo. S/N— strategies unavailable in any one species alone CCS—IB—BIOMAR—Lab de Microbiologia—BLOCO A (Anexo) when challenged by environmental perturbations. A3—sl 102, Cidade Universita´ria, Rio de Janeiro CEP 21941-599, The holobiont concept, first applied to corals after RJ, Brazil. E-mail: [email protected] they were found to host abundant and species- Received 25 March 2013; revised 23 June 2013; accepted 4 July specific microbial communities (Rohwer et al., 2013; published online 29 August 2013 2002; Reshef et al., 2006; Rosenberg et al., 2007), Rhodolith physiologic and metagenomic attributes GS Cavalcanti et al 53 has since been extended to the benthic alga (Barott ecological roles that rhodoliths have in the marine et al., 2011). These holobionts are diverse and realm. Rhodoliths may be key factors in a range of distinct from the microbiota in the surrounding invertebrate-recruitment processes, functioning as seawater and biofilms on abiotic surfaces (Barott autogenic ecosystem engineers by providing three- et al., 2011; Burke et al., 2011; Barott and Rohwer, dimensional habitat structures. A better understand- 2012). A wide range of beneficial and detrimental ing of the composition of and ecological interactions interactions have been described between macro- within the rhodolith holobiont would be a powerful algae and their microbiomes based on the exchange tool for the conservation and sustainable use of of nutrients, minerals and secondary metabolites these biological resources. This understanding (Hollants et al., 2012). could also provide insights into how cooperation Rhodoliths are biogenic calcareous structures and job partitioning between constituents contribute primarily formed by encrusting coralline algae to holobiont fitness, influence holobiont viability, (CCA; Corallinales, Rhodophyta). They provide and consequently affect associated ecosystems and highly biodiverse and heterogeneous habitats and the ubiquitous rhodolith worldwide distribution. are distributed worldwide (Foster, 2001; Steller We aimed to perform a metagenomic characteriza- et al., 2003; Nelson, 2009). Despite our vast knowl- tion of the Abrolhos rhodoliths to determine the edge concerning the ecophysiology and biogeogra- major taxonomic and functional components of phy of rhodoliths, some aspects of the biology of these organisms. We also examined the associated rhodoliths remain completely unknown. For fauna diversity and physiologic aspects (photosyn- instance, information on the microbial commu- thetic capacity and dissolved organic carbon (DOC) nities, the microbial metabolic strategies associated productivity) of the rhodoliths. with biogeochemical cycles and the biomineraliza- tion of CaCO3, and the photosynthetic productivity potential of rhodoliths is absent. Materials and methods Rhodolith beds constitute one of the Earth’s four Study site and sample collection major macrophyte-dominated benthic communities, This study was carried out in the Abrolhos Shelf off along with kelp beds, seagrass meadows and eastern Brazil. Rhodoliths were collected by scuba coralline algal reefs. Our group has mapped the diving in December 2010 from three different sites largest rhodolith bed in the world, which covers near two recently described sinkhole-like structures B 2 1 0 1 0 20 900 km of the Abrolhos Shelf (16 50 –19 45 S) called Buracas (Bastos et al., 2013). Buracas are cup- (Amado-Filho et al., 2012). The Abrolhos Bank is shaped depressions on the seafloor and their one of the largest marine CaCO3 deposits in the suggested function is to trap and accumulate organic world, with a production of 25 megatons of CaCO3 matter, thus functioning as productivity hotspots in per year (Amado-Filho et al., 2012). Nevertheless, the mid- and outer shelf of the central portion of the our limited knowledge of rhodolith biology hinders Abrolhos Bank (Cavalcanti et al., 2013). Seven our ability to develop a broader systemic under- rhodoliths were sampled as follows: two individual standing of their ecological role in the Abrolhos rhodoliths from the shallower portion (27 m) Bank. There are pressing concerns relating to the (17.813301 S/38.237441 W) outside the first Buraca; future of rhodolith beds as global carbon budgets, three from the inner region of the same Buraca (43 m and ocean acidification may interfere with rhodolith deep) (17.813991 S/38.243061 W) and two from a functioning and biogeochemical stability (Webster deeper point (51 m) (17.913611 S/37.909361 W) near et al., 2011; Whalan et al., 2012). In addition, another sinkhole-like structure (Supplementary because rhodoliths can be applied agriculturally to Table S1) away from the first (Figure 1). Mono- improve soil pH, rhodolith environments have specific rhodolith-forming CCA were selected by been suffering extensive commercial pressure. visual inspection. Immediately after collection, the In addition, rhodoliths are a non-renewable resource specimens were frozen in liquid nitrogen in the because of their extremely slow growth rates (Dias, field. For comparison purposes, surrounding water 2001; Barbera et al., 2003; Wilson et al., 2004). samples (8 l) collected using sterivex 0.2-mm filters Rhodoliths form biogenic matrices with complex at exactly the same points over the rhodolith beds at structures in which the interlocking branched thalli the first Buraca (inside—43 m; outside—27 m) can create microhabitats for diverse eukaryotic and outside the deeper Buraca (51 m) were used. assemblages, including epiphyte algae, microalgae A detailed description of the region of the Buracas, and different types of invertebrates