The ISME Journal (2012) 6, 564–576 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges Susanne Schmitt1,2,12, Peter Tsai3, James Bell4, Jane Fromont5, Micha Ilan6, Niels Lindquist7, Thierry Perez8, Allen Rodrigo3,9, Peter J Schupp10,13, Jean Vacelet8, Nicole Webster11, Ute Hentschel2 and Michael W Taylor1 1Centre for Microbial Innovation, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand; 2Julius-von-Sachs Institute for Biological Sciences, University of Wuerzburg, Julius-von-Sachs Platz 3, Wuerzburg, Germany; 3Bioinformatics Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand; 4Centre for Marine Environmental and Economic Research, School of Biological Sciences, Victoria University of Wellington, Kelburn Pde, Kelburn Campus, Wellington, New Zealand; 5Western Australian Museum, Locked Bag 49, Welshpool DC, Western Australia, Australia; 6Department of Zoology, Tel Aviv University, Tel Aviv, Israel; 7Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, NC, USA; 8Centre d’Oce´anologie de Marseille, Aix-Marseille Universite´, CNRS UMR 6540 DIMAR, Station Marine d’Endoume Rue Batterie-des-Lions Marseille 13007, France; 9Biology Department, Duke University, Durham, NC, USA; 10Marine Laboratory, University of Guam, Mangilao, GU, USA and 11Australian Institute of Marine Sciences, PMB 3, Townsville Mail Center, Queensland, Australia Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world’s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations. The ISME Journal (2012) 6, 564–576; doi:10.1038/ismej.2011.116; published online 13 October 2011 Subject Category: microbe–microbe and microbe–host interactions Keywords: biogeography; candidate phyla; core microbiota; bacterial diversity; marine sponges; 454-pyrosequencing Correspondence: S Schmitt, Department of Earth and Environmental Sciences, Paleontology and Geobiology, Ludwig-Maximilans- Universita¨t in Munich, Richard Wagner Str. 10, 80333 Munich, Germany. E-mail: [email protected] 12Current address: Department of Earth and Environmental Sciences, Paleontology and Geobiology, Ludwig-Maximilians-University in Munich, Richard-Wagner-Street 10, 80333 Munich, Germany. 13Current address: Institute for Chemistry and Biology of the Marine Environment, Postfach 2503, University of Oldenburg, 26111 Oldenburg, Germany. Received 1 December 2010; revised 15 July 2011; accepted 28 July 2011; published online 13 October 2011 Assessing the complex microbiota in marine sponges S Schmitt et al 565 Introduction their origin (host sponge and/or sampling location). The term ‘sponge-specific’ was introduced for these The recent advent of massively parallel sequencing clusters to describe a phylogenetically complex com- technologies has revolutionized microbial diversity munity that is repeatedly detected in sponges around and ecology studies. Deep sequencing of 16S the world but that is different from microbial seawater ribosomal RNA (rRNA) gene amplicon libraries communities (Hentschel et al., 2002; Taylor et al., showed that microbial communities in many envir- 2007). Interestingly, sponge-specific microbes are onments including marine habitats, soil, plants and mainly found in high microbial abundance sponges humans, are much more diverse than previously whereas low microbial abundance sponges usually thought (Huber et al., 2007; Costello et al., 2009; have a much lower microbial diversity and a different Turnbaugh et al., 2009; Andersson et al., 2010; microbial profile (Hentschel et al., 2006; Kamke et al., Hollister et al., 2010; Teixeira et al., 2010). Many of 2010). Given the observed uniformity of sponge- these newly detected microbes represent the rare specific communities, the aim of this study was to biosphere (Sogin et al., 2006). They are not abun- identify geographical or host-dependent bacterial sub- dant but collectively represent most of the diversity populations in sponges and to define the bacterial core within a sample. A recent 454 pyrosequencing study community that is shared among most sponges as well showed that marine sponges host bacterial commu- as the species-specific community that is unique to a nities with a diversity that is unparalleled in any certain sponge species. invertebrate host (Webster et al., 2010). Sponges are the oldest and most primitive of the Materials and methods metazoan phyla with a global distribution in essentially all aquatic habitats. They are ecologically Sponge collection, sample preparation and 454 important and a rich source of novel, biotechnolo- pyrosequencing gically relevant natural products. The association of Tissue samples from three individuals of each of 31 some species with dense and complex microbial sponge species and from a single individual of an consortia has long been recognized (Vacelet 1975; unidentified sponge S1 (Table 1) were collected at Vacelet and Donadey 1977; Wilkinson et al., 1981) eight different locations in the Indian and Pacific and these sponges were termed bacteriosponges or Ocean, the Caribbean, Mediterranean and Red Sea at ‘high microbial abundance’ sponges to distinguish depths between 1. 5 and 18 m while SCUBA diving them from co-occurring sponges in the same habitat (for sampling details see Supplementary Table S1). that lack dense microbial communities (which were All samples were frozen at À80 1Cforatleast24h, thus termed ‘low microbial abundance’ sponges) then freeze-dried for another 24 h and kept at À20 1C (Reiswig 1981; Hentschel et al., 2003). In high until further processing. DNA was extracted using microbial abundance sponges, microbes can com- a hexadecyltrimethylammonium bromide (CTAB)- prise as much as 40% of the sponge biomass and based protocol (Taylor et al., 2004) from each represent a variety of different morphotypes (Vacelet individual of all sponges except sponge S1 where 1975). Phylogenetic studies identified members of three different tissue samples of one individual were 26 different bacterial phyla (Taylor et al., 2007; used. Briefly, cells were disrupted by bead-beating in Webster et al., 2008, 2010; Sipkema et al., 2009) an ammonium acetate extraction buffer containing including the candidate phylum ‘Poribacteria’ that chloroform:isoamyl alcohol (24:1). DNA was precipi- is almost exclusively found within sponges (Fieseler tated with 3 M sodium acetate and isopropanol, then et al., 2004; Lafi et al., 2009). Sponge symbionts are washed in 70% ethanol, dried and redissolved in capable of diverse metabolic processes such as H2Odd. Purity and quantity of DNA was determined nitrification, nitrogen fixation, sulfate reduction with a Nanodrop 1000 spectrophotometer (Thermo and photosynthesis (Wilkinson 1979; Hoffmann Scientific, Wilmington, DE, USA) and only high et al., 2006, 2009; Bayer et al., 2008; Mohamed quality DNA was used for subsequent PCR reactions. et al., 2010) and probably contribute to sponge PCRs were performed using the FastStart high fidelity nutrition (Weisz et al., 2007). Additionally, certain PCR system (Roche Diagnostics N.Z. Ltd, Auckland, sponge symbionts produce secondary metabolites that New Zealand) and the modified primer pair 338f and 0 might be involved in the chemical defense of their 533r (338fdeg:5-ACW CCT ACG GGW GGC WGC 0 0 0 hosts (Kennedy et al., 2007; Siegl and Hentschel AG-3 , 533rdeg:5-TKA CCG CRG CTG CTG GCA C-3 ) 2010; Thomas et al., 2010a). Genomic information is to amplify a ca 145-bp fragment of the 16S rRNA gene currently being used to further characterize features