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Home , Poribacteria, Vertical transmission

The ISME Journal (2013) 7, 438–443 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej SHORT COMMUNICATION ‘Sponge-specific’ are widespread (but rare) in diverse marine environments

Michael W Taylor1, Peter Tsai2, Rachel L Simister1, Peter Deines1,4, Emmanuelle Botte3, Gavin Ericson3, Susanne Schmitt1,5 and Nicole S Webster3 1Centre for Microbial Innovation, School of Biological Sciences, University of Auckland, Auckland, New Zealand; 2Bioinformatics Institute, School of Biological Sciences, University of Auckland, Auckland, New Zealand and 3Australian Institute of Marine Science, PMB 3, Townsville Mail Centre, Queensland, Australia

Numerous studies have reported the existence of -specific 16S ribosomal RNA (rRNA) sequence clusters, representing bacteria found in but not detected in other environments, such as seawater. The advent of deep-sequencing technologies allows us to examine the rare microbial biosphere in order to establish whether these bacteria are truly sponge specific, or are more widely distributed but only at abundances below the detection limit of conventional molecular approaches. We screened 412 million publicly available 16S rRNA gene pyrotags derived from 649 seawater, sediment, hydrothermal vent and coral samples from temperate, tropical and polar regions. We detected 77 of the 173 previously described sponge-specific clusters in seawater or other non-sponge samples, albeit generally at extremely low abundances. Sequences representing the candidate phylum ‘Poribacteria’, previously thought to be largely restricted to sponges, were recovered from 46 (out of 411) seawater and 41 (out of 129) sediment samples. While the presence of an organism does not imply that it is active in situ, our results do suggest that many ‘sponge- specific’ bacteria occur more widely outside of sponge hosts than previously thought. The ISME Journal (2013) 7, 438–443; doi:10.1038/ismej.2012.111; published online 4 October 2012 Subject Category: microbe–microbe and microbe–host interactions Keywords: marine sponge; bacteria; specificity; 16S rRNA pyrosequencing

Marine sponges form relationships with a diverse sampled sponges at the time of sponge spawning range of microbes (Taylor et al., 2007; Webster and and thus a sponge origin for these bacteria could not Taylor, 2012), and many of these associations are be unequivocally ruled out. To clarify this funda- highly host specific (Schmitt et al., 2012). Numerous mental issue in sponge symbiont biology, we studies have reported the existence of monophyletic examined 412 million 16S rRNA gene pyrotags sponge-specific 16S ribosomal RNA (rRNA) gene (V6 region) generated under the auspices of the sequence clusters, representing bacteria found in International Census of Marine Microbes (ICoMM; sponges but not detected in other environments, http://icomm.mbl.edu/). These sequences were such as seawater (Hentschel et al., 2002; Taylor derived from a range of seawater, sediment, hydro- et al., 2007; Simister et al., 2012). In a recent 16S thermal vent and coral samples obtained from rRNA gene tag pyrosequencing study (Webster et al., temperate, tropical and polar regions (Figure 1). 2010), we revealed the presence of sequences The absence of ‘sponge-specific’ bacteria from these affiliated with ‘sponge-specific’ clusters in seawater. samples would provide strong additional evidence While this finding suggests that ‘sponge-specific’ for their host specificity, whereas their widespread bacteria can in fact reside outside of these hosts, the occurrence among non-sponge samples would seawater was collected only 10 m away from the imply that they are capable of at least surviving in a free-living state. We downloaded Bacteria-derived 16S rRNA gene Correspondence: MW Taylor, Centre for Microbial Innovation, pyrotag sequences that were publicly accessible via School of Biological Sciences, University of Auckland, Private the ICoMM website in May 2011. All sequence reads Bag 92019, Auckland 1142, New Zealand. had previously been de-replicated into 3% opera- E-mail: [email protected] 4Current address: Institute of Natural Sciences, Massey University, tional taxonomic units by ICoMM, and all singleton Private Bag 102904, Auckland 0745, New Zealand. operational taxonomic units (that is, those opera- 5Current address: Ludwig-Maximilians Universita¨t in Munich, tional taxonomic units represented in the entire Department of Earth and Environmental Sciences, Palaeontology ICoMM data set by only a single sequence read) were and Geobiology, Richard-Wagner-Street 10, 80333 Munich, Germany. removed before analysis. This yielded a total of Received 31 May 2012; revised 8 August 2012; accepted 9 August 12 312 433 sequences obtained from 649 samples in 2012; published online 4 October 2012 42 different studies (see Supplementary Table S1 for Specificity of marine sponge bacteria MW Taylor et al 439 detailed sample information). These sequences were (SC157, comprising 1.96% of sequence reads in the screened against a manually curated SILVA 16S hydrothermal vent sample ALR_0008_2005_05_04; rRNA database (Pruesse et al., 2007) containing Supplementary Table S1), (SC16, 366 026 bacterial sequences, using a phylogenetic comprising 1.28% of sequences in the coastal sea- assignment procedure based on those described water sample LCR_0002_2009_11_13), and a lineage previously (Webster et al., 2010; Schmitt et al., of uncertain affiliation (sponge-associated unidenti- 2012). One-hundred and seventy-three sponge-spe- fied lineage (Schmitt et al., 2012)) related to the cific clusters (SC1–SC173) and 32 sponge-coral- –– super- specific clusters (SCC 1–SCC 32) were identified in phylum (SC169, comprising 1.07% of sequences in an earlier study (Simister et al., 2012) and were the coral sample CCB_0001_2008_03_26; 0.36% in represented in the SILVA database. In brief, each the seawater samples HOT177_5 and HOT186_175). ICoMM-derived pyrotag was subjected to a BLAST One important caveat to keep in mind when (Altschul et al., 1990) search against the curated considering these data is that, while 16S rRNA gene SILVA database, and the 50 best hits were aligned in pyrotags may be represented in many samples, this order to determine sequence similarities. The most does not necessarily mean that the exact same similar pyrotag sequence to the respective reference microorganisms are so widespread. This is, of course, sequence was then assigned (or not) to an SC or SCC an acknowledged limitation of the 16S rRNA gene as cluster based on application of a 75% sequence a phylogenetic marker and is not unique to this study. similarity threshold (that is, a sequence read was It is also worth bearing in mind that the original only assigned to a cluster if it was more similar to definition of SC is based on phylogenetic reconstruc- the members of that cluster than to other sequences tions, whereas our assignment of ICoMM pyrotags to outside the cluster and its similarity to the most these clusters is based on sequence similarity. similar sequence within that cluster was above Although we do not believe that this has a substantial 75%). In cases where the assignment of the most effect on our data or its interpretation, we do similar sequences was inconsistent, a majority rule acknowledge that some sequence tags that are was applied, and the pyrotag was only assigned to assigned to a given cluster could actually fall adjacent an SC or SCC if at least 60% of the reference to (rather than within) that cluster. sequences were affiliated with this cluster. The candidate phylum ‘Poribacteria’ occurs in We detected 77 of 173 previously described SC in many marine sponges, often at very high abun- seawater or other non-sponge samples, albeit gen- dances (Fieseler et al., 2004; Lafi et al., 2009). erally at extremely low abundances (for example, Having never been detected outside of sponges 2 sequence reads out of 20 000 for a given sample) using conventional molecular approaches such as (Figure 2; Supplementary Table S1). Twelve of these 16S rRNA gene library construction or fluorescence 77 clusters were identified only in coral samples in situ hybridisation, ‘Poribacteria’ were generally (Caribbean Coral Bacteria, CCB study). Of 32 considered to be restricted to sponge hosts. How- previously described SCC, 27 were recorded outside ever, their recent detection in seawater by pyrotag of either sponges or corals (Supplementary Table (Webster et al., 2010) and fosmid (Pham et al., 2008) S1). Those that were present in higher numbers sequencing suggested their wider occurrence in included clusters within the non-sponge habitats. In this study, we detected

Figure 1 Geographic distribution of samples from the ICoMM, which were investigated in this study. Each line links together samples from a given ICoMM study. Colour codes represent sample types as shown in Supplementary Table 1: blue ¼ seawater samples; grey ¼ sediment; red ¼ hydrothermal vent; green ¼ host-associated; yellow ¼ biofilm/microbial mat; brown ¼ salt marsh.

The ISME Journal Specificity of marine sponge bacteria MW Taylor et al 440

SC 1 2.6% SC 2 SC 3 SC 4 SC 5 SC 6 SC 7 SC 8 SC 9 SC 10 SC 11 SC 12 SC 13 SC 14 SC 15 SC 16 Actinobacteria SC 17 SC 18 SC 19 1.3% SC 20 SC 21 SC 22 SC 23 SC 24 SC 25 SC 26 SC 27 SC 28 SC 29 SC 30 SC 31 SC 32 SC 33 SC 34 SC 35 SC 36 >0% SC 37 SC 38 0% SC 39 SC 40 SC 41 SC 42 SC 43 SC 44 SC 45 SC 46 SC 47 SC 48 SC 49 SC 50 SC 51 SC 52 SC 53 SC 54 SC 55 SC 56 SC 57 SC 58 SC 59 SC 60 SC 61 SC 62 SC 63 SC 64 SC 65 SC 66 SC 67 SC 68 SC 69 SC 70 SC 71 SC 72 Planctomycetes SC 73 SC 74 “Poribacteria” SC 75 SC 76 SC 77 SC 78 SC 79 SC 80 SC 81 SC 82 SC 83 SC 84 SC 85 SC 86 SC 87 SC 88 SC 89 SC 90 SC 91 SC 92 SC 93 SC 94 SC 95 SC 96 SC 97 SC 98 SC 99 SC 100 SC 101 SC 102 SC 103

The ISME Journal Specificity of marine sponge bacteria MW Taylor et al 441 sequences affiliated with ‘Poribacteria’ (cluster ultimately reveal their presence outside of sponge SC75) in 46 seawater, 41 sediment, 14 hydrothermal hosts. vent, 14 salt marsh and 3 coral samples collected The detection of an organism’s DNA in a sample from around the world (Figure 2, Supplementary does not necessarily mean that this organism is Table S1). Invariably, numbers were low, never active in situ. Hence, the finding of ‘sponge-specific’ comprising 40.19% of sequences in seawater or bacteria in diverse habitats outside of sponges could sediment samples. Interestingly, the highest non- theoretically reflect the presence of metabolically sponge occurrence of ‘Poribacteria’ was in a coral, inactive dispersal stages for these bacteria. This type where they comprised 0.25% of sequence reads in of rare biosphere ‘seed bank’ for colonisation of the sample CCB_0001_2008_03_26 (Supplementary sponges has been suggested before (Webster et al., Table S1). SC vary greatly in size, with clusters such 2010) and could explain the apparent lack of co- as the ‘Poribacteria’ (SC75) containing 136 speciation in sponge–microbe associations. It is sequences whereas other, smaller clusters such as often suggested that cellular rRNA concentrations the Planctomycetes cluster SC74 contain as few as are correlated with growth rate and activity, hence three sequences. To determine whether the size of a rRNA may reflect which members of the community sponge-specific cluster increases the likelihood of a are active (Kamke et al., 2010). To explore whether non-sponge-derived sequence being assigned to it, these ‘sponge-specific’ bacteria were metabolically we performed a simple analysis to compare SC/SCC active outside of their host we examined the only size with the number of matching ICoMM pyrotags ICoMM study to have included both RNA- and (data not shown). Cluster size was a very poor DNA-derived samples (Coral Reef Sediment, (CRS); predictor of sequence tag assignment (R2 ¼ 0.0124), Gaidos et al., 2011). Of the sponge-specific and indicating that this factor has only a negligible sponge-coral-specific clusters identified in the CRS effect. study, 25 contained CRS sequences found only in This study provides compelling evidence for the DNA-derived samples, 9 contained sequences in occurrence of many so-called sponge-specific bac- both DNA- and RNA-derived samples and only 1 teria in other marine environments. However, even contained sequences exclusively from RNA-derived upon examination of more than 12 million samples. While the majority of sponge-specific sequences, 96 of the 173 previously defined SC sequences were recovered only from the reef sedi- were undetected outside of sponges and, therefore, ment DNA fraction, the finding of any sponge- remain putatively sponge specific. These include specific bacteria within the RNA fraction suggests clusters within the Acidobacteria, Actinobacteria, that at least some of these microorganisms may be Chloroflexi, Cyanobacteria, Gemmatimonadetes, active outside the confines of the host. Sponge- Alphaproteobacteria and Gammaproteobacteria specific bacteria in the environment could also (Figure 2). If these bacteria are indeed absent outside originate from adult sponge tissue via damage and/ of sponges, then the symbiotic association must be or release of reproductive stages, although this maintained solely via vertical . The would seem unlikely for many of the environments transmission of diverse microbial communities included in this study such as deep-sea hydrother- between sponge generations is well documented mal vents and open ocean waters. (Sharp et al., 2007; Schmitt et al., 2008; Webster The occurrence of putatively sponge-specific et al., 2010). It is worth noting that the absence of bacteria in so many non-sponge samples strongly these clusters from the analysed ICoMM data set suggests that they are capable of surviving outside of does not confirm their complete absence from the sponge hosts. The analysed data demonstrate the environment and further sequencing could power of next-generation sequencing technologies

Figure 2 Occurrence of ‘sponge-specific’ 16S rRNA gene sequence clusters in ICoMM samples. Heatmap showing the distribution of representatives of previously described ‘sponge-specific’ 16S rRNA gene sequence clusters (Simister et al., 2012) among the V6 pyrotag sequences downloaded from the ICoMM website. Clusters with an SC prefix contain sequences previously reported only from sponges; SCC prefix signifies clusters containing only sponge- and coral-derived sequences. Displayed data for a given project represent all samples from that project pooled together; data for individual samples is provided in Supplementary Table S1. Black arrow at the top denotes the ‘SPO’ study which included 12 marine sponge samples from the Great Barrier Reef (Webster et al., 2010). White arrow denotes the ‘CCB’ study of Caribbean corals (Sunagawa et al., 2010). The colours indicate relative abundance of sequence reads, from blue (low abundance) via black to red (high abundance); white indicates that no sequences were assigned to the respective cluster. ICoMM project names: ABR, Active But Rare; ACB, Arctic Chuki Beaufort; AGW, Amazon-Guianas Water; ALR, Lau Hydrothermal Vent; AOT, Atlantic Ocean Transect; ASA, Amundsen Sea Antarctica; ASV, Azorean Shallow Vents; AWP, Azores Waters Project; BMO, Blanes Bay Microbial Observatory; BSP, Baltic Sea Proper; BSR, Black Sea Redox; CAM, Census Antarctic Marine; CAR, Cariaco Basin; CCB, Caribbean Coral Bacteria; CFU, Deep Subseafloor Sediment; CMM, Coastal Microbial Mats; CNE, Coastal New England; CRS, Coral Reef Sediment; DAO, Deep Arctic Ocean; Eel, Eel Pond Winter Pilot Study; FIS, Frisian Island Sylt; GMS, Guaymas Methane Seeps; GOA, Gulf of Aqaba; HCW, Hood Canal Washington; HOT, Hawaiian Ocean Time Series; ICR, IOMM Cooperative Run; KNX, Station KNOX South Pacific Gyre Ocean Drilling Project; LCR, LaCAR Cooperative Run; LCY, Lost City; LSM, Salt Marsh study; MPI, Helgoland; NADW, North Atlantic Deep Water Flow; NZS, New Zealand Sediment; ODP, Ocean Drilling Project; PML, English Channel; PSM, Pilot Project— Seamounts; RIP, Dead Sea Project; SMS, Station M Sediments; SMT, Hydrothermal seamounts; SPO, sponges; SSD, Spatial Scaling Diversity; VAG, Humboldt Marine Ecosystem.

The ISME Journal Specificity of marine sponge bacteria MW Taylor et al 442

2.6% SC 104 SC 105 Alphaproteobacteria SC 106 (cont…) SC 107 SC 108 SC 109 SC 110 SC 111 SC 112 SC 113 SC 114 SC 115 SC 116 SC 117 SC 118 SC 119 SC 120 SC 121 1.3% SC 122 SC 123 SC 124 SC 125 SC 126 SC 127 SC 128 SC 129 SC 130 SC 131 SC 132 SC 133 SC 134 SC 135 SC 136 SC 137 SC 138 >0% SC 139 SC 140 SC 141 0% SC 142 SC 143 SC 144 Gammaproteobacteria SC 145 SC 146 SC 147 SC 148 SC 149 SC 150 SC 151 SC 152 SC 153 SC 154 SC 155 SC 156 SC 157 SC 158 SC 159 SC 160 SC 161 SC 162 SC 163 SC 164 SC 165 SC 166 SAUL SC 167 SC 168 SC 169 SC 170 SC 171 TM6 SC 172 TM7 SC 173 SCC 1 SCC 2 SCC 3 Acidobacteria SCC 4 SCC 5 SCC 6 SCC 7 SCC 8 Bacteroidetes SCC 9 SCC 10 SCC 11 SCC 12 Chloroflexi SCC 13 SCC 14 SCC 15 Gemmatimonadetes SCC 16 Nitrospira SCC 17 Planctomycetes SCC 18 SCC 19 SCC 20 SCC 21 SCC 22 Alphaproteobacteria SCC 23 SCC 24 SCC 25 SCC 26 SCC 27 SCC 28 Deltaproteobacteria SCC 29 SCC 30 SCC 31 Gammaproteobacteria SCC 32

Figure 2 Continued.

The ISME Journal Specificity of marine sponge bacteria MW Taylor et al 443 for improving our understanding of marine symbioses Pham VD, Konstantinidis KT, Palden T, DeLong EF. and should enable further identification of hitherto (2008). Phylogenetic analyses of ribosomal DNA unknown reservoirs for these bacteria. containing bacterioplankton fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre. Environ Microbiol 10: 2313–2330. Acknowledgements Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, We gratefully acknowledge funding support from Peplies J et al. (2007). SILVA: a comprehensive online University of Auckland Faculty Research Development resource for quality checked and aligned ribosomal Fund grants (3609286 and 3622989) to MWT, a German RNA sequence data compatible with ARB. Nucleic Research Foundation (DFG) grant (SCHM 2559/1-1) to SS, Acids Res 35: 7188–7196. and a Feodor Lynen Research Fellowship from the Schmitt S, Angermeier H, Schiller R, Lindquist N, Alexander von Humboldt Foundation to PD. L Taylor is Hentschel U. (2008). Molecular microbial diversity thanked for insightful comments on the manuscript. We survey of sponge reproductive stages and mechanistic also thank ICoMM for the public availability of 16S rRNA insights into vertical transmission of microbial sym- gene sequence data. bionts. Appl Environ Microbiol 74: 7694–7708. Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N et al. (2012). Assessing the complex sponge micro- biota: core, variable and species-specific bacterial References communities in marine sponges. ISME J 6: 564–576. Sharp KH, Eam B, Faulkner DJ, Haygood MG. (2007). Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990). Vertical transmission of diverse microbes in the Basic local alignment search tool. J Mol Biol 215: 403–410. tropical sponge Corticium sp. Appl Environ Microbiol Fieseler L, Horn M, Wagner M, Hentschel U. (2004). 73: 622–629. Discovery of the novel candidate phylum "Poribac- Simister RL, Deines P, Botte ES, Webster NS, Taylor MW. teria" in marine sponges. Appl Environ Microbiol 70: (2012). Sponge-specific clusters revisited: a compre- 3724–3732. hensive phylogeny of sponge-associated microorgan- Gaidos E, Rusch A, Ilardo M. (2011). Ribosomal tag isms. Environ Microbiol 14: 517–524. pyrosequencing of DNA and RNA from benthic coral Sunagawa S, Woodley CM, Medina M. (2010). Threatened reef : community spatial structure, rare corals provide underexplored microbial habitats. PloS members and nitrogen-cycling guilds. Environ Micro- One 5: e9554. biol 13: 1138–1152. Taylor MW, Radax R, Steger D, Wagner M. (2007). Sponge- Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, associated microorganisms: , ecology, and Hacker J et al. (2002). Molecular evidence for a biotechnological potential. Microbiol Mol Biol Rev 71: uniform microbial community in sponges from differ- 295–347. ent oceans. Appl Environ Microbiol 68: 4431–4440. Webster NS, Taylor MW. (2012). Marine sponges and their Kamke J, Taylor MW, Schmitt S. (2010). Activity profiles for microbial symbionts: love and other relationships. marine sponge-associated bacteria obtained by 16S rRNA Environ Microbiol 14: 335–346. vs 16S rRNA gene comparisons. ISME J 4: 498–508. Webster NS, Taylor MW, Behnam F, Lu¨ cker S, Rattei T, Lafi FF, Fuerst JA, Fieseler L, Engels C, Goh WW, Whalan S et al. (2010). Deep sequencing reveals Hentschel U. (2009). Widespread distribution of exceptional diversity and modes of transmission for Poribacteria in Demospongiae. Appl Environ Microbiol bacterial sponge symbionts. Environ Microbiol 12: 75: 5695–5699. 2070–2082.

Supplementary Information accompanies the paper on The ISME Journal website (http://www.nature.com/ismej)

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