Coral Microbiome Database: Integration of Sequences Reveals High Diversity and Relatedness of Coral- Associated Microbes

Environmental Microbiology Reports (2019) 11(3), 372–385 doi:10.1111/1758-2229.12686

Brief Report

Coral microbiome database: Integration of sequences reveals high diversity and relatedness of coral- associated microbes

Megan J. Huggett1,2* and Amy Apprill3* timely given the escalated need to understand the role 1School of Environmental and Life Sciences, University of the coral microbiome and its adaptability to changing of Newcastle, Ourimbah, NSW, 2258, Australia. ocean and reef conditions. 2School of Science, Edith Cowan University, Joondalup, WA, Australia. Introduction 3Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, Corals are complex ‘holobiont’ organisms (Knowlton and MA, USA. Rohwer, 2003), hosting multi-species microbial interactions which sustain their productivity and growth in oligo- trophic reef waters. It is well known that corals rely on Summary endosymbiotic microalgae (Symbiodinium)tosupply Coral-associated microorganisms are thought to play a sugars, amino acids, lipids and other nutrients (Trench, fundamental role in the health and ecology of corals, but 1971), but corals also host an assemblage of other micro- understanding of specificcoral–microbial interactions organisms including endolithic algae, bacteria, archaea, are lacking. In order to create a framework to examine fungi and viruses (Rohwer et al., 2002; Knowlton and coral–microbial specificity, we integrated and phyloge- Rohwer, 2003; Ainsworth et al., 2017). Of these microor- netically compared 21,100 SSU rRNA gene Sanger- ganisms, bacteria and archaea are thought to play a criti- produced sequences from bacteria and archaea associ- cal role in the cycling and regeneration of nutrients for the ated with corals from previous studies, and accompany- coral holobiont (Rohwer et al., 2001, 2002; Beman et al., ing host, location and publication metadata, to produce 2007; Kelly et al., 2014). There is also evidence that coral- the Coral Microbiome Database. From this database, we associated bacteria are capable of producing antibiotic identified 39 described and candidate phyla of Bacteria and other secondary metabolite compounds, which could and two Archaea phyla associated with corals, demon- provide protection to the coral (Kelman, 2004; Ritchie, strating that corals are one of the most phylogenetically 2006; Raina et al., 2009). Understanding the individual diverse animal microbiomes. Secondly, this new phylo- contribution of these disparate organisms to the coral holo- genetic resource shows that certain microorganisms are biont, and if and how these microorganisms enhance the indeed specific to corals, including evolutionary distinct resistance and resilience of corals to changing ocean and hosts. Specifically, we identified 2–37 putative monophy- reef conditions, are current challenges within this field. letic, coral-specific sequence clusters within bacterial Over the past 15 years, numerous review articles have genera associated with the greatest number of coral reflected upon the relationship between corals and bacte- species (Vibrio, Endozoicomonas and Ruegeria)aswell ria and archaea (Rosenberg and Ben-Haim, 2002; as functionally relevant microbial taxa (“Candidatus Rohwer and Kelley, 2004; Mouchka et al., 2010; Bourne Amoebophilus”, “Candidatus Nitrosopumilus” and under et al., 2016). These reviews repeatedly demonstrate that recognized cyanobacteria). This phylogenetic resource coral–microbes are taxonomically distinct from cells provides a framework for more targeted studies of residing in the seawater, and there is general recognition corals and their specific microbial associates, which is of shared microbial groups over coral species and reefs (i.e., Roseobacter clade, Vibrio spp.). However, although most of the coral–microbial data are publically available, Received 17 January, 2018; accepted 4 August, 2018. *For corre- these data from multiple investigators and study environ- spondence. E-mail [email protected]; Tel. +61 2 4348 fi 4025; Fax +61 2 4349 4404; E-mail [email protected]; Tel. 01-508-289- ments data are not integrated. In other elds of study, 2649; Fax 01-508-457-2164. combining existing data provides valuable resources for

© 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Coral microbiome database 373 the community and especially for students and new advance investigations of the functional role of the coral investigators to these fields (e.g., Simister et al., 2012). microbiome and specificcoral–microbial interactions. Integration of coral–microbial data is critical in the current framework of limited understanding about coral–microbial associations and during a time when many corals and reefs Results and discussion are experiencing disturbances from climate and anthropo- Overview of coral-specific microbial sequence database genic sources (Hoegh-Guldberg et al., 2007; Carpenter et al., 2008; Hughes et al., 2017, 2018). A growing commu- The Coral MD includes 21,100 sequences derived from nity of scientists is pursuing studies aimed at understanding coral-associated bacteria and archaea which originated the resistance, resilience and impact of these events on from public databases, and are compiled in the ARB soft- coral microbiomes (e.g., Lee et al., 2015; Webster et al., ware environment within the framework of the Silva refer- 2016). For example, studies have shown that coral- ence database (Quast et al., 2013) (Supporting associated microbial communities demonstrate taxonomic Information Methods). The database is available for shifts in composition in response to temperature, nutrient download at BCO-DMO (https://www.bco-dmo.org/ and pH-based stressors (Littman et al., 2010; Meron et al., dataset/724355). The mean and median lengths of 2011; Shaver et al., 2017). The current research trend is to sequences in the database are 893 and 750 bp respec- produce copious partial SSU rRNA gene sequences using tively. There are 6833 nearly full-length sequences next generation sequencing methods targeted at bacteria greater than or equal to 1200 bp, and 5811 sequences and archaea to inform questions about how microbial diver- are short reads, less than or equal to 600 bp. The sity, community structure and taxonomic composition sequences span 37 described and candidate phyla of changes under different environmental scenarios. However, Bacteria (Proteobacteria considered as single phylum) sequences generated by these approaches are typically and 2 phyla of Archaea (Euryarchaeota and Thaumarch- not long enough to allow for in-depth phylogenetic analyses aeota) (Fig. 1). Of the bacteria, sequences are most com- or primer and probe design. Further, taxonomic assignment monly affiliated with the Proteobacteria (especially is generally facilitated using reference databases such as Gammaproteobacteria and Alphaproteobacteria), Bacteroi- Silva (Quast et al., 2013) or Greengenes (DeSantis et al., detes, Cyanobacteria and Firmicutes. Fewer sequences 2006), and these databases include sequences from were associated with candidate divisions, including BRC1, biomedical-based studies and it is often difficult to identify OD1,OP11,OP3,OP8,SR1,TM7andWS3. sequences originating from corals, thus hindering the devel- In order to examine if the phyla-based distribution of opment of coral-specific analyses and resources, such as sequences in this database are representative of those specific primers and probes. available in next-generation sequencing studies, we In order to examine relatedness of microorganisms examined phyla-based diversity of 715,114 coral– detected by sequencing approaches across numerous microbial sequences spanning nine studies and 23 coral studies of corals, we followed an approach utilized by Sim- species from the Alcyonacea as well as complex and ister et al. (2012) to examine the phylogeny of sponge- robust Scleractinian lineages available within the Coral associated microbes. Specifically, we utilized publically Microbiome Portal (CMP) (https://vamps2.mbl.edu/portals/ available Sanger produced sequence data from worldwide CMP) (Sunagawa et al., 2010; Barott et al., 2011; Morrow studies of corals to produce the Coral Microbiome Data- et al., 2012; Apprill et al., 2013; Bayer et al., 2013a, b; base (Coral MD), a coral-specific SSU rRNA gene data- Bourne et al., 2013; Šlapeta and Linares, 2013; Lesser base hosting 21,100 coral–microbial sequences, housed and Jarett, 2014). We identified high consistency within the ARB software (Ludwig et al., 2004). We utilized between the two types of sequence data, with three line- the Coral MD to examine the phylogenetic diversity of ages represented in the next-generation studies that coral-associated bacteria and archaea and these findings were not present in the Sanger database (Candidate divi- were confirmed in a subset of next-generation sequencing sion OP10, TG-1 and WS6), and five lineages in the data available for corals. Secondly, we identified putative Sanger sequencing based Coral MD database (BD1-5, monophyletic, coral-specific sequence clusters and exam- Elusimicrobia, NPL-UPA2, SHA-109 and WCHB1-60) ined the distribution of these clusters among the Alcyona- which were absent from the CMP. While examination of cea (soft corals) and the two main phylogenetic lineages of additional next-generation sequencing based studies Scleractinia (robust/short and complex/long), which are sep- may further increase the phyla-based diversity of coral arated by approximately 250 million years of divergence microbes, these findings are, to our knowledge, the first (Romano and Palumbi, 1996; Medina et al., 2006; Fukami to suggest such high (~ 39 Sanger from Coral MD; et al., 2008). Our phylogenetic-framed findings and the 42 from Coral MD + CMP database) phyla-based micro- Coral MD provide a novel resource for the community of bial diversity associated with corals. This diversity is simi- scientists studying coral-associated microbes, and may lar to the number of phyla observed in sponges,

6000 Cultured Uncultured

5000

4000

3000 Number of sequences 2000

1000

0 TM6 TA06 TA06 BD1-5 * * Chlorobi SHA-109 Firmicutes Nitrospirae Chloroflexi Tenericutes Tenericutes NPL-UPA2 NPL-UPA2

Chlamydiae * WCHB1-60 Unclassified Fusobacteria Spirochaetae Synergistetes Fibrobacteres Bacteroidetes

* Lentisphaerae Acidobacteria Elusimicrobia Cyanobacteria Euryarchaeota * * * Actinobacteria * Deferribacteres

* Planctomycetes * * * * * Verrucomicrobia * Thaumarchaeota * * * * Armatimonadetes * * Betaproteobacteria * * Deltaproteobacteria Gemmatimonadetes Alphaproteobacteria

* Other Proteobacteria Unclassified Bacteria Epsilonproteobacteria

* Gammaproteobacteria Deinococcus-Thermus * * * Candidate division SR1 Candidate division OP3

Candidate division OD1 * Candidate division OP8 Candidate division WS3

Candidate division TM7 Candidate division * Candidate division OP11 Candidate division OP11 * Candidate division BRC1 Candidate division * * * * * * * * * * Archaea Bacteria

Fig. 1. Sequences in the Coral Microbial Database. (21,100 Sanger-produced sequences from archaeal and bacterial SSU rRNA genes) classi- ﬁed to phyla, candidate phyla and phyla-level lineages from cultivated cells and environmental samples (uncultivated), based on the Silva reference taxonomy (Pruesse et al., 2007). Phyla that are also represented in next-generation sequencing data available in the Coral Microbiome Portal are denoted (*).

41 (Thomas et al., 2016), but higher than many other Table S1). The abundant groups included Vibrio (1217 host systems including the human skin, 19 phyla (Grice sequences), found in 42 coral species, Endozoicomonas, et al., 2009), termite, 10 phyla (Rahman et al., 2015) and with 943 sequences found in 35 coral species and an honeybee, 5 phyla (Moran et al., 2012). It is interesting uncultured group of the Rhodobacteraceae family that such high similarity exists between the coral and (Roseobacter clade) with 890 sequences from 37 coral sponge microbial phyla-based diversity, as corals are species. Other lineages with large numbers of sequences host to more biochemical microenvironments of varying included Marine Group II Euryarchaeota (493 sequences, light, oxygen and pH, which, compared with sponges, 5 coral species), Marine Group I Thaumarchaeota which may provide numerous habitats for microorgan- (443 sequences, 7 coral species), and Ruegeria isms (Kühl et al., 1995; Helmuth et al., 1997). Bacteria (356 sequences, 36 coral species) (Fig. 2). Lineages with are known to reside in the tissues (Lesser et al., 2004; over 200 sequences represented in the database include: Ainsworth et al., 2006), outer protective mucus layer Paenibacillus, Mycoplasma, Pseudoalteromonas, Fusi- (Ducklow and Mitchell, 1979), and skeleton (Crossland bacter, Marinifilum, Bacillus, ‘Candidatus Nitrosopumi- and Barnes, 1976) of hard (scleractinian) corals, the lus’, “Candidatus Amoebophilus” and undescribed major host type among the sequences examined. Scler- groups associated with Caldilineaceae, Flavobactera- actinian corals harbour several distinct tissue layers ceae and Alteromonadaceae families (Fig. 2), and including an external protective ectoderm and an endo- sequences from most of these microbial groups were dermal gastrovascular cavity that is used for digestion from less than 20 coral species. The abundant bacterial and also houses Symbiodinium, and these niches pro- groups identified here corroborate with those identified by vide further habitat differentiation for microorganisms Mouchka et al. (2010), from 32 SSU rRNA gene studies (Ainsworth et al., 2006; Rosenberg et al., 2007). with Sanger produced sequence data. Their dataset of The Coral MD was queried to describe the most abun- 5711 sequences similarly found high observations of dant coral–microbial lineages, represented by more than Rhodobacteraceae, Vibrio, Oceanospirillales, as well as 100 sequences in the database, and these lineages Cyanobacteria, associated with corals of various health spanned 14 phyla (Fig. 2, Supporting Information states, but only reported 11 bacterial phyla.

Number of sequences 0 200 400 600 800 1000 1200 1400 Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio Gammaproteobacteria;Oceanospirillales;Hahellaceae;Endozoicomonas Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae Euryarchaeota;Thermoplasmata;Thermoplasmatales;Marine Group II Thaumarchaeota;Marine Group I Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;Ruegeria Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Paenibacillus Tenericutes;Mollicutes;Mycoplasmatales;Mycoplasmataceae;Mycoplasma Chloroflexi;Caldilineae;Caldilineales;Caldilineaceae;uncultured Gammaproteobacteria;Alteromonadales;Pseudoalteromonadaceae;Pseudoalteromonas Firmicutes;Clostridia;Clostridiales;Family XII;Fusibacter Bacteroidetes;unclassified Bacteroidetes;Cytophagia;Cytophagales;Flammeovirgaceae;"Candidatus Amoebophilus" Gammaproteobacteria;unclassified Gammaproteobacteria;Alteromonadales;Alteromonadaceae Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae Bacteroidetes;Bacteroidia;Bacteroidales;Marinilabiaceae;Marinifilum Thaumarchaeota;Marine Group I;"Candidatus Nitrosopumilus" Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus Cyanobacteria;SubsectionIII;FamilyI Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;uncultured Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae Gammaproteobacteria;Vibrionales;Vibrionaceae;Photobacterium Alphaproteobacteria;Rickettsiales;SAR116 clade Alphaproteobacteria;Rhodospirillales;Rhodospirillaceae;uncultured Deltaproteobacteria;Desulfovibrionales;Desulfovibrionaceae;Desulfovibrio Alphaproteobacteria;Rhodospirillales;Rhodospirillaceae;Thalassospira Bacteroidetes;Cytophagia;Cytophagales;Flammeovirgaceae Firmicutes;Clostridia;Clostridiales Alphaproteobacteria; unclassified Gammaproteobacteria;Oceanospirillales Cyanobacteria;SubsectionIII;FamilyI;Oscillatoria Gammaproteobacteria;Alteromonadales;Alteromonadaceae;Alteromonas Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Arcobacter Gammaproteobacteria;Pseudomonadales;Moraxellaceae;Acinetobacter Alphaproteobacteria;Rhodospirillales;Rhodospirillaceae Number of coral species Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;NS5 marine group Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;Shimia > 40 Cyanobacteria;Cyanobacteria Gammaproteobacteria;Xanthomonadales;JTB255 marine benthic group 30 - 39 Proteobacteria;Alphaproteobacteria;Rhodobacterales;Rhodobacteraceae;Thalassobius Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;Pseudomonas 20 - 29 Alphaproteobacteria;SAR11 clade;Surface 1 Gammaproteobacteria;Pseudomonadales;Moraxellaceae;Psychrobacter 10 - 19 Actinobacteria;Actinobacteria;Propionibacteriales;Propionibacteriaceae;Propionibacterium Cyanobacteria;SubsectionI;FamilyI;Synechococcus < 10

Fig. 2. Abundances of archaeal and bacterial SSU rRNA gene sequences in the Coral Microbial Database according to most detailed taxonomic lineage. The colours reﬂect the representation of these sequences across different species of coral.

Coral-specific microbial sequence clusters this time that data is not compiled in a way that can be querried. Therefore, we identify these lineages as puta- In order to identify microbial groups that may be specific tively monophyletic to corals. to the coral holobiont, we interrogated the most common Bacteria from the genus Vibrio have disparately been taxonomic groups to determine the presence of mono- shown to be highly abundant (Bourne and Munn, 2005; phyletic clusters originating only from corals. This Koren and Rosenberg, 2006) or only a small component approach follows that used by Simister et al. (2012) for (Apprill et al., 2013; Roder et al., 2014) of the coral associ- sponge-associated microorganisms. Clusters were ated microbial community. They are frequently associated assigned where three or more high-quality sequences consistently formed a monophyly across neighbour join- with diseases including white syndrome, white band dis- ing, maximum likelihood and maximum parsimony ease, yellow band disease and bleaching (see Tout et al., methods of phylogenetic reconstruction (Supporting Infor- 2015 and reference therein). Our Vibrio phylogenetic anal- mation Methods). The taxa examined included those ysis also included the closely related genera Photobacter- associated with the largest number of coral species, ium, Salinivibrio, Enterovibrio and Grimonita. Collectively including Vibrio, Endozoicomonas and Ruegeria, as well there were 1461 coral associated sequences in the data- as sequence clusters with relatives with known functions base that belonged to these genera, with 1217 assigned including the eukaryotic parasite “Candidatus Amoebo- to Vibrio. Within the genus Vibrio, 37 putative monophy- philus”, Thaumarchaeota including the ammonia oxidizing letic coral associated clusters (denoted VC1-37) were “Candidatus Nitrosopumilus” and under recognized Cya- identified, and 15 of these clusters were associated with nobacterial lineages belonging to subsection III, Family both the complex and robust phylogenetic lineages of the I. We recognize that there may be next-generation Scleractinia (Fig. 3). Cluster VC31, associated with Den- sequencing data available from other non-coral environ- dronephthya, was exlusive to the Alcyonacea. The largest ments which are associated with these lineages, but at cluster (VC22) contained 37 sequences exclusively from

VC1 Montastraea(Orbicella) (9) VC2 Colpophelia, Diploria, Favia, Montastraea(Orbicella) (7) VC3 Colpophelia, Diploria, Montastrea(Orbicella), Mussismilia, Turbinaria (17) VC4 Colpophelia, Diploria, Favia, Montastraea(Orbicella), Montipora (6) Clone sequence from seawater (HM032018) Vibrio harveyi ATCC strain 35084 (EU130475) VC5 Acropora (3) VC6 Acropora, Favites, Pocillopora (12) VC7 Acropora, Favites, Pocillopora (10) VC8 Acropora, Pocillopora (5) VC9 Host genera unrecorded (7) Isolate sequence from the coral Turbinaria mesenterina (EU276981) Vibrio rotiferianus type strain LMG 21460T (AJ316187) Clone sequence from the coral Oculina patagonica Vibrio alginolyticus ATCC strain 17749 (X56576) VC10 Acropora, Favites, Montastrea(Orbicella), Oculina, Pocillopora (19) VC11 Montastrea(Orbicella), Turbinaria (9) Clone sequence from the coral Turbinaria mesenterina (EU267662) VC12 Montastraea(Orbicella) (6) Clone sequence from the coral Montastraea(Orbicella) faveolata (EU854878) VC13 Montastraea(Orbicella), Oculina (4) Clone sequence from the coral Montastraea(Orbicella) faveolata (EU854849) Clone from the coral Acropora eurystoma (GU319733) VC14 Acropora (4) Vibrio owensii strain DY05 (GU018180) Clone sequence from the coral Millepora dichotoma (HQ288770) VC15 Diploria, Montastraea(Orbicella), Oculina, Turbinaria (30) Isolate sequence from the coral Acropora digitifera (EU660311 ) Vibrio parahaemolyticus ATCC strain 17802 (AF388389) Vibrio natriegens ATCC strain 14048 (HM771343) VC16 Acropora, Favites, Pocillopora (5) VC17 Host genera unrecorded (6) Isolate sequence from the coral Fungia echinata (JQ409372) VC18 Host genera unrecorded (4) Isolate sequence from the coral Acropora hyacinthus (GU903236) VC19 Acropora, Montipora, Pocillopora (16) Isolate sequence from the coral Acropora digitifera (EU660325) VC20 Acropora, Pocillopora (6) Clone sequence from the coral Acropora tenuis (GU184362) VC21 Dendronephytha, Montastraea(Orbicella), Oculina, Turbinaria (34) Vibrio chagasii strain R-3712 (AJ316199) Vibrio crassostreae strain CAIM 1405 (EF094887) Clone sequence from the coral Acropora eurystoma (GU319758) VC22 Montastraea(Orbicella) (37)

VC23 Fungia, Montastraea(Orbicella) (6) Clone sequence from coral Montastraea(Orbicella) faveolata (EU517640) Isolate sequence from the coral Fungia echinata (JQ409371) Clone sequence from the coral Mussismilia hispida (GU199894) VC24 Platygrya (3) Isolate sequence from the coral Lophelia pertusa (HQ640863) Unpublished clone sequence from coral with white plague disease Vibrio pectenicida strain LMG 19642 (JN039139) Vibrio orientalis ATCC strain 33934 (ACZV01000001) VC25 Acropora, Montipora (7) Vibrio coralliilyticus ATCC strain BAA−450 (JN039154) Isolate sequence from diseased Pseudopterogorgia americana (GQ391983) VC26 Montipora, Oculina, Pachyseris, Platygrya (9) Vibrio coralliilyticus ATCC strain BAA−450 (ACZN01000012) Clone sequence from the coral Porites astreoides (JN694921) Vibrio furnissii strain CIP 102972 (ACZP01000018) VC27 Acropora (4) Vibrio hepatarius type strain LMG 20362T (AJ345063) VC28 Montipora, Oculina, Pachyseris (9) Vibrio tubiashii ATCC strain 19109 (HQ890464) VC29 Acropora, Favites, Pocillopora (9) Clone sequence from the coral Platygyra carnosus (JF411528) Clone sequence from the coral Acropora tenuis (GU184499)

VC30 Acropora (21) VC31 Dendronephthya (6) Isolate sequence from the coral Acropora millepora (JN119271) Scleractinia, complex clade Vibrio strain LS12 (EF584071) VC32 Acropora, Montipora, Pseudopterogorgia (4) Scleractinia, robust clade Isolate sequence from a marine sponge (HM771338) VC33 Turbinaria (11) Scleractinia, complex & robust Isolate sequence from the coral Turbinaria mesenterina (EU276998) Clone sequence from the coral Montipora hispida (JX022501) Alcyonacea VC34 Turbinaria mesenterina (5) Alcyonacea & complex Scleractinia Isolate sequence from seawater (FJ457344) VC35 Host genera unrecorded (4) Isolate sequence from the coral Turbinaria mesenterina (EU267613) VC36 Montastraea(Orbicella), Oculina (4) Isolate sequence from reef biofilm (HQ439529) Vibrio agarivorans strain 289T(AJ310647) Clone sequence from Montipora hispida (JX022538) Isolate sequence from sediment (EU082035) VC 37 Favites, Pocillopora (5) VC38 Acropora, Montastraea(Orbicella), Platygyra, Pocillopora (10)

To outgroups Isolate sequence from seawater (JN968377) VC39 Tubinaria, Platygyra (7) Isolate sequence from the coral Turbinaria mesenterina (EU267624) Photobacterium leiognathi strain ATCC 25521T (X74686) Clone sequence from the coral Platygyra carnosus (JF411428)

Photobacterium (61)

Salinivibrio costicola strain ATCC 35508T (X74699) Clone sequence from the coral Acropora tenuis (GU184388) VC41 Acropora, Favites (11) Salinivibrio costicola strain H-178 (X95532) Salinivibrio proteolyticus strain AF-2004 (DQ092443) Enterovibrio (17) Grimontia (17) Photobacterium damselae strain NCIMB 2058 (X78105) Isolate sequence from the coral Acropora millepora (JN119268) VC40 Acropora (5) 0.10

Fig. 3. Phylogeny of coral-speciﬁc clusters within the Gammaproteobacteria genus Vibrio and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral-associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool in ARB. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 10% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters, and microbial species name in bold and described or type species in culture. Accession numbers for speciﬁc sequences are shown in brackets, where accession numbers are absent none were available. ATCC, American Type Culture Collection.

© 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology Reports, 11, 372–385 Coral microbiome database 377 Montastraea faveolata (now referred to as Orbicella faveo- Ruegeria species, with 1 cluster (denoted RC1) related lata) (Budd et al., 2012). Additional putative monophyletic to sequences from the genus Phaeobacter (Fig. 5). coral associated clusters were associated with the genera The Bacteroidetes genus “Candidatus Amoebophilus” Photobacterium (VC38-VC40) and Salinivibrio (VC41) and was originally described as freshwater intracellular proto- were exclusive to the Scleractinia. zoan symbionts (Horn et al., 2001). Whole genome anal- The Gammaproteobacterial genus Endozoicomonas ysis has uncovered a suite of mechanisms by which “Ca. and closely related Endozoicomonas-like sequences are Amoebophilus” are thought to interact with their host, one of the most recognized coral-associated bacteria including a high number of eukaryotic protein domains (Bayer et al., 2013b; Neave et al., 2016, 2017b) and (Schmitz-Esser et al., 2010). Interestingly, members of abundances of Endozoicomonas sequences have been this genus are dominant members of the microbiome of recorded at over 85% per colony (Morrow et al., 2012; Caribbean corals, and are specifically associated with Apprill et al., 2016). These cells are thought to play a role coral tissue (but not mucus) in relative abundances of up within host-associated marine communities in protein and to 70% of affiliated bacterial and archaeal sequences carbohydrate transport and cycling (Neave et al., 2017a) (Apprill et al., 2016). Both the current phylogenetic analy- although their specific function is still undetermined. We sis, and that conducted previously (Apprill et al., 2016) identified 918 coral-associated Endozoicomonas-like place the coral-associated sequences in a separate line- sequences in the database and our phylogenetic analysis age, adjacent to protozoan intracellular endosymbionts placed 798 of these into 10 monophyletic coral- (Fig. 6), and into two monophyletic clusters, AC1 and associated clusters, denoted EC1-EC10 (Fig. 4). The AC2, which were exclusive to the Scleractinia and found largest of these (EC10) contained 357 sequences span- across both robust and complex clades. Sequence clus- ning both the robust and complex lineages of Scleracti- ter AC1 contained 232 sequences from various coral nia, and the most closely related sequences are from fish species including Montastraea/Orbicella faveolata, Mon- and sponges (Fig. 4). The second largest (EC1) con- tastraea/Orbicella annularis, M. franksi, Siderastrea stel- tained 289 sequences and were widely associated with lata, Diploria strigosa, Porites astreoides and Montipora diverse hosts, including both clades of Scleractinia as hispida. Cluster AC2 contained 13 sequences and was well as the soft corals. Specific sequence clusters exclu- closely related to a seaweed endosymbiont (Fig. 6). It is sive to the Alcyonacea include EC6, found in Gorgonia. unclear if these sequences are endosymbionts of the Clusters exclusive to the robust clade of Scleractinia corals themselves or if instead they may be associated include EC4, EC8 and EC9, but no putative monophyletic with other members of the holobiont such as a coral- Endozoicomonas sequence clusters were exclusive to associated protozoan which is more prevalent in stony the complex Scleractinia, which includes the corals with compared with soft corals. often the highest relative abundance of Endozoicomo- The Thaumarchaeota Marine Group II, including the nas-like sequences in their microbiome including Porites ammonia oxidizing “Candidatus Nitrosopumilus”, were astreoides and Stylophora pistillata (Morrow et al., 2012; represented in the database from scleractinian corals Bayer et al., 2013b; Apprill et al., 2016). This suggests spanning both complex and robust clades, including that the complex clade corals may be colonized by Endo- Siderastrea stellata, Montipora hispida, Montastraea/ zoicomonas-like cells which are broad generalists for Orbicella faveolata, Fungia sp., Favia sp. and Acantas- many distantly related corals, rather than specialists to trea sp. (Fig. 7). Previous studies show that the Archaeal related corals. portion of the coral-associated microbial community can The paraphyletic alphaproteobacterial genus Ruegeria be dominated by sequences from this genus (Siboni has been identified as one of the more successful line- et al., 2008) and suggest that these microbes may play a ages of bacteria inhabiting a variety of marine environ- role in nitrogen cycling via ammonia oxidation due to the ments, equipped with a large diversity of metabolic presence of archaeal ammonia monooxygenase genes capabilities and regulatory pathways (Luo and Moran, (Beman et al., 2007; Siboni et al., 2012). Our phyloge- 2014). We analysed 353 coral-associated sequences netic analysis of sequences in this genus uncovered identified taxonomically as Ruegeria and also found a 12 monophyletic coral-associated clusters that were con- number of paraphyletic groups, with some more phyloge- sistent between all three treeing methods, denoted netically related to sequences from the Roseobacter NC1-NC12, with NC11 from the robust Scleractinia, clade, including Phaeobacter, Marinovum and Thalasso- including sequences from Acantastrea, Favia and Fungia coccus (Fig. 5). We identified 9 small monophyletic coral most closely related to the ammonia oxidizing Nitrosopu- associated clusters containing between 3 and milus maritimus (Könneke et al., 2005) (Fig. 7). Interest- 7 sequences, represented in either one or both clades of ingly, the majority of the sequence clusters were Scleractinia, and absent from soft corals. Eight of these exclusively associated with robust clade corals (NC1, clusters (denoted RC2-RC10) were related to described NC6, NC7, NC10 and NC11), suggesting that these

EC1 Acantastrea, Alcyonium, Eunicella, Favia, Fungia, Gorgonia, Montipora, Paramuricea (289) Clone sequence from coral Oculina patagonica Clone sequence from the coral Stylophora pistilla (JN635437) Clone sequence from the coral Acropora tenuis (GU184547) Clone sequence from unidentified coral (GQ924719) Clone sequence from the coral Acropora tenuis (GU184686) DGGE sequence from the coral Acropora millepora (JF834212) Clone sequence from the coral Favites abdita (GU184707) Clone sequence from the coral Acropora muricata (HQ180160) EC2 Acropora, Gorgonia (10) Clone sequence from the coral Stylophora pistilla (JN635425) Scleractinia, complex clade EC3 Favites, Pocillopora (46) Scleractinia, robust clade Clone sequence from the coral Pocillopora damicornis Clone sequence from Acropora tenuis (GU184581) Scleractinia, complex & robust Clone sequence from unidentifid coral (GQ924718) Clone sequence from the coral Acropora muricata (HQ180161) Alcyonacea Clone sequence from marine sediment (EU799933) Clone sequence from the tunicate Cystodytes dellechiajei (DQ884169) Alcyonacea & complex Scleractinia Alcyonacea & both clades of Scleractinia EC4 Colpophyllia (22) Endozoicomonas elysicola strain MKT110 (AB196667) Clone sequence from the coral Stylophora pistilla (JN635420) Clone sequence from the coral Stylophora pistilla (JN848830) Loripes lacteus (bivalve) gill symbiont (GQ853555)

EC5 Favites, Pocillopora (26) EC6 Gorgonia (9) Clone sequence from the sponge Ianthella basta (JN388027) Clone sequence from the coral Acropora muricata (HQ180156) Clone sequence from unidentified coral Clone sequence from the coral Alcyonium gracillimum (JF925005) Isolate sequence from the coral Fungia granulosa Clone sequence from the coral Alcyonium gracillimum (JF925006) Clone sequence from an unidentified Acropora coral Clone sequence from unidentified coral (GQ924728) Clone sequence from unidentified coral (GQ924726) Clone sequence from unidentified coral (GQ924704) Clone sequence from an unidentified Acropora coral Clone sequence from an unidentified coral (GQ924734) Clone sequence from an unidentified Acropora coral Clone sequence from the coral Muricea elongata (DQ917877) Clone sequence from an unidentified coral (GQ924730)

EC7 Acropora, Erythropodium, Gorgonia, Muricea (17) Endozoicomonas montiporae strain CL−33 (FJ347758) Clone sequence from the coral Erythropodium caribaeorum (DQ889911) Clone from the octocoral Eunicea fusca (EF657844) Clone sequence from an unidentified coral (GQ924731) EC8 Diploria, Montastraea(Orbicella) (12) Clone sequence from the coral Montipora hispida (GQ204922) Clone sequence from the coral Montipora hispida (JX022272) Clone sequence from the sponge Petrosia ficiformis (AM990755) Clone sequence from an unidentified Haplosclerida sponge (AB695089) Clone sequence from an unidentified sponge (FJ457274) Clone sequence from the sponge Halichondria okadai (AB054136) Clone sequence from the sponge Tsitsikamma favus (HQ287901) Isolate sequence from mucusthe coral Fungia granulosa Clone sequence from the sponge Chondrilla nucula (AM259915) Clone sequence from the mussel Bathymodiolus spp. (FM162189) EC9 Diploria (10) Clone sequence from the tunicate Cystodytes dellechiajei (DQ884170) Clone sequence from the coral Montipora hispida (GQ204921) To outgroups

EC10 Acropora, Colpophyllia, Montastraea(Orbicella), Porites (357)

Clone sequence from the fish Pomacanthus sexstriatus (EU884930) Clone sequence from the sponge Suberites domuncula (JN624846) Isolate sequence from the coral Alcyonium antarcticum Clone sequence from the sponge Ianthella basta (GU784983) Clone sequence from the sponge Tsitsikamma favus (HQ241774) Clone sequence from the sponge Tsitsikamma favus (HQ241786)

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Fig. 4. Phylogeny of coral-speciﬁc clusters within the Gammaproteobacteria genus Endozoicomonas and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral-associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 5% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters, and microbial species name in bold and described or type species in culture. Accession numbers for speciﬁc sequences are shown in brackets, where accession numbers are absent none were available.

Marinovum cluster (44)

Thalassococcus cluster (12) Clone sequence from the coral Montastraea(Orbicella) faveolata Clone sequence from seawater (GU061966) Clone sequence from the coral Porites astreoides (JN694924) Clone sequence from the coral Pocillopora damicornis (AY700622) Ruegeria mobilis strain NBRC 101030 (EU977137) Clone sequence from unidentified coral (JQ23593) Clone sequence from seawater (HQ338128) Clone sequence from seawater (AJ810843) Ruegeria scottomollicae type strain LMG 24367T (AM905330) Clone sequence from the coral Montipora hispida (GQ204807) RC1 Acropora, Montipora, Oculina (5) Clone sequence from the coral Diploria strigosa Clone sequence from marine sediment (FJ716898) Phaeobacter daeponensis type strain TF-218 (DQ981486) Phaeobacter caeruleus type strain LMG 24369T (AM943630) Clone sequence from seawater (HM031990) Clone sequence from Montipora hispida (JX022328) Clone sequence from deep sea vent (AM268741) Clone sequence from the coral Montastraea(Orbicella) annularis Clone sequence from the coral Montastraea(Orbicella) faveolata (FJ403103) Clone sequence from the sea urchin Tripneustes gratilla (AM930431) RC2 Montastraea(Orbicella), Montipora (7) Isolate sequence from the coral Turbinaria mesenterina (EU267637) Clone sequence from the coral Favia sp. (GQ455159) RC3 Montastraea(Orbicella) (4) Clone sequence from seawater (HM116852) Oceanobacterium insulare strain NBRC 102018 (AB681665) Clone sequence from the coral Porites astreoides (JN694832) Clone sequence from deep sea vent (JN874367) Clone sequence from Acropora tenuis (GU184732) Isolate sequence from marine sediments (GU176617) Isolate sequence from unidentified coral (JF411503) Clone sequence from the coral Montastraea(Orbicella) annularis (HM768686) Isolate sequence from a marine sponge (HQ908721) Clone sequence from the coral Acropora tenuis (GU174659) RC4 Diploria (2)

RC5 Sidastrea (5) Clone sequence from the coral Montastraea(Orbicella) faveolata (FJ425599) Isolate sequence from a marine sponge (HQ908680) Clone sequence from the coral Platygyra carnosus (JF411445) Clone sequence from the coral Diploria clivosa Isolate sequence from marine sediment (EU624444) Clone sequence from the coral Montipora hispida (JX022136) RC6 Platygyra (2) Isolate sequence from the coral Montastraea(Orbicella) annularis (FJ952790) RC7 Montastraea/(Orbicella)(6) Isolate sequence from the coral Montastraea(Orbicella) annularis (FJ952800) RC8 Acropora(4) Clone sequence from the sponge Ianthella basta (JN388032) Clone sequence from the coral Diploria strigosa (GU118286) Isolate sequence from the sponge Tedania anhelans (FM180519) Clone sequence from the coral Acropora tenuis (GU174650) Clone sequence from salt marsh (DQ421656) RC9 Montipora (3) Clone sequence from the coral Montipora hispida (JX022486) Ruegeria pomeroyi strain DSS−3 (CP000031) Isolate sequence from seawater (GU551937) Clone sequence from hydrothermal plume (JN874182) Clone sequence from the coral Montipora hispida (GQ204835) Clone sequence from the abalone Haliotis diversicolor (HQ393411) Scleractinia, complex clade Cone from the coral Pocillopora damicornis (GU185207) Scleractinia, robust clade Clone sequence from the coral Favia sp. (GQ455154) Ruegeria lacuscaerulensis isolate sequence (DQ915630) Scleractinia, complex & robust Isolate sequence from sponge Gelliodes carnosa (FJ937894) Clone sequence from the coral Favites abdita (GU185064) Isolate sequence from the coral Turbinaria mesenterina (EU276977) Clone sequence from freshwater biofilm (HQ326304) RC10 Host genera unrecorded (3) Clone sequencefrom the coral Millepora dichotoma (HQ288796) Ruegeria atlantica isolate sequence (D88527) Clone sequence from the coral Acropora digitifera (FJ695549) Isolate sequence from the coral Turbinaria mesenterina Clone sequenced from unidentified coral (HQ456721) Isolate sequence from the coral Turbinaria mesenterina (EU276972) Ruegeria atlantica sequence (HE584803) Clone sequence from the coral Montipora hispida (JX022408) Clone sequence from the squid Sepioteuthis lessoniana (AJ633976) Clone sequence from the coral Montipora hispida (GQ204840) Clone sequence from the coral Acropora tenuis (GU174668) Clone sequence from ballast water (FJ666154) Clone sequence from hypersaline mat (JN430927) To outgroups

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Fig. 5. Phylogeny of coral-speciﬁc clusters within the Alphaproteobacteria genus Ruegeria and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral-associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 10% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters, and microbial species name in bold and described or type species in culture. Accession numbers for speciﬁc sequences are shown in brackets, where accession numbers are absent none were available. ATCC, American Type Culture Collection. NBRC, NTE (National Institute of Technol- ogy and Evaluation) Biological Resource Centre.

AC1 Acropora, Diploria, Montastraea(Orbicella), Montipora, Pocillopora, Porites, Siderastrea (232) Scleractinia, complex & robust AC2 Diploria, Galaxea (13) Clone sequence from the alga Bryopsis sp. (HE648942) Clone sequence from unidentified coral (JQ235893) “Candidatus Amoebophilus asiaticus” (AB506780) Endosymbiont of the protozoan Acanthamoeba sp. ATCC 30868 (AY549546) “Candidatus Amoebophilus asiaticus” (AF366581) Endosymbiont of the protozoan Acanthamoeba sp. KA/E21 (EF140637) Clone sequence from soil (GQ264283) Endosymbiont of the protozoan Acanthamoeba sp. (AF215634) Clone sequence from soil (HQ397140) Clone sequence from hypersaline mat (JN522434) Clone sequence from hypersaline mat (JN527296) Clone sequence from hypersaline mat (JN435020) Cardinium endosymbiont of the insect Plagiomerus diaspidis (AY327472) Endosymbiont of the insect Brevipalpus lewisi (AB116515) Cardinium endosymbiont of the insect Tetranychus cinnabarinus (DQ369962) Endosymbiont of the insect Metaseiulus occidentalis (AY753170 ) Cardinium endosymbiont of the insect Culicoides peregrinus (AB506779) Clone sequence from intestinal tract of the fish Panaque nigrolineatus (JF681750) “Candidatus Paenicardinium endonii” (DQ314214) Clone sequence from from the intestinal tract of the fish Naso tonganus (HM630174) Flexibacter roseolus (AB078063) To outgroups

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Fig. 6. Phylogeny of coral-specific clusters within the Bacteroidetes genus “Candidatus Amoebophilus” and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral- associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 10% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters, and microbial species name in bold and described or type species in culture. Accession numbers for specific sequences are shown in brackets, where accession numbers are absent none were available. corals may harbour more specialized relationships with present in both soft corals and both clades of stony these microbes. Whole genome sequence analyses of corals. Leptolyngbya-related sequences from marine Nitrosopumilus strains indicate that members of A. eurystoma were found to persist even when exposed the genus are ammonia oxidizing autotrophs but vary in to reduced pH (Meron et al., 2011), suggesting that this other metabolic traits, possibly promoting niche segrega- group may be tolerant of a range of environmental tion and thus influencing nitrification rates in various envi- conditions. ronments (Bayer et al., 2015). Cyanobacteria are abundant primary producers in the Cultured isolates obtained from corals waters surrounding corals (Sorokin, 1973), and have been shown to fix dinitrogen in some coral species Of the 21,100 sequences in the Coral MD, only 1378 (Lesser et al., 2004). Despite the potential functional (6.5%) were generated from cultured isolates (Supporting importance of these cells, the vast majority of cyanobac- Information Table S2). A further 18,400 (87.4%) were terial sequences recovered from sequencing surveys of identified as uncultured (either from clone libraries or corals are not well-resolved taxonomically. We examined other PCR based methods), and the remaining 1271 the Cyanobacterial Family I lineage and identified sequences (6.1%) did not have enough associated data 17 monophyletic coral-specific sequence clusters span- to confirm their source (cultured or uncultured). Of the ning several described genera (Fig. 8). The largest clus- 41 identified taxonomic groups (which includes divisions ter (denoted CC14) was most closely related to clusters of the Proteobacteria), 14 contained cultured representa- CC13 and CC15, all from Montipora corals and related to tives (Fig. 1). Gammaproteobacteria (870 sequences), Hydrocoleum lyngbyaceum and Trichodesmium ery- dominated by sequences from the genera Vibrio thraeum (Fig. 8). The filamentous Phormidium/Leptolyng- (401 sequences), Pseudoalteromonas (149 sequences) bya genera contained 8 monophyletic clusters (denoted and Photobacterium (110 sequences) were the most CC1–8), with up to 14 sequences in each cluster, and abundant cultured organisms in the database followed by mostly spanning complex clade Scleractinia with CC8 Bacillus (Firmicutes, 71 sequences), Shewanella

NC1 Acantastrea, Fungia, Favia (8) Clone sequence from coral (FJ215907) NC2 Host genera unrecorded (4) Clone from Montipora hispida (JX021768) NC3 Favia, Montipora (15) NC4 Host genera unrecorded (6) Clone sequence from Montipora hispida (JX021894) NC5 Host genera unrecorded (7) Clone sequence from the coral Favia sp. (EU377086) Clone sequence from the coral Diploria labyrinthiformis (AY380688) NC6 Acantastrea, Fungia (11)

NC7 Acantastrea, Fungia, Favia (6) Clone sequence from the coral Acantastrea sp. (EU377042) Clones sequence from the coral Favia sp. (EU377331) NC8 Siderastrea (15) Clone sequence from a marine sponge (AY192644) Clone sequence from the coral Diploria labyrinthiformis (AY380677) Clone sequence from the coral Favia sp. (EU377358) Clone sequence from marine sediment (JQ258629) Clone sequence from the coral Acantastrea sp. (EU377045) Clone sequence from the coral Diploria strigosa (AY380697) Clone sequence from estuarine sediment (EF367469) Clone sequence from the coral Fungia sp. (EU377251) Clone sequence from the coral Acantastrea sp. (EU377265) Clone sequence from Montipora hispida (JX021977) Clone sequence from marine sediment (JQ258641) Clone sequence from estuarine sediment (EF367456) Clone sequence from the coral Acantastrea sp. (EU377016) Clones sequence from the coral Favia sp. (EU377360) Clone sequence from the coral Fungia sp. (EU377211) Clone sequence from the ascidian Cystodytes dellechiajei (EU283426) NC9 Acantastrea, Favia, Fungia (11) Clone sequence from the coral Fungia sp. (EU377219) Clone sequence from the coral Acantastrea sp. (EU377279) Clones sequence from the coral Favia sp. (EU377362) Clone sequence from deep marine sediment (AY505053) Clone sequence from the coral Montipora hispida (JX021971) Clone sequence from the coral Montastraea faveolata (JQ514898) Clone sequence from the coral Favia sp. (EU377177) Clone from Montipora hispida (JX021930) Clone sequence from estuarine sediment (DQ641792) Clone sequence from marine sediment (GU137368) Clone sequence from the coral Montipora hispida (JX021908) Clone sequence from marine sediment (FJ571709) Clone sequence from marine sediment (FJ571750) Clone sequence from deep marine sediment (EF069381) Clone sequence from the coral Montastraea(Orbicella) faveolata (JQ514915) Clone sequence from the coral Fungia sp. (EU377224) Clone sequence from the marine sponge Axinella polypoides (DQ299272) Clone sequence from the coral Montipora hispida (JX021896) Clone sequence from the coral Montastraea(Orbicella) annularis (AY380590) Clone sequence from the marine sponge Phakellia sp. (DQ299273) Clone sequence from marine sediment (GU137382) Clone sequence from the coral Montipora hispida (JX021973) Clone sequence from hypersaline groundwater (JF747757) Clone sequence from the coral Favia sp. (EU377194) Clone sequence from marine sediment (HM171809) Clone sequence from the coral Acantastrea sp. (EU377043) NC10 Montastraea(Orbicella)(5) Clone sequence from the coral Acantastrea sp. (EU377270) Clone sequence from estuarine sediment (DQ641805) Clone sequence from the coral Fungia sp. (EU377413) Clone sequence from estuarine sediment (EF367478) Clone from the coral Montipora hispida (JX021901) Clone sequence from estuarine sediment (DQ641832) Clone sequence from the coral Favia sp. (EU377087) Clone sequence from wetland (EU420707) Clone sequence from estuarine sediment (EF367660) Clone sequence from the coral Acantastrea sp. (EU377313) Clone sequence from a marine sponge (DQ299263) Clone sequence from the marine sponge Plakortis sp. (EF076092) Clone sequence from the coral Montipora hispida (JX021923) Clone sequence from the coral Fungia sp. (EU377420) Clone sequence from seawater (JQ227647) NC11 Acantastrea, Favia, Fungia (6) Clone sequence from the coral Favia sp. (EU377335) Clone sequence from estuarine sediment (DQ641746) Clone sequence from the coral Acantastrea sp. (EU377022) Clone sequence from the coral Montipora hispida (JX021976) Clones sequence from the coral Fungia sp. (EU377242) Nitrosopumilus maritimus isolate sequence (DQ085097) Clone sequence from seawater (EU817631) Clone sequence from the coral Fungia sp. (EU377393) Clone sequence from seawater (JQ221178) Clone sequence from sperm whale fall (AB426393) Clone sequence from the coral Montipora hispida (JX021820) Clone sequence from marine sediment (GU270164) Isolate sequence from strain BD31 (AEXL01000085) Clone sequence from deep sea sediment (HM103770) Clone sequence from the coral Acantastrea sp. (EU377297) Clone sequence from the marine sponge Luffariella (EU049820) Clone sequence from wetland (EU420706) Clone sequence from the coral Acantastrea sp. (EU377288) Clone sequence from marine sediment (HM171793) Clone sequence from the coral Favia sp. (EU377336) Clone sequence from marine sediment (HQ267323) NC12 Favia, Fungia (3) Scleractinia, complex clade Clone sequence from the coral Favia sp. (EU377340) Clone sequence from a marine sponge (DQ299265) Scleractinia, robust clade Clone sequence from marine sediment (FJ971113) Clone sequence from a marine sponge (JN162067) Scleractinia, complex & robust Clone sequence from the coral Fungia sp. (EU377207) Clone sequence from the sponge Axinella polypoides (DQ299266) Clone sequence from a marine sponge (AY192645) Clone sequence from the coral Montipora hispida (JX021935) Clone sequence from a marine sponge (DQ299268) Clone sequence from the coral Fungia sp. (EU377227) Clone sequence from the sponge Astrosclera willeyana (JN113061) Clone sequence from the sponge Luffariella sp. (EU049816) Clone sequence from the sponge Luffariella sp. (EU049817) Isolate sequence from the coral Diploria strigosa (AY380715) Cenarchaeum symbiosum from a marine sponge (U51469) Clone sequence from hydrothermal vent (AB019717) Clone sequence from hydrothermal vent (AB007303) To outgroups 0.10

Fig. 7. Phylogeny of coral-speciﬁc clusters within the Thaumarchaeota Marine Group II “Candidatus Nitrosopumilus” and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral-associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 10% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters, and microbial species name in bold and described or type species in culture. Accession numbers for speciﬁc sequences are shown in brackets, where accession numbers are absent none were available.

CC1 Acropora (5) Phormidium persicinum SAG 80.79 (EF654085) Leptolyngbya sp. PCC 7375 (AB039011) Clone sequence from the coral Porites sp. (EF372581) . Leptolyngbya sp. clone sequence ( DQ917847) CC2 Acropora (9) Clone sequence from the coral Montastraea(Orbicella) faveolata CC3 Acropora, Montastraea(Orbicella) (8)

CC4 Montastraea(Orbicella) (3) Clone sequence from the coral Acropora eurystoma (GU319449) Clone sequence from the coral Turbinaria mesenterina Leptolyngbya sp. RS03 (JF518829) Clone sequence from the coral Diploria strigosa (HM768618) Clone sequence from a marine macroalga (GU451384) Clone from the coral Siderastraea stellata (HM216518) Clone sequence from the coral Acropora eurystoma (GU319317) Clone sequence from the coral Montastraea(Orbicella) faveolata (JQ516288) CC5 Acropora (10) Clone sequence from the coral Pocillopora damicornis (GU185724) Filamentous cyanobacterium FLK9 (EU196364) Bacterial clone from the coral Sidastrea siderea (EF372582) Phormidium priestleyi strain ANT.LACV5.1 (AY493586) Clone sequence from Antarctic microbial mat (AY151722) CC6 Turbinaria (4) Phormidium sp. MBIC10818 (AB183571) Phormidium/Leptolyngbya Phormidium sp. F3 (AF091110) Clone sequence from the coral Montipora hispida (GQ204794) CC7 Turbinaria (4) Leptolyngbya sp. isolate sequence from limestone cliff (GQ162218) Leptolyngbya sp. isolate sequence from Antarctic quartz (AF170757) Clone sequence from marine macroalga (GU451406) Clone sequence from unidentified coral (JQ236258) Pseudophormidium sp. strain ANT.LPE.3 isolated from Antarctica (AY493587) Clone sequence from the coral Montastrea faveolata (FJ202667) CC8 Acropora, Erythropodium, Montastraea(Orbicella), Siderastraea (14) Isolate sequence ‘Gollwitz Poel’ (EF654035) Clone sequence from the coral Acropora eurystoma (GU319364) Clone sequence from the coral Montastraea cavernosa Isolate sequence from the Phormidium strain MBIC100709 (AB058219) Isolate sequence from strain LLi71 from a hot volcanic stream (DQ786167) CC9 Dendrogyra, Montastraea(Orbicella) (6) Isolate sequence from a salt lake (GQ243430) Clone sequence from unidentified coral (JQ236033) Clone sequence from the deep sea (HM799099) Clone sequence from unidentified coral Isolate sequence from the sponge Petrosia ficiformis (GU220365) Clone sequence from the coral Acropora eurystoma (GU319174) Clone sequence from unidentified coral Clone sequence from an alkaline lake (JN825332) Clone sequence from the coral Montastraea(Orbicella) faveolata Clone sequence from the coral Acropora eurystoma (GU319250) Clone sequence from unidentified coral (GU118152) Clone sequence from an alkaline lake (JN825330) Isolate sequence from strain PCC 790 (AF216946) CC10 Diploria, Montipora (4) Clone sequence from hypersaline mat (JN435649) Clone sequence from wetland sediment (J516843) Clone sequence from the coral Gorgonia ventalina (HM768630) Oscillatoria corallinae strain CJ1 SAG8.92 (X84812) Clone sequence from the coral Siderastraea stellata (HM216501)

Clone sequence from the anthozoan Muricea elongata. (DQ917838) Oscillatoria Oscillatoria spongeliae isolated from a marine sponge (AF420446) Clone sequence from tropical marine habitat (JF26.)2062 CC11 Gorgonia, Montastraea(Orbicella) (19) Spirulina sp. strain PCC 6313 (AM709631) Spirulina subsalsa strain PD2002/gca (AY575935) Spirulina sp. strain MPI S4 (Y1.)8792 Clone sequence from marine microbial mat (GQ441247) Clone sequence from the coral Gorgonia ventalina (HM768633) Clone sequence from unknown coral Clone sequence from the coral Gorgonia ventalina (HM768659) Spirulina Spirulina subsalsa strain IAM M−223 (AB003166) Clone sequence from hot spring (AF445678) Spirulina sp. strain CCC Snake .P. Y-85 (Y18793) Clone sequence from unidentified coral CC12 Acropora, Diploria (11) Schizothrix PNG5−22 (EU445293) Symploca sp. strain HPC−3 (AY430150) Symploca atlantica strain CCY9617 (GQ402026) Clone sequence from unidentified coral Symploca sp. strain VP642b (AY032933) Clone sequence from unidentified coral Symploca Clone sequence from dust storm (EF683073) CC13 Montipora (5) Lyngbya sp. strain BAN TS04 (HQ419196) Clone sequence from Montipora hispida (JX022377) Hydrocoleum lyngbyaceum strain HBC7 (EU249124) Clone sequence from the coral Porites lutea (AY123040) Oscillatoria sp. strain PAL08−3.2 (HM585025) Clone sequence from the coral Montipora hispida (JX022250) Trichodesmium erythraeum IMS101 (CP000393CP000393) Clone sequence from the coral Montipora hispida (JX022175)

CC14 Montipora (26)

CC15 Montipora (19) Planktothrix rubescens strain NIVA−CYA 18 (AB045925) Planktothrix agardhii (AB045900) Clone sequence from freshwater (JN398120) Scleractinia, complex clade CC16 Gorgonia (19) Oscillatoria acuminata (AB039014) Scleractinia, robust clade Phormidium sp. strain CENA135 (HQ730084) Clone sequence from unidentified coral Phormidium sp. strain ETS−05 (AJ548503) Scleractinia, complex & robust Planktothricoides raciborskii strain NIES−207 (AB045960) Clone sequence from the coral Porites sp. Alcyonacea Clone sequence from the coral Siderastrea siderea Clone sequence from a marine bivalve (FM995184) Alcyonacea & robust Scleractinia Clone sequence from the coral Favites abdita (GU185007) Clone sequence from coastal sediment (FM242281) Alcyonacea & both clades of Scleractinia Oscillatoriales cyanobacterium srain WBK15 (GQ324967) Tychonema bourrellyi strain CCAP 1459/11B (AB045897) CC17 Porites, Siderastrea (7) Clone sequence from unidentified coral. with black band disease (EF154084) Clone sequence from mixed Cyanobacteria consortium (FN794239) Phormidiaceae cyanobacterium strain SAG 31.9 (EF654088) Clone sequence from unidentified coral (JQ236160) Isolate sequence from marine cyanobacterium strain VP0401 (AY599507) Clone sequence from hot spring (AF445719) Clone sequence from salt lake (HQ425199) To outgroups 0.10

Fig. 8. Phylogeny of coral-speciﬁc clusters within the Cyanobacterial ‘Family I’ and related sequences, based on 16S ribosomal RNA genes. The displayed tree is a maximum likelihood tree constructed based on long (> 1200 bp) sequences only; shorter coral-associated sequences (indicated by dashed lines) were added using the ARB Parsimony (Quick add marked) tool. Filled circles indicate bootstrap support (maximum parsimony, with 1000 resamplings) of > 90%, and open circles represent > 75% support. Bar indicates 10% sequence divergence. The numbers of sequences in each cluster are indicated in brackets and the coral host genera for each cluster are listed adjacent to clusters. Accession numbers for speciﬁc sequences are shown in brackets, where accession numbers are absent none were available.

(Gammaproteobacteria, 27 sequences) and Ruegeria and healthy Fungiidae corals. Environ Microbiol 15: (Alphaproteobacteria, 25 sequences) (Fig. 1). A high 2063–2072. number of Actinobacteria (114 sequences) were repre- Apprill, A., Weber, L., and Santoro, A. (2016) Distinguishing sented by cultures, primarily from the genera Streptomy- between microbial habitats unravels ecological complexity in coral microbiomes. mSystems 1: e00143-00116. ces, Micromonospora, Kocuria, Micrococcus and Barott, K.L., Rodriguez-Brito, B., Janouškovec, J., Brachybacterium. A number of key taxa were dominant Marhaver, K.L., Smith, J.E., Keeling, P., and Rohwer, F.L. – in the database notably Euryarchaeota, Thaumarch- (2011) Microbial diversity associated with four functional aeota, Chloroflexi, Tenericutes, Spirochaetae and Gem- groups of benthic reef algae and the reef-building coral matimonadetes – yet contained no cultured Montastraea annularis. Environ Microbiol 13: 1192–1204. representatives (Fig. 1). These groups denote important Bayer, T., Arif, C., Ferrier-Pagès, C., Zoccola, D., targets for future culture studies, along with highly abun- Aranda, M., and Voolstra, C.R. (2013a) Bacteria of the dant groups with minimal cultured representatives such genus Endozoicomonas dominate the microbiome of the as Endozoicomonas (4 cultured sequences) and Paeni- Mediterranean gorgonian coral Eunicella cavolini. Mar Ecol Prog Ser 479:75–84. bacillus (1 cultured representative) (Fig. 1). Bayer, T., Neave, M.J., Alsheikh-Hussain, A., Aranda, M., Yum, L.K., Mincer, T., et al. (2013b) The microbiome of the Red Sea coral Stylophora pistillata is dominated by Utility of the coral MD tissue-associated Endozoicomonas bacteria. Appl Environ – The Coral MD provides a number of support features that Microbiol 79: 4759 4762. may advance coral microbiology studies. The sequences Bayer, B., Vojvoda, J., Offre, P., Alves, R.J., Elisabeth, N.H., Garcia, J.A., et al. (2015) Physiological and genomic char- and their placement in ARB software are an ideal sce- acterization of two novel marine thaumarchaeal strains nario for primer and probe design. The database provides indicates niche differentiation. ISME J 10: 1051–1063. fi a reference taxonomic framework for sequence classi - Beman, J.M., Roberts, K.J., Wegley, L., Rohwer, F., and cations for amplicon sequence datasets produced from Francis, C.A. (2007) Distribution and diversity of archaeal corals. Additionally, the database offers descriptive infor- ammonia monooxygenase genes associated with corals. mation about the coral-associated microbes in a phyloge- Environ Microbiol 73: 5642–5647. netic structure. The numerous taxonomic groups of Bourne, D.G., and Munn, C.B. (2005) Diversity of bacteria bacteria and archaea associated with corals can be over- associated with the coral Pocillopora damicornis from the – whelming to newcomers in the field, and the database Great Barrier Reef. Environ Microbiol 7: 1162 1174. provides a context to familiarize the user with phyloge- Bourne, D.G., Dennis, P.G., Uthicke, S., Soo, R.M., Tyson, G.W., and Webster, N. (2013) Coral reef inverte- netic groups that frequently associate with corals. Over- brate microbiomes correlate with the presence of photo- all, the database has the potential to facilitate coral symbionts. ISME J 7: 1452–1458. microbiology research toward the goal of understanding Bourne, D.G., Morrow, K.M., and Webster, N.S. (2016) the role of coral-associated microbes in the health and Insights into the coral microbiome: underpinning the health ecology of corals. and resilience of reef ecosystems. Annu Rev Microbiol 70: 317–340. Budd, A.F., Fukami, H., Smith, N.D., and Knowlton, N. ACKNOWLEDGEMENTS (2012) Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia). 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