Sponge-Specific Phylotypes

One of the most striking features of sponge microbial ecology is the identification of sponge-specific phylotypes as defined by Hentschel (Hentschel et al., 2002). Numerous sequence clusters identified in this study can be classified as sponge- specific. From the sponge R. ramosa 17 sequence clusters, representing 2.8% of quality-filtered reads, constitute 2 novel sponge-specific clusters. One cluster of four sequences represents a novel sponge-specific cluster in the phylum Cyanobacteria (Figure 2.7) while 16 R. ramosa-derived clusters representing 391 sequences represent a novel sponge-specific cluster in the α- class of Proteobacteria (Figure 2.9). From S. stuposa, 18 sequence clusters representing 26% of sequences from that sponge align to ‘sponge cluster 23’ in the phylum Nitrospira (Figure 2.8) using the cluster numbering system used by Webster (Webster et al., 2010). Notable sponge-

specific clusters from numerous phyla (Chloroflexi, Bacteroidetes,

Gemmatimonadetes, Verrucomicrobia, Planctomycetes, Lentisphaerae,

Poribacteria, Spirochaetes, γ-Proteobacteria) identified in other sponge species were absent from the sponges examined here. In addition, Poribacteria-specific PCR primers failed to amplify a product from metagenomic DNAs of the sponges examined here (data not shown).

131 Stelligera_cluster_87(37) Stelligera_cluster_85(40) Stelligera_cluster_112(21) Stelligera_cluster_62(72) Stelligera_cluster_60(74) Stelligera_cluster_48(130) Stelligera_cluster_157(6) Stelligera_cluster_96(32) Stelligera_cluster_58(70) Stelligera_cluster_83(41) Stelligera_cluster_190(3) Stelligera_cluster_1(2420) Stelligera_cluster_41(169) Stelligera_cluster_94(33) Stelligera_cluster_142(8) Stelligera_cluster_194(3) Stelligera_c luster_195(3) Stelligera_cluster_185(4)

Agela dilatata clone EF076129 Agelas dilatata clone EF076152 Agelas dilatata c lone EF076168

Sponge Cluster 23

Theonella swinhoei clone AF186449 Aplysina aerophoba clone AJ347039 Theonella swinhoei clone AF186451 coral as sociated c lone AF365703 Theonella sp. clone AF434964 Theonella swinhoei clone AF186456 Theonella swinhoei clone AF186454 Theonella swinhoei clone AF186446 Plak toris sp. c lone EF076109

sediment c lone AY592123 Nitrospira marina Nb-295 X82559 Raspailia_clus ter_496(4)

deep sea sediment c lone AY375077 deep sea sediment c lone AB015550 marine sediment clone DQ351808

deep sea octac oral c lone DQ395470 Nullarbor caves clone AF317764

Nullarbor caves clone AF317762 Nitros pira moscoviensis X82558

fluidized bed reactor c lone AJ224039 nitrifying biofilm clone AB117711 fluidized bed reac tor clone AJ224041 Cymbastela concentrica clone AY942775

Cy mbas tela concentrica c lone AY942757

Leptos pirillum ferrooxidans AF356837 Leptospirillum ferrooxidans AF356829

Bac teroides fragilis M61006 hot spring clone AF027001

Thermodesulfovibrio islandicus X96726 Stelligera_cluster_129(11) Pseudomonas aeruginosa M34133

deep sea sediment c lone AY373422 Borrelia burgdorferi U03396

Magnetobacterium bavaricum X71838

Agrobacterium tumefasciens D14504 Stelligera_cluster_115(19) Chlamydia trachomatis U68443 Bifidobacterium bifidum S83624

0.05

Figure 2.8: Maximum Likelihood phylogenetic tree of bacteria from the phylum

Nitrospira including 18 clusters (3,166 sequences) derived from the marine sponge Stelligera stuposa recruiting to sponge cluster 23 according to the numbering system of Webster et al, 2010.

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sargasso sea metagenome AACY01000000AACY01008274 Roseobacter gallaeciensis AY881240

sargasso sea metagenome AACY01072860 Pseudoceratina clavata isolate DQ227659 Pseudoceratina clavata DQ227660 H.panicea isolateZ88566 H.panicea isolateZ88567 H.panicea isolateZ88569 H.panicea isolateZ88578 H.panicea isolateZ88582 L.chlorea cloneAY845238 L.chlorea cloneAY845239 L.chlorea cloneAY845240 L.chlorea cloneAY845241 L.chlorea cloneAY845242 L.chlorea cloneAY845243 L.chlorea cloneAY845244 L.chlorea cloneAY845245 sponge isolateAY362009 sponge isolateAY362016 sponge isolateAY368512 sponge isolateAY368574 sponge isolateAY371428 sponge isolateAY371430 sponge isolateAY369982 sponge isolateAY369984 sponge isolateAY369990 H.panicea cloneAY948354 H.panicea cloneAY948355 H.panicea cloneAY948358 H.panicea cloneAY948370 S.lacustris cloneAY598784 sponge isolateAB026194

Hyphomonas jannaschiana AJ227814 Trojanella thessalonices AF069496 Holospora obtusa X58198 Silicibacter pomeroyi AF098491 cuttlefish symbiont AJ244786 Ruegeria lacuscaerulensisU77644 cuttlefish symbiont AJ244791 Ruegeria atlantica D88526 Roseobacter sp. AY332662 Roseobacter sp. AY349460 hydrothermal vent clone AF254107 hypersaline mat isolate AF344287 lake isolate AY345413

D.dissoluta clone AY897099 mangrove clone DQ234106

Aegean sea clone AF406520 Plaktoris sp. clone EF076060

sediment clone AY221078 Plaktoris sp. cloneEF076066

Plaktoris sp. clone EF076069 Plaktoris sp. clone EF076081 Plaktoris sp. clone EF076084 Plaktoris sp. cloneEF076106 Sulfitobacter brevis Y16425

Roseobacter denitrificans M96746 Roseobacter litoralis X78312 Arctic sea ice clone AF468373 Octadecabacter artcticus U73725 dinoflagellate isolate AY258099 dinoflagellate isolateAY258100 sediment clone AY375143 marine clone AJ240910 brittle star symbiontU63548 marine bacterium AF365993 alga isolate U37762

diseased coral clone AF473915 Black Sea isolateAJ133762 Roseobacter algicola X78315 Ruegeria gelatitovora D88523 Roseobacter sp. AY167339 Roseobacter sp. AF026463 sediment isolate AF254105 mill effluent isolate AF388307 Roseobacter gallaeciensis Y13244 marine isolate AY239008 Erythrobacter citreus AF118020 Erythrobacter litoralis AB013354 R.odorabile clone AF384141 sponge isolateAB026492

Suberites domuncula isolateAY265806 Halichondria panicea isolate AY948375 Halichondria panicea isolate AY948367 sponge isolate AY367757

sponge isolate AY370007 sponge isolate AY370003 sponge isolate AY369983 sponge isolate AY369980 sponge isolate AY369979 sponge isolate AY369978 sponge isolate AY368572 Geodiidae sp. isolate AF489286 Halichondria sp. isolate Z88581 whale fall metagenome AAFZ01011523 coral isolate AY654831

HicBalt21390bprnaX52909 Cymbastella concentrica clone AY942776 sponge isolate AY542761

Chondrilla nucula clone DQ079037 Chondrilla nucula clone DQ079035

Azospirillum irakense X79737 Rhodovulum imhoffii AM180953 Rhodovulum euryhalinum D16426 Rhodovulum strictum D16419 Jannaschia helgolandensis AJ438157 sargasso sea isolate L15345 Spongilla lacustris clone AY368522 Spongilla lacustris clone AY598791

Halichondria panicea isolateZ88572

sargasso sea metagenome AACY01099207 Roseomonas cervicalis AY150047

sponge isolate AY369981 sponge isolate AY362010

sponge isolate AF414882 sponge isolate AY362017 sponge isolate AY368505 sponge isolate AY371414 sponge isolate AY369996 sponge isolate AY369997 sponge isolate AY367744 sponge isolate AY367758 Ircinia stroboloina isolate AY584527 Theonella swinhoei clone AF434978 Rhodobium marinum D30790 Rhodobium marinum D14434

Chondrosia reniformis isolate AY562560 Chondrosia reniformis isolate AY562567 Chondrosia reniformis isolate AY562568 Fulvimarina pelagi AY178860

coral isolate AY065627 Mesorhizobium loti D14514

Mesohizobium mediteraneum L38825 Mesorhizobium sp. AY258089 Rhizobium giardinii U86344

Agrobacterium tumefaciens AJ389903 Rhizobium pisi AY509899

Stappia stellulata D88525 oyster isolate AF246614 dinoflagellate clone Y10915 Stappia aggregata D88520 marine isolate AY007682 mangrove isolate AB012760 Roseibium hamelinense D85836 Roseibium denhamense D85832 sediment isolate AY221043 sponge isolate AF414881

sponge isolate AY370010 sponge isolate AF414886 Stelligera_cluster_201(3)

Lamellodysidea herbacea isolate AY845236 Dysidea avara isolate DQ274151

Lamellodysidea herbacea isolate AY845237 sponge isolate AY368536

Sphingomonas paucimobilis U20776 Rhodobacter veldkampii D16421 Rhodobacter sphaerides X53854 Paracoccus aminovorans D32240 Paracoccus yeeii AY014179 dinoflagellate isolate AJ534213 Roseovarius tolerans Y11551 dinoflagellate isolate AY258091 reservoir clone AF418955

sponge clone DQ274149 sponge clone DQ274148 Erythrobacter flavus AF500004 sponge isolate AY367756 sponge isolate AY371411 sponge isolate AY371410 Erythrobacter sp. AJ849370 Phyllospongia papyracea clone AY845233 Chondrilla nucula clone DQ079038

Halichondria panicea clones (26) plus sargasso sea metagenome (1)

sponge isolate AY371399 sponge isolate AY368535

133

Figure 2.9 continued

s ponge is olate AY371399 s ponge is olate AY368535 Spongilla lac ustris clone A Y466480 s ediment c lone AY 375070 s ediment c lone AY 375078 s ponge is olateAY368534

Ras pailia_c luster_204(12) Ras pailia_c luster_243(10)

'orange sponge' c lones (13)

Agelas dilatata clone EF076136

Bac teroides fragilis M61006 C. nucula c loneDQ079030

hot spring cloneA Y555804 C. nucula c loneDQ079036 C. nucula c loneDQ079033 C. nucula c loneDQ079031 Rc ketts ia ric ketts ii L36217

sponge isolate AY371443

Aegean Sea cloneA F406523 Phyllos pongia papy rac ea c lone AY 845234 Alphaprot eobact erium MGP-68 AF530148 Halic hondria panic ea isolateZ88576

Dis codermia diss oluta clone A Y897108 s ponge is olate AY362018

s ponge is olate AY364594

s ponge is olate AY371429 s eawat er is olate DQ167235 s ponge is olate AY371436 marine c lone AF143834 sponge isolate AY371451 gutless worm clone AJ890093 Antartic lak e mic robial mat is olate AJ441010 sediment is olat e AF530152

Halic hondria panic ea c lone Z88570 Cy mbastela c oncent ric a clone A Y942774 Dis c odermia dis s oluta c lone AY897100 Cy mbastela c oncent ric a clone A Y942778 Aply sina aerophoba clone AJ347032 Aply sina aerophoba clone AJ347054 Aply sina aerophoba clone AJ347073 Phyllospongia papy racea clone A Y845235 sponge isolate AY368533

Sphingomonas sp. s ponge is olate AY369991 sponge isolate AY369992

Spongilla lac us tris c lone AY 466481 Spongilla lac us tris c lone AY 466482 Theonella swinhoei clone AF434963 Aply sina aerophoba clone AJ347050 Sphingomonas ec hinoides AB021370 Sphingomonas sp. AF131296 Sphingomonas suberifac iens D13737 Sphingopyx is terrae D13727 biofilter clone AJ318143 sludge isolate AJ132378 Brucella melitens is L26166

Cymbas tella c oncentric a clone A Y942764 bas alt glas s clone DQ070813

My c ale lax is sima c lone DQ098850 My c ale lax is sima c lone DQ098832 sponge isolate AY364600 sponge isolate AY367745

Halic hondria panic ea c lone AY 948356 insect larva clone A J459874 Caulobac ter vibrioides AJ009957 Brevundimonas ves ic ularis AJ007802 Brevundimonas vis ic ularis AJ007801 Pannonibac ter phragmetitus AJ400704 Methylobac terium mes ophilicum D32225

A plys ina aerophoba c lone AJ347030 Theonella s winhoei c lone AF434985 sediment c lone AJ567552

S pongilla lacust ris c lone AY466478 Halichondria panic ea clone AY948360 Dis codermia dis soluta clone AY897109

s ediment clone AY375135 Spongilla lac us tris c lone AY598785

soil is olate AF498728 Halic hondria panic ea c lone Z88580 Methyloc ys tis echinoides AJ458473 soil clone AY080914

soil is olate AF498711 Rhodos pirillum rubrum M32020

Magnetos pirillum magnetotac ticum Y10110 soil clone AY192272

Theonella swinhoei clone A F434956 Theonella swinhoei clone A F434950 Aplys ina aerophoba clone A J347025 Halic hondria panic ea c lone Z88584

Ras pailia_c luster_162(14) Ras pailia_c luster_363(6) Raspailia_clus ter_439(5)

Raspailia_clus ter_406(5)

Halic hondria ok adai clone AB054144 Ras pailia_c lust er_450(4)

Ras pailia_c lust er_427(5)

Ras pailia_c luster_501(4)

Ras pailia_c luster_474(4) Ras pailia_c luster_275(8)

Ras pailia_c lust er_459(4) Ras pailia_c lust er_202(12) Ras pailia_c lust er_90(30) Ras pailia_c lust er_130(20) Ras pailia_c lust er_641(3) Ras pailia_c lust er_65(64) Ras pailia_c lust er_34(204)

Novel sponge specific cluster

seawater c lone DQ071157

Ehrlichia c anis M73221

A canthamoeba sp. s ymbiont AF069962

Cymbas tela concentric a c lone AY 942762 Chlamy dia t rac homitis U68443

Rumen isolate AB009205 Spongilla lac us tris c lone AY 466476 Mesorhiz obium sp. AB 098586

Mesorhiz obium sp. AY 368521 Devos ia riboflavina D49423

Maricaulis maris AJ007807 Porphyrobac ter neutonens is L01785 Ax inella verruc os a clone A J581348 sponge c lone AY 542783

Halic hondria panicea c lone Z88590 Cy mbas tela concent ric a c lone AY 942756

Plak toris sp. c lone EF076075 s argass o s ea c lone AA CY01099937 s ponge is olate AF414879

Halichondria panicea clone Z88583 Halichondria panicea clone Z88591 bac terioplankt on clone AY033300 bac terioplankt on clone AY033316

Chondrilla nuc ula c lone DQ079039 P elagibac ter ubique A F510191

S uberites s p. c lone AJ810784 marine c lone U70679

s ediment clone A Y373415

B orrelia burgdorferi U03396 Spongilla lac ustris clone AY466479

Spongilla lac ustris clone AY466475 Spongilla lac ustris clone AY466474

Spongilla lac us tris c lone AY 466477

Pelodic ty on c lat hratiforme Y08106 Chlorobium limic ola Y 10113

Ant ho chartacea c lone EF076242 Halichondria ok adia c lone AB 054163

Cymbas tela conc entric a c lone AY942765 c oral clone DQ312248

coral isolate DQ416557

Spongilla lac us tris c lone AY598790 sargas so s ea clone AACY01026023 My cale lax is sima c lone DQ097238 sea squirt isolate AB210280

marine is olat e A Y576766 sea squirt isolate AB175663

Rhabdastrella globos tellata is olate AY372908 Pseudoceratina clavata is olate AY372915 Pseudoceratina clavata is olate AY372903 Pseudoceratina clavata is olate AY372904 Rhabdastrella globos tellata is olate AY372905 Rhabdastrella globos tellata is olate AY372906 Rhabdastrella globos tellata is olate AY372907 Rhabdastrella globos tellata is olate AY372909 Rhabdastrella globos tellata is olate AY372910 Rhabdastrella globos tellata is olate AY372911 Rhabdastrella globos tellata is olate AY372912 Pseudoceratina clavata is olate AY372914 My c ale lax is sima islate DQ097237 My c ale lax is sima isolate DQ097239 My c ale lax is sima isolate DQ097240 My c ale lax is sima isolate DQ097241 My c ale lax is sima c lone DQ098829 My c ale lax is sima c lone DQ098846 Monanchora unguifera is olat e DQ097242 Monanchora unguifera is olat e DQ097243 Monanchora unguifera is olat e DQ097244 Pseudovibrio denitrificans AY486423 Alphaproteobact erium AB 106120 marine c lone AF382114

Chondrosia reniformis isolate AY562566 Chondrosia reniformis isolate AY562565 Chondrosia reniformis isolate AY562564 Chondrosia reniformis isolate AY562563 Chondrosia reniformis isolate AY562562 Chondrosia reniformis isolate AY562561 Chondrosia reniformis isolate AY562559 sponge isolate AB012864

sponge isolate AF295099

Aplys ina aerophoba is olate AF218241 'orange sponge 13' is olate AY542795 Halic hondria panicea isolate AY948383 Halic hondria panicea isolateAY948382 sponge isolate AY370009

sponge isolate AY364592 Halic hondria panicea c lone Z88575 sponge isolate AF414887 sponge isolate AF414885 sponge isolate AF414884 sponge isolate AF414883 sponge isolate AF414880

Pseudoceratina clavata is olate DQ227658 Pseudoceratina clavata is olate DQ227657 Pseudoceratina clavata is olate DQ227656 Pseudoceratina clavata is olate DQ227655 Microc iona prolifera is olate DQ097264 Microc iona prolifera is olate DQ097263 Axinella corrugata is olate DQ097262 Axinella corrugata is olate DQ097261 Axinella corrugata is olate DQ097260 My c ale lax is sima isolate DQ097259 My c ale lax is sima isolate DQ097258 My c ale lax is sima isolate DQ097257 Niphates digit alis is olate DQ097256 sponge isolate DQ097255 sponge isolate DQ097254

Acanthost rongly ophora sp. isolateDQ097253 sponge isolate DQ097252

Acanthost rongylophora sp. isolate DQ097251 Acanthost rongylophora sp. isolate DQ097250 Didis cus ox eata is olat e DQ097249

Didis cus ox eata is olat e DQ097248 Monanchora unguifera is olat e DQ097247 Monanchora unguifera is olat e DQ097246 Monanchora unguifera is olat e DQ097245

0.02

Figure 2.9: Maximum Likelihood phylogenetic tree of bacteria from the α- class of

Proteobacteria including 16 clusters (391 sequences) derived from the marine sponge Raspailia ramosa forming a monophyletic novel sponge specific cluster.

134