Phylogenetic Diversity and Spatial Distribution of the Microbial Community Associated with the ...

16 Pages·2008·1.06 MB·English

MicrobEcol(2008)56:306–321
DOI10.1007/s00248-007-9348-5
ORIGINAL ARTICLE
Phylogenetic Diversity and Spatial Distribution
of the Microbial Community Associated with the Caribbean
Polymastia corticata
Deep-water Sponge cf. by 16S rRNA,
aprA amoA
, and Gene Analysis
Birte Meyer&Jan Kuever
Received:9July2007/Accepted:12November2007/Publishedonline:10January2008
#SpringerScience+BusinessMedia,LLC2007
Abstract Denaturinggradientgelelectrophoresis(DGGE)- recent studies that demonstrated the sponge species speci-
based analyses of 16S rRNA, aprA, and amoA genes ficity of the associated microbial community while the
demonstrated that a phylogenetically diverse and complex biogeography of the host collection site has only a minor
microbial community was associated with the Caribbean influence on the composition. In P. cf. corticata, the
deep-water sponge Polymastia cf. corticata Ridley and specificity of the sponge–microbe associations is even
Dendy,1887.Fromthe38archaealandbacterial16SrRNA extended to the spatial distribution of the microorganisms
phylotypes identified, 53% branched into the sponge- within the sponge body; distinct bacterial populations were
specific, monophyletic sequence clusters determined by associated with the different tissue sections, papillae, outer
previous studies (considering predominantly shallow-water andinnercortex,andchoanosome.Thelocaldistributionof
sponge species), whereas 26% appeared to be P. cf. a phylotype within P. cf. corticata correlated with its (1)
corticata specifically associated microorganisms (“special- phylogenetic affiliation, (2) classification as sponge-
ists”); 21% of the phylotypes were confirmed to represent specific or nonspecifically associated microorganism, and
seawater- and sediment-derived proteobacterial species (3) potential ecological role in the host sponge.
(“contaminants”) acquired by filtration processes from the
host environment. Consistently, the aprA and amoA gene-
based analyses indicated the presence of environmentally Introduction
derived sulfur- and ammonia-oxidizers besides putative
sponge-specificsulfur-oxidizingGammaproteobacteriaand Spongesconstitutethe phylumPorifera, which is taxonom-
Alphaproteobacteria and a sulfate-reducing archaeon. A ically divided into the three classes: Calcarea, Hexactinel-
sponge-specific, endosymbiotic sulfur cycle as described lida, and Demospongiae with the latter encompassing
for marine oligochaetes is proposed to be also present in P. approximately 85% of the recognized species [23]. They
cf. corticata. Overall, the results of this work support the are benthic, sessile filter feeders that depend on (dissolved)
organic particle uptake from a continuous stream of water
Electronicsupplementarymaterial Theonlineversionofthisarticle passing through their aquiferous canal system of channels
(doi:10.1007/s00248-007-9348-5)containssupplementarymaterial, and chambers. Particles including bacteria and single-celled
whichisavailabletoauthorizedusers. eukaryotes can be captured by epithelial pinacocytes (form-
:
B.Meyer J.Kuever ing the ectosome) at the sponge surface but are predomi-
Max-Planck-InstituteforMarineMicrobiology, nantlytrapped fromthe filteredwater within the choanocyte
Celsiusstrasse1,
chambers-containing region of the sponge, the choanosome
28359Bremen,Germany
(or endosome). The food particles are transferred via trans-
Presentaddress: cytosistothe spongecellsofthe interiormesohylmatrix. In
J.Kuever(*) some sponge species, a specialized area of the mesohyl that
BremenInstituteforMaterialsTesting,
lacks choanocyte chambers but contains mineral deposits or
Paul-Feller-Strasse1,
collagenfibrils islocated immediately belowthe exopinaco-
28199Bremen,Germany
e-mail:[email protected] derm, the cortex. As part of the ectosome, the cortex has
PhylogeneticDiversityandSpatialDistribution... 330077
been presumed to act as a special supportive device for the aim of the present study was to examine the phylogenetic
openingsofthecanalsystemandasaprotectivelayerofthe composition and spatial distribution of the archaeal and
endosomeagainstsuperficialinjuryorstrongcurrents[1,56]. bacterialcommunityassociatedwithadeep-waterspongeP.
The genus Polymastia Bowerbank, 1864 (Demospongiae, cf. corticata by DGGE-based 16S rRNA and functional
Tetractinomorpha, Hadromerida, Polymastiidae) with ap- geneanalyses.Theanalysesoffunctionalgenesthatencode
proximately 50 described species is characterized by their key enzymes of the dissimilatory sulfate reduction, sulfur
encrusting,spherical-orcushion-shapedspongesalwayswith oxidation, and ammonia oxidation pathway, e.g., aprA and
pore-bearing, inhalant and exhalant papillae and a cortex of amoA (coding for the alpha subunits of the dissimilatory
theectosomalskeletonthatiscomposedofatleasttwolayers APS reductase, AprA, and ammonia monooxygenase,
[4, 43]. The species Polymastia cf. corticata Ridley and AmoA), allow diversity surveys of certain physiological
Dendy, 1887, is distinctive by its dense and leathery cortex groups, e.g., sulfate-reducing, sulfur-oxidizing, and ammo-
(2 mm thickness; two distinct layers; intermediary layer nia-oxidizing prokaryotes. The polyphyly of these physio-
collagenous), 100 inhalant and 4 to 5 exhalant papillae of logical groups (see [5, 11, 13, 24, 28, 29, 50, 73] and
approximately 8 mm height, and choanosomal bundles of references therein) restricts the concomitant detection of all
spicules that stop below the cortex (the latter can be easily recognized members by the use of single 16S rRNA gene-
detachedfromtheendosome).Thespecieshasbeencollected targetingprobesorprimerpairsandlimitstheidentification
offthecoastofBrazilandeastoftheAzoresinabathymetric of novel lineages in environmental analyses. In addition,
range from 200 to 1,385 m [4, 43]. the analysis of 16S rRNA genes cannot provide an
Early microscopic and culture-based examinations dem- unambiguous link between the genetic identity of an
onstrated that large numbers of microbes populate the uncultured microorganism and its physiological or meta-
mesohyl matrix of many demosponges (termed “bacterio- bolic capacity. Analyses of functional genes like aprA and
sponges”) forming up to 40% of the sponge volume with amoA circumvent these limitations and (although compli-
densities of 108 to 1010 bacteria per gram of sponge wet cated by lateral gene transfer events of aprA [39, 40]) have
weight [64, 72]. The hypothesis of a widespread, sponge- beensuccessfullyappliedforbiodiversitystudies[2,14,31,
specific microbial community that is distinctly different 41, 46, 47]. They were used to determine the phylogenetic
from the marine bacterioplankton that was postulated by complexity of the P. cf. corticata-associated microbial
Hentschel et al. [15, 16] and has been supported by others communities putatively involved in sulfur and nitrogen
(for recent reviews, see [17, 19, 59, 67]). In contrast, the cycling within the sponge.
results of several recent diversity surveys contradict the
existence of a general uniform sponge-associated microbial
community regardless of sponge species and location. Materials and Methods
Instead, they provided evidence that the composition of
thesponge-inhabiting microbial consortium depends onthe Sampling
hostspecies[18,22,33,60–63,68].Acorrelation between
thepresenceofcertainmajorbacterialtaxonomicgroupsin Thedeep-waterspongewascollectedbyachainbagdredge
the sponge microbiota and the geographical location of the from a depth of 1,127 m at the Kahouanne Basin (Lesser
host sponge was proposedbyHilletal.[18].However,the Antilles, Caribbean Sea) (16°28.80′ N, 61°58.66′ W) in
presence of distinct microbial consortia in different sponge January 2001 (RV Sonne cruise SO-154). Ambient seawa-
species sharing the same habitat indicated a low impact of ter (temperature 5°C) was sampled from the surface of
the surrounding marine plankton for the sponge–bacteria undisturbed sediment cores with a sterile syringe; all
associations [60, 62, 63, 68]. As recently demonstrated by sampleswereimmediatelyfrozenandstoredat−20°Cuntil
Thiel et al. [63], the specificity of sponge–microbe further molecular investigation. In the laboratory, the
associations extends to the spatial distribution of microbial sponge was washed carefully three times in autoclaved
populations within the sponge body: Distinct bacterial artificial seawater before cutting. Small subsamples (2–
communities were found to inhabit the endosome and 3cm3tissuecubes)werefixedin4%formaldehyde,placed
cortex of marine sponge Tethya aurantium; specifically in 70% ethanol, and sent to the Senckenberg Museum,
associated phylotypes (e.g., cortex-associated members of Frankfurt am Main, Germany, for sponge identification.
the Bacteroidetes and Alphaproteobacteria) were identified Examination of its general morphological features includ-
for both regions. ingspiculesgeometryofthetissuesample(depositedunder
The current knowledge about sponge–microbe associa- the registration number SMF 9633) classified this deep-
tionsisrestrictedtoinvestigations ofshallow-water species waterspecimenasP.cf.corticataRidleyandDendy,1887,
with the only exception of the bacterial 16S rRNA gene- a member of the Polymastiidae (Demospongiae, Hadro-
basedanalysispublishedbyOlsonandMcCarthy[48].The merida) (D. Janussen, pers. comm.). For molecular analy-
308 B.Meyer,J.Kuever
sis, two sponge tissue subsamples were separated into sequences and detailed thermocycling conditions see
papillae, outer cortex (including ectopinacoderm), inner Table S1 of the Electronic Supplementary Material).
cortex, and endosome (choanosome) sections (Fig. 1) with
a sterile scalpel. The tissue sections were gently rinsed in PCR Amplification of Partial 16S rRNA, aprA, and amoA
autoclaved artificial seawater to remove loosely attached genesforSubsequentDoubleGradientDenaturingGradient
bacteria from the surfaces and cut into small pieces before Gel Electrophoresis Analysis
DNAextraction.Twoseawatersamples(each200ml)were
filtered through a Sterivex filter (0.2 μM pore size, Ingeneral,PCRmixtures(50μltotalvolume)contained1x
Millipore). REDTaq PCR reaction buffer (Sigma-Aldrich, St. Louis,
Missouri, USA), 0.3 mg ml−1 bovine serum albumin
Genomic DNA Extraction (BSA), 200 μM deoxynucleoside triphosphates (dNTPs)
mixture, 1 μM of each primer, 0.05 U μl−1 REDTaq DNA
Total genomic DNAwere extracted from the four different Polymerase, and 100 ng genomic DNA as template
sponge tissue sections in duplicate and purified using the (negative controls with water). Partial 16S rRNA gene
DNeasy® Tissue Kit (Qiagen, Hilden, Germany) following amplification for subsequent double gradient denaturing
the manufacturer’s protocol for Gram-positive bacteria and gradient gel electrophoresis (DG-DGGE) analysis was per-
animal tissue. Genomic DNA from both filtered seawater formedusingtheprimersets(1)GM5F-GCclamp(341F)and
samples was extracted after five cycles of thawing at 30°C 907RforBacteria[45],(2)Arch516F-GCclamp(K.Knittel,
andfreezinginliquidnitrogenusingtheprotocolofZhouet unpublished) and Arch958R [7] for Archaea, and (3)
al. [74]. The DNA concentrations were estimated spectro- CTO189f-GC clamp and CTO654r specific for betaproteo-
photometrically, whereas its integrity was examined visu- bacterial ammonia-oxidizers [30]. An approximately 0.4-kb
ally by gel electrophoresis on 0.8% (w/v) agarose gels run aprA gene fragment was amplified using the primer set
in 1x Tris–borate–EDTA (TBE) buffer followed by ethi- AprA-1-FW and AprA-5-RV with GC clamp [41]. An
dium bromide staining (0.5 mg l−1). The extracted DNA approximately 0.5-kb amoA gene fragment was yielded
(dissolved in 10 mM Tris–HCl, 1 mM EDTA, pH 7.5) was applying the primer pair AmoA-1F and AmoA-2R-TC [46]
stored at −20°C until further analysis. (for details see Table S1 of the Electronic Supplementary
Material). Duplicate amplifications were performed from
PCR Amplification of Partial 28S rRNA Gene each sponge tissue section and seawater DNA sample. The
PCR products were visually analyzed by electrophoresis of
Besides its morphological identification, 28S rRNA gene aliquots (10% of the reaction volume) on 2% agarose gels
fragments(C1,D1,C2,andD2domainsatthe5′-end)were (w/v) run in 1x TBE buffer stained with ethidium bromide
amplifiedfromthesponge-derivedgenomicDNAusingthe (0.5 mg l−1) to verify correct amplicon size. If necessary,
primers and polymerase chain reaction (PCR) protocol of amplicons of the expected gene fragment size were purified
Chombard et al. [6] to confirm the former morphological before further analysis using either the QIAquick gel
classification by molecular systematics (for PCR primer extraction kit (Qiagen, Hilden, Germany) or the Perfectprep
gel cleanup sample kit (Eppendorf, Hamburg, Germany)
following the supplier’s recommendations.
DG-DGGE Analysis
in-/exhalant papillae
The DG-DGGE analyses of the aforementioned duplicate
16S rRNA, aprA, and amoA amplicons from each sponge
outer cortex section
inner cortex section ectosome tissue type and seawater DNA sample were performed
using the D-GENE™ and D-CODE™ system (Bio-Rad,
Munich, Germany). DG-DGGE gels (1.0 mm thick) were
choanosome poured with an polyacrylamide gradient from 6% to 8% of
acrylamide/bis-acrylamidestocksolution,37.5:1(v/v)(Bio-
Rad) superimposed over a colinear denaturant gradient
[100% denaturant corresponds to 7 M urea and 40% (v/v)
Figure 1 Photograph of a cross-section of Polymastia cf. corticata formamide, deionized with AG501-X8 mixed bed resin
showing the examined morphologically different tissue regions, (1)
(Bio-Rad)],whichvariedforthedifferentgenesanalyzedin
papillae, (2) outer section of thecortex including theexopinacoderm
this study (see Table S2 of the Electronic Supplementary
and (3) inner (collagenous) section of the cortex (comprising the
ectosome),and(4)thechoanosome(endosome) Material).GradientswereformedusingaBio-RadGradient
PhylogeneticDiversityandSpatialDistribution... 330099
FormerModel385.TwentymicrolitersofthePCRsamples calculatedbasedonthenearfull-length16SrRNAreference
were mixed with 6 μl of dye solution [0.1% bromphenol sequences received from the database; their robustness was
blue (w/v), 70% glycerol (v/v)] and applied to the gels. tested by bootstrap analysis with 100 resamplings. Subse-
Triplicate gel runs were carried out as reported elsewhere quently, thesetrees wereimportedintoARB andtheDGGE
(for references see Table S2 of the Electronic Supplemen- analysis-derivedshortsequenceswereindividuallyaddedby
tary Material) followed by ethidium bromide staining using the QUICK_ADD parsimony tool of ARB without
(0.5 mg l−1) for 15 min and subsequent destaining in allowing changes in the overall tree topology. The phyloge-
double-distilled water for 10 min. The DNA bands were netic positions of the short sequences were additionally
visualized on a UV transillumination table (Biometra, verified by bootstrap analysis with 100 resamplings (maxi-
Göttingen, Germany); persisting and dominant bands were mum-likelihoodmethod).Todeterminethespecificityofthe
excisedfrommultiplelanesofthepolyacrylamidegelswith microbial associations, the detected phylotypes from P. cf.
ethyl alcohol-sterilized scalpel, incubated in 50 μl Tris– corticata were classified as (1) “specialists”, (2) “sponge
HCl, pH 8.0, overnight at 4°C, and reamplified using 1 μl associates”, or (3) “generalists” depending on their presence
of the eluate as template and PCR conditions as described in (1) this host species only, (2) several sponge species but
above. The purity and migration behavior of the ream- absent from seawater, or (3) several sponge species and
plification products of the bands were checked by DG- seawaterinaccordancetoTayloretal.[60];theenumeration
DGGE. The reamplification products were purified from ofthesponge-associatedbacterial(SAB)andarchaeal(SAA)
free PCR primers using either the QIAquick gel extraction phylogenetic clusters is based on the previous studies of
kit (Qiagen) or the Perfectprep gel cleanup sample kit Hentschel et al. [16], Thiel et al. [63], Holmes and Blanch
(Eppendorf) following the supplier’s recommendations. [22], and Lee et al. [32].
The aprA and amoA nucleotide sequence data obtained
Nucleotide Sequencing from DGGE analysis were assembled and manually cor-
rectedusingtheBioedit(version7.0.5)sequencealignment
All reamplification products of the DG-DGGE bands were editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
sequenced directly in both directions using the respective The sequences were maintained separately according to
amplification primers and the ABI Prism BigDye termina- their source (sponge tissue type, seawater); each set of
tor cycle sequencing ready reaction kit (Applied Biosys- sequences was grouped into phylotypes based on a >99%
tems, Foster City, USA) according to the manufacturer’s identity cutoff. Only one sequence per OTU and tissue
instructions. Sequencing reactions were run on an ABI type/seawater was used for further analysis. BLAST
PRISM® 3100 Genetic Analyzer (Applied Biosystems). searches in the public databases for homologous sequences
ofthepartialAmoAsequenceswereperformed.Thepartial
Phylogenetic Analysis and complete AmoA sequences were automatically aligned
using the Web server Tcoffee@igs (http://igs-server.cnrs-
The partial 16S rRNA sequences obtained from DGGE mrs.fr/Tcoffee/);theinitialalignmentwasrefinedmanually.
analysis were checked for chimeras with the program The partial AprA sequences were integrated into the
CHECK_CHIMERA of the Ribosomal Database Project, persisting Apr alignment of sulfate-reducing and sulfur-
addedtothe16SrRNAsequencedatabaseoftheTechnical oxidizing reference strains [39,40] including all full-length
University Munich (Germany) using the automatic align- Apr sequences available from the public databases. The
ment function ARB_Align implemented in the ARB AmoA and AprA data sets were phylogenetically analyzed
software program package (http://www.arb-home.de), and using PhyML. Regions of insertions and deletions (indels)
manually corrected. The sequences were maintained sepa- wereomitted.Themaximum-likelihoodmethod-basedphy-
rately according to their source (sponge tissue type, logenetictreeswereconstructedusingtheglobalrearrange-
seawater); each set of sequences was grouped into ment and randomized species input order options and the
phylotypes,i.e.,operationaltaxonomicunits(OTUs),based JTT matrix as amino acid replacement model. Statistical
ona>99%identitycutoff.OnlyonesequenceperOTUand supportisgivenbybootstrapanalysiswith100resamplings.
tissue type/seawater was used for further analysis. The
closest phylogenetic relatives of each phylotype were GenBank Accession Numbers
identified by comparison to the National Center for
Biotechnology Information (NCBI) GenBank database The nucleotide sequence data reported in this article are
using the Basic Local Alignment Search Tool (BLAST) availableundertheGenBankaccessionnumbersEU005552
analysis tools (www.ncbi.nlm.nih.gov/BLAST/). For phy- (28S rRNA gene), EU005553-EU005595 and EU005641-
logeneticanalysis,theonlineversionofPhyML(http://atgc. EU005648 (16S rRNA gene), EU005596-EU005620 (aprA
lirmm.fr/phyml) was used. Maximum-likelihood trees were gene), and EU005621-EU005640 (amoA gene).
310 B.Meyer,J.Kuever
Figure 2 DGGE banding pat- a b
ternsofamplified16SrRNA,
SAS Pa OC IC Ch SAS Pa OC IC Ch
aprA,andamoAgenefragments
usingDNAsamplesfrom
spongetissuesections,papillae
24 15
(Pa),outercortex(OC),inner 50
cortex(IC),andchoanosome 2 1 41 45 30
(Ch)ofCaribbeanP.cf.corti- 3 183 19 51 1
cataandspongeambientsea- 6 2 42 37 52
water(SAS)collectedatthe 38 53
3 2
KahouanneBasin(seeFig.1). 8 1 2 3 4
16SrRNAgene-specificdiver-
sityanalysiswasperformedap- 56
plyinguniversalbacterial(a), 11
universalarchaeal(b),and 4 43 48 54
betaproteobacterialammonia-
49 28 32
oxidizer-specific(b)gene-
targetingprimerpairsinPCR. 55
Diversityanalysisoffunctional
c d
geneswasperformedusing
SRP-andSOB-specificaprA SAS Pa OC IC Ch SAS Pa OC IC Ch
gene-targetingprimers(d)and 46
betaproteobacterialammonia-
oxidizer-specificamoAgene- 38 44
targetingprimers(e).Numbered
4 53
DGGEbandswereexcisedfrom
replicategelsandsuccessfully
sequenced(phylogeneticanaly- 41
9 18
sFeisgso.f3r,e4tr,ieavnedd5s)equences,see 111012 1 3 5 7 5567 6432
13 2 4 6 8 19
3
10
5 5 66
6 54
11 55 62
14 32 21
e
SAS Pa OC IC Ch
28 1 5 10 18
11
2 6 12 15
3 7 13 16
29
25 4 9 14 17
Results tissue type were identical, indicating no genomic DNA
extractionandPCRamplificationbias.Thebandingpatterns
DGGE of DGGE analyses using universal archaeal, bacterial, and
aprA gene-targeting primers showed the presence of
DGGE was used to fingerprint the microbial community in different microbial communities in the inner and outer
P. cf. corticata (Fig. 1). The banding patterns of the same regionsofP.cf.corticata(Fig.2a,b,d).Inaddition,alltissue
PhylogeneticDiversityandSpatialDistribution... 331111
FFiigguurree 33 PPhhyyllooggeenneettiicctrtereeessbabsaesdedonontheth1e6S16rSRNrARNsAequseenqcueesnocebstainedbafcrtoermialthaendP.arccfh.aceoarlti1c6aStar-RasNsoAcicaltuesdtemrsicarroebhiaiglhcloigmhtmedunbiytyliagnhdt-ghroasyt
sopbotanigneedsufrroromunthdeinPg.csef.awcoartteircawtait-hassthoeciathterdeem1i6crSobrRiaNlcAomgmenuen-tiatyrgaentidng prbiomxeers.sTehtseuPs.ecdf.cinortthiciastas-tudderyivaenddserqeulaetnecdessaerqeucelnacsesisfiebdelionntogicnlgustteorsthoef
Aholpshtaspproontegoebasucrteroriuan,diBnegtasperaowteaotebracwteirthia,thaendthrDeeelt1a6pSrorteRoNbAactegreinae-(a);PG.cafm.mcoarptircoatteaobspaecctiearliiasts((b“)P;mACcoidsopbeacciatelirsita”),agnedneNrailtr“ossppoinrgae-(acs)s;ocainatd-
tAacrtgientoinbagctperriimaearndseAtrschuaseead(din). Stheiqsuesntcuedsyobatnadinerdelafrtoemd Pse.qcuf.enccoertsicataeda”reBsahcotwerniainanbdolAdrcthypaeeaan(SdAhBig,hSliAghAt)edanbdy“dgaernk-egrarlaiystsb”ox(e“sg,enwehraelriesat”s
tbheolosengoibntgainteodtfhreomAltphheahporsottespoobnagceteraima,biBenettaspearowtaetoebraacrteeruinad,earlnindedD.eTl-hemgornooupphsyfleotrimc,esdpobnygoet-haesrsoscpioatnegdebsapcetceireias-ldaenrdiveadrchseaqeualen1c6eSsraRreNfAramcluedstebrys
atareprhoitgeholbigahctteedribay(laig)h;t-Ggraamymbaopxeros.teTohbeaPct.ecrfi.aco(rbt)ic;aAtac-iddeoribvaecdtesreiqaueanncdesaredaclsahsesdifieldineinst)oacclucsotredrisnogfPto.cfT.acyolortricaettaaslp.ec[i6a0li]s.tsM(“aPxmimCuoms-pliekceialilhisoto”)d,
gNeintreoraspli“rsapo(ncg);e-aasnsodciaAtcetdi”noBbaacctteerriaiaanadndArAcrhcaheaaea(SA(dB),. SSAeAqu)eanncdes“genbeoraoltissttrsa”p(“regseanmerpalliinsgt”vgarlouuepssgfroeramteerdthbayno5th0e%r s(p1o0n0geresspamecpielisn-dges)rivaerde
soebqtuaiennecdesfraorme fPr.amcfe.dcobryticdaatsaheadrelisnheosw)nacicnorbdoinldgttyopeTaaynldorhiegthalilg.h[t6e0d]. Mainxdimicautmed-linkeealrihtohoednobdoeost.stTrahpe1re6sSamrRpNlinAggveanleuesseqgureenacteerotfhCanen5a0r%cha(e1u0m0
rbeysamdaprlkin-ggrsa)yarbeoixnedsi,cawtehdenreeaasrththeonseodoesb.taTinheed16fSromrRNthAegheonsetsesqpuoenngceeofsCyemnbairocshuameuwmassyumsebdioasusmouwtgarsouupserdefaesreonucteg.rTouhpesrecfaelerebnacre.coTrhreesspcoanledsbtaor
camorbreiespnotnsdesawtoat1e0r%areesutinmdaetreldinesedq.uTehnecemdoivneorpgheynlceetic, sponge-associated 10%estimatedsequencedivergence
sections of the sponge differed in their DGGE banding patterns of the betaproteobacterial ammonia-oxidizer-specific
pattern from the sponge ambient seawater (SAS) samples. 16S rRNA and amoA gene-based analyses were highly
Archaealandbacterialphylotypesspecificallyassociatedwith similar (1) between the distinct sponge tissue types and (2)
thedistinctspongeregionswererepresentedbyDGGEbands between sponge and ambient seawater (Fig. 2c and e).
that were exclusively present in all papillae, outer cortex, Phylotypes specifically associated with P. cf. corticata that
innercortex,orchoanosomesamples,respectively,butabsent were represented by DGGE bands exclusively found in the
in seawater (Fig. 2a, b, d). Other bands were found in all sponge samples (but not in seawater) were only detected by
sponge tissue samples. Contrarily, the DGGE banding the amoA gene-based analysis (Fig. 2e).
312 B.Meyer,J.Kuever
Figure3 (continued)
Phylogenetic Analysis of Partial 16S rRNA Gene phylotypes (53%) were most closely affiliated with other
Sequences sponge-derived sequences previously found in Tethya
aurantium, Rhopaloides odorabile, Theonella swinhoei,
A total of 38 OTUs was identified in P. cf. corticata Latrunculiaapicalis,Halichondriapanicea,Aplysinaaero-
forming 18 distinct sequence clusters. The sequences fell phoba, Sclerotiderma sp., Chondrilla nucula, Petrosia sp.,
into eight different archaeal and bacterial divisions, the Suberites sp., and Axinella verucosa from different geo-
Crenarchaeota, Alphaproteobacteria, Betaproteobacteria, graphical locations (predominantly shallow-water habitats).
Gammaproteobacteria,Deltaproteobacteria,Acidobacteria, These phylotypes comprised the eight sponge-specific,
Actinobacteria, and Nitrospira (Fig. 3). In agreement with monophyletic clusters SAB-Beta-I [63], SAB-Gamma-II,
microbial communities associated with other examined SAB-Delta-I and SAB-Delta-II, SAB-Acido-III, SAB-
deep-water sponges [48], no phylotype related to the Actino-III,SAB-Nitrospira-I[16],andSAA-Cren-I(sponge
cyanobacterial lineage was identified in the deep-water group C) [22, 32] (classified as “sponge associates”). It is
specimen of P. cf. corticata. Members of the latter photo- interesting to note that the detected alphaproteobacterial,
trophic group are abundant in shallow-water sponges [16, most gammaproteobacterial, actinobacterial, and crenarch-
18, 51, 60, 61, 63]. Twenty of the aforementioned 38 aeal phylotypes appeared to represent P. cf. corticata host-
PhylogeneticDiversityandSpatialDistribution... 331133
Figure3 (continued)
specific microorganisms (“specialists” PmCo-Alpha-I, andBacteroidetes(respectiveSAS-B2 16SrRNA sequence
PmCo-Gamma-I/-II, PmCo-Actino-I/-II/-III, and PmCo- is not shown in Fig. 3; closest relative is AJ567605; M.X.
Cren-I) not present in the ambient seawater and other Xu,P.Wang,F.P.Wang,andX.Xiao,unpublishedresults).
sponge species. Based on the current database, these 10
phylotypes (26%) were only moderately related to other Phylogenetic Analysis of Partial AprA and AmoA
sponge-derived sequences but affiliated to environmental Sequences
sequences received from other marine macroorganisms
(Pogonophora endosymbionts; PmCo-Gamma-II), aquatic The aprA gene-based analysis allowed the detection of six
microbial formations in Nullarbor caves [21] (PmCo- phylotypesthatareindicativeforthepresenceoffivesulfur-
Gamma-I,PmCo-Actino-I),inactivedeep-seahydrothermal oxidizing alphaproteobacterial and gammaproteobacterial
vent chimneys [57] (PmCo-Actino-II), and deep-sea sedi- species and one sulfate-reducing archaeon in the tissue of
ments [65] (PmCo-Cren-I). Indeed, by the use of betapro- P. cf. corticata (Fig. 4). The alphaproteobacterial SOB-
teobacterial ammonia-oxidizer-specific primers [30], close phylotypes were most closely related to AprA sequences of
relatives of uncultured marine Nitrosospira [10] were cultured members of the SAR11 clade [12] (PmCo-sulfur-
detected in the sponge tissue and in the host surrounding oxidizer-ItoPmCo-sulfur-oxidizer-III)andtheLGT-affected
seawater (classified as “generalists” PmCo-Beta-I and Thiobacillus plumbophilus (PmCo-sulfur-oxidizer-IV). Be-
PmCo-Beta-II). causerepresentativesofthealphaproteobacterialclustersIto
The bacterioplankton assemblage of the sponge ambient III have also been found to be abundant in deep-sea
seawater at the Kahouanne Basin (1,127 m depth) was sediments of the Kahouanne Basin and the sponge ambient
phylogenetically less diverse with respect to the complex seawater (Fig. 4), the corresponding SOB species were
microbial community associated with P. cf. corticata classified as “generalists”. The gammaproteobacterial SOB-
(Fig. 3). The detected phylotypes were most closely phylotype (PmCo-sulfur-oxidizer-V) was moderately affili-
affiliated with seawater- and deep-sea sediment-derived ated to AprA sequences of uncultured Thiothrix species,
sequences of uncultured members of the marine group-I which have been found in cold seep Beggiatoa-mats at the
Crenarchaeota (MG-I, group C1a-α) [65], Euryarchaeota HydrateRidge,Oregon[41]. All identified SOB-phylotypes
[38], Nitrosospira [10], uncultured Gammaproteobacteria could not be assigned to any detected 16S rRNA gene-
314 B.Meyer,J.Kuever
Figure3 (continued)
based OTU. Besides, a SRP-phylotype was detected that is Spatial Distribution of the Sponge-Associated Microbial
mostcloselyrelatedtotheAprAsequencesofArchaeoglobus Community
members; a corresponding euryarchaeal species was not
identified by the 16S rRNA gene-based analysis. The phylogenetic investigations of different tissue regions
The amoA gene-based analysis (Betaproteobacteria- from P. cf. corticata (Fig. 1) demonstrated high variability
specific primers) allowed the detection of nearly identical in the microbial communities associated with the sponge
phylotypes (PmCo-ammonia-oxidizer-I and PmCo- papillae/outer cortex and the inner cortex/choanosome
ammonia-oxidizer-II) in the sponge tissue and the host (Figs. 3, 4, and 5, summarized in Table 1). The 16S rRNA
surrounding seawater (Fig. 5); closely related environmental gene-based analysis indicated that the highest phylogenetic
sequences havealsobeenreportedfromdiverse sedimentor diversity is present in the inner region of the sponge, the
seawater–sedimentinterfacesamplesofotherhabitats[2,14, choanosome. From the total of 38 different 16S rRNA
31]. The AmoA sequence clusters corresponded to the gene-based phylotypes, 12 and 10 OTUs were detected in
uncultured Nitrosospira sequence clusters that have been thechoanosome and theinner cortex,respectively,whereas
identified by the group-specific 16S rRNA gene analysis only 7 and 9 OTUs were found in the outer cortex and in
(Fig. 3). the papillae tissue sections, respectively. The members of
PhylogeneticDiversityandSpatialDistribution... 331155
Figure 4 Phylogenetic tree based on the AprA sequences obtained ambientseawaterareunderlined.Thetaxonomicclassification ofthe
from the P. cf. corticata-associated microbial community, the host SRP and SOB reference strains and affiliated sponge-derived
spongesurroundingseawater,andadjacentsedimentsamplesfromthe sequences is indicated. Maximum-likelihood bootstrap resampling
KahouanneBasin.SRP-andSOB-typesequencesreceivedfromP.cf. values greater than 50% (100 resamplings) are indicated near the
corticata are shown in bold type and highlighted by light-gray and nodes. The scale bar corresponds to 10% estimated sequence
dark-gray boxes, respectively; those obtained from the host sponge divergence
the different archaeal and bacterial divisions were not sponge surface regions belonged to the Alphaproteobacteria,
evenly distributed within the sponge body but appeared to Betaproteobacteria, and Gammaproteobacteria (8 phylo-
be associated with distinct parts of the host tissue. Five of types). The OTUs comprised only two general sponge-
theeightgeneralsponge-specificclusters(SAB-Delta-I and specific clusters (SAB-Beta-I, SAB-Gamma-II) besides four
SAB-Delta-II, SAB-Acido-III, SAB-Actino-III, SAA-Cren- putative“specialists”clusters(PmCo-Alpha-I,PmCo-Gamma-
I, 11 phylotypes) and all P. cf. corticata-specifically II, PmCo-Cren-I) whose phylotypes were affiliated with
associated actinobacterial phylotypes (PmCo-Actino-I, nonsponge-derived sequences received from diverse habitats.
PmCo-Actino-II, and PmCo-Actino-III, 4 phylotypes) were The Crenarchaeota associated with the papillae and outer
exclusivelypresentintheinnercortexandthechoanosome. cortexsectionsweredistincttothosefoundintheinnercortex
Thus,themembersoftheDeltaproteobacteria,Acidobacteria, and choanosome. Notably, with the exception of PmCo-
and Actinobacteria were restricted to the inner parts of this Gamma-I,nophylotypeobtainedfromthechoanosomeorthe
sponge. In contrast, four of the five P. cf. corticata-derived inner cortex section was closely related to environmental
sequence clusters that were exclusively identified in the sequences found in seawater and sediment samples.