Microbial diversity associated with algae, ascidians and sponges from the north coast of São Paulo state, Brazil

Microbiological Research 165 (2010) 466—482

0944-5013/$ - sdoi:10.1016/j.

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Microbial diversity associated with algae,ascidians and sponges from the northcoast of Sao Paulo state, Brazil

Claudia B.A. Menezesa, Rafaella C. Bonugli-Santosa, Paula B. Miquelettoa,Michel R.Z. Passarinia, Carlos H.D. Silvaa, Mariana R. Justoa, RebecaR. Leala, Fabiana Fantinatti-Garbogginia, Valeria M. Oliveiraa, RobertoG.S. Berlinckb, Lara D. Settea,�

aDivisao de Recursos Microbianos, Centro Pluridisciplinar de Pesquisas Quımicas, Biologicas e Agrıcolas, UniversidadeEstadual de Campinas, CP 6171, CEP 13083-970, Campinas, SP, BrazilbInstituto de Quımica de Sao Carlos, Universidade de Sao Paulo, CP 780, CEP 13560-970, Sao Carlos, SP, Brazil

Received 17 August 2009; received in revised form 24 September 2009; accepted 27 September 2009

KEYWORDSMarine inverte-brates;Microbial diversity;Bacteria and fungi;Ribosomal RNA;ARDRA

ee front matter & 2009micres.2009.09.005

ing author. Tel.: +55 19ess: [email protected]

SummaryLittle is known about the microbial diversity associated with marine macroorgan-isms, despite the vital role microorganisms may play in marine ecosystems. The aimof the present study was to investigate the diversity of bacteria and fungi isolatedfrom eight marine invertebrate and one algae samples. Data derived from ARDRAand sequencing analyses allowed the identification of marine-derived microorgan-isms isolated from those samples. Microbial strains identified up to the genus levelrevealed 144 distinct ribotypes out of 256 fungal strains and 158 distinct ribotypesout of 181 bacterial strains. Filamentous fungi were distributed among 24 differentgenera belonging to Ascomycota, Zygomycota and Basidiomycota, some of which hadnever been reported in the literature as marine invertebrate-inhabiting fungi(Pestalotiopsis, Xylaria, Botrysphaeria and Cunnninghamella). Bacterial isolateswere affiliated to 41 different genera, being Bacillus, Ruegeria, Micrococcus,Pseudovibrio and Staphylococcus the most abundant ones. Results revealed anunexpected high microbial diversity associated to the macroorganisms which havebeen collected and suggested the selection of certain microbial taxonomic groupsaccording to the host. The combined data gathered from this investigation

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Microbial diversity associated with marine macroorganisms 467

contribute to broaden the knowledge of microbial diversity associated to marinemacroorganisms, including as a promising source for the discovery of new naturalproducts.& 2009 Elsevier GmbH. All rights reserved.

Introduction

The ocean covers more than 70% of Earth�ssurface and is considered as a great reservoir ofnatural resources. However, the extent of marinebiodiversity, especially of microorganisms, is barelyknown. It has been estimated that the biologicaldiversity in marine ecosystems is higher than intropical rain forests (Larsen et al. 2005). Marinemicrobial communities are composed of ubiquitousmembers that can be found not only in the surfacewaters of the sea, but also in the lower and abyssaldepths from coastal to the offshore regions, andfrom the general oceanic to the specialized nicheslike blue waters of coral reefs to black smokers ofhot thermal vents at the sea floor (Surajit et al.2006; Schafer et al. 2001).

Marine invertebrates, especially sponges, representan important source for potential active and biologi-cally functional natural products (Osinga et al. 2001).Many of these compounds exhibit cytotoxic, antibac-terial, antifungal, antiviral or anti-inflammatory activ-ities (Blunt et al. 2008; Zhang et al. 2006; Schirmer etal. 2005). Several studies have reported the discoveryof new bioactive compounds from marine organisms,focusing mainly on chemistry of secondary metabo-lites, which include now more than 15,000 structurallydiverse bioactive compounds isolated during the last30 years (Salomon et al. 2004). The secondarymetabolism of marine-derived microorganisms startedto be investigated much more recently. However, theecological associations occurring between the micro-organisms and the marine substrates have been greatlyneglected (Kurtboke 2005).

In the terrestrial and marine environment, fungiare ecologically important intermediaries of energythat flows from detritus to higher trophic levels andplay an important role in nutrient regenerationcycles as decomposers of dead and decaying organicmatter (Bugni and Ireland 2004; Prasannarai andSridhar 2001). On the other hand, marine bacteriaserve as an important source of food for a variety ofmarine organisms and may also function as biologi-cal mediators through their involvement in thebiogeochemical processes (Surajit et al. 2006).

The field of marine mycology has a relativelyshort history and has yielded so far the classifica-tion of two groups of marine fungi on the basis oftheir ability to grow and to reproduce in seawater

(Kohlmeyer and Kohlmeyer 1979). Obligate marinefungi are those that grow and sporulate exclusivelyin a marine or estuarine habitat, while facultativemarine fungi are those from freshwater or terres-trial milieus that are able to grow (and possiblysporulate) in the marine environments. About 800species of obligate marine fungi have been de-scribed, including representatives from the Basi-diomycota, Ascomycota, mitosporic fungi andyeasts (Surajit et al. 2006).

Considering that conservation and the sustain-able use of biological diversity requires a compre-hensive knowledge about the species richness,abundance and distribution and that little is knownabout the phylogenetic and functional diversity ofmarine microbial communities, the aim of thepresent study was to investigate the diversity offilamentous fungi and bacteria from differentsamples of marine macroorganisms collected atthe north coast of Sao Paulo State, Brazil.

Material and methods

Sampling of marine macroorganisms

Nine samples of marine macroorganisms, includ-ing the sponges Amphimedon viridis (AV); Axinellacorrugata (AC); Dragmacidon reticulata (DR); Geo-dia corticostylifera (GC); Mycale laxissima (ML) andMycale angulosa (MA); the ascidians Didemnumligulum (DL) and Didemnum sp. (DSP), and thealgae Sargassum sp. (AS), were collected in January2007 in beach areas named Praia Guaeca (23149S;45125W), Ilha Toque-Toque (23151S; 45131W) andIlhota da Prainha (23151S; 45124W), in Sao Sebas-tiao region, Sao Paulo State, Brazil, at depthsbetween 5 and 10 m. The samples were placed insterilized polyethylene bags containing seawaterand immediately transported to the Center ofMarine Biology of Sao Paulo University (CEBIMar).

Isolation and maintenance of microorganisms

In order to avoid external contamination duringthe microbial isolation procedure, algae, spongeand ascidian samples were firstly sterilized with0.001 g l�1 mercury chloride in 5% ethanol andthen washed twice with sterilized seawater. Two

C.B.A. Menezes et al.468

different methods were used to isolate filamentousfungi from D. reticulata, M. laxissima, Didemnumsp. and A. viridis, as follows: (1) Trituration:samples were triturated and serially diluted up to100-fold with sterilized distilled water. Aliquots of100 ml were plated onto Petri dishes containing oneof the following culture media described below,and; (2) direct plating: 1 cm3 pieces of eachmacroorganism sampled were placed over Petridishes containing one of the culture media de-scribed below. Bacterial strains were isolated onlyfrom triturated samples. Aliquots of 100 ml (10�2,10�4 and 10�6) were inoculated onto Petri dishescontaining one of the bacterial specific mediadescribed below. Agar plates were inoculated andincubated at 25 1C for 1–4 weeks. Isolation ofcolonies was conducted from the 2nd to the 30thday of plating. Pure cultures were obtained afterserial transfers to the same culture medium used toplate the macroorganism samples. The maintenanceof the isolates was performed by cryopreservationat �80 1C (10% glycerol) for both bacteria and fungiand by the Castellani method at 4 1C for filamentousfungi only.

Seven culture media supplemented with rifampicin(300 mg ml�1) were used for the isolation of filamen-tous fungi: glucose, peptone, yeast extract (GPY,glucose 1 g l�1, peptone soymeal 0.5 g l�1, yeastextract 0.1 g l�1, agar 15 g l�1, ASW 800 ml l�1);tryptic soy agar (TSA, Oxoid); malt extract (MS, maltextract 30 g l�1, peptone soymeal 3 g l�1, agar 12 gl�1, ASW 800 ml l�1, pH 5.5); Tubakii media (TM,glucose 30 g l�1, yeast extract 0.5 g l�1, peptone1 g l�1, K2HPO4 1 g l�1, MgSO4 � 7H2O 0.5 g l�1, FeS-O4 � 7H2O 0.01 g l�1, agar 15 g l�1, ASW 800 ml l�1);cellulose agar (CA, 10 g l�1, yeast extract 1 g l�1,agar 15 g l�1, ASW 800 ml l�1); oatmeal agar (OA, Oat30 g l�1, agar 20 g l�1, ASW); potato carrot agar (PCA,Oxoid, pH 8.0) and corn meal media (CM, 42 g l�1,agar 15 g l�1, ASW 800 ml l�1). Four culture mediasupplemented with cycloheximide (50 mg ml�1) wereused for bacteria isolation: GPY, TSA, M1 (solublestarch 10 g l�1, yeast extract 4 g l�1, peptone 2 g l�1,agar 15 g l�1) and marine agar (DifcoTM, USA). Allmedia were prepared with artificial seawater (ASW):KBr 0.1 g l�1, NaCl 23.48 g l�1, MgCl2 � 6H2O 10.61 gl�1, CaCl2 � 2H2O 1.47 g l�1, KCl 0.66 g l�1,SrCl2 � 6H2O 0.04 g l�1, Na2SO4 3.92 g l�1, NaHCO3

0.19 g l�1, H3BO3 0.03 g l�1.

Morphological characterization offilamentous fungi

Fungal morphology was characterized by colonyobservation with a stereoscope (Leica MZ6, Wet-

zlar, Germany) and by squash mounts stained withLactophenol and Cotton Blue using a light micro-scope (Leica DM LS, Wetzlar, Germany). Fungi wereidentified based on these observations and bymorphological criteria determined in the literature(Ellis 1971; Pitt 1979; Domsch et al. 1980).

Molecular characterization ofmicroorganisms

Genomic DNA extraction and PCR amplificationFilamentous fungi and bacteria were cultured in

the respective isolation media. Genomic DNAextraction was performed accordingly for filamen-tous fungi (Da Silva et al. 2008) and for bacteria(Pitcher et al. 1989). The 28S rRNA D1/D2 region ofthe filamentous fungi and 16S rRNA gene of thebacteria were amplified from genomic DNA by PCRusing the following sets of primers, respectively:NL�1m (50GCA TAT CAA TAA GCG GAG GAA AAG30)and NL-4m (50GGT CCG TGT TTC AAG ACG30)(O’Donnell 1993) and 27f (50AGA GTT TGA TCMTGG CTC AG30) (Lane 1991) and 1401r (50CGG TGTGTA CAA GGC CCG GGA ACG30) (Heuer et al. 1997).PCR was performed in reaction mixtures containing0.4 mM each primer, 0.2 mM dNTPs (GE Healthcare),1.5 mM MgCl2 (Invitrogen), 2.0 U Taq polymerase(Invitrogen) and 1.0� � reaction buffer (Invitro-gen), in a final volume of 25 ml for fungi (5–25 nggenomic DNA) and 50 ml for bacteria (50–100 nggenomic DNA). The PCR amplifications were carriedout using an initial denaturation step of 5 min at95 1C, followed by 30 cycles of 1 min at 94 1C fordenaturation, 1 min at 55 1C for annealing, and3 min at 72 1C for extension, with a final extensioncycle of 3 min at 72 1C in an Eppendorf thermalcycler.

ARDRA and sequencing analysesPCR products were digested using the restriction

enzymes HaeIII, RsaI and MspI (GE Healthcare) forfilamentous fungi and AluI, HhaI (GE Healthcare)and DdeI (Invitrogen) for bacteria. Restrictionreactions were carried out at 37 1C for 2 h andelectrophoresis was run at 230 V for 2.5 h on 2.5%agarose gel stained with ethidium bromide (0.5 mgml�1). Band profile analyses were carried out usingthe GelCompar software version 4.2 (AppliedMaths, Kortrijk, Belgium) and UPGMA-based den-drograms constructed from Dice’s coefficient ma-trices (Dice 1945). One isolate representative ofeach distinct ribotype (cut off Z96% similarity) wassequenced for phylogenetic inference. Amplifiedproducts were purified using GFX PCR DNA andgel band purification kit (GE Healthcare) for


subsequent sequencing using DYEnamic ET DyeTerminator Cycle Sequencing Kit for an automatedMegaBace DNA Analysis System 1000 (GE Health-care), in accordance to the manufacturer�s instruc-tions. Sequencing was performed according to DaSilva et al. (2008) for filamentous fungi andVasconcellos et al. (2009) for bacteria. Partial 28SrRNA and 16S rRNA sequences obtained from theisolates were assembled in a contig using thephred/Phrap/CONSED software. The identificationwas achieved by comparing the contiguous rRNAgene sequences obtained with rRNA sequence datafrom reference and type strains available in thepublic database GenBank (http://www.ncbi.nlm.-nih.gov) using the BLASTn routine. The sequenceswere aligned using CLUSTAL X program (Thompsonet al. 1997) and analyzed with MEGA softwareVersion 4.0 (Tamura et al. 2007). The evolutionarydistances were derived from the sequence-pairdissimilarities, calculated as implemented in MEGAusing the DNA substitution model reported byKimura (1980). The phylogenetic reconstructionwas done using the neighbour-joining (NJ) algo-rithm (Saitou and Nei 1987), with bootstrap valuescalculated from 1000 replicate runs, using theroutines included in MEGA software.

Statistical analyses based on ARDRA dataRandomization analyses based on ARDRA data

were performed using the independent samplingalgorithm, implemented in the EcoSim software(Gotelli and Entsminger 2003). This module ofEcoSim provides a computer-sampling algorithm,in which a specified number of individuals (i.e.,sub-samples of the original one) are randomlydrawn, without replacement, from the total com-munity sample, creating pseudo-communities. Var-ious sub-sample sizes were specified and for eachsub-sample size, 1000 pseudo-communities weredrawn from the original one. Data from these wereused to calculate richness and Shannon–Wiener(diversity) and Dominance indices. The resultantvalues were used to calculate the mean and the95% confidence intervals for each index at eachsub-sample size specified.

Results

Isolation and diversity of marine-derivedfilamentous fungi

A total of 256 filamentous fungal strains wereobtained from the Brazilian invertebrate samples(A. viridis, Didemnum sp., D. reticulata andM. laxissima), mainly by using the direct plating

method (75%). MS medium was the most appro-priate for fungi isolation, resulting in 37% of theisolated strains, followed by media TM (15%), PCA(12%), AC and GPY (10%), OA (9%) and CM (5%). Thenumber of marine-derived fungal strains obtainedwas quite similar among the marine invertebratessampled, being 67 from D. reticulata, 65 fromA. viridis, 63 from Didemnum sp. and 61 fromM. laxissima.

ARDRA analyses and sequencing of representativeribotypes revealed the presence of 144 distinctribotypes distributed among 24 different genera offilamentous fungi (Figure 1), which were affiliatedto the phyla Ascomycota, Basidiomycota andZygomycota. The majority of the fungi (230isolates, 134 distinct ribotypes) belonged toAscomycota and was distributed among sevendifferent orders and 18 genera (Figure 2):Botryosphaeriales (Botryosphaeria); Capnodiales(Cladosporium); Eurotiales (Aspergillus and Peni-cillium); Hypocreales (Acremonium, Bionectria,Fusarium and Trichoderma), Phyllachorales(Colletotrichum and Glomerella); Pleosporales(Alternaria and Cochliobolus) and Xylariales(Eutypella, Pestalotiopsis and Xylaria). Additionally,representatives of the genera Apiospora andArthrinium (family Apiosporaceae) and Phoma(mitosporic Ascomycota) were found. The genusTrichoderma, with 91 representatives and 32distinct ribotypes, was the most frequent one.However, a great diversity was also observed forthe genera Penicillum (17 isolates and 14 distinctribotypes), Aspergillus (18 isolates and 16 distinctribotypes) and Fusarium (33 isolates and 17 dis-tinct ribotypes) (Figure 1). The phylum Zygomycotawas represented by 13 isolates (7 distinctribotypes) affiliated to the order Mucorales(genera Cunninghamella, Mucor and Rhizopus),whereas the phylum Basidiomycota wasrepresented by four isolates (3 distinct ribotypes)affiliated to the orders Agaricales, Atheliales andPolyporales and closely related to the generaMarasmiellus, Amphinema and Trametes/Pere-nniporia, respectively (Figure 2).

Representatives of the orders Hypocreales, Euro-tiales, Pleosporales and Xylariales (phylum Asco-mycota) were found in all samples of marineinvertebrates used for fungi isolation. However,the genera Acremonium, Arthrinuium and Bothyo-sphaeria were only found associated with thesponge D. reticulata and the genera Alternaria,Pestalotiopsis and Cunninghamella were observedonly associated with the ascidian Didemnum sp.(Figure 1). In addition, the phylum Basidiomycotawas only detected in sponges A. viridis andD. reticulata (Figure 1).

http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov

Alternaria

1% 1% 1% 1% 3% 1%2%

12%

2%

17%

AlternariaAspergillusBionectriaBotryosphaeriaCladosporiumCochliobolus CunninghamelaFusariumMucorPenicillium

9%3%

2%4%

7%

2%2%2%

2%

25%

Mycale laxissima

Aspergillus Bionectria CladosporiumCochliobolus Fusarium GlomerellaPenicillium Phoma

11%

6%11%2%

29%PenicilliumPestalotiopsisPhoma Rhizopus Trichoderma Unidentified

42%

Trichoderma Verticillium Unidentified

1%11%

2%

7%

3%2%

23%

Amphimedon viridisAgaricales Aspergillus Atheliales BionectriaCladosporiumCochliobolusFusarium

1% 1%9%

3%5%

12%38%

8%

Dragmacidon reticulata Acremonium ArthtiniunAspergillus BotryosphaeriaCochliobolusFusarium GlomerellaMucor Nectria Penicillium5%

2%

7%2%35%

PenicilliumRhizopusTrichodermaUnidentified

1%6%

2%5%3%3%3%

PenicilliumPhoma PolyporalesRhizopus TrichodermaUnidentified

Didemnum sp.

Glomerella

Figure 1. Occurrence of filamentous fungi in marine macroorganism samples: ascidian Didemnum sp. (66 isolates, 47ribotypes); sponge Mycale laxissima (55 isolates, 35 ribotypes); sponge Amphimedon viridis (60 isolates, 43 ribotypes)and sponge Dragmacidum reticulata (66 isolates, 43 ribotypes).


About 19% (27 ribotypes) of the total number ofdistinct ribotypes (144) obtained in this study wereunidentified fungi. Among them, 18 ribotypes couldnot be identified due to their low quality se-quences. The remaining 9 ribotypes showedhigh sequence similarity with fungi which havenot been identified yet (isolates F169 and F186) orpresented low sequence similarity with fungalsequences available at the database, forminga separate cluster in the phylogenetic tree [isolatesF176(=F215), F197 and F229 (=F226, F184)](Figure 2).

Isolation and diversity of marine-derivedbacteria

A total of 181 bacteria were isolated from algae,sponges and ascidians in the present investigation.M1 medium was the most appropriate for bacteriaisolation, allowing the recovery of 45% of the

bacterial strains, followed by the media TSA(27%), GPY (14%) and MA (14%).

ARDRA analysis yielded 158 distinct bacterialribotypes, revealing a high bacterial diversity. Thelargest diversity was recovered from the ascidianD. ligulum, with 32 isolates and 29 distinctribotypes, followed by algae Sargassum (26 isolatesand 24 distinct ribotypes), sponges M. angulosa (21isolates and 18 distinct ribotypes), M. laxissima (19isolates and 18 distinct ribotypes) and A. viridis (19isolates and 17 distinct ribotypes), ascidian Didem-num sp. (18 isolates and 17 distinct ribotypes), andsponges G. corticostylifera (17 isolates and 17distinct ribotypes), D. reticulata (15 isolates and 11distinct ribotypes) and A. corrugata (14 isolates and14 distinct ribotypes) (Figure 3).

16S rRNA gene sequencing-based analysis showedthat marine-derived bacteria were related to 41genera distributed among the phyla Proteobacteria(35.4%), Actinobacteria (30.4%), Firmicutes (28.7%)and Bacteroidetes (1.1%). Eight isolates (4.4%)were classified as unidentified bacteria, since the


first 100 hits in Blastn analyses were unculturedbacteria. The phylum Proteobacteria included repre-sentatives of Classes Alphaproteobacteria (54 iso-lates), represented by the genera Aurantimonas,Brevundimonas, Pseudovibrio, Stappia, Ruegeria andNautella, and Gammaproteobacteria (10 isolates),

Strain F244 (1)Penicillium paxilli NRRL 2008 (EU427293)

Penicillium sumatrense CBS 416.69 (AY213621)Penicillium sp. NRRL 32575 (DQ123664)Strain F12 (2)

Strain F193 (3)Penicillium sclerotiorum NRRL 2074 (AF033404)

Strain F238Penicillium paneum NRRL 25159 (DQ339554)Penicillium chrysogenum NRRL 35688 (EF200101)

Aspergillus parasiticus NRRL 6433 (EF661568)

98

91

87

73

100

Aspergillus flavus NRRL 4998 (EF661566) Strain F234 (3)

Strain F84 (3)Aspergillus bridgeri NRRL 13000 (EF661404)Aspergillus sclerotiorum NRRL 35024 (EF661401)

Strain F19Penicillium citrinum NRRL 35459 (EF634428)

APU28822A spergillu sphoenicis NRStrain F80 (4)Aspergillus tubingensis NRRL 4875 (EF661193)

Aspergillus caesiellus NRRL 14879 (ARU29651)Strain F213Aspergillus sydowii NRRL 4768 (EF652473)

Cladosporium cladosporioides ATCC 58991 (AY342067)100

99

94

90

Eur

Cap

Strain F159 (2)Strain F225 (3)Botryosphaeria ribis (AY004336) Botryosphaeria parva CBS 110.301 (AY928046)

Strain F60 (3)Cochliobolus heterostrophus CBS 134.39 (AY544645)Strain F194Alternaria sp.CBS 174.52 (DQ678068)

Strain F233 (2)Phoma glomerata CBS 528.66 (EU754184)

Fungal sp. MC541 (EF177838)Strain F169

Sporidesmium obclavatulum (DQ408556)Fungal endophyte 9200 (EF420061)

99

81

75100

72

96

99

73

85

Ple

Bot

1Camarosporium leucadendri CBS123027(EU552106)

Strain F176 (1)Strain F229 (3)

Arthopyrenia salicis CBS 368.94 (AY538339)Pleospora leptosphaer ulinoides CBS 452.84(AY849956) Strain F192 (1)

Arthrinium sacchari ATCC76303 (AY345898)Strain F17 (2)

Apiospora setosa ATCC 58184 (AY346259)Strain F186Non-identified fungus Apiosporaceae F5 (EF564155)

Pestalotiopsis maculiformans CBS 122683 (EU552147)Pestalotiopsis photiniae PSHI 2002 (DQ657877)Strain F221 (3)

Strain F195 (4)97

7496

99

99

83

73

90 Xyl

Eutypa consobrina CBS 122677 (EU552126)Xylaria hypoxylon ATCC 42768 (AY327480)Strain F204 (1)Xylariasp. NRRL 40192 (EF157664)Strain F228 (2)Glomerella cingulata FAU 553 (AF543786)Colletotrichum gloeosporioides BBA 70071 (J301908)Acremonium furcatum CBS 122.42 (EF543831)Strain F142Acremonium antarcticum CBS987.87 (EF543830)

Strain F211 (9)Trichoderma inhamatum CBS273.78 (AF399237) Trichoderma sp.CBMAI 852 (EU088357)

Strain F137 (8)Trichoderma viride ATCC 38501 (AF127145)()

98

100

100

77

95

86

97

81 Hyp

Phy

Strain F1 (1)Nectria ipomoeae NRRL 22101 (AF178367)

Fusarium solani ATCC 56480 (FJ345352)Fusarium ambrosium NRRL 20438 (AF178366)Strain F203

Strain F224 (15)Fusarium equiseti NRRL 13405 (X80812)

Strain F275Verticillium sp. Vl-23 (AY312607)

Strain F180Hydropisphaera erubescens ATCC 44545 (AF193231)

Nomura earileyi (DQ067301)Cordyceps chlamydosporia CBS 101244 (DQ518758)

Strain F197

100

98

99

96

93Hyp

2

Clonostachys divergens CBS 967.73 (AF210677)Strain F243 (5)

Strain F155 (1)Marasmiellus palmivorus DED 6519 (AY639434)Marasmiellus sp. DMC 027 (EF160084)

Crinipellis maxima DAOM 196019 (AF042630)Peniophora cinerea CBS 404.74 (DQ094786)

Amphinema byssoides HHB-13195 (AF518597)Strain F154Strain F205

Amphinema byssoides HHB-13195 (AF518597)Trametes gibbosa Wu 9411-7 (AY351924)

Strain F232Cunninghamella echinulata NRRL1382 (AF157184)

9598

94

97

97

9099

98

Ath

Pol

Aga

0.05

3 Cunninghamella polymorpha NRRL 6441 (AF113461)Strain F124

Mucor ramosissimus ATCC 28933 (AY213715)Rhizopus oryzae CBS 395.95 (AY213624)

Strain F141 (1)Rhizopus stolonifer NRRL 1477 (AF113482)100

99

88

99

99 Muc

Cladosporiumuredinicola ATCC46649 (EU019264)

Bionectriaralfsii CBS129.87 (AF210676)

represented by Acinetobacter, Endozoicomonas, Mi-crobulbifer, Pantoea, Photobacterium, Vibrio andStenotrophomonas (Figure 4). The Firmicutes wererepresented by 9 genera, including Bacillus,Exiguobacterium, Halobacillus, Lysinibacillus, Paeni-bacillus, Paucisalibacillus, Planomicrobium, Staphy-lococcus and Terribacillus (Figure 4). The phylumActinobacteria encompassed seventeen genera ofcultivable actinobacteria, namely Agrococcus, Art-hrobacter, Brachybacterium, Brevibacterium, Curto-bacterium, Gordonia, Janibacter, Kineococcus,Knoellia, Kocuria, Marmoricola, Microbacterium,Micrococcus, Nocardia, Nocardioides, Sacharo-polyspora and Williamsia (Figure 5). Finally, thephylum Bacteroidetes was represented by only 2isolates, related to the genera Aquimarina andDokdonia.

Bacillus was the most abundant genus recovered,with 33 isolates, followed by Ruegeria with 31isolates and Micrococcus with 23 isolates. All ofthem revealed broad distribution among the marinemacroorganisms sampled (Figure 3). On the otherhand, several genera were isolated uniquely fromone host. This was the case of Dokdonia andKnoellia, recovered only from algae Sargassum;Acinetobacter, Paucisalibacillus, Paenibacillus andExigobacterium, isolated only from the ascidianD. ligulum; Aquimarina, Kineococcus, Photobacter-ium, Lysinibacillus and Terribacillus, isolated onlyfrom the sponge M. laxissima; Marmoricola, Pan-toea and Planomicrobium, recovered only from thesponge G. corticostylifera; Saccharopolyspora andAgrococcus, isolated only from the spongeA. viridis; Stenotrophomonas and Janibacter, iso-lated only from the sponge A. corrugata and thegenera Halobacillus and Gordonia, found only insponge M. angulosa and ascidian Didemnum sp.,respectively (Figure 3).

The highest bacterial richness was obtained fromthe ascidian D. ligulum, which harbored 14 bacter-ial genera, followed by the sponge G. corticostyli-fera and the algae Sargassum, with 12 and 11bacterial genera, respectively (Figure 3).

Figure 2. Phylogenetic analysis of partial 28 S rDNA gene(D1/D2 region) sequences of fungal isolates and relatedmicroorganisms belonging to the phylum: (1) Ascomyco-ta, (2) Basidiomycota and (3) Zygomycota. Evolutionarydistances were based on Kimura 2p model and treereconstruction on the neighbour joining method. Boot-strap values (1000 replicate runs, shown as %) 470% arelisted. Mucor ramosissimus ATCC 28933 was used asoutgroup. Order: Eur (Eurotiales), Cap (Capnodiales), Bot(Botryosphaeriales), Pl (Pleosporales), Xyl (Xylariales),Hyp (Hypocreales), Phy (Phyllachorales), Pol (Polypor-ales), Ath (Atheliales), Aga (Agaricales) and Muc (Mucor-ales).

22%

5%

5%22%

6%

6%6%

Didemnum sp.

Bacillus

Curtobacterium

Endozoicomonas

Gordonia

Micrococcus

Pseudovibrio

Ruegeria

11%

5%

11%

5%32%

5%5%

Mycale laxissima

Unidentified

Aquimarina

Bacillus

Kineococcus

Lysinibacillus

Micrococcus

Nautella

5%

17%6%

Stappia

Unidentified

Vibrio

5%

11%5%5%

Phbiotobacterium

Pseudovibrio

Terribacillus

Vibrio

5%

32%

5%

11%

Amphimedon viridis

Agrococcus

Pseudovibrio

Ruegeria

33%

20%

Dragmacidon reticulata

Bacillus

Brachybacterium

Brevibacterium32%

21%5%

21%Sacharopolyspora

Staphylococcus

Unidentified

Williamsia 7%

7%13%

20%

Micrococcus

Ruegeria

Staphylococcus

6%12%

Geodia corticostyliferaAurantimonasBacillus

17%

11%

6%6%6%6%

6%

6%

6%

6%

6% BrachybacteriumMarmoricolaMicrococcusNocardiaNocardioidesPantoeaPlanomicrobiumStaphylococcusUnidentified VibrioWilliamsia

3% 3%6%

3%3%

3%

10%

35%

3%3%

6%

Didemnum ligulum AcinetobacterArthrobacterBacillusBrevibacteriumCurtobacteriumExiguobacteriumKocuriaMicrococcusNocardiaPaenibacillus

4%

27%

19%

8%4%

Sargassum sp. algae Arthrobacter

Bacillus

Brevundimonas

Dokdonia

Knoellia

Kocuria

Micrococcus

13%

3%3%3%

35%PaucisalibacillusRuegeriaStaphylococusStappiaUnidentified

4%

4%4%

7%15%

4%Nocardioides

Ruegeria

Staphylococcus

Vibrio

7%7%7%

7%

Axinella corrugata

Bacillus

Brevundimonas

Janibacter

19%

Mycale angulosa

Bacillus

7%

7%

15%36%

7% Janibacter

Kocuria

Microbacterium

Micrococcus

Staphylococcus

Stenotrophomonas

Unidentified

38%

5%5%14%

19%

Halobacillus

Microbulbifer

Micrococcus

Pseudovibrio

Ruegeria

Figure 3. Occurrence of bacterial genera in marine macroorganism samples: ascidian Didemnum sp. (18 isolates, 17ribotypes); sponge Mycale laxissima (19 isolates, 18 ribotypes); sponge Amphimedon viridis (19 isolates, 17 ribotypes);sponge Dragmacidum reticulata (15 isolates, 11 ribotypes); sponge Geodia corticostylifera (17 isolates, 17 ribotypes);ascidian D. ligulum (32 isolates, 29 ribotypes); algae Sargassum sp. (26 isolates, 24 ribotypes); sponge Axinellacorrugata (14 isolates, 14 ribotypes) and sponge Mycale angulosa (21 isolates, 18 ribotypes).


73

98

9997

97

99

Strain B386Strain B106 (1)Strain B105Ruegeria sp. JC1077 (AF201086.1)Strain B504 (2)

Strain B163 (10)Ruegeria lacuscaerulensis ITI 1157 (U77644.1)Ruegeria scottomollicae LMG24367 (AM905330.1)

Ruegeria atlantica IAM14463 (D88526.1)Strain B438 (4)

Nautella italica LMG24365 (AM904562.1)Strain B173

Alpha

99

99

92

9698

79

85

96

88

99

80

85

Aurantimonas coralicida WP1 (AJ786361.1)Strain B242 (1)Brevundimonas intermedia ATCC 15262 (AJ227786.1)

Brevundimonas mediterranea LMG 22801 (AJ244706.1)Strain B385Stappia aggregata IAM 12614 (D88520.1)

Strain B323Pseudovibrio ascidiaceicola NBRC 100514 (AB175663.1)

Pseudovibrio japonicus NCIMB 14279 (AB246748.1)Strain B468 (2)Strain B505 (5)Pseudovibrio denitrificans DN34 (AY486423.1)Strain B259

Strain B466Microbulbifer maritimus TF-17 (AY377986.1)Microbulbifer thermotolerans JAMBA94 (AB124836.1)

Gamma

9299

90

99

81

77

99

97

7795

Endozoicomonas elysicola MKT110 (AB196667.1)Strain B169

Acinetobacter septicus AK001 (EF611420.1)Strain B404

Acinetobacter baumannii DSM 30007 (X81660.1)Strain B454Pantoea stewartii subsp. indologenes LMG 2632 (Y13251.1)

Photobacterium ganghwensis FR1311 (AY960847.1)Strain B463

Strain B243Vibrio vulnificus ATCC 27562 (X56582.1)

Vibrio parahaemolyticus ATCC 17802 (X74720.1)Vibrio campbellii ATCC 25920 (X56575.1)Strain B498 (1)Vibrio harveyi NCIMB1280 (AY750575.1)Strain B497

Strain B278Stenotrophomonas maltophilia ATCC 13637 (M59158.1)

99

99

93

99

99

99

85

93

77

ppPaenibacillus provencensis 4401170 (EF212893)Strain B220

Paucisalibacillus globulus 5 (EU430986.1)Strain B239 (1)

Strain B311Halobacillus litoralis SL-4 (X94558.1)Terribacillus halophilus 002-051 (AB243849.1)Strain B508

Bacillus herbersteinensis D1,5a (AJ781029.2)Bacillus boroniphilus T-15Z (AB198719.1)Strain B186

Bacillus firmus NBRC 15306 (AB271750.1)Strain B237 (4)

Exiguobacterium profundum 10C (AY818050.1)Strain B164

Bacillus licheniformis DSM 13 (X68416.1)Strain B198

Bacillussubtilis DSM10 (AJ276351.1)

Firmicutes

Bacillus pycnus NRS 1691 (AF169531.1)Strain B118

Lysinibacillus boronitolerans 10a (AB199591.)Strain B442

Planomicrobium okeanokoites IFO 12536 (NR_025864)Strain B459Bacillus beijingensis ge10 (EF371374.1)

Strain B116Strain B475Bacillus thuringiensis ATCC 10792 (AF290545.1)Bacillus weihenstephanensis DSM 11821 (AB021199.1)

Strain B119Staphylococcus caprae ATCC 35538 (NR_024665)

98

99

98

96

97

Strain B153 (1)Strain B115 (8)Bacillus flexus IFO15715 (AB021185.1)Bacillus megaterium L2S3 (EU221414.1)

Strain B389Strain B265Strain B465 (1)Strain B172 (3)Staphylococcus hominis ATCC 27844 (L37601.1)Strain B480Staphylococcus epidermidis ATCC 14990 (D83363.1)

Brevibacterium lutescens CF87 (AJ488509.1)Brevibacterium casei DSM 20657 (AJ251418.1)99

99

0.02

Alpha proteobacterium A40 (AB3025355.1)

Figure 4. Phylogenetic analysis of partial 16S rRNA gene sequences of bacterial isolates and related microorganismsbelonging to the phyla Proteobacteria and Firmicutes. Evolutionary distances were based on Kimura 2p model and treereconstruction on the neighbour joining method. Bootstrap values (1000 replicate runs, shown as %) 470% are listed.Brevibacterium lutescens CF87T and B. casei DSM 20657T were used as outgroup. Alpha=Alphaproteobacteria Class;Gamma=Gammaproteobacteria Class.


Williamsia deligens IMMIBRIV 956Fl (AJ920291.1)

Strain B138Williamsia maris JS1 (AB010909.2)

90

97

Williamsia serinedens IMMIB SR 4T (AM283464.1)

Strain B375

Strain B452

Gordonia terrae CCNAPH1296 (DQ490984.1)

Strain B204

Nocardia harenosa WS26 (DQ282122.1)

Strain B276 (1)

Strain B506

Saccharopolyspora flava AS4.1520 (AF154128.1)

Marmoricola aequoreus SST45 (AM295338.1)

Strain B374

Marmoricola aurantiacus BC361 (Y18629.1)

Nocardioides lentus KSL19 (DQ121391.1)

Strain B422

100

9896

100

72

99

100

100

100

94

86

77

Nocardioides kongjuensis A24 (DQ218275.1)

Nocardioide soleivorans DSM 16090 (AJ698724.1)

Strain B177

Strain B366

Kineococcus radiotolerans SRS30216 (AF247813.1)

Knoellia sinensis DSM 12331 (AJ294412)

Strain B175

Janibacter melonis CM2104 (NR_025805)

Strain B255

Janibacter brevis DSM 13953 (AJ310085.1)

Strain B189 (1)

Brevibacterium lutescens CF87 (AJ488509.1)

Brevibacterium casei DSM 20657 (AJ251418.1)

8692

100100

100

77

100

Brachybacterium paraconglomeratum LMG 19861 (AJ415377.1)

Strain B373 (1)

Strain B235

Brachybacterium rhamnosum LMG 19848 (AJ415376.1)

Micrococcus luteus 5N5 (EU379292.1)

Strain B470 (17)

Micrococcus lylae DSM 20315 (X80750.1)

Strain B123 (1)

Arthrobacter globiformis A2 S3 (EU221407.1)

Kocuria palustris 5N4 (EU379291.1)

Strain B502 (3)

Kocuria rosea ATCC 187T (Y11330.1)

Kocuria marina KMM 3905 (AY211385.1)

Strain B141

84

95

100

100

100

82

100

100

74

86

98

Kocuria flavus HO9041 (EF602041.1)

Strain B136

Curtobacterium flaccumfaciens pv betae DSM 20141 (AM410689.1)

Strain B216

Curtobacterium sp. HIF2 (DQ205304.1)

Strain B419

Agrococcus versicolor DSM 19812 (AM940157.1)

Strain B281

Agrococcus sp. HZBC70 (EF155550.1)

Microbacterium foliorum DSM 12966 (AJ249780.1)

Strain B307

Microbacterium oxydans 006 (AY769918.1)

Strain B305

Pseudoalteromonasspongiae UST010723006 (AY769918.1)

7899

98

99

100

87

94

99

Pseudoalteromonas viridis G364 (AB231329.1)100

0.02

Figure 5. Phylogenetic analysis of partial 16S rRNA gene sequences of bacterial isolates and related microorganismsbelonging to the phylum Actinobacteria. Evolutionary distances were based on Kimura 2p model and tree reconstructionon the neighbour joining method. Bootstrap values (1000 replicate runs, shown as %) 470% are listed.Pseudoalteromonas spongiae UST010723-006T and P. viridis G-364T were used as outgroups.



Statistical comparisons of microbial diversityamong marine invertebrates based on ARDRAdata

In order to investigate the microbial diversityderived from marine macroorganisms, diversityindices were used to calculate the microbialrichness, diversity and dominance from the ARDRAdata. Since these indices are dependent on thetotal number of ribotypes analyzed and the micro-organisms (filamentous fungi and bacteria) wereunequally sampled, randomization analyses wereused to create pseudo-communities at specifiedsubsample sizes and thus allow the comparisonamong the marine macroorganisms. Relatively highribotype richness and diversity index (Shannon–Wi-ener) values were observed for cultivable microbialcommunities recovered from marine macroorgan-isms (data not shown).

In general, the cultivable fungal and bacterialcommunities recovered did not present statisticallydifferent richness and diversity indices based uponARDRA data at all the sub-sample sizes analyzed.One exception was the bacterial community of thesponge D. reticulata (DR), which showed a ten-dency for lower values of richness and diversityindices (data not shown). However, the sample sizeof D. reticulata (DR) was substantially smaller thanthe samples recovered from the other macroorgan-isms. Therefore, further sampling would be neces-sary to allow conclusive comparisons. The steepshape of the richness curves for both bacteria andfungi, strongly indicates that much more diversitymay be encountered if larger sample efforts aremade (data not shown). The dominance index, thefraction of the community that is represented bythe most common ribotype, rapidly reached rela-tively low values for all cultivable microbialcommunities derived from marine macroorganisms(data not shown), thus revealing that these did notpresent significant dominance of any particularribotype.

Discussion

The nature and diversity of filamentous fungi andbacteria associated with marine macroorganisms isstill poorly understood. Research reports on mar-ine-derived fungi and bacteria have typicallyconcentrated on natural product chemistry, sincethese microorganisms have been shown to producenovel bioactive natural compounds that are notfound in terrestrial strains (Blunt et al. 2008, Koniget al. 2006, Jensen and Fenical 2002). In order to

achieve a broader characterization of microbialassemblages in a marine environment, a culture-dependent approach was successfully applied in thepresent investigation, resulting in the recovery of256 filamentous fungi and 181 bacteria frominvertebrate and algae samples.

Marine fungi have long been known to exist in themarine environment. Ecologically, they are impor-tant intermediaries of energy flow and play animportant role in nutrient regeneration cycles.Moreover, some marine fungi also cause diseasesof marine animals and plants while others formmutualistic symbiotic relationships with otherorganisms (Wang et al. 2006). Investigations ofthe distribution of marine-derived fungi based onchemical assays revealed that sponges have com-monly yielded the greatest taxonomic diversity(Bugni and Ireland 2004). However, in the presentstudy the ascidian Didemnum sp. presented thehighest diversity of filamentous fungi. These resultscould be explained by the fact that fungi classifiedas ‘sponge-generalists’ have shown to producenovel bioactive compounds (Bugni and Ireland2004; Wang et al. 2006; Baker et al. 2008) and,for that reason, there is a large number ofinvestigations being conducted on sponge-derivedfungi.

Marine-invertebrate-inhabiting fungi isolated inthe present work have been commonly found inboth terrestrial and marine habitats. Data derivedfrom taxonomic characterization allowed the iden-tification of 24 distinct taxa belonging to theAscomycota, Basidiomycota, Zygomycota and mi-tosporic fungi. It is worth to mention that 6% (9ribotypes) of the total number of distinct fungiribotypes may possibly represent new species offilamentous fungi, considering the high sequencesimilarity with fungi that are yet unidentified or thelow sequence similarity with known fungi from thedatabases. However, additional taxonomical ana-lyses based on a polyphasic approach should beconducted in order to confirm the new specieshypothesis.

The majority of the fungal isolates derived fromthe marine invertebrates sampled in the presentwork were shown to be related to Ascomycota.Representatives of these fungi have been isolatedfrom marine algae, cnidarians, seagrasses, spongesand tunicates in different parts of the world (Li andWang 2007; Da Silva et al. 2008). The predominanceof ascomycetes in aquatic habitats has beendiscussed in the literature. This group representsfungi that are readily cultivable (Baker et al. 2008)and could be easily recovered when culture-dependent techniques are applied. The majorhypothesis to explain this predominance is the


presence of spores with adaptation (appendages) tothe aquatic ecosystem, which facilitates buoyancyin water and substrate adherence (Prasannarai andSridhar 2001). According to Vijaykrishna et al.(2006), species of freshwater ascomycetes share amassive apical ring, which possibly facilitates thespores dispersion, explaining why certain ascomy-cetes are better adapted to aquatic habitats thanother fungal groups including the basidiomycetes.

All marine invertebrates studied in this workyielded a great number of Ascomycota fungibelonging to the genera Aspergillus, Penicillium,Trichoderma and Fusarium, which have alreadybeen reported in the literature as invertebrate-inhabiting fungi (Baker et al. 2008; Da Silva etal. 2008; Holler et al. 2000) and as a prolificsource of biologically active secondary metabo-lites, such as polyketides with antitumoral and/or antimicrobial activities (Bugni and Ireland2004). In addition, species from the genusTrichoderma are well known by their capacityto produce cellulolytic enzymes. Trichodermareesei is the main industrial source of cellulasesand hemicellulases harnessed for the hydrolysisof biomass to simple sugars, which can then beconverted to biofuels such as ethanol and otherchemicals (Le Crom et al. 2009). One explana-tion for the high number of bioactive compoundsreported from marine-derived Aspergillus andPenicillium spp. is that representative of thesegenera are salt tolerant, fast growing and easilyobtained from many substrates. On the otherhand, Aspergillus sydowii is considered thecausative agent of an epizootic disease affectingsea fan corals (Lewbart 2006) and Fusariumsolani is known for its ability to infect variousmarine crustaceans (Bugni and Ireland 2004).

Some Ascomycota recovered in the present workare not common in marine sponges and ascidians,such as Pestalotiopsis spp., which have beenisolated from Micronesia seawood (Masuma et al.2001); Eutypella spp., already isolated from aBrazilian marine zoanthid (da Silva 2008 and froma Chinese mollusk (Ciavatta et al. 2008), andXylaria spp., already isolated from a south Chinasea coast (Lin et al. 2001) and from coral reefs inPuerto Rico (Toledo-Hernandez et al. 2008). It isworth to note that these three fungal generabelong to Xylariales order, which have been provento be prolific sources of novel bioactive compounds(Ciavatta et al. 2008; Lin et al. 2001). Additionally,four Dematiaceous fungi isolated from the spongeD. reticulata (3) and from the ascidian Didemnumsp. (1) identified as Botrysphaeria sp. (Ascomycota)have only been reported as coral-derived fungi inPapua New Guinea (Alves et al. 2007).

Representatives of Zygomycota, Mucor and Rhi-zomucor, have been broadly isolated from marinesamples, including algae, cnidarians and sponges(Da Silva et al. 2008; Holler et al. 2000). However,the genus Cunninghamella was only reported inmarine water (Gomes et al. 2008). Fungi from thegenus Mucor, Rhizomucor and Cunninghamella haverepresentatives with high biotechnological poten-tial for producing bioactive compounds, such aslactic acid, lipases, proteases, a-amylases and b-gluconases (Celestino et al. 2006; Millati et al.2005).

Basidiomycota fungi are rarely isolated frommarine samples, however, in the present work,two representatives of Agaricales (with the sameribotype and from different hosts), one represen-tative of Polyporales and one representative of theAtheliales were recovered from the marine spongesD. reticulata and A. viridis. Gao et al. (2008) firstreported the presence of fungi belonging toAgaricales and Polyporales orders in the spongeSuberites zeteki. However, it is important tohighlight that this is the first report on the isolationof basidiomycete from Atheliales associated withmarine sponges. As filter feeders, sponges areexposed to pollutants present in waters, andaccumulated impurities from phytoplankton, orother suspended matters. Hence, it is reasonableto believe that some microbes in sponges producehydrolytic enzymes to convert these organic mat-ters into nutrients (Wang et al. 2006). Basidiomy-cete fungi are considered as the most efficientmicroorganisms to produce enzymes responsible forthe breaking down of lignocellulosic material andcolored pollutants. The production of these en-zymes by marine-derived fungi from ascomycota,zygomycota and basidiomycota has been reportedin the literature (Bonugli-Santos et al. 2009,Raghukumar 2008). These studies have been fo-cused mainly on lignocellulose-degrading enzymesproduction for black liquor, molasses, syntheticdyes and textile effluents decolorization and forpolycyclic aromatic hydrocarbons (PAHs) degrada-tion. Taking into account that in these processesthe salinity and pH are the main factors that caninfluence the enzyme production, marine-derivedfungi are expected to present a natural advantage(in comparison to their terrestrial counterparts) tobe used in high salt concentration processes orhabitats.

The relationship between terrestrial and aquaticfungi was reported by Vijaykrishna et al. (2006) andrevealed that, although freshwater taxa (includingmarine fungi) have evolved directly from terrestrialspecies, this evolution has been independent forseveral lineages. Opposite findings were reported


by Hibbet et al. 2001, who studied the relationshipbetween terrestrial and aquatic basidiomycetesand demonstrated that the latter was closelyrelated to the terrestrial fungi, since a specificaquatic clade was not found. The same results wereshown in the present work, once the marine-derived isolates recovered were only related toterrestrial counterparts in the phylogenetic tree(Figure 2).

In recent years, the marine environment has alsoshown a surprisingly diverse abundance of bacteria,including new species of Alphaproteobacteria (Mur-amatsu et al. 2007), Gammaproteobacteria (Bret-tar et al. 2002), Epsilonproteobacteria (Campbellet al. 2001), as well as actinomycetes (Maldonadoet al. 2005). These microorganisms have beenisolated from marine sediments, seawater andmarine invertebrates. The culture-dependent ap-proach used in this study resulted in the isolation of181 bacteria distributed in 41 genera affiliated tothe phyla Proteobacteria (Alpha- and Gammapro-teobacteria), Firmicutes, Actinobacteria and Bac-teroidetes, corroborating previous literature data.

Alphaproteobacteria have shown to be numeri-cally dominant in the water column using culture-independent molecular techniques (Venter et al.2004). Additionally, some marine invertebrates canharbor or be dominated by members of this taxon(Sfanos et al. 2005; Webster and Hill 2001).

In this work, Alphaproteobacteria accounted for30% of total bacterial isolates, encompassingmainly isolates belonging to genera Pseudovibrioand Ruegeria. The latter was one of the mostabundant genera found and widely distributedamong the hosts investigated. Bacteria related togenus Ruegeria require NaCl or sea salts for growthand have been isolated from marine environmentand sponges (Lee et al. 2007). The prevalence ofgenus Ruegeria associated to marine organismscould be related with quorum sensing systemscommonly found in Proteobacteria, which allowscell-to-cell communication and may be involved inthe symbiotic interactions between bacteria andtheir hosts (Mohamed et al. 2008). The genusPseudovibrio has only three reported species,named Pseudovibrio denitrificans, P. ascidiaceicolaand P. japonicus, all isolated from marine environ-ments (Hosoya and Yokota 2007, Fukunaga et al.2006, Shieh et al. 2004). Ruegeria spp. andPseudovibrio spp. are widely reported as present-ing potential to produce bioactive compounds,which are quite advantageous from an ecologicalpoint of view (Sertan-de Guzman et al. 2007;Mitova et al. 2004; Hentschel et al. 2001).

Gammaproteobacteria found in this study werediverse but not numerically abundant. This class

included Vibrio spp., which were isolated fromsponges, ascidians and Sargassum algae. Thesebacteria are consistently found in marine inverte-brates, mainly in oysters (Garnier et al. 2007,Comeau et al. 2005), corals (Bruck et al. 2007) andsponges (Mohamed et al. 2008). Vibrio spp. aretypically much more abundant in sediments,plankton, shellfish, oysters and corals than in thewater column (Comeau et al. 2005) and thisabundance may be related to their pathogenicity,resulting in increased oysters mortality in summer(Garnier et al. 2007) and in coral bleaching disease(Rosenberg et al. 2007).

The class Gammaproteobacteria also includedone representative of the genus Endozoicomonas,recovered from the marine invertebrate Didemnumsp. This recently described genus has only onespecies, E. elysicola. The type strain was isolatedfrom the sea slug Elysia ornate (Kurahashi andYokota 2007). Additionally, one isolate of the genusMicrobulbifer was recovered from the marineinvertebrate M. angulosa. Bacteria in the genusMicrobulbifer tend to be moderately halophilic andare often isolated from marine samples, occasion-ally marine invertebrates. These bacteria are ableto hydrolyze complex polysaccharides and somemembers are cellulolytic, what could explain theirabundance in marine macroorganisms such asascidians, which are encased in a tunic comprisedof cellulose (Riesenfeld et al. 2008).

Representatives of phylum Firmicutes, Bacillusspp., together with Ruegeria spp., were the mostabundant bacteria isolated in this work, andshowed wide distribution among the macroorgan-isms sampled. Although the majority of the Bacilliidentified in this study are usually terrestrialbacteria, studies focusing on the phylogeny andbiodiversity of marine Bacilli have shown thatB. subtilis, B. licheniformis, B. cereus, B. amylo-liquefaciens and B. pumilus are common inhabi-tants of marine environments (Gontang et al.2007). One representative of B. firmus was recov-ered from the ascidian Didemnum sp. This bacterialspecies belongs to halotolerant groups withoutbeing obligate marine bacteria, and seems to beinvolved in biogeochemical cycles and diversedegradation processes (Etoumi et al. 2009). ManyGram-positive bacteria are known to generatespores under adverse conditions, such as thoseencountered in marine ecosystems, and this isthought to ensure their survival within the marineinvertebrates (Ettoumi et al. 2009). Diverse anti-biotics including cyclic peptides, cyclic lipopep-tides and novel thiopeptides have been reportedfrom marine Bacillus sp. (Anand et al. 2006). Otherrepresentatives from Firmicutes recovered in this


study, such as Halobacillus, Paenibacillus, Staphy-lococcus, Planomicrobium and Exiguobacterium,have been previously found in marine samples aswell (Shnit-Orland and Kushmaro 2009; Gontanget al. 2007; Vishnivetskaya et al. 2006; Fiedleret al. 2005).

Actinobacteria found in the present investigationhave been already isolated from marine environ-ments, including Knoellia, Nocardia (Bredholdtet al. 2007; Zhang et al. 2006; Maldonado et al.2005), Kocuria, Marmoricola, Microbacterium,Gordonia and Williamsia (Fiedler et al. 2005),Arthrobacter (Farris and Olson 2007), Micrococcus(Cabaj and Kosakowska 2007), Janibacter (Kageya-ma et al. 2007), Kineococcus (Lee 2006), Nocar-dioides (Dastager et al. 2009, Kim et al. 2009),Brevibacterium (De et al. 2008; Lee 2008a), Agro-coccus (Lee 2008b), Saccharopolyspora (Pimentel-Elardo et al. 2008) and Brachybacterium (Chelossiet al. 2006; Montalvo et al. 2005). The genusCurtobacterium has been previously reported inmarine samples only by culture-independent methods(Gontang et al. 2007).

Surprisingly, the genus Streptomyces, reported asthe most abundant culturable actinobacteria groupfound in marine samples (Zhang et al. 2008), wasnot recovered from the marine invertebrates andalgae analyzed in this work. It should be noted thatcultivation-based approaches are limited since thehigh selectivity of isolation media and culturingconditions usually allow only a small fraction of thebacteria present within a marine sample to beisolated (Webster and Hill 2001) and further effortsare needed to improve selective procedures for theisolation of representative marine actinobacteria.

Actually, little is known about the diversity ofactinobacteria in marine habitats compared to thediverse range isolated from terrestrial environ-ments, even though these organisms have beenstudied in more detail than members of othergroups of prokaryotes due to their biotechnologicalimportance. Members of the Actinobacteria classhave considerable value as prolific producers ofbiologically active secondary metabolites, such asantibiotics and other therapeutic compounds, inaddition to vitamins and enzymes (Mincer et al.2005; Jensen et al. 2007) Like their terrestrialrelatives, marine actinobacteria may play animportant role in the breakdown of recalcitrantorganic matter and therefore in the ocean biogeo-chemical cycles (Surajit et al. 2006). Additionally,even as spores, marine Gram-positive bacteria havethe capacity to impact their surrounding chemicalenvironment, as evidenced by their capacity tooxidize metals (Gontang et al. 2007). Undoubtedly,knowledge of actinobacteria diversity and distribu-

tion in marine systems will contribute to theunderstanding of their ecology and aid to improvebioprospecting strategies (Zhang et al. 2006).

Data derived from ARDRA and sequencing ana-lyses enabled us to identify marine-derived micro-organisms and revealed a high microbial diversityassociated to the macroorganisms obtained. Repre-sentatives of filamentous fungi and bacteria iso-lated from Brazilian marine algae, ascidians andsponges samples belonged to 65 (24 fungal; 41bacterial) different genera known to be associatedwith terrestrial and marine environments, including5 groups (4 fungal; 1 bacterial) that have neverbeen reported in the literature as marine inverte-brate-derived microorganisms. The results sug-gested the selection of certain microbialtaxonomic groups according to the host. Thesedata may contribute to unravel specific ecologicalinteractions known to occur between microorgan-isms and marine macroorganisms. Specific inver-tebrate–microbial associations are suspected toplay a major role in maintaining coral health byprotecting the host from invasion of potentiallypathogenic microbes (Klaus et al. 2007; Salyers andWhitt 1994). It has been suggested that theresident microbial populations compete with in-vading microbes for nutrients and ecological nicheswithin the mucus and tissue of the corals (Klauset al. 2007). In addition, a recent study has showedthat the microbiota associated with the cnidarianHydra was conserved between samples maintainedin the laboratory for 30 years and fresh samplestaken from the marine environment (Fraune andBosch 2007).

Marine microorganisms are considered a promis-ing source of novel drugs due to their biodiversityand consequent chemodiversity, which, to date,are largely unexplored. Under varying ecologicaland physicochemical conditions in the oceans,microorganisms are able to mutate, evolve andadapt in a particular environment more readilythan higher life forms. Adaptation may include theproduction of specific secondary metabolites thatare important in their survival either as free-livingorganisms in the water or sediment, or in associa-tion with other marine organisms (Jensen andFenical 1996). The microbiological richness of themacroorganism environment indicates that cultiva-tion studies are highly relevant and are likely toresult in the discovery of new microbes and novelmetabolic pathways.

In conclusion, this investigation represents anemerging view of the filamentous fungi andbacterial diversity from Brazilian marine macro-organisms, since, to our knowledge, there wereno previous reports on fungi and/or bacteria


associated with sponges A. viridis; A. corrugata;D. reticulata; G. corticostylifera; M. laxissima;M. angulosa; the ascidians D. ligulum and Didem-num sp., and the algae Sargassum sp. anywhereelse in the world. Additionally, marine-derivedfilamentous fungi and bacteria recovered in thepresent work may represent a promising source ofmicrobial genetic resources to be biotechnologi-cally explored. The marine-derived isolates arepart of the research collection associated to theBrazilian Collection of Microorganisms from Envir-onment and Industry (CBMAI) and are currentlybeing investigated in the search for bioactivecompounds, such as antitumoral, antimicrobialand enzymes (lipases, cellulases and ligninases).Special attention will be given to the isolatesrepresenting putative new species, since theymight show ability to produce yet undescribedbioactive compounds.

Acknowledgements

The authors thank to Professor Marcio dos ReisCustodio (Instituto de Biociencias, Universidade deSao Paulo) for the assistance in the collection andidentification marine macroorganisms, as well as toDr. Alvaro Esteves Migotto and the technical staff ofthe Centro de Biologia Marinha (Universidade deSao Paulo) for logistical support. Financial supportand scholarships provided by FAPESP (05/60175-2)and CNPq and also gratefully acknowledged.

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