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ORIGINAL ARTICLE

Diversity and bioprospecting of fungal communitiesassociated with endemic and cold-adapted

macroalgae in AntarcticaValeria M Godinho1, Laura E Furbino1, Iara F Santiago1, Franciane M Pellizzari2,Nair S Yokoya3, Dicla Pupo3, Tania MA Alves4, Policarpo A S Junior5, Alvaro J Romanha5,Carlos L Zani4, Charles L Cantrell6, Carlos A Rosa1 and Luiz H Rosa11Departamento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,Belo Horizonte, Brazil; 2Laboratorio de Ficologia e Qualidade de Agua do Mar, Universidade Estadual doParana, Paranagua, Brazil; 3Secao de Ficologia para Nucleo de Pesquisa em Ficologia, Sao Paulo, Brazil;4Laboratorio de Qu mica de Produtos Naturais, Centro de Pesquisas ReneRachou, Fundacao Oswaldo Cruz,Belo Horizonte, MG, Brazil; 5Laboratorio de Parasitologia Celular e Molecular, Centro de Pesquisas ReneRachou, Belo Horizonte, Brazil and 6USDA-ARS, Natural Products Utilization Research Unit,Oxford, MS, USA

We surveyed the distribution and diversity of fungi associated with eight macroalgae fromAntarctica and their capability to produce bioactive compounds. The collections yielded 148 fungalisolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. Themost frequent taxa were Geomyces species (sp.), Penicillium sp. and Metschnikowia australis.Seven fungal isolates associated with the endemic Antarctic macroalgae Monostroma hariotii(Chlorophyte) displayed high internal transcribed spacer sequences similarities with the psychro-philic pathogenic fungus Geomyces destructans. Thirty-three fungal singletons (66%) were identified,representing rare components of the fungal communities. The fungal communities displayed highdiversity, richness and dominance indices; however, rarefaction curves indicated that not all of thefungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB6120, recovered from the endemic species Palmaria decipiens (Rhodophyte) and M. hariotii,respectively, yielded extracts with high and selective antifungal and/or trypanocidal activities, in

which a preliminary spectral analysis using proton nuclear magnetic resonance spectroscopyindicated the presence of highly functionalised aromatic compounds. These results suggest that theendemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungalcommunities consisting of a few dominant indigenous or mesophilic cold-adapted species, and alarge number of rare and/or endemic taxa, which may provide an interesting model of algalfungalinteractions under extreme conditions as well as a potential source of bioactive compounds.The ISME Journal (2013) 7, 14341451; doi:10.1038/ismej.2013.77; published online 23 May 2013Subject Category: Microbial ecology and functional diversity of natural habitatsKeywords: Antarctica; marine fungi; seaweeds; diversity; extremophiles

Introduction

Antarctica represents one of the most pristine

ecosystems in the world, characterised by shortfood chains dominated by microorganisms. Thefungal mats in the Antarctic exhibit complexcommunities that are able to survive under extremeenvironmental conditions, including dehydration,freeze-thaw cycles, low nutrient concentrations,

low temperatures, osmotic stress and ultravioletradiation irradiation (Fell et al., 2006; Goncalveset al., 2012). The Antarctic fungal communitiesinclude representatives of genera and species fromthe major fungal phyla Ascomycota, Basidiomy-cota, Zygomycota, Chytridiomycota and Glomero-mycota, as well as Oomycetes (Heterokontophyta,Oomycota) and slime moulds (Mycetozoa) tradi-tionally studied by mycologists (Bridge andSpooner, 2012). In recent decades, mycologicalstudies in Antarctica have mainly focused on thefungi present in soil, ice and lakes, and theirassociations with plants. However, limited atten-tion has thus far been devoted to the fungaldiversity of the Antarctic marine environment.Few existing studies address marine Antarctic

Correspondence: LH Rosa, Departamento de Microbiologia,Laboratorio de Sistematica e Biomoleculas de Fungos, Institutode Ciencias Biologicas, Universidade Federal de Minas Gerais,P O Box 486, Belo Horizonte MG CEP 31270-901, Brazil.E-mail: [email protected] 3 January 2013; revised 3 April 2013; accepted 7 April2013; published online 23 May 2013

The ISME Journal (2013) 7, 14341451

& 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13

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fungi, which have been detected in water (Fell andStatzel, 1971; Fell et al., 1973), wood baits (Grassoet al., 1997) and water and marine sediments (Vazet al., 2011). According to Bridge and Spooner(2012), only 42 fungal species have been describedfrom the Antarctic marine ecosystem.

The known marine macroalgal communities from

Antarctica are not very diverse in comparison withthat of warmer regions. However, this flora ischaracterised by a high degree of endemism andthe presence of cold-adapted species (Wiencke andClayton, 2002; Oliveira et al., 2009). In addition,macroalgae are important primary producers, pro-ducing B74 000 tons of wet biomass and having akey role in organic carbon fluxes in Antarctica(Nedzarek and Rakusa-Suszczewski, 2004). Also,macroalgae may shelter large numbers of associatedorganisms, including microbial mats survivingunder extreme conditions (Loque et al., 2010).

According to Bugni and Ireland (2004), fungirecovered from macroalgae represent the secondlargest source of marine fungi and include parasites,saprobes or mutualistic species. A number ofmacroalgal species have been studied in detailworldwide with respect to their associated fungalcommunities, which include the genera Ascophyl-lum, Ballia, Caulerpa, Ceramium, Ceratiodictyon,Cladophora, Chondrus, Dictyota, Dilsea, Egregia,Enteromorpha, Fucus, Gelidiella, Gracilaria, Grate-loupia, Halimeda, Halymenia, Hypnea, Laminaria,Lobophora, Padina, Porphyra, Portieria, Saccorhiza,Sargassum, Stoechospermum, Turbinaria and Ulva(Kohlmeyer and Volkmann-Kohlmeyer, 1991,Stanley, 1992; Zuccaro and Mitchell, 2005;

Zuccaro et al., 2008; Suryanarayanan et al., 2010).To the best of our knowledge, except for an initialcontribution by Loque et al. (2010), no other data areavailable regarding the species composition offungal communities associated with Antarcticmacroalgae. In this study, we acquire to presentinformation on the diversity and distribution offungal communities associated with endemic andcold-adapted macroalgae across latitudinal gradi-ents along the Antarctic Peninsula and their cap-ability to produce bioactive compounds.

Materials and methodsMacroalagae collectionSixty fresh thalli from each selected macroalgalspecies (four Phaeophyceae, three Chlorophyta andone Rhodophyta) were collected during December2010 and January 2011 in intertidal transects along arocky coastline that becomes ice free during theAntarctic summer. Samples of Adenocystis utricu-laris (Bory de Saint-Vicent) Skottsberg, Adenocystisspecies (sp.), Desmarestia menziesii J Agardh,Phaeurus antarcticus Skottsberg, Acrosiphoniaarcta (Dillwyn) Gain, Monostroma hariotii Gain,Ulva intestinalis Linnaeus and Palmaria decipiens

RW Ricker (Figure 1) were collected onboard theBrazilian Navy Polar Ship Almirante Maximiano(H41) along a 350-km transect through Elephant,King George and Deception Islands, in the AntarcticPeninsula (Figure 2). Physical and chemical waterparameters (temperature, salinity, conductivity, dis-solved oxygen and pH) were also recorded at each

site using a multiparameter probe Hexis TCS(Yellow Springs, OH, USA).

Macroalgae identificationComplete and fertile samples were sorted, washedand preserved in seawater formalin (4%) in theships laboratory, with the aim of performing macro-and micromorphological analyses. The identifica-tion of the macroalgal specimens was based on thepublications of Papenfuss (1964), Ricker (1987),Wiencke and Clayton (2002), Quartino et al. (2005)and Amsler et al. (2009). Nomenclatural updatesfollowed Guiry and Guiry (2012). Exsiccatae voucherswere manufactured for deposition in the SP Herbar-ium of the Jardim Botanico of Sao Paulo, Brazil.

Fungal isolationFive discs 8 mm in diameter were cut from eachmacroalgal specimen and washed twice using sterilelocal seawater for 2 min. The discs were inoculatedin Petri dishes containing marine agar (Difco,Franklin Lakes, NJ, USA) supplemented with 2%glucose and chloramphenicol (Sigma, St Louis, MO,USA) at a concentration of 200 mg ml 1 for selectiveisolation of marine fungi. All of the inoculated Petri

dishes were incubated for up to 60 days at 10 1C, andindividual colonies of fungi were purified on marineagar. Long-term preservation of fungi was carried outat 80 1C using cryotubes with sterile 15% glycerol.All of the fungal isolates examined in this work weredeposited in the Culture Collection of Microorganismsand Cells of the Universidade Federal of Minas Gerais,Brazil, under codes UFMGCB.

Fungal identificationThe protocol for DNA extraction from filamentousfungi followed Rosa et al. (2009). The internaltranscribed spacer (ITS) region was amplified with

the universal primers ITS1 and ITS4 (White et al.,1990). Amplification of the ITS region was per-formed as described by Rosa et al. (2009). Yeastswere characterised via standard methods (Yarrow,1998), and their identification was carried out usingthe taxonomic keys of Kurtzman et al. (2011). Yeastidentities were confirmed by sequencing the D1-D2variable domains of the large subunit ribosomalRNA gene using the primers NL1 and NL4, asdescribed by Lachance et al. (1999).

Amplification of the b-tubulin gene was per-formed with the Bt2a and Bt2b primers (Glass andDonaldson, 1995). PCR assays were conducted in

Fungi associated with Antarctic macroalgaeVM Godinho et al

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50ml reaction mixtures containing 1 ml of genomicDNA (10 ngml 1), 5ml of PCR buffer (100 mM Tris-HCl, 500 mM KCl, pH 8.8), 2 ml of dNTPs (10mM)plus 3ml of MgCl2 (25mM), 1 ml of each primer(50 pmolml1), 1 ml of dimethyl sulfoxide (DMSO;Merck, Billerica, MA, USA), 2 ml betaine (5 M), 0.2mlof Taq polymerase (5 U ml1 DNA) and 33.8ml ofultrapure sterile water. PCR amplifications wereperformed with the Mastercycler pro (Eppendorf,Hamburg, Germany), programmed for initial dena-turation at 94 1C for 5 min, followed by 35 cycles of

1 min of denaturation at 94 1C, primer annealing for1min at 59 1C and extension for 1.30 min at 72 1C,with a final 7-min elongation step at 72 1C. Afteramplification of the b-tubulin template, excessprimers and dNTPs were removed from the reactionmixture using a commercial GFX column with thePCR DNA Purification kit (Amersham Bioscience,Roosendaal, Netherlands). Purified PCR fragmentswere resuspended in 50 ml of Tris-EDTA buffer.

The amplified DNA was concentrated and pur-ified using the Wizard Plus SV Miniprep DNAPurification System (Promega, Madison, WI, USA)and sequenced using an ET Dynamic Terminator Kit

in a MegaBACE 1000/Automated 96 Capillary DNAsequencer (GE Healthcare, Piscataway, NJ, USA).The obtained sequences were analysed with Seq-Man P with Lasergene software (DNASTAR Inc.,Madison, WI, USA), and a consensus sequence wasobtained using Bioedit v. 7.0.5.3 software (Carlsbad,ON, Canada). To achieve species-rank identificationbased on ITS and b-tubulin data, the consensussequence was aligned with all sequences fromrelated species retrieved from the NCBI GenBankdatabase using BLAST (Altschul et al., 1997). The

consensus sequences of the algicolous fungi weredeposited into GenBank (see the accession numbersin the Table 1). However, according to Gazis et al.(2011), sequencing of the ITS region may fail torecognise some fungal genera. For this reason, theb-tubulin sequences, which are considered promis-ing for a one-gene phylogeny (Frisvad and Samson,2004), were used to elucidate the taxonomic posi-tions of the inconclusive taxa identified using ITSsequences. In addition, the followed criteria wereused to interpret the sequences from the GenBankdatabase: for query coverage and sequence identitiesX99%, the genus and species were accepted; for

Figure 1 Macroalgae collected from the Antarctic Peninsula, with information on their distributions given in parenthesis.(a) Acrosiphonia arcta (Dillwyn) Gain (cosmopolitan); (b) Adenocystis sp. (most likely endemic); (c) Adenocystis utricularis (Bory deSaint-Vicent) Skottsberg (Australia, New Zealand and Antarctica); (d) Monostroma hariotii Gain (Antarctic and Subantartic islands);(e) Palmaria decipiens RW Ricker (Antarctic and Subantarctic islands); (f) Ulva intestinalis Linnaeus (cosmopolitan); (g) Phaeurus

antarcticus Skottsberg (Antarctic and the Subantarctic islands); (h) Desmarestia menziesii J Agardh (Antarctic and the Subantarcticislands). Bars represent 1 cm.


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query coverage and sequence identities showing98%, the genus and species were accepted, but termcf. (Latin for confer compares with), used toindicate that the specimen resembles, but hascertain minor features not found in the reference

species, was included; for query coverage andsequence identities between 95% and 97%, onlythe genus was accepted; and for query coverage andsequence identities p95%, the isolates werelabelled with the order or family name or asunknown fungi. However, taxa that displayedquery coverage and identitiesp97% or an in-conclusive taxonomic position were subjected tophylogenetic ITS and b-tubulin analysis, withestimations conducted using MEGA Version 5.0(Tamura et al., 2011). The maximum compositelikelihood method was employed to estimate evolu-tionary distances with bootstrap values calculated

from 1000 replicate runs. To complete the molecularidentification, the sequences of known-type fungalstrains or reference sequences obtained from fungalspecies deposited in international culture collec-tions found in GenBank were added to improve the

accuracy of the phylogenetic analysis. The informa-tion about fungal taxonomic hierarchical follows therules established by Kirk et al. (2011) and theMycoBank (http://www.mycobank.org), and IndexFungorum (http://www.indexfungorum.org) databases.

Diversity, richness, dominance and distributionTo quantify species diversity, richness and even-ness, we used the following indices: (i) Fishers a,(ii) Margalefs and (iii) Simpsons, respectively.A rarefaction curve was calculated using the MaoTao index (Colwell et al., 2004). The similarities

Figure 2 Maps showing the positions of the islands sampled on the (a) Antarctic Peninsula across a transect of 350km. Sampling sites:D, Ulva intestinalis, Palmaria decipiens and Phaeurus antarcticus in (b)Stinker Point (61107.935S; 055125.997W) at Elephant island;D, Acrosiphonia arcta and D. menziesiiin (c), Keller Peninsula (62105.163S; 058124.784W) at Admiralty Bay at King George Island; D,Adenocystis utricularis, Monostroma hariotii, Adenocystis sp. in (d), Telefon Bay (62155.192S; 060139.797W) at Deception Island.


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Table 1 Molecular identification of fungi associated with Antarctic macroalgae: identification conducted using BLASTn searches of theof the internal transcribed spacer (ITS) region and b-tubulin gene

Macroalgaespecies

UFMGCBa

codeNo. of

isolatesTop BLAST search results(GenBank accessionnumber)

Querycoverage

(%)

Identity(%)

No. of bpsequenced

andanalysed

Proposed species or taxonomicgroup (GenBank accessionnumber)

Adenocystisutricularis D1N10.34.1 1 Debaryomyces hansenii(HE681104) 100 99 524 Debaryomyces hansenii(KC485456b)D1N10.13.1 1 Meyerozyma caribbica

(JQ686909)100 99 547 Meyerozyma caribbica

(KC485457b)Acrosiphoniaarcta

6063c 19 Geomyces pannorum(DQ189229)

98 99 729 Geomyces sp. (KC333881d)

K2.13.1 6 Metschnikowia australis(U76526)

99 99 484 Metschnikowia australis(KC485458b)

6074c,e 2 Penicillium chrysogenum(HQ652873)

99 99 662 fPenicillium sp. (KC333882d,KC823167g)

K2.6.1 1 Candida sake (EU326138) 100 99 588 Candida sake (KC485459b)6361c,e 1 Cladosporium perangustum

(JF499836)100 97 486 fCladosporium sp. (KC341715d,

KC823166g)6079 1 Cladosporium tenuissimum

(JQ246357)100 99 476 Cladosporium tenuissimum

(KC341716d)K2.9.1 1 Debaryomyces hansenii

(HQ860269)100 99 546 Debaryomyces hansenii

(KC485460b)6077c 1 Mortierella sp. (JX270406) 90 97 562 fMortierella sp. (KC341717d)6083c 1 Phoma sp. (JQ388278) 99 99 468 fPhoma sp. (KC341718d)6318 1 Thelebolus microsporus

(GU004196)99 99 495 Thelebolus globosus

(KC341719d)Desmarestiamenziesii

K1.17.1 1 Metschnikowia australis(FJ911872)

99 99 446 Metschnikowia australis(KC485461b)

6084c,e 1 Penicillium chrysogenum(JN021549)


Ulvaintestinalis

6101c,e 6 Penicillium commune(JN676122)


6092c,e 3 Penicillium solitum(JN642222)

98 99 534 fPenicillium discolor(KC485423d, KC823170g)

6090 3 Antarctomyces psychrotro-phicus (JN104511)

100 99 482 Antarctomyces psychrotrophicus(KC485424d)

CM.01.2 1 Cryptococcus victoriae[AY040653]

98 99 605 Cryptococcus victoriae[KC485462b]

6096 1 Engyodontium album(HM214540) 90 99 566 Engyodontium sp. (KC485425d

)

6097c 1 Geomyces luteus (AJ938164) 100 98 451 Geomyces luteus (KC485426d)6328 1 Helotiales sp. (HQ533820) 90 99 477 Helotiales sp. (KC485427d)6326 1 Mycoarthris corallinus

(AF128440)97 98 458 Mycoarthris cf. corallines

(KC485428d)6325c,e 1 Penicillium chrysogenum

(KC009835)98 98 507 fPenicillium sp. (KC485429d,

KC823171g)6095 1 Thelebolus microsporus

(GU004196)100 100 485 Thelebolus globosus

(KC485430d)Monostromahariotii

6120c,e 12 Penicillium chrysogenum(KC009826)


6112c 7 Geomyces pannorum(JF311913)

100 99 439 Geomyces cf. destructans(KC485432d)

D1N18.31.1 4 Meyerozyma guilliermondii(JF766631)

100 99 537 Meyerozyma guilliermondii(KC485463b)

6330 1 Cryptococcus laurentii

(FJ743631)

100 98 570 Cryptococcus cf. laurentii

(KC485433d)6130c 1 Lecanicillium araneicola

(AB378506)99 92 573 fCordycipitaceae sp.

(KC485434d)6138c 1 Paecilomyces anatarcticus

(AJ879113)100 96 475 fHelotiales sp. (KC485435d)

6316c 1 Pezizella discreta (JF908571) 99 97 487 fHyaloscyphaceae sp.(KC485436d)

D1N18.42.1 1 Rhodotorula mucilaginosa(JQ277252)

100 99 537 Rhodotorula mucilaginosa(KC485464b)

Palmariadecipiens

6034c,e 11 Penicillium chrysogenum(JN032681)

100 99 538 fPenicillium sp. (JX976546d,KC823172g)


91 99 554 Geomyces sp. (KC485437d)

6056 2 Acremonium strictum(AY138844)

92 99 565 Acremonium sp. (KC485438d)


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among fungal taxa from different areas were esti-mated using the Sorensen coefficient and Bray-Curtis measures. All of the results were obtainedwith 95% confidence, and bootstrap values were

calculated from 1000 iterations. All diversity andsimilarity indices, rarefaction curves and the princi-pal components analysis calculations were per-formed using the computer Program PAST, version1.90 (Hammer et al., 2001). Further information aboutthese indices can be found in Magurran (2011).

Fungal cultivation and preparation of extracts forbiological assaysAll fungal isolates were cultivated using solid statefermentation. Five-millimetre-diameter plugs fromeach filamentous fungus were inoculated into the

centres of Petri dishes (60 mm diameter, with 20 mlof marine agar). The plates were incubated at102 1C for 15 days, and the cultured materialsfrom each Petri dish were then cut and transferred to

50-ml vials containing 35 ml of ethanol. After 48 h atroom temperature, the organic phase was decanted,and the solvent was removed under a vacuumcentrifuge at 35 1C (Santiago et al., 2012). An aliquotof each dried extract was dissolved in DMSO(Merck) to prepare a 100-mg ml 1 stock solution,which was stored at 20 1C.

Assay for antimicrobial activitySusceptibility testing against Escherichia coliATCC 11775, Staphylococcus aureus ATCC 12600,Pseudomonas aeruginosa ATCC 10145, Candida

Table 1 (Continued )

Macroalgaespecies

UFMGCBa

codeNo. of

isolatesTop BLAST search results(GenBank accessionnumber)

Querycoverage

(%)

Identity(%)

No. of bpsequenced

andanalysed

Proposed species or taxonomicgroup (GenBank accessionnumber)

6050 2 Fusarium oxysporum(HQ248198)

91 99 524 Fusarium sp. (KC485439d)

E2N5.42.1 2 Yamadazyma mexicana(FJ455104)

100 99 500 Yamadazyma mexicana(KC485465b)

6047 1 Aspergillus candidus(FJ441637)

93 99 548 Aspergillus sp. (KC485440d)

6053 1 Chaetomium globosum(JN689341)

66 99 751 Chaetomium sp. (KC485441d)

6140c,e 1 Penicillium raistrickii(FR670335)

78 97 665 fPenicillium sp.(KC485442d,KC823173g)

6048c,e 1 Penicillium spinulosum(DQ132828)

74 99 697 fPenicillium spinulosum(KC485443d, KC823174g)

Phaeurusantarcticus

6141c,e 13 Penicillium chrysogenum(JQ665262)



100 99 495 Geomyces sp. (KC485445d)

6153c,e 2 Penicillium raistrickii(FR670335)


6164c,e

1 Aspergillus terreus(JQ070071)

100 100 536f

Aspergillus terreus (KC485447d

,KC823163g)

6167 1 Eurotium herbariorum(JN942870)

100 99 429 Eurotium herbariorum(KC485448d)

6327 1 Eurotium repens (AY373890) 100 99 482 Eurotium repens (KC485449d)6320c,e 1 Penicillium steckii

(HM469415)100 99 467 fPenicillium steckii(KC485450d,

KC846137g)Adenocystissp.

6323c,e 3 Penicillium chrysogenum(JQ665262)


6106 1 Aspergillus conicus(HE578068)

99 99 426 Aspergillus conicus(KC485452d)

6110c 1 Geomyces pannorum(JF311913)

100 99 451 Geomyces sp. (KC485453d)

6109c,e 1 Penicillium citrinum(FJ765031)

93 100 507 fPenicillium citrinum(KC485454d, KC823177g)

6322c,e 1 Penicillium commune(FJ499454)


When the number of isolates was 41, 50% of the isolates were sequenced and the best sequence was deposited in the GenBank database.aUFMGCBCulture of Microorganisms and Cells from the Federal Universidade of Minas Gerais.bD1/D2 sequences deposited.cTaxa subjected to phylogenetic analysis based on the ITS region for elucidation of taxonomic positions.dITS sequences deposited.eTaxa subjected to phylogenetic analysis based on the b-tubulin regions for elucidation of taxonomic positions.fTaxonomic position suggested by the phylogenetic analyses.gb-tubulin sequences deposited.


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albicans ATCC 18804, Candida krusei ATCC 6258and Cladosporium sphaerospermum CCT 1740 wasperformed using a protocol established by Carvalhoet al. (2012). All extracts (dissolved in DMSO) werediluted to a final concentration of 250 mg ml1 foruse in the antimicrobial assays. The results areexpressed as the percent inhibition in relation to

controls without drugs. All antimicrobial assayswere performed in duplicate.

In vitro assays with intracellular amastigote forms ofTrypanosoma cruziIn vitro assays with amastigote forms ofT. cruziwereperformed according to protocols established byBuckner et al. (1996) with some modifications.Trypanosoma cruzi (Tulahuen strain) expressingthe E. coli b-galactosidase gene were grown on amonolayer of mouse L929 fibroblasts. Cultures to beassayed for b-galactosidase activity were grown inRoswell Park Memorial Institute 1640 medium(pH 7.2-7.4) without phenol red (Gibco BRL,Cergy-Pontoise, France), plus 10% foetal bovineserum and glutamine. L929 fibroblasts were seededinto 96-well tissue culture microplates at a concen-tration of 4.0103 per well in a volume of 80 ml andincubated overnight. b-galactosidase-expressing try-pomastigotes were then added at a concentration of4.0104 per well in a volume of 20 ml. After 2 h, themedium containing trypomastigotes that did notpenetrate into the cells was discarded and replacedwith 200ml of fresh medium. After 48 h, the mediumwas discarded again and replaced with 180ml offresh medium and 20ml of the test extracts. Each

extract was tested in triplicate. After 7 days ofincubation, chlorophenol red b-D-galactopyranoside(100mM final concentration) and Nonidet P-40(Sigma-Aldrich, St. Louis, MO, USA) at finalconcentration of 0.1% were added to the plates,followed by incubation overnight at 37 1C, and theabsorbance was measured at 570 nm in an auto-mated microplate reader. Benznidazole at its 50%inhibitory concentration (IC50; 1 ml ml

1) was usedas a positive control. The results were expressed asthe percentage of growth inhibition. All assays wereperformed in triplicate.

Minimal inhibitory concentration and IC50The crude antimicrobial extracts were subjected todetermination of a minimal inhibitory concentration(MIC) and the trypanocidal extracts were subjectedto determination of a IC50. MIC and IC50 representthe lowest concentrations of a crude extract(or compound) that inhibits the functions of a target(in this study, microorganisms and T. cruzi) by 100%and 50%, respectively. This quantitative measureindicates how much of a particular crude extract isneeded to inhibit a given biological process by 50%.SoftMax Pro 5.3 (Sunnyvale, CA, USA) was used tocalculate MIC values via nonlinear curve fitting of

two or more independent experimental data sets to afour-parameter logistic doseresponse model. Noconstraints were applied to the curve-fitting calcula-tions. All assays were performed in duplicate.

Nuclear magnetic resonance spectroscopy

Bioactive extracts were analysed via nuclear mag-netic resonance (NMR) spectroscopy in a VarianINOVA 600 MHz spectrometer (Varian Inc., PaloAlto, CA, USA). 1H-NMR spectra were recorded inDMSO-d6. The samples were prepared at a concen-tration of B10mgml1, and the results wererecorded in 3 mm NMR tubes using a standard 1Hpulse programme.

Results

Macroalgae collection and fungal identificationA total of 148 fungal isolates were recovered from391 tissue fragments from the eight macroalgalspecies, which were identified by DNA sequencesof the ITS region and b-tubulin gene in 21 differentgenera within of the phyla Ascomycota, Basidiomy-cota and subphylum Mortierellomycotina (Zygomy-cota; Table 1). All of the screened macroalgaeharboured associated fungi, and 50 taxa wereidentified. However, the number of fungal taxa anddiversity indices differed among the macroalgae.Among the phyla characterised, the most repre-sented orders were Eurotiales (43.24%), Helotiales(32.43%), Saccharomycetales (11.48%), Hypo-creales (6%) and Thelebolales (4.05%). In contrast,

taxa of the orders Tremellales, Capnodiales, Mortier-ellales, Pleosporales, Sordariales and Sporidiobo-lales were found as minority components of thefungal communities with an abundance ofp1.35%.

Twenty-eight fungal taxa presented low molecularsimilarities or inconclusive information in compar-ison with known fungal ITS sequences deposited inthe GenBank database. To identify these fungi,phylogenetic trees of the ITS (Figure 3) and/orb-tubulin (Figure 4) genes were constructed toillustrate their relationship with GenBank sequences.All ITS (Figure 3i) and b-tubulin (Figure 4c)sequences of the Penicillium isolates were analysedand the taxa P. spinulosum, P. steckii, P. citrinum

and P. chrysogenum were identified. The otherPenicillium taxa displayed inconclusive sequenceanalyses when compared with the sequences of thetype species, and were identified as Penicillium sp.

Thirty-six fungal isolates displayed ITS sequencesimilarities with Geomyces sp. Among them, theseven isolates were identified as G. destructans,which showed 100% of query coverage and 98%identity; also these fungal isolates presented only1.1% of sequence difference in comparison with thetype species G. destructans (GenBank Access num-ber EU884921; Table 1; Figure 3g). All otherGeomyces isolates formed a separated cluster when


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Figure 3 Phylogenetic analysis (ai) of the sequences of fungi (in bold) associated with Antarctic macroalgae in comparison with type(T) or reference (R) sequences of the closest species, following BLAST analysis, deposited in the GenBank database. The trees wereconstructed based on the ITS region sequences using the maximum composite likelihood method.


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compared with sequences of the type speciesof G. pannorum (GenBank Access number

FJ545236) and G. asperulatus (GenBank Accessnumber AJ390390; Figure 3g); these taxa wereidentified as Geomyces sp.

In addition, after ITS (Figure 3) and b-tubulin(Figure 4) sequence analysis, 27 fungal taxa wereidentified as belonging to the genera Acremonium,Aspergillus, Chaetomium, Cladosporium, Engyo-dontium, Fusarium, Geomyces, Mortierella,Penicillium, Phoma as well as the families Cordyci-pitaceae, Hyaloscyphaceae and order Helotiales,which displayed low sequence similarities whencompared with fungal sequences deposited in theGenBank.

Diversity, richness, dominance and distributionThe most frequent fungal taxa identified werePenicillium sp. (35.8%), Geomyces sp. (24.32%)and M. australis (4.72%). However, in general, thefungal communities associated with the Antarcticmacroalgae presented high values of the diversity(Fishers a22.0), richness (Margalefs10.41) anddominance (Simpsons0.94) indices. The phylumChlorophyta displayed a Fishers a5.67 and 9.33fungal taxa per macroalgal species, which wasfollowed by Rhodophyta (Fishers a4.47 and 9fungal taxa/macroalgal species) and Phaeophyta(Fishers a2.7 and 4 fungal taxa/macroalgalspecies). However, the values of the indices differedamong the macroalgae (Table 2). The fungal

Figure 3 Continued.


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communities associated with U. intestinalis andAdenocystis sp. showed high diversity and dom-inance values, whereas the communities corre-sponding to A. utricularis and D. menziesiidisplayed the lowest diversity, richness and dom-inance values. In addition, the A. arcta fungalcommunity showed the highest richness, but mod-erate diversity and dominance. Only two fungalspecies were isolated from the macroalgae A.utricularis and D. menziesii, which exhibited the

lowest Fishers a, Margalefs and Simpsons indices.In addition to the high fungal diversity (Fishers avalues) found in the sampled macroalgae, theMao Tao rarefaction curves continued to rise anddid not reach an asymptote (Figure 5), indicatingthat not all fungal diversity had been recovered.Thirty-three fungal taxa (66%) occurred assingletons corresponding to a single macroalgalspecies, representing rare components of the fungalcommunities.

a

b

c

Figure 4 Phylogenetic analysis (ac) of the b-tubulin sequences of the UFMGCB fungi (in bold) in comparison with type (T) or reference(R) sequences of the closest species, following BLAST analysis, deposited in the GenBank database. The trees were constructed based on

the b-tubulin gene sequences using the maximum composite likelihood method.


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The fungal composition was variable among themacroalgal species, which was confirmed by thevalues of the Sorensen and Bray-Curtis similarityindices (Figure 6). The Sorensen index showed thatthe most similar fungal communities were found inP. antarcticus and Adenocystis sp., both of which arebrown macroalgal species, but with differentmorphologies. However, the Bray-Curtis similaritybetween the fungal communities associated with theendemic macroalgae P. decipiens and M. hariotiiindicated the highest similarity with the presence

and abundance of the Geomyces and Penicilliumtaxa associated with both macroalgae. In addition,Penicillium taxa occurred in association with sevenother macroalgal species, followed by Geomyces (sixmacroalgae).

The principal components analysis of the physi-cal and chemical parameters of the water where themacroalgae were sampled related to the fungaldiversity indices (Figure 7) revealed that conductivityexhibited a positive correlation with the Fishers aand Simpsons indices for the fungal assemblages

Table 2 Physico-chemical parameters of water and diversity indices of fungal communities associated with the Antarctic macroalgae

Parameters/

diversity indices

Phyla of macroalgae host species

Phaeophyta Chlorophyta Rhodophyta

Adenocystis

utricularis

Adenocystis sp. Desmarestia

menziesii

Phaeurus

antarcticus

Acrosiphonia

arcta

Monostroma

hariotii

Ulva intestinalis Palmaria

decipiens

Temperature (1C) 3.7 3.7 0.5 2.1 0.5 3.7 2.1 2.1

Conductivity

(mScm1)

49.89 49.89 27.23 50.6 27.23 49.89 50.6 50.6

Salinity (p.p.t.) 32.1 32.1 32.8 33.0 32.8 32.1 33.0 33.0

pH 7.49 7.49 7.74 7.74 7.74 7.49 7.74 7.74

Optical Density

(mg l1)

79.9 79.9 100 34.6 100 79.9 34.6 34.6

Number of fungal

taxa

2 5 2 7 10 8 10 9

Fishers a 0 (00.8)a 7.8 (0.937.82) 0 (00.8) 3 (0.863.1) 4.77 (1.614.0) 3.7 (1.273.74) 8.54 (2.218.54) 4.47 (1.744.47)

Margalefs 1.44 (01.44) 2 (0.52.1) 1.44 (01.44) 1.82 (0.61.82) 2.55 (1.132.26) 2.1 (0.92.1) 0.03 (1.353.05) 2.3 (1.182.37)

Simpsons 0.5 (00.5) 0.73 (0.20.77) 0.5 (00.5) 0.66 (0.50.75) 0.64 (0.460.75) 0.72 (0.550.8) 0.83 (0.650.86) 0.7 (0.60.82)

aThe numbers in parentheses represent the lower and upper diversity values, respectively, with 95% of confidence and bootstrap valuescalculated from 1000 iterations.

4 8 12 16 20 24 28 32

Specimens

01.22.43.64.8

67.28.49.6

10.8

Taxa

(95%

con

fidence

)

2 4 6 8 10 12 14 16 18

Specimens

1.2

2.4

3.6

4.8

6

7.2

8.4

9.6

10.8

Taxa

(95%

con

fidence

)

3 6 9 12 15 18 21 24 27

Specimens

0

1

2

3

4

5

6

7

8

9

Taxa

(95%

con

fidence

)

3 6 912

15

18

21

24

27

Specimens

0

1

2

3

4

5

6

7

8

9

Taxa

(95%

con

fidence

)

3 6 912

15

18

21

24

27

Specimens

0

0.81.62.43.2

4

4.85.66.47.2

8

Taxa

(95%

con

fidence

)

1.2

1.8

2.4 3

3.6

4.2

4.8

5.4 6

6.6

Specimens

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Taxa

(95%

con

fidence

)

Figure 5 Species accumulation curves for the fungal communities associated with Antarctic macroalgae. Acrosiphonia arcta (a),Ulva intestinalis (b), Monostroma hariotii (c), Palmaria decipiens (d), Phaeurus antarcticus (e) and Adenocystis sp. (f).


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associated with U. intestinalis; dissolved oxygenwith Margalefs index for A. arcta and D. menziesii;temperature with Adenocystis sp., M. hariotiiand A.utricularis; and salinity and pH with P. decipiensand P. antarcticus.

Biological activitiesAll fungal isolates were grown using solid statefermentation techniques to obtain crude extracts,which were then screened against bacteria, fungiand parasite targets to detect bioactive compounds.

100

43

2036 36

38

50

0 1 2 3 4 5 6 7 8 9

0

0.12

0.24

0.36

0.48

0.6

0.72

0.84

0.96

Sim

ilarity

Aa

Ui

Mh

Dm

Pa

Ad

Pd

Au

100

50

27

44

73

31

36

0 1 2 3 4 5 6 7 8 9

0

0.12

0.24

0.36

0.48

0.6

0.72

0.84

0.96

Similari

ty

Aa

Ui

Mh

Pd

Dm

Pa

Ad

Au

Figure 6 Dendrograms showing the Sorensen (a) and (b) Bray-Curtis similarity measures for the fungal communities associated with theAntarctic macroalgae. The results were obtained with 95% confidence and bootstrap values calculated from 1000 iterations. The sampledmacroalgae were Aa, Acrosiphonia arcta; Ad, Adenocystis sp.; Au, Adenocystis utricularis; Dm, Desmarestia menziesii; Pa, Phaeurusantarcticus; Mh, Monostroma hariotii; Pd, Palmaria decipiens; and Ui, Ulva intestinalis.

Temperature

Conductivity

Salinity

pH

Dissolved oxygen

Fisher s

Margalef?s

Simpson s

-3.2 -2.4 -1.6 -0.8 0.8 1.6 2.4 3.2

PCoA 1

-3.2

-2.4

-1.6

-0.8

0.8

1.6

2.4

3.2

Ulva intestinalis

Adenocystissp.

Monostroma hariotii

Adenocystis utricularis

Acrosiphonia arcta

Desmarestia menziesii

Phaeurus antarcticus

PCoa 2

Palmaria decipiens

Figure 7 Principal component analysis plot calculated among the physicochemical water parameters (temperature, salinity, pH,dissolved oxygen and conductivity) obtained where the macroalgae were collected and Fishers b (diversity), Margalefs (richness) andSimpsons (dominance) indices of the fungal communities associated with Antarctic macroalgae.


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The extracts of Penicillium sp. UFMGCB 6034 andPenicillium sp. UFMGCB 6120 recovered from theendemic macroalgae P. decipiens and M. hariotii,respectively, were able to produce bioactive extracts.Penicillium sp. UFMGCB 6120 displayed antifungalactivity against the filamentous fungus Cladosporiumsphaerospermum, producing 96% inhibitionand an MIC value of 250mg ml 1. In addition,Penicillium sp. UFMGCB 6034 and Penicillium sp.UFMGCB 6120 exhibited 100% inhibition of trypo-mastigote forms of T. cruzi, with IC50 values of 1.28and 0.45mg ml1 being obtained, respectively,which were close to or lower than the IC50 (1mg ml

1)

of the control drug (Benznidazole) used in theexperiments. In addition, these two trypanocidalextracts showed low toxicity against normal fibroblastcells.

1H NMR (600MHz, DMSO-d6) analyses of theethanol extracts from the freeze-dried culture mediaof Penicillium sp. UFMGCB 6034 and Penicilliumsp. UFMGCB 6120 were performed. Preliminaryanalysis indicated the presence of functional groupsassociated with aromatic protons, methoxy protons,unsaturated fatty-acid olefinic protons, and fatty-acid methylene and methyl protons, as indicated inFigure 8. Both Penicillium sp. UFMGCB 6034 and

13

.0

12

.5

12

.0

11

.5

11

.0

10

.5

10

.09

.59

.08

.58

.07

.57

.06

.5

Aromatic signals

Unsaturated fattyacid signals

Aromatic signalsUnsaturated fatty

acid signals

OCH3 signal Fatty acid signals

OCH3 signal

Fatty acid signals

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

f1 (ppm)

13

.0

13

.5

12

.5

12

.0

11

.5

11

.0

10

.5

10

.09

.59

.08

.58

.07

.57

.06

.56

.05

.55

.04

.54

.03

.53

.02

.52

.01

.51

.00

.5

f1 (ppm)

Figure 8 1H-NMR spectrum (600MHz, DMSO-d6) of bioactive ethanol extracts from the freeze-dried culture media of (a) Penicillium sp.UFMGCB 6034 and (b) Penicillium sp. UFMGCB 6120. Regions of interest are labelled above the corresponding signals.


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Penicillium sp. UFMGCB 6120 clearly contain thesame major components, as demonstrated by thesimilar 1H NMR signals observed.

Discussion

Macroalgae collection and fungal identificationMarine algae are known to shelter fungal species,but few taxa have been studied in detail. The initialwork of Loque et al. (2010) and the present study,which addresses the fungal communities associatedwith eight macroalgae sampled along a latitudinalgradient over 350 km on the Antarctica Peninsula,represent the first systematic analyses of this kind.Similar to other mycological studies conducted inthe Antarctic that have characterised fungi in soil(Fell et al., 2006), wood debris (Arenz et al., 2006),lakes (Goncalves et al., 2012) and in associationswith plants (Rosa et al., 2009), the fungal taxaassociated with the Antarctic macroalgae examined

in this study comprised a few dominant taxa. Ourresults agree with those of Suryanarayanan et al.(2010), who found few dominant fungal speciesassociated with 25 macroalgae occurring along thecoast of southern India. However, algicolous fungiremain relatively unexplored as a group. Thepresence of Geomyces sp., Penicillium sp. and M.australis as dominant species associated with theinvestigated Antarctic macroalgae suggests that theymay exhibit an interesting ecological relationshipwith their hosts.

Diversity, richness, dominance and distributionStudies on fungal diversity in Antarctica showinconsistencies in the discrimination of endemic,indigenous and cosmopolitan species. Endemicspecies are characterised as true psychrophilic fungithat are able to actively grow and reproduce only inAntarctica (Ruisi et al., 2007). According to Onofriet al. (2007), indigenicity is indicated by theobservation of a large number of isolates over timeas well as being commonly recorded in Antarcticafrom different sites and substrata. In addition, themajority of Antarctic fungi consist of ecotypes ofcosmopolitan species that show mesophilic-psy-chrotolerant behaviour as an adaptation to the cold

Antarctic climate (Zucconi et al., 1996). On the basisof the above criteria, the fungal species M. australisis indigenous to Antarctica, and Penicillium is acosmopolitan mesophilic-psychrotolerant groupthat shows adaptation to the cold Antarctic climate.In contrast, Antarctomyces psychrotrophicus (notfound outside of Antarctica), which was part of theminority component associated with the macroalgaU. intestinalis, has been reported to be endemic toAntarctica (Stchigel et al., 2001).

Geomyces (Helotiales, Ascomycota), found asthe most abundant taxa associated with Antarcticmacroalgae, includes about 10 named species,

which show a wide distribution (http://www.myco-bank.org/). Geomyces sp. have been frequentlyrecorded in Antarctica, are a celluloytic, keratino-philic, psychrophilic and halotolerant fungi with aubiquitous distribution in the soils of cold regionsthat is able to colonise and to utilise different carbonsources (Mercantini et al., 1989). In addition,

Geomyces taxa were found on the thalli of themacroalgae A. utricularis and D. anceps (Loqueet al., 2010), leaves of Colobanthus quitensis (Rosaet al., 2010), mosses (Tosi et al., 2002) and infreshwater lakes (Goncalves et al., 2012). Sevenfungal isolates found associated with the endemicmacroalgae M. hariotii displayed high sequencesimilarity with G. destructans. Geomyces destruc-tans is characterised as a psychrophilic pathogenicfungus able to decrease bats population in tempe-rate regions (Lorch et al., 2011). According to Lorchet al. (2012), the diversity of Geomyces taxa, alsoclassified as Gymnostellatospora and Pseudogym-nosascus, is far greater than previously recognisedbased on traditional taxonomic methods, and can beunderestimated. In addition, we found in associa-tion with Antarctic macroalgae 36 isolates identifiedas Geomyces sp., which may include new speciesand will be subject to further studies to elucidatetheir taxonomic positions.

The yeast M. australis, which has been reported tobe endemic to Antarctica, has been isolated fromAntarctic seawater (Fell and Hunter, 1968), thestomach of the Antarctic krill species Euphausiasuperba (Donachie and Zdanowski, 1998) and,recently, at a high abundance from the algal thalliofA. utricularis (Loque et al., 2010). In addition, M.

australis has been found in marine sediment andfreshwater in Antarctica (Vaz et al., 2011). Therecovery of M. australis in association with themacroalgae A. arcta and D. menziesii reinforces thenotion that this yeast may exhibit a specificassociation with Antarctic macroalgae.

The cosmopolitan genus Penicillium has beenrecovered from alpine and tundra soils as well aspermafrost layers (Gunde-Cimerman et al., 2003). InAntarctica, species of Penicillium have beendescribed from the soil (Azmia and Seppelt, 1998),lakes (Ellis-Evans, 1996), wood (Arenz et al., 2006)and on the macroalga A. utricularis (Loque et al.,2010). As an extremophile, P. chrysogenum has been

isolated as a dominant species from Arctic sub-glacial ice (Gunde-Cimerman et al., 2003, Sonjaket al., 2005). In addition, according to Bugni andIreland (2004) Penicillium represents one of themore common genera isolated from macroalgae. Inthe present study, we found different Penicilliumtaxa associated with the Antarctic macroalgae.According to Frisvad and Samson (2004), Scottet al. (2004) and Houbraken et al. (2011), thetaxonomy of the group Penicillium has beenregarded to be especially difficult and needs apolyphasic study including physiological, morpho-logical, multilocus sequence typing and chemical


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methods. For these reasons, the Penicillium sp.associated with the Antarctic macroalgae wereidentified using the term cf. or in genus rank.These Penicillium taxa will be the subject of furthertaxonomic evaluations.

The non-asymptotic species accumulation curves(Mao Tao analysis) generated for the fungal commu-

nities found in associations with the examinedAntarctic macroalgae showed that a large numberof single individuals (singletons) were recovered,which are considered rare species and often repre-sent more than half of the species within thesecommunities. Similar results were described for thefungal communities of A. utricularis, D. anceps andP. decipiens collected at King George Island,Antarctica (Loque et al., 2010).

Twenty-seven fungal taxa associated with theAntarctic macroalgae displayed a low molecularsimilarity with the sequences of known fungideposited in GenBank, suggesting that they arecandidates for new fungal species. However, not alldescribed fungal species are deposited in theGenBank database, and further taxonomic analysesincluding detailed physiological and macro-and micromorphological characterisation andmolecular sequencing of other DNA regions,together with new phylogenetic analyses, will beneeded to describe these potentially new species.According to the recent checklist of Antarctic fungipublished by Bridge and Spooner (2012), thespecimens of Meyerozyma caribbica, Mycoarthriscoralline and Yamadazyma mexicana, which werefound as minor components within the fungalcommunities associated with Antarctic macroal-

gae, represent the first records of these species inAntarctica.According to Ruisi et al. (2007), the distribution of

fungi in Antarctica is related to the distribution oftheir hosts or substrates, such as bird feathers anddung, invertebrates, vegetation, soils, rocks andlichens. However, marine fungal dispersal remainsunknown. In the present study, the calculatedsimilarity indices (Sorensen and Bray-Curtis)revealed the presence of some species (D. hansenii,Geomyces sp., Helotiales sp., M. australis,Penicillium sp. and T. globosus) in association withdifferent macroalgae and from distinct Antarcticislands. The high values of the Bray-Curtis similar-

ity index (which has priority regarding the abun-dance of common species) between the fungalcommunities associated with endemic macroalgaeP. decipiens and M. hariotii indicate that Geomycesand Penicillium sp. might show a specific associa-tion with macroalgae adapted to extreme conditions.

Fungi are one of the largest and most diversekingdoms of eukaryotes and are important biologicalcomponents of terrestrial ecosystems. According toBridge and Spooner (2012), there are many recordsof marine fungi from Antarctica, although there islittle available information about their ecologicalroles. In the present study, some of the fungal taxa

found to be associated with Antarctic macroalgaewere identified as species known to be mutualists,saprobes and parasites. Bridge and Spooner (2012)suggest that Antarctic fungi occupy many differentecological niches, but their significance in theseniches remains poorly understood. Understandinghow mutualistic, saprobic and pathogenic fungi

achieve their lifestyles is crucial for understandingtheir ecological functions and their impact onAntarctic macroalgae under the extreme conditionsof this continent. In addition, the abiotic seawaterparameters in the areas where the macroalgae werecollected revealed low temperatures and highsalinities, demonstrating that the fungi found inassociation with the macroalgae may exhibit specificmechanisms that allow them to survive in extremeconditions.

Biological activitiesIn general, fungi have been reported to representprolific sources of various compounds, includingseveral bioactive molecules. However, the potentialof extremophile fungi to produce bioactive com-pounds is poorly understood. According toSantiago et al. (2012), the ability of Antarctic fungito survive in extreme conditions suggests that theymay display unusual biochemical pathways thatallow them to generate specific or new moleculesthat could be used to develop new drugs. Ourbioprospecting results show that the extracts fromPenicillium sp. UFMGCB 6034 and Penicillium sp.UFMGCB 6120, which were recovered as dominantspecies from endemic macroalgae, P. decipiens and

M. hariotii, displayed high and selective antifungaland/or trypanocidal activities with low MIC and IC50values. The combination of low MIC and IC50 valuestogether with the 1H NMR spectral data suggestingthe presence of functionalised aromatic compoundswarrants extensive bioassay-directed fractionation tospecifically identify the constituents responsible forthe observed biological activity.

Marine fungi have been described as a promisingsource of antimicrobial compounds. Marine Peni-cillium sp. are known to produce a large variety ofcompounds with a wide range of biological andpharmacological activities. Penicillium sp. isolatedfrom the alga U. intestinalis were observed to

produce cytotoxic metabolites including commune-sins (Numata et al., 1993), penochalasius A-C(Numata et al., 1995), penostatins A-I (Takahashiet al., 1996, Iwamoto et al., 1998) and penochalasiusD-H (Iwamoto et al., 2001). The antibacterial andcytotoxic compounds di(2-ethyl hexayl) phthalateand fungisterol were isolated from P. brevicompactum,which was isolated from the associated marine algaPterocladia sp. (Atalla et al., 2011). The extracts ofPenicillium sp. UFMGCB 6034 and Penicillium sp.UFMGCB 6120 will be subjected to further systema-tic chemical bioactivity-guided fractionation toisolate their bioactive compounds.


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Conclusion

Although marine fungi have been studied for the last100 years (Jones, 2011), many aspects regarding theirtaxonomy, ecology and potential biotechnologicalapplications remain poorly documented. Our resultsare consistent with a suggestion by Bridge and

Spooner (2012) that the true diversity of Antarcticfungi may be far greater than is currently estimated.Our results also suggest that macroalgae can act as asubstrate to shelter fungi in Antarctica and con-tribute to the knowledge of the cryptic and unknownalgicolous Antarctic mycota. We showed that ende-mic and cold-adapted macroalgae living under theextreme conditions of Antarctica shelter rich,diverse and complex fungal communities consistingof few dominant indigenous or mesophilic cold-adapted species. In addition, we detected a largenumber of rare and/or endemic taxa, which mayprovide an interesting model of algalfungal inter-actions in extreme conditions as well as a potential

source of bioactive compounds. Our results rein-force the call for further studies of fungal commu-nities present across the Antarctic marineenvironment as well as their phylogenetic relation-ships with species that occur in other cold regions.Finally, the present work suggests that examinationof the fungi associated with macroalgae in Antarc-tica may provide new insights into the biologicalmechanisms underlying tolerance and adaptation toextreme marine polar conditions.

Acknowledgements

This study had financial and logistic support from the

Brazilian Antarctic Program (PROANTAR/MCT/CNPqNo. 23/2009), Marine of Brazil and Francisco Petrone fromthe Clube Alpino Paulista (CAP). We acknowledge thefinancial support from Fundacao de Amparo a Pesquisado Estado de Minas Gerais (FAPEMIG), Health-PDTIS-FIOCRUZ, PRPq-UFMG, CAPES/PGCI 036/13 and Con-selho Nacional de Desenvolvimento Cientfico e Tecnolo-gico (CNPq). This study represents the Pos-doctoral degreeof Dr VM Godinho under the supervision of Dr LH Rosa.Also, the results of this study are part of the MasterScience degree of LE Furbino within the Programa of Pos-Graduacao in Biotecnologia of UFOP. We thank Isis VGalliza and Fernanda LM Francisco for performing the

biological assays. We thank Solomon Green III fortechnical assistance. We also thank the anonymousreviewers for helpful comments to improve the qualityof the manuscript.

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