The ISME Journal (2013) 7, 1434–1451 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in

Vale´ria M Godinho1, Laura E Furbino1, Iara F Santiago1, Franciane M Pellizzari2, Nair S Yokoya3, Dicla´ Pupo3,Taˆnia MA Alves4, Policarpo A S Junior5, Alvaro J Romanha5, Carlos L Zani4, Charles L Cantrell6, Carlos A Rosa1 and Luiz H Rosa1 1Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 2Laborato´rio de Ficologia e Qualidade de A´gua do Mar, Universidade Estadual do Parana´, Paranagua´, Brazil; 3Sec¸a˜o de Ficologia para Nu´cleo de Pesquisa em Ficologia, Sa˜o Paulo, Brazil; 4Laborato´rio de Quı´mica de Produtos Naturais, Centro de Pesquisas Rene´ Rachou, Fundac¸a˜o Oswaldo Cruz, Belo Horizonte, MG, Brazil; 5Laborato´rio de Parasitologia Celular e Molecular, Centro de Pesquisas Rene´ Rachou, 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 from Antarctica and their capability to produce bioactive compounds. The collections yielded 148 fungal isolates, which were identified using molecular methods as belonging to 21 genera and 50 taxa. The most frequent taxa were species (sp.), 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 Geomyces destructans. Thirty-three fungal singletons (66%) were identified, representing rare components of the fungal communities. The fungal communities displayed high diversity, richness and dominance indices; however, rarefaction curves indicated that not all of the fungal diversity present was recovered. Penicillium sp. UFMGCB 6034 and Penicillium sp. UFMGCB 6120, 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 spectroscopy indicated the presence of highly functionalised aromatic compounds. These results suggest that the endemic and cold-adapted macroalgae of Antarctica shelter a rich, diversity and complex fungal communities consisting of a few dominant indigenous or mesophilic cold-adapted species, and a large number of rare and/or endemic taxa, which may provide an interesting model of algal–fungal interactions under extreme conditions as well as a potential source of bioactive compounds. The ISME Journal (2013) 7, 1434–1451; doi:10.1038/ismej.2013.77; published online 23 May 2013 Subject Category: Microbial ecology and functional diversity of natural habitats Keywords: Antarctica; marine fungi; seaweeds; diversity; extremophiles

Introduction low temperatures, osmotic stress and ultraviolet radiation irradiation (Fell et al., 2006; Gonc¸alves Antarctica represents one of the most pristine et al., 2012). The Antarctic fungal communities ecosystems in the world, characterised by short include representatives of genera and species from food chains dominated by microorganisms. The the major fungal phyla , Basidiomy- fungal mats in the Antarctic exhibit complex cota, Zygomycota, Chytridiomycota and Glomero- communities that are able to survive under extreme mycota,aswellasOomycetes (Heterokontophyta, environmental conditions, including dehydration, Oomycota) and slime moulds (Mycetozoa)tradi- freeze-thaw cycles, low nutrient concentrations, tionally studied by mycologists (Bridge and Spooner, 2012). In recent decades, mycological Correspondence: LH Rosa, Departamento de Microbiologia, studies in Antarctica have mainly focused on the Laborato´rio de Sistema´tica e Biomole´culas de Fungos, Instituto fungi present in soil, ice and lakes, and their de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, associations with plants. However, limited atten- P O Box 486, Belo Horizonte MG CEP 31270-901, Brazil. E-mail: [email protected] tion has thus far been devoted to the fungal Received 3 January 2013; revised 3 April 2013; accepted 7 April diversity of the Antarctic marine environment. 2013; published online 23 May 2013 Few existing studies address marine Antarctic Fungi associated with Antarctic macroalgae VM Godinho et al 1435 fungi, which have been detected in water (Fell and RW Ricker (Figure 1) were collected onboard the Statzel, 1971; Fell et al., 1973), wood baits (Grasso Brazilian Navy Polar Ship Almirante Maximiano et al., 1997) and water and marine sediments (Vaz (H41) along a 350-km transect through Elephant, et al., 2011). According to Bridge and Spooner King George and Deception Islands, in the Antarctic (2012), only 42 fungal species have been described Peninsula (Figure 2). Physical and chemical water from the Antarctic marine ecosystem. parameters (temperature, salinity, conductivity, dis- The known marine macroalgal communities from solved oxygen and pH) were also recorded at each Antarctica are not very diverse in comparison with site using a multiparameter probe Hexis TCS that of warmer regions. However, this flora is (Yellow Springs, OH, USA). characterised by a high degree of endemism and the presence of cold-adapted species (Wiencke and Clayton, 2002; Oliveira et al., 2009). In addition, Macroalgae identification macroalgae are important primary producers, pro- Complete and fertile samples were sorted, washed ducing B74 000 tons of wet biomass and having a and preserved in seawater formalin (4%) in the key role in organic carbon fluxes in Antarctica ship’s laboratory, with the aim of performing macro- (Nedzarek and Rakusa-Suszczewski, 2004). Also, and micromorphological analyses. The identifica- macroalgae may shelter large numbers of associated tion of the macroalgal specimens was based on the organisms, including microbial mats surviving publications of Papenfuss (1964), Ricker (1987), under extreme conditions (Loque et al., 2010). Wiencke and Clayton (2002), Quartino et al. (2005) According to Bugni and Ireland (2004), fungi and Amsler et al. (2009). Nomenclatural updates recovered from macroalgae represent the second followed Guiry and Guiry (2012). Exsiccatae vouchers largest source of marine fungi and include parasites, were manufactured for deposition in the SP Herbar- saprobes or mutualistic species. A number of ium of the Jardim Botaˆnico of Sa˜oPaulo,Brazil. macroalgal species have been studied in detail worldwide with respect to their associated fungal communities, which include the genera Ascophyl- Fungal isolation lum, Ballia, Caulerpa, Ceramium, Ceratiodictyon, Five discs 8 mm in diameter were cut from each Cladophora, Chondrus, Dictyota, Dilsea, Egregia, macroalgal specimen and washed twice using sterile Enteromorpha, Fucus, Gelidiella, Gracilaria, Grate- local seawater for 2 min. The discs were inoculated loupia, Halimeda, Halymenia, Hypnea, Laminaria, in Petri dishes containing marine agar (Difco, Lobophora, Padina, Porphyra, Portieria, Saccorhiza, Franklin Lakes, NJ, USA) supplemented with 2% Sargassum, Stoechospermum, Turbinaria and Ulva glucose and chloramphenicol (Sigma, St Louis, MO, (Kohlmeyer and Volkmann-Kohlmeyer, 1991, USA) at a concentration of 200 mgmlÀ 1 for selective Stanley, 1992; Zuccaro and Mitchell, 2005; isolation of marine fungi. All of the inoculated Petri Zuccaro et al., 2008; Suryanarayanan et al., 2010). dishes were incubated for up to 60 days at 10 1C, and To the best of our knowledge, except for an initial individual colonies of fungi were purified on marine contribution by Loque et al. (2010), no other data are agar. Long-term preservation of fungi was carried out available regarding the species composition of at À 80 1C using cryotubes with sterile 15% glycerol. fungal communities associated with Antarctic All of the fungal isolates examined in this work were macroalgae. In this study, we acquire to present deposited in the Culture Collection of Microorganisms information on the diversity and distribution of and Cells of the Universidade Federal of Minas Gerais, fungal communities associated with endemic and Brazil, under codes UFMGCB. cold-adapted macroalgae across latitudinal gradi- ents along the Antarctic Peninsula and their cap- ability to produce bioactive compounds. Fungal identification The protocol for DNA extraction from filamentous fungi followed Rosa et al. (2009). The internal Materials and methods transcribed spacer (ITS) region was amplified with the universal primers ITS1 and ITS4 (White et al., Macroalagae collection 1990). Amplification of the ITS region was per- Sixty fresh thalli from each selected macroalgal formed as described by Rosa et al. (2009). species (four Phaeophyceae, three Chlorophyta and were characterised via standard methods (Yarrow, one Rhodophyta) were collected during December 1998), and their identification was carried out using 2010 and January 2011 in intertidal transects along a the taxonomic keys of Kurtzman et al. (2011). rocky coastline that becomes ice free during the identities were confirmed by sequencing the D1-D2 Antarctic summer. Samples of Adenocystis utricu- variable domains of the large subunit ribosomal laris (Bory de Saint-Vicent) Skottsberg, Adenocystis RNA gene using the primers NL1 and NL4, as species (sp.), Desmarestia menziesii J Agardh, described by Lachance et al. (1999). Phaeurus antarcticus Skottsberg, Acrosiphonia Amplification of the b-tubulin gene was per- arcta (Dillwyn) Gain, Monostroma hariotii Gain, formed with the Bt2a and Bt2b primers (Glass and Ulva intestinalis Linnaeus and Palmaria decipiens Donaldson, 1995). PCR assays were conducted in

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1436

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 de Saint-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 Subantarctic islands). Bars represent 1 cm.

50 ml reaction mixtures containing 1 ml of genomic in a MegaBACE 1000/Automated 96 Capillary DNA DNA (10 ng ml À 1), 5 ml of PCR buffer (100 mM Tris- sequencer (GE Healthcare, Piscataway, NJ, USA). HCl, 500 mM KCl, pH 8.8), 2 ml of dNTPs (10 mM) The obtained sequences were analysed with Seq- plus 3 ml of MgCl2 (25 mM), 1 ml of each primer Man P with Lasergene software (DNASTAR Inc., (50 pmol ml À 1), 1 ml of dimethyl sulfoxide (DMSO; Madison, WI, USA), and a consensus sequence was Merck, Billerica, MA, USA), 2 ml betaine (5 M), 0.2 ml obtained using Bioedit v. 7.0.5.3 software (Carlsbad, of Taq polymerase (5 U ml À 1 DNA) and 33.8 mlof ON, Canada). To achieve species-rank identification ultrapure sterile water. PCR amplifications were based on ITS and b-tubulin data, the consensus performed with the Mastercycler pro (Eppendorf, sequence was aligned with all sequences from Hamburg, Germany), programmed for initial dena- related species retrieved from the NCBI GenBank turation at 94 1C for 5 min, followed by 35 cycles of database using BLAST (Altschul et al., 1997). The 1 min of denaturation at 94 1C, primer annealing for consensus sequences of the algicolous fungi were 1 min at 59 1C and extension for 1.30 min at 72 1C, deposited into GenBank (see the accession numbers with a final 7-min elongation step at 72 1C. After in the Table 1). However, according to Gazis et al. amplification of the b-tubulin template, excess (2011), sequencing of the ITS region may fail to primers and dNTPs were removed from the reaction recognise some fungal genera. For this reason, the mixture using a commercial GFX column with the b-tubulin sequences, which are considered promis- PCR DNA Purification kit (Amersham Bioscience, ing for a one-gene phylogeny (Frisvad and Samson, Roosendaal, Netherlands). Purified PCR fragments 2004), were used to elucidate the taxonomic posi- were resuspended in 50 ml of Tris-EDTA buffer. tions of the inconclusive taxa identified using ITS The amplified DNA was concentrated and pur- sequences. In addition, the followed criteria were ified using the Wizard Plus SV Miniprep DNA used to interpret the sequences from the GenBank Purification System (Promega, Madison, WI, USA) database: for query coverage and sequence identities and sequenced using an ET Dynamic Terminator Kit X99%, the and species were accepted; for

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1437

Figure 2 Maps showing the positions of the islands sampled on the (a) Antarctic Peninsula across a transect of 350 km. Sampling sites: D, Ulva intestinalis, Palmaria decipiens and Phaeurus antarcticus in (b) ¼ Stinker Point (61107.935’S; 055125.997’W) at Elephant island; D, Acrosiphonia arcta and D. menziesii in (c), Keller Peninsula (62105.163’S; 058124.784’W) at Admiralty Bay at King George Island; D, Adenocystis utricularis, Monostroma hariotii, Adenocystis sp. in (d), Telefon Bay (62155.192’S; 060139.797’W) at Deception Island. query coverage and sequence identities showing from 1000 replicate runs. To complete the molecular 98%, the genus and species were accepted, but term identification, the sequences of known-type fungal ‘cf.’ (Latin for confer ¼ compares with), used to strains or reference sequences obtained from fungal indicate that the specimen resembles, but has species deposited in international culture collec- certain minor features not found in the reference tions found in GenBank were added to improve the species, was included; for query coverage and accuracy of the phylogenetic analysis. The informa- sequence identities between 95% and 97%, only tion about fungal taxonomic hierarchical follows the the genus was accepted; and for query coverage and rules established by Kirk et al. (2011) and the sequence identities p95%, the isolates were MycoBank (http://www.mycobank.org), and Index labelled with the order or family name or as Fungorum (http://www.indexfungorum.org) databases. ‘unknown’ fungi. However, taxa that displayed query coverage and identitiesp97% or an in- conclusive taxonomic position were subjected to Diversity, richness, dominance and distribution phylogenetic ITS and b-tubulin analysis, with To quantify species diversity, richness and even- estimations conducted using MEGA Version 5.0 ness, we used the following indices: (i) Fisher’s a, (Tamura et al., 2011). The maximum composite (ii) Margalef’s and (iii) Simpson’s, respectively. likelihood method was employed to estimate evolu- A rarefaction curve was calculated using the Mao tionary distances with bootstrap values calculated Tao index (Colwell et al., 2004). The similarities

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1438 Table 1 Molecular identification of fungi associated with Antarctic macroalgae: identification conducted using BLASTn searches of the of the internal transcribed spacer (ITS) region and b-tubulin gene

Macroalgae UFMGCBa No. of Top BLAST search results Query Identity No. of bp Proposed species or taxonomic species code isolates (GenBank accession coverage (%) sequenced group (GenBank accession number) (%) and number) analysed

Adenocystis D1N10.34.1 1 Debaryomyces hansenii 100 99 524 Debaryomyces hansenii utricularis (HE681104) (KC485456b) D1N10.13.1 1 Meyerozyma caribbica 100 99 547 Meyerozyma caribbica (JQ686909) (KC485457b) Acrosiphonia 6063c 19 98 99 729 Geomyces sp. (KC333881d) arcta (DQ189229) K2.13.1 6 Metschnikowia australis 99 99 484 Metschnikowia australis (U76526) (KC485458b) 6074c,e 2 Penicillium chrysogenum 99 99 662 fPenicillium sp. (KC333882d, (HQ652873) KC823167g) K2.6.1 1 sake (EU326138) 100 99 588 Candida sake (KC485459b) 6361c,e 1 Cladosporium perangustum 100 97 486 fCladosporium sp. (KC341715d, (JF499836) KC823166g) 6079 1 Cladosporium tenuissimum 100 99 476 Cladosporium tenuissimum (JQ246357) (KC341716d) K2.9.1 1 Debaryomyces hansenii 100 99 546 Debaryomyces hansenii (HQ860269) (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 99 99 495 Thelebolus globosus (GU004196) (KC341719d) Desmarestia K1.17.1 1 Metschnikowia australis 99 99 446 Metschnikowia australis menziesii (FJ911872) (KC485461b) 6084c,e 1 Penicillium chrysogenum 100 98 486 fPenicillium sp. (KC341720d, (JN021549) KC823168g) Ulva 6101c,e 6 Penicillium commune 100 100 496 fPenicillium sp. (KC485461d, intestinalis (JN676122) KC823169g) 6092c,e 3 Penicillium solitum 98 99 534 fPenicillium discolor (JN642222) (KC485423d, KC823170g) 6090 3 Antarctomyces psychrotro- 100 99 482 Antarctomyces psychrotrophicus phicus (JN104511) (KC485424d) CM.01.2 1 victoriae 98 99 605 Cryptococcus victoriae [AY040653] [KC485462b] 6096 1 Engyodontium album 90 99 566 Engyodontium sp. (KC485425d) (HM214540) 6097c 1 Geomyces luteus (AJ938164) 100 98 451 Geomyces luteus (KC485426d) 6328 1 sp. (HQ533820) 90 99 477 Helotiales sp. (KC485427d) 6326 1 Mycoarthris corallinus 97 98 458 Mycoarthris cf. corallines (AF128440) (KC485428d) 6325c,e 1 Penicillium chrysogenum 98 98 507 fPenicillium sp. (KC485429d, (KC009835) KC823171g) 6095 1 Thelebolus microsporus 100 100 485 Thelebolus globosus (GU004196) (KC485430d) Monostroma 6120c,e 12 Penicillium chrysogenum 100 99 512 fPenicillium sp. (KC485431d, hariotii (KC009826) KC823175g) 6112c 7 Geomyces pannorum 100 99 439 Geomyces cf. destructans (JF311913) (KC485432d) D1N18.31.1 4 Meyerozyma guilliermondii 100 99 537 Meyerozyma guilliermondii (JF766631) (KC485463b) 6330 1 Cryptococcus laurentii 100 98 570 Cryptococcus cf. laurentii (FJ743631) (KC485433d) 6130c 1 Lecanicillium araneicola 99 92 573 fCordycipitaceae sp. (AB378506) (KC485434d) 6138c 1 Paecilomyces anatarcticus 100 96 475 fHelotiales sp. (KC485435d) (AJ879113) 6316c 1 Pezizella discreta (JF908571) 99 97 487 fHyaloscyphaceae sp. (KC485436d) D1N18.42.1 1 Rhodotorula mucilaginosa 100 99 537 Rhodotorula mucilaginosa (JQ277252) (KC485464b) Palmaria 6034c,e 11 Penicillium chrysogenum 100 99 538 fPenicillium sp. (JX976546d, decipiens (JN032681) KC823172g) 6049c 8 Geomyces pannorum 91 99 554 Geomyces sp. (KC485437d) (DQ189229) 6056 2 Acremonium strictum 92 99 565 Acremonium sp. (KC485438d) (AY138844)

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1439 Table 1 (Continued ) Macroalgae UFMGCBa No. of Top BLAST search results Query Identity No. of bp Proposed species or taxonomic species code isolates (GenBank accession coverage (%) sequenced group (GenBank accession number) (%) and number) analysed

6050 2 Fusarium oxysporum 91 99 524 Fusarium sp. (KC485439d) (HQ248198) E2N5.42.1 2 Yamadazyma mexicana 100 99 500 Yamadazyma mexicana (FJ455104) (KC485465b) 6047 1 candidus 93 99 548 Aspergillus sp. (KC485440d) (FJ441637) 6053 1 Chaetomium globosum 66 99 751 Chaetomium sp. (KC485441d) (JN689341) 6140c,e 1 Penicillium raistrickii 78 97 665 fPenicillium sp.(KC485442d, (FR670335) KC823173g) 6048c,e 1 Penicillium spinulosum 74 99 697 fPenicillium spinulosum (DQ132828) (KC485443d, KC823174g) Phaeurus 6141c,e 13 Penicillium chrysogenum 100 100 482 fPenicillium sp. (KC485444d, antarcticus (JQ665262) KC823176g) 6148c 8 Geomyces pannorum 100 99 495 Geomyces sp. (KC485445d) (DQ189229) 6153c,e 2 Penicillium raistrickii 81 98 631 fPenicillium sp. (KC485446d, (FR670335) KC823178g) 6164c,e 1 Aspergillus terreus 100 100 536 fAspergillus terreus (KC485447d, (JQ070071) KC823163g) 6167 1 herbariorum 100 99 429 Eurotium herbariorum (JN942870) (KC485448d) 6327 1 Eurotium repens (AY373890) 100 99 482 Eurotium repens (KC485449d) 6320c,e 1 Penicillium steckii 100 99 467 fPenicillium steckii (KC485450d, (HM469415) KC846137g) Adenocystis 6323c,e 3 Penicillium chrysogenum 95 99 504 fPenicillium sp. (KC485451d, sp. (JQ665262) KC823164g) 6106 1 Aspergillus conicus 99 99 426 Aspergillus conicus (HE578068) (KC485452d) 6110c 1 Geomyces pannorum 100 99 451 Geomyces sp. (KC485453d) (JF311913) 6109c,e 1 Penicillium citrinum 93 100 507 fPenicillium citrinum (FJ765031) (KC485454d, KC823177g) 6322c,e 1 Penicillium commune 100 98 542 fPenicillium sp. (KC485455d, (FJ499454) KC823165g)

When the number of isolates was 41, 50% of the isolates were sequenced and the best sequence was deposited in the GenBank database. aUFMGCB ¼ Culture 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. among fungal taxa from different areas were esti- centres of Petri dishes (60 mm diameter, with 20 ml mated using the Sorensen coefficient and Bray- of marine agar). The plates were incubated at Curtis measures. All of the results were obtained 10±2 1C for 15 days, and the cultured materials with 95% confidence, and bootstrap values were from each Petri dish were then cut and transferred to calculated from 1000 iterations. All diversity and 50-ml vials containing 35 ml of ethanol. After 48 h at similarity indices, rarefaction curves and the princi- room temperature, the organic phase was decanted, pal components analysis calculations were per- and the solvent was removed under a vacuum formed using the computer Program PAST, version centrifuge at 35 1C (Santiago et al., 2012). An aliquot 1.90 (Hammer et al., 2001). Further information about of each dried extract was dissolved in DMSO these indices can be found in Magurran (2011). (Merck) to prepare a 100-mg ml À 1 stock solution, which was stored at À 20 1C.

Fungal cultivation and preparation of extracts for biological assays Assay for antimicrobial activity All fungal isolates were cultivated using solid state Susceptibility testing against Escherichia coli fermentation. Five-millimetre-diameter plugs from ATCC 11775, Staphylococcus aureus ATCC 12600, each filamentous fungus were inoculated into the ATCC 10145, Candida

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1440 albicans ATCC 18804, Candida krusei ATCC 6258 two or more independent experimental data sets to a and Cladosporium sphaerospermum CCT 1740 was four-parameter logistic dose–response model. No performed using a protocol established by Carvalho constraints were applied to the curve-fitting calcula- et al. (2012). All extracts (dissolved in DMSO) were tions. All assays were performed in duplicate. diluted to a final concentration of 250 mgmlÀ 1 for use in the antimicrobial assays. The results are expressed as the percent inhibition in relation to Nuclear magnetic resonance spectroscopy controls without drugs. All antimicrobial assays Bioactive extracts were analysed via nuclear mag- were performed in duplicate. netic resonance (NMR) spectroscopy in a Varian INOVA 600 MHz spectrometer (Varian Inc., Palo Alto, CA, USA). 1H-NMR spectra were recorded in In vitro assays with intracellular amastigote forms of DMSO-d6. The samples were prepared at a concen- Trypanosoma cruzi tration of B10 mg ml À 1, and the results were In vitro assays with amastigote forms of T. cruzi were recorded in 3 mm NMR tubes using a standard 1H performed according to protocols established by pulse programme. Buckner et al. (1996) with some modifications. Trypanosoma cruzi (Tulahuen strain) expressing the E. coli b-galactosidase gene were grown on a Results monolayer of mouse L929 fibroblasts. Cultures to be assayed for b-galactosidase activity were grown in Macroalgae collection and fungal identification Roswell Park Memorial Institute 1640 medium A total of 148 fungal isolates were recovered from (pH 7.2-7.4) without phenol red (Gibco BRL, 391 tissue fragments from the eight macroalgal Cergy-Pontoise, France), plus 10% foetal bovine species, which were identified by DNA sequences serum and glutamine. L929 fibroblasts were seeded of the ITS region and b-tubulin gene in 21 different into 96-well tissue culture microplates at a concen- genera within of the phyla Ascomycota, Basidiomy- tration of 4.0 Â 103 per well in a volume of 80 ml and cota and subphylum Mortierellomycotina (Zygomy- incubated overnight. b-galactosidase-expressing try- cota; Table 1). All of the screened macroalgae pomastigotes were then added at a concentration of harboured associated fungi, and 50 taxa were 4.0 Â 104 per well in a volume of 20 ml. After 2 h, the identified. However, the number of fungal taxa and medium containing trypomastigotes that did not diversity indices differed among the macroalgae. penetrate into the cells was discarded and replaced Among the phyla characterised, the most repre- with 200 ml of fresh medium. After 48 h, the medium sented orders were (43.24%), Helotiales was discarded again and replaced with 180 mlof (32.43%), (11.48%), Hypo- fresh medium and 20 ml of the test extracts. Each creales (6%) and (4.05%). In contrast, extract was tested in triplicate. After 7 days of taxa of the orders , Capnodiales, Mortier- incubation, chlorophenol red b-D-galactopyranoside ellales, Pleosporales, Sordariales and Sporidiobo- (100 mM final concentration) and Nonidet P-40 lales were found as minority components of the (Sigma-Aldrich, St. Louis, MO, USA) at final fungal communities with an abundance of p1.35%. concentration of 0.1% were added to the plates, Twenty-eight fungal taxa presented low molecular followed by incubation overnight at 37 1C, and the similarities or inconclusive information in compar- absorbance was measured at 570 nm in an auto- ison with known fungal ITS sequences deposited in mated microplate reader. Benznidazole at its 50% the GenBank database. To identify these fungi, À 1 inhibitory concentration (IC50;1mlml ) was used phylogenetic trees of the ITS (Figure 3) and/or as a positive control. The results were expressed as b-tubulin (Figure 4) genes were constructed to the percentage of growth inhibition. All assays were illustrate their relationship with GenBank sequences. performed in triplicate. All ITS (Figure 3i) and b-tubulin (Figure 4c) sequences of the Penicillium isolates were analysed and the taxa P. spinulosum, P. steckii, P. citrinum

Minimal inhibitory concentration and IC50 and P. chrysogenum were identified. The other The crude antimicrobial extracts were subjected to Penicillium taxa displayed inconclusive sequence determination of a minimal inhibitory concentration analyses when compared with the sequences of the (MIC) and the trypanocidal extracts were subjected type species, and were identified as Penicillium sp. to determination of a IC50. MIC and IC50 represent Thirty-six fungal isolates displayed ITS sequence the lowest concentrations of a crude extract similarities with Geomyces sp. Among them, the (or compound) that inhibits the functions of a target seven isolates were identified as G. destructans, (in this study, microorganisms and T. cruzi) by 100% which showed 100% of query coverage and 98% and 50%, respectively. This quantitative measure identity; also these fungal isolates presented only indicates how much of a particular crude extract is 1.1% of sequence difference in comparison with the needed to inhibit a given biological process by 50%. type species G. destructans (GenBank Access num- SoftMax Pro 5.3 (Sunnyvale, CA, USA) was used to ber EU884921; Table 1; Figure 3g). All other calculate MIC values via nonlinear curve fitting of Geomyces isolates formed a separated cluster when

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1441

Figure 3 Phylogenetic analysis (a–i) 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 were constructed based on the ITS region sequences using the maximum composite likelihood method.

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1442

Figure 3 Continued.

compared with sequences of the type species Diversity, richness, dominance and distribution of G. pannorum (GenBank Access number The most frequent fungal taxa identified were FJ545236) and G. asperulatus (GenBank Access Penicillium sp. (35.8%), Geomyces sp. (24.32%) number AJ390390; Figure 3g); these taxa were and M. australis (4.72%). However, in general, the identified as Geomyces sp. fungal communities associated with the Antarctic In addition, after ITS (Figure 3) and b-tubulin macroalgae presented high values of the diversity (Figure 4) sequence analysis, 27 fungal taxa were (Fisher’s a ¼ 22.0), richness (Margalef’s ¼ 10.41) and identified as belonging to the genera Acremonium, dominance (Simpson’s ¼ 0.94) indices. The phylum Aspergillus, Chaetomium, Cladosporium, Engyo- Chlorophyta displayed a Fisher’s a ¼ 5.67 and 9.33 dontium, Fusarium, Geomyces, Mortierella, fungal taxa per macroalgal species, which was Penicillium, Phoma as well as the families Cordyci- followed by Rhodophyta (Fisher’s a ¼ 4.47 and 9 pitaceae, Hyaloscyphaceae and order Helotiales, fungal taxa/macroalgal species) and Phaeophyta which displayed low sequence similarities when (Fisher’s a ¼ 2.7 and 4 fungal taxa/macroalgal compared with fungal sequences deposited in the species). However, the values of the indices differed GenBank. among the macroalgae (Table 2). The fungal

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1443 a

b

c

Figure 4 Phylogenetic analysis (a–c) 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. communities associated with U. intestinalis and lowest Fisher’s a, Margalef’s and Simpson’s indices. Adenocystis sp. showed high diversity and dom- In addition to the high fungal diversity (Fisher’s a inance values, whereas the communities corre- values) found in the sampled macroalgae, the sponding to A. utricularis and D. menziesii Mao Tao rarefaction curves continued to rise and displayed the lowest diversity, richness and dom- did not reach an asymptote (Figure 5), indicating inance values. In addition, the A. arcta fungal that not all fungal diversity had been recovered. community showed the highest richness, but mod- Thirty-three fungal taxa (66%) occurred as erate diversity and dominance. Only two fungal singletons corresponding to a single macroalgal species were isolated from the macroalgae A. species, representing rare components of the fungal utricularis and D. menziesii, which exhibited the communities.

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1444 Table 2 Physico-chemical parameters of water and diversity indices of fungal communities associated with the Antarctic macroalgae

Parameters/ Phyla of macroalgae host species diversity indices Phaeophyta Chlorophyta Rhodophyta

Adenocystis Adenocystis sp. Desmarestia Phaeurus Acrosiphonia Monostroma Ulva intestinalis Palmaria utricularis menziesii antarcticus arcta hariotii decipiens

Temperature (1C) 3.7 3.7 0.5 2.1 0.5 3.7 2.1 2.1 Conductivity 49.89 49.89 27.23 50.6 27.23 49.89 50.6 50.6 (mS cm À 1) 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 79.9 79.9 100 34.6 100 79.9 34.6 34.6 (mg l À 1) Number of fungal 2527108109 taxa Fisher’s a 0 (0—0.8)a 7.8 (0.93—7.82) 0 (0—0.8) 3 (0.86—3.1) 4.77 (1.61—4.0) 3.7 (1.27—3.74) 8.54 (2.21—8.54) 4.47 (1.74—4.47) Margalef’s 1.44 (0—1.44) 2 (0.5—2.1) 1.44 (0—1.44) 1.82 (0.6—1.82) 2.55 (1.13—2.26) 2.1 (0.9—2.1) 0.03 (1.35—3.05) 2.3 (1.18—2.37) Simpson’s 0.5 (0—0.5) 0.73 (0.2—0.77) 0.5 (0—0.5) 0.66 (0.5—0.75) 0.64 (0.46—0.75) 0.72 (0.55—0.8) 0.83 (0.65—0.86) 0.7 (0.6—0.82)

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

10.8 10.8 9 9.6 9.6 8 8.4 8.4 7 7.2 7.2 6 6 6 5 4.8 4.8 4 3.6 3.6 3 2.4 2.4 2 1.2 1

Taxa (95% confidence) 1.2 Taxa (95% confidence) 0 Taxa (95% confidence) 0 4 8 12 16 20 24 28 32 2 4 6 8 10 12 14 16 18 3 6 9 12 15 18 21 24 27 Specimens Specimens Specimens

9 8 5.5 8 7.2 5 7 6.4 5.6 4.5 6 4.8 4 5 4 3.5 4 3.2 3 3 2.4 2.5 2 1.6 2

Taxa (95% confidence) 1 Taxa (95% confidence) Taxa (95% confidence) 0.8 1.5 0 0 1 6 3 9 12 18 21 24 15 27 9 6 3 6 3 12 15 21 24 27 18 4.2 4.8 6.6 1.2 5.4 Specimens 1.8 2.4 3.6 Specimens Specimens 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).

The fungal composition was variable among the and abundance of the Geomyces and Penicillium macroalgal species, which was confirmed by the taxa associated with both macroalgae. In addition, values of the Sorensen and Bray-Curtis similarity Penicillium taxa occurred in association with seven indices (Figure 6). The Sorensen index showed that other macroalgal species, followed by Geomyces (six the most similar fungal communities were found in macroalgae). P. antarcticus and Adenocystis sp., both of which are The principal components analysis of the physi- brown macroalgal species, but with different cal and chemical parameters of the water where the morphologies. However, the Bray-Curtis similarity macroalgae were sampled related to the fungal between the fungal communities associated with the diversity indices (Figure 7) revealed that conductivity endemic macroalgae P. decipiens and M. hariotii exhibited a positive correlation with the Fisher’s a indicated the highest similarity with the presence and Simpson’s indices for the fungal assemblages

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1445

Aa Ui Mh Pd Dm Pa Ad Au Aa Ui Mh Dm Pa Ad Pd Au 0.96 0.96 0.84 0.84 0.72 0.72 0.6 0.6 73 0.48 0.48

50 Similarity 0.36

Similarity 0.36 44 38 0.24 36 0.24 27 36 36 20 0.12 31 0.12 50 43 0 100 0 100 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Figure 6 Dendrograms showing the Sorensen (a) and (b) Bray-Curtis similarity measures for the fungal communities associated with the Antarctic macroalgae. The results were obtained with 95% confidence and bootstrap values calculated from 1000 iterations. The sampled macroalgae were Aa, Acrosiphonia arcta; Ad, Adenocystis sp.; Au, Adenocystis utricularis; Dm, Desmarestia menziesii; Pa, Phaeurus antarcticus; Mh, Monostroma hariotii; Pd, Palmaria decipiens; and Ui, Ulva intestinalis.

3.2

Ulva intestinalis

2.4

Salinity

pH 1.6 Simpson’ s α Palmaria decipiens Fisher’ s Phaeurus antarcticus 0.8 Conductivity

PCoa 2 -3.2 -2.4 -1.6 -0.8 0.8 1.6 2.4 3.2

Acrosiphonia arcta Temperature -0.8 Adenocystis sp. Desmarestia menziesii Margalef?s Monostroma hariotii -1.6 Dissolved oxygen

Adenocystis utricularis -2.4

-3.2

PCoA 1 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 Fisher’s b (diversity), Margalef’s (richness) and Simpson’s (dominance) indices of the fungal communities associated with Antarctic macroalgae. associated with U. intestinalis; dissolved oxygen Biological activities with Margalef’s index for A. arcta and D. menziesii; All fungal isolates were grown using solid state temperature with Adenocystis sp., M. hariotii and A. fermentation techniques to obtain crude extracts, utricularis; and salinity and pH with P. decipiens which were then screened against bacteria, fungi and P. antarcticus. and parasite targets to detect bioactive compounds.

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1446

Fatty acid signals OCH3 signal

Unsaturated fatty Aromatic signals 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 9.5 9.0 8.5 8.0 7.5 7.0 6.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 f1 (ppm)

Fatty acid signals

OCH3 signal

Unsaturated fatty Aromatic signals acid signals 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 f1 (ppm)

1 Figure 8 H-NMR spectrum (600 MHz, 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.

The extracts of Penicillium sp. UFMGCB 6034 and of the control drug (Benznidazole) used in the Penicillium sp. UFMGCB 6120 recovered from the experiments. In addition, these two trypanocidal endemic macroalgae P. decipiens and M. hariotii, extracts showed low toxicity against normal fibroblast respectively, were able to produce bioactive extracts. cells. 1 Penicillium sp. UFMGCB 6120 displayed antifungal H NMR (600 MHz, DMSO-d6) analyses of the activity against the filamentous fungus Cladosporium ethanol extracts from the freeze-dried culture media sphaerospermum, producing 96% inhibition of Penicillium sp. UFMGCB 6034 and Penicillium andanMICvalueof250mgmlÀ 1. In addition, sp. UFMGCB 6120 were performed. Preliminary Penicillium sp. UFMGCB 6034 and Penicillium sp. analysis indicated the presence of functional groups UFMGCB 6120 exhibited 100% inhibition of trypo- associated with aromatic protons, methoxy protons, mastigote forms of T. cruzi,withIC50 values of 1.28 unsaturated fatty-acid olefinic protons, and fatty- and 0.45 mgmlÀ 1 being obtained, respectively, acid methylene and methyl protons, as indicated in À 1 which were close to or lower than the IC50 (1 mgml ) Figure 8. Both Penicillium sp. UFMGCB 6034 and

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1447 Penicillium sp. UFMGCB 6120 clearly contain the which show a wide distribution (http://www.myco- same major components, as demonstrated by the bank.org/). Geomyces sp. have been frequently similar 1H NMR signals observed. recorded in Antarctica, are a celluloytic, keratino- philic, psychrophilic and halotolerant fungi with a ubiquitous distribution in the soils of cold regions Discussion that is able to colonise and to utilise different carbon sources (Mercantini et al., 1989). In addition, Macroalgae collection and fungal identification Geomyces taxa were found on the thalli of the Marine algae are known to shelter fungal species, macroalgae A. utricularis and D. anceps (Loque but few taxa have been studied in detail. The initial et al., 2010), leaves of Colobanthus quitensis (Rosa work of Loque et al. (2010) and the present study, et al., 2010), mosses (Tosi et al., 2002) and in which addresses the fungal communities associated freshwater lakes (Gonc¸alves et al., 2012). Seven with eight macroalgae sampled along a latitudinal fungal isolates found associated with the endemic gradient over 350 km on the Antarctica Peninsula, macroalgae M. hariotii displayed high sequence represent the first systematic analyses of this kind. similarity with G. destructans. Geomyces destruc- Similar to other mycological studies conducted in tans is characterised as a psychrophilic pathogenic the Antarctic that have characterised fungi in soil fungus able to decrease ’ population in tempe- (Fell et al., 2006), wood debris (Arenz et al., 2006), rate regions (Lorch et al., 2011). According to Lorch lakes (Gonc¸alves et al., 2012) and in associations et al. (2012), the diversity of Geomyces taxa, also with plants (Rosa et al., 2009), the fungal taxa classified as Gymnostellatospora and Pseudogym- associated with the Antarctic macroalgae examined nosascus, is far greater than previously recognised in this study comprised a few dominant taxa. Our based on traditional taxonomic methods, and can be results agree with those of Suryanarayanan et al. underestimated. In addition, we found in associa- (2010), who found few dominant fungal species tion with Antarctic macroalgae 36 isolates identified associated with 25 macroalgae occurring along the as Geomyces sp., which may include new species coast of southern India. However, algicolous fungi and will be subject to further studies to elucidate remain relatively unexplored as a group. The their taxonomic positions. presence of Geomyces sp., Penicillium sp. and M. The yeast M. australis, which has been reported to australis as dominant species associated with the be endemic to Antarctica, has been isolated from investigated Antarctic macroalgae suggests that they Antarctic seawater (Fell and Hunter, 1968), the may exhibit an interesting ecological relationship stomach of the Antarctic krill species Euphausia with their hosts. superba (Donachie and Zdanowski, 1998) and, recently, at a high abundance from the algal thalli of A. utricularis (Loque et al., 2010). In addition, M. Diversity, richness, dominance and distribution australis has been found in marine sediment and Studies on fungal diversity in Antarctica show freshwater in Antarctica (Vaz et al., 2011). The inconsistencies in the discrimination of endemic, recovery of M. australis in association with the indigenous and cosmopolitan species. Endemic macroalgae A. arcta and D. menziesii reinforces the species are characterised as true psychrophilic fungi notion that this yeast may exhibit a specific that are able to actively grow and reproduce only in association with Antarctic macroalgae. Antarctica (Ruisi et al., 2007). According to Onofri The cosmopolitan genus Penicillium has been et al. (2007), indigenicity is indicated by the recovered from alpine and tundra soils as well as observation of a large number of isolates over time permafrost layers (Gunde-Cimerman et al., 2003). In as well as being commonly recorded in Antarctica Antarctica, species of Penicillium have been from different sites and substrata. In addition, the described from the soil (Azmia´ and Seppelt, 1998), majority of Antarctic fungi consist of ecotypes of lakes (Ellis-Evans, 1996), wood (Arenz et al., 2006) cosmopolitan species that show mesophilic-psy- and on the macroalga A. utricularis (Loque et al., chrotolerant behaviour as an adaptation to the cold 2010). As an extremophile, P. chrysogenum has been Antarctic climate (Zucconi et al., 1996). On the basis isolated as a dominant species from Arctic sub- of the above criteria, the fungal species M. australis glacial ice (Gunde-Cimerman et al., 2003, Sonjak is indigenous to Antarctica, and Penicillium is a et al., 2005). In addition, according to Bugni and cosmopolitan mesophilic-psychrotolerant group Ireland (2004) Penicillium represents one of the that shows adaptation to the cold Antarctic climate. more common genera isolated from macroalgae. In In contrast, Antarctomyces psychrotrophicus (not the present study, we found different Penicillium found outside of Antarctica), which was part of the taxa associated with the Antarctic macroalgae. minority component associated with the macroalga According to Frisvad and Samson (2004), Scott U. intestinalis, has been reported to be endemic to et al. (2004) and Houbraken et al. (2011), the Antarctica (Stchigel et al., 2001). of the group Penicillium has been Geomyces (Helotiales, Ascomycota), found as regarded to be especially difficult and needs a the most abundant taxa associated with Antarctic polyphasic study including physiological, morpho- macroalgae, includes about 10 named species, logical, multilocus sequence typing and chemical

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1448 methods. For these reasons, the Penicillium sp. found to be associated with Antarctic macroalgae associated with the Antarctic macroalgae were were identified as species known to be mutualists, identified using the term ‘cf.’ or in genus rank. saprobes and parasites. Bridge and Spooner (2012) These Penicillium taxa will be the subject of further suggest that Antarctic fungi occupy many different taxonomic evaluations. ecological niches, but their significance in these The non-asymptotic species accumulation curves niches remains poorly understood. Understanding (Mao Tao analysis) generated for the fungal commu- how mutualistic, saprobic and pathogenic fungi nities found in associations with the examined achieve their lifestyles is crucial for understanding Antarctic macroalgae showed that a large number their ecological functions and their impact on of single individuals (singletons) were recovered, Antarctic macroalgae under the extreme conditions which are considered rare species and often repre- of this continent. In addition, the abiotic seawater sent more than half of the species within these parameters in the areas where the macroalgae were communities. Similar results were described for the collected revealed low temperatures and high fungal communities of A. utricularis, D. anceps and salinities, demonstrating that the fungi found in P. decipiens collected at King George Island, association with the macroalgae may exhibit specific Antarctica (Loque et al., 2010). mechanisms that allow them to survive in extreme Twenty-seven fungal taxa associated with the conditions. Antarctic macroalgae displayed a low molecular similarity with the sequences of known fungi deposited in GenBank, suggesting that they are Biological activities candidates for new fungal species. However, not all In general, fungi have been reported to represent described fungal species are deposited in the prolific sources of various compounds, including GenBank database, and further taxonomic analyses several bioactive molecules. However, the potential including detailed physiological and macro- of extremophile fungi to produce bioactive com- and micromorphological characterisation and pounds is poorly understood. According to molecular sequencing of other DNA regions, Santiago et al. (2012), the ability of Antarctic fungi together with new phylogenetic analyses, will be to survive in extreme conditions suggests that they needed to describe these potentially new species. may display unusual biochemical pathways that According to the recent checklist of Antarctic fungi allow them to generate specific or new molecules published by Bridge and Spooner (2012), the that could be used to develop new drugs. Our specimens of Meyerozyma caribbica, Mycoarthris bioprospecting results show that the extracts from coralline and Yamadazyma mexicana,whichwere Penicillium sp. UFMGCB 6034 and Penicillium sp. found as minor components within the fungal UFMGCB 6120, which were recovered as dominant communities associated with Antarctic macroal- species from endemic macroalgae, P. decipiens and gae, represent the first records of these species in M. hariotii, displayed high and selective antifungal Antarctica. and/or trypanocidal activities with low MIC and IC50 According to Ruisi et al. (2007), the distribution of values. The combination of low MIC and IC50 values fungi in Antarctica is related to the distribution of together with the 1H NMR spectral data suggesting their hosts or substrates, such as bird feathers and the presence of functionalised aromatic compounds dung, invertebrates, vegetation, soils, rocks and warrants extensive bioassay-directed fractionation to lichens. However, marine fungal dispersal remains specifically identify the constituents responsible for unknown. In the present study, the calculated the observed biological activity. similarity indices (Sorensen and Bray-Curtis) Marine fungi have been described as a promising revealed the presence of some species (D. hansenii, source of antimicrobial compounds. Marine Peni- Geomyces sp., Helotiales sp., M. australis, cillium sp. are known to produce a large variety of Penicillium sp. and T. globosus) in association with compounds with a wide range of biological and different macroalgae and from distinct Antarctic pharmacological activities. Penicillium sp. isolated islands. The high values of the Bray-Curtis similar- from the alga U. intestinalis were observed to ity index (which has priority regarding the abun- produce cytotoxic metabolites including commune- dance of common species) between the fungal sins (Numata et al., 1993), penochalasius A-C communities associated with endemic macroalgae (Numata et al., 1995), penostatins A-I (Takahashi P. decipiens and M. hariotii indicate that Geomyces et al., 1996, Iwamoto et al., 1998) and penochalasius and Penicillium sp. might show a specific associa- D-H (Iwamoto et al., 2001). The antibacterial and tion with macroalgae adapted to extreme conditions. cytotoxic compounds di(2-ethyl hexayl) phthalate Fungi are one of the largest and most diverse and fungisterol were isolated from P. brevicompactum, kingdoms of eukaryotes and are important biological which was isolated from the associated marine alga components of terrestrial ecosystems. According to Pterocladia sp. (Atalla et al., 2011). The extracts of Bridge and Spooner (2012), there are many records Penicillium sp. UFMGCB 6034 and Penicillium sp. of marine fungi from Antarctica, although there is UFMGCB 6120 will be subjected to further systema- little available information about their ecological tic chemical bioactivity-guided fractionation to roles. In the present study, some of the fungal taxa isolate their bioactive compounds.

The ISME Journal Fungi associated with Antarctic macroalgae VM Godinho et al 1449 Conclusion Antarctic algae: prevalence in hosts and palatability to mesoherbivores. Phycologia 48: 324–334. Although marine fungi have been studied for the last Arenz BE, Held BW, Jurgens JA, Farrell RL, Blanchette RA. 100 years (Jones, 2011), many aspects regarding their (2006). Fungal diversity in soils and historic wood taxonomy, ecology and potential biotechnological from the Ross Sea Region of Antarctica. Soil Biol Bioch applications remain poorly documented. Our results 38: 3057–3064. are consistent with a suggestion by Bridge and Atalla MM, Zeinab HK, Eman RH, Amani AY, Abeer Spooner (2012) that the true diversity of Antarctic AAEA. (2011). Physiological Studies on Some fungi may be far greater than is currently estimated. Biologically Active Secondary Metabolites from Our results also suggest that macroalgae can act as a Marine. Derived Fungus Penicillium brevicompactum 1: 1–15http://www.GAte2Biotech.com. substrate to shelter fungi in Antarctica and con- Azmia´ OR, Seppelt RD. (1998). The broad-scale distribu- tribute to the knowledge of the cryptic and unknown tion of microfungi in the Windmill Islands region, algicolous Antarctic mycota. We showed that ende- continental Antarctica. Polar Biol 19: 92–100. mic and cold-adapted macroalgae living under the Bridge PD, Spooner BM. (2012). Non-lichenized Antarctic extreme conditions of Antarctica shelter rich, fungi: transient visitors or members of a cryptic diverse and complex fungal communities consisting ecosystem? Fungal Ecol 5: 381–394. of few dominant indigenous or mesophilic cold- Buckner FS, CLMJ Verlinde, La Flamme AC, Van Voorkhis adapted species. In addition, we detected a large WC. (1996). Efficient Technique for screening drugs number of rare and/or endemic taxa, which may for activity against Trypanosoma cruzi using parasites b provide an interesting model of algal–fungal inter- expressing -galactosidase. Antimicrob Agent Chemother 40: 2592–2597. actions in extreme conditions as well as a potential Bugni TS, Ireland CM. (2004). Marine-derived fungi: a source of bioactive compounds. Our results rein- chemically and biologically diverse group of micro- force the call for further studies of fungal commu- organisms. Nat Prod Rep 21: 143–163. nities present across the Antarctic marine Carvalho CR, Gonc¸alves VN, Pereira CB, Johann S, Galliza environment as well as their phylogenetic relation- IV, Alves TMA et al. (2012). The diversity, antimicro- ships with species that occur in other cold regions. bial and anticancer activity of endophytic fungi Finally, the present work suggests that examination associated with the medicinal plant Stryphnodendron of the fungi associated with macroalgae in Antarc- adstringens (Mart.) Coville (Fabaceae) from the tica may provide new insights into the biological Brazilian savannah. Symbiosis 57: 95–107. mechanisms underlying tolerance and adaptation to Colwell RK, Mao CX, Chang J. (2004). Interpolating, extrapolating, and comparing incidence-based species extreme marine polar conditions. accumulation curves. Ecology 85: 2717–2727. Donachie SP, Zdanowski MK. (1998). Potential digestive Acknowledgements function of bacteria in krill, Euphausia superba stomach. Aquat Microb Ecol 14: 129–136. This study had financial and logistic support from the Ellis-Evans JC. (1996). Microbial diversity and function in Brazilian Antarctic Program (PROANTAR/MCT/CNPq— Antarctic freshwater ecosystems. Biodivers Conserv 5: No. 23/2009), Marine of Brazil and Francisco Petrone from 1395–1431. the Clube Alpino Paulista (CAP). We acknowledge the Fell JW, Hunter IL, Tallman AS. (1973). Marine basidio- financial support from Fundac¸a˜o de Amparo a Pesquisa mycetous yeasts (Rhodosporidium spp. n.) with tetra- do Estado de Minas Gerais (FAPEMIG), Health-PDTIS- polar and multiple alletic bipolar mating systems. Can FIOCRUZ, PRPq-UFMG, CAPES/PGCI 036/13 and Con- J Microbiol 19: 643–657. selho Nacional de Desenvolvimento Cientı´fico e Tecnolo´- Fell JW, Hunter IL. (1968). Isolation of heterothallic yeast gico (CNPq). 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