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Diversity of fungal isolates from three Hawaiian marine sponges
ARTICLE IN PRESS Microbiological Research 164 (2009) 233—241
www.elsevier.de/micres
Diversity of fungal isolates from three Hawaiian marine sponges Quanzi Li, Guangyi Wang Department of Oceanography, University of Hawaii at Manoa, Honolulu, HI, USA Received 28 February 2007; received in revised form 26 June 2007; accepted 3 July 2007
KEYWORDS Fungi; Marine sponges; Fungal diversity; Sponge–fungal association
Summary Sponges harbor diverse prokaryotic and eukaryotic microbes. However, the nature of sponge–fungal association and diversity of sponge-derived fungi have barely been addressed. In this study, the cultivation-dependent approach was applied to study fungal diversity in the Hawaiian sponges Gelliodes fibrosa, Haliclona caerulea, and Mycale armata. The cultivated fungal isolates were representatives of 8 taxonomic orders, belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. A portion of these isolates (n ¼ 15, 17%) were closely affiliated with fungal isolates isolated from other marine habitats; the rest of the isolates had affiliation with terrestrial fungal strains. Cultivated fungal isolates were classified into 3 groups: ‘sponge-generalists’—found in all sponge species, ‘sponge-associates’—found in more than one sponge species, and ‘sponge-specialists’—found only in one sponge species. Individuals of G. fibrosa collected at two different locations shared the same group of ‘sponge-specialists’. Also, representatives of 15 genera were identified for the first time in marine sponges. Large-scale phylogenetic analysis of sponge-derived fungi may provide critical information to distinguish between ‘resident fungi’ and ‘transient fungi’ in sponges as it has been done in other marine microbial groups. This is the first report of the host specificity analysis of culturable fungal communities in marine sponges. & 2007 Elsevier GmbH. All rights reserved.
Introduction Sponges are commonly known to harbor diverse prokaryotic and eukaryotic microbes (Hentschel Corresponding author. Tel.: +1 808 956 3744;
fax: +1 808 956 9225. E-mail address: [email protected] (G. Wang).
et al., 2006; Wang, 2006). Some efforts have been made to investigate structure and composition of prokaryotic microbial communities in marine sponges, using both cultivation-dependent and cultivation-independent approaches. These investigations have resulted in the identification of phylogenetically diverse, yet highly sponge-specific, prokaryotic microbial groups (for recent reviews,
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.07.002
ARTICLE IN PRESS 234 see Lee et al., 2001; Hentschel et al., 2003, 2006; Imhoff and Stoehr, 2003; Hill, 2004; Wang, 2006). Eukaryotic microbes such as zooxanthellae, zoochlorelae, dinoflagellates, and diatoms are reported to be associated with marine sponges (Saller, 1989; Rosell and Uriz, 1992; Bavestrello et al., 2000; Cerrano et al., 2000; Carballo and Avila, 2004; Webster et al., 2004). However, detailed studies of the diversity of eukaryotic communities living within sponges are rare. Filamentous fungi are ubiquitous and can be easily isolated from the inner tissue of sponges (Ko ¨nig et al., 2006; Wang, 2006). Nevertheless, most investigations of sponge-derived fungi have been focused on chemistry of natural products simply because sponge-derived marine fungi produce the highest number of novel marine fungal metabolites (Jensen and Fenical, 2002; Bugni and Ireland, 2004; Wang, 2006). In several cases, there is evidence from experiments that sponge-derived fungi are the true biosynthetic origin of spongederived secondary metabolites such as jasplakinolide (Jensen and Fenical, 2002). Hundreds of fungal strains have been isolated from marine sponges, representing three phyla of Ascomycota, Zygomycota, and mitosporic fungi (Ho ¨ller et al., 2000; Jensen and Fenical, 2002; Morrison-Gardiner, 2002; Bugni and Ireland, 2004). Diversity of fungi associated with marine sponges still remains an understudied area. So far, identification of fungi in marine sponges has been solely made using a morphology-based approach. Many microscopic fungi lack a sexual state (mitosporic or anamorphic), but can possess a surprisingly high level of genetic variation for a similar morphology (Kohn, 1995; Harrington and Rizzo, 1999; Talhinhas et al., 2002). In fact, the majority of fungi derived from marine sponges are mitosporic; about one-third of these fungi cannot be identified by using morphology-based approach (Morrison-Gardiner, 2002). The morphology-based approach, therefore, can be severely limited in identification of spongederived marine fungi. As of now, only the marine ascomycete of the genus Koralionastes is reported to have a unique physical association with crustaceous sponges (Kohlmeyer and Volkmannkohlmeyer, 1990). In addition, limited information is available on the ecological function of fungi living within marine sponges. Fungi have been reported to contribute to the localized lesions of sponges and thus may enhance disease development of sponges. Fungi are opportunistic, sometimes acting as secondary colonizers to other infections or stresses (Galstoff, 1942; Sparks, 1985; Vacelet et al., 1994). Overall, the diversity of fungal communities within sponges and the nature of
Q. Li, G. Wang sponge–fungal association remain largely unknown. Development of molecular genetics has provided a significant advancement in understanding diversity and function of microbes in diverse natural habitats. Phylogenetic taxonomy has been accepted as the appropriate way to analyze teleomorphic and anamorphic fungi and is becoming increasingly popular with mycologists (Taylor et al., 2000); hence, phylogenetic analysis can provide a better tool to assess fungal communities within sponges. In this study, we isolated fungi from three Hawaiian marine sponge species collected at two different locations on the island of Oahu, and carried out the phylogenetic analysis of their ITSrDNA sequences. Being the first isolated from marine sponges, many of these fungal isolates are valuable for further exploration of their biomedical potential. Thus, our data provide, for the first time, a look into the association nature of culturable fungal communities living within marine sponges.
Materials and methods Sampling sites and sponge collection Specimens of the marine sponges Gelliodes fibrosa, Haliclona caerulea and Mycale armata were collected snorkeling at Kuli’ou’ou Beach in Hawaii Kai (1571440 W, 211170 N) and on Coconut Island in Kane’ohe Bay (1571470 W, 211260 N), Oahu, Hawaii (Figure 1). The two sites are located 37 km apart. Three samples of each species were collected within a 20-m radius at depths of 0.5–3 m.
Oahu
Figure 1. Map of the main Hawaiian Islands and approximate locations of collection sites of marine sponges on Oahu (open circle and closed circle indicates the locations of Hawaii Kai and Coconut Island, respectively).
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges Latex gloves were worn during collection. Seawater samples from the collection sites were collected using 100-ml syringes to serve as controls. Specimens were transferred directly to Zip-lock bags containing seawater to prevent contact of sponge tissue with air. The samples were transported to the laboratory and processed immediately for the isolation and cultivation of fungi. Alternatively, sponge tissue was frozen in liquid nitrogen and then stored at 80 1C for future use.
Fungal isolation To get rid of nonspecific fungal propagules from seawater columns on sponge surfaces, sponge tissues were rinsed three times with sterile artificial seawater. The surface of the sample was disinfected with 70% ethanol. The inner tissue was taken out with a scalpel and forceps and then homogenized using a blender containing 20 ml sterile natural seawater in aseptic conditions. The resulting homogenate was diluted with sterile seawater at three dilutions (1:10, 1:100, and 1:1,000). For fungal cultivation, 100 ml of each dilution was plated in quadruplicate onto GPY plates (1.0 g glucose, 0.1 g yeast extract, 0.5 g peptone, 15 g agar, and seawater, pH 8.0), Marine agar (per liter Marine broth 2216 plus 15 g DifcoBacto agar), Gause I (starch 20 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4 7H2O 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, 15 g Difco-Bacto agar, pH 7.2-7.4), and Luria Bertani (LB) agar plates, containing the antibiotics penicillin and streptomycin (100 mg/ml each). The plates were incubated at room temperature (18–20 1C) for 1–3 weeks until the morphology of fungi could be distinguished. Each isolate was picked and transferred onto a new GPY, Marine Agar (Difco), Gause I, and LB agar plates containing streptomycin and penicillin. The resulting plates were incubated at room temperature for pure culture. All the above media were made with natural seawater.
Morphological and molecular analysis of cultivated fungi Morphological traits (e.g. morphology and color of spore and mycelia) were examined to exclude redundant fungal isolates. The rest of the isolates were cultured in GPY broth at 28 1C for 2–5 days. The mycelium was harvested using vacuum filtration and dried between two layers of paper towel. The resulting mycelial mat was ground into powder in liquid nitrogen. Fungal DNA was extracted using the procedure described by Fredricks et al. (2005)
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with modifications. In brief, the mycelial powder was transferred to a 1.5-ml Eppendorf tube containing 400–500 ml TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.3); equal volume of phenol solution was added to the tube. After brief mixing, the mixture was centrifuged at 12,000g for 10 min at 4 1C. The aqueous phase was transferred to a new microtube and sequentially extracted with phenol solution and chloroform. RNA was removed in the aqueous phase using RNaseA. The sample was then again extracted with phenol solution and chloroform. Finally, DNA was precipitated by adding two volumes of ethanol. The DNA pellet was washed with 75% ethanol and resuspended in 50–100 ml of sterile water. The resulting genomic DNA was used as a template to amplify fungal ITS-rDNA fragments using the primers ITS1 (50 -TCCGTAGGTGAACCTGCG30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) (White et al., 1990). The reaction mixture contained 5 ml of 10 reaction buffer with 15 mM MgCl2 (Promega), 2 ml of 2.5 mM dNTPs, 2 ml of 10 pmol forward primer, 2 ml of 10 pmol reverse primer, 1 ml of fungal DNA, 0.5 ml of Taq DNA polymerase (5 U ml1, Promega), and 32.5 ml of H2O. PCR conditions were as follows: initial denaturation (94 1C for 5 min); 30 cycles of denaturation (94 1C for 50 s), primer annealing (55 1C for 50 s), and elongation (72 1C for 1 min), with a final elongation at 72 1C for 10 min. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and used directly for sequencing analysis. Alternatively, if satisfactory sequencing results were not obtained from PCR products, PCR products were cloned into pGEM-T Easy Vector (Promega) and sequenced using the plasmid DNA with the universal plasmid primers T7 and SP6.
Sequence and phylogenetic analysis Sequencing analyses were performed on an ABI 3730 XL (Applied Biosystems) automated sequencer using ITS1 and ITS4 primers for PCR templates or universal plasmid primers (T3 and T7) for plasmid templates. Sequence data were edited with Chromas Lite, version 2 (Technelysium). For preliminary identifications, sequences of fungal rDNA-ITS regions were compared with those in the NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). Fungal ITS-rDNA sequences in this study and the matched sequences from GenBank were edited and aligned with Seq-Man and Megalign (DNASTAR Package). The aligned sequences were imported into PAUP 4.0b10 (Swofford, 2002). Neighbor-joining (NJ) trees were estimated using pairwise genetic distances based
ARTICLE IN PRESS 236 on the basis of all substitutions with the Jukes– Cantor distance parameter. For maximum-parsimony (MP) analyses, we used a heuristic search strategy. A strict consensus tree was drawn when multiple best trees were obtained. The quality of the branching patterns for MP and NJ was assessed by bootstrap resampling of the data sets with 1000 replications.
Nucleotide sequence accession number Fungal rDNA-ITS sequences obtained in this study were deposited in GenBank/EMBL/DDBJ under accession numbers DQ092514–DQ092533 and EF029806–EF029831.
Results Diversity of culturable fungi within sponges Cultivation of fungi from G. fibrosa, H. caerulea, and M. armata yielded a total of 235 isolates. Redundant isolates were excluded from further analysis using morphological characteristics. A total of 119 isolates was cultured for genomic DNA extraction and sequencing analysis. ITS-rDNA sequence analyses identified 86 independent isolates. These isolates matched diverse previously described fungi, and similarity of their ITS-rDNA sequences ranged from 66 to 99%. A portion of these isolates (n ¼ 15, 17%) were closely (498% sequence similarity) affiliated with fungal isolates from marine habitats, and the rest were related to terrestrial fungi. Some of these isolates (n ¼ 12, 14%) were affiliated with previously described fungal strains, which produce interesting fungal bioproducts (natural products and enzymes), the others (n ¼ 10, 12%) were closely affiliated (499% sequence similarity) with previously described pathogens (marine animal, human, and plant). Several of these isolates such as MACIP4 (identical to Lacazia loboi, a marine animal pathogen) were cultivated for the first time. Phylogenetic analysis revealed that fungal isolates from these 3 sponges clustered into 8 phylogenetic groups, corresponding to 8 taxonomic orders (Aphyllophorales, Diaporthales, Eurotiales, Hypocreales, Mycosphaerellales, Pleosporales, Saccharomycetales, and Xylariales) and belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. Figures 2 and 3 provide an overview of the phylogenetic relationship of sponge-associated culturable fungi while Figure 4 shows affiliations of individual genera with marine
Q. Li, G. Wang sponges collected at different locations. Isolates (n ¼ 24, 28%) of the Eurotiales were closely affiliated with 3 genera: Aspergillus, Eupenicillium and Penicillium. Isolates (n ¼ 23, 27%) of the Pleosporales had affiliations with 8 genera Ampelomyces, Bipolaris, Cochliobolus, Curvularia, Didymella, Leptosphaerulina, Phaeosphaeria, and Paraphaeosphaeria as well as 4 unclassified genera. Isolates (n ¼ 19, 22%) of the Hypcreales were representatives of 7 genera Bionectria, Fusarium, Hypocrea, Myrothecium, Solheimia, Trichoderma, and Tubercularia. Isolates (n ¼ 10, 12%) of the Mycosphaerellales were closely affiliated with 2 genera, Cladosporium and Lacazia. Isolates (n ¼ 4, 5%) of the Xylariales were related to 2 genera Bartalinia and Nigrospora as well as 1 unclassified genus. Isolates (n ¼ 3, 3%) of Aphyllophorales did not have affiliation with any certain genus, but were closely related to the fungal endophyte (AY456192) of Taxus mairei, a producer of the anticancer drug Taxol (Chang and Yang, 1996). One isolate of the Dothideales and the Saccharomycetales belonged to the genera Fusicoccum and Candida, respectively. Among the identified fungal genera, 15 were isolated for the first time from marine sponges. The other isolates associated with unclassified genera were potentially new fungal species. Overall, 16 genera were identified in the sponge G. fibrosa collected on Coconut Island while 6 genera were isolated from M. armata from the same location (Figure 4). G. fibrosa and H. caerulea collected in Hawaii Kai yielded 15 and 8 genera, respectively. G. fibrosa from a different location (Hawaii Kai and Coconut Island) gave a similar number of fungal genera or fungal diversity although samples of G. fribrosa from different locations did contain different genera. Finally, G. fibrosa gave overall the highest fungal diversity and M. armata the least diversity.
Association of culturable fungi with different marine sponges Association of cultivated fungal genera displayed interesting patterns (Figure 4). Three fungal genera Aspergillus, Penicillium, and Eupenicillium were found in all sponges and can be classified as ‘sponge-generalists’. Fungal genera such as Ampelomyces, Tubercularia, and Clasoprorium, which were identified in more than one sponges, can be called ‘sponge-associates’. The fungal genera such as Didymella, Fusicoccum, and Lacazia, which were found only in one sponge species, can be named as ‘sponge-specialists’. Individuals of G. fibrosa
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges
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99 MACIP4 Lacazia loboi AF035674 MACIG4 Cladosporium cucumerinum AF393696 Cladosporium oxysporum AF393720 MACIM2 100 MACIM3 Mycosphaerellales MACIP9 100 GFHK25 HCHK18 GFHK6 97 HCHK20 55 73 Cladosporium cladosporioides DQ335984 GFHK21 54 Cladosporium sphaerospermum DQ335985 HCHK9 Penicillium herquei AF033405 100 MACIP5 89 GFHK10 Eupenicillium hirayamae AF033418 Penicillium namyslowskii AF033463 MACIP6 99 MACIG2 66 GFHK13 67 Eupenicillium sp. DQ092516 87 Penicillium steckii DQ123666 94 100 GFHK14 Penicillium citrinum DQ123646 95 Penicillium roseopurpureum AF455492 MAHKP2 Penicillium griseofulvum AY373917 100 Penicillium crustosum AY373907 Penicillium commune AF455527 71 MACIB1 99 Penicillium griseoroseum AY425988 GFHK19 99 100 Penicillium olsonii DQ123662 100 GFCI23 Eurotiales GFCI24 GFCI25 100 Penicillium brevicompactum AY373897 100 HCHK1 57 Aspergillus ochraceus AY373854 58 HCHK13 100 Aspergillus flavipes AY822647 MACIP1 HCHK3 100 Aspergillus awamori DQ235784 GFCI31 GFCI28 100 Aspergillus tamarii AY373870 58 100 Aspergillus japonicus AJ876880 100 Aspergillus aculeatus U65309 GFCI27 MACIP7 Uncultured fungus clone G49 DQ279843 Uncultured fungus clone G7 DQ279847 MACIM4 MACIP3 64 Aspergillus nidulans AF455505 56 Aspergillus sydowii AM176727 100 Aspergillus versicolor AY37388 MACIP11 GFCI9 100 Fusicoccum mangiferum AY615187 Dothideales Candida parapsilosis AY227019 99
100
0.1 substitutions/site Figure 2. Neighbor-joining phylogenetic tree from analysis of ITS-rDNA (4540 bp) sequences from culturable fungal isolates clustering within the Mycosphaerellales, Eurotiales, and Dothideales. Numbers at branches indicate bootstrap values of neighbor-joining analysis (450%) from 1000 replicates. MA—Mycale armata; GF—Gelliodes fibrosa; HC—Haliclona caerulea; HK—Hawaii Kai; CI—Coconut Island; the other letter and number stand for isolate identification.
collected at 2 different locations shared 6 ‘spongespecialists’ genera. However, 3 genera (Bartalinia, Diaporthe, and Paraphaeosphaeria) and 1 unclassified genus isolated from G. fibrosa samples collected at Hawaii Kai were not present in specimens from Coconut Island. On the other hand, 6 genera (Curvularia, Bionectria, Bipolaria, Fuscicoccum, Nigrospora, and Solheimia) and 1 unclassified genus
cultivated from G. fibrosa collected on Coconut Island were not present in samples from Hawaii Kai either. The cause of this discrepancy needs to be further investigated. Also, different sponge species collected at the same location (e.g. H. caerulea vs G. fibrosa) or different location (e.g. M. armata vs H. caerulea) had their own unique ‘sponge-specialists’ genera. Seawater samples collected at the site of
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Q. Li, G. Wang GFHK4 Hypocrea lixii AY605745 GFCI11 GFHK12 Hypocrea lixii AF469189 Trichoderma inhamatum AF362101 Trichoderma aureoviride AF194015 GFCI10 GFCI16 65 Trichoderma inhamatum AF455502 GFHK40 96 GFHK41 Trichoderma tomentosum AY605718 100 GFCI12 100 GFCI2 70Trichoderma asperellum AY857244 Hypocreales 100 HCHK15 64 Bionectria ochroleuca AF106532 99 GFHK28 100 HCHK11 84 Tubercularia sp. AF422980 100 GFCI22 Solheimia costispora DQ135996 100 FFHK34 Myrothecium roridum AJ301995 56 9569 97 Myrothecium verrucaria AJ301999 GFCI5 GFHK3 Fusarium chlamydosporum AY213655 68 GFCI3 GFCI6 100 Fusarium incarnatum AY633745 GFHK5 100 GFHK37 Diaporthe phaseolorum AF001027 Diap orthales 54 100 GFHK35 Bartalinia sp. AY924288 84 55 GFCI1 84 Ascomycete sp. DQ480361 100 GFCI8 Nigrospora oryzae DQ219433 Xylariales GFHK25 100 HCHK18 Cladosporium oxysporum AF39372 100 GFHK33 50 Uncultured soil fungus DQ421204 100 Uncultured soil fungus DQ420984 67 Ampelomyces humuli AF035779 59 HCHK5 58 Didymella cucurbitacearum AY293804 GFHK1 100 HCHK22 100 HCHK8 HCHK7 56 HCHK23 98 100 Pleosporales sp. AB255299 HCHK14 100 Cochliobolus verruculosus AF071333 57 GFHK23 100 GFCI26 71 86 Curvularia affinis AF071335 100 GFCI35 100 GFCI30 Pleospor ales 51 Cochliobolus lunatus DQ836799 100 GFCI34 Bipolaris australiensis AJ853762 Phaeosphaeria oryzae AF439495 81 Fungal sp. AM231359 100 HCHK12 94 87 HCHK17 100 100 HCHK16 Leptosphaeriaceae sp. DQ342361 100 GFHK2 GFCI19 100 Foliar endophyte AY 566890 51 Uncultured ascomycete AY969713 84 62 Paraphaeosphaeria sp. AB096264 66 GFHK26 100 GFHK20 100 88 100 HCHK6 GFHK32 100 100 GFCI4 Leptosphaerulina chartarum AM231398 100 GFHK31 Fungal sp. AM231373 Fungal endophyte AY 456192 HCHK10 100 Aphyl lop horales GFHK30 98 HCHK4 MACIC19 100 Candida parapsilosis AY227019 Fusicoccum mangiferum AY615187
Saccharomyce tales
0.1 substitutions/site Figure 3. Neighbor-joining phylogenetic tree from analysis of ITS-rDNA (4540 bp) sequences from culturable fungal isolates clustering within the Hypocreales, Diaporthales, Xylariales, Pleosporales, Aphyllophorales, and Saccharomycetales. Numbers at branches indicate bootstrap values of neighbor-joining analysis (450%) from 1000 replicates. MA—Mycale armata; GF—Gelliodes fibrosa; HC—Haliclona caerulea; HK—Hawaii Kai; CI—Coconut Island; the other letter and number stand for isolate identification.
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges
Gelliode fibrosa (CI)
Bartalinia Diaporthe Paraphaeosphaeria
Cladosporium
Didymella Phaeosphaeria Haliclona caerulea (HK)
Cochliobolus Fusarium Hypocrea Myrothecium Leptosphaerulina Aspergillus Trichoderma Eupenicillium Penicillium
Ampelomyces, Tubercul aria
Gelliode fibrosa (HK)
Bionectria, Bipolaris Curvularia, Fusicoccum Nigrospora, Solheimia
Candida, Lacazia Mycale armata (CI)
Figure 4. Association of cultivated fungal genera with the Hawaii sponges collected on Coconut Island (CI) and in Hawaii Kai (HK).
Coconut Island yielded two fungal species Penicillium janthinellum and Penicillium chrysogenum and seawater samples from Hawaii Kai two fungal species Fusarium solani and P. chrysogenum. These three species were not present within sponges. Therefore, it seems that sponges harbor unique culturable fungal communities from the water column.
Discussion Marine fungi are still one of the most understudied marine ecological groups. Two groups of marine fungi have been classified on the basis of their ability to grow and to reproduce in seawater. Obligate marine fungi are those that grow and sporulate exclusively in a marine or estuarine habitat, while facultative marine fungi are those from freshwater or terrestrial milieus that are able to grow (and possibly sporulate) in the marine environments (Kohlmeyer and Kohlmeyer, 1979; Kohlmeyer and Volkmann-Kohlmeyer, 2003). In addition, facultative fungi could be those from terrestrial origins that have adapted to life in marine environments (Morrison-Gardiner, 2002). All sponge isolates of this study were able to grow on media made from natural seawater, and therefore can at least be classified as facultative fungi. Moreover, 15 isolates (17%), which were closely affiliated with marine fungal isolates, were likely obligate marine fungi. The other isolates (83%) were related to common genera to terrestrial habitats, suggesting that these isolates may be of terrestrial origin. Results of this study and two
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other investigations (Ho ¨ller et al., 2000; MorrisonGardiner, 2002) indicated that facultative fungi are frequently isolated from sponges at both nearshore and offshore locations. One important question to be addressed is whether any of these fungi simply result from terrestrial fungal spores that are trapped in sponge tissues during the filter-feeding process and remain dormant until being plated onto a suitable growth medium. But, fungal isolates, recovered in seawater samples of this study, were different from those in sponges. Also, if fungi in sponges are simply resulting from terrestrial fungal spores, sponge-derived fungi should have similar metabolic activity to their terrestrial counterparts. Nevertheless, many fungal isolates from marine sponges and other marine substrates have been shown to produce novel natural compounds that are not found in terrestrial strains (Jensen and Fenical, 2002; Proksch et al., 2003; Ko ¨nig et al., 2006). Therefore, sponge-derived fungi at least have their uniqueness of metabolic activities. Finally, before a new actinomycete taxon was identified from marine sediments through phylogenetic analysis, it was frequently assumed that actinomycetes isolated from marine samples are merely of terrestrial origin (Mincer et al., 2002). The similar scenario might hold for sponge-derived fungi. To our knowledge, this study represents the first phylogenetic study of fungi associated with marine sponges. Further large-scale phylogenetic analysis may provide solid molecular evidence regarding the difference between ‘resident fungi’ and ‘transient fungi’ in marine sponges. The availability of ITS-rDNA sequences of sponge-derived fungi in GenBank database is a major obstacle for such analyses. Fungi have commonly been isolated from marine sponges, but most studies have focused on novel metabolites of sponge-derived fungi (Jensen and Fenical, 2002; Ko ¨nig et al., 2006; Wang, 2006). Reports on fungal diversity in the ecological context of marine sponges as a habitat are scarce. In the survey of dominant filamentous fungi in Australian coral reefs, Morrison-Gardiner identified 208 isolates from 70 sponge samples using a morphology-based method (Morrison-Gardiner, 2002). Alternaria, Aspergillus, Cladosporium, Fusarium, and Penicillium were predominant fungal genera. Because sponge samples of different species collected at different locations were treated as a single source of fungal isolates and no sponge species information was given, it is difficulty to assess the fungal diversity in this study. In the second study of diversity and secondary metabolites from sponge-derived fungi, 681 fungal strains were isolated from 16 sponge species collected from the North Sea, the Mediterranean,
ARTICLE IN PRESS 240 the Caribbean and the Great Barrier Reef using the same identification approach (Ho ¨ller et al., 2000). The predominant fungal genera were ubiquitous ones: Acromonium, Arthrinium, Coniothyrium, Fusarium, Mucor, Penicillium, Phoma, Trichoderma, and Verticilium. Due to the different treatment of the sponge samples following sampling and before isolation of the fungi, the numbers of fungal isolates per sponge varied greatly between various locations. Sponges collected at two locations did not yield any fungal strain. In this study, the predominant genera were Penicillum (15%), Aspergillus (13%), and Trichoderma (11%). All 3 genera were also listed as the predominant ones in previous two studies, but the overall composition of fungal genera in Hawaiian sponges were quite different from that of the other two studies. This difference may reflect either the real difference of fungal populations living within different sponge species, the difference of different geographic locations, or the different media used in fungal isolation. One major reason for the differences may also be the different identification methods used in these studies. Whether sponge-derived fungi can be considered as symbionts of sponges or perform a specific function in this context is currently unknown. Increasing evidence appears to support a symbiotic nature of the association. An endosymbiotic yeast was identified in the sponges of the genus Chondrilla (Maldonado et al., 2005). In addition, the sponge Suberites domuncula was shown to have the ability to recognize fungi in their environments at the molecular level (Perovic-Ottstadt et al., 2004). Furthermore, the horizontal gene transfer of a mitochondrial intron from a fungus to sponges suggested a symbiotic relationship between fungi and sponges (Rot et al., 2006). Together with our results, the increasing evidence suggests that fungi may have a ‘‘true symbiotic’’ relationship with marine sponges. Clearly, further studies of fungi associated with marine sponges are needed to improve our current understanding of the nature of sponge–fungal association and phylogenetic diversity of sponge-associated fungal communities. Such efforts will contribute to exploration of novel secondary metabolites of sponge-derived fungi and to ecology of sponge–microbial symbionts.
Acknowledgments Special thanks go to Dr. Lifang Zhang for her assistance in constructing phylogenetic tree. We thank Diane Henderson for suggestions to greatly improve this manuscript and Emilie Lefait, Allison
Q. Li, G. Wang Fong, and Sang-Hwal Yoon for sponge sample collections. This work is funded by NOAA Grant NA04OAR4600196 and the University of Hawaii Sea Grant under institutional Grants NA05OAR4171048 and NA16RG2254. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies.
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www.elsevier.de/micres
Diversity of fungal isolates from three Hawaiian marine sponges Quanzi Li, Guangyi Wang Department of Oceanography, University of Hawaii at Manoa, Honolulu, HI, USA Received 28 February 2007; received in revised form 26 June 2007; accepted 3 July 2007
KEYWORDS Fungi; Marine sponges; Fungal diversity; Sponge–fungal association
Summary Sponges harbor diverse prokaryotic and eukaryotic microbes. However, the nature of sponge–fungal association and diversity of sponge-derived fungi have barely been addressed. In this study, the cultivation-dependent approach was applied to study fungal diversity in the Hawaiian sponges Gelliodes fibrosa, Haliclona caerulea, and Mycale armata. The cultivated fungal isolates were representatives of 8 taxonomic orders, belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. A portion of these isolates (n ¼ 15, 17%) were closely affiliated with fungal isolates isolated from other marine habitats; the rest of the isolates had affiliation with terrestrial fungal strains. Cultivated fungal isolates were classified into 3 groups: ‘sponge-generalists’—found in all sponge species, ‘sponge-associates’—found in more than one sponge species, and ‘sponge-specialists’—found only in one sponge species. Individuals of G. fibrosa collected at two different locations shared the same group of ‘sponge-specialists’. Also, representatives of 15 genera were identified for the first time in marine sponges. Large-scale phylogenetic analysis of sponge-derived fungi may provide critical information to distinguish between ‘resident fungi’ and ‘transient fungi’ in sponges as it has been done in other marine microbial groups. This is the first report of the host specificity analysis of culturable fungal communities in marine sponges. & 2007 Elsevier GmbH. All rights reserved.
Introduction Sponges are commonly known to harbor diverse prokaryotic and eukaryotic microbes (Hentschel Corresponding author. Tel.: +1 808 956 3744;
fax: +1 808 956 9225. E-mail address: [email protected] (G. Wang).
et al., 2006; Wang, 2006). Some efforts have been made to investigate structure and composition of prokaryotic microbial communities in marine sponges, using both cultivation-dependent and cultivation-independent approaches. These investigations have resulted in the identification of phylogenetically diverse, yet highly sponge-specific, prokaryotic microbial groups (for recent reviews,
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.07.002
ARTICLE IN PRESS 234 see Lee et al., 2001; Hentschel et al., 2003, 2006; Imhoff and Stoehr, 2003; Hill, 2004; Wang, 2006). Eukaryotic microbes such as zooxanthellae, zoochlorelae, dinoflagellates, and diatoms are reported to be associated with marine sponges (Saller, 1989; Rosell and Uriz, 1992; Bavestrello et al., 2000; Cerrano et al., 2000; Carballo and Avila, 2004; Webster et al., 2004). However, detailed studies of the diversity of eukaryotic communities living within sponges are rare. Filamentous fungi are ubiquitous and can be easily isolated from the inner tissue of sponges (Ko ¨nig et al., 2006; Wang, 2006). Nevertheless, most investigations of sponge-derived fungi have been focused on chemistry of natural products simply because sponge-derived marine fungi produce the highest number of novel marine fungal metabolites (Jensen and Fenical, 2002; Bugni and Ireland, 2004; Wang, 2006). In several cases, there is evidence from experiments that sponge-derived fungi are the true biosynthetic origin of spongederived secondary metabolites such as jasplakinolide (Jensen and Fenical, 2002). Hundreds of fungal strains have been isolated from marine sponges, representing three phyla of Ascomycota, Zygomycota, and mitosporic fungi (Ho ¨ller et al., 2000; Jensen and Fenical, 2002; Morrison-Gardiner, 2002; Bugni and Ireland, 2004). Diversity of fungi associated with marine sponges still remains an understudied area. So far, identification of fungi in marine sponges has been solely made using a morphology-based approach. Many microscopic fungi lack a sexual state (mitosporic or anamorphic), but can possess a surprisingly high level of genetic variation for a similar morphology (Kohn, 1995; Harrington and Rizzo, 1999; Talhinhas et al., 2002). In fact, the majority of fungi derived from marine sponges are mitosporic; about one-third of these fungi cannot be identified by using morphology-based approach (Morrison-Gardiner, 2002). The morphology-based approach, therefore, can be severely limited in identification of spongederived marine fungi. As of now, only the marine ascomycete of the genus Koralionastes is reported to have a unique physical association with crustaceous sponges (Kohlmeyer and Volkmannkohlmeyer, 1990). In addition, limited information is available on the ecological function of fungi living within marine sponges. Fungi have been reported to contribute to the localized lesions of sponges and thus may enhance disease development of sponges. Fungi are opportunistic, sometimes acting as secondary colonizers to other infections or stresses (Galstoff, 1942; Sparks, 1985; Vacelet et al., 1994). Overall, the diversity of fungal communities within sponges and the nature of
Q. Li, G. Wang sponge–fungal association remain largely unknown. Development of molecular genetics has provided a significant advancement in understanding diversity and function of microbes in diverse natural habitats. Phylogenetic taxonomy has been accepted as the appropriate way to analyze teleomorphic and anamorphic fungi and is becoming increasingly popular with mycologists (Taylor et al., 2000); hence, phylogenetic analysis can provide a better tool to assess fungal communities within sponges. In this study, we isolated fungi from three Hawaiian marine sponge species collected at two different locations on the island of Oahu, and carried out the phylogenetic analysis of their ITSrDNA sequences. Being the first isolated from marine sponges, many of these fungal isolates are valuable for further exploration of their biomedical potential. Thus, our data provide, for the first time, a look into the association nature of culturable fungal communities living within marine sponges.
Materials and methods Sampling sites and sponge collection Specimens of the marine sponges Gelliodes fibrosa, Haliclona caerulea and Mycale armata were collected snorkeling at Kuli’ou’ou Beach in Hawaii Kai (1571440 W, 211170 N) and on Coconut Island in Kane’ohe Bay (1571470 W, 211260 N), Oahu, Hawaii (Figure 1). The two sites are located 37 km apart. Three samples of each species were collected within a 20-m radius at depths of 0.5–3 m.
Oahu
Figure 1. Map of the main Hawaiian Islands and approximate locations of collection sites of marine sponges on Oahu (open circle and closed circle indicates the locations of Hawaii Kai and Coconut Island, respectively).
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges Latex gloves were worn during collection. Seawater samples from the collection sites were collected using 100-ml syringes to serve as controls. Specimens were transferred directly to Zip-lock bags containing seawater to prevent contact of sponge tissue with air. The samples were transported to the laboratory and processed immediately for the isolation and cultivation of fungi. Alternatively, sponge tissue was frozen in liquid nitrogen and then stored at 80 1C for future use.
Fungal isolation To get rid of nonspecific fungal propagules from seawater columns on sponge surfaces, sponge tissues were rinsed three times with sterile artificial seawater. The surface of the sample was disinfected with 70% ethanol. The inner tissue was taken out with a scalpel and forceps and then homogenized using a blender containing 20 ml sterile natural seawater in aseptic conditions. The resulting homogenate was diluted with sterile seawater at three dilutions (1:10, 1:100, and 1:1,000). For fungal cultivation, 100 ml of each dilution was plated in quadruplicate onto GPY plates (1.0 g glucose, 0.1 g yeast extract, 0.5 g peptone, 15 g agar, and seawater, pH 8.0), Marine agar (per liter Marine broth 2216 plus 15 g DifcoBacto agar), Gause I (starch 20 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4 7H2O 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, 15 g Difco-Bacto agar, pH 7.2-7.4), and Luria Bertani (LB) agar plates, containing the antibiotics penicillin and streptomycin (100 mg/ml each). The plates were incubated at room temperature (18–20 1C) for 1–3 weeks until the morphology of fungi could be distinguished. Each isolate was picked and transferred onto a new GPY, Marine Agar (Difco), Gause I, and LB agar plates containing streptomycin and penicillin. The resulting plates were incubated at room temperature for pure culture. All the above media were made with natural seawater.
Morphological and molecular analysis of cultivated fungi Morphological traits (e.g. morphology and color of spore and mycelia) were examined to exclude redundant fungal isolates. The rest of the isolates were cultured in GPY broth at 28 1C for 2–5 days. The mycelium was harvested using vacuum filtration and dried between two layers of paper towel. The resulting mycelial mat was ground into powder in liquid nitrogen. Fungal DNA was extracted using the procedure described by Fredricks et al. (2005)
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with modifications. In brief, the mycelial powder was transferred to a 1.5-ml Eppendorf tube containing 400–500 ml TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.3); equal volume of phenol solution was added to the tube. After brief mixing, the mixture was centrifuged at 12,000g for 10 min at 4 1C. The aqueous phase was transferred to a new microtube and sequentially extracted with phenol solution and chloroform. RNA was removed in the aqueous phase using RNaseA. The sample was then again extracted with phenol solution and chloroform. Finally, DNA was precipitated by adding two volumes of ethanol. The DNA pellet was washed with 75% ethanol and resuspended in 50–100 ml of sterile water. The resulting genomic DNA was used as a template to amplify fungal ITS-rDNA fragments using the primers ITS1 (50 -TCCGTAGGTGAACCTGCG30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) (White et al., 1990). The reaction mixture contained 5 ml of 10 reaction buffer with 15 mM MgCl2 (Promega), 2 ml of 2.5 mM dNTPs, 2 ml of 10 pmol forward primer, 2 ml of 10 pmol reverse primer, 1 ml of fungal DNA, 0.5 ml of Taq DNA polymerase (5 U ml1, Promega), and 32.5 ml of H2O. PCR conditions were as follows: initial denaturation (94 1C for 5 min); 30 cycles of denaturation (94 1C for 50 s), primer annealing (55 1C for 50 s), and elongation (72 1C for 1 min), with a final elongation at 72 1C for 10 min. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and used directly for sequencing analysis. Alternatively, if satisfactory sequencing results were not obtained from PCR products, PCR products were cloned into pGEM-T Easy Vector (Promega) and sequenced using the plasmid DNA with the universal plasmid primers T7 and SP6.
Sequence and phylogenetic analysis Sequencing analyses were performed on an ABI 3730 XL (Applied Biosystems) automated sequencer using ITS1 and ITS4 primers for PCR templates or universal plasmid primers (T3 and T7) for plasmid templates. Sequence data were edited with Chromas Lite, version 2 (Technelysium). For preliminary identifications, sequences of fungal rDNA-ITS regions were compared with those in the NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). Fungal ITS-rDNA sequences in this study and the matched sequences from GenBank were edited and aligned with Seq-Man and Megalign (DNASTAR Package). The aligned sequences were imported into PAUP 4.0b10 (Swofford, 2002). Neighbor-joining (NJ) trees were estimated using pairwise genetic distances based
ARTICLE IN PRESS 236 on the basis of all substitutions with the Jukes– Cantor distance parameter. For maximum-parsimony (MP) analyses, we used a heuristic search strategy. A strict consensus tree was drawn when multiple best trees were obtained. The quality of the branching patterns for MP and NJ was assessed by bootstrap resampling of the data sets with 1000 replications.
Nucleotide sequence accession number Fungal rDNA-ITS sequences obtained in this study were deposited in GenBank/EMBL/DDBJ under accession numbers DQ092514–DQ092533 and EF029806–EF029831.
Results Diversity of culturable fungi within sponges Cultivation of fungi from G. fibrosa, H. caerulea, and M. armata yielded a total of 235 isolates. Redundant isolates were excluded from further analysis using morphological characteristics. A total of 119 isolates was cultured for genomic DNA extraction and sequencing analysis. ITS-rDNA sequence analyses identified 86 independent isolates. These isolates matched diverse previously described fungi, and similarity of their ITS-rDNA sequences ranged from 66 to 99%. A portion of these isolates (n ¼ 15, 17%) were closely (498% sequence similarity) affiliated with fungal isolates from marine habitats, and the rest were related to terrestrial fungi. Some of these isolates (n ¼ 12, 14%) were affiliated with previously described fungal strains, which produce interesting fungal bioproducts (natural products and enzymes), the others (n ¼ 10, 12%) were closely affiliated (499% sequence similarity) with previously described pathogens (marine animal, human, and plant). Several of these isolates such as MACIP4 (identical to Lacazia loboi, a marine animal pathogen) were cultivated for the first time. Phylogenetic analysis revealed that fungal isolates from these 3 sponges clustered into 8 phylogenetic groups, corresponding to 8 taxonomic orders (Aphyllophorales, Diaporthales, Eurotiales, Hypocreales, Mycosphaerellales, Pleosporales, Saccharomycetales, and Xylariales) and belonging to at least 25 genera of Ascomycota and 1 of Basidiomycota. Figures 2 and 3 provide an overview of the phylogenetic relationship of sponge-associated culturable fungi while Figure 4 shows affiliations of individual genera with marine
Q. Li, G. Wang sponges collected at different locations. Isolates (n ¼ 24, 28%) of the Eurotiales were closely affiliated with 3 genera: Aspergillus, Eupenicillium and Penicillium. Isolates (n ¼ 23, 27%) of the Pleosporales had affiliations with 8 genera Ampelomyces, Bipolaris, Cochliobolus, Curvularia, Didymella, Leptosphaerulina, Phaeosphaeria, and Paraphaeosphaeria as well as 4 unclassified genera. Isolates (n ¼ 19, 22%) of the Hypcreales were representatives of 7 genera Bionectria, Fusarium, Hypocrea, Myrothecium, Solheimia, Trichoderma, and Tubercularia. Isolates (n ¼ 10, 12%) of the Mycosphaerellales were closely affiliated with 2 genera, Cladosporium and Lacazia. Isolates (n ¼ 4, 5%) of the Xylariales were related to 2 genera Bartalinia and Nigrospora as well as 1 unclassified genus. Isolates (n ¼ 3, 3%) of Aphyllophorales did not have affiliation with any certain genus, but were closely related to the fungal endophyte (AY456192) of Taxus mairei, a producer of the anticancer drug Taxol (Chang and Yang, 1996). One isolate of the Dothideales and the Saccharomycetales belonged to the genera Fusicoccum and Candida, respectively. Among the identified fungal genera, 15 were isolated for the first time from marine sponges. The other isolates associated with unclassified genera were potentially new fungal species. Overall, 16 genera were identified in the sponge G. fibrosa collected on Coconut Island while 6 genera were isolated from M. armata from the same location (Figure 4). G. fibrosa and H. caerulea collected in Hawaii Kai yielded 15 and 8 genera, respectively. G. fibrosa from a different location (Hawaii Kai and Coconut Island) gave a similar number of fungal genera or fungal diversity although samples of G. fribrosa from different locations did contain different genera. Finally, G. fibrosa gave overall the highest fungal diversity and M. armata the least diversity.
Association of culturable fungi with different marine sponges Association of cultivated fungal genera displayed interesting patterns (Figure 4). Three fungal genera Aspergillus, Penicillium, and Eupenicillium were found in all sponges and can be classified as ‘sponge-generalists’. Fungal genera such as Ampelomyces, Tubercularia, and Clasoprorium, which were identified in more than one sponges, can be called ‘sponge-associates’. The fungal genera such as Didymella, Fusicoccum, and Lacazia, which were found only in one sponge species, can be named as ‘sponge-specialists’. Individuals of G. fibrosa
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges
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99 MACIP4 Lacazia loboi AF035674 MACIG4 Cladosporium cucumerinum AF393696 Cladosporium oxysporum AF393720 MACIM2 100 MACIM3 Mycosphaerellales MACIP9 100 GFHK25 HCHK18 GFHK6 97 HCHK20 55 73 Cladosporium cladosporioides DQ335984 GFHK21 54 Cladosporium sphaerospermum DQ335985 HCHK9 Penicillium herquei AF033405 100 MACIP5 89 GFHK10 Eupenicillium hirayamae AF033418 Penicillium namyslowskii AF033463 MACIP6 99 MACIG2 66 GFHK13 67 Eupenicillium sp. DQ092516 87 Penicillium steckii DQ123666 94 100 GFHK14 Penicillium citrinum DQ123646 95 Penicillium roseopurpureum AF455492 MAHKP2 Penicillium griseofulvum AY373917 100 Penicillium crustosum AY373907 Penicillium commune AF455527 71 MACIB1 99 Penicillium griseoroseum AY425988 GFHK19 99 100 Penicillium olsonii DQ123662 100 GFCI23 Eurotiales GFCI24 GFCI25 100 Penicillium brevicompactum AY373897 100 HCHK1 57 Aspergillus ochraceus AY373854 58 HCHK13 100 Aspergillus flavipes AY822647 MACIP1 HCHK3 100 Aspergillus awamori DQ235784 GFCI31 GFCI28 100 Aspergillus tamarii AY373870 58 100 Aspergillus japonicus AJ876880 100 Aspergillus aculeatus U65309 GFCI27 MACIP7 Uncultured fungus clone G49 DQ279843 Uncultured fungus clone G7 DQ279847 MACIM4 MACIP3 64 Aspergillus nidulans AF455505 56 Aspergillus sydowii AM176727 100 Aspergillus versicolor AY37388 MACIP11 GFCI9 100 Fusicoccum mangiferum AY615187 Dothideales Candida parapsilosis AY227019 99
100
0.1 substitutions/site Figure 2. Neighbor-joining phylogenetic tree from analysis of ITS-rDNA (4540 bp) sequences from culturable fungal isolates clustering within the Mycosphaerellales, Eurotiales, and Dothideales. Numbers at branches indicate bootstrap values of neighbor-joining analysis (450%) from 1000 replicates. MA—Mycale armata; GF—Gelliodes fibrosa; HC—Haliclona caerulea; HK—Hawaii Kai; CI—Coconut Island; the other letter and number stand for isolate identification.
collected at 2 different locations shared 6 ‘spongespecialists’ genera. However, 3 genera (Bartalinia, Diaporthe, and Paraphaeosphaeria) and 1 unclassified genus isolated from G. fibrosa samples collected at Hawaii Kai were not present in specimens from Coconut Island. On the other hand, 6 genera (Curvularia, Bionectria, Bipolaria, Fuscicoccum, Nigrospora, and Solheimia) and 1 unclassified genus
cultivated from G. fibrosa collected on Coconut Island were not present in samples from Hawaii Kai either. The cause of this discrepancy needs to be further investigated. Also, different sponge species collected at the same location (e.g. H. caerulea vs G. fibrosa) or different location (e.g. M. armata vs H. caerulea) had their own unique ‘sponge-specialists’ genera. Seawater samples collected at the site of
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Q. Li, G. Wang GFHK4 Hypocrea lixii AY605745 GFCI11 GFHK12 Hypocrea lixii AF469189 Trichoderma inhamatum AF362101 Trichoderma aureoviride AF194015 GFCI10 GFCI16 65 Trichoderma inhamatum AF455502 GFHK40 96 GFHK41 Trichoderma tomentosum AY605718 100 GFCI12 100 GFCI2 70Trichoderma asperellum AY857244 Hypocreales 100 HCHK15 64 Bionectria ochroleuca AF106532 99 GFHK28 100 HCHK11 84 Tubercularia sp. AF422980 100 GFCI22 Solheimia costispora DQ135996 100 FFHK34 Myrothecium roridum AJ301995 56 9569 97 Myrothecium verrucaria AJ301999 GFCI5 GFHK3 Fusarium chlamydosporum AY213655 68 GFCI3 GFCI6 100 Fusarium incarnatum AY633745 GFHK5 100 GFHK37 Diaporthe phaseolorum AF001027 Diap orthales 54 100 GFHK35 Bartalinia sp. AY924288 84 55 GFCI1 84 Ascomycete sp. DQ480361 100 GFCI8 Nigrospora oryzae DQ219433 Xylariales GFHK25 100 HCHK18 Cladosporium oxysporum AF39372 100 GFHK33 50 Uncultured soil fungus DQ421204 100 Uncultured soil fungus DQ420984 67 Ampelomyces humuli AF035779 59 HCHK5 58 Didymella cucurbitacearum AY293804 GFHK1 100 HCHK22 100 HCHK8 HCHK7 56 HCHK23 98 100 Pleosporales sp. AB255299 HCHK14 100 Cochliobolus verruculosus AF071333 57 GFHK23 100 GFCI26 71 86 Curvularia affinis AF071335 100 GFCI35 100 GFCI30 Pleospor ales 51 Cochliobolus lunatus DQ836799 100 GFCI34 Bipolaris australiensis AJ853762 Phaeosphaeria oryzae AF439495 81 Fungal sp. AM231359 100 HCHK12 94 87 HCHK17 100 100 HCHK16 Leptosphaeriaceae sp. DQ342361 100 GFHK2 GFCI19 100 Foliar endophyte AY 566890 51 Uncultured ascomycete AY969713 84 62 Paraphaeosphaeria sp. AB096264 66 GFHK26 100 GFHK20 100 88 100 HCHK6 GFHK32 100 100 GFCI4 Leptosphaerulina chartarum AM231398 100 GFHK31 Fungal sp. AM231373 Fungal endophyte AY 456192 HCHK10 100 Aphyl lop horales GFHK30 98 HCHK4 MACIC19 100 Candida parapsilosis AY227019 Fusicoccum mangiferum AY615187
Saccharomyce tales
0.1 substitutions/site Figure 3. Neighbor-joining phylogenetic tree from analysis of ITS-rDNA (4540 bp) sequences from culturable fungal isolates clustering within the Hypocreales, Diaporthales, Xylariales, Pleosporales, Aphyllophorales, and Saccharomycetales. Numbers at branches indicate bootstrap values of neighbor-joining analysis (450%) from 1000 replicates. MA—Mycale armata; GF—Gelliodes fibrosa; HC—Haliclona caerulea; HK—Hawaii Kai; CI—Coconut Island; the other letter and number stand for isolate identification.
ARTICLE IN PRESS Diversity of fungal isolates from three Hawaiian marine sponges
Gelliode fibrosa (CI)
Bartalinia Diaporthe Paraphaeosphaeria
Cladosporium
Didymella Phaeosphaeria Haliclona caerulea (HK)
Cochliobolus Fusarium Hypocrea Myrothecium Leptosphaerulina Aspergillus Trichoderma Eupenicillium Penicillium
Ampelomyces, Tubercul aria
Gelliode fibrosa (HK)
Bionectria, Bipolaris Curvularia, Fusicoccum Nigrospora, Solheimia
Candida, Lacazia Mycale armata (CI)
Figure 4. Association of cultivated fungal genera with the Hawaii sponges collected on Coconut Island (CI) and in Hawaii Kai (HK).
Coconut Island yielded two fungal species Penicillium janthinellum and Penicillium chrysogenum and seawater samples from Hawaii Kai two fungal species Fusarium solani and P. chrysogenum. These three species were not present within sponges. Therefore, it seems that sponges harbor unique culturable fungal communities from the water column.
Discussion Marine fungi are still one of the most understudied marine ecological groups. Two groups of marine fungi have been classified on the basis of their ability to grow and to reproduce in seawater. Obligate marine fungi are those that grow and sporulate exclusively in a marine or estuarine habitat, while facultative marine fungi are those from freshwater or terrestrial milieus that are able to grow (and possibly sporulate) in the marine environments (Kohlmeyer and Kohlmeyer, 1979; Kohlmeyer and Volkmann-Kohlmeyer, 2003). In addition, facultative fungi could be those from terrestrial origins that have adapted to life in marine environments (Morrison-Gardiner, 2002). All sponge isolates of this study were able to grow on media made from natural seawater, and therefore can at least be classified as facultative fungi. Moreover, 15 isolates (17%), which were closely affiliated with marine fungal isolates, were likely obligate marine fungi. The other isolates (83%) were related to common genera to terrestrial habitats, suggesting that these isolates may be of terrestrial origin. Results of this study and two
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other investigations (Ho ¨ller et al., 2000; MorrisonGardiner, 2002) indicated that facultative fungi are frequently isolated from sponges at both nearshore and offshore locations. One important question to be addressed is whether any of these fungi simply result from terrestrial fungal spores that are trapped in sponge tissues during the filter-feeding process and remain dormant until being plated onto a suitable growth medium. But, fungal isolates, recovered in seawater samples of this study, were different from those in sponges. Also, if fungi in sponges are simply resulting from terrestrial fungal spores, sponge-derived fungi should have similar metabolic activity to their terrestrial counterparts. Nevertheless, many fungal isolates from marine sponges and other marine substrates have been shown to produce novel natural compounds that are not found in terrestrial strains (Jensen and Fenical, 2002; Proksch et al., 2003; Ko ¨nig et al., 2006). Therefore, sponge-derived fungi at least have their uniqueness of metabolic activities. Finally, before a new actinomycete taxon was identified from marine sediments through phylogenetic analysis, it was frequently assumed that actinomycetes isolated from marine samples are merely of terrestrial origin (Mincer et al., 2002). The similar scenario might hold for sponge-derived fungi. To our knowledge, this study represents the first phylogenetic study of fungi associated with marine sponges. Further large-scale phylogenetic analysis may provide solid molecular evidence regarding the difference between ‘resident fungi’ and ‘transient fungi’ in marine sponges. The availability of ITS-rDNA sequences of sponge-derived fungi in GenBank database is a major obstacle for such analyses. Fungi have commonly been isolated from marine sponges, but most studies have focused on novel metabolites of sponge-derived fungi (Jensen and Fenical, 2002; Ko ¨nig et al., 2006; Wang, 2006). Reports on fungal diversity in the ecological context of marine sponges as a habitat are scarce. In the survey of dominant filamentous fungi in Australian coral reefs, Morrison-Gardiner identified 208 isolates from 70 sponge samples using a morphology-based method (Morrison-Gardiner, 2002). Alternaria, Aspergillus, Cladosporium, Fusarium, and Penicillium were predominant fungal genera. Because sponge samples of different species collected at different locations were treated as a single source of fungal isolates and no sponge species information was given, it is difficulty to assess the fungal diversity in this study. In the second study of diversity and secondary metabolites from sponge-derived fungi, 681 fungal strains were isolated from 16 sponge species collected from the North Sea, the Mediterranean,
ARTICLE IN PRESS 240 the Caribbean and the Great Barrier Reef using the same identification approach (Ho ¨ller et al., 2000). The predominant fungal genera were ubiquitous ones: Acromonium, Arthrinium, Coniothyrium, Fusarium, Mucor, Penicillium, Phoma, Trichoderma, and Verticilium. Due to the different treatment of the sponge samples following sampling and before isolation of the fungi, the numbers of fungal isolates per sponge varied greatly between various locations. Sponges collected at two locations did not yield any fungal strain. In this study, the predominant genera were Penicillum (15%), Aspergillus (13%), and Trichoderma (11%). All 3 genera were also listed as the predominant ones in previous two studies, but the overall composition of fungal genera in Hawaiian sponges were quite different from that of the other two studies. This difference may reflect either the real difference of fungal populations living within different sponge species, the difference of different geographic locations, or the different media used in fungal isolation. One major reason for the differences may also be the different identification methods used in these studies. Whether sponge-derived fungi can be considered as symbionts of sponges or perform a specific function in this context is currently unknown. Increasing evidence appears to support a symbiotic nature of the association. An endosymbiotic yeast was identified in the sponges of the genus Chondrilla (Maldonado et al., 2005). In addition, the sponge Suberites domuncula was shown to have the ability to recognize fungi in their environments at the molecular level (Perovic-Ottstadt et al., 2004). Furthermore, the horizontal gene transfer of a mitochondrial intron from a fungus to sponges suggested a symbiotic relationship between fungi and sponges (Rot et al., 2006). Together with our results, the increasing evidence suggests that fungi may have a ‘‘true symbiotic’’ relationship with marine sponges. Clearly, further studies of fungi associated with marine sponges are needed to improve our current understanding of the nature of sponge–fungal association and phylogenetic diversity of sponge-associated fungal communities. Such efforts will contribute to exploration of novel secondary metabolites of sponge-derived fungi and to ecology of sponge–microbial symbionts.
Acknowledgments Special thanks go to Dr. Lifang Zhang for her assistance in constructing phylogenetic tree. We thank Diane Henderson for suggestions to greatly improve this manuscript and Emilie Lefait, Allison
Q. Li, G. Wang Fong, and Sang-Hwal Yoon for sponge sample collections. This work is funded by NOAA Grant NA04OAR4600196 and the University of Hawaii Sea Grant under institutional Grants NA05OAR4171048 and NA16RG2254. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies.
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