Fungal diversity in deep-sea sediments – the presence of novel fungal groups

fungal ecology 3 (2010) 316–325

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Fungal diversity in deep-sea sediments – the presence of novel fungal groups Yuriko NAGANO*, Takahiko NAGAHAMA, Yuji HATADA, Takuro NUNOURA, Hideto TAKAMI, Junichi MIYAZAKI, Ken TAKAI, Koki HORIKOSHI Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan

article info

abstract

Article history:

We investigated the diversity of fungal communities in 10 different deep-sea sediment

Received 23 August 2009

samples by PCR-mediated internal transcribed spacer (ITS) regions of rRNA gene clone

Revision received 8 January 2010

analysis. Although some of the amplified sequences were identified as common terrestrial

Accepted 14 January 2010

fungal species, the majority were novel sequences that were not closely related to previ-

Available online 24 April 2010

ously identified fungal ITS sequences in public databases. Phylogenetic analysis also

Corresponding editor: Felix Ba¨rlocher

suggested the novel phylogenetic affiliation of a group of predominant deep-sea phylotypes within the phylum Ascomycota. These phylotypes may be distributed widely in

Keywords:

global deep-sea sediments. Another novel phylotype was placed in deep branches within

Deep-sea sediments

the phylum Chytridiomycota with Rozella spp. as the closest related organisms. This study

Early evolution of fungi

revealed the possible existence of previously unknown fungal components in various

Extreme environments

depths and sites of deep-sea sedimentary habitats. It is suggested that deep-sea envi-

Fungal diversity

ronments are habitats hosting previously unexplored fungi, which may provide key insights into the early evolution of fungi and their ecological and physiological significance. ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction Deep-sea environments remain among the least explored regions of the earth. The deep-sea is recognized as an extreme environment, characterized by the absence of sunlight irradiation, predominantly low temperatures (occasionally extremely high, >400  C near hydrothermal vents) and high hydrostatic pressure (up to 110 MPa). Due to these extreme environmental conditions, the deep-sea has great potential to host a wealth of unique organisms. In particular, the discovery of hydrothermal vents, methane cold-seeps and surrounding ecosystems has resulted in completely new concepts for considering energy sources which are available for sustaining

life in deep oceans (Lonsdale 1977; Cavanaugh 1985; Grassle 1985). As with surface environments, diverse microorganisms are known to live in deep-sea environments. In contrast to a currently increasing knowledge of the phylogenetic diversity and ecophysiological importance of deep-sea bacteria and archaea (Jannasch & Mottl 1985; Prieur et al. 1995; Jorgensen & Boetius 2007), some of the most ecologically successful eukaryotic microorganisms, the fungi, have been poorly investigated. The isolation of deep-sea fungi was first reported from samples taken in the Atlantic Ocean at a depth of 4 450 m (Roth et al. 1964). Since then, there have been a few reports

* Corresponding author. Tel.: þ81 46 867 9662; fax: þ81 46 867 9645. E-mail address: [email protected] (Y. Nagano). 1754-5048/$ – see front matter ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2010.01.002

Fungal diversity in deep-sea sediments

noting the isolation of fungi, including novel species, in several deep-sea environments, for example, in the deepest sea, the Mariana Trench and hydrothermal vents (Takami et al. 1997; Gadanho & Sampaio 2005; Nagahama et al. 2006, 2008). Although the role and ecology of fungi in deep-sea environments have not yet been clarified, the barotolerance of culturable fungi retrieved from deep-sea sediments of the Indian Ocean has been reported (Raghukumar & Raghukumar 1998; Damare et al. 2006). Fungal growth under 20 MPa pressure was observed. It was suggested that fungi may be active in deep-sea extreme conditions. Due to the development of culture-independent molecular techniques, the presence of fungi has also been directly reported in deep-sea environmental DNA samples. However, universal PCR primers that amplify all eukaryotic species were employed in most of these studies, hence the emphasis was not primarily on the discovery of fungi (Lopez-Garcia et al. 2001, 2003, 2007; Edgcomb et al. 2002; Takishita et al. 2007). The reports on fungal communities detected by employing fungal-specific PCR primers were published only recently (Bass et al. 2007; Lai et al. 2007). Bass et al. (2007) employed a fungal-specific primer set targeted at the 18S rRNA gene, to investigate fungal diversity in water column and sediment samples from several deep-sea sites (depths ranged from 1 500 to 4 000 m). They demonstrated that the majority of sequences detected in samples branched close to known fungi found in surface environments. However, Bass et al. (2007) also revealed the existence of highly novel fungal sequences in environmental gene libraries of deep-sea environments, which had been detected in previous studies. Bass et al. (2007) noted that their methodology, which used a fungal-specific primer set targeting 18S rRNA gene, was not entirely fungal-specific. More than half of the clones sequenced were not fungal. Lai et al. (2007) employed a fungal-specific primer set targeting ITS regions of rRNA genes, for the detection of fungal communities from methane hydrate-bearing deep-sea marine sediments (depths ranged from 350 to 3 100 m). They also suggested the wide occurrence of previously unidentified fungal taxa in their samples. Although the use of fungal-specific primers revealed the presence of diverse fungi, including various unknown species, the distribution and diversity of fungal communities in deepsea environments are still largely unknown. In particular, deep-sea environments below 4 000 m have not yet been investigated. Interestingly, the highest fungal diversity was found in a sample from the deepest site in the study by Bass et al. (2007). In the present study, the diversity of fungi in 10 deep-sea sediment samples from several locations at depths ranging from 1 200 to 10 000 m, mainly below 7 000 m, was investigated by using three fungal-specific primer sets, targeting the ITS1–5.8S–ITS2–28S rRNA regions, in order to increase our knowledge of fungal communities in deep-sea ecosystems. Phylogenetic positions of detected unknown deep-sea fungi were examined by analyzing 5.8S rRNA gene sequences. The 5.8S rRNA region evolves relatively slowly and has been shown to contain considerable phylogenetic information, particularly in respect to deep basal branches (Hershkovitz & Lewis 1996).

317

Materials and methods Deep-sea sediment samples Deep-sea sediment samples were collected at five different sites (depths ranged from 1 200 to 10 000 m) off Japanese islands, including a sample from the deepest ocean depth, the Mariana Trench. Sediment samples were collected during KR07-17, KR07-04, KR06-15, NT01-11 and KR01-05 cruises, using the remotely operated vehicles (ROV) ABISMO, KAIKO 7000 II, KAIKO, as well as the manned submersible SHINKAI 2000. The details of the collected deep-sea sediment samples are described in Table 1 and Fig 1. Sampling sites, excluding site 4, were not associated with hydrothermal vents or coldseep sites where a large chemosynthetic assemblage was present. BO32 was collected from the bacterial mat at the periphery of the Calyptogena community at the Sagami Bay cold-seep site (site 4). Detailed information of site 4 was given by Fang et al. (2006). At site 1 (N1–N5) and site 2 (U2, U4), long sediments (110 cm and 30 cm, respectively) were collected for analysis and cut into layers at different depths from the surface of the sea floor. The sediment samples subjected to the direct extraction of DNA were kept at 80  C, until used.

DNA extraction and PCR amplification DNA was extracted from sediment samples by the employment of ISOIL beads beating kit (Nippon Gene, Japan), in accordance with the manufacturer’s instructions. Extracted DNA was stored at 20  C, prior to PCR amplification. For extractions, a negative extraction control containing all reagents minus sediment was performed. Genomic DNA was amplified with three different fungal-specific primer sets, targeting the 18S–ITS1–5.8S–ITS2–28S rRNA regions (Fig 2). For samples which were negative by primary PCR, a nested PCR was performed to improve the sensitivity of fungal detection. Primer set 1 employed ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 )/ ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) as a primary primer set and ITS3 (50 -GCATCGATGAAGAACGCAGC-30 )/ITS4 as a nested primer set (White et al. 1990). Primer set 2 employed ITS-1FS (50 -CTTGGTCATTTAGAGGAAGTAA-30 )/ITS4 as a primary primer set and ITS1/ITS4 as a nested primer set (Gardes & Bruns 1993). Primer set 3 employed ITS1/LR7 (50 -TACTACCACCAAGATCT-30 ) as a primary primer set and ITS1/LR5 (50 -TCCTGAGGGAAACTTCG-30 ) as a nested primer set (Vilgalys & Hester 1990). PCR reaction mixes (25 ml) contained: 20 mM Tris–HCl, pH 8.0, 100 mM KCl, 2.5 mM MgCl2, 200 mM dNTPs; 1.25 U of LA Taq DNA polymerase (TaKaRa, Japan), 0.2 mM (each) of a pair of primers and 1–2 ml of DNA template (10–100 ng). For the nested PCR, 0.5 ml of primary PCR products were used as a DNA template. The reaction mixtures following a ‘‘hot start’’ were subjected to the following thermal cycling parameters in a GeneAmp PCR system 9700 (Applied Biosystems): 94  C for 5 min followed by 30 cycles of 94  C for 30 s, 55  C for 30 s, 72  C for 30 s (for primer set 3, 72  C for 1 min), followed by a final extension at 72  C for 7 min. The reaction mixtures lacking template DNA in parallel were performed as a negative control, with appropriate DNA templates from Saccharomyces cerevisiae

Methane cold-seep, red bacterial mat

Cloning and sequencing All amplicons were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, U.S.A.) and eluted in molecular grade water, particularly to remove dNTPs, polymerases, salts and primers. The purified DNA fragments were cloned using the QIAGEN PCR cloningplus kit (QIAGEN Ltd., Japan), in accordance with the manufacturer’s instructions. Clones were screened according to a-complementation on Luria– Bertani agar medium containing 100 mg ampicillin, 80 mg 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) and 50 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Colony PCR was performed with M13 forward (50 -GTAAAAC GACGGCCAGTG-30 ) and M13 reverse (50 -AAACAGCTAT GACCATG-30 ) primer to amplify inserted DNA. PCR reaction mixes (25 ml) contained: 20 mM Tris–HCl, pH 8.0, 100 mM KCl, 2.5 mM MgCl2, 200 mM dNTPs; 1.25 U of Taq DNA polymerase (TaKaRa, Japan), 0.2 mM (each) of the M13F/M13R and a pick of colonies as a DNA template. The PCR conditions were presented in a GeneAmp PCR system 9700 (Applied Biosystems) as follows: following a ‘‘hot start’’, 94  C for 5 min followed by 30 cycles of 94  C for 30 s, 55  C for 30 s, 72  C for 30 s, followed by a final extension at 72  C for 7 min. All positive PCR amplicons were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, U.S.A.) and were eluted in molecular grade water prior to sequencing. Amplicons were sequenced in the forward direction employing the ITS1 primer employing the DYEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare, U.K.) on an automated DNA analyzer, Megabase 1000 (Amersham Bioscience, U.S.A.).

142 480 4000 E

9 774

KR01-05

KAIKO

Gravity core sampler Gravity core sampler Gravity core sampler Gravity core sampler Gravity core sampler Push corer Push corer Mini multiple corer Sterilized sediment samplera Sterilized sediment sampler ABISMO ABISMO ABISMO ABISMO ABISMO KAIKO 7000 II KAIKO 7000 II KAIKO SHINKAI 2000 KR07-17 KR07-17 KR07-17 KR07-17 KR07-17 KR07-04 KR07-04 KR06-15 NT01-11 9 760 9 760 9 760 9 760 9 760 7 111 7 111 10 131 1 174 E E E E E 5400 E 5400 E 7700 E 48400 E 490 490 490 490 490 520 520 330 130 142 142 142 142 142 141 141 142 139

Mud sampler Sampling vehicles Cruise no. Depth (m) Longitude

included as a positive control. The amplified products were visualized on 1.0 % [w/v] agarose gel electrophoresis.

29 100 0100 N

a Ikemoto & Kyo 1993. b cmbsf: centimeters below sea floor.

2001.4.3 BO97

5

Izu-Ogasawara Trench

N N N N N 7200 N 7200 N 3600 N 17300 N 090 090 090 090 090 470 470 240 000 29 29 29 29 29 32 32 11 35 Izu-Ogasawara Trench Izu-Ogasawara Trench Izu-Ogasawara Trench Izu-Ogasawara Trench Izu-Ogasawara Trench Izu-Ogasawara Trench Izu-Ogasawara Trench Mariana Trench Sagami Bay 2007.12.9 2007.12.9 2007.12.9 2007.12.9 2007.12.9 2007.4.11 2007.4.11 2006.12.10 2001.12.3 1 1 1 1 1 2 2 3 4 N1 N2 N3 N4 N5 U2 U4 MT BO32

Date of sampling Sampling site

Locality area

Latitude

Phylogenetic analyses

Sample name

Table 1 – Details of the deep-sea sediment samples examined in this study

0–10 cmbsfb 10–20 cmbsf 30–40 cmbsf 60–70 cmbsf 100–110 cmbsf 10 cmbsf 20 cmbsf

Y. Nagano et al.

Remarks

318

Resulting sequences were analyzed using the rRNA Database Project CHECK_CHIMERA program, to detect potential chimeric gene artefacts (Maidak et al. 2001). All confirmed sequences were compared with sequences stored in the GenBank database using the FASTA alignment software (http://fasta.ddbj.nig.ac.jp). All sequences were subsequently submitted to DDBJ and accession numbers were issued (AB507819–AB507861). The 5.8S rRNA gene coding region of all successfully obtained sequences was aligned with a comprehensive sampling of known fungal sequences (roughly based on Berbee & Taylor 2001), including the sequences with close FASTA hits for our deep-sea sequences by using the CLUSTALX program. Alignment positions that were hypervariable or high in insertions/deletions, were removed from all sequences in the alignment. Phylogenetic analyses were conducted using the neighbour-joining method and also the RAxML algorithm, with 100 bootstrap replicates with the optimal model implemented in TOPALi package ver. 2.5 (Milne et al. 2009).

Results Fungal DNA was detected in 9 out of 10 deep-sea sediment samples (N1, N2, N3, N4, N5, U2, U4, BO23 and BO97) with

Fungal diversity in deep-sea sediments

319

Fig 1 – Location of the deep-sea sediment sampling sites: 1: N1–N5 (Izu-Ogasawara Trench); 2: U2, U4 (Izu-Ogasawara Trench); 3: MT (Mariana Trench); 4: BO32 (Sagami Bay); 5: BO97 (Izu-Ogasawara Trench).

fungal-specific PCR primer sets 1 and 2, and only in 6 out of 10 samples (N1, N5, U2, U4, BO23 and BO97) with primer set 3, which targeted a longer region, including the 28S rRNA gene. In total, fungi were detected in 9 out of 10 deep-sea sediment samples. Fungi were not detected in the sample collected from the Mariana Trench (MT). This sample predominantly comprised rough sand, whereas other samples, were silty clay-like sediments. Clone libraries were constructed for the full total of 24 resulting fragments by PCR, with 2 304 clones selected randomly and sequenced. To remove identical sequences that likely originated from the same organisms in clone libraries, operational taxonomic units (OTUs) were defined by the

difference of >99 % sequence similarity. Thus, 2 304 sequences were reduced to a total of 43 unique sequences (DSF1-43, AB507819–AB507861). Rarefaction curves were constructed for ITS clone libraries from each of the 9 sampling sites (Fig 3). Rarefaction curves of sampling sites N2, N3, N4, N5, U2, U4 and BO97 showed a plateau, which indicated that these libraries covered almost all phylotypes. The numbers of unique clones obtained from these sampling sites were 1–4, which suggested a low fungal diversity in these sites. Sampling sites N1 and BO32 contained a higher number of OTUs than other sites and the rarefaction curves of these two sites did not reach a plateau. It is likely that the fungal diversity of these sites is higher than what was detected in

PCR product with primer set 1

PCR product with primer set 2

PCR product with primer set 3 ITS-F1 ITS1

ITS3

ITS1 18S rRNA gene

ITS4

LR5

LR7

ITS2 5.8S rRNA gene

28S rRNA gene

Fig 2 – Description and location of three different primer sets used in this study.

320

Y. Nagano et al.

14

Number of OTUs

12 N1 N2, U2, U4 N3 N4 N5 BO32 BO97

10 8 6 4 2 0

0

20

40

60

80

100

Number of clones Fig 3 – Rarefaction curves constructed for ITS clone libraries from each of the 9 sampling sites.

this study. 17 OTUs were amplified with both primer sets 1 and 2 (Fig 4). 8 OTUs were amplified with all three different primer sets. 12 OTUs were amplified with only primer set 3. Primer sets 1 and 2 detected similar subsets of the fungal community. Primer set 3 detected different subsets of the fungal community than primer sets 1 and 2. There were no significant differences (P > 0.05) in fungal diversity with respect to the depths of sampling sites. After comparing the different layers of sediment, it appeared that the upper layers of core sediments contained more diverse fungi. The methane cold-seep site (BO32) also harboured the most diverse fungi, including many highly novel species. The majority of amplified ITS sequences (34 out of 43) showed a very low association with known fungal sequences in the public database (Table 2), though some common fungal

Primer set 1

Primer set 2

17

3 0

8

3

0

12 Primer set 3 Fig 4 – Venn diagram showing the number of OTUs that were detected simultaneously by two or three of the primer sets and number of OTUs amplified exclusively with a single primer set.

species in surface environments, such as Penicillium (DSF22, 43), Aspergillus (DSF27, 31, 42), Trichosporon (DSF17) and Candida (DSF20, 33), were identified. Phylogenetic analysis, based on the 5.8S rRNA gene located within the ITS, was performed to estimate the phylogenic position of unknown novel fungal sequences (Fig 5). DSF13, 14 and 32 were removed from the phylogenetic tree analysis, as they were only amplified with primer set 1 and could not provide sufficient information of 5.8S rRNA sequences. As a result of phylogenetic analysis, most of the unknown sequences were grouped into phylum Ascomycota. DSF27, 31 and 42 were suggested as novel Ascomycota spp. closely related to Aspergilllus penicillioides (EF652037), with 94 % identity. Aspergillus and Penicillium appear to be ubiquitous in deep-sea environments. DSF39 was a highly novel sequence and appeared to be another novel species in Ascomycota, which was placed within the subphylum of Pezizomycotina. DSF20 and 33 were identified as Candida parapsilosis with 99 % and 100 % identity. C. parapsilosis was detected in two of the locations examined in this study. This opportunistic human pathogen had been isolated previously from water samples collected in hydrothermal fields as well (Gadanho & Sampaio 2005). A highly novel group (DSF-Group1) that was detected in almost all fungus-positive sediment samples (N1, N2, N3, N4, N5, U2, U4, BO32) belonged to phylum Ascomycota. Although this group did not have a closely related, previously identified fungal species, the uncultured fungus clone (DQ279844) detected from methane hydrate-bearing deep-sea sediments in South China Sea (Lai et al. 2007), showed high similarity to DSF-Group1. Phylogenetic analysis based on the 5.8S rRNA gene suggested the affiliation of DSF-Group1 to the genera Candida and Metschnikowia, having Candida alocasiicola (EU284099) as the most closely related identified organism. In addition, DSF-Group1 had a remarkably short ITS region. The deletion was also seen in the 5.8S rRNA gene. PCR amplicons of the DSF-Group1 with ITS1 and ITS4 primers were around 330 bp, while others were between 500 and 650 bp. Two sequences (DSF17 and 34) were placed in phylum Basidiomycota. DSF17 was identified as Trichosporon sp. with 99 % identity. DSF34 had a highly novel ITS sequence, which showed the most similarity (84 %) to Cryptococcus skinneri (AF444305) by FASTA search analysis. However, the 5.8S rRNA gene sequence of DSF34 showed a high similarity to many species, such as Cryptococcus spp. and Tremella spp., within the order Tremellales. One highly novel phylotype (DSF23) was affiliated to phylum Chytridiomycota. Interestingly, it was grouped with Rozella spp., which parasitize other members of the phylum and suggested to be on the earliest diverging branch of the fungal phylogenetic tree (James et al. 2006). Phylogenetic analysis showed that some of the highly novel sequences detected in this study may not be included in Kingdom Fungi. The phylogenetic positions of DSF26 and 29 remained unclear due to their highly novel sequences and also the lack of molecular information of other related microeukaryotes. DSF6, 8, 9, 11, 37 and 40 were related to species found within Kingdom Rhizaria. However, their phylogenetic position also remained unclear, as there are no known organisms with similar sequences. DSF7, 10, 12, 36, 38 and 41 were excluded from the phylogenetic tree analysis. As these

Sample name

Locality area

Primer set

N1

Izu-Ogasawara Trench

1, 1, 1, 1, 1, 1, 3 3 3 3 3 3

N2

Izu-Ogasawara Trench

N3

2 2, 3 2 2 2, 3 2

Clone name

Fungal species with most similar sequence

Number of bases analyzed

Percentage (%) identity

Query coverage

Submitted DDBJ accession no.

DSF1 DSF2 DSF3 DSF4 DSF5 DSF6 DSF7 DSF8 DSF9 DSF10 DSF11 DSF12

Glomerella lagenaria (AJ301970) Metschnikowia colocasiae (EU143309) Metschnikowia continentalis (DQ641238) Metschnikowia continentalis (DQ641238) Metschnikowia kamakouana (EU143312) – – – – Rhodotorula laryngis (AB078500) – –

Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota – – – – Basidiomycota – –

512 297 299 272 291 616 561 602 580 561 621 643

99 % 70 % 72 % 73 % 71 % – – – – 62 % – –

100 % 100 % 100 % 100 % 100 % – – – – 84 % – –

AB507819 AB507820 AB507821 AB507822 AB507823 AB507824 AB507825 AB507826 AB507827 AB507828 AB507829 AB507830

1 1 1, 2 1, 2

DSF13 DSF14 DSF15 DSF16

Rhizophlyctis rosea (AY997078) – Metschnikowia kamakouana (EU143312) Metschnikowia kamakouana (EU143312)

Chytridiomycota – Ascomycota Ascomycota

404 360 323 310

95 % – 67 % 70 %

84 % – 100 % 100 %

AB507831 AB507832 AB507833 AB507834

Izu-Ogasawara Trench

1, 2 1, 2

DSF17 DSF18

Trichosporon mucoides (FJ515196) Metschnikowia colocasiae (EU143309)

Basidiomycota Ascomycota

497 325

99 % 69 %

100 % 100 %

AB507835 AB507836

N4

Izu-Ogasawara Trench

1, 2 1, 2

DSF19 DSF20

Metschnikowia colocasiae (EU143309) Candida parapsilosis (EU552501)

Ascomycota Ascomycota

327 471

67 % 99 %

100 % 100 %

AB507837 AB507838

N5

Izu-Ogasawara Trench

1, 2 1, 2, 3 2

DSF21 DSF22 DSF23

Metschnikowia colocasiae (EU143309) Penicillium chrysogenum (AF034450) –

Ascomycota Ascomycota –

319 554 688

69 % 99 % –

100 % 100 % –

AB507839 AB507840 AB507841

U2

Izu-Ogasawara Trench

1, 2, 3 1, 2 3 1, 2

DSF24 DSF25 DSF26 DSF27

Metschnikowia kamakouana (EU143312) Metschnikowia kamakouana (EU143312) – Aspergillus penicillioides (EF652037)

Ascomycota Ascomycota – Ascomycota

328 326 1 003 605

67 % 69 % – 94 %

100 % 100 % – 100 %

AB507842 AB507843 AB507844 AB507845

U4

Izu-Ogasawara Trench

1, 2, 3 3 1, 2, 3 1, 2 1

DSF28 DSF29 DSF30 DSF31 DSF32

Metschnikowia colocasiae (EU143309) – Metschnikowia kamakouana (EU143312) Aspergillus penicillioides (EF652037) –

Ascomycota – Ascomycota Ascomycota –

642 557 322 568 353

74 % – 68 % 95 % –

100 % – 100 % 100 % –

AB507846 AB507847 AB507848 AB507849 AB507850

BO32

Sagami Bay

1, 2, 3 1, 2 1, 2 3 3

DSF33 DSF34 DSF35 DSF36 DSF37

Candida parapsilosis (EU552501) Cryptococcus skinneri (AF444305) Metschnikowia colocasiae (EU143309) Acaulospora laevis (FM876782) –

Ascomycota Basidiomycota Ascomycota Glomeromycota –

488 482 320 639 602

100 % 84 % 69 % 62 % –

100 % 92 % 100 % 87 % –

AB507851 AB507852 AB507853 AB507854 AB507855

Fungal diversity in deep-sea sediments

Table 2 – Sequence-based identification of detected clones from each deep-sea sediment sample

(continued on next page)

321

–: No similar known organisms. Clones that showed less than 90 % similarity to known fungal sequences are highlighted in dark gray. Only fungal species, which have the most similar sequence to unknown clones detected in this study with more than 80 % query coverage by FASTA search are listed.

AB507861 100 % 99 % Izu-Ogasawara Trench BO97

1, 2, 3

DSF43

Penicillium corylophilum (AY373906)

Ascomycota

560

88 % 100 % 89 % 95 % 100 % 62 % 74 % 64 % 61 % 94 % 3 2 3 2 1, 2

DSF38 DSF39 DSF40 DSF41 DSF42

Acaulospora laevis (FM876787) Golovinomyces cichoracearum (AB077674) Typhula ishikariensis (AF193368) Nematoctonus concurrens (EF409724) Aspergillus penicillioides (EF652037)

Glomeromycota Ascomycota Basidiomycota Basidiomycota Ascomycota

630 443 639 654 574

Query coverage Percentage (%) identity Number of bases analyzed Fungal species with most similar sequence Clone name Primer set Locality area Sample name

Table 2 – (continued).

AB507856 AB507857 AB507858 AB507859 AB507860

Y. Nagano et al.

Submitted DDBJ accession no.

322

sequences were especially highly novel, it was difficult to determine whether they are fungal in origin or from other eukaryotes. The analysis failed to yield reliable phylogenetic trees. These unanalyzed highly novel sequences were amplified mostly with primer set 3 and showed a lower similarity to fungi, compared to the sequences detected with the other two primer sets. This is probably due to the lack of fungalspecificity in LR5 and LR7 primers. Although it was difficult to determine their true position in the phylogenetic tree of life, due to the highly novel sequences and also the lack of molecular information of other eukaryotic organisms, it can be suggested that these sequences consist of novel branches and possibly, a novel eukaryotic lineage.

Discussion Fungi are one of the most ecologically successful eukaryotic lineages and occupy a wide variety of niches, by virtue of their highly versatile physiological adaptations. This study suggested that deep-sea environments, where extreme conditions are present, may be no exception. This study revealed the possible existence of fungi, including various previously unknown fungal components, in deep-sea sedimentary habitats, even at 10 000 m below sea level. Although it is hypothesized that extracted eukaryotic DNA from deep-sea sediments may be derived from fossilized DNA (Jorgensen & Boetius 2007), fungi have repeatedly been detected by researchers in deepsea environments by both conventional and molecular methods (Takami et al. 1997; Nagahama et al. 2003; Raghukumar et al. 2004; Damare et al. 2006; Bass et al. 2007; Lai et al. 2007; Burgaud et al. 2009; Le Calvez et al. 2009). It is appropriate to recognize that diverse fungi are present in deepsea environments and may play an important role in deep-sea ecosystems, just as with other environments. This study also suggested that a wide variety of fungi are present in deep-sea sediments not associated with hydrothermal vents or cold-seep sites, where high species diversity is present. Although there was no significant correlation between the detected fungal diversity and examined sites and depths, as with prokaryotes (Takami et al. 1999), highly novel sequences were most frequently detected in the methane cold-seep site (site 4, BO32). This site harbors the large vesicomyid clam, the Calyptogena soyoae community. It has been reported that mussels from deep-sea hydrothermal vents in Fiji Basin are infected with a black yeast, which is causing massive mussel mortality (Van Dover et al. 2007). The highly novel fungal phylotypes detected from the site at C. soyoae community, may also have a relationship with these unique deep-sea animals. In addition, it has been previously reported that eukaryotic organisms in deep-sea environments tend to be parasite-related phylotypes (Lopez-Garcia et al. 2003). This study also suggested that some phylotypes detected were related to parasites such as Metschnikowia spp. and Rozella spp. To obtain a better understanding of the ecology of fungi in deep-sea environments, the investigation of interactions between fungi and other organisms, such as deep-sea animals, protists and bacteria, will be an important next step. An interesting symbiosis may exist.

Fungal diversity in deep-sea sediments

323

Fig 5 – Phylogenetic tree for 5.8 rRNA sequences of environmental fungal clones from deep-sea sediments and appropriate fungal species in public databases. The fungal clones detected in this study are indicated in bold. The phylogenetic tree was constructed using the RAxML algorithm. Numbers at nodes are bootstrap indices of support (respectively ML/NJ), and only branches with bootstrap values above 50 % from an analysis of 100 bootstrap replicates are indicated. Highly novel sequences which showed low similarity to known fungal species by FASTA search, are marked with a star. Novel sequences which showed more than 90 % similarity to known fungal species are marked with a triangle.

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One of the most interesting findings of this study was the discovery of the highly novel fungal phylotypes, DSF-Group1, which was frequently observed in deep-sea sediments examined. Further investigation was carried out on this group and partial sequences of 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA were determined (AB514601). BLASTN searches for 18S rRNA sequences of DSF-Group1 revealed that the same/ similar sequences had also been detected from the deep-sea methane cold-seep of Sagami Bay, Japan at a depth of 1 170 m, as well as from anoxic white bacterial mat in the Gulf of California at a depth of 1 575 m (Takishita et al. 2007; Bass et al. 2007). It seems that DSF-Group1 is globally distributed in deep-sea environments. The detection of this group has not been reported in any other environment, except one from small zooplankton, Daphnia species in European lakes (Wolinska et al. 2009). This fungus, described as a Daphnia parasite, was first identified as Metschnikowia bicuspidate by morphology but the molecular analysis showed that it clearly differed from known Metschnikowia spp. and revealed high similarity to the sequences obtained from deep-sea environments. This report suggested a possibility of DSF-Group1 as a parasitic fungus of planktonic animals living in deep-sea environments. As DSF-Group1 has been detected from several oxygen-depleted deep-sea environments, such as methane cold-seep, anoxic bacterial mat and below the sea floor, it can be considered that they are anaerobic or facultative anaerobic fungi. The detection of this phylotype through anaerobic cultivation by using molecular methods has also been reported (Takishita et al. 2007). Another interesting finding was that deep-sea sediments at depths that reach 10 000 m harbor the highly novel fungal phylotype found within deep branching Chytridiomycota, which is related to the possible earliest diverging fungal lineage. The detection of novel fungal phylotypes related to Rozella spp. has also been reported from ‘shallow’ deep-sea environments (<1 500 m) in a previous study (Bass et al. 2007). As deep-sea sedimentary environments have the potential to harbor organisms that may be relevant to the early evolution of eukaryotes (Lai et al. 2007), the presence of a novel phylotype in deep-sea environments related to Rozella may be highly relevant. However, the isolation of Chytridiomycota from deep-sea environments by conventional methods has not yet been reported. Further investigation on these organisms is essential for understanding the evolutionary history of fungi, which may lead to the origin of fungi. As in other previous reports, some known fungi from terrestrial surfaces were also detected in deep-sea sediments in this study. As these Ascomycota are ubiquitous in diverse environments, there is always the possibility that these organisms originate from sunken cells, which are not indigenous to the environment, or brought in by contamination and are not from the samples examined. Since it is very difficult to definitely exclude these contaminations, a better understanding of the ecological role and importance of deep-sea derived fungi is essential. We note that some of the sequences detected in this study were very unusual and not represented in the database. Therefore, using the 5.8S rRNA regions data only for determining their phylogenetic position may not be satisfactory. Although targeting the 5.8S rRNA regions for PCR analysis

Y. Nagano et al.

showed an advantage in detecting fungal DNAs in deep-sea sediments, additional phylogenetic analysis based on other genes, such as 18S rRNA and 28S rRNA, is needed for more rigorous results. In addition, it is demonstrated that different fungal-specific primer sets detect different subsets of the fungal community in this study. We therefore conclude that using multi-primer sets for investigating the fungal diversity in environments is essential to understand their true diversity. In conclusion, this study suggests that a very broadly based variety of fungi, including previously undocumented groups, are present even in deep-sea environments at 10 000 m below the surface. This study also suggests the existence of highly novel fungi, which are widely distributed in deep-sea environments, as well as the possible existence of fungi that may be associated with the early evolution of fungi. Further investigation is needed to unequivocally document the existence of fungi in deep-sea environments convincingly and to understand their true abundance and role in ecosystems. We believe this will lead to a better understanding of deep-sea ecosystems, the phylogenetic history of fungi, and their mechanism of adaption to extreme environments.

Acknowledgement We thank everyone who was involved in KR07-17, KR07-04, KR06-15, NT01-11 and KR01-05 cruises for their great help in collecting the deep-sea sediment samples. We would like to give a special mention to the ABISMO development team, led by Dr. Hiroshi Yoshida and also people who were involved in test dives and cruises of the ROV ABISMO for their dedicated efforts. We also thank Prof. John E Moore and Mr. Robert Collins for valuable comments on the manuscript. This work was supported by Grant-in-Aid for Young Scientists, Japan Society for the Promotion of Science (No. 20870044).

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