Fungal diversity in deep-sea extreme environments

f u n g a l e c o l o g y 5 ( 2 0 1 2 ) 4 6 3 e4 7 1

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/funeco

Mini-review

Fungal diversity in deep-sea extreme environments Yuriko NAGANOa,*, Takahiko NAGAHAMAa,b a

Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan b Department of Food and Nutrition, Higashi-Chikushi Junior College, 5-1-1 Shimoitozu, Kokurakita-ku, Kitakyusyu, Fukuoka 800-0351, Japan

article info

abstract

Article history:

The deep-sea is one of the most mysterious and unexplored extreme environments,

Received 12 July 2011

holding great potential and interest for science. Despite extensive studies on deep-sea

Revision received 16 November 2011

prokaryotes, the diversity of fungi, one of the most ecologically important groups of

Accepted 18 December 2012

eukaryotic micro-organisms, remains largely unknown. However, the presence of fungi in

Available online 23 February 2012

these ecosystems is starting to be recognised. Many fungi have been isolated by culture-

Corresponding editors:

dependent methods from various deep-sea environments, with the majority showing

Kevin K. Newsham & Lynne Boddy

similarity to terrestrial species. However, culture-independent methods have revealed many novel fungal phylotypes, including novel fungal lineages recently described as

Keywords:

Cryptomycota, which are suspected to lack typical fungal chitin-rich cell walls. Although

Cryptomycota

true fungal diversity and its role in deep-sea environments is still unclear, the intention of

Deep-sea

this review is to assess current knowledge of the diversity of fungi in these ecosystems and

Extreme environments

to suggest future direction for deep-sea fungal research.

Fungal diversity

ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved.

Zoosporic fungi

Introduction The deep-sea is recognised as an extreme environment. It is characterised by the absence of sunlight, predominantly low temperatures (<4
C but occasionally >400
C close to hydrothermal vents) and high hydrostatic pressure (up to 110 MPa). The deep-sea normally refers to oceans greater than 200 m depth. With nearly three quarters of the Earth’s surface area being covered by ocean, the average depth of which is 3 800 m, the vast majority of our planet thus comprises deep-sea environments. Although once thought to be an uninhabitable milieu owing to its extreme conditions, the deep-sea environment is now recognised as highly dynamic, hosting

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 available for sustaining life in deep oceans (Lonsdale 1977; Cavanaugh 1985; Grassle 1985). As in other environments, micro-organisms play an important role in deep-sea ecosystems. Since the first foray into deep-sea research, with the development of advanced instrumentation for sampling and researching life at great depths, the presence and ecological importance of deep-sea bacteria and Archaea has been extensively researched (Horikoshi 1998; Jørgensen & Boetius 2007; Dubilier et al. 2008; Lauro & Bartlett 2008; Dekas et al. 2009; Takai & Nakamura

* Corresponding author. Tel.: þ81 46 867 9662; fax: þ81 46 867 9645. E-mail address: [email protected] (Y. Nagano). 1754-5048/$ e see front matter ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2012.01.004

464

Y. Nagano, T. Nagahama

2011). In contrast, fungi, one of the most extremotolerant and ecologically important groups of micro-organisms, have been relatively underexplored in deep-sea environments. Although their true abundance and importance within these ecosystems is not yet understood, this review presents recent knowledge of fungal diversity in the deep-sea.

Sauvadet et al. 2010; Singh et al. 2011; Eloe et al. 2010; Quaiser et al. 2011; Nagahama et al. 2011). The taxonomic distribution of fungal diversity in deep-sea environments from recent studies is shown in Table 1, and the sample details and methods in Table 2. Fungi reported from deep-sea environments mostly belong to the Phylum Ascomycota, with a few yeast species belonging to the Basidiomycota. At present, there are no reports of zygomycetes and Chytridiomycota having been isolated from deep-sea environments. Since zygomycetes have not been detected by culture-independent methods either, it appears likely that these fungi are very rare or non-existent in deep-sea environments. However, this cannot be concluded with any certainty, as the primers that have been used may not amplify zygomycetes from deep-sea environments, and culturing methods may not be appropriate. For instance, we have isolated a novel psychrophilic fungus whose optimum growth temperature is <4
C. Observations indicate that this fungus is most closely related to the zygomycete Mucor hiemalis (unpublished data). Chytridiomycota have been detected as one of the major fungal components in several deep-sea environments, such as hydrothermal vents and methane cold-seeps, but only by culture-independent methods (Table 1).

Fungal diversity in deep-sea environments Although the presence of fungi in deep-sea environments was not well recognised until very recently, the isolation of deepsea fungi was first reported approximately 50 yr ago from the Atlantic Ocean at a depth of 4450 m (Roth et al. 1964). Since this first report, others have been published on the isolation of fungi, including novel species (mostly yeasts) from several deep-sea environments, e.g. hydrothermal vents and the deepest of the seas, the Mariana Trench (Takami et al. 1997; Gadanho & Sampaio 2005; Nagahama et al. 2006, 2008). Although the true role and diversity of deep-sea fungi remains largely unclear, the significance of fungi in deep-sea environments is starting to be recognised, with more intensive investigations in recent years. Fungal diversity in deep-sea environments has been investigated by both conventional culture-dependent methods (Nagahama et al. 2001; Raghukumar et al. 2004; Gadanho & Sampaio 2005; Damare et al. 2006; Le Calvez et al. 2009; Burgaud et al. 2009; Connell et al. 2009; Jebaraj et al. 2010; Singh et al. 2010) and culture-independent methods (Bass et al. 2007; Lopez-Garcia et al. 2007; Lai et al. 2007; Le Calvez et al. 2009; Jebaraj et al. 2010; Nagano et al. 2010;

Ascomycota Eurotiomycetes are the most frequently detected fungal taxa from deep-sea environments within the phylum Ascomycota, followed by the classes Saccharomycetes, Dothideomycetes and Sordariomycetes. The majority of species belonging to the Eurotiomycetes are members of the Aspergillus/Penicillium

Table 1 e Fungi recorded from deep-sea extreme environments by culture-independent and culture-dependent methods Methods Reference* Ascomycota Dothideomycetes Eurotiomycetes Leotiomycetes Saccharomycetes Sordariomycetes DSF-Group1b Basidiomycota Agaricomycetes Cystobasidiomycetes Entorrhizomycetes Exobasidiomycetes Microbotryomycetes Tremellomycetes Ustilaginomycetes Wallemiomycetes Chytridiomycota Chytridiomycetes Other basal lineage Cryptomycotab Basal clone group Ib

Culture-independent 1

2

C C

C C

3

C

4

C C

5

6

7

8

C C

C C

C C

C

C C

C C

C C

C

C

Culture-dependent 9

10

C C C C C C

C C C C C

11

C C C C C

12

13

3

14

C

C C

C C

C C C

C

C

C C

C

C

C C

C

C

C

C

C

C C C

C

C

C C C C

C C

C C C

C C

C

C

C

C C

a Taxa expected from morphology. b Unknown groups containing highly novel phylotypes. * Numbered references indicated in Table 2.

C

C C

C C C C

7

16

C C

C C

C

C

C C

17

18

19 C

C C

C

C

C

C

C C C

C

C C

C C

C C C

a

15

C

C C C C

a

C a

C C C

C C

Reference no.

Author/Published year

Cultureindependent

1

Bass et al. (2007)

C

2 3 4

Lopez-Garcia et al. (2007) Le Calvez et al. (2009) Sauvadet et al. (2010)

C C C

5 6 7

Singh et al. (2010) Eloe et al. (2010) Jebaraj et al. (2010)

C C C

8 9 10 11

Quaiser et al. (2011) Nagahama et al. (2011) Lai et al. (2007) Nagano et al. (2010)

C C C C

12 13 14 15 16 17 18 19

Burgaud et al. unpublished Damare et al. (2006) Burgaud et al. (2009) Connell et al. (2009) Singh et al. (2010) Nagahama et al. (2001) Gadanho & Sampaio (2005) Burgaud et al. (2010)

C

Culturedependent

C

C

Target gene SSU rDNA

Fungi

SSU rDNA SSU rDNA SSU rDNA

Eukaryota Fungi Eukaryota

SSU rDNA/ITS SSU rDNA SSU rDNA

Fungi Eukaryota Fungi/ Eukaryota Eukaryota Fungi Fungi Fungi

SSU rDNA SSU rDNA ITS ITS ITS C C C C C C C

Target species

Fungi Fungi Fungi Fungi Fungi Yeasts Yeasts Yeasts

Source Various (including hydrothermal) Hydrothermal Hydrothermal Water and hydrothermal Sediment Water Sediment and water Sediment and water Sediment (cold seep) Sediment Sediment (including cold seep) Sediment Sediment Hydrothermal Cold hydrothermal Sediment Sediment Hydrothermal Hydrothermal

Depth

Isolation methods

<4 000 m 750e900 m 1 700 m, 2 630 m 500e3 000 m 3 992e5 377 m 6 000 m 200 m

1 atm, 25
C

Fungal diversity in deep-sea extreme environments

Table 2 e Details of methods and samples used for investigations in the references cited in Table 1

1 atm, room temp.

1 000e1 260 m 850e1 200 m 350e3 011 m 1 174e10 131 m 1 750e3 750 m 4 800e5 400 m 700e3 700 m 707e1 667 m 4 000e5 500 m 1 000e11 000 m 800e2 400 m 700e2 700 m

1e30 atms, various temp. 1 atm, various temp. 1 atm, 4
C,10
C 1e30 atms, various temp. 1 atm, various temp. 1 atm, various temp. 1 atm, various temp.

465

466

group, which are known to be globally distributed. It has been suggested that these taxa are ubiquitous in deep-sea environments, but it is doubtful that they are indigenous to the deep-sea, as they are also widespread in terrestrial habitats. However, evidence of physiological adaptation of Aspergillus species to deep-sea environments has been reported (Raghukumar & Raghukumar 1998; Raghukumar et al. 2004; Damare et al. 2006; Damare & Raghukumar 2008). Saccharomycetes are a class of ascomycetous yeasts that have often been isolated from oceanic regions by culturing approaches (Fell 1976). In deep-sea environments, the genera Candida, Debaryomyces, Kodamaea, Metschnikowia, Pichia and their relatives have been frequently reported. The Dothideomycetes, and particularly members of the order Pleosporales, are bitunicate ascomycetes that are often reported from deep-sea environments. However, these fungi are not marine Pleosporales but are species of Aureobasidium, Cladosporium or Hortaea (Damare et al. 2006; Singh et al. 2010, 2011). These frequently encountered species have common characteristics of adaptation or resistance to low temperature and high osmotic pressure, which may be essential keys to survival under deep-sea conditions. Also, species of Phoma have often appeared in clone libraries in deep-sea studies (Lai et al. 2007; Singh et al. 2010). Phoma is known to associate not only with land plants but also with marine plants, such as seaweed and seagrass, as well as with marine invertebrates, such as sponges (Kohlmeyer & Volkmann-Kohlmeyer 1991). The Sordariomycetes have also been detected in many studies. However, their phylotypes are few and are unique to the studied areas. From oxygen-depleted regions, a number of OTUs close to Fusarium oxysporum, which is known to be a denitrifying fungus, have also been reported (Jebaraj et al. 2010).

Basidiomycota The most ubiquitous classes within the phylum Basidiomycota detected in deep-sea environments are the Exobasidiomycetes, Microbotryomycetes and Tremellomycetes (Table 1). The majority of clones belonging to the class Exobasidiomycetes are related to the genus Malassezia. Members of this genus are well-known as the causative agents of skin diseases in mammals, including marine mammals, such as seals or sea lions (Guillot et al. 1998; Nakagaki et al. 2000; Pollock et al. 2000). Whereas molecular studies show the ubiquitous presence of Malassezia phylotypes in deep-sea environments, no cultures have been obtained from deep-sea water and deep-sea sediments. Since members of the genus have been reported from terrestrial soil nematodes (Renker et al. 2003), they may associate with small marine invertebrates, such as nematodes or polychaetes, which also inhabit deep-sea sediments, hampering their isolation. Importantly, this fungal group accounts for the majority of eukaryotic diversity in deep-sea subsurface sediments (Edgcomb et al. 2011). The typical marine yeast orders, Sporidiobolales and Erythrobasidiales within the class of Microbotryomycetes and Cystobasidiomycetes respectively, are also found in deep-sea environments. Rare occurrences of Erythrobasidiales in molecular studies on deep-sea waters and sediments are consistent with the results from culturing studies (Nagahama

Y. Nagano, T. Nagahama

et al. 2001). Cryptococcus-related phylotypes are a major component of the Tremellomycetes, some of which are psychrotolerant and are present in polar or alpine regions (Connell et al. 2008; Turchetti et al. 2008). In marine environments, Agaricomycete fungi are scarce and are mostly reported from mangroves (Jones et al. 2009). However, clones of Agaricomycetes have also been detected in deep-sea environments (Bass et al. 2007; Le Calvez et al. 2009; Sauvadet et al. 2010; Nagahama et al. 2011).

Chytridiomycota and other basal fungal lineages Interestingly, culture-independent analyses have revealed the presence of Chytridiomycota and other basal fungal lineages in deep-sea environments (Bass et al. 2007; Le Calvez et al. 2009; Nagano et al. 2010; Sauvadet et al. 2010; Quaiser et al. 2011; Nagahama et al. 2011). The deep-sea Chytridiomycota and other basal fungal phylotypes are considered to be almost novel, as their sequences differed remarkably from published species. Le Calvez et al. (2009) reported a new branch of the Chytridiomycota from hydrothermal vents forming an ancient evolutionary lineage, and Nagahama et al. (2011) reported the presence of novel deep-branching lineages as major fungal components in deep-sea methane cold-seeps.

Different fungal communities reported by culture-dependent and culture-independent methods Le Calvez et al. (2009) reported striking differences in their comparison of deep-sea fungal diversity assessed by culturedependent or culture-independent methods. They isolated only ascomycetes through culture-dependent methods. No ascomycetes were detected by culture-independent methods, but basidiomycetes and chytrids were detected. Jebaraj et al. (2010) also reported that none of the isolates they retrieved from the deep-sea sediments of the Central Indian Basin were identical to any of the environmental sequences obtained from the same samples. Many factors could be responsible for these different results, making it difficult to judge which method more accurately reflects true fungal diversity in deepsea environments. The frequent isolation of ascomycetes can occur through biased enrichment of relatively rare organisms in deep-sea environments by conventional cultivation methods. In contrast, chytrids and other basal fungi may be missed by culture-dependent analysis using a solid agar medium containing antibiotics, which are often employed in deep-sea fungal studies. Many chytrids cannot be isolated on solid growth media, especially those to which chloramphenicol has been added (Gleason & Marano 2011). Moreover, as the majority of the phylotypes belonging to the Chytridiomycota and other basal lineages detected by culture-independent methods are highly novel, these fungi may have a completely different physiology for survival, and thus cannot be isolated by conventional culture-dependent methods. In general, culture-independent methods are thought to be able to detect wider ranges of fungi and hence reflect more accurately true diversity in the natural environment. However, culture-independent methods are also easily biased by many processes, such as PCR primer selection and DNA

Fungal diversity in deep-sea extreme environments

extraction methods. It has often been reported that a different primer pair can amplify different fungal communities in deepsea environments (Jebaraj et al. 2010; Nagano et al. 2010; Nagahama et al. 2011). Also, many unknown basal fungal clones detected from deep-sea environments are affiliated with the Cryptomycota (Nagahama et al. 2011), a newly described clade, which are thought to lack typical fungal chitin cell walls (Jones et al. 2011). It is possible that DNA is extracted more efficiently from these micro-organisms, which lack tough cell walls.

Unknown novel phylotypes in deep-sea environments DSF-group1 within the phylum Ascomycota Within the phylum Ascomycota, OTUs of Saccharomycetes related to Metschnikowia/Candida, with very long evolutionary distances from other ascomycetes (DSF-group1, Fig 1), have appeared as hitherto uncultured taxa from several deep-sea environments (Bass et al. 2007; Lai et al. 2007; Takishita et al. 2007b; Nagano et al. 2010; Nagahama et al. 2011). DSF-Group1 has only been detected from deep-sea environments, with the exception of one report of these fungi in Daphnia, zooplankton, in European lakes (Wolinska et al. 2009). This report suggests that DSF-Group1 may parasitise planktonic animals living in deep-sea environments. As DSF-Group1 has been detected from several oxygen-depleted deep-sea environments, such as methane cold-seeps, anoxic bacterial mats and deep-sea sediments (Bass et al. 2007; Takishita et al. 2007b; Nagano et al. 2010), it is possible that they are anaerobic or facultatively anaerobic fungi. This view is supported by the data of Takishita et al. (2007b), who detected DSF-Group1 by molecular methods after enriching a growth medium under anaerobic, but not aerobic, conditions.

LKM11 related clades within the newly described Cryptomycota Some phylotypes detected from deep-sea environments belong to the clone group known as the LKM11 clade (Bass et al. 2007; Nagano et al. 2010; Nagahama et al. 2011; Fig 1). The LKM11 clade is an extensive clone group that has been retrieved mainly from oxygen-depleted aquatic environments. It has been previously recognised as the most basal branch of fungi, or as a sister group to the parasitic genus Rozella (Lara et al. 2010). Jones et al. (2011) reported that this clade is present in numerous ecosystems including soil, freshwater and aquatic sediments, and they classified the clade formally as Cryptomycota. Interestingly, it has been revealed that several clades within the Cryptomycota contain Rozella, the putative primary branch of the fungal kingdom (James et al. 2006), which do not produce a typical fungal chitin-rich cell wall (Jones et al. 2011). It will be interesting to see if the clones detected from deep-sea environments within the Cryptomycota also have the same characteristics. Since members of the Cryptomycota clade occur in anaerobic ecosystems (van Hannen et al. 1999; Takishita et al. 2005; Takishita et al. 2007a), it is likely that clones within the

467

Cryptomycota detected from deep-sea environments also occur in anoxic environments.

Basal clone group I (BCGI) The BCGI clade has been reported to be the largest clone assemblage in deep-sea methane cold-seep environments (Nagahama et al. 2011, Fig 1). It is, therefore, expected that they play important roles in these environments. This clone group occurs in marine sediments of cold ecosystems, namely deepsea sediments and arctic sediments (Bass et al. 2007; Takishita et al. 2007b; Tian et al. 2008). It is considered that BCGI represents a true fungal clade, but its phylogenetic position remains unclear. Until now, BCGI was considered to be a coherent clone group branched from a known fungal clade, in contrast to the Cryptomycota, which is an extensive clone group (Nagahama et al. 2011).

The physiology of fungi from deep-sea environments Deep-sea conditions are mainly characterised by high hydrostatic pressure, and low temperature and salt content (3.5 %). The growth characteristics of isolates of deep-sea fungi under simulated deep-sea conditions have been examined in several studies (Raghukumar & Raghukumar 1998; Damare et al. 2006; Damare & Raghukumar 2008; Singh et al. 2010). For example, the marine yeasts Rhodotorula rubra and Rhodosporidium sphaerocarpum grow at 40 MPa, corresponding to the pressure experienced at depths of 4 000 m (Lorenz & Molitoris 1997). Many deep-sea fungi grow at 20 MPa (Singh et al. 2010). However, even though most of these fungi are piezotolerant, fungi usually show better growth at low pressures. No piezophilic fungi have been reported, in contrast to the many reported piezophilic prokaryotic micro-organisms from deepsea environments (Yayanos et al. 1979; Bale et al. 1997; Kato et al. 1998; Alain et al. 2002; Nogi et al. 2004; Wang et al. 2004; Takai et al. 2009; Birrien et al. 2011). The majority of fungi isolated from deep-sea environments are halotolerant (Burgaud et al. 2009; Le Calvez et al. 2009). Halophilic yeasts have been reported from deep-sea hydrothermal vents (Burgaud et al. 2010). However, Damare et al. (2006) reported that isolates of fungi from deep-sea sediments did not have an absolute requirement for seawater for growth. Similarly, most of the recovered fungi from deep-sea environments are psychrotolerant, but isolates of fungi from deep-sea sediments grew more rapidly at 30
C than 5
C (Damare & Raghukumar 2008; Singh et al. 2010). Thus, it is plausible that there are no true indigenous fungi in deep-sea environments but they gradually adapted to deep-sea extreme conditions from terrestrial environments. However, environmental PCR analysis revealed that cultivable fungi are only the tip of the iceberg of total fungi in deep-sea environments. Therefore, the information on the physiology of fungi isolated from deep-sea environments remains very biased and limited. It should also be noted that only cultured fungi have been investigated and that isolates were obtained by almost identical methods to those applied in research on terrestrial fungi. It seems likely that we have simply not been able to isolate the real indigenous fungi in deep-sea environments.

468

Y. Nagano, T. Nagahama

Deep-sea environments are much more complex than the deep-sea conditions simulated in the laboratory, and it may hence be possible to isolate piezophilic fungi and psychrophilic fungi endemic to deep-sea environments by using unique culturing systems, such as selective culturing at high pressure, or long term culturing in bioreactors with continuous-flow. Indeed, we have successfully isolated psychrophilic fungi that are unable to grow at >4
C from deep-sea environments (unpublished data). Although piezophilic fungi have yet to be reported from the deep-sea, the effects of high hydrostatic pressure on Saccharomyces cerevisiae have been well studied (Shimada et al. 1993; Abe & Horikoshi 1998; Fernandes et al. 2004). The ability of S. cerevisiae to grow under different pressures can be extended to >50 MPa, regardless of the amino acid auxotrophy of the strain (Abe & Horikoshi 2000). Also polygalacturonases, enzymes purified from the deep-sea yeast Cryptococcus liquefaciens strain N6, remain almost unchanged up to a hydrostatic pressure of 100 MPa at 24
C (Abe et al. 2006). As deep-sea environments always have elevated hydrostatic pressure, it is important to elucidate the effects of pressure on deep-sea fungi. There is no doubt that the physiology of fungi in deep-sea environments needs to be investigated more extensively, to understand fully their presence and role in the ecosystem.

Zoosporic fungi: key organisms in deep-sea extreme environments? Recently, the dominant presence of zoosporic fungi in extreme environments, for example, in high-elevation soils of the Himalayas (Freeman et al. 2009), soils of Antarctica (Bridge & Newsham 2009), extremely acidic water with pH values as low as 2 (Amaral Zettler et al. 2002), deep-sea hydrothermal vents (Le Calvez et al. 2009) and also methane cold-seeps (Nagahama et al. 2011), has been frequently reported. It is postulated that zoosporic fungi may tolerate a wide array of environmental extremes, such as drying, anaerobic conditions, low and high temperatures or pH values and high hydrostatic pressures (Gleason et al. 2010). Some zoosporic fungi are considered to be extremophiles, and may play a crucial role within extreme environments. In general, not much is known about zoosporic fungi living in marine environments. However, zoosporic fungi are beginning to be considered as an important functional unit in microbial ecology (Gleason & Lilje 2009) and the important role of zoosporic fungi in aquatic ecosystems has been recently suggested (Wurzbacher et al. 2010; Sime-Ngando et al. 2011). Further investigation is needed on unknown but potentially highly diverse zoosporic

Fig 1 e Phylogenetic position of the unknown clone groups (DSF-group1, Cryptomycota related clade and BCG; arrowed) detected from deep-sea environments. The 18S rDNA sequences of environmental fungal clones detected from deep-sea environments and appropriate fungal species in public databases were aligned using the clustalx2 program (Chenna et al. 2003). About 800 base pairs were used for the phylogenetic analysis. The tree was inferred by the neighbour-joining method (Saito & Nei 1987), as implemented in the program.

Fungal diversity in deep-sea extreme environments

469

fungi present in deep-sea environments, to unmask their ecology and understand their role in the ecosystem. Moreover, the presence of ancestral zoosporic fungal lineages in deep-sea environments has led to a new hypothesis on the diversification of fungi. Le Calvez et al. (2009) suggested that the emergence and initial diversification of fungi occurred in marine environments, challenging the widely accepted hypothesis that the diversification of fungi occurred on land. It is believed that the ancestor of fungi was a marine unicellular organism propelled by a flagellum (Wainright et al. 1993). Zoosporic fungi are known as the most primitive fungal group, and possess ancestral characteristics, such as the presence of flagellated gametes. Such fungi are mainly aquatic, being ubiquitous in freshwater environments and also in soils. However, they were rarely found in marine environments until the recent recovery of diverse zoosporic fungi in deep-sea environments (Le Calvez et al. 2009; Nagahama et al. 2011). Investigations of fungal diversity in deep-sea environments may also provide key insights into the evolutionary history of fungi.

environments by molecular methods have been carried out by targeting small subunit (SSU) rRNA and internal transcribed spacer (ITS) regions as molecular markers (see Table 2). The use of ITS as a molecular target results in the better detection of fungi, but the region contains a poor phylogenetic signal. In contrast, SSU rRNA is a good molecular target for phylogenetic analysis, but the use of existing SSU rRNA molecular markers often lack sensitivity and specificity. Although the use of a multiple primer approach is very effective for detecting maximum fungal diversity, more sensitive and betterconserved molecular markers within SSU rRNA or other genes still need to be developed for phylogenetic studies. Also, it is important to develop other methods, such as direct microscopic observation with appropriate staining, FISH, metagenomic approaches and other powerful new tools to investigate the diversity and ecology of fungi in the deep-seas.

Conclusions and future prospects

The authors would like to thank Dr. K.K. Newsham and three anonymous reviewers for their helpful comments which substantially improved the manuscript. We also would like to acknowledge Mr. Robert Collins for his kind contribution to the manuscript.

In conclusion, deep-sea environments harbour diverse fungi, including hidden diversity, which may be endemic to these environments. Culture-dependent methods often isolate fungi (typically ascomycetes) that are closely related to terrestrial species, while culture-independent methods often detect hidden fungal diversity, including phylotypes belonging to the phylum Chytridiomycota and other basal lineages (Le Calvez et al. 2009; Nagano et al. 2010; Nagahama et al. 2011). Zoosporic fungi have been extensively detected, especially from hydrothermal vents and methane cold-seeps (Le Calvez et al. 2009; Nagahama et al. 2011). It has been suggested that zoosporic fungi are tolerant to extreme environmental conditions and may be key organisms in deep-sea environments. Thus, it is important to understand the ecological and physiological significance of these fungi, especially those that are apparently endemic in deep-sea environments. This will help provide key insights into the phylogenetic histories of fungi and their mechanisms of adaptation to extreme environments, and should provide a better understanding of little-studied deepsea ecosystems. At the same time, it is important to investigate the physiology of cultured fungal species from deep-sea environments, and their extremotolerances to, for example, pH, temperature and hydrostatic pressure. Deep-sea fungi also have high potential as a source of secondary metabolites and enzymes with novel properties, and should thus be screened for useful agents. Novel natural compounds have already been reported from these fungi (Li et al. 2009; Cai et al. 2011). Moreover, there are still few fungi that have been successfully cultured from deep-sea environments. Improved culturing methods and growth media are needed in order to isolate and culture diverse fungi from these environments, including those with unique physiologies that allow them to adapt to deep-sea extreme habitats. Similarly, it is necessary to improve culture-independent methods for the enhanced detection of deep-sea fungi, especially the development of better molecular markers for investigating true fungal diversity. Investigations of fungal diversity in deep-sea

Acknowledgements

references

Abe F, Horikoshi K, 1998. Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology. Extremophiles 2: 223e228. Abe F, Horikoshi K, 2000. Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae. Molecular Cell Biology 20: 8093e8102. Abe F, Minegishi H, Miura T, Nagahama T, Usami R, Horikoshi K, 2006. Characterization of cold- and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens strain N6. Bioscience, Biotechnology, Biochemistry 70: 296e299. Alain K, Marteinsson VT, Miroshnichenko ML, BonchOsmolovskaya EA, Prieur D, Birrien JL, 2002. Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. International Journal of Systematic and Evolutionary Microbiology 52: 1331e1339.  mez F, Zettler E, Keenan BG, Amils R, Amaral Zettler LA, Go Sogin ML, 2002. Microbiology: eukaryotic diversity in Spain’s River of Fire. Nature 417: 137. Bale SJ, Goodman K, Rochelle PA, Marchesi JR, Fry JC, Weightman AJ, Parkes RJ, 1997. Desulfovibrio profundus sp. nov., a novel barophilic sulfate-reducing bacterium from deep sediment layers in the Japan Sea. International Journal of Systematic Bacteriology 47: 515e521. Bass D, Howe A, Brown N, Barton H, Demidova M, Michelle H, Li L, Sanders H, Watkinson SC, Willcock S, Richards TA, 2007. Yeast forms dominate fungal diversity in the deep oceans. Proceedings of the Royal Society B Biological Sciences 274: 3069e3077. rellou J, Birrien JL, Zeng X, Jebbar M, Cambon-Bonavita MA, Que Oger P, Bienvenu N, Xiao X, Prieur D, 2011. Pyrococcus yayanosii sp. nov., the first obligate piezophilic hyperthermophilic

470

archaeon isolated from a deep-sea hydrothermal vent. International Journal of Systematic and Evolutionary Microbiology. Bridge PD, Newsham KK, 2009. Soil fungal community composition at Mars Oasis, a southern maritime Antarctic site, assessed by PCR amplification and cloning. Fungal Ecology 2: 66e74. Burgaud G, Arzur D, Durand L, Cambon-Bonavita MA, Barbier G, 2010. Marine culturable yeasts in deep-sea hydrothermal vents: species richness and association with fauna. FEMS Microbiology Ecology 73: 121e133. Burgaud G, Le Calvez T, Arzur D, Vandenkoornhuyse P, Barbier G, 2009. Diversity of culturable marine filamentous fungi from deep-sea hydrothermal vents. Environmental Microbiology 11: 1588e1600. Cai S, Zhu T, Du L, Zhao B, Li D, Gu Q, 2011. Sterigmatocystins from the deep-sea-derived fungus Aspergillus versicolor. Journal of Antibiotics 64: 193e196. Cavanaugh CM, 1985. Symbioses of chemoautotrophic bacteria and marine invertebrates from hydrothermal vents and reducing sediments. Bulletin of the Biological Society of Washington 6: 373e388. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD, 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31: 3497e3500. Connell L, Barrett A, Templeton A, Staudigel H, 2009. Fungal diversity associated with an active deep sea volcano: Vailulu’u seamount, Samoa. Geomicrobiology Journal 26: 597e605. Connell L, Redman R, Craig S, Scorzetti G, Iszard M, Rodriguez R, 2008. Diversity of soil yeasts isolated from South Victoria Land, Antarctica. Microbial Ecology 56: 448e459. Damare S, Raghukumar C, 2008. Fungi and macroaggregation in deep-sea sediments. Microbial Ecology 56: 168e177. Damare S, Raghukumar C, Raghukumar S, 2006. Fungi in deep-sea sediments of the Central Indian Basin. Deep-Sea Research I 53: 14e27. Dekas AE, Poretsky RS, Orphan VJ, 2009. Deep-sea archaea fix and share nitrogen in methane-consuming microbial consortia. Science 326: 422e426. Dubilier N, Bergin C, Lott C, 2008. Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nature Reviews Microbiology 6: 725e740. Edgcomb VP, Beaudoin D, Gast R, Biddle JF, Teske A, 2011. Marine subsurface eukaryotes: the fungal majority. Environmental Microbiology 13: 172e183. Eloe EA, Shulse CN, Fadrosh DW, Williamson SJ, Allen EE, Bartlett DH, 2010. Compositional differences in particleassociated and free-living microbial assemblages from an extreme deep-ocean environment. Environmental Microbiology Reports 3: 449e458. Fell JW, 1976. Yeasts in oceanic regions. In: Jones EBG (ed), Recent Advances in Aquatic Mycology. Elek Science, London, pp. 93e124. Fernandes PMB, Domitrovic T, Kao CM, Kurtenbach E, 2004. Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEBS Letters 556: 153e160. Freeman KR, Martin AP, Karki D, Lynch RC, Mitter MS, Meyer AF, Longcore JE, Simmons DR, Schmidt SK, 2009. Evidence that chytrids dominate fungal communities in high-elevation soils. Proceedings of the National Academy of Sciences of the United States of America 106: 18315e18320. Gadanho M, Sampaio JP, 2005. Occurrence and diversity of yeasts in the mid-atlantic ridge hydrothermal fields near the Azores Archipelago. Microbial Ecology 50: 408e417. Gleason FH, Daynes CN, McGee PA, 2010. Some zoosporic fungi can grow and survive within a wide pH range. Fungal Ecology 3: 31e37. Gleason FH, Lilje O, 2009. Structure and function of fungal zoospores: ecological implications. Fungal Ecology 2: 53e59. Gleason FH, Marano AV, 2011. The effects of antifungal substances on some zoosporic fungi (Kingdom Fungi). Hydrobiologia 659: 81e92.

Y. Nagano, T. Nagahama

Grassle JF, 1985. Hydrothermal vent animals: distribution and biology. Science 229: 713e717. ho E, Chermette R, 1998. Guillot J, Petit T, Degorce-Rubiales F, Gue Dermatitis caused by Malassezia pachydermatis in a California sea lion (Zalophus californianus). Veterinary Record 142: 311e312. Horikoshi K, 1998. Barophiles: deep-sea microorganisms adapted to an extreme environment. Current Opinion in Microbiology 1: 291e295. James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ, Celio G, Gueidan C, Fraker E, Miadlikowska J, Lumbsch HT, Rauhut A, Reeb V, Arnold AE, Amtoft A, Stajich JE, Hosaka K, Sung GH, Johnson D, O’Rourke B, Crockett M, Binder M, Curtis JM, Slot JC, Wang Z, Wilson AW, Schussler A, Longcore JE, O’Donnell K, Mozley-Standridge S, Porter D, Letcher PM, Powell MJ, Taylor JW, White MM, Griffith GW, Davies DR, Humber RA, Morton JB, Sugiyama J, Rossman AY, Rogers JD, Pfister DH, Hewitt D, Hansen K, Hambleton S, Shoemaker RA, Kohlmeyer J, Volkmann-Kohlmeyer B, Spotts RA, Serdani M, Crous PW, Hughes KW, Matsuura K, Langer E, Langer G, Untereiner WA, Lucking R, Budel B, Geiser DM, Aptroot A, Diederich P, Schmitt I, Schultz M, Yahr R, Hibbett DS, Lutzoni F, McLaughlin DJ, Spatafora JW, Vilgalys R, 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443: 818e822. Jebaraj CS, Raghukumar C, Behnke A, Stoeck T, 2010. Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation. FEMS Microbiology Ecology 71: 399e412. Jones EBG, Sakayaroj J, Suetrong S, Somrithipol S, Pang KL, 2009. Classification of marine Ascomycota, anamorphic taxa and Basidiomycota. Fungal Diversity 35: 1e187. Jones MD, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, Richards TA, 2011. Discovery of novel intermediate forms redefines the fungal tree of life. Nature 474: 200e203. Jørgensen BB, Boetius A, 2007. Feast and famineemicrobial life in the deep-sea bed. Nature Reviews Microbiology 5: 770e781. Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K, 1998. Extremely barophilic bacteria isolated from the Mariana Trench, Challenger Deep, at a depth of 11,000 meters. Applied and Environmental Microbiology 64: 1510e1513. Kohlmeyer J, Volkmann-Kohlmeyer B, 1991. Illustrated key to the filamentous higher marine fungi. Botanica Marina 34: 1e61. Lai X, Cao L, Tan H, Fang S, Huang Y, Zhou S, 2007. Fungal communities from methane hydrate-bearing deep-sea marine sediments in South China Sea. The ISME Journal 1: 756e762.  pez-Garcıa P, 2010. The environmental clade Lara E, Moreira D, Lo LKM11 and Rozella form the deepest branching clade of fungi. Protist 161: 116e121. Lauro FM, Bartlett DH, 2008. Prokaryotic lifestyles in deep sea habitats. Extremophiles 12: 15e25. Le Calvez T, Burgaud G, Mahe S, Barbier G, Vandenkoornhuyse P, 2009. Fungal diversity in deep sea hydrothermal ecosystems. Applied and Environmental Microbiology 75: 6415e6421. Li Y, Ye D, Chen X, Lu X, Shao Z, Zhang H, Che Y, 2009. Breviane spiroditerpenoids from an extreme-tolerant Penicillium sp. isolated from a deep sea sediment sample. Journal of Natural Products 72: 912e916. Lonsdale P, 1977. Clustering of suspension-feeding macrobenthos near abyssal hydrothermal vents at oceanic spreading centers. Deep-Sea Research 24: 857e863. Lopez-Garcia P, Vereshchaka A, Moreira D, 2007. Eukaryotic diversity associated with carbonates and fluid-seawater interface in Lost City hydrothermal field. Environmental Microbiology 9: 546e554. Lorenz R, Molitoris HP, 1997. Cultivation of fungi under simulated deep sea conditions. Mycological Research 101: 1355e1365. Nagahama T, Abdel-Wahab MA, Nogi Y, Miyazaki M, Uematsu K, Hamamoto M, Horikoshi K, 2008. Dipodascus tetrasporeus sp.

Fungal diversity in deep-sea extreme environments

nov., an ascosporogenous yeast isolated from deep-sea sediments in the Japan Trench. International Journal of Systematic and Evolutionary Microbiology 58: 1040e1046. Nagahama T, Hamamoto M, Horikoshi K, 2006. Rhodotorula pacifica sp. nov., a novel yeast species from sediment collected on the deep-sea floor of the north-west Pacific Ocean. International Journal of Systematic and Evolutionary Microbiology 56: 295e299. Nagahama T, Hamamoto M, Nakase T, Takami H, Horikoshi K, 2001. Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean. Antonie Van Leeuwenhoek 80: 101e110. Nagahama T, Takahashi E, Nagano Y, Abdel-Wahab MA, Miyazaki M, 2011. Molecular evidence that deep-branching fungi are major fungal components in deep-sea methane coldseep sediments. Environmental Microbiology 13: 2359e2370. Nagano Y, Nagahama T, Hatada Y, Nunoura T, Takami H, Miyazaki J, Takai K, Horikoshi K, 2010. Fungal diversity in deep-sea sediments e the presence of novel fungal groups. Fungal Ecology 3: 316e325. Nakagaki K, Hata K, Iwata E, Takeo K, 2000. Malassezia pachydermatis isolated from a South American sea lion (Otaria byronia) with dermatitis. The Journal of Veterinary Medical Science 62: 901e903. Nogi Y, Hosoya S, Kato C, Horikoshi K, 2004. Colwellia piezophila sp. nov., a novel piezophilic species from deep-sea sediments of the Japan Trench. International Journal of Systematic and Evolutionary Microbiology 54: 1627e1631. Pollock CG, Rohrbach B, Ramsay EC, 2000. Fungal dermatitis in captive pinnipeds. Journal of Zoo and Wildlife Medicine 31: 374e378.  pez-Garcıa P, 2011. Quaiser A, Zivanovic Y, Moreira D, Lo Comparative metagenomics of bathypelagic plankton and bottom sediment from the Sea of Marmara. The ISME Journal 5: 285e304. Raghukumar C, Raghukumar S, 1998. Barotolerance of fungi isolated from deep-sea sediments of the Indian Ocean. Aquatic Microbial Ecology 15: 153e163. Raghukumar C, Raghukumar S, Sheelu G, Gupta S, Nagendernath B, Rao B, 2004. Buried in time: culturable fungi in a deep-sea sediment core from the Chagos Trench, Indian Ocean. Deep Sea Research Part I 51: 1759e1768. Renker C, Alphei J, Buscot F, 2003. Soil nematodes associated with the mammal pathogenic fungal genus Malassezia (Basidiomycota: Ustilaginomycetes) in Central European forests. Biology and Fertility of Soils 37: 70e72. Roth FJ, Orpurt PA, Ahearn DJ, 1964. Occurrence and distribution of fungi in a subtropical marine environment. Canadian Journal of Botany 42: 375e383. Saito N, Nei M, 1987. The neighbor-joining method: a new method for constructing phylogenetic trees. Molecular Biology and Evolution 4: 406e425. Sauvadet AL, Gobet A, Guillou L, 2010. Comparative analysis between protist communities from the deep-sea pelagic ecosystem and specific deep hydrothermal habitats. Environmental Microbiology 12: 2946e2964. Shimada S, Andou M, Naito N, Yamada N, Osumi M, Hayashi R, 1993. Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology 40: 123e131. Sime-Ngando T, Lefevre E, Gleason FH, 2011. Hidden diversity among aquatic heterotrophic flagellates: ecological potentials of zoosporic fungi. Hydrobiologia 659: 5e22.

471

Singh P, Raghukumar C, Verma P, Shouche Y, 2010. Phylogenetic diversity of culturable fungi from the deep-sea sediments of the Central Indian Basin and their growth characteristics. Fungal Diversity 40: 89e102. Singh P, Raghukumar C, Verma P, Shouche Y, 2011. Fungal community analysis in the deep-sea sediments of the central Indian basin by culture-independent approach. Microbial Ecology 61: 507e517. Takai K, Miyazaki M, Hirayama H, Nakagawa S, Querellou J, Godfroy A, 2009. Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney. Environmental Microbiology 11: 1983e1997. Takai K, Nakamura K, 2011. Archaeal diversity and community development in deep-sea hydrothermal vents. Current Opinion in Microbiology 14: 282e291. Takami H, Inoue A, Fuji F, Horikoshi K, 1997. Microbial flora in the deepest sea mud of the Mariana Trench. FEMS Microbiology Letters 152: 279e285. Takishita K, Miyake H, Kawato M, Maruyama T, 2005. Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the smallsubunit rDNA phylogeny. Extremophiles: Life Under Extreme Conditions 9: 185e196. Takishita K, Tsuchiya M, Kawato M, Oguri K, Kitazato H, Maruyama T, 2007a. Genetic diversity of microbial eukaryotes in anoxic sediment of the saline meromictic Lake Namako-ike (Japan): on the detection of anaerobic or anoxic-tolerant lineages of eukaryotes. Protist 158: 51e64. Takishita K, Yubuki N, Kakizoe N, Inagaki Y, Maruyama T, 2007b. Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures. Extremophiles: Life Under Extreme Conditions 11: 563e576. Tian F, Yu Y, Chen B, Li H, Yao Y-F, Guo X-K, 2008. Bacterial, archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis. Polar Biology 32: 93e103. Turchetti B, Buzzini P, Goretti M, Branda E, Diolaiuti G, D’Agata C, Smiraglia C, Vaughan-Martini A, 2008. Psychrophilic yeasts in glacial environments of Alpine glaciers. FEMS Microbiology Ecology 63: 73e83. van Hannen EJ, Mooij W, van Agterveld MP, Gons HJ, Laanbroek HJ, 1999. Detritus-dependent development of the microbial community in an experimental system: qualitative analysis by denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 65: 2478e2484. Wainright PO, Hinkle G, Sogin ML, Stickel SK, 1993. Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260: 340e342. Wang F, Wang P, Chen M, Xiao X, 2004. Isolation of extremophiles with the detection and retrieval of Shewanella strains in deep-sea sediments from the west Pacific. Extremophiles 8: 165e168. Wolinska J, Giessler S, Koerner H, 2009. Molecular identification and hidden diversity of novel Daphnia parasites from European lakes. Applied and Environmental Microbiology 75: 7051e7059. Wurzbacher CM, Barlocher F, Grossart HP, 2010. Fungi in lake ecosystems. Aquatic Microbial Ecology 59: 125e149. Yayanos AA, Dietz AS, Van Boxtel R, 1979. Isolation of a deep-sea barophilic bacterium and some of its growth characteristics. Science 205: 808e810.