The Igneous Oceanic Crust E Earth's Largest Fungal Habitat?

Fungal Ecology 20 (2016) 249e255

Contents lists available at ScienceDirect

Fungal Ecology

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

Mini-review The igneous oceanic crust e Earth's largest fungal habitat?

* M. Ivarsson a, , S. Bengtson a, A. Neubeck b a Department of Palaeobiology and Nordic Center for Earth Evolution (NordCEE), Swedish Museum of Natural History, Box 50007, Svante Arrhenius vag€ 9, SE-104 05, Stockholm, Sweden b Deptartment of Geological Sciences, Stockholm University, Svante Arrhenius vag€ 8, SE-106 91, Stockholm, Sweden article info abstract

Article history: In recent years the igneous oceanic crust has been recognized as a substantial microbial habitat and a Received 26 November 2015 scientific frontier within Geology, Biology, and Oceanography. A few successful metagenomic in- Received in revised form vestigations have indicated the presence of Archaea and Bacteria, but also fungi in the subseafloor 13 January 2016 igneous crust. A comprehensive fossil record supports the presence of fungi in these deep environments Accepted 27 January 2016 and provides means of investigating the fungal presence that complements metagenomic methods. Available online 18 February 2016 Considering the vast volume of the oceanic crust and that it is the largest aquifer on Earth, we put Corresponding Editor: Felix Barlocher€ forward that it is the largest fungal habitat on the planet. This review aims to introduce a yet unexplored fungal habitat in an environment considered extreme from a biological perspective. We present the Keywords: current knowledge of fungal abundance and diversity and discuss the ecological role of fungi in the Deep subseafloor biosphere igneous oceanic crust. Endoliths © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND Basalt license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Fungi Extreme environments

1. Introduction studies that have shown the presence of Archaea and Bacteria in subseafloor basalts (Mason et al., 2010; Orcutt et al., 2010; Lever Coordinated scientific drilling and exploration have during the et al., 2013) the concept of a deep biosphere in subseafloor crust last three decades recognized a deep biosphere in the deep-sea is based on a fossil record (Staudigel et al., 2008; Ivarsson et al., sediments and a seafloor biota that was previously unknown 2012, 2013a; Bengtson et al., 2014). Ichnofossils in volcanic glass (Schrenk et al., 2009). So far, the emphasis has been on prokaryotes represent the most studied fossil type (Staudigel et al., 2008) but but, although neglected at the beginning, the presence of eukary- fossilized microorganisms in open pore spaces have, during the last otes including fungi in these environments is now being investi- 10 yr, become more acknowledged and investigated (Ivarsson et al., gated (Orsi et al., 2013a,b). It is evident that fungi occur in 2008a,b; Peckmann et al., 2008). Unexpectedly, a majority of the abundance and high diversity in such varied environments as the fossilized microorganisms are the remains of fungal communities. deep-sea sediments (Nagano et al., 2010; Orsi et al., 2013a,b), at The fungi seem to play an important ecological role in the igneous hydrothermal vents (Gadanho and Sampajo, 2005; Lopez-García oceanic crust as they exist in symbiosis with chemoautotrophic et al., 2007; Connell et al., 2009), and at methane cold-seeps prokaryotes, decompose organic matter from overlying sediments, (Nagano et al., 2010; Nagahama et al., 2011). dissolve and form minerals, and are involved in cycling of elements The underlying igneous oceanic crust, on the other hand, is still (Ivarsson et al., 2012, 2013a, 2015a,b,c; Bengtson et al., 2014). It is more or less unexplored in terms of biology. Considering that deep obvious that the igneous oceanic crust is a previously unrecognized sea sediments are estimated to contain the largest proportion of and unexplored fungal niche that might be of great importance Earth's microorganisms (Whitman et al., 1998; Schrenk et al., considering the distribution of fungi both spatially and in time. This 2009), it would be reasonable to assume that a significant review aims at presenting the occurrence, diversity, and ecological biosphere is hosted in deep crustal environments as well. However, role of fungi in one of Earth's most extreme environments e the owing to sampling issues, next to nothing is known about microbial igneous oceanic crust. life in the igneous crust. Apart from a few successful molecular 2. The igneous oceanic crust as a microbial habitat

* Corresponding author. The igneous oceanic crust consists of three main parts: an upper E-mail address: [email protected] (M. Ivarsson). 500e1000 m section of permeable basalts, a middle layer down to http://dx.doi.org/10.1016/j.funeco.2016.01.009 1754-5048/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 250 M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255

~1.5 km of sheeted dikes, and a deeper layer to about 4 km depth of speculative. However, in the absence of sunlight, a majority of the gabbroic rock (Fig. 1). The total rock volume is 2.3 Â 1018 m3, which deep subseafloor biosphere is thought to consist of chemo- is 6e10 times the total volume of the marine sediments. The upper autotrophs; organisms that obtain energy from inorganic sources layer is characterized by extensive fracturing, about 10% porosity, and synthesize all necessary organic compounds from inorganic À À and with permeabilities of about 10 12 to 10 15 m2 (Bach and carbon sources, in contrast to photoautotrophs that utilize solar Edwards, 2004; Orcutt et al., 2011). Fractures created by tension energy. release or quick cooling occur with varying size and frequency, as Basalts exposed at the seafloor, and thus more accessible do vesicles as a result of pressure release during magma extrusion. compared to deeper basalts, are commonly coated with biofilms Thus, subseafloor basalts contain coherent systems of micro- mainly dominated by Bacteria according to quantative molecular fractures and vesicles interconnected with each other in which studies like PCR or FISH (Edwards et al., 2003; Sudek et al., 2010; seawater and hydrothermal fluids circulate. Roughly 60% of the Templeton et al., 2009). Taxonomically, the microbial commu- oceanic crust is hydrologically active and the total fluid volume that nities are dominated by Actinobacteria, Bacteroidetes, Chloroflexi, is held within the oceanic crust corresponds to 2% of the total ocean Firmicutes, Planctomycetes, and Proteobacteria phyla (Mason et al., (Orcutt et al., 2011). The entire water volume of the ocean circulates 2007, 2008; Einen et al., 2008; Santelli et al., 2008; Santelli et al., through the oceanic igneous crust every 105e107 years, which 2009). The Archaea are much less known but tend to be domi- means that the oceanic igneous crust is the largest aquifer system nated by members of Crenarchaeota and Thaumarchaeota on Earth (Fisher and Becker, 2000; Orcutt et al., 2011). Indirectly (Thorseth et al., 2001; Fisk et al., 2003; Lysnes et al., 2004; Mason this means that the oceanic igneous crust is the largest potential et al., 2007). Eukaryotes, including fungi, have been reported microbial habitat on Earth. Microorganisms are passively trans- from dredged basalts (Connell et al., 2009) and hydrothermal vent ported or actively migrate through this system wherever pore space sites (Lopez-García et al., 2007). and fluid flow permit. The host rock and secondary mineralizations Subseafloor basalts are more difficult to access and sample than of the fracture walls are used for colonization and anchoring of seafloor-exposed basalts, and molecular studies from such envi- microbial communities (Figs. 1 and 2A,B), and the minerals of the ronments are therefore sparse. Alpha-, Beta-, and Gammaproteo- host rock can be used as energy sources for the microorganisms. bacteria lineages in gabbroic layers (Mason et al., 2010) have been Little is known of metabolic reactions in the subseafloor crust reported as well as anaerobic Archaea such as Archaeoglobus and because of restricted accessibility of live microbial communities, Methanosarcina (Cowen et al., 2003; Orcutt et al., 2010). Lever et al. and discussions on possible metabolic pathways tend to be rather (2013) reported Methanosarcinales, anaerobic methane-oxidizing

Fig. 1. Diagram showing the oceanic crust and endolithic habitats. M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255 251

Fig. 2. (A) Optical microphotograph showing a vug (small cavity inside rock) in basalt with a few calcite crystals and a fungal mycelium attached to the wall of the pore space. The vug is covered by a biofilm from which the mycelium protrudes and grows. (B) ESEM image showing close-up of the fungal mycelium in A. At the bottom is the biofilm from which the hyphae grew. The biofilm and the hyphae are mineralized as montmorillonite. In the upper right corner are the remains of microstromatolitic bacterial colonies, called Frutexites, seen as cauliflower-like structures. The bacteria were probably iron oxidizers living in symbiosis with the fungi. (C) Tomographic reconstruction showing a mycelium with symbiotic organisms; Frutexites and prokaryotic cells suspended between the hyphae in a cobweb-like fashion. Abbreviations: ba, basalt; ca, calcite; bf, biofilm; my, mycelium; hy, hyphae; fx, Frutexites;cw,“cobweb”; ze, zeolite; md, mineral dissolution. Fig. 2A is modified from Ivarsson et al. (2013a), and Fig. 2B is modified from Bengtson et al. (2014).

Archaea (ANME-1), and a cluster of microbes that falls within un- abundance, diversity and origin of these ichnofossils, but still today cultured sulfate reducers. Furthermore, studies of crustal fluids their biological origin is questioned (Lepot et al., 2009), especially collected on ocean ridge flanks have indicated the presence of the ancient ones found in ophiolites as old as 3.5 Ga (Grosch and Firmicutes (Cowen et al., 2003; Nakagawa et al., 2006), Bacteria that McLoughlin, 2014). During the last decade fossilized microorgan- do not group closely with cultivated microorganisms and whose isms have been observed in drilled cores and dredged samples from metabolism is unclear, although they have been connected with the ocean floor and in ophiolites (Schumann et al., 2004; Ivarsson nitrogen or sulfur cycling. Only one fungal isolate from a basalt drill et al., 2008a, b; 2012; Peckmann et al., 2008; Eickmann et al., core has been reported so far (Hirayama et al., 2015). The drill core 2009; Cavalazzi et al., 2011; Sakakibara et al., 2014). The microor- was collected at North Pond on the western flank of the Mid- ganisms have been found fossilized in carbonate- and zeolite-filled Atlantic ridge. The isolated fungal strain was affiliated to the veins and vesicles in the basalts or open pore spaces, and represent genus Exophiala of the order Chaetothyriales. microbial communities that once lived in these fractures. Surpris- Apart from this rather fragmentary and circumstantial evidence, ingly, a majority of these findings represents fungi (Ivarsson et al., our understanding of the biosphere of the subseafloor crust is 2012, 2013a; Bengtson et al., 2014). based on a fossil record. Granular and tubular ichnofossils (trace fossils) in volcanic glass are the most abundant type of fossil (Staudigel et al., 2008). Numerous reports have discussed the 252 M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255

3. Endolithic fungi in the igneous oceanic crust colonies. Ivarsson et al. (2013a) further reported mycelium-like networks Schumann et al. (2004) first described fossilized fungal hyphae in open veins and vesicles in subseafloor basalts (Fig. 2). The main in carbonate-filled vesicles in North Pacific Eocene basalts. difference compared to the previous studies was the mode of Morphological traits including branching, septa, and a central pore preservation in open vesicles compared to the entombment by were used to argue for a fungal interpretation of the fossilized fil- carbonates and subsequent mineralization. This type of preserva- aments. These were interpreted as endolithic fungi that were tion enabled in-detail studies of an entire fungal community and introduced to the system after the carbonate formation. Thus, they not only slices of communities visible in petrographic thin sections. were interpreted as active borers of the carbonates. Reitner et al. The fungal communities in the open vesicles were also mineralized (2006) reported fungal hyphae in dredged vesicular basalt from and preserved by a clay-phase, normally montmorillonite, but the Kolbeinsey Ridge, north of Iceland, which has later been revised lacked the high carbon content that characterized the fossils as the Vesteris Seamount in the Greenland basin (Ivarsson et al., embedded in carbonates. This is probably due to fossilization in an 2015a). Stephen et al. (2003) reported fungal hyphae in open system where microbial activity has decomposed, reminer- carbonate-filled veins from basalts collected during the Ocean alized and thus removed all remains of carbon compounds. Besides Drilling Program (ODP) 200 (Site 1224) in the Pacific Ocean outside hyphae, yeast-like growth states were also described. The yeast Hawaii. The fungal interpretation of both Reitner et al. (2006) and occurred as spherical bodies with a diameter of ~20 mm and could Stephen et al. (2003) is based on appearance only and does not rely form assemblages of hundreds of cells. Ivarsson et al. (2013a) on any characteristic fungal morphologies or fungal biomarkers. further described structures associated with the mycelium that Peckmann et al. (2008) and Eickmann et al. (2009) further they interpreted as resting structures, similar to sclerotia. described similar filamentous fossilized microorganisms in ophio- Using SRXTM Bengtson et al. (2014) revealed new information lites without interpreting them as remains of fungi, even though on the fungal communities described by Ivarsson et al. (2013a) that was discussed as one possibility. However, lack of morpho- (Figs. 2c and 3a). They showed that the fungal colonization is logical characteristics made a fungal interpretation frail. Ivarsson initiated by the formation of a biofilm that is laid down directly on et al. (2008a, b) also reported long, curvilinear filamentous fossil- the vesicle walls. The entire walls are covered by this biofilm from ized microorganisms in subseafloor basalts from the Emperor which hyphae as well as sporophores protrude. The biofilm and Seamounts in the Pacific Ocean, without interpreting the findings associated mycelium are partially overgrown by secondary calcite, as fungi. All these reports referred to similar filamentous microor- and Bengtson et al. (2014) were able to show that the fungi survived ganisms in carbonate-filled veins in basalts of varying age; the fil- this overgrowth and bored in the calcite after its formation, pro- aments were preserved and mineralized by a clay phase like ducing long tubular cavities in the calcite (Fig. 3a). The hyphal chamosite or illite (Peckmann et al., 2008), or by a composition of boring started both from the biofilm overgrown by calcite and from the end member bertierine-chamosite and illite-glauconite non-overgrown parts of the mycelium. The dissolution of the (Eickmann et al., 2009). Ivarsson et al. (2008a, b) described the calcite was most likely mediated by the production of organic acids, composition of the filamentous microorganisms as a poorly crys- which is a common trait among fungi (Gadd, 2007, 2010). A unique talline clay-like phase of Si, Al, Mg, Fe and C, and/or iron oxides. feature of these borings is that the responsible organisms, without Many of the filamentous structures contained high amounts of exception, remain in the bored structure. carbon (10e50 wt%), but also phosphates, hydrocarbons and lipids, Bengtson et al. (2014) further showed that the fungi existed in a which were used as evidence for their biogenic origin (Ivarsson close symbiotic-like relationship with two types of prokaryotes, et al., 2008b). Propidium iodide (PI), a dye that binds to cells with and that the prokaryotes used the structural framework of the damaged cell membranes and traces of DNA, were used to stain mycelia for their growth. Minute cells suspended between the fossils successfully (Ivarsson et al., 2008a). The high content of hyphae formed a “cobweb” with an ultrastructure similar to that of organic remains was explained as a result of instant entombment of Pyrodictium or Euryarchaeon SM1, common sulfate-reducing the microorganisms by the carbonate phase, and lack of diagenetic archaea found in hydrothermally active areas. Based on element processes and metamorphosis after fossilization. analyses these were interpreted as remains of chemoautotrophic The first study that was able to combine fungal morphologies prokaryotes involved in iron oxidation. The other type of prokary- with chemical data characteristic for fungi was Ivarsson et al. ote was represented by microstromatolites called Frutexites. These (2012). They described mycelium-like networks of hyphae in ve- structures also grew on the hyphal network and showed a distinct sicular basalts from the Emperor Seamounts in the Pacific Ocean growth direction in cross section. The Frutexites were interpreted as with the aid of synchrotron-radiation X-ray tomographic micro- remains of bacterial communities involved in iron oxidation as scopy (SRXTM). This technique enabled a much more detailed well. The fungi obviously gained from the close relationship with study of the fossil morphologies and spatial occurrence. The hyphae chemoautotrophic prokaryotes, which probably made the fungal were characterized by repetitive septa, anastomoses between colonization in the nutrient-poor environment possible. In this branches and a central strand. They were also able to detect chitin interpretation the chemoautotrophs fixed dissolved carbon from in the cell walls by staining with WGA-FITC under fluorescence fluids and built biomass, which the heterotrophic fungi could microscopy. Chitin is absent in prokaryotes but common among scavenge for their metabolism. There might also have been a several lineages of fungi and thus a strong indication of a fungal feedback mechanism when the fungi decomposed the carbohy- affinity. Based on the presence of chitin and morphological traits drates to CO2 accessible for the chemoautotrophs, although the Ivarsson et al. (2012) interpreted the fungi as ascomycetes or stem- extent of this feedback is unknown. group Dikarya. This study involved a re-interpretation as hyphal fungi of the filamentous microfossils previously reported by 4. Metabolic pathways and cycling of elements in the oceanic Ivarsson et al. (2008a,b) from the same samples. Ivarsson (2012) igneous crust further showed the presence of fruit bodies, spores, thallic con- idiogenesis, and hyphal tips with preserved hyphal vesicles. The presence of fungi in subseafloor igneous crust raises ques- Abundant reproductive structures indicated that the fungi do not tions regarding metabolic pathways, access to bioavailable ele- simply represent specimens randomly introduced by downward ments, metals, and carbon sources (Schumann et al., 2004; Ivarsson transport of seawater but that they existed in vital, sustainable et al., 2012). Fungi are heterotrophs and need a constant supply of M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255 253

Fig. 3. (A) Tomographic reconstruction of a fungal mycelium with symbiotic prokaryotic cells suspended in between the hyphae in a cobweb-like fashion. The mycelium is draped around the calcite surface (blue) and the hyphae penetrate the calcite creating tunnels within the mineral. (B) ESEM image of a zeolite surface on which hyphal boring is seen. Note the dissolution features on the mineral surface around the hyphae. Fig. 3B is modified from Ivarsson et al. (2015c). Abbreviations as for Fig. 2. carbohydrates for their metabolism. In a nutrient-poor environ- indicating a primary process in which fungi are involved in Mn(II) ment such as the oceanic crust, occurrence of accessible biomass oxidation and formation of subsequent Mn(IV) oxide minerals and organic compounds is poorly understood and investigated. (Ivarsson et al., 2015a). Possible sources could be the trickling down from overlying sedi- Dekov et al. (2013) reported fungal hyphae in hydrothermal ments and seawater, or chemoautotrophic communities, but sulfide-sulfate samples from seafloor and subseafloor rocks whether such carbon is evenly or patchily distributed throughout mineralized as orpiments (As-sulfides). They interpreted the fungi the oceanic crust is not known. The symbiotic-like relationship as endolithic organisms killed by As-rich pulses of fluids, with with chemoautotrophs reported by Bengtson et al. (2014) describes fungal organic matter serving as a geochemical trap for hydro- one possible way fungi can get access to biomass. Ivarsson et al. thermal As. The As reacted with S from the basement or the (2015c) describe a similar community structure from Detroit seawater and mineralized the hyphae as As2S3. Seamount, where fungi basically overgrow and graze on the pro- Considering the plethora of elements fungi are known to karyotic portion of the community. Thus, chemoautotrophic com- interact with in terrestrial environments (Gadd, 2007, 2010), it is munities seem to be a carbon source in deep basalts. In dredged probable that the geobiological impact of fungi in the oceanic samples from the Vesteris Seamount in the Greenland basin, rep- igneous crust is far from resolved and understood. One unresolved resenting depths of no more than 1 m, Ivarsson et al. (2015a) issue is the concentration of O2 in the oceanic crust, which is poorly showed how fungi feed on marine organisms such as algae, investigated, and would greatly impact on the fungal abundance which have been introduced to the basalts by ingress of seawater. and diversity since most known fungi are aerobic. Shallow igneous The fungi produce haustoria with which they attack and penetrate crustal environments appear to be oxygenated since they introduce the algae; even the silica frustules of diatoms are attacked and oxygen-rich pore waters into the anoxic bottom sediments (Orcutt degraded by the haustoria. Thus, in shallow basalts, where marine et al., 2013), but further down in the igneous portion of the crust sediments rich in organic remains are easily introduced, the main oxygen levels are far from being understood. Local areas of carbon source for fungal metabolism seems to be organic matter in oxygenated seawater can probably be introduced, but there should the sediments. At greater depths, where sediments do not reach by also be provinces poor in oxygen. In a heterogeneous environment, downward migration, the fungi need to find other accessible carbon such as the igneous crust, geochemical conditions vary on a local sources of which symbiotic chemoautotrophic communities are scale and anaerobic niches are probably common. Most fungi are one candidate (Bengtson et al., 2014; Ivarsson et al., 2015c). aerobic but anaerobic fungi are known from freshwater lakes, In addition to cycling carbon, fungi are also involved in the landfill sites (McDonald et al., 2012), and deep sea sediments cycling of metals and other elements through bioweathering of (Nagano and Nagahama, 2012) but are best known from the rumen minerals and biomineralization. The dissolution and subsequent of ruminating herbivores (Khejornsart and Wanapat, 2010; boring of tunnels in carbonates (Bengtson et al., 2014) and zeolites Liggenstoffer et al., 2010), an anaerobic environment in which (Ivarsson et al., 2015c)(Fig. 3) mobilize metals and elements such as they play an important role in energy metabolism including the Ca, Si, Al, Na, and K. The reason for the mineral boring could be formation of H2. Anaerobic fungal species have no mitochondria migration, acquisition of habitable space, response to environ- and are unable to produce energy by either aerobic or anaerobic mental stress, or trophic strategies (Bengtson et al., 2014; Ivarsson respiration (Yarlett et al., 1986; O'Fallon et al., 1991). Instead, et al., 2015c). Ca, Na, and K are essential metals for fungal growth anaerobic fungi have hydrogenosomes, organelles capable of and metabolism (Gadd, 2007, 2010), and they could thus be mined coupling the metabolism of glucose to cellular energy production. for metabolic reasons. Hydrogenosomes contain hydrogenase and produce H2,CO2, ace- Ivarsson et al. (2015b) showed fungal communities associated tate, formate, lactate, and ethanol as metabolic waste products with biogenic Mn oxides. The biogenicity of these Mn oxides was (Yarlett et al., 1986; Brul and Stumm, 1994; Khejornsart and supported by electron paramagnetic resonance (EPR) analysis, but Wanapat, 2010). Anaerobic fungi consort with methanogenic whether the fungi or an associated prokaryotic community was archaea in the rumen. Methanogens are microorganisms that responsible for the direct oxidation of Mn(II) to Mn(IV) oxide produce methane using carbon dioxide, acetate or one-carbon mineral could not be fully determined. Fungal involvement in Mn substrates as carbon sources and electron acceptors while oxidation has been shown (Tebo et al., 1997), also from exposed hydrogen functions as an electron donor (Mountfort et al., 1982; seafloor-basalts (Connell et al., 2009). In the Vesteris Seamount Hook et al., 2010). The methanogens increase the enzymatic ac- samples fungal spores mineralize by Mn(II) oxide minerals tivity in the fungi through the removal of hydrogen, which is 254 M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255 followed by a shift in the metabolic activity within the fungi to- contemporaneous with the diversification of vascular plants (James wards a production of methanogenic precursors. The deep sub- et al., 2006). The earliest subseafloor fungi reported (Ivarsson et al., seafloor basalts are inhabited by anaerobic communities utilizing 2013b) were marine fungi that inhabited an impact-generated hydrogen or formate as electron sources (Orcutt et al., 2010). Bio- paleo-hydrothermal system on the ocean floor, dated to 458 Ma. logically produced H2 by anaerobic fungi could very well serve as an Thus, subseafloor fungal communities existed at least 458 Ma ago, energy source for methanogens or other microbial communities but probably as long as marine fungi have been around. that are utilizing hydrogen within the subseafloor basalt systems, In terrestrial environments fungi are known as a powerful and in turn provide a carbon source for heterotrophic life forms geobiological force that exists in symbiotic relationships with all such as fungi. This would constitute a vital microbial consortium known kingdoms of life (Webster and Weber, 2007). Mycorrhizas that would be sustainable for long periods of time in deep anoxic for instance are a symbiotic consortium that 90% of all land plants pore space. are dependent on for their survival. Lichens are another strong form of symbiosis between fungi and cyanobacteria or algae (or both) 5. Diversity that enables colonization in various environments, including extreme ones like Arctic tundra or hot dry deserts. In subseafloor Based on the presence of chitin and morphological traits, igneous crust fungi appear to occur in symbiotic-like relationships, Ivarsson et al. (2012) interpreted fungi from the Emperor Sea- and are involved in weathering of minerals such as carbonates and mounts in the Pacific Ocean as ascomycetes or stem-group Dikarya. zeolites. At the moment we are only on the verge of understanding Fossilized fungi from the Vesteris Seamount in the Greenland Basin the full role and capacity of fungi in these environments. As the (Ivarsson et al., 2015a) were interpreted as zygomycetes based on exploration continues the full range of fungal interaction with, and the identification of zygospores and the various morphological impact on element mobilization, biogeochemical cycles, and sea- stages of the zygomycete reproduction cycle. With the exception of floor/ocean element budgets will be revealed. Even though the one fungal isolate from a basalt rock core reported from North fossil record so far has provided us with some information on Pond, Mid-Atlantic Ridge, affiliated to the genus Exophiala of the fungal communities, metagenomic investigations need to be order Chaetothyriales (Hirayama et al., 2015), there are no molec- included in the future for a more comprehensive understanding of ular studies of fungi in subseafloor igneous crust. This is probably fungi in the igneous oceanic crust. due to contamination issues involved in sampling live species at great depths in marine environments rather than reflecting an Acknowledgments absence of fungi. On seafloor-exposed basalt, where sampling is easier, fungi have been isolated. Connell et al. (2009) isolated eight We thank Marianne Ahlbom at the Department of Geological yeast-like fungal species belonging to both Ascomycota and Basi- Sciences, Stockholm University, for assistance with ESEM analyses, diomycota from seafloor-exposed basalt at Vailulu'u Seamount, Veneta Belivanova at the Department of Palaeobiology, Swedish Pacific Ocean. Fungi have also been reported from vent fluids at the Museum of Natural History and Federica Marone at the Paul Lost City, the Mid-Atlantic Ridge, where fungal strains of both Scherrer Institute, Switzerland, for assistance with SRXTM analyses. Ascomycota and Basidiomycota were reported including strains of We acknowledge funding from the Swedish Research Council anaerobic fungi (Lopez-García et al., 2007). Ascomycetes and ba- (Contracts No. 2010-3929, 2012-4364, and 2013-4290), Danish sidiomycetes have also been isolated from marine sediments (Orsi National Research Foundation (DNRF53), and Paul Scherrer Insti- et al., 2013a,b) including the deepest sediments on Earth at the tute (20130185) as well as Swedish National Space Board (Contract Mariana Trench (Takami et al., 1997). The presence of Chy- No. 100/13). tridiomycota has also been reported from hydrothermal vents (Le Calvez et al., 2009) and methane seeps (Nagahama et al., 2011). References Interestingly, most Chytridiomycota appear to be novel. At deep- sea methane-seeps a majority are novel deep-branching lineages, Bach, W., Edwards, K.J., 2004. Iron and sulphide oxidation within the basaltic ocean and Le Calvez et al. (2009), for example, reported a new branch in crust: Implications for chemolithoautotrophic microbial biomass production. e hydrothermal systems forming an ancient evolutionary lineage. Geochim. Cosmochim. Acta 67, 3871 3887. Bengtson, S., Ivarsson, M., Astolfo, A., et al., 2014. Deep-biosphere consortium of Thus, the fungal diversity seems to be high in both marine sedi- fungi and prokaryotes in Eocene sub-seafloor basalts. Geobiology 12, 489e496. ments and in subseafloor basalts represented by all major divisions; Brul, B., Stumm, C.K., 1994. Symbionts and organelles in anaerobic protozoa and Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota. fungi. Tree 9, 319e324. Cavalazzi, B., Westall, F., Cady, S.L., et al., 2011. Potential fossil endoliths in vesicular pillow basalt, coral patch seamount, eastern north Atlantic Ocean. Astrobiology 6. Concluding remarks 11, 619e632. Connell, L., Barrett, A., Templeton, A., et al., 2009. Fungal diversity associated with an active deep sea volcano: Vailulu'u Seamount, Samoa. Geomicrobiol. J. 26, The presence of fungi in the oceanic igneous crust extends the 597e605. known spatial distribution of fungi by magnitudes and introduces a Cowen, J.P., Giovannoni, S.J., Kenig, F., et al., 2003. Fluids from aging ocean crust that whole new ecological niche for fungi. Considering the vast volume support microbial life. Science 299, 120e123. fl Dekov, V.M., Bindi, L., Burgaud, G., 2013. Inorganic and biogenic As-sulfide precip- of the subsea oor crust, hosting Earth's largest aquifer, and that it is itation at seafloor hydrothermal fileds. Mar. Geol. 342, 28e38. considered to contain a substantial biomass, this could be the Edwards, K.J., Bach, W., Rogers, D.R., 2003. Geomicrobiology of the ocean crust: a largest fungal habitat on Earth. It might also be an important role for chemoautotrophic Fe-bacteria. Biol. Bull. 204, 180e185. environment for fungal evolution. The deep subseafloor crust has Eickmann, B., Bach, W., Kiel, S., et al., 2009. Evidence for cryptoendolithic life in Devonian pillow basalts of Variscan orogens, Germany. Palaeogeogr. Palaeoclim. been a microbial habitat throughout the history of life on Earth Palaeoecol. 283, 120e125. from 3.5 Ga up until today (Furnes et al., 2008). Even though crown- Einen, J., Thorseth, I.H., Øvreås, L., 2008. Enumeration of Archaea and bacteria in fl fl group fungi are considered to be much younger, estimated to be- sea oor basalt using real-time quantative PCR and uorescence microscopy. FEMS Microbiol. Lett. 282, 182e187. tween 760 Ma and 1.1 Ga according to molecular clocks (Lücking Fisher, A.T., Becker, K., 2000. Channelized fluid flow in oceanic crust reconciles heat- et al., 2009; Parfrey et al., 2011), and at least not inconsistent flow and permeability data. Nature 403, 71e74. with a meager fossil record (Porter, 2006), the crust could have Fisk, M.R., Storrie-Lombardi, M.C., Douglas, S., et al., 2003. Evidence of biological activity in Hawaiian subsurface basalts. Geochem. Geophys. Geosyst 4, 1103. been an early fungal habitat. Fungi or their ancestors were marine Furnes, H., McLoughlin, N., Muehlenbachs, K., et al., 2008. Oceanic pillow lavas and or aquatic, living prior to the major diversification on land hyaloclastites as habitats for microbial life through time-A review. In: Dilek, Y., M. Ivarsson et al. / Fungal Ecology 20 (2016) 249e255 255

Furnes, H., Muehlenbachs, K. (Eds.), Links between Geological Processes, Mi- fibrobacters. Environ. Microbiol. 14, 1077e1087. crobial Activities and Evolution of Life. Springer, pp. 1e68. Mountfort, D.O., Asher, R.A., Bauchop, T., 1982. Fermentation of cellulose to methane Gadanho, M., Sampajo, J.P., 2005. Occurrence and diversity of yeasts in the mid- and carbon dioxide by a rumen anaerobic fungus in a triculture with Meth- Atlantic ridge hydrothermal fields near the Azores Archipelago. Microb. Ecol. anobrevibacter sp. strain RA1 and Methanosarcina barkeri. Appl. Environ. 50, 408e417. Microbiol. 44, 128e134. Gadd, G.M., 2007. Geomycology: biogeochemical transformations of rocks, min- Nagahama, T., Takahashi, E., Nagano, Y., et al., 2011. Molecular evidence that deep- erals, metals and radionuclides by fungi, bioweathering and bioremediation. branching fungi are major fungal components in deep-sea methane cold-seep Mycol. Res. 111, 3e49. sediments. Environ. Microbiol. 13, 2359e2370. Gadd, G.M., 2010. Metals, minerals and microbes: geomicrobiology and bioreme- Nagano, Y., Nagahama, T., 2012. Fungal diversity in deep-sea extreme environments. diation. Microbiol. 156, 609e643. Fung. Ecol. 5, 463e471. Grosch, E.G., McLoughlin, N., 2014. Reassessing the biogenicity of Earth's oldest Nagano, Y., Nagahama, T., Hatada, Y., et al., 2010. Fungal diversity in deep-sea trace fossil with implications for biosignatures in the search for early life. PNAS sediments e the presence of novel fungal groups. Fung. Ecol. 3, 316e325. 111, 8380e8385. Nakagawa, S., Inagaki, F., Suzuki, Y., et al., 2006. Microbial community in black rust Hirayama, H., Abe, M., Miyazaki, J., et al., 2015. Data Report: Cultivation of Micro- exposed to hot ridge flank crustal fluids. Appl. Environ. Microbiol. 72, organisms from Basaltic Rock and Sediment Cores from the North Pond on the 6789e6799. Western Flank of the Mid-Atlantic Ridge, IODP Expedition 336. Proc. Int. Oc. O'Fallon, J.V., Wright, R.W., Calza, R.E., 1991. Glucose metabolic pathways in the Drill. Prog. 336. anaerobic rumen fungus Neocallimatrix frontalis EB 188. Biochem. J. 274, Hook, S.E., Wright, A.-D.G., McBride, B.W., 2010. Methanogens: methane producers 595e599. of the rumen and mitigation strategies. Archaea 2010, 1e11. Orcutt, B.N., Wheat, C.G., Rouxel, O., et al., 2013. Oxygen consumption rates in Ivarsson, M., 2012. The subseafloor basalts as fungal habitats. Biogeosciences 9, subseafloor basaltic crust derived from a reaction transport model. Nat. Comm. 3625e3635. 4, 2539. http://dx.doi.org/10.1038/ncomms3539. Ivarsson, M., Peckmann, J., Tehler, A., et al., 2015a. Zygomycetes in vesicular Orcutt, B.N., Sylvan, J.B., Knab, N.J., et al., 2011. Microbial ecology of the dark ocean basanites from Vesteris Seamount, Greenland Basin e a new type of cryp- above, at, and below the seafloor. Microbiol. Mol. Biol. Rev. 75, 361e422. toendolithic fungi. PLoS One 10 (7), e01333368. http://dx.doi.org/10.1371/ Orcutt, B.N., Bach, W., Becker, K., et al., 2010. Colonization of subsurface microbial journal.pone.01333368. observatories deployed in young ocean crust. ISME J. 5, 692e703. Ivarsson, M., Broman, C., Gustafsson, H., et al., 2015b. Biogenic Mn-oxides in sub- Orsi, W.D., Edgcomb, V.D., Christman, G.D., et al., 2013a. Gene expression in the seafloor basalts. PLoS One 10 (6), e0128863. http://dx.doi.org/10.1371/ deep biosphere. Nature 499, 205e208. journal.pone.0128863. Orsi, W.D., Biddle, J.F., Edgcomb, V.D., 2013b. Deep sequencing of subseafloor Ivarsson, M., Bengtson, S., Skogby, H., et al., 2015c. A fungal-prokaryotic consortium eukaryotic rRNA reveals active fungi across marine subsurface provinces. PLoS at the basalt-zeolite interface in subseafloor igneous crust. PLoS One 10 (10), One 8, 1e10. e014016. http://dx.doi.org/10.1371/journal.pone.0140106. Parfrey, L.W., Lahr, D.J.G., Knoll, A.H., et al., 2011. Estimating the timing of early Ivarsson, M., Bengtson, S., Skogby, H., et al., 2013a. Fungal colonies in open fractures eukaryotic diversification with multigene molecular clocks. PNAS 108, of subseafloor basalt. Geo-Mar. Lett. 33, 233e243. 13624e13629. Ivarsson, M., Broman, C., Sturkell, E., et al., 2013b. Fungal colonization of an ordo- Peckmann, J., Bach, W., Behrens, K., et al., 2008. Putative cryptoendolithic life in vician impact-induced hydrothermal system. Sci. Rep. 3, 3487. http://dx.doi.org/ Devonian pillow basalt, rheinisches Schiefergebirge, Germany. Geobiology 6, 10.1038/srep03487. 125e135. Ivarsson, M., Bengtson, S., Belivanova, V., et al., 2012. Fossilized fungi in subseafloor Porter, S.M., 2006. The proterozoic fossil record of heterotrophic eukaryotes. In: Eocene basalts. Geology 40, 163e166. Xiao, S., Kaufman, A.J. (Eds.), Neoproterozoic Geobiology, Topics in Geobiology, Ivarsson, M., Lindblom, S., Broman, C., et al., 2008a. Fossilized microorganisms vol. 27. Springer. associated with zeolite-carbonate interfaces in sub-seafloor hydrothermal en- Reitner, J., Schumann, G., Pedersen, K., 2006. Fungi in Subterranean Environments. vironments. Geobiology 6, 155e170. in: Fungi in Biogeochemical Cycles. Cambridge University Press, Cambridge, UK. Ivarsson, M., Lausmaa, J., Lindblom, S., et al., 2008b. Fossilized microorganisms from Sakakibara, M., Sugawara, H., Tsuji, T., et al., 2014. Filamentous microbial fossil from the Emperor Seamounts: Implications for the search for a subsurface fossil low-grade metamorphosed basalt in northern chichibu belt, central Shikoku. record on Earth and Mars. Astrobiology 8, 1139e1157. Jpn. Planet. Space Sci. 95, 84e93. James, T.Y., Kauff, F., Schoch, C.L., et al., 2006. Reconstructing the early evolution of Santelli, C.M., Edgcomb, V.P., Bach, W., et al., 2009. The diversity and abundance of fungi using a six-gene phylogeny. Nature 443, 818e822. bacteria inhabiting seafloor lavas positively correlate with rock alteration. Khejornsart, P., Wanapat, M., 2010. Diversity of rumen anaerobic fungi and meth- Eviron. Microbiol. 11, 86e98. anogenic archaea in swamp buffalo influenced by various diets. J. Anim. Vet. Santelli, C.M., Orcutt, B.N., Banning, E., et al., 2008. Abundance and diversity of Adv. 9, 3062e3069. microbial life in ocean crust. Nature 453, 653e657. Le Calvez, T., Burgaud, G., Mahe, S., et al., 2009. Fungal diversity in deep sea hy- Schrenk, M.O., Huber, J.A., Edwards, K.J., 2009. Microbial provinces in the subsea- drothermal ecosystems. Appl. Environ. Microbiol. 75, 6415e6421. floor. Annu. Rev. Mar. Sci. 2, 279e304. Lepot, K., Philippot, P., Benzerara, K., et al., 2009. Garnet-filled trails associated with Schumann, G., Manz, W., Reitner, J., et al., 2004. Ancient fungal life in North Pacific carbonaceous matter mimicking microbial filaments in Archean basalt. Geo- Eocene oceanic crust. Geomicrobiol. J. 21, 241e246. biology 7, 393e402. Staudigel, H., Furnes, H., McLoughlin, N., et al., 2008. 3.5 billion years of glass bio- Lever, M.A., Rouxel, A., Alt, J.C., et al., 2013. Evidence for microbial carbon and alteration: Volcanic rocks as a basis for microbial life? Earth-Sci. Rev. 89, sulphur cycling in deeply buried ridge flank basalt. Science 339, 1305e1308. 156e176. Liggenstoffer, A.S., Youssef, N.H., Couger, M.B., et al., 2010. Phylogenetic diversity Stephen, R.A., Kasahara, J., Acton, G.D., 2003. Leg 200 Summary. Proc. Oc. Drill. Prog., and community structure of anaerobic gut fungi (phylum Neo- Init. Rep.. 200, 1e73. callimastigomycota) in ruminant and non-ruminant herbivores. ISME J. 4, Sudek, L.A., Templeton, A.S., Tebo, B.M., et al., 2010. Microbial ecology of Fe(hydr) 1225e1235. oxide mats and basalts rock from Vailulu'u Seamount, American Samoa. Geo- Lopez-García, P., Vereshchaka, A., Moreira, D., 2007. Eukaryotic diversity associated microbiol. J. 26, 581e596. with carbonates and fluid-seawater interface in Lost-City hydrothermal field. Takami, H., Inoue, A., Fuji, F., et al., 1997. Microbial flora in the deepest sea mud of Environ. Microbiol. 9, 546e554. the Mariana Trench. FEMS Microbiol. Lett. 152, 279e285. Lücking, R., Huhndorf, S., Pfister, D.H., et al., 2009. Fungi evolved right on track. Tebo, B.M., Ghiorse, W.C., van Waasbergen, L.G., et al., 1997. Bacterially mediated Mycologia 101, 810e822. mineral formation: insighths into managanese(II) oxidation from molecular Lysnes, K., Thorseth, I.H., Steinsbau, B.O., et al., 2004. Microbial community diversity genetic and biochemical studies. Geomicrobiology: interactions between mi- in seafloor basalt from the Arctic spreading ridges. FEMS Microbiol. Ecol. 50, crobes and minerals. Revs. Min. 35, 225e266. 213e230. Templeton, A.S., Knowles, E.J., Eldridge, K.L., et al., 2009. A seafloor microbial biome Mason, O.U., Nakagawa, T., Rosner, M., et al., 2010. First investigation of microbi- hosted within incipient ferromanganese crusts. Nat. Geosci. 2, 872e876. ology of the deepest layer of ocean crust. PLoS One 5 (11), e15399. Thorseth, I.H., Torsvik, T., Torsvik, V., et al., 2001. Diversity of life in ocean floor Mason, O.U., Di Meo-Savoie, C.A., Van Nostrand, J.D., et al., 2008. Prokaryotic di- basalt. Earth Planet Sci. Lett. 194, 31e37. versity, distribution, and insights into their role in biogeochemical cycling in Webster, J., Weber, R.W.S., 2007. Introduction to Fungi, third ed. Cambridge Uni- marine basalts. ISME J. 3, 231e242. versity Press, Cambridge, UK. Mason, O.U., Stingl, U., Wilhelm, L.J., et al., 2007. The phylogeny of endolithic mi- Whitman, W.B., Coleman, D.C., Wiebe, W.J., 1998. Prokaryotes: the unseen majority. crobes associated with marine basalts. Environ. Microbiol. 9, 2539e2550. PNAS 95, 6578e6583. McDonald, J.E., Houghton, J.N.I., et al., 2012. The microbial ecology of anaerobic Yarlett, N., Orpin, C.G., Munn, E.A., et al., 1986. Hydrogenosomes in the rumen cellulose degradation in municipal waste landfill sites: evidence of a role for fungus Neocallimastix patriciarum. J. Biochem. 236, 729e739.