STUDIES IN MYCOLOGY 51: 1–32. 2005. Fungi at the edge of life: cryptoendolithic black fungi from Antarctic desert L. Selbmann1, G.S. de Hoog2,3, A. Mazzaglia4, E.I. Friedmann5 and S. Onofri1* 1Dipartimento di Scienze Ambientali, Università degli Studi della Tuscia, Largo dell’Università, Viterbo, Italy 2Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD, Utrecht, The Netherlands 3Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Kruislaan 315, NL-1098 SM Amsterdam, The Netherlands 4Dipartimento di Difesa delle Piante, Università degli Studi della Tuscia, Via S. Camillo de Lellis, Viterbo, Italy 5NASA Ames Research Center, Moffett Field, CA 94035-1000, U.S.A. *Correspondence: S. Onofri, [email protected] Abstract: Twenty-six strains of black, mostly meristematic fungi isolated from cryptoendolithic lichen dominated communi- ties in the Antarctic were described by light and Scanning Electron Microscopy and sequencing of the ITS rDNA region. In addition, cultural and temperature preferences were investigated. The phylogenetic positions of species recognised were determined by SSU rDNA sequencing. Most species showed affinity to the Dothideomycetidae and constitute two main groups referred to under the generic names Friedmanniomyces and Cryomyces (gen. nov.), each characterised by a clearly distinct morphology. Two species could be distinguished in each of these genera. Six strains could not be assigned to any taxonomic group; among them strain CCFEE 457 belongs to the Hysteriales, clustering together with Mediterranean marble- inhabiting Coniosporium species in an approximate group with low bootstrap support. All strains proved to be psychrophiles with the only exception for the strain CCFEE 507 that seems to be mesophilic- psychrotolerant. All had very thick melanised cell walls, the ability to produce exopolysaccharides and to grow meristematically. They are thought to be well adapted to the harsh environment of the Antarctic cold Desert. Hypotheses concerning their origin and evolution are put forward. Taxonomic novelties: Cryomyces antarcticus Selbmann, de Hoog, Mazzaglia, Friedmann & Onofri gen. sp. nov., C. minteri Selbmann, de Hoog, Mazzaglia, Friedmann & Onofri sp. nov., Friedmanniomyces simplex Selbmann, de Hoog, Mazzaglia, Friedmann & Onofri sp. nov. Key words: Antarctica, black yeasts, cryptoendolithic community, Dothideales, extremophiles, Hysteriales, ITS, licheni- colous fungi, meristematic fungi, microcolonial fungi, phylogeny, SSU, taxonomy. INTRODUCTION rather than colonising its surface lies in the signifi- cant microclimatic changes occurring over a distance Antarctic rock-inhabiting microorganisms live at the of a few millimetres within the rock substratum absolute edge of life under the most extreme condi- (Friedmann & Weed 1987). Average Winter tem- tions known on Earth. They live between the limit of peratures at rock surfaces generally lie 2 °C below adaptability and near-death, barely surviving and the outside air temperature, ranging in 1985 from – rarely reproducing (Friedmann & Weed 1987). The 47.2 °C to –19.4 °C on Linnaeus Terrace (McMurdo environmental conditions of the cold deserts in the Dry Valleys; Friedmann et al. 1987), but in Summer McMurdo Dry Valleys (Fig. 1B–D) are extremely the rock surface temperature may reach 20 °C above stressful to microbial growth. These conditions have that of outside air. Large differences in microclimatic been postulated to resemble those prevailing at early conditions are noted with exposition. Wide thermal Mars, combining extremely low temperatures with fluctuations at exposed sites contribute to the practi- extreme dryness and high solar irradiation (Onofri et cally sterile conditions at the outside, the communi- al. 2004). ties being inside the rock where the nanoclimate Rocks have a prominent role as substrata in such supplies milder and more stable conditions (Nienow cold deserts (Nienow & Friedmann 1993). When & Friedmann 1993). conditions at the rock surface are prohibitive for Antarctic cryptoendolithic microorganisms consti- microbial life, Antarctic microbial communities form tute very simple communities comprising only a few a particular cryptoendolithic ecotype (Golubic et al. species (Nienow & Friedmann 1993). The most 1981), i.e. condensed growth in microscopic niches common and extensively studied is the “lichen- under the surface of porous rock (Nienow & Fried- dominated community” in sandstone (Friedmann mann 1993). The reasons for growth inside rock 1982). This community colonises porous rocks up to 1 SELBMANN ET AL. about 10 mm deep under the rock crust. Microbial Such fungi are also found in other environments growth leads to variously coloured bands (Fig. 1F). where they are subjected to extreme conditions. This stratification is maintained because each micro- Black meristematic microfungi, producing slowly bial type has different physiological requirements and expanding cauliflower-like colonies, are morphologi- abilities (e.g., exposure to solar radiation) and/or cally barely differentiated. Their phylogeny is quite produces different antibiotic substances (Ocampo- diverse, and they have been assigned to three differ- Friedmann & Friedmann 1993). The community is ent ascomycete orders: Dothideales, Chaetothyriales organised as follows. Immediately under the silici- and Pleosporales (Sterflinger et al. 1999). They are fied, reddish brown crust (1) a black speckled zone commonly isolated from rocks in hot climates (Wol- (2) is observed, followed by a white band (3), a green lenzien et al. 1997), marble monuments (Sterflinger band (4) and sometimes a blue-green band. Fungi and & Krumbein 1997), hypersaline environments (Zalar chlorophycean algae, together forming a lichen et al. 1999a) and other substrates that are hard to association, occupy the black (2) and the white (3) colonise. Some of them, belonging to the order zones. The fungal hyphae in the dark zone are melan- Chaetothyriales, are encountered as opportunists on ised, while in the white zone they are colourless. The humans. The skin disease chromoblastomycosis has a hyaline fungi and part of the pigmented ones proba- meristematic invasive phase, the muriform cells bly represent different morphotypes of one and the (Matsumoto et al. 1984). The natural niche of these same lichen species, in which the pigmentation species is in the spines of cactus plants in semi-arid expressed in the upper layer is supposed to be a climates (Zeppenfeldt et al. 1994). In humans the response to higher light intensities. The cryptoen- thick melanised cell wall is supposed to protect the dolithic growth is a remarkable morphological adap- fungus from phagocytic action by oxygen radicals tation adopted by lichens to extreme conditions. They (Schnitzler et al. 1999). The ability to grow meris- loose their typical morphology and enter the porosity tematically, ensuring to the colonies an optimal of rocks to protect themselves to the hostile outside surface/volume ratio (Wollenzien et al. 1995), is climate. Nevertheless, they preserve the ability to hypothesized to enhance the ability to survive under form a thallus with characteristic morphology as soon conditions of stress such as high or low temperature, as they reach protected niches such as small depres- low water availability (Wollenzien et al. 1995), sions or crevices on rock surfaces, where epilithic acidity, nutrient deficiency (Sterflinger et al. 1999), structures can be observed. Thick-walled and dark- high UV exposure (Urzì et al. 1995) or high salt pigmented, non-lichenised fungi grow mixed with the concentration (Zalar et al. 1999c). In some fungi lichen-forming fungi in the black zone (2). These meristematic growth could be induced by acidifica- fungi, often showing meristematic growth in vitro, tion of the culture medium (Mendoza et al. 1993). are isolated as regular members of the lichen domi- Furthermore, black fungi that preserve meristematic nated cryptoendolithic community. The green (4) growth as a stable character are reported to tolerate zone is characterised by the presence of non- desiccation, UV exposure (Wollenzien et al. 1995) lichenised algae, among which the endemic chloro- and high temperature (Sterflinger 1998) better than phycean alga Hemichloris antarctica is predominant, lichens. and several of the most extremophilic prokaryotes Meristematic black fungi from the Antarctic known to date, viz. the cyanobacteria Chroococ- cryptoendolithic communities are fascinating and cidiopsis sp. and Gloeocapsa sp. (Friedmann 1982). poorly known microorganisms, adapted to environ- A further band has been occasionally observed when mental conditions that are even more hostile than Chroococcidiopsis sp. form a separate zone below those of the remaining rock-inhabiting fungi. Al- Hemichloris antarctica. though their actual limits of survival are not well The enzymatic abilities tested in some cryptoen- known, they seem to be, both from morphological dolithic fungi were found to be different among and physiological points of view, very well adapted species, so that micro- or nano-niches occupied by to withstand unfavourable conditions. This is exem- these fungi do not completely overlap (M. Fenice, plified by the fact that they express melanised thick pers. comm.). A conspicuous sign of microbial walls as a stable character, allowing them to resist colonisation macroscopically visible on the rock dryness and the UV irradiation that reaches them surface is the characteristic mosaic-like pattern