Potential extinction of Antarctic endemic fungal species as a consequence of global warming

Science of the Total Environment 438 (2012) 127–134

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Potential extinction of Antarctic endemic fungal species as a consequence of global warming Laura Selbmann a,⁎, Daniela Isola a, Massimiliano Fenice a, Laura Zucconi a, Katja Sterflinger b, Silvano Onofri a a b

Department of Ecological and Biological Sciences (DEB), Università degli Studi della Tuscia, Largo dell'Università, 01100 Viterbo, Italy Department of Biotechnology, Austrian Center of Biological Resources and Applied Mycology (ACBR), University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Wien, Austria

H I G H L I G H T S ► ► ► ► ►

We studied interactions among Antarctic fungi to evaluate the effects of global warming. Cryomyces spp. was parasitized and killed by Lecanicillum muscarium in co-cultures. L. muscarium lythic activities may have intriguing and new applications. L. muscarium may expand its area of distribution as a consequence of global warming. Extinction of threatened species previously living in confined niches may occur.

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Article history: Received 6 March 2012 Received in revised form 6 August 2012 Accepted 6 August 2012 Available online 13 September 2012 Keywords: Antarctica Climate change Extreme conditions Fungi Mycoparasites

a b s t r a c t Cryomyces spp. are fungi adapted to the harsh conditions of the McMurdo Dry Valleys in the Antarctic. The structure of their cell wall is one of the main factors for their uncommon ability to survive external stressors. The cells are, in fact, embedded in a thick and strongly melanised cell wall encrusted with black rigid plaques giving a supplementary protection and making them practically impregnable and refractory even to commercial enzymes including chitinases and glucanases. The Antarctic fungus Lecanicillium muscarium CCFEE 5003, able to produce an arsenal of lytic enzymes, including chitinases and glucanases, is known for its ability to degrade the cell walls of different food spoiling and opportunistic fungi as well as plant pathogenic Oomycota. Active cells of Cryomyces spp. were cultivated in dual culture with the mycoparasitic fungus both in liquid and solid media. Light microscope observations revealed that the cell walls of Cryomyces were heavily decayed. This resulted in the release of protoplasts. Hyphae penetration was evident with both scanning and transmission electron microscope observations. Due to its ecological amplitude (i.e. temperature growth range 0–28 °C), the parasitic fungus could easily expand its area of distribution as a consequence of global warming by invading new areas towards the interior of the continent. The establishment of interactions with organisms living at present in border ecosystems may lead to extinction of extremely specialized and poorly competitive entities. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The increase of greenhouse gasses principally due to the use of fossil fuels for human activities, strongly contributed to climate change on earth in the last decades. The consequent global warming has caused, as a main general effect, the movement throughout the poles, or through higher latitudes, of some habitats and associated species with a consequent disappearance of the pre-existing border ecosystems (Parmesan and Yohe, 2003; Pimm, 2008; Root et al.,

⁎ Corresponding author at: DEB, Università degli Studi della Tuscia, Largo dell'Università, 01100 Viterbo, Italy. Tel.: + 39 0761 357012; fax: + 39 0761 357097. E-mail address: [email protected] (L. Selbmann). 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.08.027

2003; Thomas et al., 2004). Meanwhile, organisms adapted to particular environmental conditions can be easily replaced by less specialized but more competitive species. Proxy data for climate from polar regions are of particular interest because global circulation models typically indicate that climate changes may be amplified in the polar regions (Roots, 1989). Over the past 50 years, for instance, portions of Antarctica and sub-Antarctic islands have experienced some of the most rapid changes in mean air temperatures on Earth, leading to increases by roughly 1 °C average over the Western Antarctic Ice Sheet (WAIS) and more than 2 °C in the maritime Antarctic (Turner et al., 2005; Steig et al., 2009), much higher than the general increase of the worldwide Earth's surface temperature that was between 0.3 and 0.6 °C over the past century (Cane et al., 1997).

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Over the same period, introductions of non-indigenous species have also increased, particularly in the sub-Antarctic islands (Frenot et al., 2005). Even in the McMurdo Dry Valleys, the widest ice-free region of the Antarctic and one of the coldest and driest terrestrial environment on Earth, the rising lake-levels of Lake Bonney, a permanently ice-covered closed basin lake in Taylor Valley over a period of about thirty years (from 1969 to 2001), indicate a recent change in the local climate (Bomblies et al., 2001). It is virtually certain that temperature increases and species' introductions will intensify and continue to influence Antarctic terrestrial ecosystems. Thus, it is important to widen our knowledge on Antarctic terrestrial ecosystems and use this information to identify ecosystem change (Hogg and Wall, 2011). The endolithic life is one of the most spectacular adaptations of microbes to the environmental pressure and the predominant life-form in the inner part of continental Antarctica (Nienow and Friedmann, 1993). One of the most complex among the endolithic communities is the lichen dominated community (Friedmann, 1982) thriving inside the porosity of sedimentary rocks, i.e. sandstone, that is particularly common in the McMurdo Dry Valleys. Climate observations in this area, basing on a network of seven valley floor automatic meteorological stations during the period 1986 to 2000, revealed that annual mean temperatures ranged from − 14.8 °C to − 30.0 °C, depending on the site and period of measurement (Doran et al., 2002). Aridity of the region is also extreme and causes sublimation to exceed precipitation (less than 100 mm water per year, Nienow and Friedmann, 1993), contributing to the ice-free nature of the area (Fountain et al., 1998). These conditions, coupled with high solar irradiation and strong thermal fluctuations, prevent microbial settlement on rock surface that are virtually abiotic: epilithic lichens occur in protected niches and endolithic life is predominant. Several prokaryotic and eukaryotic organisms take part of the cryptoendolithic lichen dominated communities living at the limit of their biological potential (De la Torre et al., 2003; Onofri et al., 2004; Ruisi et al., 2007). Black meristematic fungi are invariably present in these communities (Selbmann et al., 2005, 2008) among which the Antarctic endemic new genus and species Cryomyces antarcticus Selbmann et al. and Cryomyces minteri Selbmann et al. (Selbmann et al., 2005) are among the most‐stress-tolerant organisms known to date; they are, in fact, able to resist extremes of temperatures, UV radiations and even outer space exposure (Onofri et al., 2007a, 2007b, 2008, 2012; Selbmann et al., 2011). Despite their resistance to abiotic stresses, black rock fungi are believed to be potentially vulnerable because of their high specialization and poor competitive abilities, so they may easily disappear when conditions become more permissive as a consequence of the introduction of more competitive species (Sterflinger, 2005). Therefore, any further warming of the Antarctic continent may lead to the expansion of the areal distribution of some species with subsequent interaction of organisms spreading in segregated climatic and geographic areas that would never actually meet under natural conditions at present. We investigated the potential effects of this process by observing the interactions of C. antarcticus CCFEE 534 and C. minteri CCFEE 5187 with Lecanicillium muscarium CCFEE 5003. All these fungi were isolated in the Antarctic but their ecology is quite different: the first two live in the McMurdo Dry Valleys, and are forced to live inside of the rock because conditions on the surface are too harsh and do not allow epilithic life. The fungus L. muscarium (Petch) Zare and Gams (Zare and Gams, 2001) is an entomopathogenic species already suggested for biological control of plant pathogenic insects (Cuthbertson and Walters, 2005); the strain CCFEE 5003 was isolated from mosses collected in Kay Island, Northern Victoria Land, Antarctica (Zucconi et al., 1996). This species occurs also

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in other coastal sites of the continental Antarctic including Prior Island, Crater Cirque or in the soils heated by volcanism in Mt. Melbourne (Onofri et al., 2007a, 2007b). There, the climatic conditions are significantly milder compared to the McMurdo Dry Valleys where Cryomyces spp. thrive: monthly mean temperature may remain above 0 °C for 1–2 months during the austral summer and, during winter, ranges between −10 °C and −30 °C but temperatures may briefly rise towards freezing when winter storms bring warm air towards the Antarctic coast.. Precipitation is about 150 mm of water equivalent per year (Øvstedal and Lewis Smith, 2001). Rock surfaces in these locations are colonized by epilithic lichens and mosses with which L. muscarium is frequently associated (Onofri et al., 2007a, 2007b). The strain CCFEE 5003 is able to parasitize different food spoiling and pathogenic fungi as well as plant pathogenic oomycetes (Fenice et al., 1998; Fenice and Gooday, 2006;) thanks to beta-glucanases and chitinases production. Chitinases in particular have the rather uncommon ability to be active in a broad range of temperatures and to maintain a strong activity even at 5 °C (Fenice et al., 1997, 1998). The aim of this study was to investigate if the chitinolytic fungus L. muscarium could seriously threaten rock inhabiting fungi in case the Antarctic temperature shifts, allowing L. muscarium to occupy a wider territory including the Dry Valleys. To this aim laboratory co-cultures of meristematic fungi and L. muscarium were carried out to verify possible interactions. Effects on structure and functionality of the black fungi cell walls were documented by light and electron microscopy. 2. Materials and methods 2.1. Microorganisms C. antarcticus CCFEE 534 was isolated by R. Ocampo–Friedmann from weathered rock collected at Linnaeus Terrace (Southern Victoria Land, Antarctica) by E.I. Friedmann, during the Antarctic expedition in 1981–1982; C. minteri CCFEE 5187 was isolated by S. Pagano from weathered rocks collected by S. Onofri at Battleship Promontory (Southern Victoria Land, Antarctica) on Dec 28, 1996; L. muscarium CCFEE 5003 was isolated from moss samples collected by Del Frate on Feb 9, 1988 at Kay Island (Northern Victoria Land, Antarctica). Cultures are preserved in the CCFEE (Culture Collection of Fungi from Extreme Environments), Department of Ecological and Biological Sciences, Università degli Studi della Tuscia, Viterbo (I), in the Mycological Section of the Italian Antarctic Museum “Felice Ippolito”. 2.2. Dual cultures in liquid medium 2.2.1. Pre-culture preparation A 72 h culture of L. muscarium grown on MA (Malt Agar) slant was suspended with 5 ml of sterile distilled water and used to inoculate a 250 ml Erlenmeyer flask prepared with 50 ml of YNB (Yeast Nitrogen Base DIFCO) 1% + glucose 1%. Incubation was performed at 180 rpm, 25 °C for 72 h. 2.2.2. Dual-culture preparation After 72 h of incubation, the pre-culture of L. muscarium was washed twice and re-suspended in a 50 ml of sterile distilled water. Five ml of the suspension were used to inoculate 250 ml Erlenmeyer flasks with 50 ml of the following media: 1) 0.1% YNB 2) Sterile distilled water 3) Physiological solution (0.90% w/v of NaCl).

Fig. 1. Cryomyces minteri CCFEE 5187 and Lecanicillium muscarium CCFEE 5003 co-cultured in liquid medium after 24 h (A), 48 (C, D), 72 (F, G, H) and 96 h (L, M, N) of incubation; controls are shown in pictures B, E, I, O respectively. Protoplasts are visible after 48 h of incubation already (C) but are rather common after 96 h (L, M). After 96 h remains of the rigid plaques of the external coat are visible (N). Scale bars = 10 μm.

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1% w/v of Cryomyces' fresh biomass was added as the sole carbon source in the media to induce enzyme production, and flasks were incubated at 15 °C for 96 h. Tests were performed in triplicate. Cultures were observed daily at light microscope and documented with a Nikon 5000 camera. Pictures were acquired using Micromax Arkon software (version 8.12.05).

once met with Cryomyces' biomass (Fig. 2A). This suggests that the parasitic fungus was actively feeding on the biomass of the black fungus.

2.2.3. Co-cultures in solid medium Petri dishes (4 sectors) were prepared as follows: first layer in two adjacent sectors 0.1% glucose + agar 2%; in the other 2 sectors water agar 2%. All the sectors were filled further with a second layer prepared as follows: YNB 0.2% + agar 2%. Half of each plate was inoculated by spreading C. antarcticus or C. minteri alternatively and incubated at 15 °C for 3 months. Once grown, L. muscarium was seeded by spotting the inoculum in the half clean plate and the plates were incubated at 15 °C until the overgrowth of L. muscarium was evident.

Fig. 2B shows the black fungus C. antarcticus in pure culture (control) by scanning electron microscopy with intact cell walls coated by the external plaques. The cells were also embedded in an external layer of extracellular polymeric substances (EPS). The production of EPS in these fungi has been already reported (Selbmann et al., 2005). Observations by scanning electron microscopy of the treated samples evidenced the hyphae of the parasitic fungus growing tightly the cells of Cryomyces (Fig. 2C) and often points of entry of the hyphae into the cells of the black fungus are evident. Many cell walls of the black fungi appeared heavily damaged and the cells lost their protoplasts (2D, F); the growth of the L. muscarium hyphae inside of the cells of the black fungi were well visible (Fig. 2D, E, F). Cells of Cryomyces appeared regularly globose and the cell wall was thick and dense in the control samples when observed by the transmission electron microscope (Fig. 3A). The cells were instead heavily damaged in the treated samples: the wall appeared invariably partially hydrolyzed (Fig. 3B, C, D) and the hyphae of L. muscarium were actively penetrating the cell wall (Fig. 3C, E, F) and massively present inside the cells of the black fungi (Fig. 3D, E).

2.3. Electron microscopy Fragments 0.5 × 0.5 cm wide of the cultures in solid medium were taken and prepared for SEM observations according to Onofri et al. (1980). Samples were observed using a JEOL JSM 5200 Scanning Electron Microscope (SEM). Samples for TEM observations were fixed and dehydrated in a graded ethanol series according to Onofri et al. (1980). Specimens were then infiltrated for three days with mixtures of LRWhite resin/ ethanol. At the end of the procedure, samples were embedded in LRWhite resin. Embedded material was cut with Reichert Ultracut ultramicrotome using a diamond knife. Thin sections (60–80 nm) were collected on copper grids, stained with uranyl acetate and lead citrate, and observed with a JEOL 1200 EX II Transmission Electron Microscope. Micrographs were acquired by the Olympus SIS VELETA CCD camera equipped with iTEM software. 3. Results 3.1. Observation of liquid cultures at light microscope Pictures taken from light microscope observations are shown in Fig. 1. Already after 24 h of incubation the lytic activity of the fungus L. muscarium on the cell wall of the test fungi was clearly visible (Fig. 1A) as compared to the control (Fig. 1B); the cells of the black fungus appeared swollen and the cell wall discolored and thinned. Damages on the cell walls increased progressively after 48 and after 72 h of incubation: the cell walls were almost completely hydrolyzed with visibly emerging protoplasts (Fig. 1C, D, F, G, H). After 96 h protoplasts were rather frequent in the cultural broth (Fig. 1L, M); empty plaques constituting the supplementary outer cellular coat were found accumulated in the medium (Fig. 1N). In all controls, the cell walls of Cryomyces remained thick and strongly melanised at all incubation times (Fig. 1B, E, I, O); the cells did not swell even if they were cultivated in sterile distilled water. 3.2. Solid cultures All co-cultivations were prepared using minimal media to induce L. muscarium to produce lytic enzymes and exploit the cell wall of Cryomyces as the sole carbon source. In all conditions, the growth of the parasitic fungus on solid medium was barely visible when seeded on minimal media but gave an evident overgrowth in the same plates

3.3. Observation of solid cultures at scanning and transmission electron microscopes

4. Discussion Black meristematic fungi are specialized to extreme environments including bare rock in hot and cold deserts (Sterflinger et al., 2012). Their ecology is diverse; they may colonize plant tissues and behave as parasites (Frank et al., 2009), some are well known human pathogens (de Hoog et al., 2000), some others colonize monuments (Diakumaku et al., 1995; Sert et al., 2007; Sterflinger and Prillinger, 2001; Wollenzien et al., 1995; Zucconi et al., 2012) or live on or within natural exposed rocks (Ruibal et al., 2005) even in extreme environments in spatial association with lichens or cyanobacteria (Nienow and Friedmann, 1993; Selbmann et al., 2005, 2008). All these environments, although apparently different, require microorganisms to deal with similar injuries as extremes of temperatures, osmotic stress (Sterflinger, 1998), desiccation (Gorbushina et al., 2008) and oxygen action (Langfelder et al., 2003). Black fungi adopt similar morpho-physiological adaptations allowing them to cope with a wide selection of stresses (Ruisi et al., 2007), this leads to a marked morphological convergence despite their wide systematic diversification (Ruibal et al., 2008). One of the main adaptations of these fungi is represented by a strongly melanized and thick cell wall (Sterflinger, 2005). Black fungi seem to be mainly adapted to the cold since they react actively to low temperatures up-regulating protein expression while high temperatures cause inhibition of protein expression independently if they come from hot or cold environments (Tesei et al., in press). Melanins and thick cell walls are also the main factors allowing these fungi to shift from cold to hot conditions, since they may efficiently protect the cells from UV exposition (Selbmann et al., 2011). Adaptation to extreme environments is energy consuming for many organisms since it requires the production of specific proteins to resist cold-shocks or protective intra- and extracellular polymers like polyoles, trehalose or fatty acids (Selbmann et al., 2005). Considering the oligotrophic conditions of the natural environments

Fig. 2. Scanning electron micrographs of Cryomyces minteri CCFEE and Lecanicillium muscarium CCFEE 5003 co-cultured in solid medium where the overgrown of the parasitic fungus is evident (A). Intact cells of C. antarcticus cultivated in pure culture embedded with EPS (B). Hyphae of the parasitic fungus growing tight to the cells of the rock fungus C.antarcticus (C). The parasitic fungus inside the cells of C. minteri (D, E) and C. antarcticus (F).

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Fig. 3. Transmission electron micrographs of intact cells of Cryomyces antarcticus (A). Hyphae of the parasitic fungus Lecanicillium muscarium CCFEE 5003 penetrating (B, C, F) and massively growing (D, E) inside the cells of C. antarcticus. Scale bars = 1 μm.

of most black fungi, together with the high carbon and energy demand to produce protectants as EPSs, it is not surprising that they normally show a very slow growth rate. Consequently, the production of metabolites that are not strictly necessary must be suppressed to avoid waste of energy. The low metabolic activities and slow growth rates make them very poor competitors (Rubial, 2004; Sterflinger, 2005) confined in the extremes, occupying niches where only few other organisms may survive. The Antarctic endemic fungi C. antarcticus and C. minteri are well adapted to their natural environment of the McMurdo Dry Valleys, similar to other black fungi they focus on resistance rather than as revealed by their psychrophilic nature and high tolerance to UV irradiance; moreover they are surprisingly resistant even to conditions that they would never experience in nature such as temperature of up to 90 °C and outer space radiation (Onofri et al., 2008; Onofri et al., 2012). Therefore they might eventually survive potential environmental changes due to global warming, despite their high level of specialization. Nonetheless, their weak point is the potential invasion of more competitive species from milder areas that could easily outcompete the autochthonous specialists. Due to their ecological fitting, a process through which an organism colonizes and persists in novel environments thanks to the suite of traits it carries at the time of encountering the new condition (Agosta and Klemens, 2008), fungi

may jump to new environments and easily survive. In this respect, the ability to adapt to Antarctic conditions of alloctonous fungal species carried to the continent, is well known (Farrell et al., 2011). As reported in this study a parasitic fungus growing in a relatively milder coastal area of the Antarctica may be a candidate to invade environments colonized by endolithic fungal species. The results of this study clearly demonstrated the ability of the parasitic fungus L. muscarium CCFEE 5003 to attack the cell wall of the strains of Cryomyces spp. both in solid and liquid cultures. The cell wall of the rock fungi were significantly damaged in the test samples as compared with the controls in all the conditions studied, including cultivation in distilled water. The presence of swollen cells, even at the first interval of observation, might therefore be addressed to the action of lytic enzymes rather than to osmotic stress. The presence of glucanases in the cultural broths was not investigated in this study but L. muscarium is a fungus the degrading activity of which is well documented. The production of lytic enzymes in this fungus appears to be inducible (Fenice et al., 1998) according to the carbon sources in the cultural medium. For our tests we decided to use viable cells of Cryomyces species as the sole carbon source to induce the parasitic fungus to produce enzymes ad hoc, according to the black fungi cell wall composition. The parasitic action is quite rapid since cell walls were considerably damaged after a few hours of incubation already

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even under sub-optimal conditions of growth (15 °C) for the parasitic fungus. Being adapted to distinct environmental conditions, the strategy of L. muscarium is quite different from that of Cryomyces; it is a fast growing fungus with optimum at 25 °C but it is also able to grow in a wide range of temperatures, including 0 °C (Zucconi et al., 1996). Mesophilic-psychrotolerant behavior is a predominant feature of many Antarctic microorganisms (Montes et al., 2004; Zucconi et al., 1996) as an adaptation to the fluctuating conditions of the terrestrial Antarctic ecosystems. The activity of the lytic enzymes reflects the psychrotolerant nature of the fungus. The chitinases of this fungus are, in fact, active in a broad range of temperatures and at 5 °C still maintain 50% of the activity recorded at the best conditions of 40 °C (Fenice et al., 1998). At present these two fungi are segregated; Cryomyces spp. spread exclusively in the Dry Valleys thanks to their extremotolerance and absence of competitors, while the harsh conditions and the absence of proper substrata to which it is normally associated, as mosses and lichens, in the interior confine L. muscarium in coastal sites of the Antarctic. Even if no specific data are available for the Dry Valleys, West and East Antarctica warmed between 1957 and 2006 at the rates of 0.17 and 0.10 °C per decade, respectively; while the continent-wide trend is 0.12 °C per decade (Steig et al., 2009). If the warming trend across the continent continues in the future, an increase of water availability in the McMurdo Dry Valleys is expected. This would amplify connectivity among glaciers, streams, soils and lakes enhancing nutrient-cycle mixing across landscapes and increasing biodiversity and productivity within the ecosystem. As a consequence the proper conditions for the settlement of other microbial species such as L. muscarium would be created. Even if the main winds blow from the interior of the Antarctic to the coasts, viable propagules of cosmopolitan hyphomycetes are currently present in permafrost samples from the Dry Valleys (Zucconi et al., 2011); therefore the transportation of spores of L. muscarium in an inner area of the Victoria Land as the Dry Valleys is possible. The movement and adaptation to new conditions of this Antarctic strain of L. muscarium may be facilitated by its broad ecological amplitude related to broad temperature growth range (0 to 28 °C) and production of a wide array of extracellular lytic enzymes active also at low temperatures (Fenice et al., 1997, 1998; Fenice and Gooday, 2006). It cannot be excluded that fungi with similar potentialities and powerful destructive action may easily invade areas currently precluded for their inhospitable conditions if environmental parameters would shift from prohibitive to sub-optimal. Any change may lead to an irreversible alteration of the weak equilibrium of these border ecosystems. In addition to the ecological implications of the results presented here, the lytic activity of the fungus might very well be used as a tool to produce protoplasts of fungi that are otherwise resistant to all commercially available enzymes and enzyme cocktails as “Driselase” – a cocktail of laminariase, xylanase and cellulase, lyzing enzymes from Trichoderma harzianum Rifai or a cocktail of ß-glucanase, cellulase, protease and chitinase, as well as lyticase from the bacterium Arthrobacter luteus. The elimination of the rigid cell walls from these fungi is a necessary prerequisite for methods requiring protoplasts – as for example karyotyping – but could also help to get a better DNA and RNA quality necessary for new generation sequencing approaches. Last but not least, L. muscarium is a promising fungus to be used as antagonist in order to attack black fungi that are one of the most important biogenous agents on valuable historic monuments made of marble and other rock types.

Acknowledgments The authors thank the PNRA (Italian National Program for Antarctic Research) for funding logistical and technical support and the Italian National Antarctic Museum “Felice Ippolito” for funding CCFEE (Culture Collection of Fungi from Extreme Environments).

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References Agosta SJ, Klemens JA. Ecological fitting by phenotypically flexible genotypes: implications for species associations, community assembly and evolution. Ecol Lett 2008;11:1123–34. Bomblies A, McKnight DM, Andrews ED. Retrospective simulation of lake-level rise in Lake Bonney based on recent 21-yr record: indication of recent climate change in the McMurdo Dry Valleys, Antarctica. J Paleolimnol 2001;25:477–92. Bryan R, Jiang Z, Friedman M, Dadachova E. The effect of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanisation. Fungal Biol 2001;115: 945–9. Cane MA, Clement AC, Kaplan A, Kushnir Y, Pozdyakov D, Seager R, et al. Twentieth century sea surface temperature trends. Science 1997;275:957–60. Cuthbertson AG, Walters KF. Pathogenicity of the entomopathogenic fungus, Lecanicillium muscarium, against the sweetpotato whitefly Bemisia tabaci under laboratory and glasshouse conditions. Mycopathol 2005;160:315–9. de Hoog GS, Guarro J, Gené J, Figueras F. Atlas of Clinical Fungi. 2nd ed. Utrecht/Reus: Centraalbureau voor Schimmelcultures/Universitat Rovira I Virgili; 2000. De la Torre JR, Goebel BM, Friedmann EI, Pace NR. Microbial diversity of cryptoendolithic communities from the McMurdo Dry Valleys, Antarctica. Appl Environ Microbiol 2003;215:3858–67. Diakumaku E, Gorbushina AA, Krumbein WE, Panina L, Soukharjeski S. Black fungi in marble and limestones: an aesthetical, chemical and physical problem for the conservation of monuments. Sci Total Environ 1995;167:295–304. Doran PT, McKay CP, Clow GD, Dana GL. Valley floor climate observations from the McMurdo dry valleys. Antarctica 1986–2000. Geophys Res 2002;107(D24):4772. Farrell RL, Arenz BE, Duncan SM, Held BW, Jurgens JA, Blanchette RA. Introduced and indigenous fungi of the Ross Island historic huts and pristine areas of Antarctica. Polar Biol 2011;34:1669–77. Fenice M, Gooday GW. Mycoparasitic actions against fungi and oomycetes by a strain (CCFEE 5003) of the fungus Lecanicillium muscarium isolated in continental Antarctica. Ann Microbiol 2006;56:1–6. Fenice M, Selbmann L, Zucconi L, Onofri S. Production of extracellular enzymes by Antarctic fungal strains. Polar Biol 1997;17:275–80. Fenice M, Selbmann L, Di Giambattista R, Federici F. Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium cfr. lecanii. Res Microbiol 1998;149:289–300. Fountain AG, Dana GL, Lewis KJ, Vaughn BH, McKnight DM. Glaciers of the McMurdo Dry Valleys, Southern Victoria Land, Antarctica. In: Priscu JC, editor. Ecosystem Dynamics in a Polar Desert. Antarctic Research SeriesWashington, D.C: American Geophysical Union; 1998. p. 65–75. Frank J, Crous PW, Groenewald JZ, Oertel B, Hyde KD, Phengsintham P, Schroers H-J. Microcyclospora and Microcyclosporella: novel genera accommodating epiphytic fungi causing sooty blotch on apple. Persoonia 2009;24:93-105. Frenot Y, Chown SL, Whinam J, Selkirk PM, Convey P, Skotnicki M, et al. Biological invasions in the Antarctic: extent, impacts and implications. Biol Rev 2005;80: 45–72. Friedmann EI. Endolithic microrganisms in the Antarctic cold desert. Science 1982;215: 1045–53. Gorbushina AA, Kotlova ER, Sherstneva OA. Cellular responses of microcolonial rock fungi to long-term desiccation and subsequent rehydration. Stud Mycol 2008;61: 91–7. Hogg ID, Wall DH. Global change and Antarctic terrestrial biodiversity. Polar Biol 2011;34:1625–7. Langfelder K, Streibel M, Jahn B, Haase G, Brakhage AA. Biosynthesis of fungal melanin and their importance for human pathogenic fungi. Fungal Genet Biol 2003;38: 143–58. Montes MJ, Mercadé E, Bozal N, Guinea J. Paenibacillus antarcticus sp. nov., a novel psychrotolerant organism from the Antarctic environment. Int J Syst Evol Microbiol 2004;54:1521–6. Nienow JA, Friedmann EI. Terrestrial lithophytic (rock) communities. In: Friedmann EI, editor. Antarctic Microbiology. New York: Wiley-Liss; 1993. p. 343–412. Onofri S, de la Torre R, de Vera JP, Ott S, Zucconi L, Selbmann L, Scalzi G, Venkateswaran KJ, Rabbow E, Horneck G. Survival of rock-colonizing organisms after 1.5 years in outer space. Astrobiology 2012;12(5):508–16. Onofri S, Castagnola M, Rossi Espagnet S. L'impiego della microscopia elettronica a scansione in micologia. Micol Ital 1980;1:29–32. Onofri S, Selbmann L, Zucconi L, Pagano S. Antarctic microfungi as models for exobiology. Planet Space Sci 2004;52:229–37. Onofri S, Selbmann L, Zucconi L, de Hoog GS, de los Rios A, Ruisi S, et al. Fungal associations at the cold edge of life. In: Seckbach J, editor. Algae and cyanobacteria in extreme environments. Netherlands: Springer; 2007a. p. 735–57. Onofri S, Zucconi L, Tosi S. Continental Antarctic Fungi. Eching bei Munchen: IHM-Verlag; 2007b. Onofri S, Barreca D, Selbmann L, Isola D, Rabbow E, Horneck G, et al. Resistance of Antarctic black fungi and cryptoendolithic communities to simulated space and Martian conditions. Stud Mycol 2008;61:99-109. Øvstedal DO, Lewis Smith RI. Lichens of Antarctica and South Georgia. In: Øvstedal DO, Lewis Smith RI, editors. A guide to their identification and ecology. Studies in Polar Research, University of Cambridge; 2001. p. 4–5. Parmesan C, Yohe G. A globally coherent fingerprint of climate change impacts across natural systems. Nature 2003;421:37–42. Pimm SL. Biodiversity: climate change or habitat loss–which will kill more species? Curr Biol 2008;18:117–9. Root TL, Price JT, Hall KR, Schneider SH, Rosenzweig C, Pounds JA. Fingerprints of global warming on wild animals and plants. Nature 2003;421:57–60.

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L. Selbmann et al. / Science of the Total Environment 438 (2012) 127–134

Roots EF. Climate change: high latitude regions. Clim Chang 1989;14:223–50. Ruibal C. Isolation and characterization of melanized, slow-growing fungi from semiarid rock surfaces of central Spain and Mallorca. Dissertation, Universidad Autónoma de Madrid, 2004. Ruibal C, Platas G, Bills GF. Isolation and characterization of melanised fungi from limestone formations in Mallorca. Mycol Prog 2005;4:23–38. Ruibal C, Platas G, Bills GF. High diversity and morphological convergence among melanised fungi from rock formations in the Central Mountain System of Spain. Persoonia 2008;21:93-110. Ruisi S, Barreca D, Selbmann L, Zucconi L, Onofri S. Fungi in Antarctica. Rev Environ Sci Biotechnol 2007;6:127–41. Selbmann L, de Hoog GS, Mazzaglia A, Friedmann EI, Onofri S. Fungi at the edge of life: cryptoendolithic black fungi from Antarctic deserts. Stud Mycol 2005;51:1-32. Selbmann L, de Hoog GS, Zucconi L, Isola D, Ruisi S, Gerrits van den Ende AHG, et al. Drought meets acid: three new genera in a dothidealean clade of extremotolerant fungi. Stud Mycol 2008;61:1-20. Selbmann L, Isola D, Zucconi L, Onofri S. Resistance to UV-B induced DNA damage in extreme-tolerant cryptoendolithic Antarctic fungi: detection by PCR assays. Fungal Biol 2011;115:937–44. Sert HB, Sümbül H, Sterflinger K. Microcolonial fungi from antique marbles in Perge/Side/Termessos (Antalya/Turkey). Antonie Van Leeuwenhoek 2007;91:217–27. Steig EJ, Schneider DP, Rutherford SD, Mann ME, Comiso JC, Shindell DT. Warming of the Antarctic ice-sheet surface since the 1957 International Geophysical Year. Nature 2009;457:459–62. Sterflinger K. Temperature and NaCl-tolerance of rock-inhabiting meristematic fungi. Antonie Van Leeuwenhoek 1998;74:271–81. Sterflinger K. Black yeasts and meristematic fungi: ecology, diversity and identification. In: Seckbach J, editor. The yeast handbook. Biodiversity and ecophysiology of yeastsBerlin: Springer-Verlag; 2005. p. 501–14.

Sterflinger K, Prillinger H. Molecular taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria). Antonie Van Leeuwenhoek 2001;80(3–4):275–86. Sterflinger K, Tesei D, Zakharova K. Fungi in hot and cold deserts with particular reference to microcolonal fungi. Fungal Ecol 2012;5:379–480. Tesei D, Marzban G, Zakharova K, Isola D, Selbmann L, Sterflinger K. Monitoring of stress-related protein expression black fungi from hot and cold environments. Fungal Biol in press, http://dx.doi.org/10.1016/j.funbio.2012.06.004. Thomas CD, Cameron A, Green RE, Bakkenes M, Beaumont LJ, Williams SE. Extinction risk from climate change. Nature 2004;427:145–8. Turner J, Colwell SR, Marshall GJ, Lachlan-Cope TA, Carleton AM, Jones PD, et al. Antarctic climate change during the last 50 years. Int J Climatol 2005;25:279–94. Wollenzien U, de Hoog GS, Krumbein WE, Urzì C. On the isolation of microcolonial fungi occurring on and in marble and other calcareous rocks. Sci Total Environ 1995;167:287–94. Zare R, Gams W. A revision of Verticillium section Prostrata. IV. The genera Lecanicillium and Simplicillium gen. nov. Nova Hedwigia 2001;73:1-50. Zucconi L, Pagano S, Fenice M, Selbmann L, Tosi S, Onofri S. Growth temperature preferences of fungal strains from Victoria Land, Antarctica. Polar Biol 1996;16: 53–61. Zucconi L, Selbmann L, Buzzini P, Turchetti B, Guglielmin M, Frisvad JC, et al. Searching for eukaryotic life preserved in Antarctic permafrost. Polar Biol 2011;5:749–57. Zucconi L, Gagliardi M, Isola D, Onofri S, Andaloro MC, Pelosi C, et al. Biodeteriorigenous agents dwelling the wall paintings of the Holy Saviour's Cave (Vallerano, Italy). Int J Biodet Biodegrad 2012;70:40–6.