Extremotolerant fungi from alpine rock lichens and their phylogenetic relationships

Martin Grube

Many black meristematic fungi persist on rock surfaces—hostile and exposed habitats where high doses of radiation and periods of desiccation alternate with rain and temperature extremes. To cope with these extremes, rock-inhabiting black fungi show phenotypic plasticity and produce melanin as cell wall pigments. The rather slow growth rate seems to be an additional prerequisite to oligotrophic conditions. At least some of these fungi can undergo facultative, lichen-like associations with photoautotrophs. Certain genera presenting different lifestyles are phylogenetic related among the superclass Dothideomyceta. In this paper, we focus on the genus Lichenothelia, which includes border-line lichens, that is, associations of melanised fungi with algae without forming proper lichen thalli. We provide a first phylogenetic hypothesis to show that Lichenothelia belongs to the superclass Dothideomyceta. Further, culture experiments revealed the presence of co-occurring fungi in Lichenothelia thalli. These fungi are related to plant pathogenic fungi (Mycosphaerellaceae) and to other rock-inhabiting lineages (Teratosphaeriaceae). The Lichenothelia thallus-forming fungi represent therefore consortia of different black fungal strains. Our results suggest a common link between rock-inhabiting meristematic and lichen-forming lifestyles of ascomycetous fungi.

Rock-inhabiting fungi (RIF) are melanized, meristematic fungi which dwell on and within rocks and have adapted to withstand harsh conditions in extreme habitats worldwide. Their morphological and genetic diversity remained unknown for a long time, but in the past few years culture-dependent and molecular phylogenetic approaches have contributed to uncovering the species richness of these otherwise very inconspicuous fungi. Only a few taxa of RIF develop both sexual reproductive structure (fertile stromata and/or pycnidia) and show multiple life styles, interacting with algae and lichen thalli in different ways. The genus Lichenothelia is one of these: It is characterized by fertile stromata and pycnidia and by species which can grow on and within exposed rocks, optionally associating with algae, with some species also being lichenicolous. The genus Lichenothelia includes up to now 25 species and form a monotypic family (Lichenotheliaceae) and order (Lichenotheliales) in Dothideomyce...

Lichenicolous fungi are obligately lichen-associated organisms that have evolved many times throughout the Ascomycota and Basidiomycota. Approximately 20% of lichenicolous ascomycetes are recognized only from asexual (anamorphic) characteristics, so the phylogenetic position of many groups has never been resolved. Here we present the first molecular phylogeny of Lichenoconium, a genus of strictly asexual, obligately lichenicolous species with broad geographic distributions and diverse host ecologies. We obtained nuclear and mitochondrial rDNA sequences from fungal cultures isolated from four species in the genus, including a new species, Lichenoconium aeruginosum sp. nov., collected in France, Luxembourg and Netherlands. Our multilocus phylogeny supports the monophyly of fungi in the genus Lichenoconium, and places the genus in the Dothideomycetes, an ascomycete class made up mainly of saprobes and plant-associated endophytes and pathogens. There are only a few recognized groups of lichen-formers in the Dothideomycetes, but Lichenoconium is not supported as being closely related to any of these, nor to any other recognized order within the Dothideomycetes. Given that Lichenoconium is but one of over 100 genera of anamorphic lichenicolous fungi, most of which have never been studied phylogenetically, we suggest that asexual lichenicolous fungi may represent novel and evolutionarily significant phylogenetic groups in the Kingdom Fungi.

Fungal Diversity (2016) 76:119–142 DOI 10.1007/s13225-015-0343-8 Extremotolerant fungi from alpine rock lichens and their phylogenetic relationships Lucia Muggia 1,2 & Antonia Fleischhacker 1 & Theodora Kopun 1 & Martin Grube 1 Received: 20 March 2015 / Accepted: 6 August 2015 / Published online: 22 August 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Fungi other than the lichen mycobiont frequently co-occur within lichen thalli and on the same rock in harsh environments. In these situations dark-pigmented mycelial structures are commonly observed on lichen thalli, where they persist under the same stressful conditions as their hosts. Here we used a comprehensive sampling of lichen-associated fungi from an alpine habitat to assess their phylogenetic relationships with fungi previously known from other niches. The multilocus phylogenetic analyses suggest that most of the 248 isolates belong to the Chaetothyriomycetes and Dothideomycetes, while a minor fraction represents Sordariomycetes and Leotiomycetes. As many lichens also were infected by phenotypically distinct lichenicolous fungi of diverse lineages, it remains difficult to assess whether the culture isolates represent these fungi or are from additional cryptic, extremotolerant fungi within the thalli. Some of these strains represent yet undescribed lineages within Chaethothyriomycetes and Dothideomycetes, whereas other strains belong to genera of fungi, that are known as lichen colonizers, plant and human pathogens, rock-inhabiting fungi, parasites and saprotrophs. The symbiotic structures of the lichen thalli appear to be a shared habitat of phylogenetically Electronic supplementary material The online version of this article (doi:10.1007/s13225-015-0343-8) contains supplementary material, which is available to authorized users. * Lucia Muggia lmuggia@units.it; lucia_muggia@hotmail.com 1 Institute of Plant Sciences, University of Graz, Holteigasse 6, 8010 Graz, Austria 2 Department of Life Sciences, Università degli Studi di Trieste, Via Valerio 12/2, 34128 Trieste, Italy diverse stress-tolerant fungi, which potentially benefit from the lichen niche in otherwise hostile habitats. Keywords Black fungi . Endolichenic . Symbioses . Lichenicolous . Life style . Phylogeny Introduction Bare rock surfaces provide little comfort to life. They are poor sources of nutrients and are constantly exposed to a variety of extremes in abiotic conditions. Variations in surface temperatures and water availability can occur at very short time spans and be the source of diverse stresses (Zakharova et al. 2013; Sterflinger et al. 2012), and with enormous amplitudes. In addition, direct exposure to full sunlight includes a threatening level of energy-rich ultraviolet wavelengths. Not many organisms can cope with such surfaces at this Bedge of life^, thus these surfaces are colonized by specialists with particular adaptations (Selbmann et al. 2005, 2013; Onofri et al. 2007; Marzban et al. 2013). In fact, some fungal lineages, which are known as Bblack fungi^ or Bmicrocolonial fungi^ are among the most stress-resistant eukaryotic organisms on Earth and can occur at considerable diversity on rocks (Ruibal et al. 2005, 2009). The adaptations of these rockinhabiting fungi (RIF) include pleomorphic growth, efficient osmolyte management, melanin production, biofilm formation, and survival in cryptobiotic stage (Gostincar et al. 2010, 2011). Black fungi do not form a monophyletic lineage but are members of Dothideomycetes and Chaetothyriomycetidae (Gueidan et al. 2008; Ruibal et al. 2009) which evolved during periods of dry climate in the late Devonian and middle Triassic, respectively (Gueidan et al. 2011). At approximately the same time scale, the symbiotic association whereby a 120 fungus shelters microscopic algae or cyanobacteria in exchange for fixed carbon and nitrogen helped to ameliorate nutrient deficiencies on rocks. The lichen symbiosis was this key innovation in the evolution of fungi and lichenized mycobionts have since evolved and diversified (Lutzoni et al. 2001; Hawksworth 2015). Particularly, in alpine altitudes where conditions prevent the development of higher plants, lichen thalli express their phenotypic and phylogenetic diversity and shape the landscapes with colorful mosaics on rock surfaces. In such variably stressed situations, black fungi and lichens can occur side by side on rock, and black fungi also colonize asymptomatic lichens, especially in arid situations (Harutyunyan et al. 2008). Harutyunyan et al. (2008) showed that black fungi may opportunistically infect lichens, but do not cause damage to their host thalli. Some of the fungi resemble hyphomycetous lichenicolous fungi. However, most lichenicolous fungi have a host specific occurrence and are recognized by their phenotypic symptoms and their sexual or asexual spore-producing structures (Hawksworth 1979; Lawrey and Diederich 2003). It is not known whether black fungi, cryptically colonizing lichen thalli, are directly in contact with the photobiont to obtain nutrients. Some studies, however, suggest that black fungi indeed have some affinity to microscopic algae (Brunauer et al. 2007; Gorbushina et al. 2005). Arnold et al. (2009) also used micro-dissection followed by surface sterilization to show that more culturable fungi were associated with the algal layer compared to the medulla and cortex. In this study, we conducted a comprehensive sampling of saxicolous lichen species (as reported in Fleischhacker et al. 2015), including samples infected by symptoms-developing lichenicolous fungi from different sites of an alpine range, above the tree-line. We prepared culture isolates of the fungi and produced sequence data for phylogenetic analyses. With these we aimed at answering the following questions: i) are there patterns of co-occurrence among cryptic, black extremotolerant fungi, symptomatic lichenicolous fungi and lichen mycobionts?; ii) are lichen-associated fungal communities structured by mycobiont host? ; iii) what is the phylogenetic placement of the isolated strains? Material and methods Sampling Lichen thalli were collected on the Koralpe mountain range in the southeastern rim of the Austrian Alps , between the states Styria and Carinthia. The sampling was carried out as in Fleischhacker et al. (2015). Ten collection sites (plots), each further divided into 3 subplots, were selected in alpine habitat, above the timberline, ranging between 1800 and 2100 m a.s.l., and are characterized by big boulders and cliffs of homogeneous size of siliceous-schist/ gneissic rocks Fungal Diversity (2016) 76:119–142 separated by wide areas of pastures or dwarf shrub formations. Here winds, in particular from South and West, reach speeds over 120 km/h and the annual temperature averages 0-5 °C (http://www.umwelt.steiermark.at/cms/beitrag/10023583/ 25206/). In winter, rocks can remain covered by wind-pressed snow and ice for several weeks; alternatively, in summer the south-exposed rock surfaces receive intense solar radiation. In these sites, boulders’ surfaces are almost entirely colonized by crust-forming (90%), foliose and fruticose (10%) lichens. Crust-forming and foliose lichens were selected for the culture isolation experiment: (i) crustose thalli are composed by contiguous islands of thallus (areoles) which tightly adhere to the substrate with their entire lower surfaces; (ii) foliose thalli adhere only partly to the substrate by a central holdfast (umbilicus) or by root-like appendices (rhizines). About 10% of the lichen thalli in this region are infected by lichenicolous fungi (Fleischhacker et al. 2015). For the isolation of lichenicolous and extremotolerant fungi we selected multiple lichen thalli of different species visibly infected by different species of symptomatic lichenicolous fungi (Tables 1, 2, S3). In doing so, we aimed at including a comprehensive survey of the different lichenicolous fungus-lichen host associations occurring in the area. Within the same subplot, we selected up to four different symptomatically infected thalli. These were either lying close to each other or lying apart up to 50 cm. The lichen thalli were sampled together with their substratum by chiseling the piece of rock. We sampled on both horizontal and vertical positions and at different expositions. Culture isolation A total of 130 lichen samples, comprising 25 different lichenicolous fungus-lichen host associations, were selected for culture isolations. Thallus areoles or lobes presenting lichenicolous fungal infections were removed with a sterile razor blade and put into an Eppendorf tube. The isolation protocol followed Yamamoto et al. (2002). The pieces, about 2 mm2, were washed three times for 15 minutes in distilled sterile water on a shaking bath, followed by a 30 minutes washing step with 500 μl of 1:10 dilution of Tween 80 to remove the possible external contaminations of bacteria and yeast (Bubrick and Galun 1986). A final washing step was carried out twice in distilled sterile water for 15 minutes. The clean fragments were dissected under the stereomicroscope using a sterile razor blade and single pieces were picked with a sterile needle, moistured with distilled sterile water, and transferred into agar tubes. In order to promote the growth of different fungi we inoculated the dissected fragments on six different media: Trebouxia Medium (TM, Ahmadjian 1967), Malt Yeast Extract Medium (MY, Ahmadjian 1967), Lilly and Barnett´s Medium (LBM, Lilly and Barnett 1951), Potato Glucose Agar (PGA; Sigma), Dichloran-Glycerol 18%-Agar (DG18; Sigma), Sabouraud-Agar (SAB; Sigma). Four tubes of the same medium were inoculated, resulting in a total of 24 tubes (inocula) for each original sample. The tubes were Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A46 (LBM) A46 (SAB) A135 (KGA) A343 (LBM) A343 (SAB) A343 (SAB) A343 (KGA) A343 (TM) Tephromela atra Tephromela atra Lecanora intricata Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Taeniolella atricerebrina Taeniolella atricerebrina Muellerella - Li Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae A573 A589 A515 A859 A860 A861 A862 A893 LMCC0184 LMCC0197 LMCC0136 LMCC0208 LMCC0233 LMCC0234 LMCC0235 LMCC0217 KT263034 KT263035 KT263033 KT263036 KT263037 KT263038 KT263039 KT263040 KT263047 KT263048 KT263046 KT263049 KT263050 KT263051 KT263052 KT263053 KT263060 KT263061 KT263059 KT263062 KT263063 KT263064 KT263065 KT263066 clade I clade I clade I clade I clade I clade I clade I clade I A343 (LBM) A343 (TM) Lecanora polytropa Lecanora polytropa Lichenoconium lecanorae Lichenoconium lecanorae A916 A921 LMCC0265 LMCC0266 KT263041 KT263042 KT263054 KT263055 KT263067 KT263068 clade I clade I A343 (MY) A343 (KGA) Lecanora polytropa Lecanora polytropa Lichenoconium lecanorae Lichenoconium lecanorae A922 A936 LMCC0267 LMCC0276 KT263043 KT263044 KT263056 KT263057 KT263069 KT263070 clade I clade I A666 (SAB) A97 (KGA) Rhizocarpon geographicum Rhizocarpon geographicum Endococcus macrosporus Muellerella - Rh A1022 A944 LMCC0346 LMCC0283 KT263045 KT263072 KT263058 KT263094 KT263071 KT263110 clade I clade II A97 (KGA) A263 (SAB) A385 (TMY) A385 (MY) A46 (SAB) Rhizocarpon geographicum Rhizocarpon geographicum Rhizocarpon geographicum Rhizocarpon geographicum Tephromela atra A994 A993 A1003 A1015 A528 LMCC0332 LMCC0331 LMCC0364 LMCC0340 LMCC0148 KT263074 KT263073 KT263075 KT263076 KT263088 – KT263095 – KT263096 KT263104 KT263112 KT263111 KT263113 KT263114 KT263123 clade II clade II clade II clade II clade III A64 (TM) A64 (LBM) Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Schaereria fuscocinerea Schaerera fuscocinerea Muellerella - Rh Muellerella - Rh Muellerella - Rh Muellerella - Rh T. atricerebrina (+ Miutoexcipula tephromelae) Lichenodiplis lecanorae Lichenodiplis lecanorae Muellerella atricola Muellerella atricola Muellerella atricola Endococcus perpusillus Endococcus perpusillus L1858 L1860 L1992 L1993 L1994 A511 A570 – LMCC0513 LMCC0066 LMCC0487 LMCC0515 LMCC0132 LMCC0181 KT263086 KT263087 KT263083 KT263084 KT263085 KT263126 KT263158 KT263100 KT263101 – KT263102 KT263103 KT263171 KT263202 KT263118 KT263119 KT263120 KT263121 KT263122 KT263215 KT263246 clade III clade III clade III clade III clade III – – A65 (TM) A72 (SAB) A100 (MY) A198 (KGA) A198 (KGA) Lecanora polytropa Lecanora polytropa Umbilicaria cylindrica Lecanora polytropa Lecanora polytropa Muellerella - Lp Carbonea supersparsa Stigmidium gyrophorarum Muellerella - Lp Muellerella - Lp A891 A514 A584 A961 A1010 LMCC0253 LMCC0135 LMCC0193 LMCC0311 LMCC0367 KT270616 KT263128 KT263164 KT270661 KT263332 KT270704 KT263173 KT263208 – KT263365 KT270786 KT263217 KT263252 KT270830 KT263397 – – – – – 121 Lichen thallus ID (medium name) Fungal Diversity (2016) 76:119–142 Table 1 List of isolates recovered in Eurotiomycetes (Chaetothyriomicetidae). The isolates are identified by their DNA extraction numbers. Number of the original lichen thallus (growth medium of inoculation), name of the lichen, name of the associated lichenicolous fungus, culture collection number and the newly published NCBI accession numbers are reported. Samples in bold are those selected as representatives in the analysis of Fig. 1. The affiliation (clade name) of the other isolates is reported based on the initial analysis including all the isolates. Dash (−) indicate loss of culture due to unsuccessful subsequent growth 122 Table 1 (continued) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A267 (KGA) A267 (TM) A267 (TM) A267 (TM) A319 (TM) A319 (SAB) A329 (TM) Aspicilia Aspicilia Aspicilia Aspicilia Lecidea sp. Lecidea sp. Aspicilia Endococcus verrucosus Endococcus verrucosus Endococcus verrucosus Endococcus verrucosus Muellerella pygmaea Muellerella pygmaea Endococcus verrucosus A885 A903 A911 A949 A873 A875 A924 LMCC0248 LMCC0223 LMCC0230 LMCC0286 LMCC0242 LMCC0264 LMCC0261 KT270614 KT270624 KT270629 KT270653 KT270602 KT270604 KT270635 KT270702 KT270712 KT270717 KT2707238 KT270690 KT270692 KT270724 KT270784 KT270794 KT270799 KT270822 KT270772 KT270774 KT270804 – – – – – – – A329 (LBM) A347 (TM) Aspicilia Lecidea lapicida Endococcus verrucosus Cecidonia umbonella A939 A865 LMCC0278 LMCC0238 KT270644 KT270594 – KT270682 KT270813 KT270764 – – A347 (KGA) A390 (LBM) Lecidea lapicida Rhizocarpon geographicum Cecidonia umbonella Endococcus macrosporus A866 A956 LMCC0239 LMCC0292 KT270595 KT270658 KT270683 KT270742 KT270765 KT270827 – – A440 (MY) A23 (TM) A65 (TM) Tephromela atra Lecanora intricata Lecanora polytropa Muellerella atricola Muellerella - Li Muellerella - Lp A1053 A989 A516 LMCC0385 LMCC0330 LMCC0137 KT263356 KT270678 KT263130 KT263388 KT270760 KT263174 KT263420 KT270847 KT263218 – clade IV clade IV A65 (TM) A65 (LBM) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A522 A531 LMCC0142 LMCC0150 KT263134 KT263140 KT263178 KT263184 KT263222 KT263228 clade IV clade IV A65 (SAB) A65 (MY) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A532 A539 LMCC0151 LMCC0157 KT263141 KT263146 KT263185 KT263190 KT263229 KT263234 clade IV clade IV A65 (TM) A65 (SAB) A65 (LBM) A84 (MY) A84 (TM) A84 (TM) A84 (LBM) A135 (TM) A237 (LBM) A237 (KGA) A194 (LBM) A194 (SAB) A254 (SAB) A675 (KGA) A832 (KGA) A94 (LBM) Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Rhizocarpon geographicum Rhizocarpon geographicum Rhizocarpon geographicum Rhizocarpon geographicum Lecanora polytropa Tephromela atra Lecanora bicincta Lecanora intricata Muellerella - Lp Muellerella - Lp Muellerella - Lp Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae Muellerella - Lp Muellerella - Rh Muellerella - Rh Endococcus macrosporus Endococcus macrosporus Muellerella - Lp Taeniolella atricerebrina Arthonia varians Muellerella - Li A541 A547 A548 A517 A520 A533 A909 A950 A895 A929 A889 A918 A1045 A980 A969 A587 LMCC0158 – LMCC0163 LMCC0138 LMCC0140 LMCC0191 LMCC0228 LMCC0287 LMCC0219 LMCC0262 LMCC0251 LMCC0361 LMCC0377 LMCC0317 LMCC0300 LMCC0195 KT263147 KT263151 KT263152 KT263131 KT263132 KT263142 KT270628 KT270654 KT270619 KT270640 KT270615 KT270632 KT263349 KT270672 KT270665 KT263165 KT263191 KT263195 KT263196 KT263175 KT263176 KT263186 KT270716 KT270739 KT270707 KT270728 KT270703 KT270721 KT263381 KT270754 KT270748 KT263209 KT263235 KT263239 KT263240 KT263219 KT263220 KT263230 KT270798 KT270823 KT270789 KT270809 KT270785 KT270801 KT263413 KT270841 KT270834 KT263253 clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade IV clade V Fungal Diversity (2016) 76:119–142 Lichen thallus ID (medium name) Lichen thallus ID (medium name) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A122 (MY) A122 (LBM) A267 (MY) A267 (SAB) A307 (LBM) A307 (LBM) A309 (LBM) Aspicilia caesiocinerea Aspicilia caesiocinerea Aspicilia Aspicilia Lecanora intricata Lecanora intricata Rhizocarpon geographicum Endococcus rugulosus Endococcus rugulosus Endococcus verrucosus Endococcus verrucosus Muellerella - Li Muellerella - Li Endococcus macrosporus A521 A574 A952 A1000 A879 A884 A912 LMCC0141 LMCC0185 LMCC0289 LMCC0333 LMCC0246 LMCC0247 LMCC0256 KT263133 KT263160 KT270655 KT263328 KT270608 KT270613 KT270630 KT263177 KT263204 – KT263361 KT270696 KT270701 KT270718 KT263221 KT263248 KT270824 KT263393 KT270778 KT270783 KT270800 clade V clade V clade V clade V clade V clade V clade V A309 (TM) A309 (SAB) Rhizocarpon geographicum Rhizocarpon geographicum Endococcus macrosporus Endococcus macrosporus A914 A920 LMCC0257 LMCC0260 KT270631 KT270634 KT270719 KT270723 – KT270803 clade V clade V A329 (TM) A329 (TM) Aspicilia Aspicilia Endococcus verrucosus Endococcus verrucosus A926 A927 LMCC0304 LMCC0270 KT270637 KT270638 KT270726 – KT270806 KT270807 clade V clade V A329 (LBM) A352 (KGA) A352 (TM) Aspicilia Lecanora polytropa Lecanora polytropa Endococcus verrucosus Cercidospora epipolytropa Cercidospora epipolytropa A928 A945 A946 LMCC0271 LMCC0284 LMCC0307 KT270639 KT270649 KT270650 KT270727 KT270735 KT270736 KT270808 KT270818 KT270819 clade V clade V clade V A398 (SAB) A666 (SAB) Lecidea lapicida Rhizocarpon geographicum Cecidonia umbonella Endococcus macrosporus A955 A1025 LMCC0291 LMCC0348 KT270657 KT263337 KT270741 KT263370 KT270826 – clade V clade V A703 (SAB) A703 (LBM) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A983 A987 LMCC0302 LMCC0321 KT270674 KT270676 KT270756 KT270758 KT270843 KT270845 clade V clade V A703 (LBM) A12 (MY) A12 (SAB) A12 (TM) A12 (TM) A12 (SAB) A12 (LBM) A12 (LBM) A12 (LBM) A37 (TM) A46 (LBM) A46 (SAB) A94 (SAB) A94 (LBM) A94 (MY) A100 (TM) Lecanora polytropa Lecidea sp. Muellerella - Lp Muellerella pygmaea A974 A526 Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Tephromela atra Tephromela atra Tephromela atra Lecanora intricata Lecanora intricata Lecanora intricata Umbilicaria cylindrica Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Taeniolella atricerebrina Taeniolella atricerebrina Taeniolella atricerebrina Muellerella -Li Muellerella - Li Muellerella -Li Stigmidium gyrophorarum A527 A530 A535 A536 A544 A546 A892 A898 A525 A572 A575 A576 A581 A564 LMCC0314 LMCC0146 LMCC0147 – LMCC0153 LMCC0154 LMCC0161 – LMCC0216 – LMCC0393 LMCC0183 LMCC0186 LMCC0187 LMCC0192 LMCC0175 KT270668 KT263136 KT263137 KT263139 KT263143 KT263144 KT263149 KT263150 KT270617 KT270621 KT263135 KT263159 KT263161 KT263162 KT263163 KT263155 KT270751 KT263180 KT263181 KT263183 KT263187 KT263188 KT263193 KT263194 KT270705 KT270709 KT263179 KT263203 KT263205 KT263206 KT263207 KT263199 KT270837 KT263224 KT263225 KT263227 KT263231 KT263232 KT263237 KT263238 KT270787 KT270791 KT263223 KT263225 KT263249 KT263250 KT263251 KT263243 clade V clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI Fungal Diversity (2016) 76:119–142 Table 1 (continued) 123 124 Table 1 (continued) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A100 (KGA) A106 (SAB) A149 (TM) A173 (SAB) A173 (SAB) A173 (TM) A173 (KGA) Umbilicaria cylindrica Tephromela atra Protoparmela badia Lecanora polytropa Lecanora polytropa Lecanora polytropa Lecanora polytropa Stigmidium gyrophorarum Taeniolella atricerebrina Phacographa protoparmeliae Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae Lichenoconium lecanorae A566 A562 A555 A513 A529 A538 A943 LMCC0177 LMCC0173 LMCC0166 LMCC0134 LMCC0149 LMCC0156 LMCC0282 KT263156 KT263154 KT263153 KT263127 KT263138 KT263145 KT270648 KT263200 KT263198 KT263197 KT263172 KT263182 KT263189 KT270734 KT263244 KT263242 KT263241 KT263216 KT263226 KT263233 KT270817 clade VI clade VI clade VI clade VII clade VII clade VII clade VII A184 (MY) A184 (SAB) Tephromela atra Tephromela atra Taeniolella atricerebrina Taeniolella atricerebrina A543 A596 LMCC0160 LMCC0203 KT263148 KT263167 KT263192 KT263211 KT263236 KT263255 clade VI clade VI A184 (LBM) A184 (KGA) Tephromela atra Tephromela atra Taeniolella atricerebrina Taeniolella atricerebrina A597 A599 LMCC0204 LMCC0206 KT263168 KT263170 KT263212 KT263214 KT263256 KT263258 clade VI clade VI A198 (TM) A215 (KGA) A215 (TM) Rhizocarpon geographicum Lecanora polytropa Lecanora polytropa Endococcus macrosporus Lichenoconium lecanorae Lichenoconium lecanorae A933 A598 A594 LMCC0274 LMCC0205 LMCC0201 KT270641 KT263169 KT263166 – KT263213 KT263210 KT270810 KT263257 KT263254 clade VII clade VI clade VI A241 (KGA) A254 (LBM) Tephromela atra Lecanora polytropa Taeniolella atricerebrina Muellerella - Lp A894 A906 LMCC0218 LMCC0226 KT270618 KT270627 KT270706 KT270715 KT270788 KT270797 clade VI clade VI A254 (KGA) A254 (SAB) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A953 A962 LMCC0309 LMCC0296 KT270656 KT270662 KT270740 KT270745 KT270825 KT270831 clade VI clade VI A263 (LBM) A280 (KGA) A280 (MY) A280 (LBM) A280 (LBM) A280 (SAB) A280 (TM) A280 (SAB) A296 (MY) A296 (SAB) A319 (SAB) A319 (MY) A319 (TM) A319 (TM) A319 (SAB) A319 (LBM) Rhizocarpon geographicum Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Rhizocarpon geographicum Rhizocarpon geographicum Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Lecidea sp. Muellerella - Rh Skyttea tephromelarum Skyttea tephromelarum Skyttea tephromelarum Skyttea tephromelarum Skyttea tephromelarum Skyttea tephromelarum Skyttea tephromelarum Endococcus macrosporus Endococcus macrosporus Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea A878 A880 A881 A882 A883 A896 A900 A942 A934 A948 A869 A874 A938 A940 A941 A998 LMCC0245 LMCC0212 LMCC0213 LMCC0214 LMCC0215 – LMCC0220 LMCC0281 LMCC0275 LMCC0308 LMCC0263 LMCC0243 LMCC0277 LMCC0279 LMCC0280 LMCC0325 KT270607 KT270609 KT270610 KT270611 KT270612 KT270620 KT270622 KT270647 KT270642 KT270652 KT270598 KT270603 KT270643 KT270645 KT270646 KT270670 KT270695 KT270697 KT270698 KT270699 KT270697 KT270708 KT270710 KT270733 KT270729 KT270737 KT270686 KT270691 KT270730 KT270731 KT270732 KT270761 KT270777 KT270779 KT270780 KT270781 KT270782 KT270790 KT270792 KT270816 KT270811 KT270821 KT270768 KT270773 KT270812 KT270814 KT2708145 KT270848 clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI clade VI Fungal Diversity (2016) 76:119–142 Lichen thallus ID (medium name) Lichen thallus ID (medium name) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A329 (MY) A329 (LBM) A329 (KGA) A347 (TM) A347 (KGA) A347 (KGA) A347 (LBM) Aspicilia Aspicilia Aspicilia Lecidea lapicida Lecidea lapicida Lecidea lapicida Lecidea lapicida Endococcus verrucosus Endococcus verrucosus Endococcus verrucosus Cecidonia umbonella Cecidonia umbonella Cecidonia umbonella Cecidonia umbonella A925 A947 A1013 A864 A867 A868 A870 LMCC0269 LMCC0285 LMCC0338 LMCC0237 LMCC0240 LMCC0209 LMCC0210 KT270636 KT270651 KT263333 KT270593 KT270596 KT270597 KT270599 KT270725 – KT263366 KT270681 KT270684 KT270685 KT270687 KT270805 KT270820 KT263398 KT270763 KT270766 KT270767 KT270769 clade VI clade VI clade VI clade VI clade VI clade VI clade VI A347 (SAB) A347 (LBM) Lecidea lapicida Lecidea lapicida Cecidonia umbonella Cecidonia umbonella A871 A872 LMCC0211 LMCC0241 KT270600 KT270601 KT270688 KT270689 KT270770 KT270771 clade VI clade VI A347 (TM) A347 (KGA) Lecidea lapicida Lecidea lapicida Cecidonia umbonella Cecidonia umbonella A876 A877 – LMCC0244 KT270605 KT270606 KT270693 KT270694 KT270775 KT270776 clade VI clade VI A357 (TM) A357 (TM) A357 (SAB) Lecidea sp. Lecidea sp. Lecidea sp. Muellerella pygmaea Muellerella pygmaea Muellerella pygmaea A904 A905 A958 LMCC0224 LMCC0225 LMCC0294 KT270625 KT270626 KT270660 KT270713 KT270714 KT270744 KT270795 KT270796 KT270829 clade VI clade VI clade VI A357 (LBM) A357 (KGA) Lecidea sp. Lecidea sp. Muellerella pygmaea Muellerella pygmaea A1002 A1004 LMCC0334 LMCC0335 KT263329 KT263330 KT263362 KT263363 KT263394 KT263395 clade VI clade VI A357 (LBM) A357 (TM) Lecidea sp. Lecidea sp. Muellerella pygmaea Muellerella pygmaea A1018 A1019 LMCC0342 LMCC0343 KT263334 KT263335 KT263367 KT263368 KT263399 KT263400 clade VI clade VI A373 (KGA) A373 (TM) A408 (LBM) A408 (TM) A426 (KGA) A440 (SAB) A440 (LBM) A440 (LBM) A440 (LBM) A464 (LBM) A464 (MY) A469 (SAB) A475 (LBM) A613 (KGA) A643 (KGA) A651 (TM) Lecidea sp. Lecidea sp. Muellerella pygmaea Muellerella pygmaea A957 A1030 Rhizocarpon geographicum Rhizocarpon geographicum Lecanora polytropa Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Tephromela atra Rhizocarpon geographicum Tephromela atra Schaereria fuscocinerea Lecidea sp. Lecanora polytropa Endococcus macrosporus Endococcus macrosporus Muellerella - Lp Muellerella atricola Muellerella atricola Muellerella atricola Muellerella atricola Skyttea tephromelarum Skyttea tephromelarum Opegrapha geographicola Taeniolella atricerebrina Muellerella - Sf Muellerella pygmaea Carbonea supersparsa A901 A919 A1049 A1050 A1051 A1052 A1054 A1058 A1059 A1060 A1008 A986 A1029 A1020 LMCC0293 LMCC0352 LMCC0221 LMCC0259 LMCC0381 LMCC0382 LMCC0383 LMCC0384 LMCC0386 LMCC0390 LMCC0391 – LMCC0337 LMCC0320 LMCC0351 LMCC0344 KT270659 KT263340 KT270623 KT270633 KT263352 KT263353 KT263354 KT263355 KT263357 KT263358 KT263359 KT263360 KT263331 KT270675 KT263339 KT263336 KT270743 KT263373 KT270711 KT270722 KT263384 KT263385 KT263386 KT263387 KT263389 KT263390 KT263391 KT263392 KT263364 KT270757 KT263372 KT263369 KT270828 KT263404 KT270793 KT270802 KT263416 KT263417 KT263418 KT263419 – KT263421 KT263422 KT263423 KT263396 KT270844 KT263403 KT263401 clade VI clade VI clade VI clade VII clade VII clade VII clade VII clade VII clade VII clade VI clade VI clade VI clade VI clade VI clade VI clade VI Fungal Diversity (2016) 76:119–142 Table 1 (continued) 125 126 Table 1 (continued) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nucLSU nucSSU mt-SSU clade ID A651 (KGA) A653 (MY) A663 (LBM) A663 (TM) A670 (KGA) A670 (TM) A683 (SAB) Lecanora polytropa Tephromela atra Tephromela atra Tephromela atra Lecanora polytropa Lecanora polytropa Lecanora polytropa Carbonea supersparsa Taeniolella atricerebrina Muellerella atricola Muellerella atricola Muellerella - Lp Muellerella - Lp Muellerella - Lp A1046 A975 A981 A1042 A999 A1035 A978 LMCC0378 LMCC0315 LMCC0318 LMCC0375 LMCC0326 – LMCC0301 KT263350 KT270669 KT270673 KT263348 KT270680 KT263344 KT270670 KT263382 KT270752 KT270755 – KT270762 KT263377 KT270753 KT263414 KT270838 KT270842 KT263412 KT270849 KT263408 KT270839 clade VI clade VI clade VI clade VI clade VI clade VI clade VI A683 (LBM) A683 (TM) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A988 A1036 LMCC0303 LMCC0357 KT270677 KT263345 KT270759 KT263378 KT270846 KT263409 clade VI clade VI A683 (TM) A689 (SAB) Lecanora polytropa Tephromela atra Muellerella - Lp Taeniolella atricerebrina A1047 A979 LMCC0379 LMCC0316 KT263351 KT270671 KT263383 – KT263415 KT270840 clade VI clade VI A698 (KGA) A698 (MY) A703 (TM) Rhizocarpon geographicum Rhizocarpon geographicum Lecanora polytropa Endococcus macrosporus Endococcus macrosporus Muellerella - Lp A971 A1027 A1031 LMCC0313 LMCC0349 LMCC0353 KT270666 KT263338 KT263341 KT270749 KT263371 KT263374 KT270835 KT263402 KT263405 clade VI clade VI clade VI A703 (TM) A703 (MY) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A1032 A1034 LMCC0354 LMCC0356 KT263342 KT263343 KT263375 KT263376 KT263406 KT263407 clade VI clade VI A703 (MY) A709 (MY) Lecanora polytropa Rhizocarpon geographicum Muellerella - Lp Muellerella - Rh A1040 A1041 LMCC0373 LMCC0374 KT263346 KT263347 KT263379 KT263380 KT263410 KT263411 clade VI clade VI A832 (MY) A840 (TM) A840 (TM) A341 (TM) A100 (SAB) A100 (LBM) A94 (KGA) A670 (LBM) A678 (SAB) A97 (KGA) A651 (LBM) A56 (TM) Lecanora bicincta Lecidea sp. Lecidea sp. Pertusaria corallina Umbilicaria cylindrica Umbilicaria cylindrica Lecanora intricata Lecanora polytropa Lecanora bicincta Rhizocarpon geographicum Lecanora polytropa Lecanora intricata Arthonia varians Muellerella pygmaea Muellerella pygmaea Sclaerococcum sphaerale Stigmidium gyrophorarum Stigmidium gyrophorarum Muellerella - In Muellerella - Lp Sphaerellothecium atrinae Muellerella - Rh Carbonea supersparsa Muellerella - Li A967 LMCC0312 A968 A973 A1016 A561 A563 A512 A1044 A1033 A579 A1026 A568 LMCC0299 LMCC0396 LMCC0341 LMCC0172 LMCC0174 LMCC0133 LMCC0376 LMCC0355 – LMCC0370 LMCC0179 KT270663 KT270664 KT270667 KT263077 KT263079 KT263080 KT263078 KT263082 KT263081 KT263089 KT263090 KT263157 KT270746 KT270747 KT270750 KT263097 KT263092 KT263093 KT263091 KT263099 KT263098 KT263105 KT263106 KT263201 KT270832 KT270833 KT270836 KT263115 KT263108 KT263109 KT263107 KT263117 KT263116 KT263124 KT263125 KT263245 clade VI clade VI clade VI Sclerococcum basal to Chaetothyriaceae basal to Chaetothyriaceae Herpothrychielaceae Herpothrychielaceae Herpothrychielaceae Epibryaceae Epibryaceae incerta saedis Fungal Diversity (2016) 76:119–142 Lichen thallus ID (medium name) Lichen species Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. LSU nuSSU mtSSU Phlogenetic clade A56 (LBM) A56 (TM) A56 (SAB) A56 (SAB) A56 (MY) Lecanora intricata Lecanora intricata Lecanora intricata Lecanora intricata Lecanora intricata Muellerella - Li Muellerella - Li Muellerella - Li Muellerella - Li Muellerella - Li A545 A571 A577 A923 A959 LMCC0162 LMCC0182 LMCC0188 LMCC0268 LMCC0310 KT263458 KT263457 KT263459 KT263456 KT263460 KT263493 KT263492 KT263494 KT263491 KT263487 KT263528 KT263527 KT263529 KT263526 KT263522 Capnodiales Capnodiales Capnodiales Capnodiales Capnodiales A102 (DG) A102 (DG) Acarospora fuscata Acarospora fuscata Polycoccum microstictum Polycoccum microstictum A557 A951 LMCC0168 LMCC0288 KT263447 KT263448 KT263481 KT263482 KT263516 KT263517 Capnodiales Capnodiales A135 (LBM) A135 (KGA) Lecanora polytropa Lecanora polytropa Muellerella - Lp Muellerella - Lp A886 A887 – LMCC0249 KT263453 KT263454 KT263488 KT263489 KT263523 KT263524 Capnodiales Capnodiales A135 (SAB) A215 (SAB) Lecanora polytropa Lecanora polytropa Muellerella - Lp Lichenoconium lecanorae A888 A913 LMCC0250 – KT263455 KT263450 KT263490 KT263484 KT263525 KT263519 Capnodiales Capnodiales A224 (DG) A229 (DG) A291 (DG) Lecanora polytropa Lecanora polytropa Lecanora rupicola Carbonea supersparsa Carbonea supersparsa Arthonia varians A863 A997 A995 LMCC0236 LMCC0324 LMCC0362 KT263451 KT263449 KT263445 KT263485 KT263483 KT263479 KT263520 KT263518 – Capnodiales Capnodiales Capnodiales A393 (KGA) A709 (MY) A128 (TM) A72 (SAB) A94 (KGA) A94 (DG) A56 (SAB) A102 (DG) A160 (DG) Lecanora polytropa Rhizocarpon geographicum Lecanora bicincta Lecanora polytropa Lecanora intricata Lecanora intricata Lecanora intricata Acarospora fuscata Pertusaria lactea Cercidospora epipolytropa Muellerella - Rh Sphaerellothecium atrinae Carbonea supersparsa Muellerella - Li Muellerella - Li Muellerella - Li Polycoccum microstictum Stigmidium eucline A960 A1043 A559 A554 A569 A578 A537 A558 A542 LMCC0295 LMCC0401 LMCC0170 LMCC0165 LMCC0180 – LMCC0155 LMCC0169 LMCC0159 KT263452 KT263446 – KT263442 KT263444 KT263443 KT263430 KT263431 KT263432 KT263486 KT263480 KT263478 KT263475 KT263477 KT263476 KT263465 KT263466 KT263467 KT263521 KT263515 KT263514 KT263511 KT263512 KT263513 KT263499 KT263500 KT263501 Capnodiales Capnodiales Teratosphaeriaceae I Myriangiales Myriangiales Myriangiales Phoma (Pleosporales) Phoma (Pleosporales) Phoma (Pleosporales) A254 (TM) A23 (SAB) A56 (SAB) A100 (MY) A100 (TM) A128 (DG) A333 (KGA) A440 (TM) A678 (SAB) A333 (LBM) Lecanora polytropa Lecanora intricata Lecanora intricata Umbilicaria cylindrica Umbilicaria cylindrica Lecanora bicincta Tephromel atra Tephromel atra Lecanora bicincta Tephromel atra Muellerella - Lp Muellerella - Li Muellerella - Li Stigmidium gyrophorarum Stigmidium gyrophorarum Sphaerellothecium atrinae Muellerella atricola Muellerella atricola Sphaerellothecium atrinae Muellerella atricola A593 A583 A552 A565 A567 A595 A931 A1057 A977 A930 LMCC0200 LMCC0190 LMCC0164 LMCC0176 LMCC0178 LMCC0202 LMCC0272 LMCC0389 – LMCC0305 KT263433 KT263438 KT263436 KT263441 KT263439 KT263437 KT263435 KT263440 KT263448 KT263472 KT263470 KT263474 KT263473 KT263471 KT263449 – KT263502 KT263508 KT263506 KT263510 KT263509 KT263507 KT263505 – KT263434 KT263424 – KT263461 KT263504 KT263495 Phoma (Pleosporales) basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae basal to Lichenotheliaceae Lichenostigmatales 127 Lichen thallus ID (medium name) Fungal Diversity (2016) 76:119–142 Table 2 List of isolates recovered in Dothideomycetes as in the phylogenetic analysis of Fig. 3. The isolates are identified by their DNA extraction numbers. Number of the original lichen thallus (growth medium of inoculation), name of the lichen, name of the associated lichenicolous fungus, culture collection number and the newly published NCBI accession numbers are reported. The affiliation (clade name) of the isolates is reported. Dash (−) indicate loss of culture due to unsuccessful subsequent growth Pleosporales Pleosporales Pleosporales Pleosporales KT263496 KT263497 KT263498 KT263503 KT263462 – KT263463 KT263466 KT263425 KT263426 KT263427 KT263429 LMCC0372 – LMCC0350 LMCC0371 Lecanora polytropa Tephromel atra Tephromel atra Tephromel atra A651 (TM) A675 (SAB) A675 (MY) A675 (MY) Carbonea supersparsa Taeniolella atricerebrina Taeniolella atricerebrina Taeniolella atricerebrina A1039 A1011 A1028 A1038 LSU Lichen species Lichen thallus ID (medium name) Table 2 (continued) Lichenicolous fungus species Cultured fungus DNA extraction N. Culture collection N. nuSSU Phlogenetic clade Fungal Diversity (2016) 76:119–142 mtSSU 128 incubated in a growing chamber at 20 °C, with a light-dark regime of 14:10 hours with light intensity of 60-100 μmol photons m-2s-1 and 60% humidity. After three to five months, the inocula reached about 1-3 mm in diameter and it was possible to subculture and to prepare them for DNA extraction, sequencing and morphological analyses. The subcultures were set on agar plates using the same growth medium where the inoculum grew successfully. The cultured strains are deposited at the University of Graz in the culture collection of the first author LM and are preserved as cryostocks. Morphological analyses Morphological and anatomical characters of the cultured strains were analysed using standard microscopic techniques and documented with photographs. Analyses and photographs were performed on 10 month to one year old subcultures considering the following characters: form of growth, branching of the hyphae and melanization. Small fragments of the mycelia were taken; squashed sections were mounted in water and studied by light microscopy. Images were acquired with a ZeissAxioCam MRc5 digital camera fitted to the microscope. Both images of growth habit and hyphae structure were digitally processed using the CombineZM software (www.hadleyweb.pwp.blueyonder.co. uk/). The photos were slightly refined in sharpness and color tone with Adobe Photoshop 7.0 and the figures were prepared with CorelDRAW X4. DNA extraction, amplification and sequencing Small parts of the subcultured fungi were taken, transferred into 1.5 ml reaction tubes containing sterile beads for homogenization, frozen and ground using a TissueLyserII (Retsch). The DNA was then extracted following either the C-TAB protocol of Cubero et al. (1999) or using the DNeasy Plant Mini Kit (Qiagen, Austria). The industrial kit was used for those most melanized isolates for which the C-TAB protocol failed in extracting amplifiable DNA. The identity of the cultured fungal strains was studied with sequences of the nuclear large and partial nuclear small ribosomal subunits (nucLSU and nucSSU) and the mitochondrial small ribosomal subunit (mtSSU). The nucLSU fragment was obtained in two pieces using primers SR6R (http://www. botany.duke.edu/fungi/mycolab) and LR5 for the upstream fragment, and LR3R and LR7 (Vilgalys and Hester 1990; http://www.biology.duke.edu/fungi/mycolab/primers.htm) for the downstream fragment. The nucSSU locus was amplified using the primers NS1 (White et al. 1990) and nuSSU0852 (Gargas and Taylor 1992). The mtSSU locus was amplified with primers mtSSU1KL (Lohtander et al. 2002) and MSU7 (Zhou and Stanosz 2001) or mtSSU1 and mtSSU3R (Zoller et al. 1999). PCRs amplifications were carried out with the Illustra™ puReTaq Ready-To-Go PCR Beads (GE Healthcare, UK Limited) with a reaction volume of 25μl and a primer concentration of 0,6 pmol/μl. The Fungal Diversity (2016) 76:119–142 amplification of the genes followed touch-down PCR conditions as in previous studies (Muggia et al. 2011, 2013). PCR products were cleaned with E.Z.N.A.® Cycle Pure Kit (Omega Biotek, VWR) according to the manufacturer 's instructions. Both complementary strands were sequenced using the same PCR amplification primers by Microsynth (Sanger 3730xl from ABI, Vienna, Austria). Forward and reverse sequences were assembled into contigs and edited manually in BioEdit (Hall 1999). Alignment and phylogenetic analyses We checked the identity of the newly generated sequences with sequences available in the GenBank database by blast similarity search (Altschul et al. 1990). Taxa which closest matched our sequences for a value not lower than 95% identity and the further closest related ones (up to 90% identity) were selected for the phylogenetic analyses. As our sequences showed closest matches with representatives of the classes Eurotiomycetes (particularly in the subclasses Chaetothyriomycetidae), Dothideomycetes, Leotiomycetes and Sordariomycetes, we prepared four different datasets representing each lineage (the multilocus sequences alignments are deposit at TreeBASE). We tried to include in each dataset the widest spectrum of taxon diversity by selecting, if possible, at least three taxa representatives of different families or orders of the four classes (Table S1, S2, S3). We based our selection also on previous phylogenetic analyses which considered the aforementioned classes (e.g. Zhang et al. 2006; Wang et al. 2006; Gueidan et al. 2008, 2011; Ruibal et al. 2009; Schoch et al. 2009; Huhndorf and Miller 2011; Untereiner et al. 2011; Muggia et al. 2013; Hyde et al. 2013; Maharachchikumbura et al. 2015; Suija et al. 2015, ). The datasets of Eurotiomycetes and Dothideomycetes were prepared in summer 2014 whereas those of Leotiomycetes and Sordariomycetes in January 2015. For this reason recent sequence data published subsequently summer 2014 by Gueidan et al. (2014) and Ertz and Diederich (2015) are not included here. For each dataset, outgroup taxa were chosen from the most closely related classes. Sequence alignments for each locus (nucLSU, nucSSU and mtSSU) and for each fungal class (Eurotiomycetes, Dothideomycetes, Leotiomycetes and Sordariomycetes,) were prepared manually in BioEdit (Hall 1999). Introns and ambiguous SNPs were removed from the alignment. For a number of specimens we were unable to generate sequences for all of the selected loci and for other taxa sequences were not available in GenBank. Therefore we present here a three-locus phylogenetic inference for the classes Eurotiomycetes and Dothideomycetes, and two-locus inferences for the classes Leotiomycetes and Sordariomycetes. The final phylogenetic analyses of the Eurothiomycetes dataset included a subset of the isolates, which were selected after having estimated a first phylogeny including all the isolates. As multiple isolates shared identical sequences, we selected for the final analyses as representatives 129 those isolates obtained from different samples of the 25 lichenicolous fungus-lichen host associations which were grown on different media. Combined data of different loci, either fully or partially congruent, have been commonly considered in phylogenetics (Dettman et al. 2003). We performed, therefore, as in previous studies (Kauff and Lutzoni 2002; Miadlikowska et al. 2006; Muggia Perez-Ortega et al. 2014), both single locus and combined datasets analyses. We analysed the single locus datasets with a Maximum Likelihood (ML) approach (Meson-Gamer and Kellogg 1996; Reeb et al. 2004) and the combined dataset using both maximum likelihood (ML) and Bayesian approaches. In both approaches the combined datasets were treated in partitions by genes nucLSU, nucSSU and mtSSU. The ML analyses were performed using the program RAxML v. 7.1.3 (Stamatakis et al. 2005). The GTRMIX model was applied both for the single loci and to each partition in the combined datasets (as only a single model of molecular evolution can be used across gene partitions in RAxML), and 1000 bootstrap replicates were run. The Bayesian Markov Chain Monte Carlo (B/MCMC) analyses were run in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2003; Ronquist et al. 2005). The model of molecular evolution applied in the Bayesian analysis to each gene partition, the GTR+I+G model, was estimated in JModeltest v. 2.1.4 (Darriba et al. 2012) using the Akaike Information Criterion (Posada and Crandall 1998). The B/MCMC analyses were run with six chains simultaneously, each initiated with a random tree. Ten million generations for the Eurotiomycetes and Dothideomycetes datasets and five million generations for Leotiomycetes and Sordariomycetes datasets were run, respectively. Trees were sampled every 100 generations. The log-likelihood scores were plotted against generation time using Tracer 1.4 (Rambaut and Drummond 2007) to determine when the stationarity of likelihood values had been reached (e.g., the burn-in stage; Ronquist et al. 2005). Burn-in was set at half of the generations (the first 50,000 and 25,000 sampled trees for the two datasets groups respectively) and the majority rule consensus trees were calculated from the posterior samples of 50,001 and 25,001 trees, respectively. The convergence of the chains was confirmed by the convergent diagnostic of the Potential Scale Reduction Factor (PSRF), which approached 1 (Ronquist et al. 2005). The phylogenetic trees were visualized in TreeView (Page 1996). Results Culture isolation A total of 248 fungal cultures from 77 host lichen thalli were isolated and identified to date: 191 belong to the subclass Chaetothyriomycetidae, 36 to the class Dothideomycetes, 12 to Leotiomycetes and 9 to 130 Sordariomycetes. We obtained 21 additional isolates that corresponded to the lichen mycobionts (not shown). The majority of the strains, 24%, grew on TM, 22% grew on LBM, 20% on SAB, 16% on KGA, 13% on MY and 5% were isolated on DG media. Cultured mycobionts represented 2% of the grown isolates. From these cultures we obtained in total 710 new sequences: 244 for nucLSU rRNA gene, 237 for nucSSU rRNA gene and 229 for mtSSU rRNA gene (Table 1, Table 2, Table S3). The diversity of fungi isolated from lichen thalli, and which did not represent the mycobiont of the lichen symbiosis, varied among the 77 original thalli. The specificity of the isolated fungi neither correlates with the presence of any observed lichenicolous fungus nor with the identity of the lichen mycobiont. Fungi belonging to the same lineage were isolated from multiple thalli representing the same association of lichen and lichenicolous fungus, but also from the same lichen host species infected by different lichenicolous fungi (from hosts not growing in vicinity) and from other different associations of lichen and lichenicolous fungus (Tables 1, 2, S3). For example, we isolated up to five different lineages of fungi in two lichen individuals; fungi of four different lineages were isolated from only a single thallus, fungi of three different lineages were isolated from nine thalli. Fungi representing two different lineages were retrieved from 21 thalli, and fungi representing one lineage were obtained from 40 thalli. Phylogenetic and morphological analysis of Chaetothyriomycetidae (Fig. 1, Fig. 2, Table 1 and Table S1) The phylogenetic relationships recovered in Chaetothyriomycetidae are highly congruent with previous studies of Gueidan et al. (Gueidan et al. 2008, Gueidan et al. 2011) and Diederich et al. (2013). There were no significant incongruences between single locus (not shown) and multilocus trees. The only exception is the clade of Sclerococcum sphaerale, which is placed in our multilocus reconstruction at the base of Chaetothyriomycetidae, possibly due to the availability of only the nucLSU marker (Fig. 1). In this Sclerococcum sphaerale clade we recovered the single isolate A1016. A1016 was isolated from a thallus of Pertusaria corallina infected by Sclerococcum sphaerale, and this placement seems to confirm the identity of the lichenicolous fungus. This isolated strain forms pale pinkish, compact mycelia with thin, hyaline hyphae (Fig. 2 F1-F5). Clade I is represented by six isolates (from three host species), which together with Celothelium cinchonarum are basal to the split between Verrucariales and Chaetothyriales. These isolates are similar in morphology, forming white mycelia composed by thin, hyaline hyphae, which occasionally gather in thick, plectenchymatous strands (Fig. 2 A1-A4, B1-B4). Clade II is represented by three isolates: they come from three different thalli of the same lichen host- lichenicolous fungus association (Rhizocarpon geographicum – Muellerella Fungal Diversity (2016) 76:119–142 Fig. 1 Multilocus phylogenetic inference of Eurotiomycetes. The ML and the Bayesian phylogenetic hypotheses were inferred from the combined dataset of nucLSU, nucSSU and mtSSU loci and corresponded in their topologies; the ML analysis is shown. Bayesian posterior probabilities (PP ≥ 95 %) and ML bootstrap support values (≥ 70 %) are reported above branches (PP/bootstrap value). Newly identified clades of isolated fungi obtained from this study are highlighted in bold and are labelled as clade I to VII. Symbols indicate the different lichen host-lichenicolous fungal associations which represent the original thallus from where the fungal strains were isolated. A symbol reported multiple times for a clade indicates the number of different original thalli sharing the same lichen host-lichenicolous fungal association. Fungal life-styles are reported in parenthesis. Samples labelled with an asterisk (*) are those photographed in Fig. 2 pygmaea-Rh). These strains also present a pale pinkish mycelium, but hyphae are formed by cylindrical to semi-elliptical cells which are occasionally intercalated by roundish cells (Fig. 2 C1-C7). Two samples, A579 and A1026, are nested within Epibryaceae, the lineage formed by Epibryon and two rock-inhabiting fungi. The mycelium of these isolates is a dense aggregate of roundish, melanised cells containing inclusions, and filamentous hyphae are rarely present (Fig. 2 E1E5). Clade III represents the lichenicolous fungus Lichenodiplis lecanorae (Muggia et al. in prep.), which appears here basal to the split between the families Epibryaceae, Chaetothyriaceae and Herpotrichiellaceae. The identity of these isolates is also confirmed by the conidiomata-like structures and the conidia that are observed in the cultures (Fig. 2 D1-D4). Herpotrichiellaceae is here the most represented family of Chaetothyriales and comprises ecologically diverse fungi including human pathogens (Exophiala dermatitidis and Capronia semiimmersa), lichenicolous fungi (Capronia peltigerae and Cladophialophora parmeliae) and rock inhabiting fungi (Gueidan et al. 2008; Gueidan et al. 2011, Gueidan et al. 2014). Four newly cultured isolates are nested in this main Chaetothyriales lineage. A561 is nested in a clade with RIF and Phialophora europaea, and is morphologically similar to other previously isolated black RIFs (Fig. 2 L1-L3), having melanized hyphae frequently budding laterally and apically. Three other samples are nested in a clade with Cladophialophora parmeliae and Capronia semiimmersa. The isolates are characterized by melanized mycelia, with branching hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa (Fig. 2 K1-K5). In Chaetothyriales, the majority of the isolates group into sub clade s of a fully su ppor te d linea ge siste r to Chaetothyriales. Within this lineage we distinguished the main clades IV, V, VI and VII (as subclade of clade VI, Fig. 1), each represented by more than four isolates. The other isolates are placed on separate smaller clades in this large assemblage of branches. Clade IV and clade V include isolates from six and seven, respectively, different lichen hostlichenicolous fungus associations. Clade VI contains the majority of the isolates which come from 16 different lichen host Fungal Diversity (2016) 76:119–142 131 132 Fig. 1 (continued) Fungal Diversity (2016) 76:119–142 Fungal Diversity (2016) 76:119–142 - lichenicolous fungus associations. These include lichens infected by known but unrelated lichenicolous fungi. With the exception of few isolates, such as A514 (Fig. 2 H1-H5) and A511, which lack melanized mycelium, all the fungal strains included in this big assemblage of lineages are characterized by melanized mycelia. However, two main morphologies are observed among the strains: i) mycelia with filamentous, branching hyphae composed by cylindrical cells, usually aseptate (rarely 1-septate), intercalating by roundish cells, ii) mycelia with hyphae composed exclusively by globose, roundish cells, sometimes 1-septate, forming dense assemblages and budding. Except for the Sclerococcum clade and the clade III, we do not find clear evidence of correspondence of certain lineages with other lichenicolous fungi infecting the lichen samples. Phylogenetic and morphological analysis of Dothideomycetes – (Fig. 3, Fig. 4, Table 2 and Table S2) The phylogenetic relationships recovered in Dothideomycetes are highly congruent with previous studies of Ruibal et al. (2009), Lawrey et al. (Lawrey et al. 2011) Lawrey Diederich et al. 2012, Muggia et al. (2013); Hyde et al. (2013). Topological congruence was recovered between the Bayesian and the maximum likelihood analyses and among the single locus analyses. Also in Dothideomycetes the isolates are nested in clades together with fungi of diverse ecological niches and presenting different lifestyles (Fig. 3). The isolate A930 is recovered within Lichenostigmatales, which includes lichenicolous fungi and RIFs. A930 is morphologically identical to the Lichenostigma cultures isolated by Ertz et al. (2014), presenting yeast-like, budding, melanized cells. Four isolates form a fully supported clade nested in Pleosporales. Also in Pleosporales, four further isolates group together with lichenicolous species of the genus Phoma; however they were isolated from thalli of four different lichen host-lichenicolous fungi associations and none of them showed the symptomatic presence of Phoma species. These isolates form whitish to pale pinkish mycelia, composed by hyaline hyphae distributed to form a dense aggregate (Fig. 4 A1-A5, B1-B3). Seven isolates represent a lineage sister to Lichenotheliales; these isolates originate also from four thalli representing different lichen hosts infected by different lichenicolous fungi. The isolates comprise both melanized and non-melanized fungi (Fig. 4 D1-D6 and E1-E3). Three isolates are recovered in Myriangiales, a lineage of saprobic fungi; they present white mycelium of very thin hyaline hyphae (Fig. 4 F1-F4). The single isolate A559 is recovered as a member of Teratosphaeriaceae I. The remaining isolates group as a single lineage in Capnodiales, being nested among the clades Teratosphaeriaceae I, Teratosphaeriaceae II and Mycosphaerellaceae. In this lineage we identify three subclades, even though all isolates have a similar morphology, with dark, melanized mycelia composed by suglobose to 133 cylindrical cells with rough cell wall and sometimes constricted at the septa (Fig. 4 G1-G6, H1-H6). Phylogenetic and morphological analysis of Leotiomycetes and Sordariomycetes (Fig. S1, Fig. S2, Table S3 and Table S4) Only 15 and nine isolates have been identified as Leotiomycetes (Helotiales) and Sordariomycetes, respectively. Within Leotiomycetes none of our isolates is closely related to the lineage Encoelioideae, where recently lichenicolous fungi were identified to belong (Suija et al. 2015). Five isolates are placed with unresolved position at the base of Leotiomycetes; one isolate is closely related to Leotia lubrica (saprotroph among mosses and plant rests, Kuo 2003) and Microglossum olivaceum (a grassland species, Fleming 2001). Eight isolates obtained from three different combinations of lichen host and lichenicolous fungus are closely related to Mitrula paludosa (a species known from swamps and bogs, Wang et al. 2005). Three isolates are identified in Xylariales within the Sordariomycetes, one isolate is nested in Hypocreales (including insect parasitic species, mycoparasites, endophytes and saprotroph, Gazis et al. 2014), and five isolates, deriving from three different lichen host-lichenicolous fungi associations are recovered in Coniochaetales (saprotrophs, leaf and root endophytes, plant pathogens, Zhang et al. 2006). Strains of both Leotiomycetes and Sordariomycetes form pale pinkish to white mycelia (Fig. S3); melanization was seldom observed and was restricted only to localized parts of the culture (Fig S3 G). Discussion Rock-inhabiting alpine lichens are exposed to harsh environmental conditions, with drastic and sometimes sudden changes in temperature and hydration, as well as UV radiation. Conceivably, only fungi that tolerate such fluctuating conditions can persist or grow in lichens. In addition, these fungi must cope with the diverse and highly concentrated extracellular secondary products of their host species. We already found a surprising number of lichenicolous fungi in lichens (Fleischhacker et al. 2015), and evidence for a high number of additional, cryptically occurring fungi. Here we provided a comprehensive set of isolates of the culturable fungal fraction in lichens from an alpine habitat for a survey of their phylogenetic relationships, with special emphasis on members of Dothideomycetes and Chaetothyriomycetes. Molecular data and the morphological analyses seem to confirm the identity of only two symptomatic lichenicolous fungal species with Eurotiomycetes. The isolates obtained from thalli infected by Sclerococcum sphaerale indeed group within the lineage Sclerococcum (Diederich et al. 2013). The formation of conidiomata and conidocells was observed in 134 Fungal Diversity (2016) 76:119–142 Fungal Diversity (2016) 76:119–142 Fig. 2 Habitus of one year old, representative, cultured fungal strains belonging to Eurotiomycetes and included in the phylogenetic analysis of Fig. 1. Anatomical structures were photographed from squashed sections mounted in water. Samples are reported with their number and the clade to which they belong as in Fig. 1. A1-A4) A922 (clade I) - A1, A2 habitus of the mycelium; A3, A4 fine, hyaline hyphae. B1-B4) A1022 (clade I) - B1, B2 habitus of the mycelium; B3, B4 fine, hyaline hyphae, gathering in entangled, plectenchymatous strands. C1-C7) A993 (clade II) - C1, C2 habitus of the mycelium; C3-C7 hyaline hyphae with branching and globose cells intercalating with cylindrical cells. D1-D5) A528 (clade III) - D1, D2 habitus of the mycelium; D3, D4 brown cell structures containing conidia-like cells (arrow in D4); D5, hyaline hyphae. E1-E5) A1026 (Epibryaceae) - E1, E2 habitus of the mycelium; E3-E5 dense aggregate of roundish, melanised cells containing inclusions, filamentous hyphae rarely present (E5). F1-F5) A1016 (Sclerococcum) – F1, F2 habitus of the mycelium; F3-F5 hyaline hyphae with branching and cylindrical, more or less elongated cells. G1-G7) A1053 (single branch, basal to clade VI) – G1, G2 habit of the melanized mycelium; G3-G7 hyphae composed by melanised, single or 1-septate cells, with numerous apical and lateral buds. H1-H5) A514 (basal to clade VI) – H1, H2 habitus of the mycelium; H3-H5 melanized hyphae with cylindrical cells, apical bud with roundish cells (H4), infrequent branching. I1-I5) A986 (clade VI) – I1, I2 habitus of the melanized mycelium; I3I5 hyphae composed by globose, roundish cells, sometimes 1-septate (I3 arrow), with thick cell wall. J1-J4) A971 (clade VI) – J1 habitus of the melanized mycelium; J2-J4 melanized hyphae with cylindrical cells intercalating with roundish cells (J3 arrow), ramifications originate both from the cylindrical and the roundish cells. K1-K5) A1033 (Herpotrichiellaceae) – K1, K2 habitus of the melanized mycelium; K3K5 melanized hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa. L1-L3) A561 (basal to Chaetothyriaceae) melanized hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa, frequently laterally and apically budding. Scale bars =4 mm (D1, G1, H1, I1, K1), 3 mm (A1, B1, C1, E1, F1), 1 mm (B2, D2, I2, K2), 0.5 mm (A2, C2, E2, F2, G2, H2, J1), 50 μm (B3, D3, F3, I5), 20 μm (A3, A4, B4, C3-C7, D4, D5, F4, F5, G3-G7, H3-H5, I3, I4, J2-J4, K3, K4, L2, L3) multiple cultured fungi from different thalli with infections of Lichenodiplis lecanorae. This proved the identity of the culture with the original infection of the lichenicolous hyphomycete. Except for the above mentioned clades, we do not find clear evidence of correspondence of certain lineages with other lichenicolous fungal species infecting the lichen samples. Some of the observed lichenicolous fungi cannot be the origin of the sequenced cultures, since these belong to completely unrelated groups (e.g. Arthonia, Carbonea, Cecidonia, Opegrapha, Skyttea, Stigmidium; Ertz et al. 2009, 2014; Schmull et al. 2011; Suija et al. 2015). It is likely that the clade IV and V, and the plenty of clades with few representatives, so far correspond to lineages of still unknown fungi which may occur widespread in lichen thalli from rocks, but are unapparent to the eye. The present phylogenetic results also show that some of the detected fungi are closely related to lichenicolous fungi as well as to fungi known from diverse other ecological niches. Two isolates are closely related to the genus Epibryon, which was originally described as bryophilous (Döbbeler 1978). It is now emended by non-lichenized lichenicolous species 135 (Zhurbenko and Hafellner 1999; Sérusiaux et al. 1999), which demonstrates cross-kingdom host switches in this monophyletic genus. The host lichens of the Epibryon strain were also visibly infected by the genera Carbonea and Muellerella, respectively. Also Muellerella comprises species on bryophytes and lichens (Döbbeler and Triebel 1985), but its relationship with Epibryon requires further study. Clearly Carbonea, as a member of Lecanoraceae, is unrelated. The results suggest that Epibryon could occur also as a non-symptomatic lichen inhabitant, which agrees with the previous results of U’Ren et al. (2010), who discovered a group of fungi capable to live cryptically in both lichens and mosses. The cryptic presence of otherwise symptomatic lichenicolous fungi is also demonstrated by isolates placed with the lichenicolous lineage of the anamorph genus Phoma, and those strains which are nested with lichenicolous species of Capronia and Cladophialophora. None of these isolates, however, originated from thalli which were visibly infected by either Phoma, Capronia or Cladophialopora. The high similarity (>95%) that the new sequences showed with the already available Phoma sequences suggests that the isolated strain could represent closely related Phoma species. The majority of the isolates are melanized fungi, which closely resemble previously studied rock-inhabiting fungi (RIF) and in fact are closely related to them. The presence or absence of these fungi in hosts of the same area seems to be largely unpredictable, unspecific and facultative. Rather than indicating host specificity, they seem to be broadly tolerant species whose presence might depend more on physical parameters. Nonetheless, lineage clade VI (Chaetothyriales) seems to be rather ubiquitous in lichens. All selected fungi likely represent the same species occurring in many thalli and in combination with different lichenicolous fungi. The finding of few isolates in Myriangiales, Xylariales, Hypocreales and Coniochaetales is quite interesting, as this is the first record for lichens from rocks; members of these groups are mainly biotrophic plant-associated fungi, endophytes, saprotrophs on wood and insect parasites. Fungi in Xylariales were, though, already isolated from lichen thalli from other ecological niches (Ding et al. 2009, U’Ren et al. 2012). However, no diagnostic structure hinting at these fungi have ever been observed under the microscope. Arnold et al. (2009) first suggested that fungi may live a symptomless life in lichens and coined the term ´endolichenic fungi´ for such organisms. Arnold and co-authors (Arnold et al. 2009, U’Ren et al. 2010, 2012) also have studied lichens from different habitats, such as tropical forest, temperate, boreal and arctic locations. Though some of these lichens are of the same mycobiont genera as the species included in this study, Arnold and colleagues found a higher proportion of fungi in Leotiomycetes and Sordariomycetes, more closely related to lineages of plant endophytes, rather than to the lineages 136 Fig. 2 (continued) Fungal Diversity (2016) 76:119–142 Fungal Diversity (2016) 76:119–142 137 Fig. 3 Multilocus phylogenetic inference of Dothideomycetes. The ML and the Bayesian phylogenetic hypotheses were inferred from the combined dataset of nucLSU, nucSSU and mtSSU loci and corresponded in their topologies; the ML analysis is shown. ML bootstrap support values (≥ 70 %) and Bayesian posterior probabilities (PP ≥ 95 %) are reported above branches (bootstrap value/PP). Fungal isolates obtained from this study are highlighted in bold. Symbols indicate the different lichen host-lichenicolous fungal associations as reported in Fig. 1. Fungal life-styles are reported in parenthesis. Samples labelled with an asterisk (*) are those photographed in Fig. 4 predominantly found in this survey. It is likely that the taxonomic diversities recovered between the two surveys correlates with the local vegetation and geologic histories of the regions. The cryptic occurrence of fungi has been also found in different environments (Stergiopoulos and Gordon 2014), and even included plant pathogens (Malcolm et al. 2013). We also isolated fungi which constitute two monophyletic lineages, both closely related to orders and families of lichenicolous and lichenized genera, RIF and pathogens in Dothideomycetes: the first closely related to Lichenotheliales, the second nested in Capnodiales. The first lineage is closely related to species of the genus Lichenothelia, which are known to share multiple lifestyles on rocks (Hyde et al. 2013; Muggia et al. 2013, 2015). They dwell on bare rock surfaces, but are often found associated with free living algae also present on the rocks. Some species specialize as lichen parasites and seem to associate with the lichen photobiont (Muggia et al. 2015). Some oligotrophic fungi apparently improve their carbon supply by attaching to microscopic algae. A direct involvement of black fungi in fungalalgal interactions was earlier described as a balanced algal parasitism (Turian 1977). Several rock-inhabiting and lichen-inhabiting microcolonial fungi develop into lichenoid structures within months when co-cultured with algae 138 Fungal Diversity (2016) 76:119–142 Fig. 3 (continued) obtained from lichen thalli (Gorbushina et al. 2005; Brunauer et al. 2007). Gorbushina and Broughton (2009) showed an example with a co-culture of Nostoc and a rock-inhabiting fungus (Sarcinomyces). They observed a specific spatial arrangement of both organisms and growth alterations in the photosynthetic cyanobacteria suggested a specific interaction. Therefore black fungi that loosely associate with algae in nature might be interpreted as Blichenoids^ and are considered prime forms of symbiosis (Muggia et al. 2013). The apparent ability of black fungi to associate loosely with algae sheds an interesting light on the evolution of lichens. In fact some of the rock-inhabitants are basal to the large lichenized Ascomycete lineages Arthoniomycetes and Verrucariales (Gueidan et al. 2008; Ruibal et al. 2009). Otherwise, the lichenized life styles are scattered in various clades of Dothideomycetes (Muggia et al. 2008; Ruibal et al. 2009; Nelsen et al. 2009), where lichen thallus morphology remains generally simple. However, not all of the lineages do associate with algae or establish lichen symbioses. Some Dothideales have evolved into highly adaptable and versatile species -e.g. Aureobasidium pullulans commonly found on Fig. 4 Habitus of one year old, representative, cultured fungal strains belonging to Dothideomycetes and included in the phylogenetic analysis of Fig. 3. Anatomical structures were photographed from squashed sections mounted in water. Samples are reported with their number and the clade to which they belong as in Fig. 3. A1-A5) A537 (Phoma) – A1, A2 habitus of the mycelium; A3-A5 hyaline hyphae with inclusions forming a dense aggregate. B1-B3) A542 (Phoma) – B1, B2 habitus of the mycelium, B3 dense aggregate of hyphae. C1-C5) A930 (Lichenostigmatales) – C1, C2 habitus of the mycelium; C3-C5 yeast-like melanised cells forming dense aggregates. D1-D6) A931 (clade sister to Lichenotheliales) – D1, D2 habitus of the mycelium; D3-D6 plectenchymatous structure of hyaline hyphae with cylindrical cells, round cells as buds at the apexes of the hyphae (arrows). E1-E3) A 567 (clade sister to Lichenotheliales) – E1 habitus of the mycelium; E2, E3 melanized hyphae composed by elliptical and subcylindrical cells constricted at the septa, laterally budding. F1-F4) A554 (Myriangiales) – F1, F2 habitus of the mycelium; F3, F4 thin, hyaline hyphae. G1-G6) A559 (Teratospaheriaceae I) – G1, G2 habitus of the mycelium; G3-G6 melanized hyphae, branching and composed by cylindrical to subglobose cells. H1-H6) A960 (clade nested in Teratosphaeriaceae) H1, H2 habitus of the mycelium, hyphae develop inside the growth medium; H3-H6 melanized hyphae, branching and composed by cylindrical to subglobose cells. Scale bars =4 mm (A1, C1, D1, E1, F1, G1), 3 mm (B1), 2 mm (B2, H1), 1.5 mm (C2), 1 mm (A2, D2, F2, G2), 0.4 mm (H2), 50 μm (G3), 40 μm (C3, D3), 20 μm (A3, C5, E2, E3, F3, G4, H3-H6), 10 μm (A5, B3, D4-D6, F4, G5, G6) Fungal Diversity (2016) 76:119–142 139 140 Fungal Diversity (2016) 76:119–142 Fig. 4 (continued) leaf surfaces of plant- but have not been found to be associated with lichens. In our survey, fungi of unrelated lineages were recovered several times from individual lichen thalli. This may indicate that there is no competition between the different fungi, which complies with a concept of niche-sharing (Crous et al. 2009), and that the occurrence of certain lineages does not implicate the presence or absence of others. Lichen-associated fungi, which do not develop any diagnostic structure on the thallus host, use the host just for their own cryptic internal life, likely awaiting the most suitable substrate/host to propagate. Perhaps not all isolated fungi grow equally well in lichens, and we cannot exclude that some might be present as spores or small germlings, while others form mycelia networks in their hosts. We often see mycelia of melanized fungi on the lichens and expect their growth is well adapted to the poikilohydric lichen habitat. The symbiotic structures of the lichen thalli function as a shared habitat of phylogenetically diverse stress-tolerant fungi, some of which use their host as protection, while others use it as nutrition sources in otherwise hostile environments. Acknowledgement This work was supported by the Austrian project FWF P24114-B16. 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