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
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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
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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
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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)
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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
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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.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
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