The Lichen Connections of Black Fungi

The Lichen Connections of Black Fungi

Lucia Muggia • Cecile Gueidan •

Kerry Knudsen • Gary Perlmutter •

Martin Grube

Received: 4 September 2012 / Accepted: 2 November 2012

� Springer Science+Business Media Dordrecht 2012

Abstract 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 hypoth-

esis to show that Lichenothelia belongs to the super-

class Dothideomyceta. Further, culture experiments

revealed the presence of co-occurring fungi in Liche-

nothelia thalli. These fungi are related to plant

pathogenic fungi (Mycosphaerellaceae) and to other

rock-inhabiting lineages (Teratosphaeriaceae). The

Lichenothelia thallus-forming fungi represent there-

fore consortia of different black fungal strains. Our

results suggest a common link between rock-inhabit-

ing meristematic and lichen-forming lifestyles of

ascomycetous fungi.

Keywords Algae � Borderline lichens �

Dothideomyceta � Lichenicolous � Lichenothelia �

Symbiosis

Introduction

The rock-inhabiting oligotrophic lifestyle of melan-

ized fungi is found in the large lineages of Dothideo-

mycetes and Chaetothyriomycetidae. Rock surfaces

are the most abundant natural substrates for oligo-

trophic black fungi, being colonized in all climatic

zones, including the most hostile habitats on Earth

L. Muggia (&) � M. Grube

Institute of Plant Sciences, Karl-Franzens-University

Graz, Holteigasse 6, 8010 Graz, Austria

e-mail: [email protected]

L. Muggia

Department of Life Science, University of Trieste, Via L.

Giorgieri 10, Trieste, Italy

C. Gueidan

Department of Life Science, The Natural History

Museum, Cromwell Road, SW7 5BD London, UK

K. Knudsen

The Herbarium, Department of Botany and Plant

Sciences, University of California, Riverside, CA 92521,

USA

G. Perlmutter

UNC Herbarium, North Carolina Botanical Garden,

University of North Carolina, Chapel Hill,

NC 27599-3280, USA

123

Mycopathologia

DOI 10.1007/s11046-012-9598-8

such as Antarctic dry valleys, the Atacama desert or

summits in the Himalayas [33]. Owing to their high

desiccation-tolerance, black fungi seem to prevail

under poikilohydric conditions when water is only

temporarily available (humidity, rain and condensa-

tion). This stress-tolerant lifestyle apparently evolved

early in the Ascomycota, when exposed rocks were the

primary substrates on land. Recent phylogenetic

timing suggests that the rock-inhabiting lifestyle arose

much earlier in Dothideomycetes than in Chaetothyri-

ales [14].

It is still not well understood how rock-inhabiting

black fungi gain nutrients and energy for growth. In

many habitats, external sources of fixed carbon and

other nutrients could be derived from the atmosphere

by rain or animal droppings, but this hardly applies to

the most harsh environments where black fungi are

present, such as the McMurdo Dry Valleys or the

Makalu cliff in the Himalaya at 7,400 m altitude

(material in herbarium GZU). Alternatively, associa-

tionwith other stress-tolerantmicroorganisms could be

a symbiotic strategy to gain nutrients. Attachment of

melanized fungi to microscopic algae and cyanobac-

teria has been repeatedly observed. A direct involve-

ment of black fungi in fungal–algal interactions was

earlier described as a ‘‘balanced algal parasitism’’ [42].

When black fungi are co-cultured with algae, some

rock-inhabiting species can develop lichen-like struc-

tures in only a few months [13]. The co-culture of

Nostoc sp. with a rock-inhabiting fungus (Sarcinomy-

ces sp.) resulted in specific arrangements of both

organisms, which was seen as an indicator of a specific

interaction by Gorbushina and Broughton [11]. These

authors also regarded rock surfaces as a ‘‘symbiotic

playground’’, where detrimental interactions between

species (antibiosis) are selected against [8, 12].

Black fungi and lichens often co-occur on the same

pieces of rock, and in arid habitats, black fungi

frequently even colonize lichens. Harutyunyan et al.

[18] isolated several strains that belong to melanized

fungi of different genera, such as Mycosphaerella,

Rhinocladiella and Capnobotryella. Their closest

relatives have diverse ecological relationships and

include human and plant pathogens. Some of the

lichen-associated black fungi might also interact with

algae from their hosts. Brunauer et al. [3] axenically

co-cultured a black fungus isolated from a lichen

thallus with various lichen algae. Observations

revealed the development of a lichen-like fungal

plectenchyme covering the algae and attaching to the

algal cells, especially with those of the native lichen

host [3].

Associations of fungi and algae which do not result

in a stratified lichen thallus structure, but in a poorly

structured fungal–algal consortium, have been termed

‘‘borderline lichens’’ [13, 24]. Nonetheless, distinctive

structures and/or sexual fungal fruiting bodies may

develop in borderline lichens. Because these fruiting

bodies represent recognizable phenotypic characters,

they have been studied by lichenologists. Only few

black fungal species form characteristic morphologi-

cal thallus shapes without sexual reproduction: the

thalli of Cystocoleus ebeneus and Racodium rupestre,

in contrast to most other lichens, are not shaped by the

fungal partners. In these species, an algal thread—

representing the genus Trentepholia—is densely en-

caged by fungal hyphae with melanised cell walls.

Muggia et al. [28] found that these two sterile genera

do not form a monophyletic group but are related with

other members in Capnodiales, an order also including

human opportunists and plant pathogens.

In this paper, we present the first phylogenetic data

on the peculiar genusLichenothelia [19].Lichenothelia

is a cosmopolitan genus of rock-inhabiting melanised

fungi that encompasses currently 24 species (http://

www.mycobank.org/). Some species, which have been

found in association with algae or with lichen thalli,

produce fertile structures with asci and ascospores, but

these are not sufficient for proper phylogenetic classi-

fication and species concept in Lichenothelia is argu-

able. The phylogenetic relationships between species

of this genus and with other rock-inhabiting and lich-

enized fungal lineages have not been elucidated so far

and are the aim of the present study.

Materials and Methods

Sampling and Molecular Analyses

Lichenothelia spp. specimens were collected in the

period 2009–2011 and are stored in GZU, NCU and

UCR. Thirty specimens were selected for DNA

extraction, among which we included the generic type

Lichenothelia scopularia (Nyl.) D. Hawksw. We

selected Lichenothelia species from both lichenico-

lous (Lichenothelia convexa and L. tenuissima) and

saxicolous (Lichenothelia sp. and L. cf. calcarea)

Mycopathologia

123

http://www.mycobank.org/

http://www.mycobank.org/

habit; among the samples, six thalli were found

associated with algae. The samples which were

successfully sequenced are reported in Table 1. The

material was analyzed under stereo and light micro-

scope, and digital images of the samples were acquired

with a ZeissAxioCam MRc5 digital camera fitted to

the microscopes. Images of both herbarium samples

and cultures (Fig. 1a-d) were digitally optimized using

the CombineZM software (open source image pro-

cessing software available at www.hadleyweb.pwp.

blueyonder.co.uk/). The photographs were further

refined with Adobe Photoshop 7.0, and the plate was

prepared with CorelDRAW X4.

The samples were carefully dissected under the

stereo-microscope and prepared for DNA extraction.

Small groups of ascomata or, if these were rare or

lacking, about 0.5 cm2 of the dry crustose melanised

thalli were detached from the rock substratewith a sterile

razor blade. The fungalmaterialwas always taken froma

single area of the rock. The dry fungal material was

transferred into a 1.5-ml tube and pulverized with metal

beads using a TissueLyserII (Retsch). The DNA was

extracted according the protocol ofCubero et al. [6]. The

phylogenetic affiliation of Lichenothelia was studied

with sequences of the nuclear large and partial nuclear

small ribosomal subunits (nucLSU and nucSSU). The

nucLSU fragment was obtained in two pieces using

primers ITS1F [9] and LR5 for the first half, and LR7

[43] and LR3R for the second (http://www.biology.

duke.edu/fungi/mycolab/primers.htm). The nucSSU

locus was amplified using the primers nuSSU0072 and

nuSSU0852 [10]. Gene amplifications followed touch-

down PCR conditions as in previous studies [29, 30].

Both complementary strands were sequenced, and

sequencing was run by Macrogen Inc. (Amsterdam,

Netherlands). The sequences were assembled and edited

in BioEdit [17].

Culture Isolation

Axenic cultures of the Lichenothelia fungus were

prepared from specimens collected since up to

5 months. Three samples for which enough material

was available were selected for culture isolation

(Lichenothelia sp. L985, L986 and Lichenothelia cf.

calcarea L1323; all associated with algae).The mode

of isolation and inoculation followed the method

described by Yamamoto et al. [44] and Stocker-

Worgotter [41], with somemodifications depending on

the type of thallus. An area of the thallus of about

1–2 cm2 was selected and was washed by pipetting

with bi-distilled sterile water and Tween80 to remove

possible external contaminations such as bacteria and

yeasts [4]. The thalluswas then carefully detachedwith

a sterile razor blade, and the melanized fragments of

the hyphae and ascomata were taken with a sterile

needle and inoculated in slanted tubes or on small agar

plates. Each tube contained one single inoculum,

whereas up to four inocula were put on one petri plate.

Agar plates and tubes were sealed with parafilm to

avoid desiccation of themedium andwere incubated in

a growing chamber at 20 �C, with a light–dark regime

of 14–10 hours with light intensity of 60–100 lmol

photons m-2 s-1 and 60 % humidity. Cultures and the

following subcultures were set on the malt yeast (MY,

[1]), Trebouxia (TM, [1]) and Lilly and Barnett’s

(LBM, [25]) media. Each sample was inoculated on up

to five slanted tubes or agar plates of each medium.

Inocula were checked weekly for contamination. After

1 month, the inocula reached about 0.5 cm in diameter

and it was possible to subculture them and to prepare

them for DNA extraction and sequencing. The DNA

extraction protocol followed Cubero et al. [6]; the

identity of the cultures was checked by sequencing the

same nuclear loci (nucLSU and nucSSU) selected to

study the Lichenothelia specimens. PCR and sequenc-

ing followed as described above.

Alignment and Phylogenetic Analyses

The identity of the new generated sequences was

checked with sequences available in the GenBank

database. Sequences obtained from herbarium speci-

mens and sequences obtained from the cultures showed

a high similarity with taxa from the Dothideomycetes.

Therefore, we included in our dataset selected taxa of

the new established superclass Dothideomyceta (Doth-

ideomycetes and Arthoniomycetes, [32, 39]) in order

to cover a broad phylogenetic range of taxa (Table 2).

The majority of the selected taxa were from a recent

study by Ruibal et al. [38]. Symbiotaphrina buchneri

and S. kochii were selected as outgroups. Sequence

alignments were prepared manually in BioEdit; sepa-

rate alignments were originally prepared for nucLSU

and nucSSU. Ambiguous regions and introns were

excluded from the alignments. For a number of

specimens, we were unable to generate sequences for

both selected loci, and for other taxa, sequences were

Mycopathologia

123

http://www.hadleyweb.pwp.blueyonder.co.uk/

http://www.hadleyweb.pwp.blueyonder.co.uk/

http://www.biology.duke.edu/fungi/mycolab/primers.htm

http://www.biology.duke.edu/fungi/mycolab/primers.htm

Table 1 Origin data and sequence accession numbers of analyzed Lichenothelia specimens and black fungi newly isolated in culture

Species DNA

extraction

Collection N. Origin nucLSU nucSSU

Lichenothelia cf.

calcarea

L1296 Knudsen K. 13482

(UCR

1778KK64)

USA, California, Riverside County, Mojave

Desert, Joshua Tree National Park, 33�5702200N/

116�0005500W

KC015060 KC015080

Lichenothelia cf.

calcarea

L1323 Muggia L. (GZU) Europe, Czech Republic, South Moravia,

Moravsky Krumlov, on the hill of Kaple Svateho

Floriana.

KC015061 KC015081

Lichenothelia cf.

calcarea

L1324 Muggia L. (GZU) Europe, Czech Republic, South Moravia,

Moravsky Krumlov, on the hill of Kaple Svateho

Floriana

KC015062 KC015082

Lichenothelia cf.

calcarea

L1706 Knudsen K.

13079.2 (UCR

1782KK64)



116�1002900W

KC015063 –

Lichenothelia cf.

calcarea


(UCR

1576KK64)



116�0203500W

KC015064 –

Lichenothelia cf.

calcarea


(UCR

1503KK64)



116�0203500W

KC015065 –

Lichenothelia cf.

calcarea

L1715 Knudsen K.

13079.2 (UCR

1782KK64)



116�1002900W

KC015066 –

Lichenothelia cf.

calcarea


(UCR

1778KK64)



116�0005500W

KC015067 –

Lichenothelia

convexa


(UCR1 67675)



116�0405800W

KC015068 KC015083

Lichenothelia

convexa


(URC

1304KK64)

Europe, Czech Republic, Pitkovice, 50�0102600N/

14�3402100E

KC015069 KC015084

Lichenothelia

convexa


(URC

1304KK64)


14�3402100E

KC015070 KC015085

Lichenothelia

convexa


(URC

1304KK64)


14�3402100E

KC015071 KC015086

Lichenothelia

convexa

L1702 Knudsen K.

14252.2 (UCR

597KK64)



116�0602500W

KC015072 –

Lichenothelia

tenuissima

– Knudsen K. 10406

(UCR 197485)

USA, California, San Bernardino County, San

Bernardino National Forest, 34�1202900N/

116�4300300W

KC015073 –

Lichenothelia sp. L984 Perlmutter G. 2617

(NCU)

USA, North Carolina, Wake County, on granitic

boulder, 35�4404800N/78�2502700W

KC015074 KC015087


(NCU)


boulder, 35�4404800N/78�2502700W

KC015075 KC015088


(NCU)


boulder, 35�4404800N/78�2502700W

KC015076 KC015089

Culture from

Lichenothelia

sp. L985

L1285 Culture collection GZU – KC015090

Mycopathologia

123

not available in GenBank. To check the phylogenetic

congruence of the two loci, we analyzed first the

genetic signal of each locus separately and then we

combined the loci into a multilocus analysis as

described in previous studies [22, 27]. The congruence

was tested with both a B/MCMC Bayesian [20] and a

Maximum Likelihood (ML) approach [26, 37]. The

models of molecular evolution were estimated in

MrModeltest v. 3.7 using the Akaike Information

Criterion [36]. The concatenation of the two loci was

possible as no incongruence was found between our

two individual dataset. The final phylogenetic analysis

of the two-gene dataset was performed with RAxML

[40] using theGTRMIXmodel ofmolecular evolution,

and 1,000 bootstrap replicates applied to a two-gene

partition (nucLSU and nucSSU). The phylogenetic

trees were visualized in TreeView [34].

Results

Culture

A total of 45 inoculawere prepared. Seventy per cent of

the inoculated slanted tubes and agar plates were

discarded due to contamination by bacteria and fast

growing moulds or yeasts. Four inocula from the two

American collections of Lichenothelia sp. L985 and

L986 specimens and three inocula from the European

specimensL1323 could be isolated axenically (Fig. 1a,

b). The seven cultures were further subcultured onto

MY and LBM medium and used for molecular and

phylogenetic analyses. Besides the fungi isolated, two

isolates of algae were also obtained. They are coccoid

Chlorophyceae morphologically similar to Trebouxia-

and Coccomyxa cells (data not shown).

Sequencing, Alignments and Phylogenetic

Inference

About half of the selected specimens could be

successfully extracted for DNA and sequenced. These

are eight specimens of Lichenothelia cf. calcarea

(three of which associated with algae), five of L.

convexa, one of L. tenuissima and three of Lichenot-

helia sp. (all three associated with algae). Unfortu-

nately, the type species Lichenothelia scopularia

could not be amplified and included in the phyloge-

netic analysis. Nineteen nucLSU and 17 nucSSU new

sequences were generated. Although several attempts

were done, it was impossible to obtain sequences from

both loci for seven Lichenothelia spp. samples and 5

fungal cultures. The final alignment included 136 taxa:

17 Lichenothelia spp., 7 culture isolates and 112 taxa

Table 1 continued

Species DNA

extraction

Collection N. Origin nucLSU nucSSU

Culture from

Lichenothelia

sp. L985


Culture from

Lichenothelia

sp. L986


Culture from

Lichenothelia

sp. L986


Culture from

Lichenothelia

sp. L1323

L1775 Culture collection GZU KC015077 –

Culture from

Lichenothelia

sp. L1323

L1777 Culture collection GZU KC015078 KC015094

Culture from

Lichenothelia

sp. L1323

L1778 Culture collection GZU KC015079 KC015095

A dash indicates missing sequences

Mycopathologia

123

selected within the Dothideomyceta. Among the 112

taxa retrieved from GenBank, 13 had sequences for

only one locus. After exclusion of introns and regions

with ambiguous size, the final combined alignment

contained 1,960 nucleotide, 1,252 for nucLSU and 708

for the partial nucSSU subunit.

Our results (Fig. 2) are congruent with previous

phylogenetic inferences reported by Ruibal et al. [38],

Schoch et al. [39] and Nelsen et al. [31, 32]. Parts of the

backbonephylogeny, especially basal branches, are still

little supported, but family and order clades of

Dothideomyceta are well resolved. Myriangiales and

Dothideales are sister group (67 %), yet their sister

relationship with Capnodiales has low support. In

Capnodiales, the family Teratosphaeriaceae splits into

two monophyletic clades Teratosphaeriaceae (1) and

(2). The majority of rock-inhabiting fungi (RIF) is

placed within Capnodiales; RIF are also included in

Arthoniales, Dothideales, Myriangiales and are at the

base of the newly recovered Lichenothelia-group. Two

rock isolates TRN456 and TRN529, which formed a

separate clade, the ‘‘unknown lineage 1’’ in Ruibal et al.

[38], are nestedwithinArthoniales, supporting the sister

relationship found by Ruibal et al. [38]. The lichen

family Trypetheliaceae is on a long branch sister to

Arthoniales; this relationship has here low support and

may be an artefact, as suggested in Ruibal et al. [38].

The present phylogenetic inference shows one new

clade, although not well supported as a monophyletic

lineage, including Lichenothelia cf. calcarea, L.

convexa and Lichenothelia sp. All the specimens

associated with algae are included in this clade. Four

Fig. 1 Habit of Lichenothelia sp. and cultured black fungi. a,

b habit of Lichenothelia sp. specimens from the Lichenothelia-

group (Fig. 2; Table 1): aLichenothelia sp. L986, bLichenothelia

sp. L1296. c, d culture of black fungi isolated from specimens of

Lichenothelia sp.: c culture of black fungus L1286 of clade I

(Fig. 2), d culture of black fungus L1777 of clade II (Fig. 2).

Bar = 0.5 cm

Mycopathologia

123

Table 2 NCBI accessions of taxa included in the phylogenetic analysis of Fig. 2

Taxon Sample ID nucLSU nucSSU

Anisomeridium polypori AFTOL 101 – DQ782877

Arthonia caesia AFTOL 775 FJ469668 –

Arthopyrenia salicis CBS 368.94 AY538339 AY538333

Astrothelium cinnamomeum AFTOL 110 AY584652 AY584676

Bimuria novae-zelandae CBS 107.79/AFTOL 931 AY016356 AY016338

Botryosphaeria dothidea CBS 115476/AFTOL 946 DQ678051 DQ677998

Capnobotryella renispora CBS 214.90 EU019248 Y18698

Capnodiales sp. CBS 191364 GU323215 GU561840

Capnodium coffeae CBS 147.52/AFTOL 939 DQ247808 DQ247801

Catenulostoma abietis CBS 459.93/AFTOL 2210 DQ678092 DQ678040

Cladosporium cladosporioides CBS170.54/AFTOL 1289 DQ678057 DQ678004

Cladosporium sp. CBS180.53/AFTOL 1035 AY016367 AY016351

Columnosphaeria fagi (1) CBS 171.93/AFTOL 1582 AY016359 AY016342

Columnosphaeria fagi (2) CBS 584.75/AFTOL 912 DQ470956 DQ471004

Coniosporium apollinis CBS 100218 GU250898 GU250919

Coniosporium apollinis CBS 352.97 GU250895 GU250916


Coniosporium apollinis CBS 109867 GU250901 –




Coniosporium uncinatum CBS 123158/A35 GU250925 GU250933

Coniosporium uncinatum CBS 100219 GU250903 GU250923

Coniosporium uncinatum CBS 100212 GU250902 GU250922

Cryomyces antarcticus CCFEE 536 GU250365 GU250321

Cryomyces minteri CBS 116302/CCFEE 5187 GU250369 DQ066714

Cystocoleus ebeneus L348 (GZU, Hafellner 41566) EU048580 EU048573

Davidiella tassiana CBS 399.80/AFTOL 1591 DQ678074 DQ678022

Delphiniella strobiligena CBS 735.71/AFTOL 1257 DQ470977 DQ471029

Dendrographa leucophaea AFTOL 308 AY548810 AY548803

Dendrographa minor AFTOL 355 AF279382 AF279381

Dendryphiella arenaria CBS 181.58/AFTOL 995 DQ470971 DQ471022

Devriesia streliziae CBS 122379 GU296146 GU301810

Dothidea insculpta CBS 189.58/AFTOL 921 DQ247802 DQ247810

Dothiora cannabinae CBS 737.71/AFTOL 1359 DQ470984 DQ479933

Elasticomyces elasticus CBS 122540/CCFEE 5320 GU250376 GU250333

Elsinoe centrolobi CBS 222.50/AFTOL 1854 DQ678094 DQ678041

Elsinoe phaseoli CBS 165.31/AFTOL 1855 DQ678095 DQ678042

Farlowiella carmichaelina CBS 206.36/AFTOL 1787 AY541492 AY541482

Friedmanniomyces endolithicus CCFEE 524 GU250364 DQ066715

Gloniopsis praelonga CBS 112415 FJ161173 FJ161134

Guinardia bidwellii CBS 237.48/AFTOL 1618 DQ678085 DQ678034

Helicomyces roseus CBS 283.51/AFTOL 1613 DQ678083 DQ678032

Hysteropatella clavispora CBS 247.34/AFTOL 1305 AY541493 DQ678006

Hysteropatella elliptica CBS 935.97/AFTOL 1790 DQ767657 EF495114

Mycopathologia

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Table 2 continued


Kirschsteiniothelia aethiops (1) CBS 109.53/AFTOL 925 AY016361 AY016344

Kirschsteiniothelia aethiops (2) DAOM 231155/AFTOL 273 DQ678046 DQ677996

Laurera megasperma AFTOL 2094 FJ267702 GU561841

Lecanactis abietina AFTOL 305 AY548812 AY548805

Leptosphaeria maculans DAOM 229267/AFTOL 277 DQ4709646 DQ470993

Lophium mytilinum CBS 269.34/AFTOL 1609 DQ678081 DQ678030

Macrophomina phaseolina CBS 227.33/AFTOL 1783 DQ678088 DQ678037

Microxyphium citri CBS 451.66 GU301848 GU296177

Mycosphaerella euripotami JK 5586J GU301852 GZ479761

Mycosphaerella fijiensis OSC 100622/AFTOL 2021 DQ678098 DQ767652

Mycosphaerella graminicola CBS 292.38/AFTOL 1615 DQ678084 DQ678033

Mycosphaerella punctiformis CBS 113265/AFTOL 942 DQ470968 DQ471017

Myriangium duriaei CBS 260.36/AFTOL 1304 DQ678059 AY016347

Mytilinidion resinicola CBS 304.34 FJ161185 FJ161145

Neofusicoccum ribis CBS 115475/AFTOL 1232 DQ678053 DQ678000

Opegrapha dolomiticola AFTOL 993 – DQ883706

Patellaria atrata CBS 958.97 GU301855 GU296181

Phaeosclera dematioides CBS 157.81 GU301858 GU296184

Phaeotrichum benjaminii CBS 541.72/AFTOL 1184 AY004340 AY016348

Pleospora herbarum CBS 541.72/AFTOL 940 DQ247804 DQ247812

Pleosporales sp. CBS 101277 – GU456309

Preussia terricola DAOM 230091/AFTOL 282 AY544686 AY544726

Racodium rupestre L242 (TSB 37932) EU048582 EU048577

Rhythidhysterium rufulum CBS 306.38 FJ469672 AF164375

Roccella fuciformis AFTOL 126 AY584654 AY584678

Roccellographa cretacea AFTOL 93 DQ883696 DQ883705

Sarcinomyces crustaceus CBS 156.89 GU250893 –

Schismatomma decolorans AFTOL 307 AY548815 AY548809

Scorias spongiosa CBS 325.33/AFTOL 1594 DQ678075 DQ678024

Simoniella variegata AFTOL 80 – AY584669

Sirodesmium olivaceum CBS 395.59 GU250915 GU250904

Stylodothis puccinioides CBS 193.58 AY004342 AY016353

Sydowia polyspora CBS 116.29/AFTOL 1300 DQ678058 DQ678005

Symbiotaphrina buchneri CBS 6902 FJ176887 FJ176831

Symbiotaphrina kochii CBS 250.77 AY227719 FJ176833

Teratosphaeria associata CBS 112224 GU301874 GU296200

Tripospermum myrti CBS 437.68 GU323216 –

Trypethelium nitidiusculum AFTOL 2099 FJ267701 GU561842

Tubeufia cerea CBS 254.75/AFTOL 1316 DQ470982 DQ471034

Tubeufia paludosa CBS 245.49/AFTOL 1589 DQ767654 DQ767649

Tyrannosorus pinicola CBS 124.88/AFTOL 1235 DQ470974 DQ471025

Westerdykella cylindrica CBS 454.72/AFTOL 1037 AY004343 AY016355

Rock isolate A6 – GU250924 GU250932

Rock isolate A73 – GU250926 GU250934

Rock isolate AN1 – GU250927 GU250935

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subclades are identified; Lichenothelia cf. calcarea

and Lichenothelia convexa seem to be paraphyletic.

One additional specimen of L. cf. calcarea (L1717) is

basal to Teratosphaeriaceae (2), and L. tenussima is

basal to Myriangiales and Dothideales. These two

specimens are reported in parenthesis because they

might represent other fungal contaminants which have

been amplified instead of the true Lichenothelia

fungus. The sister relationship of the main Lichenot-

helia-group with Coniosporium apollinis- and C.

uncinatum-groups is poorly supported. Still these

three groups remain phylogenetically distinct from

other known lineages of Dothideomycetes.

In contrast, sequences obtained from cultured

specimens group in two distinct clades, clade I and

clade II: clade I represents the specimens from USA,

clade II those from Europe (Table 1). Clade I is nested

within Teratosphaeriaceae (2) and is closely related to

RIFs, to the lichenized Cystocoleus ebeneus and to the

two plant pathogens Tripospermum myrti and Devrie-

sia streliziae. Clade II is supported by a long branch in

Mycosphaerellaceae and is related to plant pathogenic

species of Mycosphaerella.

Discussion

The present study gives new insights into phylogenetic

relationships among Dothideomyceta fungi [39] with

different lifestyles, such as rock-inhabiting fungi

(RIF), melanised lichenized fungi and plant patho-

gens. Our phylogenetic inference reveals three new

fungal lineages in this class and provides for the first

time a phylogenetic placement of the enigmatic

lichen-like genus Lichenothelia. On the basis of

morphological characters, at the time of its descrip-

tion, this genus was believed to represents a link

between the dothidealean and lecanoralean fungi [19].

The results show that the majority of Lichenothelia

specimens form one clade in the Dothideomyceta

whose sister-group relationship with Coniosporium

apollinis and C. uncinatum is, however, only poorly

Table 2 continued


Rock isolate AN13 – GU250928 GU250936

Rock isolate CCFEE5211 – GU250371 GU250419

Rock isolate TRN5 CBS 118762 GU323956 GU323988

Rock isolate TRN11 CBS 118281 GU323957 –















Rock isolate TRN267 CBS 118769 – GU324043


Rock isolate TRN456 – GU323986 GU324015

Rock isolate TRN529 – GU323987 GU324016

A dash indicates missing sequences

Mycopathologia

123

Mycopathologia

123

supported. Despite the occasional presence of several

fungal species in one sample (see below), the genetic

identity of Lichenothelia seems convincing, as spec-

imens coming from different localities group all

together. In contrast, the placement of two additional

Lichenothelia samples at the base of Teratosphaeria-

cea (2) and of Myriangiales and Dothideales might be

due to the amplification of contaminating fungi. Our

preliminary results were obtained from a fairly

restricted number of taxa, and no data could so far

be obtained for the generic type species Lichenothelia

scopularia. A broader taxon and gene sampling (such

as mtSSU—for which already few sequences could be

obtained—or protein coding genes) will be needed to

corroborate the monophyly of Lichenothelia.

The phenotypically similarity between Lichenothe-

lia and the subgenera Lichenostigma and Licheno-

gramma raised the question whether they should be

recognized as distinct groups [7, 21, 23, 35]. Despite

the striking morphological similarities of these two

subgenera with Lichenothelia, they have been sepa-

rated due to subtle variations of thalli and fruiting

bodies [5, 15]. All the three genera are known to be

both lichenicolous fungi and to grow on bare rock

surfaces (such as Lichenostigma saxicola, [23]).

Therefore, common ascomata structure and spores

types, variable presence of vegetative hyphae and

shared lifestyles could represent traits assignable to

one homogenous fungal lineage. According to mor-

phological similarities, especially in the ascus struc-

ture, they were classified in the order Arthoniales, as

their own family Lichenotheliaceae [2, 5, 8, 16, 21].

The inclusion of Lichenostigma and Lichenogramma

species in future studies will clarify this unresolved

taxonomic issue. Our results, in any case, place

Lichenothelia in a new lineage within the Dothideo-

mycetes, but not in the Arthoniales.

Owing to the inconspicuous thallus these fungi

produce, the taxa are not frequently collected in the

field bymycologists or lichenologists. Therefore, fresh

collections are rarely available for molecular studies.

Even then, the fungi of interest are either directly

sequenced or isolated and cultured to obtain sufficient

amount of mycelium to extract genomic DNA.

Contaminant fungi which are cryptically associated

with the specimens grow sometimes more efficiently

in cultures than the apparently slower growing Liche-

nothelia species. These additional or co-occurring

fungi can bias the final results. Their presence is

confirmed in this study by the fungi represented in

clades I and II (Fig. 1). Both clades contain cultured

isolates from two geographic origins. The revelation

of co-occurring fungi in Lichenothelia thalli demon-

strates that thallus-forming black fungi frequently

represent consortia of different black fungal strains.

These additional fungi are related to plant pathogenic

fungi (Mycosphaerellaceae) and to other rock-inhab-

iting lineages (Teratosphaeriaceae).

Our results suggest a common link between rock-

inhabiting meristematic and lichen-forming lifestyles

in ascomycetous fungi. Lichenized lineages are scat-

tered in the Dothideomyceta [31, 32]. However,

compared to the larger and predominantly lichenized

lineages of Arthoniales and Trypeteliales, the thallus

of other lichenized species within this superclass is

usually poorly developed or simple and crust-like.

Cystocoleus and Racodium are two monospecific

lichen genera forming own lineages within Capnodi-

ales [28]. Their thallus is formed by colonies of

filamentous Trentepohlia-algae which are densely

entwined by coherent and dark-pigmented fungal

hyphae. In previous studies, we found that other black

fungi are frequently found in lichens [18] and that at

least some of these lichen-associated fungi might also

interact specifically with the lichen’s algae [3].

Meristematic growth is a common feature of pheno-

plasticity found in diverse rock-inhabiting black fungi.

We hypothesize that the ability to form coherent

cellular structures (or ‘‘pseudo-tissues’’), in some

oligotrophic rock-inhabiting fungi, could represent a

pre-adaptation in the evolution of the lichen thallus.

Lifestyle transition in fungi can be studied through

several approaches. Alternatively, to statistically eval-

uating the phylogenetic history with discrete catego-

ries, we show here, with Lichenothelia as one example,

that there could be a smooth transition from rock-

inhabiting lifestyle to lichenized lifestyle.Whether this

includes even an incipient stage of lichenicolous habit

still need further investigations. We think that in this

ecologically versatile group of oligotrophic fungi,

lifestyle expression could depend to some extent on

Fig. 2 Phylogenetic relationships of Lichenothelia spp. and

other rock-inhabiting fungi within known lineages of Dothide-

omyceta [38]: maximum likelihood analyses of the combined

nucLSU and nucSSU loci. Branches with bootstrap support

above 75 % are in bold, other bootstrap supports above 60 % are

reported above or aside the corresponding branches. Lichenized

taxa are labelled by an asterisk

b

Mycopathologia

123

ecological settings. Along with the notion of a

‘‘symbiotic playground’’ put forward by Gorbushina

and Broughton [11], mutualistic symbiotic relations

could then be selected against detrimental interactions,

especially under oligotrophic conditions. These inter-

actions then involve self-sustaining interactions with

algae or cyanobacteria, as well as the presence of black

fungal commensals in pre-formed thallus structures of

related and unrelated lichen-forming fungi.

Acknowledgments LM and MG are grateful to the Austrian

Science Foundation for financial support (FWF P24114). We

thank Cene Gostincar and Josef Hafellner for constructive

discussions and Jana Kocourkova for field co-work.

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