Fungal Diversity (2014) 66:113–137
DOI 10.1007/s13225-013-0250-9
Molecular data resolve a new order of Arthoniomycetes sister
to the primarily lichenized Arthoniales and composed of black
yeasts, lichenicolous and rock-inhabiting species
Damien Ertz & James D. Lawrey & Ralph S. Common &
Paul Diederich
Received: 2 April 2013 / Accepted: 11 June 2013 / Published online: 3 July 2013
# Mushroom Research Foundation 2013
Abstract Lichenicolous fungi belonging to the anamorphtypified genus Phaeosporobolus and to the teleomorph-typified
genus Lichenostigma were isolated in pure culture or sequenced
directly, with nuLSU and mtSSU sequences obtained. Phylogenetic analyses place the species of Phaeosporobolus in a strongly
supported clade with the generic type of Lichenostigma (L.
maureri), the genus Phaeococcomyces and several melanized
rock-inhabiting isolates. This strongly supported nonlichenized
lineage is sister to the primarily lichenized Arthoniales in the
Arthoniomycetes and is here described as the Lichenostigmatales. The new order is characterized by cells multiplying
by budding, either representing black yeasts, or species in which
conidiomata and ascomata are entirely made of an organised
agglomeration of spherical yeast-like cells. This way of life is not
only very different from all other Arthoniomycetes that exist only
in the mycelial stage, but ascomata and conidiomata representing
a dense and organised agglomeration of yeast cells might be
D. Ertz (*)
Jardin botanique national de Belgique, Domaine de Bouchout,
B-1860 Meise, Belgium
e-mail: damien.ertz@br.fgov.be
J. D. Lawrey
Department of Environmental Science and Policy, George Mason
University, 4400 University Drive, Fairfax, VA 22030-4444, USA
R. S. Common
534 Fenton St, Lansing, MI 48910, USA
P. Diederich
Musée national d’histoire naturelle, 25 rue Munster,
L-2160 Luxembourg, Luxembourg
unique amongst fungi. A further difference with the Arthoniales
is the absence of paraphysoids. Phylogenetic results suggest that
Phaeosporobolus usneae is the asexual stage of Lichenostigma
maureri. Most species of Phaeosporobolus are transferred to the
genus Lichenostigma except P. trypethelii, for which the new
genus Etayoa is described. The genus Diederimyces is reduced
into synonymy with Lichenostigma. Several other members of
Lichenostigma are placed in the Dothideomycetes and are
intermixed with Lichenothelia species.
Keywords Anamorphic fungi . Arthoniomycetes .
Dothideomycetes . Lichenostigmatales .
Phaeococcomycetaceae . Yeasts
Introduction
Lichenicolous fungi are lichen-associated organisms that live
mainly as parasites or commensals. Around 1800 species have
been described throughout the Ascomycota and Basidiomycota
(Lawrey and Diederich 2003; http://www.lichenicolous.net), and
it is estimated that 3000–5000 species will eventually be described. About 20 % of described lichenicolous ascomycetes are
known only in the asexual stage (anamorphs), most of uncertain
phylogenetic position. Molecular data are especially needed to
assess the phylogenetic position of these organisms and to make
connections between sexual (teleomorphs) and asexual stages
that are often described in different genera.
We began our study to investigate the phylogenetic placement
and hypothesized relationship between the teleomorph-typified
lichenicolous species Lichenostigma maureri and the anamorph-
114
typified lichenicolous species Phaeosporobolus usneae.
Lichenostigma maureri grows on Usnea and various other
macrolichens and forms superficial rounded ascomata containing bitunicate, subglobose asci and 1-septate, dark brown ascospores (Hafellner 1982). Phaeosporobolus usneae grows on
various macrolichens (mainly Parmeliaceae such as Bryoria,
Evernia, Letharia, Usnea, but also Ramalina) and forms tiny,
brownish, superficial, stromatic conidiomata containing
dark brown, subglobose or ellipsoid, multicellular conidia
(Hawksworth and Hafellner 1986). An anamorph-teleomorph
relationship between the two was suggested by the remarkable similarity of the ascomata of L. maureri to the
conidiomata of P. usneae (Hawksworth and Hafellner 1986;
Berger and Brackel 2011), and the fact that they sometimes
grow intermixed (Berger and Brackel 2011). The fruit bodies
of the two taxa are so similar that a microscopical examination of squash preparations is required for an accurate identification (Diederich 2004). Lichenostigma maureri was supposed to produce only ascomata whereas P. usneae only
conidiomata.
The phylogenetic position of these fungi has been discussed
but never firmly resolved. Lichenostigma has been thought by
some authors (Hafellner 1982; Hafellner and Calatayud 1999) to
belong to the Arthoniales based on ascus type, but other authors
consider it a member of the Dothideomycetes close to
Lichenothelia (Eriksson and Hawksworth 1986: 137; NavarroRosinés and Hafellner 1996; Muggia et al. 2012).
Phaeosporobolus was originally suggested to be most closely
allied to the lichenicolous genus Sclerococcum (Hawksworth and
Hafellner 1986), but Etayo (1995) has more recently suggested
that it is the asexual stage of the lichenicolous genus
Diederimyces.
Analysis of sequences obtained from L. maureri and P.
usneae yielded one expected result—that the two were conspecific, but also an unexpected one—that L. maureri is in a clade
sister to the Arthoniomycetes along with several rockinhabiting fungi (RIF) and does not group with the other
sequences of Lichenotheliaceae placed in the Dothideomycetes.
The existence of this clade had been recognized earlier by
Ruibal et al. (2008, 2009) but its composition was far from
certain. Many of the RIF in this clade were originally identified
in Ruibal et al. (2008) as Phaeococcomyces species or
“phaeococcomyces-like”, but these labels were dropped in the
subsequent paper (Ruibal et al. 2009). Nevertheless, our results
now indicated a possible unrecognized link between
Lichenostigma and Phaeococcomyces.
Given these initial results, we expanded our objectives
to include a larger number of specimens of Lichenostigma
and Phaeosporobolus in a two-locus molecular phylogenetic study designed to determine the composition of the
new clade in Arthoniomycetes, and provide formal recognition of the clade. Moreover, this study aims to test the
monophyly of the two subgenera of Lichenostigma.
Fungal Diversity (2014) 66:113–137
Materials and methods
Morphological study
Herbarium specimens are deposited in BR, KRAM, MSC, NY
and UBC, and in the private collections of P. Diederich, J. Etayo
and K. Kalb. Dry herbarium specimens were examined and
measured under a binocular microscope Leica MZ 7.5 (magnification up to 50×). Macroscopic photographs were done using a
Canon 40D camera with Nikon BD Plan 10 or Nikon ELWD 40
microscope objectives, StackShot (Cognisys) and Helicon Focus
(HeliconSoft) for increasing the depth of field. Entire
unsectioned ascomata or conidiomata were studied in water,
either without or with pressure on the coverslip. Microscopic
photographs of stromata in water were prepared using a Zeiss
Photomikroskop III or Leica DMLB microscope, a Leica EC3
camera and Helicon Focus. Measurements based on statistical
data are indicated as ðminimum–ÞX−σx –X þ σx ð−maximumÞ,
followed by the number of measurements (N). Unless otherwise
indicated, iodine reactions were done using 1 % IKI (1 g I2, 2 g
KI, 100 ml water), without (I) or with (K/I) pre-treatment with
10 % KOH, without or with pre-treatment with commercial
bleach (C).
Isolation of fungal cultures
Ascomata, conidiomata or hyphae of freshly collected material
of Lichenostigma or Phaeosporobolus were washed in sterilized water and transferred to Malt-Yeast Extract agar following
Yoshimura et al. (2002). The cultures were kept at room temperature in the laboratory of the National Botanic Garden of
Belgium and exposed to a natural day light regime. No culture
chambers were used to test whether different light or temperature conditions could improve the growth rate. All the strains
were slow growing and therefore 3 to 5 months were required
in order to obtain sufficient material for DNA extraction.
Molecular techniques
Genomic DNA was isolated from mycobiont cultures following
the procedures cited by Ertz et al. (2011). When cultures failed,
2 to 4 ascomata or conidiomata of freshly collected specimens
were added to a tube containing the PCR reaction mixture and
amplified directly as explained in Lawrey et al. (2007: 780).
Because Lichenostigma maureri and Phaeosporobolus usneae
often grow intermixed, we carefully checked under the microscope each ascoma or conidioma by crushing them in a drop of
sterile water before adding the material to the PCR tube. We
amplified and sequenced a fragment of about 1.2 kb of the
nuLSU using primers LIC15R, LR3R, LR3, LR7, and LR6
(Miadlikowska et al. 2002; Vilgalys and Hester 1990). Primers
Fungal Diversity (2014) 66:113–137
for amplification and sequencing of the mtSSU rDNA were
mrSSU1 and mrSSU3R (Zoller et al. 1999).
PCR products were purified using the QIAquick PCR
Purification Kit (Qiagen). The yield of the PCRs was verified
by running the products on a 1 % agarose gel using ethidium
bromide. Both strands were sequenced by Macrogen®. Sequences were edited and overlapping fragments were assembled in larger consensus sequences using Sequencher v4.6
(Gene Codes Corporation, Ann Arbor, Michigan). Sequences were subjected to BLAST searches to verify their
closest relatives and to detect potential contaminations.
Taxon selection and phylogenetic analyses
We obtained 51 new sequences for this study from Canada,
the Canary Islands (Gomera, Tenerife), Croatia, Florida,
France (including Corsica), Luxembourg, South Africa and
Switzerland. For the phylogenetic analyses, 126 sequences
were retrieved from GenBank in addition to the newly generated sequences. We selected members representing all major
clades in the Arthoniomycetes and Dothideomycetes and as
far as possible having both nuLSU and mtSSU sequences
(Table 1). These data were selected mainly from Diederich
et al. (2012b), Ertz and Tehler (2011), Lawrey et al. (2011),
Muggia et al. (2012) and Ruibal et al. (2009). More taxa were
retrieved from GenBank based on BLAST searches of our
newly generated sequences. Three outgroup species were
chosen to represent the Eurotiomycetes (Caliciopsis pinea
and Capronia munkii) and the Leotiomycetes (Lachnum
virgineum). The sequences of taxa listed in Table 1 were
aligned manually using MacClade 4.05 (Maddison and
Maddison 2002). Ambiguous regions and introns representing
a total of 3054 bp (1282 bp for nuLSU and 1772 bp for mtssu;
mainly due to long introns) were delimited manually and
excluded from the analyses.
Because it was not possible to complete the nuLSU and
mtSSU sequences for the same set of 97 samples, analyses for
topological incongruence among loci were carried out on the
same data sets for each gene (i.e., 80 nuLSU and 80 mtSSU
sequences; Table 1). Analyses were carried out using 1000
replicates of Neighbor-Joining bootstrapping (NJ-bs) with distance measure estimated by maximum likelihood under a sixparameter (GTR) best-fit evolutionary model for nucleotide
substitution (Cunningham et al. 1998; Liò and Goldman 1998;
Yang et al. 1994) using PAUP* 4.0b10 (Swofford 2002). Best-fit
evolutionary models were estimated for all NJ analyses using
Akaike Information Criterion (AIC) as implemented in
Modeltest v. 3.06 (Posada and Crandall 1998). The TIM+I+G
model was selected for the nuLSU data set while the GTR+I+G
model was selected for the mtSSU data set. All topological
bipartitions with NJ-bs values≥70 % were compared for the
two loci. A conflict was assumed to be significant if two different
relationships (one being monophyletic and the other being non-
115
monophyletic) for the same set of taxa were both supported with
bootstrap values≥70 % (Mason-Gamer and Kellogg 1996).
Based on this criterion, no conflict was detected and therefore
the nuLSU and mtSSU data sets were concatenated. The combined two-loci data set consisted of 97 taxa and 1803 unambiguously aligned sites, 1249 for nuLSU and 554 for mtSSU.
Bayesian analyses were carried out on the two-loci data set
using the Metropolis-coupled Markov chain Monte Carlo method (MCMCMC) in MrBayes v. 3.1.2 (Huelsenbeck and
Ronquist 2001; Ronquist and Huelsenbeck 2003) on the
CIPRES Web Portal (Miller et al. 2010). Analyses were run
under the selected models for each gene partition using a
gamma-distributed rate parameter and a proportion of invariable
sites. Two parallel MCMCMC runs were performed each using
four independent chains and 30 million generations, sampling
trees every 1000th generation. TRACER v.1.5 (Rambaut and
Drummond 2007) was used to ensure that stationarity was
reached by plotting the log-likelihood values of the sample points
against generation time. Posterior probabilities (PP) were determined by calculating a majority-rule consensus tree generated
from the 45002 post-burnin trees of the 60002 trees sampled by
the two MCMCMC runs using the sumt option of MrBayes. A
Maximum Likelihood (ML) analysis was performed on the twoloci data set using GARLI (Zwickl 2006, v.0.951 for OSX) with
default settings, and a single most likely tree was produced
(−lnL=25100.4677). One thousand bootstrap pseudoreplicates
were used to calculate a majority rule consensus tree in PAUP* to
assess the Maximum Likelihood bootstrap values (ML-bs). In
addition, the aligned sequences were subjected to a maximum
likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al.
2005; Stamatakis 2006) on the Cipres Web Portal, with parametric bootstrapping using 1000 replicates under the GTRGAMMA
model. The Bayesian tree did not contradict the ML trees topology for the strongly supported branches and hence only the
majority rule consensus tree of the ML analysis using GARLI
is shown with the posterior probabilities of the Bayesian analysis
added below the internal branches and the bootstrap support
values of the RAxML analysis added above the internal
branches. ML-bs≥70 % and PP≥95 % were considered to be
significant. Phylogenetic trees were visualized using FigTree
v1.3.1 (Rambaut 2012).
Results
The phylogenetic analyses resolved the Dothideomyceta
(Arthoniomycetes and Dothideomycetes) as a strongly supported group, but the Dothideomycetes does not form a sister group
to the Arthoniomycetes as shown in other studies using a wide
sampling in the Arthoniomycetes and Dothideomycetes
(Ruibal et al. 2009; Muggia et al. 2012, Fig. 1). Some main
groups of Dothideomycetes are strongly supported by the three
analyses, such as the Dothideales, the Capnodiales, the
116
Fungal Diversity (2014) 66:113–137
Table 1 Specimens and DNA sequences used in this study, with their respective voucher information. GenBank accession numbers (in bold) refer to
sequences (51) generated by this project. All other sequences (126 GenBank identification numbers) were obtained directly from GenBank
Name
Order
Family
Voucher information
nuLSU
mtSSU
Acrospermum compressum
Acrospermales
Acrospermaceae
M151; J. Vesterholt s.n. (TUR)
EU940084
EU940237
Alyxoria ochrocheila
Arthoniales
‘Lecanographaceae’
Ertz 7519 (BR)
EU704100
EU704072
Alyxoria varia
Arthoniales
‘Lecanographaceae’
Ertz 7570 (BR)
EU704103
EU704075
Arthonia didyma
Arthoniales
Arthoniaceae
Ertz 7587 (BR)
EU704083
EU704047
Arthonia dispersa
Arthoniales
Arthoniaceae
UPSC2583
AY571381
AY571383
Arthopyrenia salicis
Pleosporales
Arthopyreniaceae
CBS 368.94
AY538339
AY538345
Botryosphaeria dothidea
Botryosphaeriales
Botryosphaeriaceae
CBS 115476 (AFTOL-ID 946)
DQ678051
FJ190612
Botryosphaeria ribis
Botryosphaeriales
Botryosphaeriaceae
CBS 115475 (AFTOL-ID 1232)
DQ678053
AF271148
Briancoppinsia cytospora
Arthoniales
Arthoniaceae
Ertz 15244 (BR)
JF830770
JF830772
Caliciopsis pinea
Coryneliales
Coryneliaceae
AFTOL-ID 1869
DQ678097
FJ190653
Capnodium coffeae
Capnodiales
Capnodiaceae
CBS 147.52 (AFTOL-ID 939)
DQ247800
FJ190609
Capronia munkii
Chaetothyriales
Herpotrichiellaceae
AFTOL-ID 656
EF413604
FJ225723
Chiodecton natalense
Arthoniales
Roccellaceae
Ertz 6576 (BR)
EU704085
EU704051
Chrysothrix caesia
Arthoniales
Chrysotrichaceae
AFTOL-ID 775
FJ469668
FJ469671
Chrysothrix chrysophthalma
Arthoniales
Chrysotrichaceae
Ertz 10927 (S)
HQ454519
KF176966
Coniosporium apollinis
Unknown
Unknown
CBS 109860
GU250899
GU250910
Coniosporium uncinatum
Unknown
Unknown
CBS 100212
GU250902
GU250913
Cryomyces antarcticus
Unknown
Unknown
CCFEE 536
GU250365
GU250411
Cryptothecia candida
Arthoniales
Arthoniaceae
Ertz 9260 (BR)
HQ454520
EU704052
Cystocoleus ebeneus
Capnodiales
Unknown
L161 Hafellner & Muggia (GZU)
EU048578
EU048584
Dendrographa leucophaea
Arthoniales
Roccellaceae
Sparrius 7999 (BR)
AY548810
AY548811
Dimidiographa longissima
Arthoniales
Roccellographaceae
Ertz 9155 (BR)
EU704097
EU704069
Dothidea insculpta
Dothideales
Dothideaceae
CBS 189.58 (AFTOL-ID 359/921)
DQ247802
FJ190602
Dothideomycetes sp. AN13
Unknown
Unknown
AN13
GU250928
N/A
Enterographa crassa
Arthoniales
Roccellaceae
Ertz 5041 (BR)
EU704088
EU704056
Enterographa zonata
Arthoniales
Roccellaceae
Vigneron 104 (BR)
EU704109
EU704081
Erythrodecton granulatum
Arthoniales
Roccellaceae
Ertz 9908 (BR)
EU704090
EU704058
Etayoa trypethelii
Lichenostigmatales
Phaeococcomycetaceae
Common 9200-G
KF176940
KF176967
Etayoa trypethelii
Lichenostigmatales
Phaeococcomycetaceae
Common 9215-P
KF176941
KF176968
Etayoa trypethelii
Lichenostigmatales
Phaeococcomycetaceae
Common 9434-K
KF176942
KF176969
Etayoa trypethelii
Lichenostigmatales
Phaeococcomycetaceae
Mukherjee s.n. (BR)
KF176943
KF176970
Farlowiella carmichaeliana
Unknown
Unknown
CBS 206.36 (AFTOL-ID 1787)
AY541492
AY571387
Friedmanniomyces endolithicus
Capnodiales
Teratosphaeriaceae
CCFEE 5180
GU250367
GU250415
Gloniopsis praelonga
Hysteriales
Hysteriaceae
CBS 112415
FJ161173
N/A
Hortaea werneckii
Capnodiales
Teratosphaeriaceae
CBS 708.76
GU301818
GU561844
Hysteropatella clavispora
Patellariales
Patellariaceae
CBS 247.34 (AFTOL-ID 1254)
AY541493
AY571388
Kirschsteiniothelia aethiops
Unknown
Unknown
CBS 109.53 (AFTOL-ID 925)
AY016361
FJ190604
Lachnum virgineum
Helotiales
Hyaloscyphaceae
AFTOL-ID 49
AY544646
AY544745
Laurera megasperma
Trypetheliales
Trypetheliaceae
Ertz 9725 (BR) (AFTOL-ID 2094)
FJ267702
GU561847
Lecanactis abietina
Arthoniales
Roccellaceae
Ertz 5068 (BR) (AFTOL-ID 305)
AY548812
AY548813
Lichenoconium erodens
Unknown
Unknown
JL363-09
HQ174267
HQ174266
Lichenostigma alpinum
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17379
KF176944
KF176971
Lichenostigma alpinum
Lichenostigmatales
Phaeococcomycetaceae
Ertz 17522 (BR)
KF176945
KF176972
Lichenostigma alpinum
Lichenostigmatales
Phaeococcomycetaceae
Ertz 17519 (BR)
KF176946
KF176973
KF176974
Lichenostigma chlaroterae
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17329
KF176947
Lichenostigma chlaroterae
Lichenostigmatales
Phaeococcomycetaceae
Neuberg
KF176948
KF176975
Lichenostigma chlaroterae
Lichenostigmatales
Phaeococcomycetaceae
Goward 12-30
N/A
KF176976
Lichenostigma chlaroterae
Lichenostigmatales
Phaeococcomycetaceae
Goward 12-42
KF176949
KF176977
“Lichenostigma” cf. elongatum
Unknown
Lichenotheliaceae
Ertz 15255 (BR)
KF176950
N/A
Lichenostigma maureri asexual stage
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17306
KF176951
KF176978
Fungal Diversity (2014) 66:113–137
117
Table 1 (continued)
Name
Order
Family
Voucher information
nuLSU
mtSSU
Lichenostigma maureri asexual stage
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17337
KF176952
N/A
Lichenostigma maureri sexual stage
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17326
KF176953
KF176979
Lichenostigma maureri sexual stage
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17337
KF176954
KF176980
Lichenostigma sp.
Lichenostigmatales
Phaeococcomycetaceae
Diederich 17240
KF176955
KF176981
“Lichenostigma” sp.
Unknown
Lichenotheliaceae
Ertz 16122 (BR)
KF176956
KF176982
“Lichenostigma” sp.
Unknown
Lichenotheliaceae
Ertz 16340 (BR)
KF176957
KF176983
“Lichenostigma” sp.
Unknown
Lichenotheliaceae
Ertz 16455 (BR)
KF176958
KF176984
“Lichenostigma” sp.
Unknown
Lichenotheliaceae
Ertz 17457 (BR)
KF176959
KF176985
“Lichenostigma” sp.
Unknown
Lichenotheliaceae
Ertz 17540 (BR)
KF176960
KF176986
Lichenostigma sp.
Lichenostigmatales
Phaeococcomycetaceae
Ertz 17591 (BR)
KF176961
KF176987
Lichenothelia cf. calcarea 1
Unknown
Lichenotheliaceae
L1324; Muggia L.
KC015062
N/A
Lichenothelia cf. calcarea 2
Unknown
Lichenotheliaceae
L1708; Knudsen K. 12672
KC015065
N/A
KF176988
Lichenothelia convexa
Unknown
Lichenotheliaceae
Diederich P. 17491
KF176962
Lichenothelia L985
Unknown
Lichenotheliaceae
L985; Perlmutter G. 2621
KC015075
N/A
Lichenothelia rugosa
Unknown
Lichenotheliaceae
Diederich 17310
KF176963
KF176989
Lichenothelia rugosa
Unknown
Lichenotheliaceae
Ertz 16065 (BR)
KF176964
N/A
Lophium mytilinum
Mytilinidiales
Mytilinidiaceae
CBS 269.34 (AFTOL-ID 1609)
DQ678081
N/A
Mycosphaerella punctiformis
Capnodiales
Mycosphaerellaceae
CBS 113265 (AFTOL-ID 942)
DQ470968
FJ190611
Myriangium duriaei
Myriangiales
Myriangiaceae
CBS 260.36 (AFTOL-ID 1304)
DQ678059
AY571389
Opegrapha vermicellifera
Arthoniales
Opegraphaceae
Ertz 7562 (BR)
EU704105
EU704077
Opegrapha vulgata
Arthoniales
Opegraphaceae
Ertz 7564 (BR)
EU704108
EU704080
Paralecanographa grumulosa
Arthoniales
Opegraphaceae
Ertz 13565 (BR)
HQ454555
N/A
Phaeococcomyces eucalypti
Lichenostigmatales
Phaeococcomycetaceae
CPC:17606
KC005791
N/A
Phaeococcomyces nigricans
Lichenostigmatales
Phaeococcomycetaceae
CBS 652.76
AF361048
N/A
Phaeococcomyces cf. nigricans
Lichenostigmatales
Phaeococcomycetaceae
MZ107
AJ276065
N/A
Phaeotrichum benjaminii
Unknown
Phaeotrichaceae
CBS 541.72
AY004340
AY538349
Phoma cladoniicola
Pleosporales
Phaeosphaeriaceae
CBS 128025
JQ238625
JQ238624
Phyllobathelium anomalum
Unknown
Strigulaceae
Lücking s.n. (F)
GU327722
GU327698
Preussia terricola
Pleosporales
Sporormiaceae
DAOM 230091 (AFTOL-ID 282)
AY544686
AY544754
Racodium rupestre
Capnodiales
Unknown
L346 - Hafellner & Muggia (GZU)
EU048583
EU048588
Reichlingia leopoldii
Arthoniales
Arthoniaceae
Ertz 13293 (BR)
HQ454581
JF830773
Roccella fuciformis
Arthoniales
Roccellaceae
Diederich 15572 (AFTOL-ID 126)
AY584654
EU704082
rock isolate CCFEE 5284
Lichenostigmatales
Phaeococcomycetaceae
CCFEE 5284
GU250373
N/A
rock isolate D007_09
Lichenostigmatales
Phaeococcomycetaceae
D007_09
GU250402
N/A
rock isolate TRN 213
Lichenostigmatales
Phaeococcomycetaceae
TRN 213
N/A
GU324040
rock isolate TRN 452
Lichenostigmatales
Phaeococcomycetaceae
TRN 452
GU323985
GU324048
rock isolate TRN 456
Lichenostigmatales
Phaeococcomycetaceae
TRN 456
GU323986
GU324049
rock isolate TRN 529
Lichenostigmatales
Phaeococcomycetaceae
TRN 529
GU323987
GU324050
Schismatomma pericleum
Arthoniales
Roccellaceae
Tehler 7701 (S)
AF279408
AY571390
Sydowia polyspora
Dothideales
Dothioraceae s.l.
CBS 116.29 (AFTOL-ID 1300)
DQ678058
FJ190631
Trypethelium nitidiusculum
Trypetheliales
Trypetheliaceae
Ertz 9716 (BR) (AFTOL-ID 2099)
FJ267701
GU561848
Tubeufia cerea
Unknown
Tubeufiaceae
CBS 254.75 (AFTOL-ID 1316)
DQ470982
FJ190634
Tylophoron hibernicum
Arthoniales
Arthoniaceae
Ertz 11546 (BR)
JF295083
JF830778
Tyrannosorus pinicola
Unknown
Unknown
CBS 124.88 (AFTOL-ID 1235)
DQ470974
FJ190620
Venturia chlorospora
Unknown
Venturiaceae
Kruys 502 (UPS)
DQ384101
DQ384084
Xanthoriicola physciae
Capnodiales
Teratosphaeriaceae
Diederich 16713
KF176965
KF176990
Zwackhia viridis
Arthoniales
‘Lecanographaceae’
Ertz 7619 (BR)
EU704106
EU704078
Botryosphaeriales, the Pleosporales and the Trypetheliales, but
the relationships among them are not supported. Most of the
Lichenotheliaceae cluster together, but the clade is not well
supported. Two Lichenothelia specimens (Lichenothelia cf.
118
Fungal Diversity (2014) 66:113–137
Roccella fuciformis
Schismatomma pericleum
Chiodecton natalense
Lecanactis abietina
91
Dendrographa leucophaea
100
.99
Enterographa zonata
100
Enterographa crassa
1
100
1
Erythrodecton granulatum
100
1
Opegrapha vermicellifera
99
Opegrapha vulgata
1
77
1
Paralecanographa grumulosa
.99
Dimidiographa longissima
100
Alyxoria varia
100
Alyxoria ochrocheila
1
98
1
Zwackhia viridis
1
Cryptothecia candida
92
Briancoppinsia cytospora
1
Tylophoron hibernicum
100
Arthonia didyma
1
76 .98
Reichlingia leopoldii
Arthonia dispersa
100
Chrysothrix caesia
Chrysothrix chrysophthalma
1
89 Lichenostigma chlaroterae G12-42 Canada/Lecanora chlarotera s.l.
Lichenostigma chlaroterae G12-30 Canada/Lecanora chlarotera s.l.
91
Lichenostigma chlaroterae Neuberg Luxembourg/Lecanora argentata
Lichenostigma chlaroterae D17329 Switzerland/Lecanora chlarotera
Lichenostigma sp. D17240 Luxembourg/Fuscidea lightfootii
.96 77Lichenostigma alpinum E17519 France/Fuscidea cyathoides
76
Lichenostigma alpinum E17522 France/Pertusaria albescens
.99
97
Lichenostigma alpinum D17379 Croatia/Pertusaria albescens
Lichenostigma maureri D17326 sexual stage Switzerland/Usnea sp.
1
100 Lichenostigma maureri D17337 asexual stage Switzerland/Usnea sp.
Lichenostigma maureri D17337 sexual stage Switzerland/Usnea sp.
1
Lichenostigma maureri D17306 asexual stage Switzerland/Letharia vulpina
Lichenostigma sp. E17591 France/Phaeographis smithii
.99
98
Phaeococcomyces eucalypti
rock isolate TRN452
1
94
100
rock isolate TRN456
Phaeococcomyces
cf. nigricans MZ107
1
98
Phaeococcomyces nigricans CBS 652.76 (TYPE)
100 Etayoa trypethelii C9200G Florida/Graphidaceae
1
100
1 Etayoa trypethelii C9215P Florida/Graphidaceae
95
1
Etayoa trypethelii C9434K Florida/Graphis caesiella
1
Etayoa trypethelii Mukherjee South Africa/Graphis sp.
87
rock isolate CCFEE 5284
96
73
rock isolate D007 09
1
1
1
rock isolate TRN529
rock isolate TRN213
100
Laurera megasperma
Trypethelium nitidiusculum
1
Lichenothelia cf. calcarea 2
Lichenothelia L985
Dothideomycetes AN13
Coniosporium uncinatum
Lichenoconium erodens
Coniosporium apollinis
Arthopyrenia salicis
100
Preussia terricola
1
Phoma cladoniicola
.96
Lophium mytilinum
Gloniopsis praelonga
1
Acrospermum compressum
Phyllobathelium anomalum
Farlowiella carmichaeliana
100
Botryosphaeria dothidea
Botryosphaeria ribis
1
‘Lichenostigma’ sp. E16455
99
‘Lichenostigma’ sp. E17540
1
‘Lichenostigma’ sp. E17457
.99
‘Lichenostigma’ sp. E16340
Lichenothelia convexa D17491
‘Lichenostigma’ sp. E16122
Lichenothelia cf. calcarea 1
‘Lichenostigma’ cf. elongatum E15255
100
Lichenothelia rugosa D17310
Lichenothelia rugosa E16065
1
100
Venturia chlorospora
94
Tyrannosorus pinicola
1
1
Phaeotrichum benjaminii
Kirschsteiniothelia aethiops
87
Cystocoleus ebeneus
Mycosphaerella punctiformis
1
99
100
Friedmanniomyces endolithicus
1
100
Xanthoriicola physciae D16713
1
1
99
Hortaea werneckii
Capnodium coffeae
1
94
Racodium rupestre
100
Sydowia polyspora
1
79
Dothidea insculpta
1
1
Myriangium duriaei
Cryomyces antarcticus
Tubeufia cerea
Hysteropatella clavispora
Lachnum virgineum
Capronia munkii
100
1
ARTHONIALES
ARTHONIOMYCETES
LICHENOSTIGMATALES
TRYPETHELIALES
PLEOSPORALES
BOTRYOSPHAERIALES
DOTHIDEOMYCETA
97
1
100
1
LICHENOTHELIACEAE
CAPNODIALES
DOTHIDEALES
Caliciopsis pinea
0.04
Fig. 1 Phylogenetic relationships among 94 samples within Dothideomyceta (with three outgroup taxa) based on a combined data set of
nuLSU and mtSSU sequences that resulted from a maximum likelihood
analysis using Garli. Internal branches with maximum likelihood bootstrap values≥70 obtained from a Garli analysis are considered strongly
supported and represented by thicker lines. Posterior probabilities≥0.95
resulting from a bayesian analysis are shown below internal branches and
bootstrap support values from a RAxML analysis are shown above the
internal branches. Lichenized taxa are in green and lichenicolous fungi
in red (P. grumulosa might have both live styles). Collecting numbers
of the authors following the species names act as specimen and sequence identifiers. Countries and host lichens are indicated for the
lichenicolous Lichenostigmatales
calcarea 2 and Lichenothelia L985) are placed in a different
lineage as sister to ‘Dothideomycetes AN13’, but this placement is not supported either. In the combined nuLSU and
nuSSU tree by Muggia et al. (2012), the Lichenothelia clade
also lacks significant support.
The Arthoniomycetes are strongly supported by the three
analyses and are divided in two main groups corresponding to
the Lichenostigmatales and to the Arthoniales. However, the
sister relationship of the Chrysothricaceae with the rest of the
Arthoniales is not supported. As a consequence, the phylogenetic
Fungal Diversity (2014) 66:113–137
position of this family is uncertain within the Arthoniomycetes.
The relationships within the Arthoniales are strongly supported
and are congruent with the well-supported lineages in Ertz and
Tehler (2011). The Lichenostigmatales are divided in two main
lineages, a first well-supported clade including the new genus
Etayoa with some unidentified rock isolates and a second poorly
supported clade divided itself in a well-supported clade including
the genus Phaeococcomyces (with the generic type
Phaeococcomyces nigricans) and another well-supported clade
corresponding to Lichenostigma s. str.
Discussion
This is the first molecular phylogenetic study that includes
members of the genera Lichenostigma and Phaeosporobolus.
The genus Lichenostigma has been considered a member of the
Lichenotheliaceae Henssen (Eriksson and Hawksworth 1986:
137), which otherwise includes only the genus Lichenothelia.
The Lichenotheliaceae were considered to belong to the
Arthoniales by some authors (e.g., Fernández-Brime et al.
2010, Ihlen 2004, Valadbeigi and Brackel 2011), but they have
also been placed in the Dothideomycetes (e.g., Lumbsch and
Huhndorf 2010). Based on molecular data, Muggia et al. (2012)
recently suggested that the Lichenotheliaceae (Lichenostigma
not included) were part of the Dothideomycetes and not of the
Arthoniales. Our phylogenetic tree confirms the results of
Muggia et al. (2012), as most described Lichenothelia and
Lichenostigma species form a clade within the Dothideomycetes.
However, in our phylogenetic tree, the generic type
Lichenostigma maureri does not belong to this clade but occupies an isolated position in the Arthoniomycetes. Moreover, all
species sequenced here and previously accepted in the genus
Phaeosporobolus are part of the Arthoniomycetes in a lineage
with Lichenostigma maureri, black yeasts (Phaeococcomyces)
and several unidentified rock inhabiting fungi.
This well-supported clade is sister to the Arthoniales and we
are recognizing it here as a new order in the Arthoniomycetes,
the Lichenostigmatales. Previous phylogenetic studies (Ruibal
et al. 2008, 2009) hinted at the existence of this group, but its
composition and placement were somewhat equivocal. Ruibal
et al. (2008) identified several of the rock isolates in the group
as Phaeococcomyces spp. or phaeococcomyces-like species,
but most isolates have not been named. Ruibal et al. (2009)
suggested the entire group may represent an example of an
early diverging Dothideomycetes lineage, supporting the hypothesis that rock surfaces were important substrates for ancient
fungal lineages, as appears to the be case for lichens in
Verrucariales (Gueidan et al. 2008). Given its position sister
to the lichenized Arthoniales, the Lichenostigmatales, which
thus far appears to be nonlichenized, may represent a possible
transitional group between the Arthoniomycetes and the
119
Dothideomycetes, but one showing clear affinities with the
Arthoniomycetes.
The position of Lichenostigma maureri within the
Arthoniomycetes is supported by the ascus type. Calatayud
and Barreno (2003) observed a K/I+ blue ring in the lower
part of the apical dome (near the ocular chamber) of the asci
when studying material of Lichenostigma maureri. We observed that parts of the ascus tholus (not only a ring) may
become blue (Fig. 2b). This reaction, not mentioned in the
original description of the species (Hafellner 1982), supports
the placement of the generic type of Lichenostigma close to
the Arthoniales. Moreover, they observed that the outer wall
of some young hyaline ascospores of L. maureri reacted K/I+
blue (confirmed by us, see Fig. 2b), as in the gelatinous
sheath of certain Arthonia species, a diagnostic character
also supporting the close relationship with the Arthoniales.
However, the Lichenostigmatales appears to be morphologically quite distinct from the Arthoniales. Our detailed studies of
Lichenostigma maureri and the related Phaeosporobolus species
revealed that the cells forming the conidiomata and ascomata of
these taxa are spherical and multiply by budding (Figs. 2d, 3f and
7b). Only three specialized cells types of the conidiomata and
ascomata are not spherical: the asci, the ascospores and the
conidiogenous cells. The ascospores are the only one of these
having real septa and that could form a mycelial stage, although
this is rarely observed (see Taxonomy section, Fig. 4j–l). Germination of conidia is also exclusively by budding. This way of life
is very different from all Arthoniales species that exist only in the
mycelial stage (=multiplication of cells by septa), never in the
yeast stage. Moreover, to our knowledge, ascomata and
conidiomata representing a dense and organised agglomeration
of yeast cells is unique amongst fungi. The order also includes
black yeasts of the genus Phaeococcomyces that exclusively
reproduce by budding but do not agglomerate. A further difference with the Arthoniales is the absence of paraphysoids in the
ascomata of Lichenostigma maureri. Some groups within
the Dothideomycetes (e.g., Cookellaceae) are similar to
Lichenostigma s. str. by having small ascomata with scattered
globose-subglobose asci dispersed in stromatic tissue lacking
pseudoparaphyses. However, the Cookellaceae differ from the
Lichenostigmatales by having a different ecology (parasitic on
leaves, possibly fungicolous), asci lacking an ocular chamber,
muriform ascospores and non-spherical stromatic cells that probably do not multiply by budding. They were placed by von Arx
(1963) in the Myriangiales but are considered now of uncertain
order in Dothideomycetes (Lumbsch and Huhndorf 2010). Molecular data are needed here to confirm their placement in the
Myriangiales.
Interestingly, Lichenostigma maureri and Phaeosporobolus
usneae are closely related in our phylogenetic tree. Along with
the morphological similarities of the fruit bodies, our results
strongly suggest that Lichenostigma maureri is the sexual stage
of Phaeosporobolus usneae. As Lichenostigma maureri is the
120
Fungal Diversity (2014) 66:113–137
Fig. 2 Lichenostigma maureri.
a Ascomata on Usnea hirta
(Diederich 6642). b Young asci
with hyaline ascospores in
0.15 % IKI, after pre-treatment
with C and K), showing blue
reaction of ascus wall and of
ascospore wall (Miller s.n.).
c Ascoma with mature ascospores
in water (Diederich 17326).
d Stromatic cells and verruculose
ascospores in squash preparation
in water (Van Cappellen s.n.).
e Conidioma with conidia in
water (Diederich 6633).
f Magnification of e. g Conidium
in water (Diederich 17334).
h K/I-reaction of ascoma (shortly
bluish; inner part did not yet react)
(Diederich 17334). Scale bars:
a=200 μm, b–c, e=20 μm,
d,f–g=10 μm, h=50 μm
generic type of Lichenostigma and Phaeosporobolus usneae the
generic type of Phaeosporobolus, both genera are consequently
considered as synonyms, and most Phaeosporobolus species are
here newly combined in the genus Lichenostigma. Because
Phaeosporobolus trypethelii clusters with rock isolates in a
strongly supported clade sister to the Phaeococcomyces+
Lichenostigma, it is consequently accommodated in the new
genus Etayoa (see Taxonomy section).
Our phylogenetic tree clearly shows that the genus
Lichenostigma as it is currently recognized is polyphyletic, with
several described species placed in the Lichenothelia clade as
defined by Muggia et al. (2012). The genus Lichenostigma is
divided in two subgenera: the subgenus Lichenostigma characterized by rounded, cushion-like ascomata and by the absence of
visible vegetative hyphae or strands on the surface of the host
lichens, and the subgenus Lichenogramma Nav.-Ros. &
Hafellner characterized by oval to elongate or irregularly shaped
ascomata interconnected by superficial black hyphae or hyphal strands (Calatayud et al. 2004, 2002; Navarro-Rosinés
and Hafellner 1996). In our analysis, all species of
Lichenostigma subgen. Lichenogramma (type L. elongatum)
are part of the Lichenotheliaceae s. str. in the Dothideomycetes. Surprisingly, Lichenostigma rugosum, supposed to
belong to the subgenus Lichenostigma (type L. maureri) clusters with the species of the subgenus Lichenogramma far away
from Lichenostigma maureri. L. rugosum does not form the
typical superficial pigmented hyphal strands as do species of
subgen. Lichenogramma. A careful study of the ascomatal
structure showed that stromatic cells in L. rugosum divide by
septa (a character confirmed in cultures of L. rugosum), whereas those of L. maureri divide by budding (no cultures of L.
maureri available). We conclude that the main diagnostic
Fungal Diversity (2014) 66:113–137
121
Fig. 3 a–b Lichenostigma
alpinum (Diederich 7878).
a Conidiomata on Pertusaria
amara. b Conidioma
with conidia in water.
c–d Diederimyces fuscideae
(Etayo 12083, isosyntype).
c Conidiomata on Fuscidea
cyathoides. d Conidioma
and conidia in water.
e, g–j Phaeosporobolus
minutus (isotype). e Conidiomata
on Coccotrema cucurbitula.
g–h Asci with hyaline
ascospores in water. i-j Conidia
in water. f Lichenostigma cf.
alpinum on Phaeographis,
stromatic cells in squash
preparation in water, showing
cells multiplying by budding
(arrow) (Ertz 17638). Scale bars:
a, c, e=200 μm, b, d, f=20 μm,
g–j=10 μm
character to distinguish between Lichenostigma s.str. and the
species of Lichenostigma belonging to the Lichenotheliaceae is
the way cells divide, a character never highlighted before.
Based on this character, we combine Lichenostigma rugosum
in Lichenothelia even though more data are probably needed to
decide if the Lichenothelia clade represents one or several
genera. Henssen (1987) reported cells that divide by budding
in several Lichenothelia species, but these were only occasionally observed for macroconidia or some absorption hyphae
when cells from other parts, including those from ascomatal
structure, divide by septa.
It must be noted that Lichenostigma subgenus Lichenogramma, originally comprising only L. elongatum, now includes most of the described Lichenostigma species. This species assemblage is quite heterogeneous because it includes
species having plurihyphal (stromatic) superficial strands (most
of the species, including L. elongatum) and others having single
hyphal strands (= formed by a single row of cells; e.g., L.
cosmopolites, L. epipolinum, L. semiimmersum). It also includes species having submuriform ascospores (e.g., L.
amplum, L. diploiciae).
Lichenicolous fungi make up over 13 % (215 species) of the
Arthoniomycetes and over 1 % (266 species) of the
Dothideomycetes, and they appear to have evolved independently in many different lineages in each class. In our phylogenetic
tree, lichenicolous species were newly added to both the
Arthoniomycetes (Lichenostigmatales) and to the
Dothideomycetes (the Lichenothelia clade). As shown in our
phylogenetic tree, lichenicolous fungi are also represented in
the Dothideomycetes by Phoma s. lat. species (in our tree
122
Fungal Diversity (2014) 66:113–137
Fig. 4 Lichenostigma
chlaroterae (a–c, e–g, i:
Diederich 17267; h, j–l:
Diederich 17270; d Neuberg; all
in water). a–b Conidiomata on
Lecanora cf. chlaroterae.
c Conidioma and single conidia
deposited on the thallus of
Lecanora cf. chlaroterae.
d–e Conidiomata with conidia.
f Young ascus. g Ascus with
mature ascospores.
h Development of conidiomata
from conidia. i Development of
conidia from initial
conidiogenous cells (arrow
heads) (note the upper right is
still fixed to a cell of the stroma:
arrow). j Mycelium developing
in a conidioma. k Mycelium
with young conidiomata.
l Mycelium originating from
two ascospores deposited on the
host thallus. Scale bars:
a=500 μm, b=100 μm,
c–d=20 μm, e–g, i–l=10 μm,
h=50 μm
represented by Phoma cladoniicola) as part of the Pleosporales
(Lawrey et al. 2012), by Xanthoriicola physciae in the
Teratosphaeriaceae (Capnodiales) (Ruibal et al. 2011) and by
the genus Lichenoconium (Lawrey et al. 2011) that will be
described in the family Lichenoconiaceae (in prep.). The
‘Lichenostigma’ species belonging to the Lichenothelia clade
are intermixed with Lichenothelia species in our phylogenetic
tree, suggesting that transitions between rock inhabiting fungi
and the lichenicolous habit possibly occurred several times in
the evolution of closely related species. Some species may
even have both life habits as suggested by Henssen (1987)
for Lichenothelia species (i.e., Lichenothelia convexa, L.
patagonica, L. tenuissima; one species, L. solitaria, is also
facultatively fungicolous) and by Knudsen and Kocourkova
(2010) for Lichenostigma saxicola.
In the Arthoniales, a group of mainly lichenized species, the
lichenicolous life habit has also evolved independently several
times (Ertz and Tehler 2011; Diederich et al. 2012b), a conclusion supported in our phylogenetic tree by Briancoppinsia
cytospora (Arthoniaceae) and Paralecanographa grumulosa
(Opegraphaceae). The latter species starts as a lichenicolous
fungus on different hosts belonging to the Arthoniales (e.g.,
Dirina, Roccella), sometimes killing the host lichen, and eventually develops its own thallus suggesting that the transition
between the lichenicolous and lichenized habit might be easy
(Egea et al. 1993; Ertz and Tehler 2011). Many other
Fungal Diversity (2014) 66:113–137
lichenicolous taxa are expected to belong to the
Dothideomycetes (e.g., Echinothecium, Sphaerellothecium) or
to the Arthoniales (e.g., many species of Arthonia s.l. and
Opegrapha s.l.), and these will need to be included in future
phylogenetic studies to better understand the evolution of the life
habits in these groups.
Taxonomy
Lichenostigmatales Ertz, Diederich & Lawrey ord. nov.
MycoBank MB 804670
Type: Lichenostigma Hafellner
Mycelium extremely rare, brown, superficial, smoothwalled. Ascomata and conidiomata absent or present, and
then almost indistinguishable, stromatic, lichenicolous, dark
brown to blackish, subspherical to elongate, internally I– or
I+ reddish or blue, K/I– or K/I+ blue; stromatic cells
subspherical, thick-walled, multiplying by budding, exposed
cells dark brown, verrucose, internal cells hyaline to pale
brown. Ascomata without hamathecial filaments; asci developing between stromatic cells, subspherical to shortly ellipsoid, wall apically thickened when young, often with an
ocular chamber, wall I– or I+ reddish and K/I– or K/I+ pale
blue, ascoplasm I and K/I+ orange, part of the tholus may be
I+ reddish, K/I+ blue; ascospores hyaline when young, outer
wall occasionally K/I+ blue, hyaline or dark brown when
old, 1-septate, ellipsoid or elongate, with roundish or pointed
apices. Conidiomata macroscopically indistinguishable from
ascomata; conidiophores absent; conidiogenous cells pale to
medium brown, developing from spherical stromatic cells,
shortly subcylindrical to almost ellipsoid, with an indistinctly truncate base, polyblastic, seceding with conidia; conidia
multicellular, composed of the conidiogenous cell and the
conidial cells, ellipsoid, brown, smooth, but sometimes with
a verrucose to echinulate ornamentation when over-mature,
dry, single, secession schizolytic; conidial cells subspherical
to ellipsoid, c. 3–5 μm diam. Yeast stage of dark brown,
aseptate, thick-walled cells covered by mucus and reproducing by multilateral budding.
Our phylogenetic analyses show that the new order belongs to the Arthoniomycetes and is sister to the Arthoniales.
The inclusion in the Arthoniomycetes is supported by the
ascus type and the amyloid reactions of hymenium and asci
(see ‘Discussion’). It differs from the Arthoniales by the
yeast-like cells multiplying by budding, either agglomerated
in dense stromata (ascomata devoid of hamathecial filaments
or conidiomata) or not (black yeasts), and the extreme rarity
of a mycelial stage.
The new order includes the single family Phaeococcomycetaceae. As Lichenostigma species are likely to be predominant in the order, we decided to typify the new order on the
genus Lichenostigma because the name Lichenostigmatales will
123
be more informative to most mycologists than a name typified on
the genus Phaeococcomyces.
Phaeococcomycetaceae McGinnis & Schell
MycoBank MB81139
In McGinnis, Schell & Carson, Sabouraudia 23: 183
(1985). Type: Phaeococcomyces de Hoog
The family Phaeococcomycetaceae initially included ‘fungi
that occur as solitary 1- or 2-celled cells that reproduce by
budding’ (McGinnis et al. 1985), with presence of melanin in
the fungal cell wall, and included the genera Phaeoannellomyces
McGinnis & Schell and Phaeococcomyces de Hoog. Following
our phylogenetic analyses, the family is here emended to include
the black yeast genus Phaeococcomyces, two genera producing
ascomata and conidiomata, Etayoa and Lichenostigma, and one
additional unnamed clade with undescribed rock-inhabiting fungi known only from cultures.
Phaeococcomyces de Hoog
MycoBank MB9294
Taxon 28: 348 (1979). Type species: Cryptococcus
nigricans M.A. Rich & A.M. Stern
Syn.: Phaeococcus de Hoog, Stud. Mycol. 15: 122 (1977);
non Phaeococcus Borzi, Atti Congr. Int. Genova 1892: 463
(1892) (Algae). Nigrococcus E.K. Novák & Zsolt, Acta bot.
Hung. 7: 101 (1961); non Nigrococcus Castell. & Chalm.,
Man. Trop. Med.: 932 (1919) (Bacteria).
The genus Phaeococcomyces was originally described for
the single species P. nigricans (de Hoog 1979). Later, more
species were added, but some of these, such as
Phaeococcomyces catenatus (de Hoog & Herm.-Nijh.) de
Hoog, were eventually shown to be phylogenetically not
related to the generic type, but instead belong to the
Chaetothyriales (Ruibal et al. 2008; Tsuneda et al. 2011).
Currently, Phaeococcomyces eucalypti is the only additional
species that is known to be congeneric with P. nigricans,
based on molecular data.
Phaeococcomyces nigricans (M.A. Rich & A.M. Stern)
de Hoog
MycoBank MB319531
Taxon 28: 348 (1979). Basionym: Cryptococcus
nigricans M.A. Rich & A.M. Stern, Mycopath. Mycol. appl.
9: 191 (1958). Nigrococcus nigricans (M.A. Rich & A.M.
Stern) E.K. Novák & Zsolt, Acta bot. Hung. 7: 142 (1961)
(nom. inval., Art. 33.4). Melanocryptococcus nigricans
(M.A. Rich & A.M. Stern) Della Torre & Cif., in Della Torre,
Atti lst. Bot. ‘Giovanni Briosi’ 21: 9 (1964). Phaeococcus
nigricans (M.A. Rich & A.M. Stern) de Hoog, Studies in
Mycology 15: 125 (1977). Type: Isolated from paint on
storage tank, United States; ex-type culture CBS 652.76–
neotype, derived from original isolate (McGinnis et al.
1985).
124
For a description, discussion and illustrations, see Rich
and Stern (1958).
Phaeococcomyces eucalypti Crous & R. G. Shivas
MycoBank MB801769
Persoonia 29: 159 (2012). Type: Australia, Queensland,
Anderson Park Botanic Garden, Townsville, 19°17′28.5″ S,
146°47′13.5″ E, on leaf litter of Eucalyptus sp., together with
ascomata of Thyriopsis sphaerospora, 5 Aug. 2009, P.W.
Crous (CBS H-21091– holotype), ex-type cultures CPC
17606=CBS 132526, ITS sequence GenBank KC005769,
LSU sequence GenBank KC005791.
For a description, discussion and illustrations, see Crous
et al. (2012).
Lichenostigma Hafellner
MycoBank MB2854
Herzogia 6: 301 (1982). Type: Lichenostigma maureri
Hafellner
Diederimyces Etayo, Nova Hedwigia 61: 190 (1995) (not
validly published, as type species not validly published,
ICN: Art. 38.5). Type: Diederimyces fuscideae Etayo
Phaeosporobolus D. Hawksw. & Hafellner, Nova
Hedwigia 43: 525 (1986). Type: Phaeosporobolus usneae
D. Hawksw. & Hafellner
Mycelium extremely rare, brown, superficial, smooth-walled.
Stromata dispersed over the host thallus, dark brown to blackish,
subspherical when young, later often becoming elongate, frequently slightly or distinctly centrally depressed, sometimes
resembling lirellae when old, 10–160 μm diam., internally I–
or I+ reddish or blue, K/I– or K/I+ pale blue; stromatic cells
subspherical, thick-walled, multiplying by budding, exposed
cells dark brown, often with a verrucose or mosaic-like ornamentation, internal cells hyaline to pale brown. Ascomata present
or absent, without hamathecial filaments; asci developing between stromatic cells, (4–)8-spored, subspherical to shortly ellipsoid, wall apically very thick, often with a distinct ocular chamber, wall I– or I+ reddish, K/I– or K/I+ pale blue, ascoplasm I and
K/I+ orange, part of the tholus may be I+ reddish, K/I+ blue;
ascospores hyaline when young, outer wall occasionally K/I+
blue, hyaline or dark brown when old, 1-septate, ellipsoid or
elongate, with roundish or attenuated and pointed apices.
Conidiomata frequent, often intermixed with ascomata from
which they are macroscopically indistinguishable, but sometimes
smaller; conidiophores absent; conidiogenous cells pale to medium brown, developing from spherical stromatic cells, shortly
subcylindrical to almost ellipsoid, with an indistinctly truncate
base, c. 4.5–6.5 μm long and 3.5–5.5 μm diam., polyblastic,
seceding with conidia; conidia multicellular, composed of the
conidiogenous cell and the conidial cells, ellipsoid, brown,
smooth, but sometimes with a verrucose to echinulate ornamentation when over-mature, dry, single, secession schizolytic; conidial cells subspherical to ellipsoid, c. 3–5 μm diam.
Fungal Diversity (2014) 66:113–137
Notes:
1. Ascomata and conidiomata are entirely composed of globose cells that reproduce by budding (e.g., Fig. 3f). The only
septa that can be observed within an ascoma are those of the
2-celled ascospores. Thus, this represents an interesting case
in which ascomata and conidiomata are entirely made of a
compact agglomeration of yeast-like cells.
2. Mature conidia deposited on a natural substratum do not
germinate. Instead, individual cells grow in diameter, more
cells are added by budding, and the outer wall of the
external cells rapidly obtains a typical dark brown, verrucose ornamentation (Figs. 3j, 4h and 5f). They either develop into an ascoma or a morphologically almost identical
conidioma. When young, developing conidiomata reach a
size somewhat superior to the size of the conidia, e.g.,
20 μm diam., then start producing conidia in the interior
of the stroma. From some of the globose cells inside the
conidioma, specialized, subcylindrical conidiogenous cells
develop (these and the asci and ascospores are the only
cells within stromata that are not globose), and a more or
less larger number of subspherical conidial cells are produced around this conidiogenous cell. When mature, the
entire complex—i.e., the original conidiogenous cell together with the surrounding conidial cells—are released
together and act as a single multi-celled conidium (Figs. 2e–f, 3b, 4d and 5a–b). In conidia with a large number of
cells, the conidiogenous cell is often difficult to see.
3. Mature ascospores deposited on a natural substratum usually do not germinate either, but also produce new cells by
budding, developing into multicellular young stromata. On
rare occasions, ascospores may germinate, producing a
brown mycelium from which new stromata are produced.
This filamentous stage is extremely rare in Lichenostigma
and has been observed by us in one specimen of
Lichenostigma chlaroterae from Canada (Diederich
17270) (Fig. 4j–l). Sterile mycelia of other fungi often
overgrow the surface of host thalli and these should not
be confused with a mycelium of Lichenostigma.
4. Conidia are typically smooth-walled. However, in some
specimens, conidia with an echinulate ornamentation
have been observed (Fig. 3j). Brackel (2011) observed
such material on Fuscidea stiriaca and considered it as an
undescribed species that was, however, left unnamed. We
have observed similar specimens with echinulate conidia
in Lichenostigma alpinum (e.g., Diederich 7878, on
Pertusaria amara), in L. cf. alpinum (Diederich 7988b,
on Fuscidea lightfootii) and in L. chlaroterae (Diederich
15608, on Lecanora symmicta). Careful examination of
this material has shown that conidia are smooth until
maturity, but when overmature, individual cells may become larger and develop an outer layer with such an
echinulate ornamentation. Therefore, this character has
probably no diagnostic value.
Fungal Diversity (2014) 66:113–137
125
Fig. 5 a–b Lichenostigma
fellhanerae (holotype; in water).
a Conidioma with conidia.
b Conidia. c–i Lichenostigma
hyalosporum (holotype).
c Ascomata on thallus of
Haematomma eremaeum.
d Conidioma containing several
conidia in water. e–f Conidia
(the lower right is developing in
a young conidioma). g–i Asci,
in g and i with ascospores
(e–i: in diluted K/I). Scale bars:
a=20 μm, b=10 μm,
c=200 μm, d–i=10 μm
5. Young, developing ascomata and conidiomata are typically roundish, with an irregular opening to release ascospores and conidia. Older fructifications in some species
tend to become elongate, with a depressed centre and they
consequently often resemble lirellae. Their size appears to
depend largely on the age of the population and does not
represent a character that allows distinguishing species.
Lichenostigma maureri Hafellner (Fig. 2)
MycoBank MB109054
Herzogia 6: 301 (1982). Type: Austria, Steiermark, Grazer Bergland, auf Fichten an der Nordseite des Schöckels,
1400 m, auf Usnea florida, 24 Nov. 1974, W. Maurer (GZU–
holotype; UPS, hb. Hafellner 8701–isotypes, non vid.).
Phaeosporobolus usneae D. Hawksw. & Hafellner, Nova
Hedwigia 43: 526 (1986). Type: Austria, Ostalpen, Gurktaler
Alpen, Steiermark, Frauenalm S von Murau, subalpiner
Fichten-Lärchenwald ober der Murauer Hütte, 1700 m, auf
Usnea, 24 May 1981, J. Hafellner 9106 (IMI 281394–holotype; GZU–isotype, non vid.).
Mycelium unknown. Stromata (30–)50–100(−120) μm diam.;
external cells with a verrucose ornamentation. Ascomata frequent; asci (4–)8-spored, 20–25×13–18 μm; ascospores ellipsoid, 1-septate, when young hyaline and smooth, soon becoming
dark brown and verruculose, 9–12×4.5–6 μm. Conidiomata
frequent, often intermixed with ascomata from which they are
macroscopically indistinguishable; conidia subspherical to ellipsoid, (9–)14.3–22.5(−24)×(8–)10.5–16.0(−17) μm, composed
of (8–)14–36(−45) cells [in optical section (6–)10–21(−23) cells],
cells (3–)3.6–4.6(−6) μm diam.
For a detailed description and illustrations, see Hafellner
(1982) and Hawksworth and Hafellner (1986).
This species is particularly common in montane coniferous forests, growing mainly on fruticose lichens (Bryoria,
Evernia, Letharia, Pseudevernia, Ramalina, Usnea, etc.),
and is known from all continents (Kocourková 2000).
126
Notes:
1. Hawksworth and Hafellner (1986) described the
conidiomata of Ph. usneae as being “surrounded by a
pellicle-like layer of brown hyphae 2.5–3 μm wide forming
a layer 4–7 μm thick”. Alstrup and Hawksworth (1990)
described the new Ph. alpinus as differing notably by “the
absence of a pellicle-like outer hyphal layer over the stroma”. In the many specimens examined by us, we never
observed such a pellicle-like layer, and we only observed
spherical, never hyphal cells forming the stromata. Berger
and Brackel (2011) similarly explained that in ‘some’ specimens of Ph. usneae examined, the outer hyphal layer was
missing, and they wondered if that layer might appear only
at a later stage of development, resulting from collapsing of
the previously spherical outer cells.
2. Following Hawksworth and Hafellner (1986), conidia in
Ph. usneae are composed of 6–12 or more globose cells
of 4–6 μm diam. Alstrup and Hawksworth (1990) distinguished the new Ph. alpinus partly on the basis of
conidia composed of 10–15(−20) much smaller cells, 3–
4 μm diam. Berger and Brackel (2011) confirmed this
information for Ph. alpinus, but slightly modified the
data on Ph. usneae in that conidia are composed of 6–
12(−16) cells of (3–)4–6 μm. Our study of many specimens has shown that conidia in Ph. usneae are typically
larger than those of Ph. alpinus, with a larger number of
cells (and not the contrary as stated by previous authors),
but that the diameter of individual cells is statistically
more or less identical in both species.
3. In rich populations, both morphs often seem to occur
together. However, this has rarely been documented,
probably as most collectors just studied one or two
stromata, and depending on the morph observed called
the specimen either L. maureri or Ph. usneae. When
many stromata from the same host thallus are examined,
a mixture of both morphs is often observed.
4. Many lowland specimens examined by us (e.g., all specimens from Belgium and Luxembourg) only presented
the asexual stage. More studies are needed to identify
whether these represent a distinct species that is rarely or
never accompanied by a sexual stage, or if the sexual
stage of L. maureri has a more restricted area of distribution, being mainly present in montane or boreal environments, while in more temperate environments, only
the asexual stage develops.
5. Iodine reactions present in ascomata and conidiomata
are difficult to observe because of the dark pigmentation.
It was found that bleaching (with C) until the stromata
became colourless made the reactions much easier to
observe. Both reddish and blue reactions were seen in
many stromata. Sequential pre-treatment with C and K,
however, proved to be too harsh, often resulting in
removal of the reactive material. After pre-treatment
Fungal Diversity (2014) 66:113–137
with K only, blue colouration is increased and reddish
staining was not seen. It is the gelatinous material between cells which reacts, most strongly on the outer
surface of the cells. In 1 % IKI, cytoplasmic staining is
often strong, making the observation of reddish or pale
blue reactions difficult. The reactions were best seen after
C, in 0.15 % IKI. Reddish cell wall staining visible in
0.15 % IKI is lost when transferred to Melzer’s reagent.
This type of reddish reaction with iodine was termed
hemiamyloid by Baral (1987), and the blue staining seen
without K pre-treatment termed euamyloid. These observations are also compatible with the amylomycan type
reaction described by Common (1991). The reddish
colouration of the ascoplasm, and to a lesser extent other
cells of the stromata, is not converted to a blue staining
form by pre-treatment with K, so would be called
dextrinoid in the terminology of Baral.
Selected specimens examined: France: Pyrénées-Orientales:
NW of Mont-Louis, on Pinus, on Usnea hirta, 30 July 1985,
Diederich 6642 (hb. Diederich); SE of Eyne, on Pinus, on
Usnea, 31 July 1985, Diederich 6633 (hb. Diederich). Switzerland: Valais: S of Nendaz, 2 km SSW of Siviez, 150 m W of
téléphérique station Tortin, 46°06′49″ N, 7°18′03″ E, 2145 m, on
Larix, on Letharia vulpina, 26 July 2012, Diederich 17306 (hb.
Diederich); 3 km S of Haute-Nendaz, road to Siviez, between
main road and Planchouet, 46°09′07″ N, 7°18′59″ E, 1500 m, on
Larix, on Usnea, 21 July 2012, Diederich 17326 (hb. Diederich);
Aletschwald, NNW of Riederalp, 46°23′03″ N, 8°01′13″ E,
2050 m, on Larix, on Usnea, Diederich 17337 (hb. Diederich);
SE of Les Haudères, forêt de Tauge, 46°04′42″ N, 7°31′09″ E,
1500 m, on Larix, on Usnea, 23 July 2012, Diederich 17334 (hb.
Diederich); Saint-Luc, sentier des Grands Pras, 1650 m, on
Larix, on Usnea, 5 Feb. 2013, Van Cappellen s.n. (BR).
U.S.A.: California, Siskiyou Co., 6 mi. west of Gazelle,
4000 ft., 41°31′ N, 122°31′ W, 8 Oct. 2010, Miller s.n., on
Letharia vulpina (MSC).
Lichenostigma alpinum (R. Sant., Alstrup & D. Hawksw.)
Ertz & Diederich comb. nov. (Figs. 3 and 7c–d)
MycoBank MB 804671
Basionym: Phaeosporobolus alpinus R. Sant., Alstrup &
D. Hawksw., in Alstrup & Hawksworth, Meddelelser om
Grønland, Bioscience 31: 51 (1990). Type: Greenland, Disko, Qutdligssat, 1 km SW of churchyard, 70°05′N, 53°01′W,
100 m, on Ochrolechia frigida, 27 July 1950, Gelting 13333
(UPS–holotype, non vid.) [in Alstrup and Hawksworth
(1990, caption of fig. 28), another specimen, Christiansen
5507, was erroneously said to be the holotype].
Syn. (?): Diederimyces fuscideae Etayo, Nova Hedwigia
61: 190 (1995). Type: Spain, Alava, Sierra de Entzia, Opacua
pass, Fagus-wood north-oriented, on Fuscidea cyathoides,
995 m, 6 April 1994, Etayo 12083!, 12059 (MA-Lich–
Fungal Diversity (2014) 66:113–137
syntype, hb. Etayo!–isosyntype) (not validly published, as
two different specimens were designated as the holotype,
ICN: Art. 40.2).
Syn. (?): Diederimyces microsporus Etayo, in Etayo &
Sancho, Bibl. Lichenol. 98: 84 (2008). Type: Chile, Navarino,
bosquete de lengas achaparrado antes del Cerro Bandera, sobre
Pertusaria microcarpa corticícolas, 54°57′34′′S, 67°37′46′′W,
550 m, 15 Jan. 2005, J. Etayo 22265, A. Gómez-Bolea & U.
Søchting (MAF–holotype; UMAG, hb. Etayo!–isotypes) (not
validly published, as generic name not validly published).
Syn. (?): Phaeosporobolus minutus Etayo, in Etayo &
Sancho, Bibl. Lichenol. 98: 164 (2008). Type: Chile, Navarino,
bosquete de lengas achaparrado antes del Cerro Bandera, sobre
Pertusaria microcarpa y Coccotrema cucurbitula corticícolas,
54°57′34″S, 67°37′46″W, 550 m, 15 Jan. 2005, J. Etayo 22265,
A. Gómez-Bolea & U. Søchting (MAF–holotype; UMAG, hb.
Etayo!–isotypes).
Mycelium unknown. External cells of stromata medium to
dark reddish brown, with a verrucose to sometimes indistinctly
mosaic-like ornamentation. Sexual stage not certainly identified, as two kinds of ascomata have been found associated with
a L. alpinum-like asexual stage (see below). Conidiomata frequent, c. (20–)25–100(−150) μm diam.; conidia subspherical to
ellipsoid, (10–)11.2–13.6(−15)×(7–)8.5–10.6(−12) μm, composed of (4–)8–17(−22) smooth, rarely verrucose cells [in
optical section (4–)6–10(−12) cells], cells (2.9–)3.5–4.9(−5.8)
μm diam.
‘Diederimyces fuscideae’ sexual stage: Ascomata rare,
intermixed with conidiomata, 150–300 μm diam.; asci 8spored, 31–39×13–17 μm; ascospores hyaline, 1-septate, with
attenuated and pointed ends, bent spirally, 20–27×3–4 μm (description from Etayo 1995).
‘Diederimyces microsporus’ sexual stage: Ascomata extremely rare, intermixed with conidiomata, 70–100 μm
diam.; asci 8-spored, 14–17×9–12 μm; ascospores hyaline,
1-septate, ellipsoid, with rounded ends, 6.5–7×2–2.5 μm
(description from Etayo and Sancho 2008).
For a detailed description and illustrations, see Alstrup
and Hawksworth (1990), Etayo (1995) and Etayo and
Sancho (2008).
This species is rather common on terricolous and corticolous
species of Ochrolechia, Pertusaria and Varicellaria, and has also
been reported on a variety of other hosts, including Fuscidea and
Coccotrema. Following Kocourková (2000), the species is
known from Europe, Asia, North and South America and Antarctica, and additional hosts include Caloplaca, Lecanora
(possible confusion with L. chlaroterae), Physcia and
Sphaerophorus. Berger and Brackel (2011) added Anaptychia,
Baeomyces, Buellia, Cladonia, Flavocetraria, Hypogymnia,
Maronea, Megaspora, Melanohalea, Parmelia, Physconia,
Platismatia, Rhizoplaca, Stereocaulon, Umbilicaria. However,
as the taxonomic value of morphological characters allowing the
distinction of Phaeosporobolus species was often
127
misunderstood, most of these records should be revised before
being accepted. Furthermore, the existence of further,
undescribed species within the L. alpinum complex is likely.
We have additionally studied material on Graphis pulverulenta,
Ropalospora viridis and Fuscidea lightfootii that is morphologically indistinguishable from L. alpinum.
Notes:
1. L. alpinum is widespread and rather common on species
of Ochrolechia, Pertusaria and Varicellaria. Owing to
difficulties in separating the species from the asexual
stage of L. maureri (i.e., Ph. usneae) by morphological
characters, many authors based the identification of their
specimens mainly on a hypothetic host-specificity.
2. The distinction between this species and Ph. usneae (i.e.,
Lichenostigma maureri) has always been problematic. In the
original account of Ph. alpinus (Alstrup and Hawksworth
1990), both species were considered to differ by several
characters, but the discussion was rather confusing as the
authors mixed characters of both species when characterizing Ph. alpinus (first sentence after ‘Notes’). Following these
authors, Ph. usneae is distinguished by conidiomata covered
by a pellicle-like layer (a character not confirmed by us, see
notes under L. maureri), larger conidia (15–25 μm diam.),
ellipsoid rather than subglobose conidia (not confirmed by
us: length/breadth ratio 1.2–1.4 in Ph. alpinus and 1.2–1.6 in
Ph. usneae), conidia adhering in chains (not observed by us),
and much larger individual cells of conidia (4–6 μm, versus
3–4 μm in Ph. alpinus; a difference not confirmed by us, see
notes under L. maureri). Furthermore cells in the central
parts of the stroma would be more pigmented in one of the
two species, but it is not possible to understand which
species is meant (this character has not been confirmed by
us). Following our observations, the main characters that
allow distinguishing both species in the asexual stage are the
conidial dimensions and the number of cells per conidium.
3. Morphologically similar specimens have been reported
from many other host genera (see above). Specimens from
Fuscidea lightfootii and Phaeographis smithii included in
our phylogenetic analyses appear not to be conspecific with
material on Pertusaria or Fuscidea cyathoides, suggesting
that several cryptic species might be hidden in the L.
alpinum species complex. The inclusion of more specimens and the use of a more variable locus such as ITS
would be needed to solve species circumscription here.
4. The conidiomatal diameter is very variable within this
species and probably reflects the maturity of a population. For example, in specimen Ertz 17522, conidiomata
are (20–)26–40(−50) μm diam. (N=38), whilst in specimen Kalb 15574, they are (50–)59–103(−150) μm
diam. (N=39) [in the case of elongate conidiomata, the
largest diameter has been measured]. Both specimens are
from the same host, Pertusaria albescens.
128
5. Etayo (1995) considered Diederimyces fuscideae (on
Fuscidea cyathoides), characterized by narrow, elongate,
hyaline, apically attenuated ascospores with pointed ends,
spirally bent in the asci, to be the sexual stage of Ph. alpinus
(described from Ochrolechia and Pertusaria species) (Fig. 3c–d). As both have been described from different host
genera, they might represent two distinct, morphologically
similar species. However, two arguments speak in favour of
a synonymy: (1) a specimen on F. cyathoides included in
our phylogenetic analysis groups with specimens on
Pertusaria (Fig. 1); (2) Alstrup and Hawksworth (1990)
have observed and illustrated in their fig. 29 a fertile specimen on Ochrolechia frigida with hyaline ascospores
strongly resembling those of the type of D. fuscideae,
depicted by Etayo (1995). We have studied an isosyntype
(Etayo 12083), but were unable to find any ascomata.
However, we examined several conidiomata with conidia
of variable size, mostly intermediate between those of L.
alpinum and L. chlaroterae, probably representing young,
developing conidia of L. alpinum.
6. Etayo and Sancho (2008) described a second species of
Diederimyces, D. microsporus, from southern South America on corticolous Pertusaria microcarpa (Fig. 3e, g–j). That
species has narrowly ellipsoid, hyaline ascospores, and
ascomata are intermixed with an asexual Phaeosporobolus
stage that the authors described as the new Ph. minutus. This
asexual stage was characterized by particularly small
conidia, 6.5–10.5×5–8 μm, composed of (3–)4–5(−8)
smooth-walled cells, formed in small conidiomata 20–
60 μm diam., and grew on Pertusaria and Coccotrema
species. We have examined many conidiomata in an isotype
and were surprised to find much larger conidia, 15–19×11–
15 μm, composed of 13–18 cells (in optical section of 8–10
cells), and individual cells were relatively large, mainly 4.5–
6 μm diam. All morphological characters were those of Ph.
alpinus, of which Ph. minutus is possibly a synonym.
7. As some populations of Ph. alpinus s. lat. on Fuscidea
cyathoides are intermixed with ascomata of Diederimyces
fuscideae, and others on Pertusaria microcarpa [i.e., Ph.
minutus] are intermixed with ascomata of D. microsporus,
the main question is to know which of these two
Diederimyces species represents the sexual stage of Ph.
alpinus. To answer this question, more populations of Ph.
alpinus need to be sequenced, including those on unusual
hosts, and material of both Diederimyces species needs to
be sequenced. If possible, material collected close to the
type localities of Ph. alpinus and both Diederimyces species should be included in a phylogenetic analysis. For the
time being, we consider that Ph. alpinus probably represents an assemblage of several species that cannot be
distinguished by morphological characters in the asexual
stage, and that it is not possible to identify with certainty the
sexual stage of Ph. alpinus s. str. Therefore, we
Fungal Diversity (2014) 66:113–137
provisionally include both Diederimyces names and Ph.
minutus under the synonymy of Lichenostigma alpinum,
pending further studies.
8. The new species Diederimyces fuscideae was not validly
published, as two different specimens (Etayo 12083 and
Etayo 12059) were designated as the holotype (ICN: Art.
40.2). Consequently the new generic name Diederimyces,
typified on D. fuscideae is not valid (ICN: Art. 38.5) and
the new D. microsporus is not validly published as well.
9. The ICN, Art. 57.2, says that widely used teleomorphtypified names cannot be displaced by earlier anamorphtypified names. However, as the names Diederimyces
fuscideae and D. microsporus have not been widely used
– both species have never been reported after the original
description, as the anamorph-typified name Ph. alpinus
is widely used, as further the names D. fuscideae and D.
microsporus are not validly published (see above), and
as the anamorph-teleomorph relationship has not yet
been solved in this species complex, the anamorphtypified name Ph. alpinus has to be used for this
taxon.
Selected specimens examined: Croatia: Plitvice, Plitvice
Lakes National Park, west-shore of Lake Kozjak, 44°52′48″
N, 15°36′48″ E, on Fraxinus, on Pertusaria albescens, 16
Aug. 2012, Diederich 17379 (hb. Diederich). France:
Finistère: SE de Commana, Monts d’Arrée, 48°24′05″ N,
3°55′35″ W, on saxicolous Fuscidea cyathoides, 18 July
2012, Ertz 17519 (BR); centre du village de Huelgoat,
48°21′49″ N, 3°44′56″ W, on Liriodendron, on Pertusaria
albescens, 18 July 2012, Ertz 17522 (BR). Luxembourg: S of
Doncols, ruisseau de Sonlez, 49°57′ N, 5°50′ E, on Carpinus,
on Pertusaria amara, 22 March 1987, Diederich 7878 (hb.
Diederich). U.S.A.: Michigan: Chippewa Co., Tahquamenon
Falls State Park, on M-123, 0.2 miles from boundary with
Luce Co., swampy area near road, on corticolous Pertusaria,
22 July 1980, Common 4443 L (MSC).
Selected specimens of Lichenostigma alpinum s. lat.: France:
Ille-et-Vilaine: Paimpont, près de Tréhorenteuc,Val Sans Retour,
48°00′01″ N, 2°17′04″ W, on Carpinus, on Phaeographis
smithii, 23 July 2012, Ertz 17591 (BR); Paimpont, au nord de
l’étang de Paimpont, 48°01′26″ N, 2°10′22″ W, on Ilex, on
Phaeographis, 22 July 2012, Ertz 17638 (BR). Luxembourg:
Lellingen, verson droit du Lellgerbaach, op Baerel, 49°59′22″ N,
6°01′48″ E, on Sorbus, on Fuscidea lightfootii, 3 Sept. 2011,
Diederich 17240 (hb. Diederich).
Lichenostigma chlaroterae (F. Berger & Brackel) Ertz &
Diederich comb. nov. (Figs. 4 and 7a–b)
MycoBank MB 804672.
Basionym: Phaeosporobolus chlaroterae F.Berger &
Brackel, Herzogia 24: 351 (2011). Type: Austria, Oberösterreich,
Donautal, Engelhartszell, Oberranna, MTB 7548/2, 290 m, on
Fungal Diversity (2014) 66:113–137
Malus domestica, on Lecanora chlarotera, 9 Jan. 1999, Berger
12950 (LI–holotype; hb. Berger–isotype, non vid.).
Mycelium extremely rare, brown, superficial, smoothwalled, originating from the germination of ascospores. Stromata variable in diameter, in young populations frequently
less than 20 μm diam., in older populations up to 80 μm diam.
or more; external cells with a verrucose or granulose ornamentation. Ascomata extremely rare, mixed with conidiomata
from which they are macroscopically indistinguishable; asci c.
28×22 μm (1 ascus measured); ascospores ellipsoid, 1septate, brown, c. 9–9.5×4–4.5 μm (2 spores measured inside
ascus). Conidiomata frequent; conidia subspherical to ellipsoid, (5.5–)7.1–10.2(−13)×(5–)6.3–8.8(−11) μm, composed
of (3–)4–9(−16) cells that are smooth, rarely with an
echinulate ornamentation when overmature [in optical section
(3–)4–6(−8) cells] (N=57), cells (2.5–)3.3–4.5(−6) μm diam.
(N=207).
For a detailed description and illustrations, see Berger and
Brackel (2011).
This is a rather common species, widespread in Europe,
here newly reported from North America (Canada, U.S.A.)
and probably cosmopolitan, but poorly recorded as only recently described. It mainly develops over corticolous
Lecanora species, but has also been reported from other hosts,
such as Buellia griseovirens (Diederich et al. 2012a). We have
also examined specimens on Fuscidea lightfootii and Graphis
pulverulenta with similar morphological characters.
Notes:
1. Following the literature, the asexual stage of this species
can hardly be distinguished from Ph. minutus. Berger and
Brackel (2011, Table 1) gave a number of characters that
should allowing distinguishing both species. Following
these authors, conidia of L. chlaroterae are composed of
5–7(−9) cells of 3–3.5(−5) μm diam., whilst those of Ph.
minutus of (3–)4–5(−8) cells of 3–5 μm. Conidiomata
were said to be 60–80(−110) μm in L. chlaroterae, much
larger than those of Ph. minutus (20–60 μm), but this
represents a lapsus in Table 1 published by these authors,
as conidiomata of L. chlaroterae were said to be 20–
80(−110) in the species description on p. 351, a result
confirmed by us. A re-examination of an isotype of Ph.
minutus revealed much larger conidia with more cells,
similar to those of L. alpinum, and therefore no confusion
between L. chlaroterae and Ph. minutus should occur.
2. In a Canadian specimen on corticolous Lecanora cf.
chlarotera (Diederich 17267) we observed slightly larger conidia, (8.5–)8.8–10.6(−11.5)×(6.5–)6.9–8.4(−9)
μm, composed of (7–)7–14(−16) cells [in optical section
(5–)5–7(−8) cells] (N=7), cells (3–)3.3–4.0(−4.5) μm
diam. (N=34), morphologically intermediate between
L. chlaroterae and L. alpinum. The study of more
conidiomata in the same specimen revealed some with
129
exclusively very small conidia with few cells. Molecular
data clearly placed this population with other specimens
of L. chlaroterae on Lecanora. This suggests that the
infraspecific variability in L. chlaroterae is rather large,
and overlap of morphological characters with other species, especially L. alpinum, is not rare, making the identification of some specimens particularly difficult.
3. This Canadian collection is the only hitherto known
specimen of L. chlaroterae in which a sexual stage could
be found. This might be explained by a particularly
favourable climate in British Colombia, resulting not
only in the production of ascomata, but also in conidia
becoming larger than in Central European material. Unfortunately, amongst the numerous stromata examined,
only two represented the sexual stage, and all but one
asci were immature, without ascospores (Fig. 4f–g).
Additional material collected on the same trees 3 years
later, and also specimens collected a few kilometres from
this locality exclusively presented the asexual stage.
4. The frequency of L. chlaroterae on corticolous
Lecanora species, the entire absence of the species on
other lichens growing on the same trees, and the unexpected occurrence on Buellia griseovirens (three specimens published by Diederich et al. 2012a) and also on
specimens of Fuscidea lightfootii and Graphis
pulverulenta suggests that either several cryptic species
are involved or that the species has very narrow ecological affinities that are found in several non-related lichen
species or genera. Brackel (2011) also reported a specimen with small conidiomata and small, few-celled
conidia from Diplotomma alboatrum (as Ph. aff.
minutus). To answer this question, more specimens on
unusual hosts will need to be sequenced, if possible
using other genetical markers, such as ITS.
Selected specimens examined: France: Pas-de-Calais:
Au S de Boulogne-sur-Mer, forêt domaniale d’Hardelot, on
Fuscidea lightfootii, 12 Aug. 2000, Diederich 14369b (hb.
Diederich). Pyrénées-Atlantiques: Au S de Pau, à l’E de
Bielle, à 0.5 km à l’E du col de Marie-Blanque, on Graphis
pulverulenta, 28 July 1990, Diederich 9301 (hb. Diederich).
Luxembourg: Weiswampach, near lake, on Alnus, on
Lecanora argentata, 5 Febr. 2012, Neuberg (hb. Diederich).
Blaschette, Bëddelbësch, on Carpinus, on Buellia
griseovirens, 30 Oct. 1983, Diederich 3943 (hb. Diederich).
Switzerland: Valais: SE of Les Haudères, forêt de Tauge,
46°04′42″ N, 7°31′09″ E, 1500 m, on Sorbus, on L.
chlarotera, 23 July 2012, Diederich 17329 (hb. Diederich).
Canada: British Columbia: Wells Gray Provincial Park,
Clearwater valley, 26 km N of Clearwater, Edgewood Blue
(near house of T. Goward), 51°52′9″ N, 120°01′18″ W, on
Alnus, on L. chlarotera s. lat., 24 July 2008, Diederich 17267
(hb. Diederich); ibid., 14 April 2012, Goward 12–30 (UBC),
130
ibid., 9 May 2012, Goward 12–42 (UBC); ibid., Kingfishers
Wood Cottage, 51°51′23″ N, 120°00′52″ W, on Alnus, on
Lecanora, 22 July 2008, Diederich 17270 (hb. Diederich).
U.S.A.: Michigan: Chippewa Co., Tahquamenon Falls State
Park, on M-123, 0.2 miles from boundary with Luce Co.,
swampy area near road, on corticolous Lecanora, 22 July
1980, Common 4443E (MSC). South Carolina: locality
unknown, on corticolous Lecanora, 28 Dec. 1975, Common
3658 N (MSC).
Lichenostigma fellhanerae (R.C. Harris & Lendemer)
Ertz & Diederich comb. nov. (Fig. 5a–b)
MycoBank MB 804673
Basionym: Phaeosporobolus fellhanerae R.C. Harris &
Lendemer, Opuscula Philolichenum 6: 173 (2009). Type:
U.S.A. North Carolina, Haywood Co., Great Smoky Mountains National Park, 3 miles southeast of Waterville, along
Baxter Creek Trail, south of Big Creek Campsite, Mount
Sterling Ridge, lower slopes of Mount Sterling, Cove Creek
Quad., rich cove forest (Aesculus hippocastanum, Acer
saccharum, Liriodendron tulipifera, Betula lenta, Halesia
carolina) on north-facing slope with narrow rocky ravine
of Baxter creek, on Fellhanera granulosa on rock, 28 Oct.
2006, J.C. Lendemer 8087 & E. Tripp (NY–holotype!).
Mycelium unknown. External cells of stromata medium to
dark reddish brown, with a verrucose to sometimes indistinctly mosaic-like ornamentation. Sexual stage unknown.
Conidiomata (20–)40–80 μm diam., I–, K/I+ pale blue;
conidia produced in large numbers (typically more than
100 per conidioma), ellipsoid, very regular in form,
(8–)8.7–11.0(−12.5)×(6.5-)6.8–7.7(−8) μm (N=18), composed of (16–)18–23(−27) smooth cells [in optical section
9–12 cells], cells (2.5–)2.6–3.2(−3.5) μm diam. (N=18).
For a description and illustrations, see Harris and
Lendemer (2009).
The species is hitherto known only from the type specimen from the U.S.A. (North Carolina) on the thallus of
saxicolous Fellhanera granulosa.
Note: This species is unusual by producing large numbers of
very regularly formed, ellipsoid conidia. These conidia are rather
compact, with relatively small cells, contrasting with the more
loose conidia with larger cells in other Lichenostigma species,
and we wondered if the species might belong to the new genus
Etayoa described below. As no molecular data of this species are
available (the species is just known from the type collection), a
final answer to this question cannot be given now.
Lichenostigma hyalosporum Kalb & Hafellner (Fig. 5c–i)
MycoBank MB 413399
In Kalb et al., Bibl. Lichenol. 59: 208 (1995) (as L.
hyalospora). Type: Australia, Western Australia, ‘Dryandra
Forest’, wenige km NW von Narrogin, 32°45′ S, 116°56′ E,
300 m, on Haematomma eremaeum, 17 Aug. 1994, K. & A.
Fungal Diversity (2014) 66:113–137
Kalb 27456 (hb. Kalb–holotype!; GZU [hb. Hafellner]–
isotype).
Mycelium unknown. External cells of stromata with a dark
reddish brown, mosaic-like ornamentation. Ascomata roundish
to sometimes elongate, 60–160 μm diam., I– and K/I–. Asci 8spored, 15–25×13–18 μm, when ascospores fully mature, 25–
30×18–22 μm; ascospores hyaline, 1-septate, ellipsoid, 9–
15×3.5–8 μm. Conidiomata rare, intermixed with ascomata
and often smaller, 20–40 μm diam.; conidia subspherical, c.
10–15×8–13 μm, composed of c. 13 smooth cells [in optical
section 8–9 cells] (two conidia observed), cells 4–6 μm diam.
For a detailed description and illustrations, see Kalb et al.
(1995).
The species was known from three Western Australian specimens on Haematomma eremaeum. We have studied two further
specimens, one from the type locality, overgrowing other species
of corticolous crustose lichens, and one from the same area on
corticolous species of Protoparmelia and Hafellia. All localities
are from regions with a temperate climate.
Notes:
1. This was the only known Lichenostigma s. lat. species with
hyaline ascospores. Following the detailed description and
the high quality photographs given in the original account
(Kalb et al. 1995), and after re-examination of the holotype,
we can affirm that this species belongs to Lichenostigma s.
str., although no sequences are available. Stromata are
entirely composed of globose cells multiplying by budding,
and typical Phaeosporobolus-type conidia, previously unknown in this species, have been observed.
2. Ascospores were given in the original description as 12–
15×6.5–8 μm. We have examined one ascus with 8 hyaline
ascospores measuring just 9–11×3.5–4.2 μm. The variability is thus larger than initially believed. A more accurate
statistical analysis of ascospores needs additional material.
Additional specimens examined: Australia: Western Australia: Same locality and date as type, on several species of
corticolous crustose lichens, Kalb 27616 (hb. Kalb). Ca. 30
km W von Hyden und ca. 30 km E von Kondinin, 32°26′ S,
118°30′ E, 300 m, on corticolous Protoparmelia and Hafellia
spp., 18 Aug. 1994, Kalb 27617 (hb. Kalb).
Etayoa Diederich & Ertz gen. nov.
MycoBank MB 804674
Type: Phaeosporobolus trypethelii Flakus & Kukwa
Mycelium unknown. Stromata dispersed over the host
thallus, dark brown to blackish, subspherical when young,
later often becoming elongate, frequently slightly or distinctly
centrally depressed, sometimes resembling lirellae when old;
stromatic cells subspherical, thick-walled, multiplying by
budding, exposed cells dark brown, with a mosaic-like ornamentation, internal cells hyaline to pale or medium brown.
Ascomata present or absent, without hamathecial filaments,
Fungal Diversity (2014) 66:113–137
internally I+ and K/I+ blue; asci developing between
stromatic cells, 8-spored, subspherical to shortly ellipsoid,
wall apically very thick, often with a distinct ocular chamber,
wall I– and K/I–, ascoplasm I and K/I+ orange; ascospores
hyaline, 1-septate, ellipsoid, with roundish apices.
Conidiomata frequent, not intermixed with ascomata from
which they are macroscopically indistinguishable, but sometimes smaller, I– or I+ pale blue, K/I– or K/I+ pale blue;
conidiophores absent; conidiogenous cells pale to medium
brown, developing from spherical stromatic cells, shortly
subcylindrical to almost ellipsoid, with an indistinctly truncate
base, polyblastic, seceding with conidia; conidia multicellular,
composed of the conidiogenous cell and the conidial cells,
subspherical, brown, verrucose, dry, single, secession
schizolytic; conidial cells subspherical to ellipsoid.
Notes:
1. Molecular data suggest that Ph. trypethelii does not belong
to Lichenostigma s. str., but to a distinct, new genus. Such a
Fig. 6 Etayoa trypethelii.
a Conidiomata on
Phaeographis major (Common
9200G). b Ascomata on
Lecanora caesiorubella (Buck
29314). c Conidioma showing
mosaic-like ornamentation of
exposed cells, in water
(Common 9481D).
d Young conidioma showing
mosaic-like ornamentation, in
water (Common 9215P).
e Conidia in water
(1: Common 9434 K,
2: Common 9200G, 3: Harris
39452A, 4–5: holotype).
f Conidiostroma in 0.15 % IKI,
after pre-treatment with C and K
(Common 9200G). g Immature
asci in water. h Immature asci in
K/I (stroma at first entirely K/I+
blue, then becoming orange).
i Ascostroma K/I+ blue
(g–i Buck 29314). j–k Ascus
and ascospores in water.
l Ascus with ascospores in K/I
(after pre-treatment with C)
(j–l Diederich 11495). Scale
bars: a–b=200 μm, c–l=10 μm
131
new genus would be morphologically extremely similar to
Lichenostigma, and we wondered how it could be justified
on a morphological basis. The main differences are (1)
almost spherical conidia in Ph. trypethelii (they are
more ellipsoid in Lichenostigma s. str.); (2) more compact
conidia in Ph. trypethelii (compared to the more loose
conidia in Lichenostigma s. str., the only exception being
L. fellhanerae, only provisionally placed in Lichenostigma,
as no molecular data are available) with slightly smaller
cells (in those species of Lichenostigma s. str. with large
conidia, individual cells are slightly larger); and (3)
external cells of stromata with a distinct, dark
greyish brown mosaic-like ornamentation (in
Lichenostigma s.str., the ornamentation is often verrucose, sometimes mosaic-like, but then slightly paler and more reddish brown).
2. The new genus is dedicated to our excellent friend Javier
Etayo (Pamplona, Spain). Javier is one of the most productive recent explorers of lichenicolous fungi, working
132
Fungal Diversity (2014) 66:113–137
Fig. 7 Cultures of
Lichenostigma, Etayoa and
Lichenothelia species.
a–b Lichenostigma chlaroterae
(Neuberg). c–d Lichenostigma
cf. alpinum on Fuscidea
lightfootii (Diederich 17240).
e–f Etayoa trypethelii
(Common 9215P).
g–h Lichenothelia rugosa (Ertz
16065). i–k ‘Lichenostigma’ cf.
elongatum (Ertz 15255). Scale
bars: a, g, i=0.5 mm, b=5 μm,
c=2 mm, d, j=20 μm, e=1 mm,
f, h, k=10 μm
especially in South America, and he is the author of
several taxa now included in Lichenostigma s. str.
Etayoa trypethelii (Flakus & Kukwa) Diederich & Ertz
comb. nov. (Figs. 6 and 7e–f)
MycoBank MB 804695
Basionym: Phaeosporobolus trypethelii Flakus & Kukwa,
Lichenologist 44: 472 (2012). Type: Bolivia, Dept. Beni, Prov.
Ballivian, near Reyes village, 14°18′10″S, 67°18′49″W, 192 m,
savannah vegetation, on thallus of Trypethelium ochroleucum,
29 Nov. 2004, A. Flakus 3724 (KRAM–holotype!; LPB, hb.
Flakus–isotypes).
Fungal Diversity (2014) 66:113–137
External cells of stromata with a dark brown mosaic-like
ornamentation. Ascomata provisionally referred to this species (30–)38–84(−160) μm diam. (N=56) (Buck 29314), I+
and K/I+ blue; asci 8-spored, c. 25×20 μm; ascospores hyaline, 1-septate, ellipsoid, c. 8.5–13×4.5–6 μm (very few asci
and ascospores observed). Conidiomata (20–)31–69(−140) μm
diam. (N=316, from 6 specimens including the holotype), I– or
pale blue, K/I– or K/I+ pale blue; conidia subspherical,
(10.5–)11.8–14.5(−16)×(10–)11.1–14.0(−15) μm, length/width
ratio 1.0–1.1, composed of (16–)20–31(−36) verrucose cells [in
optical section (11–)12–17(−19) cells] (N=19), cells (2.5–)3.0–
4.0(−4.5) μm diam. (N=94).
For a detailed description and illustrations, see Flakus and
Kukwa (2012).
The asexual stage was described from Bolivian material on
Trypethelium ochroleucum. We have examined additional
specimens from the U.S.A. (Florida), where it appears to be
rather common on the thallus of Graphidaceae, viz. Dyplolabia
afzelii, Fissurina columbina, F. mexicana, Graphis caesiella,
G. cupei, G. lucifica, Graphis sp., Ocellularia americana,
Phaeographis major, P. inconspicua, P. schizoloma, and
Phaeographis sp., but also on Bathelium carolinianum,
Laurera megasperma and Trypethelium ochroleucum, from
Papua New Guinea on Pertusaria ramulifera, and from South
Africa on Graphis. Material representing the sexual stage that
we provisionally refer to the same species has been collected in
the U.S.A. (Florida) on Lecanora caesiorubella subsp.
glaucomodes and in Papua New Guinea on Graphis and
Pertusaria ramulifera. All known specimens from both
morphs have been collected in subtropical or tropical regions
on corticolous lichens with a crustose thallus. The species does
not visibly damage the host thallus.
Notes:
1. For many years we have studied an apparently undescribed
Phaeosporobolus species that is rather common in Florida.
Comparison with the type of the recently described Ph.
trypethelii confirmed that the Florida material belongs to that
species, and at the same time that the species is not hostspecific (many Florida specimens are on Graphidaceae,
whilst the Bolivian type is on Trypethelium). Instead, this
species appears to be confined to subtropical to tropical
environments on corticolous lichens with a crustose thallus.
Field observations in Florida have shown that the species is
rather specialized concerning host choice, and that some
species tend to be highly colonized, whereas nearby thalli
of related species may have little or no colonization. Further
recent collections from South Africa on Graphis and from
Papua New Guinea on Pertusaria confirm that the species
is widespread in tropical habitats and that it prefers
Graphidaceae hosts.
2. A specimen from Florida on corticolous Lecanora
caesiorubella is macroscopically identical to Ph. trypethelii
133
(Fig. 6b) and microscopically indistinguishable, except for
the presence of asci instead of conidia. The anatomy of
stromata, including the colour and ornamentation of the
external cells are typically those of Ph. trypethelii. It most
probably belongs to the same species, and thus represents
the sexual stage of Ph. trypethelii. As the specimen is too old
(collected in 1996), unfortunately no DNA sequences could
be obtained. Two specimens that we collected in Papua New
Guinea in 1992, one on corticolous Graphidaceae, the other
on corticolous Pertusaria ramulifera, both appear to represent the same species, but all our efforts to obtain sequences
from them also failed. On the latter host, Pertusaria
ramulifera, we also collected the asexual stage.
3. The three specimens representing the sexual stage were
compared with the type of Lichenostigma hyalosporum
and found to represent a distinct species. L. hyalosporum
differs by ascomata not becoming blue in I after K and
by the presence of typical Phaeosporobolus-type
conidia, much looser than the compact conidia of E.
trypethelii.
4. The conidiomatal size is rather variable within this species
and does not represent a taxonomically useful character.
For example, in specimen Common 9200G, conidiomata
measure (20–)30–45(−55) μm diam. (N=58), in Harris
39452A they are (25–)31–55(−80) μm diam. (N=46),
whilst in Common 9481D they measure (55–)61–
100(−140) μm (N=52) [in case of elongate conidiomata,
the largest diameter has been measured]. After bleaching
and observation in 0.15 % IKI, a pale to moderately strong
I+ blue reaction was often seen in conidiomata. Distinct
hemiamyloid staining was not seen.
Additional specimens examined: Asexual stage: Papua
New Guinea: Madang Prov.: Huon Peninsula, Finisterre
range, Yupna valley, Teptep village, trail in NNW and deep
valley in N direction, 5.95° S, 146.55° E, 2500 m, on
corticolous Pertusaria ramulifera, 30 July 1992, Diederich
10815, 12415 (hb. Diederich). South Africa: Mngazana
Estuary, on Graphis, 2013, Mukherjee (BR). U.S.A.: Florida: Collier Co., Fakahatchee Strand State Preserve, trail
north of Boardwalk, 25°56.51′ N, 81°28.16′ W, on Graphis
caesiella, 11 Nov. 2011, Common 9434 K (hb. Diederich);
Everglades City, near Everglades City Motel on Fla. 29, on
Sabal palmetto leaf bases and on fallen tree, on Bathelium
carolinianum, 29 April 1997, Common 7347L (MSC).
Hernando Co., SR 50, between CR 595 N and CR 595S,
about 4 miles east of Gulf of Mexico, wet hardwood forest,
on Ocellularia americana, 31 Dec. 1975, Common 3737Q
(MSC). Hillsborough Co., Hillsborough River State Park,
28°08.601′ N, 82°13.786′ W, on Dyplolabia afzelii and
Phaeographis major, 3 Sept. 2011, Common 9200G
(MSC, hb. Diederich); ibid., on Laurera megasperma, Dec.
1990, Common 4821 (MSC); ibid., trail from Parking area 2,
134
28°08.94′ N, 82°13.61′ W, on P. major and P. schizoloma, 2
Sept. 2011, Common 9215P (hb. Diederich); ibid., Florida
Trail, 28°08.90′ N, 82°14.01′ W, on Graphis sp., P. major
and P. inconspicua, 26 Oct 2011, Common 9244E (hb.
Diederich); ibid., 28°08.94′ N, 82°14.10′ W, on
Phaeographis sp., 15 Sept. 2011, Common 9481D (hb.
Diederich); along CR. 581, 3.2 miles S of junction with I75, hardwood and cypress area along stream, SE of bridge,
on Fissurina mexicana, 26 April 1996, Common 6895A
(MSC). Marion Co., Ocala National Forest, along Co. Rd.
316, c. 0.5 mi E of Oklawaha River bridge at Eureka, 0.2 mi
W of Forest Serv. Rd. 67, 29°22′ N, 81°43′ W, QuercusEricaceae scrub, on Lyonia, on F. columbina, 29 Dec. 1995,
Buck 28630 (NY). Pasco Co., Withlacoochee State Forest,
on Clay Sink Rd., about 1 mile from W boundary of forest,
on Trypethelium, 31 Dec. 1975, Common C3701P (MSC).
Sarasota Co., Myakka River State Park, at first crossing of
river from south entrance to park on north bank of river, on
Trypethelium ochroleucum, 1 Jan. 1976, Common 3755D
(MSC); ibid., along river near S entrance to park, on Graphis
cupei and G. lucifica, 27 April 1997, Common 7216G
(MSC). Taylor Co., Big Bend Wildlife Management Area,
Tide Swamp Unit, along C. R. 361, 4.4 mi NW of bridge in
Steinhatchee, 29°43′ N, 83°26′ W, planted Pinus clausa
forest, on P. clausa, on G. lucifica, 3 Dec. 1996, Harris
39452A (NY).
Sexual stage of E. cf. trypethelii: Papua New Guinea:
Eastern Highlands Prov.: Mount Gahavisuka Provincial
Park, 11 km N of Goroka, along trail to lookout, little
disturbed mossy mountain forest, 6°01′ S, 145°25′ E,
2400 m, on corticolous Pertusaria ramulifera, 3 Aug. 1992,
Diederich 10630 (hb. Diederich). Madang Prov.: Near Bogia,
long road Bogia-Josephstaal, near Tanggu church, 4°27′ S,
144°56′ E, 330 m, on crown and trunk of felled tree among
gardens, on Graphidaceae, 25 July 1992, Diederich 11495 (hb.
Diederich). U.S.A.: Florida: Seminole Co., along Willingham
Road, 0.5 mi N of Co. Rd. 419 at Econlockhatchee River, on W
border of Flying Seminole Ranch, 28°40′ N, 81°10′ W, oakpine-palmetto scrub, on Lecanora caesiorubella subsp.
glaucomodes, 10 Jan. 1996, Buck 29314 (NY).
Fungal Diversity (2014) 66:113–137
2(1)
3(1)
3(1)
4(3)
Key to the species of Lichenostigmatales
1
Colonies of dark brown, aseptate, thick-walled yeast
cells covered by mucus and reproducing by multilateral budding; mycelium formation, ascomata and
conidiomata unknown.............. [Phaeococcomyces] 2
1
Colonies of stromatic ascomata or conidiomata, on
rare occasions arising from an initial, brown mycelium.......................................................................... 3
2(1) Colonies jet black from the beginning; cells at first
hyaline or subhyaline, soon becoming dark olivaceous
brown, at first thin-, later usually thick-walled, finally
4(3)
5(4)
often forming an irregular, verrucose capsule (granular pigment), globose to broadly ellipsoidal, 4–6.5×4–
5 μm diam., each giving rise to similarly shaped,
slightly smaller secondary cells from 1 to 3 loci; often
the cells remain attached, forming short chains of
compact clumps; isolated from paint of storage tank
in the U.S.A..............................................P. nigricans
Colonies iron-grey; cells globose, verruculose, 4–8 μm
diam., remaining attached to one another, at colony
margin producing ellipsoid to globose, hyaline, finely
verruculose conidia-like cells covered by mucus, 3–
5×2.5–5 μm; isolated from Eucalyptus leaves in South
Africa .......................................................... P. eucalypti
Ascomata I+ and K/I+ intensively blue (but conidiomata
I+ pale blue); external cells of stromata (ascomata
or conidiomata) with a dark brown, mosaic-like ornamentation; conidia regularly spherical, length/width ratio
1.0–1.1, (10.5–)11.8–14.5(−16)×(10–)11.1–14.0(−15)
μm diam., rather compact, composed of 20–31 cells [in
optical section 12–17 cells] mainly 3–4 μm diam., surface verrucose; ascospores hyaline, ellipsoid; colonies
either representing the sexual or the asexual stage, never
mixed; on crustose, corticolous lichens in subtropical to
tropical environments..........................Etayoa trypethelii
Ascomata and conidiomata I- and K/I-, or I+ reddish or
blue and K/I+ blue; external cells of stromata medium
to dark brown, with a verrucose, rarely mosaic-like
ornamentation; conidia ellipsoid [rarely irregular in
form], length/width ratio 1.0–1.6 [if less than 1.2,
then conidia composed of less than 16 cells], more
loose, composed of cells that are often slightly larger,
3.5–4.5 μm diam. [if cells 2.5–3.5 μm diam., then
conidia either composed of less than 8 cells or regularly
ellipsoid], surface smooth, becoming echinulate
when overmature; ascospores hyaline or brown;
colonies mostly representing the asexual stage,
more rarely the sexual stage and then both stages frequently mixed; in temperate to subalpine or boreal
environments................................... [Lichenostigma] 4
Conidia produced in a very high number, typically
more than 100 per conidioma, regularly ellipsoid,
rather compact, (8–)8.7–11.0(−12.5)×(6.5-)6.8–7.7
(−8) μm, composed of (16–)18–23(−27) smooth
cells [in optical section 9–12 cells], cells 2.5–3.2
(−3.5) μm diam.; ascomata unknown; on Fellhanera
granulosa.................................................. L. fellhanerae
Conidia usually produced in a smaller number, often
more irregular in form, looser, cells larger, 3–5 μm
diam...........................................................................5
External cells of stromata with a mosaic-like ornamentation; ascomata usually present; ascospores hyaline,
ellipsoid, 1-septate, 9–15×3.5–8 μm; on crustose
corticolous lichens… ............................ L. hyalosporum
Fungal Diversity (2014) 66:113–137
5(4) External cells of stromata with a verrucose, rarely
mosaic-like ornamentation; ascomata either present
and ascospores dark brown; or ascomata extremely
rare, with hyaline ascospores that are much smaller,
6.5–7×2–2.5 μm, or elongate, attenuated and pointed
at both ends, spirally bent, 20–27×3–4 μm; or
ascomata absent ........................................................6
6(5) Conidia 6–10×5.5–9 μm, composed of 4–9 cells [in
optical section 4–5(−8) cells]; sexual stage extremely
rare; ascospores brown; on corticolous Lecanora and
other corticolous crustose lichens.........L. chlaroterae
6(5) Conidia larger, at least 10×8.5 μm, composed of at
least 8 cells; ascospores hyaline or brown...................7
7(6) Conidia mostly 14–23×10–16 μm, composed of
14–35 cells [in optical section 10–21 cells]; frequently accompanied by a sexual stage; ascospores dark brown, ellipsoid, 9–12×4.5–6 μm;
asci 20–25×13–18 μm; mainly on epiphytic fruticose macrolichens...................................L. maureri
7(6) Conidia mostly 10–15×8.5–11 μm, composed of 8–
17 cells [in optical section 6–10 cells]; sexual stage
extremely rare; ascospores hyaline; mainly on
terricolous, saxicolous or corticolous Ochrolechia,
Pertusaria, Varicellaria and Fuscidea, also on other
crustose lichens ...........................[L. alpinum s. lat.] 8
8(7) Ascospores elongate, attenuated and pointed at both
ends, spirally bent, 20–27×3–4 μm; asci 31–39×13–
17 μm; on Fuscidea cyathoides and Ochrolechia frigida
...............L. alpinum s. lat. p. p. (‘Diederimyces fuscideae’)
8(7) Ascospores ellipsoid, 6.5–7×2–2.5 μm; asci 14–
17×9–12 μm; on Pertusaria and Coccotrema
...............L. alpinum s. lat. p. p. (‘Diederimyces microsporus’)
A new combination for Lichenostigma rugosum
Lichenothelia rugosa (G. Thor) Ertz & Diederich comb.
nov. (Fig. 7g–h)
MycoBank MB 804675
Basionym: Lichenostigma rugosum G. Thor, Lichenologist 17 (3): 269 (1985) [as L. rugosa]. Type: Libya, Cyrenaica, 62 km SSW of Al Marj, 25 km SSE of Al Abyar, 32°00′
N, 20°65′ E, on Diploschistes steppicus, 26 March 1983, G.
Thor 3458 (S–holotype; BM, GZU, UPS–isotypes, non
vid.).
For a detailed description and illustrations, see Thor
(1985).
Notes:
1. This species was originally distinguished from the very
similar Lichenostigma maureri by several characters,
such as usually aggregated, black ascomata (compared
to brown to black, non-aggregate ascomata), and larger
and darker ascospores with deep irregular fissures
135
(ascospores of L. maureri are verrucose) (Thor 1985).
Our phylogenetic results show, however, that these two
species are not related. They differ entirely by the structure of the ascostroma, as stromatic cells in L. rugosum
divide by septa, whilst those of L. maureri by budding.
We have confirmed this character in cultures of L.
rugosum (Fig. 7h).
2. Phylogenetically, Lichenostigma rugosum groups with
species of Lichenothelia (Lichenotheliaceae). As the species clearly does not belong to Lichenostigma, we herewith combine it in Lichenothelia. Sequences from several
other Lichenostigma species belonging to subgen.
Lichenogramma Nav.-Ros. & Hafellner also belong to
this group, but no other transfers in Lichenothelia are
formally proposed here, as that subgenus was not the
main object of the present study. These species include
Lichenostigma elongatum, a species that also multiplies
by septa (Fig. 7i–k), and not by budding as in
Lichenostigma s.str.
Selected specimens of Lichenothelia rugosa examined:
France: Ardennes: Chooz, Rochers de Petit-Chooz sur la
rive droite de la Meuse, 200 m, affleurement de roches de
l’Emsien supérieur, sur Diploschistes scruposus, 20 Febr.
2011, Ertz 16065 (BR). Switzerland: Valais: S of Nendaz,
2 km SSW of Siviez, 46.11176° N, 7.30436° E, on
Diploschistes scruposus, 26 July 2012, Diederich 17310
(hb. Diederich).
Acknowledgments We would like to thank the curators of herbaria
cited in Materials and Methods for the loan of specimens. Ann Bogaerts,
Myriam Dehaan and Wim Baert are thanked for technical assistance.
Gisèle Van Cappellen, Trevor Goward, Nibedita Mukherjee and Paul
Neuberg kindly provided fresh specimens used in this study. Finally,
the first author acknowledges financial support from the Fonds National
de la Recherche Scientifique (FNRS) from Belgium (F.R.F.C. #
2.4567.08).
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