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Molecular support for the recognition of the Mycoblastus fucatus group as the new genus Violella (Tephromelataceae, Lecanorales)

Abstract

The crustose lichen genus Mycoblastus in the Northern Hemisphere includes eight recognized species sharing large, simple ascospores produced 1-2 per ascus in strongly pigmented biatorine apothecia. The monophyly of Mycoblastus and the relationship of its various species to Tephromelataceae have never been studied in detail. Data from ITS rDNA and the genes coding for translation elongation factor 1-α and DNA replication licensing factor mini-chromosome maintenance complex 7 support the distinctness of Mycoblastus s. str. from the core of the Tephromelataceae, but recover M. fucatus and an undescribed Asian species as strongly supported within the latter group. We propose accommodating these two species in a new genus, Violella, which is characterized by its brownish inner ascospore walls, Fucatus-violet hymenial pigment granules and secondary chemistry, and discuss the position of Violella relative to Calvitimela and Tephromela. We describe the new species Violella wangii T. Sprib. & Goffinet to accommodate a new species with roccellic acid from Bhutan, China, India and the Russian Far East. We also exclude Mycoblastus indicus Awasthi & Agarwal from the genus Mycoblastus and propose for it the new combination Malmidea indica (Awasthi & Agarwal) Hafellner & T. Sprib.
Molecular support for the recognition of the Mycoblastus
fucatus group as the new genus Violella (Tephromelataceae,
Lecanorales)
Toby SPRIBILLE, Bernard GOFFINET, Barbara KLUG,
Lucia MUGGIA, Walter OBERMAYER and
Helmut MAYRHOFER
Abstract: The crustose lichen genus Mycoblastus in the Northern Hemisphere includes eight recog-
nized species sharing large, simple ascospores produced 1–2 per ascus in strongly pigmented biatorine
apothecia. The monophyly of Mycoblastus and the relationship of its various species to Tephromelata-
ceae have never been studied in detail. Data from ITS rDNA and the genes coding for translation
elongation factor 1-and DNA replication licensing factor mini-chromosome maintenance complex 7
support the distinctness of Mycoblastus s. str. from the core of the Tephromelataceae, but recover M.
fucatus and an undescribed Asian species as strongly supported within the latter group. We propose
accommodating these two species in a new genus, Violella, which is characterized by its brownish inner
ascospore walls, Fucatus-violet hymenial pigment granules and secondary chemistry, and discuss the
position of Violella relative to Calvitimela and Tephromela. We describe the new species Violella wangii
T. Sprib. & Goffinet to accommodate a new species with roccellic acid from Bhutan, China, India and
the Russian Far East. We also exclude Mycoblastus indicus Awasthi & Agarwal from the genus
Mycoblastus and propose for it the new combination Malmidea indica (Awasthi & Agarwal) Hafellner &
T. Sprib.
Key words: ascus types, Asia, Calvitimela, EF1-gene, fatty acid, lichens, Malmidea, Mcm7 gene,
phylogeny, pigment, taxonomy
Introduction
The genus Mycoblastus is a widely distributed
group of mainly epiphytic species found in
cool temperate to arctic regions of both
hemispheres. Its type species, M. sangui-
narius (L.) Norman, is one of the common
and familiar crustose lichens of boreal conifer
forests, and is circumboreal. Despite being
easily recognized and often collected, the
genus has never been subjected to a complete
global revision. Northern Hemisphere
species concepts in Mycoblastus developed
gradually through the description of forms
and varieties of M. sanguinarius that were
later raised to species rank. More species
were added to the genus as regions of
the Southern Hemisphere became better
explored and species previously described
under Lecidea were combined into Myco-
blastus (e.g., Müller-Argoviensis 1894;
Zahlbruckner 1926). Recent European taxo-
nomic concepts and nomenclature were out-
lined by Schauer (1964), who recognized two
species, and were expanded by James (1971),
who provided a key. Recently Kantvilas
(2009) revised cool temperate Southern
Hemisphere material, recognizing eight
species, which he considered to belong to two
different species groups, the ‘M. sanguinarius
group’ which always contains atranorin, and
the ‘M. dissimulans group’, the members of
which always contain perlatolic acid.
Mycoblastus in the Northern Hemisphere is
currently considered to include eight species,
T. Spribille, B. Klug, L. Muggia, W. Obermayer and H.
Mayrhofer: Institute of Plant Sciences, University of
Graz, Holteigasse 6, A-8010 Graz, Austria. Email:
toby.spribille@uni-graz.at
B. Goffinet: Department of Ecology and Evolutionary
Biology, University of Connecticut, 75 N Eagleville
Road, Storrs, CT 06269-3043, USA.
The Lichenologist 43(5): 445–466 (2011) © British Lichen Society, 2011
doi:10.1017/S0024282911000478
namely M. affinis,M. alpinus,M. glabrescens
(Kantvilas 2009), M. sanguinarius,M. san-
guinarioides (Spribille et al. 2011), M. japoni-
cus (Müller-Argoviensis 1891), M. fucatus
(James 1971) and M. caesius (Tønsberg
1992). A dichotomy between atranorin- and
perlatolic acid-containing species is present
in the Northern Hemisphere as well, with
M. caesius containing perlatolic acid and all
other taxa containing atranorin and other
substances. The atranorin-containing Myco-
blastus species of the Northern Hemisphere
have been accorded renewed attention re-
cently with a detailed study of the M. sangui-
narius group by Spribille et al. (2011).
Specifically, these authors inferred the phylo-
genetic relationships with an emphasis on
testing monophyly of M. sanguinarius in a
phylogeny in which all known atranorin-
containing Northern Hemisphere species
were represented. Mycoblastus fucatus was
represented by a single specimen, and was
resolved to be only distantly related to the
core group of Mycoblastus.
Mycoblastus fucatus is enigmatic among the
Northern Hemisphere atranorin-containing
species, for at least two reasons. First, its
brilliant violet hymenial pigment, termed
‘Fucatus-violet’ by Kantvilas (2009), sets it
apart from other Mycoblastus species, which
contain the dull greenish to green-blue pig-
ment ‘Cinereorufa-green’. Second, it is the
common and sole host of a lichenicolous
fungus, Tremella lichenicola, which does
not invade any other Mycoblastus species
(Coppins & James 1979; Diederich 1986,
1996). Apart from James (1971), little atten-
tion has been paid to the ascocarps of M.
fucatus, in part because they are so rare; in
Norway, apothecia were observed in only
three of 103 specimens studied by Tønsberg
(1992). Sterile forms were described in
Britain as a separate species, M. sterilis
(Coppins & James 1979) until it was later
realized that they were sterile forms of M.
fucatus (Tønsberg 1992).
The recovery of Mycoblastus fucatus out-
side of the core of Mycoblastus by Spribille
et al. (2011) motivated us to expand our
sampling in line with our previous phylo-
genetic work on Tephromela s. lat. (Muggia
et al. 2008), a lineage which has repeatedly
been found to be related to Mycoblastus
(Mia˛dlikowska et al. 2006; Arup et al. 2007;
Ekman et al. 2008). We also wanted to ex-
plore the possible relationship of M. fucatus
with the saxicolous genus Calvitimela and
some of the species groups discussed by
Kantvilas (2009). Sequence motifs in M.
fucatus indeed suggested affinities to Teph-
romela or Calvitimela rather than to Mycoblas-
tus. At the same time, another taxon clearly
related to M. fucatus was collected by the first
two authors of this paper in Russia and
China, providing more fresh material and
further solidifying the concept of this as a
recognizable species group with distinct mor-
phological characters. Here, we present the
results of molecular phylogenetic and mor-
phological investigations on the M. fucatus
group and propose for it the new genus
Violella.
Materials and Methods
Taxon sampling and hypothesis testing
We designed our taxon sampling to include the core
groups of Mycoblastus for which we could obtain fresh
material, as well as representatives of major groups in
the Tephromelataceae identified by Hertel & Rambold
(1985), including Tephromela,Calvitimela and the
Lecideaaglaea group, which has been treated as
belonging to both Tephromela and Calvitimela in the past.
We also generated sequences for several taxa of Parme-
liaceae, which is a group often retrieved in BLAST
searches of Mycoblastus sequences in GenBank. We
included one specimen of Japewia (Lecanoraceae),
hypothesized as being close to Mycoblastus by Kantvilas
(2009), and spent some sequencing effort examining the
possibility of relationships to Megalaria, also proposed
as a relative of Mycoblastus by Kantvilas (2009), and
Psorinia, suggested as a possible relative to Calvitimela by
Hafellner & Türk (2001). We ultimately excluded Mega-
laria and Psorinia from our sampling because 1) morpho-
logical evidence, especially the strongly gelatinized
proper exciple of Megalaria, argues against close rela-
tionships with that genus, and 2) DNA sequence data we
obtained for single loci for both Megalaria and Psorinia
were so different from the other taxa in our dataset as to
be easily ruled out as close relatives. Heppsora indica,a
species and genus described from Tamil Nadu state,
India (Awasthi & Singh 1977; Singh & Sinha 2010:
photograph), exhibits clear morphological affinities to
Tephromelataceae (Poelt & Grube 1993). Unfortunately
we did not have access to any fresh material; a speci-
men distributed under this name in a recent exsiccate
446 THE LICHENOLOGIST Vol. 43
(Lumbsch & Feige, Lecanoroid Lichens #85) differs in
chemistry and ascocarp pigmentation and is not H.
indica. In the end, our taxon sampling (Table 1) pro-
vided a sufficient taxonomic neighbourhood to test the
hypothesis of whether Mycoblastus fucatus would be
recovered within Mycoblastus, in the vicinity of Teph-
romela, or in the vicinity of Parmeliaceae or Lecanoraceae.
Laboratory methods
Material for DNA extraction was taken from apothe-
cia if present, otherwise from parasite-free thallus frag-
ments inspected in water droplets on a microscope slide
under ×20 magnification. Prepared material was trans-
ferred into reaction tubes, dried and pulverized using a
TissueLyserII (Retsch). DNA was extracted using the
DNeasy Plant Mini Kit (Qiagen) extraction kit using the
manufacturers instructions. For Tephromela specimens
already studied by Muggia et al. (2008), existing extrac-
tions were used. Dilutions (mostly 5 × 10
−2
)ofthe
genomic DNA extractions were used as a template for
the PCR reactions. After screening potential markers
(Spribille et al. 2011), we settled on using three loci: two
protein-coding genes, namely translation elongation fac-
tor 1-(EF1-) and the DNA replication licensing fac-
tor mini-chromosome maintenance complex 7 (Mcm7),
and the nuclear ribosomal internal transcribed spacer
region (ITS). For amplification of EF1-from Myco-
blastus japonicus,weemployedaMycoblastus-specific
primer pair which will be described in detail elsewhere.
PCR reactions were performed with Illustra Ready-
To-Go RT-PCR Beads (GE Healthcare) in a thermo-
cycler (AlphaMetrix) using conditions detailed by
Spribille et al. (2011). Two l aliquots of PCR products
were viewed on 1% agarose gels stained with GelRed™
(Biotium, VWR); whole products were subsequently
purified with NucleoSpin Extract II Kit (Macherey-
Nagel). PCR product sequencing was outsourced to
Macrogen, Inc. (Seoul, South Korea). Sequence frag-
ments were obtained electronically from Macrogen and
electropherogram ambiguities checked in BioEdit (Hall
1999). All DNA sequences were submitted to GenBank
and are retrievable under the accession numbers listed in
Table 1.
Phylogenetic analyses
Alignments were performed using ClustalW
(Thompson et al. 1994) and subsequently optimized by
hand in BioEdit (Hall 1999). Non-conserved regions
and positions with missing data in >50% of sequences
were removed using Gblocks (Talavera & Castresana
2007). Candidate nucleotide substitution models were
identified for each partition using the likelihood ratio test
implemented in jModelTest (Posada 2008); likelihood
scores were then compared based on the Akaike Infor-
mation Criterion (AIC). Individual gene alignments
were analyzed using a maximum likelihood (ML) and
Bayesian Markov Chain Monte Carlo (B/MCMC)
approach. We tested for conflict between partitions by
examining frequencies of bipartitions for the same taxon
sets across all three partitions using a set of B/MCMC
gene trees; a conflict was interpreted as significant if two
well supported different relationships were detected
for the same taxon set (Kauff & Lutzoni 2002); we used
the threshold of R95%. Maximum likelihood analy-
ses were performed using the program PhyML 3.0
(Guindon et al. 2010). Bootstrapping was carried out on
500 tree replicates. B/MCMC analyses were performed
using the program MrBayes v. 3.1.2 (Huelsenbeck &
Ronquist 2001) using substitution models approxi-
mated by jModeltest (see above). For each analysis, two
runs with ten million generations each starting with a
random tree and running four simultaneous chains was
employed. Every 1000th tree was sampled and saved to
a file. The first 5 000 000 generations (5000 sampled
trees) were discarded as chain ‘burn-in’. Of the remain-
ing 5001 trees a majority consensus tree with averaged
branch lengths and annotated with posterior probability
values at every node was calculated using the sumt
command in MrBayes. The program TRACER v. 1.5
(http://tree.bio.ed.ac.uk/software/tracer/) was used to
assess whether likelihood values had reached stationarity
within the allocated burn-in window by plotting log
likelihood against the number of generations. In addi-
tion, we examined the distributions of split frequencies
using the online program AWTY (Nylander et al. 2007)
to test whether runs had converged. Only clades that
received bootstrap values R70% in ML and posterior
probabilities R0·95 were considered significantly
robust. Phylogenetic trees were visualized in TreeView
(Page 1996).
Morphological and chemical analyses
To test whether our phylogenetic results could be
matched by morphological traits, we sorted specimens
under a Leica Wild M3Z dissecting microscope and
examined anatomical sections on material mounted in
water with a Zeiss Axioskop light microscope fitted with
Nomarski differential interference contrast and outfitted
with a ZeissAxioCam MRc5 digital camera. Some
images were digitally optimized through ‘stacking’ using
CombineZM open source image processing software
(www.hadleyweb.pwp.blueyonder.co.uk/CZM/). Asco-
spore, areole, soredia and apothecia measurements
are given as (smallest absolute measurement–)smallest
average – largest average(–largest absolute measure-
ment). Ascus morphology was investigated in asci with
immature ascospores (following Hafellner 1984). In
addition, we examined specimens for chemical patterns
that could corroborate phylogenetic differentiation
using thin-layer chromatography (TLC), following
the methods of Culberson (1972) with modifications
(Culberson & Johnson 1982). We used silica-coated
glass plates (Macherey-Nagel 821 030) run their full
length in solvent systems A, Band C. Aliphatic acids
were visualized by immersing completely dried plates
post-development into a tank of water for 1–2 s, quickly
dripping off the plates and marking spots over the next
4 min. No attempt was made to separate roccellic and
angardianic acids, which are indistinguishable in TLC
(Tønsberg 1992).
2011 Molecular support for Violella gen. nov. 447
T 1.DNA vouchers and GenBank Accession Numbers of the species used in this study; bold species names and accession numbers indicate new accessions
Species Ref. number Voucher GenBank Accession Numbers
EF1-ITS Mcm7
Alectoria sarmentosa 638 Canada, British Columbia, near mouth of Halfway River
on Upper Arrow Lake, 2009, Spribille s.n. (GZU)
JN009675 JN009706 JN009737
Allantoparmelia sibirica*854 USA, Alaska, Dalton Highway, Finger Mtn., 2010,
Spribille s.n. (GZU)
JN009676 JN009707
Calvitimela armeniaca 599 Canada, Yukon, Mt. Martin, Spribille 28707 (GZU) JN009677 JN009708 JN009738
C. armeniaca 607 Austria, Carinthia, Koralpe, Hafellner 71304 (GZU) JN009678 JN009709 JN009739
C. armeniaca 836 Spain, Catalonia, Parque Nacional de Aigüestortes i
Estany De Sant Maurici, Pérez-Ortega 1321 (GZU)
JN009710 JN009740
C. armeniaca 837 Spain, Catalonia, Parque Nacional de Aigüestortes i
Estany De Sant Maurici, Pérez-Ortega 1322 (GZU)
JN009711 JN009741
C. armeniaca 856 USA, Alaska, Dalton Highway, Finger Mtn., 2010,
Spribille s.n. (GZU)
JN009679 JN009712 JN009742
C. melaleuca 150 USA, Alaska, White Pass, Spribille 26952 (KLGO) JN009680 JN009713 JN009743
C. melaleuca 838 USA, Alaska, Alaska Range, Mt. Healy, Spribille 27965-B
(GZU)
JN009714 JN009744
Cetraria sepincola 639 Slovakia, Nizke Tatry, between C
ˇertovica and Dumbierˇ,
Spribille 32131 & Wagner (GZU)
JN009681 JN009715 JN009745
Japewia subaurifera 764 USA, New Hampshire, Coos Co., ridge S of Dixville
Notch, 2009, Spribille & Wagner s.n. (GZU)
JN009682 JN009716
“Lecidea” aglaea 608 Austria, Vorarlberg, Rätikon, Hafellner 72944 (GZU) JN009683 JN009717
“Lecidea” aglaea 847 Austria, Styria, Koralpe, Hafellner 70358 (GZU) JN009684 JN009718
“Lecidea” aglaea 867 Sweden, Jämtland, Åre par., Mt. Skurdalsbergen, Nordin
6659 (UPS-183008)
JN009685 JN009719
Miriquidica instrata 852 USA, Montana, Lincoln Co., Whitefish Range, Lewis
Creek talus, 2010, Spribille s.n. (GZU)
JN009686 JN009720 JN009746
Mycoblastus affinis 90 Canada, British Columbia, Philipp Lake, 2008, Goward
& Wright s.n. (GZU)
JF744895 JF744969 JF744809
M. affinis 121 USA, Alaska, Russian River, Spribille 27371 (GZU) JF744896 JF744812
M. affinis 379 USA, Montana, Lincoln Co., Laughing Water Creek,
Spribille 30126 (GZU)
JF744898 JF744980 JF744795
M. affinis 420 Austria, Styria, near Oberzeiring, Spribille 30220 (GZU) JF744899 JF744978 JF744797
M. affinis 464 Germany, Bavaria, Bayrischer Wald, Dreisesselfels,
Spribille 32115 & Wagner (GZU)
JF744900 JF744979 JF744800
448 THE LICHENOLOGIST Vol. 43
T 1.Continued
Species Ref. number Voucher GenBank Accession Numbers
EF1-ITS Mcm7
M. affinis 465 Austria, Styria, Hörsterkogel, Spribille 32102 (GZU) JF744902 JF744977 JF744801
M. affinis 766 Canada, Nova Scotia, Cape Breton, 2009, Spribille &
Wagner s.n. (GZU)
JF744897 – JF744813
M. affinis 795 China, Yunnan, Goffinet 10030 (CONN) JN009721 JN009747
M. affinis 858 Canada, Québec, Gaspesie E of Claridorme, 2009,
Spribille & Wagner s.n. (GZU)
JN009722
M. alpinus
466 Canada, Yukon, LaBiche River area, Spribille
28541 (GZU)
JF744903 – JF744802
M. alpinus 537 Canada, Québec, Lac à Jack, 2009, Spribille & Clayden
s.n. (GZU)
JF744901 JF744976 JF744805
M. alpinus 468 USA, Alaska, White Pass, Spribille 26781 (KLGO) JF744904 JF744803
M. glabrescens 92 USA, Washington, Skamania Co., Elk Pass, Spribille
29848 (GZU)
JF744894 JF744967 JF744810
M. glabrescens 352 USA, Idaho, Shoshone Co., Hobo Cedars, Spribille
30024 (GZU)
JF744893 JF744985 JF744816
M. glabrescens 367 USA, Oregon, Linn Co., Tombstone Pass, Spribille
29899 (GZU)
JF744892 JF744984 JF744815
M. japonicus 802 South Korea, Gangwon Prov., Sorak-san National Park,
Thor 20551 (UPS)
JN009688 JF744983 –
M. sanguinarioides 250 USA, Alaska, Chilkoot Trail, Spribille 27038-A (GZU) JF744884 JF744971 JF744794
M. sanguinarioides 460 Russia, Khabarovskiy Krai, 10 km W of De Kastri,
Spribille 30655 (GZU)
JF744886 JF744974 JF744799
M. sanguinarioides
502 Japan, Hokkaido, Prov. Kushiro, Mt. O-akan, Ohmura
6740 (GZU)
JN009689 JN009723 JN009748
M. sanguinarioides 542 Canada, Nova Scotia, Advocate Harbour, 2009, Spribille
& Wagner s.n. (GZU)
JF744888 JF744981 JF744806
M. sanguinarioides 582 Australia, Tasmania, foot of Adams Peak, Kantvilas
1/09 (GZU)
JF744889 JF744972 JF744819
M. sanguinarioides
857 Japan, Honshu, Mt. Fuji, Ohmura 5996 (GZU) JN009690 JN009724
M. sanguinarius 100 Canada, British Columbia, Retallack, Spribille 30134-A
& Pettitt (GZU)
JF744879 JF744913 JF744746
M. sanguinarius 120 USA, Alaska, Russian River, Spribille 27370 (GZU) JF744827 JF744914 JF744747
M. sanguinarius 170 Norway, Hordaland, Åsane, Spribille 30237-I (GZU) JF744843 JF744905 JF744765
2011 Molecular support for Violella gen. nov. 449
T 1.Continued
Species Ref. number Voucher GenBank Accession Numbers
EF1-ITS Mcm7
M. sanguinarius 236 USA, Oregon, Wasco Co., along Hwy. 26, Spribille
29881-C (GZU)
JF744858 JF744944 JF744777
M. sanguinarius 410 Russia, Khabarovskiy Krai, Etkil-Yankanskiy Mountains,
Spribille 31330 (GZU)
JF744864 JF744949 JF744781
M. sanguinarius 436 Russia, Khabarovskiy Krai, near Lazarev, Spribille
30949 (GZU)
JN009691 JF744950 JF744782
M. sanguinarius 486 Sweden, Pite Lappmark: Arvidsjaur par., 13 km NNW of
Moskosel, Muggia (TSB-38893)
JN009692 JN009725
M. sanguinarius 493 Japan, Hokkaido, Prov. Kushiro, Mt. O-akan, Ohmura
6746 (GZU)
JF744866 JF744953 JF744786
M. sanguinarius 543 Canada, Québec, Rte. 138 N of Les Escoumins, 2009,
Spribille & Clayden s.n. (GZU)
JF744856 JF744956 JF744787
M. sanguinarius 590 Russia, Chelyabinskaya Oblast, ZyuratkulNational
Park, Khrebet Nurgushch, 31 May 2009, Urbanavichene
s.n. (GZU)
JN009693 JN009749
M. sanguinarius 598 Russia, Leningrad Oblast, 7·5 km E of Ladva Village,
2009, Stepanchikova s.n. (GZU)
JF744869 JF744961 JF744792
M. sanguinarius 605 Canada, Yukon, LaBiche River area, Spribille
28305 (GZU)
JF744877 JF744987 JN009750
M. sanguinarius GB1 Canada, Québec, Rivière Noire, Lutzoni & Mia˛dlikowska
(DUKE-47513)
DQ782898 DQ782842
M. sanguinarius MS15 Russia, Primorskiy Krai, Oblachnaya, Spribille 23583 &
Krestov (BG)
JN009694 JN009726
M. sanguinarius 772 Russia, Khabarovskiy Krai, Bureinskiy Zapovednik, near
Staraya Medvezhka, Spribille 31959 & Yakovchenko
(GZU)
JN009695 JN009727 JN009751
Protoparmelia badia 853 USA, Montana, Lincoln Co., Whitefish Range, Lewis
Creek talus, 2010, Spribille s.n. (GZU)
JN009696 JN009728 JN009752
Tephromela atra L415 Greece, Crete, Herakleion, Kameraki, Muggia (TSB
37924)
JN009697 EU558688 JN009753
T. atra L223 Italy, Campania, Napoli, Capri Island, Muggia (TSB
37119)
JN009698 EU558648 JN009754
450 THE LICHENOLOGIST Vol. 43
T 1.Continued
Species Ref. number Voucher GenBank Accession Numbers
EF1-ITS Mcm7
T. atra L228 Italy, Campania, Napoli, Capri Island, Muggia (TSB
37124)
JN009699 EU558650 JN009755
T. atra L248 Italy, Campania, Napoli, Capri Island, Muggia (TSB
37133)
EU558656 JN009756
T. atra L284 Italy, Sardinia, Nuoro, Mt. Albo, Muggia (TSB 37465) EU558661 JN009757
T. atra calcarea 628 Greece, Epirus, Tzoumerka, Spribille 15951 (GZU) JN009700 JN009729 JN009758
T. atra calcarea L403 Greece, Crete, Lasithi, Selakano forest, Muggia (TSB
37912)
EU558681 JN009759
T. atra calcarea L280 Italy, Sardinia, Nuoro, Mt. Albo, Muggia (TSB 37461) EU558660 JN009760
T. cf. pertusarioides
§
850 Russia, Khabarovskiy Krai, Bureinskiy Zapovednik, near
Staraya Medvezhka, Spribille 31797 & Yakovchenko
(GZU)
JN009701 JN009730 JN009761
Tephromela sp. Björk 18057
629 Canada, British Columbia, Fraser Canyon, Björk
18057 (UBC)
JF744875 JF744986 JF744821
Usnea intermedia 609 Austria, Styria, Gurktaler Alpen, Obermayer
11839 (GZU)
JN009702 JN009731 JN009762
Violella fucata 844 Germany, Bavaria, Bayerischer Wald, Dreisesselfels,
Spribille 32112 (GZU)
JN009732
V. fucata 600 USA, Massachusetts, Mt. Greylock, Spribille
32161 (GZU)
JN009703 JF744968 JF744818
V. fucata 835 Slovenia, Snežnik area, Spribille 30276 & Mayrhofer
(GZU)
JN009733 JN009763
V. wangii 796 China, Yunnan, Laojunshan, Goffinet 10029 (KUN) JN009704 JN009734 JN009764
V. wangii 842 China, Yunnan, Laojunshan, Goffinet 10033 (UPS) JN009735 JN009765
V. wangii 840 Russia, Khabarovskiy Krai, Chegdomyn-Sofiysk road,
Spribille 31621 & Yakovchenko (H)
JN009705 JN009736 JN009766
*first confirmed record for North America (TLC: -collatolic and alectoronic acids)
reported as M. affinis by Spribille et al. (2011), this specimen actually corresponds to the alpinus morphotype
first modern record for Japan
§
first record for Russia
previously published as T. atra s.lat. by Spribille et al. (2011), but probably an undescribed taxon
2011 Molecular support for Violella gen. nov. 451
Pigments were examined under the light microscope
and named according to Meyer & Printzen (2000),
except for Fucatus-violet, which was not treated by
those authors. Fucatus-violet would key in Meyer &
Printzens key under lead 2 as N+ violaceous as it goes
from its natural violet colour in H
2
O to a deep raspberry
red. It has the following standard reactions: K+ peacock-
blue, N+ raspberry-red, HCl− (slowly fading but main-
taining hue), C+ grey, eventually bleaching altogether;
after pretreatment with N: K greenish yellow 4HCl
completely clear. The pigment was mentioned already
by Stirton (1879) as an ‘intense violaceous colour’ and
has also been previously referred to as ‘gentian violet’
(James 1971; James & Watson 2009). We adopt the
name proposed by Kantvilas (2009).
Reference material studied for morphological comparisons.
Calvitimela armeniaca (DC.) Hafellner: Austria: Carin-
thia: Koralpe, c. 12 km NE above St. Paul in Lavanttal,
2008, Hafellner 71304 & Hafellner (GZU).
Lecideaaglaea Sommerf.: Austria: Styria:
Koralpe, c. 15·5 km WNW of Deutschlandsberg, 2007,
Hafellner 70358 (GZU); Vorarlberg, Rätikon, 2008,
Hafellner 72944 (GZU).
Mycoblastus dissimulans (Nyl.) Zahlbr.: Chile: Region
de los Lagos: Isla Grande de Chiloé, 2009, Pérez-Ortega
1186 & Etayo (GZU).
Mycoblastus sanguinarius (L.) Norm.: USA: Alaska:
Kenai Peninsula, Russian River, 2008, Spribille 27359 &
Wright (GZU).
Tephromela atra (Huds.) Hafellner: Greece: Epirus:
Tzoumerka, near Kataraktis, Shrine of Profitis Ilias,
2005, Spribille 16260 (GZU).
Results of phylogenetic analysis
We obtained 91 new DNA sequences from
43 individuals, including 30 of EF1-,31of
ITS and 30 of Mcm7. Following exclusion of
positions with missing or ambiguous data,
the sequences consisted of 852, 478 and 564
characters, respectively, for a combined total
of 1894 characters. Tests of nucleotide sub-
stitution models returned TIM3ef+I+G for
EF1-, GTR+I+G for ITS, and HKY+I+G
for Mcm7. We ran individual B/MCMC
analyses for each locus but detected no sig-
nificant conflict between the loci, and thus
combined them. Our partitioned B/MCMC
analysis employed six, six and two substitu-
tion rate categories, respectively, for the three
partitions; four rate categories, predicted in
the TIM3ef model, are not possible to imple-
ment in current software. Overall rate hetero-
geneity was modelled using a gamma density
function. ML and B/MCMC returned con-
gruent phylogenies for the concatenated data
set. Analysis of B/MCMC log likelihood out-
puts in Tracer indicated that convergence
was reached well before our burn-in thresh-
old; plotting of split frequencies between
runs in AWTY also showed stationarity had
been reached. The average standard devia-
tion across runs for splits with a frequency of
at least 0·1 was 0·003493.
We recovered two strongly supported
core groups (Fig. 1), one of which includes
Tephromela,Calvitimela s.lat. and the Myco-
blastus fucatus group (which we call here ‘core
Tephromelataceae’), and another including
Mycoblastus s.str. Both of these clades were
separated from the five taxa of Parmeliaceae at
the base of the tree and Japewia subaurifera,
which was recovered close to Miriquidica
instrata (Lecanoraceae). The combined Myco-
blastus clade consists of a strongly supported
monophyletic M. sanguinarius,M. sangui-
narioides and M. glabrescens.Mycoblastus
alpinus was recovered within a strongly
supported M. affinis clade and the single in-
dividual of M. japonicus, which for the first
time is represented by two markers in a mol-
ecular phylogeny, is recovered as strongly
supported sister to M. affinis.
The ‘core Tephromelataceae’ clade consists
of four distinct, well supported groups; the
relationships to each other are, however, not
supported. These groups correspond to
Tephromela (T. atra,T. cf. pertusarioides and
the undescribed Tephromela sp. Björk
18057), Calvitimela s.str. (C. armeniaca and
C. melaleuca), the Mycoblastus fucatus group,
interpreted here as the new genus Violella
(see below), and “Lecideaaglaea on its
own long branch separate from the rest of
Calvitimela.
Discussion
Hertel & Rambold (1985) provided an over-
view of species groups in what they consid-
ered Tephromela, and later Kantvilas (2009)
proposed a range of potential relatives for
Mycoblastus. Our results shed new light on
potential relationships and invite a reassess-
ment of meaningful morphological charac-
ters (Table 2). In his study of Lecanoralean
452 THE LICHENOLOGIST Vol. 43
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F. 1.Majority rule B/MCMC consensus tree of the concatenated EF1-, ITS and Mcm7 data set. Posterior
probabilities R95% are shown as thick branches; bootstrap support results of maximum likelihood analysis are
shown where R70%. Reference numbers refer to Table 1.
2011 Molecular support for Violella gen. nov. 453
ascus types, Hafellner (1984) implied deep
differences between Tephromela and Myco-
blastus, sufficient for him to recognize them as
belonging to different families, Tephromelat-
aceae and Mycoblastaceae. Indeed, our results
strongly support the distinctness of Myco-
blastus s. str. from a ‘core Tephromelataceae
(Fig. 1). This does not necessarily translate
to different families, however. We did not
structure the taxon sampling of our phylo-
genetic analysis to test family-level relation-
ships within a broader Lecanoralean context,
and cannot predict the outcome of such a
study. Morphologically, however, the dis-
tinction of two families would appear to be
untenable. Mycoblastus shares a similar ascus
apical apparatus with members of Tephro-
melataceae, similar development of a peculiar
thalline cushion below the apothecia (see
below), similar pycnidial development, con-
idiophores, shared ascocarp pigments and
widely overlapping thallus secondary chem-
istry. Morphologically, the only difference we
have found may relate to the basic type of
hymenial matrix formed by the paraphyses.
In ‘core Tephromelataceae’, paraphyses can be
branched and anastomosing, but more often
than not they form long, straight, multicellu-
lar ‘beams’ that separate easily in K and are
substantially thicker than the cross-bridges
(Fig. 2F). In M. sanguinarius, by contrast,
paraphyses almost never form straight seg-
ments even within a single paraphysis cell,
the anastomosing network is intricate, with
bridges often nearly as thick as the main
beams (Fig. 2E), and the entire network
enmeshes the asci; even in K, squashing of
the hymenium results in breakage of the
hymenium rather than separation of asci and
paraphyses. We never found the extreme de-
gree of branching and anastomosing without
straight beams depicted by James (1971: fig.
7) for M. fucatus but instead always found the
paraphysis beams to be much thicker than
the bridges and easily separable in K, and
thus similar to other core Tephromelataceae.
Another enigmatic structure linking Teph-
romelataceae and Mycoblastus is the so-called
thalline exciple, especially evident in Teph-
romela. Hertel & Rambold (1985) and
Kantvilas (2009) have interpreted the ‘thal-
line exciple’ of Tephromela to be homologous,
or at least worthy of providing in the same
table category, to the proper exciple in other
genera. We have, however, found apparently
homologous thalline tissue, in addition to the
presence of a rudimentary proper exciple, in
all genera of Tephromelataceae and Mycoblas-
tus. We hesitate to refer to this as an amphi-
thecium or thalline exciple because it lacks
an algal layer and consists of differentiated,
dense, prosoplectenchymatous tissue not
normally found in the thallus. Instead we will
refer to it as a ‘thalline cushion’. The thalline
cushion occasionally emerges to outer view
as a thin or thick white line in M. sanguinari-
oides (T. Spribille, unpublished data), is
visible in section in the M. fucatus group
(Fig. 3C & 3F), and in Tephromela it forms a
‘thalline rim’. However, it is even present in
Calvitimela, where it forms a dense layer
between the subhymenium and the thallus
medulla.
Our phylogenetic results re-open a dis-
cussion on the generic boundaries in Teph-
romelataceae, begun by Hertel & Rambold
(1985) and continued by Hafellner & Türk
(2001), with the description of Calvitimela.
Tephromela possesses Biatora-type asci with a
sometimes bulbous masse axiale (Fig. 2B).
Hafellner & Türk (2001) separated out
Calvitimela in part based on its Lecanora-type
ascus, though even in describing their new
genus they already anticipated that the
Lecideaaglaea group, with its Biatora-type
asci (Fig. 2C), might not be closely related to
the type species C. armeniaca (Fig. 2A). Even
so, they transferred it to Calvitimela. Our
results confirm that the two are not closely
related and we thus maintain this taxon in the
genus Lecidea in the broad sense until its
generic disposition can be resolved. To this
medley can now be added the M. fucatus
group with its Biatora-type asci (Fig. 2D).
Mycoblastus fucatus has long been recognized
for its unusual hymenium pigmentation, a
character absent from Mycoblastus s. str.
Furthermore, M. fucatus, and in particular
material from Asia that will be described here
as a new taxon, possesses a character not
known from any of the other associated gen-
era studied here, namely the tendency of the
454 THE LICHENOLOGIST Vol. 43
internal ascospore wall to turn brown. This
character was already noted by Leighton
(1879, see also below). These characters also
do not reconcile with those of Tephromela and
Calvitimela, which differ in hymenium pig-
mentation, ascus type and, in part, secondary
chemistry (Table 2). We accordingly propose
recognizing M. fucatus and this new taxon as
constituting the new genus Violella. The
alternative generic solution would require all
F. 2.Selected asci and paraphyses. A–D, ascus variation in the Tephromelataceae, showing asci with immature
ascospores; A, Calvitimela armeniaca (Hafellner 71304); B, Tephromela atra (Spribille 16260); C, “Lecideaaglaea
(Hafellner 72944); D, Violella wangii (holotype).E&F,paraphyses; E, Mycoblastus sanguinarius (Spribille 27359);
F, Violella wangii (holotype). A–D in I
Lugols
after pretreatment with K,E&FinK.Scales: A–F = 10 m.
2011 Molecular support for Violella gen. nov. 455
T 2.Characters of genera and major groups in the Tephromelataceae and Mycoblastus
Violella Calvitimela “Lecidea” aglaea
group
Heppsora*Tephromela Mycoblastus M. dissimulans
Ascospore walls
melanizing
when old
yes, in endospore no no no no no no
Ascospore walls double
apparently single apparently single apparently single apparently single double double
Ascus wall in
I
Lugols
moderately
amyloid,
internal
content visible
weakly amyloid,
internal
content clearly
visible
weakly amyloid,
internal
content clearly
visible
not studied weakly amyloid,
internal
content clearly
visible
strongly amyloid,
internal
content
concealed
except when
iodine
dissipates
strongly amyloid,
internal
content
concealed
except when
iodine
dissipates
Ascus apical
apparatus
Biatora-type Lecanora-type Biatora-to
Bacidia-type
±Lecanora-type ±Biatora-type Biatora-to
Bacidia-type
±Bacidia-type
Ascus ocular
chamber at
median
development
c. 1/4 to 1/5 of
ascus length
c. 1/5 of ascus
length
c. 1/5 of ascus
length
not studied c. 1/5 of ascus
length
c. 1/3–1/4 of
ascus length
c. 1/3–1/4 of
ascus length,
ascus often
becoming
pyriform
Number of
ascospores per
ascus
mostly 2 (1–3) 8 8 8 8 1–2 2
Paraphyses stout with thin
cross-bridges
stout with thin
cross-bridges
stout with thin
cross-bridges
not studied stout with thin
cross-bridges
netted,
cross-bridges
of similar
thickness to
main beams
netted,
cross-bridges
of similar
thickness to
main beams
456 THE LICHENOLOGIST Vol. 43
T 2.Continued
Violella Calvitimela “Lecidea” aglaea
group
Heppsora*Tephromela Mycoblastus M. dissimulans
Hymenial pig-
mentation
Fucatus-violet,
secondary
Cinereorufa-
green
Cinereorufa-
green
Cinereorufa-
green
Atra-red Atra-red Cinereorufa-
green
Cinereorufa-
green
Proper exciple reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
reduced, hyphae
similar to para-
physes
‘Thalline cush-
ion’
rudimentary to
well developed
and forming
ring around
apothecia
rudimentary, thin
layer below
proper exciple
rudimentary, thin
layer below
proper exciple
highly reduced or
appearing ab-
sent
well developed
and forming
‘thalline mar-
gin
§
rudimentary to
well developed
and forming
ring around
apothecia
rudimentary, thin
layer below
proper exciple
Conidia bacilliform bacilliform
ellipsoid to bacil-
liform
bacilliform filiform
bacilliform bacilliform
Thallus morpho-
logy
crustose crustose crustose peltate-
squamulose
crustose to fruti-
cose
**
crustose crustose
Thallus second-
ary chemistry
atranorin, fumar-
protocetraric
acid + fatty
acid
alectorialic acid,
psoromic acid,
stictic acid +
fatty acids
atranorin, usnic
acid + fatty
acids
atranorin, alecto-
ronic and
-collatolic
acid
atranorin, alecto-
ronic acid,
-collatolic
acid, physodic
acid and rarely
fatty acids
atranorin, pla-
naic, fumar-
protocetraric +
fatty acids
perlatolic acid +
fatty acids
*description based on Awasthi & Singh (1977) and Poelt & Grube (1993);
outer wall considered an epispore by Stirton (1879), but not dissolving in C;
Fucatus-violet not seen in Chilean material but reported from Tasmania by Kantvilas (2009);
§
‘exciple’ of Kantvilas (2009, p. 158: table);
illustrated by Hertel & Rambold (1985);
**
in T. siphuloides (Poelt & Grube 1993).
2011 Molecular support for Violella gen. nov. 457
taxa from Tephromela s. str. through Calvi-
timela,Violella and the “Lecideaaglaea
group to be referred to Tephromela s. ampl.
but in our opinion this would defeat the
purpose of genera to circumscribe like
species groups, and make Tephromela, which
even in its narrow definition has more than
20 described species, unnecessarily large
F. 3.Violella species, habit. A–C, V. fucata; A, fertile specimen (Tønsberg 19004); B, sterile specimen on wood
(Spribille 32161); C, section of apothecium, in water (Tønsberg 19004). D–F, V. wangii; D, (holotype); E, sorediate
morph (Spribille 31621); F, section of apothecium, in water (Goffinet 10033). Scales: A,D&E=2mm;B=1mm;
C = 100 m; F = 200 m.
458 THE LICHENOLOGIST Vol. 43
and unwieldy. We expect “Lecideaaglaea
will eventually be placed in its own genus,
possibly together with C. perlata (Haugan &
Timdal) R. Sant., which has Bacidia-type
asci, and some of the various entities cur-
rently treated as chemotypes of “Lecidea
aglaea (Haugan & Timdal 1994). Already
Andreev (2004) has postulated that these
two taxa are closely related, though he re-
tained them in Calvitimela. We leave these
problems unresolved until a better sampling
of Calvitimela s. lat. has been achieved, per-
haps including Southern Hemisphere taxa
(Fryday 2011) and Heppsora, a south Asian
genus (Awasthi & Singh 1977), for which
DNA could not be obtained for the current
study.
Taxonomy
Violella T. Sprib. gen. nov.
MycoBank No: MB 519831
Genus novum ad Tephromelataceas pertinet. Generi
Calvitimela simile sed differt pigmentis hymenialibus
violaceis (haud viridibus), ascis ut in Biatora constructis
(haud ut in Lecanora), ascosporis primum hyalinis,
demum strato interno fuscescenti (haud persistenter
hyalinis) et substanciis chimicis aliis (atranorinum vice
acidi alectorialici).
Typus: Violella fucata (Stirt.) T. Sprib.
Thallus crustose, areolate to rimose; photo-
biont chlorococcoid algae. Thallus chemistry
includes the depside atranorin, a depsidone
and a fatty acid.
Apothecia apparently biatorine, macro-
scopically black, formed on a rudimentary
thalline cushion, this prosoplectenchyma-
tous, tawny with brown streaks; proper exciple
reduced; epihymenium not differentiated as a
distinct layer, epipsamma lacking; hymenium
inspersed with violet granules (‘Fucatus-
violet’) that react N+ raspberry red, K+ pea-
cock green; paraphyses straight or slightly
curved with thinner cross-bridges; asci
Biatora-type; ascospores simple, in the known
species two per ascus (reported as occasion-
ally 1 or 3: Stirton 1879; Leighton 1879;
James 1971), initially with a single wall, even-
tually a differentiated internal wall turning
brown.
Pycnidia apparently rare, colourless or with
light brown pigment around ostiole, sunken
in thallus areoles; conidiophores Parmelia-
type; conidia bacilliform.
Etymology. Diminutive of Viola, a refer-
ence to the characteristic pigment in the
hymenium of both known species (Fig. 3).
Comments. Species of Violella are dis-
tinguished from related genera first and fore-
most by their abundant Fucatus-violet
pigment and the tendency of the inner asco-
spore walls to become brown. The latter
character appears to have been recorded only
once previously in the literature, by Leighton
(1879: 545), who noted the tendency of the
‘protoplasm’ of the ascospores of V. fucata
to turn a ‘nigro-fulvaceous colour’ in K.
However, this colour, apparently produced
in the internal ascospore wall, is present even
without treatment with K and seems to occur
in all mature ascospores in both species of
the genus (Figs. 2D, 3C & F, 4A, B & F). It
appears that many ascospores with a brown
inner wall are collapsing internally and are
possibly abortive (Fig. 4A & B). However,
healthy ascospores with brown internal walls
were also observed (Fig. 4F) and no mature
ascospores were observed in either species
that had not turned brown internally.
Kantvilas (2009) speculated that the per-
latolic acid-containing species of Mycoblas-
tus, the so-called M. dissimulans group, may
constitute a distinct genus. We were not able
to sample that group for our phylogeny, but
we note that Violella differs from M. dissimu-
lans in 1) its paraphyses linked over small
bridges to other paraphyses, as opposed to
the dense network paraphyses similar to
those in Mycoblastus s. str. formed in M.
dissimulans; 2) its ascospores, in which the
internal ascospore wall frequently becomes
brown or olivaceous (remaining hyaline in
M. dissimulans); and 3) its secondary thallus
chemistry. We suspect that M. dissimulans
will ultimately be found to cluster more
closely with Mycoblastus s. str., which also has
brittle, anastomosing paraphysis networks.
We are not aware of any existing generic
2011 Molecular support for Violella gen. nov. 459
F. 4.Microscopic characteristics of Violella apothecia and pycnidia. A, V. fucata ascospore in water (Tønsberg
19004). B–I, V. wangii internal anatomy; B, mature and immature ascospore, in water; C–F, asci at different stages
of development, ending in nearly mature ascospore, using differential interference contrast following pretreatment
with C; G, section of pycnidium in water (holotype); H, conidiophores (in water); I, conidia (in water). B–F from
Goffinet 10033, G–I from holotype. All scales = 10 m.
460 THE LICHENOLOGIST Vol. 43
name in this group that would need to be
considered before describing Violella.
Violella fucata (Stirt.) T. Sprib. comb.
nov.
MycoBank No.: MB 519832
Basionym: Lecidea fucata Stirton, Scottish Naturalist 5:
16 (1879); type: Great Britain, Scotland, Mid Perth,
Tyndrum, on wood, July 1878, Stirton s.n. (BM!—
holotype).— Megalospora fucata (Stirt.) H. Olivier, Bull.
de Géogr. Bot.21: 187 (1911; on p. 207 Olivier incor-
rectly attributes the combination to Leighton 1879).—
Mycoblastus fucatus (Stirt.) Zahlbruckner, Cat. Lich.
Univ.4: 3 (1926).
(Figs 3A–C, 4A)
The first species of the genus to be de-
scribed was Violella fucata (Stirton 1879, as
Lecidea fucata), but this taxon rarely pro-
duces apothecia. A detailed description is
provided by James (1971). Violella fucata is
widely reported from western Europe (e.g.,
Tønsberg 1992; James & Watson 2009), the
Pacific Coast of North America (British
Columbia and Washington: Tønsberg 1993;
Alaska: Spribille et al. 2010) and eastern
North America (Massachusetts: Spribille et al.
2011 and below; Newfoundland: Tønsberg
1993; New York: Schmull et al. 2002; Harris
2004). A distribution map of its obligate
parasite Tremella lichenicola (Diederich 1996:
102) includes many European and some
western North American records.
Selected specimens examined.Norway: Hordaland:
Fjell, Sotra, Tælavåg, W of Midttveit, UTM 32V, KM
766874, map 115 IV, alt. 10 m, [corticolous] on mari-
time Calluna vulgaris, 1993, T. Tønsberg 19004 (BG,
c.fr.); Sogn og Fjordane, Askvoll, W of Fure, S of
Djupevika, hill 48, UTM 32V, KP 8601, Map 1117 IV,
alt. 20–48 m, [corticolous] on Calluna vulgaris, 1989, T.
Tønsberg 11779 (BG, c.fr.).—Great Britain: Scotland:
V.C. 105, West Ross, Dundonnell, Allt aChàirn ra-
vine, on lignum of fallen Betula trunk, alt. 160 m, 1999,
A. M. & B. J. Coppins 18794 (E, c.fr.); V.C. 107, East
Sutherland, N side of Dornoch Firth, Spinningdale,
Ledmore Wood, on lignum of fallen, decorticate Quercus
trunk, alt. 10–30 m, 2001, B. J. & A. M. Coppins 20015
(E, c.fr.); V.C. 97, Westerness, N side Loch Sunart,
Coel na mara, on lignum of fallen trunk of Quercus, alt.
c. 40 m, 2004, B. J. Coppins 21427 & H. L. Andersen (E,
c.fr.).—USA: Massachusetts: Berkshire Co., Mount
Greylock, 42°38·231#N, 73°10·208#W, 1034 m alt.,
lignicolous on snags, 2009, T. Spribille 32161 &V.
Wagner (FH, GZU, NY).
Violella wangii T. Sprib. & Goffinet sp.
nov.
MycoBank No.: MB 519833
AViolella fucata areolis maioribus bullatisque, apotheciis
maioribus et substanciis chimicis aliis (atranorinum et
acidum roccellicum/angardianum vice atranorini et
acidi fumarprotocetrarici) differt. Habitat in montibus
altis Asiae extratropicae.
Typus: China, Yunnan, Lijiang Prefecture, Lijiang
Co. S of Lijiang, Jinhue village, Laojunshan Mountain,
at the border with Jianchuan Co., 26°38·538–37·936#N,
99°43·509–45·992#E, 3510–3900 m, montane forest
dominated by Abies and further up by Rhododendron,
along trail from parking lot to peak, epiphytic, 16 July
2010, B. Goffinet 10029, with L. Wang, S. L. Guo and
S.Y. Huang (KUN—holotypus; CONN, GZU—
isotypi); same locality, same date, B. Goffinet 10033,
with L. Wang, S.L. Guo and S.Y. Huang (TNS, UPS).
(Figs 3D–F, 4B–I)
Thallus crustose, covering patches as much
as 8 cm diam., consisting of discrete areoles
(0·15–)0·2–0·6(–1·5) mm diam., these some-
times confluent forming a rimose thallus;
colour white to ashen grey, surficial thallus
granules corticate, corticate surface finely
pruinose; cortex in esorediate thalli proso-
plectenchymatous, 30–55 m thick; algal
layer c.50m thick, grading into medulla
that is variably thin to as much as 200 m
thick, to 300 m thick under apothecia; sore-
dia when present borne in soralia at tips of
areoles, rarely areoles dissolving into soredia,
internal and external soredia white; soredia
roundish, (40–)64–88(–110) m diam., some-
times forming consoredia; hypothallus not
observed; photobiont chlorococcoid, cells
rounded to irregularly angular, (7–)8·4–
11·1(–17) m diam.
Apothecia always present, rounded, single
or clustered in groups of 2–3 and becoming
confluent, (0·7–)1·3–2·6(–4·1) mm diam.,
base broadly adnate, disc ± flat to weakly
convex, jet black and shiny; margin indis-
tinct, visible from above only in the youngest
apothecia, concolorous with the disc; ‘thalline
cushion’ present, rarely visible from above
and forming a thin white line, in section
prosoplectenchymatous, variable in thickness,
25–230 m thick, typically tawny brown with
streaks of darker brown pigment, clearly dif-
ferentiated from subhymenium above and
2011 Molecular support for Violella gen. nov. 461
medulla below; proper exciple similar in struc-
ture to the hymenium, hyphae radiate, simi-
lar to paraphyses, when well developed in
young apothecia to 170 m wide laterally,
filled with Fucatus-violet granules and often
suffused with Cinereorufa-green pigment;
differentiated hypothecium absent; subhyme-
nium consisting of a thin layer of ascogenous
hyphae, c. 20–50 m tall, filled like the hyme-
nium with Fucatus-violet granules but some-
times also infused with Cinereorufa-green;
hymenium highly variable in thickness even
within one and the same apothecium, (80–)
100–300(–350) m tall, strongly infused
with Fucatus-violet granules and collectively
forming a deep violet impression in section,
but hymenial gel itself hyaline in thin section;
epithecium not differentiated, epipsamma
lacking; paraphyses mostly simple, arranged
vertically and linked to each other in their
lower halves by thin bridges, the main beams
stouter than the bridges and not readily
breaking when squashed in K; paraphysis
tips not or scarcely expanded, 4–6 m wide
including gel sheaths, lumina to 1·5 m wide,
paraphyses completely coated on the outside
by Fucatus-violet granules; asci clavate, 85–
110 × 25–33 m when mature, inner and
outer walls staining blue, tholus strongly
I
Lugol
+ blue, pierced by a broad, conical
non-amyloid structure, thus similar to the
Biatora-type; ascospores 2 per ascus, beginning
colourless and apparently with a single wall,
eventually developing a secondary inner wall,
which quickly turns brown while still in the
ascus; outer wall thick, to 5 m in some cases,
the inner brown wall thin, often collapsing
(spore aborting?), live, healthy ascospores
also with brown endospore, (35–)41·7–54·2
(–65) × (15–)20·8–30·8(–35) m in water.
Pycnidia apparently rare, barely visible ex-
ternally, in small colourless bumps on the
thallus, to 60 m diam.; wall 10–20 m thick,
pigmented a pale rufous brown or hyaline;
conidiophores of Parmelia-type (type VI of
Vobis 1980), with zig-zag cells sprouting
conidia in upper part of each cell; conidia
bacilliform, c.45×1m.
Chemistry. Atranorin and roccellic/
angardianic acid detected by TLC.
Etymology. The species is named in honour
of Dr. Wang Li-Song, for his ongoing efforts
to describe the lichen diversity in western
China.
Habitat and distribution. Found on bark of
Rhododendron sp. in China (Hengduan Shan,
Yunnan) and on wood of Pinus pumila in
the Russian Far East (Bureinskiy Khrebet,
north-western Khabarovskiy Krai). Sub-
stratum was not recorded for the Indian and
Bhutanese material. Collections came from
elevations of 3500 to 4000 m in the southern
area and c. 1000 m in the northernmost col-
lection. In two of the collections it was associ-
ated with Mycoblastus affinis; one of these
specimens is included in our phylogeny.
Comments. Violella wangii is a distinct
species that seems to be widespread, if rarely
collected, in the mountains of high Asia. It
occurs in two intergrading morphs, one
esorediate with granular, corticate areoles
that can become heaped and almost phyllo-
cladioid, and another in which these areoles
remain small and erupt in apical soralia, in
one specimen even disintegrating completely
into soredia in parts of the thallus. The two
morphs exhibit no other consistent differ-
ences however and several specimens are
intermediate. The apparently fluid gradient
between esorediate and sorediate morphs
recalls the case of Mycoblastus sanguinarius
(Tønsberg 1992), in which fully leprose
morphs have not been found to be genetic-
ally distinct from esorediate morphs (T.
Spribille, unpublished data).
Violella wangii differs from the only other
species in the genus, V. fucata, in possessing
much larger thalli (frequently covering
patches 4–8 cm in diam. (rarely >3 cm diam.
in V. fucata), robust areoles 0·2–0·6 mm
across (to 0·3 mm in V. fucata), external
soredia, if present, which remain white
(often turning bluish grey in V. fucata), and
chemistry (roccellic/angardianic instead of
fumarprotocetraric acid). Ascospores aver-
age larger in V. wangii than in V. fucata;
though based on a limited number of apo-
thecia available and paucity of ascospores,
our measurements in V. fucata (38·5 ± 6·7 ×
462 THE LICHENOLOGIST Vol. 43
18·5 ± 3·3 m, n= 24) fall exactly within the
ranges given by Stirton (1879) and James &
Watson (2009). The apothecia of V. wangii
are larger than anything we have measured in
V. fucata but this may not be a reliable
character given that apothecia are rare and
often poorly developed in V. fucata, a primar-
ily sterile species.
Specimens examined (V. wangii). Bhutan: Tongsa Dis-
trict: Black Mountains NW of Nubji, 27°12#N, 90°22#E,
4040 m elev., Rhododendron thicket with Abies densa at
treeline on ridges, on Rhododendron, 2000, G. & S.
Miehe 00-13-07/06 (GZU).—India: Darjeeling: Phalut-
Dentam, 11 v 1960, Togashi et al. s.n. (TNS). Sikkim:
Jongri, elev. 4000 m, 20 v 1960, Togashi et al. s.n.
(TNS).—Russia: Khabarovskiy Krai: Chegdomyn-
Sofiysk road, high pass, watershed between Niman and
Umalta Rivers, c. 7·1 km S of the bridge over the Niman
River, 26 km (air line) SW of Sofiysk, 52°05·866#N,
133°42·433#E, Pinus pumila-Rhododendron aureum wood-
land under Larix gmelinii, on hard wood of P. pumila,
1016 m, 2009, T. Spribille 31621 & L. Yakovchenko (H).
Veˇzda (1993) issued an exsiccate of a
specimen from China under the name Myco-
blastus fucatus, but as Kantvilas (2009) has
pointed out, it is distinct from that taxon. It
was collected near the type locality of V.
wangii but is distinct from that species in its
chemistry (fumarprotocetraric instead of
roccellic/angardianic acid, in this respect re-
calling V. fucata) and thallus morphology
(larger, flatter areoles). It is also distinct from
the chemically concordant V. fucata in,
amongst other characters, developing larger
thalli, large, flattened areoles and large apo-
thecia, and apparently lacking soredia. We
regard this as probably another species dis-
tinct from V. wangii and V. fucata based on
thallus chemistry and morphology. However,
we were unable to obtain fresh material of
this species and hesitate to describe it with-
out getting a better overview of its variability.
We have seen three specimens conforming to
this morphology and chemistry, all from
China.
Specimens examined (unnamed fumarprotocetraric
acid-containing form): China: Prov. Yunnan: montes
Yulong Shan, 30 km ad septentriones ab oppido
Likiang, alt. 4000 m s.m., 25 vii 1990, Soják s.n. (Veˇzda,
Lich. Rar. Exs. 66, GZU). Prov. Sichuan: Hengduan
Shan, Daxue Shan, 57 km S of Kangding, Gongga
Shan, Hailougou glacier and forest park, 29°34#35$N,
101°59#56$E, 2940–3130 m, on Betula utilis, 2000, W.
Obermayer 08686 (GZU); ibid., northern Qionglai Shan,
Barkam, 31°57#N, 102°39#E, 4050 m, 1995, G. & S.
Miehe 94-502-2/14 & U. Wündisch (GZU).
Status of Mycoblastus indicus
A candidate name for our new taxon that
required examination was Mycoblastus indicus
(Awasthi & Agarwal 1968, as “indicum”),
described from Darjeeling district, India,
near to where Violella wangii has also been
collected. We did not receive a response to
repeated requests for type material from
Lucknow (LWU), but we did find a speci-
men of M. indicus at UPS, collected and
identified by Awasthi and Agarwal only days
before they collected the type specimen. The
specimen fits the description provided by
Awasthi & Agarwal (1968) and in habit
resembles the photograph of the holotype
provided by Singh & Sinha (2010), though
the latter appears to have more mature apo-
thecia. Mycoblastus indicus is clearly not a
member of Mycoblastus or Tephromelataceae.
Instead, detailed study of the UPS specimen
(Fig. 5) revealed brown epihymenial and
hypothecial pigments, a strongly developed
proper exciple, mostly simple, loose para-
physes, and asci with a dark apical amyloid
cylinder. We obtained an unknown phenolic
substance from the thallus, with R
f
values
similar to confluentic acid in TLC. We
concur with Awasthi & Agarwals original
statement that the species appears similar to
the group of tropical species around Lecidea
granifera Vain., for which the genus
Malmidea has been erected by Kalb et al.
(2011). We accordingly combine the species
into that genus, where it appears similar to
M. coralliformis Kalb. We note that it has
larger ascospores than any of the members of
the genus discussed by Kalb et al. (2011).
Malmidea indica (Awasthi & Agarwal)
Hafellner & T. Sprib. comb. nov.
Basionym: Mycoblastus indicum Awasthi & Agarwal,
Current Science 37: 84 (1968); type: India, Darjeeling
District, Pashkok Road, 19 March 1967, D. D. Awasthi
&M.R.Agarwal67.78 (LWU—holotype, n.v.).
Specimen examined (Malmidea indica). India: Darjeeling
District: Rangit river valley, near Lebong, alt. 1520 m, on
bark of trees, 1967, D. D. Awasthi & M. R. Agarwal
67.224 (UPS).
2011 Molecular support for Violella gen. nov. 463
F. 5.Malmidea indica (Awasthi & Agarwal 67.224, UPS). A, habit; B, section of apothecium; C, section through
hymenium showing paraphyses; D, ascus, squash preparation stained in I
Lugols
after pretreatment with K; E,
ascospore, in I
Lugols
. Scales:A=2mm;B=200m; C–E = 10 m.
464 THE LICHENOLOGIST Vol. 43
We thank J. Hafellner (Graz) for helpful ascus and
ascospore observations and for translating the diagnoses
into Latin. S. Pérez-Ortega, Y. Ohmura, Göran Thor
and Irina Urbanavichene provided fresh material for
DNA analysis and S. Clayden and L. Yakovchenko
facilitated sampling in Atlantic Canada and the Russian
Far East, respectively. The director of the Bureinskiy
Nature Reserve is thanked for permission to collect in
that reserve in 2009. Field work by BG was funded
by grant DEB-0919284 from the American National
Science Foundation and made possible by Drs Wang
Li-Song (Kunming Institute of Botany) and Guo
Shuiliang (Shanghai Normal University), for which
BG is grateful. We thank the curators of the herbaria
BG, BM, E, TNS and UPS for loans of specimens.
This project was supported by the Austrian Science
Foundation (project P21052-B16, Circumboreal
Lichen Diversification).
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Accepted for publication 24 May 2011
466 THE LICHENOLOGIST Vol. 43
... This is a small genus of crustose lichens that was recently introduced by Spribille et al. (2011) to accommodate species that were similar to Mycoblastus Norman but differed in aspects of the secondary chemistry and fruiting bodies. Only a single species is known from Delmarva. ...
... America with a range that extends south along the Appalachian Mountains (Brodo et al. 2013, Spribille et al. 2011unpublished herbarium records at NY). It belongs to the group of species that have northern and Appalachian distributions but which also occur in the Coastal Plain from Delmarva northward. ...
Article
***AVAILABLE ONLINE VIA JSTOR: https://www.jstor.org/stable/e26976761 Delmarva (also known as the Delmarva Peninsula), the name itself at once invites questions and invokes history. Just as the name is an aggregation (of Delaware, Maryland and Virginia), so is the land an aggregation of floras and faunas, of political units and cultures, of times and places. Situated between the Chesapeake Bay, America’s beleaguered estuarine jewel, the Delaware Bay, an area of critical global importance to migrating shorebirds, and the inexorably rising waters of the Atlantic Ocean, Delmarva is a vast peninsula defined by coexistence and contrasts, a meeting place of north and south, of urban and rural, of past and future. Like Delmarva, lichens are also defined by coexistence and contrast. They are among the most remarkable products of evolution: fungi that have entered into obligate symbioses with algae and cyanobacteria. Together the two form complex and striking structures unlike any other in nature. While this specialized lifestyle has allowed lichens to survive, and even thrive, in nearly every ecosystem on land, it also means these fungi cannot survive on their own. In fact, lichens are extremely sensitive to habitat loss, degradation and disturbance. Lichens have long been an integral component of the Delmarva landscape. They serve important functions in the environment, supplying food and shelter for animals, and providing ecosystem services that ultimately benefit human welfare and society. As we show in this book, while the link between lichens and Delmarva in the past is clear, how exactly they fit into the narrative for the future of Delmarva is an open question. We wrote this book with the goal of providing a resource that would open the world of these neglected species to anyone who wanted to learn about them. Our work draws on more than four years of detailed study in the region, trudging through swamps and reveling in beaches. There are many resources to learn about, identify and appreciate other groups of Delmarva biodiversity, such as birds and wildflowers. Yet to date there has never been such a resource for lichens. In this volume we treat the 299 species of lichens that have been documented from Delmarva to date, all of which are presumed to be native. This includes not just the macrolichens, some of which have been treated elsewhere, but also the microlichens that have long been neglected in North American floras. For each species, we provide a description, a discussion of its distribution and ecology, a proposed conservation rank, and a comparison with similar species. We have also assembled a discussion of Delmarva natural history, checklists that are arranged alphabetically and evolutionarily, and a complete set of identification keys. A distribution map for each species is also included that shows the range of the species in the context of the broader Washington D.C. metro region, and contrasts the historical occurrences with those from modern times. Nearly all of the species are illustrated with color photographs. Our hope is that publication of this work will prove to be a turning point for the lichens in the Mid-Atlantic Region, serving as inspiration to explore and appreciate a long-neglected component of biodiversity. While the book focuses on Delmarva, it covers the majority of species that occur along the Atlantic Coast from Norfolk, Virginia, to Cape Cod, Massachusetts. Thus the area covered by this book includes the metropolitan areas of Washington, D.C., Philadelphia and New York.
... The genus Malmidea was established together with the family Malmideaceae by Kalb et al. (2011) to accommodate the Lecidea piperis-and Lecanora granifera groups into their corresponding phylogenetic position. At present, the family Malmideaceae includes five genera, and the genus Malmidea comprises actually 55 species (Kalb, 2011;Spribille et al., 2011;Kalb et al., 2012;Schumm and Aptroot, 2012;C aceres et al., 2012Aptroot, 2013, 2014;Singh and Pinokiyo, 2014;Breuss and L€ ucking, 2015;Weerakoon et al., 2016;C aceres et al., 2017). Breuss and L€ ucking (2015) keyed out all known species of Malmidea in the world. ...
... The genus Malmidea is characterized by a crustose thallus composed of goniocysts, biatorine excipulum often encrusted with hydrophobic granules, asci without tubular structure and simple ascospores . The species are mostly found in tropical rainforests and mainly corticolous while two species viz., M. nagalandica (G.P. Sinha & Kr.P. Singh) G.P. Sinha et al. and M. trailiana (M€ ull (Spribille et al., 2011;Singh and Pinokiyo, 2014;Sinha et al., 2015;Gupta and Sinha, 2016;Sinha and Gupta, 2017). During the examinations of the specimens earlier identified as Lecidea granifera deposited in the herbarium LWG and fresh collections from Goa, the authors came across with some interesting specimens which resulted in six new records for India. ...
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... haematites, C. xerica, and C. variabilis groups) or even genera (Pyrenodesmia and Kuettlingeria). In the context of the current taxonomy of the lichenized fungi (Crespo et al., 2010;Nordin et al., 2010;Spribille et al., 2011;Ekman & Svensson, 2014;Buaruang et al., 2015), and particularly of the family Teloschistaceae (Fedorenko et al., 2012;Arup et al., 2013;Søchting et al., 2014;Kondratyuk et al., 2017b, etc.), the three groups within Pyrenodesmia sensu lato should be treated at the genus rank, as they show a considerable phylogenetical and morphological differentiation. ...
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... Mycoblastus caesius similarly has a conspicuous bluish-black prothallus; however, it differs chemically in the production of perlatolic acid instead of atranorin together with 2'-O-methylperlatolic acid (Tønsberg 1992). When fertile, M. caesius can easily be recognized by its black apothecia that bear huge, hyaline ascospores (Spribille et al. 2011). Nonetheless, fertile individuals of M. caesius are rare in the southern Appalachians and thus chemistry is the best way to distinguish between the two species. ...
Article
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We present an exhaustive analysis of the ITS barcoding marker in the genus Usnea s.lat., separated into Dolichousnea, Eumitria, and Usnea including the subgenus Neuropogon, analyzing 1,751 accessions. We found only a few low-quality accessions, whereas information on voucher specimens and accuracy and precision of identifications was of subpar quality for many accessions. We provide an updated voucher table, alignment and phylogenetic tree to facilitate DNA barcoding of Usnea, either locally or through curated databases such as UNITE. Taxonomic and geographic coverage was moderate: while Dolichousnea and subgenus Neuropogon were well-represented among ITS data, sampling for Eumitria and Usnea s.str. was sparse and biased towards certain lineages and geographic regions, such as Antarctica, Europe, and South America. North America, Africa, Asia and Oceania were undersampled. A peculiar situation arose with New Zealand, represented by a large amount of ITS accessions from across both major islands, but most of them left unidentified. The species pair Usnea antarctica vs. U. aurantiacoatra was the most sampled clade, including numerous ITS accessions from taxonomic and ecological studies. However, published analyses of highly resolved microsatellite and RADseq markers showed that ITS was not able to properly resolve the two species present in this complex. While lack of resolution appears to be an issue with ITS in recently evolving species complexes, we did not find evidence for gene duplication (paralogs) or hybridization for this marker. Comparison with other markers demonstrated that particularly IGS and RPB1 are useful to complement ITS-based phylogenies. Both IGS and RPB1 provided better backbone resolution and support than ITS; while IGS also showed better resolution and support at species level, RPB1 was less resolved and delineated for larger species complexes. The nuLSU was of limited use, providing neither resolution nor backbone support. The other three commonly employed protein-coding markers, TUB2, RPB2, and MCM7, showed variable evidence of possible gene duplication and paralog formation, particularly in the MCM7, and these markers should be used with care, especially in multimarker coalescence approaches. A substantial challenge was provided by difficult morphospecies that did not form coherent clades with ITS or other markers, suggesting various levels of cryptic speciation, the most notorious example being the U. cornuta complex. In these cases, the available data suggest that multimarker approaches using ITS, IGS and RPB1 help to assess distinct lineages. Overall, ITS was found to be a good first approximation to assess species delimitation and recognition in Usnea s.lat., as long as the data are carefully analyzed, and reference sequences are critically assessed and not taken at face value. In difficult groups, we recommend IGS as a secondary barcode marker, with the option to employ more resource-intensive approaches, such as RADseq, in species complexes involving so-called species pairs or other cases of disparate morphology not reflected in the ITS or IGS. Attempts should be made to close taxonomic and geographic gaps especially for the latter two markers, in particular in Eumitria and Usnea s.str. and in the highly diverse areas of North America and Central America, Africa, Asia, and Oceania.
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The family Parmeliaceae (Lecanorales, Ascomycota) is possibly the largest, best known and most thoroughly studied lichen family within its order. Despite this fact the relationship between Parmeliaceae and other groups in Lecanorales is still poorly known. The aim of the present study is to contribute to finding the sister group of Parmeliaceae as an aid in future studies on the phylogeny and character evolution of the group. We do this by sampling all potential relatives to the Parmeliaceae that we have identified, i.e. Gypsoplaca, Japewia, Mycoblastus, Protoparmelia, and Tephromela, a good representation of the major groups within the Parmeliaceae s. lat. and a good representation of other taxa in the core Lecanorales. We use molecular data from two genes, the large subunit of the nuclear ribosomal RNA gene (nrLSU) and the small subunit of the mitochondrial ribosomal RNA gene (mrSSU), and a Bayesian analysis of the combined data. The results show that the closest relatives to Parmeliaceae are the two genera Protoparmelia and Gypsoplaca, which are crustose lichens. Parmeliaceae in our sense is a well supported group, including also the family segregates Alectoriaceae, Hypogymniaceae, Usneaceae and Anziaceae.
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128 species in 45 genera of sorediate and isidiate, crustose, corticolous lichens are recorded from Norway. Accounts of their morphology, chemistry, and substratum preferences are presented, and a discussion of their distribution in Norway is supported by maps for a number of taxa. With few exceptions, the taxa can be distinguished on thallus characters alone. Several taxa, especially those with brown or blue-pigmented soralia, have soredia with a distinct cortex. New species are: Buellia arborea Coppins & Tønsb. (from Norway and Scotland), Fuscidea arboricola Coppins & Tønsb. (from Norway, Sweden, and Scotland), F. pusilla Tønsb. (from Norway, Sweden, and Scotland), Lecanora flavoleprosa Tønsb. (from Norway and Austria), L. flavopunctata Tønsb. (from Norway and Sweden), L. norvegica Tønsb. (from Norway), Lecidea gyrophorica Tønsb. (syn. L. epizanthoidiza auct., non Nyl.), L. praetermissa Tønsb. (from Norway and Sweden), L. subcinnabarina Tønsb. (from Norway), L. vacciniicola Tønsb. (from Norway, Sweden, and Spain), Lecidella subviridis Tønsb. (from Norway and Sweden), Lepraria elobata Tønsb. (from Norway), L. jackii Tønsb. (from Norway), L. obtusatica Tønsb. (from Norway), L. umbricola Tønsb. (from Norway, England, and Scotland), Micarea coppinsii Tønsb. (from Norway and Scotland), Rinodinaflavosoralifera Tønsb. (from Norway), R. disjuncta Sheard & Tønsb. (from Norway and the pacific coast of U.S.A. and Canada), and Schaereria corticola Muhr & Tønsb. (from Norway, Sweden and Scotland). Ochrolechia androgyna s. lat. is shown to comprise at least four distinct species. New combinations are: Cliostomum leprosum (Räsänen) Holien & Tønsb., Lepraria rigidula (B. de Lesd.) Tønsb., Mycoblastus caesius (Coppins & P. James) Tønsb., Placynthiella dasaea (Stirton) Tønsb., and Ropalospora viridis (Tønsb.) Tønsb. Lecidea turgidula var. pulveracea Fr. is raised to specific level with the new name Lecidea leprarioides Tønsb. Mycoblastus sterilis Coppins & P. James is reduced to synonymy with M. fucatus Stirton. Pertusaria borealis is new to Europe. Halecania viridescens, Lecanora farinaria, Lepraria caesioalba Laundon ined., L. eburnea Laundon ined., Megalospora tuberculosa, Opegrapha multipuncta, and Scoliciosporum gallurae are new to Scandinavia. Mycoblastus caesius, Lecidella elaeochroma “f. soralifera”, L. flavosorediata, Micarea granulans (saxicolous, not treated), Opegrapha sorediifera, and Rinodina degeliana are new to Norway. In some cases, Poelt’s species pair concept can be applied to this group of lichens. Additional secondary substances, not occurring in the primary species, sometimes occur in the soralia of the secondary species. In this case, presence of the additional substance cannot be regarded as an independent taxonomic character, and the species pair concept is still useful. However, morphologically indistinguishable specimens with different chemistry may represent different secondary species. The term consoredia is introduced to denote diaspores composed of aggregated soredia.
Article
The Lecanoromycetes includes most of the lichen-forming fungal species (>13 500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson’s classification of the Lecanoromycetes (Outline of Ascomycota—2006 Myconet 12:1–82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.