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StudieS in Mycology 64: 135–144. 2009.
Mutualism is one of the three main modes of nutrition within
Ascomycota, besides saprotrophism and parasitism. A large
number of mutualistic ascomycetes form symbiotic relationships
with algae and/or cyanobacteria, so-called lichens. Of the
64 000 species currently accepted in Ascomycota (Kirk et al. 2008),
about almost 30 % (17 600) are lichen-forming fungi (Feuerer &
Hawksworth 2007, Kirk et al. 2008). Lichenised fungi differ from all
other fungi in the formation of complex, persistent vegetative thalli,
which makes them a prime subject for evolutionary studies.
It was long believed that lichens evolved several times
independently within Ascomycota (and Basidiomycota), an idea
supported by the first molecular study testing this hypothesis
(Gargas et al. 1995). Lutzoni et al. (2001, 2004) were unable to
conclusively determine whether there were multiple gains of
lichenisation or whether an initial lichenisation event occurred deep
within Ascomycota, however, Lutzoni et al. (2001) found some
Eurotiomycetes to be secondarily de-lichenised. This is particularly
intriguing as Eurotiomycetes includes economically important fungi
in the genera Aspergillus and Penicillium that feature a complex
secondary chemistry similar to that found in lichens produced by
homologous polyketide synthase genes (Grube & Blaha 2003,
Kroken et al. 2003, Schmitt et al. 2005, Schmitt & Lumbsch 2009).
Since then, the phylogeny and classification of Ascomycota
has further advanced (Lindemuth et al. 2001, Lumbsch et al. 2001,
2002a, b, 2004, Grube et al. 2004, Lücking et al. 2004, Lutzoni
et al. 2004, Persoh et al. 2004, Wedin et al. 2005, del Prado et
al. 2006, Miadlikoswka et al. 2006, Schmitt et al. 2006, Spatafora
et al. 2006, Hibbett et al. 2007, Hofstetter et al. 2007, Lumbsch
& Huhndorf 2007a, Schoch et al. 2006, 2009a–c). Our current
understanding suggests that there were several lichenisation
events but also some major delichenisation events during the
evolution of Ascomycota (Gargas et al. 1995, Lutzoni et al. 2001,
Liu & Hall 2004, Gueidan et al. 2008, Schoch et al. 2009a). The
largest clade of lichenised fungi, Lecanoromycetes, with 14 000
accepted species, appears to be the result of a single lichenisation
event with at least one major delichenisation event in Ostropales
and several delichenisation events throughout the class (Lumbsch
et al. 2004, Persoh et al. 2004, Wedin et al. 2005, Miadlikoswka
et al. 2006, Hofstetter et al. 2007, Schoch et al. 2009a, Baloch
et al. in prep.). A similar pattern is suggested within the second
largest lichenised clade, Arthoniomycetes, with about 1 500
species (Tehler 1995, Myllys et al. 1998, Sundin 2000, Tehler &
Irestedt 2007, Ertz et al. 2008). This class was recently shown to
include the mazaediate genus Tylophoron (Lumbsch et al. 2009a),
previously considered to be related to pyrenocarpous lichens
(Aptroot et al. 2008). Arthoniomycetes is composed primarily of
lichenised fungi producing apothecia or apothecioid ascomata with
partially ascolocular development and bitunicate asci (Henssen
& Jahns 1974, Eriksson & Winka 1997). The base of this clade
was reconstructed as lichenised (Schoch et al. 2009a) and it is
presumed that non-lichenised and lichenicolous species within the
class represent reversions to the unlichenised state. One family
that has not yet been confirmed within Arthoniomycetes using
molecular data is Chrysothrichaceae, a small family of two genera
(Byssocaulon, Chrysothrix) and little over 20 species (Kirk et al.
2008). The third primarily lichenised class is Lichinomycetes (350
M.P. Nelsen1, 2, R. Lücking2, M. Grube3, J.S. Mbatchou2, 4, L. Muggia3, E. Rivas Plata2, 5 and H.T. Lumbsch2
1Committee on Evolutionary Biology, University of Chicago, 1025 E. 57th Street, Chicago, Illinois 60637, U.S.A.; 2Department of Botany, The Field Museum, 1400 South Lake
Shore Drive, Chicago, Illinois 60605-2496, U.S.A.; 3Institute of Botany, Karl-Franzens-University of Graz, A-8010 Graz, Austria; 4Department of Biological Sciences, DePaul
University, 1 E. Jackson Street, Chicago, Illinois 60604, U.S.A.; 5Department of Biological Sciences, University of Illinois-Chicago, 845 West Taylor Street (MC 066), Chicago,
Illinois 60607, U.S.A.
*Correspondence: Matthew P. Nelsen, email@example.com
Abstract: We present a revised phylogeny of lichenised Dothideomyceta (Arthoniomycetes and Dothideomycetes) based on a combined data set of nuclear large subunit
(nuLSU) and mitochondrial small subunit (mtSSU) rDNA data. Dothideomyceta is supported as monophyletic with monophyletic classes Arthoniomycetes and Dothideomycetes;
the latter, however, lacking support in this study. The phylogeny of lichenised Arthoniomycetes supports the current division into three families: Chrysothrichaceae (Chrysothrix),
Arthoniaceae (Arthonia s. l., Cryptothecia, Herpothallon), and Roccellaceae (Chiodecton, Combea, Dendrographa, Dichosporidium, Enterographa, Erythrodecton, Lecanactis,
Opegrapha, Roccella, Roccellographa, Schismatomma, Simonyella). The widespread and common Arthonia caesia is strongly supported as a (non-pigmented) member of
Chrysothrix. Monoblastiaceae, Strigulaceae, and Trypetheliaceae are recovered as unrelated, monophyletic clades within Dothideomycetes. Also, the genera Arthopyrenia
(Arthopyreniaceae) and Cystocoleus and Racodium (Capnodiales) are confirmed as Dothideomycetes but unrelated to each other. Mycomicrothelia is shown to be unrelated to
Arthopyrenia s.str., but is supported as a monophyletic clade sister to Trypetheliaceae, which is supported by hamathecium characters. The generic concept in several groups
is in need of revision, as indicated by non-monophyly of genera, such as Arthonia, Astrothelium, Cryptothecia, Cryptothelium, Enterographa, Opegrapha, and Trypethelium in
Key?words: Arthoniomycetes, Ascolocularous fungi, bitunicate fungi, Dothideomycetes, lichens, phylogeny, ribosomal DNA.
available online at www.studiesinmycology.org
nelSen et al.
The remaining lichenised fungi are primarily restricted
Chaetothyriomycetidae). Gueidan et al. (2008) demonstrated that
lichenisation may have evolved at least twice within Eurotiomycetes
(once at base of Verrucariales and once at base of Pyrenulales),
though, this is uncertain as the ancestral state of the common
ancestor to Pyrenulales, Verrucariales and Chaetothyriales, is
not unambiguously resolved (Gueidan et al. 2008, Schoch et al.
2009a). Within both Verrucariales and Pyrenulales, there appears
to be at least one loss of lichenisation each. Dothideomycetes and
Arthoniomycetes together form the rankless clade Dothideomyceta,
a name introduced by Schoch et al. (2009a, b). The ancestral state
of Dothideomyceta and Dothideomycetes nodes are not resolved
with confidence (Gueidan et al. 2008, Schoch et al. 2009a, b). In
this paper we do not aim to resolve this issue but rather attempt to
clarify, confirm or reject the placement of lichenised lineages within
Dothideomyceta, specifically Dothideomycetes.
The following families have been confirmed or are believed
to belong in either Chaeothyriomycetidae or Dothideomycetes:
Verrucariaceae (930 species), Pyrenulaceae (280 species),
Celotheliaceae (eight species),
(three species), and Pyrenothrichaceae (three species) in
Chaetothyriomycetidae (Herrera-Campos et al. 2005, del Prado
et al. 2006, Lücking 2008), and Trypetheliaceae (200 species),
Monoblastiaceae (130 species), Strigulaceae (120 species), and
Arthopyreniaceae (120 species) in Dothideomycetes (Lutzoni et al.
2004, del Prado et al. 2006, Lumbsch & Huhndorf 2007b). Most
of these families have traditionally been placed within Pyrenulales
(Poelt 1973, Henssen & Jahns 1974, Hafellner 1986, Kirk et al.
2001, Eriksson et al. 2004, Cannon & Kirk 2007), and much of the
confusion regarding previous classifications of these pyrenocarpous
lichens stems from the fact that Pyrenulales were at some point
considered synonymous with the ascolocular Melanommatales
(currently regarded synonymous with Pleosporales; Barr
1980, Harris 1984, 1990, 1991, 1995), whereas other workers
considered Pyrenulales to be ascohymenial (Henssen & Jahns
1974). The fact that Trypetheliaceae have no close relative within
Dothideomycetes was reflected in the establishment of a separate
order, Trypetheliales (Aptroot et al. 2008).
In addition to the aforementioned families, there are several
genera of uncertain position, such as Cystocoleus and Racodium,
both of which belong in Capnodiales/Dothideomycetes (Muggia
et al. 2007), as well as Julella, Mycoporum, Collemopsidium
(Pyrenocollema), and others, of unconfirmed affinities (Harris
1995). Yet other lineages, such as the recently discovered
Eremithallus (Lücking et al. 2008) or the genera Thelocarpon and
Vezdaea (Reeb et al. 2004, Lumbsch et al. 2009b) appear to fall
outside the currently accepted classes known to contain lichen-
forming fungi. The current phylogeny of Chaetothyriomycetidae
suggests that the two large lichen-forming families in this subclass
may have emerged from distinct lichenisation events, however,
this could not be resolved with confidence (see node 18 in fig. 1
and table 1 of Gueidan et al. 2008, Schoch et al. 2009a). It thus
appears that Dothideomycetes, the largest class of Ascomycota
with an estimated number of 19 000 species (Kirk et al. 2008),
a class that has largely been neglected when assessing the
phylogeny of lichenised fungi, might be the only class within
Ascomycota containing several lineages that evolved through
independent lichenisation. In addition to Trypetheliaceae, at least
two other families, which exhibit substantial radiation accompanied
with morphological variation at the generic and species level
(Monoblastiaceae and Strigulaceae) have been suggested to
to Eurotiomycetes (subclass
belong to Dothideomycetes. The only sequenced species of
Strigula has been suggested to belong to Eurotiomycetes (Schmitt
et al. 2005); however, re-examination of the specimen used in
this study showed that it belonged in Verrucariaceae. Therefore
the phylogenetic position of Strigulaceae remains unresolved.
In addition, Anisomeridium polypori (Monoblastiaceae) was
suggested to belong to Dothideomycetes (James et al. 2006).
In this paper, we are using nuclear large subunit (nuLSU)
and mitochondrial small subunit (mtSSU) rDNA data, to construct
a phylogeny of lichenised fungi with bitunicate asci, focusing
on Dothideomyceta. We also present novel data that require
adjustments in the systematic classification of taxa within both
classes. A further objective was to begin to examine generic
concepts within the family Trypetheliaceae, which is comprised of
11 genera (Lumbsch & Huhndorf 2007b) and approximately 200
species (Harris 1984, Aptroot 1991b, del Prado et al. 2006).
Representatives of lichenised Dothideomyceta taxa were obtained
through recent field work in the U.S.A., Central and South
America, Europe, India, Thailand, and Fiji. Newly generated
sequences were supplemented with other lichenised and non-
lichenised Dothideomyceta from GenBank plus additional taxa in
Pezizomycetes, Leotiomycetes, Sordariomycetes, Eurotiomycetes,
and Lecanoromycetes, chiefly from a previous alignment published
by Schoch et al. (2009a). In total, we analysed 162 operational
taxonomic units (OTUs) representing 152 species and 111 genera.
All OTUs included in the analyses, along with GenBank accession
numbers and collection information for newly sequenced samples,
are listed in Table 1 - see online Supplementary Information.
The Sigma REDExtract-N-Amp Plant PCR Kit (St. Louis, Missouri,
U.S.A.) was used to isolate DNA, following the manufacturer’s
instructions, except only 10 µL of extraction buffer and 10 µL
dilution buffer were used, following Avis et al. (2003). Dilutions of
these extractions (rather than the stock DNA solution) were found
to work best for PCR (C. Andrew, pers. comm. 2009), and a 20×
DNA dilution was then used in subsequent PCR reactions.
Samples were PCR amplified and/or sequenced using the
mrSSU1, mrSSU2, mrSSU2r and mrSSU3r primers (Zoller et al.
1999) for the mitochondrial small subunit (mtSSU) and the AL2R
(Mangold et al. 2008), LR3R, LR3, LR5, LR6, LR7 (Vilgalys &
Hester 1990) primers for the nuclear ribosomal large subunit rDNA
(nuLSU). The 10 µL PCR reactions consisted of 5 µM of each
PCR primer, 3 mM of each dNTP, 2 µL of 10 mg/mL 100x BSA
(New England BioLabs, Ipswich, Massachusetts, U.S.A.), 1.5 µL
10× PCR buffer (Roche Applied Science, Indianapolis, Indiana,
U.S.A.), 0.5 µL Taq, approximately 2 µL diluted DNA, and 2 µL
water. The PCR cycling conditions were as follows: 95 °C for 5 min,
followed by 35 cycles of 95 °C for 1 min, a locus-specific annealing
temperature for 1 min, and 72 °C for 1 min, followed by a single
72 °C final extension for 7 min. An annealing temperature of 53 °C
was used for mtSSU, while 57 °C was used for nrLSU.
Samples were visualised on a 1 % ethidium bromide-stained
agarose gel under UV light and bands were gel extracted, heated
at 70 °C for 5 min, cooled to 45 °C for 10 min, treated with 1 µL
unravelling the phylogenetic relationShipS of licheniSed fungi in dothideoMyceta
GELase (Epicentre Biotechnologies, Madison, WI, U.S.A.) and
incubated at 45 °C for at least 24 h. The 10 µL cycle sequencing
reactions consisted of 1–1.5 µL of Big Dye v. 3.1 (Perkin-Elmer
Applied Biosystems, Foster City, California, U.S.A.), 2.5–3 µL of
Big Dye buffer, 6 µM primer, 0.75–2 µL Gelased PCR product and
water. The cycle sequencing conditions were as follows: 96 °C for
1 min, followed by 25 cycles of 96 °C for 10 s, 50 °C for 5 s and
60 °C for 4 min. Samples were precipitated and sequenced in an
Applied Biosystems 3730 DNA Analyser (Foster City, California,
U.S.A.), and sequences assembled in Sequencher 4.9 (Gene
Codes Corporation, Ann Arbor, Michigan, U.S.A.).
The alignment of Schoch et al. (2009a) was used as a starting
point, from which a large number of sequences were removed.
Newly generated sequences were added and manually aligned
(nuLSU), or were separately aligned, added to the Schoch et al.
(2009a) alignment, and manually adjusted (mtSSU). In addition to a
representative set of dothideomycetous fungi, members of several
Ascomycota classes were retained and Pezizomycetes taxa were
used as the outgroup. The entire set of sequences generated in the
present study plus those from GenBank were aligned in Se-Al v.
2.0a11 (Rambaut 1996) and BioEdit 7.0.9 (Hall 1999). An iterative
procedure was used for the nuLSU in which ambiguous regions
were aligned with Muscle 3.6 (Edgar 2004) through Mesquite 2.71
(Maddison & Maddison 2009); the alignment was again manually
refined and other portions realigned with Muscle. After a final
manual refinement, ambiguous regions and introns were removed
and the alignment was deposited in TreeBase.
Alignments for each gene were concatenated in Mesquite 2.71
(Maddison & Maddison 2009) and analysed under the maximum
likelihood (ML) optimality criterion in RAxML 7.0.4 (Stamatakis
2006). The data set was partitioned by locus and the GTRMIXI
model with twenty-five rate parameter categories (default) was
used for each partition. In addition, support was estimated by
performing 1000 bootstrap replicates, and clades with bootstrap
support of 70 % or greater were considered strongly supported.
Additionally, the data sets were analyzed in GARLI 0.96 (Zwickl
2006) using the GTR-gamma-invariant model which is similar to the
model used in RAxML.
The final alignment consisted of 1 915 unambiguously aligned
characters (1 199: nuLSU; 716: mtSSU). Both ML analyses recovered
the major class-level ingroup nodes (Fig. 1) corresponding to other
recent studies (Leotiomycetes, Sordariomycetes, Eurotiomycetes,
Arthoniomycetes and Dothideomycetes form a strongly supported
sister-group relationship, corresponding to Dothideomyceta.
Individual gene phylogenies suggested some incongruence
between loci (unpubl. data), however, the topology in the combined
analysis is in agreement with previously reported phylogenies and
we did not exclude taxa.
The phylogeny of Arthoniomycetes (Arthoniales) largely
confirmed previous analyses, with Chrysothrichaceae forming an
additional family within this clade (Fig. 1). Arthoniaceae s. l. and
Roccellaceae s. l. are both monophyletic and well separated.
However, several smaller lineages that eventually could be
reinstated at the family level show strong support: Arthoniaceae
s. str., Cryptotheciaceae (Cryptothecia-Herpothallon), the
Tylophoron clade, Roccellaceae s. str., Opegraphaceae s. str.,
and possibly Chiodectonaceae (as Chiodecton sphaerale is
closely related to Erythrodecton and Dichosporidium whereas the
sequenced C. natalense is apparently not a Chiodecton s. str.).
Surprisingly, Arthonia caesia clustered with Chrysothrichaceae
and not Arthoniaceae. Herpothallon rubrocinctum is nested within
Cryptothecia s. l.
Six distinct, lichenised lineages were confirmed as belonging
to Dothideomycetes (Fig. 1): the order Trypetheliales, the families
Arthopyreniaceae, Monoblastiaceae, and Strigulaceae, and the
genera Cystocoleus and Racodium. The latter two (Cystocoleus
and Racodium) are members of the order Capnodiales, whereas
Arthopyreniaceae, represented by the species Arthopyrenia
salicis, was confirmed as clustering within Pleosporales. However,
Arthopyreniaceae as currently defined, including the genera Julella
(not sequenced) and Mycomicrothelia, is not monophyletic, as
the sequenced species of Mycomicrothelia appeared outside
Pleosporales and form a sister-group to Trypetheliaceae.
Strigulaceae is represented by five samples of the three genera
Flavobathelium, Phyllobathelium, and Strigula, which formed a
supported monophyletic clade sister to Kirschsteiniothelia aethiops,
but without support. Monoblastiaceae was strongly supported
and included four genera with one species each in this analysis:
Acrocordia subglobosa, Anisomeridium ubianum, Megalotremis
verrucosa, and Trypetheliopsis (syn. Musaespora) kalbii. Initially
we also included a GenBank sequence of Anisomeridium polypori
in the data set, but the nuLSU sequence was recovered in
Eurotiomycetes and the taxon was excluded from the final analysis.
It is possible that this sequence is derived from a contaminant or
that it was confused with a similar species in an unrelated lineage.
Trypetheliaceae was strongly supported as monophyletic,
being sister to the genus Mycomicrothelia. There was no support
for the traditional separation into the perithecial and ascospore core
genera Astrothelium, Laurera, and Trypethelium, as species of
these genera were found scattered over the Trypetheliaceae clade.
This is the first molecular phylogenetic study that includes
presumably all major lichenised lineages within Dothideomyceta.
This rankless taxon was informally introduced by Schoch et
al. (2009a, b) for the clade including Arthoniomycetes and
Dothideomycetes. The sister group of Dothideomyceta is not yet
resolved but Ruibal et al. (2009; this volume) demonstrated an
unnamed lineage of melanised rock-inhabiting fungi to be basal to
Arthoniomycetes (not included in our sampling).
Arthoniomycetes is the second largest class of primarily
lichenised Ascomycota and exhibits considerable morpho-
anatomical variation (Fig. 2). The molecular phylogeny
presented here confirms the current classification of
lichenised Arthoniomycetes in three families: Arthoniaceae,
Chrysothrichaceae, and Roccellaceae (Tehler 1995, Grube 1998,
Tehler & Irestedt 2007). The morphological concept used to classify
the single order included few large genera, with Arthonia and
Opegrapha having the highest number of species (500 and 300,
respectively). The infrageneric relationships of these species were
repeatedly discussed and there was common agreement that these
genera were not monophyletic and include morphologically distinct
groups. Similarly the relationships of other genera with fewer
species or of monospecific genera in the family Roccellaceae was
nelSen et al.
Fig.1.?The ML tree from RAxML maximum likelihood analysis with bootstrap percentages equal to or greater than 70 are plotted above or below branches. Lichenised taxa are
in green, while non-lichenised taxa are in black.
unclear. Along with previous data (Tehler 1995, Myllys et al. 1998,
Tehler & Irestedt 2007) and recent results by Ertz et al. (2009), the
present tree is a further step to resolve these questions based on
Little can be said regarding generic concepts of most genera,
as the taxon sampling is still far too incomplete for this group, but
it appears that some of the traditional concepts based on fruit body
structure are not supported, which suggests some degree of parallel
evolution. An example is the Chiodecton-Enterographa complex:
while the sequenced Chiodecton natalense appears to be unrelated
to the morphologically and anatomically similar Dichosporidium
and Erythrodecton (Thor 1990), Enterographa and the similar
Schismatomma (Sparrius 2004) were found in three different
clades related to either Chiodecton natalense (Schismatomma),
(Enterographa anguinella), respectively. This is in agreement with
crassa), and Opegrapha
unravelling the phylogenetic relationShipS of licheniSed fungi in dothideoMyceta
Fig.?2. Select lichenised Arthoniomycetes. A. Chrysothrix xanthina; B. C. septemseptata; C. Arthonia caesia; D. A. cyanea; E. A. pulcherrima; F. A. rubrocincta; G. Cryptothecia
candida; H. Herpothallon rubrocinctum; I. Tylophoron crassiusculum (teleomorph); J. T. crassiusculum (anamorph); K. Opegrapha filicina; L. O. astraea; M. Enterographa
anguinella; N. Syncesia glyphysoides; O. S. byssina; P. Lecanactis epileuca; Q. Chiodecton sphaerale; R–S. Erythrodecton granulatum; T. Dichosporidium boschianum; U. D.
nigrocinctum (ascomata); V. D. nigrocinctum (isidia); W. Mazosia rotula; X. Roccella spec. Photo credits: R. Lücking.
Ertz et al. (2009), who showed that Enterographa is not monophyletic
and groups either with the core Opegrapha clade (here represented
by O. lithyrgica), or with Chiodecton-like species (Dichosporidium
and Erythrodecton). Consequently, Ertz et al. (2009) tranferred
Enterographa anguinella to Opegrapha. Not surprisingly, neither
Arthonia nor Opegrapha are monophyletic. Ertz et al. (2009) showed
convincingly that despite different ascomatal structure, Opegrapha
atra and O. calcarea (with distinct excipulum) are closely related
to Arthonia radiata (lacking an excipulum), which is confirmed by
similarities of ascus structure and pigment type. Subsequently, Ertz
et al. (2009) suggested these two Opegrapha species be recognised
as belonging to Arthonia. Opegrapha varia and O. celtidicola form
another monophyletic lineage together with Simonyella variegata.
Most likely this branch also includes other Opegrapha species,
according to the results of Ertz et al. (2009). Opegrapha s. str. forms
a further lineage including O. lithyrgica, which is closely related to the
type species O. vulgata (Ertz et al. 2009), the foliicolous O. filicina, as
well as Combea mollusca and Roccellographa cretacea.
nelSen et al.
Herpothallon rubrocinctum is now confirmed as an ascomycete
in Arthoniomycetes. This seems trivial as the species also
morphologically shows clear affinities with Cryptothecia (Aptroot
et al. 2008), but the position of this taxon was questioned long
ago and was even considered a basidiomycete (see discussion
in Withrow & Ahmadjian 1983, Aptroot et al. 2008). Our analysis
shows Herpothallon nested within Cryptothecia, supporting the
previous hypothesis that byssoid-isidiate species within this
complex are indeed members of Cryptothecia rather than forming
a separate genus, as proposed by Aptroot et al. (2008). However,
a larger taxon sampling is needed to resolve the Cryptothecia-
Herpothallon complex, especially considering that there are other
genera such as Stirtonia involved and even further new genera
have been segregated recently (Aptroot et al. 2009, Frisch & Thor
2010). The fruticose Roccella species form a clearly monophyletic
branch together with several crustose species representing various
genera; this assemblage of core Roccellaceae has already been
recognised previously (Tehler 1995, Myllys et al. 1998, Tehler &
Irestedt 2007). The placement of Tylophoron, a genus that has
passive spore dispersal and was previously assigned to Caliciales,
is here confirmed as a member of Arthoniaceae s. l., in agreement
with Lumbsch et al. (2009a).
The strongly supported placement of Arthonia caesia within
Chrysothrix is unexpected; however, fertile species of Chrysothrix
are very similar to Arthonia in ascoma morphology and anatomy,
and particularly A. caesia and allies can be easily perceived as
non-pigmented species of Chrysothrix in apothecial anatomy and
morphology and thallus structure (including the chlorococcoid
photobiont). Similar Arthonia species include A. cupressina,
which is closely related to A. caesia. Further studies are needed
to elucidate which additional Arthonia taxa need to be placed in
Chrysothrix. The latter genus was variously placed in its own family
Chrysothrichaceae mainly due to the presence of pulvinic acids as
secondary metabolites but also in Arthoniaceae due to similarities
in ascus characters (Grube 1998). The present data strongly
support Chrysothrichaceae as a separate family, especially as it
is sister to all remaining Arthoniales and not to Arthoniaceae. It
is therefore necessary to transfer Arthonia caesia (which lacks
pulvinic acids) and related species to this family. The other Arthonia
species sampled group form a fairly well supported monophyletic
group, which includes a species formerly assigned to Arthothelium,
i.e. Arthonia ruana, because of its muriform ascospores; however,
it has been known for some time that most species with muriform
ascospores are more closely related to Arthonia than to the type
of Arthothelium, A. spectabile (Tehler 1990, Sundin & Tehler 1998,
Cáceres 2007, Grube 2007), which has not yet been sequenced.
Notably, Arthonia didyma and A. rubrocincta, two species with
reddish pigments, form a weakly supported group. If future efforts
confirm this grouping, the name Coniocarpon could be used for this
clade (Cáceres 2007).
In contrast to Arthoniomycetes, the overwhelming majority
of Dothideomycetes species are non-lichenised. In addition
to Arthopyreniaceae, Trypetheliaceae and Cystocoleus and
Racodium (Muggia et al. 2007), this study confirms the placement
of Monoblastiaceae and Strigulaceae within Dothideomycetes.
Although our support for the Dothideomycetes node is weak, the
included non-lichenised taxa are well supported within this class in
other studies (Schoch et al. 2006, 2009a, b); in addition, placement
within Dothideomyceta is strongly supported. Both, Monoblastiaceae
and Strigulaceae are comparatively large with over 100 accepted
species each and show substantial morphological and ecological
radiation (Fig. 3); both are chiefly tropical. The mostly corticolous
Monoblastiaceae range from barely lichenised forms with exposed
perithecia (many species of Anisomeridium) to taxa with well-
developed, corticate thalli (Anisomeridium p.p., Megalotremis,
Trypetheliopsis). Ascospores vary from small to large and thick-
walled but are always simple or transversely septate only (Harris
1995). Substantial variation is found in the conidiomata, and many
species, particularly in the genera Caprettia, Megalotremis, and
Trypetheliopsis (= Musaespora) have developed unique pycnidia
that in part are similar to campylidia or hyphophores found in
certain Lecanoromycetes (Aptroot & Sipman 1993, Lücking et al.
1998, Aptroot et al. 2008, Lücking 2008). Secondary substances
are few, including lichexanthone and anthraquinones. All species
of Monoblastiaceae in which conidiomata are known share a
particular synapomorphy: the conidia are always embedded in a
strongly coherent, gelatinous matrix. Thus, besides the uniform
hamathecium and ascus anatomy, there is substantial phenotypic
evidence for monophyly of this family, now confirmed by molecular
Strigulaceae share many characteristics with Monoblastiaceae,
specifically the ascus type and the mostly 1- or 3-septate ascospores,
although some species have muriform ascospores (Harris 1995,
Aptroot et al. 2008, Lücking 2008). Species in this family are found
on a variety of substrata, including rocks, bark, and living leaves.
Poorly developed thalli are found in corticolous species with barely
lichenised thalli and exposed perithecia (Strigula p.p.), whereas the
genera Flavobathelium, Phyllobathelium, and Phyllocratera include
taxa with well-developed, corticate thalli. Also in this family, the most
characteristic synapomorphy are the conidia, which feature terminal
gelatinous appendices (Harris 1995, Lücking 2008). Unfortunately,
our taxon sampling of this family is poor but sufficient to confirm
its monophyly and its placement in Dothideomycetes. This is the
first molecule-based support for the inclusion of Phyllobatheliaceae
within Strigulaceae, a concept first presented by Harris (1995).
The largest lichenised family within Dothideomycetes,
Trypetheliaceae, contains members that are typically lichen-
forming and tropical to subtropical in distribution, with some taxa
extending into temperate regions (Aptroot 1991, Harris 1995, Brodo
et al. 2001, Aptroot et al. 2008). The species are almost exclusively
corticolous, forming a crustose, endo- or epiperidermal thallus with
algae belonging to Trentepohliaceae; however, Anisomeridium
is often found lignicolous and Aptrootia grows on bryophytes.
Detailed studies in Costa Rica suggest Trypetheliaceae to occur
primarily on trunks and branches of trees in exposed habitats
of lowland to lower montane (200–1000 m) rain and dry forests
and savannas with rather distinct dry season (Aptroot et al.
2008, Rivas-Plata et al. 2008). Trypetheliaceae species are quite
variable in perithecial morphology (Fig. 3) but have a rather uniform
hamathecium composed of thin, anastomosing pseudoparaphyses
embedded in a stiff gelatinous matrix. The most characteristic
synapomorphy are the usually hyaline ascospores with internal wall
thickenings that cause more or less diamond-shaped septa, but
these wall thickenings are often reduced or absent in species with
multiseptate or muriform ascospores (Harris 1984, 1990, 1995,
Aptroot 1991b, Aptroot et al. 2008). The secondary chemistry is
equally simple, with lichexanthone and pigments as most common
substances, i.e. polyketide derived aromatic compounds produced
through the acetyl-polymalonyl pathway (Elix & Stocker-Wörgötter
2008). However, the number of species with substances present
is much higher in Trypetheliaceae than any other lineage within
Dothideomycetes: more than 70 species are known to produce
secondary substances in this family. The core genera Astrothelium,
Campylothelium, Cryptothelium, Laurera, and Trypethelium, are
unravelling the phylogenetic relationShipS of licheniSed fungi in dothideoMyceta
separated primarily on the basis of perithecial arrangement and
ostiolar orientation (solitary vs. aggregate, apical vs. excentric) and
ascospore septation (transverse vs. muriform; Harris 1990, 1995,
del Prado et al. 2006). Because of the schematic classification,
Harris (1995) suggested that these genera may be polyphyletic, and
del Prado et al. (2006) subsequently illustrated the non-monophyly
of Trypethelium. Aptroot et al. (2008) echoed Harris’s (1995)
sentiment and stated that generic concepts in Trypetheliaceae are
in need of revision.
Surprisingly, Mycomicrothelia was recovered as sister to
Trypetheliaceae. Mycomicrothelia has traditionally been considered
a sister genus to Arthopyrenia with brown ascospores (Harris 1995).
However, the hamathecium at least of the sequenced species is
identical to that found in Trypetheliaceae, whereas Arthopyrenia has
thicker and less branched and anastomosing pseudoparaphyses.
Moreover, the ascospores are of a different type, often with internal
wall thickenings. It remains to be tested whether Arthopyrenia and
Mycomicrothelia in their current circumscriptions are monophyletic
Fig.?3. Select lichenised Dothideomycetes; A. Arthopyrenia cinchonae; B. Mycomicrothelia modesta; C. Anisomeridium subprostans; D. Anisomeridium spec. (pycnidia); E. A.
foliicola (pycnidia); F. Caprettia amazonensis (pycnidia); G. Megalotremis cauliflora (pycnidia); H. Trypetheliopsis (= Musaespora) coccinea (campylidia); I. Strigula viridiseda;
J. S. laureriformis (pycnidia); K. S. smaragdula; L. Flavobathelium epiphyllum; M. Phyllobathelium firmum; N. P. leguminosae (pycnidia); O. Pseudopyrenula subnudata;
P. Trypethelium tropicum; Q. T. platystomum; R. Bathelium degenerans; S. Laurera purpurina; T. Astrothelium cinnamomeum; U. A. eustomum; V. Trypethelium nitidiusculum;
W. Laurera megasperma; X. Campylothelium spec. Photo credits: R. Lücking.
nelSen et al.
genera or whether at least some species currently assigned to
these genera perhaps represent further lichenised lineages within
Dothideomycetes. Whether Mycomicrothelia should be included
within Trypetheliaceae or receive its own family rank is open to
question. Mycomicrothelia has primarily thin-walled, dark brown
ascospores, whereas in Trypetheliaceae they are primarily thick-
walled with diamond-shaped lumina and hyaline (brown only in
Aptrootia and Architrypethelium). Understanding the phylogenetic
position of Polymeridium, which also has thin-walled ascospores,
will hopefully help clarify this.
In spite of the many characters in parallel with Monoblastiaceae
and Strigulaceae, also the Trypetheliaceae plus Mycomicrothelia
(Trypetheliales) are quite unique genetically and there is no
evidence that the three families would be related to each other
or with Arthopyreniaceae. This supports the notion of several
shifts in lichenisation within the Dothideomycetes (Aptroot 1991a,
1998). However, the often barely lichenised thalli in certain
species of Anisomeridium, Arthopyrenia, Julella, Mycomicrothelia,
Mycoporum, Pseudopyrenula, and Strigula (Aptroot 1991a, Aptroot
1998, Harris 1995) suggest that these species can possibly switch
between being (almost) non-lichenised to distinctly lichenised,
a situation also found in the unrelated genus Stictis within
Lecanoromycetes (Wedin et al. 2004).
The present study clarifies the systematic position of further
pyrenocarpous lichenised lineages within the Ascomycota and
shows that previous concepts in part diverged widely from our
present understanding but also came suprisingly close even
without molecular evidence (Table 2). This study emphasises
that pyrenocarpous lichens with bitunicate asci are not only
not monophyletic, but belong to at least two different classes
(Dothideomycetes and Eurotiomycetes) and several different
orders and families; the data at hand also suggest that these
Genus Zahlbruckner?1926 Barr?1987 harris?1995current
(as Leptorhaphis)PleosporalesMelanommatales Pyrenulales
Pyrenocarpeae PyrenulaceaePyrenulaceae Pyrenulaceae
(as Arthopyrenia) Melanommatales Melanommatales incertae sedis
Pyrenulaceae Acrocordiaceae Monoblastiaceae Monoblastiaceae
Pyrenocarpeae Loculoascomycetes LoculoascomycetesDothideomycetes
StrigulaceaeChaetothyriales Melanommatalesincertae sedis
PyrenocarpeaeLoculoascomycetes Loculoascomycetes Dothideomycetes
Pyrenocarpeae TrypetheliaceaeTrypetheliaceae Trypetheliaceae
(as Microthelia)Pleosporales PleosporalesTrypetheliales
Table?2.?Systematic placement of selected pyrenocarpous lichens according to different concepts.
unravelling the phylogenetic relationShipS of licheniSed fungi in dothideoMyceta
represent several independent lineages of lichenisation. Although
we consider this study a contribution to clarify the systematic
position of pyrenocarpous lichens and the evolution of lichenisation
within Dothideomycetes, much remains to be done, considering that
at present only a fraction of the presumably 600 species of lichens
belonging in this class have been studied using DNA sequences.
In particular, clarifying the generic and species concepts within
Monoblastiaceae, Strigulaceae, and Trypetheliaceae, speciose
families that are important elements of crustose lichen communities
especially in the tropics, will be a major challenge in the near future.
Material used in this study was collecte in the framework of three NSF grants to The
Field Museum: DEB 0206125 “TICOLICHEN” (PI Robert Lücking), DEB 0516116
“Phylogeny and Taxonomy of Ostropalean Fungi, with Emphasis on the Lichen-
forming Thelotremataceae” (PI Thorsten Lumbsch), and DEB 0715660 “Neotropical
Epiphytic Microlichens – An Innovative Inventory of a Highly Diverse yet Little Known
Group of Symbiotic Organisms” (PI Robert Lücking). We also thank Z. Palice, G.
Perlmutter & D.G. Zimmerman for collections used in this study and K. Feldheim
for discussions on laboratory techniques. Most work was performed in the Pritzker
Laboratory at the Field Museum of Natural History.
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Ph.D. dissertation - The University of Texas at Austin, U.S.A.
Unravelling the phylogenetic relationships of lichenised fUngi in dothideomyceta
Acrocordia subglobosa (HTL940) Palice s.n., Poland (F) GU327681
Amphisphaeria umbrina FJ176863FJ713609
Anisomeridium ubianum (94) Lumbsch 19845j, Fiji (F)GU327709 GU327682
Aptrootia terricola DQ328995
Arthonia caesiaFJ469668 FJ469671
Arthonia didyma EU704083EU704047
Arthonia dispersaAY571381 AY571383
Arthonia radiate EU704048
Arthonia ruana (79B)Zimmerman 1117, Germany (F)GU327683
Arthonia rubrocincta (129) Nelsen 4010, U.S.A. (F) GU327684
Arthopyrenia salicis AY538339 AY538345
Ascobolus crenulatusAY544678 FJ713607
Astrothelium cinnamomeum AY584652 AY584632
Astrothelium confusum (98) Nelsen 4004a, Peru (F) GU327710GU327685
Bacidia schweinitzii DQ782911 DQ972998
Bathelium degenerans DQ328987
Bimuria novae-zelandiae AY016356FJ190605
Botryosphaeria tsugae DQ767655
Botryotinia fuckeliana AY544651AY544732
Caliciopsis orientalis DQ470987 FJ190654
Caliciopsis pinea DQ678097FJ190653
Camarops ustulinoides DQ470941FJ190588
Capnodium coffeae DQ247800FJ190609
Cercospora beticola DQ678091FJ190647
Cheilymenia stercorea AY544661AY544733
Chiodecton natalenseEU704085 EU704051
Chlorociboria aeruginosaAY544669 AY544734
Chrysothrix flavovirens (L466)Perlmutter 786, U.S.A. (NCU) GU327711 GU327686
Chrysothrix xanthina (126)Nelsen 4005, U.S.A. (F) GU327712GU327687
Cladosporium cladosporioides DQ678057 FJ190628
Cochliobolus heterostrophus AY544645AY544737
Cochliobolus sativus DQ678045FJ190589
Combea molluscaAY571382 AY571384
Coniothyrium palmarum DQ767653FJ190638
Cordyceps capitata AY489721 FJ713628
Cryptothecia assimilis (86B) Lumbsch 19815l, Fiji (F) GU327688
Table 1. Taxa included in this study with GenBank accession numbers and collection information. Numbers following taxon names are DNA
identification numbers used in this study.
nelsen et al.
Cryptothecia candida EU704052
Cryptothelium amazonum (47)Nelsen 4000a, Peru (F)GU327713 GU327689
Cryptothelium sepultum (63C) Nelsen 4001a, Peru (F) GU327714GU327690
Cudoniella cf. clavus DQ470944FJ713604
Cystocoleus ebeneus EU048578EU048584
Delitschia winteriDQ678077 FJ190644
Dendrographa alectoroides (100)Lumbsch 19914g, U.S.A. (F)GU327715 GU327691
Dendrographa leucophaea f. minorAF279382 AY548811
Dendryphiella arenaria DQ470971 FJ190617
Diaporthe eresAF408350 FJ190607
Dichosporidium boschianum (89B) Lumbsch 19815a, Fiji (F)GU327716GU327692
Dothidea insculpta DQ247802FJ190602
Dothidea sambuciAY544681 AY544739
Dothiora cannabinaeDQ470984 FJ190636
Eleutherascus lectardii DQ470966FJ190606
Elsinoe centrolobiDQ678094 FJ190651
Elsinoe veneta DQ767658FJ190650
Enterographa anguinella EU704086 EU704054
Enterographa crassa EU704088EU704056
Erythrodecton granulatum EU704090 EU704058
Eupenicillium javanicum EF413621FJ225778
Exophiala salmonis EF413609FJ225745
Flavobathelium epiphyllum (67)Lücking s.n. Panama (F)GU327717
Glomerella cingulataAF543786 FJ190626
Glyphium elatum AF346420AF346425
Guignardia gaulteriae DQ678089FJ190646
Herpothallon rubrocinctum (128)Nelsen 4006, U.S.A. (F)GU327693
Herpotrichia diffusaDQ678071 DQ384076
Hypocrea lutea AF543791 FJ713620
Hysteropatella cf. ellipticaDQ767657 FJ190649
Lachnum virgineum AY544646 AY544745
Laurera megasperma FJ267702
Lecanactis abietinaAY548812 AY548813
Lecanora hybocarpa DQ782910DQ912273
Macrophomina phaseolina DQ678088 FJ190645
Table 1. (Continued).
Unravelling the phylogenetic relationships of lichenised fUngi in dothideomyceta
Megalotremis verrucosa (104)Lücking 26316, Colombia (F) GU327718GU327694
Monilinia laxa AY544670AY544748
Mycomicrothelia hemispherica (102)Lücking 28641, Nicaragua (F)GU327719 GU327695
Mycomicrothelia miculiformis (101B)Lücking 28637, Nicaragua (F) GU327720GU327696
Mycomicrothelia obovata (95) Nelsen 4007a, Peru (F) GU327721GU327697
Mycosphaerella fijiensis DQ678098FJ190656
Mycosphaerella punctiformisDQ470968 FJ190611
Nectria cinnabarina U00748FJ713622
Opegrapha celtidicolaEU704094 EU704066
Opegrapha filicina EU704095EU704067
Opegrapha lithyrga EU704096EU704068
Opegrapha variaEU704103 EU704075
Ophionectria trichospora AF543790FJ713626
Peltigera degeniiAY584657 AY584628
Penicillium freiiAY640958 AY584712
Pertusaria dactylinaDQ782907 DQ972973
Phoma herbarum DQ678066FJ190640
Phyllobathelium anomalum (242)Lücking s.n., Panama (F) GU327722 GU327698
Phyllobathelium firmum (HTL3175)Lücking s.n., Panama (F)GU327723
Pleospora herbarum var. herbarumDQ247804 FJ190610
Pseudopyrenula subgregaria (106)Lücking 24079, Thailand (F) GU327724GU327699
Pyrenophora phaeocomesDQ499596 FJ190591
Pyrenophora tritici-repentisAY544672 FJ713605
Pyrgillus javanicus DQ823103FJ225774
Ramichloridium ancepsDQ823102 FJ225752
Roccella canariensis AY779328
Roccella fuciformisAY584654 EU704082
Roccella montagnei (109) Lumbsch 19700a, India (F) GU327725GU327700
Roccellographa cretacea DQ883696FJ772240
Schismatomma decoloransAY548815 AY548816
Schismatomma pericleumAF279408 AY571390
Scorias spongiosaDQ678075 FJ190643
Sporormiella minimaDQ678056 FJ190624
Table 1. (Continued).
144-S4 Download full-text
nelsen et al.
Staurothele frustulenta DQ823098FJ225702
Strigula nemathora (72)Lücking s.n., Costa Rica (F) GU327701
Strigula schizospora (73)Lücking s.n., Costa Rica (F) GU327702
Stylodothis puccinioides AY004342 AF346428
Sydowia polyspora DQ678058FJ190631
Syncesia farinacea EF081452
Trematosphaeria pertusaDQ678072 FJ190641
Trimmatostroma abietisDQ678092 FJ190648
Trypetheliopsis kalbii (243)Lücking s.n., Panama (F)GU327703
Trypethelium eluteriae DQ328989
Trypethelium eluteriae (111) Lumbsch 19701a, India (F)GU327726 GU327704
Trypethelium marcidum DQ329007
Trypethelium marcidum (132)Nelsen 4008, U.S.A. (F)GU327727 GU327705
Trypethelium nitidiusculum (139) Nelsen 4002a, U.S.A. (F)GU327728 GU327706
Trypethelium papulosum (97)Nelsen 4009a, Peru (F)GU327729GU327707
Trypethelium tropicum (25) Nelsen 4003, Thailand (F)GU327730GU327708
Tubeufia cereaDQ470982 FJ190634
Tyrannosorus pinicola DQ470974 FJ190620
Vibrissea truncorumFJ176874 FJ190635
Xylaria hypoxylonAY544648 AY544760
Table 1. (Continued).