f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 5 8 4 e5 9 8
journal homepage: www.elsevier.com/locate/funbio
Starting from scratch: Evolution of the lichen
thallus in the basidiolichen Dictyonema
(Agaricales: Hygrophoraceae)
Manuela DAL-FORNOa, James D. LAWREYa, Masoumeh SIKAROODIa,
Smriti BHATTARAIa, Patrick M. GILLEVETa, Marcelo SULZBACHERb,
c,
€
*
Robert LUCKING
a
Department of Environmental Science and Policy, George Mason University, 4400 University Drive, Fairfax,
VA 22030-4444, USA
b
Departamento de Micologia/CCB, Universidade Federal de Pernambuco, Av. Prof. Nelson Chaves, s/n, Recife,
Pernambuco CEP 50670-901, Brazil
c
Botany, The Field Museum, 1400 South Lake Shore Drive, Chicago, IL 60605-2496, USA
article info
abstract
Article history:
Phylogenetic studies indicate that the basidiolichen genus Dictyonema s.lat., often thought
Received 28 March 2013
to represent only a single genus with few species, includes several well-supported genus-
Accepted 31 May 2013
level clades, all of which form associations with a unique lineage of obligately lichenized
Available online 28 June 2013
cyanobacteria (Rhizonema). In an attempt to elucidate the evolution and genus- and
Corresponding Editor:
species-level diversification in Dictyonema s.lat., we generated 68 new sequences of the nu-
Martin Grube
clear large subunit rDNA (nuLSU), the internal transcribed spacer (ITS), and the RNA polymerase II subunit (RPB2), for 29 species-level lineages representing all major clades of
Keywords:
Dictyonema s.lat. and most of the species currently known. The multilocus phylogeny ob-
Basidiocarp
tained via maximum likelihood and Bayesian approaches indicates the presence of five
Basidiolichens
genus-level groups: a basal clade, Cyphellostereum, that is sister to the rest of the species,
Cyanolichens
a paraphyletic grade representing Dictyonema s.str., and three clades representing the gen-
Mushrooms
era Acantholichen, Cora, and Corella. To determine the evolutionary transformations of the
lichenized thallus in the group, ancestral character state reconstruction was done using
six characters (lichenisation, thallus type, cortex type, hyphal sheath and haustorial
type, photobiont morphology, and basidiocarp type). Our analysis indicates a progressive
development of the lichenized thallus from loosely organized filamentous crusts with separate, cyphelloid basidiocarps in Cyphellostereum, to filamentous crusts with derived hyphal
sheath and cyphelloidestereoid basidiocarps partially incorporated into the lichen thallus
in Dictyonema, to squamuloseefoliose thalli with corticioid basidiocarps entirely supported
by the lichen thallus in Cora. These results indicate a remarkable evolutionary integration
of lichenized and reproductive tissues in Dictyonema s.lat., supporting the hypothesis that,
at least in this case, lichenized thalli may have evolved from reproductive structures in
their nonlichenized ancestors.
ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ1 312 665 7154; fax: þ1 312 665 7158.
E-mail addresses: mdalforn@gmu.edu (M. Dal-Forno), marcelo_sulzbacher@yahoo.com.br (M. Sulzbacher), rlucking@fieldmuseu€ cking).
m.org (R. Lu
1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.funbio.2013.05.006
Evolution in Dictyonema
Introduction
Lichens represent the most remarkable and successful of living symbiotic organisms in terms of numbers of species, diversity and complexity of symbiotic morpho-anatomical
features, and the number of lineages that have achieved this
type of symbiosis (Ahmadjian 1993; Kirk et al. 2008; Nelsen
et al. 2009; Schoch et al. 2009). Yet, the origin of lichenisation
is a matter of debate, as are the evolutionary steps that have
led to the development of the integrated lichen thallus
(Gargas et al. 1995; Lutzoni et al. 2001; Eriksson 2005;
€ cking et al. 2009; Schoch et al. 2009).
Hawksworth 2005; Lu
One theory suggests that, because the lichen thallus is
structurally complex, whereas the mycelium of nonlichenized
fungi is usually undifferentiated, the lichen thallus evolved
from fungal stromata or reproductive fungal tissue (Poelt &
Wunder 1967; Moser-Rohrhofer 1969; Jahns 1988). Indeed, in
lichens such as Cladonia, the vertical thallus is derived from reproductive tissue (Jahns 1970, 1988). Several studies have
shown that the photobiont can affect lichen thallus morphogenesis, to the point that the same lichen fungus is able to
form morphologically distinct thalli with different photosynthetic partners (James & Henssen 1976; Brodo & Richardson
1978; Jahns 1988; Armaleo & Clerc 1991; Tschermak-Woess
1995; Sanders 2001a; Tønsberg & Goward 2001; Stocker€ rgo
€ tter 2002; Takahashi et al. 2006). These theories focus
Wo
on lichenized Ascomycota, but except for supported sistergroup relationships between rock-inhabiting fungi and lichenized forms in the Arthoniales and Verrucariales (Gueidan
et al. 2008; Ruibal et al. 2009), no lineages are known in this
phylum that show a direct transition from nonlichenized to
lichenized forms.
While nearly all of the approximately 18 000 species of
lichenized fungi belong in Ascomycota, few members of Basidiomycota are lichenized, being chiefly found in the orders
Agaricales, Cantharellales, Hymenochaetales, and Lepidostromatales (Oberwinkler 1970, 2012; Hibbett et al. 2007; Lawrey
€ cking 2013). Most lichenized Basiet al. 2009; Hodkinson & Lu
diomycota concentrate in the family Hygrophoraceae in the
Agaricales, forming two diverse clades: Lichenomphalia s.lat.
and Dictyonema s.lat. (Lawrey et al. 2009). Despite their phylogenetic affinities, the two differ in nearly every possible way.
Lichenomphalia forms agaricoid-omphalinoid mushrooms
that arise from lichenized granules or squamules containing
a green algal (Coccomyxa) photobiont, whereas Dictyonema
forms cyphelloid to stereoidecorticioid basidiocarps and
a lichenized, filamentous to squamuloseefoliose thallus containing a cyanobacterial photobiont (Oberwinkler 1970, 2012;
Redhead et al. 2002; Chaves et al. 2004; Lawrey et al. 2009).
Historically, some authors considered Dictyonema s.lat. to
represent several different genera in different families, based
on features related to growth form, presence of clamp connections, and nature of the photobiont (Hariot 1891, 1892; Metzner
1934). Foliose forms were usually treated as Cora, whereas filamentous forms were assigned to either Laudatea, Dictyonema,
or Rhipidonema, depending on appressed or shelf-like growth
and the absence or presence of clamp connections. The other
extreme was the inclusion of different growth forms into a sin€ ller
gle species as ontogenetic and ecological variation (Mo
585
1893; Oberwinkler 1970; Larcher & Vareschi 1988). Parmasto
(1978) chose a middle ground in accepting five ‘lichen’ species
in a single genus, but he stated that only two fungal species
were involved. This is inconsistent from a nomenclatural
point of view, since names of lichens always apply to the fungal component [ICN Art. 13.1(d)] and hence there cannot be
two ‘fungal’ and five ‘lichen’ species at the same time.
Zmitrovich et al. (2006) even went as far as including Dictyonema in the nonlichenized genus Byssomerulius based on
shared mycological characters, disregarding the fact that
Byssomerulius, based on its type species, belongs in the unrelated Polyporales (Larsson et al. 2004; Larsson 2007;
Oberwinkler 2012). Oberwinkler (1970, 1980, 1984, 2001, 2012)
provided several morphological and anatomical treatments
of Dictyonema and related groups, discussing the value of certain characters for taxonomy and pointing out that filamentous (Dictyonema s.str.), squamulose (Acantholichen), and
foliose (Cora) forms share the same type of unique haustoria
(Roskin 1970; Slocum & Floyd 1977; Slocum 1980;
Oberwinkler 1980, 1984, 2001, 2012).
Until recently, Dictyonema s.lat. was best known by the two
supposedly common and widespread tropical montane species, the foliose Dictyonema glabratum (¼Cora pavonia) and the
filamentous Dictyonema sericeum (Oberwinkler 1970, 1984,
2001; Parmasto 1978), with the first being a popular object
for biological studies (Mitidieri et al. 1964; Feige 1969; Coxson
1987a, 1987b, 1987c; Fritz-Sheridan & Portecop 1987; Iacomini
et al. 1987; Fritz-Sheridan 1988; Wolf 1993; Lange et al. 1994;
Thomas et al. 1997; Azenha et al. 1998; Trembley et al. 2002a,
2002b). In addition to these two, Parmasto (1978) accepted
three more ‘lichen’ species, focussing on mycological characters and regarding variation in thallus morphology as infraspecific and habitat-induced. However, taxonomic and
phylogenetic studies suggest that Dictyonema s.lat. is comprised of a fairly large number of species, many undescribed
nez
€ cking 2008; Lawrey et al. 2009; Ya
(Chaves et al. 2004; Lu
et al. 2012). This has implications on the commonly used epithets glabratum and pavonia for foliose forms, for which
Hawksworth (1988) pointed out that glabratum is the correct
usage. However, based on our studies, including revision of
€ cking et al.
most types of names described in this clade (Lu
2013a), glabratum and pavonia are different species, only pavonia being widespread and common.
The first detailed molecular study suggested that this
group can be divided into at least four genera, Cyphellostereum,
Dictyonema s.str., Acantholichen, and Cora (Lawrey et al. 2009),
and this concept was employed for a treatment of Galapagos
nez et al. 2012). The putative genera are
basidiolichens (Ya
well-distinguished morphologically and anatomically, as
also noted by Oberwinkler (1984, 2012): Cyphellostereum and
Dictyonema have filamentous thalli with the mycobiont forming a hyphal sheath around the cyanobacterial filaments, the
sheath consisting of irregular hyphae (lacking haustoria) in
Cyphellostereum and of jigsaw-puzzle-shaped cells (forming
haustoria) in Dictyonema s.str.. In addition, Cyphellostereum
has cyphelloid basidiocarps emerging from the undifferentiated lichenized thallus, whereas Dictyonema s.str. has
stereoidecorticioid basidiocarps that develop from the
underside of the lichenized thallus. Acantholichen forms
586
M. Dal-Forno et al.
a microsquamulose thallus, while species of Cora have
folioseemacrosquamulose thalli forming distinct layers
(cortex, photobiont layer, medulla) and producing corticioid
basidiocarps on the lobe underside. Corella brasiliensis, a basidiolichen morphologically similar to Cora (Vainio 1890;
Metzner 1934; Xavier Filho & Vicente 1979), had not yet been
sequenced, but was assumed to be in or near the Cora clade
based on morphological evidence (Oberwinkler 1970, 1984). Although morphologically and anatomically quite distinct from
D. glabratum, Parmasto (1978) even included C. brasiliensis in
the synonymy of the latter, regarding it a juvenile or underdeveloped form of no taxonomic value.
The transition from unstructured to complex vegetative
thalli and the simultaneous gradual incorporation of the
basidiocarps into thallus formation lend support to the hypothesis that the lichen thallus evolved from fruiting body
structures of nonlichenized ancestors in these basidiolichens. This view was already expressed by Oberwinkler
(1970), who regarded the Dictyonema thallus as lichenized
fruiting body in his depictions of thallus sections.
Oberwinkler (1984: 748) also regarded the foliose thallus
(Cora) as ‘. ecologically very well adapted symbiotic
structure’. Since Dictyonema s.lat. is hitherto the only lichenized lineage for which direct, extant, nonlichenized ancestors have been established in the paraphyletic genus Arrhenia
(Lawrey et al. 2009), this clade is an excellent model to
investigate the evolution of the lichen thallus. We therefore
expanded our sampling of this group both taxonomically
and geographically and used a combination of the large subunit (nuLSU) and the internal transcribed spacer (ITS) partition of the nuclear ribosomal DNA and sequences between
the conserved domains 6 and 7 of the protein-coding second
largest subunit of the RNA polymerase II gene (RPB2) to expand our previous phylogenetic analyses (Lawrey et al.
2009). Our objectives were to: (1) produce a comprehensive
multigene phylogeny of Dictyonema s.lat. using representatives of all the major clades, including for the first time also
the genus Corella; (2) test hypotheses about the evolution of
the lichen thallus in the clade using ancestral character state
reconstructions; and (3) examine current genus concepts.
Material and methods
Taxon sampling
The dataset consisted of 29 ingroup species-level terminals
plus the outgroup Eonema pyriforme (Lawrey et al. 2009), including 68 new sequences obtained from specimens of Acantholichen, Cyphellostereum, Cora, Corella, and Dictyonema species
collected from 13 countries in North, Central, and South
America, Europe, and southeast Asia (Table 1).
Table 1 e Specimens, collection information, and GenBank accession numbers of fungi used in this study.
Species
Eonema pyriforme
Cyphellostereum imperfectum
Cyphellostereum nitidum
Cyphellostereum phyllogenum
Cyphellostereum pusiolum
Cyphellostereum sp.
Dictyonema aeruginosulum
Dictyonema hernandezii
Dictyonema interruptum
Dictyonema irpicinum
Dictyonema metallicum
Dictyonema obscuratum
Dictyonema phyllophilum
Dictyonema schenkianum 1
Dictyonema schenkianum 2
Dictyonema sericeum 1
Dictyonema sericeum 2
Dictyonema sericeum 3
Acantholichen pannarioides
Corella brasiliensis
Cora arachnoidea
Cora aspera
Cora byssoidea
Cora hirsuta
Cora inversa
Cora minor
Cora pavonia
Cora reticulifera
Cora squamiformis
Cora strigosa
Strain or Herbarium
accession number
Collection
location
Genbank
number ITS
Genbank
number nuLSU
Genbank
number RBP2
Hjm 18581
€ cking 25588
Lu
Rivas Plata 1130
Lumbsch s.n.
€ cking s.n.
Lu
Rivas Plata 2183b
Nelsen 3754
€ cking 26258
Lu
Ertz 10475
Lumbsch 19837e
€ cking 26255
Lu
€ cking 23025
Lu
Lumbsch 19821
€ cking 30062
Lu
€ cking 17200
Lu
Wilk 8868
Fuentes 4788
€ cking 25551b
Lu
Bungartz 5593
Dal-Forno 1271
ndez 1779
Herna
€ cking 29128
Lu
€ cking s.n.
Lu
€ cking s.n.
Lu
€ cking s.n.
Lu
Navarro s.n.
€ cking s.n.
Lu
€ cking 26201
Lu
Wilk 7577
Paz 3
Sweden
Guatemala
Philippines
Fiji
Costa Rica
Philippines
Costa Rica
Ecuador
Madeira
Fiji
Ecuador
Brazil
Fiji
Brazil
Costa Rica
Bolivia
Bolivia
Guatemala
pagos
Gala
Brazil
Venezuela
Bolivia
Colombia
Colombia
Colombia
Costa Rica
Ecuador
Ecuador
Bolivia
Peru
EU118605
KF443218
e
KF443219
EU825976
KF443220
EU825955
KF443221
e
e
KF443222
KF443223
KF443224
KF443225
EU825972
KF443226
KF443227
KF443228
EU825953
KF443229
KF443232
KF443230
KF443234
KF443235
KF443236
EU825968
KF443238
KF443239
KF443240
KF443241
EU118605
KF443243
EU825970
KF443244
EU825976
KF443245
EU825955
KF443246
EU825967
KF443247
KF443248
KF443249
KF443250
KF443251
EU825972
KF443252
KF443253
KF443254
EU825953
KF443255
KF443256
KF443257
KF443258
KF443259
KF443260
EU825968
KF443261
KF443262
KF443263
KF443264
e
KF443277
KF443278
e
KF443279
e
KF443280
KF443281
KF443282
KF443283
KF443284
e
e
KF443285
KF443286
e
KF443287
e
KF443265
KF443276
KF443266
KF443267
KF443268
KF443270
KF443271
KF443272
KF443275
KF443269
KF443273
KF443274
Evolution in Dictyonema
Light microscopy
All sequenced specimens were examined with an LEICA MS5
(Wetzlar, Germany) and an OLYMPUS SZX12 (Shinjuku, Japan)
dissecting microscope and a ZEISS Axioskop 2 (Jena, Germany)
and an OLYMPUS BH-2 (Shinjuku, Japan) compound microscope. Sections of the thallus including the photobiont were
studied in water without any staining or dyes, as well as in lactophenol cotton blue, Lugol solution, and 10 % potassium hydroxide. Microphotographs were taken with DAGE MTI DC-330
3CCD (Michigan City, IN, USA) and JENOPTIK ProgRes C3 and
C5 (Jena, Germany) digital microscope cameras attached to
the aforementioned microscopes. Macrophotos were taken
in situ with CANON Powershot SX20IS (Ota, Japan) and NIKON
F301 (Tokyo, Japan) digital cameras.
In addition to sequenced specimens, to test for variation
and consistently in morphological, anatomical, and chemical
features, we studied several hundred specimens of Dictyonema
s.lat. both directly in the field (Mexico, Costa Rica, Colombia,
Ecuador, Galapagos, Bolivia, Brazil, Thailand, Philippines)
and deposited as herbarium material in BM, F, NY, PC,
UDBC, US, W, and herb. Kalb, plus type material of most of
the names described in this group (Parmasto 1978).
587
type (crustose filamentous ¼ Laudatea type, shelf-like
filamentous ¼ Dictyonema type, microsquamulose ¼ Acantholichen type, macrosquamuloseefoliose ¼ Cora type), (3) thallus
cortex (absent, loose corticiform with palisadic ‘medullary’
layer ¼ Cora type, compact paraplectenchymatous ¼ Corella
type), (4) photobiont morphology (distinctly filamentous ¼
‘Scytonema’ type, in irregular, coiled threads or groups ¼
‘Chroococcus’ type), (5) hyphal sheath and haustorial type (irregular hyphae lacking haustoria ¼ Cyphellostereum type,
closed sheath of jigsaw-puzzle-shaped cells forming tubular
haustoria ¼ Dictyonema s.lat. type), and (6) integration of the
basidiocarps into the thallus (basidiocarp with hymenophore
separated from basal thallus by a stipe ¼ Cyphellostereum
type, basidiocarp partially integrated into thallus with upper
part overgrown by cyanobacterial filaments ¼ Laudatea type,
basidiocarp fully supported underneath the vegetative thallus
structure ¼ Cora type). For species in which basidiocarps are
yet unknown (including Acantholichen and Corella), character
6 was scored as missing data (‘?’). We did not use the character
clamp connections, since this is a strictly mycological feature
not related to thallus morphology per se and species with
clamp connections occurred very scattered in our dataset.
Chemical data
Morphological data
Each sequenced species, including the outgroup, was scored
for six morpho-anatomical characters that determine the development of the lichen thallus (Table 1; see also Fig 3): (1)
lichenisation (absent, present, and with Rhizonema), (2) thallus
In addition to morphology and anatomy, we analysed 64 samples of Dictyonema (8), Acantholichen (1), Cora (50), and Corella (5)
by means of thin-layer chromatography (Orange et al. 2001) to
detect the possible presence of secondary substances, as suggested by an earlier study (Piovano et al. 1995).
Fig 1 e Best-scoring ML tree obtained from a three-gene dataset via RAxML. Supported branches are indicated by thick lines
and bootstrap support values as well as posterior probabilities from a separate Bayesian analysis are given. The five genuslevel clades and grades (Cyphellostereum, Dictyonema, Acantholichen, Corella, Cora) are highlighted. The columns to the right
indicate the CMI reflecting thallus development and the number of nodes from the root to the terminal leaves.
588
Molecular data
Genomic DNA was extracted from lichenized thalli using the
Bio 101 Fast DNA Spin Kit for tissue (Qbiogene, Illkirch, France)
according to the manufacturer’s protocol with slight modifications. About 10 ng of extracted DNA were subjected to a standard PCR in a 25 mL reaction volume using either Taq Gold
polymerase (Applied Biosystems, Foster City, CA, USA) or
Bio-X-Act Long Mix (Bioline USA, Taunton, MA, USA) according to manufacturer’s protocols. Sequence data were obtained
from ITS (ITS1, 5.8S, and ITS2), and nuLSU nuclear rDNA (approximately 600 and 1400 bp, respectively), as well as RPB2 (between the 6 and 7 conserved domains). After visualising the
PCR products on a 1 % agarose gel with ethidium bromide
and confirming the size, the products were purified with magnetic beads (Agencourt Bioscience, Beverly, MA, USA). The purified PCR products were used in standard sequencing
reactions with BigDye Terminator Ready Reaction Mix v3.1
(Applied Biosystems). The primers used were LR0R, LR3R,
LR5, LR7, LR16, ITS4, and ITS5 (http://www.biology.duke.edu/
fungi/mycolab/primers.htm) and basidiomycete specific
primers bRPB2-6F or bRBP2-5F and bRBP2-7R, bRBP2-7R2 or
bRPB2-7.1R (Denton et al. 1998; Liu et al. 1999; Matheny 2005).
The sequencing reactions were then purified using Sephadex
G-50 (SigmaeAldrich, St. Louis, MO, USA), dried in a speedvac,
denatured in HiDi Formamide (Applied Biosystems) and run
on an ABI3130-xl capillary sequencer (Applied Biosystems).
The data collected were analysed using ABI software, and
500e700 bases were collected for each primer used. These sequences were then assembled together with the software
Sequencher version 5.0 (Gene Codes, Ann Arbor, MI, USA) for
manual corrections in base calling and to make contiguous
alignments of overlapping fragments.
M. Dal-Forno et al.
We used the Shimodaira-Hasegawa (SH) test incorporated
in RAxML 7.2.6 to test hypotheses about putative monophyly
of certain groups, particularly Dictyonema s.str..
The Heads-or-Tails (HoT) scores provided by the GUIDANCE web server (Penn et al. 2010a 2010b) were used to quantify differences in evolution of ITS length variation between
clades. For this purpose, the ITS alignment for each clade
was separately submitted to the GUIDANCE web server and
both the overall alignment confidence scores and the confidence scores for each sequence were retrieved. The scores
were compared among clades using a nonparametric KruskaleWallis ANOVA in STATISTICA 6.0.
Ancestral character state reconstruction and lichen thallus
development
Ancestral character state reconstruction was done using the
morphological dataset and both the best-scoring ML tree and
the Bayesian tree sample (1500 trees). The Trace Character
History and Trace Characters over Tree functions in MESQUITE 2.75 (Maddison & Maddison 2012) were used to visualize character evolution, employing likelihood ancestral
character states.
In addition, we applied a linear correlation test between
the combined score of all morphological characters for each
species (CMI ¼ combined morphology index) and the number
of nodes for each species from the root to the terminal leaf in
the best-scoring ML tree, to test whether the lichen thallus is
progressively more complex along the phylogeny of the clade.
The linear correlation was done in STATISTICA 6.0 employing
both the parametric Pearson and the nonparametric Spearman rank correlation coefficients.
Sequence alignment and phylogenetic analysis
Results
Newly generated sequences were assembled with sequences
from Genbank using BIOEDIT 7.09 (Hall 1999) and automatically aligned with the program MAFFT using the eauto option
(Katoh & Toh 2005). The individual nuLSU and ITS alignments
were subjected to analysis of ambiguously aligned regions using the GUIDANCE web server (Penn et al. 2010a, 2010b) and introns and regions aligned with low confidence (below 0.90),
particularly in the ITS, were removed. This resulted in an
alignment length of 1327 for the nuLSU, 600 for the ITS, and
1021 for the RPB2 partition, for a total of 2948 sites in the combined dataset. The alignments were subjected to maximum
likelihood (ML) search using RAxML 7.2.6 (Stamatakis et al.
2005; Stamatakis 2006), with parametric bootstrapping using
500 replicates under the GTRGAMMA model. Each gene was
first analysed separately and all data were eventually combined since no conflict was detected after removal of ambiguously aligned regions.
The dataset was also analysed under a Bayesian framework using MrBAYES 3.1.2 (Huelsenbeck & Ronquist 2001),
with two independent runs, a total chain length of one million
generations, and four separate chains each, resampling every
1000 trees and generating a majority rule consensus tree from
the tree sample after discarding 25 % burnin to obtain posterior probability estimates.
Phylogenetic analysis
ML analysis of the combined three-gene dataset of the selected species sequences resulted in a well-supported topology in which Cyphellostereum was monophyletic and sister to
the remaining species (Fig 1). Dictyonema s.str. formed a paraphyletic grade with Acantholichen, Cora, and Corella emerging
as a supported, monophyletic clade. Acantholichen plus Corella
formed a supported, monophyletic clade sister to Cora. No
support was found in the backbone of the Dictyonema s.str.
grade and, consequently, monophyly of Dictyonema s.str. could
not be rejected by the SH test (at both the 0.05 and 0.01 level).
Bayesian analysis of the same dataset resulted in a congruent topology with strong support for the aforementioned
clades (tree not shown but posterior probabilities plotted on
the ML tree in Fig 1). Thus, the phylogenetic analysis supports
the recognition of up to five genera: Cyphellostereum, Dictyonema s.str., Acantholichen, Corella, and Cora.
Ancestral character state reconstruction and lichen thallus
development
Ancestral character state analysis showed a well-supported
progression of morphological and anatomical features in
Evolution in Dictyonema
Fig 2 e Ancestral character state reconstruction based on
the best-scoring ML tree. Bayesian tree sample analysis (not
shown) resulted in identical reconstructions.
the clade, both based on ML tree reconstruction (Fig 2) and
Bayesian tree sampling (results identical, not shown). The
lichenized thallus progresses from appressed, crustose filamentous forms in Cyphellostereum (Fig 3A, B) and Dictyonema
(Fig 3EeI) to microsquamulose thalli in Acantholichen (Fig 3M,
N) and macrosquamulose to large foliose forms in Corella
(Fig 3O) and Cora (Fig 3Q, R, W, X). Cora develops a loose,
corticiform layer of basally anticlinal and peripherally perpendicular hyphae (Fig 3U), while Corella has a true,
589
paraplectenchymatous cortex (Fig 3P). The photobiont maintains its filamentous morphology in the filamentous thallus
types (Fig 3C, K, L) and breaks into irregular groups of cells
in the squamulose and foliose forms (Fig 3P, V). The hyphal
sheath in Cyphellostereum is composed of irregular, cylindrical
hyphae leaving large interspaces (Fig 3C, D), whereas in all
other taxa it forms a closed, paraplectenchymatous layer
composed of jigsaw-puzzle-shaped cells (Fig 3L, P, V). This correlates with the absence of tubular intracellular haustoria in
Cyphellostereum and their presence in the other genera. Coincidentally, the cyanobacterial hyphae are narrow in Cyphellostereum (5e8 mm) and broad in the other genera [8e12(e20) mm].
Cyphellostereum has cyphelloid basidiocarps emerging from
the otherwise undifferentiated lichenized thallus (Fig 3A),
with the entire basidiocarp free of photobiont filaments; instead, the hyphae associated with the cyanobacterial filaments appear to represent vegetative hyphae, as they are
thinner and different from the generative hyphae forming
the basidiocarp. In contrast, most species of Dictyonema have
stereoidecorticioid basidiocarps emerging from the underside
of the lichenized thallus and usually at least partly overgrown
with photobiont filaments (Fig 3G, H); the hyphae associated
with the cyanobacterial filaments are thicker (4e6 mm) than
in Cyphellostereum (2e3 mm) and indistinguishable from the
generative hyphae supporting the hymenophore. Most species of Cora and also Dictyonema sericeum have flat, corticioid
hymenophores developing on the lobe underside and completely incorporated into the thallus (Fig 3J, S, T), meaning
that the thallus entirely supports the hymenophore but is in
itself not transformed in shape when fertile.
The progressive development of the lichen thallus, as represented by the CMI, is strongly and significantly correlated
with the number of nodes from the root to the terminal leaves;
i.e., early-diverging species closer to the root have a lessdeveloped thallus, with clear differentiation of thallus and
basidiocarps, compared to late-diverging species far from
the root (Fig 4).
In addition to the phylogenetic progression in morphologicaleanatomical features, the three major clades and grades
also exhibit distinctive patterns of sequence evolution, particularly in the ITS1 and ITS2 regions (Fig 5). Species currently
assigned to Cyphellostereum have extremely variable, partially
unalignable sequence portions in both the ITS1 and ITS2 region (overall alignment confidence score ¼ 0.784). Species contained within Dictyonema s.str. also have highly lengthvariable ITS1 and ITS2 regions but their alignment is less ambiguous (overall alignment confidence score ¼ 0.862). Species
classified as Cora have substantially less length variation in
the ITS and the level of ambiguity is significantly lower (overall alignment confidence score ¼ 0.954). The differences are
highly significant at the p ¼ 0.0001 level (Fig 6).
Discussion
Lichens, especially ascolichens, exhibit a remarkable diversity
of thallus morphologies, and this variation has traditionally
provided the basis for delimiting taxonomic groups. However,
it is now apparent that thallus morphology is not always
a good indicator of phylogenetic relationships among
590
M. Dal-Forno et al.
Fig 3 e Thallus development and morphological and anatomical characters in Dictyonema s.lat.. (A) Cyphellostereum pusiolum
(Brazil, Sulzbacher & Coelho 1480), filamentousecrustose thallus with emerging, nonlichenized, cyphelloid basidiocarps. (B)
€ cking 15207a), filamentousecrustose thallus enlarged. (C) C. phyllogenum (Costa Rica, Lu
€ cking
C. phyllogenum (Costa Rica, Lu
15207a), cyanobacterial filaments with loose hyphal sheath formed by irregular hyphae. (D) C. imperfectum (holotype), dense
€ cking s.n.), filamentousecrustose
hyphal sheath formed by irregular hyphae. (E) Dictyonema phyllophilum (Costa Rica, Lu
thallus enlarged. (F) D. hernandezii (holotype), filamentousecrustose thallus enlarged. (G, H) D. schenkianum (Costa Rica,
€ cking 17200), filamentousecrustose thallus with emerging, partially integrated, cyphelloidestereoid basidiocarps. (I)
Lu
€ cking 25551b), filamentous, shelf-like thallus. (J) D. sericeum (Guatemala, Lu
€ cking 25551b), filaD. sericeum (Guatemala, Lu
mentous, shelf-like thallus with fully supported, corticioid basidiocarps on the underside. (K) D. phyllophilum (Costa Rica,
€ cking 17252i), cyanobacterial filaments with heterocysts. (L) D. schenkianum (Brazil, Lu
€ cking 30060), cyanobacterial filaments
Lu
with hyphal sheath of jigsaw-puzzle-shaped cells. (M) Acantholichen pannarioides (Costa Rica, Chaves 3910), microsquamulose
thallus competing with other lichens. (N) A. pannarioides (Costa Rica, Sipman 48329), squamules enlarged. (O) C. brasiliensis
€ cking 35314), section through
(Costa Rica, Dal-Forno 1768), macrosquamuloseefoliose thallus. (P) C. brasiliensis (Colombia, Lu
thallus showing groups of photobiont cells wrapped in paraplectenchymatous sheath and paraplectenchymatous true cor€ cking s.n.), terrestrial thallus between
€ cking s.n.), epiphytic thallus. (R) C. pavonia (Ecuador, Lu
tex. (Q) Cora aspera (Colombia, Lu
Evolution in Dictyonema
Fig 4 e Linear correlation between the number of nodes
between root and terminal leaf for each species and the
CMI.
lichenized fungi (Grube & Hawksworth 2007; Rivas Plata &
Lumbsch 2011; Lumbsch & Leavitt 2011; Gaya et al. 2012).
Even among basidiolichens that generally have simpler and
less variable thalli, identical morphologies arose independently in unrelated groups, such as Multiclavula, Lepidostroma,
and Lichenomphalia (Oberwinker 1970, 1984, 2012; Ertz et al.
€ cking 2013). In
2008; Sulzbacher et al. 2012; Hodkinson & Lu
the Dictyonema clade, however, thallus morphology is strongly
correlated with phylogeny: ancestral character state reconstructions indicate a noticeable progression from a simple,
undifferentiated vegetative thallus with separate cyphelloid
basidiocarps in the early-diverging Cyphellostereum to forms
in which basidiocarps themselves appear to dominate the lichen thallus, with individual hymenophores regularly dispersed over the thallus underside, in the late-diverging Cora.
This suggests that the structure of the basidiocarp and its
gradual incorporation into the lichen thallus might be responsible for thallus formation in these lichenized species, and it
may also explain why the most derived species of Cora and
Corella closely resemble shelf-like stereoid macrofungi.
Oberwinkler (1970) regarded the Dictyonema s.lat. thallus as
lichenized fruiting body, a view shared by other workers
(Parmasto 1978; Slocum 1980; Ryan 2002; Trembley et al.
mençon et al. (2004) remarked that hyme2002b). Similarly, Cle
nium development in Cora glabrata may be interpreted as an
aggregation of simple cyphelloid basidiomes (the hymenophore) on the undersurface of a lichenized ‘stroma’. This
interpretation is supported by the observation that in Dictyonema s.str., Acantholichen, Corella, and Cora, the hyphae forming the ‘vegetative’ thallus (cortex, medulla, photobiont
591
layer) resemble those producing the hymenophore, and hence
can be interpreted as generative hyphae, whereas in Cyphellostereum, the hyphae associated with the photobiont filaments
strongly differ from the generative hyphae forming the basidiocarp. As a consequence, it is no longer possible to attribute
morphological differentiation in this clade as ontogenetic or
€ ller
ecological variation, as done by previous workers (Mo
1893; Oberwinkler 1970, 2001; Parmasto 1978; Larcher &
Vareschi 1988). Instead, this ‘variation’ reflects distinct evolutionary patterns resulting in a large number of species- and
genus-level clades, a view also accepted by Oberwinkler
(2012).
The observed trends in thallus evolution, along with the
morphological transformation of the basidiocarps, are restricted to lichenized forms and therefore likely caused by
lichenisation. The immediate, nonlichenized ancestors of Dictyonema, Eonema pyriforme, and the genus Arrhenia s.lat.
(Redhead et al. 2002; Barrasa & Rico 2003; Lawrey et al. 2009),
have mostly dorsiventral, arrhenioid (with gills) or cyphelloid
(without gills) basidiocarps, the latter resembling those of
Cyphellostereum. Most other members of the family Hygrophoraceae have radially symmetrical, agaricoid-omphalinoid
basidiocarps, including the lichenized genus Lichenomphalia,
which forms only crustose to microsquamulose thalli in
which the basidiocarps are not integrated into the lichen thallus (Oberwinkler 1970, 2001, 2012; Lutzoni & Vilgalys 1995;
Lutzoni 1997; Redhead et al. 2002; Lawrey et al. 2009; Lodge
et al., in preparation). We conclude that the evolution of dorsiventral basidiocarps in Arrhenia and Eonema facilitated the
subsequent formation of an integrated lichen thallus in Dictyonema. Our reasoning is that flattened, dorsiventral basidiocarps can incorporate photobionts readily into dorsal sterile
portions exposed to light. In radially symmetrical basidiocarps
(as in Lichenomphalia), the hymenophore separates the sterile
cap from the sterile stipe, and hence also from the substrate
where the interaction between mycobiont and photobiont occurs. The structurally complex thallus in Cora was regarded by
Oberwinkler (1984: 748) as ‘. ecologically very well adapted symbiotic structure’, which is reflected in the abundance of these lichens compared to their mushroom-like relatives (i.e.,
Cyphellostereum and Lichenomphalia). In tropical paramo regions and comparable habitats, Cora lichens occur in dozens
to hundreds of individuals per m2 over extensions of many
km2 (Fig 7A), values never observed for any other known basidiolichen. While most basidiolichens are pioneer species on
open soil, Cora lichens compete successfully with ascolichens
with similar morphologies occurring in the same habitats,
such as Coccocarpia, Coenogonium, Hypotrachyna, Lobariella, Normandina, Peltigera, and Sticta, among others (Bigelow 1970;
bryophytes and small vascular plants. (S) C. strigosa (Peru, Paz 3), thallus underside showing fully supported hymenophore.
€ cking 21017), hymenophore enlarged. (U) Cora aff. squamiformis (Colombia, Lu
€ cking 35312),
(T) C. arachnoidea (Costa Rica, Lu
section through thallus showing loose upper cortex. (V) Same material, section through thallus showing groups of photo€ cking s.n.), undescribed epiphytic species
biont cells wrapped in paraplectenchymatous sheath. (W) Cora sp. (Costa Rica, Lu
€ cking s.n.), undescribed terrestrial species forming
with green thallus forming concentric ridges. (X) Cora sp. (Colombia, Lu
€ cking except A (Marcelo Sulzbacher). Scale in A, I, Q, R,
small white squamules with brownish margin. Photographs by R. Lu
W [ 20 mm, in J, M, S [ 10 mm, in G, H, O, X [ 5 mm, in B, E, F, N [ 1 mm, in U [ 50 mm, in C, D, K, L, P, V [ 10 mm. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
592
M. Dal-Forno et al.
Fig 5 e Selected ITS alignments obtained via the GUIDANCE web server for Cyphellostereum, Dictyonema, and Cora. Purple
colours denote high and blue colours denote low confidence scores, also expressed by the columns (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.).
Barrasa & Rico 2001; Brodo et al. 2001; Chaves et al. 2004; Smith
et al. 2009).
Within Dictyonema s.lat., all species associate with Scytonema-like Rhizonema cyanobionts as primary photobionts,
and these appear to be obligately lichenized and largely tropical, occurring also in other, unrelated but ecologically similar
€ cking et al. 2009). In terms of the level of uniquelichens (Lu
ness, lichenisation appears to differ in each genus. Species
in Cyphellostereum, although always having Rhizonema as primary photobiont, commonly harbour admixtures of cyanobacteria and even unicellular chlorophytes, suggesting a
more loose association of the fungus with diverse
photobionts. Oberwinkler (2001, 2012) mentioned green algae
as primary photobionts of certain Cyphellostereum species.
However, in the abundant fresh and herbarium material revised and sequenced by us, all species feature cyanobacterial
Rhizonema as primary photobionts (Lawrey et al. 2009; Lawrey
€ cking et al. 2013b). Most likely, forms
et al., in preparation; Lu
with a primary green algal photobiont represent species of
Semiomphalina, which is more closely related to Lichenomphalia
than to Dictyonema (Dal-Forno et al., in preparation). Such
a specimen was in fact erroneously depicted as Dictyonema
sp. by us (Lawrey et al. 2009: fig 9D). Dictyonema s.str., Acantholichen, Cora, and Corella species always form associations with
Evolution in Dictyonema
Fig 6 e KruskaleWallis ANOVA of the variation of the individual ITS sequence alignment confidence scores for Cyphellostereum (n [ 4), Dictyonema (n [ 9), and Cora (n [ 10).
a single, dominant photobiont, as apparent from 454 sequencing data (Dal-Forno et al., in preparation). Morphologically,
photobionts in Cyphellostereum and Dictyonema s.str. resemble
free-living Scytonema trichomes, whereas in the derived Cora
and Corella clades, the trichomes are coiled and broken, forming coiled aggregates of more rounded cells somewhat similar
mençon
to Chroococcus (Parmasto 1978; Chaves et al. 2004; Cle
€ cking et al. 2009). This difference was discussed
et al. 2004; Lu
€ ller 1893; Ma
€ gdefrau & Winkler 1967;
by other authors (Mo
Tomaselli & Caretta 1969; Oberwinkler 1970; Ryan 2002) and
even led to the establishment of a separate lichenized genus,
Wainiocora, supposedly differing from Cora by a Chroococcus
photobiont (Tomaselli 1950, 1951). Oberwinkler (1970),
Parmasto (1978), and Chaves et al. (2004) suggested that these
morphotypes represent the same photobiont, which was confirmed by molecular phylogenetic analysis showing that this
variation is not due to a photobiont switch but caused by
fungal-specific morphogenetic effects on the photobiont
€ cking et al. 2009, 2013b). Thus, in addition to progressive
(Lu
thallus development, the Dictyonema s.lat. clade also exhibits
a gradual evolution towards stable symbiotic interactions between its bionts, with an increased morphological dominance
of the mycobiont over the photobiont, also expressed by the
nature of the haustoria.
The particular and unique haustoria of Dictyonema s.lat.
have been described and discussed in detail by several authors
(Roskin 1970; Slocum & Floyd 1977; Slocum 1980; Oberwinkler
1980, 1984, 2001, 2012). They are present in Dictyonema s.str.,
Acantholichen, Cora, and Corella, but not in Cyphellostereum.
We also found a strict correlation between the presence of
haustoria, the shape of the hyphal sheath around the photobiont cells, and photobiont morphology. Taxa with haustoria
always feature a hyphal sheath formed by jigsaw-puzzleshaped cells, with the sheath being tubular around the photobiont filaments in Dictyonema s.str. and orbicular around the
irregular photobiont cell groups in Acantholichen, Corella, and
Cora. Both haustoria and sheath are absent in Cyphellostereum.
Remarkably, the cyanobacterial filaments are much narrower
in Cyphellostereum than in the other genera, which we initially
interpreted as a different photobiont species. However, 454 sequencing data indicate the primary photobiont to represent
593
the same clade in all observed lichens (Lawrey et al., in preparation). This apparently conflicting result is explained by the
effect of the intracellular haustoria: apparently, the cyanobacterial filaments in Cyphellostereum, which lack haustoria, represent the natural cell width, whereas the haustorial hyphae
in Dictyonema, Acantholichen, Corella, and Cora cause lateral ‘inflation’ of the penetrated photobiont cells. On the other hand,
even in taxa with haustoria present, the cell width of the pho€ cking et al. 2013a,
tobiont can vary according to species (Lu
2013b).
A differentiated cortex is a particular achievement of lichen thalli, as it provides a certain level of protection (Jahns
1988; Honegger 2001; Sanders 2001b). In many lichens, this
correlates with the production of particular secondary metabolites in the cortex acting as sun-screens, such as atranorin,
usnic acid, parietin, and melanin (Rundel 1978; Lawrey 1986;
ndez et al. 1996; Gauslaa &
Solhaug & Gauslaa 1996; Ferna
Solhaug 2001; Bjerke & Dahl 2002; Gauslaa 2009). In the Dictyonema clade, only the highly evolved foliose forms represented
by the genera Cora and Corella feature a cortex. The cortex is
different in both genera, suggesting independent evolution,
which is supported by their phylogeny, with Corella being sister to the squamulose, ecorticate Acantholichen, and Cora forming a separate clade. The unique, medullary upper cortex in
Cora and the differences compared to Corella were already
noted by earlier workers (Zahlbruckner 1926; Metzner 1934;
Tomaselli 1950; Ozenda 1963; Tomaselli & Caretta 1969;
Xavier Filho & Vicente 1979). Oberwinkler (1970) interpreted
the paraplectenchymatous cortex of Corella as ‘. kollabierte
Hyphen .’ (collapsed hyphae) and Parmasto (1978) did not recognize the difference as significant or of taxonomic importance. Our results show that this difference is structural and
highly consistent, and the phylogeny, with Corella being sister
to Acantholichen, even suggests that the foliose thalli of Corella
might have evolved independently from those of Cora. The
particular cortex of Corella species appears to be directly derived from the paraplectenchymatous sheath enclosing the
photobiont, forming an extra, continuous layer on the surface
directly above the photobiont layer.
Piovano et al. (1995) reported the presence of atranorin
and tenuiorin in material of Dictyonema glabratum, now in
the genus Cora. These are substances commonly found in
large macrolichens in the Ascomycota, such as Lobariaceae,
Parmeliaceae, and Peltigeraceae (Huneck & Yoshimura 1996;
Miadlikowska & Lutzoni 2000; Moncada et al. 2013), and their
presence in Cora would imply a remarkable level of parallel
evolution in completely unrelated lineages with comparable
ecology. However, our analysis of 64 samples of Cora, Corella,
Acantholichen, and Dictyonema, resulted in the complete absence of acetone-soluble compounds deposited extracellularly in the hyphal walls. This is supported by the colour
change in wettened Cora lichens, which become much darker
than in dry state, very different from Lobariaceae and Parmeliaceae that have atranorin as cortical compound. We therefore consider the result by Piovano et al. (1995) as artifactual,
since it is highly unlikely that only a species or population in
Chile would produce these substances, whereas samples
from Mexico, Costa Rica, Colombia, Ecuador, Bolivia, and Brazil studied by us contained no secondary compounds. Another
chemical study (Xavier Filho et al. 1980) reported
594
Fig 7 e (A) A Cora species growing abundantly on the ground in
the Colombian paramo, competing with bryophytes, small
vascular plants, and other lichens. (B) Cyphellostereum basidiocarps emerging from inconspicuous, terricolous thalli. (C) Cora
cyphellifera from Ecuador (holotype), showing cyphelloid hymenophores similar to those of Cyphellostereum. (D) Cladonia
squamules growing in the centre of a cyanobacterial Coccocarpia
species from the Philippines, resembling a photosymbiodeme
and demonstrating how different lichens growing intermingled
can misguide even the experienced observer. Photographs by
€ cking except C (Manuela Dal-Forno). Scale [ 10 mm.
R. Lu
M. Dal-Forno et al.
phytohaemagglutinin from Dictyonema sericeum, D. glabratum,
and Corella brasiliensis, but these are intracellular substances
not comparable to extracellular secondary compounds in lichens. The same applies to the proteins, lipids, and carbohydrates reported by Elifio et al. (2000), Sassaki et al. (2001), and
Carbonero et al. (2002) from D. glabratum. Phytohaemagglutinin, found for example in legumes, triggers blood agglutination and is also assumed to play a role in early stages of
lichen symbiosis (Lockhart et al. 1978). The occurrence of
this substance has not been much studied, but there are reports from Peltigera (Lockhart et al. 1978), which makes it unlikely that the shared occurrence in Dictyonema, Cora, and
Corella has any phylogenetic significance.
The Dictyonema clade is a prime example of how interpretation of morphological differentiation as ontogenetic or ecological variation, even if based on detailed field observations, can
lead to misinterpretations about the evolution and classification of a group of organisms. In this case, we refer to the stud€ ller (1893) and Larcher & Vareschi (1988), discussed
ies by Mo
by others (Oberwinkler 1970, 2001, 2012; Parmasto 1978) as potential evidence for ecomorphological and ontogenetic varia€ ller (1893) provided a lengthy
tion in these lichens. Mo
account on the ontogeny and seasonal variation of Dictyonema
s.lat. lichens depending on ecological conditions, based on
field observations over several years. While this study is
unique in its approach, it merges different taxa, at the time
unknown to the author, to document ‘variation’. For example,
the nonlichenized, terricolous basidiomata considered by
€ ller (1893) to represent ‘free-living’ Cora mushrooms are
Mo
in reality species of Cyphellostereum, which often cooccur
with Cora in the same habitat (Fig 7B). He also mentioned
that certain Cora lichens, with bluish thalli, produced the
same cyphelloid basidiocarps, considering this evidence for
the conspecificity of all these elements. These are likely to rep€ cking
resent Cora cyphellifera, a new species described by us (Lu
€ ller (1893) also
et al. 2013a) from northern Ecuador (Fig 7C). Mo
observed foliose Cora thalli growing out of filamentous Dictyonema and concluded that one and the same fungus was involved and simply changed its morphology due to switching
from a Scytonema-like to a Chroococcus-like photobiont. While
such statements were revolutionary for his time and actually
hold true in several lichen lineages, such as the photosymbiodemes in Lobariaceae and Pannariaceae (James & Henssen
1976; Brodo & Richardson 1978; Jahns 1988; Armaleo & Clerc
1991; Tschermak-Woess 1995; Sanders 2001a; Tønsberg &
€ rgo
€ tter 2002; Takahashi et al. 2006),
Goward 2001; Stocker-Wo
in this particular case they are incorrect and based on accidental observation of two different lichens growing together. Such
phenomena are not rare; for example, we observed Cladonia
squamules growing in the centre of a Coccocarpia thallus suggesting a photosymbiodeme (Fig 7D). However, in the hundreds of collections and field individuals seen by us, we
never found evidence of Cora-like thalli developing from Dictyonema-like forms, and the phylogenetic data clearly do not
support this idea: in instances where we collected specimens
of Cora, Dictyonema or Cyphellostereum growing closely together, sequence data always showed that they represented
different taxa.
A similar case is the study by Larcher & Vareschi
(1988), who attributed morphological differentiation in four
Evolution in Dictyonema
populations of what they identified as a single species, D. glabratum, as habitat-induced. Unfortunately, one of their populations, actually labelled by them (Larcher & Vareschi 1988: 272)
as f. brasiliense, represents C. brasiliensis and thus not only a different species, but a different genus. They correctly observed
the differences in cortex type between true Cora and Corella,
but their interpretation of this difference as habitat-induced
is misguided. We also suspect that the other populations studied by these authors represent three different Cora species, as
€ cking et al.
this genus is highly speciose in the Andes (Lu
2013a), but without revising their material, this is difficult to
ascertain.
The observed variation in ITS sequences among the different clades, with reduced variation in more derived clades, suggests that Cyphellostereum represents a group that diverged
earlier, whereas Cora is a relatively young clade. A dating
€ cking et al.
study employing a relaxed molecular clock (Lu
2013c) suggests the early divergence of Dictyonema s.lat. to
have taken place about 45 mya during the Eocene, with the
Cyphellostereum crown node estimated at 35 mya in the late Eocene, whereas Cora diversified during the early Miocene, about
20 mya. These results support our hypothesis that the morphologically primitive Cyphellostereum species are relicts
from an earlier radiation, with many species now extinct,
whereas Cora represents a more recent radiation, with unrecognized and partially cryptic species diversity. The relatively
young age of the Dictyonema clade, together with the evidence
of progression in thallus morphology and anatomy, has been
viewed as witnessing the ‘birth’ of lichenisation in this clade
€ cking et al. 2013c). Notably, there are
of Basidiomycota (Lu
some lineages in Ascomycota, specifically in class Dothideomycetes, which also form filamentous lichens and produce
hyphal sheaths comparable to those of Dictyonema s.str.; these
are the genera Cystocoleus, Racodium, and Racoleus (Gauckler
1960; Nelsen et al. 2009; Hawksworth et al. 2011). If our viewpoint is correct, these might represent other recently emerging lichenized lineages on their way to evolving competitive
lichen thalli. It should, however, be pointed out that basidiolichens, such as the clade studied here, cannot be used as
model for thallus evolution in ascolichens, since the underlying conditions are different. In Basidiomycota, the dikaryotic
mycelium can live in a vegetative stage and eventually produce basidiocarps, which make the formation of a thallus by
integrating the basidiocarp a logical evolutionary step. In
Ascomycota, the vegetative mycelium is always formed by
a haploid mycelium, whereas dikaryotic hyphae only produce
ascocarps. We therefore have to assume that thallus evolution
in ascolichens followed different pathways.
Acknowledgements
Financial support for this study was provided by grant DEB
0841405 from the National Science Foundation to George Mason University: ‘Phylogenetic Diversity of Mycobionts and
Photobionts in the Cyanolichen Genus Dictyonema, with Emphasis on the Neotropics and the Galapagos Islands’ (PI: J.
€ cking, P. Gillevet). Material was also colLawrey; CoPIs: R. Lu
lected as part of grant DEB-0715660 to The Field Museum:
595
‘Neotropical Epiphytic Microlichens e An Innovative Inventory of a Highly Diverse yet Little Known Group of Symbiotic
€ cking) and DEB-0206125 to The Field MuOrganisms’ (PI: R. Lu
€ cking), as well as two lichen
seum: ‘TICOLICHEN’ (PI: R. Lu
courses as part of the Organization for Tropical Studies
ndez, Thorsten
s Herna
(OTS) speciality courses syllabus. Jesu
Lumbsch, Elias Paz, Luis Salcedo, and Karina Wilk provided
some of the material used in this study. We thank William
Sanders for fruitful discussions on the evolution of the lichen
thallus and we are indebted to Franz Oberwinkler for critical
revision of a previous version of this manuscript, which
helped to improve it considerably.
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