Content uploaded by Zuzana Ferencova
Author content
All content in this area was uploaded by Zuzana Ferencova
Content may be subject to copyright.
A discussion about reproductive modes of Pseudevernia
furfuracea based on phylogenetic data
Zuzana FERENCOVA, Ruth DEL PRADO,
Israel PÉREZ-VARGAS, Consuelo HERNÁNDEZ-PADRÓN and
Ana CRESPO
Abstract: Two asexual reproductive strategies of the common lichen Pseudevernia furfuracea are
described. Although the species propagates mainly by isidia, some specimens also show the develop-
ment of soralia. Morphological, chemical and molecular analyses were performed on three such
sorediate specimens from the Canary Islands, Morocco and Turkey. Maximum parsimony, maximum
likelihood and Bayesian analyses indicate that: a) sorediate samples represent only a morphological
variant of the reproductive mode and b) the separation of taxa (at species level or below) on the basis
of their containing either olivetoric acid or physodic and oxyphysodic acids is not appropriate. In
addition, a phylogenetic reconstruction of the genus Pseudevernia is presented for the first time. The
tree shows two sister monophyletic clades, one containing American species (P. intensa, P. cladonia,
P. consocians), and the second encompassing the P. furfuracea samples (including sorediate specimens).
The biological and taxonomic significance of soralia in sorediate samples is discussed.
Keywords: isidia, lichens, Parmeliaceae, soredia, species pairs, vegetative modes
Introduction
Lichens are stable symbiotic organisms in
which only the fungal partner is able to carry
out sexual reproduction. The development
of special dispersal structures containing
both symbionts, such as soredia or isidia,
together with thallus fragmentation, allow
the efficient propagation of the symbiotic
dual organism. Soredia have been inter-
preted as being vegetative propagules se-
lected during the evolutionary history of
lichens (Poelt 1970, 1972) and have been
accorded significant status as taxonomic
characters. Earlier investigations defined
‘species pairs’ in lichens as two species, one
fertile without vegetative propagules (pri-
mary species) and the other with soredia or
isidia (secondary species); the latter being
derived from the former. Hale (1965) pre-
sumed in his work on Parmelia (subgenus
Amphigymnia) that sorediate species evolved
from either isidiate or nonsorediate–
nonisidiate ancestors, naming the two or
three taxa involved ‘counterparts’.
The classic interpretation among li-
chenologists supports taxonomic separation
at the species level for both members of a
species pair. However, studies based on
morphological and ecological information
(Tehler 1982) or phylogenetic analysis
(Lohtander et al. 1998a, b; Myllys et al. 1999,
2001; Articus et al. 2002; Molina et al. 2002;
Cubero et al. 2004; Buschbom & Mueller
2006) show, at least for sorediate/
nonsorediate species pairs, that these forms
do not separate into monophyletic lineages.
Consequently, pairs have been interpreted as
representing two dispersal strategies and the
specific status of each member of the pair has
not been corroborated. Few cases of isidiate/
Z. Ferencova, R. Del Prado and A. Crespo (correspond-
ing author): Departamento de Biolog ı´a Vegetal II,
Facultad de Farmacia, Universidad Complutense de
Madrid, E-28040 Madrid, Spain.
Email: acrespo@farm.ucm.es
I. Pérez-Vargas and C. Hernández-Padrón: Departa-
mento de Biologı´a Vegetal, Facultad de Farmacia, Uni-
versidad de La Laguna, E-38071, La Laguna, Tenerife,
Canary Islands, Spain.
The Lichenologist 42(4): 449–460 (2010) © British Lichen Society, 2010
doi:10.1017/S0024282909990739
non-isidiate pairs have been studied, and
recent results based on molecular techniques
do not provide evidence of any clear ‘sister’
phylogenetic relationship for members of
such ‘species pairs’ (Argüello et al. 2007).
It appears that the occurrence of isidia or
soredia, particularly the latter, is a character
that has been gained and lost in the phy-
logeny of lichens (Lohtander et al. 1998a, b;
Myllys et al. 1999, 2001; Articus et al. 2002;
Molina et al. 2002; Cubero et al. 2004;
Buschbom & Mueller 2006). In many groups
of lichens there are species bearing isidia or
soredia but there are few, if any, examples of
lichens bearing both kinds of structures at the
same time. One vegetative dispersal strategy
usually predominates in evolutionary
monophyletic branches in the Parmeliaceae.
Vegetative propagating members of the gen-
era Xanthoparmelia (Hale 1990), Melanelixia
(Blanco et al. 2004), Parmelina (Hale 1976)
and Pseudevernia (Hale 1968) mostly pro-
duce isidia, whereas the majority of com-
monly sterile species in Parmotrema and
Flavoparmelia (Hale 1965) disperse by
means of soredia.
The genus Pseudevernia (a member of the
family Parmeliaceae), which is thought to be
closely related to Brodoa and Hypogymnia
(Crespo et al. 2007), comprises only a small
number of species. Depending on the taxo-
nomic concepts applied, either six species
(Hale 1968) or only four species (recent con-
cepts) are accepted. Three well defined
species containing lecanoric acid [P. conso-
cians (Vain.) Hale & W. Culb. with isidia,
P. cladonia (Tuck.) Hale & W. Culb. and
P. intensa (Nyl.) Hale & W. Culb. without
isidia] occur in North America (see distri-
bution map in Hale 1968). Three other isidi-
ate taxa, namely P. furfuracea (L.) Zopf (with
physodic and oxyphysodic acids), P. oliveto-
rina (Zopf) Zopf (with olivetoric acid) and
P. soralifera (Bitt.) Zopf (with physodic and
oxyphysodic acids; with soralia in addition to
isidia), which were treated by Hale (1968)
as separate species, are classified by several
European lichenologists (e.g., Clerc 2004)
within a single entity, P. furfuracea.
Pseudevernia furfuracea (L.) Zopf is a com-
mon and widespread species in Eurasia and
North Africa; there are some very occasional
reports from Mexico, Central America,
Bolivia and Uganda (Hale 1968). It is rarely
infertile in several localities in Europe
(Obermayer 2008) and has abundant
cylindrical, dark-brown-tipped, sometimes
branched, isidia on the upper surface. In rare
cases, in addition to isidia, capitate soralia
have been found. Such sorediate specimens
have been considered by Hale (1968) as a
different species, P. soralifera. Recent studies
tend to reject any systematic category for
these specimens (Hafellner & Obermayer
2004; Clerc 2004), but no phylogenetic
studies have been performed until now to
support such an opinion.
During the last 30 years, populations of
P. furfuracea from different regions of
Europe have been the subject of chemical
analyses. Three chemical races or chemo-
types have been described and their distri-
bution in relation to their ecology has been
widely discussed (Hawksworth & Chapman
1971; Culberson et al. 1977; Halvorsen &
Bendiksen 1982; López & Manrique 1989;
Rikkinen 1997). In many European floras,
the chemotypes are treated as varieties of
such species: P. furfuracea var. furfuracea
with physodic and oxyphysodic acids (corre-
sponding to chemical race I) and P. furfuracea
var. ceratea with olivetoric acid in addition,
or not, to physodic acid (corresponding to
chemical races II and III, respectively).
All specimens of P. furfuracea collected by
us bearing soralia were infected by a filamen-
tous fungus, growing both internally and ex-
ternally. This infection was observed not
only in sorediate/isidiate samples but also in
exclusively isidiate samples.
The aim of our investigation was to study
the morphology and phylogeny of sorediate
specimens of P. furfuracea in order to
evaluate the biological and taxonomic
significance of propagules in this species.
Material and Methods
Taxon sampling
Sorediate samples of P. furfuracea were collected from
the Canary Islands (North West Africa), the mountains
450 THE LICHENOLOGIST Vol. 42
of the north of Morocco and the west of Turkey. Speci-
mens with soralia are rare and only four specimens
were found as isolated sorediate thalli in populations
of P. furfuracea; no separate populations of sorediate
P. furfuracea were found. Non-sorediate samples of P.
furfuracea were also collected both from the same or
localities nearby and from several other regions of
Europe. The details of the material, area of collection,
location of voucher specimens and their GenBank acces-
sion number are presented in Table 1. The material is
deposited in the herbarium at the Facultad de Farmacia
of the Complutense University of Madrid, Spain (MAF)
and in the University of La Laguna, Tenerife, Canary
Islands, Spain (TFC).
Morphological and anatomical studies
The morphology of the specimens of P. furfuracea
(both non-sorediate and sorediate) listed in bold in
Table 1 was studied under a dissecting microscope
(Leica Wild M 8; Leica, Wetzlar, Germany).
Internal structure was studied in hand-cut trans-
verse sections of thalli and vegetative propagules (isidia
and soredia). Sections were stained with unheated
lactophenol cotton blue (Panreac, Barcelona, Spain)
and observed and photographed using a light
microscope (Nikon Eclipse 80i; Nikon, Badhoevedrop,
Netherlands).
Chemical analysis
Chemical analysis was carried out on the 16 samples
of Pseudevernia listed in bold in Table 1. Chemical
constituents were identified by thin-layer chromatog-
raphy (TLC) using solvent systems A [benzene: dioxane
: acetic acid, 180:45:5], B’ [hexane : methyl t-butyl ether
: formic acid, 140:72:18] and C [toluene : acetic
acid, 85:15] (Culberson 1972; Culberson et al. 1981;
Culberson & Johnson 1982; Elix & Ernst-Russell 1993;
Orange et al. 2001).
DNA extraction, PCR and sequencing
Total DNA was extracted from freshly collected
material (Table 1), using the DNeasy Plant Mini Kit
(Qiagen, Hilden, Germany) following the manufac-
turer’s instructions, with slight modifications described
in Crespo et al. 2001. Primers for amplification were: (1)
for the nu-ITS rDNA: ITS1F (Gardes & Bruns 1993)
and ITS4 (White et al. 1990); (2) for the mt-LSU
rDNA: ML3 and ML4 (Printzen 2002). Amplifications
were performed in 50 µl volumes containing a reaction
mixture of 5 µl 10 × DNA polymerase buffer (Biotools,
Madrid, Spain; containing 2 mM MgCl
2
, 10 mM Tris-
HCl, pH 8·0, 50 mM KCl, 1 mM EDTA, 0·1% Triton
X-100), 1 µl deoxyribonucleotide triphosphate (dNTP)
(Biotools), containing 10 mM of each base, 2·5 µl of
each primer (10 µM) (Sigma Aldrich), 1·25 µl DNA
polymerase (1 U/µl) (Biotools), and 27·75 µl distilled
water. Finally, 40 µl of this mixture were added to 10 µl
of DNA of each sample.
Amplifications were carried out in an automatic ther-
mocycler (Techne Progene Jepson Bolton & Co. Ltd.,
Watford, Herts, UK) under the following conditions:
initial denaturation at 94°C for 5 min, followed by 30
cycles of 94°C for 1 min, 54°C (nu ITS primers) or 47°C
(mt LSU primers) for 1 min and 72°C for 1·5 min, and
a final extension at 72°C for 5 min. Amplification prod-
ucts were viewed on 1% agarose gels stained with ethid-
ium bromide and subsequently purified using the
Bioclean Columns kit (Biotools, Madrid, Spain) accord-
ing to the manufacturer’s instructions. Fragments were
sequenced using the Big Dye Terminator reaction kit
(ABI PRISM, Applied Biosystems, Foster City, CA,
USA). Sequencing and PCR amplifications were per-
formed using the same sets of primers. Cycle sequencing
was carried out as follows: initial denaturation at 94°C
for 3 min, followed by 25 cycles of 96°C for 10 s, 50°C
for5sand60°Cfor4min.Sequencingreactions were
electrophoresed on a 3730 DNA analyser (Applied Bio-
systems). Sequence fragments obtained were assembled
with SeqMan 4.03 (DNAStar Madison) and adjusted
manually.
Sequence alignments and phylogenetic analysis
We generated 17 new nu ITS and 14 new mt LSU
rDNA sequences for this study. Details of the samples
analysed are listed in Table 1. Seven nu ITS sequences
were downloaded from GenBank (http://
www.ncbi.nlm.nih.gov).
Two analyses were performed. 1) A single-gene
analysis of the nu ITS sequences of Pseudevernia furfura-
cea (including three sequences of sorediate samples) in
which 13 newly obtained sequences were aligned with
three sequences obtained from GenBank (Table 1); one
new sequence of P. cladonia and two new sequences of
P.affintensa were included as the outgroup. 2) A com-
bined analysis of nu ITS and mt LSU rDNA sequences
of P. furfuracea and species of Pseudevernia from Mexico
and USA. Two data matrices were assembled for this
analysis: 2a) 14 samples using the mt LSU rDNA
region; 2b) 17 samples using those 14 sequences of nu
ITS region for which sequences of the mt LSU rDNA
region were available, and three sequences of P. cladonia,
P. consocians and P. intensa whose mt LSU sequences
failed; two taxa of Hypogymnia were included as out-
group since this genus is shown to be closely related
(Crespo et al. 2007).
A single-gene analysis was also presented as mt LSU
extraction failed in most cases of European P. furfuracea.
Thus, the nu ITS rDNA data set allows us to study a
larger taxon sampling of this species, and the combined
analysis was performed to show the phylogenetic hy-
pothesis for all species of the genus Pseudevernia.
Each data set was aligned separately using Clustal W
(Thompson et al. 1994). Regions that could not be
aligned with statistical confidence were excluded from
the phylogenetic analysis. Ambiguously aligned regions
were delimited manually (Lutzoni 2000).
The topology of the 95% majority rule consensus tree
of the two Bayesian single-partition analyses for matrices
(2a) and (2b) was congruent (data not shown), justifying
their subsequent combined analysis. The alignment of
the combined data set was analysed using maximum
2010 Molecular analysis of sorediate Pseudevernia furfuracea—Ferencova et al. 451
T 1. Species and specimens used in the current study (taxa studied morphologically and newly obtained sequences are indicated in bold)
Species Locality Collector(s) Herbarium
accession no.
GenBank accession numbers
nuITS mtLSU
Hypogymnia farinacea Spain: Guadarrama P.K. Divakar MAF 15592 GU300776 GU300798
H. physodes AF058036 ––––
H. physodes Spain: La Rioja Crespo & Argüello MAF 12543 GU300799
Pseudevernia cladonia USA: NC, Mitchell Co. AF297736 ––––
P. consocians USA: NC, Mitchell Co AF297735 ––––
P. intensa 1 USA: CO, Saguache Co., AF297737
P. intensa 2 USA: AZ, Chiricahua Mountains S. Pérez-Ortega GU300792 GU300797
P.aff.intensa 1 Mexico: Mexico L.G. Sancho et al. MAF 15625 GU300777 GU300796
P.aff.intensa 2 Mexico: Mexico L.G. Sancho et al. MAF 15626 GU300778 GU300795
P. furfuracea 1 Austria: Stiermark AF297734 ––––
P. furfuracea 2
†
Canary Islands: La Palma I. Perez-Vargas TFC 7027 GU300779 ––––
P. furfuracea 3*
†
Morocco: Ifrane A. Crespo et al. MAF 15595 GU300780 GU300800
P. furfuracea 4
†
Morocco: Ifrane A. Crespo et al. MAF 15594 GU300781 GU300801
P. furfuracea 5 Spain: Madrid E. Gonzáles Burgos MAF 14178 GU300782 GU300802
P. furfuracea 6 Spain: Madrid M. B. Gros Otero MAF 14129 GU300783 GU300803
P. furfuracea
†
Slovakia: Roznava Z. Ferencova MAF 15600 GU300784 GU300804
P. furfuracea 8 Finland: Regio Aboensis AF451768 ––––
P. furfuracea 9 Spain: Madrid AY611112 ––––
P. furfuracea 10
†
Slovakia: Liptovský. Mikulás Z. Ferencova MAF 15601 GU300785 GU300805
P. furfuracea 11*
†
Morocco: Ifrane A. Crespo et al. MAF 15596 GU300786 ––––
P. furfuracea 12 Spain: Castellón A. Crespo et al. MAF 15599 GU300787 ––––
P. furfuracea 13 Slovakia: Poprad Z. Ferencova MAF 15602 GU300788 ––––
P. furfuracea 14 Slovakia: Spisská Nová Ves Z. Ferencova MAF 15603 GU300789 GU300806
P. furfuracea 15 Turkey: Eskisehir A. Crespo et al. MAF 15604 GU300790 GU300794
P. furfuracea 16* Turkey: Eskisehir A. Crespo et al. MAF 15605 GU300791 GU300793
P. furfuracea 17*
†
Canary Islands: La Palma I. Perez-Vargas TFC 5875 –––– ––––
*sorediate specimens; †indicates presence of olivetoric acid; all others contain physodic and oxyphysodic acids.
452 THE LICHENOLOGIST Vol. 42
parsimony (MP), maximum likelihood (ML) and a
Bayesian approach (B/MCMC). MP analyses were per-
formed using the PAUP* program (Swofford 2003). A
heuristic search with 2000 random taxon addition repli-
cates, using the general time-reversible nucleotide sub-
stitution model (Rodriguez et al. 1990) and assuming a
gamma shape parameter of 0·5 was conducted with
TBR branch swapping and the MulTrees option se-
lected. Bootstrapping (Felsenstein 1985) was performed
on the basis of 2000 pseudoreplicates with the same
settings as for the heuristic search.
ML analyses were performed using PhyML on the
program’s online web server, http://atgc.lirmm.fr/phyml
(Guindon et al. 2005), assuming the general time-
reversible model of nucleotide substitution (Rodriguez
et al. 1990) and a discrete gamma distribution with six
rate categories for the combined data set. The bootstrap
analysis was run with 500 pseudoreplicates.
The B/MCMC analyses were conducted using the
MrBayes 3.1.2 program (Huelsenbeck & Ronquist
2001), adopting the general time-reversible model of
nucleotide substitution (Rodrı´guez et al. 1990) includ-
ing estimation of invariant sites, assuming a discrete
gamma distribution with six rate categories and allowing
site-specific rates (GTR+I+G). The nucleotide substi-
tution model was selected using jModelTest (Posada
2008).
The combined data set was divided into the two parts
(nu ITS and mt LSU), each of which was allowed to
have its own parameters as suggested by Nylander et al.
(2004). No molecular clock was assumed. Parallel runs
were made with 3 000 000 generations starting with a
random tree and employing 12 simultaneous chains
each. Trees were sampled every 200 generations for a
total of 30 000 trees. The first 300 000 generations (i.e.,
the first 3000 trees) were discounted as burn-in for the
chain.
The log-likelihood scores of sample points were plot-
ted against generation time using TRACER 1.0 (http://
evolve.zoo.ox.ac.uk/software.html/tracer/) to ensure
that stationarity was achieved after the first 300 000
generations by checking whether the log-likelihood
values of the sample points had reached a stable equilib-
rium (Huelsenbeck & Ronquist 2001). For the remain-
ing 54 000 trees (27 000 from each parallel run) a
majority-rule consensus tree with average branch
lengths was calculated using the sumt option in
MrBayes.
We used a Bayesian approach to examine the hetero-
geneity in phylogenetic signal among the two data par-
titions (DeQueiroz 1993; Buckley et al. 2002). Posterior
probabilities (PPs) were approximated for each data set
by sampling trees using an MCMC method. The PPs of
each branch were calculated by counting their occur-
rence in trees that were visited during the course of the
MCMC analysis. PPs were obtained for each clade.
Only clades with PPs R 95 % were considered as
strongly supported. If no conflict was evident, it was
assumed that the two data sets were congruent and
could be combined.
In the combined data set, a conservative approach for
interpreting support values was considered. Only clades
that received bootstrap support R 70% in MP and ML
analyses and PPs R 0·95 were considered as strongly
supported.
The alignment of 19 samples of nu ITS (1) was
analysed using a Bayesian approach (B/MCMC). Phylo-
genetic trees were drawn using TREEVIEW (Page
1996).
Results
Morphological observations
Sorediate thalli looked weaker, were
shorter and showed reduced vitality com-
pared with non-sorediate specimens. Isidia
F. 1. Pseudevernia furfuracea, transverse sections of the thallus and vegetative propagules of a sorediate specimen.
A, parasitic fungal hyphae growing on and inside an isidium; B, uninfected soredia. Scales:A&B=100µm.
2010 Molecular analysis of sorediate Pseudevernia furfuracea—Ferencova et al. 453
F. 2. A 50% majority-rule consensus tree of nu ITS sequences of Pseudevernia furfuracea. This tree is based on
27 000 trees from a B/MCMC tree sampling procedure. Branches with posterior probabilities R 0·95 are indicated
in bold. Sorediate specimens are indicated in bold. Specimens with olivetoric acid are indicated with †, all others
contain physodic and oxyphysodic acids. Different haplotypes are indicated by an ‘H’ followed by a number.
454 THE LICHENOLOGIST Vol. 42
were morphologically similar in both kinds of
thalli but, in the sorediate specimens, were
less abundant and poorly developed. Soralia
were regularly capitate, substipitate, whitish,
laminar and marginal and were occasionally
also present on the lower surface of the thalli.
All sorediate samples were infected by
at least one filamentous fungus (Fig. 1A).
Fungal hyphae grew on the surface of the
lichen thallus including the isidia (cortical
and medullar inner part). They also pen-
etrated into the whole thalline cortex and
reached the medulla. Interestingly soralia
were not infected, at least not in the central
zone where they are produced (Fig. 1B).
Chemical TLC analysis
All samples of the morphospecies Pseudev-
ernia furfuracea contained atranorin. Seven
samples, marked (†) in Table 1 and Fig. 2,
contained olivetoric acid (hitherto treated in
many European lichen floras as P. furfuracea
var. ceratea). The other seven samples had
oxyphysodic and physodic acids (named as
P. furfuracea var. furfuracea). Specimens with
mixed chemistry (containing both physodic
and olivetoric acid), described from Spain
by Culberson et al. (1977) and López &
Manrique (1989), were not found. Samples
with olivetoric acid did not form a mono-
phyletic group and could not be treated
as a separate valid taxon, P. olivetorina,as
suggested by some authors (e.g., Hale
1968).
Phylogenetic analysis
We generated 17 new nu ITS and 14 new
mt LSU rDNA sequences for this study
(Table 1). Two analyses were performed: 1)
single gene analysis of the nu ITS sequences
of Pseudevernia furfuracea, in which the data
matrix had 525 unambiguously aligned
nucleotide positions, 48 of which were vari-
able; 2) a combined analysis of nu ITS and
mt LSU rDNA sequences where the data
matrix had 476 unambiguously aligned
nucleotide positions in the nu ITS and 809 in
the mt LSU rDNA partitions. Eighty-seven
characters were variable in the nu ITS and 27
in the mt LSU data set. Since the topologies
of the MP, ML and B/MCMC analyses
did not show any supported conflicts, only
the 50% majority-rule consensus tree of
Bayesian tree sampling is shown (Fig. 3), the
nodes in bold being those that received
strong support in all three analyses (i.e., PP
R 0·95 in the B/MCMC analysis and
R 70% for the MP and ML bootstraps).
In the single-gene analysis (Fig. 2), the
likelihood parameters of the sample had the
following mean values (variance): LnL =
−1076·7 (0·10), base frequencies (A) =
0·225 (0·0002), (C) = 0·28 (0·0002),
(G) = 0·254 (0·0002), (T) = 0·242
(0·0002), rate matrix r(AC) = 0·089
(0·0012), r(AG) = 0·242 (0·0018), r(AT) =
0·143 (0·0014), r(CG) = 0·0109 (0·0011),
r(CT) = 0·382 (0·0018), r(GT) = 0·035
(0·0008), gamma shape parameter alpha =
0·097 (0·0003) and proportion of invariable
sites, p(invar) = 0·762 (0·0004).
In the two-gene analysis (Fig. 3), in the
B/MCMC analysis, the likelihood para-
meters in the sample had the following mean
values for the nu ITS partition (variance):
LnL = −2381·69 (0·09), base frequencies
(A) = 0·214 (0·0002), (C) = 0·283
(0·0002), (G) = 0·263 (0·0002), (T) =
0·24 (0·0002), rate matrix r(AC) = 0·075
(0·0004), r(AG) = 0·228 (0·0008), r(AT) =
0·09 (0·0006), r(CG) = 0·065 (0·0003),
r(CT) = 0·495 (0·001), r(GT) = 0·046
(0·0004), gamma shape parameter alpha =
0·097 (0·0003) and the proportion of invari-
able site p(invar) = 0·55 (0·002). The fol-
lowing means were obtained for the mt
LSU partition (variance): LnL = −2381·69
(0·09), base frequencies (A) = 0·369
(0·0002), (C) = 0·143 (0.0001), (G) =
0·189 (0·0002), (T) = 0·3 (0·0002), rate
matrix r(AC) = 0·071 (0·0014), r(AG) =
0·174 (0·0017), r(AT) = 0·213 (0 ·0017),
r(CG) = 0·186 (0·0025), r(CT) = 0·26
(0·0028), r(GT) = 0·095 (0·0013), gamma
shape parameter alpha = 0·068 (0·0004) and
p(invar) = 0·912 (0·0002).
The majority-rule consensus tree of the
ITS data set included sequences from
European and North African samples and is
shown in Fig. 2. All the samples of P. furfura-
cea from Central and Western Europe,
2010 Molecular analysis of sorediate Pseudevernia furfuracea—Ferencova et al. 455
F. 3. A 50% majority-rule consensus tree of the phylogenetic relationships in the genus Pseudevernia based on
54 000 trees from a B/MCMC tree-sampling procedure from a combined data set of nu ITS and mt LSU rDNA.
Branches that received strong support in all three analyses (i.e., PP R 0·95 in B/MCMC analysis and R 70%inMP
and ML bootstraps) are indicated in bold; the branch that received strong support in MP and PhyML is indicated by
an empty rectangle. Specimens with olivetoric acid are indicated with †, all others contain physodic and oxyphysodic
acids.
456 THE LICHENOLOGIST Vol. 42
Turkey and North Africa (Morocco) nested
in a monophyletic group resolved in a pecti-
nated topology. One monophyletic clade that
includes the two samples from Turkey is
supported. Three sequences of sorediate
specimens of P. furfuracea were obtained
but the sorediate samples from the Canary
Islands remained unamplified, most likely
because of the ‘unhealthy’ state of the
material and the heavy fungal infection. All
specimens are monophyletic (Fig. 3) but
six haplotypes were noted. Three of these
haplotypes (H1, H3 and H5) included both
sorediate and nonsorediate samples.
The phylogenetic relationships for all
species of the genus (Fig. 3) shows that the
New and Old World samples are separated
into two sister monophyletic clades. The
American species (P. intensa (Nyl.) Hale
& W. Culb., P. cladonia (Tuck.) Hale &
W. Culb., P. consocians (Vain.) Hale &
W. Culb. and P. aff. intensa) form a
sister group to the European P. furfuracea
(including sorediate samples).
Discussion
Sorediate populations of Pseudevernia fur-
furacea are rare. Nevertheless, our collections
are from distant localities around the
Mediterranean basin and the Canary Islands,
so it is notable that such a rare phenomenon
is so widespread. A common origin for the
sorediate specimens would be a straight-
forward hypothesis. However, no mono-
phyletic separate lineage of sorediate
specimens was found within the phylogenetic
tree, and the same haplotypes were found in
sorediate and non-sorediate samples. In
other words, our phylogenetic analyses do
not suggest that P. soralifera is a valid taxon
(Hale 1968), but rather that it should be
included within P. furfuracea (as already has
been done, for example, by Clerc 2004). A
previous study based on biological observa-
tions of a population in the Austrian Alps also
concluded that the sorediate collections do
not merit recognition as a separate taxon
(Hafellner & Obermayer 2004).
Similarly, none of our studied samples
containing olivetoric acid represents a phylo-
genetically supported lineage. Thus, the in-
clusion of the taxon P. olivetorina in P.
furfuracea, as already proposed by López &
Manrique (1989) and Rikkinen (1997) and
implemented by Clerc (2004), is supported.
The presence of well-defined soralia char-
acterized by a constant capitate form appears
to be an important morphological feature for
differentiating morphospecies (Taylor et al.
2000), particularly when molecular data are
not available. However, the status of P. soral-
ifera had already been discussed by Hale
(1968), who reported the rare simultaneous
occurrence of isidia and soralia in his synop-
sis of the species. He suspected that this
feature was “an abnormal condition of
otherwise normal P. furfuracea” and was
prompted to reduce the status of the species.
Recently, Hafellner & Obermayer (2004)
were more conclusive in their study of
Austrian sorediate material, stating that “it is
not convenient to apply a taxon name to
sorediate Pseudevernia specimens.” More-
over, although a considerable number of
sorediate ‘species’ persist in taxonomic index
lists (www.indexfungorum.org), it has been
demonstrated repeatedly in lichens that dif-
ferentiation based on the presence of soralia
does not merit acceptance of sorediate forms
as valid species (Tehler 1982). In addition,
all available data from molecular markers
concur in demonstrating that sorediate coun-
terparts in ‘species pairs’ do not appear as an
isolated evolutionary monophyletic lineage
that can be described as a single separate
species (Lohtander et al. 1998a, b; Myllys
et al. 1999, 2001; Articus et al. 2002; Molina
et al. 2002; Cubero et al. 2004; Buschbom &
Mueller 2006). There are examples anal-
ogous to these appealing capitate soralia,
such as the profuse marginal soralia of Phy-
sconia perisidiosa, where the use of molecular
markers did not indicate that the sorediate P.
perisidiosa is a monophyletic species separate
from the non-sorediate P. venusta (Cubero
et al. 2004).
However, Pseudevernia soralifera is not a
typical case of a ‘species pair’ but unusual in
the occasional simultaneous presence of
2010 Molecular analysis of sorediate Pseudevernia furfuracea—Ferencova et al. 457
isidia and soredia. Pseudovernia furfuracea,
probably propagating by means of isidia, is
an extremely common species in most Euro-
pean regions and shows notable ecological
plasticity. Therefore, isidia appear to be
efficient propagules and the presence of capi-
tate soralia in these populations of Pseudever-
nia may not be simply an additional
reproductive strategy.
The three collections of sorediate speci-
mens studied here all had regular capitate
soralia, showed more or less reduced vitality
and were infected or colonized by at least one
fungal mycelium. The fungus was visible su-
perficially but anatomical studies demon-
strated that it also penetrated the upper
cortex and that most of the isidia were also
infected internally and externally. Interest-
ingly, infection did not appear to penetrate
the soralia. These morphological similarities
among all sorediate samples from widely
separated populations lead us to suggest that
soralia arise as a consistent reaction to a
similar agent, the lichenicolous or parasitic
fungi, and that it may be a coevolved repro-
ductive strategy.
The fungal infection in the lichen thallus
covers the isidia, so their subsequent disper-
sal would also spread the fungal infection or
possibly make the isidial propagules inviable.
Interestingly, soralia are free of infection and
their production could be an alternative re-
productive strategy by P. furfuracea to ensure
survival when the health of isidia is compro-
mised by fungal infection.
Finally, the reconstruction of the phy-
logeny of Pseudevernia reveals two mono-
phyletic sister branches of the genus. The
two branches separate the American species
(P. intensa, P. cladonia, P. consocians), from
the Eurasiatic and North African P. furfura-
cea (including both sorediate samples and
samples with olivetoric acid).
We thank Dr W. Obermayer and an anonymous re-
viewer for their suggestions, which have improved the
quality of our work and Dr A. Green for his kind help in
revising the English language of the manuscript. This
project was funded by the Banco Bilbao Vizcaya
Argentaria (BBVA) Foundation. We are grateful to the
Spanish Ministerio de Educación y Ciencia for the FPU
grant to ZF (CGL2007- 64652). Sequencing, for which
we thank Maria Isabel Garcı´a Saez, was carried out
at the Unidad de Genómica (Parque Cientı´fico de
Madrid).
R
Argüello, A., Del Prado, R., Cubas, P. & Crespo, A.
(2007) Parmelina quercina (Parmeliaceae, Lecano-
rales) includes four phylogenetically supported
morphospecies. Biological Journal of the Linnean
Society 91: 455–467.
Articus, K., Mattsson, J.-E., Tibell, L., Grube, M. &
Wedin, M. (2002) Ribosomal DNA and beta-
tubulin data do not support the separation of the
lichens Usnea florida and U. subfloridana as distinct
species. Mycological Research 106: 412–418.
Blanco, O., Crespo, A., Divakar, P. K., Esslinger, T. L.,
Hawksworth, D. L. & Lumbsch, H. T. (2004)
Melanelixia and Melanohalea, two new genera segre-
gated from Melanelia (Parmeliaceae) based on mol-
ecular and morphological data. Mycological Research
108: 873–884.
Buckley, T. R., Arensburger, P., Simon, C. &
Chambers, G. K. (2002) Combined data, Bayesian
phylogenetics, and the origin of the New Zealand
cicada genera. Systematic Biology 51: 4–18.
Buschbom, J. & Mueller, G. M. (2006) Testing ‘species
pair’ hypotheses: evolutionary processes in the
lichen-forming species complex Porpidia flavocoeru-
lescens and P. melinodes. Molecular Biology and Evo-
lution 23: 574–586.
Clerc, P. (2004) Les champignons lichénisés de Suisse.
Catalogue bibliographique complété par des don-
nées sur la distribution et l’écologie des espèces.
Cryptogamica Helvetica 19: 1–320.
Crespo, A., Blanco, O. & Hawksworth, D. L. (2001)
The potential of mitochondrial DNA for establish-
ing phylogeny and stabilising generic concepts in
the parmelioid lichens. Taxon 50: 807–819.
Crespo, A., Lumbsch, H. T., Mattsson, J.-E., Blanco,
O., Divakar, P. K., Articus, K., Wiklund, E.,
Bawingan, P. A. & Wedin, M. (2007) Testing
morphology-based hypotheses of phylogenetic rela-
tionships in Parmeliaceae (Ascomycota) using three
ribosomal markers and the nuclear RPB1 gene.
Molecular Phylogenetics and Evolution 44: 812–824.
Cubero, O. F., Crespo, A., Esslinger, T. L. & Lumbsch,
H. T. (2004) Molecular phylogeny of the genus
Physconia (Ascomycota, Lecanorales) inferred from
a Bayesian analysis of nuclear ITS rDNA se-
quences. Mycological Research 108: 498–505.
Culberson, C. F. (1972) Improved conditions and new
data for the identification of lichen products by a
standardized thin-layer chromatographic method.
Journal of Chromatography 72: 113–125.
Culberson, W. L., Culberson, C. F. & Johnson, A.
(1977) Pseudevernia furfuracea-olivetorina relation-
ships: chemistry and ecology. Mycologia 69: 604–
614.
Culberson, C. F., Culberson, W. L. & Johnson, A.
(1981) A standardized TLC analysis of ß-orcinol
depsidones. Bryologist 84: 16–29.
458 THE LICHENOLOGIST Vol. 42
Culberson, C. F. & Johnson, A. (1982) Substitution of
methyl tert.-butyl ether for diethyl ether in the
standardized thin-layer chromatographic method
for lichen products. Journal of Chromatography 238:
483–487.
DeQueiroz, A. (1993) For consensus (sometimes). Sys-
tematic Biology 42: 368–372.
Elix, J. A. & Ernst-Russell, K. D. (eds) (1993) A Cata-
logue of Standardized Thin Layer Chromatographic
Data and Biosynthetic Relationships for Lichen
Substances. 2nd ed. Canberra: Australian National
University.
Felsenstein, J. (1985) Confidence-limits on phylogenies
– an approach using the bootstrap. Evolution 39:
783–791.
Gardes, M. & Bruns, T. D. (1993) ITS primers
with enhanced specificity for basidiomycetes –
application to the identification of mycorrhizae and
rust. Molecular Ecology 2:113–118.
Guindon, S., Lethiec, F., Duroux, P. & Gascuel, O.
(2005) PHYML Online–awebserver for fast
maximum likelihood-based phylogenetic inference.
Nucleic Acids Research 33(Web Server Issue):
W557–W559.
Hafellner, J. & Obermayer, W. (2004) Beobachtungen
an einer sorediösen Population von Pseudevernia
furfuracea. Herzogia 17: 45–50.
Hale, M. E. (1965) A monograph of Parmelia subgenus
Amphigymnia. Contributions from the United States
National Herbarium 36: 193–358.
Hale, M. E. (1968) A synopsis of the lichen genus
Pseudevernia. Bryologist 71: 1–11.
Hale, M. E. (1976) A monograph of the lichen genus
Parmelina Hale (Parmeliaceae). Smithsonian Contri-
butions to Botany 33: 1–60.
Hale, M. E. (1990) A synopsis of the lichen genus
Xanthoparmelia (Vainio) Hale (Ascomycotina,
Parmeliaceae). Smithsonian Contributions to Botany
74: 1–250.
Halvorsen, R. & Bendiksen, E. (1982) The chemical
variation of Pseudevernia furfuracea in Norway.
Nordic Journal of Botany 2: 371–380.
Hawksworth, D. L. & Chapman, D. S. (1971) Pseudev-
ernia furfuracea (L.) Zopf and its chemical races in
the British Isles. Lichenologist 5: 51–58.
Huelsenbeck, J. P. & Ronquist, F. (2001) MRBAYES:
Bayesian inference of phylogenetic trees. Bioinfor-
matics 17: 754–755.
Lohtander, K., Kallersjö, M. & Tehler, A. (1998a)
Dispersal strategies in Roccellina capensis (Arthoni-
ales). Lichenologist 30: 341–350.
Lohtander, K., Myllys, L., Sundin, R., Kallersjö, M. &
Tehler, A. (1998b) The species pair concept in
the lichen Dendrographa leucophaea (Arthoniales):
analyses based on ITS sequences. Bryologist 101:
404–411.
López, F. & Manrique, E. (1989) Pseudevernia furfuracea
(L.) Zopf: Razas quı´micas y su distribución en la
Penı´nsula Ibérica. Anales del Jardín Botánico de
Madrid 46: 295–305.
Lutzoni, F. Wagner, P., Reeb, V. & Zoller, S. (2000)
Integrating ambiguously aligned regions of DNA
sequences in phylogenetic analyses without violat-
ing positional homology. Systematic Biology 49:
628–651.
Molina, M. C., Crespo, A., Blanco, O., Hladun, N. &
Hawksworth, D. L. (2002) Molecular phylogeny
and status of Diploicia and Diplotomma, with obser-
vations on Diploicia subcanescens and Diplotomma
rivas-martinezii. Lichenologist 34: 509–519.
Myllys, L., Lohtander, K., Källersjö, M. & Tehler, A.
(1999) Sequence insertions and ITS data provide
congruent information on Roccella canariensis and
R. tuberculata (Arthoniales, Euascomycetes) phy-
logeny. Molecular Phylogenetics and Evolution 12:
295–309.
Myllys, L., Lohtander, K. & Tehler, A. (2001) Beta-
tubulin, ITS and group I intron sequences challenge
the species pair concept in Physcia aipolia and
P. caesia. Mycologia 93: 335–343.
Nylander, J. A. A., Ronquist, F., Huelsenbeck, J. P. &
Nieves-Aldrey, J. L. (2004) Bayesian phylogenetic
analysis of combined data. Systematic Biology 53:
47–67.
Obermayer, W. (2008) Fotografische Dokumentation
einer ungewöhnlich reich fruchtenden Auf-
sammlung von Cetraria islandica (L.) Ach. (mit
einem historischen Abriss zur Darstellung fertiler
Thalli, Anmerkungen zur Gestalt der Pycnosporen
und einigen Notizen zum Gebrauch des, Kramper-
ltees’). Mitteilungen des Naturwissenschaftlichen
Vereines für Steiermark 138: 113–158.
Orange, A., James, P. W. & White, F. J. (eds) (2001)
Microchemical Methods for the Identification of
Lichens. London: British Lichen Society.
Page, R. D. M. (1996) Treeview: an application to
display phylogenetic trees on personal computers.
Computer Applications in the Biosciences 12: 357–358.
Poelt, J. (1970) Das Konzept der Artenpaare bei den
Flechten. Berichte der Deutschen Botanischen Gesells-
chaft 4: 187–198.
Poelt, J. (1972) Die taxonomische Behandlung von
Artenpaaren bei den Flechten. Botaniska Notiser
125: 77–81.
Posada, D. (2008) jModelTest: phylogenetic model
averaging. Molecular Biology and Evolution 25:
1253–1256.
Printzen, C. (2002) Fungal specific primers for PCR-
amplification of mitochondrial LSU in lichens. Mol-
ecular Ecology Notes 2: 130–132.
Rikkinen, J. (1997) Habitat shifts and morphological
variation of Pseudevernia furfuracea along a topo-
graphical gradient. In Lichen Studies Dedicated to
Rolf Santesson (Symbolae Botanicae Upsalienses)(L.
Tibell & I. Hedberg, eds): 223–245. Uppsala: Acta
Universitatis Upsaliensis.
Rodrı´guez, F., Oliver, J. F., Martı´n,A.&Medina,J.R.
(1990) The general stochastic model of nucleotide
substitution. Journal of Theoretical Biology 142: 485–
501.
Swofford, D. L. (2003) PAUP*. Phylogenetic Analysis
Using Parsimony (*and Other Methods). Version 4.
Sunderland, Massachusetts: Sinauer Associates.
2010 Molecular analysis of sorediate Pseudevernia furfuracea—Ferencova et al. 459
Taylor, J. W., Jacobson, D. J., Kroken, S., Kasuga, T.,
Geiser, D. M., Hibbett, D. S. & Fisher, M. C.
(2000) Phylogenetic species recognition and species
concepts in fungi. Fungal Genetics and Biology 31:
21–32.
Tehler, A. (1982) The species pair concept in lichenol-
ogy. Taxon 31: 708–714.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994)
Clustal W: improving the sensitivity of progressive
multiple sequence alignment through sequence
weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Research 22:
4673–4680.
White, T. J., Bruns, T. D., Lee, S. & Taylor, J. (1990)
Amplification and direct sequencing of fungal ribos-
omal RNA genes for phylogenies. In PCR Protocols:
a Guide to Methods and Applications (M. A. Innis,
D. H. Gelfand, J. J. Sninsky & T. J. White, eds):
315–322. San Diego: Academic Press.
Accepted for publication 30 November 2009
460 THE LICHENOLOGIST Vol. 42
