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Two Trebouxia algae with different physiological
performances are ever-present in lichen thalli of
Ramalina farinacea. Coexistence versus Competition?emi_2386 1..13
Leonardo M. Casano,1Eva M. del Campo,1*
Francisco J. García-Breijo,2,3 José Reig-Armiñana,2
Francisco Gasulla,2Alicia del Hoyo,1
Alfredo Guéra1,2 and Eva Barreno2
1Department of Plant Biology, University of Alcalá,
28871-Alcalá de Henares, Madrid, Spain.
2Universitat de València, Botánica, ICBIBE -Jardí
Botánic, Fac. C. Biológicas, C/ Dr Moliner 50.
46100-Burjassot. Valencia; Spain.
3Dpto. Ecosistemas Agroforestales, Universidad
Politécnica de Valencia, Camino de Vera s/n,
46022-Valencia, Spain.
Summary
Ramalina farinacea is an epiphytic fruticose lichen
that is relatively abundant in areas with Mediter-
ranean, subtropical or temperate climates. Little is
known about photobiont diversity in different lichen
populations. The present study examines the phyco-
biont composition of several geographically distant
populations of R. farinacea from the Iberian Penin-
sula, Canary Islands and California as well as the
physiological performance of isolated phycobionts.
Based on anatomical observations and molecular
analyses, the coexistence of two different taxa of
Trebouxia (working names, TR1 and TR9) was deter-
mined within each thallus of R. farinacea in all of the
analysed populations. Examination of the effects of
temperature and light on growth and photosynthesis
indicated a superior performance of TR9 under rela-
tively high temperatures and irradiances while TR1
thrived at moderate temperature and irradiance.
Ramalina farinacea thalli apparently represent a
specific and selective form of symbiotic association
involving the same two Trebouxia phycobionts. Strict
preservation of this pattern of algal coexistence
is likely favoured by the different and probably
complementary ecophysiological responses of each
phycobiont, thus facilitating the proliferation of this
lichen in a wide range of habitats and geographic
areas.
Introduction
Lichen thalli represent a relatively well-balanced symbi-
otic system that can be regarded as a self-contained
miniature ecosystem (Honegger, 1991). Lichen symbio-
genesis involves the close morphological and physiologi-
cal integration of a fungus (mycobiont) and at least a
population of green algae and/or cyanobacteria (photo-
bionts), resulting in a unique entity or holobiont (Barreno,
2004). More recently, it was proposed that lichens are
more complex symbiotic systems than thought previously
including non-photosynthetic bacterial communities as
multifunctional partners in the holobiont (Barreno et al.,
2008; Grube et al., 2009). Lichen symbioses are cyclical
processes comparable to other symbioses such as corals,
mycorrhiza, etc. Lichenization is a successful symbiosis
as evidenced by the fact that lichens are found in almost
all terrestrial habitats and geographic areas. The green
algae in the genera Trebouxia and Asterochloris occur in
at least 35% of all lichens but are rarely found in a free-
living state (Skaloud and Peksa, 2010). Gene sequence
comparisons of the nuclear-encoded small subunit RNA
(18S rRNA gene) and ultrastructural findings support the
inclusion of Trebouxia in a distinct Trebouxiophyceae
clade within Chlorophyta (Skaloud and Peksa, 2010). In
addition, the diversity of Trebouxia and Asterochloris phy-
cobionts has been extensively investigated on the basis of
internal transcribed spacers (ITS) and actin sequence
polymorphisms (DePriest, 2004; Doering and Piercey-
Normore, 2009; Skaloud and Peksa, 2010). More
recently, the use of plastid 23S rRNA gene has been
proposed as a complementary tool for the identification
and phylogenetic analysis of lichen algae (Del Campo
et al., 2010).
Patterns of fungal–algal association can be described
in terms of selectivity and specificity (Yahr et al., 2004).
Selectivity is defined by the association frequency of com-
patible partners, and specificity by the taxonomic range of
acceptable partners, which could be influenced by the
environment (Rambold et al., 1998; DePriest, 2004).
Received 16 July, 2010; accepted 20 October, 2010. *For correspon-
dence. E-mail eva.campo@uah.es; Tel. (+34) 91 8856432; Fax (+34)
91 8855066.
Environmental Microbiology (2010) doi:10.1111/j.1462-2920.2010.02386.x
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
Based on Combes’ filter model (Euzet and Combes,
1980), Yahr and colleagues (2006) proposed a pattern for
mycobiont-photobiont interactions and the mechanisms
that structure them. The latter may differ considerably
depending on the lichen species. Several mycobionts
associate with a single species, or even a single clade of
photobiont (Beck et al., 1998; Kroken and Taylor, 2000;
Romeike et al., 2002; Piercey-Normore, 2006). However,
the same photobiont may associate with several different
lichen fungi (O’Brien et al., 2005). In addition, most of the
studies on population structure have reported the pres-
ence of a single primary photobiont per thallus (e.g. Yahr
et al., 2004; Muggia et al., 2008; Nelsen and Gargas,
2008). In other cases, multiple algal genotypes have been
found in a single thallus (e.g. Guzow-Krzeminska, 2006;
Ohmura et al., 2006), which may confer advantages in the
lichen’s ability to respond to environmental changes or to
occupy diverse microenvironments (Piercey-Normore,
2006). Further support for this argument is obtained by
considering lichen thalli as microecosystems in which the
fungus is the host and the photobiont(s) the primary pro-
ducer(s). In nature, there are multiple examples demon-
strating that positive interactions among potential
competitors can sustain the stable coexistence of multiple
species (Gross, 2008; Haruta et al., 2009; Loarie et al.,
2009).
Here we present the results of an in-depth morphologi-
cal, molecular, and physiological study of the phycobiont
composition of the fruticose lichen Ramalina farinacea
and its diversity. The growth pattern of fruticose species
constitutes an important advantage in the separation of
individuals prior to DNA extraction because growing thalli
emerge from a single punctual holdfast. Microscopic
observations and molecular markers were used to inves-
tigate the diversity of phycobiont composition in lichen
thalli obtained from geographically distant localities, as
Iberian Peninsula, Canary Islands and California, charac-
terized by diverse environmental conditions within the
context of Mediterranean and temperate climates. These
studies were carried out on phycobionts within the thallus
as well as those isolated in axenic culture. Using chloro-
phyll (Chl) afluorescence analyses, we identified several
important photosynthetic traits in axenic cultures of phy-
cobionts. Overall, the results provided evidence that there
are two different taxa belonging to the genus Trebouxia.
They differ in their morphologies as well as in physiology,
and successfully coexist within the same lichen thallus.
Results
Morphological analysis of phycobionts from
Ramalina farinacea
In this study, LM and TEM were used to characterize the
structure and ultrastructure of R. farinacea photobionts.
Thalli from all the studied populations of R. farinacea
always contained two types of Trebouxia phycobionts
(working names: TR1 and TR9). These photobionts are
structurally well characterized. The main morphological
features of these algae, as observed in the thalli and in
cell culture, are compared in Table 1 and Figs 1 and 2.
TR1 and TR9 phycobionts were usually grouped by mor-
photype and located near the chondroid tissue, in close
contact with the hyphae solely in the photobiont layer
(Fig. 1A, B and F). Additionally, TEM demonstrated the
joint occurrence of the two algae (Fig. 1B) although
groups of a single photobiont type are more frequent.
Table 1. Main morphological features of the algae TR1 and TR9.
Morphological attributes TR1 TR9
Cell size Cell diameter (mm) 9.76 ⫾0.12* 15.26 ⫾0.10*
Cell diameter (mm) (in culture) 6.37 ⫾0.11* 10.01 ⫾0.10*
Cell wall Thickness (nm) 304.50 ⫾6.10* 594.16 ⫾5.37*
Thickness (nm) (in culture) 147.36 ⫾3.58* 412.67 ⫾6.49*
L1. 50 L1. 70–90
Thickness of cell wall L2. 75–90 L.2. 90–110
layers (L) (nm) L3. 50–60 L3. 400–500
L4. 160–200
Pyrenoid Pyrenoid matrix +-
Pyrenoglobules +-
Thylakoids invaginations +-
Chloroplast Shape Lobated Lobated
Stacked thylacoids ⱕ3 Numerous
Large electron dense vesicles Number Few (ⱕ4) Numerous (>5)
Size (mm) <0.6 ⱖ0.6–1.5 mm
Aplanospores ++
Zoospores (in culture) ++
Unless otherwise stated, measurements were performed in thallus, from a random selection of R. farinacea specimens from Sierra El Toro,
Castellón, Spain (n=15). The data are the means of 150 measurements ⫾the standard error of the means (SEM). All measurements were
performed by TEM, except those corresponding to cell diameter, which were by LM. Values with asterisks are significantly different at P<0.01
(comparisons made with independent Student’s t-test). +: presence; -: absence.
2L. M. Casano et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
LM and TEM anatomy of TR1 and TR9 phycobionts
Photobiont identification was based on the following mor-
phological features: (i) vegetative-cell size and cell-wall
thickness; (ii) pyrenoid structure; (iii) electron-dense
vesicles; and (iv) chloroplast shape and grana type. Two
different taxa of the genus Trebouxia were found to be
consistently present in R. farinacea thalli. As seen using
LM, TR1 and TR9 phycobiont cells differ in size both in
thallus and in culture, being smaller in the later environ-
ment (Table 1). On TEM, the cell walls of TR1 algae varied
in thickness (Table 1), and were made up of four layers
(Fig. 2C). The cell walls of TR9 were thicker than in TR1
(Table 1) and consisted of only three well-differentiated
layers (Fig. 2F, Table 1). These structures in culture con-
ditions were maintained but exhibiting thinner walls, prob-
ably due to the slower growth rate and lower nutrient
availability. Both phycobionts had a central massive single
star-shaped and lobed chloroplast. In TR1 cells, the lobes
extending to the cellular margins were loosely packed.
Thylakoids were often closely associated in stacks of
three at the most (Figs 1E, 2A and B). A large central part
of the chloroplast was occupied by the pyrenoid (Figs 1D
and E, 2A and B); the outer lamellae of the thylakoid
stacks included tubules that penetrated the pyrenoid
matrix and were either long or short depending on
the section (Fig. 2B); osmiophilic pyrenoglobules were
associated with these finger-like straight and not
Fig. 1. A, C and F. Location by LM of the two Trebouxia algae, TR1 and TR9, in transverse sections of a Ramalina farinacea thallus stained
with toluidine blue.
B, D, E, G and H. Anatomy of TR1 and TR9 by TEM of ultrathin sections of a R. farinacea thallus.
B. Location of TR1 and TR9 in the photobiont layer.
C–E. TR1 phycobiont, filled with a chloroplast containing a large central pyrenoid.
F–H. TR9 phycobiont, with a lobated chloroplast filling the protoplast and containing numerous large electron-dense vesicles.
Abbreviations: Aut, autospores; Chl, chloroplast; ChT, chondroid Tissue; Co, cortex; CT, cytoplasmic tufts; CW, cell wall; EV, electron-dense
vesicles; Hy, hyphae; N, nucleus; PhL, photobiont layer; Py, pyrenoid; SS, secretion space.
Successful coexistence of two algae in R. farinacea lichens 3
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
branched invaginations, consistent with the impressa-
type described by Friedl (1989). In TR9 cells, the
chloroplast occupied most of the cell volume; the most
peripheral thylakoids were grouped in stacks shaped by
numerous membranes, similar to the grana in vascular
plants (Figs 1H and 2E). Large spherical vesicles
(ⱖ0.6–1.5 mm) with an electron-dense content made
up of lipids were seen throughout the cytoplasm and
were especially abundant at the periphery and near the
mitochondria (Fig. 2D). These vesicles were observed
within the lichen thallus (Fig. 1G and H) and in culture
(Fig. 2D–F). The pyrenoid matrix was usually absent or
4L. M. Casano et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
could not be clearly differentiated and pyrenoglobules
were not observed. Only two Asterochloris species are
known to share this feature (Friedl, 1989). Zoospores with
distinct flagella have been recorded in both TR1 and TR9,
but only in cultures (Table 1).
Molecular analyses of phycobionts from R. farinacea
confirm the coexistence of two different Trebouxia taxa
In previous work, we sequenced a portion of the
chloroplast-encoded 23S rRNA gene in several Tre-
bouxia and Asterochloris algae, including a Trebouxia
phycobiont isolated from a population of Ramalina fari-
nacea collected in Sierra del Toro (Castellón, Spain) (Del
Campo et al., 2010). The obtained sequence spanned
position 759–2596 in the homologous Escherichia coli
23S rRNA gene and revealed the presence of distinct
and species-specific group I introns within the gene (Del
Campo et al., 2009; 2010). This first characterized Tre-
bouxia photobiont corresponded to the present TR9 phy-
cobionts. TR9 algae were initially named Trebouxia sp.
(accession EU600236, Del Campo et al., 2010). In this
work, we sequenced the same portion of the 23S rRNA
gene of the isolated TR1 phycobionts of R. farinacea,
which was shown to be highly homologous to that of
Trebouxia jamesii UTEX 2233 (accession EU352794,
Del Campo et al., 2010). The number, sequence,
and distribution of introns within the same portion
of 23S rRNA gene differed greatly between TR1 and
TR9.
In the present study, the photobiont composition of 36
thalli of R. farinacea collected from different Mediterra-
nean and temperate regions was analysed (Table S1). In
accordance to the above-mentioned information and in
order to determine the presence of TR1 and/or TR9 phy-
cobionts in these thalli, two distinct group I introns were
amplified, each present in one phycobiont but absent in
the other (see Experimental procodures). Figure 3B and
C shows the products of a representative amplification
experiment performed with the DNA extracted from indi-
vidual thalli and either the cL2263F/cL2263R or the
cL781F/cL781R primer pair. Significantly, both primer
pairs amplified all assayed samples. Sequencing of the
amplified products confirmed the presence of TR1 and
TR9 phycobionts in all studied lichen thalli independent of
its native geographical localization.
In addition, we designed two different primer pairs, ITS-
TR1f/ITS-TR1r and ITS-TR9f/ITS-TR9r, homologous to
the nrITS sequences of the isolated phycobionts TR1 and
TR9 respectively. PCRs were carried out with DNA
obtained from each lichen thallus (Rf) and the phyco-
bionts TR1 and TR9 isolated in culture. Figure 3D depicts
a gel electrophoresis of the reaction products, showing
that the primer pair ITS-TR1f/ITS-TR1r amplified DNA
from both the lichen thallus and the TR1 phycobiont but
not from the TR9 phycobiont. By contrast, the primer pair
ITS-TR9f/ITS-TR9r amplified DNA from both the lichen
thallus and the TR9 phycobionts but not from the TR1
phycobionts. These findings support our initial hypothesis
of the presence of two different coexisting Trebouxia
species, corresponding to TR1 and TR9, within the same
lichen thallus.
Growth and photosynthetic behaviour of Ramalina
farinacea photobionts
Morphological and molecular results suggest a highly
specific and selective pattern of association of the
lichen-forming fungus R. farinacea, since the same two
phycobionts, TR1 and TR9, were found in all lichen thalli
studied. To search for physiological differences between
the two R. farinacea phycobionts, the effects of tempera-
ture and light on the growth and photosynthetic traits of
Fig. 2. Ultrastructure of TR1 and TR9 phycobionts in culture by TEM micrographs of ultrathin sections.
A, B, C. TR1 phycobionts.
A. Cell with a central massive single lobated chloroplast and a central pyrenoid.
B. The chloroplast is loosely packed with thylakoidal membranes closely associated in stacks of three at the most. The outer lamellae of the
thylakoid stack develop tubules penetrating the pyrenoid matrix as finger-like invaginations. Osmiophilic pyrenoglobules are associated with
these tubules. Pyrenoid morphology corresponds to the impressa-type.
C. Cell wall and secretion space. The cell wall shows four layers. The outermost (1) is thin and electron-opaque; followed by an
electron-transparent layer (2), an electron-opaque layer (3), and finally, towards the interior (4), a heavier layer made up from the materials
contained in the secretion space. In this space, several secretion vesicles coming from the cytosol can be observed.
D, E, F. TR9 phycobionts.
D. Cell with a large lobated chloroplast containing numerous electron-dense vesicles.
E. Chloroplast in the peripheral zone. The outer thylakoids are grouped in stacks made up by numerous membranes, as in the grana of
vascular plants. Several starch granules, a mitochondrion, and electron-dense vesicles are well stand out.
F. Cell wall and secretion space. The cell wall shows three layers. The outermost (1) is thin and dense while the intermediate layer (2) is
clearer. The dense internal layer (3) is formed from the materials contained in the secretion space.
Abbreviations: Chl, chloroplast; ChM, chloroplast membrane; CM, cellular membrane; CW, cell wall; Cy, cytosol; EV, electron-dense vesicles;
Gr, grana; Mit, mitochondria; N, nucleus; Nu, nucleolus; Pg, pyrenoglobules; Py, pyrenoid; S, starch; SS, secretion space; SV, secretion
vesicles; T, tubules; TM, thylakoids membranes.
Successful coexistence of two algae in R. farinacea lichens 5
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Fig. 3. A. Genetic maps of the sequenced portion of plastid LSU rDNA in TR1 and TR9 phycobionts (position 755–2535 in E. coli). The
dark-grey boxes represent exons. Introns are depicted as light-grey boxes between exons. Intron names (according to Johansen and Haugen,
2001) are indicated above each intron. Open reading frames (ORFs) encoding putative homing endonucleases are depicted as black boxes
within introns. Primer positions are indicated below each map with arrows.
B. Agarose gel electrophoresis of PCR amplification products (1.5% agarose) obtained with the cL781F/cL781R primer pair and DNA extracted
either from lichen thalli collected at different geographic locations (As, Asturias; Le, León; Ca, Castellón, Tf, Tenerife, MH, Monte Hamilton; SF,
San Francisco) or from isolated and axenically propagated TR1 and TR9 phycobionts. Molecular size markers are indicated on the right.
C. PCR amplification products obtained with the cL2263F/cL2263R primer pair and DNA extracted either from lichen thalli collected in different
geographic locations or from isolated TR1 and TR9 phycobionts. Molecular size markers are indicated on the right.
D. PCR amplification products obtained with the ITS-TR1f/ITS-TR1r and ITS-TR9f/ITS-TR9r primer pairs and DNA extracted either from a
lichen thallus (Rf) collected at Sierra El Toro (Castellón, Spain) or from isolated TR1 and TR9 phycobionts. Molecular size markers are
indicated on the left.
6L. M. Casano et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
isolated and cultured TR1 and TR9 algae were studied.
It should, however, be noted that while the physiological
performance of algae in culture was assumed to be rep-
resentative of that in symbiosis, further experiments
with entire thalli (e.g. transplantation of thalli to higher or
lower irradiances) are needed to confirm this assump-
tion. Figure 4A shows the fresh weight reached by TR1
and TR9 after 30 days at 17°C or 20°C and 15–
100 mmol m-2s-1of PAR. Both species grew better at
the lower temperature; however, at 20°C the negative
impact of the high temperature was less on TR9 than on
TR1, whose growth was almost inhibited. This general
trend was similarly influenced by light intensity. At 17°C,
the maximum biomass of TR1 and TR9 was obtained
at an irradiance of 30 and 50 mmol m-2s-1respec-
tively. While the highest irradiance (at 17°C) negatively
affected the growth of both species, the biomass accu-
mulation of TR1 decreased by c. 56% with respect to
the maximum value but only by c. 6% in the case of
TR9.
The effects of light on photosynthesis were studied
through modulated fluorescence analyses. Since the low
algal biomass at 20°C significantly increased the experi-
mental error of fluorescence measurements, rendering
non-reliable data, the experiments were carried out only
in cultures growing at 17°C. The fluorescence parameter
Fv/Fm, provides an estimate of the maximum quantum
efficiency of PSII photochemistry, showing normal values
of about 0.8 for healthy leaves of vascular plants, inde-
pendently of tissue structure, cell number or total Chl
content (Björkman and Demmig, 1987; Baker and Oxbor-
ough, 2004). Registered values of Fv/Fmfor lichen thalli
are lower, usually above 0.7, but there are also normal
values as low as 0.67 for unstressed lichens with green
phycobionts (Demmig-Adams et al., 1990). Decrease of
Fv/Fmcould result from a decrease in the fraction of PSII
centres that are capable of photochemistry and/or an
increase of non-photochemical quenching (NPQ) (Baker
and Oxborough, 2004). Fv/Fmwas greater in TR9 than
in TR1 at all culture irradiances (Fig. 4B). The Fv/Fm
values in TR9 remained nearly constant. In contrast, TR1
was more sensitive to changes in culture irradiance,
as a maximum value of Fv/Fm(0.678) recorded at
30 mmol m-2s-1decreased to 0.642 at 100 mmol m-2s-1,
indicating the appearance of a slight photoinhibition when
TR1 was cultured out of a narrow light intensity range. As
shown in Figure 5, there was a clear difference in the
photosynthetic light response of TR1 and TR9 cultured at
different light intensities. TR1 showed similar values of the
relative quantum yield of electron transfer at PSII (FPSII,a
measure of the overall efficiency of PSII reaction centres
in the light, Fig. 5A) and relative electron transport rate
(ETR, Fig. 5B), independent of the light during culture.
However, TR9 exhibited increasing FPSII and ETR values
with increasing culture light intensity. Electron transport
flow was saturated at approximately the same irradiance
(c. 700 mmol m-2s-1) in both species and under all culture
conditions. However, photosynthetic activity was higher
in TR9 than in TR1.
Non-photochemical quenching, NPQ, is an estima-
tion of the non-radiative dissipation of excitation energy
and can thus be considered as photo-protective
(Demmig-Adams and Adams III, 1996). Figure 5C shows
the photosynthetic light response of NPQ in TR1 and TR9
cultured at different light intensities. The photosynthetic
photon flux density (PPDF) response curves showed a
correlation between NPQ and the culture light condition.
Fig. 4. Effects of temperature and irradiance during culture on
growth and photosynthesis in isolated TR1 and TR9 phycobionts.
A. TR9 and TR1 biomass accumulation after 30 days of culture at
17°C or 20°C and the indicated irradiances. Grey and white circles
represent TR1 and TR9 phycobionts cultured at 17°C respectively.
Grey and white squares represent TR1 and TR9 phycobionts
cultured at 20°C respectively. Data are the mean values of five
independent replicates (+or -SD).
B. Maximum quantum yield of PSII (Fv/Fm) of TR1 and TR9
phycobionts cultured at 17°C and four different light intensities
during 30 days. Data are the mean values of five independent
replicates (⫾SD). Grey and white circles represent TR1 and TR9
phycobionts respectively. Different normal and italic letters indicate
significant differences among culture conditions for TR1 and TR9
phycobionts respectively (LSD test).
Successful coexistence of two algae in R. farinacea lichens 7
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
The NPQ values increased with increasing light intensity
up to 50 mmol m-2s-1, without further changes in either
TR1 or TR9. Interestingly, the highest NPQ values
were observed in strains of TR9 cultured at higher light
intensities. In addition, we examined the parameter 1-qP
(Fig. 5D), which is a measure of the reduction state
of the first electron acceptor of PSII (QA); high 1-qP
values are considered indicative of light stress (Weis and
Berry, 1987). In both algae, lower values of 1-qPwere
observed at low and moderate PPDF. The lowest
1-qPvalues occurred in TR9 cultured at higher light
intensities.
Discussion
Two Trebouxia taxa are ever-present in lichen
thalli of Ramalina farinacea
To date, most of the studies on the population structure
and patterns of symbiotic association in lichens have
reported the presence of a single species of phycobiont
per thallus. These findings are in agreement with the
Gause’s Principle (Gause, 1934), which postulates that
the presence of closely related species with similar
resource requirements within the same ecological niche
often results in competitive exclusion(s) and the preva-
lence of the competitor that can maintain itself with the
lowest level of the limiting resource. In the case of lichens,
the thalli can be considered as self-contained miniature
ecosystems (Honegger, 1991), with the algal layer as the
single primary producer. However, there is increasing evi-
dence that in some lichens, including Evernia mesomor-
pha (Piercey-Normore, 2006) and Protoparmeliopsis
muralis (Guzow-Krzeminska, 2006), more than one algal
genotype is present within the same thallus. The present
study has demonstrated the coexistence within each
lichen thallus of the same two Trebouxia algae (TR1 and
TR9) analysed from geographically distant populations of
the lichen R. farinacea. This is the first report of this type
of symbiotic association in a lichen species supported by
morphological, molecular, and physiological evidences.
Unlike previous reports, in our study the simultaneous
presence of TR1 and TR9 phycobionts was not restricted
to a few analysed specimens but was instead found
to occur in all lichen thalli, independent of their native
geographic location. Moreover, the two coexisting algae
Fig. 5. Effects of irradiance during culture on fluorescence parameters in isolated TR1 and TR9 phycobionts. PPFD response curves of the
actual quantum yield of the PSII (A, FPSII), relative electron transport rate (B, ETR), non-photochemical quenching (C, NPQ), and reduction
state of the QA(D, 1-qP) for TR1 (grey) and TR9 (white) algae cultured at 17°C and at 15 mmol m-2s-1(circles), 30 mmol m-2s-1(squares),
50 mmol m-2s-1(triangles) and 100 mmol m-2s-1(rhombus). Data are the mean values of five independent replicates, with standard deviations
(not shown) ranging from 0.5% to 3.2% of the corresponding mean values.
8L. M. Casano et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
clearly belonged to two different Trebouxia taxa, as deter-
mined by differences in key morphological traits (Table 1,
Figs 1 and 2) and in a large portion of the plastid-encoded
23S rRNA gene (Fig. 3A). It should be pointed out that the
rate of evolution of the chloroplast genome is slower than
that of the nuclear genome and that there is a single
sequence for each gene (even in the case of genes
repeated in different locations). Therefore, the detection of
two different sequences of the same plastid-encoded
gene in the same thallus clearly indicates the presence of
two different taxa. On the other hand, when we analysed
the algal nrITS associated with each lichen thallus
employing the primer pair ITS1T-5′/ITS4T-3′(Kroken and
Taylor, 2000), only one ITS sequence was found in all
cases (data not shown). However, the use of two different
primer pairs homologous to the ITS sequences of the
isolated photobionts TR1 and TR9 allowed us to corrobo-
rate the presence of these Trebouxia species coexisting
within the same lichen thallus (Fig. 3D). Therefore, care
must be taken when ‘general’ primers are employed and
only a single algal nrITS sequence is detected. It may be
indicative of the predominant photobiont while others in
minority are not adequately amplified.
Environmental conditions and the physiological
performance of each algal species may modulate
coexistence within the thallus
Studies on other better known symbiotic systems where
the host is heterotrophic and constitutes the habitat
for photoautotrophs, such as reef-building corals, initially
considered a single algal endosymbiont species per host.
However, it is now recognized that several large and
genetically diverse algal groups often co-occur within a
single host species or colony (Wegley et al., 2007). Little
and colleagues (2004) demonstrated dynamic variations
of coral-algal associations according to environmentally
related changes in the host’s physiological needs. More
recently, Jones and colleagues (2008) described dramatic
alterations in the algal symbiont community of some coral
species in response to environmental stress. Such poly-
morphic symbioses suggest that the identity of the algal
partner(s) is as significant as that of the host in determin-
ing the physiology of the holobiont. In the case of lichen
symbiosis, there is increasing experimental evidence
that the association between phycobionts and mycobionts
increases tolerance to stress conditions (e.g. Kranner
et al., 2005; Kosugi et al., 2009). In our study, the
observed physiological responses (growth and photosyn-
thesis, Fig. 4) to temperature and light conditions indi-
cated that TR9 performs better under relatively high
temperature and irradiances whereas TR1 thrives under
more temperate and shady conditions. These conclusions
are supported by studies of biomass accumulation and
photosynthetic traits, including the photochemical effi-
ciency of PSII, the reduction state of QA, and the NPQ
(Fig. 5). Non-photochemical quenching is an important
process under stress conditions, transforming excess
light energy that cannot be used in photosynthesis into
heat. In vascular plants, it is associated with the xantho-
phyll cycle (Niyogi et al., 2005). However, the role of this
cycle in lichen photobionts is thought to be limited, as
alternative mechanisms of light dissipation are employed
(Kopecky et al., 2005; Gasulla et al., 2009). In addition,
recent results from our lab (Catalá et al., 2010) support
an important role for NO in the photo-protection of phy-
cobionts in R. farinacea. The diversity of habitats and
local climates characteristic of the geographic locations
(Table S1) sampled in the present study, along with the
variety of ecological contexts in which R. farinacea prolif-
erates, reflects the ecophysiological plasticity of this sym-
biosis as a mechanism allowing the lichen to cope and
thus to adapt to changing and often stressful environ-
ments. Moreover, a positive interaction between the two
phycobionts cannot be ruled out. According to Gross
(2008) and Angert and colleagues (2009), positive inter-
actions among competitors may produce stable and
species-rich communities. Therefore, we propose that
the constant presence of both Trebouxia phycobionts
in R. farinacea is favoured by the different and probably
complementary physiological behaviour of each algal
species, thus improving the ecological fitness of the
holobiont.
Vegetative reproduction of Ramalina farinacea seems
to maintain a specific and selective association
among symbionts
In addition to environmental conditions, an important
mechanism controlling symbiotic associations may be the
symbiont’s reproductive mode. Lichens can reproduce
sexually and asexually. Specifically, mycobionts produce
either sexual spores, requiring ‘de novo’ associations with
photobionts in each generation, or vegetative propagules
containing fungal tissues and photobiont cells (Zoller and
Lutzoni, 2003; Yahr et al., 2004; 2006). Lichens that
depend on the cyclical establishment of fungal-photobiont
associations to colonize varied wide-ranging habitats
might require a relatively higher flexibility in the specificity
and ecological selection of their photobionts. This flexibil-
ity would facilitate successful relichenizations by allowing
for alternative partnerships in each habitat (Romeike
et al., 2002). Among the lichens that disperse through
soredia or other vegetative propagules, mycobiont and
photobionts are jointly propagated within the same repro-
ductive structure (Nelsen and Gargas, 2008). This repro-
duction strategy should allow to a strict preservation of the
relationship among symbionts, although maintenance of
Successful coexistence of two algae in R. farinacea lichens 9
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
symbiotic associations seems to be an option rather than
a strict consequence of joint symbiont dispersal in lichens
(Wornik and Grube, 2009). Ramalina farinacea propa-
gates through vegetative propagules (soredia), which is
consistent with the overall pattern of coexistence of the
same two algal Trebouxia species in all sampled thalli.
Our future work will deal with the origin and maintenance
of genetic variability within R. farinacea phycobionts (and
the possible relationship with mycobiont genotypes). We
will try to discriminate between alternative hypotheses
whether TR1 and TR9 are packed in each soredium or are
gathered from the environment.
In conclusion, R. farinacea represents a novel pattern
of lichen symbiosis that is maintained through joint
propagation of the mycobiont and the same two Tre-
bouxia phycobiont taxa. The preservation of this pattern
seems to be favoured by the distinct and probably
complementary ecophysiological responses of each phy-
cobiont, which in turn permits the lichen to proliferate in
very different habitats.In addition, these lichen provide
an interesting model system to be studied within the
context of hologenome theory of evolution (Rosenberg
et al., 2009), which emphasizes the cooperation and
competition among symbionts and their hosts, and to
monitor the responses of organisms to current and
future environmental changes.
Experimental procedures
Ramalina farinacea sampling, photobiont isolation
and culture
Specimens of Ramalina farinacea (L.) Ach. were collected
from seven different locations in the Iberian Peninsula and
Canary Islands and from two sites in California (USA), as
detailed in Table S1. Samples were dried out in the shaded
open air for 1 day and then stored at -20°C until needed. For
photobiont isolation, R. farinacea thalli were collected in the
air-dried state on Quercus rotundifolia Lam. at Sierra El Toro
(Castellón, Spain). Samples were frozen at -20°C until the
isolation experiment, 3 months after collection. TR1 and TR9
phycobionts were isolated in our laboratories according to
Gasulla and colleagues (2010). Isolated phycobionts were
cultured in liquid or semisolid Bold 3N medium (Bold and
Parker, 1962) in a growth chamber at 15°C, under a 14 h/10 h
light/dark cycle (light conditions: 25 mmol m-2s-1).
Morphological analysis of ‘in thallus’ lichen photobionts
and isolated algae
Pieces of rehydrated R. farinacea thalli [from Sierra El Toro
(Castellón, Spain)] were used to examine by TEM the TR1
and TR9 algae inside thallus. They were fixed in 2% Kar-
novsky fixative for 2 h at 4°C. The specimens were then
washed three times with 0.01 M PBS, pH 7.4, for 15 min each
and fixed with 2% OsO4in 0.01 M PBS, pH 7.4, for 2 h at 4°C.
Thereafter, specimens were washed in 0.01 M PBS,
pH 7.4, for 15 min and then dehydrated at room temperature
in a graded series of ethanol, starting at 50% and increasing
to 70%, 95% and 100% for no less than 20–30 min for each
step. The fixed and dehydrated samples were embedded
in Spurr’s resin according to manufacturer’s instruc-
tions (http://www.emsdiasum.com/microscopy/technical/
datasheet/14300.aspx). To analyse isolated algae, cultured
colonies were suspended in liquid Bold 3N medium then
added to an equal volume of 2% glutaraldehyde in the same
medium. After 1 h at 4°C, the samples were washed in
0.01 M PBS buffer, pH 7.4, and post-fixed with 1% OsO4in
0.01 M PBS, pH 7.4 (2 h at 4°C), then gently centrifuged,
washed again, dehydrated through an ethanol series, and
embedded in Spurr’s resin as previously described for in
thallus observations.
For light microscopy (LM) analyses, 1–2 mm sections were
cut from samples (R. farinacea thalli or isolated algae)
embedded in Spurr’s resin using a diamond knife (DIATOME
Histo 45°) and an ultramicrotome (Ultratome Nova LKB
Bromma). The sections were stained with 1% toluidine blue
and observed with an Olympus Provis AX 70 microscope
equipped with an Olympus Camedia C-2000 Z camera. For
transmission electron microscopy (TEM), 90 nm sections
were cut with a diamond knife (DIATOME Ultra 45°) using an
ultramicrotome (Ultratome Nova LKB Bromma), mounted on
copper grids of 100 mesh, and post-stained with 2% (w/v)
aqueous uranyl acetate and 2% lead citrate. The prepared
sections were observed with a JEOL JEM-1010 (80 kV) elec-
tron microscope, equipped with a MegaView III digital camera
and ‘AnalySIS’ image acquisition software, at the SCSIE
service of the University of Valencia.
Nucleic acids extraction, purification
and PCR amplification
Total DNA was extracted from each lichen thallus and from
isolated Trebouxia phycobionts following the procedure of
Cenis (1992). Isolated DNA was PCR-amplified with specific
primers under the cycling conditions described in Del Campo
and colleagues (2010). Amplification products were sub-
jected to agarose electrophoresis and then purified and
sequenced according to Del Campo and colleagues (2010).
All oligonucleotides used in the PCRs and in the subsequent
sequencing of the chloroplast 23S rDNA were designed
based on the nucleotide sequence of the chloroplast
23S rRNA gene from Trebouxia aggregata (DDBJ/EMBL/
GenBank Accession No. L43542; Pombert et al., 2006) and
on partial sequences obtained in our laboratory. The primers
used for amplifying and sequencing of nrITS from isolated
TR1 and TR9 were obtained from Kroken and Taylor (2000).
The primers ITS-TR1f and ITS-TR1r, ITS-TR9f and ITS-TR9r,
specific for each isolated photobionts, were designed based
on the obtained global sequences of nrITS from isolated TR1
and TR9.
List of oligonucleotides:
cL2263R: 5′-GTAATACTACATTGGTGCGGAC-3′
cL2263F: 5′-AGCACATCACAGAGAAGCTG-3′
cL781F: 5′-TCATAACGGTGAAACCTAAGGC-3′
cL781R: 5′-CAGAACGCCAAACCATATACCG-3′
10 L. M. Casano et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
ITS-TR1f: 5′-ACACACGTCAAGCAATCAACTC-3′
ITS-TR1r: 5′-CTGACCGGCAACCCGAAG-3′
ITS-TR9f: 5′-AACATACCTTAAGCAATTAATTC-3′
ITS-TR9r: 5′-TGACCGGCTAGCATTCAG-3′
The cL2263F/cL2263R primer pair (Fig. 3A) is included
within the group IB4 intron Tja.cL2263 of the chloroplast-
encoded 23S rRNA gene (Del Campo et al., 2010). This
intron is exclusive to TR1 phycobionts. The cL781F/cL781R
primer pair (Fig. 3a) is included within the group IA3 intron
Tsp.cL781 of the chloroplast-encoded 23S rRNA gene (Del
Campo et al., 2010) and is exclusive to TR9 phycobionts.
Measurements of growth and chlorophyll afluorescence
A50ml aliquot of actively growing algae resuspended in liquid
Bold 3N medium (1 ¥106cells ml-1) was inoculated on
cellulose–acetate disks placed on agarized medium and then
cultured for 30 days at 17°C and 20°C with an irradiance of
15, 30, 50 or 100 mmol m-2s-1under a 14 h/10 h light/dark
cycle. The influence of culture conditions on algal growth was
measured as biomass accumulation at the end of the culture
period.
Chlorophyll afluorescence was measured at room tem-
perature using a pulse modulation fluorometer (PAM-2000,
Walz, Effeltrich, Germany). Isolated algae grown for 3 weeks
on cellulose–acetate membranes placed on semisolid culture
medium were layered on filter paper that was kept moist with
distilled water in order to maintain the cells in a fully hydrated
state. The membranes were placed in the dark for 30 min
after which the minimal fluorescence yield (Fo) was obtained
by exciting the phycobionts with a weak measuring beam
from a light-emitting diode. A saturating pulse (800 ms) of
white light (at a photosynthetic photon fluence rate of
8000 mmol m-2s-1over a wavelength band of 400–700 nm),
closing all reaction centres, was then applied to obtain
maximal fluorescence (Fm). Variable fluorescence in dark-
adapted samples (Fv) was calculated as Fm-Fo. The
maximum quantum yield of photosystem II (PSII) was calcu-
lated as Fv/Fm(Schreiber et al., 1986). Subsequently, a series
of 30-s actinic light pulses (44, 65, 87, 139, 210, 315, 460,
700, 1100, 1700 mmol m-2s-1) and further saturated pulses of
white light were applied to determine: (i) maximum fluores-
cence yield during actinic illumination (Fm′); (ii) Chl afluores-
cence yield during actinic illumination (Fs); and (iii) the level of
modulated fluorescence during a brief interruption (3 s) of
actinic illumination in the presence of 6 mmol m-2s-1far-red
light (Fo′). The non-photochemical dissipation of absorbed
light energy (NPQ) was determined at each saturating pulse
according to the equation NPQ =(Fm-Fm′)/Fm′(Bilger and
Björkman, 1991). The coefficient for photochemical quench-
ing, qp, was calculated as (Fm′-Fs)/(Fm′-Fo′) (Schreiber
et al., 1986). The quantum efficiency of PSII photochemistry,
FPSII, closely associated with the quantum yield of non-cyclic
electron transport, was estimated from (Fm′-Fs)/Fm′(Genty
et al., 1989).
Acknowledgements
This study was funded by the Spanish Ministry of Education
and Science (CGL2006-12917-C02-01/02), the Spanish Min-
istry of Science and Innovation (CGL2009-13429-C02-01/
02), the AECID (PCI_A/024755/09) and the Generalitat
Valenciana (PROMETEO 174/2008 GVA). We are grateful to
Dr J. Gimeno-Romeu (University of California, Davis, USA)
and to Dr P.J.G. de Nova (IREC, Ciudad Real, Spain), who
were the first to isolate DNA from Ramalina farinacea thalli in
our group. Wendy Ran revised the manuscript in English.
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Location for collections of Ramalina farinacea
samples used in this study.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing mate-
rial) should be directed to the corresponding author for the
article.
Successful coexistence of two algae in R. farinacea lichens 13
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Supporting Table. Location for collections of Ramalina farinacea samples used in this study.
Locality / geographic coordinates / altitude / bioclimatic belt / collection data Phorophyte n
Spain, Puerto de Ventana - AS-228 (Teverga, Asturias) / 43º 4’ 33’’ N - 6º 1’ 54’’ / 1200 m / montane
belt / (leg. Barreno & Temple 25/09/2000) Fagus sylvatica 3
Spain, Villamayor, Puerto de Maravio – AS-311 (Teverga, Asturias) / 43º 13’ 1.73’’ N - 6º 7’ 59.66’’ W /
650 m / coline belt / (leg. Vázquez 20/03/2006) Quercus pyrenaica 5
Spain, close to Modino – N-625 (León) / 42º 45’ 31.57’’ N - 5º 8’ 54.69’’ W / 1300 m /
supramediterranean belt / (leg. Barreno & Herrera-Campos 27/09/2007) Quercus pyrenaica 5
Spain, Mas del Pastor, Sierra del Toro (Castellón) / 39º 57’ 32.34” N - 0º 46’ 35.51” W / 1150 m /
mesomediterranean belt / (leg. Barreno & Gasulla 27/11/2007) Quercus rotundifolia 6
Spain, Villanueva de la Fuente, Valdepeñas de Alcaraz – CM-412 (Ciudad Real, Castilla-La Mancha)
38º 40’ 40.89” N - 2º 39’ 16.48” W / 820 m / mesomediterranean belt / (leg. Gómez de Nova
18/04/2006
)
Quercus rotundifolia 5
Spain, Pinar de la Guancha (Tenerife, Canary Islands) / 28º 20’ 48’’ N - 16º 39’ 43’’ W / 900 m /
supramediterranean belt / (leg. Barreno & Santos 30/01/1997) Pinus canariensis 5
Spain, Los Realejos - Zona Recreativa Parque de Corona Forestal (Tenerife, Canary Islands,) / 28º 20’
51’’ N - 16º 35’ 27’’ W / 1300 m / supramediterranan belt / (leg. Barreno & Santos 02/02/1997) Pinus canariensis 5
USA, Joseph Grant Co. Park / Route 130 from San José to Mount Hamilton / Santa Clara Co. (CA,
USA) / 37° 19’ 23’’ N - 121° 40’ 9’’ W / 580 m / mesomediterranean belt / (leg. Barreno, Sánchez Mata,
Sancho & Tretiach 24/07/2008
)
Quercus kellogii 1
USA, Hill around the San Francisco Botanical Garden, San Fancisco City and Co. (CA, USA) / 37º 46’
00’’ N - 122º 28’ 08’’ W / 230 m / mesomediterranean belt / (leg. Barreno & Clerc 11/07/2008) Pinus sp. 1
