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Ramalina farinacea is an epiphytic fruticose lichen that is relatively abundant in areas with Mediterranean, subtropical or temperate climates. Little is known about photobiont diversity in different lichen populations. The present study examines the phycobiont composition of several geographically distant populations of R. farinacea from the Iberian Peninsula, 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 determined 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 relatively 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.
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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 3 Numerous
Large electron dense vesicles Number Few (4) Numerous (>5)
Size (mm) <0.6 0.6–1.5 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
(0.6–1.5 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
... Among them, Trebouxia lynnae Barreno has emerged as the Trebouxia research model because its ultrastructure has been largely analysed and its cells have been described in detail by different approaches [8,9]. Furthermore, its nuclear, chloroplast, and mitochondrial genomes have been sequenced and annotated, and more physiology information is gathered every year [8][9][10][11][12][13][14][15]. ...
... In particular, a diversity of Trebouxia phycobionts within different lichen species has been reported across niche gradients [16][17][18][19][20][21]. In addition, the intrathalline coexistence of various Trebouxia species-level lineages has been frequently reported, and it has been suggested that the thallus morphology and growth stage affect the diversity of the associated phycobionts [12,17,[22][23][24][25][26][27][28]. Inter-and intrathalline phycobiont diversity has been mainly assessed by molecular techniques [19,21,22,[24][25][26][27][28][29]. ...
... Mitochondria were observed in high numbers in LTSEM (Figure 7), although their internal structures were difficult to discern ( Figure 7A,B). TEM images allowed us to observe the internal structures of the mitochondria in T. lynnae ( Figure 7C), as already reported by Casano et al. [12]. As expected, every Trebouxia species-level lineage analysed by TEM presented well-developed mitochondria (Supplementary Figure S4). ...
Article
Full-text available
The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucle-olar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.
... In the past decades, several studies have proven the coexistence of multiple Trebouxia species-level lineages within a single lichen thallus, shedding light on diverse patterns of photobiont-mycobiont associations [20][21][22][23][24][25][26]. The pioneer studies in this field were those of del Campo et al. [27,28], which revealed the coexistence of multiple Trebouxia taxa in the individual thalli of the lichen Ramalina farinacea. ...
... Since then, Trebouxia sp. TR9 and Trebouxia jamesii were recurrently found in the thalli of both R. farinacea and other lichen species using culture isolations and Sanger sequencing (e.g., [22,23,[27][28][29]31,32]). These species have been successfully maintained as viable in vitro culture for over 10 years. ...
... This microalga presents a pyrenoid impressa-type and a shallowly lobed-type of chloroplast [15]. It is photosynthetically better performing at higher temperature and irradiance [22], and shows novel inducible responses against abiotic stresses. In particular, Trebouxia sp. ...
Article
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Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont–photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.
... Surprisingly, multiple algal species have been observed in association with the same thallus in a large number of studies [24,31,32]. For example, Backor et al. [33] confirmed the presence of multiple algal genopypes in a single lichen thallus, Casano et al. [34] found that Ramalina farinacea thalli represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts, and Del Campo et al. [35] concluded that ecological diversification and speciation of lichen symbionts in different habitats could include a transient phase consisting of associations with more than one photobiont in individual thalli. This pattern of algal coexistence is probably promoted by their different and complementary ecophysiological responses which facilitate the proliferation of the lichen in a wide range of habitats and geographic areas [34]. ...
... For example, Backor et al. [33] confirmed the presence of multiple algal genopypes in a single lichen thallus, Casano et al. [34] found that Ramalina farinacea thalli represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts, and Del Campo et al. [35] concluded that ecological diversification and speciation of lichen symbionts in different habitats could include a transient phase consisting of associations with more than one photobiont in individual thalli. This pattern of algal coexistence is probably promoted by their different and complementary ecophysiological responses which facilitate the proliferation of the lichen in a wide range of habitats and geographic areas [34]. ...
... However, the lack of experimental evidence regarding the lichen microbiome hindered our ability to reveal the network of interactions within this holobiont. As opposed to what was traditionally believed, the photobiont should not be limited to a single strain of algae [34] and protists and even viruses can form symbiotic associations with lichens [62,63] (Figure 2). ...
Article
Full-text available
Lichens have long been considered as composite organisms composed of algae and/or cyanobacteria hosted by a fungus in a mutualistic relationship. Other organisms have been gradually discovered within the lichen thalli, such as multiple algal species, yeasts, or even viruses. Of pivotal relevance is the existence of the lichen microbiome, which is a community of microorganisms that can be found living together on the lichen surface. This community performs a growing number of functions. In this entry, we explore the journey of lichens being considered from a dual partnership to a multi-species symbiotic relationship.
... The enhancement of molecular methods and development of specific primers for algal gene loci has improved the knowledge of the diversity and variability of photosynthetic partners (Grube and Spribille, 2012). The questions which can be asked are if they are merely epibionts or only distributed in low abundance within the lichens or only spotted in certain parts of the thallus (Guzow-Krzemi nska, 2006; Grube and Muggia, 2010;Casano et al., 2011). A study hypothesized, by analysing both washed and unwashed lichen samples (Muggia et al., 2013), that the epithalline algae communities host numerous algal species, and if not separately considered, might lead to an overestimation of photobiont diversity in lichens in general and a direct improper function in the lichen symbiosis. ...
... A study hypothesized, by analysing both washed and unwashed lichen samples (Muggia et al., 2013), that the epithalline algae communities host numerous algal species, and if not separately considered, might lead to an overestimation of photobiont diversity in lichens in general and a direct improper function in the lichen symbiosis. But as emphasized, on the other hand, it may confer advantages in the lichen's ability to counteract environmental changes or to occupy extreme environments mixing strategies to fine-tune their association (Casano et al., 2011;Molins et al., 2018). Indeed, a higher variety of symbiotic associations could be helpful when changing environments and therefore might be the rule in lichens living on a wide variety of substrates, as R. geographicum, and in diverse habitats like the very hostile environment of the Britain Atlantic Coast. ...
... Indeed, a higher variety of symbiotic associations could be helpful when changing environments and therefore might be the rule in lichens living on a wide variety of substrates, as R. geographicum, and in diverse habitats like the very hostile environment of the Britain Atlantic Coast. They could finally correspond to a habitat-adapted symbiosis (Rodriguez et al., 2008;Casano et al., 2011) and different partnerships can be tested at low risk for the entire thallus structure (Muggia et al., 2013). Even if the Sanger direct sequencing approach gave clues about fungusalgal association, distribution patterns and diversity in lichens, it also can lead to oversimplify diversity (Voytsekhovich and Beck, 2016), thus, underestimating all the complexity of the symbiotic association and underdetecting less abundant co-occurring photobiont partner (Moya et al., 2017). ...
Article
Full-text available
Recently, the study of the interactions within a microcosm between hosts and their associated microbial communities drew an unprecedented interest arising from the holobiont concept. Lichens, a symbiotic association between a fungus and an alga, are redefined as complex ecosystems considering the tremendous array of associated microorganisms that satisfy this concept. The present study focuses on the diversity of the microbiota associated with the seashore located lichen Rhizocarpon geographicum, recovered by different culture‐dependent methods. Samples harvested from two sites allowed the isolation and the molecular identification of 68 fungal isolates distributed in 43 phylogenetic groups, 15 bacterial isolates distributed in five taxonomic groups and three microalgae belonging to two species. Moreover, for 12 fungal isolates belonging to 10 different taxa, the genus was not described in GenBank. These fungal species have never been sequenced or described and therefore non‐studied. All these findings highlight the novel and high diversity of the microflora associated with R. geographicum. While many species disappear every day, this work suggests that coastal and wild environments still contain an unrevealed variety to offer and that lichens constitute a great reservoir of new microbial taxa which can be recovered by multiplying the culture‐dependent techniques.
... Co-occurrence of Trebouxia photobionts within the same lichen thallus has been recognized to be a common phenomenon for many years (Muggia et al., 2008;Casano et al., 2011;Català et al., 2016;Moya et al., 2017). It was speculated that multiple photobionts may serve in the lichen symbiosis as strategic, additional partners to cope with changeable environmental conditions and to help the mycobiont widen its ecological distribution (Casano et al., 2011). ...
... Co-occurrence of Trebouxia photobionts within the same lichen thallus has been recognized to be a common phenomenon for many years (Muggia et al., 2008;Casano et al., 2011;Català et al., 2016;Moya et al., 2017). It was speculated that multiple photobionts may serve in the lichen symbiosis as strategic, additional partners to cope with changeable environmental conditions and to help the mycobiont widen its ecological distribution (Casano et al., 2011). However, this has not been demonstrated on a wide scale. ...
... The multiplicity of algal partners inside a thallus may be interpreted in the light of different symbiotic strategies and flexibility of the symbionts. Indeed, the potential of a lichen mycobiont to host multiple intrathalline photobionts (Casano et al., 2011) led to the possibility to build the best habitat-adapted symbiosis (Rodriguez et al., 2008). This would allow also for photobiont demographic variation within the thallus to ideally match the ecological conditions in which the thallus develops. ...
Article
Full-text available
Fungal–algal relationships—both across evolutionary and ecological scales—are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra, to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000–2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia “A52.” Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia “A52” and three other previously recognized lineages, i.e., Trebouxia “A02,” T. vagua “A04,” and T. vagua “A10,” which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes.
... Since the report of the coexistence of two Trebouxia species in Ramalina farinacea (Del Campo et al. 2010, 2013, the multiple microalgae coexisting pattern in a lichen was prevalently found in recent studies (Blaha et al. 2006;Dal Grande et al. 2018;Engelen et al. 2016;Lohtander et al. 2003;Molins et al. 2018Molins et al. , 2020Moya et al. 2017;Muggia et al. 2014;Onut-Brannstrom et al. 2018;Paul et al. 2018), which greatly challenged the traditional paradigm that a lichen consists of one lichen-forming fungus and a chlorobiont or a cyanobiont or both photobionts. Apart from their major photosynthetic partners, lichens may also choose different algae to respond and adapt to environmental changes (Casano et al. 2011;Castillo and Beck 2012;Dal Grande et al. 2018). ...
... Clade II LM-2014) in samples HO2, HO5 and HO6. Regardless of the effects of identification bias, a potential explanation for the change in photobionts might be host specificity or a way to respond to microclimatic conditions, micro niches or potential photobiomemicrobial interactions (Casano et al. 2011;Castillo and Beck 2012;Dal Grande et al. 2018). In addition, some shared taxa, such as Hem. ...
... The genetic background of the host-symbiont could establish and maintain symbiotic partnerships , therefore the lichen-forming fungus is key for determining the stable algal association patterns. Meanwhile, as the photobionts of lichens contribute greatly to the carbon source, the photobionts diversity may also influence the ability of lichens to respond or adapt to environmental changes, and to occupy diverse ecological niches (Casano et al. 2011;Castillo and Beck 2012;Dal Grande et al. 2018). In this study, it is interesting to find that various predominant algae were detected among different individuals of the same lichen host. ...
Article
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The dual nature of fungal-algal lichen symbioses is extended by other microbial associations. Increasing evidence has confirmed that lichens are successful holobionts composed of complex and multiple species. Specific interactions between these microbes contributed to the lichens’ health, growth and fitness. Previous studies suggested that the composition of microorganisms in lichens was potentially influenced by the genetic background of the host-symbiont, large-scale geography or different photobiont-types (cyanobiont and chlorobiont). However, our knowledge of the interactions between the main symbiotic partners under a certain ecological condition and how horizontal acquisition of these microorganisms contributes to endolichenic microbiome diversity remains limited. In the present study, using amplicon sequencing, we investigated the complex diversity and community composition of fungi, bacteria, and microalgae within Heterodermia obscurata from a similar niche. We found that endophytic bacteria displayed greater diversity than fungi and microalgae. Although preferences for core taxa varied among the different definitions, all analyses support that lichen-forming genus Heterodermia and green alga Trebouxia sp. OTU A15 were the main symbionts, and the bacterium Beijerinckiaceae was the core microbiome in H. obscurata. Significantly, we found that different alga species (Trebouxia) from H. obscurata are accompanying with a shift in composition and function of endolichenic bacteria and fungi. This finding suggested that besides host- or habitat specificity as well as photobiont-types, the shifts of dominant alga may also contribute to the taxonomical and functional differences of microbiomes within lichens.
... In the case of lichens, symbiont switching is commonly considered as the most obvious similarity with corals, with several studies reporting on the ability of lichen-forming fungal hosts to associate with different, potentially locally-adapted algae as a strategy to increase the ecological amplitude of the lichen holobiont [13][14][15][16]. Furthermore, lichen individuals may carry multiple algal strains with different physiological properties, which has been interpreted as a strategy to increase ecological flexibility of the holobiont [17,18]. Interestingly, Trebouxiophyceaen photobionts in lichens have specialized reaction centers of photosystem II to prevent damages in case of abrupt changes in light intensity [19,20]. ...
... Our data confirm the results from previous studies that different algae can display different photosynthetic performance. Casano et al. [17] showed that two common photobionts of the lichen Ramalina farinacea, often co-inhabiting the lichen thallus, have clearly distinct physiological optima. The authors suggested that these physiological differences might be a key factor in increasing the niche space of the lichen. ...
Article
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Corals and lichens are iconic examples of photosynthetic holobionts, i.e., ecological and evolutionary units resulting from the tightly integrated association of algae and prokaryotic microbiota with animal or fungal hosts, respectively. While the role of the coral host in modulating photosynthesis has been clarified to a large extent in coral holobionts, the role of the fungal host in this regard is far less understood. Here, we address this question by taking advantage of the recent discovery of highly specific fungal–algal pairings corresponding to climatically adapted ecotypes of the lichen-forming genus Umbilicaria. Specifically, we compared chlorophyll a fluorescence kinetics among lichen thalli consisting of different fungal–algal combinations. We show that photosynthetic performance in these lichens is not only driven by algal genotype, but also by fungal host species identity and intra-host genotype. These findings shed new light on the closely intertwined physiological processes of fungal and algal partners in the lichen symbiosis. Indeed, the specific combinations of fungal and algal genotypes within a lichen individual—and the resulting combined functional phenotype—can be regarded as a response to the environment. Our findings suggest that characterizing the genetic composition of both eukaryotic partners is an important complimentary step to understand and predict the lichen holobiont’s responses to environmental change.
... Lichen-forming fungi are also able to switch photosynthetic partners to form a stable symbiosis with a more compatible partner; incompatible symbiosis impedes thallus development and lichen growth (Beck et al., 2002;Insarova & Blagoveshchenskaya, 2016). The photobiont type can also determine the lichenized fungus's fitness by impacting its tolerance to ecological conditions (Hyvärinen et al., 2002;Casano et al., 2011;Ertz et al., 2018). A lichenized fungus's flexibility in choosing a suitable partner might therefore promote wide geographic distributions and it might also affect the establishment of the symbiosis within its environment Magain & Sérusiaux, 2014;Ertz et al., 2018). ...
Article
Photosymbiodemes are a special case of lichen symbiosis where one lichenized fungus engages in symbiosis with two different photosynthetic partners, a cyanobacterium and a green alga, to develop two distinctly looking photomorphs. We compared gene expression of thallus sectors of the photosymbiodeme‐forming lichen Peltigera britannica containing cyanobacterial photobionts with thallus sectors with both green algal and cyanobacterial photobionts and investigated differential gene expression at different temperatures representing mild and putatively stressful conditions. First, we quantified photobiont‐mediated differences in fungal gene expression. Second, because of known ecological differences between photomorphs, we investigated symbiont‐specific responses in gene expression to temperature increases. Photobiont‐mediated differences in fungal gene expression could be identified, with upregulation of distinct biological processes in the different morphs, showing that interaction with specific symbiosis partners profoundly impacts fungal gene expression. Furthermore, high temperatures expectedly led to an upregulation of genes involved in heat shock responses in all organisms in whole transcriptome data and to an increased expression of genes involved in photosynthesis in both photobiont types at 15 and 25 °C. The fungus and the cyanobacteria exhibited thermal stress responses already at 15 °C, the green algae mainly at 25 °C, demonstrating symbiont‐specific responses to environmental cues and symbiont‐specific ecological optima.
... In all thalli of Ramalina farinacea were two unnamed Trebouxia spp. found with different physiological properties (Casano et al. 2011;del Hoyo et al. 2011). In 45% of Evernia mesomorpha were multiple genotypes of one Trebouxia species found (Piercey-Normore 2006). ...
Chapter
This chapter gives an overview on (1) lichen-forming fungi, lichen photobionts and peculiarities of lichen symbiosis such as gains and losses of lichenization, species concepts, specificity, morphodemes and morphotype pairs, non-lichen mutualistic fungal interactions with unicellular algae and cyanobacteria and mycophycobioses; (2) the mycobiont–photobiont interface, water relations and gas exchange, mycobiont-derived secondary metabolites and the accumulation of heavy metals or radionuclides; (3) the microbiome of lichen thalli, i.e. the bacteriome (epi- and endolichenic bacteria), lichenicolous and endolichenic fungi, lichenicolous lichens and the virome of lichens and their allies; (4) fossil lichens and their microbiome; (5) lichen–animal interactions such as the micro- and mesofauna of lichen thalli, lichenivory in invertebrates and vertebrates, endo-and epizoochory; (6) lichenomimesis in animals and flowering plants.
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Exposure to high light induced a quantitatively similar decrease in the rate of photosynthesis at limiting photon flux density (PFD) and of photosystem II (PSII) photochemical efficiency, FV/FM, in both green and blue-green algal lichens which were fully hydrated. Such depressions in the efficiency of photochemical energy conversion were generally reversible in green algal lichens but rather sustained in blue-green algal lichens. This greater susceptibility of blue-green algal lichens to sustained photoinhibition was not related to differences in the capacity to utilize light in photosynthesis, since the light-and CO2-saturated rates of photosynthetic O2 evolution were similar in the two groups. These reductions of PSII photochemical efficiency were, however, largely prevented in lichen thalli which were fully desiccated prior to exposure to high PFD. Thalli of green algal lichens which were allowed to desiccate during the exposure to high light exhibited similar recovery kinetics to those which were kept fully hydrated, whereas bluegreen algal lichens which became desiccated during a similar exposure exhibited greatly accelerated recovery compared to those which were kept fully hydrated. Thus, green algal lichens were able to recover from exposure to excessive PFDs when thalli were in either the hydrated or desiccated state during such an exposure, whereas in blue-green algal lichens the decrease in photochemical efficiency was reversible in thalli illuminated in the desiccated state but rather sustained subsequent to illumination of thalli in the hydrated state.
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Exposure to high light induced a quantitatively similar decrease in the rate of photosynthesis at limiting photon flux density (PFD) and of photosystem II (PSII) photochemical efficiency, FV/FM, in both green and blue-green algal lichens which were fully hydrated. Such depressions in the efficiency of photochemical energy conversion were generally reversible in green algal lichens but rather sustained in blue-green algal lichens. This greater susceptibility of blue-green algal lichens to sustained photoinhibition was not related to differences in the capacity to utilize light in photosynthesis, since the light-and CO2-saturated rates of photosynthetic O2 evolution were similar in the two groups. These reductions of PSII photochemical efficiency were, however, largely prevented in lichen thalli which were fully desiccated prior to exposure to high PFD. Thalli of green algal lichens which were allowed to desiccate during the exposure to high light exhibited similar recovery kinetics to those which were kept fully hydrated, whereas bluegreen algal lichens which became desiccated during a similar exposure exhibited greatly accelerated recovery compared to those which were kept fully hydrated. Thus, green algal lichens were able to recover from exposure to excessive PFDs when thalli were in either the hydrated or desiccated state during such an exposure, whereas in blue-green algal lichens the decrease in photochemical efficiency was reversible in thalli illuminated in the desiccated state but rather sustained subsequent to illumination of thalli in the hydrated state.
Article
The temperature dependence of the rate of de-epoxidation of violaxanthin to zeaxanthin was determined in leaves of chilling-sensitive Gossypium hirsutum L. (cotton) and chilling-resistant Malva parviflora L. by measurements of the increase in absorbance at 505 nm (ΔA 505) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between ΔA 505 and the decrease in the epoxidation state of the xanthophyll-cycle pigment pool was obtained over the range 10–40° C. The maximal rate of de-epoxidation was strongly temperature dependent; Q10 measured around the temperature at which the leaf had developed was 2.1–2.3 in both species. In field-grown Malva the rate of de-epoxidation at any given measurement temperature was two to three times higher in leaves developed at a relatively low temperature in the early spring than in those developed in summer. Q10 measured around 15° C was in the range 2.2–2.6 in both kinds of Malva leaves, whereas it was as high as 4.6 in cotton leaves developed at a daytime temperature of 30° C. Whereas the maximum (initial) rate of de-epoxidation showed a strong decrease with decreased temperature the degree of de-epoxidation reached in cotton leaves after a 1–2 · h exposure to a constant photon flux density increased with decreased temperature as the rate of photosynthesis decrease. The zeaxanthin content rose from 2 mmol · (mol chlorophyll)−1 at 30° C to 61 mmol · (mol Chl)−1 at 10° C, corresponding to a de-epoxidation of 70% of the violaxanthin pool at 10° C. The degree of de-epoxidation at each temperature was clearly related to the amount of excessive light present at that temperature. The relationship between non-photochemical quenching of chlorophyll fluorescence and zeaxanthin formation at different temperatures was determined for both untreated control leaves and for leaves in which zeaxanthin formation was prevented by dithiothreitol treatment. The rate of development of that portion of non-photochemical quenching which was inhibited by dithiothreitol decreased with decreasing temperature and was linearly related to the rate of zeaxanthin formation over a wide temperature range. In contrast, the rate of development of the dithiothreitol-resistant portion of non-photochemical quenching was remarkably little affected by temperature. Evidently, the kinetics of the development of non-photochemical quenching upon exposure of leaves to excessive light is therefore in large part determined by the rate of zeaxanthin formation. For reasons that remain to be determined the relaxation of dithiothreitolsensitive quenching that is normally observed upon darkening of illuminated leaves was strongly inhibited at low temperatures.
Article
Chlorophyll fluorescence is now widely employed to investigate electron transport and C02 assimilation in leaves and algae. A brief description of how the application of fluorescence parameters has developed for the investigation of electron transport, and C02 assimilation in situ, is initially presented and followed by a consideration of the current appropriate fluorescence terms for such studies. The relationships between the operating efficiency of PS II and the efficiencies of PS I and C02 assimilation are then examined. It is concluded that fluorescence can be a valuable tool in assessing plant photosynthetic performance; however, serious problems can arise when attempting to estimate absolute rates of electron transport and C02 assimilation.