Alcobiosis, an algal-fungal association on the threshold of lichenisation

Abstract

Alcobiosis, the symbiosis of algae and corticioid fungi, frequently occurs on bark and wood. Algae form a layer in or below fungal basidiomata reminiscent of the photobiont layer in lichens. Identities of algal and fungal partners were confirmed by DNA barcoding. Algal activity was examined using gas exchange and chlorophyll fluorescence techniques. Carbon transfer from algae to fungi was detected as ¹³C, assimilated by algae, transferred to the fungal polyol. Nine fungal partners scattered across Agaricomycetes are associated with three algae from Trebouxiophycae: Coccomyxa sp. with seven fungal species on damp wood, Desmococcus olivaceus and Tritostichococcus coniocybes, both with a single species on bark and rain-sheltered wood, respectively. The fungal partner does not cause any obvious harm to the algae. Algae enclosed in fungal tissue exhibited a substantial CO2 uptake, but carbon transfer to fungal tissues was only detected in the Lyomyces-Desmococcus alcobiosis where some algal cells are tightly enclosed by hyphae in goniocyst-like structures. Unlike lichen mycobionts, fungi in alcobioses are not nutritionally dependent on the algal partner as all of them can live without algae. We consider alcobioses to be symbioses in various stages of co-evolution, but still quite different from true lichens.

Full text

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Alcobiosis, an algal‑fungal
association on the threshold
of lichenisation
Jan Vondrák 1,2, Stanislav Svoboda
1,2, Lucie Zíbarová
3, Lenka Štenclová 2,4, Jan Mareš 2,4,
Václav Pouska
5, Jiří Košnar 1,2 & Jiří Kubásek 6*
Alcobiosis, the symbiosis of algae and corticioid fungi, frequently occurs on bark and wood. Algae
form a layer in or below fungal basidiomata reminiscent of the photobiont layer in lichens. Identities
of algal and fungal partners were conrmed by DNA barcoding. Algal activity was examined using gas
exchange and chlorophyll uorescence techniques. Carbon transfer from algae to fungi was detected
as 13C, assimilated by algae, transferred to the fungal polyol. Nine fungal partners scattered across
Agaricomycetes are associated with three algae from Trebouxiophycae: Coccomyxa sp. with seven
fungal species on damp wood, Desmococcus olivaceus and Tritostichococcus coniocybes, both with a
single species on bark and rain‑sheltered wood, respectively. The fungal partner does not cause any
obvious harm to the algae. Algae enclosed in fungal tissue exhibited a substantial CO2 uptake, but
carbon transfer to fungal tissues was only detected in the Lyomyces-Desmococcus alcobiosis where
some algal cells are tightly enclosed by hyphae in goniocyst‑like structures. Unlike lichen mycobionts,
fungi in alcobioses are not nutritionally dependent on the algal partner as all of them can live without
algae. We consider alcobioses to be symbioses in various stages of co‑evolution, but still quite
dierent from true lichens.
Premises
(1) Denition of symbiosis. Symbiosis is a commonly used term in biology, but traditionally has two distinct
meanings1. In what follows, we use it in the sense of de Bary2 to refer to the close and long-term coexistence
of two dierent organisms. Symbiosis in this sense need not involve any benecial or harmful relationship,
merely close co-existence.
(2) Denition of lichen. Lücking etal.3 provided a chronologically ordered list of lichen denitions. None of
these is entirely satisfactory for our purposes, so here we dene a lichen as follows. A lichen is an association
of a fungus (mycobiont) and an alga or cyanobacterium (photobiont) with the following characteristics: (i)
e mycobiont is nutritionally dependent on its photobiont. (ii) e mycobiont is not obviously harmful to
its photobiont. (iii) e photobiont occurs within the mycobiont thallus. (iv) Mycobionts and photobionts
usually cannot persist over a long period outside the symbiosis.
Mutualistic relationship between photoautotrophs and fungi arose many times in evolution, had paramount
importance in the development of terrestrial life and still remains essential in the present-day ecosystems4. A
agship of these relationships is lichen symbiosis, a highly elaborated cooperation between fungi and green
algae and/or cyanobacteria where the fungal partner is nutritionally dependent on its photoautotroph5. Lichen
symbiosis has multiple independent origins6 and its complexity and the stage of lichenisation diers considerably
OPEN
1Institute of Botany of the Czech Academy of Sciences, Zámek 1, CZ‐252 43 Průhonice, Czech
Republic. 2Department of Botany, Faculty of Science, University of South Bohemia CZ, 370 05 České Budějovice,
Czech Republic. 3Independent Researcher, Resslova 26, 400 01 Usti Nad Labem, Czech Republic. 4Biology Centre
of the Czech Academy of Sciences, Institute of Hydrobiology, 370 05 České Budějovice, Czech Republic. 5Faculty of
Forestry and Wood Science, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Praha-Suchdol, Czech
Republic. 6Department of Experimental Plant Biology, Faculty of Science, University of South Bohemia, CZ‐370
05 České Budějovice, Czech Republic. *email: jirkak79@gmail.com
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in various examples7. In some cases, a single fungal species may be either lichenised or saprophytic depending
on conditions8,9, and the same is truth for algae10.
Numerous fungi are apparently associated with, and nutritionally dependent on algae, but their thallus is
inconspicuous, not stratied into the typical lichen thallus6. A list of such fungi, so called “semilichens”, has been
recently provided by Vondrák etal.11. Apart from semilichens, other algal-fungal symbioses that do not fully
meet the denition of a lichen exist12. A remarkable but overlooked one, is linked to corticioid fungi, tradition-
ally dened as basidiomycetes with fruiting bodies (basidiomata) appressed to the substrate with supercial
non-poroid hymenium (extended denition in13). ese at basidiomata (“crusts” in further text) usually cover
wood or bark.
Albertini & Schweinitz14 described one of the wood-dwelling corticioid fungi as Hydnum bicolor, currently
named Resinicium bicolor. e epithet bicolor reected the contrast between the white surface of fruiting bodies
and the rusty brown tips of hymenial spines (the coloration frequently observed in older basidiomata). is epi-
thet additionally matches an even more distinctive colour contrast—the white (or partly translucent) fungal crust
is regularly green beneath. e green tinge is caused by an algal layer formed directly below the white fungal coat.
A detailed investigation of various corticioid fungi in European woodlands revealed living algal cells thriving
below or inside the crusts of several unrelated fungal species. We propose to name these alliances “alcobioses”
(singular “alcobiosis”), such as alga and corticioid fungus in symbiosis. Whereas some alcobioses form unstable
associations where the algal cells are few in scattered colonies, others apparently have a tight relationship where
algae form a lichen-like algal layer15. Only a few algal taxa have previously been reported in an association with
corticioid fungi, mainly unicellular members of the green-algal class Trebouxiophyceae such as Coccomyxa
glaronensis15 and undetermined species of Coccomyxa and Elliptochloris16,17. ese algae are ubiquitous in ter-
restrial habitats and exhibit a general tendency to enter lichen-like symbioses18. However, their symbiotic and
free-living members are morphologically uniform, and separation of the cryptic lineages requires molecular
data19,20 not available for alcobioses yet.
e similarity of alcobioses and crustose lichens is remarkable, but the former have received little and only
supercial attention. e question of whether alcobioses have a nutritional character, as in lichens21, has not been
addressed. Here we provide the most comprehensive morphological and taxonomical assessment so far concern-
ing both symbiotic partners. We also demonstrate that algal cells in these consortia are alive and metabolically
and photosynthetically active even when fully embedded in the fungal crust. And we also studied carbon transfer
from algal polyols (ribitol and sorbitol in our cases) into fungal mannitol that would conrm the nutritional
relationship of the fungus to the algae.
Results
Alcobioses are stratied systems with an internal algal layer. Algal cells were observed enclosed
either in the lower part of crustose basidiomata (subiculum) or in the substratum below the crusts, mostly rot-
ten wood, however they never covered the crust surface. e density of algal cells varied considerably among
infraspecic individuals and among species from an entire absence to a distinct thick continuous layer. e
thickness of the algal layer varied, but frequently exceeded 100µm. Whereas the algal layer was formed only
occasionally in some species (Exidiopsis calcea and Tubulicrinis subulatus), it was found regularly in Lyomyces
sambuci (Fig.1), Resinicium bicolor (Fig.2), Skvortzovia furfuracea (Fig.3) and some Xylodon spp. All these
fungi, however, were also recorded living separately, without an internal algal layer. In all cases, the colonies of
algal cells were enclosed in fungal tissue (although they are also found in thesubstrate below the crust Fig.3E).
Nevertheless, a truly close contact where algae are encircled by fungal hyphae was mostly not observed. e only
exception was Lyomyces sambuci which occasionally formed spherical goniocyst-like structures (20–40µm in
diameter) where a group of algal cells was tightly enclosed within the hyphal network (Fig.1F).
Diverse fungal partners are associated with several ecologically distinct algae. e fungal part-
ners appear to be more diverse in alcobioses than the associated algae. We found nine fungal species dispersed
across the phylogeny of Agaricomycetes (TableS1, Fig.S3) and only three algal partners from the class Trebouxi-
ophycae (TableS1, Fig.4, FiguresS4, S5, S6). e vast majority of fungi involved (i.e. seven species) entered
the symbiosis with a single Coccomyxa species (FiguresS4, S6A,B,C,D). All these fungi have similar ecology,
occurring in temperate forests on decaying wood, especially on fallen spruce trunks, in shaded conditions (Fig-
ures2 and 3; TableS1). e associated Coccomyxa sp. has a rbcL sequence almost identical (> 99% identity) to a
lichen photobiont of Sticta22 and to a symbiont in an allegedly lichenised Schizoxylon albescens9. Both symbioses
have quite distinct ecology: the former is a tropical lichen from Cuba and the latter is known from aspen bark
in Northern Europe. In addition, the same algal genotype labelled Coccomyxa sp. “cort06” in terms of the rbcL
sequence (~99.8% identity) was found to colonize the bark of trees with no report of association with fungi23.
e single fungus, Lyomyces sambuci, was regularly associated with another green algal species Desmococcus
olivaceus, as determined both by morphological identity (Fig.S6E,F,G,H) and high rbcL sequence similarity
(~99.5%; Fig.S5) to a typical strain of this species—SAG 1.9424. is alcobiosis diers in ecology from the
associations with Coccomyxa. It is more tolerant of drying, occurs in lighter sites and has a strong anity to
bark and wood of living or dying Sambucus shrubs. e alliance Lyomyces-Desmococcus is apparently the most
intimate among the observed alcobioses as it forms goniocyst-like structures and the algal carbon is undoubtedly
transferred to the fungus (see below). However, again, the same species of Desmococcus also occurs free-living
(Fig.1A,24) and its symbiosis with Lyomyces is clearly facultative.
Finally, a green alga matching Tritostichococcus coniocybes, both in morphology (Fig.S6H,I) and the rbcL
sequence (97–99% identity; Fig.S5), was detected in alcobiosis with a single fungus, Kneiella abieticola. It was
observed on so rotten wood of spruce snag in a microsite sheltered from rain. Tritostichococcus coniocybes is a
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lichen photobiont detected in Chaenothecopsis spp.24, Arthonia thoriana and Chaenotheca spp. (Fig.4), and also
was observed free-living (our data). It always occurred in rain-sheltered microhabitats.
Algae thrive in the association. Absolute uorescence intensity (e.g. Fo) is very variable and demon-
strates heterogeneity in algal chlorophyll abundance (Fig.5A,D,G). e maximal quantum yield of photosystem
II (Fv/Fm) is very homogeneous (0.55 to 0.75) for well hydrated alcobioses studied (Fig.5, Fig.S7). Moreover, Fv/
Fm for alcobioses and uncovered algal layer does not dier substantially (Fig.5C,F,I). In contrast, dry systems
have Fv/Fm close to zero and recover quickly aer rehydration (FiguresS7, S8). Minimal uorescence (Fo) was
increased to some extent when fungal crusts were removed by razor blade from algal layer. It may demonstrate a
shielding eect of the fungal partner on algae, particularly in Lyomyces-Desmococcus system (Fig.5J,K,L,M,N,O).
CO2 exchange of alcobioses was also very variable but easily detectable, and it conrms the viability and
physiological activity of the partners. Typical light response curves of CO2 assimilation for ve alcobioses,
Figure1. Association of Lyomyces sambuci and Desmococcus olivaceus (GPS: 48.9409975N, 14.5175219E;
voucher: PRA-JV25262). (A) bark of Sambucus nigra covered by a free-living Desmococcus algal crust which is
largely overgrown by Lyomyces; (B) vertical section of the Lyomyces crust with a distinct algal layer; (C) vertical
section with the red chlorophyll autouorescence; (D) algal colonies incorporated in a loose hyphal tissue, below
the cover of compact fungal tissue; (E) Desmococcus in the algal layer; (F) Desmococcus and Lyomyces form
lichen-like goniocysts. Scales: (B, C), 100µm; (D, E, F), 20µm.
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free-living terrestrial alga and foliose lichen are in Fig.S9. e following features were common for all systems:
(1) Respiration of just rehydrated alcobioses was much higher (typically ve to tenfold) than of those hydrated for
a long time, equilibrating to steady-state in hours to days. (2) All alcobioses respond to light, and photosynthesis
exceeded overall respiration in some cases (particularly under elevated [CO2]). (3) Photosynthesis is saturated
under unusually dim light (typically 50 to 100µmol photons m−2 s−1) in systems with Coccomyxa algae, but
may be still increasing in the others, under full sun intensity (ca 1800µmol m−2 s−1) and elevated CO2, Fig.S5).
Moreover, lower temperatures are benecial for the net carbon gain (Fig.S10).
Algal carbon is absorbed by fungi in some alcobioses. Pilot (HPLC–MS, GC–MS) experiments did
not detect any signicant carbon transfer from algal polyols (sorbitol and ribitol) to fungal substances (mannitol
and ergosterol) in Resinicium bicolor-Coccomyxa, Skvortzovia furfuracea-Coccomyxa and Xylodon asper-Cocco-
myxa. In contrast, the control lichens, Hypogymnia physodes and Multiclavula mucida, expressed a clear pattern
of 13C transfer from algal ribitol to fungal mannitol aer two hours of assimilation in the 13C-enriched air. Inter-
mediate results were obtained for Lyomyces sambuci-Desmococcus where mannitol expresses some degree of 13C
enrichment, but barely signicant in our set-up (data not shown).
Figure2. Association of Resinicium bicolor and Coccomyxa (GPS: 48.6679583N, 14.7053083E; voucher:
PRA-JV25257). (A) typical habitat–vertical surface of rotten spruce trunks; (B) R. bicolor crust; (C) vertical
section with a distinct algal layer below the fungal coat; (D) the red chlorophyll autouorescence indicates
locations of Coccomyxa cells in the vertical section. Scales: (B) 5mm; (C, D) 50µm.
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Subsequent detailed studies using isotope ratio mass spectrometry (IRMS), much more sensitive for isotope
abundances, were performed (Method S1). ese experiments delivered two strikingly dierent results: (1) e
Skvortzovia furfuracea-Coccomyxa system did not display any algal-fungal carbon transport. e principal polyols
were algal ribitol and fungal mannitol (Fig.6A). TMS-ribitol was highly 13C enriched (3.90 ± 0.26 At%, t = 24.4,
P < 0.001, N = 5) aer 18h of labelling whereas the fungal TMS-mannitol remained very close to the natural 13C
abundance (1.07 ± 0.03 At%, t = − 0.8, P = 0.45, N = 5; Fig.6D). (2) e algal-fungal carbon transfer was conrmed
in the Lyomyces sambuci-Desmococcus alliance, where sorbitol is the principal algal polyol (Fig.6C). We recorded
a substantial 13C signal in TMS-sorbitol (2.15 ± 0.07 At%, t = 28.8, P = 0.001; N = 4) and a slightly lower but still
very signicant signal in TMS-mannitol (1.48 ± 0.10 At%, t = 8.2, P = 0.004, N = 4; Fig.6F).
Two specimens of Botryobasidium-Coccomyxa, measured in parallel, having low biomass and, thus, low
polyol abundances (Fig.6B), delivered very convincing negative result. Its TMS-ribitol 13C enrichment was
high and consistent (3.70 ± 0.14 At%, t = 25.9, P = 0.024; N = 2) but mannitol invariant from natural abundance
(1.11 ± 0.04 At%, t = 1.4, P = 0.39, N = 2; Fig.6E), very similar to Skvortzovia furfuracea-Coccomyxa system. See
also chromatograms (FiguresS11, S12).
Snails rejuvenate alcobioses and produce isidia‑like diaspores. Literature says little about the per-
sistence of the corticioid fungal crusts involved in this study. According to our eld observations, crusts of
Lyomyces sambuci, Resinicium bicolor and Skvortzovia furfuracea can persist over several years. Persistence of
alcobioses is apparently assisted by snail grazing. Large areas of fungal crusts including algae are frequently
removed by snails and the algal layer is uncovered (Fig.7A). Grazed spots are quickly overgrown by rejuvenated
fungal hyphae (Fig.7B). erefore, fungal crusts, especially of Skvortzovia furfuracea, typically form a continu-
ous mosaic of younger and older (i.e. thinner and thicker) patches.
Snail excrements form distinct structures accompanying alcobioses. ey have a granular or caterpillar-like
shape (Fig.7C) and, when lying on grazed fungus, they are quickly overgrown by young hyphae and incorpo-
rated into the fungal crust (Fig.7D). Fresh excrements are green, densely lled by living algal cells (Fig.7E) in
a mixture with remnants of fungal hyphae (Fig.7F). e algal-fungal content makes excrements an analogous
structure to vegetative propagules (e.g. isidia) of lichens and may serve for vegetative reproduction when being
Figure3. Association of Skvortzovia furfuracea and Coccomyxa (GPS: 48.6679583N, 14.7053083E; voucher:
PRA-JV25255). (A) typical habitat – shaded surface of rotten spruce trunks; (B) S. furfuracea crust grazed by
snails; (C, E) vertical sections of S. furfuracea crust. Fungal tissues coloured by lactoglycerol cotton blue. A
distinct algal layer is visible below a dark blue fungal coat; (D) the red chlorophyll autouorescence indicates
locations of Coccomyxa cells in the vertical section; (F) Coccomyxa loosely integrated in the fungal tissue. Scales:
(B) 1cm; (C, D) 50µm; (E) 20µm; (F) 10µm.
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placed/translocated outside the current fungal crust. e same function was described at mite excrements that
contained viable fungal and algal cells of the lichen Xanthoria parietina25.
Discussion
Corticioid fungi associated with algae. Corticioid fungi are generally considered saprophytic, rarely
parasitic and mycorrhizal; the frequent association with algae has largely been ignored in most monographs26.
However, Parmasto27, already in 1967, observed an alcobiosis in the newly described fungus Phlebia lichenoides,
as he refers to an internal algal layer containing Chlorococcus-like cells. Hjortstam etal.28 considered Phlebia
lichenoides as a synonym of P. subcretacea (currently Cabalodontia subcretacea) and dismissed its association
with algae.
Poelt & Jülich15 provided the most comprehensive study so far on alcobiosis revealed in Resinicium bicolor,
and refer to the presence of an algal layer formed, allegedly, of Coccomyxa glaronensis. (is algal species was
described as a symbiont in the lichen Solorina saccata29). Poelt & Jülich15 observed algae in all surveyed specimens
Figure4. Linkage between algal and fungal partners in alcobioses. Algae are arranged in rbcL phylogenetic
trees of Trebouxiophyceae; only parts with Stichococcus s.lat., Desmococcus and Coccomyxa depicted. Fungi are
arranged in the ITS tree of selected Agaricomycetes. Symbionts in alcobioses are in grey rectangles. Detailed
trees are available on FiguresS3, S4, S5.
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and algal colonies were either restricted below fungal crusts or only slightly expanded out of the fungal spots
and formed a surrounding green rim (which corresponds with our observations; Fig.5A,B). e authors classi-
ed the algal-fungal contact as intimate, but without obvious appressoria or haustoria. Subsequent short notes
in literature13,30–32 have only repeated the ndings by Poelt & Jülich. Whereas Oberwinkler speculated in 1970
that R. bicolor represents a basidiolichen30, he omitted this fungus from his later overview of basidiolichens33.
Two related recent conference contributions refer to associations of corticioid Hyphodontia s.lat. (namely
Lyomyces crustosus, Hyphodontia pallidula and Xylodon brevisetus) with algae Coccomyxa and Elliptochloris, but
Figure5. Chlorophyll uorescence imaging of fungal-algal associations. Minimal uorescence (Fo), visual
frame and maximal quantum yield of photosyntem II (Fv/Fm) for Resinicium bicolor-Coccomyxa (A, B, C, J, K),
Skvortzovia furfuracea-Coccomyxa (D, E, F, L, M) and Lyomyces sambuci-Desmococcus (G, H, I, N, O). Each
sample contains both algal patches and alcobiosis where fungal crust completely covers the algae. Bottom pairs
of frames demonstrate shielding eect of fungal crusts. Upper frames are intact alcobioses (J, L, N), whereas
fungal part of the system was carefully removed by razor blade in red frames of the bottoms (K, M, O).
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without molecular sequence data16,17. It is not clear from the published meeting abstracts, if both algal genera
were observed co-occurring in an association with a single fungal crust, or if each algal-fungal system involved
only a single algal partner. We have only observed the latter case. e anatomical observations reported by
Voytsekhovich etal.17 allegedly revealed appressoria and haustoria in hyphae attached to algal cells and, on
this basis, the authors consider these fungi to be optionally lichenised. Physiological relationships between the
corticioid fungi and their algae were not studied. Our data conrmed that some species of Hyphodontia s.lat.
are frequently involved in alcobioses, but we do not consider them lichens (see below). Whereas Coccomyxa is
undoubtedly frequent in alcobioses, we did not detect Elliptochloris in specimens collected in the current study.
Associations of corticioid fungi and algae sometimes have a typically parasitic character. It was demonstrated
for Athelia epiphyla15 where fungal hyphae form haustoria penetrating algal cells. is fungus causes bleaching
of corticolous algae, i.e. forms characteristic pale-grey rounded spots on otherwise green tree bark. Although A.
epiphyla is apparently a parasite, Jülich34 and Oberwinkler33 refer to symbiotic relationships between epiphytic
algae and some species of Athelia and Athelopsis. Some of these cases may be close to our concept of alcobiosis.
Parallel research has been conducted on associations of polypore fungi and their epiphytic algae. e surface
of polypore fruiting bodies is a suitable substrate for numerous algal (and cyanobacterial) species35,36. However,
these associations do not show any specicity–a single polypore is oen covered by a community of several
Figure6. Abundance and 13C enrichment in trimethylsilyls of principal algal and fungal polyols aer 18h
assimilation of alcobioses in 13CO2 atmosphere. Algal polyols: ribitol (R) and sorbitol (S); fungal mannitol
(M). Median (central point), 25% and 75% quantiles (box) and min–max (whiskers) are shown. Dotted line
represents natural 13Cabundance (1.07 At %) and t-test was performed against it; NS: not signicant (P > 0.05),
*: 0.05 < P > 0.01, **: 0.01 < P > 0.001, ***: P < 0.001.
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algal species with broader niches (not restricted to polypores). Carbon transfer from epiphytic algae to polypore
fungal tissues was repeatedly reported using 14C tracer37,38, but we are skeptical of that result as the algae usually
grow on/in dead polypore tissues. us, this association seems to be very dierent from alcobioses with algae
embedded within the viable fungal tissue.
e taxonomic composition of algae found in alcobioses is in general not surprising. Unicellular trebouxi-
ophyte algae such as Coccomyxa and Stichococcus sensu lato are ubiquitous in terrestrial habitats and have been
long known to create various types of fungal-algal consortia39. In our study we employed molecular barcoding of
the algal partners in alcobioses for the rst time, which allowed us to place all three detected taxa into narrowly
dened and strongly supported monophyletic clades of Coccomyxa sp., Desmococcus olivaceus, and Tritostichoc-
occus coniocybes. Each of these clades probably corresponds to a single algal species, judging from their uniform
morphology and high rbcL gene sequence identity (Fig.4, FiguresS3, S4, S5). e Coccomyxa sp. found in our
samples evidently represents a species new to science, but its transfer to pure culture, deposition of a holotype,
and formal description was beyond the scope of the current study. As well as living in alcobioses, we know from
previously published sequences that each of these three genospecies can live as a free-living terrestrial alga23,24.
In the case of D. olivaceus, our results are the rst reliable observation of this species in symbiotic association
with fungi, but S. coniocybes and the particular clade of Coccomyxa have previously been reported as genuine
lichen photobionts22,24.
Ecophysiological implications. We conrmed the viability of algae in alcobioses via chlorophyll a
uorescence40 and gas exchange measurement. e maximal quantum yield of photosystem II (Fv/Fm) is a well-
accepted measure of vitality and impact of stress. In the case of alcobioses, Fv/Fm conrms that the algae are not
stressed under the fungal layer, having Fv/Fm comparable to values for adjacent free-living algae (Fig.5C,F,I).
Photochemistry recovered within minutes to tens of minutes to values > 0.4 aer dry alcobioses were rehydrated
(FiguresS7, S8), being considered “physiologically active”, in accordance with studies on terrestrial algae41.
Figure7. Snail-grazed Skvortzovia furfuracea. (A) a green area of exposed algal layer formed by snail
grazing; (B) regenerated mycelium overgrowing grazed areas. Algal cells are indicated by the red chlorophyll
autouorescence. (C) fresh snail excrement; (D) excrements are quickly overgrown by regenerated mycelium;
(E) the red chlorophyll autouorescence indicates high density of Coccomyxa cells in an excrement; (F) a
mixture of Coccomyxa and remnants of fungal tissue in an excrement. Scales: (A) 2mm; (B) 0µm; (C, D) 1mm;
(E, F) 20µm.
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e minimal uorescence (Fo), in turn, shows a low shielding eect of fungal crusts to incident light, particu-
larly for shade-adapted alcobioses formed by Coccomyxa and fungi with rather thin basidiomata (Fig.5J,K,L,M).
A substantial shielding eect was detected at the thicker and more light-scattering crust of Lyomyces sambuci,
growing in more-light exposed sites, which may be photoprotective for Desmococcus algae in this association
(Fig.5 N,O).
e CO2 exchange, reecting the real photosynthetic performance and production process, has not been
studied in alcobioses yet, but reference data are available for lichens42–44. Here we provide gas-exchange data for
four alcobiosis systemes in the context of lichen symbiosis (represented by Parmelia sulcata) and a free-living
algal crust (Trentepohlia aurea). FiguresS9 and S10 demonstrate relationships of the CO2 exchange to an incident
light intensity, gaseous CO2 concentration and ambient temperature. Respiration of the fungal partner plus co-
occurring microbiomes was usually higher in shade-adapted Coccomyxa-based alcobioses (Fig.S9). us, net
carbon balance of the system was mostly negative and algal photosynthesis could not serve as a principal source
of carbon for the fungus. Saturating light intensity was also unusually low (50 to 100µmol photons m−2 s−1)
suggesting a strong shade adaptation of the algae involved. In contrast, free-living Trentepohlia aurea, the lichen
Hypogymnia physodes and the alcobiosis Lyomyces sambuci-Desmococcus occurring in more light-exposed con-
ditions, had higher maximal CO2 assimilation and higher light saturation intensity and the carbon gain of these
systems was frequently positive. CO2 assimilation rate rising even close to full sun intensity under elevated CO2
suggests high diusional limitation of the photobionts and their high photosynthetic capacity as well (Fig.S9).
In addition, the overall carbon balance of those systems is strongly temperature dependent. Lower temperatures
promote carbon gain of alcobioses, as demonstrated on Xylodon system (Fig.S10). is can be explained by higher
temperature dependency of dark respiration (Rdark) than gross photosynthetic capacity (Agross). Comparable
magnitudes of Rdark and Agross will lead to substantial eect of temperature on carbon gain45.
We observed a signicant alga-to-fungus 13C transfer in only one of the systems studied. We selectively meas-
ured algal (ribitol, sorbitol) and fungal (mannitol) compounds. Polyols are not suitable for gas chromatography,
therefore their trimethylsilyls (TMS-) were measured46. at is, the ve-carbon ribitol was converted to the
twenty-carbon TMS-ribitol (C20H52O5Si5). Similarly, six-carbon mannitol and sorbitol led to the formation of
24-carbon TMS-derivatives (C24H62O6Si6). us, the 13C enrichment (indicating carbon transfer) is four-times
more pronounced in mother molecules than in TMS derivatives measured here and thus the negative result for
Skvortzovia furfuracea-Coccomyxa is very convincing (Figs.7and S11). e isotopic precision of IRMS is better
than 0.01 At% of 13C for isotope ratios close to natural (< 5 At% of 13C). Mannitol 13C is clearly unchanged from
its natural value in this system, whereas TMS-ribitol is highly enriched up to 4.6 At% 13C (by 3.5 At% compared
to natural). is means that the parent ribitol should have up to 15.1 At% 13C (4 × 3.5 + 1.1). In contrast, the
Lyomyces-Desmococcus TMS-mannitol was signicantly enriched by approximately 0.4 At% (Figs.7andS12).
us, mannitol should be enriched up to 2.7 At% (1.1 + 4 × 0.4 At%). Despite the clear statistical signicance,
more work is needed to decipher the timing, environmental dependencies and, thus, ecological signicance of
those carbon transfers.
Symbiosis on the threshold of lichenisation. e lichen is dened above in the Premise 2 as a symbio-
sis of alga or cyanobacterium (photobiont) and fungus (mycobiont) with following specics: (1) e mycobiont
is nutritionally dependent on its photobiont47. (2) e mycobiont is not obviously harmful to its photobiont48.
(3) e photobiont occurs within the mycobiont thallus49. (4) Mycobionts and photobionts usually cannot per-
sist over a long period outside the symbiosis50,51.
Alcobioses do comply with points (2) and (3). ey have an internal lichen-like algal layer (Figs.1, 2, 3) and
the algae thrive in the symbiosis (Fig.5, FiguresS3, S4, S5, S6). Only Lyomyces-Desmococcus partly complies
with point (1); we conrmed carbon transfer from alga to fungus (Fig.6, FiguresS11, S12). We suggest however
that Lyomyces is not fully dependent on algal assimilates, because it has been observed occasionally without the
algal symbiont. Consequently, Lyomyces-Desmococcus does not meet point (4), because both symbionts may live
apart. Surprisingly, Desmococcus olivaceus is absent from the list of lichen photobionts39 and is mostly reported
free-living, forming extensive epiphytic or epilithic algal crusts52. Another member of the genus, Desmococcus
vulgaris, was found overgrowing fruiting bodies of the polypore Fomes fomentarius53. Lyomyces sambuci, like
other Hyphodontia s. lat., is believed to cause white rot, i.e. is saprophytic and, remarkably, the frequently present
algal layer in this common fungus was not mentioned or illustrated in the monographs26,28. Point (4) is partly met
in some alcobioses with the Coccomyxa species known from lichens9,22. At our sampling sites, the alga appears
to occur only inside and below the fungal crusts (Fig.5A,B). Contrary to our observations, an almost identical
genotype of Coccomyxa was found free-living on the bark of live pine and oak trees in the study of corticolous
algae by Kulichová etal.23. erefore, the lifestyle of this alga seems to be diverse, and investigations on the popu-
lation level are needed to elucidate the potential specic life strategies of individual strains. Fungi associated with
Coccomyxa were observed to live without algae, but the alcobiosis is almost omnipresent in Resinicium bicolor and
Skvortzovia furfuracea. e nature of this symbiosis remains enigmatic as it probably has no nutritional character.
In conclusion, the photosynthetic potential of algal partners is clearly substantial, but their direct nutritional
importance for fungi (and whole alcobioses) is still obscure, even in Lyomyces-Desmococcus, and needs more
study. Simultaneously, the insignicant carbon exchange observed in most systems implies that there must be
other ecological advantages keeping the partners in a stabile association (e.g. exchange of bioactive substances
under stress conditions such as drought).
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Materials & methods
In the period 2016–2021, we recorded 58 specimens with alcobiosis (TableS1). Corticioid fungi were identi-
ed to species from their morphology and 27 specimens were barcoded by ITS nrDNA sequences. Algae were
determined to genus according to their morphology and 20 specimens were barcoded by sequencing the ribulose
bisphosphate carboxylase large subunit gene (rbcL). e NCBI accession numbers of the sequences obtained
are provided in TableS1. Field collections from 2019–2021 were employed in morphological and anatomical
observations (uorescent microscope Olympus BX 61 in bright eld and uorescent mode) and in physiological
measurements that were done within one week aer collection. Before that, samples were hydrated if necessary,
put in Petri-dishes and accommodated in LED illuminated cultivating room under 20°C, irradiation about
100µmol m−2 s−1 and photoperiod of 12h for at least two days.
DNA barcoding & phylogenetic analysis. Fresh specimens were used for DNA extraction of both fun-
gal and algal partners. DNA was extracted with a cetyltrimethylammonium bromide (CTAB)-based protocol54.
ITS nrDNA locus was found to be a useful fungal barcode sequence, easily ampliable, and moreover with
a sucient number of references in the NCBI database. In the case of algae, we sequenced nrDNA ITS and
18S regions and the rbcL gene. e latter was found as best amplied and therefore selected for barcoding and
phylogenetic analysis. Polymerase chain reactions were performed in a reaction mixture containing master mix
consisting of 2.5mmol/L MgCl2, 0.2mmol/L of each dNTP, 0.3μmol/L of each primer, 0.5 U Taq polymerase
in the manufacturer’s reaction buer (Top-Bio, Praha, Czech Republic), and milli-Q water to make up a nal
volume of 10μL. e primers used for PCR and the cycling conditions are summarized in TableS2. Successful
amplications were sent for Sanger sequencing (GATC Biotech, Konstanz, Germany). Sequences were edited
using BioEdit v.7.0.9.055 and Geneious Prime 2022.0 (https:// www. genei ous. com).
Sequences of our specimens were supplemented by relevant sequences from GenBank—NCBI database.
Sequences were aligned by MAFFT v.756; available online at http:// ma. cbrc. jp/ align ment/ server/) using the
Q-INS-i algorithm and adjusted manually. e best-t model of sequence evolution was selected using the Akaike
information criterion calculated in jModelTest v.0.1.157. Relationships were assessed using Bayesian inference as
implemented in MrBayes v.3.1.258. Two runs starting with a random tree and employing four simultaneous chains
each (one hot, three cold) were executed. e temperature of a hot chain was set empirically to 0.1, and every
100th tree was saved. e analysis was considered to be completed when the average standard deviation of split
frequencies dropped below 0.01. e rst 25% of trees were discarded as the burn-in phase, and the remaining
trees were used for construction of a 50% majority consensus tree.
Gas exchange. CO2 exchange was measured using the portable photosynthetic system LI-6400XT (Li-
Cor, Lincoln, NE, USA) connected to a custom-made peltier-conditioned gas exchange chamber described
elsewhere59,60, see also Figure. S1. e chamber allows to accommodate samples up to 64 cm2 of ground area and
1cm in thickness. It is of high sensitivity, as we have demonstrated in the past by successfully measuring the very
small gas exchange of moss capsules61. Algae-containing basidiomata were attached to the razor-thinned native
substrate to minimise microbial respiration. “White” PAR irradiation was supplied by a LI6400-18 RGB system
source controlled by LI-6400XT. Temperature was maintained at 20°C (except when measuring the response
to varying temperature) and airow at 250µmol mol−1. e light response of CO2 assimilation was measured in
continual-logging mode (each 5s), at least 3min in each light intensity. Aer a steady state was reached, data
were collected, and light intensity changed to the next value. e temperature curve relied on Peltier-cooling of
the cryptogamic chamber. At least 15min was allowed to pass before data on gas exchange was taken, to allow
equilibrium to be reached. Finally, background (empty chamber response) was measured and subtracted.
Chlorophyll a uorescence imaging. 2D uorescence measurements were performed by the FluorCam
FC800 instrument (PSI, Drásov, Czech Republic). Red LEDs (λ = 660nm) were used for both, measuring light
(10, 20 or 33µs ashes, in average < 1µmol m−2 s−1) and actinic light (continuous, photosynthesis driving, about
100µmol m−2 s−1) photon sources. White LEDs (≈ 2500µmol m−2 s−1) delivered saturation pulses of 1s in dura-
tion. e camera has 720 × 560 px resolution, 12-bit data depth and is equipped with a zoom objective imaging
frame down-to ca 10 × 7.5cm (≈ 0.13mm per pixel).
Metabolite transfer measurement. Stable carbon 13C was chosen to trace photobiont assimilated car-
bon. Substrate-attached sporocarps with active algal partner (Li-6400XT-proved, Figure. S1) were enabled to
assimilate 13CO2 enriched air in a water-sealed 3L inverse Petri dish with internal fan (See Figure. S2). e label-
ling device is described in detail elsewhere61. [13CO2] was about 1000µmol mol−1. Atmospheric CO2 was previ-
ously replaced by CO2 free synthetic air and then ca 3mL of 13CO2 (> 99 atom %, Sigma-Aldrich, Luis., USA) was
injected. Samples assimilated for two to 18h under about 200µmol m−2 s−1 of white LED light. en fungal/algal
crusts were scratched down by razor and killed in boiling methanol. Homogenised and ltered metOH extracts
were used for analysis of polyols and ergosterol (see Methods S1 for further details).
We employed three approaches to trace 13C enriched metabolites. (1) HPLC–MS to separate algal polyols
(ribitol and sorbitol) from fungal mannitol and to measure their 13C content. Although mannitol was detected
in some algal groups62,63, it is not produced by algae in our systems, i.e., Coccomyxa and Desmococcus64. (2) GC-
IRMS to separate trimethylsilyl derivatives of those polyols (TMS-ribitol, TMS-mannitol and TMS-sorbitol) and,
again, to quantify their 13C enrichment. And (3) HPLC–MS to separate fungal specic ergosterol and to measure
if it is 13C enriched. For more details see Method S1.
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Data availability
e datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request.
Received: 7 May 2022; Accepted: 3 February 2023
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Acknowledgements
Linda in Arcadia kindly revised the manuscript. Ondřej Peksa and Zdeněk Palice kindly consulted some points.
Our research received support by a long-term research development grant RVO 67985939andby the Technology
Agency of the Czech Republic, grant TH03030469.
Author contributions
J.V. designed the research, J.V. and S.S. conducted eldwork and sample studies, J.K. performed physiological
experiments, L.Z., V.P., L.S. and J.M. provided algological and mycological expertise. J.K. analysed DNA sequence
data. All authors contributed to the writing of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 29384-4.
Correspondence and requests for materials should be addressed to J.K.
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