The intertidal marine lichen formed by the pyrenomycete fungus Verrucaria tavaresiae (Ascomycotina) and the brown alga Petroderma maculiforme (Phaeophyceae): thallus organization and symbiont interaction

The thallus formed by the marine pyrenomycete fungus Verrucaria tavaresiae and the phaeophycean alga Petroderma maculiforme was studied to elucidate the organization of the symbionts, determine the type of cellular contacts between them, and evaluate the status of the symbiosis as a lichen. Hand-sectioned and resin-embedded samples were examined with light and transmission electron microscopy. Within the uppermost portion of the cellular fungal tissue, separate algal filaments were arranged anticlinally. Protrusions of the fungal cell wall penetrated into adjacent algal walls but did not enter the cell lumen. A striking feature of these penetrations was the frequent separation of algal cell wall layers and insertion of fungal wall material between them. Algal filaments grew downward intrusively between fungal cells, often penetrating deeply into the fungal cell wall. Despite the exceptional nature of the phycobiont involved, the Verrucaria tavaresiae-Petroderma maculiforme symbiosis unequivocally fits the prevailing concept of a lichen. The distinctive interpenetrations observed between symbionts may be related to the integration of their different growth forms within a coherent tissue regularly subject to mechanical stresses. Periclinal cell divisions within and just below the algal layer may serve to replenish surface tissues lost to abrasion and herbivory.     

ULTRASTRUCTURE OF THE LICHEN COENOGONIUM INTERPLEXUM NYL.

Coenogonium interplexum Nyl. is a green to yellow-orange filamentous lichen commonly found on tree bark, rocks, and soil. The mycobiont is the ascomycetous fungus Coenogonium. The ultrastructure of the lichenized phycobiont, Trentepohlia, closely resembles that of the non-lichenized form, a filamentous subaerial green alga. The mycobiont has a typical fungal ultrastructure, and the cell wall sometimes appears thinner at points of contact with the phycobiont wall. Several branched fungal hyphae are usually randomly arranged around a Trentepohlia filament, and may in some cases completely ensheath the alga. Although no haustoria were observed, this relationship may still be termed a lichen since there is some modification of the alga and the lichen is structurally distinct from the two symbionts.     

Effect of low water potential on photosynthesis in intact lichens and their liberated algal components

Earlier experiments (T.D. Brock 1975, Planta124, 13–23) addressed the question whether the fungus of the lichen thallus might enable the algal component to function when moisture stress is such that the algal component would be unable to function under free-living conditions. It was concluded that the liberated phycobiont in ground lichen thalli could not photosynthesize at water potentials as low as those at which the same alga could when it was present within the thallus. However, our experience with lichen photosynthesis has not substantiated this finding. Using instrumentation developed since the mid-1970's to measure photosynthesis and control humidity, we repeated Brock's experiments. When applying “matric” water stress (equilibrium with air of constant relative humidity) we were unable to confirm the earlier results for three lichen species including one of the species,Letharia vulpina, had also been used by Brock. We found no difference between the effects of low water potential on intact lichens and their liberated algal components (ground thallus material and isolated algae) and no indication that the fungal component of the lichen symbiosis protects the phycobiont from the adverse effects of desiccation once equilibrium conditions are reached. The photosynthetic apparatus of the phycobiont alone proved to be highly adapted to water stress as it possesses not only the capability of functioning under extremely low degrees of hydration but also of becoming reactivated solely by water vapor uptake.     

The cryoprotective role of polyols in lichens: Effects on the redistribution of RNase in Evernia prunastri thallus during freezing

The effect of low temperatures on the distribution of RNase (EC 3.1.26.1) in the lichen Evernia prunastri (L.) Ach. has been studied in laboratory conditions. Freezing of lichen thalli produces solubilization of part of the particulate enzyme from the cell wall of both mycobiont and phycobiont to the corresponding cytoplasm. A supply of exogenous ribitol (naturally produced by the algal partner) totally prevents the solubilization of the enzyme whereas mannitol (naturally produced by the fungal partner) impedes the enzyme solubilization to a minor extent. RNase is preferably located in the phycobiont cells in terms of specific activity. Ribitol also impedes the solubilization of algal enzyme whereas mannitol strongly promotes the loss of RNase from algal cell wall to the soluble fraction. Solubilization of fungal enzyme is enhanced by both polyols, with a preference for ribitol.     

INVESTIGATIONS ON LICHEN SYNTHESIS

Ahmadjian , V. (Clark U., Worcester, Mass.) Investigations on lichen synthesis. Amer. Jour. Bot. 49(3): 277–283. Illus. 1962.—Separated fungal and algal components of the lichen, Acarospora fuscata (Nyl.) Am., were recombined under controlled laboratory conditions to form structures comparable to those of the naturally occurring lichen thallus. The primary condition for this artificial synthesis was the absence of organic and inorganic supplements to the agar substrate. Thus, in effect, the mycobiont was forced into union with the phycobiont. It was demonstrated that both alga and fungus derived benefit from the lichenized association.     

Exploring symbiont management in lichens

Lichens are unique among fungal symbioses in that their mycelial structures are compact and exposed to the light as thallus structures. The myriad intersections of unique fungal species with photosynthetic partner organisms (green algae in 90% of lichens) produce a wide variety of diverse shapes and colours of the fully synthesized lichen thallus when growing in nature. This characteristic complex morphology is, however, not achieved in the fungal axenic state. Even under ideal environmental conditions, the lichen life cycle faces considerable odds: first, meiotic spores are only produced on well-established thalli and often only after achieving considerable age in a stable environment, and second, even then in vivo resynthesis requires the presence of compatible algal strains where fungal spores germinate. Many lichen species have evolved a way around the resynthesis bottleneck by producing asexual propagules for joint propagation of symbionts. These different dispersal strategies ostensibly shape the population genetic structure of lichen symbioses, but the relative contributions of vertical (joint) and horizontal (independent) symbiont transmission have long eluded lichen evolutionary biologists. In this issue of Molecular Ecology, Dal Grande et al. (2012) close in on this question with the lung lichen, Lobaria pulmonaria, a flagship species in the conservation of old growth forests. By capitalizing on available microsatellite markers for both fungal and algal symbionts, they show that while vertical transmission is the predominant mode of reproduction, horizontal transmission is demonstrable and actively shapes population genetic structure. The resulting mixed propagation system is a highly successful balance of safe recruitment of symbiotic clones and endless possibilities for fungal recombination and symbiont shuffling.     

The lichen-forming ascomycete Evernia mesomorpha associates with multiple genotypes of Trebouxia jamesii

The epiphyte Evernia mesomorpha forms a lichen association with green algae in the genus Trebouxia. Little is known about the population structure of E. mesomorpha. Here, population structure of the algal and fungal symbionts was examined for 290 lichen thalli on 29 jack pine (Pinus banksiana) trees in Manitoba. Through phylogenetic analysis of internal transcribed spacer (ITS) nuclear ribosomal DNA (rDNA) sequences, five algal genotypes were detected that were nested within T. jamesii. Two fungal genotypes were detected that formed a clade with two other Evernia species. The genus Evernia was paraphyletic with E. prunastri, sister to Parmelia saxatilis. Restriction fragment length polymorphism (RFLP) of ITS rDNA showed multiple algal genotypes in 45% of the 290 lichen thalli collected, whereas all thalli only contained one fungal genotype. Low population subdivision of algal and fungal genotypes among trees suggested that the algal symbiont was being dispersed in the lichen soredium. Low fungal specificity for multiple algal genotypes and a hypothesized algal switch may be important life history strategies for E. mesomorpha to adapt to changing environmental conditions.     

Community Analysis Reveals Close Affinities Between Endophytic and Endolichenic Fungi in Mosses and Lichens

Endolichenic fungi live in close association with algal photobionts inside asymptomatic lichen thalli and resemble fungal endophytes of plants in terms of taxonomy, diversity, transmission mode, and evolutionary history. This similarity has led to uncertainty regarding the distinctiveness of endolichenic fungi compared with endophytes. Here, we evaluate whether these fungi represent distinct ecological guilds or a single guild of flexible symbiotrophs capable of colonizing plants or lichens indiscriminately. Culturable fungi were sampled exhaustively from replicate sets of phylogenetically diverse plants and lichens in three microsites in a montane forest in southeastern Arizona (USA). Intensive sampling combined with a small spatial scale permitted us to decouple spatial heterogeneity from host association and to sample communities from living leaves, dead leaves, and lichen thalli to statistical completion. Characterization using data from the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS-LSU rDNA) provided a first estimation of host and substrate use for 960 isolates representing five classes and approximately 16 orders, 32 families, and 65 genera of Pezizomycotina. We found that fungal communities differ at a broad taxonomic level as a function of the phylogenetic placement of their plant or lichen hosts. Endolichenic fungal assemblages differed as a function of lichen taxonomy, rather than substrate, growth form, or photobiont. In plants, fungal communities were structured more by plant lineage than by the living vs. senescent status of the leaf. We found no evidence that endolichenic fungi are saprotrophic fungi that have been “entrapped” by lichen thalli. Instead, our study reveals the distinctiveness of endolichenic communities relative to those in living and dead plant tissues, with one notable exception: we identify, for the first time, an ecologically flexible group of symbionts that occurs both as endolichenic fungi and as endophytes of mosses.     

Two Trebouxia algae with different physiological performances are ever‐present in lichen thalli of Ramalina farinacea. Coexistence versus Competition?

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.     

On optimal propagule size and developmental time

A negative relationship between propagule size and development time is often imposed as a constraint on the evolution of optimal propagule size. Here I argue that when mortality is size-dependent, the optimisation of propagule size is in fact independent of the length of the ensuing developmental period. Furthermore, I show that whether larger or smaller propagules are favoured in response to factors that affect growth and mortality rates will depend on which of these variables scale more sharply with size. I use marine fish to exemplify some of the points raised in this note.     

A new method to isolate lichen algae by using Percoll®gradient centrifugation

Abstract:A rapid method to isolate intact functional algae from the lichens Evernia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll^®gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.     

HEAVILY LICHENIZED PHYSOLINUM (CHLOROPHYTA) FROM A DIMLY LIT CAVE IN MISSOURI1

Heavily lichenized Physolinum monile (De Wildem.) Printz from damp limestone walls in a dimly lit cave located in Missouri was studied from fresh collections and specimens fixed in situ, and from cultures. The narrow (7-13 μm wide thallus), profusely branched plant consisted of filaments of the alga P. monile ensheathed by clear fungal cells (5-8 in a single layer) that adhered tightly to each other and completely covered the algal cells. Cells of P. monile filaments were uninucleate, each containing a single massive chloroplast with numerous tightly packed thylakoids and lipid droplets and surrounded by a thin layer of cytoplasm. No plasmodesmata occurred in the cellulosic crosswalls between adjacent cells. The ensheathing fungal cells contained concentric bodies, produced haustoria that penetrated the algal cells, and developed hyphae (the tips of which formed clusters of conidia). Ensheathing fungal cells were well situated and constructed to concentrate light on the algal cells. Colonies of blue-green algae were firmly attached to the surface of the fungal cells. The association was slow growing but frequently produced and released aplanospores from the algal cells. Aplanospores were single (not attached to each other) with smooth walls or united in groups of two or more. Structures resembling lichen soredia, composed of aplanospore-like cells attached to one or more comdia-like cells, commonly occurred among the lichenized Physolinum filaments. The single chloroplast that occupies most of the cell's volume, the numerous, tightly packed thylakoids, and light focusing by ensheathing fungus cells may enable the organism to survive in a dimly lit environment. Because the filamentous alga reproduces only by aplanospores, we propose resurrection of the genus Physolinum. The lichenized Physolinum somewhat resembles the lichens Coenogonium moniliforme Tuck. and Cystocoleus Thwaites.     

The complexity of symbiotic interactions influences the ecological amplitude of the host: A case study in Stereocaulon (lichenized Ascomycota)

Symbiosis plays a fundamental role in nature. Lichens are among the best known, globally distributed symbiotic systems whose ecology is shaped by the requirements of all symbionts forming the holobiont. The widespread lichen‐forming fungal genus Stereocaulon provides a suitable model to study the ecology of microscopic green algal symbionts (i.e., phycobionts) within the lichen symbiosis. We analysed 282 Stereocaulon specimens, collected in diverse habitats worldwide, using the algal ITS rDNA and actin gene sequences and fungal ITS rDNA sequences. Phylogenetic analyses revealed a great diversity among the predominant phycobionts. The algal genus Asterochloris (Trebouxiophyceae) was recovered in most sampled thalli, but two additional genera, Vulcanochloris and Chloroidium, were also found. We used variation‐partitioning analyses to investigate the effects of climatic conditions, substrate/habitat characteristic, spatial distribution and mycobionts on phycobiont distribution. Based on an analogy, we examined the effects of climate, substrate/habitat, spatial distribution and phycobionts on mycobiont distribution. According to our analyses, the distribution of phycobionts is primarily driven by mycobionts and vice versa. Specificity and selectivity of both partners, as well as their ecological requirements and the width of their niches, vary significantly among the species‐level lineages. We demonstrated that species‐level lineages, which accept more symbiotic partners, have wider climatic niches, overlapping with the niches of their partners. Furthermore, the survival of lichens on substrates with high concentrations of heavy metals appears to be supported by their association with toxicity‐tolerant phycobionts. In general, low specificity towards phycobionts allows the host to associate with ecologically diversified algae, thereby broadening its ecological amplitude.     

European phylogeography of the epiphytic lichen fungus Lobaria pulmonaria and its green algal symbiont

In lichen symbiosis, fungal and algal partners form close associations, often codispersed by vegetative propagules. Due to the particular interdependence, processes such as colonization, dispersal or genetic drift are expected to result in congruent patterns of genetic structure in the symbionts. To study the population structure of an obligate symbiotic system in Europe, we genotyped the fungal and algal symbionts of the epiphytic lichen Lobaria pulmonaria at eight and seven microsatellite loci, respectively, and analysed about 4300 L. pulmonaria thalli from 142 populations from the species' European distribution range. Based on a centroid approach, which localizes centres of genetic differentiation with a high frequency of geographically restricted alleles, we identified the South Italy–Balkan region as the primary glacial refugial area of the lichen symbiosis. Procrustean rotation analysis and a distance congruence test between the fungal and algal population graphs indicated general concordance between the phylogeographies of the symbionts. The incongruent patterns found in areas of postglacial recolonization may show the presence of an additional refugial area for the fungal symbiont, and the impact that horizontal photobiont transmission and different mutation rates of the symbionts have on their genotypic associations at a continental scale.     

Haustorienbefall und inäquale Teilungen desNostoc-Phycobionten vonLempholemma botryosum (Lichinaceae)

In fully developed parts of thalli ofLempholemma botryosum a relatively high number of algal cells is attacked by haustoria. They differ specifically in form and effect from haustoria of otherLempholemma species. Cells of theNostoc phycobiont attacked by hyphae developing into haustoria may divide to give inequal besides equal daughter cells.

     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

VISUALIZATION OF EXTRACELLULAR DEPOSITS IN RECENT AND SUBFOSSILUMBILICARIA HYPERBOREA

Field emission scanning electron microscopy was used to characterize mycobiont wall surfaces inUmbilicaria hyperboreafrom Greenland. To determine the precise intrathalline distribution of phenolics, comparisons were made of hyphal surface features and dimensions before and after acetone extraction. Stratification was evident within the medulla, as extracellular phenolics were observed only on hyphae near or within the algal zone. The outside diameter of hyphae in this region was thus significantly greater than in the remainder of the medulla. Surface deposits were also examined in 1350-year-old subfossil thalli and hyphal diameters were compared statistically to those in extant thalli. The mean hyphal diameter in the upper medulla was not significantly less in subfossil specimens than in recent thalli, suggesting that phenolic cover was maintained in spite of glaciation. However, after ice burial phenolic masses tended to be flatter than in recent specimens and finely tuberculate. The appearance of mycobiont hyphae in the cortex of subfossil thalli seemed to be the same as in extant thalli, except that there tended to be more compressible, smaller and less conglutinated filaments near the algal layer.     

Faecal pellets of lichenivorous mites contain viable cells of the lichen-forming ascomycete Xanthoria parietina and its green algal photobiont, Trebouxia arboricola

The bright yellow wall lichen, Xanthoria parietina , is usually inhabited by oribatid mites (Acari) which do not only find shelter, but also graze on selected areas of the thallus. As X. parietina does not produce symbiotic vegetative propagules and its compatible photobiont, unicellular green algae of the genus Trebouxia , are rare outside lichen thalli, we tested the hypothesis of dispersal of viable Trebouxia cells via acarine faeces. The lichenivorous mites, Trhypochtonius tectorum and Trichoribates trimaculatus , were isolated from thalli of X. parietina and cultured in the laboratory on a lichen diet. Light microscopic investigations of faecal pellets from mites that had been feeding on X. parietina indicated gut passage of intact ascospores and photobiont cells. In a series of experiments, viable algal and fungal cells contained in such faecal pellets were cultured. The taxonomic affiliation of these isolates was identified using molecular techniques, i.e. comparative investigations of nuclear ribosomal gene data (ITS 1 and 2, 5.8S rDNA) in the algal and fungal partners, and of the species-specific hydrophobin gene sequence in the fungal partner. Our culturing experiments demonstrated that the faecal pellets of both lichenivorous mites, upon feeding on X. parietina , contain viable ascospores and photobiont cells ( Trebouxia arboricola ) and thus might be a common and successful mode of vegetative short- and long-distance dispersal of this and numerous other lichen-forming ascomycetes and their photobionts. Future studies will have to elucidate the evolutionary significance of invertebrate interactions with lichens. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 76 , 259–268.     

Location and interseasonal variation in flowering, propagule setting and propagule size in mangroves species of the family Rhizophoraceae

Three species of mangroves, Rhizophora stylosa, Rhizophora mangle (synonym R. samoensis) and Bruguiera gymnorhiza in the family Rhizophoraceae were studied to understand the flowering pattern, propagule development and the propagule size at maturity prior to dispersal from the mother plant. The study was conducted in the Wet and the Dry zones over two seasons in Viti Levu, the Main Island of Fiji. The flower number, number of propagules and propagule size at maturity were significantly different among three species and within species in the Dry and the Wet zones. Only 1–2% of total flowers in each species became mature propagules. This percentage was significantly lower in the Dry zone for all three species. Rhizophora stylosa produced the biggest size of propagules followed by Rhizophora mangle and Bruguiera gymnorhiza. Relatively longer and heavier propagules were recorded in the Wet zone and shorter and lighter in the Dry zone. Inter-seasonal differences were not significant for these characters. This could be mainly due to almost similar amount of rainfall, relative humidity and temperature regimes over two seasons within each zone.     

CYANOPHYTE CEPHALODIA IN THE LICHEN GENUS NEPHROMA

The lichens, Nephroma expallidum (Nyl.) Nyl. and N. arcticum (L.) Torss., consistently have at least two symbionts in a single thallus: a green alga in the algal layer and a blue-green alga in the internal cephalodia. The cephalodia originate from algal cells in contact with the lower surface of the lichen, in the zone of rhizine formation. The rhizines surround the epiphytic algal colony and form a second cortical layer; following dissociation of the original lower cortex, further growth of the two organisms results in the cyanophyte colony being enveloped by a compact layer of fungal tissue and positioned in the lichen medulla. The colony may eventually assume a superior or inferior position in relation to the lichen thallus, depending in part on the lichen species. Nephroma anticum may have two distinct morphological forms of blue-green algae in the same thallus and occasionally in the same cephalodium. It appears that the relationship that exists between the cephalodial algae and the lichen thallus is antagonistic and results, in some cases, in the exclusion of the green algal layer and death to the cephalodial cyanophytes.