Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen‐forming family Parmeliaceae (Ascomycota)

Microbial symbionts are instrumental to the ecological and long‐term evolutionary success of their hosts, and the central role of symbiotic interactions is increasingly recognized across the vast majority of life. Lichens provide an iconic group for investigating patterns in species interactions; however, relationships among lichen symbionts are often masked by uncertain species boundaries or an inability to reliably identify symbionts. The species‐rich lichen‐forming fungal family Parmeliaceae provides a diverse group for assessing patterns of interactions of algal symbionts, and our study addresses patterns of lichen symbiont interactions at the largest geographic and taxonomic scales attempted to date. We analysed a total of 2356 algal internal transcribed spacer (ITS) region sequences collected from lichens representing ten mycobiont genera in Parmeliaceae, two genera in Lecanoraceae and 26 cultured Trebouxia strains. Algal ITS sequences were grouped into operational taxonomic units (OTUs); we attempted to validate the evolutionary independence of a subset of the inferred OTUs using chloroplast and mitochondrial loci. We explored the patterns of symbiont interactions in these lichens based on ecogeographic distributions and mycobiont taxonomy. We found high levels of undescribed diversity in Trebouxia, broad distributions across distinct ecoregions for many photobiont OTUs and varying levels of mycobiont selectivity and specificity towards the photobiont. Based on these results, we conclude that fungal specificity and selectivity for algal partners play a major role in determining lichen partnerships, potentially superseding ecology, at least at the ecogeographic scale investigated here. To facilitate effective communication and consistency across future studies, we propose a provisional naming system for Trebouxia photobionts and provide representative sequences for each OTU circumscribed in this study.     

Recognition mechanisms during the pre-contact state of lichens: II. Influence of algal exudates and ribitol on the response of the mycobiont of Fulgensia bracteata

Successful re-lichenization between the two bionts of the lichen symbiosis, the fungal mycobiont and its specific photobiont, is a process that is not well understood yet. To assess potential signalling between the two bionts during initial pre-contact, exudates of the Trebouxia photobionts of Fulgensia bracteata, Fulgensia fulgens, and Xanthoria elegans, of the Asterochloris photobiont of Lecidea lurida, and of the non-lichenizing green alga Myrmecia bisecta were investigated. The compounds identified in these exudates were tested with respect to their influence on germination and early development of the Fulgensia bracteata mycobiont. Additionally, carbohydrates (glucose, sucrose, ribitol) were tested to appraise their effect on the mycobiont growth patterns. Three hypotheses were confirmed: (i) photobionts exude various substances, (ii) the photobiont exudation pattern varies with the identity of the photobiont, and (iii) a pre-contact influence induces changes in the early development of the mycobiont of F. bracteata. This study gives comparative insight to exudates of lichen photobionts. In vitro photobionts differentially release compounds belonging to several substance classes which include indole-3-carbaldehyde, two cyclic dipeptides, and rhamnose. Two compounds had inhibitory effects on germination and germ-tube growth of the mycobiont and one other enhanced spore germination. Additionally, ribitol was found to elicit a strong effect on the mycobiont’s growth. In general, photobiont-exudation, its effect on the mycobiont, and the response to ribitol suggest that complex pre-contact signalling has a crucial role in lichen biont recognition.     

Geographic mosaic of symbiont selectivity in a genus of epiphytic cyanolichens

In symbiotic systems, patterns of symbiont diversity and selectivity are crucial for the understanding of fundamental ecological processes such as dispersal and establishment. The lichen genus Nephroma (Peltigerales, Ascomycota) has a nearly cosmopolitan distribution and is thus an attractive model for the study of symbiotic interactions over a wide range of spatial scales. In this study, we analyze the genetic diversity of Nephroma mycobionts and their associated Nostoc photobionts within a global framework. The study is based on Internal Transcribed Spacer (ITS) sequences of fungal symbionts and tRNA^Leu (UAA) intron sequences of cyanobacterial symbionts. The full data set includes 271 Nephroma and 358 Nostoc sequences, with over 150 sequence pairs known to originate from the same lichen thalli. Our results show that all bipartite Nephroma species associate with one group of Nostoc different from Nostoc typically found in tripartite Nephroma species. This conserved association appears to have been inherited from the common ancestor of all extant species. While specific associations between some symbiont genotypes can be observed over vast distances, both symbionts tend to show genetic differentiation over wide geographic scales. Most bipartite Nephroma species share their Nostoc symbionts with one or more other fungal taxa, and no fungal species associates solely with a single Nostoc genotype, supporting the concept of functional lichen guilds. Symbiont selectivity patterns within these lichens are best described as a geographic mosaic, with higher selectivity locally than globally. This may reflect specific habitat preferences of particular symbiont combinations, but also the influence of founder effects.     

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.     

Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist‐mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad‐scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub‐species‐level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen‐forming fungi to broaden their ecological niche. The extent and direction of photobiont‐mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub‐species‐level resolution across the mycobiont's Europe‐wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiont‐photobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub‐species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiont‐mediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.     

Sizing the fungal and algal genomes of the lichen Cladonia grayi through quantitative PCR

Using a method based on quantitative PCR, we determined that the nuclear genome sizes for the mycobiont and photobiont of the lichen Cladonia grayi are 28.6 Mb and 106.7 Mb, respectively. This is the first genome size determination for lichens, and suggests that between 20,000 and 25,000 genes function in C. grayi. The mycobiont genome size is near the middle of the range observed within the Pezizomycota, the subphylum containing all known ascomycete lichen fungi. The genome size of the photobiont (the green alga Asterochloris sp.) is near the lower end of its class, the Trebouxiophyceae. Genomes in this size range can be sequenced at relatively low cost with current pyrosequencing-based methods. The genome sizing method requires very small amounts of precisely quantified DNA and should be applicable to any lichen whose symbionts can be reliably isolated from one another. Since the symbionts used in this project were isolated from soredia, the lichen’s vegetative propagules, we also describe a method for the establishment of axenic symbiont cultures from large numbers of soredia.     

Combined observational and experimental data provide limited support for facilitation in lichens

It is increasingly recognized that facilitative interactions can shape communities. One of the mechanisms through which facilitation may operate is when one species facilitates the colonization of another through the exchange of shared symbionts. Lichens are symbiotic associations composed of a mycobiont (lichenised‐fungus) and one or two photobionts (algae or cyanobacteria). Different lichen species may have overlapping specificity for photobionts, creating the possibility that facilitation drives lichen community assembly. To investigate whether facilitation occurs in lichens, we combined an observational study (a) with a manipulative field experiment (b). For (a), we quantified the effect of local patch conditions, facilitation and the size of the surrounding metapopulation on colonizations of an epixylic lichen species (Cladonia botrytes) in an area of managed boreal forest. This was done by twice surveying lichens on 293 stumps, located in stands of three age classes. For (b), we treated unoccupied surfaces of 56 cut stumps with algal mixtures of an Asterochloris photobiont and recorded C. botrytes colonizations over three years. In (a), colonization rates of C. botrytes increased with increasing abundance of other lichen species with specificity for Asterochloris photobionts, consistent with an effect of facilitation. However, in the field experiment (b), colonizations of the focal species did not provide support for facilitation. We conclude that our study provides limited support for facilitation in green‐algal lichens, underscoring the importance of combining observational studies with experiments when studying species interactions.     

Proteins from the liehenXanthoria parietina which bind to phycobiont cell walls Localization in the intact liehen and cultured mycobiont

Summary A protein fraction, previously isolated from the lichenXanthoria parietina and known to bind to the appropriate culturedTrebouxia phycobiont, was visualized in the intact lichen thallus and cultured mycobiont by an indirect immunoperoxidase assay. The protein was localized in both the upper and lower cortices of the lichen thallus; it was also present in the cell walls of the mycobiont culturedin vitro. The possible role of this protein in the recognition, or initial interaction, between separated lichen symbionts is discussed.     

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.     

Differential effects of the crustose Diploschistes diacapsis and the squamulose Fulgensia bracteata on the establishment of a Mediterranean grass species

The interest of the scientific community in biological soil crusts has grown exponentially over the last decades. One of the scientific research interests is the study of the effect of these crusts on plant establishment. Findings in this topic have been controversial, and some differences were attributed to crust types. Biological soil crusts dominated by lichens are common components of Stipa tenacissima steppes in arid and semi‐arid environments of the southern Mediterranean. In the current study, we conducted growth chamber experiments to investigate the differential effects of two lichen species with continuous crustose thalli (Diploschistes diacapsis) and with squamulose semicontinuous thalli (Fulgensia bracteata) on seed germination, root penetration, shoot emergence and seed viability of the tussock grass species S. tenacissima. Our results showed that under laboratory conditions, two distinct lichen species had significantly different effects on the establishment of S. tenacissima. Our findings clearly demonstrated that D. diacapsis significantly decreased germination, root penetration and shoot emergence of S. tenacissima compared to F. bracteata. This can be related to differences in morphological and physiological characteristics between crustose and squamulose lichens. Overall, we suggest that D. diacapsis and crustose lichens generally can act as natural barrier to the establishment of S. tenacissima.     

The impact of nitrogen deposition on photobiont‐mycobiont balance of epiphytic lichens in subtropical forests of central China

Excessive nitrogen (N) deposition can impact lichen diversity in forest ecosystems, and this is a particular situation in China. Here, we examined the N uptake, assimilation, and the impact of excessive N deposition on the symbiotic balance of dominant epiphytic lichens in the subtropical forests in the Mts. Shennongjia of central China. The results show that lichen species took up, assimilated and utilized more ammonium than nitrate in a species‐specific way, following the increase of N availability. The photobiont of the lichens decreased with the increase of N concentration following an initial increase, while the mycobiont response to the N addition was not apparent. Considerable variation in response to excessive N deposition exists among the lichen species. Usnea longissima could regulate its N uptake, resulting in a stable photobiont‐mycobiont ratio among N treatments. In contrast, the photobiont‐mycobiont ratio of other four lichens increased initially but decreased when N concentration exceeded a certain level, and N stress may have broken the balance between photobiont and mycobiont of these lichens. Our results suggest that most epiphytic lichens in subtropical forest of central China could uptake and assimilate more ammonium than nitrate and that the balance between photobiont and mycobiont of many epiphytic lichens might change with the increasing N deposition load, which could impact the lichen diversity of this forest ecosystem.     

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.     

Can Antarctic lichens acclimatize to changes in temperature?

The Antarctic Peninsula, a tundra biome dominated by lichens and bryophytes, is an ecozone undergoing rapid temperature shifts. Such changes may demand a high physiological plasticity of the local lichen species to maintain their role as key drivers in this pristine habitat. This study examines the response of net photosynthesis and respiration to increasing temperatures for three Antarctic lichen species with different ecological response amplitudes. We hypothesize that negative effects caused by increased temperatures can be mitigated by thermal acclimation of respiration and/or photosynthesis. The fully controlled growth chamber experiment simulated intermediate and extreme temperature increases over the time course of 6 weeks. Results showed that, in contrast to our hypothesis, none of the species was able to down‐regulate temperature‐driven respiratory losses through thermal acclimation of respiration. Instead, severe effects on photobiont vitality demonstrated that temperatures around 15°C mark the upper limit for the two species restricted to the Antarctic, and when mycobiont demands exceeded the photobiont capacity they could not survive within the lichen thallus. In contrast, the widespread lichen species was able to recover its homoeostasis by rapidly increasing net photosynthesis. We conclude that to understand the complete lichen response, acclimation processes of both symbionts, the photo‐ and the mycobiont, have to be evaluated separately. As a result, we postulate that any acclimation processes in lichen are species‐specific. This, together with the high degree of response variability and sensitivity to temperature in different species that co‐occur spatially close, complicates any predictions regarding future community composition in the Antarctic. Nevertheless, our results suggest that species with a broad ecological amplitude may be favoured with on‐going changes in temperature.     

Fungal farmers or algal escorts: lichen adaptation from the algal perspective

Domestication of algae by lichen‐forming fungi describes the symbiotic relationship between the photosynthetic (green alga or cyanobacterium; photobiont) and fungal (mycobiont) partnership in lichen associations ( Goward 1992 ). The algal domestication implies that the mycobiont cultivates the alga as a monoculture within its thallus, analogous to a farmer cultivating a food crop. However, the initial photobiont ‘selection’ by the mycobiont may be predetermined by the habitat rather than by the farmer. When the mycobiont selects a photobiont from the available photobionts within a habitat, the mycobiont may influence photobiont growth and reproduction ( Ahmadjian & Jacobs 1981 ) only after the interaction has been initiated. The theory of ecological guilds ( Rikkinen et al. 2002 ) proposes that habitat limits the variety of photobionts available to the fungal partner. While some studies provide evidence to support the theory of ecological guilds in cyanobacterial lichens ( Rikkinen et al. 2002 ), other studies propose models to explain variation in symbiont combinations in green algal lichens ( Ohmura et al. 2006 ; Piercey‐Normore 2006 ; Yahr et al. 2006 ) hypothesizing the existence of such guilds. In this issue of Molecular Ecology, Peksa & ?kaloud (2011) test the theory of ecological guilds and suggest a relationship between algal habitat requirements and lichen adaptation in green algal lichens of the genus Lepraria. The environmental parameters examined in this study, exposure to rainfall, altitude and substratum type, are integral to lichen biology. Lichens have a poikilohydric nature, relying on the availability of atmospheric moisture for metabolic processes. Having no known active mechanism to preserve metabolic thallus moisture in times of drought, one would expect a strong influence of the environment on symbiont adaptation to specific habitats. Adaptation to changes in substrata and its properties would be expected with the intimate contact between crustose lichens in the genus Lepraria. Altitude has been suggested to influence species distributions in a wide range of taxonomic groups. This is one of the first studies to illustrate an ecological guild, mainly for exposure to rainfall (ombrophiles and ombrophobes), with green algal lichens.     

Algal and Fungal Diversity in Antarctic Lichens

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7–100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen‐associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen‐associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen‐associated fungi was not evident.     

Survival analyses of symbionts isolated from <Emphasis Type="Italic">Endocarpon pusillum</Emphasis> Hedwig to desiccation and starvation stress

This work deals with the survival analyses of the symbionts isolated from the lichen E. pusillum under desiccation and starvation stress. The mycobiont of the symbionts was under the desiccation in combination with starvation stress, and under starvation stress alone as well. The phycobiont of the symbionts was under desiccation stress alone. The experiments were detected by means of the biomass size, weight and cell density, deformity of the hyphae and cells, and metabolic activity through SEM (scanning electron microscopy), TEM (transmission electron microscopy), FM (fluorescence microscopy), spectrophotometry, and FCM (flow cytometry). The results show that the mycobiont can survive for seven months under desiccation stress in combination with starvation stress, and for eight months under starvation stress alone. The phycobiont can survive for two months under desiccation stress. It can provide a scientific basis for further research of the reproduction biology of lichens and arid desert biocarpet engineering to fix sand and carbon.     

Genetic diversity and structure of the epiphytic foliose lichen Lobaria pindarensis in the Himalayas depends on elevation

The epiphytic lichen Lobaria pindarensis is a Himalayan endemic species with little information on distribution, genetic diversity and structural complexity. During an intensive survey in the Nepal Himalayas, we collected 1256 thallus fragments from 45 phorophyte species to study their distribution and population genetics along an elevational gradient. We quantified genetic diversity and population structure of each symbiont at 17 fungus specific and 9 alga specific microsatellite loci. The Bayesian clustering identified three and two distinct gene pools for mycobiont and photobiont. We found that genetic diversity, allelic richness and gene pool composition and distribution were significantly influenced by elevation. We discovered both clonally and sexually reproduced repeated genotypes of the symbionts.     

Nitrogen deposition drives lichen community changes through differential species responses

Nitrogen (N) deposition has increased globally over the last 150 years and further increases are predicted. Epiphytic lichens decline in abundance and diversity in areas with high N loads, and the abundance of lichens decreases along gradients of increased deposition. Thus, although N is an essential nutrient for lichens, excessive loads may be detrimental for them. However, these gradients include many correlated pollutants and the mechanisms behind the decline are thus poorly known. The aim of this study was to assess effects of N deposition, alone, on the epiphytic lichen community composition in a naturally N‐poor boreal forest. For this purpose, whole spruce trees were fertilized daily with N at five levels, equivalent to 0.6, 6, 12.5, 25, and 50 kg N ha^?1 yr^?1, during four consecutive growing seasons (2006–2009), and changes in the abundance of lichens were monitored each autumn from the preceding year (2005). The studied lichen communities were highly dynamic and responded strongly to the environmental perturbation. N deposition detectably altered the direction of succession and reduced the species richness of the epiphytic lichen communities, even at the lowest fertilization application (6 kg N ha^?1 yr^?1). The simulated N deposition caused significant changes in the abundance of Alectoria sarmentosa, Bryoria spp., and Hypogymnia physodes, which all increased at low N loads and decreased at high loads, but with species‐specific optima. The rapid decline of A. sarmentosa may have been caused by the added nitrogen reducing the stability of the lichen thalli, possibly due to increases in the photobiont: mycobiont ratio or parasitic fungal attacks. We conclude that increases in nitrogen availability, per se, could be responsible for the reductions in lichen abundance and diversity observed along deposition gradients, and those community responses may be due to physiological responses of the individual species rather than changes in competitive interactions.