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.     

Congruent genetic structure in the lichen-forming fungus Lobaria pulmonaria and its green-algal photobiont

The extent of codispersal of symbionts is one of the key factors shaping genetic structures of symbiotic organisms. Concordant patterns of genetic structure are expected in vertically transmitted symbioses, whereas horizontal transmission generally uncouples genetic structures unless the partners are coadapted. Here, we compared the genetic structures of mutualists, the lichen-forming fungus Lobaria pulmonaria and its primary green-algal photobiont, Dictyochloropsis reticulata. We performed analysis of molecular variance and variogram analysis to compare genetic structures between symbiosis partners. We simulated the expected number of multilocus-genotype recurrences to reveal whether the distribution of multilocus genotypes of either species was concordant with panmixia. Simulations and tests of linkage disequilibrium provided compelling evidence for the codispersal of mutualists. To test whether genotype associations between symbionts were consistent with randomness, as expected under horizontal transmission, we simulated the recurrence of fungal-algal multilocus genotype associations expected by chance. Our data showed nonrandom associations of fungal and algal genotypes. Either vertical transmission or horizontal transmission coupled with coadaptation between symbiont genotypes may have created these nonrandom associations. This study is among the first to show codispersal and highly congruent genetic structures in the partners of a lichen mutualism.     

Strong fungal specificity and selectivity for algal symbionts in Florida scrub Cladonia lichens

Symbiosis is a major theme in the history of life and can be an important force driving evolution. However, across symbioses, it is difficult to tease apart the mechanisms that structure the interactions among potential partners. We used genetic similarity and frequency-based methods to qualitatively and quantitatively examine the patterns of association among several co-occurring Cladonia lichen fungi and their algal photobionts in six disjunct Florida scrub sites. The patterns of association were described by the degree of specificity, i.e. the phylogenetic range of associated partners, and of selectivity, i.e. the frequency of association among partners. Six fungal species associated with only one algal internal transcribed spacer clade, with the remaining two fungi being associated with two algal clades. In all cases, the fungi associated in unequal frequencies with the observed algal photobiont genotypes within those clades--suggesting that both specificity and selectivity were higher than expected. Fungal species can be grouped into three significantly different specificity classes: photobiont specialists, intermediates and generalists. In contrast to the pronounced specificity for photobionts among fungal species, the different Florida scrub sites do not harbour distinct photobiont pools, and differential photobiont availability cannot explain the patterning of lichen associations at this spatial scale. Therefore, we conclude that fungal specificity and selectivity for algal photobionts are major factors in determining the local composition of symbiotic partnerships.     

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.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus <Emphasis Type="Italic">Tephromela atra</Emphasis> from the Mediterranean region

Recent DNA sequence analyses have revealed the diversity of algal partners in lichen symbioses. Although morphologically similar, different genetic lineages of photobionts are detected in wide geographic ranges of the same lichen fungal species. We studied the photobiont of the genus Trebouxia, which are known as partners of diverse lichen-forming fungal species in the Mediterranean region. We studied the phylogeny of these algae with a multilocus dataset including three loci: ITS, rbcL, and actin type I gene. The two lineages found, informally named Trebouxia sp. 1 and Trebouxia sp. 2, are related to Trebouxia arboricola/decolorans. The cultivation under axenic conditions succeeded only for one of them so far. We used light microscopy, confocal laser scanning microscopy and transmission electron microscopy for phenotypic characterisation. The ultrastructural characters currently used to describe species in the genus do not support the segregation of Trebouxia sp.1 from Trebouxia arboricola. The preferential presence in Mediterranean climates of these strains suggests eco-physiological adaptation. Despite their asexuality in long living lichen symbioses, coccoid algal lichen partners have apparently diversified genetically and physiologically.     

Identity and genetic structure of the photobiont of the epiphytic lichen Ramalina menziesii on three oak species in southern California

Lichens, a classic example of an obligate symbiosis between fungi and photobionts (which could be algae or cyanobacteria), are abundant in many terrestrial ecosystems. The genetic structure of the photobiont population found in association with a lichen-forming fungal species could be affected by fungal reproductive mode and by the spatial extent of gene flow in the photobiont. Using DNA sequences from one nuclear ribosomal and two chloroplast loci, we analyzed the genetic structure of the photobiont associated with the fungus Ramalina menziesii at an oak woodland study site in southern California. We had previously shown that the fungus exhibited no genetic structure among four local sites or three phorophyte species. Our goals were to identify the photobiont species and assess its genetic structure. We found that R. menziesii was highly specific in its photobiont choice and associated with one alga, Trebouxia decolorans. In contrast to the fungal population, we found significant differentiation among the algae sampled on three oak species and little genetic structure among the sites for two of the three algal loci. We hypothesize that R. menziesii is locally adapted to the phorophyte species through habitat specialization in the algal partner of the symbiosis.     

Microsatellite markers for Dictyochloropsis reticulata (Trebouxiophyceae), the symbiotic alga of the lichen Lobaria pulmonaria (L.)

We isolated and characterized eight microsatellite markers for Dictyochloropsis reticulata, the primary photosynthetic partner of the epiphytic lichen Lobaria pulmonaria. These are the first microsatellite loci reported for a lichen symbiotic alga. These polymorphic markers will be useful for investigating spatial genetic structure, biogeography and dispersal of this eukaryotic alga and will generally shed light on the coevolution of the green-algal lichen symbioses.     

Population structure of mycobionts and photobionts of the widespread lichen Cetraria aculeata

Lichens are symbioses between fungi (mycobionts) and photoautotrophic green algae or cyanobacteria (photobionts). Many lichens occupy large distributional ranges covering several climatic zones. So far, little is known about the large‐scale phylogeography of lichen photobionts and their role in shaping the distributional ranges of lichens. We studied south polar, temperate and north polar populations of the widely distributed fruticose lichen Cetraria aculeata. Based on the DNA sequences from three loci for each symbiont, we compared the genetic structure of mycobionts and photobionts. Phylogenetic reconstructions and Bayesian clustering methods divided the mycobiont and photobiont data sets into three groups. An amova shows that the genetic variance of the photobiont is best explained by differentiation between temperate and polar regions and that of the mycobiont by an interaction of climatic and geographical factors. By partialling out the relative contribution of climate, geography and codispersal, we found that the most relevant factors shaping the genetic structure of the photobiont are climate and a history of codispersal. Mycobionts in the temperate region are consistently associated with a specific photobiont lineage. We therefore conclude that a photobiont switch in the past enabled C. aculeata to colonize temperate as well as polar habitats. Rare photobiont switches may increase the geographical range and ecological niche of lichen mycobionts by associating them with locally adapted photobionts in climatically different regions and, together with isolation by distance, may lead to genetic isolation between populations and thus drive the evolution of lichens.     

In situ development of the foliicolous lichen Phyllophiale (Trichotheliaceae) from propagule germination to propagule production

The vegetative cycle of the foliicolous lichen Phyllophiale, from propagule germination to propagule production, was studied by light microscope observation of thalli colonizing plastic cover slips placed within a lowland tropical forest. Discoid propagules germinated by growth of radially arranged fungal cells and developed directly into lichen thalli. The young lichen comprised a single disc of closely branched, radiating filaments of the algal symbiont Phycopeltis, covered by a network of fungal hyphae extending onto the substrate as a prothallus. The prothallic hyphae incorporated additional Phycopeltis thalli encountered on the substrate. The phycobiont formed a single layer, with individual algal thalli clearly distinguishable within the lichen. Radial growth ceased at points of contact between adjacent phycobiont thalli. The visible shape of the crustose lichen thallus corresponded to the perimeter of the phycobiont thalli within. Propagules were initiated at points corresponding to the margins of the phycobiont thalli, by vertical reorientation of horizontal algal filaments surrounded by fungal hyphae. The lichenized alga produced intercalary gametangia. Degeneration of propagules unsuccessful in lichen establishment sometimes resulted in free growth of the phycobiont. The alga generally maintained its shape, growth pattern, and reproductive independence within the lichen, while also participating in the formation of unique symbiotic propagules.     

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.     

Algal switching among lichen symbioses

Lichens are intimate and long-term symbioses of algae and fungi. Such intimate associations are often hypothesized to have undergone long periods of symbiotic interdependence and coevolution. However, coevolution has not been rigorously tested for lichen associations. In the present study we compared the nuclear internal transcribed spacer (ITS) phylogenies of algal and fungal partners from 33 natural lichen associations to test two aspects of coevolution, cospeciation and parallel cladogenesis. Since statistically significant incongruence between symbiont phylogenies rejected parallel cladogenesis and minimized cospeciation events, we conclude that switching of highly selected algal genotypes occurs repeatedly among these symbiotic lichen associations.     

Relationship between the algal partners and the growth of lichen-forming fungus Porpidia crustulata

In recent years, examinations based on morphological characters and phylogenetic analyses have begun to reveal the diversity of photobionts in lichen symbioses. However, still little is known regarding genetic diversity and ecological adaptation of algal partners in lichen symbioses. In this study, we investigated the photobiont Chlorella “sp. GC” (Trebouxiophyceae), a partner of Porpidia crustulata from the Guancen Mountains, China. We examined the relationship between photobiont layer thickness and Porpidia crustulata growth over a 6-year period (2007–2012). Although Porpidia crustulata exhibited moderate growth rates (0.4–0.62 mm year^?1), photobiont layer became increasingly thinner over the six-year period. We speculate that prolonged exposure to sunlight and desiccation may deleteriously alter photobiont morphology and physiology. Porpidia crustulata may be forced into a state of physiological dormancy.     

Joint Dispersal Does Not Imply Maintenance of Partnerships in Lichen Symbioses

Dispersal of symbiotic partners by joint propagules is considered as an efficient strategy to maintain successful associations and to circumvent low symbiont availability. Joint dispersal is widespread in diverse symbioses and a particularly common reproductive mode in lichens. We were interested in the implications of joint symbiont dispersal on population genetic structure and investigated patterns of symbiont association in populations of two closely related lichen species in the genus Physconia, with similar range of compatible algal partners. One of the lichen species is characterized by joint dispersal of both symbionts, whereas the other species propagates by meiotic fungal spores alone. The latter species must re-establish the symbiotic stage with appropriate algae sampled from the environment. Both fungal species have specialized on photobionts representing a monophyletic lineage of the algal genus Trebouxia. The results indicate no correlated association of symbiont genotypes in the species with joint symbiont dispersal. We rather show that algal gene diversity in populations of lichenized fungi with different propagation strategies is not necessarily different. The association with algae that differ from the co-dispersed genotypes during the vegetative development of the thalli is the most likely explanation for the observed pattern. Maintenance of symbiotic associations is an option but not a strict consequence of joint symbiont dispersal in lichens.     

Characteristics of lichen lectins and their role in symbiosis

Lectins are proteins or glycoproteins of non-immune origin which bind reversibly to carbohydrates that are exposed on cellular surfaces and mediate cellular recognition processes in a variety of biological interactions. Though initially discovered in plants, lectins from various sources including lichens, have been extensively studied by researchers all over the world. The symbiotic interaction between a fungus (mycobiont) and its photosynthetic partner (photobiont), usually an alga, constitutes a lichen. Some lichen lectins displays activity to human or animal erythrocytes. Although only a few lichen lectins have been examined to date, their characteristics suggest that they play an important role in the symbiotic interactions of this association. Lectin binding and the related enzymatic activity with respect to algal cell recognition illustrates a finely tuned mechanistic system which involved in the lichen symbiosis. This review provides an overview of the characteristics of lichen lectins and an insight into lectin-mediated symbiotic interactions and the galectin encoding genes. Future prospects for lichen lectin research in different areas are also highlighted.     

Differential responses of lichen symbionts to enhanced nitrogen and phosphorus availability: an experiment with Cladina stellaris

BACKGROUND AND AIMS: Lichens can be both nitrogen- (N) and phosphorous- (P) limited and thus may be susceptible to nutrient enrichment. Nutrient enrichment with N and P may have differing impacts on the lichen structure because of different physiological responses of fungal and algal partners to these nutrients. The hypothesis was tested that the differential responses of lichen symbionts to enhanced availability of N and P is reflected in the lichen thallus structure and the wall-to-wall interface between the algal and fungal cells. METHODS: Lichen cushions of Cladonia stellaris were treated with one P and two N concentrations alone and in combination that yielded total depositions of approx. 300 (moderate) and 1000 (high) mg N m(-2) and 100 (high) mg P m(-2) over an experiment lasting 14 weeks. The effects of N and P inputs on the relative volumes of fungal and algal cell in the medullary tissue and on the thallus structure were studied using light microscopy. The interface between algal and fungal cell walls was examined using transmission electron microscopy. KEY RESULTS: The influence of excess P on the lichen thallus structure was stronger than that of additional N. Addition of P reduced the N : P ratio in podetia, the proportion of the medullary layer volume occupied by the algal cells, the thallus volume occupied by the internal lumen, and the algal cell-wall area covered by fungal hyphae. CONCLUSIONS: Ecologically realistic changes in the availability of key macronutrients can alter the growth of symbionts. Reduction in the proportion of photobiont cells indicates that the application of P either stimulates fungal hyphal growth in the medullary tissue or impairs the cell division of the algal cells. The results suggest that both the N and P availability and thallus N : P ratio affect the growth rates of lichen symbionts.     

Compatibility and thigmotropism in the lichen symbiosis: A reappraisal

The development of many complex stratified lichen thalli is made through stages of complex phenotypic interactions between a filamentous fungus (the mycobiont), and a trebouxioid alga (the photobiont). Typically, the second stage of this symbiotic development is marked by the envelopment of the photobiont by the mycobiont through increased lateral hyphal branching and the formation of appressoria. Previously, the mycobiont’s envelopment of photobiont cells was considered thigmotropic (a growth response due to shape) as a mycobiont can envelop algal sized objects in its environment. However, after growing the mycobiontCladonia grayi with various phototrophs and glass beads, we conclude that the mycobiont does not show this characteristic second stage morphological response when grown in non-compatible pairings. Instead,C. grayi displays a distinctive morphological growth response only in compatible symbiotic pairings, such as with its natural photobiontAsterochlor’is sp.     

Local genetic structure in a North American epiphytic lichen, Ramalina menziesii (Ramalinaceae)

Epiphytic lichens possess unique life history traits that can have conflicting effects on genetic structure: symbiotic mutualism between a fungus with its algal or cyanobacterial photobiont, association with a host plant, and ability to reproduce sexually and asexually. Our study species, Ramalina menziesii, has small ascospores that can facilitate long-distance gene movement, and it is capable of clonal reproduction. The goals of this study are to test whether different haplotypes were differentially distributed across host plant species, to look for evidence of asexual vs. sexual reproduction, and to assess the local genetic structure of the population. We sampled individuals from multiple trees of three oak species in four lichen subpopulations within a savanna ecosystem. Using DNA sequence data from four fungal nuclear loci, we found no tendency for host specialization. Alleles were randomly distributed across subpopulations. The frequency of multilocus genotypes was consistent with a randomly mating population. Sexual reproduction involving relichenization appeared to be the predominant mode of reproduction of R. menziesii at this study site. We found no significant local genetic structure suggesting widespread gene flow at the local scale. The genetic structure of this lichen is comparable to that of widely distributed epiphytic plants.