The development of characteristic thallus structures in lichen-forming fungi requires the association with suitable photoautotrophic partners. Previous work suggests that fungi have a specific range of compatible photobionts and that selected algal strains are also correlated with the habitat conditions. We selected the rock-inhabiting crust lichen Protoparmeliopsis muralis, which exhibits high flexibility in algal associations. We present a geographically extended and detailed analysis of algal association patterns including thalli which host superficial algal colonies. We sampled 17 localities in Europe, and investigated the photobiont genotypic diversity within and between thalli and compared the diversity of intrathalline photobionts and externally associate algal communities between washed and unwashed thalli by single-strand conformation polymorphism analyses and ITS sequence data. The results show that (1) photobiont population within the lichen thalli is homogeneous; (2) multiple photobiont genotypes occur within single areoles and lobes of individual lichens; and (3) algal communities which superficially colonize the lichen thalli host taxa known as photobionts in unrelated lichens. Photobiont association patterns are extremely flexible in this ecologically versatile crust-forming lichen. We suggest that lichen surfaces represent a potential temporary niche for free-living stages of lichen photobionts, which could facilitate the establishment of further lichens in the proximal area.

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.

The genus Asterochloris represents one of the most common, widespread, and diverse taxa of lichen photobionts. In this report, we describe and characterize six new species (A. echinata, A. friedlii, A. gaertneri, A. leprarii, A. lobophora, and A. woessiae) that were identified during our recent investigation of photobiont diversity. We found that the species differed genetically, morphologically, ecologically, and with respect to their mycobiont partners. Statistical analyses revealed significant morphological differentiation of all six newly described species, as well as their separation from previously described Asterochloris species. Chloroplast morphology represented the best morphological marker for species delineation. In fact, each species can be recognized by the dominance and unique assemblage of particular chloroplast types. Although genetically well recognized by rapidly evolving internal transcribed spacer rDNA and actin intron markers, all 13 investigated Asterochloris species shared identical small subunit rDNA sequences. We therefore demonstrated that morphologically and ecologically diverse species can frequently be grouped into a single taxonomic unit in whole-transcriptome sequencing studies, considerably affecting the resulting estimates of species diversity. Finally, we demonstrated the presence of isogamous sexual reproduction in Asterochloris, disputing the current symbiotic dogma of the loss of sexual reproduction in algal symbionts.

The microalgae of the genus Asterochloris are the preferential phycobionts in Cladonia, Lepraria and Stereocaulon lichens. Recent studies have highlighted the hidden diversity of the genus, even though phycobionts hosting species of the genus Cladonia in Mediterranean and Canarian ecosystems have been poorly explored. Phylogenetic analyses were made by concatenation of the sequences obtained with a plastid -LSU rDNA -and two nuclear -internal transcribed spacer (ITS) rDNA and actin -molecular markers of the phycobionts living in several populations of the Cladonia convoluta-Cladonia foliacea complex, Cladonia rangiformis and Cladonia cervicornis s. str. widely distributed in these areas in a great variety of substrata and habitats. A new strongly supported clade was obtained in relation to the previously published Asterochloris phylogenies. Minimum genetic variation was detected between our haplotypes and other sequences available in the GenBank database. The correct identification of the fungal partners was corroborated by the ITS rDNA barcode. In this study we provide a detailed characterization comprising chloroplast morphology, and ultrastructural and phylogenetic analyses of a novel phycobiont species, here described as Asterochloris mediterranea sp. nov. Barreno, Chiva, Moya et Š kaloud. A cryopreserved holotype specimen has been deposited in the Culture Collection of Algae of Charles University in Prague, Czech Republic (CAUP) as CAUP H 1015. We suggest the use of a combination of several nuclear and plastid molecular markers, as well as ultrastructural (transmission electron and confocal microscopy) techniques, both in culture and in the symbiotic state, to improve novel species delimitation of phycobionts in lichens.

This paper describes a new genus of lichen photobionts, Vulcanochloris, with three newly proposed species, V. canariensis, V. guanchorum and V. symbiotica. These algae have been discovered as photobionts of lichen Stereocaulon vesuvianum growing on slopes of volcanos and lava fields on La Palma, Canary Islands, Spain. Particular species, as well as the newly proposed genus, are delimited based on ITS rDNA, 18S rDNA and rbcL sequences, chloroplast morphology, and ultrastructural features. Phylogenetic analyses infer the genus Vulcanochloris as a member of Trebouxiophycean order Trebouxiales, in a sister relationship with the genus Asterochloris. Our data point to the similar lifestyle and morphology of these two genera; however, Vulcanochloris can be well distinguished by a unique formation of spherical incisions within the pyrenoid. Mycobiont specificity and geographical distribution of the newly proposed genus is further discussed.