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Lichens provide valuable systems for studying symbiotic interactions. In lichens, these interactions are frequently described in terms of availability, selectivity and specificity of the mycobionts and photobionts towards one another. The lichen-forming, green algal genus Trebouxia Puymaly is among the most widespread photobiont, associating with a broad range of lichen-forming fungi. To date, 29 species have been described, but studies consistently indicate that the vast majority of species-level lineages still lack formal description, and new, previously unrecognized lineages are frequently reported. To reappraise the diversity and the evolutionary relationships of species-level lineages in Trebouxia, we assembled DNA sequence data from over 1600 specimens, compiled from a range of sequences from previously published studies, axenic algal cultures, and lichens collected from poorly sampled regions. From these samples, we selected representatives of the currently known genetic diversity in the lichenized Trebouxia and inferred a phylogeny from multi-locus sequence data (ITS, rbcL, cox2). We demonstrate that the current formally described species woefully underrepresent overall species-level diversity in this important lichen-forming algal genus. We anticipate that an integrative taxonomic approach, incorporating morphological and physiological data from axenic cultures with genetic data, will be required to establish a robust, comprehensive taxonomy for Trebouxia. The data presented here provide an important impetus and reference dataset for more reliably characterizing diversity in lichenized algae and in using lichens to investigate the evolution of symbioses and holobionts.

[EN] Three vagrant (Circinaria hispida, Circinaria gyrosa, Circinaria sp. `paramerae¿) and one crustose (semi-vagrant, Circinaria sp.`oromediterranea¿) growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp.`oromediterranea¿ and Circinaria sp. `paramerae¿ formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp. where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for HTS analyses: 22 Trebouxia OTUs were detected, ten of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51 and T. cretacea) whose ultrastructure is described here in detail for the first time, HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species., Supported by the Ministerio de Economia y Competitividad (MINECO and FEDER, Spain; CGL2016-79158-P), Excellence in Research (Generalitat Valenciana, Spain; PROMETEO/2017/039). We want to thank the technicians (Ma Teresa Minguez and Nuria Cebrian) from the Servicio de Microscopia Electronica, SCSIE, and Jardi Botanic (Universitat de Valencia) who helped us to perform the TEM process, and Santiago Catala for the pyrosequencing analyses. Daniel Sheerin revised the English manuscript.

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. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd., This study was funded by the Spanish Ministry of Education and Science (CGL2006-12917-C02-01/02), the Spanish Ministry of Science and Innovation (CGL2009-13429-C02-01/02), the AECID (PCI_A/024755/09) and the Generalitat Valenciana (PROMETEO 174/2008 GVA). We are grateful to Dr J. Gimeno-Romeu (University of California, Davis, USA) and to Dr P. J. G. de Nova (IREC, Ciudad Real, Spain), who were the first to isolate DNA from Ramalina farinacea thalli in our group. Wendy Ran revised the manuscript in English.

FOS, S., CALATAYUD, A. & BARRENO, E. 2001. Lichen diversity associated with post-fire colonization in the Valenciano-castellonense cork-oak forests. Bot. Complutensis 25: 103113. 103 Simón Fos et al. Diversidad liquénica asociada a fenómenos post-incendio... The post-fire colonization of the burned barks of cork-oaks (Quercus suber L.) by epiphytic lichens affected by fire episodes has been studied in different areas of the Valenciano-castellonense cork-oak forests (Asplenio onopteridis-Querco suberis sigmetum). These forests, which have been affected by fires of different intensity, are located in Espadán and Calderona mountains and in the Desierto de Las Palmas and differ with respect to their bioclimate and forest structure. This facts result in a different floristic composition of the epiphytic communities prior to the occurrence of fires. The aim of this paper is to determine the recolonization state and to identify the factors involved in the process. Specific richness and coverage of the epiphytic communities show differences between areas. These differences are related with the proximity to undisturbed areas and with its bioclimatic characteristics. 40 species have been identified: 18 crustose (45%), foliose (45%) and 4 fruticose lichens (10%). Secondary succession is dominated by species with wide ecological tolerance with soredia or isidia. Hyperphyscia adglutinata, Physcia adscendens, Ph. tenella and Scoliciosporum chloroccocum show the greatest colonization success; others, such as Evernia prunastri, P. tiliacea, P. soredians, P. subrudecta, Ramalina farinacea, etc., show higher frequency and thallus development in forests which were affected by less intense fires and with subhumid ombroclimate. FOS, S., CALATAYUD, A. & BARRENO, E. 2001. Diversidad liquénica asociada a fenómenos post-incendio en los alcornocales valenciano-castellonenses. Bot. Complutensis 25: 103-113. Se estudia la colonización post-incendio de las cortezas quemadas de los alcornoques (Quercus suber L.), por líquenes epífitos,en los diferentes núcleos del alcornocal valenciano-castellonense (Asplenio onopteridis-Querco suberis sigmetum). Estos bosques, afectados por incendios diferentes, se localizan en las Sierras de Espadán y Calderona y en el Desierto de las Palmas y difieren en sus condiciones bioclimáticas, en la estructura del bosque y, consecuentemente, en la composición florística de las comunidades previas a los incendios. El objetivo de este trabajo es conocer el estado de la recolonización e identificar que factores podrían estar implicados en el proceso. La riqueza específica y la cobertura de las comunidades epífitas muestra asimetrías importantes entre áreas. Estas diferencias se relacionan con la proximidad de zonas inalteradas, que deben actuar como inóculo, y con las características bioclimáticas de cada territorio. En total, se han identificado 40 especies liquénicas: 18 crustáceas (45%), 18 foliáceas (45%) y 4 fruticulosas (10%). La sucesión secundaria está dominada por especies de amplia tolerancia ecológica que poseen estructuras de multiplicación vegetativa. Hyperphyscia adglutinata, Physcia adscendens, Ph. tenella y Scoliciosporum chloroccocum son las que aparecen más tempranamente y las más ampliamente representadas; otras, como Evernia prunastri, P. tiliacea, P. soredians,P. subrudecta, Ramalina farinacea, etc., aparecen con mayor frecuencia y desarrollo en los alcornocales que sufrieron un incendio menos intenso y con ombroclima subhúmedo.

Trebouxiophyceae is a wide class of green algae comprising coccoid and elliptic unicells, filaments, blades and colony-forming species that occur in diverse terrestrial and aquatic environments. Within this class, the genus Trebouxia Puymaly is among the most widespread lichen phycobionts worldwide. However, the 29 formally described species based on the combination of morphological traits and genetic diversity woefully underrepresented the overall species-level diversity recognized in the genus. In Trebouxia, reliable differentiation and characterization of the species-level lineages can be achieved by studying the diversity of key diagnostic features of pyrenoid ultrastructure and chloroplast morphology of axenically grown algal cultures. Here, we used transmission electron microscopy (TEM) coupled with confocal laser scanning microscopy (CLSM) to analyze the pyrenoid and the chloroplast of 20 Trebouxia species-level lineages grown directly on solid agar medium and on cellulose-acetate discs laid over the agar medium. With the new, detailed morphoanatomical characterization of these species-level lineages, we reappraise Trebouxia taxonomy in light of the most recent phylogenetic delimitation provided by Muggia et al. (2020).

Fungal–algal relationships—both across evolutionary and ecological scales—are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra, to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000–2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia “A52.” Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia “A52” and three other previously recognized lineages, i.e., Trebouxia “A02,” T. vagua “A04,” and T. vagua “A10,” which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes.

The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones), and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to ecophysiological and evolutionary studies to understand the roles of the multiple photobionts in lichen symbioses.

The present monitoring study was undertaken to analyze the impact of atmospheric pollution on the lichens found near a power station in northern Spain. Data from 565 releves gathered on 113 sites were analyzed. In areas near the power station the lichen species belong to the Xanthorion community. In areas away from the power station and that are not under the influence of dominant winds, the lichenic diversity increases and the species present belong to the Parmelion community. In intermediate areas between the two, there is a great increase in lichen diversity, in these areas there is a replacement of the species that typically belong to the Xanthorion community, by species typical of the Parmelion community. Because of this we propose the analysis of the number of lichen species together with the distribution of bioindicator taxa as a complement to lichen diversity analysis in biomonitoring studies.