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.

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Abstract:Myrmecia israeliensis has been traditionally considered as a green coccoid free-living microalga. This microalga was previously suggested as the primary phycobiont in the lichens Placidium spp., Heteroplacidium spp., and Psora decipiens. However, due to the absence of ITS rDNA sequences (barcode information) published along with these investigations, the symbiotic nature of M. israeliensis might be confirmed by using the DNA barcoding and different microscopic examinations both in the symbiotic state and in culture. The aim of this study was to settle the presence of M. israeliensis as the primary microalga in squamulose lichens growing in terricolous communities (Psora spp., Placidium spp. and Claviscidium spp.) in 32 localities within European and Canary Island ecosystems by using both molecular and ultrastructural techniques. The lichen-forming fungi were identified using ITS rDNA as a barcode, and in the case of P. decipiens specimens, the mycobiont analyses showed an unexpected variability. Phycobiont phylogenetic analyses were made using both chloroplast (LSU rDNA) and nuclear (ITS rDNA) molecular markers. Our results proved that M. israeliensis is the primary symbiotic microalga in all the chosen and analyzed lichens. In addition, fluorescence microscopy, transmission electron microscopy and scanning electron techniques were used to characterize M. israeliensis. Finally, the presence of this microalga in lichen thalli was verified using different microscopic observations. A combination of different techniques, both molecular and microscopic, allowed for the accurate identification of this symbiotic microalga, beforehand mainly known as free living. Here, we suggest the combination of these techniques to prevent incorrect identification in microalgal lichen studies.

Buellia zoharyi is a crustose placodioid lichen, usually occurring on biocrusts of semiarid ecosystems in circum-Mediterranean/Macaronesian areas. In previous work, we found that this lichenized fungus was flexible in its phycobiont choice in the Canary Islands. Here we test whether geography and habitat influence phycobiont diversity in populations of this lichen from the Iberian Peninsula and Balearic Islands using Sanger and high throughput sequencing (HTS). Additionally, three thallus section categories (central, middle and periphery) were analyzed to explore diversity of microalgal communities in each part. We found that B. zoharyi populations hosted at least three different Trebouxia spp., and this lichen can associate with distinct phycobiont strains in different habitats and geographic regions. This study also revealed that the Trebouxia composition of this lichen showed significant differences when comparing the Iberian Peninsula with the Balearics thalli. No support for differences in microalgal communities was found among thallus sections; however, several thalli showed different predominant Trebouxia spp. at each section. This result corroborate that thallus parts selected for DNA extraction in metabarcoding analyses are key to not bias the total phycobiont diversity detected. This study highlights that inclusion of HTS analysis is crucial to understand lichen symbiotic microalgal diversity.

The Canary Islands are famous for their extraordinary biodiversity; however, lichenized algae have only been studied partially. Buellia zoharyi is a circum-Mediterranean/Macaronesian species that usually occurs in semi-arid areas of the Mediterranean, but occasionally some interesting communities of this species grow on basaltic lava flows in Lanzarote, Fuerteventura and Tenerife. Those three locations showed similar ecological conditions, but different mean annual temperatures. Here we applied a multidisciplinary approach to describe microalgae diversity from B. zoharyi covering the entire described range of distribution in the Canary Islands. Photobionts were characterized in symbiosis using molecular and microscopic techniques. Different Trebouxia spp. were detected as primary photobiont in each island (Trebouxia cretacea-Fuerteventura, T. asymmetrica-Lanzarote and Trebouxia sp.`arnoldoi´-Tenerife). Coexistence of various Trebouxia spp. within a thallus were detected by using specific primers-PCR. Those three photobionts were isolated and cultured under laboratory conditions. Different phytohormone profiles were obtained in the isolated strains which suggest different internal signalling needs. In addition, we characterized the response of the isolated strains to different temperatures using chlorophyll fluorescence. T. asymmetrica did not modify their F v /fm values with respect to temperature acclimation. In contrast, Trebouxia sp.`arnoldoi'and T. cretacea were more sensitive to changes in growing temperature decreasing Fv/fm at 17°C. Our results indicate that B. zoharyi is flexible regarding the photobiont choice depending on the region, and suggest that bioclimatic factors could influence the myco/photobiont association patterns.