Molecular phylogeny and ultrastructure of the lichen microalga Asterochloris mediterranea sp. nov. from Mediterranean and Canary Islands ecosystems.

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 Škaloud. 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.