So far, phycobiont diversity in lichen thalli has been mainly investigated by Sanger sequencing; this traditional technique may fail to detect the less represented photobionts and may lead to an underestimation of the whole microalgal diversity. Recently, lichen-associated phycobiont communities within several lichen spp. were tested using environmental DNA metabarcoding approaches; however, the crustose terricolous lichen species has not been explored in depth. Buellia zoharyi Galun is a circum-Mediterranean/ Macaronesian lichen species that usually occurs in gypsum biocrusts in semi-arid areas. In this study, 153 thalli of B. zoharyi, collected from 18 geographical populations, were analysed to assess microalgal diversity both by Sanger and high-throughput sequencing (HTS). Selected fragments from different parts of each thallus were randomly excised and pooled together (MIX). To confirm the identity of the primary phycobiont in all of the 153 lichen, two algal loci were amplified. In all the samples, three Trebouxia species were recognized: Trebouxia cretacea, Trebouxia asymmetrica and Trebouxia sp. OTUA25. Moreover, 36 selected MIX_DNAs from different locations were sequenced using Illumina. To corroborate the hypothesis of differential distribution of the microalgae throughout the specimens, eight thalli were further divided into three parts: central, middle and periphery and then analysed by HTS. A total of 21 Trebouxia lineages were recorded from the 60 samples studied. T. cretacea and Trebouxia sp. OTUS52 showed the highest number of reads. To illustrate differences in the composition of Trebouxia microalgae between locations and regions, ? and ß-diversity indexes were conducted. The Trebouxia communities of thalli collected from the Iberian Peninsula vs Balearic Islands showed significant dissimilarities. Regarding whether the central, middle and peripheral portions of thalli hosted different Trebouxia communities, no support was found for differences in microalgal communities among the three thallus portions; however, six thalli showed nonidentical predominant Trebouxia spp. within each part. Funding: PROMETEO/2017/039
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