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Taxonomic identifications in some groups of lichen-forming fungi have been challenge largely due to the scarcity of taxonomically relevant features and limitations of morphological and chemical characters traditionally used to distinguish closely related taxa. Delineating species boundaries in closely related species or species complexes often requires a range of multisource data sets and comprehensive analytical methods. Here we aim to examine species boundaries in a group of saxicolous lichen forming fungi, the Aspiciliella intermutans complex ( Megasporaceae ), widespread mainly in the Mediterranean. We gathered DNA sequences of the nuclear ribosomal internal transcribed spacer (nuITS), the nuclear large subunit (nuLSU), the mitochondrial small subunit (mtSSU) ribosomal DNA, and the DNA replication licensing factor MCM7 from 80 samples mostly from Iran, Caucasia, Greece and eastern Europe. We used a combination of phylogenetic strategies and a variety of empirical, sequence-based species delimitation approaches to infer species boundaries in this group. The latter included: the automatic barcode gap discovery (ABGD), the multispecies coalescent approach *BEAST and Bayesian Phylogenetics and Phylogeography (BPP) program. Different species delimitation scenarios were compared using Bayes factors species delimitation analysis. Furthermore, morphological, chemical, ecological and geographical features of the sampled specimens were examined. Our study uncovered cryptic species diversity in A . intermutans and showed that morphology-based taxonomy may be unreliable, underestimating species diversity in this group of lichens. We identified a total of six species-level lineages in the A . intermutans complex using inferences from multiple empirical operational criteria. We found little corroboration between morphological and ecological features with our proposed candidate species, while secondary metabolite data do not corroborate tree topology. The present study on the A . intermutans species-complex indicates that the genus Aspiciliella , as currently circumscribed, is more diverse in Eurasia than previously expected.

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Background Due to their huge biodiversity and the capability to produce a wide range of secondary metabolites, lichens have a great potential in biotechnological applications. They have, however, hardly been used as cell factories to date, as it is considered to be difficult and laborious to cultivate lichen partners in pure or co-culture in the laboratory. The various methods used to isolate lichen fungi, based on either the ascospores, the conidia, or the thallus, have so far not been compared or critically examined. Therefore, here we systematically investigate and compare the known methods and two new methods to identify the most suitable technology for isolation of fungi from lichens. Results Within this study six lichen fungi species were isolated and propagated as pure cultures. All of them formed colonies within one month. In case of lichens with ascocarps the spore discharge was the most suitable method. Spores were already discharged within 2 days and germinated within only four days and the contamination rate was low. Otherwise, the soredia and thallus method without homogenization, as described in this work, are also well suited to obtain pure fungal cultures. For the isolation of algae, we were also successful with the thallus method without homogenization. Conclusion With the methods described here and the proposed strategic approach, we believe that a large proportion of the lichen fungi can be cultivated within a reasonable time and effort. Based on this, methods of controlled cultivation and co-cultivation must now be developed in order to use the potential of lichens with regard to their secondary metabolites, but also for other applications.

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