Isolation and characterization of melanized fungi from limestone formations in Mallorca

Abstract

Melanized fungi were isolated from limestone surfaces in upland and coastal environments in the Mediterranean island of Mallorca. One hundred seventeen isolates were recovered from two topographically distinct sites. Due to the difficulty in distinguishing among isolates based on morphological criteria, microsatellite-primed PCR techniques were used to group isolates into genotypes that were assumed to represent species. Seventeen genotypes were characterized from one site and twenty six from the other, with four genotypes common to both. Classical and molecular methods were used to identify representative strains. Morphological methods rarely provided a reliable identification; only three isolates, Hortaea werneckii, Trimmatostroma abietis and Aureobasidium pullulans were identified with certainty, and the identification was confirmed by molecular data. Morphological characters that were widespread among the isolates included scarce micronematous conidial states or non-sporulating sterile mycelia, mature mycelia with dark olive green or black hyphae, mycelia with torulous hyphae, whose cells developed one or more transverse septa. In many of these fungi, the cells of mature hyphae disarticulated, suggesting asexual reproduction by a thallic micronematous conidiogenesis or by simple propagative fragmentation. Sequencing of the internal transcribed spacers (ITS1, ITS2) and 5.8S ribosomal gene, as well as the 18S rDNA ribosomal gene were employed to investigate the isolates’ phylogenetic affinities. The majority of the isolates could be grouped in two main classes of Ascomycetes, Dothideomycetes and Chaetothyriomycetes, although many others did not correspond with any sequence deposited in public databases, suggesting they could be of unknown genera that did not correspond with any well-defined Ascomycete order.