Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

• Klíčová slova
• Laboulbenia, Laboulbeniales, Barcoding, DNA extraction, PCR amplification,
• Publikační typ
• časopisecké články MeSH

Fungi are among the least known organisms on earth, with an estimated number of species between 1.5 and 10 million. This number is expected to be refined, especially with increasing knowledge about microfungi in undersampled habitats and increasing amounts of data derived from environmental DNA sequencing. A significant proportion of newly generated sequences fail to match with already named species, and thus represent what has been referred to as fungal "dark taxa". Due to the challenges associated with observing, identifying, and preserving sporophores, many macro- and microfungal species are only known from a single collection, specimen, isolate, and/or sequence-a singleton. Mycologists are consequently used to working with "rare" sequences and specimens. However, rarity and singleton phenomena lack consideration and valorization in fungal studies. In particular, the practice of publishing new fungal species names based on a single specimen remains a cause of debate. Here, we provide some elements of reflection on this issue in the light of the specificities of the fungal kingdom and global change context. If multiple independent sources of data support the existence of a new taxon, we encourage mycologists to proceed with formal description, irrespective of the number of specimens at hand. Although the description of singleton-based species may not be considered best practice, it does represent responsible science in the light of closing the Linnean biodiversity shortfall.

• Klíčová slova
• Integrative taxonomy, Morphology, Rarity, Sequencing, Singletons, Species descriptions,
• Publikační typ
• časopisecké články MeSH