2.4.1 Single locus species delimitation using the standard fungal DNA barcode
To compare species boundaries inferred using the standard fungal barcoding marker with inferences from genome-scale data, we used Assemble Species by Automatic Partitioning (ASAP; Puillandre et al., 2021). ASAP is a recently developed method that circumscribes species partitions using an implementation of a hierarchal clustering algorithm based on pairwise genetic distances from single-locus sequence alignments (Puillandre et al., 2021). The pairwise genetic distances are used to build a list of partitions ranked by a score, which is computed using the probabilities of groups to define panmictic species. ASAP provides an objective approach to circumscribe relevant species hypotheses as a first step in the process of species delimitation. Therefore, we used ASAP to circumscribe candidate Niebla species from a multiple sequence alignment of the ITS barcoding region. ITS sequences generated for this study were combined with those from Spjut et al. (2020) and aligned using the program MAFFT v7 (Katoh et al. 2005, Katoh and Toh 2008). We implemented the G-INS-i alignment algorithm and ‘1PAM / K=2’ scoring matrix, with an offset value of 0.1, the ‘unalignlevel’ = 0.2, and the remaining parameters were set to default values. The multiple sequence alignment was analyzed using the ASAP Web Server (https://bioinfo.mnhn.fr/abi/public/asap/), with the ‘asap-score’ considered to select the optimal number of species partitions (Puillandre et al.).