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.).