Compensatory Base Changes in ITS2 Secondary Structure Alignment, Modelling, and Molecular Phylogeny: An Integrated Approach to Improve Species Delimitation in (Basidiomycota).

Background: The delimitation of species of has been extensively studied, mainly at the morphological (sexual and asexual states) and molecular levels-showing ambiguity between them. An integrative species concept that includes characteristics such as molecular, ecology, morphology, and other information is crucial for species delimitation in complex groups such as .

Objectives: The aim of this study is to test evolutionary relationships using a combination of alignment-based and alignment-free distance matrices as an alternative molecular tool to traditional methods, and to consider the secondary structures and CBCs from ITS2 (internal transcribed spacer) sequences for species delimitation in .

Methodology: Three phylogenetic approaches were plotted: (i) alignment-based, (ii) alignment-free, and (iii) a combination of both distance matrices using the DISTATIS and pvclust libraries from an R package. Finally, the secondary structure consensus was modeled by Mfold, and a CBC analysis was obtained to complement the species delimitation using 4Sale.

Results And Conclusions: The phylogenetic tree results showed delimited monophyletic clades in spp., where all 142 sequences were divided into two main clades A and B and assigned to seven species (, , , , ), supported by bootstrap values from 72% to 100%. From the 2D secondary structure alignment, three types of consensus models with helices and loops were obtained. Thus, belongs to type I; , , and belong to type II; and , , , and spp. (GER) belong to type III; each type contains four to six domains, with nine CBCs among these that corroborate different species.