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

Abstract

Background: The delimitation of species of Tulasnella 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 Tulasnella.

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 Tulasnella.

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 Tulasnella spp., where all 142 Tulasnella sequences were divided into two main clades A and B and assigned to seven species (T. asymmetrica, T. andina, T. eichleriana ECU6, T. eichleriana ECU4 T. pinicola, T. violea), 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, T. albida belongs to type I; T. eichleriana, T. tomaculum, and T. violea belong to type II; and T. asymmetrica, T. andina, T. pinicola, and T. spp. (GER) belong to type III; each type contains four to six domains, with nine CBCs among these that corroborate different species.

Keywords: ITS2; alignment-based; alignment-free; compensatory base changes; secondary structure; species delimitation.

Figure 1

Flowchart summary to delimit species boundaries in Tulasnella genus. (i) Download Tulasnella sequences with total region ITS-5.8S from Genbank database [31]; ITS2 region extraction with ITSx v1.0.11 software [17,32]; (ii) Obtain two distance matrices from Alignment-based and Alignment-free; (iii) Combine the distance matrices by Distatist [9], (iv) Plot the resulting phylogenetic tree using the pvclust library (R package) [34]; (v) Secondary structure modelling and; (vi) Define the CBCs as additional molecular markers to delimit Tulasnella species.

Figure 2

(a) Uncultured Tulasnellaceae (Genbank FJ786646) secondary structure for the 26 partial ITS-5.8, sequence clone PA195. Helices were numbered I–IV from 5′ to 3′ direction (b) Secondary structure of the ITS2 transcript of Colletotrichum gloeosporioides (Genbank AF444327).

Figure 3

ITS2 sequence structure-based alignment generated by LocaRNA-P. Dot-bracket notation represents unpaired bases and matching parenthesized positions represent paired bases. The red regions indicate structure reliability, the yellow regions represent sequence reliability, and the thin line shows the combined column-reliability.

Figure 4

(a). AB tree built with Opal [42] and MEGA [38] tools, showing 11 clades, and (b). AF tree built with VRNAdistance [37] and MEGA [38] tools, with 9 clades. AB tree matrix carries information about the evolutionary changes in the ITS2 sequences and the AF matrix carries information about changes in their secondary structure.

Figure 5

The M-GMYC phylogenetic tree displays the Tulasnella species division into different clades and subclades distinguished by colors: T. violea (in red), T. eichleriana (in grey), T. tomaculum (in green), T. sp_GER (in yellow), T. pinicola (in light blue), T. albida (in grey), T. asymmetrica (in blue), and T. andina (in brown), with two main monophyletic clades (A) (A1,A2) and (B) (B1,B2[I–III],B3). (A1,A2,B1,B2-I,B2-II,B2-III,B3) represent monophyletic clades identified by M-GMYC analysis using the R tool (see Table 1).

Figure 6

A consensus secondary structures of ITS2 based on minimum free energy (MFE). The colors indicate structural conservation according to the key in the figure, where the homologous regions highlighted in red contain the highest probability of compatibility, with a radiating central and internal loop interconnected with unpaired nucleotides (colorless) in the helices.