DOI: 10.5061/dryad.tht76hf1x
title: UnFATE: A comprehensive probe set and bioinformatics pipeline for phylogeny reconstruction and multilocus barcoding of filamentous ascomycetes (Ascomycota, Pezizomycotina)

description: The subphylum Pezizomycotina (filamentous ascomycetes) is the largest
 clade within Ascomycota. Despite the importance of this group of fungi,
 our understanding of their evolution is still limited due to insufficient
 taxon sampling. Although next-generation sequencing technology allows us
 to obtain complete genomes for phylogenetic analyses, generating complete
 genomes of fungal species can be challenging, especially when fungi occur
 in symbiotic relationships or when the DNA of rare herbarium specimens is
 degraded or contaminated. Additionally, assembly, annotation, and gene
 extraction of whole-genome sequencing data require bioinformatics skills
 and computational power, resulting in a substantial data burden. To
 overcome these obstacles, we designed a universal target enrichment probe
 set to reconstruct the phylogenetic relationships of filamentous
 ascomycetes at different phylogenetic levels. From a pool of single-copy
 orthologous genes extracted from available Pezizomycotina genomes, we
 identified the smallest subset of genetic markers that can reliably
 reconstruct a robust phylogeny. We used a clustering approach to identify
 a sequence set that could provide an optimal trade-off between potential
 missing data and probe set cost. We incorporated this probe set into a
 user-friendly wrapper script named UnFATE
 (https://github.com/claudioametrano/UnFATE) that allows phylogenomic
 inferences without requiring expert bioinformatics knowledge. In addition
 to phylogenetic results, the software provides a powerful multilocus
 alternative to ITS-based barcoding. Phylogeny and barcoding approaches can
 be complemented by an integrated, pre-processed, and periodically updated
 database of all publicly available Pezizomycotina genomes. The UnFATE
 pipeline, using the 195 selected marker genes, consistently performed well
 across various phylogenetic depths, generating trees consistent with the
 reference phylogenomic inferences. The topological distance between the
 reference trees from literature and the best tree produced by UnFATE
 ranged between 0.10 and 0.14 (nRF) for phylogenies from family to
 subphylum level. We also tested the in vitro success of the universal
 baits set in a target capture approach on 25 herbarium specimens from ten
 representative classes in Pezizomycotina, which recovered a topology
 mostly congruent with recent phylogenomic inferences for this group of
 fungi. The discriminating power of our gene set was also assessed by the
 multilocus barcoding approach, which outperformed the barcoding approach
 based on ITS. With these tools, we aim to provide a framework for a
 collaborative approach to build robust, conclusive phylogenies of this
 important fungal clade.
description: # UnFATE: A Comprehensive Probe Set and Bioinformatics Pipeline for
 Phylogeny Reconstruction and Multilocus Barcoding of Filamentous
 Ascomycetes (Ascomycota, Pezizomycotina) The repository includes the
 representative sequences of the UnFATE 195 genes and the baits designed
 from them, the single locus trees, alignments and final phylogenies for
 the proof of concept Pezizomycotina phylogeny inferred using the universal
 probe set and the pipeline we developed (files ending in
 "Pezizo_pilotTE"). It also includes the supermatrices and single
 locus alignments generated by mining the 195 genes of our gene set from
 publicly available genome, used in published phylogenomic inferences. ---
 **File description:** the following **tar.gz** files contain the the
 reference sequences (unfate_markers_reference_sequences_DNA.tar.gz)
 obtained from the clustering approach adopted to find the best
 representative sequences to build the universal bait set (baits.tar.gz).
 See Ametrano et al. 2025 (Systematic Biology journal) for comprehensive
 methods. **File list:** * unfate_markers_reference_sequences_DNA.tar.gz *
 baits.tar.gz --- **File description:** the following **tar.gz** files
 contain the analysis performed using the UnFATE pipeline on existing
 genome assembly datasets used in previously published phylogenomic
 inferences (e.g. Parmeliaceae metagenome dataset from Pizarro et al.,
 2019). Both the single locus alignments and the concatenated
 (block-filtered) alignments are provided. In single locus alignment
 archives: "AA" stands for amino-acid "NT" for
 nucleotides, if the -gb suffix is present, the alignments were block
 filtered with Gblocks. "macsed" stands for aligned using MACSE2
 pipeline, the beginning of the file name describes the gene name and the
 taxon code from OrthoDB (e.g., 1627at4890). In supematrix archives: FcC
 stands for Fastconcat (the tool used to perform concatenation).
 "AA" stands for amino-acid "NT" for nucleotides, if
 the -gb suffix is present, the alignments were block filtered with
 Gblocks. **File list:** *
 Aspergillus_Penicillium_Steenwyk_2019_supermatrices.tar.gz *
 Aspergillus_Penicillium_Steenwyk_2019_single_locus_alignments.tar.gz *
 Dothideomycetes_Haridas_2020_supermatrices.tar.gz *
 Dothideomycetes_Haridas_2020_single_locus_alignments_gblocked.tar.gz *
 Parmeliaceae_Pizarro_2019_single_locus_alignments_gblocked.tar.gz *
 Parmeliaceae_Pizarro_2019_supermatrices.tar.gz *
 Pezizomycotina_Li_2021_single_locus_alignments.tar.gz *
 Pezizomycotina_Li_2021_supermatrices.tar.gz --- **File description:** the
 following **tar.xz** files contain the analysis performed using the UnFATE
 pipeline on the newly generated Pezizomycotina target enrichment (TE in
 the file name) data captured with the universal bait set we developed (see
 the manuscript). Single locus alignments, supermatrices (DNA and AA),
 single locus trees and final trees (Astral DNA and AA phylogeny,
 Maximum-Likelihood DNA and AA) are provided. **File list:** *
 **Supermatrix_Pezizo_pilotTE.tar.xz**: in the archive there are the two
 supermatrix folders (amino-acids and nucleotides) in each of them there
 all the files produced by Fasconcat (supermatrix, partition file and
 statistics) and the subsequent IQTREE2 ML run using the supermatrix itself
 (see IQTREE2 manual for additional details). *
 **Final_trees_Pezizo_pilotTE.tar.xz**: within the archives, species trees
 produced by Astral summary method have "astralspeciestree" as
 prefix, the species trees from concatenation and ML inference with IQTREE
 have "FcC_supermatrix*_partition" as prefix, "blocked"
 means that alignment were blocked before concatenation or single tree
 inference, "AA" or "aa" stands for amino-acid,
 "dna" or "NT" stands for nucleotides.
 "SPECIES_NAME" is in the tree files where the accession numbers
 are complemented with species name (if the sample is from an NCBI
 assembly). Files whose name starts with "Accession" contain the
 taxonomic information about the species name present in the tree files. *
 **Alignments_Pezizo_pilotTE.tar.xz**: within the archive "AA"
 stands for amino-acid "NT" for nucleotide, "trimal"
 means alignments were block filtered with TrimAl. "macsed"
 stands for aligned using MACSE2 pipeline, the beginning of the file name
 describes the gene name and the taxon code from OrthoDB (e.g.,
 1627at4890). "Cleaned" is reported in the name of fasta files
 where sequences with &gt; 75% missing data (gaps) were deleted. *
 **Single_locus_trees_Pezizo_pilotTE.tar.xz**: contains the IQTREE2 output
 for each single locus inference performed to generate single gene trees
 (see IQTREE2 manual for additional details).