UnFATE: A Comprehensive Probe Set and Bioinformatics Pipeline for Phylogeny Reconstruction and Multilocus Barcoding...

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 (WGS) 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 Universal Filamentous Ascomycete Target Enrichment (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 Internal transcribed spacer (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 (normalized Robinson-Foulds) 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 10 representative classes in Pezizomycotina, which recovered a topology 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.