## 2 Materials and Methods
### Type I Iterative PKS Alignment  
We performed a phylogenetic analysis of all TI-PKSs identified in twenty-three studied genomes of Lecanoromycetes, to assess the relationship of non-reducing PKSs (NR-PKS) and reducing PKSs (R-PKS) (including partly reducing PKSs). First, we prepared a list of all identified TI-PKS based on our functional annotations. We retrieved the sequences based on the corresponding gene ID numbers included in the gff3 file of each genome using a custom python script (select_transcript.py: [[https://github.com/reslp/genomics/blob/master/select_transcripts.py](https://github.com/reslp/genomics/blob/master/select_transcripts.py)]([https://github.com/reslp/genomics/blob/master/select_transcripts.py](https://github.com/reslp/genomics/blob/master/select_transcripts.py)); accessed on 13 October 2021). We included predicted  
Figure 1: Two alternative topologies used for comparison in the topology test. Topology 1, recovering _Bacidia_ + Cladoniaceae as monophyletic; Topology 2, recovering Cladoniaceae + Parmeliaceae as monophyletic.  
ketoacyl synthase (KS) amino acid alignment reported by [PERSON] et al. [14] to enhance our dataset. The inclusion of reference PKSs enables us to compare the tree topology proposed in [PERSON] et al. [14] to our larger sampling of putative PKS genes.  
All TI-PKS sequences were combined into a single file and aligned using MAFFT v7.480 [47]. We chose the E-INS-i alignment strategy because it performs better when aligning sequences with several conserved motifs interspersed in long, unalignable regions. We trimmed the alignment using trimAl v.1.4.rev15 [58]. Initial testing of different trimming settings (e.g., -gappyout, -strict, and -automated), showed that parameter combination (-gt 0.70 -resoverlap 0.70 -seqoverlap 60) is a good trade-off between removing ambiguously aligned sites and keeping phylogenetically informative sites of the PKS sequences. The final data matrix consisted of 624 amino acid sequences with 1049 amino acid sites, 1049 distinct patterns, and 1043 parsimony-informative sites from 61 taxa (23 studied genomes and 38 other fungal and bacterial taxa from [PERSON] et al. [14]). In addition, PKS23 from _Stereoacuoln aluminum_ (QXF68953) and PKS16 from _Cladonia grayi_ (ADM79459) from NCBI were included. The alignment is available on figshare ([[http://doi.org/10.6084/m9.figshare.19487837](http://doi.org/10.6084/m9.figshare.19487837)]([http://doi.org/10.6084/m9.figshare.19487837](http://doi.org/10.6084/m9.figshare.19487837))).  
We selected the best-fitting substitution model (LG+F+G4) according to the Akaike and Bayesian Information Criteria using the ModelFinder implemented in IQ-TREE [49] on our TI-PKS alignment. We calculated a phylogenetic tree using maximum-likelihood (ML) analysis implemented in IQ-TREE v2.1.4 [48], with 1000 ultrafast bootstrap replicates [59]. We also calculated a tree using RAxML-NG v1.0.3 [60] with parameters -all -bs-trees 100 -model LG+F+G4 -threads 16 -data-type AA. The inferred phylogenetic tree was then rooted with Bacterial Type II polyketide sequences using phyx [61].  
To compare the congruence of the trees, we utilized Dendroscope v3.7.6 [62] using Tanglegram (Algorithms). The resulting tree was visualized using FigTree 1.3.1 [55] and a custom R script, with additional annotations added in Adobe Illustrator v24.0.3.