## 2 Materials and Methods
### Genome Annotation  
The publicly available genomes were sequenced with different sequencing technologies at different times and are thus of varying quality. To make annotations comparable, we performed ab initio gene calling and functional annotations for all of them. The downloaded genomes were analyzed using the smsi-funannotate ([[https://github.com/reslp/smsi-funannotate](https://github.com/reslp/smsi-funannotate)]([https://github.com/reslp/smsi-funannotate](https://github.com/reslp/smsi-funannotate)); git commit 398a144; accessed on 4 June 2021) pipeline based on funannotate v.1.8.7 [39]. First, we removed duplicated identical contigs (funannotate clean) in each assembly. To avoid long contig/scaffold names, we sorted our assembly by contig length and then renamed the fasta headers (funannotate sort). Afterwards, we made a soft repeat masking of the assembly using tantan (funannotate mask) [40]. The following steps included gene prediction using the gene-callers Augustus v3.3.2 [41], snap [42], GlimmerHMM v3.0.4 [43] and Genemark ES v4.68 [44]. For Augustus, we used _Aspergillus nidulans_ as a pre-trained species. All the pipelines were run on the cluster of the University of Graz and our in-house Linux Server at LMU Munich. The genome statistics are summarized in Table 4 (See Section 3.2. Biosynthetic Gene Composition in Twenty-Three Annotated Fungal Genomes). The output files of the newly annotated genomes from NCBI and JGI used in this study are 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))).  
\begin{table}
\begin{tabular}{l c c c c} \hline \hline
**Nuclear Genome** & _Bacidia rubella_ & _Bacidia gigantensis_ & _Ramalina intermedia_ & _Ramalina pernuiana_ \\ \hline
**Substrate** & bark & bark & rock & twig \\ \hline
**Growth form** & crustose & crustose & fruticose & fruticose \\ \hline
**Source** & Axenic culture & Whole thallus & Whole thallus & Axenic culture \\ \hline
**Sequencing method** & Illumina NovaSeq SP; & PromethION 24 & Illumina MiSeq & Illumina MiSeq \\ \hline
**Raw reads produced** & 17 Gbp; 22 Gbp & 32 Gbp & 13.33 Gbp & - \\  & (roughly) & & & \\ \hline
**Coverage** & 500\(\times\) & 500\(\times\) & 290\(\times\) & 2.0\(\times\) \\ \hline
**Assembly size (Mb)** & 33.52 & 33.11 & 26.19 & 25.53 \\ \hline
**Largest scaffold (bp)** & 2,353,056 & 3,530,911 & 898,913 & 694,821 \\ \hline
**Average scaffold (bp)** & 657,358 & 1,379,912 & 148,821 & 25,764 \\ \hline
**Number of scaffolds** & 51 & 24 & 176 & 991 \\ \hline
**N50** & 1,771,855 & 1,807,239 & 282,362 & 43,940 \\ \hline
**GC (\%)** & 45.28 & 44.67 & 51.90 & 50.58 \\ \hline
**Num of predicted genes** & 8773 & 8451 & 7405 & 6756 \\ \hline
**Num of predicted proteins** & 8728 & 8400 & 7355 & 6706 \\ \hline
**Number of unique proteins** & 2514 & 2343 & 1099 & 1088 \\ \hline
**tRNAs** & 45 & 51 & 50 & 50 \\ \hline
**Proteins with at least** & 5860 & 5711 & 6111 & 5467 \\
**one ortholog** & 2345 & 2345 & 2345 & 2345 \\ \hline
**Secreted proteins (SignalP)** & 678 & 612 & 464 & 384 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Starting material, sequencing strategy and genome quality statistics of Ramalinaceae genomes analyzed in this study.