## 3 Results and Discussion
### General Characteristics of De Novo Bacidia rubella Genome
#### 3.1.1 Atranorin  
Atranorin is the main secondary compound known in _B. rubella_[64], and its biosynthetic pathway has received attention in previous studies on lichen-forming fungi [26, 29]. Our phylogenetic results--which included previously identified sequences of genes involved in atranorin production from _Cladonia_ species and _Stereocaulon alpinum_--revealed a putative PKS23 homolog in _B. rubella_. This is consistent with the phylogenetic investigation of [PERSON] et al. [29], where PKS23 sequences were reported to group together with sequences from atranorin-producing lichen-forming fungi (see Section 3.3 Type I PKS phylogeny). The domain configuration of _B. rubella_ PKS23 also supports our phylogenetic results, showing the same organization of putative atranorin BGC in _B. rubella_ as has been reported for _Cladonia rangiferina_ (Figure 2). The _B. rubella_ PKS23 cluster contains a cytochrome P450 domain (_atr_2) required for oxidation, as well as an O-methyltransferaseerase (OMT) domain (_atr_3) and transporter gene (_atr_4), which are involved in atranorin biosynthesis [29]. A BLASTp search of the OMT domain (_atr_3) in _B. rubella_ PKS23 resulted in 36% protein sequence identity to Trt5 (UniProtKB accession no. QQC8A3). This level of similarity is consistent with previous findings by [PERSON] et al. [29] for C. _rangiferina_ PKS23. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline
**Species** & **Species** & **Genome** & **No of** & **Type I** & **Type I** & **Type I** & **PR-PKS** & **Hybrid** & **NRPS/** & **Metabolites** \\
**(NCBI)** & **Tag** & **(Mb)** & **Clusters** & **(Total)** & **NR-PKS** & **R-PKS** & **R-PKS** & **NRPS** & **NRPS** & **NRPS** & **NRPS** \\ \hline \hline \end{tabular} \begin{tabular}{c c c c c c c c c c c} \hline _Letharia lupina_ (Leup\_v1.1) & **letluppred** & 49.2 & 48 & 18 & 11 & 7 & 0 & 2 & 3/11 & \begin{tabular}{c} Vulinic acid and **atranorin** in the \\ cortex, with \\ hypernium of the \\ apotheca \\ \end{tabular} \\ \hline _Pentevernia_ (Pfur\_oli\_TBG\_2151) & psefurpred & 37.8 & 48 & 26 & 8 & 18 & 0 & 3 & 3/11 & \begin{tabular}{c} **Atranorin**, physocid \\ acid and \\ oxyphysodic acid \\ \end{tabular} \\ \hline _Ramulina internedia_ (RamVal02\_v1.0) & ramintrepred & 26.2 & 54 & 31 & 13 & 17 & 1 & 3 & 5/9 & \begin{tabular}{c} Usanic acid (major); \\ medulla with \\ homosekikic acid \\ (major), 4’-O- \\ methylnornowcesikikic acid (minor) \\ \end{tabular} \\ \hline _Ramulina peruviana_ (RamVal01\_v1) & ramperpred & 25.5 & 43 & 17 & 9 & 7 & 1 & 1 & 4/10 & \begin{tabular}{c} Usanic acid, \\ homosekikic acid \\ (major), 4’-O- \\ methylnornowcesikikic acid \\ \end{tabular} \\ \hline _Lysnen haknensis_ (Uhk\_1.0) & usnhakpred & 40.4 & 70 & 23 & 10 & 12 & 1 & 3 & 5/22 & \
\begin{tabular}{c} Usanic and nonstictictic \\ acids \\ \end{tabular} \\ \hline _Xantharia elegans_ (ASMI13630v1) & xaneleppred & 44.2 & 63 & 25 & 7 & 18 & 0 & 1 & 7/17 &
\begin{tabular}{c} Parieitin (major), \\ fallacinal, emodin, \\ teloschistian and \\ parietilina acid \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Cont._The newly identified, putative PKS23 sequence of _B. rubella_ was recovered as a sister to PKS23 sequences from _C. rungiferina_ and _S. alipinum_, suggesting a sister-group relationship of _Bacidia_ with Cladoniaceae (See Figure 3 in Section 3.3: Type I PKS Phylogeny), whereby this clade formed a sister-group to the PKS23 sequences from Parmeliaceae (schematically shown in Figure 1). This is in contrast to the family-level taxonomy previously established, e.g., by [PERSON] et al. [66] and [PERSON] et al. [50], where Cladoniaceae is more closely related to Parmeliaceae than to Ramalinaceae. To test the monophyly of the clade comprising _Bacidia_ + Cladoniaceae compared to a group comprised of Parmeliaceae and Cladoniaceae, we performed a phylogenetic tree topology test on the PKS23 dataset.