###### Abstract  
Fungi involved in lichen symbioses produce a large array of secondary metabolites that are often diagnostic in the taxonomic delimitation of lichens. The most common lichen secondary metabolites--polyketides--are synthesized by polyketide synthases, particularly by Type I PKS (TI-PKS). Here, we present a comparative genomic analysis of the TI-PKS gene content of 23 lichen-forming fungal genomes from Ascomycota, including the de novo sequenced genome of _Bacidia rubella_. Firstly, we identify a putative atranorin cluster in _B. rubella_. Secondly, we provide an overview of TI-PKS gene diversity in lichen-forming fungi, and the most comprehensive Type I PKS phylogeny of lichen-forming fungi to date, including 624 sequences. We reveal a high number of biosynthetic gene clusters and examine their domain composition in the context of previously characterized genes, confirming that PKS genes outnumber known secondary substances. Moreover, two novel groups of reducing PKSs were identified. Although many PKSs remain without functional assignments, our findings highlight that genes from lichen-forming fungi represent an untapped source of novel polyketide compounds. l +
Footnote †: journal: Fungi  
comprises non-reducing (NR) PKSs, which lack reductive domains and mainly produce aromatic polyketides [13; 15]. The second subgroup contains partially reducing (PR) PKSs, typically having a single KR or a KR and DH domain. Finally, reducing (R) PKSs contain a complete set of reductive domains, viz. KR, DH, and ER. All three subgroups can be found in lichen-forming fungi. Previous work suggests various ecological roles of polyketides in lichens ranging from light-screening and chemical weathering to allebotatic effects and herbivore defense [3; 6; 16; 17]. More broadly, secondary metabolite profiles including polyketides are often characteristic of taxonomic groups and are thus extensively used to identify lichens. The first PKS gene from a lichen-forming fungus was cloned and analyzed by [PERSON] et al. in 2011 [18], and in recent years secondary metabolite research has benefited from genome mining approaches in bacteria, fungi, and plants, uncovering hidden diversity (e.g., [19; 20; 21]). The fact that genes encoding natural product biosynthetic pathways are often clustered in the genome [2] facilitates the identification of biosynthetic gene clusters (BGCs) in whole genome sequences. In many cases, the chemical structure of their products can be predicted from the biosynthetic logic of enzymes encoded in a BGC and their similarity to known counterparts [22; 23; 24; 25]. Despite this progress, the biosynthetic genes for most secondary metabolites of lichen-forming fungi remain uncharacterized. Observations that lichen-forming fungi contain more biosynthetic genes than characterized chemical compounds also complicates clear gene assignments to particular metabolites. Consequently, previous studies have focused only on a few well-known compounds, e.g., usnic acid, grayanic acid, and atranorin (e.g., [25; 26; 27; 28; 18; 29]). However, the increasing availability of lichen-forming fungal genomes provides a largely untapped resource for identifying additional biosynthetic genes [30]. Here, we investigate the diversity of BGCs in the de novo sequenced _Bacidia rubella_ (Hoffm.) [PERSON]. (Ramalinaceae) genome within a two-level comparative genomic framework. We aim to identify genes involved in the biosynthesis of atranorin, a secondary metabolite present in many lichens including _B. rubella_. To achieve this, we examine the secondary metabolite biosynthetic potential of _B. rubella_ by comparing its genome to that of Ramalinaceae species: _Bacidia gigantensis, Ramalina intermedia_ and _R. peruviana_. The secondary metabolites of these four closely-related species are known, and the chemical substance profiles overlap. This facilitates comparison of the presence, absence and structure of PKS genes, and enables the evaluation of BGCs for previously characterized genes, including those encoding for atranorin biosynthesis.