## 3 Results and Discussion
### Groups NR-III and NR-IV  
The sister groups NR-III and NR-IV contain sequences of proteins producing large polyktetide chains, such as conidial yellow pigment Alb1 (NR-III) or aflatoxin/ sterigmatocystin of _A. nidulans_ (NR-IV) [14; 71; 82]. NR-III additionally contains characterized PKSs producing duclauxin (35-71% similarity) and naphthopyrone (100% similarity). Duclauxins are dimeric, heptacyclic fungal polyktetides with various activities [83], while bis-naphthopyrones act in herbivore defense in filamentous ascomycetes [84]. Several genes recovered in the NR-IV group are potentially involved in producing different fungal pigments. The genes from _Ramalina intermedia_ (ramintpred_005964) and _Cladonia grayi_ (clagrapred_006320) were assigned to an FSR1 gene, which is involved in producing highly pigmented naphthoquinones fusarubins responsible for fruiting body coloration of _Fusarium fujikuroi_[85]. A BLAST search of the _R. intermedia_ gene (ramintpred_005964) revealed 72.3% similarity to a PKS of _Cladonia metacoralifera_ (GenBank: QIX11499) that is involved in the biosynthesis of the red compound cristazarin, characterized by antibacterial and antitumor activity [86]. Sequences of _Bacidia rubella_ and several _Cladonia_ species producing red pigments were recovered sister to the putative _Ramalina intermedia FSR1_ gene. Interestingly, _R. intermedia_, at the same time, lacks red pigments [87]. This close phylogenetic proximity raises the possibility that combinations of substances synthesized by different PKS genes could be responsible for red-colored pigments (see also discussion on monascorburn above), particularly for the red fruiting bodies in _B. rubella_. According to the previous studies, NR-V contains PKSs without the TE domain [71]; however, in the characterized PKSs in our phylogeny, the TE or R domains were present in all clades. The genes in the NR-V group are suggested to be involved in the production of different mycotoxins, such as desertorin (_Aspergillus nidulans_) and atrochrysone (_Aspergillus fumigatus_) [71; 88; 89]. The crown clades in the NR-V group include the annotated PKS81 orthologs from _Cladonia metacorallifera_, _C. macilenta_, _Lasalia pustulata_ and _L. hispanica_ as sister to a PKS19 sequence from _Cochliobolus heterostrophus_ and two clades with characterized Agnpks1 also from several lichen-forming fungi. Agnpks1 (Atrochrysone carboxylic acid synthase) was originally described from _Penicillium divaricatum_ and is involved in the biosynthesis of agnestins and dihydroxy-xanthone metabolites [90]. Xanthones and related benzophenones are produced by various filamentous fungi. They exhibit insecticide, antioxidant, antibacterial, anti-inflammatory, and anticancer activities [91]. Examples include desmethyl-sterigmatocystin, a key intermediate of the aflatoxin group of mycotoxins produced by _Aspergillus flavus_, and a norlichexanthone from the lichen-forming _Lecanora straminea_[90]. An additional BLAST search of norlichexanthone synthase from _L. straminea_ (GenBank: D7 PI15) against our annotated fungal sequences showed at least 37% similarity to sequences from the clade containing PKS81 and Agnpsks1. The relationship between these two genes remains unclear, but given our results, PKS81 might belong to or have evolved from Agnps1. Additional closer investigations will be necessary to show the role of PKS81 and Agnpsk1 orthologs in lichen-forming fungi and their possible role in xanthone biosynthesis. Subclade II of NR-PKSs (Including Groups NR-VI, NR-VII, and NR-IX)  
The second largest clade of NR-PKSs includes the previously recovered groups NR-VI, NR-VII, and the recently defined group NR-IX [29] (Figure 3). The generalized domain arrangement in these groups is SAT-KS-AT-DH-ACP-[ACP-][HTH]-CMeT-[ADH/NAD]-[Peptides9], deviating from the generalized arrangement for other NR-PKS (see above). Although previously reported from all NR-PKS groups except group NR-V [69; 71], we did not observe TE domains in groups NR-VI, NR-VII, and NR-IX.