# Trepenoid Biosynthesis Dominates among Secondary Metabolite Clusters in _Mucoromycotina_ Genomes
## 3 Results and Discussion
### SMC Sequence Clustering  
A network of SMC nodes, created on basis of the MultiGeneBlast all-against-all search, showed the relatedness of SMCs belonging to one type and the interconnectedness of SMCs without prior categorisation (Figure 3; for MultiGeneBlast all-against-all scores, see Supplementary Table S8). While only a few SMCs lacked clear similarity to any other SMC (\(n=3\)), this similarity was contingent on only a single matching gene in a further 176 cases and thus would be undetectable with a default MultiGeneBlast run (this was particularly frequent among siderophore biosynthetic regions, where 59 out of 155 showed no other genes in the matching region). In general, the similarity of core genes was the main driver for cluster similarity (a tendency also corroborated by the results of candidate family analysis, where there were relatively few large gene families shared by multiple SMCs). Based on AntiSmash labelling, the SMC similarity network (Figure 3A) was dominated by terpene (\(n=503\), including 10 hybrid clusters) and NRPS-like SMCs (\(n=421\); 16 hybrid clusters). There were at least five major SMCs groups visible in the network, corresponding to: siderophores, NRPS and NRPS-like, and two related to terpen. All of them are formed mainly by _Mucorales_ sequences. NRPS-like sequences formed two distant groupings connected to the network of terpene SMCs and a separated NRPS cluster formed by _Morierellales_ sequences. Siderophore sequences grouped together, forming a well-separated cluster. Bacteriocins formed a separate subgraph, whereas PKS, betalactone and fungal-RiPP SMCs were located in the centre of the network, interconnected with terpene and NRPS clusters. Notably, the identity of visible SMC groups was properly refined by parallel annotation of the candidate gene families (Figure 3B), where conservation of core genes such as squalene synthase homologues was shown to explain the visible groupings (Figure 3B). Notably, in the unsupervised Markov Cluster Algorithm (MCL) (see Supplementary Table S2 for mapping of candidate families to individual clusters) clustering of all proteins encoded by constituent SMC member genes, no unambiguous core genes were detected in 141 cases. While the clusters as a whole passed the rule-based check of signature activities (e.g., presence of condensation and adenylation domains), an inspection of individual gene annotations (see Supplementary Table S5) typically pointed to distantly related gene families. For example, in NRPS-like clusters, the fatty acyl-AMP ligases (MCL0047, FAAL) fulfilled the detection rule requirements for nine clusters in the absence of a canonical adenylate reductase signature. We decided to retain such cases among the results, as, according to [43], their involvement in the biosynthesis of a particular metabolite is a likely prediction (e.g., unique complex lipids). The presence/absence of common SMC types for the analysed genomes is summarised in Figure 4. Notably, the only cases of missing even common SMCs is limited to genomes lacking any form of GFF annotation (five genomes of _Rhizopus irregularities_, 2-_Cunningham bertholletiae_, 1-_Clomus cerebriforme_, 1-_Cehlia diaphana_; a further raw assembly of _Cunningham elegans_ B9769 contains only a single terpene-like cluster candidate).