Comparative genomics reveals the origin of fungal hyphae and multicellularity

Hyphae represent a hallmark structure of multicellular fungi. The evolutionary origins of hyphae and of the underlying genes are, however, hardly known. By systematically analyzing 72 complete genomes, we here show that hyphae evolved early in fungal evolution probably via diverse genetic changes, including co-option and exaptation of ancient eukaryotic (e.g. phagocytosis-related) genes, the origin of new gene families, gene duplications and alterations of gene structure, among others. Contrary to most multicellular lineages, the origin of filamentous fungi did not correlate with expansions of kinases, receptors or adhesive proteins. Co-option was probably the dominant mechanism for recruiting genes for hypha morphogenesis, while gene duplication was apparently less prevalent, except in transcriptional regulators and cell wall - related genes. We identified 414 novel gene families that show correlated evolution with hyphae and that may have contributed to its evolution. Our results suggest that hyphae represent a unique multicellular organization that evolved by limited fungal-specific innovations and gene duplication but pervasive co-option and modification of ancient eukaryotic functions.


Supplementary Information
Comparative genomics reveals the origin of fungal hyphae and multicellularity Kiss et al.

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Supplementary

Supplementary Note 1
A complete genome of Fonticula alba, a representative of the sister group of Fungi became available recently. Fonticula forms simple multicellular fruiting bodies which arise by the aggregation of individual cells (termed cellular slime mold habit 1-5 ). As such, its multicellularity differs from hyphal multicellularity in fungi, but it is nevertheless interesting if any of the genes we examined in fungi also show expansions/contractions in Fonticula. The genome of Fonticula alba became available after the completion of our analyses, but considering its importance -its sister position to fungi and aggregative multicellular lifestyle -we complemented our analysis with its genome and performed copy numberbased analyses using the same methods as for fungi.
First we investigated Ser/Thr and histidine kinases, G-protein coupled receptors (GPCRs) and adhesive proteins. We found that Fonticula has much fewer Ser/Thr kinases (323) than metazoans (mean 644) and it was more similar to gene copy numbers of non-fungal opisthokonts (mean 392) than to fungi (mean 260). Considerably fewer histidine kinases were found in Fonticula (10) than in multicellular fungi (mean 23)(Suppl. Fig. 3). We detected no GPCRs in Fonticula (Fig. 2b). The adhesion gene set (6), in turn, was similar to that of protists, early fungi and the Mucoromycotina, although it should be noted that all these groups have poor adhesion gene repertoires.
We also focused on the main hyphal multicellularity-related functional categories and analyzed their copy numbers in Fonticula alba compared to the other 71 species (Suppl. Data 2). In "cell wall biogenesis" and "transcriptional regulation" categories, where we found significant gene family expansions potentially related to hyphal multicellularity, Fonticula had low gene copy numbers, resembling protists and animals rather than fungal lineages. In terms of "polarity maintenance" genes, Fonticula was most similar to other non-fungal eukaryotes like Monosiga and Capsaspora, and to plesiomorphically unicellular fungi (Rozella, Catenaria, Batrachochytrium species). On the other hand, in gene groups that were more diverse in non-fungal lineages (e.g. vesicle transport, actin cytoskeleton), Fonticula showed transitional copy numbers between non-fungal protists and fungi. For example in "vesicle transport" genes, Fonticula showed the same pattern as fungi compared to non-fungal species. In "actin cytoskeleton" genes we observed a reduction of gene repertoire in Fonticula (27) compared to fungi (mean 34), metazoa (mean 47) and protists (mean 45) Microtubule transport-related genes were generally more diverse in Metazoa (mean 101), protists (mean 51), early fungi (mean 70), Mucoromycota (mean 56) and Agaricomycotina (mean 77), than in Fonticula (25). The genetic toolkit for signaling was small in Fonticula (52) compared to animals (mean 148), protists (mean 85) and most of fungi (mean 77). Phagocytosis-related genes were conserved in Fonticula alba, including two families (WAVE and WASH) that were lost in fungi, indicating an overall similarity to non-fungal protists rather than fungi (Fig. 4.).
Taken together, these analyses reveal that in the examined multicellularity-related genes Fonticula resembles the non-fungal protists more than it does fungi, which accords well with the independent origin of a different type of multicellularity (aggregative) in Fonticula 2 . Supplementary Fig. 1. Phylogenetic trees inferred from 455 single-copy orthologs using three different methods.