Microsporidia evolved from ancestral sexual fungi

Abstract

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Figure 1. The sex locus is conserved between zygomycetes and microsporidia

(A) Three zygomycetes and three microsporidia harbor a syntenic sex locus encoding a triose phosphate transporter (TPT), HMG domain protein, and an RNA helicase. In zygomycetes, the orientation of sexP of P. blakesleeanus is opposite to the other HMG protein genes and the P. blakesleeanus (+) allele contains a repetitive element. The TPT gene of R. oryzae is inverted with respect to the P. blakesleeanus and M. circinelloides sex alleles and an additional gene encoding a BTB/Ankyrin/RCC1 repeat protein is present. Microsporidia also have an additional hypothetical protein gene in the extended sex locus, and the RNA helicase and TPT genes are inverted with respect to the zygomycetes. Notably, in A. locustae, the RNA helicase gene lies at a different genomic location. (B) Dot plot analysis of the sex locus alleles of M. circinelloides and P. blakesleeanus illustrates (+) and (−) specific DNA sequences at the sex locus. X axis is the (+) and Y axis the (−) sex alleles. Sequences shown here are 7,103 bp and 6,511 bp for (+) and (−) strains of M. circinelloides and 9,971 bp and 7,631 bp for (+) and (−) strains of P. blakesleeanus. The unique sequences at the sex locus span 1,541 bp and 1,463 bp for (+) and (−) strains of M. circinelloides and 5,830 bp and 3,494 bp for (+) and (−) strains of P. blakesleeanus. The dot plot was performed with a 17 bp window with no mismatches allowed. HP: hypothetical protein.

Figure 2. Examples of conserved synteny and relaxed synteny found in zygomycetes and microsporidia

(A) Conserved synteny among P. blakesleeanus, R. oryzae, and the microsporidia. Syntenic 60S (L21) and 40S (S9) protein genes are conserved throughout fungi (see Supplementary Fig. 6). Another synteny of ribosomal protein and kinase genes is observed in two zygomycete genomes and two microsporidian genomes (E. cuniculi and E. bieneusi). (B) Conserved synteny between P. blakesleeanus and the three microsporidia. (C) Conserved synteny between R. oryzae and the three microsporidia. White arrows indicate additional non-syntenic genes. With the exception of two ribosomal proteins (L21 and S9), none of the syntenic gene clusters shown in panels (A), (B) and (C) were conserved in other ascomycete (A. nidulans), basidiomycete (C. neoformans), or chytridiomycete (B. dendrobatidis) fungi. The genomes of P. blakesleeanus and R. oryzae were compared to the E. cuniculi genome using BLASTp. Homologs in E. bieneusi and A. locustae were identified using tBLASTn.