Intracellular obligate parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because intermediate forms are hardly ever found. Microsporidia are highly derived intracellular parasites that are related to fungi. We describe the evolutionary history of a new microsporidian parasite found in the hindgut epithelium of the crustacean Daphnia and conclude that the new species has retained ancestral features that were lost in other microsporidia, whose hallmarks are the evolution of a unique infection apparatus, extreme genome reduction, and loss of mitochondrial respiration. The first evolutionary steps leading to the extreme metabolic and genomic simplification of microsporidia involved the adoption of a parasitic lifestyle, the development of a specialized infection apparatus, and the loss of diverse regulatory proteins.

Intracellular parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because the parasites and their known free-living relatives are so divergent from one another. Microsporidia are intracellular parasites of humans and other animals, which evolved highly specialized morphological structures, but also extreme physiologic and genomic simplification. They are suggested to be an early-diverging branch on the fungal tree, but comparisons to other species are difficult because their rates of molecular evolution are exceptionally high. Mitochondria in microsporidia have degenerated into organelles called mitosomes, which have lost a genome and the ability to produce ATP. Here we describe a gut parasite of the crustacean Daphnia that despite having remarkable morphological similarity to the microsporidia, has retained genomic features of its fungal ancestors. This parasite, which we name Mitosporidium daphniae gen. et sp. nov., possesses a mitochondrial genome including genes for oxidative phosphorylation, yet a spore stage with a highly specialized infection apparatus—the polar tube—uniquely known only from microsporidia. Phylogenomics places M. daphniae at the root of the microsporidia. A comparative genomic analysis suggests that the reduction in energy metabolism, a prominent feature of microsporidian evolution, was preceded by a reduction in the machinery controlling cell cycle, DNA recombination, repair, and gene expression. These data show that the morphological features unique to M. daphniae and other microsporidia were already present before the lineage evolved the extreme host metabolic dependence and loss of mitochondrial respiration for which microsporidia are well known.

Microsporidia are intracellular parasites that represent the extreme of known genome simplification and size reduction among eukaryotes (1). There are more than 1,200 described microsporidia species (2), with several having an economic impact by causing disease in animals such as fish and honey bees, and are a problem to human health, in particular since the AIDS pandemic. Microsporidia are currently placed on an ancestral branch within the fungi, although this placement has only recently been worked out, due to the absence of clear morphological and physiological connections to other eukaryotes and the profound changes resulting from adaptations to an intracellular lifestyle (3⇓⇓–6). The evolutionary history of microsporidia is marked by the loss of several eukaryotic features, such as mitochondria (7), a typical Golgi apparatus (8), a flagellum (3), and the evolutionary innovation of an infection apparatus, the polar tube. Microsporidia evolved distinctive genetic features, such as massive loss of genes, leading to the smallest known eukaryotic genomes (1). Remnants of mitochondria appear in microsporidian cells as DNA-free organelles called mitosomes (9, 10) that perform simplified versions of the original mitochondrial functions, such as the assembly of iron-sulfur clusters (11), but no ATP production via citrate cycle and oxidative phosphorylation (12, 13). For example, the human parasite Enterocytozoon bieneusi seems to have no fully functional pathway to generate ATP from glucose (12), relying on transporters to import ATP from its host. These ATP transporters have been acquired by horizontal gene transfer (HGT) from intracellular parasitic bacteria (13). Despite major progress in the understanding of microsporidian biology, it is still unclear how this clade of extreme parasites evolved.

The key to understanding the sequence of events leading to extreme parasitism is a well-resolved phylogeny. Microsporidia pose a problem in this regard due to their phenomenal molecular rate acceleration, possibly related to loss of cell cycle control genes (14). A phylogenetic analysis of 53 conserved concatenated genes supports a topology in which microsporidia is the most basal branch in the fungal tree (5). Another phylogenetic analysis based on 200 genes included the endoparasite Rozella allomycis, a representative of the recently discovered basal fungal lineage Cryptomycota (15, 16), and placed microsporidia and Cryptomycota together on the most basal fungal branch (4). One of the shared genomic elements between microsporidia and R. allomycis is the nucleotide transporter that is used by microsporidia for stealing energy in the form of ATP from their hosts; however, Rozella harbors a mitochondrion containing a genome, its proteome has not undergone major contraction, and it does not show the typical infection apparatus of microsporidia: the polar tube. However, the sequence of events that occurred at the root of microsporidian evolution is still unclear. Here we describe the evolutionary history of an unusual intracellular parasite of the hindgut of D. magna that morphologically resembles gut-inhabiting microsporidia (17, 18). Ultrastructural examination reveals profound morphological similarities to microsporidia, including a polar tube. However, the genome of the new species surprisingly differs from known microsporidia by the presence of a mitochondrial genome and genes coding for ATP production from glucose via citrate cycle and oxidative phosphorylation. Its genome sequence allows us to produce a well-resolved phylogeny, which shows that the new parasite, Mitosporidium daphniae, is the most early diverging microsporidian described to date. Here we provide a full morphological description of the new species, analyze its genome, and use the underlying data to make inferences about the order of events that occurred during the evolution of microsporidia.

Results and Discussion

Species Description. The new species, which we formally name Mitosporidium daphniae gen. et sp. nov., is known from northwestern Europe (Belgium, Germany, and United Kingdom) and has been used in earlier studies, without formal classification, where it was shown to negatively affect the fitness of the host (19). Most distinctly, this gut parasite has a three-layered spore wall with an endospore, exospore, and plasma membrane and contains a structure similar to the microsporidian infection apparatus with polar sac, polar filament, and polaroplast (Fig. 1). Briefly, the new genus and species are described as follows. Mitosporidium gen. nov.: all stages have isolated nuclei. A multinucleate sporogonial plasmodium generates a great and irregular number (>20) of ovoid or slightly bent, uninucleate spores in a sporophorus vesicle lacking prominent inclusions of the episporontal space. Spores have a thin, electron opaque exospore and a wide, electron lucent endospore, a uniform, weakly developed polaroplast of concentrically arranged lamellae, and an anisofilar polar filament with three regions of posteriad reduced width. Polar filament connects anteriorly to a curved polar sac. M. daphniae sp. nov.: with the basic character of the genus and the following additions: spore size range, 2.31–2.67 × 1.05–1.29 µm (live material, n = 40); polar filament arranged as two steeply tilted coils (coiling starts the posterior pole of the spore, angle of tilt about 35°), wide anterior part twice as wide as the narrowest coil; polaroplast reaches the equator of the spore. Type host: Daphnia magna (Crustacea, Cladocera). A full description is presented in SI Text and Figs. S1–S4. Fig. 1. Light (A and B) and transmission electron microscopic (C–E) pictures of M. daphniae. (A) Part of the posterior gut of Daphnia magna with vesicles of M. daphniae. (B) Individual mature spores of M. daphniae. (C and D) Longitudinal sections of immature (C) and mature (D) spores. A three-layered spore wall with exo-and endospore layers is present. A structure similar to the microsporidian infection apparatus with polar sac, polar filament, and polaroplast is clearly visible. (E) Plasmodium interdigitating with the host cell (not a microsporidian character). In the proximity of the nucleus a mitochondrium-like structure with a double membrane can be seen (enlarged in the Inset and marked with double arrowheads).

Genome Sequencing, Annotation, and Phylogenetics. Using shotgun sequencing of DNA extracted from isolated parasite spores, we generated a draft genome of M. daphniae (SI Materials and Methods) assembling into 612 contigs totaling 5.64 Mbp with an N50 of 32.031 kbp (350× average coverage). A total of 3,300 proteins were predicted, for a coding density of 0.585 proteins/kbp, with an average of 1.27 introns per protein. Despite showing a twofold difference in size, the M. daphniae genome is only slightly more compact than that of the water mold parasite R. allomycis (11.86 Mbp; 0.535 proteins/kbp). The number of predicted proteins in M. daphniae is roughly half that of R. allomycis and on par with the 3,266 ORFs predicted in 8.5+ Mbp genome of the microsporidian human pathogen Trachipleistophora hominis (20). The relative levels of similarity observed for the M. daphnia proteins suggest that it is closely related to both other microsporidian species and to R. allomycis (Table S1). A phylogeny based on 53 conserved orthologous genes (5) gives strong support for placing M. daphniae as the most early diverging microsporidian and, together with the microsporidia, sister to the cryptomycete R. allomycis (Fig. 2). The statistical robustness of this phylogenetic placement was tested by rearranging the position of M. daphniae to every other possible position on the tree, and using this rearranged tree as a constraint for a maximum likelihood search in which the relationships between the other taxa were free to vary. We statistically rejected (P ≤ 0.001) each of the 50 possible alternative placements of M. daphniae as a likely alternative placement using the approximately unbiased test (21). Because concatenation may produce incorrect phylogenetic relationships due to systematic biases in the data (22), we evaluated the phylogenetic relationships suggested for each protein partition. The basal position of M. daphniae among microsporidia was observed in 37% of the individual genes, similar to the support for Dikarya (40%) or Mucoromycotina + Mortierellomycotina (37%). Our tree, therefore, provides strong evidence that M. daphniae is the earliest-diverging microsporidian sequenced to date and suggests that microsporidia are derived from within or are sister to the Cryptomycota. A phylogenetic analysis using solely the fast-evolving ribosomal RNA genes recovers a phylogeny consistent with this finding (Fig. S5). Furthermore, the M. daphniae rRNA operons contain eukaryotic-sized rRNA subunits that lack the characteristic 5.8S/23S gene fusion observed in derived microsporidia (Fig. 3) (21, 23). Fig. 2. M. daphniae is the most early diverging microsporidian genome sequenced to date. Phylogeny based on concatenated analysis of 53 conserved orthologs identified by Capella-Gutiérrez et al. (5). Tree shown is the maximum-likelihood tree found using RAxML 7.2.8a (47). Indicated at each node is the bootstrap percentage/Bayesian posterior probability/percentage of individual genes containing clade (see SI Materials and Methods for full details). Because of missing data, not every taxon is present in each protein alignment, and therefore the percentage of genes reflects only a proportion from which the relationship could be evaluated. The placement of Allomyces was different in the Bayesian consensus tree in which it formed the sister taxon to the Dikarya + Zygomycota sl. Fig. 3. Evolution of microsporidia-derived features. Inferred gains and losses of morphological characters, genomic features, and protein encoding genes in the evolutionary lineage leading to microsporidia are shown in green and red backgrounds, respectively. The following proteins are found in most other fungi but not predicted from microsporidia genomes and therefore inferred as gene losses (National Center for Biotechnology Information accessions in parenthesis): tumor supressor gene RB (NP_525036), Tfb1 (P41895), Tfg1 (P32776), Mcd1 (NP_010281), and chitin synthase with myosin domain (cd00124). Orthologous proteins containing domains found in other organisms but showing a microsporidian-specific distribution are inferred as gains (OrthoMCL database accessions in parenthesis): RtcB-like ligase (OG_12746), kinesin motor domain-containing protein (OG5_127467), homeobox KN domain-containing protein (OG5_181516), mechanosensitive ion channel (OG5_182073), and exocyst complex component Sec6 (OG_188308). For more information, see Tables S1 and S2.

Comparative Genomics. We analyzed the M. daphniae genome to identify genes that it shares uniquely with microsporidia or other fungi. Foremost among microsporidia traits is the absence of a mitochondrial genome, yet the genome assembly of M. daphniae revealed a single contig of 14,043 bp with a sequencing coverage of 765× representing a mitochondrial genome (Fig. S6). This mitochondrial genome seems to be linear, because it has terminal inverted repeats of 477 bp. Linear mitochondrial chromosomes with terminal inverted repeats are known in fungi and supposedly represent an evolutionary transition between circular and “true” linear (with telomeric ends) mitochondrial genomes (24). The mt-genome of the cryptomycete R. allomycis is circular, but otherwise, the M. daphniae and R. allomycis mt-genomes have an nearly identical gene content, and a phylogenetic analysis of mt-genome encoded proteins shows that the two genomes are closely related (Fig. S7). The M. daphniae mitochondrial genome encodes 17 tRNAs, small and large ribosomal subunits, and six proteins belonging to complexes II, III, and IV of the electron transport chain (ETC). The mitochondrial genome is also apparently functional; intact reading frames for the mitochondrial RNA and DNA polymerases are found in the nuclear genome (Table S1). Genes encoding proteins involved in pathways related to energy metabolism are more numerous in M. daphniae compared with the well-studied microsporidium Encephalitozoon cuniculi (Fig. S8) or other microsporidian species. On the other hand, M. daphniae lacks all genes of respiratory chain complex I (NADH dehydrogenase), just as observed in R. allomycis (Fig. 3). However, the M. daphniae nuclear genome encodes two genes, internal NADH dehydrogenase and alternative oxidase, that are presumably capable of moving electrons through the ETC without pumping protons. R. allomycis differs from M. daphniae in having an additional dehydrogenase, external NADH dehydrogenase that presumably projects into the intermembrane space (4). This partial oxidative phosphorylation pathway combined with a complete citrate cycle suggest that M. daphniae mitochondria are capable of producing ATP, although without generating as much energy as a complete ETC (Fig. 3 and Fig. S9). Surprisingly, we did not find an ATP transporter gene that is common to all microsporidia and used to steal ATP from the host, which was also detected in the Rozella genome. This suggests that the ATP transporter gene has either been lost in M. daphniae, was horizontally transferred independently to microsporidia and Rozella from the same lineage of intracellular parasitic bacteria (Chlamydia), or is not present in the current assembly of the M. daphniae genome. Because our genome has about 350× sequencing coverage, failing to assemble the gene is unlikely. However, the ATP transporter gene might have been lost or diverged to become unrecognizable in the genome of M. daphniae. Additional cryptomycete and microsporidian genomes will be needed to work this out. The presence of an ATP transporter gene in the early stages of microsporidia evolution, when mitochondria were still functional, might have been transient. Fungal genomes encode a diverse set of chitin synthase genes that are involved in producing the polymer that provides structure to the fungal cell wall. Among the known chitin synthase genes, one is restricted to fungi, known as the division II chitin synthases (25). Moreover, only fungi possess a chitin synthase gene with a myosin domain—the class V, division II chitin synthases (26)—with the exception of the choanozoan Corallochytrium limacisporum (27). The myosin motor domain of the chitin synthase is believed to function by depositing chitin at particular regions of the plasma membrane via the cytoskeletal highway and is associated with polarized secretion and apical growth in fungi (28). The genome of M. daphniae encodes four chitin synthases, all of division II; however, there is no class V chitin synthase containing a myosin domain (Fig. 3 and Table S1). Because microsporidia germinate via a highly specialized polar tube extrusion mechanism (29) and grow in vivo by schizogony, the myosin domain might no longer have been necessary as there is no polarized growth phase. If this hypothesis is correct, the related group aphelids, which like Rozella appear to grow into their hosts through the formation of a germ tube (30) with a cell wall, would be predicted to have a chitin synthase with a myosin domain. M. daphniae and other microsporidia presumably solely require chitin for the development of the cell wall of the resting spore. Resting spores disperse passively without motility, unlike Rozella and aphelids, which use a flagellum for dispersal. Indeed, none of the proteins that are found in all eukaryotes exhibiting flagellar movement (31) are found in the proteomes of M. daphniae and other microsporidia (Fig. 3 and Table S1). The proteome of M. daphniae, albeit smaller, resembles to a large extent the fungi (Fig. S8A). We identified 3,330 proteins, of which 2,200 belong to 1,878 gene families, or orthologous groups (OGs). Thus, about 34% of the predicted proteome corresponds to proteins without detectable similarity to any other available sequences. We compared M. daphniae OGs to those of its most closely related nonmicrosporidian fungus (R. allomycis), as well as OGs from the well-studied fungus Saccharomyces cerevisiae and the microsporidium Enc. cuniculi. The largest fraction of all M. daphniae protein families (65%) is either shared with R. allomycis, S. cerevisiae, and Enc. cuniculi (619/1,878 OGs) or with only R. allomycis and S. cerevisiae (606/1,878). The majority of protein families involved in basic cellular processes or features, such as repair and recombination, transcription, ribosome biogenesis and function, and chromosome structure are shared with R. allomycis, S. cerevisiae, and Enc. cuniculi. Most OGs related to mitochondrial biogenesis, carbon metabolism, and cellular transport are not found in Enc. cuniculi (Fig. S8B). The M. daphniae proteome also contains most of the so-called microsporidian-specific domains, which appear in all microsporidia with a known genome and are shared with some other eukaryotes, but not with fungi other than Rozella (18) (Table S1). Thus, we conclude that the metabolic profile of M. daphniae is not as simplified as in other microsporidia, and yet already shows microsporidia-specific features. Given that M. daphniae is the earliest diverging microsporidian parasite, we expected to find orthologs that had already evolved specifically in the microsporidian lineage that were coincident with the evolution of some of the morphological features, such as the polar filament. However, we found only four orthologs that were shared between M. daphniae and more derived microsporidia, but not with other fungi (Fig. 3 and Table S2); one (OG5_127467; PF01139) encodes a RtcB-like ligase involved in tRNA splicing and repair (32), which is ubiquitous in all kingdoms, except the fungi. Another ortholog (OG5_181516) contains a DNA-binding homeodomain of the Knotted (KN) family (PF05920) that is common in plants (33). The other two are microsporidian-specific orthologs (OG5_182073 and OG5_188308): a protein containing a mechanosensitive (MS) ion channel domain (PF00924) and another containing a Sec6 exocyst component domain (PF06046). MS ion channels have a variety of roles in the physiology of eukaryotic cells, such as osmotic gradients, cell swelling, gravitropism, and control of cellular turgor (34). We speculate that the microsporidian-specific MS channel could be used for spore germination, which involves increased intrasporal osmotic pressure (29). The exocyst complex, which is well known in yeast, functions by the interaction of four subunits—Sec6, Sec8, Sec10, and Exo70—delivering proteins that are essential for cell separation after division (35). Blast searches using the Schizosaccharomyces pombe Sec6 (NP_587736) as query identified a single protein in M. daphniae (OG5_188308; Fig. 3) and in all other microsporidian proteomes that we examined (Table S1). S. pombe has three paralogous genes encoding proteins with a Sec6 domain (PF06046)—OG5_128060, OG5_129076, and OG5_129499—but none of them is orthologous to the microsporidian Sec6-like proteins. No blast hits are found in the proteomes of any microsporidia using other S. pombe exocyst proteins as queries. Nonetheless, the genomes of Enc. cuniculi, Enc. intestinalis, and Enterocytozoon bieneusi contain one microsporidian-specific ortholog gene (OG5_196856) encoding a Sec8 (PF04048) domain-containing protein, but no homologous gene or domain was found in M. daphniae. These results illustrate the difficulties in performing microsporidian comparative genomics, because distinguishing gene loss from extreme molecular divergence is not straightforward. Indeed, about one third of the M. daphniae proteome could not be assigned to any OG. The involvement of Sec6 and Sec8 in microsporidian schizogony deserves more evaluation in the future.