Elsevier

Gene

Volume 375, 21 June 2006, Pages 103-109
Gene

Assessing the microsporidia-fungi relationship: Combined phylogenetic analysis of eight genes

https://doi.org/10.1016/j.gene.2006.02.023Get rights and content

Abstract

Microsporidia are unicellular eukaryotes that are obligate parasites of a variety of animals. For many years, microsporidia were thought to be an early offshoot of the eukaryotic evolutionary tree, and early phylogenetic work supported this hypothesis. More recent analyses have consistently placed microsporidia far from the base of the eukaryotic tree and indicate a possible fungal relationship, but the nature of the microsporidian–fungal relationship has yet to be determined. The concatenated dataset employed in this analysis consists of eight genes and contains sequence data from representatives of four fungal phyla. A consistent branching pattern was recovered among four different phylogenetic methods. These trees place microsporidia as a sister to a combined ascomycete + basidiomycete clade. AU tests determined that this branching pattern is the most likely, but failed to reject two alternatives.

Introduction

Microsporidia are a fascinating group of organisms from both a medical and an evolutionary point of view. These unicellular eukaryotes infect at least 1200 species of animals from every major evolutionary lineage, from crustacean to mammal, with a large proportion infecting insects (Wittner and Weiss, 1999). Microsporidia first came to the attention of humans when a strange parasite decimated lucrative European silkworm populations in the 19th century, but the drive to investigate these organisms was drastically increased by the more recent discovery of microsporidian infections in immuno-compromised humans, such as AIDS, organ transplant and cancer patients in the 1980s and 1990s.

Microsporidia alternate between two life forms — the spore and the meront, but only the spores are viable outside an animal host. Although diverse in size and shape, microsporidian spores have a typical morphology that includes a thick, protective proteinaceous coat containing chitin, and the polar filament, which is specialized for host invasion. This filament, also called the polar tube, is perhaps the most recognized microsporidian feature. It is attached to a plate at one terminus of the spore, is wound around the spore contents, and eventually ends near the posterior vacuole at the other end of the spore. Both the polar tube and the posterior vacuole play integral roles in spore germination and host infection. For a comprehensive review of the microsporidian life-cycle, see Keeling and Fast (2002).

Although microsporidia contain unique infective organelles, they lack several structures that are usually considered hallmarks of eukaryotic life, such as typical mitochondria, peroxisomes and centrioles. They also possess several seemingly “prokaryotic” characteristics, such as 70S ribosomes, tiny genomes and a fused 5.8S and 28S rRNA. For these reasons, microsporidia were long thought to be a primitive or ancestral eukaryotic lineage, that diverged from the universal eukaryotic ancestor before the gain of the α-proteobacterial endosymbiont, which eventually became the mitochondrion (Cavalier-Smith, 1991). This hypothesis placed microsporidia into Kingdom Archezoa, a group of organisms defined by their primitive lack of mitochondria (Cavalier-Smith, 1991).

Initially, molecular data seemed to support the inclusion of microsporidia in the Archezoa. Notably, analyses of ribosomal RNA (Vossbrinck et al., 1987), elongation factor 1-alpha (EF1-α) and elongation factor 2 (EF-2) sequences (Kamaishi et al., 1996a, Kamaishi et al., 1996b) placed microsporidia at the base of the eukaryotic tree. Later, α- and β-tubulin phylogenies (Edlind et al., 1996, Keeling and Doolittle, 1996, Keeling et al., 2000) indicated a close relationship to fungi, in stark contradiction to the data supporting microsporidia as members of the Archezoa. Additional analyses conducted on mitochondrial Hsp70 (Germot et al., 1997, Hirt et al., 1997, Peyretaillade et al., 1998), TATA-box binding protein (Fast et al., 1999), the largest subunit of RNA polymerase II (RPB1) (Hirt et al., 1999), and pyruvate dehydrogenase subunits E1α and β (Fast and Keeling, 2001) bolstered the proposed microsporidia–fungi relationship. In line with the discovery of mitochondrion-derived genes in microsporidian genomes and a fungal ancestry for microsporidia, Williams et al. identified a cryptic mitochondrion in the microsporidian Trachipleistophora hominis, by immunolocalization of Hsp70 (Williams et al., 2002).

In addition, re-analysis of certain molecules, such as EF-2 (Hirt et al., 1999, Van de Peer et al., 2000) and LSU rRNA (Fischer and Palmer, 2005), did not support a basal origin of microsporidia when using a larger taxon set. As phylogenetic methodology advanced, shortcomings were also found with other previous analyses that had originally supported a basal position for microsporidia. For instance, EF1-α is not a suitable molecule to use in many phylogenetic analyses due to its mutation saturation and covarion behaviour, particularly in microsporidian sequences (Hirt et al., 1999, Inagaki et al., 2004).

Microsporidia share physiological and biochemical characteristics with fungi as well. Both have a similar meiotic mechanism, utilizing a closed spindle formation (Flegel and Pasharawipas, 1995), and they also share a common mRNA capping mechanism (Hausmann et al., 2002). However, these characteristics are not exclusive to fungi and microsporidia. Taken together, the phylogenetic, cytological and biochemical evidence indicate that microsporidia do have some tie with fungi, but the exact nature of this relationship has remained elusive, partially due to inadequate fungal representatives in analyses.

Two more recent analyses attempted to remedy this situation and came to differing conclusions. Tanabe et al. conducted analyses of RPB1 and EF1-α (Tanabe et al., 2002). These analyses included a wider sampling of fungal sequences, including representatives from four fungal phyla (ascomycetes, basidiomycetes, zygomycetes and chytrids), and results did not indicate a strong relationship between microsporidia and fungi. Instead, the microsporidia were placed at the base of a combined fungal/animal clade. In contrast to the Tanabe analysis, a phylogenetic study performed by Keeling not only strongly supported a relationship between microsporidia and fungi, but also proposed that microsporidia evolved from a zygomycete ancestor (Keeling, 2003). This is an important issue to be addressed herein: If microsporidia are related to fungi, as the majority of data indicate, are microsporidia the descendants of an actual fungus, or did they share a common ancestor with extant fungi?

Unfortunately, the evolutionary history of any lineage is difficult, if not impossible, to ascertain from the analysis of any single gene. In general, support values for branching patterns in trees are low with any dataset of restricted length. This is also true for phylogenies including microsporidia. By increasing the length of the dataset and the number of fungal species, our aim is to generate a more robust tree. Here we attempt to resolve the nature of the microsporidia–fungi relationship using a concatenated alignment of eight genes, containing 1666 amino acid characters.

Section snippets

Gene and taxon selection

In order to clarify the relationship between microsporidia and fungi, genes were chosen for analysis based on the recent work of Thomarat et al. (Thomarat et al., 2004). Thomarat's group analyzed the genome of Encephalitozoon cuniculi, and conducted four types of phylogenetic analysis on each of several dozen genes. Each gene was annotated as branching at a specific point between fungi, animals and plants. Genes that consistently branched with fungi were selected for this study and the final

Tree topologies and fungal phyla

Until recently, the lack of available sequences from both fungi and microsporidia prevented a large-scale phylogenetic analysis. In general, single gene phylogenies addressing the microsporidia–fungi relationship were not robust. Although microsporidian sequences consistently branched with fungal sequences in these analyses, the lack of sampling or poor phylogenetic resolution prevented the exact nature of the microsporidia–fungi relationship from being pinpointed. In 2003, Keeling's combined

Conclusions

  • (1)

    A combined maximum likelihood analysis of 8 genes (1666 characters) placed microsporidia within the fungal clade, as a sister to the ascomycetes + basidiomycetes.

  • (2)

    This placement of microsporidia is supported by bootstrap values of 81% and 73% for maximum likelihood and maximum likelihood–distance methods, respectively.

  • (3)

    AU tests revealed that the ML/ML–distance/Bayesian tree is the most likely, however two alternative topologies could not be rejected at a significance level of 5%. These trees place

Acknowledgements

We would like to thank J. Leigh at Dalhousie University for her assistance with AU tests and provision of helpful programs, M. Berbee and M. Rogers for helpful discussions, and P. Keeling and R. Lee for providing comments on the manuscript. EEG's work is supported by a Canada Graduate Scholarship from NSERC and research in the Fast lab is funded by NSERC Discovery Grant No. 262988 to NMF.

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