Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins

Abstract

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.

Figure 1

Phylogenetic analyses of RPB1. Effects on bootstrap support of ISR or FSR were considered by using protein ML. The tree shown is the ML consensus tree topology from analysis of 760 aligned positions for 15 RPB1 sequences and 1 outgroup RPA1 sequence. Values report bootstrap support from ML analyses for all 760 sites (ALL sites), 669 sites (FSR, where the fastest evolving sites common to the ML tree and a tree where Microsporidia are at the base of the eukaryotes were removed), and 645 sites (ISR). Where only a single bootstrap value is shown, support was 100% in all analyses. The scale bar represents 10% estimated sequence divergence under the JTT-F model.

Figure 2

Phylogenetic analyses of EF-2. (A) Protein ML tree with outgroups removed and FSR (72 sites). The scale bar represents 10% estimated sequence divergence under the JTT-F model. (B–E) Phylogenetic analyses of EF-2 DNA codon positions 1 + 2 using LogDet and investigating the influence of constant sites and choice of outgroup on bootstrap support. The DNA alignment was derived from a 542-aa alignment. (B) Bootstrap consensus tree topology for LogDet distances estimated from 812 variable sites (93% of constant sites removed as invariant) indicating support for M + F. The effect of incremental removal of observed constant sites on bootstrap support for different relationships in the presence or absence of all 3 outgroups (C), with Halobacterium halobium as outgroup (D), and with Methanococcus vannielii as outgroup (E). The dashed vertical lines represent the ML estimates of invariant sites: 93%, 92%, and 93% of constant sites for C, D, and E, respectively (see text for discussion).