Unlike plants and animals, most sexual fungi are isogamous and so do not suffer from the "two-fold cost" of producing male gametes that do not contribute resources to the offspring [8]. All sexual fungi are also able to reproduce asexually, allowing the direct cost of sexual reproduction to be minimised by engaging in sex only when conditions are optimal [9]. Furthermore, experiments have demonstrated that sexual strains of the yeast Saccaromyces cerevisiae are able to out-compete asexual strains under a range of environmental conditions [10,11]. Given the demonstrable benefits and apparently low costs of sex in fungi, one might reasonably predict that fungi should demonstrate a high incidence of sexuality. Indeed population genetics studies using molecular markers have revealed genetic signatures of sex in most fungal species that have been tested, several of which were previously thought to be asexual [12]. However, ancient asexual lineages also exist among the fungi. In the arbuscular mycorrhizal (AM) fungi, an ancient and diverse group of plant symbionts, ancient asexuality was indicated by the relaxation of concerted evolution acting upon multi-copy ribosomal RNA (rRNA) genes [13,14].

The existence of ancient asexual lineages has also been implied in the microsporidia, a diverse group of intracellular fungal parasites that infect a wide range of vertebrate and invertebrate animal hosts. Some of the most intensively studied microsporidia belong to the genus Nosema, parasites of arthropods that have attracted attention both as causes of disease in honey bees and silk moths and as potential biological control agents for insect pests. Microsporidian life cycles typically involve the alternation of diplokaryotic (binucleate, diploid) stages and monokaryotic (uninucleate, haploid) stages (Figures ​(Figures11 and ​and2).2). Members of the genus Nosema appear to have lost the monokaryotic cycle, remaining diploid throughout their life cycles, and are therefore considered to be asexual. Molecular evidence supporting asexuality in Nosema has been provided by variable rRNA gene sequences amplified from single spores of N. bombi [15], indicating relaxation of concerted evolution, a finding similar to that supporting asexuality in the AM fungi [13]. Nosema contains over 100 described species, with several new species added every year, and its host range spans the Arthropoda, including insects, arachnids and crustaceans. Furthermore, many of the physiological interactions between Nosema parasites and their hosts are complex and host-specific, indicating a high degree of host-parasite coevolution. These include many incidences of transovarial transmission [16] and, most dramatically, the feminization of genetically male crustacean hosts by the parasite N. granulosis [17]. Given the assumption of ancient asexuality within Nosema, the high level of species diversity and high degree of host-specialisation within this genus presents a major challenge to the doctrine of limited evolution within asexual eukaryotic lineages.

Morphologically based taxonomies of the microsporidia have generally assumed that changes of life cycle, such as the loss of the monokaryotic cycle, correspond to major evolutionary transitions [18-21]. It was on this basis that all microsporidian species that remained diplokaryotic throughout their life cycles and lacked a sporophorous vesicle were placed within Nosema [19]. Recently, the taxonomy of Nosema has been extensively revised on the basis of phylogenetic analyses using the sequence of the small subunit ribosomal RNA (16S rRNA) gene. These molecular phylogenies have shown "Nosema" species scattered widely throughout the Phylum Microspora [22], suggesting that the monokaryotic cycle has been lost on many separate occasions. On the basis of 16S rRNA phylogenies, several microsporidian species have been transferred from Nosema to newly created genera (e.g. Antonospora locustae, Paranosema grylli, Brachiola algerae), while several species formerly assigned to the genus Vairimorpha (including Vairimorpha's type species, V. necatrix) have been tentatively placed within Nosema [23]. Following the notation of Baker et al. [23], I will henceforth refer to the monophyletic group containing the Nosema type species N. bombycis and the Vairimorpha type species V. necatrix as Nosema/Vairimorpha. Most of the "Vairimorpha" species within this group possess complete sexual life cycles, with alternating monokaryotic and diplokaryotic stages. The exception is V. imperfecta, in which meiosis is followed by an abortive monokaryotic sporogony, indicating an intermediate phase in the loss of sex [24]. The presence of these sexual species within Nosema/Vairimorpha suggests that the sexual, monokaryotic cycle has been lost since the origin of the genus, perhaps on several separate occasions.

Phylogenetic analyses based solely upon 16S rRNA sequences are, however, of limited use when comparing closely related microsporidian species. This is because the microsporidian small ribosomal RNA subunit is substantially shorter than those of other fungi and lacks many of the more variable regions [25-27]. In fact, both the small and large ribosomal RNA subunits of microsporidia are even shorter than those of most prokaryotes and consist of little more than a core of highly conserved sequences. Consequently, microsporidian 16S rRNA sequence alignments rarely contain sufficient phylogenetically informative sites to unambiguously assign a topology to trees consisting of closely related species [24,28-30]. However, despite their short length and low sequence variability, the rRNA genes of Nosema/Vairimorpha vary exceptionally in the order in which they occur within the ribosomal repeat unit. In non-microsporidian fungi, and most other eukaryotes, the order of rRNA genes from 5' to 3' is 18S, 5.8S, 25S, with internal transcribed spacers between the 18S and 5.8S subunit genes (ITS1) and between the 5.8S and 25S subunit genes (ITS2) (Figure ​(Figure3a).3a). In the Nosema species N. apis [31] and N. bombi [15] and in all known non-Nosema microsporidian species [32-36] the 18S subunit is reduced in size to 16S, the 5.8S and 25S subunit genes are reduced and the ITS2 spacer is absent, producing the gene order 5'-16S, 18S-3' (Figure ​(Figure3b).3b). In N. bombycis an additional rearrangement has occurred, placing the 18S subunit gene upstream of the 16S subunit gene [37]. A 5S subunit is also positioned downstream of the 16S subunit of N. bombycis to give the order 5'-18S, 16S, 5S-3' (Figure ​(Figure3c)3c) [37]. This deviates from the arrangement seen in the model microsporidium Encephalitozoon cuniculi, in which no 5S subunits occur in the vicinity of the larger ribosomal repeat unit [38]. The unusual ribosomal subunit gene order demonstrated by N. bombycis also occurs in N. spodopterae [39], N. plutellae (direct submission to Genbank: AY960987) and N. antheraeae [40], which, like N. bombycis, are parasites of Lepidoptera and which fall close to N. bombycis in phylogenetic trees based on the 16S rRNA gene sequence [29]. Nosema/Vairimorpha species possess multiple copies of the ribosomal RNA repeat unit which can show intragenomic variation in sequence [15,31] and some isolates of N. bombycis also possess fragmented copies of rRNA genes (Figure 3d, e) [41] which are transcriptionally active and coexist with intact rRNA copies within the same genome. The fact that, within Nosema/Vairimorpha, the rRNA repeat unit exists in multiple copies with intragenomic variation in gene order, integrity and sequence is a potential source of confusion in phylogenies based entirely upon 16S rRNA gene sequences.

There is currently a trend towards describing new microsporidian species on the basis of incomplete life cycle data or even solely on the basis of their 16S rRNA sequences. The evidence presented here indicates that the life cycles of Nosema/Vairimorpha species can evolve very rapidly relative to evolution at the molecular level. This means that a newly discovered species will not necessarily share life cycle characteristics with a model species, even if their DNA sequences are extremely similar. The evidence presented here also suggests that rRNA sequences are unreliable indicators of the phylogenetic relationships of closely related species and so the phylogenetic information yielded by comparison of rRNA sequences is of limited value. On this basis, it is proposed that taxonomic descriptions of microsporidian species should describe every stage of the life cycle and should include molecular phylogenetic information based on the sequences of several genes.