Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.     

Phylogenetic Analysis of the Complete Genome Sequence of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> Supports the Fungal Origin of Microsporidia and Reveals a High Frequency of Fast-Evolving Genes

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites. Lacking mitochondria, they were initially considered as having diverged before the endosymbiosis at the origin of mitochondria. That microsporidia were primitively amitochondriate was first questioned by the discovery of microsporidial sequences homologous to genes encoding mitochondrial proteins and then refuted by the identification of remnants of mitochondria in their cytoplasm. Various molecular phylogenies also cast doubt on the early divergence of microsporidia, these organisms forming a monophyletic group with or within the fungi. The 2001 proteins putatively encoded by the complete genome of Encephalitozoon cuniculi provided powerful data to test this hypothesis. Phylogenetic analysis of 99 proteins selected as adequate phylogenetic markers indicated that the E. cuniculi sequences having the lowest evolutionary rates preferentially clustered with fungal sequences or, more rarely, with both animal and fungal sequences. Because sequences with low evolutionary rates are less sensitive to the long-branch attraction artifact, we concluded that microsporidia are evolutionarily related to fungi. This analysis also allowed comparing the accuracy of several phylogenetic algorithms for a fast-evolving lineage with real rather than simulated sequences.This article contains online supplementary material.Reviewing Editor: Dr. Wen-Hsiung LiSupplementary material is available at     

Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies

Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and diplomonad lineages, depending on the phylogenetic method employed. Tubulin gene phylogenies were in general agreement with mitochondrial gene phylogenies and ultrastructural data in indicating that the "jakobids" may be polyphyletic. Relationships with the putatively deep-branching amitochondriate diplomonads remain uncertain.     

α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

Covarion shifts cause a long-branch attraction artifact that unites microsporidia and archaebacteria in EF-1alpha phylogenies

Microsporidia branch at the base of eukaryotic phylogenies inferred from translation elongation factor 1alpha (EF-1alpha) sequences. Because these parasitic eukaryotes are fungi (or close relatives of fungi), it is widely accepted that fast-evolving microsporidian sequences are artifactually "attracted" to the long branch leading to the archaebacterial (outgroup) sequences ("long-branch attraction," or "LBA"). However, no previous studies have explicitly determined the reason(s) why the artifactual allegiance of microsporidia and archaebacteria ("M + A") is recovered by all phylogenetic methods, including maximum likelihood, a method that is supposed to be resistant to classical LBA. Here we show that the M + A affinity can be attributed to those alignment sites associated with large differences in evolutionary site rates between the eukaryotic and archaebacterial subtrees. Therefore, failure to model the significant evolutionary rate distribution differences (covarion shifts) between the ingroup and outgroup sequences is apparently responsible for the artifactual basal position of microsporidia in phylogenetic analyses of EF-1alpha sequences. Currently, no evolutionary model that accounts for discrete changes in the site rate distribution on particular branches is available for either protein or nucleotide level phylogenetic analysis, so the same artifacts may affect many other "deep" phylogenies. Furthermore, given the relative similarity of the site rate patterns of microsporidian and archaebacterial EF-1alpha proteins ("parallel site rate variation"), we suggest that the microsporidian orthologs may have lost some eukaryotic EF-1alpha-specific nontranslational functions, exemplifying the extreme degree of reduction in this parasitic lineage.     

Phylogenetic Position of Amblyospora Hazard & Oldacre (Microspora: Amblyosporidae) Based on Small Subunit rRNA Data and Its Implication for the Evolution of the Microsporidia

ABSTRACT. Sequences of the small subunit rRNA genes of Amblyospora californica and an Amblyospora sp. from Culex salinarius were determined. These sequences were compared phylogenetically with 16 other microsporidia. The results suggest Amblyospora forms a sister taxon to the rest of the microsporidia examined. The basal position of Amblyospora is discussed with respect to the evolution of microsporidian life cycles. These sequences represent the longest microsporidian small subunit rRNA genes sequenced to date, 1,359 and 1,358 bp, respectively. Structural features and GC content (49% for both) are comparable to those of other microsporidia which have been sequenced.     

Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness

Microsporidia are obligatory intracellular parasites related to fungi and since their discovery their classification and origin has been controversial due to their unique morphology. Early taxonomic studies of microsporidia were based on ultrastructural spore features, characteristics of their life cycle and transmission modes. However, taxonomy and phylogeny based solely on these characteristics can be misleading. SSU rRNA is a traditional marker used in taxonomical classifications, but the power of SSU rRNA to resolve phylogenetic relationships between microsporidia is considered weak at the species level, as it may not show enough variation to distinguish closely related species. Overall genome relatedness indices (OGRI), such as average nucleotide identity (ANI), allows fast and easy-to-implement comparative measurements between genomes to assess species boundaries in prokaryotes, with a 95% cutoff value for grouping genomes of the same species. Due to the increasing availability of complete genomes, metrics of genome relatedness have been applied for eukaryotic microbes taxonomy such as microsporidia. However, the distribution of ANI values and cutoff values for species delimitation have not yet been fully tested in microsporidia. In this study we examined the distribution of ANI values for 65 publicly available microsporidian genomes and tested whether the 95% cutoff value is a good estimation for circumscribing species based on their genetic relatedness.     

Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

Hypothesis of parasitic stress in parasitized insects caused by microsporidia

Pathological alterations being similar to those that can be seen while hormonal dysbalance, particularly the increase of juvenile hormone (JH) titre, is one of the consequences of microsporidian infections. Though the increase of JH in insects infected with microsporidia has not been shown directly, there are many indirect proofs of this. It has been believed that JH is produced by microsporidia. But this has not been shown for microsporidia or for other endoparasites. In this article we want to propose another hypothesis. We suppose that during microsporidiosis the following events develop: exhaustion of host nutrition stores and other destructive consequences of microsporidian dwelling in host cells lead to the decrease of host biosynthetical and reparation activity in the infected cells and then to destructive alterations that can be seen by electro-microscopic methods. The infected cells are stressed and then the typical answer for many physiological stresses follows. Secretion of prothoracicotropic hormone by brain neurosecretory cells is inhibited and as a result the production and release of ecdysone is also inhibited and ecdysteroid titre decreases. The activity of JH-esterases is decreased and as a result the JH titre is increased. If microsporidian infection causes the stress in the host cells, the endocrine system will undoubtedly answer to this stress and this answer will definitely be the same as for all other stresses. Thus, in any case JH titre will be increased in infected insects independently of whether microsporidia produce JH or not. So, hormonal alterations in infected insects should be the consequence not of the microsporidian JH production but of the host response reaction to infection. We suppose that microsporidia do not differ from other parasites of insects and that they can not produce JH.     

Should the microsporidian spores be treated as dormant stages?

Spores of bacteria, fungi, microsporidia and other protists are traditionally treated as dormant stages, intended to the long-term survival in the environment and to activation of parasitic forms during the infestation of a new host. However, in the process of examination of insect microsporidia at the molecular cellular levels and also at the level of organisms and populations, we came to a conclusion that spores are very active developmental stages with the entire potential directed to the rapid and successful infestation of new hosts during contact with the later. The work summarizes the original data demonstrating (1) the necessity of the rapid activation of microsporidian spores during host contact, (2) hopelessness of the long retaining of viability by spores of many microsporidia in the environment after leaving host organism; and (3) specific accumulation of metabolic ferments in "dormant" spores, but not in actively proliferating prespore developmental stages. On the basis of these data we conclude that microsporidian spores tend to shorten the period when they stay outside host organism to the maximal degree. The probability of host infestation within the limited time period increases due to diverse modes of transmission of pathogens, accumulation of maximally possible volume of infective spores, and the rapid mobilization of the extrusion apparatus.     

Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1α genes

True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1α (tef) from 16 zygomycetes (total of 96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera – Umbelopsis and Micromucor – previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.     

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.     

Ribosomal RNA Sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: Phylogenetic Construction and Structural Correspondence

ABSTRACT. The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5'-GTGCCATCCAGCCGCGG-3') was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5'-GGTCCGTGTTTCAAGACGG-3') was in the large subunit rRNA (LSU-rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590–650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.     

柞蚕微孢子虫Nosema pernyi微管蛋白基因的克隆及系统发育分析

【目的】柞蚕微粒子病的病原为柞蚕微孢子虫Nosemapernyi,为解明柞蚕微孢子虫微管蛋白基因的序列信息,明确柞蚕微孢子虫的系统分类学地位。【方法】采用RT-.PCR、3′RACE(Rapid amplification ofcDNAends)等技术克隆得到了柞蚕微孢子虫的α、β和y-微管蛋白基因,并利用α、β-微管蛋白序列,分别采用NJ、ML法构建进化树。【结果】将克隆得到的基因序列提交NCBI(GenBank登录号:KF154086、KF023271、KF740389)。构建的系统发育树显示,微孢子虫类以一个独立群位于真菌群体中,与真菌的虫霉门关系较近,且与担子菌、球囊菌、壶菌、接合菌及部分子囊菌互为姐妹群。从部分微孢子虫的系统发育分析结果可以看出,20种微孢子虫分为2个分支,柞蚕微孢子虫与其他Nosema属聚为一类。【结论】本研究克隆得到了柞蚕微孢子虫α、β和y-微管蛋白基因,系统发育分析为更进一步了解柞蚕微孢子虫奠定了基础。     

Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements

Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.     

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.     

Molecular characteristics and physiology of microsporidia

A survey of the molecular features of microsporidia is presented which attempts to comment on unresolved questions concerning the physiology of these amitochondrial intracellular parasites. Various transports of host-derived molecules can be predicted and trehalose appears as a potential reserve of glucose for energy metabolism. Significant insights into membrane lipids, polyamine metabolism and sporogony-specific proteins have been gained. Some species, such as Encephalitozoon cuniculi, are heterogeneous entities and harbor a small genome. Although showing a variation in genome size of 8.5-fold, microsporidia share reduced rDNA genes. Finally, data on gene organization and a possible evolutionary relationship with fungi are considered.