Ivan Fiala

History of myxozoan character evolution on the basis of rDNA and EF-2 data

BackgroundPhylogenetic relationships among myxosporeans based on ribosomal DNA data disagree with traditional taxonomic classification: a number of myxosporeans with very similar spore morphology are assigned to the same genera even though they are phylogenetically distantly related. The credibility of rDNA as a suitable marker for Myxozoa is uncertain and needs to be proved. Furthermore, we need to know the history of myxospore evolution to understand the great diversity of modern species.ResultsPhylogenetic analysis of elongation factor 2 supports the ribosomal DNA-based reconstruction of myxozoan evolution. We propose that SSU rDNA is a reliable marker for inferring myxozoan relationships, even though SSU rDNA analysis markedly disagrees with the current taxonomy. The analyses of character evolution of 15 morphological and 5 bionomical characters show the evolution of individual characters and uncover the main evolutionary changes in the myxosporean spore morphology and bionomy. Most bionomical and several morphological characters were found to be congruent with the phylogeny. The summary of character analyses leads to the simulation of myxozoan ancestral morphotypes and their evolution to the current species. As such, the ancestor of all myxozoans appears to have infected the renal tubules of freshwater fish, was sphaerosporid in shape, and had a spore with polar capsules that discharged slightly sideways. After the separation of Malacosporea, the spore of the common myxosporean ancestor then changed to the typical sphaerosporid morphotype. This species inhabited the marine environment as a parasite of the gall bladder of marine fish and ultimately separated into the three main myxosporean lineages evident today. Two of these lineages re-entered the freshwater environment, one as a myxosporean with Chloromyxum and another with a primitive sphaerosporid morphotype. The common ancestor of all marine myxosporeans had a ceratomyxid shape of spore.ConclusionsWe support rDNA based myxozoan phylogeny by the analysis of a protein coding gene and demonstrate the reliability of rDNA as a marker explaining myxozoan relationships. Our tracing the history of myxozoan character evolution discloses ancestral morphotypes and shows their development over the course of evolution. We point out several myxozoan characters that are to a certain extent congruent with the phylogeny and determined that the discrepancy between phylogeny and current taxonomy based on spore morphology is due to an extreme myxospore plasticity occurring during myxozoan evolution.

Perkinsiella amoebae-like endosymbionts of Neoparamoeba spp., relatives of the kinetoplastid Ichthyobodo

Eukaryotic endosymbionts (“parasomes”) were studied in Neoparamoeba strains of different origin including two strains of N. pemaquidensis (Page, 1970), the agent of amoebic gill disease (AGD). Comparative study of the endosymbionts revealed their mutual ultrastructural similarity and also similarity with the endosymbiont Perkinsiella amoebae Hollande, 1980 described from Janickina chaetognathi and J. pigmentifera. Morphological features supported Hollande’s hypothesis on the kinetoplastid origin of P. amoebae but the first conclusive results were obtained in this study using marker genes. The recognition of euglenozoan spliced leader RNA (SL RNA) gene sequences in the genomic DNA of endosymbionts from five Neoparamoeba strains together with the acquisition of one SSU RNA gene sequence allowed us to specify the relationship of the endosymbionts under study with kinetoplastids. Phylogenetic analyses of SSU rRNA gene sequence data currently available revealed close relationship of the first sequenced Perkinsiella amoebae-like organism with Ichthyobodo necator. Sequence comparisons disclosed that P. amoebae-like organisms possess SL RNA genes with a significant level of divergence from other kinetoplastids. However, the sequences are more closely related to kinetoplastids and Diplonema spp. than to euglenids. Three types of the SL RNA gene sequences obtained from P. amoebae-like organisms were congruent with phylogeny of their Neoparamoeba host strains.

Diversity of Insect Trypanosomatids Assessed from the Spliced Leader RNA and 5S rRNA Genes and Intergenic Regions1

Abstract We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.

A Suspected Parasite Spill-Back of Two Novel Myxidium spp. (Myxosporea) Causing Disease in Australian Endemic Frogs Found in the Invasive Cane Toad

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.

Classification and Phylogenetics of Myxozoa

Myxozoans evolved as an endoparasitic radiation of cnidarians exploiting invertebrate and vertebrate (primarily fish) hosts in freshwater and marine environments. Currently, there are some 2,200 nominal species classified to 64 genera and 17 families. These groupings are mainly based on spore morphology. This chapter provides an updated spore-based taxonomic key that takes into account the recent recognition of the cnidarian origin of myxozoans as well as important revisions at generic, family and suborder levels over the last decade. A list of generic synonyms is also reviewed here. Myxozoan molecular phylogenies largely disagree with traditional spore-based classification systems, probably due to extreme plasticity in myxospore morphologies that has resulted in extensive convergence. Molecular phylogenies of myxozoans (based on all existing SSU sequences and those available for species with known actinospore-producing stages) resolve the malacosporeans, the freshwater myxosporeans, the marine myxosporeans and the sphaerosporid lineages. Within these clades species can be resolved according to definitive host type and, partially, according to host environment. Numerous exceptions are indicative of several recursions of species into freshwater or marine environments. Further resolution within clades identifies groups of species according to tissue tropism in their vertebrate hosts. We suggest future studies of myxozoan systematics to address persisting taxonomic and phylogenetic discrepancies and make recommendations for describing taxa in the absence of sequence data or when sequence and morphological data are incongruent.

Molecular evidence for the existence of cryptic species assemblages of several myxosporeans (Myxozoa)

Myxosporeans Chloromyxum cristatum, Chloromyxum fluviatile and Zschokkella nova (Myxozoa) are common gall bladder parasites of the cyprinid fishes frequently persisting as co-infections. Despite the fact that they are believed to be innocuous endocommensals, C. cristatum clearly displays the potential of a serious pathogen since it may pervade fish liver parenchyma and cause its necrosis. Employing the comparison of genetic distances among the myxosporean rDNA sequences and performing phylogenetic analyses, we demonstrate that cryptic species assemblages exist in C. fluviatile and Z. nova. Sequence comparison revealed that Chloromyxum legeri, previously assigned as a junior synonym of C. fluviatile, is a valid species. The same method is used to display the distinction of Z. nova isolates from China and the Czech Republic. We show that C. cristatum is not an assemblage of more species, and our results support the synonymy of Chloromyxum cyprini with C. cristatum. We have developed a multiplex PCR as an effective tool for the detection and discrimination of Z. nova, C. cristatum, and C. fluviatile. It is especially advantageous for the distinction of the non-mature plasmodia of both Chloromyxum species. This method also helped to assess the exact prevalence of these parasites in examined samples and enabled to select single-infected host samples for the intended population studies.

Sphaerospora sensu stricto: Taxonomy, diversity and evolution of a unique lineage of myxosporeans (Myxozoa)

Myxosporeans (Myxozoa) are eukaryotic parasites, primarily of fish, whose classification is in a state of flux as taxonomists attempt to synthesize the traditional morphology-based system with emerging DNA sequence-based phylogenies. The genus Sphaerospora Thélohan, 1892, which includes pathogenic species that cause significant impacts on fisheries and aquaculture, is one of the most polyphyletic taxa and exemplifies the current challenges facing myxozoan taxonomists. The type species, S. elegans, clusters within the Sphaerospora sensu stricto clade, members of which share similar tissue tropism and long insertions in their variable rRNA gene regions. However, other morphologically similar sphaerosporids lie in different branches of myxozoan phylogenetic trees. Herein, we significantly extend taxonomic sampling of sphaerosporids with SSU+LSU rDNA and EF-2 sequence data for 12 taxa including three representatives of the morphologically similar genus Polysporoplasma Sitjà-Bobadilla et Álvarez-Pellitero, 1995. These taxa were sampled from different vertebrate host groups, biogeographic realms and environments. Our phylogenetic analyses and statistical tests of single and concatenated datasets revealed Sphaerospora s. s. as a strongly supported monophyletic lineage, that clustered sister to the whole myxosporean clade (freshwater+marine lineages). Generally, Sphaerospora s. s. rDNA sequences (up to 3.7 kb) are the longest of all myxozoans and indeed metazoans. The sphaerosporid clade has two lineages, which have specific morphological, biological and sequence traits. Lineage A taxa (marine Sphaerospora spp.) have a single binucleate sporoplasm and shorter AT-rich rDNA inserts. Lineage B taxa (freshwater/brackish Sphaerospora spp.+marine/brackish Polysporoplasma spp.) have 2-12 uninucleate sporoplasms and longer GC-rich rDNA inserts. Lineage B has four subclades that correlate with host group and habitat; all Polysporoplasma species, including the type species, cluster together in one of these subclades. We thus suppress the genus Polysporoplasma and the family Polysporoplasmidae and emend the generic diagnosis of the genus Sphaerospora. The combination of morphological, biological and DNA sequence data applied in this study helped to elucidate an important part of the taxonomic puzzle within the phylum Myxozoa.

Synopsis of the species of Myxidium Bütschli, 1882 (Myxozoa: Myxosporea: Bivalvulida)

A synopsis of the species of Myxidium Bütschli, 1882 (Myxozoa: Myxosporea: Bivalvulida) is presented. It includes a total of 232 nominal species, whose principal morphological and morphometric characteristics, site of infection within the host, and original hosts and locality are indicated in a tabulated format. A diagrammatic illustration of a spore of most of the species is also provided.

Evolutionary origin of Ceratonova shasta and phylogeny of the marine myxosporean lineage.

In order to clarify the phylogenetic relationships among the main marine myxosporean clades including newly established Ceratonova clade and scrutinizing their evolutionary origins, we performed large-scale phylogenetic analysis of all myxosporean species from the marine myxosporean lineage based on three gene analyses and statistical topology tests. Furthermore, we obtained new molecular data for Ceratonova shasta, C. gasterostea, eight Ceratomyxa species and one Myxodavisia species. We described five new species: Ceratomyxa ayami n. sp., C. leatherjacketi n. sp., C. synaphobranchi n. sp., C. verudaensis n. sp. and Myxodavisia bulani n. sp.; two of these formed a new, basal Ceratomyxa subclade. We identified that the Ceratomyxa clade is basal to all other marine myxosporean lineages, and Kudoa with Enteromyxum are the most recently branching clades. Topologies were least stable at the nodes connecting the marine urinary clade, the marine gall bladder clade and the Ceratonova clade. Bayesian inference analysis of SSU rDNA and the statistical tree topology tests suggested that Ceratonova is closely related to the Enteromyxum and Kudoa clades, which represent a large group of histozoic species. A close relationship between Ceratomyxa and Ceratonova was not supported, despite their similar myxospore morphologies. Overall, the site of sporulation in the vertebrate host is a more accurate predictor of phylogenetic relationships than the morphology of the myxospore.

Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): coevolution demonstrated by SSU rRNA gene phylogenies.

The molecular phylogeny of Neoparamoeba spp. based on SSU rDNA was updated by including new sequences of strains isolated from an invertebrate and an alga. In total, 59 sequences of strains representating N. pemaquidensis, N. branchiphila, N. aestuarina and N. perurans were analysed. Sequences of SSU rDNA of eukaryotic endosymbionts (Perkinsela amoebae-like organisms) were prepared from 34 samples of genomic DNA of strain-representatives of N. pemaquidensis, N. branchiphila and N. aestuarina. Comparison of phylograms reconstructed from corresponding SSU rDNA sequences of host amoebae and their symbionts revealed a high level of congruence, which argues very strongly for coevolution of these two eukaryotic organisms.