Miloslav Jirků

CBOL Protist Working Group: Barcoding Eukaryotic Richness beyond the Animal, Plant, and Fungal Kingdoms

A group of protist experts proposes a two-step DNA barcoding approach, comprising a universal eukaryotic pre-barcode followed by group-specific barcodes, to unveil the hidden biodiversity of microbial eukaryotes.

On the phylogenetic positions of the Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eucestoda) inferred from 18S rRNA.

A phylogenetic analysis of tapeworms (Eucestoda) based on complete sequences of the 18S rRNA genes of 43 taxa (including new sequences of 12 species) was carried out, with the emphasis on the groups parasitising teleost fish and reptiles. Spathebothriidea and Trypanorhyncha (the latter group being paraphyletic) appeared as basal groups of the Eucestoda but their position was not stable. The tetrafossate orders (Litobothriidea, Lecanicephalidea, Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea) were well separated from the remaining groups. Results supported polyphyly of the Pseudophyllidea formed by two distinct clades: one with diphyllobothriids (Diphyllobothrium, Schistocephalus, Spirometra and Duthiersia) and another including Abothrium, Probothriocephalus, Eubothrium and Bothriocephalus. The former pseudophyllidean clade formed a separate branch with the Caryophyllidea (Khawia and Hunterella) and Haplobothriidea (Haplobothrium), the latter taxon being closely related to either caryophyllideans or diphyllobothriids in different analyses. Proteocephalideans formed a monophyletic group in all analyses and constituted a clade within the Tetraphyllidea thus rendered paraphyletic. Within the Proteocephalidea, the Acanthotaeniinae (Acanthotaenia from reptiles in Africa) and Gangesiinae (Gangesia and Silurotaenia from silurid fish in the Palearctic Region) were separated from parasites of freshwater fish and mammals. The family Proteocephalidae was found to be paraphyletic due to the placement of a monticelliid species, Monticellia sp., in a clade within the former family. The genus Proteocephalus appeared as an artificial assemblage of unrelated taxa which is congruent with previous molecular analyses.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

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.

A NEW SPECIES OF MYXIDIUM (MYXOSPOREA: MYXIDIIDAE), FROM THE WESTERN CHORUS FROG, PSEUDACRIS TRISERIATA TRISERIATA, AND BLANCHARD'S CRICKET FROG, ACRIS CREPITANS BLANCHARDI (HYLIDAE), FROM EASTERN NEBRASKA: MORPHOLOGY, PHYLOGENY, AND CRITICAL COMMENTS ON AMPHIBIAN MYXIDIUM TAXONOMY

During March 2001–April 2004, 164 adult anurans of 6 species (47 Rana blairi, 35 Rana catesbeiana, 31 Hyla chrysoscelis, 31 Pseudacris triseriata triseriata, 11 Bufo woodhousii, and 9 Acris crepitans blanchardi) from Pawnee Lake, Lancaster County, Nebraska, were surveyed for myxozoan parasites. Of these, 20 of 31 (65%) P. triseriata triseriata and 1 of 9 (11%) A. crepitans blanchardi were infected with a new species of Myxidium. Myxidium melleni n. sp. (Myxosporea) is described from the gallbladder of the western chorus frog, P. triseriata triseriata (Hylidae). This is the second species of Myxidium described from North American amphibians. Mature plasmodia are disc-shaped or elliptical 691 (400–1,375) × 499 (230–1,200) × 23 (16–35) μm, polysporic, producing many disporic pansporoblasts. The mature spores, 12.3 (12.0–13.5) × 7.6 (7.0–9.0) × 6.6 (6.0–8.0) μm, containing a single binucleated sporoplasm, are broadly elliptical, with 2–5 transverse grooves on each valve, and contain 2 equal polar capsules 5.2 (4.8–5.5) × 4.2 (3.8–4.5) μm positioned at opposite ends of the spore. Myxidium melleni n. sp. is morphologically consistent with other members of Myxidium. However, M. melleni n. sp. was phylogenetically distinct from other Myxidium species for which DNA sequences are available. Only with improved morphological analyses, accompanied by molecular data, and the deposit of type specimens, can the ambiguous nature of Myxidium be resolved. Guidelines for descriptions of new species of Myxidium are provided.

A Model for Taxonomic Work on Homoxenous Coccidia : Redescription, Host Specificity, and Molecular Phylogeny of Eimeria ranae Dobell, 1909, with a Review of Anuran-Host Eimeria (Apicomplexa: Eimeriorina)

ABSTRACT. We attempt to extend knowledge of anuran Eimeria, and to provide a model for a complex approach to studies on coccidia. New host and geographic records of coccidia in European Anura are provided. In the second part, Eimeria ranae Dobell, 1909 is redescribed from European terrestrial frogs of the genus Rana based on light microscopic and ultrastructural data on both exogenous and endogenous developmental stages, host specificity, and molecular phylogenetic data. Results of experimental transmissions show for the first time that the host specificity of E. ranae is restricted to the genus Rana and that isolates from tadpoles and adults are conspecific. Disappearance of infection during metamorphosis was confirmed experimentally, suggesting that infections in adults result from reinfections. Poikilotherm‐host Eimeria species possessing a Stieda body (SB) are for the first time included in a molecular phylogenetic analysis. Eimeria ranae and Eimeria arnyi from a colubrid snake form together a well‐supported clade, basal to other SB‐bearing coccidia. The other analysed reptile–host eimerians, Eimeria tropidura and Choleoeimeria sp., which possess bivalved sporocysts and lack a SB, represent a distinct basal lineage of the eimeriid clade. The third part of the article reviews anuran‐host Eimeria. Three distinct oocyst morphotypes, apparently correlating with the character of endogenous development, are recognized and characterized among anuran eimeriids.

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.

Goussia Labbe ´ , 1896 (Apicomplexa, Eimeriorina) in Amphibia: Diversity, Biology, Molecular Phylogeny and Comments on the Status of the Genus

We provide new data on morphology, host specificity, life history, pathology and phylogeny of the coccidian genus Goussia in European anurans. Divergence in the SSU rDNA sequences (3-4%) of three Goussia isolates from three anuran hosts suggests that the isolates represent three distinct species. The isolate from Pelophylax kl. esculentus was determined as Goussia neglecta. The isolates from Rana dalmatina and Rana temporaria are considered conspecific and are, on the basis of host specificity, light microscopical, ultrastructural, and molecular phylogenetic data, described as a new species, Goussia noelleri. The new Goussia species from Bufo bufo remains unnamed. During the hosts metamorphosis the infection disappears, however, oocysts are retained in the liver of post-metamorphic frogs. Molecular phylogenetic analysis showed that anuran-host Goussia form a well-supported monophyletic clade, which together with a clade represented by piscine Goussia metchnikovi, constitute basal lineages of the Eimeriidae. The two lineages show polytomy, likely reflecting undersampling of the basal eimeriid taxa. Goussia janae represents a distinct lineage, sister to the clade containing the other eimeriorinid taxa, suggesting a paraphyly of the genus Goussia. The identity of G. neglecta, the status of the genus Goussia, the presence of cryptic species in anuran-host Goussia and their ecological peculiarities are discussed.

Another chloromyxid lineage: molecular phylogeny and redescription of Chloromyxum careni from the Asian horned frog Megophrys nasuta.

ABSTRACT. Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 μm were localized in Bowmans space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0–8.5 × 5.0–6.5 μm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0–4.0 × 2.5–3.0 μm and a single sporoplasm. Each valve possesses 14–24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6–10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU‐rich inserts. In all SSU and LSU rDNA‐based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists

Significance Amphibians are among the most threatened animal groups. Population declines and extinctions have been linked, in part, to emerging infectious diseases. One such emerging disease has been attributed to Perkinsea-like protists causing mass mortality events in the United States. Using molecular methods, we evaluated the diversity of Perkinsea parasites in livers sampled from a wide taxonomic collection of tadpoles from six countries across three continents. We discovered a previously unidentified phylogenetically distinct infectious agent of tadpole livers present in a broad range of frogs from both tropical and temperate sites and across all sampled continents. These data demonstrate the high prevalence and global distribution of this infectious protist. The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a “marine” protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.