Molecular Phylogenetic Relatedness of Frenkelia spp. (Protozoa,Apicomplexa) to Sarcocystis falcatula Stiles 1893: Is the Genus Sarcocystis Paraphyletic?

ABSTRACT The coccidians Frenkelia microti and F. glareoli (Apicomplexa: Sarcocystidae) form tissue cysts in the brain of small rodents (intermediate hosts) while oocysts are formed in the intestine of final hosts, buzzards of the genus Buteo. The inclusion of the small subunit ribosomal RNA gene sequences (SSU rRNA) of both Frenkelia species into the SSU rRNA trees of other, tissue cyst-forming coccidia strongly supports paraphyly of the genus Sarcocystis. Frenkelia spp. exhibit close relatedness to Sarcocystis falcatula Stiles 1893, a bird-opossum parasite, recognized under its junior synonym S. neurona Dubey et al. 1991, as the causative agent of equine protozoan myeloencephalitis on the American continent. As the definition of the genus Frenkelia is based on a plesiomorphic character (affinity to the neural tissue) of supposedly low phylogenetic value, the synonymization of the genus Frenkelia with Sarcocystis is proposed. This renders the genus Sarcocystis monophyletic.     

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.     

Multiple origin of the dihomoxenous life cycle in sarcosporidia

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.     

Protozoan parasites of British small rodents

Thirty-five species of protozoan parasites belonging to thirteen genera have now been recorded for British small rodents. These include species of Entamoeba, Giardia, Spironucleus, Trichomonas, Chilomastix, Eimeria and Cryptosporidium in the gut; Trypanosoma, Hepatozoon and Babesia in the blood; and Toxoplasma, Frenkelia and Sarcocystis in the tissues. Recent advances have progressed along two lines, the elucidation of the life-cycles of the species of Frenkelia and Sarcocystis , which are now known to involve a carnivore as the final host, and laboratory studies on those parasites that can be maintained in laboratory animals. It is now possible to draw up a definitive list of hosts and parasites and this should serve as a basis for studies on the epidemiology of these parasites and their possible effects on their hosts.     

Evolutionary relationships among cyst-forming coccidia Sarcocystis spp. (Alveolata: Apicomplexa: Coccidea) in endemic African tree vipers and perspective for evolution of heteroxenous life cycle

Cyst-forming coccidia of the genus Sarcocystis (Alveolata: Apicomplexa: Coccidea) parasitize vertebrates worldwide. Data from the small subunit rRNA genes (SSU) and the D2 domain of the large subunit rRNA genes were used to reconstruct phylogeny for all species in the Sarcocystidae for which sequences are currently available. We have focused on the evolutionary history of species that circulate between snakes as definitive hosts and rodents as intermediate hosts. Trees were reconstructed using maximum parsimony, minimum evolution, maximum likelihood and the bayesian phylogenetics. Our reconstructions support monophyly of Sarcocystidae but fail to robustly resolve the relationship within clades. Using a concatenated dataset of available rDNAs, the "isosporoid" coccidia Neospora, Toxoplasma, Besnoitia, Isospora and Hyaloklossia form a sister group to the monophyletic Sarcocystis. Moreover, we show that Sarcocystis from arboreal vipers of the genus Atheris, which are endemic to the mountain rain forests of the Equatorial Africa, are monophyletic, with sister species parasitizing the desert viper Pseudocerastes persicus from the Near East. We report the co-evolution of Sarcocystis spp. with their final snake hosts. The geological history of the African continent, mountain ranges, forests and general SSU rDNA rates were used to construct a linearized tree. Possible origin of the heteroxenous life cycle of Sarcocystis is discussed.     

Comparative review of the developmental biology of the genera Sarcocystis, Frenkelia, Isospora, Cystoisospora, Hammondia, Toxoplasma and Besnoitia (author's transl)]

A review is given of the advances in our knowledge of the developmental biology of the so-called cyst-forming coccidia in the years from 1974 to 1978. Until 1970 only 6 Isospora species were known to occur in cats, dogs and men. After the discovery of the coccidian nature of the genera Toxoplasma, Sarcocystis, Besnoitia and Frenkelia, and after the discovery of the new genus Hammondia the number of known species rose to over 30. In addition it could be shown that also birds of prey, owls and reptiles serve as final hosts for several Sarcocystis and Frenkelia species. The coccidia with isosporoid oocysts can be classified into two major groups: Species with gamogony and sporogony in the final host (Sarcocystis, Frenkelia) and species with schizogony and gamogony in the final host and sporogony on the ground (Isospora, Cystoisospora, Hammondia, Toxoplasma, Besnoitia). The subdivision of the first group into the genera Sarcocystis and Frenkelia based on the localization of their cysts in the musculature and in the brain, respectively, cannot be upheld in the future. Their classification into organisms with small cystozoites of about 7 microm with birds or reptiles as final hosts (Sarcocystis and Frenkelia species of rodents) and those with large cystozoites of about 15 microm and mammals as final hosts (Sarcocystis spp. of domestic animals and rodents) would be more significative. The second group can be subdivided into monoxenous species (Isospora), species with an optional intermediate host in which no or only slight multiplication occurs (Cystoisospora) and in genera with a multiplication in two phases in the intermediate host (Hammondia, Toxoplasma, Besnoitia). The nomenclature of single species is very controversial. As an example the controversial apprehension of the taxonomy of the Sarcocystis species of cattle is discussed. An application has been submitted to the International Commission for the Zoological Nomenclature to delcare a number of names as nomina dubia and to introduce unambiguous names for those organisms for which type specimens are available.     

Phylogenetic Analysis of Coccidia Based on 18S rDNA Sequence Comparison Indicates that Isospora Is Most Closely Related to Toxoplasma and Neospora

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa. continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis , and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.     

Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae)

The blood flukes of turtles (Digenea: Spirorchiidae) and the blood flukes of crocodilians, birds and mammals (Digenea: Schistosomatidae) have long been considered as closely related, but distinct evolutionary lineages. Recent morphological and molecular studies have considered these families as sister taxa within the Schistosomatoidea. Representatives of both families have similar furcocercous cercariae and similar two-host life cycles, but have different definitive hosts, distinct reproductive patterns and different morphologies. Sequences including approximately 1800 bases of the small subunit ribosomal DNA and 1200 bases of the large subunit ribosomal DNA were generated from representatives of eight spirorchiid genera. These sequences were aligned with pre-existing sequences of Schistosomatidae and other representatives of the Diplostomida and analysed for phylogenetic signal using maximum parsimony and Bayesian inference. These analyses revealed that the Spirorchiidae is paraphyletic and that the turtle blood flukes are basal to the highly derived schistosomatids. Three genera of spirorchiids from marine turtles form a sister group to the Schistosomatidae and five genera of spirorchiids from freshwater turtles occupy basal positions in the phylogeny of tetrapod blood flukes. Marine turtles are considered to be derived from freshwater turtles and the results of the current study indicate that the spirorchiid parasites of marine turtles are similarly derived from a freshwater ancestor. The close relationship of the marine spirorchiids to schistosomatids and the basal position of the marine transmitted Austrobilharzia and Ornithobilharzia in the schistosomatid clade suggests that schistosomatids arose after a marine turtle blood fluke ancestor successfully colonised birds.     

Identification of Synapomorphic Characteris in the Genus Sarcocystis Based on 18S rDNA Sequence Comparison

ABSTRACT. In order to further investigate synapomorphic characters in the genus Sarcocystis , the small subunit ribosomal RNA gene sequences of Sarcocystis capracanis and Sarcocystis moulei were determined and used to infer the phylogenetic position of these two organisms within the cyst-forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated that S. capracanis groups with Sarcoystis tenella and Sarcocystis arieticanis as a clade that shares the characteristic of using canids as their definitive host. S. moulei was shown to group with Sarcocystis gigantea and Sarcocystis fusiformis as a clade that shares the characteristic of using felids as their definitive host.     

Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.     

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.     

Experimental transmission of Sarcocystis from icterid birds to sparrows and canaries by sporocysts from the opossum.

Cowbirds (Molothrus ater) and grackles (Cassidix mexicanus) infected with muscle cysts of Sarcocystis were fed to opposums (Didelphis virginiana) and fecal sporocysts from the latter were given to sparrows (Passer domesticus, Family Ploceidae), canaries (Serinus canarius, Family Fringillidae) and ducks (Anas platyrhynchos, Family Anatidae). Asexual parasites were found in the endothelium of sparrows and canaries but not in ducks. When birds were kept 10 weeks or more after infection, muscle cysts were found grossly and microscopically in the majority of sparrows, and in 1 canary, but not in ducks. Muscle zoites were found in digests of all sparrows and canaries but not in that of ducks. Metrocytes and forms dividing by endodyogeny also were found in the digest. Thus, avian Sarcocystis was transmitted experimentally from 2 genera of 1 family (Icteridae) to 2 different families of passerine intermediate hosts by sporocysts from the definitive host. This is the broadest intermediate host spectrum known for a species of Sarcocystis.     

The phylogeny of Goussia and Choleoeimeria (Apicomplexa; Eimeriorina) and the evolution of excystation structures in coccidia

The phylogenetic relationships of Goussia janae and Choleoeimeria sp. were analyzed using the small subunit ribosomal RNA gene (SSU rDNA). This is a first attempt to study the molecular phylogeny of coccidian genera parasitizing strictly poikilotherm hosts. The biliary Eimeria-like coccidia of reptiles classified into the genus Choleoeimeria form a sister clade to the family Eimeriidae, which confirms the separate generic status of the genus Choleoeimeria. The position of Goussia is less robustly resolved, since it forms a trichotomy with the Eimeriidae and Sarcocystidae, or alternatively constitutes the earliest branch of the coccidian lineage. Morphological similarities, namely the extracytoplasmic location of the endogenous stages, and the presence of sutures in the sporocyst wall are discussed in the context of the traditional classification of eimeriids. In contrast to the morphology-based systematics, the monophyly of Goussia and Choleoeimeria is not supported by the SSU rDNA data.     

Systematic position of Pseudocorynosoma and Andracantha (Acanthocephala, Polymorphidae) based on nuclear and mitochondrial gene sequences

Species of Pseudocorynosoma are North and South American acanthocephalans that use waterfowl as definitive hosts and amphipods as intermediate hosts, whereas species of Andracantha occur in fish-eating birds with a worldwide distribution. Pseudocorynosoma and Andracantha were originally described as Corynosoma (now restricted to endoparasites of marine mammals). Morphologically, Andracantha is distinct from other genera of Polymorphidae in possessing 2 fields of spines on the trunk, whereas Corynosoma and Pseudocorynosoma have a single field. A recent phylogenetic hypothesis based on morphological characters suggested that Andracantha is closely related to Corynosoma, whereas Pseudocorynosoma was of uncertain phylogenetic position within the Polymorphidae. To test the systematic affinities of these 3 genera, we sequenced 2 nuclear genes (SSU and LSU ribosomal DNA) and 1 mitochondrial gene (cytochrome c oxidase subunit 1; cox 1) of species representing Corynosoma, Andracantha, and Pseudocorynosoma and analyzed the data, including available sequences of other polymorphids. Maximum parsimony (MP), maximum likelihood (ML), and Bayesian analyses of the combined (SSU + LSU) sequences and the concatenated data of 3 genes (SSU + LSU + cox 1) placed Andracantha as the sister taxon to Corynosoma with robust support values. All analyses also showed that Pseudocorynosoma is an independent lineage that does not share a common ancestry with Andracantha and Corynosoma. These phylogenetic hypotheses suggest that birds were the ancestral hosts of polymorphids and that the association of Corynosoma with marine mammals represents a subsequent episode of colonization.     

Phylogenetic analysis of the class Sporozoea (phylum Apicomplexa Levine, 1970): evidence for the independent evolution of heteroxenous life cycles

A phylogenetic analysis of representative genera in the class Sporozoea was undertaken using biological and morphological features to infer evolutionary relationships among the widely recognized groups in the class. Gregarines were used as a functional outgroup to the remaining sporozoa (adeleids, eimeriorins, haemosporinids, and piroplasms). The piroplasms were shown to be closely related to the adeleid parasites. Species of Babesia and Theileria were shown not to form a sister group to the haemosporinids as has been frequently suggested. The data indicate that the biologically diverse family Haemogregarinidae should be divided into at least 3 families (Haemogregarinidae Neveu-Lemaire, 1901, containing the genera Haemogregarina and Cyrilia; Karyolysidae Wenyon, 1926, containing the genus Karyolysus; Hepatozoidae Wenyon, 1926, containing the genus Hepatozoon) because the 4 genera currently within the family do not form a monophyletic group. Correlation between parasite phylogeny and taxonomic affinities of their definitive hosts suggests that the definitive hosts of heteroxenous sporozoa are their ancestral hosts. Heteroxenous sporozoan life cycles apparently have evolved independently to adapt to changes in the feeding behaviors of their definitive hosts.     

A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.     

Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay

The ITS1 sequences for C. suis, C. belli, C. rivolta, C. felis, and C. ohioensis-like oocysts were determined and a diagnostic PCR-RFLP assay specific for Cystosisopora species was developed. Phylogenetic analysis of ITS1 sequences of Cystosisopora species along with ITS1 sequences for Toxoplasma, Neospora, Sarcocystis and Eimeria spp. using distance, minimum evolution and parsimony-based methods confirmed previous studies, which suggested that the genus Cystoisospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae.