Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.     

Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences

Investigating the evolutionary relationships of the major groups of Apicomplexa remains an important area of study. Morphological features and host-parasite relationships continue to be important in the systematics of the adeleorinid coccidia (suborder Adeleorina), but the systematics of these parasites have not been well-supported or have been constrained by data that were lacking or difficult to interpret. Previous phylogenetic studies of the Adeleorina have been based on morphological and developmental characters of several well-described species or based on nuclear 18S ribosomal DNA (rDNA) sequences from taxa of limited taxonomic diversity. Twelve new 18S rDNA sequences from adeleorinid coccidia were combined with published sequences to study the molecular phylogeny of taxa within the Adeleorina and to investigate the evolutionary relationships of adeleorinid parasites within the Apicomplexa. Three phylogenetic methods supported strongly that the suborder Adeleorina formed a monophyletic clade within the Apicomplexa. Most widely recognized families within the Adeleorina were hypothesized to be monophyletic in all analyses, although the single Hemolivia species included in the analyses was the sister taxon to a Hepatozoon sp. within a larger clade that contained all other Hepatozoon spp. making the family Hepatozoidae paraphyletic. There was an apparent relationship between the various clades generated by the analyses and the definitive (invertebrate) host parasitized and, to lesser extent, the type of intermediate (vertebrate) host exploited by the adeleorinid parasites. We conclude that additional taxon sampling and use of other genetic markers apart from 18S rDNA will be required to better resolve relationships among these parasites.     

Phylogenies based on combined mitochondrial and nuclear sequences conflict with morphologically defined genera in the eimeriid coccidia (Apicomplexa)

Partial mitochondrial (mt) cytochrome c oxidase subunit I (COI) and near-complete nuclear (nu) 18S rDNA sequences were obtained from various eimeriid coccidia infecting vertebrates. New and published sequences were used in phylogenetic reconstructions based on nu 18S rDNA, mt COI and concatenated sequence datasets. Bayesian analyses of nu 18S rDNA sequences used secondary structure-based alignments with a doublet nucleotide substitution model; the codon nucleotide substitution model was applied to COI sequences. Although alignment of the mt COI sequences was unambiguous, substitution saturation was evident for comparisons of COI sequences between ingroup (eimeriid) and outgroup (sarcocystid) taxa. Consequently, a combined dataset applying partition-specific analytical and alignment improvements was used to generate a robust molecular phylogeny. Most eimeriid parasites that infect closely related definitive hosts were found in close proximity on the resulting tree, frequently in a single clade. Whether this represents coevolution or co-accommodation or a combination remains an open point. Unlike host associations, basic oocyst configuration (number of sporocysts per oocyst and sporozoites per sporocyst) was not correlated with phylogeny. Neither ‘Eimeria-type’ nor ‘Isospora-type’ oocyst morphotypes formed monophyletic groups. In the combined dataset tree (representing only a tiny fraction of described eimeriid coccidia), at least 10 clades of Eimeria spp. would need to be re-assigned to nine distinct genera to resolve their paraphyly. The apparent lack of congruence between morphotype and genotype will require taxonomists to balance nomenclatural stability and diagnostic ease against the ideal of monophyletic genera. For now, recognition of paraphyletic eimeriid genera defined by basic oocyst configuration may be necessary for reasons of taxonomic stability and diagnostic utility. Future taxonomic revisions to produce monophyletic eimeriid genera will ultimately require the identification of reliable phenotypic characters that agree with the molecular phylogeny of these parasites or, less optimally, acceptance that genotyping may be needed to support monophyletic supraspecific taxonomic groups.     

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.     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

Identification of two novel coccidian species shed by California sea lions (Zalophus californianus)

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using the full ITS-1 region, partial small subunit 18S rDNA sequence, and the Apicomplexa rpoB region identified 2 distinct sequence clades, referred to as Coccidia A and Coccidia B, and placed them in the Sarcocystidae, grouped with the tissue-cyst-forming coccidia. Both sequence clades resolved as individual taxa at ITS-1 and rpoB and were most closely related to Neospora caninum. Coccidia A was identified in 11 and Coccidia B in 4 of 12 sea lion oocyst samples successfully sequenced (3 of those sea lions were co-infected with both parasites). Shedding of Coccidia A oocysts was not associated with age class, sex, or stranding location, but yearlings represented the majority of shedders (8/15). This is the first study to use molecular phylogenetics to identify and describe coccidian parasites shed by a marine mammal.     

Phylogenetic inference regarding Parergodrilidae and Hrabeiella periglandulata ('Polychaeta', Annelida) based on 18S rDNA, 28S rDNA and COI sequences

Parergodrilidae and Hrabeiella periglandulata are Annelida showing different combinations of clitellate-like and aclitellate characters. Similarities between both of these taxa and Clitellata have widely been regarded as the result of convergent evolution due to similar selection pressures. The position of the three taxa in the phylogenetic system of Annelida is still in debate. However, in analyses based on 18S rDNA sequences a close relationship of Parergodrilidae with Orbiniidae and Questidae was suggested. To infer their phylogeny the sequences of the 28S rDNA and of the cytochrome oxidase I (COI) gene of Stygocapitella subterranea , Parergodrilus heideri and H. periglandulata were determined. The data were extended by sequences of various species including species from Clitellata and Orbiniidae. Prior to tree reconstruction the dataset was analysed in detail for phylogenetic content by applying a sliding window analysis, a likelihood mapping and Modeltest V.3.04. Subsequently, generalized parsimony and maximum likelihood methods were employed. Clade robustness was estimated by bootstrapping. In addition, combined analyses of the sequences of 18S rDNA and 28S rDNA as well as of 18S rDNA, 28S rDNA and COI were performed. The combination of the data of the two structure genes and a mitochondrial gene improved the resolution obtained with the single datasets slightly. These analyses support a close relationship of Parergodrilidae and Orbiniidae but cannot resolve the position of H. periglandulata . In every analysis Clitellata cluster within 'Polychaeta', confirming previous investigations.     

Molecular characterization and phylogenetic analysis of Eimeria from turkeys and gamebirds: implications for evolutionary relationships in Galliform birds

In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.     

Systematic position of the pelagic Thecosomata and Gymnosomata within Opisthobranchia (Mollusca, Gastropoda) – revival of the Pteropoda

The complete 18S (SSU) rRNA as well partial 28S (LSU) rRNA and partial mitochondrial COI sequences have been used to reconstruct the phylogenetic relationships within Opisthobranchia with special focus on the pelagic orders Thecosomata and Gymnosomata. Maximum parsimony, maximum likelihood, distance as well as Bayesian analysis of a combined dataset of the three genes reveals that Thecosomata and Gymnosomata are sister groups and together are closely related to Anaspidea. Possible sister taxon to Thecosomata, Gymnosomata and Anaspidea is Cephalaspidea s. str . Analysis of a taxon-extended dataset of partial 28S sequences supported a basal position of Limacina within Euthecosomata. Within Cavolinidae, Creseis is basal to the other taxa. Other phylogenetic implications from the present results are also discussed. Investigation of the morphology and histology of Thecosomata and Gymnosomata as well as several other opisthobranch taxa helped to identify autapomorphies for Thecosomata and Gymnosomata as well as apomorphies for the clades including these taxa.     

Molecular phylogeny of Besnoitia and the genetic relationships among Besnoitia of cattle, wildebeest and goats

Knowledge on parasites of the genus Besnoitia is sparse, which are classified in the subfamily Toxoplasmatinae of the phylum Apicomplexa. This arrangement hypotheses that Besnoitia represents the sister group to species such as Toxoplasma gondii and Hammondia hammondi. In order to test this hypothesis, phylogenetic analyses of 18S ribosomal DNA (rDNA) from Besnoitia, Hammondia, Isospora, Frenkelia, Eimeria, Neospora, Sarcocystis and Toxoplasma were performed. The 18S rDNA of Besnoitia besnoiti, Besnoitia jellisoni and Eimeria alabamensis were amplified by PCR and sequenced. Phylogenetic analyses by parsimony and maximum-likelihood methods showed Besnoitia to be reproducibly the sister group to a clade containing Hammondia, Neospora and Toxoplasma. Furthermore, Besnoitia of cattle, wildebeest and goats had identical ITS1 rDNA sequences, which questions the use of the taxon Besnoitia caprae to describe the Besnoitia found in goats.     

Speciation studies with Eimeria acervulina and Eimeria mivati.

Eimeria mivati was described as a new species of chicken coccidia in 1964 by Edgar and Seibold, but recently some British workers have relegated its status to that of a variety of Eimeria acervulina. Using strains supplied by Dr. Edgar, we have prepared lines of E. acervulina resistant to methyl benzoquate, sulfaquinoxaline and robenidine and a line of E. mivati resistant to methyl benzoquate. Genetic transfer of resistance between the various lines of E. acervulina to produce doubly-resistant coccidia has been demonstrated, but no such transfer could be obtained between E. mivati resistant to methyl benzoquate and the resistant lines of E. acervulina. Although some immunological relationship between E. acervulina and E. mivati has been demonstrated, we conclude that this failure of the 2 organisms to interbreed lends support to the status of E. mivati as a distinct species.     

Aggregata polibraxiona n. sp. (Apicomplexa: Aggregatidae) from Octopus bimaculatus Verrill, 1883 (Mollusca: Cephalopoda) from the Gulf of California,Mexico

The Apicomplexa Aggregata spp. are intracellular parasites of cephalopods that infect the intestinal tract of commercially important species such as Octopus bimaculatus, which sustains the octopus fishery in Baja California (B.C.), Mexico. In this study, Aggregata polibraxiona n. sp. was described from the cecum of O. bimaculatus collected from Bahia de Los Angeles, B. C. Light and electron microscopy revealed that oocysts and sporocysts were spherical to ovoid in shape. Sporulated oocysts (293–835 × 177–688 μm) contained 135–674 sporocysts (12–24 × 11–22 μm). The sporocyst wall was covered by tubular projections (0.55–2.19 μm in length) bifurcated in the top, unevenly distributed, covered by a thin membrane. Each sporocyst contains 11–13 sporozoites (16–26 × 1.20–3 μm). Three partial sequences of the 18S rDNA gene were obtained, and two phylogenetic approaches were performed according to Bayesian inference and Maximum Likelihood. In both phylogenetic reconstructions, the sequences of A. polibraxiona n. sp. were recovered as a monophyletic group within the genus Aggregata and placed as a sister group to Aggregata octopiana Lineage II. Aggregata polibraxiona n. sp. is the first Apicomplexa described from a cephalopod host from Mexico and extends the geographical range of Apicomplexa infecting cephalopods.     

Description of the two strains of turkey coccidia Eimeria adenoeides with remarkable morphological variability

Although oocyst morphology was always considered as a reliable parameter for coccidian species discrimination we describe strain variation of turkey coccidia, Eimeria adenoeides, which remarkably exceeds the variation observed in any other Eimeria species. Two strains have been isolated - the first strain maintains the typical oocyst morphology attributed to this species - large and ellipsoidal - while the second strain has small and ovoid oocysts, never described before for this species. Other biological parameters including pathogenicity were found to be similar. Cross-protection between these 2 strains in 2 immunization and challenge experiments was confirmed. Sequencing and analysis of 18S and ITS1 ribosomal DNA revealed a close relationship according to 18S and a relatively distant relationship according to ITS1. Analysis of 18S and ITS1 sequences from commercial turkey coccidiosis vaccines Immucox?-T and Coccivac?-T revealed that each vaccine contains a different strain of E. adenoeides and that these strains have 18S and ITS1 sequences homologous to the sequences of the strains we have isolated and described. These findings show that diagnostics of turkey coccidia according to oocyst morphology have to be carried out with caution or abolished entirely. Novel PCR-based molecular tools will be necessary for fast and reliable species discrimination.     

Further comment on the coccidian nature of cryptosporidia (Sporozoa: Apicomplexa)

The coccidian nature of the genus Cryptosporidium was undoubtedly accepted by Tyzzer who was the first to describe this sporozoan parasite in 1907. Electron microscopic studies made in 70-90s demonstrated the intracellular, although extracytoplasmic localization of Cryptosporidium spp. The pattern of Cryptosporidium life cycle fits well that of other intestinal homogeneous coccidian genera of the suborder Eimeriina: macro- and microgamonts develop independently, a microgamont gives rise to numerous male gametes, oocysts serving for parasite's spreading in the environment. Along with these characters, Cryptosporidium spp. demonstrate some secondary peculiarities (an endogenous phase of development in microvilli of epithelial surfaces, two morphofunctional types of oocysts, the smallest number of sporozoites per oocyst, a multi-membraneous "feeder" organelle etc.), which may be due presumably to their early acquisition of specialization in the course of evolution. The recent studies based on molecular sequence data (18S rRNA) applied to 8 eimeriid and isosporid coccidian genera (Morrison, Ellis, 1997), suggested that the subclass Coccidia (class, according to Morrison and Ellis) be considered monophylic if Cryptosporidium were excluded, and this genus was regarded as the sister group to the rest of the Apicomplexa, or as the sister to the suborder (class) Hematozoa within the Apicomplexa. Either of these placements of Cryptosporidium definitely conflicts with both the generally accepted taxonomic scheme by Levine (1982) and the phenotypically based phylogeny of the phylum Apicomplexa (Barta e. a., 1990). The author's opinion is that the differences between the examined eimeriid and isosporid coccidia, on the one hand, and Cryptosporidium, on the other hand, provided by molecular sequence data, may testify primarily to the well known morphofunctional dissimilarities between the compared organisms, rather than cast doubt on the coccidian nature of Cryptosporidium. Again, these data can hardly prove that Cryptosporidium does not belong to the coccidia. Thus, the modern molecular sequence data, despite their obvious scientific value, would make sense for phylogeny estimation only, if they are critically analysed and considered in combination with results of the relevant basic research.     

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers

• Intracellular parasites of the genus Eimeria are described as tissue/host‐specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species.
• With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non‐Mus isolates, even with this high‐resolution data.
• In conclusion, we show that species‐level resolution should not be assumed for COI and 18S markers in coccidia. Host–parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

     

Comparing the usefulness of distance, monophyly and character-based DNA barcoding methods in species identification: a case study of neogastropoda

Background

DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.

Methodology/Principal Findings

Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.

Conclusions/Significance

This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species.     

A phylogenetic study of chthamaloids (Cirripedia; Thorcica; Chthamaloidae) based on 16S rDNA and COI sequence analysis

The sequences of gene fragments encoding cytochrome  c oxidase subunit I (COI) and 16S rDNA were obtained and used to construct phylograms of eight taxa of chthamaloid barnacles using the scalpelloid Calantica as an out-group. The phylograms support the basal position of Catomerus within the chthamaloids . Analysis of 16S rDNA shows that Octomeris and the four-plated barnacle Chamaesipho are located on the same clade, while Chthamalus , Euraphia and Tetrachthamalus are located on a second clade, indicating that reduction in the number of shell plates occurred twice in the evolution of the chthamaloids. The topology of phylograms based on COI sequences is poorly resolved: 93% of third position nucleotides in this fragment are polymorphic while the amino acid sequences are strictly conserved. We assume that in the chthamaloids, at least at the generic level, polymorphism in the COI gene is saturated beyond phylogenetic information and cannot resolve the phylogenetic relationships within this superfamily.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 39–45.