135 The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence
M.C.JENKINS"*, J.T.ELLIS#, S.LIDDELL",C.RYCE#, B.L.MUNDAY$, D.A.MORRISON# and J. P. DUBEY" " Parasite Biology and Epidemiology Laboratory, Agricultural Research Service, USDA, BARC-EAST, Building 1040, Beltsville, MD 20705, USA # Molecular Parasitology Unit, Department of Cell and Molecular Biology, University of Technology, Sydney, Westbourne Street, Gore Hill, NSW 2065, Australia $ Department of Aquaculture, University of Tasmania at Launceston, PO Box 1214, Launceston, Tasmania 7250, Australia
(Received 30 December 1998; revised 18 February 1999; accepted 18 February 1999)
The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.
Key words: Hammondia, Apicomplexa, phylogenetic analysis.
relationship of H. hammondi to other cyst-forming coccidia and (2) the monophyly of the genus Due to the paucity of structural features to differen- Sarcocystis. tiate among apicomplexan parasites, the sequence of H. hammondi is a coccidian parasite that has an genes encoding slowly evolving ribosomal RNA has obligatory two-host life-cycle (Frenkel & Dubey, been utilized for phylogenetic reconstructions 1975; Dubey, 1993). The phylogenetic relationship (Johnson et al. 1987, 1991; Barta, Jenkins & of this parasite to other cyst-forming coccidia is Danforth, 1991; Gajadhar et al. 1991; Gagnon et al. unknown, but it is thought to be most closely related 1993; Fenger et al. 1994; Holmdahl et al. 1994; Ellis to T. gondii (Frenkel & Dubey, 1975; Sheffield, et al. 1995; Relman et al. 1996). Previous studies Melton & Neva, 1976; Melhorn & Frenkel, 1980; using SSU rDNA from a wide range of apicom- Araujo, Dubey & Remington, 1984; Riahi et al. plexan parasites showed that cyst-forming hetero- 1995, 1998). For instance, both H. hammondi and T. xenous coccidia formed a monophyletic group sep- gondii oocysts are shed by cats after ingestion of arate from the homoxenous coccidia, such as Eimeria tissue cysts present in intermediate hosts such as and Cryptosporidium. Phylogenetic reconstructions rodents. In contrast to Sarcocystis, metrocytes are based on alignment of SSU rDNA sequences showed not formed in H. hammondi nor in T. gondii, but that T. gondii and N. caninum formed a monophyletic bradyzoites within cysts arise from endodyogeny. clade distinct from pathogenic and non-pathogenic Sporogony in both H. hammondi and T. gondii Sarcocystis spp. (Ellis et al. 1994; Holmdahl et al. occurs outside the definitive host in contrast to 1994; Escalante & Ayala, 1995; Ellis & Morrison, endogenous sporogony observed in pathogenic and 1995; Jeffries et al. 1997). The purpose of this study non-pathogenic Sarcocystis spp. (see Tenter & was to examine 2 hypotheses regarding (1) the Johnson, 1997). However, H. hammondi and Sarco- cystis spp. of felids (S. muris and S. gigantea) share * Corresponding author: Parasite Biology and Epidemi- several features that are absent in T. gondii. For ology Laboratory, Agricultural Research Service, USDA, example, H. hammondi, S. muris, and S. gigantea B-1040\Rm-100, BARC-EAST, Beltsville, MD 20705, USA. Tel: j301 504 8054. Fax: j301 504 5306. E-mail: oocysts are not infectious for cats, only capable of mjenkins!lpsi.barc.usda.gov infecting an intermediate host. To determine
Parasitology (1999), 119, 135–142. Printed in the United Kingdom # 1999 Cambridge University Press M. C. Jenkins and others 136 whether H. hammondi and T. gondii are mono- rDNA and to H. hammondi-specific sequences were phyletic, SSU rDNA sequences from these and employed. The DNA sequence was confirmed by at related coccidians were compared. least 3 separate reactions on both DNA strands. Evidence provided elsewhere has indicated that Sarcocystis may be paraphyletic because of the Preparation of S. mucosa DNA and amplification of monophyly of some Sarcocystis species with 18S rDNA Frenkelia (Votypka et al. 1998). Our research has provided extensive evidence to suggest that Sarco- Macroscopic gastrointestinal sarcocysts were cystis is probably composed of at least 3 groups obtained from roadkills of Tasmanian Bennetts (Holmdahl et al. 1999), 1 of which may correspond wallabies (Macropus rufogriseus) (O’Donoghue et al. to the clade sharing a common ancestor with 1987; Jakes, 1998). The cysts were lysed in DNA Frenkelia. It has also been pointed out that a extraction buffer containing 1% SDS, 10 m Tris, characteristic of this group is that they have non- pH 9n0, 100 m EDTA containing proteinase K ruminants as their intermediate host (Holmdahl et (100 µg\ml) at 65 mC for at least 2 h. The released al. 1999). Sarcocystis mucosa exists as macroscopic DNA was purified by phenol\chloroform extraction sarocysts in the gastrointestinal tract of macropodid and desalted by ethanol precipitation. The DNA was marsupials (O’Donoghue et al. 1987). It has been resuspended in 10 m Tris, pH 8n0, 0n1m EDTA suggested that dasyurid marsupials may act as the and used for PCR. The SSU rDNA was amplified definitive host in the life-cycle of this organism by universal primers in 4 overlapping fragments (as (Jakes, 1998). Addition of SSU rDNA data from this described by Holmdahl et al. 1999) and the PCR taxon, may help to support or refute the hypothesis products were sequenced directly by cycle of whether the taxa possessing non-ruminants as sequencing with the aid of an ABI automated intermediate hosts will form a monophyletic group. sequencer. A consensus sequence for the SSU rDNA was generated from at least 3 sequencing runs from each primer. Preparation of H. hammondi DNA and amplification of 18S rDNA Alignment of SSU rDNA sequences and phylogenetic reconstruction H. hammondi oocysts were obtained from faeces of experimental cats fed tissue cysts of mice infected The SSU rDNA sequences of H. hammondi with the H.H-24 strain (Riahi et al. 1995). The (GenBank accession number AF096498) and S. oocysts were isolated by sucrose gradient centri- mucosa were aligned by clustal W (with default fugation, sporulated in 2% H#SO% for 2 weeks at parameters) to SSU rDNA sequences of T. gondii, room temperature, and then stored at 4 mC. H. N. caninum, B. besnoiti (AF109678), I. felis hammondi oocysts were treated with 20% sodium (U85705), I. belli (U94787), I. suis (LSU97523), S. hypochlorite for 10 min to destroy contaminating neurona (U07812), S. muris (M64244), F. glareoli microorganisms, washed several times with distilled (AF009245), F. microti (AF009244), S. sp. H#O, centrifuged and resuspended in sterile H#O. (SSU97524), S. capracanis (AF012885), S. tenella The oocyst suspension was pipetted dropwise into (L19615), S. cruzi (AF017120), S. arieticanis liquid nitrogen and ground to a fine powder in a (L24382), S. gigantea (L24384), S. moulei sterile mortar and pestle. The extracted oocysts were (AF012884), S. buffalonis (AF01712), S. hirsuta resuspended in 0n2 Tris, pH 8n0, 0n1 EDTA, (AF017122), S. fusiformis (SFU03071), S. hominis 0n4 NaCl containing 1 mg\ml proteinase K and (AF006470), S. aucheniae (AF017123), Cyclospora 0n1% SDS and then incubated for 16 h at 50 mC. The sp. (U40261), E. tenella (U40264), E. nieschulzi DNA suspension was extracted with phenol, (U40263) and E. bovis (U40264). The alignment was phenol–chloroform, and chloroform, precipitated imported into MacClade 3.04 and minor modi- with ethanol, pelleted by centrifugation, and fications made to the alignment in order to correct resuspended in sterile 10 m Tris, pH 8n0, 1 m clearly ambiguously placed nucleotides. EDTA and stored at k20 mC. The SSU rDNA from Consensus SSU rDNA sequences were used for H. hammondi was amplified by PCR using conserved both T. gondii and N. caninum and were derived primers as described (Medlin et al. 1988) and cloned from all of the sequence entries available in GenBank into the pCRII vector (Invitrogen, San Diego, CA) for these taxa. The T. gondii (or N. caninum) using techniques provided by the manufacturer. At sequences were aligned by clustal W and a majority least 3 clones from 2 different PCR amplifications rule procedure was used to produce a consensus by were subjected to DNA sequencing analysis. eye using MacClade where different or ambiguous Sequencing was performed on plasmid DNA using character states were identified. The base found in $&S-dATP and the dideoxy chain termination the majority of the aligned sequences was chosen Sequenase kit (U.S. Biochemical, Cleveland, OH). for the consensus sequence. The phylogenetic Primers directed to conserved regions of the SSU relationships between the aligned coccidial Relationship of cyst-forming coccidia 137
neurona to Frenkelia and the relationship of S. aucheniae to the clades containing S. tenella and S. gigantea. These relationships are best summarized in the form of a majority rule consensus tree (Fig. 1) where the H. hammondi, T. gondii and N. caninum can be seen as a polytomy since the relationships between them are not resolved. Besnoitia, as dis- cussed elsewhere is the sister group to these taxa (J. T. Ellis, unpublished observations). In this tree, S. muris, S. mucosa, S. neurona and Frenkelia form a monophyletic group that is the sister group to Isospora, B. besnoiti, N. caninum, H. hammondi and T. gondii, which for convenience we define here as the Toxoplasmatinae. S. sp. (from a rattlesnake) is the sister to this combined group. A distance analysis of the aligned SSU rDNA sequences from the 28 species of coccidia is shown in Table 1. There are few nucleotide differences between the sequences of T. gondii, N. caninum and H. hammondi thus explaining the polytomy observed above. Alignments based on secondary structure Fig. 1. Phylogenetic relationships among the showed that SSU-rDNA encoding helical regions of Sarcocystidae by analysis of SSU rDNA using H. hammondi and T. gondii rRNA were identical and parsimony. A majority rule consensus tree is shown and differed from N. caninum SSU rDNA by only 3 the numbers on the branches represent the percentage nucleotides (not shown). In contrast, approximately support for that branch in the 6 most parsimonious 100 nucleotide differences were detected between trees found. Sarcocystis and H. hammondi, and approximately 200 base differences between H. hammondi and sequences were reconstructed using distance, maxi- Eimeria. Since Ellis & Morrison (1995) and Morrison mum likelihood, and parsimony methods. Distance & Ellis (1997) demonstrated that the strongest analysis was performed by neighbour-joining using phylogenetic signal is located in the helical domains the Kimura 2 parameter model in Treecon (Van de of the SSU rDNA, we conclude that the finding of Peer & De Wachter, 1994). Bootstrap analysis was nucleotide differences between N. caninum and T. performed as follows – 200 new datasets were gondii\H. hammondi in the helices is support for the randomly generated using eseqboot (Felsenstein, conclusion that H. hammondi is the sister taxon to T. 1985, 1988) on the Australian National Genome gondii. This observation is consistent with results Information Service (ANGIS) (Gaeta & Balding, presented elsewhere using ITS1 and partial LSU 1997) and converted into a distance matrix using rDNA comparisons (Ellis et al. 1999) which dnadist and the Kimura-2 parameter model. suggested that H. hammondi and T. gondii may be Ednaneighbour was used to generate the trees for the monophyletic. 200 matrices. The program consense was used to Distance analysis of the data set using neighbour- generate a majority rule consensus tree (Fig. 1). joining gave a tree (Fig. 2) showing similar Maximum likelihood was performed using the relationships to that described by parsimony analysis ednaml program on ANGIS. Global rearrangements (Fig. 1). Because of the size of the data set, bootstrap plus 5 random starts were used. Parsimony analysis analysis was performed by distance methods. Boot- involved the heuristic search procedure in PAUP strapping demonstrated strong support (200\200) 3.1.1. Tree bisection-reconnection plus 10 random for the monophyly of Sarcocystis, Frenkelia, Isospora, starts were used. B. besnoiti, N. caninum, T. gondii and H. hammondi. Strong support (196\200) was also found for the clades of (1) Sarcocystis having ruminants as in- termediate hosts as observed by others (Holmdahl et A SSU rDNA sequence alignment generated by al. 1999) and (2) the Toxoplasmatinae, thus con- clustal W containing 28 taxa and 2040 characters was firming previous findings (Carreno et al. 1998; J. T. analysed phylogenetically. Parsimony analysis using Ellis, unpublished observations). Of interest is that the heuristic search option yielded 6 most par- S. mucosa and S. neurona were monophyletic and simonious trees of 1101 steps (consistency index this conclusion is robust since 153 of 200 bootstraps 0n699; homplasy index 0n301). These trees differed in support this node. This tree differed from parsimony the relationships between H. hammondi, T. gondii analysis in the position of S. hominis, S. sp. and also and N. caninum; the relationship of S. mucosa and S. S. aucheniae. M. C. Jenkins and others 138 3 43 3658 3665 65 3851 63 5757 43 48 6469 65 54 21 50 66 69 65 56 25 49 68 68 80 55 55 77 70 74 59 61 74 72 66 61 16 82 6398 17 77 19 95 17 36 19 96 34 17 95 33 98 35 102 103 82 82 85 83 80 114 75 64 67 77 69 74 41 44 41 51 15 32 32 31 37 22 24 21 211 210200 207 197210 214 197 210 207 201 207 214 201 213 203 205 208 207 197 216 203 193 204 169 193 206 176 155 202 211 162 161 218 200 169 217 208 203 208 208 223 215 197 211 212 208 203 228 208 211 231 215 210 180 216 219 234 167 223 231 219 176 230 216 225 236 222 222 53 227 226 63 229 64 70 68 60 115 111106 111 102107 114 105 103119 116 103 105 114105 123 103 105 118 104109 121 109 106 116 106 107 111 113 114 106 109116 99 113 120 116 110 116115 89 98 120 124 118 115 112119 84 88 100 122 126 115 112 117128 89 83 126 116 113 119 125 78 99 88 92 136 117 119 126 77 92 101 83 90 91 125 127 122 122 129 78 94 100 126 131 124 90 93 90 130 137 107 87 103 93 97 98 91 138 119 132 100 97 20 94 97 111 94 100 45 58 98 116 121 96 97 100 98 103 31 47 111 127 100 102 105 109 145 98 56 97 138 118 104 97 99 104 154 96 136 124 113 95 85 89 102 104 111 86 91 93 106 16 106 111 116 90 124 70 76 110 121 67 73 74 100 121 78 82 71 125 76 121 7 33 37 Hh Tg Nc Bb Ib Is If Sm Sn Fg Fm Sm Ss Sc St Sc Sa Sg Sm Sh Sb Sh Sf Sa Cs Et Eb . 210 209 207 216 205 211 202 207 203 166 174 209 210 217 205 214 209 229 233 175 232 229 234 231 . 858785918895967760576175 hammondi caninum sp gondii besnoiti tenella bovis nieschulzi mucosa neurona glareoli microti muris sp capracanis tenella cruzi arieticanis gigantea moulei hominis buffalonis hirsuta fusiformis aucheniae felis suis belli ...... E E E S S F F S S S S S S S S S S S S S C I I Table 1. Nucleotide sequencegenerated differences using between clustal helical W regions of SSU rDNA from the Apicomplexa determined by distanceH analysis of alignments T N B I Relationship of cyst-forming coccidia 139
is the sister to the Toxoplasmatinae; S. muris is the sister to Toxoplasmatinae and the clade containing the F. microtijF. glareoli S. mucosajS. neurona. The likelihood-ratio test (Felsenstein, 1988) was performed in order to test whether the maximum likelihood tree was any better or worse than the other trees found in these analyses. It indicates that none of these trees are statistically significantly different from each other (P " 0n05).
In this study we set out to examine 2 hypotheses, the relationship between H. hammondi and T. gondii, and whether Sarcocystis species having non- ruminants as an intermediate host form a mono- phyletic group. Frenkel (1977) divided the Sarco- cystidae into 2 subfamilies, Toxoplasmatinae and Sarocystinae. Recent phylogenetic analyses have provided considerable support for this division Fig. 2. Phylogenetic relationships among the (J. T. Ellis, unpublished observations). In addition, Sarcocystidae inferred by neighbour-joining and the Eimeridae is considered to be the sister group to the Kimura 2 parameter model. The bootstrap values Sarcocystidae. Therefore the approach used in these represent the support (out of 200) for that branch. analyses was the alignment of sequences of the Sarcocystidae to 3 representative sequences of Eimeria plus Cyclospora (Relman et al. 1996). Using this approach, phylogenetic analyses by distance, maximum likelihood, and parsimony methods gave trees possessing consistent relation- ships among the taxa. Specifically, the branch leading to T. gondiijH. hammondijN. caninum is robust, although the 3 taxa yield a polytomy since the amount of variation between the 3 sequences is very small. Our recent analyses of partial LSU rDNA and ITS1 data indicated that T. gondi and H. hammondi are probably monophyletic (Ellis et al. 1999). The analyses from the SSU rDNA presented here are therefore consistent with this conclusion. Besnoitia, represented in this study by B. besnoiti, is the sister group to N. caninum, T. gondii, and H. hammondi. Previous studies on Besnoitia of cattle, wildebeest and goats also revealed strong support for this monophyletic group (J. T. Ellis, unpublished observations). The 3 species of Isospora formed a monophyletic group which was the sister to Besnoitiaj HammondiajNeosporajT. gondii. This grouping Fig. 3. Phylogenetic relationships among the supports the hypothesis for a separate genus within Sarcocystidae inferred by maximum likelihood analysis. the Sarcocystidae that contains Isospora species The branch lengths are proportional to the amount of (Frenkel, 1977). The names Cystoisospora or Levineia inferred evolutionary change, as shown by the scale bar. were originally suggested; however, one of the criteria for establishing this genus included The tree obtained using maximum likelihood (Fig. heteroxeny which is not fulfilled by the inclusion of 3) had a log likelihood of k9218n9. This tree is homoxenous Isospora (I. suis and I. belli) in this similar to the consensus majority rule tree obtained clade. from parsimony analysis (Fig. 1). H. hammondi is the The analyses of the Sarcocystidae demonstrated sister taxon to T. gondii. The positions of S. sp., S. strong support for 3 monophyletic groups. These muris and S. aucheniae on the tree are different. S. sp. groups are present in all 3 analyses and have a high M. C. Jenkins and others 140 bootstrap support in the neighbour-joining tree. dation might be to incorporate S. neuronajS. They are (1) the Toxoplasmatinae (here defined mucosajS. muris and Frenkelia into a new genus, as IsosporajBesnoitiajN. caninumjT. gondiijH. given they potentially show monophyly. hammondi), (2) a monophyletic group of ruminant Several hypotheses have been made regarding the Sarcocystis which contain species forming microcysts evolutional biology and phylogeny of the coccidia and with dogs as their definitive host, and (3) a (reviewed by Tenter & Johnson, 1997). One idea was second monophyletic group of ruminant Sarcocystis that homoxenous coccidia developed from hetero- containing those species forming macrocysts and xenous coccidia by simplifying the life-cycle; with cats as their definitive host. another was that heteroxenous coccidia evolved from The remaining group of species, containing ancestral homoxenous coccidia. Our analysis indi- Frenkelia, S. mucosa, S. neurona, S. muris and S. sp., cates that the latter hypothesis (suggested by Tadros is less robust. The placement of S. muris and S. sp., & Laarman, 1982) is more likely, as it requires only in particular, was unstable. Similar analyses were a single origin of the heteroxenous life-style, with a also performed using sequence alignments based on reversal to the homoxenous condition in Isospora, that described by Van de Peer et al. (1997), which whereas the first hypothesis requires 3 separate defines the complete secondary structure of the SSU origins of heteroxeny. Furthermore, all the available rRNA molecule. The results obtained were es- information (Holmdahl et al. 1999) and the present sentially the same as those described here. In these study suggest that the nature of the intermediate analyses the position of S. muris and S. sp. were also host (ruminant\non-ruminant) represents an im- unstable. The relatedness of S. muris and S. neurona portant influence on the evolutionary biology of is interesting. Although S. muris is transmitted via Sarcocystis. Toxoplasma and Neospora have multiple cats to mice, it is most closely related to S. neurona. intermediate hosts, and therefore have acquired Sarcocystis falcatula and S. neurona are related ruminant hosts independently of Sarcocystis. organisms utilizing opposum as the definitive host. S. neurona has as its definitive host the Virginia Although initial phylogenetic studies indicated that opposum, a marsupial (Dubey & Lindsay, 1998). there were the same organism (Dame et al. 1995; The close relationship of S. neurona to S. mucosa is Fenger et al. 1995), recent studies have shown that another compelling example supporting the concept they have unique biological, antigenic, and structural of a prehistoric Gondwana with continuous land characters (Dubey & Lindsay, 1998; Dubey, Speer connections between the present South America and & Lindsay, 1998). S. neurona causes neurological Australia (Veevers, 1991). There is evidence that disease in horses in the Americas and is not infective both these organisms, S. neurona from America and to birds. Furthermore, S. neurona causes neuro- S. mucosa from Australia, have carnivorous mar- logical disease in immunodeficient mice whereas S. supials as their definitive hosts (Dubey & Lindsay, falcatula is not infectious to immunodeficient mice 1998; Jakes, 1998). This concept is further (Marsh et al. 1997; Dubey & Lindsay, 1998). More strengthened by the observation that the marsupial data from representative taxa are needed, from S. genera Didelphis and Dasyrus are sister groups falcatula or from Sarcocystis that infect reptiles for (Kirsch & Mayer, 1998). example, to resolve the relationships among the branches in this region of the tree. The lack of resolution in this part of the tree appears to be the result of short branches, indicating , . ., , . . , . . (1984). a slow evolutionary rate. The likelihood ratio test Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi. Journal (Felsenstein, 1988), comparing the maximum like- of Protozoology 31, 145–147. lihood tree with the molecular clock to that without , . ., , . . , . . (1991). the molecular clock, rejects the molecular clock for # Evolutionary relationships between avian Eimeria these data (χ l 68n21, P ! 0n001). The short branch species among other apicomplexan protozoa: lengths occur in that part of the tree containing monophyly of the Apicomplexa is supported. Frenkelia, S. neurona, S. mucosa and S. muris. Thus Molecular Biology and Evolution 8, 345–355. there is less informative phylogenetic information , . ., , . ., , . ., , among these taxa. . ., , . . , . . (1998). It has been suggested that Sarcocystis is para- Phylogenetic analysis of coccidia based on 18S rDNA phyletic and that Frenkelia should be incorporated sequence comparison indicates that Isospora is most into the genus Sarcocystis (Votypka et al. 1998). The closely related to Toxoplasma and Neospora. Journal of Eukaryotic Microbiology 45, 184–188. data presented here show FrenkeliajS. neuronajS. , . . , . ., , . ., , . ., mucosa to be monophyletic and the sister group to , . , . . (1995). Sarcocystis falcatula the Toxoplasmatinae. If one was to include Frenkelia from passerine and psittacine birds: Synonymy with in Sarcocystis then it would be necessary to in- Sarcocystis neurona, agent of equine protozoal corporate all the other taxa mentioned into Sarco- myeloencephalitis. Journal of Parasitology 81, cystis as well. A much more practical recommen- 930–935. Relationship of cyst-forming coccidia 141
, . . (1993). Toxoplasma, Neospora, Sarcocystis, , . ., , . ., , ., and other cyst-forming coccidia of humans and , ., -, . . , . . animals. In Parasitic Protozoa,2nd Edn. Vol. 6 (ed. (1991). Ribosomal RNA sequences of Sarcocystis Kreier, J. P.), pp. 1–158. Academic Press, San Diego. muris, Theileria annulata and Crypthecodinium cohnii , . . , . . (1998). Isolation in reveal evolutionary relationships among immunodeficient mice of Sarcocystis neurona from apicocomplexans, dinoflagellates, and ciliates. opposum (Didelphis virginiana) faeces, and its Molecular and Biochemical Parasitology 45, 147–154. differentiation from Sarcocystis falcatula. International , . . , . (1997). Molecular phylogeny. Journal for Parasitology 28, 1823–1828. In ANGIS Bioinformatics Handbook, vol. 3, pp. , . ., , . . , . . (1998). 3.1–3.43. CSIRO Publishing, Collingdale, Australia. Isolation of a third species of Sarcocystis in , ., , . ., , . . , immunodeficient mice fed feces from opposums . . (1993). Molecular cloning, complete sequence of (Didelphis virginiana) and its differentiation from the small subunit ribosomal RNA coding region and Sarcocystis falcatula and Sarcocystis neurona. Journal phylogeny of Toxoplasma gondii. Molecular and of Parasitology 84, 1158–1164. Biochemical Parasitology 60, 145–148. , . ., , ., , . ., , . ., , . . ., , . ., , . , . . , . . (1994). The phylogeny , . . (1994). The phylogeny of Neospora of Neospora caninum. Molecular and Biochemical caninum and Toxoplasma gondii based on ribosomal Parasitology 64, 303–311. RNA sequences. FEMS Microbiology Letters 119, , . . , . (1995). Effects of sequence alignment on the phylogeny of Sarcocystis deduced 187–192. from 18S rDNA sequences. Parasitology Research 81, , . . ., , . ., , . . 696–699. , . . . (1999). Evolution of ruminant , . ., , ., , . ., , Sarcocystis (Sporozoa) parasites based on small ., , . . , . . (1995). subunit rDNA sequences. Molecular Phylogeny and Phylogenetic relationships between Toxoplasma and Evolution (in the Press). Sarcocystis deduced from a comparison of 18S rDNA , . . (1998). Sarcocystis mucosa in Bennetts sequences. Parasitology 110, 521–528. wallabies and pademelons from Tasmania. Journal of , . ., , . ., , ., , . ., Wildlife Diseases 34, 594–599. , . ., , ., , . . . , . ., , ., , . ., , , . . (1999). The genus Hammondia is . . , . . (1997). Identification of paraphyletic. Parasitology 118, 357–362. synapomorphic characters in the genus Sarcocystis , . . , . . (1995). Evolutionary based on 18S rDNA sequence comparison. Journal of origin of Plasmodium and other Apicomplexa based on Eukaryotic Microbiology 44, 388–392. rRNA genes. Proceedings of the National Academy of , . ., , . ., , . , Sciences, USA 92, 5793–5797. . . (1987). Rapid nucleotide sequence analysis of the , . (1985). Confidence limits on small subunit ribosomal RNA of Toxoplasma gondii: phylogenies: An approach using the bootstrap. evolutionary implications for the Apicomplexa. Evolution 39, 783–791. Molecular and Biochemical Parasitology 25, 239–246. , . (1988). Phylogenies from molecular , . ., , ., , ., ’, . . sequences: inference and reliability. Annual Reviews of , . . (1991). The phylogenetic Genetics 22, 521–565. relationships of the genus Eimeria based on , . ., , . ., , . ., comparison of partial sequences of 18S rRNA. , ., , ., , . ., Systematic Parasitology 18, 1–8. , . , . . (1994). Phylogenetic , . . , . . (1998). The platypus is not a relationships of Sarcocystis neurona to other members rodent: DNA hybridization, amniote phylogeny, and of the family Sarcocystidae based on small subunit the palimpset theory. Philosophical Transactions of the ribosomal RNA gene sequence. Journal of Parasitology Royal Society of London, B 353, 1221–1237. 80, 966–975. , . ., , . ., , ., , ., , ., , . ., , . ., , . ., , . , . . (1997). In vitro cultivation , ., , . ., , . ., and experimental inoculation of Sarcocystis falcatula , . ., , . ., , . Sarcocystis neurona , . . (1995). Identification of opposums and merozoites into budgerigars (Didelphis virginiana) as the putative definitive host of (Melopsittacus undulatus). Journal of Parasitology 83, Sarcocystis neurona. Journal of Parasitology 81, 1189–1192. 916–919. , ., , . ., , . , . . , . . (1977). Besonoitia wallacei of cats and (1988). The characterization of enzymatically rodents: With a reclassification of other cyst-forming amplified eukaryotic 16S-like rRNA-coding regions. isosporoid coccidia. Journal of Parasitology 63, Gene 71, 491–499. 611–628. , . , . . (1980). Ultrastructural , . . , . . (1975). Hammondia comparison of cysts and zoites of Toxoplasma gondii, hammondi gen. nov., sp. nov., from domestic cats, a Sarcocystis muris, and Hammondia hammondi in new coccidian related to Toxoplasma and Sarcocystis. skeletal muscle of mice. Journal of Parasitology 66, Zeitschrift fuW r Parasitenkunde 46, 3–12. 59–67. M. C. Jenkins and others 142
, . . , . . (1997). Effects of cultures. Proceedings of the Helminthological Society of nucleotide sequence alignment on phylogeny Washington 43, 217–225. estimation: a case study of 18S rDNAs of , . , . . (1982). Current concepts on Apicomplexa. Molecular Biology and Evolution 14, the biology, evolution and taxonomy of tissue cyst- 428–441. forming eimeriid coccidia. Advances in Parasitology ’, . ., , . ., ’, . ., 20, 293–468. , . , . . (1987). Sarcocystis mucosa , . . , . . (1997). Phylogeny of the (Blanchard 1885) Labbe 1889 in unadorned rock tissue cyst-forming coccidia. Advances in Parasitology wallabies (Petrogale assimilis) and Bennett’s wallabies 39, 69–139. (Macropus rufogriseus). Parasitology Research 73, , ., , ., , . , . 113–120. (1997). Database on the structure of small ribosomal , . ., , . ., , ., , ., subunit RNA. Nucleic Acids Research 25, 111–116. , ., , ., , . , . , . , . (1994). TREECON (1996). Molecular phylogenetic analysis of Cyclospora, for Windows: a software package for the construction the human intestinal pathogen, suggests that it is and drawing of evolutionary trees for the Microsoft closely related to Eimeria species. Journal of Infectious Windows environment. Computer Applications in Diseases 173, 440–445. Bioscience 10, 569–570. , ., , . ., , ., , . . , . . (1991). Phanerozoic Australia in the -, . (1995). Hammondia hammondi changing configuration of Proto-Pangea through cysts in cell cultures. Journal of Parasitology 81, Gondwanaland and Pangea to the present dispersed 821–824. continents. Australian Systematic Botany 4, 1–11. , ., , . , . . (1998). Antigenic , ., , ., , ., , ., , . similarity between Hammondia hammondi and , . (1998). Molecular phylogenetic relatedness of Toxoplasma gondii tachyzoites. Journal of Parasitology Frenkelia spp. (Protozoa, Apicomplexa) to Sarcocystis 84, 651–653. falcatula Stiles 1893: is the genus Sarcocystis , . ., , . . , . . (1976). paraphyletic? Journal of Eukaryotic Microbiology 45, Development of Hammondia hammondi in cell 137–141.