Evolutionary Relationships of Avian Eimeria Species among Other Apicomplexan Protozoa: Monophyly of the Apicomplexa Is Supported 1

John R. Barta, * Mark C. Jenkins,? and Harry D. Danforth? *Department of Pathology, University of Guelph, and tU.S. Department of Agriculture, Protozoan Diseases Laboratory

Direct, reverse transcriptase-mediated, partial sequencing of the small-subunit ( 16S- like) ribosomal RNA (srRNA) of Eimeria tenella and E. acervufina was performed. Sequences were aligned by eye with six previously published, partial or complete srRNA sequences of apicomplexan protists (Plasmodium berghei, Theileria an- nulata, Cryptosporidium sp., Toxoplasma gondii, Sarcocystis muris, and S. gigan- tea). Six eukaryotic protists (a slime mold, a yeast, two dinoflagellates, and two ciliates) acted as an outgroup for a parsimony-based phylogenetic analysis (PAUP Ver. 3.0.). The 188 phylogenetically informative sites (i.e., those positions that neither were unvaried nor had only autapomorphic substitutions) supported a single tree topology 481 steps in length with a consistency index of 0.65 in which the monophyly of the Apicomplexa was supported. The two Eimeria species and S. muris, S. gigantea, and T. gondii formed a pair of monophyletic groups that were sister groups. The two Sarcocystis species were not hypothesized to he sister taxa. The genera Plasmodium and Cryptosporidium were hypothesized to form the sister group to these five coccidia and T. annulata. A priori data-editing techniques that deleted “variable” positions prior to analysis failed to recognize the monophyly of the Apicomplexa when the same parsimony-based tree-building algorithm was used. Inability of the outgroup taxa to root the well-supported ingroup tree ( Apicomplexa) at a unique site when these taxa were used individually for this purpose reinforces the need for an appropriate, multiple-taxon outgroup in such analyses.

Introduction

The eimeriid parasites (family Eimeriidae) infecting the intestinal epithelium of Galliformes have been considered to be a relatively ancient group of Apicomplexa. Some authors have suggested that biologically similar gut parasites were the ancestors of the malarial organisms (Plasmodium spp.) and piroplasms (see Manwell 1955; Mattingly 1965; Levine 1988, p. 4). However, a recent phylogenetic analysis, based on morphological and biological characteristics (Barta 1989) of the class Sporozoea, suggested that species of Eimeria may be highly derived parasites. In the same analysis, species of Plasmodium and the piroplasms (Babesia and Theileria spp.) were found to be distantly related to the higher eimeriids. Recent phylogenetic analyses of api- complexan parasites, analyses based on the semiconserved regions of partial (Johnson et al. 1987, 1988, 1990) or complete (Gajadhar et al., accepted) sequences of small-

I. Key words: ribosomal RNA, sequencing, maximum parsimony, Apicomplexa, a priori editing, phy- logeny.

Address for correspondence: J. R. Bar& Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N 1G 2W I.

Mol. Bid. Evol. 8(3):345-355. 1991. 0 199 I by The University of Chicago. All rights reserved. 0737-4038/91/0803-0006$02.00

345

346 Barta et al.

subunit ribosomal RNA (srRNA), also suggested that the malarial parasites may not be especially closely related to the coccidian parasites of the genera Toxoplasma and Sarcocystis. Indeed, Johnson et al. ( 1990) have questioned the monophyly of the Apicomplexa. Our phylogenetic analysis based on the partial sequencing of srRNA from E. acervulina Tyzzer 1929 and E. tenella (Raillet and Lucet 189 1) Fantham 1909 supports both the results obtained from morphological and biological characters (Barta 1989) and the monophyly of the Apicomplexa. The apparent lack of support for monophyly of the Apicomplexa (Johnson et al. 1987, 1990) may be an artifact of the a priori data-editing techniques used in the latter analyses. We question the utility of such editing.

Material and Methods Preparation of RNA

Approximately 1 O9 oocysts were obtained from chickens experimentally infected with 5 X lo5 oocysts of Eimeria acervulina (API strain 12) or 5 X lo4 E. tenella (API strain 80). Oocysts of E. tenella were collected by caecal harvest. Oocysts of E. acervu- lina were acquired by fecal collection at 6-9 d postinoculation. Oocysts were purified by sugar-flotation techniques and were permitted to sporulate for -7 h prior to RNA extraction. Purified oocysts were extracted by homogenizing in the presence of RNA extraction buffer (4 M guanidine isothiocyanate, 0.1 M P-mercaptoethanol, 10 mM ethylenediaminetetraacetate, 5 mM sodium citrate, and 0.5% sodium sarcosine). The parasite extract was resuspended in 1.2 vol of cesium trifluoroacetate (density 2 g/ ml; Pharmacia) and was banded by ultracentrifugation to isolate total RNA (Zarlenga and Gamble 1987).

Sequencing

Sequencing was performed according to a method described elsewhere (Lane et al. 1985), with minor modifications. Commercial oligonucleotide primers from Boehringer Mannheim Biochemicals (Indianapolis) [corresponding to positions 5 19- 536 (primer A), 907-926 (primer B), and 1392- 1406 (primer C) of Escherichia coli as defined by Brosius et al. 198 l] were annealed to -7.0 pg of parasite RNA. An aliquot (6.5 U) of reverse transcriptase isolated from avian myeloblastosis virus (Bio- Rad, Richmond, CA) was used in the sequencing reactions. Samples were heated to 90°C for 2 min prior to separation on a 7 M urea, nongradient, 5% polyacrylamide gel operated at - 1,200 V. Samples from each reaction were run for 2, 4, and 6 h to extend the sequence available for phylogenetic analysis. A minimum of three complete sequencing reactions and electrophoresis runs were made for each primer with each species to confirm the accuracy of the partial sequences obtained.

Phylogenetic Reconstructions

Sequences obtained in the present study were aligned by eye both with published sequences for six other apicomplexan taxa (see Gunderson et al. 1986; Johnson et al. 1987, 1988, 1990; Gajadhar et al., accepted) and with complete or partial sequences from six other protists. The outgroup consisted of two dinoflagellates [ Prorocentrum micans (see Herzog and Maroteaux 1986) and Crypthecodinium cohnii (see Gajadhar et al., accepted)], a yeast [ Saccharomyces cerevisiae (see Rubtsov et al. 1980)], two ciliates [ Oxytricha nova and Stylonychia pustulata (see Elwood et al. 1985)], and a cellular slime mold [ Dictyostelium discoideum (see McCarroll et al. 1983)]. All nu- cleotide positions were unambigously aligned (fig. 1)) except in small areas adjacent

Apicomplexan Phylogeny from rRNA Sequences 347

to the lengthy inserts found with Plasmodium berghei. All nucleotide positions-and only those-that were neither unvaried nor had only autapomorphic substitutions were included. This is called the “complete data set.” Swofford’s ( 1990) parsimony- based tree-building program [ phylogenetic analysis using parsimony ( PAUP), version 3.01 was used for all analyses. A branch-and-bound algorithm guaranteed to find the most parsimonious tree(s) was used in all cases. All characters were unordered and were uniformly weighted. Autapomorphic character transformations (character states possessed by only one taxon in the analysis) were not included in the calculation of the consistency indices (CIs) for the trees but have been mapped onto the tree presented in figure 2.

Ingroup Tree Rooting

Determination of closely related sister taxa to form a taxonomic outgroup for the apicomplexan ingroup on the basis of morphological or biological characters re- mains uncertain (Barta 1989); for the Apicomplexa no sister group is recognized that can be used as a taxonomic outgroup. Therefore, six protozoan taxa were used as the taxonomic outgroup. To test the consistency and suitability of the rooting point ob- tained by using this outgroup, the dinoflagellates, ciliates, yeast, and cellular slime mold were used individually to root the ingroup network. A new character set was constructed for each phylogenetic reconstruction.

A Priori Data Editing

The utility of analyzing only semiconserved regions was examined by dividing the sequences into semiconserved and variable regions in two ways. In the first way, the semiconserved regions defined by Johnson et al. ( 1990) were accepted, the re- mainder of the sequence being,considered variable. In the second way, the variable regions were those regions that seemed to us to have a high concentration of di- and multitypic sites (see fig. 1)) the remainder of the sequence being considered s’emicon- served. In either case, the semiconserved and variable character sets are the subsets of those positions in the complete data set that were assigned according to the defined nature of the region in which they resided. By the second way, eight variable regions that corresponded to helices 15-> 15’, 16-> 16’, 17-> 17’, P21.1, 33->34->34’, 35->35’, 36->36’, and 38->38’ of the secondary-structure map for Pr. micans (see Herzog and Maroteaux 1986) were identified (see fig. 1). We used a complete character character set and each of the two variable and the two semiconserved character sets to construct phylogenies to see how editing affected the results.

Results

In figure 1 the sequences obtained from avian myeloblastosis virus/reverse tran- scriptase-mediated sequencing of Eimeria species ribosomal RNA are aligned with those of six other apicomplexan protozoa and with those of the six outgroup taxa. A total of 566 nucleotides were sequenced from the srRNA of E. tenella, and 650 from nucleotides were sequenced E. acervulina. After alignment, 188 evolutionarily infor- mative sites excluding gaps and autapomorphies were recognized and constituted the complete set for parsimony analyses; 132 additional sites contained autapomorphic base changes.

Phylogenetic analysis using the complete character set revealed a single most parsimonious tree topology of 48 1 steps, including autapomorphic positions (fig. 2), with a CI of 0.65 for informative characters only; overall, the CI for the tree was 0.73.

348 Barta et al.

A-PRIMER

Removing from the analysis all characters with missing or unknown nucleotides had no effect on the results. The 99 characters that had no missing or unknown nucleotides generated an identical tree topology. Mapping the deleted characters (those containing missing or unknown nucleotides) onto the latter tree resulted in hypothesized character- state transformations that were identical with those found when the tree had been calculated by using the complete data set.

As expected, the two avian Eimeriu species included in the present analysis were the most closely related apicomplexan taxa. The three remaining morphologically similar coccidia (Toxoplasma gondii and the two Sarcocystis spp.) formed a second monophyletic group. It is interesting that the two Sarcocystis species did not form a monophyletic group. Together, the five coccidia formed a monophyletic group. The piroplasm, Theileria annulata, was hypothesized to be the sister taxon to the five

Apicomplexan Phylogeny from rRNA Sequences 349

FIG. 1 .-Sequence alignments used in maximum-parsimony phylogenetic reconstruction. Numerals in the upper left corner of each block of sequences indicate the nucleotide position of the Plasmodium berghei sequence (see Gunderson et al. 1986). Double underlining indicates sequences used by Johnson et al. ( 1990). Letters above the aligned sequences identify autapomorphic (a), ditypic (d) , and multitypic (m) sites and regions where independent inserts are hypothesized ( ) <-- * * * --> ( ); these regions were not assigned as autapomorphies and were ignored during the generation of characters from the aligned sequences. Variable regions that corresponded to helices 15>15’, 16->16’, 17->17’, P21.1, 33->34->34’, 35->35’, 36->36’, and 38-x38’ of the secondary-structure map for Prorocentrum micuns (see Herzog and Maroteaux 1986) are indicated by single underlining below these letters. The remaining regions were considered semiconserved. A dash (-) denotes gaps added to improve alignment; a question mark (?) denotes an unknown nucleotide; and an asterisk (*) denotes that region was not sequenced.

coccidia. Members of the genus Cryptosporidium were hypothesized to be the sister taxa to the malarial parasites of the genus Plasmodium. The six outgroup taxa rooted the taxonomic ingroup between a monophyletic group consisting of the genera Plus- modium and Cryptosporidium and the monophyletic group consisting of the five coc-

350 Barta et al.

6 . ..f 34 Eimeria acervulina

29

16

L?- Sarcocystis murk 13

15 12 Sarcocystis gigantea

Toxoplasma gondii

-?. Theileria annulata

19 sp.

Plasmodium berghei

..____........._.... 52 ._....___....__ DjcQqjste/jum

FIG. 2.-Most parsimonious tree using six protists as taxonomic outgroup. Dictyostelium discoideum was used to root the remaining taxa. Horizontal distance represents the number of hypothesized character- state transformations occurring along each branch. Dotted regions indicate autapomorphic changes mapped onto the tree after the parsimony analysis. Monophyly of the eight apicomplexan taxa is supported.

cidian taxa and the piroplasms (fig. 2 ) . As predicted on morphological grounds (Levine 1988, p. 4)) the most closely related taxa among the outgroup were the dinoflagellates Prorocentrum micans and Crypthecodinium cohnii.

When the dinoflagellates, ciliates, yeast, or cellular slime mold was used to root the ingroup tree individually, the rooting point varied as illustrated in figure 3. The following three rooting locations were identified: ( 1) as in the multiple-taxon outgroup analysis between the genera Cryptosporidium and Plasmodium and the remaining apicomplexan taxa [ fig. 3(a)] ; (2) between the genus Cryptosporidium and the re- maining apicomplexan taxa [ figs. 3(b) and (c)l; and (3) between the genus Plas- modium and the remaining apicomplexan protozoa [ figs. 3 (c) and (d)] .

Characters from semiconserved regions, regardless of the a priori editing procedure used, did not support the monophyly of the Apicomplexa [ figs. 4(a) and 5 (a)]. The editing used by Johnson et al. ( 1990) in their phylogenetic analysis, which included four of the eight apicomplexan taxa in the present report, produced a condensed character set of 64 characters from which eight equally parsimonious trees [ fig. 4 (a)] were constructed (CI = 0.69; 147 steps); the strict-consensus tree (see Rohlf 1982) from these eight equally parsimonious topologies revealed few resolved branching points. Use of semiconserved regions between the variable sites (located in the helices as listed above) yielded 78 characters that produced 36 equally parsimonious trees (CI = 0.64; 170 steps); the strict-consensus tree for these trees did not support the monophyly of the apicomplexan taxa [ fig. 5 (a)] ; few branches were resolved. In contrast, the characters derived from “variable” regions (those characters excluded

Apicomplexan Phylogeny from rRNA Sequences 35 I

Ctypthecodinium Prorocentrum H=2OSteps

(a) 291 Steps; cko.71 W 285stqs;c1=0.80

Eimeria acervulina

SarwcysUs muds Sarcocystis gigantea Toxoplasma gondii

Theileria annulata

Plasmodium berghei

Eimeria acenrulina

Sarcocystis giganiea Toxoplasma gondii

Theileria annulata Cryptosporidium sp.

Plasmodium berghei

Eimeda acervulina

Sarc~b muds Sarcocysbs gigantea Toxoplasma gondii

Theileria annulata

Cryptosporidium sp. Plasmodium berghei

(c) Tree 1. 268 Steps; CI=O.75 (c) Tree 2. 268 Steps; CI=0.75 (d) 291 Steps; CI=O.T7

FIG. 3.-Effect on rooting point of ingroup taxa when outgroup taxa are used singly. Outgroup taxa used were as follows: (a), Prorocentrum micans and Crypthecodinium cohnii; (b), Stylonichia pustulafa and Oxytricha nova; (c), Saccharomyces cerevisiae; and (d), Dictyostelium discoideum. Tree (a) corresponds to the rooting point in fig. 2. Horizontal distance represents the number of hypothesized character-state transformations occurring along each branch; autapomorphic character transformations are not shown.

from the analyses above) produced single, most parsimonious trees [ figs. 4(b) and 5(b)] that supported the monophyly of the ingroup (fig. 2). In the first case (editing is as in Johnson et al. 1990), the 124 discarded characters produced a single tree (CI = 0.64; 335 steps) that supported the monophyly of the Apicomplexa but differed from the tree generated by using the entire data set in the relationships hypothesized among the apicomplexan genera Plasmodium, Theileria, and Cryptosporidium [fig. 4(b)]. In the second case, the 110 characters found within variable regions (corre- sponding to the helices listed above) produced a single most parsimonious tree (CI = 0.65; 309 steps) that was identical in topology to that generated by the entire data set [fig. 5(b)].

\

Discussion

The phylum Apicomplexa Levine 1970 consists of protists which have been grouped together on the basis of the possession of several ultrastructural components that together form the apical complex (see Levine 1988, p. 6). All members of this phylum are parasitic organisms which share a wide variety of biological, biochemical, and ultrastructural characteristics (Levine 1988, p. 6); thus, there is significant in- dependent synapomorphic support for the monophyly of the Apicomplexa. Among the eight apicomplexan taxa included in the present analysis are species that have been hypothesized, on the basis of morphological and biological characteristics, to form monophyletic groups (Barta 1989). The genus Eimeria has been hypothesized to be the sister taxon to the genera Toxoplasma and Sarcocystis (see Levine 1988; Barta 1989). Maximum-parsimony analysis supported the monophyly of eight mem- bers of the phylum Apicomplexa. The long branch lengths observed among taxa in the phylum support the concept of an ancient origin of its members.

352 Barta et al.

Eimeria acervulina

i :

Eimeria acetvulina

Eimeria tenella Eimeria tenella

Sarcocystis murk Sarcocystis muris

Sarcocystis gigantea Sarcocystis gigantea

Toxoplasma gondii Toxoplasma gondii

Theileria annulata - Cryptosporidium sp.

Crypthecodinium Theileria annulata

Prorocentrum Plasmodium berghei

Plasmodium berghei Crypthecodinium

Cryptosporidium sp. Prorocentrum

Stylonychia

4 Oxyfricha

Stylonychia

Oxytricha

Saccharomyces Saccharomyces

(a) k Dictyostelium (b) Dictyostelium

FIG. 4.-Effect of a priori editing technique of Johnson et al. ( 1990). (a), Strict consensus tree ( Rohlf 1982) of eight equally parsimonious trees (CI = 0.69; 147 steps) generated by using regions defined by Johnson et al. ( 1990) as being semiconserved. Monophyly of the Apicomplexa is not supported. (b), Single most parsimonious tree (CI = 0.64; 335 steps) generated by using variable regions not used by Johnson et al. ( 1990), supporting monophyly of apicomplexan taxa. Only branching order is shown; branch lengths are not proportional to hypothesized evolutionary change.

Hypothesized definitive host-parasite coevolution (Barta 1989) also suggests that the phylum is of impressive antiquity. On the basis of morphological criteria, sugges- tions have been made that the apicomplexan protozoa may share a most recent com- mon ancestor with the dinoflagellates (Levine 1988, p. 4) and srRNA sequences (Johnson et al, 1990; Gajadhar et al., accepted). This is supported by our analysis, where the dinoflagellates Prorocentrum micans and Crypthecodinium cohnii were identified as the most closely related outgroup taxa. The only point of divergence between the present srRNA-based phylogenetic reconstruction and one based on mor- phological/biological characters (Barta 1989) was in the relationships among the genera Plasmodium, Theileria, and Cryptosporidium. Additional intermediate species may resolve this conflict. The consensus of phylogenies that was derived from independent data sets has been promoted as strong evidence of a true phylogeny (Ruse 1979; Hillis 1987; Crowe 1988). Thus, congruence of Barta’s (1989) reconstruction based on morphological/biological characters for the coccidia with the srRNA-based phylogeny appears supportive of these relationships among the apicomplexan taxa.

The differences in results obtained in both our study and those of Johnson et al. ( 1990)) based on partial srRNA sequences, do not appear to be the result of differences in tree-building philosophies, because the global parsimony criterion was used in both cases (results of Johnson et al. that are based on distance-matrix analyses are not included in this discussion). Instead, the variation in results appears to be related to the a priori editing technique used by the latter authors and possibly also to the inclusion of inappropriate, ancient outgroup taxa. These may be related difficulties. Inclusion of taxa that are too distantly related to the taxonomic ingroup may make alignment of large portions of the sequences impossible. The inability to unambiguously align sequences in the data set may force the deletion of the variable regions that we hy- pothesize to contain the information necessary to resolve the relationships among the

Apicomplexan Phylogeny from rRNA Sequences 353

c Eimeria acervulina

Eimeria tenella

- Sarcocystis muds

c

Sarcocystis gigantea

Toxoplasma gondii

- Theileria annulata

- Cryptosporidium sp.

rl- Plasmodium berghei

Crypthecodinium

Prorocentrum

Sty/onychia

Oxytricha

Saccharomyces

Eimeria acervulina

Eimeria tenet/a

Sarcocystis muris

Sarcocystis gigantea

Toxoplasma gondii

Theileria annulata

Cryptosporidium sp.

Plasmodium berghei

Prorocentrum

(a) L Dictyostelium (W L t%tyoste/ium

FIG. 5.-Effect of removing variable regions identified in fig. 1. (a), Strict consensus tree of 36 equally parsimonious trees (CI = 0.64; 170 steps) resulting from characters from “semiconserved” regions. Monophyly of the Apicomplexa is not supported. (b), Single most parsimonious tree (CI = 0.65; 309 steps) resulting from characters found within eight variable regions. This is identical in branching order to the tree generated by using the entire data matrix (fig. 2). Only branching order is shown; branch lengths are not proportional to hypothesized evolutionary change.

terminal taxa. Although homology of the srRNA molecules under study is unques- tioned, the alignment of sites to maximize the number of identities will likely produce increasing numbers of nonhomologous characters that will be presumed homologous as the phyletic distance between taxa increases. At an as yet undetermined phyletic distance, homoplastic “noise” resulting from these nonhomologous characters appears to overwhelm the signal. This is reflected in the inability of various individual outgroup taxa to root the ingroup network at a single location. Clearly, the number of nonho- mologous characters in the various outgroup data sets exceeded the ability of the homologous characters to provide a unique rooting point even among these eukaryotic protists. The relatively closely related outgroup taxa Pr. micuns and C. cohnii rooted the ingroup tree at the same location as did the multiple-taxon outgroup. The cellular slime mold Dictyostelium discoideum and one of the two trees with Saccharomyces cerevisiae rooted the Apicomplexa between the genus Plasmodium and the remaining ingroup taxa. The inability of relatively closely related protists to root the apicomplexan ingroup network uniquely questions the utility of srRNA sequences for producing a so-called universal phylogenetic tree based on srRNA sequences. If nonhomologous sites have overwhelmed our ability to infer relationships among these closely related taxa, then perhaps the utility of srRNA molecules for inferring ancient relationships among taxa has been overestimated. Indeed, Ragan ( 1988) has suggested that the major subsets of ribosomal RNA may be nonhomologous.

Acknowledgments

Thoughtful discussions with Dr. R. Murphy and G. Klassen of the Royal Ontario Museum are appreciated. J.R.B. is a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Fellow.

354 Barta et al.

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MASATOSHI NEI, reviewing editor

Received January 30, 1990; revision received November 2 1, 1990

Accepted November 2 1, 1990