Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) Based on 18S rRNA: Evidence for Paraphyly of Trypanosoma and Six Other Genera

Austin L. Hughes*, and Helen Piontkivska{dagger}

* Department of Biological Sciences, University of South Carolina
{dagger} Institute of Molecular Evolutionary Genetics, The Pennsylvania State University


    Abstract
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic analysis of 18S rRNA sequences from the families Trypanosomatidae and Bodonidae (Eugelenozoa: Kinetoplastida) was conducted using a variety of methods. Unlike previous analyses using unrooted trees and/or smaller numbers of sequences, the analysis did not support monophyly of the genus Trypanosoma, which includes the major human parasites T. cruzi (cause of Chagas' disease) and T. brucei (cause of African sleeping sickness). The section Salivaria of the genus Trypanosoma fell outside a cluster that includes the section Stercoraria of the genus Trypanosoma, along with members of the genera Leishmania, Endotrypanum, Leptomonas, Herpetomonas, Phytomonas, Crithidia, and Blastocrithidia. The phylogenetic analysis also indicated that the genera Bodo, Cryptobia, Leptomonas, Herpetomonas, Crithidia, and Blastocrithidia are polyphyletic. The results suggested that parasitism of vertebrates has probably arisen independently a number of times within the Trypanosomatidae.

Key Words: Bodonidae • Evolution of parasitism • Salivaria • Stercoraria • Trypanosoma


    Introduction
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The family Trypanosomatidae (Eugelenozoa: Kinetoplastida) includes some of the most important protist parasites of humans in the genera Leishmania and Trypanosoma, as well as other species parasitic on a wide variety of vertebrates, invertebrates, ciliates, and plants (Stevens, Noyes, and Schofield 2001). Within the genus Trypanosoma, molecular phylogenies have generally identified two well-defined major groups: (1) the section Stercoraria, or the T. cruzi clade, including T. cruzi, the causative agent of Chagas' disease, along with certain other parasites of mammals and leech-transmitted parasites of aquatic vertebrates, and (2) the section Salivaria, or the T. brucei clade, including T. brucei, the causative agent of African sleeping sickness, along with other African parasites of mammals.

Maslov et al. (1996) presented an 18S SSU rRNA phylogeny in which T. brucei clustered outside a group that includes the T. cruzi clade, along with members of some other genera of Trypanosomatidae, thus suggesting that the genus Trypanosoma is not monophyletic. However, a relatively small number of species were used in this analysis. Furthermore, the phylogenetic tree was rooted with two species from the related kinetoplastid family Bodonidae, Bodo caudatus and Trypanoplasma borreli. This rooting is not appropriate if the family Trypanosomatidae itself is paraphyletic.

Other authors (Haag, O'hUigin, and Overath 1998; Luke et al. 1997; Stevens, Notes, and Gibson 1998; Stevens, Noyes, and Schofield 2001) presented additional SSU rRNA phylogenies that supported monophyly of Trypanosoma. In these phylogenies, the two major groups of Trypanosoma species clustered together. However, like Maslov et al. (1996), these authors rooted their phylogenies with Bodo caudatus and Trypanoplasma borreli; thus, their conclusions were subject to the same criticism as those of Maslov et al. (1996). On the other hand, Wright et al. (1999) presented a SSU rRNA phylogeny of Trypanosomatidae that was rooted with selected species of stramenopiles (Chrysophyceae and Eustigmatophyceae) and Euglenida. Since these species constitute undisputed outgroups to Kinetoplastida, rooting of Wright et al.'s (1999) phylogenetic tree could not be questioned. Wright et al.'s (1999) phylogeny supported monophyly of Trypanosoma; however, it included only a small number of species.

Some authors have addressed the evolutionary history of the genus Trypanosoma using protein-coding sequences. In all cases, the numbers of sequences available have been small. Alvarez, Cortinas, and Musto (1996) claimed support for monophyly of Trypanosoma based on analyses of three protein-coding genes, but their analyses included only five species of Trypanosomatidae and no undisputed outgroup. Hannaert, Opperdoes, and Michels (1998) also claimed support for monophyly of Trypanosoma on the basis of an analysis of glyceraldehyde-3-phosphate dehydrogenase sequences. These authors' phylogenies were rooted with a sequence from Euglena gracilis (Euglenida), an undisputed outgroup. However, they included only a single species of Stercoraria (T. cruzi); thus their conclusion may be influenced by biased taxon sampling.

In addition to the question of the relationship of the two major groups within Trypanosoma, another problem involving this genus concerns the phylogenetic position of Trypanosoma vivax. The only sequence data for this species are based on a single isolate from a zebu cow in Nigeria in 1973 (Stevens and Rambaut 2001). In most phylogenies that have included T. vivax, it has clustered as a distant member of the Salivaria clade (Stevens, Noyes, and Schofield 2001). Haag, O'hUigin, and Overath (1998) excluded this species from their analysis on the grounds that it showed a somewhat higher rate of evolution in comparison with outgroup species (Bodonidae) than did other members of Trypanosoma. In addition, Haag, O'hUigin, and Overath (1998) noted that the percent G+C in the T. vivax 18S rRNA sequence was somewhat higher than that in other Trypanosoma species that they examined. Stevens and Rambaut (2001) also claimed to have evidence of a higher evolutionary rate in T. vivax than in other Trypanosoma species, but this conclusion was not justified, because their phylogeny was not rooted with an inappropriate outgroup (including species of the genera Leishmania, Endotrypanum, and Crithidia).

All previously published phylogenies of Trypanosomatidae can be criticized on the grounds that they have not included examples of most genera within Trypanosomatidae and the related family Bodonidae. For example, the most extensive analysis published to date, that of Stevens, Noyes, and Schofield (2001), includes only four genera of Trypanosomatidae and includes Bodonidae only in the outgroup used to root the tree.

Therefore, the present paper addresses the phylogenetic relationships within Trypanosomatidae and Bodonidae by constructing a rooted phylogeny of 18S rRNA sequences from an extensive collection of species representing nine genera of Trypanosomatidae and six genera of Bodonidae. In addition, using two species of Euglenida as an outgroup, we use outgroup comparisons to test the hypothesis of a high evolutionary rate in T. vivax.


    Methods
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We aligned 18S rRNA sequences (table 1GoGoGo) using the ClustalW program (Thompson, Higgins, and Gibson 1994). The alignment is available from the authors upon request. In phylogenetic analyses and in computation of pairwise distances among sequences, any site at which the alignment postulated a gap in any sequence was removed from all comparisons so that a comparable set of sites was used for each comparison. Phylogenetic trees were reconstructed by four methods: (1) the minimum evolution (ME) method (Rzhetsky and Nei 1992), as implemented in the MEGA2 computer package (Kumar et al. 2001); (2) the maximum parsimony (MP) method, using heuristic search, as implemented in the PAUP* computer package (Swofford 2000); (3) the quartet maximum likelihood (QML) method (Strimmer and von Haeseler 1996), as implemented in the TREEPUZZLE 5.0 program; and (4) the Bayesian method, as implemented in MRBAYES version 2.01 (Huelsenbeck and Ronquist 2001). All trees were rooted using the Euglenida Euglena viridis and Khawkinea quartana as an outgroup.


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Table 1 18S rRNA Sequences Used in Analyses.

 

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Table 1 Continued.

 
ME trees were based on a number of distances, including the Kimura 2-parameter distance (Kimura 1980) and the Tamura-Nei distance (Tamura and Nei 1993). The former distance takes into account unequal rates of transition and transversion, whereas the latter takes into account both transitional and GC content biases (Nei and Kumar 2000). The Tamura-Nei model was used is the QML analysis. The significance of internal branches in the ME tree was tested by the standard error test, with standard errors of branch lengths estimated by the bootstrap method using 1,000 bootstrap samples (Kumar et al. 2001). Reliability of clustering patterns in the MP analysis was assessed by the bootstrap method using 1,000 bootstrap replicates. Reliability of clustering patterns in the QML analysis was assessed by the proportion of 25,000 puzzling steps that supported a given pattern.

In the Bayesian analysis, the general time reversible (GTR) + gamma + proportion of invariant sites nucleotide substitution model was used (Felsenstein and Churchill 1996). An uninformative prior was used. Metropolis-coupled Markov chain Monte Carlo sampling was performed with four chains (one cold and three incrementally heated, with temperature parameter value = 0.2). These chains were run for 1,000,000 generations, with trees sampled every 100 generations from the last 500,000 generations, and 5,000 sampled trees were used for inferring the Bayesian tree.

To examine the data for evidence of long-branch attraction (Hendy and Penny 1989), the mean number of nucleotide substitutions per site (d) was estimated between (1) each sequence from Trypanosomatidae and Bodonidae (table 2Go) and (2) the two outgroup sequences (Euglena viridis and Khawkinea quartana). Tamura and Nei's (1993) method was used to estimate d, and the standard error of mean d for a set of pairwise comparisons was estimated by the bootstrap method (Nei and Kumar 2000).


    Results
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic Analyses
Figure 1 illustrates the topology of the ME tree based on the Tamura-Nei distance; other distances produced similar topologies (data not shown). The ME tree included many short branches, particularly in the Stercoraria, and these were not easily displayed in a tree that included so many sequences. Therefore, figure 1 shows only topology, not branch lengths.



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FIG. 1. ME tree of 18S rRNA sequences of Trypanosomatidae and Bodonidae based on the Tamura-Nei distance. Topology only is shown, not branch lengths. Numbers on branches represent confidence levels of the standard error test of branch length; only values >=95% are shown

 
In the ME tree, the Stercoraria constituted a single cluster receiving highly significant support (fig. 1). The Stercoraria in turn clustered with a group that includes members of the genera Blastocrithidia, Herpetomonas, Phytomonas, Leptomonas, Leishmania, Endotrypanum, Crithidia, and Wallaceina; this larger cluster also was supported by a highly significant internal branch (fig. 1). Herpetomonas and Phytomonas species clustered together, supported by a significant internal branch (fig. 1). In addition, each of these two genera formed a distinct cluster supported by a significant internal branch (fig. 1).

Although species assigned to the genus Leishmania all clustered together, this cluster was not supported by a significant internal branch (fig. 1). A larger cluster that includes Leishmania, Endotrypanum, Crithidia, Wallaceina, two Leptomonas sequences, and Blastocrithidia gerricola was supported by a highly significant internal branch (fig. 1). However, Leptomonas collosoma, Blastocrithidia triatoma, and Blastocrithidia culicis fell outside this cluster. Four species of Herpetomonas formed a cluster supported by a significant internal branch (fig. 1). However, Herpetomonas cf. roitmani fell outside this cluster (fig. 1). Similarly, two species of Crithidia did not cluster together (fig. 1). Therefore, the ME tree did not support the monophyly of the genera Leptomonas, Blastocrithidia, Herpetomonas, or Crithidia.

In the genus Trypanosoma, the Salivaria, except for Trypanosoma vivax, formed a cluster supported by a highly significant internal branch (fig. 1). This cluster of Salivaria fell outside the highly significantly supported cluster of Stercoraria and members of eight other genera of Trypanosomatidae (fig. 1). Therefore, the ME tree provided strong evidence against the hypothesis that Trypanosoma is monophyletic.

Trypanosoma vivax clustered apart from all other members of Trypanosomatidae; indeed this species clustered outside even Bodonidae (fig. 1). However, the internal branch supporting this pattern was not significantly supported; thus, the position of Trypanosoma vivax was not resolved. Similarly, there was a cluster of three other members of Trypanosomatidae, which fell outside Bodonidae: Herpetomonas cf. roitmani, Crithidia oncopelti, and Blastocrithidia culicis (fig. 1). Although the cluster of these three species received highly significant support, the branch placing them outside other Trypanosomatidae and Bodonidae did not receive significant support (fig. 1). Thus, although these three species formed a clade, the exact relationship of this clade to others in the phylogeny was not well resolved.

Members of the Bodonidae were found only in a single cluster, supported by a highly significant internal branch (fig. 1). Members of the genus Bodo fell into four separate clusters within the family Bodonidae. Bodo saliens and Bodo designis formed part of a highly significantly supported cluster with Dimastigella and Rhynchomonas species (fig. 1). Bodo saltans and Bodo uncinatus clustered together, but the relationship of this cluster to other members of the family was not well resolved (fig. 1). Both Bodo sorokini and Bodo caudatus fell within a highly significantly supported cluster, along with species in the genera Cryptobia, Trypanoplasma, and Parabodo (fig. 1). However, within this group, the two Bodo species did not cluster together. Rather, Bodo caudatus clustered with Parabodo nitrophilus and Cryptobia helicis in a cluster that received highly significant support (fig. 1). Bodo sorokini fell outside this group, but its position was not well resolved (fig. 1). Thus, the phylogenetic tree did not support monophyly of the genus Bodo.

Similarly, the phylogenetic tree did not support monophyly of the genus Cryptobia. As mentioned previously, Cryptobia helicis formed part of a highly significantly supported cluster with Bodo caudatus and Parabodo nitrophilus (fig. 1). Cryptobia bullocki clustered with Trypanoplasma boreli, and this pattern also was supported by a highly significant internal branch (fig. 1).

The MP analysis found 48 equally parsimonious trees; the tree in figure 2 is the strict consensus of these trees. Most major clustering patterns revealed by MP were similar to those in the ME tree. In the MP tree as in the ME tree, the Salivaria clustered apart from the Stercoraria and outside a cluster including members of the genera Blastocrithidia, Herpetomonas, Phytomonas, Leptomonas, Leishmania, Endotrypanum, Crithidia, and Wallaceina (fig. 2). In the MP tree as in the ME tree, Trypanosoma vivax fell outside all other species analyzed from Trypanosomatidae and Bodonidae (fig. 2). Thus the MP analysis did not support the monophyly of Trypanosoma.



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FIG. 2. Strict consensus tree of 48 MP trees of 18S rRNA sequences of Trypanosomatidae and Bodonidae. Numbers on the branches represent the percentage of 1,000 bootstrap samples supporting the branch; only values >=50% are shown

 
In the MP tree as in the ME tree, the Bodonidae formed a single cluster (fig. 2). Although some details of the clustering patterns within Bodonidae differed between the two trees, the MP tree agreed with the ME tree in not supporting monophyly of Bodo or Cryptobia (figs. 1 and 2). The only striking difference between the ME and MP trees involved the position of the clade consisting of Blastocrithidia culicis, Crithidia oncopelti, and Herpetomonas cf. roitmani. In the MP tree, this clade fell within the major cluster containing other species assigned to these three genera. Nonetheless, the MP tree agreed with the ME tree in not supporting the monophyly of the genera Leptomonas, Blastocrithidia, Herpetomonas, or Crithidia.

The QML method produced a tree in which many deep branching patterns were not well resolved (fig. 3). In this tree, the Salivaria and Stercoraria formed separate clusters, and Trypanosoma vivax clustered with the Salivaria (fig. 3). Support for the Stercoraria cluster was relatively strong (92%), whereas that for the cluster of Trypanosoma vivax and Salivaria was weaker (58%) (fig. 3). Like the other trees, the QML tree did not support monophyly of the genera Leptomonas, Blastocrithidia, Herpetomonas, or Crithidia (fig. 3).



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FIG. 3. ML tree constructed by the quartet maximum likelihood method using the Tamura-Nei model. Topology only is shown, not branch lengths. Numbers on the branches represent the percentage of 25,000 puzzling steps supporting the branch

 
The Bayesian tree agreed with the QML tree in that Trypanosoma vivax clustered with the Salivaria (fig. 4). The posterior probability of this clustering pattern was only moderate (75%) (fig. 4). In this tree, Trypanosoma did not form a monophyletic group. Salivaria clustered with Bodonidae, although the posterior probability was again modest (61%) (fig. 4). Furthermore, in contrast to all other analyses, the Stercoraria themselves did not constitute a monophyletic group (fig. 4). Rather, all species assigned to the genera Leishmania, Endotrypanum, Crithidia, Blastocrithidia, Wallaceina, Leptomonas, Herpetomonas, and Phytomonas branched within the Stercoraria (fig. 4). Like the other trees, the Bayesian tree did not support monophyly of the genera Leptomonas, Blastocrithidia, Herpetomonas, or Crithidia (fig. 4).



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FIG. 4. Bayesian tree based on GTR + gamma + proportion of invariant sites model. The topology only is shown, not branch lengths. Numbers on the branches are posterior probabilities

 
Nucleotide Substitution
The mean number of nucleotide substitutions per site (d) was estimated between (1) each of 68 sequences from Trypanosomatidae and Bodonidae (table 1GoGoGo) and (2) the two outgroup sequences (Euglena viridis and Khawkinea quartana) (fig. 5A). Values of d ranged from 0.445 to 0.513; the mean was 0.464 ± 0.002 S.E. Four species showed particularly high values: Trypanosoma chelodinae (0.513), Cryptobia helicis (0.511), Dimastigella trypaniformis (0.506), and Dimastigella mimosa (0.505). These four species were not attracted to one another or to the root in the phylogenetic analyses (figs. 1–3GoGo). The value for Trypanosoma vivax was 0.471, and the mean value for the Salivaria clade (not including T. vivax) was 0.461 ± 0.022. Thus, although the value for T. vivax was slightly higher than the overall mean, it was not exceptionally high. Because the phylogenetic trees showed no evidence of long-branch attraction in the case of the taxa with the highest d values, it seems unlikely that the position of T. vivax in the ME and MP trees was the result of long-branch attraction. Furthermore, the position of the clade of Salivaria in all four trees was certainly not the result of long-branch attraction, since these species showed d values close to the mean.



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FIG. 5. (A) Frequency distribution of mean Tamura-Nei distances (d) between 18S rRNA sequences of 68 species of Trypanosomatidae and Bodonidae and the two outgroup sequences (Euglena viridis and Khawkinea quartana). (B) Frequency distribution of %GC in 18S rRNA sequences of 68 species of Trypanosomatidae and Bodonidae. (C) Scatterplot showing the relationship between d and %GC for the 68 species (r = -0.412; P < 0.001)

 
The distribution of %GC in the 68 species of Trypanosomatidae and Bodonidae was very narrow (fig. 5B). The mean value was 50.7% ± 0.1 S.E, with a minimum of 49.1% and a maximum of 51.4%. The value for Trypanosoma vivax was 49.7%, slightly below the mean. The mean for Salivaria (excluding T. vivax) was 50.9%, very close to the overall mean. There was a significant negative correlation (r = -0.412; P < 0.001) between d and %GC (fig. 5C). However, the two sequences with the lowest %GC were highly influential in this relationship. These two sequences were those of Dimastigella mimosa (49.2%) and Dimastigella trypaniformis (49.1%). When those two sequences were removed from the analysis, the correlation between d and %GC, while still negative, was no longer significant (r = -0.228; n.s.).


    Discussion
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Phylogenetic analysis of 18S rRNA sequences from the kinetoplastid families Trypanosomatidae and Bodonidae did not support the hypothesis of monophyly of the genus Trypanosoma. In ME and MP analyses, the Salivaria (not including Trypanosoma vivax) and the Stercoraria formed separate clades, with Salivaria falling outside a clade that includes Stercoraria and members of other genera, including Leishmania (figs. 1 and 2). In the ME analysis, this topology was supported by highly significant internal branches (fig. 1). In the QML analysis, the Stercoraria and Salivaria likewise formed separate clades, but T. vivax clustered with Salivaria (fig. 3). The results of all analyses imply that the two major trypanosome species causing human disease, T. brucei and T. cruzi, are not closely related to each other. Rather, the ME and MP analyses imply that T. cruzi is more closely related to the important human parasites in the genus Leishmania than it is to T. brucei.

Trypanosoma vivax clustered apart from both Stercoraria and Salivaria and was indeed basal to all other kinetoplastids in both ME and MP trees (figs. 1 and 2). However, the basal position of T. vivax was not supported by a significant branch in the ME tree (fig. 1). In the QML tree, T. vivax clustered with the Salivaria (fig. 3). Thus, whether T. vivax constitutes yet a third clade apart from the Salivaria and Stercoraria was not definitively resolved.

There was no evidence that the basal position of T. vivax in ME and MP trees was due to a long-branch attraction, since the T. vivax sequence was not exceptionally divergent with respect to the outgroup. Likewise, the position of the Salivaria outside the clade that includes Stercoraria and members of other genera in ME and MP trees could not be attributed to long-branch attraction. None of the four species that were most divergent with respect to the outgroup (Trypanosoma chelodinae, Cryptobia helicis, Dimastigella trypaniformis, and Dimastigella mimosa) showed an anomalous clustering pattern, suggesting that long-branch attraction was not a problem in these phylogenetic analyses.

Rather than long-branch attraction, which may sometimes be a problem for ME and MP methods, ML analyses can be plagued by the problem of "opposite-branch attraction" in which long branches are attracted to short branches (Nei and Kumar 2000, pp. 181–182). It might be hypothesized that opposite-branch attraction is responsible for the clustering of T. vivax with Salivaria in QML (fig. 3) and Bayesian (fig. 4) trees. However, this seems unlikely, since the divergence between T. vivax and the outgroup was not much greater than that between Salivaria and the outgroup. Likewise, none of the four species that were most divergent with respect to the outgroup (Trypanosoma chelodinae, Cryptobia helicis, Dimastigella trypaniformis, and Dimastigella mimosa) showed a strikingly different clustering pattern in the QML tree (fig. 3) or Bayesian tree (fig. 4) from those in ME (fig. 1) and MP (fig. 2) trees.

Stevens and Rambaut (2001) argued that there is a high rate of evolution in the 18S rRNA gene of T. vivax. However, this conclusion was based on comparisons with an outgroup that includes species of the genera Leishmania, Endotrypanum, and Crithidia. All four of our phylogenetic analyses did not support the hypothesis that these genera do not constitute a valid outgroup to Trypanosoma species (figs. 1–4GoGoGo). Indeed, in comparisons with an appropriate outgroup, we found no evidence of an accelerated rate of evolution in the 18S rRNA gene of T. vivax (fig. 5).

A previous phylogenetic analysis (Doleel et al. 2000) did not support monophyly of the genera Bodo and Cryptobia. The present analysis yielded a similar conclusion and also implies paraphyly of the trypanosomatid genera Leptomonas, Blastocrithidia, Herpetomonas, and Crithidia.

A number of different hypotheses have been proposed regarding origin of parasitism among Kinetoplastida (Baker 1963; Wallace 1966; Vickerman 1994; Maslov and Simpson 1995). One proposed scenario suggests that free-living kinetoplastids first became parasites of invertebrates. On this hypothesis, kinetoplastids parasitic on invertebrates evolved a life cycle that includes a vertebrate host after the invertebrate host adapted to blood feeding. A life cycle that includes a plant-parasitic stage, as seen in Phytomonas, might also have evolved by a similar pathway.

The present phylogenetic analysis suggested that, if kinetoplastid parasitism evolved by such a pathway, the same process has occurred repeatedly in the history of these organisms. In ME, MP, QML, and Bayesian trees, there was a clade that included the genera Leishmania and Endotrypanum, which are parasites of mammals with insect vectors, along with insect parasites assigned to the genera Leptomonas, Crithidia, Blastocrithidia, and Wallaceina (figs. 1–4GoGoGo). This clade was supported by a highly significant internal branch in the ME tree (fig. 1) and was well supported by all other methods (figs. 2–4GoGo). It is plausible that the ancestor of Leishmania and Endotrypanum was an insect parasite that evolved a life cycle involving parasitism on mammals. However, since species falling outside this clade include both species parasitic only on insects and species with vertebrate or plant hosts, the phylogeny suggests that other lineages of Trypanosomatidae must have adapted independently to life cycles that include a stage in a vertebrate host.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
This research was supported by grant GM34940 from the National Institutes of Heath (NIH) to A.L.H. H.P. was supported by NIH grant GM20293 to Masatoshi Nei.


    Footnotes
 
Claudia Kappen, Associate Editor Back

E-mail: austin{at}biol.sc.edu. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
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Accepted for publication December 4, 2002.