* Department of Biological Sciences, University of South Carolina
Institute of Molecular Evolutionary Genetics, The Pennsylvania State University
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Abstract |
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Key Words: Bodonidae Evolution of parasitism Salivaria Stercoraria Trypanosoma
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Introduction |
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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.
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Methods |
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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 2) 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).
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Results |
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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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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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Discussion |
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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. 181182). 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. 14). 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. 14). 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. 24
). 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.
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Acknowledgements |
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Footnotes |
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E-mail: austin{at}biol.sc.edu.
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