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Trypanosoma rangeli: sequence analysis of β-tubulin gene suggests closer relationship to Trypanosoma brucei than to Trypanosoma cruzi
Acta Tropica, 53(1993)99-105 © 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00
99
ACTROP 00267
Trypanosoma rangeli: sequence analysis of [3-tubulin gene suggests closer relationship to Trypanosoma brucei than to Trypanosoma cruzi M. Isabel Amorim, Hooman Momen and Yara M. Traub-Cseko Instituto Oswaldo Cruz, FIOCR UZ, Departamento de Bioquimica e Biologia Molecular, Rio de Janeirc Brazil Received 30 September 1992; accepted 4 December 1992) Trypanosoma rangeli, the only trypanosome besides Trypanosoma cruzi to infect humans in the Americas, shows an important geographical overlap with the agent of Chagas disease, and its taxonomic position has been the source of some controversy. This study utilizes [3-tubulin gene sequences for investigating the phylogeny of this species. All trees, produced with the different algorithms utilized, always grouped T. rangeli with Trypanosoma brucei in preference to T. cruzi. In addition evidence suggesting that the genus Trypanosorna may be polyphyletic was found. Key words: Trypanosoma rangeli; Trypanosoma cruzi; Phylogeny; 13-Tubulin sequences; Taxonomy
Introduction
Trypanosoma rangeli is the only trypanosome besides Trypanosoma cruzi, the causative agent of Chagas disease, to infect humans in the Americas. This parasite is believed not to be pathogenic to man. Currently, several South American countries have begun a joint campaign to eradicate Chagas disease and thus a better understanding of the much less studied T. rangeli, which has an overlapping geographical distribution with T. cruzi, has become imperative. The taxonomic position of T. rangeli has been the cause of some controversy. Hoare (1972), in his classic monograph, placed the organism in the subgenus Herpetosoma of the section Stercoraria, while Afiez (1982) created a new subgenus, Tejeraia for this parasite within the section Salivaria. Other authorities have also favoured this latter view (WHO, 1986). Nucleic acid sequence data are being increasingly utilized in phylogenetic studies. Within the Kinetoplastida a number of studies have been carried out using rRNA gene sequences and conflicting results have been obtained with regard to the monophyly of the genus Trypanosoma (G6mez et a1.,1991; Lake et al., 1988; Briones et al., 1992; Hernhndez et al., 1990; Campbell, 1992). These studies included only Correspondence to: Y.M. Traub-Cseko, Funda~;ao Oswaldo Cruz, Departamento de Bioquimica e Biologia Molecular, P.O. Box 926, Rio de Janeiro, RJ, 21045-900, Brazil. Phone: (021)598-4345; FAX: 55 21 590-3495; E.mail: [email protected].
100 two trypanosome species and the use of additional species as well as other molecules was suggested (Gomez et al., 1991; Briones et al., 1992). Little et al. (1984) proposed using the tubulin genes for investigating ancient divergences as these genes are very conservative and fairly easy to isolate. However, due to the problem of paralogous loci and different mutation rates, the molecule has not been widely used in phylogenetic studies. Within flagellated protists, on the other hand, the tubulin gene families are strikingly homogeneous, with the gene copies being almost identical (Silflow, 1991), thus facilitating comparison between homologous genes. Here we report the result of our studies on the systematic position of T. rangeli using nucleic acid sequence data from the ]3-tubulin gene.
Methods
Sequence analysis The sequences analysed have been published and are deposited in the EMBL Genebank with the following accession numbers: T. eruzi, M97956 (submitted for publication); T. brucei rhodesiense, Ml1748 (Kimmel et al., 1985); LeLs'hmania mexicana, M23441 (Fong & Lee, 1988); T. rangeli, M26530 (Esquenazi et al., 1989). A plot similarity using the 'Pileup' software program from the University of Wisconsin Genetic Computer Group (GCG) was made using an alignment of 20 translated amino-acid sequences of 13-tubulin from organisms as diverse as protists, fungi, plants and animals (Fig. 1). From this analysis two regions of differing overall homology were selected, the first one from position 140 to 167, and the second
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Fig. 1. Plot similarity originated from a multiple alignment of 20 translated beta tubulin published sequences from different organisms, using the program Pileup, with a window of 10 amino acids. The bars indicate the sequence regions utilized in this study.
]01
region from position 347 to 398. The nucleotide sequences corresponding to these regions were aligned by eye (Fig. 2).
Phylogenetic reconstruction Two different types of analysis for the construction of phylogenetic trees were used, distance matrix methods and maximum parsimony methods. All analyses were performed using the PHYLIP software package (Felsenstein, 1989). Euglena gracilis was used as an outgroup in the analyses.
Results
Distance methods Two different distance matrices were calculated. The method of Jukes and Cantor (1969) gives the same weight to transitions (substitution of purines by purines and pyrimidines by pyrimidines) and transversions (substitution ofpurines by pyrimidines and vice versa). The distances calculated by this method are given in the bottom half of the matrix shown in Table 1. The second method was that of Kimura's (1980) 2-parameter model which allows different weights to be given to transitions and transversions. The distances calculated with this method assumed a rate of transitions that was double that of transversions and the results are shown in the top half of the matrix in Table 1. The results given by the two methods were in close agreement; however, the distances obtained by the Kimura 2-parameter model were always slightly greater. The matrices were transformed into trees using both the UPGMA algorithm of Tcr'uzi Trange]i Tbrucei Lmexicana Euglena
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Fig. 2. Beta tubulin nucleotide sequence regions used in the present analysis. The dashes represent the gap between the two regions chosen. The boxed regions show sequence conservation.
I02 TABLE 1 Distance matrix obtained by the Jukes-Cantor (below the diagonal) and Kimura 2 parameters (above the diagonal) methods, from analysis of/~-tubulin gene sequences L. mexicana L. mexicana T. rangeli T. cruzi T. brucei Eugh,na
O.1406 0.[203 0.1510 O.1996
T. rangeli
T. cruzi
T. hrucei
Eu~lena
O.1466
O.1233 O.I 190
O.1557 O.l 136 0.1382
0.2108 0.2137 0.2224 0.1987
0.1153 O.1103 O.1996
O.1355 0.2108
O.1885
Sneath and Sokal (1973) and the neighbor joining algorithm of Saitou and Nei (1987). The U P G M A m e t h o d is the classic m e t h o d of producing phenetic trees by sequencial agglomeration of clusters and assumes a constant molecular clock, while the neighbor joining algorithm uses a phylogenetic a p p r o a c h by analysing connections between nodes and allows different rates o f evolution along the branches. The four different combinations o f the two distance methods and two tree producing algorithms all p r o d u c e d a d e n d r o g r a m o f the same t o p o l o g y with sligtly differing branch lengths. A representative tree is shown in Fig. 3. Maximum parsimony methods
M a x i m u m parsimony analysis was carried out using the Wagner algorithm (Kluge and Farris,1969) which allows both reversals and parallel mutations, and a branch and b o u n d algorithm (Hendy and Penny, 1982) which for small sample sizes guarantees to find the most parsimonious trees. T w o equally parsimonious trees of 91 steps were found (Fig. 4). A bootstrap analysis which resampled the data 100 times was unable to show any significant preference between the two topologies.
.....
T.rangeli
.....
T.brucei
....
• L.mexieana T.cruzi .... E . g r a e i l i s
Fig. 3. Dendrogram obtained from analysis of the Distance Matrix given in Table I. The dendrogram shown is from a neighbor joining analysis of the Kimura 2 parameter matrix. Branch lengths are proportional to the calculated distance.
103
Fig. 4. Topologiesof two cladograms obtained from maximum parsimony analysis of the beta tubulin nucleotide sequence. 4a and 4b show two equally parsimonious trees obtained. Discussion
The sequences of the [3-tubulin genes examined showed sufficient positional homology for the sequences to be aligned by eye. Although tubulin gene sequences are known for their overall very high level of conservation, there is an internal variation of this level, with the C-terminus of the molecule showing the lowest homology. For these studies, sequences of very high or very low homologies were avoided, the regions of the molecule chosen being close to the 80% average similarity observed among a wide range of organisms (Fig. 1). The genetic distances calculated from the sequences were similar in range to those calculated between different trypanosomatids using small subunit rRNA genes (Briones et al. 1992). For example these authors obtained a genetic distance of 0.1398 between T.cruzi and T. brucei using the Kimura parameter which compares with a value of 0.1382 obtained in the present study. A marked difference was noted however in the distances between the trypanosomatids and Euglena. In the rRNA study the distances were greater than 0.5 while in the present study the distances were about 0.2. These results probably reflect the fact that the tubulin genes are more conserved between these organisms than the rRNA genes. Briones et al. (1992) found that the distance values produced by the Jukes and Cantor parameter were always equal or greater than the values produced by the Kimura 2-parameter method. In our results, the distance found by the Kimura 2-parameter model was always slightly greater. This difference may be due to the use of differing weighting in the Kimura method, by these authors. All the trees produced by analyzing the nucleotide sequence data from the 13tubulin genes with the different algorithms grouped T. rangeli with T. brucei in preference to T. cruzi. This analysis is in agreement with the arrangement of the tubulin genes in these organisms which shows greater similarity between T.rangeli and T. brucei (Esquenazi et al., 1989). Campbell (1992) also reported a close relationship between these two species by analysis of rRNA nucleotide sequences. Historically, there has been considerable controversy over the evolutionary origin of the genus Trypanosoma with most authorities favouring a monophyletic origin (Hoare, 1972; Baker, 1974). The analysis of nucleotide sequence data has provided a new tool to examine this question. The results of the distance method analyses presented here question the monophyly of the genus Trypanosoma. They are in
104 a g r e e m e n t w i t h the results o f G 6 m e z et al. (1991) a n d the s p e c u l a t i o n s o f B a k e r (1974) r e g a r d i n g a c l o s e r r e l a t i o n s h i p b e t w e e n S c h i z o t r y p a n u m a n d Leishmania. T h e results f r o m the p a r s i m o n y a n a l y s e s w e r e n o t so c l e a r in this respect, b u t i n d i c a t e t h a t A m e r i c a n t r y p a n o s o m e s d o n o t f o r m a m o n o p h y l e t i c g r o u p a n d t h u s limit the c o n c l u s i o n s o f C a m p b e l l (1992) r e g a r d i n g the close r e l a t i o n s h i p o f A m e r i c a n a n d African trypanosomes. T h e a n a l y s i s o f the n u c l e o t i d e s e q u e n c e d a t a i n d i c a t e t h a t the v a l i d i t y o f the g e n u s T r y p a n o s o m a s h o u l d be r e e x a m i n e d p a r t i c u l a r l y in v i e w o f the w i d e l y differing life cycles o f s a l i v a r i a n a n d s t e r c o r i a n t r y p a n o s o m e s a n d the s u g g e s t i o n by M o l y n e u x (1986) t h a t the a p p e a r e n c e o f t r y p o m a s t i g o t e s is the result o f c o n v e r g e n t e v o l u t i o n .
References Afiez, N. (1982) Studies on Trypanosoma rangeli Tejera (1920) IV. A reconsideration of its systematic position. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 77, 405-415. Baker, J.R. (1974) The evolutionary origin and speciation of the genus Trypanosoma. In: Evolution in the Microbial World. Cambridge University Press, Cambridge. pp.343 366. Eds. Carlile, M.J. and Skekel, J.J., Soc. Gen. Microbiol. Symposium 24. Briones, M.R.S., Nelson, K., Beverley, S.M., Affonso, H.T., Camargo, E.P. and Floeter-Winter, L.M. (1992) Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene. Mol. Biochem. Parasitol. 53, 121-128. Campbell, D. A. (1992) Bodo caudatus medRNA and 5S RNA genes: Tandem arrangements and phylogenetic analyses. Biochem. Biophys. Res. Commun. 182:1053-1058. Esquenazi, D., Morel, C.M. and Traub-Cseko, Y.M. (1989) Charaterization of tubulin genes in Trypanosoma rangeli. Mol. Biochem. Parasitol. 34, 253 260. Fong, D. and Lee, B. (1988) Beta tubulin gene of the parasitic protozoan Leishmania mexicana. Mol. Biochem. Parasitol. 31:97-106 Felsenstein, J. (1989) PHYLIP-Phylogeny inference package (version 3.2). Cladistics 5, 164-166. G6mez, E., Vald6s, M.V., Pifiero, D. and Herflandez, R. (1991) What is a genus in the trypanosomatidae family? Phylogenetic analysis of two small rRNA sequences. Mol. Biol. Evol. 8, 254-259. Hendy, M.D. and Penny, D. (1982) Branch and bound algorithms to determine animal evolutionary trees. Mathem. Biosc. 59, 277-290. Hern~ndez, R., Rios, P., Vald~s, A.M. and Pifiero, D. (1990) Primary structure of Trypanosoma cruzi small-subunit ribosomal RNA coding region: comparison with other trypamosomatids. Mol. Biochem. Parasitol. 41,207-212. Hoare, C.A. (1972) In: Trypanosomes of mammals. A zoological monograph. Blackwell, Oxford and Edinburgh. Jukes, T.H. and Cantor, C.R. (1969) Evolution of protein molecules. In: Mammalian Protein Metabolism. Academic Press, New York, pp.21-132. Kimmel, B.E., Samson, S., Wu, J., Hirchberg, R. and Yarbrough, L.R. (1985) Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense: nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene 35, 237-248. Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111 120. Kluge, A.G. and Farris, J.S. (1969) Quantitative phyletics and the evolution of anurans. Syst. Zool. 18, 1 32 Lake, J.A., De la Cruz, V.F., Ferreira, P.C.G., Morel, C. and Simpson, L. (1988) Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences. Proc. Natl. Acad. Sci. USA 85, 4779-4783. Little, M.H., Luduefia, R.F., Morejohn, L., Asnes, C. and Hoffman, E. (1984) The tubulins of animals. plants, fungi and protists: implications for metazoan evolution. Origins Life 13, 169 176. Molyneux, D. H. (1986) Evolution of Trypanosomatidae: Considerations of polyphyletic origins of
105 mammalian parasites. In: Le&hman&. Taxonomie et phylogen6se. Applications 6co-6pid6miologiques. IMEE, Montpellier. pp. 231-240. Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425. Silflow, C.D. (1991) Why do tubulin gene families lack diversity in flagellate/ciliate protists? Protoplasma 164, 9-11. Sheath, P.H.A. and Sokal, R.R. (1973) Numerical taxonomy. W.H.Freeman, San Francisco. WHO (1986) La trypanosomiase africaine: bpidbmiologie et lutte Rapport d'un comit6 d'experts de I'OMS. OMS-S6rie de Rapports Techniques, 739,p. 16.
99
ACTROP 00267
Trypanosoma rangeli: sequence analysis of [3-tubulin gene suggests closer relationship to Trypanosoma brucei than to Trypanosoma cruzi M. Isabel Amorim, Hooman Momen and Yara M. Traub-Cseko Instituto Oswaldo Cruz, FIOCR UZ, Departamento de Bioquimica e Biologia Molecular, Rio de Janeirc Brazil Received 30 September 1992; accepted 4 December 1992) Trypanosoma rangeli, the only trypanosome besides Trypanosoma cruzi to infect humans in the Americas, shows an important geographical overlap with the agent of Chagas disease, and its taxonomic position has been the source of some controversy. This study utilizes [3-tubulin gene sequences for investigating the phylogeny of this species. All trees, produced with the different algorithms utilized, always grouped T. rangeli with Trypanosoma brucei in preference to T. cruzi. In addition evidence suggesting that the genus Trypanosorna may be polyphyletic was found. Key words: Trypanosoma rangeli; Trypanosoma cruzi; Phylogeny; 13-Tubulin sequences; Taxonomy
Introduction
Trypanosoma rangeli is the only trypanosome besides Trypanosoma cruzi, the causative agent of Chagas disease, to infect humans in the Americas. This parasite is believed not to be pathogenic to man. Currently, several South American countries have begun a joint campaign to eradicate Chagas disease and thus a better understanding of the much less studied T. rangeli, which has an overlapping geographical distribution with T. cruzi, has become imperative. The taxonomic position of T. rangeli has been the cause of some controversy. Hoare (1972), in his classic monograph, placed the organism in the subgenus Herpetosoma of the section Stercoraria, while Afiez (1982) created a new subgenus, Tejeraia for this parasite within the section Salivaria. Other authorities have also favoured this latter view (WHO, 1986). Nucleic acid sequence data are being increasingly utilized in phylogenetic studies. Within the Kinetoplastida a number of studies have been carried out using rRNA gene sequences and conflicting results have been obtained with regard to the monophyly of the genus Trypanosoma (G6mez et a1.,1991; Lake et al., 1988; Briones et al., 1992; Hernhndez et al., 1990; Campbell, 1992). These studies included only Correspondence to: Y.M. Traub-Cseko, Funda~;ao Oswaldo Cruz, Departamento de Bioquimica e Biologia Molecular, P.O. Box 926, Rio de Janeiro, RJ, 21045-900, Brazil. Phone: (021)598-4345; FAX: 55 21 590-3495; E.mail: [email protected].
100 two trypanosome species and the use of additional species as well as other molecules was suggested (Gomez et al., 1991; Briones et al., 1992). Little et al. (1984) proposed using the tubulin genes for investigating ancient divergences as these genes are very conservative and fairly easy to isolate. However, due to the problem of paralogous loci and different mutation rates, the molecule has not been widely used in phylogenetic studies. Within flagellated protists, on the other hand, the tubulin gene families are strikingly homogeneous, with the gene copies being almost identical (Silflow, 1991), thus facilitating comparison between homologous genes. Here we report the result of our studies on the systematic position of T. rangeli using nucleic acid sequence data from the ]3-tubulin gene.
Methods
Sequence analysis The sequences analysed have been published and are deposited in the EMBL Genebank with the following accession numbers: T. eruzi, M97956 (submitted for publication); T. brucei rhodesiense, Ml1748 (Kimmel et al., 1985); LeLs'hmania mexicana, M23441 (Fong & Lee, 1988); T. rangeli, M26530 (Esquenazi et al., 1989). A plot similarity using the 'Pileup' software program from the University of Wisconsin Genetic Computer Group (GCG) was made using an alignment of 20 translated amino-acid sequences of 13-tubulin from organisms as diverse as protists, fungi, plants and animals (Fig. 1). From this analysis two regions of differing overall homology were selected, the first one from position 140 to 167, and the second
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Fig. 1. Plot similarity originated from a multiple alignment of 20 translated beta tubulin published sequences from different organisms, using the program Pileup, with a window of 10 amino acids. The bars indicate the sequence regions utilized in this study.
]01
region from position 347 to 398. The nucleotide sequences corresponding to these regions were aligned by eye (Fig. 2).
Phylogenetic reconstruction Two different types of analysis for the construction of phylogenetic trees were used, distance matrix methods and maximum parsimony methods. All analyses were performed using the PHYLIP software package (Felsenstein, 1989). Euglena gracilis was used as an outgroup in the analyses.
Results
Distance methods Two different distance matrices were calculated. The method of Jukes and Cantor (1969) gives the same weight to transitions (substitution of purines by purines and pyrimidines by pyrimidines) and transversions (substitution ofpurines by pyrimidines and vice versa). The distances calculated by this method are given in the bottom half of the matrix shown in Table 1. The second method was that of Kimura's (1980) 2-parameter model which allows different weights to be given to transitions and transversions. The distances calculated with this method assumed a rate of transitions that was double that of transversions and the results are shown in the top half of the matrix in Table 1. The results given by the two methods were in close agreement; however, the distances obtained by the Kimura 2-parameter model were always slightly greater. The matrices were transformed into trees using both the UPGMA algorithm of Tcr'uzi Trange]i Tbrucei Lmexicana Euglena
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23~ 234 234 e34 231
Fig. 2. Beta tubulin nucleotide sequence regions used in the present analysis. The dashes represent the gap between the two regions chosen. The boxed regions show sequence conservation.
I02 TABLE 1 Distance matrix obtained by the Jukes-Cantor (below the diagonal) and Kimura 2 parameters (above the diagonal) methods, from analysis of/~-tubulin gene sequences L. mexicana L. mexicana T. rangeli T. cruzi T. brucei Eugh,na
O.1406 0.[203 0.1510 O.1996
T. rangeli
T. cruzi
T. hrucei
Eu~lena
O.1466
O.1233 O.I 190
O.1557 O.l 136 0.1382
0.2108 0.2137 0.2224 0.1987
0.1153 O.1103 O.1996
O.1355 0.2108
O.1885
Sneath and Sokal (1973) and the neighbor joining algorithm of Saitou and Nei (1987). The U P G M A m e t h o d is the classic m e t h o d of producing phenetic trees by sequencial agglomeration of clusters and assumes a constant molecular clock, while the neighbor joining algorithm uses a phylogenetic a p p r o a c h by analysing connections between nodes and allows different rates o f evolution along the branches. The four different combinations o f the two distance methods and two tree producing algorithms all p r o d u c e d a d e n d r o g r a m o f the same t o p o l o g y with sligtly differing branch lengths. A representative tree is shown in Fig. 3. Maximum parsimony methods
M a x i m u m parsimony analysis was carried out using the Wagner algorithm (Kluge and Farris,1969) which allows both reversals and parallel mutations, and a branch and b o u n d algorithm (Hendy and Penny, 1982) which for small sample sizes guarantees to find the most parsimonious trees. T w o equally parsimonious trees of 91 steps were found (Fig. 4). A bootstrap analysis which resampled the data 100 times was unable to show any significant preference between the two topologies.
.....
T.rangeli
.....
T.brucei
....
• L.mexieana T.cruzi .... E . g r a e i l i s
Fig. 3. Dendrogram obtained from analysis of the Distance Matrix given in Table I. The dendrogram shown is from a neighbor joining analysis of the Kimura 2 parameter matrix. Branch lengths are proportional to the calculated distance.
103
Fig. 4. Topologiesof two cladograms obtained from maximum parsimony analysis of the beta tubulin nucleotide sequence. 4a and 4b show two equally parsimonious trees obtained. Discussion
The sequences of the [3-tubulin genes examined showed sufficient positional homology for the sequences to be aligned by eye. Although tubulin gene sequences are known for their overall very high level of conservation, there is an internal variation of this level, with the C-terminus of the molecule showing the lowest homology. For these studies, sequences of very high or very low homologies were avoided, the regions of the molecule chosen being close to the 80% average similarity observed among a wide range of organisms (Fig. 1). The genetic distances calculated from the sequences were similar in range to those calculated between different trypanosomatids using small subunit rRNA genes (Briones et al. 1992). For example these authors obtained a genetic distance of 0.1398 between T.cruzi and T. brucei using the Kimura parameter which compares with a value of 0.1382 obtained in the present study. A marked difference was noted however in the distances between the trypanosomatids and Euglena. In the rRNA study the distances were greater than 0.5 while in the present study the distances were about 0.2. These results probably reflect the fact that the tubulin genes are more conserved between these organisms than the rRNA genes. Briones et al. (1992) found that the distance values produced by the Jukes and Cantor parameter were always equal or greater than the values produced by the Kimura 2-parameter method. In our results, the distance found by the Kimura 2-parameter model was always slightly greater. This difference may be due to the use of differing weighting in the Kimura method, by these authors. All the trees produced by analyzing the nucleotide sequence data from the 13tubulin genes with the different algorithms grouped T. rangeli with T. brucei in preference to T. cruzi. This analysis is in agreement with the arrangement of the tubulin genes in these organisms which shows greater similarity between T.rangeli and T. brucei (Esquenazi et al., 1989). Campbell (1992) also reported a close relationship between these two species by analysis of rRNA nucleotide sequences. Historically, there has been considerable controversy over the evolutionary origin of the genus Trypanosoma with most authorities favouring a monophyletic origin (Hoare, 1972; Baker, 1974). The analysis of nucleotide sequence data has provided a new tool to examine this question. The results of the distance method analyses presented here question the monophyly of the genus Trypanosoma. They are in
104 a g r e e m e n t w i t h the results o f G 6 m e z et al. (1991) a n d the s p e c u l a t i o n s o f B a k e r (1974) r e g a r d i n g a c l o s e r r e l a t i o n s h i p b e t w e e n S c h i z o t r y p a n u m a n d Leishmania. T h e results f r o m the p a r s i m o n y a n a l y s e s w e r e n o t so c l e a r in this respect, b u t i n d i c a t e t h a t A m e r i c a n t r y p a n o s o m e s d o n o t f o r m a m o n o p h y l e t i c g r o u p a n d t h u s limit the c o n c l u s i o n s o f C a m p b e l l (1992) r e g a r d i n g the close r e l a t i o n s h i p o f A m e r i c a n a n d African trypanosomes. T h e a n a l y s i s o f the n u c l e o t i d e s e q u e n c e d a t a i n d i c a t e t h a t the v a l i d i t y o f the g e n u s T r y p a n o s o m a s h o u l d be r e e x a m i n e d p a r t i c u l a r l y in v i e w o f the w i d e l y differing life cycles o f s a l i v a r i a n a n d s t e r c o r i a n t r y p a n o s o m e s a n d the s u g g e s t i o n by M o l y n e u x (1986) t h a t the a p p e a r e n c e o f t r y p o m a s t i g o t e s is the result o f c o n v e r g e n t e v o l u t i o n .
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