Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA *

Transcription

1 International Journal for Parasitology 34 (2004) Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA * Patrick B. Hamilton a, Jamie R. Stevens b, Michael W. Gaunt c, Jennifer Gidley a, Wendy C. Gibson a, * a School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK b School of Biological Sciences, University of Exeter, Exeter EX4 4PS, UK c Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK Received 5 July 2004; received in revised form 26 August 2004; accepted 26 August 2004 Abstract The genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major have been sequenced, but the phylogenetic relationships of these three protozoa remain uncertain. We have constructed trypanosomatid phylogenies based on genes for glycosomal glyceraldehyde phosphate dehydrogenase (ggapdh) and small subunit ribosomal RNA (SSU rrna). Trees based on ggapdh nucleotide and amino acid sequences (51 taxa) robustly support monophyly of genus Trypanosoma, which is revealed to be a relatively late-evolving lineage of the family Trypanosomatidae. Other trypanosomatids, including genus Leishmania, branch paraphyletically at the base of the trypanosome clade. On the other hand, analysis of the SSU rrna gene data produced equivocal results, as trees either robustly support or reject monophyly depending on the range of taxa included in the alignment. We conclude that the SSU rrna gene is not a reliable marker for inferring deep level trypanosome phylogeny. The ggapdh results support the hypothesis that trypanosomes evolved from an ancestral insect parasite, which adapted to a vertebrate/insect transmission cycle. This implies that the switch from terrestrial insect to aquatic leech vectors for fish and some amphibian trypanosomes was secondary. We conclude that the three sequenced pathogens, T. brucei, T. cruzi and L. major, are only distantly related and have distinct evolutionary histories. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosoma; Trypanosomatidae; Phylogeny; 18S rrna; Evolution; GAPDH 1. Introduction Trypanosomes (genus Trypanosoma) are widespread blood parasites of vertebrates, usually transmitted by arthropod or leech vectors. Several trypanosome species are agents of disease in humans and/or livestock particularly in the tropics. For example, Trypanosoma brucei causes * Note: Nucleotide sequence data reported in this paper are available in GenBank, EMBL and DDBJ databases under accession numbers: ggapdh gene sequences AJ620245, AJ620247, AJ AJ620253, AJ AJ620264, AJ AJ620270, AJ620272, AJ620273, AJ AJ620278, AJ AJ620291, AJ SSU rrna gene sequences: AJ620547, AJ620548, AJ620555, AJ620557, AJ * Corresponding author. Tel.: C ; fax: C address: w.gibson@bristol.ac.uk (W.C. Gibson). human African trypanosomiasis or sleeping sickness, while Trypanosoma cruzi causes Chagas disease in South and Central America. The genus Trypanosoma is in the phylum Euglenozoa (Eukaryota; Excavata), which comprises three orders, Diplonemida, Euglenida and Kinetoplastida, and a taxon of uncertain placement, Postgaardi (Cavalier-Smith, 1993; Simpson, 1997). Lifestyles within the Euglenozoa range from autotrophic photosynthesizers, such as Euglena gracilis, to free-living heterotrophs, such as Bodo saltans, and facultative or obligate parasites, such as genus Trypanosoma (Sleigh, 1989). Trypanosomes are an easily recognizable group, because they all share vertebrate parasitism and have a characteristic morphology the trypomastigote form in the vertebrate bloodstream. Two different evolutionary origins for trypanosomes have been proposed: vertebrate first, whereby they /$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi: /j.ijpara

2 1394 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) evolved from gut parasites of vertebrates, or invertebrate first, whereby they evolved from an invertebrate trypanosomatid parasite (Hoare, 1972; Vickerman, 1994). Central to the resolution of these theories is the issue of whether trypanosomes had a single evolutionary origin and/or gave rise to any other trypanosomatids, i.e. are they monophyletic? The first molecular phylogenetic studies, based on comparisons of genes encoding mitochondrial and nuclear ribosomal RNAs (rrna), showed trypanosomes to be paraphyletic (Gomez et al., 1991; Fernandes et al., 1993; Landweber and Gilbert, 1994; Lukes et al., 1994; Maslov and Simpson, 1995; Maslov et al., 1996). However, the inclusion of more taxa from a broader range of host species in subsequent studies based on rrna genes provided support for monophyly (Lukes et al., 1997; Haag et al., 1998; Stevens et al., 1998, 1999, 2001; Wright et al., 1999; Simpson et al., 2002), as did studies based on protein-coding genes (Hannaert et al., 1992; Hashimoto et al., 1995; Wiemer et al., 1995; Alvarez et al., 1996; Adjé et al., 1998; Hannaert et al., 1998; Simpson et al., 2002). Doubt has now been cast on this consensus by a reanalysis of SSU rrna gene sequences (Hughes and Piontkivska, 2003b). Hughes and Piontkivska contend that previous SSU rrna gene trees do not adequately prove monophyly of trypanosomes, because they either include an inadequate number and selection of taxa, or are rooted inappropriately (Hughes and Piontkivska, 2003b). In recent SSU rrna gene trees, trypanosomes and trypanosomatids appear paraphyletic (Hughes and Piontkivska, 2003a,b); in particular, placement of Trypanosoma vivax is problematic and it appears outside the main group of trypanosome species in some trees. This conflicts with the previous consensus on the taxonomic position of T. vivax, in which it is firmly established as part of the African tsetse-transmitted group characterised by antigenic variation (Hoare, 1972; Gardiner, 1989). Although existing phylogenies based on protein-coding genes show trypanosomes to be monophyletic (Hannaert et al., 1992, 1998; Hashimoto et al., 1995; Wiemer et al., 1995; Alvarez et al., 1996; Adjé et al., 1998), they include too few taxa to be reliable (Hughes and Piontkivska, 2003a). Most of the previous phylogenies of kinetoplastids have been based on analysis of variation in SSU rrna genes, multi-copy genes that evolve by concerted evolution. As single-copy, protein-coding genes are under a very different set of evolutionary constraints, analysis of such genes is likely to complement analysis based on the SSU rrna gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous and essential glycolytic enzyme and GAPDH genes have a slow rate of molecular evolution making them suitable for studying evolution over large time-scales (Fothergill-Gilmore and Michels, 1993). GAPDH gene organization has been studied in members of all three orders of the Euglenozoa, including three trypanosomatids, one bodonid (Wiemer et al., 1995), one euglenid (Henze et al., 1995), and two diplonemids (Qian and Keeling, 2001) and several gene sequences are available. In the three trypanosomatids studied [T. brucei (Michels et al., 1986); T. cruzi (Kendall et al., 1990); Leishmania mexicana (Hannaert et al., 1992)], there are three GAPDH genes, two of which encode the glycosomal enzyme (ggapdh), while the other encodes the cytosolic enzyme (cgapdh) (Michels et al., 1986; Kendall et al., 1990; Hannaert et al., 1992, 1998). Cytosolic GAPDH genes are more closely related to bacterial GAPDH genes than eukaryotic GAPDH genes and thus form a separate lineage (Wiemer et al., 1995; Hannaert et al., 1998). The two ggapdh genes from T. brucei, T. cruzi and L. mexicana are in a tandem repeat and are identical in sequence (Michels et al., 1986; Kendall et al., 1990; Hannaert et al., 1992). Likewise the bodonid Trypanoplasma borreli has two ggapdh genes in a tandem repeat, but the two predicted proteins differ by 17 amino acids (5%) (Wiemer et al., 1995). The euglenid, E. gracilis, also possesses two GAPDH enzymes, GapA, involved in the Calvin-cycle in the chloroplasts and GapC in glycolysis in the cytosol (Hallick et al., 1993). The GapA gene is similar to Gap2, a gene found in cyanobacteria (Hallick et al., 1993), and no homologue has been detected in kinetoplastids (Wiemer et al., 1995). Although E. gracilis has no glycosomes, the GapC gene is orthologous to trypanosomatid ggapdh (Henze et al., 1995). ggapdh gene orthologues have not been found in representative diplonemids (two species of Diplonema and one species of Rhynchopus)(Qian and Keeling, 2001). Thus ggapdh would appear to be a suitable candidate gene for reconstruction of trypanosome phylogeny, as it evolves slowly and under a different set of evolutionary constraints to the SSU rrna gene. In addition, several gene sequences are already available from previous studies. Here we have used ggapdh data to re-examine the questions of monophyly of trypanosomes and their evolutionary origins: invertebrate first or vertebrate first. 2. Materials and methods 2.1. Sequence analysis SSU rrna and ggapdh genes were amplified from trypanosome DNA by PCR. Details of trypanosome strains and origins are listed in Table 1. SSU rrna gene PCR and sequencing was as described (Stevens et al., 1999). The ggapdh gene was amplified with the primers shown in Table 2. Degenerate primers G3 G7 were designed from an alignment of the ggapdh sequences of Crithidia fasciculata AF047493, L. mexicana (X65226) and T. brucei (X59955). Expand High Fidelity PCR System (Roche) was used for all PCR reactions. The primers and PCR cycle described (Hannaert et al., 1998) were used for amplification of approximately 600 bp fragments of the ggapdh gene. Approximately 900 bp segments of the ggapdh gene were amplified using various combinations of primers G1 or G3 (forward) and G4a, G4b or G5 (reverse) with

3 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Table 1 Information on the origins of new Trypanosoma sequences in this study Trypanosome species Isolate code Origin Accession number Host Location ggapdh SSU rrna Fish Trypanosoma boissoni ITMAP 2211 Senegal marine ray Zanobatus atlanticus Senegal AJ a U39580 T. granulosum Portugal Eel Anguilla anguilla Portugal AJ a Trypanosoma sp. (CLAR) CLAR Catfish Clarias angolensis Import Africa AJ a AJ a Amphibia Trypanosoma mega ATCC African toad Bufo regularis Africa AJ a AJ Trypanosoma rotatorium B2-II Bullfrog Rana catesbeiana Canada AJ a AJ Reptile Trypanosoma sp. Gecko Gecko Tarentola annularis Senegal AJ a AJ a Trypanosoma varani V54 Monitor lizard Varanus exanthematicus Senegal AJ a AJ Bird Trypanosoma avium Chaffinch Chaffinch Fringilla coelebs Czech Republic AJ a AJ Trypanosoma sp. AAT Currawong Strepera sp. Australia AJ a AJ a Mammal Trypanosoma binneyi AAW Platypus Ornithorhynchus anatinus Australia AJ a,b Trypanosoma conorhini USP Rat Rattus rattus Brazil AJ a AJ T. cruzi marinkellei B7 Bat Phyllostomum discolor Brazil AJ a AJ Trypanosoma lewisi L32 Rat Rattus rattus? AJ a Trypanosoma microti TRL132 Vole Microtis agrestis England AJ a AJ Trypanosoma pestanai Lem 110 Badger Meles meles France AJ a AJ Trypanosoma sp. H25 Kangaroo Macropus giganteus Australia AJ a AJ Trypanosoma sp. AAP Wombat Vombatus ursinus Australia AJ a AJ a Trypanosoma sp. ABF Wallaby Wallabia bicolor Australia AJ a AJ a Trypanosoma sp. R5 Rabbit Oryctolagus cuniculus Australia AJ a,b Trypanosoma theileri K127 Ox Bos taurus Germany AJ a AJ T. vespertilionis P14 Bat Pipistrellus pipistrellus England AJ a AJ T. brucei rhodesiense 058 Human Homo sapiens Zambia AJ a T. congolense kilifi WG 5 Goat Capra capra Kenya AJ a AJ T. congolense savannah GAM 2 Ox Bos taurus The Gambia AJ a Invertebrate T. congolense forest ANR 3 Tsetse fly Glossina palpalis gambiensis The Gambia AJ a T. congolense Tsavo 114 Tsetse fly Glossina pallidipes Tanzania AJ a Trypanosoma cruzi C8 clone 2 Triatomine bug Triatoma infestans Bolivia AJ a Trypanosoma cruzi VINCH 89 Triatomine bug Triatoma infestans Chile AJ a AJ Trypanosoma grayi ANR4 Tsetse fly Glossina palpalis gambiensis The Gambia AJ a AJ Trypanosoma grayi BAN1 Tsetse fly Glossina palpalis gambiensis The Gambia AJ a Trypanosoma simiae KEN 2 Tsetse fly Glossina morsitans submorsitans The Gambia AJ a AJ Trypanosoma sp. F4 Tsetse fly Glossina pallidipes Kenya AJ a AJ a Trypanosoma sp. K&A Leech Piscicola geometra England AJ a,b AJ Trypanosoma sp. TL.AQ.22 Haemadipsid leech Philaemon sp. Australia AJ a,b a Obtained in this study. b Obtained by nested PCR. the following program: 95 8C, 180 s followed by 30 cycles of 60 s at 95 8C, 30 s at 55 8C, 60 s at 72 8C and a final extension period of 72 8C for 400 s. Nested PCR, using primers G3 and G5 in the first round and G1 and G4a or G1 and G4b in the second round, was used for samples containing little DNA. Amplified fragments were cloned into a plasmid vector (pcr2.1-topo, Invitrogen). Plasmid DNA was sequenced from M13 forward and reverse primers and the internal primers G6 and G7 using an automated sequencer. Consensus sequences were constructed for each clone using AutoAssembler 2.0 (ABI). Primer sequences were removed. The translate tool ( or MacClade version 3.05 (Maddison, D.R., Maddison, W.P., MacClade: Analysis of Phylogeny and Character Evolution, Version 3.0, Sinauer Associates Sunderland, MA) was used to convert nucleotide to amino acid sequences, using the universal genetic code.

4 1396 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Table 2 Primers used for amplifying and sequencing trypanosome ggapdh genes Code Primer sequence G1 a G2 a G3 G4a G4b G5 G6 G7 CGCGGATCCASGGYCTYMTCGGBAMKGAGAT CGCGGATCCCCBACVGCYTTSGCSCGRCCAGT TTYGCCGYATYGGYCGCATGG GTTYTGCAGSGTCGCCTTGG CCAMGASACVAYCTTGAAGAA ACMAGRTCCACCACRCGGTG GYGGTKTCSVTSAAGGACTG CSCCRGTBGTGCTBGGRATG a Primers from Hannaert et al. (1998); IUPAC/IUB base codes: RZAorG; YZCorT;MZAorC;SZCorG;KZGorT;BZC, G or T; VZA, C or G. The accession numbers of other GAPDH and SSU rrna gene sequences (italics) obtained from GenBank were: Non-Trypanosoma: E. gracilis, L21903; Bodo designis, AF209856; B. designis, AF464896; Bodo saliens, AF174379; Bodo edax, AY028451; Blastocrithidia culicis, L29266; Trypanoplasma borreli, X74535; Blastocrithidia gerricola, AF322391; Blastocrithidia triatoma, AF153037; C. fasciculata AF053739, AF047493, Y00055; Crithidia luciliae, AF053740; Crithidia oncopelti, L29264; Crithidia oncopelti, AF038025; Endotrypanum monterogei, X53911; Herpetomonas megaseliae, U01014; Herpetomonas mariadeanei, U01013; Herpetomonas muscarum, L18872; Herpetomonas cf. roitmani, AF267738; Herpetomonas samuelpessoai, AF047494, U01016; Leishmania major, AF047497, X53915; L. mexicana, X65226; Leishmania sp. AF303938; Leishmania tarentolae, M84225; Leptomonas collosoma, AF153038; Leptomonas lactosovorans, AF053741; Leptomonas peterhoffi, AF322390, AF153039; Leptomonas pyrrhocoris, AY029072; Leptomonas seymouri, AF053738, AF047495, AF153040; Leptomonas sp., AF339451, AF153043; Leptomonas sp., AF375664; Leptomonas sp. (Cfm), AF320820; Leptomonas sp., X53914; Phytomonas serpens, AF016323; Phytomonas sp. AF047496, AF016322, L35076; Trypanosomatidae (G755), U59491; Trypanosomatidae (Eva), AF071866; Wallaceina brevicula, AF316620; Wallaceina inconstans, AF Genus Trypanosoma: T. avium, AF416559; T. bennetti, AJ223562; T. binneyi, AJ132351; T. brucei brucei, X59955; T. brucei gambiense, AF047499, AJ009141; T. brucei rhodesiense, AJ009142; T. congolense, AJ009144; T. congolense forest, U22319; T. congolense savannah, AJ009146; T. congolense Tsavo U22318; T. cruzi, AF359467, X52898, AJ009147; T. cyclops, AJ131958; T. dionisii, AJ009151; T. evansi, AF053743, D89527; T. equiperdum, AJ223564; T. godfreyi, AJ009155; T. lewisi, AJ009156; T. rangeli, AF053742, AJ009160; T. scelopori, U67182; T. simiae Tsavo, AJ404608; T. sp. (bat Rousettus), AJ012418; T. sp. (wombat H26), AJ009169; T. sp. (OA6), AF416562; T. therezieni, AJ223571; T. triglae, U39584; T. vivax, AF053744, U Alignment Sequences were first aligned using program ClustalX (Thompson et al., 1997) using the default settings and final adjustments by eye. Six alignments were compiled as follows (see Table 3): (1) an alignment of SSU rrna gene sequences (69 taxa); (2.1) a published alignment of SSU rrna gene sequences (Hughes and Piontkivska, 2003b) (70 taxa); (2.2) alignment 2.1 with non-kinetoplastid sequences removed (68 taxa); (2.3) alignment 2.2 with T. brucei clade sequences removed (61 taxa); (3) 900 bp of the ggapdh gene (51 taxa); (4) translation of the same set of sequences. For all alignments, positions at which gaps were postulated were excluded from analyses. Alignments are available by anonymous FTP from FTP.EBI.AC.UK in directory/pub/databases/embl/align or via the EMBLA- LIGN database via SRS at under accessions ALIGN_ to Phylogenetic analysis Maximum likelihood (ML), maximum-parsimony (MP) and distance analysis of nucleotide alignments and MP analysis of amino acid alignments were carried out using PAUP* version 4.0b10 (Swofford, D.L., PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4, Sinauer Associates Sunderland, MA). The ML model consisted of a generalised reversible rate mutation matrix, a four-category gamma distribution and a parameter to accommodate the proportion of invariant sites. Maximum likelihood model parameters for all nucleotide alignments were estimated by an automated reiterative heuristic search, using the tree branch reconnection (TBR) method. A reconnection limit of eight was used except with alignment 2.1, which was terminated after 27,648 rearrangements. The nucleotide base composition was incorporated empirically into all cases. For ML bootstrapping, ML parameter values were calculated from the tree and were set for the analysis. A minimum of 100 bootstrap replicates were performed. Maximum likelihood analyses of amino acid sequences were carried out using the program TREE-PUZZLE-5.0 (Schmidt et al., 2002). The James, Taylor and Thornton (JTT) amino acid substitution matrix (Jones et al., 1992) was used with estimate parameter values for a gamma distribution plus invariable sites model of among-site rate variation. The gamma distribution parameter alpha was estimated from data with eight rate categories using the slow exact method. The tree was calculated with 1000 puzzling steps. For MP analyses, heuristic searches were performed with 100 random addition replicates and TBR branch swapping. A strict consensus was made of the shortest trees. One thousand bootstrap replicates were calculated, using the simple addition algorithm. Bayesian analyses were carried out using the program MRBAYES version 3.0b4 (Huelsenbeck and Ronquist,

5 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Table 3 Analyses undertaken and bootstrap support for three major clades Alignment No. of sequences No. included sites (informative) Outgroup(s) Method a Ztree shown Support for clades C trypanosomatids J trypanosomes K T. brucei clade SSU rrna (322) Bodonids ML a Bodonids Bayesian Bodonids MP Bodonids MLdist (HCP) (592) Euglenids ML a UN UN! (HCP) 70 Euglenids Bayesian UN UN (HCP) 70 Euglenids MP UN UN UN 2.1 (HCP) 70 Euglenids MLdist UN UN (HCP) (384) Bodonids ML a 100 UN (HCP) 68 Bodonids Bayesian UN UN (HCP) 68 Bodonids MP 98 UN (HCP) 68 Bodonids MLdist 100 UN (HCP) (328) Bodonids ML 100 UN 2.3 (HCP) 61 Bodonids Bayesian 100 UN 2.3 (HCP) 61 Bodonids MP a (HCP) 61 Bodonids MLdist ggapdh 3 (nt 1C2) (166) E. gracilis ML a E. gracilis Bayesian E. gracilis MP E. gracilis MLdist (aa) (98) E. gracilis ML E. gracilis Bayesian a E. gracilis MP !50, clade present but with bootstrap support of less than 50%; UN, clade absent;, not applicable; HCP, alignment derived published alignment (Hughes and Piontkivska, 2003b). a Shown in paper. 2001). The general time reversiblecgammacproportion of invariant sites nucleotide substitution model was used for analyses of nucleotide sequence alignments. The JTT amino acid substitution matrix (Jones et al., 1992) was used for analyses of amino acid alignments. Four chains were run for 1,000,000 generations with trees sampled every 1000 generations, and 500 sampled trees were used for inferring the Bayesian tree. Priors were left at default settings. Constrained ML trees were constructed in PAUP* version 4.0b10 (Swofford, D.L., PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4, Sinauer Associates Sunderland, MA). Each constrained tree was constructed using ML parameter values calculated from the optimal ML tree from the same dataset. The Shimodaira Hasegawa (SH) test (Shimodaira and Hasegawa, 1999), as implemented in PAUP*, was used to compare constrained with the optimal trees, using 1000 RELL bootstrap replicates. The ML models of evolution used assume that sites have not reached saturation. A matrix of uncorrected p-distances generated using the third codon position from ggapdh alignment gave a maximum value of , indicating that it may be saturated, as would be indicated by values of approximately Trees based on ML analysis can mislead if parameters that are assumed to be constant across the phylogeny (such as the ratio of base frequencies) actually vary among lineages in the true phylogeny (Swofford et al., 1996). The alignment required the removal of the third codon position to obtain nucleotide base homogeneity c 2 -test, P!0.01 for all positions and PZ 1.00 for codon positions one and two only. Thus, the third codon position was excluded for all analyses. 3. Results 3.1. Sequence variation Five new SSU rrna gene sequences and 39 ggapdh sequences were obtained. For 17 of the ggapdh sequences, two to four clones from independent PCR reactions were sequenced. For eight of these trypanosome samples there was no difference between the cloned sequences; however, for the other samples, nucleotide differences, sometimes giving rise to amino acid differences, were observed, with a maximum nucleotide sequence divergence of 0.6% and amino acid sequence divergence of 1.3%. These sequence differences may have resulted from PCR errors or differences between

6 1398 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) the four ggapdh alleles. However, inter- and intraspecific sequence differences were greater, e.g. nucleotide sequence divergences of 17.1% between Trypanosoma rangeli and Trypanosoma grayi, and 3.4% between Trypanosoma avium isolates, and 1.7% between T. grayi isolates. As variation within individual isolates is relatively minor, it is unlikely to have a significant effect on the phylogenies produced. A single 900 bp sequence from each isolate was used for phylogenetic analyses SSU rrna gene trees To investigate if SSU rrna gene sequence data support monophyly, we constructed trees from two alignments using ML, Bayesian, MP and distance methods. Alignment 1 contains a diverse range of trypanosomatids. Trees based on this alignment (e.g. Fig. 1) divide the taxa into three groups: (1) free-living bodonid outgroups (four taxa); (2) nontrypanosome trypanosomatids (23 taxa), which are either insect-only, or insect-transmitted parasites of vertebrates (Leishmania and Endotrypanum) or plants (Phytomonas); and (3) trypanosomes (42 taxa). The human pathogenic trypanosomes fall in two different clades, as previously reported (e.g. Stevens et al., 1999). Trypanosoma cruzi falls in the T. cruzi clade, which contains trypanosomes from South American mammals, Old World bats (e.g. Trypanosoma vespertilionis) and a trypanosome from an Australian kangaroo, whereas T. brucei falls in the T. brucei clade, Fig. 1. Maximum likelihood tree, based on alignment 1 of SSU rrna gene sequences, including 69 taxa. Kln LZ Single values at nodes are ML bootstrap values (%: 250 replicates). Multiple values at nodes are in order: ML bootstrap value, Bayesian support value, MP bootstrap value and ML distance bootstrap value.

7 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) with other trypanosomes of African mammals that are transmitted by tsetse flies. Monophyly of trypanosomes was robust to the method of tree construction, with strong statistical support in ML and Bayesian trees (e.g. bootstrap support of 88% in the ML tree; Fig. 1) and weaker in the MP and distance trees (e.g. bootstrap support of 51% in the MP tree; not shown). In contrast, trees derived from alignment 2.1 (Hughes and Piontkivska, 2003b) did not support monophyly of trypanosomes, confirming previous findings (Hughes and Piontkivska, 2003b). We found that in trees constructed using this alignment, such as the ML tree in Fig. 2A, the branch leading to the kinetoplastids was approximately 30 times longer than the longest internal branch within the kinetoplastids. Similar results were obtained previously using another SSU rrna gene alignment (Simpson et al., 2002). Since the use of distant outgroups increases the risk of longbranch attraction, which can result in artifactual rooting within long ingroup branches (Swofford et al., 1996; Philippe, 2000), we constructed further trees from alignment 2.1 excluding the euglenid outgroups (alignment 2.2). Although trypanosomes were paraphyletic in these trees (e.g. Fig. 2B), clades disrupting monophyly in ML, MP and distance trees were only weakly supported (bootstrap values!50%). Nevertheless, according to the SH test, a constrained ML tree in which trypanosomes were forced to be monophyletic was significantly worse than the optimal tree (PZ0.006). The ML tree from this alignment shown in Fig. 2B, shows the deepest split within trypanosomatids between the T. brucei clade and the other trypanosomatids. It also shows the trypanosomatids, excluding trypanosomes, to be monophyletic. An eight-fold higher rate of evolution of SSU rrna genes of T. brucei clade trypanosomes, compared with those of other trypanosomes, has previously been reported (Stevens and Rambaut, 2001). To investigate if inclusion of these divergent sequences artifactually alters the tree topology by long-branch attraction (Felsenstein, 1978), we constructed trees excluding both the T. brucei clade and the euglenid outgroups (alignment 2.3). In these trees, monophyly of trypanosomes depended on the method of analysis used. Excluding these sequences did not significantly alter the topology of the ML and Bayesian trees, and trypanosomes appeared paraphyletic and the other trypanosomatids monophyletic, in agreement with trees from alignment 2.2 (e.g. Fig. 2B). In contrast MP and distance trees were significantly altered and both supported monophyly of trypanosomes (e.g. Fig. 2C). Thus artifacts caused by the inclusion of divergent T. brucei clade and outgroup sequences do not fully explain failure to demonstrate monophyly of trypanosomes in trees derived from alignment 2.1, although their inclusion does significantly alter the MP and distance trees. The two different alignments of SSU rrna gene sequences suggest very different relationships within the trypanosomatids. Indeed, using alignment 1, a tree constrained to an approximation of the ML tree topology of alignment 2.2 (Fig. 2B) was significantly suboptimal (P!0.001). Likewise, using alignment 2.2, a tree constrained to an approximation of the ML tree from alignment 1 (Fig. 1) was significantly suboptimal (PZ0.003). To investigate the differences between the two alignments, they were aligned with each other. The 1431 characters included in alignment 2 are unambiguously aligned in alignment 1. However, alignment 2 has 46% more informative sites than alignment 1, largely resulting from the inclusion of divergent euglenids and bodonids; for example, removal of the two euglenid sequences decreases the number of informative sites from 592 to 384. Thus, differences in the number and range of informative characters may explain why the two alignments give significantly different trees. Nevertheless, trees based on both alignments agree that the vertebrate parasites Leishmania and Trypanosoma had separate evolutionary origins ggapdh gene trees To resolve which of the SSU rrna gene sequence tree topologies is correct, we sequenced 900 bp of the ggapdh genes of 37 trypanosome taxa and constructed phylogenies based on alignments of the ggapdh DNA and derived protein sequences from a wide selection of trypanosomatids. Few gaps needed to be introduced for optimal alignment of these sequences, e.g. only four in the amino acid sequence alignment, compared with 792 in alignment 1 of SSU rrna gene sequences. Thus, more confidence can be placed in the ggapdh than SSU rrna gene alignments. All trees based on ggapdh data support monophyly of trypanosomes and show them as a relatively late-evolving lineage within the Trypanosomatidae, which is also monophyletic (e.g. Fig. 3). Significantly, this result is robust regardless of the method of tree construction (ML, Bayesian, MP or distance methods), or whether the alignment used nucleotide or amino acid sequences. Trypanosome monophyly generally received high statistical support, e.g. ML bootstrap support of 86% and Bayesian support value of 100 in trees based on DNA sequences (Table 3, Fig. 3). These trees were rooted outside the kinetoplastids, using the E. gracilis GapC gene, which is considered an appropriate taxon for testing for monophyly of trypanosomes (Hughes and Piontkivska, 2003b). This gene (codon positions 1 and 2) should be a reliable root, because its ratio of base frequencies does not differ significantly from that of other ggapdh genes in the alignment and it differs from the other sequences in the alignment by a maximum of 21.7% (uncorrected p- distance); therefore positions are unlikely to be saturated. Use of the SH test to compare the ML ggapdh tree (Fig. 3) to constrained trees show that this tree is robust, providing additional support for monophyly (Table 4). A constrained tree in which the trypanosomatid vertebrate parasites (Trypanosoma and Leishmania) were forced into a clade was rejected (PZ0.013). Constrained trees in which

8 1400 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Fig. 2. Trees based on the SSU rrna gene alignment 2. A: Unbootstrapped ML tree including all 70 taxa. KLnZ B: ML tree from alignment 2 excluding the euglenid sequences (68 taxa). Kln LZ Values at nodes are bootstrap values (%: 100 replicates). C: MP tree excluding both euglenids and the T. brucei clade (61 taxa). One of 42 most parsimonious trees of lengthz1424, CIZ0.536, RIZ Values at nodes are bootstrap values (%: 1000 replicates). the T. brucei clade was forced into a clade with bodonids, as seen in the tree in Fig. 2A, and a tree in which T. vivax was forced outside the other kinetoplastids, as seen in distance trees from alignment 2 (Hughes and Piontkivska, 2003b), were rejected (P!0.001). Likewise, a constrained tree in which the deepest split within trypanosomatids was between the T. brucei clade and the rest, as seen the tree in Fig. 2B, was also rejected (P!0.001). The ggapdh data therefore support placement of T. vivax in the T. brucei clade, which itself falls within the monophyletic trypanosome clade Paraphyly of non-trypanosome trypanosomatids In all ggapdh DNA trees, non-trypanosome trypanosomatids branch paraphyletically at the base of trypanosomes. Significantly, a constrained ggapdh tree, in which monophyly of the non-trypanosome trypanosomatids was forced, as seen in two of the SSU rrna gene trees shown here (Fig. 1, Fig. 2B), was significantly suboptimal, according to the SH test (P!0.001). However constrained ggapdh trees in which each of the trypanosomatid lineages E (Phytomonas), F or J (trypanosomes) were forced to be the basal trypanosomatid clade, were not rejected. Significantly, trees based on ggapdh protein sequences also showed paraphyly, with H. samuelpessoai branching before the clade with Leishmania, although the trees differed in the placement of Phytomonas. The Bayesian protein tree (not shown) was identical to the DNA tree shown in Fig. 3, with branch H weakly (support value 92) and branch I strongly (support value 99) supported. In ML and MP phylogenies, Phytomonas fell in clade D or in a polytomy with clade F, respectively, but these alternate positions were weakly supported. It is noteworthy that in trees from alignment 2.3, in which trypanosome monophyly was supported (Fig. 2C), the clade with H. samuelpessoai also branched more deeply than the clade with Leishmania.

9 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Fig. 3. Maximum likelihood tree based on an alignment of the 1st and 2nd codon positions of ggapdh and rooted on the corresponding gene of Euglena gracilis. Single values at nodes are ML bootstrap values (%; 500 replicates). Multiple values at nodes are in order: ML bootstrap value, Bayesian support value, MP bootstrap value and ML distance bootstrap value). Kln LZ Branches and clades A-O were used in construction of constrained trees-see Table Discussion 4.1. Monophyly of trypanosomes Our phylogenetic analyses of ggapdh data support monophyly of trypanosomes. When considered together with published hsp90 trees (Simpson et al., 2002), a strong case for monophyly of trypanosomes is made that is independent of the SSU rrna gene data: in both trees monophyly receives robust statistical support and the outgroups are undisputed. In addition, sufficiently wide selections of the two kinetoplastid groups, trypanosomatids and bodonids, are included in the ggapdh and hsp90 trees respectively. The SSU rrna gene data neither strongly support nor reject trypanosome monophyly, as different alignments give

10 1402 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Table 4 Comparison of constrained maximum-likelihood trees based on ggapdh alignment 2 using the Shimodaira Hasegawa (S H) test Constraint Tree -Ln Diff -ln L SH test P None (Fig. 3) (best) Trypanosoma and Leishmania monophyletic (A,B,D,E, F-G (G,J)) a T. vivax outside other kinetoplastids ((A,tv)(B, C-tv )) a T. vivax outside other trypanosomes (A,B,D,E,F,tv( J-tv )) T. brucei clade sister to bodonids (A,(B,K) C-K ) a T. brucei clade basal trypanosomatid ((A,B,K) C-K ) a Fig. 2B topology ((A,B,K)(L(O(M(D,E,F))))) a Fig. 1 topology (A,B,(D,E,F)(J)) a Clade F basal trypanosomatids ((A,B,F)(D,E,J)) Brach E (Phytomonas) basal trypanosmatid ((A,B,E)(D,I)) Trypanosomes basal trypanosomatids ((A,B,J)(D,E,F)) a P!0.05 Clades and branches A O are shown in Fig. 3; tv, T. vivax; C-tv, clade C excluding T. vivax, etc. different tree topologies. The range of taxa and characters included in analyses seems most likely to account for the different tree topologies. More trypanosomatid sequences were included in alignment 1: 65 compared with 53, whereas more non-trypanosomatids were included in alignment 2 (Hughes and Piontkivska, 2003b): 15 compared with four. In other respects alignments were similar: they were constructed using similar methods, all positions with gaps were excluded from subsequent analyses, and they had similar numbers of informative positions within trypanosomatids: 280 in alignment 1 compared with 227 in alignment 2 (Hughes and Piontkivska, 2003b). The placement of T. vivax proved particularly problematic in some SSU rrna gene trees (e.g. Hughes and Piontkivska, 2003b), whereas the ggapdh data unequivocally placed T. vivax in the T. brucei clade, in agreement with trees based on our SSU rrna gene alignment and the majority of recent phylogenies based on the same gene (e.g. Lukes et al., 1997; Haag et al., 1998; Stevens and Gibson, 1999; Stevens et al., 1999, 2001). Indeed, in our SSU rrna gene trees, T. vivax only fell outside the T. brucei clade when distant euglenid outgroups were used. In common with T. vivax, other trypanosome species in this group, such as T. brucei, Trypanosoma congolense and Trypanosoma simiae, occur in Africa and are transmitted via the saliva of tsetse flies (Hoare, 1972). Like T. vivax, all T. brucei clade trypanosomes are mammalian parasites and are the only trypanosomes known to undergo antigenic variation in the vertebrate host (Barry, 1986; Gardiner, 1989; Myler, 1993). Therefore, placement of T. vivax within the T. brucei clade makes biological sense: adaptations requiring complex developmental pathways and sophisticated genetic machinery are unlikely to have evolved independently in different trypanosome lineages Evolutionary origin of trypanosomes All trypanosomes are vertebrate blood parasites. The evidence presented here suggests that they are monophyletic and thus had a single evolutionary origin. The most likely ancestor is an insect trypanosomatid parasite, because in ggapdh trees the trypanosome clade arises from within the wider group of trypanosomatids, and these are insect parasites, some of which have a vertebrate or plant host in addition. From a single origin, trypanosomes have radiated into all vertebrate classes, and have a wide variety of bloodsucking vectors, including both terrestrial and aquatic leeches. The relationships of Leishmania and Endotrypanum, which are also vertebrate parasites, suggest that they do not share a common ancestor with trypanosomes and evolved from an insect-only trypanosomatid(s) comparatively recently. It is clear that T. brucei, T. cruzi and L. major, for which complete genome sequences are now available, are distantly related and have distinct evolutionary histories. In view of the results presented here, comparison of trypanosome genomes may reveal ancestral genetic mechanisms for survival in vertebrates, which will not be shared by Leishmania. On the other hand, these trypanosomatids may share ancestral mechanisms for survival within their insect vectors. A second implication of monophyly is that trypanosomes did not give rise to other trypanosomatid lineages by for example adapting to an invertebrate-only form of parasitism. This is surprising, as many trypanosome species undergo varied and complex life cycles in their invertebrate vectors. Instead, some trypanosomes have partly or completely lost dependency on the invertebrate host. For example, although usually transmitted by bloodsucking triatomine bugs, T. cruzi can be transmitted during breastfeeding (Miles, 1979) and Trypanosoma equiperdum is transmitted during coitus between equines (Hoare, 1972). There is further support for the insect parasite origin of genus Trypanosoma from consideration of the phylogenetic position of the leech-transmitted trypanosomes of fish and amphibia. If leech-transmitted trypanosomes of fish were the first to evolve as has been suggested (Woo, 1970; Baker, 1994; Vickerman, 1994), we would expect them to branch paraphyletically at the base of the trypanosome clade. This is not the case: the phylogenies presented here show all fish trypanosomes in a single, well-supported clade.

11 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) In ggapdh, and most published SSU rrna gene trees (Lukes et al., 1997; Haag et al., 1998; Stevens et al., 1998, 1999, 2001; Wright et al., 1999; Simpson et al., 2002) this clade falls in the aquatic clade, which includes all aquatic leech-transmitted trypanosomes. Although we have no evidence whether the ancestor of the aquatic clade trypanosomes was leech or insect-transmitted, it is clear that the majority of insect-transmitted trypanosomes did not evolve from this clade. Taken together, this evidence suggests a scenario whereby an early trypanosomatid, perhaps a parasite of a blood-sucking insect, adapted to and became dependant on vertebrate parasitism, giving rise to trypanosomes, as previously suggested (Hoare, 1972). The switch from an insect-only to an insect-transmitted trypanosome would have been a terrestrial event, assuming that extinct bloodsucking insects fed on land vertebrates as today. There are no reliable dates for the origins of most extant groups of blood-sucking insects; the earliest possible date is approximately 370 million years ago after the first land vertebrates appeared (Kardong, 2002). The adaptation to transmission by aquatic leeches of fish and some amphibian trypanosomes would therefore have been secondary, as previously suggested (Maslov et al., 1996). Intriguingly, Trypanosoma binneyi, the trypanosome of the amphibious platypus, appears in the aquatic clade. Taken together with the fact that T. binneyi is a large trypanosome, similar in morphology to the trypanosomes of fish and amphibia (Mackerras, 1959), it is plausible that the trypanosome species of this primitive mammal has been acquired from aquatic leeches carrying fish trypanosomes. Since these in turn probably had ancestors that were transmitted between land vertebrates by insects, this provides an example of how trypanosomes have switched hosts. Thus, trypanosomes have not strictly coevolved with their vertebrate hosts; instead, a key factor in the evolutionary radiation of trypanosomes appears to have been transmission between vertebrates in shared environments. As noted by Haag et al. (1998), the lack of host specificity of many leeches and arthropods vectors, may have facilitated host switching Evolutionary origin of trypanosomatids Invertebrate-only trypanosomatids are restricted to 10 insect orders in which they are gut parasites (Wallace, 1966). It is unlikely that they were parasites of the earliest insects, which appeared approximately 400 million years ago (Gaunt and Miles, 2002), as living representatives of the most primitive insect orders (Odonata, Ephemoptera) do not carry trypanosomatids (Wallace, 1966). Evidence from hsp90 phylogenies, and kinetoplast DNA structure suggests that trypanosomatids evolved from a free-living bodonid (Simpson et al., 2002) rather than a leech-transmitted vertebrate parasite, such as Trypanoplasma. Today free living bodonids are common and widespread in aquatic environments including soil (Dolezel et al., 2000). A likely scenario is that bodonids consumed accidentally by insects adapted to parasitism of the insect gut, giving rise to trypanosomatids. Acknowledgements We thank Peter Holz and other staff at Healesville sanctuary, Melbourne, Australia, and Harry Noyes, Melody Serena, Geoff Williams, Steve Williams and Brian Cooke for provision of the various Australian trypanosome samples used in this study. Other trypanosomes and DNA samples were kindly provided by Jiri Lom, Iva Dykova, Julius Lukes, Angela Russell, Zhao Rong Lun, Sherwin Desser, Andy Tait, J.-P. Dedet, Michel Tibayrenc, Marta Teixeira, and Sylvain Brisse. We thank Gary Barker for help with computer analysis and The Wellcome Trust for funding. PBH was funded by a Wellcome Trust Biodiversity studentship. References Adjé, C.A., Opperdoes, F.R., Michels, P.A., Molecular analysis of phosphoglycerate kinase in Trypanoplasma borreli and the evolution of this enzyme in kinetoplastida. Gene 217, Alvarez, F., Cortinas, M.N., Musto, H., The analysis of protein coding genes suggests monophyly of Trypanosoma. Mol. Phylogenet. Evol. 5, Baker, J.R., The origins of parasitism in the protists. Int. J. Parasitol. 24, Barry, J.D., Antigenic variation during Trypanosoma vivax infections of different host species. Parasitology 92, Cavalier-Smith, T., Kingdom protozoa and its 18 phyla. Microbiol. Rev. 57, Dolezel, D., Jirku, M., Maslov, D.A., Lukes, J., Phylogeny of the bodonid flagellates (Kinetoplastida) based on small-subunit rrna gene sequences. Int. J. Syst. Evol. Micr. 50, Felsenstein, J., Cases in which parsimony or compatibility methods will be positively misleading. Syst. Zool. 27, Fernandes, A.P., Nelson, K., Beverley, S.M., Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism. Proc. Natl Acad. Sci. USA 90, Fothergill-Gilmore, L.A., Michels, P.A.M., Evolution of glycolysis. Prog. Biophys. Mol. Biol. 59, Gardiner, P.R., Recent studies of the biology of Trypanosoma vivax. Adv. Parasitol. 28, Gaunt, M.W., Miles, M.A., An insect molecular clock dates the origin of the insects and accords with palaeontological and biogeographic landmarks. Mol. Biol. Evol. 19, Gomez, E., Valdes, A.M., Pinero, D., Hernandez, R., What is a genus in the Trypanosomatidae family? Phylogenetic analysis of two small rrna sequences. Mol. Biol. Evol. 8, Haag, J., O huigin, C., Overath, P., The molecular phylogeny of trypanosomes: evidence for an early divergence of the Salivaria. Mol. Biochem. Parasitol. 91, Hallick, R.B., Hong, L., Drager, R.G., Favreau, M.R., Monfort, A., Orsat, B., Spielmann, A., Stutz, E., Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res. 21,

12 1404 P.B. Hamilton et al. / International Journal for Parasitology 34 (2004) Hannaert, V., Blaauw, M., Kohl, L., Allert, S., Opperdoes, F.R., Michels, P.A., Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana. Mol. Biochem. Parasitol. 55, Hannaert, V., Opperdoes, F.R., Michels, P.A., Comparison and evolutionary analysis of the glycosomal glyceraldehyde-3-phosphate dehydrogenase from different Kinetoplastida. J. Mol. Evol. 47, Hashimoto, T., Nakamura, Y., Kamaishi, T., Adachi, J., Nakamura, F., Okamoto, K., Hasegawa, M., Phylogenetic place of kinetoplastid protozoa inferred from a protein phylogeny of elongation factor 1 alpha. Mol. Biochem. Parasitol. 70, Henze, K., Badr, A., Wettern, M., Cerff, R., Martin, W., A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc. Natl Acad. Sci. USA 92, Hoare, C.A., The Trypanosomes of Mammals. Blackwell Scientific Publications, Oxford. Huelsenbeck, J.P., Ronquist, F., MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, Hughes, A.L., Piontkivska, H., 2003a. Molecular phylogenetics of Trypanosomatidae: contrasting results from 18S rrna and protein phylogenies. Kinetoplastid Biol. Dis. 2, 15. Hughes, A.L., Piontkivska, H., 2003b. Phylogeny of Trypanosomatidae and Bodonidae (Kinetoplastida) based on 18S rrna: evidence for paraphyly of Trypanosoma and six other genera. Mol. Biol. Evol. 20, Jones, D.T., Taylor, W.R., Thornton, J.M., The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, Kardong, K.V., Vertebrates: Comparative Anatomy, Function, Evolution. McGraw-Hill, New York. Kendall, G., Wilderspin, A.F., Ashall, F., Miles, M.A., Kelly, J.M., Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the hotspot topogenic signal model. Eur. Mol. Biol. Org. J. 9, Landweber, L.F., Gilbert, W., Phylogenetic analysis of RNA editing: a primitive genetic phenomenon. Proc. Natl Acad. Sci. USA 91, Lukes, J., Arts, G.J., Van den Burg, J., de Haan, A., Opperdoes, F., Sloof, P., Benne, R., Novel pattern of editing regions in mitochondrial transcripts of the cryptobiid Trypanoplasma borreli. Eur. Mol. Biol. Org. J. 13, Lukes, J., Jirku, M., Dolezel, D., Kral ova, I., Hollar, L., Maslov, D.A., Analysis of ribosomal RNA genes suggests that trypanosomes are monophyletic. J. Mol. Evol. 44, Mackerras, M.J., The haematozoa of Australian mammals. Aust. J. Zool. 7, Maslov, D.A., Lukes, J., Jirku, M., Simpson, L., Phylogeny of trypanosomes as inferred from the small and large subunit rrnas: implications for the evolution of parasitism in the trypanosomatid protozoa. Mol. Biochem. Parasitol. 75, Maslov, D.A., Simpson, L., Evolution of parasitism in kinetoplastid protozoa. Parasitol. Today 11, Michels, P.A., Poliszczak, A., Osinga, K.A., Misset, O., Van Beeumen, J., Wierenga, R.K., Borst, P., Opperdoes, F.R., Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei. Eur. Mol. Biol. Org. J. 5, Miles, M.A., Transmission cycles and the hetrogeneity of Trypanosoma cruzi, in: Lumsden, W.H.R., Evans, D.A. (Eds.), Biology of the Kinetoplastida, vol. 2. Academic Press, London, pp Myler, P.J., Molecular variation in trypanosomes. Acta Trop. 53, Philippe, H., Opinion: long branch attraction and protist phylogeny. Protist 151, Qian, Q., Keeling, P.J., Diplonemid glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prokaryote-to-eukaryote lateral gene transfer. Protist 152, Schmidt, H.A., Strimmer, K., Vingron, M., von Haeseler, A., TREE- PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, Shimodaira, H., Hasegawa, M., Multiple comparisons of loglikelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16, Simpson, A.G.B., The identity and composition of the Euglenozoa. Arch. Protistenkd. 148, Simpson, A.G.B., Lukes, J., Roger, A.J., The evolutionary history of kinetoplastids and their kinetoplasts. Mol. Biol. Evol. 19, Sleigh, M.A., Protozoa and other Protists. Edward Arnold, London pp Stevens, J., Gibson, W., The molecular evolution of trypanosomes. Parasitol. Today 15, Stevens, J., Noyes, H., Gibson, W., The evolution of trypanosomes infecting humans and primates. Mem. I. Oswaldo Cruz 93, Stevens, J., Noyes, H.A., Dover, G.A., Gibson, W.C., The ancient and divergent origins of the human pathogenic trypanosomes, Trypanosoma brucei and T. cruzi. Parasitology 118, Stevens, J., Noyes, H.A., Schofield, C.J., Gibson, T.J., The molecular evolution of Trypanosomatidae. Adv. Parasitol. 48, Stevens, J., Rambaut, A., Evolutionary rate differences in trypanosomes. Infect. Genet. Evol. 1, Swofford, D.L., Olsen, P.J., Waddell, P.J., Hillis, D.M., Phylogenetic inference, in: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.), Molecular Systematics. Sinauer Associates, Inc., Massachusetts, pp Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, Vickerman, K., The evolutionary expansion of the trypanosomatid flagellates. Int. J. Parasitol. 24, Wallace, F.G., The trypanosomatid parasites of insects and arachnids. Exp. Parasitol. 18, Wiemer, E.A., Hannaert, V., van den, I.P.R., Van Roy, J., Opperdoes, F.R., Michels, P.A., Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida. J. Mol. Evol. 40, Woo, P.T.K., Origin of mammalian trypanosomes which develop in the anterior station of blood-sucking arthropods. Nature 228, Wright, A.D., Li, S., Feng, S., Martin, D.S., Lynn, D.H., Phylogenetic position of the kinetoplastids, Cryptobia bullocki, Cryptobia catostomi, and Cryptobia salmositica and monophyly of the genus Trypanosoma inferred from small subunit ribosomal RNA sequences. Mol. Biochem. Parasitol. 99,