Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo;
Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro;
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo
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Abstract |
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Introduction |
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Here we studied the evolution of T. cruzi strains, based on maximum-likelihood analysis of 18S rDNA sequences from 20 strains of T. cruzi, and identified four subgroups which we named as Riboclades. Also, we expanded the analysis of the highly variable region, D7, within the 24S rDNA (D7-24S) by sequencing the corresponding segments from four strains of the zymodeme 3 subgroup (Miles and Cibulskis 1986
; Mendonça et al. 2001
). Maximum-likelihood parameters and the molecular clock of corresponding phylogenies were optimized and tested by likelihood ratio tests. Our analysis suggests that divergence times can be estimated from particular 18S rDNA subsets. Given the external calibration of the rDNA clock from metazoan 18S rDNA, the divergence times estimated here support our proposed hypothesis of geographical isolation of the two major groups of strains in the last 84 Myr and their possible coevolution with the mammalian fauna of the Americas during the Cenozoic (Briones et al. 1999
). If the 18S rRNA gene in T. cruzi is assumed to evolve twofold to fourfold faster than in other organisms, a scenario consisting of a more recent divergence (1816 MYA) is favored (Machado and Ayala 2001)
.
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Materials and Methods |
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Sequencing of 18S rDNA and D7-24S rDNA
The 18S rDNA was amplified using universal eukaryotic primers and cloned into pUC18 using the Sureclone kit (Amersham-Pharmacia). D7-24S rDNA sequences were amplified using specific primers as described earlier. Templates were sequenced in a ABI377/96 automated sequencer (Applied Biosystems) using fluorescent BigDye dideoxy-chain terminators. Chromatograms, derived from complete sequences of at least three clones from each strain, were used for contiguous assembly and finishing of high quality sequences (Phred scores above 30, which correspond to less than 1 estimated error per 1,000 bases sequenced) using Phred + Phrap + Consed (Gordon, Abajian, and Green 1998
). Chromatograms are available upon request by e-mail (marcelo@ecb.epm.br). Sequences determined in this study were deposited in GenBank as follows: 18S rDNA sequences: T. cruzi MT3869, AF303660; T. cruzi Silvio X10 cl1, AF303659; T. cruzi Y, AF301912; T. cruzi YuYu, AF245380; T. cruzi MT4166, AF292942; T. cruzi MT4167, AF288661; T. cruzi MT3663, AF288660; T. cruzi NRcl3, AF228685; T. cruzi CL Brener, AF245383; T. cruzi Dm28c, AF245382; T. cruzi CA-1, AF245381; T. cruzi G, AF239981; T. cruzi Colombiana, AF239980; and T. cruzi Sc43cl1, AF232214. The D7-24S
rDNA sequences here determined were: T. cruzi 4167, AF288665; T. cruzi 4166, AF288664; T. cruzi 3869, AF288663; and T. cruzi 3663, AF288662. Details on the host and geographic origin of the strains can be found elsewhere (Souto et al. 1996
).
Phylogenetic Inference
Alignments were made manually using the sequences determined in this study and other sequences downloaded from GenBank: T. cruzi Peru X53917 (Fernandes, Nelson, and Beverley 1993
); T. cruzi Silvio X10, AJ009147; T. cruzi Vinch89, AJ009149; T. cruzi CANIII AJ009148; T. cruzi marinkellei, AJ009150 (Stevens, Noyes, and Gibson 1998
); and T. rangeli San Agustin, AF065157 (Briones et al. 1999
). Sequences were aligned by eye using seaview sequence editor for UNIX (Galtier, Gouy, and Gautier 1996
). Alignments are available upon request by e-mail (marcelo@ecb.epm.br). Phylogenies were inferred using maximum-likelihood estimates using PAUP 4.0 (Swofford 1998
). Maximum-likelihood parameters, such as model, base frequencies, shape of gamma distribution, and proportion of invariant sites, were optimized using the hierarchical likelihood test with Bonferroni correction implemented in Modeltest 3.0 (Posada and Crandall 1998
). Bootstrap analysis to test for monophyly was done using PAUP 4.0 with removal of invariant and gapped positions, and parameters were estimated for each bootstrap replicate. Likelihood ratio tests for the molecular clock were performed as described (Felsenstein 1988
; Swofford et al. 1996
) and PAUP 4.0 was used to obtain the likelihood scores of each phylogeny. Confidence intervals for divergence time estimates were obtained using QDate 1.11 (Rambaut and Bromham 1998
), where the divergence time estimates of quartet pairs in the input file were calculated using percent divergence from fossil calibrations as described by Escalante and Ayala (1995)
. QDate input run parameters were obtained by Modeltest analysis.
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Results |
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Discussion |
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The analysis presented here also suggests that Riboclade 2 is a sister group of the Riboclades that contain representatives of zymodeme Z3 (Miles et al. 1978
) and group 1/2 (Souto et al. 1996
). The subtree (Riboclade 2 [Riboclade 3, Riboclade 4]) (fig. 1A
) has an internal divergence which can be noticed also in the D7-24S rDNA tree (fig. 1B
). This contrasts with the previous proposition that the Riboclade 1 group has more variability than Riboclade 2 (Brisse, Barnabe, and Tibayrenc 2000a
).
Possibly, the variability found among Riboclades 2, 3, and 4 might be explained by the evolution and distribution of specific mammalian hosts, and of the insect vectors, reduviid bugs, that evolved in South America during the Cenozoic (Marcilla et al. 2001)
. Group 1/2, here represented by isolates NRcl3 and S03cl5, is an interesting group of strains. This group has been characterized by Souto et al. (1996)
based on the structure of the rDNA cistrons, the intergenic region of the mini-exon gene, and RAPD profiles. We demonstrated that the genome of these isolates contains two types of rDNA cistrons (1 and 2) and that copies of type 2 are 10-fold more abundant than copies of type 1 (Souto et al. 1996
). We have also observed that only type 2 rRNA genes are transcribed in group 1/2 strains. In the present study, we compared the D7-24S rDNA sequences of the two types of rDNA cistrons of SO3cl5. In the phylogeny shown in figure 1B
, we observe that D7-24S rDNA type 1 fragment (SO3cl5[1]) branched along with other Riboclade 1 sequences, whereas D7-24S rDNA type 2 fragment (SO3cl5 [2]) branched along with Riboclade 3 sequences, which also contains parasites that have only the D7-24S rDNA type 2 gene. Accordingly, the analysis of the 18S rRNA gene of type 2 cistron of NRcl3, also belonging to group 1/2, places this sequence in Riboclade 3 in the phylogeny shown in figure 1A.
The origin of group 1/2 is still a matter of debate. Analysis of the structure of the intergenic region of the mini-exon gene and that of 5060 anonymous loci generated by RAPD places this group of strains in lineage 1 (corresponding to ribodeme 1) (Souto et al. 1996
). In that study we hypothesized that group 1/2 could have been originated by an eventual transfer of rDNA from individuals of group 2 to group 1, either by mating or by horizontal gene transfer. Analysis of other genetic markers would indicate if other sets of genes (besides rDNA) have also been transferred in group 1/2. Supporting this hypothesis, three reports suggest the formation of hybrid organisms in sylvatic T. cruzi populations (Carrasco et al. 1996
), in sympatric clinical isolates (Bogliolo, Lauria-Pires, and Gibson 1996
), and in experiments, as dual-resistant T. cruzi biological clones could be obtained following copassage of parents carrying single episomal drug-resistant markers (Stothard, Frame, and Miles 1999
). Recently, Machado and Ayala (2001) concluded
, based on nucleotide sequences from two nuclear genes that encode the enzymes trypanothione reductase and dihydrofolate reductase-thymidilate synthetase and a region from the mitochondrial genome, that two widely distributed isoenzyme typesone corresponding to group 1/2are hybrids.
Typing based on D7-24S rDNA was improved also with the identification of D7-24S rDNA type 3. Because of a small difference in size between the 125- and the 117-bp D7-24S rDNA amplicons, some strains originally typed as rDNA type 1 (Souto et al. 1996
) represent another group corresponding to our Riboclade 4.
In conclusion, in the absence of fossil records of trypanosomatids, the only way to estimate the divergence times is through molecular data. Therefore, we have tested the molecular clock in the 18S rDNA and identified sequence subsets which reconstruct phylogenies that can be used to estimate divergence times because they passed the likelihood ratio test, and an evolutionary rate for the entire 18S rDNA has been estimated (Escalante and Ayala 1995
). Our estimates of divergence times were, therefore, calibrated by other taxonomic groups whose respective divergence times were dated from the fossil record (Escalante and Ayala 1995
). Other methods to date and estimate confidence intervals were proposed, although they require that quartets in the data set are dated by the fossil record (Rambaut and Bromham 1998
). The fossil record estimates should be taken in the perspective that these estimates indicate that a particular group is as old as the estimate but do not exclude that it can be older. Variations of evolutionary rates among lineages are tested by the likelihood ratio test, and the phylogeny presented in figure 3 passed the test. Another problem is that the 18S rDNA could be evolving in the whole T. cruzi clade faster than observed for the other groups from which the rate of 0.85% sequence divergence per 100 Myr was obtained. Because of this problem we estimated our divergence times also using two other rates (2% and 4% sequence divergence per 100 Myr) (Ochman and Wilson 1987
; Wilson, Ochman, and Prager 1987
; Moran et al. 1993
), which accommodate for a 2.4-and 4.7-fold rate increase, respectively. Even with a 2.4-fold rate increase, the divergence would date back to approximately 36 MYA, when rodents and primates first entered into South America (Simpson 1980
). Our proposition is that Riboclades 2, 3, and 4 were evolving in South American marsupials, isolated from North American placentals, parasitized by Riboclade 1, during most of the Cenozoic period, which corresponds approximately to the divergence time estimated here (fig. 4
) (Penny and Hasegawa 1997
). The hypothetical ancestor of Riboclades 1, 2, 3, and 4 (fig. 3
) was spread over South and North America 84.4 MYA, still during the period when the geographical isolation of South America from the Old World was not a barrier to mammalian migration. After the isolation of South America in the Cenozoic, its mammalian fauna was composed of marsupials, edentates, and ungulates. Interestingly, T. cruzi does not infect large ungulate mammals, such as cows and horses (Wendel et al. 1992
), which possibly restricted T. cruzi in South America to marsupial and edentate hosts. According to this hypothesis, T. cruzi Riboclade 1 might have entered South America either during the Oligocene, along with rodents and primates that came via island hopping, or during the great faunal exchange during the Pliocene-Pleistocene, when South and North America were connected via the Panama isthmus. In North America the association between Riboclade 1 and placental mammals and Riboclade 2 and marsupials is very strong and might be evidence for independent evolution of these two clades for a long period (Clark and Pung 1994
). The North American marsupial Didelphis virginiana is of South American origin; possibly ancestors of these mammals introduced T. cruzi Riboclade 2 into North America during the faunal exchange (Briones et al. 1999
). In our study we are trying to estimate events that are related to the speciation time, which is not exact, and the very slow separation of South America from Africa. This is estimated to have occurred during the late Jurassic into the late Cretaceous, and even in the late Cretaceous (approximately 100 Myr) there was a narrow seaway between Africa and South America (Futuyma 1998
, p. 182). An alternative is to assume a faster rate of evolution (4% divergence per 100 Myr) for the 18S rDNA in T. cruzi. This would suggest that the divergence of strains occurred recently (less than 18 Myr) and that it might be a result of either coevolution with insect vectors or geographical barriers within South America that isolated marsupials and placental mammals carrying T. cruzi II. This latter scenario is favored by our estimates (table 2 in Briones et al. 1999
) and recent estimates using dihydrofolate reductase and trypanothione reductase, with a superimposed divergence between T. cruzi and T. brucei of 100 Myr (Machado and Ayala 2001)
. This more recent study argues that if the divergence is as deep as 8837 MYA, the silent substitution rates for these two protein-coding genes would be an order of magnitude smaller than the average synonymous substitution rates of other organisms (Machado and Ayala 2001)
. However, codon bias and expression levels of particular protein-coding genes are known to affect the rate of synonymous substitutions in such orders of magnitude (Akashi 1
994). In fact, the very broad range of confidence intervals of our estimates, as calculated by quartet analysis (QDate) (table 2
), still do not allow us to decide if the divergence among T. cruzi strains occurred 1816 MYA (Briones et al. 1999
; Machado and Ayala 2001
) or 8837 MYA (Briones et al. 1999
).
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Acknowledgements |
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Footnotes |
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Keywords: Chagas' disease
Trypanosoma cruzi
evolution
rRNA gene
molecular clock
maximum-likelihood
Address for correspondence and reprints: Marcelo R. S. Briones, Disciplina de Microbiologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu, 862, 3° andar, CEP 04023-062, São Paulo, S.P., Brazil. marcelo{at}ecb.epm.br
.
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