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Classification and phylogeny of Trypanosoma cruzi

PB. Hamilton and J.R. Stevens University of Exeter, Exeter, United Kingdom

Chapter Outline

Application of molecular phylogenetics to trypanosome taxonomy and understanding evolution 321

Origin of trypanosomes and the relationship between T. cruzi and T. brucei 323 Relationships within the genus Trypanosoma 325

Molecular phylogenetics and traditional taxonomy of mammalian trypanosomes 327

The main groups of trypanosomes recognized in molecular phylogenetic

analyses 328

The T. cruzi clade 330

The origin of the T. cruzi clade 333

Outlook 335

Glossary 335

References 336

Application of molecular phylogenetics to trypanosome taxonomy and understanding evolution

All trypanosomes (genus Trypanosoma) are vertebrate parasites and have a characteristic morphology in the vertebrate bloodstream. Trypanosomes are diverse and successful, being found in all classes of vertebrate—fish, amphibians, birds, reptiles, and mammals (monotremes, marsupial, and placental)1—and in all continents. The vast majority of trypanosome species are transmitted by leeches and arthropods (mostly insects), although a few species can be passed directly between vertebrates. There are no free-living stages. Although several species are associated with important diseases of humans and domestic livestock, there is little evidence for health impacts on their wild vertebrate hosts, with the exception of some Australian trypanosomes which have been linked to poor health of marsupials.2,3

There has been considerable interest in the evolutionary origin of trypanosomes (genus Trypanosoma) and the relationships of species within the genus since the early 1900s.1,4-6 In particular, the relationship between the two human pathogens, Trypanosoma cruzi and Trypanosoma brucei has received considerable attention.7-12 However, due to the absence of a fossil record, and with few morphological features,

American Trypanosomiasis Chagas Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-801029-7.00015-0

Copyright © 2017 Elsevier Inc. All rights reserved.

testing evolutionary hypotheses has only become possible since the late 1980s/early 1990s with the advent of molecular phylogenetics. Molecular phylogenetic trees (phylogenies) are constructed through comparisons of DNA sequences (or amino acid sequences inferred from them) from a range of organisms. Phylogenies can be used to trace the evolutionary history of a group of organisms, thus providing robust frameworks for testing evolutionary hypotheses.

It is now relatively straightforward to sequence short (100—2000 bp) stretches of DNA from trypanosomes. First the chosen gene is amplified using the polymerase chain reaction (PCR), followed by sequencing of purified PCR products. Computer programs can then be used to align sequences from different taxa (e.g., species) and for subsequent construction of evolutionary trees. Increased computing power has allowed advanced computer-intensive tree building methods, such as maximum- likelihood and Bayesian methods, to be applied to sequences from many organisms, and even sequence data from whole genomes.11 Although the first studies of this nature relied on DNA isolated from cultured parasites, many more recent studies have used DNA extracted directly from the host tissue, such as blood, or from insect guts. The development of such methods led the way to larger-scale surveys of parasite diversity, which have transformed our understanding of the diversity of trypanosome species and their host ranges.

A range of genes have been used for phylogenetic and taxonomic studies. The majority of early studies that examined relationships between species used nuclear ribosomal DNA markers, in particular, 18S rDNA (also known as the small subunit (SSU) rDNA), and to a lesser extent 28S rDNA (also known as the large subunit (LSU) rDNA). The 18S rRNA gene has both conserved regions, suitable for primer design and for resolving relationships between distantly related species, and faster- evolving regions, suitable for deducing evolutionary relationships between closely related species and at the subspecies level. The V7—V8 region of 18S rDNA is the most variable and is often called the “barcoding” region, because it is useful for species identification. It has also been used to develop species-specific PCR primers. The noncoding internal transcribed spacer (ITS) regions, ITS1 and ITS2, also have fast evolutionary rates, and have proved useful for studying within-species diversity. Protein-coding genes have also been used; in particular, glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) for phylogenetic placement of newly described species and for resolving some “difficult” relationships within the genus. Due to the ease of alignment of sequences from distantly related species, and because it is often possible to amplify the majority of the gene in a single PCR using DNA from uncultured parasites, gGAPDH has sometimes been used in preference to 18S rDNA. The faster-evolving kinetoplast (mitochondrial) cytochrome b (Cyt b) gene has also been used for examining within-species diversity.13,14

Phylogenies based on gGAPDH and 18S rDNA sequences both separately and combined have resolved many of the relationships within the genus and in most cases are sufficient for taxonomic placement of new trypanosomes. However, relationships between some of the major groups remain poorly resolved. This is likely to change in the near future as it has become relatively straightforward to sequence complete genomes of trypanosome species using next generation sequencing technologies.15 The use of such data enables phylogenies to be constructed

using vastly more information than using just one or two genes, providing greater resolution. For instance, analysis of the full genome sequence of T. grayi, a tseste- fly transmitted trypanosome of African crocodiles demonstrated unequivocally that it is more closely related to T. cruzi than to T. brucei, indicating separate origins of tsetse-fly transmission in Africa for this crocodile trypanosome and the T. brucei group.15 Previously, in gGAPDH and 18S rDNA trees, the relationship of this crocodile parasite to other trypanosomes of terrestrial mammals had been only poorly resolved.

Molecular phylogenetics has also informed taxonomy.16 Taxonomic groups should have evolutionary relevance, and arguably names should only be applied to monophyletic groups. Molecular studies have questioned the validity of some species that were previously described using only morphological and lifecycle data. Molecular methods have also raised new questions, such as whether new species should be named on the basis of sequence information alone, and the degree of genetic divergence necessary to classify lineages as the same or different species.17 At the same time, surveys of trypanosomatid diversity that use rapid methods for species identification based on sequencing , or length differences within regions of ribosomal DNA , have led to the description of new species. While many of these new species were found in previously unstudied hosts, such as Australian marsupials,25-29 some potentially pathogenic species have also been found in well-studied groups, such as the African tsetse fly-transmitted group of trypanosomes.19,20,30 Analysis of DNA from Giemsa-stained microscope slides revealed that one of the “new” trypanomes described recently using molecular methods from tsetse flies in Tanzania20 and the Central African Republic19 was T. suis. This pig pathogen was first described in the 1950s but was subsequently largely forgotten.31 It is clear that much trypanosomatid diversity is yet to be discovered.

 
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