American Trypanosomiasis || Classification and Phylogeny of Trypanosoma cruzi

13Classification and Phylogeny ofTrypanosoma cruzi

Patrick B. Hamilton* and Jamie R. Stevens

School of Biosciences, University of Exeter, Exeter, UK

13.1 Application of Molecular Phylogenetics to the studyof Trypanosome Taxonomy and Evolution

Trypanosomes (genus Trypanosoma) all share vertebrate parasitism and have a

characteristic morphology in the vertebrate bloodstream. Trypanosomes are highly

successful, being found in all classes of vertebrate (fish, amphibians, birds, reptiles,

and mammals) and in all continents. The vast majority of trypanosome species are

transmitted by arthropods (mostly insects) and leeches, although a few species can

be passed directly between vertebrates. Most do not appear to harm their hosts,

although several species are associated with important diseases of humans and

domestic livestock.

There has been considerable interest in the evolutionary origin of genus

Trypanosoma and the relationships within the genus; in particular, the relationship

between the two human pathogens, Trypanosoma cruzi and T. brucei, has received

considerable attention (Haag et al., 1998; Stevens et al., 1999b; Hughes and

Piontkivska, 2003b; Hamilton et al., 2004). However, in the absence of a fossil

record, and with few morphological features, testing evolutionary hypotheses has

only become possible within the last 20 years with the advent of molecular phyloge-

netics. Molecular phylogenetic trees (phylogenies) are constructed by comparing

DNA sequences from a range of organisms. Phylogenies allow the history of a

group of organisms to be traced, thus providing robust frameworks for testing evolu-

tionary hypotheses. Phylogenetic studies have led to an improved understanding of

the origins of the parasitic life history strategy, the relationship between the two try-

panosomatid genera that infect vertebrates (Trypanosoma and Leishmania), and the

origins of trypanosomes of medical and veterinary importance.

It is now relatively straightforward to obtain DNA sequences from trypanoso-

mes. First the chosen gene is amplified by using the polymerase chain reaction (PCR),

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followed by sequencing of purified PCR products. Computer programs can then be

used to align sequences and for subsequent tree construction. 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. Although the first studies of this nature relied on DNA isolated from

cultured parasites, many recent studies have used DNA isolated directly from the

host tissue such as blood, or from insect guts. This has led the way to large-scale

surveys of parasite diversity, which have transformed our understanding of the

diversity of trypanosome species and their host ranges. These studies have

benefited from the availability of gene sequences from a wide range of trypano-

somes and related taxa, which has facilitated the design of parasite-specific pri-mers, enabling amplification of trypanosomal genes, while avoiding amplification

of host DNA.

A range of genes have been used for phylogenetic and taxonomic studies. Most

studies examining relationships between species have used nuclear ribosomal DNA(rDNA) markers, in particular 18S rDNA (also called small subunit [SSU] rDNA),

and to a lesser extent 28S rDNA (also called large subunit [LSU] rDNA). The 18S

rRNA gene has both conserved regions, suitable for primer design and 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 identifi-

cation. The noncoding internal transcribed spacer (ITS) regions, ITS1 and ITS2,

have a faster evolutionary rate, so have been useful for studying within-species

diversity. More recently, protein-coding genes have been used; in particular, glyco-

somal glyceraldehyde phosphate dehydrogenase (gGAPDH) for phylogenetic place-

ment of newly described species and for resolving relationships within the genus.

The faster-evolving kinetoplast (mitochondrial) cytochrome b (Cyt b) gene has

been used for examining within-species diversity (Cavazzana Jr et al., 2010). At

the time of writing, complete gGAPDH and 18S rDNA sequences are available in

sequence databases for more than 100 taxa, from a wide diversity of different try-

panosome species.

Molecular phylogenetics has also informed taxonomy (Stevens and Brisse,

2005). Taxonomic groups should have evolutionary relevance, and arguably names

should only be applied to monophyletic groups. Molecular studies have questioned

the existence of some species that were previously described using morphological

and life-cycle data. They have also raised new questions, such as whether new spe-

cies 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.

At the same time, the development of rapid methods for species identification

(Adams and Hamilton, 2008; Hamilton et al., 2008; Adams et al., 2009) as well as

sequence-based surveys of trypanosomatid diversity (Votypka et al., 2010) have led

to the description of new species. While many of these new species were found in

previously unexplored hosts, such as Australian marsupials (Noyes et al., 1999;

322 American Trypanosomiasis Chagas Disease

McInnes et al., 2009), some potentially pathogenic species have also been found in

well-studied groups, such as the African tsetse fly-transmitted group of trypano-

somes (Hamilton et al., 2008). It is now clear that much trypanosomatid diversity

is yet to be discovered.

13.2 Origin of Trypanosomes—Relationship ofT. cruzi with T. brucei

Molecular phylogenetic studies have provided valuable insights into the origin of

trypanosomes, the relationship between Trypanosoma and Leishmania (which also

includes species that are human pattosens), and the relationship between the two

human pathogens, T. brucei and T. cruzi. Two groups of theories have dominated

debate regarding the origin of the genus Trypanosoma. Vertebrate-first theories

proposed that the ancestral trypanosome evolved from a gut parasite of vertebrates

(Minchin, 1908), while invertebrate-first theories proposed that trypanosomes

evolved from a single-host (monogenetic) invertebrate parasite, such as the trypa-

nosomatids that infect insect guts (Leger, 1904; Hoare, 1972; Vickerman, 1994).

Resolving the relationships between trypanosomes and their close relatives is the

key to understanding of the origin of the genus. Trypanosomes (genus Trypanosoma)

are trypanosomatids (family Trypanosomatidae), all of which are parasitic at all stages

of their life cycle. Most trypanosomatid genera (e.g., Blastocrithidia, Crithidia,

Herpetomonas, Leptomonas, Sergeia,Wallaceina) are single-host (monogenetic) para-

sites of insects. Trypanosoma and Leishmania are two-host (digenetic) vertebrate

parasites. The existence of another digenetic vertebrate parasite, Endotrypanum, has

not been verified using molecular techniques, as the isolates of this genus examined

have turned out to be Leishmania (Cupolillo et al., 2000; Noyes et al., 2002).

Phytomonas is a digenetic parasite of plants, which is transmitted by insects.

Central to the debate of the origin of trypanosomes is whether they are mono-

phyletic. A monophyletic group is a collection of organisms, which form a single

clade comprising an ancestor and all its descendants. Monophyly of trypanosomes

would indicate that all described taxa within the genus had a single common origin

and gave rise to no other group of trypanosomatids. The monophyly debate has

also been central to resolving whether the two groups of trypanosomes, that include

the human pathogens T. brucei and T. cruzi respectively, evolved vertebrate parasit-

ism independently; monophyly would indicate that these species might share some

common, ancestral adaptations necessary for survival in vertebrates. Understanding

such ancestral adaptations could aid the rational design of therapeutics that target a

broad range of pathogenic trypanosome species. Many of the early phylogenetic

trees, based on comparisons of genes encoding ribosomal RNAs, showed trypano-

somes to be paraphyletic (Gomez et al., 1991; Fernandes et al., 1993; Landweber

and Gilbert, 1994). Often the evolutionary trees obtained from these studies sug-

gested that the clade including T. brucei and related parasites (the T. brucei clade)

had an origin independent to that of the rest of the trypanosomes, including T. cruzi

323Classification and Phylogeny of Trypanosoma cruzi

and related species. Such a tree topology indicated an independent evolution of ver-

tebrate parasitism in T. brucei and related species.

On the other hand, most later studies, also using ribosomal RNA genes, but with

increased taxon-sampling, showed monophyly (Lukes et al., 1997; Haag et al.,

1998; Stevens and Gibson, 1999; Wright et al., 1999; Stevens et al., 2001; Simpson

et al., 2002; Hamilton et al., 2004), although several supported paraphyly (Hughes

and Piontkivska, 2003a,b). The issue was resolved using taxon-rich trees based on

protein-coding genes, which strongly supported monophyly of the genus (Hamilton

et al., 2004, 2007; Simpson and Roger, 2004; Simpson et al., 2006), confirming

earlier studies using protein-coding genes that included fewer taxa (Wiemer et al.,

1995; Adje et al., 1998; Hannaert et al., 1998; Simpson et al., 2002).

Molecular trees have also provided clues to the ancestor of trypanosomes. For

example, gGAPDH trees, constructed using robust maximum-likelihood and

Bayesian techniques, have suggested that Trypanosoma evolved from an insect-

only trypanosomatid (Hamilton et al., 2004). The recent discovery of an insect-only

trypanosomatid closely related to Leishmania supports the idea that the genus

evolved independently from another insect-only trypanosomatid (Yurchenko et al.,

2006). Thus, we do not expect T. cruzi and other trypanosomes to share common,

ancestral adaptations to vertebrate parasitism with Leishmania. Leishmania appears

to have evolved considerably more recently than Trypanosoma, which may par-

tially explain its comparatively narrow vertebrate (mammals, reptiles) and inverte-

brate (sandflies) host range.

The evolution of trypanosomes from a monogenetic insect parasite would indi-

cate that the first trypanosomes were insect-transmitted trypanosomes of terrestrial

mammals. Therefore, trypanosomes of amphibia and fish that are transmitted by

leeches must have evolved later. The position of leech-transmitted fish trypano-

somes in phylogenetic trees supports this idea (Haag et al., 1998); they all fall in a

subclade of the aquatic clade (Stevens et al., 1999b), indicating that they were not

the first to evolve, as previously hypothesized (Vickerman, 1976). Furthermore,

while the deepest split within the genus is between the aquatic clade and the terres-

trial clade (Figure 13.1A), both clades contain trypanosomes that are insect trans-

mitted. Adaptation to transmission by aquatic leeches enabled trypanosomes to

colonize many aquatic vertebrates, such as freshwater and marine fish.

13.3 Relationships within the Genus Trypanosoma

Studies have also examined the relationships within the genus Trypanosoma. The

composition of the clade that includes T. cruzi (the T. cruzi clade) has provided

valuable clues to the origin of the species. The most taxon-rich phylogenetic trees

are based on alignments of 18S rDNA and gGAPDH genes (Haag et al., 1998;

Stevens et al., 1999b, 2001; Wright et al., 1999; Overath et al., 2001; Martin et al.,

2002; Hughes and Piontkivska, 2003b; Hamilton et al., 2004, 2005a, 2007). It is

clear that T. brucei and T. cruzi are in different clades and evolved human parasit-

ism independently (Stevens et al., 1999b). While T. brucei falls in the T. brucei clade


with other tsetse fly-transmitted trypanosomes from Africa, indicating a long history

largely confined to Africa (Stevens et al., 1999b), the evolutionary history of T. cruzi

and other related trypanosomes in the T. cruzi clade is less certain and is discussed

later (see Section 13.7).

There are now approximately 10 well-established clades within the genus

Trypanosoma (see Figure 13.1A), all of which are found on more than one continent.

Mapping the hosts of the trypanosomes onto the phylogenetic tree has revealed

some associations between clades and certain types of vertebrate and invertebrate

hosts (Hamilton et al., 2007). For example, two groups are restricted to birds, one to

crocodilians, and there are several mammalian clades. Other clades appear to be

largely restricted to a particular type of invertebrate, such as the T. brucei clade, in

which most species are transmitted by tsetse flies. Overall, cospeciation with either

vertebrates or invertebrates appears to have played little role in trypanosome evolu-

tion. Instead, adaptations to host types and opportunities appear to have played a

more important role in determining the host range of trypanosome clades (Hamilton

et al., 2007). Analyses based on combined datasets of 18S rDNA and gGAPDH gene

sequences have provided increased resolution, combining some of these clades into

Leishmania

Insect-only parasites

Leptomonas costaricensis

Lizard/snake clade

T. brucei clade

Crocodilian clade

T. cruzi clade

T. lewisi clade

Unnamed mammalian clade

Lizard trypanosome

T. avium clade

T. corvi clade

Koala and bird trypanosomes

Fish clade

Amphibian clade

T. theileri clade

Phytomonas

Insect-only parasites

Trypanosomes

Bodonids – mostly free-living

Australian kangaroo

T. rangeli South American mammals

T. conorhini rat

T. cruzi marinkellei New World bats

T. dionisii Old and New World bats

T. vespertilionis Old World bats

African bat

African monkey

African civet

T. cruzi cruzi New World mammals

T. cruzi clade (detail)

(A)

I

Mammalian

clade

Aquatic clade

Avian clade

Terrestrial clade

v

vTrypanoso-

matids

P

(B)

Figure 13.1 (A) Phylogenetic relationships of kinetoplastids, showing the relationships

between the main trypanosomatid lineages. Groups connected by vertical lines are descended

from a common ancestor. I5 origin of single-host (monogenetic) insect parasitism;

V5 origins of two-host (digenetic) vertebrate parasitism; P5 origin of digenetic plant

parasitism. (B) Detail of relationships within the T. cruzi clade. Taxa in gray are hosts.

Sources: Stevens et al. (1999b); Hamilton et al. (2004, 2007, 2009); Simpson et al. (2006);

Yurchenko et al. (2006); Viola et al. (2009a,b).


“superclades.” These analyses have resolved the deepest split within the genus,

which is between the aquatic clade and the terrestrial clade, comprising all the other

clades. Importantly, both T. cruzi and T. brucei are in the terrestrial clade, and so are

more closely related to each other than had been previously suggested; some earlier

studies showed the T. brucei clade branching at the periphery of the main trypano-

some group, indicating that they were some of the first to evolve (Haag et al., 1998).

Another of these superclades, the mammalian clade, includes the T. cruzi clade and

two other mammalian clades (see Figure 13.1A).

13.4 Molecular Phylogenetics and Traditional Taxonomy ofMammalian Trypanosomes

Traditionally, taxonomy of trypanosomes has been based on comparisons of mor-

phology, life cycle, and disease data. The most commonly used taxonomy of Hoare

(1972) divides mammalian trypanosomes into two sections. The section Salivaria

comprises trypanosomes in which the infective forms are passed in the saliva of the

insect. The section Stercoraria comprises species in which the developmental cycle

in the insect vector is completed in the hindgut, and transmission is through contact

with infective forms in the feces. In this taxonomy, T. cruzi was placed within the

subgenus Schizotrypanum within the section Stercoraria. This taxonomy is given

below; type species of each subgenus are in parentheses:

Section: Stercoraria

Subgenera Herpetosoma (T. lewisi)

Megatrypanum (T. theileri)

Schizotrypanum (T. cruzi)

Section: Salivaria

Subgenera Duttonella (T. vivax)

Nannomonas (T. congolense)

Pycnomonas (T. suis)

Trypanozoon (T. brucei)

Molecular phylogenetic studies have largely supported the Salivaria and its sub-

genera. In contrast, the Stercoraria, and its subgenera have generally not received

support. Indeed Stevens et al. (1999a) proposed that the use of the names

Herpetosoma and Megatrypanum be discontinued because they are polyphyleticand so lack taxonomic and evolutionary relevance, whereas Schizotrypanum should

be expanded to include all trypanosomes that fall in the T. cruzi clade (see

Section 13.6). All trypanosomes originally placed within the subgenus

Schizotrypanum, that have been analysed using molecular phylogenetics, fall within

this suggested clade, and the clade now also includes species originally placed

within Herpetosoma (T. lewisi, T. rangeli) and Megatrypanum (T. conorhini).


13.5 The Main Groups of Trypanosomes Recognized inMolecular Phylogenetic Analyses

A brief description of the main trypanosome clades follows (see Figure 13.1A).

� The aquatic clade: This clade comprises trypanosomes of mainly aquatic and amphibious

vertebrates, including all fish trypanosomes and several species from amphibia. The ver-

tebrate hosts also include reptiles (turtle, chameleon) and a mammal (platypus). Many of

these species are transmitted by proboscid leeches (Bardsley and Harmsen, 1973; Desser

et al., 1973; Khan, 1976; Martin and Desser, 1990; Jones and Woo, 1991; Siddall and

Desser, 1992; Karlsbakk, 2004). There is evidence that some of the trypanosomes from

amphibious vertebrates are transmitted by insects (Desser et al., 1973), and the chameleon

trypanosome presumably has an insect vector.� The T. cruzi clade: (see Section 13.6 of this chapter).� An unnamed mammalian clade: This clade contains trypanosomes from Australian marsu-

pials, the Eurasian badger and ticks from Japan (Noyes et al., 1999; Stevens et al., 1999b;

Hamilton et al., 2005a; Thekisoe et al., 2007; McInnes et al., 2009).� The T. lewisi clade: This clade contains trypanosomes from a wide range of rodents; it

also contains trypanosomes from a lagomorph and insectivores (Hamilton et al., 2005b;

Sato et al., 2005; Bray et al., 2007). The trypanosomes in this clade are thought to be spe-

cific to their vertebrate hosts. The vast majority of the vectors of these trypanosomes are

fleas, although the life cycles of some species are not completely known (Hoare, 1972).

The clade contains T. lewisi, type species of the subgenus Herpetosoma, thus the name

Herpetosoma is occasionally used for this clade (Stevens and Brisse, 2005).� An unnamed clade: This clade contains trypanosomes from an Australian marsupial

(koala), an American kestrel (McInnes et al., 2009), and T. minasense from South

American primates (red-handed tamarind, Saguinus midas) imported into Japan (Sato

et al., 2008). See Section 13.6 (T. rangeli) for further discussion on this species. The pres-

ence of both mammalian and avian trypanosomes within this clade may demonstrate

host-switching between different vertebrate classes.� The crocodilian clade: This clade comprises crocodilian trypanosomes from Africa and

South America and includes T. grayi, a tsetse fly-transmitted trypanosome from Africa

(Minter-Goedbloed et al., 1993; Viola et al., 2009a).� The T. brucei clade: This clade contains mostly African mammalian trypanosomes that

are transmitted by tsetse flies. Two subspecies of T. brucei—T. b. gambiense and

T. b. rhodesiense—are also human pathogens. Many of these species are pathogens of

domestic livestock (T. brucei, T. congolense, T. evansi, T. simiae, and T. vivax). The

majority of trypanosomes in the T. brucei clade are transmitted by tsetse flies (genus

Glossina) via the saliva. The clade includes two trypanosomes from South America,

which are believed to have been accidentally introduced into the continent in domestic

animals relatively recently: T. evansi and T. vivax (Hoare, 1972; Cortez et al., 2006).� The T. theileri clade: This clade contains trypanosomes from marsupial and placental

mammals (deer, cattle, primates). It includes T. theileri, a trypanosome of artiodactyls

that is commonly found in domestic cattle across the world (Hamilton et al., 2005a;

Rodrigues et al., 2005). T. theileri is the type species for the subgenus Megatrypanum, so

the name Megatrypanum is sometimes used for this clade (Rodrigues et al., 2005;

Stevens and Brisse, 2005). T. theileri is known from South American cattle and buffalo,

with distinct strains in each (Rodrigues et al., 2005). Tabanid flies act as the principal


vectors of T. theileri, although ticks also have capacity to transmit this trypanosome

(Hoare, 1972; Burgdorfer et al., 1973; Bose et al., 1987a,b; Rodrigues et al., 2005). This

clade also contains trypanosomes from terrestrial bloodsucking leeches (Haemadipsidae)

that are closely related to a South East Asian primate trypanosome, T. cyclops. These

leeches are common in tropical forests across Asia and Australia, and it has been sug-

gested that they are important vectors of mammalian trypanosomes in Asia and Australia

(Hamilton et al., 2005a).� The lizard/snake clade: This clade contains trypanosomes of lizards and snakes (Viola

et al., 2009b). Sandflies are the only known vectors of trypanosomes in this clade (Ayala,

1970; Minter-Goedbloed et al., 1993).� The avian clade: This clade combines two subclades of avian trypanosomes, the T. avium

and the T. corvi clades (Votypka et al., 2002, 2004). A diversity of insect vectors transmit

the trypanosomes in this clade, including black flies (Desser et al., 1975; Votypka and

Svobodova, 2004), hippoboscid flies, and mosquitoes (Baker, 1956).

13.6 The T. cruzi Clade (Subgenus SchizotrypanumChagas, 1909)

This clade contains trypanosomes that are parasitic in a diverse range of mammals,

including two human-infective parasites: T. cruzi and T. rangeli, which are both

restricted to the New World. It also contains trypanosomes from Chiropteran (bat)

hosts from both the Old and New Worlds (T. cruzi marinkellei, T. dionisii, T. ves-

pertilionis). Other trypanosomes within the clade are T. conorhini, a rat trypano-

some, two trypanosomes from African terrestrial mammals (Njiokou et al., 2004;

Hamilton et al., 2009) and a trypanosome from an Australian kangaroo (Noyes

et al., 1999; Stevens et al., 1999b). The only known invertebrate vectors of these

trypanosomes are bugs (suborder Heteroptera; order Hemiptera), although the

invertebrate hosts of several of these trypanosomes are not yet known. Three spe-

cies are transmitted by triatomine bugs: T. rangeli by the genus Rhodnius, T. cruzi

cruzi by a wide range of species and T. conorhini by Triatoma rubrofasciata. The

bat trypanosomes are also thought to be transmitted by bat-feeding bugs. For

instance, infections of T. cruzi marinkellei have been described in the bat-feeding

triatomine bugs of genus Cavernicola (Marinkelle, 1982), while the cimicid bugs

in the genus Cimex are frequently found infected with trypanosomes and have been

implicated in the transmission of three species of bat trypanosome: T. dionisii,

T. incertum, and T. vespertilionis (Paterson and Woo, 1984; Gardner and

Molyneux, 1988a,b). The vectors of the kangaroo trypanosome, the trypanosomes

of African terrestrial mammals, and other bat trypanosomes are as yet unknown.

The T. cruzi clade contains several trypanosomes originally placed within the

subgenus Schizotrypanum. The close relationship between trypanosome species

originally placed in the subgenus Schizotrypanum is not surprising, as the subgenus

has well-defined morphology and development within the vertebrate host. The

subgenus comprises small trypanosomes that are very difficult to distinguish


morphologically from the type species T. cruzi. They have a voluminous kineto-

plast and typically curved bloodstream forms in the form of a C or S, with a short,

pointed posterior end, which constitute distinctive morphological characters

(Hoare, 1972). Within the vertebrate host, multiplication occurs within various tis-

sues and organs, rather than in the blood (like many other trypanosome species).

Nevertheless, the life cycles of several of the bat-infecting species are not

completely known. Moreover, as these trypanosomes are difficult to distinguish

morphologically, several of the morphologically described species may also be

synonyms of T. cruzi. For instance, several T. cruzi-like trypanosomes from non-

bat South American wild mammals have been described using traditional parasito-

logical techniques: T. (S.) lesourdi Leger and Porry, 1918 from a spider monkey;

T. (S.) prowazeki Berenberg-Gossler, 1908 from a Uakari (a species of New World

monkey); and T. (S.) sanmartini Garnham and Gonzales-Mugaburur, 1962 from a

squirrel monkey (Hoare, 1972). However, molecular studies have failed to demon-

strate the existence of trypanosomes related to T. cruzi in South American terres-

trial mammals, other than T. rangeli and T. conorhini. Therefore, these other

morphologically described species may represent synonyms of T. cruzi. Likewise, a

range of bat trypanosomes have been classified within the subgenus

Schizotrypanum using parasitological techniques, including two from Australia,

T. (S.) pteropi Breinl, 1921 and T. (S.) hipposideri Mackerras, 1959; and T. (S.)

hedricki, T. (S.) myoti, and T. (S.) dionisii from elsewhere within the Old World.

Morphological similarities between these bat trypanosomes have made it difficult

to delineate species and to match molecular data with old parasitological descrip-

tions. Therefore, the diversity of bat trypanosomes within this group may be con-

siderably under- or overestimated.

Trypanosomes within the T. cruzi clade are listed (see Figure 13.1B).

� T. cruzi cruzi: (also called T. cruzi sensu stricto).� T. cruzi marinkellei: This bat trypanosome is apparently restricted to South America and

was sufficiently divergent to warrant subspecies status (Baker et al., 1978). Its close rela-

tionship to T. cruzi cruzi has been verified using sequences of 18S rDNA (Stevens et al.,

1999b), gGAPDH genes (Hamilton et al., 2004, 2007), and kinetoplast Cyt b genes

(Cavazzana Jr et al., 2010).� T. dionisii: This bat trypanosome was first described from Europe, but recently distinct

strains of this species have been found in South America (Maia da Silva et al., 2009;

Cavazzana Jr et al., 2010).� Trypanosoma species (civet) and Trypanosoma species (monkey): These trypanosomes

were recently isolated from a study that examined trypanosome diversity in a wide range

of wild vertebrates in Cameroon, West Africa (Njiokou et al., 2004). In that study, they

remained unidentified, but later characterization by sequence analysis of their 18S rDNA

and gGAPDH genes provided the first proof that the T. cruzi clade is present in non-bat

mammalian hosts in the Old World (Hamilton et al., 2009).� T. conorhini: A trypanosome found worldwide in rats and transmitted by the triatomine

bug Triatoma rubrofasciata (Hoare, 1972).� T. vespertilionis: This is a widely distributed trypanosome of bats. The single isolate

included in phylogenetic trees is from Europe (Stevens et al., 1999b; Hamilton et al.,

2004, 2007).


� T. rangeli: This trypanosome is restricted to South America and has a wide mammalian

host range including humans, although it is not pathogenic to human hosts. A high preva-

lence of T. rangeli in humans has been reported in Central America and northwestern

South America (D’Alessandro and Saravia, 1992). The only known vectors are triatomine

bugs of the genus Rhodnius. The inclusion of this species within the T. cruzi clade

(Stevens et al., 1999a) resolved the considerable debate regarding the classification of this

species; its ability to develop in saliva of triatomine bugs (although it is also transmitted

via feces) had led to it being classified within the Salivaria, while resemblance of the

bloodstream forms to T. lewisi led to classification within the subgenus Herpetosoma.

Recent comparisons of a wide range of isolates of this species using sequences of the

spliced-leader gene, 18S rDNA, and ITS regions have revealed four lineages in terrestrial

mammals (Lineages A, B, C, and D) and Lineage E, which is apparently restricted to bats

(Maia da Silva et al., 2004, 2007). Lineage divergence appears to be associated with spe-

cies of Rhodnius, without any clear association of trypanosome lineages with particular

vertebrate hosts. Phylogenetic studies revealed some isolates previously classified as

T. minasense and T. leeuwenhoeki to be T. rangeli (Stevens et al., 1999a). Similarly, com-

parison of strains of T. saimirii-like trypanosomes (from squirrel monkeys) using sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that T. sai-

mirii is a synonym of T. rangeli (Ziccardi et al., 2005). However, characterization of a

range of isolates of T. minasense by SDS-PAGE has indicated that it is a separate species

to T. rangeli, as only 9 of 20 polypeptides were shared between the two species (Ziccardi

et al., 2005) and 18S rDNA gene trees demonstrated that T. minasense isolates from South

American monkeys imported to Japan were unrelated to T. rangeli (Sato et al., 2008).� An unnamed bat trypanosome: This trypanosome was isolated from a fruit bat Rousettus

aegyptiacus from Gabon in West Africa. Interestingly, sequence analysis based on 18S

rDNA indicated that it was only distantly related to the other bat trypanosomes in the

clade (Stevens et al., 1999a, 2001).� Trypanosome species (kangaroo): This trypanosome was isolated in Australia (Noyes

et al., 1999) and related trypanosomes have subsequently been found in a native

Australian rodent, Rattus fuscipes (Averis et al., 2009). Analysis by Stevens et al. (1999b)

provided strong support for placing this taxon within, but at the extremity of, the T. cruzi

clade; as such, this taxon played a major part in hypothesizing the origin of the T. cruzi

clade (see Section 13.7).

13.7 The Origin of the T. cruzi Clade

There are two main hypotheses regarding the origin of the T. cruzi clade and

T. cruzi cruzi itself (Stevens et al., 1999b; Stevens and Rambaut, 2001; Hamilton

et al., 2009). As yet, further information is required to resolve the debate.

One hypothesis is that the T. cruzi clade originated in the southern super-

continent comprising present day Antarctica, Australia, and South America when

marsupials were the dominant mammalian fauna, more than 45 million years ago

(Stevens et al., 1999b). This hypothesis is supported by the placement of a trypano-

some from an Australian kangaroo on the periphery of the clade (Stevens et al.,

1999b). It also receives support from the existence of two species that are found in

terrestrial mammals in South and Central America: T. cruzi cruzi and T. rangeli.

As both species are genetically diverse, they are likely to have had a long history


within the New World. According to this hypothesis, the ability of bats to disperse

by flying is responsible for spreading bat-trypanosome lineages within the clade to

the Old World (Stevens and Gibson, 1999; Stevens et al., 1999b). This hypothesis has

provided some useful dates for calibrating the evolutionary tree of trypanosomes. The

split between the T. brucei and T. cruzi clades has been dated using the separation of

Africa and South America, which occurred approximately 100 million years ago

(Stevens et al., 1999b; Stevens and Rambaut, 2001). The separation of Australia from

Antarctica/South America, which occurred approximately 45�80 million years ago,

has been used to date the split between the kangaroo trypanosome from the rest in the

T. cruzi clade (Stevens et al., 2001).

Another hypothesis is that several of the lineages that infect terrestrial mammals

within the T. cruzi clade evolved from bat trypanosomes, possibly including T. cruzi

cruzi (Hamilton et al., 2009). This idea receives support from studies that have

demonstrated that bat trypanosomes within the clade are diverse and polyphyletic

and several are related to terrestrial lineages. In addition, the recent discovery of

trypanosomes in the T. cruzi clade originating from African vertebrates, a monkey,

and civet (Figure 13.1B), demonstrates that members of the T. cruzi clade are found

in Old World terrestrial mammals (Hamilton et al., 2009). The monkey trypano-

some characterized in this study was closely related to the bat trypanosome, T. ves-

pertilionis. Thus, dispersal by bats and the jumping of trypanosomes between

terrestrial and bat hosts could have led to the current wide distribution of this para-

site clade. Although it is clear that further data are required to resolve the issue,

these African trypanosomes do not represent a single lineage that was recently

introduced from South America; the monkey trypanosome is most closely related

to the bat trypanosome T. vespertilionis, while the civet trypanosome appears to be

more closely related to T. conorhini. Thus, there could have been two relatively

recent introductions, or it may indicate that this African group evolved indepen-

dently in Africa for a considerable period of time (Hamilton et al., 2009). Evidence

that trypanosomes within the T. cruzi clade frequently move between bats and ter-

restrial mammals has come from recent surveys of trypanosomes of bats in South

America. These have revealed bat-specific strains of T. cruzi cruzi (Maia da Silva

et al., 2009; Marcili et al., 2009; Cavazzana Jr et al., 2010) and T. rangeli (Maia da

Silva et al., 2009). Furthermore, some T. cruzi cruzi lineages once thought to be

restricted to terrestrial mammals have been found in bats (Lisboa et al., 2008;

Cottontail et al., 2009; Maia da Silva et al., 2009; Cavazzana Jr et al., 2010), sug-

gesting that transmission of trypanosomes between bats and terrestrial mammals is

in fact relatively frequent. One implication of this hypothesis is that the clade could

have evolved relatively recently, as bats could facilitate the spread of the trypano-

somes in the clade between continents.

The idea that T. cruzi clade trypanosomes are widely distributed in terrestrial

mammals in the Old World receives support from some early parasitological stud-

ies. Trypanosome species have been described in Indonesian primates that resemble

T. conorhini (Weinman, 1977) as they developed in the T. conorhini vector, the

triatomine bug, Triatoma rubrofasciata (Weinman et al., 1978). This led Hoare

(1972) to argue that these Indonesian trypanosomes were primate-adapted strains of


T. conorhini. Indeed, although T. conorhini was thought to be restricted to the rat,

Rattus rattus, it has been shown to be capable of transiently infecting mice (Mus

musculus) and macaque monkeys (Macaca species) in laboratory experiments

(Hoare, 1972). T. rubrofasciata infected with T. conorhini has been found in South

America, Mauritius, India, Taiwan, and Malaysia (Hoare, 1972). On the other

hand, Weinman et al. (1978) argued the Indonesian monkey trypanosomes are

unlikely to be T. conorhini, as T. rubrofasciata is rat-specific and has been found

in the cities, but never in the tropical rainforests of Asia. Likewise, T. cruzi-like

trypanosomes have been described in the slow loris (a primate) in Malaysia (Kuntz

et al., 1970). Thus, it is clear that molecular phylogenetic studies are required to

reveal the true distribution of this clade.

13.8 Outlook

Molecular phylogenetic studies have provided a useful framework for understand-

ing many aspects of trypanosome biology and evolution. In addition, this research

can also be of direct relevance in applied studies, for example, research into dis-

eases of humans and livestock. In particular, one goal of the trypanosomatid

genome sequencing projects is to identify genes involved in pathogenicity, so com-

parisons with closely related, but nonpathogenic species can be informative;

molecular�taxonomic studies have been instrumental in identifying such species.

Knowledge of trypanosome diversity can also aid in the development of diagnostic

tools (e.g., to distinguish pathogenic and nonpathogenic species); such diagnostics

and the capacity they provide to distinguish species are essential for understanding

the epidemiology of pathogenic species and to identify vectors and reservoirs of

infection. Finally, an improved understanding of the origins of the trypanosomes

that are capable of causing diseases of humans and domestic animals can provide

insights into the likely sources of new and emerging pathogens. For instance, we

now know that three primate-infective species are closely related to trypanosome

species found in bats and thus bats may be regarded as (at least) potential sources

of novel human parasites in the future. Thus, understanding the evolution and

diversity of trypanosomes has the potential to deliver real benefits in applied

science, epidemiology, and medicine.

Glossary

Clade a group of biological taxa or species that comprises a common ancestor and all its

descendents (if the placement of all taxa within a clade is robust and no unrelated taxa

are included within the clade, then the group can be referred to as monophyletic).

Digenetic a parasitic life cycle involving hosts of two different species. The two host spe-

cies are essential for the completion of a particular parasite’s life cycle.

Monogenetic a parasitic life cycle involving only a single species of host.


Monophyletic group a group of taxa that derive from a single common ancestor; specifi-

cally, the group includes all the descendants of a common ancestor and no unrelated taxa

(see also Clade).

Paraphyletic refers to a group of taxa that derive from a single common ancestor; the group

includes all the descendants of a common ancestor, plus additional apparently unrela-

ted taxa.

Polyphyletic refers to a collection of taxa derived from more than one ancestor (i.e., taxa do

not share a single common ancestor).

PCR (polymerase chain reaction) a method used in molecular biology to amplify a region

of DNA, generating large quantities of a particular DNA region using oligonucleotide pri-

mers and a thermostable DNA polymerase.

Primer a short oligonucleotide from which DNA replication can initiate. Primers used for

PCR are synthetically made and are designed to anneal to the template DNA.

Phylogenetic tree (phylogeny) a diagram (often referred to as a tree) illustrating relation-

ships of evolutionary lineages among organisms (taxa).

rDNA (ribosomal DNA) DNA sequences encoding ribosomal RNA molecules that form

subunits that together form the structure of a ribosome.

Taxon (pl. taxa) any grouping within the classification of organisms such as species, genus,

family, order, etc.

Taxonomy the science and methodology of classifying and naming organisms based on

similarities.

Type species the first recorded described specimen of a species; the specimen to which the

binomial name of the species (genus name and species name) is permanently attached.

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