The evolution of trypanosomatid taxonomy

Trypanosoma spp. are ubiquitous, infecting almost all vertebrate classes [13], with vectors ranging from leeches, to biting flies and bugs [8, 14]. The tsetse fly (genus: Glossina) and bugs of the Triatominae subfamily (i.e. “kissing” bugs) are the natural vectors of Trypanosoma brucei and Trypanosoma cruzi, respectively [8] (Fig. 1). Human African Trypanosomiasis is endemic in 36 sub-Saharan countries with studies estimating that 61 million people are at risk of contracting the disease through the bite of an infected tsetse fly [15]. Domestic and wild animals serve as reservoirs for T. brucei infection, with the reported human prevalence varying between communities [2]. Between 2004 and 2014, the number of new cases reported has significantly dropped from 17,616 to only 3,796 with the total number of estimated annual cases plummeting from 50,000–70,000 to less than 15,000 [15]. Chagas disease is endemic in Central and South America, with an estimated 8 million individuals infected [16]. Humans are an incidental host of Trypanosoma cruzi, which is a zoonotic parasite predominantly infecting native wildlife, domestic dogs and cats, which are reservoirs of Chagas disease [17].

Female phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are natural vectors for Leishmania transmission [18, 19], and roughly 70 animal species serve as reservoirs for human pathogenic Leishmania species, including rodents [20], dogs [21] and other mammals [22]. Australia and Antarctica had long been considered the only continents free of endemic Leishmania though the discovery of an Australian macropod-infecting species and its midge vector, Forcipomyia (Lasiohelea) spp. [20, 22, 23], overturned this and called into question the exclusivity of the sand fly-Leishmania interaction.

Compared to their dixenous cousins, the life-cycle and habits of monoxenous trypanosomatids are obscure. By 2001, monoxenous trypanosomatids had been identified from roughly 350 insect species only, while more than 900 vertebrate hosts had been identified for the dixenous genera which are far fewer in number [24]. Due to their limited impact on human and animal health, the monoxenous trypanosomatids have received little attention from parasitologists historically. Despite this, interest in the monoxenous species has revived in recent years [25–28]. From a taxonomic perspective, trypanosomatids are now amongst the most extensively studied protozoans, as reflected by the recent surge in publications on this topic that form the basis of the trypanosomatid taxonomic system [26, 27, 29].

Trypanosomatid systematics was traditionally based on host preferences and specialised life-cycle stages, characterised by the presence or absence of several defined morphotypes (Fig. 2) [30–32]. Under the classical system of trypanosomatid taxonomy, the various Leishmania species were differentiated by their respective specialised proliferative stages [18]. Advances in molecular biology revolutionised the field by providing genetic evidence for true ancestral relationships [27, 33]. As a consequence, major flaws were identified in the classical system which underwent a series of major revisions [26–29]. However, the field would continue to benefit from studies aiming to isolate new species. Of the estimated one million known insect species described, only 2,500 of these have been studied closely for the presence of trypanosomatids [24, 26, 29, 34]. The isolation and molecular characterisation of new species will help to decipher relationships between trypanosomatids with greater resolution [24].

During a blood meal the infected sand-fly injects metacyclic promastigotes into the vertebrate host [34]. In the vertebrate host, these promastigotes invade macrophages and then differentiate into non-motile amastigotes, which multiply by binary fission [34]. Leishmania replicates within macrophages at various sites depending on the species [10], giving rise to three distinct clinical forms of leishmaniasis; cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL or Kala Azar) [4]. These clinical forms result from parasite development within the reticuloendothelial system of either the skin (CL), nasopharynx (MCL) or viscera (VL) [35] (Figs. 3, 4).

CL is the most common clinical form of the disease, presenting as a myriad of manifestations affecting the skin [8]. It is characterised by the presence of one or more skin lesions with varying morphologies that can result in highly disfiguring scars [8]. Mucocutaneous leishmaniasis is a severely disfiguring form of leishmaniasis with 90% of MCL patients having suffered from a case of CL that had healed 1–5 years prior to the onset of MCL [36]. MCL involves the ulceration of the nasal region, accompanied by symptoms of fever and hepatomegaly [36]. Later progression involves ulceration to the oronasopharyngeal mucosa, resulting in erythema and edema of the affected regions. VL is the most severe manifestation of the disease [8]. VL is a systemic disease, characterised by the incidence of irregular bouts of fever, significant weight loss, splenomegaly and anaemia [8]. If untreated, fatality is almost certain as a result of haemorrhaging or co-infections with bacteria or viruses [4].

Human infective trypanosomes differ significantly in their route of infection, proliferative stages, and preferred sites of infection. Trypanosoma cruzi is transmitted in the faeces of their triatomine vector which defecates on the victim’s skin during a blood meal. The action of biting causes the victim to unknowingly scratch the bite area rubbing metacyclic trypomastigotes of T. cruzi from the faeces into the bite wound or into micro-abrasions caused by scratching [37]. These motile metacyclics invade host cells where they differentiate into amastigotes [4]. These intracellular amastigotes multiply by binary fission, causing tissue damage and host cell apoptosis [38]. Trypanosoma cruzi favours cells of the cardiovascular system, though also affects cells of the nervous and muscular reticuloendothelial systems [3, 39]. Additionally, the oral mode of T. cruzi transmission is emerging as a major route of infection for humans and animals in some endemic regions [40]. Oral transmission occurs via the ingestion of food contaminated with infected triatomine bugs or their faeces. Between 1968 and 2000, 50% of acute cases of Chagas disease recorded from the Brazilian Amazon were attributed to micro-epidemics of the disease caused by oral transmission [40]. Experimental evidence indicates that the infectivity of metacyclic trypomastigotes increases upon contact with gastric juices, a process that is thought to be modulated by certain trypomastigote surface glycoproteins [40, 41]. Alternatively, recent rodent studies suggest that the membranes of the oral mucosa are the most important site of T. cruzi entry into the host via the oral route [42].

Chagas disease has two phases, an acute and chronic phase [5]. In most cases of acute infection, patients are asymptomatic or may experience a wide-range of general symptoms including fever, headaches, heart inflammation, difficulty in breathing, diarrhoea and enlarged lymph glands [16]. Twenty to forty percent of those infected will suffer from severe, irreversible complications during the chronic phase including fatal cardiomyopathy, gastrointestinal and neurological problems [16, 17]. It is important to note that recently, T. cruzi has been divided into six discrete taxonomic units (DTUs) which represent distinct lineages [43, 44]. Recent studies suggest that these DTUs differ in their geographical distribution, transmission and clinical manifestation [44], implying that Trypanosoma cruzi represents a species complex.

Transmission of T. brucei occurs through the bite of the tseste fly, where metacyclic trypomastigotes are injected into the bite wound [4]. Following infection, T. brucei metacyclics transform into blood stream trypomastigotes where they undergo multiplication by binary fission, travelling throughout the blood stream and lymphatic system [45]. Unlike T. cruzi, T. remains extracellular throughout its entire life-cycle.

Human African Trypanosomiasis has two distinct forms of infection, chronic and acute, which are caused by two distinct subspecies of T. brucei [46]. The chronic form (aetiology: Trypanosoma brucei gambiense), is endemic in western and central Africa and is the most common form of Human African Trypanosomiasis (HAT), with humans considered the main reservoir for the disease [12]. The acute infection (aetiology: Trypanosoma brucei rhodesiense), is endemic in eastern and southern Africa and is predominately a zoonotic disease that occasionally affects humans [12]. The clinical manifestations of both acute and chronic HAT are often similar but vary in incubation period and severity. During the early stages of infection the parasites reside in the lymphatic system and blood stream causing fever, general malaise, weakness, lymphadenopathies, endocrine disturbances, musculoskeletal pains, and hepatosplenomegaly [12]. In the acute rhodesiense form, the early stage is often fatal as one tenth of patients do not have access to treatment and die from myocardial involvement as a consequence [46]. In the gambiense form, early stage symptoms are often non-specific including lymphadenopathy and hepatosplenomegaly [46]. The second, later stage of infection occurs following an incubation period of weeks and months in rhodesiense and gambiense infection, respectively. In this stage of the infection, the blood–brain-barrier is compromised, allowing the movement of parasites into the brain where it causes severe neurological manifestation including chronic encephalopathy and finally, coma and death [46].

The genus Phytomonas includes several plant pathogens that are transmitted by phytophagous insects. The biology and life-cycle of Phytomonas spp. is a poorly understood area of science. Phytomonas has been isolated from 24 different plant families, where they primarily occur as promastigotes and less frequently as choanomastigotes (Fig. 2) [47], while it assumes the form of a slim promastigote in the insect vector [48]. Phytomonas species can infect more than 100 plant species including lactiferous plants, tomato fruits, the coffee tree, coconut and oil palms [49, 50]. Phytomonas spp. have been isolated from a wide-range of tissues including the fruit, flower, seeds and phloem of plants [51]. The genus Phytomonas is restricted to trypanosomatids infecting plants [52], though the ability of Leptomonas seymouri to multiply in plants following experimental infections suggests that this taxonomic criterion should be used with caution and highlights the requirement for molecular evidence when making taxonomic assignments [53].

Infections with ‘monoxenous’ trypanosomatids have also been reported in vertebrates other than humans [86]. For example, an unnamed trypanosomatid was isolated from rats and stray dogs in Egypt in 1989 by Morsey et al. (1988) [93]. A later study by Podlipaev et al. [94] confirmed this parasite as a member of the ‘monoxenous’ genus Herpetomonas, and a very close relative of Herpetomonas ztiplika; a parasite that infects biting midges [94, 95].

Light and electron microscopic studies show that trypanosomatids possess a general ultrastructure that is universally conserved across the family [32]. Trypanosomatids possess an interlaced network of circular DNA known as the kinetoplast DNA (kDNA), associated with the base of a single flagellum that is attached to a slender cell body [32]. Recently, Wheeler et al. [32] conveniently divided trypanosomatids into two distinct morphological superclasses that are supported by molecular phylogeny; the ‘juxtaform’ superclass which includes epimastigote and trypomastigote morphotypes, defined by the presence of a laterally attached flagellum (e.g. Trypanosoma), and the ‘liberform’ superclass which possess a free flagellum (e.g. Leishmania and Leptomonas), which includes the opisthomastigote, choanomastigote and promastigote morphotypes [32] (Fig. 2). The spherical amastigote morphotype of trypanosomatids occurs in both morphological superclasses [32]. Therefore, the presence or absence of amastigotes cannot be used to inform evolutionary relationships. Along with the morphotypes described above, several others have been defined [26].

Liberform trypanosomatids such as Leptomonas, Zelonia (Fig. 7) and Leishmania predominately exist as promastigotes in standard culture, though axenic amastigotes of Leishmania can be induced in vitro [96]. Regardless, these species are highly pleomorphic, shifting between morphotypes depending on their growth phase, host, and host compartment they are occupying [32, 34, 68, 87, 97, 98]. Promastigotes of Leishmania are divided into five morphological categories which include: (i) procyclic promastigotes - the replicating form in the sand fly; (ii) nectomonad promastigotes - the elongated promastigote stage; (iii) haptomonad promastigotes - a stage possessing a disc-like expansion at the flagellar tip; (iv) leptomonad promastigotes - the stage that secretes PSG and is a precursor to; (v) metacyclic promastigotes - the form infective to the vertebrate host (Fig. 5) [70].

Generally, the trypanosomatid morphotypes are distinguished by their cell shape, the relative position of their nucleus to the kinetoplast [33], and flagellum positioning and attachment to the cell body [32, 74, 75]. These and other subtleties once served as taxon-defining characteristics under the classic system, and are now considered to a very minor extent given the inadequacies of this system revealed by genetics [26, 68, 97]. Phylogenetic analyses have culminated in the renaming of several species due to various instances of polyphyly introduced by the classic system, and are now considered mandatory for making accurate taxonomic assignments for new isolates [27, 28, 97, 99, 100].

As new trypanosomatid species are discovered, discernible features that were once taxon defining are becoming less suitable for this purpose. The discovery and description of Novymonous esmeraldas and characterisation of its bacterial endosymbiont provides a recent example [87]. Bacterial endosymbionts were once confined to the Strigomonadinae which occupy a single phylogenetic clade [26]. However, N. esmeraldas is a distantly related trypanosomatid of the subfamily Leishmaniinae, making bacterial endosymbiosis a polyphyletic trait. However, the endosymbionts of the Strigomonadinae are only distantly related to those of Novymonas, suggesting these relationships developed independently in the two trypanosomatid subfamilies [87]. In any case, the main confusion with trypanosomatid systematics lies in the discordance between their visually discernible characteristics and their molecular biology.

Advances in molecular biology and phylogenetics have revealed a lack of taxonomic concordance between gene sequence similarity and species demarcation, most notably within the clinically relevant genera Leishmania and Trypanosoma [7]. For example, small subunit ribosomal RNA (SSU rRNA) sequences of T. cruzi and T. brucei are separated by a genetic distance of approximately 12% while different species within the genus Leishmania possess SSU rRNA sequences separated by a distance of less than 1% in some cases [101]. Similarly, T. cruzi isolates can be separated into several discrete typing units [102, 103], which could easily constitute different species if they were held to the same species demarcation criteria as the genus Leishmania. This problem has been raised by previous investigators, who offer sensible solutions to this issue, including delineation of new species based on a 90% sequence similarity threshold [27].

Based on current understanding Leishmania possesses a sexual or parasexual cycle that allows recombination between distinct lineages, and in some cases different species [80]. Instances of hybridisation have been known from studies of trypanosomatid field isolates for nearly two decades [104]. A study involving isoenzyme analysis and molecular karyotyping of two Leishmania strains isolated from wild animals in Saudi Arabia identified a suspected hybrid isolate distinct from other Leishmania species, possessing characteristics of both Leishmania major and Leishmania arabica [105]. Banuls et al. [106] identified suspected Leishmania hybrids in stocks isolated from humans in Ecuador, representing potential crosses between L. braziliensis and L. panamensis/guyanensis. More recently, whole genome sequencing was used to confirm hybridisation in 11 unique isolates of Leishmania infantum from sand flies and one CL patient [107]. Additionally, crosses between L. major and L. infantum have been achieved under experimental conditions [108]. The limited genetic divergence between Leishmania species (discussed above), in conjunction with reports of hybridization, indicate that species delineation in this genus could be relaxed. However, echoing the points made by previous investigators [27], collapsing the many Leishmania species into a few would only generate confusion, particularly for clinicians. The grouping of Leishmania species into phylogenetically supported subfamilies as per Espinosa et al. [28] seems an elegant solution as hybridization seems to only occur within subfamilies and not between them, though this requires further investigation.

Cases of locally acquired Australian cutaneous leishmaniasis were first reported in 2004, in captive red kangaroos (Macropus rufus) from a wildlife park near Darwin, in Australia’s Northern Territory [20, 109]. In 2009, the same species was isolated from three additional macropod species, Macropus robustus woodwardi (northern wallaroo), Macropus bernardus (black wallaroo) and Macropus agilis (agile wallaby) [22]. This was the first report confirming Australia’s endemicity for leishmaniasis, albeit a form restricted to native animals, with no evidence for human infection. Of the 18 known Australian phlebotomine species, little is known about their biological capacity as vectors of leishmaniasis and Leishmania has never been detected in these species [23, 110]. Australian phlebotamines are thought to feed solely on small mammals, birds and reptiles which led to the speculation that this species (recently dubbed Leishmania macropodum [68]), might be transmitted via an alternative vector [22]. Later investigations identified a day feeding midge, Forcipomyia (Lasiohelea) (Diptera: Ceratopogonidae), as the likely vector [23]. Leishmania macropodum was prevalent in ~15% of Forcipomyia (L.) midges tested and similar patterns of promastigote migration to the midgut were observed between Forcipomyia and Leishmania, when compared to those observed in the Leishmania-phlebotomine interaction [23]. In addition to Forcipomyia, other insects may be capable of supporting Leishmania replication (at least temporarily) based on evidence from experimental infections and molecular evidence from naturally infected insects. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is capable of developing late stage infections with Leishmania enriettii [111]. Culicoides nubeculosus supported L. infantum infection for up to 7 days post blood meal [72]. Leishmania infantum DNA has also been detected in naturally infected Culicoides spp. collected in Tunisia [112], while DNA from Leishmania amazonensis and Leishmania braziliensis was detected in naturally infected Culicoides spp. from Brazil [113]. These studies call into question the exclusivity of the Leishmania-phlebotomine interaction, particularly in the case of the Australian species which naturally infects Forcipomyia and reportedly produces a PSG plug in this insect [23].

Phylogenetic evidence has confirmed on multiple occasions that naming trypanosomatid taxa based on classic systems often gives rise to polyphyly [99, 114]. This is due to divergence in DNA sequences that give rise to few changes in morphology, host range and other biological characteristics [7, 97]. The absence of adequate boundaries relating to morphological and host-based criteria highlight the requirement for a phylogenetic solution to trypanosomatid taxonomy [115]. Estimating rates of evolutionary divergence is attributed to Zuckerkandl and Pauling’s concept of the molecular clock which suggests that differences between amino acid sequences are relatively proportional to evolutionary events of divergence [116–118]. This approach assumes that rates of genetic change are constant amongst species of common descent, allowing estimates of rates to be extrapolated across phylogenetic trees [119]. Subsequent studies suggested that if these clocks could infer evolutionary timescales, the differences between sequences must be exclusive to sites of neutrality to equal the rate of mutation, supporting Kimura’s theory of neutral molecular evolution [120, 121].

Slow evolving (SE) genes are characterised by a slower divergence rate, undergoing fewer nucleotide substitutions over time. Sequences of SE genes are most suitable for investigating evolutionary relationships over larger time-scales [122]. Phylogenetic studies using only the small subunit ribosomal RNA (SSU rRNA) genes may not provide reliable phylogenetic inference for species within the same taxonomic family for example, as the rRNA genes evolve very slowly [123, 124]. Instead, a middle ground must be reached for resolving relationships between closely related organisms. Several housekeeping genes encoding proteins involved in basic cellular functions have provided the most useful information on relationships between the various Leishmania species [125]. Gene sequences of the RNA polymerase II largest subunit (RPOIILS) [97, 123], DNA polymerase α catalytic polypeptide (POLA) [123], glyceraldehyde-3-phosphate dehydrogenase (GADPH) [18, 97], heat-shock protein 20 [125], and heat-shock protein 70 [126], are preferred targets for analysing phylogenetic relationships between Leishmania species and their close relatives.

Despite the power of phylogenetic inference there are some important caveats to be considered. For example, the structure of phylogenetic trees can change markedly depending on the locus used, affecting the way relationships between taxa are interpreted. Using concatenated sequences from multiple phylogenetically informative loci has been suggested as a means to counteract this problem and improve the robustness of phylogenetic trees [27]. Similarly, the structure of phylogenetic trees can change markedly depending on the outgroup selected, or if certain taxa are included or excluded from the analysis [27]. The robustness of phylogenetic predictions is also dependent on the number of appropriate taxa included in the analysis. An important example involves the early debate surrounding the most basal Leishmania subgenus (touched upon previously herein) [19, 67, 68, 127–130]. This was considered pertinent to selection of the most appropriate outgroup, and would have implications relating to Leishmania biogeography and even its first vertebrate hosts. If the Sauroleishmania were the earliest branching group, this would implicate reptiles as the original vertebrate host of an ancestral Leishmania parasite. If Viannia were most basal, Leishmania probably evolved in the Neotropics [128]. With the addition of several other taxa, including some novel monoxenous genera that represent appropriate outgroups; phylogenetic evidence indicates that Mundinia is the most basal Leishmania subgenus [28, 68].

Discrepancies also arise when using single (or very few) trypanosomatid isolates to define new genera and establish taxonomic assignments. Zelonia costaricensis represents one such example. Originally assigned to the genus Leptomonas [131], the inclusion of several novel trypanosomatid isolates in phylogenies confirmed that Zelonia is distinct from the Leptomonas, Lotmaria and Crithidia clade, but is immediately basal to the Leishmania, Endotrypanum and Porcisia clade, warranting establishment of a new genus [28, 68]. A solution exemplified by Espinosa et al. [28] is to obtain sequence data from multiple sister isolates of a new candidate taxon before making new assignments. This will ensure that trypanosomatid taxa remain monophyletic by providing robust phylogenetic support.

Two recent studies used phylogenetics to approximately date the origin of the first ancestral Leishmania parasites with intriguing results [68, 132]. However, this type of analysis is complicated by the fact that closely related taxa may evolve at different rates due to environmental pressures, including those exerted by the host immune response. These pressures are of course markedly different for monoxenous and dixenous taxa. The accuracy of these analyses also relies on selection of a precise calibration point; that is, an accurately dated geological event (or other) that is known to have triggered a speciation event in closely related taxa. As the accuracy of these predictions are difficult to test, vicariance events dated using phylogenetic inference should be considered approximations at best.

Important developments have been made in the field of trypanosomatid taxonomy in recent years, facilitated by the identification of novel trypanosomatid species in addition to advances in molecular biology and phylogenetics. Phylogenetic inference has identified flaws in the classical system of trypanosomatid taxonomy which relied predominantly on parasite morphology and host preferences. Despite significant sequence divergence, trypanosomatids have experienced limited morphological change throughout their evolution such that the subtle morphological variations that exist do not reflect molecular phylogeny. Phylogenetics has corrected some of these issues by supporting the assignment of some trypanosomatids to different genera and establishment of new genera, ensuring that taxa remain monophyletic [27]. Despite these advances, trypanosomatid systematics still remains imperfect. There remains a lack of consensus for delineating species based on molecular divergence, and the limits differ markedly for Trypanosoma and Leishmania. While not recommended due to the confusion it would cause, molecular evidence probably supports the collapsing of some Leishmania species into one, or splitting Trypanosoma into different species or even different genera depending on the limits of molecular divergence applied. Additionally, some trypanosomatid genera (i.e. Leptomonas) remain paraphyletic [68, 133]. Transmission of L. macropodum by a day feeding midge rather than a phlebotomine sand fly and the detection of clinically important Leishmania species in hematophagous insects such as Culicoides spp., questions the exclusivity of the Leishmania-sand fly relationship. Reports of monoxenous trypanosomatid infections in vertebrates and the grouping of presumably monoxenous trypanosomatids (e.g. Zelonia and Novymonas) in dixenous clades occupied by Leishmania and Endotrypanum, also call into question the suitability of host relationships as a taxonomic criterion. Long accepted dogmas such as the ‘one-insect-one-parasite’ rule have been overturned, supported by molecular evidence. These examples highlight the importance of employing molecular biology and phylogenetics when making taxonomic assignments relating to trypanosomatids. The continued isolation and characterisation of novel trypanosomatids will ensure this field continues to develop, by allowing resolution of evolutionary relationships between members of this group with increasing accuracy. To this end, generation of high-quality genome sequences for additional species would be of huge benefit, allowing relationships to be phylogenetically scrutinised across numerous loci. The genomes of trypanosomatids that encroach on the monoxenous-dixenous boundary (e.g. Zelonia and Novymonas) may prove instrumental to understanding the path to dixenous parasitism by providing new knowledge on the innovations that evolved to allow this transition [58].