**4. Phylogenetic analysis**

Phylogenetics is the study of evolutionary relationships among various groups of organisms (e.g., species or populations). Their relatedness is evaluated through morphological and molecular sequencing data. This analysis leads to a hypothesis about the evolutionary history of taxonomic groups, their phylogeny. Regarding evolution, it is considered to be a branching process. Populations are altered with time and may split into separate branches, hybridize or be eliminated. The order in which evolutionary events are assumed to have occurred is revealed and may be visualized in a phylogenetic tree.

As mentioned before, MLEE is still regarded as the reference technique for the identification of *Leishmania* species and subspecies. The data obtained from MLEE were analyzed by phenetic and cladistic techniques and led to the construction of the first phylogenetic tree of the genus *Leishmania* [162]. The latter, revealed the monophyletic origin of the genus *Leishmania* and its subdivision into two subgenera, the *L.(Leishmania)* and the *L.(Viannia*) subgenus. *L.(Leishma‐ nia*) included the Old World species and *L.mexicana* and complexes from the New World. *L. (Viannia*) subgenus was composed from the other New World species. As *Sauroleishmania* was considered to be a separate genus, the lizard species were not included in these studies. MLEE has been applied to a great variety and amount of isolates in comparison to other molecular methods in the past 25 years, resulting in the current classification system [93,162]. Phyloge‐ netics based on different molecular methods, has confirmed the previous suggested taxonomy of the genus *Leishmania* by MLEE. However, the existence of a larger number of species has been proposed.

proaches developed so far for *Leishmania spp*, make use of sets of 14–20 unlinked microsatellite loci. Microsatellite loci with high discriminatory power and being suitable for characterizing closely related strains have been published for the *L.donovani* complex [155–158], *L.donovani* strains [127] *L.major* [159], *L.tropica* [126,160] and for species of the subgenus *L. (Viannia)* [161]. Moreover, as the genetic diversity of *L.infantum* strains has been the subject of intense interest, several studies used MLMT approaches for the evaluation of the genomic variation in *L.infantum* strains [122,135]. It is worth mentioning that when MLMT was compared with other molecular markers for strain typing of *L.infantum*, the results obtained with kDNA PCR-RFLP were comparable to MLMT. kDNA and MLMT presented the highest discriminatory power especially for the MON-1 strains discrimination and appeared to be the most adequate for strain fingerprinting. However, MLMT is advantageous over kDNA PCR-RFLP because of its better reproducibility and feasibility of inter-lab comparisons and the co-dominant character of the markers used, making MLMT suitable for population genetic studies [77]. MLMT is suitable for high-throughput analysis and the data obtained are reproducible and exchange‐ able between laboratories. Moreover, accurate, quality controlled microsatellite profiles can be stored in databases and compared between different laboratories. In contrast to MLEE, selection does not seem to act on polymorphisms in microsatellite length while also the codominant nature of these markers permits the detection of the allelic variants. MLMT can be used directly on biological samples without prior culture of the parasite. DNA extracted from specimens spotted on filter paper or glass slides or from old Giemsa stained microscope slides was successfully applied in MLMT approaches [155]. It is recommended to use a panel of 10–20 unlinked microsatellite markers in all studies for nearly every species because microsatellite sequences are prone to homoplasy. Additionally, polymorphic repeats are not

Phylogenetics is the study of evolutionary relationships among various groups of organisms (e.g., species or populations). Their relatedness is evaluated through morphological and molecular sequencing data. This analysis leads to a hypothesis about the evolutionary history of taxonomic groups, their phylogeny. Regarding evolution, it is considered to be a branching process. Populations are altered with time and may split into separate branches, hybridize or be eliminated. The order in which evolutionary events are assumed to have occurred is

As mentioned before, MLEE is still regarded as the reference technique for the identification of *Leishmania* species and subspecies. The data obtained from MLEE were analyzed by phenetic and cladistic techniques and led to the construction of the first phylogenetic tree of the genus *Leishmania* [162]. The latter, revealed the monophyletic origin of the genus *Leishmania* and its subdivision into two subgenera, the *L.(Leishmania)* and the *L.(Viannia*) subgenus. *L.(Leishma‐ nia*) included the Old World species and *L.mexicana* and complexes from the New World. *L. (Viannia*) subgenus was composed from the other New World species. As *Sauroleishmania* was considered to be a separate genus, the lizard species were not included in these studies. MLEE

conserved between different species of *Leishmania* [74,122,157].

172 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

revealed and may be visualized in a phylogenetic tree.

**4. Phylogenetic analysis**

PCR-based methods with subsequent RFLP or DNA sequence analysis of multicopy targets or multigene families, to the recently developed MLST and MLMT, have been applied for the identification of the *Leishmania* species being responsible for the disease and for epidemiolog‐ ical studies in different endemic regions, as well as for taxonomic, phylogenetic, and popula‐ tion genetic studies. These tools except from their enhanced sensitivity they are also able to distinguish *Leishmania* parasites at species and intraspecies level. As for phylogenetic studies, the sequence analysis of single-copy gene targets is preferred while also the recombination and the different mutation rates between lineages make the use of one gene less suitable for the phylogenic analysis of the Trypanosomatidae or its subgroups[163].

Several DNA targets have been used to reveal the phylogeny of the *Leishmania* genus including single-copy genes encoding the catalytic polypeptide of DNA polymerase a (polA), the largest subunit of RNA polymerase II (rpoIILS) [164] and 7SL RNA [86], the ITS [165,166], the Nacetylglucosamine-1-phosphate transferase (NAGT) gene [167], the mitochondrial cytochrome b gene (cytb) [168], and most recently, sequences of the hsp70 subfamily [83]. Sequence analysis of these targets led to the conclusion that the subgenera L. (*Leishmania*) and *L. (Viannia)* constitute distinct monophyletic clades and that species of the Old and New World are segregated within the *L. (Leishmania*) subgenus. *Sauroleishmania* species branched off between the *L.(Leishmania*) and *L.(Viannia)* subgenera as an independent taxon suggesting that lizard *Leishmania* might be derived from mammalian parasites [164] and that they should be regarded as a subgenus of *Leishmania* rather than an independent genus [99]. However, the fact that RNA and DNA polymerase genes presented higher evolution rate in the lizard *Leishmania* than in the mammalian *Leishmania* species set into question the exact taxonomic position of lizard parasites [164].

In another study, Cupolillo et al. based on various molecular criteria, suggested the division of the genus *Leishmania* into two sections, *Euleishmania* and *Paraleishmania*. *Euleishmania* consisted of the subgenera *L.(Leishmania), L.(Sauroleishmania), and L.(Viannia). Paraleishmania* included *L. hertigi, L.deanei, L.colombiensis, L.equatorensis, L.herreri,* and strains of *Endotrypa‐ num*. In the latter section, the parasites of hystricomorph rodents, *L.hertigi* and *L.deanei* and the remaining species that are mainly parasites of sloths were genetically different while strains of *Endotrypanum* formed a paraphyletic group [169].

More recently Fraga et al. analyzed the phylogeny of the genus *Leishmania* based on the hsp70 gene. In this study the isolates and strains used, were of different geographic origins. The resulting phylogeny supported that the monophyletic genus *Leishmania* consisted of three distinct subgenera, the *L.(Leishmania), L.(Viannia), and L.(Sauroleishmania).* The obtained phylogeny supported the following eight species: *L.donovani*, *L.major*, *L.tropica*, *L.mexicana*, *L.lainsoni*, *L.naiffi, L.guyanensis* and *L.braziliensis*. In some of these species, subspecies were recognized including *L.donovani infantum, L.guyanensis panamensis, and L.braziliensis peruvi‐* *ana*. The so far recognized species *L.aethiopica, L.garnhami, and L.amazonensis* did not form monophyletic clusters [83].

Several discrepancies were reported for the taxonomic status of species obtained by MLEE compared to DNA based sequences. It is worth mentioning that the existence of *L.chagasi* and *L. archibaldi* as distinct species, was not supported by any molecular analyses as *L.chagasi* cannot be distinguished from strains of *L.infantum* and should therefore be regarded as South American strains of *L.infantum* [170,171] whereas *L. archibaldi* is not a valid species [125,159]. Numerous molecular studies did not even support the monophyly of the two remaining species, *L.donovani* and *L.infantum* [83,164,168]. Therefore, it was proposed that *L.donovani* is the only species of the *L.donovani* complex [83] while *L.donovani infantum* was recognized as subspecies. Regarding other geographically defined genetic groups within *L.donovani*, it was suggested that they could be delimited. Furthermore, the status of *L.killicki* has been debated. MLEE analysis supported the classification of *L.killicki* as a separate species while other molecular methods proposed that it was identical to *L.tropica* [94,126,168]. At the same time, *L.tropica* clusters to a single branch with *L.aethiopica*, making it difficult to be distinguished by the most of the DNA-based phylogenies [83,86,168]. It was suggested that they may represent different subspecies of the species *L.tropica* which is however needed to be investigated with a larger number of strains. Another discrepancy concerns the existence of the *L.mexicana* complex species. The strains of *L.mexicana* and *L.amazonensis* species are overpresented in DNA based phylogenies while only one *L. garnhami* strain was analysed in the hsp70 trees. In the latter study, none of these species could be distinguished as a monophyletic clade and *L.mexicana* was the only recognized species [83]. These results are in agreement with previous published studies [164,172,173] whereas they are in contrast to others [86,165,168]. Thus, the *L.mexicana* complex should be investigated, including *L.venezuelensis* and *L.aristidesi* strains, in order to evaluate the species and subspecies constituting this complex. The same holds true for the *L.braziliensis* complex species. Several molecular phylogenies including hsp70, RAPD and MLEE, supported the distinction of *L.peruviana* from other strains of *L.braziliensis* [83,174] and it was recognized as a subspecies in the *L.braziliensis* complex. However, this classification was questioned by a study using monoclonal antibodies [175] and another one analyzing the microsatellite variation [161] which suggested that strains of *L.peruviana* were grouped together with strains of *L.braziliensis* from Peru and from the Acre State, a Brazilian region bordering Peru. The use of a sufficiently large number of strains from different areas of distribution is needed so as the taxonomic status of the repsesentatives of the *L.braziliensis* complex to be evaluated. Moreover, in different phylogenetic trees, strains of *L.guyanensis* and *L.panamensis* formed a monophyletic cluster which was then divided into two monophyletic subclusters. Thus, the existence of two subspecies within the species *L.guyanensis* was pro‐ posed. A possible explanation for these discrepancies reported in different studies regarding the taxonomic status of both *L.peruviana* and *L.panamensis*, is the application of different molecular markers and the analysis of different strains.

Several molecular methods including MLEE [93], PCR-RFLP of ITSrDNA [78] and PCR-RFLP and sequence analysis of the hsp70 gene [102], were also suggested the inclusion of *L.shawi* in the *L. guyanensis* group. The same applies for *L.naiffi* whereas *L.lainsoni* was confirmed to be the most divergent species inside the *L.(Viannia)* subgenus [83,102].

Noyes et al. (2002) identified a parasite isolated from human cutaneous lesions. Both stains were analysed by MLEE and found to be identical to each other and distantly related to all other *Leishmania* species. The application of other molecular methods revealed a low support for both its position basal to all *Euleishmania* and its clustering with *L.enriettii*. Thus, it was suggested that this strain may cluster with *L.*(*Leishmania*) or *L.(Viannia)* or form a novel clade within the *Euleishmania* either with or without *L.enriettii* [176]. Recently *Leishmania* species isolated from clinical samples from immunocompetent and immunosuppressed patients in Thailand [177,178] and a focal CL outbreak in Ghana [179] were identified and named as *L.siamensis*. Furthermore, novel *Leishmania* species, genetically indistinguishable, were isolated from kangaroos, wallaroos, and wallabies, living in captivity in the Northern Territory of Australia, a region that was considered free of *Leishmania* parasites [180]. Additionally, autochthonous cases of CL in German and Swiss horses and in a Swiss cow could not be classified neither as Old World nor New World *Leishmania* species while they were found to be most closely related to *L.siamensis* [181,182]. Finally, two new *L.(Viannia)* species were described and named *L.lindenbergi* [183] and *L.utingensis*. The last one was represented by only one sample isolated from a *Lutzomyia tuberculata* sand fly. Although the sequence analysis of single-copy gene targets has shown to be informative, the use of several independent genes displaying different evolutionary histories is preferable [184]. Such genes have applied in MLST and provided new insights on taxonomy and evolutionary history of *Leishmania*. MLST is currently considered the most powerful phylogenetic approach, it has been shown to have high discriminatory power, reproducibility and transportability of results between laborato‐ ries. Thus far, there are 10 published MLST targets available for the *L.donovani* complex [151,152], most of which are also applicable to other Old World *Leishmania* [185] and 4 targets for the sub-genus *Leishmania (Viannia)* [153]. This should form a complete MLST system applicable to *Leishmania* parasites.

### **5. Conclusion**

*ana*. The so far recognized species *L.aethiopica, L.garnhami, and L.amazonensis* did not form

Several discrepancies were reported for the taxonomic status of species obtained by MLEE compared to DNA based sequences. It is worth mentioning that the existence of *L.chagasi* and *L. archibaldi* as distinct species, was not supported by any molecular analyses as *L.chagasi* cannot be distinguished from strains of *L.infantum* and should therefore be regarded as South American strains of *L.infantum* [170,171] whereas *L. archibaldi* is not a valid species [125,159]. Numerous molecular studies did not even support the monophyly of the two remaining species, *L.donovani* and *L.infantum* [83,164,168]. Therefore, it was proposed that *L.donovani* is the only species of the *L.donovani* complex [83] while *L.donovani infantum* was recognized as subspecies. Regarding other geographically defined genetic groups within *L.donovani*, it was suggested that they could be delimited. Furthermore, the status of *L.killicki* has been debated. MLEE analysis supported the classification of *L.killicki* as a separate species while other molecular methods proposed that it was identical to *L.tropica* [94,126,168]. At the same time, *L.tropica* clusters to a single branch with *L.aethiopica*, making it difficult to be distinguished by the most of the DNA-based phylogenies [83,86,168]. It was suggested that they may represent different subspecies of the species *L.tropica* which is however needed to be investigated with a larger number of strains. Another discrepancy concerns the existence of the *L.mexicana* complex species. The strains of *L.mexicana* and *L.amazonensis* species are overpresented in DNA based phylogenies while only one *L. garnhami* strain was analysed in the hsp70 trees. In the latter study, none of these species could be distinguished as a monophyletic clade and *L.mexicana* was the only recognized species [83]. These results are in agreement with previous published studies [164,172,173] whereas they are in contrast to others [86,165,168]. Thus, the *L.mexicana* complex should be investigated, including *L.venezuelensis* and *L.aristidesi* strains, in order to evaluate the species and subspecies constituting this complex. The same holds true for the *L.braziliensis* complex species. Several molecular phylogenies including hsp70, RAPD and MLEE, supported the distinction of *L.peruviana* from other strains of *L.braziliensis* [83,174] and it was recognized as a subspecies in the *L.braziliensis* complex. However, this classification was questioned by a study using monoclonal antibodies [175] and another one analyzing the microsatellite variation [161] which suggested that strains of *L.peruviana* were grouped together with strains of *L.braziliensis* from Peru and from the Acre State, a Brazilian region bordering Peru. The use of a sufficiently large number of strains from different areas of distribution is needed so as the taxonomic status of the repsesentatives of the *L.braziliensis* complex to be evaluated. Moreover, in different phylogenetic trees, strains of *L.guyanensis* and *L.panamensis* formed a monophyletic cluster which was then divided into two monophyletic subclusters. Thus, the existence of two subspecies within the species *L.guyanensis* was pro‐ posed. A possible explanation for these discrepancies reported in different studies regarding the taxonomic status of both *L.peruviana* and *L.panamensis*, is the application of different

monophyletic clusters [83].

174 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

molecular markers and the analysis of different strains.

Several molecular methods including MLEE [93], PCR-RFLP of ITSrDNA [78] and PCR-RFLP and sequence analysis of the hsp70 gene [102], were also suggested the inclusion of *L.shawi* in Molecular methods have revolutionised the diagnosis of leishmaniasis. A variety of target sequences has been used and evaluated in different clinical samples of parasite hosts. Regard‐ ing PCR based assays, they were found to be rapid, sensitive and discriminative at species or even strain level. However the diagnosis of leishmaniasis remains a scientific challenge. There is a gap between the scientific advances, diagnostics and management of *Leishmania* infections in the field which should be decreased and an urgent need for standardization, optimization and simplification of PCR based applications. In this context, there is a generalized effort to make these assays available mainly in endemic areas around the world which will have an impact in disease control.

The great scientific interest for species identification may be attributed to its significance in prompt diagnosis and prognosis of the disease, decision making regarding treatment and control measures. Despite the abundance of the studies carried out and the molecular markers used so far, the species discrimination is still tough in several closely related species. Thus, molecular tools with high discriminatory power are currently under development, optimiza‐ tion and evaluation.

Many molecular tools have been used for the *Leishmania* phylogeny and the definition of its taxonomy. However, evaluation of the phylogenetic relationships of *Leishmania* species is not an easy task. Moreover, there is a need for simplification of the classification and a meaningful nomenclature of *Leishmania* genus particularly for the clinicians.
