# Evolutionary history of Leishmania killicki (synonymousous Leishmania tropica) and taxonomic implications

Dhekra Chaara, Christophe Ravel, Anne- Laure Bañuls, Najoua Haouas, Patrick Lami, Loïc Talignani, Fouad El Baidouri, Kaouther Jaouadi, Zoubir Harrat, Jean-Pierre Dedet, et al.

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Dhekra Chaara, Christophe Ravel, Anne- Laure Bañuls, Najoua Haouas, Patrick Lami, et al. Evolutionary history of Leishmania killicki (synonymousous Leishmania tropica) and taxonomic implications. Parasites & Vectors, 2015, 8 (1), pp.198. 10.1186/s13071-015-0821-6 . hal-02006181

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Distributed under a Creative Commons Attribution 4.0 International License
Evolutionary history of *Leishmania killicki*
(synonymous *Leishmania tropica*) and taxonomic
implications

Dhekra Chaara<fnref n="1" />,<fnref n="2" />,³*, Christophe Ravel<fnref n="2" />,³, Anne- Laure Bañuls³, Najoua Haouas<fnref n="1" />, Patrick Lami<fnref n="2" />,³, Loïc Talignani<fnref n="2" />,³, Fouad El Baidouri<fnref n="2" />,³,⁴, Kaouther Jaouadi<fnref n="1" />, Zoubir Harrat⁵, Jean-Pierre Dedet<fnref n="2" />,³, Hamouda Babba<fnref n="1" /> and Francine Pratlong<fnref n="2" />,³

**Abstract**

**Background:** The taxonomic status of *Leishmania* (L.) *killicki*, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the *L. tropica* complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of *L. killicki* relative to *L. tropica*.

**Methods:** Thirty-five *L. killicki* and 25 *L. tropica* strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches.

**Results:** The results of the genetic and phylogenetic analyses strongly support the hypothesis that *L. killicki* belongs to the *L. tropica* complex. Our data suggest that *L. killicki* emerged from a single founder event and that it evolved independently from *L. tropica*. However, they do not validate the hypothesis that *L. killicki* is a distinct complex. Therefore, we suggest naming this taxon *L. killicki* (synonymous *L. tropica*) until further epidemiological and phylogenetic studies justify the *L. killicki* denomination.

**Conclusions:** This study provides taxonomic and phylogenetic information on *L. killicki* and improves our knowledge on the evolutionary history of this taxon.

**Keywords:** *Leishmania killicki*, *Leishmania tropica*, Evolutionary history, Phylogeny, Isoenzymatic polymorphism

**Background**

Leishmaniases are neglected tropical diseases caused by
*Leishmania* parasites and transmitted to mammals
through bites by infected Phlebotomine sandflies of the
genus *Phlebotomus* [1]. In humans, these diseases can
have cutaneous (CL), muco-cutaneous (MCL) or visceral
(VL) clinical manifestations.

Since the first description of the genus *Leishmania*
Ross, 1903, the classification methods have considerably

evolved. Indeed, between 1916 and 1987, *Leishmania*
taxonomy followed the Linnaean classification system,
mainly based on extrinsic features, such as clinical mani-
festations, geographical distribution, epidemiological cy-
cles and behaviour in sandfly vectors. This method has
led to the subdivision of the *Leishmania* genus in the
two sub-genera *Leishmania* and *Viannia* [2,3].

In the eighties, the biochemical classification based on
the study of the parasite isoenzymatic patterns started to
be developed. This approach has evolved from the clas-
sical Adansonian to the numerical cladistic classification
method that uses isoenzymes as evolutionary markers
[4-8]. The description of several *Leishmania* complexes
in the Old and New World is based on these analyses.
Specifically, by using numerical phenetic and phylogen-
etic approaches, Rioux *et al*. [9] identified four main

<footnote>* Correspondence: chaara.dhekra@yahoo.fr</footnote>

<footnote n="1">¹Département de Biologie Clinique B, Laboratoire de Parasitologie-Mycologie Médicale et Moléculaire (code LR12ES08), Faculté de Pharmacie, Université de Monastir, Monastir, Tunisia</footnote>

<footnote n="2">²Département de Parasitologie-Mycologie, Centre National de Référence des Leishmanioses, CHRU de Montpellier, Université de Montpellier, France, 39 avenue Charles FLAHAULT, 34295 Montpellier Cedex 5, France
Full list of author information is available at the end of the article</footnote>
*Leishmania* groups in the Old World, while Thomaz *et al.* and Cupolillo *et al.* [10,11] defined eight complexes and two *Leishmania* groups in the New World.

Currently, the numerical taxonomic approach based on isoenzyme analysis is considered as the gold standard for the classification of the genus *Leishmania* and is routinely used for classification updates and for epidemiological studies [12,13]. The drawbacks of this approach are the need of bulk cultures of *Leishmania* parasites and its relatively poor discriminatory power. It is also time-consuming. Therefore, DNA-based techniques represent valuable alternatives for the identification and the classification of these parasites.

Since the nineties, several DNA-based approaches that target nuclear and/or kinetoplastid markers have been used for phenetic and phylogenetic studies of *Leishmania*, including sequencing of PCR-generated fragments (PCR-sequencing) [14], nested PCR [15], random amplified polymorphic DNA (RAPD) [16,17], single strand conformation polymorphism (SSCP) analysis [18,19], multilocus sequence typing (MLST) [20], multilocus microsatellite typing (MLMT) [21,22], restriction fragment length polymorphism analysis of PCR-amplified fragments (PCR-RFLP) [23,24], high-resolution melting (HRM) [25] and amplified fragment length polymorphism (AFLP) [26]. These techniques have improved our epidemiological knowledge and consequently also leishmaniasis control and treatment. Due to the wealth of new taxonomic data generated by these DNA-based approaches, it is currently debated whether the genus *Leishmania* classification should be revised [27,28].

MLST is one of the most appropriate approaches for taxonomic studies because it provides data on the genetic variations of housekeeping genes. This approach has been increasingly used for phylogenetic investigations to understand the epidemiological and transmission features of many *Leishmania* complexes [20,29-33]. However, because of the complexity of this genus and the lack of studies, several taxa need to be detailed further [34].

*Leishmania killicki* is a recently described taxon that causes CL in Tunisia [35], Libya [36] and Algeria [37]. *L. killicki* taxonomic status and evolutionary history relative to *L. tropica* are based on very few studies and samples. The numerical taxonomic analysis using the Multilocus Enzyme Electrophoresis (MLEE) approach first included this parasite in the *L. tropica* complex [9,38]. However, after the revision of the *Leishmania* genus classification, it was considered as a separate phylogenetic complex [39]. Recently, an update study by Pratlong *et al.* [12] confirmed the inclusion of *L. killicki* within the *L. tropica* complex. Phenetic and phylogenetic studies using MLMT [40], PCR-sequencing [41] and MLST [31] also classified *L. killicki* within the *L. tropica* complex and suggested a closer genetic link with *L. tropica* from Morocco. However, these data were obtained using only seven *L. killicki* strains: two strains were analyzed by Schwenkenbecher *et al.* [40], two by Chaouch *et al.* [41] and three by El Baidouri *et al.* [31]. Therefore, the present study wanted to analyze by MLST a large number of *L. killicki* and *L. tropica* strains in order to precisely determine the evolutionary history and the taxonomic status of *L. killicki*.

# Methods

## Origin of strains

For this study, strains of *L. killicki* (n = 35), *L. tropica* (n = 25), *L. major* (n = 1) and *L. infantum* (n = 1) from different geographic areas and with various zymodeme patterns were included (total = 62 strains). These strains were from human cutaneous lesions, except the *L. infantum* strain that was isolated from a patient with VL. Most strains (n = 53) were selected from the Cryobank of the Centre National de Référence des Leishmanioses (CNRL) (Montpellier, France) and nine *L. killicki* strains were collected by the team of the Laboratoire de Parasitologie - Mycologie Médicale et Moléculaire (Monastir, Tunisia) during epidemiological investigations.

Forty-eight strains, among which 34 *L. killicki* strains (six from Algeria, one from Libya and 27 from Tunisia) and 14 *L. tropica* strains from Morocco were analyzed by MLST for the first time during this study. The eleven remaining *L. tropica* strains were from several countries (one from Egypt, one from Greece, two from Israel, two from Jordan, three from Kenya and two from Yemen) and were previously typed by MLST. Their sequences were published in Genbank under the following accession numbers: KC158621, KC158637, KC158643, KC158677, KC158682, KC158683, KC158690, KC158696, KC158711, KC158722 and KC158761 (see [31]). One *L. killicki* strain (LEM163) MHOM/TN/80/LEM163 had also already been analyzed by MLST (Genbank accession number KC158820 (see [31]).

The *L. major* (LEM62) MHOM/YE/76/LEM62 and *L. infantum* (LEM75) MHOM/FR/78/LEM75 strains, previously typed by MLST, were used as outgroups [31].

## Isoenzymatic identification

All studied strains were identified by MLEE, according to Rioux *et al.* [9], using 15 enzymatic systems: malate dehydrogenase (MDH, EC 1.1.1.37), malic enzyme (ME, EC 1.1.1.40), isocitrate dehydrogenase (ICD, EC 1.1.1.42), phosphogluconate dehydrogenase (PGD, EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), glutamate dehydrogenase (GLUD, EC 1.4.1.3), NADH diaphorase (DIA, EC 1.6.2.2), nucleoside purine phosphorylase 1 and 2 (NP1, EC 2.4.2.1 and
<table><caption>Table 1 Details about the origin, taxon and zymodeme of the 62 strains under study</caption><thead><tr><th>CNRL code</th><th>WHO code</th><th>Origin</th><th>Taxon</th><th>Zymodeme</th></tr></thead><tbody><tr><td>LEM95</td><td>MHOM/TU/79/LEM95</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM160</td><td>MHOM/TN/80/LEM160</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM163</td><td>MHOM/TN/80/LEM163</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM174</td><td>MHOM/TN/80/LEM174</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM177</td><td>MHOM/TN/80/LEM177</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM179</td><td>MHOM/TN/80/LEM179</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM180</td><td>MHOM/TN/80/LEM180</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM181</td><td>MHOM/TN/80/LEM181</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM182</td><td>MHOM/TN/80/LEM182</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM183</td><td>MHOM/TN/80/LEM183</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM184</td><td>MHOM/TN/80/LEM184</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM185</td><td>MHOM/TN/80/LEM185</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM186</td><td>MHOM/TN/80/LEM186</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM193</td><td>MHOM/TN/80/LEM193</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM194</td><td>MHOM/TN/80/LEM194</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM904</td><td>MHOM/TN/80/LEM904</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM1013</td><td>MHOM/TN/80/LEM1013</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM4390</td><td>MHOM/TN/2002/LSL65</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM4741</td><td>MHOM/TN/2004/CRE139</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM5420</td><td>MHOM/TN/2007/LPN306</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6175</td><td>MHOM/TN/2010/MET315</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6226</td><td>MHOM/TN/2004/PLC3</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6228</td><td>MHOM/TN/2003/LC39</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6229</td><td>MHOM/TN/2006/SSC36</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6230</td><td>MHOM/TN/2006/SSC37</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6231</td><td>MHOM/TN/2005/LC24 bras</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6423</td><td>MHOM/TN/2012/NAS12</td><td>Tunisia</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM6173</td><td>MHOM/TN/2010/MET300</td><td>Tunisia</td><td>L. killicki</td><td>MON-317</td></tr><tr><td>LEM6227</td><td>MHOM MN/2005/PLC5</td><td>Libya</td><td>L. killicki</td><td>MON-8</td></tr><tr><td>LEM4995</td><td>MHOM/DZ/2005/LIPA07</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM6404</td><td>MHOM/DZ/2005/LIPA11</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM6416</td><td>MHOM/DZ/2011/LIPA283</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM6418</td><td>MHOM/DZ/2005/LIPA14</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM6420</td><td>MHOM/DZ/2011/LIPA281</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM6421</td><td>MHOM/DZ/2011/LIPA282</td><td>Algeria</td><td>L. killicki</td><td>MON-301</td></tr><tr><td>LEM1623</td><td>MHOM/MA/89/LEM1623</td><td>Morocco</td><td>L. tropica</td><td>MON-102</td></tr><tr><td>LEM1663</td><td>MHOM/MA/89/LEM1663</td><td>Morocco</td><td>L. tropica</td><td>MON-102</td></tr><tr><td>LEM2017</td><td>MHOM/MA/90/LEM2017</td><td>Morocco</td><td>L. tropica</td><td>MON-102</td></tr><tr><td>LEM5276</td><td>MHOM/MA/2000/INHW02</td><td>Morocco</td><td>L. tropica</td><td>MON-102</td></tr><tr><td>LEM5506</td><td>MHOM/MA/2007/INHS10</td><td>Morocco</td><td>L. tropica</td><td>MON-102</td></tr><tr><td>LEM1591</td><td>MHOM/MA/89/LEM1591</td><td>Morocco</td><td>L. tropica</td><td>MON-109</td></tr><tr><td>LEM1880</td><td>MHOM/MA/90/LEM 1880</td><td>Morocco</td><td>L. tropica</td><td>MON-109</td></tr><tr><td>LEM1922</td><td>MHOM/MA/89/LEM 1922</td><td>Morocco</td><td>L. tropica</td><td>MON-109</td></tr><tr><td>LEM1879</td><td>MHOM/MA/89/LEM 1879</td><td>Morocco</td><td>L. tropica</td><td>MON-112</td></tr><tr><td>LEM1918</td><td>MHOM/MA/89/LEM 1918</td><td>Morocco</td><td>L. tropica</td><td>MON-112</td></tr></tbody></table>

<table><caption>Table 1 Details about the origin, taxon and zymodeme of the 62 strains under study (Continued)</caption><tr><td>LEM1778</td><td>MHOM/MA/89/LEM1778</td><td>Morocco</td><td>L. tropica</td><td>MON-113</td></tr><tr><td>LEM5283</td><td>MHOM/MA/2000/INHW19</td><td>Morocco</td><td>L. tropica</td><td>MON-113</td></tr><tr><td>LEM5295</td><td>MHOM/MA/2000/INHW20</td><td>Morocco</td><td>L. tropica</td><td>MON-113</td></tr><tr><td>LEM3015</td><td>MHOM/MA/95/LEM3015</td><td>Morocco</td><td>L. tropica</td><td>MON-264</td></tr><tr><td>LEM0617</td><td>MHOM/IL/80/SINGER</td><td>Israel</td><td>L. tropica</td><td>MON-54</td></tr><tr><td>LEM955</td><td>MHOM/YE/86/LEM955</td><td>Yemen</td><td>L. tropica</td><td>MON-71</td></tr><tr><td>LEM1015</td><td>MHOM/YE/86/LEM1015</td><td>Yemen</td><td>L. tropica</td><td>MON-71</td></tr><tr><td>LEM1904</td><td>MHOM/GR/88/LA615</td><td>Greece</td><td>L. tropica</td><td>MON-114</td></tr><tr><td>LEM1824</td><td>MHOM/KE/86/EB103</td><td>Kenya</td><td>L. tropica</td><td>MON-119</td></tr><tr><td>LEM2313</td><td>IGUG/KE/91/000</td><td>Kenya</td><td>L. tropica</td><td>MON-119</td></tr><tr><td>LEM2454</td><td>MHOM/KE/92/EB000</td><td>Kenya</td><td>L. tropica</td><td>MON-119</td></tr><tr><td>LEM2001</td><td>MHOM/EG/90/LPN65</td><td>Egypt</td><td>L. tropica</td><td>MON-137</td></tr><tr><td>LEM3956</td><td>MHOM/IL/96/LRC-L691</td><td>Israel</td><td>L. tropica</td><td>MON-137</td></tr><tr><td>LEM2869</td><td>MHOM/JO/93/JH67</td><td>Jordan</td><td>L. tropica</td><td>MON-200</td></tr><tr><td>LEM3322</td><td>MHOM/JO/96/JH-88</td><td>Jordan</td><td>L. tropica</td><td>MON-265</td></tr><tr><td>LEM62</td><td>MHOM/YE/76/LEM62</td><td>Yemen</td><td>L. major</td><td>MON-26</td></tr><tr><td>LEM75</td><td>MHOM/FR/78/LEM75</td><td>France</td><td>L. infantum</td><td>MON-1</td></tr></table>

NP2, EC 2.4.2*), glutamate oxaloacetate transaminase 1 and 2 (GOT1 and GOT2, EC 2.6.1.1), phosphoglucomutase (PGM, EC 5.4.2.2), fumarate hydratase (FH, EC 4.2.1.2), mannose phosphate isomerase (MPI, EC 5.3.1.8) and glucose phosphate isomerase (GPI, EC 5.3.1.9).

## DNA extraction

Genomic DNA from cultured parasites was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer's recommendations and eluted in 150 µl.

## Analysis by Multilocus sequence typing (MLST)

The *L. killicki* (n = 34) and *L. tropica* (n = 14) strains that had not been previously assessed by MLST were typed using the MLST approach based on the analysis of seven loci coding for single-copy housekeeping genes that was developed and optimized by El Baidouri *et al.* [31]. Genomic DNA was amplified by real-time PCR using the SYBR Green method (Light cycler 480 II, Roche). The amplified products were sequenced on both strands (Eurofins MWG Operon, Germany) and the obtained sequences were aligned and checked in both directions using the CodonCode Aligner software, v.4.0.1 (Codon Code Co., USA). For each strain, polymorphic sites (PS) and ambiguous positions corresponding to heterozygous sites (HS) were identified in each locus using the same software. The DnaSP software v.5 [42] was used to calculate the number of haplotypes from the concatenated sequences.
<table><caption>Table 2 Isoenzyme patterns for the 15 enzyme systems of the four *Leishmania killicki* zymodemes</caption><thead><tr><th rowspan="2">Taxon</th><th rowspan="2">Zymodeme</th><th colspan="15">Enzyme profiles</th></tr><tr><th>MDH</th><th>ME</th><th>ICD</th><th>PGD</th><th>G6PD</th><th>GLUD</th><th>DIA</th><th>NP1</th><th>NP2</th><th>GOT1</th><th>GOT2</th><th>PGM</th><th>FH</th><th>MPI</th><th>GPI</th></tr></thead><tbody><tr><td rowspan="4">L. killicki</td><td>MON-317</td><td>100</td><td>100</td><td>100</td><td>93</td><td>82</td><td>110</td><td>100</td><td>300</td><td>100</td><td>127</td><td>90</td><td>100</td><td>110</td><td>110</td><td>76</td></tr><tr><td>MON-8</td><td>100</td><td>100</td><td>100</td><td>93</td><td>82</td><td>110</td><td>100</td><td>300</td><td>100</td><td>127</td><td>90</td><td>100</td><td>100</td><td>110</td><td>76</td></tr><tr><td>MON-301</td><td>112</td><td>93, 66</td><td>100</td><td>93</td><td>82</td><td>110</td><td>100</td><td>300</td><td>100</td><td>140</td><td>85</td><td>100</td><td>100</td><td>110</td><td>76</td></tr><tr><td>MON-306</td><td>112</td><td>100</td><td>100</td><td>93</td><td>82</td><td>110</td><td>100</td><td>300</td><td>100</td><td>140</td><td>85</td><td>100</td><td>100</td><td>110</td><td>76</td></tr></tbody></table>

Phylogenetic relationships were inferred using a Bayesian approach implemented with the MrBayes software v. 3.2.3 [43]. The concatenated duplicated sequence alignments of the seven loci for the 32 *Leishmania* strains representing all the identified haplotypes and the two outgroup strains (n = 34 in total) were used to run two independent chains for 10,000,000 generations each and trees sampled every 1000 generations. The burn-in period was set to 200,000 generations to fit the first 20% of the analyses. Analyses were conducted using the General time reversible model of substitution with a proportion of invariable sites and gamma distribution estimated by the program (GTR + G + I).

The chain convergence was assessed using the average standard deviation of split frequencies (ASDSF). If two runs converge onto the stationary distribution, the ASDSF is expected to approach zero, reflecting the fact that the two tree samples become increasingly similar. An average standard deviation below 0.01 is thought to be a very good indication of convergence (below 0.004 in our analysis). The consensus tree was constructed using 1000 trees sampled from the stationary phase. The MEGA 5.10 software [44] was used to identify amino acid variations between *L. killicki* and *L. tropica*.

## Results

### Isoenzymatic identification of *Leishmania* strains

Among the 62 strains under study, 53 had been previously characterized by MLEE at the Centre National de

Référence des Leishmanioses. The nine strains collected by the team of the Laboratoire de Parasitologie - Mycologie Médicale et Moléculaire (Monastir, Tunisia) were identified for the first time in this study using the same technique [12,35-38,45-47]. Nevertheless, all the strains were analyzed again by MLEE at the Centre National de Référence des Leishmanioses (Montpellier, France).

Seventeen zymodemes were identified: three for *L. killicki*, 12 for *L. tropica* and a single zymodeme for each *L. major* and *L. infantum* strain (Table 1). For *L. killicki*, besides the two known zymodemes MON-8 (n = 28) and MON-301 (n = 6), a new zymodeme (MON-317) was characterized in a single Tunisian strain (LEM6173: MHOM/TN/2010/MET300). The zymodeme MON-317 differed from MON-8 by only a single enzyme (FH) [35]. On the other hand, the MDH, ME, GOT1, GOT2 and FH profiles were different in the zymodemes MON-317 and MON-301 [37], and the MDH, GOT1, GOT2 and FH profiles allowed discriminating between MON-317 and MON-306 (a zymodeme described in Algeria, but not included in our sample collection) [48] (Table 2). For *L. tropica*, all the identified zymodemes were already known [12,45]: MON-54 (n = 1), MON-71 (n = 2), MON-102 (n = 5), MON-109 (n = 3), MON-112 (n = 2), MON-113(n = 3), MON-114 (n = 1), MON-119 (n = 3),

<table><caption>Table 3 Genetic diversity indices calculated from the MLST data considering the seven loci and all the *Leishmania killicki* (n = 35) and *Leishmania tropica* (n = 25) strains</caption><thead><tr><th>Locus</th><th>Length (bp)</th><th>No of PS (% of length)</th><th>No of HS (% of length)</th></tr></thead><tbody><tr><td>03.0980</td><td>678</td><td>18 (2,65%)</td><td>11 (1,62%)</td></tr><tr><td>04.0580</td><td>711</td><td>15 (2,1%)</td><td>9 (1,26%)</td></tr><tr><td>10.0560</td><td>636</td><td>12 (1,88%)</td><td>5 (0,78%)</td></tr><tr><td>12.0010</td><td>579</td><td>12 (2,07%)</td><td>4 (0,69%)</td></tr><tr><td>14.0130</td><td>642</td><td>11 (1,71%)</td><td>8 (1,24%)</td></tr><tr><td>31.0280</td><td>810</td><td>21 (2,6%)</td><td>16 (1,97%)</td></tr><tr><td>31.2610</td><td>486</td><td>6 (1,23%)</td><td>6 (1,23%)</td></tr><tr><td>Concatenated</td><td>4542</td><td>95 (2,09%)</td><td>59 (1,3%)</td></tr></tbody></table>

PS: polymorphic sites, HS: heterozygous sites.

<table><caption>Table 4 Comparison of the MLST data for the *Leishmania killicki* (n = 35) and *Leishmania tropica* (n = 25) strains at the seven loci under study</caption><thead><tr><th rowspan="2">Locus</th><th rowspan="2">Length (bp)</th><th colspan="2">No of PS (% of length)</th><th rowspan="2">No of HS (% of length)</th></tr><tr><th>L. killicki*</th><th>L. tropica</th></tr></thead><tbody><tr><td>03.0980</td><td>678</td><td>2 (0,29%)</td><td>16 (2,36%)</td><td>9 (1,33%)</td></tr><tr><td>04.0580</td><td>711</td><td>2 (0,29%)</td><td>13 (1,81%)</td><td>7 (0,98%)</td></tr><tr><td>10.0560</td><td>636</td><td>1 (0,16%)</td><td>11 (1,72%)</td><td>4 (0,63%)</td></tr><tr><td>12.0010</td><td>579</td><td>0</td><td>12 (2,07%)</td><td>4 (0,69%)</td></tr><tr><td>14.0130</td><td>642</td><td>1 (0,15%)</td><td>10 (1,56%)</td><td>7 (1,09%)</td></tr><tr><td>31.0280</td><td>810</td><td>2 (0,24%)</td><td>19 (2,36%)</td><td>14 (1,73%)</td></tr><tr><td>31.2610</td><td>486</td><td>3 (0,61%)</td><td>6 (1,23%)</td><td>3 (0,36%)</td></tr><tr><td>Concatenated</td><td>4542</td><td>11 (0,24%)</td><td>87 (1,91%)</td><td>48 (1,06%)</td></tr></tbody></table>

S: polymorphic sites, HS: heterozygous sites.

*For L. killicki PS=HS.
<table><caption>Table 5 Amino acid variations in *Leishmania killicki* and *Leishmania tropica* sequences at the seven loci assessed by MLST</caption><thead><tr><th>Locus</th><th>Position</th><th>Amino acid</th><th></th></tr><tr><td></td><td></td><td><i>L. killicki</i></td><td><i>L. tropica</i> (number of strains/total number of strains)</td></tr></thead><tbody><tr><td rowspan="4">03.0980</td><td>24</td><td>G</td><td>G</td></tr><tr><td>231</td><td>L</td><td>L</td></tr><tr><td>623</td><td>V</td><td>V (0.2), A(0.8)</td></tr><tr><td>630</td><td>S</td><td>S</td></tr><tr><td rowspan="6">04.0580</td><td>6</td><td>V</td><td>V</td></tr><tr><td>36</td><td>H</td><td>H (0.96), R (0.04)</td></tr><tr><td>258</td><td>I</td><td>I (0.96), V (0.04)</td></tr><tr><td>261</td><td>V</td><td>V (0.92), A(0.08)</td></tr><tr><td>421</td><td>V</td><td>V (0.44), I (0.56)</td></tr><tr><td>463</td><td>L</td><td>L</td></tr><tr><td>711</td><td>R</td><td>R</td></tr><tr><td rowspan="10">10.0560</td><td>24</td><td>F</td><td>F</td></tr><tr><td>42</td><td>G</td><td>G</td></tr><tr><td>51</td><td>V</td><td>V</td></tr><tr><td>54</td><td>A</td><td>A</td></tr><tr><td>61</td><td>A</td><td>S (0.6), A (0.4)</td></tr><tr><td>496</td><td>D</td><td>D (0.96), N (0.04)</td></tr><tr><td>535</td><td>Y</td><td>Y (0.88), N (0.12)</td></tr><tr><td>545</td><td>I</td><td>I (0.96), V (0.04)</td></tr><tr><td>619</td><td>T</td><td>T (0.64), A (0.36)</td></tr><tr><td>633</td><td>E</td><td>E</td></tr><tr><td rowspan="10">12.0010</td><td>72</td><td>D</td><td>D</td></tr><tr><td>75</td><td>G</td><td>G</td></tr><tr><td>120</td><td>S</td><td>S</td></tr><tr><td>156</td><td>K</td><td>K</td></tr><tr><td>249</td><td>R</td><td>R</td></tr><tr><td>261</td><td>S</td><td>S</td></tr><tr><td>344</td><td>A</td><td>A (0.56), V (0.44)</td></tr><tr><td>438</td><td>N</td><td>N (0.96), K (0.04)</td></tr><tr><td>507</td><td>G</td><td>G</td></tr><tr><td rowspan="6">14.0130</td><td>77</td><td>L</td><td>L (0.88), Q (0.12)</td></tr><tr><td>85</td><td>S</td><td>P (0.52), S (0.48)</td></tr><tr><td>246</td><td>E</td><td>E</td></tr><tr><td>303</td><td>T</td><td>T</td></tr><tr><td>364</td><td>M</td><td>V (0.52), M (0.48)</td></tr><tr><td>367</td><td>R</td><td>R (0.36), H (0.48), C (0.16)</td></tr><tr><td rowspan="4">31.0280</td><td>7</td><td>L</td><td>L (0.52), I (0.48)</td></tr><tr><td>107</td><td>N</td><td>N (0.48), S (0.52)</td></tr><tr><td>110</td><td>I</td><td>I (0.48), S (0.52)</td></tr><tr><td>120</td><td>A</td><td>A</td></tr></tbody></table>

<table><caption>Table 5 Amino acid variations in *Leishmania killicki* and *Leishmania tropica* sequences at the seven loci assessed by MLST (Continued)</caption><tr><td></td><td></td><td></td><td></td></tr><tr><td>187</td><td>I</td><td>I (0.36), V (0.64)</td><td></td></tr><tr><td>216</td><td>A</td><td>A</td><td></td></tr><tr><td>229</td><td>T</td><td>T (0.88), A (0.12)</td><td></td></tr><tr><td>239</td><td>D</td><td>D (0.92), G (0.08)</td><td></td></tr><tr><td>417</td><td>E</td><td>E</td><td></td></tr><tr><td>553</td><td>Q</td><td>Q (0.92), K (0.08)</td><td></td></tr><tr><td>649</td><td>F</td><td>F (0.92), L (0.08)</td><td></td></tr><tr><td>717</td><td>S</td><td>S</td><td></td></tr><tr><td>807</td><td>V</td><td>V</td><td></td></tr><tr><td rowspan="5">31.2610</td><td>133</td><td>L</td><td>L</td></tr><tr><td>162</td><td>A</td><td>A</td></tr><tr><td>210</td><td>A</td><td>A</td></tr><tr><td>327</td><td>I</td><td>I</td></tr><tr><td>368</td><td>L</td><td>P (0.68), L (0.32)</td></tr></table>

MON-137 (n = 2), MON-200 (n = 1), MON-264 (n = 1) and MON-265 (n = 1) (Table 1).

## Sequence analysis

The sequences of the *L. killicki* (n = 34) and *L. tropica* (n = 14) strains were submitted to GenBank (accession numbers from KM085998 to KM086333). The sizes of the seven loci under study were identical to those reported by El Baidouri et al. [31], except for locus 12.0010 (only 579 pb instead of 714 pb), leading to a total length of 4542 pb for the concatenated sequences (Table 3). All chromatograms were clearly readable. Polymorphic sites (PS) and heterozygous sites (HS), which corresponded to ambiguous positions with two peaks, were easily identified. No tri-allelic site was detected.

## Genetic polymorphisms in *L. killicki* and in *L. tropica*

Ninety-five (2.09%) PS of which 59 (1.3%) were heterozygous positions (HS) were identified in the 60 *L. killicki* and *L. tropica* strains. The number of PS varied from six (1.23%) for locus 31.2610 to 21 (2.6%) for locus 31.0280 (Table 3).

In the *L. killicki* strains, 11 (0.24%) PS were identified and they corresponded only to HS. Locus 31.2610 was the most polymorphic with three (0.61%) PS, whereas locus 12.0010 had none. In the *L. tropica* strains, 87 (1.91%) PS among which 48 (1.06%) HS were found. The number of PS varied from six (1.23%) for locus 31.2610 to 19 (2.36%) for locus 31.0280 (Table 4).

Assessment of the presence of mutations in the seven loci under study in the *L. killicki* and *L. tropica* (heterozygous mutations were excluded from the analysis)
identified 55 mutations of which 29 were silent substitu-
tions and 26 resulted in altered amino acid residues
(Table 5). All *L. killicki* mutations corresponded to a sin-
gle amino acid change. Conversely, in the *L. tropica*
strains, mutations could lead to more than one amino
acid change.

Phylogenetic analysis of *L. killicki*

In total, 32 different haplotypes were identified: 10 for the 35 *L. killicki* strains and 22 for the 25 *L. tropica* strains. Twenty-six haplotypes were unique (eight for *L. killicki* and 18 for *L. tropica*) and the two taxa did not share any haplotype. The *L. killicki* MON-317 (strain LEM6173) had its own haplotype (Table 6). The Bayesian consensus tree using 32 strains representing all the identified haplotypes was constructed based on the concatenated sequences and duplicated nucleotide sites to avoid the loss of genetic information in ambiguous positions (Figure 1). The phylogenetic tree showed that *L. killicki* formed a separate group, although it belonged to the *L. tropica* complex. The *L. killicki* cluster showed low structuring and low polymorphism (see Figure 1). In contrast, *L. tropica* was highly polymorphic with strong structuring supported by high bootstrap values and some links with the country of origin, especially for strains from Kenya and Yemen. The larger and main clade was composed by all the Moroccan strains with the addition of other strains from other countries.

Discussion

Previous studies using a small number of strains and differ-
ent molecular tools and analytic methods [9,12,31,38,40]
included *L. killicki* in the *L. tropica* complex, except the
study by Rioux and Lanotte [39] in which it was consid-
ered as a separate phylogenetic complex. The present
study wanted to improve the knowledge on *L. killicki*
phylogeny and its evolutionary history relative to *L. tro-
pica* by using a larger sample of *L. killicki* strains from
different countries.

The phylogenetic analyses performed in this study
confirm the position of this taxon within *L. tropica* in
agreement with the previous biochemical and genetic
findings. The close phylogenetic relationships between
these taxa were also confirmed by the low number of
polymorphic sites compared to those found between
various *Leishmania* species [30,31]. The phylogenetic
tree shows that *L. killicki* creates an independent group
within *L. tropica* with high bootstrap value and no com-
mon haplotypes between them. Nevertheless, this taxon
is included in the *L. tropica* complex and our data indi-
cate that the species status of *L. killicki* is not justified.
Furthermore, based on the *L. tropica* complex diversity
and the multiple monophyletic branches in this complex,
if *L. killicki* were to be considered as a species, the

<table><caption>**Table 6 Haplotypes of Leishmania killicki and Leishmania tropica strains based on the concatenated sequences of the seven loci used for the MLST analysis**</caption>
  <thead>
    <tr>
      <th>Taxon</th>
      <th>Haplotype</th>
      <th>Number of strains</th>
      <th>CNRL code</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td rowspan="11">L. killicki</td>
      <td rowspan="11">1</td>
      <td rowspan="11">24</td>
      <td>LEM95, LEM160, LEM163, LEM174, LEM179, LEM183, LEM184, LEM186</td>
    </tr>
    <tr>
      <td>LEM194, LEM904, LEM1013, LEM4390, LEM4741, LEM4995, LEM6226, LEM6229</td>
    </tr>
    <tr>
      <td>LEM6230, LEM6231, LEM6404, LEM6416, LEM6418, LEM6420, LEM6421, LEM6423</td>
    </tr>
    <tr>
      <td>LEM0177, LEM0182, LEM5420</td>
    </tr>
    <tr>
      <td>LEM180</td>
    </tr>
    <tr>
      <td>LEM181</td>
    </tr>
    <tr>
      <td>LEM185</td>
    </tr>
    <tr>
      <td>LEM193</td>
    </tr>
    <tr>
      <td>LEM6173</td>
    </tr>
    <tr>
      <td>LEM6175</td>
    </tr>
    <tr>
      <td>LEM6227</td>
    </tr>
    <tr>
      <td>LEM6228</td>
    </tr>
    <tr>
      <td rowspan="15">L. tropica</td>
      <td>11</td>
      <td>2</td>
      <td>LEM1591, LEM1922</td>
    </tr>
    <tr>
      <td>12</td>
      <td>1</td>
      <td>LEM1623</td>
    </tr>
    <tr>
      <td>13</td>
      <td>2</td>
      <td>LEM1663, LEM5506</td>
    </tr>
    <tr>
      <td>14</td>
      <td>1</td>
      <td>LEM1778</td>
    </tr>
    <tr>
      <td>15</td>
      <td>2</td>
      <td>LEM1879, LEM1918</td>
    </tr>
    <tr>
      <td>16</td>
      <td>1</td>
      <td>LEM1880</td>
    </tr>
    <tr>
      <td>17</td>
      <td>2</td>
      <td>LEM2017</td>
    </tr>
    <tr>
      <td>18</td>
      <td>1</td>
      <td>LEM3015</td>
    </tr>
    <tr>
      <td>19</td>
      <td>1</td>
      <td>LEM5276</td>
    </tr>
    <tr>
      <td>20</td>
      <td>1</td>
      <td>LEM5295</td>
    </tr>
    <tr>
      <td>21</td>
      <td>1</td>
      <td>LEM5283</td>
    </tr>
    <tr>
      <td>22</td>
      <td>1</td>
      <td>LEM617</td>
    </tr>
    <tr>
      <td>23</td>
      <td>1</td>
      <td>LEM2869</td>
    </tr>
    <tr>
      <td>24</td>
      <td>1</td>
      <td>LEM1904</td>
    </tr>
    <tr>
      <td>25</td>
      <td>1</td>
      <td>LEM0955</td>
    </tr>
    <tr>
      <td>26</td>
      <td>1</td>
      <td>LEM1015</td>
    </tr>
    <tr>
      <td>27</td>
      <td>1</td>
      <td>LEM3322</td>
    </tr>
    <tr>
      <td>28</td>
      <td>1</td>
      <td>LEM2001</td>
    </tr>
    <tr>
      <td>29</td>
      <td>1</td>
      <td>LEM3956</td>
    </tr>
    <tr>
      <td>30</td>
      <td>1</td>
      <td>LEM1824</td>
    </tr>
    <tr>
      <td>31</td>
      <td>1</td>
      <td>LEM2313</td>
    </tr>
    <tr>
      <td>32</td>
      <td>1</td>
      <td>LEM2454</td>
    </tr>
  </tbody>
</table>

*L. tropica* complex would be composed of many spe-
cies. Therefore, we suggest calling this taxon *L. killicki*
(synonymous *L. tropica*) as it was previously done before
for *L. chagasi* (synonymous *L. infantum*) [9,11,49,50].
<figure><img src="image_3.png" /></figure>

Further epidemiological and clinical studies in the different countries where this taxon has been reported will say whether the *L. killicki* denomination should be maintained.

From an evolutionary point of view, these data strongly suggest that *L. killicki* descends from *L. tropica* following only one founder event. This hypothesis is supported by the structure of the phylogenetic tree and by biochemical and genetic data. Indeed, the isoenzymatic characterization showed a low number of *L. killicki* zymodemes compared to those of *L. tropica*. This low polymorphism in *L. killicki* was confirmed by the low numbers of PS, HS and haplotypes and amino acid variations in the sequence of the different strains. The analysis of the phylogenetic tree suggests that *L. killicki* could have originated from an *L. tropica* ancestor from the Middle East. This ancestor would have separated into *L. tropica* in Morocco and other countries and into *L. killicki* in several other countries.

Finally, the lack of shared haplotypes and the identification of the new zymodeme MON-317 and its own haplotype suggest that *L. killicki* is now evolving independently from *L. tropica*, probably due to their different transmission cycles (zoonotic for *L. killicki* [51,52] and both anthroponotic and zoonotic for *L. tropica* [45,53,54]).

As the *L. killicki* strains showed low structuring and low polymorphism, we could not determine the precise evolutionary history of this taxon and particularly the country in which it emerged for the first time. Based on the epidemiological data, the higher genetic diversity and especially the relatively high number of described cases in Tunisia compared to the other countries [35,55-57], it is likely that this taxon has emerged for the first time in Tunisia and then has spread in other North-African countries. Nevertheless, this should be further investigated.

## Conclusion

The present work brings new insights into the evolutionary history of *L. killicki* and its taxonomic classification relative to *L. tropica*. However, more investigations need to be carried out on this model and particularly a detailed population genetics analysis to better understand the epidemiology and population dynamics of this parasite in comparison to *L. tropica*.

## Competing interests

The authors declare that they have no competing interests.

## Authors' contributions

This work was accomplished by the contribution of all authors cited: DC carried out all the technical experiments and drafted the manuscript; CR contributed to the analysis, interpretation of data and was involved in the
revision of the manuscript; ALB participated in the analysis, interpretation of data and has contributed to the draft and revision of the manuscript; NH has been involved in the revision of the manuscript; PL and LT have participated in the technical experiments; FEB has contributed to data analysis; KJ and ZH have participated in sample collection; JPD was involved in the revision of the manuscript; HB directed the study; FP directed the study, revised and approved the manuscript. All authors read and approved the final manuscript.

## Acknowledgements

This study was funded by the Institut Français de Coopération de Tunisie fellowship for PhD students. We thank Elisabetta Andermarcher for editing the English.

## Author details

¹Département de Biologie Clinique B, Laboratoire de Parasitologie-Mycologie Médicale et Moléculaire (code LR12ES08), Faculté de Pharmacie, Université de Monastir, Monastir, Tunisia. ²Département de Parasitologie-Mycologie, Centre National de Référence des Leishmanioses, CHRU de Montpellier, Université de Montpellier, France, 39 avenue Charles FLAHAULT, 34295 Montpellier Cedex 5, France. ³UMR MIVEGEC (CNRS 5290-IRD 224-Université de Montpellier), Montpellier 34394, France. ⁴School of Life Sciences University of Lincoln, Joseph Banks Laboratories, Green Lane, Lincoln LN6 7DL, UK. ⁵Laboratoire d'éco-épidémiologie Parasitaire et Génétique des Populations, Institut Pasteur d'Algérie, Dely Ibrahim, Algeria.

Received: 18 September 2014 Accepted: 21 March 2015

Published online: 01 April 2015

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