https://orcid.org/0000-0003-2007-7198
Figures
Abstract
Background
Leishmania is a neglected tropical parasite that may affect millions of people worldwide, causing infections that range from subclinical to life-threatening in many animal species, including humans. The four subgenera described (i.e., Leishmania, Mundinia, Sauroleishmania and Viannia) exhibit distinct biological and ecological characteristics. The genus likely originated during the Mesozoic era, with hypotheses suggesting a Palearctic, Neotropical, or Supercontinent origin. Understanding the evolutionary history of Leishmania parasites may clarify host specificity, geographic distribution, and vector associations across subgenera.
Methodology and key findings
In this study, we analyzed Leishmania evolutionary associations with vertebrate hosts as well as sand fly vectors using a combination of cophylogenetic and maximum parsimony approaches. A significant phylogenetic congruence was found between Leishmania and its vertebrate hosts using PACo (p = 0.0001, m2XY=0.5419) and ParaFit (p = 0.0034, ParaFit Global statistic = 0.012), with several host-switching and duplication events (p = 0.0298), particularly involving Leishmania-humans associations. A cospeciation event was also predicted at a higher taxonomic level, corresponding to the divergence of mammals from reptiles and the subsequent separation of the Leishmania, Viannia and Mundinia subgenera from Sauroleishmania. Similarly, significant cophylogenetic signals were observed between Leishmania and sand fly hosts using PACo (m2XY=1.3988, p = 0.0001) and ParaFit (ParaFit Global = 0.062, p = 0.0001) functions. Cospeciation events were again predicted at higher taxonomic levels in the divergence of New World and Old World sand flies, with Viannia species and Leishmania, Sauroleishmania and Mundinia species associated with the latter (p = 0.01).
Conclusions
Our findings support the Supercontinent hypothesis and emphasize the relevance of the historical biogeography in Leishmania diversification. This comprehensive cophylogenetic analysis enhances our understanding of Leishmania origins and diversification, offering insights into host specificity, vector adaptation, and the long-term maintenance of its digenetic life cycle.
Author summary
This study highlights cospeciation as central for Leishmania parasites and sand fly coevolution, while host-switching and duplication are more common with vertebrates. Strong phylogenetic congruence was observed between the Viannia subgenus and humans, the Sauroleishmania subgenus and lizards, and the Leishmania subgenus and rodents, supporting both recent and ancient co-divergence. Our findings support the Supercontinent hypothesis and emphasize the role of historical biogeography in Leishmania diversification. Mundinia species demonstrated adaptability to both sand flies and biting midges, suggesting a broader vector range. Overall, this analysis clarifies Leishmania origins, host specificity, and vector adaptation, leading to the development of targeted interventions to reduce infections in vulnerable groups
Citation: Rojas A, Mendoza-Roldan JA, Alfaro-Segura P, Solano-Barquero A, Otranto D (2025) Partners in time: Cospeciation and host-switching shape the evolution of Leishmania parasites. PLoS Negl Trop Dis 19(12): e0013814. https://doi.org/10.1371/journal.pntd.0013814
Editor: Sarman Singh, Advanced Centre for Chronic and Rare Diseases, INDIA
Received: July 17, 2025; Accepted: December 2, 2025; Published: December 23, 2025
Copyright: © 2025 Rojas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Data can be accessed on Zenodo repository at the following link once the article has been published https://doi.org/10.5281/zenodo.16039991.
Funding: This study was supported by the EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT to AR, JAM-R and DO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Leishmania is a genus of vector-borne parasites (family Trypanosomatidae) comprising Leishmania, Sauroleishmania, Mundinia and Viannia subgenera. Of the 53 Leishmania spp. described [1], approximately 20 are considered pathogenic to humans [2], causing neglected tropical diseases in 98 countries. About 350 million people are at risk of infection with 1.3 million cases occurring every year [2] in tropical and subtropical regions [3]. Moreover, this parasite can infect a myriad of mammal hosts with dogs, cats, rodents and other wild animal species acting as main reservoirs of the most pathogenic Leishmania spp. to humans [4].
Leishmaniasis is a zoonotic infection that occurs when, during a bloodmeal of a dipteran sand fly, parasites’ promastigotes are injected into host tissues. These then develop into their non-flagellated stage in the reticuloendothelial cells [4]. In particular, while Leishmania, Mundinia and Viannia subgenera have been associated with mammal hosts, Sauroleishmania is mostly linked to herpetofauna. These parasites have different niches inside their vertebrate hosts, leading to asymptomatic infections, cutaneous manifestations and even life-threatening conditions [5,6]. Accordingly, five main clinical presentations are recognized in humans: i) visceral leishmaniasis or kala-azar (VL) mostly caused by Leishmania (Leishmania) infantum; ii) post kala-azar dermal leishmaniasis (PKDL) produced mainly by Leishmania (Leishmania) donovani, but also by L. infantum [7]; iii) cutaneous leishmaniasis (CL) associated with several Leishmania spp. of the Viannia, Mundinia or Leishmania subgenus, including L. tropica [8]; iv) diffuse cutaneous leishmaniasis (DCL) produced by different species of the Leishmania subgenus, including Leishmania mexicana or Leishmania aethiopica [9]; and v) mucocutaneous leishmaniasis (MCL) induced by several species of the Viannia subgenus [8]. On the other hand, little is known about the clinical consequences of Sauroleishmania species in their reptilian hosts. Experimental infections with Leishmania (Sauroleishmania) adleri and Leishmania (Sauroleishmania) tarentolae have been conducted in various reptile species with no pathological effects observed [10], suggesting a long-term adaptation with these hosts.
Biogeographical segregation is apparent in Leishmania spp. distribution, since the Viannia subgenus is present predominantly in the New World and infects the hindgut of New World sand flies [4,11], whereas species of the Sauroleishmania subgenus have been restricted to the Old World [12], with few reports in South America [13]. Parasites of the Sauroleishmania subgenus use Old World sand flies such as Sergentomyia and Phlebotomus as invertebrate hosts [14]. On the other hand, the Mundinia subgenus is less understood, and has been reported in Australia, Ghana, Thailand and Martinique [15] where it is vectored by sand flies and biting midges. Finally, species of the Leishmania subgenus infect mostly the midgut of Old World sand flies but have also adapted to New World sand fly species [16]. The Leishmania subgenus mostly circulates in the Old World, except for L. mexicana, Leishmania amazonensis and L. infantum [12]. Interestingly, two variants of L. infantum have been described: one from the Old World and another from the New World [17]. The latter was likely introduced to the Americas 500 years ago, during the European colonization [18,19]. Therefore, an important question in Leishmania biology concerns its origin and evolution, which in turn, could provide insights into its association with specific vertebrate and invertebrate hosts, its geographic distribution, as well as predicted expansion in the Anthropocene, which is characterized by a changing environment.
According to the results of isoenzyme analyses, the origin of the Leishmania genus occurred during the Mesozoic era (i.e., before the separation of Gondwana) [20], at the same time that the divergence in sand flies of the New World and Old World sand flies occurred (i.e., 180–200 Million years ago (Mya)) [12]. More recent research using 200,000 informative sites from the parasite’s genomes, estimated the divergence of this genus from other trypanosomatids, such as Trypanosoma cruzi and Trypanosoma brucei, to have occurred 90–100 Mya [21]. Therefore, several hypotheses have been raised regarding the origin of Leishmania. Firstly, the “Palearctic hypothesis” suggests that Leishmania originated from Cretaceous lizards found in the Palearctic region, with Sauroleishmania proposed as the ancestral lineage to all other species [22]. In this scenario, Cretaceous lizards migrated to the Nearctic and Neotropics through land bridges and is supported by biogeographic and fossil data. The “Neotropical hypothesis” is based on sequence-based phylogenies and indicates that a predecessor of L. donovani and Leishmania major evolved from monoxenous parasites of insects in South America (46–36 Mya). In this hypothesis, the Viannia subgenus is ancestral to all species since the Leishmania subgenus may have originated in the Neotropics and spread approximately 24–14 Mya through the Bering Land Bridge [23]. According to the latter hypothesis, Sauroleishmania would have originated from mammal-associated Leishmania spp. Finally, the “Supercontinent or Multiple Origins hypothesis” indicates that Leishmania genus originated in Gondwana and later split into Viannia to the Neotropics and Leishmania and Sauroleishmania in Africa, approximately 90–100 Mya [24]. This is supported by multi-locus phylogenetic reconstructions [21]. Given that multiple lines of evidence have produced conflicting hypotheses about the origin of Leishmania, it is pertinent to examine the relationships between this parasite and its hosts from a coevolutionary perspective.
This study explores the evolutionary relationships between Leishmania and its vertebrate and invertebrate hosts by using three different strategies: i) cophylogenetic analysis based on patristic distances from host and parasite phylogenies, ii) cophylogenetic analysis using phenetic distances and iii) estimation of the coevolutionary events occurring between Leishmania and its vertebrate and invertebrate hosts. Our aim is to evaluate whether these biological and ecological relationships are supported by statistical testing of global fit and event-based methods. Understanding the evolutionary history of Leishmania is crucial for informing public health strategies aimed at controlling its spread.
Materials and methods
Sequence availability analysis
To determine which DNA markers are most widely represented among Leishmania species in GenBank, sequences originating from various cellular sources were systematically retrieved from database records. This approach facilitated the selection of markers for subsequent phylogenetic analysis. Leishmania sequences of nuclear, mitochondrial, ribosomal and kinetoplast origin were mined from GenBank under the following search criteria: RNA polymerase subunit II (POLR2), internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2), small subunit ribosomal, 18S, 5.8S, minicircle kinetoplast, cytochrome b, cytochrome oxidase subunit 1 (cox1) and heat shock protein 70 (hsp70), all listed in S1 Fig. These sequences were searched for parasites of the subgenus Leishmania (i.e., Leishmania aethiopica, Leishmania amazonensis, Leishmania arabica, Leishmania donovani, Leishmania ellisi, Leishmania garnhami, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica and Leishmania turanica), Mundinia (i.e., Leishmania chancei, Leishmania enriettii, Leishmania macropodum, Leishmania martiniquensis, Leishmania orientalis and Leishmania procaviensis), Sauroleishmania (i.e., Leishmania adleri, Leishmania gymnodactyli, Leishmania hoogstraali and Leishmania tarentolae) and Viannia (i.e., Leishmania braziliensis, Leishmania guyanensis, Leishmania lainsoni, Leishmania naiffi, Leishmania panamensis, and Leishmania peruviana). In addition, the number of genomes available for each of the species above mentioned was also surveyed in GenBank database. The number of sequences and genomes for each Leishmania spp. were recorded and visualized in PowerBI (Microsoft, USA). All sequences analyzed in this study were exclusively obtained from the GenBank database. No new sequence data were generated as part of this research. Additionally, information pertaining human or animal patients was not included, as the primary objective was to investigate the phylogenetic relationships between Leishmania parasites and their respective hosts.
Phylogenetic analysis
Phylogeny with 18S, kDNA and POLR2.
By running the sequence availability survey, it was found that POLR2, ITS1 and 18S were the most represented molecular markers among Leishmania spp., as well as sequences most frequently aligned as a block. Therefore, Bayesian Inference phylogenetic trees were built with all those Leishmania sequences larger than 300 bp derived from different vertebrate hosts and that aligned as a block with other sequences as summarized in Fig 1. Sequences included in the analysis took into account all those Leishmania spp. for which a specific host was reported, fragments were larger than 300 bp and aligned to the fragment of majority of sequences. Moreover, sequences of the same Leishmania spp. were added if they met the above criteria and several different hosts were reported in GenBank.
Created in BioRender. Mora, J. (2026) https://BioRender.com/is02atg.
Two datasets were analyzed: i) Leishmania sequences isolated from vertebrate hosts as verified from metadata in GenBank and ii) Leishmania sequences associated with sand fly hosts, which have been reported in the literature as competent vectors. This search strategy was applied to sand fly hosts since very few Leishmania spp. have specific sand fly hosts annotated in their GenBank metadata. In addition, Leishmania vertebrate and invertebrate hosts cytochrome b sequences larger than 300 bp were retrieved from GenBank, for constructing the host’s phylogenetic trees.
Sequences were aligned in MEGA7 [25] using the CLUSTALW algorithm [26] and the best nucleotide substitution model was estimated in JModelTest2 [27] using 11 substitution schemes, four processors, including invariant sites and gamma distribution, Maximum Likelihood optimized and Nearest Neighbour Interchange base tree search. Endotrypanum monterogeii (AF009158) was used as an outgroup for the Leishmania spp. POLR2 tree, Leptomonas costoris (JQ359728) for the 18S, Trypanosoma cruzi (AF401100) for the kDNA, Petromyzon marinus (NC_001626) for the vertebrate host tree and Culex quinquefasciatus (MN540366) for the sand fly invertebrate host trees. The Jukes-Cantor with gamma distribution was chosen for the 18S dataset, the Tamura-Nei with gamma distribution for the POLR2 alignment and the Tamura 3-parameter with gamma distribution for the kDNA. In addition, the Time-reversible with gamma distribution was selected for the sand fly phylogenetic tree, whereas the General Time Reversible with gamma distribution was chosen for the vertebrate host tree. Bayesian Inference phylogenetic trees of Leishmania spp. and their associated hosts were built using the BEAST2.5 package [28]. Briefly, alignments were uploaded in BEAUTi v2.6.7 and 108 Monte Carlo Markov Chains (MCMC) were set with sampling every 103 trees and 25% of the trees were discarded as burning. Chain convergence was verified with effective sample sizes of each parameter larger than 300 in Tracer v1.7.2. Finally, trees were compiled in TreeAnnotator v2.6.4 and visualized in FigTree v1.4.4.
Cophylogenetic analysis
Two distinct strategies were applied to assess the congruence between Leishmania parasites and their vertebrate and sand fly hosts using global fit methods (Fig 1), namely the Procrustes Approach to Cophylogeny (PACo) [29] and ParaFit [30]. These methods typically detect cophylogenetic signals, indicating that closely related parasites tend to associate with closely related hosts, though not necessarily in strict phylogenetic congruence. Additionally, we used the event-based method eMPRess [31] to infer the most likely coevolutionary events shaping the phylogenetic relationships between Leishmania species and their hosts. A limitation of the study was the use of different markers for building host and parasite phylogenies, which could affect divergence estimates. However, similar strategies have been used in other studies yielding reliable results [32,33]. In addition, only those Leishmania-host links with available POLR2 and complete information in GenBank were tested. For sand fly-associated Leishmania species, we also used Jane [34], as this algorithm accommodates parasites with multiple hosts, unlike eMPRess.
Each of these analyses was performed on three different relationships between Leishmania spp. and the following host categories, based on the available number of host-parasite associations: i) Leishmania and confirmed vertebrate hosts according to GenBank metadata, ii) Leishmania and confirmed vertebrate hosts as observed in GenBank metadata of each parasite sequence, with the addition of L. gymnodactyli and the Caspian bent-toed gecko Tenuidactylus caspius (syn. Cyrtopodion caspium); this association may be possible due to the phylogenetic closeness of this reptile with the confirmed hosts T. caspius (syn. Gymnodactylus caspius) and Paralaudakia caucasia (syn. Agama caucasica), which did not have available cytB sequences deposited in GenBank. This analysis was done to increase the number of Sauroleishmania spp. in the study, and iii) Leishmania sequences and sand fly species which have been reported as suitable hosts in the literature (S1 Table).
For PACo implementation, each Leishmania taxon and its corresponding host were assigned a unique identifier available in S1 and S2 Tables. The input included two phylogenetic trees (hosts and parasites in newick format) and a binary matrix encoding host-parasite associations (1 = associated, 0 = not associated). PACo calculated patristic distance matrices, which were then transformed into principal coordinate (PCo) matrices. The parasite PCo coordinates were superimposed onto the host PCo coordinates, which effectively controls for host phylogeny. Phylogenetic congruence was assessed using the Procrustes global fit statistic (m2XY) in asymmetrical mode (sym = FALSE). Significance was evaluated using 1,000 random permutations of the association matrix. In asymmetrical mode, significant host constraint on parasite phylogeny is inferred if fewer than 5% of the randomized m2XY values are lower than the observed value. The contribution of each host-parasite link to the global fit was determined via jackknife resampling, taking one link away from each randomization. P values lower than 0.05 regarded an overall cophylogenetic congruence in the whole dataset. Moreover, a host-parasite link with a squared residual value lower than the global fit was considered as an association with potential cophylogenetic congruence. PACo was implemented in R using the ape [35] and vegan packages [36].
ParaFit, which addresses the fourth-corner problem [30], was performed using the same input data as PACo. Patristic distance matrices were transformed into PCo ordinations and crossed with the host-parasite association matrix. A fourth-corner matrix (D) was generated to compute the ParaFitGlobal statistic. The significance of both the global statistic and individual associations (via ParaFitLink1) was tested with 9,999 permutations and the Cailliez correction for negative eigenvalues, using the Parafit function in the ape R package. Similarly, p values lower than 0.05 suggested an overall significant cophylogenetic association between the studied hosts and parasites. Sample size was not calculated since statistical significance is given by posterior probability values of phylogenetic trees, as well as randomizations and jack-knife procedures for PACo analysis.
Event-based analysis
To further investigate host-parasite coevolution, we used the event-based software eMPRess to estimate the number and type of evolutionary events (e.g., cospeciation, duplication, host switches, and losses) needed to explain the observed associations. This analysis was carried out for the host-parasite groupings with vertebrate and sand fly hosts. Event costs were defined as follows: loss = 1.00, cospeciation = 0.00, and duplication and transfer were determined based on the Costscape plot. These values corresponded to the histogram region with the highest number of most parsimonious reconstructions (MPRs) and the lowest duplication and transfer costs. Input files included.newick trees and a.mapping file of host-parasite associations. The output consisted of visualizations of host and parasite trees with potential coevolutionary scenarios. In addition to eMPRess, Jane was used for computing Leishmania-sand fly host coevolutionary events since this dataset included several parasites with multi-host associations. In this case, different Leishmania isolates were added to the analysis to avoid false failure-to-diverge estimations. Different host switch event costs (2.00, 3.00, 4.00 and 5.00) were tested in Jane while keeping the loss = 1.00, cospeciation = 0.00, and duplication = 1.00 values constant, with 300 generations and 100 population size. Significance was tested with the costs obtained in 500 solutions.
Results
Sequence availability analysis
Since 2010, a total of 26,716 articles related to Leishmania have been published in PubMed. The Leishmania subgenus accounts for 88.12% (n = 23,542) of these publications, followed by Viannia with 10.14% (n = 2,709), Sauroleishmania with 0.96% (n = 258) and Mundinia with 0.77% (n = 207) (S1a Fig and S3 Table). When considering sequence search of specific ribosomal, kDNA and nuclear markers, L. infantum had a total of 3,948 sequences deposited in GenBank (S1d Fig), followed by L. donovani (n = 2,434), L. tropica (n = 1,922), L. major (n = 1,716) and L. braziliensis (n = 1,438). Species with less than 10 ribosomal, kDNA or nuclear markers belonged mainly to the Mundinia and Sauroleishmania subgenus. Among the searched markers, the ITS1, 5.8S and kDNA had the highest number of deposited sequences (n = 3,302, 3,190 and 2,971, respectively) (S4 Table) and varied among Leishmania subgenera (S1c Fig). For instance, kDNA has been widely used in studies of the Viannia subgenus, while ITS1 has been more commonly applied to Mundinia. In contrast, ITS1 and 5.8S loci have been used in roughly equal proportions for investigating the Leishmania and Sauroleishmania subgenera.
Phylogenetic trees of 18S, ITS1 and POLR2
Phylogenetic trees shown in Fig 2 were built using 18S, kDNA and POLR2 data, since these markers had the largest number of Leishmania spp. represented in GenBank and sequences aligned as a block to the other ones Fig 2). The POLR2 dataset included 26 Leishmania spp. of the four subgenera (Fig 2a). Even though sequence OR695081 was available for L. ellisi, it could not be included in the analysis since it did not overlap with the same gene fragment. In addition, several sequences of other Leishmania spp. (e.g., L. major, L. infantum and L. aethiopica) were added in the alignment since they were obtained from different vertebrate hosts such as rodents, dogs or mustelids. The POLR2 phylogenetic tree clustered the sequences according to the Leishmania subgenera into Leishmania, Sauroleishmania, Mundinia and Viannia. The latter was basal to the other subgenera, whereas the Sauroleishmania was paraphyletic to Leishmania.
Posterior probability (PP) values lower than 0.6 are not shown. Node circle size and color are proportional to the PP. Created in BioRender. Mora, J. (2026) https://BioRender.com/is02atg.
Eighteen Leishmania spp. could be included in the phylogenetic analysis of the 18S since not all sequences overlapped as a block (i.e., L. garnhami, L. aethiopica, L. martiniquensis and L. peruviana) or were not available in GenBank for others (i.e., L. ellisi, L. killicki, L. turanica, L. chancei, L. macropodum or L. martiniquensis) (Fig 2b). Furthermore, only L. enriettii and L. siamensis for the Mundinia subgenus and six parasites of the Leishmania subgenus were available for the phylogenetic analysis. The Leishmania subgenus had a polytomy in the phylogenetic tree with the Viannia and Mundinia subgenera between them. Additionally, the Sauroleishmania subgenus was the most diverged group, whereas L. infantum and L. donovani were located basally in the tree.
The kDNA dataset included 12 Leishmania spp. that met the inclusion criteria for the analysis (Fig 2c). Unfortunately, no sequences of the Mundinia subgenus were available in GenBank, five of the Leishmania subgenus could align as a block and only one species of the Sauroleishmania (i.e., L. tarentolae) could be analyzed. The tree topology was similar to the one obtained with the POLR2, with the Viannia subgenus basal and Sauroleishmania paraphyletic to the Leishmania subgenus
The POLR2 dataset was chosen for further cophylogenetic analyses for several reasons. Firstly, it contained the highest numbers of represented Leishmania spp. Second, the obtained phylogenetic tree showed a resolution equal to other ribosomal markers and compatible with other studies [11,37]. Third, each subgenus was separated between them with robust posterior probability values and lastly, the available sequences could overlap to the majority.
Analysis with vertebrate hosts
Two analyses were conducted to confirm the cophylogenetic relationships between Leishmania spp. and their vertebrate hosts. The first analysis included 25 different Leishmania spp. and a total of 28 host-parasite links confirmed in GenBank metadata (S2a Fig). The second analysis included 26 Leishmania spp. due to the addition of L. gymnodactyli associated with the Caspian bent-toed gecko (Fig 3a) and 32 host-parasite links.
A. Tanglegram showing host-parasite associations according to GenBank metadata. B. Procrustean superimposition plot between the principal coordinates derived from patristic distances of the RNA Polymerase II of Leishmania spp. and their vertebrate host phylogenies. Each parasite and host are denoted as circles and arrow heads, respectively. Leishmania spp. are color coded according to the subgenus. Close host and parasite positions in the PCo may indicate cophylogenetic associations C. Contribution of each Leishmania-vertebrate host link to the global phylogenetic congruence. Each bar represents the squared residual of each association and are color-coded according to the Leishmania subgenus. Error bars correspond to 95% confidence intervals of the squared residuals. The median squared residual is indicated as a dotted line. Asterisks at the top of each bar represent a significant ParaFitLink1 value and daggers to significant ParaFitLink2 values. Squared residual values lower than the median squared value suggests cophylogenetic congruence between that host-parasite association. D. Coevolutionary reconstruction of the host (black lines) and parasite (blue lines) phylogenies with the lowest global cost according to eMPRess. Values in top of nodes correspond to the support of the predicted event. E. Total cost distribution of random solutions. Created in BioRender. Mora, J. (2026) https://BioRender.com/is02atg.
Confirmed Leishmania-host parasite links.
Global-fit analysis: An overall significant phylogenetic congruence was obtained between the confirmed Leishmania-vertebrate host links with a p value of 0.0001 and a m2XY=0.5109, summarized in Table 1. The superposed PCA of host and parasite genetic distances showed the Sauroleishmania spp. (i.e., L. adleri, L. hoogstraali and L. tarentolae) separated from the other species and closer to the reptile hosts Latastia longicaudata, Hemidactylus bowringii and Tarentola mauritanica, indicating a closer cophylogenetic relationship. On the other hand, species of the Leishmania, Viannia and Mundinia subgenera were located in a separate cluster with their respective mammal hosts (S2b Fig). Five host-parasite links were below the median squared residual value, specifically the associations between Homo sapiens and L. braziliensis, L. guyanensis, L. lainsoni, L. panamensis and L. peruviana. The highest squared residual values corresponded to the association between L. turanica and the great gerbil Rhombomys opimus and L. hoogstraali and the oriental leaf-toed gecko Hemidactylus bowringii (S2c Fig).
The Parafit function revealed a significant cophylogenetic relationship, with a p value of 0.005 and a ParaFitGlobal statistic of 0.009. In addition, twelve host-parasite links were considered to contribute to the significant pattern of coevolution as obtained in ParaFitLink1 (F1) p values (Additional Fig 1c). These associations concerned all those with the Viannia subgenus (n = 6/6), four out of six links of the Mundinia subgenus, one from the Leishmania subgenus (L. major with Rhombomys opimus) and one of the Sauroleishmania subgenus (L. hoogstraali with H. bowringii).
Event-based analysis: The event-based analysis using eMPREss found a significant coevolutionary association between Leishmania parasites and its vertebrate hosts mainly driven by duplication (i.e., speciation of the parasite without host divergence; Additional Fig 1d and 1e, p = 0.0396). Seventeen duplication events were predicted with high confidence, all of them concerning Leishmania spp. parasitizing humans, like L. amazonensis, L. donovani, L. mexicana, L. killicki, and species of the Mundinia (with the exception of L. procaviensis) and Viannia subgenus (S2d Fig). Host switching or transfer was calculated in 10 situations. Some examples were the potential switches of L. tarentolae from T. mauritanica to generate L. adleri in the common long-tailed lizard L. longicaudata, the transfer of L. turanica from R. opimus to the European badger Meles meles, the switch of L. major from R. opimus to humans, L. procaviensis from humans to the hyrax Procavia capensis, or L. infantum from humans to the domestic dog Canis lupus familiaris. In addition, three cospeciation events were observed, namely in the divergence of L. tarentolae with the Moorish gecko Tarentola mauritanica and L. hoogstraali and H. bowringii, and two events involving the separation of L. major from L. turanica and L. arabica with their rodent hosts. Finally, no losses were observed, meaning that hosts did not diverge without concomitant parasite speciation.
Leishmania-host parasite links with L. gymnodactyli.
Global-fit analysis: The second analysis of Leishmania spp. with its vertebrate hosts including L. gymnodactyli (Fig 3a) improved the resolution of the analysis and confirmed the results obtained in the strict vertebrate host database S2 Fig. An overall significant cophylogenetic correspondence was obtained in PACo (p = 0.0001, m2XY=0.5419) and ParaFit (p = 0.0034, ParaFit Global statistic = 0.012) (Table 1). As in Additional Fig 1, two clusters were observed in the PCoA (Fig 3b): one with Sauroleishmania spp. and its reptile hosts and a second group with Leishmania, Viannia and Mundinia spp. with its mammal hosts. The residual cost plot showed the same host-parasite links with a value lower than the median square residual value (Fig 3c), like Viannia and Mundinia spp. with their hosts, except for L. martiniquensis with humans and L. procaviensis with P. capensis, and L. gymnodactyli with T. caspius. Furthermore, the host-parasite links with the highest squared residuals values corresponded to L. aethiopica and L. procaviensis associated with the hyrax Procavia capensis. Even though L. infantum and L. tropica have also been associated with P. capensis, POLR2 sequences obtained from infected hyraxes could not be obtained.
The ParaFit function demonstrated a strong cophylogenetic relationship with 11 significant links according to the ParaFitLink1 (F1) p value and 18 with the ParaFitLink2 (F2) p values (Fig 4c). The significant links according to ParaFitLink1 corresponded to the same host-parasite links obtained for the strict vertebrate host database (S2 Fig). In addition to these, the ParaFitLink2 determined as significant host-parasite associations from the Viannia, Mundinia and Sauroleishmania and three links of the Leishmania subgenus, i.e., L. major with Meriones shawi and R. opimus and L. turanicus with R. opimus (Fig 3c).
A. Tanglegram showing possible host-parasite associations according to the literature. B. Procrustean superimposition plot between the principal coordinates derived from patristic distances of the RNA Polymerase II of Leishmania spp. and their invertebrate host phylogenies. Each parasite and host are denoted as circles and arrow heads, respectively. Leishmania spp. are color coded according to the subgenus. Close host and parasite positions in the PCo may indicate cophylogenetic associations C. Contribution of each Leishmania-invertebrate host link to the global phylogenetic congruence. Each bar represents the squared residual of each association and are color-coded according to the Leishmania subgenus. Error bars correspond to 95% confidence intervals of the squared residuals. The median squared residual is indicated as a dotted line. Asterisks at the top of each bar represent a significant ParaFitLink1 value and daggers to significant ParaFitLink2 values. Squared residual values lower than the median squared value suggests cophylogenetic congruence between that host-parasite association. D. Coevolutionary reconstruction of the host (black lines) and parasite (blue lines) phylogenies with the lowest global cost obtained with Jane taking into consideration different Leishmania spp. isolates when having multi-host parasites. Values on top of nodes correspond to the support of the predicted event E. Total cost distribution of random solutions obtained with Jane. Red dotted line indicated the cost of the analyzed solution. Animal icons were created with Biorender.com.
Event-based analysis: The coevolutionary solution provided in the analysis between Leishmania spp. with its vertebrate hosts, including L. gymnodactyli, suggested that duplication and host-switching were the most frequent events (Fig 3d). Moreover, the solution provided was significant by taking into consideration the costs obtained from 500 random samples (p = 0.0298) (Fig 3e). Duplications were predicted in seven host-parasite associations involving Leishmania parasites with humans. In addition, host switches were suggested in L. aethiopica from hyraxes to humans, L. infantum from humans to dogs or L. turanica from the European badger to rodents. Interestingly, a host switch was predicted, with L. gymnodactyli (associated with T. caspius) transitioning to become L. tarentolae (associated with T. mauritanica). Indeed, a cospeciation event resulted in the divergence between L. gymnodactyli from L. tarentolae and L. hoogstraali with their reptile hosts. In addition, a cospeciation event was predicted at a higher taxonomical level in the split of mammals from lizards with the consequent separation of Leishmania, Viannia and Mundinia subgenus from Sauroleishmania. Furthermore, another cospeciation was predicted in the divergence of L. turanica, L. aethiopica, L. major and L. infantum with domestic dogs, the European badger and hyraxes of the other Viannia, Leishmania and Mundinia spp. cospeciating with rodents and humans. One loss was estimated in L. major with the speciation of the rodents R. opimus from M. shawi, without leading to an additional Leishmania spp.
Analysis with invertebrate hosts
The study of the association between Leishmania and its invertebrate hosts (i.e., sand flies, biting midges) resulted in a strong cophylogenetic congruence. The tanglegram shows the association of Leishmania spp. distributed in the Americas, like species of the Viannia subgenus, L. infantum, L. amazonesis and L. mexicana, to the New World sand fly genera Lutzomyia, Nyssomya, Trichophoromya and Psychodopygus (Fig 4a). Conversely, biting midges were linked to the Mundinia subgenus, whereas Old World sand flies of the Phlebotomus and Sergentomyia subgenera to Sauroleishmania and Leishmania (Fig 4a).
Global-fit analysis.
As with vertebrate hosts, the PACo (m2XY=1.3988, p = 0.0001) and ParaFit functions (ParaFit Global = 0.062, p = 0.0001) showed a cophylogenetic congruence between Leishmania and invertebrate hosts (Table 1). The PCoA superimposing host and parasite genetic distances in Fig 4b mirrored the tanglegram observations and grouped host-parasite links into three clusters: i) species of the Viannia subgenus, together with L. mexicana, L. amazonensis and L. infantum associated to New World sand flies, ii) Mundinia parasites associated to mosquitoes and biting midges and iii) species of the Leishmania, Mundinia and Sauroleishmania subgenera associated to Old World sand flies. The squared residual analysis revealed eight host-parasite links below the mean squared residual value (Fig 4c). Accordingly, the association between L. aethiopica and L. killicki with Phlebotomus sergenti, L. braziliensis and L. guyanensis with Nyssomyia neivai, L. guyanensis with Nyssomyia whitmani, L. lainsoni with Trichophoromyia ubiquitalis, Leishmania naiffi with Psychodopygus squamiventris, and L. panamensis with Lutzomyia yuilli. Conversely, the highest squared residual values were obtained with L. donovani and L. adleri with Phlebotomus argentipes.
The Parafit function found 39 significant links according to the ParaFitF1 statistic: 70% (14/20) corresponding to the Leishmania subgenus, 75% (6/8) to the Mundinia, 78% (7/9) in Sauroleishmania, and 69% (11/16) of Viannia. Non-significant host-parasite corresponded to those with L. mexicana and New World sand flies Lutzomyia olmeca and Lutzomyia ayacuchensis, as well as L. amazonensis with Nyssomyia flaviscutellata, L. infantum with Lu. longipalpis and Lutzomyia cruzi, L. enriettii with Lu. longipalpis, L. adleri with P. argentipes, L. tarentolae with Sergentomyia minuta, L. braziliensis with Lutzomyia migonei, as well as L. panamensis with the sand flies Lutzomyia gomezi, Lutzomyia hartmanni, and Lutzomyia peruensis.
Event-based analysis.
Coevolutionary events were predicted first with eMPRess, which estimated 8 cospeciations, 2 duplications, 11 transfers and 15 losses (S3a Fig). These results were statistically significant (p = 0.049), meaning that the total cost of the obtained solution did not fall into other randomly calculated scenarios (S3b Fig). Interestingly, this solution failed to include those associations for multi-host Leishmania spp., like L. infantum linked with Phlebotomus ariasi, Phlebotomus perniciosus, Lu. longipalpis and Lutzomyia cruzi. For this reason, Jane algorithm was employed to accurately analyze multi-host parasites, and estimated 19 cospeciations, 9 duplications, 24 transfers and 38 losses (Fig 4d).
Cospeciation events were predicted at higher taxonomical levels in the divergence of New World sand flies from the Old World sand flies with Viannia subgenus species and Leishmania, Sauroleishmania and Mundinia subgenus species, respectively, or the divergence of Sergentomyia sand flies with L. tarentolae, L. martiniquensis and L. orientalis with the other sand fly-Leishmania links. Moreover, duplications were observed in those Leishmania spp. from the same sand flies such as L. guyanensis and L. braziliensis in Psychodopygus carrerai, or L. gymnodactyli and L. tarentolae in P. papatasi. In addition, host-switching was predicted for multi-host parasite species, including L. infantum (in P. perniciosus, Lu. longipalpis and P. ariasi), L. panamensis (from Lu. yuilli to Lu. gomezi, Lu. harmanni and Lu. trapidoi), or L. tarentolae (from P. papatasi to P. perniciosus and Sergentomyia minuta). Finally, parasite losses were estimated at higher taxonomical levels where branching of sand flies was observed without divergence of Leishmania. This solution was significant since the cost was lower than the randomly obtained solutions (Fig 4e).
Discussion
Data suggest that Leishmania spp. coevolved with their vertebrate and invertebrate hosts, being mostly characterized by events of parasite-sand fly cospeciation, as well as host switching and duplication in their associations with vertebrate hosts. Overall, findings support the Supercontinent hypothesis enhancing current understanding of Leishmania origins and diversification, with an emphasis to the adaptation occurred amongst vertebrate hosts and vectors, under the frame of a complex digenetic life cycle.
The most abundant and represented molecular markers for Leishmania (i.e., POLR2, 18S and kDNA loci) shared the same characteristics, having a general congruence (i.e., similar rate of evolution between them) in the inferred phylogenetic trees built with markers of different cellular origin (i.e., nuclear, mitochondrial and ribosomal). Choosing the right marker is essential during phylogenetic reconstructions since markers with low intra-specific variation, such as HSP70, may yield low resolution tree branching and lead to improper conclusions during cophylogenetic analyses. On the other hand, highly polymorphic loci like kDNA provide more information regarding species divergence [38]. In this context, mutation rates of POLR2 have not been reported; however, the species branching patterns in the phylogenetic trees shown in Fig 2 were comparable to those obtained with kDNA, demonstrating a similar phylogenetic resolution for both markers.
Phylogeny based on POLR2 was similar with trees constructed before [21,39,40], also, this dataset included 26 species, compared to the 18S gene, which comprised 18 species and only 12 species in the kDNA. In this study, as well as in those above referred, Leishmania and Sauroleishmania were identified as the most divergent groups. However, Viannia was basal in our phylogenetic tree, as opposed to other reports [40], where Mundinia has been basal to the other subgenera, probably due to different sequence lengths, type of dataset used and methodological approach, in each analysis. For instance, POLR2 sequences used by Kwakye-Nuako, Mosore (40) were 1,200 long, the tree built by Sapp, Low (39) concatenated other nuclear and mitochondrial markers, whereas the one by Harkins, Schwartz (21) contained 200,000 informative sites across Leishmania genomes. Nevertheless, cophylogenetic analyses consider phenetic or patristic distances between matched host and parasite taxa [29,30], therefore, the differential positioning of a cluster should not bias the results.
An overall significant phylogenetic congruence was obtained between Leishmania spp. and their vertebrate hosts, which might have derived from the cospeciation between the Sauroleishmania subgenus and lizards, and mammals with the Viannia, Leishmania and Mundinia subgenera. In particular, cophylogenetic analysis suggests a strong relationship between species of the Viannia subgenus and humans, since all host-parasite links were significant. Accordingly, humans should be the origin source of this subgenus in the New World and of the Leishmania subgenus in the Old World, representing the main reservoir of the parasites in domestic cycles [41]. Nonetheless, a comprehensive cophylogenetic analysis is impaired by the absence of POLR2 sequences of Viannia species in two-toed sloths (Choloepus hoffmanni), domestic dogs as well as opossums, all species found positive for this subgenus [42]. This cophylogenetic signal would have provided valuable insights into the role of sloths as ancient hosts of Leishmania, as the Neotropical hypothesis of Leishmania origin suggests that these animals served as the primary reservoirs of the parasites [43]. Therefore, the current findings highlight the phylogenetic congruence of Viannia parasites and humans, but their role as main reservoirs of Leishmania (Viannia) needs further investigations. Hence, reservoir competence and infectiousness studies in different animal hosts deserve to be explored [44].
In addition, data herein obtained discard the Palearctic hypothesis, which postulates that Sauroleishmania is basal to [22] and an ancestor of other Leishmania spp., according to ecological, biochemical and biogeographical evidence [45]. The above is explained by two reasons: i) POLR2 phylogeny places the Sauroleishmania cluster as a sister group to Leishmania and Mundinia, as observed in other studies [21,39,40], while our data place Viannia basal to the other groups; ii) our results show the cospeciation of lizards with L. adleri, L. tarentolae, L. gymnodactyli and L. hoogstraali, probably supported by long-term associations. Therefore, if Sauroleishmania emerged from mammals, as the Palearctic hypothesis states, the associations with herpetofauna would be recent and thus, parasites less adapted to these hosts. Nonetheless, this study and epidemiological evidence shows Sauroleishmania infecting primarily reptiles [46]. In addition, infection by this group is transient [47] and asymptomatic in rodents [48], dogs [14] and humans [49], suggesting the low adaptation of Sauroleishmania to mammal hosts.
A significant cophylogenetic signal (e.g., between L. major with M. shawi and R. opimus and between L. turanicus with R. opimus) was obtained between parasites of the Leishmania subgenus and rodent hosts. Accordingly, many rodents (subfamily: Arvicolinae, Callosciurinae, Cricitinae, Gerbillinae, Murinae and Sciurinae) may be infected by species of the Leishmania subgenus, with prevalence of up to 39.1% [50]. This may suggest that Leishmania subgenus is more established in rodents, as these were one of the first placental mammals developing during the Paleocene (~61.7-62.4 Mya), after the split of the supercontinent Gondwana in the late Mesozoic era [12,51,52]. Indeed, the Supercontinent hypothesis supports that Leishmania started infecting rodents during the Mesozoic era on the supercontinent Gondwana [53] (Fig 5). Thus, data herein reported and illustrated in Fig 5 supports this observation since long-term host-parasite associations usually have better co-adaptations, as observed in other protozoa infections [54]. Conversely, non-significant cophylogenetic relationships were obtained between species of the Leishmania subgenus and other mammalian hosts (i.e., human, mustelid, canid, felid or hyrax), which suggests more recent host-parasite relationships [12]. For instance, mustelids originated around 33 Mya [55], canids 40 Mya [56], and felids around 15–10 Mya [57], possibly indicating that the subgenus Leishmania initially infected rodents and later switched or duplicated to these other hosts, as confirmed in our study. After the split of Gondwana, approximately 180–50 Mya, species of the genus Leishmania, Sauroleishmania and Viannia suffered from geographical isolation, and therefore, started to quickly diversify over the years [53] (Fig 5).
A. During the Mesozoic era in the Jurassic period, before the breakup of the supercontinent Gondwana, Leishmania parasites may have used ancient rodent species to co-diversify, while ancient sand fly species may have emerged earlier. B. After the split of Gondwana in the Cretaceous period, different Leishmania subgenera arose, i.e., Viannia in South America, Leishmania and Sauroleishmania in Africa and Mundinia spread all over the world. With time, speciation in vertebrate hosts occurred by host-switching or duplication, where ancestral parasite species adapted to permissive hosts giving rise to new Leishmania spp. and parasites cospeciated with ancestors of the New and Old World sand flies. C. Dispersal of Leishmania spp. by using different vertebrate and invertebrate host gave rise to the colonization of new geographical regions. For instance, it has been suggested that rodents infected with L. major migrated through the Nearctic by the Bering Land Bridge to North America, originating L. mexicana in autochtonous rodent species, which further migrated to the South giving rise to L. amazonensis, L. venezuelensis or L. waltoni. Moreover, L. aethiopica was established in East Africa with hyraxes, as well as L. (Mundinia) procaviensis, and then switched to humans. Species of the Viannia subgenus spread throughout South and Central America using humans as a main reservoir, but according to other studies, sloths and porcupines may have played an important role in the maintenance of ancestral Viannia spp. In addition, species of the Sauroleishmania subgenus spread with herpetofauna to North Africa, East Asia and Europe, without having been reported in the Americas. Finally, species of the Mundinia subgenus have been described in diverse and widely separated geographical regions, suggesting that their distribution may date back to before the breakup of Gondwana. Created in BioRender. Mora, J. (2026) https://BioRender.com/jzxajbr.
The Supercontinent hypothesis is also supported by the fact that hyraxes serve as reservoirs for L. aethiopica in Ethiopia and Kenya, reinforcing the idea of an African origin for the Leishmania subgenus [24]. Indeed, a cospeciation event was predicted in our study between the hyrax P. capensis and L. aethiopica as well as L. procaviensis. The origin of hyraxes is estimated to have occurred between 30–25 Mya [58], which aligns with the divergence of L. aethiopica around 10 Mya [21] and the origin of L. procaviensis. Hyraxes have also been found with L. tropica in other countries, such as in Israel [59], which might reveal a potential host-switching from humans to these hosts. A hypothesis that needs to be confirmed as target sequences from L. tropica infecting hyraxes were not available for the study. The role of hyraxes as natural reservoirs of the parasite was also inferred by the host switching events of L. aethiopica from hyraxes to humans, though a clear cophylogenetic signal between hyraxes and L. (Leishmania) aethiopica and L. (Mundinia) procaviensis was not obtained. This may be explained by the different phylogenetic positions of these two Leishmania spp. to distinct subgenera, or to the relatively recent divergence of L. aethiopica [21], resulting in a short host-parasite coevolutionary history. These findings highlight the temporal alignment between hyrax evolution and the origin of L. aethiopica and support a broader pattern of host–parasite co-divergence.
The present study estimated that cospeciation was the primary driver for coevolution between Leishmania and sand flies with 19 calculated events, a strong cophylogenetic signal with most host-parasite links and a clear clustering of Old World sand flies with Leishmania, Sauroleishmania or Mundinia parasites, and New World sand flies with the Viannia subgenus (Fig 4b). More than 800 sand fly species have been described in the world, 464 Neotropical species [60] and 375 recognized in the Old World [61]. Sand flies exhibit clear biogeographical differences, with the genera Phlebotomus, Sergentomyia and Chinius found in the Palearctic, Africa, the Mediterranean, Asia and Australia, whereas the Lutzomyia, Brumptomyia and Warileya genera are mainly restricted to the Americas [12]. Of these, only a few sand fly species are permissive to Leishmania development, as an effect of the adaptation to specific metabolic or physiological characteristics of these arthropods, such as salivary protein families [62], or protein ligands in the sand fly midgut [63,64]. This geographical segregation and intrinsic biological differences between sand flies, may contribute to the significant cophylogenetic relationship.
According to our findings, phylogenies between Leishmania spp. and their sand fly hosts were globally congruent. The separation of New and Old World sand flies has been estimated to have occurred after the breakup of Gondwana, ~ 200 Mya, (Fig 5). However, other studies have suggested that the split of these two groups occurred before, during the Triassic period when Pangea was divided into Laurasia and Gondwana giving rise to Old and New World sand flies, respectively [65]. If this was the case, parasites of the Leishmania, Sauroleishmania or Mundinia could have used New World sand fly species as hosts and coadapted to them during the Mesozoic era. However, we found no cophylogenetic congruence between L. infantum, L. mexicana and L. amazonensis and New World sand flies according to the individual squared residual values. On the other hand, host-switching of L. infantum was estimated from Lutzomyia spp. (Lu. longipalpis and Lu. gomezi) to Phlebotomus spp. (P. perniciosus and P. ariasi), suggesting the adaptation of Leishmania subgenus first to New World sand flies. As a further support to the latter observation, the New World sand fly Lu. longipalpis has been demonstrated as a competent host of the Old World L. major, possibly facilitated by the involvement of unspecific host-parasite interactions [66]. To unravel the origin of sand flies and their potential association with Leishmania spp., further time-calibrated phylogenetic trees using additional loci or genomic data should be rigorously analyzed.
Leishmania enrietti and L. martiniquensis, belonging to the Mundinia subgenus, were found with phylogenetic congruence with biting midges such as Culicoides sonorensis and Culicoides peregrinus, as well as Forcipomyia velox. In addition, L. (Mundinia) macropodum has been reported in the literature in the biting midge Forcipomyia sp. [67] and L. orientalis in C. sonorensis [68]. The evolutionary history of Mundinia remains largely unknown due to its recent classification following its description [15]. Nevertheless, parasites may be adapted to New and Old World sand flies, as well as biting midges, because of the ancient global distribution of Mundinia since before the breakup of Gondwana [53] with the subsequent adaptation to various arthropod species (Fig 5). This may have led to the non-specific attachment of the parasites to arthropod-associated molecules in the gut, favoring some vectors over others, such as the case of L. martiniquensis, L. orientalis and L. siamensis, which successfully colonize C. sonorensis, rather than Phlebotomus argentipes [69]. Importantly, Mundinia subgenus is still unexplored in terms of the pathogenic mechanisms it induces in hosts, host-parasite interactions and its phylogeographic distribution, all of which are crucial for gaining deeper insights into the origin of Leishmania.
Overall, coevolution between Leishmania and vertebrate hosts was estimated to be mainly driven by host-switching and duplication, whereas cospeciation appeared to be the most frequent event occurring in the Leishmania-invertebrate host. Host-switching occurs when a parasite adapts to a new host species, gradually reducing its reproductive interaction with the original host [70]. This process may result from ecological fitting, where pre-existing traits allow parasites to infect new permissive hosts, leading to novel associations [71]. If the new relationship proves successful and genetic exchange with the ancestral population decreases, speciation can occur, a process that often spans hundreds of thousands of years [72]. The divergence between Paraleishmania and Euleishmania spp. was estimated at approximately 90 Mya [21], and the association with ancient rodents and sand flies dates back to the Mesozoic era, between 280–100 Mya (Fig 5) [53], a time span sufficient to account for the current diversification of Leishmania spp. On the other hand, speciation by duplication occurs without a host change, as parasites diverge within the same host due to mutational changes that, over time, lead to the formation of new taxa [73]. In this sense, some invertebrate and vertebrate hosts have been reported with various Leishmania spp. [4]. For instance, humans are proven efficient hosts, producing overall mild clinical manifestations that, depending on the Leishmania spp., usually resolve with treatment [8,74], and other animal hosts may be infected without associated clinical signs [75]. The mild clinical presentation associated to leishmaniasis may stem from the evolutionary and immunological balance between parasites and hosts, where limited damage benefits the parasite’s effective transmission, as demonstrated with L. tarentolae [76–78]. In addition, L. infantum has been associated with post-kala azar dermal signs in human patients from the Old World [79], but these manifestations have never been reported in the Americas. This suggests that additional players, such as genetic hybridization and recombination between populations [80], are taking part in host-parasite interactions together with molecular adaptations and coevolution.
Cophylogenetic analyses are subject to inherent limitations, particularly concerning the availability of GenBank sequences of Leishmania spp. Moreover, only host–parasite associations with clearly annotated host origins could be incorporated, thereby constraining the number of taxa included in the analysis. Ideally, all taxa should be included in global-fit and event-based analysis, as well as comprehensive genomic datasets from both hosts and parasites should be employed to achieve more robust evolutionary inferences, as genes may diverge at different rates [29,81]. Nonetheless, such an approach would further restrict the number of host–parasite associations available for analysis, given that numerous species have been not yet sequenced to date. In addition, having the geographical source for each Leishmania spp. clearly specified would have provided us relevant information regarding the parasite distribution. Finally, even though host and parasite phylogenies were reconstructed with markers having different evolutionary rates, global-fit methods rely on patristic distances between taxa. In both phylogenies, high branch resolution was observed, indicating that gene origin might have not influenced the results, as described in a previous study with Hepatozoon spp. Even though these limitations exist, the conclusions obtained in this study are supported by robust statistical data. Cospeciation was regarded for a long time as the sole mechanism for host-parasite coevolution and supposes the orchestrated diversification of host and parasite phylogenies [70]. The high number of cospeciation events estimated in Leishmania-sand fly phylogenies, as opposed to those with vertebrates, suggests that a combination of intrinsic and environmental factors influences parasite attachment, multiplication or transfer to the next host [4]. Moreover, host encounter rates and availability, as well as topographical and climatic conditions may have affected sand flies and ancient mammals differently [22]. In addition, another challenging aspect that may hindrance our understanding of the evolution and divergence of Leishmania, is the possibility of facultative sexual reproduction, rather than the well-known cloning [82]. This cryptic, sexual reproduction, apart from complicating the evolutionary models needed to infer the relationship between Leishmania an its vertebrate hosts and its invertebrate vector, may lead to the generation of genotypes with unpredictable phenotypes in tissue tropism, virulence, or the emergence of drug resistance [83].
Gaining knowledge of the relationships between Leishmania and specific host orders is crucial for effective disease monitoring and control. Screening parasites in sympatric and phylogenetically related species will facilitate the identification of new hosts that may serve as reservoirs or vectors of Leishmania, thereby strengthening epidemiological surveillance and reducing the risk of parasite spread into new geographical areas or populations. Altogether, this study demonstrates that, despite millions of years of evolutionary divergence, Leishmania remains a highly adaptable parasite, capable of overcoming both biological and environmental challenges to maintain its digenetic life cycle. This comprehensive cophylogenetic analysis enhances our understanding of Leishmania origins and diversification, offering valuable insight into host specificity, vector adaptation, and the long-term maintenance of its digenetic life cycle, leading to the development of targeted interventions to mitigate the impact of Leishmania infections in at-risk populations.
Supporting information
S1 Table. List of sand fly species that have been reported as suitable hosts for Leishmania spp. with the codes used for subsequent analyses.
https://doi.org/10.1371/journal.pntd.0013814.s001
(XLSX)
S2 Table. List of Leishmania spp. with their strain codes and GenBank accession numbers that have been isolated from various vertebrate species.
These species are accompanied by the codes used for subsequent analyses.
https://doi.org/10.1371/journal.pntd.0013814.s002
(XLSX)
S3 Table. Number of scientific publications indexed in Pubmed according to Leishmania spp. from 2010 to 2024.
https://doi.org/10.1371/journal.pntd.0013814.s003
(XLSX)
S4 Table. Number of genomes and sequences of various nuclear, mitochondrial and ribosomal markers associated with different Leishmania spp.
https://doi.org/10.1371/journal.pntd.0013814.s004
(XLSX)
S1 Fig. Analysis of the Leishmania spp. sequence availability on GenBank to determine the most suitable markers to conduct the analysis.
A. Number of publications using Leishmania keywords according to year and subgenus. B. Number of genomes available (light blue bars), total number of sequences (blue lines) and total number of publications (orange lines) according to Leishmania spp. C. Number of deposited sequences according to molecular marker and Leishmania subgenus. D. Number of deposited sequences according to molecular marker and Leishmania spp.
https://doi.org/10.1371/journal.pntd.0013814.s005
(TIF)
S2 Fig. Global-fit and event-based cophylogenetic analysis between Leishmania spp. and its annotated vertebrate hosts.
A. Tanglegram showing host-parasite associations according to GenBank metadata. B. Procrustean superimposition plot between the principal coordinates derived from patristic distances of the RNA Polymerase II of Leishmania spp. and their vertebrate host phylogenies. Each parasite and host are denoted as circles and arrow heads, respectively. Leishmania spp. are color coded according to the subgenus. Close host and parasite positions in the PCo may indicate cophylogenetic associations. C. Contribution of each Leishmania-vertebrate host link to the global phylogenetic congruence. Each bar represents the squared residual of each association and are color-coded according to the Leishmania subgenus. Error bars correspond to 95% confidence intervals of the squared residuals. The median squared residual is indicated as a dotted line. Asterisks at the top of each bar represent a significant ParaFitLink1 value and daggers to significant ParaFitLink2 values. Squared residual values lower than the median squared value suggests cophylogenetic congruence between that host-parasite association. D. Coevolutionary reconstruction of the host (black lines) and parasite (blue lines) phylogenies with the lowest global cost according to eMPRess. Values in top of nodes correspond to the support of the predicted event. E. Total cost distribution of random solutions. Created in BioRender. Mora, J. (2026) https://BioRender.com/is02atg.
https://doi.org/10.1371/journal.pntd.0013814.s006
(TIF)
S3 Fig. Event-based analysis of Leishmania spp. and its possible invertebrate hosts according to the literature.
A. Coevolutionary reconstruction of the host (black lines) and parasite (blue lines) phylogenies with the lowest global cost obtained with eMPRess. Values in top of nodes correspond to the support of the predicted event. B. Total cost distribution of random solutions obtained with eMPRess. Red dotted line indicated the cost of the analyzed solution.
https://doi.org/10.1371/journal.pntd.0013814.s007
(TIF)
Acknowledgments
The authors would like to thank Dr. Ran Libeskind-Hadas for his help with the analysis of Leishmania spp. associated with sand flies using the Jane software.
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