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All issues Volume 30 (2023) Parasite, 30 (2023) 18 Full HTML
Open Access
Issue
Parasite
Volume 30, 2023
Article Number 18
Number of page(s) 10
DOI https://doi.org/10.1051/parasite/2023018
Published online 24 May 2023

© S. Zapata et al., published by EDP Sciences, 2023

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Leishmaniases are widespread vector-borne diseases caused by about 20 species [32] of the genus Leishmania, transmitted by the bite of Phlebotomine sandflies. They exhibit three main clinical forms: visceral (VL), cutaneous (CL), and mucocutaneous (MCL) leishmaniasis, with a global annual incidence estimated to be between 0.7 and 1 million new cases [9]. In the Americas, 15 species of Leishmania are known to affect humans [17]. Among these species, only L. infantum is responsible of VL in the New World, with an incidence estimated between 4,500 and 6,800 cases per year. The incidence of both CL and MCL is much higher: between 187,200 and 300,000 new cases yearly [9], mostly caused by L. braziliensis. Sandfly vectors have been identified for 12 of these 15 species, with the remaining three (L. venezuelensis, L. lindenbergi, and L. martiniquensis) not having proven vectors [2, 14, 29, 32].

Due to long co-evolution [2, 32], there is a strong link between the Leishmania species and the groups of sandflies transmitting them. In the Old World, L. infantum is mostly transmitted by sandflies belonging to the subgenus Larroussius, whereas L. major is transmitted by members of the subgenus Phlebotomus. In the New World, all the proven vectors of VL belong to Lutzomyiina, whereas most of the vectors of CL and MCL belong to Psychodopygina, especially the genera Psychodopygus and Nyssomyia, the main vectors of L. braziliensis (Table 1).

Table 1

Proven vectors of American Leishmania according to Killick-Kendrick (1990), Maroli et al. (2013) and Akhoundi et al. (2016).

The New World Phlebotomine sandflies include more than 500 species distributed throughout North, Central, and South America [25]. Sandfly systematics, established based on morphological characters, were for a long time divided into four genera: Warileya Hertig, 1948, Hertigia Fairchild, 1949, Brumptomyia França & Parrot, 1921, and Lutzomyia França, 1924 [47]. A phylogenetic classification, also based on morphological characters was proposed in 1995 by Galati [22], which has since been adopted by most medical entomologists and epidemiologists. It divides the Phlebotomine sandflies into two tribes, Hertigiini Abonnenc & Léger, 1976 and Phlebotomini Rondani & Berte, 1840 [23, 25]. The Hertigiini is a small tribe that includes only nine New World species [4]. It has no proven role in the transmission of Leishmania despite the discovery of DNA corresponding to Leishmania (Viannia) sp. in the anthropophilic Warileya rotundipennis [35]. On the other hand, the Phlebotomini tribe includes all the New World vectors. It corresponds to approximately 500 species divided into four subtribes and 20 genera (Fig. 1): i) Brumptomyiina (Brumptomyia, Oligodontomyia Galati, 1995), ii) Sergentomyiina partim (Deanemyia Galati, 1995 and Micropygomyia Barretto, 1962), iii) Lutzomyiina (Sciopemyia Barretto, 1962, Lutzomyia, França, 1924, Migonemyia Galati, 1995, Pintomyia Costa Lima, 1932, Dampfomyia Addis, 1945; Expapillata Galati, 1995, Pressatia Mangabeira, 1942, Trichopygomyia Barretto, 1962 and Evandromyia Mangabeira, 1941), and iv) Psychodopygina (Psathyromyia Barretto, 1962, Viannamyia, Mangabeira 1941, Martinsmyia Galati, 1995, Bichromomyia Galati, 2003, Psychodopygus Mangabeira, 1941, Nyssomyia Barretto, 1962 and Trichophoromyia Barretto, 1962). Although a large number of molecular studies have been published on these groups, the main focus has been to compare closely related species [11]. Regarding the studies including species belonging to different genera, only three have been published to infer phylogenetic relationships between genera of Phlebotomine sandflies [3, 7, 13].

thumbnail Figure 1

Simplified cladograms based on morphological characters (sensu Galati, 1995) representing relationships among Phlebotomini divided into 4 subtribes (A) and among Psychodopygina divided into 7 genera, 3 subgenera, series and groups (B).

Here we provide for the first time a molecular phylogeny of the Psychodopygina subtribe to compare it with the current morphological classification in use. This work allows for a better understanding of the evolutionary relationships within this group.

Material and methods

Sandfly collection

Sandflies were captured using CDC miniature light traps in Mexico, Nicaragua, French Guiana, Ecuador, Brazil, and Bolivia. A total of 48 taxa representing six genera of the Psychodopygina (Psychodopygus, Nyssomyia, Trichophoromyia, Psathyromyia, Bichromomyia, and Martinsmyia) and one species of the genus Evandromyia as an outgroup representative species of the Lutzomyiina subtribe were analyzed (Table 2).

Table 2

List of specimens analyzed in this study, with country of collection, sex, and GenBank sequence accession numbers.

Head, wings, and genitalia from specimens were cleared and mounted between a slide and cover slide [1]. The thorax related to each specimen was stored at −20 °C or in 96% ethanol before DNA extraction. Some specimens were dried and stored at −20 °C or room temperature waiting for processing. Routinely, two specimens were processed for each taxon, except for scarce species for which only one specimen was processed, and for Ny. richardwardi (four specimens processed).

DNA extraction, PCR amplification, and sequencing

DNA was extracted using a QIAmp® DNA Mini Kit (Qiagen, Hilden, Germany), following a classical protocol [12]. Polymerase chain reactions (PCR) were performed in a 50 μL volume using 5 μL of DNA extracted solution and 50 pmol of primers C3B-PDR/NIN-PDR [18], and C1’/D2 [41] described previously to amplify partial cytochrome b (cytb) from mitochondrial DNA and domains D1–D2 from ribosomal DNA 28S, respectively. PCR products were sequenced in both directions using the primers for DNA amplification. Sequences obtained are available in GenBank and accession numbers are indicated in Table 2.

Sequence alignment and phylogenetic analyses

Sequences were edited and aligned using Muscle software [16] and then manually checked for compliance with the criteria: (1) minimizing the number of inferred mutations; (2) favoring substitutions over insertions and deletions; and (3) favoring transitions over transversions due to the higher probability of their occurrence.

The TVM+I+G model of molecular evolution was determined for both data partitions out of 88 possible models with JModeltest v0.1.0. [9, 39] via the Akaike Information Criterion (AIC) [8]. The AIC values for these models in JModeltest were 7062.889520 for cytb and 11236.768300 for D12, both with a delta AIC of zero. Phylogenetic reconstruction was conducted using Bayesian (BA) inference in MrBayes [43]. Bayesian analyses on the combined two-genes dataset were performed using a mixed model with a partition by gene assigning independent models of evolution to each partition. All parameters were unlinked between partitions, except topology and branch lengths. Analyses consisted of two runs of four simultaneous Markov chains each for 10 million generations, sampling a tree every 1,000 generations and applying a 25% burn-in after checking for convergence using TRACER v1.4 [40] and AWTY [36] to confirm that the standard deviation of split frequencies approached zero. The resulting trees were kept, calculating posterior probabilities in a 50% majority-rule consensus tree. The species Evandromyia (Aldamyia) walkeri, a taxon belonging to the Lutzomyiina, was used as outgroup in our analyses because this subtribe is the sister group of Psychodopygina (Fig. 1).

Results

Sequencing

Sequence fragments for D1 and D2 amplified segments, including the flanking conserved domains of rDNA28S were 687 to 690 base pairs (bp). Their alignment included 690 nucleotide positions: 535 constant, 153 variable but parsimony uninformative, and 86 were parsimony informative. The lengths of partial cytochrome b sequences obtained ranged from 501 to 545 bp. The final alignment dataset included 545 nucleotide positions: 288 were constant, 235 were variable but parsimony uninformative, and 176 were parsimony informative. No intraspecific variation was observed for both markers.

Phylogenetic reconstruction

The Bayesian analysis of the combined two-gene dataset (1,334 bp) recovered highly supported monophyletic groups with posterior probabilities (PP > 0.9) for most of the clades (Fig. 2). Some of these same clades were not recovered using both genes independently as nodal support was not strong (PP < 70) (Supplementary Figures S1 and S2).

thumbnail Figure 2

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on the sequences of D1 and D2 domains of 28SrDNA and cytochrome b gene of mtDNA. The highlighted groups and dotted lines correspond to the six genera analyzed. The numbers in the nodes represent posterior probabilities.

The recovered phylogenetic reconstruction supports the monophyly of the genus Psathyromyia, sister to all other Psychodopygina members. The genus Psathyromyia was composed of three subgenera: Forattiniella, Psathyromyia, and Xiphopsathyromyia. The subgenera Forattiniella and Xiphopsathyromyia were found to be monophyletic, whereas the subgenus Psathyromyia appeared as paraphyletic.

The monophyly of the genus Martinsmyia was not confirmed because we processed only one species. Moreover, the genus Martinsmyia was composed of the groups alphabetica (here represented by Mt. brisolai) and gasparviannai from which no sample was analyzed. The genus Trichophoromyia was recovered as paraphyletic because it included one species belonging to the genus Nyssomyia (Ny. richardwardi) (Fig. 2), rendering the genus Nyssomyia also paraphyletic. The position of Ny. richardwardi varied depending on the selected marker. According to D1D2 28S rDNA, it was recovered within the genus Trichophoromyia, sister to Th. reburra. On the other hand, the analysis of cyt b placed this species sister to Mt. brisolai (Supplementary Figures S1 and S2). If we excluded Ny. richardwardi from the combined dataset as well as from each dataset independently (cytb or rDNA), the genera Trichophoromyia and Nyssomyia were recovered monophyletic with high confidence (PP > 0.9).

The genus Psychodopygus was found to be monophyletic and divided into six groups. The group guyanensis, represented here by 4 of its 7 species (Ps. corossoniensis, Ps. luisleoni, Ps. francoisleponti, and Ps. geniculatus) and the group davisi (Ps. davisi and Ps. claustrei) appeared monophyletic with high nodal support (Fig. 2). The rest of the group had lower posterior probabilities, from which the group panamensis was found to be paraphyletic with Ps. hirsutus, Ps. ayrozai, Ps. nocticolus, and Ps. llanosmartinsi placed in one clade that excluded the congener Ps. panamensis. Furthermore, the groups chagasi, arthuri, and bispinosus were represented by only one species (Ps. squamiventris squamiventris, Ps. lloydi and Ps. bispinosus, respectively) and their position was less supported.

Discussion

To our knowledge, there is only one morphological phylogeny study, apart from the studies by Galati [22], that focus on the subgenus Lutzomyia in New World sandflies [38]. However, a large number of molecular studies related to Phlebotomine sandflies have been carried out [11], but mostly focused on closely related species. On the other hand, few studies concerning the deep phylogeny of Phlebotomine sandflies, that is comparing several genera, have been published. For instance, the work by Depaquit et al. [13] and Aransay et al. [3] focused primarily on Old World sandflies and only included a few New World species. The most comprehensive analysis and the one that included the largest number of taxa is that of Grace-Lema et al. [27]. But despite the extension of several loci, this work suffered from a lack of data that could explain the dubious position of the genus Brumptomyia or the paraphyly of the genus Nyssomyia. Most molecular studies on New World sandflies have focused either on several closely related species of the same genus or subgenus [6, 10, 44, 46] or on the population level (i.e. population genetics) [19, 28, 37, 45]. From the eight publications available to date based on several genera of New World sandflies (Table 3), only the work by Beati et al. (2004) combined cytb with partial 28S rDNA sequences [7]. The rest of these phylogenies are exclusively based on one mitochondrial marker (cytb, COI, 12S or 16S) limiting alternative phylogenetic exploration. Consequently, Beati et al. [7] mostly focusing on Lutzomyiina from Andean regions partially tested Galati’s hypotheses. Here we summarize some of the results in light of their findings and to contrast with our proposed phylogeny. Dutari & Loaiza [15] focused primarily on sandflies from Panama recovering Pi. ovallesi and Mi. trinidadensis as part of the Psychodopygina clade. This position is probably artefactual due most likely to the effect of long-branch attraction. The study by Kocher et al. [30], based on 16S mtDNA of 40 species of Lutzomyiina, Psychodopygina, and Brumptomyiina exclusively from French Guiana, revealed that Ny. sylvicola is distantly related to the other Nyssomyia lineages. This conflicting result could be most likely related to the resolution of this marker, not ideal at species-level phylogenies. Next, Cohnstaedt et al. [10] based its phylogeny on cytb marker and 10 species of Pintomyia (Pifanomyia) belonging to the groups verrucarum, serrana, and townsendi, produced a non-surprising phylogeny considering the analyzed species were closely related ones.

Table 3

Molecular studies carried out on New World Sandflies, comparing several genera.

Psychodopygus was raised for the first time to the genus level by Forattini [20, 21] which at that time, comprised the greatest number of the species now included in the genera Bichromomyia, Psychodopygus, Nyssomyia, Trichophoromyia and Psathyromyia, partim of Psychodopygina by Galati [22]. Our study is strongly in agreement with Galati’s hypotheses (Fig. 3). However, Forattini considered Viannamyia as a distinct genus, but Galati 1981’s classification [24] based on the morphology and implantation level of the larvae antennae, included the Viannamyia subgenus in Psychodopygus. We were not able to test this hypothesis given the lack of samples from Viannamyia. On the other hand, Ready et al. [42] considered only Psychodopygus as a distinct genus from Lutzomyia, arguing that the particular spermathecae of the females (with imbricated triangular rings) would justify its differentiation. Our phylogenetic reconstructions strongly support the monophyly of the genus Psychodopygus sensu Ready et al. [42] and Galati [22] as shown by both the concatenated dataset (ribosomal and mitochondrial, Fig 2.) and by each marker (Supplementary figures). The groups (formerly called series in the literature), guyanensis and davisi are monophyletic except for the group panamensis. This paraphyly is caused by the position of the group davisi separating Ps. panamensis from the other members of the group.

thumbnail Figure 3

Phylogenetic relationships within the Psychodopygina according to Galati’s hypothesis (A) and according to the molecular data of the present study (B).

The genus Nyssomyia, considered monophyletic by Galati [23] appears paraphyletic considering the position of Ny. richardwardi grouped within the genus Trichophoromyia. If we exclude the latter species of Nyssomyia, it could be considered as a clade subdivided into two well-supported monophyletic groups: one clade including the related species Ny. trapidoi and Ny. ylephiletor and a second clade including Ny. yuilli pajoti, Ny. umbratilis and Ny. antunesi grouped with the closely related species Ny. whitmani, Ny. intermedia and Ny. neivai. Additionally, introgression has been described for Ny. whitmani and Ny. intermedia [31, 33, 34] and both male and female hybrids between Ny. intermedia and Ny. neivai were observed [26]. We confirm the phylogenetic position of Ny. richardwardi was not due to a sequencing or laboratory error because four specimens were processed and sequenced at different times obtaining 100% identical data each time. Following the morphological identification keys of Galati [23–25], Ny. richardwardi and Ny. shawi form a cohesive group within the genus Nyssomyia with some morphologic characters shared across the genera Nyssomyia and Trichophoromyia (Table 4). For example, both species present simple setae on all flagellomeres, and in the males, the internal spine of the gonostyle is implanted close to its base. These characteristics are present in Trichophoromyia but not in Nyssomyia. However, the absence of Newstead sensilla on the palpomere II and spermathecae with less than 20 rings are present in Nyssomyia but not in Trichophoromyia. Further, both species exhibit cerci longer than wide in females from Nyssomyia but different from those of Trichophoromyia. In the morphological analysis, the clade constituted by both genera Nyssomyia and Trichophoromyia share more synapomorphies with Psychodopygus than with Bichromomyia in Galati’s cladogram (Fig. 1); however, in our phylogenetic tree (Fig. 2) the genus Bichromomyia appears as the sister group of Nyssomyia.

Table 4

Morphological characters of Ny. richardwardi shared with the Nyssomyia and Trichophoromya genera.

The genus Psathyromyia was found to be monophyletic as well as the subgenera Forattiniella and Xiphopsathyromyia. However, the position of Pa. dasymera as sister to all other Psathyromyia species, including the members of the groups shannoni and lanei, renders the genus Psathyromyia paraphyletic.

Unfortunately, it was not possible to include samples belonging to the genus Viannamyia in our analysis. As a consequence, the phylogenetic position of Viannamyia within the Psychodopygina subtribe remains unsolved. In the future, exploring the relationships between the New World Leishmania agents of tegumentary leishmaniasis belonging to the genera Leishmania, Viannia, and Mundinia [5] and their vectors will prove to be essential in understanding specific vector associations and disease ecology. We suggest expanding on these phylogenetic explorations to clarify the taxonomic assessment of these vectors, strongly related to human and animal health.

Acknowledgments

The authors wish to thank François Le Pont, María Lorena Mejía, and Moises Gualapuro for their valuable support in the fieldwork and the provision of some specimens processed in the present work. This work was supported by Secretaria Nacional de Ciencia y Tecnología (SENESCYT) and Spanish collaboration funded by the Red de Investigación de Centros de Enfermedades Tropicales – RICET (Project No. RD16/0027/0023 of the PN de I+D+I, ISCIII-Subdirección General de Redes y Centros de Investigación Cooperativa RETICS), Ministry of Health and Consumption, Madrid; by CIBER de Enfermedades Infecciosas Project CB21/13/00056, ISCIII, Ministry of Science and Education, Madrid, Spain; and by Projects No. 2016/099 and 2021/004 of the PROMETEO Program, Programa of Ayudas para Grupos de Investigación de Excelencia, Generalitat Valenciana, Valencia, Spain.

Supplementary material

thumbnail Figure S1:

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on sequences of the D1 and D2 domains of 28SrDNA, the numbers in the nodes representing posterior probabilities.

thumbnail Figure S2:

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on sequences of the cytochrome b gene of mtDNA, the numbers in the nodes representing posterior probabilities.

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Cite this article as: Zapata S, Galati EAB, Chaves JA, Artigas P, Gantier J-C, Bargues MD, Mas-Coma S & Depaquit J. 2023. Molecular phylogeny of Psychodopygina (Diptera, Psychodidae) supporting morphological systematics of this group of vectors of New World tegumentary leishmaniasis. Parasite 30, 18.

All Tables

Table 1

Proven vectors of American Leishmania according to Killick-Kendrick (1990), Maroli et al. (2013) and Akhoundi et al. (2016).

In the text
Table 2

List of specimens analyzed in this study, with country of collection, sex, and GenBank sequence accession numbers.

In the text
Table 3

Molecular studies carried out on New World Sandflies, comparing several genera.

In the text
Table 4

Morphological characters of Ny. richardwardi shared with the Nyssomyia and Trichophoromya genera.

In the text

All Figures

thumbnail Figure 1

Simplified cladograms based on morphological characters (sensu Galati, 1995) representing relationships among Phlebotomini divided into 4 subtribes (A) and among Psychodopygina divided into 7 genera, 3 subgenera, series and groups (B).
In the text
thumbnail Figure 2

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on the sequences of D1 and D2 domains of 28SrDNA and cytochrome b gene of mtDNA. The highlighted groups and dotted lines correspond to the six genera analyzed. The numbers in the nodes represent posterior probabilities.
In the text
thumbnail Figure 3

Phylogenetic relationships within the Psychodopygina according to Galati’s hypothesis (A) and according to the molecular data of the present study (B).
In the text
thumbnail Figure S1:

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on sequences of the D1 and D2 domains of 28SrDNA, the numbers in the nodes representing posterior probabilities.
In the text
thumbnail Figure S2:

Phylogenetic reconstruction of the Psychodopygina subtribe from Bayesian analysis based on sequences of the cytochrome b gene of mtDNA, the numbers in the nodes representing posterior probabilities.
In the text

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