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PLOS Neglected Tropical Diseases logoLink to PLOS Neglected Tropical Diseases
. 2021 Jul 19;15(7):e0009479. doi: 10.1371/journal.pntd.0009479

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers

Astrid Cruaud 1,#, Véronique Lehrter 2,#, Guenaëlle Genson 1, Jean-Yves Rasplus 1,, Jérôme Depaquit 2,‡,*
Editor: Remi N Charrel3
PMCID: PMC8425549  PMID: 34280193

Abstract

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9–8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3–5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3–2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.

Author summary

Phlebotomine sand flies are tiny insects transmitting unicellular parasites called Leishmania worldwide. They are bad flyers and are known to be a group dispersing according to historical events. No invasive species are known within Phlebotomine sandflies. Their taxonomy includes about 40 genera. In the Old World, the subgenus Paraphlebotomus includes the main vectors of Leishmania tropica, the agent of a cutaneous leishmaniasis mostly in North Africa visceral and the Middle West. The goal of this study was to explore the phylogeny and the biogeographical history of this subgenus using a new generation sequencing technique. We successfully amplified DNA copies from the selected DNA extracts, some of them being more than 20 years old. We fully resolved the phylogeny of the subgenus Paraphlebotomus who was not recovered as monophyletic, meaning it does not include all the ascendants of last common ancestor. Consequently, the taxonomy was revised with the creation of a new subgenus. We also suggest that Paraphlebotomus originated about 10 million years ago. After a first split isolating North African species, the Messinian crisis caused by the aridification of the Tethys (the ancestor of the Mediterranean Sea) occurring 7 million years ago allowed the divergence of the main lineages including that of Phlebotomus sergenti the most important vector of the group. Most recent radiation events occurred during the Pleistocene. The historical biogeography of these Phlebotomine sandflies are closely related to that of the Leishmania they transmit.

Introduction

Phlebotomine sand flies (Diptera, Psychodidae) are the main natural vectors of Leishmania, a genus of protozoan parasites which cause visceral and tegumentary neglected tropical diseases worldwide. Of the approximately 1,000 species of sand flies, about 70 are vectors of Leishmania [1,2]. Leishmaniases are endemic in nearly 100 countries, mainly in Africa, Asia and Latin America [3]. The estimated annual incidence of cutaneous leishmaniases in humans is 0.7 to 1 million cases and 50,000 to 90,000 cases of visceral leishmaniases were reported in 2017 [4]. However, most leishmaniases are zoonotic diseases affecting wild or domestic animals. Canine leishmaniasis is for instance a serious veterinary problem in many countries [14].

As for any vector-borne disease, an accurate systematics of the vectors is critical for a better understanding of the epidemiology of diseases. However, although phlebotomine sand flies are the subject of many research programs, their taxonomy and evolutionary history remain poorly studied [5], which limits our progress to reduce the incidence of leishmaniases.

Phlebotomine are minute flies (2–3 mm), which makes their identification to species difficult, especially for non-specialists. Diagnostic characters are almost exclusively located on the head and genitalia of both sexes and their observation require that specimens are slide mounted, which further complicates taxonomic work. The small size of the phlebotomine sand flies is also a technical challenge for molecular work, which may explain why only few molecular studies were conducted so far. Because species complexes may exist, the sequencing of a pool of specimens is hazardous and DNA extracted from single specimens should be used for molecular work. In addition, as dissection of the head and genitalia is required for identification, DNA extraction must be performed only on remains of the body. As a result, DNA yield is low, which drastically limits the number of genes that can be amplified and sequenced from a single specimen using PCR and Sanger approaches. Thus, the amount of genetic information that can be analysed to infer robust relationships and highlight possible species complexes is low.

In the Old World, most vectors of Leishmania belong to the genus Phlebotomus [5], and some are included in the genus Sergentomyia [6]. Eleven subgenera have been described in the genus Phlebotomus [7] among which, four are involved in the transmission of Leishmania, namely Larroussius, Phlebotomus, Euphlebotomus and Paraphlebotomus, and two others are suspected or local vectors (Adlerius and Synphlebotomus) (Table 1).

Table 1. Subgenera of Phlebotomus sand flies involved in Leishmania transmission.

Subgenus Species Main Leishmania species transmitted (suspected or proven) References Included in the study
Adlerius Ph. (Ad.) simici
Ph. (Ad.) arabicus
L. infantum
L. tropica
[1]
[17]
x
Euphlebotomus Ph. (Eu.) argentipes L. donovani [1, 94] /
Larroussius Ph. (La.) neglectus L. infantum [95] x
Paraphlebotomus Ph. (Para.) alexandri L. donovani [1, 96] x
L. infantum [97]
Ph. (Para.) andrejevi L. major [98] /
Ph. (Para.) caucasicus L. major [99] x
Ph. (Para.) chabaudi / / x
Ph. (Para.) gemetchi / / /
Ph. (Para.) jacusieli / / x
Ph. (Para.) kazeruni L. major [1, 100] x
Ph. (Para.) mireillae / / x
Ph. (Para.) mongolensis / / x
Ph. (Para.) nuri / / /
Ph. (Para.) riouxi / / x
Ph. (Para.) saevus L. tropica [101] x
Ph. (Para.) sergenti L. tropica [13, 14, 101] x
L. aethiopica [101]
Ph. (Para.) similis / / x
Phlebotomus Ph. (Ph.) duboscqi L. major [102] x
Ph. (Ph.) papatasi L. major [103] x
Synphlebotomus Ph. (Sy.) ansarii L. major [104] x

Based on morphological characters [8], Paraphlebotomus, Phlebotomus and Synphlebotomus are closely related (presence of a basal lobe on gonocoxite, short aedeagal or spermathecal ducts, short parameral sheath, annealed spermathecae without neck), while Larroussius (lack of basal lobe on gonocoxite, long aedeagal or spermathecal ducts, long parameral sheath, annealed spermathecae with neck) appears to be part of another evolutionary lineage also including Adlerius, Euphlebotomus, Anaphlebotomus or Transphlebotomus. Two studies based on morphological characters consider these two groups of subgenera as separate clades [9,10]. Studies based on ribosomal DNA, highlighted that the subgenera Paraphlebotomus and Phlebotomus belong to the same clade [11,12] but no molecular study has been published so far that included members of these related subgenera.

Here, we focus on the subgenus Paraphlebotomus that currently includes 14 species (Table 1), four of which being demonstrated vectors of Leishmania spp. The most important vector is undoubtfully Ph. sergenti, which is the main vector of L. tropica, the etiological agent of an anthroponotic cutaneous leishmaniasis occurring in a large area from Pakistan to Morocco [13,14]. However, L. tropica can be locally transmitted by other vectors belonging to subgenera Larroussius in Kenya [15,16] and Adlerius in Israel/Palestine [17]. So far, only three molecular studies have included enough species of Paraphlebotomus to provide preliminary hypotheses about its systematics. Depaquit et al. [18] have conducted the only molecular study that attempted to resolve phylogenetic relationships within Paraphlebotomus using nine ingroup and two outgroup species. However, a single marker (Internal transcribed spacer 2, ITS2) was used and the resolution was low. Besides, we cannot exclude that possible paralogs of ITS2 may have biased results. A second study [19], whose goal was to describe the diversity of sand flies in military camps in Afghanistan using mitochondrial Cytochrome b gene (Cytb), generated data for five species of Paraphlebotomus. In this study, Paraphlebotomus appeared as a polyphyletic assemblage. Ph. alexandri grouped with species from the subgenus Phlebotomus (Ph. papatasi and Ph. bergeroti) with poor support. Finally, Grace-Lema et al. [20] used a patchy matrix of 7 genes (65% missing data, ca 45% of the species with more than 85% missing data) to infer a phylogeny of phlebotomine sand flies (113 terminals). However, as for the rest of the tree, relationships between the four species of Paraphlebotomus were poorly resolved. Thus, the systematics of the subgenus is still unclear.

Most species of Paraphlebotomus have restricted distribution (Fig 1; see also [21]). Ph. mireillae is found in Kenya and southern Ethiopia [22]; Ph. saevus occurs in Arabian Peninsula and in the Horn of Africa; Ph. nuri is a Middle-East endemic and Ph. gemetchi is reported from Ethiopia [23]. Ph. chabaudi and Ph. riouxi are Ibero-Maghrebian and Ph. similis occurs in the north-east of the Mediterranean basin, with recent records from central Turkey [24], Iran [25] and Ukraine [26]. Ph. jacusieli is distributed from eastern Mediterranean to eastern Iran [27]. Ph. caucasicus, Ph. andrejevi and Ph. mongolensis are located in central Asia or Middle-east, but the range of Ph. mongolensis extents to East China sea (Suizhong, Liaoning province) [28]. To the opposite, a few species have extremely large distribution. Ph. sergenti and Ph. alexandri occur from Europe and North Africa to Middle and Far East. Ph. kazeruni is almost always a rare species, mainly recorded from Iran, but recent records in Turkey [29], southern Algeria [30] and Pakistan [31,32] have widened its known range. As suggested by Depaquit et al. [18], the ancestor of the subgenus Paraphlebotomus may have originated in Eastern Africa or in the Arabic peninsula, however this needs to be formally tested.

Fig 1. Distribution maps and sampling sites for the different species of Paraphlebotomus sensu Lewis [7] and Depaquit [54].

Fig 1

Ranges are delimited by color lines. Lines are dotted when the presence is expected but has never been confirmed (lack of sampling, doubtful taxonomic status). Sampling sites and type localities are respectively represented by dots and stars. The background map was in the public domain and has been downloaded from https://commons.wikimedia.org/wiki/File:BlankMap-World.svg.

With technical advances, it is now possible to generate unbiased copies of whole genomes (Whole Genome Amplification, WGA) to circumvent low DNA amount issues [3335]. Although this technic is still rarely used for such purpose, it opens new avenues for the sequencing of pangenomic markers to resolve relationships among tiny species. Here we used WGA and RAD (Restriction site Associated DNA) sequencing [36] of single specimens of phlebotomine sand flies to infer a robust phylogeny of the subgenus Paraphlebotomus and five outgroups. We also performed divergence time estimates and probabilistic inference of historical biogeography of the subgenus.

Materials and methods

Taxonomic sampling

Twenty-three ingroup specimens representing 11 species (78% of the known diversity) were selected as ingroup. In average, one or two specimens per species were analysed. We failed to collect two rare species (Ph. nuri and Ph. gemetchi) and to process old DNA extracts of Ph. andrejevi. For demonstrated vectors, four to five specimens collected in several locations were included. Nine outgroup specimens (five species) were chosen in the subgenera Phlebotomus, Synphlebotomus, Larroussius and Adlerius. Sampling was carried out during the last twenty years, from a large area extending from Senegal to India (west-east axis) and from Italy to Kenya (north-south axis) (Fig 1 and S1 Table). Samples were collected using CDC trap, sticky trap, or came from colonies reared at lab. Specimens were preserved in 95% ethanol at room temperature or at -20°C until dissection. Some samples were dried freeze at -80°C or in liquid nitrogen for longer conservation. After thawing, specimens were dissected in 95% ethanol. For morphological identification under stereomicroscope, head and genitalia were mounted in chloral gum or in Euparal between slide and cover slide after clarification in boiling Marc André solution, washing and dehydration. The rest of the body was dried freeze at -20°C for molecular biology analyses. Identification to species was performed by JD and vouchers (slides + body remains after DNA extractions) have been deposited in the University of Reims Champagne-Ardenne, collection of J. Depaquit.

DNA extraction and whole genome amplification

DNA extractions were performed with either CTAB/Chloroform-Isoamyl Alcohol or extraction kits. For the CTAB/Chloroform-Isoamyl Alcohol extraction, samples were crushed and incubated at 60°C in a solution containing CTAB buffer [37]. Proteins were eliminated by the addition of chloroform-Isoamyl Alcohol. RNase (0.5 units) was added to the aqueous phase, which was then incubated at 37°C for 30 min to remove RNA. Total genomic DNA was precipitated by the addition of two-thirds of the total volume of isopropanol and stored at 4°C overnight. After centrifugation, the pellet was washed, dried and suspended in 50 μL of ultrapure water. Genomic DNA was also extracted using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s instructions. DNA was eluted using 100 μL to 160 μL of elution buffer.

DNA quantity was measured using the Qubit double-stranded DNA High Sensitivity Assay kit (Thermofisher Scientific) following the manufacturer’s instructions. DNA extracts were then subjected to Whole Genome Amplification using the GenomiPhi V2 DNA Amplification kit (GE Healthcare). Ethanol precipitation of DNA was performed prior to WGA to fit with the recommendations of the GenomiPhi protocol (1μL DNA input at 10 ng/uL). 1/10 volume of sodium acetate 3 M pH 5.2 was added to DNA. Then, 2 volumes of cooled absolute ethanol were added to the mix. The mix was incubated at -20°C overnight. The pellet of DNA extract obtained by centrifugation (30 min, 13 000 rpm, 4°C) was washed by 500 μL of cooled 70% ethanol. After a new centrifugation (15 min, 13 000 rpm, 4°C), the pellet was dried at room temperature, and suspended in 4 μL of sterile molecular biology ultrapure water. Whole Genome Amplification (WGA) was then performed on 1 to 2 μL of concentrate DNA following the manufacturer protocol.

DNA barcoding

The standard COI barcode fragment was sequenced for all specimens to obtain calibrations for divergence time estimates (see below). For each sample, the cocktail of M13 tailed primers and the PCR conditions described in Germain et al. [38] were used on 4μL of DNA obtained with WGA. Unpurified PCR products were sent to Eurofins Genomics for Sanger sequencing. Forward and reverse strands were assembled using Geneious 10.2.2 [39]. Consensuses were aligned with MAFFT-7.313 (linsi option) [40] and translated to amino acids using MEGA 7 [41] to detect frame-shift mutations and premature stop codons, which may indicate the presence of pseudogenes. Sequences were merged with all barcodes available in BOLD [42] for the genus Phlebotomus (last access December 4, 2019) and IQ-TREE v1.6.7 [43] was used to infer a maximum likelihood phylogenetic tree. The best-fit evolutionary model was selected with ModelFinder as implemented in IQ-TREE [44] and branch supports were assessed with ultrafast bootstrap [45] and SH-aLRT test [46] (1000 replicates).

Preparation and sequencing of RAD library

In silico digestion of the genome of Ph. papatasi available on GenBank (AJVK00000000.1) was performed using the recognition site of the PstI enzyme to determine the number of expected cut-sites and calculate the maximum number of samples to be sequenced on a single lane of an Illumina HiSeq 2500. 51,712 cut sites (103,424 RAD markers) were inferred. Considering that ca 150,000,000 passing filter reads would be usable to construct RAD loci according to our experience (after removal of reads with mutation in barcodes /restriction sites and PCR duplicates), 48 samples were included in the library to get a sequencing depth per marker of ca 30X (only 32 samples were analysed for this study, the 16 others were sequenced for another purpose). Library construction followed by Baird et al. [36] and Etter et al. [47] with modifications detailed in Cruaud et al. [48]. The quantity of P1 adapters to be added to saturate restriction sites (result = 2 μL) as well as the optimal time for DNA sonication in a Covaris S220 ultrasonicator (result = 70s) that are both specific to the studied group were evaluated in a preliminary experiment. After tagging with barcoded P1 adapters and prior to sonication, specimens were pooled eight by eight (six pools were thus obtained and tagged with barcoded P2 adapters). 2*125nt paired-end sequencing of the library was performed at MGX-Montpellier GenomiX on one lane of an Illumina HiSeq 2500 flow cell.

RAD data cleaning and phylogenetic inference

The quality of the raw data was assessed using FastQC (Babraham Institute, Bioinformatics Group http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). Raw data were processed using the Perl wrapper RADIS [49] that relies on Stacks [50,51] for demultiplexing of data, removing PCR duplicates and building individual and catalog loci. For data cleaning (RADIS_step1_data_cleaning.pl), parameters in the RADIS.cfg files were set as follows (radis_readlen = 125; radis_nttrim_read1_5p = 5 to remove the complementary overhang of the restriction site; radis_nttrim_read1_3p = 0; radis_nttrim_read2_5p = 0, radis_nttrim_read2_3p = 0).

Individual RAD loci were built using m (minimum depth of coverage to create a stack) = 3 and, M (maximum distance allowed between stacks) = 2; with removal (r) and deleveraging (d) algorithms enabled. Six data sets were then built to test a possible impact of analytical parameters on our phylogenetic analyses. Catalogs of loci were built with n (number of mismatches allowed between sample tags when generating the catalog) set to 6, 8 or 10. Loci / samples selection was performed as follows: only samples with more than 100 RAD loci in the catalog were kept (radis_nloci_min), only locus for which at least either 50% or 75% of the samples had sequences were kept (radis_nsample_min) and loci for which at least one sample had 3 sequences or more were removed to discard possible paralogs (radis_npbloci_cutoff). Phylogenetic inferences were performed using RAxML 8.2.9 (multi-threaded version with AVX extensions) [52] and a rapid bootstrap search with 100 replicates, followed by a thorough ML search (GTR GAMMA), without partitioning of the data set. Trees were visualized and annotated with TreeGraph 2.13.0-748beta [53]. Percentage of missing data and percentage of identity between sequence pairs were calculated using Geneious 10.2.2 [39].

Species distribution, divergence time estimation and ancestral range estimation

Species distribution for subgenus Paraphlebotomus were estimated from maps of two precedent studies [7,54] and a review of recent papers (see results for an exhaustive list). There is no fossil record of Phlebotomus and paleogeographic events that could be used as calibrations are unknown. Consequently, we used calibration priors based on mitochondrial DNA. We estimated minimum and maximum pairwise sequence divergences among COI sequences of sampled belonging to the two species pairs recovered both in the RAD and COI trees (i.e. Ph. chabaudi-Ph. riouxi and Ph. papatasi-Ph. duboscqi) using MEGA7 (evolutionary model = K2P). Divergences were then divided by the COI clock rate for insects estimated by Papadopoulou et al. [55] (i.e 3.54 ± 0.38% divergence per Myr) to obtain minimum and maximum ages for the split between the two species of each pair following Godefroid et al. [56]. A timetree was built with the RelTime method [57] as implemented in MEGA X [58] using the RelTime-ML option, a GTR+G evolutionary model (with 4 discrete Gamma categories as for the RAxML analysis) and all nucleotide sites (including gaps and missing data). The RAxML RAD tree obtained with the cstacks n = 10, radis_nsample_min = 50% of the samples (Table 2; ie the tree obtained with the largest data set) was used as input and Ph. (Adlerius) simici and Ph. (Larroussius) neglectus were considered as outgroups. Uniform distributions were used as calibration densities.

Table 2. Properties of the RAD data sets obtained with different parameters combinations.

n = number of mismatches allowed when merging individual loci in cstacks, radis_nsample_min = minimum number of samples that should have sequences for a RAD locus to be included in the analysis. In all data sets, loci for which at least one sample had three or more sequences (i.e. possible paralogs) were removed (radis_npbloci_cutoff = 3).

Combination of parameters # RAD markers # bases in alignment % missing data % variable sites % parsimony informative sites % GC RAxML tree
n = 6, radis_nsample_min = 50% of the samples 5,050 580,750 34.6 12.0 8.40 45.4 S2A Fig
n = 6, radis_nsample_min = 75% of the samples 1,423 163,645 16.7 11.5 8.20 45.9 S2B Fig
n = 8, radis_nsample_min = 50% of the samples 7,194 827,310 34.1 14.7 10.6 45.8 S2C Fig
n = 8, radis_nsample_min = 75% of the samples 2,086 239,890 16.6 14.4 10.6 46.4 S2D Fig
n = 10, radis_nsample_min = 50% of the samples 8,906 1,024,190 33.1 16.9 12.4 46.1 S2E Fig
n = 10, radis_nsample_min = 75% of the samples 2,808 322,920 16.7 16.9 12.7 46.7 S2F Fig

We inferred the biogeographic history of the subgenus Paraphlebotomus using the R package BioGeoBEARS 1.1.1 [59]. The chronogram built with RelTime was used as input but only one specimen per species was included in the analysis and outgroups (including Ph. alexandri) were removed to avoid artefacts (taxa were pruned from the chronogram with the R package ape [60]). We compared the fit of the models DEC, BAYAREALIKE, and DIVALIKE with AICc but did not consider the jump parameter for founder events (+J) following recent criticisms [61]. Geographic ranges of the species were coded using six geographical subregions of the West Palearctic: A: West Mediterranean; B: Saharo-Arabian; C: Sudanian; D: Somalian; E: East Mediterranean; F: Irano-Turanian. The maximum number of areas species may occupy was set to 6. To consider the main geologic events that occurred during the last 15 Myr in the peri-mediterranean area, we defined two time periods with different dispersal rate scalers: 1) from the Mid Miocene climatic optimum (ca 15 Ma) to the end of the Messinian crisis (5.3 Ma); 2) from the Zanclean flood (5.3 Ma) to present. The matrix of geographical ranges for the different species as well as the dispersal multipliers for each time period are available as S1 Text.

Computational resources

Analyses were performed on the Genotoul Cluster (INRAE, France, Toulouse).

Results

DNA barcoding and calibrations

COI sequences were successfully obtained from all samples but one (Ph. saevus for which all DNA was included in the RAD library). The maximum likelihood tree confirmed that COI is not informative enough to solve relationships among species of phlebotomine sand flies (S1 Fig). To avoid issues due to mitochondrial introgression and putative paralogs (heteroplasms, coding pseudogenes) that could be contained in the COI data set, only splits between highly supported species pairs that were recovered both in the RAD (S2 Fig) and the COI trees were used to calibrate our dating analysis, namely Ph. chabaudi-Ph. riouxi and Ph. papatasi-Ph. duboscqi. Minimum and maximum interspecific K2P distances resulted in minimum and maximum ages of 2.42–3.64 Myr for the Ph. chabaudi-Ph. riouxi split (rounded to 2.0–4.0 Myr for the RelTime analysis) and 2.83–4.27 Myr for the Ph. papatasi-Ph. duboscqi split (rounded to 2.0–5.0 Myr for the RelTime analysis).

RAD data sets and phylogenetic inferences

In average, 97,054 RAD loci were obtained per specimen (ustacks, S1 Table). Depending on the value of the parameters n (number of mismatches allowed when merging individual loci) and radis_nsample_min (minimum number of samples that should have sequences for a RAD locus to be included in the analysis) data sets included 1,423 to 8,906 RAD loci of 115 bp each and contained only 16.6–34.6% missing data (Table 2).

All RAxML analyses yielded the same fully resolved tree topology (Figs 2 and S2). The subgenus Paraphlebotomus was paraphyletic. Ph. alexandri was recovered sister to the rest of Paraphlebotomus species and a clade consisting of members of the Phlebotomus (Ph. papatasi and Ph. duboscqi) and Synphlebotomus (Ph. ansarii) subgenera. The remaining Paraphlebotomus species clustered into a strongly supported clade in which relationships between species were fully resolved.

Fig 2. RelTime timetree.

Fig 2

Bootstrap supports lower than 100 are reported at nodes (100 replicates, RAD data set used: cstacks n = 10, radis_nsample_min = 50% see Table 2 and S2 Fig). Species belonging to subgenus Paraphlebotomus sensu Seccombe et coll., 1993 are shown in green but terminals are annotated following the new classification proposed in this study with Ph. alexandri belonging to Artemievus subg. nov. Depaquit. Divergence times are not displayed in the outgroup (Ph. (Adlerius) simici and Ph. (Larroussius) neglectus) because RelTime uses evolutionary rates from the ingroup to calculate divergence times and does not assume that evolutionary rates in the ingroup clade apply to the outgroups. The image shows Ph. papatasi and has been modified from the image #10275 of the Centers for Disease Control and Prevention’s Public Health Image Library (PHIL: https://phil.cdc.gov/; Content provider: CDC/ Frank Collins. Photo credit: James Gathany; last access January 22, 2021) that is in the public domain and thus free of any copyright restrictions.

Species validity and intraspecific variability

When multiple conspecific specimens were analysed, they formed well-supported clades that were deeply separated from their nearest relative, suggesting that morphology alone mostly enables accurate identification of species. This was the case for the analysed specimens belonging to Ph. chabaudi and Ph. riouxi with an average pairwise percentage dissimilarity between specimens of the two clades of 1.7% for the n = 10, radis_nsample_min = 50% data set (i.e. on 1,024,190 nt). Significant genetic divergence was observed between individuals of species with large distribution ranges (Ph. alexandri–up to 1.6% dissimilarity and Ph. sergenti–up to 1.2%).

Divergence-time estimates

RelTime analysis inferred that the split between Ph. alexandri and all other lineages has occurred about 15 Ma (14.7 Ma, confidence interval–CI = 10.6–20.2) and that the subgenus Paraphlebotomus (excluding Ph. alexandri) originated in the Middle—late Miocene between 12.9 (CI = 9.3–17.9; most ancient ancestor) and 8.5 Ma (CI = 5.7–12.8; most recent common ancestor MRCA) (Fig 2). Within Paraphlebotomus, the “Ph. chabaudi / Ph. riouxi” lineage split from all other Paraphlebotomus sand flies about 8.5 Ma by the end of Miocene. All deep splits between geographical lineages occurred between 6.8 and 5.1 Ma, although with relatively large confidence intervals. Finally, most splits between sister species occurred during the Pliocene between 4.6 Ma (Ph. sergenti / Ph. kazeruni) and 2.3 Ma (Ph caucasicus / Ph. mongolensis). The main split within Ph. alexandri also occurred during this time period (ca 4.0 Ma).

Biogeography

AICc values show that the DEC and DIVALIKE models have an equally significant fit (S3 Text). The ancestral range of Paraphlebotomus is equivocal with Irano-Turanian + West Mediterranean subregions as the most likely range (Fig 3 and S4 Text). Each of the five species pairs (namely riouxi-chabaudi; mongolensis-caucasicus; saevus-mireillae; jacusieli-similis and kazeruni-sergenti) originated in a different subregion of the West-Palearctic (respectively West-Mediterranean, Irano-Turanian, Somalian, East-Mediterranean and Saharo-Arabian), suggesting an origin by vicariant fragmentation. The only difference between the DEC and DIVALIKE models is the most likely range for the MRCA of the clade mongolensis-caucasicus; saevus-mireillae; jacusieli-similis and kazeruni-sergenti, which is Somalian + Irano-Turanian subregions for DEC and Irano-Turanian only for DIVALIKE (S4 Text).

Fig 3. Ancestral range estimation for the subgenus Paraphlebotomus sensu nov.

Fig 3

Geographic areas as delineated on the map are represented with colored boxes. A: West Mediterranean region; B: Saharo-Arabian region; C: Sudanian region; D: Somalian region; E: East Mediterranean region; F: Irano-Turanian. Most probable ancestral states (areas or combination of areas) as inferred by DEC and DIVALIKE are indicated at each node of the phylogeny. When states are different between the two models, the most probable state as inferred by DIVALIKE is indicated first. Input data (matrix of geographic ranges for species included in the analysis, time periods and dispersal multipliers); ancestral range estimations for all tested models (DEC, DIVALIKE, BAYAREALIKE), and results of model testing are available in S1S4 Text. The background map was in the public domain and has been downloaded from Natural Earth (http://www.naturalearthdata.com).

Discussion

Description of a new subgenus of Phlebotomus Rondani & Berté

Ph. alexandri did not cluster with other species of Paraphlebotomus, which questions its current classification. An early divergence of Ph. alexandri [18] as well as the paraphyly of the subgenus Paraphlebotomus have already been acknowledged [19] and is confirmed here with high statistical support. Based on morphology, Ph. alexandri greatly differs from other species of Paraphlebotomus [54]. The first flagellomere (f1 = antennal segment AIII) is extremely short and the basal lobe of the coxite is wider than long. The pharyngeal armature is poorly extended and composed by a network of conspicuous scaly teeth. These apomorphies lead us to propose a new subgenus for Ph. alexandri.

Artemievus subg. nov. Depaquit. Type-species: Phlebotomus alexandri Sinton, 1928.

Artemievus subg. nov. is defined by i) a short (<170μm) flagellomere 1 (= A III), shorter than labrum and shorter than the combined length of flagellomeres 2 and 3 (= AIV + AV) in both genders, ii) in females, a rectangular pharyngeal armature occupying less than a quarter of the pharynx, spermathecae with seven to 10 rings, ascoids reaching the next articulation, iii) in males, basal lobe of gonocoxite not incurved, style with four spines, the distal one carried by a long process, paramere simple and spoon-like and a narrow sperm pump (Figs 4 and 5).

Fig 4. Comparison of Ph. alexandri, the type-species of Artemievus subg. nov. and Ph. sergenti, the type-species of the subgenus Paraphlebotomus.

Fig 4

Ph. alexandri: genitalia of the lectotype from Amara, Iran (A), female from Syria: flagellomeres 1, 2, 3 (B), pharynx (C) and spermatheca (D). Ph. sergenti syntypes from Mac Mahon, Algeria: male genitalia (E) and detail of the basal lobe of the coxite (F); female pharynx (G), flagellomeres 1, 2, 3 (H) and spermathecae (I). Drawings by J. Depaquit.

Fig 5. Morphological variability of the basal lobe of the gonocoxite of Ph. alexandri specimens collected in different localities.

Fig 5

Drawings by J. Depaquit.

This subgenus includes only Ph. (A.) alexandri, a widespread and polymorphic species extending from Morocco to Mongolia and from Ethiopia to southern Europe (Fig 1).

Systematics of the subgenus Paraphlebotomus sensu nov

Our phylogenomic analysis (Figs 2 and S2) recovers fully resolved relationships within subgenus Paraphlebotomus sensus nov. that corresponds well to morphological affinities. The first clade formed by Ph. chabaudi and Ph. riouxi is supported by several morphological characters. Females of these North African species exhibit a typical spermathecae with a bellflower-like distal ring and males differ from all other species by their parameral sheaths that are apically pointed. Ph. chabaudi and Ph. riouxi can be differiented by the size of the basal lobes of their gonocoxites and the number of setae they harbor. Based on dubious molecular evidences [and despite the tree presented in Fig 4 of their 2014 paper shows that specimens of the two species belong to two different clades], Tabbabi et al. [62,63] considered these species as synonyms. This result was acknowledged by Grace-Lema et al. [20], who wrongly interpreted a highly supported sister-taxa relationship between Ph. chabaudi and Ph. riouxi as a support for their synonymy, although they noticed the long branch between the two species. The high genetic distance is mis-interpreted by the author as a consequence of missing data, which is an inconsistent statement as other analysed species show the same level of divergence but are not considered as synonyms. The present study confirms the validity of Ph. riouxi, that was already assessed with Sanger data and morphological evidence by Lehrter et al. [64]. Therefore Ph. riouxi stat. rev. is reinstated as a valid species, from its status of junior synonym of Ph. chabaudi.

The clade grouping all other species of Paraphlebotomus is subdivided into four pairs of sister taxa that exhibit strong morphological affinities:

  1. Females of the Afrotropical Ph. saevus and Ph. mireillae mainly differ by the presence of spines on spermathecal ducts. Males, which are extremely close, differ by the distal part of the parameral sheath (thicker in Ph. saevus than in Ph. mireillae), and the hairs of the gonocoxite basal lobe (longer in Ph. mireillae than in Ph. saevus). These two species are morphologically closely related to Ph. gemetchi and Ph. nuri, two rare species occurring respectively in Ethiopia and Pakistan, that we could not include in the present study.

  2. Males of Ph. caucasicus and Ph. mongolensis are closely related but recognizable by the shape of gonostyle or of basal lobe of the gonocoxite. To the contrary, females are undistinguishable and share mitochondria [65], a putative introgression that supports the close relationships observed in our topologies. Ph. andrejevi, another species that could not be included in our analysis certainly belongs to this clade as females of Ph. andrejevi are impossible to discriminate from Ph. caucasicus and Ph. mongolensis.

  3. Males of Ph. similis and Ph. jacusieli share many characters (shape of the parameral sheath or the flagellomere lengths) but differ by the longer process carrying the most distal spine of the gonostyle in Ph. jacusieli and the thick pharyngeal teeth of the female (occupying the whole pharynx in Ph. similis versus restricted to the central and anterior part of the armature in Ph. jacusieli).

  4. Males of Ph. sergenti and Ph. kazeruni share a short and thin curved basal lobe of the coxite and similar gonostyle, whereas females share a similar pharyngeal armature. Females of Ph. kazeruni differs from those of Ph. sergenti by the lowest number of spermathecal rings (only one or two). Ph. sergenti + Ph. kazeruni cluster with Ph. similis + Ph. jacusieli in a strongly supported clade, in agreement with their morphological affinities (shape of the parameral sheath, flagellomeres lengths, size of the basal lobe of the gonocoxite).

Evolutionary history and biogeography of Paraphlebotomus sensu nov

From the Mid-Miocene Climate Optimum (16.4–14.7 Ma) to present time, the Mediterranean region and the adjacent Middle Eastern areas experienced multiple geologic and climatic events that have strongly affected the distribution of organisms. Our analyses indicate that the most ancient ancestor of present Paraphlebotomus species originated ca 12.9 Ma at a time of global cooling and strong environmental changes (Fig 2). DEC and DIVALIKE models indicate that the range of the MRCA is ambiguous and suggest that the most likely range is composed of two disjoint areas: West-Mediterranean + Irano-Turania, which seems implausible (Fig 3 and S1 Text). This result can be partly explained by the fact that i) some lineages were lost; ii) distribution data are still patchy; iii) biogeographic studies for the subgenera Phlebotomus and Synphlebotomus are lacking and it was not possible to estimate ancestral range for these two subgenera in this study because very few species were included. Two different hypotheses could be considered for the MRCA of Paraphlebotomus: a restricted distribution or a large distribution (like Ph. sergenti or other phlebotomine sandflies). DEC and DIVALIKE appear to favor a largely distributed MRCA which is compatible with paleogeographical and paleoclimatic events.

During the Early and Middle Miocene, closure of the gateway of Proto-Mediterranean Sea [66] followed by the Messinian Salinity Crisis [67] and coupled with the uplift of the Atlas and Zagros Mountains [68], have strongly affected climatic conditions in the area under consideration. These mountain ranges, as well as the uplift of the Qinghai-Tibetan Plateau on the East, have acted as topographic barriers to moisture and have largely contributed to the aridification of the area. As a consequence, the vegetation of the area dominated by forests in the Early Miocene, underwent a transition to open grassland vegetation towards the late Miocene. Grasslands may have favored the expansion of the most ancient common ancestor of present Paraphlebotomus, enabling it to range from the Maghrebian mountains to central Asia.

The expansion of the Sahara as an arid belt has long been dated back to the Quaternary Ice age (ca 2.5 Ma) [69] or to the Pliocene (5.33–2.58 Ma) [70,71]. Recent studies [72,73] have demonstrated that arid desert conditions existed repetitively since 7 Ma (Late Miocene). Zhang et al [74] used climate model simulations and confirmed that the Late Miocene (ca 7–11 Ma) has been the pivotal period for triggering north African aridity and creating the Sahara Desert. An early origin of the Sahara Desert is also suggested by the work of Böhme et al [75] who dated a saline aeolian dust deposit from Sahara in Greece back to 7.37–7.11 Ma. Therefore, the basal split between the Maghrebian clade (Ph. riouxi + Ph. chabaudi) and other Paraphlebotomus species, ca 8.5 Myr ago, may have resulted from the rise of the Saharan belt and the aridification of the region located between the Atlas Mountains and the Turanian subregion.

During the Messinian, the Mediterranean region and the Middle East underwent rapid and dramatic climatic changes. Multiple factors probably operated simultaneously, the expansion of Antarctic ice-sheet [76], the rise of Sahara [77], the closure of the connection between the Atlantic Ocean and the Mediterranean sea that leaded to the nearly complete desiccation of the Mediterranean sea (Messinian salinity crisis) [67]. Consequently, steppic vegetation [78] and desert areas [79] expanded in the sub-desert belt south of Mediterranean region and extending to Middle and Central Asia. Therefore, the disjunct occurrence of the ancestors of the four lineages present in the East Mediterranean (Ph. similis + Ph. jacusieli), Saharo-Arabian (Ph. sergenti + Ph. kazeruni), Somalian (Ph. mireillae + Ph. saevus, possibly also Ph. gemetchi) and Turanian subregions (Ph. caucasicus + Ph. mongolensis, possibly also Ph. nuri and Ph. andrejevi) is probably the result of a Messinian vicariant event in which the extreme prolonged drought has been instrumental.

Our biogeographical analyses dated the last episode of speciation within the subgenus Paraphlebotomus during the Pleistocene (between 3 and 2 Ma). At that time, the Middle East areas and the East African subregion have experienced cycles of wet periods followed by periods of strong droughness [80]. Interestingly, these cycles have also favored the diversification of the rodents, in the burrows of which Paraphlebotomus larvae develop [8183]. In arid natural areas, the presence of sand fly species is strongly limited by the low moisture of the soil [84]. Paraphlebotomus species therefore occur close to sites where larvae can develop. Rodent burrows maintain greater humidity and Paraphlebotomus larvae can develop upon organic matter (feces) that accumulate in the moist atmosphere of the deepest burrows [84]. Burrows also provide good shelter to adults during the warmer hours of the day. This coupled to the relatively low dispersal abilities of Phlebotomus species (less than 2 kilometers, [85]) and the relative scarceness of suitable larval habitats in the dryer and warmest areas, make sand fly population scanty and prone to local extinction in desert and sub-desert habitats. Therefore, cycles of dry-wet periods may have resulted in reduction and separation of sand fly populations and finally by the formation of the current species. Subsequently, a few species have experienced large expansion in the last million years and have evolved toward anthropophily (i.e. Ph. sergenti).

Putative species complexes that required further studies

The widely distributed species Ph. alexandri and Ph. sergenti exhibit high intra-specific divergences, which suggests that these species may represent complexes of closely related entities. Two subclades are recovered in Ph. alexandri (Figs 2 and S2). The genetic distance between these subclades (1.6% on 1,024,190 nt) is similar to the distance observed between other pairs of sister species present in our topology (e.g. 1.6% between Ph. similis and Ph. jacusieli and 1.7% between Ph. chabaudi and Ph. riouxi). Ph. alexandri exhibits a high variability in the number of setae on the basal lobe of the gonocoxite and this variability appears to be linked to the geographical origin of the studied specimens (Fig 5). In the same manner, the genetic distance observed between individuals of Ph. sergenti from the West Mediterranean area (Portugal, Algeria, Morocco) and those from Egypt and Afghanistan are respectively of 1.1% and 1.2%. The divergence observed within Ph. sergenti is slightly lower than the one observed in Ph. alexandri but however remains substantial. Such a high intraspecific divergence within these two species has already been observed [8688] and deserves further studies to clarify the exact status of the subclades observed within Ph. alexandri and Ph. sergenti. This is of prime importance as it may have strong consequences on the epidemiology of Leishmania infantum, L. major and to a lesser extent of L. donovani. Importantly, our results highlight the power of nuclear pangenomic markers over mitochondrial genes to analyse genetic differentiation and phylogeography of phlebotomine sand flies. Indeed, several species-complexes of Phlebotomus may have experienced mitochondrial introgression and share haplotypes between species, even when nuclear gene flow is restricted [65,89,90]. In these complex, frequent mito-nuclear discordance due to introgression, incomplete lineage sorting, endosymbionts or mis-identification renders species delimitation difficult. This is probably where reliable markers for identification are mostly wanted but this is also where barcoding approaches (based on COI or Cytb genes) may be deficient and have to be used cautiously.

RADseq to improve our understanding of the epidemiology of arthropod borne diseases

RAD sequencing is now widely used for population genetics and phylogenetics. Notably, the method has been shown effective to decipher species complexes or to detect introgression [91]. However, it is still rarely used to study minute insects due to the limiting DNA amount that can be extracted. Here, we confirm that multiple displacement amplification method can be used to generate unbiased copies of genomes to make possible the sequencing of RAD markers from tiny specimens [34]. Coupled with continuous progress in high-throughput sequencing technology, ever larger numbers of samples can be processed at the same time which make possible large-scale studies. For instance, with the PstI enzyme used here, about 400 individuals of phlebotomines could be sequenced on a S1 NovaSeq flow cell.

Several studies have shown that members of phlebotomine species complexes can display contrasted differences in terms of biology and vector capacity [92]. Therefore, deciphering species-complexes with such a high-throughput and powerful method may contribute to better identify biological entities of importance for the transmission of Leishmania. Further, RADseq characterization of species complexes may help to i) better delimit the geographical distribution of the different species; ii) identify phenotypic traits relevant to the disease epidemiology and iii) analyse intraspecific population structure within entities and genetic exchanges, if any, between them.

Once thoroughly delimitated molecularly, the specific level of these entities could be validated by crossbreeding experiments and/or other analyses focusing on chemical and behavioral characters (partner courtship, attraction by sex pheromones, etc). Subsequently, species need to be characterized morphologically, if possible, and named. The accurate species delimitation done using WGA and RAD markers, coupled with non-destructive extraction (see [93]), may facilitate the identification of diagnostic morphological characters. Finally, “barcoding-like” markers that enable accurate molecular identification of the species within analysed complexes could then be developed to enable fast and massive identification to survey large areas. The WGA+RAD approach developed here, that can be used on single specimens, may therefore represent a powerful tool to improve our understanding of the epidemiology of leishmaniases and possibly also other arthropod borne human diseases.

Supporting information

S1 Text. Input data and results of the ancestral range estimation using BioGeoBears.

Matrix of geographic ranges and map of geographic regions

(DOCX)

S2 Text. Input data and results of the ancestral range estimation using BioGeoBears.

Dispersal multipliers matrices and Time Periods

(DOCX)

S3 Text. Input data and results of the ancestral range estimation using BioGeoBears.

Results of model testing

(DOCX)

S4 Text. Input data and results of the ancestral range estimation using BioGeoBears.

Ancestral range estimations for all tested models as inferred by BioGeoBears

(DOCX)

S1 Fig. Phylogenetic tree obtained for the COI data set.

(PNG)

S2 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 6; radis_nsample_min = 50%; 5,050 loci: RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

(BMP)

S3 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 6; radis_nsample_min = 75%; 1,423 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tagsData sets are described in Table 2.

(BMP)

S4 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 8; radis_nsample_min = 50%; 7,194 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

(BMP)

S5 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 8; radis_nsample_min = 75%; 2,086 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

(BMP)

S6 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 10; radis_nsample_min = 50%; 8,906 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tagsData sets are described in Table 2.

(BMP)

S7 Fig. Phylogenetic tree obtained for the RAD data sets.

cstacks n = 10; radis_nsample_min = 75%; 2,808 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags Data sets are described in Table 2.

(BMP)

S1 Table. Information about samples used in this study and results of the RAD-seq experiment.

(XLSX)

Acknowledgments

The authors thank Carlos Alves-Pires, Azzedine Bounamous, Vit Dvořák, Nabil Haddad, Hanafi Hanafi, Najoua Haouas, Vladimir Ivovic, Kaouther Jaouadi, Mireille and Bob(†) Killick-Kendrick, Andreas Krüger, Nicole Léger, Michele Maroli, Laor Oshan, Yusuf Özbel, Jean-Antoine Rioux(†), and Petr Volf for providing specimens and/or for fruitful discussions about the distribution of Ph. similis and Ph. sergenti in Turkey and in the Balkans. We thank Zoe Adams (NHM, London) and Zoubir Harrat (Pasteur Institute of Algeria) who arranged the loans of type-specimens of Ph. alexandri and Ph. sergenti, respectively. We also are grateful to Morad Belbagra for the technical assistance in data processing, to Laure Sauné for advices during library preparation, to Montpellier GenomiX (MGX, France) for sequencing and to the genotoul bioinformatics platform Toulouse Occitanie (Bioinfo Genotoul, doi: 10.15454/1.5572369328961167E12) for providing computing resources.

Data Availability

Fastq reads for analysed samples are available as a NCBI Sequence Read Archive (PRJNA725143). COI sequences were uploaded in Genbank IDs #MZ049642-MZ049672. Data sets and trees have been uploaded on Zenodo (https://doi.org/10.5281/zenodo.4721254).

Funding Statement

The authors received no specific funding for this work.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009479.r001

Decision Letter 0

Jaap J van Hellemond, Remi N Charrel

15 Apr 2021

Dear Dr. Depaquit,

Thank you very much for submitting your manuscript "Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Psychodidae) inferred using restriction-site associated DNA markers." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Remi N. Charrel

Associate Editor

PLOS Neglected Tropical Diseases

Jaap van Hellemond

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes.

Hypotheses - With the use of advanced molecular techniques that make it possible to obtain impartial copies of the entire genome when there is a low amount of DNA and obtain sequencing of pangenomic markers for tiny species, as is the case of sandflies to be used in phylogenetic analysis with results robust?

The objectives - To investigate for the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotominae), constituted by important vector species of Leishmania that cause visceral and cutaneous leishmaniasis in the Old World, the phylogeny of the group, as well as divergence time estimates and probabilistic inference of historical biogeography of the subgenus.

-Is the study design appropriate to address the stated objectives?

Yes

The design of the study involved obtaining the sequences (Whole Genome Amplification, WGA) and RAD (Restriction site Associated DNA) of most species of the subgenus to develop the phylogenetic analysis and also of specimens from most of the areas where they occur. Therefore, I consider the design appropriate to meet the objectives.

-Is the population clearly described and appropriate for the hypothesis being tested?

Studies focusing on the systematics of groups generally use few individuals. In the present case, this is a great differential, since from the genomic information of a few individuals it was possible to obtain robust inferences about the phylogeny of the group. The information for the specimens is adequate.

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

See comments above

-Were correct statistical analysis used to support conclusions?

Yes

The statistics used to infer phylogeny have often been adopted in studies of phylogenetic systematics. To estimate the time of divergence between the clades they adopted a recently proposed method - RelTime method, relative rate framework, which presents results very close to those of more conventional methods, for example Bayesian framework.

-Are there concerns about ethical or regulatory requirements being met

Not applicable

Reviewer #2: The objectives are clear, the study is well design.

I just have a comment: L268-L271. I do not think this part is necessary.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan?

The analysis plan was presented and commented on for each topic.

-Are the results clearly and completely presented?

Phylogenetic trees are illustrated and commented using COI which, like other studies of the marker, does not present good phylogeny resolution. On the other hand, trees with RAD sequences considering the parameters that could limit the results confirm the same clades for all sets of parameters, with great robustness. On the other hand, the trees clearly show the paraphilia of one of the species that until then comprised the subgenus Paraphlebotomus and for which a new subgenus has been proposed. The trees that show the clades' divergence time as well as the probable locations that each clade appears according to the regions of the Old World are illustrated and commented.,

-Are the figures (Tables, Images) of sufficient quality for clarity?

With the exception of Table S1, which was not available, all tables and images are of high quality.

Reviewer #2: The analysis presented match the analysis plan, they are clear and completely presented.

The figures are good and clear.

I just have a question, L293: “16.6–34.6% missing data”. Do the authors have any idea why this rate? Is it really low 1/3 of missing data?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented?

Yes.

-Are the limitations of analysis clearly described?

Yes

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

The methodological approach used to infer phylogeny is discussed and, most likely, it will generate an impact among researchers for systematic studies in obtaining phylogenies, defining more faithfully the monophyletic groups and the possibility of retrieving the evolutionary history of the groups.

-Is public health relevance addressed?

The relevance of the study was commented by the authors, in the sense that the group comprises several vector species of Leishmania that cause visceral and cutaneous leishmaniasis in the Old World. Knowing the relationship of the focus group with its sister groups and between species within the group, can also help to understand the agent-vector-host relationship.

Reviewer #2: The article is well written, interesting and complete.

It could be a plus to have pictures of the described specimens especially for people having less notion in identification of sandflies. Example L 349 « Artemievus subg. nov. Depaquit. Type-species: Phlebotomus alexandri Sinton, 1928. »

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor corrections to be made.

–line 27 Ph. alexandri, replace by Phlebotomus alexandri.

Line 32 – give space after 5.3

Line 142 – Nine outgroups replace with Nine outgroup specimens

Lines 163 to 177 – replace µl with µL

line 331 and (italic); it shoud be non italic

In table 1. Standardize the number of letters for the subgenera. Only Paraphlebotomus has four letters.

Make table S1 available.

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Excellente work. As suggestion regarding the new subgenus proposed, I think it would add quality to the work if illustrations were presented on the morphological characters that make the distinction between Paraphlebotomus and Artemievus possible. I suggest illustrations of the type-species of Paraphlebotomus and Phlebotomus alexandri.

Reviewer #2: As a summary comments, I can only say that it is a good article.

--------------------

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Reviewer #2: No

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009479.r003

Decision Letter 1

Jaap J van Hellemond, Remi N Charrel

15 May 2021

Dear Dr. Depaquit,

We are pleased to inform you that your manuscript 'Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Psychodidae) inferred using restriction-site associated DNA markers.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Remi N. Charrel

Associate Editor

PLOS Neglected Tropical Diseases

Jaap van Hellemond

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009479.r004

Acceptance letter

Jaap J van Hellemond, Remi N Charrel

13 Jul 2021

Dear Dr. Depaquit,

We are delighted to inform you that your manuscript, "Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Text. Input data and results of the ancestral range estimation using BioGeoBears.

    Matrix of geographic ranges and map of geographic regions

    (DOCX)

    S2 Text. Input data and results of the ancestral range estimation using BioGeoBears.

    Dispersal multipliers matrices and Time Periods

    (DOCX)

    S3 Text. Input data and results of the ancestral range estimation using BioGeoBears.

    Results of model testing

    (DOCX)

    S4 Text. Input data and results of the ancestral range estimation using BioGeoBears.

    Ancestral range estimations for all tested models as inferred by BioGeoBears

    (DOCX)

    S1 Fig. Phylogenetic tree obtained for the COI data set.

    (PNG)

    S2 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 6; radis_nsample_min = 50%; 5,050 loci: RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

    (BMP)

    S3 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 6; radis_nsample_min = 75%; 1,423 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tagsData sets are described in Table 2.

    (BMP)

    S4 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 8; radis_nsample_min = 50%; 7,194 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

    (BMP)

    S5 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 8; radis_nsample_min = 75%; 2,086 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags. Data sets are described in Table 2.

    (BMP)

    S6 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 10; radis_nsample_min = 50%; 8,906 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tagsData sets are described in Table 2.

    (BMP)

    S7 Fig. Phylogenetic tree obtained for the RAD data sets.

    cstacks n = 10; radis_nsample_min = 75%; 2,808 loci; RAxML tree (unpartitioned data set); Bootstrap values at nodes (100 replicates); %missing = % of missing RAD tags Data sets are described in Table 2.

    (BMP)

    S1 Table. Information about samples used in this study and results of the RAD-seq experiment.

    (XLSX)

    Attachment

    Submitted filename: reply-to-reviewers.doc

    Data Availability Statement

    Fastq reads for analysed samples are available as a NCBI Sequence Read Archive (PRJNA725143). COI sequences were uploaded in Genbank IDs #MZ049642-MZ049672. Data sets and trees have been uploaded on Zenodo (https://doi.org/10.5281/zenodo.4721254).


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