J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 36 http://jad.tums.ac.ir Published Online: March 31, 2023 Original Article Descriptive and Geometric Morphometry of the Wings of Phlebotomus sergenti Populations in Central Morocco Fatima-Zahra Abou-Elaaz1, *Denis Sereno2, Oumnia Himmi1, Mohamed Ghamizi3, *Souad Guernaoui4 1Geophysics, Natural Patrimony and Green Chemistry Research Centre (GEOPAC), Geo-Biodiversity and Natural Patrimony Laboratory (GEOBIOL), Scientific Institute, Mohammed V University in Rabat, Morocco 2InterTryp, Parasite Infectiology and Public Health Research Group, MiVegec, IRD, Montpellier University, Montpellier, France 3Water, Bidiversity and Climate Change Laboratory, FSSM, Museum of Natural History of Marrakesh, Cadi Ayyad University, Marrakesh, Morocco 4Biotechnology, Conservation and Valorisation of Natural Resources laboratory, Faculty of Sciences Dhar El Mahraz, Sidi Mohamed Ben Abdellah University, Fez, Morocco *Corresponding authors: Prof Souad Guernaoui, E-mail: souad.guernaoui@usmba.ac.ma; Dr Denis Sereno, E-mail: denis.sereno@ird.fr (Received 27 Sep 2022; accepted 14 Feb 2023) Abstract Background: Phlebotomus sergenti, the proven vector of Leishmania tropica, the causative agent of anthroponotic cu- taneous leishmaniasis, is widely distributed in Morocco. Previous works using molecular markers (Internal Transcribed Spacer 2 rDNA and Cytochrome B mtDNA) hypothesized the existence of multiple closely related populations of sand fly species (cryptic species) that would exhibit distinct vectorial capacities. This work studies morphotypic diversity using traditional and geometric morphometry analyses carried out on Ph. sergenti's wings from central Morocco, where active L. tropica transmission occurs for 30 years. Methods: Descriptive characteristics (size and shape) of the right wings were measured in Ph. sergenti’s specimens collected from fourteen stations in central Morocco. Both traditional and geometric morphometry methods were used to analyse geographic variations in Ph. sergenti wing’s size and shape. Results: These analyses support the existence of distinct Ph. sergenti populations, enlightening significant phenotypic variations of Ph. sergenti’s wings, regarding their size and shape, depending on geographic origin. In addition, tradi- tional and geometric morphometric analyses of the wing’s length, centroid size, β, ɵ, and γ distances allowed clear dis- crimination of Ph. sergenti sub-populations. Conclusion: These data pinpoint the adaptative ability of Ph. sergenti to local environmental conditions. Additional studies are now required to further shed light on the genetic structure of Ph. sergenti populations in Morocco. Keywords: Phlebotomus sergenti; Wing traditional morphometry; Wing geometric morphomery; Morocco Introduction Sand flies (Diptera, Psychodidae) are vectors of human pathogenic micro-organisms, including parasites of the genus Leishmania, bacteria (Bar- tonella bacilliformis), and various arboviruses belonging to the genera Phlebovirus and Vesic- ulovirus (1–3). They represent, therefore, a pub- lic health scourge. In Morocco, the phlebotomine fauna encompasses 24 species. Six species are proven, or suspected vectors involved in vis- ceral or cutaneous leishmaniases (4). Variability in genetic, biochemical, morpho- metric, or reproductive features between allopat- ric populations of the subfamily Phlebotominae are frequent and probably favored by their large Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ mailto:souad.guernaoui@usmba.ac.ma mailto:denis.sereno@ird.fr https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 37 http://jad.tums.ac.ir Published Online: March 31, 2023 geographic distribution and the limited disper- sal capacity of populations with reproductive activity near their breeding site (5). These would lead to speciation processes not detectable with dichotomic keys (6). Phlebotomus sergenti, Par- rot, 1917, is a proven Leishmania tropica, vec- tor, the causative agent of anthroponotic cuta- neous leishmaniasis (ACL) (1, 7–9). Phleboto- mus sergenti is distributed from the southern to the western Mediterranean basins. It is re- placed in the northeast of this region by Ph. similis Perfiliev, 1963 (10). Its distribution ex- tends eastward into Iran, the former USSR, Pakistan, and India. Such geographic distribu- tion vastly exceeds that of L. tropica (10). The genetic diversity of Ph. sergenti investigated with Internal Transcribed Spacer 2 (ITS2) rDNA and/or Cytochrome B (Cyt b) mtDNA light- ened the possibility of closely related sand fly species (cryptic species) that may have distinct vectorial capacities (11-15). Phlebotomus ser- genti is widely distributed throughout Moroc- co, with a preference for semi-arid habitats (16, 17). In ACL foci, Ph. sergenti is closely associated with the human habitat and involves a domestic and peridomestic transmission of L. tropica (17, 18). The analysis of the popu- lation's genetic structure of Ph. sergenti from Moroccan foci of Taza, Azilal, and Essaouira, using Cyt b mtDNA, disclosed the presence of three mitochondrial lineages with a striking genetic diversity in the Azilal focus (12). Geometric morphometry (GM) has been applied to analyze morphological variations of insect wings (19). It has a taxonomic value for specimens belonging to puzzling morpho- logical taxa (20) and highlights Spatio-tem- poral dynamics of some Phlebotomus popula- tions; Ph. tobbi Adler and Theodor 1930, Ph. papatasi (Scopoli, 1786), Ph. ariasi Tonnoir 1921 and Ph. sergenti (21–25). In this work, traditional and geometric morphometry anal- yses were carried out on Ph. sergenti's wings from central Morocco, where active L. tropica transmission occurs for 30 years (26). Materials and Methods Study area The studied area locates in central Moroc- co (Fig. 1), where cutaneous leishmaniases (CL) are widespread (26); the first CL case due to L. tropica was reported in 1986 in Azilal (27). The environment is diverse, with altitudes var- ying from 491m to 1650m above sea level. It encompasses the Tadla plain, the phosphate plateau, and the High and Middle Atlas Moun- tains (Fig. 1, Table 1). The climate is continental, with a wet sea- son (November to March) and a dry season (April to October). The annual average tem- perature is 18 °C but ranges from 2 °C to 40 °C. The temperatures recorded during the cap- ture period are given in Table 1. Precipitation varies from 100mm to 1200mm. Béni Mellal- Khénifra region endow the natural forest of holm oak (Quercus rotundifolia), junipers (Ju- niperus phoenicea), thuja (Tetraclinis articu- lata), pines (Pinus halepensis, Pinus pinaster), cedar (Cedrus atlantica), poplar (Populus nigra), and carob tree (Ceratomia siliqua). This re- gion also has agricultural activity; cereals, ol- ives, citrus fruits, sugar beets, grenadines, al- monds, apples, carobs, sesame, and vegeta- bles (28). Sandflies sampling and identification Sampling was carried out using sticky pa- per traps placed in domestic, peridomestic, and wild biotopes in fourteen stations between May and October 2017. Traps were left in the field for a single night to avoid the deterioration of wings. In the laboratory, each specimen was dissected under a binocular. We first separat- ed the wing for each male, and then the head and genitalia were dissected and mounted in Canada balsam. Next, we identified specimens by examining the morphology of the external genitalia. Identification was performed using published keys and descriptions (10, 29). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 38 http://jad.tums.ac.ir Published Online: March 31, 2023 Wings preparation Well-preserved wings of specimens iden- tified as Ph. sergenti were prepared following the described protocol (30), with modifications by reducing the incubation time to 10min in 5% KOH, in Methylene Blue to 10min. This is sufficient to visualize veins and ribs without damaging the wings. After treatment, wings are mounted in Canada balsam on marked slides and photographed with a digital camera (Leica microsystem CH) connected to a microscope (Leica) in the Museum of Natural History of Marrakech Cadi Ayyad University, Morocco. Measurements Measures were carried out using the Gryphax 12.0 software (Jenoptik) (31). In addition, pic- tures were processed with tps-UTIL 32 ver- sion 1.74 and tps-Dig 2 version 2.30 software (32). We considered the 17 landmarks already used in previous studies of other sand fly spe- cies (21, 22). These landmarks are located at the wing veins’ intersections with their mar- gin and the crossroads of the transverse veins with the prominent veins, as depicted in Fig. 2. The size of Ph. sergenti wings is given by measures of length (5–17), width (1–9), and centroid size (CS) (Fig. 2). The CS is defined by the square root of the sum of the squared distances between the centre of the configura- tion of landmarks and each landmark. It was extracted from each matrix using MorphoJ 1.07a version 1.8.0_251 software (33). The analysis of the wing shape is based on the coordinates of the 17 landmarks and seven distances α (2-12), β (12-11), δ (1-11), ɛ (3- 12), ɵ (4-11), π (10-11) and γ (11-14) (5, 25). Data analysis Traditional Morphometry Traditional morphometry aims to describe the wing’s morphology and delineate quanti- tative morphological characteristics that allow discrimination of Ph. sergenti subpopulations. Firstly, we performed a descriptive analysis of the length, width, CS, and seven distances. Then, the mean, standard deviation, median, mode, and variation ratio were calculated. Next, the normality and variance homogeneity assump- tions were analysed for each variable using normalized plots. Secondly, for each charac- ter, the Student t-test was used to compute the significance of the differences at a significance level of 0.05. Finally, the link between each measure (size, shape) (quantitative variable) and station (qualitative variable) was examined by estimating the correlation ratio (r) that is used for allometric analysis to test the link between the wing’s shape and size (CS, length, width). Univariate and bivariate statistical analyses were performed using Microsoft Excel 10. In addition, a typology of wing morphs (size, shape) according to the geographical origin was made using canonical analysis, carried out with the STATISTICA software (34). Geometric morphometry The geometric morphometric analysis al- lows the visualization and comparison of the geometric configurations of Ph. sergenti wings. First, Ward's method (35) is used to construct the hierarchical classification dendrograms for wing size and shape, using Past 3.23 software (36). Then, the Ph. sergenti wing geometric configurations were investigated using a Gen- eralized Procrustes Analysis (GPA) (37). Af- ter GPA, shape variables were measured and analyzed by the principal component of the “relative warps” scores calculated using the Past 3.23 software (36). Results This study used descriptive and geometric morphometry analyses of Ph. sergenti wings. It investigates the size and shape of a set of stations from central Morocco differing in their ecological characteristics. Ninety-two wings of Ph. sergenti were collected in 14 stations in cen- tral Morocco (Table 1). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 39 http://jad.tums.ac.ir Published Online: March 31, 2023 Wing size and shape As described in the Material and methods section, the wing's size measured is presented in Supplementary data Table 3. Overall, in males, the length of the right wings is 2137.01±124.05µm, the width is 738.17±69.78 µm, and the CS is 3236.79±160.66µm. The shape of Ph. sergenti wings is charac- terized by seven distances: α, β, δ, ɛ, ɵ, π, and γ (Supplementary data Table 4). The wing shape varies mainly in the β (12-11), ɵ (4-11), and γ (11-14) distances. All measurements are log-normally distributed. Allometric analysis was performed to as- sess the link between wing size and shape by estimating the correlation coefficient. As a re- sult, a positive correlation is recorded particu- larly between, on the one hand, the 3 distanc- es ɵ, α, and β, and on the other hand, the size parameters, mainly the CS and the width, as shown in Table 2. Wings variability analysis To investigate the spatial variations of Ph. sergenti’s wing parameters from stations where the Ph. sergenti density is low, we gathered samples from stations 2, 3, 4, 5, 7, 10, 12, 13, and 14 into the label “other stations” as shown in (Figs. 3 and 4). The size of the right wing shows signifi- cant differences linked to their geographic origin (Fig. 3), as demonstrated by the correlation ra- tios computed between the origin and the length, width, or CS (0.68 and 0.56 and 0.74, respec- tively). Similarly, the seven distances characterizing the wing shape showed differences according to the stations (Fig. 4), with β, ɵ, and γ as the most variable distances. The correlation ratios between the collection site and the distances α, β, δ, ɛ, ɵ, π and γ are 0.39, 0.49, 0.37, 0.39, 0.55, 0.31 and 0.46, respectively. Wings of Ph. sergenti of Afourer exhibit peculiar characteristics, as shown in Figs. 3 and 4. Differences were noticed, especially for the CS, β, and ɵ distances. Furthermore, the CS in Afourer is significantly different from all oth- er stations, even the neighbouring ones. The student t-test are; Bzou (t= -10,626), Foum Ja- maa (t= -23,531), Tiski (t= -4,281), Ksiba (t= -10,747). As for results from the descriptive analy- sis, canonical analyses of the size and shape of Ph. sergenti wings, illustrated in figure 5, point to substantial variation according to the wings' geographical origin (sampling site). Ca- nonical analysis of the size (Fig. 5A) discloses that the plane (1-2) explains 92.57% of the to- tal inertia (canonical variate 1: 76.43% and canonical variate 2: 16.14%). These results in- dicate that the wing size differs from station to station. First, specimens from Afourer and Tiski are grouped on the right of the canonical analysis, followed by those from Bzou, Foum Jamaa, and the other stations (stations 2, 3, 4, 5, 7, 10, 12, 13, and 14). For the wing shape, the projection in the plane (1-2) explains 63.06% of the total inertia (canonical variate 1: 43.23% and canonical variate 2: 19.83%) (Fig. 5B). As for the parameter of wing size, the canonical analysis reveals gradual variations according to the stations. The hierarchical single-link classification trees for the measured parameters of size (Fig. 6A) and wing shape (Fig. 6B) demonstrate a clustering according to the geographic origin, which again reinforces the results of the ca- nonical analysis. But, again, and particularly for wing size, two groups stand out; the pop- ulations of Afourer and Tiski with smaller wings according to measured parameters vary from those of Foum Jamaa, Bzou, or the other sta- tions. Likewise, the mean configurations of the wing shapes vary according to the geograph- ical origin, with marked differences in the position of landmarks 2, 9, 10, and 11, as pre- sented in Fig. 7. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 40 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 1. Stations sampled for Phlebotomus sergenti in central Morocco during the period May–October 2017 *Lack of data. **(26) CL: Cutaneous leishmaniasis Table 2. Correlation coefficient (r) between Phlebotomus sergenti wing’s parameters (shape and size) during the peri- od May–October 2017 Shape Size CS Length Width ɵ 0.69 0.54 0.70 α 0.55 0.32 0.66 β 0.47 0.30 0.48 ɛ 0.34 0.26 0.28 π 0.32 0.14 0.27 δ 0.26 0.08 0.29 γ 0.08 0.19 0.0007 Table 3. Morphometry of Phlebotomus sergenti male’s right-wing collected in the study region in central Morocco during the period May–October 2017 Numerical parameters Length Width Centroid size Mean±SD (µm) (Min-Max) (µm) 2137.01±124.05 (1880-2340) 738.17±69.78 (598-935) 3236.79±160.66 (2855-3668) Mode (µm) 1990.00 700.00;725.00; 740.00 3136.00;3149.00; 3181.00 Median (µm) 2170.00 725.50 3205.00 Variation ratio (%) 5.80 9.45 4.96 Stations N° Longitude (W) Latitude (N) Altitude (m) Temperature (°C) Mean (Min-Max) Urbanization CL cases** (2009 to 2015) Specimens number Afourer 1 06°30´00´´ 32°13´00´´ 491 26 (18-34) Urban 433 13 Ait Imloul 2 06°38´18´´ 32°11´00´´ 1118 * Rural * 6 Aguelmouss 3 05°50´35´´ 33°09´30´´ 1217 * Rural * 2 Béni Ayyat 4 06°34´01´´ 32°12´31´ 535 * Rural 37 1 Béni Mellal 5 06°22´32´´ 32°20´32´´ 507 27 (23-31) Urban 90 2 Bzou 6 07°03´18´´ 32°05´09´´ 429 26.5 (19-34) Rural 489 11 El Kbab 7 05°31´01´´ 32°44´27´´ 1198 * Rural * 1 El Ksiba 8 06°01´58´´ 32°33’54´´ 1003 * Urban 5 8 Foum Jamaa 9 06°59´26´´ 31°37´54´´ 813 24.5 (17-32) Rural 386 21 Tagzirt 10 06°12´01’’ 32°26´08´´ 594 * Rural 33 3 Tiski 11 06°46´12´´ 32°06´44´´ 677 25.5 (18-33) Rural 178 16 Zaouiat Cheikh 12 05°55´01´´ 32°38’38´´ 798 * Urban 202 1 Zaouiat Ahansal 13 06°06´15´´ 31°49´59´´ 1629 * Rural 2 3 Benssarou 14 06° 09´42´´ 32°21´16´´ 1650 * Rural * 4 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 41 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 4. Numerical characteristics of the distances α, β, δ, ε, ɵ, π and γ of Phlebotomus sergenti male’s right-wing collected in central Morocco during the period May–October 2017 Numerical parameters α β δ ɛ ɵ π γ Mean±SD (µm) (Min-Max) (µm) 540.19±70.91 (399-782) 375.61±86.86 (230-835) 173.84±54.86 (108-502) 367.30±60.60 (255-787) 604.79±44.55 (510-690) 260.5±37.65 (200-488) 621.92±104.47 (385-984) Mode (µm) 510.00; 525.00 420.00 118.0;144.0; 161.0;171.0; 182.0; 203.0 320.00; 350.00 560.00; 600.00 243.00 515.00; 560.00; 610.00 Median (µm) 530.00 359.00 168.0 360.00 605.00 253.00 610.00 Variation ratio (%) 13.13 23.12 31.55 16.50 7.36 14.45 16.80 Fig. 1. Location of the sampling area for Phlebotomus sergenti populations in central Morocco (Source Google Earth) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 42 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 2. Location of the 17 landmarks used in this study on the right-wing of Phlebotomus sergenti Fig. 3. Boxplots of the lengths, widths, and centroid sizes of Phlebotomus sergenti male’s right wings in the various station of central Morocco during the period May–October 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 43 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 4. Boxplots representation of the distances α, β, δ, ε, ɵ, π, and γ of the right wings of Phlebotomus sergenti’s males in each station of central Morocco during the period May–October 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 44 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 5. Canonical analysis of the size variations (A) (92.57% of the total inertia) and the shape (B) (63.06% of the total inertia) of Phlebotomus sergenti’s male right wings depending on the geographical origin of central Morocco during the period May–October 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 45 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 6. Hierarchical classifications tree of Phlebotomus sergenti right-wings size (A) and shape (B) of central Morocco Fig. 7. Superposition of the right-wing landmarks (mean measures) of Phlebotomus sergenti males from Afourer (A), Tiski (B), El Ksiba (C), Bzou (D), Foum Jamaa (E), and other stations (F). (Arrows of a given color show variations in the position of a given landmark) of central Morocco during the period May– October 2017 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 46 http://jad.tums.ac.ir Published Online: March 31, 2023 Discussion Phlebotomus sergenti is a proven and pri- mary vector of L. tropica in Saudi Arabia, Mo- rocco, Iran and Israel (1, 7–9, 38). Previous studies have considered Ph. sergenti a species complex regarding the tremendous genetic di- versity recorded over its distribution (11–13, 39). On the other hand, the cross-mating study showed that there is no reproductive barrier between Ph. sergenti from different geograph- ical areas (24), and no statistically important differences were found in wing morphology for all local populations of Ph. sergenti, suggest- ing that the barriers are not sufficient to stop gene flow among local populations of sand flies (25). Despite this ambiguous context, no mor- photype indicator is currently available to probe Ph. sergenti population diversity. Therefore, the wing's geo-morphometric analyses would help shed light on the population's diversity of Ph. sergenti. This has been used successfully on several sand fly species, like Ph. tobbi, Ph. pa- patasi, or Ph. ariasi (21–23, 30), and has pro- vided evidence of the separation of Ph. papa- tasi populations between the southern and north- ern slopes of the High Atlas Mountains in Mo- rocco (21). This was further delineated via a genetic analysis of these populations (40). There- fore, we applied descriptive and geometric mor- phometry analyses to probe Ph. sergenti popu- lations' diversity in central Morocco for the first time. The morphometric parameters of Ph. ser- genti’s wings sampled in central Morocco show significant variability. The size is variable in length, width, and CS. The shape also showed substantial variations, particularly at the dis- tances; β, ɵ, and γ. In addition, allometric anal- yses demonstrate a strong link between size and shape variability. A correlation is record- ed between CS or wing width on one side and ɵ, α or β, on the other side. Descriptive analyses point to a link be- tween Ph. sergenti wing’s size and shape and the geographical origin. Length, width, CS, and the seven distances characterizing the wing’s shape differ significantly between stations, with correlation ratios higher than 50%. Population from Afourer locality is notably different, with relatively smaller wings. Multivariate analyses further supported these results. Canonical stud- ies reveal, in addition to the variability, the existence of a gradual variation in size and shape (92.57% and 63.06% of the total inertia, respectively). This population’s succession in the factorial plan could testify to a cline varia- tion of the measured parameters. The results of the geometric analyses cor- roborate those of the descriptive ones. Hierar- chical classifications trace the gradual varia- tion and define two types based on the geo- graphic origin; the Foum Jamaa and Bzou types mainly differ from the Afourer and Ti- ski types. Relatively smaller wings character- ize these last two populations. In addition, su- perimpositions of the average configurations of Ph. sergenti male right wings indicate sig- nificant variations in landmarks 2, 9, 10, and 11. Previous works have focused on the rela- tionship with altitude regarding the origin of the variability of sand fly wings in the natural environment. However, altitude is not an eco- logical factor, but it can express a variety of climatic factors. In this study, we conducted a bi-monthly sampling to integrate variations in connection with climatic factors in central Mo- rocco. In areas where Ph. sergenti is abun- dant, it was active for only two months, July and August, during the dry period. Therefore, no significant temporal variation in wing pa- rameters (size and shape) was recorded, prob- ably due to the short active period. Wings of Ph. ariasi (France), Ph. papatasi and Ph. tobbi (Turkey) showed variations in response to an altitudinal gradient (22, 23, 30). But, even via an indirect effect, altitude can- not explain the recorded gradual variation in Ph. sergenti from central Morocco. The indi- vidualized populations of Afourer and Tiski http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 36–50 FZ Abou-Elaaz et al.: Descriptive and … 47 http://jad.tums.ac.ir Published Online: March 31, 2023 locate in the same altitudinal zone as Bzou and Foum Jamaa. Furthermore, the altitudinal structuring of sand flies observed in the High Atlas Mountains concerned Ph. papatasi, Ph. ariasi, and the species of the Ph. perniciosus complex (41). The density and distribution of Ph. sergenti did not follow any altitudinal gradient (41). Altitude can also act indirectly through the slope effect. This has been observed in Ph. papatasi on the southern and northern slopes of the Moroccan High Atlas (21) and in Ph. tobbi (22) and Ph. ariasi (23). But this slope effect cannot be involved in the variation we detected in Ph. sergenti; the stations compared to Afourer, Bzou, Tiski, and Foum Jamaa are located on the same High Atlas Mountains slope. Moreover, the stations which show Ph. sergenti wing variations are located in the same zone of the High Atlas Mountains, at a maximum of 60km apart. Therefore, they are subject to a similar climate, and considering the low dispersal potential of male sand flies, this adds evidence for a microenvironmental adaptation. This phenomenon is also reported in Ph. ariasi in the Oiselette massif in the south of France (23). Furthermore, local habitat adaptation of Ph. sergenti populations was observed in the west- ern Moroccan High Atlas Mountains and other L. tropica foci. Although Ph. sergenti is a wide- ly distributed species, its abundance is linked to a particular microhabitat where shelters are present (areas in basins) and where the dwell- ings (construction material) are not made of cement (17). Conclusions In conclusion, our results underline the di- versity of Ph. sergenti in Morocco. Descrip- tive and geometric morphometry analyses are exciting tools for quantifying phenotypic var- iability in Ph. sergenti populations. They also point out the ability of Ph. sergenti to adapt to local environmental conditions. The correlation between the phenotypic differentiation and the genetic structure of Ph. sergenti in Morocco must be further investigated. 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