Statistical analyses of morphological variations of three <i>Larroussius</i> (Diptera: Psychodidae) sister species collected in leishmaniasis endemic foci of Adana in Türkiye

Sand flies are arthropod vectors responsible for transmitting Leishmania parasites to humans. Among them, Phlebotomus (P.) major, P. syriacus and P. neglectus are closely related sister species that play an important role in disease transmission in Türkiye. This study aimed to statistically analyse the morphological measurements of these three species and to identify reliable diagnostic characters. Seven morphometric traits were measured in both female and male specimens. Statistical analyses, including analysis of variance (ANOVA), discriminant function analysis (DFA) and principal component analysis (PCA), were performed to determine significant variables contributing to species differentiation. A total of 1729 sand flies were collected, comprising P. tobbi (44.58%), P. papatasi (21.26%), P. similis (20.30%), P. neglectus (8.50%), P. major (2.88%), P. syriacus (2.19%) and Sergentomyia (S.) fallax (0.27%). The female-to-male ratio was 1.42. ANOVA revealed significant interspecific differences (p < 0.001) in antennal, pharyngeal and genital characters. In females, P. neglectus exhibited shorter antennal segments A3 and A4 + A5, P. syriacus had the shortest pharynx (PHX), and P. major showed the longest epipharynx (EPI). In males, P. major had the longest coxite (CX), whereas P. neglectus displayed the shortest second style segment (S2) (p < 0.05). DFA confirmed clear species separation, with antennal and pharyngeal traits primarily driving differentiation in females, and genital characters being most informative in males. PCA explained 78.37% and 90.24% of the total morphometric variation in females and males, respectively, highlighting sex-specific patterns of morphological variation. Overall, these statistically supported morphometric differences provide robust diagnostic features that can complement molecular approaches, improving species identification and enhancing taxonomic resolution in sand fly vector studies.