Hidden diversity in anthropophilic sand flies of the Monticola Series (Diptera, Psychodidae)

The Monticola series comprises two anthropophilic and widely distributed species in Brazil: Pintomyia (Pifanomyia) monticola (Costa Lima, 1932) and Pintomyia (Pifanomyia) misionensis (Castro, 1959). They mainly occur in the Atlantic Rainforest, and it is known that Pi. monticola comprises at least two well-structured genetic lineages regarding a fragment of the cytochrome c oxidase subunit I (COI) gene. Here, we aim to elucidate the taxonomic status of this group using integrative taxonomy tools. Collections were performed in nine localities of four Brazilian states, and COI fragments were sequenced and merged with publicly available data. Several single-locus species delimitation algorithms, genetic distance metrics, phylogenetic trees, and haplotype networks were used to uncover cryptic diversity and population structure within Pi. monticola and Pi. misionensis. The resulting genetic clusters were then tested for morphological differences through linear and geometric morphometry of several characters. We analyzed 152 COI sequences, comprising 48 haplotypes. The maximum intraspecific p distances were 8.21% (mean 4.17%) and 9.12% (mean 4.4%) for Pi. monticola and Pi. misionensis, respectively, while interspecific ones ranged from 10.94 to 14.09% (mean 12.33%). Phylogenetic gene trees showed well-supported clades for both species, with clear structuring patterns within them. Species-delimitation algorithms split our dataset into at least three putative species for each taxon. Moreover, population structure analysis showed a strong correlation between Atlantic Forest areas of endemism as sources of molecular variation in Pi. monticola. Morphometric analyses were significant for wing shape variation and some linear measurements (mainly of the head) when comparing specimens of different genetic clusters for both taxa. These results indicate strong genetic structuring of Monticola series species, confirmed by morphometry, indicating two possible cryptic species complexes.