Multiple genetic divergences and population expansions of a Mediterranean sandfly, Phlebotomus ariasi, in Europe during the Pleistocene glacial cycles

Abstract

Phlebotomus ariasi is one of the two sandflies transmitting the causative agent of zoonotic leishmaniasis, Leishmania infantum, in France and Iberia, and provides a rare case study of the postglacial re-colonization of France by a Mediterranean species. Four DNA sequences were analysed-mitochondrial cytochrome b (cyt b), nuclear elongation factor-1α (EF-1α) and two anonymous nuclear loci-for 14-15 French populations and single populations from northeast Spain, northwest Spain, Portugal and Morocco. The presence of cryptic sibling species was not revealed by phylogenetic analyses and testing for reproductive isolation between sympatric populations defined by the two most divergent cyt b haplogroups. No locus was shown to be under positive directional or balancing selection and, therefore, molecular variation was explained demographically. Each nuclear locus showed shallow isolation by distance from Portugal to the French Pyrenees, but for both cyt b and EF-1α there was then a step change to the upland Massif Central, where leading-edge populations showed low diversity at all loci. Multiple genetic divergences and population expansions were detected by analyses of cyt b and dated to the Pleistocene. Endemicity of one cyt b sub-lineage suggested the presence of a refuge north of the Pyrenees during the last glacial period. Monopolization of the Massif Central by genetically differentiated populations of P. ariasi might possibly hinder the northwards spread of leishmaniasis.

Figure 1

Digital elevation map of the western Mediterranean region, showing locations where 19 populations of P. ariasi were sampled for molecular characterization. Ellipses demarcate the labelled regions mentioned in the text. Additional information on locations is given in Supplementary Table A (ESI).

Figure 2

Bayesian phylogeny of the haplotypes of the 3' end of cyt b (714 bp) from Phlebotomus species. Branches for subgenera, species complexes, some species, and the haplogroups of P. ariasi are labelled. Haplotypes obtained from GenBank contain the accession number in their code. Codes for species—papa: P. papatasi; cauc: P. caucasicus; masc: P. mascittii; brev: P. brevis; hale: P. halepensis; chad: P. (chadlii); aria: P. ariasi; negl: P. neglectus; majo: P. major; perf: P. perfiliewi; lang: P. langeroni; tobb: P. tobbi; pern: P. perniciosus; long: P. longicuspis; orie: P. orientalis. Cyt b was partitioned by codon positions, each with an independent substitution model selected by MRMODELTEST v2.3. Posterior probability values on branches (and to the right of haplogroups A, C, E) indicate the support for nodes (significant for posterior probability >0.7).

Figure 3

Parsimony network (TCS v1.21) showing the genealogical relationships between the 92  cyt b haplotypes from 452 P. ariasi. These haplotypes are shown as shaded or open circles with sizes proportional to their frequency of occurrence, which is given if >5. Smallest filled circles denote missing haplotypes. The lettered haplogroups or sub-haplogroups (B) are followed by the code of their modal haplotype (CBNN) along with their geographical distributions. All most parsimonious pathways are shown. C, central; E, eastern, NE, northeast; NW, northwest.

Figure 4

Plots and regression of genetic distance (F_ST/(1−F_ST)) on geographical distance (km) between pairs of P. ariasi populations, for nuclear loci: (a) EF-1α, (b) AAm20, and (c) AAm24. Symbols of pairwise population comparisons denote: ▪, Morocco versus all others; ▪▪▪, Portugal versus all others except Morocco (that is, France, Spain); +, NW (northwest) Spain versus all others except Morocco and Portugal (that is, France, northeast Spain); ▴, northeast Spain versus all others except Morocco, Portugal and northwest Spain (that is, France); •, France only. Ringed data points indicate from left to right: Lot (LNP and RME) with northeast Spain, Portugal and Morocco for EF-1α Lot (LNP and RME) with northwest Spain, Portugal and Morocco for AAm24.