Spatio-temporal genetic variation in sympatric and allopatric Mediterranean Cicada species (Hemiptera, Cicadidae) SOFIA G. SEABRA 1,2 *, JOSÉ A. QUARTAU 1 and MICHAEL W. BRUFORD 2 1 Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de Ciências de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal 2 School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3TL, UK Received 30 October 2007; accepted for publication 27 April 2008 Cicada barbara (Stål) and Cicada orni L. are two Mediterranean cicadas, very similar in morphology, that produce distinct acoustic mating signals and that have partially overlapping distribution ranges in the Iberian Peninsula, occurring in sympatry in several locations. In the present study, six microsatellite loci were analysed in C. barbara, four of which were also analysed in C. orni. Geographical and temporal genetic variation in these species was studied. No evidence of hybridization was found, enabling us to infer that the isolating barriers between these species are efficient. Partitioning of geographic variation in each species, revealed the following patterns: Iberian Peninsula and Northwestern African populations of C. barbara showed higher differentiation between than within each region, supporting C. barbara subspecific taxonomy (C. barbara lusitanica in the Iberian Peninsula and C. barbara barbara in Northwestern Africa) and highlighting isolation coincident with the presence of physical barriers to gene-flow; differentiation between populations of C. orni from both sides of the Pyrenees was very low, and this mountain range may not constitute a significant barrier for the dispersal of this species; Greek populations of C. orni were found to be highly differentiated from Western European populations; and no pattern of isolation-by-distance was found in either species within the Iberian Peninsula. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 249–265. ADDITIONAL KEYWORDS: Aegean islands – Cicadoidea – France – Iberian Peninsula – insects – micro- satellites – North Africa – null alleles – population structure. INTRODUCTION Cicadas have often been subjects for evolutionary studies of speciation, isolating mechanisms, mate rec- ognition systems, and sexual selection (Williams & Simon, 1995; Sueur & Aubin, 2003; Cooley & Mar- shall, 2004; Gogala & Trilar, 2004; Villet, Barker & Lunt, 2004; Sueur et al., 2007). This is mainly due to the ease of study of their calling songs, which are usually distinct between species and have an impor- tant role in mate attraction and species isolation (Villet, 1992; Daws, Hennig & Young, 1997; Cooley & Marshall, 2001). Cicadas may also constitute model species to study biogeographic recolonization patterns from ice age refugia, according to the three criteria defined by Schmitt (2007): as flying insects, they have high ability to disperse into suitable habitats; despite this, individuals are mostly sedentary (as seen in cicada species that aggregate in singing choruses); populations attain large numbers, thereby potentially retaining their phylogeographic signal. Additionally, being thermophilic species, cicadas must have been restricted to particular warm climate refugia during ice ages. The evolutionary history of north American periodical cicadas has been the focus of intensive studies and their biogeographic patterns are thought to reflect climatic processes during glaciations (Williams & Simon, 1995; Simon et al., 2000). In the Mediterranean region, the evolution of the genus Cicada was also influenced by Pleistocene climate *Corresponding author. E-mail: sgseabra@fc.ul.pt Biological Journal of the Linnean Society, 2009, 96, 249–265. With 5 figures © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 249–265 249