Systematic Entomology (2010), 35, 318–328 DOI: 10.1111/j.1365-3113.2009.00510.x Molecular phylogeny of the Mediterranean species of Philaenus (Hemiptera: Auchenorrhyncha: Aphrophoridae) using mitochondrial and nuclear DNA sequences ANNA MARYA ´ NSKA-NADACHOWSKA 1 , SAKIS DROSOPOULOS 2 , DOROTA LACHOWSKA 3 , LUKASZ KAJTOCH 1 and V A L E N T I N A G. KUZNETSOVA 4 1 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Krak´ ow, Poland, 2 Department of Agricultural Biotechnology, Agricultural University, Athens, Greece, 3 Deparment of Entomology, Institute of Zoology, Jagiellonian University, Krak´ ow, Poland and 4 Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia Abstract. The phylogenies of all eight European species of Philaenus were estimated from cytochrome oxidase subunit I, cytochrome B and internal transcribed spacer 2 (ITS2) fragments of DNA using phylogenetic reconstruction methods: maximum par- simony (MP), maximum likelihood (ML) and Bayesian inference (BI) analyses. Based on the topologies of all obtained phylogenetic trees, the monophyly of Philaenus is well supported, being congruent with morphological, ecological and chromosomal data. Three phylogenetic lineages were distinguished in the mitochondrial and com- bined (mtDNA with ITS2) trees. The first lineage is represented by only one species, Philaenus maghresignus, which inhabits Maghreb and southern Spain. Clade A includes three species: P. tarifa (Southern Iberia), P. italosignus (Sicily and Southern Italy) and P. signatus (the Balkans and Middle East). In clade B two subclades were recognized: B1 represented by P. loukasi (Southern Balkans) and P. arslani (Middle East), and B2 comprising P. spumarus (the most widespread Palaearctic species) and P. tesselatus (from Southern Iberia and Maghreb). These clades were also retrieved in trees reconstructed from nuclear sequences. However, four species (P. maghresignus, P. tarifa, P. italosignus and P. signatus ) showed unresolved polytomy at the base of the nuclear tree. Clade A together with P. maghresignus clustered with the ‘signatus ’ group defined from morphology, and clade B with the ‘spumarius ’ group; these might be considered separate subgenera. Genetic distances in mitochondrial DNA between ingroup species ranged from 14.0% between P. signatus and P. spumarius to 2.4% between P. tesselatus and P. spumarius. By contrast, genetic divergence of ITS2 between ingroup species was very low, at most 2.1%. The divergence of Philaenus species is estimated to have occcurred between 7.9 and 0.6 Ma. Possibly three main speciation events occurred: the first at the Miocene/Pliocene boundary (c . 5.5 Ma) for deeper splits; the second between 4.2 and 2.5 Ma in the Pliocene, when pairs of more closely related species diverged; and the most recent during the Pleistocene glaciations, when the separation of P. tesselatus and P. spumarius took place. The species status of all Philaenus species is confirmed except for P. tesselatus. Correspondence: Anna Marya´ nska-Nadachowska, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Slawkowska 17, 31-016 Krak´ ow, Poland. E-mail: maryanska@isez.pan.krakow.pl © 2010 The Authors 318 Journal compilation © 2010 The Royal Entomological Society