1 Department of Animal Biology and Marine Ecology, University of Messina, Messina; 2 Department of Evolutionary Biology, University of Siena, Siena, Italy Deep genetic divergence in the darkling beetle Pimelia rugulosa (Coleoptera, Tenebrionidae) reflects Plio-Pleistocenic paleogeographic history of Sicily Stefania Stroscio 1 ,Cosimo Baviera 1 ,Francesco Frati 2 ,Giuseppe Lo Paro 1 and Francesco Nardi 2 Abstract The genus Pimelia is represented in Sicily by the two species P. grossa and P. rugulosa, the latter with the two subspecies P. r. rugulosa and P. r. sublaevigata. A third subspecies, P. r. apula, is present only in Puglia. The phylogeographic history of this genus in Sicily was reconstructed using sequences of the complete cox2 gene, applying phylogenetic and molecular dating methods. Phylogenetic analysis confirmed the monophyly of the two species, as well as the subdivision of P. rugulosa in the three morphological subspecies. One previously undetected and deeply divergent lineage was described that is geographically related to the Hyblean massif. It is therefore suggested that the deep divergence of the two major P. rugulosa clades in Sicily reflects an ancient separation between the Hyblean massif and the other land masses of Sicily. Dating analysis confirmed that the timing of P. rugulosa genetic divergence is compatible with the formation of the Hyblean massif. Furthermore, P. rugulosa populations of the Hyblean massif, whose taxonomy has been historically problematic, display levels of genetic divergence comparable to those observed at the species level. The possibility that this new lineage might deserve species rank is presented. Key words: mtDNA – Biogeography – cryptic species – morphological variation – molecular dating Introduction Beetles of the genus Pimelia Fabricius, 1775 (Tenebrionidae, Coleoptera) are flightless and saprophagous insects that live in xerophilic habitats and display a circum-Mediterranean distri- bution. Pimelia sensu stricto, which is one of the five recognized subgenera, is a polyphyletic assemblage that has undergone an extensive adaptative radiation. Based on a morphological phylogeny of the genus as well as a comparison with the geological history of the area, Kwieton (1977) proposed a Middle East origin of the group and a subsequent radiation in north-western Africa followed by the colonization of Morocco and the Iberian peninsula about 6–12 Mya. Several taxonomic studies have been conducted on this genus, including molecular investigations (Juan et al. 1995, 1996, 1997; Bruvo et al. 2003; Contreras-Diaz et al. 2003; Pons et al. 2004; Bruvo-Madaric et al. 2007), physiological researches (e.g. Duncan et al. 2002) and karyological studies (e.g. Pons 2004). Seven species and thirteen subspecies of genus Pimelia have been reported in Italy, mostly in Sardinia (Ruffo and Stoch 2005). Two species are present in Sicily: Pimelia (Pimelia) grossa Fabricius, 1792 and Pimelia (Pimelia) rugulosa Germar, 1824, the latter being subdivided in three subspecies (Solier 1836; Gridelli 1950). Pimelia grossa has a fairly large distribution and has been reported in Sardinia, North Africa and Sicily, where it is distributed prevalently on sandy dunes along the southern coast. Pimelia rugulosa rugulosa is distributed across eastern Sicily, the Aeolian islands and the south-western Italian Peninsula (Campania and Calabria). Conversely, Pimelia rugulosa sublaevigata Solier, 1836 and Pimelia rugulosa apula Gridelli, 1950 are endemic of Sicily and Puglia, respectively. Pimelia grossa and P. rugulosa differ with regard to their origin and geographic distribution and likely have colonized Sicily through different routes: P. grossa, of North African origin, probably reached Sicily in the late Miocene, when the two land masses were connected (Bonfiglio et al. 2002). Pimelia rugulosa, on the other hand, presumably reached Sicily from Sardinia during the Pontic period (7.2–5.3 Mya), like Centaurea tauromenitana Guss, 1844 and possibly differ- entiated locally (Marcuzzi 1969). The major distinctive morphological character between P. grossa and P. rugulosa is the laterally compressed medium and posterior tarsi in the former, which led taxonomists to assume that the two taxa belong to different groups (Solier 1836; Se´nac 1884). In P. rugulosa, the identification of the subspecies is based on differences in elytral sculpture. In the subspecies P. r. rugulosa, all elytral carinae are present and developed, intercarina intervals have a dense sculpture and the two dorsal carinae merge backwards. Pimelia r. apula differs from the nominal subspecies in the peculiar elytral sculpture, with characteristic structures on the intercarina intervals. In P. r. sublaevigata, elytral carinae are very small and interval sculptures strongly reduced, making the surface appearance often smooth and shiny (See Fig. S1). Nevertheless, the aforementioned patterns of elytral sculptures used to differen- tiate the three subspecies of P. rugulosa can sometimes display a continuous distribution in individuals from the same population, therefore complicating taxonomic assignment (Marcuzzi 1969; Aliquo` and Leo 1999). Subspecies identifica- tion has been particularly problematic in southern Sicily, where some transitional forms have been reported and specimens collected in close vicinity have been variously assigned to different subspecies (Marcuzzi 1970; Canzoneri 1977; Aliquo` and Leo 1999). The island of Sicily has undergone extensive geological changes over the Plio-Pleistocene period, a fact that determined Corresponding author: Stefania Stroscio (sstroscio@unime.it) Contributing authors: Cosimo Baviera (sbaviera@unime.it), Francesco Frati (frati@unisi.it), Giuseppe Lo Paro (loparog@unime.it), Fransesco Nardi (nardifra@unisi.it) Ó 2011 Blackwell Verlag GmbH Accepted on 8 February 2011 J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2011.00617.x J Zool Syst Evol Res (2011) 49(3), 196–203