Systematic Entomology (2015), 40, 268–287 Host-associated genetic divergence and taxonomy in the Rhinusa pilosa Gyllenhal species complex: an integrative approach IVO TOŠEVSKI 1,2 , ROBERTO CALDARA 3 , JELENA JOVI ´ C 2 , GERARDO HERNÁNDEZ-VERA 4 , COSIMO BAVIERA 5 , ANDRE GASSMANN 1 and B R E N T C. EMERSON 6 1 CABI, Delémont, Switzerland, 2 Department of Plant Pests, Institute for Plant Protection and Environment, Zemun, Serbia, 3 Milan, Italy, 4 Instituto de Botánica, Departamento de Botánica y Zoología, Universidad de Guadalajara-CUCBA, Jalisco, Mexico, 5 Dipartimento di Biologia Animale ed Ecologia Marina, Università degli Studi di Messina, Messina, Italy and 6 Island Ecology and Evolution Research Group, IPNA-CSIC, La Laguna, Spain Abstract. A combined taxonomic, morphological, molecular and biological study revealed that stem-galling weevils from the genus Rhinusa associated with toadlaxes from the genus Linaria (Plantaginaceae) are composed of three different species: Rhinusa pilosa, Rhinusa brondelii and Rhinusa rara sp.n. The authentic ield host plants are respectively, Linaria vulgaris, Linaria purpurea and Linaria genistifolia/ Linaria dalmatica. These weevil species can be distinguished from each other by a few subtle morphological characteristics, mainly in the shape of the rostrum and of the integument. An analysis of the mitochondrial [cytochrome oxidase subunit II gene (COII ) and 16S ribosomal RNA gene (16S)] and nuclear (elongation factor-1, EF-1) sequence data revealed high genetic divergence among these species. Uncorrected pairwise distances on mtCOII gene were 14.3% between R. pilosa and R. brondelii, 15.7% between R. pilosa and R. rara, while R. brondelii and R. rara were approximately 11% divergent from each other. Divergences obtained on 16S and nuclear EF-1 genes were congruent. However, substantial intraspeciic mitochondrial divergence was recorded for all studied populations of R. pilosa s.s. showing two mtDNA lineages, with estimated COII and 16S divergences of 4% and 1.6%, respectively. Nuclear pseudogenes (Numts) and Wolbachia inluence, although recorded within both lineages, were excluded as possible causatives of the mtDNA divergence, while EF-1 indicated absence of lineage sorting. Species from the R. pilosa complex are estimated to have diverged from each other approximately 7.2 million years ago (mya; late Miocene), while R. brondelii and R. rara diverged from each other about 4.7 mya (early Pliocene). This published work has been registered in ZooBank, http://zoobank.org/urn:lsid: zoobank.org:pub:EEDD6248-01DB-4B4A-B79D-C5606393E3AA. Introduction The weevil genus Rhinusa Stephens (Curculionidae, Mecinini) is composed of approximately 40 species, all with a Palearctic distribution (Reitter, 1907; Caldara, 2001, 2013). Based on morphological characters, all species from the genus Rhinusa have been divided into 10 species groups (Caldara et al., 2010). A phylogenetic analysis based on morphology suggests that Correspondence: Ivo Toševski, CABI, 1 Rue des Grillons, 2800 Delémont, Switzerland. E-mail: tosevski_ivo@yahoo.com the genus Rhinusa is monophyletic and sister to the Palaearctic species of the genus Gymnetron Schoenherr, 1825 (Caldara, 2001). However, a recent comprehensive molecular phyloge- netic analysis does not support monophyly for either genus, but does suggest an origin for all taxa from an ancestral Gymnetron species from southern Africa. It was concluded that subse- quent range expansion from southern Africa was followed by extensive species radiation in the western Palaearctic, with the earliest divergence estimated to date back to circa 27 million years ago (mya) (Hernández-Vera et al., 2013). 268 © 2015 The Royal Entomological Society