Pre- and post-mating reproductive barriers drive divergence of five sympatric species of Naryciinae moths (Lepidoptera: Psychidae) JELMER A. ELZINGA 1 *, JOHANNA MAPPES 2 and LAURI KAILA 3 1 Department of Biological and Environmental Science, University of Jyväskylä, PO Box 35, FI-40014 Jyväskylä, Finland 2 Department of Biological and Environmental Science, Centre of Excellence in Biological Interactions, University of Jyväskylä, PO Box 35, FI-40014 Jyväskylä, Finland 3 Finnish Museum of Natural History, Zoology Unit, University of Helsinki, P.O. Box 17, FI-00014 Helsinki, Finland Received 29 November 2013; revised 31 January 2014; accepted for publication 31 January 2014 The biological species concept suggests that species can be separated on the basis of reproductive isolation. However, because natural interbreeding capabilities are often unknown, differences in morphology are generally used to separate species. Alternatively, genetic dissimilarity is used to separate morphologically similar species. Many genetic markers, including the maternally inherited mitochondrial cytochrome oxidase I sequence, cannot show interbreeding and therefore species status of groups may remain unresolved. In species of the genera Dahlica and Siederia (Lepidoptera: Psychidae: Naryciinae) the lack of morphological distinction and unknown interbreed- ing has led to unclear and unresolved taxonomic status. Mitochondrial DNA sequences suggest five sexual species to occur in Finland. However, their species status remains unconfirmed, due to a lack of knowledge on interbreed- ing, unclear morphological distinction and the limited variation in mitochondrial DNA. We combine three methods, a cross-mating experiment, an analysis of mitochondrial and nuclear DNA, and a detailed male genital morpho- logical examination, to establish the species status of the five suspected species. All suspected species exhibit intraspecies mating preference, although several interspecies pairs readily produce offspring. The genetic analysis, however, fails to show hybrids or introgression, suggesting that both pre- and post-copulation mechanisms isolate the species reproductively. Morphological analysis of the male genitalia confirms that the species have diverged. Our results highlight the need of combining behavioural, morphological and genetic methods to determine species status in challenging taxonomic groups. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, ••, ••–••. ADDITIONAL KEYWORDS: bagworm moth – CAD – COI – genitalia – hybridization – isolation mechanism – morphometrics – phylogeny – speciation. INTRODUCTION One of the most influential ideas in evolutionary biology and conservation biology is the biological species concept (Mayr, 1942). According to this concept a species is a ‘group or groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups’. This concept is based on the notion that during the speciation process barriers of reproduction appear which isolate groups of individuals from each other, after which they will evolve separately (Dobzhansky, 1937; Mayr, 1942). Mechanisms of pre- and post- copulatory reproductive barriers can range from pre- zygotic temporal (e.g. different reproductive timing), ecological (e.g. different habitats), behavioural (e.g. through pheromones), mechanical (e.g. genitalia mis- match) to post-zygotic genetic (e.g. hybrid inviability or infertility) barriers (Dobzhansky, 1970). Often a *Corresponding author. E-mail: jelmerelzinga@hotmail.com Biological Journal of the Linnean Society, 2014, ••, ••–••. With 6 figures © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, ••, ••–•• 1