Abstract

Cryptic species represent a challenge for documenting global biodiversity. Even in well-studied groups, such as European butterflies, the application of integrative approaches has allowed the recognition of an unexpected number of cryptic taxa. Here, we combine the analysis of mitochondrial (cytochrome c oxidase I, COI) and nuclear (internal transcribed spacer 2, ITS2) markers with geometric morphometrics of the male genitalia to study diversity within the butterfly Muschampia proto. The nuclear marker reveals three well-supported and deeply diverged lineages, which are also detected based on mitochondrial DNA, although the latter recovers one of them as paraphyletic with poor support. These lineages also present distinct male genital characters, which allow blind assignment of > 97% of specimens when applying a jackknife procedure. We conclude that M. proto comprises three cryptic species that started to differentiate ~2 Mya: M. proto, distributed in northern Africa, the Iberian Peninsula and southern France; Muschampia alta comb. & stat. nov., occurring in southern Italy and the Balkan Peninsula; and Muschampia proteides, present in the easternmost part of Europe, the Near East and Iran. This discovery adds two new species to the European butterfly fauna and highlights the necessity to continue investigating potential cryptic diversity.