Molecular Ecology (2008) 17, 1732–1744 doi: 10.1111/j.1365-294X.2007.03652.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd Defining reproductively isolated units in a cryptic and syntopic species complex using mitochondrial and nuclear markers: the brooding brittle star, Amphipholis squamata (Ophiuroidea) E. BOISSIN, J. P. FÉRAL and A. CHENUIL UMR 6540 — DIMAR, CNRS/Université de la Méditerranée, Aix-Marseille II, Centre d’Océanologie de Marseille, Station Marine d’Endoume, Chemin de la Batterie des Lions, 13007 Marseille, France Abstract At a time when biodiversity is threatened, we are still discovering new species, and particularly in the marine realm. Delimiting species boundaries is the first step to get a precise idea of diversity. For sympatric species which are morphologically undistinguishable, using a com- bination of independent molecular markers is a necessary step to define separate species. Amphipholis squamata, a cosmopolitan brittle star, includes several very divergent mitochondrial lineages. These lineages appear totally intermixed in the field and studies on morphology and colour polymorphism failed to find any diagnostic character. Therefore, these mitochondrial lineages may be totally interbreeding presently. To test this hypothesis, we characterized the genetic structure of the complex in the French Mediterranean coast using sequences of mitochondrial DNA (16S) and for the first time, several nuclear DNA markers (introns and microsatellites). The data revealed six phylogenetic lineages corre- sponding to at least four biological species. These sibling species seem to live in syntopy. However, they seem to display contrasted levels of genetic diversity, suggesting they have distinct demographic histories and/or life-history traits. Genetic differentiation and isolation- by-distance within the French Mediterranean coasts are revealed in three lineages, as expected for a species without a free larval phase. Finally, although recombinant nuclear genotypes are common within mitochondrial lineages, the data set displays a total lack of heterozygotes, suggesting a very high selfing rate, a feature likely to have favoured the formation of the species complex. Keywords: marine realm, microsatellites, mitochondrial lineages, reproductive isolation, speciation, species complex Received 1 September 2007; revision accepted 17 November 2007 Introduction An accurate assessment of species level diversity appears as an essential goal to systematic research in order to apply correct conservation policy. Cryptic species, which by definition are difficult to distinguish morphologically, represent a major problem to this goal. During the last decades, more and more sibling species have been brought to light in many animal taxa (Bickford et al. 2007). Despite an apparent homogeneity of the marine environment, marine cryptic species seem particularly abundant (Knowlton 1993, 2000). Such species are at best distinguishable by subtle differences such as preferential habitat (Muths et al. 2006), life histories (Byrne et al. 2003), or behaviour (Shaw 2000). Molecular approaches, particularly mitochondrial DNA have proved extremely useful to help in the delimitation of species (Knowlton 2000). However, when no independent marker is available, the occurrence of a deep divergence between mitochondrial lineages does not prove that there are distinct noninterbreeding species. Such a pattern (i) may have arisen after foundation-expansion events or particular events of selective sweeps, and (ii) would persist even after a secondary contact between ancient vicariant lineages Correspondence: Emilie Boissin, Fax: 0491041635; E-mail: emilie.boissin@univmed.fr