Mitochondrial DNA phylogeography reveals the existence of an Evolutionarily Significant Unit of the sand goby Pomatoschistus minutus in the Adriatic (Eastern Mediterranean) Sergio Stefanni a,b, * and Joseph L. Thorley a,c a School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK b DOP-UAc ß, Centro do IMAR-Instituto do Mar da Universidade dos Ac ßores, PT-9901-862, Horta, Azores, Portugal c Fisheries Research Service, Freshwater Laboratory, Faskally, Pitlochry, Perthshire, PH16 5LB, UK Received 25 September 2002; revised 22 January 2003 Abstract The sand goby Pomatoschistus minutus is a major component of marine shelf and estuarine food webs and an important study organism in behavioural research. Yet, despite the sand gobyÕs significance, its past and present patterns of migration and gene flow are poorly understood. Here we use the mtDNA control region and parts of the flanking tRNA genes of 63 fish from six localities in the Adriatic (Eastern Mediterranean), Western Mediterranean, Atlantic, and North Sea to investigate the phylogeography of this gobiid. Phylogenetic analyses and population genetics statistics reveal the existence of an Evolutionarily Significant Unit, sensu Moritz (1994), in the Adriatic and another in the Western Mediterranean, Atlantic, and North Sea. A possible biogeographical scenario for the separation of the ancestral population is that sand gobies in the Adriatic and Western Mediterranean split between 10,000 and 5000 years ago when due to the rise in sea temperature they migrated northwards and were bisected by the Italian peninsula. A testable prediction of this scenario is that sand gobies from the Western Mediterranean, Adriatic, and Aegean form three reciprocally monophyletic groups which are the descendants of a three-way diversification event. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Sand goby; Pomatoschistus minutus; mtDNA; ESU; Phylogeography; Adriatic 1. Introduction The sand goby Pomatoschistus minutus (Pallas, 1770) is a small, epibenthic, marine fish, which, due to its high fecundity, long breeding season and a complex mating system, has become a popular study organism among sexual selection researchers. Sand goby breeding be- haviours include male competition for nest sites (Lind- str€ om, 1988), male behavioural (Forsgren, 1997a) and acoustic (Lindstr€ om and Lugli, 2000) courtship displays, female choice (Forsgren, 1997a,b; Forsgren et al., 1996; Kangas and Lindstr€ om, 2001; Kvarnemo and Forsgren, 2000; Lindstr€ om, 1992), sneaky matings (Jones et al., 2001a), paternal care (Lindstr€ om and Wennstr€ om, 1994), and filial cannibalism (Lindstr€ om, 1998). More recently, the sand goby has been used to study the re- lationship between mating strategy, in this case sneak or nest, and ecology in Ôgeographically distinct populationsÕ (Jones et al., 2001b). The sand goby is also a major component of the food web in European marine shelf and estuarine ecosystems (Doornbos and Twisk, 1987). Yet, despite the sand gobyÕs importance as a model or- ganism and energy transducer and despite growing in- terest in its geographical variation, the phylogeography of this small gobiid is poorly understood. The sand goby inhabits the European inshore waters of the Atlantic Ocean, from Norway to Morocco, as well as the Baltic, North, Mediterranean, and Black Seas (Fig. 1). A division of the sand goby into Pomatoschistus minutus minutus from the Atlantic and Pomatoschistus minutus elongatus (Canestrini, 1861) from the Mediter- ranean and Black Seas was proposed by an earlier anonymous researcher (reported by Miller, 1986) on the basis of breast pigmentation and a dark spot on the Molecular Phylogenetics and Evolution 28 (2003) 601–609 www.elsevier.com/locate/ympev MOLECULAR PHYLOGENETICS AND EVOLUTION * Corresponding author. Fax: +351-29-22-00-411. E-mail address: sstefanni@horta.uac.pt (S. Stefanni). 1055-7903/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(03)00054-X