Molecular Ecology (2005) 14, 1939–1954 doi: 10.1111/j.1365-294X.2005.02569.x © 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal N. MESQUITA,* B. HÄNFLING,† G. R. CARVALHO † and M. M. COELHO * *Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Animal/Centro de Biologia Ambiental, Campo Grande, Bloco C2–3° Piso, 1749–016 Lisboa, Portugal, †Molecular Ecology & Fisheries Genetics Laboratory, Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK Abstract The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a char- acteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cyto- chrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Φ-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than iso- lation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottle- neck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local inter- mittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species. Keywords: endangered endemic Cyprinidae, gene flow, microsatellites, mitochondrial DNA, phylogeography, Squalius aradensis Received 5 January 2005; revision accepted 1 March 2005 Introduction The circum-Mediterranean region has been classified by Myers et al . (2000) as one of the 25 ‘biodiversity hotspots for conservation priorities’, and as such, is characterized by the presence of an exceptional number of endemic species that are currently endangered by habitat loss. One such species is the endemic and recently described Squalius aradensis (Coelho et al . 1998), currently included in the Critically Endangered category of the IUCN Red List (Instituto da Con- servação da Natureza unpublished data, http://www.icn.pt/ documentos/livro_vermelho/Classif_Crit_Peixes.pdf). Squalius aradensis is found only in the small Mediterranean- type drainages of Portugal (SMTDP) located in the extreme southwestern region of the Iberian Peninsula (Fig. 1). These drainages, though representing a small hydrological area of the Iberian Peninsula, contain considerable ichthyofaunistic value, with the presence of local endemic species, typically exhibiting a highly restricted distribution (Mesquita & Coelho 2002). The habitats of the SMTDP are characterized by a heterogeneous annual hydrological cycle, typical of the circum-Mediterranean region. In these intermittent systems the occurrence of flooding events during the winter, Correspondence: N. Mesquita, Fax: + 351 217500028; E-mail: npmesquita@fc.ul.pt