Molecular evidence of the survival of subterranean amphipods (Arthropoda) during Ice Age underneath glaciers in Iceland ETIENNE KORNOBIS,* SNÆBJO ¨ RN PA ´ LSSON,* BJARNI K. KRISTJA ´ NSSON† and JO ¨ RUNDUR SVAVARSSON* *Department of Biology, University of Iceland, Askja, Sturlugata 7, 101 Reykjavik, Iceland, †Department of Aquaculture and Fish Biology, Ho ´lar University College, Ha ´eyri 1, 550 Sauða ´rkro ´kur, Iceland Abstract Two endemic groundwater arthropod crustacean species, Crangonyx islandicus and Crymostygius thingvallensis, were recently discovered on the mid-Atlantic volcanic island of Iceland. The extent of morphological differences from closest relatives, endemism, along with the geographic isolation of Iceland and its complete coverage by glaciers 21 000 years ago, suggests that these two species have survived glaciation periods in sub-glacial refugia. Here we provide strong support for this hypothesis by an analysis of mitochondrial genetic variation within Crangonyx islandicus. Our results show that the species is divided into several distinct monophyletic groups that are found along the volcanic zone in Iceland, which have been separated by 0.5 to around 5 million years. The genetic divergence between groups reflects geographic distances between sampling sites, indicating that divergence occurred after the colonization of Iceland. The genetic patterns, as well as the dependency of genetic variation on distances from the tectonic plate boundary and altitude, points to recent expansion from several refugia within Iceland. This presents the first genetic evidence of multicellular organisms as complex as crustacean amphipods which have survived glaciations beneath an ice sheet. This survival may be explained by geothermal heat linked to volcanic activities, which may have maintained favourable habitats in fissures along the tectonic plate boundary in Iceland during glaciations. Keywords: Crustacea, glaciation, phylogeography, sub-glacial refugia, subterranean, volcanism Received 25 January 2010; revision received 29 March 2010; accepted 3 April 2010 Introduction Pleistocene glaciations are known to have shaped both the distribution and the genetic diversity of species at high latitudes (Hewitt 2004). During glacial expansions, populations either became extinct or managed to sur- vive in ice-free refugia. High-latitude areas and for- merly glaciated northern parts of Europe and North America are, thus, characterized by low endemism and species diversity, shallow genetic clades and little diver- sity within species (Sadler 1999; Hewitt 2004). This is expected because of recent colonization and bottlenecks preceding expansion of populations after the Weichs- elian glaciation period. Conversely, the refugia are char- acterized by high taxonomic and genetic diversity among and within regions (Hewitt 2004). During the last glacial maximum (LGM) around 21 000 BP, Iceland, as well as most of northern Europe and North America (Ehlers & Gibbard 2007), was almost completely covered by glaciers which extended far offshore so that only small ice-free areas may have been present along coastal mountains, mainly in eastern and northern regions (Geirsdo ´ ttir et al. 2007). The low species diversity and lack of endemism in the current Icelandic terrestrial biota (e.g. Buckland et al. 1986) can be explained by the short time since the retreat of the Correspondence: Etienne Kornobis; E-mail: etk1@hi.is Ó 2010 Blackwell Publishing Ltd Molecular Ecology (2010) 19, 2516–2530 doi: 10.1111/j.1365-294X.2010.04663.x