Zoologischer Anzeiger 255 (2015) 32–46 Contents lists available at ScienceDirect Zoologischer Anzeiger jou rn al h om epage : w ww.elsevier.com/locate/jcz Phylogeny and biogeography of three new species of Niphargus (Crustacea: Amphipoda) from Greece Alexandros Ntakis a , Chryssa Anastasiadou a,∗ , Valerija Zakˇ sek b , Cene Fiˇ ser b a Department of Biological Applications and Technology, University of Ioannina, University Campus, 45110 Ioannina, Greece b Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana SI-1000, Slovenia a r t i c l e i n f o Article history: Received 8 October 2014 Received in revised form 4 February 2015 Accepted 4 February 2015 Available online 7 February 2015 Keywords: Amphipoda Groundwater Niphargus Greece Zoogeography Phylogeny a b s t r a c t Three new freshwater Amphipods of the family Niphargidae G. Karaman, 1962 are described from sub- terranean waters of Greece, Niphargus aitolosi sp. nov., Niphargus karkabounasi sp. nov. and Niphargus koukourasi sp. nov. Phylogenetic analysis based on 28S rDNA and H3 genes suggests that N. aitolosi sp. nov. belongs to N. longicaudatus species-complex. Species from that complex are distributed along both sides of Adriatic Sea and share roughly similar morphology, including the newly described species from Greece. Niphargus karkabounasi sp. nov., the second new species, belongs to a clade distributed broadly across Europe and the Middle East. Many species of this group, including N. karkabounasi, are small and likely live in small crevices. The third species, Niphargus koukourasi sp.nov. is phylogenetically related and morphologically similar to N. sanctinaumi and N. maximus from Lake Ohrid on the border of the Former Yugoslav Republic of Macedonia and Albania. In addition, this group of species may be related to species collected from Iran. © 2015 Elsevier GmbH. All rights reserved. 1. Introduction The amphipod genus Niphargus Schiöde, 1849 is the largest genus among freshwater Amphipods (Väinöla et al., 2008), dis- tributed in all types of subterranean waters of the Western Palearctic (Fiˇ ser et al., 2009a) and one of the key taxa of European groundwater fauna (Zagmajster et al., 2014). The revision of the genus is far from complete and new species are constantly discov- ered and described (e.g., Karaman et al., 2010; Hekmatara et al., 2013). Nevertheless, two generalities seem to be arising. Firstly, phy- logenetic analyses suggest that morphological differentiation does not follow phylogenetic relationships. Closely related species may be morphologically either very different (Fiˇ ser et al., 2008; Trontelj et al., 2012), or morphologically indistinguishable from each other (e.g., Trontelj et al., 2009; Meleg et al., 2013). Molecular analy- ses revealed also the second important characteristic of Niphargus, i.e., that small distributional ranges are rather rule than exception (Trontelj et al., 2009). Their poor dispersal ability is reflected also on a higher taxonomic level, as many clades are related to distinct biogeographic regions (Fiˇ ser et al., 2008). ∗ Corresponding author. Tel.: +30 69 58602103; fax: +30 2651007358. E-mail addresses: ntakisal@gmail.com (A. Ntakis), chanasta@cc.uoi.gr (C. Anas- tasiadou), valerija.zaksek@bf.uni-lj.si (V. Zakˇ sek), cene.fiser@bf.uni-lj.si (C. Fiˇ ser). In general a high degree of endemism suggests that efficient reg- istration of extant species requires high sampling effort across the entire genus range. In Niphargus, however, this is still far from real- ity. Many areas within the genus range harbour only few species and it is likely that some of these areas have not been sufficiently explored (Hekmatara et al., 2013). The unexplored areas may con- tain the missing puzzle pieces needed for reconstruction of species diversity and evolutionary history of the genus. This study is one of the steps needed to complete description of diversity of this important genus. From a biological point of view, Greece is considered as one of the most biologically diverse countries of Europe (Kryˇ stufek and Reed, 2004; Griffiths, 2006). It is the crossroad between Europe, Mediterranean, Black Sea and the Middle East (B˘ an˘ arescu, 2004) and demonstrates a complex isolated freshwater system, espe- cially in the west and the south (Economidis, 1995). Despite the numerous phylogeographic studies in the area, many taxa remain unstudied and need revision (Poulakakis et al., 2014). Furthermore, Greek freshwaters exhibit high richness of epigean species and high endemism (Médail and Quézel, 1999; Oikonomou et al., 2014), which reflects its habitat diversity but it could also be the reflection of absence of massive Pleistocene extinctions (Hewitt, 2011). Broad comparative analyses suggest decline of subterranean species rich- ness in southern-most parts of Europe (Zagmajster et al., 2014). Indeed, up to date only seven Niphargus species (Karaman, 1934; http://dx.doi.org/10.1016/j.jcz.2015.02.002 0044-5231/© 2015 Elsevier GmbH. All rights reserved.