Org Divers Evol (2016) 16:597–611 DOI 10.1007/s13127-016-0262-x ORIGINAL ARTICLE Who lives where? Molecular and morphometric analyses clarify which Unio species (Unionida, Mollusca) inhabit the southwestern Palearctic Elsa Froufe1 & Duarte V. Gonçalves2,3,4 & Amílcar Teixeira5 & Ronaldo Sousa1,6 & Simone Varandas 7 & Mohamed Ghamizi 8 & Alexandra Zieritz9 & Manuel Lopes-Lima1 Received: 21 October 2015 /Accepted: 3 January 2016 /Published online: 19 January 2016 # Gesellschaft für Biologische Systematik 2016 Abstract Many doubts still exist about which freshwater first distributed across Iberia and corresponding to mussel Unio species inhabit Northwest Africa. While U. delphinus, and the second distributed across some authors refer to the presence of Unio delphinus Morocco, corresponding to U. foucauldianus.Thesere- in the Atlantic North African basins of Morocco, a re- sults were corroborated by the analysis of ten newly cent International Union forConservationofNature developed microsatellite loci as well as shell morphom- (IUCN) assessment performed on Moroccan Unio spe- etry. We suggest that the IUCN critically endangered cies, recognised the existence of a distinct species, Unio conservation status of U. foucauldianus should be foucauldianus, with a critically endangered conservation revised and probably down-listed since its actual distri- status. The present study delivered new genetic, mor- bution is much wider than previously described. phological, and geographical distribution data on two Phylogenetic relationships with the other Unio species Unio species (i.e. U. delphinus and U. foucauldianus) were resolved, showing that U. delphinus and greatly increasing the almost non-existent data on these U. foucauldianus fall inside the pictorum lineage. The taxa. Bayesian phylogenetic analysis revealed two high- estimated molecular rate reported herein (0.265 ± 0.06 % ly supported geographically concordant clades, which per million years) represents the first for the Unionida diverged by 3.2 ± 0.6 % (uncorrected p distance): the and could be used as a reference in future studies. Electronic supplementary material The online version of this article (doi:10.1007/s13127-016-0262-x) contains supplementary material, which is available to authorized users. * Elsa Froufe 5 CIMO-ESA-IPB-Mountain Research Centre, School of Agriculture, [email protected] Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-854 Bragança, Portugal 6 CBMA-Centre of Molecular and Environmental Biology, 1 CIIMAR/CIMAR-Interdisciplinary Centre of Marine and Department of Biology, University of Minho, Campus de Gualtar, Environmental Research, University of Porto, Rua dos Bragas 289, Braga, Portugal 4050-123 Porto, Portugal 7 CITAB-UTAD-Centre for Research and Technology of 2 CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Agro-Environment and Biological Sciences, Forestry Department, Genéticos da Universidade do Porto, R. Padre Armando Quintas, University of Trás-os-Montes and Alto Douro, Apartado 1013, 4485-661 Vairão, Portugal 5001-811 Vila Real, Portugal 3 Departamento de Biologia da Faculdade de Ciências da Universidade 8 Muséum d’Histoire Naturelle de Marrakech, Faculté des Sciences, do Porto, Rua Campo Alegre, 4169-007 Porto, Portugal Université Cadi Ayyad, SemlaliaB.P. 2390Marrakech, Morocco 4 Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), 9 School of Geography, University of Nottingham Malaysia Campus, Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain Jalan Broga 43500, Semenyih, Malaysia 598 E. Froufe et al. Keywords Freshwater molluscs . Unionidae . distributions. U. pictorum is the most widely distributed spe- Microsatellites . Messinian . Molecular clock cies, occurring from the UK extending South to Greece and Turkey and East to Russia (Van Damme 2011), while U. elongatulus is only present in Northern Italy and Croatia Introduction (Riccardi et al. 2016)andU. caffer is restricted to river systems across southern Africa (Kristensen et al. 2010). U. delphinus is Freshwater mussels (Unionida) are among the most threatened present in most Western Iberian basins (Araujo et al. 2009a). fauna, globally in decline (Strayer 2008), and particularly vul- U. ravoisieri is confined to two locations (one small basin and nerable to habitat loss and fragmentation, changes in flow a small lake) in Spain (Araujo et al. 2009a) and Northwest regimes, pollution, climatic disturbances and introduction of Africa (Khalloufi et al. 2011). U. mancus is restricted to invasive species (Strayer et al. 2004). Despite their endan- Mediterranean basins, from Eastern Iberia (Araujo et al. gered status and well-recognised ecological importance, 2005) through France and Italy to the South-Eastern unionoid conservation is often impaired due to a lack of infor- European countries (Haas 1969; Cuttelod et al. 2011). mation and knowledge in many fields including ecology, Many doubts still exist about which Unio species inhabit physiology and genetics (Lopes-Lima et al. 2014). This in- Northwest Africa. Although Araujo et al. (2009a) refer to the cludes a lack of a basic understanding of intraspecific genetic presence of U. delphinus in both Iberia and North Africa, a structure of most Unionida species (but see Froufe et al. 2014 subsequent IUCN assessment of the species restricted its oc- and Lopes-Lima et al. 2016a, for recent European examples), currence to the Iberia Peninsula (Araujo 2011a). However, the as well as unresolved phylogenetic relationships between assumption that this species is widespread in Atlantic unionoid taxa (Lopes-Lima et al. 2016b). To a large part, this Morocco, launched by Araujo et al. (2009a), has since become is due to the high phenotypic plasticity within unionoid spe- widely accepted (see, e.g. Morais et al. 2013; Machordom cies, rendering traditional conchological characters of limited et al. 2015). Araujo et al. (2009a) also recognised the presence use for identifying taxonomic units (Ortmann 1912;Zieritz of Unio gibbus Spengler 1793 in the Atlantic North African and Aldridge 2009; Zieritz et al. 2010) and making species basins of Morocco. On the other hand, recent IUCN assess- definitions in freshwater mussels a persistent and contentious ments recognised the existence of U. gibbus and a distinct problem. The emergence of genetic tools to detect evolution- species, Unio foucauldianus Pallary 1936 in Morocco (Van ary significant lineages has therefore led to broad taxonomic Damme and Ghamizi 2010; Araujo 2011c). The taxonomic revisions in bivalves (Plazzi and Passamonti 2010). status and the presence of U. gibbus have already been con- The genus Unio is particularly notorious for its extreme firmed for Morocco, Tunisia and Southern Iberia (Araujo et al. intraspecific phenotypic plasticity and regional variations in 2009b; Khalloufi and Boumaïza 2009 ; Khalloufi et al. 2011). shell shape. Up to the twentieth century, >1000 described Therefore, the present study will focus on clarifying which species were included within the genus (Graf 2007). Haas Unio species (i.e. U. delphinus, U. foucauldianus or both) (1969) considered this genus as a series of 12 ‘fundamental’ inhabit Moroccan Atlantic rivers. Unio species, each comprising different ‘races’ or incipient Numerous phylogeographic studies have confirmed the species, mostly based on conchology. Recent molecular works great importance of both Iberia and the Northwest African re- have updated the taxonomy of the genus, which resulted in 13 gion (i.e. the Maghreb) as glacial refugia during the currently recognised extant Unio species (Graf and Pleistocene (Gómez and Lunt 2007; Husemann et al. 2012). Cummings 2015). Phylogenetically, Unio belongs to the larg- Although there have been several studies using molecular est freshwater bivalve family, the Unionidae, accounting for tools on European invertebrates, few have included freshwater 674 out of 840 species (80 %) of the order Unionida (Graf and mussels from Iberia and even less from North Africa (but see Cummings 2007). The phylogeny of this genus encompasses Araujo et al. 2005, 2009a, b; Khalloufi et al. 2011;Reisetal. four main lineages: the pictorum, crassus, tumidus and gibbus 2013;Froufeetal.2014; Lopes-Lima et al. 2016a). After a lineage (Lopes-Lima et al. 2016b). Over the last decade, the land connection established around 5.96 Mya, the reopening pictorum lineage was divided into six species: Unio pictorum of the Strait of Gibraltar around 5.33 Mya (Krijgsman et al. (Linnaeus 1758), Unio mancus Lamarck 1819, Unio 1999) constituted a major vicariant event for many terrestrial delphinus Spengler 1793, Unio ravoisieri Deshayes 1848, and aquatic species. Since then, some secondary contact Unio elongatulus Pfeiffer 1825 and Unio caffer Krauss 1848 events took place, and at present, there is a high variety of (Van Damme 1984; Appleton 1996;Araujoetal.2005; colonisation patterns described across the Strait for different Khalloufi et al. 2011;Priéetal.2012;Reisetal.2013;Prié organisms. These patterns range from similar genetic lineages and Puillandre 2014). between northern Maghreb populations and those from Iberia While there are still uncertainties about the taxonomic status (e.g. Schmitt et al. 2006; Carranza et al. 2006; Gaubert et al. of the Unio species within this lineage in many regions, here 2010) to strong genetic splits (e.g. Steinfartz et al. 2000; we present the current evaluation of their geographical García-París et al. 2003; Fonseca et al. 2009; Miraldo