Journal of Fish Biology (2011) 79, 1950–1960 doi:10.1111/j.1095-8649.2011.03136.x, available online at wileyonlinelibrary.com Genetic divergence among native trout Salmo trutta populations from southern Balkans based on mitochondrial DNA and microsatellite variation A. P. Apostolidis*†, M. Th. Stoumboudi‡, E. Kalogianni‡, G. Cote§ and L. Bernatchez§ *Lab of Ichthyology and Fisheries, Department of Animal Production, Faculty of Agriculture, Aristotle University of Thessaloniki, Greece, ‡Hellenic Centre for Marine Research, Institute of Inland Waters, P.O. Box 712, 19013 Anavyssos, Greece and §Institut De Biologie Int´egrative et des Syst`emes (IBIS), Universit´e Laval, Qu´ebec, G1V 0A6, Canada (Received 8 June 2010, Accepted 20 September 2011) The genetic structure and the phylogenetic relationships among five Balkan populations of trout Salmo trutta that have been classified earlier into five different taxa were studied, using microsatellite and mitochondrial DNA (mtDNA) analyses. The pattern of population differentiation observed at microsatellites differed to that depicted by mtDNA variation, yet both methods indicated a very strong partitioning of the genetic variation among sampling locations. Results thus suggest that conservation strategies should be directed towards preserving the genetic integrity and uniqueness of each population. © 2011 The Authors Journal of Fish Biology © 2011 The Fisheries Society of the British Isles Key words: genetic diversity; Greece; Salmonidae; taxonomy. INTRODUCTION The considerable confusion regarding the taxonomy of brown trout Salmo trutta L. 1758 species complex originated from the complicated and incompletely described evolutionary history of the taxa. This is more evident in regions such as the Balkan Peninsula harbouring the most diverse phenotypic variation among Salmo spp. pop- ulations (Kottelat & Freyhof, 2007; Simonovic et al., 2007). Thus, numerous Salmo taxa have been described within this important region, and it is still debatable if these taxa should be considered as phenotypic variants or true Linnean species. On the other hand, it is well established that the use of molecular markers can provide clues regarding the evolutionary origin and useful tools for the conservation and management of populations and species (Apostolidis et al., 2008a). Previous surveys, based on the analysis of allozymic loci, mitochondrial (mt) DNA and microsatellites, revealed that southern Balkans comprise a region where Salmo †Author to whom correspondence should be addressed. Tel.: +30 2310 991719; email: apaposto@ agro.auth.gr 1950 © 2011 The Authors Journal of Fish Biology © 2011 The Fisheries Society of the British Isles GENETIC DIVERGENCE OF BALKAN SALMO TRUTTA 1951 spp. populations exhibit particularly high levels of genetic variation (Apostolidis et al., 2008a; Snoj et al., 2009). Indeed, mtDNA analyses revealed that four of the five major evolutionary lineages identified throughout the species range (Bernatchez, 2001) were present in the region. Moreover, most of the populations examined were genetically highly divergent, possessing private genotypes, indicating that they may represent distinct and potentially locally adapted gene pools (Apostolidis et al., 2008a). The aim of this work was to examine, using both bi-parentally and uni-parentally inherited molecular markers, the levels of genetic diversity and the patterns of genetic differentiation in two native wild Salmo spp. samples from Greece (Louros and Arapitsa) that have never been analysed at the molecular level so far. With respect to taxonomic status, the population from Louros was initially classified together with other Greek Salmo spp. populations from Epirus as Salmo dentex (Heckel 1851), then was considered to belong to a unique taxon (Salmo sp. Louros; Kottelat & Freyhof, 2007) and recently has been described as Salmo lourosensis (Delling, 2010). The taxonomic status of the Arapitsa population is unresolved. In order to juxtapose the present results with previous studies performed on Salmo spp. populations from the region, three more native wild populations, i.e. Agios Germanos (Lake Prespa), Aoos and Moravica were reanalysed at nuclear and mtDNA level, as well. These three Salmo spp. populations were selected on the basis that they inhabit waterbodies assumed to be unaffected by stocking activities, they represent three major mtDNA lineages, i.e. Adriatic (AD), Mediterranean (ME) and Danubian (DA) and, according to standard morphological methods of identification, they belong to three distinct taxa, namely, Salmo peristericus Karaman 1938, S. dentex and Salmo labrax Pallas 1814 (Kottelat & Freyhof, 2007) (Table I). MATERIALS AND METHODS Fin clips of 51 Salmo spp. originating from the Louros River (29) and the Arapitsa River (22) were collected in 2008. In addition, specimens originating from three locations (Aoos, Prespa and Moravica) previously analysed for mitochondrial and microsatellite variation (Apostolidis et al., 2008a, b) were reanalysed for this study, to increase the number of Table I. Description of the southern Balkan Salmo spp. populations analysed Sample size Sample mtDNA Number Population code Basin lineage Taxon mtDNA Microsatellite 1 Louros LOU Adriatic– AD lourosensis 29 20 Ionian Sea 2 Arapitsa NAO Aegean Sea AD pelagonicus? 22 20 3 Agios PRE Adriatic Sea AD peristericus 11 11 Germanos* 4 Aoos AOO Adriatic Sea ME dentex 22 14 5 Moravica MOR Danube DA labrax?2211 River *, Lake Prespa. © 2011 The Authors Journal of Fish Biology © 2011 The Fisheries Society of the British Isles, Journal of Fish Biology 2011, 79, 1950–1960 1952 A. P. APOSTOLIDIS ET AL. 5 Adriatic Sea 3 2 4 1 Aegean Sea Ionian Sea Fig. 1. Map of the south-western Balkans showing Salmo spp. sampling locations: 1, Louros River; 2, Arapitsa (Aliakmonas River); 3, Agios Germanos (Lake Prespa); 4, Aoos River; 5, Moravica River (Danube River system). character states and to allow standardization with previous studies. Detailed information of the samples is provided in Table I, and the approximate geographical location of the sampled populations is indicated in Fig. 1. Total genomic DNA was extracted using protocols reported in Apostolidis et al. (2008a). Microsatellite polymorphism was quantified at nine loci using fluorescently labelled primers: Ssad58, SsaD190, SsaD237 (King et al., 2005), SsaD85, SsaD170 (T. King, unpubl. data), CA040261, CA053307, CA060177 (Vasemagi¨ et al., 2005) and SSsp2213 (Paterson et al., 2004). Loci were PCR amplified and analysed on an Applied Biosystems 3100 auto- mated sequencer (www.appliedbiosystems.com). Mitochondria1 DNA variation was analysed both by restriction fragment length polymor- phism (RFLP) and sequencing performed on PCR amplified products. The RFLP analysis was performed on two adjacent PCR-amplified segments. One encompassed the complete ND5/6 region (c. 2·4 kb) and the other segment (c.2·1 kb) comprised the cytochrome b gene and the control region (CR). The amplified ND5/6 segment was digested with the restriction endonucleases HaeIII, MspI, Ava II, Hinf IandAluI and the CR segment with HaeIII, AluI, BcnIandMspI. Primers, amplifications, restriction digest and electrophoresis procedures were as described in Bernatchez & Osinov (1995). A 300 bp fragment at the 5 end and a 441 bp fragmentatthe3 end of the CR were sequenced on representatives of each composite hap- lotype identified with the PCR–RFLP analysis. The primers LN20 and HN20 (Bernatchez, 2001), which amplify the whole CR, were used for PCR, whereas primers H2 (Bernatchez, 2001) and HN20 in conjunction with a newly designed internal sequencing primer H3 (5- TGAATTCCAGAGAACCCATGT-3) were used for sequencing the 5 and 3 end of the CR, respectively. Technical procedures of mtDNA purification, amplification and sequencing are detailed in Apostolidis et al. (2008b). The control region variants observed were deposited in the GeneBank database under accession numbers FJ821448–FJ821452. © 2011 The Authors Journal of Fish Biology © 2011 The Fisheries Society of the British Isles, Journal of Fish Biology 2011, 79, 1950–1960 GENETIC DIVERGENCE OF BALKAN SALMO TRUTTA 1953 Microsatellite allele frequencies, number of alleles per locus and observed and unbiased expected heterozygosities were determined using the programme GENETIX v.4.04 (Belkhir, 2000). Tests of conformation to Hardy–Weinberg equilibrium (HWE) and genotypic linkage disequilibrium were carried out using Markov chain methods. Tests for differences of allele frequencies between pairs of populations were conducted by Fisher’s exact test. These anal- yses were all performed using GENEPOP 3.4 (Raymond & Rousset, 1995). Allelic richness (AR) and FIS values were calculated using the programme FSTAT 2.9.3.2 (Goudet, 2001). Global and pair-wise FST and their associated P values were estimated using the θ estimator (Weir & Cockerham, 1984) of Wright’s FST. The significance levels for the overall and the pair-wise values were determined after 10 000 permutations. As an alternative measure of the degree of genetic differences among populations, an assignment test was used to deter- mine how unique individual Salmo spp. genotypes were to the locality from which they were sampled. Fishes were individually assigned according to the Bayesian method of Rannala & Mountain (1997), with a probability threshold of 5% using the software GeneClass2 ver- sion 2.0 (Piry et al., 2004). The occurrence of a population bottleneck was tested using the software BOTTLENECK 1.2 (Cornuet & Luikart, 1996), assuming an infinite allele