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Introduction

Brown trout Salmo trutta L. is one of the best studied nativeSalmonids of Europe. The species is composed of numer-ous distinct geographical forms and shows considerablevariability and plasticity in many aspects of its mor-phology, ecology and behaviour. This variability led earlyworkers to recognize at least 50 species which are nowconsidered to belong to a single species, Salmo trutta(Behnke 1986; Elliot 1989). This ‘lumping’ approach fails toreflect the extensive interpopulation genetic variabilitywhich is found within this species (Ferguson 1989).Molecular analysis of genetic variation in natural popula-tions can provide data on population structure which,when combined with data from biogeographical informa-tion can identify important trends in population structureand dynamics (Avise 1994).

Early studies on the genetic structure of brown troutwere based on electrophoretic analysis of allozyme varia-tion (reviewed in Ferguson 1989; Guyomard 1989).Collectively, these studies have shown the distinctivenessof north-western ‘Atlantic’ and south-eastern populations,with at least two different lineages in the latter grouping(Apostolidis et al. 1996a).

Mitochondrial DNA (mtDNA) provided the first exten-sive and readily accessible data suitable for robustgenealogical inference at the intraspecific level (Avise et al.1987). The advantages of mtDNA as a tool for populationgenetics have been extensively reviewed (Moritz et al.1987; Harrison 1989), as well as the ability of mtDNA toretain a history of past isolation, even in the event of con-temporary admixture of groups that evolved in allopatry(Avise 1994). Geographical surveys of mtDNA within several freshwater fish species have also demonstrated theimportance of both historical biogeography and contem-porary gene flow in shaping intraspecific populationgenetic structure (Avise et al. 1987). Further, mtDNA

Mitochondrial DNA sequence variation and phylogeography among Salmo trutta L. (Greek brown trout) populations

A . P . A P O S T O L I D I S , C . T R I A N T A P H Y L L I D I S , A . K O U V A T S I and P . S . E C O N O M I D I S *Department of Genetics, Development and Molecular Biology, and *Department of Zoology, Laboratory of Ichthyology, School ofBiology, Aristotle University, GR-540 06 Thessaloniki, Macedonia, Greece

Abstract

To investigate the phylogenetic relationships and geographical structure among browntrout S. trutta L. populations from the South Adriatic–Ionian and Aegean sea basins,mitochondrial DNA nucleotide sequence comparisons were used. A 310-base-pair (bp)segment of the control region (D-loop), and an additional 280-bp segment of thecytochrome b gene were sequenced from representatives of 13 brown trout populations.Phylogenetic analyses, conducted after combining the data presented with publisheddata from other Eurasian brown trout, revealed four major phylogenetic groups, three ofwhich were found widely distributed within the southern Balkan region. The phylogeo-graphical patterns revealed by mtDNA represent one of the few cases where phylogeneticdiscontinuity in a gene tree exists without obvious geographical localization within aspecies’ range and has most likely resulted from the differentiation of the major mtDNAclades during Messinian or early Pleistocene times. Finally, the genetic relationshipsamong the populations suggested by mtDNA were generally not in accordance witheither allozyme or morphological data.

Keywords: Salmo trutta, mitochondrial DNA, phylogeography, population structure, conservation

Received 10 September 1996; revision accepted 3 January 1997

Molecular Ecology 1997, 6, 531–542

© 1997 Blackwell Science Ltd

Correspondence: C. Triantaphyllidis Tel: +30-31-998309, Fax: 30-31-998374, E-mail: [email protected]

can assist in determining the taxonomic distinctiveness of individual populations and therefore aid in setting priorities for conservation programmes (Moritz 1994).

Previous surveys of mtDNA sequence variation amongbrown trout populations from various locations haverevealed the existence of five major geographically dis-junct phylogenetic groups (Bernatchez et al. 1992; Giuffraet al. 1994; Bernatchez & Osinov 1995). However, thesestudies have not focused on the relationships and originsof trout from the southern Balkans, and specifically fortrout living in the Ionian and Aegean sea basins, an area thought to harbour several distinct subspecies whose taxonomic status and distribution remain unclear (Economidis 1991). Greek brown trout belong totwo different Ichthyogeographical zones: the southAdriatic–Ionian, which includes rivers of western andsouth-western Greece and Albania, and the Ponto-Aegean,which includes rivers of northern and north-easternGreece (Economidis & Banarescu 1991). Previous analysesof isozyme variation (Apostolidis et al. 1996a) clustered allGreek brown trout populations into two different lineagescorresponding to the delimitation of the two differentichthyogeographical zones (with the exception of Prespa’spopulation which clustered separately). However, a morerecent PCR–RFLP study of mtDNA variation in the samepopulations (Apostolidis et al. 1996b) revealed the exis-tence of four phylogenetic groupings and showed littlegeographical structure.

In this paper, we report on DNA sequence variation insegments of the mitochondrial control region and

cytochrome b. Furthermore, we combine the data present-ed with results obtained in previous mtDNA sequenceanalyses from brown trout to propose a biogeographicalhypothesis explaining the pattern of genetic differentia-tion observed among contemporary populations in thesouthern Balkans.

Materials and methods

Sample collections

Samples of brown trout were collected from 11 differentGreek streams (Fig. 1). In addition, one more sample wasobtained from the River Garonne (Pyrennes, France), anAtlantic drainage and another sample from the LakeOhrid (Albania). The last sample is considered to belong tothe subspecies S. t. letnica. The drainages of Nestos,Tripotamos and Venetikos belong to the Ponto-Aegeanzone, while all the rest including the Ohrid lake, belong to Adriatic–Ionian zone. All samples (except these ofNestos) were obtained from natural populations where nostocking activities have been reported. The population of Nestos is the only known population to have been stocked 20 years ago with 20 000 fingerlings of Salmo trutta ssp., originating from a fish farm at Acheloos-1 drainage.

Additional data for our analyses were obtained from:(i) a previous RFLP study of the same individuals per-formed on PCR amplified mtDNA segments (Apostolidiset al. 1996b), and (ii) published sequences from earlier

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 531–542

532 A . P . A P O S T O L I D I S E T A L .

Fig. 1 Sampling sites: NES, Nestos; TRI,Tripotamos; PRE, Ag. Germanos(Prespa); OHR, Ohrid; VEN, Venetikos;VOI, Voidomatis; THY, Thyamis; AC1,Acheloos-1; AC2, Acheloos-2; EVI,Evinos; MOR, Mornos; ALF, Alfios. Thepopulation of Garonne(Pyrenees–France) is not indicated.

mtDNA sequence analyses on brown trout populationsacross Europe (Bernatchez et al. 1992; Giufra et al. 1994).

DNA amplification and sequencing

DNA was extracted from liver tissues following the proto-col of mtDNA extraction of Bernatchez et al. (1988). A 310-bp segment of the mtDNA control region including a13-bp segment of the proline tRNA gene was sequencedfor 76 individuals. A segment of 280 bp of the S. truttacytochrome b mtDNA gene was also sequenced. PCRamplifications were performed in a thermal cycler(Techne) using the primers L19, H17 described inBernatchez et al. (1992) and H15149, L14841 described inKocher et al. (1989) and modified by McVeigh et al. (1991)for the control region and cytochrome b segments, respec-tively, using protocols reported in Apostolidis et al.(1996b).

Amplified DNA was purified with the SequenaseTM

PCR + Product Sequencing Kit according to the supplier’sprotocol. Double-stranded DNA sequencing reactionswere prepared with the Sequenase kit (Version 2.0, USBiochemical) according to the manufacturer’s directions.Sequencing of the 5′ end of the control region was carriedout using an internal primer H2 described in Giuffra et al.(1994). For the cytochrome b segment both light and heavystrand primers were used to obtain complementarysequences. Sequences were separated on 40-cm, 6% polyacrylamide (19 : 1 BIS), 7-M urea gels and visualizedby autoradiography as described by Bernatchez et al.(1992).

Sequence analysis

Sequence data obtained for both mtDNA segments wereanalysed for distance and character-based variation usingP H Y L I P (Version 3.5; Felsenstein 1993). The distance mea-sure used was the estimate of nucleotide substitution calculated under the Kimura 2-parameter model (Kimura1980) using for the transition/transversion ratio the 2.0default option of the D N A D I S T program. The resulting distance matrices of pairwise distance comparisons wereused to construct trees using the Neighbour-Joining algo-rithm (Saitou & Nei 1987) in the program N E I G H B O R.Sequence data were used to generate phylogenetic treesaccording to a maximum parsimony criterion using theD N A P A R S program. Majority-rule consensus trees wereconstructed using the C O N S E N S E program and confidencestatements on branches were estimated by running D N A P A R S on 1000 bootstrap replications obtained by theS E Q B O O T program. In all cases, trees were rooted usingAtlantic salmon Salmo salar sequences obtained from theGenBank (accession numbers: M97987 and X76253 for thesegments of D-loop and cytochrome b, respectively).

We then combined distinct single endonuclease pat-terns generated by a previous RFLP analysis on themtDNA ND-5/6 segments of the same individuals(Apostolidis et al. 1996b), with our results from the D-loopsequence, to describe RFLP/sequence composite geno-types (Table 3). As there was no overlap of mutationalsites detected by RFLP and D-loop sequence analyses,RFLP and sequence mutational sites detected amongmtDNA genotypes were pooled (Bernatchez & Osinov1995) in order to generate phylogenetic trees according toa maximal parsimony criterion using the program M I X

and again the C O N S E N S E and S E Q B O O T programs of theP H Y L I P package. Sequence divergence values betweengenotypes were estimated by using the average pairwisedistance measures obtained from RFLP and control regionsequence analyses.

Levels of intra- and interpopulation genetic diversitywere estimated from the frequency distribution of mtDNAgenotypes and their pairwise divergence estimates by themaximum likelihood estimation of the average number ofnucleotide substitutions per site within and between pop-ulations (Nei 1987). A matrix of net interpopulationnucleotide divergence was constructed and used to built aU P G M A phenogram relating all populations analysed. Thedegree of geographical heterogeneity of mtDNA haplo-type distributions was assessed using a χ2 statistic asdescribed by Roff & Bentzen (1989). The significance levelwas obtained by 10 000 Monte Carlo randomizations usingthe R E A P package (McElroy et al. 1991). NST (Lynch &Crease 1990) were used to estimate the degree of popula-tion subdivision at the nucleotide level. The resultingindex gives the ratio of the average genetic distancebetween genes from different populations relative to thatamong genes in the population. Values of NST range from0 (no population subdivision) to 1 (complete populationsubdivision). Distance matrices constructed fromallozyme and mtDNA sequence data were comparedusing the Mantel test (1967) incorporated in the N T S Y S

computer program (Rohlf 1990).

Results

The sequence of a 310-bp segment at the 5′ end of controlregion (including a 13 bp of the tRNAPro segment) wasdetermined for 76 individuals from 13 populations (Table1). In order to perform a more complete phylogeneticanalysis of coding and noncoding mtDNA sequences, a280-bp segment of cytochrome b was also sequenced for 18individuals representing all control region genotypic variants. A total of 12 variable nucleotide sites and 10genotypes were identified for the control region segment,while six variable nucleotide positions were found incytochrome b (Table 2, Fig. 2). Of the 10 control regionvariants detected, a number were found in individual


m t D N A S E Q U E N C E V A R I A T I O N I N G R E E K B R O W N T R O U T 533

cytochrome b variants, whereas in one case (genotype B2)where individuals with identical control region sequence(genotype B) differed in cytochrome b sequence. By com-bining the variable positions for D-loop and cytochrome bsegments a total of 11 genotypic variants were detected(Table 2).

A major difference in the transition : transversion ratio(TS/TV) was found in coding and noncoding segments.TS/TV in the D-loop was 2 : 1 (8 : 4), and 7 : 0 in the codingsegments (6 : 0 in the cytochrome b segment and one more transition in the proline tRNA segment). Five transi-tions occurred at the third base of a codon, and only

one involved a first position (position 190 at Fig. 2, Table2). None of the transitional changes required an aminoacid replacement and therefore represent silent sub-stitutions. The A + T content found in the cytochrome bsegment was 57%. In contrast to the cytochrome b gene,the 310-bp segment of the D-loop (including 13 bp of thetRNAPro) showed a strong bias in base content, with 68%A + T.

Pairwise sequence divergence estimates amongmtDNA variants in the D-loop segment varied from 0.32to 2.31% while when the cytochrome b segment was addedthese values varied from 0.17 to 1.72%.



D-loop sequence genotypes

Population Basin n A B C D E F G H I J

Acheloos-1 (Gr) Io 7 1(1) 6(1)Acheloos-2 (Gr) Io 5 5(1)

Alfios (Gr) Io 3 3(1)Evinos (Gr) Io 4 4(2)Garrone (Fr) At 5 5(1)Mornos (Gr) Io 5 5(1)Nestos (Gr) Ae 9 4(1) 5(1)Ohrid L. (Al) Ad 6 5(1) 1(1)Prespa (Gr) Ad 5 5(1)Thyamis (Gr) Io 6 6(1)

Tripotamos (Gr) Ae 6 1 5(1)Venetikos (Gr) Ae 9 1 8(1)

Voidomatis (Gr) Ad 6 5(1) 1(1)

Table 1 Origin of fish populations, sample sizes (n)and absolute frequency distribution of 10 mtDNAvariants detected among 76Salmo trutta control regionsequences. Basin: Io, Ionian;Ad, Adriatic; At, Atlantic; Ae,Aegean. Nestos has beenstocked about 20 years agowith domestic strains originating from a local nativefish-farm at Acheloos-1.Numbers in parentheses referto individuals used forcytochrome b gene sequenceanalyses. Letters in paren-theses refer to the following:Gr, Greece; Al, Albania; Fr,France.

Table 2 Genotype designation and variable nucleotide positions of proline tRNA (number 1), control region (2–12) and cytochrome b gene(13–18) sequence analysis among 11 Salmo trutta mtDNA genotypes. Numbers refer to positions identified in Fig. 2. The nucleotide at eachposition is given for genotype At1 described in Bernatchez et al. (1992). For all other genotypes, variable nucleotides are indicated whileidentity is indicated by slashes. Asterisks indicate deletions

Proline Control region Cytochrome btRNA

Genotype 1 2 3 4 5 6 7 8 9 10 11 12 13–14 15 16 17 18

Leu Tyr Asp Arg TyrAt1 T T T A T * G G A A T G TTA TAC GAT CGA TATA – – – – – – – – – G G – – – – – – – – – – – – – – – –B1 – – C – – – – – – – – C – – G – – – – – C – – – – – –B2 – – C – – – – – – – – C – – G – – – – – C – – – C – –C – – C – C – – – – – – C – – G – – – – – C – – – – – –D – – C – – – A – – – – C – – G – – – – – C – – – – – –E – – – – – – – – – – – C – – G – – – – – C – – – – – –F – – C G – – A – – – – C – – G – – – – – C – – – – – –G – – C – – A – – – – – A – – G – – – – – C – – – – – –H – – C – – – A – C – – – C – G – – – – – C – – – – – –I C – C – – – A – C – – – C – G – – – – – C – – – – – –J – C A – – – – A – – G – – – G – – T – – C – – G – – –

Classification of new mtDNA genotypes to majorphylogenetic groupings

The 11 mtDNA genotypes found in this study clustered(Fig. 3) into four main mtDNA clades (the Greek geno-types classified into three groups) corresponding to thosedescribed earlier (Bernatchez et al. 1992). Genotypes A andJ were not previously observed and belonged to IV(Atlantic) and III (Danube) groups, respectively, as theyshared identical states at the diagnostic positions for thesegroupings and none with those of others [except characterstate 11 (Fig. 3, Table 2) of genotype A]. Genotypes H andI belong to group I (Mediterranean), while all the restgenotypes belong to group II (Adriatic). However, within

group II, genotypes G, MA2 and MA3 consist of aninternal group IIA, supported at the 77% level in themajoity-rule consensus tree. Genotypes G, H and B1 (Table2) were identical with those named as MA1, ME2 andAD 4, respectively, defined previously in sequencinganalysis of the same segments (Giuffra et al. 1994). It isinteresting to note that despite genotypes E, D and F shar-ing identical character states for this grouping (Adriatic),they also share one identical character state with groupsIV and I, respectively. The distance values and the derivedtrees in the present case were not affected when, instead ofthe 2.0 default option, we used different possible valuesfor the TS/TV ratio.

Phylogenetic relationships and sequence divergenceamong new mtDNA genotypes

The clustering of the 11 genotypes resulting from distanceand parsimony analysis of both D-loop and cytochrome bwas identical to that obtained from the control regionanalysis alone. In a previous mtDNA RFLP study of theND-5/6 segments of the same individuals (Apostolidis etal. 1996b) eight synapomorphic sites were identifiedamong nine distinct mtDNA haplotypes. These eightsynapomorphies were used in combination with thesynapomorphies found in sequence analysis of the D-loopto generate an overall majority-rule tree. The overall con-sensus tree was constructed by pooling both RFLP andsequence data sets (Bernatchez & Osinov 1995) and usinggenotype At4 which was found only in the Atlantic popu-lation (Garonne) as an outgroup, in order to assessphylogenetic relationships within Greek brown trout pop-ulations (Fig. 4). The combination of both data sets addedfour additional genotypes to the 10 identified by D-loopsequences (Table 3). To avoid confusion the nomenclaturepresented is consistent with that used in earlier studies(Bernatchez et al. 1992; Giuffra et al. 1994; Bernatchez &Osinov 1995), e.g. as 21 different genotypes belonging tothe Danube group were previously identified the one newreported here was named as Da 22. The 14 mtDNA genotypes found, clustered (Fig. 4) into four highly differ-entiated phylogenetic groupings (net sequence divergenceestimates among groupings varying from 0.0317 to 0.0188)identical to those revealed in Fig. 3. These values are high-er than those observed in sequence analysis alone. Allbootstrapping values on major branches were increased bypooling both data sets, providing an additional indicationof their congruence. The branching pattern between groupII and its clonal subgroup IIA (separated by sequencedivergence value of 0.0056) still remained unresolved.

Apart from lineage III (Danube) the remaining lineagesshow little distinct geographical structure with genotypesfrom groups I (Mediterranean) and II (Adriatic) found inboth Adriatic–Ionian and Ponto-Aegean zones.



Fig. 2 DNA sequences (L. strand) for the 5´-end of a 310-bp seg-ment of the mtDNA control region (Fig. 2 upper) and for a seg-ment of 280-bp of the cytochrome b (Fig. 2 bellow), from Salmotrutta type At1 and S. salar as reported by Bernatchez et al. (1992)and Giuffra et al. (1994), respectively. Nucleotides are given for S.salar when different from S. trutta, while identity is indicated bydots. The first sequence (5´ end) includes part of the proline tRNAgene. Variable positions among brown trout mtDNA variantsreported here are underlined and numbered. Asterisks indicatedeletions. The sequences presented in this paper (cf. Table 2) havebeen entered into GenBank under the accession numbersU63786–U63795 and U63888–U63892, for the D-loop andcytochrome b segments, respectively.

Population gene diversity

The distance matrix of net interpopulation nucleotide diver-gence (Table 4) was used to construct a UPGMA tree relatingthe 13 populations studied (Fig. 5). The populations clus-tered into five distinct groups, largely reflecting racial parti-tioning. These clusters are: A, Acheloos-1 and 2, Mornosand Nestos populations; B, Alfios, Thyamis, Ag. Germanos(Prespa), Evinos, Tripotamos and Ohrid populations; C,Atlantic drainage population (Garonne, France); D,Venetikos population and E, the population fromVoidomatis. The UPGMA analysis of nucleotide divergenceamong populations (Fig. 5) shows also little geographicalstructure. Statistically significant differences in genotypefrequencies among all populations were observed(χ2 = 662.85, P < 0.001). The test for differences among thetwo ichthyogeographical zones (i.e. samples within zoneswere pooled) resulted in a significant outcome (χ2 = 49.25,P < 0.001). Within the south Adriatic–Ionian zone and thePonto-Aegean zone significant differences between popula-tions were also observed (χ2 = 271.43, P < 0.001 and χ2 = 48,P < 0.001, respectively). Yet, significant substructuring wasobserved within cluster B (χ2 = 140, P < 0.001), while the dif-ferences between the four populations in cluster A werefound to be nonsignificant (χ 2 = 14.16).

Contrary to the high interpopulation diversityobserved (mean = 1.44 ± 0.00%), intrapopulation diversity

was low in most cases (0.13 ± 0.00%). Thus, based on theNST estimate of 0.91, less than 10% of the overall geneticdiversity observed was attributed to intrapopulationdiversity as opposed to 91% for among populations. TheNST estimate found without the French population wassimilar (0.89).

The Mandel test, employing 10 000 random permuta-tions, showed no correlation (r) between genetic distancematrices (Table 4) found with allozymes and mtDNAmethods (r = 0.05, P = 0.412). Furthermore, there was nocorrelation between levels of intrapopulation diversityobserved in the mitochondrial and nuclear genome. Forexample, whereas no mtDNA diversity was found in thepopulation of Garonne it displayed the highest value ofnuclear heterozygosity (H = 0.098). Reciprocally, highmtDNA diversity was found in the population ofTripotamos (h = 0.33) where nuclear heterozygosity waszero (H = 0.0).

Discussion

Sequence variation in mitochondrial D-loop andcytochrome b regions

Despite the extensive variation found among the popula-tions we studied, for at least some S. trutta populations D-loop appears to evolve at a lower rate than some other



Fig. 3 Neighbour-Joining tree relatingfour trout mtDNA phylogenetic groups(20 mtDNA genotypes), resulting fromthe combination of 11 mtDNA geno-types revealed in this study (Table 2)with nine mtDNA genotypes describedpreviously by Giuffra et al. (1994), basedon sequence analysis of 310 bp of D-loopand 280 bp of cytochrome b. Romannumbers refer to group designationfrom those studies. Character states ofdiagnostic positions analysed in thisstudy are indicated bellow branchesboth for the D-loop and cytochrome bsequences (capital letters indicatingnucleotides). Numbers refer to variablepositions of Fig. 2. Bootstrap estimatesresulting from the majority-ruleconsensus tree among these genotypes(as a percentage) are indicated abovebranches for clades with 50% support.The arrow indicates the root position(Salmo salar).

regions of the mtDNA. For example, no variation wasfound in brown trout from the Atlantic basin by sequenc-ing 310 bp and 330 bp of the tRNAPro and tRNAPhe ends ofthe D-loop, respectively (Bernatchez et al. 1992), whereasextensive variation among Atlantic brown trout has beenfound in the NADH dehydrogenase genes using RFLP(Hansen 1994; Ferguson et al. 1995). Aside from a newgenotype found in the only Atlantic population studied,our results are concordant with this view. The levels ofdivergence we observed among 10 control region haplo-types (range 0.32–2.31%) are lower than those observedamong nine haplotypes revealed in a mtDNA RFLP studyof the ND-5/6 genes (Apostolidis et al. 1996b) among thesame individuals (range 0.38–4.68%). The estimates based

on the segment of control region alone were slightly high-er than those reported by an earlier study (Bernatchez et al.1992) for a 640-bp segment of the D-loop (0.16–1.92%).

Our results also corroborate previous observations onthe pattern of mutation observed in the mtDNA codingand noncoding regions of brown trout (Giuffra et al. 1994).The ratio of transitions to transversions found in coding(7 : 0) and noncoding regions (8 : 4) are comparable tothose previously reported (Bernatchez et al. 1992) in browntrout populations (17 : 0–17 : 6, respectively). Bernatchezand Danzmann (1993) observed a similar ratio of transi-tions to transversions (8 : 3) in the control region of thebrook charr Salvelinus fontinalis and suggest that structuralor functional needs of maintaining a given A + T -



Fig. 4 Majority rule consensus tree relating the 14 brown trout mtDNA genotypes identified from the combination of 10 synapomorphicsites detected in sequence analysis of the control region and eight synapomorphic sites detected in PCR–RFLP analysis of the ND-5/6 seg-ments among 76 individuals. The capital letters in parentheses indicate the haplotype designations revealed in sequence analysis of 590 bpin Fig. 3. The principal mtDNA clades are identified with vertical lines. Character states of diagnostic positions for each group are indi-cated below branches both for the D-loop sequence and restriction site data (+ = site gain, – = site loss). For restriction site data, numbersrefer to distinct enzymes: AluI (1, 2), AvaII (3, 4), HaeIII (5, 6), HinfI (7), HpaII (8) reported in Apostolidis et al. (1996b). Bootstrap estimates(as a percentage) are indicated above branches. Branch lengths are expressed in nucleotide substitution per site according to estimates fromNeighbour-Joining analysis. Genotype At4 was used as an outgroup taxon in assessing phylogenetic relationships within Greek browntrout populations.

composition in the control region of salmonids could leadto this lower probability of transitions. In addition, a simi-lar phenomenon regarding patterns of substitution in thispart of the control region was also observed in rainbowfish, where the control region had a strong bias towardA + T (70%), whereas cytochrome b had an average con-tent of 53% A + T, suggesting that apparent saturation ofTS at low levels of sequence divergence in the controlregion was due to biased base content, as in insects (Zhu etal. 1992). Thus, considering the correspondence with A + Tcontent found in our study, the same effect of composi-tional constraint on base substitution may operate in thesalmonid control region as well.

The present study revealed the existence of four high-ly differentiated mtDNA phylogenetic groups (three inGreece and one from France) in brown trout populationsstudied. This indicates an extensive subdivision of theancestral S. trutta group. It has been established thatbrown trout represent one of the most highly geneticallystructured fish species studied (Bernatchez et al. 1992).However, the identification of at least three different lineages in a restricted geographical area such as Greece issurprising and demonstrates the wealth of genetic diver-sity present in brown trout from the Southern Balkans.Thus, Greece appears as a region of rather high mtDNAdiversity among trout populations, because the NST valuesfound in this country (NST = 0.89) is, at least, equivalent tothat found across all Western Europe (NST = 0.76,Bernatchez et al. 1992). Furthermore, most populationsexamined had a unique genetic profile without sharing

genotypes with the other populations, even those belong-ing to the same cluster. This significant differentiationamong the populations may be due to a long period of isolation coupled with bottleneck and subsequent geneticdrift phenomena. In such cases, common mtDNA geno-types among populations may have become rare orextinct through stochastic lineage loss (Pamilo & Nei1988). However, this phylogeographical pattern, in whicha deep phylogenetic discontinuity in the mtDNA genetree exists without clade localisation (category II in theclassification scheme of Avise et al. 1987) is consideredunusual, having been reported previously in only a fewspecies (Avise 1994). Normally, mtDNA clades distin-guished by large sequence gaps are localized and geo-graphically orientated in ways suggestive of originsthrough long-term zoogeographical impediments to geneflow (Avise et al. 1992). Examples of phylogeographicalcategory II are considered to result from secondaryadmixture between allopatrically evolved populationsfrom which deep splits in the gene tree derive (Avise et al. 1987, 1992; Dodson et al. 1995). Yet, it is also possiblethat nucleotide saturation of TS in the D-loop segmentcould also have reduced the informativeness of thisregion and may be the reason for the lack of geographicallocalization in our analyses. We cannot also ignore thepotential stochastic effects of low sample sizes whichcould be important for mtDNA. This could be partly responsible for the extremely high numbers of private genotypes and for the phylogenetic discontinuityobserved.



Table 3 Fourteen mtDNA joint genotypes resulting from the combination of 10 mtDNA genotypes from D-loop sequence analysis withnine PCR–RFLP restriction genotypes (haplotypes) from ND-5/6 segments restriction analysis and their frequency distribution among 13brown trout populations. Joint genotypes designation refers to nomenclature used in Bernatchez et al. (1992); Atlantic (At), Adriatic (Ad),marmoratus (Ma), Mediterranean (Me) and Danube (Da). The order of fragment patterns corresponds to restriction enzymes AluI, AseI,AvaII, HaeIII, HinfI, HpaII and TaqI

D-loop ND-5/6Sequencing Restriction JointGenotypes Genotypes Genotype AC1 AC2 ALF EVI GAR MOR NES OHR PRE THY TRI VEN VOI

A DABABBB At4 1.00B AAAAAAA Ad5 0.143 1.00 0.167B AABCAAA Ad6 1.00B AAACAAA Ad7 1.00B FAAAAAA Ad8 0.833C AABCAAA Ad9 1.00D AAAAAAA Ad10 0.833E AAAAAAA Ad11 0.444F FAAAAAA Ad12 0.167G BAABABA Ma1 0.857 1.00 1.00 0.444G BAABAAA Ma4 0.112H CBCACCA Me2 0.111 0.833I CBCACCA Me3 0.167J ECCAADC Da22 0.899

Divergence times of brown trout phylogenetic groupsand relation with paleogeographical events

Earlier sequence analysis of coding and noncoding seg-ments of the mtDNA of brown trout populations distributed across a broad geographical range revealedfive different lineages (Bernatchez et al. 1992; Giuffra et al.1994; Bernatchez & Osinov 1995). According to theseanalyses all brown trout from the Atlantic basin belong tothe same group, namely ‘Atlantic’, whereas all populations

related with the Black, Caspian and Aral sea basins andsome with the Danube system belong to the Danubegroup. The remaining genotypes found in populationsthroughout the Mediterranean basin, from Spain toTurkey belong to either one of the three other major phylogenetic groups AD (Adriatic), ME (Mediterranean)and MA (marmoratus). The Neighbour-Joining and themajority rule consensus trees presented here (Figs 3 and4), suggest that three of these five major phylogeneticgroups are found in Greece.



Table 4 Pairwise number of nucleotide substitutions per site averaged for mtDNA PCR–RFLP and control region sequence analysis (belowmain diagonal), and pairwise estimates of Nei’s standard genetic distance for allozymes (above main diagonal) among the 13 brown troutpopulations

Pop. AC1 AC2 ALF EVI GAR MOR NES OHR PRS THY TRI VEN VOI

AC1 0.004 0.079 0.005 0.145 0.021 0.064 0.078 0.105 0.032 0.092 0.061 0.037AC2 0.0000 0.069 0.010 0.119 0.008 0.057 0.054 0.112 0.013 0.070 0.046 0.039ALF 0.0116 0.0138 0.080 0.109 0.075 0.049 0.018 0.101 0.083 0.052 0.034 0.040EVI 0.0054 0.0076 0.0058 0.146 0.016 0.048 0.078 0.096 0.036 0.074 0.055 0.038GAR 0.0216 0.0230 0.0228 0.0206 0.109 0.091 0.070 0.176 0.108 0.076 0.067 0.106MOR 0.0000 0.0000 0.0138 0.0076 0.0230 0.047 0.046 0.122 0.006 0.047 0.039 0.056NES 0.0007 0.0018 0.0081 0.0021 0.0188 0.0018 0.035 0.054 0.070 0.015 0.012 0.026OHR 0.0074 0.0096 0.0077 0.0019 0.0225 0.0096 0.0041 0.108 0.045 0.019 0.010 0.043PRS 0.0076 0.0098 0.0038 0.0020 0.0230 0.0098 0.0042 0.0039 0.152 0.094 0.076 0.060THY 0.0100 0.0122 0.0016 0.0042 0.0211 0.0122 0.0065 0.0061 0.0022 0.061 0.049 0.074TRI 0.0065 0.0087 0.0069 0.0011 0.0217 0.0087 0.0033 0.0025 0.0031 0.0053 0.007 0.047VEN 0.0221 0.0236 0.0226 0.0212 0.0249 0.0236 0.0203 0.0194 0.0237 0.0209 0.0221 0.021VOI 0.0248 0.0263 0.0251 0.0236 0.0281 0.0263 0.0235 0.0250 0.0263 0.0234 0.0220 0.0247

Fig. 5 U P G M A phenogram clustering 13Salmo trutta populations according to thedistance matrix resulting from the esti-mation of interpopulation nucleotidediversity.

One of the major assets of molecular phylogenetic datais the potential application of a molecular clock for esti-mating branching times of divergent assemblages.However, estimates of divergence time (t) are very proneto error arising from typically inadequate calibrations.Nevertheless, if there is no important deviation from amolecular clock model, crude estimates of the separationtime can be given, provided that the relationship betweengenetic distance and t has been calibrated before (Giuffraet al. 1996). Thus, if the substitution rate of 0.5–0.9% permillion year, which has been estimated in salmon from aprevious RFLP analysis of mtDNA coupled with fossildates (Martin and Palumbi 1993), holds for other salmonidspecies, as well as for sequencing data, the divergencetime among the four phylogenetic groups describedabove, can be estimated. Therefore, using results from acombination of both RFLP and sequence analyses, thosegroups must have diverged from a common ancestor sometime ago. It would appear (Fig. 3) that the isolation of allgroups occurred at similar geological times and the diver-gence time between these groups (Fig. 4) would be duringMessinian or early Pliocene times (2.5–6.0 Myr ago). Atthis time two important geological events occurred(Bianco 1990): First, the isolation of the Mediterranean seafrom the Atlantic Ocean, while the connections betweenMediterranean and Paratethys (which included the Black,Caspian and Aral seas) were also interrupted. BothmtDNA and allozymes data (Apostolidis et al. 1996a,b)suggest that the isolation of Atlantic from Mediterraneanbrown trout populations occurred during this time.Secondly, at the end of the Messinian, both Mediterraneanand Paratethys were reduced to a network of lakes knownas the ‘Lago Mare’ phase of the Mediterranean (Hsu 1978).The ‘Lago Mare’ stage probably played an essential rolefor the isolation of Mediterranean brown trout popula-tions – as initial panmictic populations were separated indistinct lakes – and for a latter dispersion in peri-Mediterranean river systems. It seems probable that theisolation among the Mediterranean lineages of browntrout occurred during this time. The closer relationshipbetween groups II and IIA suggests that these two groupsbecame isolated more recently, their time of separationestimated at 0.6–1.1 Myr ago (Pleistocene). According tothis scenario, the present mtDNA phylogeny probablyrepresents clades that differentiated in refugia and thencame into secondary contact. Alternatively, Slatkin &Hudson (1991) showed that large stable populations canalso generate phylogenetically discontinuous gene trees.

As Greek brown trout populations are native and nostocking activities (with the exception of Nestos) havebeen reported, no mtDNA genotypes of the AT (Atlantic)grouping were found among the Mediterranean pop-ulations in this study. The considerable geographical distribution of group II not only in Greece but in the whole

Mediterranean region (Bernatchez et al. 1992; Giuffra et al.1994) might indicate that this represents the founderpopulation in the area. Group I, which is also present inthe same area, indicates the existence of at least a secondphylogenetic group highly divergent from group II in thisregion. The internal group IIA which comprises geno-types Ma1 and Ma4 is confined to the Adriatic basin and is derived from trout populations that may have sur-vived in that region. The presence of these genotypes inthe Nestos population most likely reflects the repeatedstocking with fish-farm stocks originating from theAcheloos River.

Sequence data have also revealed that genotypeJ(Da22), found in the population of Venetikos (Ponto-Aegean zone), is associated with the phylogenetically distinct group III ‘Danube’. Although we cannot entirelyrule out the case of unrecorded introductions, it couldhave entered the area in recent years (late Pliocene toPleistocene) by one of two routes: through the Black Seaand/or more probably directly from the north by means ofrivers captures, from the Morava, a tributary of theDanube to Axios (Vardar) river and then to Venetikos(Economidis & Banarescu 1991). Given the paucity of his-torical and archaeological information it is difficult todetermine the exact sequence of events and therefore thegeographical origin of the three Mediterranean phyloge-netic groups.

Levels of congruence between allozyme and mtDNAvariation

Results obtained from the analysis of mtDNA andallozyme variation illustrated both concordance and dis-cordancies between the two methods. Both data setsdemonstrated the differentiation of the Atlantic popula-tion (Garonne) from the Mediterranean ones. The extent ofthis dichotomy was also emphasized by the magnitude ofgenetic distance values. Both mitochondrial and allozymeanalyses have shown that the largest proportion of genet-ic variability in brown trout is distributed among and notwithin populations. In addition, both allozymes(Apostolidis et al. 1996a) and the present results do notsupport the idea that the Ohrid population of S. t. letnica isa separate subspecies derived from an ancient commonancestor, but indicate a much more recent divergence.Conversely, major discrepancies between mtDNA andallozyme analyses were observed in the extent of bothintra- and interpopulation levels of genetic variation. Lackof congruence between mitochondrial and nuclear basedphylogenies can result from the different modes of trans-mission and evolution of these genetic systems. Similardiscrepancies between the two methodologies have fre-quently been reported in the literature (e.g. Bernatchez etal. 1992; Hansen 1994; Bernatchez & Osinov 1995).



Lack of congruence between genotypic and phenotypicvariation

It is widely acknowledged that the use of morphologicalcriteria to assess phylogenetic relationships in salmonids,such as body colour, meristic counts or body proportion ishampered by their phenotypic plasticity (Allendorf et al.1987). Nevertheless, several brown trout populations fromthe Greek waters have been reported as subspecies, basedsolely on morphological variation (Economidis 1991). Forexample, the Ohrid trout S. trutta letnica and the popu-lation from Prespa S. trutta peristericus have been recognized as different subspecies (Karaman 1937). In thecase of Prespa’s population allozyme data (Apostolidis etal. 1996a) further support its taxonomic distinctiveness.Yet the Ohrid and Prespa lakes have the same geologicalorigin and consequently one would expect trout popula-tions from both lakes to share a similar origin. Indeed, thisstudy argues against the subdivision into separate subspecies and suggests that both populations evolvedrecently and belong to the same evolutionary lineage(group II-‘Adriatic’) as other Greek brown trout popula-tions. In contrast, a number of populations which werefound to be morphologically similar clustered into differ-ent groupings. It is interesting to note the clustering witha relatively high bootstrap value (77%) of genotype H,found in the Acheloos and Mornos samples, to MA1 andMarmoratus group (IIA, Fig. 3). However, the identificationof haplotype G to marmoratus is questionable because it isbased on the occurrence of only two substitutions at posi-tions 126 and 275 (Fig. 2, numbers 6 and 12, respectively).Furthermore, as marmoratus has the same nucleotide (A)with S. salar at position 275 this could be the ancestral stateso, only position 126 could have some phylogenetic value.Secondly, previous morphological studies (Economidis1991) and allozyme data (Apostolidis et al. 1996a) have notindicated morphs (Sommani 1961) or alleles (Giuffra et al.1996) considered characteristic of the marmoratus group.

In conclusion, many of the proceeding arguments arerelevant to conservation efforts for brown trout popula-tions. Indeed, the loss of native trout has been widespreadthis century as a result of habitat destruction, pollutionand overexploitation (Ferguson 1989). In a number ofreports, mtDNA has proved useful for resolving popula-tion groupings in cases where morphological analyseswhere either inadequate or controversial (Bowen et al.1992), and therefore assisted in determining priorities forconservation. However, our results, while reinforcing theutility of molecular genetic data for the determination ofthe history and origins of brown trout populations,emphasize the importance of considering a number ofgenetic systems and possibly techniques when addressingsuch questions. The use of only one system or techniquecould lead to erroneous results or/and an incomplete

picture and the subsequent misinterpretation of events(Dizon et al. 1992). Thus, further mtDNA as well as nuclearDNA analyses, including fish populations from variouslocations such as north Balkans and Turkey, will be neces-sary to determine the origins and to illuminate thephylogenetic relationships among S. trutta groups.

Acknowledgements

The authors are indebted to Dr P. Berrebi (Montpellier) for pro-viding samples, to Dr R. T. Loftus for his valuable advice and hisfriendly co-operation; to Dr C. Moritz and to two anonymous ref-erees for useful suggestions on the manuscript. Financial supportfrom the European Commission within the frameworkEV5VCT920097 project is gratefully acknowledged.

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This paper is part of an ongoing collaborative effort by Europeangeneticists to apply molecular techniques to the population genet-ics and ecology of brown trout. The paper is dedicated to thememory of the late Y. Karakousis who participated in the initialsteps of the programme. This work is part of A. P. Apostolidis’sDoctorate thesis. The work was carried out in the laboratory ofProfessor C. Triantaphyllidis whose research interests, amongothers, include the genetics of fishes. Assist. Professor A. Kouvatsiis working in the same laboratory. Professor P. S. Economidis spe-cializes, among others, in the study of Greek freshwater fishfauna.

