THE EVOLUTIONARY HISTORY OF BROWN TROUT (SALMO …072) Bernatchez_Evol_01.pdf · Elucidating the evolutionary history of extant species is an important objective of any research program

351

q 2001 The Society for the Study of Evolution. All rights reserved.

Evolution, 55(2), 2001, pp. 351–379

THE EVOLUTIONARY HISTORY OF BROWN TROUT (SALMO TRUTTA L.) INFERREDFROM PHYLOGEOGRAPHIC, NESTED CLADE, AND MISMATCH ANALYSES OF

MITOCHONDRIAL DNA VARIATION

LOUIS BERNATCHEZDepartement de Biologie, GIROQ, Pavillon Alexandre-Vachon, Universite Laval, Sainte-Foy, Quebec, G1K 7P4, Canada

E-mail: [email protected]

Abstract. Phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA (mtDNA) variation wereused to infer the temporal dynamics of distributional and demographic history of brown trout (Salmo trutta). Bothnew and previously published data were analyzed for 1794 trout from 174 populations. This combined analysis improvedour knowledge of the complex evolutionary history of brown trout throughout its native Eurasian and North Africanrange of distribution in many ways. It confirmed the existence of five major evolutionary lineages that evolved ingeographic isolation during the Pleistocene and have remained largely allopatric since then. These should be recognizedas the basic evolutionarily significant units within brown trout. Finer phylogeographic structuring was also resolvedwithin major lineages. Contrasting temporal juxtaposition of different evolutionary factors and timing of major de-mographic expansions were observed among lineages. These unique evolutionary histories have been shaped both bythe differential latitudinal impact of glaciations on habitat loss and potential for dispersal, as well as climatic impactsand landscape heterogeneity that translated in a longitudinal pattern of genetic diversity and population structuringat more southern latitudes. This study also provided evidence for the role of biological factors in addition to that ofphysical isolation in limiting introgressive hybridization among major trout lineages.

Key words. Europe, fish, mismatch, mitochondrial DNA, nested clade, phylogeography, Salmo.

Received March 7, 2000. Accepted September 18, 2000.

Elucidating the evolutionary history of extant species isan important objective of any research program that seeks tounderstand population divergence and, ultimately, speciation.This history is also directly relevant to conservation biologybecause historical contingencies have been largely respon-sible for creating the most important genetic subdivisions inmany, if not most extant taxa (e.g., Zink 1996; Avise et al.1998; Hewitt 2000). The phylogeographic approach has beenused to test the congruence between distributional historiesagainst paleo-environmental settings and determining thechronology of evolutionary diversification (Avise 1998; Mo-ritz and Bermingham 1998). Comparative phylogeographyhas also emerged as a powerful method to address broaderecological and evolutionary issues.

In northern temperate freshwater fishes, comparative phy-logeography revealed predictive trends in phylogeographicstructure, genetic diversity, and speciation rates among spe-cies inhabiting formerly glaciated and unglaciated regions ofNorth America (Bernatchez and Wilson 1998). Further gen-eralizations of the effect of Pleistocene glaciations on fresh-water fish fauna could be gained by comparing phylogeo-graphic data from other regions. Contrasts between Eurasianand North American freshwater fish phylogeographic struc-ture (e.g., Bernatchez et al. 1989) would be particularly in-formative, given the much less extensive glacial advances inEurasia and its contrasting landscape with that of NorthAmerica (Hewitt 2000). However, the phylogeographic struc-ture of the Eurasian fish fauna is still largely unknown (Ber-natchez and Dodson 1994; Durand et al. 1999a; Nesbo et al.1999; Englbrecht et al. 2000).

The brown trout (Salmo trutta L.) is the most widely dis-tributed freshwater fish native to the Palearctic region. Itsnatural range extends from northern Norway and northeastern

Russia, southward to the Atlas Mountains of North Africa.From west to east, its range spans from Iceland to the head-waters of Aral Sea affluents in Afghanistan. Salmo trutta alsoexhibits considerable morphological diversity and life-his-tory variation, including specializations for anadromous, flu-viatile, and lacustrine modes of life. Large-scale patterns ofgenetic diversity in brown trout have been studied extensivelyover the last two decades, using both allozymes (e.g., Ryman1983; Ferguson 1989; Guyomard 1989; Osinov 1990; Garcıa-Marın and Pla 1996; Largiader and Scholl 1996) and mito-chondrial (mtDNA) analyses (Bernatchez et al. 1992; Giuffraet al. 1994; Hynes et al. 1996; Osinov and Bernatchez 1996;Apostolidis et al. 1997; Hansen and Mensberg 1998; Weisset al. 2000). Yet, the analysis of brown trout phylogeographicstructure is still lacking over important geographic areas,such as North Africa and eastern Europe. Patterns of post-glacial recolonizations, finer phylogeographic structure with-in major trout lineages, and demographic history have notbeen rigorously assessed or remain controversial (e.g., Ham-ilton et al. 1989; Osinov and Bernatchez 1996; Garcıa-Marınet al. 1999). This ambiguity may be attributed to both limitedanalytical resolution and statistical treatments.

Previous phylogeographic analyses of brown trout haverelied upon the use of haplotype trees and geographic dis-tribution to make biological inference by visual inspectionof how geography overlays haplotype relationships. This maynot make full use of all historical information contained ingene genealogies. Namely, this does not allow the estimationof the dynamic structure and temporal juxtaposition of dif-ferent evolutionary factors that are most likely compatiblewith the patterns of genetic diversity observed in extant pop-ulations. The recent development of statistical nested cladeanalysis of phylogeographic data offers a potentially useful

352 LOUIS BERNATCHEZ

TABLE 1. Number of populations, sample sizes, and haplotype diversity (h) within major trout evolutionary lineages. For populations, thefirst number indicates the number of pure populations for a particular lineage and the number in parentheses indicates the number of admixedpopulations but predominated in relative abundance by the particular lineage.

LineageNumber ofpopulations

Numberof fish

Number of haplotypes

Sequence RFLP CombinedHaplotype diversity

(SE)

Atlantic (AT)Danubian (DA)Adriatic (AD)Mediterranean (ME)marmoratus (MA)Total

55 (1)48 (1)37 (5)12 (1)14 (0)

166 (8)

993191298104205

1791

81410

33

38

142811

22

57

163517

34

75

0.582 (0.011)0.931 (0.005)0.851 (0.009)0.523 (0.011)0.511 (0.007)0.679 (0.008)

framework in that respect (Templeton et al. 1995; Templeton1998). Yet, the use of this method has been limited thus far(Templeton et al. 1995; Durand et al. 1999b; Nesbo et al.1999; Turner et al. 2000). The traditional phylogeographicapproach is also limited in inferring the dynamics of de-mographic history. The analysis of mismatch distributionprovides a way to estimate the magnitude and age of pop-ulation growth by statistically comparing the distribution ofintrapopulation molecular diversity with that expected underhypotheses of equilibrium (Rogers and Harpening 1992; Rog-ers 1995; Schneider and Excoffier 1999). However, the useof mismatch distribution has almost entirely been limited tohuman studies (but see Lavery et al. 1996; Merila et al. 1997;Petit et al. 1999).

In this study, I performed a genetic diversity analysis ofmtDNA and combined phylogeographic, nested clade, andmismatch analyses to infer the temporal dynamics of distri-butional and demographic history of the brown trout through-out its native geographic range. New results and previouslypublished data were combined to document the overall geo-graphic distribution of major trout evolutionary lineages. Iinfer the temporal juxtaposition of factors most compatiblewith the differential patterns of genetic diversity observedamong these lineages and discuss these factors against paleo-environmental settings encountered by brown trout in dif-ferent parts of its range.

MATERIALS AND METHODS

Samples

This study combines new analyses and previously pub-lished results (Appendix). A total of 277 fish representing 92sampling sites had never been analyzed. Samples previouslyanalyzed for sequence variation of the mtDNA control region(D-loop) (Bernatchez et al. 1992) were reanalyzed using poly-merase chain reaction–restriction fragment length polymor-phism (PCR-RFLP) to increase the number of character statesand allow standardization with previous studies. We also in-cluded the results of PCR-RFLP and sequence analyses per-formed by Giuffra et al. (1994), Bernatchez and Osinov(1995), Osinov and Bernatchez (1996), and Apostolidis et al.(1996a, 1997). The results obtained by Hynes et al. (1996)were standardized by performing the same sequence andPCR-RFLP analyses used for the other fish on representativesof all mtDNA haplotypes identified by these authors usingRFLP analyses over the entire mtDNA genome. Sixteen fish

from the Mediterranean basin that possessed the mtDNA lin-eage of Atlantic origin were discarded because they mostlikely represented the results of stocking, thus potentiallyblurring historical signals (Guyomard 1989; Bernatchez etal. 1992). Given these exclusions and that several populationsand samples overlapped among some of these studies (Ap-pendix), this analysis was based on the genetic informationobtained for 1794 trout representing 174 populations (Table1).

Laboratory Analyses

MtDNA variation was analyzed using both sequencing andRFLP performed on PCR-amplified products. Sequencingwas done on the same 310-bp segment (now 313 due to newindels) located at the 59 end of the control region studiedpreviously in brown trout (Bernatchez et al. 1992; Apostolidiset al. 1997). The primers LN20 (59-ACC ACT AGC ACCCAA AGC TA-39) and HN20 (59-GTG TTA TGC TTT AGTTAA GC-39), which amplify the whole control region, wereused for PCR, whereas primer H2 (59-CGT TGG TCG GTTCTT AC-39) was used for sequencing (Bernatchez and Danz-mann 1993). Technical procedures of mtDNA purification,amplification, and sequencing are detailed in Bernatchez etal. (1992). RFLP analysis was performed using six restrictionenzymes (HinfI, HpaII, MboI, NciI, RsaI, and TaqI) on twoadjacent PCR-amplified segments; the complete ND-5/6 re-gion (;2.4 kb), and a second segment (;2.1 kb) comprisingthe whole cytochrome oxydase b gene and the D-loop. Prim-ers, amplifications, restriction digests, and electrophoresisprocedures were as described in Bernatchez and Osinov(1995). Distinct single endonuclease patterns were identifiedby a specific letter in order of appearance and used in com-bination with sequence variation to define sequence/RFLPcomposite genotypes.

Genetic Diversity Analyses

The combined information of three previous studies basedon a small number of individuals but higher genetic resolutionthan used here resolved five major mtDNA phylogenetic lin-eages in brown trout (Fig. 1). Based on the origin of the firsthaplotypes identified, these were named Atlantic (AT), Dan-ubian (DA), Mediterranean (ME), marmoratus (MA), andAdriatic (AD) lineages. Each of these lineages were differ-entiated by eight to 12 apomorphies and were supported bybootstrap values of .99%. Instead of performing an overall

353EVOLUTIONARY HISTORY OF BROWN TROUT

FIG. 1. Consensus tree based on maximum-parsimony analysis relating five major trout mtDNA evolutionary lineages. Relationshipsamong major lineages and their bootstrap values (as a percentage) are derived from the combined information of three studies (Bernatchezet al. 1992; Giuffra et al. 1994; Bernatchez and Osinov 1995) based on sequence analysis of 1250 nucleotides and PCR-RFLP analysesof an estimated 576 additional sites resolved with 19 restriction enzymes. The rooting position of Atlantic salmon (Salmo salar) is derivedfrom the sequence analysis of Giuffra et al. (1994). The numbers in parentheses refer to the number of apomorphies unique to eachlineage. See Bernatchez and Osinov (1995) for further details. Relationships among haplotypes within each lineage was based on thesequence analysis of a 313-bp segment located at the 59 end of the control region and PCR-RFLP analysis performed using six restrictionenzymes (HinfI, HpaII, MboI, NciI, RsaI, and TaqI) on two adjacent PCR-amplified segments: the complete ND-5/6 region (approximately2.4 kb) and a second (approximately 2.1 kb), comprising the whole cytochrome oxydase b gene and the D-loop. Composite haplotypesare defined in Tables 2 and 3. Branch lengths were scaled using number of mutations, the shortest corresponding to a single mutation.


phylogenetic analysis in the present study, we first assignedhaplotypes (with confidence .99%) to these different groupsbased on their combined composition at 20 synapomorphicpositions (13 resolved by RFLP analyses, seven by sequenc-ing) that can unambiguously diagnose haplotypes belongingto any of the five trout lineages (detailed in Bernatchez andOsinov 1995). The phylogenetic relationships among hap-lotypes within each lineage was first assessed by parsimonyanalysis, as detailed in Bernatchez and Osinov (1995). Amore detailed analysis was then achieved by the nested cladeanalysis (see below).

Levels of genetic diversity within and among the five majortrout lineages were compared using the haplotype diversityand the maximum-likelihood estimation of the average num-ber of nucleotide substitutions per site within and amonggroups (Nei 1987) using REAP, version 4 (McElroy et al.1992). A hierarchical analysis of molecular variance (AMO-VA; Excoffier et al. 1992) was performed using Arlequin,version 2.0 (Schneider et al. 1999) to compare the componentof genetic diversity imputable to the variance among the fivetrout lineages to that observed among populations within eachof them. The significance of the variance components asso-ciated with the different levels of genetic structure were testedusing 1000 permutations.

Mismatch Analysis

A mismatch analysis was performed using Arlequin tocompare the demographic history of the five major trout lin-eages. Because the main objective was to compare the his-torical demography of the five major trout lineages and pop-ulation structure has a limited effect on the mismatch dis-tribution (Rogers 1995), pure samples were pooled withineach lineage. Following the method of Schneider and Ex-coffier (1999), we quantified the moment estimators of timeto the expansion t, the mutation parameter before (u0 5 2mN0)and following (u1 5 2mN1) expansion, expressed in units ofmutational time, where N0 and N1 are the female effectivepopulation sizes before and following an expansion that oc-curred t generations ago. These estimators were used to plotthe expected distribution of probabilities of observing S dif-ferences between two randomly chosen mtDNA haplotypes(Watterson 1975). We also computed the raggedness indexof Harpening (1994). The comparison of the sum of squaredeviation (SSD) between the observed and estimated mis-match distribution was used as a test statistic for the estimatedstepwise expansion models (Schneider and Excoffier 1999).Confidence intervals for those parameters were obtained byMonte Carlo simulations of 1000 random samples using thecoalescent algorithm of Hudson (1990) as modified bySchneider and Excoffier (1999). We finally used the rela-tionships t 5 mt (where m is the mutation rate and t is theexpansion time in generations) to compare the timing of pos-sible demographic expansion among trout lineages.

Nested Clade Analysis

The probability of a parsimonious relationship among hap-lotypes within each lineage was assessed as described in Tem-pleton et al. (1992), using the program ProbPars kindly pro-vided by A. R. Templeton (Washington University, St. Louis,

MO). MtDNA haplotypes differing by up to four mutationshad a probability .95% of being connected in a parsimoniousmanner. Because the number of mutations among adjacenthaplotypes never exceeded four mutational steps, we thenconstructed a minimum spanning network for each trout lin-eage using Minspnet (Excoffier and Smouse 1994). The re-sulting networks were then converted into a nested designfollowing the rules of Templeton et al. (1987) and Templetonand Sing (1993). The clade distance, Dc (which measures thegeographical range of a particular clade), the nested cladedistance, Dn (which measures how a particular clade is geo-graphically distributed relative to other clades in the samehigher-level nesting category), and contrasts of these mea-sures between tip and interior clades (Itc [y], Itn [y]) werequantified by first calculating the geographical centers of eachclade resolved at all hierarchical levels (Templeton et al.1995). Secondly, the geographical distances (in kilometer)between individuals and clade centers were calculated asgreat circle distances. The various distances were recalculatedafter each of 1000 random permutations of clades and/orhaplotypes against sampling locality to statiscally test at a5 0.05 for significantly large and small distances and interior-tip contrasts for each clade with respect to the null hypothesisof no geographic association within each of the nested clades.The model of population structure and historical events thatwas most suitable with the pattern of genetic structure ob-served within each clade showing statistically geographicalassociations was identified using the inference key providedas an appendix to Templeton (1998). To reduce bias in pa-rameters estimation and permutations procedures due to largediscrepancies among sample sizes (Appendix), those werelimited to a maximum of 10 by respecting the observed hap-lotype frequencies in the total sample (but with a minimumabsolute haplotype frequency of n 5 1). All calculations wereperformed using Excell (Microsoft, vers. 97) spreadsheetsdeveloped by J. E. Stacy (University of Oslo, Norway), asused in Nesbo et al. (1999).

RESULTS

Relationships among Major Trout Lineages and OverallPatterns of Genetic Diversity

Figure 1 illustrates the branching topology among the fivemajor trout lineages resolved from the combined informationof previous studies. The salient feature of this tree was therooting position of the outgroup, Atlantic salmon (Salmo sa-lar), which suggested the ancestral divergence of the Atlanticlineage from all others (Giuffra et al. 1994). The combinedsequence and RFLP analyses resolved 75 composite haplo-types (Tables 2, 3, Figure 2). These could unambiguously beassigned to either one of the five major trout lineages, basedon their composition at 13 RFLP and seven sequence syn-apomorphic positions, as detailed in Bernatchez and Osinov(1995).

The overall level of haplotype diversity was high (h 50.856), but the extent of nucleotide diversity (p 5 0.0133)was comparable to that observed in other northern temperatefreshwater fishes of similar latitudinal distribution (Bernatch-ez and Wilson 1998). The level of genetic diversity, however,was highly variable among the five trout lineages (Tables 1,


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TABLE 3. Definitions of 75 composite sequence/RFLP (sn/rn) mtDNA haplotypes in Salmo trutta: sn refers to sequence definition in Table 1,and rn refers to the composite fragment patterns resolved for restriction enzymes HinfI, HpaII, MboI, NciI, RsaI, and TaqI.

Haplotype RFLP Haplotype RFLP Haplotype RFLP

Atlantic1

AT-s1/r1AT-s1/r2AT-s1/r3AT-s6/r4

AAAAAABAAAAFBHAEAFQAAAAA

DanubianDA-s1/r1DA-s2/r2DA-s3/r3DA-s4/r4

CBABBCCBCCBCGAAAHCCEAAHC

MediterraneanME-s1/r1ME-s2/r2ME-s3/r1

DAADCD

AT-s7/r5AT-s1/r6AT-s1/r7AT-s1/r8AT-s1/r9

KAAAAABAAGAFBAAAAAAAAAJABAAAJF

DA-s5/r5DA-s6/r6DA-s9/r7Da-s10/r8Da-s7/r9

CEAFBCHEAGHCJEAAHCCEAAGCCEAABC

MarmoratusMA-s1/r1MA-s2/r1MA-s3/r1MA-s1/r2

EACADDEACADDEACADDEKCADD

AT-s1/r10AT-s1/r11AT-s5/r12AT-s1/r13AT-s8/r14

OAAAAFLAAAAFAAAALABHAAAFBIABAF

DA-s1/r10DA-s1/r11DA-s1/r12DA-s9/r13DA-s1/r143

JEAGHCIAAAHCJAAAICCAAAHCNA---C

AT-s3/r3AT-s4/r2

AdriaticAD-s1/r1AD-s3/r3

CCBACECDBECE

DA-s1/r15DA-s1/r16DA-s1/r15DA-s1/r16DA-s2/r17DA-s1/r18

CEAGHCMEAFBCCEAGHCMEAFBCFBABBCCEAABD

AD-s5/r4AD-s1/r5AD-s1/r6AD-s3/r7AD-s4/r8AD-s1/r92

CDBEEECDBACEPCBAMECDBECGCCBAMECC---E


CBACBDCBACBCCBABKCCBABBDCADABDCAAABC

AD-s1/r102

AD-s1/r112

AD-s1/r3AD-s2/r1AD-s6/r1AD-s7/r9

CC---ECC---E


CFACFCHJAGHCCBABOCCEAAPE

AD-s8/r1AD-s9/r1AD-s10/r11

DA-s14/r1DA-s1/r4Da-s1/r3DA-s1/r6DA-s11/r1

1 Correspondance to haplotype definition of Hynes et al. (1996a) : AT-s1/r1 (IX, X), AT-s2/r2 (I, III, VI, VIII, XIII, XIV), AT-s1/r6 (V), AT-s1/r10(IV), AT-s1/r11 (XI), AT-s1/r14 (XX), AT-s4/r2 (II), AT-s3/r3 (XII).

2 RFLP patterns described in Apostolidis et al. (1996a): r9, r10, and r11 corresponds to Type 3, 2, 4 of these authors, respectively. Data for MboI,NciI, RsaI were missing, but these haplotypes had new mutations at other enzymes (either AluI, AvaII, HaeIII) not seen previously.

3 r14 corresponds to Type 9 of Apostolidis et al. (1996a).

4). The DA lineage was the most genetically diverse, fol-lowed by the AD and AT lineages. Both ME and MA lineagesexhibited extremely reduced levels of diversity. Assumingthat the rooting position of S. salar indicates the ancientdivergence between two ancestral lineages (AT vs. one fromwhich diverged all others), it is noteworthy that the geneticdiversity within the AT lineage one was highly reduced com-pared to the other. The net nucleotide divergence among thefive trout lineages varied between 1.21% and 2.19% (Table4).

Geographic Distribution of Major Trout Lineages

Contrasting patterns of geographic distribution were ob-served among the five major trout lineages (Fig. 3A–E), asonly eight populations out of 174 (4.6%) with more than onelineage were observed (Appendix). All populations from theAtlantic basin and Morocco were fixed for the AT lineage.The DA lineage was almost exclusively associated with

drainages of the Black, Caspian, and Aral Sea basins, as wellas the Persian Gulf. The other three lineages (AD, ME, MA)showed little overlap in distribution with the other two andexhibited a differential pattern in their overall geographicdistribution within the Mediterranean. The MA lineage wasalmost strictly associated with the Adriatic basin. The MElineage was predominantly found in tributaries draining inthe western basin, whereas the AD lineage predominated inthe eastern part of the Mediterranean basin (Appendix).

Hierarchical Genetic Diversity Analysis

Most of the mtDNA molecular variance observed in nativetrout populations was imputable to differences among line-ages (Table 5). The amount of genetic variance imputable tothe among-populations within-lineage component was ap-proximately one order of magnitude less than the among-lineage components (8.942 vs. 0.873). Yet, populations werehighly structured within lineages, with an overall FST of


FIG. 2. Sequence of the 59-end, 313-bp segment of the mtDNA control region; type AT-s1 for Salmo trutta. The sequence shown isfor the light strand, and includes 10 nucleotides of the proline tRNA gene. Asterisks and numbers above sequence indicate the 30variable positions among the 38 resolved sequences. The AT-s1 sequence is entered into GeneBank under accession number U18198.

TABLE 4. Average number of nucleotide substitutions per site within(main diagonal) and net nucleotide divergence (below main diagonal)among the five major evolutionary lineages of brown trout.

Lineage AT DA AD ME MA

Atlantic (AT)Danubian (DA)Adriatic (AD)Mediterranean (ME)marmoratus (MA)

0.00200.01620.01850.01750.0188

0.00450.02040.02030.0219

0.00180.01210.0142

0.00050.0146 0.0005

0.776. The amount of molecular variance among populationswas highest for the DA and AD lineages, two to three timeslower for the AT lineage, and extremely reduced for both theME and the MA lineages. This also translated into variableFST values depending on lineages.

Mismatch Distribution and Demographic History

The overall mismatch distribution was clearly bimodal, onemode corresponding to the number of differences among ma-jor lineages (;20) and the other to differences among in-dividuals within lineages (;2; Fig. 4). The mismatch distri-

bution, however, differed substantially among trout lineages,with the mean number of differences being the lowest in ME(0.53) and MA (0.54) lineages, followed by AT (1.3), AD(2.1), and the highest value observed in DA (4.7) lineage.The mismatch distribution within AT, AD, and DA lineagesfitted well the predicted distribution under a model of suddenexpansion, but this was refuted for both ME and MA lineages(Table 6). Considering the 95% confidence interval generatedby the simulations, the observed number of polymorphic sitesvalues were consistent with simulated sites, meaning that theparameters estimated by the model were sufficiently accurateto account for the observed polymorphism in AT, AD, andDA lineages. The observed values of the age expansion pa-rameter (t) differed substantially among these three lineages.Confidence intervals on u1 values were too large to alloweven rough estimates of the magnitude of expansions.

Nested Clade Analysis

Contrasting patterns of nested clade design, both in num-bers of haplotypes and levels of clades was observed amongthe five trout evolutionary lineages (Figs. 5–7). The null hy-pothesis of no association between the position of haplotypes


→

FIG. 3. Geographic distribution of the five major trout mtDNA evolutionary lineages: Atlantic (AT), Danubian (DA), marmoratus (MA),Mediterranean (ME), and Adriatic (AD). Symbols were positioned with the exact latitudinal and longitudinal coordinates of each sampleusing the software MapInfo, and symbols of geographically proximate samples may overlap. A detailed description of lineage distributionamong all populations surveyed in provided in the Appendix.

in a cladogram with geographic position was rejected for atleast several clades at any nesting level for AT, DA, or DAlineages (Tables 7–9). Thus, we identified the likely causesfor the geographic association using the inference key ofTempleton (1998).

A temporal juxtaposition of past fragmentation at all cladelevels best explained the overall genetic structure observedwithin the AD lineage (Table 7). This pattern was mainlydriven by more easterly distributed populations. One-stepclades were highly structured geographically in the easternpart of the distribution range (from southern Italy and east-ward), whereas a more homogeneous pattern was observedamong western populations, where only two clades, largelyoverlapping in distribution, were represented (Fig. 5B).

The geographic pattern of genetic diversity observed with-in the AT lineage was best explained by the temporal jux-taposition of past fragmentation and range expansion (Table8). This was mainly driven by the strong geographic struc-turing observed among more southern populations (Fig. 6C).However, clade AT1–4 (corresponding to single haplotypeATs1/r1) was widely distributed among northern populations.Although clades AT1–1 and AT1–2 broadly overlapped indistribution among northern populations, a significantly smallDc values was observed for both (Table 8). Namely, therewas a tendency for clade AT1–1 to be mainly found in west-central populations, being almost absent from more northernpopulations (Iceland, northern Scandinavia, northern Russia).A pattern of either contiguous range expansion or dispersalimplying gene flow with isolation by distance best explainedthe geographic pattern observed among single haplotypeswithin AT1–1 and AT1–2 clades. In contrast, a signal of pastfragmentation best explained the geographic distribution ofhaplotypes within clade AT1–5 found in southern France andnorthern Spain.

The geographic pattern of genetic diversity observed with-in the DA lineage was best explained by a complex patternof temporal juxtaposition of various historical events (Table9). Past fragmentation best explained the geographic distri-bution of three-step clades. A strong geographic discontinuitywas particularly clear in more easterly distributed popula-tions, where those from the Caspian and Aral Sea basins weretypically characterized by clade DA3–3 and those surround-ing the Black Sea area by clade DA3–2 (Fig. 7C). CladeDA3–1 generally had a more western distribution, where itoverlapped extensively with DA3–2. At the two-step level,different historical events best explained the clade distribu-tion, depending on geographic areas. The distribution ofclades within DA3–1 (DA2–1 and DA2–2), which were foundin the most western part of the range, was best explained bya history of either range expansion and/or restricted dispersalwith gene flow (Table 9). In contrast, the distribution ofclades within either DA3–2 or DA3–3 clades better fit a his-

tory of fragmentation. Within DA3–2, clade DA2–3 mainly(but not exclusively) characterized populations from theBlack Sea area, whereas DA2–4 was confined to the upperreaches of the Danube drainage (Austria and Slovenia, Fig.7B). Within DA3–3, populations from the Caspian Sea basinwere characterized by clade DA2–8, whereas DA2–5, DA2–6, and DA2–7 were largely confined to the Aral Sea basin.A geographic east-west dichotomy between a history of frag-mentation and dispersal was also apparent at the one-step andzero-step clade levels (Table 9).

Nested clade analysis was not warranted for either ME orMA lineages, which were characterized by the predominanceof single haplotypes with a relatively broad geographic dis-tribution within both lineages (Appendix).

DISCUSSION

The combined use of traditional phylogeography, nestedclade, and mismatch analyses of mtDNA diversity revealedcomplex patterns in the distribution and demography ofbrown trout. The following discussion treats the species’ evo-lutionary history in hierarchical order to infer a temporaljuxtaposition of different paleo-environmental settingsthroughout its distribution range that is most compatible withthe differential patterns of genetic diversity observed amongand within major evolutionary lineages.

Choice of a Molecular Clock

Clearly, there are many ambiguities in the application ofa molecular clock; consequently, this clock was used in con-junction with estimated phylogenetic and demographic pat-terns only to provide an approximate time frame to evaluatephylogeographic hypotheses. Estimates in the range of 1–2%sequence divergence per million years were chosen for thefollowing reasons. A previous indirect calibration derivedfrom fossil records led to an average estimate of mtDNAmutation rate of 1% per million years for salmonids (Smith1992). Correlations reported between phylogeographic pat-terns and successions of Pleistocene glaciation events havepreviously been reported in other salmonids when using suchclock calibration (e.g., Bernatchez and Dodson 1991; Wilsonet al. 1996). In a more formal attempt to calibrate a fishmtDNA molecular clock using physically isolated geminatesby the Isthmus of Panama, Donaldson and Wilson (1999)estimated a divergence rate of 1% per million years in theND 5/6 region and 3.6% per million years for the controlregion, suggesting an average mutation rate for the wholemitochondrial genome intermediate of these values (the con-trol region represented approximately 40% of the subsamplednucleotides in our study). Finally, the oldest fossils reportedfor S. trutta date from the early Pleistocene (2 million yearsago; discussed in Osivov and Bernatchez 1996). Consequent-


FIG. 3. Continued.


FIG. 3. Continued.


FIG. 3. Continued.

TABLE 5. Analysis of molecular variance in brown trout using thefive major evolutionary lineages as the top level of grouping. Eightpopulations admixed for different lineages, and populations with sam-ple size ,5 were omitted for this analysis. The molecular varianceand related F-statistics imputable to the among-populations compo-nent is provided first for the overall system and then within eachlineage. All F-statistics values are significant at a 5 0.0001.

Source ofvariation df

Variancecomponents

%variation f-statistics

Among phylogeneticgroups

Among populationswithin groups

ATDAADMEMA

4

1254333271012

8.942

0.8730.6761.87311.2030.2370.251

88.83

8.6762.4278.4893.7375.0790.42

0.8883

0.77660.62420.78480.93730.75070.9042

ly, the extent of divergence among the deepest clades inbrown trout should fit within this time frame, which is thecase if one considers the extent of divergence among the fivemajor trout lineages (1.4–2.0%) and a molecular clock of 1–2% per million years.

Pleistocene Origins of Five Major Evolutionary Lineages inBrown Trout

Extending analyses to its whole distributional range con-firmed that the S. trutta complex is composed of five majorevolutionary lineages and basically ruled out the possibilityof additional subdivisions of similarly deep divergence.Theselineages most likely identify ancestral populations of troutthat evolved independently as a result of ancient allopatricfragmentation. Results of previous studies of nuclear genevariation are also congruent with these lineages. The samepopulations characterized here by different major mtDNAlineages or with the same geographic distribution were alsohighly differentiated either by diagnostic alleles or strongallele frequency differences (Guyomard 1989; Bernatchezand Osinov 1995; Giuffra et al. 1996; Apostolidis et al.1996b). A broad-scale geographic survey of microsatelliteloci variation recovered four of the five major mtDNA lin-eages (Presa 1995). Finally, Berrebi (1995) showed that Cor-sican trout populations originated from two distinct lineages,most likely corresponding to the ME and AD lineages.

Given their overall extent of divergence, the time frameinvolved in the lineages’ separation would span between 0.5million and 2 million years. This indicates that major phy-logeographic subdivisions in brown trout are associated withthe climatic and environmental changes that occurred duringPleistocene glaciations events. The most ancient separationwould have involved allopatric fragmentation between thethree major drainage subdivisions: the Atlantic (AT lineage),


FIG. 4. Frequency distributions of pairwise number of mutationaldifferences between trout individuals observed in all trout mtDNAevolutionary lineages combined (top panel) and separately in thefive major trout lineages defined by mitochondrial analysis: Atlantic(AT), Adriatic (AD), Danubian (DA), Mediterranean (ME), andmarmoratus (MA). Diamonds represent the observed data, the boldcurve is the model fitted to the data, and dashed lines delineate the2.5 and 97.5 percentile values of 1000 simulated samples.

TABLE 6. Observed (S) and 95% confidence intervals (CI) of simulated number of polymorphic sites, demographic parameters (95% CI),among major trout lineages. t, u0, and u1 are the age of expansion and population size before and following expansion, expressed in units ofmutational time. P(SSDobs) is the probability of observing by chance a less good fit between the observed and the mismatch distribution for ademographic history of the lineage defined by the estimated t, u0, and u1 parameters. P(Ragobs) is the probability of observing by chance ahigher value of the raggedness index than the observed one, under the hypothesis of population expansion.

Lineage S 95% CI t u0 u1 P(SSDobs) Raggedness P(Ragobs)

Atlantic (AT)Adriatic (AD)Danubian (DA)Mediterranean (ME)marmoratus (MA)

191937

23

4–2118–5134–9722–4251–80

0.5 (0.0–5.6)2.4 (0.5–5.7)5.5 (2.0–8.2)0.7 (0.3–1.0)0.7 (0.4–0.89)

1.36 (0–2.6)0.001 (0–1.9)0.004 (0–3.5)0 (0–0.7)0 (0–0.5)

3.8 (0.5–2681)9.7 (1.5–6792)

25.2 (10.4–6491)807 (1–3645)555 (1.6–3444)

0.3930.7690.3950.000010.00001

0.1200.0220.0150.2350.233

0.5900.8370.4380.0020.00001

Ponto-Caspian (DA), and Mediterranean followed by sub-sequent and possibly simultaneous fragmentation within theMediterranean basin, which led to the divergence of the ME,MA, and AD lineages. The most drastic climatic changesover the last 3 million years occurred approximately 700,000years ago (Webb and Bartlein 1992; Andersen and Borns1994); it is also likely that the first large isolation of severallarge river systems now draining into different basins (e.g.,Rhone, Rhine, and Danube) occurred during that period (Vil-linger 1986). Consequently, it is plausible that the most im-portant genetic subdivisions within the brown trout complexare associated with major climatic changes and basin isola-

tion that occurred in Europe between the early to the uppermid-Pleistocene.

Considering the paleo-environmental settings during thePleistocene and the overall geographic pattern of distributionand genetic diversity within each of them, it is possible toinfer hypothetical centers of origins for the five major troutevolutionary lineages. Given its clear association with theAtlantic basin, the center of origin of AT lineage is obviouslyassociated with drainages of this system. Trout from the At-lantic basin could survive in refuges marginal to ice sheetsduring the most recent glaciation events (Ferguson and Flem-ing 1983; Hamilton et al. 1989; Osinov and Bernatchez 1996).Given the much more severe climate, however, and the moreimportant glacial expansion both on land and on the oceanduring earlier glacial cycles of the Pleistocene (Webb andBartlein 1992; Andersen and Borns 1994), it is more likelythat the ancestral center of origin of the AT lineage wasfurther south, perhaps in coastal tributaries of the IberianPeninsula or even North Africa. This is corroborated by themore pronounced pattern of clade diversity and divergenceamong southern populations (see also Weiss et al. 2000).

Locating the center of origin for the DA lineage is muchmore problematic, given the less severe direct impacts ofhabitat loss during glacial advances in the Ponto-Caspianbasin and the very complex pattern of expansion, contraction,and interconnection of the Black, Caspian, and Aral Sea ba-sins (Arkhipov et al. 1995). Nevertheless, the localization ofearly trout fossils in the Caucasus area and the central po-sition of clade 3–2, both geographically and in the DA clad-ogram (intermediate between 3–1 and 3–3), suggest that theancestral populations from which all extant populations ofthe DA lineage originated inhabited from drainages associ-ated with the Black Sea.

The differential pattern of geographic distribution of thethree other lineages (ME, MA, and AD) broadly corroboratetraditionally recognized Mediterranean refugial areas: asouthwest (Ibero-Mediteranean), central (Adriato-Mediter-ranean or Italian), and an eastern (Balkans/Anatolia) refuge(Keith 1998). The ME lineage was predominantly associatedwith tributaries draining in the western basin of the Medi-terranean Sea, suggesting that it originated from this region.Persat and Berrebi (1990) proposed that a restricted area ofsouthern France (Rousillon region) that was isolated by thePyrenees to the south and by severe environmental conditionsprevailing during glacial advances to the east may haveserved as a Pleistocene refuge for other fishes. The MA lin-eage, typical of the phenotypically and ecologically distinct


TABLE 7. Nested geographical analysis of the Adriatic (AD) lineage following the inference key of Templeton (1998). Nested design, haplotypeand clade designations are given in Figure 5A. Geographic distribution of clades is provided in Figures 5B and 5C. Following the name ofhaloptypes or clade number are the clade (Dc) and nested clade (Dn) distances. For those clades containing both interior (bold) and tip nestedclades, the average difference between interior versus tip clades for both distance measures is given in the row labelled I-T. A superscript Smeans that the distance measure was significantly small at the 5% level, and a superscript L means that the distance measure was significantlylarge at a 5 0.05. At the bottom of a nested set of clades in which one or more sets of the distance measures was significant is a line indicatingthe biological inference using the same number and letter codes as those provided in Templeton (1998). Abbreviation: past frag.; past frag-mentation.

Haplotypes

No Dc Dn

One-step clades

No Dc Dn

Two-step clades

No Dc Dn

ADs1r1ADs6r1ADs2r1ADs8r1ADs9r1

I-T

6200S

00S

0S

620L

6751996L

705761916

2418S

1-2-3-5-15 No: past frag. 1-1 757L 790L

ADs1r9ADs7r9ADs1r10

I-T

0S

0S

0S

0

17S

176172

2158S

1-2-3-4-9 No: past frag. 1-2 17S 598ADs1r6ADs4r8ADs1r11ADs10r11

0S

14900

0000

1-31-41-5

I-T

0S

149S

0S

594L

524235S

419S

312L

1-2-3-4-9 No: past frag. 2-1 665S 670ADs1r3ADs1r5ADs3r3ADs3r7

I-T

5630

50S

0546L

1152L

1200445S

150616

1-2-3-5-15 No: past frag. 1-6 676 702 2-2 742 746Ads5r4 0 0 1-7 0S 1575L I-T 277.6 276.8

I-T 676L 2873S 1-2-3-4-9 No: past frag.1-2-3-5-15 No: past frag.

marble trout, was mainly confined to the Po River basin, butincluded drainages from Croatia and Slovenia, which couldall interconnect during phases of maximal interglacials (Bian-co 1990). Previous allozyme studies also support the hy-pothesis of a North Adriatic origin for the marble trout (Giuf-fra et al. 1996). Finally, the species’ predominance in trib-utaries of the eastern Mediterranean basin relative to bothME and MA lineages, along with the higher level of cladediversity observed in populations from the Balkans, indicatedthat the AD lineage most likely originated from the Balkans/Anatolia refuge.

Physical Versus Ecological Constraints to Dispersal andSecondary Intergradation

Given S. trutta’s dispersal potential, the variety of habitatsit occupies, and the evidence for interconnections among ma-jor basins during the Pleistocene, it is remarkable that majorevolutionary lineages remained largely allopatric in distri-bution for hundreds of thousands of generations. For instance,interconnections between the Atlantic and Ponto-Caspian ba-sins have been possible during different glacial phases dueto the development of either intermittent fluvial connectionsor large ice-dam lakes at the ice margins that dischargedsouthward into the Ponto-Caspian basin (Grosswald 1980;

Gibbard 1988; Arkhipov et al. 1995). Recent phylogeograph-ic (Durand et al. 1999a; Nesbo et al. 1999a) and traditionalbiogeographic studies (Banarescu 1990) confirmed that otherfishes originating from Ponto-Caspian refuges have indeedrecolonized the Atlantic basin following the most recent gla-ciations. Similarly, fish dispersal between the Ponto-Caspianand the Mediterranean basins was also possible via connec-tions between the Danube and rivers draining to the Medi-terranean, increased water discharge from the north thatdrained into the Black Sea and overflowed to the Mediter-ranean, or sea level lowering and reduced salinity that al-lowed fish movements across the Agean Sea basin (Bianco1990; Economidis and Banarescu 1991; Arkhipov et al.1995). The identification of a few trout populations charac-terized by mtDNA lineages not representative of the basinin which they were found confirms that trout could also usesuch connections. Yet, introgressive hybridization (excludingthat due to stocking or contemporary human-induced habitatdisturbance) remained relatively limited, as exemplified instudies at nuclear genes (Bernatchez and Osinov 1995; Giuf-fra et al. 1996).

This indicates that, in addition to physical isolation, bio-logical factors have contributed to limiting dispersal and in-trogressive hybridization among major trout lineages. One of


FIG. 5. (A) Minimum spanning network for 17 mitochondrial composite haplotypes resolved in the trout Adriatic lineage. Each line inthe network represents a single mutational change. A zero indicates an interior node in the network that was not present in the sample.Composite haplotypes are defined in Tables 2 and 3. Dotted-lined polygons indicate one-step clades nested together into two-step clades,and lined-shaded polygons indicate haplotypes grouped together into one-step clades. (B) Geographic distribution of two-step AD clades.


FIG. 5. Continued.

←

(C) Geographic distribution of one-step AD clades. Other lineages refer to haplotypes not belonging to the Adriatic lineage. Symbolswere positioned with the exact latitudinal and longitudinal coordinates of each sample using the software MapInfo, and geographicallyproximate samples may overlap. A detailed description of single haplotype distribution is provided in the Appendix.

these factors could be the differential dynamics of demo-graphic expansion between pioneer dispersers (presumablythose issued from the closest refuge) and later migrants innewly colonized habitats. Simulation studies showed that pi-oneers would be able to establish and expand rapidly in newlyavailable habitats (Ibrahim et al. 1996). Later migrants wouldcontribute little because they would be entering populationsat or near carrying capacity with only limited replacementdynamics. According to this scenario, trout already presentin a refuge located in a given basin or geographic area wouldbe the first to expand and occupy available ecological niches,leaving only little probability of establishment for popula-tions that survived in a more remote refugial area. This pro-cess would be enhanced if time of geographic isolation and/or differential selection in different environments (in termsof climate, physical environments, and species communities)have been sufficiently important for the accumulation of ge-netic and ecological differences. For salmonids, it has beenshown in lake whitefish (Coregonus clupeafomis) that geo-

graphic isolation of evolutionary lineages during the last gla-ciations led to the development of their partial genetic in-compatibility, resulting in a much higher embryonic mortalityrate of hybrid compared to pure progeny (Lu and Bernatchez1998). This, along with the potential for occupying distincttrophic niches, apparently explains the maintenance of re-productive isolation between populations of different evo-lutionary lineages when found in sympatry (Bernatchez et al.1999; Lu and Bernatchez 1999). Ecological and/or geneticconstraints to gene flow among trout lineages have been sug-gested by studies in which different lineages were found inparapatry (Giuffra et al. 1996; Largiader and Scholl 1996;Poteaux et al. 1998).

Mismatch Analysis: Differential Timing of MajorDemographic Expansions among Lineages

A model of sudden demographic expansion was statisti-cally supported for the AT, AD, and DA lineages. Because



FIG. 6. Continued.

←

FIG. 6. (A) Minimum spanning network for 16 mitochondrial composite haplotypes resolved in the trout Atlantic lineage. Each line inthe network represents a single mutational change. A zero indicates an interior node in the network that was not present in the sample.Composite haplotypes are defined in Tables 2 and 3. Heavier-lined polygons indicate two-step clades nested together into three-stepclades, dotted-lined polygons indicate one-step clades nested together into two-step clades, and lined-shaded polygons indicate haplotypesgrouped together into one-step clades. (B) Geographic distribution of two-step AT clades. (C) Geographic distribution of one-step ATclades. Other lineages refer to haplotypes not belonging to the Atlantic lineage. A detailed description of single haplotype distributionis provided in the Appendix.

the effects of less important demographic changes may bemasked by that of the most important one (Rogers 1995;Lavery et al. 1996), the variable estimates of the age expan-sion parameter should be interpreted as indicating that themost important demographic expansion of each lineage oc-curred at different evolutionary times. The most recent de-mographic expansion was detected within the AT lineage.Because the Atlantic basin was the most directly affected byglaciations in terms of habitat loss, one would predict moreimportant reduction of population abundance during glacialadvances in this area, as is generally reported in north tem-perate fishes differentially affected by glaciations (Bernatch-ez and Wilson 1998). This was also supported by the morereduced mtDNA diversity in AT relative to both the AD andDA lineages. Although they should be interpreted cautiouslygiven their large 95% CI, absolute estimates obtained by

considering the two different mutation rates (1% and 2% permillion years) suggested that the time of the most importantdemographic expansion of the AT lineage (13,400 years agoat 2% per million years, 26,800 years ago BP at 1% permillion years) roughly coincided with the onset of the lastglacial retreat (approximately 18,000 years ago).

In contrast, the most important demographic expansion thatoccurred in the DA lineage was much older (mean values 5154,500–309,000 years ago). This is congruent with geolog-ical evidence that the geographic range occupied by this lin-eage was much less directly affected by recent glacial ad-vances compared to the Atlantic basin. This is also reflectedby its much higher mtDNA genetic diversity. Several pos-sibilities of large demographic expansion occurred in thisregion, namely through interconnections that developedamong expanding Black, Caspian, and Aral Sea basins. How-



FIG. 7. Continued

←

FIG. 7. (A) Minimum spanning network for 35 mitochondrial composite haplotypes resolved in the trout Danubian evolutionary lineage.Each line in the network represents a single mutational change. A zero indicates an interior node in the network that was not present inthe sample. Composite haplotypes are defined in Tables 2 and 3. Heavier-lined polygons indicate two-step clades nested together intothree-step clades, dotted-lined polygons indicate one-step clades nested together into two-step clades, and thinned-lined polygons indicatehaplotypes grouped together into one-step clades. (B) Geographic distribution of three-step DA clades. (C) Geographic distribution oftwo-step DA clades. Other lineages refer to haplotypes not belonging to the Atlantic lineage. A detailed description of single haplotypedistribution is provided in the Appendix.

ever, the most important interconnections and sea expansionmost likely developed approximately 270,000–290,000 yearsago (Arkhipov et al. 1995). It is thus plausible that the majordemographic expansion within the DA lineage was associatedwith the opportunities of large-scale dispersal that occurredat that time.

The timing of demographic expansion within the AD lin-eage (67,300–134,600 years ago) was intermediate betweenthat of the AT and DA lineages. Trout populations fromdifferent parts of the Mediterranean basin were likely dif-ferentially affected by glacial advances, with habitats of thosefrom the western part of the range being more compresseddue to more severe climatic changes and the sea barrier, as

compared to more eastern populations (Hewitt 2000). Thisis reflected by the more important genetic diversity amongeastern than western populations. The most important de-mographic expansion within the AD lineage could thus beassociated with westward dispersal that occurred during theearly Wurm or late Riss glacial period.

A model of sudden demographic expansion was rejectedfor both the ME and MA lineages. This could reflect a tem-porally more stable demography and near mutation-drift equi-librium conditions for these lineages (Rogers and Harpening1992). In such a case, however, an overall high level ofgenetic diversity, as well as pronounced population structureamong physically isolated populations would be expected.


TABLE 8. Nested clade analysis of the Atlantic (AT) lineage following the inference key of Templeton (1998). Nested design, haplotype andclade designations are given in Figure 6A. The geographic distribution of clades is provided in Figures 6B and 6C. See Table 7 for furtherdetails. Abbreviations: rest. gene flow; restricted gene flow, IBD; isolated by distance., cont. range ext.; contiguous range expansion.

Haplotypes

No Dc Dn

One-step clades

No Dc Dn

Two-step clades

No Dc Dn

Three-step clades

No Dc Dn

ATs1r2ATs1r10ATs4r2ATs1r6ATs1r7

I-T

1084S

00S

0S

01084L

1083942942942

1678243

1-2-3-4 No: restr. gene flow, IBD 1-1 1081S 1085S

ATs1r3ATs3r3ATs1r13

I-T

834S

0738465S

834S

86777811

1-2-11-12 No: cont. range exp. 1-2 832S 830S

ATs8r4 0 1-3I-T

0S

658L

529S

397L

1-2-3-5-15 No: past frag. 2-1 1008 1008 3-1 1008S 1082S

ATs1r1ATs1r8ATs1r9

I-T

10940

256S

2256

1094133S

106S

26.4

1-4 1094S 1091S

1-2-3-5-15 No: past frag. 1-5 109S 1258I-T 984L 21661-2-3-5-15 No: past frag. 2-2 1110S 1103S

ATs6r4ATs7r5ATs5r12

4400

000

1-61-71-8I-T

44S

0S

0S

44

59S

43S

29S

231-2-3-5-15 No: past frag. 2-3 47S 2017L

I-T 1063L 2913S

1-2-3-5-15 No: past frag. 3-2 1240 1231I-T 232 2491-2-3-5-15 No: past frag.

Clearly, this was not the case, as illustrated by the lineages’extremely reduced diversity and molecular variance imput-able to population structuring. Instead, the predominance ofsingle haplotypes found over a wide geographic area is morecompatible with nonequilibrium conditions caused by a se-vere and relatively recent bottleneck. This is also congruentwith the evidence for more reduced habitat availability inareas occupied by refugial populations of the ME and MAlineages. Extremely reduced diversity may in turn hamperthe detection of demographic signals in the genetic signatureof populations (e.g., Lavery et al. 1996). As such, geneticdiversity observed for the ME and MA lineages do not followa typical north-south pattern of glaciation effects on popu-lation demography and illustrate that conditions favorable totrout survival may have been more severe in those southernareas than further north during the last glacial advance.

Nested Clade Analysis: Differential Dynamics of PopulationStructuring among Lineages

The nested clade analysis provided new insights into theresolution of finer evolutionary lineages within major line-ages, from which new hypotheses of brown trout evolutionary

history can be inferred and contrasted with previous inter-pretations.

Atlantic lineage: latitudinal contrasts in historicalpopulation structuring

The more southern distribution of clade AT3–2, along withits more complex pattern of clade diversity suggest that itrepresents the ancestral lineage from which lineage AT3–1derived following isolation in more northern latitudes. Itslong history of fragmentation also reflects a more temporallystable population structuring relative to more northern pop-ulations, as would be predicted from the glacial history ofthe different regions. In contrast, the nested clade analysisrevealed that northern trout populations were historically sub-divided into two ancestral lineages that intermixed exten-sively since their recent range expansion throughout most ofthe north Atlantic region following the last glacier retreat.Given their differential pattern of geographic distribution, itis most likely that AT1–1 characterizes a trout lineage thatsurvived in a refuge located in the more northwestern partof the Atlantic range of distribution, whereas lineage AT1–2 identifies a lineage that survived in a northeastern refuge.


In summary, three trout lineages, all from AT origins, wereinvolved in the recolonization process of the North Atlanticbasin; one from a southern refuge and more ancient origin(characterized by haplotype ATs1r1 belonging to lineageAT3–2) that first intergraded with an northern ancestral lin-eage (AT3–1 or AT2–1), then two lineages originating fromthis admixed group that later evolved in isolation in a west-central and a northeastern refuge.

The origin and postglacial history of brown trout in theNorth Atlantic basin has been the subject of divergent in-terpretations. Most researchers agreed on the fact that theNorth Atlantic was recolonized by different trout evolution-ary lineages. However, interpretations varied substantially asto their number, center of origin, and timing of dispersal.The broader geographic coverage in this study, along withthe ability of the nested-clade analysis to interpret the tem-poral juxtaposition of historical processes, partly reconcilesprevious interpretations. First, these results support the ex-istence of a northwestern refuge, as first proposed by Fer-guson and Fleming (1983), then reiterated by Hamilton et al.(1989), Osinov and Bernatchez (1996), and Garcıa-Marın etal. (1999). Second, they also support the existence of a north-eastern refuge first proposed by Osinov and Bernatchez(1996) and provide evidence for the contribution of a south-ern refuge, as originally proposed by Hamilton et al. (1989)and later by Garcıa-Marın et al. (1999). Unlike, these inter-pretations, however, the present results implied that northerncolonization by this southern group occurred prior to the lastglaciation. They also refute the view of a contribution of aPonto-Caspian lineage (Garcıa-Marın et al. 1999) becausethis would imply the complete disappearance of DA haplo-types in northern Europe, along with all known allozymealleles private to the DA lineage (see also Weiss et al. 2000).Finally, as previously hypothesized, the present results sta-tistically confirmed that these evolutionary lineages inter-graded extensively following the last glacier retreat.

Mediterranean lineages: longitudinal contrasts in historicalpopulation structuring

The overall geographic pattern of genetic diversity ob-served among Mediterranean lineages revealed a longitudinalpattern of reduced population structuring from east to west.The predominance of single haplotypes with a relativelybroad geographic distribution within both ME and MA lin-eages is most compatible with nonequilibrium conditions re-sulting from recent range expansion, which eventually re-sulted in partial geographic overlap between these and theAD lineage. An east-west pattern of reduced population struc-turing was also illustrated within the AD lineage. Thus, thepattern of population structuring among western populationsof the AD lineage was more similar to that observed withinME and MA lineages than among eastern populations of thissame lineage. This indicated that, in contrast to highly frag-mented structure among eastern populations, the observedpopulation structuring among western populations was morecompatible with a history of recent range expansion. It isnoteworthy that the Balkans, despite its relatively small size,is the area harboring the most diverse phenotypic diversityamong trout populations, which may be the outcome of their

long-term isolation in different environments compared toother parts of the species range of distribution (Kottelat1997).

Danubian lineage: complex juxtapositions of varioushistorical events

The geographic area occupied by the DA lineage has beenthe least investigated previously. The present study providednew insights into the existence of finer phylogeographic sub-divisions within this lineage and allowed us to propose hy-potheses regarding its evolutionary history. Previous allo-zyme studies did not show any pattern of major geographicdiscontinuity in the distribution of genetic diversity (Ber-natchez and Osinov 1995). The mtDNA anaylsis of Bernatch-ez and Osinov (1995) provided slight, but inconclusive ev-idence for genetic discontinuity among sea basins. The nestedclade analysis, however, statistically supported such a struc-ture. These results support the long-standing hypothesis pro-posed on the basis of morphological variation that popula-tions of each sea basin should be recognized as distinct evo-lutionary lineages (Berg 1948).

Conclusions

The combined use of traditional phylogeographic, nested-clade, and mismatch analyses of mtDNA diversity proved tobe very efficient in improving our knowledge of the complexevolutionary history of brown trout throughout its nativerange of distribution. This confirmed the existence of fiveevolutionary lineages that evolved in geographic isolationduring the Pleistocene and have remained largely allopatricsince then. These should be recognized as the basic evolu-tionary significant units within brown trout (Bernatchez1995). In addition to physical isolation, biological factorsmust have contributed to limiting their dispersal and intro-gressive hybridization among them. The unique evolutionaryhistories of each lineage have been shaped both by the thedifferential latitudinal impact of glaciations on habitat lossand potential for dispersal, as well as climatic impacts andlandscape heterogeneity that translated in a longitudinal pat-tern of genetic diversity and population structuring at moresouthern latitudes.

ACKNOWLEDGMENTS

I am indebted to numerous individuals that made this studypossible. I am particularly grateful to J. Schoffman for pro-viding unique tissue samples from throughout the distributionrange of brown trout. Thank you also to A. Ferguson, P.Berrebi, and R. Guyomard for sharing samples; J.E. Stacyand C.L. Nesbo for providing the Excell sheets to performthe nested clade analysis; A.R. Templeton for the programProbPars; T. Gosselin for producing the illustrations; and N.Tessier, G. Lu, S. Martin, and M. Parent for their technicalassistance. This paper was improved by the constructing com-ments of J. Turgeon and two anonymous reviewers. The re-search program of LB on the ecological and genetic deter-minism of northern fish evolution is supported by NSERC(Canada) research grants.


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Wilson, C. C., P. D. N. Hebert, J. D. Reist, and J. B. Dempson.1996. Phylogeography and postglacial dispersal of arctic charrSalvelinus alpinus in North America. Mol. Ecol. 5:187–197.

Zink, R. M. 1996. Comparative phylogeography in North Americanbirds. Evolution 50:308–317.

Corresponding Editor: R. Burton


AP

PE

ND

IX.

Sam

ple

size

,lo

cati

ons,

and

dist

ribu

tion

ofco

mpo

site

hapl

otyp

esam

ong

nati

vepo

pula

tion

sof

brow

ntr

out.

Ref

eren

ces:

1.A

post

olid

iset

al.

(199

6a,

1997

);2.

Ber

natc

hez

and

Osi

nov

(199

5);

3.B

erna

tche

zet

al.

(199

2);

4.G

iuff

raet

al.

(199

4);

5.H

ynes

etal

.(1

996)

;6.

Osi

nov

and

Ber

natc

hez

(199

6);

7.th

isst

udy.

No.

Lin

eage

Pop

ulat

ion

N

Coo

rdin

ates

Lat

itud

eL

ongi

tude

Bas

inS

ampl

ing

Com

posi

teha

plot

ypes

(num

bers

)R

efer

-en

ce

1 2 3 4 5

AD

AD

AD

AD

AD

Zrm

anja

R.

Mus

kovc

iR

.K

rupa

R.

Tohm

aR

.Z

aman

tiR

.

4 3 3 3 3

4480

89N

4481

29N

4481

09N

3884

79N

3884

59N

1681

09E

1584

59E

1585

29E

3685

59E

3683

09E

Adr

iati

cS

eaA

dria

tic

Sea

Adr

iati

cS

eaP

ersi

anG

ulf

Med

iter

rane

anS

ea

1996

1996

1996

1993

1993

AD

s4r8

AD

s4r8

AD

s4r8

AD

s6r1

AD

s1r3

7 7 7 7 76 7 8 9 10

AD

AD

AD

AD

AD

Cey

han

R.

Ana

mur

R.

Gok

suR

.K

opru

R.

Pre

psa

L.

1 1 2 3 26

3784

59N

3681

59N

3684

09N

3781

49N

4085

09N

3684

09E

3285

09E

3283

89E

3181

09E

2181

59E

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Adr

iati

cS

ea

1993

1993

1993

1993

1993

AD

s1r1

AD

s1r1

AD

s5r4

AD

s1r3

AD

s1r1

0

7 7 7 7 1,7

11 12 13 14 15

AD

AD

AD

AD

AD

Tara

voR

.O

hrid

L.

Dra

nce

R.

San

gro

R.

Fib

reno

L.

8 38 8 1 6

4185

29N

4180

09N

4682

49N

4184

59N

4184

29N

0980

79E

2084

09E

0683

09E

1385

09E

1384

09E

Med

iter

rane

anS

eaA

dria

tic

Sea

Med

iter

rane

anS

eaA

dria

tic

Sea

Tyr

rhen

ian

Sea

(Med

.)

1991

1991

1990

1996

1996

–199

7

AD

s3r3

AD

s1r1

1(32

)-A

Ds1

0r11

(6)

AD

s1r1

(7)-

AD

s2r1

AD

s4r8

AD

s1r1

3,7

1,3,

73,

77 7

16 17 18 19 20

AD

AD

AD

AD

AD

Sin

niR

.P

ardu

R.

Osu

R.

Lia

mon

eR

.F

angu

R.

2 2 1 1 1

4080

59N

3985

09N

4183

59N

4281

59N

4283

09N

1585

59E

0983

09E

0982

09E

0884

59E

0883

89E

Ioni

anS

ea(M

ed.)

Sar

dini

aC

orsi

caC

orsi

caC

orsi

ca

1996

1996

1996

1996

1996

AD

s4r8

AD

s3r3

AD

s1r1

AD

s1r1

AD

s3r3

7 7 7 7 721 22 23 24 25

AD

AD

AD

AD

AD

Asc

uR

.L

ouro

sR

.D

roso

pigi

(Lou

dhia

sR

.)S

kopo

sR

.(L

oudh

ias

R.)

Val

bona

R.

(Alb

ania

)

2 1 5 8 5

4283

39N

3982

09N

4080

09N

4085

09N

4282

09N

0980

59E

2085

09E

2182

59E

2184

09E

2080

59E

Cor

sica

Adr

iati

cS

eaA

egea

nS

eaA

egea

nS

eaA

dria

tic

Sea

1996

1993

1994

1994

1993

AD

s3r3

AD

s1r3

AD

s1r6

AD

s1r3

AD

s1r1

7 7 7 7 726 27 28 29 30

AD

AD

AD

AD

AD

Ese

Ver

acul

ungu

Gol

oC

astr

ila

Krk

icR

.S

tura

diD

emon

te

4 5 4 4 15

4185

79N

4185

29N

4282

29N

4480

59N

4482

59N

0980

39E

0980

99E

0980

89E

1681

29E

0781

09E

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Med

iter

rane

anS

eaA

dria

tic

Sea

Adr

iati

cS

ea

1996

1996

1996

1997

1991

AD

s3r3

AD

s3r3

AD

s3r3

(3)-

AD

s3r7

AD

s1r1

AD

s1r1

7 7 7 7 431 32 33 34 35

AD

AD

AD

AD

AD

Alfi

osE

vino

sT

hyam

isT

ripo

tam

osD

imca

yR

.

5 6 25 26 1

3783

79N

3882

79N

3985

09N

4083

59N

3683

49N

2182

79E

2184

09E

2083

09E

2281

59E

3281

79E

Ioni

anS

eaIo

nian

Sea

Ioni

anS

eaA

egea

nS

eaM

edit

erra

nean

Sea

— — — — 1998

AD

s7r9

AD

s1r1

AD

s1r9

AD

s1r1

(4)-

Ads

8r1(

22)

AD

s2r1

1 1 1 1 736 37 38 39 40

AD

AD

-AT

AD

-MA

AD

AD

-MA

-ME

Ado

ur(G

aube

)A

rtes

iaga

R.

Krk

aR

.N

esto

sR

.G

arda

L.

5 9 4 25 23

4285

09N

4380

39N

4385

09N

4182

09N

4585

09N

0080

99W

0183

39W

1585

89E

2484

09E

1084

09E

Atl

anti

cA

tlan

tic

Adr

iati

cS

eaA

egea

nS

eaA

dria

tic

Sea

1996

1992

1996

1993

1990

–199

1

AD

s1r1

(4)-

AD

s1r5

AD

s1r1

(5)-

AT

s1r1

(3)-

AT

s1r8

AD

s1r1

-AD

s4r8

-MA

s1r1

(2)

AD

s9r1

(11)

AD

s1r1

(15)

-MA

s2r1

(5)-

MA

s3r1

-Mes

2r1(

2)

7 7 7 1,7

3,4,

7

41 42 43 44 45 46 47

AD

-MA

-ME

AD

-ME

AT

AT

AT

AT

AT

Chi

sone

R.

Vec

chio

R.

Aub

onne

R.

Red

onR

.T

sna

R.

Bre

sle

R.

Saa

rR

.

23 6 5 3 6 8 8

4485

09N

4281

39N

4682

79N

4684

09N

5782

09N

4685

09N

4980

09N

0781

09E

0981

29E

0682

39E

0780

09E

3480

09E

0183

59E

0780

09E

Adr

iati

cS

ea

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Med

iter

rane

anS

eaC

aspi

anS

eaA

tlan

tic

Atl

anti

c

1990

–199

1

1991

1990

1990

1994

1990

1990

AD

s1r1

(12)

-MA

s2r1

(8)-

ME

s2r1

(3)

AD

s3r3

(5)-

ME

s2r1

AT

s1r1

AT

s1r1

AT

s1r1

(3)-

AT

s1r2

(2)-

AT

s1r7

AT

s1r1

(4)-

AT

s1r2

(4)

AT

s1r1

(6)-

AT

s1r2

(2)

3,4,

7

3,7

3 3 6 3,7

3,7

377EVOLUTIONARY HISTORY OF BROWN TROUTA

PP

EN

DIX

.C

onti

nued

.

No.

Lin

eage

Pop

ulat

ion

N

Coo

rdin

ates

Lat

itud

eL

ongi

tude

Bas

inS

ampl

ing

Com

posi

teha

plot

ypes

(num

bers

)R

efer

-en

ce

48 49 50 51 52 53

AT

AT

AT

AT

AT

AT

Vit

ava

R.

Slu

psk

R.

Sw

ibno

R.

Tri

ashe

noL

.N

il’m

aR

.M

edja

R.

8 4 2 14 7 8

4982

09N

5484

09N

5484

59N

6980

09N

6683

09N

5681

59N

1481

09E

1780

09E

1885

09E

3684

09E

3381

59E

3284

09E

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

Bar

ents

Sea

Whi

teS

ea

1991

1990

1990

1993

1993

1993

AT

s1r3

AT

s1r2

(2)-

AT

s1r1

-AT

s1r1

3A

Ts1

r1A

Ts1

r2A

Ts1

r1(6

)-A

Ts1

r2A

Ts1

r2(5

)-A

Ts1

r3(3

)

3,7

3,7

3,7

2,6

2,6

254 55 56 57 58 59

AT

AT

AT

AT

AT

AT

Via

shen

oL

.V

orob

’yev

R.

Luv

en’g

aR

.Je

gess

lerg

-dra

mm

erD

alal

ven

R.

Lek

saR

.

2 3 6 7 8 10

6980

09N

6682

59N

6782

09N

6081

09N

6083

09N

6382

09N

3687

09E

3382

09E

3281

09E

1182

09E

1581

09E

1085

59E

Bal

tic

Sea

Bar

ents

Sea

Whi

teS

eaW

hite

Sea

Atl

anti

cA

tlan

tic

1994

1994

1994

1992

1992

1994

AT

s1r2

AT

s1r1

AT

s1r2

(4)-

AT

s1r1

(2)

AT

s1r1

AT

s1r3

(6)-

AT

s1r2

(2)

AT

s1r1

(2)-

AT

s1r2

(7)-

AT

s1r3

6 6 6 3,7

3,7

760 61 62 63 64 65

AT

AT

AT

AT

AT

AT

Tavl

aaR

.Jy

llan

d(D

enm

ark)

Kal

lsjo

nF

alpf

jall

t-ja

mar

naB

lank

tjar

nL

eba

10 30 10 10 5 18

6482

79N

5681

09N

6384

09N

5983

59N

5983

59N

5880

09N

1183

69E

0980

09E

1380

09E

1485

09E

1482

59E

1980

09E

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

1994 — — — — —

AT

s1r1

(4)-

AT

s1r2

(6)

AT

s1r1

AT

s1r2

AT

s1r2

AT

s1r1

(2)-

AT

s1r3

(3)

AT

s1r1

(5)-

AT

s1r2

(11)

-A

Ts1

r3(2

)

7 5 5 5 5 5

66 67 68 69 70

AT

AT

AT

AT

AT

Cru

mli

n

Oue

dB

erre

mB

ouH

amed

Oue

del

Kan

arO

ued

Ziz

56 4 2 2 5

5382

59N

3284

09N

3581

39N

3581

49N

3282

59N

1080

59E

0484

79W

0580

19W

0580

59W

0484

59W

Atl

anti

c

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Med

iter

rane

anS

eaA

tlan

tic

— 1995

1995

1995

1992

AT

s1r2

(3)-

AT

s1r1

(6)-

Ats

8r14

(17)

AT

s5r1

2A

Ts1

r1A

Ts1

r1A

Ts6

r4(2

)-A

Ts7

r5(3

)

5 7 7 7 771 72 73 74 75 76

AT

AT

AT

AT

AT

AT

Oue

dO

umer

Rbi

aA

riza

kum

R.

Tru

bia

R.

Elo

rnR

.R

iabh

aich

Icel

and

(13

pop)

6 7 7 8 113

3

3380

59N

4381

59N

4381

59N

4882

79N

5083

09N

6482

09N

0581

29W

0284

09W

0680

09W

0481

69W

0484

09W

1883

09W

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

1992

1992

1992

1990

1994

1994

AT

s6r4

AT

s1r9

AT

s1r1

(5)-

AT

s1r9

(2)

AT

s1r2

AT

s1r3

AT

s1r2

7 7 7 3,7

7 577 78 79

AT

AT

AT

Mel

vin

L

Gly

nnR

.B

asqu

ere

gion

140

102 5

5483

09N

5484

59N

4381

79N

0881

09W

0585

09W

0183

39W

Atl

anti

c

Atl

anti

cA

tlan

tic

1994

1994

1996

AT

s1r2

(108

)-A

Ts1

r10(

3)-

AT

s4r2

(16)

-AT

s1r6

(9)-

AT

s1r1

(4)

AT

s1r2

(101

)-A

Ts1

r3A

Ts1

r1(4

)-A

Ts1

r9

5 5 780 81 82 83 84

AT

AT

AT

AT

AT

Gle

nari

ffR

.

Car

nlou

ghG

lenc

loy

Sut

herl

and

Loc

hN

ess

151 54 15 32 15

5580

59N

5580

09N

5485

59N

5883

09N

5783

09N

0680

59W

0680

09W

0680

09W

0485

09W

0485

09W

Atl

anti

c

Atl

anti

cA

tlan

tic

Atl

anti

cA

tlan

tic

1994 — — — —

AT

s1r3

(107

)-A

Ts1

r2(3

9)-

AT

s1r1

(3)-

AT

s1r1

3(2)

AT

s1r2

(50)

-AT

s1r3

(4)

AT

s1r2

(9)-

AT

s1r3

(6)

AT

s1r3

AT

s1r1

(14)

-AT

s1r2

5 5 5 5 585 86 87 88 89 90

AT

AT-

DA

DA

DA

DA

DA

Ern

eE

ulen

back

R.

Cet

ina

R.

Kok

raR

.G

urk

R.

Boh

ing

L.

50 6 4 3 5 1

5482

59N

4882

09N

4384

29N

4682

39N

4685

09N

4681

79N

0785

09W

1180

09E

1684

49E

1482

99E

1480

39E

1385

09E

Atl

anti

cB

lack

Sea

Adr

iati

cS

eaB

lack

Sea

Bla

ckS

eaB

lack

Sea

— 1990

1996

1996

1991

1991

AT

s1r2

AT

s1r1

(4)-

DA

s2r2

(2)

DA

s12r

22D

As1

r1D

As7

r9D

As2

r2

5 3,7

7 7 7 391 92 93

DA

DA

DA

Aba

ntL

.C

oruh

R.

Fyr

atR

.(E

uphr

ates

)

2 2 3

4083

89N

4083

09N

3983

59N

3181

69E

4183

09E

3884

09E

Bla

ckS

eaB

lack

Sea

Per

sian

Gul

f

1993

1992

1992

DA

s1r1

7D

As7

r9D

As5

r5

7 7 7


AP

PE

ND

IX.

Con

tinu

ed.

No.

Lin

eage

Pop

ulat

ion

N

Coo

rdin

ates

Lat

itud

eL

ongi

tude

Bas

inS

ampl

ing

Com

posi

teha

plot

ypes

(num

bers

)R

efer

-en

ce

94 95 96

DA

DA

DA

Sav

aR

.L

avan

tB

r.V

ella

chS

trea

m

1 1 6

4682

69N

4780

49N

4683

59N

1385

39E

1483

69E

1483

59E

Bla

ckS

eaB

lack

Sea

Bla

ckS

ea

1993

1993

1994

1DA

s11r

18D

As7

r9D

As1

r1-D

as7r

9(2)

-DA

s1r1

8(3)

7 7 797 98 99 10

010

1

DA

DA

DA

DA

DA

Sav

aR

.

Rad

ovna

R.

Vil

san

R.

Doa

mne

iR

.To

polo

gR

.

6 2 5 4 3

4682

89N

4682

39N

4581

79N

4582

19N

4582

89N

1384

69E

1482

09E

2484

69E

2484

99E

2483

09E

Bla

ckS

ea

Bla

ckS

eaB

lack

Sea

Bla

ckS

eaB

lack

Sea

1994

1995

1995

1995

1995

DA

s12r

19-D

As1

1r18

-D

As1

2r22

(3)-

DA

s13r

23D

As1

1r18

DA

s2r2

0D

As1

r18

DA

s2r2

1(2)

-DA

s1r1

7 7 7 7 710

210

310

410

510

610

7

DA

DA

DA

DA

DA

DA

Gor

enR

.M

ende

res

R.

Kol

paR

.Z

avrs

nica

R.

Beg

unje

R.

Arp

aR

.

1 1 1 1 2 8

3984

59N

3984

59N

4583

09N

4682

49N

4682

39N

3984

09N

2781

09E

2685

09E

1580

09E

1481

09E

1481

49E

4583

59E

Aeg

ean

Sea

Aeg

ean

Sea

Bla

ckS

eaB

lack

Sea

Bla

ckS

eaC

aspi

anS

ea

1993

1993

1996

1996

1996

1982

DA

s5r5

DA

s2r2

5D

As1

r24

DA

s1r1

8D

As1

r18

DA

s1r1

5

7 7 7 7 7 210

810

911

011

111

2

DA

DA

DA

DA

DA

Tere

kR

.A

psha

kR

.K

uter

ziR

.(U

ral)

Sev

anL

.K

odor

iR

.

10 14 7 6 9

4384

59N

5383

09N

5384

09N

4083

59N

4381

09N

4482

89E

5883

09E

5883

09E

4580

09E

4183

09E

Cas

pian

Sea

Cas

pian

Sea

Cas

pian

Sea

Cas

pian

Sea

Bla

ckS

ea

1982

–199

119

9419

9419

79–1

982

1982

DA

s1r1

0(8)

-DA

s1r1

1-D

As1

r12

DA

s1r1

5D

As1

r15

DA

s1r6

(5)-

DA

s6r6

DA

s5r5

(3)-

DA

s7r5

(3)-

DA

s1r1

6(3)

2 6 6 2 2

113

114

115

116

DA

DA

DA

DA

Cri

mea

(dif

fere

nttr

ibut

arie

s)S

arda

mia

naR

.

Sofi

daro

nR

.

Ner

etva

R.

5 7 6 2

4583

09N

3980

89N

3885

09N

4384

09N

3482

09E

6881

59E

6985

59E

1880

09E

Bla

ckS

eaA

ral

Sea

Ara

lS

ea

Adr

iati

cS

ea

1992

1987

1987

1991

DA

s7r9

(4)-

DA

s1r1

6D

As3

r3-D

As8

r3-D

As1

r3(2

)-D

As1

r14(

3)D

As4

r4-D

As9

r7-D

As1

r8-

DA

s9r1

3-D

As1

r4(2

)D

As1

r1

2 2 2 3,7

117

118

119

120

121

122

DA

DA

DA

DA

DA

DA

Dra

vaR

.L

udra

naR

.B

istr

aR

.To

pla

R.

Dra

vaR

.L

udra

naR

.

1 2 2 4 1 2

4683

79N

4682

59N

4682

39N

4683

09N

4683

79N

4682

59N

1580

59E

1485

59E

1485

09E

1484

59E

1580

59E

1485

59E

Bla

ckS

eaB

lack

Sea

Bla

ckS

eaB

lack

Sea

Bla

ckS

eaB

lack

Sea

1997

1997

1997

1997

1997

1997

DA

s1r1

8D

As7

r9D

As1

2r22

DA

s7r9

-DA

s12r

22(3

)D

As1

r18

DA

s7r9

7 7 7 7 7 712

312

412

512

612

7

DA

DA

DA

DA

DA

Bis

tra

R.

Bal

ikG

olu

L.

Gol

bele

nR

.F

irti

naR

.B

alkl

iR

.

2 2 3 4 1

4682

39N

3985

09N

4180

59N

4180

09N

3985

59N

1485

09E

4383

09E

4380

59E

4180

09E

4080

09E

Bla

ckS

eaC

aspi

anS

eaC

aspi

anS

eaB

lack

Sea

Per

sian

Gul

f

1997

1997

1997

1997

1997

DA

s12r

22D

As2

r27

DA

s1r2

6D

As5

r5D

As7

r9

7 7 7 7 712

812

913

013

113

2

DA

DA

DA

DA

DA

Gor

enR

.Z

eyti

nili

R.

Man

asti

rR

.To

pkha

naR

.K

apuz

basi

R.

1 2 2 9 1

3984

59N

3984

09N

3984

09N

3681

19N

3784

69N

2781

09E

2685

99E

2685

09E

7085

79E

3582

59E

Aeg

ian

Sea

Aeg

ian

Sea

Aeg

ian

Sea

Ara

lM

edit

erra

nean

Sea

1997

1997

1997

1998

DA

s5r5

DA

s5r5

DA

s1r2

6D

As1

r28-

DA

s1r4

(8)

DA

s1r1

7 7 7 7 713

313

3b13

413

513

613

7

DA

DA

MA

MA

MA

MA

Mez

aR

.K

yzyl

suS

ocha

R.

Pel

lice

R.

Toce

R.

Hud

daG

rapp

a

2 3 4 27 18 3

4682

79N

3984

09N

4682

09N

4485

09N

4681

09N

4681

09N

1484

39E

7380

09E

1384

09E

0783

89E

0881

09E

1480

09E

Bla

ckS

eaA

ral

Adr

iati

cS

eaA

dria

tic

Sea

Adr

iati

cS

eaM

edit

erra

nean

Sea

1998

1999

1994

1990

–199

119

9019

96

DA

s1r1

Das

1r4

MA

s1r1

MA

s2r1

MA

s1r1

(9)-

MA

s2r1

(9)

MA

s1r1

7 7 7 3,4,

73,

4,7

713

813

9M

AM

AZ

adla

scic

aS

tura

diL

anzo

5 1946

8109

N45

8069

N13

8509

E07

8449

EA

dria

tic

Sea

Adr

iati

cS

ea19

9619

91M

As1

r1M

As2

r17 4


AP

PE

ND

IX.

Con

tinu

ed.

No.

Lin

eage

Pop

ulat

ion

N

Coo

rdin

ates

Lat

itud

eL

ongi

tude

Bas

inS

ampl

ing

Com

posi

teha

plot

ypes

(num

bers

)R

efer

-en

ce

140

141

142

143

MA

MA

MA

MA

Bre

nta

R.

Ges

soR

.S

arca

R.

Stu

radi

Dem

onte

(Vin

adio

)

8 16 8 15

4585

09N

4482

49N

4585

29N

4482

09N

1184

09E

0783

39E

1085

29E

0782

09E

Adr

iati

cS

eaA

dria

tic

Sea

Adr

iati

cS

eaA

dria

tic

Sea

1991

1991

1991

1991

MA

s2r1

MA

s2r1

MA

s2r1

MA

s2r1

4 4 4 414

414

514

614

714

814

9

MA

MA

MA

MA

ME

ME

Ach

eloo

sM

orno

sId

rija

R.

Zal

aR

.V

oido

mat

isR

.R

ioL

lobr

egat

48 15 1 2 27 2

3882

09N

3882

59N

4680

09N

4585

59N

3985

59N

4281

59N

2180

69E

2185

09E

1385

59E

1480

59E

2084

09E

0185

79E

Ioni

anS

eaIo

nian

Sea

Adr

iati

cS

eaA

dria

tic

Sea

Adr

iati

cS

eaM

edit

erra

nean

Sea

— — 1998

1998

1993

1995

AD

s1r1

(5)-

MA

s1r1

(43)

MA

s1r1

MA

s1r1

MA

s1r1

ME

s2r1

(25)

-ME

s3r1

(2)

ME

s1r1

1 1 7 7 1,7

715

015

115

215

315

415

5

ME

ME

ME

ME

ME

ME

Rev

erot

teR

.Te

sR

.C

alor

eR

.C

aren

caA

ude

Sor

gue

(Vau

clus

e)

6 6 1 5 4 3

4781

59N

4385

09N

4084

69N

4282

69N

4284

59N

4385

59N

0684

09E

0381

59E

1580

09E

0281

39E

0280

89E

0581

09E

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

Tyr

rhen

ian

Sea

(Med

.)M

edit

erra

nean

Sea

Med

iter

rane

anS

eaM

edit

erra

nean

Sea

1986

1990

1996

1996

1996

1996

ME

s2r1

ME

s1r1

(4)-

ME

s2r1

(2)

ME

s1r1

ME

s1r1

ME

s1r1

ME

s1r1

3,7

3,7

7 7 7 715

615

715

815

916

016

1

ME

ME

ME

ME

ME

-AD

ME

-DA

Hau

tG

olo

Fon

tana

ccia

R.

Tagl

iole

R.

Rio

Ara

Rip

aR

.V

enet

ikos

4 8 8 2 6 22

4281

89N

4481

29N

4481

29N

4283

09N

4485

79N

4080

39N

0885

39E

1084

59E

1083

79E

0081

09W

0684

79E

2183

39E

Med

iter

rane

anS

eaA

dria

tic

Sea

Adr

iati

cS

eaM

edit

erra

nean

Sea

Adr

iati

cS

eaA

egea

nS

ea

1996

1991

1991

1992

1991 —

ME

s1r1

ME

s1r1

ME

s1r1

ME

s1r1

ME

s2r1

(5)-

AD

s1r1

ME

s2r1

(2)-

DA

s14r

1(20

)

7 4 4 7 4 1

Documents

THE EVOLUTIONARY HISTORY OF BROWN TROUT (SALMO …072) Bernatchez_Evol_01.pdf · Elucidating the evolutionary history of extant species is an important objective of any research program