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Introduction

Present patterns of genetic variation within species havebeen influenced by many factors. Of major importance isthe influence of the last period of glaciation and subse-quent migration of taxa from glacial refugia. Ice agesoccur at regular intervals of 100 000 years with warminterglacial periods lasting 15Ð20 000 years as a result ofinstabilities in the earthÕs climate caused by theMilankovitch cycles (Bennett 1990). Many tree speciescommon in northern Europe today survived these glacialperiods in small, low-density populations in refugia in themountains of southern Europe (Bennett et al. 1991).

The slower rate of evolution of chloroplast DNA(cpDNA) compared to nuclear DNA in plants (Wolfe et al.1987) has limited its use in population studies at theintraspecific level (Palmer 1987). Several papers have beenpublished describing varying levels of intraspecificcpDNA variation in a wide range of plant species(reviewed in Soltis et al. 1992). cpDNA is maternally inher-ited in the majority of flowering plants and therefore pro-vides a seed-specific marker. In species where seed flow ismuch less than pollen flow, it is predicted that organelle

genes (chloroplast and mitochondrial) will be highly struc-tured when compared to nuclear genes (Petit et al. 1993).

Recent studies on European and North American treespecies have shown that refugial areas and postglacialmigration routes can be identified using DNA markers.Fossil pollen maps for European deciduous oaks indicaterefugia in southern Spain, southern Italy and the BalkanPeninsula (Huntley & Birks 1983; Bennett et al. 1991). Twoseparate studies based on cpDNA variation have con-firmed the existence of these three refugia (Dumolin-Lap�gue et al. 1997; Ferris et al. 1998) and also identifyareas of northern Europe colonized by oaks from eachrefugium. Similar studies have also been carried out onFagus sylvatica in Europe (Demesure et al. 1996) andLiriodendron tulipifera in North America (Sewell et al. 1996).

Alnus glutinosa (black alder) is a wind-pollinated, self-incompatible tree species of riparian and water-loggedhabitats (McVean 1953). It is common in Europe and theMediterranean, and extends as far as the mountains ofTurkey and North Africa. Seed dispersal is most effectiveby water, but seeds may also be dispersed by wind up to30 m from the parent tree (McVean 1953). Huntley & Birks(1983) provide evidence for at least three glacial refugiafor Alnus based on fossil pollen data, including Corsica,the Carpathian Mountains and southwestern Russia, and

Molecular Ecology (1998) 7, 1151Ð1161

© 1998 Blackwell Science Ltd

Chloroplast DNA phylogeography of Alnus glutinosa(L.) Gaertn.

R. ANDREW KING and COLIN FERRISDepartment of Biology, University of Leicester, Leicester, LE1 7RH, UK

Abstract

Traditionally, information on the postglacial history of plant species has been gainedfrom the analysis of fossil pollen data. More recently, surveys of present patterns ofgenetic variation have given valuable insights into species phylogeography. The genusAlnus, based on fossil data, is known to have had at least four glacial refugia. A survey ofchloroplast DNA (cpDNA) diversity in populations of black alder (A. glutinosa) wasundertaken in order to gain more insight into its postglacial history. This revealed a highdegree of structuring of 13 cpDNA haplotypes on a European scale which indicated thatmost of northern and central Europe was colonized from a refuge in the CarpathianMountains. Based on the distribution of two common cpDNA haplotypes, colonizationroutes from this refuge can be determined. The locations of other previously identifiedrefugia are confirmed and two formerly unconfirmed refugial areas for alder (southernSpain and Turkey) are proposed.

Keywords: Betulaceae, black alder, PCRÐRFLP, polymorphism, postglacial history, refugia

Received 5 December 1997; revision received 17 February 1998; accepted 2 March 1998

Correspondence: R. A. King. Fax: +44 (0) 1162522791; E-mail:[email protected]

the Bay of Biscay region. To this may be added refugia insouthern Italy and Greece (Bennett et al. 1991). Alderpollen has also been found in late glacial deposits fromsouthwest Turkey (Van Zeist et al. 1975) and northern Iran(Van Zeist & Bottema 1977).

A previous study of isozyme variation in A. glutinosa(Prat et al. 1992) demonstrated strong differentiationbetween populations that was attributed to both ecologi-cal and historical events affecting population evolution.This contrasts with the results of Bousquet et al. (1990)who found very little population differentiation in theNorth American A. sinuata and A. crispa.

The extent to which the different putative refugia havecontributed to the present European distribution of alderis unknown and thus it was decided to study the chloro-plast DNA phylogeography of A. glutinosa using aPCRÐRFLP (polymerase chain reactionÐrestriction frag-ment length polymorphism) approach. Using theseresults, it should be possible to identify glacial refugia andrelate observed patterns of cpDNA variation to possiblepostglacial migration routes.

Materials and methods

Plant material

Two sources of Alnus glutinosa were used in the study. Seedmaterial was obtained from the International Alder SeedBank at Geraardsbergen, Belgium. Seeds were germinatedon damp compost and harvested for DNA extraction when4Ð6-weeks old. Alternatively, fresh leaf material was col-lected in the field and either snap-frozen in liquid nitrogenor dried over silica gel (Chase & Hills 1991). Where possi-ble, a minimum of three nonadjacent trees per populationwere sampled. The total sample consisted of 217 individualtrees from 101 populations covering the entire naturalrange of the species within Europe. Details of site locationand sample size per site are given in Appendix 1.

DNA extraction

DNA was extracted from frozen or dried material usingthe CTAB method of Doyle & Doyle (1990) with the addi-tion of fine sand to aid grinding of the leaf material andsubstituting PVPP (polyvinyl polypyrrolidone) in placeof PVP. For seedling material, a miniprep modification ofthe CTAB method was used. Leaf material (0.1 g) wasground with fine sand in liquid nitrogen, added to 500 µLof 2× CTAB in a 1.5 mL centrifuge tube and incubated at60 ¡C for 30Ð60 min. A volume of 500 µL of 24:1 chloro-form:iso-amyl alcohol was added, the tubes were mixedand spun at 13 000 rpm for 10 min in a bench-top cen-trifuge. The top layer was then pipetted into a clean tubeto which 250 µL of isopropanol was added. The tubes

were rocked gently to aid precipitation of the DNA. TheDNA was washed in an ethanol wash buffer (76%ethanol, 10 mM ammonium acetate), air dried at roomtemperature for a few minutes and then dissolved in50Ð100 µL of TE (Tris-EDTA buffer, pH 8).

PCR amplification

cpDNA was amplified using the universal primers ofTaberlet et al. (1991) and Demesure et al. (1995) (Table 1).Reactions were carried out in a total volume of 25 µL con-sisting of 17.55 µL of double-distilled water, 2.5 µL of 10×PCR buffer (Bioline), 1.25 µL of dNTPs (2 mM), 1 µL ofMgCl2 (50 mM), 0.5 µL of each of the forward and reverseprimers (10 µM), 1 unit of BIOTAQ DNA polymerase(Bioline) and 1.5 µL of genomic DNA. PCR amplificationswere performed in a DNA thermal cycler (Perkin ElmerCetus). An initial 5 min denaturation at 94 ¡C was fol-lowed by 30 cycles of 94 ¡C for 30 s, annealing at 54Ð62 ¡Cfor 30 s and extension at 72 ¡C for from 90 s to 3 mins.Annealing temperature was dependent upon primersused and extension time was dependent on the length ofthe PCR product (Table 1). Reactions were given a final10 min extension time at 72 ¡C.

RFLP analysis

PCR product (10 µL) was restricted overnight with eitherone or two restriction enzymes following the methods ofFerris et al. (1993, 1995). Initially, each of the nine PCRproducts from six individuals of A. glutinosa weredigested with six 4-bp cutting and two 6-bp cuttingrestriction enzymes (AluI, CfoI, EcoRI, HaeIII, HindIII,HinfI, MboI and RsaI; Gibco BRL). Restriction digests wererun on either 6% polyacrylamide or 1.6% agarose gels andvisualized by staining with ethidium bromide(0.5 µg/mL). Polymorphisms were scored visually andnumbered in order of decreasing molecular weight.Bands were scored as presence (1) vs. absence (0) of theband. In order to determine the nature of each mutation,amplicons were digested with several restriction enzymecombinations. Size variation was assumed when similarpatterns were observed with different enzymes.

Data analysis

Each polymorphism was scored as an unordered multi-state character and subjected to phylogenetic analysisusing the heuristic search option of PAUP version 3.1.(Swofford 1993). The number of mutational differencesbetween haplotypes was calculated and analysed usingM I N S P N E T (Excoffier & Smouse 1994) to produce aminimum-spanning tree of haplotypes found. This proce-dure is used to connect points, in this case haplotypes, by

1152 R. A KING AND C. FERRIS

© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1151Ð1162

direct links having the smallest possible total length (Prim1957). Minimum-spanning networks are alternatives toWagner parsimony trees, but better convey the connec-tions between haplotypes (Excoffier & Smouse 1994).

The level of population subdivision for a cytoplasmi-cally inherited genome using unordered alleles (GSTc) wascalculated following the method of Pons & Petit (1995)using the computer program H A P L O I D I V. NSTc, the levelof population subdivision for ordered alleles was calcu-lated using the program H A P L O N S T (Pons & Petit 1996).

A ratio of seed to pollen flow was calculated using theequation:

(1/GSTb Ð 1) Ð 2 (1/GSTc Ð 1)(pollen flow/seed flow = ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

(1/GSTc Ð 1)

were GSTb is the level of population subdivision based onbiparentally (nuclear) inherited genomes. For this study,GSTb is taken from Prat et al. (1992) This is a modificationof the equation of Ennos (1994) with the substitution ofGST values for FST values.

Results

An initial screen of nine pairs of universal chloroplastprimers with eight restriction enzymes revealed variationin six Alnus glutinosa fragments (Table 1). Only thoseprimerÐenzyme combinations that gave easily scorablevariation were used in the full survey. Four of these

primers pairs proved sufficient to identify all haplotypesfound. Numerous mutations were detected (Table 2,Fig. 1). In all, a total of 13 cpDNA haplotypes were foundand these are described in Table 3.

The geographical distribution of these haplotypes ishighly structured (Fig. 2). Southeast Europe is a major areafor cpDNA variation with a total of seven haplotypes beingfound in an area covering Bulgaria, Greece, Turkey,Georgia and the Ukraine. Southern Europe possesses a fur-ther three haplotypes (one each in southern Italy, Corsicaand Spain). Most of central and northern European popula-tions are comprised of one or the other of two commonhaplotypes, with a third rare haplotype restricted to a sin-gle population in central Norway. The area aroundHungary and northern Croatia is polymorphic.

Due to the low number of variable and phylogeneti-cally informative mutations and the presence of homo-plasy in the data set a reliable phylogeny of haplotypescould not be found using parsimony analysis. Using PAUP

(Swofford 1993), a total of 638 trees of length 17(C.I. = 0.842) were found. Haplotype relatedness was rep-resented using the M I N S P N E T (Excoffier & Smouse 1994)program (Fig. 3) which clearly indicates four groupings.The seven southeast European haplotypes (Fig. 2) are splitinto two groups, one containing haplotypes A, B, C and D,the other L, M and N. The Corsican (K) and Spanish (J)haplotypes form their own grouping, as do the remainingEuropean haplotypes (E, F, G and H).

PHYLOGEOGRAPHY OF BLACK ALDER 1153


Table 1 Details of primers used in this study

Annealing Extension In thisPrimers Code temperature time Variable study Reference

trnH [tRNA-His (GUG)] - A 62 ¡C 2 min Yes Yes Demesure et al. (1995)trnK1 [tRNA-Lys (UUU) 3′ exon]

trnC [tRNA-Cys (GCA)] - B 58 ¡C 3 min Yes Yes Demesure et al. (1995)trnD [tRNA-Asp (GUC)]

trnD [tRNA-Asp (GUC)] - C 54 ¡C 2 min Yes No Demesure et al. (1995)trnT [tRNA-Thr (GGU)]

psbC [psII 44 kd protein] - D 57 ¡C 2 min No No Demesure et al. (1995)trnS [tRNA-Ser (GGA)]

trnS [tRNA-Ser (UGA)] - E 62 ¡C 2 min Yes Yes Demesure et al. (1995)trnfM [tRNA-fMet (CAU)]

trnM [tRNA-Met (CAU)] - G 59 ¡C 3 min No No Demesure et al. (1995)rbcL [RuBisCo large subunit]

trnK1 [tRNA-Lys (UUU) 3′ exon] - K 53 ¡C 3 min Yes Yes Demesure et al. (1995)trnK2 [tRNA-Lys (UUU) 5′ exon]

trnS [tRNA-Ser (GGA)] - S 57 ¡C 2 min No No Demesure et al. (1995)trnT [tRNA-Thr (UGU)]

trnT [tRNA-Thr (UGU)] - T 55 ¡C 90 s Yes No Taberlet et al. (1991)trnL2 [tRNA-Leu (UAA) 3′ exon]

A total of 217 individual trees from 101 populations wasanalysed. Only populations where three or more individ-uals could be obtained were used in the H A P L O I D I V andH A P L O N S T analyses. As single plants represented popu-lations from Istanbul, Kvam and southern Italy their rep-resentative haplotypes were omitted from the analysis. Ofthe 43 populations used, seven were polymorphic. Thelevel of population subdivision within A. glutinosa washigh, GSTc = 0.866 (hs = 0.103; ht = 0.773). For the NSTc anal-ysis, a distance matrix derived from the pairwise numberof mutational differences between haplotypes was used.Again, the level of population subdivision was high,NSTc = 0.905 (vs = 0.190: vt = 2.00).

Combining GSTc with the value of subdivision for nuclearmarkers (GSTb = 0.204; Prat et al. 1992) gives effective geneflow via pollen ≈ 23 times greater than that via seed.

Discussion

A general knowledge of the postglacial history of alder isknown from fossil pollen analysis. Restricted to south-eastern Europe at 13 000 BP, Alnus glutinosa migrated

northward and westward from this refugial area, reach-ing western Europe by 10 000 BP (Huntley & Birks 1983),and arrived in Fennoscandia around 8500 BP (Tallantire1974). Colonization of Britain took place about 8000 BP

(Birks 1989). No data are available on the postglacial his-tory of alder in Spain or Turkey. We undertook this pre-sent study to compare molecular phylogenetic data withwhat is known from fossil analysis and to gain furtherinsights into the postglacial history of black alder. Bycombining both approaches we get a very good picture ofthe glacial/postglacial history of species.

The geographical distribution of the 13 alder haplotypesis highly structured (Fig. 2). From the present study, the dis-tribution of cpDNA haplotypes confirms that most ofnorthern Europe was colonized from a refuge in the area ofthe Carpathian Mountains (present-day Hungary andRomania). The initial westward migration identified byHuntley & Birks (1983) apparently involved haplotype G,while the migration into northern Europe andFennoscandia comprised haplotype F. The presence of hap-lotype F in central and southwest France and haplotype Gin southern Norway is thought to be due to long-distance



Table 2 Summary of sizes and RFLP differences found in cpDNA PCR fragments of European Alnus glutinosa and restriction enzymesused in this study. Primer codes correspond to those given in Table 1

Primer Approx. size of Total variable Band Total no. code PCR product (bp) Enzymes Gel type RFLP bands no. of mutations

A 1600 bp HaeIII/HinfI 6% polyacrylamide 2 I 4A 1600 bp HaeIII/HinfI 6% polyacrylamide II 1B 3000 bp AluI/CfoI 6% polyacrylamide 1 I 6E 1300 bp HaeIII/HinfI 6% polyacrylamide 3 I 4E 1300 bp HaeIII/HinfI 6% polyacrylamide II 1E 1300 bp HaeIII/HinfI 6% polyacrylamide III 1K 2500 bp MboI/RsaI 6% polyacrylamide 1 I 1K 2500 bp HaeIII/HinfI 1.6% agarose 1 I 1

Fig. 1 Restriction digests showingvariation in the Alnus glutinosa cpDNAPCR fragment trnH-K1 digested withHaeIII/HinfI. Lanes: 1 and 21, DNAmarker (pBR322 HaeIII digest; Sigma); 2,Millstatt, Austria; 3, Somogys�rd,Hungary; 4Ð7, Malaga, southern Spain; 8,Courbaisse, southern France; 9,Lagonegro; 10, Lasila (both Calabria, S.Italy); 11, Istanbul, Turkey; 12 and 13,Barazar, northern Spain; 14, Ponte Rosso,Corsica; 15, Jarlsberg, Norway; 16,Kurowo, Poland; 17, River Elbe, CzechRepublic; 18, Borcka, Turkey; 19, Barlston,UK; 20,: East Anatolia, Turkey.



Table 3 Catalogue of cpDNA haplotypes for Alnus glutinosa. Mutations are scored in order of decreasing molecular weight. A 0 representsthe complete absence of a restriction fragment from its expected position on a gel

trnH-trnK1 trnH-trnK1 trnK1ÐtrnK2 trnK1ÐtrnK2 trnC-trnD trnS-trnfM trnS-trnfM trnS-trnfMHaeIII/HinfI HaeIII/HinfI MboI/RsaI HaeIII/HinfI AluI/CfoI HaeIII/HinfI HaeIII/HinfI HaeIII/HinfI

Haplotype band I band II band I band I band I band I band II band III

A 1 1 2 1 3 3 1 1B 3 1 1 1 3 3 1 1C 3 1 1 1 4 3 1 1D 4 1 1 1 5 3 1 1E 3 0 2 1 4 2 1 1F 3 0 2 1 1 2 1 1G 3 0 2 1 2 2 1 1H 3 0 2 0 2 2 1 1J 2 0 2 1 3 2 1 1K 2 0 2 1 0 1 1 1L 1 0 2 1 2 2 2 1M 1 0 2 1 3 2 2 1N 1 0 2 1 3 2 2 0

Fig. 2 Geographic distribution of 13cpDNA haplotypes identified in Alnusglutinosa. For clarity symbol size does notrepresent sample size. (a) Europeandistribution. A simplified version of theminimum-spanning network tree (Fig. 3)is included for comparison. (b) Detail ofnortheast Turkey. For polymorphicpopulations shading is proportional tohaplotype proportion.

dispersal. In addition, the high number of haplotypes foundin southeast Europe would indicate that this was also a refu-gial area, supporting fossil pollen evidence (Bennett et al.1991). The existence of a Corsican refuge, as proposed for A.cordata and A. viridis ssp. suaveolens (Huntley & Birks 1983),is confirmed for A. glutinosa, which has a unique haplotypehere. From the failure to find any Corsican-type aldercpDNA on mainland Europe, it is apparent that alder isunlikely to have been able to migrate from the island. It wasnot possible to obtain alder from the neighbouring island ofSardinia and therefore we can only speculate that theCorsican haplotype would also be found there. This studyproposes the location of two further refugia for alder.Spain and Turkey apparently housed populations duringthe last period of glaciation. The high level of cpDNAdiversity found in Turkey, combined with the knowledgethat Alnus pollen has been found in late glacial deposits insouthwest Turkey (Van Zeist et al. 1975) and northern Iran(Van Zeist & Bottema 1977), indicates that this was per-haps a refugial area. Alder also grows as a native in themountains of North Africa, but we were unable to obtainsamples from here.

Of particular interest is the splitting of the seven south-east European cpDNA types into two groups; A, B, C, D,and L, M, N (Fig. 3). These two groups are well supportedin a PAUP bootstrap analysis. There are an average of 5.75mutational differences between these groups, indicatingdivergence in the order of several hundred thousandyears. The mixing of two divergent cpDNA lineages mayreflect patterns that have been established over several iceages. The present situation could represent an amalgama-tion of haplotypes from alder refugia from previous glacialcycles.

A common feature of the Alnus, Fagus (Demesure et al.1996) and Quercus (Dumolin-Lap�gue et al. 1997) phylo-geographies is the higher levels of cpDNA diversityfound in southern as compared to northern populations.Dumolin-Lap�gue et al. (1997) found that of the 28cpDNA haplotypes found in southern Europe in thewhite oak (Quercus) species complex, only a subset ofthese were found in the north. In the present case, 12 ofthe 13 alder haplotypes have been found south of the45¡ 00′ N latitude. Only the rare haplotype from centralNorway is peculiar to the north and is assumed to havearisen during range expansion, in a similar way to theEast Anglian mutation in Q. robur (Ferris et al. 1995). Thisthinning of haplotypes from south to north during expan-sion from refugia has been predicted for species thatundergo leptokurtic (long-distance) as opposed to normaldispersal (Hewitt 1996). By contrast, alder in refugialareas is more likely to have migrated by normal dispersal,thus maintaining diversity.

Data on the location of refugia and possible colonizationroutes available for four European tree taxa has beenreviewed by Taberlet et al. (1998). Each of the taxa havetheir own individual pattern of colonization, althoughthere are some similarities in the pathways taken. Forexample, the pattern for the common beech is similar tothat of alder. Both these species shared a refuge in theCarpathian mountains from which most of Europe was col-onized. Also, cpDNA types of both species from a southernItalian (Calabria) refuge have been prevented from coloniz-ing regions of northern Europe, presumably by their inabil-ity to infiltrate areas already populated by alder and beech.Northern Italy would have been colonized more easilyfrom a refuge in the Carpathian Mountains than fromsouthern Italy. This is in direct contrast to oaks, wherecpDNA haplotypes that originated in an Italian refugewere able to expand into extensive areas of northernEurope (Dumolin-Lap�gue et al. 1997; Ferris et al. 1998).

Black alder has been shown to have a relatively highdegree of geographical structuring of its nuclear genome.Prat et al. (1992) were able to resolve two groupings, basedon isozyme analysis; one containing populations fromsoutheast Europe, the other from northern and westernpopulations. They demonstrated that this population sub-division caused a deviation from random mating(GSTb = 0.204). Other wind-pollinated tree species havemuch lower values of population subdivision, e.g.GSTb = 0.05 in A. rugosa (Bousquet et al. 1988); GSTb = 0.025in Q. petraea (Zanetto & Kremer 1995). Due to its ecologi-cal preference for water-logged habitats, alder will tend tohave a patchy distribution of populations, reducing thechance of interpopulation gene flow via pollen.

As seen from the geographical distribution of haplo-types (Fig. 2), cpDNA diversity in alder is highly struc-tured. This is confirmed with the calculation of population



Fig. 3 Minimum-spanning network of 13 cpDNA haplotypesfound in European Alnus glutinosa. The major links between hap-lotypes are represented as bold lines. Other possible links identi-fied by M I N S P N E T (Excoffier & Smouse 1994) are shown asdotted lines. Superimposed on the network are the number ofmutational differences between haplotypes.

subdivision for a cytoplasmically inherited genome, forboth ordered and unordered alleles, where the values forNSTc of 0.905 and GSTc of 0.866 indicate a high degree ofisolation between populations in terms of seed flow. Thesignificantly higher value of NSTc relative to GSTc indi-cates some correspondence between haplotype phy-logeny and the geographical distribution of haplotypes(Pons & Petit 1996).

The high degree of isolation between populations isunexpected due to the high potential for seed dispersal inalder via water. Although on some islands of the UK alderis now extinct, fossil pollen and macrofossil evidence, e.g.Fossitt (1996), have shown that it was able to disperseover 70 km of open sea and colonize these islands in post-glacial times (Bennett 1995). Alder seedlings are shadeintolerant (McVean 1956) and will not readily invadewooded areas despite having the capability to disperseover long distances.

Range expansion can establish patterns of genetic vari-ation that can persist for hundreds or thousands of gener-ations (Nichols & Hewitt 1994). Initially, both nuclear andchloroplast genomes will exhibit the same geographicalpatterns of variation. Over time, especially in species withhigh pollen-flow to seed-flow ratios, nuclear patterns willbecome much less distinct. The present highly structuredpattern of cpDNA diversity in species such as oak andalder has been maintained due to the lower effectivemigration rate of organelle genes relative to nuclear genes(Birky et al. 1989).

For alder, long-distance dispersal along river systemsmay have played an important part in determining thepatterns of cpDNA diversity observed. During its expan-sion and colonization phase after the end of the last iceage, alder migrated extremely quickly with a migrationrate, based on fossil pollen analysis, of 500Ð2000 m/year(Huntley & Birks 1983). Such a rate probably reflects lep-tokurtic dispersal. Under present-day stable conditions,alder seed may only be dispersed up to 30 m from thematernal tree (McVean 1953). Hewitt (1996) describes thesituation where long-distance dispersants set up coloniesahead of the main wave of expansion. Surrounding areasare then colonized from these foci. Later migrants con-tribute little to the gene pool of these colonies owing tothe difficulty of invading already established populations(Ibrahim et al. 1996). Such a scenario could well haveplayed an important role in the postglacial migration andcolonizations of alder.

Knowing the values of GSTb and GSTc it is possible toestimate the relative rate of pollen flow to seed flow(Ennos 1994). This ratio is ≈ 23 in alder and is intermediateto those calculated by El Mousadik & Petit (1996) forseven other forest tree species. Of course, the GSTb andGSTc values for alder were derived from different popula-tions and the pollen-flow to seed-flow ratio needs to be

taken with caution. This is the first reported pollen/seedflow value for a tree species adapted to aquatic seed dis-persal. Higher pollen-to-seed-flow values have so far onlybeen found in Pinus contorta (24), Fagus sylvatica (84), Q.robur (286) and Q. petraea (500). Not surprisingly, these areall wind-pollinated species.

The findings of this study clearly demonstrate that amolecular phylogenetic approach can compliment thefindings of earlier fossil-based studies on plant popula-tion history. With better sampling in southeast Europe,the Ukraine and North Africa we will be able to gain aclearer picture of the migration and colonization of alderin these areas after the last ice age.

Acknowledgements

Financial support from the University of Leicester is gratefullyacknowledged. The authors wish to thank K. Ashburner, J. Bailey,R. Brooks, P. Catalan, P. H. Salvesen, C. Stace and R. V�in�l� forcollection of material for this study, T. Glover for computingassistance, B. Michiels of the International Alder Seed Bank andthe Royal Botanic Garden, Kew and Ness Botanical Gardens,Wirral for access to their living collections.

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SampleSite size Haplotype

AustriaBodensdorf 1 1 GDrosendorf 1 1 GGloggnitz 3 3 GMillstatt 2 2 GRetz 1 1 FWaidhofen 1 1 G

BelgiumEan Noir 3 3 GLa Molign�e 1 1 F

BulgariaPlackovci 2 2 ATeteven 3 3 AVoneshta Voda 1 1 A

CorsicaAsco 1 1 KCarazzi 1 1 KCardo-Torgia 3 3 KCorte 3 3 KPonte Rosso 2 2 KSagone 3 3 KTufo 3 3 K

CroatiaDurdevac 3 1 F, 2 GHladna Voda 3 3 FKupinje 1 1 GNovoselec 1 1 GPopovaca 3 3 F

Czech RepublicPodebrady, Central Bohemia 3 3 F

EstoniaLaiksaare 2 2 FMeeksi 1 1 F

EnglandGlenridding, Cumbria 1 1 FHapton, Norfolk 5 5 FRiver Itchen, Southampton 3 3 G

FranceCaulnes, Bretagne 4 4 GCourbaisse, N. of Nice 1 1 GFraimbois, Meurthe et Moselle 2 2 GGranville, Normandie 3 3 GLezay, Deux S�vres 3 2 F, 1 GMessein, Le Petit Etang, Meurthe et Moselle 1 1 GNeuvion en Thierache 3 3 GSt Paul en Born 1 1 FSoustons, Landes 3 3 GVall�e de la Charente, Angoul�me 3 1 F, 2 GVall�e de lÕYonne, Clemecy 3 3 F

FinlandUusimaa, Kirkkonummi 1 1 FPorvoo, Sann�s 1 1 F

GeorgiaAchaldabu 2 2 C

GermanyDiessen 1 1 GPfaueninsel, Berlin 1 1 FRott 1 1 G

Appendix 1 Sample and haplotype detailsof European Alnus glutinosa presented inFig. 2. For clarity sites in Fig. 2 are notlabelled. Lettering of haplotypescorresponds to those given in Fig. 3.



Wasserburg 1 1 GGreece

Sperkihos River Valley 4 2 B, 2 DHungary

Homokszenty 1 1 FMike 1 1 FNagykorp�d 3 1 F, 2 G�tv�skrp�d 3 3 G,Somogys�rd 2 2 G

ItalyLagonegro 1 1 ELasila 1 1 ES. Antonio di Susa 3 3 GViterbo 3 3 G

LatviaBalofi 2 2 F

LithuaniaBenderka Forest, Dzirmiskes 4 4 FKaunas Lagoon, Girionys 1 1 F

NetherlandsKorenburgerveen 3 3 FLg. Singraven, Denekamp 1 1 FWageningen, Binnenveld 3 3 GWeerribben 1 1 G

NorwayEides�sen, Odda, Hordaland county 1 1 FFron, Kvam 1 1 HGilje, Sandnes, Rogaland county 1 1 GJarlsberg, T¯nsberg, Vestfold county 5 5 GJ¯rstad, Sn�sa, Nord-T¯ndelag county 1 1 FLaukhammer, Tysnes, Hordaland county 3 3 FLyngneset, Tysnes, Hordaland county 2 2 F

PolandBiala Podlaska, Kloda 1 1 FForest Karcz, Sulecha 1 1 FKurowo, Wlolawek 4 4 FWichrowo, Smolajny 4 4 FWyszkow, Fidest 1 1 F

RomaniaSatu Mare, Tibenn 1 1 G

ScotlandGlen Barrisdale, Knoydart 3 3 F

SlovakiaGabcokovo 2 1 F, 1 G

SpainAsturias, Covadonga 2 2 JMalaga 4 4 JVizcaya-Bizkaia, Col Barazar 3 3 F

SwedenBoserup 1 1 FBubbetorp 1 1 FUppsala 1 1 FVanneberga 1 1 F

TurkeyDereli, Giresun 5 2 L, 3 NEast Anatolia 1 1 LGatak, Giresun 3 3 LG�ktas, Artvin 1 1 C

SampleSite size Haplotype

Appendix 1 Continued



Hopa, Artvin 4 1 C, 3 LIstanbul 1 1 MKapili Dag River, N.E. of Borcka 1 1 CKesap, Giresun 1 1 NMa�ka, Trabzon 4 4 LMesudiye, Ordu 4 4 LTirebolu, Giresun 3 3 LUlubey, Ordu 4 4 LVakfikebir, Trabzon 3 3 C

UkraineYalta 2 2 C

WalesRiver Twyi 2 2 G

Appendix 1 ContinuedSample

Site size Haplotype