Molecular Ecology (1998) 7, 1151Ð1161

Chloroplast DNA phylogeography of Alnus glutinosa

(L.) Gaertn.
R. ANDREW KING and COLIN FERRIS
Department of Biology, University of Leicester, Leicester, LE1 7RH, UK

Abstract

Traditionally, information on the postglacial history of plant species has been gained
from the analysis of fossil pollen data. More recently, surveys of present patterns of
genetic variation have given valuable insights into species phylogeography. The genus
Alnus, based on fossil data, is known to have had at least four glacial refugia. A survey of
chloroplast DNA (cpDNA) diversity in populations of black alder (A. glutinosa) was
undertaken in order to gain more insight into its postglacial history. This revealed a high
degree of structuring of 13 cpDNA haplotypes on a European scale which indicated that
most of northern and central Europe was colonized from a refuge in the Carpathian
Mountains. Based on the distribution of two common cpDNA haplotypes, colonization
routes from this refuge can be determined. The locations of other previously identified
refugia are confirmed and two formerly unconfirmed refugial areas for alder (southern
Spain 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

Introduction genes (chloroplast and mitochondrial) will be highly struc-

tured when compared to nuclear genes (Petit et al. 1993).
Present patterns of genetic variation within species have
Recent studies on European and North American tree
been influenced by many factors. Of major importance is
species have shown that refugial areas and postglacial
the influence of the last period of glaciation and subse-
migration routes can be identified using DNA markers.
quent migration of taxa from glacial refugia. Ice ages
Fossil pollen maps for European deciduous oaks indicate
occur at regular intervals of 100 000 years with warm
refugia in southern Spain, southern Italy and the Balkan
interglacial periods lasting 15Ð20 000 years as a result of
Peninsula (Huntley & Birks 1983; Bennett et al. 1991). Two
instabilities in the earthÕs climate caused by the
separate studies based on cpDNA variation have con-
Milankovitch cycles (Bennett 1990). Many tree species
firmed the existence of these three refugia (Dumolin-
common in northern Europe today survived these glacial
Lap•gue et al. 1997; Ferris et al. 1998) and also identify
periods in small, low-density populations in refugia in the
areas of northern Europe colonized by oaks from each
mountains of southern Europe (Bennett et al. 1991).
refugium. Similar studies have also been carried out on
The slower rate of evolution of chloroplast DNA
Fagus sylvatica in Europe (Demesure et al. 1996) and
(cpDNA) compared to nuclear DNA in plants (Wolfe et al.
Liriodendron tulipifera in North America (Sewell et al. 1996).
1987) has limited its use in population studies at the
Alnus glutinosa (black alder) is a wind-pollinated, self-
intraspecific level (Palmer 1987). Several papers have been
incompatible tree species of riparian and water-logged
published describing varying levels of intraspecific
habitats (McVean 1953). It is common in Europe and the
cpDNA variation in a wide range of plant species
Mediterranean, and extends as far as the mountains of
(reviewed in Soltis et al. 1992). cpDNA is maternally inher-
Turkey and North Africa. Seed dispersal is most effective
ited in the majority of flowering plants and therefore pro-
by water, but seeds may also be dispersed by wind up to
vides a seed-specific marker. In species where seed flow is
30 m from the parent tree (McVean 1953). Huntley & Birks
much less than pollen flow, it is predicted that organelle
(1983) provide evidence for at least three glacial refugia
Correspondence: R. A. King. Fax: +44 (0) 1162522791; E-mail: for Alnus based on fossil pollen data, including Corsica,
rak5@leicester.ac.uk the Carpathian Mountains and southwestern Russia, and

© 1998 Blackwell Science Ltd

1152 R. A KING AND C. FERRIS
the Bay of Biscay region. To this may be added refugia in
southern Italy and Greece (Bennett et al. 1991). Alder
pollen has also been found in late glacial deposits from
southwest 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 differentiation
between 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 the
North American A. sinuata and A. crispa.
The extent to which the different putative refugia have
contributed to the present European distribution of alder
is unknown and thus it was decided to study the chloro-
plast DNA phylogeography of A. glutinosa using a
PCRÐRFLP (polymerase chain reactionÐrestriction frag-
ment length polymorphism) approach. Using these
results, it should be possible to identify glacial refugia and
relate observed patterns of cpDNA variation to possible
postglacial migration routes.
Materials and methods
Plant material
Two sources of Alnus glutinosa were used in the study. Seed
material was obtained from the International Alder Seed
Bank at Geraardsbergen, Belgium. Seeds were germinated
on damp compost and harvested for DNA extraction when
4Ð6-weeks old. Alternatively, fresh leaf material was col-
lected in the field and either snap-frozen in liquid nitrogen
or dried over silica gel (Chase & Hills 1991). Where possi-
ble, a minimum of three nonadjacent trees per population
were sampled. The total sample consisted of 217 individual
trees from 101 populations covering the entire natural
range of the species within Europe. Details of site location
and sample size per site are given in Appendix 1.
DNA extraction
DNA was extracted from frozen or dried material using
the CTAB method of Doyle & Doyle (1990) with the addi-
tion of fine sand to aid grinding of the leaf material and
substituting PVPP (polyvinyl polypyrrolidone) in place
of PVP. For seedling material, a miniprep modification of
the CTAB method was used. Leaf material (0.1 g) was
ground with fine sand in liquid nitrogen, added to 500 µL
of 2× CTAB in a 1.5 mL centrifuge tube and incubated at
60 ¡C for 30Ð60 min. A volume of 500 µL of 24:1 chloro-
form:iso-amyl alcohol was added, the tubes were mixed
and spun at 13 000 rpm for 10 min in a bench-top cen-
trifuge. The top layer was then pipetted into a clean tube
to which 250 µL of isopropanol was added. The tubes
were rocked gently to aid precipitation of the DNA. The
DNA was washed in an ethanol wash buffer (76%
ethanol, 10 mM ammonium acetate), air dried at room
temperature for a few minutes and then dissolved in
50Ð100 µL of TE (Tris-EDTA buffer, pH 8).
PCR amplification
cpDNA was amplified using the universal primers of
Taberlet 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 of
MgCl2 (50 mM), 0.5 µL of each of the forward and reverse
primers (10 µM), 1 unit of BIOTAQ DNA polymerase
(Bioline) and 1.5 µL of genomic DNA. PCR amplifications
were performed in a DNA thermal cycler (Perkin Elmer
Cetus). An initial 5 min denaturation at 94 ¡C was fol-
lowed by 30 cycles of 94 ¡C for 30 s, annealing at 54Ð62 ¡C
for 30 s and extension at 72 ¡C for from 90 s to 3 mins.
Annealing temperature was dependent upon primers
used and extension time was dependent on the length of
the PCR product (Table 1). Reactions were given a final
10 min extension time at 72 ¡C.
RFLP analysis
PCR product (10 µL) was restricted overnight with either
one or two restriction enzymes following the methods of
Ferris et al. (1993, 1995). Initially, each of the nine PCR
products from six individuals of A. glutinosa were
digested with six 4-bp cutting and two 6-bp cutting
restriction enzymes (AluI, CfoI, EcoRI, HaeIII, HindIII,
HinfI, MboI and RsaI; Gibco BRL). Restriction digests were
run on either 6% polyacrylamide or 1.6% agarose gels and
visualized by staining with ethidium bromide
(0.5 µg/mL). Polymorphisms were scored visually and
numbered in order of decreasing molecular weight.
Bands were scored as presence (1) vs. absence (0) of the
band. In order to determine the nature of each mutation,
amplicons were digested with several restriction enzyme
combinations. Size variation was assumed when similar
patterns were observed with different enzymes.
Data analysis
Each polymorphism was scored as an unordered multi-
state character and subjected to phylogenetic analysis
using the heuristic search option of PAUP version 3.1.
(Swofford 1993). The number of mutational differences
between haplotypes was calculated and analysed using
M I N S P N E T (Excoffier & Smouse 1994) to produce a
minimum-spanning tree of haplotypes found. This proce-
dure is used to connect points, in this case haplotypes, by
© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1151Ð1162
 PHYLOGEOGRAPHY OF BLACK ALDER 1153

Table 1 Details of primers used in this study

Annealing Extension In this

Primers 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]

direct links having the smallest possible total length (Prim
1957). Minimum-spanning networks are alternatives to
Wagner 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) was
calculated following the method of Pons & Petit (1995)
using the computer program H A P L O I D I V . NSTc, the level
of 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 the
equation:
(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 on
biparentally (nuclear) inherited genomes. For this study,
GSTb is taken from Prat et al. (1992) This is a modification
of the equation of Ennos (1994) with the substitution of
GST values for FST values.
Results
An initial screen of nine pairs of universal chloroplast
primers with eight restriction enzymes revealed variation
in six Alnus glutinosa fragments (Table 1). Only those
primerÐenzyme combinations that gave easily scorable
variation were used in the full survey. Four of these
primers pairs proved sufficient to identify all haplotypes
found. Numerous mutations were detected (Table 2,
Fig. 1). In all, a total of 13 cpDNA haplotypes were found
and these are described in Table 3.
The geographical distribution of these haplotypes is
highly structured (Fig. 2). Southeast Europe is a major area
for cpDNA variation with a total of seven haplotypes being
found in an area covering Bulgaria, Greece, Turkey,
Georgia and the Ukraine. Southern Europe possesses a fur-
ther three haplotypes (one each in southern Italy, Corsica
and Spain). Most of central and northern European popula-
tions are comprised of one or the other of two common
haplotypes, with a third rare haplotype restricted to a sin-
gle population in central Norway. The area around
Hungary 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 haplotypes
could 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 split
into 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 remaining
European haplotypes (E, F, G and H).

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1154 R. A KING AND C. FERRIS

Table 2 Summary of sizes and RFLP differences found in cpDNA PCR fragments of European Alnus glutinosa and restriction enzymes
used 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 4

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

Fig. 1 Restriction digests showing

variation in the Alnus glutinosa cpDNA
PCR fragment trnH-K1 digested with
HaeIII/HinfI. Lanes: 1 and 21, DNA
marker (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, Czech
Republic; 18, Borcka, Turkey; 19, Barlston,
UK; 20,: East Anatolia, Turkey.

A total of 217 individual trees from 101 populations was
analysed. Only populations where three or more individ-
uals could be obtained were used in the H A P L O I D I V and
H 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. Of
the 43 populations used, seven were polymorphic. The
level of population subdivision within A. glutinosa was
high, GSTc = 0.866 (hs = 0.103; ht = 0.773). For the NSTc anal-
ysis, a distance matrix derived from the pairwise number
of 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 nuclear
markers (GSTb = 0.204; Prat et al. 1992) gives effective gene
flow via pollen ≈ 23 times greater than that via seed.
Discussion
A general knowledge of the postglacial history of alder is
known 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 (Tallantire
1974). 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 with
what is known from fossil analysis and to gain further
insights into the postglacial history of black alder. By
combining both approaches we get a very good picture of
the glacial/postglacial history of species.
The geographical distribution of the 13 alder haplotypes
is highly structured (Fig. 2). From the present study, the dis-
tribution of cpDNA haplotypes confirms that most of
northern Europe was colonized from a refuge in the area of
the Carpathian Mountains (present-day Hungary and
Romania). The initial westward migration identified by
Huntley & Birks (1983) apparently involved haplotype G,
while the migration into northern Europe and
Fennoscandia comprised haplotype F. The presence of hap-
lotype F in central and southwest France and haplotype G
in southern Norway is thought to be due to long-distance

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 PHYLOGEOGRAPHY OF BLACK ALDER 1155

Table 3 Catalogue of cpDNA haplotypes for Alnus glutinosa. Mutations are scored in order of decreasing molecular weight. A 0 represents
the 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-trnfM

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

Fig. 2 Geographic distribution of 13

cpDNA haplotypes identified in Alnus
glutinosa. For clarity symbol size does not
represent sample size. (a) European
distribution. A simplified version of the
minimum-spanning network tree (Fig. 3)
is included for comparison. (b) Detail of
northeast Turkey. For polymorphic
populations shading is proportional to
haplotype proportion.

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1156 R. A KING AND C. FERRIS
Fig. 3 Minimum-spanning network of 13 cpDNA haplotypes
found 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 as
dotted lines. Superimposed on the network are the number of
mutational differences between haplotypes.
dispersal. In addition, the high number of haplotypes found
in 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 haplotype
here. From the failure to find any Corsican-type alder
cpDNA on mainland Europe, it is apparent that alder is
unlikely to have been able to migrate from the island. It was
not possible to obtain alder from the neighbouring island of
Sardinia and therefore we can only speculate that the
Corsican haplotype would also be found there. This study
proposes the location of two further refugia for alder.
Spain and Turkey apparently housed populations during
the last period of glaciation. The high level of cpDNA
diversity found in Turkey, combined with the knowledge
that Alnus pollen has been found in late glacial deposits in
southwest 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 the
mountains of North Africa, but we were unable to obtain
samples 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 supported
in a PAUP bootstrap analysis. There are an average of 5.75
mutational differences between these groups, indicating
divergence in the order of several hundred thousand
years. The mixing of two divergent cpDNA lineages may
reflect patterns that have been established over several ice
ages. The present situation could represent an amalgama-
tion of haplotypes from alder refugia from previous glacial
cycles.
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 diversity
found in southern as compared to northern populations.
Dumolin-Lap•gue et al. (1997) found that of the 28
cpDNA haplotypes found in southern Europe in the
white oak (Quercus) species complex, only a subset of
these were found in the north. In the present case, 12 of
the 13 alder haplotypes have been found south of the
45¡ 00′ N latitude. Only the rare haplotype from central
Norway is peculiar to the north and is assumed to have
arisen during range expansion, in a similar way to the
East Anglian mutation in Q. robur (Ferris et al. 1995). This
thinning of haplotypes from south to north during expan-
sion from refugia has been predicted for species that
undergo leptokurtic (long-distance) as opposed to normal
dispersal (Hewitt 1996). By contrast, alder in refugial
areas is more likely to have migrated by normal dispersal,
thus maintaining diversity.
Data on the location of refugia and possible colonization
routes available for four European tree taxa has been
reviewed by Taberlet et al. (1998). Each of the taxa have
their own individual pattern of colonization, although
there are some similarities in the pathways taken. For
example, the pattern for the common beech is similar to
that of alder. Both these species shared a refuge in the
Carpathian mountains from which most of Europe was col-
onized. Also, cpDNA types of both species from a southern
Italian (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 easily
from a refuge in the Carpathian Mountains than from
southern Italy. This is in direct contrast to oaks, where
cpDNA haplotypes that originated in an Italian refuge
were able to expand into extensive areas of northern
Europe (Dumolin-Lap•gue et al. 1997; Ferris et al. 1998).
Black alder has been shown to have a relatively high
degree of geographical structuring of its nuclear genome.
Prat et al. (1992) were able to resolve two groupings, based
on isozyme analysis; one containing populations from
southeast Europe, the other from northern and western
populations. They demonstrated that this population sub-
division caused a deviation from random mating
(GSTb = 0.204). Other wind-pollinated tree species have
much lower values of population subdivision, e.g.
GSTb = 0.05 in A. rugosa (Bousquet et al. 1988); GSTb = 0.025
in Q. petraea (Zanetto & Kremer 1995). Due to its ecologi-
cal preference for water-logged habitats, alder will tend to
have a patchy distribution of populations, reducing the
chance 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
© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1151Ð1162

subdivision for a cytoplasmically inherited genome, for
both ordered and unordered alleles, where the values for
NSTc of 0.905 and GSTc of 0.866 indicate a high degree of
isolation between populations in terms of seed flow. The
significantly 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 is
unexpected due to the high potential for seed dispersal in
alder via water. Although on some islands of the UK alder
is now extinct, fossil pollen and macrofossil evidence, e.g.
Fossitt (1996), have shown that it was able to disperse
over 70 km of open sea and colonize these islands in post-
glacial times (Bennett 1995). Alder seedlings are shade
intolerant (McVean 1956) and will not readily invade
wooded areas despite having the capability to disperse
over 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 and
chloroplast genomes will exhibit the same geographical
patterns of variation. Over time, especially in species with
high pollen-flow to seed-flow ratios, nuclear patterns will
become much less distinct. The present highly structured
pattern of cpDNA diversity in species such as oak and
alder has been maintained due to the lower effective
migration rate of organelle genes relative to nuclear genes
(Birky et al. 1989).
For alder, long-distance dispersal along river systems
may have played an important part in determining the
patterns of cpDNA diversity observed. During its expan-
sion and colonization phase after the end of the last ice
age, alder migrated extremely quickly with a migration
rate, 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 the
maternal tree (McVean 1953). Hewitt (1996) describes the
situation where long-distance dispersants set up colonies
ahead of the main wave of expansion. Surrounding areas
are then colonized from these foci. Later migrants con-
tribute little to the gene pool of these colonies owing to
the difficulty of invading already established populations
(Ibrahim et al. 1996). Such a scenario could well have
played an important role in the postglacial migration and
colonizations of alder.
Knowing the values of GSTb and GSTc it is possible to
estimate the relative rate of pollen flow to seed flow
(Ennos 1994). This ratio is ≈ 23 in alder and is intermediate
to those calculated by El Mousadik & Petit (1996) for
seven other forest tree species. Of course, the GSTb and
GSTc values for alder were derived from different popula-
tions and the pollen-flow to seed-flow ratio needs to be
© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1151Ð1162
PHYLOGEOGRAPHY OF BLACK ALDER 1157
taken with caution. This is the first reported pollen/seed
flow value for a tree species adapted to aquatic seed dis-
persal. Higher pollen-to-seed-flow values have so far only
been found in Pinus contorta (24), Fagus sylvatica (84), Q.
robur (286) and Q. petraea (500). Not surprisingly, these are
all wind-pollinated species.
The findings of this study clearly demonstrate that a
molecular phylogenetic approach can compliment the
findings 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 a
clearer picture of the migration and colonization of alder
in these areas after the last ice age.
Acknowledgements
Financial support from the University of Leicester is gratefully
acknowledged. The authors wish to thank K. Ashburner, J. Bailey,
R. Brooks, P. Catalan, P. H. Salvesen, C. Stace and R. VŠinšlŠ for
collection of material for this study, T. Glover for computing
assistance, B. Michiels of the International Alder Seed Bank and
the Royal Botanic Garden, Kew and Ness Botanical Gardens,
Wirral for access to their living collections.
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© 1998 Blackwell Science Ltd, Molecular Ecology, 7, 1151Ð1162
 PHYLOGEOGRAPHY OF BLACK ALDER 1159

Appendix 1 Sample and haplotype details

Sample
of European Alnus glutinosa presented in
Site size Haplotype
Fig. 2. For clarity sites in Fig. 2 are not
labelled. Lettering of haplotypes
Austria
corresponds to those given in Fig. 3.
Bodensdorf 1 1G
Drosendorf 1 1G
Gloggnitz 3 3G
Millstatt 2 2G
Retz 1 1F
Waidhofen 1 1G
Belgium
Ean Noir 3 3G
La MolignŽe 1 1F
Bulgaria
Plackovci 2 2A
Teteven 3 3A
Voneshta Voda 1 1A
Corsica
Asco 1 1K
Carazzi 1 1K
Cardo-Torgia 3 3K
Corte 3 3K
Ponte Rosso 2 2K
Sagone 3 3K
Tufo 3 3K
Croatia
Durdevac 3 1 F, 2 G
Hladna Voda 3 3F
Kupinje 1 1G
Novoselec 1 1G
Popovaca 3 3F
Czech Republic
Podebrady, Central Bohemia 3 3F
Estonia
Laiksaare 2 2F
Meeksi 1 1F
England
Glenridding, Cumbria 1 1F
Hapton, Norfolk 5 5F
River Itchen, Southampton 3 3G
France
Caulnes, Bretagne 4 4G
Courbaisse, N. of Nice 1 1G
Fraimbois, Meurthe et Moselle 2 2G
Granville, Normandie 3 3G
Lezay, Deux S•vres 3 2 F, 1 G
Messein, Le Petit Etang, Meurthe et Moselle 1 1G
Neuvion en Thierache 3 3G
St Paul en Born 1 1F
Soustons, Landes 3 3G
VallŽe de la Charente, Angoul•me 3 1 F, 2 G
VallŽe de lÕYonne, Clemecy 3 3F
Finland
Uusimaa, Kirkkonummi 1 1F
Porvoo, SannŠs 1 1F
Georgia
Achaldabu 2 2C
Germany
Diessen 1 1G
Pfaueninsel, Berlin 1 1F
Rott 1 1G

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

1160 R. A KING AND C. FERRIS

Appendix 1 Continued
Sample
Site size Haplotype

Wasserburg 1 1G
Greece
Sperkihos River Valley 4 2 B, 2 D
Hungary
Homokszenty 1 1F
Mike 1 1F
Nagykorp‡d 3 1 F, 2 G
…tvšskrp‡d 3 3 G,
Somogys‡rd 2 2G
Italy
Lagonegro 1 1E
Lasila 1 1E
S. Antonio di Susa 3 3G
Viterbo 3 3G
Latvia
Balofi 2 2F
Lithuania
Benderka Forest, Dzirmiskes 4 4F
Kaunas Lagoon, Girionys 1 1F
Netherlands
Korenburgerveen 3 3F
Lg. Singraven, Denekamp 1 1F
Wageningen, Binnenveld 3 3G
Weerribben 1 1G
Norway
EidesŒsen, Odda, Hordaland county 1 1F
Fron, Kvam 1 1H
Gilje, Sandnes, Rogaland county 1 1G
Jarlsberg, T¯nsberg, Vestfold county 5 5G
J¯rstad, SnŒsa, Nord-T¯ndelag county 1 1F
Laukhammer, Tysnes, Hordaland county 3 3F
Lyngneset, Tysnes, Hordaland county 2 2F
Poland
Biala Podlaska, Kloda 1 1F
Forest Karcz, Sulecha 1 1F
Kurowo, Wlolawek 4 4F
Wichrowo, Smolajny 4 4F
Wyszkow, Fidest 1 1F
Romania
Satu Mare, Tibenn 1 1G
Scotland
Glen Barrisdale, Knoydart 3 3F
Slovakia
Gabcokovo 2 1 F, 1 G
Spain
Asturias, Covadonga 2 2J
Malaga 4 4J
Vizcaya-Bizkaia, Col Barazar 3 3F
Sweden
Boserup 1 1F
Bubbetorp 1 1F
Uppsala 1 1F
Vanneberga 1 1F
Turkey
Dereli, Giresun 5 2 L, 3 N
East Anatolia 1 1L
Gatak, Giresun 3 3L
Gšktas, Artvin 1 1C

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

 PHYLOGEOGRAPHY OF BLACK ALDER 1161

Appendix 1 Continued
Sample
Site size Haplotype

Hopa, Artvin 4 1 C, 3 L
Istanbul 1 1M
Kapili Dag River, N.E. of Borcka 1 1C
Kesap, Giresun 1 1N
Ma•ka, Trabzon 4 4L
Mesudiye, Ordu 4 4L
Tirebolu, Giresun 3 3L
Ulubey, Ordu 4 4L
Vakfikebir, Trabzon 3 3C
Ukraine
Yalta 2 2C
Wales
River Twyi 2 2G

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