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Molecular Ecology (2002) 11, 1409 – 1418
Molecular analysis of the Pleistocene history of Saxifraga
oppositifolia in the Alps
Blackwell Science, Ltd
R . H O L D E R E G G E R , * ‡ I . S T E H L I K † and R . J . A B B O T T *
*Division of Environmental and Evolutionary Biology, School of Biology, Sir Harold Mitchell Building, University of St Andrews, St
Andrews, Fife, KY16 9TH, UK, †Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich,
Switzerland
Abstract
A recent circumpolar survey of chloroplast DNA (cpDNA) haplotypes identified Pleistocene glacial refugia for the Arctic-Alpine Saxifraga oppositifolia in the Arctic and, potentially, at more southern latitudes. However, evidence for glacial refugia within the ice sheet
covering northern Europe during the last glacial period was not detected either with
cpDNA or in another study of S. oppositifolia that surveyed random amplified polymorphic DNA (RAPD) variation. If any genotypes survived in such refugia, they must have
been swamped by massive postglacial immigration of periglacial genotypes. The present
study tested whether it is possible to reconstruct the Pleistocene history of S. oppositifolia
in the European Alps using molecular methods. Restriction fragment length polymorphism
(RFLP) analysis of cpDNA of S. oppositifolia, partly sampled from potential nunatak areas,
detected two common European haplotypes throughout the Alps, while three populations
harboured two additional, rare haplotypes. RAPD analysis confirmed the results of former
studies on S. oppositifolia; high within, but low among population genetic variation and no
particular geographical patterning. Some Alpine populations were not perfectly nested in
this common gene pool and contained private RAPD markers, high molecular variance or
rare cpDNA haplotypes, indicating that the species could possibly have survived on icefree mountain tops (nunataks) in some parts of the Alps during the last glaciation. However, the overall lack of a geographical genetic pattern suggests that there was massive
immigration of cpDNA and RAPD genotypes by seed and pollen flow during postglacial
times. Thus, the glacial history of S. oppositifolia in the Alps appears to resemble closely
that suggested previously for the species in northern Europe.
Keywords: Alpine species, cpDNA RFLPs, phylogeography, Pleistocene glacial survival, RAPDs,
Saxifraga oppositifolia
Received 13 December 2001; revision received 10 April 2002; accepted 15 April 2002
Introduction
Recent research on the phylogeography and evolution of
Arctic and/or Alpine plant species has focused largely on
locations of refugia during Pleistocene glaciations and
migration routes during the postglacial period (Comes
& Kadereit 1998; Gugerli & Holderegger 2001). Much of
this research has centred on Arctic-Alpine plants with
Correspondence: Rolf Holderegger. ‡Present address: Division of
Ecological Genetics, WSL Swiss Federal Research Institute,
Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland. Fax:
+ 41 1 7392215; E-mail: [email protected]
© 2002 Blackwell Science Ltd
geographical distributions that often include the European Alps (e.g. Abbott et al. 1995; Brochmann et al. 1998;
Tollefsrud et al. 1998); however, detailed analysis of
populations from the Alps has often been lacking (but see
Bauert et al. 1998). Only recently has this omission begun to
be rectified (e.g. Stehlik et al. 2001a; Zhang et al. 2001; Kropf
et al. 2002).
Several studies on the phylogeography of Arctic species
have been conducted on the holarctic (Webb & Gornall
1989) purple saxifrage, Saxifraga oppositifolia (Abbott et al.
1995, 2000; Gabrielsen et al. 1997). An important question
was whether this species survived glaciation on ice-free
mountain tops (nunataks) within Pleistocene ice sheets or
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1410 R . H O L D E R E G G E R , I . S T E H L I K and R . J . A B B O T T
whether it re-immigrated from periglacial refugia during
the postglacial period. Gabrielsen et al. (1997) inferred high
levels of migration from clinal variation in random amplified polymorphic DNA (RAPD) data of S. oppositifolia in
Scandinavia and Svalbard. They concluded that potentially in situ surviving, resident genotypes were swamped
by massive re-immigration of periglacial genotypes after
glaciation and that glacial survival in refugia within this
region did not matter. Subsequently, a restriction fragment
length polymorphism (RFLP) analysis of chloroplast DNA
(cpDNA) was conducted on individuals from almost the
entire circumpolar distribution range of S. oppositifolia
(Abbott et al. 2000). High cpDNA diversity was found in
Arctic areas known to have remained ice-free during the
last ice age (e.g. Alaska and the Taymyr region of north–
central Siberia), indicating that these were major refugia
for the species during this period. In contrast, areas that
had been glaciated contained only a few common cpDNA
haplotypes. Abbott et al. (2000) concluded that glacial refugia for S. oppositifolia most likely existed in ice-free areas to
the north, south, east and west of the ice sheets that covered
much of North America and a large part of Eurasia during
the Pleistocene glaciations. However, their results, like
those of Gabrielsen et al. (1997), indicated that the offspring
of plants, which may have survived on nunataks within
glaciated regions, have not contributed significantly to the
presence of the species in these areas today.
Based on pollination experiments and high RAPD variation within local populations, S. oppositifolia is mainly outbred in the Alps (Gugerli 1997; Gugerli et al. 1999), where
it is widely distributed, occurring commonly between 1800
and 3800 m above sea level (a.s.l.). It is also found far below
the timberline in Alpine valleys and sometimes as low as
580 m a.s.l. (Kaplan 1995). At the other altitudinal extreme,
S. oppositifolia belongs to those few Alpine plants, which are
able to grow on the highest summits at more than 4000 m
a.s.l. The species’ habitats are rather diverse, ranging from
outcrops, rocks, glacier forefields and moraines to gravel,
boulder slopes, or landslides (Webb & Gornall 1989). On
wind-swept ridges, S. oppositifolia can survive winter temperatures of −40 °C (Kaplan 1995). The species occurs on
both siliceous and calcareous bedrock.
The wide distribution of S. oppositifolia, both in longitudinal and altitudinal terms, as well as its wide ecological
amplitude designate this species as a candidate for in situ
glacial survival within the Alps. This leads to the biogeographic question concerning where Alpine S. oppositifolia
might have survived the ice ages. Did it persist on isolated
northern Alpine or on high Alpine, ice-free nunataks, or
did it re-immigrate from major southern refugia or even
from the periglacial lowlands? Because S. oppositifolia is
distributed almost throughout the Alpine arc and because
fossils of the species are known from the adjacent lowlands
from late glacial or early postglacial periods only (Kaplan
1995; Burga & Perret 1998), it has not been possible to state
explicitly the putative locations of glacial refugia or nunatak areas of S. oppositifolia within the Alps. Thus, the principal question addressed in the present study was: is it
possible using molecular methods to deduce the Pleistocene history of a widespread and common Alpine species
such as S. oppositifolia, whose present-day ecology and distribution do not allow any exact hypotheses on its glacial
history in the Alps?
Materials and methods
The species
Saxifraga oppositifolia L. (Saxifragaceae) is a long-lived,
evergreen, runner-forming cushion plant (Webb & Gornall
1989). It occurs almost throughout the European mountains, where it is a member of a group of closely related
taxa. In the Alps, this group comprises S. biflora, S. blepharophylla, S. oppositifolia, S. retusa and S. rudolphiana (Kaplan
1995). Saxifraga oppositifolia is diploid (2n = 26) within its
Alpine distribution (Küpfer & Rais 1983). Its stems usually
bear single, outbred protandrous flowers, but selfing is
possible (Gugerli 1997).
Sampling
Former molecular genetic studies on S. oppositifolia from
the Alps either did not sample in potential nunatak areas
(Abbott et al. 2000; Fig. 1) or covered only a limited geographical region (Canton of Grisons, Switzerland; Gugerli
et al. 1999).
The middle part of the Alps is particularly suitable for
studies on the phylogeography and glacial history of
Alpine plants (Stehlik 2000). This part of the Alpine arc has
potentially been colonized by (re-)immigration waves
from western, southern and/or eastern major glacial refugia of Alpine plants, e.g. from the Grajic, Cottic, Bergamasc, or eastern Austrian Alps. In addition, peripheral
northern Alpine nunataks (e.g. the mountains Vanil Noir
and Pilatus in Switzerland) and even high Alpine nunataks
(e.g. in the Penninic or Rhaetic Alps) are known in this part
of the Alps (Stehlik 2000). Therefore, we sampled leaf
material (dried in silica gel) from 13 populations of S.
oppositifolia from the middle, Swiss part of the Alps and
from one additional population each from the French
(Ecrins) and Italian Alps (Passo Tonale; Fig. 1). Nine of
these 15 populations were located in areas described in the
literature either as major southern refugia, northern Alpine
nunataks, or high Alpine nunataks (Stehlik 2000). The
other six populations were sampled at high elevations
between the above populations (Table 1, Fig. 1). No population included in this study had been part of any former
investigations on Alpine S. oppositifolia.
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G L A C I A L H I S T O R Y O F S A X I F R A G A O P P O S I T I F O L I A 1411
Fig. 1 Location of investigated populations
of Saxifraga oppositifolia from the middle
part of the Alps and distribution of cpDNA
haplotypes within populations based on
RFLP analysis using Southern hybridization. Large symbols represent populations
studied in this study (for abbreviations
see Table 1), while small symbols indicate
populations formerly studied by Abbott
et al. (2000). Populations TA, SP, SS, BR and
PC (grey shaded area) formed one of the
two groups used in the amovas; all other
populations formed the other group (Table 3,
Fig. 3). Solid, haplotype A; hatched, B;
dotted, D; shaded, R.
Table 1 Abbreviation and location of the investigated populations of Saxifraga oppositifolia from the Alps and designation of the sampling
sites in the biogeographic literature (Stehlik 2000)
Abbreviation
Location
Elevation
(m a.s.l.)
Co-ordinates
Designation
N
EV
CN
VV
TA
SP
SS
BR
LP
PC
AG
CT
FF
DF
BL
PP
Ecrins, France
Rochers de Nayes, Switzerland
Vanil Noir, Switzerland
Augstbordpass, Switzerland
Saas Fee, Switzerland
Seehorn, Switzerland
Ritterpass, Switzerland
Pilatus, Switzerland
Passo Cristallina, Switzerland
Gemsstock, Switzerland
Piz Tom, Switzerland
Fuorcla Faller, Switzerland
Flüelapass, Switzerland
Piz Lagalp, Switzerland
Passo Tonale, Italy
2680
1720
1680
2890
2640
2380
2400
1840
2600
2820
2360
2900
2400
2950
2750
06°11′00″E/44°49′05″N
06°58′45″E/46°27′19″N
07°08′03″E/46°32′36″N
07°45′10″E/46°12′32″N
07°56′37″E/46°05′16″N
08°06′48″E/46°10′58″N
08°08′58″E/46°18′18″N
08°15′06″E/46°58′44″N
08°29′48″E/46°28′15″N
08°36′41″E/46°36′02″N
08°40′29″E/46°32′53″N
09°35′12″E/46°27′37″N
09°56′50″E/46°45′05″N
10°01′27″E/46°25′53″N
10°38′15″E/46°18′35″N
major southern refugia
northern Alpine nunatak
northern Alpine nunatak
no particular designation
high Alpine nunatak
high Alpine nunatak
no particular designation
northern Alpine nunatak
no particular designation
no particular designation
no particular designation
high Alpine nunatak
no particular designation
high Alpine nunatak
major southern refugia
3/1/3
5/1/10
5/1/10
5/1/10
6/1/10
5/1/10
6/1/10
5/1/10
6/1/10
6/1/10
6/1/10
6/1/10
5/1/10
6/1/10
5/1/10
N, number of investigated individuals per population in the cpDNA RFLP analysis using Southern hybridization, in PCR-RFLP of cpDNA
and with RAPDs.
DNA extraction
RFLP analysis of cpDNA
Genomic DNA was extracted from 80 mg of dried leaf
material using the protocol of Whittemore & Schaal (1991)
plus modifications and cleaning steps given in Milne et al.
(1999). Total DNA was additionally cleaned with 7.5 m
ammonium acetate and a Wizard DNA clean up column
(Promega).
We followed strictly the protocol of Abbott et al. (2000),
details of which are given in Milne et al. (1999). We used the
same four principal probe × enzyme combinations, i.e.
mung bean MB3 × EcoRI, MB3 × BclI, MB7 × EcoRI and
MB7 × BclI. The haplotypes of some individuals were
additionally verified using the combination MB3 × DraI.
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1412 R . H O L D E R E G G E R , I . S T E H L I K and R . J . A B B O T T
Haplotype designation was the same as in Abbott et al.
(2000). Five to six individuals per population were investigated (except for population Ecrins; Table 1).
Since only four different haplotypes were detected in the
above analysis (see Results section), we examined the two
common haplotypes A and B further using polymerase
chain reaction (PCR)-RFLP. We selected one individual
per population (Table 1), comprising eight individuals
with haplotype A and seven individuals with haplotype B.
PCR reactions followed the protocol of Demesure et al.
(1995) with only minor modifications. Noncoding regions
of cpDNA were amplified with Taq polymerase A
(Promega) using the primer pairs trnH/trnK1, trnK1/
trnK2, psbC/trnS, psaA/trnS and trnM/rbcL (Demesure
et al. 1995). Each PCR product was digested with each of
five restriction enzymes (AluI, CfoI, HaeIII, HpaII and RsaI)
according to the manufacturer’s instructions (Promega).
RFLP profiles were analysed on 8% polyacrylamide gels
stained with ethidium bromide.
RAPD analysis
Ten individuals per population (except for population Ecrins; Table 1) were used in the RAPD analysis.
We followed the methods of Gabrielsen et al. (1997) as
closely as possible, using the same Taq polymerase and the
same make of thermocycler (MJ Research PTC 100/96).
Nevertheless, some modifications were necessary. We
used 0.75 U of Super Taq polymerase (HT Biotechnology)
with 1 ng genomic DNA per reaction volume, and the
thermocycler was programmed for 180 s at 94 °C, followed
by 40 cycles of 60 s at 94 °C, 60 s at 40 °C and 90 s at 72 °C.
Final extension lasted for 300 s at 72 °C. PCR products were
separated on 1.4% agarose gels and visualized with
ethidium bromide. We considered six of the 12 Operon
Technology primers used by Gabrielsen et al. (1997), A01,
A11, A19, F01, F05, F12, and all four primers used by
Gugerli et al. (1999), B04, B07, D02, D03, resulting in a total
of 10 primers. All individual RAPD profiles were verified
in at least two independent PCR amplifications.
Statistical analysis
For each of the two data sets, the cpDNA RFLP analysis
using Southern hybridization and the RAPDs, the presence
or absence of fragments was recorded in a binary matrix.
We applied a set of diversity measurements to the RAPD
data often used to determine potential glacial refugia (Stehlik
et al. 2001a; Widmer & Lexer 2001): i.e. diversity of multilocus phenotypes per population, M, number of private
(restricted to a single population) and rare (occurring in no
more than 10% of all individuals) RAPD markers per
population and, as a measure of genetic diversity, the molecular variance [SS/(N − 1), where N is the number of
individuals investigated per population, and SS is the sum
of squares obtained in amova; see below; Landergott et al.
2001]. Additionally, we applied the concept of nestedness
used in biodiversity research (Ganzhorn & Eisenbeiss
2001). The concept of nestedness represents a null model
that measures the order in presence–absence matrices, i.e.
randomness of deviations. The model assumes that there
existed a single source of species assemblage, that habitat
islands are uniform with no significant clinal ecological
gradients and that islands are geographically equally
isolated. For instance, species distribution patterns in
fragmented habitat islands often exhibit pronounced
nestedness (Atmar & Patterson 1993). Nestedness analysis
thus tests whether assemblages of species (marker bands
within populations in the present case) represent nested
subsets of a common species pool (gene pool). Species
assemblages (populations) not fitting the hypothesis of
nestedness, hence, by definition, differing from randomness, are referred to as ‘idiosyncratic’. Therefore, genetic
idiosyncrasy points to divergent biogeographic histories
of particular populations. The corresponding analysis
based on the above binary RAPD matrix was performed
using nestedness temperature calculator (Atmar &
Patterson 1993). Significance was tested with 100 Monte
Carlo permutations.
A correspondence analysis using Euclidean distances
among individual multiband phenotypes and a neighbour
joining clustering of pairwise FST values among populations taken from amova (see below) were calculated for the
RAPD data set with ntsys-pc 2.02i (Rohlf 1997). The significance of clusters in the neighbour-joining tree was tested
by 100 bootstrap replicates in paup 4.0b3a (Swofford 1999)
and by a co-phenotic correlation coefficient calculated with
ntsys pc 2.02i (Rohlf 1997). As an estimate of isolation by
distance, a Mantel test was used to investigate the relationship
between genetic Euclidean distance and geographic distance
among samples with r package 4.0 (Casgrain & Legendre
1999) and 999 permutations applied for significance testing.
Convential amovas were performed on both the cpDNA
RFLP and the RAPD data sets. amovas were based on pairwise squared Euclidean distances between multiband phenotypes and were performed using amova prep (Miller 1998)
and amova 1.55 (Excoffier 1993). For hierachical variance
partitioning, populations were grouped according to the
two main clusters found in the neighbour-joining tree
(see above). Significance tests were based on 999 permutations. To quantify the relative proportions of pollen and
seed migration, we calculated measurements of overall
genetic differentiation among populations, FST, for both the
maternally inherited cpDNA markers and the mainly
nuclear, biparentally inherited RAPD markers using corresponding variation components from amova. The relative
rates of gene flow through pollen or seed were then determined according to Ennos (1994).
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G L A C I A L H I S T O R Y O F S A X I F R A G A O P P O S I T I F O L I A 1413
Fig. 2 Scatter plot showing the first two
axes of a correspondence analysis using
Euclidean distances based on RAPD profiles between individuals of Saxifraga
oppositifolia from the Alps (for population
abbreviations see Table 1). Western populations are indicated by solid symbols,
eastern populations by open symbols.
Results
RFLP analysis of cpDNA
RFLP analysis of cpDNA using the Southern hybridization
method revealed four different haplotypes in Saxifraga
oppositifolia from the middle part of the Alps (Fig. 1).
Haplotype A was most common, occurring in all except
two populations and having an overall frequency of 0.71.
Haplotype B, differing from haplotype A in an MB7 ×
EcoRI site mutation, was less abundant with a frequency of
0.25 but still widespread (Fig. 1). There was no obvious
geographical pattern in the distribution of haplotypes A
and B. Two rare haplotypes were also found: (i) haplotype
D (frequency 0.03), characterized by a MB7 × BclI site
mutation from haplotype A, occurred in the Seehorn and
Gemsstock populations, and (ii) haplotype R (frequency
0.01) was detected in the Flüelapass population only
(Fig. 1). Haplotype R represented a ≈ 80 base-pair (bp)
MB3 length mutation of haplotype A and was not found in
the circumpolar study on S. oppositifolia by Abbott et al.
(2000). Two populations sampled from potential major
southern refugia (Table 1), Ecrins and Passo Tonale, were
monomorphic either for haplotype A or B. Populations
monomorphic for haplotype A also occurred in the central
Alps. One of the two most diverse populations, each
harbouring three different haplotypes, stemmed from a
well known high Alpine nunatak area (Seehorn population) in the Valais; the other represented an Alpine population not specifically designated in the biogeographic
literature (Flüelapass; Table 1). In the potential northern
Alpine nunataks, the Rochers de Nayes, Vanil Noir and
Pilatus populations (Table 1), both common haplotypes A
and B were found (Fig. 1).
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1409–1418
PCR-RFLP analysis of cpDNA using 25 primer pair ×
restriction enzyme combinations did not generate further
haplotypic variation. It did not even distinguish haplotype
A from haplotype B. The low level of haplotype diversity
resolved in the Southern hybridization analysis was thus
fairly robust.
RAPD fingerprinting
The 10 RAPD primers yielded 154 scorable marker bands
of which only three were monomorphic in the whole data
set. Genetic diversity in Alpine S. oppositifolia was high;
each of the 143 individuals sampled exhibited a unique
RAPD multiband phenotype (M = 100%).
No consistent pattern among the investigated populations was found with respect to the different statistical
measurements used to detect glacial refugia or nunatak
areas. Populations that were characterized by the occurrence of private markers or a high number of rare markers
often had low molecular variances or were perfectly nested
within the whole sample set. Thus, they did not show specific peculiarities in their RAPD profiles (Table 2). Qualitatively, none of the three populations that had private
markers (Seehorn, Passo Cristallina and Piz Tom) was
characterized by a particularly high number of rare markers,
and only one of them (Passo Cristallina) was defined as idiosyncratic (overall nestedness: T = 44.6, P ≤ 0.001; Table 2).
Quantitatively, molecular variance measurements ranged
within narrow limits (14.33–20.30; Table 2). Only the
Augstbordpass population harboured both a high number
of rare markers and high molecular variance. Nevertheless,
genetic diversity in this population was a common subsample of the overall gene pool, i.e. the population was not
idiosyncratic (Table 2). In more detail, the four populations
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1414 R . H O L D E R E G G E R , I . S T E H L I K and R . J . A B B O T T
Pop.
Private markers
Rare markers
Molecular variance
Idiosyncratic population
EV*
CN
VV
TA
SP
SS
BR
PC
LP
AG
CT
FF
DF
BL
PP
0
0
0
0
0
2
0
1
0
0
1
0
0
0
0
2
29
19
30
23
20
22
25
17
21
19
27
16
23
29
14.33
15.91
17.91
20.09
18.14
19.86
17.12
20.30
19.08
18.93
20.06
13.37
18.86
16.84
16.93
no
no
no
no
no
no
yes
yes
no
yes
no
no
no
no
yes
Table 2 Statistical measurements of genetic diversity based on RAPD data in
populations of Saxifraga oppositifolia from
the Alps (for population abbreviations see
Table 1), i.e. the number of private markers
(only occurring in a single population), the
number of rare markers (occurring in up to
10% of all individuals), molecular variance
(from amova) and idiosyncratic (i.e. not
perfectly nested) populations based on
nestedness analysis
Pop., population.
*Only three individuals were investigated in population EV, while 10 individuals were
analysed in all other populations.
sampled from high Alpine nunatak areas (Saas Fee, Seehorn, Fuorcla Faller and Piz Lagalp; Table 1) did not stand
out from the whole data set, although the Seehorn population exhibited two private fragments and relatively high
molecular variance (Table 2).
Correspondence analysis revealed a genetic transgression from the western towards the eastern populations
along the first axis, while separation along the second axis
was low (Fig. 2). The first two axes explained only a small
amount of the total variation in the data set (8.68% and
7.50%). Concordantly, the overall Mantel test value of
rm = 0.137 (P ≤ 0.001) indicated a significant, though low
increase of genetic distance with increasing geographic
distance among samples. The shift along the first axis of the
correspondence analysis could nevertheless not be interpreted in a strictly clinal way, as individuals from the most
western populations (Table 1) were situated near to the
centre of the scatter plot. In particular, we could not find
any clearly separated entities or groups in the correspondence analysis (Fig. 2). Rather, the correspondence analysis
gave the picture of a large common nuclear gene pool, only
slightly changing from the west to the east.
The neighbour-joining clustering based on pairwise FST
values between populations revealed two major clusters
(Fig. 3). One cluster comprised four populations (Seehorn,
Ritterpass, Augstbordpass and Saas Fee) from a well
known high Alpine nunatak area in the Valais (Table 1),
and the adjacent population of Passo Cristallina in the
Ticino (Figs 1, 3). All other populations were placed in a
second cluster with eastern, western and northern Alpine
populations (Fig. 3). However, there was no bootstrap support above 50% for any of the branchings, and the low co-
Fig. 3 Neighbour-joining tree based on pairwise FST values
between populations (taken from amova) of RAPD data on
Saxifraga oppositifolia from the Alps (for population abbreviations
see Table 1; western populations are indicated by solid symbols,
eastern populations by open symbols; see Fig. 2). Bootstrap
support was < 50% for all bifurcations; co-phenetic correlation
coefficient: r = 0.50.
phenetic correlation coefficient (r = 0.50) indicated that the
overall clustering was not robust (Rohlf 1997).
Comparison of RAPD and RFLP data
amovas on RAPD and on cpDNA data led to a generally similar partitioning of genetic variance components
(Table 3). Very low, and in the case of cpDNA nonsignificant, genetic variation was detected among groups
of populations (4.4% for RAPDs and 4.14% for cpDNA
RFLPs; Table 3). This pointed to either no or, at best, weak
regional differentiation in the two data sets (groups of
populations were defined according to the two main
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Marker
Source of variation†
RAPDs
among groups
among populations within groups
within populations
cpDNA RFLP
d.f.
SS
Percentage of
variance component
1
13
103.96
524.62
4.40***
10.82***
128
2366.77
84.98***
among groups
among populations within groups
1
13
1.82
12.38
4.14ns
17.20***
within populations
65
28.57
78.58***
Table 3 amova of RAPD and cpDNA
RFLP data on Saxifraga oppositifolia from the
Alps
***P ≤ 0.001; ns not significant.
†For grouping of populations see Figs 1 and 3.
clusters in the neighbour-joining tree; Fig. 3). This was also
evident from the rather low, similar measurements of
overall genetic differentiation among populations, although
the latter was somewhat higher in cpDNA (FST = 0.213,
P ≤ 0.001) than in the RAPD data (FST = 0.152, P ≤ 0.001).
Most genetic variation was found within populations
(84.98% in RAPDs and 78.58% in cpDNA RFLPs), while the
genetic variation among populations within groups
differed somewhat more between the two marker types
(10.82% in RAPDs and 17.20% in cpDNA RFLPs, Table 3).
The absolute estimate of the relative proportion of pollen
and seed migration, using the above FST measurements,
equalled 0.49. This indicates that gene flow by the two dispersal agents were of similar magnitude (Whitlock &
McCauley 1999).
Discussion
Despite the application of a whole set of statistical tools
including nestedness analysis, neither the cpDNA RFLP
nor the RAPD data of Saxifraga oppositifolia from the middle
part of the Alps gave unequivocal evidence on where
representatives of the species in this region survived the
Pleistocene glaciations, i.e. whether plants re-immigrated
from periglacial refugia or whether populations survived
in situ within the Alps. Moreover, it was not possible to
deduce potential re-colonization routes.
Evidence from cpDNA data
Two widespread, common cpDNA RFLP haplotypes, A
and B, were found in Alpine populations of S. oppositifolia.
Two additional, rare haplotypes also occurred in just three
populations (Fig. 1). In their circumpolar study on S.
oppositifolia, Abbott et al. (2000) found only haplotypes
A and B among the 67 individuals surveyed from the
Alps (Fig. 1). The frequencies of these haplotypes given
in Abbott et al. (2000), i.e. A, 0.87 and B, 0.13, were not
significantly different from those recorded in the present
investigation (χ2 = 6.079, P = 0.108). In both studies, haplotypes A and B were found to occur throughout the Alps
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1409–1418
without showing any particular spatial pattern (Fig. 1).
Abbott et al. (2000) did not find any haplotypes other
than A and B in Europe including northwestern Russia,
Svalbard and Iceland.
It was not possible to differentiate further the two
cpDNA haplotypes A and B using PCR-RFLPs. Since these
two haplotypes are common throughout Europe, they cannot be used to deduce the existence or locations of potential
glacial refugia in the Alps or neighbouring regions. Both
haplotypes could have survived on northern Alpine,
almost periglacial nunataks and subsequently re-colonized
the Central Alps. Alternatively, they could have resisted
the ice ages on high Alpine nunataks in the central chains
and expanded their distribution after the retreat of the glaciers. Equally, they might have re-immigrated after glaciation from major southern refugia. Even the lowlands north
of the Alps could have served as refugia, because late glacial or early postglacial fossils of S. oppositifolia have been
found there (Burga & Perret 1998).
Do the two rare cpDNA haplotypes D and R from the
potential nunatak Seehorn population and from the two
Gemsstock and Flüelapass populations allow more specific
conclusions? Haplotype D, together with A and B, belongs
to a Eurasian clade (Abbott et al. 2000). Prior to this
study, haplotype D had only been recorded in the Siberian
Taymyr region. Disregarding potential homoplasy, the
occurrence of this haplotype in the Alps could point to a
glacial refugium there. However, the question remains
why this haplotype did not disperse further after glaciation
and is nowadays so rare and geographically restricted in
the Alps. The conclusions of Gabrielsen et al. (1997) for nordic S. oppositifolia, that the genetic traces of genotypes
potentially having survived in situ were erased by massive
immigration of periglacial genotypes after glaciation,
appear to also fit the patterns of the present cpDNA
haplotype distribution in the Alps. Haplotype R was only
found once in the Alps, and it is not known from any
other population (Abbott et al. 2000). Assuming an average
mutation rate of cpDNA (Hartl & Clark 1997), haplotype
R might simply represent a local, perhaps even postglacial, length mutation of haplotype A, thus, not being
MEC_1548.fm Page 1416 Tuesday, July 16, 2002 11:21 PM
1416 R . H O L D E R E G G E R , I . S T E H L I K and R . J . A B B O T T
particularly informative with respect to the glacial history
of S. oppositifolia.
A high level of postglacial seed dispersal, which could
have extinguished evidence of glacial refugia within the
Alps, is suggested by the finding that gene flow measurements for both cpDNA and mainly nuclear RAPDs were of
the same magnitude. Usually, estimates of gene flow via
seed, based on cpDNA variation, are much lower than
those via pollen and seed, based on nuclear variation
(Ennos 1994). Note, however, that Nm values derived from
molecular genetic FST values should only be treated as
rough estimates of gene flow (Whitlock & McCauley 1999).
Although no attempt has been made to obtain a direct
measure of seed dispersal in S. oppositifolia so far, the species’
seeds are thought to be easily dispersed by wind (Kaplan
1995). Accordingly, Von Flüe et al. (1999) recorded the rapid
formation of morphological and isozyme variation within
a recently colonized Alpine population of S. oppositifolia,
which they attributed to recurring seed migration at the
landscape level and the mainly outcrossing breeding system of the species (Gugerli 1997).
Evidence from RAPD markers
In accordance with the studies of Gabrielsen et al. (1997)
and Gugerli et al. (1999), we found high amounts of RAPD
variation in S. oppositifolia. By using 10 RAPD primers,
a unique multiband genotype was assigned to every
individual investigated in the present study. Gabrielsen
et al. (1997) detected 86% of the genetic variation within
populations of S. oppositifolia from southern Norway (an
area comparable in size with that of the present study),
which is almost identical to the corresponding 85% found
in our Alpine data set (Table 3). Gugerli et al. (1999)
observed 95% RAPD variation among populations in a
geographically restricted Alpine data set on S. oppositifolia.
It is possible that the differences within the three studies were partly caused by the well-known laboratory
dependence of RAPD markers (Nybom & Bartish 2000).
While Gabrielsen et al. (1997) only considered a mean of
2.9 polymorphic markers per primer, a rather conservative approach, Gugerli et al. (1999) used 21 polymorphic
markers per primer, whereas we recorded 15.1 polymorphic markers per primer. Despite these methodical
differences, we interpret the low genetic variation among
regions of 4.4% (Table 3) as a lack of distinct geographical
patterns, which is in line with the interpretation of a
corresponding value of 8.58% by Gabrielsen et al. (1997).
RAPD studies on S. oppositifolia all failed to find distinct
geographical patterns. While Gabrielsen et al. (1997) found
weak upgma clustering of individuals into geographical
regions, our study showed that individuals could be
assigned to a continuous Alpine gene pool because of (i) a
rather low among-population component of genetic vari-
ation as compared with other species (Nybom & Bartish
2000), (ii) nestedness of most populations, (iii) an only
slight transgression of genotypes from the west to the east
as shown in the correspondence analysis (Fig. 2) and (iv) a
low, though significant, Mantel correlation between geographical and genetic distances pointing to only weak isolation by distance.
Nevertheless, a relatively distinct cluster of five populations, four from the central Alpine Valais and one from an
adjacent high Alpine area, was detected in our survey
(Fig. 3). Several regions of the Penninic Valais represent
well-known nunatak areas based on floristic richness or
numbers of Alpine endemites (Stehlik 2000). Although the
above group of populations was statistically neither supported by bootstrap values in the neighbour-joining tree
(Fig. 3) nor by a co-phenetic correlation analysis, one of
these populations, Seehorn, harboured three different
cpDNA haplotypes (including type D, see above), contained two private RAPD markers and showed comparably high molecular variance (Table 2). Whether these
findings indicate in situ glacial survival of S. oppositifolia on
this putative high Alpine nunatak is unclear, bearing in
mind that the different estimates of RAPD diversity
applied in this study did not generally identify genetically
unique populations.
In summary, the present study revealed a similarly high
level of RAPD variation and a similar genetic variance partitioning to former studies on S. oppositifolia. There were no
distinct geographical patterns of RAPD variation, although
populations from the central Alpine Valais exhibited some
peculiarities, which might be interpreted as weak evidence
for in situ glacial survival of the species within the Alps.
Conclusions
In the present study, both cpDNA RFLP and RAPD data
on Alpine S. oppositifolia showed low variation among
populations and regional groups of populations, but high
within-population variance components (Table 3). Both
data sets provided, at best, weak evidence for in situ glacial
survival of S. oppositifolia within the Alps. The strongest
point in favour of survival on high Alpine nunataks was
the occurrence of two rare cpDNA haplotypes, one of them
hitherto only known from Siberia (Abbott et al. 2000). The
present study thus failed to re-construct the Alpine
Pleistocene history of this common and widespread ArcticAlpine plant species; an which is important result with
regard to the growing body of phylogeographic studies on
mountain plants (Gugerli & Holderegger 2001).
In general, the phylogeographic signal detected in haplotypic cytoplasmic DNA in plants, as revealed by RFLPs or
sequence analysis, has been found to be much stronger
than that detected in nuclear DNA using various techniques such as isozymes, RAPDs, amplified fragment
© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1409 – 1418
MEC_1548.fm Page 1417 Tuesday, July 16, 2002 11:21 PM
G L A C I A L H I S T O R Y O F S A X I F R A G A O P P O S I T I F O L I A 1417
length polymorphisms (AFLPs) or inter-simple sequence
repeats (ISSRs) (for nuclear sequences see Hare 2001). This
is because the former does not recombine and is usually
uniparentally inherited, while the latter is recombining
and distributed both by seed and pollen (Schaal et al. 1998;
Newton et al. 1999). Since gene flow by pollen is usually
much higher than by seed (Ennos 1994), historical geographical patterns tend to be erased more rapidly for
nuclear markers than for cytoplasmic markers.
In contrast to the present investigation, some recent
studies on Alpine plants using both cpDNA analysis and
nuclear DNA markers such as isozymes or AFLPs have
succeeded in proving the long-standing hypothesis of high
Alpine nunataks (e.g. Draba aizoides: R. Füchter & A. Widmer,
ETH Zurich, unpublished data; Eritrichum nanum: Stehlik
et al. 2001a; Stehlik et al. unpublished data; also see Stehlik
et al. 2001b). It is feasible that such proof is more difficult to
obtain for a widespread, common and nonspecialist Alpine
species such as S. oppositifolia.
In conclusion, we could neither prove nor falsify the
hypothesis of in situ glacial survival of S. oppositifolia on
Alpine nunataks. It is possible that what happened in the
Alps was similar to what may have occurred in northern
Europe (Gabrielsen et al. 1997), i.e. resident genotypes surviving glaciation in situ were swamped by massive immigration of periglacial genotypes after glaciation.
Acknowledgements
We thank Maria von Balthazar, Kurt Eichenberger, Basil Holderegger
and Nadine Müller for help during sampling, Anette Becher,
David Forbes, Amanda Gillies, Richard Milne and Lisa Smith for
their help in the laboratory and Urs Landergott for help with
statistical analyses. Felix Gugerli, Urs Landergott, Alex Widmer
and two anonymous reviewers provided valuable comments on
the manuscript. Financial support by the Swiss National Science
Foundation, the Julius Klaus Foundation for Genetic Research and
the Novartis Foundation to RH is greatly acknowledged.
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© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 1409 – 1418