Biological Journal of the Linnean Society, 2009, 97, 403–412. With 2 figures
High mountains of the Japanese archipelago as refugia
for arctic–alpine plants: phylogeography of Loiseleuria
procumbens (L.) Desvaux (Ericaceae)
HAJIME IKEDA1*, KEI SENNI2, NORIYUKI FUJII3 and HIROAKI SETOGUCHI1
1
Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho,
Sakyo-ku, Kyoto 606-8501, Japan
2
Makino Herbarium, Graduate School of Science, Tokyo Metropolitan University, Hachioji,
Tokyo 192-0397, Japan
3
Graduate School of Science and Technology, Kumamoto University, Kurokami2-39-1, Kumamoto,
Kumamoto 860-8555, Japan
Received 8 September 2008; accepted for publication 13 September 2008bij_1177
403..412
According to previous phylogeographic studies, high mountains at low latitudes are important areas for the study
of the evolutionary history of arctic–alpine plants in surviving the Pleistocene climatic oscillations. To evaluate this
hypothesis, we elucidated the genetic structure of the arctic–alpine plant, Loiseleuria procumbens, in the Japanese
archipelago, which corresponds to one of the southernmost limits of its distribution, using 152 individuals from 17
populations that covered the entire distribution of the Japanese archipelago and Sakhalin, in addition to samples
from Sweden. Based on 854 bp of chloroplast DNA, we detected eight haplotypes. Along with haplotype distribution, strong genetic differentiation between populations in central and northern Japan was elucidated by a
neighbour-joining tree (100%) and spatial analysis of molecular variance (79%), which is consistent with other
alpine plants in Japan, regardless of the species’ range. In addition, the southernmost populations from northern
Japan showed specific genetic structure, although the remaining areas of northern Japan and Sakhalin harboured
an homogenous genetic structure. Our results suggest that the populations in central Japan persisted for a long
time during the Pleistocene climatic oscillation and that genetic divergence occurred in situ, supporting our
hypothesis in conjunction with a previous study of another arctic–alpine plant, Diapensia lapponica subsp.
obovata. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412.
ADDITIONAL KEYWORDS: alpine plants – cpDNA – pleistocene climatic oscillations – SAMOVA.
INTRODUCTION
Pleistocene climatic oscillations highly influenced the
current ranges of species, resulting in genetic heterogeneity throughout their ranges that can be elucidated using molecular phylogeographic studies (Avise,
2000; Hewitt, 2000). Comparison of intraspecific geographic structure amongst various species can reveal
its consistency across biota, from which the history of
the flora or fauna may be inferred because of the
influence and subsequent range shifts that extend
*Corresponding author.
E-mail: [email protected]
not only across the individual species, but also for
regional biota as a whole. This comparative phylogeographic approach has been applied particularly to
European flora and detailed histories of range shifts
in deciduous trees and alpine plants have been
inferred (e.g. Petit et al., 2003; Schönswetter, Stehlik
& Tribsch, 2005). In deciduous tree taxa, most species
survived in southern refugia during the glacial
periods and formed their current distribution by
northward recolonization during the postglacial warm
period (Lumaret et al., 2002; Palmé & Vendramin,
2002; Petit et al., 2002; Grivet & Petit, 2003), whilst
several cold-adapted species could also survive at
higher latitudes during climatic oscillations (Palmé,
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
403
404
H. IKEDA ET AL.
Semerikov & Lascoux, 2003; Rendell & Ennos, 2003).
Furthermore, European alpine plants survived in
peripheral glacial areas and/or in islands on glaciers
(nuntak) during the last glacial period (Schönswetter
et al., 2005).
In contrast to the detailed histories of European
flora, less is known about the consistent geographic
structure of arctic–alpine plants. Long-distance dispersal throughout the Circum-Arctic (Abbott & Brochmann, 2003; Alsos et al., 2007) and the existence of
refugia at higher latitudes (Abbott et al., 2000) has
made genetic structures inconsistent across species,
resulting in inferences of the species’ specific history
(Després, Loriot & Gaudeul, 2002; Schönswetter
et al., 2003; Schönswetter, Popp & Brochmann, 2006).
However, recent studies have revealed that populations of arctic–alpine plants located on high mountains at low latitudes, which correspond to the
southernmost limits of their distribution, harbour a
distinct genetic structure from those in other regions
(e.g. European Alps, Rocky Mountains and East
African mountains; Alsos et al., 2005; Albach, Schönswetter & Tribsch, 2006; Skrede et al., 2006; Ehrich
et al., 2007; Eidesen et al., 2007), although the genetic
structure of arctic–alpine plants on southern mountains has not been much focused. This specific genetic
structure may indicate persisting populations over
a long period of time, suggesting that arctic–alpine
plants survived the Pleistocene climatic oscillations in
mountainous regions located within the southernmost
limits of their distribution and subsequently diverged
genetically in those areas. In East Asia, this hypothesis was confirmed with Diapansia lapponica subsp.
obovata in the Japanese archipelago (Ikeda et al.,
2008a). Nevertheless, because that study used lowlevel variations of chloroplast DNA (cpDNA), stochastic variance following demographic changes in
populations may have resulted in the current phylogeographic structure (Hey & Machado, 2003; Stehlik,
2003). Therefore, further phylogeographic investigations of arctic–alpine plants in this region could
strengthen the hypothesis.
The Japanese archipelago is located at the eastern
edge of Asia and harbours alpine flora that represents
one of the southernmost boundaries of arctic–alpine
plants. The alpine flora comprises 440–580 species
(Shimizu, 1983) and extends throughout the high
mountains between 1500 and 2600+ m above sea level
from central to northern Japan. Nearly half of these
species are also distributed in Arctic regions [e.g.
Dryas octopetala (Rosaceae), Loiseleuria procumbens
(Ericaceae), Diapensia lapponica (Diapensiaceae)];
therefore, the alpine plants in Japan are thought to
have originated from Arctic plants that migrated to
the archipelago during glacial periods (Koidzumi,
1919; Toyokuni, 1981).
Previous phylogeographic studies (Fujii et al., 1997,
1999; Senni et al., 2005; Fujii & Senni, 2006; Ikeda
& Setoguchi, 2006; Ikeda et al., 2006; Ikeda &
Setoguchi, 2007; Ikeda et al., 2008a, b, c) detected
strong genetic differentiations between populations in
central and northern Japan in most species investigated, including arctic–alpine Diapensia lapponica
subsp. obovata. Therefore, regardless of the current
range of species, alpine plants in Japan experienced a
history of vicariance between populations in central
and northern Japan during the Pleistocene climatic
oscillations. However, arctic–alpine plants were distributed throughout large areas with little morphological difference and could disperse over long
distances. Thus, a range-expansion event during the
last glacial and postglacial periods would be sufficient
for this distribution to occur. In this scenario, homogenous genetic structure could be detected throughout
the range of a species, including the limits of that
distribution. Given that the phylogeographic structure of arctic–alpine plants in the Japanese archipelago was elucidated only in D. lapponica subsp.
obovata, further phylogeographic studies of other
arctic–alpine plants of the region are needed to
describe a consistent history of the high mountains as
one of the southernmost distribution limits of arctic–
alpine plants.
Loiseleuria procumbens (L.) Desvaux is an arctic–
alpine plant that is distributed throughout the high
mountains of the Northern Hemisphere and the Arctic
(Yamazaki, 1993). The genus Loiseleuria includes only
this species and belongs to the tribe Phyllodoceae of
the family Ericaceae. This species is frequently found
amongst arctic–alpine plants in Japan along with D.
lapponica subsp. obovata and forms cushions with
Arcterica nana and D. lapponica subsp. obovata, especially on the mountains of northern Japan. Given that
capsules may not disperse seeds across long distances,
L. procumbens has not developed any adaptation to
long-distance seed dispersal.
In this study, we attempted to evaluate the hypothesis that high mountains at low latitudes which also
correspond to the southernmost distribution limits of
arctic–alpine plants were important areas for the
survival of arctic–alpine plants during the Pleistocene
climatic oscillations. In particular, we hypothesized
that populations of L. procumbens persisted on the
high mountains of the Japanese archipelago and subsequent genetic divergence occurred in situ. We elucidated and compared the phylogeographic structure
of L. procumbens to previous studies of D. lapponica
subsp. obovata as well as other Japanese alpine
plants. Thereby, the genetic structure of L. procumbens was also assessed to determine whether it was
consistent across species regardless of the widespread
species distribution.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
PHYLOGEOGRAPHY OF ARCTIC–ALPINE PLANTS IN JAPAN
MATERIAL AND METHODS
SAMPLING
Leaf material of L. procumbens was sampled from 16
populations in the Japanese archipelago, as well as
one population each from Sakhalin and Sweden
(Table 1); the total number of samples was 160 individuals. All leaf samples were collected from individuals as distant from each other as possible, dried in
silica gel and stored at 23–25 °C (room temperature).
Voucher specimens of all populations were deposited
in the Makino Herbarium of Tokyo Metropolitan University (MAK) and in the herbarium of Kyoto University (KYO).
DNA
EXTRACTION AND SEQUENCING
The dried leaf materials were freeze-dried using
liquid nitrogen and then ground into fine powder.
After the polysaccharides were removed from the
sample with HEPES buffer (pH 8.0; Setoguchi &
Ohba, 1995), DNA was extracted by the cetyl trimethylammonium bromide (CTAB) method (Doyle &
Doyle, 1990). The extracted DNA was dissolved in
100 mL TE buffer and used as a template for the
polymerase chain reaction (PCR). Two pairs of
405
primers were used to amplify the trnL intron (Taberlet et al., 1991) and trnS–G spacers (Hamilton, 1999).
PCR amplification was conducted in a total reaction
volume of 25 mL containing 14.7 mL of autoclaved ionexchanged water, 4 mL of 2.5 mM deoxyribonucleotide
triphosphate (dNTP) mixture, 5 mL of 10 ¥ Ex Taq
Buffer (Takara Ex Taq; Takara, Kyoto, Japan),
0.625 U of Ex Taq (Takara), 0.2 mM of each primer and
1.25 mL of DNA. Amplification was performed with an
initial denaturation of 2 min at 94 °C followed by 30
cycles of denaturation of 1 min at 94 °C, annealing of
1 min at 50 °C and an extension of 1 min at 72 °C.
Following amplification, products were visualized
on 0.5% tris-acetate EDTA (TAE)–agarose gels
stained with ethidium bromide. PCR products
were sequenced using the standard methods of
the BigDyeTM Terminator Cycle Sequencing Ready
Reaction kit (Applied Biosystems, Foster City, CA,
USA) using three primers [trnL(c), Taberlet et al.,
1991; trnS, Hamilton, 1999] on an ABI Model 3100
Genetic Analyzer (Applied Biosystems).
DATA
ANALYSIS
All sequence data were analysed and aligned using
an Auto Assembler (Applied Biosystems). cpDNA
Table 1. Number of populations, location, coordination, numbers of studied individuals (N) and haplotype composition of
18 populations of Loiseleuria procumbens
Haplotype compositon
No.
Location
Coordination
1
Sakhalin
46°50′N/142°40′E
9
3
6
Northern Japan
2
Taisetsusan
3
Furanodake
4
Poroshiridake
5
Shokanbetsudake
6
Hakkoudasan
7
Gassan
8
Iidesan
43°38′N/142°55′E
43°23′N/142°38′E
42°43′N/142°41′E
43°43′N/141°31′E
40°39′N/140°52′E
38°33′N/140°1′E
37°51′N/139°41′E
9
7
9
2
10
10
9
1
8
7
9
2
10
Central
9
10
11
12
13
14
15
16
17
36°9′N/136°46′E
36°45′N/137°45′E
36°19′N/137°43′E
35°47′N/137°48′E
35°53′N/137°29′E
36°6′N/137°33′E
35°59′N/138°22′E
35°27′N/138°9′E
35°43′N/138°11′E
10
10
7
10
10
10
10
10
10
Honshu
Hakusan
Shiroumadake
Jyonendake
Kisokomagatake
Ontakesan
Norikuradake
Yatsugatake
Akaishidake
Senjyoudake
Sweden
18
Kebnakaise, Sweden
Total No. of samples
18°30-40′N/67°50-55′E
N
A
8
160
A′
B
C
D
D′
E
G
10
9
10
8
7
2
10
3
8
7
1
1
10
6
10
4
40
3
7
36
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
23
42
1
8
8
406
H. IKEDA ET AL.
ing the methods of Pons & Petit (1995, 1996) using
the program PERMUT (http://www.pierroton.inra.fr/
genetics/labo/Software/Permut). Parameters included
the mean within-population gene diversity (hS), total
gene diversity (hT) and the coefficient of genetic differentiation over all populations (GST), as well as the
equivalent parameters (vS, vT and NST) obtained by
taking into account the similarities between haplotypes (i.e. the number of mutations between haplotypes). Population 5-Shokanbetsu was excluded as
the number of samples (N = 2) was smaller than that
required for analysis (N = 3). Two differentiation
parameters (GST, NST) were compared by a permutation test using 1000 permutations that estimated
whether each population comprised closely related
haplotypes. All parameters were estimated for the
entire population of Japan, including Sakhalin, and
for the individual populations in northern Japan
(with Sakhalin) and central Japan.
haplotypes were determined based on these aligned
sequences and a parsimony network was constructed
using the programme TCS1.06 (Clement, Posada &
Crandall, 2000). All indels were treated as substitutions. Population relationships were determined
using neighbour-joining (NJ) trees obtained from
NTSYS–PC 2.02 (Rohlf, 1998). Pairwise population
distances were estimated from the Euclidean distance
amongst populations based on the frequency of all
segregating sites. The significance of the NJ trees
was estimated by 500 bootstraps of individual
re-samplings from each population.
To reveal the geographic structure of the populations in the Japanese archipelago and Sakhalin,
spatial analyses of molecular variance (SAMOVA)
were performed to assess the genetic differentiation amongst groups of adjacent populations using
SAMOVA 1.0 (Dupanloup, Schneider & Excoffier,
2002). Given the number of groups (K), the highest
differentiation amongst groups (FCT) and the population configuration were calculated using a simulated annealing procedure. Assuming that the final
configuration was influenced by the initial configuration, 100 initial conditions were used as recommended by Dupanloup et al. (2002). Although the K
with the highest FCT represents the best number of
groups and the best population configuration, it does
not consistently represent a significant configuration. In particular, the final configurations of K with
one or more single population groups could not
derive the group structure (Heuertz et al., 2004).
Therefore, the number of groups (K) and the geographic structure were inferred from the configuration with the highest FCT that did not contain any
single population group.
Parameters of population diversity (hS, hT, vS, vT)
and differentiation (GST, NST) were estimated follow-
RESULTS
PHYLOGENETIC
RELATIONSHIPS AMONGST
HAPLOTYPES AND GEOGRAPHIC DISTRIBUTION
OF HAPLOTYPES
Based on 852–854 bp of two non-coding regions of
cpDNA, six substitutions and two indels were
detected amongst sequences and eight haplotypes
were identified from the composition of two noncoding regions in all samples (Table 2). All haplotype
sequences were deposited in DDBJ/EMBL/GenBank
(accession numbers AB440674–AB440682). The
phylogenetic relationships amongst the haplotypes
were revealed in the parsimony network (Fig. 1).
All haplotypes of the Japanese archipelago were
distinguished from adjacent ones by one or two
Table 2. Location of polymorphic sites and types of mutations in two regions of cpDNA
trnL intron (419–421 bp)
trnS–G (433 bp)
Haplotype
89–90
+T
151
G/A
214
+AT
258
G/A
265
C/A
403
G/T
41
G/C
269
C/G
A
A′
B
C
D
D′
E
G
+1
0
+1
+1
+1
+2
+1
+1
G
G
G
A
G
G
G
G
0
0
0
0
0
0
0
1
G
G
G
G
G
G
A
G
C
C
C
C
A
A
C
A
G
G
G
G
G
G
G
T
G
G
C
G
G
G
G
G
C
C
C
C
G
G
C
G
The number of base pairs in parentheses after the name of each region represents the total number of sequences analysed.
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
PHYLOGEOGRAPHY OF ARCTIC–ALPINE PLANTS IN JAPAN
D´
D
G
407
1-Sakhalin
2
5
A´
B
A
3
4
E
C
6
10
11
7
9
8
N
14
15
17
16
13
12
0
100
200
300
400 500km
Figure 1. Geographic distribution of cpDNA haplotypes detected in Loiseleuria procumbens and the parsimony network
of haplotypes. Pie charts represent the haplotype frequency of each population (chart colours correspond to those in the
network), numbers alongside the pie charts indicate the population number and the size of a pie chart represents the
number of samples per population. A black dot in the parsimony network represents a missing haplotype.
substitutions. Two groups of haplotypes were discerned by two substitutions. One group consisted of
haplotypes A, B, C and E with A′. These haplotypes
were found solely in populations of central Japan.
Haplotype A was found exclusively in the northern
part of central Japan, whereas the southern part was
dominated by haplotype C (Table 1; Fig. 1). In addition, haplotypes B and E were the private haplotypes
of 13-Ontakesan and 14-Norikuradake, respectively.
Haplotype A′, which lacks one of the 12 repeats
of thymine found in haplotype A, was found on
two mountains (i.e. 10-Shiroumadake and 14Norikuradake). However, another group comprised
the haplotypes of northern Japan, Sakhalin and
Sweden (D, D′, G). All populations in northern Japan
were fixed by a single haplotype (D or D′), except for
populations 1-Sakhalin and 2-Taisetsusan, which harboured both haplotypes. Haplotype G was unique to
Sweden. According to an outgroup comparison using a
sequence from Phyllodoce nipponica, which belongs to
the same tribe, haplotype D was considered ancestral
(sequence homology 89.2%; data not shown).
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
408
H. IKEDA ET AL.
1-Sakhalin
97
2-Taisetsu
80
3-Furano
4-Poroshiri
62
100
5-Shokanbetsu
6-Hakkouda
100
100
7-Gassan
8-Iide
18-Sweden
63
100
9-Hakusan
10-Shirouma
11-Jyounen
87
14-Norikura
13-Ontake
100
12-Kisokoma
99
15-Yatsugatake
95
17-Senjyou
16-Akaishi
0.00
0.25
0.51
0.76
1.02
Euclidian distance
Figure 2. Population relationships represented by a neighbour-joining tree. Distances between populations were estimated with Euclidean distances based on haplotype frequency and relationships. Numbers above branches indicate
bootstrap values higher than 50% (500 replicates). Numbers at the tips of branches represent population numbers.
POPULATION
DIFFERENTIATION AND
GEOGRAPHIC STRUCTURE
Relationships amongst populations are represented
as an NJ tree that revealed two major clusters (100%;
Fig. 2). As expected from the haplotype distribution,
populations in central Japan formed significant
clusters, whereas other clusters were formed by
populations in northern Japan, Sakhalin and Sweden.
Further geographic structures expected from the haplotype distribution within regions were significantly
supported by the NJ tree. Populations of the northern
(9–11, 13, 14) and southern (12, 15–17) parts of
central Japan formed two distinct clusters (> 85%),
whereas the southernmost populations of northern
Japan (7, 8), which were dominated by haplotype D,
formed distinct clusters (100%) from the remaining
populations in northern Japan, including Sakhalin
(97%). The Swedish population was distinguished
from populations of central Japan and clustered with
those of northern Japan as expected from haplotype
similarity (100%).
Results of the SAMOVA are presented in Table 3.
Most of the variation (91.9%, P < 0.001) was explained by differences amongst populations. The
number of groups with the highest FCT that included
no single population group was four (K = 4). The final
configuration of the populations in K = 4 revealed
groups from northern (9–11, 13, 14) and southern (12,
15–17) parts of central Japan, groups from the southernmost populations of northern Japan (7, 8) and of
more northern populations (1–6), explaining 89.9% of
the total variation (P < 0.001). This configuration was
consistent with the population clusters depicted in
the NJ tree (Fig. 2). When K = 2, populations of northern Japan and central Japan formed two geographic
groups, respectively, explaining 78.8% of the total
variation (P < 0.001). Moreover, additional grouping
(K = 3) divided the population of central Japan into
two geographic groups that corresponded to the
northern and southern parts of central Japan, which
explained 84.1% of the total variation (P < 0.001).
Calculated parameters of diversity and differentiation are presented in Table 4. The value of NST (0.93)
was significantly higher than that of GST (0.82)
throughout the overall populations (P < 0.01). The
same result was also found in central Japan [NST
(0.75) >> GST (0.71)]. In comparing the two regions,
haplotype diversity throughout regions and within
populations was higher in central Japan (hT = 0.66,
hS = 0.19) than in northern Japan (hT = 0.47, hS = 0.09).
DISCUSSION
Our analyses demonstrated the specific genetic structure of L. procumbens within central Japan. This
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
PHYLOGEOGRAPHY OF ARCTIC–ALPINE PLANTS IN JAPAN
409
Table 3. Results of spatial analyses of molecular variance (SAMOVA) of cpDNA sequence data from populations of
Loiseleuria procumbens in the Japanese archipelago and Sakhalin
Source of variation
d.f.
Among populations (total)
16
Sum of
squares
Variance
components
Percentage of
variation (%)
131.27
P-value
0.91146
91.94
Within populations
135
10.789
0.07992
8.06
< 0.001
K = 2 (1–8) vs. (9–17)
Among regions
Among populations within group
Within populations
1
15
135
95.772
35.498
10.789
1.25407
0.25718
0.07992
78.81
16.16
5.02
< 0.001
< 0.001
< 0.001
K = 3 (1–8) vs. (9–11, 13, 14) vs. (12, 15–17)
Among regions
Among populations within group
Within populations
2
14
135
113.853
17.417
10.789
1.1214
0.1319
0.07992
84.11
9.89
5.99
< 0.001
< 0.001
< 0.001
K = 4 (1–6) vs. (7, 8) vs. (9–11, 13, 14) vs. (12, 15–17)
Among regions
Among populations within group
Within populations
3
13
135
125.062
6.208
10.789
1.11657
0.04522
0.07992
89.92
3.64
6.44
< 0.001
< 0.001
< 0.001
Population numbers are shown in parentheses.
Table 4. Gene diversity and differentiation parameters calculated for all studied populations of Loiseleuria procumbens
in the Japanese archipelago and Sakhalin, including those from northern and central Japan
Region
hS
hT
GST
vS
vT
NST
Northern region,
1–8*
Southern region†,
9–17*
Total†
0.090 (0.0647)
0.474 (0.1219)
0.809 (0.1552)
0.090 (0.0647)
0.474 (0.1219)
0.809 (0.1552)
0.193 (0.0780)
0.658 (0.0548)
0.707 (0.1136)
0.122 (0.0492)
0.485 (0.0719)
0.749 (0.0918)
0.144 (0.0513)
0.801 (0.0337)
0.820 (0.0636)
0.062 (0.0222)
0.854 (0.0440)
0.927 (0.0264)
Standard error is given in parentheses.
*Population numbers.
†Indicates that the value of NST was significantly larger than that of GST.
All analysis excluded 5-Shyokanbetsu because the sample size was smaller than three.
indicates that populations persisted for a long time
during the Pleistocene climatic oscillations in this
region and have been isolated from the populations of
the more northerly regions. Therefore, regardless of
the widespread range of this arctic–alpine species, the
existence of a persisting population in central Japan
during the Pleistocene climatic oscillations is consistent with results reported in previous phylogeographic studies of the Japanese archipelago (Fujii
et al., 1997, 1999; Senni et al., 2005; Fujii & Senni,
2006; Ikeda & Setoguchi, 2006; Ikeda et al., 2006;
Ikeda & Setoguchi, 2007; Ikeda et al., 2008a, b, c).
Furthermore, this study, along with the previous findings of D. lapponica subsp. obovata (Ikeda et al.,
2008a), suggests that the high mountains of the
Japanese archipelago, which correspond to one of the
southernmost distribution limits of arctic–alpine
plants, could be an important area for the survival of
these plants during the Pleistocene climatic oscillations. In addition, the long persistence may cause
genetic divergence, resulting in the specific genetic
structure within central Japan. Consequently, high
mountains in central Japan may be sanctuaries for
genetic diversity of arctic–alpine species, which indicates special importance for conservation. In contrast,
these southernmost populations may not have been
involved in the range expansion of this widespread
species.
Added to the consistent genetic structure within
central Japan, the present study found that the
southernmost populations of northern Japan harboured a distinct geographic structure (Table 3),
which was characterized by the dominant occurrence
of haplotype D (Fig. 1). As recent phylogeographic
© 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 97, 403–412
410
H. IKEDA ET AL.
analysis of Cardamine nipponica, an endemic alpine
plant in Japan, significantly demonstrated the existence of persisting populations in this area (Ikeda
et al., 2008b), this geographic structure might represent long-time persisting populations in northern
Japan. However, the present study was insufficient to
evaluate this hypothesis because too few samples
outside the Japanese archipelago were included to
discard the possibility that haplotype D is a widespread haplotype of this arctic–alpine species. In fact,
remaining populations in northern Japan exclusively
harboured haplotype D′ and a homogenous genetic
structure extended throughout northern Japan as
well as Sakhalin. This homogenous genetic structure
observed throughout a wide range of regions may
have been shaped by the last range expansion,
perhaps during the last glacial period. Previous
studies have also shown that most species harboured
homogenous genetic structure throughout northern
Japan (Ikeda & Setoguchi, 2006; Ikeda et al., 2006;
Ikeda & Setoguchi, 2007; Ikeda et al.2008b). Thus,
this homogenous genetic structure was consistent
with those of previous studies, suggesting that the
majority of the distribution of alpine flora in northern
Japan was formed by recent range expansion.
However, the phylogeographic history in northern
Japan should be further evaluated, including samples
covering the entire range of L. procumbens as well as
other arctic–alpine species.
Given that current populations of alpine plants in
Japan correspond to the location of refugia during the
postglacial warm period, most populations could have
persisted during the warm periods and should
harbour some evidence of persisting populations. This
hypothesis was significantly supported by a previous
study of C. nipponica (Ikeda et al., 2008b). That study,
which used three nuclear genes as well as cpDNA,
unambiguously revealed the specific genetic structure
of most populations in northern Japan and demonstrated a population history that most populations in
northern Japan persisted for a long time. Thus, using
only cpDNA as a marker would not be sufficient to
unambiguously depict the evolutionary history of a
single species. Our study of L. procumbens, along
with previous phylogeographic studies based on
cpDNA, could not uncover significant evidence of
long-time persisting populations in northern Japan,
although a consistent history of vicariance between
central and northern Japan was shown. Therefore, a
comparative study based on a single locus of cpDNA
could demonstrate the overall history of the flora,
whereas it cannot depict the detailed history, perhaps
because of the recent range expansion that overwhelmed the pre-existing genetic structure. Therefore, in addition to the overall history detected by
using cpDNA, several genetic structures from nuclear
DNA may be needed to elucidate the detailed history
of other flora in further comparative phylogeographic
studies as well as the evolutionary history of populations. Although the details of the history of these
populations remain unclear, Japanese alpine flora
experienced vicariance between central and northern
Japan regardless of the species’ range. Hence, arctic–
alpine plants would have survived the Pleistocene
climatic oscillations on the high mountains of central
Japan, which correspond to one of the southernmost
limits of the arctic species, isolated from other regions
and resulting in genetic divergence. Further studies
in other areas corresponding to the southernmost
boundaries of arctic–alpine plant distributions are
needed to generalize the hypothesis of their survival
during the Pleistocene climatic oscillations in the
high mountains at low latitudes and their resulting
genetic divergence in each region.
ACKNOWLEDGEMENTS
This study was supported by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan and The Japan Society for the
Promotion of Science (13575011).
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