J Plant Res (2012) 125:207–221
DOI 10.1007/s10265-011-0428-8
REGULAR PAPER
Comparative phylogeography of four component species
of deciduous broad-leaved forests in Japan based
on chloroplast DNA variation
Takaya Iwasaki • Kyoko Aoki • Akihiro Seo •
Noriaki Murakami
Received: 29 September 2009 / Accepted: 17 April 2011 / Published online: 16 June 2011
 The Botanical Society of Japan and Springer 2011
Abstract A phylogeographic study of four tree species
(Padus grayana, Euonymus oxyphyllus, Magnolia hypol-
euca, and Carpinus laxiflora) growing in Japanese decid-
uous broad-leaved forests was conducted based on
chloroplast DNA (cpDNA) variations. Using nucleotide
sequences of 702–1,059 bp of intergenic spacers of
cpDNA, 20, 27, eight, and eight haplotypes were detected
among 251, 251, 226, and 262 individuals sampled from
67, 79, 75, and 71 populations of the above species,
respectively. The geographical pattern of the cpDNA
variations was highly structured in each species, and the
Electronic supplementary material The online version of this
article (doi:10.1007/s10265-011-0428-8) contains supplementary
material, which is available to authorized users.
T. Iwasaki
Department of Biology, Faculty of Science, Chiba University,
1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Present Address:
T. Iwasaki (&)
Graduate School of Arts and Sciences, The University of Tokyo,
3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan
e-mail: takaya.iwasaki11@gmail.com
K. Aoki
Graduate School of Human and Environmental Studies,
Kyoto University, Yoshida-Nihonmatsu-cho, Sakyo-ku,
Kyoto 606-8501, Japan
A. Seo
Research Institute for Humanity and Nature,
457-4 Kamigamomotoyama, Kita-ku, Kyoto 603-8047, Japan
N. Murakami
Makino Herbarium, Tokyo Metropolitan University,
1-1 Minamiosawa, Hachioji, Tokyo 192-0397, Japan
following three regional populations were genetically
highly differentiated among all four species: (1) the Sea of
Japan-side area, (2) the Kanto region, and (3) southwestern
Japan. Based on some interspecific similarities among the
phylogeographic patterns, the following migration scenario
of Japanese deciduous broad-leaved forests was postulated.
During the last glacial maximum (LGM), the forests were
separately distributed in six regions. After LGM, as the
climate warmed, the forests in eastern Japan separately
expanded from each of the refugia along the Sea of Japan-
side or along the Pacific Ocean-side. In contrast, those in
southwestern Japan retreated and moved to high altitudes
from each of the continuous forests.
Keywords Chloroplast DNA  Deciduous broad-leaved
forest  Glacial refugia  Intraspecific variation  Japanese
archipelago  Phylogeography
Introduction
Severe climatic oscillations during the Quaternary period
produced great changes in species distribution (Hewitt
2000, 2004). Recent molecular phylogeographic studies
have revealed the genetic structure of extant populations,
providing insights into the history of changes in species
distribution (Avise 2000). In general, the response of spe-
cies to climatic oscillations is considered a highly dynamic
process consisting of repeated retreats into refugia during
glacial periods and range expansions from the refugia
during interglacial periods.
The Japanese archipelago is located on the eastern edge
of East Asia and consists of four main islands: Hokkaido,
Honshu, Shikoku, and Kyushu. The archipelago stretches
3,000 km from northeast to southwest over a wide range of
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climatic zones from subarctic to subtropical. It also has a
complex mountainous topography along the central axis of
Honshu. The cool temperate zone in Japan is now domi-
nated by deciduous broad-leaved tree species, primarily
Fagus crenata and Quercus crispula. The deciduous broad-
leaved forests in northeastern Japan grow from sea level to
an altitude of more than 1,000 m, whereas those in
southwestern Japan are often isolated from each other in
the form of small patchy forests at elevations over 700 m.
During the last glacial maximum (LGM) around
23,000–18,000 years ago, the mean annual temperature
was 5–9C cooler and the precipitation was lower than
present levels (Tsukada 1988). Large areas of the Japanese
archipelago are considered to have remained unglaciated
even during the ice ages of the Quaternary period, owing to
the archipelago’s location in mid-latitudes and its maritime
climate (Ono 1984). Based on palynological data, historical
changes in the vegetation of Japan since LGM have been
studied in detail over the last several decades (Takahara
et al. 2000; Tsukada 1988; Yasuda and Miyoshi 1998). In
response to the climatic changes, temperate plant species in
Japan have generally migrated along the Pacific Ocean-
side, the Sea of Japan-side, or mountain slopes of the
archipelago (Tsukada 1988). These temperate species
either migrated southward along the coasts or to lower
altitudes into refugia during glacial periods, and they either
expanded northward or to higher altitudes during inter-
glacial periods (Tsukada 1988). The principal mountains
might have acted as geographical barriers to the migration
and gene flow of many plant species during the Quaternary
period. However, interpretations based only on fossil pol-
len records might be misleading, since trace amounts of
pollen resulting from long-distance dispersal may lead to
overestimates of the species’ distribution range during
LGM. Similarly, the absence of fossil pollen records does
not automatically indicate the absence of these species in
the sampled range (McLachlan and Clark 2004). Therefore,
other evidence is needed to reconstruct the biogeographic
history of extant species (Tremblay and Schoen 1999).
Since chloroplast DNA (cpDNA) in angiosperms is
maternally inherited without any recombination (Corriveau
and Coleman 1988), it is useful for determining the route of
seed migration and identifying locations of important
refugia during LGM (Abbott et al. 2000; Heuertz et al.
2004; McCauley 1995; Newton et al. 1999). Over the past
decade, the geographical distributions of intraspecific
cpDNA variation of various plant species in Japan have
been examined to elucidate their postglacial migration
history (Aoki et al. 2004; Fujii and Senni 2006; Fujii et al.
1997; Ikeda and Setoguchi 2007). In particular, deciduous
broad-leaved tree species, F. crenata (Fujii et al. 2002;
Okaura and Harada 2002), Stachyurus praecox (Ohi et al.
2003), and Q. crispula and related species (Kanno et al.
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J Plant Res (2012) 125:207–221
2004; Okaura et al. 2007) have been examined phylogeo-
graphically. In addition, some phylogeographic studies
have been conducted for F. crenata using other DNA
markers; for example, nuclear-encoded allozyme poly-
morphisms (Tomaru et al. 1997), mitochondrial DNA
variations (Tomaru et al. 1998), and nuclear microsatellite
markers (Hiraoka and Tomaru 2009). These three taxa
(F. crenata, S. praecox, and Quercus species) are
co-distributed in the deciduous broad-leaved forests of
Japan. However, some discrepancies have been observed
among the geographical patterns of genetic differentiation
in the three taxa. For example, based on the distribution
patterns of cpDNA variations or nuclear alleles, distinct
regional groups in the Sea of Japan-side area were genet-
ically recognized for F. crenata and S. praecox, but not for
Quercus species. In addition, the plant populations dis-
tributed in the Pacific Ocean-side area could be subdivided
into many regional groups, but locations of the phylogeo-
graphical breaks among the regional groups were not
identical among these three taxa. Thus, a consensus on the
postglacial migration history of the deciduous broad-leaved
forests in Japan has not yet been reached. Moreover, most
previous studies could not reveal the detailed locations of
refugia in Japan during LGM. However, these previous
studies relied only on palynological data for their discus-
sions of the locations of refugia.
On the other hand, to examine migration history since
LGM, previous studies based on cpDNA variation (Fujii
et al. 2002; Ohi et al. 2003) used the geographical distri-
bution of cpDNA haplotypes as well as genealogical rela-
tionships of the haplotypes. However, because of the slow
rate of molecular evolution of cpDNA (1.0–3.0 9 10-9
substitutions per site per year) (Wolfe et al. 1987), most
cpDNA haplotypes observed in these plant species proba-
bly originated long before LGM. Therefore, it may be
misleading to discuss their migration history after LGM
based on phylogenetic relationships among the detected
haplotypes. In the present study, our phylogeographical
discussion will be based only on the geographical distri-
bution patterns of the haplotypes, not on their phylogenetic
relationships.
In addition to the effects of climatic changes, the genetic
diversity and population genetic structure of each plant
species are influenced by various factors (Hamrick and
Godt 1989; Hamrick et al. 1992). Discussing the migration
history of plant species based only on the phylogeographic
pattern of a single species might be misleading. In Japan,
the vegetation zones are determined mainly by mean
temperature, because the amount of rainfall throughout the
entire country is sufficient (Kira 1977, 1991). Plant species
growing together in Japan might have reacted to past
environmental changes, such as low temperatures, in a
similar manner and could survive in the same refugia.
J Plant Res (2012) 125:207–221
A common geographic pattern among several co-distrib-
uted taxa existing over the same area would indicate the
influence of common historical factors such as climate
changes during glacial and interglacial periods. In Europe,
careful comparison was made of the intraspecific phylog-
eographic patterns of 10 taxa to search for congruent
geographic patterns of genetic variation (Taberlet et al.
1998). Although only a small degree of congruence was
found, these results indicated that the likely colonization
routes exhibit some similarities.
The present study investigated intraspecific cpDNA
variations in four plant species growing together in Japa-
nese deciduous broad-leaved forests over a common geo-
graphic area. These investigations used the same sampling
strategy and the same methods of analysis. Furthermore,
common phylogeographic patterns among genetic struc-
tures in four plant species were determined. The migration
history of deciduous broad-leaved forests is discussed in
terms of these patterns.
The following four species were selected as plant
materials for this study: Padus grayana (Maxim.)
C. K. Schneid. (Rosaceae), Euonymus oxyphyllus Miq.
(including E. oxyphyllus var. magnus Honda) (Celastra-
ceae), Magnolia hypoleuca Siebold et Zucc. (Magnolia-
ceae), and Carpinus laxiflora (Siebold et Zucc.) Blume
(Betulaceae). All these species grow in deciduous broad-
leaved forests over a similar geographic distribution range
(from the highlands of Kyushu to the lowlands of Hokka-
ido) and exhibit relatively large amount of intraspecific
cpDNA variation (Iwasaki et al. 2006). In E. oxyphyllus,
one variety, var. magnus Honda, distributed in the Sea of
Japan-side area, has been recognized based on a combi-
nation of morphological characters (relatively large leaves
and fruits, and thick branches) (Murata 1972). However, it
is often difficult to identify these intraspecific taxa due to
their clinal morphological variation. Accordingly, these
varieties were not distinguished in this study.
Our specific goals were (1) to investigate the levels and
distribution of intraspecific cpDNA variation in the four
plant species growing in Japanese deciduous broad-leaved
forests, (2) to search for common phylogeographic patterns
among them, and (3) to discuss the locations of refugia and
migration routes based on the phylogeographical data
obtained.
Materials and methods
Plant materials
Our sampling strategy was to collect a few individuals
from many localities to sufficiently cover the whole geo-
graphic range of each species in Japan. A total of 251
209
individuals of P. grayana from 67 populations, consisting
of one–eight individuals per population, were collected. In
the same way, 251 individuals of E. oxyphyllus (including
E. oxyphyllus var. magnus) from 79 populations, consisting
of one–six individuals per population, 226 individuals of
M. hypoleuca from 75 populations, consisting of one–six
individuals per population, and 262 individuals of C. lax-
iflora from 71 populations, consisting of one–nine indi-
viduals per population were collected. Silica gel-dried
leaves were collected from these sites. Geographical
information on the 106 total populations is shown in
Fig. 1a and Table S1. Voucher information is given in
Table S1. The voucher specimens have been preserved
in the Makino Herbarium (MAK), Tokyo Metropolitan
University.
DNA extraction, polymerase chain reaction
amplification, and sequencing
Prior to genomic DNA extraction, leaf tissue was washed
using 2-[4-(2-hydroxyethyl)-1-piperanzinyl] ethanesulfonic
acid (HEPES) buffer (pH 8.0; Setoguchi and Ohba 1995) to
remove polysaccharides. Genomic DNA was extracted
from the washed leaf pellets using a cetyltrimethylammo-
nium bromide (CTAB) method (Doyle and Doyle 1987)
with a slight modification. As a preliminary screening, the
following 13 noncoding regions of cpDNA were analyzed
for each species: the rps16 intron, trnG(UCC) intron, petB
intron, rpl16 intron, psbC-trnS(UGA) intergenic region,
trnG(GCC)-trnfM(CAU), trnW(CCA)-trnP(UGG), petD-
rpoA (Nishizawa and Watano 2000), trnL(UAA)30 exon-
trnF(GAA) intergenic region, trnT(UGU)-trnL(UAA)50
exon (Taberlet et al. 1991), atpB-rbcL intergenic region
(Terachi 1993), trnH(GUG)-psbA intergenic region and
trnS(GCU)-trnG(UCC) (Hamilton 1999).
In consideration of the amount of intraspecific sequence
variation, two or three regions were selected for each
species individually (data not shown). Selected regions and
their polymerase chain reaction (PCR) primers (some
primers were modified for each species) are summarized in
Table S2. The PCR mixture consisted of 0.63 units of
Blend Taq polymerase (Toyobo, Osaka, Japan), 2.5 lL of
10% Blend Taq Buffer [25 mmol/L TAPS (pH 9.3),
50 mmol/L KCl, and 10 mmol/L MgCl2], 2 lL of 2 mmol/
L dNTP solution, 1.25 lL of 10 pmol/L of each primer,
and 10–30 ng of genomic DNA, to a total volume of
25 lL. The PCR cycle conditions were 94C (2 min); then
35 cycles of 94C (45 s), 55C (45 s), 72C (1.5 min), and
finally 72C (7 min). After confirming PCR amplification
on an agarose gel, amplified products were treated with
ExoSAP-IT reagent (Amersham Biosciences, Tokyo,
Japan) to remove excess primers and dNTPs. Purified DNA
fragments were used as templates for sequencing reactions
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210 J Plant Res (2012) 125:207–221

Fig. 1 Population sites

sampling in this study (a).
Detailed voucher information is
shown in Table S1. The 57 grid
cells and nine regions defined in
this study (b)

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using the ABI PRISM Big Dye Terminator Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA,
USA) with the same primers used for PCR amplification.
The resultant mixture was analyzed using an Applied
Biosystems 3100 genetic analyzer. The sequences obtained
were aligned using ChromasPro version 1.34 (http://www.
technelysium.com.au/ChromasPro.html) and BioEdit ver-
sion 7.0.5.2 software (Hall 1999).
Haplotype classification
In this study, cpDNA variations based on inversions and
indels, including mononucleotide repeat length variations,
and nucleotide substitutions, were detected. The sequences
differing by nucleotide substitutions, inversions and indels
were treated as different DNA types (indicated by A, B,
C,…). Those differing only by number of mononucleotide
repeats were treated as different subtypes of each DNA
type (indicated by A, A0 , A00 ,…). DNA type labels denote
relative frequency alphabetically, the A-DNA type of each
species being the most common. Similarly, the subtypes
denote relative frequency, with the A-subtype of each DNA
type being the most common subtype and the A’-subtype
being the second-most common subtype. In this study, both
different DNA types and subtypes were treated as equally
different haplotypes, except for the following haplotype
network analyses. Furthermore, haplotypes with a fre-
quency of more than 5% were defined as common haplo-
types, whereas haplotypes with a frequency of 5% or less
were defined as rare haplotypes.
Haplotype network analysis
Multiple sequence alignment was performed manually. The
simple indel coding method of Simmons and Ochoterena
(2000) was employed for gap coding. The gaps caused by
variation in the number of mononucleotide repeat units
were removed from this intraspecific haplotype network
analysis, given their susceptibility to homoplasy (Ingvars-
son et al. 2003). The haplotype network was constructed
using TCS version 1.21 software (Clement et al. 2000). The
mutations (nucleotide substitutions, indels, and inversions)
were treated as unordered and were equally weighted.
Amount of genetic diversity
To evaluate the amount of genetic diversity within each of
the four species, haplotype diversity (H) (Nei 1987),
nucleotide diversity (p), that is, the average number of
nucleotide differences per site between two sequences (Nei
1987), and the Watterson estimator (h), that is, the number
of segregating sites per site (Watterson 1975), were cal-
culated using DnaSP ver. 5.10 software (Librado and Rozas
211
2009). In this analysis, nucleotide substitutions, inversions,
and indels (including mononucleotide repeat length varia-
tions) were weighted equally.
Local areas with high genetic uniqueness
To search for local areas with high genetic uniqueness, 57
grid cells were defined following the 80-km mesh cells
provided by the Geographical Survey Institute of Japan
(Fig. 1b). Several populations distributed within each grid
cell were integrated to one grid population (Table S1), and
the proportion of rare haplotypes in each grid cell (PR) was
calculated for each of the four species. The interspecific
average values of PR among the four species were also
calculated for each grid cell. To test whether areas with
high PR values are geographically concentrated or not, the
spatial autocorrelation pattern of the PR values was
investigated by calculating Moran’s I values (i.e., a mea-
sure of spatial autocorrelation; Moran 1950) using the free
software R (R development Core Team 2010) with the
spdep (Bivand 2009) package. Furthermore, in the nine
regions defined by integrating grid cells (Fig. 1b), the
values of H (Nei 1987) and PR were calculated using the
Arlequin ver. 3.1 software (Excoffier et al. 2005) and by
manual for each of the four species, respectively. The
interspecific average values of H and PR among the four
species were also calculated for each region.
Spatial genetic structure
Spatial genetic structure was assessed by testing significant
isolation by distance using a Mantel test with 9,999 random
permutations of the relationship between the matrices of
pairwise genetic distances and those of the natural loga-
rithm of geographical distances among populations. As the
genetic distance matrix, the pairwise relative Sorensen
distance (D) (McCune et al. 2002) was used.
The relative Sorensen distance is a metric based on the
sum, over the haplotypes, of the pairwise differences in
haplotype frequencies between populations as follows:
 
1 X  y1j y2j 
p
Dðx1 ;x2 Þ ¼   
2 j¼1 Rpj¼1 y1j Rpj¼1 y2j 
where D(x1, x2) is the relative Sorensen distance between
populations x1 and x2, p is the number of different haplotypes
between the two populations, j varies from 1 to p, y1j is the
number of individuals with haplotype j in populations x1, and
y2j is the number of individuals with haplotype j in popula-
tion x2. D(x1, x2) takes values between 0, when the haplotype
frequencies are identical between two populations, and 1,
when the populations have no haplotypes in common. Dif-
fering from other types of genetic distance measures, this
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distance measure makes it possible to estimate divergence,
even when populations are fixed at different haplotypes. The
values of relative Sorensen distance were calculated using
PC-ORD version 5.10 software (McCune and Mefford
1999). A Mantel test was performed using GeneAlex version
6.2 software (Peakall and Smouse 2006).
Parameters of population differentiation (GST, NST)
were estimated following the methods of Pons and Petit
(1996), using PERMUT version 2.0 software (http://www.
pierroton.inra.fr/genetics/labo/Software/). These two
parameters were compared by using a permutation test with
10,000 permutations. GST is a population differentiation
parameter based solely on haplotype frequency within
populations, whereas NST is a parameter that includes both
haplotype frequency and mutation steps between haplo-
types. The comparison thus allows testing to determine
whether distinct haplotypes occurring in the same popula-
tion are on average more closely related than distinct
haplotypes from different populations. Some populations
were excluded from this analysis because the numbers of
samples were smaller than the minimum required for
analysis (at least three samples are required).
Spatial analysis of variance (SAMOVA) based on a
simulated annealing procedure was used to define groups of
populations that are geographically homogeneous and
maximally differentiated from each other (Dupanloup et al.
2002). The software SAMOVA version 1.0 (http://web.
unife.it/progetti/genetica/Isabelle/samova.html) iteratively
seeks the composition of a user-defined number K of
groups of geographically adjacent populations that maxi-
mizes FCT; that is, the proportion of total genetic variance
resulting from differences among groups of populations. In
the present study, this analysis was carried out based only
on haplotype composition, not taking phylogenetic rela-
tionships among haplotypes into consideration. The pro-
gram was run for 10,000 iterations for K (K values were set
between 2 and 20) from each of 300 random initial con-
ditions. For each K, the configuration with the largest FCT
value after the 300 independent simulated annealing pro-
cesses was retained as the best grouping of populations.
Two criteria were considered to select the number of
groups. First, the pattern of FCT values as a function of
K was examined. In particular, the number of groups
necessary for FCT to reach a plateau was investigated.
Second, configurations with one or more single-population
groups were excluded, because this indicates that the group
structure is disappearing (Godbout et al. 2005; Heuertz
et al. 2004; Magri et al. 2006).
Because the geographic patterns observed in SAMOVA
analysis were complicated in P. grayana and E. oxyphyllus,
similarities between SAMOVA groups were measured by
performing cluster analysis using the unweighted pair-
group method with arithmetic averages (UPGMA) (Sneath
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J Plant Res (2012) 125:207–221
and Sokal 1973). To construct the distance matrix, pairwise
relative Sorensen distances (McCune et al. 2002) between
the SAMOVA groups were used. UPGMA analysis was
performed using PC-ORD version 5.10 software (McCune
and Mefford 1999).
Results
Intraspecific cpDNA variation and haplotype network
The nucleotide sequences of noncoding regions, 694–
727 bp for P. grayana, 637–668 bp for E. oxyphyllus,
1,039–1,051 bp for M. hypoleuca, and 678–702 bp for
C. laxiflora, were determined. All the sequence data obtained
in the present study have been deposited in DDBJ under
accession numbers AB525243–AB525282 for P. grayana,
AB525283–AB525336 for E. oxyphyllus, AB525337–
AB525360 for M. hypoleuca, and AB525361–AB525376
for C. laxiflora. The aligned sequences for polymorphic
sites and frequencies of each haplotype in the four species
are shown in Tables S3, S4, S5, and S6, respectively. In
P. grayana, 14 haplotypes, three of which had two–four
subtypes, were distinguished, and 20 distinguishable
cpDNA haplotypes were recognized in all. In E. oxyphyl-
lus, 19 haplotypes were distinguished, six of which had two
or three subtypes. In all, 27 distinguishable cpDNA hap-
lotypes were found. In M. hypoleuca, eight cpDNA hap-
lotypes were recognized. In C. laxiflora, seven DNA types,
one of which had two subtypes, were found, total of eight
distinguishable cpDNA haplotypes. Common haplotypes
and their frequencies were as follows: types A (22.7%), A0
(9.6%), B (27.5%), C (11.6%), and D (9.6%) in P. grayana;
types A (30.3%), A0 (14.7%), B (12.0%), B0 (7.2%), and C
(5.2%) in E. oxyphyllus; types A (51.8%), B (25.7%), and
C (17.3%) in M. hypoleuca; and types A (42.7%), B
(28.6%), and C (25.2%) in C. laxiflora. All the frequencies
of the other haplotypes were 5% or less, and these were
treated as rare haplotypes.
The calculated values of H, p, and h are shown in
Table 1. The phylogenetic relationships among the haplo-
types in each species are shown in the parsimony network
(Fig. 2a–d). All haplotypes were distinguished from their
most closely related haplotypes by three or fewer muta-
tional steps. Large phylogenetic breaks, which would
indicate the existence of cryptic species or isolation for long
time, were not observed in the networks of the four species.
Geographic distribution patterns of haplotypes
in the four species
The geographical distributions of the cpDNA haplotypes in
the four species are shown in Fig. 2a–d. The haplotype
J Plant Res (2012) 125:207–221
Table 1 Haplotype diversity, nucleotide diversity, and Watterson
estimator observed in four component species of deciduous broad-
leaved forests
Species n nh H p
Padus grayana 251 20 0.841 0.00418
Euonymus oxyphyllus 251 27 0.860 0.00425
Magnolia hypoleuca 226 8 0.638 0.00176
Carpinus laxiflora 262 8 0.674 0.00381
n number of samples, nh number of haplotypes, H haplotype diversity
(Nei 1987), p nucleotide diversity (Nei 1987), h Watterson estimator
(Watterson 1975)
compositions in the nine regions are summarized in
Table 2. Although a few haplotypes were disjunctively
distributed, most haplotypes were geographically clearly
structured. Notably, common haplotypes in each species
were distributed in specific areas, not randomly. In
P. grayana (Fig. 2a), common haplotype A was mainly
distributed in the Tohoku and the Sea of Japan-side area. In
contrast, types A0 and D were predominant in the Kanto
region. Besides, type B was widely distributed in south-
western Japan, and type C was found in the Hokkaido and
Tohoku regions. In E. oxyphyllus (Fig. 2b), common hap-
lotype A was widely distributed along the Pacific Ocean-
side. The distribution pattern of type A0 was similar to that
of type A, but this type was confined to eastern Japan. In
contrast, types B and B0 were mainly distributed in the Sea
of Japan-side area. In M. hypoleuca (Fig. 2c), common
haplotype A was widely distributed in the eastern part of
Japan. On the other hand, type B was predominantly dis-
tributed in southwestern Japan. Type C was distributed in
the Sea of Japan-side area. In C. laxiflora (Fig. 2d), com-
mon haplotype A was widely distributed in southwestern
Japan, while type C was distributed in eastern Japan. Type
B was distributed in the Chubu region and the Sea of
Japan-side area.
Geographic structure of genetic variation
In the Mantel tests, measures of genetic distance (pairwise
relative Sorensen distance) were significantly correlated
with geographical distance in the four species (Table S7).
In P. grayana and E. oxyphyllus, calculated parameters of
population differentiation, NST (0.737 and 0.609, respec-
tively) were significantly higher than GST (0.574 and
0.464) (P \ 0.001 and P \ 0.001). NST in M. hypoleuca
(0.696) was higher than GST (0.661) but not significant
(P [ 0.05). However, NST in C. laxiflora (0.763) was
smaller than GST (0.809).
In the SAMOVA analysis, the FCT values increased with
K, and for values of K C 7 in P. grayana, K C 13 in
E. oxyphyllus, K C 4 in M. hypoleuca, and K C 4 in
213
C. laxiflora. Some groups consisted only of a single pop-
ulation. Thus, the groupings corresponding to K = 6
(FCT = 0.539), 12 (FCT = 0.522), 3 (FCT = 0.715), and 3
h (FCT = 0.848) in each of the above four species were
considered as valid and retained for interpretation
0.0042
(Table 3). Geographic distributions of the recognized
0.0053
groups in each species are shown in Figs. 3a, b and 4a, b.
0.0018
In P. grayana and E. oxyphyllus, the geographic distribu-
0.0036
tion patterns of the detected SAMOVA groups were
complicated (Fig. 3a, b). In contrast, three well-separated
groups were observed in M. hypoleuca (Fig. 4a): SAM-
OVA group I in eastern Japan, II in the Sea of Japan-side
area, and III in southwestern Japan. In C. laxiflora, three
well-separated groups were also detected (Fig. 4b): SAM-
OVA group I in eastern Japan, II in the Sea of Japan-side
area, and III in southwestern Japan.
The UPGMA dendrograms of P. grayana and E. oxy-
phyllus, based on the relative Sorensen distance matrix
between the groups of populations recognized by SAM-
OVA, are shown in Fig. 3a, b. The results revealed some
clustering and implied geographic structure. In P. grayana,
three widely distributed cluster groups and one local group
(SAMOVA group IV) were observed (Fig. 3a): the first
cluster was in southwestern Japan (SAMOVA groups I and
II), the second cluster was in the Kanto region (III), and the
third cluster was in the Sea of Japan-side area (V and VI).
In E. oxyphyllus, three widely distributed cluster groups
and six local groups (SAMOVA groups III, VIII, IX, X, XI,
and XII) were found (Fig. 3b): the first cluster was in
southwestern Japan (SAMOVA group I), the second cluster
was in eastern Japan (II), and the third cluster was in the
Sea of Japan-side area (IV, V, VI, and VII).
Local areas with high genetic uniqueness in Japan
The interspecific average values of PR among the four
species in each grid cell are shown in Fig. 5. Moran’s I was
significantly positive (I = 0.079, P \ 0.001). This finding
suggests that the overall distribution pattern of PR values is
not random but spatially autocorrelated. The PR values
were relatively high in the following seven grid cells:
PR = 0.312 in HR8 grid, 0.400 in KT6 grid, 0.404 in CB4
grid, 0.376 in KI4 grid, 0.333 in CG2 grid, 0.333 in CG6
grid, and 0.350 in CG7 grid. The haplotype compositions
of each species in these seven grids are shown in Table 4.
Many rare haplotypes were detected in Padus grayana and
Euonymus oxyphyllus, comparing to Magnolia hypoleuca
and Carpinus laxiflora. On the other hand, no grid cells
with high PR (C0.20) values were observed in the Hok-
kaido and Tohoku regions or in the northern part of the
Hokuriku region.
The values of PR and H in each of the nine local regions
are shown in Table 2. Compared with the values of the
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 J Plant Res (2012) 125:207–221
b Fig. 2 Parsimony network relationships among chloroplast DNA
haplotypes, and geographical distribution of the haplotypes observed
in a Padus grayana, b Euonymus oxyphyllus, c Magnolia hypoleuca,
and d Carpinus laxiflora. Open circles in network indicate missing
haplotypes. Solid bars, open bars and stars in networks indicate
nucleotide substitutions, indels, and inversions, respectively. Letters
with single or double quotation marks designate a haplotype differing
from the original haplotype only by number of mononucleotide
repeats. Colored letters designate a common haplotype with a
frequency of more than 5%, and black letters designate a rare
haplotype with a frequency of 5% or less. Numbers in parentheses
correspond to population number
other eight regions, those of H in the Hokkaido region were
low (actually H = 0.000 in P. grayana, M. hypoleuca, and
C. laxiflora), except for E. oxyphyllus (H = 0.765). In the
other regions, the values of H were similar to each other. In
addition, the values of PR in the Hokkaido and Tohoku
regions were also low (PR = 0.000 and 0.174 in P. gra-
yana, 0.242 and 0.200 in E. oxyphyllus, 0.000 and 0.000 in
M. hypoleuca, and 0.000 and 0.000 in C. laxiflora).
Discussion
Locations of important refugia during LGM
Petit et al. (2003) investigated geographic distribution of
cpDNA variations in 22 widespread European plant species
in order to test the hypothesis that glacial refugia harbor a
large fraction of the intraspecific biodiversity. They con-
cluded that the plant populations in the refugia showed
high genetic uniqueness rather than a high number of
haplotypes. Following their suggestion, this study chose to
regard the areas rich in rare haplotypes as possible
important refugia during LGM. Because most rare haplo-
types were specific to a single area, the interspecific
average values of PR among the four species in each grid
cell were used as indexes of genetic uniqueness in each
area. Relatively high values of PR were observed in the
following seven grids (Fig. 5): HR8 in the Hokuriku
region, KT6 in the Kanto region, CB4 in the Chubu region,
KI4 in the Kinki region, and CG2, CG6, CG7 in the
Chugoku region (Table 4). Based on palynological data,
especially from F. crenata, a major component plant spe-
cies of deciduous broad-leaved forests in Japan, the full-
glacial distribution range of F. crenata during LGM was
reported to be areas south of 38N latitude along the coastal
regions of Japan (Tsukada 1982a). Furthermore, in the area
surrounding Wakasa Bay (near the HR8 grid), the existence
of refugia during LGM for Cryptomeria japonica, one of
the most important species of Japanese cool temperate
forests, was suggested both by palynological study (Tsuk-
ada 1982b) and by phylogeographic study using micro-
satellite markers (Takahashi et al. 2005). Although the six
215
other grid areas have not been identified as special refugia
in previous studies, all seven of our grid areas were
included in the full-glacial distribution range of F. crenata.
Therefore, we suggest that these seven grid areas were very
probably important refugia for Japanese deciduous broad-
leaved forests during LGM. On the other hand, no grid cell
with high PR values was observed in the Shikoku and
Kyushu regions. Palynological studies suggested the exis-
tence of refugia in these areas during LGM (Tsukada 1988;
Yasuda and Miyoshi 1998). In these regions, the present
distribution of deciduous broad-leaved forests are limited
to high altitude areas only, and the population sizes of these
forests were relatively small. Bottleneck effects can act
strongly in small-sized populations (Nei 1987). Rare hap-
lotypes in these areas might be lost because of these effects
after LGM or during previous interglacial periods.
Furthermore, no grid cell with high PR values was
observed in the Hokkaido and Tohoku regions. The fre-
quency of rare haplotypes should decline with increasing
distance from refugia as a consequence of successive
founder events during postglacial colonization (Hewitt
1996, 2000). Therefore, this result might suggest that
refugia of deciduous broad-leaved forests did not exist in
these areas, and that most extant populations were derived
from the populations in the southern regions—for example,
the Hokuriku or Kanto regions—after LGM.
Three possible migration routes after LGM in the Sea
of Japan-side area, the Kanto region, and southwestern
Japan
The geographical pattern of the cpDNA variations was
highly structured in each species. The Mantel tests revealed
significant positive correlations between geographical and
genetic distance, indicating the existence of strong isola-
tion by distance. The results of comparison between NST
and GST indicated that distinct haplotypes occurring in the
same population are more closely related than haplotypes
from different population in P. grayana and E. oxyphyllus,
but not in M. hypoleuca and C. laxiflora. Because the rate
of molecular evolution of the cpDNA genome is very low
(Wolfe et al. 1987), these observed haplotypes may be
supposed to have originated long before LGM.
The phylogeographic patterns observed in this study
were not completely identical, but they were similar among
the four species. Based on the combination analysis of
SAMOVA and UPGMA dendrogram (Figs. 3a, b; 4a, b),
the following three regional populations showed high
genetic similarities, which were genetically highly differ-
entiated among all four species: (1) the Sea of Japan-side
area (the Sea of Japan-side area of the Tohoku region, the
Hokuriku region, and the eastern part of the Chugoku
region), (2) the Kanto region, and (3) southwestern Japan
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Table 2 Summary of chloroplast DNA haplotype composition in each region of the four deciduous broad-leaved species
Haplotype counts Region (population number)
Hokkaido Tohoku Hokuriku Kanto Chubu Kinki Chugoku Shikoku Kyushu Total
(1–12) (13–26) (27–41) (42–52) (53–63) (64–76) (77–86) (87–93) (94–106)

Padus grayana
A 18 30 7 2 57
A0 5 1 13 5 24
B 9 14 11 19 1 15 69
C 13 14 1 1 29
D 1 5 16 2 24
Rare 8 12 2 8 10 4 4 48
Total 13 46 58 39 21 19 31 5 19 251
nha 1 5 11 6 3 4 7 3 3 4.8d
Hb 0.000 0.728 0.701 0.705 0.514 0.591 0.613 0.700 0.368 0.847
PRc 0.000 0.174 0.207 0.501 0.000 0.421 0.323 0.800 0.211 0.191
Euonymus oxyphyllus
A 3 7 8 7 9 6 2 8 26 76
A0 14 11 1 10 1 37
B 1 4 19 2 4 30
B0 7 2 2 7 18
C 1 1 11 13
Rare 8 6 11 13 3 16 13 2 5 77
Total 33 30 41 30 13 25 26 11 42 251
nha 7 7 10 6 5 9 5 4 5 6.4d
Hb 0.765 0.800 0.749 0.763 0.539 0.843 0.785 0.491 0.556 0.860
PRc 0.242 0.200 0.268 0.433 0.231 0.640 0.500 0.182 0.119 0.307
Magnolia hypoleuca
A 25 30 12 27 20 3 117
B 1 4 11 15 11 16 58
C 10 22 7 39
Rare 1 1 4 6 12
Total 25 41 39 27 21 14 26 11 22 226
nha 1 3 4 1 2 2 4 1 3 2.3d
Hb 0.000 0.415 0.591 0.000 0.095 0.363 0.603 0.000 0.450 0.638
PRc 0.000 0.000 0.026 0.000 0.048 0.000 0.154 0.000 0.273 0.053
Carpinus laxiflora
A 13 20 25 12 42 112
B 3 27 4 36 1 4 75
C 3 32 8 18 4 1 66
Rare 2 2 5 9
Total 3 35 50 24 45 22 29 12 42 262
nha 1 2 5 4 4 3 2 1 1 2.6d
Hb 0.000 0.162 0.627 0.424 0.354 0.178 0.246 0.000 0.000 0.674
PRc 0.000 0.000 0.040 0.083 0.111 0.000 0.000 0.000 0.000 0.034
All species
Average of H b 0.191 0.526 0.667 0.473 0.376 0.494 0.562 0.298 0.344 0.755
Average of PRc 0.060 0.093 0.135 0.142 0.097 0.265 0.244 0.245 0.151 0.146
The nine regions were defined by integrating grid cells (Fig. 1b). Values given in bold indicate that the haplotypes were found in each regions with high
frequency more than 25%
a
Number of distinct haplotypes per region
b
Haplotype diversity
c
Proportion of rare haplotypes
d
Average

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Table 3 Results of the analyses of molecular variance (AMOVAs) performed considering the groups of populations suggested by the spatial
analyses of molecular variance (SAMOVAs) in each species
Source of variation df Sum of square Variance components Percentage of variation F-statistics

(1) Padus grayana (K = 6)

Among groups 5 104.194 0.50649 53.85 FCT = 0.53850***
Among populations within groups 61 40.562 0.08606 9.15 FSC = 0.19827***
Within populations 184 64.033 0.34801 37.00 FST = 0.63000***
Total 250 208.789 0.94056
(2) Euonymus oxyphyllus (K = 12)
Among groups 11 107.462 0.49656 52.24 FCT = 0.52240***
Among populations within groups 67 34.046 0.02607 2.74 FSC = 0.05743***
Within populations 172 73.600 0.42791 45.02 FST = 0.54983***
Total 250 215.108 0.95054
(3) Magnolia hypoleuca (K = 3)
Among groups 2 88.399 0.62445 71.55 FCT = 0.71548***
Among populations within groups 72 20.720 0.01984 2.27 FSC = 0.07991***
Within populations 151 34.500 0.22848 26.18 FST = 0.73822***
Total 225 143.619 0.87278
(4) Carpinus laxiflora (K = 3)
Among groups 2 138.404 0.80934 84.84 FCT = 0.84842***
Among populations within groups 68 8.274 -0.00863 -0.91 FSC = -0.05971***
Within populations 191 29.268 0.15324 16.06 FST = 0.83936***
Total 261 175.947 0.95394
Geographical units of each K are shown in Figs. 3 and 4
df degrees of freedom
*** P \ 0.001

(the western part of the Chugoku region, and the Kinki,
Shikoku and Kyushu regions), although the genetic com-
ponents were often continuously distributed in other areas
such as the Hokkaido, Tohoku, or Chubu regions. As a
consequence of prolonged isolation, extant plant popula-
tions derived from different refugia should be highly
divergent from each other. The three regional populations
should thus originate from several independent refugia.
These hypothetical refugia were probably located in the
seven regions with high PR values as detailed in the above
section, and contributed to the three independent migration
routes (the Sea of Japan-side area, the Kanto region, and
southwestern Japan) after LGM.
In contrast to the similarities observed for the three
specific areas cited, remarkable similarities were not
observed in the other regions; that is, Hokkaido, the
northern part of the Tohoku region, and the Chubu region.
For example, for each of the three species (P. grayana,
M. hypoleuca, and C. laxiflora) with common haplotypes
that were chiefly distributed in the Tohoku region, only one
haplotype was found in Hokkaido. The simple haplotype
composition of the three species in Hokkaido might be due
to founder effects during recent migration across the sea
from Honshu, whereas the Hokkaido population of
E. oxyphyllus might have not experienced such founder
effects. To clarify migration histories in such areas, further
phylogeographic studies using more variable markers
(e.g., nuclear markers) or using other species are needed.
Influence of past range expansion during LGM
in southwestern Japan
Some inferences can be drawn from comparisons among
the seven plant taxa that have been examined to date [four
species examined in this study and three taxa examined in
previous studies; F. crenata (Fujii et al. 2002), S. praecox
(Ohi et al. 2003), and Q. crispula and related species
(Okaura et al. 2007)]. The distribution patterns of some
rare haplotypes were similar among several species, and
their distribution range was confined to the three local areas
in southwestern Japan. The areas were: (1) the Kii Penin-
sula and nearby areas, (2) areas from the Chugoku region to
the northern part of the Kyushu region, and (3) areas from
the Kii Peninsula or Shikoku to the southern part of Kyu-
shu. For (1) the Kii Peninsula and nearby areas, five hap-
lotypes were observed in the four species: rare haplotype G

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Fig. 3 The geographical locations of SAMOVA groups and the grayana and b Euonymus oxyphyllus. The number near the point in
UPGMA dendrogram based on the relative Sorensen distance matrix the figure corresponds to population number
between groups of populations obtained by SAMOVA in a Padus

Fig. 4 The geographical

locations of SAMOVA groups
in a Magnolia hypoleuca and
b Carpinus laxiflora. Number
near point in figure corresponds
to population number

in P. grayana, type F and J in E. oxyphyllus, type F in distributed in the Kanto region). For (2) areas from the
F. crenata (although disjunctively distributed in the Kanto Chugoku region to the northern part of the Kyushu region,
region), and type I in S. praecox (although disjunctively five haplotypes were observed in four taxa: rare haplotype

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J Plant Res (2012) 125:207–221 219

D in E. oxyphyllus (although not distributed in the northern
part of the Kyushu region), type H in E. oxyphyllus, type D
in M. hypoleuca, type I in F. crenata, and type V in
Q. crispula and related species. For (3) areas from the Kii
Peninsula or Shikoku to the southern part of the Kyushu
region, five haplotypes were observed in five taxa: rare
haplotype F in P. grayana, common haplotype C in
E. oxyphyllus, rare haplotype L in F. crenata, type C in
S. praecox, and type VI in Q. crispula and related species.
Two possible considerations could explain the sharing
of rare haplotypes by nearby populations separated by
areas of sea: either these populations originated from a
large continuous population after LGM, or there currently
exists a large amount of gene flow between these
populations. Deciduous broad-leaved forests in south-
western Japan now occur only in high mountains (over
700 m). In view of this patchy distribution, the latter pos-
sibility seems unlikely. Moreover, during LGM, due to
lower sea levels (ca. 100 m below present), Shikoku and
Kyushu were continuous with Honshu, and the continental
shelf, ca. 20–30 km from the present coastline, emerged
around the Japanese archipelago (Ohta and Yonekura
1987). In addition, palynological studies have suggested
that in southwestern Japan, most of the deciduous broad-
leaved tree species migrated to the coastal areas on the
Pacific Ocean and the Sea of Japan-side during LGM
(Kamei and Research Group for the Biogeography from
Würm Glacial 1981; Tsukada 1988). As the climate
warmed, they recovered and moved to higher altitudes
(Takahara et al. 2000; Tsukada 1988). Therefore, our
results support the former hypothesis and suggest the fol-
lowing scenario. In southwestern Japan, continuously dis-
tributed deciduous broad-leaved forests existed in the
following three regions during LGM: (1) the Kii Peninsula
and nearby areas, (2) areas from the Chugoku region to the
northern part of the Kyushu region, and (3) areas from the
Kii Peninsula or Shikoku to the southern part of the Kyu-
shu region. As the climate warmed, these forests moved to
higher altitudes and were split into the several small pop-
ulations that are now observed in southwestern Japan.

Migration scenario of Japanese deciduous broad-leaved

forests during and after LGM

By taking all of the results obtained in this study, as well as

Fig. 5 Distribution pattern of the interspecific average values of PR
among the four species in each grid cell. The haplotype composition
of the nine grid cells (HR8, KT6, CB4, KI4, CG2, CG6, and CG7) are
summarized in Table 4
previous palynological data, into consideration, we can
postulate the following scenario for the migration history
of Japanese deciduous broad-leaved forests (Fig. 6). Dur-
ing LGM, separate forests occurred in the following six
regions: (I) the area surrounding Wakasa Bay (corresponds
to HR8 grid), (II) the southern part of the Kanto region and
nearby areas (corresponds to KT6 and CB4 grids), (III) the

Table 4 Haplotype composition of the nine grid cells which show relatively high PR values (PR [ 0.300)
Grid Region PR Haplotype composition
Padus grayana Euonymus oxyphyllus Magnolia hypoleuca Carpinus laxiflora

HR8 Hokuriku 0.312 AAAABBBA00 A00 A00 A00 A00 HHH BBDDIIR BBCCCCCCCCCC AAAABBBBBBBBBBBBBB
KT6 Kanto 0.400 A0 A0 E0 E0 – CCCCG
CB4 Chubu 0.404 – S AAA BBBBBBBBBBBBBBBB0 B0 DD
0
KI4 Kinki 0.376 BBFGGGGGG AACFFFF JNNP AAABBBBBBBBBB AAAAAAAAAAAAAAA
CG2 Chugoku 0.333 AB00 B00 FM AB0 B0 B0 D BCC –
CG6 Chugoku 0.333 BB – D AA
CG7 Chugoku 0.350 BBBBB DDDHH BBBDDD AAAAAA
Bold letters designate rare haplotypes with a frequency of 5% or less

123
220
Fig. 6 Migration scenario of Japanese deciduous broad-leaved
forests a during LGM and b after LGM
Kii Peninsula and nearby areas (corresponds approximately
to KI4 grid), (IV) the continuous area from the western part
of the Chugoku region to the northern part of Kyushu
(corresponds partially to CG2, CG6, and CG7 grids),
(V) Shikoku, and (VI) the southern part of Kyushu [(5) and
(6) were not supported by PR values, but suggested from
the former palynological studies] (Fig. 6a). After LGM, as
the climate warmed, the forests in eastern Japan separately
expanded from each of the refugia along the Sea of Japan-
side or along the Pacific Ocean-side. They subsequently
expanded to the northern part of the Tohoku region and the
Hokkaido region (Fig. 6b). In contrast, the forest in
southwestern Japan retreated and moved to high altitudes
from each of the continuous forests. Our suggested sce-
nario, based on comparisons among the several species
studies, should represent the major migration scenario for
Japanese deciduous broad-leaved tree species.
Acknowledgments We thank Dr. Noriyuki Fujii, Dr. Naoki Ni-
shimura, Dr. Koji Yonekura, Mr. Kozo Shibata, Dr. Tomoko Fukuda,
Dr. Wataru Shinohara, Dr. Hirotoshi Sato, Dr. Hiroaki Setoguchi, Dr.
Makoto Mochida, Mr. Tomoki Kadokawa and Dr. Hiroshi Ikeda for
their assistance with the collection of plant materials. We also thank
Dr. Takashi Sugawara, Dr. Hidetoshi Kato, Mrs. Saeko Kato, Dr.
Ikuyo Saeki, Dr. Yasuyuki Watano, Dr. Vatanaparast Mohammad,
and anonymous reviewers for their valuable advices. This study was
partly supported by Research Project ‘A new cultural and historical
exploration into human-nature relationships in the Japanese archi-
pelago’ of the Research Institute for Humanity and Nature and Grant-
in-Aid for JSPS Fellows 19-5749 to TI.
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