Molecular Ecology (2003) 12, 859–868
Nuclear gene sequences and DNA variation of Cryptomeria
japonica samples from the postglacial period
Blackwell Publishing Ltd.
N A O K I T A N I ,* Y O S H I H I K O T S U M U R A * and H I T O S H I S A T O †
*Department of Forest Genetics, Forestry and Forest Products Research Institute, Matsunosato, Tsukuba, Ibaraki 305–8687 Japan,
†Landscape and Natural Reservation Section, Environment and Life Division, Shimane Prefecture, Matsue, Shimane 690–8501 Japan
Abstract
Genomic DNA was extracted from heartwood blocks of six Cryptomeria japonica individuals
that had been buried (in an area now covered by rice fields) for about 3600 years. Attempts
were made to determine the sequences of five nuclear genes following polymerase chain
reaction amplification, using previously obtained C. japonica expressed sequence tag (EST)
information. We detected 15 nucleotide substitutions and four insertion/deletions (indels)
in a partial GapC gene sequence among 13 individuals of the buried and an extant population,
which allowed us to estimate the extent of DNA variation within the buried populations, and
the level of genetic differentiation between the buried population and the extant population
growing in a neighbouring area. For the entire haplotypes of the GapC region, π and θ
nucleotide diversity estimates were 0.0063 and 0.0010, respectively, when both populations
were included, while corresponding figures for the buried population alone were 0.0009
and 0.0017. Estimates of DNA divergence statistics (dXY = 0.0062, dA = 0.0005, FST = 0.0832
and KST = 0.0935) suggest that differentiation between the two populations was not great.
However, permutation tests gave FST and KST values rejecting the null hypothesis (that
populations were not differentiated) at the 5% and 1% probability levels, respectively. The
significant genetic differentiation between the two populations was mainly caused by differences in haplotype diversity. The significant level of haplotype diversity in the extant
population compared to the buried population might be the result of gene flow from neighbouring artificial forests. Alternatively, it is possible that we failed to detect all the DNA
variation in the buried population because of clonal growth in the buried population.
Keywords: ancient DNA, buried forest, Cryptomeria japonica, DNA variation, GapC, PCR
Received 15 August 2002; revision received 10 December 2002; accepted 10 December 2002
Introduction
The most common use of ancient DNA has been directed
at the systematics of extinct species (reviewed in
Wayne et al. 1999). For example, cytochrome b sequences
demonstrated that the extinct Tasmanian wolf was closely
related to other Australian marsupials (Thomas et al. 1989).
Krings et al. (1997) demonstrated that Neanderthals
were phylogenetically divergent from recent humans. In
contrast, little interest has been shown in less ancient DNA,
isolated from fossils of extant species, for studies of
systematics, although it has been used to distinguish
species or to help probe their geographical origins. For
Correspondence: Yoshihiko Tsumura. Fax: 81–29–8743720; E-mail:
[email protected]
© 2003 Blackwell Publishing Ltd
instance, spacer regions and genes on the chloroplast
genome have been used in two studies (Suyama et al. 1996;
Poinar et al. 2001) for identifying species, because we can
seek which chloroplast sequences on public databases are
identical with the fossil chloroplast sequences. These
sequences are the most useful for identifying species
following successful polymerase chain reaction (PCR)
amplification from fossil sources. Thus, sequencing chloroplast DNA offers enormous research possibilities in the
fields of molecular evolution and systematics. However,
it is not useful for population genetic analysis related to
aspects such as genetic differentiation between extant and
extinct populations, or for probing the genetic structures
of several hierarchic levels of extinct populations because
of the low nucleotide substitution rate of chloroplast DNA
(Curtis & Clegg 1984; Palmer 1985). For these purposes,
860 N . T A N I , Y . T S U M U R A and H . S A T O
the amplification and subsequent sequencing of genes on
the nuclear genome would be more desirable, because
nucleotide and haplotype diversities of each gene between
individuals and/or populations could then be obtained,
allowing efficient evaluation of genetic diversity in terms
of population genetics and molecular evolution.
Natural populations of Cryptomeria japonica D. Don. are
discontinuously distributed from the northern part of
Honshu (the main Japanese island) to Yaku Island, which
is located 100 km off the coast of southern Kyushu
(Hayashi 1960). Although C. japonica covers a vertical range
from 0 to 1850 m above sea level, dense natural forests of
C. japonica are restricted to a montane zone. However, the
cool period following the hypsithermal climatic interval
(2000–5000 years ago) allowed the habitat of C. japonica to
be extended to alluvial plains, as evidenced by a buried forest found under rice fields at the foothill of Sanbe volcano,
Shimane prefecture in November 1998. The cause of the
burial was a pyroclastic flow (about 3600 years ago) that
dammed the Azukihara River, leading to the forest being
rapidly covered by water, earth and sand. The wood of the
trees has been well preserved to the present day. Hence, it
is possible to investigate the genetic structure of the buried
population and assess genetic differentiation between the
buried and extant populations.
Here, we describe the successful isolation of ancient
DNA from wood tissues of the buried trees, PCR amplification and sequencing of nuclear genes. We also studied
the differentiation between the buried and extant populations, and considered the genetic structure within the
buried population. This is the first report of successful
sequencing analysis of nuclear genic regions of fossil trees.
Materials and methods
Plant materials and molecular methods
The buried forest at Sanbe–Azukihara was discovered in
1998 (Figs 1, 2). Some trees buried under rice fields have
been exhumed for investigation and an exhibition has been
mounted at Sanbe Natural History Museum by Shimane
prefecture, Japan. In our study, blocks of these trees were
taken, and immediately stored at −30 °C, until genomic
DNA was isolated using a DNeasy plant mini-kit (Qiagen
GmbH) from 400 mg of each individual wood chip
(Dumolin-Lapègue et al. 1999). The DNA samples were
then diluted 1 : 20 with water. In addition, needles were
sampled from an extant population located on the hillside
of Sanbe Volcano, at a considerably higher altitude (≈ 600–
800 m) than the level of the excavation (≈ 250 m; Fig. 1),
because no natural Cryptomeria forests currently occur at
lower elevations. Needles were collected individually,
with more than 30 m between sampled trees to avoid
erroneous sampling of half-sib and/or identical ramet
individuals. Genomic DNA of these samples was also
isolated from 5 g portions of fresh leaf tissues using the
modified CTAB method of Murray & Thompson (1980).
For preliminary screening, we tried to amplify 17
nuclear genes that give PCR product sizes of less than
600 base pairs (bp), because the DNA isolated from buried
wood tissues tends to be degraded. The primer pairs for
these 17 nuclear genes yielded a single band when genomic
DNA extracted from extant samples were used as template
samples for PCR. The sequences of the primers were
derived from expressed sequence tag (EST) information
generated by the Sugi genome project (Iwata et al. 2001) or
supplied by Professor Tachida, Kyushu University, Japan
(Table 1). Attempts were made to amplify these genes from
nine DNA samples of buried trees and eight DNA samples
of natural trees from a sympatric region under the following PCR conditions: initial denaturing at 94 °C for 5 min,
followed by 40 cycles of 94 °C for 1 min, 55 °C to 60 °C
(depending on primer pair) for 1 min and 72 °C for 1 min
30 s, with a final extension step of 72 °C for 5 min. All
reactions were performed in a Perkin Elmer thermal
cycler with a Mg2+ concentration of 1.5 mm. Subsequently,
PCR products that were confirmed as single bands in
2.0% agarose gel electrophoresis were cleaned up using
a QIAquick PCR Purification kit (QIAGEN GmbH) to
remove primers and residual dNTPs. Purified PCR products
were sequenced using a Big dye deoxy-terminator cycle
sequence kit (Perkin Elmer), following the manufacturer’s
recommendations, and the corresponding primer for each
gene on an Applied Biosystems Model 377 automated
sequencer (Applied Biosystems). When heterozygotes
were detected, PCR products were cloned into the pCRTOPO vector using TOPO™ TA Cloning® Kits (Invitrogen).
Plasmids from single colonies were prepared as templates
for cycle sequencing using Quantum prep plasmid purification kits (Bio-Rad Laboratories Inc.), and eight plasmids
per heterozygote were independently sequenced with the
M13 reverse primer, following the manufacturer’s method
for the Big dye deoxy-terminator cycle sequence kit (Perkin
Elmer). Sequences were analysed on an ABI 377 sequencer.
Newly determined sequences were deposited in the EMBL
data bank under nos AJ517805–AJ517812.
Sequence analysis
Sequences used in this study were aligned by clustal w
version 1.6 computer software (Thompson et al. 1994),
followed by manual adjustment to reduce further the
number of insertions and deletions. The polymorphism
data were analysed by dnasp version 3.50 computer
software (Rozas & Rozas 1999). To assess genetic variation
within the populations we estimated the following genetic
diversity statistics: haplotype (gene) diversity using the
methods of Nei (1973) and Nei & Chesser (1983), the
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859 –868
D N A V A R I A T I O N O F A N C I E N T C R Y P T O M E R I A J A P O N I C A 861
Fig. 1 Contour map showing the location
of Sanbe buried forest and the sampling
area of the natural extant population of
Cryptomeria japonica.
Fig. 2 Locations of buried trees at the
excavation site of Sanbe buried forest. Solid
circles indicate buried trees from which
genomic DNA was isolated and successfully sequenced. Shaded circles indicate
buried trees from which it was impossible
to isolate genomic DNA. Open circles
indicate buried trees that have not been
investigated.
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859–868
Polymophism
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859 –868
Index of
EST
marker*
Putative function
Primer
CC0254
Thaumatin-like protein precursor
CC0256
No match on database
CC0257
Ephrin type-B receptor 6 precursor
CC0288
(R)-mandelonitrile lyase isoform 1 precursor
CC0349
Harpin induced gene 1 homologue
CC0392
Eukaryotic translation initiation factor 3 p42 subunit
CC0411
Homeobox protein BIX3
CC0420
Ribose 5 phosphate isomerase
CC0498
Peptidyl-prolyl cis-trans isomerase
CC1333
GTP-binding protein
CC1348
Peptidylprolyl isomerase
CC1439
Nine-cis-epoxycarotenoid dioxygenase
CC1522
Alpha-glucosidase precursor
CC1606
R2R3-MYB transcription factor
CD1514†
Chalconsintase
CD1706†
Fedoroxine
CC2241†
Glyceraldehyde-3-phosphate dehydrogenase
5′-GGCCACAACGTTCTGCTAAAT-3′
5′-GGCGGGCGTAACCATC-3′
5′-GGCATGCCCTTCTTCAAT-3′
5′-ATGACCGGATAAAGCCAAAT-3′
5′-GCCCTCGACGATGTGTGGAC-3′
5′-CACGAAACCTTCCGCCAGTG-3′
5′-TTTGTGATTTCATGCCTGCTA-3′
5′-GAAATCCAAGCTTGCCCTAC-3′
5′-GCGGATGCCTGATGTTCTTGA-3′
5′-CAGGACGCCGGTCTGTTGAT-3′
5′-CGCGATAAACCTAAGCTACAA-3′
5′-AATGGACCAGAAGACAGGAG-3′
5′-GATGAGCCGAGTATTGGTTAT-3′
5′-GATTTCATCGCCTTGGTT-3′
5′-TGGCGGCAGCAGCATTTA-3′
5′-GCCAGGTCAATCTTCGGATGA-3′
5′-GGCCGAATCGTGATGGA-3′
5′-GGGCAACTTAGAAGGGCATTA-3′
5′-CTCCTAGATCAGCGTATCACA-3′
5′-CCCTTTCTTGACCAGCAG-3′
5′-GCCGAATCGTGATGGA-3′
5′-GCAACTTAGAAGAGGGCAACT-3′
5′-GCATGGGAGTGGCGAATG-3′
5′-AGCTGAGCTTCACCGCAGAAT-3′
5′-GGACTCTGTAGCCAAATCAG-3′
5′-AAGTTAATGTCTTGCCGAATA-3′
5′-TAACCAGCTTTGCCCTCAG-3′
5′-ATACAATTCGCGGCTACCATA-3′
5′-GGTCGGTCTGACATTCCATT-3′
5′-CGAGAAGCGTCCAAACATTA-3′
5′-ATAGGCGACGCAGGTCAAAA
5′-TCTGCGGCTGTAGTTCCAGT-3′
5′-CAGATTTCATTGGAGACAGC-3′
5′-CAAATCTATGACTCGTGAAC-3′
*For details of primers with CC and CD initials see Iwata et al. (2001).
†Primer pair supplied by Dr Tachida, Kyushu University.
‡Estimated product size was deduced by 2.0% agarose gel electrophoresis.
Expected
product
size‡
Amplification
Sequencing
Buried
population
Extant
population
250 bp
Amplified
Success
Monomorphic
1 polymorphic site
380 bp
Amplified
Success
Monomorphic
1 polymorphic site
420 bp
Multiple
—
—
450 bp
Impossible
—
—
320 bp
Impossible
—
—
450 bp
Multiple
—
—
500 bp
Multiple
—
—
400 bp
Amplified
Success
Monomorphic
600 bp
Impossible
—
—
600 bp
Amplified
Success
Monomorphic
600 bp
Multiple
—
—
1300 bp
Multiple
—
—
900 bp
Impossible
—
—
900 bp
Impossible
—
—
500 bp
Impossible
—
—
400 bp
Impossible
—
—
400 bp
Amplified
Success
Polymorphic
1 polymorphic site
1 polymorphic site
15 polymorphic sites
862 N . T A N I , Y . T S U M U R A and H . S A T O
Table 1 Screened EST markers and results of PCR amplification and subsequent sequencing
D N A V A R I A T I O N O F A N C I E N T C R Y P T O M E R I A J A P O N I C A 863
Fig. 3 A 0.8% agarose gel following genomic DNA electrophoresis and ethidium bromide staining. No DNA molecules were detected in
lanes 1–3, while fragmented DNA molecules were visible in lanes 4 and 5.
number of pairwise differences between sequences (per
base estimates of nucleotide diversity, π; Nei & Li 1979;
Tajima & Nei 1984) and scaled neutral mutation rate
(θ = 4Neµ; Watterson 1975; Nei 1987; Tajima 1993). The
extent of DNA divergence between the buried and extant
populations was estimated as the average number of
nucleotide substitutions per site between populations (dXY),
and the number of net nucleotide substitutions between
the two populations (dA; Nei 1987). When statistics of
nucleotide diversity were estimated, insertions and deletions
were ignored. To test for population differentiation between
the two populations, we used permutations described by
Hudson et al. (1992a). FST (Hudson et al. 1992b) and KST
(Hudson et al. 1992a) statistics are based on the frequencies
of individual segregation sites in two populations. For each
test, 10 000 permutations of the data were carried out, and
proseq version 2.9 software was used (Filatov 2001). To
visualize relationships among haplotypes of both buried
and extant trees, a minimum spanning network was
constructed using tcs version 1.13 (Clement et al. 2000). All
indels were ignored when the network was constructed.
Results
Isolation, PCR amplification and sequencing of DNA
from buried wood
Although 0.8% agarose gel electrophoresis showed the
isolated genomic DNAs to be severely degraded (Fig. 3),
fragmented DNA molecules could be recovered from
six out of nine excavated trees, designated nos. A-2, A-3,
A-4, A-5, A-9 and A-10, from the buried population. It
was impossible to isolate genomic DNA from the remaining three excavated trees (A-1, A-7, A-19; Fig. 2). Single
bands in subsequent 2.0% agarose gel electrophoresis
were generated by each of five pairs of primers (CC0254,
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859–868
CC0256, CC0420, CC1333 and GapC exon 4 to exon 6), and
multiple bands by another five (CC0257, CC0392, CC0411,
CC1348 and CC1439) out of 17 pairs tested (Table 1). The
other seven did not amplify any detectable fragments
(Table 1). Each PCR product from the five pairs of primers
that gave single bands was subsequently sequenced, but
no nucleotide substitutions among samples from the six
individuals in the buried forest were detected in four of the
five genes. We observed only one nucleotide substitution
(CC0254 and CC1333) or indel (CC0256 and CC0420)
between individuals within the extant population with
four of the five genes. One of the haplotypes detected in
each gene was identical with the sequence of each gene
of the buried population. Another haplotype is newly
detected with low frequency. On the other hand, DNA
polymorphisms were detected in the 391-bp GapC gene
sequence among the buried tree haplotypes.
Gene diversity and DNA variation within buried and
extant populations
The 391-bp fragment comprises two introns and one exon,
defined by comparison with the glyceraldehyde-phosphate
dehydrogenase mRNA sequence of Taxus baccata (GenBank
Acc. No. L26922; Jansson et al. 1994) and the GapC2 gene
sequence of Zea mays (GenBank Acc. No. X73151). We found
only two synonymous substitutions in exon 5, which is 80 bp
long (Fig. 4). Because we detected sufficient polymorphisms
concerning only GapC gene sequence, we only measured
gene diversity and DNA variation statistics in this gene. In
the exon 4 to exon 6 region of the GapC gene we found 19
variations within the buried and extant populations of
Cryptomeria japonica: 15 nucleotide substitutions and four
indels (Fig. 4). Three nucleotide substitutions and one indel
were singletons, and there were 15 nonsingleton variations
(12 nucleotide substitutions and three indels). Of the
864 N . T A N I , Y . T S U M U R A and H . S A T O
Fig. 4 Alignment of partial GapC region of
Cryptomeria japonica. Dots indicate identical
nucleotides to haplotype 1. Bars indicate
deletions.
nonsingleton variations, six nucleotide variations and two
indels (at positions 21, 47, 49, 67, 77, 81, 83 and 107) were
uniquely detected in haplotypes 4 and 5, creating a bimorphic pattern of DNA variation (Fig. 4).
All of the buried trees were homozygotes with respect to
the GapC sequence, except A4, which was heterozygous.
Two nucleotide substitutions at 361 bp and 376 bp and one
indel at 372 bp to 373 bp were responsible for differences
between haplotype 2 (detected in A4) and haplotype 1
(typical buried tree type; Fig. 4). In contrast, seven distinct
haplotypes were observed in the seven extant trees collected from the hillside of Sanbe volcano. Of the seven extant
trees, five and two were heterozygous and homozygous,
respectively.
Table 2 summarizes estimates of DNA variation at the
GapC gene of the buried C. japonica population and the
extant population. Estimates of nucleotide diversity of
the GapC region for the entire haplotypes, including both
buried and extant populations, were 0.0063 (π) and 0.0010
(θ), while the corresponding figures were 0.0009 and 0.0017
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859 –868
0.4083 NS
−1.7204 NS
0.8070 NS
−0.6890 NS
—
−0.4457 NS
0.4083 NS
−1.7204 NS
0.6167 NS
Tajima’s D‡
−1.3709 NS
−1.4514 NS
−0.5401 NS
−1.2239 NS
—
−0.9592 NS
−1.3709 NS
−1.4514 NS
−0.6672 NS
θ
0.0104
0.0017
0.0142
0.0057
0.0000
0.0068
0.0010
0.0017
0.0125
π
0.0063
0.0009
0.0123
0.0024
0.0000
0.0044
0.0063
0.0009
0.0104
0.665 (0.010)
0.167 (0.134)
0.890 (0.055)
0.151 (0.093)
0.000 (0.000)
0.275 (0.148)
0.665 (0.010)
0.167 (0.134)
0.890 (0.055)
DNA divergence between the buried and extant
populations and phylogenetic relationships of the
haplotypes
Three individuals of the extant population displayed the
most common haplotype of the buried population, haplotype 1, while haplotype 6 detected in the extant population differed only at the 261st nucleotide position
from a unique haplotype of the buried population,
haplotype 2 (Fig. 4). Thus, DNA polymorphism within the
extant population included nearly all of the range of DNA
polymorphisms found within the buried population. The
estimates of DNA divergence statistics (dXY = 0.0062,
dA = 0.0005, FST = 0.0832 and KST = 0.0935) suggest that
there was not much differentiation between the two
populations (Table 3). When we treated indels as single
mutations, the levels of population differentiation rose a
little. When we estimated population differentiation levels
solely for the intronic region, the values also rose. FST and
KST values derived from permutation tests rejected the
null hypothesis, that populations were not differentiated,
at the 5% and 1% probability levels, respectively. Because
11
0
11
1
0
1
12
0
12
Table 3 DNA divergence in the GapC partial seqence between
buried and extant populations of Cryptomeria japonica
Entire region
*Number of sampled accessions.
†Number of polymorphic sites.
‡ns, not significant.
2
2
2
1
0
1
3
2
3
13
2
13
2
0
2
15
2
15
Intronic region
Intronic region
Intronic region
Exonic region
Exonic region
Exonic region
Entire region
Entire region
Entire region
26
12
14
26
12
14
26
12
14
Buried and extant
Buried
Extant
Buried and extant
Buried
Extant
Buried and extant
Buried
Extant
Region
n*
Population
for the buried population, respectively, and those for the
extant population were 0.0104 and 0.0125. Most of the
DNA variations occurred in the intronic region, so nucleotide diversity in this region was greater than in the exon
(Table 2). Table 2 also summarizes results of the Tajima’s
(1989) and Fu & Li’s (1993) tests. All Tajima’s D-values were
negative, but not significantly so, against the null hypotheses
of neutrality and population equilibrium. Fu and Li’s
test also gave results that were not statistically significant. However, while Tajima’s D statistics were negative
for the pool of buried and extant populations, Fu and Li’s
D statistics were positive for these populations. The conflict between Tajima’s and Fu and Li’s Ds showed that the
D-values were almost equal to expectations in the tests.
4
1
4
0
0
0
4
1
4
Fu and Li’s D‡
Neutrality test
Haplotype
diversity
Singleton
site
Polymorphic
site
Informative
site
No. of
indels
Nucleotide
diversity
S†
Table 2 DNA polymorphisms, nucleotide diversity and neutrality tests of GapC partial sequence in C. japonica populations
D N A V A R I A T I O N O F A N C I E N T C R Y P T O M E R I A J A P O N I C A 865
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859–868
dXY
dA
FST†
KST†
Intronic region
Indels
ignored
Indels
as single
mutation
Indels
ignored
Indels
as single
mutation
Exon
0.0062
0.0005
0.0832*
0.0935**
0.0078
0.0007
0.0834
0.0935*
0.0074
0.0006
0.0846*
0.0978**
0.0090
0.0011
0.1197*
0.1332**
0.0023
0.0001
0.0513
0.0232
†The significance of these statistics was tested by
10 000 permutation tests following Hudson et al. (1992a).
*P < 0.05, **P < 0.01.
866 N . T A N I , Y . T S U M U R A and H . S A T O
fragments of at most a few hundred nucleotides (Pääbo
1985, 1989; Pääbo et al. 1988; Hagelberg et al. 1989; Lawlor
et al. 1991). Our samples were exhumed from a 3600-yearold layer, and agarose gel electrophoresis showed that the
extracted DNA was fragmented. We successfully amplified
PCR fragments less than about 500 bp long, which is
consistent with previous PCR amplifications of nuclear
genomic regions using ancient DNA.
Fig. 5 Haplotype network (cladogram) of partial GapC sequences
of the buried and extant Cryptomeria japonica constructed under
the statistical parsimony criterion. Haplotype numbers (Hap 1 and
so on) correspond to those given in Fig. 4. Each line in the network
represents a single mutational change between haplotypes. Small
solid circles indicate a haplotype not present in the sample,
but inferred to be intermediate between two nearest-neighbour
haplotypes. The size of each circle and the number at the lower
right side of the circle indicate number of individuals retaining
each haplotype. All haplotypes within the buried population are
included in Haplotype 1 and 2.
the major haplotype within the buried population was
also detected in the extant population and the extant
population retained the very similar haplotype as another
haplotype within the buried population (Fig. 4), significant
population differentiation was caused by differences
in haplotype diversity between the extant and buried
populations. An estimation of genetic relationships among
eight haplotypes was obtained by the minimum spanning
network based on nucleotide substitutions in the 391-bp
region of the GapC gene. Haplotypes 4 and 5 were located
furthest from the centre of the network. Seven nucleotide
substitutions were required to evolve haplotypes 4 and 5
from the major haplotype 1 located at the centre. The two
haplotypes observed in the buried population were not
greatly differentiated (two steps), and were similar to four
extant haplotypes around haplotypes 1 and 2 that were
detected in the buried population (Fig. 5)
Discussion
Successful PCR amplification of nuclear DNA from fossils
Based on in vitro analysis of the rate of DNA degradation,
it has been suggested that the molecule could not persist
for more than 104−105 years in most geological settings
(Pääbo & Wilson 1991; Lindahl 1993). To date, only small
fragments of nuclear DNAs (including the HLA gene and
a long nucleotide tandem repeat) have been amplified,
from fossils up to about 7500 years old (Lawlor et al. 1991;
Kurosaki et al. 1993; Fischer et al. 2001). This is because
ancient DNA is usually only available in low quantities in
Population history, from the buried population to
the extant population
Haplotype 1 was the major type of the buried population,
and it was also detected in the extant population, while
haplotype 2 was detected only in the buried population. No other haplotypes were observed in the buried
population (Fig. 4). We believe there are two possible
reasons for several haplotypes being detected only in the
extant population. Firstly, clonal growth in the buried
population may have been responsible for the limited
DNA variation detected within the buried population. In
support of this hypothesis, clonal growth has been shown
to be responsible for some propagation of Cryptomeria
japonica, according to allozyme and DNA marker analysis
in a high-altitude region with heavy snow (Taira et al. 1997;
Moriguchi et al. 2001). Global warming in the postglacial
period caused sea levels to rise, and led to the inflow of a
warm current into the Sea of Japan about 8000 years ago.
Subsequent increases in the temperature of the Sea of Japan
caused snowfall on that side of southwestern Japan to rise
(Yasuda 1982). Many pollen analyses of core samples have
shown that C. japonica forest developed on the Sea of Japan
side of southwestern Japan from 4000 years to 2000 years
ago (Miyoshi & Hada 1975; Hatanaka & Miyoshi 1980;
Tsukada 1982; Sugita & Tsukada 1983; Miyoshi & Yano
1986). Moreover, it was observed that a tree with a thin
diameter (A19) was connected to a neighbouring tree with
large diameter (A9) in the ground (Fig. 2). Therefore, clonal
growth may have occurred, in an environment with moist
and heavy snow at this time, which may have led to
extension of clonal growth in the buried population. Such
clonal growth would have hindered detection of the total
DNA variation within the buried population. If so, all the
haplotypes detected in the extant population might also
have existed in the buried population.
Alternatively, the unique haplotypes in the extant population may have originated from migration from surrounding populations. Japanese people have been clearing forest
lands to construct artificial C. japonica forests for about
400 years (Ohba 1991), and the extant population is
currently surrounded by artificial forests of C. japonica.
Tomaru et al. (1994) made an indirect estimate of the mean
number of migrants exchanged per generation (Nm) based
on allozyme data derived from 17 natural populations of
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859 –868
D N A V A R I A T I O N O F A N C I E N T C R Y P T O M E R I A J A P O N I C A 867
C. japonica, obtaining a mean value of 7.1. The estimates of
Nm suggest that gene flow may have occurred frequently,
probably even between natural and artificial forests. The
minimum spanning network indicated that haplotype 1 is
the most common allele and arguably the oldest in the samples, because haplotype 1 became central and had the most
connections. The remaining haplotypes were connected to
haplotype 1 within fewer than three steps of mutations
except for haplotypes 4 and 5 (Fig. 5). Because seven mutations were needed to evolve the remote haplotypes 4 and 5
from the central haplotype 1 in the network and four intermediate haplotypes were missed from haplotype 7 to haplotypes 4 and 5, a reasonable speculation was that these
haplotypes had migrated from some other populations.
The origin of unique haplotypes in the extant population is
unknown, so further extensive study of population genetic
analysis using DNA sequences will focus on defining the
intensity and extent of gene flow and genetic structure of
C. japonica.
Acknowledgements
The authors are particularly grateful to Dr H. Tachida, Ms J.
Kusumi, Mr T. Kado, Dr Y. Suyama, Dr H. Yoshimaru and Dr
H. Iwata for their help in collecting samples, and to Dr H. Tachida
for supplying primers to amplify the GapC gene. We also thank
two anonymous referees for their perceptive suggestions on the
latest version of the manuscript. This study was supported by a
grant from the Programme for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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N. Tani is working on the sugi (Cryptomeria japonica) genome
project. His other current research interests are a population
genetic study of five-needle pines and conservation genetics for
endangered species in oceanic islands. Y. Tsumura is the leader of
the sugi genome project, whose other current research interests
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environmental policy in Shimane prefecture and is directing the
excavation and exhibition of Sanbe buried forest.
© 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 859 –868