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Biological Journal of the Linnean Society, 2013, 108, 263–277. With 6 figures
Northern richness and southern poverty: contrasting
genetic footprints of glacial refugia in the relictual tree
Sciadopitys verticillata (Coniferales: Sciadopityaceae)
JAMES R. P. WORTH1*, SHOTA SAKAGUCHI1, NOBUYUKI TANAKA2,
MICHIMASA YAMASAKI1 and YUJI ISAGI1
1
Laboratory of Forest Biology, Division of Forest and Biomaterials Science, Graduate School of
Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku Kyoto 606-8502, Japan
2
Department of Plant Ecology, Forestry and Forest Products Research Institute, 1, Matsunosato,
Tsukuba, Ibaraki 305-8687, Japan
Received 7 April 2012; revised 8 August 2012; accepted for publication 8 August 2012
Sciadopitys verticillata is amongst the most relictual of all plants, being the last living member of an ancient conifer
lineage, the Sciadopityaceae, and is distributed in small and disjunct populations in high rainfall regions of Japan.
Although mega-fossils indicate the persistence of the species within Japan through the Pleistocene glacial–
interglacial cycles, how the species withstood the colder and drier climates of the glacials is not well known. The
present study utilized phylogeography and palaeodistribution modelling to test whether the species survived within
pollen-based coastal temperate forest glacial refugia or within previously unidentified refugia close to its current
range. Sixteen chloroplast haplotypes were found that displayed significant geographical structuring. Unexpectedly,
northern populations in central Honshu most distant from coastal refugia had the highest chloroplast diversity and
were differentiated from the south, a legacy of glacial populations possibly in inland river valleys close to its current
northern range. By contrast, populations near putative coastal refugia in southern Japan, harboured the lower
chloroplast diversity and were dominated by a single haplotype. Fragment size polymorphism at a highly variable
and homoplasious mononucleotide repeat region in the trnT-trnL intergenic spacer reinforced the contrasting
patterns of diversity observed between northern and southern populations. The divergent histories of northern and
southern populations revealed in the present study will inform the management of this globally significant
conifer. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277.
ADDITIONAL KEYWORDS: chloroplast DNA – inland refugia – Japanese temperate forest – Japanese
umbrella pine – mitochondria – mononucleotide repeats – palaeodistribution modelling – pollen-based refugia.
INTRODUCTION
Sciadopitys verticillata (Thunb.) Siebold et Zucc., an
evergreen conifer endemic to Japan, is a plant of
global significance. The single species is so morphologically and genetically diverged from all other conifers (Hayata, 1931; Schlarbaum & Tsuchiya, 1985;
Crisp & Cook, 2011) that it is considered to represent
a family (the Sciadopityaceae) of its own. Indeed,
molecular dating shows that S. verticillata is considerably older (having diverged approximately 220
*Corresponding author. E-mail: [email protected]
Mya) than other recognized ‘living fossil’ gymnosperms such as Metasequoia and Wollemia (Crisp &
Cook, 2011) and represents one of the most early
diverging plant lineages survived by a single species
in the world alongside other classic ancient plants
Amborella and Ginkgo. Although an important component of forests across the northern hemisphere
during the late Cretaceous–early Palaeogene (65–
49 Mya) (Christophel, 1973), the Sciadopityaceae disappeared from these regions in the Pliocene (van der
Hammen, Wijmstra & Zagwijn, 1971; Matthews &
Ovenden, 1990) probably as a result of the onset of
the Pleistocene glaciations (Tsukada, 1963). The
living species, S. verticillata, is currently patchily
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
263
264
J. R. P. WORTH ET AL.
Hokkaido
Altitude (m)
40N
0 − 250
251 − 500
501 − 750
751 − 1000
1001 − 2000
2001 − 3776
Population locations
Known distribution records not sampled
Fukushima
Honshu
Wakasa
Bay
Central
Kinki
35N
Chugoku
Izu Peninsula
Kii Peninsula
Kyushu
Shikoku
200
300
400
500
140E
135E
30N
130E
0 km 100
Figure 1. A map of the known distribution of Sciadopitys verticillata and the putative locations of Last Glacial Maximum
refugia for temperate forest (light purple) based on the fossil pollen record (adapted from Tsukada, 1982 and Takahashi
et al., 2005). The grey line delimits the coastline at 21 kya. The 32 populations sampled in this study are indicated by
red circles, whereas nonsampled known occurrence records are indicated by white circles.
distributed in high rainfall montane areas of Japan
within temperate forests, from central Honshu
(37.5°N) on the main island of Honshu to the southern island of Kyushu (32°N) (Fig. 1). Fossil evidence
demonstrates that although S. verticillata persisted
in Japan throughout the Pleistocene the species was
strongly impacted by the glacial–interglacial cycles
that characterized this period (Heusser, 1990), probably resulting in its extinction from northern Honshu
(Tsukada, 1963), and the contraction of its range in
other regions (Uemura, 1986). However, major gaps
in our knowledge of the species remain, particularly
in terms of where the species survived during the
Pleistocene glacials, including during the cold and dry
climate of the Last Glacial Maximum (LGM) 22 000–
19 000 years ago. During the LGM, the climate of
Japan compared to present had approximately 50%
lower rainfall (Tsukada, 1986) and was a mean of
approximately 7 °C degrees cooler during summer in
central Honshu (35 °N) (Morita, Choi & Hbino, 1998).
Pollen-based evidence suggests that cool mixed forest,
a forest type now distributed only in the most northern island of Hokkaido, dominated the vegetation
across Japan at this time (Tsukada, 1983; Takahara
et al., 2000; Gotanda et al., 2002; Gotanda & Yasuda,
2008). Temperate forests, which are presently extensively distributed across the Japanese archipelago in
coastal and inland regions from southern Kyushu
to southern Hokkaido (Gotanda et al., 2002), are
assumed to have been confined to coastal areas
during the LGM along the Pacific and Japan Sea
coastlines (Kamei & Research Group for the Biogeography from Würm Glacial, 1981; Aoki, Kato &
Murakami, 2011), including along the exposed sea
floor (Tsukada, 1986) (Fig. 1). From these restricted
coastal refugia temperate forest species are thought
to have expanded to their present widespread ranges
during the Holocene (Tsukada, 1982; Tomaru et al.,
1998). Currently, three of the major occurrences of
S. verticillata in Kyushu, Shikoku, and the Kii Peninsula are found within (or near to) pollen-based coastal
refugia (Fig. 1). However, fossil-based evidence for the
persistence of S. verticillata within these temperate
refugial regions has not been found (Gotanda &
Yasuda, 2008). The only conclusive pollen evidence for
LGM survival of the species comes from the Pacific
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
PHYLOGEOGRAPHY OF A RELICTUAL CONIFER
Coast near the Izu Peninsula (Kanauchi et al., 1989)
(Fig. 1), where the species probably became extinct in
the early Holocene (Kanauchi, 2005).
Sciadopitys verticillata also occurs within two
regions outside the pollen-based refugial locations in
inland central Honshu, where a relatively large
number of populations occur, and a narrow and isolated range in Chugoku. Strong uncertainty remains
regarding the origin of these S. verticillata populations
with available pollen-based evidence uninformative.
For example, small amounts of S. verticillata in LGM
pollen records from central Honshu (Sakai, Nakajima
& Sumida, 1979; Miyagi et al., 1981) could equally be
interpreted as being derived from local populations or
distant coastal sources. Over the last decade, it has
become increasingly recognized that many temperate
species (Stewart & Lister, 2001; McLachlan, Clark &
Manos, 2005; Magri et al., 2006; Worth et al., 2009)
were able to survive the LGM not only in the expected
refugial locations, but also in sparse populations (Birks
& Willis, 2008) located outside these areas that were
undetected by the pollen record (McLachlan & Clark,
2004). Indeed, in Japan, although the fossil pollenbased coastal refugia model of LGM survival remains
the dominant paradigm, in recent years, molecular
evidence has arisen that cannot be reconciled with the
pollen-based coastal refugia hypothesis. For example,
Takahashi et al. (2005) discovered a high frequency of
rare and private nuclear SSR alleles in the most
northern populations of Cryptomeria japonica in
northern Honshu similar to that found in populations
close to (or within) southern putative coastal refugia.
In addition, organellular DNA studies show that
several temperate tree species (Tomaru et al., 1998;
Yamanaka, Kobayashi & Setoguchi, 2008; Iwasaki
et al., 2012; Sugahara et al., 2011) harbour haplotypes
whose distributions are confined to inland and/or
northern regions of Honshu.
The present study utilized organellular phylogeographical data and palaeodistribution modelling
aiming to better understand the locations of glacial
survival of the relictual conifer S. verticillata and how
the climatic changes of the Pleistocene may have
shaped patterns of genetic differentiation and diversity of the species. Specifically, the present study
tested whether S. verticillata conforms to the pollenbased coastal refugia hypothesis by undergoing extensive migrations from coastal refugia northwards and
towards inland regions during the climate warming of
the Holocene to occupy its current range (Tsukada,
1982; Tomaru et al., 1998) or, alternatively, if in situ
inland survival may have been possible for this
species. Two main results are possible: (1) central
Honshu populations in the northern part of the
species range share a subset of the organellular diversity within the nearest putative refugia on the Kii
265
Peninsula and/or Shikoku island as a result of
Holocene migration as predicted by the coastal
refugia model or (2) central Honshu populations are
differentiated from other parts of the species range
indicating inland glacial survival.
MATERIAL AND METHODS
THE SPECIES
Sciadopitys verticillata is an evergeen, monoecious
and wind pollinated tree reaching heights of 45 m,
diameters of up to 2 m (Eckenwalder, 2009), and ages
possibly of over 1000 years. The seeds are winged
(Eckenwalder, 2009) and dispersed by gravity
(Yamamoto, 1988) and, considering there light weight
(under 5 mg; J. R. P. Worth, unpubl. data), presumably also by wind (Benkman, 1995). The genus is
known to have occurred in Japan since the Miocene
(Tsukada, 1963), whereas the species, S. verticillata,
has been recognized in the Japanese fossil record
from the Pliocene (Uemura, 1986). The species occurs
on ridgetops and steep rocky slopes within evergreen
and deciduous temperate forests, usually with other
conifers. However, S. verticillata is absent from cool
conifer forests of the subalpine zone that occur at
altitudes over approximately 1500 m a.s.l. (Yoshino,
1978) probably as a result of the species poor cold
tolerance compared to other Japanese conifers (Sakai
& Larcher, 1987). The species is classified as vulnerable to extinction (Conifer Specialist Group, 1998),
with declines as a result of the past utilization of the
species for wood (Tsukada, 1963) and extensive
replacement of natural forests by plantations (Nakamura, DellaSala & Alaback, 2011).
POPULATION
SAMPLING
In total, 32 populations of S. verticillata representing
almost the full known natural range of the species
were sampled (Fig. 1; see also Supporting information, Table S1). This included 11–12 samples each
from seven populations of the nuclear SSR study by
Kawase et al. (2010) and nine to 12 samples each
from 25 additional populations collected for the
present study. The distance between sampled trees
varied between sites according to the size of stands,
with some populations consisting of less than 20 trees
and therefore sampled close together (ⱖ 1 m apart),
whereas individuals in larger stands were sampled at
least 10 m apart.
DNA
EXTRACTION, POLYMERASE CHAIN REACTION
(PCR),
AND SEQUENCING
Genomic DNA was extracted from approximately
40 mg of leaf tissue using a modified CTAB protocol
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
266
J. R. P. WORTH ET AL.
(Milligan, 1992). Both the chloroplast and mitochondria, which are paternally inherited in S. verticillata
(Gianordoli, 1973; J. R. P. Worth & Yuji Isagi, unpubl.
data), were screened for genetic variation. A total of 21
of 35 chloroplast fragments and three of 23 mitochondrial fragments tested were successfully amplified and
used for a preliminary screening for variation using 16
samples representative of the species range (see Supporting information, Table S2). DNA variation was
discovered in six chloroplast fragments (atpI-rpoC2,
trnD-trnT: Grivet et al., 2001; rpl16 intron: Baum,
Small & Wendell, 1998; petN-psbM: Lee & Wen, 2004;
ndhA intron: Shaw et al., 2007; trnT-trnL, Taberlet
et al., 1991) and one mitochondrial fragment [the small
subunit ribosomal RNA gene (SSUB); Duff & Nickrent,
1999]. One chloroplast fragment (trnT-trnL) was found
to contain a highly variable single mononucleotide
repeat (MNR) region. All six variable chloroplast
regions were amplified for 365 individuals (mean 11.4
samples per population) and the single mitochondrial
fragment was amplified for 375 individuals (11.7 per
population). PCR amplification was undertaken in a
10-mL reaction volume containing 4.2 mL of Mastermix
(Qiagen), 1.68 mM of each primer and approximately
20 ng of gDNA. Thermocycling conditions for each
variable chloroplast and mitochondrial fragment are
provided in the Supporting information (Table S3).
PCR products were purified using Exonuclease
(Takara) and TSAP enzymes (Promega), sequenced
using the BigDye Terminator Cycle Sequencing Kit
(Applied Biosystems) and run on an ABI PRISM 3100
Genetic Analyzer. DNA sequencing was performed in
one direction using the forward primers petN-psbMSciF, ndhAx1, a, and SSUB-SciF and the reverse
primers trnT, rpl16R 1516, and atpI-rpoC2-SciR2.
DNA sequences were aligned using GENEIOUS PRO,
version 5.4.6 (Drummond et al., 2011). The single MNR
region in the trnT-trnL fragment had seven fragment
size variants and displayed significant levels of
homoplasy. However, contrasting diversity patterns
between central Honshu and other regions were
evident (Fig. 2). Therefore, rather than excluding the
MNR region from all analyses, as is common in phylogeographical studies, all analyses were undertaken
excluding (referred to as dataset 1) and including the
MNR region (dataset 2) following the recommendation
of Vachon & Freeland (2011). Short repeat indels,
particularly those with high levels of homoplasy, are
commonly associated with thermodynamically stable
hairpins involving inverted or direct repeats (Morton
& Clegg, 1993; Mes et al., 2000). Therefore, all indel
regions identified in both chloroplast and mitochondria
with more than one variant were investigated for the
presence of hairpin structures using OLIGOANALYZER, version 3.1 (http://www.idtdna.com/analyzer/
Applications/OligoAnalyzer/).
HAPLOTYPE
RELATIONSHIPS
Haplotypes were defined using the ‘find haplotypes’
option in GENALEX, version 6.5 (Peakall & Smouse,
2006). A median-joining network, with equal weighting of all characters, was constructed using
NETWORK, version 4.6 (Bandelt, Forster & Rohl,
1999) for all of S. verticillata haplotypes and mitotypes. For all analyses, both chloroplast datasets 1
and 2 were used.
POPULATION
AND REGIONAL GENETIC DIVERSITY
Genetic diversity of the chloroplast was assessed at
the population and regional levels with six regions
defined as: central Honshu (ten populations),
Chugoku (three populations), Kinki (two populations),
Kii Peninsula (five populations), Kyushu (five populations), and Shikoku (seven populations) (Fig. 1).
Because the Fukushima population shared a chloroplast haplotype otherwise only found in central
Honshu, this disjunct population was included with
central Honshu for all analyses. Nucleotide diversity
(p), indel diveristy [p(I)], and haplotype diversity (HD)
were calculated using DNASP, version 5.10 (Librado
& Rozas, 2009) for each population, each region, and
all samples. Indel diversity was calculated using the
multi-allelic option. Haplotype richness (HR) and
private haplotype richness (HP) were obtained with
ADZE, version 1.0 (Szpiech, Jakobsson & Rosenberg,
2008) using a rarefaction procedure to account for
differing sample sizes of populations. Regional and
population level genetic diversity was not assessed for
the three mitochondrial haplotypes.
POPULATION
AND REGIONAL GENETIC
DIFFERENTIATION
To analyze the partitioning of chloroplast and mitochondrial DNA variation between populations and
regions, analysis of molecular variance was performed using ARLEQUIN, version 3.5.1.2 (Excoffier,
Laval & Schneider, 2005), with significance tests in
all cases conducted using 10 000 permutations. To
assess the geographical structure of chloroplast
haplotypes spatial analysis of molecular variance
(SAMOVA) was undertaken using SAMOVA, version
1.0 (Dupanloup, Schneider & Excoffier, 2002). The
method is based on a simulated annealing procedure
that aims to maximize the proportion of total genetic
variance as a result of differences between groups of
populations (FCT) resulting in the identification of
groups of populations (K) that are geographically
homogenous and maximally differentiated from one
another (Dupanloup et al., 2002). The identification
of the correct number of groups (K) has relied on
two methods: the K with the highest value of FCT
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
PHYLOGEOGRAPHY OF A RELICTUAL CONIFER
Central
Kinki
n = 24
Hr = 3.00
n = 114
Hr = 5.02
Kii Peninsula
Shikoku
n = 81
Hr = 3.18
n = 58
Hr = 3.38
Frequency
267
Chugoku
Kyushu
n = 56
Hr = 2.63
n = 32
Hr = 3.50
Size (bp)
Figure 2. The frequency of each different fragment size (bp) of the mononucleotide (MNR) repeat region observed within
each geographic region in Sciadopitys verticillata. The number of individuals within each region (n) and haplotype
richness (HR) of haplotypes based on variation at the MNR region when rarified to the smallest population size (n = 24)
are shown.
(Dupanloup et al., 2002) and the value of K before
identified groups of populations are collapsed into
groups consisting of single populations (Heuertz
et al., 2004). GST and NST for chloroplast and mitochondrial data sets were assessed using PERMUT,
version 1.0 (Pons & Petit, 1996) with the significance
of NST being assessed using 1000 permutations. Jost’s
D, an unbiased estimator of divergence (Jost, 2008),
was calculated using SPADE (http://chao.stat.nthu.
edu.tw/softwareCE.html).
PALAEODISTRIBUTION
MODELLING
Palaeodistribution modelling using climatic variables
was used to investigate the potential range of S. verticillata during the LGM. To increase the robustness of
predictions, two methods were used: a presence-only
method using a maximum entropy algorithm implemented in MAXENT, version 3.3.3a (Phillips, Anderson & Schapire, 2006) and a presence-absence method
using a general additive model (GAM) implemented in
MGCV, version 1.7-2 (Wood, 2006) of R version 2.12.0
(R Development Core Team, 2010). Presence data were
compiled using data from the present study, Kawase
et al. (2010), and the Phytosociological Releve’ Data
Base (PRDB) (Tanaka, 2007) resulting in 104 presence
records after duplicate records within each 1-km
square were deleted. Absence data were sourced from
the PDRB database (Tanaka, 2007). Modern climate
data were obtained from the Worldclim database
(Hijmans et al., 2005). Five climatic variables were
used in all analyses: minimum temperature of the
coldest period, mean temperature of the warmest
quarter, annual precipitation, precipitation of the
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
268
J. R. P. WORTH ET AL.
Phillips & Dudík (2008). Each model was ran with 100
replicates and cross validation and with logistic
output. For the GAM analysis, species presence or
absence was modelled as a sum of nonlinear smooth
functions of the bioclimatic variables. The binomial
error distribution was designated for the presence–
absence data, with the logit as link function. Estimation of smoothing parameters was performed based on
a generalized cross-validation method.
warmest quarter, and precipitation of the coldest
quarter. These precipitation variables are considered
to be key climatic controls of the species’ current
distribution (Igarashi & Oba, 2006), whereas mean
temperature of the warmest month and minimum
temperature influence the distribution of temperate
forests worldwide (Jobbagy & Jackson, 2000; Woodward, Lomas & Kelly, 2004). LGM climate data were
obtained from two general circulation models: the
Community Climate System Model 3.0 (CCSM 3.0)
(Collins et al., 2006) and the Model for Interdisciplinary Research on Climate 3.2 (MIROC 3.2) (Hasumi &
Emori, 2004), provided by the Palaeoclimate Modelling
Intercomparison Project. The LGM palaeoclimate surfaces were prepared in 30 arc-s resolution sensu Sakaguchi et al. (2010). For both presence-only (MAXENT)
and presence-absence (GAM) methods, the present
climatic species distribution model was constructed
under the modern climate and subsequently projected
onto the two LGM climate scenarios. The default
parameter setting of Maxent were used (maximum
number of background points 10 000; regularization
multiplier 1; auto features; maximum iterations 500;
convergence threshold 0.00001), as suggested by
RESULTS
CHLOROPLAST DNA VARIATION
In total, 12 685 bp of the chloroplast genome was
sequenced for 16 S. verticillata individuals during
screening for chloroplast DNA variation. Dataset 1
(aligned length of 2213 bp) contained five single
nucleotide polymorphisms (SNPs) and four indel
regions, resulting in 16 haplotypes (Table 1). Dataset
2 (aligned length of 2513 bp) had a total of 20 characters resulting in 31 haplotypes. For GenBank accession numbers of all fragments, see the Supporting
information (Table S4). Species wide nucleotide diversity (p) was 0.00017, whereas indel diversity (p (I))
Table 1. All variable sites defining the 16 chloroplast DNA haplotypes observed in Sciadopitys verticillata
1
2
3
Haplotype
N
petN-psbM
trnD-trnT-AT
repeat region
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H13
H14
H15
H16
267
10
9
7
3
1
1
1
23
19
17
1
2
1
2
1
T
–
–
–
–
–
–
–
G
G
G
G
G
–
G
G
(AT)x3
(AT)x3
(AT)x3
(AT)x1
(AT)x3
(AT)x3
(AT)x3
(AT)x3
(AT)x3
(AT)x3
(AT)x5
(AT)x5
–
(AT)x4
(AT)x4
(AT)x2
–
–
–
–
–
Ia1
–
–
–
–
–
–
–
–
–
–
4
5
rpl16 intron
–
–
–
–
D*
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Ib2
Ib2
Ic3
Ib2
–
–
–
6
C
–
–
–
–
–
–
T
–
–
–
–
–
–
–
–
7
8
9
ndhA
intron
atpI-rpoC2
Mononucleotide repeat
region size variants
(bp)
G
A
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C
–
A
–
–
–
–
–
–
–
–
–
–
–
–
–
12,
13
13
12,
13
13
14
13
12,
11,
11,
12
10,
14
13,
13
G
–
–
–
–
–
A
–
–
–
–
–
–
–
–
–
13, 14, 15, 16
13, 14
13, 14, 15
12, 13
12, 13
11
14
These consisted of five single nucleotide polymorphisms (characters 1, 6, 7, 8, and 9) and four indel regions (2, 3, 4, and
5). Indel region 3 had six different states based on the number of dinucleotide (AT) repeats, whereas indel region 5 had
three different states. All variable sites are shown in comparison to the most frequent haplotype H1, with dashes
indicating congruence with haplotype (H1). The different fragment sizes observed at the single mononucleotide repeat
region within samples of each of the 16 haplotype are also shown.
D* = ATCAATTT; Ia1 = A; Ib2 = AATA; Ic3 = AATAAATA.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
PHYLOGEOGRAPHY OF A RELICTUAL CONIFER
H12
(A)
H14
H15
H8
(B)
H7
H9
H10
M3
M2
H6
H11
269
H1
FUK
H5
H16
H2
OTK
H4
H3
H13
OTK
AKS
ABZ
ABZ
OGW
OOI
NAG
SRT
ORI
BND
SHI
ISH
UNE
HSY
OMG
YSK
YAS
MRY
SBY
MIE
IMY
HSY
YAS
HRN
ISH
MIE
IMY
ORI
TJS
BND
OMG
NAG
SRT
NGD
SHG
RKK
TJS
SHI
OGW
OOI
NGD
SHG
RKK
AKS
HGS
HGS
UNE
HRN
SAS
ZAK
SBY
YSK
MRY
SAS
ZAK
OYB
OYB
FKY
OSZ
FKY
0 km
65
130
260
390
OSZ
520
Figure 3. Distribution of (A) the 16 chloroplast haplotypes (dataset 1) and (B) three mitotypes observed in 32 populations
of Sciadopitys verticillata. Each pie diagram represents the frequency of haplotypes observed within each population. The
median-joining networks for both chloroplast and mitochondria are also shown, with the area of the circles being
proportional to the frequency of each haplotype. Lines connecting each haplotype represent one chloroplast mutation with
multiple connections for some haplotypes representing inferred homoplasies. Chloroplast spatial analysis of molecular
variance groups I and II when K = 3 are encircled by blue lines.
was higher at 0.00026 (0.00071 for dataset 2). All
three chloroplast indel repeat regions investigated
were not associated with hairpins structures and had
low DG-values (trnD-trnT = -2.15 kcal mol-1, the rpl16
intron = -1.12 and the MNR repeat of the trnT-trnL
fragment = -1.55).
CHLOROPLAST
HAPLOTYPE NETWORK
AND DISTRIBUTION
For dataset 1, the chloroplast haplotype relationships
were clearly resolved, with most haplotypes differing
by a single change from the most common haplotype
H1 (Fig. 3). Haplotypes that were most commonly
observed (H9) or only observed in central Honshu,
(H10–H11), formed a separate haplotype group. The
multiple connections of the internal haplotype, H1,
along with its widespread distribution, indicate that
H1 is likely the ancestral haplotype. H1 (observed in
73.2% of all individuals) was found in all 32 populations and was the sole haplotype in nine populations
(Fig. 3). Only in central Honshu was haplotype H1
relatively infrequent, where it comprised 46.5% of
samples compared to over 80% of individuals in
the putative refugia regions of the Kii Peninsula,
Shikoku, and Kyushu (Table 2). Central Honshu contained eight rare haplotypes (i.e. observed in less
than 6% of all samples), with six being endemic, of
which three H9 (N = 23), H10 (N = 19), and H11
(N = 17), comprised 51.7% of all samples in this
region. Outside of central Honshu, rare haplotypes
were found in the SHI population in Chugoku (H2,
N = 10), Kyushu (H5, N = 3; H8, N = 1), whereas H3
(N = 9) was restricted to Kyushu and the nearest
population in Shikoku (SAS). The haplotype relationships for dataset 2 haplotypes could not be resolved,
with each haplotype having a mean of 1.36 connections compared to 1.05 for haplotypes of dataset 1
(result not shown). Fifteen of all 31 dataset 2 haplotypes were observed only in central Honshu (see
Supporting information, Table S5).
CHLOROPLAST DNA
DIVERSITY
Central Honshu had the greatest chloroplast diversity
of all regions for all diversity measures for both
datasets 1 and 2 (Table 2). Apart from Kyushu, rarefied haplotype richness (HR) was almost twice the
magnitude of other regions for both datasets (Fig. 4,
Table 2), whereas private haplotype richness (Hp)
was six- to three-fold greater (Table 2). Similar patterns of haplotype richness were observed at the
population level for both datasets, with most populations having increased richness when including the
MNR region. All central Honshu populations (except
SRT) and the single FKY population in Kyushu harboured haplotype richness values greater than the
species-wide mean for both datasets (Fig. 4; see also
the Supporting information, Table S6).
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
Values for dataset 2 are shown italicized in parenthesis adjacent to the values for dataset 1.
*Central Honshu includes the single population (FUK) from Fukushima.
N, sample size; NA, observed number of chloroplast haplotypes; HR, rarefaction standardized haplotype richness; HP, rarefaction standardized private allelic
richness; HD, haplotype diversity; p, nucleotide diversity; and p(I), indel diversity. †Rarefied values for both HR and HP were calculated using the minimum regional
sample size of 24.
35.9 (40.3)
0 (12.5)
1.7 (5.2)
1.2 (4.9)
31.2 (31.3)
7.1 (8.9)
53.5
12.5
6.9
11.1
31.3
19.6
0.59 (1.14)
0 (0.54)
0.03 (0.40)
0.04 (0.43)
0 (0.20)
0.06 (0.23)
[0.01]
[0.00]
[0.02]
[0.02]
[0.03]
[0.03]
0.22
0.00
0.04
0.05
0.19
0.11
0.71
0.23
0.13
0.21
0.44
0.34
3.16
0.27
0.65
0.31
1.00
1.84
5.19
2.00
2.48
3.11
2.00
3.65
(20)
(4)
(7)
(8)
(5)
(7)
9
2
4
5
2
5
114
24
58
81
32
56
Central*
Kinki
Kii Peninsula
Shikoku
Chugoku
Kyushu
NA
Region
N
HR
(10.45)
(4.00)
(4.60)
(5.30)
(4.49)
(5.02)
HP
(6.29)
(1.00)
(1.24)
(0.97)
(1.26)
(2.01)
HD
(0.89)
(0.57)
(0.48)
(0.59)
(0.68)
(0.50)
p ¥ 1000 (SD)
p(i) ¥ 1000
Haplotypes other
than H1 (%)
Haplotypes endemic
to the region (%)
J. R. P. WORTH ET AL.
Table 2. Chloroplast haplotype diversity based on both datasets 1 and 2 by geographical region for Sciadopitys verticillata, including the percentage of individuals
possessing haplotypes other than the most common haplotype (H1) for dataset 1 and the percentage of individuals possessing haplotypes endemic to the region
270
GEOGRAPHICAL
STRUCTURE OF
CHLOROPLAST VARIATION
Geographical structuring was evident at the chloroplast for both datasets. For dataset 1, the observed
GST across the whole species range was 0.251 and was
lower than NST of 0.300, whereas Jost’s D was 0.175.
GST and NST were lower for dataset 2, with GST of
0.206 and NST of 0.218, whereas the value for Jost’s D
increased to 0.355, as a result of the differing effects
of rare population specific haplotypes on these measures of geographical structuring (Jost, 2008). For
dataset 1, AMOVA showed that 30.2% of the variation
was explained by differences among populations
(P < 0.001) and 25.5% among geographical regions
(P < 0.001) (see Supporting information, Table S7),
with most of the variation explained by differences
within populations (69.8%). For dataset 2, differentiation among populations decreased to 22.0% and
between regions to 15.6% with 78 % of the variation
explained by differences within populations. For
SAMOVA, the value of FCT for dataset 1 was found to
decline gradually with an increasing number of K
groups (K = 2–9), with the highest FCT of 0.50 and FST
of 0.63 for K = 2. These two groups consisted of a
single population almost fixed for a private haplotype
SHI from Chugoku (group 1) and all other 31 populations (group 2). The second highest FCT value of 0.48
and FST of 0.49 was observed for K = 3, consisting of
eight populations from central Honshu (group 1), the
SHI population (group 2), and all remaining populations (group 3), including FUK and ORI from central
Honshu that had a high frequency of the H1 haplotype. At higher K (4–9), group 1 was progressively
split into smaller and smaller groups, whereas group
3 was retained for all K groups. K = 3 was retained
because it had the second highest FCT and FST and
corresponds best with the observed geographical distribution of chloroplast haplotypes. The SAMOVA
algorithm produced contrasting results for dataset 2,
with FCT values plateauing at K = 4 with an FCT of
0.3495 and the highest FST of 0.36. K = 4 found the
same grouping as K = 3 for dataset 1, except that a
single population ISH was separated because of the
near fixation of a dataset 2 haplotype that was elsewhere rare (see Supporting information, Table S5). K
groups 5–8 were similar, with FCT values in the range
0.3488–0.3505 although they had lower FST values.
MITOCHONDRIAL DNA
In total, 2525 bp of mitochondrial sequence was
sequenced during screening. The small subunit ribosomal RNA gene contained two indels resulting in three
mitotypes. This short repeat region was found to
bordered by two inverted repeat regions with a high DG
of -11.2 kcal/mol-1. One mitotype (M3) was found in a
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
PHYLOGEOGRAPHY OF A RELICTUAL CONIFER
7
Pop. Hr excluding MNR
region (
mean)
Pop. Hr including
MNR region (
)
6
5
Hr
271
4
3
2
1
SH
G
0
Central
Kinki
Kii Pen.
Shikoku
Chugoku
Kyushu
Figure 4. Population level haplotype richness (HR) (Szpiech et al., 2008) of the chloroplast calculated with and without
the mononucleotide region for all 32 Sciadopitys verticillata populations. The mean HR for dataset 1 (thick dashed line)
and dataset 2 (thin dashed line) are shown.
single individual from SRT in central Honshu,
whereas the other mitotypes were both common, with
M1 being observed in 65.5% of individuals and M2 in
34.5% (Fig. 2). These common mitotypes were found
throughout the species range and occurred together in
30 of 32 populations with low geographical structuring
(as reflected in the low GST value of 0.101, NST of 0.106
and Jost’s D of 0.078). A total of 9.8% of the variation
was explained between populations and 90.2% within
populations (see Supporting information, Table S7).
The SAMOVA algorithm was unable to identify group
structure, with the value of FCT declining with an
increasing value of K. K = 2 was found to have the
highest FCT value with the SRT population comprising
one group and all remaining populations comprising
the other.
PALAEODISTRIBUTION
MODELS
For the MAXENT analysis, the mean area under the
curve (AUC) value was 0.928 (SD = 0.017). This is
above the value of 0.7 that is considered acceptable
for AUC values (Pearce & Ferrier, 2000), indicating
good model performance. The predicted modern
distribution of S. verticilliata was largely congruent
with the known distribution of the species (Fig. 5).
However, two regions where the species is absent
were predicted to be suitable habitat, on the Izu
Peninsula and adjacent areas of mainland Honshu
and in northern Kyushu, whereas the models poorly
predicted the occurrence of some isolated populations
near the Japan Sea coast (OOI) and in Fukushima
(FUK). The GAM present model was similar to the
Maxent model, although predicted occurrences, especially for Shikoku, Chugoku, and the Izu peninsula,
were lower (Fig. 5).
All models of LGM distribution showed an overall
reduction in the distribution of the species into discrete refugial areas. Three of the four models had
strong to moderate predictions for parts of the Kii
Peninsula, Shikoku, and southern Kyushu, where
temperate forest refugia are assumed to have existed.
Interestingly, three of the four models showed strong
to moderate predictions for central inland Japan
refugia (Fig. 6B, C, D), where the strongest predictions were observed in river valleys at between 100–
300 m in altitude. The Maxent CCSM 3.0 model was
the only model with a weak prediction of LGM occurrence for central Honshu (Fig. 6A). Notable regions
with poor or no LGM prediction were Chugoku, Kinki,
and Fukushima (Fig. 6).
DISCUSSION
Chloroplast DNA data and palaeodistribution modelling provides convincing evidence that S. verticillata
maintained populations within its northern range in
central Honshu and possibly also in Chugoku outside
the pollen-based temperate forest refugial areas
(Fig. 1). The evidence for the genetic divergence of
central Honshu populations from elsewhere is particularly solid given the intensive sampling of almost
the entire species range and the high number and
frequency of endemic haplotypes observed in central
Honshu, including the remarkable high richness at
the single mononucleotide repeat region. Palaeodistribution modelling corroborated the genetic evidence
for inland survival predicting an isolated area of
suitable climate in this region during the LGM
(Fig. 6). Unexpectedly, apart from a single population
in the southernmost part of the species range in
Kyushu, populations within, or near to, pollen-based
refugial locations Shikoku, the Kii Peninsula, and
Kyushu contained low diversity, being dominated by a
widespread ancestral haplotype (Fig. 3) and also
having markedly lower diversity at the MNR region
(Fig. 2). By contrast to the clear genetic structure of
the chloroplast, weak genetic structure was observed
at the mitochondria, a finding that could be explained
by repeated indel evolution as a result of an association with hairpin structures (Mes et al., 2000).
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
272
J. R. P. WORTH ET AL.
(A) Maxent−Present
40N
0.7 − 1.0
0.6 − 0.7
0.5 − 0.6
0.4 − 0.5
0.3 − 0.4
0.2 − 0.3
0.1 − 0.2
0 − 0.1
135E
140E
135E
140E
130E
35N
(B) GAM−Present
40N
0.1 − 1.0
0.08 − 0.1
0.06 − 0.08
0.04 − 0.06
0.02 − 0.04
0.005 − 0.02
0.001 − 0.005
0 − 0.001
130E
35N
Figure 5. The predicted present distribution of Sciadopitys verticillata based on current climatic data, estimated
using MAXENT (A) and GAM (B). The most likely areas of
occurrence are represented by warmer colours. The
current known distribution of the species is represented by
black filled circles.
NORTHERN
RICHNESS IN INLAND GLACIAL REFUGIA
Central Honshu was genetically diverged from the
rest of the species’ range and was the centre of
diversity for the species at the chloroplast DNA level.
Considering the slow evolution of the chloroplast
(Wolfe, Li & Sharp, 1987), the divergence of inland
central Honshu populations most likely represents a
signature of continued presence of S. verticillata
within the region through multiple glacials, whereby
populations in inland central Honshu have diverged
via the in situ evolution and fixation of newly evolved
haplotypes and the subsequent limited dispersal of
these haplotypes to other parts of the species range
(Neigel & Avise, 1986). The prevalence of rare haplotypes in the region, which are strongly effected by
genetic drift (Nei, Maruyama & Chakraborty, 1975),
would not be predicted based on the palaeodistribution models that indicated a significant decline in
suitable climate compared to present for the species
in central Honshu during the LGM. This apparent
conflict may be explained by recent evidence suggesting that the modern climatic ranges of species only
represents a portion of the full potential climates in
which they can occur (Veloz et al., 2012). This possibility is heightened given that rapid adaptation to
differing climate, even over one generation, has been
observed in conifers (van Zonneveld et al., 2009).
An alternative explanation of the divergence and
high diversity of central Honshu involving a postglacial migration origin from more southerly populations
must invoke extensive haplotype extinction across the
southern range of S. verticillata during the Holocene.
However, this is highly improbable considering that
this would require the extinction outside of central
Honshu of six SNP and indel based haplotypes and
three MNR fragment lengths endemic to this region
(Fig. 2), at the same time as retaining five rare haplotypes that were not observed in central Honshu.
Other explanations to explain the present differentiation of central Honshu populations could involve
migration from coastal refugia that are assumed to
have occurred along the lowered coast line on both the
Japan Sea and Pacific sides and are considered to
have contained scattered populations of the cold
hardy temperate trees C. japonica and Fagus crenata
(Fig. 1). However, these refugia are not likely to been
important refugial sources for the recolonization of
S. verticillata in central Honshu palaeodistribution
modelling had extremely low predictions for the
Japan Sea temperate forest refugia area at Wakasa
Bay (Fig. 6) and the sole population of S. verticillata
close to the putative refugia (OOI) contained no rare
or endemic haplotypes. Similarly, the refugia for
S. verticillata that occurred on the Pacific side at Izu
Peninsula is also unlikely to have been a major source
for the postglacial recolonization of inland central
Honshu considering the probable early extinction of
the species from this region approximately 12.5–9.7
kya (Kanauchi, 2005) and the fact that the central
Honshu populations with the highest diversity (ABZ,
AKS, OTK) were observed farthest away from the Izu
region.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
PHYLOGEOGRAPHY OF A RELICTUAL CONIFER
(A) Maxent−CCSM
273
(B) GAM−CCSM
40N
40N
0.7 − 1.0
0.1 − 1.0
0.08 − 0.1
0.06 − 0.08
0.04 − 0.06
0.02 − 0.04
0.005 − 0.02
0.001 − 0.005
0 − 0.001
0.6 − 0.7
0.5 − 0.6
0.4 − 0.5
0.3 − 0.4
0.2 − 0.3
0.1 − 0.2
0 − 0.1
135E
140E
135E
140E
(C) Maxent−MIROC
130E
140E
135E
35N
130E
35N
(D) GAM−MIROC
40N
40N
0.7 − 1.0
0.1 − 1.0
0.6 − 0.7
0.5 − 0.6
0.4 − 0.5
0.3 − 0.4
0.2 − 0.3
0.1 − 0.2
0 − 0.1
0.08 − 0.1
0.06 − 0.08
0.04 − 0.06
0.02 − 0.04
0.005 − 0.02
0.001 − 0.005
0 − 0.001
130E
140E
135E
35N
130E
35N
Figure 6. Potential distribution of Sciadopitys verticillata during the Last Glacial Maximum estimated using MAXENT
(A, C) and GAM (B, D) based on the species current climatic range using Community Climate System Model 3.0 (A, B)
and the Model for Interdisciplinary Research on Climate 3.2 (C, D).
LOW DIVERSITY IN THE SOUTHERN RANGE
Unexpectedly, populations in southern areas near
putative pollen-based temperate refugia, Kii Peninsula, Shikoku, and southern Kyushu, mostly harboured low diversity of chloroplast variation and were
dominated by a single chloroplast haplotype. This
result was particularly unexpected given the strong
predictions for suitable habitat during the LGM in
these regions, and studies of other plant species
where high diversity and/or endemic haplotypes have
been observed in these southern refugial areas (Aoki
et al., 2004; Iwasaki et al., 2012; Sugahara et al.,
2011). However, congruence between genetic and palaeodistribution modelling cannot always be expected
when examining the slowly evolving chloroplast
because the genome is not a ‘perfect recorder’ of past
history as a result of the persistence of ancestral
haplotypes across the ranges of species, even in
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277
274
J. R. P. WORTH ET AL.
populations that have been isolated for multiple glacials (Magri et al., 2006). Nevertheless, southern
populations of S. verticillata are most likely to have
arisen from separate glacial refugia given the differentiation of these populations from central Honshu
and the occurrence of rare haplotypes only found
there. In particular, the relatively high number of
rare and endemic haplotypes observed in Kyushu
(mostly in the population FKY) suggests that this
area may have been an important refugia for the
species, as also suggested by the nuclear study conducted by Kawase et al. (2010). However, the origin of
populations in the Kii Peninsula and Shikoku is less
certain, with further genetic evidence using faster
evolving genetic markers being required.
CONCLUSIONS
The relictual conifer S. verticillata withstood the cool
climates of the LGM, and possibly previous glacials,
in multiple parts of its range in Japan, leaving contrasting genetic ‘signatures’ of survival in the chloroplast genome. The northern populations in inland
central Honshu were found to be the area of highest
chloroplast DNA diversity in the species, a region that
was previously considered to be too cold for temperate
forest species during the LGM in Japan based on the
fossil pollen-record. Palaeodistribution modelling suggests that inland river valleys provided suitable
habitat for the species. On the other hand, southern
populations near putative temperate forest refugia
had low diversity, contrasting with expectations
of ‘southern richness and northern purity’ in northern hemisphere temperate species (Hewitt, 1996).
Overall, the present study provides previously unexpected insights into the Pleistocene history of the
relictual conifer S. verticillata, with the high chloroplast richness and endemism of northern populations
giving particular significance to this region for the
conservation of this globally significant tree species.
ACKNOWLEDGEMENTS
The authors thank H. Ando, S. Harasawa,
Y. Hatanaka, D. Kawase, L. Mizusawa, T. Oda,
T. Mizuno, N. Tomaru, Y. Yamazaki, and M. Yokogawa
for providing samples and their assistance in the
field. We also thank the Field Science Centre for
Northern Biosphere, Hokkaido University; Kyushu
University Forest; Seto Regional Forest Office; Kiso
Regional Forest Office; Mima City Government Office;
and Omogo Mountain Museum for sampling assistance. We thank G. Jordan, J. Marthick, Y. Tsumura,
and two anonymous reviewers for their valuable comments. This research was supported by a Japanese
Society for the Promotion of Science research fellow-
ship awarded to J.R.P.W. and by the program of the
Global Environmental Research of Japan (S-8) and
the Ministry of the Environment.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article:
Table S1. Geographical information of the 32 populations of Sciadopitys verticillata sampled. Population
samples sourced from Kawase et al. (2010) are indicated in bold.
Table S2. Primer pairs successfully amplified in Sciadopitys verticillata and screened for chloroplast and
mitochondrial variation with GenBank Accession numbers.
Table S3. Polymerase chain reaction (PCR) thermocycle for each variable chloroplast and mitochondrial
fragment. New primer pairs developed are also described below. To improve reliability of PCR amplification for
atpI-rpoC2 and petN-psbM, and the SSUB mitochondrial fragment, new primers were developed and are shown
below the table.
Table S4. GenBank accession numbers and aligned sequence length of all chloroplast and mitochondrial
fragments.
Table S5. Frequency of each of the 31 Sciadopitys verticillata chloroplast haplotypes observed when including
both snp, indel characters and the mononucleotide repeat region within each of the 32 populations sampled.
Table S6. Chloroplast haplotype diversity based on both datasets 1 and 2 by population for Sciadopitys
verticillata, including the percentage of individuals possessing haplotypes other than the most common
haplotype (H1) for dataset 1. Values for dataset 2 are shown italicized in parenthesis below the values for
dataset 1.
Table S7. Results of both analysis of molecular variance (AMOVA) and spatial analysis of molecular variance
(SAMOVA) based on the chloroplast (both dataset 1 and 2) and mitochondrial DNA data of Sciadopitys
verticillata. The geographical region based analysis uses the same regions as in Table 2 with population
numbers the same as in Table S1.
© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 263–277