DSpace at VNU: Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs

MEC_1399.fm Page 2697 Wednesday, October 24, 2001 6:39 PM

Molecular Ecology (2001) 10, 2697–2710

Phylogeography of Kandelia candel in East Asiatic
mangroves based on nucleotide variation of chloroplast
and mitochondrial DNAs

Blackwell Science Ltd

T. Y. C H I A N G , * Y. C . C H I A N G , † Y. J . C H E N , † C . H . C H O U , ‡ S . H AVA N O N D , § T. N . H O N G ¶
and S . H U A N G †
*Department of Biology, Cheng-Kung University, Tainan 701, Taiwan, †Department of Biology, National Taiwan Normal University,
Taipei, Taiwan 116, ‡Institute of Botany, Academia Sinica, Taipei 115, Taiwan, §Silvicultural Research Division, Royal Sorest
Department, Bangkok, 10900, Thailand, ¶Centre Forest Natural Resources and Environmental Studies, Vietnam National
University, Vietnam

Abstract
Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to
long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia
candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast
DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA
(mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated
lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-ChinaSea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak
population was closely related to the Ranong population of western Peninsula Malaysia
instead of other South-China-Sea populations, indicating its possible origin from the
Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic
differentiation between and within mainland and island populations (ΦCT = 0.015,
ΦSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations
as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF
spacer within the northern region. Instead, relative ages of alleles contributed to nonrandom chlorotype–mitotype associations and tree inconsistency. Widespread distribution

and random associations (χ2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes
indicated the panmixis of populations of the northern geographical region as a whole. In
contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.
Keywords: cpDNA, Kandelia candel, locus association, migration, minimum spanning network,
mtDNA, phylogeography, relative ages
Received 15 May 2001; revision received 5 August 2001; accepted 5 August 2001

Introduction
Kandelia candel, a monotypic genus of the Rhizophoraceae,
is one of the major mangrove species in East Asia
Correspondence : S. Huang.

© 2001 Blackwell Science Ltd

Fax: + 886-2-29312904;

E-mail:

(Tomlinson 1986; Mabberley 1997). Mangrove forests are
characteristic of tropical and subtropical coastlines of the
world. Over the last decades, due to human’s overexploitation, the genetic diversity in mangroves has been
deprived, especially from coastal ecosystems in Asia.
Many countries have categorized the sustainable management of mangroves as major priorities in biodiversity


MEC_1399.fm Page 2698 Wednesday, October 24, 2001 6:39 PM

2698 T . Y . C H I A N G E T A L .
conservation (Maguire et al. 2000). According to biogeographical evidence, the genus Kandelia underwent a
severe extinction phase during the upper Tertiary after the

closure of the Tethys Seaway (Schwarzbach & Ricklefs
1998). Since then populations of K. candel have been
restricted to the tropical Malesia and East Asia, including areas around the South China Sea and the East
China Sea.
Like many other mangrove species, K. candel is characterized by vivipary, the precocious growth of the progeny
when still attached to the maternal parent. Vivipary is
an adaptive feature for mangrove plants to colonize and
expand populations at intertidal estuary habitats. Precocious seedlings of K. candel, growing up to 47 cm long and
1.3 cm wide, are buoyant, which may allow long-range dispersal via ocean currents (Lugo & Snedaker 1974; Maxwell
1995). However, viviparous seedlings have also been considered as a hindrance to long-distance dispersal due to the
lack of protection and nutritional support from the maternal tissue (Duke 1995; Elmqvist & Cox 1996). Correspondingly, extremely various population structures have been
discovered in different mangrove species. High level of
genetic differentiation among populations as well as geographical subdivision, due to restricted gene flow across
populations, was lately detected in Avicennia marina (Avicenniaceae) (Duke 1995; Maguire et al. 2000), a result close
to the population structure of a nonviviparous species,
Acanthus ilicifolius (Lakshmi et al. 1997). In contrast, Australian populations of Rhizophora stylosa were found barely
differentiated (Goodall & Stoddart 1989). Recent allozyme
investigations revealed low level of genetic differentiation
(GST = 0.064, Sun et al. 1998) among Hong Kong populations as well as between two populations from Taiwan
(FST = 0.04, Huang 1994), both indicating that the seedling dispersal in K. candel was not as limited as previously
suggested.
Although frequent gene flow has been detected in K.
candel at the local scale, long-range dispersal across oceans
remains unknown. Apparently, the dispersal extent of
K. candel is not only regulated by the orientation of ocean
currents in the South China Sea and East China Sea, but also
constrained by the duration and survivorship of viviparous seedlings in the high saline conditions. The isolationby-distance model is thus a hypothesis to be tested. In
addition, when geographical distance increases for investigation, effects of vicariance will become more prominent
and may confound the isolation-by-distance model (Bossart
& Prowell 1998). Geological history inevitably plays

another critical role in determining the phylogeographical
pattern. For example, geographically close populations
along western and eastern coasts of the Peninsula Malaysia
were significantly differentiated due to a vicariance event
of approximately 60 – 220 million years before present
(Yamazaki 1998), which led to the geographical subdivision

of Bruguiera gymnorrhiza in Asia. According to palaeoceanographic evidence, due to latitude and temperature
differences, southern and northern banks of the South
China Sea, where K. candel is distributed, went through
different geological histories (Wang et al. 1995) over past
glacial cycles. Furthermore, populations of the Ryukyu
islands and northern Taiwan (Taipei) along coasts of
the East China Sea shared a unique geological history from
those of the South China Sea (Ota 1998; Chou et al. 2000).
In light of geological histories, genetic differentiation of
K. candel among the above three geographical regions
would be expected.
As generally known, in estimating population structure
and gene flow, some level of variance of loci is required.
Molecular markers with low resolution usually are incapable of providing information (Bossart & Prowell 1998) in
distinguishing coancestry from migration (Schaal et al.
1998). For surveying population structure within a small
geographical scale, e.g. K. candel in Hong Kong (Sun et al.
1998) and Taiwan (Huang 1994), allozymes due to their
conserved nature (cf. Bossart & Prowell 1998) might not be
able to adequately estimate gene flow among local populations. In addition, the biparental inheritance of allozymes
makes the estimations of seedling dispersal difficult, as the
effects of pollen dispersal between neighbouring populations cannot be ruled out. Recently, many noncoding
spacers of organelle DNAs have been widely applied to

phylogeographical studies (Schaal et al. 1998). With merits
of maternal inheritance in most angiosperms (Harris &
Ingram 1991), including Kandelia (Chen 2000), and nearly
neutral and fast evolution, these markers are likely to be
able to provide information in estimating the extent of
long-range seedling dispersal and reconstructing phylogeographical patterns.
In this study, we sequenced two noncoding spacers, i.e.
atpB-rbcL and trnL-trnF of chloroplast DNA (cpDNA), and
the internal transcribed spacer (ITS) of mitochondrial DNA
(mtDNA) and used them as markers to estimate the phylogeographical pattern as well as population and geographical structure of K. candel. As physically linked loci in the
chloroplast genome, atpB-rbcL and trnL-trnF should reveal
comparable phylogenies. Likewise, as being maternally
inherited, chloroplasts and mitochondria were thought to
remain associated and behave as if they are completely
linked (Schnabel & Asmussen 1989). Consistent phylogenetic patterns of cpDNA and mtDNA are thus expected
(cf. Dumolin-Lapègue et al. 1998).
However, evolutionary forces, such as lineage sorting
effects (Hoelzer et al. 1998), and frequent recurrent mutations (Desplanque et al. 2000), can result in systematic
inconsistency and thus lead to wide variance of FST values
among loci and a weak correlation between FST and
number of migrants per generation (Nm) as well (cf. Bossart
& Prowell 1998). Under such circumstances, difficulties in
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2699 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2699
interpreting phylogeography and population structure will
be inevitably encountered. In other words, when discrepant estimates are obtained from two or more loci, these estimates are not necessarily indicative of gene flow. Explicit

analysis of associations between alleles of different loci
(Desplanque et al. 2000) coupled with nested clade analysis
(cf. Schaal et al. 1998; Templeton 1998; Chiang 2000) will be
required to clarify historical and recurrent events.
K. candel, as widespread in East Asia, is a biological
model that is suited for testing the association between
vicariance and geographical structure; effects of ocean
currents in long-distance seedling dispersal; and the usefulness of cpDNA and mtDNA as population genetic
markers, as well as allele associations. Several aims are
pursued in this study: (i) to test the possibility and level of
long-distance seedling dispersal by estimating population
structure and gene flow; (ii) reconstruct the phylogeographical pattern and examine the association between
geological/geographical events and the extent of genetic
differentiation among three geographical regions; (iii) to

examine the phylogenetic consistency between atpB-rbcL
and trnL-trnF noncoding spacers of cpDNA as well as
between chloroplast and mitochondrial genomes; and
(iv) to investigate associations between alleles of cp- and
mtDNAs and to deduce the relative age of their alleles
based on spanning networks.

Materials and methods
Sample collection
Kandelia candel is a mangrove species that is widespread
in eastern coasts of mainland Asia and continental islands,
including Taiwan, and the Ryukyu (Fig. 1). One hundred
and eighty-seven samples were collected from 13 major
populations in East Asia, ranging from Bako (Sarawak,
01°40′ N) to Yakushima Islet ( Japan, 30°20′ N) (Table 1,

Fig. 1). Ten to 15 individuals, which were approximately
70–100 m apart, were sampled from each population. In
addition, 15 individuals of a population at Ranong

Fig. 1 Kandelia candel sample locations and distribution. Frequency of cytotypes (cpDNA trnL-trnF–mtDNA ITS associations) in each
population is indicated in pie diagrams. Abbreviations of populations are given in Table 1.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2700 Wednesday, October 24, 2001 6:39 PM

2700 T . Y . C H I A N G E T A L .
Table 1 Materials of Kandelia candel collected from different populations used for organelle DNA sequencing. Location, locality, sample
size, and cytotypes (trnL-trnF spacer chlorotype and mtITS mitotype associations) of each population are indicated

Localities

Area

Sampling
size (n)

Symbol

Coordinate

CC
HA
HK
QN

21°34′ N, 108°37′ E
19°54′ N, 110°20′ E
22°32′ N, 114°05′ E
20°53′ N, 106°46′ E

15

Guangxi, China
Taiwan
Fukien, China
Guangxi, China

SK
TN
XM
ZJ

21°28′ N, 109°43′ E
22°59′ N, 120°12′ E
24°26′ N, 118°04′ E
21°04′ N, 110°09′ E

10
15
15
15

Ryukyu, Japan

AM

28°15′ N, 130°40′ E

15

Ryukyu, Japan
Taiwan
Ryukyu, Japan

IR
TP
YK

24°19′ N, 123°54′ E
25°09′ N, 120°16′ E
30°20′ N, 130°30′ E

15
14
15

IB (6), IIB (3), IIIB (2), IIIC (1), IVB (2),
IVC (1)
IA (2), IB (4), IIB (3), IVB (6)
IB (9), IIB (3), IIIB (2)
IA (1), IB (8), IC (2), IIB (2), IVB (2)

Southern Region
Southern South-China-Sea Region

Bako
Sarawak

BK

01°40′ N, 110°25′ E

15

VIIbF

Indian Ocean Rim (outgroup)
Ranong
Thailand

RN

09°55′ N, 98°30′ E

15

IIaF (13), VIIaG (2)

Northern Region
Northern South-China-Sea Region:
Chinchou
Guangxi, China
Haikou
Hainan, China
Hong Kong

China
Quang Ninh
Vietnam
Shankou
Tainan
Xiamen
Zhangjiang
East-China-Sea Region:
Amami-O-Shima Islet
Irimote Islet
Taipei
Yakushima Islet

(Thailand) of the western coast of Peninsula Malaysia were
included in the analysis as outgroups. In total, this study
encompasses 14 populations (202 individuals). No materials
were collected from the Philippines, since no natural
populations are distributed in this area (Hou 1958). In
addition, during the last decade, most populations of
Vietnam have been removed for the use of inshore
fisheries (Huang & Chen 2000). Only one population at
Quang Ninh was available. Besides, samples of six
populations (from Chinchou to Xiamen) of China, one
population from Sarawak, two populations of Taiwan,
and three populations from the Ryukyu Islands were
included in this investigation. Based on the orientation of
ocean currents (Huang et al. 1997), three geographical
regions are recognized: southern South-China-Sea (S
region, including a single population of Sarawak),
northern South-China-Sea (N region, including eight

populations of Vietnam, China, and Tainan), and the
East-China-Sea (E region, including three populations of
Ryukyu and Taipei) regions. Three populations along the
Tonkin Bay, i.e. Chinchou, Shankou, and Quang Ninh, are
further grouped as the Ns region, while others of the N
region as the Nn region. Young and healthy shoots were
collected in the field, rinsed with tap water and dried in
silica gel. All samples were stored at –70 °C until they
were processed.

15
15

Cytotypes

IB (12), IC (1), IIB (1), IIIB (1)
IB (6), IIB (3), IIIB (3), VB (3)
13 IB (9), IVB (2), VB (2)
IIB (2), IIE (1), IVB (2), IVE (1), VB (3),
VIB (4), VIC (1), VIE (1)
IB (1), IIB (1), IID (1), IIIB (6), IIID (1)
IB (13), IC (1), IIIC (1)
IB (2), IIB (9), IIC (1), IVB (3)
IIIB (7), IVB (1), IVC (1), VIB (6)

DNA extraction and polymerase chain reaction
Leaf tissue or embryo of the above materials was ground to
powder in liquid nitrogen and stored in a –70 °C freezer.
Genomic DNA was extracted from the powdered tissue
following the CTAB procedure (Murray & Thompson

1980). Noncoding spacers of atpB-rbcL and trnL-trnF of the
cpDNA and the ribosomal DNA (rDNA) ITS of mtDNA
were amplified and sequenced. Universal primers for
amplifying atpB-rbcL spacer (Chiang et al. 1998), trnL-trnF
spacer (Taberlet et al. 1991), and mtDNA rITS (Chao et al.
1984) were synthesized. The polymerase chain reaction
(PCR) amplification was carried out in a volume of 100 µL
reaction using 10 ng of template DNA, 10 µL of 10× reaction
buffer, 10 µL MgCl2 (25 mm), 10 µL dNTP mix (8 mm), 10
pmole of each primer, 10 µL of 10% NP-40, and 2 U of Taq
polymerase (Promega, Madison, USA). The reaction was
programmed on a MJ Thermal Cycler (PTC 100) as one
cycle of denaturation at 95 °C for 4 min, 30 cycles of 45 s
denaturation at 92 °C, 1 min 15 s annealing at 52 °C, and
1 min 30 s extension at 72 °C, followed by 10 min extension
at 72 °C. Template DNA was denatured with reaction
buffer, MgCl2, NP-40 and ddH2O for 4 mins (first cycle),
and cooled on ice immediately. A pair of primers, dNTP
and Taq polymerase were added to the above ice-cold mix.
Reaction was restarted at the first annealing at 52 °C.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2701 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2701

T-A cloning and nucleotide sequencing
PCR products were purified by electrophoresis on a 1.0%
agarose gel using 1× TAE buffer. The gel was stained with

ethidium bromide and the desired DNA band was cut and
eluted using agarose gel purification (QIAGEN). Purified
DNA was ligated to a pGEM-T easy vector (Promega).
Plasmid DNA was selected randomly with five clones
and purified using plasmid mini kits (QIAGEN). Purified
plasmid DNA was sequenced in both directions by
standard methods of the Taq dyedeoxyterminator cycle
sequencing kit (Perkin Elmer) on an Applied Biosystems
Model 377A automated sequencer. Primers for sequence
determination were T7-promoter and SP6-promoter
located on p-GEM-T easy vector termination site.

Sequence alignments and phylogenetic analyses
Nucleotide sequences were aligned with the program
Genetics Computer Group (gcg) Wisconsin Package
(Version 10.0, Madison, Wisconsin). Neighbour-joining
(NJ) analysis by calculating Kimura 2-parameter distance
(Kimura 1980) was also performed using Data Analysis in
Molecular Biology and Evolution (dambe, version 3.5.19,
Xia 1999). Indels were treated as the fifth character.
Confidence of the clades reconstructed was tested by
bootstrapping (Felsenstein 1985) with 1000 replicates using
unweighted characters. The nodes with bootstrap values
greater than 0.70, as a rule of thumb, are significantly
supported with ≥ 95% probability (Hillis & Bull 1993). The
number of mutations between DNA genotypes in pairwise
comparisons, which were calculated using mega (Kumar
et al. 1993), was used to construct a minimum spanning
network with the aid of minspnet (Excoffier & Smouse
1994) in an hierarchical manner (cf. Chiang & Schaal 1999).

After linking the related haplotypes into a clade, closely
related clades were linked further to form higher level
groups and thereby a network.

Population genetic analysis of the cpDNA and
mtDNA sequence variation
Levels of genetic diversity within populations were
quantified by estimates of nucleotide divergence (θ)
(Watterson 1975) using dnasp (Version 3.14, Rozas &
Rozas 1999). Patterns of geographical subdivision and
gene flow were also estimated hierarchically with the aid
of dnasp. Gene flow within and among regions (populations) was approximated as Nm, the number of female
migrants per generation between populations, and was
estimated using the expression FST = 1/(1 + 2 Nm) where N
is the female elective population size and m is the female
migration rate (Slatkin 1993). We also used amova version
1.55 (Excoffier et al. 1992; Excoffier 1993) to deduce the
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710

significance of geographical divisions both among regions
and populations. The statistics of molecular variants ΦCT
(among regions), ΦST (among populations), and ΦSC
(among populations within regions), were estimated.

Results
Extent of nucleotide and haplotype diversity
In this study, trnL-trnF and atpB-rbcL noncoding spacers of
cpDNA and the rITS of mtDNA in Kandelia candel were
PCR amplified and sequenced. All sequences were registered with EMBL accession numbers AJ305472 –AJ305673
(for mtDNA ITS), AJ305674–AJ305875 (for trnL-trnF

spacer), and AJ305876–AJ306077 (for atpB-rbcL spacer).
At all three loci, no within-individual variation was
detected.
Differences between mitochondrial sequences of a
consensus length of 725 bp were mainly ascribed to point
mutations (18 sites, 2.4%). Four indel events also occurred
at sites (353–357) (592–599) (632–635) and 640. For the
atpB-rbcL spacer of the chloroplast genome, only two sites
of 781 bp (0.3%) were polymorphic. Populations of BK and
RN shared a C at site 160, while others have a T. A 1-bp
deletion at site 577 occurred in the RN population. At the
trnL-trnF spacer of cpDNA, high levels of length polymorphism, ranging from 375 bp to 415 bp, were detected. Two
deletions, at sites (17–28) and (242–268) made the spacer
shorter in populations of BK and RN. Most indels (37)
occurring in the noncoding spacer involved a 1-bp loss.
Like most noncoding regions (cf. Li 1997), A + T contents
were high, with 53.7%, 72.5%, and 76.9% at mtITS, atpBrbcL spacer, and trnL-trnF spacer, respectively.
Three haplotypes of the atpB-rbcL noncoding spacer
(h = 0.268 0.0015, θ = 0.00051 0.00008) were determined. In
contrast to other studies (Small et al. 1998; Fujii et al. 1999),
the atpB-rbcL noncoding spacer possessed a much lower
level of genetic variation than the trnL-trnF spacer in K.
candel (Table 2). In total, 28 haplotypes of the trnL-trnF
spacer of cpDNA and seven haplotypes of the mtDNA ITS
were determined. Apparently, the molecular evolution of
the mtDNA ITS was much more constrained compared to
that of the trnL-trnF spacer of cpDNA (Table 2).
At the population level, except for the lack of genetic variation at the cpDNA atpB-rbcL spacer, the level of genetic
variation varied among populations at two other organelle
loci (Table 2). High levels of nucleotide variation at both

loci, with θ-values ranging from 0.0018 to 0.0062 at trnLtrnF spacer and from 0.0020 to 0.0029 at mtITS, occurred in
populations of IR, QN, and YK, while low cpDNA variation, ranging from 0.0003 to 0.0013, was detected in populations of TN, CC, and HK. Stark contrast of the level of
genetic variation between the two loci occurred in populations of BK, HA, TP, and XM (Table 2).


MEC_1399.fm Page 2702 Wednesday, October 24, 2001 6:39 PM

2702 T . Y . C H I A N G E T A L .

cpDNA

Table 2 Haplotype diversity (h) and nucleotide diversity (θ) of the cpDNA trnLtrnF spacer and the mtDNA ITS within
populations of Kandelia candel

mtDNA

Populations

h

θ

h

θ

Total
Amami-O-Shima
Bako
Chinchou

Haikou
Hong Kong
Irimote
Quang Ninh
Ranong
Shankou
Tainan
Taipei
Xiamen
Yakushima
Zhangjiang

0.908
0.857
0.857
0.257
0.771
0.513
0.686
0.771
0.600
0.778
0.133
0.560
0.733
0.457
0.848

0.02652 ± 0.00526
0.00623 ± 0.00044

0.00615 ± 0.00092
0.00125 ± 0.00005
0.00329 ± 0.00003
0.00135 ± 0.00044
0.00310 ± 0.00164
0.00492 ± 0.00071
0.00246 ± 0.00004
0.00248 ± 0.00001
0.00034 ± 0.00018
0.00304 ± 0.00082
0.00278 ± 0.00049
0.00178 ± 0.00066
0.00457 ± 0.00040

0.406
0.248
0.000
0.133
0.000
0.000
0.248
0.448
0.248
0.467
0.248
0.000
0.133
0.362
0.133

0.00205 ± 0.00026
0.00034 ± 0.00018
0.00000
0.00018 ± 0.00015
0.00000
0.00000
0.00292 ± 0.00060
0.00255 ± 0.00034
0.00034 ± 0.00018
0.00064 ± 0.00018
0.00034 ± 0.00018
0.00000
0.00018 ± 0.00015
0.00200 ± 0.00139
0.00018 ± 0.00015

rn

bk

0

N+E

atpB-rbcL network
Fig. 2 Minimum spanning network generated using method of
Excoffier & Smouse (1994) for haplotypes of atpB-rbcL spacer of
cpDNA of populations of Kandelia candel. Each arrow indicates one
mutational change. ‘0’ indicates hypothetical ancestor.

Gene genealogies and associations between cpDNA
and mtDNA lineages
In this study we reconstructed the phylogeographical
pattern of K. candel based on gene genealogies of organelle
loci. A minimum spanning network of the cpDNA atpBrbcL spacer was reconstructed based on mutational
changes between haplotypes (Fig. 2). The BK population is
closely related to the RN population, while no variation
was detected among populations of N and E regions. An
NJ tree was recovered based on the nucleotide sequence
variation of the trnL-trnF noncoding spacer of cpDNA.
Eight clades (chlorotypes) of 28 haplotypes were identified
in this cpDNA gene tree (Fig. 3). Two major lineages of
S + RN and N + E were recognized and significantly
supported, with a bootstrap of 0.98 (P < 0.01). Four common chlorotypes I–IV of 152 sequences in total (75.2%)
were widespread in populations of N and E regions, while
types of VIIa and VIIb of 30 sequences were restricted to the
S region and RN population (Table 3). Two rare alleles of V
(4.0%) and VI (5.9%) were distributed in the N region only.
A minimum spanning network of the trnL-trnF noncoding spacer was constructed (Fig. 4). Eight clades of the

network, corresponding to those of the NJ tree, were divided
into two geographical groups (i.e. S + RN and N + E) 34
mutations apart. Within the network, closely related chlorotypes were mostly linked by single mutations. Chlorotypes
I, II and III were nested in the network as interior nodes,
while types IV and V connecting to type I, and type VI
connecting to III or IV were exterior.
In the NJ tree of mtDNA ITS sequences, two major
clades (A–E) and (F, G), of seven variants (mitotypes)
were identified (Fig. 5A). Two common mitotypes B and
C of 164 sequences (81.2%) were widespread in populations of N and E regions (Table 3), as mitotypes of F and

G were distributed in the S region and RN population.
Three rare alleles were distributed restrictedly: types A
(1.5%) in YK and IR populations, D (1.0%) in SK, and E
(1.5%) in QN. A minimum spanning network of the
mtDNA ITS was constructed (Fig. 5B). Within the clade of
N + E regions, mitotype B was nested in the network as
the interior node, connecting to other types independently with 1 – 9 mutations. Within the clade of S + RN
regions, mitotype G was linked to the interior node of
type F with two mutations, which was linked to type B
with four mutations.
Phylogenies of cpDNA and mtDNA are completely
consistent at the level of geographical regions (i.e. S + RN
vs. N + E). The atpB-rbcL noncoding spacer provided no
information in resolving the phylogeny within N + E
regions. Apparently, the gene tree of the trnL-trnF spacer of
cpDNA largely contradicted the mtDNA ITS tree. No clade
correspondence was found between the two trees. For
example, although most chlorotype III sequences (87.5%)
corresponded to the mitotype B, sequences of am4 and
tn12 were associated with mitotype C and sequence sk2
was associated with the mitotype D.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2703 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2703

Fig. 3 Neighbour-joining tree of representative sequences (haplotypes) of trnL-trnF of cpDNA in Kandelia candel. Numbers at nodes indicate
bootstrap values. Chlorotypes (I–VIIb) are labelled on clades.

Table 3 Distribution of chlorotypes (I–VIIb) and mitotypes (A–G) among populations of Kandelia candel. Regions are indicated: Indian
Ocean Rim (I), southern South-China-Sea region (S), northern South-China-Sea region (N), and East-China-Sea region (E)
Regions:

I
II
III
IV
V
VI
VIIa
VIIb
A
B
C
D
E
F
G

I

S

N

E

rn

bk

qn

cc

sk

3

13
1
1

1
2
7

3
3
6

zj

7
2

xm

ha

hk

tn

tp

2
10

6
3
3

9

14

9
3

6
3

2

6

14

2
13

3

ir

1

3

2
2

15

13

am

yk

6
3
3
3

11
2
2

6

15
15
11
1

14
1

8

14
1

14
1

2
3
13
2

15

Nevertheless, associations between cpDNA and mtDNA
haplotypes were nonrandom. Seventeen (instead of 30;
χ2 = 1.03, P = 0.00018) and three (instead of four; χ2 =
0.044, P = 0.0001) cpDNA (trnL-trnF)-mtDNA associated

© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710

13
2

13
2

1
12
2

Total
77 (38.2%)
30 (14.9%)
24 (11.9%)
21 (10.3%)
8 (4.0%)
12 (5.9%)
15 (7.4%)
15 (7.4%)
3 (1.5%)
154 (76.2%)
10 (5.0%)
2 (1.0%)
3 (1.5%)
28 (13.9%)
2 (1.0%)

cytotypes were observed in N + E and S + RN regions of K.

candel, respectively (Table 4). All sequences of the mitotype
A were exclusively associated with the chlorotype I;
and sequences of the mitotype D were associated with the


MEC_1399.fm Page 2704 Wednesday, October 24, 2001 6:39 PM

2704 T . Y . C H I A N G E T A L .

Fig. 4 Minimum spanning network generated using method of Excoffier & Smouse (1994) for haplotypes of trnL-trnF spacer of cpDNA of
populations of Kandelia candel. Each arrow indicates one mutational change. Number of mutational change is indicated when more than
one step. ‘0’ indicates hypothetical ancestor. The replicate number of haplotypes is also indicated when more than one.

Fig. 5 (A) Neighbour-joining tree of haplotypes (A –G) of rITS of mtDNA in Kandelia
candel. Numbers at nodes indicate bootstrap
values. ( B) Minimum spanning network
generated using method of Excoffier &
Smouse (1994) for haplotypes of mtDNA
ITS of populations of K. candel. Mutational
changes are indicated at nodes.

© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2705 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2705
Table 4 Associations between chlorotypes and mitotypes of Kandelia candel. Distribution range of each chlorotype and mitotype is indicated
in square brackets. Percentage of each cytotype is indicated in parentheses. W: widespread. Other symbols see Table 1

chlorotype:

mitotype:

I [W]

A
[ir + yk]

B
[W]

C
[W]

3
(1.5%)

70
(34.6%)
27
(13.4%)
21
(10.4%)
18
(8.9%)
8
(4.0%)
10
(5.0%)

4
(2.0%)
1
(0.5%)
2
(1.0%)
2
(1.0%)

II [W]
III [W]
IV [W]
V [qn, hk, ha]
VI [qn, zj]

D
[sk]

E
[qn]

1
(0.5%)
1
(0.5%)

154

Total

1
(0.5%)

30
24

1
(0.5%)

21
8

1
(0.5%)

1
(0.5%)

VIIb [bk]
3

G
[bk]

77

VIIa [rn]

Total

F
[rn + bk]

10

2

3

12
13
(6.4%)
15
(7.4%)
28

2
(1.0%)

15
15

2

202

Table 5 Pairwise FST/ Nm estimates between geographical regions (E, N, Ns and Nn) based on genetic variation of mtDNA ITS (above the
diagonal) and cpDNA trnL-trnF spacer (below the diagonal). Nucleotide diversity within each region is indicated in the parenthesis

cpDNA
E
(θ = 0.00503 ± 0.00054)
N
(θ = 0.00445 ± 0.00033)
Ns
(θ = 0.00312 ± 0.00037)
Nn
(θ = 0.00492 ± 0.00041)

mtDNA:

E
(θ = 0.00138 ± 0.00048)

N
(θ = 0.00049 ± 0.00007)

Ns
(θ = 0.00120 ± 0.00049)

Nn
(θ = 0.00135 ± 0.00007)

—

0.020/23.95

0.023/21.50

0.021/ 23.14

0.026/18.41

—

—

—

0.023/21.09

—

—

0.011/45.67

0.031/15.41

—

0.013/39.00

—

chlorotypes II and III. Likewise, the chlorotype V was exclusively associated with the most dominant mitotype B, and
most sequences of chlorotype I were associated with the
mitotype B, while some other sequences were mitotypes A
or C. Within the N + E region, cytotypes BI (40.7%) and BII

(15.7%) were most dominant in composition. In contrast,
cytotypes of CII, DII, EII, CIII, DIII, CIV, EIV, CVI, and EVI
were relatively rare (5.8% in total). Likewise, within the
S + RN region, FVIIa and FVIIb were dominant (93.3%),
while the cytotype GVIIa was rare (6.7%).
The cytotype composition varied among populations. In
Fig. 1 the genetic composition in each population was indicated. Higher number of cytotypes occurred in populations
AM (six types), QN (eight types), and YK (five types),
while a single type was detected in the BK population and
two types were detected in the RN population (Table 1).
Within the N + E regions, populations possessing a higher
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710

number of cytotypes appeared to be located at margins
(Fig. 1). In contrast, most geographically ‘central’ populations had three or four cytotypes.

Population differentiation and geographical divisions
Population and geographical structure of K. candel was
assessed based on genetic variation of the organelle loci.
Estimates of FST (= 0.93–0.95) and Nm (= 0.03 – 0.04) based
on the cpDNA trnL-trnF spacer and mtDNA ITS, indicated
significant differentiation between regions S + RN, and
N + E. In contrast to the consistent estimations of
population structure between the above two loci, higher
number of migrants (Nm = 0.10) per generation was
deduced from atpB-rbcL spacer sequences, although the
genetic differentiation was still significant (FST = 0.828).
Hierarchical analyses of sequence difference under amova

MEC_1399.fm Page 2706 Wednesday, October 24, 2001 6:39 PM

2706 T . Y . C H I A N G E T A L .
indicated that the proportion of molecular variance was
attributed to difference between geographical regions
(ΦCT = 0.860, P < 0.001). The relative contribution of difference among populations to molecular variance was
small (ΦST = 0.087). In contrast, no geographical subdivision (FST = 0.020 – 0.026 and Nm = 18.41–23.95) between
N and E was detected (Table 5).
An analysis of molecular variance (amova) based on the
trnL-trnF spacer of cpDNA also suggested low levels of
genetic differentiation between populations of mainland
and continental islands (ΦCT = 0.015) as well as among
populations within each region (ΦSC = 0.037). Genetic
variation of the mtDNA ITS yielded a similar pattern of
the genetic apportionment among geographical regions
and populations. The deduced Nm of 39.00 – 45.67 indicated
frequent gene flow between Ns and Nn regions (Table 5).
In contrast to the invariable atpB-rbcL spacer, pairwise
comparisons showed high variances in genetic estimates of
structure and genetic differentiation between cpDNA and
mtDNA loci, except for those between BK + RN and other
populations. Low level of genetic differentiation was usually detected among populations within the E + N regions.
Nearly all Nm values, ranging from 1.75 (between SK and
HK) to 622.55 (between CC and YK), deduced from the
mtDNA ITS were greater than those from the cpDNA trnLtrnF spacer. Lower Nm values, less than one, were mostly
restricted to comparisons with ZJ as well as SK based on
cpDNA variation. Some extremely high Nm values were
obtained, such as 150 between AM and CC. High variance
in deducing FST and Nm was also encountered in comparisons between BK and RN. Based on the trnL-trnF spacer,
an FST value of 0.42 and an Nm of 0.68 were deduced, while

a lower level of genetic differentiation (FST = 0.13, Nm =
3.50) was detected from the mtITS.

Discussion
Genetic variability and low level of homoplasious
mutations in cpDNAs and mtDNAs of Kandelia candel
The usefulness of molecular markers in indirect estimates
of population structure and gene flow depends on the level
of resolution, and locus-to-locus consistency (Bossart &
Prowell 1998), and is also affected by the level of recurrent
mutations (cf. Desplanque et al. 2000). Recurrent mutations
(identity by state), which are frequently encountered in the
mitochondrial genome due to limited conformations in
molecular structure (Fauron et al. 1995), will inevitably blur
the level of migration between populations. Technically,
nucleotide sequencing can simply rule out the length homoplasies, which occur usually in restriction fragment length
polymorphism (RFLP) and PCR-based fingerprints (cf. Parker
et al. 1998; Desplanque et al. 2000), from the data scoring.
In this study, as a very strong linkage disequilibrium was

estimated between mitotypes and chlorotypes both within
N + E and S + RN regions (Table 4), a high rate of recurrent
mutations can be excluded for organelle genomes of
K. candel (cf. Desplanque et al. 2000).
In this study, genetic variation of mtDNA ITS and
cpDNA trnL-trnF spacer existed both within and between
populations. Nevertheless, the haplotype diversity of the
mitochondrial DNA, with seven haplotypes out of 202
plants screened, was lower compared to other flowering
plants, such as Beta vulgaris ssp. maritima (20 mitotypes

from 414 individuals; Desplanque et al. 2000), Daucus carota
ssp. carota (25 variants based on mtDNA RFLP from 80
plants; Ronfort et al. 1995), Thymus vulgaris (50 mitotypes
from about 400 plants; Manicacci et al. 1996), and Hevea
brasiliensis (212 mtDNA RFLP variants in 395 accessions
screened; Luo et al. 1995).
For the chloroplast genome, K. candel possessed a higher
level of haplotype diversity (28 haplotypes) at the trnL-trnF
spacer, which is close to 13 cpDNA haplotypes in Beta vulgaris ssp. maritima (Desplanque et al. 2000), 11 haplotypes
in Argania (El Mousadik & Petit 1996), 23 haplotypes in
white oaks (Dumolin-Lapègue et al. 1997), and 13 haplotypes in Alnus (King & Ferris 1998). Although comparisons
of haplotype diversity among taxa, which were examined
using various molecular methods at different loci, may be
somewhat misleading, nucleotide diversity of the trnLtrnF spacer in K. candel (θ = 0.02710) appeared to be higher
than that of other plants as well, such as Cunninghamia
(θ = 0.01018, Lu et al. 2001) and Begonia (θ = 0.003, Liu
1999). Apparently, both loci in this study provided sufficient resolution at the geographical region level and
yielded consistent estimates of gene flow (Nm = 0.03 – 0.04)
and population structure (FST = 0.93 – 0.95) between S + RN
and N + E populations of K. candel. To the contrary, lack of
variability, due to its conserved nature (cf. Chiang & Schaal
2000a,b), has made the atpB-rbcL noncoding spacer locus
powerless in estimating the interpopulation migration
within the N + E regions. At interregions level, as a result
of having difficulties in distinguishing coancestry from
migration, higher Nm value (= 0.10) between S + RN and
N + E regions was thereby deduced based on this spacer.
Likewise, at the population level, the more conserved
mtDNA ITS always yielded higher values of Nm and lower
levels of FST, than the cpDNA trnL-trnF spacer. In this

investigation, due to its higher resolution, the trnL-trnF
might have higher probabilities of yielding estimates that
approximate the current population structure of K. candel.

Phylogeographical patterns of K. candel in East Asia
In this study, we investigated the phylogeography of the
viviparous species, K. candel. Both the mtDNA ITS and
the cpDNA trnL-trnF spacer suggested noticeable longdistance seedling dispersal. However, as extensive gene
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2707 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2707
flow across oceans via seedlings was detected among
populations along a 2700 km transect (between QN and
YK) in the regions of northern South-China-Sea and EastChina-Sea, gene genealogies of three organelle loci
revealed consistent geographical structure. Accordingly,
the population of Sarawak along the bank of the South
China Sea in East Asia, which is about 2000 km from
the QN (Fig. 1), was phylogenetically grouped with the
Ranong population of the Peninsula Malaysia at the
northeastern rim of the Indian Ocean.
This unique geographical structure, consistently suggested by organelle loci (Figs 2, 4 and 5) as well as allozyme
data (Huang & Chen 2000), not only indicated the origin
of the Sarawak population derived from the Indian
Ocean Rim, but also reflected a phylogeographical pattern
associated with the vicariance history. According to
palaeoceanographic evidence, during the last glacial maximum of the Pleistocene, the global sea level dropped by
some 100 –120 m. Meanwhile, with the closure of all the

southern connections to the ocean, the South China Sea
nearly became an enclosed basin, with the Bashi Strait as its
only water pathway to the Pacific Ocean (Wang et al. 1995),
and was completely isolated from the Indian Ocean. Sarawak,
although geographically located along the southern coast
of the modern South China Sea, has long been the northern
edge of the Sunda shelf since the late Quaternary. According to another estimation of Yamazaki (1998), populations
of the South China Sea and the East China Sea may have
been isolated from those of the Bay of Bengal of the Indian
Ocean for at least 0.6 – 2.2 million years, a duration sufficient for coalescence at most loci. Apparently, monophyly
of F + G alleles of the mtDNA and VIIa + VIIb alleles of the
cpDNA supported the long isolation hypothesis. In addition, based on the paucity of genetic variation at two
organelle loci and the phylogenetic affinity to the RN
population, a small number of founders through long-distance
dispersal from populations of the Indian Ocean may have
been involved in the colonization of the BK population. Nevertheless, the level of ongoing gene flow between BK and RN
appeared to be low, according to the deduced Nm of 0.00–
0.68 from the atpB-rbcL and trnL-trnF, respectively, a result
agreeing with the orientation of summer ocean currents.
According to the association between unique genetic
structure and vicariance events, the Sarawak population
must also have been shaped by limited recurrent gene flow
to other populations of the South China Sea, which in turn
is constrained by seasonal orientations of current flows.
Sea surface circulation in the modern South China Sea is
basically driven by the monsoon in East Asia. In summer,
the season during which most seedlings are detached from
maternal plants of K. candel and disperse (Tomlinson 1986;
Huang & Chen 2000), surface water of the tropical Indian
Ocean flows northward into the South China Sea and then

through the Bashi Strait (the strait between Taiwan and
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710

the Philippines) into the Pacific (cf. Wang et al. 1995).
Because populations are not distributed in the Philippines
(Hou 1958), somewhat indicating no suitable habitats for
K. candel, most seedlings of the Sarawak are likely to be
discharged into oceans, having limited probability of
colonizing territory of the northern South China Sea. With
limited gene flow with other populations, the Sarawak
population has long maintained its own identity.
Despite the isolation between South-China-Sea and EastChina-Sea regions during the glacial maximum, subsequent
ongoing gene flow via ocean currents has homogenized
between-region and between-population variation in K.
candel. In agreement with previous allozyme investigations
(Huang 1994; Sun et al. 1998; Huang & Chen 2000), both
chloroplast (trnL-trnF spacer) and mtDNAs indicated no
hindrance to the long-distance seedling dispersal between
mainland and continental islands via ocean currents
(Table 5). Although the isolation-by-distance model was
not met at the population level, gene flow within region
(i.e. between Ns and Nn, Table 5) was apparently more
frequent than between E and N regions. In addition,
according to the higher nucleotide diversity in the Nn
populations at both organelle loci (Table 5), a preferential
northward migration (from Ns to Nn) seemed to exist due
to the orientation of ocean currents in summer. Nevertheless,
some local topographical barriers may have blocked the
interpopulation gene flow. According to the estimates
based on the trnL-trnF spacer, limited gene flow occurred

in populations ZJ and SK, both located on opposite coasts
of the Leizhou Peninsula (Fig. 1), to other populations. In
addition, although no general rule can be generated, Hong
Kong seemed to have more frequent gene flow with neighbouring populations, including TN, TP, and IR, than with
distant populations.

Relative ages of alleles of cpDNAs and mtDNAs
In resolving phylogeographical pattern and phylogenetic
ambiguities, the nested cladogram provides sufficient
information, complementary to conventional cladograms
(cf. Crandall & Templeton 1993). Based on their interior
positions in the minimum networks, the ancestry of eight
dominant and widespread cytotypes was suggested: BI,
BII, BIII, BIV, CI, CII, CIII, and CIV (Table 4), which would
have a greater probability of producing mutational
derivatives (cf. Donnelly & Tavaré 1986; Golding 1987;
Crandall & Templeton 1993). In contrast to the strong
linkage disequilibrium among chlorotypes of I–VI and
mitotypes of A–E, nearly random association (χ2 = 0.822,
P = 0.189) was detected in these common cytotypes,
indicating genetic equilibrium and panmixis within the
N + E region as a whole.
The strong linkage disequilibrium may simply stem
from effects of lineage sorting, due to relative ages of alleles


MEC_1399.fm Page 2708 Wednesday, October 24, 2001 6:39 PM

2708 T . Y . C H I A N G E T A L .
of each locus (Chiang 2000). According to the exterior positions in the networks and their restricted spatial distribution, alleles V and VI of the cpDNA trnL-trnF spacer, and

alleles of A, D, and E of the mtDNA may have evolved
recently. As generally known, newly evolved cpDNA alleles,
since having a low frequency in the gene pool, were
likely to be ‘attracted’ to dominant mitotypes (B and C, in
this study), and vice versa. Probabilities of associations
between two rare alleles would be extremely low, thereafter leading to strong linkage disequilibrium.
Interestingly, almost all rare alleles occurred at marginal
populations of the E + N regions, such as allele A restricted
in IR and YK (Ryukyu), D exclusively in SK, and E in QN
only. Technically, the absence of these rare alleles in
‘central’ populations may be simply because of failure of
detection due to their low frequency. On the other hand,
some preferential directional migration due to the microgeographical hindrance might also account for the apportionment of rare alleles. Nonetheless, with ceaseless and frequent gene flow, central populations, exposed to migrants
from all neighbouring populations, would have a large
probability of maintaining genetic variation. Fluctuation
of genetic structure resulting from a limited number of
founders (cf. Sun et al. 1998) would seldom occur. The low
level of genetic variation detected in Taiwan and Hong
Kong (Huang 1994; Sun et al. 1998) may simply come from the
conserved nature of the markers themselves. For example,
in this study, high cpDNA variation (θ = 0.00304) vs. no
mtDNA variation was detected in Taipei (Table 2). On the
other hand, the organelle DNA diversity of Taiwan (with θ
of 0.00173 at cpDNA trnL-trnF locus, and of 0.00018 at the
mtITS locus) and Hong Kong (θ of 0.00135 at cpDNA locus
vs. no mtDNA variation) was lower than most other populations (Table 2). Habitat destruction in recent decades due
to human activities may have largely contributed to the loss
of genetic diversity (Yipp et al. 1995; Chiang & Hsu 2000).

Conclusions

Kandelia candel, as a viviparous species of mangroves,
provides an ideal model for testing the possibility and
extent of long-range seedling dispersal. In agreement with
previous allozyme investigations, the geographical and
population structure of the species, which adapts to the
intertidal habitats with precocious growth of seeds, was
determined both by vicariance and ongoing gene flow.
Significant genetic differentiation between populations of
northern and southern banks of the South China Sea plus
the attainment of monophyly of alleles of both cpDNAs
and mtDNAs at geographical region level indicated a long
isolation. In contrast, recurrent gene flow via long-distance
dispersal, indicated by high deduced Nm values, have
contributed to the genetic homogeneity among populations of the region of northern South China Sea and East

China Sea. Gene genealogies, which trace phylogenetic
relationships among alleles in a geographical context, of
different loci coupled with locus–locus associations,
helped clarify historical and recurrent evolutionary events.

Acknowledgements
This study was supported by NSC grants of NSC- 85 – 2311-B-003 –
006-B17, NSC- 86–2311-B-003–005-B17 and NSC87–2311-B-003 –
004-B17 to S Huang. We are indebted to Drs CI Peng, H Ota, and
HQ Fan for the assistance with sampling.

References
Bossart JL, Prowell DP (1998) Genetic estimates of population
structure and gene flow: limitations, lessons and new directions. Trends in Ecology and Evolution, 13, 202 – 206.
Chao S, Sederoff R, Levings CS III (1984) Nucleotide sequence

and evolution of the 18S ribosomal RNA gene in maize mitochondria. Nucleic Acids Research, 12, 6629 – 6644.
Chen YJ (2000) Study on the genetic variation among populations of
Kandelia candel (L.) Druce in East Asia. Master Thesis, Department of Biology, Taiwan Normal University, Taipei.
Chiang TY (2000) Lineage sorting accounting for the disassociation between chloroplast and mitochondrial lineages in oaks of
southern France. Genome, 43, 1090–1094.
Chiang TY, Hsu TW (2000) Wetland biodiversity: proceedings of Symposium of Biodiversity in Wetlands. Taiwan Endemic Species
Research Institute, Nantou, Taiwan.
Chiang TY, Schaal BA (1999) Phylogeography of North American
populations of the moss species Hylocomium splendens based on
the nucleotide sequence of internal transcribed spacer 2 of
nuclear ribosomal DNA. Molecular Ecology, 8, 1037 – 1042.
Chiang TY, Schaal BA (2000a) Molecular evolution of the atpB-rbcL
noncoding spacer of chloroplast DNA in the moss family Hylocomiaceae. Botanical Bulletin of Academia Sinica, 41, 85 – 92.
Chiang TY, Schaal BA (2000b) Molecular evolution and phylogeny
of the atpB-rbcL spacer of chloroplast DNA in the true mosses.
Genome, 43, 417–426.
Chiang TY, Schaal BA, Peng CI (1998) Universal primers for
amplification and sequencing a noncoding spacer between atpB
and rbcL genes of chloroplast DNA. Botanical Bulletin of
Academia Sinica, 39, 245–250.
Chou CH, Chiang YC, Chiang TY (2000) Genetic variability and
phytogeography of Miscanthus sinensis var. condensatus, an
apomictic grass, based on RAPD fingerprints. Canadian Journal
of Botany, 78, 1262–1268.
Crandall KA, Templeton AR (1993) Empirical tests of some predictions from coalescent theory with applications to intraspecific
phylogeny reconstruction. Genetics, 134, 959 – 969.
Desplanque B, Viard F, Bernard J et al. (2000) The linkage disequilibrium between chloroplast DNA and mitochondrial DNA
haplotypes in Beta vulgaris ssp. maritima (L.): the usefulness of
both genomes for population genetic studies. Molecular Ecology,
9, 141–154.

Donnelly P, Tavaré S (1986) The ages of alleles and a coalescent.
Advances in Applied Probability, 18, 1– 19.
Duke NC (1995) Genetic diversity, distributional barriers and rafting continents-more thoughts on the evolution of mangroves.
Hydrobiologia, 295, 167–181.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710


MEC_1399.fm Page 2709 Wednesday, October 24, 2001 6:39 PM

P H Y L O G E O G R A P H Y O F K A N D E L I A 2709
Dumolin-Lapègue S, Demesure B, Fineschi S, Le Corre V, Petit RJ
(1997) Phylogeographic structure of the white oaks throughout
the European continent. Genetics, 146, 1475–1487.
Dumolin-Lapègue S, Pemonge MH, Petit RJ (1998) Association
between chloroplast and mitochondrial lineages in oaks. Molecular Biology and Evolution, 15, 1321 – 1331.
El Mousadik A, Petit RJ (1996) Chloroplast DNA phylogeography
of the argan tree of Morocco. Molecular Ecology, 5, 547–555.
Elmqvist T, Cox PA (1996) The evolution of vivipary in flowering
plants. OIKOS, 77, 3 – 9.
Excoffier L (1993) Analysis of Molecular Variance, Version 1.5.5. Genetics and Biometry Laboratory, University of Geneva, Switzerland.
Excoffier L, Smouse PE (1994) Using allele frequencies and geographic subdivision to reconstruct gene trees within a species:
Molecular variance parsimony. Genetics, 136, 343–359.
Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular
variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction
data. Genetics, 131, 479 – 491.
Fauron CMR, Moore B, Casper M (1995) Maize as a model on
higher plant mitochondrial genome plasticity. Plant Science, 112,
11 – 32.
Felsenstein J (1985) Confidence limits on phylogenies: an
approach using the bootstrap. Evolution, 39, 783–791.

Fujii N, Ueda K, Watano Y, Shimizu T (1999) Further analysis of
intraspecific sequence variation of chloroplast DNA in Primula
cuneifolia Ledeb. (Primulaceae): implications for biogeography
of the Japanese alpine flora. Journal of Plant Research, 112, 87–95.
Golding GB (1987) Multiple substitutions create biased estimates
of divergence times and small increases in the variance to mean
ratio. Heredity, 58, 331 – 339.
Goodall JA, Stoddart JA (1989) Techniques for the electrophoresis
of mangrove tissue. Aquatic Botany, 35, 197–207.
Harris SA, Ingram R (1991) Chloroplast DNA and biosystematics:
the effects of intraspecific diversity and plasmid transmission.
Taxon, 40, 393 – 421.
Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a
method assessing confidence in phylogenetic analysis. Systematic Biology, 41, 182 – 192.
Hoelzer GA, Wallman J, Melnick DJ (1998) The effects of social
structure, geographical structure, and population size on the
evolution of mitochondrial DNA. II. Molecular clocks and
the lineage sorting period. Journal of Molecular Evolution, 47, 21–
31.
Hou D (1958) Rhizophoraceae. In: Flora Malesiana (ed. van Steenis CGGJ),
Series I, Vol. 5, pp. 429 – 493. Djakarta Press, Noordhoff-Kolff.
Huang S (1994) Genetic variation of Kandelia candel (L.) Druce
(Rhizophoraceae) in Taiwan. In: Proceedings: International Symposium on Genetic Conservation and Production of Tropical Forest
Tree Seed (eds Drysdale RM, John SET, Yapa AC), pp. 165–172.
ASEAAN-Canada Forest Tree Seed Centre Project, Muaklek,
Saraburi, Thailand.
Huang S, Chen YC (2000) Patterns of genetic variation of Kandelia
candel among populations around South China Sea. In: Wetland
Biodiversity: Proceedings of Symposium of Biodiversity in Wetlands
(eds Chiang TY, Hsu TW), pp. 59 – 64. Taiwan Endemic Species

Research Institute, Nantou, Taiwan.
Huang CY, Wu SF, Zhao M et al. (1997) Surface ocean and
monsoon climate variability in the South China Sea since the
last glaciation. Marine Micropaleontology, 32, 71–94.
Kimura M (1980) A simple method for estimating evolutionary
rates of base substitutions through comparative studies of
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710

nucleotide sequences. Journal of Molecular Evolution, 10, 111 –
120.
King RA, Ferris C (1998) Chloroplast DNA phylogeography of
Alnus glutinosa (L.) Gaertn. Molecular Ecology, 7, 1151–1161.
Kumar PS, Tamura K, Nei M (1993) MEGA: Molecular Evolutionary
Genetics Analysis, Version 1.01. The Pennsylvania State University,
PA.
Lakshmi M, Rajalakshmi S, Parani M, Anuratha CS, Parida A
(1997) Molecular phylogeny of mangroves I. Use of molecular
markers in assessing the intraspecific genetic variability in
the mangrove species Acanthus ilicifolius Linn. (Acanthaceae).
Theoretical and Applied Genetics, 94, 1121 – 1127.
Li WH (1997) Molecular Evolution. Sinauer Associates Inc.,
USA.
Liu SL (1999) Multiple origins of Begonia formosanus based on nucleotide variation of organelle and nuclear ribosomal DNAs. Master
Thesis, Department of Biology, Cheng-Kung University,
Tainan.
Lu SY, Peng CI, Cheng YP, Chiang TY (2001) Chloroplast DNA
phylogeography of Cunninghamia konishii (Cupressaceae), an
endemic conifer of Taiwan. Genome, 44, 797– 807.
Lugo AE, Snedaker SC (1974) The ecology of mangroves. Annual
Review of Ecology and Systematics, 5, 39– 64.

Luo H, van Coppenolle B, Seguin M, Boutry M (1995) Mitochondrial DNA polymorphism and phylogenetic relationships in
Hevea brasiliensis. Molecular Breeding, 1, 51 – 63.
Mabberley DJ (1997) The Plant-Book. A Portable Dictionary of the
Vascular Plants, 2nd edn. Cambridge University Press, Cambridge.
Maguire TL, Saenger P, Baverstock P, Henry R (2000) Microsatellite analysis of genetic structure in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae). Molecular Ecology, 9,
1853–1862.
Manicacci D, Couvet D, Belhassen E, Gouyon PH, Atlan A (1996)
Founder effects and sex ratio in the gyndioeciuos Thymus vulgaris L. Molecular Ecology, 5, 63–72.
Maxwell GS (1995) Ecogeographic variation in Kandelia candel
from Brunei, Hong Kong and Thailand. Hydrobiologia, 295, 59–65.
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321 – 4325.
Ota H (1998) Geographic patterns of endemism and speciation in
amphibians and reptiles of the Ryukyu Archipelago, Japan,
with special reference to their paleogeographical implications.
Researches on Population Ecology, 40, 189 – 204.
Parker PG, Snow AA, Schug MD, Booton GC, Fuerst PA (1998)
What molecules can tell us about populations: choosing and
using a molecular marker. Ecology, 79, 361 – 382.
Ronfort J, Saumitou-Laprade P, Cuguen J, Couvet D (1995) Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp. carota. Theoretical and Applied Genetics,
91, 150–159.
Rozas J, Rozas R (1999) dnasp version 3.0: an integrated program
for molecular population genetics and molecular evolution
analysis. Bioinformatics, 15, 174–175.
Schaal BA, Hayworth DA, Olsen KM, Rauscher JT, Smith WA
(1998) Phylogeographic studies in plants: problems and prospects. Molecular Ecology, 7, 465–474.
Schnabel A, Asmussen JA (1989) Definition and properties of
disequilibrium within nuclear-mitochondrial-chloroplast and
other nuclear-dicytoplasmic systems. Genetics, 123, 199 – 215.
Schwarzbach AE, Ricklefs RE (1998) Historical biogeography of
mangroves in the family Rhizophoraceae. American Journal of

Botany, 85 (Suppl.), 174–175.


MEC_1399.fm Page 2710 Wednesday, October 24, 2001 6:39 PM

2710 T . Y . C H I A N G E T A L .
Slatkin M (1993) Isolation by distance in equilibrium and nonequilibrium populations. Evolution, 47, 264–279.
Small RL, Ryburn JA, Cronn RC, Seelanan T, Wendel JF (1998) The
tortoise and the hare: choosing between noncoding plastome and
nuclear adh sequences for phylogeny reconstruction in a recently
diverged plant group. American Journal of Botany, 85, 1301–1315.
Sun M, Wong KC, Lee JSY (1998) Reproductive biology and
population genetic structure of Kandelia candel (Rhizophoraceae),
a viviparous mangrove species. American Journal of Botany, 85,
1631 – 1637.
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers
for amplification of three non-coding regions of chloroplast
DNA. Plant Molecular Biology, 17, 1105–1109.
Templeton AR (1998) Nested clade analyses of phylogeographic
data: Testing hypotheses about gene flow and population
history. Molecular Ecology, 7, 381 – 397.
Tomlinson PB (1986) The Botany of Mangroves. Cambridge University Press, Cambridge.
Wang P, Wang L, Bian Y, Jian Z (1995) Late Quaternary paleoceanography of the South China Sea: surface circulation and carbon
cycles. Marine Geology, 127, 145 – 165.

Watterson GA (1975) On the Number of segregating sites in genetical models without recombination. Theoretical Population
Biology, 7, 256–276.
Xia X (1999) Estimating the frequency of litters with multiple
paternity by using molecular data. In: The Application, Methods
and Theories in Molecular Ecology (eds Zhu YG, Sun M, Le K),

pp. 136–151. CHEP-Springer, Hong Kong.
Yamazaki T (1998) Molecular polymorphism and population
structure of several tropical tree species in southeast Asia.
American Journal of Botany, 85 (Suppl.), 62 – 63.
Yipp MW, Hau CH, Walthew G (1995) Conservation evaluation of nine Hong Kong mangals. Hydrobiologia, 295, 323 –
333.

The results reported here are from a collaboration between
S. Huang and T. Y. Chiang’s laboratories. Work in T. Y. Chiang’s
group is on data analysis. Work in S. Huang’s laboratory focuses
on field collection, ecological observations, and nucleotide
sequencing of mangrove trees.

© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710