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

Molecular Ecology (2001)

10

, 2697–2710

© 2001 Blackwell Science Ltd

Blackwell Science Ltd

Phylogeography of

Kandelia candel

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

T. Y. CHIANG,* Y. C . CHIANG,† Y. J . CHEN,† C . H. CHOU,‡ S . HAVANOND,§ T. N. HONG¶ and S . HUANG†

*

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 tolong-range dispersal. To examine the extent of seedling dispersal across oceans, we invest-igated the phylogeny and genetic structure among East Asiatic populations of

Kandeliacandel

based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplastDNA (cpDNA)

atp

B-

rbc

L spacer, cpDNA

trn

L-

trn

F spacer, and mitochondrial DNA(mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 indi-viduals. Three data sets suggested consistent phylogenies recovering two differentiatedlineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawakpopulation was closely related to the Ranong population of western Peninsula Malaysiainstead of other South-China-Sea populations, indicating its possible origin from theIndian Ocean Rim. No geographical subdivision was detected within the northern geo-graphical region. An analysis of molecular variance (

AMOVA

) revealed low levels of geneticdifferentiation between and within mainland and island populations (

Φ

CT

= 0.015,

Φ

SC

= 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Signi-ficant linkage disequilibrium excluded the possibility of recurrent homoplasious mutationsas the major force causing phylogenetic discrepancy between mtDNA and the

trn

L-

trn

Fspacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype–mitotype associations and tree inconsistency. Widespread distributionand random associations (

χ

2

= 0.822,

P

= 0.189) of eight hypothetical ancestral cytotypesindicated the panmixis of populations of the northern geographical region as a whole. Incontrast, rare and recently evolved alleles were restricted to marginal populations, reveal-ing 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

(Tomlinson 1986; Mabberley 1997). Mangrove forests arecharacteristic of tropical and subtropical coastlines of theworld. Over the last decades, due to human’s over-exploitation, the genetic diversity in mangroves has beendeprived, especially from coastal ecosystems in Asia.Many countries have categorized the sustainable manage-ment of mangroves as major priorities in biodiversity

Correspondence: S. Huang. Fax: + 886-2-29312904; E-mail:[email protected]

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

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conservation (Maguire

et al

. 2000). According to bio-geographical evidence, the genus

Kandelia

underwent asevere extinction phase during the upper Tertiary after theclosure of the Tethys Seaway (Schwarzbach & Ricklefs1998). Since then populations of

K. candel

have beenrestricted to the tropical Malesia and East Asia, includ-ing areas around the South China Sea and the EastChina Sea.

Like many other mangrove species,

K. candel

is charac-terized by vivipary, the precocious growth of the progenywhen still attached to the maternal parent. Vivipary isan adaptive feature for mangrove plants to colonize andexpand populations at intertidal estuary habitats. Preco-cious seedlings of

K. candel

, growing up to 47 cm long and1.3 cm wide, are buoyant, which may allow long-range dis-persal via ocean currents (Lugo & Snedaker 1974; Maxwell1995). However, viviparous seedlings have also been con-sidered as a hindrance to long-distance dispersal due to thelack of protection and nutritional support from the mater-nal tissue (Duke 1995; Elmqvist & Cox 1996). Correspond-ingly, extremely various population structures have beendiscovered in different mangrove species. High level ofgenetic differentiation among populations as well as geo-graphical subdivision, due to restricted gene flow acrosspopulations, was lately detected in

Avicennia marina

(Avi-cenniaceae) (Duke 1995; Maguire

et al

. 2000), a result closeto the population structure of a nonviviparous species,

Acanthus ilicifolius

(Lakshmi

et al

. 1997). In contrast, Aus-tralian populations of

Rhizophora stylosa

were found barelydifferentiated (Goodall & Stoddart 1989). Recent allozymeinvestigations revealed low level of genetic differentiation(

G

ST

= 0.064, Sun

et al

. 1998) among Hong Kong popula-tions as well as between two populations from Taiwan(

F

ST

= 0.04, Huang 1994), both indicating that the seed-ling dispersal in

K. candel

was not as limited as previouslysuggested.

Although frequent gene flow has been detected in

K.candel

at the local scale, long-range dispersal across oceansremains unknown. Apparently, the dispersal extent of

K. candel

is not only regulated by the orientation of oceancurrents in the South China Sea and East China Sea, but alsoconstrained by the duration and survivorship of vivipar-ous seedlings in the high saline conditions. The isolation-by-distance model is thus a hypothesis to be tested. Inaddition, when geographical distance increases for invest-igation, effects of vicariance will become more prominentand may confound the isolation-by-distance model (Bossart& Prowell 1998). Geological history inevitably playsanother critical role in determining the phylogeographicalpattern. For example, geographically close populationsalong western and eastern coasts of the Peninsula Malaysiawere significantly differentiated due to a vicariance eventof approximately 60–220 million years before present(Yamazaki 1998), which led to the geographical subdivision

of

Bruguiera gymnorrhiza

in Asia. According to palae-oceanographic evidence, due to latitude and temperaturedifferences, southern and northern banks of the SouthChina Sea, where

K. candel

is distributed, went throughdifferent geological histories (Wang

et al

. 1995) over pastglacial cycles. Furthermore, populations of the Ryukyuislands and northern Taiwan (Taipei) along coasts ofthe East China Sea shared a unique geological history fromthose 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 regionswould be expected.

As generally known, in estimating population structureand gene flow, some level of variance of loci is required.Molecular markers with low resolution usually are incap-able of providing information (Bossart & Prowell 1998) indistinguishing coancestry from migration (Schaal

et al

.1998). For surveying population structure within a smallgeographical scale, e.g.

K. candel

in Hong Kong (Sun

et al

.1998) and Taiwan (Huang 1994), allozymes due to theirconserved nature (cf. Bossart & Prowell 1998) might not beable to adequately estimate gene flow among local popula-tions. In addition, the biparental inheritance of allozymesmakes the estimations of seedling dispersal difficult, as theeffects of pollen dispersal between neighbouring popula-tions cannot be ruled out. Recently, many noncodingspacers of organelle DNAs have been widely applied tophylogeographical studies (Schaal

et al

. 1998). With meritsof maternal inheritance in most angiosperms (Harris &Ingram 1991), including

Kandelia

(Chen 2000), and nearlyneutral and fast evolution, these markers are likely to beable to provide information in estimating the extent oflong-range seedling dispersal and reconstructing phylo-geographical patterns.

In this study, we sequenced two noncoding spacers, i.e.

atp

B-

rbc

L and

trn

L-

trn

F of chloroplast DNA (cpDNA), andthe internal transcribed spacer (ITS) of mitochondrial DNA(mtDNA) and used them as markers to estimate the phylo-geographical pattern as well as population and geograph-ical structure of

K. candel

. As physically linked loci in thechloroplast genome,

atp

B-

rbc

L and

trn

L-

trn

F should revealcomparable phylogenies. Likewise, as being maternallyinherited, chloroplasts and mitochondria were thought toremain associated and behave as if they are completelylinked (Schnabel & Asmussen 1989). Consistent phylo-genetic 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 muta-tions (Desplanque

et al

. 2000), can result in systematicinconsistency and thus lead to wide variance of

F

ST

valuesamong loci and a weak correlation between

F

ST

andnumber of migrants per generation (

Nm

) as well (cf. Bossart& Prowell 1998). Under such circumstances, difficulties in


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interpreting phylogeography and population structure willbe inevitably encountered. In other words, when discrep-ant estimates are obtained from two or more loci, these esti-mates are not necessarily indicative of gene flow. Explicitanalysis 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 berequired to clarify historical and recurrent events.

K. candel

, as widespread in East Asia, is a biologicalmodel that is suited for testing the association betweenvicariance and geographical structure; effects of oceancurrents in long-distance seedling dispersal; and the use-fulness of cpDNA and mtDNA as population geneticmarkers, as well as allele associations. Several aims arepursued in this study: (i) to test the possibility and level oflong-distance seedling dispersal by estimating populationstructure and gene flow; (ii) reconstruct the phylogeo-graphical pattern and examine the association betweengeological/geographical events and the extent of geneticdifferentiation among three geographical regions; (iii) to

examine the phylogenetic consistency between

atp

B-

rbc

Land

trn

L-

trn

F noncoding spacers of cpDNA as well asbetween chloroplast and mitochondrial genomes; and(iv) to investigate associations between alleles of cp- andmtDNAs and to deduce the relative age of their allelesbased on spanning networks.

Materials and methods

Sample collection

Kandelia candel

is a mangrove species that is widespreadin eastern coasts of mainland Asia and continental islands,including Taiwan, and the Ryukyu (Fig. 1). One hundredand eighty-seven samples were collected from 13 majorpopulations 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 approximately70–100 m apart, were sampled from each population. Inaddition, 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 eachpopulation is indicated in pie diagrams. Abbreviations of populations are given in Table 1.


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(Thailand) of the western coast of Peninsula Malaysia wereincluded in the analysis as outgroups. In total, this studyencompasses 14 populations (202 individuals). No materialswere collected from the Philippines, since no naturalpopulations are distributed in this area (Hou 1958). Inaddition, during the last decade, most populations ofVietnam have been removed for the use of inshorefisheries (Huang & Chen 2000). Only one population atQuang Ninh was available. Besides, samples of sixpopulations (from Chinchou to Xiamen) of China, onepopulation from Sarawak, two populations of Taiwan,and three populations from the Ryukyu Islands wereincluded in this investigation. Based on the orientation ofocean currents (Huang

et al

. 1997), three geographicalregions are recognized: southern South-China-Sea (Sregion, including a single population of Sarawak),northern South-China-Sea (N region, including eightpopulations of Vietnam, China, and Tainan), and theEast-China-Sea (E region, including three populations ofRyukyu and Taipei) regions. Three populations along theTonkin Bay, i.e. Chinchou, Shankou, and Quang Ninh, arefurther grouped as the Ns region, while others of the Nregion as the Nn region. Young and healthy shoots werecollected in the field, rinsed with tap water and dried insilica gel. All samples were stored at –70

°

C until theywere processed.

DNA extraction and polymerase chain reaction

Leaf tissue or embryo of the above materials was ground topowder in liquid nitrogen and stored in a –70

°

C freezer.Genomic DNA was extracted from the powdered tissuefollowing the CTAB procedure (Murray & Thompson1980). Noncoding spacers of

atp

B-

rbc

L and

trn

L-

trn

F of thecpDNA and the ribosomal DNA (rDNA) ITS of mtDNAwere amplified and sequenced. Universal primers foramplifying

atp

B-

rbc

L spacer (Chiang

et al

. 1998),

trn

L-

trn

Fspacer (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

µ

Lreaction using 10 ng of template DNA, 10

µ

L of 10

×

reactionbuffer, 10

µ

L MgCl

2

(25 m

m

), 10

µ

L dNTP mix (8 m

m

), 10pmole of each primer, 10

µ

L of 10% NP-40, and 2 U of

Taq

polymerase (Promega, Madison, USA). The reaction wasprogrammed on a MJ Thermal Cycler (PTC 100) as onecycle of denaturation at 95

°

C for 4 min, 30 cycles of 45 sdenaturation at 92

°

C, 1 min 15 s annealing at 52

°

C, and1 min 30 s extension at 72

°

C, followed by 10 min extensionat 72

°

C. Template DNA was denatured with reactionbuffer, MgCl

2

, NP-40 and ddH2O for 4 mins (first cycle),and cooled on ice immediately. A pair of primers, dNTPand

Taq

polymerase were added to the above ice-cold mix.Reaction was restarted at the first annealing at 52

°

C.

Table 1 Materials of Kandelia candel collected from different populations used for organelle DNA sequencing. Location, locality, samplesize, and cytotypes (trnL-trnF spacer chlorotype and mtITS mitotype associations) of each population are indicated

Localities Area Symbol CoordinateSampling size (n) Cytotypes

Northern RegionNorthern South-China-Sea Region:

Chinchou Guangxi, China CC 21°34′ N, 108°37′ E 15 IB (12), IC (1), IIB (1), IIIB (1)Haikou Hainan, China HA 19°54′ N, 110°20′ E 15 IB (6), IIB (3), IIIB (3), VB (3)Hong Kong China HK 22°32′ N, 114°05′ E 13 IB (9), IVB (2), VB (2)Quang Ninh Vietnam QN 20°53′ N, 106°46′ E 15 IIB (2), IIE (1), IVB (2), IVE (1), VB (3),

VIB (4), VIC (1), VIE (1)Shankou Guangxi, China SK 21°28′ N, 109°43′ E 10 IB (1), IIB (1), IID (1), IIIB (6), IIID (1)Tainan Taiwan TN 22°59′ N, 120°12′ E 15 IB (13), IC (1), IIIC (1)Xiamen Fukien, China XM 24°26′ N, 118°04′ E 15 IB (2), IIB (9), IIC (1), IVB (3)Zhangjiang Guangxi, China ZJ 21°04′ N, 110°09′ E 15 IIIB (7), IVB (1), IVC (1), VIB (6)

East-China-Sea Region:Amami-O-Shima Islet Ryukyu, Japan AM 28°15′ N, 130°40′ E 15 IB (6), IIB (3), IIIB (2), IIIC (1), IVB (2),

IVC (1)Irimote Islet Ryukyu, Japan IR 24°19′ N, 123°54′ E 15 IA (2), IB (4), IIB (3), IVB (6)Taipei Taiwan TP 25°09′ N, 120°16′ E 14 IB (9), IIB (3), IIIB (2)Yakushima Islet Ryukyu, Japan YK 30°20′ N, 130°30′ E 15 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)


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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 withethidium bromide and the desired DNA band was cut andeluted using agarose gel purification (QIAGEN). PurifiedDNA was ligated to a pGEM-T easy vector (Promega).Plasmid DNA was selected randomly with five clonesand purified using plasmid mini kits (QIAGEN). Purifiedplasmid DNA was sequenced in both directions bystandard methods of the Taq dyedeoxyterminator cyclesequencing kit (Perkin Elmer) on an Applied BiosystemsModel 377A automated sequencer. Primers for sequencedetermination were T7-promoter and SP6-promoterlocated on p-GEM-T easy vector termination site.

Sequence alignments and phylogenetic analyses

Nucleotide sequences were aligned with the programGenetics 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 inMolecular Biology and Evolution (dambe, version 3.5.19,Xia 1999). Indels were treated as the fifth character.Confidence of the clades reconstructed was tested bybootstrapping (Felsenstein 1985) with 1000 replicates usingunweighted characters. The nodes with bootstrap valuesgreater than 0.70, as a rule of thumb, are significantlysupported with ≥ 95% probability (Hillis & Bull 1993). Thenumber of mutations between DNA genotypes in pairwisecomparisons, which were calculated using mega (Kumaret al. 1993), was used to construct a minimum spanningnetwork with the aid of minspnet (Excoffier & Smouse1994) in an hierarchical manner (cf. Chiang & Schaal 1999).After linking the related haplotypes into a clade, closelyrelated clades were linked further to form higher levelgroups and thereby a network.

Population genetic analysis of the cpDNA and mtDNA sequence variation

Levels of genetic diversity within populations werequantified by estimates of nucleotide divergence (θ)(Watterson 1975) using dnasp (Version 3.14, Rozas &Rozas 1999). Patterns of geographical subdivision andgene flow were also estimated hierarchically with the aidof dnasp. Gene flow within and among regions (popu-lations) was approximated as Nm, the number of femalemigrants per generation between populations, and wasestimated using the expression FST = 1/(1 + 2 Nm) where Nis the female elective population size and m is the femalemigration rate (Slatkin 1993). We also used amova version1.55 (Excoffier et al. 1992; Excoffier 1993) to deduce the

significance of geographical divisions both among regionsand 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 ofcpDNA and the rITS of mtDNA in Kandelia candel werePCR amplified and sequenced. All sequences were regis-tered with EMBL accession numbers AJ305472–AJ305673(for mtDNA ITS), AJ305674–AJ305875 (for trnL-trnFspacer), and AJ305876–AJ306077 (for atpB-rbcL spacer).At all three loci, no within-individual variation wasdetected.

Differences between mitochondrial sequences of aconsensus length of 725 bp were mainly ascribed to pointmutations (18 sites, 2.4%). Four indel events also occurredat sites (353–357) (592–599) (632–635) and 640. For theatpB-rbcL spacer of the chloroplast genome, only two sitesof 781 bp (0.3%) were polymorphic. Populations of BK andRN shared a C at site 160, while others have a T. A 1-bpdeletion at site 577 occurred in the RN population. At thetrnL-trnF spacer of cpDNA, high levels of length polymor-phism, ranging from 375 bp to 415 bp, were detected. Twodeletions, at sites (17–28) and (242–268) made the spacershorter 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 contentswere high, with 53.7%, 72.5%, and 76.9% at mtITS, atpB-rbcL 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. Incontrast to other studies (Small et al. 1998; Fujii et al. 1999),the atpB-rbcL noncoding spacer possessed a much lowerlevel of genetic variation than the trnL-trnF spacer in K.candel (Table 2). In total, 28 haplotypes of the trnL-trnFspacer of cpDNA and seven haplotypes of the mtDNA ITSwere determined. Apparently, the molecular evolution ofthe mtDNA ITS was much more constrained compared tothat of the trnL-trnF spacer of cpDNA (Table 2).

At the population level, except for the lack of genetic vari-ation at the cpDNA atpB-rbcL spacer, the level of geneticvariation varied among populations at two other organelleloci (Table 2). High levels of nucleotide variation at bothloci, with θ-values ranging from 0.0018 to 0.0062 at trnL-trnF spacer and from 0.0020 to 0.0029 at mtITS, occurred inpopulations of IR, QN, and YK, while low cpDNA vari-ation, ranging from 0.0003 to 0.0013, was detected in popu-lations of TN, CC, and HK. Stark contrast of the level ofgenetic variation between the two loci occurred in popula-tions of BK, HA, TP, and XM (Table 2).


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


Gene genealogies and associations between cpDNA and mtDNA lineages

In this study we reconstructed the phylogeographicalpattern of K. candel based on gene genealogies of organelleloci. A minimum spanning network of the cpDNA atpB-rbcL spacer was reconstructed based on mutationalchanges between haplotypes (Fig. 2). The BK population isclosely related to the RN population, while no variationwas detected among populations of N and E regions. AnNJ tree was recovered based on the nucleotide sequencevariation of the trnL-trnF noncoding spacer of cpDNA.Eight clades (chlorotypes) of 28 haplotypes were identifiedin this cpDNA gene tree (Fig. 3). Two major lineages ofS + RN and N + E were recognized and significantlysupported, with a bootstrap of 0.98 (P < 0.01). Four com-mon chlorotypes I–IV of 152 sequences in total (75.2%)were widespread in populations of N and E regions, whiletypes of VIIa and VIIb of 30 sequences were restricted to theS 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 noncod-ing spacer was constructed (Fig. 4). Eight clades of the

network, corresponding to those of the NJ tree, were dividedinto two geographical groups (i.e. S + RN and N + E) 34mutations apart. Within the network, closely related chloro-types were mostly linked by single mutations. ChlorotypesI, II and III were nested in the network as interior nodes,while types IV and V connecting to type I, and type VIconnecting to III or IV were exterior.

In the NJ tree of mtDNA ITS sequences, two majorclades (A–E) and (F, G), of seven variants (mitotypes)were identified (Fig. 5A). Two common mitotypes B andC of 164 sequences (81.2%) were widespread in popula-tions of N and E regions (Table 3), as mitotypes of F andG 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 themtDNA ITS was constructed (Fig. 5B). Within the clade ofN + E regions, mitotype B was nested in the network asthe interior node, connecting to other types independ-ently with 1–9 mutations. Within the clade of S + RNregions, mitotype G was linked to the interior node oftype F with two mutations, which was linked to type Bwith four mutations.

Phylogenies of cpDNA and mtDNA are completelyconsistent at the level of geographical regions (i.e. S + RNvs. N + E). The atpB-rbcL noncoding spacer provided noinformation in resolving the phylogeny within N + Eregions. Apparently, the gene tree of the trnL-trnF spacer ofcpDNA largely contradicted the mtDNA ITS tree. No cladecorrespondence was found between the two trees. Forexample, although most chlorotype III sequences (87.5%)corresponded to the mitotype B, sequences of am4 andtn12 were associated with mitotype C and sequence sk2was associated with the mitotype D.

cpDNA mtDNA

Populations h θ h θ

Total 0.908 0.02652 ± 0.00526 0.406 0.00205 ± 0.00026Amami-O-Shima 0.857 0.00623 ± 0.00044 0.248 0.00034 ± 0.00018Bako 0.857 0.00615 ± 0.00092 0.000 0.00000Chinchou 0.257 0.00125 ± 0.00005 0.133 0.00018 ± 0.00015Haikou 0.771 0.00329 ± 0.00003 0.000 0.00000Hong Kong 0.513 0.00135 ± 0.00044 0.000 0.00000Irimote 0.686 0.00310 ± 0.00164 0.248 0.00292 ± 0.00060Quang Ninh 0.771 0.00492 ± 0.00071 0.448 0.00255 ± 0.00034Ranong 0.600 0.00246 ± 0.00004 0.248 0.00034 ± 0.00018Shankou 0.778 0.00248 ± 0.00001 0.467 0.00064 ± 0.00018Tainan 0.133 0.00034 ± 0.00018 0.248 0.00034 ± 0.00018Taipei 0.560 0.00304 ± 0.00082 0.000 0.00000Xiamen 0.733 0.00278 ± 0.00049 0.133 0.00018 ± 0.00015Yakushima 0.457 0.00178 ± 0.00066 0.362 0.00200 ± 0.00139Zhangjiang 0.848 0.00457 ± 0.00040 0.133 0.00018 ± 0.00015

Table 2 Haplotype diversity (h) and nucleo-tide diversity (θ) of the cpDNA trnL-trnF spacer and the mtDNA ITS withinpopulations of Kandelia candel

rn bk N + E0

atpB-rbcL networkFig. 2 Minimum spanning network generated using method ofExcoffier & Smouse (1994) for haplotypes of atpB-rbcL spacer ofcpDNA of populations of Kandelia candel. Each arrow indicates onemutational change. ‘0’ indicates hypothetical ancestor.




Nevertheless, associations between cpDNA and mtDNAhaplotypes 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

cytotypes were observed in N + E and S + RN regions of K.candel, respectively (Table 4). All sequences of the mitotypeA were exclusively associated with the chlorotype I;and sequences of the mitotype D were associated with the

Fig. 3 Neighbour-joining tree of representative sequences (haplotypes) of trnL-trnF of cpDNA in Kandelia candel. Numbers at nodes indicatebootstrap 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: IndianOcean Rim (I), southern South-China-Sea region (S), northern South-China-Sea region (N), and East-China-Sea region (E)

Regions: I S N E

rn bk qn cc sk zj xm ha hk tn tp ir am yk Total

I 13 1 2 6 9 14 9 6 6 11 77 (38.2%)II 3 1 2 10 3 3 3 3 2 30 (14.9%)III 1 7 7 3 1 3 24 (11.9%)IV 3 2 3 2 2 6 3 2 21 (10.3%)V 3 3 2 8 (4.0%)VI 6 6 12 (5.9%)VIIa 15 15 (7.4%)VIIb 15 15 (7.4%)

A 2 1 3 (1.5%)B 11 14 8 14 14 15 13 13 14 13 13 12 154 (76.2%)C 1 1 1 1 2 2 2 10 (5.0%)D 2 2 (1.0%)E 3 3 (1.5%)F 13 15 28 (13.9%)G 2 2 (1.0%)


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 ofpopulations of Kandelia candel. Each arrow indicates one mutational change. Number of mutational change is indicated when more thanone step. ‘0’ indicates hypothetical ancestor. The replicate number of haplotypes is also indicated when more than one.

Fig. 5 (A) Neighbour-joining tree of haplo-types (A–G) of rITS of mtDNA in Kandeliacandel. Numbers at nodes indicate bootstrapvalues. (B) Minimum spanning networkgenerated using method of Excoffier &Smouse (1994) for haplotypes of mtDNAITS of populations of K. candel. Mutationalchanges are indicated at nodes.




chlorotypes II and III. Likewise, the chlorotype V was exclus-ively associated with the most dominant mitotype B, andmost sequences of chlorotype I were associated with themitotype B, while some other sequences were mitotypes Aor 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 EVIwere relatively rare (5.8% in total). Likewise, within theS + RN region, FVIIa and FVIIb were dominant (93.3%),while the cytotype GVIIa was rare (6.7%).

The cytotype composition varied among populations. InFig. 1 the genetic composition in each population was indic-ated. Higher number of cytotypes occurred in populationsAM (six types), QN (eight types), and YK (five types),while a single type was detected in the BK population andtwo types were detected in the RN population (Table 1).Within the N + E regions, populations possessing a higher

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

Population differentiation and geographical divisions

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

Table 4 Associations between chlorotypes and mitotypes of Kandelia candel. Distribution range of each chlorotype and mitotype is indicatedin square brackets. Percentage of each cytotype is indicated in parentheses. W: widespread. Other symbols see Table 1

chlorotype: mitotype:A [ir + yk]

B [W]

C [W]

D [sk]

E [qn]

F [rn + bk]

G [bk] Total

I [W] 3 70 4 77(1.5%) (34.6%) (2.0%)

II [W] 27 1 1 1 30 (13.4%) (0.5%) (0.5%) (0.5%)

III [W] 21 2 1 24(10.4%) (1.0%) (0.5%)

IV [W] 18 2 1 21 (8.9%) (1.0%) (0.5%)

V [qn, hk, ha] 8 8 (4.0%)

VI [qn, zj] 10 1 1 12 (5.0%) (0.5%) (0.5%)

VIIa [rn] 13 2 15 (6.4%) (1.0%)

VIIb [bk] 15 15 (7.4%)

Total 3 154 10 2 3 28 2 202

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

cpDNA 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)

E (θ = 0.00503 ± 0.00054)

— 0.020/23.95 0.023/21.50 0.021/23.14

N (θ = 0.00445 ± 0.00033)

0.026/18.41 — — —

Ns (θ = 0.00312 ± 0.00037)

0.023/21.09 — — 0.011/45.67

Nn (θ = 0.00492 ± 0.00041)

0.031/15.41 — 0.013/39.00 —


2706 T . Y . C H I A N G E T A L .


indicated that the proportion of molecular variance wasattributed to difference between geographical regions(ΦCT = 0.860, P < 0.001). The relative contribution of dif-ference among populations to molecular variance wassmall (ΦST = 0.087). In contrast, no geographical sub-division (FST = 0.020–0.026 and Nm = 18.41–23.95) betweenN and E was detected (Table 5).

An analysis of molecular variance (amova) based on thetrnL-trnF spacer of cpDNA also suggested low levels ofgenetic differentiation between populations of mainlandand continental islands (ΦCT = 0.015) as well as amongpopulations within each region (ΦSC = 0.037). Geneticvariation of the mtDNA ITS yielded a similar pattern ofthe genetic apportionment among geographical regionsand populations. The deduced Nm of 39.00–45.67 indicatedfrequent gene flow between Ns and Nn regions (Table 5).

In contrast to the invariable atpB-rbcL spacer, pairwisecomparisons showed high variances in genetic estimates ofstructure and genetic differentiation between cpDNA andmtDNA loci, except for those between BK + RN and otherpopulations. Low level of genetic differentiation was usu-ally detected among populations within the E + N regions.Nearly all Nm values, ranging from 1.75 (between SK andHK) to 622.55 (between CC and YK), deduced from themtDNA ITS were greater than those from the cpDNA trnL-trnF spacer. Lower Nm values, less than one, were mostlyrestricted to comparisons with ZJ as well as SK based oncpDNA variation. Some extremely high Nm values wereobtained, such as 150 between AM and CC. High variancein deducing FST and Nm was also encountered in compar-isons between BK and RN. Based on the trnL-trnF spacer,an FST value of 0.42 and an Nm of 0.68 were deduced, whilea 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 estimatesof population structure and gene flow depends on the levelof resolution, and locus-to-locus consistency (Bossart &Prowell 1998), and is also affected by the level of recurrentmutations (cf. Desplanque et al. 2000). Recurrent mutations(identity by state), which are frequently encountered in themitochondrial genome due to limited conformations inmolecular structure (Fauron et al. 1995), will inevitably blurthe level of migration between populations. Technically,nucleotide sequencing can simply rule out the length homo-plasies, which occur usually in restriction fragment lengthpolymorphism (RFLP) and PCR-based fingerprints (cf. Parkeret 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 withinN + E and S + RN regions (Table 4), a high rate of recurrentmutations can be excluded for organelle genomes ofK. candel (cf. Desplanque et al. 2000).

In this study, genetic variation of mtDNA ITS andcpDNA trnL-trnF spacer existed both within and betweenpopulations. Nevertheless, the haplotype diversity of themitochondrial DNA, with seven haplotypes out of 202plants screened, was lower compared to other floweringplants, such as Beta vulgaris ssp. maritima (20 mitotypesfrom 414 individuals; Desplanque et al. 2000), Daucus carotassp. carota (25 variants based on mtDNA RFLP from 80plants; Ronfort et al. 1995), Thymus vulgaris (50 mitotypesfrom about 400 plants; Manicacci et al. 1996), and Heveabrasiliensis (212 mtDNA RFLP variants in 395 accessionsscreened; Luo et al. 1995).

For the chloroplast genome, K. candel possessed a higherlevel of haplotype diversity (28 haplotypes) at the trnL-trnFspacer, which is close to 13 cpDNA haplotypes in Beta vul-garis ssp. maritima (Desplanque et al. 2000), 11 haplotypesin Argania (El Mousadik & Petit 1996), 23 haplotypes inwhite oaks (Dumolin-Lapègue et al. 1997), and 13 haplo-types in Alnus (King & Ferris 1998). Although comparisonsof haplotype diversity among taxa, which were examinedusing various molecular methods at different loci, may besomewhat misleading, nucleotide diversity of the trnL-trnF spacer in K. candel (θ = 0.02710) appeared to be higherthan that of other plants as well, such as Cunninghamia(θ = 0.01018, Lu et al. 2001) and Begonia (θ = 0.003, Liu1999). Apparently, both loci in this study provided suffi-cient resolution at the geographical region level andyielded consistent estimates of gene flow (Nm = 0.03–0.04)and population structure (FST = 0.93–0.95) between S + RNand N + E populations of K. candel. To the contrary, lack ofvariability, due to its conserved nature (cf. Chiang & Schaal2000a,b), has made the atpB-rbcL noncoding spacer locuspowerless in estimating the interpopulation migrationwithin the N + E regions. At interregions level, as a resultof having difficulties in distinguishing coancestry frommigration, higher Nm value (= 0.10) between S + RN andN + E regions was thereby deduced based on this spacer.Likewise, at the population level, the more conservedmtDNA ITS always yielded higher values of Nm and lowerlevels of FST, than the cpDNA trnL-trnF spacer. In thisinvestigation, due to its higher resolution, the trnL-trnFmight have higher probabilities of yielding estimates thatapproximate the current population structure of K. candel.

Phylogeographical patterns of K. candel in East Asia

In this study, we investigated the phylogeography of theviviparous species, K. candel. Both the mtDNA ITS andthe cpDNA trnL-trnF spacer suggested noticeable long-distance seedling dispersal. However, as extensive gene




flow across oceans via seedlings was detected amongpopulations along a 2700 km transect (between QN andYK) in the regions of northern South-China-Sea and East-China-Sea, gene genealogies of three organelle locirevealed consistent geographical structure. Accordingly,the population of Sarawak along the bank of the SouthChina Sea in East Asia, which is about 2000 km fromthe QN (Fig. 1), was phylogenetically grouped with theRanong population of the Peninsula Malaysia at thenortheastern rim of the Indian Ocean.

This unique geographical structure, consistently sug-gested by organelle loci (Figs 2, 4 and 5) as well as allozymedata (Huang & Chen 2000), not only indicated the originof the Sarawak population derived from the IndianOcean Rim, but also reflected a phylogeographical patternassociated with the vicariance history. According topalaeoceanographic evidence, during the last glacial max-imum of the Pleistocene, the global sea level dropped bysome 100–120 m. Meanwhile, with the closure of all thesouthern connections to the ocean, the South China Seanearly became an enclosed basin, with the Bashi Strait as itsonly 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 coastof the modern South China Sea, has long been the northernedge of the Sunda shelf since the late Quaternary. Accord-ing to another estimation of Yamazaki (1998), populationsof the South China Sea and the East China Sea may havebeen isolated from those of the Bay of Bengal of the IndianOcean for at least 0.6–2.2 million years, a duration suffi-cient for coalescence at most loci. Apparently, monophylyof F + G alleles of the mtDNA and VIIa + VIIb alleles of thecpDNA supported the long isolation hypothesis. In addi-tion, based on the paucity of genetic variation at twoorganelle loci and the phylogenetic affinity to the RNpopulation, a small number of founders through long-distancedispersal from populations of the Indian Ocean may havebeen involved in the colonization of the BK population. Never-theless, the level of ongoing gene flow between BK and RNappeared to be low, according to the deduced Nm of 0.00–0.68 from the atpB-rbcL and trnL-trnF, respectively, a resultagreeing with the orientation of summer ocean currents.

According to the association between unique geneticstructure and vicariance events, the Sarawak populationmust also have been shaped by limited recurrent gene flowto other populations of the South China Sea, which in turnis constrained by seasonal orientations of current flows.Sea surface circulation in the modern South China Sea isbasically driven by the monsoon in East Asia. In summer,the season during which most seedlings are detached frommaternal plants of K. candel and disperse (Tomlinson 1986;Huang & Chen 2000), surface water of the tropical IndianOcean flows northward into the South China Sea and thenthrough the Bashi Strait (the strait between Taiwan and

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 forK. candel, most seedlings of the Sarawak are likely to bedischarged into oceans, having limited probability ofcolonizing territory of the northern South China Sea. Withlimited gene flow with other populations, the Sarawakpopulation has long maintained its own identity.

Despite the isolation between South-China-Sea and East-China-Sea regions during the glacial maximum, subsequentongoing gene flow via ocean currents has homogenizedbetween-region and between-population variation in K.candel. In agreement with previous allozyme investigations(Huang 1994; Sun et al. 1998; Huang & Chen 2000), bothchloroplast (trnL-trnF spacer) and mtDNAs indicated nohindrance to the long-distance seedling dispersal betweenmainland and continental islands via ocean currents(Table 5). Although the isolation-by-distance model wasnot met at the population level, gene flow within region(i.e. between Ns and Nn, Table 5) was apparently morefrequent than between E and N regions. In addition,according to the higher nucleotide diversity in the Nnpopulations at both organelle loci (Table 5), a preferentialnorthward migration (from Ns to Nn) seemed to exist dueto the orientation of ocean currents in summer. Nevertheless,some local topographical barriers may have blocked theinterpopulation gene flow. According to the estimatesbased on the trnL-trnF spacer, limited gene flow occurredin populations ZJ and SK, both located on opposite coastsof the Leizhou Peninsula (Fig. 1), to other populations. Inaddition, although no general rule can be generated, HongKong seemed to have more frequent gene flow with neigh-bouring populations, including TN, TP, and IR, than withdistant populations.

Relative ages of alleles of cpDNAs and mtDNAs

In resolving phylogeographical pattern and phylogeneticambiguities, the nested cladogram provides sufficientinformation, complementary to conventional cladograms(cf. Crandall & Templeton 1993). Based on their interiorpositions in the minimum networks, the ancestry of eightdominant and widespread cytotypes was suggested: BI,BII, BIII, BIV, CI, CII, CIII, and CIV (Table 4), which wouldhave a greater probability of producing mutationalderivatives (cf. Donnelly & Tavaré 1986; Golding 1987;Crandall & Templeton 1993). In contrast to the stronglinkage disequilibrium among chlorotypes of I–VI andmitotypes of A–E, nearly random association (χ2 = 0.822,P = 0.189) was detected in these common cytotypes,indicating genetic equilibrium and panmixis within theN + E region as a whole.

The strong linkage disequilibrium may simply stemfrom effects of lineage sorting, due to relative ages of alleles


2708 T . Y . C H I A N G E T A L .


of each locus (Chiang 2000). According to the exterior posi-tions in the networks and their restricted spatial distribu-tion, alleles V and VI of the cpDNA trnL-trnF spacer, andalleles of A, D, and E of the mtDNA may have evolvedrecently. As generally known, newly evolved cpDNA alleles,since having a low frequency in the gene pool, werelikely to be ‘attracted’ to dominant mitotypes (B and C, inthis study), and vice versa. Probabilities of associationsbetween two rare alleles would be extremely low, there-after leading to strong linkage disequilibrium.

Interestingly, almost all rare alleles occurred at marginalpopulations of the E + N regions, such as allele A restrictedin IR and YK (Ryukyu), D exclusively in SK, and E in QNonly. Technically, the absence of these rare alleles in‘central’ populations may be simply because of failure ofdetection due to their low frequency. On the other hand,some preferential directional migration due to the micro-geographical hindrance might also account for the appor-tionment of rare alleles. Nonetheless, with ceaseless and fre-quent gene flow, central populations, exposed to migrantsfrom all neighbouring populations, would have a largeprobability of maintaining genetic variation. Fluctuationof genetic structure resulting from a limited number offounders (cf. Sun et al. 1998) would seldom occur. The lowlevel of genetic variation detected in Taiwan and HongKong (Huang 1994; Sun et al. 1998) may simply come from theconserved nature of the markers themselves. For example,in this study, high cpDNA variation (θ = 0.00304) vs. nomtDNA variation was detected in Taipei (Table 2). On theother hand, the organelle DNA diversity of Taiwan (with θof 0.00173 at cpDNA trnL-trnF locus, and of 0.00018 at themtITS locus) and Hong Kong (θ of 0.00135 at cpDNA locusvs. no mtDNA variation) was lower than most other popu-lations (Table 2). Habitat destruction in recent decades dueto human activities may have largely contributed to the lossof 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 andextent of long-range seedling dispersal. In agreement withprevious allozyme investigations, the geographical andpopulation structure of the species, which adapts to theintertidal habitats with precocious growth of seeds, wasdetermined both by vicariance and ongoing gene flow.Significant genetic differentiation between populations ofnorthern and southern banks of the South China Sea plusthe attainment of monophyly of alleles of both cpDNAsand mtDNAs at geographical region level indicated a longisolation. In contrast, recurrent gene flow via long-distancedispersal, indicated by high deduced Nm values, havecontributed to the genetic homogeneity among popu-lations of the region of northern South China Sea and East

China Sea. Gene genealogies, which trace phylogeneticrelationships among alleles in a geographical context, ofdifferent 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, andHQ Fan for the assistance with sampling.

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The results reported here are from a collaboration betweenS. Huang and T. Y. Chiang’s laboratories. Work in T. Y. Chiang’sgroup is on data analysis. Work in S. Huang’s laboratory focuseson field collection, ecological observations, and nucleotidesequencing of mangrove trees.


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