Botanical Studies (2009) 50: 193-204.

3

All authors contributed equally.

*

Corresponding author: E-mail: yuchung@mail.npust.edu.

tw; Tel: +886-8-7703202 ext. 6364 (Yu-Chung Chiang);

E-mail: biofv017@ntnu.edu.tw; Tel: +886-2-29326234 ext.

229; Fax: +886-2-29312904 (Jenn-Che Wang).

INTRODUCTION

Geographic isolation is considered to be a major

cause of population differentiation (Braillet et al., 2002;

Roy et al., 2006; Liao et al., 2007) and speciation (Near

and Benard, 2004; Stevens and Hogg, 2004; Hoskin

et al., 2005; Hayashi and Kawata, 2006; Starrett and

Hedin, 2007; Sheue et al., 2009). The best-known cases

are marine organisms that are isolated by isthmuses.

For example, the Isthmus of Panama, which emerged

approximately 3.5 Mya (Coates et al., 1992), caused

substantial differentiation of populations and divergence

of species in the Atlantic and Pacific Oceans (Steeves

et al., 2005; Smith et al., 2006). Similar barriers have

Gene flow of Ceriops tagal (Rhizophoraceae) across the

Kra Isthmus in the Thai Malay Peninsula

Pei-Chun LIAO

1

, Yu-Chung CHIANG

2,

*, Shong HUANG

1

, and Jenn-Che WANG

1,

*

1

Department of Life Science, National Taiwan Normal University, No. 88, Sec. 4, Tingzhou Rd., Wenshan District, Taipei

11677, Taiwan, Republic of China

2

Department of Life Science, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, R.O.C.

(Received April 21, 2008; Accepted November 24, 2008)

ABSTRACT.

The Malay Peninsula (formerly ancient Sundaland) is regarded as a barrier that isolates

organisms of the South China Sea from those of the Bay of Bengal. During the interglacial period,

approximately 5 Mya, sea levels rose and organisms migrated across the narrowest part of this peninsula, the

Kra Isthmus. In the present study, we examine the chloroplast genomes of Ceriops tagal along the coasts of

both sides of the Kra Isthmus to retrace divergence events and to evaluate the probability of previous long

distance dispersal. The haplotype distributions support the hypothesis that the Kra Isthmus was an effective

geographic barrier that caused genetically differentiated populations. Based on comparison of the chloroplast

genomes, the estimated time of divergence between the two populations is consistent with the emergence time

of the Kra Isthmus. However, ancient and recent gene flow obscures the phylogenetic relationships between

eastern and western haplotypes. We used nested clade analysis (based on user-defined-distances corresponding

to the distances across the peninsula and the sea route around it) and provide evidence of pre-isthmus range

expansion and restriction of gene flow that resulted from geographic isolation. Trans-isthmus long distance

dispersal probably occurred at the pre-isthmus region ~5 Mya via the southern Malay Peninsula. Our results

indicate that the Malay Peninsula has had separate populations on opposite sides of the Kra Isthmus since

its formation, but that interglacial migration at the Strait of Malacca may have provided a corridor for gene

flow. This is an instance of arrested allopatric speciation due to genetic homogenization via rare long distance

dispersal.

Keywords: Ceriops tagal; Dispersal; Divergence time; Kra Isthmus; Land barrier; Vicariance.

been documented in the Malay Peninsula (Karns et al.,

2000; Lessios et al., 2001; Heatwole et al., 2005) and the

Baja California Peninsula (Muniz-Salazar et al., 2005).

During short periods of geological history, organisms that

are isolated by such isthmuses can rapidly accumulate

genetic differences and drive population differentiation.

However, population differentiation will not occur if there

is insufficient time for accumulation of genetic differences

(i.e. genetic drift has not completed to fix to one allele)

or if occasional dispersal and migration maintains

connections between the populations (Trakhtenbrot et al.,

2005; Berthier et al., 2006; Wiens et al., 2006).

Most plant seeds cannot disperse over long distances,

but long distance dispersal (LDD) and colonization can

prevent speciation of geographically isolated populations

(Cain et al., 2000). Thus, population divergence generally

proceeds slowly, often via short incremental steps over

long periods of time. For instance, the average seed

dispersal rates of forest understory species generally

range from 0.0 to 2.5 m/yr (Cain et al., 2000). However,

POPUlATION GeNeTICS

194

Botanical Studies, Vol. 50, 2009

for plants near oceans, a small number of seeds can be

dispersed over very long distances (Portnoy and Willson,

1993), especially by sea currents.

Salinity can harm seeds or fruits that have prolonged

contact with the sea, as originally discussed by Darwin

(1859). Even mangrove seeds, commonly considered to

be salt-tolerant, may be harmed by prolonged soaking in

saline solutions, e.g. only 8.3¡Ó3.3% of the propagules of

Ceriops tagal developed roots after 23 days of soaking

(Clarke et al., 2001). In addition, establishment success

and niche availability are important determinants of long-

distance colonization (Moore and Elmendorf, 2006; Viard

et al., 2006).

The globally discontinuous distribution of mangrove

species (Duke et al., 2002) indicates that they can

overcome problems associated with LDD and can

effectively colonize geographically distant areas (Cain et

al., 2000). However, mangrove LDD and colonization is

likely to be stochastic and unpredictable (Duke et al., 2002;

Minchinton, 2006). Many researchers have examined the

disjunct geographic distribution of mangroves (Duke,

1995; Duke et al., 2002) and discontinuities in their of

genetic composition (Duke et al., 1998; Chiang et al.,

2001; Tan et al., 2005; Su et al., 2006, 2007; Liao et al.,

2007). However, little research has been devoted to the

underlying processes (e.g. Chiang et al., 2001; Liao et al.,

2007).

Our previous study examined chloroplast genetic

differentiation in C. tagal between populations of the

South China Sea (SCS) and the Bay of Bengal (BOB)

(Liao et al., 2007). Ceriops tagal typically grows in

inner mangroves and is geographically widespread from

East Africa through India and Malaysia to South China

(Tomlinson, 1986). Seeds of C. tagal germinate in its

viviparous fruit (hypocotyl), which is defined as the

propagule. These are slender, long, and sharply angular.

We found that ancient Sundaland (which connected the

Philippines and the islands of Borneo, Java, and Sumatra

with the Thai-Malay Peninsula during the glacial epochs)

was a geographic barrier that hindered dispersal of

mangrove propagules (Liao et al., 2007). Based on Voris¡¦s

description of the ancient basin of Sundaland (Voris,

2000), the Sundaland river system provided dispersal

routes for mangroves, and this explains their present

distribution. Recently, a large-scale phylogeographic

study of Ceriops (Huang et al., 2007) indicated that the

limited ability for LDD in Ceriops tagal propagules led to

low genetic diversity and substructured populations. This

is consistent with the field experiments of McGuinness

(1997). Furthermore, based on analysis of inter-simple

sequence repeats (ISSR), the phylogenetic split of C. tagal

populations between SCS and BOB was not completed

(Huang et al., 2007). The population substructure indicates

these populations have been sorted well. However, the

unresolved phylogenetic relationships and incomplete

lineage sorting suggest that propagule dispersal across the

Malay Peninsula has occurred recently, perhaps during

the interglacial period. If recent LDD between the SCS

and BOB has occurred, it may have been via the Strait of

Malacca (which connects the SCS and BOB) or via the

Kra Isthmus (the narrowest and lowest part of the Thai-

Malay Peninsula) when the sea level rose during the

warmer interglacial climate.

The Thai-Malay Peninsula appears to be a dispersal

barrier for mangroves, but it is unclear whether it can

stop all mangrove gene flow between the SCS and BOB,

which may occur via the Kra Isthmus or around the Malay

Peninsula. In this paper, we use Ceriops tagal (Perr.) C. B.

Rob., a species present on both sides of the Kra Isthmus, to

investigate the divergence of high-diversity cpDNA neutral

spacers (atpB-rbcL and trnL-trnF) among these separate

populations, and we consider various mechanisms of

trans-isthmus LDD of mangroves. The cpDNA properties

of high variability and maternal inheritance are useful in

tracing the propagule dispersal of mangroves.

MATeRIAlS AND MeTHODS

Sample collection and cpDNA analysis

We sampled leaves from a total of 89 C. tagal

individuals on the east and west coasts of the Malay

Peninsula (Table 1 and Figure 1). There were four

populations on the east coast and five populations on the

west coast. All sampled individuals were separated by at

least 10 m. The collected leaves were dried immediately

with silica gel prior to DNA extraction. Total genomic

Figure 1. Map of the sampling sites. The sum of the dis-

ta nce s of dot ted li nes is the shorte st distance bet ween the

most southern populations of the east and west Kra Isthmus

around the Malay Peninsula and is roughly equal to 2,100 km

(a+b +c+d +e¡Ü2,100 km). This distance was used for the sea-

route-distance based NCA. Location abbreviations are given

in Table 1.

LIAO et al. ¡X Gene flow in

Ceriops tagal

195

DNA was isolated from samples using a commercial DNA

extraction kit (BIOMAN Co.), dissolved in TE buffer (10

mM Tris, 0.1 mM EDTA, pH 8.0), and stored at -20¢XC

until analysis. Two chloroplast regions (atpB-rbcL and

trnL-trnF) were selected for amplification and sequencing.

The primers, PCR conditions, and sequencing protocols

are described in Liao et al. (2007). Sequences obtained

in this study have been deposited in the NCBI database

under the accession numbers: DQ983274-DQ983276,

DQ983280-DQ983281 and DQ983284-DQ983307 (trnL-

trnF); DQ983241, DQ983245, DQ983248, DQ983250-

DQ983252, DQ983257-DQ983260 and DQ983262-

DQ983273 (atpB-rbcL).

Population genetic analyses

The local alignment with default settings was performed

by ClustalX (Thompson et al., 1997). After sequence

alignment, nucleotide diversity (£k), haplotype diversity

( Hd), and £c (based on the total number of mutations

[£b] and segregating sites [S]) were estimated for each

population using DnaSP 4.0 (Rozas et al., 2003). Pairwise

population differentiation was estimated by calculating

F

ST

with 1000 permutations using ARLEQUIN v. 3.0.1

(Excoffier et al., 2005), and the index of gene flow ( Nm)

was calculated from Nm=(1-F

ST

)/2F

ST

. A neighbor-joining

tree, based on the matrix of pairwise F

ST

values, was

drawn to visualize the magnitude of genetic differentiation

and relationships among the populations using MEGA 3.1

(Kumar et al., 2004). The overall magnitude of population

differentiation (£X

ST

) and the variance of the hierarchical

structure of populations were measured by Analysis of

Molecular Variance (AMOVA) using ARLEQUIN v. 3.0.1

(Excoffier et al., 2005).

Phylogeographic analyses

For phylogeographic analyses, every long-fragment

indel was treated as arising from a single evolutionary

event and recoded as described by Liao et al. (Liao et al.,

2007). The phylogenetic relationships among haplotypes

were determined with the unrooted neighbor-joining

method performed by TOPALi, version 2.17 (Milne et

al., 2004). The model was selected for F84+Gamma

with the transition/transversion ratio=0.51, alpha shape

parameter=0.10, and Kappa parameter=1.22. The

statistical significance of phylogenetic groups in the

resulting tree was tested by bootstrap resampling (1000

replicates in each case), using MEGA 3.1.

A haplotype network was constructed by the minimum

spanning network method, using ARLEQUIN v. 3.0.1

(Excoffier et al., 2005). The genetic data and geographic

information of the populations were entered into GEODIS

v. 2.0 (Posada et al., 2000) to assess the significance of

associations between genetic and geographic distributions.

This was accomplished by examining the clade distance,

D

C

(average distance of an individual from the geographic

center of all individuals within the same nesting clade) and

nested clade distance, D

N

(relative geographic distribution

to other clades in the same higher-level nesting clade),

generated from the population data (Templeton et al.,

1995; Templeton, 2001). Two nested clade analyses

(NCAs) were performed, one using geographic (great

circle) distances, and the other using sea-route distances.

The great circle distances among populations were

determined from latitudinal and longitudinal coordinates

(Table 1), and the sea-route distances were determined as

described in Figure 1. The approximate sea-route distance

from an eastern Kra population (pop E) to a western Kra

population (pop W) is:

D

E-W

= D

E-ST

+ 2,100 km + D

W-PK

,

where ST and PK are the most southerly populations of the

eastern and western Kra Isthmus, respectively, D

E-ST

is the

distance from pop E to pop ST, and D

W-PK

is the distance

from pop W to pop PK. Templeton¡¦s (2004) inference

key was then applied to the results to determine the NCA

outcome. In this analysis, we ignored C. tagal populations

that were present in the southern Malay Peninsula, so

some answers from the inference key may yield the result

"Sampling Design Inadequate."

Table 1. Sampling sites.

Population

Abbreviation Longitude

Latitude

Geographic area

Ao Phangna National Park

AP

08¢X24¡¦ N

98¢X31¡¦ E

West Kra, Bay of Bengal

Phang-Nga

PN

08¢X24¡¦ N

98¢X30¡¦ E

West Kra, Bay of Bengal

Southern Phuket island

PK

07¢X52¡¦ N

98¢X23¡¦ E

West Kra, Bay of Bengal

Ranong

RN

09¢X55¡¦ N

98¢X37¡¦ E

West Kra, Bay of Bengal

Sirinat National Park

SN

08¢X11¡¦ N

98¢X17¡¦ E

West Kra, Bay of Bengal

Mu Ko Chumphon National Park

MK

10¢X22¡¦ N

99¢X10¡¦ E

East Kra, Gulf of Thailand

Surat Thani (Korn Nan)

ST

09¢X07¡¦ N

99¢X20¡¦ E

East Kra, Gulf of Thailand

Thungkra-Swi Amphur Sawi

SW

10¢X15¡¦ N

99¢X05¡¦ E

East Kra, Gulf of Thailand

Tumethong Amphur Patew

PT

10¢X02¡¦ N

99¢X08¡¦ E

East Kra, Gulf of Thailand

196

Botanical Studies, Vol. 50, 2009

ReSUlTS

Genetic diversity

We used sequence data of the atpB-rbcL and trnL-

trnF cpDNA spacers for all of the genetic analyses in

this study. We obtained 1198 base-pair length fragments

after alignment, containing 110 polymorphic sites,

which included 10 long-fragment indels and several

substitutions and one-base indels. When we recorded

each long-fragment indel as a single mutation event (site),

there were 78 variable sites, including 20 singletons and

58 informative mutations. Among the 89 samples, we

obtained 43 haplotypes and an overall haplotype diversity

of 0.974. The diversity within individual populations

ranged from 0.400 (AP) to 1.000 (RN). Overall nucleotide

diversity (£k ) was 0.00949 and ranged from 0.00037

(AP) to 0.01861 (RN) within populations. Estimates for

£c ranged from 0.00044 (AP) to 0.01589 (MK) within

populations and had an overall value of 0.01703.

The average number of nucleotide differences between

populations on the east and west sides of the isthmus was

9.479, and nucleotide substitutions averaged 0.00918 per

site (K) between them. Eastern and western populations

shared 28 mutations, excluding gaps. Thirty-seven

mutations were polymorphic in western populations but

monomorphic in eastern populations; 24 mutations were

polymorphic in eastern populations but monomorphic

in western populations. Thus, western populations had

slightly higher genetic diversity than eastern populations.

This result is supported by the £k and £c estimates (Table 2).

Population differentiation and gene flow

All of the pairwise F

ST

values were significant at the

P<0.05 level, except for those in the following four pairs

of populations: RN-PN, MK-ST, SW-PT, and PT-ST. Both

members of each of these pairs were located on the same

side of the Kra Isthmus (Table 3). The pairwise Nm values,

estimated from the reciprocal F

ST

values between eastern

populations, were mostly higher than 1.0. This indicates

that gene flow via propagule dispersal of C. tagal has

occurred frequently among eastern populations. We also

obtained high Nm values in several pairwise comparisons

of western populations (AP-RN, PN-RN, AP-SN, PN-SN,

and RN-SN). However, these Nm values were greater than

Table 3. Matrix of F

ST

(below) and Nm (above) between nine populations of Ceriops tagal based on chloroplast atpB-rbcL and

trnL-trnF spacers. Bold values indicate comparisons between populations on the two coasts of the Kra Isthmus.

¡@

East Kra populations

West Kra populations

TR Kh MK SW PT ST

PN PK SN AP RN

TR 0.756 1.279 0.426 0.462 0.744 0.227 0.253 0.614 0.246 0.475

Kh 0.398

24.500 0.184 0.173 2.064 0.069 0.064 0.546 0.031 0.975

MK 0.281 0.020 2.293 3.021 28.912 0.592 0.559 0.870 0.779 1.273

SW 0.540 0.731 0.179

9.704 8.272 0.097 0.052 0.352 0.052 0.772

PT 0.520 0.743 0.142 0.049* 28.912 0.095 0.050 0.377 0.043 0.901

ST 0.402 0.195 0.017* 0.057 0.017*

0.520 0.306 0.573 0.498 1.332

PN 0.688 0.878 0.458 0.838 0.840 0.490 0.127 0.601 0.230 2.104

PK 0.664 0.886 0.472 0.906 0.909 0.620 0.797

0.759 0.081 1.006

SN 0.449 0.478 0.365 0.587 0.570 0.466 0.454 0.397 0.614 0.789

AP 0.670 0.942 0.391 0.905 0.921 0.501 0.685 0.860 0.449

2.606

RN 0.513 0.339 0.282 0.393 0.357 0.273 0.192* 0.332 0.388 0.161

*Denotes insignificant statistical support from 1023 permutations (P>0.05).

Table 2. Genetic diversity of Ceriops tagal populations,

according to estimates of haplotype diversity (Hd), nucleotide

diversity (£k), and genetic diversity index (£c) (estimated by

segregating sites using the equation 10 from Tajima, 1996). N

is the sample size and H is the number of haplotypes.

Population ¡@

atpB-rbcL + trnL-trnF

N H Hd £k

£c

MK

9 8 0.972 0.01104 0.01589

SW

10 8 0.956 0.00217 0.00232

PT

8 7 0.964 0.00191 0.00181

ST

9 7 0.917 0.00410 0.00410

RN

10 10 1.000 0.01861 0.01546

AP

5 2 0.400 0.00037 0.00044

SN

21 13 0.957 0.00940 0.00940

PK

8 3 0.607 0.00215 0.00250

PN

9 5 0.806 0.00268 0.00239

E-Kra

36 22 0.967 0.00584 0.01228

W-Kra

53 26 0.966 0.01160 0.01381

Total

89 43 0.974 0.00949 0.01703

LIAO et al. ¡X Gene flow in

Ceriops tagal

197

1.0 for just two of the pairwise comparisons of eastern and

western populations (MK-RN and RN-ST). These results

indicate that the cpDNA gene flow (propagule dispersal)

was higher within eastern and western populations than

between these populations. Thus, the Kra Isthmus appears

to reduce dispersal of mangroves between the east and

west coasts, but it is not an absolute barrier that prevents

all dispersal.

Population genetic structure

The results of the hierarchical AMOVA indicate

that within-population variation accounts for 52.0% of

the observed genetic variation and variation between

populations on the east and west sides of the isthmus

accounts for 31.6% of the variation. Variation among

populations within these groups accounted for 16.5% of

the total variation. The hierarchical £X statistics revealed

the same pattern. It showed that most of the genetic

variation is within populations (£X

ST

= 0.480), followed

by variation among groups (£X

CT

= 0.316), and variation

among populations within groups (£X

SC

= 0.241). All of the

£X statistics and differences in percentages of variation are

statistically significant (p<0.005, Table 4). The grouping

of populations in the NJ tree derived from the pairwise F

ST

values is consistent with their geographical distribution

although the short branch length between the east and west

populations indicates minor divergence between these

groups (Figure 2). Taken together, the AMOVA results and

NJ population tree indicate that the Kra Isthmus has a hand

in structuring the C. tagal populations although the genetic

distance between populations on the east and west sides is

short.

Phylogeographic inferences from NCA

The apparent discrepancies between the groupings in

the haplotype phylogenetic tree and actual geographic

distribution result in unresolved, ambiguous, and

statistically weak relationships of gene-tree topology.

This tree indicates recent gene flow (Bernardi et al.,

2003) but provides no evidence of population history. By

contrast, the minimum spanning network separates the

east and west clades although only by one or a few steps

(Figure 4). Roughly speaking, eastern populations were

located in the interior clades and western populations in

the terminal clades. The central clades, with presumably

the most ancient evolutionary information, included

Figure 2. Neighbor-Joining tree of Ceriops tagal const ruct-

ed by pairwise F

ST

. The scale ba r indicates F

ST

differences

and divergence time. The time scale, based on the estimated

time of divergence of the Gulf of Thailand and the Bay of

Bengal populations, is approximately 5 Mya.

Table 4. Summary of results of molecular variance (AMOVA)

for East and West Kra areas of Ceriops tagal. The significance

(P) of the variance was based on 1000 permutations.

Source of Variation

d.f. % total

variance £X P

Among groups

1 31.56 0.31560 <0.005

Among populations within

groups

7 16.48 0.24079 <0.0001

Within populations

80 51.96 0.48040 <0.0001

Figure 3. Unrooted Neighbor-Joining t ree of Ceriops tagal

haplot ypes. Numbers on branches a re bootst ra p values of

1,000 replicates. Diamonds indicate haplotypes on the east

side of the isthmus, and squares indicate haplotypes on the

west side of the isthmus. The scale bar indicates the inferred

frequency of substitutions per nucleotide site.

198

Botanical Studies, Vol. 50, 2009

eastern and western haplotypes (subclades I-7 and I-16).

Furthermore, several haplotypes located in the terminal

clades were loosely connected with the long-missing links

(Figure 3), suggesting a longer evolutionary separation

of these haplotypes than merely an accumulation of a

few step mutations. Great-circle and sea-route distance-

based analyses detected insignificant nested-clade distance

associations among lower level clades. This indicates that

the null hypothesis of no association between haplotype

and geographical distribution should not be rejected for

these clades.

However, great-circle-distance and sea route distance-

based nested clade analyses generated four clades that

showed significant associations (permutational chi-

squared probabilities for geographic structure less than

0.05) with D

C

, D

N

or I-T values (Table 5). Both analyses

yielded similar inferences for three of these four clades.

We infer a restricted gene flow with isolation by distance

(IBD) evolutionary scenario for Clade II-10. This indicates

that within this centrally located clade of the western Kra

Isthmus, short-distance dispersal may have occurred,

but only rarely. In contrast, we infer that Clade II-5 (an

eastern clade connected to two western haplotypes by

single missing links), arose from long distance dispersal

Figure 4. Nesting scheme for one-step (1

st

class) and higher (2

nd

and 3

rd

class) clades. Small open circles in the 1

st

class clades

are inferred missing haplotypes we did not observe in the dataset. The haplotypes or clades observed in the east Kra Isthmus

are presented in white and those observed in west Kra Isthmus are black. In the higher class clades, the size of circles represents

sample size. The Arabic numbers within the 1

st

-class-clades circles are haplotype numbers; the Rom an numbers followed by

Arabic numbers are assigned numbers for hierarchical clades.

(LDD), according to the analyses based on both types

of geographic-distance. Clade III-3 is a clade of western

haplotypes, with many long missing links (inferred to be

consequences of "contiguous range expansion") and one

eastern haplotype.

In contrast, the inferences from the total cladograms

generated using the great circle and sea route distances are

distinctly different. The great circle distance-based results

indicate there was "inadequate sampling" to discriminate

IBD from LDD. The sea route distance-based results

indicate a restricted gene flow with an IBD scenario. The

highest-clade inferences indicate a better use of the sea

route distance mode on tracking the evolutionary events of

mangrove dispersal (Table 5).

DISCUSSION

The Thai-Malay Peninsula as a barrier

Previously, we showed a significant genetic

difference between the SCS and the BOB populations

o f C. tagal and speculated that the Sundaland served

as a geographic barrier (Liao et al., 2007). Research on

mangrove population genetics by Shi and colleagues

(Tan et al., 2005; Huang et al., 2007; Su et al., 2006,

LIAO et al. ¡X Gene flow in

Ceriops tagal

199

2007) also concluded that a biogeographic event

caused differentiation of SCS and BOB populations.

Population genetic studies of starfish (Benzie, 1999)

and vetigastropods (Imron et al., 2007) also revealed an

isolation of populations in the western Pacific and the

eastern Indian Oceans. It thus seems that the Thai-Malay

Peninsula (formerly Sundaland) is an effective geographic

barrier to the migration of species.

However, oceanographic data (Woodruff, 2003) and a

biogeographic study of populations of the giant freshwater

prawn (Macrobrachium rosenbergii) between northern

and southern sites of the Kra Isthmus (de Bruyn et al.,

2005) suggest that species have migrated between the SCS

and BOB in the past. In the case of C. tagal, the similar

genetic composition of eastern and western Kra Isthmus

populations implies that gene flow may have occurred

across the peninsula, before and/or after the formation

of the Kra Isthmus. According to Wolfe et al.¡¦s (1987)

estimation of the evolutionary rate of cpDNA (1.0-3.0¡Ñ

10

-9

per site per year), the divergence time between east

and west Kra Isthmus populations was about 4.59-1.53

Mya. This is similar to the estimated time of formation

of the Thai-Malay Peninsula land barrier after the last

marine transgressions during the Pliocene era, 5.5-4.5

Mya (Woodruff, 2003). Although dating the variation

of cpDNA markers includes a large margin of error, the

approximate agreement between the dates of the genetic

and paleoclimatic data suggests that geographical and

climatic changes caused a divergence of eastern and

western populations of C. tagal.

The geographic isolation imposed by the Kra Isthmus

contributes to about one-third of the total genetic variation

present in the C. tagal populations that we studied (Table

4). This divergence apparently began in the Pliocene,

when a land barrier emerged that separated the eastern

and western populations. In addition, 30% of the pairwise-

compared Nm values between the eastern and the western

populations are higher than 0.8, and 56.7% are higher than

0.5 (Table 3). This indicates that the Thai-Malay Peninsula

might allow some gene flow across the Kra Isthmus

(Figure 3).

The haplotypes located at the center of a network

are usually considered the most ancestral. Thus, clades

II-3 and II-10 (Figure 4) probably include the most

ancient haplotypes of eastern and western populations

and presumably carry the maximum amount of genetic

information regarding the ancestral populations of C.

tagal ~5 Mya. In addition, the star-like topology that

we observed, with short interior branches that lead to

long terminal clades with missing links, is indicative

of exponential growth events (Anderson et al., 2003).

Populations of other organisms in the Indo-Malay

region, such as the mollusc Haliotis asinina (Imron et

al., 2007), also seem to have rapidly expanded ~5 Mya.

The rapid expansion of C. tagal populations is supported

by the unresolved splitting of phylogenetic relationships

among haplotypes (Figure 3). The unresolved haplotype

relationships of the eastern and western populations

may be due to ancient gene flow and incomplete lineage

sorting, caused by climatic changes that led to fluctuations

of sea level.

Comparison of geographic distance-based

NCAs

The two geographic distance-based NCAs yielded the

same inferences for the lower-class network, but different

inferences for the total cladograms. It may be that in our

network the lower class clades are almost exclusively

composed of populations on the same side of the isthmus,

between which great-circle and sea-route distances are

similar. For the highest clade, a different interpretation

is necessary due to the substantial differences between

the great-circle and sea-route distances that separate the

eastern and western populations. The great-circle distance-

based analysis failed to discriminate between IBD and

LDD. This may be due to the short great-circle distances

between populations on the two sides of isthmus and

between populations on the same sides of the isthmus.

Thus, cross-isthmus dispersal of propagules cannot be

distinguished from around-peninsula dispersal using great-

circle distances. However, using the more realistic sea

route-based geographic distances, we infer a scenario

Table 5. Summary of nested clade phylogeographic analyses for clades showing statistically significant associations between

haplotypes and geographical distances.

Clade nesting

Great circle distance

Sea-route distance

Chain of inference

Inference

Chain of inference

Inference

Clade II-5 1¡÷2¡÷11¡÷12¡÷13¡÷

21Yes

Long-distance movement

1¡÷2¡÷11¡÷12¡÷13¡÷

21Yes

Long-distance movement

Clade II-10 1¡÷2¡÷3¡÷4No Restricted gene flow with isolation

by distance

1¡÷2¡÷3¡÷4No Restricted gene flow with

isolation by distance

Clade III-3 1¡÷2¡÷11¡÷12No Contiguous range expansion 1¡÷2¡÷11¡÷12No Contiguous range expansion

Total

cladogram

1¡÷2¡÷3¡÷5¡÷6¡÷7¡÷

8No

Sampling design inadequate to

discriminate between isolation

by distance (short distance

movements) versus long distance

dispersal

1¡÷2¡÷3¡÷4No Restricted gene flow with

isolation by distance

200

Botanical Studies, Vol. 50, 2009

with restricted gene flow and IBD. This is supported by

the absence of tight connections between haplotypes

representing eastern and western populations within all but

two clades (Clades II-5 and II-7).

This pattern excludes the possibility of propagule

exchange across the Kra Isthmus mediated by changes in

stream drainage patterns or rising sea levels. Indications

of migration around the peninsula in the 5 million years

since the Malay Peninsula emerged are also provided by

the high pairwise-Nm values derived from the F

ST

data

(Table 4). Since different inferences can be drawn from

analyses using the two types of distances, Fetzner and

Crandall (2003) suggested utilization of both types of

distances to provide insight to historical and contemporary

processes. However, the sea-route-distance mode provides

more precise inferences of mangrove dispersal that

reflect the difficulties of LDD, and are consistent with

the field experiment results of McGuinness (1997). We

cannot completely exclude the possibility of LDD. In

particular, when verifying other analytic results (i.e. Nm)

and geological evidence (i.e. glacial/interglacial cycles),

it is realistic to consider rare instances of LDD. We

conclude that (a) dispersal events have occurred between

populations on the same side of the Kra Isthmus and

between populations on different sides of the isthmus;

(b) cross-side dispersal has been far less frequent than

within-side dispersal; and (c) since its emergence, the Kra

Isthmus has played an important role in differentiating and

structuring the populations of C. tagal.

Potential means of dispersal across the Kra

Isthmus

Our network analysis (Figure 4) revealed unexpected

associations of eastern and western populations in

subclades II-5 and II-7. Dates based on the estimated

evolutionary rate of cpDNA indicate that the contiguous

range expansion of subclade II-7 (III-3) occurred ca.

7.04-2.35 Mya, and the LDD of subclade II-5 occurred

0.49-0.17 Mya. Thus, contiguous range expansion of

III-3 occurred at about the same time as the Kra Isthmus

formed. This indicates that haplotype exchanges may have

occurred before the isthmus formed. In other words, the

NCA concluded that rapid range expansion of C. tagal

occurred ~5 Mya, at which time rising sea levels promoted

frequent gene flow between mangrove populations situated

on the SCS and BOB coasts. However, LDD events also

seem to have occurred less than one million years ago,

indicating that propagules have dispersed across the Kra

Isthmus via a sea route or land bridge.

There are two possible mechanisms by which C. tagal

may have dispersed from one side of the Kra Isthmus to

the other. The first is directly across the isthmus, perhaps

through openings in the Kra Isthmus during interglacial

periods or through human-assisted introductions. There

is no evidence that sea levels have risen by more than

20 meters above present levels in the past 0.5 million

years (Rohling et al., 1998). This is much less than the

100 meters required for such openings. Human-mediated

dispersal also seems unlikely because ancient "Heidelberg

Man" (Homo heidelbergensis) and/or their relatives

living in the area roughly 0.2 to 0.6 Mya had no apparent

uses for mangrove seedlings or reasons to aid dispersal.

Although we cannot exclude the possibility of animal-

mediated dispersal, there is no evidence that mangroves

are dispersed this way. Thus, human- or animal-mediated

LDD is unlikely to account for the apparent cross-isthmus

gene flow that we detected.

The second mechanism of dispersal across the isthmus

is LDD around the Malay Peninsula, presumably via the

Malacca Strait. Straits are generally considered to provide

corridors for species migration in the waters they connect.

A classic example is the Strait of Gibraltar, in which

organisms passed between the Atlantic and Mediterranean

(Seidenkrantz et al., 2000; Betzler et al., 2006; Gonzalez-

Wanguemert et al., 2006). Similarly, the Malacca Strait

plays an important role in the passage of marine organisms

(and merchant vessels) between the Indian Ocean and the

SCS. In addition, the eastern and western populations of C.

tagal may have been isolated during glacial periods (when

the Sunda Shelf emerged above sea level) and reconnected

during interglacial periods. The likely dates for the LDD

interglacial events are apparently associated with subclade

II-5 of C. tagal and range from 0.49-0.17 Mya (including

the last part of the Gunz-Mindel, ~ 0.62-0.45 Mya and

all of the Mindel-Riss ~0.3-0.2 Mya). Thus, despite the

uncertainty of molecular dating, it is consistent with the

proposal that interglacial sea-level rises facilitated trans-

isthmus gene flow of mangroves.

On the other hand, irrespective of the routes involved,

we provided evidence of a few rare LDD events between

eastern and western populations, and these appear to have

hindered allopatric speciation. The unresolved nature of

our haplotype tree, which make determining the time of

divergence between haplotypes of the two coasts difficult,

also indicates recent contact between eastern and western

populations (Figure 3). A similar phenomenon has been

reported for Baja California¡¦s marine fishes (Halichoeres

semicinctus, Semicossyphus pulcher, Hermosilla azurea,

and Sebastes macdonaldi). Bernardi et al. (2003)

suggested that the lack of tree topology resolution for

these species was probably due to recent or ongoing

dispersal, with high levels of gene flow (although they

could not exclude the possibility of insufficient time for

lineage sorting due to the use of slowly evolving markers).

Thus young isthmuses, such as Baja California and the

Kra Isthmus, appear to inhibit migration and dispersal but

do not entirely prevent low-frequency LDD.

In summary, our results suggest that populations of

C. tagal diverged by a vicariance mechanism during

formation of the Kra Isthmus. The formation of this land

barrier is considered to have prevented gene exchange

between SCS and BOB populations of other organisms,

including sea snakes (Karns et al., 2000; Heatwole et

al., 2005), sea urchins (Lessios et al., 2001), and other

LIAO et al. ¡X Gene flow in

Ceriops tagal

201

mangrove species (Tan et al., 2005; Su et al., 2006).

However, we detected gene flow between the eastern

and western sides of the Kra Isthmus after increasing

sample sizes. We consider two types of gene flow: (a)

ancient gene flow by direct exchange across a leak in the

Kra area approximately 5 Mya, and (b) infrequent but

repeated LDD around the Malay Peninsula and through

the Strait of Malacca during interglacial periods. These

ancient and recent gene flows caused a mixed haplotype

distribution and unresolved phylogenetic relationships

among the eastern and western populations of C. tagal.

In other words, the Malay Peninsula does not completely

prevent gene flow of mangroves between the SCS and

BOB. Thus, in agreement with previous studies (Tan et

al., 2005; Su et al., 2006; e.g. Su et al., 2007; Liao et al.,

2007) our results challenge the common belief that the

Thai-Malay Peninsula has stopped the LDD of mangroves

between SCS and BOB. Most researchers have ignored the

significance of rare LDD and have only focused on "major

events," such as the vicariance caused by Sundaland.

However, some minor dispersal events can have large

impacts of population structure and even retard or impede

the process of allopatric speciation. Our study of C. tagal

provides important insight into the significant role of rare

LDD in the prevention of allopatric speciation.

Acknowledgements. We are grateful to Dr. Tzen-Yuh

Chiang for valuable suggestions on this manuscript. We

also thank Dr. Suhua Shi, Dr. Chiou-Rong Sheue, and Dr.

Sonjai Havanond for kindly providing the plant material

used in this study. This research was supported by an

Academia Sinica Thematic Grant (2001-2004), NSC92-

2311-B003-005 to S. Huang, and a partial NSC grant to

Y. C. Chiang from the National Science Council, ROC.

We thank two anonymous reviewers for their valuable

comments on this work.

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