Int J Plant Sci.167(2):291–298 2006 Ó 2006 by The University of Chicago All rights reserved 1058-5893/2006/16702-0012$15.00 GENETIC VARIATION OF TWO MANGROVE SPECIES IN KANDELIA (RHIZOPHORACEAE) IN VIETNAM AND SURROUNDING AREA REVEALED BY MICROSATELLITE MARKERS Le H Giang,* Gretel L Geada,* Phan N Hong,y Mai S Tuan,y Nguyen T H Lien,y Sosaku Ikeda,* and Ko Harada1 ,* *Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan; and yMangrove Ecosystem Research Center, Center for Natural Resources and Environmental Studies, Vietnam National University, 22, Ngo Luong Su B, Quoc Tu Giam Street, Hanoi, Vietnam Genetic variation of the mangrove genus Kandelia (Rhizophoraceae) in the South China Sea region, in four populations in Vietnam and in one population each in Iriomote, Japan, and Bako, Borneo, was evaluated using microsatellite markers A total of 54 alleles in the six populations were detected by using four microsatellite loci The two northern Vietnamese populations (Don Rui and Xuan Thuy) showed a high allelic diversity (40 alleles in total) and a high level of gene diversity (HE ¼ 0:773 on average) In contrast, the two southern Vietnamese populations (Can Gio and Ngoc Hien) showed low allelic diversity (11 alleles in total) and a low level of gene diversity (HE ¼ 0:244 on average) There was only one allele common to the two regions The Iriomote population was genetically related to the northern Vietnamese populations, while the Bako population was related to the southern populations The findings and the morphological observations indicate that these two genetically differentiated vicariant lineages represent two different species groups, Kandelia obovata Sheue, Liu, and Yong for northern Vietnam and Japan and Kandelia candel (L.) Druce for southern Vietnam and Borneo The difference in the amount of genetic variation shows that these two species experienced a different adaptive process during the past glacial ages Keywords: genetic variation, mangrove, microsatellite, Kandelia candel, Kandelia obovata, Rhizophoraceae, South China Sea Introduction projects, however, genetic variation and local adaptation of mangrove plants have seldom been considered One reason for this is that appropriate methods for measuring genetic variation have been developed only recently Analysis of genetic variation should reveal its extent and distribution within a species, indicating the range of local adaptation This information could be used for choosing germplasm for regeneration and planning sustainable management of mangrove forests Kandelia Wight and Arnold 1834, a genus in the mangrove family Rhizophoraceae, was known as a monotypic genus with the single species, Kandelia candel (L.) Druce Kandelia candel was said to be distributed from the Ganges Delta, Myanmar, through Southeast Asia to south China, the Ryukyu Islands, and south Japan (Tomlinson 1986) The eastern limit is northern Borneo (Tomlinson 1986) Recent studies on leaf anatomy (Naskar and Mandal 1999), physiological adaptation (Nakagoshi and Nehira 1986; Maxwell 1995), chromosome numbers (Yoshioka et al 1984; Das et al 1995), and molecular phylogeography (Huang and Chen 2000; Chiang et al 2001) have shown that the species can be separated into two well-distinguished geographical groups, with the border at the northern end of the South China Sea Taking these results and the detailed morphological comparisons of vegetative and reproductive characters into account, Sheue et al (2003) concluded that K candel could be split into two vicariant lineages representing the two geographically differentiated species groups Because K candel was first named for the species in India (Nicolson et al 1988), the Mangroves are constituent plants of tropical and subtropical intertidal forest communities, typical for the estuaries of large rivers that run over a shallow continental shelf They occupy two separate hemispheric regions but are more abundant in the Old World than in the New World tropics (Tomlinson 1986) Vietnam is rich in mangrove species, especially along the coast of the Gulf of Tonkin in the north and along the coast of Mekong Delta in the south Thirty-four true mangrove species have been identified in the area (Hong and San 1993) In Vietnam, mangrove forests have been used for supplying diverse commercial and traditional products However, the mangrove forests, especially in the southern part of Vietnam, were seriously damaged by chemical warfare from 1962 to 1971 (Stellman et al 2003) Although the forests have since largely regenerated, conversion of mangrove forests to agricultural and aquaculture farms and clearance for settlements have caused rapid degradation and decline of the remaining forests in the area Since mangrove forests are important not only economically but also environmentally, reforestation and rehabilitation projects have been carried out in many countries, including Vietnam (Kogo and Kogo 1997) In these Author for correspondence; phone 81-89-946-9870; fax 81-89946-9868; e-mail kharada@agr.ehime-u.ac.jp Manuscript received November 2004; revised manuscript received October 2005 291 This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) INTERNATIONAL JOURNAL OF PLANT SCIENCES 292 populations north of the South China Sea were named Kandelia obovata Sheue, Liu, and Yong (Sheue et al 2003) Specimens collected at the Gulf of Tonkin were identified as K obovata (Sheue et al 2003), but no specimens have been collected from other areas of Vietnam Kandelia is one of the main mangroves used for afforestation in coastal areas of Vietnam, and propagules from the northern part of Vietnam have often been used as a seed source for the southern part of Vietnam Genetic information on the geographical structure of Kandelia populations is needed for an appropriate choice of propagules We collected specimens of Kandelia from four Vietnamese populations, two in the northern region and two in the southern region, and additionally from one population each in the Ryukyu Islands, Japan, and in Borneo, and we examined genetic variation, using microsatellite markers Microsatellites are codominant markers and are known to be rich in genetic variation from high mutation rates (Levinson and Gutman 1987; Wolff et al 1991) These characteristics make the markers useful for genome mapping, paternity testing, and individual identification (Jeffreys et al 1985; Nakamura et al 1987; Silver 1992) The usefulness of the markers for population genetics studies on mangrove species has also been demonstrated in Avicennia marina (Maguire et al 2000; Giang et al 2003) The aim of this study was to investigate how much genetic variation is maintained in populations of Kandelia in Vietnam, the Ryukyu Islands, and Borneo, and how they are related each other These findings should give us clues for understanding the adaptive process of the two species in different climatic conditions Material and Methods Sample Collection and DNA Isolation Leaf samples from natural populations of Kandelia were collected from two populations in the northern part of Vietnam (Dong Rui and Xuan Thuy), two populations in the southern part of Vietnam (Can Gio, Ngoc Hien), one population in Iriomote Island (Urauchi River) in the Ryukyu Islands, and one population in Bako, Borneo (table 1; fig 1) Sampling was planned so as to collect ca 30 adult individuals at each location, separated from each other by at least 30 m in order to avoid collecting half-sibs and to maximize the probability of selecting diverse genotypes The leaf sam- ples were individually sealed in airtight plastic bags and placed on ice in the field before freezing at ÿ20°C About 0.5 g of leaf tissue was ground to powder in liquid nitrogen Total genomic DNA was extracted by the modified CTAB method (Doyle and Doyle 1990) Genomic DNA was further purified using a DNA binding matrix of the FastDNA kit (Qbiogene, Montreal) Microsatellite Analysis Primer sequences specific for four microsatellite loci described by Sugaya et al (2002) were used (table 2) The PCR amplification reaction mix was reaction buffer (20 mM TrisHCl, pH 8.3, 50 mM KCl, mM MgCl2, 0.001% gelatin), mM dNTP, 0.5 mM forward and reverse primers, 0.3 units Taq DNA polymerase (Sigma-Aldrich, St Louis) and 10 ng genomic DNA in a total volume of 10 mL The forward primers were fluorescent-labeled with FAM, VIC, and NED (Applied Biosystems, Foster City, CA) Microsatellite amplification was carried out using the following cycling parameters: preheating for at 94°C, followed by 35 cycles of denaturing at 90°C for 30 s, annealing at primer-specific temperatures of 55°–60°C for 30 s, and extension for at 72°C Reactions were completed by incubation at 72°C for and holding at 4°C The PCR products were denatured for at 95°C and then separated by capillary electrophoresis on a 310 Genetic Analyzer (Applied Biosystems) GeneScan software (Applied Biosystems) was used for microsatellite analysis Because these loci were cloned from Kandelia from a northern population (Amami in the Ryukyu Islands), we subsequently subcloned the four microsatellite loci from individuals sampled from two southern populations, Can Gio and Bako, and determined the sequence in order to assess whether these loci are conserved across the populations One individual from each population was sampled The microsatellite regions were amplified using COD-Plus DNA polymerase (Toyobo, Osaka, Japan) and subcloned using Zero Blunt TOPO Cloning Kit (Invitrogen, Carlsbad, CA) Plasmid DNA was extracted and sequenced on a 310 Genetic Analyzer (Applied Biosystems) using Big Dye Terminator, version 1.1 (Applied Biosystems) Data Analysis Population genetics parameters were computed using the GDA program (ver 1.1) (Lewis and Zaykin 1999) The following diversity statistics in each population were calculated: average number of alleles, unbiased expected heterozygosity Table Collection Sites of the Mangrove Genus Kandelia in Northern and Southern Regions of the South China Sea Population Northern region: Iriomote Dong Rui Xuan Thuy Southern region: Can Gio Ngoc Hien Bako Location Latitude (N) Longitude (W) Forest area (ha) Sample size Ryukyu Quang Ninh Province Nam Dinh Province 24°199 21°139 20°149 123°459 107°229 106°339 Ca 100 Ca 3500 Ca 1600 25 22 23 Ho Chi Minh City Ca Mau Province Borneo 10°229 8°509 2°369 106°469 105°099 110°199 Ca 4700 Ca 51,500 Ca 16,000 28 25 30 This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) GIANG ET AL.—GENETIC VARIATION IN TWO KANDELIA SPECIES 293 the sequences were determined (accession nos AB236960– AB236963 for Can Gio and AB236964–AB236967 for Bako) In all four loci, primer regions were completely matched with those reported by Sugaya et al (2002) A total of four nucleotide substitutions (one in Kc09, one in Kc11, and two in Kc34) and four indels (three with a length of 10 bp, 12 bp, and a single nucleotide in Kc11 and one with a length of bp in Kc34) were detected in a total length of 922 bp of franking regions Franking sequences were identical in Can Gio and Bako in all four loci Interestingly, three indels (of 10, 12, and bp) were found on one or both sides of the dinucleotide repeats Large differences were found in the number of dinucleotide repeats For Kc04, the TC unit repeated 35 times in Sugaya’s sequence, whereas it repeated 11 and 12 times in the clones from Can Gio and Bako, respectively Similarly, for Kc11, the TC unit repeated 34 times in Sugaya’s sequence, whereas it repeated nine and eight times in Can Gio and Bako, respectively For Kc09 and Kc34, the GA and AG units repeated 19 and 26 times, respectively, in Sugaya’s sequence, whereas they repeated 14 and 10 times, respectively, in both of Can Gio and Bako In all of the loci, the repeat number was much smaller in southern populations These findings indicate that the microsatellite loci are conserved across the northern and southern populations, although they are considerably differentiated Fig Collection sites of the mangrove genus Kandelia in the South China Sea region The line in the bottom represents the equator Allelic Diversity Four microsatellite loci were consistently resolved in the six populations of Kandelia A total of 54 alleles, 17 for Kc04, 12 for Kc34, for Kc09, and 18 for Kc11, were detected Among them, a total of 50 alleles were found in the Vietnamese populations Allele frequency distribution for each locus is graphically shown in figure Many of the same alleles were found in the two northern Vietnamese populations A total of 40 alleles were found in this region Some of these alleles were also found in the Iriomote population A total of 11 alleles were found in the two southern Vietnamese populations, and seven of them were shared between the populations There is only one common allele between the southern and northern Vietnamese populations, Kc09-283 A total of five alleles were detected in Bako, and all of them were also found in the southern populations in Vietnam These results show that populations in the northern and southern parts of Vietnam are clearly differentiated The northern populations are genetically related to the Iriomote population, while the southern populations are related to the Bako population (HE), and observed proportion of heterozygotes (HO) Unbiased estimates of HE and F statistics (FIS, FIT, and FST) were estimated for each locus The 95% confidence interval (CI) of F statistics was determined by bootstrap analysis using 1000 replicates The outcrossing rate was calculated as Fị=1 ỵ FÞ (Weir 1996) Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were evaluated, and the significant levels were determined using the Markov chain method with GENEPOP, version 3.4 (Raymond and Roussett 1995) The default parameters in GENEPOP with 100 batches of 1000 iterations each were used for the test Results Conservation of the Microsatellite Loci Four microsatellite loci were subcloned from two individuals, one each from the Can Gio and Bako populations, and Table Genetic Variation of Microsatellite Loci in Six Populations of the Mangrove Genus Kandelia Northern Vietnam Dong Rui Southern Vietnam Xuan Thuy Can Gio Ryukyu Ngoc Hien Borneo Iriomote Bako Locus Repeat unit A HE HO A HE HO A HE HO A HE HO A HE HO A HE HO Kc04 Kc34 Kc09 Kc11 (CT)35 (AG)26 (GA)20 (CT)34 9 14 0.850 0.747 0.634 0.911 0.591 0.636 0.667 0.818 10 0.866 0.704 0.623 0.850 0.652 0.348 0.545 0.652 2 0.457 0.382 0.000 0.249 0.321 0.071 0.000 0.286 2 0.275 0.040 0.042 0.509 0.130 0.040 0.042 0.040 3 0.676 0.584 0.085 0.523 0.370 0.458 0.000 0.148 1 0.000 0.444 0.000 0.000 0.000 0.357 0.000 0.000 Note A ¼ total number of alleles; HE ¼ expected heterozygosity; HO ¼ observed heterozygosity This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) 294 INTERNATIONAL JOURNAL OF PLANT SCIENCES Fig Allele frequencies at four microsatellite loci, Kc04, Kc34, Kc09, and Kc11 Numbers in the graphs refer to the nucleotide lengths of amplified fragments Eleven private alleles were detected in the two northern Vietnamese populations: nine (Kc34-260, Kc34-274, Kc09283, Kc09-297, Kc11-190, Kc11-192, Kc11-216, Kc11-240, and Kc11-248) in Dong Rui and three (Kc04-324, Kc11-218, and Kc11-234) in Xuan Thuy Four private alleles were detected in the two southern populations: one (Kc04-306) in Can Gio and three (Kc04-300, Kc04-304, and Kc11-180) in Ngoc Hien A total of 12 alleles were found in the Iriomote population, and four (Kc04-344, Kc04-360, Kc04-362, and Kc34-250) of them were private alleles Genetic Diversity The observed and expected heterozygosities were determined for each locus (table 2) The loci with longer dinucleotide repeats (Kc04 and Kc11) commonly revealed greater values of heterozygosity than did the two other loci (Kc34 and Kc09) The expected heterozygosity was greater than the observed heterozygosity across all of the loci and all of the populations except Kc09 in Don Rui and Kc11 in Can Gio The average and the effective numbers of alleles (na and ne, respectively), together with the expected and observed heterozygosities over all loci (HE and HO, respectively), were calculated for each population (table 3) The number of alleles was much larger in the northern Vietnamese populations than in the southern populations The Dong Rui population showed the largest na, and the Ngoc Hien population showed the smallest na The average number of alleles in the north was 8.38, while that in the south was 2.25 The greatly reduced number of effective alleles in the north (4.78) indicates that many rare alleles are involved The HO was high in the two northern Vietnamese populations, 0.549 in Xuan Thuy and 0.678 in Dong Rui, and the value was much lower in the southern populations, 0.063 in Ngoc Hien and 0.170 in Can Gio It is notable that both This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) GIANG ET AL.—GENETIC VARIATION IN TWO KANDELIA SPECIES 295 Table Genetic Variation of Six Populations of Mangrove Genus Kandelia over All Microsatellite Loci Population Sample size na ne HE HO F Outcrossing rate 22 23 9.50 7.25 8.38 5.22 4.57 4.78 0.785 0.761 0.773 0.678 0.549 0.614 0.140* 0.282*** 0.210 0.754 0.560 0.657 28 25 2.25 2.25 2.25 1.43 1.40 1.43 0.272 0.216 0.244 0.170 0.063 0.116 0.380*** 0.713*** 0.528 0.449 0.168 0.309 25 30 3.00 1.25 2.18 1.19 0.467 0.111 0.244 0.089 0.482*** 0.199 0.345 0.593 Northern Vietnam: Dong Rui Xuan Thuy Mean Southern Vietnam: Can Gio Ngoc Hien Mean Outside Vietnam: Iriomote Bako Note na ¼ average number of alleles per locus; ne ¼ effective number of alleles per locus; HE ¼ expected heterozygosity; HO ¼ observed heterozygosity; F ¼ inbreeding coefficient à Significant departure from Hardy-Weinberg equilibrium (HWE): P < 0:05 ÃÃà Significant departure from HWE: P < 0:0001 allelic diversity and gene diversity were lowest in Bako, where three of the four microsatellite loci were monomorphic Gene diversity in Iriomote was lower than that in the two northern Vietnamese populations Observed heterozygosity was lower than expected heterozygosity in all of the populations, leading to positive inbreeding coefficients Highly significant departure from HWE was detected in Xuan Thuy, Can Gio, Ngoc Hien, and Iriomote populations The inbreeding coefficient (F) was low in the two northern populations, the average value being 0.210, while the values were higher in the two southern populations, varying from 0.380 (Can Gio) to 0.713 (Ngoc Hien) Consequently, the outcrossing rate was high in the north (0.560 in Xuan Thuy and 0.754 in Dong Rui) and low in the south (0.168 in Ngoc Hien and 0.449 in Can Gio) (table 3) CIs of 0.086 and 0.175 between the two southern populations, indicating moderate genetic differentiation The FST was also calculated among the populations in the northern South China Sea region (Dong Rui, Xuan Thuy, and Iriomote) and the southern South China Sea region (Can Gio, Ngoc Hien, and Bako) (table 4) Relatively large genetic differentiation was found among the populations in the northern region (FST ¼ 0:207), while differentiation was larger in the southern region (FST ¼ 0:30) A UPGMA tree was constructed on the basis of Nei’s genetic distance (Nei 1972) It clearly divided the populations into two clusters, one consisting of the two northern Vietnamese populations and Iriomote and the other of the two southern Vietnamese populations and Bako (fig 3) These groupings were supported by high bootstrap values Genetic Differentiation among the Populations Linkage Disequilibrium The fixation index, FST, was calculated between the two northern populations and the two southern populations in Vietnam (table 4) The value was 0.014, with 95% CIs of 0.000 and 0.050 between the two northern populations, indicating no genetic differentiation and suggesting the populations to be panmictic, while the value was 0.134 with 95% Linkage disequilibrium was tested for six pairwise combinations of the four microsatellite loci in each population using Fisher’s exact test (table 5) Highly significant disequilibrium was detected in two combinations in Iriomote (Kc04Kc11 and Kc34-Kc11) Slightly significant or insignificant disequilibrium was detected for the rest of the populations Table F Statistics of Four Microsatellite Loci in the Mangrove Genus Kandelia for Each Region Northern Vietnam: Dong Rui and Xuan Thuy Locus Kc04 Kc34 Kc09 Kc11 Overall Upper CIa Lower CIa a Southern Vietnam: Can Gio and Ngoc Hien Northern South China Sea: Dong Rui, Xuan Thuy, and Iriomote Southern South China Sea: Can Gio, Ngoc Hien, and Bako FIS FIT FST FIS FIT FST FIS FIT FST FIS FIT FST 0.279 0.331 0.038 0.169 0.216 0.303 0.114 0.273 0.320 0.109 0.183 0.223 0.294 0.151 0.000 0.000 0.073 0.016 0.014 0.050 0.000 0.377 0.747 ÿ0.002 0.548 0.517 0.756 0.359 0.475 0.790 0.002 0.589 0.582 0.772 0.456 0.158 0.169 0.003 0.090 0.134 0.175 0.086 0.336 0.297 0.104 0.316 0.282 0.331 0.170 0.446 0.447 0.349 0.435 0.431 0.459 0.382 0.164 0.241 0.273 0.174 0.207 0.262 0.167 0.376 0.466 ÿ0.003 0.547 0.456 0.523 0.357 0.515 0.688 0.001 0.636 0.620 0.683 0.495 0.222 0.415 0.004 0.196 0.301 0.412 0.188 Confidence interval of the 95% probability calculated for overall F statistics by the bootstrapping method This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) INTERNATIONAL JOURNAL OF PLANT SCIENCES 296 Fig UPGMA tree based on Nei’s genetic distance showing the relationships among Kandelia populations in Vietnam, Iriomote, and Borneo Discussion Genetic Differentiation of Kandelia in Vietnam The genetic analysis using microsatellite markers displayed a large divergence between the northern populations and the southern populations These loci are conserved across the populations in the north and the south A total of 50 alleles were detected in the four Vietnamese populations by using four microsatellite loci Forty of those 50 alleles were distributed in the northern populations, and 11 were in the southern populations Of these, one allele (Kc09-283) was found in both regions This allele was distributed restrictively in Dong Rui and Ngoc Hien, which are more than 1700 km apart, and had a very low frequency (0.02 in each population), indicating homoplasious occurrence of the allele The two northern Vietnamese populations shared a large number of frequent alleles (70% of common alleles), while the two southern Vietnamese populations shared 54.5% of the alleles Frequent alleles in Iriomote were disseminated in both of the northern populations, with some alleles occurring at high frequencies Five alleles found in Bako were all found in the southern Vietnamese populations Moreover, the UPGMA tree indicated that the two populations in northern Vietnam and Iriomote can be grouped together and differentiated from the other group in southern Vietnam and Bako (fig 3) Correspondingly, Kado et al (2004) examined 1526 bp of the matK gene region in the chloroplast DNA (cpDNA) in 15 individuals from Don Rui and in 15 and 10 individuals from Can Gio and Ngoc Hien, respectively, and they found clear genetic differentiation between the two regions Cang Gio and Ngoc Hien populations were fixed in the same haplotype, while four haplotypes appeared in Don Rui, none of which were found in Can Gio and Ngoc Hien Furthermore, there are 12 nucleotide substitutions and three indels between haplotypes in the northern and southern regions Interestingly, the haplotype in cpDNA on Okinawa Island (Ryukyus) was identical to the most frequent haplotype of Dong Rui (Kado et al 2004) The nucleotide diversity between the two regions was 0.0046 (Kado et al 2004), and this is comparable to that found between species in the matK gene in Fagopyrum (Ohsako and Ohnishi 2001) and Clintonia (Hayashi et al 2001) Accordingly, Chiang et al (2001) also found that there were two lineages corresponding to the geographically isolated populations, using chloroplast DNA and mitochondrial DNA of Kandelia They showed that a northern Vietnamese population (Quang Ninh) was clustered with a population in the Ryukyus, while the population in Bako, Borneo, was clustered with a population in Ranong on the west side of the Malay Peninsula Moreover, the most pronounced difference was seen in the minimum spanning network constructed from the trnL-trnF spacer (Chiang et al 2001) Eight clades of the network were divided into two geographical groups with 34 mutational changes; the first group included populations in the northern South China Sea region, and the other consisted of the Sarawak (Bako) and Ranong populations Within the network, closely related chlorotypes were mostly linked by single mutations In Vietnam, Kandelia is distributed in the north and the south but is interrupted in the vast central area (Hong and San 1993) The central coast ranges from Lach Truong to Vung Tau Cape The seacoast is parallel to the Truong Son Range Most of the rivers here, unlike those in the coastal regions of the north and the south, originate in the Truong Son Range and are generally precipitous The geographical structure in the central coast prevents mangroves from developing large populations along the seashore, so that they often exist as small, patchy, discontinuous populations along the narrow strips of brackish water (Hong and San 1993) Although Kandelia vegetation was reported in this area by Hong and San (1993), we could not find Kandelia in our survey at some places in the central region (Canh Duong and Lang Co, near Hue) One of us (P N H.) found that Kandelia in the north varies in height, from small trees branching at ca m to a maximum of ca m Flowering time lasts from early June Table Linkage Disequilibrium between Microsatellite Loci Northern Vietnam Southern Vietnam Ryukyu Borneo Locus pair Dong Rui Xuan Thuy Can Gio Ngoc Hien Iriomote Bako Kc04-Kc34 Kc04-Kc09 Kc04-Kc11 Kc34-Kc09 Kc34-Kc11 Kc09-Kc11 1.000 0.490 1.000 0.841 0.175 1.000 0.843 0.033* 0.488 0.112 0.935 0.629 0.049* 1.000 0.224 1.000 0.821 1.000 0.220 0.090 0.239 1.000 0.392 0.082 0.054 0.467 0.003** 0.646 0.009** 1.000 1.000 1.000 1.000 1.000 1.000 1.000 à Significant departure from linkage equilibrium: P < 0:05 Significant departure from linkage equilibrium: P < 0:01 Ãà This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) GIANG ET AL.—GENETIC VARIATION IN TWO KANDELIA SPECIES to July, propagules appear on the trees from July on, and mature fruits remain until the following May Kandelia in the south, however, is taller, with trees branching from ca to 12 m Flowering time lasts from September to late November Propagules appear from November on, and fruits remain until the following September He also found that the leaves of Kandelia in the north are elliptic-obovate in shape and small in size, whereas the leaves in the south are usually elliptic-oblong and larger Other obvious morphological and anatomical differences in Kandelia between the two regions were also found in flowers, ovules, cotyledons, and seeds The new species, Kandelia obovata, is distributed in the northern South China Sea and East China Sea regions, from the Gulf of Tonkin to the Ryukyu Islands and southern Japan, while Kandelia candel is distributed from western India, Burma, and Thailand to the Malay Peninsula and northern Borneo (Sheue et al 2003) Sheue et al (2003) also reported that specimens collected in northern Vietnam at the Gulf of Tonkin were K obovata Considering the finding of Sheue et al (2003) together with the genetic and morphological differentiation between Kandelia in the northern and southern parts of Vietnam, it is obvious that there are two species of Kandelia in Vietnam, K obovata in the north and K candel in the south Genetic Diversity Expected heterozygosity (HE) or gene diversity (Nei 1973) over all loci (table 3) in the two northern Vietnamese populations was high (HE ¼ 0:785 in Dong Rui and 0.761 in Xuan Thuy, with an average of 0.773), whereas gene diversity was much lower in the two southern populations (HE ¼ 0:272 in Can Gio and 0.216 in Ngoc Hien, with an average of 0.244) This tendency was consistent with the allelic diversity observed in each population (table 3) Gene diversities in the northern populations (K obovata) were higher than those reported for Avicennia marina worldwide (HE ¼ 0:494; Maguire et al 2000) and in Vietnam (HE ¼ 0:322; Giang et al 2003) Coastline currents in the northern South China Sea and East China Sea are in a northerly direction in summer (Wang et al 1995) Corresponding to the maturation of propagules of K obovata at the Gulf of Tonkin in June to July, those populations with high genetic diversity are thought to be potential sources of genetic variation northward In contrast, the average value of genetic variation in the two southern populations was much lower than that in the two northern populations It is surprising that an even smaller level of genetic variation was found in Bako in Borneo, where there is a large estuary area with diverse mangrove species The low level of genetic variation in K candel in Vietnam and Borneo suggests that these populations experienced a severe bottleneck or were founded very recently In addition, great genetic differentiation occurred among the populations in southern Vietnam and Bako This indicates either that the effective population size is small or that gene flow is limited Since the most frequent alleles are common among these populations in all four microsatellite loci, it is thought that they are derived from a common ancestral population with limited genetic variation 297 Results of paleoceanographical studies have shown that regions in Southeast Asia were connected, resulting in the formation of Sundaland, when the sea level was lowered during glacial ages (Tjia 1980; Wang et al 1995) In the last glacial maximum (LGM), the sea level was lowered by 100–120 m, and the South China Sea closed and changed into a semienclosed basin, with Bashi Strait as its only water passage to the Pacific Ocean (Wang et al 1995) The relatively large genetic variation retained in K obovata indicates that this species remained in coastal areas of the South China Sea region as fairly large panmictic populations The winter sea surface temperature in the LGM was estimated to be not more than 23°C in the South China Sea region (Wang et al 1995) Since the sea surface temperature is critical for the survival of mangroves, K candel, which can adapt to a high temperature (Maxwell 1995), retreated to the south, and the remainder of the species was probably extinguished in the closed South China Sea during the LGM, while the main body of K candel was retained in the Indian Ocean rim (the Ranong population probably being one of them) This is plausible because the current sea surface temperature in winter in the southern part of Vietnam (the northern limit of K candel) is around 25°C (Wang et al 1995) Kandelia candel could have extended to Southeast Asian regions after the LGM, when regions were disconnected by the rise in sea level mediated by the ocean current through the Strait of Malacca to the South China Sea Greatly reduced genetic variation in K candel in Vietnam and Borneo could be explained by a founder effect Reproductive Systems In this study, all four populations of Kandelia in Vietnam showed significant departure from HWE because of heterozygosity deficits It is notable that the two northern populations showed higher outcrossing rates and lower levels of inbreeding (F ¼ 0:140 in Dong Rui, 0.282 in Xuan Thuy) These values are parallel to the value reported in K obovata populations in Hong Kong (Sun et al 1998) In contrast, the two southern populations, especially the southernmost population in Ngoc Hien, exhibited much higher levels of inbreeding (F ¼ 0:713) Actually, in Can Gio and Ngoc Hien, the habitats are restricted to small patches and separated from each other High levels of inbreeding may be due to the Wahlund principle (Hartl and Clark 1997) and/or geitonogamous pollination, as reported by Chen (2000) in Fuijan, China A high level of inbreeding also occurred in Iriomote A geomorphological survey on Iriomote Island showed that seismic uplift occurred at intervals of 1000–2000 yr, with huge earthquakes inducing fluctuation between destruction and regeneration of mangrove forests (Miyagi and Fujimoto 2003) The last seismic uplift occurred ca 1000 yr ago (Miyagi and Fujimoto 2003) This indicates that the population at Urauchi River was established very recently by colonization by a limited number of individuals from the surrounding area This explains the high inbreeding rate and the significant linkage disequilibrium detected among the microsatellite loci Conclusions We have demonstrated that two obviously different species, Kandelia obovata and Kandelia candel, exist in the This content downloaded from 155.198.030.043 on July 24, 2016 14:24:47 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c) 298 INTERNATIONAL JOURNAL OF PLANT SCIENCES north and the south in Vietnam, respectively With our limited sampling, it is not known whether these two species have a clear boundary or overlap in the central region, although the paleogeographical discussion indicates the latter Much smaller genetic variation has been indicated in the populations of K candel in the South China Sea region This indicates the vulnerability of this species to climate changes Comparative studies of genetic variation in K candel of this region with that of the Indian Ocean region are required to clarify the route of migration and to understand the adaptive process of this species in the South China Sea region after the last glacial age Acknowledgments We thank J J Kendawang and J Augustine for collecting samples of Kandelia in Bako, Sarawak, Malaysia This work was partly supported by grant-in-aid 13575001 from the Japan 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(http://www.journals.uchicago.edu/t -and- c) GIANG ET AL. GENETIC VARIATION IN TWO KANDELIA SPECIES 295 Table Genetic Variation of Six Populations of Mangrove Genus Kandelia over All Microsatellite Loci Population Sample... much genetic variation is maintained in populations of Kandelia in Vietnam, the Ryukyu Islands, and Borneo, and how they are related each other These findings should give us clues for understanding