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

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

MEC_1399.fm Page 2697 Wednesday, October 24, 2001 6:39 PM Molecular Ecology (2001) 10, 2697–2710 Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs Blackwell Science Ltd T Y C H I A N G , * Y C C H I A N G , † Y J C H E N , † C H C H O U , ‡ S H AVA N O N D , § T N H O N G ¶ and S H U A N G † *Department of Biology, Cheng-Kung University, Tainan 701, Taiwan, †Department of Biology, National Taiwan Normal University, Taipei, Taiwan 116, ‡Institute of Botany, Academia Sinica, Taipei 115, Taiwan, §Silvicultural Research Division, Royal Sorest Department, Bangkok, 10900, Thailand, ¶Centre Forest Natural Resources and Environmental Studies, Vietnam National University, Vietnam Abstract Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e northern South-China-Sea + East-ChinaSea region and southern South-China-Sea region (Sarawak) Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim No geographical subdivision was detected within the northern geographical region An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (ΦCT = 0.015, ΦSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region Instead, relative ages of alleles contributed to nonrandom chlorotype–mitotype associations and tree inconsistency Widespread distribution and random associations (χ2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration Keywords: cpDNA, Kandelia candel, locus association, migration, minimum spanning network, mtDNA, phylogeography, relative ages Received 15 May 2001; revision received August 2001; accepted August 2001 Introduction Kandelia candel, a monotypic genus of the Rhizophoraceae, is one of the major mangrove species in East Asia Correspondence : S Huang biofv057@scc.ntnu.edu.tw © 2001 Blackwell Science Ltd Fax: + 886-2-29312904; E-mail: (Tomlinson 1986; Mabberley 1997) Mangrove forests are characteristic of tropical and subtropical coastlines of the world Over the last decades, due to human’s overexploitation, the genetic diversity in mangroves has been deprived, especially from coastal ecosystems in Asia Many countries have categorized the sustainable management of mangroves as major priorities in biodiversity MEC_1399.fm Page 2698 Wednesday, October 24, 2001 6:39 PM 2698 T Y C H I A N G E T A L conservation (Maguire et al 2000) According to biogeographical evidence, the genus Kandelia underwent a severe extinction phase during the upper Tertiary after the closure of the Tethys Seaway (Schwarzbach & Ricklefs 1998) Since then populations of K candel have been restricted to the tropical Malesia and East Asia, including areas around the South China Sea and the East China Sea Like many other mangrove species, K candel is characterized by vivipary, the precocious growth of the progeny when still attached to the maternal parent Vivipary is an adaptive feature for mangrove plants to colonize and expand populations at intertidal estuary habitats Precocious seedlings of K candel, growing up to 47 cm long and 1.3 cm wide, are buoyant, which may allow long-range dispersal via ocean currents (Lugo & Snedaker 1974; Maxwell 1995) However, viviparous seedlings have also been considered as a hindrance to long-distance dispersal due to the lack of protection and nutritional support from the maternal tissue (Duke 1995; Elmqvist & Cox 1996) Correspondingly, extremely various population structures have been discovered in different mangrove species High level of genetic differentiation among populations as well as geographical subdivision, due to restricted gene flow across populations, was lately detected in Avicennia marina (Avicenniaceae) (Duke 1995; Maguire et al 2000), a result close to the population structure of a nonviviparous species, Acanthus ilicifolius (Lakshmi et al 1997) In contrast, Australian populations of Rhizophora stylosa were found barely differentiated (Goodall & Stoddart 1989) Recent allozyme investigations revealed low level of genetic differentiation (GST = 0.064, Sun et al 1998) among Hong Kong populations as well as between two populations from Taiwan (FST = 0.04, Huang 1994), both indicating that the seedling dispersal in K candel was not as limited as previously suggested Although frequent gene flow has been detected in K candel at the local scale, long-range dispersal across oceans remains unknown Apparently, the dispersal extent of K candel is not only regulated by the orientation of ocean currents in the South China Sea and East China Sea, but also constrained by the duration and survivorship of viviparous seedlings in the high saline conditions The isolationby-distance model is thus a hypothesis to be tested In addition, when geographical distance increases for investigation, effects of vicariance will become more prominent and may confound the isolation-by-distance model (Bossart & Prowell 1998) Geological history inevitably plays another critical role in determining the phylogeographical pattern For example, geographically close populations along western and eastern coasts of the Peninsula Malaysia were significantly differentiated due to a vicariance event of approximately 60 – 220 million years before present (Yamazaki 1998), which led to the geographical subdivision of Bruguiera gymnorrhiza in Asia According to palaeoceanographic evidence, due to latitude and temperature differences, southern and northern banks of the South China Sea, where K candel is distributed, went through different geological histories (Wang et al 1995) over past glacial cycles Furthermore, populations of the Ryukyu islands and northern Taiwan (Taipei) along coasts of the East China Sea shared a unique geological history from those of the South China Sea (Ota 1998; Chou et al 2000) In light of geological histories, genetic differentiation of K candel among the above three geographical regions would be expected As generally known, in estimating population structure and gene flow, some level of variance of loci is required Molecular markers with low resolution usually are incapable of providing information (Bossart & Prowell 1998) in distinguishing coancestry from migration (Schaal et al 1998) For surveying population structure within a small geographical scale, e.g K candel in Hong Kong (Sun et al 1998) and Taiwan (Huang 1994), allozymes due to their conserved nature (cf Bossart & Prowell 1998) might not be able to adequately estimate gene flow among local populations In addition, the biparental inheritance of allozymes makes the estimations of seedling dispersal difficult, as the effects of pollen dispersal between neighbouring populations cannot be ruled out Recently, many noncoding spacers of organelle DNAs have been widely applied to phylogeographical studies (Schaal et al 1998) With merits of maternal inheritance in most angiosperms (Harris & Ingram 1991), including Kandelia (Chen 2000), and nearly neutral and fast evolution, these markers are likely to be able to provide information in estimating the extent of long-range seedling dispersal and reconstructing phylogeographical patterns In this study, we sequenced two noncoding spacers, i.e atpB-rbcL and trnL-trnF of chloroplast DNA (cpDNA), and the internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA) and used them as markers to estimate the phylogeographical pattern as well as population and geographical structure of K candel As physically linked loci in the chloroplast genome, atpB-rbcL and trnL-trnF should reveal comparable phylogenies Likewise, as being maternally inherited, chloroplasts and mitochondria were thought to remain associated and behave as if they are completely linked (Schnabel & Asmussen 1989) Consistent phylogenetic patterns of cpDNA and mtDNA are thus expected (cf Dumolin-Lapègue et al 1998) However, evolutionary forces, such as lineage sorting effects (Hoelzer et al 1998), and frequent recurrent mutations (Desplanque et al 2000), can result in systematic inconsistency and thus lead to wide variance of FST values among loci and a weak correlation between FST and number of migrants per generation (Nm) as well (cf Bossart & Prowell 1998) Under such circumstances, difficulties in © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2699 Wednesday, October 24, 2001 6:39 PM P H Y L O G E O G R A P H Y O F K A N D E L I A 2699 interpreting phylogeography and population structure will be inevitably encountered In other words, when discrepant estimates are obtained from two or more loci, these estimates are not necessarily indicative of gene flow Explicit analysis of associations between alleles of different loci (Desplanque et al 2000) coupled with nested clade analysis (cf Schaal et al 1998; Templeton 1998; Chiang 2000) will be required to clarify historical and recurrent events K candel, as widespread in East Asia, is a biological model that is suited for testing the association between vicariance and geographical structure; effects of ocean currents in long-distance seedling dispersal; and the usefulness of cpDNA and mtDNA as population genetic markers, as well as allele associations Several aims are pursued in this study: (i) to test the possibility and level of long-distance seedling dispersal by estimating population structure and gene flow; (ii) reconstruct the phylogeographical pattern and examine the association between geological/geographical events and the extent of genetic differentiation among three geographical regions; (iii) to examine the phylogenetic consistency between atpB-rbcL and trnL-trnF noncoding spacers of cpDNA as well as between chloroplast and mitochondrial genomes; and (iv) to investigate associations between alleles of cp- and mtDNAs and to deduce the relative age of their alleles based on spanning networks Materials and methods Sample collection Kandelia candel is a mangrove species that is widespread in eastern coasts of mainland Asia and continental islands, including Taiwan, and the Ryukyu (Fig 1) One hundred and eighty-seven samples were collected from 13 major populations in East Asia, ranging from Bako (Sarawak, 01°40′ N) to Yakushima Islet ( Japan, 30°20′ N) (Table 1, Fig 1) Ten to 15 individuals, which were approximately 70–100 m apart, were sampled from each population In addition, 15 individuals of a population at Ranong Fig Kandelia candel sample locations and distribution Frequency of cytotypes (cpDNA trnL-trnF–mtDNA ITS associations) in each population is indicated in pie diagrams Abbreviations of populations are given in Table © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2700 Wednesday, October 24, 2001 6:39 PM 2700 T Y C H I A N G E T A L Table Materials of Kandelia candel collected from different populations used for organelle DNA sequencing Location, locality, sample size, and cytotypes (trnL-trnF spacer chlorotype and mtITS mitotype associations) of each population are indicated Localities Area Sampling size (n) Symbol Coordinate CC HA HK QN 21°34′ N, 108°37′ E 19°54′ N, 110°20′ E 22°32′ N, 114°05′ E 20°53′ N, 106°46′ E 15 Guangxi, China Taiwan Fukien, China Guangxi, China SK TN XM ZJ 21°28′ N, 109°43′ E 22°59′ N, 120°12′ E 24°26′ N, 118°04′ E 21°04′ N, 110°09′ E 10 15 15 15 Ryukyu, Japan AM 28°15′ N, 130°40′ E 15 Ryukyu, Japan Taiwan Ryukyu, Japan IR TP YK 24°19′ N, 123°54′ E 25°09′ N, 120°16′ E 30°20′ N, 130°30′ E 15 14 15 IB (6), IIB (3), IIIB (2), IIIC (1), IVB (2), IVC (1) IA (2), IB (4), IIB (3), IVB (6) IB (9), IIB (3), IIIB (2) IA (1), IB (8), IC (2), IIB (2), IVB (2) Southern Region Southern South-China-Sea Region Bako Sarawak BK 01°40′ N, 110°25′ E 15 VIIbF Indian Ocean Rim (outgroup) Ranong Thailand RN 09°55′ N, 98°30′ E 15 IIaF (13), VIIaG (2) Northern Region Northern South-China-Sea Region: Chinchou Guangxi, China Haikou Hainan, China Hong Kong China Quang Ninh Vietnam Shankou Tainan Xiamen Zhangjiang East-China-Sea Region: Amami-O-Shima Islet Irimote Islet Taipei Yakushima Islet (Thailand) of the western coast of Peninsula Malaysia were included in the analysis as outgroups In total, this study encompasses 14 populations (202 individuals) No materials were collected from the Philippines, since no natural populations are distributed in this area (Hou 1958) In addition, during the last decade, most populations of Vietnam have been removed for the use of inshore fisheries (Huang & Chen 2000) Only one population at Quang Ninh was available Besides, samples of six populations (from Chinchou to Xiamen) of China, one population from Sarawak, two populations of Taiwan, and three populations from the Ryukyu Islands were included in this investigation Based on the orientation of ocean currents (Huang et al 1997), three geographical regions are recognized: southern South-China-Sea (S region, including a single population of Sarawak), northern South-China-Sea (N region, including eight populations of Vietnam, China, and Tainan), and the East-China-Sea (E region, including three populations of Ryukyu and Taipei) regions Three populations along the Tonkin Bay, i.e Chinchou, Shankou, and Quang Ninh, are further grouped as the Ns region, while others of the N region as the Nn region Young and healthy shoots were collected in the field, rinsed with tap water and dried in silica gel All samples were stored at –70 °C until they were processed 15 15 Cytotypes IB (12), IC (1), IIB (1), IIIB (1) IB (6), IIB (3), IIIB (3), VB (3) 13 IB (9), IVB (2), VB (2) IIB (2), IIE (1), IVB (2), IVE (1), VB (3), VIB (4), VIC (1), VIE (1) IB (1), IIB (1), IID (1), IIIB (6), IIID (1) IB (13), IC (1), IIIC (1) IB (2), IIB (9), IIC (1), IVB (3) IIIB (7), IVB (1), IVC (1), VIB (6) DNA extraction and polymerase chain reaction Leaf tissue or embryo of the above materials was ground to powder in liquid nitrogen and stored in a –70 °C freezer Genomic DNA was extracted from the powdered tissue following the CTAB procedure (Murray & Thompson 1980) Noncoding spacers of atpB-rbcL and trnL-trnF of the cpDNA and the ribosomal DNA (rDNA) ITS of mtDNA were amplified and sequenced Universal primers for amplifying atpB-rbcL spacer (Chiang et al 1998), trnL-trnF spacer (Taberlet et al 1991), and mtDNA rITS (Chao et al 1984) were synthesized The polymerase chain reaction (PCR) amplification was carried out in a volume of 100 µL reaction using 10 ng of template DNA, 10 àL of 10ì reaction buffer, 10 àL MgCl2 (25 mm), 10 µL dNTP mix (8 mm), 10 pmole of each primer, 10 µL of 10% NP-40, and U of Taq polymerase (Promega, Madison, USA) The reaction was programmed on a MJ Thermal Cycler (PTC 100) as one cycle of denaturation at 95 °C for min, 30 cycles of 45 s denaturation at 92 °C, 15 s annealing at 52 °C, and 30 s extension at 72 °C, followed by 10 extension at 72 °C Template DNA was denatured with reaction buffer, MgCl2, NP-40 and ddH2O for mins (first cycle), and cooled on ice immediately A pair of primers, dNTP and Taq polymerase were added to the above ice-cold mix Reaction was restarted at the first annealing at 52 °C © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2701 Wednesday, October 24, 2001 6:39 PM P H Y L O G E O G R A P H Y O F K A N D E L I A 2701 T-A cloning and nucleotide sequencing PCR products were purified by electrophoresis on a 1.0% agarose gel using 1× TAE buffer The gel was stained with ethidium bromide and the desired DNA band was cut and eluted using agarose gel purification (QIAGEN) Purified DNA was ligated to a pGEM-T easy vector (Promega) Plasmid DNA was selected randomly with five clones and purified using plasmid mini kits (QIAGEN) Purified plasmid DNA was sequenced in both directions by standard methods of the Taq dyedeoxyterminator cycle sequencing kit (Perkin Elmer) on an Applied Biosystems Model 377A automated sequencer Primers for sequence determination were T7-promoter and SP6-promoter located on p-GEM-T easy vector termination site Sequence alignments and phylogenetic analyses Nucleotide sequences were aligned with the program Genetics Computer Group (gcg) Wisconsin Package (Version 10.0, Madison, Wisconsin) Neighbour-joining (NJ) analysis by calculating Kimura 2-parameter distance (Kimura 1980) was also performed using Data Analysis in Molecular Biology and Evolution (dambe, version 3.5.19, Xia 1999) Indels were treated as the fifth character Confidence of the clades reconstructed was tested by bootstrapping (Felsenstein 1985) with 1000 replicates using unweighted characters The nodes with bootstrap values greater than 0.70, as a rule of thumb, are significantly supported with ≥ 95% probability (Hillis & Bull 1993) The number of mutations between DNA genotypes in pairwise comparisons, which were calculated using mega (Kumar et al 1993), was used to construct a minimum spanning network with the aid of minspnet (Excoffier & Smouse 1994) in an hierarchical manner (cf Chiang & Schaal 1999) After linking the related haplotypes into a clade, closely related clades were linked further to form higher level groups and thereby a network Population genetic analysis of the cpDNA and mtDNA sequence variation Levels of genetic diversity within populations were quantified by estimates of nucleotide divergence (θ) (Watterson 1975) using dnasp (Version 3.14, Rozas & Rozas 1999) Patterns of geographical subdivision and gene flow were also estimated hierarchically with the aid of dnasp Gene flow within and among regions (populations) was approximated as Nm, the number of female migrants per generation between populations, and was estimated using the expression FST = 1/(1 + Nm) where N is the female elective population size and m is the female migration rate (Slatkin 1993) We also used amova version 1.55 (Excoffier et al 1992; Excoffier 1993) to deduce the © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 significance of geographical divisions both among regions and populations The statistics of molecular variants ΦCT (among regions), ΦST (among populations), and ΦSC (among populations within regions), were estimated Results Extent of nucleotide and haplotype diversity In this study, trnL-trnF and atpB-rbcL noncoding spacers of cpDNA and the rITS of mtDNA in Kandelia candel were PCR amplified and sequenced All sequences were registered with EMBL accession numbers AJ305472 –AJ305673 (for mtDNA ITS), AJ305674–AJ305875 (for trnL-trnF spacer), and AJ305876–AJ306077 (for atpB-rbcL spacer) At all three loci, no within-individual variation was detected Differences between mitochondrial sequences of a consensus length of 725 bp were mainly ascribed to point mutations (18 sites, 2.4%) Four indel events also occurred at sites (353–357) (592–599) (632–635) and 640 For the atpB-rbcL spacer of the chloroplast genome, only two sites of 781 bp (0.3%) were polymorphic Populations of BK and RN shared a C at site 160, while others have a T A 1-bp deletion at site 577 occurred in the RN population At the trnL-trnF spacer of cpDNA, high levels of length polymorphism, ranging from 375 bp to 415 bp, were detected Two deletions, at sites (17–28) and (242–268) made the spacer shorter in populations of BK and RN Most indels (37) occurring in the noncoding spacer involved a 1-bp loss Like most noncoding regions (cf Li 1997), A + T contents were high, with 53.7%, 72.5%, and 76.9% at mtITS, atpBrbcL spacer, and trnL-trnF spacer, respectively Three haplotypes of the atpB-rbcL noncoding spacer (h = 0.268 0.0015, θ = 0.00051 0.00008) were determined In contrast to other studies (Small et al 1998; Fujii et al 1999), the atpB-rbcL noncoding spacer possessed a much lower level of genetic variation than the trnL-trnF spacer in K candel (Table 2) In total, 28 haplotypes of the trnL-trnF spacer of cpDNA and seven haplotypes of the mtDNA ITS were determined Apparently, the molecular evolution of the mtDNA ITS was much more constrained compared to that of the trnL-trnF spacer of cpDNA (Table 2) At the population level, except for the lack of genetic variation at the cpDNA atpB-rbcL spacer, the level of genetic variation varied among populations at two other organelle loci (Table 2) High levels of nucleotide variation at both loci, with θ-values ranging from 0.0018 to 0.0062 at trnLtrnF spacer and from 0.0020 to 0.0029 at mtITS, occurred in populations of IR, QN, and YK, while low cpDNA variation, ranging from 0.0003 to 0.0013, was detected in populations of TN, CC, and HK Stark contrast of the level of genetic variation between the two loci occurred in populations of BK, HA, TP, and XM (Table 2) MEC_1399.fm Page 2702 Wednesday, October 24, 2001 6:39 PM 2702 T Y C H I A N G E T A L cpDNA Table Haplotype diversity (h) and nucleotide diversity (θ) of the cpDNA trnLtrnF spacer and the mtDNA ITS within populations of Kandelia candel mtDNA Populations h θ h θ Total Amami-O-Shima Bako Chinchou Haikou Hong Kong Irimote Quang Ninh Ranong Shankou Tainan Taipei Xiamen Yakushima Zhangjiang 0.908 0.857 0.857 0.257 0.771 0.513 0.686 0.771 0.600 0.778 0.133 0.560 0.733 0.457 0.848 0.02652 ± 0.00526 0.00623 ± 0.00044 0.00615 ± 0.00092 0.00125 ± 0.00005 0.00329 ± 0.00003 0.00135 ± 0.00044 0.00310 ± 0.00164 0.00492 ± 0.00071 0.00246 ± 0.00004 0.00248 ± 0.00001 0.00034 ± 0.00018 0.00304 ± 0.00082 0.00278 ± 0.00049 0.00178 ± 0.00066 0.00457 ± 0.00040 0.406 0.248 0.000 0.133 0.000 0.000 0.248 0.448 0.248 0.467 0.248 0.000 0.133 0.362 0.133 0.00205 ± 0.00026 0.00034 ± 0.00018 0.00000 0.00018 ± 0.00015 0.00000 0.00000 0.00292 ± 0.00060 0.00255 ± 0.00034 0.00034 ± 0.00018 0.00064 ± 0.00018 0.00034 ± 0.00018 0.00000 0.00018 ± 0.00015 0.00200 ± 0.00139 0.00018 ± 0.00015 rn bk N+E atpB-rbcL network Fig Minimum spanning network generated using method of Excoffier & Smouse (1994) for haplotypes of atpB-rbcL spacer of cpDNA of populations of Kandelia candel Each arrow indicates one mutational change ‘0’ indicates hypothetical ancestor Gene genealogies and associations between cpDNA and mtDNA lineages In this study we reconstructed the phylogeographical pattern of K candel based on gene genealogies of organelle loci A minimum spanning network of the cpDNA atpBrbcL spacer was reconstructed based on mutational changes between haplotypes (Fig 2) The BK population is closely related to the RN population, while no variation was detected among populations of N and E regions An NJ tree was recovered based on the nucleotide sequence variation of the trnL-trnF noncoding spacer of cpDNA Eight clades (chlorotypes) of 28 haplotypes were identified in this cpDNA gene tree (Fig 3) Two major lineages of S + RN and N + E were recognized and significantly supported, with a bootstrap of 0.98 (P < 0.01) Four common chlorotypes I–IV of 152 sequences in total (75.2%) were widespread in populations of N and E regions, while types of VIIa and VIIb of 30 sequences were restricted to the S region and RN population (Table 3) Two rare alleles of V (4.0%) and VI (5.9%) were distributed in the N region only A minimum spanning network of the trnL-trnF noncoding spacer was constructed (Fig 4) Eight clades of the network, corresponding to those of the NJ tree, were divided into two geographical groups (i.e S + RN and N + E) 34 mutations apart Within the network, closely related chlorotypes were mostly linked by single mutations Chlorotypes I, II and III were nested in the network as interior nodes, while types IV and V connecting to type I, and type VI connecting to III or IV were exterior In the NJ tree of mtDNA ITS sequences, two major clades (A–E) and (F, G), of seven variants (mitotypes) were identified (Fig 5A) Two common mitotypes B and C of 164 sequences (81.2%) were widespread in populations of N and E regions (Table 3), as mitotypes of F and G were distributed in the S region and RN population Three rare alleles were distributed restrictedly: types A (1.5%) in YK and IR populations, D (1.0%) in SK, and E (1.5%) in QN A minimum spanning network of the mtDNA ITS was constructed (Fig 5B) Within the clade of N + E regions, mitotype B was nested in the network as the interior node, connecting to other types independently with – mutations Within the clade of S + RN regions, mitotype G was linked to the interior node of type F with two mutations, which was linked to type B with four mutations Phylogenies of cpDNA and mtDNA are completely consistent at the level of geographical regions (i.e S + RN vs N + E) The atpB-rbcL noncoding spacer provided no information in resolving the phylogeny within N + E regions Apparently, the gene tree of the trnL-trnF spacer of cpDNA largely contradicted the mtDNA ITS tree No clade correspondence was found between the two trees For example, although most chlorotype III sequences (87.5%) corresponded to the mitotype B, sequences of am4 and tn12 were associated with mitotype C and sequence sk2 was associated with the mitotype D © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2703 Wednesday, October 24, 2001 6:39 PM P H Y L O G E O G R A P H Y O F K A N D E L I A 2703 Fig Neighbour-joining tree of representative sequences (haplotypes) of trnL-trnF of cpDNA in Kandelia candel Numbers at nodes indicate bootstrap values Chlorotypes (I–VIIb) are labelled on clades Table Distribution of chlorotypes (I–VIIb) and mitotypes (A–G) among populations of Kandelia candel Regions are indicated: Indian Ocean Rim (I), southern South-China-Sea region (S), northern South-China-Sea region (N), and East-China-Sea region (E) Regions: I II III IV V VI VIIa VIIb A B C D E F G I S N E rn bk qn cc sk 13 1 3 zj xm hk tn 10 3 14 6 14 13 ir 2 15 13 am yk 3 11 2 15 15 11 14 14 14 13 15 Nevertheless, associations between cpDNA and mtDNA haplotypes were nonrandom Seventeen (instead of 30; χ2 = 1.03, P = 0.00018) and three (instead of four; χ2 = 0.044, P = 0.0001) cpDNA (trnL-trnF)-mtDNA associated © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 13 13 12 Total 77 (38.2%) 30 (14.9%) 24 (11.9%) 21 (10.3%) (4.0%) 12 (5.9%) 15 (7.4%) 15 (7.4%) (1.5%) 154 (76.2%) 10 (5.0%) (1.0%) (1.5%) 28 (13.9%) (1.0%) cytotypes were observed in N + E and S + RN regions of K candel, respectively (Table 4) All sequences of the mitotype A were exclusively associated with the chlorotype I; and sequences of the mitotype D were associated with the MEC_1399.fm Page 2704 Wednesday, October 24, 2001 6:39 PM 2704 T Y C H I A N G E T A L Fig Minimum spanning network generated using method of Excoffier & Smouse (1994) for haplotypes of trnL-trnF spacer of cpDNA of populations of Kandelia candel Each arrow indicates one mutational change Number of mutational change is indicated when more than one step ‘0’ indicates hypothetical ancestor The replicate number of haplotypes is also indicated when more than one Fig (A) Neighbour-joining tree of haplotypes (A –G) of rITS of mtDNA in Kandelia candel Numbers at nodes indicate bootstrap values ( B) Minimum spanning network generated using method of Excoffier & Smouse (1994) for haplotypes of mtDNA ITS of populations of K candel Mutational changes are indicated at nodes © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2705 Wednesday, October 24, 2001 6:39 PM P H Y L O G E O G R A P H Y O F K A N D E L I A 2705 Table Associations between chlorotypes and mitotypes of Kandelia candel Distribution range of each chlorotype and mitotype is indicated in square brackets Percentage of each cytotype is indicated in parentheses W: widespread Other symbols see Table chlorotype: mitotype: I [W] A [ir + yk] B [W] C [W] (1.5%) 70 (34.6%) 27 (13.4%) 21 (10.4%) 18 (8.9%) (4.0%) 10 (5.0%) (2.0%) (0.5%) (1.0%) (1.0%) II [W] III [W] IV [W] V [qn, hk, ha] VI [qn, zj] D [sk] E [qn] (0.5%) (0.5%) 154 Total (0.5%) 30 24 (0.5%) 21 (0.5%) (0.5%) VIIb [bk] G [bk] 77 VIIa [rn] Total F [rn + bk] 10 12 13 (6.4%) 15 (7.4%) 28 (1.0%) 15 15 202 Table Pairwise FST/ Nm estimates between geographical regions (E, N, Ns and Nn) based on genetic variation of mtDNA ITS (above the diagonal) and cpDNA trnL-trnF spacer (below the diagonal) Nucleotide diversity within each region is indicated in the parenthesis cpDNA E (θ = 0.00503 ± 0.00054) N (θ = 0.00445 ± 0.00033) Ns (θ = 0.00312 ± 0.00037) Nn (θ = 0.00492 ± 0.00041) mtDNA: E (θ = 0.00138 ± 0.00048) N (θ = 0.00049 ± 0.00007) Ns (θ = 0.00120 ± 0.00049) Nn (θ = 0.00135 ± 0.00007) — 0.020/23.95 0.023/21.50 0.021/ 23.14 0.026/18.41 — — — 0.023/21.09 — — 0.011/45.67 0.031/15.41 — 0.013/39.00 — chlorotypes II and III Likewise, the chlorotype V was exclusively associated with the most dominant mitotype B, and most sequences of chlorotype I were associated with the mitotype B, while some other sequences were mitotypes A or C Within the N + E region, cytotypes BI (40.7%) and BII (15.7%) were most dominant in composition In contrast, cytotypes of CII, DII, EII, CIII, DIII, CIV, EIV, CVI, and EVI were relatively rare (5.8% in total) Likewise, within the S + RN region, FVIIa and FVIIb were dominant (93.3%), while the cytotype GVIIa was rare (6.7%) The cytotype composition varied among populations In Fig the genetic composition in each population was indicated Higher number of cytotypes occurred in populations AM (six types), QN (eight types), and YK (five types), while a single type was detected in the BK population and two types were detected in the RN population (Table 1) Within the N + E regions, populations possessing a higher © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 number of cytotypes appeared to be located at margins (Fig 1) In contrast, most geographically ‘central’ populations had three or four cytotypes Population differentiation and geographical divisions Population and geographical structure of K candel was assessed based on genetic variation of the organelle loci Estimates of FST (= 0.93–0.95) and Nm (= 0.03 – 0.04) based on the cpDNA trnL-trnF spacer and mtDNA ITS, indicated significant differentiation between regions S + RN, and N + E In contrast to the consistent estimations of population structure between the above two loci, higher number of migrants (Nm = 0.10) per generation was deduced from atpB-rbcL spacer sequences, although the genetic differentiation was still significant (FST = 0.828) Hierarchical analyses of sequence difference under amova MEC_1399.fm Page 2706 Wednesday, October 24, 2001 6:39 PM 2706 T Y C H I A N G E T A L indicated that the proportion of molecular variance was attributed to difference between geographical regions (ΦCT = 0.860, P < 0.001) The relative contribution of difference among populations to molecular variance was small (ΦST = 0.087) In contrast, no geographical subdivision (FST = 0.020 – 0.026 and Nm = 18.41–23.95) between N and E was detected (Table 5) An analysis of molecular variance (amova) based on the trnL-trnF spacer of cpDNA also suggested low levels of genetic differentiation between populations of mainland and continental islands (ΦCT = 0.015) as well as among populations within each region (ΦSC = 0.037) Genetic variation of the mtDNA ITS yielded a similar pattern of the genetic apportionment among geographical regions and populations The deduced Nm of 39.00 – 45.67 indicated frequent gene flow between Ns and Nn regions (Table 5) In contrast to the invariable atpB-rbcL spacer, pairwise comparisons showed high variances in genetic estimates of structure and genetic differentiation between cpDNA and mtDNA loci, except for those between BK + RN and other populations Low level of genetic differentiation was usually detected among populations within the E + N regions Nearly all Nm values, ranging from 1.75 (between SK and HK) to 622.55 (between CC and YK), deduced from the mtDNA ITS were greater than those from the cpDNA trnLtrnF spacer Lower Nm values, less than one, were mostly restricted to comparisons with ZJ as well as SK based on cpDNA variation Some extremely high Nm values were obtained, such as 150 between AM and CC High variance in deducing FST and Nm was also encountered in comparisons between BK and RN Based on the trnL-trnF spacer, an FST value of 0.42 and an Nm of 0.68 were deduced, while a lower level of genetic differentiation (FST = 0.13, Nm = 3.50) was detected from the mtITS Discussion Genetic variability and low level of homoplasious mutations in cpDNAs and mtDNAs of Kandelia candel The usefulness of molecular markers in indirect estimates of population structure and gene flow depends on the level of resolution, and locus-to-locus consistency (Bossart & Prowell 1998), and is also affected by the level of recurrent mutations (cf Desplanque et al 2000) Recurrent mutations (identity by state), which are frequently encountered in the mitochondrial genome due to limited conformations in molecular structure (Fauron et al 1995), will inevitably blur the level of migration between populations Technically, nucleotide sequencing can simply rule out the length homoplasies, which occur usually in restriction fragment length polymorphism (RFLP) and PCR-based fingerprints (cf Parker et al 1998; Desplanque et al 2000), from the data scoring In this study, as a very strong linkage disequilibrium was estimated between mitotypes and chlorotypes both within N + E and S + RN regions (Table 4), a high rate of recurrent mutations can be excluded for organelle genomes of K candel (cf Desplanque et al 2000) In this study, genetic variation of mtDNA ITS and cpDNA trnL-trnF spacer existed both within and between populations Nevertheless, the haplotype diversity of the mitochondrial DNA, with seven haplotypes out of 202 plants screened, was lower compared to other flowering plants, such as Beta vulgaris ssp maritima (20 mitotypes from 414 individuals; Desplanque et al 2000), Daucus carota ssp carota (25 variants based on mtDNA RFLP from 80 plants; Ronfort et al 1995), Thymus vulgaris (50 mitotypes from about 400 plants; Manicacci et al 1996), and Hevea brasiliensis (212 mtDNA RFLP variants in 395 accessions screened; Luo et al 1995) For the chloroplast genome, K candel possessed a higher level of haplotype diversity (28 haplotypes) at the trnL-trnF spacer, which is close to 13 cpDNA haplotypes in Beta vulgaris ssp maritima (Desplanque et al 2000), 11 haplotypes in Argania (El Mousadik & Petit 1996), 23 haplotypes in white oaks (Dumolin-Lapègue et al 1997), and 13 haplotypes in Alnus (King & Ferris 1998) Although comparisons of haplotype diversity among taxa, which were examined using various molecular methods at different loci, may be somewhat misleading, nucleotide diversity of the trnLtrnF spacer in K candel (θ = 0.02710) appeared to be higher than that of other plants as well, such as Cunninghamia (θ = 0.01018, Lu et al 2001) and Begonia (θ = 0.003, Liu 1999) Apparently, both loci in this study provided sufficient resolution at the geographical region level and yielded consistent estimates of gene flow (Nm = 0.03 – 0.04) and population structure (FST = 0.93 – 0.95) between S + RN and N + E populations of K candel To the contrary, lack of variability, due to its conserved nature (cf Chiang & Schaal 2000a,b), has made the atpB-rbcL noncoding spacer locus powerless in estimating the interpopulation migration within the N + E regions At interregions level, as a result of having difficulties in distinguishing coancestry from migration, higher Nm value (= 0.10) between S + RN and N + E regions was thereby deduced based on this spacer Likewise, at the population level, the more conserved mtDNA ITS always yielded higher values of Nm and lower levels of FST, than the cpDNA trnL-trnF spacer In this investigation, due to its higher resolution, the trnL-trnF might have higher probabilities of yielding estimates that approximate the current population structure of K candel Phylogeographical patterns of K candel in East Asia In this study, we investigated the phylogeography of the viviparous species, K candel Both the mtDNA ITS and the cpDNA trnL-trnF spacer suggested noticeable longdistance seedling dispersal However, as extensive gene © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 MEC_1399.fm Page 2707 Wednesday, October 24, 2001 6:39 PM P H Y L O G E O G R A P H Y O F K A N D E L I A 2707 flow across oceans via seedlings was detected among populations along a 2700 km transect (between QN and YK) in the regions of northern South-China-Sea and EastChina-Sea, gene genealogies of three organelle loci revealed consistent geographical structure Accordingly, the population of Sarawak along the bank of the South China Sea in East Asia, which is about 2000 km from the QN (Fig 1), was phylogenetically grouped with the Ranong population of the Peninsula Malaysia at the northeastern rim of the Indian Ocean This unique geographical structure, consistently suggested by organelle loci (Figs 2, and 5) as well as allozyme data (Huang & Chen 2000), not only indicated the origin of the Sarawak population derived from the Indian Ocean Rim, but also reflected a phylogeographical pattern associated with the vicariance history According to palaeoceanographic evidence, during the last glacial maximum of the Pleistocene, the global sea level dropped by some 100 –120 m Meanwhile, with the closure of all the southern connections to the ocean, the South China Sea nearly became an enclosed basin, with the Bashi Strait as its only water pathway to the Pacific Ocean (Wang et al 1995), and was completely isolated from the Indian Ocean Sarawak, although geographically located along the southern coast of the modern South China Sea, has long been the northern edge of the Sunda shelf since the late Quaternary According to another estimation of Yamazaki (1998), populations of the South China Sea and the East China Sea may have been isolated from those of the Bay of Bengal of the Indian Ocean for at least 0.6 – 2.2 million years, a duration sufficient for coalescence at most loci Apparently, monophyly of F + G alleles of the mtDNA and VIIa + VIIb alleles of the cpDNA supported the long isolation hypothesis In addition, based on the paucity of genetic variation at two organelle loci and the phylogenetic affinity to the RN population, a small number of founders through long-distance dispersal from populations of the Indian Ocean may have been involved in the colonization of the BK population Nevertheless, the level of ongoing gene flow between BK and RN appeared to be low, according to the deduced Nm of 0.00– 0.68 from the atpB-rbcL and trnL-trnF, respectively, a result agreeing with the orientation of summer ocean currents According to the association between unique genetic structure and vicariance events, the Sarawak population must also have been shaped by limited recurrent gene flow to other populations of the South China Sea, which in turn is constrained by seasonal orientations of current flows Sea surface circulation in the modern South China Sea is basically driven by the monsoon in East Asia In summer, the season during which most seedlings are detached from maternal plants of K candel and disperse (Tomlinson 1986; Huang & Chen 2000), surface water of the tropical Indian Ocean flows northward into the South China Sea and then through the Bashi Strait (the strait between Taiwan and © 2001 Blackwell Science Ltd, Molecular Ecology, 10, 2697–2710 the Philippines) into the Pacific (cf Wang et al 1995) Because populations are not distributed in the Philippines (Hou 1958), somewhat indicating no suitable habitats for K candel, most seedlings of the Sarawak are likely to be discharged into oceans, having limited probability of colonizing territory of the northern South China Sea With limited gene flow with other populations, the Sarawak population has long maintained its own identity Despite the isolation between South-China-Sea and EastChina-Sea regions during the glacial maximum, subsequent ongoing gene flow via ocean currents has homogenized between-region and between-population variation in K candel In agreement with previous allozyme investigations (Huang 1994; Sun et al 1998; Huang & Chen 2000), both chloroplast (trnL-trnF spacer) and mtDNAs indicated no hindrance to the long-distance seedling dispersal between mainland and continental islands via ocean currents (Table 5) Although the isolation-by-distance model was not met at the population level, gene flow within region (i.e between Ns and Nn, Table 5) was apparently more frequent than between E and N regions In addition, according to the higher nucleotide diversity in the Nn populations at both organelle loci (Table 5), a preferential northward migration (from Ns to Nn) seemed to exist due to the orientation of ocean currents in summer Nevertheless, some local topographical barriers may have blocked the interpopulation gene flow According to the estimates based on the trnL-trnF spacer, limited gene flow occurred in populations ZJ and SK, both located on opposite coasts of the Leizhou Peninsula (Fig 1), to other populations In addition, although no general rule can be generated, Hong Kong seemed to have more frequent gene flow with neighbouring populations, including TN, TP, and IR, than with distant populations Relative ages of alleles of cpDNAs and mtDNAs In resolving phylogeographical pattern and phylogenetic ambiguities, the nested cladogram provides sufficient information, complementary to conventional cladograms (cf Crandall & Templeton 1993) Based on their interior positions in the minimum networks, the ancestry of eight dominant and widespread cytotypes was suggested: BI, BII, BIII, BIV, CI, CII, CIII, and CIV (Table 4), which would have a greater probability of producing mutational derivatives (cf Donnelly & Tavaré 1986; Golding 1987; Crandall & Templeton 1993) In contrast to the strong linkage disequilibrium among chlorotypes of I–VI and mitotypes of A–E, nearly random association (χ2 = 0.822, P = 0.189) was detected in these common cytotypes, indicating genetic equilibrium and panmixis within the N + E region as a whole The strong linkage disequilibrium may simply stem from effects of lineage sorting, due to relative ages of alleles MEC_1399.fm Page 2708 Wednesday, October 24, 2001 6:39 PM 2708 T Y C H I A N G E T A L of each locus (Chiang 2000) According to the exterior positions in the networks and their restricted spatial distribution, alleles V and VI of the cpDNA trnL-trnF spacer, and alleles of A, D, and E of the mtDNA may have evolved recently As generally known, newly evolved cpDNA alleles, since having a low frequency in the gene pool, were likely to be ‘attracted’ to dominant mitotypes (B and C, in this study), and vice versa Probabilities of associations between two rare alleles would be extremely low, thereafter leading to strong linkage disequilibrium Interestingly, almost all rare alleles occurred at marginal populations of the E + N regions, such as allele A restricted in IR and YK (Ryukyu), D exclusively in SK, and E in QN only Technically, the absence of these rare alleles in ‘central’ populations may be simply because of failure of detection due to their low frequency On the other hand, some preferential directional migration due to the microgeographical hindrance might also account for the apportionment of rare alleles Nonetheless, with ceaseless and frequent gene flow, central populations, exposed to migrants from all neighbouring populations, would have a large probability of maintaining genetic variation Fluctuation of genetic structure resulting from a limited number of founders (cf Sun et al 1998) would seldom occur The low level of genetic variation detected in Taiwan and Hong Kong (Huang 1994; Sun et al 1998) may simply come from the conserved nature of the markers themselves For example, in this study, high cpDNA variation (θ = 0.00304) vs no mtDNA variation was detected in Taipei (Table 2) On the other hand, the organelle DNA diversity of Taiwan (with θ of 0.00173 at cpDNA trnL-trnF locus, and of 0.00018 at the mtITS locus) and Hong Kong (θ of 0.00135 at cpDNA locus vs no mtDNA variation) was lower than most other populations (Table 2) Habitat destruction in recent decades due to human activities may have largely contributed to the loss of genetic diversity (Yipp et al 1995; Chiang & Hsu 2000) Conclusions Kandelia candel, as a viviparous species of mangroves, provides an ideal model for testing the possibility and extent of long-range seedling dispersal In agreement with previous allozyme investigations, the geographical and population structure of the species, which adapts to the intertidal habitats with precocious growth of seeds, was determined both by vicariance and ongoing gene flow Significant genetic differentiation between populations of northern and southern banks of the South China Sea plus the attainment of monophyly of alleles of both cpDNAs and mtDNAs at geographical region level indicated a long isolation In contrast, recurrent gene flow via long-distance dispersal, indicated by high deduced Nm values, have contributed to the genetic homogeneity among populations of the region of northern South China Sea and East China Sea Gene genealogies, which trace phylogenetic relationships among alleles in a geographical context, of different loci coupled with locus–locus associations, helped clarify historical and recurrent evolutionary events Acknowledgements This study was supported by NSC grants of NSC- 85 – 2311-B-003 – 006-B17, NSC- 86–2311-B-003–005-B17 and NSC87–2311-B-003 – 004-B17 to S Huang We are indebted to Drs CI Peng, H Ota, and HQ Fan for the assistance with sampling References Bossart JL, Prowell DP (1998) Genetic estimates of population structure and gene flow: limitations, lessons and new directions Trends in Ecology and 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Discussion Genetic variability and low level of homoplasious mutations in cpDNAs and mtDNAs of Kandelia candel The usefulness of molecular markers in indirect estimates of population structure and. .. Noordhoff-Kolff Huang S (1994) Genetic variation of Kandelia candel (L.) Druce (Rhizophoraceae) in Taiwan In: Proceedings: International Symposium on Genetic Conservation and Production of Tropical