Genetic diversity and population structure of Indian Isoëtes dixitei Shende based on amplified fragment length polymorphisms and intron sequences of LEAFY

Aquatic Botany 113 (2014) 1–7

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Genetic diversity and population structure of Indian Isoëtes dixitei Shende based on amplified fragment length polymorphisms and intron sequences of LEAFY Jongduk Jung a , Sarvesh Kumar Singh b , Harish Chandra Pande c , Gopal Krishna Srivastava b , Hong-Keun Choi a,∗ a

Department of Biological Science, Ajou University, Suwon 443-749, South Korea Department of Botany, University of Allahabad, Allahabad 211002, India c Botanical Survey of India, Deharadun, India b

a r t i c l e

i n f o

Article history: Received 9 January 2013 Received in revised form 30 September 2013 Accepted 11 October 2013 Available online 8 November 2013 Keywords: Isoëtes dixitei Genetic diversity Population structure AFLP LEAFY

a b s t r a c t Isoëtes dixitei Shende is an endemic plant that grows in the Western Ghats, India. To evaluate the genetic diversity and the population structure of five I. dixitei populations from Maharashtra, we used amplified fragment length polymorphism (AFLP) markers and second intron of LEAFY sequences. Four AFLP-selective primer combinations generated 756 bands, of which 97% (735) from 154 individuals were polymorphic. In the analysis of the second intron of LEAFY, 103 sequences were generated and three types and 91 subtypes were obtained on considering the shared insertions/deletions and sequence similarity. The level of genetic diversity was high based on AFLP markers (mean Nei’s unbiased expected heterozygosity [HE ] = 0.207) and the second intron of LEAFY sequences (HE = 0.293). Estimated genetic differentiation among the populations was relatively low ( B = 0.082 on AFLP and 0.038 on LEAFY), compared with other Isoëtes species. A substantial amount of gene flow among populations was observed (Ne m = 2.566 on AFLP and 2.855 on LEAFY). Moreover, a positive marginally significant correlation between genetic distances (˚ST ) and geographical distances was detected by performing the Mantel test (r = 0.598, P = 0.062) based on AFLP markers. Isoëtes dixitei populations showed equilibrium of gene flow and genetic drift due to old-standing separation with sufficient gene flow. Two distinct groups were confirmed based on the genetic relationship: one contained two populations of Bhilar tableland (MB) and Wilson point (MW) while the other included three populations of Panchgani tableland (MP), Khingar tableland (MK), and Dandeghar tableland (MD). © 2013 Elsevier B.V. All rights reserved.

1. Introduction Isoëtes L. is an ancient genus with a long evolutionary line. About 350 species of the genus have been reported from various geographical locations around the world (Hickey et al., 2003), and to date, 16 species have been reported from different geographical regions in India (Srivastava et al., 1993). The Western Ghats is the abode of a number of rare plants, including the genus Isoëtes with six species reported from this region. These are I. sahyadriensis Mahabale, I. dixitei Shende, I. panchganiensis Srivastava, Pant and Shukla, I. udupiensis Shukla, Srivastava, Shukla and Rajagopal, I. divayadarshanii Shukla, Srivastava,

∗ Corresponding author at: Department of Biological Science, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea. Tel.: +82 31 219 2618; fax: +82 31 219 1795. E-mail addresses: [email protected], [email protected] (H.-K. Choi). 0304-3770/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2013.10.009

and Shukla and I. coromandelina L.f. Isoëtes dixitei is endemic quillwort reported form the Maharashtra and Karnataka states of India (Srivastava et al., 1993; Shukla et al., 2005). Most of the populations are found growing along the margins of small ditches and ponds except in Dandeghar and Khingar tablelands where plants are partly submerged in water (Wagai et al., 1997). Different populations exhibit subtle differences in the ornamentation of megaspores (Wagai et al., 2008). This morphological difference is expressed in genetic diversity because phenotypes related to various alleles. Cytologically, plants of the same or different populations may be diploids, tetraploids, and haxaploids (Srivastava, 2005; Shukla et al., 2007). Thus, morphological and cytological analyses have revealed a high genetic diversity of I. dixitei. Analyses of population genetics of Isoëtes species have been performed using various genetic markers such as amplified fragment length polymorphism (AFLP) markers, random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR) (Chen et al., 2005; Kang et al., 2005; Kim et al., 2009; Chen et al., 2010;

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J. Jung et al. / Aquatic Botany 113 (2014) 1–7

Gentili et al., 2010; Li et al., 2013). As another approach based on DNA sequences, non-coding regions having high substitution rates have been used for determining the relationships between taxa and populations (Hoot et al., 2004; Taylor et al., 2004; Kim et al., 2010). One of these non-coding regions, known as the second intron of LEAFY, has shown sufficient variation within each Isoëtes species (Taylor et al., 2004; Kim et al., 2010). In this study, we employed analyses using AFLP markers and the second intron of LEAFY, to genetically characterize five I. dixitei populations from Maharashtra, India. The evaluated genetic information such as a level of genetic diversity, gene flow, and correlation between genetic and geographic distance, could provide a better understanding of the relationships among the populations and the structure of the populations. 2. Materials and methods 2.1. Sampling and DNA extraction This study included five populations of I. dixitei from the high elevation tableland of Maharashtra, India (Table 1). For genetic analysis, we collected sporophylls of a total of 154 individuals, with sample sizes varying from 14 to 53 in each population (Table 1). The sampled sporophylls were dried using silica gel before DNA extraction. Total genomic DNA was extracted from one sporophyll by using the modified cetyltrimethylammonium bromide (CTAB) method (Chen and Ronald, 1999). 2.2. AFLP analysis, PCR, and cloning AFLP analysis was performed as described by Vos et al. (1995), except that fluorescent-labeled primers and capillary electrophoresis were used for detection and separation of amplified fragments respectively. The detailed procedure of AFLP analysis is outlined in the study by Kim et al. (2009). Briefly, total genomic DNA (2 ␮g) was digested using EcoRI and MseI endonuclease in a total volume of 50 ␮L. After adaptor ligation, pre-amplification was performed using EcoRI + C and MseI-A primers. The preamplified products were used for selective amplification using four selective primer combinations (HEX-EcoRI + ACT/MseI + CTC, HEXEcoRI + ACT/MseI + CGT, 6FAM-EcoRI + AAC/MseI + CTC, and 6FAMEcoRI + AAT/MseI + CGT). The amplified fragments were separated on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with an internal size standard GeneScan-500 LIZ (Applied Biosystems). Detected fragments of 60–500 bp were scored using GeneMarker (SoftGenetics, State College, PA, USA) with default value of parameters, but minimum threshold of intensity was set as 70 for samples with slightly low intensity. Ambiguous bands were manually checked. For amplification of the second intron of LEAFY, the 30F and 1190R primers were used (Taylor et al., 2004). The reactions were performed in 20 ␮L volumes containing 50–100 ng of template DNA, 1 unit of Taq DNA polymerase (SolGent, Daejeon, South Korea), 0.5 ␮M of each primer, 1× Taq buffer with 1.5 mM MgCl2 , and 0.25 mM of each deoxyribonucleotide triphosphate (dNTPs). PCR amplification was initiated with a pre-denaturation step at

95 ◦ C for 2 min, followed by 35 cycles at 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 1 min, with a final extension step at 72 ◦ C for 8 min, using a PTC-200 PCR machine (MJ Research, Watertown, MA, USA). The amplified products were purified using Gel & PCR Purification System (SolGent) and cloned into a pGEM-Teasy vector (Promega, San Luis Obispo, CA, USA). Plasmid DNA was purified using a plasmid mini-prep kit (Solgent). The purified plasmids were sequenced using a BigDye® Terminator v3.1 ready reaction mix (Applied Biosystems) and were run on an ABI 3730xl DNA analyzer (Applied Biosystems). 2.3. Data analyses AFLP bands were scored as ‘1 (present)’ or ‘0 (absent)’ in a binary matrix, and polymorphic bands were only used in further analyses after discarding monomorphic bands across all individuals (Keiper and McConchie, 2000). To estimate genetic diversity and population differentiation, the percentages of polymorphic loci (PPL, at the 0.99 level) and Nei’s (1978) unbiased expected heterozygosity (HE ) were estimated using TFPGA 1.3 (Miller, 1997). Shannon and Weaver’s (1949) index (I) was estimated using POPGENE 1.31 (Yeh et al., 1997). A Bayesian approach implemented in Hickory 1.1 (Holsinger and Lewis, 2005) was also performed to determine the genetic diversity (hs, analogous to HE ) and population differentiation ( B ). To ensure consistency of results, we used five runs for each of the three models (full model, f = 0 model, and f-free model). We used the deviance information criterion (DIC) to determine the most suitable model for our data, summarized from 50,000 generations after a burn-in of 10,000 generations. An analysis of molecular variance (AMOVA; Excoffier et al., 1992) was conducted using the Arlequin 3.5 (Excoffier and Lischer, 2010) to assess hierarchical genetic structure. The amount of gene flow (Ne m, Ne is effective population size and m is migration rate) among five populations was estimated based on the genetic differentiation (GST ; Nei, 1973) using the formula, Ne m = (1 − GST )/4GST . A Mantel test (Mantel, 1967) was conducted to identify any correlations between straight-line geographical distance and genetic distance (pairwise ˚ST ) among the five populations, and the significance of the test was determined with 5000 permutations by Arlequin 3.5 (Excoffier and Lischer, 2010). A scatterplot and linear regression were used to illustrate the correlation between the two distances using SigmaPlot 10.0 (Systat, San Jose, CA, USA). A neighbor joining dendrogram was generated based on the genetic distance (pairwise ˚ST ) matrix using PAUP* 4.0b10 (Swofford, 2002). To detect number of clusters, a model-based clustering method was implemented by STRUCTURE ver. 2.3 (Pritchard et al., 2000). Simulations of Markov chain Monte Carlo (MCMC) were carried out by varying the number of clusters (K) from 1 to 10 because number of predefined populations is 5. Ten replicates of simulation were run with 20,000 MCMC steps after a burn-in of 20,000 iterations under an admixture model with correlated allele frequency, for each K value. The rate of change in the log probability (K) was calculated to find the correct estimate of K by the formula of Evanno et al. (2005). A vertical bar plots to represent the ten simulations at the selected K was generated using CLUMPP 1.1 (Jakobsson and Rosenberg, 2007) and DISTRUCT 1.1 (Rosenberg, 2004). The obtained sequences of the

Table 1 Information on the samples from the five populations of Isoëtes dixitei Shende obtained from Maharashtra, India. Population

Number of individuals

Locality

Latitude (N)

Longitude (E)

Altitude (m)

MB MW MP MK MD Total

14 34 38 53 15 154

Bhilar tableland Wilson point Panchgani tableland Khingar tableland Dandeghar tableland –

17◦ 55 00.6 17◦ 55 14.0 17◦ 55 36.7 17◦ 54 41.3 17◦ 55 48.2 –

73◦ 44 59.7 73◦ 40 31.4 73◦ 48 31.3 73◦ 49 11.5 73◦ 49 28.2 –

1341 1432 1312 1290 1334 –

J. Jung et al. / Aquatic Botany 113 (2014) 1–7

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second intron of LEAFY were collated using DNA Baser v3 (HeracleSoftware, Lilienthal, Germany). Multiple-sequence alignment was performed by Clustal X v1.8 (Thompson et al., 1997) with the default alignment parameters. Sequence types and frequencies of the second intron of LEFY were determined by DnaSP 4.20 (Rozas et al., 2003). Uninformative gaps and bases were ignored in the determination of sequence types and genetic analyses. Each nucleotide was considered as a distinct locus in the estimation of genetic diversity, genetic distance, and population differentiation. The estimations of each statistic were conducted using conditions similar to those used for the analyses of AFLP data. Fig. 1. Representation of the three types of second intron of LEAFY from Isoëtes dixitei Shende. The three types were determined based on sequence similarities and shared gaps. The Gaps 1–7 indicate insertions/deletions shared by sequences within each type. Gaps 3 and 5 are identical in both types B and C. Scale represents position at multiple alignments.

3. Results 3.1. Polymorphism of AFLP markers The four primer combinations in the AFLP analysis generated a total of 756 bands, of which 735 (97%) were polymorphic across 154 individuals from five populations (Table 2). The number of bands generated by each primer combination varied from 164 (6FAM-EcoRI + AAT/MseI + CGT) to 223 (HEXEcoRI + ACT/MseI + CGT), and the proportion of polymorphic bands ranged from 93% (HEX-EcoRI + ACT/MseI + CGT) to 99% (HEXEcoRI + ACT/MseI + CTC) (Table 2). The mean number of bands per sample by each primer combination varied from 49 (6FAMEcoRI + AAC/MseI + CTC) to 79 (HEX-EcoRI + ACT/MseI + CGT), and the mean number of total bands per sample was 238 (Table 2).

subtypes in type C) based on substitutions and insertions/deletions (Table 3). Type A and B presented similar frequencies in the whole population (type A = 47% and type B = 45%), but the proportion of the two types was biased in the MB population (type A = 75% and type B = 25%) (Table 4). A low frequency of type C (8%) was observed, and all the sequences of this type were cloned from two populations – MP (3 clones) or MK (5 clones) (Table 4).

3.3. Genetic diversity 3.2. Genetic variation of the second intron of LEAFY A total of 103 second intron of LEAFY fragments were cloned from 24 individuals from five populations (Table 3; GenBank accession number KC460421–KC460523). Lengths of the sequences varied from 963 to 1068 bp, and the aligned length was 1096 bp (Table 3). Three genetic types were determined on the basis of similarities and shared gaps among 103 sequences: (i) Type A included 49 varied sequences from 1053 to 1068 bp in length and shared three gaps (gap 1: 5 bp in size and located at 289–293 bp in the aligned length; gap 4: 4 bp, located at 569–572 bp; gap 6: 11 bp, located at 702–712 bp); (ii) Type B contained 46 constant sequences 1048 bp in length and shared two gaps (gap 3: 27 bp, located at 425–451 bp and gap 5: 5 bp, located at 647–651 bp); (iii) Type C included eight sequences ranging from 963 to 966 in length and shared four gaps (gap 2: 81 bp, 323–403 bp, gap 3, gap 5, and gap 7: 6 bp, 1030–1036 bp. Gap 3 and 5 were identical with those of type B) (Fig. 1). In addition, a total of 103 sequences were grouped into 91 subtypes (41 subtypes in type A, 42 subtypes in type B, and 8

The statistics for genetic diversity within populations were estimated based on AFLP markers and the second intron of LEAFY. With AFLP markers, the percentage of polymorphic loci (PPL) ranged from 62% (MB) to 93% (MP), with a mean value of 79% (Table 5). The highest level of genetic diversity was exhibited in the MP population (HE = 0.207, hs = 0.266, I = 0.320), while that of the MB population was lowest (PPL = 62%, HE = 0.170, hs = 0.241, I = 0.257) (Table 5). Similar levels of genetic diversity were estimated within the other populations (MW, MK, and MD). Significant correlation between genetic diversity and sample size (N, number of individuals) was not detected. In the analysis of genetic diversity based on LEAFY, PPL ranged from 68% (MB) to 85% (MK), with a mean value of 76% (Table 5). Otherwise, the MP population exhibited the highest genetic diversity in other estimates (HE = 0.331, hs = 0.302, I = 0.487), and the lowest value was estimated from the MK population (HE = 0.233, hs = 0.281, I = 0.348) (Table 5). A significant correlation between genetic diversity and sample size (N, number of clones) was not detected.

Table 2 Polymorphic bands generated by AFLP primer combinations in five populations of Isoëtes dixitei Shende from Maharashtra, India. Primer combination

Total bands

HEX-EcoRI + ACT/MseI + CTC HEX-EcoRI + ACT/MseI + CGT 6FAM-EcoRI + AAC/MseI + CTC 6FAM-EcoRI + AAT/MseI + CGT

197 223 172 164

Mean

189.0

Total

756

Mean number of bands per sample

Polymorphic bands

Polymorphism (%)

54.2 79.2 48.9 56.1

196 207 171 161

99 93 99 98

59.8

183.8

98

238.4

735

–

Table 3 Characteristics of three types of second intron of LEAFY cloned from Isoëtes dixitei Shende. Characteristics

Type A

Type B

Type C

Total

Number of clones (number of subtypes) Range of length Number of polymorphic nucleotides/aligned length

49 (41) 1053–1068 32/1071

46 (42) 1048 31/1048

8 (8) 963–966 4/969

103 (91) 963–1068 182/1096

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Table 4 Proportion of three types of second intron of LEAFY within five populations of Isoëtes dixitei Shende. Population

Number of individuals

Type A (%)

Type B (%)

4 4 4 8 4

16 19 16 34 18

12 (75) 11 (58) 6 (37) 13 (38) 7 (39)

4 (25) 8 (42) 7 (44) 16 (47) 11 (61)

– – 3 (19) 5 (15) –

24

103

49 (47)

46 (45)

8 (8)

MB MW MP MK MD Total

Number of clones

Type C (%)

Table 5 Genetic diversity based on AFLP markers and the second intron of LEAFY within five populations of Isoëtes dixitei Shende. Population

AFLP

LEAFY

N

PPL (%)

HE

hs

I

N

PPL (%)

HE

hs

I

MB MW MP MK MD

14 34 38 53 15

62 79 93 80 79

0.170 0.200 0.239 0.210 0.218

0.241 0.260 0.296 0.260 0.274

0.257 0.312 0.374 0.329 0.329

16 19 16 34 18

68 72 81 85 71

0.233 0.293 0.331 0.321 0.288

0.281 0.289 0.302 0.302 0.291

0.348 0.421 0.487 0.483 0.416

Mean

31

79

0.207

0.266

0.320

21

76

0.293

0.293

0.431

N, sample size (number of individuals and clones in AFLP and LEAFY data respectively); PPL, percentage of polymorphic loci; HE , Nei’s (1978) unbiased expected heterozygosity; hs, genetic diversity using Bayesian approach; I, Shannon and Weaver’s (1949) index.

3.4. Population differentiation, gene flow, and genetic structure

assignment to two clusters based on the second intron of LEAFY but the clusters did not correspond to any division of the pre-defined populations (Supplementary Fig. 2B).

Population differentiation ( B ) was determined using the Bayesian approach by using three different models. The full model with smallest deviance information criterion (DIC) was selected based on the two types of genetic data we generated, AFLP markers and second intron of LEAFY (DIC = 15844.6 on AFLP and 3371.1 on LEAFY) (Table 6). The  B values under the full model were estimated as 0.082 and 0.038 for AFLP markers and the second intron of LEAFY respectively (Table 6). As an additional approach for determining population differentiation, we analyzed the GST values and found that GST values were 0.089 on AFLP markers and 0.081 on the second intron of LEAFY. Gene flow (Ne m) was calculated based on GST and was estimated as 2.566 on AFLP and 2.855 on LEAFY. Analysis of molecular variance (AMOVA) was performed with two hierarchical levels, among and within populations. AMOVA based on AFLP markers showed no significant differentiation among populations because 11% of the total variation was distributed among populations and 89% was within the populations (P < 0.001) (Table 7). Based on the second intron of LEAFY, significant differentiation among populations was not detected because 4% and 96% of the total variation was attributed to differentiation among and within populations respectively (P = 0.059) (Table 7). In the clustering analysis, the highest K was found at the simulation with K = 6 based on AFLP markers and with K = 2 based on the second intron of LEAFY (Supplementary Fig. 1). Estimated population structure based on AFLP markers presented complexity of the pre-defined populations (natural populations) including admixture of individuals with high probability to two or more clusters (Supplementary Fig. 2A). Each clones had a high probability of

3.5. Correlation between genetic distances and geographic distances Genetic distance was represented as ˚ST for each pairwise combination of populations, based on AFLP markers and the second intron of LEAFY. Genetic distances (˚ST ) based on AFLP markers were highly significant (P < 0.001) and varied from 0.060 (between populations MP and MK) to 0.166 (between MB and MD), with a mean distance of 0.111 (Supplementary Table 1). Based on the second intron of LEAFY, genetic distances between MB and MD were furthest (˚ST = 0.168, 0.001 < P < 0.05). Genetic distances among the three populations MP, MK, and MD were regarded as zero with further analysis because negative values were estimated (P > 0.05). Geographic distances among populations varied from 1.7 km (between MP and MD) to 16.4 km (between MW and MK) (Supplementary Table 1). To illustrate the genetic relationship among the five populations, neighbor-joining dendrograms were generated using ˚ST , based on the two types of genetic data obtained. Two groups, populations MB-MW and MP-MK-MD, were consistently identified in the two dendrograms (Fig. 2). Positive correlation between genetic distances (˚ST ) and geographical distances (km) was detected using the Mantel test (r = 0.598, P = 0.062) (Fig. 3A), based on the AFLP markers. It was marginally significant Based on the second intron of LEAFY, the correlation was weakly positive but was not significant (r = 0.142, P = 0.280) (Fig. 3B).

Table 6 The differentiation ( B ) of Isoëtes dixitei Shende populations, calculated with three different models using the Bayesian approach. Model

AFLP

Full f=0 Free a b

LEAFY

f



0.970 0 0.491

0.082 0.048 0.075

a

Inbreeding index within populations. Deviance information criterion.

B

DIC

fa

B

DICb

15844.6 15889.5 17178.2

0.378 0 0.491

0.038 0.031 0.080

3371.1 3437.5 3643.0

b

J. Jung et al. / Aquatic Botany 113 (2014) 1–7

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Table 7 Summary of analysis of molecular variance (AMOVA) within/among five populations of Isoëtes dixitei Shende, based on AFLP markers and the second intron of LEAFY. Source of variation

AFLP df

LEAFY Variance component (%)

Among populations Within populations

4 149

11.77 (11) 99.42 (89)

Total

153

111.19 (100)

a

Pa <0.001 <0.001

df 4 98 102

Variance component (%)

Pa

2.08 (4) 44.42 (96)

0.059 0.059

46.50 (100)

–

Levels of significance are based on 1000 interaction steps.

Fig. 2. Genetic relationships among five populations of Isoëtes dixitei Shende. (A) Neighbor joining dendrogram based on genetic distance (˚ST ) using AFLP markers. (B) Neighbor joining dendrogram based on genetic distance (˚ST ) using the second intron of LEAFY.

4. Discussion 4.1. Polymorphism of AFLP markers and variation of the second intron of LEAFY Genetic markers are effective tools for investigating genetic diversity and population structure. AFLP markers have high polymorphism across overall genome and have been previously employed to determine genetic diversity of Isoëtes species populations (Kang et al., 2005; Kim et al., 2009; Gentili et al., 2010). Likewise, AFLP markers in this study provided sufficient polymorphic bands to evaluate genetic characteristics of I. dixitei populations. On average, 98% (735 bands) of the total number of bands (756) was polymorphic (Table 2).

Sequences from non-coding regions, such as nuclear ITS, second intron of LEAFY, chloroplast trnS-psbC, and atpB-rbcL, have been used for determining relationships among taxa and for testing the hypothesis for the speciation process in Isoëtes (Hoot and Taylor, 2001; Hoot et al., 2004; Taylor et al., 2004; Kim et al., 2010). In case the second intron of LEAFY had high substitution rates, two types of second intron of LEAFY sequences were identified based on a length difference of about 100 bp in Isoëtes species and the longer type was sequenced more often (Hoot and Taylor, 2001; Taylor et al., 2004; Kim et al., 2010). An example of variation in the longer type was observed in a study by Kim et al. (2010), where 23 sequence subtypes were obtained from 40 clones isolated from two populations of I. coreana. In our study, three types of the second intron of LEAFY were defined by considering the length and

Fig. 3. Correlations between genetic distances and geographical distances among five populations of Isoëtes dixitei Shende. (A) Scatterplot of genetic distances (˚ST based on AFLP markers) against geographical distances (km), with correlation (r = 0.598, P = 0.062) by Mantel test. (B) Scatterplot of genetic distances (˚ST based on the second intron of LEAFY) against geographical distances (km), with weak correlation (r = 0.142, P = 0.280) by Mantel test.

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similarity from five populations of I. dixitei (Table 3 and Fig. 1). The type A (1053–1068 bp) and B (1048 bp) corresponded to the longer type and type C to the shorter type observed in a previous study (Hoot and Taylor, 2001). One of the longer types, type B was more similar to the shorter type C because they shared gaps 3 and 5 (Fig. 1). The relationship between types B and C was supported by group mean pairwise distances among the three types; distances between types A and B, A and C, and B and C were 0.092, 0.097, and 0.049 respectively. The second intron of LEAFY within I. dixitei was more variable than those of species in previous studies (Hoot et al., 2004; Taylor et al., 2004; Kim et al., 2010). The high level of variation of the second intron of LEAFY in this study suggests availability as a genetic marker in the analysis of populations. Various sequence types of the trnS-psbC region have been observed within and among Isoëtes species (Kim et al., 2010). In our preliminary study, variation of sequence was not confirmed in the sequences of chloroplast atpB-rbcL (679 bp) and trnS-psbC (999 bp) from 24 individuals isolated from the five populations of I. dixitei (data not shown). 4.2. High level of genetic diversity within the populations Various levels of genetic diversity have been reported by employing different genetic markers (such as AFLP, RAPD, and ISSR) on Isoëtes species (Chen et al., 2005; Kim et al., 2009; Chen et al., 2010; Li et al., 2013). In the case of AFLP markers, PPL values ranged from 22% (I. japonica) to 49% (I. asiatica) in seven East Asian species (Kang et al., 2005; Kim et al., 2009). The high percentage of PPL values from I. dixitei (79%) is similar to the levels observed in one European species, I. malinverniana (74%) (Gentili et al., 2010). In addition, the high level of genetic diversity within I. dixitei populations is supported by other estimates, such as Nei’s (1978) unbiased expected heterozygosity (HE = 0.207), genetic diversity using Bayesian approach (hs = 0.266), and Shannon and Weaver’s (1949) index (I = 0.320) (Table 5). These high levels of genetic diversity of I. dixitei are similar to those of I. malinverniana (Gentili et al., 2010). A high level of genetic diversity within populations is often explained by a lack of genetic drift, outcrossing in breeding behavior, and/or overlapping generations by long-lived individuals (Loveless and Hamrick, 1984; Muir et al., 2004). The populations of I. dixitei may not undergo serious genetic drift by stable environment at tropical highlands. The heterosporous and perennial habits of I. dixitei would favor outbreeding and overlapping generations respectively. In contrast, low level of genetic diversity was investigated from populations of East Asian Isoëtes species, which have the similar habits with I. dixitei (Kim et al., 2009). Their low genetic diversity was explained by genetic drift and inbreeding in small population, and recent speciation by a few polyploidy individuals (Kim et al., 2009). The level of genetic diversity estimated using the second intron of LEAFY was higher than that obtained based on AFLP markers, but both genetic diversities showed similar patterns; the genetic diversity of the MB population was significantly lower, and a high level of genetic diversity in the MP population was revealed. 4.3. Low level of differentiation and relationships among the populations The degree of genetic differentiation is influenced by the relative strength of gene flow and genetic drift, and the status of populations can be inferred by testing for correlation between genetic and geographical distances (Slatkin, 1987; Hutchison and Templeton, 1999). The genetic differentiation estimated from populations of I. dixitei was significantly low ( B = 0.082 and GST = 0.089 on AFLP, and  B = 0.038 and GST = 0.081 on LEAFY) compared with that of other Isoëtes species, such as I. coreana ( B = 0.730 on AFLP and

0.742 on RAPD), I. jejuensis ( B = 0.368 on AFLP), I. sinensis ( B = 0.607 and GST = 0.608 on AFLP), and I. malinverniana (GST = 0.220 on AFLP + ISSR) (Kang et al., 2005; Kim et al., 2009; Gentili et al., 2010). However, I. taiwanensis ( B = 0.074 on AFLP) showed similarly low differentiation (Kim et al., 2009). The result of AMOVA also supported the hypothesis that I. dixitei populations were not significantly differentiated, because a small portion (11% and 5% based on AFLP and LEAFY respectively) of the total variation was attributed among populations (Table 7). The gene flow among I. dixitei populations was estimated to be Ne m = 2.566 using AFLP and 2.855 using LEAFY. Such a level of gene flow implies that genetic drift will not result in substantial differentiation (Slatkin, 1987). This high gene flows among populations were supported by the model based clustering analysis using AFLP markers (Supplementary Fig. 2A). In the clustering analysis represented as a bar plot, the high frequency of admixed individuals was observed in the pre-defined populations. The admixed individuals had a high probability of each cluster and were contained in the related populations. For example, ‘pink’ and ‘light green’ cluster (in Supplementary Fig. 2A) had high probability in the admixed individuals of three related populations (MP, MK, and MD in Fig. 2). Furthermore, a marginally significant positive correlation between genetic and geographical distance was revealed using Mantel test of AFLP markers. The correlation between both distances explains that I. dixitei populations are situated in equilibrium of gene flow and genetic drift due to old-standing separation with sufficient gene flow (Hutchison and Templeton, 1999). Spore dispersal is inferred as one of the factors for the sufficient gene flow between the I. dixitei populations and can be promoted by animals preferring to visit wetland, such as birds. The dispersal of microspore with the wind can be occurred when the wetland is annually dry up. The five populations of I. dixitei were divided into two groups (MB-MW and MP-MK-MD) in the neighbor-joining dendrogram based on both molecular data sets obtained (Fig. 2). The MB population is located between MW and MP-MK-MD geographically. However, the MB population has a genetically closer relationship with MW (˚ST = 0.101 on AFLP and 0.022 on LEAFY) and a remote relationship with MP-MK-MD (mean of ˚ST = 0.141 on AFLP and 0.151 on LEAFY) (Supplementary Table 1). The MB population is inferred as a population formed from MW after division of the two groups.

Acknowledgements We thank Dr. Paramjit Singh (Director of Botanical Survey of India) for his support to collect samples. We have lost one of the fine pteridologists and one of the authors, late Dr. Harish Chandra Pande (BSI, Deharadun, India) who had passed away on Sept. 21st, 2013. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Education, Science and Technology [MEST]) (NRF-2011-0016929).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.aquabot.2013.10.009.

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