African Journal of Agricultural Research Vol. 6(20), pp. 4760-4768, 26 September, 2011 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR10.611 ISSN 1991-637X ©2011 Academic Journals

Full Length Research Paper

Genetic diversity and molecular discrimination of the closely related Taiwanese Ulmaceae species Celtis sinensis Persoon and Celtis formosana Hayata based on ISSR and ITS markers

Shih-Chieh Lee 1, Chi-Feng Chang 2 and Kuen-Yih Ho 2*

1Department of Bio-Industry Technology, Da-Yeh University, Changhua, 51591, Taiwan. 2Department of Forestry and Nature Resources, National Chiayi University, Chiayi, 60054, Taiwan.

Accepted 14 September, 2010

Celtis sinensis Persoon and Celtis formosana Hayata belong to the Ulmaceae family. These closely related species are native to Taiwan. In the present study, 120 samples from 18 natural habitats in Taiwan were studied. The genetic diversity of these two species was determined by comparing the inter-simple sequence repeat (ISSR) and internal transcribed spacer (ITS) regions; these data were also used for the molecular identification of each species. Among the 71 bands amplified by PCR using nine ISSR primers, 51 exhibited polymorphism (71.8%). The population genetic variation analysis (POPGENE) revealed a genetic differentiation (Gst) of 0.3814 and a gene flow (Nm) of 0.8110. AMOVA showed that interspecies differences accounted for 57.38% of the variance (p < 0.0001). Despite their high morphological similarity, C. sinensis and C. formosana can be discriminated and classified into two independent species at the molecular level.

Key words: Celtis , genetic diversity, molecular discrimination, inter-simple sequence repeat (ISSR), internal transcribed spacer (ITS).

INTRODUCTION

Celtis sinensis Persoon grows in the piedmont plain side; the petioles are about 1 cm long (Liu, 1985). This regions throughout the entire island of Taiwan. This plant tree species is commonly planted near beaches or grows rapidly in a variety of soil types under various around villages to provide shade and wind resistance. conditions, from moist, fertile areas to hot, dry locations in The bark and leaves of C. sinensis can be used as the full sun. C. sinensis Persoon is wind and drought medicine to treat various maladies, including urticaria and tolerant once established. It may be propagated through lumbago (Liu et al., 1994). Celtis formosana grows in seeds or cuttings. C. sinensis is a large deciduous tree both the lowlands and highlands of Taiwan and is found in that can grow up to 20 m in height; its bark is smooth and mainland China, the Ryukyu Islands, India, and Indochina gray. This species features chartaceous leaves that are (Liu et al., 1994). This species variant survives easily in ovate to ovate-oblong, obtuse to acute, obliquely broad- hot, humid environments and tolerates drought and salt. It cuneate at their base, and crenated-serrated toward the is very similar to the model species C. sinensis, except base. The leaves are dark green and smooth above and that C. formosana has a smoother bark surface, a are glabrous and slightly glaucous beneath. There are tapering leaf apex, and a fruit diameter of 6 to 9 mm. Both three nerves at the base, and three-four lateral veins per species are used in similar ways (Yang and Lu, 1996). The ISSR (inter-simple sequence repeat) molecular fingerprinting technology developed by Zietkiewicz et al. (1994) utilizes random priming to amplify chromosomal *Corresponding author. E-mail: [email protected]. Tel: DNA fragments that can then be used as molecular +886-5-2717463. Fax: +886-5-2717467. markers. This method can discriminate between particular

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Table 1. The taxon, collection site, elevation, and source codes of C. sinensis and C. formosana samples collected from 18 natural habitats in Taiwan.

Taxon Collection site Elevation (m) Temp./ Year (°C) Rainfall/year (mm) Source code Zhongli (Taoyuan County) 151 21.6 1820 S-jl1 − S-jl5 Xinfeng (Hsinchu County) 25 21.8 1845 S-sf1 − S-sf10 Huoyenshan (Miaoli County) 250 22.0 1813 S-hy1 − S-hy5 Xinshe (Taichung County) 613 22.2 1742 S-ss1 − S-ss7 C. sinensis Pakuashan (Changhua County) 180 22.6 1820 S-bg1 − S-bg6 Ssuhu (Yunlin County) 23 23.5 2305 S-shu1 − S-shu7 Potzu (Chiayi County) 33 23.1 2200 S-pt1 − S-pt5 Xinhua (Tainan County) 60 24.2 2030 S-sh1 − S-sh5 Tashan (Kinmen County) 48 22.6 1825 S-ts1 − S-ts10

Fuxing (Taoyuan County) 482 22.4 2987 F-fs1 − F-fs10 Wufeng (Hsinchu County) 1290 19.3 2745 F-wf1 − F-wf10 Henglungshan (Miaoli County) 850 20.3 2697 F-hl1 − F-hl5 Kukuan (Taichung County) 1045 18.9 2953 F-gg1 − F-gg5 C. Jenlun (Nantou County) 1001 18.7 3126 F-rl1 − F-rl5 formosana Shifmengku (Chiayi County) 1750 17.4 3522 F-sm1 − F-sm5 Tatungshan (Chiayi County) 1241 18.5 3342 F-dd1 − F-dd10 Nantzuhsienhsi (Kaohsiung County) 1850 19.6 3523 F-nt1 − F-nt5 Lilungshan (Pingtung County) 724 20.6 3207 F-ll1 − F-ll5

genotypes and directly reflects the genetic differences MATERIALS AND METHODS between organisms. Furthermore, a considerable number of gene loci can be studied due to the large numbers of Plant materials

fragments that are amplified by these primers. Therefore, Samples of C. sinensis Persoon and C. formosana Hayata were this method has been widely used for species gathered from 27 sites across Taiwan and its adjacent islands identification and phylogenetic analyses as well as in (Figure 3). Between 20 and 40 specimens of each species were studies of plant diversity (Petros et al., 2008; Zong et al., sampled for leaf morphology analysis (Table 2), and 20 to 60 2008). The internal transcribed spacers in ribosomal DNA specimens of each species were sampled for molecular marker analysis (ITS and ISSR) (Table 2). In addition, 5 to 10 mature, non- (rDNA ITS) have been used as molecular markers to diseased leaf blade samples were dehydrated, aliquoted into air- study the genetic relationships among closely related permeable bags, and stored in dry, sealed containers. Sample taxonomic groups by examining the diversity of ITS collection sites and DNA sample codes are listed in Table 1. The fragments. Bellarosa et al. (2005) investigated the genetic identification of all of the leaves was authenticated by Dr. Fu-Yuan relationships among the subgenera in the genus Quercus Lu in the Department of Forestry and Nature Resources at National spp. of the Fagacease family using ITS. Won and Renner Chiayi University. Voucher specimens were deposited in the Department of Forestry and Nature Resources at National Chiayi (2005) studied 25 species from the genus Gnetum in University. South America, Africa, and Asia through an analysis of ITS diversity. Traditional plant taxonomy has been based on DNA extraction differences in external morphology. However, plant morphology might be affected by changes in the external DNA was extracted using the CTAB extraction method published by Kobayashi et al. (1998), with slight modifications. DNA was environment and by endogenous factors, such as visualized by agarose gel electrophoresis followed by staining with genetics. The interplay between environmental and ethidium bromide (Sambrook et al., 1989). Known amounts of genetic factors often causes difficulties in taxonomic lambda DNA (MBI, Fermentas, Hanover, MD, USA) were included studies. The present study aimed to investigate the on the gel to facilitate quantification of the DNA. genetic diversity in natural populations of C. formosana

and C. sinensis in Taiwan and to use molecular data (that ISSR amplification is, ISSR and ITS sequences) to discriminate between these two closely related species. One hundred random primers (UBC 801-900, Canada) were

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Table 2. The nine UBC primers and the number of corresponding fragments used in the ISSR analysis.

Number of Number of Percentage of Annealing UBC primer No. Sequence 5’- 3’) polymorphic monomorphic polymorphic ((( temperature(°C) fragments fragments fragments (%) UBC 823 TCTCTCTCTCTCTCTCC * 52 6 3 66.7 UBC 825 ACACACACACACACACT 53 5 2 71.4 UBC 834 AGAGAGAGAGAGAGAGYT 56 9 0 100 UBC 886 VDVCTCTCTCTCTCTCT 57 5 2 71.4 UBC 887 DVDTCTCTCTCTCTCTC 56 8 0 100 UBC 888 BDBCACACACACACACA 56 5 2 71.4 UBC 889 DBDACACACACACACAC 56 7 3 70.0 UBC 890 VHVGTGTGTGTGTGTGT 56 3 4 42.9 UBC 891 HVHTGTGTGTGTGTGTG 56 3 4 42.9

*B = ( C, G, T ); D = ( A, G, T ); H = ( A, C, T ); V = ( A, C, G ); Y = ( C, T ).

screened for PCR. Among them, a total of nine optimal primers the gel were visualized and photographed using the EZlab Uni- were selected for use in amplification (Table 2). Each of the ISSR photo system. PCR reaction mixture contained 16.5 l ddH 2O, 2.5 l 10× PCR buffer, 1.6 l 2.5 mM dNTPs, 2 l primers (10 ng/ l), 0.4 l Taq DNA polymerase (1 U), and 2 l DNA template (20 ng/ l) in a total Data analysis based on ISSR marker profiles volume of 25 l. The template DNA was denatured at 94°C for 5 min and then subjected to 35 cycles of 94°C for 30 s, 52 to 56°C for The pairwise similarities between samples were calculated by the 50 s, and 72°C for 2 min. The final cycle included an extension at simple matching formula in NTSYS (Rohlf, 1993) to establish the 72°C for 10 min. similarity matrix. POPGENE (Yeh et al., 1997; Yeh et al., 1999) was used to calculate the H values of genetic diversity between different populations (Nei, 1973), the G st values of genetic differentiation ITS amplification (Nei, 1973), and the genetic distance (Nei, 1978). The G st values were used to estimate the Nm values, which represent gene flow Five samples each from C. sinensis and C. formosana (S-sf1, S- (Slatkin and Barton, 1989). hy1, S-pt1, S-sh1, and S-ts1; F-hl1, F-rl1, F-sm1, F-dd1, and F-ll1) were selected for amplification of the 18S, 5.8S, and 26S rDNA H = Nei's (1973) gene diversity; Nm ＝ 0.5 × (l – Gst) / Gst internal transcribed spacer (ITS) sequences, using the primers ITS1 and ITS2. ITS1 (5’-ACCTGCGGAAGGATCATTG-3’) and ITS2 (5’- The intraspecies and interspecies variance components were NNTAAATTCAGCGGGTAGC-3’) were designed according to the calculated using the AMOVA program (Excoffier et al., 1992), and published Celtis laevigata sequence (AF174621) retrieved from the significance was tested by 9999 random permutations. The NCBI (National Center for Biotechnology Information). An annealing SAHN program in NTSYS and the UPGMA method were used for temperature of 57°C was used to amplify the ITS region of the cluster analysis to generate phylogenetic trees. rDNA of C. sinensis . The ITS PCR reaction mixture included 61 l ddH 2O, 10 l 10× PCR Buffer, 6 l 2.5 mM dNTPs, 3 l 20 mM MgCl 2, 3 l 5% DMSO, 2 l each primer (10 ng/ l), 1 l Taq DNA Phylogenetic tree construction based on ITS sequences polymerase (1 U), and 12 l DNA template (10 ng/ l) in a total volume of 100 l. The template DNA was denatured at 94°C for 5 The DNA sequences were first aligned using the ClustalV method min and then subjected to 30 cycles of 94°C for 1 min, 57°C for 50 (Higgins and Sharp, 1989) with MegAlign software (DNASTAR, s, and 72°C for 1 min. The final cycle included an extension at 72°C Lasergene). Following alignment, the base composition of the for 10 min. sequences was analyzed by MEGA v. 4.0 (Tamura et al., 2007). Phylogenetic trees were generated using the maximum parsimony (MP) method (Fitch, 1971). The support for the consensus trees Purification and sequence determination of ITS PCR products obtained from the MP and NJ methods was examined (Felsenstein, 1985). DNA bands with the correct molecular weights were excised, and the DNA was recovered and purified using a DNA Purification Kit (Gene Spin TM V2). The purified DNA was sequenced using a fully automated high-throughput DNA sequencer (ABI PRISM 3730). RESULTS AND DISCUSSION

Selection of ISSR primers and reproducibility Electrophoresis

The amplified PCR products were run on 1.2% agarose gels and One hundred random primers (UBC 801-900, Canada) then stained with 0.5 g/ml ethidium bromide. The DNA bands on were used for PCR. PCR optimization was performed

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Table 3. POPGENE analysis of genetic diversity, genetic differentiation, and gene flow in C. sinensis and C. formosana populations from 18 natural habitats in Taiwan.

Prov. code Sample number (N) 1) Genetic diversity (H) 2) Genetic differentiation (Gst 3) Gene flow (Nm 4) S- Zhongli 5 0.0924 S- Xinfeng 10 0.1479 S- Huoyenshan 5 0.0556 S- Xinshe 7 0.0814 S- Pakuashan 6 0.0845 S- Ssuhu 7 0.0968 S- Potzu 5 0.0197 S- Xinhua 5 0.0228 S- Tashan 10 0.0878 S total 60 0.2521 0.6887 0.2260 F- Fuxing 10 0.0980 F- Wufeng 10 0.0376 F- Henglungshan 5 0.0000 F- Kukuan 5 0.0932 F- Jenlun 5 0.0225 F- Shifmengku 5 0.0416 F- Tatungshan 10 0.0408 F- Nantzuhsienhsi 5 0.0262 F- Lilungshan 5 0.0068 F total 60 0.1579 0.7409 0.1748 18 populations total 120 0.3339 0.8200 0.1097 S and F total 120 0.3339 0.3814 0.8110

1) N = Sample number; 2) H = Nei’s genetic diversity; 3) Gst = genetic coefficient of differentiation; 4) Nm = gene flow, Nm = 0.5(1 − Gst) / Gst.

using a temperature gradient with a thermocycler to 0.1579, the genetic differentiation coefficient (Gst) was determine the optimal annealing temperature. Among the 0.7409, and the gene flow value (Nm) was 0.1748. These primers, nine (Table 2) produced robust amplification values indicate that there is a low level of gene flow and a patterns and were selected for the population genetic high level of genetic differentiation among the C. analysis in this study. Among the 71 bands amplified formosana populations in different regions. Among the 18 using these nine primers, 51 were polymorphic (71.8%). sampling sites tested, the overall genetic diversity (H) was highest (H = 0.1479) in the populations from the Hsinfeng area and lowest (H = 0.0000) in the populations Analysis of genetic variation (POPGENE) from the Henglong Mountain area; the low diversity in the Henglong Mountain area could be due to the geographic The POPGENE analysis (Table 3) revealed that the isolation imposed by the tall mountains in this particular genetic diversity parameter (Nei’s gene diversity; H) of C. area. sinensis was highest (H = 0.1479) in the populations from The overall H for the populations in these 18 regions the Hsinfeng area and lowest (H = 0.0197) in the was 0.3339, the Gst was 0.820, and the Nm was 0.1097, populations in the Puzih region. The overall genetic suggesting a low level of gene flow and a high level of diversity index (H) of C. sinensis was 0.2521, the genetic genetic differentiation among the populations in all 18 differentiation coefficient (Gst) was 0.6887, and the gene regions. Gene flow can affect genetic structure, and the flow (Nm) value was 0.2260. These values indicate that more prominent the gene flow, the lower the degree of there is a low level of gene flow and a high level of genetic differentiation. Wright (1931) suggested that genetic differentiation among the C. sinensis populations genetic structures are prone to equalization when the in different regions. The genetic diversity (H) of C. gene flow among populations (Nm) is greater than one. formosana was highest (H = 0.0932) in the populations On the contrary, an Nm value less than one indicates a from the Kukuan area and lowest (H = 0.0001) in the gene flow that has been partially hindered by populations from the Henglong Mountain area. The geographical conditions. Gene flow allows exchange overall genetic diversity index (H) of C. formosana was between gene pools in two regional populations, which

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Table 4. AMOVA of C. sinensis and C. formosana from the 18 sample sites.

Source of variation df SSD MSD Variation component Total var. (%) p-value Sample interval variation 17 762.31 44.84 6.5236 80.11 < 0.0001 Sample area variation 102 165.19 1.62 1.6195 19.89 < 0.0001

Table 5. Hierarchical analysis of molecular variance (AMOVA) of C. sinensis and C. formosana from 18 natural habitats in Taiwan.

Source of variation df SSD MSD Variation component Total var. (%) p-value Interspecific variation 1 379.71 379.71 6.2511 57.38 < 0.0001 Intraspecific variation 118 547.78 4.64 4.6422 42.62 < 0.0001

leads to a decrease in the degree of population population groups. A larger distance between two species differentiation (Slatkin, 1987). The overall genetic indicates a lower degree of similarity. As shown by the diversity index (H) between C. sinensis and C. formosana ISSR cluster map, C. sinensis and C. formosana are was 0.3339, the genetic differentiation (Gst) was as high separated into two large population groups with a as 0.3814, and the gene flow value (Nm) was 0.8110. distance coefficient of 50. The above data demonstrate a significant differentiation between these two species, with low gene flow and high genetic diversity likely due to a geographical barrier. ITS sequence analysis

Electrophoresis of the amplified fragments indicated a Analysis of molecular variance (AMOVA) results length of approximately 680 bp. The sequence alignments indicated sequences of 584 to 589 bp, The genetic variance components were analyzed with encompassing ITS1, 5.8S rDNA, and ITS2. ITS1 was AMOVA for C. sinensis and C. formosana in the 18 approximately 182 bp, ITS2 was 243 to 248 bp, and the sample regions. The results show that the variance 5.8S rDNA was 159 bp. The total length of the sequence, components among the sample regions accounted for obtained from the alignment of the sequences from these 80.11% total source of variation (p < 0.0001), and the two species using the ClustalV program in MegAlign, was variance components within each sample region 589 bp. A total of 63 variable base pairs were found in the accounted for 19.89% total source of variation (p < sequence, accounting for 10.70% of the full length of the 0.0001). The variation between sampling sites was an sequence. Among these variable sites, 24 were important source of variation (Table 4). parsimony-informative sites, constituting 4.07% of the full The genetic variance components between these two length. Base composition analysis of the sequence species were also analyzed by AMOVA. These results revealed that A accounted for 18.0% of the sequence, G indicate that the variance between the species was for 30.7%, C for 34.1%, and T for 17.25%. The genetic 57.38% (p < 0.0001) and within the species was 42.62% similarity of the ITS fragments from C. sinensis and C. (p < 0.0001). The variation between species was the formosana was as high as 0.969 (Table 7). major source of variation (Table 5).

Phylogenetic analysis of ITS sequences Molecular relationship and cluster analysis The sequences of the amplified ITS fragments from the The genetic distance matrix ( Φst) between C. sinensis samples were processed with the maximum parsimony and C. formosana was calculated using the POPGENE method in MEGA, and the MP tree and NJ tree (Figure 3) software package (Table 6). A phylogenetic tree was were obtained from 1000 bootstrap resampling replicates. generated through cluster analysis with SAHN and the The consensus tree and the cluster analysis results show UPGMA method in the NTSYSpc v. 2.0 software package that C. sinensis and C. formosana tended to separate into (Figure 2). A cophenetic correlation coefficient (r) of two population groups, with some degree of differen- 0.92448 was obtained by comparing the distance matrix tiation in their genetic relationships. Because the and the phylogenetic tree, indicating that the cluster map sequence evolution rate is not assumed to be constant in was not distorted and could reflect the actual status of the the NJ method, the NJ trees here are unrooted. Only the

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Table 6. Nei's genetic distance ( Φst), based on ISSR data, of C. sinensis and C. formosana from 18 natural habitats in Taiwan.

Area S-jl S-sf S-hy S-ss S-bg S-shu S-pt S-sh S-ts F-fs F-wf F-hl F-gg F-rl F-sm F-dd F-nt F-li groups S-jl 0 S-sf 0.313 0 S-hy 0.171 0.188 0 S-ss 0.224 0.208 0.094 0 S-bg 0.153 0.235 0.131 0.087 0 S-shu 0.224 0.296 0.118 0.133 0.160 0 S-pt 0.405 0.332 0.163 0.155 0.241 0.188 0 S-sh 0.510 0.318 0.240 0.271 0.323 0.295 0.131 0 S-ts 0.456 0.229 0.279 0.224 0.306 0.207 0.162 0.220 0 F-fs 0.585 0.685 0.675 0.588 0.443 0.641 0.712 0.631 0.712 0 F-wf 0.535 0.741 0.597 0.424 0.382 0.524 0.549 0.595 0.635 0.096 0 F-hl 0.476 0.633 0.562 0.393 0.364 0.512 0.522 0.566 0.628 0.152 0.099 0 F-gg 0.509 0.600 0.541 0.465 0.435 0.486 0.532 0.601 0.483 0.174 0.156 0.135 0 F-rl 0.471 0.641 0.576 0.536 0.450 0.627 0.625 0.593 0.645 0.144 0.137 0.113 0.124 0 F-sm 0.533 0.595 0.556 0.434 0.415 0.510 0.505 0.554 0.548 0.136 0.116 0.088 0.145 0.168 0 F-dd 0.501 0.620 0.560 0.456 0.343 0.580 0.608 0.675 0.681 0.114 0.105 0.128 0.185 0.181 0.081 0 F-nt 0.518 0.503 0.540 0.480 0.368 0.524 0.583 0.615 0.496 0.216 0.295 0.178 0.153 0.210 0.165 0.222 0 F-li 0.475 0.627 0.557 0.388 0.354 0.490 0.447 0.539 0.565 0.204 0.144 0.041 0.175 0.161 0.089 0.129 0.169 0

consensus trees obtained from the NJ method has occurred between these two species. C. relatively lower in C. formosana Hayata. The were generally consistent with the consensus formosana Hayata was confirmed to be an overall values of H, Gst, Nm (including C. sinensis trees established through the MP method. By individual species with bootstrap values of 85 and Persoon and C. formosana Hayata) were 0.3339, integrating the clustering results of C. sinensis 90% in the MP and NJ trees, respectively. 0.820, and 0.1097, respectively. These results and C. formosana , MP yielded a bootstrap value In contrast, a higher level of within-species show that the gene flow among populations in of 11% for C. sinensis , showing a relatively high divergence was observed for C. sinensis Persoon. distinct regions is low. degree of differentiation in this species; The ISSR analysis of C. sinensis Persoon Theoretically, higher gene flow between species furthermore, MP yielded a bootstrap value of 85% conducted in POPGENE indicated that the values should lead to a lower degree of genetic differen- for C. formosana , indicating a relatively low level for genetic diversity (H), genetic differentiation tiation. The high level of genetic differentiation of genetic differentiation. Based on the PCR- (Gst), and gene flow (Nm) were 0.2521, 0.6887, between C. sinensis Persoon and C. formosana amplified ITS1-5.8S-ITS2 nucleotide sequences, and 0.2260, respectively (Table 3). These results Hayata observed in our experiments may have C. sinensis Persoon and C. formosana Hayata show that the gene flow between individual resulted from geographical barriers. were clustered into two individual groups in both populations of C. sinensis Persoon is low, that is, The AMOVA analysis of C. sinensis Persoon of the MP and NJ pedigree diagrams (Figure 3). there are high levels of genetic differentiation and C. formosana Hayata at the 18 sample sites These results indicated that some differentiation between regional groups. These values were showed that interspecific and intraspecific

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Figure 1. Geographic locations of the 18 sampled natural habitats of C. sinensis and C. formosana (S: C. sinensis , F: C. formosana ).

variations contributed 57.38% (p<0.0001) and 42.62% Hayata are genetically closely related to each other. (p<0.0001) of the total variation, respectively. These However, AMOVA analysis based on ISSR markers results indicate that the majority of the variation between showed that the majority of the variation among samples the two species was due to interspecific variation. from the two species was due to interspecific variation, Overall, the ISSR and ITS sequence analyses suggest and POPGENE showed a very clear differentiation that the differentiation of C. sinensis Persoon and C. between species. formosana Hayata may have occurred due to The Mantel test revealed a significant correlation geographical isolation, coupled with the continuing impact between genetic distance and the elevation of the of morphological variation. ITS sequence analysis collection site. Field studies have found that C. sinensis indicates that C. sinensis Persoon and C. formosana Persoon is mainly distributed at low elevations and that

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Figure 2. UPGMA dendrogram of C. sinensis and C. formosana based on ISSR bands (S: C. sinensis , F: C. formosana ).

Figure 3. Phylogenetic tree of C. sinensis and C. formosana generated with the (A) MP; and (B) NJ methods, based on ITS analysis (S: C. sinensis , F: C. formosana ).

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