Biologia, Bratislava, 61/3: 295—298, 2006 Section Cellular and Molecular Biology DOI: 10.2478/s11756-006-0054-4

Genetic diversity among geographically separated populations of mirabilis

Arunrat Chaveerach1*, Alongkod Tanomtong1, Runglawan Sudmoon2 & Tawatchai Tanee2

1Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, ; phone: ++ 66 4334 2908, fax: ++ 66 4336 4169, e-mail: [email protected] 2Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

Abstract: The distribution of Nepenthes mirabilis ranges from Northeast (NE) to South (S) Thailand. Eleven individuals from NE, S and Suen Jatujak market in Bangkok, Central (C) Thailand, were collected and divided into four populations according to their geographical areas. These four populations were analyzed to determine a genetic diversity profile using thirteen inter-simple sequence repeat markers. The individuals produced 75.18% polymorphic banding profiles. The Shan- non’s index was used to estimate genetic diversity. Total genetic diversity (HT) and inter-population genetic diversity (HS) were 0.854 and 0.678, respectively. The degree of genetic differentiation (GST) of the species populations is 0.206, whereas the gene flow (Nm) among all the various geographical area populations is 1.016. Both the dendrogram and the results of the Shannon’s diversity index suggest great genetic diversity. These results support the broad range of distribution sites of Nepenthes mirabilis, which would require high genetic diversity to adapt to the environmental variations that can be found between NE, C, and S Thailand. ANOVA shows that the genetic diversity in each population is not significantly different (P>0.05). Mantel tests reveal that geographical distance is an important factor for affecting the genetic distances among populations. Key words: genetic diversity, ISSR analysis, Nepenthes mirabilis, Shannon’s index.

Introduction lishing effective and efficient conservation and breeding practices. Nepenthes mirabilis Druce of the genus Nepenthes is Traditionally, morphological characters have been a dioecious with high economic value due to its used to characterize levels and patterns of diversity. unique carnivorous and attractive pitchers. The func- Since these traits represent only a small portion of tion of the pitcher, which forms from a swelling at the the plant genome and are influenced by environmen- tip of the mid-vein in the leaf, is to attract, kill, and tal factors, they have limited utility for describing the digest insects and absorb the breakdown products with potentially complex genetic structure, which may ex- which the plant augments its nutrient uptake from soil ist within and between taxa (Avise, 1994). Various (Moran, 1995). molecular approaches have been devised to overcome On account of their fascinating beauty, wild Ne- these constraints (Soltis &Soltis, 1990). A number penthes spp. are often taken from the forest to the of PCR-based DNA markers, including the random am- market place and hybridized to produce a diversity of plified polymorphic DNA (RAPD), simple sequence re- pitcher characters. Natural hybrids are possible when peat (SSR), inter-simple sequence repeat (ISSR), and two species flower at the same time and the pollen of amplified fragment length polymorphism (AFLP), have one species fertilizes the ovary of the other. However, been used widely to investigate population genetics. hybrid offspring rarely succeed to develop into a popu- ISSR marker has been proven to be extremely variable lation of their own (Clarke, 2002). As a result, a large and sensitive enough to differentiate cultivars and nat- number of wild Nepenthes spp. have been lost and are ural populations (Wolfe et al., 1998). These markers rarely found in local forests. Genetic diversity allows of genetic variation are generally independent of envi- species to adjust to a changing world, which is caused ronmental factors and more numerous than phenotypic by both natural and human factors. Understanding ge- characters, thereby providing a clearer indication of the netic diversity within the genus is essential for estab- underlying variation in the genome.

* Corresponding author

c 2006 Institute of Molecular Biology, Slovak Academy of Sciences 296 A. Chaveerach et al.

Table 1. A summary of ISSR primers, number of bands scored, number of polymorphic bands, and percentage of polymor- phisms for amplification profiles of four populations of Nepenthes mirabilis.

Primer sequence No. of band No. of poly- Percentage of 5’ to 3’ scored morphic bands polymorphism

(CA)6GG 12 8 66.67 (CA)6AC 11 11 100 (CA)6AG 10 9 90 (CT)8AC 11 11 100 (CT)8TG 10 6 60 (GA)6GG 10 6 60 (GA)6CC 11 4 36.36 (GT)6CC 10 6 60 (CAC)3GC 7 5 71.43 (CTC)3GC 10 7 70 (GACA)4 9 7 77.78 CCCC(GT)6 14 13 92.86 (GAG)3GC 12 10 83.33

Total 137 103 75.18

with 0.8% agarose gel electrophoresis stained with ethidium bromide and the quality and quantity of the DNA samples were determined by gel document instrument. Then, the DNA samples were diluted to a final concentration of about 20 ng in TE, and these dilutions were used as the DNA templates in PCR reactions.

ISSR analysis Amplifications were carried out in 25 µL reactions consisting Fig. 1. Map of Thailand indicating sampling provinces of popu- of Taq reaction buffer, 2 mM MgCl2, 0.3 mM of each dNTP, lation 1 (Bangkok), population 2 (Ranong), population 3 (Phang 0.3 µM primers, 1.25 units of Taq DNA polymerase (Invit- Nga) and population 4 (Nong Khai). rogen), and 5 ng DNA template. Thirty-five ISSR primers were used in PCR reactions. The 13 primers that success- fully amplified clear bands are listed in Table 1. ◦ This study aims to use the ISSR marker to assess The reaction mixture was incubated at 94 Cfor3min and the amplification was performed with the following ther- genetic diversity and divergence within and among four mal cycles: 35 cycles of denaturation for 1 min at 94 ◦C, 2 populations of Nepenthes mirabilis. Additionally, the min annealing temperature Tm −5 ◦C, 2 min at 72 ◦C, and gene flow is calculated to measure the total number of 7 min final extension at 72 ◦C. Amplification products were migrants per generation. detected by agarose gel electrophoresis in TAE buffer and visualized by ethidium bromide staining. All amplification reactions were repeated at least two times using a PCR ma- Material and methods  chine (GeneAmp PCR system 9700, Applied Biosystems).

Plant materials ISSR data analysis Young leaves of eleven individuals of N. mirabilis were The total number of ISSR bands discerned from the agarose collected from populations in various geographical areas, gel was documented as diallelic characters, present=1, ab- namely Suen Jatujak, a famous market in Bangkok, Cen- sent=0; the ISSR are considered the dominant markers. The tral Thailand (C), Nong Khai province, Northeast Thailand resulting bands were used to reconstruct a dendrogram fol- (NE), Ranong and Phang Nga provinces, South Thailand lowing the UPGMA with the DNA fingerprinting II pro- (S). The distances of Nong Khai to Bangkok, Bangkok to gram, version 3.0. The outgroup species is Ranong, and Ranong to Phang Nga provinces are 635, 555 Korth. The percentage of polymorphic loci was calculated and 230 km, respectively. The province locations are shown with the Shannon’s diversity index for each population, HO on a map of Thailand (Fig. 1). Leaf materials were immedi- = −Σ pi ln pi,wherepi is the frequency of a given ISSR ately dried using silica gel, then transported to the lab, and band and HO is diversity. HO was calculated at two levels: ◦ stored at −70 C until DNA extraction. the average genetic diversity within each population (HS) and the total genetic diversity (HT) in the pooled popula- DNA extraction tion. HT and HS were calculated using the modified formula: 2 Genomic DNA was extracted from dried leaves using the HT =1− Σ pi (NEI, 1978). The genetic diversity between DNeasy Plant Mini Kit (Qiagen). The DNA was evaluated populations was estimated using GST =(HT − HS)/HT Genetic diversity among populations of Nepenthes mirabilis 297

Table 2. Shannon’s index of genetic diversity (HO)withinfour populations of Nepenthes mirabilis from different geographical locations.

Primer sequence Pop.1 Pop. 2 Pop. 3 Pop. 4 5’ to 3’

(CA)6GG 0.6870 0.6853 0.7184 0.6701 (CA)6AC 0.6806 0.5623 0.6606 0.6585 (CA)6AG 0.6931 0.6730 0.6560 0.6888 (CT)8AC 0.5799 0.6902 0.7238 0.5998 (CT)8TG 0.6775 0.6365 0.7307 0.6768 (GA)6GG 0.6914 0.6914 0.7125 0.6916 (GA)6CC 0.6881 0.6616 0.7311 0.6931 (GT)6CC 0.6918 0.6829 0.7279 0.6897 (CAC)3GC 0.6902 0.6616 0.7324 0.6931 Fig. 2. A sample of ISSR banding pattern for primer (CAC)3GC. (CTC)3GC 0.6870 0.6870 0.7399 0.6906 (GACA)4 0.6853 0.6931 0.6371 0.6931 CCCC(GT)6 0.6931 0.6555 0.5840 0.6120 (NEI, 1973). The gene flow (Nm) was evaluated indirectly (GAG)3GC 0.6581 0.6920 0.7397 0.6271 by measuring the total number of migrants per generation, Average 0.6770 0.6670 0.6980 0.6680 Nm=1−FST/4FST (WRIGHT, 1931), where standard vari- ation FST is considered equivalent to GST (NEI, 1973). Anal- ysis of variance (ANOVA) was estimated using SPSS pro- gram. Mantel tests (MANTEL, 1976) were performed to ana- lyze the effects of geographical distance on genetic distance which shows close genetic relationships between popu- by using formula lation 1 and population 2, from Ranong province, and n n population 3, from Phang Nga province; genetic dis- 2 (xij − x¯) (yij − y¯) r = , tance of 22.7 to 26.9, respectively (Fig. 3). The genetic n(n − 1) Sx Sy i=1 j=1 relationships are significantly close, which suggests that the species is N. mirabilis (Chaveerach et al., 2002) where x and y are geographical and genetic distances at and from Ranong province. locations i and j, n is the number of data entries in the Genetic distances in the studied species ranges distance matrices, Sx and Sy are standard deviations for from 22.7 to 36.1. Population 3 from the S and popula- variable x and y. tion 4 from the NE have the great genetic distance and geographical distance between the collection areas. The Results lowest is between population 1 and 2, which are both from Ranong province in S Thailand. Accordingly, ge- The study reports that thirteen informative ISSR netic distance between population 1 or 2 and 3 is higher, primers generated a total of 137 bands, ranging in at 28.8 and 26.9, which agrees with the greater distance size from 100-2500 bp and averaging at 10.54 bands of geographical collection areas. Remarkably, the out- per primer. Of these bands, 75.18% (103 bands) were group, N. gracilis, from Phu Wua Wildlife Sanctuary in polymorphic. Percentages of polymorphic bands (PPB) Nong Khai province, NE Thailand, is closely related to for each primer ranged from 36.36 to 100%. Primer N. mirabilis from Bueng Khonglong non-hunting area CCCC(GT)6 produced the highest number of bands in the same province. This suggests an accordance of (14). The minimum number of bands (7) was produced geographical area. by anchored repeat (CAC)3GC. The primers (CA)6AC and (CT)8AC produced all 11 bands (Tab. 1). Figure Genetic differentiation 2 shows an example of ISSR DNA fingerprinting which Shannon’s index of phenotypic diversity was used to demonstrates the banding pattern differences between class the constituents within and between populations. individuals and populations of Nepenthes mirabilis. The results show that the genetic diversity within indi- viduals from varying geographical areas directly relates Clustering of individuals to the different primers and populations. The genetic di- A dendrogram was reconstructed based on the ISSR versity (HO) within these populations range from 0.562 scoring data and using UPGMA cluster analysis. N. of primer (CA)6AC in population 2 to 0.732 of primer gracilis is separated as outgroup with a bootstrap value (CAC)3GC in population 3 (Tab. 2). Between different of 97. The four populations are shown to be clustered populations, population 3 indicates a slightly greater according to geographical areas. average diversity of 0.698 and population 2 shows the Population 1 is an immature specimen from Suen least average diversity at 0.667. Within populations, the Jatujak market in Bangkok, C Thailand, which was means of the genetic diversity (HS) is 0.678, the total stated to originate from Ranong province, S Thai- genetic diversity (HT) is 0.854, and the degree of ge- land. This information is supported by the dendrogram, netic differentiation (GST) is 0.206. The gene flow or 298 A. Chaveerach et al.

Fig. 3. Dendrogram indicating genetic distance and depicting the thirteen ISSR primers produced by UPGMA analysis that is used to classify the four populations of Nepenthes mirabilis. the total number of migrants per generation among all among genetic diversity in each population. Mantel test populations (Nm) is 1.016. ANOVA shows that there is confirms the significant positive correlation among all no significant difference among genetic diversity in each four populations. In conclusion, the geographical dis- population (P>0.05). Mantel test shows a significant tances between populations affect the genetic distances. positive correlation between geographical and genetic While the study sample is small, a broad genetic di- distances among all four populations (r = 0.0606). versity is represented because the collection sites cover a wide range of areas from NE to S Thailand. Accord- Discussion ingly, the resulting values agree with conclusions found in the cited studies. These factors suggest that the re- The dendrogram and results of the genetic diversity sults of this study may be considered valid. within and between all four populations show good fac- tors for conservation and management of genetic diver- sity of the species. Additionally, the genetic diversity of References population 1 was identified as N. mirabilis from Ranong province, S Thailand. AVISE, J.C. 1994. Molecular Markers, Natural History and Evo- lution. Chapman and Hall, New York. The genetic diversity using ISSR markers indicates CHAVEERACH, R.A., KUNITAKE,H.,NUCHADOMRONG,S., a high polymorphic value of 75.18% in the geographi- SATTAYASAI,N.&KOMUTSU, H. 2002. RAPD patterns as a cally separated populations. The genetic differentiation useful tool to differentiate Thai Piper from morphologically alike Japanese Piper. Science Asia 28: 221–226. coefficient between all populations (GST) is 0.206. The CLARKE, C. 2002. A Guide to the Pitcher of Peninsular low value indicates that the proportion of total genetic Malaysia. Natural History Publications (), Kota Kin- diversity to population differentiation is low. There is abalu. a high total number of migrants per generation among FRANKEL, O.H., BROWN, A.H.D. & BURDEN, J.J. 1995. The all populations. This is interpreted from an indirect es- Conservation of Plant Biodiversity. Cambridge University Press, London. timate of gene flow (Nm) based on the degree of genetic MANTEL, N. 1976. The detection of disease clustering and gen- differentiation (GST). It demonstrates a high value of eralized regression approach. Cancer Res. 27: 209–220. 1.016 among the isolated geographical populations and MORAN, J.A. 1996. Pitcher dimorphism, prey composition and Ne- indicates high movement of genes from one population the mechanisms of prey attraction in the pitcher plant penthes rafflesiana in Borneo. Ecology 84: 515–525. to another, causing them to become more similar. The NEI, M. 1973. Analysis of gene diversity in subdivided popula- dendrogram supports the total genetic diversity (HT) tions. Proc. Natl. Acad. Sci. USA 70: 3321–3323. values of all populations and the means of genetic di- NEI, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: versity (HS), which are 0.854 and 0.678, respectively. 589–590. This shows a great genetic distance of 22.7 to 36.1 in SOLTIS,D.E.&SOLTIS, P.S. 1990. Isozymes in Plant Biology. a species. These values infer that the extended ecosys- Chapman and Hall, London. tems, NE to S Thailand, are reflected in the genetic drift WOLFE, A.D., XIANG,Q.Y.&KEPHART, S.R. 1998. Diploid hy- Penstemon in N. mirabilis, which tends towards higher allelic diver- brid speciation in (Scrophulariaceae). Proc. Natl. Acad. Sci.USA 94: 5112–5115. sity. Such a drift is a serious threat to the genetic vari- WRIGHT, S. 1931. Evolution in Mendelian populations. Genetics ability of natural populations (Frankel et al., 1995). 16: 97–159. ANOVA shows that there is no significant difference Received November 9, 2005 Accepted December 2, 2005