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Genetic differentiation in endangered

Gynostemma pentaphyllum (Thunb.)
Makino based on ISSR polymorphism and
its implications for conservation
ARTICLE in BIOCHEMICAL SYSTEMATICS AND ECOLOGY SEPTEMBER 2008
Impact Factor: 0.97 DOI: 10.1016/j.bse.2008.07.004

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Biochemical Systematics and Ecology 36 (2008) 699705

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Biochemical Systematics and Ecology

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Genetic differentiation in endangered Gynostemma pentaphyllum

(Thunb.) Makino based on ISSR polymorphism and its
implications for conservation
Chong Wang a, b, Hao Zhang a, b, Zeng-Qiang Qian c, Gui-Fang Zhao a, b, *
a

Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education), Northwest University, Xian 710069, PR China
College of Life Sciences, Northwest University, Xian, Shaanxi Province 710069, PR China
c
School of Marine and Tropical Biology, James Cook University, Townsville, QLD 4811, Australia
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 January 2008
Accepted 26 July 2008

Studies were performed to investigate the genetic variation of 14 natural populations of

Gynostemma pentaphyllum (Thunb.) Makino, an outcrossing clonal plant species in China,
using inter-simple sequence repeat (ISSR) markers. Fourteen selected primers were used to
amplify DNA samples from 140 individuals, and totally 194 loci were detected. The
percentage of polymorphic bands (PPBs) showed that the genetic diversity was pretty high
at the species level (PPB 96.39%) but quite low at the population level (PPB 1.03
25.26%). Shannons information index (I) and Neis gene diversity (h) displayed a similar
trend to PPB. According to the hierarchical analysis of molecular variance (AMOVA) and
Neis analysis of gene diversity, the percentages of genetic variation among populations
were 88.66 and 88.94%, respectively, indicating a high level of inter-population genetic
differentiation. The low levels of genetic diversity within populations and high genetic
differentiation among populations were assumed to result from the limited gene ow, the
clonal nature and genetic drift. Based on the genetic data, effective conservation strategies
were proposed for conserving this traditional Chinese medicinal herb. Concerning the
management of G. pentaphyllum, we suggested that in situ conservation be an important
and practical measure for maintaining the genetic diversity and that a possibly maximum
number of populations be conserved. Populations EMS and HLT, in which particularly low
levels of genetic variation were characterized, should be given the priority for ex situ
conservation.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Gynostemma pentaphyllum (Thunb.) Makino
Genetic diversity
Conservation
Inter-simple sequence repeats (ISSRs)

1. Introduction
Long-term survival and evolution of species depend on the maintenance of sufcient genetic variability within and among
populations to accommodate new selection pressures exerted by environmental changes (Barrett and Kohn, 1991). Loss of
genetic diversity usually threatens the survival of natural populations, and has dramatic effects on the evolutionary potential
of species (Reed and Frankham, 2003). Genetic diversity maintained in a plant species would be inuenced by many
processes, such as the long-term evolutionary history and the characteristics of the species, including genetic drift, gene ow,

* Corresponding author. College of Life Sciences, Northwest University, Xian, Shaanxi Province 710069, PR China. Tel.: 86 29 88305207; fax: 86 29
88303572.
E-mail address: guifang.zhao@gmail.com (G.-F. Zhao).
0305-1978/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2008.07.004

700

C. Wang et al. / Biochemical Systematics and Ecology 36 (2008) 699705

reproductive mode and mating system (Hamrick and Godt, 1989). Thus, an accurate estimate of genetic diversity of
endangered species would provide insights into the evolutionary process as well as information useful for developing
conservation plans to preserve genetic diversity (Falk and Holsinger, 1991).
Gynostemma pentaphyllum (Thunb.) Makino, a perennial creeping herb of the family Cucurbitaceae, widely disperses
throughout China, India, Myanmar, Korea and Japan (Blumert and Liu, 1999; Chen, 1995), and occurs naturally in forests, scrub
and bushes with an elevation of 2003200 m. In China, the natural populations of G. pentaphyllum distribute from drainage
areas of the Yangtze River to Yunnan Province (Chen, 1995). It consists of slender stems of thin, soft leaves arranged like
ngers on a hand, bearing 39 leaves (usually 57 leaves). The owers of G. pentaphyllum are dioecious and the pollen is
dispersed by wind. The fruit consists of a smooth, globular berry about 56 mm in diameter and two ovoid-heart shaped
seeds inside (Gao et al., 1995; Razmovski-Naumovski et al., 2005). This plant exhibits both sexual reproduction and clonal
growth by rhizomes or bulbils (Gao et al., 1995; Liu et al., 2001). As a traditional Chinese medicinal herb, G. pentaphyllum has
a variety of important biological functions, such as inhibiting tumor cell growth, anti-ulceration and enhancing immunity
(Zhou and Wang, 1998; Lin et al., 2000), and has been extensively used to treat chronic diseases. For example, the dammarane
saponins isolated from it, also known as gynosaponins, are believed to be the active components responsible for its various
biological activities and reported clinical effects (Razmovski-Naumovski et al., 2005). Phytochemical studies also identied in
it approximately 84 dammarane-type saponin glycosides (Yin et al., 2004), which are structurally similar to glycosides found
in Panax ginseng C.A. Mey. (Liu et al., 2005). The male-based sex ratio and the low rate of seed recruitment were also reported
(He and Zhong, 2000; Liu et al., 2005). With the growth of commercial demands in recent years, excessive exploitation has
shrunk the natural resource of G. pentaphyllum to a narrow distribution, and its survival has been seriously threatened.
Nowadays, it is considered to be rare and threatened in China and has been listed among Grade II Key Protected Wild Plant
Species (Yu, 1999). To date, previous studies have mainly focused on the resource distribution, its morphological characteristics, dynamics and pharmacological properties (Blumert and Liu, 1999; He and Zhong, 2000; Razmovski-Naumovski et al.,
2005), and no efforts have been reported on its genetic diversity. In order to formulate effective conservation strategies of G.
pentaphyllum, assessment of its genetic diversity and differentiation within and among populations is necessary.
Among various marker systems, inter-simple sequence repeats (ISSRs) use repeat-anchored primers to amplify DNA
sequences between two inverted SSRs (Zietkiewicz et al., 1994; Culley and Wolfe, 2001). Because of higher annealing
temperature and longer sequence of ISSR primers, they can yield reliable and reproducible bands, and the cost of the analyses
is relatively lower than that of some other markers such as AFLPs. Therefore, ISSRs have established wide applications in
genetic diversity studies in a wide range of plant species (Ge et al., 2005; Chen et al., 2006; Han et al., 2007).
In the present study, using ISSR markers, we aimed to: (1) estimate the genetic diversity in G. pentaphyllum populations;
(2) reveal the partitioning of the genetic diversity within and among populations; and (3) suggest the conservation strategies
for this endangered medicinal herb.
2. Materials and methods
2.1. Plant sampling
According to the previous eld survey, a total of 14 natural populations of G. pentaphyllum were sampled across nine
provinces in China, which represented a wide geographic distribution, and the distribution range is from 132 to 2560 m in
elevation (Table 1; Fig. 1). Ten individuals were collected randomly from each population, and the distances between
neighboring samples were at least 5 m apart to avoid sampling from the same rhizomes. Young leaf tissues were stored in ziplock bags with silica gel and transported back to the laboratory for DNA extraction. In addition, corresponding to each
population, longitude and latitude were determined and recorded for further analysis.
2.2. DNA extraction
Genomic DNA was extracted using a modication of the protocol of Doyle and Doyle (1987). DNA quality and quantity were
determined in 0.7% agarose gels. The DNA samples were diluted to 20 ng/ml and stored at 20  C for use.
2.3. ISSRPCR amplication
ISSR primers used in this study were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co.,
Ltd., according to the primer set published by University of British Columbia (UBC) (http://www.michaelsmith.ubc.ca/
services/NAPS/Primer_Sets/Primers_Oct2006.pdf). Fifty-seven primers were initially screened for PCR amplication, and 14
of them, which yielded clear and reproducible banding patterns, were selected for nal analysis (Table 2). PCRs were performed in a 20 ml reaction volume containing 20 ng of genomic DNA, 1.0 U of Taq polymerase, 2.0 mM MgCl2, 0.2 mM dNTP,
1.0 mM primer and 1  PCR buffer, which were determined after comparison and optimization.
PCR amplications were performed in a PTC-200 thermocycler (MJ Research), with the following program: an initial step
of 5 min at 94  C, followed by 40 cycles of 30 s at 94  C, 45 s at appropriate annealing temperature (see Table 2 for details) and
2 min at 72  C, and a nal 2 min extension step at 72  C. The amplication products were separated in 1.5% agarose gels
stained with ethidium bromide, visualized in ultraviolet light, and photographed with Kodak Electrophoresis Documentation

C. Wang et al. / Biochemical Systematics and Ecology 36 (2008) 699705

701

Table 1
Sampling details of G. pentaphyllum populations in the present study
Populations
CS
EMS
FJS
FX
GGJ
HLT
LH
LY
QCS
SWD
TYD
XX
YFG
ZJS

Sampling locations

Latitude (N )

Longitude (E )

Altitude (m)

Sample size

Mt. Cang, Dali, Yunnan Province

Mt. Emei, Sichuan Province
Mt. Fanjing, Guizhou Province
Fang Town, Hubei Province
Gaoguang Village, Jishou, Hunan Province
Heilongtan, Kunming, Yunnan Province
Linghu, Lingbao, Henan Province
Lingyin, Hangzhou, Zhejiang Province
Mt. Qingcheng, Chengdu, Sichuan Province
Mt. Saiwudang, Shiyan, Hubei Province
Taoyuandong Forest, Zhuzhou, Hunan Province
Mt. Funiu, Xixia Town, Henan Province
Yaofu Village, Ankang, Shaanxi Province
Mt. Zijin, Nanjing, Jiangsu Province

25 410

100 070
103170
108 460
110 300
110 040
102 440
110 850
120 050
103 340
110 450
114 010
112 120
109 18
118 490

2560
882
538
700
840
1943
469
132
924
936
651
800
482
133

10
10
10
10
10
10
10
10
10
10
10
10
10
10

29 350
27 500
31540
28 390
25 080
34 520
30 140
30 540
32 260
26 290
32 480
32 2300
32 030

and Analysis System 120 (Eastman Kodak Company). Molecular weights were estimated using the DNA ladder DL2000
(Takara Biotech Co., Ltd.).
2.4. Data analysis
Amplied bands were scored in a size range from 200 to 2000 bp. ISSR bands were scored as presence (1) or absence (0) for
each DNA sample. The resulting binary data matrix was analyzed using POPGENE Version 1.32 (Yeh et al., 1997). Genetic
diversity within and among populations was measured by the percentage of polymorphic bands (PPBs), the effective number
of alleles (ne), observed number of alleles (na), Neis gene diversity (h) and Shannons information index (I). Genetic differentiation among populations was estimated by gene differentiation coefcient (Gst) (Nei, 1973). Corresponding estimates of
gene ow (Nm), i.e. the average per generation number of migrants exchanged among populations, was related to Gst
according to the equation Nm (1/Gst  1)/4 (Nei, 1973). All these calculations assumed that populations are in Hardy
Weinberg equilibrium. Genetic divergence between populations was investigated using Neis (1978) unbiased genetic
distances and genetic identities. Neis unbiased genetic distances were calculated for all population pairs and used to
construct a UPGMA dendrogram.

Fig. 1. Geographic distribution of the 14 sampled populations of G. pentaphyllum in China. For population abbreviations, see Table 1 for details.

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C. Wang et al. / Biochemical Systematics and Ecology 36 (2008) 699705

Table 2
ISSR Primers used in this study and analysis of ISSR-generated banding patterns
Primers

Sequence (50 / 30 )

Annealing temperature ( C)

No. of loci scored

Percentage of polymorphic
bands (PPB, %)

UBC826
UBC854
UBC857
UBC861
UBC862
UBC864
UBC865
UBC873
UBC880
UBC887
UBC888
UBC889
UBC891
UBC895

(AC)8C
(TC)8RG
(AC)8YG
(ACC)6
(AGC)6
(ATG)6
(CCG)6
(GACA)4
(GGAGA)3
DVD(TC)7
BDB(CA)7
DBD(AC)7
HVH(TG)7
AGA GTT GGT AGC TCT TGA TC

49
51
47
63
57
54
77
51
48
51
49
49
45
54

11
13
13
14
14
10
12
15
19
14
18
14
12
15

90.91
100
100
100
92.86
100
91.67
100
78.95
100
100
100
100
100

Note: R A/G; Y C/T; D A/G/T; V A/C/G; B C/G/T; H A/C/T.

The additional measurement for partitioning genetic variation was obtained with the hierarchical analysis of molecular
variance (AMOVA) analysis, using WINAMOVA 1.55 (Excofer et al., 1992). To test the correlation between genetic and
geographic distances (in km) among populations, Mantel test was performed using the program Mantel 2.0 (Liedloff, 1999).
3. Results
3.1. Genetic diversity
Totally, 14 primers amplied 194 loci, with an average of 13.86 per primer. The size of the ISSR fragments ranges from 200
to 2000 bp. At the population level, the percentages of polymorphic bands (PPBs) ranged from 1.03 to 25.26%, with an average
of 8.91%, while at the species level, this value rose sharply to 96.39% (Table 3). Neis gene diversity (h) varied between 0.0040
and 0.0842, with an average of 0.0257, and Shannons information index (I) ranged from 0.0061 to 0.1288, with an average of
0.0405. The values of h and I showed a similar trend to PPB. When calculated at the species level, the h and I values equaled
0.2624 and 0.4074, respectively. Among the 14 populations investigated, the highest and lowest levels of genetic variability
occurred in population ZJS (PPB: 25.26%; h: 0.0842; I: 0.1288) and population EMS (PPB: 1.03%; h: 0.0040; I: 0.0061),
respectively.
3.2. Genetic differentiation
According to Neis analysis of gene diversity and the AMOVA analysis (Table 4), the percentages of genetic variation among
G. pentaphyllum populations were 88.94% (Gst) and 88.66% (vst), respectively, both of which indicated a high inter-population
genetic differentiation. This was also conrmed by the extremely limited gene ow among populations (Nm 0.0622).
Mantel test revealed no signicant correlation between genetic and geographic distance matrices (r 0.1628, P > 0.05).
3.3. Genetic relationships
To further reveal the relationships among populations, cluster analysis (UPGMA) was used to generate a dendrogram
based on Neis genetic distances (Fig. 2). Fourteen populations of G. pentaphyllum were clustered into four groups. Group I
comprised six populations (population XX, FX, LY, QCS, LH and SWD) from higher latitudes and population FJS from lower
latitude, while populations CS, EMS, HLT, GGJ and TYD from lower latitudes formed Group II. Populations ZJS and YFG, which
showed relatively distant genetic relationship with the others, formed the other two separate clusters.
4. Discussion
4.1. Genetic diversity and differentiation
The present study revealed a relatively higher level of genetic diversity in G. pentaphyllum compared with other Cucurbitaceae species based on ISSR markers. At the species level, a higher PPB value was detected in this species (PPB 96.39%)
than in Momordica charantia L. (PPB 74.7%) (Behera et al., 2008), in Cucumis melo L. (PPB 65.51%) (Liu et al., 2002) and in
Siraitia grosvenorii (Swingle) C. Jeffery (PPB 82.0%) (Peng et al., 2005). Compared with the high level of inter-population
genetic variability, relatively low genetic diversity occurred within populations. At the population level, the PPB values ranged

C. Wang et al. / Biochemical Systematics and Ecology 36 (2008) 699705

703

Table 3
Genetic diversity within populations of G. pentaphyllum
Populations

Observed no.
of alleles (na)

Effective no.
of alleles (ne)

Percentage of polymorphic
bands (PPB, %)

Neis gene
diversity (h)

Shannons information
index (I)

ZJS
FJS
QCS
XX
LH
SWD
YFG
GGJ
TYD
CS
FX
LY
HLT
EMS

1.2737
1.1804
1.1889
1.1495
1.0979
1.0979
1.0789
1.0549
1.0610
1.0361
1.0361
1.0309
1.0206
1.0118

1.1410
1.0869
1.0695
1.0620
1.0412
1.0412
1.0172
1.0172
1.0146
1.0180
1.0107
1.0159
1.0191
1.0072

25.26
18.04
17.53
14.95
9.79
9.28
6.19
5.15
5.15
3.61
3.61
3.09
2.06
1.03

0.0842
0.0536
0.0444
0.0395
0.0268
0.0323
0.0135
0.0125
0.0113
0.0109
0.0075
0.0094
0.0099
0.0040

0.1288
0.0831
0.0717
0.0626
0.0426
0.0502
0.0241
0.0208
0.0199
0.0167
0.0127
0.0144
0.0139
0.0061

Average
Species level

1.0953
1.9639

1.0410
1.4264

8.91
96.39

0.0257
0.2624

0.0405
0.4074

from 1.03 to 25.26%, displaying a similar trend to the parameters h and I. Besides, Neis analysis of gene diversity and AMOVA
analysis revealed that the vast majority of genetic variation occurred among populations (Gst 0.8894, fst 0.8866).
The genetic structure of plant populations reects the interactions of various processes such as the long-term evolutionary
history of the species (e.g. shifts in distribution, habitat fragmentation, and population isolation), mutation, gene ow, genetic
drift and selection (Schaal et al., 1998). In population genetics, a value of gene ow (Nm) < 1.0 is generally regarded as the
threshold quantity beyond which signicant population differentiation occurs (Slatkin, 1987). In this study, the effective gene
ow for G. pentaphyllum (Nm 0.0622) was extremely lower than one successful migrant per generation, which might partly
result from low seed production and the large geographic distances among populations.
On one hand, as a dioecious species, G. pentaphyllum exhibits both sexual reproduction and clonal growth, but the rate of
seed recruitment is quite low (Gao et al., 1995; Liu et al., 2001). The unbalanced sex ratio may contribute to the low rate of
seed recruitment and the migration by seeds would be relatively rare under natural conditions. Furthermore, the unbalanced
sex ratio means that part of the individuals lose the chance for sexual reproduction, which leads to a shrinking effective
population size (Ne). Two major long-run genetic consequences of small effective population sizes are high levels of genetic
drift and inbreeding (Ellstrand and Elam, 1993), in agreement with the vast majority of genetic variation among populations.
On the other hand, the low levels of gene ow are also consistent with the geographic isolation of its populations. In this
study, the smallest inter-population distance was 126 km, which largely impeded the exchange of seeds or pollen among
populations. The harsh climate and mountainous environments are probable barriers to pollinator migrations and seed
dispersal. The pairwise genetic distances between the 14 populations were not correlated with geographic distances
(r 0.1628, P > 0.05), indicating that there was no clear geographic tendency in the distribution of the genetic variability and
that the isolation-by-distance model could not explain diversity patterns detected in this study. Besides, the low genetic
variation within populations may be partly attributed to the clonal propagation of this species. G. pentaphyllum exhibits both
sexual reproduction and clonal growth by rhizomes or bulbils (Gao et al., 1995; Liu et al., 2001). Expansion to new sites by
vegetative means is highly unlikely (Esselman et al., 1999), but the number and spatial distribution of genets could decide the
genetic structure of species. Based on the ISSR data and our eld survey of habitats, the low levels of heterozygosity within
populations and high genetic differentiation among populations probably resulted from limited gene ow, clonal nature of
this species and genetic drift.
4.2. Implications for conservation
Successful management and preservation of populations of threatened species depend on a good understanding of the
species, including levels and distribution of genetic variation (Francisco-Ortega et al., 2000; Wallace, 2002). Our ndings give
a gloomy implication for G. pentaphyllum genetic structure.

Table 4
Analysis of molecular variance (AMOVA) within/among populations of G. pentaphyllum
Source of variation

d.f.

Variance component

Percentage

fst

P value

Among populations
Within populations

1
119

23.61
3.01

88.66
11.34

0.8866

<0.001
<0.001

Note: Levels of signicance are based on 1000 iteration steps, data include degrees of freedom (d.f.), variance component estimates, percentage of total
variance (% total) contributed by each component and signicance of variance (P value).

704

C. Wang et al. / Biochemical Systematics and Ecology 36 (2008) 699705

Fig. 2. UPGMA dendrogram based on Neis (1978) unbiased genetic distances from ISSR data.

Considering the high genetic differentiation and the limited gene ow in this species, the conservation should ensure that
most of the populations are preserved to conserve the vast majority of variations. In situ conservation consists of a set of
measures aimed at restoring the maximum evolutionary potential, which pays more attention to restoring the suitable
habitat and the effective population size. For those populations with high levels of genetic variation such as ZJS and FJS, we
suggest that their habitats be protected and that human disturbance be avoided to allow them to increase in size through
natural regeneration. Ex situ conservation can also be achieved, e.g. by transplanting seedlings from different populations to
articially increase the gene ow among populations. According to the eld survey, in the past decades, the over-exploitation
of natural populations and the extensive loss of habitats have seriously threatened the survival of populations EMS and HLT,
which should be given the highest priority for the ex situ conservation plan.
Acknowledgements
The authors would like to thank Prof. Shu-Kun Chen of the Chinese Academy of Sciences for his valuable comments and
suggestions. We are also grateful to Prof. Yan-Sheng Chen for sample identication, and to Dr. Shi-Biao Liu for his assistance in
eldwork. The research was nancially supported by the Program for Changjiang Scholars and Innovative Research Team in
University (PCSIRT).
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