全 文 ：Received 4 Jun. 2003 Accepted 22 Sept. 2003
Supported by the grants from the National Ministry of Education Foundation for Ph.D Program (20010558004).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 515-521
Genetic Diversity of Sonneratia alba in China Detected by Inter-simple
Sequence Repeats (ISSR) Analysis
LI Hai-Sheng 1, 2*, CHEN Gui-Zhu 1
(1. Institute of Environmental Sciences, Zhongshan University, Guangzhou 510275, China;
2. Department of Biology, Guangdong Education Institute, Guangzhou 510310, China)
Abstract: Sonneratia alba J. Smith. is a widely distributed mangrove species. Leaf samples of one
hundred individuals from four natural and one introduced populations in Hainan Island, China, were collected.
Genetic variation among the five populations was evaluated using inter-simple sequence repeats (ISSR)
markers. Eleven ISSR primers gave rise to 133 discernible DNA fragments of which 103 (77.44%) were
polymorphic, indicating a considerable genetic variation at the species level. However, the polymorphism at
the population level was relatively low, with the percentage of polymorphic bands from 51.88% to 65.41%.
The mean expected heterozygosity and Shannon’s information index were 0.227 1 and 0.348 9
respectively at the species level, 0.183 7 and 0.277 5 at the population level. Based on Nei’s Gst value, a
large proportion of genetic variance (81.02%) was resided among individuals within populations, however,
only 18.98% genetic variance was resided among populations. AMOVA analysis also indicated a similar
genetic structure. The genetic identity between populations was 0.934 2 on average. UPGMA cluster
analysis based on Nei’s genetic distance divided the populations into two main groups: Lingshui population
(LS) and Sanya population (SY) formed one group, Qionghai population (QH), Wenchang population (WC) and
Dongzhai Harbor population (DZ) were in the other group. The Mantel test showed that genetic distance
was significantly correlated with geographical distance.
Key words: Sonneratia alba ; genetic diversity; ISSR; Hainan Island; conservation
Mangroves are trees and shrubs growing in the inter-
tidal zone of tropical and subtropical coastlines of the world.
There are about 70 mangrove species belonging to 30 gen-
era and 20 families（Duck, 1992）. They play an impor-
tant role in coastline wetland ecosystems. They can help to
stabilize shores and prevent excessive shifting of coast-
lines and soil erosion resulting from tidal currents and wave
action. Mangroves also buffer the destructiveness of wind
and storm tides during hurricane and typhoons. In addi-
tion to their ecological importance, they also provide many
forest products, such as firewood, timber, materials for
making boats and paper, edible fruits, and feeding grounds
for fish, prawns and shellfish. Mangrove forests, all over
the world are heavily exploited for wood and fish-pond
operations, as well as other activities. These problems are
most serious in China. This overexploitation of mangroves
has led to losses of valuable plant resources, that in turn
may lead to losses of genetic diversity as well. In order to
have a successful mangrove conservation program, breed-
ing program, or afforestation scheme, attention needs to be
paid to the genetic diversity and structure within and among
populations.
Sonneratia alba, a widely distributed mangrove species,
extends from east Africa and Madagascar to Southeast Asia,
the Malay Archipelago to the Philippines, and tropical Aus-
tralia to Micronesia, the New Hebrides and New Caledonia
(Tomlinson, 1986). In China, it is the most widely distrib-
uted species of the Sonneratia species. From north to south,
it is distributed along the eastern coastline of Hainan Island.
However, S. alba has become a threatened species owing
to people’s activities, especially for fish-pond operations
(Yu et al., 1998).
Inter-simple sequence repeats (ISSR) is a newly devel-
oped modification of SSR-based marker systems
(Zietkiewicz et al., 1994). It offers a lot of advantages, such
as low quantities of template DNA required, no need of
sequence data for primer construction, random distribu-
tion throughout the genome, generation of many informa-
tive bands per reaction, etc. ISSR has been proved to be
useful in population genetic diversity studies (Esselman et
al., 1999; Ge and Sun, 1999; Huang and Sun, 2000; Camacho
and Liston, 2001; Li and Ge, 2001). Therefore, in the present
study, the ISSR technique was used to investigate the ge-
netic diversity of S. alba in China in order to provide the
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004516
baseline information for the development of a conserva-
tion program for the species.
1 Materials and Methods
1.1 Sampling
A total of 100 individuals of Sonneratia alba J. Smith.,
representing four natural populations (Lingshui Popula-
tion (LS), Sanya Population (SY), Qionghai Population (QH),
Wenchang Population (WC)) and one introduced popula-
tion (Dongzhai Harbor Population (DZ)), were sampled
throughout the species’ entire range in China (Table 1; Fig.
1). Young leaf materials from selected trees were collected
in each population and were stored with silica gel in zip-
lock plastic bags until use. In 1994, the seeds of S. alba
were collected from Qinglan Mangrove Reserve and nursed
in the nursery of Dongzhai Harbor. Two years later, the
seedlings were planted in Linjiawan, Dongzhai Harbor with
other Sonneratia species. The individual number of the
population (DZ) is about 30, with 4 m in height on average.
1.2 DNA isolation and PCR amplification
Total DNA was isolated from silica gel dried leaves by
the CTAB method (Doyle and Doyle, 1987). One hundred
ISSR primers from the Biotechnology Laboratory, Univer-
sity of British Columbia (UBC set No.9) were initially
screened. Eleven of the primers (Table 2) were used to pro-
vide polymorphic markers for genetic diversity. Each 10 µL
amplification reaction consisted of 3 mmol/L MgCl2, 300
µmol/L dNTPs, 0.3 µmol/L primer, 1 U Taq DNA polymerase
(Genda Tech. Corp., Toronto, Canada) and 10 ng template
DNA. Amplification was performed in a PTC100TM Program-
mable Thermal Controller (MJ Research Inc., USA) under
the following cycle profile: 5 min at 94 ℃, followed by 45
cycles of 45 s at 94 ℃, 45 s annealing at 52.0-53.5 ℃
(depending on primers used) (Table 2), and 1.5 min exten-
sion at 72 ℃, and ended with 7 min at 72 ℃ for a final
extension. The amplification products were eletrophoresed
Table 1 The general situation of Sonneratia alba sampling populations
Population code Population location Latitude Longitude No. of samples
SY Qingmei Harbor, Sanya 18º13′43″N 109°36′57″E 20
LS Xincun Harbor, Lingshui 18º26′16″N 110°01′58″E 21
QH Futian, Qionghai 19º13′58″N 110°36′16″E 19
WC Xiachangcun, Wenchang 19º37′06″N 110°47′52″E 20
DZ Linjiawan, Dongzhai Harbor 19º57′11″N 110°35′11″E 20
DZ, Dongzhai Harbor Population; LS, Linshui Population; QH, Qionghai Population; SY, Sanya Population; WC, Wenchang Population.
Fig.1. Map showing the five sampled population locations (shown with circles) of Sonneratia alba included in this study. Abbreviations
are the same as in Table 1.
LI Hai-Sheng et al.: Genetic Diversity of Sonneratia alba in China Detected by Inter-simple Sequence Repeats (ISSR) Analysis 517
on 1.5% agarose gels buffered with 0.5×TBE, and detected
by staining ethidium bromide. Band size was estimated from
a 100 bp DNA ladder (Shenggong Inc., Shannghai, China).
1.3 Data analysis
ISSR bands were used to assign loci for each primer and
scored as presence (1) and absence (0). The band pres-
ence/absence data matrix was analyzed by POPGENE ver-
sion 1.31 (Yeh et al., 1999), making the assumption that the
populations were in Hardy-Weinberg equilibrium at these
ISSR marker loci. The estimates included the percentage of
polymorphic bands (P), observed number of alleles (Na),
effective number of alleles (Ne), the mean expected het-
erozygosity (h) (Nei, 1973), Shannon’s information index of
diversity (I). At the species level, genetic diversity mea-
sures total gene diversity (Ht), gene diversity within popu-
lation (Hs), coefficient of gene differentiation (Gst), aver-
age gene identity (I), and gene flow (Nm). A dendrogram
was generated with Nei’s genetic distance (Nei, 1972) and
the UPGMA method using the software NTSYS pc2.02
(Rohlf, 1998). The analysis of molecular variance (AMOVA)
was also used to partition the total phenotypic variance
into within and among populations (Excoffier et al., 1992).
The input files for AMOVA were prepared by the aid of
AMOVA-PREP version 1.01 (Miller, 1998). A Mantel test
(Mantel, 1967) was performed to estimate the association
between two independent dissimilarity matrices: genetic
distance and geographical distance.
2 Results
2.1 ISSR profile
For the 100 individuals of S. alba from the five
populations, 11 primers produced a total of 133 replicated
bands, of which 101 (77.44%) were polymorphic. The bands
per primer produced ranged from 8 to 18, and average band
loci per primer was 12.1 (133/11, Table 2). The size of the
amplified fragments ranged from 270 bp to 2 000 bp. Each of
the 100 individuals presented a unique ISSR genotype, in-
dicating an extensive genetic variation in the populations
studied. Figure 2 shows the ISSR PCR fingerprints of 23
samples using primer 868.
2.2 Genetic diversity and differentiation
The percentage of polymorphic bands (P) within popu-
lations ranged from 51.88% (QH) to 65.41% (LS), with an
average of 57.74% (Table 3). Interestingly, the P value of
the introduced population (DZ) in Dongzhai Harbor was
higher (59.40%) than that of its source population (WC,
54.14%). The mean expected heterozygosity values ranged
from 0.169 7 (SY) to 0.210 1 (LS). The Shannon’s informa-
tion index of diversity (I) also shows an identical trend
(Table 3). In addition, total gene diversity (Ht), gene diver-
sity within population (Hs), and between populations (Dst),
coefficient of gene differentiation (Gst), average gene iden-
tity (I), and gene flow (Nm) are shown in Table 4
respectively. The distribution of gene diversity revealed a
large proportion of gene differentiation (81.02%) based on
the difference of individuals within populations, whereas
only 18.98% among populations based on their location
sites. The AMOVA analysis provides a similar result (Table
5). Nei’s genetic identity and genetic distance are shown in
Table 6. The values of the gene identity range from 0.897 3
to 0.962 6 (Table 6) with a mean of 0.934 2 (Table 4).
Table 2 Name of inter-simple sequence repeats (ISSR) primers, sequence, annealing temperature and bands after amplification
Primer Sequence of primer Annealing temperature (℃) No. of bands scored No. of polymorphic bands
808 (AG)8C 53.5 10 8
809 (AG)8G 52.0 12 8
835 (AG)8(CT)C 53.5 12 9
836 (AG)8(CT)A 52.0 8 4
840 (GA)8(CT)T 53.0 13 11
842 (GA)8(CT)G 53.0 12 9
847 (CA)8(AG)C 53.5 8 7
857 (AC)8(CT)G 52.0 18 17
864 (ATG)6 53.5 14 12
868 (GAA)6 52.5 13 8
887 (AGT)(ACG)(AGT)(TC)7 52.0 13 8
Total 133 101
Fig.2. Inter-simple sequence repeats (ISSR) PCR fingerprints
of 23 samples of Sonneratia alba using primer 868. 1-6, samples
from Sanya Population; 7-23, samples from Linshui Population;
M, 100 bp ladder.
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004518
2.3 Cluster analysis
On the basis of the genetic distances, a dendrogram of
five populations was generated by using UPGMA cluster
analysis. The five populations were divided into two main
groups: LS and SY were in one group, QH, WC and DZ
were in the other group (Fig.3). The Mantel test showed
that genetic distance was significantly correlated with geo-
graphical distance (r = 0.934 8, P = 0.021 0).
3 Discussion
3.1 Genetic diversity
Compared with other mangrove species studied (P:
Table 3 The genetic variation statistics among populations of Sonneratia alba (Mean ± SD)
Population Na Ne h I P
SY 1.578 9±0.495 6 1.281 7±0.347 0 0.169 7±0.188 6 0.260 7±0.269 9 57.89
LS 1.654 1±0.477 4 1.357 6±0.371 0 0.210 1±0.198 8 0.316 7±0.281 1 65.41
QH 1.518 8±0.501 5 1.289 7±0.362 3 0.170 4±0.196 6 0.256 8±0.281 7 51.88
WC 1.541 4±0.500 2 1.299 0±0.373 3 0.173 8±0.199 8 0.261 5±0.284 1 54.14
DZ 1.594 0±0.501 5 1.336 0±0.382 1 0.194 5±0.202 9 0.291 8±0.286 9 59.40
Average 1.577 4±0.052 2 1.312 8±0.032 5 0.183 7±0.017 9 0.277 5±0.026 0 57.74
All locus 1.774 4±0.419 5 1.377 9±0.358 0 0.227 1±0.185 5 0.348 9±0.257 7 77.44
h, mean expected heterozygosity; I , Shannon’s information index; Na, observed number of alleles; Ne, effective number of alleles; P, the
percentage of polymorphic bands. Abbreviations are the same as in Table 1.
Table 4 Genetic diversity estimated by inter-simple sequence
repeats (ISSR) markers
Parameters Mean ± SD
Percentage of polymorphic loci (P) 77.44%
Total gene diversity (Ht) 0.266 7±0.034 4
Gene diversity within population (Hs) 0.183 7±0.023 6
Gene diversity between populations (Dst) 0.083 0±0.010 8
Coefficient of gene differentiation (Gst) 0.189 8
Average gene identity (I) 0.934 2±0.023 7
Gene flow (Nm*) 1.067 4
*, Nm = (1-Gst)/4Gst.
Table 5 Analysis of molecular variance (AMOVA) for 100 individuals in five populations of Sonneratia alba using 133 inter-simple
sequence repeats (ISSR) markers
Source of variation d.f. SSD MSD Variance component Total (%) P-value*
Among population 4 268.54 67.135 2.815 20.61 <0.001
Within population 95 1 030.36 10.846 10.846 79.39 <0.001
*, significance tests after 1 000 permutations.
Table 6 Nei’s (1972) genetic identity (above diagonal) and genetic distance (below diagonal) of Sonneratia alba
Population SY LS QH WC DZ
SY - 0.961 1 0.916 2 0.907 0 0.897 3
LS 0.039 7 - 0.931 5 0.922 0 0.933 2
QH 0.087 5 0.070 9 - 0.953 3 0.958 2
WC 0.097 6 0.081 0 0.047 8 - 0.962 6
DZ 0.108 4 0.069 2 0.042 7 0.038 1 -
Abbreviations are the same as in Table 1.
7.7% for Rhizophora stylosa, Goodall and Stoddart, 1989;
P: 20.0%, h: 0.04 for Kandelia candel, Sun et al., 1998; P:
4.76%, h: 0.024 for Aegeceras corniculatum, Ge and Sun,
1999), results from the present study using ISSR markers
revealed a relatively higher diversity in S. alba . The per-
centage of polymorphic bands at species level was 77.44%,
at population level ranged from 51.88% to 65.41%, and av-
eraged 57.74%. Genetic diversity was 0.348 9 using
Shannon’s information index and 0.266 7 for Nei’s genetic
diversity measure (Ht). Firstly, the result was associated
with the method detected. ISSR markers involve the PCR
amplification of DNA using a single primer composed of
Fig.3. Dendrogram of UPGMA cluster analysis based on Nei’s
(1972) genetic distances among five populations of Sonneratia
alba. Abbreviations are the same as in Table 1.
LI Hai-Sheng et al.: Genetic Diversity of Sonneratia alba in China Detected by Inter-simple Sequence Repeats (ISSR) Analysis 519
microsatel lite sequences. These primers target
microsatellites that are abundant throughout the eukary-
otic genome (Tautz and Renz, 1984; Kijas et al., 1995) and
evolve rapidly (Levinson and Gutman, 1987). So, the marker
are much more informative and powerful tools to detect
polymorphism than other molecular markers (Nagoka et al.,
1997; Tani et al., 1998; Esselman et al., 1999; Qian et al.,
2001). On the basis of its life history characteristics, S.alba
is a widespread, long-lived, woody, perennial species, and
the combination of these life history traits enable the spe-
cies to maintain a high level of genetic diversity. However,
based on ISSR, Ge and Sun (1999) examined genetic diver-
sity of Ceriops tagal from Thailand and China, and found
that P and h were 8.96% and 0.016, respectively. ISSR di-
versity for A. corniculatum was also very low at the specifio
level (P: 16.18%, h: 0.039) (Ge and Sun, 2001). Lakshmi et al.
(1997) suggested that different species of mangrove were
likely to display varying degrees of polymorphism depend-
ing on their edaphic preferences and adaptations.
3.2 Population genetic structure
Based on Gst value and AMOVA analysis, most of the
genetic variations of S. alba were within populations
(81.02% and 79.39% respectively), similar to some other
mangroves using the same ISSR molecular marker, such as
A. corniculatum (82.2%) (Ge and Sun, 1999) and Heritiera
littoralis (76.06%) (Jian et al., 2002). The population ge-
netic structure of a species is affected by a number of evo-
lutionary factors including its mating system, gene flow,
seed dispersal, and its mode of reproduction as well as its
natural selection (Hamrick and Godt, 1990). The mating sys-
tem plays a critical role for the population genetic structure.
Although ISSR markers can potentially distinguish many
individuals, they unfortunately do not provide direct infor-
mation on the mating-system due to their dominant inherit-
ance (Wolfe and Liston, 1998). However, according to
allozyme-based Gst values, average Gst<19% for outcross-
ing species compared with 51% for inbreeders (Hamrick
and Godt, 1990). RAPD-based Gst values are available for
35 plants species, with an average of 15.5% for 27 out-
crossing species, and 59.6% for eight inbreeding species
(Bussell, 1999). Therefore, compared with the proportion of
genetic diversity found among populations of outcrossing
species as presented above, S.alba is probably an out-
crossing species. S.alba can be pollinated by bats, birds
(Stafford-Deitsch, 1996), hawk moths (Primack et al., 1981)
etc., which may also to some extent show that S. alba has
an outcrossing mating system. For S.alba, the indirect es-
timation of the level of gene flow based on Gst was moder-
ate (Nm = 1.067 4), which means that the numbers of
migrants per generation are greater than one successful
migrant, and the level of genetic diversity maintained within
a population is less susceptible to genetic drift. A migration
rate of 0.5 was considered sufficient to overcome the diver-
sifying effects of random drift (Ellstrand and Elam, 1993).
The Nm value of 1.067 4 is near to the average value re-
ported for outcrossed animal-pollinated species (Nm =
1.154) and higher than that of mixed — mating species (Nm
= 0.727) (Hamrick and Godt, 1990). Gene flow between popu-
lations occurs when migrant genes arriving by pollen or
seed become established in new genets. Though S.alba is
frequently visited by insects and bats that lead to pollen
exchange, there is no information suggesting that S. alba
has a long-distance pollinator. From the Mantel test, it can
be seen that genetic distance between populations was
significantly correlated to the geographical distance be-
tween them. Thus geographical distance barrier could
largely affect the gene flow. S.alba is one of the most
widely distributed mangrove species. Although in the
present study, all samples were limited to China, if materials
from other countries and regions could be included in the
study, it is expected that an increased genetic differentia-
tion among populations would be detected resulting from
an increased geographical distance. A possible long-dis-
tance dispersion by ocean currents could be further deeply
investigated.
3.3 Conservation consideration
S. alba in Hainan Island is widely distributed, and al-
though it is not endangered at present, it faces great pres-
sures subsequent to people’s economic activities, espe-
cially fish-pond and prawn-pond operations. Their overall
number is steadily decreasing. If some conservation mea-
sures are not adopted, it will become endangered some
day. Genetic diversity is very important for the conserva-
tion and management of rare and endangered species, and
is also important for the long-term survival and evolution-
ary process of these tropical intertidal area plants. Because
a large proportion of genetic variance of S. alba was found
among individuals within populations, a considerable
amount of genetic variation of the species could be ob-
tained when sampling a larger number of plants from one or
two populations rather than smaller collections from many
different sites. It should be noticed that more polymorphism
in introduced population (DZ) was detected than in its
source population (WC), that may be the result of recurrent
hybridization and introgression between S.alba and other
Sonneratia species and people’s selection. As for in situ
conservation, the LS harbors a significant amount of the
genetic diversity within S. alba. Moreover, in the field, we
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004520
noticed S. alba grew well in Linshui, while, in other
populations, the trees of the species did not, since we of-
ten found many black spots on leaves. So, the LS could be
a priority for conservation action. In addition, according to
our investigation, the seedlings of S.alba were less visible,
most were mature trees. The reason for this may be that the
fruits and seeds of S. alba are vulnerable to crabs and ants,
and perhaps they lack the conditions for seed germination
in their growing places. Such a type of population struc-
ture is unfavorable for population survival. In order to suc-
cessfully conserve this threatened species, reproductive
biology and other factors which may affect the population
structure should be further studied.
Acknowledgements: We thank ZHENG Xin-Ren, CHEN
Jian-Hai, ZHONG Cai-Rong from Hainan Dongzhai Harbor
National Nature Reserve, LIANG Yong from Qinglan Man-
grove Reserve Station, YAN Gui-Xin from Qionghai For-
estry Bureau, LI Zhi-Dong from Sanya Forestry Bureau,
YANG Ke-Ren from Lingshui Forestry Bureau for their help
in sampling, TAN Feng-Xiao, TIAN Cun-Jie, YU Yan for
their technical assistance, JIAN Shu-Guang, YUAN Chang-
Chun, ZHOU Ren-Chao and NAN Peng for their help in
data analysis. We also thank Prof. SHI Su-Hua for her help
in technique and writing. This research was completed in
the Key Laboratory of Gene Engineering of the Ministry of
Education, Zhongshan University.
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