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Biochemical Genetics, Vol. 43, Nos. 7/8, August 2005 ( C
2005)
DOI: 10.1007/s10528-005-6778-y
Genetic Evaluation of the Efficacy of In Situ
and Ex Situ Conservation of Parashorea chinensis
(Dipterocarpaceae) in Southwestern China
Qiaoming Li,1Tianhua He,2,3and Zaifu Xu1
Received 8 June 2004—Final 30 September 2004
The majority of research in genetic diversity yields recommendations rather than
actual conservation achievements. We assessed the efficacy of actual in situ and
ex situ efforts to conserve Parashorea chinensis (Dipterocarpaceae) against the
background of the geographic pattern of genetic variation of this species. Samples
from seven natural populations, including three in a nature reserve, and one ex
situ conservation population were studied. Across the natural populations, 47.8%
of RAPD loci were polymorphic; only 20.8% on average varied at the population
level. Mean population genetic diversity was 0.787 within natural populations
and 1.410 for the whole species. Significant genetic differentiation among re-
gions and isolation by distance were present on larger scales (among regions).
AMOVA revealed that the majority of the among-population variation occurred
among regions rather than among populations within regions. Regression analy-
sis, Mantel test, principal coordinates analysis, and cluster analysis consistently
demonstrated increasing genetic isolation with increasing geographic distance.
Genetic differentiation within the region was quite low compared to that among
regions. Multilocus spatial autocorrelation analysis of these three populations
revealed random distribution of genetic variation in two populations, but genetic
clustering was detected in the third population. The ex situ conserved population
contained a medium level of genetic variation compared with the seven natural
populations; it contained 77.1% of the total genetic variation of this species and
1Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, People’s
Republic of China.
2Department of Environmental Biology, Curtin University of Technology, P.O. Box U1987, Perth,
Western Australia 6845, Australia.
3To whom correspondence should be addressed; e-mail: thhe@bgpa.wa.gov.au.
387
0006-2928/05/0800-0387/0 C
2005 Springer Science+Business Media, Inc.
388 Li, He, and Xu
91% of the moderate to high frequency RAPD fragments (f>0.05). Exclusive
bands were detected in natural populations, but none were found in the ex situ
conserved population. The populations protected in the nature reserve contained
most of the genetic variation of the whole species, with 81.4% of the total genetic
variation and 95.7% of the fragments with moderate to high frequency (f>0.05)
of this species conserved. The results show that the ex situ conserved popula-
tion does not contain enough genetic variation to meet the need of release in the
future, and that more extensive ex situ sampling in natural populations TY, NP,
HK, and MG is needed. The in situ conserved population contains representative
genetic variation to maintain long-term survival and evolutionary processes of
P. chinensis.
KEY WORDS: genetic evaluation; biodiversityconservation; isolation by distance; multilocus spatial
autocorrelation analysis; Parashorea chinensis.
INTRODUCTION
Geographic differentiation of genetic variation results from limited gene disper-
sal in many plant species and is summarized in isolation by distance, increased
geographic distance leading to increasing genetic isolation of population (Wright,
1943). An extensive investigation of genetic structure over different geographic
distances would demonstrate the consequences of isolation by distance and gene
flow. Examination of patterns of genetic variation, particularly in combination
with ecological and geographic data, can provide insights into the species’ recent
evolutionary and biogeographic history. In the case of rare or endangered plant
species, the analysis of population genetic structure and diversity over differ-
ent geographic distances can provide important information for the development
of a conservation program (e.g., Kang and Chung, 1997; Chung et al., 1998;
Gemmill et al., 1998; Godt and Hamrick, 1998; Li et al., 2002; Jin et al., 2003).
The number of investigations of genetic diversity is large, but the majority of
that research yields recommendations rather than actual conservation achieve-
ments. Few attempts have been made to use such genetic information to evaluate
and guide actual conservation efforts (e.g., Manuder et al., 1999; Storme et al.,
2004).
Parashorea chinensis (Dipterocarpaceae) is a rare dipterocarp restricted to
southwest China and adjacent areas of Laos and Vietnam (Fig. 1). On favorable
sites, P. chinensis can reach diameters of 1.5m and heights of more than 80 m
(Xu and Yu, 1982; Zhu, 1992). Seeds of this species are dispersed mainly by
gravity and germinate quickly after falling to the ground, and the seedlings require
light for establishment (Ying and Shuai, 1990). P. chinensis produces high-quality
timber but has a highly fragmented distribution and is currently subject to specific
Conservation Genetics of Parashorea chinensis 389
Fig. 1. Relative locations of populations of Parashorea chinensis sampled for this study. Ovals
encircle the three main distribution ranges of this species.
protection and management plans (Fu, 1992). A comprehensive conservation pro-
gram has been in place to protect this species since the 1950s, when a national
nature reserve was established in Mengla County, protecting 60% of the individ-
uals of P. chinensis and many other endangered and rare wildlife species. In the
1980s an ex situ conserved population was established with the transplantation
of more than 200 seeds and 50 seedlings in Xishuangbanna Tropical Botanical
Garden (China). Little is known, however, about the genetic profile of this ex situ
population or about the pattern of genetic variation in the entire species; therefore,
the long-term survival of the ex situ population and the efficacy of the in situ con-
servation efforts are uncertain, leaving further conservation of this species in the
dark. More broadly, the spatial and temporal scales at which genetic processes oc-
cur in tropical forest ecosystems are largely unknown. The complexity of species
composition and ecological diversity can be used for sampling strategies for moni-
toring genetic diversity of an in situ conservation area and the ex situ conservation
of rare and threatened species. There is increasing interest in the management
and preservation of genetic diversity in such tropical forest ecosystems. A better
understanding of gene flow and genetic isolation within this species will assist in
defining the relationships between populations as well as estimating their potential
contribution to future evolution. As a result, information on the genetic structure
and ecology of P. chinensis will be fundamental to the development of long-term
efficient conservation strategies.
390 Li, He, and Xu
The aim of this investigation was to assess the geographic pattern of genetic
variation within and among populations of P. chinensis and to reveal the genetic
consequences of geographic isolation at different spatial scales. The ultimate aim
is to evaluate the efficiency of current conservation strategies by identifying the
amount of genetic variation contained in ex situ and in situ conserved populations
and then inferring strategies for further conservation efforts, e.g., determination
of in situ conservation site variation and the need for further material collection to
reinforce ex situ populations. For this purpose, randomly amplified polymorphic
DNA (RAPD) markers were used (Williams et al., 1990). The use of RAPDs as
markers in population genetic studies for tree species has been well established
(Chalmers et al., 1992; Yeh et al., 1995; Gillies et al., 1997; Allnutt et al., 1999).
The ease and efficiency of this method makes it a desirable option when appropriate
statistical analyses are used (Lynch and Milligan, 1994; Stewart and Excoffier,
1996; Parker et al., 1998; Smouse and Peakall, 1999), and it is recommended
for use in small low-tech laboratories (Kjolner et al., 2004). It is particularly
attractive because DNA sequence information is not required prior toinvestigating
a previously unstudied species, which facilitates its application to a wide range
of taxa that are currently threatened with extinction worldwide (Newton et al.,
1999).
MATERIALS AND METHODS
Field Sampling
Seven natural populations and one ex situ conserved population were sampled
from three main distribution ranges of P. chinensis in southwestern China (Fig. 1
and Table I). Leaves were harvested from plants with a diameter at breast height
(dbh) greater than 5 cm for each population (Table I). Leaves were dried in the
field in silica gel and transported to the laboratory for DNA extraction. Population
Tab l e I. Location of Parashorea chinensis Populations Analyzed in This Study
Locality Code NnLatitude, Longitude Altitude (m)
Tianyang, Guangxi AR TY 120 24 23◦42N, 106◦54E 460
Napo, Guangxi AR NP 45 24 23◦18N, 105◦52E 650
Hekou, YP HK 35 24 22◦47N, 103◦58E 600
Maguan, YP MG 50 24 23◦01N, 104◦24E 600
Nanshahe, Mengla, YP NSH 32 24 21◦31N, 101◦35E 850
Huiduhe, Mengla, YP HDH 45 34 21◦32N, 101◦34E 850
Huiyinghe, Mengla,YP HYH 80 40 21◦30N, 101◦36E 850
Menglun, Mengla, YP ML 205 24 21◦55N, 101◦06E 850
Note. N: estimated population size. n: sample size. AR: Autonomous Region. YP: Yunnan
Province.
Conservation Genetics of Parashorea chinensis 391
size was estimated roughly by counting individuals with dbh greater than 5cm.
Three populations from the south Yunnan region (NSH, HDH, and HYH) were
chosen to investigate the spatial genetic variation in fine geographic scale (i.e.,
within-population genetic structure). In these three populations, P. chinensis forms
small, dominant populations, providing a unique opportunity to undertake spatial
analysis. The relative physical position of each sampled tree was recorded for
those three populations.
RAPD Amplification
Genomic DNA was isolated using a modified CTAB extraction method (Doyle
and Doyle, 1990). Twenty arbitrary primers that yielded reproducible and clear
amplification products were selected from 132 primers (Shengong Inc.) and were
employed in PCR amplification. DNA amplification was performed in a Rapid-
cycler 1818 (Idaho Tech.), programmed for an initial 1 min at 94◦C, 10 s at 35◦C,
20 s at 72◦C for two cycles, followed by 40 cycles of 0 s at 94◦C, 0 s at 35◦C,
and 1 min at 72◦C, and a final step for 7 min at 72◦C (0 s at a temperature means
that the target temperature is reached but not maintained for any length of time).
Reactions were carried out in a volume of 10 µL containing 50 mM Tris-HCl,
pH 8.3, 500 µg/mL BSA, 10% Ficoll, 1 mM Tartrazine, 2 mM MgCl2, 200 µM
dNTP, 1 µM primer, 5ng of DNA template, and 0.5 U Ta q polymerase. Ampli-
fication products were analyzed by electrophoresis on 1.5% agarose gels stained
with ethidium bromide and imaged by Bio-Rad imaging devices (Gel Doc 2000
Gel Documentation System) supported by Quantity One (version 4.2). With soft-
ware and manual verification, fragment size was estimated using the 100–3000 bp
DNA ladder as a marker. All PCR reactions were prepared in sterile conditions,
and a negative control (in which DNA was omitted) was included with each
PCR run.
Data Analysis
Only RAPD bands that could be unequivocally scored were counted in the analysis.
Amplified products were scored as a discrete banding state (1 as present and 0
as absent) for each individual tree. Shannon’s index of phenotypic diversity (H0),
estimated as −Pilog 2Pi, where Piis the frequency of the band’s presence
or absence, was used to quantify the degree of population diversity. Shannon’s H
is frequently used for RAPD studies because the index is insensitive to bias that
may be introduced into data by undetectable heterozygosity (Black-Samuelsson
and Andersson, 1997; Parani and Parida, 1997; Gustafson et al., 1999; Maki and
Horie, 1999).
We used the AMOVA procedure (Excoffier et al., 1992) to estimate the
variance of components of RAPD phenotypes associated with the geographic
392 Li, He, and Xu
nested structure of natural populations, with the partitioning of variation in the
genetic diversity among individuals within populations, among populations within
regions, and among regions. A further principal coordinates analysis based on
genetic distance (Euclidean distance) was used to illustrate the pattern of genetic
variation within and among populations (GenAlEx V5.1, Peakall and Smouse,
2001).
Pairwise unbiased genetic distances (Nei, 1978) calculated by Popgen V3.22
(Yeh et al., 1999) were used to construct a neighbor-joining tree using the program
MEGA 2 (Kumar et al., 2001). To test the hypothesis of isolation by distance, geo-
graphic distances were obtained with a global positioning system (GPS). Pairwise
FST values were linearly transformed [FST/(1 −FST)] and regressed on pairwise
natural logarithm transformation of geographic distances. This transformation
was made because populations are not distributed along a linear transect (Rousset,
1997). Since population pairs are not independent, a Mantel test was used to evalu-
ate if the significance was consistent. A Mantel test on the transformed matrices of
pairwise FST and geographic distance was conducted by GenAlEx V5.1 (Peakall
and Smouse, 2001).
For within-population spatial genetic structure analysis, we employed a multi-
variate approach to the microspatial autocorrelation analysis developed by Smouse
and Peakall (1999) for multiallelic dominant loci such as RAPD. Unlike classical
spatial autocorrelation analysis, which is usually executed one allele at a time, the
procedure is intrinsically multivariate, avoiding the need for an allele-by-allele,
locus-by-locus analysis. By combining alleles and loci, this approach strength-
ens the spatial signal by reducing stochastic noise (Smouse and Peakall, 1999;
Peakall et al., 2003). In brief, pairwise individual-by-individual genetic distances
for the dominant RAPD loci were calculated via the method of Smouse and
Peakall (1999). Genetic distance matrices for each locus were summed across
loci, under the assumption of statistical independence. A linear pairwise geo-
graphic matrix was calculated as the Euclidean distance between xand ycoordi-
nates at a site. The spatial autocorrelation coefficient, r, was calculated according
to Smouse and Peakall (1999). Then 1000 random permutations were performed
to test for statistical significance and to define the upper and lower bounds of the
95% confidence interval. Multilocus spatial autocorrelation analyses were con-
ducted for all three populations separately. All spatial genetic autocorrelations
were performed using the software package GenAlEx V5.1 (Peakall and Smouse,
2001).
To evaluate the efficiency of genetic conservation, the amount of genetic
diversity in the ex situ conserved population (ML) was compared with that in
seven natural populations (NSH, DH, HYH, HK, MG, TY, NP), and the genetic
diversities of in situ conserved populations (NSH, HDH, HYH) were compared
with that of the entire species.
Conservation Genetics of Parashorea chinensis 393
RESULTS
RAPD Profile and Genetic Diversity Estimates in Natural Populations
The 20 primers revealed polymorphic bands ranging between 0 and 13. The size
of amplified bands ranged between 160 and 2080 bp. Of the 253 reliable bands
generated by the 20 chosen primers, 121 bands (47.8%) were polymorphic across
the seven populations, and the percentage of polymorphic bands varied from 15.4
to 30.0% (with an average of 20.8%) at the population level (Table II).
The highest genetic diversity values were obtained with primer S307, in
the population NP; the lowest values were obtained with primer S314, which
amplified only monomorphic bands across the seven populations. Interestingly,
primer S366 amplified only monomorphic bands in seven populations, while the
size of amplified bands varied among populations. The highest and lowest genetic
diversity values were obtained in the populations NP (H0=1.464) and HDH
(H0=0.473), respectively. The mean diversity within the seven populations of P.
chinensis was Hpop =0.787, and the total diversity was Hsp =1.410 (Table II).
AMOVA Partition
Populations of P. chinensis were grouped into three regions, south Yunnan (NSH,
HYH, HDH), southeast Yunnan (HK, MG), and southwest Guangxi (TY, NP). Of
the total molecular variance, 37.7% was partitioned to regional diversity, 11.4%
to the population differences within regional diversity, and 50.9% to individual
differences within populations. When the total variance was partitioned without
considering the regional distribution of the populations, 43.4% was attributed to
population divergence and 56.7% to individual differences within populations. On
TableII. Summary of Genetic Diversity in Eight
Populations of Parashorea chinensis
Population PPB (%) H0
TY 19.8 0.7656
NP 30.0 1.4640
HK 18.6 0.6870
MG 20.2 0.8133
NSH 17.4 0.5721
HDH 15.4 0.4730
HYH 24.5 0.7215
ML 20.6 0.7029
Population average 20.7 0.7870
Species 47.8 1.4100
Note. PPB: percentage of polymorphic bands,
H0: Shannon’s index of phenotypic diversity.
394 Li, He, and Xu
Table III. Analysis of Molecular Variance of Seven Populations
of Parashorea chinensis
Variance % Total
Source of variation component variance
Nested analysis
Variance among groups 0.0265 37.7
Variance among populations 0.0080 11.4
within groups
Variance within populations 0.0359 50.9
Analysis among populations
Variance among populations 0.0274 43.4
Variance within populations 0.0359 56.7
Analysis among groups
Variance among groups 0.0300 42.5
Variance within groups 0.0406 57.5
Note. Total of 194 individuals sampled from seven populations,
and 253 RAPD markers employed. Nested analysis was carried
out on all populations.
the other hand, if the total variance was partitioned considering only the regional
distribution of individuals, 42.5% would be attributable to regional divergence and
57.5% to individual differences within regions (Table III).
Genetic Consequences of Isolation at Large Scale of Spatial Distance
Regression analyses indicated a significant positive correlation between trans-
formed FST and geographic distances (Fig. 2; r2=0.7109, p<0.001). In
Fig. 2. Plot of transformed FST and geographic distance matrices showing
the pattern of isolation by distance.
Conservation Genetics of Parashorea chinensis 395
Fig. 3. Neighbor-joining tree of genetic distance, showing the
distinctive branches corresponding to the three distribution areas.
addition, the results from the Mantel test indicated that genetic and geographic
distance were significantly related (r=0.853, p=0.008). Cluster analysis with
neighbor-joining approach of pairwise genetic distance was performed; three dis-
tinctive branches, corresponding to three regions, were identified (Fig. 3). A further
principal coordinate analysis illustrated the pattern of within- and between-regions
RAPD diversity; two principal coordinates distinguished the genetic diversity from
three regions, and populations NSH, HYH, and HDH (South Yunnan region) could
not be distinguished (Fig. 4).
Size of Individual Tree in Three Populations
and Within-Population Genetic Structure
Frequency distributions of tree size in populations NSH and HDH were similar
(Fig. 5). Population HYH has more individuals in the second size category but
very few in the bigger category. Only two trees (5%) in population HYH were
assigned to the biggest group; eight trees (26%) in population NSH and nine trees
(27%) in HDH were assigned to the biggest group.
Multilocus spatial autocorrelation analysis revealed different patterns of ge-
netic structure within population in three populations (Fig. 6). The multilocus
autocorrelation coefficient r-values were not significantly positive for the first
distance class in either population NSH or population HDH. Although not sta-
tistically significant, positive rvalues extend to 25 m in population NSH and
38 m in population HDH, indicating weak spatial clustering of genetic variation
396 Li, He, and Xu
Fig. 4. Principal coordinates analysis showing the genetic pattern of RAPD variation within and
among populations. The ellipses indicate the relativity of the three groups and do not represent levels
of confidence.
in both populations. Though both correlograms show oscillation of high and low
autocorrelation, the clustering patterns are not statistically significant.
In population HYH, rvalues are positive and significant for the first two
distance classes (17 and 27 m), with an xintercept at 36 m, which suggests
significant genetic clustering in this population. The correlogram shows clearly
the correlation coefficient ras a function of distance, and there is no apparent
Fig. 5. Frequency of size of sampled trees in three populations. The standard
for age classification is arbitrary.
Conservation Genetics of Parashorea chinensis 397
Fig. 6. Genetic correlation ras a function of distance in three populations, 95% CI, about the
null hypothesis of a random distribution of genotypes shown by dashed line, and 95% confi-
dence error bars about ras determined by bootstrapping. (A) Autocorrelation for population
NSH. (B) Autocorrelation for population HDH. (C) Autocorrelation for population HYH.
oscillation of high and low autocorrelation. Instead, there is a general decline in r
with distance. Consistently negative rvalues are found after 36 m.
Genetic Evaluation of ex situ and in situ Conservation
The ex situ population ML generated a total of 195 bands, ranging from 160
to 2080 bp. Of those, 26.7% (52 out of 195) were polymorphic. If the genetic
data from the seven natural populations are considered, however, the ex situ
population ML represents intermediate genetic variation compared with seven
natural populations, with 20.6% of bands (52 out of 253) being polymorphic.
The percentage of polymorphic bands in population ML was lower than that in
populations NP and HYH, but higher than in populations TY, HK, MG, NSH, and
HDH.
398 Li, He, and Xu
Of the 253 bands amplified in the entire species, 195 were detected in the
ex situ population ML, suggesting that 77.1% of the total genetic variation is
conserved in that population. Also, 187 bands had frequencies greater than 0.05,
and 171 were detected in ML, indicating that 91.44% of the alleles with moderate to
high frequency ( f>0.05) were conserved in that ex situ population. In population
TY, 192 bands were generated, of which 170 occurred in population ML; 167
TY bands had frequencies greater than 0.05, and 156 ( f>0.05) occurred in
population ML, implying that 88.5% of the total population genetic variation
and 93.4% of alleles with moderate to high frequency were conserved in ex situ
population ML (Table IV). In the other six natural populations, the percentage of
the total population genetic variation conserved in ex situ population ML ranged
from 82.03% to 92.5%, and the percentage of alleles with moderate to high
frequency conserved in this population ranged from 87.6% to 98.8% (Table IV).
Of the low-frequency RAPD bands ( f<0.05), the natural population TY had 25
bands, and 14 bands occurred in population ML, indicating that about 56.0% of the
rare loci were also conserved in population ML (Table IV). The frequencies of the
low-frequency alleles of the other six natural populations conserved in population
ML ranged from 37.5 to 55.6% (Table IV).
RAPD analysis also revealed exclusive bands in natural populations. Seven
RAPD primers produced 16 exclusive bands in populations TY, NP, MG, NSH,
and HYH, and the frequencies of exclusive bands ranged from 0.0211 to 0.5000.
None, however, was detected in population ML.
Genetic diversities of the populations conserved in the nature reserve (NSH,
HYH, and HDH) were lower than those of the populations (TY, NP, HK, and
MG) outside the reserve (Table V). In NSH, HYH, and HDH, 30.8% RAPD bands
were polymorphic; 44.7% of bands were polymorphic in the populations outside
the nature reserve (Table V). Considering the whole species, 221 of 253 RAPD
bands were detected in populations NSH, HYH, and HDH, implying that 87.4%
of the total genetic variation was conserved in in situ populations. Among the
Tab le IV. The Amount of Genetic Variation of Natural Populations of Parashorea chinensis Conserved
in Artificial Population ML
Total Bands detected Bands with Bands >0.05 Bands with Bands <0.05
Population bands in ML (%) f>0.05 in ML (%) f<0.05 in ML (%)
TY 192 170 (88.54) 167 156 (93.41) 25 14 (56.00)
NP 217 178 (82.03) 193 169 (87.56) 24 9 (37.50)
HK 196 171 (87.24) 173 159 (91.91) 23 12 (52.17)
MG 199 176 (88.44) 179 164 (91.62) 20 11 (55.00)
NSH 190 174 (91.58) 167 162 (97.01) 23 12 (52.17)
HDH 187 173 (92.51) 164 161 (98.17) 23 12 (52.17)
HYH 206 186 (90.29) 170 168 (98.82) 36 20 (55.56)
Mean 198 175 (88.38) 173 163 (94.07) 25 13 (51.51)
Species 253 195 (77.08) 187 171 (91.44) 66 25 (37.88)
Conservation Genetics of Parashorea chinensis 399
Tab l e V. Genetic Variation Conserved in In Situ Populations of Parashorea chinensis
Population % Bands NbNd
(sample size) N(PPB) detected Na(% of species) Nc(% of species)
Out of reserve (96) 246 (44.7%) 97.23 189 186 (99.47) 57 60 (90.91)
In reserve (98) 221 (30.8%) 87.35 168 179 (95.72) 53 42 (63.64)
Species (194) 253 (48.2%) — 187 — 66 —
Note. N (PPB): total number of bands (percentage of polymorphic bands). % Bands detected: Per-
centage of bands detected compared to the whole species. Na: Number of bands with frequency >
0.05. Nb: Number of bands >0.05 detected. Nc: Number of bands with frequency <0.05. Nd: Number
of bands <0.05 detected.
total of 253 bands, 187 had frequencies greater than 0.05, and 179 occurred in in
situ conserved populations, indicating that 95.7% of alleles with moderate to high
frequency ( f>0.05) were conserved. Of the low-frequency alleles ( f<0.05),
66 RAPD bands were detected, with 42 bands occurring in in situ conserved
populations, indicating that about 63.6% of the rare loci were also conserved
(Table V).
DISCUSSION
Geographic Pattern of Genetic Variation in P. chinensis
Increasing geographic distance will lead to increasing genetic isolation of popula-
tions; this is clearly illustrated in our investigation of the genetic pattern along dif-
ferent spatial scales. Isolation by distance is obviously responsible for the genetic
distance patterns over large geographic distance found in P. chinensis. Signifi-
cant genetic differentiation is the result of limited capability of long-distance seed
dispersal and perhaps subsequent genetic drift in geographically isolated regions.
Seeds of P. chinensis are gravity-dispersed, with most of them falling within a
short distance of the tree crown and with little secondary dispersal. Seeds of P.
chinensis are recalcitrant and remain viable for only several days (Ying and Shuai,
1990), which further limits effective seed dispersal. Understandably, successful
long-distance seed dispersal events will probably be extremely limited. Recent
timber harvesting by humans would affect the population size negatively, and
small populations are more likely to experience genetic drift than large popula-
tions (Ellstrand and Elam, 1993). In addition, adaptation to a different habitat is
probably another cause of significant regional genetic differentiation. A conclusion
can be drawn from the amplified pattern of primer S366, which amplified different
monomorphic bands in different populations. Moreover, ecological and morpho-
logical differences between different regions were demonstrated in this species
(Zhu, 1992). Supportably, relatively high levels of regional genetic differentiation
are not uncommon in tropical tree species with a wide geographical range. For
400 Li, He, and Xu
instance, Spanish cedar (Cedrela odorata) is a highly valued timber species na-
tive to the American tropics. Analysis indicated that 55% of the total variation
recorded was maintained among rather than within populations using RAPDs
(Gillies et al., 1997). Studies of the leguminous tree Gliricidia sepium have also
recorded a relatively high degree of population differentiation, where 60% of the
variation recorded was among, rather than within, populations (Chalmers et al.,
1992).
The results of the AMOVA suggest considerable genetic differentiation
among regions, but little genetic divergence among populations within regions.
Furthermore, random distribution of genetic variation in fine scale was detected in
two (out of three) populations of P. chinensis. Though no detailed work has been
done on the pollination biology of P. chinensis, this species is a canopy tree and
its flowers are 20–30 m above the secondary canopy, which would be a distinctive
attraction for the pollinators over long distances. The present results indicate that
despite the high level of fragmentation and small size of subpopulations in P.
chinensis, there was an extensive network of genetic exchange over the spatial
scale of the study (within 3–5 km).
Genetic clustering within populations was expected in populations of P.
chinensis. Seed dispersal in this species is limited, with seeds being primarily
gravity dispersed (Ying and Shuai, 1990). Limited patterns of seed dispersal have
been shown to have a significant effect on patterns of spatial genetic structure
within plant populations (Chung et al., 2000). The existing randomness of genetic
variation in P. chinensis may result from overlapping seed shadows. Because adult
trees are 50–80 m tall, seed may fall some distance away from the maternal trees,
and variation in the density of the adults could explain the different patterns of
spatial genetic structure in three of the populations. Population HYH has a much
lower density of large trees than the other two sites, thus there should be less overlap
of seed shadow and greater relatedness at short distance. In addition, these results
may be influenced by the exclusion of seedlings with a dbh smaller than 5 cm
from the analysis. The significant spatial clustering of genetic variation detected
in population HYH may reflect the high ratio of young to old trees recorded
in this population. Many tree species exhibit strong genetic structure in young
stages, and juvenile cohorts were more structured than adult cohorts (Hamrick
et al., 1993; Hamrick and Nason, 1996; Doligez and Joly, 1997; Epperson and
Alvarez-Buylla, 1997; Aldrich et al., 1998; Ueno et al., 2000). Such results may
be anticipated due to extensive mortality and selection between seedling and adult
stages. Theoretical simulations by Doligez et al. (1998) showed that the occurrence
of symmetric overdominance selection at a few loci might reduce spatial genetic
structure at all loci, as would be expected if selection against inbred individuals
were involved. No information is available regarding the distance of pollen flow in
P. chinensis, although pollen dispersal is less likely to influence the spatial pattern
of genetic structure compared to seed dispersal (Chung et al., 2000).
Conservation Genetics of Parashorea chinensis 401
The total diversity level of P. chinensis (percentage of polymorphic bands
PPB =47.8%) and the mean population diversity (PPB =20.8%) are substantially
lower than those of other dipterocarps in tropical areas. Recent studies based on
analysis of variation at isozyme loci have revealed considerable genetic variation
in natural populations of many dipterocarps. In Stemonoporus oblongifolius, the
percent of polymorphic loci ranges from 88.9 to 100% (Murawski and Bawa,
1994). Similarly, a high level of genetic variation has been observed in Shorea
megistophylla (Murawski et al., 1994) and Hopea odorata (Wickneswari et al.,
1994). Moreover, considerable variation was found within populations of many
Malaysian species of Hopea and Shorea using RAPD (Wickneswari et al., 1996;
Harada et al., 1994). Recently, a high level of genetic variation has been observed
in Shorea leprosula (Lee et al., 2000b) and Dryobalanops aromatica (Lee et al.,
2000a; Lim et al., 2002). Although it is difficult to compare different studies, espe-
cially those using different methods of data generation, in our study relatively low
levels of genetic variation were revealed in populations of P. chinensis. Population
genetic structure has a close link to evolutionary history, geographic distribution,
and life history. Many researchers (e.g., Ashton, 1982; Xu and Yu, 1982; Zhu,
1996) have suggested that dipterocarps originated from the old Gondwana conti-
nent in the early Tertiary and that the Guinea continent was the center of diversity
for this family. Currently P. chinensis is mainly distributed throughout the south
and southeast of Yunnan, southwest of Guangxi Autonomous Region, and scat-
tered in adjacent Laos and Vietnam, where the northern margin of the range of
the dipterocarps occurs. The current pattern of distribution could therefore be the
result of postglacial recolonization, resulting in reduced genetic variability as a
consequence of repeated bottleneck events (Seitz, 1995; Boulton et al., 1998).
Efficiency of Genetic Conservation of P. chinensis
The goal of ex situ conservation programs is to maintain the species in captivity
until habitat restoration allows its release back to nature. Such restoration could
take decades or even centuries; therefore, captive populations must be managed
as a long-term, multigenerational breeding program. The restored environments
will undoubtedly differ from the original habitats and communities. It is therefore
critical that the released populations have sufficient genetic variability to provide
adaptive flexibility in an uncertain future (Templeton, 1982, 1991).
Assuming that a population’s short-term viability has been assured, its long-
term viability will probably depend, in part, on the amount of genetic variability it
retains. The importance of the absolute level of genetic variability for conservation
is debatable. Some suggest that all genetic variation within a species should be
captured (Hawkes, 1976), but as Brown and Briggs (1991) have pointed out, this
is unrealistic, and it is also unnecessary. First, many low-frequency alleles are un-
conditionally deleterious and are maintained only as a result of recurrent mutation,
402 Li, He, and Xu
many low-frequency alleles might actually contribute to genotypes that lower the
average viability of individuals, and low-frequency alleles are likely to be lost in
just a few generations. Second, most adaptively significant variation is contained
in alleles found at moderate to high frequency. In short, maintenance of long-term
population viability requires an attempt to preserve a representative sample of
moderate- to high-frequency alleles, whether the population is managed in its nat-
ural habitat or samples are collected for off-site preservation (Templeton, 1991).
The current local rarity of such alleles implies their insignificant contribution to
present adaptation.
Marshall and Brown (1975) suggested that the objective of genetic conser-
vation is to maintain the population that will contain 95% of all the alleles with
a frequency greater than 0.05 at a random locus occurring in the target popula-
tion. In our study, the ex situ population ML conserves only part of the genetic
variation of the whole species (77.1% of the total genetic variance of the species,
and 91.4% for the alleles with frequency greater than 0.05), which is lower than
the Marshall and Brown standard. Our results implied that the amount of genetic
variation conserved in population ML is not enough to sustain long-term and
multigenerational survival of P. chinensis. To meet the needs of future releases,
more genetic variation must be included in this population. On the other hand,
the in situ conserved populations maintain most of the genetic variation of the
entire species (87.4% of the total genetic variation of the species, and 95.7% for
the alleles with frequency greater than 0.05), indicating that the in situ conserved
populations in the nature reserve contain sufficient representative genetic variation
to maintain the long-term survival and evolutionary process of P. chinensis.
Exclusive bands occurring in natural populations were not detected in the
ex situ population ML. Five out of 16 exclusive bands occurred in the natural
populations with moderate to high frequency. The populations that are distinctive
in their DNA traits should have high conservation status (Avise, 1989; Dizon
et al., 1992). Consequently, to add those exclusive traits to the ex situ population
and to assure adaptation to the future restoration environment, more extensive
sampling is needed from populations TY, NP, HK, and MG. Considering the high
genetic diversity of the populations outside the nature reserve and the extensive
population differentiation among the regions, more attention should also be paid
to the populations outside the reserve.
Ex situ conservation of rare tropical trees is thought to be difficult, because
only a limited number of large woody plants can be cultivated in botanical gardens
(Bawa and Ashton, 1991). In our study, even with as many as 200 individuals
surviving in botanical gardens, the genetic diversity of P. chinensis was not fully
represented. On the other hand, limited facilities are available, and inevitable
genetic changes from random genetic drift and selection in artificial environments
may make it difficult for captive populations to be reestablished in the wild (Soul´
e,
1987). Although many species are being rescued by ex situ methods, and then being
Conservation Genetics of Parashorea chinensis 403
reintroduced, the primary method for their conservation must be in situ protection
and management. It conserves not only the plant species and the habitat in which
it lives but also the associated animals on which it may depend for pollination
and dispersal of its diasporas, and also the animals, particularly insects, that might
depend on the plant species. As suggested in the present work, in situ conservation
maintains genetic diversity more efficiently than the ex situ strategy.
Implications for Further Efforts in Conservation of P. chinensis
The geographic pattern of genetic variation in P. chinensis has important practical
implications for conservation and management efforts. First, strategies for the
conservation of genetic diversity need to consider not only current threats to a
particular region but also the level of diversity in an area. Our data indicate that
the area of population NP contains a significant amount of the genetic diversity
within P. chinensis and that this area should be a priority for conservation, either
for traditional in situ approaches or ex situ collecting to reinforce the genetic
variation in the ex situ conserved population. In the case of ex situ collection,
the distribution pattern of genetic variation within the population could provide
guidance. Meanwhile, materials going into an ex situ population could be screened
to select individuals that would increase the overall diversity. Second, significant
genetic differentiation was revealed between regions in P. chinensis. Outcrossing
depression may be an important risk in ex situ conservation of this species, which
suggests that ex situ collection of enough individuals from different regional pop-
ulations to assure possible matings between individuals from the same ecological
type is needed to avoid outbreeding depression. Third, extensive gene flow within
populations and among adjacent populations on a small geographic scale (<4km)
implies that policymakers need to be aware of the importance and complemen-
tary role that remnant forest patches and trees play in providing connectivity and
enhancing population variability when designing the nature reserve.
ACKNOWLEDGMENTS
The authors thank Prof Yuping Zou and Dr Shiliang Zhou for assistance in RAPD
techniques, and Dr. Anthony Janet and Luke Barrett for English improvement.
This work was supported by the Key Project of the Chinese Academy of Sciences
(KSCX2-SW-104), chinese Academy of Sciences’ Training Qualified Plan “Hope
of the Western China” and Yunnan Province Project (2002C0018Q).
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