全 文 ：·Short Communication·
A Preliminary Study on Conservation Genetics of Endangered
Vatica guangxiensis (Dipterocarpaceae)
LI Qiao_Ming1 , 2 , XU Zai_Fu1 , HE Tian_Hua3＊
(1.Xishuangbanna Tropical Botanical Garden , The Chinese Academy of Sciences , Mengla 666303 , China;2.Kunming Institute of Botany , The Chinese Academy
of Sciences , Kunming 650204, China;3.Laboratory of Systemat ic and Evolutionary Botany , Institute of Botany , The Chinese Academy of Sciences , Beijing
100093, China)
Key words:　Vatica guangxiensis;RAPD;genetic diversity ;conservation biology
　　Vatica guangxiensis is a characteristic tree species of
tropical rain forest in Southern Yunnan , it is also an im-
portant timber tree species
[ 1] .This species has limited
number of individuals , only with three natural populations
distributed in Nanshahe and Maochaoshan , Mengla Coun-
ty of Southern Yunnan , and Liushaoshan , Napo County of
Guangxi , and it was listed as an endangered plant species
in China[ 2] .From 1980s , Xishuangbanna Tropical Botan-
ical Garden , the Chinese Academy of Sciences (XTBG)
began to carry out conservation program and research on
this species.Since the evolutionary potential of species
and populations is determined to a great extent by the lev-
el of genetic diversity and the pattern of genetic varia-
tion[ 3] the well understanding of genetic diversity and pop-
ulation genetic structure is a prerequisite for conservation
and management of rare and endangered species.Al-
lozyme analysis has ever been applied to detect the genetic
diversity of V.guangxiensis , but the limited loci could
only detect low genetic diversity , which could provide
limited information for conservation and management of
this species[ 4] .Therefore , in this study , with an intent to
obtain more detailed information of population genetic
structure of this endangered plant , the more sensitive ran-
dom amplified polymorphic DNA(RAPDs)assay[ 5 ,6] were
applied to analyze the genetic diversity and population ge-
netic structure.
1　Materials and Methods
Samples of Vatica guangxiensis X.L.Mo (Diptero-
carpaceae)were collected from the three remaining natu-
ral populations(NS , NP , NX)and one cultivated popu-
lation (ML) conserved in the Xishuangbanna Tropical
Botanical Garden.The population locations and sample
sizes are presented in Table 1.The fresh leaves were
dried quickly by using silica gels in field , and stored at
room temperature for further use.
Total DNA was isolated according to the protocol of 2
×CTAB[ 7] , and was dissolved in 0.1×TE (1 mmol/L
Tris_HCl(pH 8.0), 0.1 mmol/L EDTA(pH 8.0))and
subjected to PCR amplification after adjusting concentra-
tion.Twenty arbitrarily primers that could amplify repro-
ducible and clear DNA bands were selected from 138
primers(Shengong Inc.)for further amplification.
DNA amplification was performed in a Rapidcycler
1818(Idaho Tech.), programmed for an initial 1 min at
94 ℃, 10 s at 35 ℃, 20 s at 72 ℃ for 2 cycles , fol-
lowed by 40 cycles of 0 s at 94 ℃, 0 s at 35 ℃, and 1
min at 72 ℃, and ended with 7 min at 72 ℃[ 8] .Reac-
tions were carried out in a volume of 10μL containing 50
mmol/L Tris_HCl(pH 8.3), 500μg/mL BSA , 10%Fi-
coll , 1 mmol/L Tartrazine , 2 mmol/L MgCl2 , 200μmol/L dNTP , 1μmol/L primer , 5 ng of DNA template
and 0.5 U Taq polymerase.Amplification products were
analyzed with electrophoresis (TBE electrophoresis)on
1.5% agrose gel stained with ethidum bromide , and im-
aged on the Bio_Rad imaging devices(Gel Doc 2000 Gel
Documentation System)supported by Quantity One (ver-
sion 4.2).Molecularweights were estimated using 100-
3 000 bp DNA Ladder.　　The bands were marked by Quantity One software ,
and the bands were watched at the same position(molec-
ular weights).Amplified fragments were scored for the
presence (1)absence (0)of homologous bands and the
matrix of the RAPD phenotypes was assembled for the fol-
lowing analysis:Genetic diversity as measured by the per-
centage of polymorphic bands(PPB), Shannon diversity
index(I), population gene diversity (Ht), subpopula-
tion gene diversity (Hs), and subpopulation differentia-
tion (Gst)by using POPGENE[ 9] .RAPDistance pro-
gram[ 10] was used to calculate Jaccard similarity coeffi-
cients for further analysis of AMOVA[ 11] .With AMOVA
we calculated variance components which were partitioned
among individuals within populations , among populations
within regions(Yunnan and Guangxi), and between re-
gions.
2　Results
Twenty primers were used for amplification in 95 in-
dividuals in 4 populations of V.guangxiensis.A total of
231 bands ranged from 170-1 995 bp were scored , cor-
responding to 6-18 bands per primer.The bands ampli_
fied by primer S317 ,S226 ,S246 , S501 are shown in
Received:2001-07-04　Accepted:2001-08-08
Supported by the Great Project of the Chinese Academy of Sciences(KZ951_A1_104).
＊Author for correspondence.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　

Acta Botanica Sinica　2002 , 44(2):246-249
Table 1　Population location and sample size
Population Code Sample sites Estimated population size Sample size
1.NS Nanshahe , Mengla county , Yunnan 21°30′N , 101°35′E, 800-1 100 m Alt. 100 27
2.NP Liushaoshan , Napo county , Guangxi 23°07′N , 105°42′E , 500-600 m Alt. 40 30
3.NX Maocaoshan , Mengla county , Yunnan 21°37′N , 101°50′E , 750-1 000 m Alt. 50 10
4.ML XTBG , Menglun , Mengla county , Yunnan 21°54′N , 101°18′E , 600 m Alt. 90 28
Fig.1.　Amplification products of Vatica guangxiensis using primer S317(A), primer S226(B), primer S246 (C)and primer S501(D).
Fig.1.Of the 231 bands , 124 were polymorphic bands(PPB), accounted for 53.68%(Table 2).According to
the PPB , the amount of genetic variation within these 4
populations was NS>NP>ML>NX(from high to low),
and the Shannon diversity index gave another result:NS>ML>NP >NX ,which differed slightly from the results
of PPB .By using POPGENE , the observed number of
alleles(na), effective number of alleles(ne), and Nei
s gene diversity index (h)were calculated (Table 3).
The genetic diversity of population NS was the highest ,
which was in accordance with the results measured by
Shannon diversity index.
V.guangxiensis was grouped into 2 regions , Yun-
nan(NS 、ML 、NX)and Guangxi (NP).The analysis by
AMOVA implied that the genetic variation within popula-
tions(55.09%)was higher than that among populations(44.91%);and the variation among regions (51.18%)
was higher than that within regions(48.82%).On the
whole , the variation within populations (45.13%)was
higher than that among regions(45.08%), and the vari-
ation(9.79%)among populations of region was low.The
Gst of this species was 0.374 6 , population Ht was
0.164 6 , and subpopulation gene Hs was 0.103 0.Table
4 shows the genetic distance and genetic identity among
populations.The population NS and NX , both located in
Mengla County of Yunnan , are close in spatial distance(about 30 km).Accordingly , the genetic identity be-
tween these two populations was high(0.962 0), and the
genetic distance was 0.038 8.On the contrary , compared
with population NP located at Napo County of Guangxi ,
the spatial distance betweenNS andNP is about 500 km ,
the genetic distance was 0.148 0 between population NS
and NP , and 0.165 3 between NX and NP , suggesting
that geographic isolation has profound effects on popula-
tion genetic differentiation.
Table 2　RAPD polymorphism of populations of Vatica guangxiensis
Populations N b N pb PPB(%) N pb/ p I
NS 213 89 38.53 4.45 0.178 8
NP 201 73 31.60 3.65 0.149 2
NX 199 63 27.27 3.15 0.142 8
ML 204 71 30.74 3.55 0.150 1
Total 231 124 53.68 6.20 0.254 3
N b , the sum of all bands;Npb , the sum of polymorphic bands;PPB , the
ratio of polymorphic bands;N pb/ p , the ratio of polymorphism per primer;I ,
Shannon diversity index.
Table 3　Statistical analysis of genetic variation for all loci of Vati-
ca guangxiensis
Populations na ne h
NS 1.385 3 1.201 8 0.118 0
NP 1.316 0 1.165 8 0.098 3
NX 1.272 7 1.163 7 0.095 5
ML 1.307 4 1.171 8 0.100 1
Total 1.536 8 1.287 8 0.168 6
na , observed number of alleles;ne , effective number of alleles;h , Nei s
gene diversity.
Table 4　Nei s unbiased measures of genetic identity and genetic
distance(POPGENE)
Populations NS NP NX ML
NS ＊＊＊＊ 0.862 4 0.962 0 0.974 4
NP 0.148 0 ＊＊＊＊ 0.847 6 0.857 8
NX 0.038 8 0.165 3 ＊＊＊＊ 0.963 1
ML 0.026 0 0.153 4 0.037 6 ＊＊＊＊
Nei s genetic identity (above diagonal)and genetic distance(below diago-
nal).
LI Qiao_Ming et al:A Preliminary Study on Conservation Genetics of Endangered Vatica guangxiensis(Dipterocarpaceae) 247　
3　Discussion
RAPD analysis revealed low level of genetic diversity
and high level of population differentiation in the endan-
gered V.guangxiensis , which was consistent with the re-
sults from allozyme analysis[ 4] , however RAPD detected
more detailed data on the amount and distribution of ge-
netic diversity and population genetic structure than the
latter.The population genetic structure of a species was
the result of long_term evolution , and has close link to its
evolutionary history , geographic distribution , life history ,
etc.Xu and Yu[ 12] and Zhu[ 13] suggested that the dipte-
rocarps originated from the old Gangwana Continent in
early Tertiary , and the Guyita Continent was the diversity
center of this family.The current V.guangxiensis is only
distributed in Mengla County , South of Yunnan , and
Napo County , Southwest of Guangxi , which are the north-
ern margins of the range of dipterocarp , therefore , its
current distribution pattern could be the results of the
glacial migration.In the Quaternary , as the glacials ap-
peared on the globe , some thermophilous plants had lived
through an unfavourable period in “ refuge” , and some
others migrated to the south.When the temperature ele-
vated in the interglacial , the plants migrated to the North
and the regions of high elevation gradually.After many
repeats , there remained some groups , or some new groups
derived from these remains.As a result , it is reasonable
that the low leveled genetic diversity of V.guangxiensis
could be attributed to the effect of the population bottle-
neck resulting from the advance and retreat of glacial in
its evolutionary history.On the other hand , geographic
isolation of small populations and genetic drift enlarged
the genetic differentiation among populations.Moreover ,
the gravity dispersal of seeds of V.guangxiensis also
partly contributed to the high subpopulation differentia-
tion.Considering its big fruits and tropical static wind cli-
mate , the dispersal distance was so limited that the high
genetic relatives of neighbor plants and result , and selfing
frequently occurred in population , which contributed to
the high genetic differentiation among populations.
The information of population genetic diversity is of
critical importance for conservation and management of
rare and endangered plants , including assessment of the
conservation value and state of special populations , ex
situ collection and conservation[ 14-16] .Because of the low
level of genetic diversity and high population differentia-
tion in V.guangxiensis , promoting gene flow (via seed
and seedling)among populations would be practical ef-
forts of conservation.Furthermore , the results also have
special implication for the ex situ conservation of this en-
dangered plant.The cultivated population (ML)did not
contain the highest genetic diversity , indicating that this
population could not represent the whole genetic variation
of this species.Thus , more extensive ex situ collections
are needed to conserve the whole genetic variation of this
species.Considering the high genetic variation within
populations and among populations of V.guangxiensis ,
ex situ collection should sample enough individuals , and
take all populations into account.The results showed that
the genetic diversity of cultivated populationML was lower
than that of population NS and NP (PPB), consequent-
ly , more emphasis should be given to these two popula-
tions in ex situ collection afterwards.The results also
showed that cultivated population ML had high genetic
similarity to population NS , but had high differentiation to
population NP , therefore , in order to conserve the more
genetic diversity of V.guangxiensis , we need to give
more attentions to population NP in whenever in situ con-
servation of original habitat and collecting samples supple-
mentally for ex situ conservation.
Acknowledgment:The authors thank Prof.ZOU Yu_
Ping and Dr.ZHOU Shi_Liang (Institute of Botany , the
Chinese Academy of Sciences)for their kind helps in
DNA techniques and data analysis;Prof.GE Song for his
valuable suggestions and comments on this manuscript.
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濒危植物版纳青梅保护遗传学研究初报
李巧明1 ,2　许再富1　何田华3＊
(1.中国科学院西双版纳热带植物园 , 勐腊 666303;2.中国科学院昆明植物研究所, 昆明 650204;
3.中国科学院北京植物研究所系统与进化植物学开放研究实验室 , 北京 100093)
摘要:　运用 20 个 10 碱基随机引物 , 对中国龙脑香科(Dipterocarpaceae)特有的珍稀濒危植物版纳青梅(Vatica
guangxiensis X.L.Mo)进行了 RAPD多态性分析。 3个自然居群和 1 个迁地保护居群(分布于云南和广西)共扩增出
231 个位点 ,多态位点所占比例(PPB)为 53.68%;观察等位基因数 na=1.536 8 , 有效等位基因数 ne=1.287 8 , Nei
基因多样性指数 h 为0.168 6 , 居群内的遗传多样性水平较低。基于 AMOVA 和POPGENE的结果均表明居群内的遗
传变异大于居群间的遗传变异。居群内的遗传变异为 55.09%,居群间的变异为 44.91%(AMOVA);基因分化系数
Gst为 0.374 6 (POPGENE), 表明居群间存在高水平的遗传分化。研究结果对该濒危植物的保护有重要意义。考虑
到低水平的遗传多样性和高水平的居群分化 ,通过居群间种子和幼苗的交换来促进基因流是可行的保护方案。迁
地保护居群(ML)不具最高的遗传多样性 ,表明为了保护此濒危物种的全部遗传变异 , 需要进一步采集更多个体补
充到迁地保护居群中。
关键词:　版纳青梅;RAPD;遗传多样性;保护生物学
中图分类号:Q941+.3　　　文献标识码:A　　　文章编号:0577-7496(2002)02-0246-04
收稿日期:2001-07-04　接收日期:2001-08-08
基金项目:中国科学院重大项目(KZ951_A1_104)。
＊通讯作者。
(责任编辑:李长复)
LI Qiao_Ming et al:A Preliminary Study on Conservation Genetics of Endangered Vatica guangxiensis(Dipterocarpaceae) 249