全 文 :珍稀濒危植物硬叶兜兰的遗传多样性及遗传结构研究∗
黄家林1ꎬ2ꎬ 李树云1ꎬ 胡 虹1∗∗
(1 中国科学院昆明植物研究所ꎬ 云南 昆明 650201ꎻ 2 中国科学院大学ꎬ 北京 100049)
摘要: 由于人为采集、 走私贩卖以及生境的破坏ꎬ 分布于中国西南石灰岩地区的野生硬叶兜兰居群受到严
重的干扰与威胁ꎮ 为有效地保护这种珍稀野生植物ꎬ 本研究采用 ISSR和 SRAP 两种分子标记对 15个硬叶
兜兰野生居群进行遗传多样性及遗传结构的研究ꎮ 结果表明ꎬ 硬叶兜兰在物种水平上具有较高的遗传多样
性 (ISSR: PPB= 91 66%ꎬ He = 0 3839ꎻ SRAP: PPB= 99 29%ꎬ He = 0 2806)ꎮ 硬叶兜兰居群间存在一定程
度的遗传分化 ( ISSR: Gst = 0 2577ꎻ SRAP: Gst = 0 2383)ꎬ 可能由于较低的基因流 ( ISSR: Nm = 0 7201ꎻ
SRAP: Nm= 0 7991) 所致ꎮ UPGMA聚类分析以及主成分分析均把 15 个居群分成 2 个主要分支ꎮ 居群间
的地理距离和海拔差距是引起居群遗传分化的自然因素ꎮ
关键词: 资源保护ꎻ 分子标记ꎻ 遗传多样性ꎻ 遗传分化ꎻ 硬叶兜兰
中图分类号: Q 16 文献标识码: A 文章编号: 2095-0845(2014)02-209-10
ISSR and SRAP Markers Reveal Genetic Diversity and Population
Structure of an Endangered Slipper Orchidꎬ Paphiopedilum
micranthum (Orchidaceae)
HUANG Jia ̄Lin1ꎬ2ꎬ LI Shu ̄Yun1ꎬ HU Hong1∗∗
(1 Key Laboratory of Economic Plants and Biotechnologyꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ
Kunming 650201ꎬ Chinaꎻ 2 University of Chinese Academy of Sciencesꎬ Beijing 100049ꎬ China)
Abstract: Paphiopedilum micranthum is an endangered pink slipper orchid mainly distributed in the limestone areas
of southwestern China. Wild populations of this species have been seriously threatened by excessive collectionsꎬ ram ̄
pant smuggling for exportꎬ and habitat destruction. We used 15 ISSR markers and 11 SRAP markers to investigate
the genetic diversity and structure of 15 natural populations. A high degree of diversity was observed at the species
level (ISSR: PPB= 91 66%ꎬ He = 0 3839ꎻ SRAP: PPB= 99 29%ꎬ He = 0 2806). Certain degree of genetic differ ̄
entiation among populations (ISSR: Gst = 0 2577ꎻ SRAP: Gst = 0 2383) was detected maybe caused by low gene
flow (ISSR: Nm= 0 7201ꎻ SRAP: Nm= 0 7991). Consistent with the results of Principal Coordinate Analysisꎬ the
UPGMA dendrogram analysis divided the 15 populations into two main clades. In addition to geographic distanceꎬ
the difference in elevation was another natural factor contributing to this differentiation. Knowledge about genetic di ̄
versity and structure gained from our study will be beneficial for the development of reasonable and efficient strategies
to conserve this endangered species.
Key words: Conservationꎻ DNA markersꎻ Genetic diversityꎻ Genetic differentiationꎻ Paphiopedilum micranthum
The genus Paphiopedilum Pfitz.ꎬ a primitive group within Orchidaceaeꎬ is distributed from southern China
植 物 分 类 与 资 源 学 报 2014ꎬ 36 (2): 209~218
Plant Diversity and Resources DOI: 10.7677 / ynzwyj201413085
∗
∗∗
Funding: The Basic Research Program of Yunnan Province (2007C001Z) and the Large ̄Scale Scientific Facilities of the Chinese Academy
of Sciences (2009 ̄LSF ̄GBOWS ̄01)
Author for correspondenceꎻ E ̄mail: huhong@mail kib ac cn
Received date: 2013-04-11ꎬ Accepted date: 2013-09-04
作者简介: 黄家林 (1974-) 男ꎬ 助理研究员ꎬ 主要从事兰科植物的资源保护与开发利用ꎮ E ̄mail: huangjialin@mail kib ac cn
to New Guinea in tropical and subtropical Asia (Liu
et al.ꎬ 2009). These slipper orchids are named for
the shape of the deeply saccate lip of their flowers.
Growers prize them for their very great ornamental
valueꎬ particularly because these small plants have
spectacularꎬ large flowers ( Cribbꎬ 1998 ). More
than 20 000 slipper orchid hybrid grexes have been
registeredꎬ demonstrating their remarkable rise in
popularity (Zeng et al.ꎬ 2010). Provinces in south ̄
western Chinaꎬ including southeastern Yunnanꎬ
northern and western Guangxiꎬ and southwestern
Guizhouꎬ are known as diversity and evolutionary
hotspots for this genus ( Luo et al.ꎬ 2003ꎻ Liu et
al.ꎬ 2010). There are 25 of 80 species grow on lime ̄
stone hills in that region. In addition to threats from
rapid habitat destruction associated with faster eco ̄
nomic growth and rural development during the past
three decadesꎬ wild slipper orchids are facing de ̄
structive collecting pressures due to large horticultural
and commercial demands in China (Luo et al.ꎬ 2003ꎻ
Liu et al.ꎬ 2009). This vulnerability by Paphiopedi ̄
lum species to environmental change can be also
traced to the limited distribution of many species in
the wild and their occurrence in populations that often
contain only a few plants each (Cribb and McGoughꎬ
1997). It is urgent to take proper strategies to con ̄
serve these endangered species.
Paphiopedilum micranthum is restricted to the
limestone hills of southwestern China. In the early
1980sꎬ plants were exported from China and deserv ̄
edly received First Class Certificates from the Ameri ̄
can Orchid Societyꎬ the Royal Horticultural Societyꎬ
and many other awarding groups because these beau ̄
tiful plants have pink flowers that are larger than
those previously known (Cribbꎬ 1998). Since thenꎬ
this species has been cultivated in large quantities
while wild populations have declined sharply (Li et
al.ꎬ 2002b). To preserve and exploit P micranthum
and its allied speciesꎬ multiple studies have been
carried out including the effects of conservation ef ̄
forts (Liu et al.ꎬ 2004ꎻ 2006)ꎬ physiological ecolo ̄
gy (Chang et al.ꎬ 2011)ꎬ pollination biology (Shi et
al.ꎬ 2007)ꎬ and breeding strategies ( Chen et al.ꎬ
2004ꎻ Liao et al.ꎬ 2011ꎻ Chung and Choiꎬ 2012).
Clarifying genetic diversity and population
structure of extant populations not only provides in ̄
sights into the evolutionary and demographic history
of threatened species (Hamrick et al.ꎬ 1982ꎻ Ham ̄
rick and Godtꎬ 1990)ꎬ but also facilitates the design
of effective conservation and management strategies
(Jian et al.ꎬ 2006). Howeverꎬ only a few studies
have been conducted because of the difficulty associ ̄
ated with collecting population samples for slipper
orchidsꎬ including one that used random amplified
polymorphic DNA (RAPD) markers to examine the
genetic diversity within four naturally distributed
P micranthum populations (Li et al.ꎬ 2002b).
Inter ̄simple sequence repeats ( ISSR) and se ̄
quence ̄related amplified polymorphism (SRAP) are
relatively simple and highly reproducible marker
technologies that do not rely on prior information a ̄
bout a DNA sequenceꎬ and which require very little
starting DNA template (Zietkiewicz et al.ꎬ 1994ꎻ Li
and Quirosꎬ 2001). These two systems have proven
to be powerful and efficient techniques for analyses
of population genetics diversityꎬ molecular taxonomic
classificationꎬ and marker ̄assisted breeding in many
orchid species (Smith et al.ꎬ 2002ꎻ Wallaceꎬ 2003ꎻ
Ding et al.ꎬ 2008ꎻ George et al.ꎬ 2009ꎻ Wang et
al.ꎬ 2009ꎻ Cai et al.ꎬ 2011). We thus apply these
two markers to investigate the genetic diversity and
population structure of P micranthum in southwest ̄
ern China. Our main goals were to 1) determine the
level of genetic variation within and among popula ̄
tionsꎬ 2) characterize the extent of genetic differen ̄
tiation between populationsꎬ and 3 ) identify the
causes for this observed differentiation.
1 Materials and methods
1 1 Sampling and plant material collection
We collected 407 individuals of Paphiopedilum
micranthum from 15 wild populations in three prov ̄
inces of southwestern China (Fig 1ꎻ Table 1). Be ̄
cause this species is rhizomatous and forms clumps
012 植 物 分 类 与 资 源 学 报 第 36卷
of multiple plants in the wild (Tsi et al.ꎬ 1999)ꎬ we
tried to avoid collecting ramets of only a single geno ̄
type by randomly selecting individuals that were
spaced at least 3 m apart. Young leaves were harves ̄
ted and dried by silica gel for further DNA isolation.
The genomic DNA of each sample was extracted from
leaves by the standard CTAB (cetyltrimethyl ammo ̄
nium bromide) method of Doyle and Doyle (1987).
Fig 1 Sampling sites for wild populations of Paphiopedilum
micranthum in southwestern China. Population
codes are explained with Table 1
1 2 ISSR fingerprinting
The sequences for our ISSR primers were pro ̄
vided by the Biotechnology Laboratoryꎬ University of
British Columbiaꎬ Canada. There were 15 (Table 2)
from 90 arbitrary primers showing good repetitionꎬ
special bandsꎬ and distinct polymorphism. Each IS ̄
SR reaction mixture ( 20 μL) contained 50 ng of
template DNAꎬ 8 μL of 2 × Taq PCR Master Mix
(0 1 U of Taq polymerase per μLꎬ 0 5 mmolL-1
dNTPꎬ 20 mmolL-1 Tris ̄HClꎬ 100 mmolL-1 KClꎬ
and 3 mmolL-1 MgCl2)ꎬ plus 2% formamideꎬ 100
nmolL-1 primerꎬ and double ̄distilled water. The
PCR program included an initial denaturation at 94 ℃
for 5 minꎻ then 40 cycles of 94 ℃ for 45 sꎬ 53 ℃ for
1 minꎬ and 72 ℃ for 2 minꎻ followed by a final ex ̄
tension at 72 ℃ for 7 min. The ISSR ̄PCR products
were separated in 2 0% agarose gels buffered with
0 5 × TBE. A 100 bp DNA ladder (Fermentas) was
used as a size marker. After staining with ethidium
bromideꎬ the DNA fragments were identified by image
analysis software for gel documentation (Lab Works
Softwareꎬ version 3 0ꎻ UVPꎬ Uplandꎬ CAꎬ USA).
1 3 SRAP fingerprinting
The SRAP marker analysis was performed as de ̄
scribed by Li and Quiros (2001). From 88 arbitrary
primer pairsꎬ we selected 11 combinations that showed
distinct polymorphism (Table 2). Each SRAP reaction
mixture (20 μL) contained 40 ng of template DNAꎬ
8 μL of 2 × Taq PCR Master Mixꎬ 100 nmolL-1
each for forward and reverse primersꎬ and double ̄
distilled water. Amplification was performed under the
following conditions: 94℃ / 3 minꎻ five cycles of 94 ℃
/ 1 minꎬ 35 ℃ / 1 minꎬ 72 ℃ / 1 minꎻ then 30 cycles
Table 1 Sampling information for 15 Paphiopedilum micranthum populations
Population code Location Number of samples Elevation / m Longitude (E) Latitude (N)
YW Wenshanꎬ Yunnan 36 1550 104°18′42″ 23°10′22″
YG Guangnanꎬ Yunnan 36 1530 105°04′15″ 24°11′31″
YT Tianbaoꎬ Yunnan 24 1350 104°44′26″ 23°03′41″
YX Xichouꎬ Yunnan 27 1540 104°31′51″ 23°20′13″
YF Fadouꎬ Yunnan 29 1580 104°45′19″ 23°24′11″
YJ Jinchangꎬ Yunnan 24 1490 104°49′17″ 23°07′47″
YM Maguanꎬ Yunnan 21 1438 104°22′20″ 22°56′48″
XL Leyeꎬ Guangxi 23 1020 106°21′14″ 24°48′54″
XN Napoꎬ Guangxi 24 1077 105°57′21″ 23°24′29″
XY Linyunꎬ Guangxi 19 1050 106°44′13″ 24°34′14″
GC Cehengꎬ Guizhou 29 1273 105°39′59″ 24°59′22″
GN Nidangꎬ Guizhou 29 1479 104°48′24″ 24°49′49″
GW Wangmoꎬ Guizhou 28 1274 106°23′12″ 25°14′54″
GZ Zhaojiaduiꎬ Guizhou 30 1375 105°00′23″ 25°01′57″
GB Bajieꎬ Guizhou 28 1182 104°59′41″ 24°54′39″
1122期 HUANG Jia ̄Lin et al.: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of
Table 2 Sequences of 15 ISSR primers and 11 SRAP Primers
ISSR primers Sequences SRAP primer combinations Forward primer Reverse primer
UBC811 (GA) 8C Me1 ̄Em9 TGAGTCCAAACCGGATA GACTGCGTACGAATTCGA
UBC814 (CT) 8A Me2 ̄Em3 TGAGTCCAAACCGGAGC GACTGCGTACGAATTGAC
UBC815 (GA) 8G Me3 ̄Em11 TGAGTCCAAACCGGAAT GACTGCGTACGAATTCCA
UBC822 (TC) 8A Me5 ̄Em2 TGAGTCCAAACCGGAAG GACTGCGTACGAATTTGC
UBC823 (TC) 8C Me5 ̄Em5 TGAGTCCAAACCGGAAG GACTGCGTACGAATTAAC
UBC825 (AC) 8T Me5 ̄Em7 TGAGTCCAAACCGGAAG GACTGCGTACGAATTCAA
UBC827 (TC) 8G Me5 ̄Em9 TGAGTCCAAACCGGAAG GACTGCGTACGAATTCGA
UBC834 (AG) 8YT Me6 ̄Em3 TGAGTCCAAACCGGTAA GACTGCGTACGAATTGAC
UBC835 (AG) 8YC Me6 ̄Em4 TGAGTCCAAACCGGTAA GACTGCGTACGAATTTGA
UBC844 (CT) 8RC Me7 ̄Em10 TGAGTCCAAACCGGTCC GACTGCGTACGAATTCAG
UBC845 (CT) 8RG Me8 ̄Em3 TGAGTCCAAACCGGTGC GACTGCGTACGAATTGAC
UBC853 (TC) 8RT
UBC857 (AC) 8YG
UBC859 (CG) 8RC
UBC873 (GACA) 4
of 94 ℃ / 1 minꎬ 50 ℃ / 1 minꎬ 72 ℃ / 1 minꎻ and a fi ̄
nal extension of 72 ℃ / 10 min. All PCR reactions
were run in the ABI 2720 Thermal Cycler. The SRAP ̄
PCR products were analyzed on 6% non ̄denatured
polyacrylamide gels in 1 × TBE buffer running at 380 V
constant voltage for 1 0 h. Afterwardꎬ silver ̄staining
was done as reported by Bassam et al. (1991).
To ensure the reliability of the genotypeꎬ nega ̄
tive controls were run at each step to check for exog ̄
enous contamination for ISSR and SRAP. The exper ̄
iment was repeated twiceꎬ and only data from in ̄
tensely stainedꎬ unambiguous bands were used for
statistical analysis.
1 4 Data analysis
Amplified bands were scored according to the
presence (1) or absence (0) of homologous bands
for all samplesꎬ and were displayed as part of a bi ̄
nary matrix. These data were analyzed by POPGENE
version 1 32 (Francis and Yangꎬ 2000) to estimate
the degree of genetic diversity in P micranthum.
Some essential diversity parametersꎬ e g.ꎬ percent ̄
age of polymorphic bands (PPB)ꎬ Shannon’s infor ̄
mation index I (Shannon and Weaverꎬ 1949)ꎬ and
Nei’s genetic diversity He (Neiꎬ 1978)ꎬ were eval ̄
uated at both the population and species levels. Ge ̄
netic differentiation between populations was esti ̄
mated by the coefficients for genetic differentiation
(Gst ) and gene flow (Nm ) ( Slatkin and Bartonꎬ
1989). To examine the genetic relationship among
populationsꎬ we generated a genetic distance map
via POPGENE. We also constructed a dendrogram
per Nei’s (1978) genetic distance methodꎬ using
the unweighted pair ̄group method of averages (UP ̄
GMA) and 1 000 permutations of bootstrappingꎬ
with TFPGA version 1 3 (Millerꎬ 1997). A Mantel
test was performed to estimate any correlations between
the matrices of genetic distances and geographical
distancesꎬ using GenAlEx version 6 5 (Peakall and
Smouseꎬ 2006). Principal Coordinate Analysis (PCoA)
was also conducted with GenAlEx version 6 5ꎬ
based on the calculated Jaccard’s similarity coeffi ̄
cients. Correlations between elevational differences
and genetic distances were estimated by PASSAGE
version 2 (Rosenberg and Andersonꎬ 2011).
2 Results
2 1 Genetic diversity in populations
A total of 88 different bands were scored from
15 ISSR primersꎬ of which 81 were polymorphic
(92 04%). Our 11 SRAP primer combinations pro ̄
duced 280 bandsꎬ from 100 to 500 bp longꎬ across
all 407 individuals. Of thoseꎬ 278 were polymorphic
(99 29%). A summary of the ISSR and SRAP data
from each Paphiopedilum micranthum population is
212 植 物 分 类 与 资 源 学 报 第 36卷
presented in Table 3. The ISSR analyses resulted in
a He value of 0 3839 and an I value of 0 5646 at the
species level. Within each populationꎬ the PPB var ̄
ied from 64 89% (YJ and XL) to 89 36% (YW).
The mean He was 0 2847ꎬ ranging from 0 1975
(XL) to 0 3238 (YW). Values for I showed similar
trendsꎬ ranging from 0 3024 ( XL ) to 0 4799
(YW). The SRAP analyses produced a He value of
0 2806 and an I of 0 4359 at the species level.
Within each populationꎬ the PPB varied from
62 54% (XL) to 82 69% (YW). Values for He
were 0 1963 to 0 2410 ( mean 0 2166)ꎻ for Iꎬ
0 2974 to 0 3701 (mean 0 3301). These data from
both ISSR and SRAP showed thatꎬ among the 15
populationsꎬ the genetic diversity of P micranthum
was richest within YW and poorest within XL.
2 2 Genetic differentiation within and among
populations
AMOVA analysis with GenAlEx 6 5 software
presented significant genetic variation (P < 0 001)
among and within the 15 populations. Based on ISSR
analysisꎬ the main component (69%) within the to ̄
tal molecular variance was attributed to differences
between individuals within populationsꎬ with the re ̄
mainder ( 31%) coming from among populations
(Table 4). This was consistent with the POPGENE
results (Gst = 0 2577). Similarlyꎬ when the SRAP
data were analyzedꎬ moderate genetic differentiation
(Gst =0 2383) was found among populations. The av ̄
erage number of individuals exchanged between popu ̄
lations per generation (Nm) was 0 7201 based on IS ̄
SR markers and 0 7991 when SRAP markers were
used (Table 3). This indicated that limited pollen
and seed dispersal occurred among populations.
2 3 Genetic distances among populations
The Mantel tests showed significant positive cor ̄
relations between genetic and geographic distances
for both ISSRs ( r= 0 455ꎻ P= 0 001) and SRAPs
Table 3 Genetic diversity of Paphiopedilum micranthum based on ISSR and SRAP analyses
Population
ISSR
PPB / % He I Gst Nm
SRAP
PPB / % He I Gst Nm
YW 89 36 0 3238 0 4799 82 69 0 2410 0 3701
YG 87 23 0 3087 0 4598 68 90 0 2015 0 3076
YT 77 66 0 2873 0 4239 64 66 0 2084 0 3159
YX 80 85 0 2831 0 4196 75 27 0 2177 0 3346
YF 75 53 0 2787 0 4113 70 67 0 2154 0 3274
YJ 64 89 0 2314 0 3448 63 60 0 2053 0 3106
YM 87 23 0 3220 0 4767 67 14 0 2040 0 3095
XL 64 89 0 1975 0 3024 62 54 0 1963 0 2974
XN 75 51 0 2602 0 3907 64 32 0 2199 0 3289
XY 77 66 0 2851 0 4219 69 26 0 2136 0 3301
GC 81 91 0 3026 0 4468 71 38 0 2227 0 3384
GN 87 23 0 3177 0 4694 74 91 0 2273 0 3479
GW 82 98 0 2838 0 4255 79 86 0 2362 0 3650
GZ 88 30 0 3035 0 4506 79 95 0 2384 0 3651
GB 82 98 0 2912 0 4312 67 49 0 2018 0 3077
Mean 80 28 0 2847 0 4236 70 67 0 2166 0 3301
Species level 91 66 0 3839 0 5646 0 2577 0 7201 99 29 0 2806 0 4359 0 2383 0 7991
Table 4 AMOVA results for genetic variance within and among populations by ISSR and SRAP
Markers Source of variation df Sum of squares Mean squares Variation components Total variation / % P ̄value
ISSR Among populationsWithin populations
14
392
2425 072
5086 606
173 219
12 976
5 918
12 976
31
69
<0 001
<0 001
SRAP Among populationsWithin populations
14
392
4569 319
12709 875
326 380
32 423
10 857
32 423
25
75
<0 001
<0 001
3122期 HUANG Jia ̄Lin et al.: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of
(r= 0 421ꎻ P = 0 003) (Table 5). To examine fur ̄
ther why genetic distances formed among these popu ̄
lationsꎬ we used a Mantel test with PASSAGE to in ̄
vestigate any correlation between elevational differ ̄
ences and genetic distances. Hereꎬ positive correla ̄
tions between those two components were identified
from both ISSR analysis ( r = 0 4356ꎻ P = 0 0006)
and SRAP analysis ( r= 0 3953ꎻ P= 0 0025).
The UPGMA analyses applying both ISSRs
(Fig 2A) and SRAPs ( Fig 2B) clearly resolved
the 15 populations into two major clusters. Cluster I
Table 5 Mantel tests to evaluate genetic distancesꎬ geographic
distancesꎬ and elevational differences for 15
Paphiopedilum micranthum populations
Genetic distance versus
Geographic distance
P r
Genetic distance versus
Elevational difference
P r
ISSR 0 455 0 001 0 436 0 001
SRAP 0 421 0 003 0 395 0 003
included 12 populations from Guizhouꎬ Guangxiꎬ
and Yunnan provinces while Cluster II comprised the
XLꎬ GWꎬ and XY populations from Guizhou and
Guangxi provinces. The PCoA results of ISSR and
SRAP data were shown in Figure 3A and Figure 3Bꎬ
respectively. Consistent with the UPGMA dendro ̄
gramꎬ PCoA also revealed that the 15 populations
could be separated into two main clusters.
3 Discussion
3 1 Genetic diversity
We collected 407 samples of P micranthum from
native 15 populations distributed in southwestern Chi ̄
na and analyzed their genetic variation in this study.
The ISSR and SRAP markers revealed higher genetic
diversity than those of other orchidsꎬ e g.ꎬ Cymbidi ̄
um goeringiiꎬ Goodyera proceraꎬ Changnienia amoe ̄
naꎬ and Cypripedium flavum (Table 6). Moreoverꎬ
Fig 2 A. UPGMA dendrogram based on ISSR dataꎻ B. UPGMA dendrogram based on SRAP data
Fig 3 A. Principal Coordinate Analysis of 407 individualsꎬ based on ISSR dataꎻ
B. Principal Coordinate Analysis of 407 individualsꎬ based on SRAP data
412 植 物 分 类 与 资 源 学 报 第 36卷
Table 6 Comparisons of values for genetic diversity (PPB and He) and differentiation (Gst) between
Paphiopedilum micranthum and other orchid species
Species PPB / % He Gst Markers References
Paphiopedilum micranthum 91 6699 29
0 3839
0 2806
0 2577
0 2383
ISSR
SRAP
This paper
This paper
P micranthum 73 30 0 2170 ——— RAPD Li et al.ꎬ 2002b
Cypripedium flvum 82 69 0 2884 0 1540 AFLP Cai et al.ꎬ 2008
Cypripedium calceolus 36 40 0 1490 0 0590 Allozyme Brzosko et al.ꎬ 2011
Cymbidium goerigii 88 19 0 2628 0 2440 ISSR Gao and Yangꎬ 2006
Goodyera procera 97 03 0 2930 0 3900 RAPD Wong and Sunꎬ 1999
Changnienia amoena 76 50 0 1941 ——— RAPD Li et al.ꎬ 2002a
Orchidaceae (average) ——— ——— 0 1870 ——— Forrest et al.ꎬ 2004
the degree of diversity for P micranthum found here
was greater than that calculated when RAPD markers
were used with four previous populations of that spe ̄
cies (PPB=73 3%ꎻ He = 0 217) (Li et al.ꎬ 2002b).
The primary reason for this discrepancy may lie in
the choice of markers. The polymorphism of products
was higher with ISSRs and SRAPs than with RAP ̄
Dsꎬ thereby providing more information about a par ̄
ticular genome. Moreoverꎬ ISSR and SRAP molecu ̄
lar markers are considered more stable and reliable
when compared with the RAPD technique ( Dirle ̄
wanger et al.ꎬ 1998ꎻ Esselman et al.ꎬ 1999ꎻ Gilbert
et al.ꎬ 1999). Another explanation is that we used
more populations and individual samples than were
involved in an earlier investigation by Li et al.
(2002b). Some genetic parameters (He) are sensi ̄
tive to sample sizeꎬ thus influencing how well those
parameters can be estimated (Li et al.ꎬ 2000).
Plants of P micranthum are perennial and herba ̄
ceous. Their decades ̄long life spans may contribute
to their high genetic diversity (Nybomꎬ 2004). Mo ̄
reoverꎬ the breeding mode is an important factor af ̄
fecting genetic diversity (Hamrickꎬ 1982ꎻ Hamrick
and Godtꎬ 1990). This species reproduces by both
sexually and asexuallyꎬ which has been considered
as a strategy to maximize heterozygosity and repro ̄
ductive success (Yan et al.ꎬ 1999). One capsule of
P micranthum contains more than 5 000 seedsꎬ which
can ensure a large gene pool that can provide abun ̄
dant heterozygotes (Luo et al.ꎬ 2003). In additionꎬ
P micranthum has strong capacity of clonal reproduc ̄
tionꎬ developing rhizomes that often form numerous
ramets and support an apparent plexiform distribution
(Tsi et al.ꎬ 1999). Although a pollination system has
not been reported for P micranthumꎬ Cribb and Mc ̄
Gough (1997) has classified it as essentially an out ̄
breeding species that utilizes insect pollination.
Thenꎬ its out ̄crossing strategy might also contribute
to its higher diversity comparing with that of inbreed ̄
ing orchid species (Ehlers and Pedersenꎬ 2000).
3 2 Population genetic structure
Based on the assumption of Hardy-Weinberg e ̄
quilibriumꎬ we detected moderate genetic differentia ̄
tion among our populations of P micranthum (Gstꎬ
0 2577 by ISSRꎻ 0 2383ꎬ by SRAP). AMOVA ana ̄
lysis also showed that 31% (ISSR) and 25% (SRAP)
genetic variation existed among populationsꎬ as well
as 69% (ISSR) and 75% (SRAP) variation within
populations (Table 4). This differentiation is higher
comparing with affinitiveꎬ insect ̄pollinated orchid
speciesꎬ such as Cypripedium calceolus (Gst = 0 059ꎻ
Brzosko et al.ꎬ 2011) and C flavum (Gst = 0 154ꎻ
Cai et al.ꎬ 2008)ꎬ for which seeds are wind ̄disper ̄
sed. Forrest et al. (2004) have reported that orchid
Gst values range from 0 012 to 0 924 ( average
0 187)ꎬ and that variations in population genetics
differentiation are huge between orchid species. As
shown hereꎬ the Gst values for P micranthum (0 2577
and 0 2383) were higher than the average of 0 187
calculated for other orchid species.
The genetic structure of a plant species is usu ̄
ally influenced by factors such as mating system and
extent of gene flow (Hamrick and Godtꎬ 1990). The
theory of population genetics suggests that low Nm
5122期 HUANG Jia ̄Lin et al.: ISSR and SRAP Markers Reveal Genetic Diversity and Population Structure of
values (i e.ꎬ <1) cannot prevent the differentiation
between populations that is caused by genetic drift
(Wrightꎬ 1931ꎻ Hartl and Clarkꎬ 1989). Our study
produced Nm values of 0 7201 (ISSR) and 0 7991
(SRAP)ꎬ both less than 1ꎬ which aggravated the
differentiation between populations. The distribution
of pollen and seeds is a major determinant of gene
flow in natural populations (Li and Chenꎬ 2004).
Because most orchid species rely primarily on wind
dispersalꎬ their seeds can move across great dis ̄
tances (Swamy et al.ꎬ 2004ꎬ 2007). In contrast to
related species of Paphiopedilum and Cypripediumꎬ
howeverꎬ seeds of P micranthum are not carried as
far (Zhangꎬ 2012). This might explain why genetic
differentiation between populations is relatively high
in that species. Another factor in the reduction of
gene flow between populations is anthropogenic. Be ̄
cause of their high ornamental valueꎬ most of the ol ̄
derꎬ flowering plants have been collected from wild
populationsꎬ leaving behind only the younger speci ̄
mens (Luo et al.ꎬ 2003).
3 3 Genetic relationships among populations
Both the UPGMA and PCoA evaluations divid ̄
ed the 15 geographical populations of P micranthum
into two clustersꎬ with one comprising 12 popula ̄
tions in Yunnan and Guizhou and the other contai ̄
ning two populations in Guanxi plus one in Guizhou.
PCoA revealed high genetic similarity in Cluster Iꎬ
whereas Cluster II showed evidence of genetic isola ̄
tion among its three populations. Results of Mantel
tests based on data from ISSR ( r = 0 455ꎬ P =
0 001) and SRAP (r = 0 421ꎬ P= 0 003) indicated
that genetic distance was correlated with geographic
distanceꎬ a phenomenon that is commonly found in
endemic and endangered species (Godt et al.ꎬ 2005ꎬ
Luan et al.ꎬ 2006). Although P micranthum is not
itself endemic to Chinaꎬ it is considered endangered
and protected there.
Genetic differentiation between natural popula ̄
tions is usually related to geographical barriersꎬ such
as high mountains and riversꎬ which make gene ex ̄
change almost impossible to achieve from one popu ̄
lation to another (Godt et al.ꎬ 2005). Hereꎬ howev ̄
erꎬ we could not identify any such barrierꎬ other
than elevational differenceꎬ between Clusters I and
II. Our Mantel tests detected a remarkable correla ̄
tion (P<0 05) between genetic distance and eleva ̄
tion. Many studies have shown that elevation ̄associ ̄
ated temperature variations play an important role in
directing genetic diversity and differentiation ( Ba ̄
yerꎬ 1992ꎻ Li and Chenꎬ 2004ꎻ Jiang et al.ꎬ 2005).
For exampleꎬ the genetic variation among Rhodiola
angusta populations at Changbai Mountain increases
as the temperature decreases due to elevation (Yan
et al.ꎬ 1999). Likewiseꎬ apart from geographical
distanceꎬ differences in elevation may be an impor ̄
tant factor that divided our P micranthum popula ̄
tions into two genetic clusters.
3 4 Conservation recommendations
For endangered speciesꎬ the goals of conserva ̄
tion are to ensure the continued survival of popula ̄
tions and to maintain their evolutionary potential
(Hamrick and Godt 1990ꎻ Wong and Sun 1999).
Given our findingsꎬ individuals of Cluster II com ̄
prised the XLꎬ GWꎬ and XY populations from
Guizhou and Guangxi provinces have relatively low
genetic variation. Thereforeꎬ we recommend that
these populations be prioritized for conservation pro ̄
tection. Meanwhileꎬ it is necessary to carry out the
ex ̄situ conservation and the artificial reproduction of
P micranthum as soon as possibleꎬ to store up mass
seedlings of artificial reproduction prepared for wild
re ̄introductionꎬ to renew this endangered species
and to make it thriving.
4 Conclusion
Using ISSR and SRAP molecular markersꎬ we
determined that plants of P micranthum exhibit a
high degree of genetic diversityꎬ and that genetic dif ̄
ferentiation is moderate among natural populations.
When our sample populations were assigned to two
clustersꎬ the one representing sites in Yunnan and
Guizhou showed high diversity while that of the Guan ̄
gxi sites had low diversity. The genetic differentiation
612 植 物 分 类 与 资 源 学 报 第 36卷
between populations was related to variations in biolog ̄
ical characteristicsꎬ such as capacity for seed dispers ̄
alꎬ as well as associations with geographical distance
and elevational differences. Our results will be benefi ̄
cial to managers who can develop reasonable strategies
for conserving this endangered slipper orchid.
Acknowledgments: The authors thank Yang Jun ̄Boꎬ Li
Hong ̄Taoꎬ and Zhang Zhi ̄Rong for help with the laboratory
work and data analysis.
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