全 文 ：Received 22 Dec. 2003 Accepted 24 May 2004
Supported by the Natural Science Foundation of Yunnan Province, China (2001 C0057M), the Knowledge Project from the Ministry of
Science and Technology of China (2001DEA10009) and the Knowledge Innovation Program of The Chinese Academy Sciences.
* Author for correspondence. Tel.: +86 (0)871 5223630; Fax: +86 (0)871 5216345; E-mail:.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1163-1169
High Genetic Diversity in a Rare, Narrowly Endemic Primrose Species:
Primula interjacens by ISSR Analysis
XUE Da-Wei1, GE Xue-Jun2, HAO Gang2, ZHANG Chang-Qin1*
(1. Kunming Institute of Botany, The Chinese Academy of Sciences, Kunming 650204, China;
2. South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China)
Abstract: Primula interjacens Chen (Primulaceae) is a rare and narrow endemic species of central-
south of Yunnan Province in China. This species consists of two varieties: P. interjacens var. interjacens
known with only one population, and P. interjacens var. epilosa with two populations. Inter-simple sequence
repeat (ISSR) marker was used to detect the genetic diversity of the three extant populations. We
expected a low genetic diversity level, but our results revealed a high level of intraspecific genetic diversity
(at population level: P = 59.75%, HE = 0.236 8, and Hpop = 0.345 9; at species level: P =75.47%, HT = 0.320 5,
and Hsp = 0.461 8), probably resulting from floral heteromorphism and preferring outcrossing. A moderate
level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis
(26.13%) and Shannon’s diversity index (25.09%). Although P. interjacens var. interjacens and P. interjacens
var. epilosa were morphologically distinct, UPGMA cluster analysis showed that the two varieties had no
distinct genetic differentiation and may be treated as a single taxon. Conservation measures are suggested,
including in situ and ex situ strategies, based on the observed population genetic information.
Key words: Primula interjacens ; endemic; ISSR; genetic diversity; variety; conservation strategy
Of the ca. 425 species in Primula, 75% are concentrated
in the Himalayan mountain chain and western China
(Richards, 1993). In Yunnan Province alone, there are about
150 Primula species (Hu and Kelso, 1996). P. interjacens is
a rare dwarf perennial, diploid (2n = 2x = 18; Xue and Zhang,
2003) with woody rhizomes endemic to the limestone crev-
ices of Mt. Wu-Liang-Shan, central-south of Yunnan
Province. Its current known range is a narrow corridor of
approximately 25 km×3 km (Fig.1). This plant is of poten-
tial horticulture value by its larger attractive flowers and
beautiful leaves. The scapes are purplish brown, umbels
are in one or two whorls with 3-8 flowers. The limb of the
rose flower is ca. 2 cm wide. Based on the character of
plants being whether pubescent or glabrous, two varieties
were recognized (Hu and Kelso, 1996): P. interjacens var.
interjacens is pubescent and is found with only one popu-
lation of approximately 2 500 individuals, whereas P.
interjacens var. epilosa is glabrous and known with only
two populations: population Bingbu with approximate
15 000 individuals, and population Wangjiaqing with about
1 200 individuals (personal observation).
Biologists have long been fascinated with rare and nar-
rowly endemic species. In recent years, this fascination
has turned to urgency, as ever more species dwindle to-
ward extinction (Gitzendanner and Soltis, 2000). Rare and
endangered species are susceptible to loss of genetic varia-
tion through genetic drift in small populations. Manage-
ment decisions for the conservation of rare taxa ideally ne-
cessitate an understanding of their biology and other
factors, including genetic variability. Successful manage-
ment of many rare plant populations has been greatly im-
proved by genetic data (Ellstrand and Elam, 1993). The gath-
ering of data on population genetic structure of rare spe-
cies has become a common prelude to conservation plan-
ning (Archibald et al., 2001). Moreover, knowledge of the
genetic diversity within and among populations might also
contribute to understanding of the interpopulation rela-
tionships (Martin et al., 1997).
An abundance of genetic markers allows for a more ac-
curate fingerprinting that is proved to be useful with a wide
range of applications in plant population studies, such as
the detection of genetic variation within and between
populations, the characterization of clones and the analy-
sis of breeding systems (Weising et al., 1995). ISSR is a
PCR based technique that has been successful in the stud-
ies of population genetic structure (Esselman et al., 1999;
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041164
Ge and Sun, 1999; Culley and Wolfe, 2001; Qian et al., 2001).
ISSR reaction is more specific than RAPD reaction due to
the longer SSR-based primers, thus enabling higher-strin-
gency amplifications (Wolfe and Liston, 1998). The high-
stringency results in fewer problems with reproducibility, a
common criticism against the low-stringency RAPD assay
(Yang et al., 1996).
In the present study, we conducted an ISSR analysis of
P. interjacens, with the following two aims in mind: (1) to
investigate its genetic diversity, and (2) to provide elemen-
tary information for the conservation strategies.
1 Materials and Methods
1.1 Population sampling
Twenty-two individuals were sampled randomly from
the only one population of Primula interjacens var.
interjacens in Ximei (PX) (100º3354 E, 24º3411 N, 1 900 m
a.s.l.); 22 individuals from the population of P. interjacens
var. epilosa in Bingbu (PB) (100º3428 E, 24º3452 N, 2 050
m a.s.l.) and 23 in Wangjiaqing (PW) (100º3330 E, 24º3653
N, 2 000 m a.s.l.) (Fig.1) were obtained. Fresh leaves of
individual plants were collected and dried with silica gels.
1.2 DNA extraction and PCR amplification
Genomic DNA was isolated following the CTAB proce-
dure (Doyle, 1991). DNA quality and quantity were deter-
mined in 1% agarose gel buffered with 0.5× TBE. One
hundred primers of 15-23 nucleotides in length (UBC primer
set # 9, Biotechnology Laboratory, University of British
Columbia) were screened. PCR amplification was carried
out in a volume of 20 mL, containing approximately 20 ng
template DNA, 2.0 mmol/L MgCl2, 2.0 mL 10×PCR buffer,
2% formamide, 0.1 mmol/L dNTPS, 0.225 mmol/L primer, and
1 U Taq DNA polymerase. PCR reactions were performed
using an MJ Research 96-well thermal cycler with hot bon-
net following the conditions: 5 min at 94 ºC followed by 35
cycles of 30 s at 94 ºC, 45 s annealing at 50-52 ºC, and 1.5
min extension at 72 ºC, a final extension cycle of 7 min at 72
ºC. Amplification products were electrophoresed on 1.8%
agarose gel buffered with 0.5×TBE. A 100 bp DNA ladder
(New England Biolabs) was used as a size marker (100-
1 000 bp). DNA fragments were identified by image analy-
sis software for gel documentation system (LabWorks Soft-
ware Version 3.0; UVP, Upland, CA 91786, USA) following
staining with ethidium bromide.
1.3 Data analysis
ISSR bands were scored as present (1) or absent (0).
The resulting present/absent data matrix was analyzed us-
ing POPGENE v. 1.31 (Yeh et al., 1999). Assuming Hardy-
Weinberg equilibrium, the percentage of polymorphic loci
(P) and gene diversity (HE) were estimated both at popula-
tion level and at the species level. Coefficient of gene dif-
ferentiation (GST) and the level of gene flow (Nm) were mea-
sured using Nei’s (1973) gene diversity statistics. Nei’s
(1972) unbiased genetic identity (I) and genetic distance
(D) between populations were also computed. A dendro-
gram was constructed based on the matrix of genetic dis-
tance using UPGMA (unweighted pair-group method us-
ing arithmetic average) using software packages NTSYS-
pc 2.0 (Rohlf, 1998).
Genetic diversity was also estimated using Shannon’s
information measure (Lewontin, 1972) Ho = -åpi log2 pi,
where pi is the frequency of a given ISSR fragment. Ho was
calculated at two levels: the average diversity within popu-
lations (Hpop), and the total diversity (Hsp). Then the
Fig.1. Locations of the three known populations of Primula interjacens. Its entire range is indicated by the spot on the map of China.
PB, population of P. interjacens var. epilosa from Bingbu (22 individuals); PW, population of P. interjacens var. epilosa from Wangjiaqing (23
individuals); PX, population of P. interjacens var. interjacens from Ximei (22 individuals).
XUE Da-Wei et al.: High Genetic Diversity in a Rare, Narrowly Endemic Primrose Species: Primula interjacens by ISSR
Analysis 1165
proportion of diversity among populations was estimated
as (Hsp-Hpop)/Hsp.
As the absence of ISSR fragments may not necessarily
imply shared ancestry, Jaccard’s coefficient was also used
to analyze the presence/absence data. Jaccard’s similarity
coefficient was estimated using NTSYS-pc software (Rohlf,
1998). UPGMA clustering was performed from the SHAN
option of NTSYS-pc and a dendrogram representing the
relationship between all individuals tested was derived from
the TREE option.
2 Results
Of 100 primers screened, 13 primers were chosen for
further analysis (Table 1). A total of 106 different ISSR bands
were scored, corresponding to an average of 8.15 bands
per primer. Among the 106 loci, 80 (75.47%) were polymor-
phic at the species levels (Table 2). The percentages of
polymorphic loci (P) for a single population ranged from
56.6% (PW) to 64.15% (PB) with an average of 59.75%.
Assuming Hardy-Weinberg equilibrium, the average
gene diversity was estimated to be 0.236 8 within popu-
lations (HE), and 0.320 5 at the species level (HT). The
Shannon indices ranged from 0.324 2 to 0.376 3, with an
average of 0.345 9 at the population level (Hpop) and
0.461 8 at the species level (Hsp). Among the three
populations, PB population exhibits the greatest level of
variability (P: 64.15%, HE: 0.258 8, Hpop: 0.376 3,
respectively), whereas the PW population exhibits the
lowest level of variability (P: 56.6%, HE: 0.220 9, Hpop:
0.324 2) (Table 3). These values are close to that of the
widespread P. ovalifolia (Nan et al., 2002) and P. obconica
(Nan et al., 2003).
The coefficient of genetic differentiation between popu-
lations (GST) was 0.261 3 as estimated by partitioning of the
total gene diversity. The Shannon’s diversity index analy-
sis partitioned 25.09% of the total variation between
populations, in broad agreement with the result of genetic
differentiation analysis. Genetic identities (I) between popu-
lations varied from 0.813 7 to 0.873 3 with a mean of 0.835 7
± 0.033. The average of genetic distance is 0.179 9 ±
0.038 71 (Table 3). The level of gene flow (Nm) was esti-
mated to be 0.71 individual per generation between
populations.
A dendrogram of all 67 individuals (Fig.2) and the three
populations (Fig.3) of P. interjacens were generatedly us-
ing the UPGMA cluster method. In Fig.2, all individuals of
each population form a distinct cluster, suggesting that
there is genetic differentiation among populations.
However, in both Fig.2 and Fig.3, the PB population falls in
a clade grouping with the PX population, rather than with
the PW population, meaning that there is no distinct ge-
netic differentiation between P. interjacens var. interjacens
and P. interjacens var. epilosa.
3 Discussion
According to Hamrick and Godt (1989), there are strong
associations between geographical range and genetic
diversity. There is evidence that a low level of genetic di-
versity is a common feature of endemic plant species (Karron,
1991; Ellstrand and Elam, 1993; Gitzendanner and Soltis,
2000). An almost complete absence of genetic diversity ei-
ther within or between populations has been revealed by
allozyme and RAPD in the Scottish endemic P. scotica
Table 1 Primers used for ISSR amplification and number of
bands per primer
Primer Sequence No. of
Polym-
bands
orphic
bands
807 (AG)8T 6 4
808 (AG)8C 11 10
811 (GA)8C 10 9
813 (CT)8T 6 4
823 (TC)8C 5 5
834 AGA GAG AGA GAG AGA G(CT)T 6 5
835 AGA GAG AGA GAG AGA G(CT)C 11 9
836 AGA GAG AGA GAG AGA G(CT)A 8 4
840 GAG AGA GAG AGA GAG A(CT)T 9 7
855 ACA CAC ACA CAC ACA C(CT)T 10 9
857 ACA CAC ACA CAC ACA C(CT)G 7 5
888 (CGT)(AGT)(CGT)CAC ACA CAC ACA CA 11 5
889 (AGT)(CGT)(AGT)ACA CAC ACA CAC AC 6 4
Table 2 Genetic variability within populations of P.
interjacens detected by ISSR analyses
Population HE Hpop P (%)
P X 0.230 6 (0.213 0) 0.337 1 (0.302 7) 58.49
PB 0.258 8 (0.214 1) 0.376 3 (0.301 4) 64.15
P W 0.220 9 (0.210 4) 0.324 2 (0.300 6) 56.60
Mean (SD) 0.236 8 (0.019 7) 0.345 9 (0.027 1) 59.75
HE, expected heterozygosity; Hpop, Shannon’s Information index;
P, percentage of polymorphic loci; Abbreviations are the same as
in Fig.1.
Table 3 Nei’s (1972) genetic identity and distance between
pairs of populations
Pop ID P X PB P W
P X **** 0.873 3 0.820 1
PB 0.135 5 **** 0.813 7
P W 0.198 3 0.206 1 ****
Nei’s genetic identity (above diagonal) and genetic distance (below
diagonal). Abbreviations are the same as in Fig.1.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041166
(Glover and Abbott, 1995). In contrast to our expectation of
low genetic diversity in P. interjacens, high levels of ge-
netic variation was revealed in all of the three populations
of P. interjacens. Its genetic diversity is comparable with
the widespread congeners P. ovalifolia (Nan et al., 2002)
and P. obconica (Nan et al., 2003). Thus P. interjacens
belongs to the subset of endemics possessing high levels
of genetic variability (Smith and Pham, 1996). This is
noteworthy, given the extremely small geographic range of
this species, less than 20 km between the PW and PX popu-
lations (Fig.1).
In recent years, a lot of studies have found high levels
of genetic variability in rare or narrow endemic species
(Richter et al., 1994; Smith and Pham, 1996; Ge et al., 1997;
Ayres and Ryan, 1999; Kang et al., 2000; Helenurm, 2001;
Zawko et al., 2001; González-Astorga and Castillo-Campos,
2004). Factors such as recent speciation from a more wide-
spread species, recent changes in distribution or habitat,
breeding system, somatic mutations, multiple founder
events, or Pleistocene refugia have been invoked to ex-
plain high levels of genetic diversity in rare plants (Ranker,
1994; Lewis and Crawford, 1995; Maguire and Sedgley, 1997;
Zawko et al., 2001). Among these factors, breeding system
is one of the important factors that often cause the counter-
theoretical results. Outcrossing species commonly have
higher levels of genetic diversity and lower differentiation
between populations than selfing and clonal plants. Study
on genetic diversity of 29 species of genera Schiedea and
Alsinidendron revealed outcrossing hermaphroditic and
dimorphic species had significantly higher genetic variability
than species with autogamous breeding systems (Weller et
al., 1996). The Primulaceae is one of the six or so families of
flowering plants that are characterized by the widespread
rather than occasional occurrence of hetero-morphic self-
incompatibility (Gibbs and Talavera, 2001). The high ge-
netic diversity maintained in P. interjacens is probably due
to outcrossing facilitated by distylous floral morphs
(Clapham et al., 1987; Kery et al., 2000). The pin flowers
and thrum flowers are generally self-incompatible and re-
ciprocally pollinated by insects. Floral dimorphism facili-
tates pollen exchange among different individuals. This
breeding system will tend to: (1) maintain the genetic diver-
sity of individuals and populations; and (2) promote gene
flow within populations, increasing the effective popula-
tion size and reducing the effects of drift (Hamrick and Godt,
1989). On the other hand, as a narrow endemic of subtropi-
cal region, P. interjacens may have a recent speciation from
a more widespread species that harbors high genetic
diversity. Among the 18 primrose species recorded in Mt.
Wu-Liang-Shan (Peng, 1998), P. malacoides is a widespread
species and assumedly have a close relationship with P.
interjacens. Both species belong to the section
Monocarpicae Franch. ex Pax (Hu and Kelso, 1996).
However, the genetic diversity of P. malacoides is still
Fig.2. UPGMA dendrogram of Primula interjacens individuals
based on ISSR data. Abbreviations are the same as in Fig.1.
Fig.3. The UPGMA dendrogram based on Nei’s (1972) genetic
distance. Abbreviations are the same as in Fig.1.
XUE Da-Wei et al.: High Genetic Diversity in a Rare, Narrowly Endemic Primrose Species: Primula interjacens by ISSR
Analysis 1167
unknown. Further studies are needed to confirm this
speculation.
Typically inbreeding species maintain relatively more of
their genetic diversity among populations rather than within
populations than do outcrossers (Brown, 1979). In their
recent review on estimates of genetic diversity obtained
with RAPD markers, Nybom and Bartish (2000) compiled
mean GST values of 0.59, 0.19 and 0.23 for selfing, mixed
mating and outcrossing plant species, respectively. Com-
pared with these values, the populations of P. interjacens
is less differentiated than that of selfing, but close to that
with a outcrossing breeding system. This moderate level of
genetic differentiation found among populations may be
due to the limited seed dispersal ability of P. interjacens. In
the meantime, the detected genetic differentiation in this
study may represent an overestimation of the true level of
genetic differentiation of this species. The estimates of
genetic differentiation among populations based on mo-
lecular markers are generally higher than estimates based
on allozymes (Ayres and Ryan, 1999).
The present study failed to offer clear genetic evidence
to separate P. interjacens var. interjacens and P. interjacens
var. epilosa. According to the UPGMA dendrogram (Figs.
2, 3), PB population of P. interjacens var. epilosa is clus-
tered with PX population of P. interjacens var. interjacens,
other than with PW population of P. interjacens var.
epilosa. Because of the geographical proximity among the
three populations, the gene flow among them could pre-
vent significant differentiation of populations. Taking into
account that pubescent or glabrous is the only diagnostic
character of these two varieties, and their ecological condi-
tions are similar, the infraspecific classification of P.
interjacens needed to be reevaluated.
From a conservation perspective, the high genetic di-
versity maintained within populations is encouraging. The
high level of genetic variation, unusual for so narrow an
endemic, indicates a lack of genetic bottlenecking in this
species and suggests that crosses between members of
the same population would be unlikely to lead to inbreed-
ing depression that might result from combining geneti-
cally related gametes (Williamson and Werth, 1999). In the
short term, it is necessary to protect existing natural popu-
lations in order to preserve as much genetic variety as
possible. In the long term, the most suitable strategy for
the conservation of P. interjacens is the protection of its
habitat. In addition, because of its highly horticultural value,
the successful artificial propagation of this species in fu-
ture could not only guarantee its ex situ conservation and
sustainable survival, but also enhance the in situ
conservation.
Acknowledgements: We thank Prof. GE Song for valuable
comments on this manuscript, and YU Yan for technical
assistance.
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