全 文 ：植 物 分 类 学 报 45 (4): 488–496(2007) doi:10.1360/aps06214
Acta Phytotaxonomica Sinica http://www.plantsystematics.com
———————————
Received: 28 December 2006 Accepted: 6 March 2007
Supported by the National Natural Science Foundation of China, Grant Nos. 30470125, 40671066, and the Knowledge
Innovation Project of Chinese Academy of Sciences, Grant No. KSCX2-SW-132.
* Author for correspondence. E-mail: haogang@scau.edu.cn; Tel.: 86-20-38635898; Fax: 86-20-85282180.
Phylogeographic structure of Primula obconica
(Primulaceae) inferred from chloroplast
microsatellites (cpSSRs) markers
1,4YAN Hai-Fei 2PENG Ching-I 1HU Chi-Ming 3HAO Gang*
1(South China Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650, China)
2(Research Center for Biodiversity, Academia Sinica, Nankang, Taipei 115, China)
3(College of Life Sciences, South China Agricultural University, Guangzhou 510642, China)
4(Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
Abstract Primula obconica has been cultivated widely as a popular garden plant. In order to
discover the pattern of genetic diversity and the evolutionary process, a total of 278
individuals from 17 populations throughout its distribution in China were analyzed using
chloroplast microsatellites (cpSSRs) markers. Four loci and a total of 14 haplotypes were
identified by our data set. The total gene diversity (HT =0.971) is high, while gene diversity
within populations (HS=0.028) is low. Analysis of molecular variance (AMOVA) shows that
about 98% variation is among populations. The results suggest that past fragmentation and
limited dispersal ability of seeds might play important roles in forming the present genetic
structure. A significantly higher value of Nst than that of Gst indicates that closely related
haplotypes are often found in the same area, and we found two different groups in the
minimum spanning tree (MST), which occupy different geographic regions. Furthermore,
older haplotypes were detected in the two groups, respectively. Possible refugia are inferred in
western Hubei Province and SW China during the glacial period.
Key words Primula obconica, genetic diversity, chloroplast microsatellite, glacial period,
refugia.
Primula obconica Hance is a perennial herb first introduced to Britain from China in
1880 (Connolly et al., 2004). Since then it has been cultivated by horticulturist as popular
houseplants. So far, P. obconica has been widely accepted as an ornamental primrose plant.
However, it was also claimed as one of the most allergenic species among primroses and
known as a significant source of allergic contact dermatitis in Europe, especially in England,
Germany, and the Scandinavian countries (Christensen & Larsen, 2000). Surprisingly, allergic
compounds were not found in wild plants of this species, suggesting the wild ones will be
ideal resource for horticultural use (Nan et al., 2002, 2003).
Primula obconica is widely distributed in the area south of Yangtze River in China,
mainly growing at 500–3000 m elevation in moist thickets, forests and rocks in mountain
woods (Hu, 1990; Hu & Kelso, 1996). In contrast to its behavior in cultivation, this species
shows exceptional variations in morphological traits in the wild (Richards, 2002). Six
subspecies had been taxonomically recognized, viz., ssp. obconica, ssp. begoniiformis
(Petitm.) W. W. Smith & Forr., ssp. parva (I. B. Balfour) W. W. Smith & Forr., ssp.
werringtonensis (Forr.) W. W. Smith & Forr., ssp. nigroglandulosa (W. W. Smith & Fletcher)
C. M. Hu, and ssp. fujianensis C. M. Hu & G. S. He (Hu, 1990; Hu & Kelso, 1996; He & Hu,
No. 4 YAN et al.: Phylogeographic structure of Primula obconica inferred from cpSSRs markers 489
2002). Little was known about the genetic diversity of this species in the wild despite its
important horticultural value, until Nan et al. (2003) conducted a genetic variation study for a
small number of populations in central and southwest China using inter-simple sequence
repeats (ISSR) markers.
The organelle genome based molecular markers are useful tools for evolutionary studies
since they are nonrecombinant and uniparentally inherited (Provan et al., 2001; Zhang et al.,
2005a). The chloroplast genome is always inherited maternally in angiosperms (Palmé et al.,
2003). A half/quarter effective population size of the chloroplast genome of that for the
nuclear genome makes it more sensitive to the effect of historic events (Schaal et al., 1998;
Freeland, 2005). Additionally, high mutation rates in simple sequence repeats give a good
chance to address the issue of genetic diversity (Hardy et al., 2003). These features make
chloroplast microsatellites (cpSSRs) a useful tool for studies in plant ecology and evolution,
especially as several conserved primers have been developed. To date, cpSSRs has been
widely used to detect genetic diversity, to understand crop plant evolution and domestication,
and even to elucidate the phylogeny of species with taxonomic problem (Echt et al., 1998;
Chaïr et al., 2005; Cubas et al., 2005).
Here we employed cpSSRs to survey intraspecific polymorphisms of P. obconica in
China. We aimed to infer the evolutionary history of this species based on the recovered
distribution of genetic patterns.
1 Material and methods
1.1 Sampling of plant materials
A total of 17 populations of P. obconica were sampled for the present study. Fresh leaves
were collected and dried in silica gel immediately in the field. The sampling strategy was to
cover the natural range of the species, and to include all the subspecies. Unfortunately, we
were unable to collect P. obconica ssp. parva and P. obconica ssp. nigroglandulosa, which
are believed very rare now in the wild. The sampling information is indicated in Table 1.
1.2 DNA extraction, amplification, and screening
Total DNA was isolated with a modified CTAB protocol (Doyle, 1991). Polymerase
chain reaction (PCR) assays were performed in a total volume of 20 µL, containing 10 ng of
genomic DNA, 0.15 µmol/L of each primer, 100 µmol/L of dNTPs, 2.0 mmol/L of MgCl2,
and 1 U Taq polymerase. PCR amplification was performed on a MJ Research PTC-100
Thermocycler programmed with an initial denaturation step at 94 for 3 min, 30 cycles℃
denaturation at 94 for 30 s, the annealing temperature indicated in T℃ able 2 for 30 s, and
extension at 72 for 30 ℃ s, followed by a final extension step at 72 ℃ for 7 min.
A total of twenty sets of primers (ccmp and ccSSR) were used in a preliminary study
(Weising & Gardner, 1999; Chung et al., 2003). One individual per population was selected
randomly, and amplified using these primers. PCR products stained with ethidium bromide
were checked in 1% (w/v) agarose gel electrophoresis. When the amplified fragments were
detected, the products mixed with loading buffer (7 µL) were loaded on vertical
polyacrylamide gel, and electrophoresed in 1×TBE buffer at constant voltage 125 V for
approximately 2 h 30 min. Bands with the help of fragment size standards were discriminated
visually in silver stained gel. The primers with polymorphic bands were used in whole
populations for further analysis. Presence or absence of polymorphic bands was scored with 1
or 0, respectively.
1.3 Data analysis
Because of the haploid characteristic of chloroplast genomes, the chloroplast haplotype
is defined as the unique combination of polymorphic scores obtained from each individual.
Acta Phytotaxonomica Sinica Vol. 45 490
Table 1 Sampling subspecies, populations, and individuals of Primula obconica Hance
Code Subspecies Location Voucher Sample
size
Haplotype
NP ssp. fujianensis C. M. Hu &
G. S. He
(福建报春)
Nanping, Fujian, China
(福建南平)
F. Y. Wang (王凤
英) s.n. (IBSC)
20 H6
LC ssp. obconica
(鄂报春)
Lechang, Guangdong, China
(广东乐昌)
H. G. Ye (叶华谷)
s.n. (IBSC)
3 H7
RY ssp. obconica
(鄂报春)
Ruyuan, Guangdong, China
(广东乳源)
G. Hao (郝刚) 392
(IBSC)
20 H1, H14
SZ-1 ssp. obconica
(鄂报春)
Sangzhi, Hunan, China
(湖南桑植)
G. Hao (郝刚) 297
(IBSC)
15 H2
SZ-2 ssp. obconica
(鄂报春)
Sangzhi, Hunan, China
(湖南桑植)
L. C. Tian (田连成)
17987 (IBSC)
8 H6
YC ssp. obconica
(鄂报春)
Yichang, Hubei, China
(湖北宜昌)
G. Hao (郝刚) 295
(IBSC)
17 H1, H5
EMS ssp. obconica
(鄂报春)
Emeishan, Sichuan, China
(四川峨眉山)
G. Hao (郝刚) 432
(IBSC)
22 H1, H5
LD ssp. obconica
(鄂报春)
Luding, Sichuan, China
(四川泸定)
G. Hao (郝刚) 448
(IBSC)
14 H8
DJY-1 ssp. Obconica
(鄂报春)
Dujiangyan, Sichuan, China
(四川都江堰)
G. Hao (郝刚) 422
(IBSC)
20 H4
DJY-2 ssp. obconica
(鄂报春)
Dujiangyan, Sichuan, China
(四川都江堰)
G. Hao (郝刚) 443
(IBSC)
6 H4
ML-1 ssp. werringtonensis (Forr.)
W. W. Smith & Forr.
(波叶鄂报春)
Muli, Sichuan, China
(四川木里)
G. Hao (郝刚) 540
(IBSC)
21 H11
ML-2 ssp. werringtonensis
(波叶鄂报春)
Muli, Sichuan, China
(四川木里)
G. Hao (郝刚) 565
(IBSC)
20 H12
LQ ssp. begoniiformis (Petitm.)
W. W. Smith & Forr.
(海棠叶鄂报春)
Luquan, Yunnan, China
(云南禄劝)
G. Hao (郝刚) 378
(IBSC)
20 H3
DL ssp. obconica
(鄂报春)
Dali, Yunnan, China
(云南大理)
G. Hao (郝刚) 524
(IBSC)
21 H10
WX ssp. obconica
(鄂报春)
Weixi, Yunnan, China
(云南维西)
G. Hao (郝刚) 516
(IBSC)
20 H9
BS-1 ssp. Obconica
(鄂报春)
Baoshan, Yunnan, China
(云南保山)
G. Hao & H. F. Yan
(郝刚, 颜海飞) 580
(IBSC)
19 H13
BS-2 ssp. begoniiformis
(海棠叶鄂报春)
Baoshan, Yunnan, China
(云南保山)
G. Hao & H. F. Yan
(郝刚, 颜海飞) 584
(IBSC)
12 H13


Several population genetic parameters were computed for each population: haplotype
frequency in all individuals (P), the effective number of haplotypes (Ne) computed as
Ne=(∑pi2)–1, and unbiased haplotype diversity (HE) according to the formula: HE=[n(n–1)–1]
(1–∑pi2), where n is the number of individuals analyzed and p is the frequency of the i-th
haplotype in a population (Nei, 1987).
In addition, estimates of average diversity over populations (HS), the average total gene
diversity (HT), and the value of genetic differentiation among populations (Gst and Nst) were
calculated by PERMUT 1.0 (developed by R. J. Petit, available at http://www.pierroton.
inra.fr/genetics/labo/Software/Permut/)(Pons & Petit, 1996). Genetic variance components
within and among populations between and within groups were inferred by a hierarchical
analysis of molecular variance (AMOVA) using Arlequin Ver. 3.0 (Excoffier et al., 2005). A
minimum spanning tree (MST) computed from the matrix of pairwise distances calculated
between all pairs of haplotypes using a modification of the algorithm described in Rohlf
No. 4 YAN et al.: Phylogeographic structure of Primula obconica inferred from cpSSRs markers 491
(1973) was estimated by the Arlequin package.
2 Results
By analyzing twenty primer pairs, only 13 loci were successfully amplified (10 loci were
ccmp series, 3 belonged to ccSSR series). Four loci (ccmp1, ccmp4, ccSSR-5, ccSSR-7) were
polymorphic and used for further analyses. All of four fragments presented a bright band by
silver staining. We analyzed 278 individuals using these four primer pairs (Table 2).
Consequently, 16 alleles were identified, and a total of 14 haplotypes was detected throughout
all individuals identified as H1–14 (Table 1).

Table 2 The list of chloroplast microsatellite primers used in the present study
Loci Location Sequence (5′-3′) Tm (℃)
ccmp1 trnK intron F: CAGGTAAACTTCTCAACGGA
R: CCGAAGTCAAAAGAGCGATT
50
ccmp4 atpF intron F: AATGCTGAATCGAYGACCTA
R: CCAAAATATTBGGAGGACTCT
58
ccSSR-5 rps2-rpoC2 F: TCTGATAAAAAACGAGCAGTTCT
R: GAGAAGGTTCCATCGGAACAA
55
ccSSR-7 psbC-trnS F: CGGGAAGGGCTCGKGCAG
R: GTTCGAATCCCTCTCTCTCCTTTT
58

The genetic diversity of P. obconica is shown in Table 3. Haplotype frequencies (P) for
all individuals range from 0.011 to 0.122. Populations EMS, YC, and RY are the divergent
populations revealed by all parameters, where H1 is shared with the highest haplotype
frequency (0.122). In contrast, H7 and H14 are the lowest frequency haplotypes, which are
only found in three samples, respectively. Five of the 14 haplotypes (H1, H4, H5, H6, and
H13) are shared by two or three populations, while the rest of them are population-specific.
The “private” haplotypes are about 60% in all haplotypes, and most of them are located in SW
China (Sichuan and Yunnan provinces).

Table 3 Haplotype frequencies and diversity assessed by cpSSRs in Primula obconica
Population code Haplotype
YC SZ-1 LQ RY DJY-1 EMS SZ-2 LC LD DJY-2 NP WX DL ML-1 ML-2 BS-1 BS-2
P
H1 16 17 1 0.122
H2 15 0.054
H3 20 0.072
H4 20 6 0.094
H5 1 21 0.079
H6 8 20 0.101
H7 3 0.011
H8 14 0.050
H9 20 0.072
H10 21 0.076
H11 21 0.076
H12 20 0.072
H13 19 12 0.112
H14
n

17

15

20
3
20

20

22

8

3

14

6

20

20 21

21

20

19

12
0.011

Ne 1.125 1 1 1.342 1 1.094 1 1 1 1 1 1 1 1 1 1 1
HE 0.118 0 0 0.268 0 0.090 0 0 0 0 0 0 0 0 0 0 0
n, the number of sampling per population; Ne, the effective number of haplotypes; HE, unbiased haplotype diversity; P, the
frequency of haplotypes in all individuals.

Acta Phytotaxonomica Sinica Vol. 45 492
The mean total gene diversity, HT=0.971, is extraordinarily high, while the average
diversity within populations (HS=0.028) is very low. Only three populations (YC, RY, and
EMS) are composed of different haplotypes and present higher polymorphism than average
located in central and southeast China. AMOVA analysis shows that 98.55% variation is
among populations, only 1.45% is within population. When AMOVA analysis is conducted
for two groups (Fig. 1), the apportionment of the variation is that: 39.48% is due to
differences between two groups, 59.35% is within groups, and only 1.07% to difference
within populations (Table 4).



Fig. 1. A minimum spanning tree (MST) for 14 haplotypes based on cpSSRs data. The haplotype names are given in
pane. Two groups are indicated. Numbers between panes represent mutational step.


Table 4 Results of molecular variance analysis (AMOVA) for populations of Primula obconica
Source of variation df Variance components Variation (%) P
Group I
Among populations 7 1.90291 95.85 0
Within populations 108 0.08233 4.15 0
Group II
Among populations 8 1.59873 100 0
Within populations 153 0 0 0
Two groups
Among groups 1 1.14570 39.48 0
Within groups 15 1.72251 59.35 0
Within populations 261 0.03407 1.07 0
All the populations
Among populations 16 2.31896 98.55 0
Within populations 261 0.03407 1.45


A high level of genetic differentiation among populations (Gst=0.971) was found, while
the value of Nst (0.988) was significantly higher than that of Gst (P<0.05). This indicates a
high degree of geographic structure of genetic distribution. Phylogenetic relationship of the 14
haplotypes constructed by a minimum spanning tree (MST) is shown in Fig. 1. Most of the
haplotypes differ from one another by two mutational steps. A tip haplotype H4, however, is
separated by four mutation steps from H7. The MST shows that the haplotypes of P. obconica
are clustered into two groups. H5 shows the connection between two groups. Haplotypes from
the group II are mainly restricted to the SW China, whereas central and southeast China is
No. 4 YAN et al.: Phylogeographic structure of Primula obconica inferred from cpSSRs markers 493
occupied by the other, except for the haplotype H4 (from the north of Sichuan province). The
central position of the two groups is occupied by H10 and H1, respectively, while other tips
enclose them, forming a star-like topology.
3 Discussion
In the present study, low average diversity within populations (HS=0.028), and high
diversity among populations (HT=0.971), are recovered in P. obconica. In addition, the level
of differentiation among populations (Gst=0.971) is remarkably high as compared to the value
(Gst=0.5197) assessed by ISSR (Nan, 2002). The unique genetic structure (high population
differentiation but low within-population diversity) of this species as well as the distinct
discrepancy with ISSR measure was mainly caused by the following four reasons. Firstly, the
uniparentally inherited chloroplast genome has a twofold smaller effective population size
than biparentally nuclear genomes. Therefore, this chloroplast marker is more susceptible to
historical events, such as genetic drift, bottlenecks, and founder effects, and the differentiation
based on its variation is obviously larger than those from bi-parental nuclear markers (Cruzan
& Templeton, 2000; Powell et al., 1995; Petit et al., 1997). Secondly, the limited ability of
seed dispersal of this species may similarly lead to increased differentiation between
populations, as also revealed in other species, such as Saxifraga hirculus (Oliver et al., 2006)
and Carpinus betulus (Grivet & Petit, 2003). By our observation in the field, the seeds of this
species were dispersed mainly by the opening power of the ripened capsules. Furthermore, the
patch-like populations separated by high mountains and valleys undoubtedly reduce the
opportunity for gene exchange through seeds. This geographical isolation was also proposed
to account for the high differentiation in Amentotaxus argotaenia that has a similar
distribution pattern with P. obconica (Ge et al., 2005). Finally, climate oscillations could
affect the distribution of plants and animals, especially in the ice age, which became
increasingly severe through the Pleistocene (Hewitt, 1999). Glacial-induced downward
migrations of some alpine species occurred in central and west of Yunnan Province during the
Pleistocene (Li, 1998). Given the small size of population and the unstable temperature during
the Pleistocene ice-age, these repeatedly occurred expansion-contraction processes, as well as
geographical barriers, may have reduced the polymorphism within populations as well
increasing the population differentiation.
A higher Nst than Gst indicates a significant geographic structure in this species (Zhang et
al., 2005b) and our results further suggest that the closely related haplotypes are often found
in the same area (Fig. 1; Table 1). Two groups with special geographic regions in the MST
were detected. Based on the coalescent theory, ancestral alleles with high frequency and broad
distribution have a greater probability of becoming interior haplotypes (Posada & Crandall,
2001). Notably, the haplotype H1 with highest frequency and wide distribution occupies the
central position of group I in the MST. This result implies that haplotype H1 may be one of
the ancestral haplotypes of P. obconica. In addition, western Hubei and the adjacent Sichuan
might be one of the predicted refugia of tertiary flora with many of the primitive temperate
genera and endemic relicts (Ying et al., 1979). As expected, the population YC located in this
region is mainly occupied by H1. Thus, a rational prediction is that the ancestral haplotype
might be preserved in this region during the glacial periods. The downward movement of
alpine species will expand to previously isolated habitats during glacial periods, despite that
such an expansion may be slow due to habitat constraints (the phalanx model; cf. Chiang &
Schaal, 2006). The fact that the old haplotype H1 is located in the north, and tip haplotypes
are scattered in the south China suggests that a migration may have occurred from the North
during cold times. The average temperature in SE China during the LGM (13000 a BP) was
Acta Phytotaxonomica Sinica Vol. 45 494
lower about 4–6 than ℃ that of the present (Pu, 1991). It afforded an opportunity for P.
obconica to migrate southward, and retreat to mountains following the temperature arising,
and finally forming the present distribution.
The other group is mainly restricted to SW China. This region is the assumed center of
origin of Primulaceae, and includes the mountain regions of Yunnan, the south of Guizhou,
the west of Guangxi of China, and Vietnam, Myanmar, and the northern part of Thailand (Hu,
1994). Because of the special geographic condition, SW China was affected slightly during
the Pleistocene glaciations (Ying, 2001; Shen et al., 2005), and was considered as a key area
for resolving the issue of the origin of the flora of northern temperate zone (Wang, 1992).
Additionally, its climate was warm and humid during the rigor time of glaciations (Li, 1998).
Those special environments give the chances for species in refugia evolving rapidly and
spreading along the high mountain ranges (Hu, 1994). Therefore, it is assumed that this region
may also be the origin place or at least the evolutionary center of P. obconica. Populations of
P. obconica were saved in different refugia when the glacial periods advanced, since many
private haplotypes were fixed.
H4, remarkably, in the north of Sichuan Basin, is isolated from H7 by four unique
mutations. Uncertain relationships between the two haplotypes (H4 and H7) should be given
more attention, since there is a distance of more than 1000 km between the two sampling
sites. Another ambiguity occurs in Hunan Province, with two close populations (SZ-1 and
SZ-2) belonging to two different groups in MST respectively (Fig. 1). It is plausible that this
region may be the potential contact area of the species P. obconica derived from different
ancestors. More sampling sites included in this region or the adjacent areas are required for
further phylogeographic study of P. obconica.
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基于叶绿体微卫星研究鄂报春谱系遗传结构
1, 4颜海飞 2彭镜毅 1胡启明 3郝 刚*
1(中国科学院华南植物园 广州 510650)
2(中研院生物多样性研究中心 台北 115)
3(华南农业大学生命科学学院 广州 510642)
4(中国科学院研究生院 北京 100049)

摘要 鄂报春Primula obconica作为一种广泛栽培的园艺植物, 其野生居群的遗传多样性及遗传结构
的研究还少见报道。本文通过叶绿体微卫星分析了17个鄂报春野生居群(共278个个体), 共发现4个多态
性位点(16个等位基因), 得到14个单倍型。结果表明鄂报春具有很高的总基因多样性(HT=0.971)和极低
的居群内基因多样性(HS=0.028); 分子方差分析(AMOVA)显示98%的变异存在于居群间。这些结果说
明早期的生境片断化及有限的种子传播能力是造成当前遗传结构的重要原因。Nst值显著大于Gst值, 表
明关系相近的单倍型会出现在相同的地区内, 同时最小生成树(MST)的分析结果证实了这样的结论。我
们在最小生成树的两个组中推断出一些古老单倍型, 并推测在冰期时湖北和我国的西南地区可能是该
物种的避难所。
关键词 鄂报春; 遗传多样性; 叶绿体微卫星; 冰期; 避难所