Journal of Heredity 2008:99(1):22–33
The American Genetic Association. 2007. All rights reserved.

doi:10.1093/jhered/esm080 For permissions, please email: journals.permissions@oxfordjournals.org.

Advance Access publication September 28, 2007

Population Genetic Variation and

Structure of the Invasive Weed Mikania
micrantha in Southern China:
Consequences of Rapid Range Expansion
TING WANG, YINGJUAN SU, AND GUOPEI CHEN
From the Wuhan Institute of Botany, the Chinese Academy of Sciences, Wuhan, Hubei 430074, China (Wang and Chen); the
School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China (Su and Chen); and the Department
of Biology, Indiana University, Bloomington, IN 47405 (Su).

Downloaded from http://jhered.oxfordjournals.org/ by guest on March 25, 2012

Address correspondence to Y. Su at the address above, or e-mail: suyj@mail.sysu.edu.cn.

Abstract
Invasive plants such as Mikania micrantha provide valuable opportunities for studying population genetic consequences of
rapid range expansion. Twenty-eight populations of M. micrantha throughout its introduced range in southern China were
examined by using intersimple sequence repeat markers. Population genetic parameters were estimated by Bayesian
approaches as well as conventional methods. Bottleneck signature, multilocus linkage disequilibrium, character
compatibility, and cluster analyses were conducted to assay the factors that may act to shape population variability. High
levels of genetic variation and differentiation were detected in the introduced populations of M. micrantha. All populations
experienced severe bottlenecks. Most of them demonstrated significant linkage disequilibrium and matrix compatibility.
Populations were mainly clustered into 2 groups, and those from different regions intermingled in the unweighted pair
group method with arithmetic mean (UPGMA) dendrogram. No geographical signature was found in the pattern of
population genetic variation. This research indicates that during M. micrantha invasion, multiple introductions mitigated the
loss of genetic variation associated with bottlenecks. Nonetheless, bottlenecks enhanced the population differentiation.
Human-mediated long-distance dispersal events of seeds or propagules explain the lack of geographic structure in genetic
variation. Although asexual reproduction is the predominant mating mode in M. micrantha, it has little effect on the
population genetic composition.

Biological invasions are recognized as a leading threat to
global biodiversity (Sala et al. 2000). Some invasive species
cause severe economic loss in agriculture and forestry
(Wilcove et al. 1998; Pimentel et al. 2000; Sakai et al. 2001;
Bossdorf et al. 2005). However, invasions also provide
significant opportunities to study genetic consequences of
populations with expanding range (Sakai et al. 2001; Hassel
et al. 2005). Generally, colonization of new areas involves
considerable founder effects with genetic drift that reduce
within-population genetic variation and increase genetic
differentiation among populations (Husband and Barrett
1991; Novak and Welfley 1997; Amsellem et al. 2000). This
is especially the case for selfing and apomictic weedy plant
species (Husband and Barrett 1991; Amsellem et al. 2000;
Walker et al. 2003). But exceptions, such as high genetic
variation and low genetic differentiation (Jørgensen and
Mauricio 2004; Refoufi and Esnault 2006), high genetic
variation and high genetic differentiation (Meekins et al.
2001; Walker et al. 2003; Chapman et al. 2004; Durka et al.
2005), as well as low genetic variation and low genetic
differentiation (Amsellem et al. 2000), have been recorded.
Mechanisms proposed to explain the maintenance of genetic
variation or depauperation of diversification in introduced
regions include multiple introductions, admixture or
hybridization of different source populations, and selection
realignment (Carson 1989; Ellstrand and Schierenbeck 2000;
Moody and Les 2002; Kolbe et al. 2004; Lindholm et al.
2005; Parisod et al. 2005).
From an applied perspective, the knowledge of pop-
ulation genetic structure of invasive plants is essential for
improving the efficacy of biological control programs
(Burdon and Marshall 1981; Chapman et al. 2004; Gaskin
et al. 2005). Intraspecific genetic variation has been ob-
served in plant defense to biological control agents (Jarosz

22
 Wang et al.  Population Genetic Consequences of Range Expansion in Mikania micrantha
and Burdon 1990; Bruckart et al. 2004). A mixture of both
resistant and nonresistant genotypes within an invasion may
hinder biological control efforts because plants resistant to
the biological control agent may become predominant
(Burdon et al. 1981, 1984). Moreover, analyses of molecular
genetic variation can characterize the roles that local and
long-distance dispersal events have played in invasions
(Walker et al. 2003).
Mikania micrantha H.B.K. (Tribe Eupatorieae, Family
Asteraceae), a perennial creeping vine commonly called
"mile-a-minute," is one of the top 10 worst weeds in the
world (Holm et al. 1977). It originates from tropical Central
and South America (Kong et al. 2000) and has become
a major pest of crops and forests across Asia and Africa.
The earliest record of the species in the eastern hemisphere
is from 1884 when it was cultivated at Hong Kong Zoo-
logical and Botanical Gardens (voucher number: HK14738)
(Wang et al. 2003). Its naturalization in Hong Kong dates to
at least 1919, and it is believed to have started to spread
during the 1950–1960s (Wang et al. 2003). The earliest
collection of M. micrantha in mainland China (voucher
number: IBSC545255) is in 1984 at Yinhu, Shenzhen,
Guangdong Province (Kong et al. 2000). By the late 1980s
and early 1990s, the weed had spread over Guangdong,
colonizing agricultural land, orchards, nurseries, lawns, man-
groves, secondary forests, scrubland, waste ground, ponds,
and seashores (Zan et al. 2000; Zhang et al. 2004). Once
established, M. micrantha smothers and displaces native
vegetation.
Mikania micrantha reproduces readily through both sexual
and vegetative means (Swarmy and Ramakrishnan 1987;
Zhang et al. 2004). It produces copious amounts of fine,
fluffy seeds with mean seed production ranging from 23 673
to 52 616/0.25 m 2 (Zhang et al. 2003). Seeds are dispersed
by wind or water; but the most efficient mechanism is to
become attached to clothing, animals, and machinery. When
reproducing vegetatively, M. micrantha produces shoots from
small stem fragments and rosettes (Swarmy and Ramakrishnan
1987; Wen et al. 2000).
Intersimple sequence repeats (ISSR) have been used to
characterize genetic diversity in plants (e.g., Tsumura et al.
1996; Camacho and Liston 2001; Deshpande et al. 2001;
Barth et al. 2002; King et al. 2002; Meloni et al. 2006). The
technique may provide a higher reproducibility of bands
than some polymerase chain reaction (PCR)–generated markers
such as randomly amplified polymorphic DNA (RAPD)
(Nagaoka and Ogihara 1997; Wolfe et al. 1998). However,
for population genetic studies, a major drawback of ISSR
markers is their dominance (Zietkiewicz et al. 1994). To
mitigate this problem, Holsinger et al. (2002), Holsinger and
Wallace (2004) developed a Bayesian approach to partition
genetic variation with data derived from dominant markers.
Interestingly, it has also been reported that the Shannon
diversity index is insensitive to the bias generated by the
dominance of molecular markers in the assays of population
genetic differentiation of plant species such as Gliricidia
sepium (Dawson et al. 1995), Alliaria petiolata (Meekins et al.
2001), and Platanthera leucophaea (Holsinger et al. 2002).
Although some research has been conducted on
ecophysiological aspects as well as eradication methods of
M. micrantha (Hu and But 1994; Yang et al. 2003; Deng et al.
2004; Yang et al. 2005), comprehensive analyses of molec-
ular genetic composition of introduced populations are
lacking. Because all populations of M. micrantha in southern
China are derived from recent colonization events, several
hypotheses can be formulated that are testable with ISSR
markers. First, if expansion of M. micrantha is a result of
easily dispersed and sexually produced seeds, little genetic
differentiation will occur among populations and the initial
bottleneck event may hardly have an effect on population
genetic diversity due to effective dispersals. Second, if
independent introductions are frequent because of anthro-
pogenic activities (e.g., intensive human-mediated transport),
no association between geographical and genetic structure
may be detected. Third, if sexually reproduced seeds are the
main agent of dispersal, little linkage disequilibrium is
expected among loci; alternatively, high linkage disequilib-
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rium is expected if asexual reproduction is dominant.
Materials and Methods
Plant Material
Samples were collected from 28 populations of M. micrantha
throughout its introduced range in southern China (Figure 1
and Table 1). The physical boundaries of each population
were well delineated, and most populations were separated
by more than 20 km. A population (Mikania cordata from
Xinglong [MCXL]) of another Mikania species, Mikania cordata,
was also sampled in Xinglong, Hainan Province, China. This
taxon is primarily distributed in tropical Asia and closely
related to M. micrantha (Kong et al. 2000; Wang et al. 2001).
Twelve to 19 plants (intervals at  10 m) were sampled
from each population (Table 1). For each plant, approxi-
mately 3.0 g of fresh leaf tissue was collected using silica gel
to dry and preserve samples (Meekins et al. 2001). Voucher
specimens (CGP1–409) have been deposited at the
herbarium of Sun Yat-sen University, Guangzhou, China.
DNA Extraction
Total genomic DNA was extracted from ground tissue
following the modified CTAB protocol (Su et al. 2005).
DNA concentration and purity were determined by
measuring Ultraviolet (UV) absorption using a Pharmacia
2000 UV/Visible Spectrophotometer. The quality of DNA
was determined by 0.8% agarose gel electrophoresis.
ISSR Amplification and Examination
PCR amplification of ISSR loci was carried out in 20 ll
volumes containing 50 mM KCl, 10 mM Tris–HCl (pH 9.0),
0.1% Triton X-100, 2.0 mM MgCl2, 0.2 mM each
deoxynucleoside triphosphate, 1 U Taq DNA polymerase,
0.3 lM primer, 30 ng genomic DNA, and DNA-free water.
Each reaction mixture was overlaid with 20 ll of PCR grade
23
Journal of Heredity 2008:99(1)
Figure 1. Map of populations of Mikania micrantha sampled in the introduced range of southern China.
paraffin oil. PCR was performed in an MJ-Research PTC-
100TM Peltier thermal cycler. The thermocycling program
was set for 5 min at 94 C, 40 cycles of 40 s at 94 C, 35 s at
53 C, 70 s at 72 C, and 5 min at 72 C. Negative controls
where all reagents but DNA were added to the reaction mix
were included with each experiment in order to verify the
absence of contamination. Genotyping was performed with
24 ISSR primers (Table 2) that were selected based on the
results of initial screening of 80 ISSR primers (UBC ISSR
Primers 802, 805, 807–881, 885, 886, and 888, University
of British Columbia, Vancouver, Canada) against a set of
representative samples of M. micrantha. When amplifications
for the whole set of samples were conducted, one reaction
mix per primer was prepared for all samples to reduce possible
inconsistencies among different amplification runs. Duplicate
reactions were run to determine the reproducibility of banding
patterns. In the few cases where the 2 replicates were not
identical, PCR reactions were repeated at least 3 times to
confirm the banding pattern. Amplification products were
separated by electrophoresis in 1.8% agarose gels containing
ethidium bromide and then photographed under UV light.
Data Analysis
Data were scored according to the presence and absence of
bands, and only those bands that are clear and reproducible
were used to construct data matrices. Percentage of poly-
morphic loci, Nei’s (1973) gene diversity, and coefficient of
gene differentiation, GST, were computed with POPGEN32
24
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software (Yeh and Yang 1999). An unweighted pair group
method with arithmetic mean (UPGMA) dendrogram based
on Nei’s (1978) unbiased genetic distances was constructed
to illustrate relationships among populations. Shannon’s P
index of phenotypic diversity was calculated as S 5 pi
ln pi, where pi is the frequency of a given ISSR band in the
population (Lewontin 1973). In line with Allnutt et al.
(1999), we here rather denote Shannon’s measure as "S"
than "H " in order to avoid confusion with other measures
of diversity such as heterozygosity. S was calculated for 2
levels: the average diversity within population (Spop) and
the diversity within species (Ssp). Its 95% confidence
interval was estimated by a sampled randomization test
(Sokal and Rohlf 2003).
Holsinger et al. (2002) proposed a Bayesian approach to
infer allele frequency and population structure from banding
data of dominant markers. Its parameters, f and hB, are
analogues to the inbreeding coefficient (FIS) and the fixation
index (FST) of F-statistics (Wright 1951), respectively. The
posterior distributions of f and hB were approximated
numerically through Markov Chain Monte Carlo methods
by running HICKORY v1.0 (Holsinger et al. 2002). The
‘‘f-free’’ model option was used due to its lower deviance
information criterion and pD (measure of model complex-
ity) values than for other models. Point estimates of hB as
well as their 95% credible intervals were achieved with
a burn-in of 50 000 iterations and a sampling run of 250 000
iterations from which every 50th sample is retained for
posterior calculations. Three runs were conducted to ensure
 Wang et al.  Population Genetic Consequences of Range Expansion in Mikania micrantha

Table 1. Mikania micrantha populations evaluated from 6 regions in southern China. The sample size, invasion year, and altitude
are listed

Region Population Location Sample size Year of invasion Altitude (m)

a
Hong Kong HK81 Hong Kong Island, Stubbs Road, in shrubs 15 1957 74
HK82 Hong Kong Island, Barker Road, in tussock 13 1960a 300
HK84 Hong Kong Island, Mount Gough, in tussock 16 1919a 315
HK85 Hong Kong Island, Victoria Peak, in tussock 15 1965a 503
HK87 Hong Kong Zoological and Botanical Gardens, 12 1889a 372
under bamboo forest
HK88 New Territories, Hok Tau, in tussock 15 1980sa 46
HK89 New Territories, Luk Keng, Pat Sin Leng 12 1980sa 8
Country Park, rivulet-side
HK90 Kowloon, Hong Kong Baptist University, slope 19 25
Macao MA101 Hác Sá Beach, wasteland 15 1990sb 3
MA105 Hác Sá Village, roadside 15 1990sb 1
MA106 Hác Sá Beach, building site 14 1990sb 8
Shenzhen SZ34 Fairy Lake Botanical Garden, Liang Yi Ting, in 14 1988c 88
tussock
SZ35 Fairy Lake Botanical Garden, Desert Plant 16 1989c 42
Section, roadside

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SZ64 The Office of Mangrove Natural Reserve, 11 19
roadside
SZ72 Meilin Park, Huanshan Road, slope 16 83
SZ75 Lotus Hill Park, the Kite Square, in tussock 14 38
SZ77 Lotus Hill Park, under the Eucalyptus forest 16 46
Neilingding NLD1 Management station, roadside 15 3
NLD10 Dong Jiao Zui Wan, slope, in shrubs 14 2001c 51
NLD15 Dong Jiao Zui Wan, slope, under spiny date 17 145
palms
NLD26 Nan Wan, in shrubs 14 2000c 3
NLD29 Dong Wan, ravine 15 1998c 15
NLD30 Bei Wan Ma Guan, in shrubs 15 3
NLD31 Management Station East, ravine 14 1990sb 8
Zhuhai ZH43 Qi’ao Island, roadside 15 1990sb 2
ZH50 Qi’ao Island, Nong Jia Le, roadside 12 1990sb 2
Dongguan DG91 Da Ling Shan Forestry Centre, Shan Zhu Wo 16 1990sb 174
DG92 Da Ling Shan Forestry Centre, Chang Keng 15 1990sb 114
Kou
a
Wang et al. (2003).
b
Zan et al. 2000).
c
Our own data.

that the results were consistent. In order to report allele
frequency at each locus, a ‘‘Set reportFrequencies’’ state-
ment was specified in the hickory block of data files. Alleles
were considered rare if their frequencies were 0.05 in the
sampled populations (Marshall and Brown 1975).
Linkage disequilibrium and character compatibility
analysis were carried out to assess whether marker
distributions originated from sexual or asexual reproduction
in the introduced populations. Multilocus linkage disequi-
librium was estimated using the index of association IA
(Brown et al. 1980; Maynard Smith et al. 1993; Haubold
et al. 1998) and its modified measure rd (Agapow and Burt
2001) to remove the dependency of sample size. The
program Multilocus v1.2 (http://www.bio.ic.ac.uk/evolve/
software/multilocus/) was used to calculate statistics and
test their significance by randomization. With compatibility
methods, for a pair of biallelic markers, no more than 3 of
the 4 possible character combinations should be observed.
Once all 4 character combinations are present, this means an
incompatibility and will be explained by recombination.
Thus, the sum of incompatible counts over all pairwise
comparisons can be used as a measure of recombination
(Mes 1998; van der Hulst et al. 2000; Wilkinson 2001). In
this research, a summary statistic, the incompatibility excess
ratio (IER) (Wilkinson 2001) was estimated based on
comparison of the observed incompatibility count for the
original data and mean incompatibility count for randomly
permuted data. Asexual reproduction is expected to cause
none or a small fraction of incompatible loci (Chapman
et al. 2004; Hassel et al. 2005).
The program BOTTLENECK (Luikart and Cornuet
1999) was used to test whether populations have recently
passed through a bottleneck. Both the stepwise mutation
model (SMM) and the infinite allele model (IAM) were
run to calculate the heterozygosity (Heq) expected at
mutation–drift equilibrium because ISSR markers may

25
Journal of Heredity 2008:99(1)
Table 2. Primers used in ISSR analyses of Mikania micrantha (R 5 A, G; Y 5 C, T)
Primers Sequences (5#/3#)
UBC808 AGA GAG AGA GAG AGA GC
UBC810 GAG AGA GAG AGA GAG AT
UBC815 CTC TCT CTC TCT CTC TG
UBC817 CAC ACA CAC ACA CAC AA
UBC818 CAC ACA CAC ACA CAC AG
UBC820 GTG TGT GTG TGT GTG TC
UBC822 TCT CTC TCT CTC TCT CA
UBC825 ACA CAC ACA CAC ACA CT
UBC826 ACA CAC ACA CAC ACA CC
UBC827 ACA CAC ACA CAC ACA CG
UBC834 AGA GAG AGA GAG AGA GYT
UBC835 AGA GAG AGA GAG AGA GYC
evolve under a true model intermediate between them (Di
Rienzo et al. 1994; Godwin et al. 1997; Hassel et al. 2005).
The sign test was conducted to determine the significance of
heterozygosity excess (Cornuet and Luikart 1996).
A Euclidean squared distance matrix was calculated in
ARLEQUIN 2.0 (Schneider et al. 2000) and used as the
input file for an analysis of molecular variance (AMOVA)
described by Excoffier et al. (1992). In this analysis, the
total variance in the ISSR data set was partitioned into regional,
among-population, and within-population components. Using
the same software, a Mantel test (Mantel 1967) was conducted
to determine the correlation between the matrix of pairwise hB
estimates and the matrix of geographic distances.
Results
Population Genetic Variation
A pilot experiment was performed to evaluate primer
suitability. From an initial screening of 80 primers, 24 (Table 2)
were identified that produce clear and reproducible banding
patterns. Only reproducible bands obtained from the
selected primers were scored to construct data matrices
for subsequent statistical analyses. Figure 2 presented ex-
amples of ISSR profiles produced with primer UBC817.
The 24 primers generated 209 bands, and each detected
polymorphic loci, with 73.14% of polymorphic loci overall
(Table 3). The number of bands per primer ranged from 2 to
15 with an average of 8.71 bands/primer. No identical ISSR
pattern was found to be shared among sampled individuals.
At the regional scale, all the genetic diversity statistics
consistently indicated that Hong Kong and Neilingding
maintained the highest level of variation (Table 3). As for
regions of Hong Kong, Shenzhen, Macao, and Neilingding,
each contained populations like HK84, HK87, SZ35, SZ64,
SZ72, MA105, MA106, and NLD10 that possessed con-
siderably lower amounts of genetic variation than the others
in the same region (Table 3).
Allele Frequency Distribution
In total, 22 alleles were identified as rare at the species level.
Across regions, 16, 11, and 8 rare alleles were detected in
26
Primers Sequences (5#/3#)
UBC841 GAGAGAGAGAGA GAG AYC
UBC842 GAG AGA GAG AGA GAG AYG
UBC846 CAC ACA CAC ACA CAC ART
UBC847 CACACA CAC ACA CAC ARC
UBC848 CAC ACA CAC ACA CAC ARG
UBC855 ACACAC ACA CAC ACA CYT
UBC856 ACA CAC ACA CAC ACA CYA
UBC857 ACACACACA CAC ACA CYG
UBC859 TGT GTG TGT GTG TGT GRC
UBC866 CTC CTC CTC CTC CTC CTC
UBC880 GGA GAG GAG AGG AGA
UBC881 GGG TGG GGT GGG GTG
Hong Kong, Neilingding, and Shenzhen, respectively;
whereas no rare alleles were found in Zhuhai, Dongguan,
and Macao. Populations in Neilingding and Macao tended
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to possess more rare alleles. Specifically, population NLD10
possessed the highest number of rare alleles (51), NLD30
and MA106 the second (50), NLD1 and NLD31 the third
(47), and NLD26 and MA105 the fourth (46). In contrast,
population ZH43 possessed the least (Table 4). These
results indicated that certain alleles were rare in certain
populations, but not in others.
Linkage Disequilibrium and Character Compatibility
By testing the index of association (IA and rd ) and
IER, significant linkage disequilibrium and considerable
matrix compatibility were observed within each region
(Table 4). At the population scale, significant linkage
disequilibrium and matrix compatibility were likewise
detected in populations NLD1, NLD26, NLD31, SZ34,
SZ35, SZ77, ZH43, ZH50, HK81, HK82, HK84, HK85,
HK87, MA101, MA106, and DG91; but not in NLD10,
SZ75, HK89, and HK90 (Table 4). For the remaining
populations, either significant linkage disequilibrium or
matrix incompatibility was obtained.
Bottleneck Signature Test
Our data demonstrate that M. micrantha has gone through
severe bottlenecks across all the populations and regions.
Each data set revealed a heterozygosity excess/deficiency
ratio that significantly deviated from the expected ratio (1:1)
at mutation–drift equilibrium when either the SMM or the
IAM was assumed (P , 0.05, data not shown). Moreover,
a significant bottleneck signature was also detected over the
whole range (Figure 3).
Population Genetic Differentiation and Relationship
A run of f-free analysis in Hickory yielded an overall esti-
mate for hB 5 0.2797 (95% credible interval: 0.2540–0.3038).
For comparisons, the degree of population genetic
differentiation was also quantified by other parameters.
AMOVA analysis indicated that UST 5 0.3226; 67.74% of
 Wang et al.  Population Genetic Consequences of Range Expansion in Mikania micrantha

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Figure 2. ISSR patterns produced with primer UBC817 for representative individuals from 28 introduced populations of
Mikania micrantha and a population of Mikania cordata. M 5 100 bp DNA ladder. Population codes as indicated in Table 1.

the variation was partitioned within populations, 23.83%
was attributed to differences among populations within
regions, and only 8.43% of the variation was due to
regional differences (all 3 hierarchical levels were signifi-
cant with P , 0.001) (Table 5). When individual pairs of
populations were compared, all pairwise UST values
derived from AMOVA were significant (P , 0.001) except
between populations DG91 and DG92 (P 5 0.100),
highlighting remarkable differences between populations.
Meanwhile, values of GST (Nei 1973) and the compo-
nent of genetic diversity among populations (1 Spop/
Ssp) based on Shannon’s index were 0.4422 and 0.2911,
respectively.
Pairwise estimates of hB between populations showed
nonsignificant associations with geographical distances (r 5
0.139, P 5 0.123) for the full data set. Similar results were
derived from the data sets of regions. Thus, Mantel tests
failed to detect any evidence of ‘‘isolation by distance’’
(Wright 1943) at either the regional or the entire range level.
UPGMA analysis (Figure 4) revealed that 28 populations
mainly fell into 2 groups: Group I was composed of HK84,
HK87, SZ35, SZ64, SZ72, NLD10, MA105, and MA106,

27
Journal of Heredity 2008:99(1)

Table 3. Summary statistics revealed by using 24 ISSR primers to detect populations of Mikania micrantha

Number of Percentage of Shannon’s index

Population Number of loci polymorphic loci polymorphic loci Nei’s gene diversity (95% confidence interval)
HK81 158 76 0.4810 0.1610 0.1999 (0.1659, 0.2349)
HK82 164 80 0.4878 0.1747 0.1972 (0.1647, 0.2314)
HK84 157 55 0.3503 0.1186 0.1595 (0.1237, 0.1954)
HK85 161 75 0.4658 0.1734 0.1985 (0.1646, 0.2325)
HK87 149 42 0.2819 0.0952 0.1233 (0.0923, 0.1577)
HK88 156 77 0.4936 0.1725 0.2170 (0.1801, 0.2517)
HK89 167 85 0.5090 0.1783 0.2061 (0.1738, 0.2393)
HK90 170 87 0.5118 0.1868 0.2129 (0.1813, 0.2456)
Hong Kong 200 146 0.7300 0.2144 0.2437 (0.2135, 0.2697)
MA101 162 73 0.4506 0.1700 0.1799 (0.1472, 0.2145)
MA105 144 46 0.3194 0.1172 0.1156 (0.0857, 0.1467)
MA106 139 36 0.2590 0.0908 0.1100 (0.0764, 0.1441)
Macao 171 98 0.5731 0.1913 0.2006 (0.1682, 0.2333)
SZ34 160 66 0.4125 0.1521 0.1751 (0.1429, 0.2106)
SZ35 150 53 0.3533 0.1282 0.1472 (0.1147, 0.1826)
SZ64 161 59 0.3665 0.1363 0.1573 (0.1240, 0.1905)
SZ72 139 40 0.2878 0.1179 0.1208 (0.0889, 0.1532)

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SZ75 169 83 0.4911 0.1715 0.2016 (0.1644, 0.2362)
SZ77 175 96 0.5486 0.1896 0.2284 (0.1960, 0.2597)
Shenzhen 195 136 0.6974 0.2157 0.2377 (0.2070, 0.2658)
NLD1 152 74 0.4868 0.1807 0.1982 (0.1614, 0.2319)
NLD10 139 36 0.2590 0.0977 0.1083 (0.0770, 0.1391)
NLD15 162 75 0.4630 0.1799 0.1844 (0.1499, 0.2162)
NLD26 144 55 0.3819 0.1518 0.1467 (0.1127, 0.1843)
NLD29 157 83 0.5287 0.1942 0.2059 (0.1747, 0.2406)
NLD30 141 56 0.3972 0.1394 0.1581 (0.1239, 0.1931)
NLD31 151 66 0.4371 0.1645 0.1671 (0.1334, 0.1985)
Neilingding 191 144 0.7539 0.2248 0.2296 (0.2015, 0.2566)
ZH43 176 105 0.5966 0.2075 0.2475 (0.2170, 0.2792)
ZH50 168 85 0.5060 0.1836 0.2146 (0.1792, 0.2472)
Zhuhai 190 128 0.6737 0.2213 0.2544 (0.2250, 0.2844)
DG91 173 96 0.5549 0.1923 0.2367 (0.2032, 0.2693)
DG92 152 63 0.4145 0.1572 0.1812 (0.1450, 0.2173)
Dongguan 180 109 0.6056 0.2007 0.2387 (0.2072, 0.2707)
Total 209 179 0.8565 0.2404 0.2518 (0.2248, 0.2792)

whereas Group II consisted of the remaining populations.
Populations from different regions intermingled in the
dendrogram, except for Zhuhai and Dongguan, suggesting
a lack of geographical pattern.
Discussion
Compared with population genetic estimates based on ISSR
analyses of other invasive weed species (Table 6), substantial
amounts of intraspecific and within-population variation
were revealed across the introduced range of M. micrantha in
southern China (Table 3). The findings are striking
considering all the populations have passed through severe
bottlenecks. Kolbe et al. (2004) suggest that high genetic
variation in introduced populations can be created via
multiple introductions by transforming among-population
variation from different geographical sources into within-
population variation. Here, multiple introductions are
inferred among populations of M. micrantha. As shown with
cluster analysis, closely related populations often are from
different geographical locations (Figure 4), whereas individuals
from the same location do not group together (data not
shown). Therefore, it is reasonable to deduce that multiple
introductions have played a role in the spread of M. micrantha.
In addition, M. micrantha populations that are genetically
more similar appear to maintain equivalent level of genetic
variation. For example, populations HK84, HK87, SZ35,
SZ64, SZ72, NLD10, MA105, and MA106 are all members
of Group I, and they unvaryingly possess less variation than
those populations that constitute Group II (Figure 4 and
Table 3). The result further indicates that levels of
population genetic variation are affected by introduced
individuals.
Holm et al. (1977) suggest that M. micrantha mainly
propagates by seeds, but others report that its local spread
results mostly from vegetative propagation (Swarmy and
Ramakrishnan 1987; Wen et al. 2000). We find that
significant linkage disequilibrium and locus compatibility
were identified in a majority (16 out of 28) of the
M. micrantha populations (Table 4), suggesting that asexual
reproduction is the predominant mating system in the

28
 Wang et al.  Population Genetic Consequences of Range Expansion in Mikania micrantha

Table 4. Number of rare alleles and parameters derived from

linkage disequilibrium and character compatibility analysis for
Mikania micrantha

Population R IA rd IER

HK81 35 2.337*** 0.0312*** 0.180***
HK82 40 1.424*** 0.0180*** 0.054*
HK84 31 9.747*** 0.1806*** 0.557***
HK85 39 1.5467* 0.0209* 0.199***
HK87 44 3.0107*** 0.0735*** 0.373***
HK88 42 0.344 0.0045 0.076*
HK89 39 0.3254 0.0039 0.048
HK90 35 0.2956 0.0034 0.036
Hong Kong 16 1.269*** 0.0092*** 0.048***
MA101 41 2.264** 0.0315** 0.186***
MA105 46 2.044** 0.0454** 0.067
MA106 50 1.8660** 0.0533** 0.304***
Macao 0 3.622*** 0.0384*** 0.172***
SZ34 42 1.699** 0.0261** 0.141**
SZ35 45 2.890** 0.0556** 0.314***
SZ64 43 1.921** 0.0331** 0.059

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Figure 3. Heterozygosity excess at each of 179 polymorphic
SZ72 44 4.0520** 0.1040** 0.001
SZ75 39 0.4973 0.0061 0.050
loci in Mikania micrantha. The horizontal dashed line represents
SZ77 29 3.617** 0.0381** 0.052* the heterozygosity excess expected at equilibrium. Loci with
Shenzhen 8 1.353*** 0.0105*** 0.061*** a heterozygosity excess scatter above the dashed line. ¤IMM,
NLD1 47 2.588** 0.0355** 0.152*** hSMM.
NLD10 51 0.0438 0.0013 0.039
NLD15 41 1.343** 0.0182** 0.015
NLD26 46 4.813** 0.0892** 0.260*** and Richardson 1986; Barrett and Husband 1990; Pellegrin
NLD29 42 1.941** 0.0237** 0.010
NLD30 50 6.029** 0.1097** 0.046
and Hauber 1999; Amsellem et al. 2001). However, in 4
NLD31 47 5.243** 0.0807** 0.116** other populations NLD10, SZ75, HK89, and HK90,
Neilingding 11 1.467*** 0.0110*** 0.082*** M. micrantha indeed reproduces sexually and generates sub-
ZH43 28 1.448** 0.0139** 0.149*** stantial recombinations (Table 4). And contrary to general
ZH50 30 4.557** 0.0542** 0.176*** expectation that sexual reproduction tends to increase
Zhuhai 0 1.388*** 0.01105*** 0.081*** population genetic variation (Godt and Hamrick 1991;
DG91 33 1.819** 0.0192** 0.143*** Hamrick and Godt 1996; Pappert et al. 2000), these 4
DG92 38 4.340** 0.0701** 0.043
Dongguan 0 1.163* 0.0102* 0.046** populations possess either less or similar degree of genetic
variation to those with predominantly asexual reproduction
R, number of rare alleles; IA, index of association; rd , modified index of in the same region (Table 3). Reproductive system seems
association. not to have been a determining factor in the level of
*
P , 0.05 population genetic variation during the range expansion of
**
P , 0.01 M. micrantha.
***
P , 0.001. A relatively high degree of genetic differentiation was
detected among introduced populations of M. micrantha in
comparison with ISSR inferences from other invasive weeds
populations. This is in accordance with the findings that (Table 6), which was further highlighted by the findings that
most invasive plants are selfing or asexual (Price and Jain pairwise UST values between populations were all significant
1981; Husband and Barrett 1991; Amsellem et al. 2001), and (P , 0.001) except between DG91 and DG92. Such
some plants can even switch to higher levels of inbreeding a population genetic structure is not unexpected in view of
or vegetative reproduction during their invasions (Barrett each population having experienced a severe bottleneck.

Table 5. AMOVA analysis of ISSR variation for 28 Mikania micrantha populations in 6 regions: Hong Kong, Shenzhen, Zhuhai,
Macao, Neilingding, and Dongguan

Degrees of Sum of Variance Percentage

freedom squares components of total variation P U-statistics
Among regions 5 480.681 2.002 8.43 ,0.001 UCT 5 0.0843
Among populations within regions 22 1048.670 5.655 23.83 ,0.001 USC 5 0.2602
Within populations 128 2058.450 16.082 67.74 ,0.001 UST 5 0.3226

The P value was calculated by a permutation procedure based on 1023 replicates.

29
Journal of Heredity 2008:99(1)

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Figure 4. UPGMA dendrogram, derived from Nei’s (1978) unbiased genetic distances, showing the relationships among 28
examined populations of Mikania micrantha. A population of Mikania cordata MCXL was used as the root. Branch lengths are
proportional to genetic distances (see scale at the bottom of figure).

The rationale behind the bottleneck signature test is that
a population that has suffered a recent bottleneck will
transiently disrupt the mutation–drift equilibrium across loci
and generate a heterozygosity excess (Luikart and Cornuet
1999). Our results show that all the sampled populations
and regions exhibit a significant bottleneck pattern under
both the SMM and the IAM. Generally, bottlenecks can
enhance genetic differentiation among newly established
populations by causing rapid losses of rare alleles as well as
significant changes in allelic frequencies (Hedrick 1999,
2000). Besides M. micrantha, high genetic differentiations
have also been recorded in the populations of other invasive
weeds such as Heracleum mantegazzianum (Walker et al. 2003),
Hieracium lepidulum (Chapman et al. 2004), and A. petiolata
(Durka et al. 2005). In those studies, effects of multiple
independent introductions are invoked to account for the
strong population genetic structure. Similar reasoning may
apply to M. micrantha. Additionally, given the results that 16
populations are dominant for asexual reproduction, whereas
4 are prevalent for sexual reproduction, impact of mating
systems on population differentiation were further exam-
ined. AMOVA analyses, using the data sets established from
sexually reproduced and asexually reproduced populations
separately, demonstrated that equivalent amounts (28.71%

30
 Wang et al.  Population Genetic Consequences of Range Expansion in Mikania micrantha
Table 6. Percentage of polymorphic loci (PL), Shannon’s diversity within species (Ssp), Nei’s (1973) total gene diversity (HT), GST,
and UST obtained from the introduced populations of different invasive weed species based on ISSR data
Species PL Ssp HT
Chromolaena odorata 12.31 0.0623 0.0406
Hieracium lepidulum 88 — —
Pueraria lobata — 0.221 — —
Leersia hexandra 94.3 — 0.261
Mikania micrantha 85.65 0.2518 0.2404
and 29.53%) of among-population variation are partitioned
(data not presented).
At either regional or entire range scale, no geographical
signature is revealed in the pattern of ISSR variation among
introduced populations of M. micrantha. The result may be
mainly attributed to long-distance dispersals of seeds or
propagules mediated by humans. During the past 2 decades,
business trade, transport, and tourism have dramatically
increased in the area including the Pearl River Delta, Hong
Kong, and Macao. It has been noticed that intensive
economic activities do assist the expansion of M. micrantha in
the region (Zhang et al. 2004). Nonanthropogenic mecha-
nisms also play a role in generating the nongeographical
nature of population divergence. Typhoons, tornadoes, and
thunderstorms occur in the spring in the coastal area of
Guangdong Province, precisely at the time that fruits of
M. micrantha are maturing and seeds are being released (Hu
and But 1994). Because M. micrantha seeds are small and light
(8.80–10.20  10 5 g) (Zhang et al. 2003), the violent winds
are potentially able to uplift and transport them several
hundred kilometers (Zhou and Huang 2001), facilitating
gene flow among distant populations. And finally, repeated
or secondary introductions induced by both mechanisms
would further complicate the geographical consequences of
population genetic variation.
F-statistics developed by Wright (1951, 1965) are widely
used for describing population genetic structure. Sub-
sequently, Nei (1973, 1977) has improved Wright’s method
in 2 respects: making it independent of the number of alleles
at a locus and of the pattern of evolutionary forces such as
mutation, selection, and migration (Nei 1973). Nevertheless,
in the case of only 2 alleles at a locus, Nei’s coefficient of
gene differentiation GST becomes identical with FST (Nei
1973; Nei and Kumar 2000). Thus, values of GST and FST
should not be different when derived from our ISSR data.
Another type of F-statistic analogues, U-statistics, has been
established within an AMOVA framework (Excoffier et al.
1992; Stewart and Excoffier 1996). In the present research,
calculated from ISSR data of M. micrantha populations, Nei’s
(1973) method and AMOVA give GST 5 0.4422 and UST 5
0.3226, respectively. Both are substantially larger than
Bayesian estimate hB 5 0.2797 (95% credible interval:
0.2540–0.3038). However, when the data were analyzed
phenetically, Shannon’s index partitioned 29.11% of the
genetic diversity among populations, which is consistent
with hB estimate. This study provides further evidence that
GST UST Reference
— 0.736 Ye et al. (2004)
— 0.54 Chapman et al. (2004)
0.187 Sun et al. (2005)
0.417 0.401 Song et al. (2006)
0.4422 0.3226 This Study
Shannon diversity is insensitive to the skewing effects
caused by the dominance of molecular markers, such as
ISSR and RAPD (Dawson et al. 1995; Meekins et al. 2001;
Holsinger et al. 2002).
Funding
Downloaded from http://jhered.oxfordjournals.org/ by guest on March 25, 2012
Key Pilot Project of Department of Science and Technology
of Guangdong Province, China (2004B33301020); the
Scientific Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry of China ([2005]546);
and the ‘‘100 Talent Project’’ of the Chinese Academy of
Sciences (05052903).
Acknowledgments
We thank Dr Troy E. Wood at the Department of Biology, Indiana
University for advice and revision of English.
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Received May 16, 2007
Accepted August 28, 2007
Corresponding Editor: Lisa J Rowland
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