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Population genetics of Ageratina adenophora using

inter-simple sequence repeat (ISSR) molecular markers
in China
a b c a c a c
Fu-Rong Gui , Prof. Fang-Hao Wan & Jian-Ying Guo
a
The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences , Beijing, China
b
College of Plant Protection, Yunnan Agricultural University , Kunming, China
c
Center for Management of Invasive Alien Species, Ministry of Agriculture , Beijing, China
Published online: 07 Aug 2008.

To cite this article: Fu-Rong Gui , Prof. Fang-Hao Wan & Jian-Ying Guo (2008) Population genetics of Ageratina
adenophora using inter-simple sequence repeat (ISSR) molecular markers in China, Plant Biosystems - An International
Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 142:2, 255-263, DOI:
10.1080/11263500802150399

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 Plant Biosystems, Vol. 142, No. 2, July 2008, pp. 255 – 263

Population genetics of Ageratina adenophora using inter-simple

sequence repeat (ISSR) molecular markers in China

FU-RONG GUI1,2,3, FANG-HAO WAN1,3, & JIAN-YING GUO1,3

1
The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,
Chinese Academy of Agricultural Sciences, Beijing, China, 2College of Plant Protection, Yunnan Agricultural University,
Kunming, China, and 3Center for Management of Invasive Alien Species, Ministry of Agriculture, Beijing, China
Downloaded by [University of Connecticut] at 16:29 08 October 2014

Abstract
Understanding distribution and diversity of invasive weeds is essential for the development of efficient control measures
against it. In the present study, inter-simple sequence repeat (ISSR) markers were used to assess the biogeographic
relationships among populations of the invasive Crofton weed (Ageratina adenophora (Spreng.)) during 2004–2006 in China.
A total of 100 ISSR primers with di-, tri-, tetra- and penta-nucleotide repeats were screened, from which 20 polymorphic and
informative primers were selected. Amplification of the 20 primers generated a total of 479 polymorphic bands among the 64
weed populations, and a high level of genetic diversity (HE ¼ 0.1541 + 0.0193) was detected in A. adenophora. Neighbor-
joining (NJ) cluster analysis based on genetic distances among populations grouped the populations according to their
geographical origin, i.e. (1) populations of southwestern Guizhou, (2) populations of Liangshan city in Sichuan, (3)
populations of western Guizhou, (4) Guangxi populations plus Chongqing populations, (5) populations of southern
Yunnan, and (6) populations of Yangtze River Valleys in Sichuan plus populations of western Yunnan. A significant positive
correlation between geographical and genetic distance was detected by the Mantel test (r ¼ 0.183, p ¼ 0.0012). Based on the
divergence relationships revealed by ISSR markers, it was assumed that A. adenophora mainly dispersed through wind and
water in China.

Key words: Ageratina adenophora, genetic relationship; dispersal; ISSR-PCR

century (Paxton 1849). It spread from Europe to

Introduction
Australia and Asia. Ageratina adenophora was first
Invasive alien species are the most important introduced into Yunnan province of China from
indicators of environmental change worldwide Myanmar at the end of the 1940s; from there it
(Mooney & Hobbs 2000). It may result in environ- rapidly spread to other southern and southwestern
mental degradation, loss of biodiversity, food and provinces of China including Sichuan, Guizhou,
water shortage, and high possibility and severity of Guangxi and Chongqing. The weed-invaded areas
natural disasters (Reaser et al. 2003). Interest in were estimated to be 30 million hm2 (Qiang 1998).
management of biological invasion has grown rapidly Ageratina adenophora rapidly became the dominant
in the recent years. The Crofton weed Ageratina flora of the invaded areas, where it threatened the
adenophora (Spreng.) King & Robinson (1970) native biodiversity and ecosystem, and caused
(synonymous with Eupatorium adenophorum Spreng.) serious economic losses. Some biological control
has caused ecological and economic threats in many agents (e.g. Procecidochares utilis Stone, Cercosporo
countries, including Australia, New Zealand, South eupatoric and Alternaria altemata) have been used
Africa and China (Auld 1966). Ageratina adenophora to control the weed since the 1980s in China.
is an aggressive perennial shrub of the Asteraceae However, the spread of the weed was not notice-
family. It was native to Mexico, but was introduced ably limited mainly due to: (1) the ineffective
into Europe as an ornamental plant in the nineteenth coordination of implementing the control strategy,

Correspondence: Prof. Fang-Hao Wan, Institute of Plant Protection (South Campus), Chinese Academy of Agricultural Sciences, 12 South Street,
Zhongguancun, Beijing 100081, People’s Republic of China. E-mail: [email protected]
ISSN 1126-3504 print/ISSN 1724-5575 online ª 2008 Società Botanica Italiana
DOI: 10.1080/11263500802150399
 256 F.-R. Gui et al.

and (2) the lack of basic knowledge (including Information was lacking about the spatial varia-
population structure and diversity) about the weed. bility of A. adenophora in southern and southwestern
Wang and Wang (2006) stated that a sound under- China. The present study aimed to utilize ISSRs for
standing of the genetic diversity, dispersal pattern, examining the genetic structure as well as diversity
and environmental adaptability of the weed is of among the invasive populations of A. adenophora in
utmost importance for the development and im- China. The main objectives of the study were to
plementation of a more effective strategy for its assess bio-geographic relationships and genetic simi-
management. larities across several A. adenophora populations in
DNA-based molecular markers have been intro- southern and southwestern China. The results of this
duced in the last two decades, and currently are used study increase our understanding of the dispersal
for a wide range of taxa. Inter-simple sequence pattern, invasion routes and settlement habitats of
repeat (ISSR) markers are cost-effective and robust, A. adenophora in China.
and are highly useful for assessing genetic variability
in plant species. ISSRs take advantage of simple
sequence repeats (SSR) or microsatellites, which are Materials and methods
abundant in all eukaryotic genomes. However, unlike
Plant material and DNA extraction
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SSR markers, the ISSRs do not require any prior

knowledge of the genome sequence. This technique For the different experiments, a total of 64 populations
is rapid as well as quite sensitive, and capable of of A. adenophora were sampled from five southern
differentiating between closely related individuals and southwestern provinces of China during 2004–
(Zietkiewicz et al. 1994). In contrast to other 2006 (Figure 1; Appendix S1: www.informaworld.com/
dominant markers, such as Random Amplified mpp/uploads/tplb_315205_supplementary_material.
Polymorphic DNA (RAPD), the ISSR technique doc). Ten plants were harvested from each field
uses longer primers, thus allowing the use of a higher population, and transplanted in the greenhouse.
annealing temperature, thereby resulting in greater From each plant, 200 mg of young healthy leaves
reproducibility of the DNA fragments. The ISSRs were sampled, and the CTAB (cetyltrimethyl-
have been used to detect intra-specific polymorph- ammonium bromide) protocol used to extract
isms, and to characterize genetic diversity in various DNA. The purified DNA was quantified using an
crop species, such as citrus (Poncirus trifoliata (L.) UV2550 Spectrophotometer (Shimadzu, Japan).
Raf.), peanut (Arachis hypogaea L.), and wild rice Working DNA solutions (15 ng/ml) were made with
(Oryza granulata L.). sterile double-distilled water, and bulked DNA

Figure 1. Locations of the 64 Ageratina adenophora populations during 2004–2006 in China. Numbers represent the different populations
described in appendix S1 in the supplementary material (www.informaworld.com/mpp/uploads/tplb_315205_supplementary_material.doc).
 Population genetics of Ageratina adenophora 257

solutions were prepared by mixing equal volumes of where fi was the frequency of the ith band. For each
working DNA solutions of 10 individual plants from pair of populations being compared, the number of
each population. polymorphic markers per gel lane (marker index,
MI) was calculated according to Powell et al. (1996).
The ISSR molecular data were elaborated using the
ISSR-PCR amplification
NTSYS-pc (Numerical Taxonomy System) version
One hundred ISSR PCR primers of 15–23 nucleo- 2.10 computer program. The SIMQUAL (similarity
tides in length were selected from the ISSR primer for qualitative data) program was used to calculate
set (UBC primer set #9) developed by the University the genetic similarities. Pairwise comparisons be-
of British Columbia Biotechnology Laboratory tween samples were performed using Dice and
(Vancouver, BC, Canada; www.biotech.ubc.ca) and Jaccard similarity indices with the NTSYS-pc pro-
synthesized by Sangon Biological Engineering Tech- gram, according to the following equations:
nique & Service, Co. Ltd. (Shanghai, China). These
primers were screened using DNA from 10 A. Dice coefficient ¼ 2NAB =ð2NAB þ NA þ NB Þ ð2Þ
adenophora samples, and the 20 primers that pro-
duced the most robust and clear amplification Jaccard’s coefficient ¼ NAB =ðNAB þ NA þ NB Þ
Downloaded by [University of Connecticut] at 16:29 08 October 2014

profiles were utilized for subsequent experiments. ð3Þ

Each PCR reaction mixture consisted of one unit
of Taq DNA polymerase, 16PCR buffer, 2.5 mM
MgCl2, 0.25 mM of each dNTP (all PCR reagents where NAB was the number of bands shared by
were obtained from New England Biolabs Ltd., samples, NA represented total scorable amplified
Beijing, China), 1.25 mM of a single ISSR primer bands in sample A, and NB represented total scorable
and 30 ng of DNA template in a total volume of amplified bands in sample B. Similarity matrices of
20 ml; 2.0% of deionized formamide was added to Dice and Jaccard were then converted into distance
the PCR mixture to increase band clarity. PCR was matrices (distance ¼ 1 – similarity). Based on these
performed in a PTC-200 thermocycler (MJ Re- matrices, dendrograms were constructed using the
search, Inc., USA) with the following thermal Neighbor-Joining (NJ) method. In addition to NJ
profile: one cycle at 958C for 2 min; five cycles at cluster analysis, UPGMA (unweighted pair-group
948C for 30 s and 558C for 45 s, 728C for 2 min, method with arithmetic averages) was performed on
with a reduction of 18C for each cycle, followed by the same data sets. All the computations were
40 cycles with the annealing temperature maintained performed using NTSYS-pc software.
at 508C; finally, one cycle of 7 min at 728C ended The bootstrap analysis was performed with
the thermal profile. The PCR products were 500 replicates in NJ trees using the FreeTree soft-
separated electrophoretically and compared with a ware (available at: http://www.natur.cuni.cz/*flegr/
100-bp NEB DNA ladder size standard (New freetree.htm). In order to estimate the congruency
England Biolabs Ltd., Beijing, China). The electro- among dendrograms, cophenetic values (rcp) based
phoresis was performed at 5 volts/cm for 3 h on 2% on the results of the NJ cluster and UPGMA cluster
agarose gels (containing 0.5 mg/ml ethidium bro- analysis were calculated to measure the quality of the
mide) in 0.56TBE buffer. At the end of the clustering (Rohlf & Sokal 1981). The cophenetic
electrophoresis, gels were photographed on an UV matrices for each index type were computed and
transilluminator (Bio-Rad Ltd., USA). compared using the Mantel matrix correspondence
Amplified fragments were scored for the presence test. This test yielded a product moment correlation
or absence of bands (1 ¼ present; 0 ¼ absent; 9 ¼ not (r) that provided a measurement of relatedness
amplified, missing value), and each score was treated between two matrices.
as an independent character. Bands with different The geographical distance of pairs of populations
intensity were treated equally, but only clear and was calculated using the latitude, longitude and
consistently reproducible bands were scored and elevation of each population. The Mantel Z-statistic
analyzed. was used to test the correlation between geographical
as well as genetic distances. The genetic isolation was
tested by geographic distance for the entire data set,
Data analysis
for populations prevalent in more than two pro-
The genetic diversity of A. adenophora populations vinces, as well as for regions that contained more
was estimated by the average polymorphic informa- than 15 populations. The significance of Z was
tion content (PIC) as the algorithm: determined by comparing the observed Z value with
Xn a critical Z value obtained from permutation. As one
PIC ¼ 1  fi2 ð1Þ of the most important methods of ordination
i¼1 analysis, principal coordinate analysis (PCOA) was
 258 F.-R. Gui et al.

performed using the NTSYS-pc version 2.10 soft- information content (PIC) varied from 0.21(UBC
ware. It constructed a new set of orthogonal 864) to 0.83 (UBC834), whereas the marker index
coordinate axes such that the projections of points (MI) ranged from 3.07 (UBC855) to 35.1
onto them have maximum variance in as few (UBC844). The mean PIC and MI of the 20 ISSR
dimensions as possible. primers were 0.56 and 17.4, respectively. The ISSRs
that exhibited a high PIC value, together with a
higher multiplex ratio, were likely to be efficient for
Results the analysis of intra-specific genetic variation in a
species like A. adenophora for which no prior
ISSR polymorphism
sequence information was available.
The results revealed that a total of 100 ISSR primers
were screened and 20 of them (16 di-, 2 tri- and 2
Cluster and coordinate analysis
tetra-ISSRs) were selected on the basis of clarity,
informativeness and repeatability of their banding The similarity matrices calculated from the 479
patterns (Table I). The representative banding polymorphic ISSR bands showed highly variable
patterns are shown in Figure 2. Among the 20 genetic distances among the different populations
Downloaded by [University of Connecticut] at 16:29 08 October 2014

primers, 8 were poly (AG) or poly (GA) primers, (Figure 2). A high level of genetic diversity (expected
indicating the high frequency of the poly (GA) motif heterozigosity, HE ¼ 0.1541 + 0.0193) was detected
in the A. adenophora genome. in each A. adenophora population. The genetic
From the 20 selected primers, a total of 479 distance was highest (0.486) between the Lincang
polymorphic bands were amplified from among the population (site no. 52, located in Yunnan province)
64 A. adenophora populations. The number of bands and the Zhenning population (site no. 11, located in
simultaneously analyzed per primer, i.e. the multi- Guizhou province), and lowest (0.145) between
plex ratio, was 28.9 and 24 for the total bands and eastern district populations of Ningnan and Panzhi-
polymorphic bands, respectively. The size of the hua (site no. 28 and no. 38, respectively; both
polymorphic bands ranged from 100 to 2170 bp. The located in Sichuan province).
primers differed greatly in their potential informa- The dendrogram generated from the NJ cluster
tiveness as indicated by the number of scorable analysis represented the genetic relationships among
amplified bands, e.g. the primer UBC844 produced the 64 A. adenophora populations (Figure 3). The NJ
as many as 45 bands, while primer UBC855 only trees generated from both the Dice and the Jaccard
amplified 12 bands. The average polymorphic distances were the same (r ¼ 1.0, p ¼ 0.0001).

Table I. Inter-simple sequence repeat (ISSR) primers, together with their core sequences, anchoring nucleotides, numbers and size of their
amplified bands, and their polymorphisms, polymorphic information content (PIC) value and marker index (MI) for Ageratina adenophora
populations during 2004–2006 in China.

Primer* Core sequence Anchoring nucleotides Recorded bands Polymorphic bands Size range (bp) PIC MI

UBC836 (AG)8 YA 23 20 220–850 0.63 14.51

UBC809 (AG)8 G 28 24 170–1150 0.44 12.42
UBC834 (AG)8 YT 36 33 140–940 0.83 29.88
UBC835 (AG)8 YC 41 39 170–1860 0.64 26.09
UBC810 (GA)8 T 29 25 110–1150 0.57 16.65
UBC812 (GA)8 A 21 16 150–780 0.35 7.33
UBC840 (GA)8 YT 40 39 110–1790 0.79 31.56
UBC842 (GA)8 YG 33 25 100–1240 0.60 19.65
UBC855 (AC)8 YT 12 5 270–810 0.26 3.07
UBC816 (CA)8 T 19 12 140–650 0.42 8.00
UBC846 (CA)8 RT 21 15 240–1960 0.56 11.74
UBC847 (CA)8 RC 26 24 210–1020 0.61 15.77
UBC886 (CT)7 VDV 17 7 180–1240 0.24 4.00
UBC844 (CT)8 RC 45 44 160–1240 0.78 35.10
UBC891 (TG)7 HVH 26 15 210–960 0.34 8.96
UBC859 (TG)8 RC 34 28 210–2170 0.58 19.74
UBC864 (ATG)6 – 23 11 300–1350 0.21 4.79
UBC866 (CTC)6 – 33 33 320–2020 0.80 26.56
UBC873 (GACA)4 – 33 31 190–1150 0.77 25.38
UBC876 (GATA)2(GACA)2 – 37 33 210–1770 0.75 27.68

*Primer set 9, University of British Columbia (Canada). R ¼ (A, G), Y ¼ (C, T), D ¼ (A, G, T) (i.e. not C), H ¼ (A, C, T) (i.e. not G),
V ¼ (A, C, G) (i.e. not T).
 Population genetics of Ageratina adenophora 259

Figure 2. ISSR PCR amplification patterns of 16 Ageratina adenophora populations using primer (GACA)4. (M: 100 bp DNA ladder, the
Downloaded by [University of Connecticut] at 16:29 08 October 2014

smallest band is 200 bp; lane 1–16 same as the site number of appendix S1 in supplementary material: www.informaworld.com/mpp/
uploads/tplb_315205_supplementary_material.doc) during 2004–2006 in China.

Figure 3. Neighbour-joining analysis of the 64 Ageratina adenophora populations generated from the Dice distance matrix during 2004–2006
in China. The symbols refer to appendix S1 in supplementary material (www.informaworld.com/mpp/uploads/tplb_315205_supplementary_
material.doc). The numbers at the forks indicate the confidence limits for grouping of those populations. Only bootstrap values over 50% are
shown.

The Mantel test revealed a highly significant groups (Figure 3): (1) southwestern populations of
correlation between Dice’s distance matrix and NJ Guizhou, (2) populations of Liangshan city in
dendrogram (r ¼ 0.916, p ¼ 0.0001), which indicated Sichuan, (3) western populations of Guizhou, (4)
accuracy of the dendrogram of the Dice’s distance Guangxi populations plus Chongqing populations,
matrix. The NJ cluster analysis grouped the popula- (5) southern populations of Yunnan, and (6)
tions according to their geographical origins. The populations of Yangtze River Valleys in Sichuan
populations collected from similar geographic re- plus western populations of Yunnan. Within these
gions were generally grouped in the same cluster or six groups, there were found two subgroups, i.e. one
nearby clusters. The NJ tree results grouped the A. with populations of Yangtze River Valleys in Sichuan
adenophora populations into the following six major and the other with western populations of Yunnan.
 260 F.-R. Gui et al.

The UPGMA tree showed a similar pattern of the southwestern populations of Guizhou were split
clusters with the NJ tree (dendrogram not shown), into the third and the fourth clusters in the PCOA.
and the results of the Mantel test indicated a The lack of fit of these populations into any group
highly significant (r ¼ 0.808, p ¼ 0.0001) cophenetic was also reflected in the results of the Mantel test,
correlation between the NJ tree and the UPGMA where the geographical and genetic matrices did not
tree. show an overall correlation (Table II). Moreover, the
The results of principal coordinate analysis Mantel test of the populations within each province
(PCOA) showed four main clusters in the two- showed that there was no significant genetic isolation
dimensional PCOA (Figure 4). The first cluster by distance.
included populations from Yunnan province (Group The Mantel test of the geographical pattern, as
5 in the NJ cluster). The second cluster included suggested by the NJ and PCOA clusters, indicated
populations from Yangtze River Valleys in Sichuan that the genetic distance between populations in-
province (Group 6 in the NJ cluster). The third creased with increasing of geographical distance.
cluster included populations from Guangxi, Eleven of the 15 Mantel test comparisons were
Chongqing, and Liangshan city of Sichuan province statistically significant (Table II). The positive
(Group 2 plus Group 4 in the NJ cluster), and the correlation between geographical distance and ge-
Downloaded by [University of Connecticut] at 16:29 08 October 2014

fourth cluster consisted of the population from netic distance ranged from 0.175 for the populations
western Guizhou province (Group 3 in the NJ of Yunnan, Guizhou, and Guangxi to 0.493 for the
cluster). The first two components of the PCOA populations of Yunnan, Sichuan, and Guizhou. A
expressed 14.7% of the total variation, and the first significant positive correlation was found among all
three components revealed 19.1% of the total geographical regions (r ¼ 0.183, p ¼ 0.0012). The
variation (data not shown). There were a few correlation became highly significant when popula-
populations that did not fit into any group (e.g. site tions from Guangxi province and Chongqing muni-
no. 9 did not fall into group 3 in the NJ cluster), and cipality were excluded from the calculations.

Figure 4. Association among the 64 Ageratina adenophora populations revealed by PCOA analysis performed on Dice genetic distance index
calculated from ISSR data during 2004–2006 in China. The site numbers refer to appendix S1 in supplementary material (www.
informaworld.com/mpp/uploads/tplb_315205_supplementary_material.doc).
 Population genetics of Ageratina adenophora 261
Dispersal pattern of A. adenophora in China
from site no. 53 (the site where the weed first
The routes and dispersal pattern of A. adenophora in appeared in China) (Figure 1), which was an out-
China was analyzed using the population collected group to the cluster of other populations. The results
confirmed the positive correlation between geogra-
Table II. Mantel test results for the correlation between phical distance and genetic distances (Figure 5). By
geographical region and genetic distance of Ageratina adenophora combining the clustering results and the known
populations during 2004–2006 in China. of biological characteristics of A. adenophora, it can
be assumed that the main dispersal mode for
Total
Comparison populations r p value A. adenophora in China was through wind, followed
by water.
All samples together 64 0.183 0.0012*
All samples excluding 62 0.270 0.0001**
Chongqing Discussion
Yunnan, Sichuan, Guizhou 54 0.493 0.0001**
Yunnan, Sichuan, Guiangxi 46 0.329 0.0001** Among the 20 selected ISSR primers, 80% were di-
Yunnan, Guizhou, Guangxi 43 0.175 0.0067* nucleotide ISSRs, while half were poly (AG) or poly
Sichuan, Guizhou, Guangxi 43 0.220 0.0009** (GA) primers. Present results are in accordance with
Yunnan, Sichuan 38 0.238 0.0017*
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findings on other plant species (e.g. conifers, sugi,

Sichuan, Guizhou 35 0.389 0.0001**
Yunnan, Guizhou 35 0.279 0.0002**
wheat and rice), where the poly (AG) and poly (GA)
Yunnan, Guangxi 27 0.376 0.0001** repeats were the most polymorphic and informative
Sichuan, Guangxi 27 0.251 0.0031* (Tsumura et al. 1996; Nagaoka & Ogihara 1997;
Guizhou, Guangxi, Chongqing 26 0.177 0.0650 Blair et al. 1999). Also, the good amplification of the
Yunnan 19 0.328 0.0107 tetra-nucleotide repeat (GACA)4 primers in A.
Sichuan 19 0.152 0.0813
Guizhou 16 0.194 0.0376
adenophora supported our results. However, the poly
(AT) and poly (TA) primers were not amplified over
(9000 iterations, levels are indicated by *p  0.01; **p  0.001). a range of annealing temperatures, regardless of the

Figure 5. Dendrogram of the 63 Ageratina adenophora populations using the NJ method (population no. 53 was used as an outgroup to root
the tree) during 2004–2006 in China. The symbols refer to appendix S1 in supplementary material (www.informaworld.com/mpp/uploads/
tplb_315205_supplementary_material.doc).
 262 F.-R. Gui et al.

anchoring nucleotides; these primers were likely to of genetic variation within and among A. adenophora
self-anneal, which might have caused amplification population invasion pathways.
problems (Blair et al. 1999). Results similar to ours The present study was the first attempt of its kind
have also been reported in some other plant species assessing the genetic diversity of A. adenophora using
including loblolly pine and Douglas-fir (Tsumura the ISSR technique. The results clearly revealed the
et al. 1996), trifoliate orange and wheat (Nagaoka & validity and suitability of using ISSR markers in
Ogihara 1997), rice (Blair et al. 1999) and Chilean detecting genetic variation among A. adenophora
Nothofagus (Mattioni et al. 2002). populations from different regions. It also provided
Between the NJ and UPGMA clustering method, an overall understanding of population dynamics. In
we preferred the dendrogram derived from the addition to proving the usefulness of ISSR markers
former, because it minimized the sum of branch for DNA profiling, analysis of the genetic structures
lengths at each stage of clustering the operational among A. adenophora populations enabled us to infer
taxonomic units and started with a star-like tree, its evolutionary relationships.
which was less affected by the presence of admixture
among populations (Ruiz-Linares 1994). The ‘‘out-
Acknowledgements
liers’’ resulting from the PCOA analysis suggested
Downloaded by [University of Connecticut] at 16:29 08 October 2014

that there was genetic heterogeneity within each We thank Dr Dapeng Zhang from Sustainable
geographical region to some extent, which further Perennial Crops Laboratory, Agricultural Research
suggested that A. adenophora in China came from Service, USDA and Prof. Dr Imtiaz Ali Khan from
different sources, e.g. via different routes or at NWFP Agricultural University Peshawar, NWFP,
different times. Pakistan, for reviewing and editing the original
Wang and Wang (2006) stated that A. adenophora manuscript. We also thank Dr Dan Johnson from
expanded its range mainly along tributary streams University of Lethbridge, Alberta, Canada, Dr
and highways in river valleys. From the clustering Zhengyue Li from Yunnan Agriculture University,
results and A. adenophora distribution in China, it China, and Mr Lambert Motilal from the Cocoa
was assumed that A. adenophora mainly dispersed in Research Unit, The University of West Indies,
China through wind and water flow. Our hypothesis Trinidad and Tobago, for their valuable comments
on the dispersal route of A. adenophora in China are on the manuscript. This research was funded by
in accordance with the findings of Wang and Wang the National Basic Research and Development
(2006). Since A. adenophora was introduced in the Program, China (Grant No. 2002CB111400 and
southwestern region of Yunnan Province from 2006CB100204).
Myanmar in the 1940s, it must have spread toward
the north and the east at the same time. The
northward dispersal led to it becoming established
in the western part of Yunnan province, and the References
eastward spread led to colonization in the southern Auld BA. 1966. The distribution of Eupatorium adenophorum
and southeastern part of the province. From there, Spreng. on the far north coast of New South Wales. New Zeal
the populations in western Yunnan spread north- Sci 102:159–161.
Blair MW, Panaud O, McCouch SR. 1999. Inter-simple sequence
ward to southern Sichuan, and further dispersed to
repeat (ISSR) application for analysis of microsatellite motif
the Yangtze River valley in Sichuan by water flow. frequency and fingerprinting in rice (Oryza sativa). Theor Appl
Several southeastern populations spread eastward to Genet 98:780–792.
northwestern Guangxi province, and others spread King RM, Robinson H. 1970. Studies in the Eupatorieae
north to the central part of Yunnan, and northeast to (Compositae). XXIX. The genus Chromolaena. Phytologia
the western part of Guizhou province. 20:196–209.
Mattioni C, Casasoli M, Gonzalea M, Ipinza R, Villani F. 2002.
The role of natural forces such as wind and water Comparison of ISSR and RAPD markers to characterize three
in the dispersal of invasive weeds seems to be Chilean Nothofagus species. Theor Appl Genet 104:1064–
relatively small as compared with human mediated 1070.
dispersal. However, the presence of pappus hairs and Mooney HA, Hobbs RJ. 2000. Invasive species in a changing
the tiny seed size of A. adenophora make its dispersal world. Washington, DC: Island Press. pp 1–30.
Nagaoka T, Ogihara Y. 1997. Applicability of inter-simple
by wind and water easier. Wind not only plays a sequence repeat polymorphisms in wheat for use as DNA
primary role in local dispersal, but also plays a markers in comparison to RFLP and RAPD markers. Theor
secondary role in long-distance dispersal of plants. Appl Genet 95:597–602.
From the present results, it was assumed that the Paxton J. 1849. A pocket botanical dictionary. 2nd ed. London:
natural pathways (e.g. wind and water flow) were the Bradbury and Evans. 354 pp
Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S,
main dispersal sources of A. adenophora in China. Rafalski A. 1996. The comparison of RFLP, RAPD, AFLP and
But, the role of human mediated dispersal cannot be SSR (microsatellite) markers for germplasm analysis. Mol
excluded. Further studies should focus on partition Breed 2:225–238.
 Population genetics of Ageratina adenophora 263

Qiang S. 1998. The history and status of the study on the crofton Tsumura Y, Ohba K, Strauss SH. 1996. Diversity and inheritance
weed (Ageratina adenophora), a worst worldwide weed. J of inter-simple sequence repeat polymorphisms in Douglas-fir
Wuhan Bot Res 16:366–372 (in Chinese with an English (Pseudotsuga menziesii) and sugi (Cryptomeria japonica). Theor
abstract). Appl Genet 93:40–45.
Reaser JK, Yeager BB, Phifer PR, Hancock AK, Gutierrez AT. Wang R, Wang YZ. 2006. Invasion dynamics and potential spread
2003. Environmental diplomacy and the global movement of of the invasive alien plant species Ageratina adenophora
invasive alien species: A U.S. perspective. In: Ruiz GM, (Asteraceae) in China. Div Dist 12(4):397–408.
Carlton JT, editors. Invasive species: Vectors and management Zietkiewicz E, Rafalski A, Labuda D. 1994. Genome fingerprint-
strategies. Washington, DC: Island Press. pp 362–381. ing by simple sequence repeat (SSR)-anchored polymerase
Rohlf FJ, Sokal RR. 1981. Comparing numerical taxonomic chain reaction amplification. Genomics 20:176–183.
studies. Syst Zool 30:459–490.
Ruiz-Linares A. 1994. Analysis of classical and DNA markers for
reconstructing human population history. In: Brenner S,
Hanihara K, editors. The origin and past of modern humans
as viewed from DNA. Singapore: World Scientific. pp 123–
148.
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