Genetic Diversity And Population Structure Of Ageratina Adenophora Spreng. (Asteraceae) In China

Crofton weed, Ageratina adenophora Spreng. [Eupatorium adenophorum (Spreng.)] (Asteraceae), is a worldwide disasterous weed. It is native to Mexico and was introduced into Europe as an ornamental plant in the 19th century. Now it can be found in more than thirty countries and causes ecological and economic threats in many countries. At the end of 1940s, it invaded into Yunnan province of China from neighboring Myanmar. Then it spread rapidly to other southern and southwestern provinces of China, became dominant in local environment, threatened the native biodiversity and ecosystem, and thus caused serious economic loss in the invaded areas. It is essential to understand the diversity and distribution of invasive weeds for the development of efficient control measures. The genetic diversity and population structure of A. adenophora were studied, the biogeographic relationships among populations of crofton weed within its introduced range in China were assessed, and the origin of crofton weed invaded in China and its dispersal pathways were discussed. This study is also important for the development of effective control methods of A adenophora, and for the revealment of its evolutionary processes during invasion. The main results of this study are formulated as follows:1. Simple sequence repeats (SSR) are conserved in the family of Asteraceae, few SSR primers developed from sunflower can be used in crofton weed.2. Inter-simple Sequence Repeats (ISSR) are very useful molecular markers in detecting genetic diversity of A. adenophora. Among the 100 screened ISSR primers, 49 failed to amplify in Crofton weed genomic DNA, the poly-(AT) and poly-(TA) primers could not amplify over a range of annealing temperatures, regardless of the anchored nucleotides. Penta-nucleotide repeats and mixed nucleotide primers also failed in amplification. Twenty primers (16 di-, 2 tri- and 2 tetra-ISSRs) were selected from the 100 primers based on their clear, informative and repeatable banding patterns, and poly-(AG) and poly-(GA) repeats were the most polymorphic and informative. High level of genetic diversity was detected in A. adenophora. Amplification of the 20 primers generated a total of 479 polymorphic bands among the Crofton weeds which selected from 64 collecting sites, and 46% of them were generated by poly-(AG) and poly-(GA) repeats primers. Different types of ISSR primers have different amplifying capacities, and the average amplified bands of di-, tri- and tetra-ISSR were 28, 28 and 35, respectively. Generally, number of polymorphic bands amplified by 5' anchored primers fewer than those by 3' anchored primers.3. With the sustaining influence of various habitats' climates, genetic variations were detected for the nucleus DNA among the Chinese distributed populations of A. adenophora. Twelve polymorphic ISSR primers were employed to investigate the genetic variation and structure of A. adenophora populations in China, and 256 individual plants of 32 populations were examined. Most individuals (99%) displayed a unique ISSR fingerprint, and a total of 446 bands were amplified, 93.5% of them were polymorphic. AMOVA, Wright's F-Statistics, Shannon'sindex, and Nei's gene diversity all indicated that genetic differentiations are exist among populations of A. adenophora invaded in China.They are FST = 0.3140 in analysis of molecular variance (AMOVA), GST = 0.3453 in Wright's F-statistics, 7/^=0.2330 in Shannon's information index and HE = 0.1541 in Nei's gene diversity. At species level, Nei's gene diversity and Shannon's information index were 0.2354 + 0.0265 and 0.3716 ± 0.2194, respectively. At the population level, Nei's gene diversity were between 0.1244 and 0.2041 with an average of 0.1541 ± 0.0193, Shannon's information index were between 0.1873 and 0.3086 with an average of 0.2330 ± 0.0289. At regional level, populations from Guangxi Province exhibited the greatest level of variability (P%: 55.5%, HE: 0.1866 ± 0.0196, //pop: 0.2822 ± 0.0277), and those from Sichuan Province possessed the lowest genetic variation (P%: 42.9%, HE: 0.1417 ± 0.0127, //pop: 0.2148 ± 0.0196). PCR-RFLP polymorphism analysis of chloroplast DNA (cpDNA) indicated evolution of cpDNA of A. adenophora is very slow. Among the 18 universal primer pairs of cpDNA tested, 14 showed good amplifications, which represented about 30.7 kb of the chloroplast genome;the amplified product of each primer was of the same size in the 256 investigated A. adenophora individuals. When each of the 14 amplified fragments was digested by 12 restriction enzymes, a total of 168 primer-enzyme combinations were detected. Forty-six of them failed to digest the related target amplified fragments while the rest combinations produced a total of 752 restriction sites. However, all those sites resulted in a monomorphic banding profile for all of the 256 individuals, revealing no variation of cpDNA, both within and among the populations. It indicates that the 256 individuals analyzed may have the same chloroplast genotype and their maternals are close.4. Isolation caused by environmental factors is one of the main ingredients inducing genetic differentiation. Genetic diversity of Chinese distributed populations of A. adenophora decreased along with the increased latitude, (R2 = 0.1311, P< 0.05), whereas increased along with the longitude shift from west to east (R2 = 0.1677, P< 0.05). Significant differences were detected among vary altitude regions, the level of genetic diversity of crofton weed reduced along with the increased altitude, Nei's gene diversity and Shannon's information index decreased with the increased altitude. Genetic diversity of crofton weeds highly correlated with the annual mean temperatures of its habitat (R2= 0.2731, p < 0.01), whereas only a weak correlation was found between genetic diversity and annual mean rainfall (R = 0.0749, p > 0.1). The Mantel Z-statistic test showed that the genetic distance between populations increased significantly with geographic distance (R2 = 0.2940, p < 0.001), indicated that isolation by distance is a main block of gene flow. In a nested AMOVA analysis based on the five regions (provinces), 8.82% of the total variation was found among the regions, 22.60% was found among populations within the regions and 68.58% was found within populations;while in the analysis among regions, 87.87% of total variance was found within the regions. In an AMOVA among populations, 30.08% of the total molecular variance was attributable to population divergence and 69.92% to individual differences within populations.5. As growing in various climate regions, populations of crofton weeds distributed in China have formed different geographical patterns. They are basically divergence into two subgroups: the first including the populations growing in the vegetation region of tropic seasonal rain forest, for instance, southern populations of Yunnan, Guangxi populations and southwestern populations of Guizhou;thesecond subgroup including the populations growing in the west perennial shrub subregion of the vegetation region of subtropical perennial shrub, such as western populations of Yunnan, Sichuan populations and the populations of Yangtze River Valleys.6. The subsistent geographical patterns of A. adenophora reflect the history of this exotic weed invading in China. Cluster analysis grouped the populations generally according to their geographical origins. Populations collected from similar geographic regions were generally grouped in the same cluster or nearby clusters. Based on the results of cluster analysis combined with the location information of Chinese distributed populations of crofton weed, we speculated that the main dispersal mode of A. adenophora in China was wind dispersal, followed by water flow. After being introduced into the Cangyuan County of Yunnan, the Crofton weed spread toward the north and the east simultaneously. The northward dispersal established in the west part of Yunnan province, the populations in western Yunnan spread northward to Panzhihua prefecture, which locating southern of Sichuan, and further dispersed northward to Liangshan prefecture of Sichuan by wind and to the Yangtze River valley by water flow. The eastward dispersal populations colonized in the southern and southeastern Yunnan, such as Simao, Xishuangbanna and Wenshan prefecture, some of them spread eastward to Baise prefecture of Guangxi province, synchronously, mixed with the populations spread from the borders of Chinese- Vietnam and the borders of Chinese-Laos, then spread northeastward to the northwest parts of Guangxi province and the west parts of Guizhou province.