| This paper was supported by National Natural Science Foundation of China (Grant No. 30370208). The amplified fragment length polymorphism (AFLP) reaction system was firstly used to investigate the genetic diversity and the degree of the genetic differentiation of wolf spider Pardosa pseudoannulata, distributed in south rice field of China. It was discussed that the influence of long-period pesticide force on genetic polymorphism of dominant wolf spider and its adaptive mechanism. The aim of this paper was to offer the scientific evidence for protecting the spiders to control the pest insects, reducing pesticide, recovering the polluted environment and keeping the sustainable development of farm ecosystem.(1) The estabilishment and optimization of AFLP system for population genetics of wolf spiderAFLP was firstly applied to study the population genetics of spider. The results showed AFLP that was a sensitive and reliable molecular marker technique for studying genetic polymorphism of wolf spider. Eight primer pairs generated high polymorphic and accurate bands. Based on previous study, one-step enzyme ligation method was established and the detection method of selective amplified products was modified.In the process of template DNA preparation, enzymolysis and ligation were accomplished by one step. The progress was simple and easy. The probability of fragment self-connection was decreased by one-step enzyme ligation method. The method could improve the work efficiency one more times, and AFLP system was optimized.The detection effect of fluorescence tag analyses technique was better than that of silver-staining. Its cost was higher than that of silver-staining, but it was excellent intelligent in data acquisition and processing by the Genescan analysis software. By fluorescence, the error was decreased and the data was more accurate. The work efficiency was improved. Therefore, the optimized AFLP system was suitable to study the population genetics of spider.(2) Analysis of the population genetic diversity of P.pseudoannulata and key affecting factorsBased on the known distribution of P pseudoannulata, 6 characteristic geographical populations were selected. With eight pairs of AFLP selective primers, 1038 AFLP loci were recorded, of which 86.66% were polymorphic. All individuals showed unique AFLP pattems. From the parameters such as Shannon's index and Nei's gene diversity index, the P. pseudoannulata showed a high genetic diversity either species level (P=86.62%, H=0.2622, I=0.3101) or populations level(P=73.0%, H=0.2155, I=0.2554) and had obvious genetic differentiation. The genetic diversity of P. pseudoannulata was higher in northern south China (Hunan,Hunan and Jiangxi) than southern south China(Yunnan and Hainan). The intra-polulation genetic variation was largest in Leifeng town and lowest in Gaotigong mountain of Yunan. From the population variation source, 35.77% genetic variation came from intra-polutation and 64.23% from inter-population (Nm=0.898).The analysis results of phylogeography showed that there was obviously negative correlation between genetic diversity and annual average temperature and altitude (r=-0.7292, -0.5188; P＜0.05). The polymorphic loci, Shannon's diversity index and Nei's gene diversity index of each population decreased with the elevation of annual average temperature and altitude. Cluster analysis indicated that there was standing genetic differentiation between Fugong population of Yunan with high altitude (1780m) and other population. The correlation was not obvious between inter-population genetic distance and geographical distance (r=0.07104, P＞0.1). The geographical isolation had not obvious effect on gene differentiation. From above the habitat heterogeneity was the important factor resulting in the population genetic differentiation of P. pseudoannulata.Sum up, external factor (intense environmental stress, et al.) and intrinsic factor (genetic response, et al.)resulted in genetic differentiation of P. pseudoannulata.(3) Genetic response mechanism of P. pseudoannulata under long-period pesticide forceIt was found that long-period pesticide stress could change genetic diversity of P pseudoannulata. Distance of the two habitats mentioned in this paper, Tianding town and Leifeng village, was shorter than 2km and their climate factors were similar to each other. Tianding village was an unpolluted rice cultivating base in Hunan Province, where pesticide had been forbidden for several decades, while organ phosphorous pesticides were used in Leifeng village frequently. The AFLP result indicated that:①the distribution of gene frequency of amplified loci had obvious difference between each other. The low gene frequency main existed in TD population, but the high main existed in LF population. At 0 frequency zone, the amplified loci of TD population were much more than that of LF population. It meaned the genetic structure of two populations occured the significant chang.②symbol loci: Twelve dominant loci of LF population were absent in the AFLP pattern of TD population amplified by E-AAG/M-CTG,E-ACA/M-CAG. They were considered to be the adaptive loci to long-period pesticide stress.③The genetic diversity index of LF population (P=-64.29%; H=0.2692) were higher than that of TD population (P=-50.51%; H=0.1821) LF population showed strong resist-pesticide adaptability. The author thought the allelic variation and the recessive homozygous loci pattern of the dominant wolf spider species were an ecological evolution strategy under a long term pesticide force。Due to the fact that there was no significant isolated patches between the two villages, we thought that pesticide (human farming activities) should be responsible for the genetic differentiation of the P. pseudoannulata populations. In other words, long-period pesticide force had speeded up the genetic differentiation of P. pseudoannulata in paddy fields, which was more dominant than natural ecological factors.It suggested that the climatic variation(such as altitude, annual average temperature etc.) resulted in adaptive eco-geographic differentiation, and it was the long-period pesticide force that speeded up the genetic differentiation of P. pseudoannulata and changed the genetic diversity of the population.
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