Genetic Diversity And Ecological Adaption In The Genus Oxya

Oxya Serville, which is in Oedipodidae, Orthoptera, Insecta, is a worldwide agricultural pest. In recent years, most researches of Oxya genus are focus on Oxya chinensis using different methods, such as morphology, cytology, biochemistry and molecular biology. The study of other Oxya species is still limited.In this study, genetic diversity, genetic structure and phylogeography of three Oxya species were examined using DNA sequences, AFLP technology and morphological marker. The results were analyzed combined with informations of biogeography to discuss the genetic differnciation and ecological adaption. The main contents and conclusions are as follows:1) In this study, we used AFLP marker to investigate the genetic diversity and population structure of rice grasshoppers collected from south China, with emphasis on testing the hypothesis that there was significant genetic differentiation among grasshopper populations associated with different hosts (i.e. wild vs. cultivated rice). Seven populations consisting of 104 individuals were sampled from Hainan Island and the mainland of south China. Eight primer combinations produced 564 reliable bands, of which 563 were polymorphic. Oxya japonica showed considerable genetic variation at population level, with gene diversity (HE) ranging from 0.1103 to 0.2035. Genetic diversity were studied on seven populations, and generally three populations from wild rice had higher levels of genetic diversity (HE=0.1635) than the other four populations feeding on cultivated rice (HE=0.1327). We observed high population differentiation, with Fst ranging from 0.4172 to 0.7652 among the seven populations. However, Mantel test detected no significant correlation between genetic distance and geographical distance (r=0.3541; p=0.0689). By contrast, we found significant genetic differentiation between groups collected from different hosts. These data suggested that the anthropogenic activity in cultivated rice fields (i.e. pesticides, fertilization and cultivation) could have played an important role in shaping the genetic structure of O. japonica.2) 100 individuals of Oxya japonica were analyzed by using mitochondrial cytochrome oxidase subunit one (COI) DNA sequences collected in four Southeast Asian countries, to examine whether phylogeographic pattern and population genetic is related to past geological events and/or climatic oscillations. Four populations of Philippines have higher haplotype diversity and nucleotide diversity than populations from mainland. Five geographic groups were identified which congruent with the geographically separated. It is notable that networks presented deep genetic divergence pattern.Several unobserved haplotypes were existed between ach two adjacent geographical groups. AMOVA and FST value showed that high genetic variance among groups were found which indicating a relatively strong geographic structure for this species. LAMARC analysis showed that there had been frequent historical gene flow of O. japonica among local populations within region, while low gene flow existed among regions. Networks and high FST values all suggested that all clades diverged in allopatry from a common ancestor, and deep divergences were happened related to specific geologic events. Significantly negative values of Fu’s Fs tests and unimodal mismatch distributions of the frequencies of pairwise differences were presented in TC and north Philippines groups, which all indicated these populations had passed through recent demographic expansions after the bottleneck. The expansion times of TC and north Philippines groups were 92820 years ago and 70940 years ago respectively. The divergence time between north Philippines group and south Philippines group and Malaya group and south China group was 0.74 and 1.13 Mya respectively. The time between the Philippines populations and mainland populations was 1.55 Mya while the time between TC and other populations was dating back to 2.13 Mya during the late Miocene to early Pliocene.3) Population genetic diversity and structure of this Oxya species was examined using both DNA sequences and AFLP technology. The samples of 12 populations were collected from four Southeast Asian countries, among which 175 individuals were analysed using mitochondrial DNA cytochrome c oxidase subunit I (COI) sequences, and 232 individuals were examined using amplified fragment length polymorphisms (AFLP) to test whether the phylogeographical pattern and population genetics of this species are related to past geological events and/or climatic oscillations. No obvious trend of genetic diversity was found along a latitude/longitude gradient among different geographical groups. Phylogenetic analysis indicated three deep monophyletic clades that approximately correspond to three geographical regions separated by high mountains and a deep strait, and TCS analysis also revealed three disconnected networks, suggesting that spatial and temporal separations by vicariance, which were also supported by AMOVA as a source of the molecular variance presented among groups. Gene flow analysis showed that there had been frequent historical gene flow among local populations in different regions, but the networks exhibited no shared haplotype among populations. In conclusion, the past geological events and climatic fluctuations are the most important factor on the phylogeographical structure and genetic patterns of O. hyla intricata in Southeast Asia. Habitat, vegetation, and anthropogenic effect may also contribute to gene flow and introgression of this species. Moreover, temperature, abundant rainfall and a diversity of graminaceous species are beneficial for the migration of O. hyla intricata. High haplotype diversity, deep phylogenetic division, negative Fu’s Fs values and unimodal and multimodal distribution shapes all suggest a complicated demographic expansion pattern of these O. hyla intricata populations, which might have been caused by climatic oscillations during glacial periods in the Quaternary.4) Morphology of 12 Oxya hyla intricata populations and 10 Oxya japonica populations from China and Philippines was analyzed. Length of body, length of elytra, length of pronotum, length of femur, width of femur were chosen, measured by electronic vernier caliper and analyzed by using SPSS 11.5 software. Morphological difference between male and female individuals, morphological difference of different populations and the correlations among body measurement indices and between body measurements and elevation were analyzed in order to demonstrate the relation between morphology and environment. Female individuals were obviously larger than male individuals. The results of O. hyla intricata indicated that three populations (TC, XZ, WN) from China have have significant difference with all populations from Philippines,. The results of O. japonica indicated significant difference was exist between Chinese and Philippines populations. Based on the above results, we proposed that (i) Geographical barrier was the major reason for significant differentiation of morphological characters between Chinese and Philippines populations and (ii) these two Oxya species with large body and high mobile ability were benefit for survival in the high elevation area.5) Morphological description and phylogenetic analysis were conducted to indentify a species of the genus Oxya collected form Tengchong, Yunnan province. This species has most characters of genus Oxya, but differns in:1). the length of elytra was much smaller than other Oxya specie; 2). the epiproct was similar with O. hyla intricata but without wart at the edge.3). Female subgenital plate has no central fovea and edge teeth. Mitochondrial cytochrome b gene fragment sequence was analyzed and compared with other Cytb gene sequence of download from GenBank, and three phylogenetic trees were conducted base on NJ, MP and ML method. Phylogenetic tree indicated 12 individuals of Oxya sp. form a monophyletic group and suggested that this species belongs to genus Oxya.6) Oxya-specific RAPD band was screened out from lots of RAPD-PCR results, the specific band was cloned and sequenced, a pair of primer was designed based on homologue region after sequence alignment. PCR was performed by using designed specific primers, genomic DNA from different Oxya species and other Acridoidea species were used as template. A clear and reproductive band about 650 bp amplified from random primer S823 was present in different Oxya species, sequence analysis showed high nucleotide homology (92.3%-96.6%), each sequence determined consisted of 651-667 nucleotides, with G+C content >15% and repetitive A or T regions. Sequence characterized amplified region (SCAR) primers for specific PCR were developed, a expected specific band (550 bp) can be amplified using SCAR primers in seven species of Oxya examined but absent in Pseudoxya diminuta and other Acridoidea species. Oxya-specific SCAR molecular marker was identified, SCAR primers can be used for rapid identification of Oxya species.