Studies On Ecological Characteristics And Intraspecific Differentiation Of Blaps Rhynchopetera Fairmaire (Coleoptera: Tenebrionidae)

Blaps rhynchopetera Fairmaire belongs to the family Tenebrionidae, the order Coleoptera. The secretion of adult insect, as a natural medicine, has been commonly used for anti-bacteria, diminish inflammation and anti-cancer by local people in Yunnan province. It was researched in this dissertation that distribution and habitat, the ecological characteristics, cytological, intraspecific differentiation from morphology level to molecular level of this beetle. The intra- and inter- species phylogeographical relationships were analyzed too. The studies were significant important for sustainable conservation and utilization of the beetle. The research also laid a scientific foundation for artificial breeding. The main results of the study are as follows:1. Ecological characteristics and its distribution. The age-specific life table of experimental population indicated that survivorship curve of B. rhynchopetera resembled typeⅢ. The death rate of generations was 65.17%. The death rate of larva period was highest, which was 51.43%. The egg stage was next. The larva and egg stages were critical stages, as they were important in the whole population fluctuation process of quantity. Density restriction effects occurred on every instars of larvae development stage. The sensitivity of larvae to density was strengthened as increasing of instars. The highest survival rate occurs in 5/cm3 (77.70±4.49%) among 4 density groups from 0.3/cm3 to 10/cm3 in early-aged larvae. There was an inverse ratio between body weight and population density. Among 6 density groups from 0.01/cm3 to 0.6/cm3 in middle-aged larvae, survival rate dropped down when density increased. Survival rate was over 60.00% for all groups. There was more than 70% pupation rate in low density groups (0.002/cm3 and 0.005/cm3) , the time for pupation were less than 51 days among 4 density groups from 0.002/cm3 to 0.005/cm3 in the mature larvae. The average weight of pupae had a negative correlated with density. 92.26% old larva pupate at the centre of the receptacle, the pupal chambers were mainly oval shape.B. rhynchopetera was distributed in about 19 regions in Yunnan, Sichuan, Guizhou and Guangxi provinces of southwestern China, from 22°40′~28°20′N to 97°40′~107°20′E. B. rhynchopetera found in Yunnan mainly, where vertical distribution was between 708 and 3 110 m, and mainly distributioned from 1 000 to 2 100 m. The habitat of the beetle was house, which was found around the villages. The annual average temperature was 15.65±2.55℃. Accumulated temperature of≥10℃was 4972.40±1230.48℃. Annual precipitation was 1020.63±211.79 mm. Relative humidity was 73.08±7.24% in the suitable living area. Principal components analyses showed that the factors of temperature, sunlight & low temperature, moisture had critical importance on the geographical distribution of B. rhynchopetera.2. Chromosome. Cytological preparations of chromosomes were carried out according to revised air-drying protocol. Testes were dissected and treated with a 0.025% colchicine solution for 4h, then in 0.075 M KC1-hypotonic solution for 30 min, fixed in a methyl alcohol/acetic acid solution(3:1) for at least 18h. Some drops of cell suspension were splashed on cold precleaned slides and flame dried. B. rhynchopetera's chromosome isolated from the testes was dyed by Giemsa solution. The number of male chromosome was 2n=38, including 17 pairs of autosomes, 3 X chromosomes and 1 Y chromosome. Xyp complex was parachute-like. Sex-chromosome X1, X2 and X3 tied together to form the head, then getting together with Y-chromosome which was located in the pole. It was speculated that female chromosome was 2n=40.3. Comparison among morphological characteristics. Paired t-test of inter- populational morphological variations of 114 morphological characteristics among 7 geographic populations demonstrated that variation was significant among Xichang & Ludian populations and other populations (P<0.01). The variations were non-significant among Shilin, Maguan and Weng'an, Langping and Weng'an poppulation. Analyses and comparisons of 9 exterior and interior characteristics such as defensive glands, genitalia, wing and seta etc. indicated that body shape indexes of Xichang population were different from other populations. There was highly morphological polymorphism in populations of the insect. But no any single law or trend have been found in the morphological variance, which changed in different characteristics. Six components were picked up by principal components analyses. The first one contributed 41.23% to the whole variance, the body factors was dominant. The proportion of abdomen features was dominant in second and third ones. UPGMA clustering analysis indicated that there was distinct differentiation among populations. Ludian and Xichang population formed a clade which differed from other populations. Shilin, Weng'an, Lanping and Maguan populations clustered together in another clade. There were significantly positive correlation relation between 40 morphological characteristics and 13 environmental factors.4. Intra-species AFLP analysis. 10 primer pairs were used to indicate genetic differentiation among 13 geographic populations. The ratio of polymorphic bands amounts to 97.32%. Genetic diversity level of eastern populations was lower than that of western populations. AMOVA analysis showed that among- and between- populations'variances contribute 53.22% and 46.78% to the whole genetic variance. UPGMA clustering analysis based on Nei (1972) indicated that there were two clades among 13 geographic populations, which were separated according to eastern and western distributions of these geographic populations. Shannon's index had negative correlation relationship of longitude(r=-0.681,P<0.05). There was positive correlation relationship between genetic and geographic distances (r=0.435,P=0.004<0.01), which suggested that genetic differentiation mainly caused by geographical isolation.5. COI and COII segments of mtDNA gene. The combined data of COI and COII regions was 1303 bp, the variance rate was 11.36%. 34 haplotypes were generated from 115 individuals. There were only two shared haplotypes (B and F from eastern region). Individuals from eastern and western regions respectively were significantly separated on the MP and NJ phylogenetic tree of these haplotypes. The AMOVA revealed that the majority genetic variation (43% and 39%) of the total were due to variation among regions (eastern and western) and among populations within regions respectively. Only 18% genetic variance was found within individual populations, so there was significant genetic differentiation between eastern and western populations. The Mantel test suggested that genetic differentiation came mainly from geographical isolation. Nested clade analysis of haplotype net indicated that 6 nested clades had a significantly positive correlation between genetic and geographical distances. Four clades among 6 nested clades explained the reasons of differentiation occurred among the western populations. Those are allopatric fragment and free gene flow had been blocked. The pattern of eastern populations should be originated from human being migration, after the development of agriculture culture. There were no significant expanding history among populations according to neutral tests and mismatch tests. 6. Relationships among B. rhynchopetera and related species in geography. Analyses including AFLP, NJ and MP phylogenetic trees of mtDNA region COI and COII affirmed the taxonomic status of the genera Blaps, Prosoblapsia (B. apicecostata and B. allardiana allardiana) and Coelocnemodes. In the subgenus Blaps, B. sztschwana and Blap sp. had closer relationship with B. rhynchopetera comparing to that with B. kolbei. The taxonomy of B. tschitscherini should be further clarified.To sum up, B. rhynchopetera had narrow distributions, mainly found in Yunnan and circumjacent provinces. This area had a varied topography and climate. But eastern and western populations were fairly separated in these distributions according to morphological and molecular analyses. Two evolutionarily significant units(ESU) were defined by differentiation between eastern and western populations. The two ESUs might need conservation. The differentiation level of western region was higher than that of eastern region. There was significant genetic differentiation among different geographic populations. The reasons of genetic differentiation should be population history, geographic isolation, weak dispersal ability confined by its biological characteristics and habitat stability. It was inferenced that two refugial areas for B. rhynchopetera in northern range of distribution had been existed in the history, Hengduan mountains region in the northwestern and Zhaotong region in the northeastern of Yunnan Province.