1 Comparison of morphological characters
1.1 Collection of A.mali
E.lanigerum aphids were collected from apple orchards in Tai’an, Shandong province and Dalian, Liaoning province, in mid-August, 2014. Parasitized aphids were noted and placed in petri dishes and held atca25°Cfor parasitoid emergence. Adult A. mali were removed each day and placed in 100% ethanol and stored at 20°C.
1.2 Determination of sex of parasitoids
Male and female A. mali were separated based on the larger body size of females, their antennal shape (the third section of the female’s antenna at the funicular joint is square, while that of the male is rectangular)[30], and the abdominal shape (females have a short, thick abdomen, while the male’s abdomen is slender)[8].
1.3 Morphology measurements
Images of body regions or parts were taken with the universal video imaging system(LY-WN-HPCCD (10),Chengdu Li Yang Precision Mechanical and Electrical Co., Ltd., China). This system was used to capture photograph and measure the length of the body and abdomen, the length and width of front and hind wing, the length of the leg, tibial, tarsal, anterior tarsal, and calcar of the anterior, middle and hind legs, the length and width of the first, the rod, the second, and the third sections of the antenna. One hundred A. mali(50M and 50F)of two sample areas were measured.
2 Mitochondrial genome sequencing
2.1 Collection of Aphelinus mali
Aphids parasitized by A. mali were collected from one abandoned apple orchard in Tai'an, Shandong Province and another in Huludao, Liaoning province. At each site, the five-point sampling method were used, sampling ten trees at each of the five points. On each of these 50 trees per location, five branches bearing blackened E. lanigerum aphids that were parasitized by A. mali were collected and put into a 25°Cincubator in the laboratory for A. mali emergence. One hundred adults of A. mali were obtained from each site and placed individually in 1.5 ml centrifuge tubes containing absolute ethanol and sent to Wuhan transduction bio co., LTD for sequencing.
2.2 Experimental reagents
DNA extraction was done with the Takara Genomic extraction Kit, using the specific Primer, LA Taq Polymerase, a MgCl2 solution, and a pMD18-T Vector.
For extraction, we used the Takara Genomic extraction Kit, specific Primer LA Taq Polymerase, MgCl2 solution and pMD18-T Vector. For sequencing we used the following procedure. First, primers were designed by the general conserved region to amplify the short regions of the 16s, 12s, cox1, atp6, cox3, nad4, cytb, and nad1 genes, and then specific primers were designed to amplify the remaining sequences according to the amplified conserved sequence. The resulting long fragments were then amplified using LA Taq polymerase, and the PCR program was run through 35 cycles (specific process: 94°C for 30 s, and annealed at 50°C for 30s, then followed the extension at 72°C for 1 min per 1kb.). The PCR product was then cloned into a PMD-18T (Takara, JAP) vector or sequenced directly with PCR product in ABI3730 automatic sequencer. The sequencing results were then spliced and assembled by DNASTAR software, and the complete mitochondrial sequence was obtained by manual analysis and adjustment. For amplification of the product, we then added 0.5μL TaKaRa LA Taq (5U/μL), 5μL 10×LA Taq BufferII (Mg2+Plus), 8 μL dNTP Mixture (2.5 mM each), 60 ng Template, 0.2 -1.0μm (final conc.), Primer F and Primer R, 0.2 -1.0μm (final conc.), adding ddH2O to make up the solution to 50 μL.
3 Hybridization experiment
3.1 Collection of Aphelinus mali
We selected Qingdao population of Shandong clade and Qinhuangdao population of Liaoning clade as experimental objects, and identified similar species by interspecific hybridization[9].
Live adults of A. mali were collected for this experiment in the same locations and using the same methods as described above. We choose an apple orchard with less management, protected the apple trees which have colony of E. lanigerum by screens(1m×1m×2m), pick out the parasitic aphids every day.
3.2 Experimental design methods
Crosses and reciprocal crosses between clades were made between the Shandong and Liaoning clades, using within clade crosses in each population as the control group (Liaoning♀×Shandong♂,Liaoning♂×Shandong♀); all crosses were repeated 15-20 times. For each cross, we recorded the number of host attacks and parasitoid offspring for each female, as well as the sex ratio of the offspring (% female).
The F1 generation of each hybrid cross were backcrossed with the parents of each population, with a within population back cross as the control group, with 3-5 replicates per crossing. We recorded the number of host attacks and parasitoid offspring for each female in all crosses, as well as the sex ratio of the progeny (as % female).
In each cross, the numbers of male and female progeny were counted and the mean sex ratio (MSR) for that cross was calculated as the percentage of female progeny. The relative compatibility of an intercultural cross (A x B) is expressed as[31,32]:
4 Statistical analysis
The average data of morphological measurements, hybridization and backcross were calculated as the mean ± standard deviation (SD) using Statistical Product and Service Solutions(SPSS) 19.0 and significant differences were tested using one-way analysis of variance(ANOVA) corrected by SPSS 19.0. The independent samples t-test was used to analyze the parthenogenesis between two clades by SPSS 19.0
For genomic analysis, we used MITOS Webserver (http:// mitos. bioinf. uni-leipzig. de/index. py) to annotate and analyze the mitochondrial genome sequence and obtain the linear alignment of the mitochondrial genome sequence. The AT-skew and GC-skew were calculated using DNASTAR as follows: AT-skew = (A% -T%)/ (A% +T%) and GC-skew = (G%-C %)/ (G%+C%).