Initially, the development of resistance to Eo NPV, possibly caused by selection pressure in some populations, was considered as an explanation .
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However, there were no heavy applications of Eo NPV in any of our seventeen collection sites. In addition, we hypothesize that this divergence was induced by geographic, ecological or other factors, rather than Eo NPV selection pressure. Examining divergence levels by means of molecular markers and inheritance analysis is the first step toward exploring a possible mechanism.
Mitochondrial DNA mtDNA has been widely used in insect taxonomy and molecular systematics to elucidate the phylogenetic relationships of insects  —  , especially among some sibling species and cryptic species that are difficult to distinguish solely by morphology  , . The widely distributed neotropical skipper butterfly Astraptes fulgerator was formerly recognized as at least 10 species in northwestern Costa Rica, but morphological analysis and a DNA barcoding study of COI showed that whilst A.
In this study, we collected seventeen populations from eight main tea-producing provinces of China. Single-pair cross analysis was also conducted to verify any species-level diversification and to understand the biological effects of divergence among populations.
No specific permits were required for this study. All samples were collected in open tea plantations and not from any national parks or protected areas. Two outgroup species of Buzura suppressaria Guenee and Scopula subpunctaria Herrich-Schaeffer were collected from a tea plantation in Hangzhou, Zhejiang province. All insect specimens were first identified under a stereo microscope following the current keys based on characters such as antennae, forewing and venation before further analysis  ,  , . A total of specimens were sequenced for this study, representing 17 different populations Table 1.
Total DNA was extracted from individual larvae after air-drying for 20 min. The specimen tissue was first ground with a flame-sealed pipette tip in a 1. The PCR products were detected by electrophoresis on a 1. Genetic diversity was assessed using DnaSP v5. Phylogenetic tree searches were conducted using neighbor-joining NJ and maximum-parsimony MP methods by MEGA 5 program with bootstrap replications. The software Arlequin 3.
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The evolutionary network was constructed via a median joining method as implemented in the Network 4. All the tea geometrid populations used were first inspected using a microscope for morphological analysis. Taxonomic keys including antennae, forewing, wing, and genitalia characters were used for diagnosing specimens as E. Three populations susceptible or insensitive to virus were randomly selected for cross-breeding and maintained at least two generations prior to the start of cross-breeding and propagated in the laboratory as Xi et al. The male and female pupae of each population were isolated and reared for mating.
Based on the genetic divergence and molecular phylogenetic analysis of the mitochondrial COI gene, the populations were divided into two distinct groups. We randomly selected several populations from each group for further crossing experiments.
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All reciprocal crosses were conducted simultaneously and under the same laboratory conditions as the parental colonies were maintained  , . Each cross was set up between one virgin female and one male in a transparent colorless glass cage, and intra-population mating was used as the control treatment. Two pairs were used for one repeat and twelve experimental repeats were for all cross-breeding or mating tests. Each pair was observed daily to record egg number, hatching rate, survival to adult, the proportion of normal adults, and the sex-ratio.
The F 1 offspring were self-crossed using the conditions mentioned above. The self-crosses of the intra-population of the F 1 generation were the same as for the population in the control treatment, and the eggs, hatching rate and larvae development of F 2 were simultaneously recorded. The sex-ratios across replicates were calculated by dividing the total number of females by the overall number of males in each treatment. We cloned and sequenced the COI gene for all specimens from seventeen tea geometrid populations and 20 samples from two outgroup populations.
The sequence alignment contains polymorphic sites and parsimony informative sites.
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A total of 50 COI haplotypes were observed. Sequences of 50 haplotypes for E.
The overall haplotype diversity index Hd was 0. The mean nucleotide diversity index Pi and mean gene flow Nm among the 17 populations were 0. The other 48 haplotypes were each found in only one population. Among the 17 populations, the LY population had just one haplotype, and other sixteen populations had more than one haplotype.
The populations with the highest haplotype diversity were WH, which had seven haplotypes, and LX, which had eight haplotypes. The mean genetic divergences D and nucleotide diversity Pi differed significantly between the two groups Table 2. The genetic distances among seventeen populations were calculated using the Kimura 2-parameter model in MEGA 5 Table 3. The genetic divergences for the COI genes among populations of E.
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They were obviously less than those between E. The pairwise distances between populations from two groups were 3. These results showed that the seventeen E. Based on the 50 haplotypes and two outgroups of B. The phylogenetic trees were highly consistent, and both showed that all haplotypes within any population fell into a single group.
The E. Because two haplotypes of QZ1 and HZ2 distributed universally within their respective clade, and the analysis of median joining network also supported they were predominant ones, they may be the original haplotypes. The phylogenetic results were consistent with the genetic distance analysis that concluded that E. Numbers above the nodes indicate bootstrap support. The outgroups used were B. All the crossed pairs either inter-clade or intra-clade, produced eggs.
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All eggs produced in the intra-population and inter-population intra-clade crosses were fertile. To detect reproductive isolation level between the two E. Highly unbalanced sex-ratios and few emerged normal adults reduced the opportunities for selfcrosses and backcrosses of the F1 hybrids.
Therefore, the inter-clade cross-breeding for E. In addition to using the self-cross intra-population as controls, we also conducted one intra-clade cross. Furthermore, their F 2 and F 3 generations also developed and produced normal offspring after self-crosses. Tea is an evergreen bush with a long growing season from March to September.
Growers have to spray several times to minimize herbivore damage. The development of resistance to chemical insecticides is very common in many tea insects  ,  , possibly driven by selection pressure . Although Eo NPV is not a chemical insecticide, resistance may still develop in target pest populations. Our previous study showed significant variations up to fold in susceptibility to Eo NPV in field populations of E.
Therefore, we surveyed the level of Eo NPV used in those locations. Because the only Eo NPV pesticide product used in China was developed by our institute and an agricultural chemical company, we have a clear application record of this virus in tea plantation. Considering the high specificity of the virus and the vast geographic variation in tea ecosystems, our second hypothesis for this study was that E. In this study, we first conducted phylogenetic analysis of the COI region of E. All 50 haplotypes assembled from sequences from seventeen E. Within a clade, populations showed close relationships, with genetic distances less than 0.
In contrast, the genetic divergences between clades were 3. These molecular data strongly indicated that E. Moreover, the lack of shared haplotypes also indicated a certain degree of reproductive isolation between the clades . To test species divergence and reproductive isolation, we selected three populations from each clade and conducted inter-clade crossing and F 1 self-crossing tests.
Although the number of eggs produced by inter-clade crossing was not significantly different from the in-clade self-crossed control, the egg hatchability, survival rates to adult and percentage of normal adults were significantly lower along with an abnormal sex ratio than those of the self-crossing controls.
The selfcross of the F 1 generation from inter-clade hybrids produced sterile eggs or no eggs, while the control group F 1 , F 2 and F 3 generations obtained from crossings between populations within a clade could grow, develop and produce normal progenies. The cross-breeding results further support the suggestion of reproductive isolation between two clades of E. Cryptic species complexes, virtually identical in appearance but nonetheless having reproductive isolation, are very common in nature  ,  — .
The biological characteristics of the tea plant and decreasing artificial disturbance after initial planting turn tea plantations into a shaded and stable ecosystem that is consequently a permanent habitat for herbivores. Thus, not all insects in tea plantations migrate long distances, and thus, these insects can easily colonize and develop diversified evolution in adaptive surroundings  , . Furthermore, few tea plant varieties require replacement during production due to their long life, which also decreases the genetic exchanges among E.
Many tea plantations are also located on mountains with highly diversified geography and climate, which might drive the divergence. The locations of the four populations within Clade I HZ, YH, YX and LX are close to each other and possess the same geographic characteristics and climate, while the Clade II locations are in the mountains with diversified geography and changeable climate within each of them.
As a result, these possible factors may drive the divergent evolution in relatively isolated E.