**6. Determination of non-local** *M. alternatus*

78 Zoology

occurred between the two groups, 21.86% occurred within populations, and only 5.60% occurred among populations within groups (Table 3). The relatively high variation within populations was also mirrored by the haplotype assemblage that most of the populations possessed multiple haplotypes, especially the three populations from southwestern Yunnan

Between groups 1 1.725 72.54 *F*CT = 0.73 0.025 Among populations within groups 5 0.133 5.60 *F*SC = 0.21 0.001 Within populations 54 0.520 21.86 *F*ST = 0.78 0.000 Total 60 2.378 100 -- --

Fig. 6. The terrain of Yunnan and the grouping result of seven populations of *M. alternatus* determined by SAMOVA and AMOVA analyses with major geographical barriers marked (Ailao Mountains, Wuliang Mountains, and Nushan Mountains). SPG, the southwestern population group; RPG, the remaining population group. Population codes correspond to

**components**

**Percentage** 

**of variation** *F P* 

rich in unique haplotypes (Table 1; Fig. 3).

those in Fig. 2.

**Source of variation** *d. f.* **Variance** 

Table 3. AMOVA analysis results. Data source: Fu et al. (2010).

The experiment provided molecular evidence strongly supporting the following two hypotheses: 1) the genetic structure divides the seven *M. alternatus* populations from Yunnan into two groups, the southwestern population group (SPG) and the remaining population group (RPG), and 2) it is most likely that there were non-local individuals of *M. alternatus* in the three populations sampled from southwestern Yunnan, as these populations shared one of the dominant haplotypes of the reference population from Zhejiang. However, in an attempt to finally determine the identity of non-local *M. alternatus*, the relationship between such genetic divergence and the natural geographical characteristics of Yunnan should be considered thoroughly.

Genetic divergence has been frequently discussed in terms of natural geographical barriers (Yagi et al., 2001; Shoda-Kagaya, 2007; Shi et al., 2010). Yunnan is a typical LLPR with complex terrain, where numerous population phylogenetic studies on different insects have revealed the role that geographical barriers play in obstructing the gene flow among populations from various localities (Shi & Ye, 2004; Liu et al., 2007; P. Chen & Ye, 2008; Shi et al., 2010). Basically, the mountain ranges in the LRGR portion of Yunnan are considered effective geographical barriers, which restrict gene flow in the latitudinal direction.

Since the natural environment of Yunnan is able to cause genetic divergence, the separation of two population groups could be either the result of natural geographical barriers or the consequence of introduced non-local sources. The seven sampling sites in Yunnan were separated by great mountain ranges. Ruili, Wanding, and Lianghe were separated from other populations by Nushan Mountains, Pu'er was separated from Stone Forest, Huaning, and Yongsheng by Wuliang and Ailao Mountains (Fig. 6). But it is interesting to note that the genetic distances among populations from the RPG were limited to 0.0014 to 0.0031 (Table 2), the while genetic variation among them comprised only 5.6% of the total divergence (Table 3), and they shared all of the haplotypes defined by the samples taken from them (Table 1; Fig. 3). Hence, the geographical barriers such as Wuliang and Ailao Mountains had not caused sufficient genetic divergence among these populations. Judging from the phylogenetic tree (Fig. 5), these four populations were latest diverged, which may represent a short inhabitation history of *M. alternatus* in Yunnan under natural condition. To the contrast, the genetic distances between populations from the SPG and the RPG were much higher than that within each group, varied from 0.0064 to 0.0101 (Table 2), the genetic variation reached 72.54% (Table 3), and most of the unique haplotypes were defined (Table 1). Given the discussion above, the geographical barriers can not explain such divergence.

Assuming that the SPG and the RPG were two genetically independent sources, there must be gene flows among these populations in the course of evolution, which will be detected by shared haplotypes. It is noticeable that population from Lianghe shared two haplotypes with populations from the RPG; however, no haplotype from the SPG was shared by any other populations. Given that the individuals from the RPG were able to "migrate" into the SPG, the same phenomenon should have happened the other way around. Another assumption was that the genetic divergence between the SGP and the RPG was caused by introduction of non-local *M. alternatus*. The haplotype distribution demonstrated that about 90% individuals of the southwestern populations shared haplotype 5 with population from Zhejiang (Table 1; Fig. 3), and the phylogenetic tree indicated a close genetic connection

Detecting Non-Local Japanese Pine Sawyers

thoroughly.

**9. Acknowledgements** 

of Yunnan University (21134025).

*Forest Entomology*, 2 (3): 217-223.

*Entomology*, 132 (7): 530-537.

**10. References** 

129-140.

Chinese]

Chinese]

International.

in Yunnan, Southwestern China via Modern Molecular Techniques 81

colleagues are conducting a panoramic research on the population phylogeny of *M. alternatus* sampled from multiple localities in mainland China. The result of this research is to partially answer the long-asked question regarding the dispersal pattern of *M. alternatus* during the spread of the pine wilt disease in China. And as it is now possible to test the genetic profiles of *B. xylophilus* and *M. alternatus* together, future studies on the dispersal pattern and original sources of the pine wood nematodes in China can be revealed more

The related researches were conducted under a joint cooperation of the following persons and faculties: Z. Zhang (Chinese Academy of Forestry), Y.Z. Pan (Southwest Forestry University), H.P. Liu (Forest Disease and Pest Control and Quarantine Bureau of Yunnan Province), P.Y. Zhou (FDPCQB of Dehong Prefecture), R.J. Rui and W.C. Cai (Forest Disease and Pest Control and Quarantine Station of Yongsheng), Z.Q. Li and B.C. Liu (FDPCQS of Wanding), Z. Nong (FDPCQS of Ruili), H. Zhou (Forestry Bureau of Pu'er), D.D. Chen, X.Y. Ma, S.N. Ge, and C. Wu (Yunnan University), and X. Zhang (Kunming Institute of Zoology). The related research programs were conducted with the financial supports of the Key Program of State Forestry Administration (2006BAD08A19105), the International S&T Cooperation Program of China (2006DFA31790), the Key Scientific Project Fund of the Science and Technology Bureau of Yunnan Province (2005NG03), and the "211" Key Project

Anbutsu, H. & Togashi, K. (2000). Deterred ovipostion response of *Monochamus alternatus*

Braasch, H., Tomiczek, C., Metge, K., Hoyer, U., Burgermeister, W., Wulfert, I., & Schönfeld,

CABI & EPPO. (1997). *Quarantine Pests for Europe (2nd edition)*. Wallingford: CAB

Cesari, M., Marescalchi, O., Francardi, V., & Mantovani, B. (2005). Taxonomy and phylogeny

Chen, P. & Ye, H. (2008). Relationship among five populations of *Bactrocera dorsalis* based on

Chen, S.Y. & Zhang, Y.P. (2006). The genetic approaches and applications in the research of

Chen, Z.Y. (2001). *The Pandect of Yunnan Climate*. Beijing: China Meteorological Press. [in

Davis, E.E., Albercht, E.M., & Venette, R.C. (2008). *Monochamus alternatus*. In: Venette, R.C.

*Zoological Systematics and Evolutionary Research*, 43 (1): 1-7.

*Exotic Pine Pests: Survey Reference*, USDA Forest Service.

(Coleoptera: Cerambycidae) to oviposition scars occupied by eggs. *Agricultural and* 

U. (2001). Records of *Bursaphelenchus* spp. (Nematoda, Parasitaphelenchidae) in coniferous timber imported from the Asian part of Russia. *Forest Pathology*, 31 (3):

of European *Monochamus* species: first molecular and karyological data. *Journal of* 

mitochondrial DNA sequences in western Yunnan, China. *Journal of Applied* 

the origin of domesticated animals. *Chinese Science Bulletin*, 51 (21): 2469-2475. [in

between haplotype 5 and those unique haplotypes (Fig. 5), which had been regarded as a symbol of establishment of non-local populations (Slatkin & Hudson, 1991; J. Hu et al., 2008).

As mentioned previously, the pine wilt disease in Wanding was reported four years after the arrival of electronics with wood packaging materials from eastern China where the pine wood disease was most severe. Considering the characteristics of the immature stages of *M. alternatus* and its association with *B. xylophilus*, it was possible that the un-treated, infested wood packaging materials disposed casually near the construction site became the source of infestation. This is not only would help to explain the significant genetic divergence between the SPG and the RPG in Yunnan, but also can explain the genetic connection between the SPG and the population from Zhejiang instead of Hubei.
