**5.4 Population genetic structure**

SAMOVA and AMOVA analyses were applied to verify the genetic structure of the seven populations from Yunnan. When the number of groups (*K*) grows and the *F*CT value reaches the plateau, the SAMOVA analysis yields the optimal result. In this research, the *F*CT value reached the maximum (*F*CT = 0.73) when *K* = 2, with populations from Pu'er, Huaning, Stone Forest, and Yongsheng being one group and populations from Ruili, Wanding, and Lianghe being the other (Fig. 6), supporting the grouping result mentioned previously. The grouping was then tested by the AMOVA analysis, which indicated that 72.54% of the variation

Detecting Non-Local Japanese Pine Sawyers

Yunnan should be considered thoroughly.

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

in Yunnan, Southwestern China via Modern Molecular Techniques 79

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

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

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

effective geographical barriers, which restrict gene flow in the latitudinal direction.

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


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

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 those in Fig. 2.
