**5.1 Haplotype assemblage**

Eighteen haplotypes were defined by 22 polymorphic sites in the 565 bp *cox2* sequence, with haplotypes 1, 2, and 5 showing much higher frequencies. The distribution pattern of these 18 haplotypes was locality related: haplotypes 1 to 4 were widely distributed, dominating most of the central, northern, and southern Yunnan; haplotypes 6 to 18 were found within single localities. Haplotype 5 was shared by populations from southwestern Yunnan and Zhejiang; however, haplotypes 1 to 4 were neither shared by population from Hubei nor population from Zhejiang; and haplotypes 12 to 15 were only found in Hubei population. Further analysis of the haplotypes derived from the three populations in southwestern Yunnan discovered a high ratio of haplotype 5: with 69.2%, 70.0%, and 71.4% in Ruili, Wanding, and Lianghe populations, respectively. The matrix of shared haplotypes showed a boundary between the three populations from southwestern Yunnan and the remaining populations, with the shared haplotypes being confined within each population group and the unique

The same PCR primers developed by Roehrdanz (1993) and Kawai et al. (2006) were applied, and the PCR reaction was performed by the protocols described by Kawai et al. (2006). Bioinformatic and statistic software like DAMBE 5.0.7 (Xia & Xie, 2001), MEGA 4.0 (Tamura et al., 2007), SAMOVA 1.0 (Dupanloup et al., 2002), AMOVA 3.1 (Excoffier et al., 2005), and SSPS 13.0 (SPSS Inc., Illinois, US) were applied to analyze haplotype assemblage, Kimura two-parameter (K2P) distance (Kimura, 1980), NJ phylogenetic reconstruction (Saitou & Nei, 1987), population grouping, and multidimensional scaling (MDS) (Lessa, 1990). The mapping of haplotype distribution was performed in AcrView 3.3 (ESRI, USA).

Fig. 2. The sampling sites of *M. alternatus* in Yunnan, Hubei, and Zhejiang. Dots on the map

Eighteen haplotypes were defined by 22 polymorphic sites in the 565 bp *cox2* sequence, with haplotypes 1, 2, and 5 showing much higher frequencies. The distribution pattern of these 18 haplotypes was locality related: haplotypes 1 to 4 were widely distributed, dominating most of the central, northern, and southern Yunnan; haplotypes 6 to 18 were found within single localities. Haplotype 5 was shared by populations from southwestern Yunnan and Zhejiang; however, haplotypes 1 to 4 were neither shared by population from Hubei nor population from Zhejiang; and haplotypes 12 to 15 were only found in Hubei population. Further analysis of the haplotypes derived from the three populations in southwestern Yunnan discovered a high ratio of haplotype 5: with 69.2%, 70.0%, and 71.4% in Ruili, Wanding, and Lianghe populations, respectively. The matrix of shared haplotypes showed a boundary between the three populations from southwestern Yunnan and the remaining populations, with the shared haplotypes being confined within each population group and the unique

of China represent the capital cities of the three provinces. After Fu et al. (2010).

**5. Validating the hypothesis – Molecular evidences** 

**5.1 Haplotype assemblage** 

haplotypes being presented only in two of the populations from southwestern Yunnan (Table 1). When mapping haplotype frequencies into pie charts, a more obvious population boundary as well as the existence of non-local individuals in three populations from southwestern Yunnan can be observed (Fig. 3).


Table 1. Matrix of shared haplotypes of the nine *M. alternatus* populations (below diagonal) and the numbers of unique haplotypes (last row). Population codes correspond to those in Fig. 2.

Fig. 3. Mapping of haplotype frequencies of all nine populations of *M. alternatus*.
