**2.3 Genetic diversities among 3 species of** *Dendrolimus* **by ISSR**

A set of optimized response system of ISSR has been developed, which provides a powerful tool for investigation of genetic variation of 9 natural populations(*D. punctatus spectabilis*; *D. punctatus tabulaeformis*; *D. superans*) (figure4-8,table7). Shannon index shows highest genetic diversity in Weichang population(*D. superans*) and lowest genetic diversity in Shenyang population(*D. spectabilis*), which is identical with Nei′s index(table8-10).

Different geographical groups in the same populations has a genetic divergence in some extent. Genetic similarities and cluster analysis shows that inter-species heredity difference is obviously bigger than the homogeneous between populations in the heredity difference, which reflects the heredity difference degree is consistent from the DNA level with the phenotype level. *D. punctatus spectabilis* has nearer genetic distance with *D. punctatus tabulaeformis* than *D. superans*, which is identical with that researched in sexual information of pine caterpillars(figure9).

Genetic Diversity and Population Differentiation of Main Species

Fig. 8. ISSR profile amplified by primer 35

order Sequence ofprimer(5′-3′) Total

Primer

Note: V= (G/A/C)

of *Dendrolimus* (Lepidoptera) in China and Influence of Environmental Factors on Them 327

1~14: Shenyang population of D. Punctatus spectabilis M: molecular marker(DL3000bp DNA marker)

 AGAGAGAGAGAGAGAGG 11 9 81.82 GAGAGAGAGAGAGAGAC 17 16 94.12 ACCACCACCACCACCACC 14 13 92.86 AGCAGCAGCAGCAGCAGC 17 16 94.12 CACACACACACACACAG 11 11 100.00 ATGATGATGATGATGATG 16 14 87.50 GGATGGATGGATGGAT 14 13 92.86 VDVCTCTCTCTCTCTCT 14 14 100.00 108 CACACACACACAAC 16 15 93.75 112 AGAGAGAGAGAGAGAGTA 15 15 100.00 GTCGTCGTCGTCGTCGTC 17 16 94.12 GAGGAGGAGGAGGC 18 18 100.00 121 GTGCGTGCGTGCGTGC 15 14 93.33

Table 7. Result of ISSR on adults of 3 species of *Dendrolimus* with 13 primers

bands

all locus 195 184 94.36%

population *Na Ne h I PPB* TC 1.1949±0.3971 1.0983±0.2359 0.0605±0.1371 0.0927±0.2035 19.49 JC 1.2256±0.4191 1.1383±0.2989 0.0794±0.1624 0.1182±0.2345 22.56 SC 1.2051±0.4048 1.0913±0.2293 0.0565±0.1310 0.0883±0.1944 20.51 QC 1.2103±0.4085 1.1158±0.2647 0.0691±0.1484 0.1047±0.2176 21.03 ZL 1.3846±0.4878 1.2055±0.3245 0.1230±0.1791 0.1873±0.2608 38.46 WL 1.4308±0.4965 1.2605±0.3588 0.1518±0.1959 0.2264±0.2824 43.08 CY 1.2256±0.4191 1.1287±0.2805 0.0759±0.1557 0.1143±0.2271 22.56 PY 1.1846±0.3890 1.1159±0.2805 0.0662±0.1517 0.0984±0.2190 18.46 SY 1.2205±0.4157 1.1561±0.3244 0.0869±0.1736 0.1268±0.2482 22.05 Average 1.2535±0.4264 1.1456±0.2886 0.0855±0.1594 0.1286±0.2319 25.35 All locus 1.9436±0.2313 1.4926±0.3278 0.2955±0.1610 0.4495±0.2127 94.36

Note: CY: *D. tabulaeformis* (Chengde population) ; PY:*D.tabulaeformis* (Pingquan population) ; SY: *D.tabulaeformis* (Shenyang population) QC:*D.spectabilis* (Qingdao population) ; TC :*D.spectabilis* (Taian population) ; SC: *D.spectabilis* (Shenyang population) ; JC: *D.spectabilis* (Jinzhou population) ; ZL:

*D.superans* (Zhangjiakou population) ; WL: *D. superans* (Weichang population) Table 8. The genetic variation among 9 populations of *Dendrolimus* 

Polymorphic bands

Percentage of Polymorphic bands

Sequence (from left to right): WL1~WL14,Marker, SC1~SC14

Fig. 4. ISSR-PCR fingerprints of 14 samples of *Dendrolimus superans* from WL and *Dendrolimus punctatus spectabilis* from SC populations using primer 106

1~14: Qingdao population of D. punctatus Spectabilis M: molecular marker(DL3000bp DNA marker) Fig. 5. ISSR profile amplified by primer 30

1 ~ 14: Qingdao population of D. punctatus spectabilis M: molecular marker(DL3000bp DNA marker) Fig. 6. ISSR profile amplified by primer 32

1~14: Weichang population of D.superans M:molecular marker(DL3000bp DNA marker) Fig. 7. ISSR profile amplified by primer 33

1~14: Shenyang population of D. Punctatus spectabilis M: molecular marker(DL3000bp DNA marker) Fig. 8. ISSR profile amplified by primer 35


Note: V= (G/A/C)

326 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

Fig. 4. ISSR-PCR fingerprints of 14 samples of *Dendrolimus superans* from WL and

1~14: Qingdao population of D. punctatus Spectabilis M: molecular marker(DL3000bp DNA marker)

1 ~ 14: Qingdao population of D. punctatus spectabilis M: molecular marker(DL3000bp DNA marker)

1~14: Weichang population of D.superans M:molecular marker(DL3000bp DNA marker)

*Dendrolimus punctatus spectabilis* from SC populations using primer 106

Sequence (from left to right): WL1~WL14,Marker, SC1~SC14

Fig. 5. ISSR profile amplified by primer 30

Fig. 6. ISSR profile amplified by primer 32

Fig. 7. ISSR profile amplified by primer 33

Table 7. Result of ISSR on adults of 3 species of *Dendrolimus* with 13 primers


Note: CY: *D. tabulaeformis* (Chengde population) ; PY:*D.tabulaeformis* (Pingquan population) ; SY: *D.tabulaeformis* (Shenyang population) QC:*D.spectabilis* (Qingdao population) ; TC :*D.spectabilis* (Taian population) ; SC: *D.spectabilis* (Shenyang population) ; JC: *D.spectabilis* (Jinzhou population) ; ZL: *D.superans* (Zhangjiakou population) ; WL: *D. superans* (Weichang population)

Table 8. The genetic variation among 9 populations of *Dendrolimus* 

Genetic Diversity and Population Differentiation of Main Species

diversity, *Ht*

Table 9. Genetic differentiations among 3 species of *Dendrolimus*

Note: Nei's genetic identity (above diagonal) and genetic distance (below diagonal)

**2.4 Genetic diversities among 3 species of** *Dendrolimus* **by AFLP** 

with the *D. punctatus spectabilis* Butler together (figure10).

different species of the tested *Dendrolimus*

Table 10. Genetic identity and genetic distance among 9 populations of *Dendrolimus*

The genetic structure and diversity among the 3 natural populations of the *D. punctatus tabulaeformis* Tsai *et* Liu*, D. punctatus spectabilis* Butler and *D. punctatus* Walker were tested with AFLP technique(table11). At population level, gene diversity with in populations (Hs) was 0.0895; coefficient of population differentiation (Gst) was 0.7623. Genetic variation among populations accounted for the genetic diversity of the total population of 76.23%, few portions of variation exist in the population (23.77%). Gene flow between populations Nm (0.1559) on the exchange of genes between populations is not strong, can not be effectively offset by genetic drift caused by population differentiation(table12,13). Clustering results show that *D. punctatus tabulaeformis* Tsai and *D. punctatus* Walker together with a category,

Primer combination Total bands Polymorphic bands Polymorphism(%)

 P1-T4 46 42 91.30 P2-T3 39 33 84.62 P2-T4 62 59 95.16 P2-T5 56 52 92.86 P7-T9 33 33 100.00 P8-T1 40 39 97.50 P8-T3 40 38 95.00 P8-T4 34 31 91.18 P8-T5 34 31 91.18 P8-T6 34 33 97.06 Table 11. List of AFLP primer labels,primer sequences and amplification results of 3

species Total gene

of *Dendrolimus* (Lepidoptera) in China and Influence of Environmental Factors on Them 329

*D.punctatus spectabilis* 0.1143 0.0664 0.4192 0.6927 *D.punctatus tabulaeformis* 0.1132 0.0763 0.3257 1.0351 *D.superans* 0.1626 0.1374 0.1550 2.7265

populations TC JC SC QC ZL WL CY PY SY TC \*\*\*\* 0.9492 0.9293 0.9305 0.6429 0.6434 0.6729 0.6735 0.6501 JC 0.0521 \*\*\*\* 0.9512 0.9219 0.6104 0.6239 0.6833 0.6839 0.6654 SC 0.0733 0.0500 \*\*\*\* 0.9224 0.6446 0.6535 0.7072 0.7071 0.6967 QC 0.0720 0.0813 0.0808 \*\*\*\* 0.6154 0.6209 0.6648 0.6605 0.6510 ZL 0.4418 0.4936 0.4392 0.4855 \*\*\*\* 0.9473 0.6970 0.6872 0.6784 WL 0.4410 0.4717 0.4254 0.4766 0.0541 \*\*\*\* 0.6969 0.6954 0.7076 CY 0.3961 0.3808 0.3464 0.4083 0.3609 0.3612 \*\*\*\* 0.9772 0.9259 PY 0.3952 0.3799 0.3466 0.4147 0.3751 0.3632 0.0230 \*\*\*\* 0.9292 SY 0.4306 0.4074 0.3614 0.4292 0.3880 0.3459 0.0769 0.0735 \*\*\*\*

Within population gene diversity, *Hs*

Genetic differentiation among populations, *Gst*

*Nm*

Fig. 9. Dendrogram of all D. punctatus spectabilis, D. punctatus tabulaeformis and D.superans clustered by NTSYS


Table 9. Genetic differentiations among 3 species of *Dendrolimus*

328 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

Fig. 9. Dendrogram of all D. punctatus spectabilis, D. punctatus tabulaeformis and

D.superans clustered by NTSYS

*D. punctatus spectabilis* 

*D. superans* 

*D. punctatus tabulaeformis* 


Note: Nei's genetic identity (above diagonal) and genetic distance (below diagonal)

Table 10. Genetic identity and genetic distance among 9 populations of *Dendrolimus*
