Title Page

Contents

ABSTRACT 8

Ⅰ. INTRODUCTION 9

1. General introduction 9

1.1. Invasive species 9

1.2. Reptile invasion 10

1.3. Freshwater turtles 11

2. Ecology and introduction of target species in Jeju Island. 13

2.1. Trachemys scipta elegans 13

2.2. Pseudemys peninsularis 14

2.3. Mauremys sinensis 15

3. Environmental DNA (eDNA) 16

4. Study purposes 18

Ⅱ. MATERIALS AND METHOD 20

1. Sample collection 20

1.1. Preliminary survey for housed laboratory individuals of Pseudemys peninsularis. 20

1.2. Study site 21

1.3. Visual observation 23

1.4. Pond sampling 23

1.5. Abiotic measurement 24

2. Filtration. 25

3. DNA Extraction 26

4. Polymerase Chain Reaction (PCR) detection 28

5. Statistical Analysis 29

Ⅲ. RESULTS 30

1. Preliminary survey for housed laboratory individuals of Pseudemys peninsularis. 30

2. Biotic factors 31

3. Abiotic factors 33

4. Environmental DNA detection 35

4.1. Detection of Trachemys scripta elegans 40

4.2. Detection of Mauremys sinensis 43

4.3. eDNA and visual encounter survey 46

Ⅳ. DISCUSSION 47

1. Preliminary survey for housed laboratory individuals of Pseudemys peninsularis. 47

2. Environmental DNA detection 49

2.1. Detection of Trachemys scipta elegans 49

2.2. Detection of Mauremys sinensis 51

2.3. eDNA and visual encounter survey 51

3. Recommendations for further studies 52

Ⅴ. CONCLUSION 54

REFERENCES 56

Table 1. Specific primers developed and used in this study to detect T. s. elegans, M. sinensis and P. peninsularis at Yeonhwaji, Jeju Island, South Korea. 28

Table 2. Surrounding biotic factors observed at study site. 32

Table 3. Two-way ANOVA statistical test for variations between basking site and non-basking site on abiotic variables. 34

Table 4. Two-way ANOVA statistical test for variations among weather conditions on abiotic variables. 34

Table 5. Target species identified by eDNA and visual encounter sampling method. 36

Figure 1. Location map of Yeonhwaji, Aewol-eup, Jeju Island, South Korea. 22

Figure 2. Surrounding habitat of study site - Yeonhwaji in Jeju Island, South Korea. 22

Figure 3. Field site work. (A) Collecting 1 liter of water sample at basking site; and (B) Setting up for measurement of abiotic factors in water samples collected. 25

Figure 4. General procedure for eDNA detection of invasive freshwater turtle in this study. 29

Figure 5. PCR product from Pseudemys peninsularis eDNA at lab setting over time after removing genetic sources 1, 4, 7, 14, 21, 28, 29 and 30 days. Product was run against 1Kb ladder. Negative... 30

Figure 6. Observed turtle species at basking site. A group of Trachemys genus at basking site. 31

Figure 7. Target species identified by eDNA and visual encounter sampling method at Yeonhwaji, Jeju Island. (A) Trachemys scripta elegans and, (B) Mauremys sinensis. 38

Figure 8. The ratio of positive and negative detection of 3 target species. TSE - Trachemys scripta elegans, MS - Mauremys sinensis, PP - Pseudemys peninsularis. 39

Figure 9. PCR results of Trachemys scripta elegans in a total of 30 samples. 16 water samples were successfully amplified using the eDNA of target species 40

Figure 10. The detection rate of Trachemys scripta elegans among sites and weather conditions. 42

Figure 11. PCR results of Mauremys sinensis in a total of 30 samples. 4 water samples were successfully amplified the eDNA of target species. 43

Figure 12. The detection rate of Mauremys sinensis among sites and weather conditions. 45