Luận án: Biến đổi gen, diet metabarcoding & bảo tồn linh trưởng Colobine tại VN

GENETIC VARIABILITY, DIET METABARCODING, AND CONSERVATION OF COLOBINE PRIMATES IN VIETNAM by ANDIE ANG B.), National University of Singapore, 2008 M., National University of Singapore, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Anthropology 2016 This thesis entitled: Genetic Variability, Diet Metabarcoding, and Conservation of Colobine Primates in Vietnam written by Andie Ang has been approved for the Department of Anthropology ____________________________________ Herbert H. Covert, Committee Chair ____________________________________ Steven R. Leigh, Committee Member ____________________________________ Michelle Sauther, Committee Member ____________________________________ Robin M. Bernstein, Committee Member ____________________________________ Barth Wright, Committee Member Date __________________ The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline.

ii Ang, Andie (Ph., Anthropology) Genetic Variability, Diet Metabarcoding, and Conservation of Colobine Primates in Vietnam Thesis directed by Professor Herbert H. Covert This dissertation examines the genetic variability and diet of three colobine species across six sites in Vietnam: the endangered black-shanked douc (Pygathrix nigripes, BSD) in Ta Kou Nature Reserve, Cat Tien National Park, Nui Chua National Park, and Hon Heo Mountain; endangered Indochinese silvered langur (Trachypithecus germaini, ISL) in Kien Luong Karst Area (specifically Chua Hang, Khoe La, Lo Coc and Mo So hills); and critically endangered Tonkin snub-nosed monkey (Rhinopithecus avunculus, TSNM) in Khau Ca Area. A total of 395 fecal samples were collected (July 2012-October 2014) and genomic DNA was extracted. This research provides the first information on their mitochondrial hypervariable region I variability and also pioneers the characterization of their diet using DNA metabarcoding in association with next-generation sequencing.

BSDs exhibit high variability but no gene flow between populations. Similarly, ISLs showed high variability but only the subpopulation in Khoe La, the site under mining disturbance, retains most of the remaining genetic diversity in the species. Zero mitochondrial variability was found in TSNMs, the lowest ever reported for primate species in the wild. Diet sequences of colobines were matched to plant databases for identifications.

Forty plant families were recorded for BSDs, including new records from 18 families, 15 genera and 13 species. There was little overlap in their diet with only six taxa found across four populations. They were also selective by feeding on less abundant species. Twenty-five families were recorded for ISLs, including new records from nine families, 18 genera and 14 species.

Moraceae dominated their diet as retrieved from fecal samples, and was also the top family as revealed by field observations, demonstrating the significance of this plant family iii in their diet. Eighteen families were identified for TSNMs, including new records from three families, five genera and three species. The dominant taxon belonged to Polyalthia (Annonaceae) and they were also the dominant genus and family respectively within Khau Ca Area. This research highlights the importance of using genetic methods to complement field observations so as to better understand population viability and dietary profiles in order to identify priority actions for the conservation of colobine primates.

(350 words) iv ACKNOWLEDGEMENTS I am grateful for the day that I attended my first International Primatological Society (IPS) Congress at the Kyoto University, Japan, six years ago. With a stack of CV in my hands, I was excited and nervous by the prospect of approaching potential Ph. If not for Quyet, I would not have had the great fortune of meeting Prof. Bert, thank you so much for all the advice and guidance, without which I would not have the slightest chance of embarking and finishing this dissertation.

This research was also exactly what I wanted to do, and I could not imagine doing it, and would not wish to do it, without you. A central part of this dissertation relied on the enormous support from various collaborators and institutions in and outside Vietnam; thank you so much Bert for giving me the opportunity to join this network of great people. Thank you Sherri for always being so sweet and nice. I hope it won’t be long until we sit by the roadside stalls in Vietnam eating ban mi chuong and drinking ca fe sua da! Thank you so much Profs.

Steve Leigh, Michelle Sauther, Robin Bernstein and Barth Wright for being such a supportive committee and for helping to shape this dissertation into a work that I am proud of. You have also given me a lot more ideas on how to improve the contents for future publications and I appreciate it. Boss Rudolf Meier, I had promised that I would study flies in the unfortunate event that all non-human primates went extinct. I guess the time has not come yet.

(Phew!) Thank you for putting me on the path of primatology and conservation (!) and for the generous advice and time for a student who finished and left your lab. Despite leaving, I always know I’ll be coming back to harass the lab (again against your wish, oops). Time for Two-Chefs! This time my treat. As a graduate student in the department, I learned a great deal from the faculty and fellow students, particularly Quyet, Jonathan, Amy, Griette, Eric, Erin, Robin, Dawa, Jeff, Willi, Ben and all my friends in the department.

Quyet, thank you for introducing me to the v Tonkin snub-nosed monkeys and their oh-my-gosh-challenging habitat. Thanks for taking care of me in the field and for being the good friend who bought my earliest beer in the day ever at 10am in Hanoi (and I think you broke the record again with 7am in Khau Ca). Jonathan, thanks for being the big brother that I never had, and for being such a nice friend. I miss speaking Mandarin with you already.

I wish we would have the opportunity to work in the field together sometime. Come to Singapore! Griette Lady D., you are such a weird lady, whom I adore. I’ll miss your crazy laughter (but not the scares you frequently gave me from behind my back. Can’t wait for when you come to Asia and we are going to go look for monkeys! Thank you so much Valerie and Lesa for always being there to help with my progress and for making sure that I was on track with the requirements of the department and the graduate school; you both make everything easier and smoother.

I would like to thank everyone whom I had the good fortune to work with in Vietnam, particularly Duc, Long, Bach, Bang, Thao, Hung, Minh, Luc (at Ta Kou), Thuan (at Cat Tien), Khoan (Khau Ca), his team and their families, friends at the Southern Institute of Ecology, Vietnam Administration of Forestry, and management boards of the protected forests. I miss you Tho and your wonderful family. Thanks for hosting me while I was in Vietnam! Thank you so much Amrita for your kind advice and for being ever so generous and willing to help. Miss monkey chitchatting with you! Oh dearest Evo Labbies, thank you YC, Kathy, Sujatha, Maosheng, Wendy, Gowri, Wing Hing, Bilge, Theo, Darren, David, Mindy, Yujie, Diego, Rebecca, Jonathan, Denise, Sheng Rong, Gaurav, Gwynne, Weisong, and friends and colleagues at NUS.

Thanks Susan Tsang for the nice meals and skype calls; can’t wait for you to return to Asia for field work! Thanks Amy Klegarth for the wonderful time while you were in Singapore. The macaques miss you lady! All these would not have been possible without the first primate training course that I attended in December 2010 in Vietnam. Thank you Chia Tan and Sylvia Atsalis for being vi such wonderful mentors and for introducing me to like-minded young researchers who then became my really good friends: Kefeng, Bach, Little Minh and many others. Thank you Vilma and friends at Jane Goodall Institute (Singapore), Prof.

Tommy Koh, Sonja, John Sha, Jonathan Clayton, Tuan, Neahga, Russ Mittermeier, Ben Rawson, Noel Rowe, Marc Myers for the support. There were so many people whom I met in Boulder and for whom I am grateful: Tee Tong, Wee Kiat, Harng Luh, Wee Soon, Mahir, Nigel, Shun, Yuma, Brad and Betsy (and her kitty cats Zoe and Maeve). I miss hanging out! To my besties Fern, Janelle and Yixin, so many changes have occurred while I was away but I’m grateful that I participated in most of the major events in your lives while I was back. I appreciate the support that all of you gave me.

Dickson, thank you for the sacrifices. Thank you for flying 14,601 km, twice, with me (and another 29,202 km on your own). 爸爸 ，妈妈，谢谢你们这么支持我，让我能够好好地念书。这五年来，当别家同龄的小孩挣钱养家 ，而我还不能让你们舒适地过生活时，你们不但没有给我压力，反而不断地问我: “吃得饱吗 ，玩得好吗?” 世上只有爸妈好。Thank you little sister for taking care of them and ah huat while I was away and not able to help out. You have been so reliable, and made me feel assured that they were in good hands.

This dissertation would not have been possible without the generous support from the University of Colorado Boulder (Department of Anthropology Pre-Dissertation Grant, Beverly Sears Graduate Student Research Grant, Scott Ferris Graduate Research Award, Haskell-Houghtelin Scholarship Fund, and Willena D. Cartwright Anthropology Fund), Evolutionary Biology Lab of National University of Singapore, Wildlife Reserves Singapore Conservation Fund, Mohamed bin Zayed Species Conservation Fund, and National Geographic Waitt Grant. vii This dissertation is dedicated to ah boy. I know there is no more chance to see you again.

I hope that you remembered me, and I miss you. Singapore, 2000 Zambia, 2005 viii TABLE OF CONTENTS ABSTRACT. v TABLE OF CONTENTS. ix LIST OF TABLES.

xiii LIST OF FIGURES. xv LIST OF APPENDICES. xvii LIST OF PUBLICATIONS. xviii CHAPTER I: INTRODUCTION.

Primate Conservation in Vietnam. Research Questions and Hypotheses. 20 CHAPTER II: STUDY SUBJECTS, SITES, AND SAMPLE COLLECTION. The Black-shanked Douc (BSD; Pygathrix nigripes).

Ta Kou Nature Reserve. Cat Tien National Park. Nui Chua National Park. Hon Heo Mountain.

The Indochinese Silvered Langur (ISL; Trachypithecus germaini). Kien Luong Karst Area. The Tonkin Snub-nosed Monkey (TSNM; Rhinopithecus avunculus). Khau Ca Area.

Fecal Sample Collection. 31 CHAPTER III: GENETIC VARIABILITY OF COLOBINE PRIMATES. DNA Amplification and Sequencing. Mitochondrial Genomes and Nuclear DNA of TSNM.

Mitochondrial Genomes and Nuclear DNA of TSNM. Genetic Variability of the Black-shanked Douc. Genetic Variability of the Indochinese Silvered Langur. Genetic Variability of the Tonkin Snub-nosed Monkey.

54 CHAPTER IV: DIET METABARCODING. DNA Amplification and Sequencing. Taxonomic Reference Databases. Diet Metabarcoding Dataset.

64 CHAPTER V: DIETARY PROFILE OF BLACK-SHANKED DOUCS. Feeding Observations from the Field. Floristic Diversity in Ta Kou and Cat Tien. Diet Metabarcoding Results.

Number of Plant Identifications. Taxonomic Levels of Plant Identifications. Plant Identifications for Samples. From Ta Kou Nature Reserve.

From Cat Tien National Park. From Nui Chua National Park. From Hon Heo Mountain. Comparison between Metabarcoding and Feeding Observations.

Comparison between Dietary Profile and Habitat Diversity. Inter-population Differences. 102 CHAPTER VI: DIETARY PROFILE OF INDOCHINESE SILVERED LANGURS 104 6. Feeding Observations from the Field.

Floristic Diversity in Kien Luong. Diet Metabarcoding Results. Number of Plant Identifications. Taxonomic Levels of Plant Identifications.

Effect of Habitat Disturbances. Comparison between Metabarcoding and Feeding Observations. Comparison between Dietary Profile and Habitat Diversity. Effect of Habitat Disturbances.

123 CHAPTER VII: DIETARY PROFILE OF TONKIN SNUB-NOSED MONKEYS. Feeding Observations from the Field. Floristic Diversity and Seasonality in Khau Ca Area. Diet Metabarcoding Results.

Number of Plant Identifications. Taxonomic Levels of Plant Identifications. Diet Transition between Seasons. Comparison between Metabarcoding and Feeding Observations.

Comparison between Dietary Profile and Habitat Diversity. Diet Transition between Seasons. 145 CHAPTER VIII: CONSERVATION IMPLICATIONS AND CONCLUSIONS. Conservation Implications and Recommendations for Future Studies.

179 xii LIST OF TABLES Table 1.