A BIOGEOGRAPHICAL CASE STUDY OF DIDERMA HEMISPHAERICUM (MYXOMYCOTA) predicting local habitat suitability in changing climate scenarios and assessing genetic diversity across southeast asian populations

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY KING JOSHUA ALMADRONES REYES A BIOGEOGRAPHICAL CASE STUDY OF DIDERMA HEMISPHAERICUM (MYXOMYCOTA): PREDICTING LOCAL HABITAT SUITABILITY IN CHANGING CLIMATE SCENARIOS AND ASSESSING GENETIC DIVERSITY ACROSS SOUTHEAST ASIAN POPULATIONS BACHELOR THESIS Study Mode: Major: Faculty: Batch: Full-time Environmental Science and Management Advanced Education Program Office K46 N01-AEP Thai Nguyen, 15/09/2018 Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Science in Environmental Science and Management Student name Student ID Thesis Title King Joshua Almadrones Reyes DTN1454290094 A BIOGEOGRAPHICAL CASE STUDY OF DIDERMA HEMISPHAERICUM (MYXOMYCOTA): Predicting local habitat suitability in changing climate scenarios and assessing genetic diversity across Southeast Asian populations Supervisor(s) Dr.rer.nat Nikki Heherson A Dagamac & Mr Do Tuan Tung Abstract: Biogeographic and molecular studies are currently the trends for biodiversity assessments to address issues related to conservation and biological resource management Since these areas are underexplored for economically important terrestrial protist like the myxomycetes, two research frameworks using the cosmopolitan Diderma hemisphaericum as the biological model were designed for this study: (1) predicting suitable niches for the species under different climate change scenarios and (2) assessing genetic diversity of the species population between Philippines and Vietnam using partial 18s rDNA gene marker The first framework showed: (i) temperature seasonality and isothermality to be the most important bioclimatic predictors and (ii) widespread increase of suitable habitats for D hemisphaericum in A2 and B1 scenarios This is the first report of species distribution modeling for the Southeast Asian myxomycetes which can be used for finding priority areas for conservation in the Philippines The second framework revealed: (i) that gene flow exist yielding a total of 27 ribotypes coming from suspected putative biospecies and (ii) geographical barriers are not the most significant driver in the speciation events for the D hemisphaericum populations supporting now the “Everything is everwhere” model of biogeographic distribution This finding provides groundbase information for myxomycetes biodiversity at the genetic level in Southeast Asian region Keywords: Biogeography –climate change –environmental niche –funguslike protists –molecular diversity Number of pages: 102 Date of Submission: September 15, 2018 ii ACKNOWLEDGEMENT The King would like to give its deepest gratitude to the people who made this one hell of a scientific work:  To Dr rer nat Nikki Heherson Aldea Dagamac (University of Greifswald) and my Vietnamese adviser Mr Do Tuan Tung (TUAF) for their supervision  To Prof Dr Martin Schnittler and the International Office in Greifswald, thank you very much for the financial assistance during my research internship in Greifswald, Germany  To the AEP Office, especially Chi Hong, thank you very much for all the assistance you’ve extended from Visa applications and all the questions you enthusiastically entertains Also, huge thanks to my roommate Tu Quang Tu  To Tita Dory Zapanta and Tito Joey Zapanta, thank you for all the support you have been extending to me and my family from my high school years until now that whom without, I will be probably in a very different path  To Tita Marge Parulan, thank you very much for your good heart and support for me and my family And to all the many people who helped me and my family through thick and thin, thank you very much!  I would also like to give thanks to the General Botany and Plant Systematics working group of the Institute of Botany and Landscape Ecology for the scientific discussions, barbecue parties and table tennis on a very hot German summer – Frau Anja Klahr, Dr Manuela Bog, Oleg Shchepin, Jan Woyzichovski, Melanie Zacharias and Paul Lamkowski  Huge thanks to my Lola Flor, Kuya Etang, Kuya Eric and Kuya Erwin for always checking my condition everytime  To the Hood - Niecer, JD, Aaron and Ian thank you for not forgetting to send memes during my stay in Germany, I really appreciate yall doing that iii  A huge shoutout to Nami, Blindie and Kaneki You’re my favorite pets that kept me smiling even though I am far away from Home  To Yani and Enzo – we survived the MyxoGod  I would also like to give thanks to the people that I’ve met in Greifswald - Kasia, Oleg, Jan, Mori, Virna, Nadine, Felix 1, Linh, Felix 2, Benjamin, Julia, Jonas, Lukas, Christoph, Paul, Robin, Oriana, Andrea, Laura 1, Laura 2, Prakash for the parties, hotpot, picnics, volleyball and board game nights we shared Vielen dank for making me feel at home in that one small medieval-ish town See ya fellow adventurers and guardians (I have golds)!  Also, very big thanks to Sir Tom, Ate Jeane and Baby Jillian dela Cruz, for the warm welcome in Berlin  Next, to my Asian friends, Lorna, Lita, and Fibi, thanks for the Asian company and for the reminder to try “new” things in life  Moreover, to these people, first, Nikki, thank you for the memorable experience while hanging out in Europe I really enjoyed the sisig you cooked while staying in your flat (still it’s my recipe) Second, to William, thanks for the fun while travelling in Prague and continously telling me to go back to my island Also to Ward, who always have good jokes (well, sometimes), thank you for reminding me to behave like I’m not Thanks to the three of you for making my Greifswald (and Domburg) experience complete See you all next time and let’s go to a church someday  Also to my partner, Maria Yssabella Sinfuego You have been my fuel and inspiration Thank you for all the times you checked my level of insanity and your never ending encouragement and support from the start  To my MENTOR, I couldn’t express the gratitude for all the guidance and patience you gave me during my stay in Germany I have learned so many things, not just in the field of science but also about the many perspectives in life Lastly, thank you for believing and seeing the potential in me when no one else can iv This masterpiece is dedicated to my supportive parents: My mom, Jocelyn Almadrones Reyes, and My dad, Efren Martin Reyes Your unconditional love all throughout my journey of ups and down made me the person I am right now I am blessed that I was raised by the best parents in the world who has been my inspiration to every life decisions I need to make and I will make in the future I hope I can make you both proud on the MAN that I have become! “You can anything you set your mind to” - Eminem “Jesus loves you more than you will know” - King 2k18 v TABLE OF CONTENTS List of Figures List of Tables List of Abbreviations 10 Part I Introduction 11 1.1 Research Rationale 11 1.2 Research Questions and Hypotheses 14 1.2.1 Predicting local habitat suitability in changing climate scenarios 14 1.2.2 Assessing gene flow of Diderma hemisphaericum across regional geographical barrier 15 1.3 Research Objectives 17 1.4 Scope and Limitations 18 1.5 Definition of terms 19 Part II Literature Review 22 2.1 Biogeography of myxomycetes 22 2.2 Species Distribution Modelling (SDM) 25 2.3 DNA Barcoding 28 Part III Methodology 32 3.1 Predicting habitat suitability in changing climate snenario 32 3.1.1 Gathering the distribution records of D hemisphaericum in the Philippines 32 3.1.2 Obtaining current and future (A2 and B1) environmental layers 32 3.1.3 Species Distribution Modelling 33 3.2 Assessing genetic diversity across regional populations 35 3.2.1 Obtaining samples of Diderma hemisphaericum .35 3.2.2 DNA extraction, amplification and sequencincing 35 3.2.3 Quality filtering and sequence alignment 37 3.2.4 Phylogenetic tree and ribotype networking 38 vi 3.2.5 Analysis of the genetic diversity 39 Part IV Results and Discussion 40 4.1 Predicting local habitat suitability in changing climate scenarios 40 4.1.1 Results 40 4.1.2 Discussion 44 4.2 Assessing genetic diversity across regional populations 46 4.2.1 Results 46 4.2.2 Discussion 55 PART V Summary, Conclusion and Recommendation 62 References 67 Appendices 83 vii LIST OF FIGURES Figure 1: Fructification of Diderma hemisphaericum viewed under a compound stereo microscope 13 Figure 2: A diagram that shows major steps for constructing a robust correlative species distribution model 34 Figure 3: Species distribution models for Diderma hemisphaericum showing map of the Philippines and the predictive suitable habitat areas under current conditions and two future changing climate scenarios for the year 2080 43 Figure 4: The rooted consensus tree based on the maximum likelihood algorithm constructed on successfully amplified DNA products from specimens coming from Philippines and Vietnam based on partial 18S rDNA sequences The scale bar indicates the evolutionary distance per site and the black dot indicates the branch Grey triangles indicate the collapsed specimens having the same ribotype assignment 50 Figure 5: The constructed ribotype network constructed using TCS that also showed division into four different clades The circle size indicates the number of sequences represented by the ribotypes The connecting line segments indicate mutational steps between alleles and the small circle in between represent the hypothetical transitional ribotypes 51 Figure 6: Species accumulation curve based on the Chao estimates in (A) Vietnam and (B) Philippines The dark blackline shows the Chao mean and the grey highlight indicates the 5% and 95% limits (C) A smooth species accumulation curve based on Coleman rarefaction 57 LIST OF TABLES Table 1: Number of species occurrence record used for the models, AUC- Values, percentage contribution, permutation importance and training gain (with and without respective variable) for the most contributory predictors 42 Table 2: Summary of the polymorphic site analysis comparing the two countries generated by the DNAsp v.5.1: (i) Philippines and (ii) Vietnam 47 Table 3: AMOVA (Analaysis of molecular variance) displays the computed FST index (0.083), with (0) indicating genetic material sharing and (1) as no sharing 47 Table 4: Correlation using mantel test between the genetic and geographic distance of the samples from Philippines and Vietnam 49 Table 5: List of samples of Diderma hemisphaericum with their corresponding; location, collector, ribotype number, and corresponding coordinates 52 LIST OF ABBREVIATIONS AMOVA Analysis of Molecular Variance BLAST Basic local alignment search tool COI Cytochrome c oxidase DNA Deoxyribonucleic Acid EF1a Elongation factor alpha gene EiE Everything is everywhere GBIF Global Biodiversity Information Facility MAFFT Multiple alignment using fast Forier transform ME Moderate endemicity MEGA Molecular evolutionary genetic analysis PCR Polymerase chain reaction SDM Species distribution modelling SEA Southeast Asia SSU Small subunit TAE Tris base, acetic acid, and EDTA 10 The following picture shows the omission rate and predicted area as a function of the cumulative threshold The omission rate is is calculated both on the training presence records, and (if test data are used) on the test records The next picture is the receiver operating characteristic (ROC) curve for the same data This implies that the maximum achievable AUC is less than 88 The following picture shows the results of the jackknife test of variable importance The environmental variable with highest gain when used in isolation is _bio_4_ph The next picture shows the same jackknife test, using test gain instead of training gain 89 Lastly, the same jackknife test, using AUC on test data These curves show how each environmental variable affects the Maxent prediction The curves show how the predicted probability of presence changes as each environmental variable is varied, keeping all other environmental variables at their average sample value 90 Appendix C MaxEnt results of Diderma hemisphaericum in B1 climate scenario for the year 2080 in the Philippine archipelago MaxEnt representation for D hemisphaericum showing warmer colors with better prediction 91 The following picture shows the omission rate and predicted area as a function of the cumulative threshold The omission rate is is calculated both on the training presence records, and (if test data are used) on the test records The next picture is the receiver operating characteristic (ROC) curve for the same data This implies that the maximum achievable AUC is less than 92 The following picture shows the results of the jackknife test of variable importance The environmental variable with highest gain when used in isolation is _bio_4_phb or temperate seasonality The next picture shows the same jackknife test, using test gain instead of training gain Lastly, the same jackknife test, using AUC on test data 93 These curves show how each environmental variable affects the Maxent prediction The curves show how the predicted probability of presence changes as each environmental variable is varied, keeping all other environmental variables at their average sample value 94 Appendix D Here is the list of 46 coordinates that has been collected based on published literatures where Diderma hemisphaericum has been found in the Philippines SPECIES LONGITUDE LATITUDE D hemisphaericum 120.74 15.2 D hemisphaericum 121.8236111 14 D hemisphaericum 120.9054444 13.48361111 D hemisphaericum 120.05225 15.14686111 D hemisphaericum 122.9516667 14.10527778 D hemisphaericum 124.0955556 12.755 D hemisphaericum 123.4352778 13.44555556 D hemisphaericum 124.1728056 9.813361111 D hemisphaericum 124.0750833 9.66425 D hemisphaericum 123.7527778 9.546891667 D hemisphaericum 121.1158333 13.96027778 D hemisphaericum 121.9838889 14.72222222 D hemisphaericum 124.1728056 9.813361111 D hemisphaericum 123.7513611 9.580555556 D hemisphaericum 121.1158333 13.96027778 D hemisphaericum 121.0416667 13.92083333 D hemisphaericum 121.9555 14.74919444 D hemisphaericum 120.9271944 13.51091667 D hemisphaericum 120.8950556 13.49802778 D hemisphaericum 120.9128889 13.47680556 D hemisphaericum 120.0107778 15.337 D hemisphaericum 119.9793333 15.56552778 D hemisphaericum 120.2817222 15.75388889 D hemisphaericum 122.9638889 14.06083333 D hemisphaericum 123.4272222 13.65138889 D hemisphaericum 123.7402778 13.30388889 D hemisphaericum 123.8822222 12.95333333 D hemisphaericum 124.1713889 9.794138889 D hemisphaericum 123.9650833 9.69725 D hemisphaericum 123.6846948 9.517194 D hemisphaericum 121.1158333 13.96027778 D hemisphaericum 120.9927778 14.11666667 D hemisphaericum 121.0416667 16.28333333 D hemisphaericum 120.2558333 13.77086111 D hemisphaericum 120.15375 13.75905556 D hemisphaericum 120.1511111 13.79033333 D hemisphaericum 121.9631111 14.74036111 D hemisphaericum 121.9544722 14.72883333 D hemisphaericum 121.1 14.9 D hemisphaericum 121.08 14.7 D hemisphaericum 120.9216667 12.80527778 D hemisphaericum 120.3333333 14.8 D hemisphaericum 121.2333333 15.03333333 D hemisphaericum 120.6166667 14.2 D hemisphaericum 119.9688889 16.28333333 D hemisphaericum 120.04 16.18333333 95 Appendix E Here is the full alignment of the 27 different ribotypes of Diderma hemisphaericum that were manually checked using MEGA 96 97 Appendix F The phylogenetic tree of D hemisphaericum that was constructed based on Maximum likelihood algorithm 98 Appendix G Laboratory works in the Institute of Botany and Landscape Ecology in Greifswald, Germany 99 Some photos of the machines the were used in this study; (a) Robot ThermoFlex for DNA extraction (b) UV-Transilluminator for DNA visualization (c) Thermocycler for DNA Amplification (d) Thermofischer Nanodrop for DNA quantification 100 Appendix H Photos of the certificates were received during the stay at the Institute of Botany and Landscape Ecology, Greifswald, Germany 101 102 ... DTN1454290094 A BIOGEOGRAPHICAL CASE STUDY OF DIDERMA HEMISPHAERICUM (MYXOMYCOTA): Predicting local habitat suitability in changing climate scenarios and assessing genetic diversity across Southeast Asian. .. barriers in a clear-cut population of myxomycetes 1.2.1 Predicting local habitat suitability in changing climate scenarios Background: Modeling the species distribution of an organisms are becoming a. .. modeling provides many advantages Like for an instance, in finding populations of a known species (Pearson et al., 2007; Guisan et al., 2006), a study of Wasof et al (2015) revealed that SDM can