SHOULD i STAY OR SHOULD i GO insinuation of spore dispersal in local medata of myxomycetes assemblages from laguna, philippines using models for community ecology

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY JOHN LORENZO M BERNARDO SHOULD I STAY OR SHOULD I GO?: INSINUATION OF SPORE DISPERSAL IN LOCAL METADATA OF MYXOMYCETES ASSEMBLAGES FROM LAGUNA, PHILIPPINES USING MODELS FOR COMMUNITY ECOLOGY BACHELOR THESIS Study Mode: Major: Faculty: Batch: Full-time Environmental Science and Management International Programs Office 2015-2018 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 John Lorenzo M Bernardo DTN1454290110 Thesis Title SHOULD I STAY OR SHOULD I GO?: Insinuation of Spore dispersal in local medata of myxomycetes assemblages from Laguna, Philippines using models for Community Ecology Supervisor (s) Dr Do Ngoc Oanh & Dr rer.nat Nikki Heherson A Dagamac Supervisors’ signature Abstract: In spite of the increasing number of myxomycetes studies in the tropical Southeast Asia over the last decades, many forest patches are still left unexplored for the region, in particular in the Philippine archipelago Thus, an assessment of myxomycetes diversity, occurrence, and composition was carried out in forest fragments from two municipalities of the province of Laguna, Philippines From the 12 established 5x5 m plots, a total of 240 moist chamber cultures was set-up for this study and yielded 42 myxomycetes species belonging to 14 genera Rarefaction curves and several heterogeneity indices revealed higher species diversity in Los Baños than in Calauan Moreover, the taxonomic diversity index also showed that the taxonomic diversity of myxomycetes in Los Baños is more intuitively diverse than in Calauan In terms of community analysis between the two municipalities, it showed that myxomycetes communities clustered between different microhabitats ii High values of Coefficient of Community and Percentage Similarity indices indicates that spore dispersal in forest fragments in closer proximities may cause high number of shared species (60%) With an addition of Cribraria lepida as a new record for the country, this study now updates the myxomycetes profile for Laguna, Philippines Fisher’s alpha, intermediate disturbance hypothesis, microbial Keywords: biodiversity, plasmodial slime moulds, S/G ratio, species richness Number of pages: 102 pages Date of Submission: November 15, 2018 iii ACKNOWLEDGEMENT First of all, I would like to thank the almighty God for giving me the opportunity and strength to finish this work Proverbs 9:10 “The fear of the Lord, is the beginning of wisdom.” I would also like to extend my deepest gratitude to my mentor/adviser, Dr Nikki Heherson A Dagamac (Hedwig for short), thank you sir for the patience, support, encouragement, words of wisdom, learnings, and of course for the laughters and corny jokes we shared I won’t be able to finish this work without your unending help Just like I told you, I admire you not just for being a very passionate person, but also because of your values and characteristics Because of who you are as a person Thank you again sir Also, to Dr Do Ngoc Oanh, thank you ma’am for the comments and honest suggestions, and I would also like to extend my deepest gratitude to the AEP office and University of Greifswald To my family, Anicio Bernardo, Aurea Bernardo, Gio Coronado and Mary Coronado, you guys are my source of strength and happiness, I love you all Thank you for giving me everything that you can, you guys are one of the many reasons why I have given so much for this work You all sacrificed so much, so this one is for all of you To M&M’s/Blue rats (Luis, Lester, Vea, Pau, Jessica, Joy, AJ, France, and Kristina) thank you guys for the never ending support! My thesis mates, Yani and King, it was awesome working with you guys, thank you for the help, from the substrate collection to writing our publication Cheers! to all the memories we shared and failed experiments we faced iv To the working group, Oleg, Professor Martin, Jan, Anja, Manuela, Linh, Orianna and Paul, thank you guys for the warm welcome and help, hoping to see you guys again Of course to my Greifswald friends, Lorna, Virna, Kashia, Morvarid, Nadine, William, and Ward, thank you all for the bbq parties, movie nights, and sincere friendship You guys made my journey in Germany truly unforgettable To Professor Steingrube, thank you professor for the financial support you have granted to us, I am extremely grateful To our new born baby, John Fender, you’re a wonderful gift from God, let this work be a warning of what college life is To all the people involved in this work, thank you so much Remember to believe in yourself, and that you can impossible things The world has so much to offer, learn to look at the bigger picture Life is short, learn to value it Learn to see kindness to all the people you’ll meet and know that you have a bigger God #allforHisglory #calauanlosbanosmyxo #bepassionate John Lorenzo M Bernardo Researcher 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 1.3 Research Objectives 16 1.4 Scope and Limitations 17 1.5 Definition of terms 18 Part II Literature Review 23 2.1 Myxomycetes Life Strategies 23 2.2 Taxonomy of Myxomycetes 25 2.3 Ecological Distribution of Myxomycetes 27 2.4 2.5 Roles of Myxomycetes in Nature 30 Advantages and Disadvantages of Classical Method in Myxomycetes Research 32 Modern Molecular Methods 35 2.6 Part III Methodology 40 3.1 Phase 1: Patterns of myxomycetes communities in two disturbed forest fragments of Laguna Philippines using “fructification only” data 40 3.1.1 3.1.2 3.1.3 3.1.4 3.2 Study Sites 40 Substrate Collection 41 Characterization and Identification of Fruiting Bodies 41 Data Evaluation 42 Phase 2: Plasmodia and sclerotia identification and storage experiment study on plasmodia 45 3.2.1 DNA Isolation 45 3.2.2 Sequence-based Identification 47 vi Part IV Results and Discussion 48 4.1 Phase 1: Patterns of myxomycetes communities in two disturbed forest fragments of Laguna Philippines using “fructification only” data 48 4.1.1 Results 48 4.1.2 Discussion 56 4.2 Phase 2: Plasmodia and sclerotia identification and storage experiment study on plasmodia 62 4.2.1 Results 62 4.2.2 Discussion 63 PART V Conclusion 68 References 71 Appendices 84 vii LIST OF FIGURES Figure 1: Map of the study area showing the two municipalities of Laguna 40 Figure 2: (a) Rarefaction curve for the two municipalities and the generated sample based, species accumulation curve based from Chao estimator for (b) Los Baños and (c) Calauan The thick line shows the Chao 1-mean, and the thin-dotted lines shows the 5% and 95% confidence interval 48 Figure 3: Species abundance distribution curve following the Zipf-Mandlebrot model, showing the ranked-abundant species (x-axis) and the relative abundance (y-axis) 51 Figure 4: Boxplot showing the three different species diversity indices between the two municpalities 52 Figure 5: Clustering analysis using Bray Curtis index 53 Figure 6: Bar chart displaying the percentage occurrence of each species in both municipalities 54 Figure 7: Venn diagram showing the distribution of the 39 myxomycetes species reported in this study and the values of the coefficient of community and percentage similarity indices between the two municipalities 55 LIST OF TABLES Table 1: The table exhibits the alphabetically arranged list of myxomycete species found on moist chamber cultures The average pH± and min.-max were also measured from every positive moist chambers, but for MCs that yielded record (rare) of fruting bodies, pH value was recorded only The abundance index (AI) in accordance to Stephenson et al., (2011), total number of records and its breakdown for each municipality, total number of species, and total number of genera were further reported in this table The taxonomic diversity index (TDI) was calculated as the ratio of the number of species to the number of genera (S/G) 49-50 Table 2: Description of the sequences using the specific primer pair for dark-spored species of myxomycetes from the plasmodia (Pls) and sclerotia isolated from moist chamber cultures Moist chamber code and municipality means the specific culture and location where these stages were harvested and collected The storage shows plasmodia that are stored with RNAlater® The species assignment and the % similarity tells the identity and similarity from the BLAST hit in NCBI of the successful sequences 62-63 LIST OF ABBREVIATIONS AL aerial litter BK bark BLAST Basic Local Alignment Search Tool CC Coefficient of Community DGGE denaturing gradient gel electrophoresis DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate GL ground litter HTS High-throughput sequencing NCBI National Center for Biotechnology Information MC moist chamber PCR polymerase chain reaction PS Perecentage Similarity RNA ribonucleic acid SAC species accumulation curve SAD species abundance distribution SSU Small subunit TAE Tris-Acetate-EDTA TRFLP Terminal Restriction Fragment Length Polymorphism TW twigs 10 Appendix D Example photos of the initial identification of myxomycetes 88 Appendix E Example photos of fruting body: (1) Perichaena depressa (2) Arcyria denudata (3) Comatricha tenerrima (4) Physarum bitectum (5) Stemonitis fusca (6) Physarum oblatum (7) Didymium squamulosum (8) Physarum album (9) Hemitrichia serpula 89 Appendix F Sample machines and tools used for molecular works Centrifuge Analytical Balance PCR machine Vortex Incubator 90 Electrophoresis Biostep: for Gel documentation Centrifuge 91 Mortar and pestel HiBind Pipette Tips RNAlater solution and plasmodia in microtube 92 Appendix G Chemicals and kit used for molecular identification of plasmodia and sclerotia 93 S1 dNTPs SU19 94 Appendix H Photos of laboratory works for plasmodial and sclerotial identification, and storage experiment 95 Appendix I Photos of plasmodia alignment, all plasmodia matched the species Physarum melleum Plasmodia (SSUE6) 96 Plasmodia (SSUE24) 97 Plasmodia (SSU41) 98 Appendix J Example R script used in R program for models in Community Ecology Sys.setenv(LANG = "en") require(vegan) require(mvabund) library(hydroTSM) require(labdsv) require(e1071) require(nortest) require(MASS) require(lattice) require(stats) library(akima) library(car) library(BiodiversityR) library(simba) library(Biostrings) library(seqinr) library(RFLPtools) library(picante) library(indicspecies) require(mgcv) require(PCNM) require(cluster) require(plotrix) require(reshape) #####also set your working directory(it means, where to spec and env file!!!) setwd("/Users/Nikki/Desktop/Myxo_Enzo/R") ##### making some colours brown.transp