Tài liệu 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 do 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 ...................................................................................................................... 8 List of Tables ....................................................................................................................... 9 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 1 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 8 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 1 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 9 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 PART I. INTRODUCTION 1.1. Research Rationale General patterns of community structure for terrestrial macroorganisms are relatively well known, but, similar information on microorganisms remains fragmented and limited mainly due to their small size, unique life cycle, and dispersal capacity (Martiny et al., 2006; Novozhilov et al., 2017). Fortunately, the mathematical method of community ecology can be applied in myxomycetes using the morphological species concept. Furthermore, it was only in the 1980s when the first comprehensive ecological studies were carried out, analyzing the occurrence of fruiting bodies in relation to environmental gradient, which includes the first studies of niche breadth and niche overlap (Rojas et al., 2008; Schnittler, 2001). Approximately only 60% of all myxomycetes morphospecies can be detected in the field, while the remaining percentage needs moist chamber culture technique and examination using microscope. But recently, the ecological studies of myxomycetes were limited by the detection and identification of the fruiting bodies, since, trophic stages of this species lacks the characteristics for intial identification. With that, the challenge now is to determine the factors that constrain species distribution. Ecological niches was applied as the main theoretical concept for myxomycetes distribution studies (Broennimann et al., 2012; Soberón, 2007) using habitat and microhabitat as its describing parameters. Even though macroclimate and habitat limits the myxomycetes distribution, evidences have occurred supporting the idea that microhabitat availability strongly affects the species distribution (Rojas et al., 2014). 11 Myxomycetes also known as plasmodial slime moulds or true slime moulds are phagotrophic eukaryotes, abundantly thriving in terrestrial ecosystem (Dagamac et al., 2010; Dagamac et al., 2015b; Kuhn et al., 2013; Massingill & Stephenson, 2013). Morphologically, there are ca. 1000 species known and describe worldwide (Lado 20052018). They are usually reported in many forested regions of the world, especially in areas with mass of decaying logs, twigs, and leaf litter (Dagamac & dela Cruz, 2015). Their placement in the tree of life became controversial due to their complex life cycle. They were once classified in Kingdom Animalia, Kingdom Plantae, and Kingdom Fungi because of the seemingly high similarity of characteristics. However, based on the molecular evidences reported during the recent studies they were classified in the Kingdom Protista (Amoebozoans). In terms of their environmental role, they are neither pathogens nor decomposers but rather as “microbial predators”, consuming many microorganisms in their surrounding (Keller et al., 2008; Corpuz et al., 2012). Hence, they are considered to play an important role in maintaining the balance in soil ecosystem (Feest 1987; Foissner 1999; Stephenson et al., 2011), soil biochemistry and biology. Furthermore, insects also depend on slime moulds for food (Keller & Snell 2002). However, despite its relevance to nature, studies of myxomycetes biodiversity is still limited and unexplored especially in the Southeast Asian Paleotropics where higher species diversity can be expected (Dagamac et al., 2014). The Philippines is considered to be one of the world’s most biologically rich tropical countries. Even though it is taking the lead on myxomycetes studies among the 12 Southeast Asian countries, it is still limited and remain unexplored (Dagamac et al., 2012; Rea-Maminta et al., 2015). Over the last decades, there was a significant increase on studies about myxomycetes of the Philippines. The earliest study was dated way back 1973, when Uyenco (1973) claimed to have published the first report on Philippine myxomycetes. Here, she has successfully collected 314 species of myxomycetes from Luzon (Quezon City and Laguna) and Mindanao (Basilan and Zamboanga). But according to Dagamac & dela Cruz (2015), Dogma (1975) has stated that Martin and Alexopoulos (1969) have already credited the Philippines with 22 species in their book “The Myxomycetes”. However, it was Don Reynolds (1981) who has successfully made the most remarkable findings of myxomycetes in the Philippines, after he reported a total of 107 species collected from Mindanao (Davao, Cotabato, and Zamboanga) by E.B. Copeland; from Luzon (Benguet) by A.B.E. Elemer; and Bataan, Manila, Cavite, and Laguna by E.P. Merill. Since then, surveys and publications about myxomycetes have remained stagnant (Dagamac & dela Cruz, 2015) for nearly 30 years. Apparently, Moreno et al., (2009) identified new species (Craterium retisporum) in Anda. Furthermore, five new species were found by Dagamac et al, (2012) during his studies in Mount Arayat National Park (see Dagamac et al., 2014). Another, two additional records by dela Cruz et al., (2011) were added in the records of Philippine myxomycetes. In addition to these, another seven new records were added (see Macabago et al., 2012) from Lubang Island, three new records from Hundred Island, Pangasinan (see Kuhn, 2013), and another 17 new records by dela Cruz et al. (2014) from Bataan, Cavite, and Subic. Moreover, Bicol Peninsula was successfully assessed and produced 8 new records 13 (see Dagamac et al., 2015c). Recently, Macabago et al. 2017 has recorded 8 new more species during their collection in the islands of Bohol, increasing the number of records for the Philippines to a total of 158. Thus, the Philippines has outnumbered data from areas in other Paleotropic Southeast Asia, like Thailand with 145 taxa (Ko Ko et al., 2010; Dagamac & dela Cruz, 2015), Indonesia with 119 (Farr, 1990; Rosing et al., 2011), Republic of Singapore with 76 (Rosing et al., 2011), Myanmar with 67 (Ko Ko et al., 2013), Laos with 44 (Ko Ko et al., 2013) and Vietnam with 131 (Redeña Santos et al., 2018). 1.2. Research Questions and Hypotheses The major goal of this research study is to establish a comparative assessment of myxomycete diversity and distribution between two municipalities having strongly exposed and openly disturbed forest patches. To achieve such goal, a two-fold research phases was designed for the whole study. 1.2.1. Phase 1: Patterns of myxomycetes communities in two disturbed forest fragments of Laguna Philippines using “fructification only” data Background: The two municipalities in Laguna (Calauan and Los Banos) have been exposed to many fragmented forests and deforestation due to the increasing urbanization of the province. This has been the major challenge not just for the locals, but most especially for researchers, as habitats tend to be highly affected, resulting to the degradation of both macroorganisms and microorganisms. However, despite the high probability of organisms’ existence in the two municipalities, low number of studies had 14 been conducted, especially for the municipality of Calauan wherein no records of myxomycete diversity studies have ever been conducted. Thus, the first phase of this study intends to address the following question: Question: How different are the myxomycete communities between Los Baños and Calauan? Hypothesis: Prima facie null hypothesis that the communities between the disturbed fragments are the same due to the capability of the spores to disperse via long distance. 1.2.2. Phase 2: Plasmodia and sclerotia identification and storage experiment study on plasmodia Background: Ever since the myxoycetes studies started, only fruting body was used in the analysis of data, neglecting its plasmodial and sclerotial stages. This is due to the fact that there are no visible morphological characteristics that can be used to assign them any specific species name. This makes many assessments heavily bias on fruiting bodies only. Moreover, it is such a difficulty to store plasmodia for a long period of time especially if they are in the moist chamber cultures. This is because, they easily become a sclerotia (resting stage) or they can ultimately disappear in the cultures without any trace. To address this common challenge in many tropical moist chambers, it is necessary to test methods of storage. Thus, the second phase of this research study wants to address the following questions: (i) Can the harvested plasmodia and sclerotia be successfully amplified, sequenced, and identified? 15 (ii) Can the commercially produced RNAlater® solution, used to store animal tissues be effectively used to store plasmodia that are found on the moist chamber cultures? Hypothesis: A new method of storage is successfully developed for plasmodial preservation that would guarantee good quality of DNA for future molecular studies. 1.2. Research Objectives Since this research study has two phases, this part is divided into two components: The Phase 1 of this research study specifically aims to: • harvest the fruiting bodies and transfer to Herbarium boxes • characterize and identify the occurring fruiting bodies on moist chamber cultures • estimate the myxomycetes species between the two municipalities by generating species accumulation curve • measure the taxonomic diversity, species richness and community similarities between the two localities The Phase 2 of this research study specifically aims to: • collect active plasmodia directly on moist chamber cultures • asceptically transfer the plasmodia to RNAlater® • test the effectivity of the long storage (2 weeks) experiment by subjecting them to column based DNA isolation and amplification 16 1.3. Scope and Limitations This study was divided in two phases in order to address the question/s and test several hypotheses raised in this study. The first phase of this study was mainly conducted to investigate (i) the variation of myxomycete species in two different municipalities of Laguna, Philippines, and (ii) the species richness and diversity using models for community ecology between the two sites having different community structure, one being managed yielding diverse plant species that are inmost of the cases, creating closed canopies and the other one being exposed to heavily grazed human activities making the vegetation subjected to lesser plant diversity and open understory forest types. Meanwhile, the Phase 2 of this study was constructed for two different reasons, it wanted (i) to assess first the identification of myxomycete species using its plasmodial and sclerotial stages, and (ii) to test the effectivity of RNAlater® solution as a storage compound of plasmodia. The whole study was conducted from January 2018 to July 2018. The collection of substrates was carried out in the month of January 2018 in two municipalities of Laguna, Philippines. Then, the process of setting up the myxomycetes moist chamber culture, molecular methods, and data analysis was carried out at the Institute of Botany and Landscape Ecology, University of Greifswald in Greifswald, Germany from February 2018 to July 2018. Even though this research has reached its goals, there were still some unavoidable limitations for the study. For Phase 1, (1) with the amount of time and money provided for this study, the data was acquired only from the moist chamber cultures, and no 17 fruiting body was collected, (2) there was no comparison of species richness, diversity, and taxonomic diversity between the 4 substrates (AL, GL, TW, and BK), since the study focused more on community structure and not on the microhabitat. For the second phase, (3) only 12 samples of plasmodia and sclerotia were subjected to the experiment, because other samples either disappear or shifted to another stage, (4) no phylogenetic tree was constructed due to lack of samples, (5) samples that resulted to negative bands were not subjected to primers for bright-spored species, because of the expenses for molecular materials and components, and (6) the biodiversity assessment of myxomycetes was carried out rapidly at a local-scale. 1.5. Definition of terms Listed below are unfamiliar terms essential for this study, to better understand the goals and purposes of this research. Basic Local Alignment Search Tool (BLAST) refers to a program that finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Chromatograms pertains to a visible record (such as a graph) showing the result of separating the components of a mixture by chromatography. 18 Closed canopy forest pertains to a dense growth of trees in which the top branches and leaves form a ceiling, or canopy, that light can barely penetrate to reach the forest floor. Community represents the population of all species living and interacting in an area at a particular time Dispersal/Biological dispersal, in biology, the dissemination, or scattering, of organisms over periods within a given area or over the Earth, unlike migration, it is defined as the movement of individuals and not of group. DNA amplification pertains to a process that increase the freuquency of replication of a DNA segment using universal or specific primers such as the combination of forward and reverse primers. DNA sequencing means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. This sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. Forest structure is the horizontal and vertical distribution of layers in a forest including the trees, shrubs, and ground cover (which includes vegetation and dead and down woody material). Forward primer is a primer that attaches to one side of the strand in the DNA during the PCR process. Together with the reverse primer, this makes the DNA doubles strand again. 19 Fruiting body refers to a macroscopic reproductive structure produced by some organisms such as fungi (for example, mushrooms) and some bacteria (for example, myxobacteria). They are are distinct in size, shape, and colouration for each species, and produce spores. Genus/Genera is a principal taxonomic category that ranks above species and below family. Metadata refers to set of data that describes and gives information about other data Microhabitat refers to a habitat that is of small or limited extent Morphospecies is a group of biological organisms that differs in some morphological respect from all other groups. Moist chamber pertains to a container that can hold a high humidity atmosphere within itself for a long period of time and keeps a specimen moist. Municipality refers to a city or town that has corporate status and local government (Los Baños and Calauan) Open canopies pertains to collection of tall trees that have not grown together to shield the sun away from the vegetation below. Petri dish refers to a shallow, circular, transparent dish with a flat lid, used for the culture of microorganisms. 20