THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY JOHN CARLO REDEÑA SANTOS THE FIRST REPORT OF MYXOMYCETES (AMOEBOZOA) IN THAI NGUYEN CITY, NORTHERN VIETNAM: RAPID BIODIVERSITY ASSESSMENT USING TOOLS IN MODERN MOLECULAR ERA BACHELOR THESIS Study Mode: Full-time Major: Environmental Science and Management Faculty: International Programs Office Batch: K45-AEP Thai Nguyen, 20/11/2017 c Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Science in Environmental Science and Management Student name John Carlo Redeña Santos Student ID DTN1454290051 Thesis Title The first report of Myxomycetes (Amoebozoa) in Thai Nguyen City, Northern Vietnam: Rapid biodiversity assessment using tools in modern molecular era Supervisor (s) Dr. Duong Van Thao & Dr. Dagamac Abstract: Myxomycetes are phagotrophic, fungus-like protists abundantly occurring as microbial predators in soil ecosystems. However, in spite of their cosmopolitan distributions, limited studies were conducted in Southeast Asia.
In particular, gaps are found in Northern Vietnam which has no single reported studies that annotates myxomycetes. In order to fill the missing voids, a three-phase rapid biodiversity assessment employing purely moist chamber cultures was carried out in Thai Nguyen City, Northern Vietnam using (i) traditional taxonomic survey, (ii) classical diversity measurement and estimation utilizing sophisticated statistical programming pipelines and (iii) modern molecular technique using DNA barcoding (partial 18s rRNA gene) technology. These efforts showed: (1) agricultural plantations with specified leaf textural morphology are good microhabitat for leaf- litter inhabiting myxomycetes, (2) protected and unprotected forests shares myxomycetes assemblages, (3) anthropogenic activities plays a role in spore dispersal, (4) confirmation that both at community and genetic level, dispersal ecology of myxomycetes are not affected by management strategies or forest type, ii c and (5) 54 species newly recorded for Northern Vietnam, 5 of which are new records for the whole country, with 1 possible species new to science. As such, this study provided the very first baseline information on the biodiversity of myxomycetes for Northern Vietnam.
α and β diversity, barcoding, dispersal, forest management, Keywords slime molds, tropical plants Number of pages 143 pages Date of Submission September 25, 2017 Supervisor‘s signature iii c ACKNOWLEDGEMENT This body of work was made possible by a lot of people who participated in this endeavor in very different but significant ways. Firstly, I would like to acknowledge my whole family for the unconditional support, especially to my parents (Nanay Annie & Tatay Jun), to whom I am beyond grateful as they invested not just money but also time and sweat for my education. To my brothers (PJ, Paulo, Third) and my sister (Darla) in blood, a humongous shout-outs to the four of you. I would also like to include a special note of ―hindi matatawarang pasasalamat‖ to Dr.
Dagamac for not just being an adviser and fostering me for three months, but also for being my mentor in every aspect of life. Without his untiring efforts and never-ending patience, I would not have finished this beautiful piece of work. I would also like to express my gratitude to Prof. Martin Schnittler (University of Greifswald), Prof.
Wilhelm Steingrube (SusEnMan Project, University of Greifswald) and Frau Katharina Schmitt (International Office, University of Greifswald) for the research scholarship, internship and field collection fundings, and to Dr. Duong Van Thao, Frau Anja Klahr (Laboratory coordinator, Allgemeine und Spezielle Botanik), Dr. Manuela Bog, Dr. Hoang Hai Thanh, Dr.
Nguyen Dang Cuong (TUAF), Oriana Sanchez-Mahecha and Nguyen Thuy Linh for the technical assistance. Big thanks to my friends (Nicole, Jose, Hong, Thao, Keraia, Pons, Tina, Colleene, Kenneth, Alex, Mishel, Kathleen, Jamba, Bojo, Tintin, CJ, Edwin) for the personal support, and to my Greifswald friends (Rafa, Kasia, Kemani, Oleg, Tim, Lukas, iv c Franzi, Jinny, Carlos, Brook, Christian, Paul, Sabine, Lorna, Dani, Julia) for keeping me stable during my research and internship in Germany. Lastly, a very special mention to Ikkin, Ward and William for the Pizza party and Jenga session we shared during my stay in Germany. v c TABLE OF CONTENTS List of Figures.
8 List of Tables. 10 List of Abbreviations. Research Questions and Hypotheses. Occurrence of myxomycetes in homogenous vegetation.
α and β diversity in a heterogeneous vegetation. Scope and Limitations. Definition of terms. Natural Classification history of myxomycetes.
Economic and ecological importance. Cutting edge technologies applied in studying the diversity and ecology of myxomycetes. Occurrence of myxomycetes in homogenous vegetation. Collection of substrates and preparation of moist chambers.
Characterization and identification of fruiting bodies. Calculation of productivity and myxomycete occurrence. α and β diversity in a heterogeneous vegetation. Collection of substrates, laboratory isolation method and myxomycetes characterization.
DNA Extraction, amplification and sequencing. Sequence alignment, tree construction and genetic analysis. Results and Discussion. Occurrence of myxomycetes in homogenous vegetation.
α and β diversity in a heterogeneous vegetation. 104 vii c LIST OF FIGURES Figure 1: The study area: Thai Nguyen City, Thai Nguyen Province showing the sampling location of the three agricultural plantations .37 Figure 2: Schematic diagram for the methodology of the first phase of this research study.40 Figure 3: Sampling locations for the two community forests in Thai Nguyen City, Thai Nguyen Province .41 Figure 4: Schematic diagram for the methodology of the second phase of this research study. 46 Figure 5: Schematic diagram for the methodology of the third phase of this research study. 50 Figure 6: The 2D bar graph shows (a) percentages of positive moist chambers between the aerial (AL) and ground leaf (GL) litter substrates in three different agricultural plantations and (b) percentages of positive moist chambers between Superorder Lucisporidia (bright-spored) and Superorder Fuscisporidia (dark-spored) in three different agricultural plantations .51 Figure 7: (a) Rarefaction curves for two different forest type and generated species accumulation curves from Chao 1 estimator for the (b) both community forests; (c) for protected forest; (d) for unprotected forest .58 Figure 8: Box plot showing the comparison of six different diversity indices (Alpha = Fisher‘s alpha; Shannon = Shannon‘s H index; Simpson = Simpson‘s diversity index; N0 = species richness only, N1 = exponent of the Shannon diversity, N2= inverse of the Simpson diversity) .71 8 c Figure 9: Non-metric multidimensional scaling (NMDS) of species occurrence between two community forests.
Black dots represent the position of myxomycetes species in the ordination space. Colored circles represent the forest type; colored ellipses denote dispersion based on standard deviation of point scores .72 Figure 10: Phylogenetic tree of Diderma hemisphaericum: based on partial SSU sequences of the 13 successfully amplified specimens.79 9 c LIST OF TABLES Table 1: Occurrence of myxomycetes: The table shows the list of myxomycetes and their average pH ±, min - max values as measured on every positive moist chamber. A species recorded as rare (1 record) uses only the pH value of the sole moist chamber where it was found. The color on the left side of the species indicates their Superorder (yellow=bright-spored, Superorder Lucisporidia; brown=dark-spored, Superorder Fucisporidia).
The abundance index (AI) in accordance to Stephenson et al. (2013), and their total number of records is further reported on this table. The distribution of records for substrates (AL or GL) and agricultural plants are also enumerated.52 Table 2: Computed values using Analysis of Molecular Variance .80 10 c LIST OF ABBREVIATIONS AL aerial litter AMOVA Analysis of molecular variance BK bark CMC carboxymethyl cellulose DGGE denaturing gradient gel electrophoresis DNA deoxyribonucleic acid GL ground litter HPLC High Performance Liquid Chromatography HTS High-throughput sequencing MAFFT Multiple Alignment using Fast Fourier Transform MC moist chamber NGS Next generation sequencing NMDS Non-metric multidimensional scaling PCR polymerase chain reaction PERMANOVA Permutation analysis of variance SSU Small subunit TRFLP Terminal Restriction Fragment Length Polymorphism TW twigs 11 c PART I. Research Rationale The concept of biodiversity has a long history of usage which can be dated back to the publications of Lovejoy (1980 a,b) where the author defined the term as the number of species present.
However, it was Norse et al. From which the present definition of biological diversity, the variability among living organisms from all sources including, inter alia, terrestrial, marine and other aquatic systems part, was derived. We are aware of the immense potential of various life-forms existing on Earth since we largely depend on biological resources, their diversity and the ecosystem that gives us essential goods and services. Yet, as much as we understand their importance, our knowledge of what they are is still limited.
In fact, only ~1.26 million species of animals and ~300,000 species of plants) out of more than estimated 50 million species of plants, animals and microorganisms have been identified so far. However, compared to plant and animal groups, there is still a large gap to fill for the underexplored microbial world (~28,000 identified species). Assessing the biodiversity of these microorganisms is very important, since they play a vital role in maintaining life on earth such as fixing gases and breaking down dead plant and animal matter into simpler substances that are used at the beginning of the food chain. Fortunately, 12 c intensely known eukaryotic microorganisms that can fulfill such roles are the myxomycetes.
Myxomycetes, also known as plasmodial slime molds, are small group of fungus- like organisms abundant in terrestrial ecosystems, with 1 000 morphological species described worldwide (Lado, 2017). Their life cycle is characterized by two distinct trophic stages (one microscopic and one macroscopic): (1) a uninucleated amoebae, with or without flagella, and (2) a distinctive multinucleated structure called the plasmodium (Everhart & Keller, 2008). They can also occur in any of their three dormant stages namely the (i) spores, (ii) microcysts, and (iii) sclerotium. Moreover, their plasmodial stage can also develop into fruiting bodies carrying haploid spores when the environmental condition and food resources become unfavorable.
These fruiting bodies exhibit delicate structures and colors that serve as their diagnostic character for identification (Schnittler & Mitchell, 2000). This species concept popularly used for myxomycetes diversity assessments is known as the morphological species concept. However, during the last recent years, the molecular age for myxomycetes developed rapidly wherein fruiting bodies are subjected to DNA barcoding for identification at the genetic level. Moreover, molecular studies paved the way to confirm the biological species concept (Clark & Haskins, 2013) for myxomycetes that involved reproductively isolated units (Feng et al., 2016; Shchepin et al.
2016; Dagamac et al. In terms of their ecological importance, these organisms play an active role in the soil biota as ―microbial predators‖ as they consume 13 c bacterial cells along with yeasts and other fungi associated with decomposing plant tissues (Ing, 1994; Keller et al. Whereas, these microbial predators serves a huge part in both maintaining the ecosystem balance and in nutrient cycling (Urich et al., 2008; Stephenson et al. However, in spite of their fascinating mechanisms, myxomycetes are still underexplored in many parts of the world especially in the Southeast Asian region.
Although known with rich biodiversity, Southeast Asia is still an understudied area for myxomycetes diversity. Some of the Southeast Asian countries that produced myxomycetes species records includes Philippines with 158 species (Dagamac & dela Cruz, 2015; Macabago et al., 2017), Thailand with 145 (Ko Ko et al., 2010; Dagamac et al., 2017b), 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., 2013a), and Laos with 44 (Ko Ko et al. In similarity, few myxomycetes studies have been conducted in Vietnam. The first myxomycete report in the country came from Van Hoof (2009) that initially annotated 23 species where one species, Cribraria tecta, is reported as a species new for science.
Additionally, two new species of Diderma namely, Diderma cattiense and Diderma pseudotestaceum were reported by Novozhilov et al. This is then followed by an ecological study in three lowland tropical forests in Vietnam by Tran et al. Recently, the most comprehensive study ever conducted for the country was the biodiversity assessment study of myxomycetes conducted in Southern Vietnam by Novozhilov et al.