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

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.. With an addition of Cribr

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

Thai Nguyen, (15/09/2018)

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Science in Environmental Science and

Management Student name John Lorenzo M Bernardo

Student ID 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

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

Keywords:

Fisher’s alpha, intermediate disturbance hypothesis, microbial biodiversity, plasmodial slime moulds, S/G ratio, species richness

Number of pages: 102 pages

Date of

Submission:

November 15, 2018

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

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

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 Roles of Myxomycetes in Nature 30

2.5 Advantages and Disadvantages of Classical Method in Myxomycetes Research 32

2.6 Modern Molecular Methods 35

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 Study Sites 40

3.1.2 Substrate Collection 41

3.1.3 Characterization and Identification of Fruiting Bodies 41

3.1.4 Data Evaluation 42

3.2 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

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

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

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

DGGE denaturing gradient gel electrophoresis

dNTP deoxynucleotide triphosphate

HTS High-throughput sequencing NCBI National Center for Biotechnology

PCR polymerase chain reaction

SAC species accumulation curve SAD species abundance distribution

TRFLP Terminal Restriction Fragment Length

Polymorphism

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)

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 2005-2018) 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

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

(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

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

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?

(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

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 ofRNAlater® 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

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

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

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

Plasmodia is a colorless or brightly colored vegetative body, non-cellular,

multinucleate, jellylike, amoeboid, and assimilative stage of the myxomycetes

Polymerase Chain Reaction (PCR) is the process that splits the DNA into single

strand by the action of heat and primers

Rarefaction is a technique that allows the calculation of species richness for a

given number of individual samples, based on the construction of so-called rarefaction curves

Rarefaction curve is a plot of the number of species as a function of the number

of samples

Reverse primer pertains to primer during the PCR process that attach to one side

of DNA, thus with the help forward primer makes the DNA double strand again

RNAlater® is a trade name for an aqueous, non-toxic tissue storage reagent that

rapidly permeates tissues to stabilize and protect cellular RNA

Species accumulation curve is a graph recording the cumulative number of

species of living things recorded in a particular environment as a function of the cumulative effort expended searching for them

Species composition refers to the species that occur on a site or in a successional

stage of a plant community

Species diversity pertains as the number of species and abundance of each species

that live in a particular location

Substrate refers to the surface or material on or from which an organism lives,

grows, or obtains its nourishment

PART II LITERATURE REVIEW

2.1 Myxomycetes Life Strategies

The complex yet fascinating life cycle of myxomycetes (Keller & Schoknecht, 1989) make them a perplexing group of biological organisms Their life cycle usually starts with many dispersed spores in the environment that would eventually germinate an amoeba This amoeba will then transform based on the conditions of the environment where they landed In terrestrial environment these amoebas, usually become swarm cells

or myxamoebas The myxamoeba is capable of undergoing multiple cell divisions through mitosis called binary fusion while the swarm cells do not undergo such cell division (Everhart & Keller, 2008; Stephenson & Stempen, 1994; Fiore-Donno et al., 2005) On the other hand, amoebas that landed on aquatic environments usually develop flagella to help them propel on such wet conditions (Clark & Haskins, 2016) Either the amoeboflagellated cells or the swarm cells turns to its resting stage called the microcyst once their conditions become unfavourable i.e lack of food, overcrowding, accumulation of toxic metabolic byproducts, drought or drier conditions, wide temperature fluctuations, or too much water

Sexual reproduction of myxomycetes then begins when two compatible mating haploid amoebal types fused by random chance that would eventually form a diploid zygote (Gray and Alexopolous, 1968; Everhart & Keller, 2008; see review by Clark & Haskins, 2013) The formed zygote will then undergo a period of development and through chemical signalling will form a multinucleated network of giant cell mass

capable of cytoplasmic streaming called the plasmodia (Everhart & Keller, 2008; Clark & Haskins, 2016) This giant cell mass then starts to be the animal-like feature in its whole life cycle as it move across different substrate to prey on microorganisms (Gray & Alexopoulos, 1968; Dagamac & dela Cruz, 2015) This predatory nature of the plasmodium makes them a good limiting factor that maintains the balance of soil ecosystems (Feest, 1987; Foissner, 1999; Dagamac & dela Cruz, 2015)

There are three morphological types of plasmodia: phaneroplasmodium, aphanoplasmodium, protoplasmodium (Gray & Alexopoulos, 1968; Keller & Braun, 1999; Keller et al., 2008), and an intermediate type called the trichiaceous plasmodium (Gray & Alexopolous, 1968; Keller 1971) First, the phaneroplasmodium usually shapes into a fan-like plasmodium which forms hundreds of fruiting bodies or one large aethalium up to 70 cm (Keller & Braun, 1999) Second, the aphanoplasmodium needs free water to form a vein-like reticulate pattern that lacks distinct antero-posterior polarity (Keller et al., 2008) Then, the protoplasmodium exhibits the most irregular shape among the types, and unlike other types it does not develop a vein-like network characteristic and produce only a single stalked sporangium Lastly, trichiaceous plasmodium exhibits the merging morphological characteristics of both aphano- and phanero- plasmodium

which requires free water, yet also shows an antero-posterior polarity, Perichaena depressa and P quadrata are examples of this type (Keller et al., 2008)

Likewise, plasmodia embodies the capability to be in a dormant stage under harsh, stress, strains and unfavorable condition, called the sclerotia, but can also return to active

plasmodia once the environment becomes favorable again (Krzywda et al., 2008; Martin

& Alexopoulos, 1969) Interestingly, plasmodia can also decide not to go dormant but rather shift into a more stable reproductive strategy by transforming into fungus-like fructification that would again develop the haploid spores through a reductive division called meiosis (Rollins & Stephenson, 2011)

The fruiting bodies are what makes the myxomycetes appear to be like fungi, because besides being found on similar habitats where fungi are usually thriving (Everhart & Keller, 2008; Dagamac et al., 2015a), myxomycetes also follow its dispersal ecology Fruiting bodies may vary in different forms which can either be sessile or stalked sporangium that has definite size, shape, or color (Keller & Everhart, 2006, 2010) These limited characters are the commonly used diagnostics to determine a particular myxomycete species Moreover, in spite of having distinct morphological features, comprehensive microscopic examinations should be done in order to examine other micro-morphological structures like the spores and capillitium for further identification of myxomycetes species

2.2 Taxonomy of the myxomycetes

Carl Linnaeus (1763) first classified myxomycetes as gasteromycetes fungi because of the miniature puffball appearance of Lycogala (Keller, 2012) However, it was Heinrich Anton de Bary (1831–88) the man behind the first demonstration of spore germination as well as the development of other life cycle that removed the myxomycetes from the fungal gasteromycete and coined the name Mycetozoa (Martin 1958; Everhart &

Keller 2008) Furthermore, it was classified in the Kingdom Plantae (Class Myxomycota)

as well as in Kingdom Animalia belonging to Class Mycetozoa (Martin & Alexopoulos, 1969)

However, since myxomycetes are usually found in habitat that is similar to fungi (Keller & Braun, 1999), mycetozoa were then proposed to be classified in Kingdom Fungi (Class Myxomycetes) and therefore later on coined as Myxomycetes However, according to Fiore-Donno et al., 2010 the current genetic tool that uses the analysis of the 18S rDNA gene already indicated that the group does not belong to the fungal grouping, instead it forms a more monophyletic clade in the Kingdom Protista and are classified more closely to the division Amoebozoa This is supported by the first phylogenies of the group that was published during the last decade (Baldauf & Doolittle, 1997)

In traditional sense, there are five orders that are commonly applied for myxomycetes taxonomy These orders are divided into two supergroups: the Lucisporidia

or the so called bright-spored myxomycetes and the Fucisporidia or the so-called spored myxomycetes Belonging to the supergroup Lucisporidia are the order Trichiales and the order Liceales, and so far this order has the least information among other orders

dark-as supported by (Fiore-Donno et al., 2009, 2013) Supergroup Fucisporidia hdark-as the three other orders namely the Stemonitales, the Physarales and the Echinosteliales The first order is characterized to have longer fruiting bodies but its distinguishing feature is the presence of the columella or the extended structure of the stalk Physarales is the largest order and is easily recognized by having the presence of lime node crystals surrounding

its sporocarp Echinosteliales are defined by light, rarely dark colored spores produced in

a minute sporocarp usually having a stipe, columella and capillitium (Clark & Haskins, 2014)

2.3 Ecological distribution of myxomycetes

The understanding of ecological dispersal and speciation pattern is the way to understand species distribution and diversity patterns (Schnittler et al., 2017) In the case

of the myxomcyetes, only a portion of its life cycle, the fruiting body, can be analyzed due to its morphological features that can be used to identify them (Lister & Lister, 1925; Martin & Alexopoulos, 1969; Schnittler & Mitchell, 2000; Keller & Everhart, 2008, 2010; Stephenson, 2011; Schnittler et al., 2012;) Furthermore, these fruiting bodies contain numerous spores that can disperse through wind and germinate eventually Thus, spores is the key factor for myxomcyetes distribution (Kuhn et al., 2013) But, some factors constrains the dispersal of these spores, (i) spore size, applying the Stoke’s law, perhaps, smaller spores have higher dispersal abilities compare to larger spores, (ii) the starting point of spores, fruiting bodies with longer stalk, tends to reach farther distance and (iii) the spore ornamentation, classified into three different ornamentation types: spiny, reticulate and smooth surfaces; spores with spines half-sink into the water surface, but they eventually float on the surface While, reticulate spores show super-hydrophobic effects and are refractive to the water surface Spores without ornamentation sink after contact with water (Hoppe & Schwippert, 2014)

In addition to these, animals’ feeding behavior and environmental factors like pH, water, and moisture can also constrain spores dispersal (Novozhilov et al., 2006) Although spores can be dispersed across the globe, myxomcyetes exhibit a biogeographical pattern Globally, climate and vegetation highly attributes the distribution pattern, while, locally, ecological differences attributes the distribution Current evidence shows that myxomcyetes do not follow the conventional “species richness increases with the decreasing latitude” gradient that is the case for many other organisms (Rollins & Stephenson, 2011) Despite the fact that myxomcyetes occurs in many places around the world, ecological studies were mostly carried out in temperate regions such as Southwestern Virginia (Stephenson, 1989) and in Everglades National Park, Florida (Keller, 1973) both in the United States, and in the Lower Volga River Basin in Russia (Novozhilov et al., 2006), while tropic regions remain understudied (Rojas & Stephenson, 2007; Kuhn et al., 2013), in fact, few studies were carried out in Neotropic areas (Schnittler et al., 2002; Lado et al., 2003; Rojas et al., 2010) and few more for Paleotropic areas (Alfaro et al., 2015; Dagamac et al., 2010, 2011, 2012, 2014, 2017a; Macabago et al., 2010, 2012, 2016 ; Novozhilov et al., 2017; Rea-Maminta et al., 2015; Tran et al., 2006, 2008 )

Furthermore, temperate regions cover a wide variety of forest ecosystems Like for example the case of temperate deciduous forests that support the most diverse and abundant myxomycetes communities in the world (Ing, 1994; Novozhilov et al., 2017; Stephenson et al., 2001; Takahashi, 2004) For instance is the temperate forests of the

Great Smoky Mountain National Park, being one of the most diverse assemblages of myxomycetes in the world (Stephenson et al., 2001) This diversity is perhaps caused by wide variation in elevation and topography, giving rise to numerous different types of plant communities that provide many ecological settings as well as a diversity of suitable substrate for myxomycetes (Rollins & Stephenson, 2011) However, despite this diversity, species of myxomycetes in temperate forests are not uniformly distributed For instance, the sharp contrast between the assemblages of myxomycetes associated with high elevation coniferous forests and the surrounding hard wood forests found at lower elevations within the temperate forests zone (Stephenson, 2004) In general, both species diversity and abundance tend to acquire greatest values near the wetter end of the moisture gradient (Rollins & Stephenson, 2011; Schnittler et al., 2017)

Another type of forest where myxomycetes most likely loves to thrive are the areas in the tropical ecozones Stephenson et al., (2004) have summarized information of Neotropical Myxomycetes, suggested that three major trends seem to exist, (i) compare to temperate forests, myxomycetes species richness and abundance appear to be lower in tropical forests, (ii) species abundance and richness is inversely proportional to moisture,

as the moisture increases, abundance and richness decreases (Schnittler & Stephenson,

2000), (iii) species such as A bombarda and C sphaerosperma requires high temperature

for development, limited to tropical climates Hence, microhabitats with no equivalents in temperate regions support assemblages of myxomycetes Furthermore, compared to other areas in Paleotropic, better studies were carried out in Southern Asian Paleotropics

(Dagamac et al., 2017a; Estrada-Torres et al., 2009) For tropical region, moist chamber cultures provide less species diversity and fruiting bodies per species found compared to the same microhabitat from temperate regions that obtains opposite results (Stephenson et al., 1999) Moreover, studies in Puerto Rico (Novozhilov et al., 2000), a moisture gradient along a volcano in Costa Rica (Schnittler & Stephenson, 2000) and western slopes of the Andes (Schnittler et al., 2002) obtained patterns of decreasing species richness with increasing elevation and moisture

However, despite the success of these ecological studies, a portion only of the myxomcyetes life cycle was used to carry out these studies Thus, other stages remain fragmented, therefore, our knowledge about them is still limited

2.4 Roles of Myxomycetes in Nature

2.4.1 Myxomycetes as biocontrol agents

Myxomycetes (swarm cells, myxamoeba, and plasmodia) are phagotrophic, bacteriovores and fungivores that feed on spores with certain size range (Baba & Sevindik, 2018) For such reason, soil-dwelling myxamoeba and swarm cells plays an important role as secondary saprotrophs (Adl & Gupta, 2006) Some fungivorous secondary saprotrophs i.e gymnamoebae and Leptomyxa could be useful biocontrol agents (Adl, 2003), reducing the number of spores of the soil dwelling plant pathogens However, despite this, it is less likely that they could be useful biocontrol agents, because they lack specificity in their food source and are not capable to penetrate hyphae

Interestingly, studies of aethalium of Fuligo septica containing billions of spores inside

of a thick calcium carbonate crust also has a large yellow plasmodium, which serve as an environmental model to study the uptake and concentration of heavy metals such as the hyper-accumulation of zinc (Baba & Sevindik, 2018) Additionally, biochemical

detoxification mechanism of highly toxic levels of zinc in F septica and the genes

involved might be used in plants with greater biomass for bioremediation of polluted soils (see Rea-Maminta et al., 2015)

2.4.2 Bioactive compounds from Myxomycetes

Bioactive compounds from myxomycetes were intensively studied during the period between the 1950s to the 1970s But the studies were largely discontinued until the early part of the 21st century due to the unavailability of samples to process

Over the next years, myxomycetes have shown great potential to have anti-cancer compounds especially the alkaloid groups For instance, alkaloid arcyriaflavin exhibited high potential as anticancer drugs Another group of alkaloids, the makaluvamines,

specifically Makaluvamines A and B isolated from the fruiting bodies of D bahiense

(Ishibashi et al., 2001), have shown to exhibit in vitro cytotoxicity against the human colon tumor cell line HCT 116 and ovarian cancer (Dembitsky et al., 2005) In addition to these alkaloids, two novel rearranged triterpenoid lactones, tubiferal A and tubiferal B were extracted and purified from fruiting bodies of Tubifera dimorphotheca Tubiferal A showed anti-tumor activity against vincristine-resistant KB cell lines (Kamata et al., 2004) In addition, Nakatani et al., (2005) reported that a 90% MeOH and 90% acetone

crude extract from Physarum melleum exhibited antimicrobial activity against Bacillus

subtillis

However, the most remarkable study regarding myxomycetes cancer treatment is with the discovery of the Polycefin This drug is synthesized from purified Poly β-L-

malic acid extracted from a famous model organism, Physarum polycephalum

Preliminary testing showed that fluorescently labelled Polycefin can be injected in to the tail vein of a mouse and accumulates within breast and brain tumor cells (Ljubimova et al., 2008; Nguyen et al., 2017)

2.5 Advantages and Disadvantages of Classical Methodologies used in Myxomycetes Research

Despite the recent advances in the use of modern methods, it is undeniable that classical methods have advantages for studying microorganism diversity (Schmit & Lodge, 2004) Below are common classical methods, tackling the advantages and

disadvantages of these techniques

2.5.1 Field Survey

Field-based survey is the most common classical method applied in most myxomycete diversity studies (see Dagamac et al., 2017a) In many rapid diversity assessments of myxomycetes in many parts of the world, this method requires meticulous planning especially on predicting the right time and the right spots in the field where to find myxomycete fructifications This is due to the fact that seasonal and environmental

variation of the study areas creates artefacts associated with phenological variation in sporulation activity of myxomycetes A good case study was conducted by Tran et al., (2006) that examined fruiting phenomenology with seasonality in tropical forest Their findings showed 62 species were successfully recovered from 5 study site in Thailand during the rainy season, and additional 2 species were collected during dry season In addition, the first biodiversity studies conducted by Wrigley de Basanta et al., 2012 in Madagascar have successfully produced 124 species from 22 different genera The 761 collection include: 1 species new for science, 21 new species record for Africa and 106 recorded for the first time for Madagascar Moreover, the recently concluded study of Redeña- Santos et al., 2018 in Vietnam has produced 5 new records for the country

However, field based surveys are not as effective as moist chambers in arid regions especially in winter-cold deserts that lacks succulent plants This was supported

by studies in Kazakhstan (Schnittler & Novozhilov, 2000), Western Mongolia (Novozhilov & Schnittler, 2008), Lower Volga River basin of Russia (Novozhilov et al., 2006), Tarim Basin in China (Schnittler et al., 2013), Sultanate of Oman (Schnittler et al., 2015) where in almost all specimens were obtained from moist chamber cultures

2.5.3 Moist Chamber Cultures

Gilbert and Martin (1933) introduced the first moist chamber culture techniques, supplementing the information obtained from collecting specimens that have fruited in the field under natural conditions The substrata are usually placed on a moist paper towel, and put in container with lid like MCs The samples were then monitored and

check every 2 or 3 weeks The technique substantially increases the yield of field collection in all surveys and to give a more complete picture of the real biodiversity of a particular locality Furthermore, it is a method for obtaining relatively easy, inexpensive, reproducible, comparative results from different habitats, substrates, conditions or areas, and it also give major source of data needed for diversity studies of the myxomycetes (Eliasson & Lundqvist, 1979; Härkönen, 1977; Stephenson, 1985; Wrigley de Basanta et al., 2002)

However, this technique inherent bias since it only favors to species with short

developmental cycle and small fruiting bodies Species such as Tubifera or Lycogala that

have large compound fruiting bodies appears rarely in moist chambers than in the field (Novozhilov et al., 2017) Additionally, the long lasting period of moisture can produce dormant stages that do not or rarely form fruiting bodies (Novozhilov et al., 2017) This seems to be always the case in many studies in the Philippine archipelago that employs higher productivity in the moist chamber cultivation

Nonetheless, this technique is helpful to study ecological groups with very small fruiting bodies, such as corticolous, due to the fact that these myxomycetes usually thrive

in dry habitat (Everhart & Keller, 2008) or coprophilous (fimicolous) myxomycetes where fruiting bodies are rarely encountered in the field (Shchepin et al., 2017) Additionally, this is still the most common applied technique on biodiversity studies, since it is simple yet effective, requiring only Petri dishes, filter paper, and a good dissecting microscope

2.5.4 Agar Cultures

Cultivation of myxomycetes from spores dates back well over 100 years Lister (1901) reported the first spore-to-spore cultures of myxomycetes, using agar culture or, the classical approach of physiological and autoecological studies of myxomycetes At the moment, only about 100 myxomycete morphospecies (about 10% of morphospecies known worldwide) have been cultured from spore-to-spore on agar media (Haskins and Wrigley de Basanta, 2008; Liu et al., 2010)

Despite the lower productivity (positive culture) of agar culture compared to moist chamber cuture, this method can be effective in detecting minute myxomycetes (Schnittler et al., 2015) and protostelids (Spiegel et al., 2004) especially when a relatively weak, resource-poor agar i.e water agar is used since it keeps other competing fungi at minimum growth However, myxomycetes with larger fruiting bodies and/or colonies are usually absent in these cultures or at certain occasions may develop only a few fruiting bodies (Novozhilov et al., 2017)

2.6 Modern Molecular Methods

Myxomycetes studies used the classical method such as field observations and moist chamber cultures to study ecological patterns and biodiversity analysis However, though this method is simple and cost-effective it would not account for all life cycle of myxomycetes, hence, limiting our information (Walker & Stephenson, 2016) A study of Fiore-Donno et al (2010) revealed some species of myxomycetes lost the ability to mate

and to form fruiting bodies, thereby, leaving them undetected The molecular era for myxomycetes started quite late from the first phylogenies described in the group (see Fiore-Donno et al., 2008, 2011, 2012) up to the current barcoding gap assignment of species delimitations (see Borg Dahl et al., 2017) Enumerated hereafter are some molecular tools that have been used in studying myxomycetes

2.6.1 Terminal Restriction Fragment Length Polymorphism (TRFLP)

TRFLP is another molecular methodology that differentiates microbial populations based on the terminal restriction fragment size It allows a quick and economical way to compare different communities; it has been used to investigate prokaryotic and fungal communities (Hoppe & Schnittler, 2015) This methodology has high throughput capability and is very sensitive in regards to discriminating microbial communities, but there are limits to the accurate prediction of the microbiota present

2.6.2 Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) is a technique that has been used

to separate a mixture of DNA fragments according to their melting point, to analyze microbial communities without cultivation This technique was first used for investigations of prokaryotic communities (Muyzer et al., 1993) but was later adopted to detect the presence of airborne myxomycetes (Kamono &Fukui, 2006) and to investigate myxomycete communities inhabiting the soil (Kamono &Fukui, 2006) This method not only makes visual comparison of communities, but also well-separated bands can be

extracted from the agarose gel can be later sequenced and identified As an example, Ko

ko et al (2009) conducted their study in northern Thailand, here, genomic DNA extraction from the environmental samples on which myxomcyetes were not apparent was successfully done, meaning, molecular diversity studies of myxomycetes even without its fruiting body is possible

However, researchers find the technique time consuming and clumsy Additionally, with the improving sequencing technologies, the method is declining as more researchers turn to HTS

2.6.3 High-Throughput Sequencing

From 1977 to 2016, three generation of the sequencing technologies of various types have been developed Second and third generation sequencing technologies referred commonly to as next generation sequencing technology, has evolved significantly with increase in sequencing speed, decrease in sequencing cost, since its inception in 2004 For instance, HTS technologies being utilized to study myxomycetes are only now emerging but shows great promise

However, there are still limitations with the use of HTS and environmental sequencing that need to be considered and improved upon, such as PCR bias and a limited number of reference sequences Nonetheless, the technique will unfold unknown information about communities and genetic diversity

2.6.4 DNA Barcoding

Unicellular protists constitute the most abundant soil eukaryotes (Ekelund & Rønn, 1994), influencing nutrient cycling and energy flow (Bonkowski, 2004) Together with the advances in high-throughout sequencing of marker genes (Jamman et al., 2016) and with the updated and improved sequence database for accurate taxon annotation more understanding and discoveries of the hidden world of microorganisms can be expected However, as of thee moment, there is still no universal primer that can be developed for the whole group Therefore, due to multicopy and homologous nature of some sequences

of 18S rRNA gene, it was suggested as a temporary universal eukaryotic DNA marker This goes also with myxomcyetes, as mentioned before, diversity studies conducted for myxomcyetes were based on its fruiting bodies and their morphological determination only Since plasmodia and sclerotia lacks morphological traits for intial identification But, through molecular methods like the emerging DNA barcoding, gaps in studies of myxomycetes especially its plasmodial and scletorial stages can be filled (Borg Dahl et al., 2017) The recently concluded study of Shchepin et al (2017) successfully identified the plasmodia and sclerotia of myxomycetes that appeared in the moist cambers of Vietnam with the used of DNA barcoding As a result, DNA barcoding annotate 45 of 70 sequences obtained from sclerotia and plasmodia at species level This result is surprisingly high considering the results obtained by Borg Dahl et al., (2017) , where in it only contains sequences of not more than 14% of the ca 1000 morphospecies currently accepted (Lado 2005–2018) Furthermore, 50 samples of plasmodia and sclerotia

harvested from the moist chambers of Vietnam were successfully identified by Shchepin

et al (2017) Here, most of the species were assigned as Physarum melleum

PART III METHODOLOGY

3.1 Phase 1: Patterns of myxomycetes communities in two disturbed forest

fragments of Laguna Philippines using “fructification only” data

3.1.1 Study Site

Figure 1: Map of the study area showing the two municipalities of Laguna

The province of Laguna (Fig.1) belongs to the southern Luzon part of the Philippines For convenience purposes, unmanaged forest fragments from two nearby municipalities were chosen as collecting localities for this study namely, Los Baños (14⃘° 9’34" N, 121°