The first report of myxomycetes amoebozoa in thai nguyen city northern vietnam rapid biodiversity assessment using tools in modern molecular era

Research Questions and Hypotheses Since this thesis is designed to be the first intensive baseline information for Thai Nguyen City with regards to myxomycetes biodiversity, three indep

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

THE FIRST REPORT OF MYXOMYCETES (AMOEBOZOA)

IN THAI NGUYEN CITY, NORTHERN VIETNAM: RAPID BIODIVERSITY ASSESSMENT USING TOOLS IN MODERN

MOLECULAR ERA

BACHELOR THESIS

Thai Nguyen, 20/11/2017

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 Nikki Heherson A 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,

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

Keywords

α and β diversity, barcoding, dispersal, forest management, slime molds, tropical plants

Number of pages 143 pages

Date of Submission September 25, 2017

Supervisor‘s signature

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 Nikki Heherson A 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 Dr Martin Schnittler (University of Greifswald), Prof Dr 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,

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

TABLE OF CONTENTS

List of Figures 8

List of Tables 10

List of Abbreviations 11

Part I Introduction 12

1.1 Research Rationale 12

1.2 Research Questions and Hypotheses 15

1.2.1 Occurrence of myxomycetes in homogenous vegetation 15

1.2.2 α and β diversity in a heterogeneous vegetation 16

1.2.3 Genetic diversity 17

1.3 Research Objectives 18

1.4 Scope and Limitations 20

1.5 Definition of terms 21

Part II Literature Review 25

2.1 Natural Classification history of myxomycetes 25

2.2 Economic and ecological importance 26

2.3 Cutting edge technologies applied in studying the diversity and ecology of myxomycetes 31

Part III Methodology 37

3.1 Occurrence of myxomycetes in homogenous vegetation 37

3.1.1 Collection of substrates and preparation of moist chambers 37

3.1.2 Characterization and identification of fruiting bodies 38

3.1.3 Calculation of productivity and myxomycete occurrence 39

3.2 α and β diversity in a heterogeneous vegetation 40

3.2.1 Collecting localities 40

3.2.2 Collection of substrates, laboratory isolation method and myxomycetes characterization 41

3.2.3 Data evaluation 43

3.3 Genetic diversity 46

3.3.1 Specimen acquisition 46

3.3.2 DNA Extraction, amplification and sequencing 47

3.3.3 Sequence alignment, tree construction and genetic analysis 49

Part IV Results and Discussion 51

4.1 Occurrence of myxomycetes in homogenous vegetation 51

4.1.1 Results 51

4.1.2 Discussion 55

4.2 α and β diversity in a heterogeneous vegetation 57

4.2.1 Results 58

4.2.2 Discussion 73

4.3 Genetic diversity 78

4.3.1 Results 78

4.3.2 Discussion 80

PART V Conclusion 83

References 88

Appendices 104

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 forestsin 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

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

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

PERMANOVA Permutation analysis of variance

TRFLP Terminal Restriction Fragment Length

Polymorphism

PART I INTRODUCTION

1.1 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 (1986) who considered

‗biodiversity‘ as synonymous with ‗biological diversity‘ and expanded this usage to describe biological diversity at three levels: genetic (within species), species (species numbers) and ecological (community) diversity 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 ~1.75 million species (~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,

intensely known eukaryotic microorganisms that can fulfill such roles are the

myxomycetes

Myxomycetes, also known as plasmodial slime molds, are small group of 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

fungus-et al 2016; Dagamac fungus-et al., 2017a) In terms of their ecological importance, these organisms play an active role in the soil biota as ―microbial predators‖ as they consume

bacterial cells along with yeasts and other fungi associated with decomposing plant

tissues (Ing, 1994; Keller et al., 2008) 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., 2011) 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., 2013b) 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 (2014) This is then followed by an ecological study in three lowland

tropical forests in Vietnam by Tran et al (2014) 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 (2017) which

accounted a total of 69 new records for Vietnam These studies now resulted to a total

of 126 myxomycete species for the country Although, the number of myxomycetes records for Vietnam is rapidly increasing, much of these myxomycetes species are reported only in Southern Vietnam However, no profiling of myxomycete species was reported in Northern Vietnam, a region with rich and diversified vegetation, and contrasting seasonality

1.2 Research Questions and Hypotheses

Since this thesis is designed to be the first intensive baseline information for Thai Nguyen City with regards to myxomycetes biodiversity, three independent research components were developed to address different focal research frameworks

1.2.1 Occurrence of myxomycetes in homogenous vegetation

Background: Since myxomycetes are known to be living on ecosystems where there is

a high decaying organic plant material such as leaf litter, they are considered to be playing an important role in nutrient cycling of forest detritus (Rayner & Boddy, 1988) This is probably the reason why much of the previous reports regarding the distribution

of foliicolous myxomycetes in Asia were described from diversity studies conducted mainly on forest floor litters In spite of the fact that the major economic resources in Southeast Asia are coming from agriculture, there is relatively limited information about foliicolous myxomycetes that associate it with monotypic vegetation like an

agricultural plantation which could also serve as an ideal source of leaf litter substrates Thus, this study is intended to answer the question:

Question: Can monotypic vegetation like an agricultural plantation harbor myxomycetes? Specifically, the researcher wants to know:

a What myxomycetes will grow on three types of common agricultural plants in

Thai Nguyen City namely Camellia sinensis (L.) Kuntze, Dimocarpus longan (Lour.) and Psidium guajava (L.)?

b Can these plants serve as a good microhabitat for foliicolous myxomycetes? Hypothesis: Myxomycetes can successfully occur on decaying aerial and ground leaf

litter of Camelia sinensis, Dimocarpus longan, and Psidium guajava

1.2.2 alpha and beta diversity in heterogeneous vegetation

Background: Although taxonomic studies about myxomycetes are steadfastly growing for the past decades, relatively few studies have been conducted for the sake of understanding their ecology (Ndiritu et al., 2009; Coelho & Stephenson, 2012; Rojas et al., 2016) Hence, this rapid biodiversity assessment using the moist chamber technique in Thai Nguyen City wanted to contribute on the current knowledge about their ecology by investigating myxomycetes diversity and community structure in two different community forests

Question: What is the diversity and distribution of myxomycetes in two different community forests? Specifically the researcher wants to know:

a What is the species (i) occurrence, (ii) diversity and (iii) distribution of myxomycete communities between protected forest and unprotected forest in Thai Nguyen City, Northern Vietnam?

Hypothesis: This study wants to test the prima facie null hypothesis which states that

there is no significant difference of species diversity between protected and unprotected community forest This hypothesis best explains that despite differences in vegetation types and substrates availability, spore dispersal in localities around Thai Nguyen City and not the heterogeneity of plant communities may play a substantial factor in myxomycetes distribution in a local setting

1.2.3 Genetic diversity

Background: Since common morphologies of myxomycetes species has been found worldwide due to their cosmopolitan distribution, the morphological species concept has been appropriate in identifying myxomycetes However, recent molecular studies show that although similar morphology, intraspecific variation exists within cosmopolitan species of myxomycetes (Winsett & Stephenson, 2011; Dagamac et al., 2017a) Therefore, to test if there is variation of genes within a species, the most

abundant and morphologically unmistakable species from three vegetation types was

subjected for a phylogenetic investigation

Question: What is the genetic diversity of selected myxomycetes found in different vegetation types? Specifically, the research wants to know:

a What is the genetic diversity of Diderma Hemisphaericum populations among

the agricultural, protected and unprotected forests?

Hypothesis: This study wants to test the null hypothesis which states that there is no

significant difference among populations of Diderma hemispahericum collected from

different forest types

1.3 Research Objectives

The major objectives of this study were to investigate:

(i) if monotypic vegetation, particularly agricultural plantation, can serve as microhabitat for myxomycetes; to specifically answer this first major objective, this research phase specifically aims to:

 collect aerial and ground litter in a monotypic (homogenous) vegetation

 assign an occurrence index on clearly determined myxomycetes species

 compare the distribution of bright-spored and dark spored myxomycetes among the three agricultural plantation

(ii) if the species diversity and community distribution of myxomycetes between two different forest types are significantly differentiable; to specifically answer this second major objective, this research phase specifically aims to:

 collect aerial leaf litter, ground leaf litter, twigs and barks substrates from three predetermined forest types around Thai Nguyen City, Northern Vietnam

 assemble moist chamber cultures using the collected substrates in the field

 characterize and identify the species of myxomycetes collected directly

on the moist chambers

 assess the relative abundance and the diversity of myxomycetes

 calculate the taxonomic diversity between the two forest type

 estimate the number of myxomycetes species by constructing an extrapolated species accumulation curves

 compute for the α and β diversity indices between the two forest type

And (iii) if at the molecular level, genetic diversity of a cosmopolitan

myxomycetes species is structurally distinguishable based on three different vegetation

or forest types; to specifically answer this third major objective, this research phase specifically aims to:

 extract, amplify, visualize, purify and sequence DNA from specimens collected in different vegetation types

 construct a phylogenetic tree from the sequenced DNA samples

 calculate the FST index among Diderma hemisphaericum populations

1.4 Scope and Limitations

The whole study was conducted from March 2017 to September 2017 The collection of samples was carried out in the month of March – April 2017 in Thai Nguyen City, Vietnam while the process of setting up the myxomycetes culture, molecular methods, and data analysis was carried out at the Institute of Botany and Landscape Ecology, University of Greifswald in Greifswald, Germany from May 2017

to September 2017

Although the research has reached its aims, there were some unavoidable limitations for the study: (1) considering the amount of time and money provided for field collections, only the data coming from the moist chamber technique were used for this study No fruiting bodies found on extensive field surveys were included for this research work; (2) only leaf litters were collected in the first phase of the study, since the researcher only wants to assess foliicolous myxomycetes among three agricultural plantations; (3) no comparison of myxomycetes assemblages among agricultural plantations was done in the first phase of the study since the researcher only wanted to assess if monotypic vegetation, agricultural plantation in particular, can harbor myxomycetes; (4) no comparison of myxomycete assemblages among substrates in the second phase of the study was done; (5) only the pH level of the substrates and no other environmental data (during field collections) i.e temperature, humidity, etc were gathered since the present study did not attempt to investigate what abiotic factor influences myxomycete growth, but rather what ecosystem management would have an

influence on the composition and distribution of myxomycetes in a local landscape setting; (6) the pH range for every myxomycetes species reported in this study came from the average of the positive moist chambers where the species was found However, no discussion about the correlation of myxomycete occurrence with respect

to a certain pH range was included for this study since it has been confirmed by other researches that there are myxomycetes that are associated with a certain pH level; (7)

only the myxomycetes species Diderma hemisphaericum, and only 5 specimens of

each for every forest type were subjected for phylogenetic investigation due to lack of fruiting bodies in a collection, and also because of the expenses for molecular materials and components; (8) the constructed phylogenetic tree only shows intraspecific variation among populations with an intent to test the hypothesis of dispersal ecology

at a local setting, not in resolving evolutionary species hypothesis; (9) the biodiversity assessment of myxomycetes was carried out rapidly and in a local-scale

1.5 Definition of terms

For the purpose of clarity and understanding, the following terms were defined

within the context on how the terms are used in the study

α diversity refers to the species diversity of each forest type (local species pool)

β diversity refers to the differences in species composition between the forest type

(community analysis)

Diversity pertains to a variety of species; typically measured by a species count

(richness) and sometimes with an evenness index

DNA amplification pertains to the process that artificially increases the number of

copies of a particular DNA fragment into millions of copies through replication of a target segment using universal or specific primer pairs (forward primer and reverse primer) combinations

DNA sequencing pertains to the process of determining the precise order of

nucleotides within a DNA molecule It includes any method or technology that is used

to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in

a strand of DNA For this study the DNA sequencing technology used was the Sanger sequencing methodology

Foliicolous refers to a distinct guild of myxomycetes that thrive on leaf litter

substrates

Fruiting body refers to the part of the myxomycetes that holds the microscopic

strutures like the spores and capillitium

Genetic diversity refers to the variety of genes within a given species

Genotypes pertains to the set of genes in a DNA which is responsible for a

particular trait; the genetic makeup of an organism

Herbarium box refers to a container in which a dried myxomycetes fruiting body

is stored and preserved

Homogenous vegetation pertains to vegetation communities with uniform

composition of plant species all throughout

Heterogeneous vegetation pertains to vegetation communities with various

species composition of plant species

Microhabitat pertains to a small, localized habitat within a larger ecosystem in

which the myxomycetes are adapted to its conditions such as leaf litters, twigs and barks

Moist chamber pertains to a container which holds a high humidity atmosphere

within itself for a long period of time and is used for keeping a specimen moist

Monotypic Plantations refers to the localities that are characterized of having a

single grown plant species in the field

Occurrence pertains to the presence of myxomycetes in a particular microhabitat

Petri dish refers to a shallow, circular, transparent dish with a flat lid that is used

for the culture of microorganisms

Phylogenetic tree is a branching diagram or "tree" showing the inferred

evolutionary relationships among various biological species

Plasmodia/Plasmodium is a colorless or brightly colored vegetative body,

non-cellular, multinucleate, jellylike, amoeboid, and assimilative stage of the myxomycetes

Population refers to organisms of the same species that lives in a particular area

Protected forest refers to forests with some amount of legal or constitutional

protection These forests are commonly managed by an authority

Rarefaction pertains to the technique that assesses the richness of species from the

results of sampling

Rarefaction curves pertains to the plot of the number of species as a function of

the number of samples

Species accumulation curve refers to a graph indicating the exhaustiveness of a

species survey in a particular area

Species composition refers to the identity of all myxomycetes species that make

up a community

Spore morphology pertains to a particular form, shape or structure of the

myxomycetes fruiting body

Substrate refers to the medium of microhabitat in which myxomycetes lives and

grows In this case, substrates are aerial leaf litters, ground leaf litters, bark, and twigs

Unprotected forest refers to forests that are more open, and freely accessible to

human-mediated disturbances

PART II LITERATURE REVIEW 2.1 Natural classification history of myxomycetes

Myxomycetes (plasmodial slime molds or myxogastrids) are a group of living terrestrial heterotrophs which are characterized by two distinct trophic stages: (i)

free-a uninuclefree-ated free-amoebfree-ae with or without flfree-agellfree-a (collectively known free-as the

―amoeboflagellate‖ stage) and a (ii) multinucleate ―plasmodium‖ stage that was developed via sexual fusion (Everhart & Keller, 2008) The two trophic stages in their life cycle are usually cryptic, but the fruiting bodies are often large enough to be observed directly in nature (Schnittler et al., 2012)

The first report of myxomycetes can be dated back in the middle of the

seventeenth century after the German mycologist published on his book Herbarium

Portatile, oder behendes Kräuter- und Gewächsbuch the fruiting bodies of Lycogala epidendrum (Panckow, 1654) Afterwards, the myxomycetes have been variously

classified in different kingdoms, depending on the phase of the life cycle that the taxonomist emphasized (Everhart & Keller, 2010) They were previously classified in the Kingdom Animalia, Kingdom Protista, Kingdom Plantae, and Kingdom Fungi (de Bary, 1887; Schroeter, 1889; Hawksworth, 1983; Martin, 1960, Olive, 1975)

Anton deBary (1887) classified myxomycetes in the Kingdom Animalia, because they were more closely related to amoeboid protozoa than to fungi To

emphasize his point, he proposed the term Mycetozoa (as Mycetozoan) (mykes = mushroom, fungus + zoon = animal) which literally translates to ―fungus animals‖

Schroeter (1889) termed them ‗Myxothallophyta‘, a subdivision of thallophytes traditionally described as ―thalloid plants, ―relatively simple plants‖ or ―lower plants‖, which is a defunct division of Kingdom Plantae

Dr G.W Martin (1960) classified them in Kingdom Fungi because of its fungal nature He then gave strong arguments supporting the relationship of the myxomycetes

to the protozoa since myxomycetes have protozoan ancestry because of (i) their amoeboid, naked somatic phase, and (ii) their phagotrophic nutrition

According to Hawskworth et al (1983), myxomycetes show characters of both

plants and animals, and constitute the Mycetozoans of Olive (1975), which is a polyphyletic assemblage that Olive classifies in the Phylum Gymnomyxa under the Kingdom Protista

However, recent development in molecular phylogenetic analysis of highly conserved elongation factor 1-alpha (EF-1α) gene sequences by Baldauf and Doolittle (1997) had revealed that myxomycetes are not fungi, and the work of Fiore – Donno et

al (2010) about genetic analysis supported the classification of myxomycetes in the

Kingdom Protista

2.2 Ecological and economic importance of myxomycetes

Myxomycetes are non-pathogenic organisms with promising economic and ecological potentials Ing (1994) assumed myxomycetes as ‗microbial predators‘ that are utilized in the soil ecosystem by feeding on microorganisms i.e yeasts, bacteria,

and fungal spores during their amoeboid stage and served as an important secondary saprotrophs (Adl & Gupta, 2006) Thus, concluding that this organism plays a key role

in nutrient cycling by regulating the flow of nutrients through the ecosystem (Urich et al., 2008; Stephenson et al., 2011)

Moreover, myxomycetes are also known to serve as a key organism for degradation of wood and litters; and in order to investigate the ability of plasmodia to degrade the said substrates, a 3-pronged study was conducted mainly by Raghu (2001):

(1) A study of myxomycete occurrence on logs of wood in the forest, in relation

to the state of decay of the wood and the presence of higher fungi, (2) The ability of monoxenic plasmodia to actually degrade wood and litter, as assessed from loss of dry weight and changes in nitrogen content and (3) the actual production of extracellular enzymes by the plasmodia to degrade wood polymers

Results from the first study (Raghu & Kalyanasundaram, 1997) indicated that myxomycetes are found mostly on moderately decayed logs in coniferous wood, while

in angiospermous wood they are found on logs in moderate as well as in advanced stages of decay However, it has been observed that undecayed and slightly decayed wood rarely harbor myxomycetes which led them to the conclusion that myxomycetes cannot attack or degrade intact wood as primary invaders, but once the wood is partially degraded by basidiomycetes, they can colonize it

In the second study, Raghu (2001) showed that by incubating several different

substrates with the plasmodium Physarum gyrosum, the plasmodium could degrade

some substrates (pine wood, and rice straw in this case) A definite reduction in dry

weight and a convincing increase in the nitrogen content of the substrates were also recorded This phenomenon seems to occur in nature also, because samples of wood bearing plasmodia showed a much higher nitrogenase activity than wood without plasmodia

Lastly, the culture of Physarum gyrosum was then subjected to preliminary

screening for the production of wood degrading enzymes to degrade lignin, carboxymethyl cellulose (CMC) and xylan through agar media Positive tests were obtained with CMC and xylan The extracellular CMC-ase and xylanase were then incubated with the substrate and subjected to HPLC analysis The identification of glucose and xylose made it clear that the enzymes were active on the respective substrates In conclusion, they found myxomycetes to be a potential source for bioremediation Several studies during the last years also affirmed the potentials of

myxomycetes to bio-remediate pollutants in the soil From the study of Zhulidov et al (2002), they observed that the myxomycetes Fuligo septica has the ability to hyper-

accumulate zinc (395-3,600 mg/kg based on dry matter) The same myxomycetes species was also observed to have significant traces of other polluting elements of barium, cadmium, manganese and strontium (Stijve & Andrey, 1999) In a more recent study of Rea-Maminta et al (2015), they tested several myxomycetes species for chromium and manganese content and hypothesized that the ability of myxomycetes to phagocytize food bacteria in forested areas with soil laden of heavy metal causes the high yield of Mn7 in the fruiting bodies of myxomycetes

Additionally, because of the intricate life cycle of the myxomycetes, they are

used as a model organism for cancer researches Using the model organism Physarum

polycephalum, Cummins and Rusch (1968) demonstrated that the animal like creeping

amoeba functions as a giant single unit of cell with distinct events involved with growth, mitosis, differentiation and cytokinesis The mitotic synchronous cycle of the myxomycetes could be assumed as a product of certain biological timer found inside the nucleus This mechanism is deemed important and may have a direct relevance when compared to the unregulated cell divisions that have lost tract of the cellular internal control leading to the formation of cancerous or tumor cells (Keller & Everhart, 2010) Moreover, many chemical studies investigated the potential of myxomycetes to become sources of many novel bioactive metabolites that can be used as anticancer or antimicrobial agents

Early studies of myxomycetes for the production of compounds with biological activity often focused on isolating and testing crude extract of the plasmodium or fruiting bodies For example, in the study of Nakatani et al (2005), crude extract of

Physarum melleum exhibited antimicrobial properties against Bacillus subtilis but the

extracts, melleumin A and B, did not show antimicrobial activity against B subtilis,

and therefore, it was thought that the compounds found in the crude extract that show antimicrobial activity are unstable yellow pigments in the plasmodium In addition, Iwata et al (2003) used the crude extract from the collected fruiting bodies of

Cribraria cancellata The crude extract also showed antimicrobial activity against

Bacillus subtilis, but the secondary compound, cribrarione B, did not exhibit

antimicrobial activity

Ishibashi et al (2001) isolated two compounds (makaluvamine A and B) from

Didymium bahiense which have considerable cytotoxic activity on the human colon

carcinoma cell line HCT 116 and displayed inhibition activity on topoisomerase in vitro (Barrows et al., 1993) In addition, the compounds makaluvamine A and C have been reported to show inhibition activity against colon cancer and human ovarian cells

in vitro (Barrows et al., 1993; Matsumoto et al., 1999)

Furthermore, in the study of Steglich (1989), the Arcyriacyanin A isolated from

Arcyria nutans has shown an inhibitory activity against a number of human cancer cell

lines, inhibit protein kinase C, and inhibits protein tyrosine kinase Further studies like the work of Murase et al (2000) also discovered that Arcyriacyanin A was possible to

be used to inhibit cancer cell growth where the latter author conducted an in vitro

assays on 39 human cancer cell lines, including those taken from tissues in the lung, stomach, colon, ovary, central nervous system, breast, renal, skin, and prostate

However, the most remarkable example of myxomycetes enabling cancer treatment was by Ljubimova et al (2008) wherein the study was conducted with a non-toxic, non-immunogenic, and biodegradable nanoconjugate drug delivery system called

Polycefin Polycefin is synthesized from purified Poly (β-L-malic acid) (by Physarum

polycephalum) and is subsequently modified for directed delivery of morpholino

antisense oligonucleotides (gene silencing therapy), antibodies, and anti-tumor drugs to certain tumor cells Preliminary testing showed that fluorescently labeled Polycefin can

be injected in to the tail vein of a mouse and accumulates within breast and brain tumor cells

Although studies carried out to date have proved myxomycetes to be a potential source of natural active products that have unique chemical structures, additional investigations are still required since the number of species that was used in the previous studies is still relatively small Nonetheless, with the growing studies about myxomycetes unique life cycle, more useful metabolites are expected to be discovered

2.3 Cutting edge technologies applied in studying the diversity and ecology of

do not account for the array of complex life histories found throughout the group nor

the reproductive isolation within morphospecies i.e some species of myxomycetes

may have lost the ability to mate and form fruiting bodies altogether (Fiore-Donno et al., 2010) or species that are uncultivable and inaccessible for systematic studies

(Epstein, 2013) Thus, understanding of myxomycete diversity and ecology has been limited by these factors

The first major attempt towards molecular studies in myxomycetes was dated back in late 1960s with the objective of using isozyme profiles to investigate relationship between closely related species, particularly to distinguish between similar morphospecies and even between different strains of a single morphospecies (Franke, 1967; Franke et al., 1968; Franke & Berry, 1972; Betterley & Collins, 1983) For

example, Berry and Franke (1973) investigated the species Fuligo septica where it has

been observed that distinct isozyme patterns could be detected between white and yellow strains within a single morphospecies Following this discovery, they also outlined and limited information available for other species and strains Furthermore, in the study of Betterley and Collins (1983), isozyme patterns of 44 isolates of the species

Diderma iridis correlated with three distinct heterothallic breeding groups and many

reproductively isolated nonheterothallic isolates However, although the technique made some early contributions, it was eventually abandoned due to the interversus intraspecific variation repeatedly found within morphospecies that limits its usefulness

in species delimitation outright (Franke & Berry, 1972; Franke, 1973; El Hage et al., 2000)

The most significant development towards molecular methods started in the 1980s through the use of DNA sequencing for the purpose of investigating the phylogeny of myxomycetes (Otsuka et al., 1983; Cavalier-Smith, 1993; Rusk et al., 1995; Baldauf & Doolittle, 1997; Baldauf, 1999) The ability to decode DNA

sequences is an essential tool in biological researches since it can be utilized to gather variety of information, such as biogeochemical properties and phylogenetic relationships However, these early studies were limited by the technology at that time and the availability of myxomycete DNA Nevertheless, with the rapid advancing of molecular tools and applications, more and more sequences emerged and obtaining myxomycetes DNA (either from individual isolates or from communities represented

in environmental samples) became much easier Moreover, several applications are utilized in studying the myxomycetes biodiversity or ecology such as Terminal Restriction Fragment Length Polymorphism (TRFLP), Denaturing Gradient Gel Electrophoresis (DGGE), and High-Throughput Sequencing (HTS)

Terminal Restriction Fragment Length Polymorphism

Terminal restriction fragment length polymorphism is a technique that allows a quick and economical way to investigate communities of prokaryotic microorganisms (Liu et al., 1997), wherein fluorescently labeled polymerase chain reaction (PCR) primers are designed to target and amplify a target gene of interest Once the resulting amplicons are subjected to digestion with a restriction enzyme, which identifies sequence polymorphisms and results in fragments of various lengths, the fluorescently labeled fragments are then fractionated using a DNA sequencer An example of TRFLP application on myxomycetes was the study of Hoppe and Schnittler (2015) in which the molecular technique was adapted to compare mycomycete communities based on genomic DNA extracted from two soil samples As a result of their study, differences

in the composition of the respective myxomycetes communities were shown by comparison of the generated fragment length pattern community Moreover, they described TRFLP to be cost-efficient alternative that reveals differences between ecological communities with low effort compared to DGGE which is more complex and needs more processing time

Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis is a technique used for separating DNA fragments according to their melting point, to analyze microbial communities without cultivation Similar to TRFLP, primers are first designed to amplify target gene sequences from the samples of interest Afterwards, PCR products are run along a urea gradient in a polyacrylamide gel to separate fragments Melted products then move slowly through the gel, allowing the detection of sequence variation Muyzer et al (1993) was the first to use the technique to investigate prokaryotic communities and was later adopted by Kamono and Fukui (2006) to detect the presence of airborne myxomycetes and also, to investigate the myxomycetes communities inhabiting the soil In addition, a study of Ko Ko et al (2009), which extracted genomic DNA directly from environmental samples on which myxomycetes were not apparent, but showed that DGGE can be used to obtain data on the presence of myxomycetes on their primary microhabitats without the need of a fruiting body Although the technique seems to have considerable potential in understanding myxomycetes diversity and

ecology, many consider the technique to be time-consuming Thus, more and more researchers are turning now into HTS which is more time and cost efficient

High-Throughput Sequencing

High-throughput sequencing (HTS), also known as next generation sequencing (NGS), is a term to describe a number of different modern sequencing technologies

(e.g Illumina sequencing, Ion torrent, SOLiD sequencing) that are capable of

sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time (Kircher & Kelson, 2010) The first published example of HTS of myxomycetes was by Fiore-Donno et al (2016), wherein the authors investigated the diversity and distribution of two major ubiquitous groups of

soil amoeba, the genus Acanthameoba and the myxomycetes From the 150 grassland

soil samples in three Biodiversity Exploratories study regions in Germany, the authors obtained more than 900,000 reads which have 338 operational taxonomic units (OTUs) that represented 35 unique blast hits From these hits, 10 myxomycete genera and 2

unidentified taxa were identified Furthermore, their study revealed that Acanthameoba

and dark-spore myxomycetes communities were widely distributed, highly diverse and non-randomly structured in grassland soils In addition, they found three distinct communities between the three studied regions As a result of their study, implementation of HTS particularly with environmental samples showed a promising potential in gathering information about hitherto unknown communities, genetic diversity and undescribed species (uncultivable and rare or non-fruiting species)

In general, these cutting edge technologies advanced many myxomycetes researches in ecological sense since after using these technologies many hidden diversities of myxomycetes population were revealed (see studies of Novozhilov et al

2013 in Caucasus Mts) Since much of the underlying current knowledge rely only on fruiting body based morphological concepts, the ecological role they fulfill as of now can be categorically described as the ―tip of the iceberg‖ and traditional ecological survey coupled with molecular tools will be the step forward as exampled from the survey of Feng and Schnittler (2015) in temperate forests of Northeastern Germany

PART III METHODOLOGY

3.1 Occurrence of myxomycetes in homogenous vegetation

3.1.1 Collection of substrates and preparation of moist chambers

Aerial leaf litters (AL) and ground leaf litter (GL) substrates were randomly

collected from three different agricultural plantations (Camellia sinensis, Dimocarpus

longan, and Psidium guajava) in Thai Nguyen City (21° 35' 39.19" N, 105° 50' 53.41"

E), a municipality situated in the Northeast region of Vietnam (see Appendix A)

Figure 1: The study area: Thai Nguyen City, Thai Nguyen Province showing the sampling location of the three agricultural plantations

Three 5 x 5 m plots were initially established for each agricultural plantation Five samples of AL and GL were then randomly collected for each plot making a total now of 15 AL and 15 GL per agricultural plantations All of the collected substrates were initially placed inside dry paper bags and were then immediately transported in the laboratory The moist chamber (MC) cultures were set up following the protocol of Stephenson and Stempen (1994) The MCs were then soaked overnight in sterile distilled water After 24 hours, the pH of each substrate was checked 3 times with a pH-meter and was maintained under ambient light conditions, at room temperature (22 – 25 0

C) for up to 10 weeks The MCs were regularly checked every week for the presence of myxomycetes plasmodia and/or fruiting bodies To keep the MCs moist,

distilled water was added in certain occasions

3.1.2 Characterization and identification of fruiting bodies

Moist chambers that produced myxomycetes fruiting bodies were initially segregated The substrates inside those moist chambers were then placed inside herbarium matchboxes and were cooled under freezing condition overnight The fruiting body characteristics (type, shape, presence of lime, height, color) were then described and were used for initial determination using a stereo microscope (Zeiss Stemi DV4) Internal structure of the fruiting bodies was also noted such as presence or absence of columella, appearance of the hypothallus, attachment to the calyculus and layers of peridium Microscopic characterizations of the fruiting bodies were also conducted by carefully transferring a single fruiting body on a slide with a drop of

Hoyer‘s medium After 24 hours, the mounted slides were viewed using a light compound microscope (Zeiss Axiolab A1) Structures such as the spore (size, appearance, color) and capillitium (morphology, presence of free ends) were then characterized to verify the identity of a fruiting body The specimens were then determined using published literatures (Poulain et al., 2011) and with web-based identification key (http://slimemold.uark.edu/) Current names were counter-checked using an online nomenclatural database for the eumycetozoans (http://nomen.eumycetozoa.com) For specimens that could not be fully identified with strong certainty due to some malformations but with distinguishing character enough to separate it as a species, the abbreviation ―cf‖ was used in the taxon name All vouchers

of collected specimens were deposited under the private collection of the last author in

the Institute of Landscape Ecology and Botany, Greifswald University

3.1.3 Calculation of productivity and myxomycete occurrence

The productivity of the moist chambers for each agricultural plantation was generated in accordance to the calculation of the percentage yield of Dagamac et al (2012) A moist chamber that exhibited either plasmodial and/or fruiting body growth was considered as positive record for myxomycetes, and therefore, was noted as one positive collection For all of the foliicolous myxomycetes collected in this study, an abundance index was used following the categories of Stephenson et al (1993) which

is based upon the proportion of a species to the total number of records: R– rare (< 0.5%), O–occasional (> 0.5–1.5%), C–common (> 1.5–3%), A–abundant (> 3%)

Figure 2: Schematic diagram for the methodology of the first phase of this research study

3.2 α and β diversity in heterogeneous vegetation

3.2.1 Collecting Localities

Two community forests within the vicinities of Thai Nguyen City (21° 35' 39.19" N, 105° 50' 53.41" E) were classified as protected and unprotected forests Both forest types are characterized as plantation forest mainly dominated by the non-

indigenous plant species Acacia mangium (Wild.) with natural regeneration of native

species under forest canopy The collecting localities for protected forest were within the well-managed Nui Coc Lake which serves as an ecotourism park for the city The localities in the protected forest are only exposed with moderate human activities that