A Molecular Phylogeny of Bornean Tree Frogs (Amphibia: Anura: Rhacophoridae)

from Genus Polypedates.

Sarina Mat Yasin (22247)

This thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of Science with Honours

in Animal Science and Management

Department of Zoology Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA WAK 2011

ACKNOWLEDGEMENTS

First and foremost, praise to Allah S.W.T for giving me inner strength to continue pursuing

my research work despite the hardship faced. Secondly, I would like to express my sincere

appreciation towards my supervisor, Dr. Rarnlah Binti Zainudin for all the guidance,

advices, suggestion and support throughout the process of completing this thesis. My

appreciation to Professor Dr. Mohd Tajuddin Abdullah, Dr. Yuzine Esa, and other

lecturers involved for sharing their valuable information and thoughts.

I would like to thank the staff of Department of Zoology, namely, Mr. Wahap Marni, Mr.

Huzal Irwan Husin, Mr. Nasron Ahmad, Mr. Isa Sait and Mr. Trevor for every drop of

their sweat, helping me collecting samples. I would also like to thank the Director of

Sarawak Forestry for approving my permit, which enables me to collect samples for my

study.

I would also like to extend my appreciation to the post graduate students in the Molecular

Ecology Laboratory, namely, Elvy Quatrin Deka, Roberta Chaya Tawie, Mohd Isham

Mohd Azhar, Muhammad Fadzil Arnrarn, Ridwan Rahman, Fidzl Sidq, Ikhwan Idris, Nur

Aida Tarnrin, Zahirunisa Abdul Rahim, Hanif Ridzuan Mat Daud, Madinah Adrus and

Klshen Bunya for all the knowledge sharing, guidance, support during my ups and downs

and companionship throughout these years in UNIMAS.

•

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I would like to extend my gratitudes to my coursemates especially Shamsudilaila Gambet,

Nurasyikin A.Rahim, Siti Noralizah Radi, Nurul Aina Ab.Razak, Siti Faizah Ismail, Nurul

Afiqah Mahadi, Normasarah Rahman Muhammad Adib Yusoff and my best friends,

Afifahtul Akmal Nor and Muhammad Salleh Rahim for their assistance given,

companionship, and dulcet word of supports throughout the years.

Above and beyond, I would like to give a special thanks to the angels of my heart, Mr. Mat

Yasin Ismail and Madam Rekiah Ismail, also my brothers and sisters for their loves,

encouragement and supports throughout the entire duration of my study.

11

Table of Contents

Acknowledgements...... ............ ...... ... .................. ......................... I

Declaration...... ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . III

Table of Contents.................................................................... .... IV

List of Abbreviations... . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. VI

List of Figures........ ............. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . ... VII

List of Tables ... ................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. VIII

Abstract................................. ... ................... ............ ... ........... ... ...... 1

1.0 Introduction......................................................................... 2

1.1 Problems statement. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. 5

1.2 Research question and hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 6

1.3 Research objectives..... ............................................... .... 6

2.0 Literature Reviews. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. .. . . . 7

3.0 Materials and Methods.......... ..................... ....................... ......... 13

3.1 Field Samling............................................................ ..... 13

3.1.1 Study sites....... ................................................................ 14

3.2 Molecular works............................................................ 17

3.2.1 Total genomic of DNA extraction............................. 17

3.2.2 Amplification of Targeted Sequence.................................. 18

3.2.2.1 Polymerase Chain Reaction (PCR)................ 18

3.2.2.2 Purification................................. .......... 20

3.3 Data Analysis................................................................ 20

3.3.1 DNA Sequence Analysis ......... ... ............. ... ...... ...... 20

3.3.2 Phylogenetic Analysis.. ............. .................. .......... 21

IV

4.0 Result...... ................................... ............ .................................... 23

4.1 DNA extraction.......................... ........................... ... 23

4.2 Polymerase Chain Reaction (PCR)............... . . . . . . . . .. . ..... 24

4.2 Purification................. ............................................... .... 25

4.4 Sequence Analysis... ......... ... ............ .............................. 26

4.4.1 Pairwise Genetic Distance.................. ... ...... ... 26

4.5 Phylogenetic Analysis........................... ........................ 27

5.0 Discussion......... ...... ......................................................................... .... 34

5.1 DNA extraction. .......... ... .......................................... .... 34

5.2 Polymerase Chain Reaction (PCR).................. ............ 35

5.3 Phylogenetic Analysis................. ............................. ..... 36

6.0 Conclusion and Recommendations... ................................................... 38

7.0 References.................... .................................................................... .... 39

8.0 Appendix... .................................................................... ... ........... ... ...... 44

v

I

16S rRNA

Bp

BPP

et al.

-1 g

I.e.

Kimura2P

mL-1

ML

Mm

MP

mtDNA

NJ

NMDS

No.

pH

PAUP

P.

Unimas

LIST OF ABBREVIATIONS

Degree Celsius

16 S ribosomal Ribonucleic acid

base pairs

Bayesian posterior probabiJities

and other people

per gram

in other words

Kimura 2 Parameter

per millilitre

Maximum likelihood

millimetre

Maximum parsimony

Mitochondrial Deoxyribonucleic acid

Neighbor-joining

Non-metric dimensional scaling

Number

A measurement of the acidity or alkalinity of solution

[p stands for "potenz" (this means the potential to be) and

H stands for Hydrogen]

Phylogenetic Analysis Using Parsimony

Polypedates

Universiti Malaysia Sarawak

VI

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List of Figures

Figure Page

1.0 Study sites and locations for preserved tissues. 14

2.0 The position of l6S-rRNA genes in mtDNA genome. 18

3.0 Gel photo of DNA visualization for DNA extraction 23

4.0 Gel photo of DNA visualization for PCR 24

5.0 Gel photo of DNA visualization for purification 25

6.0 Bootstrap 50% majority-rule consensus Neighbor Joining tree 30 (NJ) of mitochondrial ribosomal l6S genes of genus Polypedates.

7.0 Bootstrap (50% majority-rule) consensus of Maximum Parsimony 31 Tree (MP), mitochondrial ribosomal l6S genes of genus Polypedates.Bootstrap values are indicated above branch. Tree branch is 293 with the consistency index (CI) =0.8362 and retention index (RI) =0.9375

8.0 Bootstrap (50% majority-rule) consensus Maximum Likelihood 32 tree (ML) of mitochondrial ribosomal l6S genes of genus Polypedates. Bootstrap values are indicated above and below

branch.

9.0 Bayesian inference (50% majority rule) consensus tree 33 of mitochondrial ribosomal l6S genes of genus Polypedates using program PAUP version 4.0blO. Values of Bayesian posterior probabilities (BPP) are indicated besides the branch nodes.

VB

List of Tables

•

Table Page

1.0 Details of tissues samples used in this study (include preserved samples from molecular laboratory and fresh tissues samples). 16

2.0 Oligonucleotide primer pairs and its peR annealing temperature 18 (Tm) used in this study.

3.0 Amplification profile. 19

4.0 Master mix preparation for 1 time reaction. 19

5.0 Pair-wise distance matrix of genus Polypedates (including 44 outgroups) of 16S rRNA gene using Kimura two- parameter (Kimura, 1980).

6.0 Aligned partial of l6S rRNA of mitochondrial DNA (excluded 50 sites with missing/ambiguous data and gaps) for genus Polypedates.

VIII

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A Molecular Phylogeny of Bornean Tree Frogs (Amphibia: Anura: Rhacophoridae) from Genus Polypedates.

Sanna Mat Yasin Animal Resource Science and Technology

Department of Zoology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT The evolutionary biology of genus Polypedates was not well understood. Thus, molecular phylogeny of the genus Polypedates were studied by using mitochondrial ribosomal of partial 16S gene, in order to elucidate the phylogenetic relationship among members of genus. Tissues samples were collected from six different localities, namely UNIMAS East Campus, Bario, Similajau National Park, Bako National Park, Matang Wildlife Centre and Kubah National Park. A total of 489 bp of mitochondrial ribosomal of partial 16S gene from 25 samples were analysed using molecular technique approach (DNA extraction, PCR amplification, and direct sequencing). From this study, there are two major monophyletic groups were appeared and sister to each other supported by highly bootstrap values of 100% in Neighbour-Joining (NJ), Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Analysis. Hence, Polypedates otilophus was identified as basal by outgroup rooting which fully supported by highly bootstrap values of NJ, MP, ML and Bayesian Analysis. Moreover, Polypedates leucomystax, Polypedates macrotis, Polypedates colletti and Polypedates otilophus were was sister groups. Mitochondrial ribosomal 16S gene is a useful genetic marker for species divergence for all tree frogs and this study should be extends to elucidate the phylogenetic analysis all species in the genus including new species of Polypedates chlorophthalmus. Key words: Genus Polypedates, molecular phylogeny, mitochondrial ribosomal of partial 16S gene.

ABSTRAK Evolusi biologi dar; genus Polypedates tidak difahami dengan baik. Dengan demikian, filogeni molecuZ dari genus Polypedates dipeZajari dengan menggunakan ribosom mitokondria dari sebahagian gen 16S, untuk menjelaskan hubungan keakraban diantara genus. Sampel tisu dikumpul aripada en am lokasi berbeza, Kampus Timur UNIMAS, Bario, Taman Negara Similajau. Taman Negara Bako, Pusat Hidupan Liar Matang dan Taman Negara Kubah. Sebanyak 489 bp ribosom mitokondria dari sebahagian gen 16S, 25 sampel dianalisis dengan menggunakan pendekatan teknik molekul (DNA ekstraksi, ampl~fikasi peR, and sekuensing). Dari kajian ini, dua kumpulan besar monofiletik muncul dan adik beradik satu sama lain dengan nilai bootstrap yang tinggi 100% untuk Neighbour-Joining (NJ), Maksimum Parsimoni (MP), Maksimum Likelihood (ML) dan Analisis Bayesian. Oleh itu, Polypedates otilophus dikenalpasti sebagai basal oleh kumpulan luar perakaran yang sepenuhnya disokong oleh nilai bootstrap yang tinggi bag; NJ, MP, ML dan Analisis Bayesian. Tambahan itu, Polypedates leucomystax. Polypedates macrotis. Polypedates colletti and Polypedates otilophus adalah kumpulan adik. Gen 16S ribosom mitokondria merupakan penanda genetik yang berguna untuk variasi species· unluk katak pokok dan kajian ini perlu diperluaskan untuk menjelaskan hubungan keakraban antara semua spesies kalak pokok termasuk spesies baru Polypedates chlorophthalmus. Kata kunci: Genus Polypedates, filogeni molekul, ribosom mitokondria dari sebahagian gen 16S.

1

1.0 INTRODUCTION

•

Frogs are amongst the most familiar animal, wherever they occur in the world and easy to

recognize. Frogs are classified under the Order Anura (Amphibia) which exactly means

without tail when adult (Inger and Stuebing, 2005). Their distinctive features include no

tail, a short(often stocky body), long hind legs and short front ones, large bulging eyes, and

a very wide mouth (Inger and Stuebing, 2005; Garbutt and Prudent a, 2006 ). Frogs are

unique among terrestrial vertebrates in that they depend on two separate environments

within their life cycle. While as adults they live on lands and breathe air, their breeding

techniques are dependent upon an aqueous environment (Inger and Tan, 1996). However,

during the larvae stage, they do live in water and have tail for swimming and moving.

Borneo is the second largest tropical Island in the world after New Guinea. There are rich

biodiversity in the island of Borneo especially in the group of herpetofauna (Das and

Ghazally, 2001). Herpetofauna can be divided into two classes which are amphibians

(frogs) and reptiles. A total of 150 species of frog were reported from Borneo (Inger and

Stuebing, 2005) in which 89 species that are endemic. In Borneo, there are six families of

frogs such as Bufonidae, Dicroglossidae, Megophryidae, Microhylidae, Ranidae and the

last one is Rhacophoridae.

The genus Polypedates came from family Rhacophoridae (Brown and Alcala, 1994).

There are four species of the genus have been reported from Borneo (Inger and Stuebing,_

2005; Inger and Tan, 1996), such as P.leucomystax, P. macro tis, P. colletti, and P.

otilophus. Most species live in primary or secondary forest in the 10wlands(P. macrotis, P.

2

I

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colletti, and P. otilophus) and some live almost everywhere except primary forest such as

in towns, villagers, agricultural fields, road sides, and cleared forest (P. leucomystax).

Inger and Stuebing (2005) stated that adults spend their time above ground in shrubs and

trees, though the actual tree height is not known (P. macrotis, P. colletti, and P. otilophus).

However, P. leucomystax is an exception, as it is often found at the ground level, either in

grass or under, or even in houses. Inger and Tan (1996), reported that P. leucomystax is

widely distributed in Brunei, Kalimantan, Sabah, Sarawak, Southeast Asia and adjacent

islands. P. macrotis distributed in Kalimantan, Sabah, Sarawak, Sumatra, Natuna Islands,

and Philippine Islands. Meanwhile P. otilophus is known to be distributed in Kalimantan,

Sabah, and Sarawak (endemic) and P. colletti is distributed in Sabah, Sarawak, Peninsular

Thailand (Smith, 1930), Sumatra (Boulenger, 1890), and Natuna Islands.

Four species from this genus have similar breeding habits. Males call around bodies of

shallow standing water. When clasped by a male, the female produces a foam nest that is

made by churning mucus with the hind limbs and that's attached to leaves of shrubs

overhanging water (P. macrotis, P. colletti, and P. otilophus) (Inger and Stuebing, 2005).

The foam nest of P. leucomystax is often placed on the ground water's edge. After

hatching, the tadpole's wriggles free of the nest and ifthe foam mass has not already fallen

on to the pool's surface, drop into the water (Inger and Stuebing, 2005).

Why use mitochondrial ribosomal 16S genes? The mitochondrial ribosomal 16S genes

fulfill the requirements for a universal DNA barcoding marker in amphibians (Vences et

3

I

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aI., 2005). Yu et aI. (2008), they use partial sequences of three mitochondrial (l2S rRNA,

l6S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-I, rhodopsin exon 1,

and tyrosinase exon 1) genes from 57 ingroup taxa and eight outgroup taxa to propose a

hypothesis for phylogenetic relationships within Rhacophoridae.

Previous studies have proven that mitochondrial ribosomal l6S genes were reliable for

further molecular analysis of the Sarawak Rana, since they are easy to isolate respectively

(Ramlah, 2009). Therefore, mitochondrial ribosomal l6S genes were used in elucidate the

phylogeny of genus Polypedates.

There is most valuable benefit of using PCR in isolating and amplifying the site region in

order to study molecular phylogeny of genus Polypedates. This is because this method is

less time consuming to perform rapid duplication by only screening for variation between

numerous numbers of individuals. In addition, PCR uses only a small amount or quantity

of DNA and the procedure is much easier to automate than by cloning the gene.

The importance of this study are, it could elucidate the information on phylogenetic

relationship of the genus Polypedates such as sister taxon of each species under this genus

and any existence of genetic break. This will enhance our knowledge on the taxonomy,

systematic, and the phylogenetics of this particular group of species.

4

I

1.1 PROBLEMS STATEMENTS

•

Inger and Stuebing (2005) reported that the amphibians of Borneo itself were quite diverse

with 150 species to date and the number of species keeps increasing with more systematic

complexity within species. Therefore, studies on systematic anurans, the taxonomic, and

the phylogenetic relationships of anurans should be more extensive.

Amphibians have existed on earth for over 300 million years, yet in just the last two

decades there have been an alarming number of extinctions, nearly 168 species are

believed to have gone extinct and at least 2,469 (43%) more have populations that are

declining (Stuart et aI., 2004). This indicates that the number of extinct and threatened

species will probably continue to rise. Thus, serious action should be taken to ensure the

phylogeny.

There were lack of study on the genetic of the Bornean tree frog and the phylogeny for the

Rhacophoridae was not well constructed. For this, mitochondrial DNA (mtDNA) genes

were used to infer relationships among closely related taxa, nuclear DNA (nuDNA), with a

slower rate of evolution, were more useful for deciphering older relationships (Simmons et

al., 2004). Further study on molecular phylogenetic of Rhacophoridae and genus under

this family should be constructed to evaluate the evolutionary relationship.

5

I

,

1.2 RESEARH QUESTIONS AND HYPOTHESIS

In this study, the mitochondrial ribosomal 16S genes was used based on Vences et al.

(2005) whom stated that the mitochondrial ribosomal l6S genes fulfill the requirements for

a universal DNA barcoding marker in amphibians and the previous study; Ramlah (2009)

reported that high levels of parsimony informative sites and sequence divergences indicate

that the mitochondrial genes of 16S is a good marker to infer phylogenies of species within

genus Rana. Therefore one question arose:

• Is mitochondrial ribosomal 16S gene a good marker for species divergens for

tree frogs and are they consist of four monophyletic group as listed in the

classification?

In answering these questions, it is hypotesizeds that all the phylogenetic trees obtained

consisted of four monophyletic group which consisting of the four species within genus

Poiypedates.

1.3 RESEARCH OBJECTIVES

The objectives of the study were as the following:

1) To elucidate the interspecific phylogenetic relationship among four species within

genus Polypedates by using mitochondrial ribosomal 16S genes.

2) To observe the interspecific genetic divergences of four species from genus I

Polypedates such P. leucomystax, P. macrotis, P. colletti, and P. otilophus. I

~ I t

6

2.0 LITERATURE REVIEW

Genus Polypedates

There were six families of frogs in Borneo, namely Bombinatoidae (one genera),

Megophryidae (four genera), Bufonidae (six genera), Microhylidae (seven genera),

Ranidae (eight genera), and Rhacophoridae with five genera (Inger and Stuebing 2005).

Liem (1970) made an extensive morphological study of the Old World tree frogs and

established taxonomy to split members of the family Rhacophoridae into 10 genera. Since

then several new suprageneric classifications have been proposed by subsequent authors

(e.g., Channing, 1989; Dubois, 1981, 1992; Bossuyt and Dubois, 2001), and many new

species have been added to some genera (e.g., Rhacophorus. Poiypedates, and Philautus:

Inger et al., 1999; Man amendra-Arachc hi and Pethiyagoda, 2001; Orlov et al., 2001;

Ziegler und Kohler, 2001). One of the genera under family of Rhacophoridae is genus

Polypedates.

The genus Polypedates (Anura: Rhacophoridae), as defined by Brown and Alcala (1994),

is known to contain 16 nominal species, of which 10 occur in Southeast Asia (Glaw et ai .•

2000; Frost, 1985, Frost, 2004; Iskandar and Colijn, 2000). The members of the genus are

distributed from southern China, Sri Lanka and south western and north eastern India south

to Indo-China and Indo-Malaya (Frost, 1985). Of these, four species have been reported

from Borneo (Inger and Stuebing, 2005; Inger and Tan, 1996). Ramlah (2006) reported

that 12 species representing 8.5 % of Bornean frogs have been recorded in peat swamp

forest of Kota Samarahan and none of these are endemic to peat swamp and Ramlah and

Lizanah (2000) reported that 14 species were caught in the peat swamp forest in Borneo

and family Rhacophoridae were most found in the peat swamp.

7

Although the genus Polypedates has been considered synonymous with Rhacophorus by

some authorities, based on morphology (Dubois, 1992) or acoustic data (Matsui and Wu,

1994), and phylogenies of the group, the result show support on the validity of the genus

(Channing, 1989; Wilkinson and Drewes, 2000; Wilkinson et aI., 2(02). There were two

features known to separate these Polypedates species from the genus of Rhacophorus. The

adults of Polypedates have no webbing on the fingers, and the tadpoles of Polypedates

have the eyes set at the side. All adults Rhacophorus species have noticeable webbing on

the hand and in all tadpoles eyes were set on top of the head, not at the sides.

Mitochondrial DNA Genes (mtDNA)

Mitochondrion was the power generator or energy transformer of the cell. Mitochondria

could be found in cytoplasm but maximally dispersed near nucleus. Mitochondrion

composes of 70% protein, 25-30% lipids, approximately 1 % RNA and less than 1% of

DNA (Rajendra and Akhi1esh, 2005). There were many small particles which called

oxysosomes located on the cristae of mitochondria. The oxysosome contains ATP

synthetase enzyme which function in the process of oxidative phosphorylation (Tielens et

ai., 2(02). In this process, oxygen was needed with additional net effect (protons)

transported across the mitochondrial inner membrane. Apart from these compositions,

other protein also present in mitochondria such as cytochrome (Rajendra and Akhilesh,

2002; Russell, 1990).

Mitochondrial DNA was known ar.; genetic material which composed of about 37 genes, all

of which were essential for normal mitochondrial function. Thirteen of these genes were

8

providing instructions for making enzymes involved in oxidative phosphorylation also for

making molecules called transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which

were chemical cousins of DNA. Hillis et aI., (1996) stated that the mitochondrial DNA

(mtDNA) sequences that received the most attention were genes of ribosomal RNA (128

and 168), and control region. It has been one of the most widely used molecular markers

for phylogenetic studies in animals, because of its simple genomic structure (A vise, 2004).

They included the small size of the molecule; the high rate of evolution of mitochondrial

gene sequences, which is 5-1O-fold faster than in nuclear loci; haploid maternal

inheritance; and lack of recombination (8eminaet aI., 2007). Mitochondrial DNA

sequences have usually served as sequence markers to date, which derive their power from

the ability to infer relationship between alleles (Michael, 2009).

However, mitochondrial DNA sequences were frequently utilized for inferring

phylogenetic relationship between genus Polypedates as example in family

Rhacophoridae, due to their properties of having a large copy number, faster evolutionary

rate, maternal inheritance, smaller molecular weight, and a lack of introns (Brown et al.,

1979; Moritz et al., 1987).

Mitochondrial ribosomal16S gene (16S rRNA)

In this study, a mitochondrial ribosomal 168 gene which was a large subunit ribosomal

RNA gene in mtDNA was used. Hillis et aI., (1996) stated that a mitochondrial ribosomal

168 gene was fairly conserved in sequence. The amplified fragment of the 168ar and

168br primers were larger than the 128 fragment (about 550bp compared to 400bp) and

9

seem to evolve more slowly than the mitochondrial genome as a whole. Hence, it was

slightly more useful in phylogenetic reconstruction.

The mitochondrial 16S rRNA gene fullfils the requirements for a universal DNA

barcoding marker in amphibians (Vences et ai., 2005). This comparative sequencing of

small subunit rRNA was now the method of choice for constructing higher level

classifications (Woese, 1987). The 16S and 12S rRNA genes have been selected as

standard markers for phylogeny reconstruction in amphibians, which lead to a near­

complete global dataset of amphibian 16S sequences in the near future.

Suzuki et al., (2010) stated the 16S mitochondrial genes were maternally inherited as a

single unit, they acknowledge that their phylogeny specifically tracks the evolutionary

history of the mitochondrial genome, and the mitochondrial phylogeny provided a

strongly-supported phylogenetic framework for future comparative evolutionary and

genetic analyses.

10

..... ,...

Molecular phylogeny

The advance of molecular technique in this present day has been found the solution to use

mitochondrial DNA which was faster than nuclear DNA and indeed, maternally inherited

(Strachan, 1992). Molecular data obtained from molecular findings was more ~ompleted

and specific and could be used to determine species characters as well as the species

natural history (Baker and Bradley, 2006).

Jackman et al. (1999), described DNA extraction and amplification was conducted. In this

study the mitochondria gene was used. Hillis (1987), in the past two decades, molecular

investigations of systematic problems have progressed from uncommon curiosities to a

standard means of elucidating phylogenetic history_

Bossuyt and Milinkovitch (1999) stated that adaptive radiation has been used as a major

concept in evolutionary biology_ In this context, convergences in morphological,

ecological, and physiological characteristics were usually regarded as occasional

curiosities, i.e., the exception rather than the rule. However, most features whose radiation

has been studied were also the major characters used in the systematic classification of the

organisms bearing them. Characters can therefore be circularly interpreted as showing lack

of convergence through a phylogenetic hypothesis inferred partly from these very same

characters. The development of molecular phylogenetic has contributed a major

breakthrough in the study of these radiations.

Furthermore, a primary objective of phylogenetic studies was to reconstruct the

evolutionary history of a group of organisms. Because the organisms under study have a

11

single history, systematic studies of any set of genetically determined characters should be

congruent with other such studies based on different set of characters in the same

organisms (Bossuyt and Milinkovitch, 1999).

Moreover, most of these phylogenetic hypotheses were based on one or a very few

representative species of each genus, and characters that can be used to distinguish each

genus as a whole have not been presented beyond Liem's original 36 characters (Liem,

1970; Wilkinson and Drewes, 2000). Much of the recent taxonomic reshuffling of known

Rhacophorid species and naming of species has been based mainly on a few superficial

morphological characters that mayor may not represent phylogeny (Chou, 1993; Inger et

aI., 1999), So in this study, the phylogenetic analysis was conducted as to prove it.

12

,. ...

3.0 MATERIALS AND METHODS

3.1 Field sampling

A total of 25 samples (included preserved tissues samples from molecular ecology

laboratory) were collected based on three methods namely stream transect, forest floor

quadrate and forest transect from six different localities which are Matang Wildlife Centre,

Kubah National Park, Bako National Park, Similajau National Park, Bario, east campus,

UNIMAS as shown in Figure 1. Fresh samples were collected from east campus, UNIMAS

(8 individuals). Details of tissues used in this study are shown in Table 1. Those samples

were identified using Inger and Stuebing (2005) and Inger et ai., (1985). The size (snout

vent length), weight of frogs and the microhabitat of the frogs were recorded. Next, the

tissue of fresh samples was cut from the frog thigh muscles. The tissue samples were

stored in -20°C freezer for long term storage. The preserved tissue samples from previous

collection from other selected study sites were also included in this analysis.

13

3.1.1 Study Sites

Figure 1.0 : Study sites and locations for preserved tissues

Study sites and locations for preserved tissues:

a) Matang Wildlife Centre

Matang wildlife center is located about 22km west of Kuching center and generally

consist of flat and lowland forest.

b) Kubah National Park

Kubah National Park is situated within the Matang Ranges about 22 km west of

Kuching City. There is a natural frog pond within the park area.

c) Bako National Park

Bako National Park lies on a rocky headland, the Muara Tebas Peninsula, about 30

km north of Kuching City. The park consists of eight vegetation types; Kerangas

forest, open shrubland, mixed dipterocarp forest, riverine forest, mangrove forest,

beach forest, cliff vegetation and cultivated land and secondary vegetation.

d) Similajau National Park

Similajau National Park is situated about 20 km northeast of Bintulu Town. This

park consist of small areas of mangrove forest, dipterocarp forest, riverine and

kerangas forest.

e) Bario

Bario also known as the Kelabit Highlands, is situated at the upper north of

Sarawak Borneo. The area is a plateau with an altitude of approximately 1,200

meters a.s.l., and formed the uppermost catchments of Sg Baram watershed.

1) East Campus,Unimas

Unimas is located at Kota Samarahan and it is about 30 minutes drive from Kuching

city, Sarawak. Unimas generally consist of peat swamp forest.

15

Table 1.0: Details of tissues samples used in this study (including preserved samples from molecular laboratory and fresh tissues samples).

No. Locality ID sample Accession Total Number number of

saml!les,N 1. Matang Wildlife Centre RZ 038 P.otilophus JF951972 3

(preserved samples) 1232 P.macrotis JF951983 RZ 217 P.otilophus JF951992

2. Kubah National Park RZ05 P.otilophus JF951974 5 (preserved samples) N006 P.otilophus JF951993

N 014 P.otilophus JF951994 N 018 P.otilophus JF951996 N 019 P.otilophus JF951991

3. Bako National Park BNP 061 P.leucomystax JF951978 2 (preserved samples) BNP 074 P.colletti JF951973

4. Bario RZ 9 P.macrotis JF951975 3 (preserved samples) RZ 40 P.macrotis JF951995

RZ 41 P.macrotis JF951979 5. Similajau National Park SNP 001 P.colletti JF951990 4

(preserved samples) SNP 002 P.colletti JF951997 SNP 009 P.colletti JF951977 SNP 012 P.colletti JF951976

6. East Campus, Unimas UE 139 P.leucomystax JF951984 8 (fresh tissues samples) UE 141 P.leucomystax JF951982

UE147 P.leucomystax JF951988 UE 222 P.leucomystax JF951981 UE 225 P.leucomystax JF951986 UE 226 P.leucomystax JF951980 UE 228 P.leucomystax JF951987 UE229 P.leucomystax JF951985

Total of Saml!les 25 *P.leucomystax (Polypedates leucomystax). P.macrotis (Polypedates macrotis), P.colletti ( Polypedates colletti). P.otilophus (Polypedates otilophus)

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