MATCHING PHYLOGENETIC RELATIONSHIP WITH

ADVERTISEMENT CALL CHARACTERISTICS OF THE MALE BORNEAN

HYLARANA BARAMICA

MELYNDA CHEOK KA YI

Bachelor of Science with Honours

(Animal Resource Science and Management)

2012

Faculty of Resource Science and Technology

Matching Phylogenetic Relationship with Advertisement Call Characteristics of the

Male Bornean Hylarana baramica

MELYNDA CHEOK KA YI

This project is submitted in fulfilment of the requirements for the degree of

Bachelor of Science with Honours

(Animal Resource Science and Management)

Department of Zoology

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2012

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an

application for another degree or qualification at this or other university or institutions of

higher learning.

______________________

Melynda Cheok Ka Yi

23992

Animal Resource Science and Management Programme

Department of Zoology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

I

ACKNOWLEDGEMENT

First and foremost, I would like to express gratitude to God for giving me the

strength and wisdom in finishing this final year project. I would also thank my supervisor,

Dr. Ramlah Zainudin for her patience in guiding me to fulfil the requirement of this project.

I kindly thank the postgraduate students and lab assistants, especially to Miss Elvy Quatrin

and Mr. Muhammad Fadzil Amram for their help, guidance, efforts, as well as experiences

in completing this project.

For those who helped me with my work, most sincere gratitude to them for their

constructive critics and excellent advice during preparation of project. I really appreciate

the collaboration with many colleagues, especially to Khatijah Ismail, Nur Alwanie Maruji,

Sharizzaty Rais, Syafiq Zahari, Esther Sheren, Siti Nor Baizurah Malik Chang, Kirupaliny

Susiee, Amaziasizamoria Jumail, Nurziehan Muhamed, Mariana Abdullah, Dellroy Donny

and for whom I have great regard during the project.

Finally, I would like to say thank you to all my family and friends, especially to my

mother for her loving support and encouragement.

II

TABLE OF CONTENTS

Acknowledgement .................................................................................................................. I

Table of Contents .................................................................................................................. II

List of Abbreviations ........................................................................................................... IV

List of Tables ........................................................................................................................ V

List of Figures ..................................................................................................................... VI

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

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

1.1 Problem Statement ........................................................................................... 3

1.2 Objectives ......................................................................................................... 3

1.3 Hypothesis ........................................................................................................ 4

2.0 Literature Review ........................................................................................................... 5

2.1 Phylogeny of anurans ....................................................................................... 5

2.2 Polymerase Chain Reaction (PCR) .................................................................. 6

2.3 Advertisement call characteristics .................................................................... 7

2.4 Call and Genetic Variation Study for Anurans ................................................ 8

2.5 Hylarana baramica .......................................................................................... 9

3.0 Materials and Methods ................................................................................................. 11

3.1 Sample Location ............................................................................................. 12

3.2 Sample Collection .......................................................................................... 14

3.3 DNA Laboratory Work .................................................................................. 15

3.3.1 DNA Extraction of Tissue Samples .................................................. 15

3.3.2 Polymerase Chain Reaction (PCR) ..................................................... 16

3.3.3 PCR Product Purification and Sequencing ........................................ 18

3.3.4 Phylogenetic Analysis ......................................................................... 19

3.4 Call Analysis ................................................................................................. 20

3.5 Matching Analysis of DNA and Call Characteristics ..................................... 22

III

4.0 Results .......................................................................................................................... 23

4.1 Tissue Sample Collection ............................................................................... 23

4.2 DNA Extraction .............................................................................................. 23

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

4.4 DNA Purification ........................................................................................... 26

4.5 Sequence Analyses ......................................................................................... 28

4.6 Phylogenetic Analyses .................................................................................. 28

4.7 Call Characteristic Analyses .......................................................................... 32

5.0 Discussion .................................................................................................................... 38

5.1 DNA Extraction .............................................................................................. 38

5.2 Polymerase Chain Reaction (PCR) ................................................................ 38

5.3 DNA Sequence .............................................................................................. 40

5.4 Phylogenetic Analyses .................................................................................. 40

5.5 Call Characteristic Analyses .......................................................................... 41

6.0 Conclusion and Recommendation ................................................................................ 43

7.0 References .................................................................................................................... 44

Appendices

iv

List of Abbreviations

mtDNA: Mitochondrial deoxyribonucleic acid

DNA: Deoxyribonucleic acid

CTAB: Cetyltrimethylammonium bromide

NAOAc: Sodium acetate

NaCl: Sodium chloride

rpm: Revolutions per minute

ddH2O: Double-distilled water

HCl: Hydrogen chloride

KCl: Potassium chloride

NH42SO4: Ammonium sulfate

MgCl2: Magnesium chloride

dNTPs: Deoxyribonucleotide triphosphates

µL: Microlitter

MEGA: Molecular Evolutionary Genetics Analysis

COI: Cytochrome Oxidase I

bp: base pair

AGE: Agarose Gel Electrophoresis

UPGMA: Unweighted Pair Group Method with Arithmetic Mean

MWC:Matang Wildlife Center

BNP: Bako National Park

KS: Kota Samarahan (UNIMAS)

MHQ: Mulu National Park (Headquarters)

v

List of Tables

Page

Table 1 PCR master mix of reaction mixture ................................................................ 16

Table 2 Primers for partial COI ..................................................................................... 17

Table 3 PCR profile to amplify the DNA ...................................................................... 17

Table 4 Measured characteristics list of call variables in this study ............................. 20

Table 5 Pairwise genetic distance between species and outgroup ................................ 29

Table 6 The nucleotide composition (%) of the 6 individuals. ...................................... 29

Table 7 Base frequencies of nucleotide composition of 6 individuals ......................... 29

Table 8 The meaningful variables of 27 call characteristics of Hylarana baramica ... 37

vi

List of Figures

Page

Figure 1 Olympus Linear PCM Recorder LS-11 ....................................................... 11

Figure 2a KE-EN TM 02 Type K thermometer .......................................................... 11

Figure 2b Insertion probe of the thermometer ............................................................. 11

Figure 3 Map of Sarawak .......................................................................................... 13

Figure 4 Localities of the three study sites from Kuching ........................................ 13

Figure 5 The PCR cycle of amplification .................................................................. 17

Figure 6 AGE showing the extraction using CTAB protocol. .................................. 24

Figure 7 AGE showing the PCR product ................................................................... 25

Figure 8 AGE showing the PCR product ................................................................... 25

Figure 9 AGE showing the purification product ....................................................... 26

Figure 10 AGE showing the purification product ....................................................... 27

Figure 11 AGE showing the purification product ....................................................... 27

Figure 12 Neighbour-joining (NJ) tree ........................................................................ 31

Figure 13 Maximum-parsimony (MP) tree ................................................................. 32

Figure 14a-l The descriptive measurements obtained by SoundRuler for the call

characteristics ........................................................................................ 33-35

Figure 15 Euclidean distances in UPGMA clustering method ................................... 36

1

MATCHING PHYLOGENETIC RELATIONSHIP WITH ADVERTISEMENT

CALL CHRACTERISTICS OF THE MALE BORNEAN HYLARANA BARAMICA

Melynda Cheok Ka Yi

Animal Resource Science and Management Program

Department of Zoology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

The study is done with samples from four different locations, which are the east campus of

Universiti Malaysia Sarawak (UNIMAS), Matang Wildlife Centre, Bako National Park, and Mulu

National Park. The advertisement calls of male anurans are different between species as it is a

motivation for them to reproduce their next generation, and also in ensuring the survivorship of the

particular species by maintaining reproductive isolation among population. In this study, the

phylogenetic relationship of different Hylarana baramica population were determine by using

partial mitochondrial DNA COI gene. DNA extraction protocol was used for the extraction of

DNA obtained from the muscle tissue samples. Whereas annealing temperature in PCR

amplification of the species is 50°C. The phylogenetic relationship was analysed using

Phylogenetic Analysis Using Parsimony (PAUP) version 4.0. As for the analysis of advertisement

call characteristics, SoundRuler Acoustic Analysis version 0.9.6.0. was used to analyse 38

characters from the recorded call for all of the individuals. While, MultiVariate Statistical Package

(MVSP) version 3.1 was used to generate cluster analysis of the call characteristics. The call

characteristic analysis showed no specific clustering group of each population. However, the

phylogenetic data are insufficient to match the phylogenetic relationship with advertisement call

characteristics of the different population of Hylarana baramica.

Key terms: phylogenetic relationship, advertisement call, PAUP, MVSP, Hylarana baramica

ABSTRAK

Kajian telah dibuat dengan sampel daripada empat lokasi yang berlainan, iaitu kampus timur

Universiti Malaysia Sarawak (UNIMAS), Pusat Hidupan Liar Matang, Taman Negara Bako, dan

Taman Negara Mulu. Panggilan iklan daripada anuran jantan adalah berbeza antara spesis

kerana itu adalah salah satu motivasi bagi mereka untuk reproduksi bagi generasi yang akan

datang, di samping memastikan kelangsungan hidup spesis tertentu dengan mengekalkan

pengasingan pembiakan antara populasi. Dalam kajian ini, hubungkait filogenetik untuk populasi

Hylarana baramica yang berlainan telah ditentukan menggunakan gene mitokondria DNA COI.

Protokol pengesktrakan DNA telah digunakan untuk mengekstrak DNA daripada sample tisu otot.

Manakala suhu annealing bagi spesis ini dalam PCR adalah 50°C. Hubungkait filogenetik telah

dianalisa dengan menggunakan “Phylogenetic Analysis Using Parsimony” (PAUP) versi 4.0. Bagi

analisa panggilan iklan pula, “SoundRuler Acoustic Analysis” versi 0.9.6.0. telah digunakan untuk

mengkaji 38 ciri-ciri daripada rekod panggilan individu yang sama. Manakala, “MultiVariate

Statistical Package” (MVSP) versi 3.1 telah digunakan untuk menghasilkan analisis kluster bagi

ciri-ciri panggilan. Analisa daripada ciri-ciri panggilan telah menunjukkan bahawa tiadanya

kumpulan kluster mengikut populasi. Walau bagaimanapun, data filogenetik adalah tidak

mencukupi untuk menghubungkaitkan filogenetik dengan ciri-ciri panggilan iklan bagi Hylarana

baramica daripada populasi yang berbeza.

Kata kunci: hubungkait filogenetik, panggilan iklan, PAUP, MVSP, Hylarana baramica

2

1.0 INTRODUCTION

Anurans are the first tetrapods emerged onto land 360 million years ago at the end

of Devonian period, evolving from the sacropterygian (lobe-finned) fishes through

selective forces being applied upon them (Stocker, 2000). According to Frost et al. (2011),

anurans are the most successful, abundant and diverse of living amphibians which

compromised 32 families, 372 genera, and nearly 6000 species.

The family Ranidae (True Frogs) are distributed worldwide except Australia, and

contains seven subfamilies which include Diacroglossinae, Petropedetinae, Ptychadeninae,

Pyxicephalinae, Raninae, Ranixalinae, and Tomopterninae (Zug et al., 2001). By

comparing all of the anurans, family Ranidae has the widest distribution of all frog families

with 54 genera and 772 species, and is the largest family in Borneo where its members are

dwellers of river, stream, and forest floor (Inger and Stuebing, 2005).

There are at least 150 species of Bornean frogs have been recorded by Inger and

Stuebing (2005), which represented by seven families: Bombinatoridae, Megophryidae,

Bufonidae, Microhylidae, Ranidae, Dicroglossidae, and Rhacophoridae (Haas and Das,

2012). Haas and Das (2012) states that the current definition of Ranidae is 26 Bornean

species, with many species are endemic in Malaysia (Inger and Stuebing, 2005). According

to Inger (2005), there are five genera of Ranidae occur in Borneo, namely, Micrixalus,

Ooeidozyga, Staurois, Amolops, and Rana. The Bornean species of genus Rana have very

diverse habits and form, with sizes range from small to large (Inger and Stuebing, 2005).

The purpose of this study is to determine whether there are relationship between the

evolutionary development and diversification of anuran to their advertisement call. For this,

Bornean male Hylarana baramica was chosen as their loud and distinct advertisement call

can be easily distinguished from the call of another anuran species. With the extensive peat

3

swamp cover in Sarawak (Ministry of Natural Resource and Environment, 2006), the near

coast swampy peat forest where the H. baramica inhibits can be found in the west part of

Borneo island, hence making this study possible to be conducted.

1.1 Problem Statement

Hylarana baramica, being one of the species from the family Ranidae is studied for their

phylogenetic relationship and comparison to their advertisement call. The male‟s

advertisement call of H. baramica is usually loud and fast series of pulses with increasing

intensity (Haas and Das, 2012), and can be distinguish from the call of another species.

Hence, it is important to define whether there are concordance in their phylogeny and

advertisement call as this can be used as the main key information of species identification,

besides to determine if there are any differences in call characteristics among the

populations of the same species.

1.2 Objectives

This study was undertaken with the following objectives:

i. To document the call of the male Bornean Hylarana baramica from different

populations.

ii. To infer the molecular phylogeny (mtDNA) of the male Bornean H. baramica

using partial COI primer.

iii. To match the advertisement call with the DNA of male Bornean H. baramica by

comparing their phylogenetic and call characteristics analysis.

4

1.3 Hypothesis

The hypotheses for this project are:

H0: There are no concordant relationships between the advertisement call of male Bornean

Hylarana baramica with their DNA evolution.

HA: There are concordant relationships between the advertisement call of the male Bornean

Hylarana baramica with their DNA evolution.

5

2.0 LITERATURE REVIEW

2.1 Phylogeny of Anurans

Ryan and Rand (1999) evaluated how phylogenetic models can influence studies of

behavioural evolution, in which they determine the sensitivity of the results and conclusion

of the call character value at ancestral nodes by using the ancestral call for Physalaemus

pustulosuss species group and some close relative. The data obtained supported their

previous conclusions about the range of female preferences, discrimination between calls,

and responses of the female to the same signal variation in discrimination and recognition

experiments.

While according to Masta et al. (2002), the interspecific hybridization will not

affect the genealogical reconstruction of mitochondrial sequences and their relationships

can be depicted by bifurcating genealogies as the mitochondrial genes do not recombine.

Thus, mtDNA is ideal for inferring the maternal genealogy of animal species that are

known to hybridize as it appears to be maternally inherited and not recombining in most

metazoans.

The study of phylogeny of the New World true frogs (rana) by Hillis and Wilcox

(2004) examined the phylogeny and diversification of Rana in the New World based on

mitochondrial DNA, and tests the significance of differences between current and previous

estimates of New World Rana phylogeny. They concluded that the species of New World

Rana vary in other aspects of call production, and the study should serve as an ideal model

system for the study of frog call evolution.

6

2.2 Polymerase Chain Reaction (PCR)

PCR is a deceptively simple technique where repetitive bidirectional DNA

synthesis via primer extension of a specific region of nucleic acid (Dieffenbach and

Dveksler, 2003). They explained that the processes are functionally programmed into a

PCR machine so that each reaction can proceeds through each step in an orderly manner.

There are studies on phylogenetic relationships where they are done by using PCR

as the amplifier of DNAs. The analysis from the study by Garcia-Paris et al. (2003) had

supported phylogenetic relationship of ancient anurans using two mitochondrial genes.

Whereas Mauro et al. (2004) have used PCR to determine the phylogenetic relationships of

frogs, based on mtDNA and nuclear genes.

PCR has revolutionised the molecular biology, where the number of DNA

molecules can be efficiently increased in a logarithmic and controlled fashion (Viljioen et

al., 2005). According to McPherson and Moller (2006), PCR provides the tool for the

research scientist; where routine and repetitive DNA analyses are adapted to meet specific

needs in which speed and accuracy are important factors.

Campbell et al. (2008) had stated that PCR has been used to amplify DNA from a

variety of sources. The study by Marosi et al. (2010) had extracted DNA from toe clips of

adult frogs and tail clips of tadpoles. While frog thigh muscles were also used to extract

mtDNA genome for PCR amplification in the genetic study of Hylarana erythraea by

Ramlah et al. (2010).

7

2.3 Advertisement Call Characteristics

The acoustic characteristics of advertisement calls can vary among populations and

individuals although they are species-specific (Capranica et al., 1973; Nevo and Capranica,

1985; Sullivan, 1985; Ryan and Wilczynski, 1988, 1991; Wagner, 1989; Keddy-Hector et

al., 1992; Wilczynski et al., 1992). It is the intraspecific diversity in call characteristics that

allows females to discriminate among potential conspecific mates on the basis of some of

the same acoustic parameters used for species identification (Ryan et al., 1992).

Essentially all male frogs incorporate some form of advertisement call in their

vocalization that is usually a necessary precursor to successful courtship and mating,

although there are situation where animals vocalize differently among species (Wells, 1988;

Rand, 1988; Gerhardt, 1988). Acoustic communication serves as an important tool in the

social behaviour of most anuran amphibians; advertisement call with unique temporal and

spectral parameters enabling females to identify and select conspecific males for mating

(McClelland et al., 1996).

Frogs in multispecies communities were faced with the problem of interspecific

acoustic interference; therefore effective communication is done by necessary modification

of calls (Duellman and Trueb, 1994). They stated that there are possibilities to utilize

vocalization in some limited analyses of phylogenetic relationships. The systematic studies

of frogs have also shown that species sharing morphological and/or biochemical attributes

also have structurally similar advertisement calls.

According to Ramlah et al. (2009), the differences in calling characteristics

between species is shown to have modification system in producing sound, and every call

produced by an individual must have their own characteristics. From the study, the

8

characteristic of Hylarana baramica is consistent with the study done by Leong et al.

(2003) in terms of calling note, dominant frequency and harmonic calling.

Ramlah et al. (2010) also revealed that advertisement call characteristics are good

character in species recognition, and genetic distance can be used to infer speciation and

reproductive isolation. The study also provides a baseline data on advertisement call

characteristics of Bornean frogs. They stated that with sufficient data, advertisement call

can be a tool for discriminating species.

2.4 Call and Genetic Variation Study for Anurans

Ryan et al. (2007) examined how female preferences for mating calls vary with

genetic distances from within the population to between species. Their study shows that

there is significant genetic variation and mating call variation among population of tungara

frogs. However, there is no relationship displayed between the mate preference and genetic

distance within the population.

Sheridan et al. (2010) found that the taxonomic conclusion done by call and DNA

data from both Thailand and Singapore populations differs in three Southeast Asia anurans.

They found that the calls of Polypedates leucomystax indicate that the populations have

been diverged, although the genetic variations for 16S rRNA are relatively small. As for

Microhyla heymonsi and Hylarana erythraea, similar calls indicate that the two

populations are conspecific, but the genetic distances are larger than those observed in

Polypedates leucomystax.

Cryptic diversity in frog species can also be determined by using the mtDNA and

nDNA (nuclear DNA) sequences, morphological and bioacoustics traits, as done by Funk

9

et al.(2012) for the study of uncovering cryptic species in the Amazon basin. From the

intensive sampling for genetic and call data, they revealed that the frog diversity is much

higher than the estimation of previous study.

2.5 Hylarana baramica

H. baramica can be recognized by their colouration: dark brown dorsal, lighter on

sides, dark spots scattered on the dorsal and sides, cream-coloured venter, more or less

heavily spotted with dark brown, and limb with dark crossbar dorsally (Inger, 1966; Inger,

1990). This species was describe by Inger (1966) as a “body stout to slender, leg slender,

adults were ranged 40-67mm, head obtusely pointed, snout longer than eye, projecting

slightly in profile, nostril much nearer to tip of snout than to eye, interorbital equal to or

wider than upper eyelid and tympanum conspicuous”. The skin of H. baramica has

scattered, small, and rounded bump at the back and sides (as shown in Appendix 1.0)

which had been described by Inger and Stuebing (2005).

Adult H. baramica sizes around 40mm in males and up to 67mm in females (Haas

and Das, 2012). Inger has noted that the females of this species are larger than males.

However, the males have relatively larger tympanum and shorter tibia. According to Inger

and Stuebing (2005), the males of this species sing in a repetitively low chirp during

breeding period. There are two major groups of frogs which are found close to human

inhabitation and frogs that are confined in forest area (Inger and Stuebing, 2005). H.

baramica lies in the later where they are distributed in swampy forests.

From the ICUN Red List Species Account, the range description of the species H.

baramica is known from a number of localities in Borneo (Sabah and Sarawak - Malaysia,

10

and Kalimantan – Indonesia), Johor and Selangor States in Peninsular Malaysia, and also

from the island of Singapore (as shown in Appendix 2.0). It also states that the Rana

species is known from a variety of lowland floodplain situation, including peat swamp

forest and swampy flatland primary forest at low elevations. Although it is known to breed

in water, but its breeding habits are poorly known. The population trend of this species is

decreasing due to the major threats of habitats loss (Haas and Das, 2012).

11

3.0 MATERIALS AND METHODS

Materials used for documenting the calls of H. baramica were Olympus Linear

PCM Recorder LS-11 (Figure 1), while their body temperatures were detected using KE-

EN TM 02 Type K thermometer (Figure 2a and 2b). Molecular laboratory analyses were

used as in Ramlah et al. (2010).

Figure 1 Olympus Linear PCM Recorder LS-11

Figure 2a KE-EN TM 02 Type K

thermometer

Figure 2b Insertion probe of the

thermometer

12

3.1 Sample Location

The field survey had been done in UNIMAS campus, Matang Wildlife Center,

Bako National Park and Mulu National Park. Four populations of the H. baramica were

studied to identify their genetic and calling variation among the chosen populations. The

four locations were chosen due to their swampy areas which are most likely to be inhabited

by H. baramica.

UNIMAS (1°28‟28.90” N, 110°25‟57.74” E) is located in Kota Samarahan, where

it is about 30 km south east of Kuching (capital city of Sarawak). The lands in Samarahan

are mostly non-hilly, flat and low-lying. Most of the soils found here are peat soils.

Matang Wildlife Centre (1°36‟37.04” N, 110°9‟36.13” E) is located in Matang,

about 30 km from Kuching City and it takes about 40 minutes to reach the Centre. It is

situated at the western corner of the Kubah National Park, consisting of about 179 hectares

of lowland forest.

Bako National Park (1°43‟05.42” N, 110°27‟45.41” E) has a variety of plant

species and vegetation. There are seven complete eco-systems in Bako: Beach vegetation,

Cliff vegetation, Kerangas or heath Forest, Mangrove Forest, Mixed Dipterocarp Forest,

Padang or Grasslands Vegetation and Peat Swamp Forest. It covers an area of 27.27 square

kilometres, and is approximately 40 kilometres away by road from Kuching.

Mulu National Park (4°2‟17.76” N, 114°55‟12.93” E) is located near Miri, which

serves as the main gateway. The park contains eight different types of forests, which

include peat swamp and mixed dipterocarp, moss forest and stunted upper monane

vegetation.

13

Figure 3 and Figure 4 show the locations of the four populations. Populations from

UNIMAS, Bako National Park and Matang Wildlife Centre are within 40km radius from

Kuching, whereas Mulu National Park is located in the much further up to the north-east of

Kuching.

Figure 3 Map of Sarawak retrieved from Google Earth ver2010 (A = UNIMAS; B = Mulu

National Park; C = Bako National Park; D = Matang Wildlife Center).

Figure 4 Localities of the three study sites from Kuching retrieved from Google Earth

ver2010 (A = UNIMAS; C = Bako National Park; D = Matang Wildlife Center).

14

3.2 Sample Collection

The collection of samples were done by using forest transect in all of the study sites,

which was conducted starting from October 2011 until February 2012. Collection was

done at night and took about two hours which started at around 1900 hours until 2100

hours along the forest trail. The location of the H. baramica were first located by their

vocalization, which was then followed by the recording of their call using Olympus Linear

PCM Recorder LS-11 for about two minutes long.

After recording, the frogs were captured by hand and their body temperature is

taken using a thermometer. The probe of the thermometer (Figure 2b) was inserted into the

anal, and the first reading was taken. A pair of gloves was worn during the capture to avoid

direct contact with the frog. Otherwise, the body temperature of the calling frogs will

immediately increase due to body heat transfer from handling it.

Finally, the captured frogs were retained in 6cm x 6cm plastic bag with some

leaves and water before proceed to processing in the laboratory. Their thigh muscle tissue

sample were taken, which were immediately put in a vial tube containing absolute ethanol.

The vial was labelled to match the specimen tags, along with the species name, date and

location of collection.

The tissue samples were preserved in ethanol, as Bucklin and Allen (2003)

mentioned that ethanol preserved samples have higher yield and larger fragments

compared to those preserved in formalin. Study by Zimmermann et al. (2008) had shown

that preservation of tissue samples in formaldehyde produces low DNA yield, where the

lysis process of formalin stored tissue is the main obstacle in obtaining DNA.

15

The vials containing sample tissue were then stored in -20° freezer before

continued to the extraction process. Meanwhile, the body of the individuals were fixed in

buffered formalin for a few days before being preserved in 70% ethanol as voucher

specimens.

3.3 DNA Laboratory Work

3.3.1 DNA Extraction of Tissue Samples

The partial length of COI gene in the mtDNA genome from the tissue samples were

extracted using modified CTAB DNA extraction protocol (Grewe et al., 1993).

About one cubic millimeters of tissue sample was grinded in a 1.5ml eppendorf

tube with 100 µl CTAB buffer and 10 µl Proteinase K (20mg/ml). Next, another 600 µl of

CTAB buffer was added into the tube and mixed well. The tube was incubated in the water

bath at 65°C until the tissue completely dissolved before adding 600 µl of chroloform-

isoamyl alcohol (24:1) and shook for 2-3 minutes. It was then centrifuged at 13000rpm for

20 minutes. An equal volume of cold absolute ethanol (99%) was added and mixed well.

Next, the tube was spun at 13000rpm for 20 minutes. The ethanol was discarded but DNA

pellet was made sure to be still intact at the bottom of the tube. An equal volume or more

of cold 70% ethanol and 25 µl of 3M NaCl was added again. It was then spin at 13000rpm

for 20 minutes. After the ethanol was discarded, the excess solution was left to dry at room

temperature. The remaining pellet was then re-dissolved in ddH2O.

To ensure that there is any DNA obtained before proceeding to the PCR

amplification process, the extraction product was visualized by loading 2µL of the product

into Agarose Gel Electrophoresis (AGE) containing ethidium bromide. Two microliter of