POPULATION GENETICS AND DISTRIBUTION
OF TWO SYMPATRIC FROG SPECIES IN
PENINSULAR MALAYSIA, Fejevarya cancrivora
(Gravenhorst, 1829) AND Fejevarya limnocharis
(Boie, 1834)
AMIRAH HURZAID
UNIVERSITI SAINS MALAYSIA
2013
POPULATION GENETICS AND DISTRIBUTION OF TWO SYMPATRIC
FROG SPECIES IN PENINSULAR MALAYSIA, Fejevarya cancrivora
(Gravenhorst, 1829) AND Fejevarya limnocharis (Boie, 1834)
By
AMIRAH HURZAID
Thesis submitted in fulfilment of the requirements
for the degree of
Masters of Science
November 2013
This piece of work is a token of dedication to my beloved mom,
Khairun Mahmood.
A special dedication…
My late father
Allahyarham Hurzaid Hj. Mohamad Isa
Who passed away on 18th February 2001.
-1st April 2013-
ACKNOWLEDGEMENTS
‘On no soul doth Allah Place a burden greater than it can bear’
[Al Baqarah: 286]
Alhamdulillah, all praises and thanks to Allah the Almighty for His consent and blessing this study is finally completed.
First and foremost, I would especially like to express my heartfelt thanks and appreciation to both my supervisor and co supervisor, Prof. Ibrahim Jaafar and Prof. Siti
Azizah Mohd Nor who have always being supportive, encouraging and understanding of my difficulties in completing this thesis. I would also give my gratitude to Universiti
Sains Malaysia for funding this project under grant no.: USM- RU- PRGS
1001/PJJAUH/834059 and Ministry of Higher Education, Malaysia and USM for the
USM Academic Staff Training Scheme (ASTS) scholarship award.
A special thanks goes to Ana, Zaza, Syaida, Syaibah, Daniel, Wan, Semah,
Dilla, Amer, En. Shara, Pija, En. Shahfiz, Ika and Amet for their vital encouragement, understanding and assistance. Without them, this research would not be completed successfully. I would like to thank my dear labmates Yap Chee Hui, Kak Naz, Jam, Dr.
Tan Min Pau, Kak Adel, Lim, Lutfi, Su Yin, Balkhis, Elham, Su, Katy, Dr/Om Icin, Dr.
Abdullah, Leen, Zai and Rina who were always there when I needed them. To all my beloved friends and those that have not been mentioned here, I appreciate their support and sharing of joy. Last, but not least, I thank my beloved sister, Yafroh Hurzaid for her spirit and strength that really inspired me to complete this thesis and of course, to all my family members especially my mother for their patience, encouragement and nonstop absolute support I received along the way. Thank you for their endless love and always being by my side. I am indebted for the rest of my life.
ii
TABLE OF CONTENTS
Acknowledgements ii
Tables of Contents iii
List of Tables viii
List of Figures x
List of Plates xii
List of Symbols and Abbreviations xiii
Abstrak xiv
Abstract xvi
CHAPTER 1 INTRODUCTION
1.1 Introduction 1
1.2 Objectives 5
CHAPTER 2 LITERATURE REVIEW
2.1 Amphibians in general 6
2.2 Threats to amphibians 7
2.3 Taxonomy and Species Description
2.3.1 Taxonomic status of Fejevarya cancrivora 9
2.3.2 Taxonomic status of Fejevarya limnocharis 12
2.4 Morphology 14
2.4.1 Fejevarya cancrivora 15
iii
2.4.2 Fejevarya limnocharis 16
2.5 Distribution, Habitat and Biology
2.5.1 Fejevarya cancrivora 17
2.5.2 Fejevarya limnocharis 19
2.6 Status of Fejevarya cancrivora and F. limnocharis 19
conservation in Asia
2.7 Genetic Variation 21
2.7.1 Genetic marker: Mitochondrial DNA (mtDNA) in 22
general
2.7.2 Application of mtDNA in amphibian research 26
2.7.3 D-loop analysis 27
2.8 Application of Geographic Information System (GIS) in 28
amphibians
CHAPTER 3 MATERIALS AND METHODS
3.1 Sampling Site 31
3.1.1 Sampling of Fejevarya cancrivora 31
3.1.2 Sampling of Fejevarya limnocharis 34
3.2 Sampling Method 35
3.3 Analysis in Amphibian Laboratory 35
3.4 Population Genetic Analysis of Two Sympatric Frog Species 36
in Peninsular Malaysia based on partial mitochondrial
D-loop Gene
iv
3.5 Sample preparation and preservation 37
3.6 DNA Extraction 39
3.7 Quality and Quantity of DNA Analysis by spectrophometry 40
3.8 Analysis of mtDNA D-loop Gene
3.8.1 Polymerase Chain Reaction (PCR) Procedures 42
3.8.2 PCR Optimisation 43
3.8.2.1 Annealing temperature optimisation 43
3.8.3 Agarose Gel Preparation 43
3.8.4 Analysis of PCR Product 44
3.8.5 Purification of PCR products 44
3.8.6 Sequencing 45
3.8.7 Data Analysis of mtDNA D-loop Gene 46
3.8.7.1 Multiple Sequence Alignment and 46
Identification of Haplotypes in Fejevarya
cancrivora and F. limnocharis Populations
3.8.7.2 Genetic variability within and between
populations 46
3.8.7.3 Phylogenetic Tree 47
3.8.7.4 Minimum Spanning Network 48
3.9 GIS mapping of Fejevarya cancrivora and F. limnocharis 48
v
CHAPTER 4 RESULTS
4.1 Genomic DNA Extraction & Purity of Genomic DNA 55
4.2 Mitochondrial DNA Sequencing of D-loop Gene 55
4.2.1 Amplification of D-loop Gene 55
4.2.2 Purification of PCR Product 55
4.2.3 Sequencing of Purified PCR Product 57
4.3 Data Analysis of partial mtDNA D-loop Gene of
Fejevarya cancrivora populations
4.3.1 Intrapopulation Genetic Diversity 57
4.3.2 Interpopulation Genetic Variability 60
4.3.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony (MP) 61 Analysis
4.3.2.2 Analysis of Molecular Variance (AMOVA) 63
4.3.2.3 Minimum Spanning Network 64
4.4 Data Analysis of partial mtDNA D-loop Gene of
Fejevarya limnocharis populations
4.4.1 Intrapopulation Genetic Diversity 66
4.4.2 Interpopulation Genetic Variability 70
4.4.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony (MP) 72 Analysis
vi
4.4.2.2 Analysis of Molecular Variance 74 (AMOVA)
4.4.2.3 Minimum Spanning Network 79
4.5 GIS mapping of Fejevarya cancrivora and F. limnocharis 81
CHAPTER 5 DISCUSSION
5.1 Genetic Diversity of Fejevarya cancrivora 87
5.2 Population Structure of Fejevarya cancrivora 88
5.3 Genetic diversity of Fejevarya limnocharis 90
5.4 Population structure of Fejevarya limnocharis 95
5.5 Implications for conservation 99
5.6 GIS mapping on Fejevarya cancrivora and F. limnocharis 100
CHAPTER 6 CONCLUSION 102
References 104
Appendices
List of publications
vii
LIST OF TABLES
Page
Table 3.1 Summary of sampling areas based on habitat specificity of 33
Fejevarya cancrivora and F. limnocharis.
Table 3.2 Location, region, coordinate and sample size for 34
Fejevarya cancrivora populations in northern Peninsular
Malaysia.
Table 3.3 Location, region, coordinates and sample size for 35
Fejevarya limnocharis populations in Peninsular Malaysia.
Table 3.4 Primers used to amplify D- loop mtDNA gene. 43
Table 3.5 PCR profile used to amplify D-loop mtDNA gene. 43
Table 3.6 Primary data of Fejevarya cancrivora. 50
Table 3.7 Primary data of Fejevarya limnocharis. 51
Table 3.8 Secondary data of Fejevarya cancrivora. 52
Table 3.9 Secondary data of Fejevarya limnocharis. 53
Table 4.1 Haplotypes identified in six (6) populations of F. cancrivora. 58
Table 4.2 Distribution of nine (9) observed haplotypes, number of 59
polymorphic sites, nucleotide diversity (π), number of
haplotypes and haplotype diversity (ɦ) among 16
populations of F. cancrivora.
Table 4.3 Mean pairwise genetic distance among six (6) 61
F. cancrivora populations.
Table 4.4 Results of AMOVA of F. cancrivora populations. 64
viii
Table 4.5 Population differentiation (FST) among six (6) F. cancrivora 64
samples based on mtDNA D- loop sequence.
Table 4.6 Haplotypes identified in 16 populations of F. limnocharis. 68
Table 4.7 Distribution of 14 observed haplotypes, number of 69
polymorphic sites, nucleotide diversity (π), number of
haplotypes and haplotype diversity (h) among 16 populations
of F. limnocharis.
Table 4.8 Mean pairwise genetic distance index between (below 71
diagonal) and within (bold), and geographical distance
of 16 F. limnocharis populations (above diagonal).
Table 4.9 AMOVA results of 16 F. limnocharis populations partitioned 76
into three groups according to regions (northwest, central west
and east).
Table 4.10 AMOVA results of 16 F. limnocharis populations after 76
partitioning into two groups (group 1: northwest populations;
group 2: central west and east populations combined).
Table 4.11 Population divergence (FST) between F. limnocharis populations 78
based on mtDNA D- loop sequence.
ix
LIST OF FIGURES
Page
Figure 1.1 General anatomy of frogs (Berry, 1975). 15
Figure 2.1 The gene organization of the Fejervarya cancrivora 24
mitochondrial genome.
Figure 2.2 The gene organization of Fejevarya limnocharis mitochondrial 25 genome.
Figure 3.1 Sampling sites of both species in Peninsular Malaysia. 32
Figure 3.2 Flow-chart of population DNA analysis. 38
Figure 3.3 Extraction Protocol for DNeasy Kit (QIAGEN). 41
Figure 3.4 Methodology flowchart. 49
Figure 4.1 NJ tree of six (6) F. cancrivora populations. 62
Figure 4.2 MP tree of six (6) F. cancrivora populations. 63
Figure 4.3 Minimum spanning network for six (6) F. cancrivora 65
populations.
Figure 4.4 NJ tree of 16 F. limnocharis populations. 73
Figure 4.5 MP tree of 16 F. limnocharis populations. 74
Figure 4.6 Minimum spanning network of 16 F. limnocharis 80
populations.
x
Figure 4.7 A minimum spanning network of 14 haplotypes obtained from 81
16 F. limnocharis populations classified according to region.
Figure 4.8 The distribution of Fejevarya cancrivora and F. limnocharis in 82
Peninsular Malaysia.
Figure 4.9 The distribution of Fejevarya cancrivora and F. limnocharis in 83
Figure 4.10 The distribution of Fejevarya cancrivora and F. limnocharis in 84
Perak, Selangor and Kuala Lumpur.
Figure 4.11 The distribution of Fejevarya cancrivora and F. limnocharis in 85
Melaka, Negeri Sembilan and Johor.
Figure 4.12 The distribution of Fejevarya cancrivora and F. limnocharis in 86
Kelantan, Terengganu and Pahang.
xi
LIST OF PLATES
Page
Plate 2.1 Fejevarya cancrivora (Gravenhorst, 1829) 11
Plate 2.2 Fejevarya limnocharis (Boie, 1834) 14
Plate 4.1 DNA extraction results of F. cancrivora and F. limnocharis 56
on a 0.8% (w/v) agarose gel.
Plate 4.2 PCR products run on a 1.7% (w/v) agarose gel. 56
Plate 4.3 Purified D-loop PCR products run on a 1.7% (w/v) agarose 57
gel.
xii
LIST OF SYMBOLS AND ABBREVIATIONS
π nucleotide diversity h haplotype diversity AMOVA Analysis of Molecular Variance bp Basepair DNA Deoxyribonucleic Acid dNTP Dinucleotide triphosphate EDTA Ethylenediamine tetra-acetic acid EtBr Ethidium Bromide EtOH Ethanol IUCN International Union for the Conservation Nature kb Kilobase mM Milimolar mtDNA Mitochondrial DNA MW Mouth Width OD Optical density rpm rotations per minute SVL Snout –Vent Length TBE Tris borate EDTA TD Tympanum Diameter TE Tris EDTA TL Tibia Length UV Ultraviolet
xiii
GENETIK POPULASI DAN TABURAN DUA SPESIES KATAK SIMPATRIK DI
SEMENANJUNG MALAYSIA, Fejevarya cancrivora (Gravenhorst, 1829) DAN
Fejevarya limnocharis (Boie, 1834)
ABSTRAK
Satu kajian genetik populasi dua spesies katak simpatrik di Semenanjung
Malaysia, Fejevarya cancrivora dan F. limnocharis telah dijalankan dengan menggunakan segmen D-loop yang berevolusi tinggi. Tinjauan genetik telah dijalankan ke atas enam populasi (26 individu) F. cancrivora di kawasan utara Semenanjung
Malaysia dan 16 populasi (106 individu) F. limnocharis melibatkan kawasan Barat Laut,
Barat Tengah dan Timur Semenanjung Malaysia. Secara umumnya, F. cancrivora menunjukkan genetik variasi yang sangat tinggi bagi populasi Pulau Langkawi dan ketiadaan variasi bagi populasi Jitra. Pohon filogeni ‘Neighbour Joining’ (NJ) dan
‘Maximum Parsimony’ (MP) menunjukkan topologi yang sama dimana populasi Pulau
Langkawi membentuk klad tersendiri. Oleh itu, dicadangkan bahawa populasi Pulau
Langkawi mencapah jauh dengan kadar mutasi yang tinggi dari haplotip utama disebabkan oleh lanjutan masa pengasingan populasi yang telah mengalami pengecutan
(bottleneck) diikuti dengan hanyutan genetik. Bagi spesies F. limnocharis, min kepelbagaian haplotip (h) dan kepelbagaian nukleotida (π) secara puratanya adalah rendah dalam semua sampel (h = 0.471 ± 0.27; π = 0.004 ± 0.00005). Pembiakbakaan dalaman dan pencemaran oleh racun perosak telah dicadangkan sebagai punca yang
xiv membawa kepada kepelbagaian genetik yang agak rendah dalam spesies ini. Analisis filogenetik yang dijalankan menggunakan kaedah NJ dan MP gagal menunjukkan sebarang penstrukturan geografi antara populasi pantai timur dan pantai barat. Analisis
AMOVA menggabungkan kumpulan barat laut dan barat tengah serta kumpulan timur menunjukkan bahawa kebanyakan varian genetik bertabur di antara kumpulan (Φ=
54.81%) yang menunjukkan bahawa populasi barat tengah adalah lebih berkaitan dengan populasi timur berbanding dengan populasi barat laut. Di samping itu, kajian awal yang menggunakan pendekatan GIS untuk memeta taburan kedua-dua spesies ini di
Semenanjung Malaysia telah menunjukkan bahawa kedua-dua spesies berkelompok dan kebanyakannya hanya bertumpu di kawasan utara Semenanjung Malaysia. Kajian yang lebih terperinci terhadap penanda mitokondria DNA dan nuklear DNA ke atas saiz sampel yang lebih besar serta merangkumi seluruh taburan geografi spesies-spesies ini dicadangkan untuk merancang pengurusan pemuliharaan yang efektif. Usaha daripada ahli herpetologi di Malaysia amat diperlukan untuk mengisi jurang pengetahuan yang sedia ada pada kedua-dua spesies ini serta spesies amfibia asal yang lainnya.
xv
POPULATION GENETICS AND DISTRIBUTION OF TWO SYMPATRIC
FROG SPECIES IN PENINSULAR MALAYSIA, Fejevarya cancrivora
(Gravenhorst, 1829) AND Fejevarya limnocharis (Boie, 1834)
ABSTRACT
A population genetic study of two sympatric frog species in Peninsular Malaysia,
Fejevarya cancrivora and F. limnocharis was conducted using the highly evolving D- loop segment of mtDNA. The genetic survey was conducted on six populations (26 individuals) of F. cancrivora from northern Peninsular Malaysia and 16 populations
(106 individuals) of F. limnocharis from northwest, central west and east of Peninsular
Malaysia. In general, F. cancrivora showed very high genetic variation for Pulau
Langkawi population and lack of variation for the Jitra population. The constructed
Neighbour Joining (NJ) and Maximum Parsimony (MP) phylogenetic trees showed similar topology, with the Pulau Langkawi population forming its own clade. It is suggested that the deep branching of Pulau Langkawi populations with high mutation rates from major haplotypes may be caused by the extension time of isolation of populations which had experienced bottleneck followed by genetic drift. As for F. limnocharis, mean haplotype and nucleotide diversity was low over all samples (h =
0.471 ± 0.27; π = 0.004 ± 0.00005). Inbreeding and pollution by pesticides are suggested as the causes which have led to the comparatively low genetic variability detected in this
xvi species. Phylogenetic analysis based on NJ and MP methods failed to show any geographic structuring between the east and west coast populations. AMOVA analysis between northwest group with combined central west and east populations group, showed that the majority of genetic variance was distributed between groups (Φ=
54.81%) which indicated that the central west was more related to the eastern populations than it is to the northwest populations. Additionally, the preliminary use of
GIS approach to map the distribution of both species in Peninsular Malaysia revealed that both species were clustered mostly in the northern area of Peninsular Malaysia.
More detailed study of mitochondrial DNA and nuclear DNA markers on larger sample sizes, throughout the geographical distributions of these species are suggested in planning an effective conservation management. Efforts from herpetologists in Malaysia are much needed to fill the existing knowledge gap on these species.
xvii
CHAPTER 1
INTRODUCTION
1.1 Introduction
Amphibians are unique vertebrates comprising over 6,700 known species
(Frost, 2011), found in diverse habitats around the world, except in very high latitudes in the hemispheres, most oceanic islands and the most low-lying, arid regions (Pough et al. 2004; Hillman et al. 2009). Amphibians are recognised as important components of natural ecosystems and they play important roles in the ecological process of the ecosystem. They are the mid-level consumers in the food chain of many tropical ecosystems, meaning that they consume insects, other arthropods, large invertebrates and small vertebrates. Likewise, amphibians are important prey for numerous predators, such as snakes, wading birds, lizards and small mammals in the food web (Ibrahim, 2004).
Amphibians have existed since the Devonian period, nearly 350 million years ago having evolved from lobe-finned fish (Crossopterygia) that used their strong, bony fins to radiate into most habitats on earth (Duellman & Trueb, 1986; Hickman et al. 2006). In doing so, they have acquired spectacular and sometimes unusual physiological, morphological, behavioural, and ecological attributes that form their innovative life histories. Their successful adaptation and resilience contribute to their survival from the past until present. However, sadly even though they had overcome many catastrophes in the recent decades, they are now facing serious threats due to human activities.
1
Biologists have divided the class Amphibia into three major groups namely
Anura (frogs and toads), Caudata (salamanders and newts) and Gymnophiona
(caecilians). Among these, the order Anura is the largest and most diverse with approximately 5,100 species. Unfortunately, nearly one-third (32%) of the world’s amphibian species are threatened, representing more than 1,800 species (Stuart et al.
2004). Various factors are known to cause the decline including habitat loss, alteration and fragmentation (Fisher & Shaffer, 1996; Davidson et al. 2001; Marsh &
Trenham, 2001; Norhayati et al. 2005a), introduction of competitors or predators
(Kats & Ferrer, 2003; Norhayati et al. 2005a) over-harvesting (Blaustein & Wake,
1990, Lannoo et al. 1994), climate change (Pounds et al. 1999; Kiesecker et al. 2001;
Carey & Alexander, 2003), increased UV-B radiation, chemical pollution (Hayes et al. 2002; Blaustein et al. 2003) and emerging infectious diseases (Daszak et al.
2003). Thus, their continued survival depends on our concern and assistance.
Kiew (1984) found that forest frog species in Malaysia are threatened by extensive logging and development and hence they are susceptible to extinction. To further aggravate the problem, Ibrahim (2004) stated that paddy field frogs are suffering from pesticide pollution and over collecting such that their numbers are declining and dwindling. It is worrisome that if no progress is made in the near future with respect to measures on amphibian conservation, many species will be lost even before they are discovered, recorded and studied.
Malaysia is purported to be an amphibian hotspot and the fourth on the list of countries having the largest amphibian populations in Asia, which currently stands at
218 species (IUCN, 2009). There are 12 families of Anura worldwide and Malaysia harbours seven, namely Bombinatoridae, Megophryidae, Bufonidae, Microhylidae,
Ranidae, Dicroglossidae and Rhacophoridae. Fejevarya limnocharis and F. 2
cancrivora are two frogs from the Dicroglossidae family commonly found around human habitation, lowland plains and agricultural areas in Malaysia (Ibrahim, 2004).
Fejevarya cancrivora is also known as ‘Crab-Eating Frog’ and it is the only
Malaysian frog that can tolerate saline habitats (Inger & Stuebing, 1989). According to The World Conservation Union (IUCN) report published in 2009, amphibian status in Malaysia is still under control and there is no extinction report for the two species investigated.
These two frog species always occur sympatrically in paddy fields area.
However, F. cancrivora is bigger, which allows them to feed on different- sized prey items, thus avoiding competition for food (Ibrahim, 2004; Wong, 2007). The main food items for both species are insects and about 80% of these insects are pests of rice. Hence, these frogs are considered as important biological control organisms in the paddy field habitat (Ibrahim, 2004).
In Malaysia, F. limnocharis is utilized for bait in sport fishing or as food for carnivorous aquarium fishes in the ornamental fish industry. In contrast, F. cancrivora is exploited for food consumption by Chinese townspeople in Malaysia.
They claimed that the meat of this frog is sweet, tender and tasty and even described them as “paddy chicken” (Ibrahim, 2004). Thus, over-harvesting is a potential threat to this species. Since both of these frogs inhabit the paddy field area, another very important threat to their existence is habitat degradation due to pollution by pesticides and chemical fertilizer used on crops. They are also common prey at the tadpole and adult stages to water birds, fishes and snakes. Other possible reasons for their decline are general habitat alteration and loss, urbanization, prolonged drought, habitat fragmentation and habitat modification from deforestation, or logging related activities. Although there is no reported data on the effects of the factors mentioned 3
above, immediate management steps should be implemented before any of the declining populations reaches extirpation in Malaysia.
O’Brien (1994) and Bowen and Karl (2007) stated that in a holistic management action, genetic information is now recognised as a crucial factor in all biological management programs. According to Schierwater et al. (1994), genetic variability data can reveal information on individual identity, breeding patterns, degree of relatedness and disturbances of genetic variation in populations. Genetic diversity data is also important in the assessment of molecular ecology, such as connectivity of population and characterization of geographic structure (Sivasundar et al. 2001; Barroso et al. 2005). Besides, it can also be utilized to determine the extinction risk of populations (Schierwater et al. 1994).
In addition to the use of molecular data in assessing extinction risk of populations, Geographical Information System (GIS) data can also be applied in the conservation and management of amphibian populations. For the past twenty years, utility and availability of GIS has increased greatly. A major example is the work by
Patla and Peterson (2002), which used GIS in addressing amphibian diversity, distribution and habitat in the Yellowstone Lake Basin, United States to conserve and restore amphibian populations. In addition, Ray et al. (2002) also used GIS approach to analyse amphibian’s habitat, which is very important in biological conservation.
In Malaysia, limited data is available for both approaches. The genetic variation of amphibians in Malaysia using mtDNA sequencing methods have been reported by Ramlah (2009) and Ramlah et al. (2010). Their studies mainly involved amphibian populations from Borneo and Sarawak with limited samples from
Peninsular Malaysia. However, they have provided a foundation for further studies in addressing population subdivision of the Malaysian amphibians. This study was 4
focused on two important amphibian species in Peninsular Malaysia as models of the application of population genetic studies for conservation; F. cancrivora and F. limnocharis. While F. cancrivora is found in disturbed but generally natural, wild habitats, F. limnocharis inhabits paddy field, hence it is vulnerable to pollution by pesticides.
In contrast, to date, there is no reported data on GIS application on amphibian distributions in Peninsular Malaysia. Nevertheless, in this study, the application of the GIS is solely used to map the distribution of two sympatric frog species in
Peninsular Malaysia, Fejevarya limnocharis and F. cancrivora in order to provide better understanding of their occurrence in Peninsular Malaysia.
1.2 Objectives:
With the above considerations, the objectives of this study were:
1) To determine the genetic variation of F. cancrivora from Northern
Peninsular Malaysia using mitochondrial D-loop gene.
2) To determine the genetic variation of F. limnocharis from Peninsular
Malaysia based on mitochondrial D-loop gene.
3) To map the distribution of F. cancrivora and F. limnocharis in
Peninsular Malaysia using GIS approach.
5
CHAPTER 2
LITERATURE REVIEW
2.1 Amphibians in general
Amphibians are found on all continents in the world, except in Antartica (Van der Meijden et al. 2005). To date, over 6,700 known species (Frost, 2011) have been reported and more are being described yearly. The class Amphibia comprised three orders namely Anura (Salientia), Caudata (Urodela), and Gymnophiona (Apoda).
There are several distinct characteristics that differentiate each order from the others.
Nevertheless, the taxonomic classification of each order is still under debate
(Duellman & Trueb, 1986).
Frogs and toads are members of the order Anura, which is further divided into approximately 40 families and more than 5,900 species (Frost, 2011). Generally, anurans are jumping amphibians with the presence of four limbs and no tails
(excluding all larval anurans and male of Ascaphus truei), which differentiate them from caudates and caecilians (Heyer et al. 1994). The hind limbs are typically larger that are specifically modified for leaping or climbing. Anurans are also unique in terms of their ability to vocalization, and produce an array of sounds from squeaks to barking noises (Miller, 2012). Vocalization is a pivotal component of the reproductive behavior for majority of frogs (Heyer et al. 1994). Unlike the majority of salamanders and caecilians, most anurans display external fertilization, have aquatic eggs and feeding larvae known as tadpoles (Heyer et al. 1994; Miller, 2012).
6
2.2 Threats to amphibians
Amphibians are thought to be good biological indicators as they are inhabitants of both terrestrial and aquatic environments. Thus, they are the first organism to be exposed to climatic changes and habitat pollution. As a result, amphibian populations have suffered widespread declines and extinctions in recent decades (Kiesecker et al. 2001; Beebee, 2005; Frost, 2011). Stuart et al. (2004) reported that amphibians have become more threatened than birds and mammals.
In the last few years, demand for frog legs for food consumption has increased drastically. The main reason of wild frog exploitation to support this trade is because of its large body (Altherr et al. 2011) and delicious taste (Ibrahim, 2004). In several
Asian, African and Latin American countries, frogs are hunted for subsistence or local consumption (Altherr et al. 2011). Some of these countries, are also involved in the commercial trade of frogs and frog products, supplying markets in the European
Union (EU) and the United States of America (USA). As a consequence, local frog populations have seriously declined (Lannoo et al. 1994).
Indonesia has been the world’s leading exporter of frog legs in the last 20 years followed by China, Taiwan and Vietnam (Altherr et al. 2011). The majority of frogs are caught from their natural habitats on the island of Java - particularly the
Crab-eating Frog, F. cancrivora (75%) and the Giant Javan Frog, Limnonectes macrodon (19%) (Kusrini & Alford, 2006). India and Bangladesh had in the past dominated this frog leg trade until their frog populations collapsed, leading to the reduction of a major natural control agent for agricultural pests and mosquitoes
(Abdulali, 1985; Oza, 1990). This unfortunate scenario could just likely impact our local frog populations if no conservation efforts are made in the near future. 7
Numerous studies have been conducted around the globe to investigate the effects of pesticides on amphibians. Liu et al. (2011) reported that the broad-spectrum herbicide, butachlor, depresses survival, development, and time to metamorphosis in
F. limnocharis in subtropical Taiwan. Butachlor is genotoxic to toad and frog tadpoles where it induces DNA strand break in erythrocytes (Geng et al. 2005; Yin et al. 2008). Atrazine, one of the most widely used pesticide in the world, create havoc with the sex attributes of adult male frogs, emasculating three-quarters of them and turning one in 10 into females according to a study by Sanders (2010). Atrazine acts by inhibiting production of testosterone (the male sex hormone) and induces estrogen production (the female sex hormone), resulting in imbalance between these two hormones. As a result, this atrazine-exposed male will possess both chemical castration (demasculinization) and feminization (Hayes, 2005).
Jayawardena et al. (2010) discovered that exposure of propanyl on Common
Hourglass Tree Frog (Polypedates cruciger), not only affected the survival and growth of tadpoles, but also led to malformation. Exposed tadpoles took a longer period to metamorphose and were smaller in size as compared to tadpoles in natural conditions. The longer periods of metamorphosis and smaller size of adults can have many consequences in nature (Jayawardena et al. 2010) as the ability to escape from predators and defending territories (Bridges, 1999) and survival (Shenoy et al. 2009) are compromised. Other pesticides known to cause devastation in frog populations include carbaryl (Relyea & Mills, 2000; Boone et al. 2004; Relyea, 2006), 2, 4-D butyl ester (Pérez-Coll & Herkovits, 2006) and carbofuran (Bacchetta et al. 2007;
Jayatillake et al. 2011). In Malaysia, among the commonly used pesticides in rice fields are propanyl, carbaryl, 2, 4-D butyl ester (Abdul Rani, 2002).
8
This situation highlights the dire need for conservation. One increasingly popular approach is the generation of genetics data from amphibian populations.
Numerous molecular markers have been developed for this purpose (Beebee, 2005).
Knowledge on the genetic diversity and population structure is one the fundamental areas for conservation and restoration of species and ecosystem diversity.
Conservation of genetic variability is important to the overall health of populations because decreased genetic variability leads to increased levels of inbreeding, and reduced fitness. Amphibians are good models for investigating animal genetic population for several reasons. They have limited mobility, widely distributed in most ecosystems and many are easy to sample because they congregate at specific localities for reproduction (Beebee, 1996). As anurans (frogs and toads) undergo external fertilization, controlled crosses are amenable under laboratory conditions (Beebee,
2005).
2.3 Taxonomy and Species Description
2.3.1 Taxonomic status of Fejevarya cancrivora
Fejevarya cancrivora is also known as the Crab-eating Frog, Mangrove Frog,
Rice Field Frog and Asian Brackish Frog (Plate 2.1). It was initially called Rana cancrivora by Gravenhorst (1829) due to its freshwater crab-eating habit. More recently, it was placed as a member of the genus Fejevarya (Iskandar, 1998; Dubois
& Ohler, 2000). Currently, the taxonomic classification of this species is represented by the following:
9
Kingdom: Animalia
Phylum: Chordata
Subphylum: Vertebrata
Class: Amphibia
Order: Anura
Family: Dicroglossidae
Genus: Fejevarya
Species: Fejevarya cancrivora (Gravenhorst, 1829)
10
Plate 2.1: Fejevarya cancrivora (Gravenhorst, 1829) (Photographed by Shahriza
Shahrudin)
Even though the Crab-eating Frog, F. cancrivora is one of the most widely distributed frog species in the Asian region, taxonomic relationships among different populations is still unclear. Several attempts have been made by various researchers
(Sumida et al. 2002; Islam et al. 2008; Kurniawan et al. 2010; Kurniawan et al. 2011) to elucidate the taxonomic status of this species using morphological characteristics, allozyme and molecular analysis. Kurniawan et al. (2010) suggested that F. cancrivora from Asian populations could be divided into three types, namely, the mangrove, large, and Pelabuhan Ratu/ Sulawesi-types, with the last two types showing the most similarity in morphology. The authors discovered that the mangrove-type was distributed in the Asian mainland and the Philippines, the large- type in Sundaland area and the Pelabuhan Ratu/Sulawesi-type in Pelabuhan Ratu,
11
Java Island and Sulawesi Island of Indonesia. Kurniawan et al. (2011) proposed the large-type as F. cancrivora, the mangrove-type, F. moodiei, and the Sulawesi-type, an undescribed species.
2.3.2 Taxonomic status of Fejevarya limnocharis
Fejevarya limnocharis is also known as the Asian Grass Frog, Common Pond
Frog, Field Frog and Indian Rice Frog (Plate 2.2). Fejevarya limnocharis closely resembles F. cancrivora in external morphology. However, according to Gravenhorst
(1829), F. cancrivora is larger than F. limnocharis, and the name F. cancrivora has been consistently applied to larger individuals in the F. limnocharis complex occurring in Java and neighbouring regions (Kurniawan et al. 2010).
As in the case of F. cancrivora, the taxonomic status of F. limnocharis is also still controversial (Fei et al. 2002). According to Toda et al. (1998), F. limnocharis is a species complex of frogs. Kotaki et al. (2008) stated that F. limnocharis has been conventionally regarded as a single species because of few morphological differences. However, recent detailed analyses by Dubois & Ohler (2000) have revealed that there is a degree of genetic differentiation within F. limnocharis, and therefore it has been proposed that F. limnocharis contains several cryptic species.
Sumida et al. (2002) revealed that there is no reproductively isolating mechanism between populations of F. limnocharis as shown by hybridization experiments. Nevertheless, phylogenetic tree based on mitochondrial DNA sequences of 12S and 16S rRNA genes discovered three clades of F. limnocharis in the East Asian populations. The first clade comprising populations from Japan
Islands; the second, Sakishima Island and the third, the Okinawa Island and Taiwan
12
populations. Based on crossing experiments and molecular data, Sumida et al. (2002) regarded the Sakishima-Island populations as a distinct species of F. limnocharis.
Djong et al. (2007) and his colleagues conducted morphological observation, phylogenetic reconstruction of mitochondrial DNA of 16S and cytochrome b genes, backcrossing experiments, histology and spermatogenesis studies among Indonesian,
Malaysian, and Japanese populations of F. limnocharis. They concluded that the
Malaysian population of F. limnocharis and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japanese population as a distinct species. The current taxonomic classification of this species can be represented as follows.
Kingdom: Animalia
Phylum: Chordata
Subphylum: Vertebrata
Class: Amphibia
Order: Anura
Family: Dicroglossidae
Genus: Fejevarya
Species: Fejevarya limnocharis (Boie, 1834)
13
Plate 2.2: Fejevarya limnocharis (Boie, 1834) (Photographed by Shahriza Shahrudin)
2.4 Morphology
The frog is unique as it has no tail in the adult but is present in the tadpole.
Most frogs have long hind legs, elongated ankle bones, webbed toes, no claws, large eyes and a smooth or warty skin. Besides, they also have a short vertebral column, with no more than ten free vertebrae and a fused tailbone. The general anatomy of frog is shown in Fig. 1.1. Morphological characteristics of F. cancrivora and F. limnocharis have been described by Berry (1975), Inger and Stuebing (1997),
Ibrahim (2004), Kurniawan (2010) and Norhayati (2012a) and the description of each species is given below.
14
Figure 1.1: General anatomy of frogs (Berry, 1975)
2.4.1 Fejevarya cancrivora
Fejevarya cancrivora is a medium-sized frog with a long snout and well- muscled hind limbs (Inger & Stuebing, 1997). The total length of this frog is between
50 mm–85 mm (Inger & Stuebing, 1999), while Berry (1975) gave the snout vent length (SVL) as 50-75 mm. Females are usually larger and their bodies more sturdy and stocky as compared to males. Inger and Stuebing (1999) stated that the total length for female is between 52.9 mm – 82.0 mm, while male is between 51.0 mm to
70.9 mm. Inger and Stuebing (1997) recorded the female to be between 53 and 82 mm in length and male between 51-70 mm. Kurniawan et al. (2010) observed total
15
length of females and males to range from 58.0–87.5 mm and 59.3–71.2 mm, respectively. Ibrahim (2004) also found many females with mean sizes of 66.0 ± 7.1 mm compared to 61.0 ± 7.1 mm in males.
According to Berry (1975), vomerine teeth are present in this species with its head as broad as or slightly broader than long and the snout is rounded or obtusely pointed. The colour of this frog is grey or brown dorsally with irregular dark markings, often in the form of a ‘W’. The skin is irregular with longitudinal ridges on the back and the tympanum is conspicuous about 1/2 to 2/3 of eye diameter
(Norhayati et. al. 2012a). The limbs are with dark crossed bars or irregular dark markings. The stomach is creamy white, while the underside of the head is either white or with dark mottling which sometimes extends to the chest area in some individuals (Ibrahim, 2004). Males have black patches under the corner of the jaw on the skin overlying the vocal sacs (Inger & Stuebing, 1997).
2.4.2 Fejevarya limnocharis
Fejevarya limnocharis is a small frog with a long narrow head and a slender, oval body (Inger & Stuebing, 1997). According to Berry (1975), snout vent lengths
(SVL) of the females are 48-60 mm and 32-50 mm for males. Inger and Stuebing
(1997) also reported SVL of 49-58 mm in females and 32- 50 mm for males. Ibrahim
(2004) recorded the mean sizes of males as 39.9 ± 2.77 mm and 46.8 ± 4.72 mm in females. Djong et al. (2007) stated that male sizes are between 35.5- 41.7 mm while females between 46.3- 47.9 mm. The total length of this species, however, is smaller than F. cancrivora.
16
Berry (1975) recorded the presence of vomerine teeth in two oblique series between the choanae, a moderate head and snout more or less pointed in F. cancrivora. This species is grey or brownish in colour with dark spots or blotches often with a yellow creamy vertebral stripe on the dorsal side. The skin is warty dorsally with distinct tympanum about 3/5 diameter of their eye (Norhayati et. al.
2012a). There is usually a W-shaped marking across the shoulders. The back of the thighs are yellowish in colour while the limbs have dark cross bars (Ibrahim, 2004).
The lips are barred brown and white, while the undersides of the female are completely white. Unlike other species, which are difficult to differentiate between the sexes, the presence of a black, M-shaped band across the male throat is the key sex marker in this species (Inger & Stuebing, 1997; Ibrahim, 2004).
2.5 Distribution, Habitat and Biology
2.5.1 Fejevarya cancrivora
The crab-eating frog, F. cancrivora is a widely distributed frog known to inhabit many parts of Asian region. Its geographic range extends from the coast of
Southern China in Guangxi and the north-eastern coast of Hainan Island, China, through to Vietnam, the Andaman and Nicobar Islands (India), Peninsular Thailand,
Malaysia, Singapore, the Greater Sundas, the Philippines, and the Lesser Sundas as far as Flores. It has also been intentionally introduced to Sorong and Jayapura,
Papua, Indonesia (Kurniawan et al. 2010). In New Guinea, introduced populations are known from the Sorong, Manokwari, Nabire and Jayapura areas of Papua,
Indonesia (Frost, 2011). The presence of this species was reported in the Pondicherry
Mangroves, Bay of Bengal-India for the first time by Satheeshkumar (2011).
17
In Malaysia, F. cancrivora is usually found in disturbed habitats, cultivated areas, along the coast, lower reaches of large river basins, semi brackish, swampy areas close to the sea or in freshwater swamps beyond tidal influence (Inger &
Stuebing, 1989; Norhayati et. al. 2012a). Although it occurs in coastal rice fields in
Peninsular Malaysia, the presence of this species from such places in Borneo is not known (Inger & Stuebing, 1997).
Among the amphibian species in Malaysia, F. cancrivora is the only frog that can constantly live in saline habitat and tolerate marine environments. Salinity tolerance in this Crab-eating Frog has been conducted by several researchers (Gordon et al. 1961; Schmidt-Nielsen & Lee, 1962; Chew et al. 1972; Wright, 2004). Gordon et al. (1961) and Schmidt-Nielsen and Lee (1962) suggested that the major feature for the high salinity tolerance in the adults of F. cancrivora is their osmoregulatory mechanisms. The adults can respond to high external salinities with an increase in internal osmotic concentration, largely due to accumulation of urea (Gordon &
Tucker, 1965; Wright, 2004). Gordon et al. (1961) found that adults of F. cancrivora can tolerate environmental salinities as high as 28% at 30°C, while the tadpoles tolerated salinities as high as 39% at the same temperature.
Gordon and Tucker (1965) conducted an experiment on salinity tolerance particularly on F. cancrivora tadpoles, and the results supported previous study by
Pearse (1911) that tadpoles of this species had greater tolerance for high salinities than the adults. Wright (2004) stated that F. cancrivora is able to withstand salinity of
75% of seawater which is about 25 parts per thousand (ppt) and higher. The unusually high salinity tolerance of F. cancrivora is of great interest to researchers even though this special feature is not unique to them. Balinsky et al. (1972) reported that Xenopus
18
laevis and Bufo viridis have also been known to withstand 20 ppt and 26 ppt of salinity, respectively. However, F. cancrivora is probably the most tolerant among the known amphibian species (Balinsky et al. 1972).
2.5.2 Fejevarya limnocharis
The Rice Frog, F. limnocharis is a common and widespread amphibian among Southeast Asian frogs. This species can be found in all Southeast Asian and the rest of the Asian region extending from western Japan, Taiwan, south-western
China, the Malay Peninsula, Bangladesh, Nepal, the Philippines, Indonesia, Sri
Lanka, and India to Pakistan (Sumida et al. 2002). In China, this species is also distributed throughout a wide range of altitude (2–2000 m) (Fei & Ye, 2001). In
Malaysia, the species only exist in disturbed habitats associated with human activities including paddy fields, roadsides, lawns, agricultural fields and football fields (Inger & Stuebing, 1997; Ibrahim, 2004; Norhayati et. al. 2012a).
Similar to F. cancrivora, Wu and Kam (2009) observed that F. limnocharis tadpoles display higher salinity tolerance compared to tadpoles of most species studied to date. They found that more than 50% tadpoles could survive in 9 ppt salinities for over a month, and a few individuals could survive in 11 ppt salinities for up to 20 days. Tadpoles metamorphosed earlier at a smaller size as salinity increased, suggesting the existence of adaptive developmental plasticity in F. limnocharis in response to osmotic stress (Wu & Kam, 2009).
2.6 Status of Fejevarya cancrivora and F. limnocharis conservation in Asia
Currently, F. cancrivora and F. limnocharis have been placed under Least
Concern category by the International Union for Conservation of Nature (IUCN).
19
Even though there is no report on the decline of this species, they are facing serious threats due to human activities in their natural habitats, globally. According to
Zhigang et al. (2009) over-harvesting, habitat alteration, wood harvest from mangrove forests, human settlement expansion and road kill may threaten the F. cancrivora populations. Recently, the global emergence of a chytrid fungus that causes chytridiomycosis in amphibians, Batrachochytrium dendrobatidis (Bd) has gained public awareness about this disease (Fisher et al. 2009).
This chytrid fungus was first described in 1998 and 1999 (Berger et al. 1998;
Longcore et al. 1999) and is now the known cause of this potentially fatal disease, chytridiomycosis. Up to date, Bd has been reported in 36 countries where wild amphibian populations exist (Savage et al. 2011). Skerratt et al. (2007) reported that
Bd has infected over 350 amphibian species and of these 200 are in decline. In
Peninsular Malaysia, infection by this chytrid fungus has been reported in ten species from four families namely Megophryidae, Microhylidae, Ranidae and Rhacoporidae
(Savage et al. 2011). Even though there is no data about this infection on F. cancrivora, it is still susceptible to the disease through infection by invasive frog species. It has been documented that one of the reasons of Bd infection in Peninsular
Malaysia is the introduction of non-native amphibians (Lithobates catesbeianus and
Holoplobatrachus rugulosus) in the frog farming industry.
Frost (2011) stated that F. limnocharis is adversely affected by pesticide pollution of chemicals used on the crops and falls prey at the tadpole and adult stages to water visiting birds, fishes and snakes. About 10, 000 years ago, rice (Oryza sativa
L.) was first cultivated in Asia (Vaughan et al. 2008) and grown in paddy fields (Liu et al. 2011). Since paddy field is characterized by both aquatic habitats and dry lands,
20
it harbours a rich biological diversity (Bambaradeniya & Amarasinghe, 2003). Among them, frogs and toads (anurans) have benefited from the creation of paddy fields, which have become an important breeding habitat (Liu et al. 2011).
However, in order to ensure the good quality of rice produced and enhances rice production; farmers tend to use huge amounts of pesticides and fertilizers. The presence of pesticides in the environment has become a global issue. Field studies have shown that the reproduction, growth and development of wildlife species, invertebrates, amphibians, reptiles, fish, birds and mammals may have been impacted by chemicals that interact with the endocrine system (Khan & Law, 2005). Pesticides at low concentrations may act as blockers of sex hormones, causing abnormal sexual development, abnormal sex ratios and unusual mating behavior. Pesticides can also interfere with other hormonal processes, such as thyroid and its influence on bone.
Although, both species are not experiencing conservation threat, determining the genetic structure of these species would be a preemptive measure to arrest any possibility of decline.
2.7 Genetic variation
Genetic variation or diversity is a basic component of biodiversity, forming the basis of species and ecosystem survival. The three major sources of genetic variation are mutation, gene flow and sexual reproduction (Understanding Evolution,
2012). It is well known that a decrease in genetic variation can lead to reduce fitness and lack of adaptability to a changing environment (Allentoft & O’Brien, 2010).
Populations of species with low genetic variation are exposed to a higher probability of becoming genetically inbred, with the potential consequence of lowered fitness. In contrast, wide genetic variation helps improve the species ability to survive in a 21
changing environment, as the chances that some individuals will tolerate a particular change are increased (Martin & Hine, 2000). For instance, the extremely low genetic diversity in Ranodon sibiricus (Amphibia: Caudata) was taken to imply an absence of genetic variation for adaptive quantitative characteristic, hence, enhanced risk of extinction due to genetic homogeneity (Chen et al. 2012). Besides the importance of genetic diversity in adaptability to a changing environment, information on genetic diversity also helps in ascertain pedigrees, reconstructing phylogenies and estimating migration frequencies (Nicod et al. 2004; Nguyen et al. 2006). Various types of markers such as mtDNA and nuclear DNA markers are utilised to assess genetic variation and hence address issues of conservation and management of wildlife.
2.7.1 Genetic marker: Mitochondrial DNA (mtDNA) in general
Mitochondria are organelles specialized in energy conservation reactions and appear to have endosymbiotic origin in eukaryotic cells (Gray, 1992). They play an important role in metabolism, apoptosis, illness, and aging (Boore, 1999; Cao et al.
2006). For instance, the formation of adenosine-5'-triphosphate (ATP) by oxidative phosphorylation occurs in these organelles. They also possess their own double- stranded circular mitochondrial DNA (mtDNA), which are inherited independently of the nuclear genome.
Animal mtDNA is a double-stranded, closed circular molecule. This extrachromosomal genome is small and usually ranges from 16-20 kilobase in length
(Boore, 1999; Kucuktas & Liu, 2007). MtDNA differs from nuclear DNA in the sense that mtDNA is maternally inherited and non-recombining (Avise, 1994;
Nguyen et al. 2006), thus rendering it suitable for tracing maternal lineage. MtDNA also has high rates of nucleotide substitution as compared with nuclear DNA (Moritz, 22
1987; Pesole et al. 1999) with an evolutionary rate 5 to 10 times faster than the nuclear genes (Avise, 1994; Castro et al. 1998). These characteristics make mtDNA suitable for population level studies.
In general, animal mitochondrion contains only 37 genes; 13 protein-coding genes, two ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs) and a noncoding control region known as the D-loop containing the signals for regulation and initiation of mtDNA replication and transcription (Wolstenholme, 1992). Studies of the whole amphibian genome have been conducted by several researches (Roe et al. 1985; Zardoya & Meyer, 2000; Sumida et al. 2001; Zhang et al. 2003; Liu et al.
2005; Ren et al. 2009). To date, the complete mtDNA sequences are available for 26 anuran species (Ren et al. 2009).
Figures 2.1 and 2.2 show the organisation of F. cancrivora and F. limnocharis genome based on the studies by Ren et al. (2009) and Liu et al. (2005), respectively. The complete nucleotide sequence of mtDNA of F. cancrivora is
17,843 bp in length and contains 13 protein-coding genes (ATP6, ATP8, COI–III,
ND1–6 and 4L and Cyt b), two ribosomal RNAs (12S and 16S rRNA), and 23 transfer RNAs genes (Ren et al. 2009). In contrast, the complete mtDNA sequence of
F. limnocharis is 17,717 bp in length containing 13 protein-coding genes, 2 rRNA genes and 23 tRNAs genes and noncoding region (Liu et al. 2005). Although almost all animal mtDNAs encode 22 tRNA genes, the F. cancrivora and F. limnocharis mtDNA possesses an extra copy of tRNAMet. The genome organization of F. cancrivora is identical with F. limnocharis, suggesting that the unique gene arrangement occurred in the common ancestor of the genus of these two species.
23
Figure 2.1: The gene organization of the Fejervarya cancrivora mitochondrial genome. The protein-coding genes are designated using abbreviations. The tRNA genes are denoted by the single-letter amino acid code. O-L represents the replication origin of the L-strands; and B is the intergenic spacer (Ren et al. 2009).
Abbreviations: A6, ATPase subunit 6; A8, ATPase subunit 8; COI–III, cytochrome c oxidase subunit; CR, control region; Cyt b, cytochrome b; ND1–6 and ND4L, NADH dehydrogenase subunits 1–6.
24
Figure 2.2: The gene organization of Fejevarya limnocharis mitochondrial genome. tRNA genes are denoted by the single-letter amino acid code. All protein coding genes are H-strand-encoded with the exception of ND6 indicated by an arrow. OH and OL represent the replication origins of H- and L-strands, respectively. M’ shows the extra copy of tRNAMet (Liu et al. 2005).
Abbreviations: ATP6, ATPase subunit 6; ATP8, ATPase subunit 8; COI-III, cytochrome c oxidase subunit I–III; Cyt b, cytochrome b; D-loop, displacement loop; H-strand, heavy strand; L-strand, light strand; ND1-6, NADH dehydrogenase subunit 1–6.
25
2.7.2 Application of mtDNA in amphibian research
In the last few decades, mtDNA analysis has become established as a powerful tool for evolutionary and population studies of animals (Moritz et al.
1987). Animal mtDNA has been extensively studied to address problems in population genetics, as well as systematics of closely related species and individuals within a species. Moreover, mtDNA analysis can also provide an insight of population structure and gene flow, hybridization, biogeography and phylogenetic relationships (Moritz et al. 1987).
Application of mtDNA in studies of amphibians in Malaysia has been limited. Ramlah (1998; 2003; 2005) carried out a study on population genetics of three species of Sarawak forest frogs based on mitochondrial CO1 gene sequences, which revealed a genetic break between the western Sarawak populations (Gading,
Matang and Padawan) and east of Batang Lupar (Batang Ai, Mendolong, and
Danum, Sabah) for Limnonectes kuhlii and L. leporinus, but not for populations of
Rana chalconota. Genetic structure of the commensal species, Hylarana erythraea
(Ramlah et al. 2010) from Sarawak, Malaysian Borneo (Borneo Heights of Padawan,
Sadong Jaya, and Bario) and central Peninsular Malaysia (Tasik Chini of Pahang) suggested that populations of H. erythraea were subdivided, where populations in central Peninsular Malaysia and western Borneo were more closely related than those in western Borneo were to those of eastern Borneo. The study implied that a feature in the landscape of Borneo (the Lupar line) created a greater barrier than repeated intervening ocean between glacial periods during the Pleistocene epoch. However, studies on frog populations based on mitochondrial DNA are still lacking in
Peninsular Malaysia.
26
2.7.3 D-loop analysis
The various regions of the mitochondrial genome evolve at different rates
(Saccone et al. 1991), thus lending different regions suitable for different types of studies. Sumida and Ogata (1998) noted that the gene cytochrome b (Cytb) and D- loop has been efficient in resolving phylogenetic relationships, ambiguities between populations of the same species and closely related species. The control region or displacement loop (D-loop) gene is the only noncoding segment in vertebrate mtDNA (Faber & Stepien, 1998) and encompasses the sites of initiation for H- strands replication and both H- and L- strand transcription (Sumida et al. 2000). The
D-loop segment evolves much faster than the average mitochondrial gene (Brown,
1985), but it also has short sequence elements conserved among most vertebrates studied (Wolstenholme, 1992). Because of its ability for rapid change, the D-loop region is an ideal choice for addressing population-level genetic questions (Hoelzel et al. 1991).
The wide utilisation of the D-loop segment has elevated its status as a universal marker in resolving population genetic ambiguities. Several studies involving D-loop as a marker includes studies on the freshwater terrapin
(Norkarmila, 2009), marine fishes (Bernardi, 2000; Bernardi et al. 2003; Aboim et al.
2005; Rodrigues et al. 2008; Jamsari et al. 2010), snakes (Ashton & Queiroz, 2001), lizards (Rest et al. 2003) and anurans (Zhong et al. 2008). Several detailed comparisons of the D-loop region in mammalian mtDNA have also been recorded
(Saccone et al. 1991; Hoelzel et al. 1991). Moritz et al. (1987) and Rand (1993) found substantial length variation in the D-loop region of mammalian and fish mtDNA. However, there is still a dearth of information available about the structure,
27
function and evolution of the D-loop region in all but a few amphibian species
(Sumida et al. 2000).
Zhong et al. (2008) utilised the mtDNA control region to study the systematics and phylogeographic relationships among the rice frog species in China,
Fejevarya multistriata. Chen et al. (2012) used the D-loop region to investigate genetic diversity of the Siberian salamander, Ranodon sibiricus (Amphibia: Caudata) and its implication on phylogeography.
2.8 Application of Geographic Information System in Amphibians
The Global Positioning System (GPS) is a relatively new and modern technology used in determining correct locations and coordinates on or near the earth by calculating the signals received from a cluster of satellites (Ruslan & Noresah,
1998). In 1978, the first Global Positioning System (GPS) satellite (Navstar 1) was launched and only designed for US military applications (Sturdevant, 2007).
However, the system was made available for civilian uses in mid 1980s after realizing its importance in aviation safety following a civilian airliner tragedy (Al-
Ghamdi et al. 2007). In April 1991, there were 20 satellites available in the Earth’s orbit and this number increased to 24 in 1994. These satellites transmit radio signals into a three- dimensional space, namely longitude, latitude and altitude (Azizul,
2010).
As GPS applications have been rapidly growing, so have Geographic
Information Systems (GIS) (Al-Ghamdi et al. 2007). Geographic Information System
(GIS) is a system designed to capture, store, manipulate, analyse, manage and present all types of geographical data. The geographical information science or
28
geospatial information studies are the other terms used referring to the academic discipline or career of working with geographic information systems (ESRI, 2012).
Since the development of the GIS, spatial models of species distributions have become a common tool in wildlife management (Penman et al. 2007). One of the most common applications of spatial modelling in this area is the prediction of species distributions on a landscape level (Penman et al. 2007). Research in the area of geographic distribution using GIS comprised many different methods such as the point pattern analysis which is used to assess the physical distribution of point events and reveal whether there is a significant clustering of points in a particular area or otherwise (Cromley & McLafferty, 2002; Narimah et al. 2010). According to
Robinson (1998), point pattern analysis is aimed at finding patterns in data composed of points in a spatial region and allows for the measurement of the location of individuals relative to each other. In the past, point pattern analysis has been widely used in biology or ecology to detect and estimate the distribution patterns of species
(Narimah et al. 2010). The GIS has also been applied in other fields of study, such as epidemiology. For instance, Moore and Carpenter (1999) used point pattern analysis to study the distribution and determinants of diseases and injuries in human populations and factors that influence their distribution.
In recent years, the use of GIS in modelling amphibian distribution is increasingly being introduced. Lai (2009) conducted Maximum Entropy Species
Distribution Model (MaxEnt) to predict distribution of 18 amphibian species in
Poland by using species presence-only observation data together with environmental variables. From this model, Lai (2009) revealed that precipitation and soil temperature variables are the most important factors that influenced amphibian
29
species distribution in Poland. Ray et al. (2002) used GIS to predict occurrence of two amphibian species, the common toad (Bufo bufo) and the alpine newt (Triturus alpestris) by using information on land use surrounding breeding ponds at the State of Geneva. In their study, they used generalized additive models (GAMs) to test the ability of the migration zones to enhance predictions of species occurrence and several landscape variables to determine the presence of amphibians. Their study indicated the usefulness and importance of GIS in the functional analysis of a landscape, with potential applications in biological conservation aside from the need for enhancing our knowledge of habitat use by animals.
In addition, Patla and Peterson (2002) had conducted investigation on amphibian species occurrence, distribution, and habitat-use patterns in the
Yellowstone Lake area using GIS methods. They concluded that GIS helps in integrated information system that Yellowstone National Park could be used for environmental analysis, project planning, monitoring, research, evaluation of ecosystem health, and education (Patla & Peterson, 2002). Other than the advantages of utilizing GIS for amphibian distributions as explained above, distribution models may be most valuable for the management of rare or cryptic species (Penman et al.,
2007). Generally, rare and cryptic species are often difficult to detect and most of the data sets from these species are scarce and rely only on chance observations rather than stratified surveys (Penman et al. 2004). Thus, by successfully predicting sites or areas of occurrence, conservation efforts could be better directed to those habitats which are important for the species (Penman et al. 2007).
30
CHAPTER 3
MATERIALS AND METHODS
3.1 Sampling Site
Sampling of the two species, F. cancrivora and F. limnocharis were conducted at 19 locations in Peninsular Malaysia; 17 on the west coast and two on the east coast (Figure 3.1). The West Coast locations were Pulau Langkawi, Jitra,
Lembah Bujang, Sedim, Ulu Paip, Air Itam, Sungai Burung, Jerejak Island, Sungai
Dua, Sungai Acheh, Bukit Panchor, Kuala Kangsar, Pusing, Langkap, Sabak
Bernam, Tanjong Karang and Kepong. The East Coast populations comprised
Kubang Kerian and Besut. However, not all the sampling sites supported the presence of both species. Selection of sampling areas was based on the habitat specificity of each species (Table 3.1).
3.1.1 Sampling of Fejevarya cancrivora
Disturbed habitats, cultivated areas and mangrove swamps were believed to support the existence of this species (Inger & Stuebing, 1989). Thus, sampling activities were conducted at paddy field areas (Jitra, Pulau Langkawi and Sungai
Dua), mangrove swamps (Sungai Burung) and human settlements (Air Itam and
Jerejak Island) (Table 3.2). However, only 26 individuals were obtained from the six sampling sites, which were all located in the northern region of Peninsular Malaysia.
31
Figure 3.1: Sampling sites of both species in Peninsular Malaysia. Key: F. cancrivora, F. limnocharis. Both species. Titiwangsa mountain range. 1. Pulau Langkawi, 2. Jitra, 3. Lembah Bujang, 4. Sedim. 5. Ulu Paip, 6. Sungai Burung, 7. Air Itam, 8. Jerejak, 9. Sungai Dua, 10. Sungai Acheh, 11. Bukit Panchor, 12. Kuala Kangsar, 13. Pusing, 14. Langkap, 15. Sabak Bernam, 16. Tanjong Karang, 17. Kepong, 18. Kelantan, 19. Terengganu.
32
Table 3.1: Summary of sampling areas based on habitat specificity of Fejevarya cancrivora and F. limnocharis.
No. Habitat Population F. cancrivora F.limnocharis 1 Cultivated areas Pulau Langkawi x x Jitra x x Sungai Burung (paddy x field) Sungai Dua x Sungai Acheh x Kuala Kangsar x Pusing x Langkap x Sabak Bernam x Tanjong Karang x Besut x 2 Human settlement Lembah Bujang x Air Itam x Jerejak x Kubang Kerian x Recreational x 3 forest Sedim Ulu Paip x Bukit Panchor x Kepong x 4 Mangrove swamp Sungai Burung x
33
Table 3.2: Location, region, coordinate and sample size for F. cancrivora populations in northern Peninsular Malaysia.
No. Location Region Coordinate Sample size (n) 1 Pulau Langkawi North N6.30771°,E 99.72456° 4 2 Jitra North N5.27898°,E100.39253° 2 3 Sungai Burung North N5.48346°,E100.20916° 8 4 Air Itam North N5.39346°,E100.25826° 8 5 Jerejak North N5.1848°,E100.31036° 2 6 Sungai Dua North N5.47386°,E102.40032° 2 TOTAL 26
3.1.2 Sampling of Fejevarya limnocharis
According to Berry (1975) and Inger and Stuebing (1989), F. limnocharis can be found in cleared areas where human has destroyed the original vegetation, such as the lowlands along the coasts, the lower reaches of larger rivers, in vegetable gardens, botanic gardens, paddy fields and rural villagers. Thus, the sampling sites for this species were centred at cultivated areas (Pulau Langkawi, Jitra, Sungai Aceh,
Kuala Kangsar, Pusing, Langkap, Sabak Bernam, Tanjong Karang and Besut), lower reaches of larger rivers (Sedim, Bukit Panchor Ulu Paip and Kepong) and human settlements (Lembah Bujang and Kubang Kerian) (Table 3.3). The samples were also classified into regions; north, central west and east for F. limnocharis. A total of 106 individuals were sampled.
34
Table 3.3: Location, region, coordinates and sample size for F. limnocharis populations in Peninsular Malaysia.
Sample size No. Location Region Coordinate (n) 1 Pulau Langkawi North N6.30771°, E99.72456° 4 2 Jitra North N5.27898°, E100.39253° 4 3 Lembah Bujang North N5.69463°, E100.45208° 3 4 Sedim North N5.40291°, E100.78146° 7 5 Ulu Paip North N5.40393°, E100.66981° 3 6 Sungai Burung North N5.48346°, E100.20916° 4 7 Sungai Acheh North N5.16082°, E100.42729° 6 8 Bukit Panchor North N5.15951°, E100.54862° 8 9 Kuala Kangsar Central West N5.44477°, E100.20560° 5 10 Pusing Central West N4.47719°, E101.00009° 7 11 Langkap Central West N4.08367°, E101.14022° 8 12 Sabak Bernam Central West N3.73179°, E100.93846° 7 13 Tanjong Karang Central West N3.42405°, E101.1845° 16 14 Kepong Central West N3.238525°, E101.6250° 5 15 Kubang Kerian East N5.10089°, E102.28493° 6 16 Besut East N5.63671°, E102.48339° 13 TOTAL 106
3.2 Sampling Method
Sampling activities were conducted from July 2010 until November 2011 using a random sampling method. Field parties consisting of 3–6 people would first scour the area from 2000 hours to 2200 hours. Specimens were captured by hand or fishnets with the help of handheld battery powered torchlights to locate hiding sites.
Individuals were brought back to the laboratory in separate plastic bags for analysis.
3.3 Analysis in Amphibian Laboratory
Upon reaching the Amphibian Laboratory, located near Hamzah Sendut library, Universiti Sains Malaysia, each captured specimen was immediately identified by referring to Berry (1975) and Norhayati et al. (2009). All specimens
35
were injected with benzocaine solution into its dorsal lymph sacs to humanely kill them before dissection. Solution was made up of 20 mg benzocaine powder/ specimen weight (g) in 20 ml of 70% ethanol (EtOH). The procedure was made according to the standard method approved by the Animal Ethics Committee,
Universiti Sains Malaysia. Specimens were then weighed to the nearest 0.1 g using an ANDElectronic Precision Balance Model EK-1200A (1200 g capacity). Basic morphometric data; snout –vent length (SVL), mouth width (MW), tibia length, (TL) and tympanum diameter (TD) were measured to the nearest 0.01 mm using dial calipers (± 0.05 mm). Each specimen was then dissected and a small quantity of liver tissue (1 cm3) was excised from the fresh specimen using a pair of sterile scissors before they were placed in 95% ethanol (EtOH) in 1.5 ml Eppendorf tube. For reference voucher, specimens were then immersed in 10% formalin for overnight, washed with distilled water and transferred into a bottle containing 70% EtOH
(Matsui, 1984). The bottle was then labelled with scientific name, date and location of sampling and was then deposited at the Herpetological Collections in the School of Distance Education, Universiti Sains Malaysia, for future reference.
3.4 Population Genetic Analysis of Two Sympatric Frog Species in
Peninsular Malaysia based on partial mitochondrial D-loop Gene
Summary of general procedure used in evaluating the population status of the two species investigated inferred from partial D-loop mtDNA gene is illustrated in
Fig. 3.2.
36
3.5 Sample preparation and preservation
A total of 132 samples from both species were analysed for the population studies. The specimens were obtained from 19 locations: 17 from West Peninsular
Malaysia and two from East Peninsular Malaysia as previously mentioned in section
3.1.
Approximately 1 cm3 of liver tissue were cut from fresh specimens through sterile procedure to avoid cross contamination among specimens. The tissues were then placed in 95% ethanol (EtOH). To ensure the EtOH concentration was maintained at 95% (the wet samples contain moisture that would dilute the initial concentration of the solution), the EtOH preservation solution was periodically changed; 1) after two hours 2) after 24 hours and 3) after 72 hours of placing the specimen tissues. Samples were preserved for two weeks at room temperature prior to DNA extraction.
37
Field sampling
Specimen
Sample preparation & preservation
95% EtOH
Change of EtOH solution after 2, 24 & 72 hours of placing specimen tissues
DNA extraction using DNeasy Tissue Kit (QIAGEN)
PCR OUTPUT: Genetic distance, Agarose gel electrophoresis Phylogenetic tree, Haplotypes and nucleotide diversity, MSN & PCR product visualisation tree under UV light
Sequence editing & analyses using PCR product MEGA 4.0, Clustal W, dnaSP 4.50.3,
Arlequin 3.1 & Network 4.2.0.1 4.0
Purification using PCR PCR product sequencing Purification Kit (PROMEGA)
Figure 3.2: Flow-chart of population DNA analysis.
38
3.6 DNA Extraction
DNA extraction of liver sample was carried out using DNeasy Tissue Kit
(QIAGEN) following the manufacturer’s manual (Fig. 3.3). Approximately 10 mg of liver tissue from each specimen was cut and placed in a sterile 1.5 ml micro centrifuge tube. 180 µl of Buffer ATL (tissue lysis buffer) and 20 µl proteinase K was added into the tube. The mixture was then briefly vortexed, followed by three- hour incubation at 56°C in a hot block. Since thicker liver samples needed a longer time to lyse, the incubation time was extended until the samples were completely lysed. The tube was inverted once every 30 minutes to evenly mix the samples and the buffer solution. After the incubation, a volume of 200 µl Buffer AL (lysis buffer) was added into the tube, followed by 200 µl EtOH (96-100%). The mixture was again vortexed to mix. The contents were then transferred into a DNeasy Mini Spin
Column attached to a 2 ml collection tube and centrifuged at 8,000 rpm for 1 minute.
Collection tube including the flow-through was discarded. The spin column was then placed in a new 2 ml collection tube before 500 µl Buffer AW1 (wash buffer 1) was added into the spin column followed by centrifugation at 8,000 rpm for 1 minute.
Collection tube including the contents was disposed. Spin column was placed in a new 2 ml collection tube and 500 µl Buffer AW2 (wash buffer 2) was added into the column before centrifuged at 13,300 rpm for 3 minutes. The spin column was then carefully removed and transferred into a sterile 1.5 ml microcentrifuge tube. The flow through and the collection tubes were discarded. A volume of 200 µl Buffer AE
(elution buffer) was then added into the tube for elution and left to stand for 3 minute incubation at room temperature. The tube was then centrifuged at 8,000 rpm for 1
39
minute. This step was repeated for maximum yield. The DNA obtained was stored in
-20°C prior to use.
Electrophoresis was conducted on a 0.8 % (w/v) agarose gel at 100 volt for an hour to check the presence of DNA. Lambda Hind III was used as a molecular marker. The agarose gel was then stained with ethidium bromide for 10- 20 minutes and visualized under UV light in a gel documentation system (GENE Flash). The presence of DNA was shown by the appearance of single band on the gel. The quality and quantity of DNA were assessed using a spectrophotometer, Ultrospec
2000 (Pharmacia Biotech). High quality DNA extract was then PCR amplified using partial D-loop mtDNA gene primers.
3.7 Quality and Quantity of DNA Analysis by spectrophometry
The OD260, OD280 and OD320 values for each sample were used to calculate the quality and quantity of DNA extractions. One unit of OD is equivalent to 50 µg/ ml the concentration of double stranded DNA. The DNA extract can be considered to have a high level of purity (85-95%), if the values lie between 1.7-1.9 and is at optimum stage for analysis.
DNA quality: OD260 Reading
OD280 Reading
DNA quantity: OD260 Reading X 50 µg X Cuvette Volume (µl)
1000 µl Sample Volume (µl)
40
10 mg of liver tissue was cut and place in 1.5 ml microcentrifuge tube + 180 µl Buffer ATL + Proteinase K & vortexed
Incubated 56°C, 3 hrs (vortexed occassionally)
200 µl Buffer AL was added & vortexed
200 µl EtOH (96-100%) was added & vortexed
Transferred into DNeasy Mini Column in a 2 ml collection tube
Centrifuged (8,000 rpm, 1 min.), flow through discarded
DNeasy Mini Spin Column was placed in new 2 ml collection tube
500 µl Buffer AW1 was added
(8,000 rpm, 1 min.), flow through discarded
DNeasy Mini Spin Column was placed in new 2 ml collection tube
500 µl Buffer AW2 was added
13,300 rpm, 3 min
Flow through discarded
DNeasy Mini Spin Column was placed in new 1.5 ml microcentrifuge tube
200 µl Buffer AE (for elution) was added
Incubate for 1 min at room temperature, 8,000 rpm, 1 min
Stored in -20°C prior to use
Figure: 3.3: Extraction Protocol for DNeasy Kit (QIAGEN).
41
3.8 Analysis of mtDNA D-loop Gene
3.8.1 Polymerase Chain Reaction (PCR) Procedures
PCR was conducted in a G Storm thermal cycler (Gene Technologies Ltd.,
Braintree Essex, UK). All steps were conducted under sterile conditions in order to avoid any cross contamination. Table 3.4 shows the primers used to amplify mtDNA
D- loop gene (Zhong et al. 2008). PCR reagents used in this study were 10X PCR
Buffer (TOYOBO), 2mM dNTPs (TOYOBO), 5 pmol of each primer, 327-L and
885-H, 1.25 U Taq DNA polymerase (TOYOBO) and 25ng of template DNA. The mixture was brought up to a final volume of 25 µl with deionised distilled water, ddH20. A negative control tube containing all PCR reagents except the DNA template was included to detect contamination through the presence of a band
(despite the absence of genomic DNA). The PCR profile (Table 3.5) was according to the protocol provided by PCR reagent supplier (Blend Taq, TOYOBO), but optimised for the annealing temperature.
42
Table 3.4: Primers used to amplify D- loop mtDNA gene.
Primer Code Sequence Direction D-loop 327-L 5’ CTG TCC ATA TCA TGA CTA CTT G 3’ Forward 885-H 5’ GGT CTT AGC TTG TAG AGA GGT C 3’ Reverse
Table 3.5: PCR profile used to amplify D-loop mtDNA gene.
Temperature Duration
(°C) (minute/s) Pre Denaturation 94°C 1 Denaturation 94°C 1 Annealing 48.3°C 1 30 cycles Extension 72°C 2 Hold 4°C 12 hrs
3.8.2 PCR Optimisation
Optimisation is vital in order to obtain satisfactory amplified products which shown by a strong, bright band. In this study, only one parameter was optimised which was the annealing temperature.
3.8.2.1 Annealing temperature optimisation
A total of eight 0.2 ml PCR tubes containing all reagents for PCR were prepared in a final volume of 25 µl. Optimisation of annealing temperature were tested at 44.6°C, 44.8°C, 46.1°C, 48.3°C, 51.3°C, 53.3°C, 54.5°C and 54.6°C.
Annealing temperature at 48.3°C was found to be the most suitable annealing temperature for all samples studied.
3.8.3 Agarose Gel Preparation
Agarose gel was used to confirm the presence of PCR products. 1.7% gel concentration was used for PCR product detection. To produce a 70 ml, 1.7% (w/v)
43
gel, 1.19 grams of biotechnology grade agarose (BST Tech Lab) powder and 70 ml of 0.5 X TBE Buffer (Appendix A) were mixed and stirred. The mixture was then melted in a microwave oven for 2 minutes at medium level power. The melted agarose was slowly poured into a gel cast with a comb attached to ensure a smooth gel without air bubbles. The agarose gel was left at 27 ºC to solidify.
3.8.4 Analysis of PCR Product
Four µl of PCR product was mixed with 1.2 µl of loading dye and electrophoresed at 100 volts, 500 current and 250 power for 1 hour. After completion, the gel was soaked in ethidium bromide (EtBr) for about 5 minutes. The gel was then washed in distilled water or under running tap water to remove excess
EtBr from the gel surface. The gel was viewed and documented under UV transilluminator, a digital camera and an Electrophoresis Documentation and
Analysis System 120 (Kodak). High intensity bands indicated that high quality DNA was obtained and vice versa. The amplified D-loop mtDNA gene obtained was approximately 520 base pairs when aligned with a 100 bp DNA Ladder Plus
(Fermentas).
3.8.5 Purification of PCR products
The PCR products were purified prior to sequencing to remove excess primers, nucleotides, polymerases and salts as these compounds may inhibit the sequencing process. ‘Wizard® SV Genomic DNA Purification Kit (Promega,
Corporation, Madison, USA) was used for purification, according to the protocol recommended by the manufacturer. The purification procedure is described as follows. An equal volume of Membrane Binding Solution was added into the sample.
44
The contents were vortexed to mix completely. SV Mini column was then inserted into a 2 ml collection tube. Next, the contents in microcentrifuge tube were transferred into the SV Mini Column followed by incubation at 27 ºC for 1 minute.
The samples were centrifuged at 13,000 rpm for 1.5 minutes. Flow-through was discarded. The SV Mini Column was placed back into the same tube and the sample was washed with 700 µl Membrane Wash Solution and centrifuged at 13,000 rpm for
1.5 minutes. The flow-through was then discarded. The SV Mini Column was placed back into the same tube and the sample was again washed with 500 µl Membrane
Wash Solution followed by centrifugation at 13,000 rpm for 5.5 minutes. The flow- through was discarded. The SV Mini Column was placed back into the same tube and centrifuged at 13,000 rpm for 1.5 minutes. The SV Mini Column was then placed into a new, clean 1.5 ml microcentrifuge tube. Finally, a total of 50 µl
Nuclease Free Buffer was added at the centre of the SV Mini Column membrane to elute the purified DNA from the column which was then incubated at 27 ºCfor 2 minutes. The sample was then centrifuged at 13,000 rpm for 1.5 minutes. Purified
PCR products was then stored in -20°C prior to sequencing.
3.8.6 Sequencing
Purified PCR product was sent to a service provider, First Base Laboratory for sequencing. The purified PCR products were sequenced in the forward direction.
The sequencing technique employed the Sanger method based on fluorescently labelled DNA fragments. Fragments were detected using a tunable laser during electrophoresis. The process is automated and the sequence results were documented as a chromatogram.
45
3.8.7 Data Analysis of mtDNA D-loop Gene
3.8.7.1 Multiple Sequence Alignment and Identification of Haplotypes in
Fejevarya cancrivora and F. limnocharis Populations
The sequence results were analysed using several softwares. In the first step, sequences were edited and aligned using Chromas ver. 2.33 and Molecular
Evolutionary Genetic Analysis ver. 4 Software (MEGA4 - Tamura et al. 2007).
Primer sequences were trimmed of ‘noisy’ bases at both ends from the data prior to multiple alignments. All edited sequences were then multiply- aligned using
Alignment Explorer/CLUSTAL with ClustalW (1.6) DNA weight matrix in MEGA4.
All aligned sequences were then imported into Basic Local Alignment Search Tool
(BLAST; http://www.ncbi.nlm.nih.gov/blast) software to confirm the identity of the samples. Haplotype sequences were then generated using DNA Sequences
Polymorphism (DnaSP) program ver. 5.10.01 (Rozas et al. 2010).
3.8.7.2 Genetic variability within and between populations
The level of mtDNA D-loop gene variability within populations of both frog species were examined by computing the nucleotide diversity, π (the probability that two randomly chosen homologous nucleotides are different) and haplotype diversity, h (the probability that two randomly chosen haplotypes are different) indices using
Arlequin ver. 3.1 (Excoffier et al. 2005). The pairwise FST values for each combination of populations were also calculated and significance of this estimate was calculated using 1000 permutations of the data at P = 0.05. The mean pairwise distances within and between populations was calculated using Kimura 2-parameter model (Kimura, 1980) that takes into account nucleotide transition and transversion
46
substitution rates. MEGA4 software was also employed to estimate these genetic distances of within and between populations in both species.
Analysis of molecular variance, AMOVA (Excoffier et al. 1992) was also carried out using the same software based on 1000 permutations of the data matrix.
Samples of F. limnocharis were also analysed based on a hierarchical AMOVA considering the geography of the region (North, Central West and East of Peninsular
Malaysia). However, due to no sample obtained from Central west and East region of
F. cancrivora population as in the case of F. limnocharis, AMOVA analysis was performed without grouping them into region. The pairwise FST assessed with 10000
Markov chain steps, (followed by Bonferroni corrections for multiple comparisons to correct for ‘false positives’), was also applied to estimate the significant levels of population differentiation.
3.8.7.3 Phylogenetic Tree
The haplotype data were subjected to phylogenetic tree reconstruction using distance-based method, Neighbor Joining (NJ) and character- based method,
Maximum Parsimony (MP) in MEGA4. In this study, NJ phylogenetic trees were constructed using Kimura’s two-parameter (K2P) substitution model with 10,000 bootstrap replicates to assess the level of confidence. The NJ method takes into account transition and transversion substitution rates and differences in substitution rates among sites of nucleotide sequences. Nei and Kumar (2000) clearly stated that if the average pairwise Jukes-Cantor (JC) distance is > 1.0, calculated by computing the overall mean distance of the sequences, then the data is not suitable for generating NJ trees, and therefore an alternative method will be used.
47
MP is part of a class of character- based tree estimation methods, which take into account a matrix of discrete phylogenetic characters to infer one or more optimal phylogenetic trees for a set of taxa (Tamura et al. 2007). MP is based on the assumption that the most likely tree is the one that requires the least evolutionary change to explain the alignment data. The basic premise of parsimony is that taxa sharing a common characteristic will be grouped together because they inherited that characteristic from a common ancestor. In this study, Close- Neighbour- Interchane
(CNI) methods with 1,000 bootstrap replicates were used to obtain most equally parsimonious trees.
3.8.7.4 Minimum Spanning Network
The relationships among haplotypes for both species were also constructed by creating a minimum spanning tree using Network ver. 4.6.1.0 (Fluxus, 2011), connecting all haplotypes as nodes in a network connected by the least number of substitutions. Network calculation was based on median-joining by Network ver. 4.6 software.
3.9 GIS mapping of Fejevarya cancrivora and F. limnocharis
The study maps the distribution of Fejevarya cancrivora and F. limnocharis of Peninsular Malaysia. Figure 3.4 shows the methodology flowchart. The data for this project came from two sources, which were defined as follows: (1) primary GPS data collected during the sampling of both species conducted from July 2010 until
November 2011 (Tables 3.6 and 3.7); (2) secondary data taken from literature
(journals, proceedings, books) on the two species (Tables 3.8 and 3.9). For the secondary data, in cases where authors did not mention the coordinates of their study
48
locations, the coordinates were determined via the Google Earth software. Data for
Peninsular Malaysia were gathered and consolidated into databases and mapped using the ArcGIS 9 software ver. 9.3 (Esri, 2012).
Figure 3.4: Methodology flowchart.
49
Table 3.6: Primary data of Fejevarya cancrivora.
Sub-district No. Location (mukim) District State 1 Sedim Sedim Kulim Kedah 2 Bukit Perangin Malau Kubang Pasu Kedah 3 Gunung Jerai Yan Yan Kedah 4 Sungai Teroi Yan Yan Kedah 5 Kampung Pulau Nyior Jitra Kubang Pasu Kedah 6 Ulu Paip Karangan Kulim Kedah 7 Pantai Kok Padang Mat Sirat Padang Mat Sirat Kedah 8 Sungai Dua Mukim 10 SPU SPU Penang 9 Titi Kerawang Telok Bahang Barat Daya Penang 10 Durian Valley Paya Terubung Timur Laut Penang 11 Jerejak Paya Terubung Timur Laut Penang 12 Sungai Burong Sungai Burong Barat Daya Penang 13 Sungai Cempias Kampung Buaya Kuala Kangsar Perak
50
Table 3.7: Primary data of Fejevarya limnocharis.
Sub-district No Location (mukim) District State 1 Kampung Kedawang Kedawang Langkawi Kedah 2 Kampung Pulau Nyior Jitra Kubang Pasu Kedah 3 Lembah Bujang Bujang Kuala Muda Kedah 4 Sedim Sedim Kulim Kedah 5 Ulu Paip Karangan Kulim Kedah 6 Gunung Jerai Yan Yan Kedah 7 Sungai Teroi Yan Yan Kedah 8 Ulu Paip Karangan Kulim Kedah 9 Lata Mengkuang Sik Sik Kedah 10 Pantai Kok Padang Mat Sirat Padang Mat Sirat Kedah 11 Telaga Tujuh Padang Mat Sirat Padang Mat Sirat Kedah 12 Sungai Burong Sungai Burong Barat Daya Penang 13 USM Trans Kerian Mukim 9, SPS SPS Penang 14 Bukit Panchor Mukim 10 SPS SPS Penang 15 Sungai Acheh Mukim 10 SPS SPS Penang 16 Titi Kerawang Telok Bahang Barat Daya Penang 17 Sungai Tukun Telok Bahang Barat Daya Penang 18 Durian Valley Paya Terubung Timur Laut Penang 19 Sungai Dua Mukim 10 SPU SPU Penang 20 Sungai Cempias Kampung Buaya Kuala Kangsar Perak 21 Pusing Pusing Kinta Perak 22 Langkap Changkat Jong Hilir Perak Perak 23 FRIM Batu Gombak Selangor 24 Tanjong Karang Tanjong Karang Kuala Selangor Selangor 25 Sabak Sabak Sabak Bernam Selangor 26 USM Kubang Kerian Kubang Kerian Kota Bharu Kelantan 27 Gunung Stong Dabong Dabong Kelantan 28 Kampung Raja Kubang Bemban Besut Terengganu
51
Table 3.8: Secondary data of Fejevarya cancrivora.
No. Location Sub-district District State Source (mukim) 1 Bukit Tebing Wang Perlis Perlis Berry (1975) Tinggi Bintong 2 Taman Negeri Titi Tinggi Perlis Perlis Ibrahim et al. Wang Kelian (2001) 3 MADA Kuala Kedah Kota Setar Kedah Ibrahim et al. (2009) 4 Sungai Junjung Kulim Kulim Kedah Mohd Hamidi (2010), Ahmad Ridzwan (2010) 5 Pulau Singa Kedawang Langkawi Kedah Lim et al. Besar (2010) 6 Pulau Tuba Kuah Langkawi Kedah Berry (1975) 7 Bukit Panchor Mukim 10 SPS Pulau Ibrahim et al. Pinang (2012) 8 Jerejak Paya Timur Pulau Ibrahim et al. Terubung Laut Pinang (2013) 9 Bukit Jana Kamunting Larut dan Perak Shahriza & Matang Ibrahim (2012a) 10 Klang Coast Klang Klang Selangor Berry (1975) 11 Bukit Lagong Sungai Buluh Petaling Selangor Berry (1975) 12 Ampang New Ampang Ulu Selangor Berry (1975) Village Langat 13 Bukit Belata Jeram Kuala Selangor Lim et al. Selangor (2008) 14 Peta Area of Labis Segamat Johor Shahriza et al. Endau-Rompin (2012b) National Park 15 Bukit Bauk Jerangau Dungun Terengganu Ibrahim et al. (2008) 16 Pulau Tioman Tioman Rompin Pahang Berry (1975) 17 Bangkong Lepar Pekan Pahang Berry (1975)
52
Table 3.9: Secondary data of Fejevarya limnocharis.
No. Location Sub-district District State Source (mukim) 1 Kaki Bukit Beseri Perlis Perlis Berry (1975) 2 Kg. Wang Beseri Perlis Perlis Berry (1975) Kelian 3 Chuping Chuping Perlis Perlis Berry (1975) 4 Bukit Tebing Wang Perlis Perlis Berry (1975) Tinggi Bintong 5 MADA Kuala Kedah Kota Setar Kedah Ibrahim et al. (2009) 6 Sungai Kulim Kulim Kedah Mohd Hamidi Junjung (2010) 7 Lata Bukit Baling Baling Kedah Shahriza et al. Hijau (2011a) 8 Bukit Wang Jitra Kubang Kedah Berry (1975) Pasu 9 Taman Negeri Titi Tinggi Perlis Perlis Ibrahim et al. Wang Kelian (2001) 10 Ayer Hangat Ayer Hangat Langkawi Kedah Berry (1975) 11 Pulau Tuba Kuah Langkawi Kedah Berry (1975) 12 Beris Valley Kulim Kulim Kedah Shahriza et al. (2011b) 13 Alor Setar Kota Setar Kota Setar Kedah Berry (1975) 14 Hutan Simpan Sik Sik Kedah Norhayati et al. Ulu Muda (2005b) 15 Bukit Panchor Mukim 10 SPS Pulau Ibrahim et al. Pinang (2012) 16 Jerejak Paya Timur Pulau Ibrahim et al. Terubung Laut Pinang (2013) 17 Bukit Jana Kamunting Larut dan Perak Shahriza & Matang Ibrahim (2012a) 18 Pulau Pangkor Lumut Manjung Perak Chan et al. (2010), Norhayati et al. (2012b) 19 Lake Durian Pipit Ulu Perak Perak Berry (1975) Chenderoh 20 Sungai Kulim Kulim Kulim Perak Berry (1975) 21 Sungai Grik Grik Ulu Perak Perak Berry (1975) 22 Taman Negeri Grik Ulu Perak Perak Norhayati et al. Diraja Belum (2011) 23 Field Study Setapak Gombak Selangor Berry (1975) Centre 24 Bukit Lagong Sungai Buluh Petaling Selangor Berry (1975) 25 Templer's Park Rawang Gombak Selangor Berry (1975) 26 Bukit Belata Jeram Kuala Selangor Lim et al. 53
Selangor (2008) 27 Lake Garden Bandar Kuala Kuala Kuala Berry (1975) Lumpur Lumpur Lumpur 28 Jasin District Chabau Jasin Melaka Berry (1975) 29 Petaling Kuala Jelebu Negeri Berry (1975) Kelawang Sembilan 30 Ulu Bendul Terachi Kuala Negeri Norhayati et al. Pilah Sembilan (2010) 31 Peta Area of Labis Segamat Johor Berry (1975) Endau-Rompin National Park 32 Mawai Estate Sedhili Besar Kota Johor Berry (1975) Tinggi 33 Pantai Melawi Melawi Bachok Kelantan Belabut et al. (2010) 34 Gunung Stong Dabong Dabong Kelantan Norhayati et al. (2005c) 35 Bukit Bauk Jerangau Dungun Terengganu Ibrahim et al. (2008) 36 Sungai Tekam Tembeling Jerantut Pahang Berry (1975) 37 Gunung Ulu Cheka Jerantut Pahang Berry (1975) Benom 38 Pahang Kuala Jerantut Pahang Berry (1975) National Park Tembeling 39 Tasek Bera Bera Bera Pahang Berry (1975) 40 Janda Baik Bentong Bentong Pahang Berry (1975) 41 Rompin Rompin Rompin Pahang Berry (1975) 42 Taman Rimba Kuala Lipis Lipis Pahang Norhayati et al. Kenong (2007) 43 Bukit Fraser Teras Raub Pahang Imbun et al. (2010)
54
CHAPTER 4
RESULTS
4.1 Genomic DNA Extraction & Purity of Genomic DNA
A total of 132 samples of the two investigated species, 26 of F. cancrivora and 106 of F. limnocharis from various locations were successfully extracted using
DNeasy Tissue Kit (QIAGEN). Representative images of DNA extractions run on
0.8% (w/v) agarose gel are shown in Plate 4.1. The extracted DNA was then checked for purity and quantification using a spectrophotometer. The spectrophotometric ratio values obtained ranging from 1.7- 2.1 were considered to be satisfactory.
4.2 Mitochondrial DNA Sequencing of D-loop Gene
4.2.1 Amplification of D-loop Gene
All 132 samples were successfully amplified (Plate 4.2) using the optimised
PCR conditions previously determined. The DNA samples with amplified D-loop fragments were then purified.
4.2.2 Purification of PCR Product
Purification of PCR products was performed to eliminate any enzymatic reactions generated during the amplification process. Only PCR products with clear, high intensity bands were purified. Plate 4.3 shows the purification results of the D- loop PCR products. All successfully purified products were then sent for sequencing.
55
M 1 2 3 4 5 6 7 23 130 bp
Plate 4.1: DNA extraction results of F. cancrivora and F. limnocharis on a 0.8% (w/v) agarose gel. Legend: Marker λ Hind III (M), samples Lane 1-4: Representative DNA fragments of F. limnocharis. Lane 5-7: Representative DNA fragments of F. cancrivora.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
600 bp
bp
Plate 4.2: PCR products of D- loop gene run on a 1.7% (w/v) agarose gel. Legend: Marker 100 bp Plus DNA Ladder (M), Lane 3 - 8: Representative fragments of F. limnocharis. Lane 10-17: Representative fragments of F. cancrivora. Lane 19: Negative control.
56
M 1 2 3 4 5 6 7 8 9 10 11
600 bp
bp
Plate 4.3: Purified D-loop PCR products run on a 1.7% (w/v) agarose gel. Legend: Marker 100 bp Plus DNA Ladder (M). Lane 2- 6: Representative fragments of F. limnocharis. Lane 7- 9: Representative fragments of F. cancrivora. Lane 11: Negative control.
4.2.3 Sequencing of Purified PCR Product
The sequencing results were highly satisfactory with good, clean and high peaks as shown on the sequence chromatogram (Appendix B). The sequences generated were approximately 600 bp. BLAST analysis in the National Center for
Biotechnology Information (NCBI) confirmed that the sequences obtained were D- loop gene of F. cancrivora and F. limnocharis. Data analysis was then independently conducted for both species as stated in Section 3.8.7.
4.3 Data Analysis of partial mtDNA D-loop Gene of Fejevarya cancrivora
populations
4.3.1 Intrapopulation Genetic Diversity
The D-loop gene was sequenced for 26 individuals of F. cancrivora from six populations, all from the northwest. Owing to the lack of D-loop sequences of
Fejervarva species in public databases, three Genbank sequences of related species 57
Limnonectes bannaensis (GenBank accession no: NC012837.1), L. fujianensis
(GenBank accession no: NC007440) and Rana nigromaculata (GenBank accession no: NC002805) were used as outgroups. The aligned sequences of 495 bp contained
19 (3.8%) variable positions, 13 (2.6%) of which were parsimony informative and 6 were singletons. Among the 26 individuals sequenced, nine unique haplotypes were detected in this species (Table 4.1). The common haplotype, namely Haplotype 1 was observed in all populations, except in Sungai Dua (SGDs). Most haplotypes, however, differed by only one and two bases from the major sequence (Haplotype 1), except for Haplotype 2 (Table 4.2). All haplotypes were private, specific to different populations, except Haplotype 1.
Table 4.1: Haplotypes identified in six (6) populations of F. cancrivora.
HAPLOTYPE LKWs JITs SBGs ARTs JERs SGDs TOTAL 1 1 2 6 6 1 16 2 3 3 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 1 TOTAL 4 2 8 8 2 2 26
Key: LKWs= Pulau Langkawi, JITs= Jitra, SBGs= Sungai Burung, ARTs= Air Itam, JERs= Pulau Jerejak, SGDs= Sungai Dua.
58
Table 4.2: Distribution of nine (9) observed haplotypes, number of polymorphic sites, nucleotide diversity (π), number of haplotypes and haplotype diversity (ɦ) among 16 populations of F. cancrivora.
Nucleotide sites Haplotype 1 1 1 2 2 2 2 3 4 4 4 frequency 3 3 4 6 6 6 4 5 9 1 3 4 5 4 4 4 4 Haplotype 4 9 5 7 7 2 5 8 3 5 4 8 3 7 2 3 1 6 9 LKWs JITs SBGs ARTs JERs SGDs 1 A A T T T C C C A T C T T T T G T G C 0.2 1 0.8 0.8 0.5 2 . . . C C T . T G C T C C C C . . . . 0.8 3 ...... A . . . 0.1 4 ...... A . . 0.1 5 G C ...... A . . 0.1 6 . . C ...... 0.1 7 ...... T ...... 0.5 8 . . C ...... A 0.5 9 ...... A . 0.5 No. of samples (n) 4 2 8 8 2 2 No. of polymorphic sites 11 0 3 5 1 4 Nucleotide diversity (π) 0.011 0 0.002 0.003 0.002 0.008 No. of haplotypes 2 1 3 3 2 2 Haplotype Diversity (h ) 0.500 0.000 0.464 0.464 1.000 1.000 Key: LKWs= Pulau Langkawi, JITs= Jitra, SBGs= Sungai Burung, ARTs= Air Itam, JERs= Pulau Jerejak, SGDs= Sungai Dua.
59
Intrapopulation genetic diversity ranged from very low to high. Nucleotide diversity of 0.000 was observed in Jitra (JITs) and slightly higher in Sungai Burung
(SBGs) and Jerejak (JERs) of 0.002, and in Air Itam (ARTs), 0.008. The Langkawi
(LKWs) population was highest at 0.011 followed by Sungai Dua (SGDs) population at 0.008. ARTs and SBGs had haplotype diversity of 0.464 while LKWs, 0.5. The
JITs population showed no variation (0.0). The highest haplotype diversity was shown by JERs and SGDs with 1.000. Trend of intrapopulation variability was also supported by the genetic distances observed based on Kimura-2-parameter. The mean intrapopulation genetic divergence ranged from 0.0% for Jitra (JITs) to 1.1 % for Langkawi (LKWs) (Table 4.3).
4.3.2 Interpopulation Genetic Variability
Interpopulation genetic divergence (Table 4.3) ranged from 0.1 to 2.0 %.
Overall, population from LKWs was distantly related from other populations.
Genetic divergence between LKWs with JITs, SBGs, ARTs, JERs and SGDs were
1.7%, 1.7%, 1.8%, 1.8% and 2.0%, respectively. Genetic divergence of SGDs population with JITs, SBGs, ARTs and JERs showed a slight level differentiation of
0.4%, 0.5%, 0.5% and 0.5%, respectively. JITs was closely related to SBGs, ARTs and JERs with a value of 0.1%.
60
Table 4.3: Mean pairwise genetic distance among six (6) F. cancrivora populations.
LKWs JITs SBGs ARTs JERs SGDs LKWs 0.011 JITs 0.017 0.000 SBGs 0.017 0.001 0.002 ARTs 0.018 0.001 0.002 0.003 JERs 0.018 0.001 0.002 0.002 0.002 SGDs 0.020 0.004 0.005 0.005 0.005 0.008
4.3.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony
(MP) Analyses
Nei and Kumar (2009) stated that if the average pairwise Jukes-Cantor (JC) distance is > 1.0, then the data is not suitable for constructing Neighbour-Joining
(NJ) trees, and an alternative method should be used. Since the mean pairwise distance was < 1.0 (0.005) the NJ analysis was performed based on Kimura 2- parameter with 10000 pseudoreplicates (Figure 4.1).
The NJ tree bifurcated into two clades with very high (100%) bootstrap support (Fig. 4.1). The first clade comprised all (but one) populations namely JITs,
SBGs, ARTs, JERs, SGDs and LKWs, while the second clade represented LKWs only. The first clade was further divided into two sub clade with moderately high bootstrap support (68%), where the first sub clade consists of mixed population from all locations, while the second sub clade comprised Penang Island populations only
(ARTs and SBGs).
The phylogenetic trees of Maximum Parsimony (MP) analysis also shown the same set of major nodes as in NJ analysis with highly supported bootstrap values of
99% (Fig. 4.2). The first clade comprised all populations namely JITs, SBGs, ARTs,
JERs, SGDs and LKWs, while the second clade represented LKWs only as in the NJ analysis.
61
Figure 4.1: NJ tree of six (6) F. cancrivora populations. Key: Pulau Langkawi, mixed populations from all locations, except Sungai Dua, Sungai Burung, Air Itam, Pulau Jerejak, Sungai Dua.
62
Figure 4.2: MP tree of six (6) F. cancrivora populations. Key: Pulau Langkawi, mixed populations from all locations except Sungai Dua, Sungai Burung, Air Itam, PulauJerejak, Sungai Dua.
4.3.2.2 Analysis of Molecular Variance (AMOVA)
Since all the samples of this species were collected at the northern part of
Peninsular Malaysia, AMOVA was performed on all populations without grouping them into regions (Table 4.4). AMOVA analysis revealed that the majority of variability was within populations (76.7%, p < 0.05) with a smaller degree of the variation among populations (23.3%). The overall pattern of population differentiation was low and not significant (Table 4.5). Nevertheless, only the pairwise FST between LKWs with SGDs and JERs were significant with FST = 0.4120 and FST = 0.2453 (p< 0.05) respectively.
63
Table 4.4: Results of AMOVA of F. cancrivora populations.
Source of Variation d.f Variance components Percentage of variation Among populations 6 0.076 23.2 Within population 20 0.25 76.7
Table 4.5: Population differentiation (FST) among six (6) F. cancrivora samples based on mtDNA D- loop sequence.
LKWs JITs SBGs ARTs JERs SGDs LKWs * JITs 0.5294 * SBGs 0.3443 0.5000 * ARTs 0.4120 -0.2455 0.4176 * JERs 0.4120 -0.2455 0.4176 -0.0612 * SGDs 0.2453 0.0000 0.0000 0.0109 0.0109 *
Significant probabilities (P<0.05) based on 1000 permutations of haplotype frequencies among samples are indicated as bold.
4.3.2.3 Minimum Spanning Network
The minimum spanning network of nine F. cancrivora haplotypes revealed one major haplotype (Haplotype 1) that was represented by five populations (Fig.
4.3). The peripheral haplotypes branched out from the ancestral haplotypes by one to three mutations. Haplotype 2 comprising only LKWs population was highly differentiated out from the major presumed ancestral haplotype with high mutational differences. The minimum spanning network with a description of evolutionary relationships based on substitutions among haplotypes was concordant with the NJ and MP phylogenetic trees.
64
Figure 4.3: Minimum spanning network for six (6) F. cancrivora populations. Keys: Langkawi, Jitra, Sungai Burung, Air Itam, Jerejak, Sungai Dua.
65
4.4 Data Analysis of partial mtDNA D-loop Gene of F. limnocharis
populations
4.4.1 Intrapopulation Genetic Diversity
Multiple sequence alignments were performed for all 106 F. limnocharis samples using MEGA4 ClustalW (1.6) DNA weight matrix. Owing to the lack of D- loop sequences of Fejervarya species in public databases, three Genbank sequences of closely related species; Limnonectes bannaensis (GenBank accession no:
NC012837.1), L. fujianensis (NC007440) and Rana nigromaculata (NC002805) were used as outgroups. The aligned sequences of 519 bp contained 13 (2.5%) variable positions, six (1.2%) of which were parsimony informative and seven were singletons.
Among the 106 sequences, only 14 haplotypes were found in this study
(Table 4.6). The two common haplotypes, namely Haplotype 1 and Haplotype 5 were observed in almost equal frequency (35 and 38%, respectively). Haplotype 1 was shared among twelve of 16 populations, which included Pulau Langkawi (LKWs),
Jitra (JITs), Lembah Bujang (LBGs), Sedim (SEDs), Ulu Paip (ULPs), Sungai
Burung (SBGs), Bukit Panchor (BPRs), Sungai Acheh (SGAs) (northwest), Langkap
(LKPs), Sabak Bernam (SBKs), Tanjong Karang (TKs) (central west) and
Terengganu (TRGs) (east). Haplotype 5 was shared by nine populations, which included population from Sedim (SEDs) (northwest), Pusing (PUSs), Langkap
(LKPs), Kuala Kangsar (KKs), Sabak Bernam (SBKs), Tanjong Karang (TKs),
Kepong (KEPs) (central west), Kelantan (KLNs) and Terengganu (TRGs) (east).
Interestingly, Haplotype 1 was common to all three regions, although were missing in a few populations in central west and east. In contrast, Haplotype 5 comprised
66 populations mainly from central west and the east region. PUSs, KKs, KEPs and
KLNs were populations that did not contain the common Haplotype 1, but were made up of only Haplotype 5. Haplotypes 2 and 4 were singletons and specific to populations in northwest, while the singletons 6-10 were specific to populations in central west and 11-14 to east Peninsular Malaysia.
Haplotype diversity (h) ranged from 0.000 in LBGs, SBGs and PUSs to 0.833 in JITs with TRGs second in rank at 0.744, while nucleotide diversity (π) ranged from 0.000 in LBGs, SBGs and PUSs (northwest) to 0.006 in TRGs (Table 4.7).
Trend of intrapopulation variability was also supported by the genetic distances observed based on Kimura-2-parameter. Intrapopulation genetic distances (Table
4.8) ranged from 0.0 to 0.3% in northwest, 0.0 to 0.4% in central west and 0.1 to
0.6% in the east. These values are in agreement with other genetic variability values
(haplotype and nucleotide diversities) earlier described where LBGs, SBGs and
PUSs were the lowest and TRGs the highest (Table 4.8). Overall, the genetic diversity did not show any regional trend i.e. there were both high and low variability populations in all three regions.
67
Table 4.6: Haplotypes identified in 16 populations of F. limnocharis.
Northwest Central West East HAP. LKWs JITs LBGs SEDs ULPs SBGs BPRs SGAs PUSs LKPs KKs SBKs TJGs KEPs KLNs TRGs TOTAL 1 3 2 3 4 2 4 3 3 1 4 3 5 37 2 1 1 3 1 2 1 5 3 1 13 4 1 1 5 1 7 6 4 1 7 4 5 5 40 6 1 1 7 1 1 8 1 1 9 1 1 10 6 6 11 1 1 12 1 1 13 1 1 14 1 1 TOTAL 4 4 3 7 3 4 8 6 7 8 5 7 16 5 6 13 106 Key: LKWs = Pulau Langkawi, JITs = Jitra, LBGs = Lembah Bujang, SEDs = Sedim, ULPs = Ulu Paip, SBGs = Sungai Burung, BPRs = Bukit Panchor, SGAs = Sungai Acheh, PUSs = Pusing, LKPs = Langkap, KKs = Kuala Kangsar, SBKs = Sabak Bernam, TJGs = Tanjong Karang, KEPs = Kepong, KLNs = Kelantan, TRGs = Terengganu.
68
Table 4.7: Distribution of 14 observed haplotypes, number of polymorphic sites, nucleotide diversity (π), number of haplotypes and haplotype diversity (h) among 16 populations of F. limnocharis.
Nucleotide sites Haplotype frequency 1 2 3 3 3 3 4 4 5 1 3 7 9 4 5 0 1 5 9 6 6 0 Northwest Central West East Haplotype 5 2 0 8 9 8 2 2 0 5 1 4 9 LKWs JITs LBGs SEDs ULPs SBGs BPRs SGAs PUSs LKPs KKs SBKs TJGs KEPs KLNs TRGs 1 T A C A A C A A A A C C G 0.75 0.5 1 0.571 0.667 1 0.375 0.5 0.125 0.571 0.188 0.385 2 . . T ...... 0.25 3 . . . G ...... 0.25 0.286 0.333 0.625 0.5 0.2 4 ...... G . . . . 0.25 5 . . . G . T . . G . T T . 0.143 1 0.75 0.8 0.143 0.438 0.8 0.833 0.385 6 . . . G . T C . G . T T . 0.125 7 . T . G . T . . G . T T . 0.2 8 . . . . . T ...... 0.143 9 . . . . G ...... 0.143 10 . . . . . T . . G . T T . 0.375 11 C . . G . T . . G . T T . 12 ...... G . . . . . 0.167 0.077 13 . . . G . T . . G . T T A 0.077 14 . . . G . T . . G C T T A 0.077 No. of sample (n) 4 4 3 7 3 4 8 6 7 8 5 7 16 5 6 13 No. of polymorphic sites 1 2 0 5 1 0 1 1 0 6 1 6 5 4 1 8 Nucleotide diversity (π) 0.001 0.002 0.000 0.003 0.001 0.000 0.001 0.001 0.000 0.003 0.001 0.004 0.004 0.004 0.001 0.006 No. of haplotypes 2 3 1 3 2 1 2 2 1 3 2 4 3 2 2 5 Haplotype Diversity (h ) 0.500 0.833 0.000 0.667 0.667 0.000 0.536 0.600 0.000 0.464 0.400 0.714 0.675 0.400 0.333 0.744 Key: LKWS= Pulau Langkawi, JITs= Jitra, LBGs= Lembah Bujang, SEDs= Sedim, ULPs= UluPaip, SBGs= Sungai Burung, BPRs= Bukit Panchor, SGAs= Sungai Acheh, PUSs= Pusing, LKPs= Langkap, KKs= Kuala Kangsar, SBKs= Sabak Bernam, TJGs= Tanjong Karang, KEPs= Kepong, KLNs= Kelantan, TRGs= Terengganu.
69
4.4.2 Interpopulation Genetic Variability
The northwest populations were closely related to each other with genetic divergence ranging from 0.0 to 0.2%, similar in magnitude to the intrapopulation genetic distances. The same was observed for the interpopulation genetic divergence of the central west region, which ranged from 0.0 to 0.4% (the latter only between
KEP-TJG), except for comparisons with Sabak Bernam (SBK), which had higher values of 0.7 to 0.9% with other populations. The genetic divergence between the two eastern populations of Kelantan and Terengganu was 0.5% of similar magnitude to the intradivergence of Terengganu population. Inter-regional divergence was generally high between northwest and central west populations, except for Sabak
Bernam (SBKs) which was surprisingly, more closely related to the northwest population (0.2 to 0.3%) than it was within its own central west region of 0.4%. The eastern populations of Kelantan and Terengganu showed different patterns of relationships. While the Kelantan population was very closely related to the central western populations (0.0 to 0.3%), but distantly related to the northwest Peninsular populations (0.9 to 1.1%), the Terengganu population was uniformly (0.5 to 0.6%) related to all populations in all regions. Nevertheless, generally low mean pairwise population genetic distance was observed.
70
Table 4.8: Mean pairwise genetic distance index between (below diagonal) and within (on diagonal) populations, and geographical distance of 16 F. limnocharis populations (above diagonal).
Region Northwest Central West East Population LKWs JITs LBGs SEDs ULPs SBGs BPRs SGAs PUSs LKPs KKs SBKs TJGs KEPs KLNs TRGs LKWs 0.001 72 104 141 133 121 158 150 247 292 217 316 359 405 281 313 JITs 0.001 0.002 32 69 61 106 128 125 211 258 178 289 287 368 209 242 LBGs 0.000 0.001 0.000 37 29 49 68 65 154 201 122 230 271 313 209 229 SEDs 0.002 0.002 0.002 0.003 8 55 42 48 117 163 83 196 235 274 187 198 ULPs 0.001 0.001 0.001 0.002 0.001 48 42 45 121 168 88 200 239 279 193 206 SBGs 0.000 0.001 0.000 0.002 0.001 0.000 42 31 129 173 101 196 238 285 241 252 BPRs 0.002 0.002 0.001 0.002 0.001 0.001 0.001 13 89 135 60 163 203 247 219 222 SGAs 0.001 0.001 0.001 0.002 0.001 0.001 0.001 0.001 98 143 71 168 210 255 229 234 PUSs 0.010 0.009 0.010 0.008 0.009 0.010 0.009 0.009 0.000 46 33 83 119 158 228 209 LKPs 0.009 0.008 0.009 0.007 0.008 0.009 0.008 0.008 0.001 0.003 80 45 73 112 256 228 KKs 0.011 0.009 0.010 0.008 0.009 0.010 0.009 0.009 0.000 0.002 0.001 116 152 191 208 196 SBKs 0.002 0.003 0.002 0.003 0.002 0.002 0.003 0.003 0.008 0.008 0.009 0.004 44 98 301 272 TJGs 0.008 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.003 0.003 0.003 0.007 0.004 57 320 285 KEPs 0.009 0.007 0.008 0.007 0.008 0.008 0.007 0.007 0.002 0.003 0.002 0.007 0.004 0.003 327 285 KLNs 0.011 0.009 0.010 0.008 0.009 0.010 0.009 0.009 0.000 0.002 0.001 0.009 0.003 0.002 0.001 55 TRGs 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.005 0.005 0.005 0.006 0.005 0.005 0.005 0.006 Key: LKWs= Pulau Langkawi, JITs= Jitra, LBGs= Lembah Bujang, SEDs= Sedim, ULPs= UluPaip, SBGs= Sungai Burung, BPRs= Bukit Panchor, SGAs= Sungai Acheh, PUSs= Pusing, LKPs= Langkap, KKs= Kuala Kangsar, SBKs= Sabak Bernam, TJGs= TanjongKarang, KEPs= Kepong, KLNs= Kelantan, TRGs= Terengganu.
71
4.4.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony
(MP) Analyses
The NJ tree bifurcated into two clades with high support (100%) (Figure 4.4).
The first clade included all populations from the northwest region, comprisinG
Penang, Kedah and Perak populations and a single central west (SBK) population with an individual from the eastern region of Terengganu and Kelantan. A closer inspection and comparison with Table 4.7 showed that these are populations which have the predominant haplotype (Haplotype 1) and those haplotypes varying only by a single nucleotide from it.
The second clade comprised all samples from the central west and eastern populations of Kelantan and Terengganu and a single representative from the northern region in Kedah (Sedim). These are the populations with predominantly
Haplotype 5 and its variants. This shows that the central west and northwest regions were fairly differentiated from each other, while the eastern Kelantan and
Terengganu populations had haplotypes closely related to either of these two regions, but mainly to central west and do not form their own discrete clade.
The phylogenetic tree of Maximum Parsimony (MP) showed the same set of major nodes as in NJ tree with high bootstrap values of 100% as presented in Figure
4.5. The first clade included all populations from the northwest region and a single central west (SBK) population with an individual from the eastern region of
Terengganu and Kelantan. On the other hand, the second clade comprised all samples from the central west and eastern populations of Kelantan and Terengganu and a single representative from the northern region in Kedah (Sedim) which is similar to the NJ tree.
72
CLADE 1
CLADE 2
Figure 4.4: NJ tree of 16 F. limnocharis populations. Key: Mixed populations from northwest, central west and east; mixed populations from northwest and central west; populations from northwest region; populations from central west region; populations from east region.
73
Figure 4.5: MP tree of 16 F. limnocharis populations. Key: Mixed populations from northwest, central west and east; mixed populations from northwest and central west; populations from northwest region; populations from central west region; populations from east region.
4.4.2.2 Analysis of Molecular Variance (AMOVA)
The analysis of molecular variance (AMOVA) showed that the level of variation among groups partitioned to northwest, central west and east groups was -
4.64% (indicated as 0%); among population within groups, 31.89% and within population, 72.74% (Table 4.9). Analysis of F- statistics revealed that the variance among populations relative to the total variance was FST = 0.273, p < 0.005. The variance among populations within groups was FSC = 0.305, p < 0.005 and FCT = -
0.047, p > 0.005 for the variance among groups relative to the total variance. The results revealed most of variation occurred within population. The fairly low and insignificant level of regional or group differentiation may be largely attributed to the
74 fact that although high differentiation of central west population to northwest population was detected, this was negated by the low differentiation of central west to eastern populations.
Considering the earlier results described above, a reanalysis was conducted.
The AMOVA, repeated with all the populations partitioned into two groups (group 1: northwest populations; group 2: central west and east populations) (Table 4.10) according to haplotype data, revealed different results. The analysis showed that
54.81% of the total variance resided among groups i.e. among regions (FCT =
0.54806, P <0.05), 9.99% of the variance resided within groups i.e. between populations (FSC = 0.22107, P <0.05) and 35.2% of the total genetic variance resided within populations (FST = 0.64797, P <0.05). The higher among group variation and significant F- statistic results supported the earlier observation that the central west populations were closely related to the eastern populations.
75
Table 4.9: AMOVA results of 16 F. limnocharis populations partitioned into three groups according to regions (northwest, central west and east).
Source of variation d. f Variance Components Percentage of Variation Fixation Indices P value Among groups 2 -0.0168Va -4.63 ФCT = -0.047 0.658 Among population within groups 13 0.116Vb 31.89 ФSC = 0.305 0 Within population 90 0.264Vc 72.74 0
Table 4.10: AMOVA results of 16 F. limnocharis populations after partitioning into two groups (group 1: northwest populations; group 2: central west and east populations combined)
Source of Variation d.f Variance components Percentage of variation Fixation Indices P value Among groups 1 1.04850 Va 54.81 ФCT = 0.54806 0.00069 Among population within groups 14 0.19114 Vb 9.99 ФSC = 0.22107 0.0001 Within populations 90 0.67347 Vc 35.2 0
76
The low pairwise FST value among populations in northwest region (Table
4.11) indicated low genetic differentiation for this region. P value after Bonferroni correction revealed that pairwise FST values involving central west and east regions with northern populations were highly differentiated and significant. However, SBKs
(Sabak Bernam, central west) population indicated low genetic differentiation with the northwest population in agreement with genetic distance and AMOVA data described previously.
77
Table 4.11: Population divergence (FST) between F. limnocharis populations based on mtDNA D- loop sequence.
Region Northwest Central west East Population LKWs JITs LBGs SEDs ULPs SBGs BPRs SGAs PUSs LKPs KKs SBKs TJGs KEPs KLNs TRGs LKWs * JITs 0 * LBGs -0.09091 -0.09091 * SEDs 0.05114 -0.14361 -0.03279 * ULPs 0.4032 0.10187 0.41463 -0.0231 * SBGs 0.25773 -0.04348 0.25 -0.09045 -0.13386 * BPRs 0 0 0 0.03448 0.46067 0.31429 * SGAs 0.01449 -0.26829 0 -0.21307 -0.08525 -0.25806 0.11111 * PUSs 0.96793* 0.92412* 1* 0.78571 0.9343* 0.93913* 1* 0.96354 * LKPs 0.75416 0.67785* 0.75403 0.57292 0.74751* 0.73084 0.77645 0.70016 -0.01818 * KKs 0.91868 0.85941* 0.94907 0.72295 0.89399* 0.89142* 0.95619 0.89905 0.07285 -0.00427 * SBKs -0.0257 -0.10253 -0.12299 -0.0813 0.17012 0.06275 -0.05461 -0.1274 0.77778* 0.57706 0.71608* * TJGs 0.62238* 0.5507* 0.6095 0.473 0.64255* 0.61929* 0.63044 0.57845 0.19867 0.03436 0.16295 0.45462* * KEPs 0.74905 0.64899 0.75435* 0.51444 0.73435 0.71486 0.78581 0.67367 0.07285 -0.14615 0 0.52988 0.04498 * KLNs 0.92675 0.87359* 0.95427 0.742* 0.90088* 0.9* 0.95988* 0.9106 0.02778 -0.02666 0.00339 0.73565* 0.17985 0.0255 * TRGs 0.28794 0.17752 0.24404 0.11833 0.30013 0.26373 0.28683 0.18053 0.28218 0.11832 0.23478 0.13368 0.06565 0.04215 0.25677 * Significant probabilities (P<0.05) based on 1000 permutations of haplotype frequencies among samples are indicated as bold. *Significant population differentiation via exact test (after Bonferroni corrections), P<0.05. Key: LKWS= Pulau Langkawi, JITs= Jitra, LBGs= Lembah Bujang, SEDs= Sedim, ULPs= Ulu Paip, SBGs= Sungai Burung, BPRs= Bukit Panchor, SGAs= Sungai Acheh, PUSs= Pusing, LKPs= Langkap, KKs= Kuala Kangsar, SBKs= Sabak Bernam, TJGs= Tanjong Karang, KEPs= Kepong, KLNs= Kelantan, TRGs= Terengganu.
78
4.4.2.3 Minimum Spanning Network
The evolutionary relationships and number of substitutions among haplotypes is displayed in the minimum spanning network according to populations (Fig. 4.6) and according to regions (Fig 4.7). The percentage contribution of each population or region for a particular haplotype is given by the area of coloured region. Size of the circle is proportional to frequencies of the haplotype. The red diamond dots are median vectors which represent sample that should be present but could not be detected during sampling.
The minimum spanning network of 14 F. limnocharis haplotypes revealed two major haplotypes from which lower frequency haplotypes radiated. Clade A consisted of haplotypes of populations from all three regions although not detected in a few populations in central west and east while Clade B was occupied by the majority of central west and eastern populations. The ancestral haplotypes for Clade
A was Haplotype 1 whereas Haplotype 5 was the ancestral haplotype for Clade B.
79
CLADE A
CLADE B
Figure 4.6: Minimum spanning network of 16 F. limnocharis populations. Key: Langkawi, Jitra, LembahBujang, Sedim, UluPaip, Sungai Burung, Bukit Panchor, Sungai Acheh, Pusing, Langkap, Kuala Kangsar, SabakBernam, TanjongKarang, Kepong, Kelantan, Terengganu.
80
CLADE A
CLADE B
Figure 4.7: A minimum spanning network of 14 haplotypes obtained from 16 F. limnocharis populations classified according to region. Key: Northwest, Central west, East
4.5 GIS mapping of Fejevarya cancrivora and F. limnocharis
As described in section 3.9, the data collected for both of the species in
Peninsular Malaysia were gathered and consolidated into databases and mapped using the ArcGIS 9 software ver. 9.3 (Esri, 2012). Figure 4.8 shows the distribution of F. cancrivora and in Peninsular Malaysia. Figures 4.9 - 4.12 show the close-up map of the distributions of both species for each state.
81
Figure 4.8: The distribution of Fejevarya cancrivora and F. limnocharis in Peninsular Malaysia.
82
Figure 4.9: The distribution of Fejevarya cancrivora and F. limnocharis in Perlis, Kedah and Penang.
83
Figure 4.10: The distribution of Fejevarya cancrivora and F. limnocharis in Perak, Selangor and Kuala Lumpur.
84
Figure 4.11: The distribution of Fejevarya cancrivora and F. limnocharis in Melaka, Negeri Sembilan and Johor.
85
Figure 4.12: The distribution of Fejevarya cancrivora and F. limnocharis in Kelantan, Terengganu and Pahang.
86
CHAPTER 5
DISCUSSION
Amphibians are very sensitive to environmental and climatic changes; hence their genetic diversity at population levels can provide useful information for tracking historical environmental variation (Zhong et al. 2008). Thus, investigating its genetic structure at the population level may reveal some very interesting insights.
The present study has utilized the most highly evolving gene in the mitochondrial genome of vertebrates, namely the D-loop to investigate population structures of two common frog species of the paddy fields, the Crab-eating Frog, F. cancrivora in
Northern Peninsular Malaysia and the Asian Grass Frog, F. limnocharis in
Peninsular Malaysia. In addition, the GIS approach was also applied in this study to map the presence and distributions of both species in Peninsular Malaysia.
5.1 Genetic diversity of Fejevarya cancrivora
Due to the very low sample sizes, the results obtained for this species could only be considered as preliminary at this stage and any inference should be noted with caution. Generally, all genetic variability parameters - nucleotide and haplotype diversity and genetic distances showed very high genetic variation for Pulau
Langkawi (π = 1.1%, ɦ = 0.5) and lack of variation for the Jitra population (π = 0%, ɦ
= 0), while the other populations showed divergences of 0.2 to 0.3%. These were low and of the same magnitude as most populations of F. limnocharis.
87
5.2 Population structure of Fejevarya cancrivora
The major finding of this investigation conducted on the limited number of F. cancrivora populations was the genetic isolation of Pulau Langkawi and its comparatively high diversity. The second point to consider was the isolation-by- distance pattern of genetic variability of this species, in which the Pulau Langkawi population was more closely related to the mainland Kedah population. According to
Wright’s island model of migration, gene flow is higher in closely related island but a longer distance would result in a dispersal barrier for restricted dispersal species
(Wright, 1969). On the other hand, the Penang Island populations of Sungai Dua,
Sungai Burung, Air Itam and Jerejak formed another genetic cluster, but were more closely related to each other unlike the Pulau Langkawi population with its mainland
Kedah populations. One plausible explanation of this pattern is that F. cancrivora is hunted for its juicy, tender meat for Chinese townspeople consumption in Malaysia
(Ibrahim, 2004). Since the Penang state comprised a Chinese majority (45.6%)
(Department of Statistics, 2010), it is assumed that the demand of frog meat in this area is high (particularly in Penang Island), which enables the transportation and movement of this species throughout the Penang state.
The Minimum Spanning Network (MSN) indicated that F. cancrivora population produced star-like mtDNA genealogies (Fig. 4.3) as a result of a recent expansion from the major maternal lineage (Mäkinen & Merilä, 2007). Under the coalescent theory, the central haplotype (Haplotype 1) is the most variable haplotype that is likely to be older and ancestral to the tip haplotypes (Crandall & Templeton,
1996) as observed in this species.
88
The tip haplotypes were unique to their own population (excluding JITs population), which showed limited gene flow between populations. Pulau Langkawi population had two sets of distantly related haplotypes (Haplotype 1 and Haplotype
2). Haplotype 2, which was confined to the Pulau Langkawi population, was derived from Haplotype 1, but with high mutational steps.
Imsiridou et al. (1998) postulated that the deep branching of populations from major haplotypes with high mutation rates may be caused by the extension time of isolation of populations, which had experienced bottleneck, followed by genetic drift.
This could explain the contemporary pattern in Pulau Langkawi, which is geographically isolated and geologically different from Kedah mainland. According to Mohd Shafeea (2010), rock outcrops of Pulau Langkawi are among the best known in Malaysia comprising various types of sedimentary rocks formed throughout the Palaeozoic Era and granitic rocks of Late Triassic age. Thus, it can be postulated that amphibians on Pulau Langkawi had evolved independently to adapt to this geographic condition and contributed to this kind of pattern. This also agrees with the expected lack of human mediated movement for a seemingly low economically important species in the predominantly Malay state of Kedah.
The evolutionary history of Pulau Langkawi formation could also account for the present genetic relationships observed. The rate of seawater tolerance in amphibians varies among species and long-distance movements by rafting have been suggested as a possible way of colonization for continental islands (Vences et al.
2003; Measey et al. 2007; Velo-Anto´n et al. 2012). This indicated that seawater is not an effective barrier to gene flow (Velo-Anto´n et al. 2012). However, in this study, the genetic differentiation between Pulau Langkawi and its mainland population (including the Penang Island population) is comparatively high (FST =
89
0.2453-0.5294, Table 4.5), which suggest that the Pulau Langkawi population had become isolated from the mainland with the increased sea level during the last glaciation time (Pleistocene – 18 kya ago). The same pattern of differentiation was also observed between island and coastal populations in Iberian insular fire salamanders, Salamandra salamandra (FST = 0.181–0.287) as a result of rising sea levels approximately 8000–9000 years ago (Velo-Anto´n et al. 2012) and in slender salamanders, Batrachoseps, on the islands of San Francisco Bay (Martı´nez-Solano
& Lawson, 2009) which share similar age (9000 years) and distances among islands and mainland population. Nevertheless, this hypothesis remains speculative as more samples and populations are required to qualify any inference made to explain the genetic differentiation of F. cancrivora population in this study.
5.3 Genetic diversity of Fejevarya limnocharis
In general, mean haplotype diversity (h) was low over all samples (0.471 ±
0.27) and varied among local populations from 0.000 to 0.833 ± 0.07. The mean nucleotide diversity (π) was 0.004 ± 0.00005 ranging from 0.00 to 0.006 ± 0.002 in the populations. This is however, comparable to the study conducted by Zhong et al.
(2008) on F. multistriata also based on the D-loop mtDNA gene. Their study showed high level of nucleotide diversity, π = 0.969 ± 0.005 but low level of haplotype diversity, ɦ = 0.017 ± 0.009. In contrast, a study on the Patagonian frog, Eupsophus calcaratus, using D-loop showed relatively low nucleotide diversity π = 0.048, but high level of haplotype diversity, ɦ = 0.999 (Nuñez et al. 2011). Nevertheless, none of the previous research based on the D-loop on frogs have shown low levels of both nucleotide and haplotype diversities.
90
The present study is consistent with those reported by Farjallah et al. (2012) on the pattern of the genetic diversity of the North African Green Frog, Pelophylax saharicus in Tunisia. Using partial Cytb gene, they suggested that the low levels of both nucleotide (π = 0.02) and haplotype diversities (ɦ = 0.7) shown by this species may be due to a recent population expansion from a small founder population. Grant
& Bowen (1998) hypothesised that populations with low values of both parameters
(π = < 0.5%, ɦ < 0.5) is a feature of recent population bottleneck or founder event by a single or a few mtDNA lineages. According to Chen et al. (2012), the extremely low genetic diversity of the Siberian salamander populations, Ranodon sibiricus in
China based on the mtDNA D-loop segment (π = ranging from 0.000 to 0.00091; ɦ = ranging from 0.000 to 0.526) was the result of the isolation of R. sibiricus populations that occurred recently due to human activity and/or climatic changes that adversely affected their habitats.
Bay and Caley (2011) stressed that maintaining genetic diversity should be the main focus of a conservation biology initiative. This is because genetic diversity is the raw material, which enables species populations to adapt to environmental fluctuations. Thus, reduced levels of genetic diversity is of major concern because it can lead to inbreeding (Frankham, 1998) and can lead to extinction risk for populations and species (Reed et al. 2002). It has been proven that inbreeding depression in captive (Frankham, 1995) and wild populations (Newman & Pilson,
1997; Saccheri et al. 1998) is directly linked with population declines and extinction.
Fejevarya limnocharis is a unique species, which can tolerate a broad range of habitats (van Dijk et al. 2013). Thus, the relatively low levels of genetic variation at the D-loop region in most populations of F. limnocharis observed in this study is unexpected as the general trend for species that has a broad geographic, climatic,
91 habitat, and ecological ranges is that they can harbour higher amounts of genetic diversity (Nevo, 1988). The very low level of genetic variability detected in this species could be attributed to high inbreeding. Amphibians are philopatric, with limited mobility, which can lead to self- mating or kin-mating among species in populations. The effect of inbreeding has also been reported in previous amphibian studies (Rower & Beebee, 2005; Halverson et al. 2006; Frantz et al. 2009).
It is widely accepted that inbreeding has significant effects on the viability of small and/or isolated animal and plant populations (Frankham, 1995; Keller &
Waller, 2002). Inbreeding has an impact in decreasing the level of the average heterozygosity of the offspring, which can further reduce survival and fecundity.
Decreased heterozygosity or increased homozygosity can directly decrease fitness and, furthermore, individuals may become homozygous for deleterious recessive alleles (Wedekind et al. 1995; Halverson et al. 2006).
The effect of inbreeding in amphibians in the wild and under laboratory condition has been examined by Halverson et al. (2006) using the wood frog, Rana sylvatica as a model. In their study, they revealed that inbreeding had no influence on growth or development in the wild and in the laboratory. However, inbreeding was shown to have a bigger effect on fitness in the wild than in captivity. As a result, the measurements of survival are more sensitive than measures of growth or development by inbreeding. Rowe and Beebee (2005) found that inbred larvae grew more slowly than outbred larvae, where in mixed populations the survival of outbred larvae was 3 to 10-fold higher than that of inbred larvae. Inbreeding is usually associated with endangered population, which requires prompt action to be taken to avoid extinction. In addition, individuals in isolated and small effective population size are typically genetic impoverished with high inbreeding depression (Bataillon &
92
Kirkpatrick, 2000). However, a study by Frantz et al. (2009) on two remnant populations of the natterjack toad, Bufo calamita in Luxembourg indicated that genetic diversity in both populations were relatively high and evidence for inbreeding for small and isolated population was rejected.
Another possible factor that may influence the low genetic variation in the studied species is pollution by pesticides as the majority of individuals were collected at paddy field areas. Application of pesticides on rice fields is practiced in order to ensure production of high quality rice (Rola & Pingali, 1993; Gianessi,
2009). Unfortunately, it will also lead to devastation of other biota that inhabits the same area. Among all, amphibians are more prone to be impacted by water pollutants because of their semipermeable skin and, therefore, caused high mortality leading to population shrinking (bottleneck). Many species also begin life in water, where they risk contaminant exposure during their most vulnerable years. Gillespie and Guttman
(1989) discovered that increased levels of chemical pollution were correlated with reduced levels of genetic diversity in the central stoneroller, Campostoma anomalum collected from industrial activities at Paddy’s Run, near Ross, Ohio. Similarly,
Guttman (1994), also found extreme lackof genetic variation in the yellow perch
(Perca flavescens) from the western and central basins of Lake Erie due to extensive contamination by heavy metals including mercury, copper and zinc. In support, Ma et al. (2000) found low genetic diversity in two intertidal invertebrate species,
Mytilus galloprovincialis and Balanus glandula due to the pollution at the bay sites in southern California. Maes et al. (2005) observed reduced genetic variability in
European eel, Anguilla anguilla (L.) exposed to high pollution. This highlights the understanding that the effects of pollutants on the genome is of crucial importance to preserve the evolutionary potential of natural populations. However, the ability to
93 react to environmental pollution may vary among individuals (Bridges & Semlitsch,
2001; Eeva et al. 2006). For instance, chemical pollution may also lead to high nucleotide diversity due to mutations as observed in the wild bird, Parus major
(Eeva et al. 2006), local bird (Ellegren et al. 1997) and rodent populations (Matson et al. 2000). Thus, the relatively low and zero diversity observed in several populations in this study might be attributed to inbreeding coupled with bottleneck effect due to pesticide pollution.
Slatkin and Hudson (1991), Roger and Harpending (1992) and Lim (2009) stated that the star like pattern of haplotype relatedness in the Minimum Spanning
Network as shown in this study is produced when there is a history of population bottlenecks and expansions. Population bottlenecks, as well as large reductions in effective population size can lead to reduced genetic variation, which in turn compromises the ability of a population to respond to environmental change (Amos
& Balmford, 2001; Spear et al. 2006). Factors for this phenomenon may be attributed to anthropogenic and environmental changes. Anthropogenic factor, such as over exploitation by humans for baits in sport fishing may lead to population collapse. On the other hand, pollution may be the main factor of any environmental change. The west coast of the Peninsular Malaysia is the most commercially important paddy area and has been listed as the lead granary areas in Peninsular Malaysia. The granary areas (such as in Kedah and Tanjong Karang) refer to major irrigation schemes (areas greater than 4,000 hectares) and recognized by the Government in the National
Agricultural Policy as the main paddy producing areas (Department of Agriculture,
2010). Thus, for optimal production, application of pesticides is expected to be high at these sites, presumably causing environmental degradation adversely affecting the frog inhabitants.
94
5.4 Population Structure of Fejevarya limnocharis
In general, the genetic differentiation among F. limnocharis populations assessed by FST pairwise comparisons were relatively high between the northwest population with central west (excluding Sabak Bernam population) and each population comparisons between the two regions showed significant genetic differentiation and limited gene flow. Several populations within northwest region population, pairwise FST statistics were negative, which indicated remarkably high gene flow intra-regionally. Wright (1943) was the first to propose the isolation-by- distance model and predicted that populations, which were geographically closer to each other should be separated by smaller phenotypic and genetic distances because the homogenizing effects of gene flow are related to the geographic distance between populations. On the other hand, as geographic distance increases, differences between populations will increase stochastically (Avise, 1994; Bernal et al. 2005).
However, in the presence of physical barriers to dispersion, gene flow between populations has been viewed as a limited force (Slatkin, 1987).
AMOVA analysis did not show any evidence of genetic structuring related to the three initial predefined groups (northwest, central west and east Peninsular
Malaysia) with variation of -4.64% (indicated as 0%). The majority of variability was found within populations (72.74%) with a smaller degree of the variation among populations (31.89%). The structuring between the combined two west coast groups and east coast group were unresolved and no significant phylogeographic structure was apparent among these two major groups. However, when the central west and east populations were pooled together, the majority of genetic variance was distributed between groups at 54.81% while 9.99% were distributed among populations within groups and 35.2% distributed within populations. This indicated
95 that the central west was more related to the eastern populations than it is to the northwest populations.
Amphibians, in this case, frogs have limited mobility and low capability of dispersal. Thus, the present results, which revealed the close relationship between the central west and eastern populations was unexpected (in contrast to the above – northwest vs central west). Considering the large geographical distance, presence of five mountain ranges (Kedah-Singgora, Bintang, Keledang, Benom, Tahan) and particularly the Titiwangsa mountain range, which separates the west coast and east coast, it was very surprising that the central west populations were pooled together with the eastern populations.
In concordance with the previous analysis, the evolutionary relationships among haplotypes were further supported by the Minimum Spanning Network
(MSN) analysis. The low number of missing expected haplotypes (mv) linking actual observed haplotypes revealed that sufficient number of samples (both numbers of individuals and locations within the distribution range) had been gathered for the phylogeographical analysis and therefore conclusions should be considered well supported. The high frequency of the common haplotype (Haplotype 1) among populations in Clade A produced a pattern of population relationships associated with the ancestry of the haplotypes found within each population with Haplotype 1 being ancestral. Similarly, Haplotype 5 was ancestral to Clade B which represented all east coast and central west populations.
It is not possible to compare the pattern of population structuring with other
Malaysian amphibians as to date only limited molecular data (Ramlah et al. 2010) is available on the amphibians in Peninsular Malaysia. However, many studies have
96 shown the genetic demarcation of various biota including the snakehead, (Channa striata), Tan et al. (2011); freshwater terrapins, Batagur spp. (Norkarmila, 2009); marble goby, Oxyeleotris marmoratus (Ruzainah, 2008) between the Titiwangasa divide.
The present data is in agreement with the previous reports when considering the northwest and eastern regions (with respect to the east-west boundary). However, the close relationships between the central west and the eastern populations suggest that there are other factors influencing the pattern observed. Although, all northwest populations harboured Haplotype 1, it was also shared by other regions, including the eastern population of Terengganu and vice versa the northwest population of Sedim also contained Haplotype 5, the major haplotype of the eastern region. Three hypotheses could be proposed for this divergence. Firstly, is the occurrence of gene flow between separated regions. Thus, the clustering of the central west populations with the east coast populations could be attributable to human mediated translocation events. Fejevarya limnocharis is commonly used as bait for sport fishing or as food for bigger fishes in Peninsular Malaysia (Ibrahim, 2004). Thus, one plausible explanation is that this species had been actively translocated to fulfill the demand of this trade. According to Sahabat Alam Malaysia (personal communication), professional frog-catchers will collect between 2,000 - 5,000 frogs nightly and sell them to middle men in the area of Tanjung Karang. This frog is then sold in the market at 30 to 40 cents per individual. It is very highly likely that such activities are also occurring within the central western populations of Sabak Bernam, Langkap and other areas which share genetic similarity with the eastern region.
However, the above hypothesis requires that transportation occurs from the eastern into the central western region. Logistically, this is not a cost effective
97 activity as the frogs could be much easier obtained from neighbouring areas.
Furthermore, localities such as Pusing (PUSs), Kuala Kangsar (KKs) did not contain
Haplotype 1 although they are nearer to the northwest region than Sabak Bernam
(SBKs) and Tanjung Karang (TKs) which harbour Haplotype 1, and Kepong (KEPs), the most southern locality investigated which had Haplotype 3, a closely derived variant of Haplotype 1. Thus, a hypothesis which attributes the genetic differentiation between the northwest and central west as due to a barrier between the northwest and central west breaks down.
A second hypothesis is the interplay of the food chain within the distributional range of this species. As described previously, the frog is the second consumer in the food web chain. They tend to eat small vertebrates, arthropods and insects and, in turn, become prey for natural predators including snakes and wading birds. The Great Egret is known to be natural predators on paddy field frogs in
Malaysia, thus, it is possible that some live specimens or viable eggs may still attached to the predators and trans regional migration occurs. However, this hypothesis requires high occurrences of such events and does not account for the differentiation between the northwest and central regions.
Therefore, an alternative hypothesis should also be considered. The third hypothesis is evidence of ancestral signatures. The hypothesis postulates that both
Haplotypes 1 and 5 were ancestral haplotypes of Peninsular Malaysia, but due to stochastic events one or the other has become more established at certain sites and regions. This is in tandem with the inherent lack of migratory ability of this species leading to genetic drift towards fixation of certain alleles. The presence of both major haplotypes (Haplotypes 1 and 5) at certain localities irrespective of regions; Sabak
Bernam, Tanjung Karang, Terengganu and Sedim and closely derived haplotypes
98 provide stronger evidence to this. Furthermore, the presence of population-specific tip haplotypes indicated that each population was evolving independently with restriction to gene flow and supported this third hypothesis – that the occurrences of
Haplotypes 1 and Haplotypes 5 inter-regionally were due to their ancestry and not free gene flow. More intensive sampling on the investigated sites could confirm this or sampling of more sites particularly in the east coast. A result showing presence of both major haplotypes (or closely related derived haplotypes) at randomly distributed geographical areas would prove that the third hypothesis is reflective of the pattern.
The two species studied, F. cancrivora and F. limnocharis are varied somewhat in their differentiation pattern despite the fact that both species are sympatric in Pulau Langkawi. Differentiation of the Pulau Langkawi F. cancrivora population was significantly greater than in F. limnocharis, which suggested that F. limnocharis is a better disperser than F. cancrivora. An explanation for the superior dispersal ability in F. limnocharis might be attributed to the habits and habitat of this frog, which is confined to disturbed habitats associated with the activities of man, such as in the paddy fields, which make them more subjected to translocation from one place to another. Alternatively, since F. cancrivora is a frog of disturbed habitats near the coast and mangrove, thus the ability of this frog species remain stable in an area unless they are transferred to the other areas in order to support the demand of their meats.
5.5 Implications for conservation
Genetic structure data of a population has serious implications for management and conservation (Mockford et al. 2005). Because of the high level of genetic homogeneity among geographically close populations where interbreeding is
99 likely to occur, both species are prone to reduction of fitness when the environment changes dramatically. Recently, habitat alteration by anthropogenic factors are causing global decline of amphibian population. Thus, it is vital for amphibians to possess high genetic variance for tolerance to environmental changes. Bridges and
Semlitscht (2001) revealed that even though genetic responses may be inadequate to to challenge environmental changes that increased rapidly or are severe, populations with larger amounts of high genetic variance that inhabit habitats experiencing slow changes may have enhanced ability to adapt to these changes.
The generated information from this study is a very important contribution to the genetic database for Peninsular Malaysia frogs. The information of the amount of genetic variance within a population will help in identifying high-risk populations before irreversible decline can take place and will be useful in making conservation decisions, such as in restoration efforts (Bridges & Semlitscht, 2001). Clearly, genetic variation is very low in both species, although may not have reached threatening levels. Amphibians are highly philopatric and poor dispersers, which make them suitable for studying the effects of man-made habitat fragmentation based on genetic variation and population differentiation (Blaustein et al. 1994; Seppa &
Laurila, 1999). This study is a serious warning of the deteriorating condition of our environment. Therefore, a crucial step in the conservation of these two sympatric frog species is to protect their habitats in addition to the direct protection of the frogs itself from being exploited for their flesh and as bait.
5.6 GIS mapping on Fejevarya cancrivora and F. limnocharis
It is well accepted that both the species are known to be present in most habitats. However, this study shows that they are mainly distributed in northern
100
Peninsular Malaysia. This was probably due to the intensive survey that was focused mostly in northern Peninsular Malaysia and inclusive of most of the habitats of the two species namely human settlements, agricultural areas and recreational forests.
Thus, more individuals were screened in this area compared to other parts of the
Peninsular Malaysia resulting in biasness of reporting. Nevertheless, this study has shown very interesting insights particularly with respect to the relatively limited or lack of investigations in the other areas of Peninsular Malaysia- southern and east coast. Amphibians are good indicators of ecosystem degradation (Ibrahim, 2011; Hu et al. 2012). Thus, the understanding of their spatial distribution is vital for the development of integrative conservation strategies and elucidation of geographic patterns of amphibians (Hu et al. 2012).
101
CHAPTER 6
CONCLUSION
The aim of this study was to determine the genetic variation of two sympatric frog species inhabiting Northern Peninsular Malaysia namely F. cancrivora and F. limnocharis, the latter species focused in Peninsular Malaysia. Genetic variabilities were generally low for both studied species, but were not at alarming levels, presumably due to their large population sizes and wide distributions. Despite the limited number of populations surveyed for F. cancrivora populations, the study revealed the genetic isolation of Pulau Langkawi and its comparatively high diversity. Another interesting factor was the close relationship of Penang Island and its mainland population in contrast to Pulau Langkawi with its mainland Kedah population. It is proposed that the consumption and demand of F. cancrivora is high in Penang state. Thus, two hypotheses are suggested to explain the present day genetic pattern in F. cancrivora; 1) anthropogenic introductions from mainland populations as in the case of Pulau Pinang, and 2) population isolation before island formation which occurred during the last glaciation time (Pleistocene – 18 kya ago) with increased sea level as in the case of Pulau Langkawi.
In the case of F. limnocharis, based on partial D-loop segment of mtDNA revealed that their populations were genetically heterogenous throughout Peninsular
Malaysia with high similarities between several central west and eastern populations.
Again, two hypotheses are proposed to explain the genetic pattern observed; 1) the clustering of the central west populations with the east coast populations is a result of a human mediated translocation event since F. limnocharis have long been used as
102 bait for sport fishing or as food for bigger fish in Peninsular Malaysia, and 2)
Haplotypes 1 and 5 were ancestral to the whole of Peninsular Malaysia, but due to stochastic events one or the other has become more established at certain sites and regions. Based on the present data, the second hypothesis seemed more plausible.
As aforementioned, anthropogenic factor is one of the main causes that contribute to the above patterns of distribution for both species. Recently, anthropogenic factors are altering habitats and causing amphibian populations to decline worldwide. This study highlights the importance of genetic data in planning future management and conservation strategies of both species as well as other amphibians in Malaysia.
In conclusion, more thorough and detailed studies using both mtDNA and nuclear markers (such as microsatellite loci) with more intensive and extensive sampling throughout the species distributions are needed to confirm any hypothesis and to conduct more effective conservation effort throughout Malaysia.
The distribution map generated from the GIS approach showed that the distributions of both species were clustered mostly in the northern area of Peninsular
Malaysia. The study highlights that more work needs to be done by herpetologists, especially in the east coast and southern parts of Peninsular Malaysia to fill the existing knowledge gap. This study also shows the glaring lack of workers and herpetologists in Malaysia.
103
104
REFERENCES:
Abdulali, H. (1985). On the export of frog legs from India. Journal of the Bombay Natural History Society, 2, 347-375. Abdul Rani, A. (2002). Pesticides use in Malaysia: Trends and impacts. In J. Everaarts, F. P. Carvalho, M. D. Taylor, S. J. Klaine, & D. Barcelo. Pesticide Residues in Coastal Tropical Ecosystems Distribution, Fate and Effects (pp. 159-179). Malaysia: CRC Press. Aboim, M. A., Menezes, G. M., Schlitt, T., & Rogers, A. D. (2005). Genetic structure and history of populations of the deep‐sea fish Helicolenus dactylopterus (Delaroche, 1809) inferred from mtDNA sequence analysis. Molecular Ecology, 14(5), 1343-1354. Ahmad Ridzwan, D. (2010). Kajian Kepelbagaian dan kelimpahan bandingan komuniti amfibia di Hulu Sungai Junjung, Kulim, Kedah (Bachelor’s Thesis). Universiti Sains Malaysia. Allentoft, M. E., & O’Brien, J. (2010). Global amphibian declines, loss of genetic diversity and fitness: a review. Diversity, 2(1), 47-71. Altherr, S., Goyenechea, A., & Schubert, D. J. (2011). Canapés to Extinction: The International Trade in Frog's Legs and Its Ecological Impact. ProWildlife. Al-Ghamdi, Y., Bowhay, M., & Abulleif, T. (2007). GPS for GIS: What’s Really Involved? Amos, W., & Balmford, A. (2001). When does conservation genetics matter? Heredity, 87(3), 257-265. Ashton, K. G., & Queiroz, A. D. (2001). Molecular Systematics of the Western Rattlesnake, Crotalus viridis (Viperidae), with Comments on the Utility of the D-Loop in Phylogenetic Studies of Snakes. Molecular Phylogenetics and Evolution, 21(2), 176-189. Avise, J. C. (1994). Molecular Markers, Natural History and Evolution. New York: Chapman & Hall. Avise, J. C. (2000). Phylogeography, the History and Formation of Species. Massachusetts: Harvard University Press. Azizul, A. (2010). Aplikasi Sistem Maklumat Geografi (Gis) Dalam Menganggar Keluasan Petempatan Awal Tapak Arkeologi Lembah Lenggong, Hulu Perak. Pulau Pinang (Master’s thesis). Universiti Sains Malaysia.
104
Bacchetta, R., Mantecca, P., Andrioletti, M., Vismara, C., & Vailati, G. (2008). Axial–skeletal defects caused by Carbaryl in Xenopus laevis embryos. Science of the Total Environment, 392(1), 110-118. Balinsky, J. B., Dicker, S. E., & Elliott, A. B. (1972). The effect of long-term adaptation to different levels of salinity on urea synthesis and tissue amino acid concentrations in Rana cancrivora. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 43(1), 71-82. Bambaradeniya, C. N., & Amarasinghe, F. P. (2004). Biodiversity associated with the rice field agroecosystem in Asian countries: A brief review. International Water Management Institute. Barroso, M. R., Hilsdorf, A. W., Moreira, H. L., Cabello, P. H., & Traub-Cseko, Y. M. (2005). Genetic diversity of wild and cultured populations of Brycon opalinus (Cuvier, 1819) (Characiforme, Characidae, Bryconiae) using microsatellites. Aquaculture, 247(1), 51-65. Bataillon, T., & Kirkpatrick, M. (2000). Inbreeding depression due to mildly deleterious mutations in finite populations: size does matter. Genetical Research, 75(01), 75-81. Bay, L. K., & Caley, M. J. (2011). Greater genetic diversity in spatially restricted coral reef fishes suggests secondary contact among differentiated lineages. Diversity, 3(3), 483-502. Beebee T. J. C. (1996). Ecology and Conservation of Amphibians. London: Chapman & Hall. Beebee, T. J. C. (2005). Conservation genetics of amphibians. Heredity, 95(6), 423- 427. Belabut, D., Rosli, H., Yong, H. S., Zaleha, M. D., Norhayati, A. & Lim, B. L. (2010). A Note on the Amphibian Fauna at Pantai Melawi, Bachok, Kelantan. Malaysian Journal of Science, 29, (Special Issue), 111-115. Berger, L., Speare, R., Daszak, P., Green, D., Cunningham, A., Goggin, C., Slocombe, R., Ragan, M., Hyatt, A., Mcdonald, K., Hines, H., Lips, K., Marantelli, G. & Parkes, H. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences, 95(15), 9031-9036. Bernal, X. E., Guarnizo, C., & Lüddecke, H. (2005). Geographic variation in advertisement call and genetic structure of Colostethus palmatus (Anura, Dendrobatidae) from the Colombian Andes. Journal Information, 61(4). Bernardi, G. (2000). Barriers to gene flow in Embiotoca jacksoni, a marine fish lacking a pelagic larval stage. Evolution, 54(1), 226-237.
105
Bernardi, G., Findley, L., & Rocha‐Olivares, A. (2003). Vicariance and dispersal across Baja California in disjunct marine fish populations. Evolution, 57(7), 1599-1609. Berry, P. Y. (1975). The Amphibian Fauna of Peninsular Malaysia. Kuala Lumpur: Tropical Press. Blaustein, A. R. & Wake, D. B. (1990). Declining Amphibian Populations: A Global Phenomena? Trends in Ecology and Evolution, 5, 203-204. Blaustein, A. R., Wake, D. B., & Sousa, W. P. (1994). Amphibian declines: judging stability, persistence, and susceptibility of populations to local and global extinctions. Conservation Biology, 8(1), 60-71. Blaustein, A. R., Romansic, J. M., Kiesecker, J. M., & Hatch, A. C. (2003). Ultraviolet radiation, toxic chemicals and amphibian population declines. Diversity and Distributions, 9(2), 123-140. Boone, M. D., Semlitsch, R. D., Fairchild, J. F., & Rothermel, B. B. (2004). Effects of an insecticide on amphibians in large-scale experimental ponds. Ecological Applications, 14(3), 685-691. Boore, J. L. (1999). Animal mitochondrial genomes. Nucleic Acids Research, 27(8), 1767-1780. Bowen, B. W., & Karl, S. A. (2007). Population genetics and phylogeography of sea turtles. Molecular Ecology, 16(23), 4886-4907. Bridges, C. M. (1999). Predator-prey interactions between two amphibian species: effects of insecticide exposure. Aquatic Ecology, 33(2), 205-211. Bridges, C. M., & Semlitsch, R. D. (2001). Genetic variation in insecticide tolerance in a population of southern leopard frogs (Rana sphenocephala): implications for amphibian conservation. Copeia, 2001(1), 7-13. Brown, W. M. (1985). The mitochondrial genome of animals. Molecular Evolutionary Genetics, 95. Cao, S. Y., Wu, X. B., Yan, P., Hu, Y. L., Su, X., & Jiang, Z. G. (2006). Complete nucleotide sequences and gene organization of mitochondrial genome of Bufo gargarizans. Mitochondrion, 6(4), 186-193. Carey, C., & Alexander, M. A. (2003). Climate change and amphibian declines: is there a link? Diversity and Distributions, 9(2), 111-121. Castro, J. A., Picornell, A., & Ramon, M. (2010). Mitochondrial DNA: a tool for populational genetics studies. International Microbiology, 1(4), 327-332. Chan, K. O., van Rooijen, J., Grismer, L. L., Belabut, D., Abdul Muin, M. A., Hamidi, J., Gregory, R., & Norhayati, A. (2010). First report on the herpetofauna of Pulau Pangkor, Perak, Malaysia. Russian Journal of Herpetology, 17(2), 139 – 146.
106
Chen, S. Y., Zhang, Y. J., Wang, X. L., Sun, J. Y., Xue, Y., Zhang, P., Zhou, H. & Qu, L. H. (2012). Extremely Low Genetic Diversity Indicating the Endangered Status of Ranodon sibiricus (Amphibia: Caudata) and Implications for Phylogeography. PloS one, 7 (3), 1-11. Chew, M. M., Elliott, A. B., & Wong, H. Y. (1972). Permeability of urinary bladder of Rana cancrivora to urea in the presence of oxytocin. The Journal of Physiology, 223(3), 757-772. Crandall, K. A. & Templeton, A. R. (1996). Application of intraspecific phylogenetics. In P. H. Harvey, A. J. Leigh Brown, S. J. Maynard, & S. Nee, (Eds). New Uses for New Phylogenies, (pp. 81-99). Oxford: Oxford University Press. Cromley, E., & McLafferty, S. (2002). GIS and Public Health. New York: The Guilford Press. Daszak, P., Cunningham, A. A., & Hyatt, A. D. (2003). Infectious disease and amphibian population declines. Diversity and Distributions, 9(2), 141-150. Davidson, C., Bradley Shaffer, H., & Jennings, M. R. (2001). Declines of the California red-legged frog: climate, UV-B, habitat, and pesticides hypotheses. Ecological Applications, 11(2), 464-479. Department of Agriculture. (2010). Paddy. Retrieved December 16, 2012 from http://www.statistics.gov.my on 16th Dec 2012. Department of Statistics. (2010). Population distribution and basic demographic characteristics. Retrieved January 5, 2013 from http://www.statistics.gov.my/portal/download_Population/files/census2010/Ta buran_Penduduk_dan_Ciri-ciri_Asas_Demografi.pdf Djong, T. H., Matsui, M., Kuramoto, M., Belabut, D. M., Yong, H. S., Nishioka, M. & Sumida, M. (2007). Morphological Divergence, Reproductive Isolating Mechanism,and Molecular Phylogenetic Relationships Among Indonesia,Malaysia, and Japan Populations of the Fejervarya limnocharis Complex (Anura, Ranidae). Zoological Science, 24, 1197–1212. Dubois, A. & Ohler, A. (2000). Systematics of Fejervarya limnocharis (Gravenhorst, 1829) (Amphibia, Anura, Ranidae) and related species. 1. Nomenclatural status and type-specimens of the nominal species Rana limnocharis Gravenhorst, 1829. Alytes, 18, 15–50. Duellman, W. E., & Trueb, L. (1986). Biology of Amphibians. New York: McGraw- Hill Book Co. Eeva, T., Belskii, E., & Kuranov, B. (2006). Environmental pollution affects genetic diversity in wild bird populations. Mutation Research, 608(1), 8-15. Eisen, E. J. (1975). Population size and selection intensity effects on long-term selection response in mice. Genetics, 79(2), 305-323.
107
Ellegren, H., Lindgren, G., Primmer, C. R., & Møller, A. P. (1997). Fitness loss and germline mutations in barn swallows breeding in Chernobyl. Nature, 389(6651), 593-596. Esri. (2012). Understanding our world. Retrieved August 6, 2012 from www.esri.com/products. Excoffier, L., Smouse, P. E., & Quattro, J. M. (1992). Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics, 131(2), 479-491. Excoffier, L., Laval, G., & Schneider, S. (2005). Arlequin ver. 3.0: An Integrated Software Package for Population Genetics Data Analysis. Evolutionary Bioinformatics Online, 2, 47–50. Faber, J. E., & Stepien, C. A. (1998). Tandemly Repeated Sequences in the Mitochondrial DNA Control Region and Phylogeography of the Pike-Perches Stizostedion. Molecular Phylogenetics and Evolution, 10 (3), 310-322. Farjallah, S., Amor, N., Merella, P., & Said, K. (2012). Pattern of genetic diversity of North African green frog Pelophylax saharicus (amphibia) in Tunisia. Pakistan Journal of Zoology, 44(4), 901-907. Fei, L., & Ye, C. Y. (2001). The Colour Handbook of Amphibians of Sichuan. Beijing: China Forestry Publishing House. Fei, L., Ye, C. Y., Jiang, J. P., & Xie, F. (2002). On taxonomic status of Rana limnocharis group with revision of nomenclature of the rice frog from China. Herpetologica Sinica, 9, 88-96. Fisher, R. N., & Shaffer, H. B. (1996). The decline of amphibians in California's Great Central Valley. Conservation Biology, 10, 1387-1397. Fisher, M. C., Garner, T. W., & Walker, S. F. (2009). Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. Annual Review of Microbiology, 63, 291-310.
Fluxus. (2011). Network 4.6.1.0. Retrieved April 3, 2012 from http://Fluxus- engineering.com. Frankham, R. (1995). Inbreeding and extinction: a threshold effect. Conservation Biology, 9(4), 792-799. Frankham, R. (1998). Inbreeding and extinction: island populations. Conservation Biology, 12(3), 665-675. Frantz, A. C., Proess, R., Burke, T., & Schley, L. (2009). A genetic assessment of the two remnant populations of the natterjack toad (Bufo calamita) in Luxembourg. The Herpetological Journal, 19(2), 53-59. Frost, D. R. (2011). (31 January, 2011). Amphibian Species of the World: an Online Reference. [Web log post] Retrieved from http://research.amnh.org/vz/ herpetology/amphibia/American Museum of Natural History, New York, USA.
108
Geng, B. R., Yao, D., & Xue, Q. Q. (2005). Genotoxicity of pesticide dichlorvos and herbicide butachlor in Rhacophorus megacephalus tadpoles. Acta Zool Sin, 51, 447–454. Gianessi, L. (2009). The Benefits of Insecticide Use: Rice. Washington: CropLife Foundation. Gillespie, R. B., & Guttman, S. I. (1989). Effects of contaminants on the frequencies of allozymes in populations of the central stoneroller. Environmental Toxicology and Chemistry, 8(4), 309-317. Gordon, M. S., Schmidt-Nielsen, K., & Kelly, H. M. (1961). Osmotic regulation in the crab-eating frog (Rana cancrivora). Journal of Experimental Biology, 38(3), 659-678. Gordon, M. S., & Tucker, V. A. (1965). Osmotic regulation in the tadpoles of the crab-eating frog (Rana cancrivora). Journal of Experimental Biology, 42(3), 437-445. Grant, W. A. S., & Bowen, B. W. (1998). Shallow population histories in deep evolutionary lineages of marine fishes: insights from sardines and anchovies and lessons for conservation. Journal of Heredity, 89(5), 415-426. Gray, M. W. (1992). The endosymbiont hypothesis revisited. International Review of Cytology, 141, 233–357. Guttman, S. I. (1994). Population genetic structure and ecotoxicology. Environmental Health Perspectives, 102(Suppl 12), 97- 100. Halverson, M. A., Skelly, D. K., & Caccone, A. (2006). Inbreeding Linked to Amphibian Survival in the Wild but Not in the Laboratory. Journal of Heredity, 1-9. Hayes, T. B., Collins, A., Lee, M., Mendoza, M., Noriega, N., Stuart, A. A., & Vonk, A. (2002). Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences, 99(8), 5476-5480. Hayes, T. B. (2005). Welcome to the revolution: integrative biology and assessing the impact of endocrine disruptors on environmental and public health. Integrative and Comparative Biology, 45(2), 321-329. Heyer, W. R., Donnelly, M. A., McDiarmid, R. W., Hayek, L. C., & Foster, M. S. (1994). Measuring and Monitoring Biological Diversity Standard Method for Amphibians. Washington and London: Smithsonian Institution Press. Hickman, C. P. Jr., Roberts, L. S., Larson, A., I’Anson, H., & Eisenhour, D. J. (2006). Integrated Principles of Zoology (13th Ed.). New York: McGraw-Hill. Hillman, S. S., Withers, P. C., Drewes, R. C., & Hillyard, S. D. (2009). Ecological and Environmental Physiology of Amphibians. New York: Oxford University Press.
109
Hoelzel, A. R., Hancock, J. M., & Dover, G. A. (1991). Evolution of the cetacean mitochondrial D-loop region. Molecular Biology and Evolution, 8(4), 475-493. Hu, J., Li, C., Xie, F., & Jiang, J. (2012). Endemic amphibians and their distribution in China. Asiatic Herpetological Research, 3(2), 163–171. Ibrahim, J., Ektella, M. A., & Shahrul Anuar, M. S. (2001). Diversity of Amphibians at Taman Negeri Perlis Wang Kelian. In I. Faridah- Hanum, O. Kasim, & A. Latiff (Eds.), Kepelbagaian Biologi dan Pengurusan Taman Negeri Perlis: Persekitaran Fizikal dan Biologi Wang Kelian (pp. 123- 127). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Ibrahim, J. (2004). Aspects of Biology and Ecology of Two Sympatric Frog Species in Malaysia. Sungai Petani: Yusran Publishing House. Ibrahim, J., Wong, L. H., Nur Ziana, A. S., Muin, M. A., Ismail Ishaq, I., & Shahrizan M. A. A., (2008). Diversity and Distribution of Anurans at Rimba Bandar Bukit Bauk. In U. Razani, H. L. Koh, R. Mohd. Rahim, J. Na’aman, I. Faridah- Hanum, & A. Latiff (Eds.), Rimba Bandar Bukit Bauk: Terengganu, Pengurusan Hutan, Persekitaran Fizikal dan Kepelbagaian Biologi (pp. 286- 292). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Ibrahim, J., Teoh, C. C., Shahrul Anuar, M. S., & Abdul Muin, M. A. (2009). Checklist and Simple Identification Key for Frogs and Toads from District IV of The MADA Scheme, Kedah, Malaysia. Tropical Life Sciences Research, 20(2), 49–57. Ibrahim, J., Wong, J., Mohd Fazlin, M. S., Fatan, H.Y., Siti Hadijah, Y., & Norhaslinda, S. (2011). Amphibian Assemblage of Bubu Permanent Forest Reserve, Perak, Malaysia. Malaysian Applied Biology, 40 (1), 1-6. Ibrahim, J., Shahriza, S., Ismail Ishaq, I., & Amirudin, A. (2012). An Annotated Checklist of the Amphibians and Reptiles of Bukit Panchor State Park, Penang. In A. R. Abd. Rahman, A. H. Mohd. Nasir, D. Mohd Puat, A. Muhamad, & A. Latiff (Eds.), Taman Negeri Bukit Panchor dan Pulau Jerejak, Pulau Pinang: Pengurusan Hutan, Persekitaran Fizikal dan Kepelbagaian Biologi (pp. 203- 210). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Ibrahim, J., Amirah, H., Shahriza, S., Nur Hafizah, I., Zalina, A., Yap, C. H., Nurliza, A. M., & Nur Hafizah, C. B. (2013). Additions to the herpetofauna of Jerejak Island, Penang, Peninsular Malaysia. Malayan Nature Journal, 64(4): 213- 232. Imbun, P., Dusun, S., & Hamidi, J. (2010). Taburan Herpetofauna di Rizab Hidupan Liar Bukit Fraser, Pahang. In Y. Mohd. Nawayai, M. N. Shukor, I. Rahmah, S. M. K. Syarifah Khadiejah, B. M. Mohamad Rufino, & T. S. Frankie (Eds.), Proceeding of National Biodiversity Seminar (pp. 17- 26). Kuala Lumpur: Department of Wildlife and National Parks. Imsiridou, A., Apostolidis, A. P., Durand, J. D., Briolay, J., Bouvet, Y., & Triantaphyllidis, C. (1998). Genetic differentiation and phylogenetic relationships among Greek Chub Leuciscus cephalus L. (Pisces, Cyprinidae) 110
populations as revealed by RFLP analysis of mitochondrial DNA. Biochemical Systematics and Ecology, 26(4), 415-429. Inger, R. F., & Stuebing, R. B. (1989). Frogs of Sabah. Kota Kinabalu: Sabah Park Trustees. Inger, R. F. and Stuebing, R. B. (1997). A Field Guide to the Frogs of Borneo. Kota Kinabalu: Natural History Publications. Inger. R. F., & Stuebing, R. B. (1999). Panduan Lapangan Katak-katak Borneo. Kota Kinabalu: Natural History Publications. Iskandar, D. (1998). The Amphibians of Java and Bali. Research and Development Centre for Biology Lipi. Islam, M. M, Kurose, N., Khan, M. M. R., Nishizawa, T., Kuramoto, M., Alam, M. S., Hasan, M., Kurniawan, N., Nishioka, M., & Sumida, M. (2008). Genetic divergence and reproductive isolation in the genus Fejervarya (Amphibia: Anura) from Bangladesh inferred from morphological observations, crossing experiments, and molecular analyses. Zoological Science 25, 1084–1105. IUCN. (2009). International Union for Conservation of Nature. Retrieved March 23, 2012 from http://www.iucn.org. Jamsari, A. F. J., Muchlisin, Z. A., Musri, M., & Azizah, M. S. (2010). Remarkably low genetic variation but high population differentiation in the climbing perch, Anabas testudineus (Anabantidae), based on the mtDNA control region. Genetics and Molecular Research, 9(3), 1836-1843. Jayatillake, B. A. D. M. C., Wijesinghe, M. R., Ratnasooriya, W. D., & Lakraj, G. P. (2011). Toxic effects of Carbofuran on Duttaphrynus Melanostictus Larvae. International Journal of Environmental Science, 2 (2), 1060-1070. Jayawardena, U. A., Rajakaruna, R. S., Navaratne, A. N., & Amerasinghe, P. H. (2010). Toxicity of agrochemicals to common hourglass tree frog (Polypedates cruciger) in acute and chronic exposure. Internatianal Journal of Agriculture and Biology, 12, 641-648. Kats, L. B., & Ferrer, R. P. (2003). Alien predators and amphibian declines: review of two decades of science and the transition to conservation. Diversity and Distributions, 9(2), 99-110. Keller, L. F., & Waller, D. M. (2002). Inbreeding effects in wild populations. Trends in Ecology & Evolution, 17(5), 230-241. Khan, M. Z., & Law, F. C. (2005). Adverse effects of pesticides and related chemicals on enzyme and hormone systems of fish, amphibians and reptiles: a review. Proc. Pakistan Acad. Sci, 42(4), 315-323. Kiesecker, J. M., Blaustein, A. R., & Belden, L. K. (2001). Complex causes of amphibian population declines. Nature, 410(6829), 681-684.
111
Kiew, B. H. (1984). Conservation status of the Malaysian fauna. III. Amphibians. Malayan Naturalist, 37(4), 6-10. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111-120. Kotaki, M., Kurabayashi, A., Matsui, M., Khonsue, W., Djong, T. H., Tandon, M., & Sumida, M. (2008). Genetic divergences and phylogenetic relationships among the Fejervarya limnocharis complex in Thailand and neighboring countries revealed by mitochondrial and nuclear genes. Zoological Science, 25(4), 381- 390. Kucuktas, H., & Liu, Z. (2007). Allozyme and Mitochondrial DNA Markers. In: Liu, Z. First Edition. Aquaculture Genome Technology. Victoria: Blackwell Publishing Ltd. Kurniawan, N., Islam, M., Djong, T. H., Igawa, T., Belabut, D. M., Yong, H. S., Wanichanon, R., Khan, M. M. R., Iskandar, D. T., Nishioka, M., & Sumida, M. (2010). Genetic Divergence and Evolutionary Relationship in Fejervarya cancrivora from Indonesia and Other Asian Countries Inferred from Allozyme and MtDNA Sequence Analyses. Zoological Science, 27, 222–233. Kurniawan, N., Djong, T. H., Islam, M. M., Nishizawa, T., Belabut, D. M., Yong, H. S., Wanichanon, R., Yasir, I., & Sumida, M. (2011). Taxonomic Status of Three Types of Fejervarya cancrivora from Indonesia and Other Asian Countries Based on Morphological Observations and Crossing Experiments. Zoological Science, 28 (1), 12-24. Kusrini, M. D., & Alford, R. A. (2006). Indonesia's exports of frogs' legs. Traffic Bulletin, 21, 13-24. Lai, J. (2009). Amphibian Species Distribution Modelling in Poland (Master’s thesis). International Institute for Geo-Information Science and Earth Observation Enschede. Lannoo, M. J., Lang, K., Waltz, T., & Phillips, G. S. (1994). An altered amphibian assemblage: Dickinson County, Iowa, 70 years after Frank Blanchard's survey. American Midland Naturalist, 311-319. Lim, H. C. (2009). Genetic structure of Japanese threadfin bream, Nemipterus japonicus (Bloch, 1791) along the coast of Peninsular Malaysia (Master’s thesis). Universiti Sains Malaysia, Penang. Lim, B. L., Chai, K. S., Yong, D., Lim, C. C., & Norhayati, A. (2008). Herpetofauna of Bukit Belata and Raja Muda Forest Reserve. In M. Azahar, H. L. Koh, N. M. Nik Mohd. Shah, N. Samsul Anuar, & A. Latiff (Eds.), Bukit Belata, Selangor: Pengurusan, Persekitaran Fizikal, Kepelbagaian Biologi dan Sosio- ekonomi (pp. 241- 252). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia.
112
Lim, B. L., Noor, A. W. D., Chan, K. O., Belabut, D., & Norhayati, A. (2010). An updated checklist of herpetofauna of Pulau Singa Besar, Langkawi, Peninsular Malaysia. Malaysian Applied Biology, 39(1), 13-23. Liu, W. Y., Wang, C. Y., Wang, T. S., Fellers, G. M., Lai, B. C., & Kam, Y. C. (2011). Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan. Ecotoxicology, 20(2), 377-384. Liu, Z., Wanga, Y., & Su, B. (2005). The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia: Anura): a new gene order in the vertebrate mtDNA. Gene, 346, 145–151. Longcore, J. E., Pessier, A. P., & Nichols, D. K. (1999). Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia, 219-227. Ma, X. L., Cowles, D. L., & Carter, R. L. (2000). Effect of pollution on genetic diversity in the bay mussel Mytilus galloprovincialis and the acorn barnacle Balanus glandula. Marine Environmental Research, 50, 559-63. Maes, G. E., Raeymaekers, J. A. M., Pampoulie, C., Seynaeve, A., Goemans, G., Belpaire, C., & Volckaert, F. A. M. (2005). The catadromous European eel, Anguilla anguilla (L.) as a model for freshwater evolutionary ecotoxicology: Relationship between heavy metal bioaccumulation, condition and genetic variability. Aquatic Toxicology, 73(1), 99-114. Measey, G. J., Vences, M., Drewes, R. C., Chiari, Y., Melo, M., & Bourles, B. (2007). Freshwater paths across the ocean: molecular phylogeny of the frog Ptychadena newtoni gives insights into amphibian colonization of oceanic islands. Journal of Biogeography, 34, 7–20. Mäkinen, H. S., & Merilä, J. (2008). Mitochondrial DNA phylogeography of the three-spined stickleback (Gasterosteus aculeatus) in Europe—Evidence for multiple glacial refugia. Molecular Phylogenetics and Evolution, 46(1), 167- 182. Marsh, D. M., & Trenham, P. C. (2001). Metapopulation dynamics and amphibian conservation. Conservation Biology, 15, 40-49. Martin, E., & Hine, R. S. (2000). A Dictionary of Biology. New York: Oxford University Press. Martı´nez-Solano, I., & Lawson, R. (2009). Escape to Alcatraz: evolutionary history of slender salamanders (Batrachoseps) on the islands of San Francisco Bay. BMC Evol Bio, 9, 38. Matson, C. W., Rodgers, B. E., Chesser, R. K., & Baker, R. J. (2000). Genetic diversity of Clethrionomys glareolus populations from highly contaminated sites in the Chornobyl region, Ukraine. Environmental Toxicology and Chemistry, 19(8), 2130-2135.
113
Matsui, M. (1984). Morphometric variation analyses and revision of the Japanese toads (Genus Bufo, Bufonidae). Contributions from the Biological Laboratory, Kyoto University, 26, 209–428. Miller, J. J. (2012). Amphibian Biology. Retrived December 20, 2012 from http://www.amphibiainfo.com/biology/. Mockford, S. W., McEachern, L., Herman, T. B., Synder, M., & Wright, J. (2005). Population genetic structure of a disjunct population of Blanding’s turtle (Emydoidea blandingi) in Nova Scotia, Canada. Biological Conservation, 123, 373-380. Mohd Hamidi, I. (2010). Kajian kepelbagaian dan kelimpahan bandingan komuniti katak dan kodok di hilir Sungai Junjung, Simpang Ampat, Seberang Perai Selatan, Pulau Pinang (Bachelor’s Thesis). Universiti Sains Malaysia. Mohd Shafeea, L. (2010). Geoheritage Conservation in Langkawi Geopark, Malaysia. Akademika, 80, 19-30. Moore, D. A., & Carpenter, T. E. (1999). Spatial analytical methods and geographic information systems: use in health research and epidemiology. Epidemiologic Reviews, 21(2), 143-161. Moritz, C., Dowling, T. E., & Brown, W. M. (1987). Evolution of animal mitochondrial DNA: relevance for population biology and systematics. Annual Review of Ecology and Systematics, 18, 269-292. Narimah, S., Dina, J., Norul Shahira, M., Aishah Knight, A. S., Azizah, A. M., & Yasmin, S. (2010). Using a Geographic Information System (GIS) in evaluating the accessibility of health facilities for breast cancer patients in Penang state, Malaysia. Kajian Malaysia, 28 (1), 103-122. Nei, M., & Kumar, S. (2000). Molecular Evolution and Phylogenetics. New York: Oxford University Press. Nevo, E. (1988). Genetic diversity in nature: patterns and theory. Evolutionary Biology, 23, 217–246. Newman, D., & Pilson, D. (1997). Increased probability of extinction due to decreased genetic effective population size: experimental populations of Clarkia pulchella. Evolution, 354-362. Nguyen, T., Hurwood, D., Mather, P., Na-Nakorn, U., Kamonrat, W., & Bartley, D. (2006). Manual on application of molecular tools in aquaculture and inland fisheries management. Part 2. Laboratory protocols and data analysis. Nicod, J. C., Wang, Y. Z., Excoffier, L., & Largiader, C. R. (2004). Low levels of mitochondrial DNA variation among central and southern European Esox lucius populations. Journal of Fish Biology, 64(5), 1442-1449. Norhayati, A., Juliana, S., & Lim, B.L. (2005a). Amphibians of Ulu Muda Forest Reserve, Kedah. Kuala Lumpur: Forestry Department Peninsular Malaysia.
114
Norhayati, A., Juliana, S., Sharma, D. S., Shukor, M. N., Ahmad Zafir, A. W., & Surin, S. (2005b). Fauna Amfibia di Sungai Lasor, Hutan Simpan Ulu Muda. In M. I. Shaharudin, H. Che Hashim, D. Mohd. Puat, M. S. Jalil, Y. Norhaidi, & A. Latiff (Eds.), Hutan SImpan Ulu Muda, Kedah: Pengurusan, Persekitaran Fizikal dan Biologi (pp. 173- 184). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Norhayati, A., Shukor, M. N., & Juliana, S. (2005c). Inventory of Herpetofauna at Gunung Basor Forest Reserve, Kelantan. In M. I. Shaharudin, T. Dahalan, S. Abdullah Sani, M. S. Jalil, I. Faridah- Hanum, & A. Latiff (Eds.), Taman Negeri Gunung Stong, Kelantan: Pengurusan, Persekitaran Fizikal, Biologi dan Sosio- ekonomi (pp. 191- 205). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Norhayati, A., Juliana, S., Daicus, B., & Chan, K. O. (2007). Amphibian Diversity of Taman Rimba Kenong. In M. Azahar, A. R. Azman, H. Che Hashim, H. L. Koh, A. Norhayati, & A. Latiff (Eds.), Taman Rimba Kenong, Pahang: Pengurusan, Presekitaran Fizikal dan Kepelbagaian Biologi (pp. 234- 242). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Norhayati, A., Belabut, D.M., Juliana, S., & Chan, K. O. (2009). Amphibians and Reptiles of Malaysia. Retrieved December 22, 2009 from http://www.herpweb.net. Norhayati, A., Mohamad Shahril- Lizan, A., Juliana, S., Sharma, D. S. K., Chan, K. O., & Daicus, B. (2011). Herpetofaunal Records from Royal Belum State Park. In A. R. Abdul Rahman, H. L. Koh, A. Muhamad, & A. Latiff (Eds.), Taman Negeri Diraja Belum, Perak: Pengurusan Hutan, Persekitaran Fizikal, Kepelbagaian Biologi dan Sosio- ekonomi (pp. 272- 279). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Norhayati, A., Belabut, D. M., & Chan, K. O. (2012a). Amphibians and Reptiles of Peninsular Malaysia. Retrieved January 12, 2012 from http://www.amphibia.my. Norhayati, A., Chan, K. O., Grismer, L. L., Muin, M. A., Hamidi, J., & Daicus, B. (2012b). Amphibians of Pulau Pangkor, Perak. In A. R. Abdul Rahman, M. S. Masran, B. H. Mohd Nasir, A. Muhammad, & A. Latiff (Eds,). Hutan Pulau Pangkor, Perak: Pengurusan Hutan, Persekitaran Fizikal dan Kepelbagaian Biologi (pp. 262- 265). Kuala Lumpur: Jabatan Perhutanan Semenanjung Malaysia. Norkarmila, D. (2009) Morphometric and Genetic Variability of River Terrapin, (Batagur baska) and Painted Terrapin, (Batagur borneonsis) (Master’s thesis). Universiti Sains Malaysia, Penang. Nuñez, J. J., Wood, N. K., Rabanal, F. E., Fontanella, F. M., & Sites Jr, J. W. (2011). Amphibian phylogeography in the Antipodes: Refugia and postglacial colonization explain mitochondrial haplotype distribution in the Patagonian frog Eupsophus calcaratus (Cycloramphidae). Molecular Phylogenetics and Evolution, 58(2), 343-352.
115
O'Brien, S. J. (1994). A role for molecular genetics in biological conservation. Proceedings of the National Academy of Sciences, 91(13), 5748-5755. Oza, G. M. (1990). Ecological effects of the frog's legs trade. Environmentalist, 10(1), 39-42. Patla, D. A., & Peterson, C. R. (2002). Amphibian Diversity, Distribution, and Habitat Use in the Yellowstone Lake Basin. Yellowstone Lake: Hotbed of Chaos or Reservoir of Resilience, 179. Pearse, A. S. (1911). Concerning the development of frog tadpoles in sea water. Philippine Journal of Science, 6, 219-220. Penman, T., Lemckert, F., & Mahony, M. (2004) Two hundred and ten years looking for the Giant Burrowing Frog. Australian Journal of Zoology, 32, 597–604. Penman, T. D., Mahony, M. J., Towerton, A. L., & Lemckert, F. L. (2007). Spatial models of giant burrowing frog distributions. Endangered Species Research, 3(2), 115-124. Pérez-Coll, C. S., & Herkovits, J. (2006). Synergistic effects of copper and butylic ester of 2, 4-dichlorophenoxyacetic acid (Esternon Ultra) on amphibian embryos. International Journal of Environmental Research and Public Health, 3(4), 343-347. Pesole, G., Gissi, C., De Chirico, A., & Saccone, C. (1999). Nucleotide substitution rate of mammalian mitochondrial genomes. Journal of Molecular Evolution, 48(4), 427-434. Pough, F. H., Andrews, R. M., Cadle, J. E., Crump, M. L., Savitzky, A. H., & Wells, K. D. (2004). Herpetology, 3rd (Ed.). New Jersey: Prentice Hall. Pounds, J. A., Fogden, M. P., & Campbell, J. H. (1999). Biological response to climate change on a tropical mountain. Nature, 398(6728), 611-615. Ramlah, Z. (1998). Microgeographic variation of mitochondrial DNA in populations of Sarawak riparian frogs (Master’s thesis). University of Illinois, Chicago. Ramlah, Z. (2003). High levels of mitochondrial DNA diversity in populations of Sarawak Limnonectes leporinus. In M. Maryati, M. Matsui, & K. H. Boon (Eds). Herpetology and The Future, (pp.64-71). Proceedings of the International Conference on Sarawak Herpetology. Ramlah, Z. (2005). Mitochondrial DNA diversity in populations of the Sarawak White- Lipped frog, Rana chalconota. Proceedings of the Second Regional Symposium on Environment & Natural Resources. Vol 2, pp. 148-153. Ramlah, Z. (2009). Ecology and molecular phylogenetics of frogs from the genus Rana Linnaeus 1758 in Sarawak (Doctoral dissertation). Universiti Malaysia Sarawak.
116
Ramlah, Z., Shukor, M. N., Norhayati, A., & Md-Zain, B. M. (2010). Genetic Structure of Hylarana erythraea (Amphibia: Anura: Ranidae) from Malaysia. Zoological Studies, 49(5), 688-702. Ray, N., Lehmann, A., & Joly, P. (2002). Modeling spatial distribution of amphibian populations: a GIS approach based on habitat matrix permeability. Biodiversity & Conservation, 11(12), 2143-2165. Reed, D. H., Briscoe, D. A., & Frankham, R. (2002). Inbreeding and extinction: the effect of environmental stress and lineage. Conservation Genetics, 3(3), 301- 307. Relyea, R. A., & Mills, N. (2001). Predator-induced stress makes the pesticide carbaryl more deadly to gray treefrog tadpoles (Hyla versicolor). Proceedings of the National Academy of Sciences, 98(5), 2491-2496. Relyea, R. A. (2006). The effects of pesticides, pH, and predatory stress on amphibians under mesocosm conditions. Ecotoxicology, 15(6), 503-511. Ren, Z., Zhu, B., Ma, E., Wen, J., Tu, T., Cao, Y., Hasegawa, M., & Zhong, Y. (2009). Complete nucleotide sequence and gene arrangement of the mitochondrial genome of the crab-eating frog Fejervarya cancrivora and evolutionary implications. Gene, 441(1), 148-155. Rest, J. S., Ast, J. C., Austin, C. C., Waddell, P. J., Tibbetts, E. A., Hay, J. M., & Mindell, D. P. (2003). Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome. Molecular Phylogenetics and Evolution, 29(2), 289-297. Robinson, G. M. (1998). Methods and Techniques in Human Geography. Chichester: John Wiley & Sons. Rodrigues, R., Schneider, H., Santos, S., Vallinoto, M., Sain-Paul, U., & Sampaio, I. (2008). Low levels of genetic diversity depicted from mitochondrial DNA sequences in a heavily exploited marine fish (Cynoscion acoupa, Sciaenidae) from the Northern coast of Brazil. Genetics and Molecular Biology, 31(2), 487- 491. Roe, B. A., Ma, D. P., Wilson, R. K., & Wong, J. F. (1985). The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. Journal of Biological Chemistry, 260(17), 9759-9774. Rogers, A. R., & Harpending, H. (1992). Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution, 9(3), 552-569. Rola, A. C., & Pingali, P. L. (1993). Pesticides, Rice Productivity and Farmers’ Health: an Economic Assessment. Philippines: International Rice Research Institute. Rowe, G., & Beebee, T. J. (2005). Intraspecific competition disadvantages inbred natterjack toad (Bufo calamita) genotypes over outbred ones in a shared pond environment. Journal of Animal Ecology, 74(1), 71-76.
117
Rozas, J., Librado, P., Sánchez-DelBarrio, J. C., Messeguer, X., & Rozas, R. (2010). DnaSP, DNA Sequence Polymorphism version 5.10.1. Retrieved May 5, 2012 from http://www.ub.edu/dnasp. Ruslan, R., & Noresah, M. S. (1998). Sistem Maklumat Geografi. Kuala Lumpur: Dewan Bahasa dan Pustaka. Ruzainah, A. (2008). Molecular Genetic Studies of Marble Goby, Oxyeleotris marmoratus (Bleeker, 1852) in Malaysia (Master’s thesis). Universiti Sains Malaysia, Penang. Saccheri, I., Kuussaari, M., Kankare, M., Vikman, P., Fortelius, W., & Hanski, I. (1998). Inbreeding and extinction in a butterfly metapopulation. Nature, 392(6675), 491-494. Saccone, C., Pesole, G., & Sbisá, E. (1991). The main regulatory region of mammalian mitochondrial DNA: structure-function model and evolutionary pattern. Journal of Molecular Evolution, 33(1), 83-91. Sanders, R. (2010). Pesticide atrazine can turn male frogs into females. ScienceDaily. Retrieved March 1, 2010 from http://sciencedaily.com. Satheeshkumar, P. (2011). First record of a mangrove frog Fejervarya cancrivora (Amphibia: Ranidae) in the Pondicherry mangroves, Bay of Bengal- India. World Journal of Zoology, 6(3), 328-330. Savage, A. E., Grismer, L. L., Shahrul Anuar, M. S., Chan, K. O., Grismer, J. L., Quah, E., Muin, M. A., Ahmad, N., Lenker, M., & Zamudio, K. R. (2011). First record of Batrachochytrium dendrobatidis infecting four frog families from Peninsular Malaysia. EcoHealth, 8(1), 121-128. Schierwater, B., Streit, B., Wagner, G. P., & Desalle, R. (1994). Molecular Ecology and Evolution, Approaches and Applications. Switzerland: Birkhauser Verlag. Schmidt-Nielsen, K., & Lee, P. (1962). Kidney function in the Crab-eating Frog (Rana cancrivora). Journal of Experimental Biology, 39(1), 167-177. Seppä, P., & Laurila, A. (1999). Genetic structure of island populations of the anurans Rana temporaria and Bufo bufo. Heredity, 82(3), 309-317. Shenoy, K., Cunningham, B. T., Enfroe, J. W., & Crowley, P. H. (2009). Growth and survival of northern leopard frog (Rana pipiens) tadpoles exposed to two common pesticides. Environmental Toxicology and Chemistry, 28(7), 1469- 1474. Sivasundar, A., Bermingham, E., & Orti, G. (2001). Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers. Molecular Ecology, 10(2), 407-417. Skerratt, L. F., Berger, L., Speare, R., Cashins, S., McDonald, K. R., Phillott, A. D., Hines, H. B., & Kenyon, N. (2007). Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth, 4, 125-134. 118
Shahriza, S., Ibrahim, J., & Shahrul Anuar, M. S. (2011a). The amphibian fauna of Lata Bukit Hijau, Kedah, Malaysia. Russian Journal of Herpetology 18(3), 221 – 227.
Shahriza, S., Ibrahim, J., Nurul Dalila, A. R., & Abdul Muin, M. A. (2011b). An Annotated Checklist of the Herpetofauna of Beris Valley, Kedah, Malaysia. Tropical Life Sciences Research, 22(1), 13–24.
Shahriza, S., & Ibrahim, J. (2012a). The Amphibian Diversity of Bukit Jana, Taiping, Perak. Tropical Life Sciences Research, 23(2), 49–57. Shahriza, S., Ibrahim, J., Shahrul Anuar, M. S., & Abdul Muin, M. A. (2012b). Herpetofauna of Peta Area of Endau-Rompin National Park, Johor, Malaysia. Pertanika Journal of Tropical Agricultural Science, 35(3), 553 – 567. Slatkin, M. (1987). Gene flow and geographic structure of natural populations. Science, 236, 787–792. doi: 10.1126/science.3576198. Slatkin, M., & Hudson, R. R. (1991). Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations. Genetics, 129(2), 555-562. Spear, S. F., Peterson, C. R., Matocq, M. D., & Storfer, A. (2006). Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park. Conservation Genetics, 7(4), 605-611. Stuart, S. N., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S., Fischman, D. L., & Waller, R. W. (2004). Status and trends of amphibian declines and extinctions worldwide. Science, 306(5702), 1783-1786. Sturdevant, R. W. (2007). NAVSTAR, the Global Positioning System: A Sampling of Its Military, Civil, and Commercial Impact. In S. J. Dick & R. D. Launius (Eds.), Societal Impact of Spaceflight (pp. 331-351). Washington D.C.: NASA Office of External Relations, History Division. Sumida, M., & Ogata, M. (1998). lntraspecific differentiation of the Japanese brown frog Rana japonica inferred from mitochondrial DNA sequences of the cytochrome b gene. Zoological Science, 15, 989–1000. Sumida, M., Kaneda, H., Kato, Y., Kanamori, Y., Yonekawa, H., & Nishioka, M. (2000). Sequence variation and structural conservation in the D-loop region and flanking genes of mitochondrial DNA from Japanese pond frogs. Genes & Genetic Systems, 75(2), 79-92. Sumida, M., Kanamori, Y., Kaneda, H., Kato, Y., Nishioka, M., Hasegawa, M., & Yonekawa, H. (2001). Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata. Genes & Genetic Systems, 76(5), 311-325. Sumida, M., Kondo, Y., Kanamori, Y., & Nishioka, M. (2002). Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and
119
the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes. Molecular Phylogenetics and Evolution, 25(2), 293–305. Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24(8), 1596-1599. Tan, M. P. (2011). Population genetics of the striped snakehead, Channa striata (Bloch, 1793) in Malaysia (Master’s thesis). Universiti Sains Malaysia, Penang. Toda, M., Matsui, M., Nishida, M., & Ota, H. (1998). Genetic divergence among Southeast and East Asian populations of Rana limnocharis (Amphibia: Anura), with special reference to sympatric cryptic species in Java. Zoological Science, 15(4), 607-613. Understanding Evolution. (2012). Genetic variation. Retrieved September 21, 2012 from http://evolution.berkeley.edu/evolibrary/article/evo17. van der Meijden, A., Vences, M., Hoegg, S., & Meyer, A. (2005). A previously unrecognized radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 37(3), 674-685. van Dijk, P. P., Iskandar, D., Inger, R., Lau, M. W. N., Ermi, Z., Baorong, G., Dutta, S., Manamendra-Arachchi, K., de Silva, A., Bordoloi, S., Kaneko, Y., Matsui, M., & Khan, M. S. (2013). Fejervarya limnocharis. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2. Retrived January 12, 2013 from www.iucnredlist.org. Vaughan, D. A., Lu, B. R., & Tomooka, N. (2008). The evolving story of rice evolution. Plant Science, 174(4), 394-408. Velo-Antón, G., Zamudio, K. R., & Cordero-Rivera, A. (2011). Genetic drift and rapid evolution of viviparity in insular fire salamanders (Salamandra salamandra). Heredity, 108(4), 410-418.
Vences, M., Vieites, D. R., Glaw, F., Brinkmann, H., Kosuch, J., Veith, M., & Meyer, A. (2003). Multiple overseas dispersal in amphibians. Proceedings of the Royal Society B: Biological Sciences, 270, 2435– 2442. doi:10.1098/rspb.2003.2516. Wedekind, C., Seebeck, T., Bettens, F., & Paepke, A. J. (1995). MHC-dependent mate preferences in humans. Proceedings of the Royal Society of London. Series B: Biological Sciences, 260(1359), 245-249. Wolstenholme, D. R. (1992). Animal mitochondrial DNA: structure and evolution. International Review of Cytology, 141, 173-216. Wong, L. H. J. (2007). Kajian Pemakanan dan Kewujudan Bersama Dua Spesies Katak Simpatrik, Fejevarya cancrivora dan Fejevarya limnocharis di Pulau Pinang, Malaysia (Master’s thesis). Universiti Sains Malaysia. 120
Wright, S. (1943). Isolation by distance. Genetics, 28, 114–138. Wright, S. (1969). Evolutions and the Genetics of Populations, Vol. 2, the Theory of Gene Frequencies. Chicago: The University of Chicago Press. Wright, P., Anderson, P., Weng, L., Frick, N., Wong, W. P., & IP, Y. K. (2004). The crab‐eating frog, Rana cancrivora, up‐regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity. Journal of Experimental Zoology Part A: Comparative Experimental Biology, 301(7), 559-568. Wu, C. S., & Kam, Y. C. (2009). Effects of salinity on the survival, growth, development, and metamorphosis of Fejevarya limnocharis tadpoles living in brackish water. Zoological Science, 26(7), 476-482. Yin, X. H., Li, S. N., Zhang, L., Zhu, G. N., & Zhuang, H. S. (2008). Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay. Ecotoxicology, 17(4), 280-286. Zardoya, R., & Meyer, A. (2000). Mitochondrial evidence on the phylogenetic position of caecilians (Amphibia: Gymnophiona). Genetics, 155(2), 765-775. Zhang, P., Chen, Y. Q., Zhou, H., Wang, X. L., & Qu, L. H. (2003). The complete mitochondrial genome of a relic salamander, Ranodon sibiricus (Amphibia: Caudata) and implications for amphibian phylogeny. Molecular Phylogenetics and Evolution, 28(3), 620-626. Zhigang, Y., Zhao, E., Haitao, S., Diesmos, A., Alcala, A., Brown, R., Afuang, L., Gee, G., Sukumaran, J., Yaakob, N., Ming, L. T., Chuaynkern, Y., Thirakhupt, K., Das, I., Iskandar, D., Mumpuni and Inger, R. (2009). Fejervarya cancrivora. In: IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. Retrieved September 9, 2011 from www.iucnredlist.org. Zhong, J., Liu, Z. Q., & Wang, Y. Q. (2008). Phylogeography of the rice frog, Fejervarya multistriata (Anura: Ranidae), from China based on mtDNA D- loop sequences. Zoological Science, 25(8), 811-820.
121
APPENDICES
Appendix A: Preparation of buffers; TE buffer and 10X TBE
TE (Tris-EDTA) Buffer, pH 8.0, per liter
1M Tris, pH 8.0 10.00 ml 0.5 M EDTA (pH 8.0) 2.00 ml
ddH20 top up to 1 liter
10X TBE (Tris-borate-EDTA), pe r liter
Tris base 108.00 g Boric acid 55.00 g 0.5M EDTA (pH 8.0) 40.00 ml
ddH20 top up to 1 liter
pH 8.3
to get 0.5 X TBE/liter: 50 ml of 10X TBE was diluted in 950 ml ddH20
Appendix B: Sequence chromatogram of Fejevarya limnocharis.
LIST OF PUBLICATIONS
1. Shahriza, S., Ibrahim, J., Shahrul Anuar, M. S., Nur Hafizah, I., Amiruddin,
I., Amirah, H., & Zalina, A. (2013). An Addition of Reptiles of Gunung
Inas, Kedah, Malaysia. Russian Journal of Herpetology, 20 (3): 171- 180.
2. Amirah, H., Yap, C. H., Mohd Azmeer, A. B., Ahmad Ridzuan, Y. A., &
Ibrahim, J. (2013). Diversity and Density of Amphibians at Sungai Enam,
Temengor Forest Reserve, Perak, Malaysia. In A. Latiff, B. K. Daniel, K.
Zainon, A. K. Zulfadhlan, I. Nurul Irdayu, N. R. Eza Feizaty, S. Alizah, M.
Mohd Syaiful, H. Mohd Najmi, & Y. A. Ahmad Ridzuan. Proceedings of the
2nd Temengor Scientific Expedition 2012. Paper presented at The 2nd
Temengor Scientific Expedition 2012, Pulau Banding Foundation, Pulau
Banding, 22- 25 August (pp. 353- 364). Petaling Jaya, Selangor: Pulau
Banding Foundation.
3. Amirah, H., Mohd Azmeer, A. B., Dionysius, S., Nurolhuda, N., Reuben, S.,
Ahmad Ridzuan, Y. A., & Ibrahim, J. (2013). An Updated Checklist of the
Herpetofauna of the Belum- Temengor Forest Reserves, Hulu Perak,
Peninsular Malaysia. In A. Latiff, B. K. Daniel, K. Zainon, A. K. Zulfadhlan,
I. Nurul Irdayu, N. R. Eza Feizaty, S. Alizah, M. Mohd Syaiful, H. Mohd
Najmi, & Y. A. Ahmad Ridzuan. Proceedings of the 2nd Temengor Scientific
Expedition 2012. Paper presented at The 2nd Temengor Scientific Expedition
2012, Pulau Banding Foundation, Pulau Banding, 22- 25 August (pp. 365-
388). Petaling Jaya, Selangor: Pulau Banding Foundation.
4. Ibrahim, J., Amirah, H., Shahriza, S., Nur Hafizah, I., Zalina, A., Nurliza, A.
M., & Nur Hafizah, C. Z. (2013). Updated Checklist of Amphibians of Pulau Jerejak, Penang, Peninsular Malaysia. Procedia- Social and Behavioral
Sciences (in press).
5. Ibrahim, J., Amirah, H., Shahriza, S., Nur Hafizah, I., Zalina, A., Yap, C. H.,
Nurliza, A. M., & Nur Hafizah, C. B. (2013). Additions to the Herpetofauna
of Jerejak Island, Penang, Peninsular Malaysia. Malayan Nature Journal,
64(4): 213- 232.
6. Zalina, A., Amirah, H., Ibrahim, J., & Siti Azizah, M. N. (2013). DNA
Barcoding of Ranidae Frogs of Peninsular Malaysia. Poster presented at The
5th International Barcode of Life Conference, 27th- 31st October 2013,
Kunming, China.
7. Ibrahim, J., Zalina, A., Shahriza, S., Shahrul Anuar, M. S., Nur Hafizah, I.,
Amirah, H., Nurul Dalila, A. R., Muin, M. A., & Amirudin, I. (2012).
Checklist of the Herpetofauna of Bukit Perangin Forest Reserve, Kedah,
Malaysia. Sains Malaysiana, 41 (6): 691- 696.
8. Ibrahim, J., Amirah, H., Shahriza, S., Nur Hafizah, I., Zalina, A., Nurliza, A.
M., & Nur Hafizah, C. B. (2012). Additions to the herpetofauna of Jerejak
Island, Penang, Peninsular Malaysia. USM-PSU International Conference on
Art and Sciences 2012 (ICAS 2012). Transforming Research for Sustainable
Community. 2nd-4th December 2012.
9. Yap, C. H., Zalina, A., Amirah, H., Belabut, D., & Ibrahim, J. (2012).
Diversity and Density of Amphibians at Sungai Kampi, Teluk Kampi, Penang
National Park, Penang, Malaysia. (Seminar Biodiversiti Perhilitan 2012, 20th-
23rd November 2012, Ipoh, Malaysia). Jabatan Perhilitan.
10. Belabut, D., Norhayati, A., Amalina, I., Zalina, A., Yap, C. H., Mohd
Asyraff, M. S., Amirah, H., Ibrahim, J., Paul, Y., Tan, P. E., Norfariza, M. K., Osman, N., Tang, T. K., Ahmad, S., Helpis, I., & Yong, H. M. (2012).
Amphibian Fauna Diversity and Endemism in the Pulau Tioman and Taman
Negara Pulau Pinang, Peninsular Malaysia. (Seminar Biodiversiti Perhilitan
2012, 20th-23rd November 2012, Ipoh, Malaysia). Jabatan Perhilitan.
11. Ibrahim, J., Sim, E. C., & Amirah, H. (2012). Development of eggs and
larvae of the Common Swallowtail Butterfly, Papilio polytes (L.)
(Lepidoptera: Papilionidae) in Malaysia. International Conference of
Multidisciplinary Research 2012, 1st- 3rd November 2012, USM, UniSYiah,
Unikuala.