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Molecular Analysis of the Breeding Biology

of the Asian Arowana (Scleropages formosus)

Chang Kuok Weai Alex

Thesis submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Department of Biological Science

National University of Singapore

2010

Supervisor: Associate Professor Laszlo Orban

PhD committee members : Emeritus Professor Lam Toong Jin

Dr Hong Yan

Dr Gregory Jedd

Associate Prof Laszlo Orban

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Acknowledgements

Throughout my project, Prof. Laszlo Orban, my supervisor, was generous with his time and knowledge and on target with his counsel and instilling in me a real appreciation for the scientific method. . I gratefully acknowledge the essential contributions of my Supervisory Committee, Emeritus Professor Toong Jin Lam (chair), Dr Yan Hong and Dr Gregory Jedd, and I also thank Professor Lam for chairing my Examination Committee. I commend the administration of the Temasek Life Sciences Laboratory (TLL) and Qian Hu Fish Farm for facilitating the educational pursuits of their employees I also thank Yap Kim Choon and his staff at Qian Hu Fish Farm believing in me and providing me such a important platform to work on this wonderful species and I am grateful to Kenny Yap for providing invaluable early assistance. I am grateful to my colleagues, Woei Chang Liew, Hsiao Yuen Kwan, Rajini Srineevasan, Felicia Feng, Dr. Xingang Wang, Dr. Richard Bartfai for help and collaborative work; and to Chin Heng Goh, Dr. Patrick Gilligan, and Dr. Gen Hua Yue, who quickly and patiently replied to questions relating to material herein. I thank the contribution of several attachment students, Wee Kee, Qi Feng, Zi Jie, Say Aik, Serene, and Daniel who brightened up the sky every time they were around. In addition, I fondly acknowledge the help and support of numerous TLL colleagues and PIs. Also, special thanks to Dr Robert Brooks, who patiently guided me through the advanced analysis of some of my results (in understanding mating systems. I also thank Aaron Chuah and Graham Wright, who provided computing advice and code, Prof Rudolf Meier and Prof Tan Heok Hui, who helped me in understanding the interesting phylogenetic studies. Lastly, I thank my Dad for getting me hooked on fishes and fisheries science early on.

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Dedication

To my family, especially my dad and mum, my wife Cynthia, my son Andre Jacob and friends, for their unwavering kindness, patience, and support.

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TABLE OF CONTENT

ABSTRACT ...... 11

List of Tables ...... 12

List of Figures ...... 13

List of Abbreviations ...... 23

1 INTRODUCTION ...... 27

1.1 Taxonomy of bonytongues ...... 27

1.2 The general biology of Asian arowana ...... 31

1.3 The Asian arowana has at least six main colour strains ...... 34

1.4 Sex and strain identification of teleosts using classical tools ...... 42

1.5 Mating system and parental care in fishes ...... 46

1.6 Molecular approaches in fish biology and aquaculture research ...... 50

1.7 The aims of my research ...... 62

2 MATERIALS AND METHODS ...... 63

2.1 The origin of fish studied ...... 63

2.2 Arowana breeding and holding facilities in QH ...... 64

2.3 Field Observations ...... 64 4 | P a g e

2.4 Sample collection ...... 65

2.5 DNA isolation ...... 66

2.6 Isolation and genotyping of microsatellites ...... 68

2.7 Detection of steroid hormone levels using an Enzyme Immunosorbent Assay kit ……………………………………………………………………………...69

2.8 Bradford total soluble protein (TSP) Assay ...... 69

2.9 Amplified Fragment Length Polymorphism (AFLP) ...... 70

2.10 Fluorescent Motif Enhanced Polymorphisms (FluoMEP) ...... 72

2.11 Determining single nucleotide polymorphisms in the mitogenome to confirm the gender of the mouthbrooder ...... 72

2.12 Computational and statistical analysis ...... 74

2.12.1 Microsatellite genotyping ...... 74

2.12.2 Analysis of AFLP-based phylogenetic relationship between the colour

strains …………………………………………………………………………75

2.12.3 Analysis of kinship between the brooders for understanding egg thievery

event …………………………………………………………………………75

2.12.4 Study of the genetic similarity among the colour strains ...... 76

2.13 Morphometric measurements ...... 76

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2.13.1 External morphometric differences ...... 76

2.13.2 Internal morphometric differences – moulding/roughness index and

touch-based examination ...... 77

2.13.3 Morphometry of the eggs and larvae ...... 79

3 RESULTS ...... 80

3.1 The reproduction biology of Asian arowana is very different from that of

most teleosts ...... 80

3.1.1 Observation of mating - temporal and spatial data ...... 80

3.2 Differences in the productivity and survival rate of colour strains and

dependence of breeding events on environmental factors ...... 84

3.2.1 There were significant differences in offspring number per brooder and

their survival between several colour strains ...... 84

3.3 The early development of Asian arowana larvae is a slow process ...... 87

3.3.1 Fertilised eggs ...... 88

3.3.2 Juveniles ...... 89

3.4 Sexing the brooders with classical and molecular tools ...... 91

3.4.1 External morphometric measurements on sexually mature adults showed

significant differences between the two sexes ...... 91

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3.4.2 Internal morphometry – The surface of the buccal cavity transformed in

mouthbrooding males ...... 92

3.4.3 Hormonal measurements from mucus ...... 95

3.5 Identification of the sex of the mouthbrooding parent in the MG variety of

Asian arowana ...... 96

3.6 Genotyping data reveals complex relationships between the brooders in the ponds ……………………………………………………………………………...99

3.7 Change in breeding pattern following the loss of a male in pond WH001..105

3.8 No signs of possible inbreeding or incompatibility avoidance were observed

…………………………………………………………………………….106

3.9 The Asian arowanas display an unusual phenomenon of egg thievery ...... 107

3.10 Transient morphological modification of the surface of buccal cavity in

Asian arowana during mouthbrooding ...... 111

3.11 Most colour variants of Asian arowana can be differentiated from the others using molecular analysis ...... 113

3.11.1 Differentiation of the colour strains using microsatellite-based

genotypes ...... 113

3.11.2 FluoMEP was able to differentiate between two commercially important

colour strains ...... 115

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3.11.3 The microsatellite-based phylogenetic tree of the colour variants of

Asian arowana was congruent with geographical reconstruction of prehistoric

events in South-East Asia ...... 117

3.11.4 Bayesian clustering analysis allowed for differentiation of most colour

strains ………………………………………………………………………..119

3.12 Pairwise comparison of the FST value indicated that the colour strains are

likely to be one species ...... 121

4 DISCUSSION ...... 123

4.1 Microsatellites allow for accurate parentage analysis and reveal breeding

relationships of Asian arowana ...... 123

4.2 The Asian arowana male protects its eggs by transient morphological

modification of the surface of its buccal cavity during mouthbrooding ...... 130

4.3 The advantages of being able to sex the adult Asian arowanas ...... 132

4.4 The change in the breeding relationships in a pond after the death of a highly

productive male indicates the presence of a complex hierarchical breeding system

…………………………………………………………………………….138

4.5 Our data do not show inbreeding or incompatibility avoidance, indicating

unique mate choice and strategy ...... 139

4.6 Egg thievery: Why do Asian arowanas steal each other‟s eggs? ...... 141

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4.7 Asian arowanas provide another example for a positive correlation between

egg size and parental care ...... 144

4.8 Genetic confirmation of a paternal care in the Malaysian golden variety of

Asian arowana ...... 145

4.9 Strong positive effect of a mating strategy involving multiple mates, number

of reproductive events (broods) and lack of difference between the sexes ...... 146

4.10 Genetic analysis detects distinct differences between Asian arowana

strains ………………………………………………………………………….150

4.11 The divergence of the different colour strains seems to be consistent with

the change of the land mass configuration of South East Asia ...... 152

4.12 The different colour strains are likely to be geographically isolated

populations and not different species ...... 154

5 POSSIBILITIES FOR THE FUTURE ...... 156

5.1 Selective breeding program – production of the first hybrids in preparation

for linkage mapping ...... 156

6 REFERENCE ...... 158

7 SUPPLEMENTARY TABLES ...... 178

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ABSTRACT

The dragonfish or Asian arowana (Scleropages formosus Müller & Schlegel, 1844) is one of the few living, „near-basal‟ teleosts belonging to the family Osteoglossidae.

This CITES-protected species possesses a fascinating collection of biologically interesting characters that could be important for the study of basal vertebrate breeding biology, mating behavior and mate preference. We report here, to the best of our knowledge, the most detailed documentation of the breeding behavior, including mate choice and observed mating strategy, of S. formosus. For the analysis of mate choice, we created the “genetic mating map” for three ponds containing the total of more than 60 brooders over a 3 yr period, using 12 highly polymorphic microsatellites. Our data indicated that there were no multiple paternities, only single paternity in the 100 clutches of offspring sampled. The interspawning interval ranged from 2 months to 17 months. Parentage assignment, together with identification of the maternal and paternal genotypes using mitochondrial haplotyping, demonstrated that

Asian arowana practiced both polygamy and monogamy. We also reported the unusual form of egg thievery practiced by the Asian arowana and a transient modification of the buccal cavity in the male brooders in preparation for the mouthbrooding event.

Our data are important not only for better understanding the breeding biology of this unusually interesting bonytongue, but also for their potential to improve the existing aquaculture programs.

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List of Tables

Table 1: Members of the family Osteoglossidae and their geographical distribution . 30

Table 2: The different colour strains of Asian arowana found across the islands in

Southeast Asia and one of their hybrids ...... 37

Table 3：The mating systems and types of parental care observed in the family

Osteoglossidae are variable ...... 49

Table 4: Types of DNA markers, their characteristics and potential applications...... 52

Table 5: The productivity and offspring survival of different colour strains in the farm

...... 85

Table 6 : The modification of buccal cavity surface is transient and occurs in mouthbrooding males only...... 95

Table 7: The mitochondrial gene-based SNP profiles of the offspring are identical with those of their non-mouthbrooding genetic parent ...... 98

Table 8: Characterization of microsatellites and genetic diversity in 230 Asian arowana brooders ...... 103

Table 9: Average FST values of the different colour varieties of Asian arowana and two Australian arowana species ...... 122

Table 10: The fecundity of some females from WH001 ...... 130

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List of Figures

Figure 1: The phylogenetic analysis of the Osteoglossids and other teleosts by using concatenated mitochondrial protein-coding genes. The data set consist of a total of

3,675 amino acid positions concatenated from 12 protein sequences for each species.

The phylogenetic relationship of Asian arowana with repect to representatives from

Actinopterygii and Sarcopterygii taxa using dogfish shark as outgroup was performed by maximum parsimony (MP), maximum likelihood (ML) and Bayesian interferences

(BI) methods. Tree topology produced by the different methods was similar.

Bootstrap values are in parentheses and in MP/ML/BI order. Diagram obtained from figure 5 of Yue et al., BMC Genomics 7:242 , 2006; [10]...... 28

Figure 2: The seven members of the Osteoglossidae family show phenotypic similarity. Panels: A) Arapaima; B) Black arowana; C) Silver arowana; D) African arowana; E) Asian arowana; F) Pearl arowana; G) Red spotted arowana...... 29

Figure 3: The architecture of the Asian arowana‟s buccal cavity and the tongue bite apparatus (TBA). TBA is thought to be a derived feature of the Osteoglossomorpha used for anchoring the prey to the buccal cavity. Labels: A) Teeth on the medial edge of entopterygoid; B) Basihyal/basibranchial toothplate...... 34

Figure 4: The colour varieties are located in different geographical locations in

Southeast Asia. Labels: RG1 – Red Grade 1 (red); MG – Malaysian Golden (blue);

IG- Indonesian Gold (brown); RG2 – Red Grade 2 (black); GR- Green (green); Other than the GR, the other colour varieties are endemic only to one geographical location.

The GR is found in Cambodia, Peninsular Malaysia and Indonesia ...... 36

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Figure 5: The presence of different natural colour varieties of Asian arowana is unique compared to the rest of the subfamily Osteoglossinae. Panels: A) RG1; B) RG2; C)

Malaysian Gold (MG); D) Indonesian Gold (IG); E) Tong Yan Hybrid (TY); F) Green

(GR). Please see Figure 4 for labels...... 38

Figure 6: The juveniles of all the colour stains are phenotypically very similar. Panels:

A) RG1; B) RG2; C) MG; D) IG; E) Tong Yan hybrid (TY); F) GR. Please see Figure

4 for labels...... 39

C) IG; D) GR. Second set of panels with the respective close-up using a DLR camera‟s macro function of the scales: E) RG1; F) MG; G) IG; H) GR...... 40

DNA Analyzer (ABI, Foster City, CA, USA) and peak profiles were presented using a software GeneMapper software V3.5 (Applied Biosystems). From the peak profiles,

Offspring #1 have inherited the larger allele from parent A and the smaller allele of parent B leading to a homozygous genotype, where else offspring #2 inherited the smaller allele of parent A and larger allele of parent B resulting in a heterozygous genotype...... 56

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Figure 9: A schematic representation showing the difference between RAPD and

FluoMEP. RAPD typically uses one short, unlabelled primer (half-arrow). The amplified band pattern is separated on agarose gel, detected by ethidium bromide staining and analyzed by visually comparing the patterns. The typical number of bands varies between one and a dozen. FluoMEP uses fluorescently labeled “common primers” (half-arrow with star) with the potential ability of targeting frequent motifs

(rectangle) in the genome together with the short, unlabelled RAPD primers. The PCR product of FluoMEP contains three different kinds of bands amplified by: i) the common primer only; ii) the common primer and the RAPD primer together; and iii) the RAPD primer alone. The pattern is separated by capillary gel electrophoresis, and only the fluorescently labeled bands at one or both ends are detected. The typical number of bands varies between 10 and 100. (Figure 1 of Chang et al.,

Electrophoresis 28; 525-534, 2007 [153]...... 61

Figure 10: External morphometric measurements used when searching for phenotypic sex markerson fully mature, adult specimen of Asian arowana. Labels; SL - Standard

Length, HL - Head Length, GPH - Gill Plate Height, GPL - Gill Plate Length, and ML

- Mouth Length...... 77

Figure 11: The breeding process of Asian arowana captured on video by a hobbyist.

Panel A: During the start of the mating process, the two partners would lay close to the bottom beside each other and swirl in a circular motion; Panel B: this swirling movement will stop and both the fish will stay motionless for about 30 seconds and after which there would be a coordinated release of milt and eggs (see arrowhead).

Panel C: The release of the milt and eggs, the female floats towards the surface of 14 | P a g e

water. The male would also leave the fertilized eggs and move to the surface for a gulp of air (Panels C-D). The male slowly collected them into his mouth by gentle suction (Panels E-F). The pictures were taken from a video publicly available at

Youtube: http://www.youtube.com/watch?v=5ltmk6oHXg8 ...... 83

Figure 12: The breeding cycle of the Asian arowana in the farm. The graph shows the total production in terms of number of batches（dotted line）and number of offspring

(solid line) for the three experimental ponds in relation to the rainfall (bar) data from

January 2003 to November 2006...... 87

Figure 13: The development of Asian arowana embryos, larvae and juveniles. Panels:

A) Fertilized egg at 0 dpf; B) Embryo at 3 dpf; C) Larva at 7 dpf; D) 9 dpf; E) 5 dpf;

F) 13 dpf; G) 15 dpf; H) 25 dpf; I) 28 dpf; J) Juvenile at 33 dpf; K) 36 dpf; L) 40 dpf; and M) 45 dpf. The bar indicates 5 mm on every panel...... 90

Figure 14: Significant morphometric differences between male and female individuals of the Asian arowana. Male; Female; * = denotes significant difference between males and females (student t test; P<0.0005); n=40; SL - Standard length; HL - Head length; ML - Mouth Length; GPL - Gill plate Length ...... 92

Figure 15: Scanning electron micropgraphs of the parasphenoid (pharyngeal teeth) do not reveal differences in teeth morphology between males and females. Panels ; A -

Male; B - Female; C - Teeth that are found on the endopterygoid (relative position to the entopterygoid teeth is indicated by the red box) of both males and females. Bars represent 100 micron...... 94

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Figure 16: Smooth buccal cavity can be found only in some Asian arowana brooders.

A total of 144 brooders (sex of the fish is not determined yet) were examined for the roughness using touch-based examination and the results were compared to their breeding records. On the graph, a smooth surface is indicated with a green bar and indicated as (“-”), a intermediate roughness is given a maroon bar and indicated as

“+/-”, and a rought surface is given a blue bar and is indicated as “+”...... 94

Figure 17: Comparing the 11-KT levels in the mucus of males to females in pond

WH001 over a period of 26 months. Hormone levels were determined by ELISA essay and normalized by the total soluble protein content determined by Bradford essay. The bars show the average values with their standard deviation...... 96

The three boxed IDs in the bottom left corner indicate inactive brooders (i.e. individuals that we have not collected any offspring from and also did not have any breeding relationships based on genotyping since the start of the experimentation) without known sex. Lines represent breeding connections that were determined by microsatellite genotyping. Each circle (○) on the line represents one breeding event that occurred between the two brooders, whereas the number beside the circle represents the number of offspring that was collected from the mouthbrooding 16 | P a g e

individual. The column on the left indicates the date of the collection of the offspring from the buccal cavity of the brooder...... 100

Figure 19: Fewer active brooders appeared to form less complicated breeding reelationships. The breeding relationship chart between the brooders in pond WH002.

For labels see Figure 18...... 101

Figure 20: There were no monogamous pairs in the third pond. The breeding relationship chart between the brooders in pond WH003. For labels see Figure 18.. 101

Figure 21: The loss of a fecund male resulted in long cessation of reproduction, followed by rearrangements of breeding connactions pond WH001. For labels see

Figure 18. Blue lines indicate terminated breeding connections, whereas red lines newly formed connections following the re-initiation of breeding after a half-a-year break, black lines indicates breeding connections that are resumed following the hyatus...... 106

Figure 22: There is no correlation between the genetic similarity of female brooders and their male breeding partners in the pond. The chart shows the dissimilarity indices (Nei‟s genetic distances) between 4 females (A2, A6, A8 and B2) and all their potential male breeding partners in the pond, respectively. The indices were calculated using the software package NTSYS [166]. The red bar indicates at least one breeding event with the female. Panels; A: Female A2, B: Female A6, C: Female A8, D:

Female B2 ...... 108

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Figure 23: There is no correlation between the genetic similarity of male brooders and their female breeding partners in the pond. The chart shows the dissimilarity indices

(Nei‟s genetic distances) between 4 males (B5, B9, A1 and C1) and all their potential female breeding partners in the pond, respectively. The red bar indicates at least one breeding event with the male. The indices were calculated using the software package

NTSYS [166]. Panels; A: Male B5, B: Male B9, C: Male A1, D: Male C1 ...... 108

Figure 24: Egg thievery occured in Asian arowana and in this pond all the thievery events were linked to one female. A green line connects a mouthbrooding individual that is mouthbrooding a batch of offspring where it is not the genetic parent; to the genetic parents breeding event and the triangle (∆) represents the mouthbrooding event by the “thief” (alpha numeric name encircled) and number shows the number of offspring collected (For rest of the labels, see Fig. 18)...... 110

Figure 25: Kinship coefficient data of the thief and the genetic parents of stolen eggs did not show any indication of kin selection. We tested the relatedness between pairs of brooders available in the pond and calculates the ratio of the primary hypothesis that they are related (full-sibs or half-sibs) to the null hypothesis that they are unrelated. k refers to the kinship coefficient where higher k value indicates higher chances of the pair being kin of each other (relatedness), where k of 1 indicates full siblings while 0 shows no relation. We compared the k of the thief (male)/genetic parents versus the ave k value amongst the brooders, we assume that if the k value of the thief/genetic parents is higher than the ave k, then there is a possibility of kin- selection occurring in the pond where kins are helping each other in parental care.

The grey bar indicates the k value of the thief with the genetic father where the white 18 | P a g e

bar refers to the k value of the thief with the genetic mother. The triangle symbol shows the ave k value...... 110

Figure 26: Observing the changes in the entopterygoid teeth profile of a male before, during and after mouthbrooding by confocal microscopy-based analysis of dental moulds. Panels: A) A planar representation of the entopterygoid teeth profile of a male at day 0 (larvae removed after mouthbrooding); B) 42 days later (day 42); C) 3- dimensional representation of the profile shown in A; D) 3-dimensional representation of the profiles shown in B; E) and F) Graphical representation of the changes in surface profile in the above male and a female, respectively...... 112

Neighour-Joining algorithm (NJ) using NTsys package 2.02e. An unrooted phylogenetic tree was fitted to the chord distance matrix by using the neighbor-joining

(NJ) algorithm as implemented in MEGA (ver3.1.2). TreeView was used to visualize the tree. The subcluster shaded in blue contains all Malaysian golden individuals. 114

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C117+BH05 with a peak at 137bp which is present in RG1 but absent in RG2. Panel

B shows the primer combination C117+F04 which produces a peak at 314bp position for RG1 and 294bp position for RG2...... 116

B) 40m contour, 10,000 yrs ago and C) 20m contour, 10,000 yrs ago. D) Genetic distance tree generated from 12 microsatellite genotypes by Neighbor-Joining (NJ) method. The branching points labeled with 1 and 2 indicate events that separated some colour varieties; 1 and 2 indicates the clade that is formed by the colour strains.

(Source of Paleogeographical figures – Harold K.V., 27; 1153-1157, 2000 [180]) .. 118

Figure 30: Bayesian clustering analysis using microsatellite and AFLP data enabled the differentiation of most colour strains including the hybrids. Bayesian model– based clustering method implemented in Structure v 2.1 was used to investigate the genetic structure of the different population [171,172] using data from 8 polymorphic

MS loci and 225 AFLP markers, this helped in the determination of the most likely number of genetically distinct clusters (K) of populations...... 120

Figure 31: The Asian arowana fits in the proposed correlation of mating system, sexual selection in sexual dimorphism. Pictorial definitions of four genetic mating

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systems possible in fishes. Lines connecting males and females indicate spawning partners that produced offspring. Also shown are the theoretical gradients in sexual- selection intensities and the degrees of gender dimorphism in secondary sexual traits

(epigamic features) often associated with these mating systems. (picture obtained from Avise JC et. al. 36; 19-25, 2002 [16]...... 129

Figure 32: There is an advantage in having more mates in both Asian arowana sexes.

Total offspring produced were plotted against the number of unique mating partners and brooder has mated with and also against the number of separate broods of offspring. Regression analysis shows that there is a strong positive effect indicating more mates and more reproductive events (broods) equates to more offspring and when we compared the interaction term which formally test the different regression slopes, the effect is not different between the sexes. (This analysis was done by Dr

Robert Brooks of UNSW, Sydney Australia, using our data.) ...... 150

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List of Abbreviations

11-KT 11-ketotestosterone

AFLPs Amplified Fragment Length Polymorphisms

ANOVA Analysis of Variance

ART Alternative Reproductive Tactics atp6 ATP synthase subunit 6

BI Bayesian Interferences bp Basepair

CE Capillary Electrophoretic

CI Confidence Intervals

CITES Convention on International Trade in Endangered Species of Wild Fauna and Flora cob cytochrome oxidase b cox1 cytochrome c oxidase subunit I cox2 cytochrome c oxidase subunit II cox3 cytochrome c oxidase subunit III dNTPs Deoxynucleoside Triphosphates dpf days post fertilisation

E2 Estradiol-17β

EIA Enzyme Immunosorbent Assay

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ELISA Enzyme-Linked Immunosorbent Assay

ESA Endangered Species Act

ESTs Expressed Sequence Tags f Coefficient index fluoMEP Fluorescent Motif Enhanced Polymorphism

FST Genetic Similarity

GPH Gill Plate Height

GPL Gill Plate Length

GR Green

GSI Gonadal Somatic Index

H Genetic diversity

He Expected heterozygosity

HL Head Length

Ho Observed heterozygosity

IG Indonesian Golden

IUCN International Union for the Conservation of Nature and Natural Resources k Pairwise Kinship Coefficient

KCl Potassium Chloride

MCMC Markov Chain Monte Carlo

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MG Malaysian Golden

MgCl2 Magesium Chloride

MHC Major Histocompatibility Complex

MJK Mensiku strain

ML Maximum Likelihood

ML Mouth Length

MP Maximum Parsimony mtDNA mitochondrial DNA nad1 NADH dehydrogenase subunit 1 nad2 NADH dehydrogenase subunit 2 nad4 NADH dehydrogenase subunit 4 nad5 NADH dehydrogenase subunit 5

NEA National Environmental Agency

NJ Neighbor-Joining

OSR Operational Sex Ratio

PCR Polymerase Chain Reaction

PIT Passive Integrated Transponder

QH QianHu

QTL Quantitative Trait Loci

RAPD Random Amplified Polymorphic DNA

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RFLPs Restriction Fragment Length Polymorphisms

RG1 Red Grade 1

RG2 Red Grade 2

SL Standard Length

SNPs Single Nucleotide Polymorphisms

SSC Standard Saline Citrate

SSCP Single-Stranded Conformation Polymorphism

SSR Simple Sequence Repeat

STRs Short Tandem Repeats

TBA Tongue Bite Apparatus

TL Total Length

Tris-HCl Tris Hydrochloric Acid

TSP Total Soluble Protein

TY Tong Yan

VTNR Variable Number of Tandem Repeat

WRG1 Wild Red Grade 1

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1 INTRODUCTION

1.1 Taxonomy of bonytongues

Asian arowana or dragonfish (Scleropages formosus) [1], is one of the few living members of the family Osteoglossidae within the order Osteoglossiformes.

There are altogether seven species described from the family Osteoglossidae

[2,3,4,5,6,7,8,9] which consists of two subfamilies: Heterotidinae and Osteoglossinae

(Figs. 1-2 and Table 1). Using a molecular phylogenetic tree[8], under the subfamily

Heterotidinae, there are two genera, each consisting of only one species, namely, the arapaima or pirarucu (Arapaima gigas; Cuvier, 1892) from South America and the

African arowana (Heterotis niloticus; Cuvier, 1892). Under the subfamily

Osteoglossinae, there are two genera, Osteoglossum and Scleropages. The genus

Osteoglossum consists of the silver arowana (Osteoglossum bicirrhosum; Cuvier,

1892) and the black arowana from South America (Osteoglossum ferreirai;

Kanazawa, 1966). Under the genus Scleropages, the three species include the Asian arowana (Scleropages formosus; Müller, 1844) the pearl arowana (Australia, Northern

Territory; Papua New Guinea) (Scleropages jardinii; Saville-Kent, 1892) and the

Australian spotted arowana (Australia, Central Queensland) (Scleropages leichardti;

Günther, 1864).

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Figure 1: The phylogenetic analysis of the Osteoglossids and other teleosts by using concatenated mitochondrial protein-coding genes. The data set consist of a total of 3,675 amino acid positions concatenated from 12 protein sequences for each species. The phylogenetic relationship of Asian arowana with respect to representatives from Actinopterygii and Sarcopterygii taxa using dogfish shark as out-group was performed by maximum parsimony (MP), maximum likelihood (ML) and Bayesian interferences (BI) methods. Tree topology produced by the different methods was similar. Bootstrap values are in parentheses and in MP/ML/BI order. Diagram obtained from figure 5 of Yue et al., BMC Genomics 7:242 , 2006; [10]. 27 | P a g e

Table 1: Members of the family Osteoglossidae and their geographical distribution

Common name Latin Name Location Other names References

Arapaima Arapaima gigas South America Pirarucu [11]

Black arowana Osteoglossum South America Black belt [11] ferreirai

Silver arowana Osteoglossum South America Silver belt [11] bicirrhosum

African arowana Heterotis Africa African [11] niloticus bonytongue

Asian arowana, Scleropages Southeast Asia Dragonfish, [7] formosus Asian bonytongue, Siluk, Arwana, Kayangan, Naga, Kelesa

Australian pearl Scleropages Australia, Papua Northern [12] arowana jardinii New Guinea, saratoga, Irian Jaya Northern spotted barramundi, Jardine's saratoga

Australian red Scleropages Australia Spotted [12] spotted arowana leichardti barramundi, spotted saratoga, spotted bonytongue

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1.2 The general biology of Asian arowana

The Asian arowana is known to grow to 100 cm in total length and live up to at least

43 years of age [13] and is morphologically distinct from the “typical teleosts” due its few salient features. It has a laterally compressed fusiform body and a strong caudal fin which allow the fish to move rapidly in water and hunt for prey. The species also possesses a posterior dorsal fin located at the posterior third of the body, an elongated anal fin, a lunate caudal fin and a pair of extended pectoral fins that are located near the ventral end of the operculum.

The presence of mandibular barbels at the lower jaw allows the fish to sense for prey in the tannin-filled, dark waters of the South East Asian Rivers. The terminal, superior mouth has sharp premaxillary and maxillary teeth which are located at the edges of the upper jaw. Together with the entopterygoid teeth and the toothed bonytongue it holds a struggling prey until it is weakened before consuming it and the entopterygoid teeth prevents the prey from escaping from the throat. The body is protected by large cosmoid scales (about 50 mm in diameter for an adult of 90 cm total length or TL). They are also known as bonytongues due to the presence of large tooth plates on the tongue (basihyal) and basibranchials that bite against the roof of the mouth cavity or the parasphenoid [14] (Fig. 3). Asian arowana is the top predator in its natural habitat and thus, probably plays an important role in its ecosystem.

Asian arowanas possess a fascinating collection of interesting characters that are important for the study of breeding biology, mating behavior and mate preference of this species. In contrast to most other teleost breeding systems [15,16,17], the species exhibits probably the most extreme and unique form of high parental care. Asian 30 | P a g e

arowanas produce relatively few (20-80), extremely large eggs (12-15 mm in diameter) and they are mouth-brooded by one of the parents for 40-50 days. It is not surprising why the Asian arowana is not well-studied: its late maturation (36- 60 months), long generation time, low fecundity, lack of sexual dimorphism [18] and restriction in sample collection in the wild and most importantly, the difficulties involved with its culture all make the analysis of the species very difficult. In addition to this, conventional breeding occurs regularly only in earthen outdoor ponds and not in artificial holding tanks, with extreme rare exceptions [19]. Moreover, documentation through direct observations of the temporal and spatial scope of the arowana breeding activities is almost impossible in the dense pond water. There is no documentation of artificial fertilization of this species, probably due to the large eggs and low fecundity and lack of available specimens. Such lack of basic biological information of the Asian arowana creates a bottleneck for the study of its phylogeny, behaviour, population structure and genetics, demographics and most importantly its reproductive biology. These also indirectly compelled us to study the mating system using molecular tools.

The Asian arowana is one of only four freshwater teleost species that are protected under CITES (Convention on International Trade in Endangered Species of Wild

Fauna and Flora) Appendix I [20], and the International Union for the Conservation of

Nature and Natural Resources (IUCN) Red List of Threatened Species. It is also listed as endangered in the Endangered Species Act (ESA) [21] and is the only teleost species under CITES appendix I that is commercially cultured in CITES-licensed

31 | P a g e

farms solely for ornamental purpose. Even to this day, importation of the Asian arowana to Australia and North-America is still prohibited [22].

The study of teleost breeding behaviours has been of interest mainly with regards to mate choice and mating strategy. Molecular analyses on mate choice, mating strategies and parental care in teleost fishes have primarily been focusing on mating behaviour involving parental care [16] and most studies have been focusing on

“model” teleosts, such as three-spined stickleback (Gasterosteus aculeatus,

Gasterosteidae), guppy (Poecilia reticulate, Poecilidae) and members of the family

Cichlidae [16,23]. To the best of our knowledge, no similar work has been done on the other three CITES appendix I protected freshwater teleost, namely, the Isok barb

(Probarbus jullieni), the Cui-ui suckerfish (Chasmistes cujus) and the Mekong river catfish (Pangasianodon gigas). Similarly, there are no detailed genetic studies of mate choice, mating system, and parental care in any primitive teleosts, particularly the subfamily Osteoglossinae. We hope that our work will enhance the understanding of the evolution of the breeding biology of teleost, and provide a genetic glimpse into the biology of ancient teleosts using molecular tools.

32 | P a g e

Figure 3: The architecture of the Asian arowana’s buccal cavity and the tongue bite apparatus (TBA). TBA is thought to be a derived feature of the Osteoglossomorpha used for anchoring the prey to the buccal cavity. Labels: A) Teeth on the medial edge of entopterygoid; B) Basihyal/basibranchial toothplate.

1.3 The Asian arowana has at least six main colour strains

One interesting and distinct difference between the Asian arowana and its related species in the family Osteoglossidae is the presence of naturally occurring colour strains in the former. Unlike the rest of the family that only have one colour form, the

Asian arowana possesses at least six known naturally occurring colour strains (Table

2). These colour strains have distinct phenotypic differences after maturation and in their natural habitat and they are isolated geographically (see Fig. 4 and Table 2). The

33 | P a g e

colour strains include firstly, Red Grade 1 (RG1) (Fig. 5A and 6A). Mature individuals of this strain show an overall distinct red colouration on all scales (Fig. 7A and 7E) and fins and they are probably the largest among all in terms of attainable overall size and weight. Interestingly, they are only found in Kalimantan (Indonesia), which is part of an island called Borneo and explicitly in the Kapuas River and the

Sentarum Lake. The Red Grade 2 (RG2; Fig. 5B and 6B), found only in Banjarmasin district on Kalimantan island (Indonesia), received its name as it is very similar phenotypically to RG1 during the first 3-5 months. Surprisingly, at adulthood, the

RG2 turns to a dull brown matt colouration with light thin pink striations throughout the body and the finnage will develop to a yellowish red colouration. Thus, it is also known as the “fake” RG1 arowana. The RG2 is often passed off as RG1 by unethical arowana dealers, especially when illegal trading of this fish is concerned.

The Malaysian golden (MG; Fig. 5C, 6C, 7B and 7F), also known as the crossback golden, is native to Bukit Merah Lake in Taiping, Perak (Malaysia) [24]. Maximum attainable weight and size for the MG is probably the smallest among the colour strains. They are characterized by the presence of “golden” scales throughout the body and gold striations on all the finnages. The “golden” scales are formed by the reflective yellow iriodiophores present in the scales (Fig. 7B). The Indonesian golden

(IG; Fig. 5D, 6D, 7C and 7G), also known as the red-tail golden arowana is found in

Pekanbaru, on Sumatra island (Indonesia). IG is phenotypically very similar to the

MG, the only distinct difference is the distribution of the iridiophores on the scales of the fish (compare Fig. 7G and 7H), the IG only has “golden” scales up to the fourth rows of scales (from bottom up), whereas the MG has the golden scales “crossing”

34 | P a g e

over the dorsal side of the fish, thus, it is also known as the crossback golden

(compare Fig. 5D and 5C).

Figure 4: The colour varieties are located in different geographical locations in Southeast Asia. Labels: RG1 – Red Grade 1 (red); MG – Malaysian Golden (blue); IG- Indonesian Gold (brown); RG2 – Red Grade 2 (black); GR- Green (green); Other than the GR, the other colour varieties are endemic only to one geographical location. The GR is found in Cambodia, Peninsular Malaysia and Indonesia

35 | P a g e

Table 2: The different colour strains of Asian arowana found across the islands in Southeast Asia and one of their hybrids

Colour Abbreviation Country Known Location/s Region/s Reference/s strains

Green GR Cambodia/ Cambodia/Peninsular Koh Kong/Trengganu, [19,25,26,27] Malaysia/ Malaysia/Sumatra/ Johor, Pahang/Jambi/ Indonesia Kalimantan Samarinda

Malaysian MG Malaysia Peninsular Malaysia Taiping, Perak [24,27] golden

Indonesian IG Indonesia Sumatra Pekanbaru [7,28] golden

Red grade RG1 Indonesia Kalimantan Pontianak, Kapuas river [7,28] one

Red grade RG2 Indonesia Kalimantan Banjarmasin [29] two

Mensiku MJK Indonesia Kalimantan Pontianak, Kapuas River [19]

Hybrid* TY Singapore Singapore Singapore [19]

36 | P a g e

A D

B E

C F

Figure 5: The presence of different natural colour varieties of Asian arowana is unique compared to the rest of the subfamily Osteoglossinae. Panels: A) RG1; B) RG2; C) Malaysian Gold (MG); D) Indonesian Gold (IG); E) Tong Yan Hybrid (TY); F) Green (GR). Please see Figure 4 for labels.

37 | P a g e

A D

B E

C F

Figure 6: The juveniles of all the colour stains are phenotypically very similar. Panels: A) RG1; B) RG2; C) MG; D) IG; E) Tong Yan hybrid (TY); F) GR. Please see Figure 4 for labels. 38 | P a g e

Figure 7: The scales of each colour variety contain different chromatophores which strongly contribute to their unique phenotype. Scales of four different colour varieties were viewed under stereomicroscope (20X magnification). Panels: A) RG1; B) MG; C) IG; D) GR. Second set of panels with the respective close-up using a DLR camera‟s macro function of the scales: E) RG1; F) MG; G) IG; H) GR.

In the early 90‟s breeders in Singapore have successfully hybridized the RG1 and the MG and produced a hybrid named Tong Yan (TY; Fig. 5E and 6E). This hybrid is fertile and some of its individuals combine the desirable phenotypes from the two colour strains: huge attainable size, big and long fins of the RG1 and the intense colouration of the MG.

One of the shortfalls of the hybrid is the large variation of phenotypes produced in their offsprings which is common in hybridisation. (Unfortunately, a paper was published a few years ago stating that the colour strains are species [178]. However, as the data used by them is limited and the results do not seem to be convincing, therefore I will still consider them as colour strains.) Green arowana (GR; Fig. 5F, 6F, 7D and 7H), which has the widest geographical distribution, is found throughout Pekanbaru and Sumatra in

39 | P a g e Indonesia, Terengganu, Pahang and Johor in Peninsular Malaysia, Thailand, Vietnam and

Myanmar [7]. The GR phenotype is characterized by the matt grayish green overall colouration and the presence of green “horse shoe” markings on the scales (see Fig. 7H for a close up of the scale). According to farm operator, there are slight phenotypic differences between the GR from difference geographical locations [19].

Within the colour strains of the Asian arowana, there are large differences in terms of their commercial value as an ornamental fish, ranging from S$ 100-S$ 5,000 per fish at market size (ca. 15 cm TL). The commercial value of young juveniles is in the following order: RG1, MG and TY are the most expensive, followed by IG, RG2 and GR. The time of evolutionary divergence for the different colour strains has not been fully elucidated.

Kumazawa and Nishida [30] used a few samples of unknown origin to study the phylogenetic relationship between Asian arowana and the other family members, thus their study does not provide indication on the phylogenetics of the colour strains. This is therefore an interesting aspect that is worth working on. Small isolated populations due to geographical barriers and adaptation to new environmental conditions may be the cause of the distinct phenotypic changes observed in each strain, including fins shape, body shape, and scale colour. One important point to note is that the colour strains are able to interbreed and produce fecund F1 generations [19].

Due to the rise in the number of arowana farms in the region during the recent years, most of the colour strains can be found anywhere in the world in captivity. As for the wild population, there is no data to show whether the alien strains have infiltrated into

40 | P a g e their non-endemic habitats nor do we have information on the current status of the

endemic population.

At the juvenile stage (TL: 14-18 cm) when the fishes are normally sold, the different

colour strains are very similar, virtually undistinguishable morphologically (see Fig. 6).

In addition, arowanas change their body and finnage colouration to merge with the

surrounding aquatic environment quite readily. For example, in response to a white

background with strong lighting, the fish will “bleach-out”, forming a very white based

body and finnage colour, thereby adding to the difficulty in recognizing the colour strains.

Records have shown that occasionally deliberate or accidental mix up of the juveniles

happens. These pose a serious problem to the consumers as well as the farmers, resulting

in monetary losses. Commercially, there is therefore a need to develop a molecular

marker set for the differentiation of the major strains in order to assist in their

identification and prevent unnecessary dispute. Such strain-specific molecular markers

may also help to establish a molecular based barcoding system to ensure legal trading of

this endangered species into countries otherwise not permitted to be imported.

1.4 Sex and strain identification of teleosts using classical tools

Asian arowanas lack distinctive morphological signs of sexual dimorphism through all of

their life stages. Farmers observed subtle differences in the mature adults: males are

typically bigger than females, and males tend to have a longer and wider head, providing

a bigger buccal cavity to hold the eggs. These observations have not been verified

scientifically and are based on assumptions that the male is the mouthbrooder and the

dominant sex. More often than not, these predictions are not more than fortunate guesses.

41 | P a g e The lack of an easy and reliable sexing method is one of the bottlenecks in the culture of the species. Without knowing the sex of the fish, broodstock selection and management becomes skewed to selection by desired phenotype, such as colour and body shape. In addition, the sex ratio cannot be determined in the breeding ponds, leading to decreased and unpredictable production rate (or absence) in many cases. There are two approaches that are used by the regional farmers: one is to stock relatively large ponds of about 1-2 hectares in size with 100-200 brooders and harvest them twice a year. This practice is common in Indonesia and some parts of Malaysia. The other method involves the use of smaller ponds with fewer brooders (20-30), harvesting once every two months and

“rearranging” the brooders after one year if there is no production (Singapore, Malaysia and Indonesia). Neither of these methods is economical and practical, and no breeding program or broodstock enhancement can be performed for the species due to the reasons mentioned above. Therefore, there is a strong commercial need to develop a viable sexing method, preferably a molecular sex marker that can be used at any stage during the development of the fish. For basic research, it is also important to have a sex marker as knowing the gender will assist in our studies, especially in experimental setups.

1.4.1. Morphometry

Morphometric measurements have been used widely to compare isolated populations of individuals of the same species [31,32,33], predicting biomass of fishes [34], and also along with other parameters to determine sex and maturity of fishes. Although the Asian arowana is known to be sexually monomorphic, nonetheless subtle signs of sexual morphometric dimorphism still exist. The species is thought to practice paternal

42 | P a g e mouthbrooding and farmers have made assumptions that due to the fact that the male needs to hold the huge, delicate eggs in the buccal cavity, there should be morphological difference on the head region.

The unique presence of parasphenoid-tongue bite apparatus (TBA) in the

Osteoglossomorpha was first described by Greenwood et. al [35] and is thought to be a derived feature for Osteoglossomorph fishes [36,37]. TBA encompasses two main groups of teeth (see Fig. 3), one that is located on the base of the skull and palatal region

(also known as entopterygoid teeth), and the other located on the hyobranchial apparatus or parasphenoid (or commonly known as the bonytongue) which is located at the floor of the mouth [36]. The TBA enables the arowana to use the toothed palatal bone

(basibranchial toothplate or entopterygoid teeth) and bite against the parasphenoid tongue

(see Fig. 3) when shearing a prey. Although TBA is not restricted to Osteoglossomorpha, this is the only family where members have a sharp dentition on the parasphenoid [38].

1.4.2. Biochemical methods

There are several types of changes that occur in teleosts in preparation for mating, ranging from physiological to behavioural ones. Most processes leading to these changes are initiated by secreted molecules called pheromones and many of the fishes use these to establish hierarchical division in social groups, to achieve dominance and to obtain the right to mate. This is especially true in an environment where visual cues are limited and sound frequency is relatively impaired [39,40,41]. These pheromones are known to be hormonally controlled and water-soluble steroids [42,43]. The sexual maturation of teleosts marks the endpoint for the gonad development during sexual differentiation

43 | P a g e [44,45] and sex steroids are known to be directly involved in the differentiation process as morphogenic and induction factors [46]. During sexual maturation, sex steroids also become activational factors [47].

Sex steroids act through specific receptors in targeted cells and these are well documented in fishes [45,48]. For example, estrogen receptors have been cloned from species like tilapia [49] and goldfish [50], whilst androgen receptors from trout [51] and zebrafish [52]. The level of sex steroids that circulate in the fish are maintained by catabolic and anabolic processes, as well as solubilization and excretion into the environment [53].

The two classes of sex steroids, namely androgens [54]and estrogens [55]are important for the development of secondary sexual characteristics and regulation of reproductive behaviour in teleosts [45] and they are not exclusively found in one gender only. There is also another class, the progestogens or progestins [55,56,57,58], which may be involved in the regulation of reproductive behavior in teleosts and several pheromones belongs in this class. 11-ketotestosterone (11-KT) is the major androgenic steroid with an important role in testicular development, particularly in spermatogenesis and secondary sexual characteristics in male teleost species [58]. It is also known that 11-KT is synthesized from testosterone via two enzymes that act sequentially in Leydig cells [48,59].

Literature shows that the 11-KT peaks at the height of the spawning season for many teleosts [56,60]. Estrogen is the feminizing hormone in teleost and endogenous level of oestrogens is involved and responsible for the differentiation of the ovary and again. The aromatase enzyme is a member of the cytochrome P450 superfamily whose function is to

44 | P a g e produce estrogens by aromatizing androgens. As such, it is an important factor in sexual

development and plays a central role in the differentiation process [58]. Since androgens

and estrogens are known to be excreted into the environment, we decided to test the

presence of these androgens or estrogens in the surface mucus of the fish using a

commercial ELISA (enzyme-linked immunosorbent assay) kit.

1.5 Mating system and parental care in fishes

There are more than 25,000 species of teleosts existing in diverse aquatic habitats [61]

from marine water (58%), to freshwater (41%) and the rest (1%) migrating between the

two (also known as euryhaline or anadromous/catadromous migratory fishes). The

freshwater species occupy only 0.01% of the world‟s total water supply, thus their

volume-based species diversity is almost 7500 times more than the marine species [62].

The diversity of mating systems in teleosts is immense. They range from monogamous

mating system to group spawning involving cooperation between the parents [16].

Parental care (post-zygotic service) is absent in 79% of the taxonomic families, only

approximately 90 out of the 413 taxonomic families exhibit some form of parental care.

This phenomenon is in contrast to that observed in mammals and birds in which parental

care predominates (70% ) [16,23]. The “variety” of parental care seen in teleosts is

probably the most diverse of all vertebrate groups, ranging from mouthbrooding,

viviparous, ovoviviparous to constructed nest [16,17].

To date, numerous species‟ mating systems have been evaluated in teleosts, but none of

the species falls in the superorder Osteoglossomorpha [16]. A large proportion of the

work has been done on sunfish (Lepomis sp.) [63,64], livebearers [65,66], and cichlids

45 | P a g e [67,68]; this is due to their short life cycle and their ability to be easily contained in a laboratory tank setup for experimentation.

1.5.1 Types of mating systems

A mating system refers to the system in which an animal population or society structures itself in terms of sexual behaviour and reproduction [69]. The mating system specifies which males mate with which females under which circumstances. A mating system encompasses three components: pre-fertilisation reproductive behaviour (eg. competition in obtaining a mating partner), mating modes (monogamy, etc) and parental care [70].

Monogamy, also referred to as “pair bonding”, is a mating system where a male and a female have only one exclusive breeding partner. Polygamy refers to one or more males having one or more female breeding partners [71]. There are three main types of polygamous relationships. Polygyny, which is most common in vertebrates, refers to a mating system where one male mates with two or more females. Polyandry refers to one female mating with two or more males. Polygyandry refers to two or more males mating with two or more females where the numbers can be unequal. Lastly, promiscuity refers to a system where any male can mate with any of the available females [15]. Some species of reptiles show different mating systems in different circumstances, for example in painted lizards (Ctenophorus pictus), different colour morphs employ different mating systems [72] and in St. Peter‟s fish (Sarotherodon galilaeus), the male employs different mating systems that correlates to its androgen levels [41].

46 | P a g e 1.5.2 Parental care in teleosts

Parental care in this context refers to the post-fertilization care given to the offspring, in order to increase its fitness [73] and presumably, to increase the chance for passing on its genetic inheritance to the next generation. One important point to note is that the parental care involved is tightly linked to the mating system the fish exhibits [16,70,74]. A wide variety of parental care exists in teleosts, and often these investments involve high “cost” to the parents of both sexes [75]. Typically, parental care involves paternal care [16], but care can also be given maternally [68,76] or biparentally [17,77]. Whilst these are all logical steps to ensure fitness for the next generation, there are also alternative reproductive tactics [16,17,70,77,78,79] which are plastic, depending on the availability of resources [74] or type of threats present (eg. predation), the age [70,78] or size [80] of the parents and alloparental care where “foster” parental care is provided [74,81,82].

Almost all species under the family Osteoglossidae practice some form of parental care

(Table 3), ranging from nest guarding in Heterotis [83,84,85,86] and Arapaima

[84,85,86] to mouthbrooding in the genera Osteoglossum [87,88] and Scleropages

[5,6,87,88,89,90,91,92,93]. Despite of their evolutionary and biological significance, no extensive study has been performed on the mouthbrooding practices of the members of this family. Only a few short statements have been mentioned by farmers through visual observation and deduction from experience.

As far as the literature and personal communication with the farmers are concerned (see

Table 3), the gender of the mouthbrooder parent in Asian arowana is not confirmed and it is also interesting to note that the closest relative of the Asian arowana [8,10,30], the two

47 | P a g e Australian species, S. jardinii and S. leichardti, both practice female mouthbrooding

[90,91].

Table 3：The mating systems and types of parental care observed in the family

Osteoglossidae are variable

Species Parental care/gender of References mouthbrooder

Scleropages Mouthbrooding/unknown, male or [89,94] formosus female

Scleropages Mouthbrooding/female [6,90,91] jardinii

Scleropages Mouthbrooding/female [90,91,93] leicharti

Osteoglossum Mouthbrooding/male [87] bicirrhosum

Osteoglossum Mouthbrooding/unknown [88] ferrarai

Heterotis niloticus Nest guarding [83,84,85,86]

Arapaima gigas Nest guarding [95,96]

48 | P a g e 1.6 Molecular approaches in fish biology and aquaculture research

The combination of classical long-term field studies and the use of polymorphic DNA markers could significantly improve our understanding of the breeding behaviour of the Asian arowana. DNA markers substituted allozymes [97] as the most powerful tools in genetic research in the mid 1980s, due to the higher variability of the genome at the DNA level. Fish geneticists rapidly adopted them as one of the most powerful tools in the genomic research of aquatic organisms [98].

Genetic variations can include: base substitutions, commonly referred to as single nucleotide polymorphisms (SNPs), insertions or deletions of nucleotide sequences within a locus, inversion of a segment of DNA within a locus, and rearrangement of

DNA segments around a locus of interest [99]. Large deletions and insertions (known as indels) result in the size change of DNA fragments containing these regions following restriction digestion. These mutations can be detected easily by electrophoresis of the fragments on an agarose gel, whereas for the identification of smaller changes (e.g. SNPs), more elaborate techniques such as capillary electrophoresis used for DNA sequencing will be required [100].

DNA-based molecular marker types include restriction fragment length polymorphisms (RFLPs), variable number of tandem repeat (VTNR) markers, single- stranded conformation polymorphism (SSCPs)[101], amplified fragment length polymorphisms (AFLPs)[102], random amplified polymorphic DNA (RAPD)

[103,104,105,106,107], simple sequence repeat (SSR) markers or microsatellites, single nucleotide polymorphisms (SNPs) and expressed sequence tags (ESTs)

[17,77,108]. The ultimate aim of using these markers is to detect differences in DNA

49 | P a g e sequence between homologous genomic regions [109]. Table 4 summarizes the basic properties of the most popular DNA markers.

These DNA-based techniques have already been used for a wide range of aquaculture species and applications, including species/strain identification and pedigree tracing

[110], confirmation of isogenicity in products of chromosome-set manipulations and clones [111,112], linkage mapping [113], identification of QTLs[114], and other uses in breeding programs [109].

These techniques provide useful tools for studying sex linkage in fish since DNA structure will not change with altered physiology or environment. Furthermore, examination of DNA sequence organization on sex chromosomes can provide insights into the evolutionary processes that are operating to influence sex chromosome structure, and ultimately, can yield information on the conservation (or lack thereof) of sex-determining processes among species. Due to the variability in sex chromosome organization in fish, the number and type of loci involved in sex determination can be variable among and within species [45,115,116] and, accordingly, sex linked markers are laborious to isolate [117].

50 | P a g e Table 4: Types of DNA markers, their characteristics and potential applications.

Marker type Acronym Require Locus under Likely allele Polymorphism Major applications prior investigation no or power molecular info Restriction Fragment RFLP Yes Single 2 Low Linkage mapping Length Polymorphism Random Amplified RAPD No Multiple 2 Intermediate Fingerprinting for population Polymorphic DNA studies, hybrid identification Amplified Fragment AFLP No Multiple 2 High Linkage mapping, population Length Polymorphism studies

Fluorescent Motif FluoMEP No Multiple 2 High Population studies, isolation of Enhanced biomarkers Polymorphism Microsatellites SSR Yes Single Multiple High Linkage mapping, population studies, paternity analysis Expressed Sequence EST Yes Single 2 Low Linkage mapping, physical Tags mapping, comparative mapping

Single Nucleotide SNP Yes Single 2, but up to 4 High Linkage mapping, population Polymorphism studies

Insertions/deletions Indels Yes Single 2 Low Linkage mapping,

51 | P a g e Recently, the first sex-determining gene has been identified from a non-mammalian vertebrate, the teleost fish medaka [118,119]. The role of this gene, called dmY/dmrt1Y, is comparable to that of the Sry mammalian testis-determining gene.

However, it is not the universal primary sex-determining gene in fish [115]. A repeat sequence termed Bkm, isolated as a minor satellite DNA component from banded krait snake [120] and associated with the W and Y chromosomes of several organisms, has been used as a probe to look for sex-linked markers in fish. Similarly to previous markers, it is present in the genome of rainbow trout [121], channel catfish [122] and a coral reef fish, Anthias squamipinnis [123], but is not sex-associated. Other repeat sequences that have been found to be tightly associated with the sex determination locus are a microsatellite marker in brown trout, Salmo trutta [124] and lake charr

Salvelinus namaycush [125].

A second approach widely used in fish is the screening of whole genomes for sex or colour-strain associated DNA markers, by using basically two techniques, which do not require prior information about the organism. The Random Amplified

Polymorphic DNA (RAPD) [126,127] and the Amplified Fragment Length

Polymorphism (AFLP) [128] techniques, have allowed the identification of sex-linked markers in several fish species such as rainbow trout [117,129], African catfish [130],

Nile tilapia (Oreochromis niloticus) [105] and three-spined stickleback [131].

Genetic mapping studies are beginning to provide a wealth of markers for segregation studies in fish, allowing for the localization of DNA sex-associated markers which could indicate the localization of the sex-determining region. Genetic linkage maps are constructed by assigning polymorphic DNA markers (such as microsatellites,

SNPs or AFLPs) to chromosome configurations based on their segregation

52 | P a g e relationships [100]. This requires two elements: polymorphic DNA markers, and families in which these markers segregate. Once a linkage map has been constructed for a given species, it can be used in combination with studies of breeding and assessment of quantitative traits to identify markers that are closely associated

(linked) to the quantitative trait loci (QTL) of interest, thus allowing the QTL to be positioned on the linkage map [132]. In tilapia, for instance, QTLs associated with sex have been identified recently [133].

Even though application of these methods in teleosts is less frequent than in mammalian and avian taxa, several teleosts that are commercially important food fish are being studied. Microsatellites have been shown to be extremely well suited for the study of parentage and also in the evolutionary aspect of breeding patterns in fishes,

They have been proven to be a powerful approach to resolve genetic mating systems, mate choice and kinship in teleost [16].

1.6.1 Microsatellites

Microsatellite loci (Short Tandem Repeats, or STRs) are short, tandemly repeated

DNA sequences that are distributed randomly throughout the genome

[108,134,135,136]. They have been shown to be excellent candidates for the study of parental care, genetic mating system, parentage analysis and kinship studies [16,17].

Microsatellites consist of short, usually 1-6 bp core sequences in tandem repeats (eg.

CA repeats, GATA repeats) or in arrays [99] (Fig. 8). Microsatellites are thought to be very abundant and well distributed throughout the whole genome including coding and non-coding regions. Their frequent occurrence (one SSR every 10 kb) makes their isolation easier. Isolation methods often involve an enrichment step before cloning, followed by sequencing the “enriched” sequences and designing primers that target 53 | P a g e the flanking regions of these SSRs [136]. Amplified microsatellites are often small,

100-500 bp fragments; this characteristic allows them to be favoured in a PCR reaction where reagents are relatively expensive and time consuming. Polymorphism in microsatellites is related to the length of the repeats contained; the more polymorphic ones tend to be generally higher in number of repeats. Thus, an individual‟s polymorphism is based on the size differences due to the different number of repeats at a given locus. These differences in the number of repeats are thought to be caused by DNA slippage which involves transient dissociation of the replicating DNA strands with subsequent re-association during DNA replication

[137], or due to unequal recombination during replication [134] which resulted in the hypermutability of DNA microsatellites, thus causing the hypervariability in not just species but even within populations. The microsatellite mutation rate is estimated at

10−2–10−6 per locus per generation [108], compared to 10-9 in non-repetitive DNA

[138].

One of the most important characteristic features of microsatellites is that they are inherited in a Mendelian fashion as codominant markers [100].

54 | P a g e

Figure 8: Inheritance of a single microsatellite locus from the parents to their offspring. Microsatellites are amplified by PCR, using the unique sequences of flanking regions as primers. The amplified DNA is then detected using the 3730xl DNA Analyzer (ABI, Foster City, CA, USA) and peak profiles were presented using a software GeneMapper software V3.5 (Applied Biosystems). From the peak profiles, Offspring #1 have inherited the larger allele from parent A and the smaller allele of parent B leading to a homozygous genotype, where else offspring #2 inherited the smaller allele of parent A and larger allele of parent B resulting in a heterozygous genotype.

1.6.2 Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) [126,127] is a method which employs short, arbitrary primers (8-12 primers) per reaction, at relatively low annealing temperature, separation of the PCR products on agarose gel by using a horizontal

“submarine-type” electrophoresis device and visual analysis of the resulting band pattern. A number of RAPD variations have been published showing the usefulness of

55 | P a g e this technique [127,139,140]. This method is a popular method for comparing the genome of non-model organisms. Compared to traditional PCR analysis, RAPD does not require any specific knowledge of the DNA sequence (blind screening) of the target organism. The random primers may or may not amplify a segment of DNA, depending on whether the primer positions are complementary to the primers' sequence. Thus, no fragment is produced if primers anneal too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product would not be generated, resulting in a different pattern of amplified DNA segments on the gel.

Most users of the RAPD assay use the procedure proposed by Williams and colleagues [141] but the method was actually co-discovered by Williams and colleagues and Welsh and coworkers [126,141].

Despite its usefulness, there are still limitations to the use of this assay, particularly the lack of resolution in the analysis. Improvements to this bottleneck were made by extending or varying the coverage of the assay to include an additional primer to the reaction: a second RAPD primer [142,143,144], a longer specific primer [145,146] or a microsatellite-derived primer [147,148,149]. Some findings have demonstrated the advantage of including a re-synthesized RAPD primer with fluorescent labels, allowing for separation and detection of the resulting patterns on DNA sequencers

[150]. However, none of these methods investigated the large-scale use of commercial

RAPD primers together with high-throughput procedures of separation and detection.

56 | P a g e 1.6.3 Amplified Fragment Length Polymorphism (AFLP)

The Amplified Fragment Length Polymorphism (AFLP) technique is a PCR-based, multi-locus fingerprinting method that combines the specificity, resolution and strength of Restriction Fragment Length Polymorphism technique (RFLP) [151] with the speed of detection and effectiveness of polymorphisms by PCR, such as RAPD

[126,127]. Moreover, it overcomes the problems of both methods, as it requires no probe or previous sequence information as needed by RFLP and it is reliable because of highly stringent amplification conditions in contrast to the problem of low reproducibility of RAPDs [100,152].

The molecular basis of AFLP polymorphisms includes deletions and insertions between restriction sites, base substitutions at restriction sites and also base substitutions at PCR primer binding sites. The specific feature of the technique is the ligation of synthetic double stranded linkers of known sequence (adaptors) to DNA fragments generated by digestion of whole genomic DNA. This allows for the subsequent PCR amplification of a subset of the total fragments for their easy separation by gel electrophoresis. First employed by Vos et al., 1995 [128], the technique consists mainly of four steps.

AFLP fragments are inherited in a Mendelian way, where each band is considered a bi-allelic locus and scored as present/absent, although software packages are available for co-dominant scoring of AFLP bands [100].

The power of AFLP analysis is high for revealing genomic polymorphisms, but their number depends on the relationship between the number of selective nucleotides added to the primers designed from the adaptors sequence and the complexity of the

57 | P a g e genome under study. More complex genomes require a higher number of selective nucleotides, and when the genomic complexity of the organism is not well defined, this number should be experimentally determined. Moreover, since different enzymes can be used to scan genomes, the potential power of the technique is practically endless, although a full scan using all existing primer combinations would be prohibitively time-consuming and expensive.

58 | P a g e 1.6.4 FluoMEP assay

We described a new genotyping method, called Fluorescent Motif Enhanced

Polymorphism (fluoMEP) in 2007 [153]. The fluoMEP method is based on RAPD, but combines the advantages of the large collection of unlabeled 10mer primers (ca.

5,000) from commercial sources and the power of the automated capillary electrophoretic (CE) devices used for the detection of AFLP patterns. The linkage between these two components is provided by a fluorescently labeled “common primer” that is used in a two-primer PCR together with an unlabeled RAPD primer.

The result contains a mixture of labeled and unlabeled products, out of which only the former are detected by the CE device (Fig. 9). By screening with the same “common primer” and a series of RAPD primers templates can be screened rapidly and efficiently.

59 | P a g e

Figure 9: A schematic representation showing the difference between RAPD and FluoMEP. RAPD typically uses one short, unlabelled primer (half-arrow). The amplified band pattern is separated on agarose gel, detected by ethidium bromide staining and analyzed by visually comparing the patterns. The typical number of bands varies between one and a dozen. FluoMEP uses fluorescently labeled “common primers” (half-arrow with star) with the potential ability of targeting frequent motifs (rectangle) in the genome together with the short, unlabelled RAPD primers. The PCR product of FluoMEP contains three different kinds of bands amplified by: i) the common primer only; ii) the common primer and the RAPD primer together; and iii) the RAPD primer alone. The pattern is separated by capillary gel electrophoresis, and only the fluorescently labeled bands at one or both ends are detected. The typical number of bands varies between 10 and 100. (Figure 1 of Chang et al., Electrophoresis 28; 525-534, 2007 [153].

60 | P a g e 1.7 The aims of my research

Despite the fact that Asian arowana is a highly endangered species, very little is known about their reproduction patterns and life history. My investigations aimed to improve our understanding of the reproductive biology of the Asian arowana

(Scleropages formosus) with special emphasis on the breeding relationship, mouthbrooding process and phylogenetic relationships using molecular and biochemical approaches (described above). I was particularly interested in understanding the mating system, breeding behaviour, phylogeography, as well as differences between strains, and genders of the Asian arowana and comparing them with those of its close relatives.

In view of the above, the objectives of my study were the following:

i) Use microsatellite genotyping to understand the breeding relationship between

the brooders and construct the genetic mating system of Asian arowana by

assigning parentage to the offspring produced.

ii) Develop sexing methods for adult brooders

iii) Use mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs)

to determine the gender of the mouthbrooding parent of S. formosus.

iv) To gain better understanding of the mouthbrooding process of this species.

v) To attempt to understand the phylogenetic relationship and genetic differences

between the colour strains using AFLP and microsatellite genotyping.

61 | P a g e 2 MATERIALS AND METHODS

2.1 The origin of fish studied

2.1.1. Wild stocks from Kalimantan, Indonesia

Samples from wild-caught Red Grade 1 (WRG1) individuals were collected from a

farm in Pontianak, Kalimantan. These individuals were captured 34 years ago when

the farm started operation from the Kapuas River. Since then, they were stocked as

brooders in the farm. We managed to collect the fin clips and scales from 22 adults

with permission from the owner of the farm. The fin clips and scales were kept in

absolute ethanol as described in section 2.4.

2.1.2. Broodstock from QianHu fish farm

When the project started in 2004, we started collecting fin clips from all the

broodstock of the QianHu fish farm (from here onwards, QH). The broodstocks

consisted of different colour strains, namely, RG1, Malaysian Golden (MG), Hybrids

(TY) and Indonesian Golden (IG). New broodstock recruitment also started in 2006,

when the farm started to purchase younger broodstocks (3-4 yrs old). To date, we

have collected over 600 broodstock samples with the method described in section 2.4.

There is no background information on the exact origin of these broodstocks, as they

were mainly purchased as young adults from Indonesia and Malaysia during 1990-

1994. The broodstock in QH were all sorted strictly according to the colour strains.

We have collected scale samples from all the offspring that have bred in the farm.

During harvesting, any batch of offspring harvested from the mouthbrooding

individual was verified with its Passive Integrated Transponder (PIT) tag number and

each of the offspring batches were kept isolated until they were ready to be tagged. 62 | P a g e Sample collections of the offspring were done during their tagging and the procedure was described in section 2.4. We define “brooders” as the fish that we used for the breeding in the ponds and “mouthbrooders” as the individual that has been found mouthbrooding eggs at one point of time.

2.2 Arowana breeding and holding facilities in QH

Our target individuals were kept in six earthen ponds consisting a total of 122 adult brooders (five to ten years old F1 captive bred stocks). Ponds are 25m (W) X 40m

(L) X 3m (D) with dikes that are 3 m wide and their slope is maintained at a ratio of

2:1.5. The water inlet PVC pipes of each pond are situated at the width of the pond and the outlet is situated at the diagonal end of the inlet. Ponds do not have any mechanical aeration; water is only topped up with stored rain water if the water level falls below 2 m and water changes occur only during the rain. Brooders were all tagged with a PIT tag at the dorsal muscular tissue. The identity of all individual samples was verified by a PIT tag reader during sampling or harvesting. All the 6 ponds were labeled and used for different experimentation as described in supplementary table 1.

2.3 Field Observations

Field observations were made daily starting at 0700 hrs to 0900 hrs and 1700 hrs to

1900 hrs (from June 2004 to July 2004) as this is the only time when the brooders rise to the water surface and also where the glare from the sunlight is at its minimum.

Observations were facilitated by wearing a pair of polarized sunglasses to cut off the glare from the water surface reflecting the sunlight. Briefly, the behaviour of brooders was categorized and recorded as mating approaches, descend, flaring, guarding and jerks. An approach was defined as the focal fish moving to within 5 cm beside another 63 | P a g e fish and if these two individuals sink together, it is termed as descends. Flaring is described as erecting of all the fins towards another fish. Guarding involves the mouthbrooding individual and presumably the mated partner where the mated partner circles around the mouthbrooding fish and exhibits flaring when another individual approaches. Jerks are typical courtship behaviour characterized by a distinctive approach toward another fish with abrupt starts and stops and erect fins.

Weather records from Jan 2003 to Dec 2006 were purchased from the National

Environmental Agency (NEA). The weather record contained rainfall and temperature records taken from a meteorological station situated approximately 122 m from the farming pond.

We were observing the fish under artificial conditions in captivity, therefore these observations may not truly reflect the actual behavior in theie natural habitat but all these observations are novel and very interesting and therefore worth reporting so as to have a better understanding of the biology of this fish.

2.4 Sample collection

2.4.1. Fin clips and scales collection

Caudal fin clips were collected from all the brooders using sterile scissors and kept in absolute ethanol in a 1.5ml Eppendorf tube for DNA extraction. To date, we have not experienced any mortality due to sampling. All the clutches of offspring collected from the ponds were documented and sampled by collecting one scale from each fish, which was subsequently kept in absolute ethanol and stored at 4oC. Offspring individuals were labeled by the insertion of a PIT tag during the scale collection. For

64 | P a g e the purposes of microsatellite-based genotyping, DNA was isolated from the scales as described in section 2.5 [154].

2.4.2. Mucus collection

External body mucus samples were collected from brooders of pond WH001 for the purpose of testing the level of sex steroids in it using a ELISA-based method (see section 2.7). During harvesting, brooders from the pond were collected using a plastic bag and placed in a glass tank. Fish were held against the wall of the tank by the farmer with the caudal peduncle about 10 cm above the water, and care was taken to ensure that there was no contact with the caudal peduncle. Without any anesthesia, mucus was collected by swabbing along the root of the caudal peduncle with autoclaved cotton swabs cut to 2 cm in length, immediately storing them in a 2 ml

Eppendorf tube and snap-freezing them in liquid nitrogen. The tubes were then immediately transferred to a -80oC freezer upon returning from the farm. Samples of mucus were collected (every two month once, for a period of one year) during harvesting of the pond and stored at -80oC until processing.

2.5 DNA isolation

Several methods of DNA isolation were used during the course of the study. Their use was dependent on the degree of purity of DNA required, speed of extraction and sample size.

2.5.1. Phenol-chloroform extraction method

This method was used for DNA sequencing and genomic cloning which require high degree of purity of DNA, long extraction time and small sample size. The procedure was modified from the protocol described in [155]. A pair of forceps was placed into 65 | P a g e absolute ethanol, then flamed and placed onto clean filter paper to allow the alcohol to evaporate for 15-20 sec. Fin clips or scales were removed by the flamed forceps and they were then transferred into a new tube containing 800 μl SET buffer (10 mM Tris-

HCl pH 8.0, plus 50 mM EDTA, 200 mM NaCl and 0.5% SDS), containing 250

μg/ml Proteinase K. The solution was shaken overnight at 55°C. Samples were then processed using the method described in Sambrook [155].

2.5.2. Chelex extraction method

This method was used for high-throughput genotyping and PCR as they were compatible with medium degree of purity of DNA. We used the protocol published by Yue et.al. [154] with slight modifications. Fin clips or scales (about 5 mm2 in area) were removed by flamed forceps The sample was then added into 150 μl of digestion solution containing 5% Chelex -100 resin (Bio-rad, Hercules, CA, USA) and 0.1 μg/ul of proteinase K (Roche, Mannheim, Germany) in distilled water.

The digestion solution was then incubated in a shaker incubator (Forma Scientific orbital shaker, Thermo Scientific Inc, Rockford, IL 61105 USA) at 55oC for 2 hrs, after which the solution was then heated to 95oC for 10 min to heat inactivate the proteinase K. The solution was then spun down at 14,000 rpm for 5 min and the supernatant was removed and kept separately and checked for quality and concentration by agarose gel electrophoresis.

The supernatant was kept in -20oC and could be used as a template for PCR reaction.

For each PCR reaction, 4 μl of template was used.

66 | P a g e 2.5.3. Sodium hydroxide extraction method

This method was used for genotyping and PCR as they were compatible with low degree of purity of DNA. This method was modified from Nathan et. al. [156]. Fin clips or scales (about 5 mm2 in area) were removed by flamed forceps from absolute alcohol and left on a clean filter paper for 10-15 seconds to allow the alcohol to evaporate. The tissue was then placed in an Eppendorf tube containing 200 μl of

50mM NaOH and heated to 95oC for 20 minutes. The tube was then immediately cooled to 4oC and 20 μl (1/10th volume) of 1M Tris-HCl (pH 8) was added to neutralize the solution. The tube was centrifuged at 14000 rpm for 5 min to pellet the debris and the supernatant was transferred to another clean tube and kept at -20oC for use in PCR.

2.6 Isolation and genotyping of microsatellites

Microsatellite isolation was done according to Yue et.al. [10,157] with some modifications described below. Approximately 500 ng genomic DNA was digested with RsaI, and then an adaptor produced by hybridizing 5‟-phosphorylated 25-mer and 21-mer oligonucleotides (and containing a MluI restriction site) was ligated onto the blunt ends (Edwards et al. 1996).

The ligation product was polymerase chain reaction (PCR)- amplified in 25 μL containing 10 mM Tris-HCl (pH 8.8), 150 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP, 10 pmol of the 21-mer oligonucleotide as primer, 250 ng template and 2.0 U

DyNAzyme II DNA-polymerase (Finnzymes).

The PCR products were then hybridized to a biotinylated (CA)10 probe in 6x SSC at

55°C for 20 min. One microliter of pooled sample was mixed with 10 μL Hi-Di

67 | P a g e formamide and 0.2 μL Genescan ROX-500 size standard (Applied Biosystems) and denatured for 5 minutes at 95°C. Capillary electrophoresis was performed using an

Applied Biosystems 3730×1 DNA Analyzer, and allele fragments were determined against the ROX-500 standard with GeneMapper software V3.5 (Applied

Biosystems). Only peaks that were spaced at intervals corresponding to the dinucleotide repeats were considered as possible alleles.

2.7 Detection of steroid hormone levels using an Enzyme Immunosorbent Assay

kit

After the mucus samples were collected and processed as described in section 2.4, they were thawed on ice and were diluted using 100 μl of sterile distilled cold water and centrifuged in a refrigerated centrifuge (Centrifuge 5417R, Eppendorf 10,000X g). The supernatant fluid from the mucus sample was then used for total soluble protein and 11-KT determinations.

11-KT in the surface mucus was determined using the commercial test kit for 11- ketotestosterone measurements (Cayman Chemical, Ann Arbor, MI), whereas estradiol-17beta (E2) was quantified by the Estradiol-17β ELISA test kit of the same manufacturer. The protocol provided by the kit supplier was used in both cases. The hormone levels were calculated as pg hormone per mg of total soluble protein (TSP, see section 2.8).

2.8 Bradford total soluble protein (TSP) Assay

The total soluble protein assay was performed by the Pierce Coomasie (Bradford) protein assay kit (cat # 23236, Thermo Fisher Scientific Inc, Rockford, IL 61105

USA). The protocol provided by the kit supplier was used.

68 | P a g e 5 dilutions (duplicates) of BSA standard were prepared (25μg to 2000μg) and absorbance was read at 595 nm after 10 min incubation at room temp. A standard curve was obtained by plotting absolute amounts of BSA against absorbance reading at 595 nm.

2.9 Amplified Fragment Length Polymorphism (AFLP)

AFLP analysis was performed using an AFLP plant mapping kit according to the manufacturer‟s protocol (ABI/PE, Foster City, CA, USA) with some modification

[128]. EcoRI was used as the rare cutter and MseI as the frequent cutter; selective amplification with EcoRI primer having three 3' selective nucleotides while MseI primer having either 3 or 4 selective 3' nucleotides. Several combinations of these conditions were tested, with the optimal combinations incorporating EcoRI and MseI restriction enzymes, 7 selective 3' nucleotides on MseI primer, and a selective touchdown PCR.

Genomic DNA (500 ng) was digested with 5 U each of EcoRI and MseI (New

England BioLabs [NEB], Beverly, MA) for 1 h at 37°C in a 40 µl reaction using manufacturers' recommendations. Adapters were ligated to restriction fragments in 10

µl of solution containing 5 pmol EcoRI adapter (5'-CTC GTA GAC TGC GTA CC-3' and 5'-AAT TGG TAC GCA GTC TAC-3'), 50 pmol MseI adapter (5'-GAC GAT

GAG TCC TGA G-3' and 5'-TAC TCA GGA CTC AT-'3), 1 Weiss U T4 DNA ligase

(NEB) and 1x T4 DNA ligase reaction buffer (NEB). Adapters were then added to 40

µl of restriction digest solution and incubated at 37°C overnight. Restriction fragments were amplified in 2 consecutive PCR rounds (preamplification and selective amplification) using a PTC 100 thermocycler (Bio-rad).

69 | P a g e The preamplification PCR was performed in 20 µl containing 2 µl of undiluted restriction fragment products, 1.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphates (dNTPs), 1 U Taq polymerase (Qiagen), 1x Taq buffer (Qiagen), and 75 ng each of EcoRI and MseI primers (5'-CTC GTA GAC TGC GTA CCA ATT-3' and

5'-GAC GAT GAG TCC TGA GTA AC-3', respectively) carrying one selective nucleotide. The preamplification PCR profile was as follows: 30 s at 94° C, 1 min at

56° C, and 1 min at 72° C for 26 cycles. The PCR product was diluted 1:10 with distilled H2O for subsequent use in the selective amplification.

The selective PCR amplification was carried out in 20 µl containing 3 µl of diluted template DNA, 1.5 mM MgCl2, 0.2 mM each dNTPs, 1 U Taq polymerase (Qiagen),

1x Taq buffer (Qiagen), 5 ng of fluorescently (Hex or Fam) terminally labeled 3+

EcoRI selective primers (LI-COR Inc.), and 30 ng of 3+ MseI selective primers (LI-

COR Inc). Selective amplification was performed as follows: 30 s at 94°C, 30 s at

65°C (first annealing step), and 1 min at 72°C. At each cycle the annealing temperature was decreased 0.7°C until 58°C, at which temperature 26 further cycles were conducted and labeled products were separated by capillary electrophoresis on

3730xl DNA Analyzer (ABI, Foster City, CA, USA) sequencing machine using 50 cm long 96-well capillaries filled with Pop-7 DNA analyzer polymer (ABI) gel matrix.

Samples were denatured by addition of Hi-Di formamide (ABI) at 95oC. Samples were introduced into the capillaries by using 15 sec injection time, and the separation was performed at room temperature.

Peaks were detected by the GeneMapper v3.5 software (ABI). Only the fragments between 50 and 500 bp were scored.

70 | P a g e 2.10 Fluorescent Motif Enhanced Polymorphisms (FluoMEP)

FluoMEP assays were performed as described earlier [153]. “Common primers” were designed against motifs frequently occurring in the genome. Potential target sequences included conserved splice sites [158], GATA/GACA repeats [159], and V-

SINE [160] sequences. Primers of 10-12mer length were designed against the most conserved parts of the target sequences by using the Primer Premier 5 software

(Premier Biosoft International, Palo Alto, CA, USA). All “common primers” were synthesized by 1stBase (Singapore) with either Fam or Hex fluorescent labels. RAPD primers were 10mer commercial primers from Operon Biotechnologies GmbH

(Cologne, Germany). One common primer (C117-modified V-SINE [160]) was tested against these RAPD primers ; AH03, AJ11, AK20, BH05, C05, C18, F04, H19, Y01, and Z13).

PCR products were separated either on 2% agarose gels in continuous TBE-based buffer using a horizontal submarine-type electrophoresis unit as described earlier

[161] or by capillary electrophoresis on 3730xl DNA Analyzer (ABI, Foster City, CA,

USA) sequencing machine using 50 cm long 96-well capillaries. In the latter case the preset conditions were used for the separation with 15 sec injection time. Peaks were detected by the GeneMapper v3.5 software (ABI).

2.11 Determining single nucleotide polymorphisms in the mitogenome to confirm

the gender of the mouthbrooder

In collaboration with Woei Chang Liew, we employed a novel approach to confirm the gender of the mouthbrooder in Asian arowana, S. formosus. The approach was based on the matrilineage of the mitochondrial DNA (mtDNA) in all vertebrates, with only 5 known exceptions where biparental inheritance is seen [162,163]. The 71 | P a g e mitochondria of the offspring are maternally inherited, thus, if the female and male breeding pair had polymorphisms between their mitochondrial DNA (eg. SNP in the genes), by comparing the mtDNA SNP haplotypes of the two parents, we were able to confirm the gender of the mouthbrooder.

We have designed flanking PCR primers based on the full-length mtDNA sequence of

Asian arowana [10] for nine mitochondrial genes: NADH dehydrogenase subunit 1

(nad1), NADH dehydrogenase subunit 2 (nad2), NADH dehydrogenase subunit 4

(nad4), NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit I

(cox1), cytochrome c oxidase subunit II (cox2), cytochrome c oxidase subunit III

(cox3), ATP synthase subunit 6 (atp6), and cytochrome oxidase b (cob). These genes were chosen as they were all longer than 500 bp, giving us a higher chance of SNP differences between individuals. Coding sequences were preferred as non-coding sequences were difficult to amplify due to their high repeat content.

The nine mitochondrial gene fragments were ligated into pBluescript KS (-)

(Stratagene) vector pre-digested with SmaI (Stratagene). Nucleotide sequencing of the cloned inserts was conducted by using BigDye assay kit version 3.1 (Applied

Biosystems) and M13F/M13R sequencing primer. Flanking vector sequences were clipped automatically by using commercially available software Sequencher

(GeneCodes) with manual correction. Sequences were assembled by using the same software.

The full mitochondrial sequence was obtained from NCBI Genbank (DQ023143) and the sequenced mitochondrial genes were aligned using software Sequencher

(GeneCodes). The SNPs were manually identified. Altogether we examined a total of

32 individuals.

72 | P a g e 2.12 Computational and statistical analysis

2.12.1 Microsatellite genotyping

Allele frequencies were estimated by direct counting and inbreeding coefficient index

(f), observed heterozygosity (Ho) and expected heterozygosity (He) were calculated using the software GDA [164], Deviations from Hardy-Weinberg equilibrium were examined using chi-square test from the same software.

Parental analysis was determined by PAPA v2.0 (Package for the Analysis of Parental

Allocation) [165]. It employs the breeding likelihood method which is based on the likelihood of a parental pair producing the multilocus genotype found in the offspring being tested. Unlike methods that use exclusion-based parentage, PAPA allows for some degree of transmission errors due to genotype misreading or mutation. This was one of the reasons why this software was employed in this study. As our brooders were clearly marked and breeding data was well documented and unambiguous, there was no need for exclusion methods, which would be more efficient in deciphering wild populations where total population sampling is difficult.

Analysis of the relationship between the colour strains was performed by using twelve microsatellite markers; 219 data points were generated for each individual sample.

Cluster analysis was performed using Neighbor-Joining algorithm (NJ) using NTsys package 2.02e[166]. An unrooted phylogenetic tree was fitted to the chord distance matrix by using the NJ algorithm as implemented in MEGA (ver3.1.2) [167].

TreeView [168] was used to visualize the tree.

For comparing the dissimilarity index between brooders and their potential breeding partners Nei‟s genetic distance (Dissimilarity index) [169] was computed from the

73 | P a g e binary data from 12 polymorphic microsatellites. All pairwise combinations of each brooder were calculated with all its potential breeding partners. All the data analysis was performed using the software package NTSYS-pc [166]. Genetic diversity (H) was calculated according to the following equation [170]:

H=[n/(n!1)] . C1!+i/1 x2i D, where x is the frequency of the ith genotype in the population, and n is the number of isolates examined.

2.12.2 Analysis of AFLP-based phylogenetic relationship between the colour

strains

We used the Bayesian model–based clustering method implemented in Structure v 2.1 to investigate the genetic structure of the different populations [171,172]. This approach allowed us to determine the most likely number of genetically distinct clusters (K) of populations. We computed different hypotheses representing one to six clusters, with 10 iterations for each solution and 100,000 Markov Chain Monte Carlo

(MCMC) replicates (burn-in length: 100,000) for each iteration. The mean likelihood was calculated for each solution in order to determine the most appropriate number of clusters.

2.12.3 Analysis of kinship between the brooders for understanding egg thievery

event

We studied the relatedness and kinship among the brooders within each pond using

Kinship v1.3 [173]. Kinship tests hypotheses of relatedness [174] between pairs of brooders available in the pond and calculates the ratio of the primary hypothesis that they are related (full-sibs or half-sibs) to the null hypothesis that they are unrelated.

74 | P a g e The significance of this ratio was then determined by software simulation at three different significance levels (P=0.05, 0.01 and 0.001).

2.12.4 Study of the genetic similarity among the colour strains

For genotyping, eight polymorphic microsatellite (MS) markers [10,157] were used to compare the Genetic Similarity, FST, calculated using Fstat version 2.9.3.2 [175].

Pairwise FST values in lower diagonal were calculated from eight microsatellite loci of the various colour strains of Asian arowana and two Australian arowana species.

We also calculated bootstrap confidence intervals (CI) on FST values by resampling loci 1000 times for every pairwise comparison with the software GDA [164].

2.13 Morphometric measurements

Morphometric measurements were classified into external and internal morphometric differences. Due to the relatively large size of the specimens and the laborious harvesting procedure, external measurements can only be determined from digital photos taken from the field with a known length marker in the photo (see Fig. 10).

The photos were then analyzed with public domain free software Image J (1.33U)

[176].

2.13.1 External morphometric differences

Morphometric measurements were taken for 20 confirmed female and 20 confirmed males (sex was confirmed by genotyping, mtDNA haplotyping, and 11-KT measurements using ELISA) of the MG colour strain. Five external morphometric measurements (see Fig. 10) were made by image capture of the fish using a digital camera. A ruler in the image was digitalized (in cm) to serve as a measurement

75 | P a g e reference standard. Morphometric measurements were repeated three times using three separate photos to assess reproducibility.

External morphometry parameters used were based on information collated from experienced farmers and also physical observations of the genotyped brooders with confirmed gender.

Figure 10: External morphometric measurements used when searching for phenotypic sex markerson fully mature, adult specimen of Asian arowana. Labels; SL - Standard Length, HL - Head Length, GPH - Gill Plate Height, GPL - Gill Plate Length, and ML - Mouth Length.

2.13.2 Internal morphometric differences – moulding/roughness index and touch-

based examination

Scanning Electron Microscope was used to produce the paraspheniod images (fig 15).

Samples were prepared by an ultrathin coating of gold, deposited by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging.

Such coatings include gold, gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the scanning electron microscope.

76 | P a g e For the generation of dental moulds, the arowanas were sedated using MS-222 and bite-registration material („O-Bite‟ Chemisch-Pharmazeutische Fabrik GmbH) was automatically dispensed and mixed while extruding the two components through a mixing tip. The material was dispensed on a wooden spatula, customized to fit into the right row of the entopterygoid teeth in the buccal cavity of the arowana. The arowana was held still above water until the material fully set to avoid distortion and inaccuracies in the impressions.

Imaging of the dental moulds was performed using a Leica SP5 confocal microscope with the AOBS set to reflection mode, equipped with a Leica HCX Plan

Fluotar 5x/0.15 dry objective lens. The argon laser output was set to 20% and the 488 nm laser line was then used at 50-60% transmission. The reflected light was then collected between 482-493 nm, with the confocal pinhole set to 1 airy unit, and the

PMTs gain and offset adjusted to avoid saturation of the detector. The limited field- of-view meant that 3 Z-series had to be collected per dental mould to image the whole sample. We performed Z-series with a step size of 20 μm. 3D reconstructions were then rendered using Bitplane Imaris software, in which measurements were made of the distance from the base of the mould to the surface at regular intervals along its length.

Microsoft Excel and SigmaPlot were then used to process the data and produce graphs respectively. The variance on the surface topography in the buccal cavity was measured and compared between five males and five females over a period of 42 days

(day 0, 21 and 42 after offspring were removed from the buccal cavity of mouthbrooding individuals).

77 | P a g e Harvested brooders were also subjected to touch-based examination; this is a method where the index finger of the examiner was used to feel the presence or absence of exposed entopterygoid teeth on the upper jaw. The “roughness” at the upper jaw was scored as “+” for exposed pharyngeal teeth, “-” as absence and “+/-” to indicate an intermediate stage where the entopterygoid teeth were partially exposed. Touch-based examinations were performed throughout the whole period of data collection.

2.13.3 Morphometry of the eggs and larvae

Eggs and larvae were collected from the buccal cavity during each “harvesting” and kept in a farm-designed incubation facility. Each batch of eggs was kept in separate tanks and labelled with the tag number of the mouthbrooder. Measurements were made using an ocular micrometer and total length (TL), standard length (SL) and body depth were measured.

78 | P a g e 3 RESULTS

3.1 The reproduction biology of Asian arowana is very different from that of

most teleosts

3.1.1 Observation of mating - temporal and spatial data

The courtship of the S. formosus usually occurred in the morning (06.00 hrs-08.00 hrs) and in the evening (18.00-20.00 hrs) when the water temperature was cool (25-

28oC). The process involved brooders schooling in circles, courting pairs having the putative male (assumed on the basis of phenotypic cues observed in some individuals, like irregular scale patterns and broken barbels or pectoral fins) swimming beside the female and using the pectoral fin to rub against the abdominal region of the female.

The process also involved occasional approaches from other males as well as flaring of the courting male towards the approaching males. This was thought to stimulate ovulation for the female. The courtship normally lasted for 1-2 hrs with occasional jerks and flaring. These motions were usually repeated for 5-7 days after which the pair descended into deeper waters for the external fertilization to occur.

In a recorded video available from the web [177], we observed that the two partners laid close to the bottom side-by-side and coordinated the release of milt and eggs allowing the latter to be fertilized externally (Fig. 11A-B). Immediately after the release of the milt and eggs, the female floated towards the surface of water, we suspect that this maybe due to the sudden loss in her weight but we do not have any supporting data as there is no literature stating whether this species is physostomatous or physoclistous which would allow us to speculate further the reason for floating after releasing of the eggs. The male also left the fertilized eggs (Fig. 11C-D).

Following the breeding event, one of the parents (presumably the female) usually 79 | P a g e stayed on the surface for 2-3 hours, whereas the other one (presumably the male) took a gulp of air on the surface (the reasons for this was unknown), then returned immediately to the fertilized eggs and slowly collected them into his mouth by gentle suction (Fig. 11 E-F).

Asian arowanas are known to mouthbrood for 40-50 days before releasing the offspring into the open waters. We observed that during the first 3-5 days the other parent “guarded” the mouthbrooding partner and prevented any attacks from other members (we do not know the sex of the attackers) in the pond by flaring and bites.

During this period, the mouthbrooding parent usually stayed near the surface and

“spouted water” vigorously out of the buccal cavity, presumably to increase the flow of oxygenated water through the buccal cavity holding the eggs. Following this period, vigorous action usually ceased and the fish returned to the alert mode and descended whenever an observer approached the pond. The mouthbrooding parent was not seen consuming any food during the whole 40-50 day period and remained near the edge of the pond.

The buccal cavity of the mouthbrooding parent gradually enlarged as the eggs developed. The free-swimming larvae could be first seen only after 35-42 dpf where the large yolk sac was fully absorbed. They were typically highly alert and clustered together for protection and returned rapidly into the buccal cavity of the mouthbrooder when sensing danger. After 45-48 dpf, free-swimming larvae were released to fend for themselves; larvae sought shelter at shallow waters where aquatic and semi-aquatic vegetation provided plenty of protection and enough source of food such as worms, insects, insect larvae, small fish/fish larvae and small amphibians like frogs.

80 | P a g e There were a few marked differences observed between the MG and RG1 arowanas in terms of their breeding behaviour. In the RG1 arowana ponds, the mouthbrooding parent was typically not guarded by the breeding partner, and there were more attacks from the other members in the pond than in the case of MG. More often than not, the mouthbrooding RG1 arowana was attacked by other brooders in the pond and subsequently, the eggs were released from the buccal cavity and they were consumed by the attackers. Due to this phenomenon, the production rate of RG1 arowanas in the farm was typically much lower than that of the other colour strains.

81 | P a g e

Figure 11: The breeding process of Asian arowana captured on video by a hobbyist. Panel A: During the start of the mating process, the two partners would lay close to the bottom beside each other and swirl in a circular motion; Panel B: this swirling movement will stop and both the fish will stay motionless for about 30 seconds and after which there would be a coordinated release of milt and eggs (see arrowhead). Panel C: The release of the milt and eggs, the female floats towards the surface of water. The male would also leave the fertilized eggs and move to the surface for a gulp of air (Panels C-D). The male slowly collected them into his mouth by gentle suction (Panels E-F). The pictures were taken from a video publicly available at Youtube: http://www.youtube.com/watch?v=5ltmk6oHXg8

82 | P a g e 3.2 Differences in the productivity and survival rate of colour strains and

dependence of breeding events on environmental factors

3.2.1 There were significant differences in offspring number per brooder and

their survival between several colour strains

The six earthen ponds analyzed for production in the study included WH001 (22 MG individuals), WH002 (21 TY), WH003 (17 TY), WH004 (31 IG), WH005 (14 RG1) and WH006 (16 RG1). During the period of three years (from April 2004 to March

2007), there were a total of 237 batches of offspring produced. In total, 6,566 offspring individuals were produced, out of which 4,381 (66.72%) survived until the tagging and DNA collection stage. The highest number of offspring in the same batch was 58 (MG and TY varieties), whereas the lowest was 4 (TY variety). When the average number of offspring per brooder produced by the four varieties were compared, the values of IG and TY were significantly higher from those of MG and

RG1 (one-way ANOVA; P<0.05, Tukey's test; Table 5). One significant difference was also observed between the numbers of offspring that survived till tagging (% survival): the 54.54% value of IG was significantly lower than those of the other three strains (70.9-73.75%; one-way ANOVA; P<0.05, Tukey's test; Table 5).

83 | P a g e Table 5: The productivity and offspring survival of different colour strains in the farm

Ave Mouthbrooding Offspring Offspring offspring/brooder % Survival Colour Strains parent produced survived (+- S.D.) (+- S.D.) IG 35 1070 378 30.57 + 11.6 a 35.32 + 42 a MG 129 3331 2405 25.82 + 12.8 b 73.75+ 30.4 b TY 59 1846 1370 31.28 + 16.1 a 73.29 + 29.1 b RG1 14 319 228 22.79+ 9.1 b 70.9 + 42.4 b TOTAL 6566 4381

Legends: IG: Indonesian golden; MG: Malaysian golden; TY: Hybrid (RG1xMG); RG1: Red grade one

Values labeled with the same letter in superscript are not significantly different, whereas those labeled with different letters are significant different (one-way ANOVA; P<0.05, Tukey‟s test)

84 | P a g e 3.2.2 The breeding frequency of brooders did not show correlation with the rainfall

Our data show that Asian arowanas are fractional spawners, as they usually the whole year round, but the collection of the offsprings from the buccal cavity peak after the monsoon season (September to December). From the rainfall and average temperature data purchased from the National Environmental Agency (NEA), we evaluated the breeding frequency and the number of offspring produced against the rainfall data.

The amount of rainfall was quite random and did not show consistent differences between the monsoon and dry seasons for the period analyzed (Fig. 12). Although the trend of rainfall and breeding activity looked quite similar, there was no statistical correlation between the rainfall and the offspring production in the farm (r = 0.04, n =

48, P = 0.05). On the other hand, there was a sharp decrease in production around

June and July in some years, even when the rainfall was relatively high compared to other months in the same year (Fig. 12).

85 | P a g e

Figure 12: The breeding cycle of the Asian arowana in the farm. The graph shows the total production in terms of number of batches（dotted line）and number of offspring (solid line) for the three experimental ponds in relation to the rainfall (bar) data from January 2003 to November 2006.

3.3 The early development of Asian arowana larvae is a slow process

We have studied the early development of the Asian arowana by observing fertilized eggs (obtained from the buccal cavity of mouthbrooding parents) that were reared in fish tanks. From our observations, it seemed that only the left ovary was functional in

Asian arowanas; we were not able to locate the right ovary in any of the 20 females dissected. Eggs before the age of 3 dpf were difficult to incubate; most of these early eggs failed to survive (n=20 batches, average mortality >90%) and turned white within 60 mins after harvesting them from the buccal cavity. The eggs were extremely large (Fig. 13A); their average diameter was nearly a centimeter (mean =

0.99+0.42 cm; range = 0.78-1.20 cm; n =170). Eggs from the same brood were similar in size and differed between broods only. Older females typically produced bigger eggs. As eggs were harvested at different developmental stages from the mouth of the

86 | P a g e brooder, they had to be kept in special holding devices in the incubation facilities developed by the farmers to simulate the mouthbrooding process.

3.3.1 Fertilised eggs

The chorion was transparent and the colour of the yolk was the only visible colour observed (Fig. 13B). The perivitelline space was very narrow, whereas the chorion was clear and unsculptured (Fig. 13C). The yolk was homogeneous, unsegmented and opaque (Fig. 13D). Although its colour ranged from yellow to orange, there was no variation within the batches, i.e. all eggs from the same batch showed the same shade of colour. No oil globule was seen.

At a constant temperature of 28oC at 3-5 dpf (Fig. 13B), the vitellic vein was observed extending anteriorly and posteriorly around the large yolk sac. The blood vessel was branched at the tip and continued to extend and branched out further surrounding the yolk sac during the early development (Fig. 13D-E). At 5-7 dpf, the larvae hatched from the transparent chorion (Fig. 13C-D). The TL of larvae was about 5-8 mm and their weight was about 50 times smaller than that of the yolk sac (diameter: 15-18 mm). At this time, both the head and tail of the larvae were detached from the yolk sac (Fig. 13D-F), leaving only the exposed opening from the peritoneal cavity in contact with the yolk sac. The notochord, forebrain, pectoral fin buds and optic lobes/eyes were clearly visible and the fin fold was evident at the end of the caudal regions (Fig. 13F). Pigmentation of the larvae became first evident at 8-10 dpf as dorsal melanophores started to develop with higher accumulation at the somite region and were also evident at the dorsal head region and caudal region (Fig. 13F). A distinct tubular foregut and hindgut were observed at this stage and proceeded to develop by thickening and convoluting of the gut till 13-15 dpf (Fig. 13G).

87 | P a g e The morphological development of the larvae progressed as the yolk sac continued to be absorbed. At 13-15 dpf, the larvae were about 15-20 mm in TL, whereas the yolk sac was reduced to about 10-12 mm in diameter. At this stage, membranous fins and fin rays of the caudal, pectoral, pelvic, and anal fin were all evident (Fig. 13G). At day 15-18, the mandibular barbels at the tip of the lower jaw started to develop and at this stage the individuals still remained immobile due to the large yolk sac present

(Fig. 13H).

3.3.2 Juveniles

In captivity, the young arowanas were fed with chromis fly larvae between 20-24 dpf, and were able to digest the feed as defecation was typically observed 1-2 hours after feeding (Fig. 13I-J). At 22-25 dpf, the larvae started to learn to swim and at this stage, the diameter of the yolk sac was reduced to about 5-8 mm compared to the 35-

40 mm of the TL (Fig. 13K-L). The larvae became free-swimming by 30-35 dpf and responded to external cues by evasion (Fig. 13L). The yolk sac was usually fully absorbed by 35-40 dpf; at that time TL of the young juveniles ranged between 4.5-6.5 cm (Fig. 13M).

88 | P a g e

Figure 13: The development of Asian arowana embryos, larvae and juveniles. Panels: A) Fertilized egg at 0 dpf; B) Embryo at 3 dpf; C) Larva at 7 dpf; D) 9 dpf; E) 5 dpf; F) 13 dpf; G) 15 dpf; H) 25 dpf; I) 28 dpf; J) Juvenile at 33 dpf; K) 36 dpf; L) 40 dpf; and M) 45 dpf. The bar indicates 5 mm on every panel.

89 | P a g e 3.4 Sexing the brooders with classical and molecular tools

3.4.1 External morphometric measurements on sexually mature adults showed

significant differences between the two sexes

We have taken external morphometric measurements from 20 male and 20 female

Asian arowana brooders from ponds WH001, WH002 and WH003. The following parameters were measured: gill plate height (GPH), gill plate length (GPL); head length (HL); mouth length (ML), and standard length (SL). The measured data were used to calculate the following five ratios: SL/HL; HL/GPL; HL/ML; SL/ML; and

SL/GPL (for details, see Fig. 14 and Section 2.13.1 in Materials and Methods).

Out of the five ratios tested, the following three showed significant differences

(student unpaired t test) between the two sexes: SL/HL (male: 3.52+0.12; female:

4.29+0.27; P<0.0005); SL/ML (male: 6.16+0.37; female: 7.98+0.33; P<0.0005); and

SL/GPL (male: 8.89+0.35; female:11.14+0.87; P<0.0005; Fig. 14.). We used these three ratios to determine the sex of 50 active sexually mature adult Asian arowana brooders and the results showed a 94% agreement with those obtained with genotyping and ELISA-based hormonal measurement from the mucus (see Section

3.4.3. of Results).

90 | P a g e

3.4.2 Internal morphometry – The surface of the buccal cavity transformed in

mouthbrooding males

The Asian arowana is known to be a carnivore that predates on smaller teleosts and amphibians. The presence of the Tongue-Bite Apparatus (TBA) allows the arowanas to grab prey tightly with sets of sharp teeth in its buccal cavity, hence the name bonytongues. In order to evaluate how the Asian arowanas managed to keep the large and fragile eggs in their mouth without damaging them, we decided to examine the buccal cavity of mouthbrooding parents. A total of 40 mouthbrooding males and 110 inactive brooders (those that had not mouthbrooded for at least the previous two months at time of data collection) were sampled.

91 | P a g e First we looked at the buccal cavity of non-mouthbrooding individuals. Using scanning electron microcopy, we were able to observe the 2 rows of entopterygoid teeth along the inner top surface of buccal cavity (see Fig. 15A), while around them smaller teeth were also observed (Fig. 15B and 15C).

A total of 144 brooders (with sex unknown) were first examined for the roughness using touch-based examination and after which the results were then compared to their breeding records in a blind test. The brooders were categorized according to their mouthbrooding status and their roughness index was recorded. Those brooders that were mouthbrooding during the harvesting (MBROOD) (n=14) all had “smooth” buccal cavity surface, i.e. no entopterygoid teeth exposed. On the other hand, those that mouthbrooded before, but were not mouthbrooding during the time of harvesting

(MBRED) (n=90), as well as those that had never mouthbrooded (NONMB) (n=40), all had varied smoothness (Fig. 16). MBRED showed smooth (50%), intermediate

(29%) and rough (21%) surfaces, whereas NONMB were mostly rough (“+”) (70%) and intermermediate (“+/-”) (25%) with only 5% showing smooth surfaces (“-”).

The analysis of 90 brooders, for which the sex was confirmed by genotyping and

ELISA assay (see Table 6) showed that the smoothened surface was only found in the confirmed males and some individuals without breeding record, but not in the females. In the first group, roughness varied according to the status of the males. Fully covered teeth (labeled as “-” on the figure) were found only in ca. 30% of the confirmed males and these males were mouthbrooding during the examination.

92 | P a g e Figure 15: Scanning electron micropgraphs of the parasphenoid (pharyngeal teeth) do not reveal differences in teeth morphology between males and females. Panels ; A - Male; B - Female; C - Teeth that are found on the endopterygoid (relative position to the entopterygoid teeth is indicated by the red box) of both males and females. Bars represent 100 micron.

Figure 16: Smooth buccal cavity can be found only in some Asian arowana brooders. A total of 144 brooders (sex of the fish is not determined yet) were examined for the roughness using touch-based examination and the results were compared to their breeding records. On the graph, a smooth surface is indicated with a green bar and indicated as (“-”), a intermediate roughness is given a maroon bar and indicated as “+/-”, and a rought surface is given a blue bar and is indicated as “+”.

93 | P a g e Table 6 : The modification of buccal cavity surface is transient and occurs in mouthbrooding males only. We evaluated the buccal cavity of 90 brooders (males =40; females =50) of known sex (sex is confirmed by ELISA results) and when we compared the roughness of the buccal cavity surface, the results showed that only the males that are mouthbrooding has a smooth cavity surface. The rest of the males that was not mouthbrooding during the time of testing either had intermediate or rough surface.

Sex of brooder “ –” “+/-” “+” Males (n=40) 12 12 16 Females (n=50) 0 0 50

3.4.3 Hormonal measurements from mucus

We determined the 11-KT levels in the surface mucus of mature males and females that were more than five years of age (their sex was previously confirmed by microsatellite- and mtSNP-based genotyping).

We followed the average 11-KT levels in the mucus of 21 brooders in pond WH001

(an individual died during the period of experimentation) over a period of 26 months

(from June 2005 to August 2007) and separated them into two groups, male and females (see Fig. 17). Although the average 11-KT levels in the surface mucus of males was substantially higher than those of females (see Figure 17), unfortunately there was no significant difference between them (student t test, P >0.05).

In contrast, we were not able to determine the level of E2 in most samples and those that had readings did not coincide with the genotype data. Thus, after three attempts, these experiments were terminated, and subsequently only the 11-KT levels were used.

94 | P a g e

Figure 17: Comparing the 11-KT levels in the mucus of males to females in pond WH001 over a period of 26 months. Hormone levels were determined by ELISA essay and normalized by the total soluble protein content determined by Bradford essay. The bars show the average values with their standard deviation.

3.5 Identification of the sex of the mouthbrooding parent in the MG variety of

Asian arowana

To confirm the gender of the mouthbrooder in Asian arowana, in collaboration with

Woei Chang Liew, we cloned and sequenced a total of ~ 8kb fragments from six mitochondrial genes using four breeding pairs (eight different individuals from the

MG variety) kept in WH001 pond. Altogether, there were eight mtSNPs identified from six mitochondrial genes (nad1, nad5, cox2, cox3, atp6, cob; Table 7). Then, we randomly selected five offspring individuals from each breeding pair for mtSNP genotyping. All the offspring from the four families showed an mtSNP haplotype identical (except from family 3 where there was one difference in the cytb SNP, see

Table 7) with that of their non-mouthbrooding parent (identified earlier by microsatellite genotyping). The difference seen in family 3 (in the cytb gene) could be

95 | P a g e due either to a de novo mutation or to sequencing error, but the rest of the data (29/30

SNPs) showed complete agreement with the corresponding haplotype of the non- mouthbrooding parent. As the inheritance of mitochondria is known to be matrilinear in all animals, with the exception of a few unique cases [162,163], and we had no reason to assume that Asian arowana belongs to the exceptions, therefore we confirmed that the mouthbrooder in the MG variety of Asian arowana is the male.

This approach also allows us to sex the brooders with the SNP genotypes from the offspring. This approach was very precise although it was not a very practical approach to sex the brooders.

96 | P a g e Table 7: The mitochondrial gene-based SNP profiles of the offspring are identical with those of their non-mouthbrooding genetic parent

Mt gene atp6 nd1 nd5 cox2 cox2 cox3 cox3 cytb SNP position (bp) 147 666 1389 309 501 198 336 429

Family 1 Labels: MB (A1) C A G A T T T A NMB (A2) A G A G C C C G MB: mouthbrooding O/S A G A G C C C G NMB: non-mouthbrooding Family 2 MB (B3) C A G A T T T A ND : Non-determined NMB (A9) A G A G C C C G O/S A G A G C C C G O/S: offspring

atp6: ATP synthase subunit 6 Family 3 MB (B9) A G G G C C C G nd1: NADH dehydrogenase subunit 1 NMB (B2) C A G A T T T A O/S C A ND A T T T G nd5: NADH dehydrogenase subunit 5

Family 4 cox2: cytochrome oxidase subunit II MB (B9) A G G G C C C G NMB (C2) C A G A T T T A cox3: cytochrome oxidase subunit III O/S C A ND A T T T A cytb: cytochrome b

*data obtained in collaboration with Woei Chang Liew

97 | P a g e 3.6 Genotyping data reveals complex relationships between the brooders in the

ponds

For the study of the breeding relationship between brooders, we used individuals in ponds WH001 (containing 22 brooders from the MG strain) (Fig. 18), WH002 (21

TY) (Fig. 19) and WH003 (17 TY) (Fig. 20). These brooders had been kept within their respective ponds since the start of the project till the genotyping experiments have ended. Although the ponds were subjected to minimal human disturbance to ensure highest possible simulation of the natural environment, nonetheless these observations were still made in an artificial setting, thus might not necessarily reflect the conditions affecting fishes in the wild.

On a few occasions, we encountered apparent null alleles in the offspring when an expected heterozygote was scored as a homozygote (one of the alleles was not amplified). In these cases, as other loci did not show a similar result, the issue was often resolved after re-amplification (that is, this null allele was detected due to PCR errors). When re-amplification did not work, that would indicate that a null allele was present. In those cases when a homozygote did not show any allelic product, and the results remained the same after re-amplification, we treated the results as missing data. From our results, we did encounter a few typing errors, such as stutter bands and PCR errors, but repeated runs of the genotyping helped to resolve the issue in all cases.

98 | P a g e

Figure 18: The breeding relationships of Asian arowana were complex and involved different mating systems. The breeding relationship chart showing the connections between the brooders in pond WH001, as revealed by genotyping with a set of highly polymorphic microsatellites. Each boxed ID represents an individual brooder in a pond; numbers in the top row represent mouthbrooding individuals, whereas those immediately below the chart are non-mouthbrooding parents identified by genotyping. The three boxed IDs in the bottom left corner indicate inactive brooders (i.e. individuals that we have not collected any offspring from and also did not have any breeding relationships based on genotyping since the start of the experimentation) without known sex. Lines represent breeding connections that were determined by microsatellite genotyping. Each circle (○) on the line represents one breeding event that occurred between the two brooders, whereas the number beside the circle represents the number of offspring that was collected from the mouthbrooding individual. The column on the left indicates the date of the collection of the offspring from the buccal cavity of the brooder.

99 | P a g e

Figure 19: Fewer active brooders appeared to form less complicated breeding reelationships. The breeding relationship chart between the brooders in pond WH002. For labels see Figure 18.

Figure 20: There were no monogamous pairs in the third pond. The breeding relationship chart between the brooders in pond WH003. For labels see Figure 18.

100 | P a g e Altogether 61 MG and TY brooders were genotyped using 12 highly polymorphic microsatellite markers [157,178,179]. A total of 149 different alleles were obtained across all loci, with the number of alleles per locus ranging from 6 (D31 and D116) to

18 (D04) (Table 8). Observed heterozygosity values were quite high across the whole stock analysed (mean = 12.4; range = 0.571-0.889). No significant linkage disequilibrium was detected in any of the pairwise comparisons of loci across the stock tested following a Bonferroni correction, thus confirming the independence of loci. After determining the genotype of the brooders, we started to analyze their offspring. Over the period of three years, 147 batches of offspring, containing the total of 2,458 individuals, were produced, and all batches were genotyped (see

Supplementary table 2 for example of the parentage assignment using the PAPA software). Initially, we genotyped every single offspring per batch to determine whether males had accidentally mouthbrooded the eggs of multiple females or had picked up eggs from other mated pairs. As the first 80 offspring batches all contained exclusively siblings, for the remaining 67 batches only five individuals per batch were sampled. In each of these cases, all five offspring individuals were full siblings.

101 | P a g e Table 8: Characterization of microsatellites and genetic diversity in 230 Asian

arowana brooders

Locus Allele no. Allele range He Ho f PHW

D04 18 165- 227 0.915 0.825 0.098 0.000** D31 6 225- 277 0.566 0.619 -0.095 0.001** D38 16 183- 225 0.780 0.778 0.002 0.002* D42 13 145- 187 0.863 0.810 0.063 0.255(ns) D92 12 146- 180 0.854 0.889 -0.041 0.000** D104 12 222- 246 0.822 0.587 0.288 0.000** D105 9 234- 270 0.767 0.587 0.236 0.000 ** D106 15 195- 233 0.885 0.873 0.013 0.000** D108 17 215- 253 0.890 0.794 0.109 0.197(ns) D109 12 196- 264 0.846 0.698 0.175 0.001** D115 13 204- 238 0.869 0.810 0.068 0.197(ns) D116 6 105- 117 0.676 0.571 0.156 0.001** Average 12.4 0.811 0.737 0.0 92

Key: ns=not significant; He: expected heterozygosity; Ho: observed heterozygosity; f: inbreeding coefficient index.

PHW: Hardy-Weinberg probability

* Loci showing significant ( P<0.01) deviation from Hardy-Weinberg equilibrium ;

** Loci showing significant ( P<0.001) deviation from Hardy-Weinberg equilibrium .

102 | P a g e Our genotyping data showed that 143 out of 147 batches of offspring were mouthbrooded by one of their genetic parents (the remaining four cases will be described at Section 3.9). The fertilized embryos within a given brood were of approximately the same developmental stage. The average number of embryos per mouthbrood was 27.6 (range: 5-58) and the SL of the mouthbrooding fathers ranged from 53 cm to 78 cm. We found no significant relationship between the SL of the father and the brood size (r = 0.14, n = 147, P = 0.05).

Combined microsatellite-based parentage analysis data from various ponds (WH001-

WH003) showed that males always brooded the eggs of a single female at a time; yet several individuals from both sexes were shown to breed with multiple mating partners over subsequent breeding events. This was evident from the variation of maternal alleles in the offspring batches carried by the same male in different breeding events and from the variation of paternal alleles found in two subsequent broods produced by the same mother. Thus, the Asian arowanas seemed to practice both monogamy (32 breeding events; see above) and polygamy (115 breeding events).

Within the polygamous relationships, all three main forms, i.e. polyandry (54 breeding events), polygyny (97 breeding events) and polygynandry (45 breeding events), could be observed. Both polygynous males and polyandrous females had a maximum of four mates each. Our genotyping results also showed that Asian arowanas practiced strict pair spawning and we have not observed any sneak or satellite mating systems. The maximum number of offspring batches from the same breeding pair was six over the period of sampling (C1 and C3 in WH001; Fig. 18).

No correlation was seen between the development stage of larvae at the time of collection and the number of offspring mouthbrooded (r = 0.18, n= 147, P=0.05).

The shortest interval between two breeding events for a given female was two 103 | P a g e months, whereas the shortest period a male was seen to mouthbrood again following the removal of the embryos from its mouth was two weeks.

3.7 Change in breeding pattern following the loss of a male in pond WH001

In December 2004, the most productive male in pond WH001 (B5; Fig. 21) was found dead. An autopsy was performed, but no sign of disease could be observed. During the next three bimonthly harvesting events, no offspring was found in the buccal cavity of any of the active males from pond WH001, although brooders showed no sign of disease symptoms and no additional mortality was observed in the pond.

The next batch of offspring was collected from the pond only in May 2005 (Fig. 21), indicating a six month lapse in the breeding events. Our genotyping data showed that after the death of B5, there was a shift in mating partners for the rest of the brooders that practiced polygamous mating. The primary breeding partner of B5, called A2, mated first with B9 (September 2005), then with B8 (June 2006), after which no mating was observed from this female. B2, the other partner of B5, continued breeding with only one of its two previous partners, called A1. C1 was a polygamous male which turned monogamous after the death of B5. On the other hand, monogamous pairs (A5-B7, B1-B6 and B3-A9) resumed their mating with the same partner after the long break.

104 | P a g e

Figure 21: The loss of a fecund male resulted in long cessation of reproduction, followed by rearrangements of breeding connactions pond WH001. For labels see Figure 18. Blue lines indicate terminated breeding connections, whereas red lines newly formed connections following the re-initiation of breeding after a half-a-year break, black lines indicates breeding connections that are resumed following the hyatus.

3.8 No signs of possible inbreeding or incompatibility avoidance were observed

To test for genetic incompatibility avoidance, or inbreeding avoidance, where brooders choose mating partners of higher genetic dissimilarity, we examined the dissimilarity index of mating pairs compared to the rest of the available breeding partners. If inbreeding avoidance was present, mating pairs should be genetically less similar compared to the average of all the breeding partners. In addition, genetically similar mating pairs should have less offspring than the more distantly related breeding pairs.

105 | P a g e We then compared the genetic dissimilarity of four males with all their potential female breeding partners (active females present in the same pond; Fig. 22) and that of four females with all their potential male breeding partners (Fig. 23), respectively.

No indication for preference towards genetically dissimilar partners was observed in either of the two sexes. For instance, although one of the four male brooders (B9) mated with the most dissimilar female available in the pond (Fig. 23A), B5 paired up with the most genetically similar female available (Fig 23B). For the females, two of the four female brooders (A2 and A8; Fig. 22C-D) bred with the 50% higher percentile dissimilar males and the other two did not show any obvious trend; one of them (A6; Fig. 22B) bred with the second least dissimilar male (A3).

3.9 The Asian arowanas display an unusual phenomenon of egg thievery

On September 16, 2003, a batch of offspring was collected from the buccal cavity of a young male, labeled A7. Genotyping with microsatellites indicated that their genetic mother was A2. To our surprise, the genotype of the offspring excluded A7 from the list of potential fathers and pointed firmly to A1. We decided to perform additional analysis on all three brooders involved and their offspring, by using six additional polymorphic microsatellites (the total of 18 markers). The results further confirmed that the true parents of that batch were A1 and A2. We had therefore documented an unusual phenomenon of “egg thievery” being displayed by individual A7 (see

Supplementary table 3).

106 | P a g e

Figure 23: There is no correlation between the genetic similarity of male brooders and their female breeding partners in the pond. The chart shows the dissimilarity indices (Nei‟s genetic distances) between 4 males (B5, B9, A1 and C1) and all their potential female breeding partners in the pond, respectively. The red bar indicates at least one breeding event with the male. The indices were calculated using the software package NTSYS [166]. Panels; A: Male B5, B: Male B9, C: Male A1, D: Male C1

107 | P a g e Over the three-year recording period, three additional egg thievery events were observed. All four cases occured in two ponds: there were three incidents in WH001

(Fig. 24), whereas one in WH003 (indicated on Fig. 20 by an asterisk *). On two occasions (one in WH001 and one in WH003), eggs from the same parent pair were found both in the mouth of the genetic father as well as the thief, indicating that the thief only managed to steal part of the batch (the results were further confirmed with six additional microsatellites in both cases).

In pond WH001, all three thievery events involved eggs from the same female (A2).

We used Kinship v1.3 software [173], to study the relatedness and kinship of the true genetic parents and the thief. We compared the pairwise kinship coefficient (k) between the thieves and both genetic parents. Using the average k value between the subject and the rest of the brooders as a reference, we assumed that if there was a stronger kinship between the thief and the parents, their k value would be higher than the average k value. Only C4 and A2 showed a significantly higher k value (0.564) than the average (0.084) (Fig. 25).

108 | P a g e

109 | P a g e 3.10 Transient morphological modification of the surface of buccal cavity in

Asian arowana during mouthbrooding

We compared the inner surface of the buccal cavity in five actively brooding males and five females by analysing the contours or “surface topography” of the endopterygoid. The height of the exposed entopterygoid teeth extending from the epidermal layer (termed “roughness index”) was either estimated by a touch-based method or determined from images taken by a confocal microscope by using moulds as templates. The measurements were taken at Day 0 (when the eggs were removed from the male‟s buccal cavity), at Day 21 and at Day 42. Figure 26 shows an example of the analysis of the confocal image taken from a dental mould where the planar

(Panel 26A for male during the mouthbrooding and Panel 26B for the same male after the mouthbrooding event) and 3-D (Panel 26C for male during the mouthbrooding and Panel 26D for the same male after the mouthbrooding event) images are converted into a graphical form (Panels 26E and 26F for male and female, respectively) for comparison.

Next, we compared the data obtained by confocal microscopy and touch-based analysis by analyzing the same set of 10 individuals in a single blind experiment.

Results obtained by the two different methods showed agreement in 8 out of 10 samples. Thus, we subsequently continued our data collection using touch-based examination only.

110 | P a g e

Figure 26: Observing the changes in the entopterygoid teeth profile of a male before, during and after mouthbrooding by confocal microscopy-based analysis of dental moulds. Panels: A) A planar representation of the entopterygoid teeth profile of a male at day 0 (larvae removed after mouthbrooding); B) 42 days later (day 42); C) 3-dimensional representation of the profile shown in A; D) 3-dimensional representation of the profiles shown in B; E) and F) Graphical representation of the changes in surface profile in the above male and a female, respectively.

111 | P a g e 3.11 Most colour variants of Asian arowana can be differentiated from the others

using molecular analysis

3.11.1 Differentiation of the colour strains using microsatellite-based genotypes

Together with Wei Kee Yap, an attachment undergraduate from Faculty of Veterinary

Science, The University of Melbourne, Australia, we compared the microsatellite- based genotypes of six colour variants of Asian arowana using the data generated for the parent-sibling analysis (12 polymorphic markers). Cluster analysis based on NJ algorithm using number of difference coefficients separated the colour variants into two main groups (Fig. 27). There was clear separation between the different colour varieties within the main groups and individuals from the same colour varieties were assigned to the same cluster, except for Indonesian gold and Malaysian gold. The dendrogram revealed clear grouping pattern that could separate all the RG1, WRG1,

GR, RG2, and MJK varieties with relatively high certainty. Although there was no clear separation between the IG and MG individuals, most of the MG individuals

(10/12) were grouped together as a sub-cluster (shaded blue in Fig. 27).

112 | P a g e

Figure 27: Microsatellite data analysis was able to differentiate the colour strains into five clusters. Dendrogram showing the cluster analysis separating the colour strains into 5 distinct clusters according to the colour strains. Twelve microsatellite markers were used to analyse the phylogenetic relationship between the colour strains, 219 data points were generated per individual. Cluster analysis was performed using Neighour-Joining algorithm (NJ) using NTsys package 2.02e. An unrooted phylogenetic tree was fitted to the chord distance matrix by using the neighbor-joining (NJ) algorithm as implemented in MEGA (ver3.1.2). TreeView was used to visualize the tree. The subcluster shaded in blue contains all Malaysian golden individuals.

113 | P a g e 3.11.2 FluoMEP was able to differentiate between two commercially important

colour strains

This work was done together with Mr QiFeng Lin, an honours student from NUS. We collected DNA samples (n =8) from two strains of Asian arowana (Red Grade 1 and

Red grade 2). {Individuals from these two strains show similar phenotypes when they are young, but they have very different commercial value (see Section 1.3).} These samples were screened using the FluoMEP method that we have developed earlier

[153]. Two markers (C117+BH05 and C117+F04, see Section 2.10) were found using this method: marker A showed up as a 137bp band in RG1 individuals only (Fig.

28A), whereas marker B produced a 314 bp band in RG1 and a 294 bp one in RG2 individuals (Fig. 28B). Out of the 8 samples tested, none of the samples deviated from the expected results.

114 | P a g e

Figure 28: fluoMEP assay allowed for the differentiation between the RG1 and RG2 colour variants at the genetic level. DNA samples (n =8) were collected from two strains of Asian arowana (Red Grade 1 and Red grade 2) and they were screened using the FluoMEP method. Panel A shows the result of primer combination C117+BH05 with a peak at 137bp which is present in RG1 but absent in RG2. Panel B shows the primer combination C117+F04 which produces a peak at 314bp position for RG1 and 294bp position for RG2.

115 | P a g e 3.11.3 The microsatellite-based phylogenetic tree of the colour variants of Asian

arowana was congruent with geographical reconstruction of prehistoric events

in South-East Asia

Paleogeographic data of South East Asia[180] shows that this region used to be a huge landmass with inter-connecting river systems (~17,000 yrs ago; Fig. 29). As the sea level rose, the sea started to divide the landmass into islands. Thus, together with

Woei Chang Liew, we analysed genotype data generated from 8 polymorphic microsatellite loci. With this approach, we were able to assign individuals of different varieties to populations according to their origin. Further analysis using genetic distance showed that the order of divergence of the different colour varieties was congruent with the timing of geographic events predicted for ancient South East Asia

[180].

From the microsatellite data (Fig. 29), the GR variety remained a basal group and we could clearly define the two major clusters of arowanas belonging to Kalimantan island (Clade1; RG1 and MJK) and Sumatra island and Peninsular Malaysia (Clade 2;

IG and MG). On the other hand, RG2 a colour strain that could only be found in

Banjarmasin river (not connected to the Kapuas river system) shares the closest relationship to the clade containing MG and IG, and forms a distinct clade.

116 | P a g e

Figure 29: The shape of the microsatellite-based genetic distance tree of the Asian arowana colour varieties appeared to be consistent with pre-historic geographic events. The change in the land mass of South East Asia shows consistency with the divergence of the different phenotype strains and their endemic location and this is also supported by the relatively similar phenotype between the strains. Panels: A) shows the 120m water level contour of the South East Asia land mass 17,000 yrs ago B) 40m contour, 10,000 yrs ago and C) 20m contour, 10,000 yrs ago. D) Genetic distance tree generated from 12 microsatellite genotypes by Neighbor-Joining (NJ) method. The branching points labeled with 1 and 2 indicate events that separated some colour varieties; 1 and 2 indicates the clade that is formed by the colour strains. (Source of Paleogeographical figures – Harold K.V., 27; 1153- 1157, 2000 [180])

117 | P a g e 3.11.4 Bayesian clustering analysis allowed for differentiation of most colour

strains

A phylogenetic tree was also produced from data generated by genotyping 12 polymorphic microsatellite loci (the same markers used in Section 3.13) and 225

AFLP markers. From the Bayesian clustering approach (Fig. 30), in both data sets, we were able to differentiate RG1 and WRG1 (cluster 2) from the rest of the clusters and also MG (cluster 4) from the IG (cluster 1). On the other hand, we were not able to separate IG, RG2 and GR from each other as they were all grouped into the same cluster (cluster 1). The Mensiku strain (MJK) formed an isolated cluster (cluster 3).

Interestingly, data from the hybrids (RG1 x MG) indicated potential contributions from cluster 2 and 4, although the hybrids were placed into cluster 4.

118 | P a g e

Figure 30: Bayesian clustering analysis using microsatellite and AFLP data enabled the differentiation of most colour strains including the hybrids. Bayesian model–based clustering method implemented in Structure v 2.1 was used to investigate the genetic structure of the different population [171,172] using data from 8 polymorphic MS loci and 225 AFLP markers, this helped in the determination of the most likely number of genetically distinct clusters (K) of populations.

119 | P a g e 3.12 Pairwise comparison of the FST value indicated that the colour strains are

likely to be one species

A previous report [181] claimed that all the colour strains of the Asian arowana were different species. We decided to re-visit this claim by performing genetic similarity comparison amongst the different colour strains.

The pairwise comparison of the average FST values of the different colour varieties and also the closest relative species S. leichardti and S. jardinii (n=15 each for all colour strains and species) was shown in Table 9. Very wide ranges in FST values were observed; pairwise comparison of FST between the samples ranged from 0.061 to

0.356. The FST values were typically 1.5–5.2 times higher between the three species than between the different colour strains of the Asian arowanas. The pairwise FST values between the two Australian species and between any Australian species and any colour variety of the Asian arowana were significantly higher than the inter- variety average of 0.124 (Table 9). Among the latter, only two (MG-WRG1 and MG-

MJK) showed significant difference from 0.237(Table 9). The smallest FST values of all pairwise comparisons were observed between repeat samples within the GR and

RG2 (0.061), TY and MG (0.065), and TY and IG (0.066) and the highest FST values were between MG and MJK (0.356); MG and WRG1 (0.337); and MG and RG1

(0.317).

120 | P a g e Table 9: Average FST values of the different colour varieties of Asian arowana and two Australian arowana species

SJ SL RG1 WRG1 IG GR RG2 MJK HYB MG SJ 0.000 SL 0.681 0.000

RG1 0.400 0.412 0.000 WRG1 0.446 0.457 0.102 0.000 IG 0.410 0.417 0.191 0.210 0.000 GR 0.339 0.356 0.149 0.211 0.096 0.000 RG2 0.369 0.376 0.169 0.208 0.072 0.061 0.000 MJK 0.480 0.474 0.240 0.243 0.192 0.187 0.191 0.000 HYB 0.449 0.432 0.163 0.194 0.066 0.138 0.134 0.264 0.000

Items shaded in yellow indicate significantly different Fst values from average value of 0.227 (p<0.05) as calculated by bootstrap test.

Labels:

SJ: Scleropages jardinii IG: Indonesian gold TY: Hybrid

SL: Scleropages leichardti GR: green MG: Malaysian gold

RG1: red grade one RG2: red grade two

WRG1: wild red grade one MJK: mensiku

121 | P a g e 4 DISCUSSION

Asian arowana (Scleropages formosus) is a bonytongue teleost belonging to the family Osteoglossidae. This species possesses many unique and fascinating characteristics; however, much remains to be discovered with regards to its breeding biology. My objective for this work was to study the basic biology of the Asian arowana using molecular and biochemical approaches in order to understand its mating system, breeding behaviour, phylogeography, genetic differences among the colour strains and gender differences in comparison to their close relatives.

To my knowledge, this is the first genetic characterization of the parentage, and mating system for any species from the subfamily Osteoglossinae. A combination of long-term classical aquaculture tools and advanced molecular genetic approaches has allowed substantial improvement of our understanding of the breeding biology and behaviour of Asian arowana. The applications of modern, molecular genetic techniques have enabled us to recognize details of the breeding biology, and behaviour of Asian arowana that would have remained undiscovered otherwise. Here,

I discuss our findings in the view of available knowledge on these subjects collected from fishes and other vertebrates.

4.1 Microsatellites allow for accurate parentage analysis and reveal breeding

relationships of Asian arowana

The use of highly polymorphic microsatellite markers has facilitated the investigation of the breeding behaviour, breeding relationship, reproductive success and most importantly, parentage assignment for many species [182,183,184,185,186,187]. Our studies have evaluated the breeding biology of the Asian arowana through the analysis of parent-sibling relationships using these polymorphic loci, which offers a

122 | P a g e straightforward and easily interpretable way of linking progeny to their parents. The high average number of alleles detected per locus (see Table 8) allowed us to accurately assign parentage to the offspring harvested from the ponds. This method can be performed with minimal level of invasiveness, and thus, would not affect the survival and most importantly the reproductive physiology of the fish. Moreover, the genotyping data will allow us to develop an individualized “genetic tag” that is permanent compared to RFID tags that can be easily tampered with. We now have the genetic tags for more than 500 brooders and over 4300 individuals of their offspring. In addition to the tags, we also have also documented breeding relationships for more than 300 pairs of Asian arowana (full set of results not presented here due to reasons of confidentiality, as requested by the farm).

Molecular typing using microsatellite DNA markers provides a powerful and reproducible approach for discovering, for the first time, the breeding relationships of

Asian arowana brooders in the pond. The analysis of mating system and genetic parentage have also benefited tremendously from these tools. Our study shows that the microsatellite loci of Asian arowana brooders analyzed were much more polymorphic (Table 8) than those of other freshwater fish species reported by De

Woody and Avise [77]. In their work, they reviewed 13 freshwater species and 75 microsatellite loci and found an average number of alleles of 9.1 and average heterozygosity of 0.54 [77]. In contrast, in our arowanas we found of the corresponding values to be 12.4 and 0.737, respectively. This may be due to the fact that our sampled population included wild-caught specimens as well as different colour strains which presumably originated from different geographical locations.

123 | P a g e In our study, we were able to assign parentage to all the 147 batches of offspring from the test ponds using the software PAPA v2.0 (Package for the Analysis of Parental

Allocation) [165] without any difficulty. This is probably due to the following three reasons: Firstly, the enclosed ponds ensured complete sampling of the pool of potential parents. Secondly, the brooder population contained mainly F0 and F1 generation of Asian arowana, resulting in a relatively higher genetic diversity compared to cultured stocks of other fishes that have been bred in captivity for many generations. Thirdly, we were using normally around 20 individuals from each batch of offspring for parentage analysis, leaving behind the remaining offspring for counter-verification of batches where parentage assignment has failed. Our results are comparable, if not better, than those obtained for many other aquatic species as most of those have a much larger parent pool compared to ours and also a higher mortality rate in the parents as well as offspring [188,189,190,191]. In a study involving parentage assignment of Alantic salmon (Salmo salar) offspring in a farm using only

4 microsatellites, 99.7% of the offspring were accurately assigned to their genetic parents [190]. This result was remarkable as the brooder sample size was much larger

(~800) than ours.

The definition of mating systems is the reproductive strategies adopted by individuals of both sexes in order to achieve mating success [73]. We have fully exploited the use of polymorphic loci to understand the breeding biology, the mating system employed and the breeding relationship by directly comparing the parental and offspring multilocus genotypes. Our data indicate that 100% of the brood carried by a mouthbrooding male contained eggs from a single female. Therefore, males that have breeding relationships with multiple partners would only breed with one of them during a breeding event, and the same is true for the females. Compared to other 124 | P a g e species with uniparental care, for example, nest tenders, pregnant species (viviparous) and brooding species (eg. Sygnathidae), all embryos of Asian arowana from the same batch were sired by the genetic parents (but see Section 3.9 on egg thievery), whereas in other species that ratio ranged from 70% to 95.5% sireship [17]. Among the mouthbrooders, Cichlidae is the most intensively studied family [68,192]: many of the cichlid species have multiple paternities within the broods [193,194].

Single paternity for each batch of Asian arowana offspring indicates a unique breeding strategy employed by the species. Data from other teleosts have shown the presence of alternative reproductive tactics (ART) where both the sexes try to maximize their breeding efficiency and sire as many offspring as possible. In the

Asian arowana, no studies could be performed on ART, as such investigations would require visual observation of the breeding event, social connections and breeding behavior. One of the most interesting observations from our study is the presence of monogamous pairs that have been together for at least five years in every pond. We have also observed territorial behavior in adults when placed in a confined area (e.g. a fish tank), where two adults would attempt to attack each other till one is badly injured and this often leads to death. Territoriality is often linked to males in a fish species where competition for territories is important for mating success [70]. This might indicate that defending against a rival for territory is important in securing an opportunity to breed. Our data support a highly variable genetic mating system where monogamy, polygyny, polyandry and polygyandry are evident. We propose that the genetic mating system of Scleropages formosus is primarily polygyandrous since this was the most frequently employed system in the ponds analyzed according to our observations.

125 | P a g e There might be some indications for a potential sex-role reversal in Asian arowana - as in the sygnathids [195,196] - as the male performs an extreme form of parental care where fertilized eggs are mouthbrooded for up to 45 days. If there were a sex-role reversal in our species studied, we should have seen a biased Operational Sex Ratio

(OSR) [75,197,198,199]. However, we did not see a female-biased OSR and we did not observe a higher intensity of secondary sexual traits in the female arowana. The fact that the Asian arowana has not evolved to be morphologically sexually dimorphic implies that secondary sexual traits or epigamic features [200] are not expected in either gender. This conforms to Avise‟s proposed correlation of the mating system

[16], sexual selection and sexual dimorphism (see Fig 31) using sygnathid species.

Our initial findings on the lack of phenotypic sexual dimorphism in the Asian arowana mating system might indicate the presence of weak or variable sexual selection.

Our genotyping data showed that the shortest interval between two breeding events performed by the same female is two months. This is based on the assumption that no mating has occurred between the two harvests. As there is no literature on the gonadal development of the Asian arowana and only one peer-reviewed publication was found for the closest relative, Arapaima gigas [201], we were not able to compare our findings to those of others. The number of mature eggs formed in the functional dissected ovary of mature adults of more than four years old ranged from 53-84 (n=8).

This raises the possibility that mothers might not release all the eggs available in a single spawn. We now have information on the productivity and survival of the offsprings according to their colour strains (see Table 5) and most importantly, fecundity of individual females (for some examples, see Table 10). These data are

126 | P a g e very important for commercial farms, allowing them to record and forecast their production.

Males are able to mouthbrood a surprisingly large number of eggs (up to 58 eggs were collected from a single male). The ability to mouthbrood is an important asset to the male, and thus may indicate a possibility of a male mate choice in this species. All mate choice involving any sex is costly [202], thus setting “rules” for choosy individuals to discriminate potential mates [203] and this involves the expenditure of time and energy. Although females are usually the choosier sex [204], but under conditions where the investment in male parental care is high (as in the Asian arowana), the opposite might happen. This is because the effect of direct fitness costs is important for both sexes [202,203,205,206,207,208] and probably can be significantly more important in the Asian arowana.

From our results, in the ponds the breeding records of Asian arowanas do not show any correlation to the rainfall data (see Fig. 12). On the other hand, we do not have any data from their natural habitat. A correlation of the monsoon to the breeding season is common in the waters of Southeast Asia, where during the dry season, the scarcity of food often leads to higher mortality. On the other hand, during and after the monsoon, more food and cleaner water become available for the juveniles. We suspect that in the farm, the environmental cues do not play as important roles as in the wild due to the fact that the fish in the farm is fed more regularly and water change is more frequent and regular compared to the wild. There are also other environmental cues that are linked to the monsoon that we do not have any data, for thus, it would be impossible for us to relate any of the breeding events to the seasons.

From our data, we have also noticed a sharp drop in breeding during the months of

127 | P a g e July and August during 2003-2005, even though the rainfall was relatively higher than the previous or later months. We suspect that the noise from the fighter planes taking off from the nearby airbase negatively affected the breeding frequency but we do not have either proof or refutation for lack of such effect in some years. Together with the fact that the feeding regime for the brooders remains unchanged the whole year round, these data indicate that spawning activities of Asian arowana may be linked to other factors, like a regular feeding regime and water change with some cues from the environment.

Figure 31: The Asian arowana fits in the proposed correlation of mating system, sexual selection in sexual dimorphism. Pictorial definitions of four genetic mating systems possible in fishes. Lines connecting males and females indicate spawning partners that produced offspring. Also shown are the theoretical gradients in sexual- selection intensities and the degrees of gender dimorphism in secondary sexual traits (epigamic features) often associated with these mating systems. (picture obtained from Avise JC et. al. 36; 19-25, 2002 [16].

128 | P a g e Table 10: The fecundity of some females from WH001

Total Number of Offspring from Female Date Female Male Offspring Number Brooder 10-Apr-03 A2 B5 53 136 10-Apr-03 A2 A1 9 25-Jun-03 A2 B5 40 1-Sep-03 A2 B5 34 23-Oct-03 A6 C1 18 68 23-Oct-03 A6 A3 31 4-Feb-04 A6 C1 19 15-Apr-04 A8 B8 22 22 1-Jun-04 A9 B3 20 20

4.2 The Asian arowana male protects its eggs by transient morphological

modification of the surface of its buccal cavity during mouthbrooding

The Asian arowana is a carnivorous teleost that impairs its prey by pressing its toothed tongue against the tooth plate on the roof of its mouth cavity. It is indeed intriguing that despite these destructive features, and the males are still able to hold dozens of large-sized delicate embryos/larvae in their buccal cavity for 40-50 days without damaging them. It is unclear how the same organ is able to perform two completely different tasks without failing in either of them.

We studied the “safety measures” adopted by the father during the mouthbrooding process to prevent damage to the eggs caused by the pharyngeal teeth. We found that the epidermal layer of the mouthbrooding male has covered the two rows of entopterygoid teeth on the dorsal part of buccal cavity. This observation led us to hypothesize that the modification is important for the mouthbrooding and that it may have been triggered by the mating event. However, the mechanism by which the modification had taken place is unknown. 129 | P a g e We attempted to quantify this important observation, but conventional methods were all too destructive (Fig. 15; for example, sacrificing an individual, removing a portion of the embedded parasphenoid and measuring its surface profile using a scanning electron microscope). Such methods would also not allow us to monitor the change in an individual temporally. We managed to utilize a procedure used to produce dental imprints (see Section 2.13.2) combined with confocal microscopy techniques. This method allowed us to monitor the change as well as visually observe the changes in the surface profile in a male after removal of the larvae (see Fig. 26).

Such transient modification in the buccal cavity has never been described in any teleost or other organism to date. This modification only takes place during the mouthbrooding process and presumably would only occur in the sex that is mouthbrooding (i.e. the male in the case of Asian arowana). We compared the surface topography of the buccal cavity of five males using the confocal microscope.

Taking into account both the stage of the offspring and the surface topography (see

Fig. 26 for as typical example), we could see the reversion from the smoothened buccal cavity to the exposure of the teeth. The initiation of the modification in the buccal cavity may be the only trigger to start off the “smoothening” process and it also implied that the reversion to the exposed form starts when the signal decreases; thus implying that the reversion is not dependent on the timepoint when the offsprings were removed by hand. The modification is presumably an important event as mouthbrooding can only start when it has been fully completed. From our visual observation, the epidermal layer appears to have swollen and this stage persists throughout the mouthbrooding period. The „swelling‟ then subsides over a period of about 40 days before it reverts back to its normal state, thereby exposing the teeth.

130 | P a g e It would be interesting to find out the biochemical and physiological pathways that trigger and cause the modification. If inflammation causes the swelling, two candidate gene families may potentially be involved in this process, the mucin [209] and the ceruloplasmin genes [210]. Genes participating in processes necessary for oedemic swelling of tissues (e.g. tachykinins and calcitonin gene-related peptide) may also be related to the transient change in the buccal cavity of the Asian arowana

[211,212]. We are currently working together with Prof. Maxey Chung (Department of Biochemistry, National University of Singapore) on the proteomics analysis of the mucus and also tissue samples obtained from the buccal cavity of mouthbrooding individuals (during mouthbrooding and non-mouthbrooding stage) and non- mouthbrooding individuals to analyse the possible difference between the two stages.

4.3 The advantages of being able to sex the adult Asian arowanas

Prior to my studies, there wasn‟t a noninvasive field procedure available that would have allowed for the reliable sexing of Asian arowana. The ability to determine the sex of individual fish is very important both for aquaculture and conservation management. However, the determination of the sex of individuals without extensive stress or disruption of their ongoing activities is challenging. Sexual dimorphism of adult fishes with specialized reproductive behavior is usually highly visible; it includes differences in body shape, coloration, fin size or nuptial tubercles [4,200].

Sexual dimorphism is less obvious in some species[213,214] and there are also species that appear to show no visible external morphological differences between adult males and females. The latter species are of course, the most challenging to assess.

131 | P a g e In Asian arowana, farmers have observed that the paired fins of males are slightly longer than that of females. However, such a criterion requires the measurement and comparison of fin lengths of a large number of fish of each sex. In addition, this criterion would not be particularly useful to assess the sex of a given individual as physical damage (eg. broken fins) culture conditions and feeding regime often changes the physical features of the fish. Thus, there seemed to be no reliable method for sexing Asian arowanas based on their external morphology (refer to Figure 10).

We have now shown that it is possible to determine the sex of live adult Asian arowana from their external morphology, namely the ratios of Standard Length (SL) to Head Length, SL to Mouth Length, and SL to Gill Plate Length. This is in contrast to earlier reports stating that Asian arowana are sexually monomorphic [94]. Our success rate for determining the sex of the fish using the external morphometric ratio that we have developed was 94% (n=50; Figure 10).

One of the major limitations of morphological characteristics is that phenotypic variation can be subjected to environmental alteration, and is therefore not entirely under genetic control. Thus, the phenotypic plasticity of fish allows them to respond adaptively to environmental changes by modifiying their physiology and behavior which eventually lead to changes in their morphology, reproduction or survival to alleviate the effects of environmental variation [215]. These types of phenotypic adaptations may not necessarily result in genetic changes in the population, and therefore, may not be considered as evidence of genetic differentiation. For example,

Swain and colleagues [216] used morphological differences for the identification of hatchery and wild populations of Coho salmon (Oncorhynchus kisutch) and found that there were significant variations, which were attributed to the rearing environment rather than to genetic differences between hatchery or wild populations.

132 | P a g e Morphological characteristics have also been used in fisheries biology to measure discreteness and relationships among various taxonomic categories [217,218].

We started to search for a non-invasive biochemical sexing method which is known to be more accurate and precise than morphometry. There are publications in the peer- reviewed literature showing that the concentrations of steroid hormones or other compounds circulating in the blood of Atlantic salmon, Salmo salar, showed significant differences between the males and females [219]. A number of studies have demonstrated that biochemical sexing by ELISA testing of the blood plasma of fish can be highly accurate depending on the source population and the individual‟s state of sexual maturity [220,221,222,223]. However, laboratory analyses are relatively expensive and time consuming and most importantly, even the least invasive approach would require anesthetizing the fish and obtaining blood samples from the dorsal aorta near the caudal fin. Such analysis would typically be performed to determine the gonad developmental stage, which can be used to assess the maturation cycle [223].

In 2004, we started to test a less invasive approach by sampling the body mucus of the fish. By using a commercial hormonal test kit, we were able to differentiate the two sexes with a low error rate by following the 11-KT levels in the body mucus thropugh several months with repeated measurements without inflicting any harm on the fish, as the removal of the mucus from a small portion of the body surface would not result in any adverse effects. Concurrently, Schultz and colleagues also reported the use of surface mucus to detect 11-KT from several different fish species

[224,225,226]. They have also evaluated the mucus-based tests against traditional plasma-based methods of ELISA assay in detecting 11-KT [224,225,226].

133 | P a g e One important point that was discussed was the use of the ELISA assay as a single approach to determine the sex or reproductive stage of the fish. From our data, we have anticipated the fact that 11-KT is not a hormone exclusive to males as in addition to its role in spermatogenesis, it has been shown to be involved in spawning migrations, secondary sexual characteristics in both the sexes as well as heart and red muscle growth stimulation [60,227]. Our results show that the 11-KT levels vary temporally and this was also shown in a marine toadfish [228]. 11-KT was also found in all our female mucus samples that we collected but the levels were generally lower than those in the males. The averages of repeated measurements through 4-6 months period allowed us to estimate the sex of individuals with a relatively low error rate.

Furthermore, our genotypes and mitochondrial SNP haplotypes matched very well with the ELISA results. In addition, autopsy results of some brooders that had died earlier (18 out of the 400) have shown a shown full agreement with the sexes predicted based on the ELISA results. While studies on Atlantic halibut have shown positive correlation between the Gonadal Somatic Index (GSI, which is an indicator for the stage of gonadal development) to the 11-KT level [229], work on other species like freshwater eels have shown no such correlation [230].

As determining the GSI would require us to sacrifice of the individual in question, this method cannot be used on the Asian arowanas, due to the high price and rarity of a good brooder. Thus, we decided to assess whether the breeding events correlate with the 11-KT level since the former is the direct outcome of spermatogenesis and mating.

Social interactions are known to influence reproduction through regulation off ovulation [231] and are negatively correlated to the exhibition of parental care

[232,233,234].

134 | P a g e We were keen to determine whether there is any decrease in the level of 11-KT (the major androgen in fish) during the mouthbrooding period compared to other periods.

In the bluegill sunfish and St. Peter‟s fish, it was shown that the androgen levels increase during the courtship and decrease prior to fertilization and continue to stay low during the parental care phase [55,233]. In addition, 11-KT level peaks in threespine stickleback and bluegill sunfish during the breeding season [56,235] and pre-spawning period [233], respectively.

From our results, we were unable to correlate the 11-KT levels to the breeding events.

These results may be due to the fact that harvesting the brooders was tedious and time consuming; thus we were unable to collect the samples as and when we needed to but rather during harvesting only. As a result, our „snapshot‟ type data may not display the actual profile of androgenic levels of the individual brooders and because of this, the correlation cannot be established.

Figure 17 shows the average 11-KT concentration/total soluble protein of all the male and female brooders in one pond (WH001) from June 2005 to August 2007 (26 months). Data from other studies indicate that dominant individuals in a hierarchical system will benefit from higher reproductive success compared to other individuals ranked at a lower level (for example in some African cichlids) [236]. According to the „challenge hypothesis‟ of Wingfield [237], the androgen stimulation resulting from the interaction with con-specific males will determine the stability of a male‟s social environment and subsequently, the formation of a dominance hierarchy

[238,239].

Accordingly, dominant male(s) should have a relatively higher androgen level as they must be more aggressive to maintain their territory and most importantly achieve 135 | P a g e higher reproductive success. However, according to our findings, there is no obvious trend in the relative levels of 11-KT in terms of hierarchical structure of Asian arowanas where certain males (presumably dominant ones) are constantly placed high in a hierarchy, in terms of the 11-KT level. Therefore, despite the fact that we can sex the adult brooders using the ELISA-based 11-KT assay, we are not able to correlate the hormone levels either to the breeding event, or to the breeding seasons.

From our results (Figure 17), we can see that the ELISA-based 11-KT assay allowed for the identification of a male if the 11-KT concentration in the mucus was high in at least one of several samplings over a period of time. When an individual was assayed a few times and has fluctuating 11-KT concentrations, we usually took the highest 11-

KT concentration and compared it to the database of 11-KT levels of confirmed males and as long as there was one result that pointed to maleness, it was tagged as a male.

Although we are not able to accurately sex the adult brooders by using the ELISA assay alone, we were able to increase the efficiency of the process by applying the

ELISA results together with those of morphometry, breeding relationships and the mtDNA haplotyping (for MG strains).

The development of our simple and non-invasive procedures for sexing the Asian arowana provides tremendous potential for the management and basic scientific studies of the species, both in the wild and in farms. There would also be a substantial advantage for conservation, stocking or other management procedures. Commercial aquaculture production of Asian arowana depends extensively on whether fish are placed in ponds at the optimum sex ratio for breeding or whether breeding pairs of known sex can be placed together to increase the chances of breeding. Similarly, the production of broodstock will be greatly enhanced upon verification of the sex of

136 | P a g e individual brooders. The stocking of fish for restoration or rehabilitation is also expected to improve once the sex of individual fish is known. By linking the sexing procedure to information regarding the activity of brooders in the pond, one can also improve the yield and/or quality of offspring by breeding more active/dominant males with the more productive females.

4.4 The change in the breeding relationships in a pond after the death of a

highly productive male indicates the presence of a complex hierarchical

breeding system

In pond WH001, there was a sudden stop in the breeding activities after the death of a highly productive male, B5 (see Fig. 21). We were not able to relate this ceasation to any disease outbreak as the rest of the fishes in the pond were all found to be healthy without showing any external or internal sign of infection.

This sudden change in the breeding frequency may be due to the presence of a hierarchical system in the pond where aggressive dominant males enjoy higher reproductive success over the rest by occupying territories and preferential mating rights over some of the females [237,239,240,241]. Thus, we assumed that after the loss of B5 male, the remaining individuals started a new competition for territorial and mating rights and that period lasted for six months before a stable social environment was established again. It is known that dominance hierarchy in some teleost species is tightly linked to the right to mate [238,242]. We propose that there is a hierarchical system in place for this species but we were unable to use conventional methods whereby fishes can be placed in tanks for observation and collection of visual data can be done via video records. Therefore, we attempted to collaborate with a RFID company to construct a system where a personalized active

137 | P a g e tag would be placed into each brooder. A tracking system would be able to record the movement of each brooder and present it in a grid system real time to show the relative interaction of the brooders. With this data, we would be able to know when the fish are mating and mouthbrooding, and most importantly, what interactions happen during the mouthbrooding and mating event. Unfortunately, this collaboration was not pursued further due to lack of funding, expertise and commitment from the

RFID company.

4.5 Our data do not show inbreeding or incompatibility avoidance, indicating

unique mate choice and strategy

From our findings, we now know that Asian arowana exhibits predominantly a polygynandric mating system. The theory of sexual selection suggests that males enhance their reproductive success by mating with multiple females, and females in turn will choose males that will provide superior genes (so as to improve the genetic quality of the progeny) or optimal resources [243,244]. This sexual selection theory has been under scrutiny as this applies to species that have a difference in gamete investment, thus leading to a differentiation in mating tactics [244]. The female will choose males that are able to provide “good genes” or provide genes that are complementary to her own based on phenotypic or other cues, an event which eventually increases the heterozygosity of the progenies [245].

Others have shown that polyandry had evolved to increase the chance of the viability of the progeny in the long term [246,247,248,249]. The “good genes as heterozygosity” hypothesis was mainly derived from inbreeding avoidance [250] which ultimately leads to inbreeding depression. Studies have been performed in

138 | P a g e Atlantic cod [251], guppies and other teleosts to understand these theories and their relation to the Teleostei group [252,253,254,255,256].

We used dissimilarity index for both sexes (see Fig. 22 and 23), in order to investigate whether Asian arowanas exhibit some form of inbreeding avoidance and thus a strategy to cope with genetic incompatibility. Our results do not support the possibility that inbreeding avoidance would be the driving force in the pre- or post- copulatory mate choice as we observed that several brooders were breeding with partners that were the most genetically similar choices from the whole pond.

There could be three main reasons why inbreeding avoidance was not seen in our species. Firstly, although our pond setup mimicked their endemic habitat and environmental cues are present, it is nevertheless an artificial enclosure where the choice of mating partners is restricted. Secondly, we have not found many concrete examples in the literature citing that inbreeding avoidance does occur in teleosts, most of them studied the effect of major histocompatibility complex

(MHC;[257,258,259,260,261,262,263,264,265]) on inbreeding avoidance/genetic incompatibility which is known to be an important genetic factor in mate choice

[202,261], sexual selection [266,267,268,269,270] and most importantly in inbreeding avoidance [265,270,271]. MHC is a large genomic region or gene family found in most vertebrates including fishes. It is the most gene-dense region of the mammalian genome and plays an important role in the immune system and autoimmunity. The diversity of MHC is important in the immune diversity in the population. Lastly and most importantly, the extreme form of parental care and the low number of extremely large embryos in the Asian arowana may indicate that they are in the midst of an evolutionary shift from one mating strategy to another, and they might be at a point

139 | P a g e where mate choice is neither solely maternal nor paternal. This could explain the lofty investment from both the genetic parents which directly affects the offspring fitness is

“balanced” compared to many other teleost species, where the female usually invests far more in the production of the next generation than the male. Many other factors such as the health status of the partners [197], availability of territories [272,273,274], adjustment of breeding strategies according to the environment or availability of resources [63,272,275,276], age-dependent [277,278,279], and size-dependent mating strategies [280,281], could all affect the breeding event and also mate choice of both sexes.

4.6 Egg thievery: Why do Asian arowanas steal each other’s eggs?

In the 147 clutches of offspring genotyped, we found four that were not mouthbrooded by their biological father (see Figs. 20 and 24). Our genotyping data showed that two out of the four clutches of “stolen” offspring were mouthbrooded solely by the thief, whereas the other two clutches were split between the genetic father and the thief (see Fig. 24).

Egg thievery is a reproductive phenomenon that has been documented for a few teleosts, mainly sticklebacks [282,283,284] and in the marine teleost blenny [285]. A review by Avise and colleagues [16] listed many possible reasons for egg thievery or piracy. They are as follows: a) Predator–dilution effect: increasing the number of eggs in a nest will decrease the predation of the guardian‟s own eggs [286]; b) Pump- priming effect: females preferentially spawn in nests already containing eggs [283]; c)

Kin selection: genetic siblings who may be more successful in rearing the offspring resulting in a overall increase in inclusive fitness for the genetic parents

[200,287,288]; d) Larder-stocking effect: stealing eggs to provide nutrition for itself at

140 | P a g e a later stage [16]; and e) Cooperative breeding: sharing of brood-care duties by helpers [74,81,289,290,291].

In the Asian arowana, (a) is unlikely as our results showed single paternity in all the

147 clutches that were genotyped. Moreover, these thieves were not mouthbrooding their own embryos and thus could not directly benefit from protecting them. For (b) it is possible that the thieves were demonstrating the ability to sire and execute paternal care to the mates as 50% of the thieves that have not bred before did so after the thievery event. Out of the four clutches, one clutch was at ca. day 10 larval stage, while three clutches were already at day 25 larval stage. This might indicate that egg thievery could be part of a complex strategy like in sand gobies [292] where parental care is used as a form of courtship. With regards to (d), the thieves could have easily consumed the eggs for nutrition since they were not mouthbrooding their own embryos, the fact that they were found mouthbrooding them instead indicates other possible reasons. In addition, mouthbrooding is a form of parental investment that comes with a hefty cost such as decrease in mating and foraging opportunities [293] and increased chances of mortality due to attacks or predation [294] and/or starvation.

These reasons do not justify an investment on the part of the thief in order to obtain nutrients. Another possible reason is that these are “mouthbrooding ready” thief might have lost its own clutch recently and have snatched other breeding pair‟s eggs.

With regards to kin selection (c), our data do not indicate that the thieves may be more closely related to either one of the genetic parents than to the rest of brooders in the pond. We compared kinship coefficient (k) of all the other brooders in the pond to that of the respective genetic parents (see Fig. 25). The k value between the thief and either one of the genetic parents were not higher than the average k value. Although

141 | P a g e we cannot rule out this reason for the egg-thievery in the Asian arowana, our findings do not seem to support it. Another possible reason for the egg thievery, as stated in

(e), is the “pay-to-stay” phenomenon [204,289,291] where subordinate groups

(helpers) will need to assist the dominant group in order to be tolerated and to survive in the territory. This is prevalent in some teleosts that exhibit territoriality [295]. We could not set up an experiment to test this hypothesis in the Asian arowana and thus we are not able to ascertain this possibility.

It is also possible that this unusal behaviour of the Asian arowana may be a form of

“preparation” and “training” for the challenging mouthbrooding period, in which the fish could possibly face starvation, risk of attack from other brooder as well as the loss of opportunity to breed. These are likely to stress the male at both physiological and psychological levels. The starvation process was described to affect the metabolic capacities of both the white and red muscles in cod (Gadus morhua) and swimming endurance also decreased by 70% [296]. Egg thievery could also simply be a form of display by the male to show off its ability to care for the offspring, thereby attracting females to breed. One point to note that is the two suggested reasons are true, we would expect to see a higher frequency of thievery happening.

Our data show that two of the “thieves” had never bred before till present and also in one pond, all the thievery events occurred with the offspring of one female from three different males (see Fig. 24). This may indicate the incapability of this particular female to be able to assist in guarding the eggs until they are taken by the genetic father for mouthbrooding.

142 | P a g e 4.7 Asian arowanas provide another example for a positive correlation between

egg size and parental care

Asian arowanas produce most likely the largest eggs with the longest larval developmental time among all freshwater teleosts. Bonislawska and colleagues [297] compared the egg diameter of various fishes and none of the freshwater teleosts showed larger egg diameter than that measured for the Asian arowana. There are many benefits of having larger eggs: it has been shown that egg size, being an important determinant of larval size and fitness, is positively correlated to the parental care, and these includes higher survival and resistance to starvation and increased mobility [298,299].

According to Sargent and colleagues [294] larger eggs should have a lower surface area to volume ratio than smaller ones and if the fish egg‟s oxygen demand is proportional to its volume, this should affect respiration by negative diffusion. Their hypothesis was later challenged by Einum and colleagues [300] who showed that at low oxygen concentration larger eggs had higher survival rates than the smaller eggs containing the same biomass. According to their explanation the increase in egg volume is not proportional to oxygen demand and increase in egg size may decrease the metabolic rate, resulting in decreased oxygen demand in larger eggs [300].

Kolm and colleagues [301] in their review paper commenting on the correlation of egg size and parental care then questioned the theory on the increase in parental care of larger eggs eventhough there is data to show that in flagfishes and sand gobies, parental males will change it fanning behavior and nest building in order to increase the oxygen available to the embryos in a low oxygen environment [302,303].

143 | P a g e One possible explanation is that larger eggs will require longer developmental time

[301] and the “safe harbour” hypothesis also states that larger eggs will benefit from a longer residence in the protected state due to the longer developmental time [304].

Thus, the observations in the Asian arowana seem to support the hypothesis where the production of large eggs is compensated with the high parental care (provided through mouthbrooding) and the long developmental time as described in my results.

4.8 Genetic confirmation of a paternal care in the Malaysian golden variety of

Asian arowana

The taxon of Osteichthyes (bony fishes) contains 422 families, out of which 89 (about

21%) exhibit some form of post-zygotic parental care [23]. Blumer [23] has indicated that nearly 70% of these 89 families shows paternal rather than maternal care, but according to his review, the family of Osteoglossidae exhibited either biparental or maternal care which recent data – including our own - have shown otherwise (see

Table 1). This might indicate that Blumer‟s comprehensive compilation is in need for an update.

Mouthbrooding is a rare form of parental care in fishes (occurring in only eight out of

89 families); however strict paternal mouthbrooding is even more unique, as it has only been observed in three families – Osphronemidae, Ariidae and Cichlidae - out of

422 [23]. One of the longstanding problems we managed to solve was the confirmation of the sex of the mouthbrooder in the Asian arowana. This issue was particularly difficult to elucidate for the following reasons: i) Asian arowanas do not exhibit obvious visual signs of sexual dimorphism; ii) the price of individual brooders is extremely high (estimated to be in the million dollar range); iii) lethal sampling of

144 | P a g e the fish for gonadal examination is not feasible due to its inclusion in numerous protected species lists; and iv) the lack of extensive research on its breeding biology.

Our novel approach using the mtDNA SNP haplotyping (see section 2.11) has confirmed that the MG colour strain of the Asian arowana exhibits paternal mouthbrooding. Due to lack of suitable samples, we have not been able to confirm the sex of the mouthbrooder in the other strains; however, we do not expect any intraspecific variation. Although earlier some breeders claimed to have observed biparental mouthbrooding in the MG strain, this information has never been scientifically verified.

Commercially, this is an important finding, as the farmers can confirm the sex of each individual in a breeding pair at the first mouthbrooding event. Moreover, this is an important advancement towards gaining a deeper understanding of the breeding biology of the species.

4.9 Strong positive effect of a mating strategy involving multiple mates, number

of reproductive events (broods) and lack of difference between the sexes

Teleosts are likely to exhibit the most vibrant variety of mating strategies and tactics among all vertebrates [16,17,305]. In this study, we refer to “mating strategy” as the set of genetically encoded “regulations” employed by an organism to guide its reproductive behavior throughout its lifetime, whereas a mating tactic/behavior is a set of conditional activities used in order to lead to successful reproduction [200].

There are many different mating tactics utilized by teleosts in order to ensure breeding success. One of the most important points to note is the difference in the contribution to the fertilized embryo in a breeding event. In many teleosts, breeding involves

145 | P a g e minimal investment from the male due to the less energetically expensive milt production compared to the high investment from the female

[202,278,306,307,308,309,310,311,312]. In the Asian arowana, the investment from both sexes seems to be in equilibrium as the female produces large eggs and the male exhibits extreme level of parental care which will last for 40-50 days. Thus, neither of them is expected to be able to mate again within a short period of time. In order to determine the presence of a mate choice and the reasons why polygyandry occurs, in collaboration with Dr. Rob Brooks (University of New South Wales, Sydney,

Australia) we have studied the effect of this mating system on the fitness of the brooders. By analyzing our data, Dr. Brooks has shown that mating in the Asian arowana does not occur by chance and that mating with the same mate is higher than the expected chance. In addition, both the sexes show some partner fidelity or at least consistency of choice (see Fig. 32). Although not all fish species seem to practice mate choice [200], male parental care and female investments rather than physical factors (e.g. mate size, parental experience, or costly epigamic features), may force both sexes to exercise mate choice in the case of the Asian arowana.

We propose that further studies on this species should look into the major histocompatibility complex (MHC)-based mate preference

[202,258,260,261,262,267,306,313,314]. In a reproductive success study [258] involving brown trout (Salmo trutta L.), using microsatellite based parentage analysis, it was found that pairs with intermediate MHC dissimilarity mated more often than expected by chance. This is in contrast with the results found in several other fish species [263,315] where mating occurs with maximal MHC dissimilarity [260]. With the help from Dr Robert Brooks, we further studied, whether a possible fitness advantage could be gained by increasing the number of offspring produced and also 146 | P a g e whether this is different between the two sexes. His results showed that there were no statistical differences between the sexes, between the different ponds sampled and also the effect of the two on the total offspring production (F test (df =2), between sexes P >0.526; between ponds P>0.323; sex by pond P>0.261). Moreover, there is statistical support for the fact that the increase in the number of mates is positively correlated to the increase in offspring production (Fig. 32).

In our case, the presence of more mating partners for a given brooder correlated with more broods of offspring. In order to exclude the possibility of a random coincidence,

Dr. Brooks tested whether both events (having more broods and more mates) were positively correlated with the offspring production. The statistical analysis showed that both the increasing number of broods as well as the increasing number of (unique) mates affected the total offspring production positively and again, this observation was independent of the sex of the fish (t test, number of unique mates P<0.003; number of separate broods P<0.001). Thus, in Asian arowana, there seems to be a cohesive and cooperative approach towards increasing the offspring production, which is important for such species where mating strategy is based on low fecundity and high parental care. Mate choice may also be strongly affected biochemically and this might shed some light on the presence of different mating systems present in the

Asian arowana. Studies involving characterization of vasopressin gene in relation to faithfulness in pairie voles showed that vasopressin has a role in male social behavior

(e.g. communication, aggression, sexual commitment and paternal care of offspring).

Vasopressin antagonists were administered into monogamous prairie voles (Microtus ochrogaster) and their promiscuous relatives, the non-monogamous meadow vole

(Microtus pennsylvanicus). When vasopressin antagonists were administered to male prairie voles, they became promiscuous, however, vasopressin administration to male 147 | P a g e meadow voles had no effect on their mating habits. The results led to the conclusion that the number of vasopressin receptors would determine the promiscuity of the species [316,317]. Preliminary data has also indicated the existence of such mechanisms in humans [318].

In species that exhibit other kinds of parental care tactics, like nest guarding, (for example, cichlids, sirulids, and anabantids), such an approach would not be as efficient. The reason for this is that the chances for the male to mate with other partners even during the parental care period would still be possible. For species that exhibit biparental care, which normally evolves from paternal care, even if the male abandons the brood, the female will still be able to tend the nest [73,319,320,321]. In

Asian arowana, there would be no logical explanation for the male to abandon the mouthbrooded offspring to look for other mating partners. Thus, this strategy of mating with more partners is optimal for the ultimate aim of passing down its genetic material to as many individuals, thereby increasing genetic diversity.

148 | P a g e

Figure 32: There is an advantage in having more mates in both Asian arowana sexes. Total offspring produced were plotted against the number of unique mating partners and brooder has mated with and also against the number of separate broods of offspring. Regression analysis shows that there is a strong positive effect indicating more mates and more reproductive events (broods) equates to more offspring and when we compared the interaction term which formally test the different regression slopes, the effect is not different between the sexes. (This analysis was done by Dr Robert Brooks of UNSW, Sydney Australia, using our data.)

4.10 Genetic analysis detects distinct differences between Asian arowana strains

The study of the genetic relationships of the colour strains of the Asian arowana increases our knowledge about the evolution and relatedness. From our preliminary data obtained using microsatellite genotypes alone (Fig. 27), and a Bayesian cluster analysis using microsatellite genotypes and AFLP markers (Fig. 30), we were clearly able to differentiate the colour strains genetically and this is crucial in their identification at juvenile stage when they are nearly identical in their colour and

149 | P a g e phenotype. AFLP markers and microsatellites have been used by others earlier in assessing genetic diversity [322,323] but with the ultimate aim of constructing a linkage map for commercially important species [324].

Cluster analysis based on the microsatellite genotypes separated the colour strains into two main groups (Fig. 27). The controversies observed with Indonesian (IG) and

Malaysian golden (MG) varieties may have been caused by the high level of similarity of these two colour strains not only genetically, but phenotypically as well.

Microsatellites are normally used for parentage assignment and they are also excellent tools for generating genetic linkage maps [113,114,325]. In our case, these genotypes allowed us to differentiate some of the colour strains that would otherwise be difficult to identify.

Until now, identification of the different colour strains was done by “experts” in the farm who look for subtle phenotypic differences. It is generally known that differentiating color strains are difficult in some cases, and that there have been some mis-classification of individuals. There are also cases where fishes were willfully altered by chemical treatment which further aggravate the problem. These mis- classifications caused problems for commercial trading as well as breeding. Thus, the highly efficient system of identification using the assignment test in our initial studies is a promising tool for future identification of different colour strains by genetic methods.

Geneotyping data produced by eight polymorphic microsatellite loci and 225 AFLP markers was also analyzed in combination. From the Bayesian clustering approach

(Fig. 32), in both data sets, we were able to differentiate the RG1 and WRG1 clusters

(cluster 2) from the rest of the clusters and also MG (cluster 4) from the IG cluster 150 | P a g e (cluster 1). On the other hand, we were not able to differentiate the IG, RG2 and GR

(cluster 1), as they were grouped into one cluster. The Mensiku strain (MJK) formed an isolated cluster (cluster 3). Interestingly, we are able to see that the hybrid showed contributions from cluster 2 and 4, even though it was placed into cluster 4.

We have managed to use a novel method developed earlier by us, FluoMEP [153] to discover two markers that are able to differentiate between RG1 and RG2. This is commercially important as the price difference between these two varieties is the highest amongst the different colour strains, at the same time phenotypically, they are the most similar during juvenile stage (see Section 1.3 in the Introduction). Our results (Fig. 28) show that FluoMEP proved to be an improved method compared to other blind screens as by using only 10 primer combinations, we were able to find two molecular markers that allowed us to detect differences between these two strains.

We expect to enhance the efficiency and speed of this method further through the targeting ability of „common primers‟ so that we can use specific primers to target sex-determining regions on the basis of conserved regions of orthologs from related species or known sex genes.

4.11 The divergence of the different colour strains seems to be consistent with the

change of the land mass configuration of South East Asia

Using bathymetric depth contours, Harold [180] estimated the changes in the shore lines of the Sunda and Sahul shelves (which mainly encompass the present South East

Asia). Using the different depth contours, he also estimated the duration and timings of these changes in the sea level, which eventually gives us an indication of the connection of the different islands in South East Asia [180].

151 | P a g e Using our microsatellite data, we constructed a dendrogram that revealed a clear grouping pattern of the Asian arowana varieties (Fig. 29D). The green arowana (GR) remained a basal group and interestingly, it is the only colour strain that is found throughout the islands. Our group is currently studying the phylogenetic relationships between all the GR isolates found in the different geographical locations, the colour strains and the species within the subfamily Osteoglossinae [326] by using extensive sequence information from mitochondrial and nuclear sequences.

By comparing our results with the geographical data from Harold [180], we can clearly define the two major clusters of Asian arowanas. The first one belongs to the

Kalimantan island (Clade1; RG 1 and MJK), the Sumatra island and peninsular

Malaysia (Clade 2; Indonesian golden and Malaysian golden). On the other hand,

RG2, which is only found in the Banjarmasin river system that is not connected to the

Kapuas river system, is not clustered to any clade. This dendrogram is consistent with the estimated changes in the land mass configurations over the past 17,000 years

[180]. This might indicate a possibility that the colour strains may have started to diverge from a common basal colour form in order to adjust to the environmental cues and restrictions or possibly due to natural selection that favored certain populations to adapt to the ecological niches. This may be the reason why different colour strains do not co-exist for the other members of the subfamily Osteoglossinae (for example, in the Amazon basin, the endemic habitat of the silver and black arowana). Although there are niche populations along the whole river system, there do not seem to be any observable phenotypic difference between these two species.

The development of the various colour strains may be a sign of speciation in progress

[322,327]. We hypothesize that the species might be at an early stage of this process

152 | P a g e as we have observed from our data that intraspecific breeding is readily achievable and due to the geographical barrier that exists uniquely to our species. Thus, colour strains may have started to evolve (in terms of size, colour, and mating tactics) in order to adapt to the harsh environment [7,29,62] of the South East Asian river systems, resulting in and phenotypically deviation from the basal phenotype. This data is important as it provides information on the current evolutionary status of the species as well as how the colour strains may have formed. Commercially, it allows breeders to take into consideration the genetic relationship between the colour strains when a breeding program is set up with the aim of genotyping hybrids.

4.12 The different colour strains are likely to be geographically isolated

populations and not different species

From our results (Table 9), we observed a significant difference in FST values between the three species (Scleropages jardini, Scleropages leichardti and Scleropages formosus) compared to the different populations of the Asian arowana. This data serves a reference for us to compare the FST of the different colour strains and to determine whether the colour strains are independent species or simply different colour forms of the same species. Some of the FST values between the colour strains were not as low as observed among others and showed significant differences from the inter-variety average of 0.237 (p<0.05; WRG1 vs. MG, MJK vs. MG, and RG1 vs.

MG). One of the reasons for this may be that although the polymorphic loci used were sufficient for our parentage assignment, they may have been insufficient for the analysis of genetic similarity [141,328].

Another possible reason for such difference in genetic similarity between the different populations of Asian arowana was found after comparing our data and the land mass

153 | P a g e divergence data [180]. The different populations may have diverged about 17,000 years ago and have been geographically isolated since. The smallest genetic differences determined by the low FST value of the GR vs. RG2 indicates a relative stability within these two strains as they are found in the same river system on the island of Sumatra. A low degree of differentiation observed between the TY vs. the two golden strains (MG and IG) were expected, as TY is actually a cross between

RG1 and either one of the golden strains. The highest average FST values when compared to the rest of the colour strains was the MG, this may be due to the isolation of this species on Peninsular Malaysia without the presence of all the other colour strains occurring naturally in the river systems.

The pairwise comparison of the FST values between the MJK and RG1 was higher than its distance to the other strains. This was unexpected, as MJK is the only other strain found in the same river system (Kapuas River) on the island of Kalimantan.

The pairwise comparison of FST value of the Asian arowana and its closest relative species, Scleropages leichardti and S. jardinii, clearly shows that most differences within the colour strains (0.124) are substantially lower than the differences between the species (0.406). This indicates that different populations of Asian arowana are unlikely to be different species, instead just isolated populations of the same species.

Farms have also shown that the colour strains interbreed readily whilst to our knowledge; there have not been successful breeding events between the three

Scleropages species. In previous publications [90,91], S. leichardti and S. jardinii were both described as female mouthbrooders and this might be one of the reasons why interspecific hybridization has not occurred yet.

154 | P a g e 5 POSSIBILITIES FOR THE FUTURE

5.1 Selective breeding program – production of the first hybrids in preparation

for linkage mapping

Based on the knowledge obtained from this study, we have started the selective

breeding program for commercial interest as well as for research purposes. We have

selected the two combinations of the most distant colour strains (IG and MJK and MG

and MJK) for hybridization. The sex of brooders was identified using the two

methods developed earlier by us (ELISA and morphometry) and ponds were stocked

with males from one colour strain and females from the other and vice versa. After

six months, we have managed to obtain the first batch of IG and MJK hybrids (n=22).

These individuals will be grown and used for of the generation of the first genetic

linkage map from the Asian arowana.

5.2. Additional tasks

With the help of molecular tools, we have unveiled many important discoveries with

regards to the biology of the Asian arowana. The data we have obtained can now be

used for further understanding of not only the reproduction, but several additional

aspects of the secretive life this species (e.g. phylogeny and conservation). In the

future, the following problems will require additional research efforts: i) the

phylogenetics and phylogeography of the species will need to be better understood,

with special emphasis on the varieties, in order to clarify their relationship to each

other; ii) the conservation of the species will have to be ensured and that requires the

determination of the current genetic diversity in the wild, as the presently available

samples from wild-caught individuals is a mixture of old and recently collected

155 | P a g e specimen; iii) cDNA libraries need to be generated and large sets of clones sequenced in order to identify genes of interest; iv) screens muts be performed in order to identify molecular sex markers allowing for rapid sexing of juveniles; and perhaps most importantly, v) biochemical and physiological processes leading to the transient modification of the buccal cavity must be studied in order to understand the regulation of these processes.

The future improvements resulting from the above research activities are expected to provide additional help for the progress of the commercial aquaculture of Asian arowana and hopefully, they will also yield improvements in the conservation of this

CITES-protected species.

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283. Jones AG, Ostlund-Nilsson S, Avise JC (1998) A microsatellite assessment of sneaked fertilizations and egg thievery in the fifteenspine Stickleback. Evolution 52: 848-858. 284. Largiader CR, Fries V, Bakker TCM (2001) Genetic analysis of sneaking and egg- thievery in a natural population of the three-spined stickleback (Gasterosteus aculeatus L.). Heredity 86: 459-468. 285. Neat F, Locatello L (2002) No reason to sneak: why males of all sizes can breed in the hole-nesting blenny, Aidablennius sphinx. Behav Ecol Sociobiol 52: 66-73. 286. Ulrika C, Heinz-Rudolf V (1998) Predator-induced nest site preference: safe nests allow courtship in sticklebacks. Anim Behav 56: 1205-1211. 287. Connor RC (1996) Partner preferences in by-product mutualisms and the case of predator inspection in fish. Anim Behav 51: 451-454. 288. Pitcher TE, Neff BD, Rodd FH, Rowe L (2003) Multiple mating and sequential mate choice in guppies: females trade up. P Roy Soc B-Biol Sci 270: 1623-1629. 289. Bergmüller R, Heg D, Taborsky M (2005) Helpers in a cooperatively breeding cichlid stay and pay or disperse and breed, depending on ecological constraints. P Roy Soc B-Biol Sci 272: 325-331. 290. Brouwer L, Heg D, Taborsky M (2005) Experimental evidence for helper effects in a cooperatively breeding cichlid. Behav Ecol 16: 667-673. 291. Taborsky M (1985) Breeder-helper conflict in a Cichlid fish With broodcare helpers: an experimental analysis. Behaviour 95: 45-75. 292. Pampoulie C, Lindstrom K, St. Mary CM (2004) Have your cake and eat it too: male sand gobies show more parental care in the presence of female partners. Behav Ecol 15: 199-204. 293. Williams GC (1975) Sex and evolution. Princeton, NJ: Princeton University Press. 195 p. 294. Sargent RC, Taylor PD, Gross MR (1987) Parental care and the evolution of egg size in fishes. Am Nat 129: 32. 295. Balshine-Earn S, Neat FC, Reid H, Taborsky M (1998) Paying to stay or paying to breed? Field evidence for direct benefits of helping behavior in a cooperatively breeding fish. Behav Ecol 9: 432-438. 296. Martinez M, Guderley H, Dutil JD, Winger PD, He P, et al. (2003) Condition, prolonged swimming performance and muscle metabolic capacities of cod Gadus morhua. J Exp Biol 206: 503-511. 297. Bonisławska M, Formicki K, Korzelecka-Orkisz A, Winnicki A (2001) Fish egg size variability: biological significance. Electron J Polish Agri Uni 4. 298. Ahnesjö I (1992) Fewer newborn result in superior juveniles in the paternally brooding pipefish (Syngnathus typhle L). J Fish Biol 41: 53-63. 299. Einum S, Fleming IA (1999) Maternal effects of egg size in brown trout (Salmo trutta): norms of reaction to environmental quality. P Roy Soc B-Biol Sci 266: 2095-2100. 300. Einum S, Fleming IA (2002) Does within-population variation in fish egg size reflect maternal influences on optimal values? Am Nat 160: 756-765.

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301. Kolm N, Ahnesjö I (2005) Do egg size and parental care coevolve in fishes? J Fish Biol 66: 1499-1515. 302. Hale RE, St. Mary CM, Lindström K (2003) Parental responses to changes in costs and benefits along an environmental gradient. Environ Biol Fish 67: 107-116. 303. Lissaker M, Kvarnemo C, Svensson O (2003) Effects of a low oxygen environment on parental effort and filial cannibalism in the male sand goby, Pomatoschistus minutus. Behav Ecol 14: 374-381. 304. Shine R (1978) Propagule size and parental care: the "safe harbor" hypothesis. J Theoret Biol 75: 417-424. 305. DeWoody JA, Fletcher DE, Wilkins SD, Nelson WS, Avise JC (2000) Genetic monogamy and biparental care in an externally fertilizing fish, the largemouth bass (Micropterus salmoides). P Roy Soc B-Biol Sci 267: 2431-2437. 306. Aeschlimann PB, Haberli MA, Reusch TBH, Boehm T, Milinski M (2003) Female sticklebacks Gasterosteus aculeatus use self-reference to optimize MHC allele number during mate selection. Behav Ecol Sociobiol 54: 119-126. 307. Albert AY (2005) Mate choice, sexual imprinting, and speciation: a test of a one- allele isolating mechanism in sympatric sticklebacks. Evolution 59: 927-931. 308. Aspbury AS, Gabor CR (2004) Differential sperm priming by male sailfin mollies (Poecilia latipinna): Effects of female and male size. Ethology 110: 193-202. 309. Head ML, Hunt J, Jennions MD, Brooks R (2005) The indirect benefits of mating with attractive males outweigh the direct costs. Plos Biol 3: 289-294. 310. Howard RS, Lively CM (2004) Good vs complementary genes for parasite resistance and the evolution of mate choice. BMC Evolution Biol 4. 311. Jordan WC, Bruford MW (1998) New perspectives on mate choice and the MHC. Heredity 81: 239-245. 312. Schutz D, Taborsky M (2000) Giant males or dwarf females: what determines the extreme sexual size dimorphism in Lamprologus callipterus? J Fish Biol 57: 1254-1265. 313. Rajakaruna RS, Brown JA, Kaukinen KH, Miller KM (2006) Major histocompatibility complex and kin discrimination in Atlantic salmon and brook trout. Mol Ecol 15: 4569-4575. 314. Sommer S (2005) Major histocompatibility complex and mate choice in a monogamous rodent. Behav Ecol Sociobiol 58: 181-189. 315. Sato A, Satta Y, Figueroa F, Mayer WE, Zaleska-Rutczynska Z, et al. (2002) Persistence of MHC heterozygosity in homozygous clonal Killifish, Rivulus marmoratus: implications for the origin of hermaphroditism. Genetics 162: 1791- 1803. 316. Lim MM, Wang Z, Olazabal DE, Ren X, Terwilliger EF, et al. (2004) Enhanced partner preference in a promiscuous species by manipulating the expression of a single gene. Nature 429: 754-757. 317. Smeltzer MD, Thomas Curtis J, Aragona BJ, Wang ZX (2006) Dopamine, oxytocin, and vasopressin receptor binding in the medial prefrontal cortex of monogamous and promiscuous voles Neurosci Letters 394: 146-151.

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318. Cherkas LF, Oelsner EC, Mak YT, Valdes A, Spector TD (2004) Genetic Influences on Female Infidelity and Number of Sexual Partners in Humans: A Linkage and Association Study of the Role of the Vasopressin Receptor Gene (AVPR1A). Twin Res Hum Genet 7: 649-658. 319. Manica A (2002) Filial cannibalism in teleost fish. Biol Rev 77: 261-277. 320. Mank JE, Promislow DEL, Avise JC (2005) Phylogenetic perspectives in the evolution of parental care in ray-finned fishes. Evolution 59: 1570-1578. 321. Reynolds JD, Goodwin NB, Freckleton RP (2002) Evolutionary transitions in parental care and live bearing in vertebrates. Philos T Roy Soc B 357: 269-281. 322. Volff JN (2004) Genome evolution and biodiversity in teleost fish. Heredity 94: 280-294. 323. Yue GH, Li Y, Chen F, Cho S, Lim LC, et al. (2002) Comparison of three DNA marker systems for assessing genetic diversity in Asian arowana (Scleropages formosus). Electrophoresis 23: 1025-1032. 324. Chistiakov DA, Tsigenopoulos CS, Lagnel J, Guo YM, Hellemans B, et al. (2008) A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L. Anim Genet 39: 623-634. 325. Sakamoto T, Danzmann RG, Gharbi K, Howard P, Ozaki A, et al. (2000) A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics 155: 1331-1345. 326. Lin QF (2009) Molecular analysis of osteoglossid teleosts. Singapore: National University of Singapore. 327. Taylor JS, Van de Peer Y, Meyer A (2001) Genome duplication, divergent resolution and speciation. Trends Genet 17: 299-301. 328. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, et al. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2: 225-238.

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7 SUPPLEMENTARY TABLES

Supplementary table 1: Master list of QH ponds and its involvement in the different experimentation.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 WH001 • • • • • • • • • • • • • • • WH002 • • • • • • • • • WH003 • • • • • • • • WH004 • • • • • • WH005 • • • • • • • • WH006 • • • • • • • •

Legend :

1 Observation of mating 2 Offspring survival between colour strains 3 Breeding frequency following rainfall 4 Embryonic and larval development 5 External morphometric study showing differences between two sexes 6 Internal morphometry of buccal cavity transformation in mouthbrooding males 7 Hormonal measurements from mucus 8 Identification of the sex of the mouthbrooding parent in the MG 9 Genotyping study on relationships in the ponds 10 Change in breeding pattern after loss of a male 11 Inbreeding or incompatibility avoidance study 12 Egg thievery 13 Differentiation of the colour strains using microsatellite-based genotypes 14 Differentiatation of two commercially important colour strains using FluoMEP 15 Microsatellite-based phylogenetic tree of the colour variants 16 Differentiation of colour trains using Bayesian clustering analysis

17 Pairwise comparison of the FST value study

177 | P a g e

Supplementary table 2: PAPA results from WH002 family.

RESULTS

Collected parents file:D:\New Folder\PAPA results\WH002\WH002 adult D0438429210811531104105106109115119.txt

Offspring file:D:\New Folder\PAPA results\WH002\ospring p1 D0438429210811531104105106109115119.txt

Global level of transmission error:0

Distribution of transmission error:0

Names of loci file: D:\New Folder\PAPA results\WH002\name of loci D0438429210811531104105106109115119.txt

Choice of loci: D04 D42 D92 D108 D115 D31 D104 D105 D106 D109 D116 D119

Offspring

DZ1 165199 000000 147165 148162 223231 204212 229233 234234 242242 209233 204214 113113 155181

Parents

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189

*************************************

Offspring

DZ2 199203 000000 165165 164164 223231 204212 229233 234234 242242 209233 214214 107107 189189

Parents

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189

*************************************

Offspring

DZ3 199199 000000 147165 164164 223223 204212 229233 000000 242242 199209 214214 113113 000000

Parents

D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

Parents

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 178 | P a g e

*************************************

Offspring

DZ10 199203 000000 147147 162164 223239 212212 229229 234234 242242 209199 206214 107113 155189

Parents

C8 199203 000000 147147 148164 239245 212204 229233 234240 242242 209199 206212 113115 181189

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189

Parents

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189

*************************************

Offspring

EA1 199199 000000 147165 148164 223223 204212 233233 232240 242242 209199 000000 107113 000000

Parents

C7 199203 000000 147165 148164 223239 212204 233229 232234 242242 209199 204212 107113 189181

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

*************************************

Offspring

EA2 165203 000000 147147 148164 223239 212212 233233 000000 242242 199209 214214 113113 155155

Parents

D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189

E4 199165 000000 147165 148148 239245 212212 233229 240234 242242 209199 204214 113115 155189

*************************************

Offspring

EA3 165199 000000 147147 148164 223239 212212 229229 234234 242242 209233 214214 107113 189189

Parents

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189

Parents

D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 179 | P a g e

E4 199165 000000 147165 148148 239245 212212 233229 240234 242242 209199 204214 113115 155189

*************************************

Offspring

EA4 199199 000000 147165 164164 231239 212212 229229 232240 242242 209199 214214 107113 155181

Parents

D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189

D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181

*************************************

Offspring

CB1 199199 000000 147165 164164 223245 212212 229233 234240 242242 209233 212212 113115 189189

Parents

D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189

D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189

*************************************

Offspring

CB2 165199 000000 147147 162164 231239 212212 229233 232234 242242 209233 204212 113113 189181

Parents

D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189

D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189

*************************************

Offspring

CB3 165203 000000 147165 162162 231239 212212 233233 234240 242242 233233 204212 113113 189189

Parents

D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189

D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189

*************************************

180 | P a g e

Supplementary table 3: The batch of offsprings were collected from brooder A7 but the genotyped results of 12 microsatellite marker showed that A7 was not one of the genetic parent. The table below showed the results of additional 6 microsatellite markers used, which still showed that the genetic parents were A1 and A2.

RESULTS

Collected parents file:D:\New Folder\PAPA results\WH001\WH001_parents.txt

Offspring file:D:\New Folder\PAPA results\WH001\WH001_offspring_p1.txt

Global level of transmission error:0

Distribution of transmission error:0

Names of loci file: D:\New Folder\PAPA results\WH001\name of loci.txt

Choice of loci: D04 D38 D42 D106 D108 D109

Offspring

DE1 203209 189199 163175 195213 000000 214238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DE2 193209 189199 000000 195213 000000 214214

Parents

A1 209215 197199 165175 203213 245253 214238

B8 193203 189197 171177 195207 225241 214214

*************************************

Offspring

DE3 203209 197199 163175 195213 000000 208214

Parents 181 | P a g e

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DE4 203215 189197 163165 195203 000000 214238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DE5 203215 197197 163165 000000 000000 208238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

************************************

Offspring

DE6 193209 000000 163165 195203 241245 208238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DE7 000000 000000 163163 207213 000000 000000

Parents none

*************************************

Offspring

DE8 203215 189199 163165 195213 241253 214214

Parents 182 | P a g e

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DE9 193215 189199 000000 195213 225255 214238

Parents none

*************************************

Offspring

DE10 193215 197197 163175 203207 225253 208238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DF1 203209 189197 171175 195203 000000 208214

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

Offspring

DF2 000000 000000 171175 000000 000000 208238

Parents

A1 203215 183201 171171 195207 219241 208210

A1 209215 197199 165175 203213 245253 214238

*************************************

Offspring

DF3 203209 000000 000000 203207 000000 208214

Parents 183 | P a g e

A1 209215 197199 165175 203213 245253 214238

C3 193203 195197 177187 203207 215217 208208

*************************************

Offspring

DF4 203209 189197 171175 195203 225245 208238

Parents

A1 209215 197199 165175 203213 245253 214238

A2 193203 189197 163171 195207 225241 208214

*************************************

184 | P a g e