MORPHOLOGICAL CHARACTERISATION OF SELECTED …studentsrepo.um.edu.my/8307/1/LLB-HGT140008-Scyphozoan.pdf · significant differences in shape among all the specimens based on PCA.

MORPHOLOGICAL CHARACTERISATION OF SELECTED

SCYPHOZOAN JELLYFISH SPECIES AND

GEOMETRIC MORPHOMETRIC ANALYSIS OF

Chrysaora chinensis IN PENINSULAR MALAYSIA

LOW LIANG BOON

DISSERTATION SUBMITTED IN FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF PHILOSOPHY

INSTITUTE OF GRADUATE STUDIES

UNIVERSITY OF MALAYA

KUALA LUMPUR

2017

ii

UNIVERSITI MALAYA

ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: Low Liang Boon

I.C/Passport No: 761110-01-5729

Registration/Matric No: HGT 140008

Name of Degree: Master of Philosophy

Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):

MORPHOLOGICAL CHARACTERISATION OF SELECTED SCYPHOZOAN

JELLYFISH SPECIES AND GEOMETRIC MORPHOMETRIC ANALYSIS OF

Chrysaora chinensis IN PENINSULAR MALAYSIA

Field of Study: Environmental Science (Marine Sciences)

I do solemnly and sincerely declare that:

(1) I am the sole author/writer of this Work;

(2) This Work is original;

(3) Any use of any work in which copyright exists was done by way of fair dealing and

for permitted purposes and any excerpt or extract from, or reference to or

reproduction of any copyright work has been disclosed expressly and sufficiently

and the title of the Work and its authorship have been acknowledged in this Work;

(4) I do not have any actual knowledge nor do I reasonably know that the making of

this work constitutes an infringement of any copyright work;

(5) I hereby assign all and every rights in the copyright to this Work to the University

of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work

and that any reproduction or use in any form or by any means whatsoever is

prohibited without the written consent of UM having been first had and obtained;

(6) I am fully aware that if in the course of making this Work I have infringed any

copyright whether intentionally or otherwise, I may be subject to legal action or any

other action as may be determined by UM.

Candidate’s Signature Date

Subscribed and solemnly declared before,

Witness’s Signature Date

Name:

Designation:

iii

ABSTRACT

Although jellyfish blooms have significant impacts to the coastal environments, the

effective management of blooms and understanding of their proliferation are often

confounded by the lack of baseline data, which includes species identification, their

biology and ecology. In Malaysia, jellyfish blooms can negatively impact human

activities such as causing beach closures, damage to fishing nets, threats of stings and

blocking power station systems. Therefore, detail characterization and documentation of

their morphology would facilitate species identification of jellyfish in the field,

especially in Malaysian waters and this region which may harbours many undiscovered

species. In this study, the morphology of nine jellyfish species found in Peninsular

Malaysia that belong to the class Scyphozoa, namely Chrysaora chinensis, Cyanea sp.,

Versuriga anadyomene, Rhopilema hispidum, Rhopilema esculentum, Phyllorhiza

punctata, Acromitus flagellatus, Lobonemoides robustus and Lychnorhiza malayensis

were characterised in detail. Sea nettle jellyfish in Malaysia was verified as C.

chinensis. The status of Cyanea sp. found in Malaysia is uncertain as it may possibly be

a new species. This study also reported the first record of Lychnorhiza malayensis. A

total of 107 specimens of C. chinensis were obtained from four coastal areas of

Peninsular Malaysia (East-Central, East-North, West-Central, and West-North) to

compare the possible morphological variation between populations using geometric

morphometric analysis. Procrustes superimposition, Principal Component Analysis

(PCA) and Canonical Variate Analysis (CVA) were applied to the images of

gastrovascular pouches of C.chinensis to extract the shape information. There were no

significant differences in shape among all the specimens based on PCA. However, CVA

showed shape variation between populations of the four areas of Peninsular Malaysia.

iv

ABSTRAK

Walaupun ledakan obor-obor boleh memberi impak yang besar kepada persekitaran

pantai, pengurusan yang berkesan dan pemahanam tentang perkembangan mereka

sering terjejas disebabkan kekurangan data asas, termasuk pengecaman spesies, biologi

dan ekologi mereka. Di Malaysia, ledakan obor-obor boleh memberi kesan negatif

kepada aktiviti manusia seperti menyebabkan penutupan pantai, kerosakan kepada jala

ikan, ancaman sengatan dan sistem stesyen kuasa tersumbat. Oleh itu, pencirian dan

dokumentasi morfologi mereka secara terperinci akan memudahkan pengecaman

spesies obor-obor di lapangan, terutamanya di perairan Malaysia dan rantau ini yang

mungkin mengandungi pelbagai spesis yang belum ditemui. Dalam kajian ini,

morfologi sembilan spesies obor-obor di Semenanjung Malaysia yang tergolong dalam

kelas Scyphozoa iaitu Chrysaora chinensis, Cyanea sp., Versuriga anadyomene,

Rhopilema hispidum, Rhopilema esculentum, Phyllorhiza punctata, Acromitus

flagellatus, Lobonemoides robustus, dan Lychnorhiza malayensis telah dicirikan secara

terperinci. Obor-obor “sea nettle” di Malaysia telah disahkan sebagai C. Chinensis.

Status Cyanea sp. yang terdapat di Malaysia tidak dapat dipastikan dan mungkin

merupakan satu spesies yang baru. Kajian ini juga melaporkan rekod pertama bagi

Lychnorhiza malayensis. Sejumlah 107 spesimen C. chinensis diambil dari empat

kawasan persisiran pantai di Semenanjung Malaysia (East-Central, East-North,

West-Central, dan West-North) untuk membandingkan variasi morfologi spesies

dengan menggunakan analisa “geometric morphometrics”. “Procrustes

superimposition”, “principal component” (PCA) dan “canonical variate” (CVA) telah

diaplikasikan terhadap imej kantung gastrovaskular C. chinensis untuk mendapatkan

informasi bentuknya. Keputusan daripada PCA tidak menunjukkan perbezaan bentuk

yang signifikan disemua specimen. Tetapi, keputusan CVA menunjukan perbezeaan

bentuk diantara populasi dari empat lokasi di Semenanjung Malaysia.

v

ACKNOWLEDGEMENTS

I would like to express appreciation to my supervisor, Dr. Mohammed Rizman Idid and

lab mate Wan Mohd Syazwan for the supports, ideas and guidance. I am grateful to the

staff at Institute of Ocean and Earth Sciences (IOES), University of Malaya for

providing constant assistance during the study. I thank University of Malaya for

providing research grants RU006E-2014 and RG104-11SUS to my supervisor.

vi

TABLE OF CONTENTS

ORIGINAL LITERARY WORK DECLARATION ii

ABSTRACT iii

ABSTRAK iv

ACKNOWLEDGEMENTS v

TABLE OF CONTENTS vi

LIST OF FIGURES viii

LIST OF TABLES xiv

CHAPTER 1 INTRODUCTION 1

1.1 Jellyfish Studies in Malaysia 1

1.2 Taxonomy Position and Problem of the Malaysia Sea Nettles 8

1.3 Introduction to Geometric Morphometric Analysis 10

1.4 Research Aims and Questions 12

1.5 Objectives of the Study 14

1.6 Significance of the Study 15

CHAPTER 2 MATERIALS AND METHODS 16

2.1 Collection of Jellyfish Samples 16

2.2 Photography of Specimens and Collection of Morphological Data 19

2.3 Geometric Morphometric Analysis of Gastrovascular Pouch 22

2.3.1 Landmark Configurations and Photography of

Gastrovascular Pouches 23

2.3.2 Formating of Images into TPS File 26

2.3.3 Digitisation of Images 26

2.3.4 Quantification of Measurement Error 27

2.3.5 Procrustes Superimposition 28

2.3.6 Principle Component Analysis Using MorphoJ 29

2.3.7 Canonical Variate Analysis Using MorphoJ 31

2.3.8 Visualization of Shape Outline 31

vii

CHAPTER 3 RESULTS 34

3.1 Overview of Major Morphological Structures of Jellyfish 34

3.1.1 Order Semaeostomeae 34

3.1.2 Order Rhizostomeae 35

3.2 Morphological Description of Chrysaora chinensis Vanhöffen 1888 52

3.3 Morphological Description of Cyanea Sp. 56

3.4 Morphological Description of Rhopilema esculentum Kishinouye 1891 60

3.5 Morphological Description of Rhopilema hispidum Vanhöffen 1888 64

3.6 Morphological Description of Lobonemoides robustus Stiasny 1920 67

3.7 Morphological Description of Versuriga anadyomene Maas 1903 71

3.8 Morphological Description of Phyllorhiza punctata von Lendenfeld 1884 75

3.9 Morphological Description of Lychnorhiza malayensis Stiasny 1920 79

3.10 Morphological Description of Acromitus flagellates Maas 1903 82

3.11 Geometric Morphometric Analysis 85

3.11.1 Procrustes Anova 86

3.11.2 Principal Component Analysis (PCA) 86

3.11.3 Canonical Variate Analysis (CVA) 88

CHAPTER 4 DISCUSSION 93

4.1 Morphology, Diversity and Importance of Jellyfish Species of

Peninsular Malaysia 93

4.2 Lion’s mane Jellyfish (Cyanea Sp.) of Malaysia 94

4.3 Sea Nettles (C. Chinensis) of Peninsular Malaysia 96

4.4 Potential Application of GMM in Jellyfish Studies 99

CHAPTER 5 CONCLUSION 101

REFERENCES 103

APPENDICES 113

viii

LIST OF FIGURES

Figure 1.1 An adult scyphozoan jellyfish, Phyllorhiza punctata

3

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Classification of Cnidarian

Life cycle of jellyfish

Bloom and stranding of Crambione mastigophora in Pulau

Ketam in April 2016

Cross section of a jellyfish

GMM used on various organisms. A: Oak leaf. B: Dog. C:

Cichlid

3

4

4

5

11

Figure 2.1 Diagram showing pompang (bag net)

18

Figure 2.2 Photography setup to float jellyfish in the tank against a

black background, with colour chart and scale. Wires were

used to suspend the specimens, and a scaled wire was also

used to aid recording of measurements.

21

Figure 2.3 Dry photography setup showing the acrylic stage, with

light source below and additional light sources at the sides.

22

Figure 2.4 An image of a subumbrella quadrant of a C. chinensis

specimen showing the 16 geometric morphometric

landmark points (indicated in red and numbered) of a

single gastrovascular pouch analysed (P1). Pouch P1 is

flanked by pouches P2 and P3. Rhopalia are indicated as R.

25

Figure 2.5 The Number of replicate for each level of the process of

specimen imaging and digitization.

28

Figure 2.6

Figure 3.1

56 landmark configurations used to create the outline of

gastrovascular pouch

Map of eight sampling sites, with number of specimen

collected for each species.

33

38

Figure 3.2

Figure 3.3

Whole medusa of an adult jellyfish, Phyllorhiza punctata

Morphology of bell of four different species of scyphozoan

jellyfish. (A): Acromitus flagellates with smooth surface

(B): Rhopilema esculentum with smooth surface (C):

Phyllorhiza punctata with warts on the surface (D):

Cyanea sp. With smooth surface

39

40

ix

Figure 3.4 Lappet of two different species of scyphozoan jellyfish.

(A): Rhopalar lappet (RL) and velar lappet (VL) of

Lychnorhiza malayensis (B): Lappet of Cyanes sp. (L).

Lappet shape broad and semi-square

40

Figure 3.5 Marginal sensory organ / rhopalium (R) of Lychnorhiza

malayensis

41

Figure 3.6 Morphology of umbrella. (A): Tentacle (T) of Chrysaora

chinensis. (B): Lychnorhiza malayensis of Rhizostomeae

without tentacles

42

Figure 3.7 Morphology of the manubrium of Chrysaora chinensis 42

Figure 3.8 Morphology of oral arm (A): Whole medusa floated in a

tank, showing four soft and curtain-like oral arms (OA).

(B): Oral arm (OA) of Rhopilema esculentum. (C): Oral

arm (OA) of Acromitus flagellatus

43

Figure 3.9 Morphology of scapulae. (A): Balde shape Scapulae of

Rhopilema hispidum. (B): Scapulae (S) attached to oral

arms of Rhopilema esculentum

44

Figure 3.10 Morphology of oral arm with terminal club. (A): Terminal

club (C) at the margin of the oral arm of Rhopilema

esculentum. (B): Terminal club (C) at the margin of the

oral arm of Phyllorhiza punctata

44

Figure 3.11 Morphology of filaments. (A): Filament (F) at the oral disk

of Phyllorhiza punctata. (B): Filament (F) at the oral arms

of Acromitus flagellatus

45

Figure 3.12 Morphology of subumbrella of Chrysaora chinensis

revealing the gastrovascular cavity (G)

46

Figure 3.13 Subumbrella morphology of subgenital fenestration (F) of

Rhopilema esculentum

47

Figure 3.14 Subumbrella morphology of papillae. (A): Three papillae

(P) at the subumbrella of Lychnorhiza malayensis. (B):

Three papillae (P) at the subumbrella of Rhopilema

hispidum

48

Figure 3.15 Network of canals of (A): Lychnorhiza malayensis injected

with red dye. (B): Phyllorhiza punctata injected with blue

dye. (C): Lobonemoides robustus injected with yellow dye

49

Figure 3.16 Subumbrella morphology of muscle. (A): Coronal muscle

(CM) of Phyllorhiza punctata. (B): Coronal muscle (CM)

and radial muscle of Cyanea sp.

50

x

Figure 3.17 Subumbrella morphology of gonad. (A): Gonad (G)

protruding from the subgenital fenestration of Cyanea sp.

(B): Gonad (G) at the base of the gastrovascular cavity of

Versuriga anadyomene

51

Figure 3.18 Gross morphology of Chrysaora chinensis. (A): Whole

medusa floated in a tank, showing four soft and curtain-

like oral arms (OA) and tentacles (T); (B): Side view of the

bell (umbrella) with reddish brown pigmentation on the

lappet (L); (C): Oral arms (OA1) without pigmentation;

(D): Oral arms (OA2) with reddish pigmentation; (E):

Subumbrella view of the central stomach (S)

54

Figure 3.19 Umbrella morphology of C.chinensis. (A): Subumbrella

view revealing 16 gastrovascular pouches, rhopalar pouch

(P1), inter-rhopalar pouch (P2), rhopalium (R), lappet (L),

tentacle (T) and oral arms (OA); (B): Subumbrellla view of

creamy white gonads (G), whereby each of the four gonads

is located inside a subgenital fenestration of the

subumbrella; (C): Subumbrella view showing the mouth

(M) bounded by four walls; (D): View of exumbrella with

numerous warts (W) densely concentrated in the centre;

(E): View of subumbrella showing lappet (L), rhopalia (R)

and three tentacles (T) following a 2-1-2 arrangement per

octant, whereby the numbers represent the ontogenetic

sequence of tentacles originating from the cleft of the

lappet

55

Figure 3.20 Gross morphology of Cyanea sp. (A): Whole medusa (B):

Numerous tentacles (T) and curtain like oral arms (OA);

(C): View of exumbrella with numerous warts (W); (D):

Subumbrella showing a group of radial muscle (RM) and a

horseshoe shaped whorl of tentacles (T) between each

group of radial muscle;. (E) Subumbrella view showing a

group of coronal muscle (CM)

58

Figure 3.21 Subumbrella morphology and structures of Cyanea sp. (A):

Subumbrella view of the medusa. (R) Rhopalium. (G)

Gonad. (T) Tentacle. (L) Lappet. (CM) Coronal muscle.

(RM) Radial muscle. (B): A whorl of tentacle (T). Radial

muscle (RM). Coronal muscle (CM), alternating between a

longer group and a shorter group. (C): Lappet shape broad

and semi-circular, with network of canal (C). (D): Gonad

(G) creamy white.

59

Figure 3.22

Gross morphology of Rhopilema esculentum (A): Whole

medusa. (B): Exumbrealla (EX) smooth. (C): Terminal

club (C). (D): 16 scapulae (S) in each medusa. (E): One

subgenital fenestration (F) for each quadrant. One papilae

(P) at the centre of the fenestration. (F): Oral arm (OA).

62

xi

Figure 3.23 Subumbrella morphology and structures of Rhopilema

esculentum (A): Gonad (G) creamy white. (B): Lappet (L).

Coronal muscle (M). Rhopalia (R). (C): Network of canals

injected with green dye

63

Figure 3.24 Gross morphology of Rhopilema hispidum (A): Whole

medusa. (B): Three papilae (P) for each quadrant where the

largest is in the centre. (F) Filaments. (C): Blade shape

scapulae (S) with numerous filaments (F). (D): Exumbrella

surface rough, with numerous warts (W). (E): Lappet (L).

(F): Network of canal (C) injected with blue dye, forming

a pyramid shape of network

66

Figure 3.25 Gross morphology of Lobonemoides robustus (A): Whole

medusa. (F) Filaments. (B): Gonad (G). (C, D): Conical

papillae on the exumbrella in water (P1), and on dry stage

(P2).

69

Figure 3.26 Morphology of Lobonemoides robustus (A): Coronal

musculatures (M) with colouration. Rhopalium (R). (B):

Spindle shape club (C). (C): Network of canal (C) with

yellow dye injected. (G) Gonad. (D): Lappet (L) elongated.

(E): Oral arm (OA) with yellow dye injected

70

Figure 3.27 Gross morphology of Versuriga anadyomene (A): Whole

medusa. Oral pillar (OP). Numerous mouthlets (MO) on

oral arms. Appendage (A). (B): Lappet (L). (C):

Portuberances (P) on exumberalla. (D): Numerous

filaments on the oral disk. Coronal muscle (M). (OA) Oral

arm.

73

Figure 3.28 Gross morphology of Versuriga anadyomene (A): Oral arm

with numerous mouthlets (M). A window on the oral arm

(W). (B): Gonad (G) brownish. (C): Network of canals,

injected with blue dye

74

Figure 3.29 Gross morphology of Phyllorhiza punctata (A): Whole

mesusa. Numerous warts (W) on the exumbrella. Terminal

club (TC). (B): Numerous mouthlets (MO) on the oral arm.

(C): Filaments (F) on oral disk. (D): Oral pillar (OP). The

white ring is a wire to suspend the jellyfish and it is not

part of the jellyfish structure

77

Figure 3.30

Gross morphology Phyllorhiza punctata (A): Coronal

musculatures (M). (R) Rhopalia (L) Lappet. (B): Warts

(W) on exumbrella. (C): Numerous appendages (A) on the

oral arms. Terminal club (C). (D): Gonad (G) brownish.

(E): Network of canals (C) injected with blue dye

78

xii

Figure 3.31 Gross morphology of Lychnorhiza malayensis (A): Whole

medusa. (OA) Oral arm. (B): Rhopalium (R). Lappet (L).

(C): Network of canals (C) injected with red dye. (D):

Gonad (G) creamy white. (P) Papillae. (E): Oral arm (OA).

(F) Papillae

81

Figure 3.32 Gross morphology of Acromitus flagelatus (A): Whole

medusa. Oral arm (OA). (B): Lappet (L) pointy.

Rhopalium (R). (C): Network of canals (C) injected with

red dye. (D): Papilae (P). Coronal muscle (M). (E): A

single terminal filament (F) on oral arm. (F): Numerous

filaments (F) on oral arms

84

Figure 3.33 Map of the sampling locations for geometric morphometric

analysis, with the number of specimens obtained. Green

circle represents East-North (EN), red circle represent

East-Centre (EC), blue represents West-Central (WC), and

purple represents West-North (WN) coastal areas of

Peninsular Malaysia

85

Figure 3.34 PCA Result – % of variation explained by components.

Two independent contrasts of the gastrovascular pouch

shape of the two main components PC1 and PC2 are

illustrated whereby light blue outline indicates the mean

shape and the dark blue outline indicates the shape change

87

Figure 3.35

Scatter plots of PC1 vs PC 2. PC1 accounted for 33.86%

and PC2 accounted for 19.41% of the total variance of the

shape change of gastrovascular pouch of specimens from

East-Central (EC), East-North (EN), West-Central (WC)

and West-North (WN) of Peninsular Malaysia.

88

Figure 3.36

Independent contrast of component using canonical variate

analysis of the gastrovascular pouch of specimens from

East-Central (EC), East-North (EN), West-Central (WC)

and West-North (WN) of Peninsular Malaysia, whereby

CV1, CV2 and CV3 accounts for 47.46%, 32.72% and

19.83% of the amount of relative between-group variation,

respectively. Light blue outline indicates the mean shape

and the dark blue outline indicates the shape change. CV1

denotes changes with blunt protuberance at the margin and

enlarging of the distal end, CV2 denotes changes with

blunt protuberance at the margin and CV3 denotes changes

with blunt protuberance at the margin and enlarging of the

distal end

90

xiii

Figure 3.37 Scatter plot of CV1 vs CV2 and illustration of mean shape

of gastrovascular pouch of specimens from four coastal

areas East-Central (EC), East-North (EN), West-Central

(WC) and West-North (WN) of Peninsular Malaysia. Plot

shows shape differences mainly between specimens of the

east and west coasts, even between those from EN and EC,

but with no distinct differences between those from WN

and WC

91

Figure 3.38 Pairwise comparisons between the pouch shape of

C.chinensis populations from East-Central (EC), East-

North (EN), West-Central (WC) and West-North (WN) of

Peninsular Malaysia

92

xiv

LIST OF TABLE

Table 2.1 The sites where samples of jellyfish were collected off the

coasts of Peninsular Malaysia, with the period, GPS and

sampling methods

16

Table 2.2 The sites where samples of C. chinensis were collected off the

coasts of Peninsular Malaysia, with the sample sizes (n) and

sampling methods. The sites are designated as coastal areas

West-North (WN), West-Central (WC), East-North (EN) and

East Central (EC) of Peninsular Malaysia

17

Table 2.3 Definition of the 16 landmark configurations

24

Table 3.1 Result of Procrustes ANOVA (SS=Sum of square, MS=Mean

Squre, df=degree of freedom)

86

Table 3.2 Mahalanobis distances among the pouch shape of C. chinensis

populations from four coastal areas designated as West-North

(WN), West-Central (WC), East-North (EN) and East Central

(EC) of Peninsular Malaysia

91

Table 4.1 Comparison of different Cyanea species 95

1

CHAPTER 1: INTRODUCTION

1.1 Jellyfish Studies in Malaysia

The term ‘jellyfish’ generally refers to free-floating gelatinous animals that belong to

the phyla Ctenophora and Cnidaria (Figure 1.1). The phylum Ctenaphora consists of

organisms such as sea gooseberry and comb jellies, whereas the phylum Cnidaria

consists of a wide range of both the sessile and floating forms of organisms that

includes jellyfish, corals and sea anemones (http://www.marinespecies.org). All

Cnidarian possess cnidae - an organelle-like capsule with eversible tubules, and it is

considered as the diagnostic feature of Cnidarian. There are three main classes under the

phylum Cnidaria: Cubozoa (46 accepted species), Hydrozoa, and Scyphozoa (187

accepted species) (Figure 1.2). All species under Scyphozoa, Hydrozoa and Cubozoa

develop the ‘medusa’ or jellyfish stage in the life cycles. Class Scyphozoa is ascribed

with four orders, namely Coronatae (crown jellyfish), Staurozoa (stalked jellyfish),

Semaeostome (sea nettle) and Rhizostomae (true jellyfish), with 65 genera and over 187

species (Mayer, 1910; Kramp, 1961; Pitt & Kingsford, 2003; Brusca & Brusca 2002;

Shao et al., 2006; Daly, 2007; Richardson et al., 2009; Bayha, 2010).

The most commonly observed jellyfish are usually those from the class

Scyphozoa, particularly from the medusa life stage. They begin their life cycle from

fertilized eggs that produces planulae. These free swimming planulae (Figure 1.3) then

settled at the substrate, becoming scyphistomas, also known as polyps. During this

sessile polyp stage, Scyphistomas asexually buds and strobilate to produce ephyra.

Strobilation is a process where each layer of the scyphistoma is separated and form a

new juvenile jellyfish, resembling a disc being liberated from a stack. This polydisc

2

strobilation only found in Scyphozoan. Although most of the scyphozoan have a polyp

stage, sometimes a direct development from planula to ephyra is also possible (Arai,

1997; Boero et al., 2008; Ceh, 2015). The adult medusa stages is dominant in the life

cycle. Therefore, this free-swimming form is most commonly seen and found stranded

on beaches (Figure 1.4).

Jellyfish are 97% water and are semi-transparent. They have two body layers,

the outer layer epidermis and the inter layer gastrodermis (Figure 1.5). Between both

layers is a thick layer of mesoglea which consists of fibres embedded in a hydrated

matrix that contains cells. These layers of tissues make up the umbrella of the jellyfish

which is usually bell shape, thus the umbrella is also known as the bell. The scyphozoan

jellyfish are tetraradially symmetrical, meaning having many structures in multiples of

four. It contains a simple gastrovascular cavity which acts as stomach. They are also

characterized by having gastric filament in the stomach. Some scyphozoan jellyfish

such as Semaestomeae contain an opening, or mouth at the subumbrella. There are four

to eight oral arms near the mouth, which functions as arms to capture and transport food

to the gastrovascular cavity. Jellyfish lack eyes, but possess many sensory receptors

capable to detect light, pressure, temperature and gravity. These sensory receptors are

concentrated in the marginal sense organ that contains the rhopalium (Nakanishi, 2015).

Not all jellyfish possess tentacles. For Semaestomeae jellyfish, tentacles can be found at

the margin of the bell or at the subumbrella whereas tentacles are absence from the

Rhizostomeae jellyfish. Jellyfish contains network of canals that usually anestomoses

with each others that formed various patterns (Hamner, 1995; Arai, 1997; Hale, 1999).

3

Figure 1.1: An adult scyphozoan jellyfish, Phyllorhiza punctata

Figure 1.2: Classification of Cnidarian

4

Figure 1.3: Life cycle of jellyfish

(http://thescyphozoan.ucmerced.edu/Biol/Ecol/LifeHistory/ScyphozoaLH.html)

Figure 1.4: Bloom and stranding of Crambione mastigophora in Pulau Ketam in April

2016 (Sin Chew Daily, 20 April 2016)

5

Figure 1.5: Cross section of a jellyfish (Hale, 1999)

The food source generated from the ocean today has been greatly reduced

compared to from decades ago, due to factors such as global warming, ocean

acidification and over fishing (Morton, 2005; Alison et al., 2009). Jellyfish has long

been a delicacy in Asia for centuries and has been increasingly important as a food

source (Hsieh et al., 2001; Pitt & Kingsford, 2003; You et al., 2007). But not all

jellyfish species are edible, and some are not economically to be produced as food due

to its size. A traditionally non edible jellyfish, Cyanea nozakii was produced into food

in China but because of the low quality and unpleasant taste, the prices of food made

from Cyanea nozakii fetched a much lower price than the more sought after Rhopilema

esculentum (Dong et al., 2010). Today, the demand of edible jellyfish is greater than

ever with more people from China, Japan, Korea, Singapore, Thailand and Malaysia

consuming it. Jellyfish are marinated with a mixture of salt and alum over a period of

approximately seven days, and the semi-dried products are sold as food ready for

6

consumption. A few commonly sought after edible jellyfish in this region are the “red

type” (Rhopilema esculentum), “white type” (Lobonemoides robustus), “river type”

(Acromitus hardenbergi), “ball type” or “sunflower type” (Crambionella annadalei),

“prigi type” (Crambione mastigophora), “sand type” (Rhopilema hispidum) (Omori &

Kitamura, 2004).

Despite its value, jellyfish on the other hand do pose treat to human and the

environment (Uye et al., 2010; Graham et al., 2015). Many jellyfish are venomous, and

some are even deadly. Beaches are forced to be closed when large quantity of venomous

jellyfish swamp the beach, causing lost in tourism revenue (Lucas, 2001; Purcell et al.,

2009; Gershwin, 2010). Jellyfish blooms have been reported extensively over the past

few decades (Brodeur, 2008; Kogovsek et al., 2010), notably the bloom of Pelagia

noctiluca throughout the Mediterranean Sea during the 1980s (Doyle et al., 2007),

bloom of Phyllorhiza punctata in the northern Gulf of Mexico in 2000 (Graham et al.,

2003), and the bloom of Nemopilema nomurai in the Sea of Japan in 2002 and 2003

(Kawahara et al., 2006). There are increasing evidence indicate that human activities

could attribute to the cause of jellyfish bloom, such as eutrophication (Purcell et al.,

2001; Parsons & Lalli, 2002; Malej et al., 2007), overfishing (Mullon et al., 2005;

Bakun & Weeks, 2006), introduction of alien species (Bolton & Graham, 2004; Graham

& Bayha, 2007; Mills, 2001), installation of artificial substrates in the ocean

(Richardson et al., 2009) and climate change (Raskoff, 2001; Attrill et al., 2007;

Gibbons & Richardson, 2008; Ruiz et al., 2012).

Beside blooms, one of the most alarming problems is the introduction of

nonindigenous jellyfish in a new region (Mills, 2001). They are believed to be

introduced to the new environment by mean of exchange of ballast water and transport

of biofouling polyps. (Richardson et al., 2009). Certain species of jellyfish previously

7

not known to other region has since appearing. These invasive species cause decline in

fisheries as they destroy the fishing net (Haddad & Nogueira, 2006).

Limited research was done on jellyfish in Malaysia. Due to the scarcity of

research effort in jellyfish, especially the morphological study of jellyfish in Malaysia

water, this region may harbor many species that yet to be discovered. Rumpet (1991)

and Daud (1998) published a field survey of scyphozoan jellyfish, whereas others

studied on their venom and toxinology (Othman & Burnett, 1990; Azila & Othman

1993; Tan et al., 1993), whereby species may have been misidentified. Jellyfish

fisheries in Malaysia are focusing on the “red type”, as it fetches the highest price due to

the high demand from China and Japan, whereas “white type”, “river type” and “sand

type” fetches relatively lower price (Omori & Nakano, 2001; Omori & Kitamura, 2004),

but these studies did not have satisfactory result on morphological descriptions and

taxonomy identification as the studies were only done on edible jellyfish. Base on

personal observations and reports, jellyfish often swamp and endanger beachgoers of

being stung, which sometimes can be lethal. They are often stuck in the cooling intake

of the power plant, causing damage to the power plant (Azila & Chong, 2010). It is also

notable that when they bloom, they were sometimes caught in the fishing net and

destroy them.

Despite their socio-economic importance in fisheries and also its treats, there

has been a lack of baseline data and documentation of jellyfish, particularly about their

diversity and ecology (Rizman-Idid et al., 2016). Jellyfish diversity studies in general

has been confounded by problems of identification. Their morphological examinations

are notoriously difficult, due to their fragile bodies, inadequate preservation and lack

of sound identification keys.

8

More recently, researchers have started using molecular genetic techniques to

facilitate the classification of jellyfish and detection of cryptic species (Dawson, 2004).

Applications of DNA sequence analysis and phylogenetics have been used to help

identify and barcode some of the Malaysian jellyfish species, including C. chinensis

(Rizman-Idid et al., 2016). Even with molecular techniques, species identification is

still based on the description of its morphology. Hence a more detailed morphological

evaluation of jellyfish in Malaysia is required.

1.2 Taxonomy Position and Problem of the Malaysia Sea Nettles

Jellyfish has a long history in evolution (Young & Hagadorn, 2010). Fossils have been

reported from as early as early Cambrian in China (Hou et al., 2005), and well-

preserved medusozoan fossils from the Middle Cambrian was reported in North

America (Cartwright et al., 2007). Ever since Linnaeus first described the popular moon

jellyfish Aurelia aurita in 1758, many studies have been done on numerous jellyfish

species throughout the years, but there are still many problems confounded with their

classification, such as the Malaysia sea nettles in the genus Chrysaora. The Chrysaora

jellyfish are classified under family Pelagidae (order Semaeostomeae) and typically

recognized by having 32-48 lappets, with eight marginal sense organs, with three or

more tentacles per octant, with 16 gastrovascular pouches, and with numerous warts on

the exumbrella (Kramp, 1961). Species of sea nettle has a worldwide distribution and

have been reported to occur in the South China Sea, North, Central and South America,

Africa, Europe and Australia (Morandini & Marques, 2010; Yap & Ong, 2012).

According to World Register of Marine Species (http://www.marinespecies.org) there

are possibly 15 -18 species, whereby 4 species have been verified; Chrysaora achlyos,

Chrysaora hysoscella, Chrysaora pacifica and Chrysaora quinquecirrha.

9

Although some regions of Southeast Asia have been reported to harbour

Chrysaora species such as - Chrysaora quinquecirrha and Chrysaora melanaster

(Kramp, 1961; Yap & Ong, 2012), there is the possibility they were misidentified base

on colour variations and warrants verification. In the past, jellyfish species were

notoriously difficult to identify as there were no reliable taxonomic keys, specimens

were badly preserved, and confounded by the existence of cryptic species complexes

that could be detected only by the application of molecular genetic techniques. For

example, Chrysaora chinensis in Malaysia has been previously identified as C.

hysoscella or C. quinquecirrha based on reports of envenomation and toxicology

studies of jellyfish stings (Azila & Othman, 1993) and those in Singapore straits as C.

melanaster (Yap & Ong, 2012) – possibly misidentified due to the colour variations.

Some features, such as nematocysts have been used to aid species identification,

whereby sea nettles from South China Sea were believed to be C. chinensis

(Morandini & Marques, 2010; Yap & Ong, 2012).

C. chinensis in Malaysia has tendencies to bloom, sometimes causing blockage

of cooling systems of coastal power plants, contamination of fishing nets and a nuisance

to fishing activities (personal observation). Beach tourism is also affected as beach

goers are often warned about its stings that are intense with painful burning sensation.

In general, jellyfish blooms have been linked to eutrophication and added nutrients to

the diet of the jellyfish (Richardson et al., 2009). Therefore, certain areas in

Malaysia were reported to have more occurances of C. chinensis. Moreover,

morphological adaptations to different localities with various ecological and

environmental conditions are well documented for jellyfish (Dawson, 2005). Thus, it

will be beneficial to study the morphological variation of C. chinensis in Malaysia.

10

1.3 Introduction to Geometric Morphometric Analysis

Shape analysis has long been an important and fundamental role in biological research

(Klingenberg, 2016). Traditionally, taxonomic classification was base mainly on

descriptive approach of morphology to formally describe species. Such shape

descriptions are sometimes ambiguous, and may not be sufficient to delineate species,

especially among closely related species that have high degree of morphological

resemblance. In the beginning of the twentieth century, researchers started employing

quantitative study of shape by meristic measurement such as length and width, and data

collected were subject to statistical analysis, using univariate, bivariate or

multivariateanalysis (Webster, 2010; Adams et al., 2013; Chen et al., 2013) to describe

the pattern of shape variation (Adams et al., 2004; Bookstein, 1998). With the

advancement of computing technology in the late 20th century multivariate

morphometrics are preferred, in which multiple measurements are analysed together

using Canonical Variates Analysis (CVA), Principal Components Analysis (PCA), and

other type of analysis (Polly et al., 2016). In the 1990’s, a new approach in studying

shape was introduced with the application of Geometric Morphometric Analysis

(GMM). GMM is the quantitative study of the biological shape, shape variation, and

covariation. Over the years, GMM has been improved and revised, with better effective

methods and softwares (Rohlf & Marcus, 1993; Adams et al., 2013). GMM have

employed outline and landmark methods.

Landmark coordinates were identified on the shape of the organism, and the

non-shape information contained in the data is removed by using the Procrustes

superimposition process. The data can then be further analyzed using statistical analysis

such as PCA and CVA.

11

One of the key advantages of GMM is that shape differences can be visually

displayed as illustrations or computer animations, making it the preferred method

compared to traditional morphometric which are usually shown as data such as length,

width and distance. (Rohlf & Marcus, 1993; Durón-Benítez & Huang, 2016). The

various methods of GMM visualization can illustrate even complex morphological

changes more effectively, making it appealing to researchers since the results are no

longer presented only as a series of statistical data but also as graphical representations

of the actual organism being studied (Rohlf & Marcus, 1993; Adams et al., 2004).

Furthermore, these visualizations provide information on morphological changes in

their immediate anatomical context (Klingenberg, 2013; Mayer et al., 2014).

GMM have been applied to a variety of organisms and structure such as oak leaf

(Viscosi & Cardini, 2011), dog (Drake & Klingenberg, 2010) and cichlid (Maderbacher

et al., 2008; Kerschbaumer & Sturmbauer, 2011) (Figure 1.6). Most organisms that

were analysed are usually rigid in structure. To date, there have yet to be any study of

shape of gelatinous organism such as jellyfish using GMM. Thus, this study is the first

of its kind to provide a more detailed morphological description of C.chinensis of

Peninsular Malaysia and to employ geometric morphometric analysis to distinguish its

populations.

Figure 1.6: GMM used on various organisms. A: Oak leaf. B: Dog. C: Cichlid

12

GMM can be performed using mathematical statistical packages such as R,

MATHEMATICA© and MATLAB©. There are also other open source integrated

software such as MorphoJ (Klingenberg, 2011), MORPHEUS (Slice, 2013) and IMP

(Sheets, 2011). R is a comprehensive GMM software because there are a number of

packages in it, and one can write script to perform analysis as wish. Thus, complex

statistical analysis can be performed in R. Since this study only require the standard

statistical analysis such as Procrustes superimposition, Canonical Variates Analysis and

Principal Components Analysis, software such as MorphoJ, MORPHEUS and IMP are

sufficient to perform the analysis needed for this study. Although these three software

have different graphical user interface, they essentially perform the same analysis with

the same numerical results. The preference of which software to use then is largely

depending on the familiarity and user-friendliness of the software. MorphoJ is choosen

to be used in this study because of the extensive training received in using the MorphoJ

from the author.

1.4 Research Aims and Questions

The aim of this research is to describe the jellyfish found in Peninsular Malaysia

water, and to compare the possible morphological variation within species or genus in

Malaysia water. Since this region may harbours many undiscovered species, detailed

documentation of morphology is required to allow for better identification and

comparison with conspecifics or congenerics in nearby waters. More recently,

applications of DNA sequence analysis and phylogenetics have been used to help

identify and barcode some of the Malaysian jellyfish species, including C. chinensis

(Rizman-Idid et al., 2016), which concurred the notion that specimens with different

colour morphs are often genetically similar and regarded as the same species. However,

13

it is important to realize that the efficiency of such DNA barcoding method relies on the

availability of reference sequences of correctly identified voucher specimens in the

Genbank database. Although the study provided 16S and ITS1 sequences, it did not

have the required cytochrome oxidase I (COI) sequences to definitively barcode C.

chinensis. Furthermore, the morphological description of the species in the study was

quite simple and preliminary, even lacking photographs, morphological illustrations and

have very simple descriptions. Thus many of previous morphological identification

needs to be reevaluated. Hence there is need to study them in more detail.

The sea nettle jellyfish of the genus Chrysaora has a worldwide distribution

and have been reported to occur in the South China Sea, North, Central and South

America, Africa, Europe and Australia (Morandini & Marques 2010; Yap & Ong

2012). Although some regions of Southeast Asia have been reported to harbour

Chrysaora species such as Chrysaora quinquecirrha and Chrysaora melanaster

(Kramp 1961; Yap & Ong 2012), there is the possibility they were misidentified base

on colour variations and warrants verification. In the past, jellyfish species were

difficult to identify as there were no reliable taxonomic keys, specimens were badly

preserved, and confounded by the existence of cryptic species complexes that could be

detected only by the application of molecular genetic techniques. Hence a more

detailed morphological evaluation of the Malaysian sea nettle (Chrysaora chinensis)

is required. Moreover, morphological adaptations to different localities with various

ecological and environmental conditions are well documented for jellyfish (Dawson,

2005). Base on personal observation, certain degrees of variations are found. For

example, gastrovascular pouches shape of C. chinensis are slightly different between

the species found in east coast and west of Peninsular Malaysia. Therefore, this

present study aims to distinguish morphologically the populations of Malaysian C.

chinensis by using geometric morphometrics, especially between populations of the

14

Straits of Malacca that are heavily impacted by anthropogenic activities from those

found in the South China Sea that has relatively better water quality. Thus, this study is

the first of its kind to provide a more detailed morphological description of

scyphozoan jellyfish in Peninsular Malaysia and to employ geometric morphometric

analysis to distinguish populations of C. chinensis.

There are two research questions in this project:

Question 1: What are the detailed morphological characteristics of different jellyfish

species in Peninsular Malaysia?

Question 2: Is there any gastrovascular pouch shape variation of Chrysaora chinensis

across different localities in Peninsular Malaysia?

1.5 Objectives of the Study

The objectives of this study are:

1. To identify, photograph and describe the detailed morphology of selected

scyphozoan jellyfish species in Peninsular Malaysia

2. To photograph the gastrovascular pouch of C. chinensis and establish its

landmark configuration for geometric morphometrics analysis

3. To discriminate between C.chinensis populations of Peninsular Malaysia by

analyzing the gastrovascular pouch shape variation using geometric

morphometrics and statistical analysis base on the configured pouch landmarks.

15

1.6 Significance of the Study

The proposed study has the following significance:

1) Some researchers, such as Kramp in his work “Synopsis of the medusae of the world

(1961)”, described various species of jellyfish in the Malayan Archipelago. But the

Malayan Archipelago encompasses a wide range of region, from Malaysia to Australia.

Thus it is unclear whether the specimen is found near Malaysia water. This research

could provide more accurate information on the jellyfish found in Malaysia water. The

findings of this study will contribute to the Malaysian checklist of marine species,

whereby baseline information of jellyfish diversity is still lacking. This study also

provide detailed description jellyfish species and relevant photographs of their

morphology that would facilitate their identification in the field.

2) This study would verify if sea nettle species in Peninsular Malaysia belong to

C.chinensis and determine if they consist of morphologically different populations.

GMM approach would also be first of its kind to be applied to jellyfish. This part of the

study would also contribute to baseline data needed for future management of sea nettle

blooms in Peninsular Malaysia.

16

CHAPTER 2: MATERIALS AND METHODS

2.1 Collection of Jellyfish Samples

Surveys and samplings in this study were carried out between April 2014 and

December 2014 from eight sites to obtain as many specimens possible that may

represent different jellyfish species and morphological variations from the east and west

coasts of Peninsular Malaysia (Table 2.1). Two sites located in the West-Central

(Sungai Janggut and Klang Power Station), three located in West-North (Pantai Kok,

Kilim and Balik Pulau), one located in East-Central (Kampung Cempaka), and two

located in East-North (Pantai Sabak, Pantai Melawi) coastal areas of Peninsular

Malaysia (Table 2.2). These sites were also chosen for the sampling of C.chinensis

samples for the GMM study. For the purpose of geometric morphometric analysis, a

minimum of 32 specimens were required, which was calculated based on 16 landmark

configurations used in the GMM.

Table 2.1: The sites where samples of jellyfish were collected off the coasts of

Peninsular Malaysia, with the period, GPS coordinates and sampling methods.

Site Period GPS Method

Sungai Janggut 9 Jul 2013* N03.16916° Bag Net

5 Dec 2013* E101.29833°

29 Apr 2014

Pantai Kok 14 Apr 2014 N06.34869° Dip Net

E99.64806°

Kilim 14 Apr 2014 N06.4766362° Dip Net E99.8042679°

Kampung. Cempaka 26 Jun 2014 N03.74365 o Dip Net

E103.32847 o

17

Table 2.1, continue

Pantai Sabak 20 Jul 2014 N06.13712o Dip Net

E102.36976o

Pantai Melawi 21 Jul 2014 N05.99612o Dip Net

E102.43714o

Klang Power 26 Jun 2014 N03.3229o Dip Net

Station E101.30108o

Balik Pulau 14 Dec 2014 N05.53972° Dip Net

E100.34888°

* Museum specimens of that was previously collected (not from this study) but

have been used for morphological examination

Table 2.2 The sites where samples of C. chinensis were collected off the coasts of

Peninsular Malaysia, with the sample sizes (n) and sampling methods. The sites are

designated as coastal areas West-North (WN), West-Central (WC), East-North (EN)

and East Central (EC) of Peninsular Malaysia.

Area Site Period GPS Method

West-North (WN) Pantai Kok 14 Apr 2014 N06.34869° Dip Net

(n = 38) (n = 5) E99.64806°

Balik Pulau 14 Dec 2014 N05.53972° Dip Net

(n = 33) E100.34888°

West-Central (WC) Sungai Janggut 9 Jul 2013* N03.16916° Bag Net

(n = 27) (n = 10 + 17*) 5 Dec 2013* E101.29833°

29 Apr 2014

East-North (EN) Pantai Sabak 20 Jul 2014 N06.13712o Dip Net

(n = 26) (n = 21) E102.36976o

Pantai Melawi 21 Jul 2014 N05.99612o Dip Net

(n = 5) E102.43714o

East-Central (EC) Kampung Cempaka 26 Jun 2014 N03.74365 o Dip Net

(n = 16) (n = 16) E103.32847 o

* Museum specimens of C. chinensis that was previously collected (not from this study)

but have been used for GMM in the present study.

18

Samples were obtained by using dip nets and bag nets. Dip nets with the mesh

size of 5mm were used to scoup out the jellyfish samples from the water surface and

kept in containers filled with seawater. Although specimens were less damaged, this

method is more labour intensive. Bag nets, also known as Pompang (Figure 2.1), were

methods used by fishermen in Sungai Janggut to collect pelagic jellyfish species.

Sixteen bag nets were deployed approximately 8m below the water surface during

spring tide and left for 6 hours before retrieving the nets. Bag nets were emptied on the

boat and jellyfish samples were sorted immediatey from the catch, and placed

temporarily into containers filled with seawater.

Figure 2.1: Diagram showing pompang (bag net)

Once at the jetty, jellyfish specimens that were relatively in good condition and

least damaged were chosen and photographed immediately to record their live

colouration by floating them in acrylic aquarium filled with seawater. Species of

19

specimens were tentatively identified in the field based on Kramp (1961). Bell diameter

and length of oral arms were measured on site using a measuring tape with the

exumbrella facing up. Specimens were cataloged and other sampling information such

as date and sampling site were recorded.

Tissue samples from oral arm and bell of each specimen were taken using

forceps and scissors that were cleaned with alcohol between each sampling to eliminate

cross contamination between samples. Tissue samples were rinsed with distilled water

and kept in vials containing 100% ethanol, cataloged and stored at 4C° for future

molecular genetic studies (not within the scope of the present study).

Each whole specimen was rinsed with distilled water to remove as much debris

as possible before being transferred into individual heavy duty plastic bag containing

5% formalin in seawater (Appendix A) and brought back to the laboratory. After seven

days of specimens being fixed in formalin, the specimens were rinsed with distilled

water to remove any remaining debris and transferred to a new container with 5%

formalin in filtered seawater to ensure better fixation.

2.2 Photography of Specimens and Collection of Morphological Data

Species identification of specimens were further verified in the laboratory based

on detailed morphological examination and taxonomic classifications of Kramp (1961)

and Morandini & Marques (2010). Prior to the examination, each specimen and its

catalog paper were removed carefully from its container, place into another container

with tap water and soaked for at least five minutes to remove the formalin as it is highly

carcinogenic. For safety measure, gloves and mask were used during handling of

specimens and examinations done in well ventilated area.

20

Morphological examinations of jellyfish specimens were done on major

structures such as bell, subumbrella, lappet, gastrovascular cavity, tentacle, muscle,

network, oral arm, scapulae, terminal club, filament and gonad. 186 detailed

morphological characters of jellyfish specimens were analysed, measured and their

respective character states recorded (Appendix B). A set of photographs were also taken

for those structures (Appendix C) to facilitate the morphological examination and

collection of morphological data. All examinations and photographs were based on a

modified data sheet guidelines of how to study the morphological characteristics of

scyphozoans, provided by courtesy of Michael Dawson and Liza Gomez Daglio from

University of California, Merced.

Morphology of specimens was photographed using a digital camera (Olympus

PEN Lite E-PL5, lense 18mm to 105mm) with additional light source, scale and colour

chart (Tiffen Q-13 Color Separation Guide) included so that measurements and the

colour code (CMYK) can be calibrated appropriately later from the digital images

(Figure 2.2). The specimens were photographed in two different methods. The first

method was by suspending the specimen using wires in a transparent glass aquarium

(50cm x 30cm) that was filled with water against a black background, so that it appears

to floating and positioned as natural as possible (Figure 2.3). The placement of the

specimen in the glass tank and the camera’s angle and distance from the specimen were

adjusted depending on the structure to be photographed, whether to obtain closeup or

overall images of the structure.

The second method is by laying the specimen on a flat transparent acrylic stage

(various sizes depending on the specimens) for dry and more detailed photography of

the morphology. A transparent acrylic glass measuring was used as the stage for

detailed photography of its morphology. A fluorescent light of 16 watt is placed 15cm

21

below the stage. Two extra light sources are placed one on each side of the stage (left

and right side) to provide extra light source. (Figure 2.3). In general, the specimen was

placed so that the orientation of rhopalia were aligned at 3, 6, 9 and 12 o’clock

accordingly, but the positioning of the specimen on the stage, the camera’s angle and

distance were adjusted depending on the various structures to be photographed.

Microsope and magnifying glass were used to observe minute structures such as

the gastric filaments. Visualisation of network of canals, gastrovascular pouches and

anastomoses in the lappets were enhanced by carefully injecting food dye (blue, green

or red) into the canals.

Figure 2.2: Photography setup to float jellyfish in the tank against a black background,

with colour chart and scale. Wires were used to suspend the specimens, and a scaled

wire was also used to aid recording of measurements.

22

Figure 2.3: Dry photography setup showing theacrylic stage, with light source below

and additional light sources at the sides.

2.3 Geometric Morphometric Analysis of Gastrovascular Pouch

Rhopalar gastrovascular pouch of C. chinensis was the chosen structure for the

geometric morphometric analysis as this internal structure often remains intact during

sampling compared to other external structures. Jellyfish are fragile organisms, and

quite often certain structures are destroyed during the sampling, such as detached

tentacles and oral arms, deformed exumbrella and others. Gastravascular pouch is

located at the underside of the bell of the jellyfish, and with the oral extending below it.

Therefore, it is relatively protected during sampling. The other reason for choosing

rhopalar gastrovascular pouch is because for Chrysaora species, they generally have two

shapes; the Pacific shape and the Atlantic shape. The Pacific septa shape is

characterized by first thinning of the tentacular pouch, then enlarging it, thus making an

“S” shape. Whereas the Atlantic septa shape is characterized by enlarging of the

tentacular pouch but gradually becomes thin, thus appearing pear shaped. Most of the

Chrysaora found in the Pacific Ocean such as C. chinensis, C. pacifica and C.

melancaster have the Pacific septa shape, whereas the Chrysaora found in the Atlantic

23

ocean such as C. quinquecirrha and C. hysoscella have the Atlantic septa shape

(Morandini & Marques, 2010). Therefore, there is reason to believe the biological

significance of the gastrovascular pouch variation base on different regions.

2.3.1 Landmark Configurations and Photography of Gastrovascular Pouches

Landmark-based geometric morphometrics methods begin with the

identification of the landmarks coordinate, either two or three-dimensional. Landmarks

are points on the Cartesian coordinates (x, y, z) that can be identified on each and every

specimen in the study to represent the shape. Although these landmark coordinates

contains information regarding the shape of the specimen, these coordinates data should

not be used directly because the effect of variation in position, orientation and size of

the specimens are still present in the coordinate. When studying shape of an organism,

the information regarding the position, orientation and size of the specimen is irrelevant.

Therefore, this non-shape information must be removed prior to the analysis of the

landmark coordinates (Klingenberg, 2013; Mitteroecker et al., 2015). Once the non-

shape variables are removed, these variables then can be used to perform statistically

analysis. The result of the analysis will be able to tell whether there is any variation in

shape comparison, in both statistical scatterplots as well as graphical representation of

the shape change (Adams et al., 2004). There are a few criteria in choosing landmarks.

Firstly, the landmarks must be of biologically significant. Secondly, landmarks must be

able to represent the morphology of the specimen, and all landmarks must be present on

all specimens, and must be reliably and repeatedly digitized for each specimen

(Klingenberg, 2013).

There are a total of 16 pouches for each specimen of C. chinensis, among them

eight rhopalar gastrovascular pouches are to the interest of this study. The reason why

24

these eight pouches were chosen over the other eight inter-rhopalar gastrovascular

pouches is because the rhopalia can be used as the guide to align the pouch more easily

along the 3 and 6 o’clock accordingly. Inter-rhopalar pouch could have been chosen to

be analised and it would have yield the same result since both pouches neighbouring

each other and shape variation on one pouch will also be shown on the neigbouring

pouch, but it will take more time to align the axis. 16 points along the edges of the

rhopalar grastovascular pouch were chosen as the landmarks for the GMM (Figure 2.4)

as those points represent the shape outline of pouch. Point 3 and 15 could have

beenomitted but they are used as the semi landmark since they are in the center of the

septa. The definition or position of the 16 landmark configurations are given in Table

2.3.

Table 2.3: Position and definition of the 16 landmark configurations.

Landmark Position

LM 1 Proximally, at the center of the rhopalar pouch nearest to the

gastrovascular cavity

LM 2 Proximally, at the upper left corner of the pouch

LM 3 Centrally, at the left septa of the pouch; half way between the top of the

pouch to the point where septa curves

LM 4 At the section where septa starting to curve outward

LM 5 Distally at the section where septa is curved at its furthest

LM 6 At the section where septa curves inward

LM 7 Distally at the lowest left point of the pouch

LM 8 Distally at the lower left of the septa next to the rhopalium

LM 9 Centrally at the rhopalium

LM 10 Distally at the lower right section of the septa next to the rhopalium

LM 11 Distally at the lowest right section of the pouch

LM 12 At the section where septa curves outward

LM 13 Distally at the section where septa is curved at its furthest

LM 14 At the section where septa starting to curve inward

LM 15 Centrally, at the right septa of the pouch; half way between the top of the

pouch to the point where septa curves

LM 16 Proximally, at the upper right corner of the rhopalar pouch

25

Figure 2.4: An image of a subumbrella quadrant of a C.chinensis specimen showing the

16 geometric morphometric landmark points (indicated in red and numbered) of a single

gastrovascular pouch analysed (P1). Pouch P1 is flanked by pouches P2 and P3.

Rhopalia are indicated as R.

Each specimen was placed on the stage with the subumbrella facing up. Oral

arms were moved away from the desired gastrovascular pouch in order to reveal a clear

view of the septa. All air bubbles trapped in the pouches were removed by pressing

gently on the pouches. The desired pouch is positioned where the rhopalar was aligned

at the centre of the lower left quadrant (Figure 2.4). A chosen quadrant of the umbrella

containing the pouch to be analysed was photographed, which would include three

gastrovascular pouches and three rhopalia. Sometimes, blue dye was injected into the

rhopalar pouch to enhance the visualization of the pouch shape when it was difficult to

see the edges. Camera was held directly above the specimen at a right angle with a

distance of approximately 40cm to 45cm between the camera and the specimen. The

pictures of the specimens were taken many times and the clearest pictures were chosen.

26

A total of 107 specimens were photographed (Appendix D). The minimum

requirement of specimen for a two-dimensional study is twice the number of the

landmarks. In this case, with 16 landmarks, the minimum specimen required is 32. Thus

a 107 specimen data size is well above the minimum requirement (Klingenberg, 2014).

For each specimen, two different pouches were photographed as replicates for each

specimen, making it a total of 214 images to be analysed.

2.3.2 Formating of Images Into TPS File

All 214 pouch images representing 107 specimens were converted into data

format in order to be processed by MorphoJ (Klingenberg, 2011) which is a software for

GMM. Images were saved in a folder in the computer. Images were converted to a TPS

file format using tpsUtil version 1.58 (Rohlf, 2010) (Appendix E).

2.3.3 Digitisation of Images

All pouch images were then digitised as an image information file in tpsDIG

(Rohlf, 2010). These digitised data contain information of the 16 defined landmarks that

were configured on the gastrovasular pouch. During the landmarking process of the

pouch in tpsDig software (Appendix F), the scale was adjusted so the sizes of images

were standardized. This was done by referring to the scale bar indicated in the images,

and calibrating the value of the scale into the tps file. Hence, image information of 214

pouch images representing 107 specimens, were digitized and imported into MorphoJ

for geometric morphometric analysis.

27

2.3.4 Quantification of Measurement Error

Measurement errors will always occur in any data collection procedure, and

errors can be attributed by various factors, such as imprecision of the measuring device,

the location of the landmarks, the rigidity of the specimen and the experience of the

researchers (Arnqvist & Martensson, 1998; Muñoz-Muñoz & Perpiñán, 2010;

Klingenberg, 2014). It is almost impossible to eliminate measurement errors

completely, thus it is important to reduce the measurement errors to as minimum as

possible.

A pilot study using a relatively small sample size can be performed to determine

the relative sizes of errors associated with each step. If the result shows that errors are

negligible, then replicate is not needed during the actual process.

One way of quantifying error in geometric morphometrics analysis study

involves imaging the specimen twice, and digitize (i.e. measurement) each image twice,

which build a 2 stages structure (i.e, imaging and measurement). For certain organisms

with symmetrical shape, another stage of “side” may be added to further investigate the

error associate with “side”. Procrustes ANOVA then can be performed to compute the

deviations of the individual values around the mean value at higher level (Figure 2.5).

The value at each level can then be used as relative sizes of errors associated with each

step. If the error is small, then replicate is not needed (Viscosi & Cardini, 2011).

28

Figure 2.5: The Number of replicate for each level of the process of specimen imaging

and digitization.

A preliminary quantification measurement error test was performed on 25

randomly chosen specimens using the Procrustes ANOVA analysis (Appendix G). For

each specimen, two gastrovascular pouches were photographed. Each pouch was

photographed twice, and each photograph was digitised twice. Therefore, three levels of

errors were measured: errors associated with “side”, imaging and the digitization of

landmarks, respectively. Hence, the total images used in Procrustes ANOVA for each

specimen was:

2 pouches (from each specimen) x 2 photographs x 2 digitizations = 8 images.

For 25 specimens, a tps file containing the image information of 200 images was

imported into MorphoJ to perform Procrustes ANOVA analysis.

2.3.5 Procrustes Superimposition

To remove the information of position, orientation and size of the specimens, a

procedure called the generalized Procrustes superimposition is performed.

(Klingenberg, 2013; Mitteroecker et al., 2013). The generalized Procrustes

superimposition is an iterative procedure that fits each configuration to the mean shape

in the sample as closely as possible. The end result of generalized Procrustes

superimposition is that only shape related information are extracted from the samples

landmark configurations.

29

Three stages were involved in the genaralized Procrustes superimposition. First,

variation in size was removed by scaling each configuration so that it has a centroid size

of 1.0. Secondly, variation in position was removed by shifting the landmark

configurations so that they shared the same centroid. The third procedure dealt with

rotations to find an optimal orientation for each configuration.

After the generalized Procrustes superimposition, every configuration in the

sample is then optimally aligned to the average configuration. Because the

configurations were aligned so that position, orientation and size were kept constant

according to the criterion for the least-squared fit, the remaining variation in landmark

positions will be solely the variation of shape only. The relative position of the

landmarks from the mean shape to another shape can then be used to detect the shape

variation. These relative position of the landmarks also provide a visualization of the

shape change by showing how the landmarks are reposition against each other after the

non-shape components variation of position, orientation and size are removed using the

Procrustes superimposition. (Klingenberg, 2013).

In this study, a generalized Procrustes superimposition was performed on the

gastrovascular pouch to eliminate the non-shape information (size, position and

orientation) of the image (Klingenberg, 2016). A covariance matrix was initially

generated before running the Procrustes analysis to produce a result set (Appendix H).

Once the Procrustes analysis was completed, two types of statistical analyses were

performed: the Principal Component Analysis (PCA) and Canonical Variate Analysis

(CVA).

30

2.3.6 Principle Component Analysis Using MorphoJ

PCA is a multivariate analysis whereby a numbers of factors, or components

that can be used to represent relationships among the data set with many possible

correlated variables are identified. In PCA, the basic principal is to identify a few major

components that explain most of the total variance. The first principal component (PC)

accounts for the most variability in the data set, the second PC accounts for the next

most variability, and follow by each succeeding PCs. Mathematically, this is done by

transforming the data to different axes so that those with the same pattern are aligned to

form a PC. In the context of geometric morphometrics analysis, the specimens can be

treated as points in a multivariate space where the viewing position of the data are

changed so that the most important components of the shape change are able to be

identified (Viscosi & Cardini, 2011).

PCA does not assume there any group membership in the data set. PCA will try

to maximize the variance on each of the component, and if the between-group variations

is bigger than the within-group variations, the scatterplots of PCA may show the group

variations. On the other hand, even if PCA failed to show any group varation, it doesn’t

necessary mean that there is no group variation in the data. (Strauss, 2010). Therefore,

PCA can be used as a preliminary review of the data, without making any assumptions

of group membership in the data set.

In this study, a PCA is performed for the general inspection of the gastravascular

pouch shape of C. chinensis (Appendix H).

31

2.3.7 Canonical Variate Analysis Using MorphoJ

The primary use of CVA in geometric morphometrics analysis is to determine

whether there is a significant difference in shape for pre-defined distinct groups in

multivariate data set. Therefore, in contrast to PCA, CVA makes the assumption that

there is group membership in the dataset. It tries to maximize the between-group

variation relative to the within-group variation. CVA is similar to PCA in the sense that

it realigns data to reconstructs new axes to form components, or factors.

There is a possibility that CVA will find distinction between group

memberships, even though a preliminary review by PCA may not indicate any

distinction between groups. Of course the underlying biological reasons to assign

specimens to different groups should be valid, or else the result generated by CVA may

not be significant (Sheets et al., 2001; Webster, 2010).

In this study, a CVA was performed on the gastrovascular pouches shape of C.

chinensis to distinguish populations by four coastal areas (Appendix H).

2.3.8 Visualization of Shape Outline

One of the main advantages of geometric morphometrics analysis is the ability

to visualize shape change. One of the widely used method for visualization is the

transformation grid. The deformation method is performed by placing a two-

dimensional rectangular grid over the first specimen, or the initial form, and the grid

was “stretched” to match the morphology of the second specimen, or the target form.

All parts of both specimens are still in the same grid cell after the transformation. The

change in the grid shows the differences in the shape of both specimens (Richtsmeier et

al., 2002). Later on, Bookstein (Bookstein, 1978), successfully adapt D’Arcy

32

Thompson’s transformation grid into thin plate spline, where he was able to construct

the transformation grids using an interpolation technique that fits the grids perfectly on

landmarks (Klingenberg, 2013).

Transformation grids by itself with only landmark configurations and grid can

be difficult for viewer to understand the morphological structure of the subject.

Furthermore, landmarks chosen do not sufficiently describe the morphology of the

subject of study. When all the landmarks are connected, the shape will usually not look

quite like the shape of the real organism as the connection between each landmark are

rather linear and sharp. Thus, to better visualize the shape, an outline drawing is used to

represent the organism. Sometimes, two outline drawings are imposed one on top of the

other to show the variation in shape. Although outline drawing is visually compelling in

displaying shape and variation in shape, it must be reminded that one must not rely

solely on outline drawing when analyzing shape and shape variation. Outline file is

merely a graphical representation of the shape of the specimen for the purpose of better

visualization. The disadvantage with outline file is that the points along the outline are

only assumptions, and may not represent the real shape of the specimen (Klingenberg,

2013).

In this study, an outline file was produced in tpsDIG using 56 landmark

coordination sets (Figure 2.6, Appendix I). They were able to represent the shape

outline of the gastrovascular pouch more closely to that of the specimen when compared

with outlines based on only 16 landmarks for the geometric morphometric analysis

process.

33

Figure 2.6: 56 landmark configurations used to create the outline of gastrovascular

pouch

34

CHAPTER 3: RESULTS

3.1 OVERVIEW OF MAJOR MORPHOLOGICAL STRUCTURES OF

JELLYFISH

Class Scyphozoa is ascribed with four orders, namely Stauromedusae Coronatae,

Semaeostomeae and Rhizostomeae, with 65 genera and over 187 species (Mayer, 1910;

Kramp 1961; Daly et al., 2007; Bayha et al., 2010). This study focuces on the order

Semaeostome and Rhizostomae only.

3.1.1 Order Semaeostomeae

The order Semaeostomeae composed of three families, four subfamilies, 18

genera and 56 species (Kramp, 1961). Semaestommeae jellyfish are characterized by

four oral arms around the mouth. Tentacles are found at the umbrella margin. (Arai,

1997). The two families of Semaeostomeae of the interest in this study are Cyaneidae

and Pelagiidae.

FAMILY CYANEIDAE

Family Cyaneidae is Semaeostomeae that central stomach gives rise to radiating

pouches, and it in turns gives rise to numerous blind canals extending towards the

marginal lappets; without a ring canal; with gonad completely folded; with tentacles

arising from the subumbrella distally from the margin.

Genus Cyanea – with eight rhopalia; with eight whorls of adradial tentacles, each

contains several rows of tentacles; radial and circular muscles in the subumbrella.

35

FAMILY PELAGIIDAE

Family Pelagiidae is Semaeostomeae that central stomach gives rise to radiating

pouches divided by a septa; without a ring canal; tentacles are formed at the cleft of the

bell margin; oral arms long and folded.

Genus Chrysaora – with 32 – 48 simple marginal lappets; with eight marginal sense

organs; with three or more tentacles for each octant; with 16 radial stomach pouches; in

the marginal area the eight rhopalar pouches are narrower than the eight tentacular

pouches, thus forming an “S” shape; exumbrella with numerous nematocyst warts.

3.1.2 Order Rhizostomeae

The order Rhizostomeae composed of two suborders, 10 families, 25 genera and

approximately 89 species (Kramp, 1961). Rhizostomeae jellyfish are characterized by

having bell margin cleft into lappet, with no tentacle on the bell margin, without a

central mouth, with eight oral arms extended from the subumbrella, where each oral

arms are bear numerous secondary mouths. Network of canals are found beyond the

stomach. (Kramp, 1961; Arai, 1997). This study focuces on the order Mastigiidae,

Versurigidae, Lychnorhizidea, Catostylidea, Lobonematidae and Rhizostomatidae.

FAMILY MASTIGIIDAE

Family Mastigiidae is charaterized with short, pyramidal, three-winged oral

arms; with numerous filaments on the oral disk (Kramp, 1961).

36

Genus Phyllorhiza - with broad oral arms, leaf-shaped, with window opening in the

oral arms, with numerous filaments at the oral arms; network of canals bounded by the

ring canal and do not anastomoses with the perradial rhopalar canals.

FAMILY VERSURIGIDAE

Family Versurigidae is charaterized with broad, leaf-shaped oral arms. (Kramp,

1961)

Genus Versuriga – with three-winged oral arms, with scapulaes, with terminal club,

with numerous filaments and clubs formed at the oral arms (Kramp, 1961).

FAMILY LYCHNORHIZIDAE

Family Lychnorhizidae is charaterized with centripetal, usually blinded end and

not anastomosing with the 16 canals; with broad and folded oral arms (Kramp, 1961).

Genus Lychnorhiza – with three-winged oral arms, without terminal club, with or

without filaments; with eight radial canals reaching bell margin, and another eight

reaching only the ring canal. (Kramp, 1961)

FAMILY CATOSTYLIDEA

Family Catostylidea is charaterized with intracircular network of canal

anastomosing with the ring canal but not always with the 16 radial canals; the eight

rhopalar canals reaching bell margin, and inter-rhopalar canals reaching only the ring

canal. Oral arms pyramidal (Kramp, 1961).

37

Genus Acromitus – Intracircular network of canals anastomosing with the ring canal

and the rhopalar canal, but not with the inter-rhopalar canals; oral arm is characterized

with a terminal club (Kramp, 1961).

FAMILY LOBONEMATIDAE

Family Lobonematidae is charaterized with intracircular network of canal

anastomosing with the ring canal and with some or all of the 16 – 32 radial canals, but

not with the stomach; with window openings in the membranes of the oral arms;

marginal lappets very elongated, tapering (Kramp, 1961).

Genus Lobonemoides – With large-meshed, intracircular network of canal

anastomosing with the ring canal and rhopalar canals.

FAMILY RHIZOSTOMATIDAE

Family Rhizostomatidae is charaterized with oral arms coalesced in proximal

portion only; without a mouth opening; with complicated network of canal system;

distal portion of the oral arms three winged, and usually with a terminal club (Kramp,

1961).

Genus Rhopilema – With large scapulae and long manubrium; oral arms with

numerous club and filaments, usually with a large terminal club; usually without a ring

canal; broad network of canal with numerous fine meshes; inter-rhopalar canal wide.

In this study, a total of 146 specimens (nine species from nine genera) were

obtained from the coast of Peninsular Malaysia (Figure 3.1 and Appendix J). Among

38

them, a total of 44 specimens (eight species from eight genera) were examined which

also included museum specimens (Appendix K). The nine species of scyphozoan

jellyfish described in this thesis were Chrysaora chinensis, Cyanea sp., Rhopilema

esculentum, Rhopilema hispidum, Lobonemoides robustus, Versuriga anadyomene,

Phyllorhiza punctata, Lychnorhiza malayensis and Acromitus flagellatus.

Figure 3.1: Map of eight sampling sites, with number of specimen collected for each

species.

Adult medusae of scyphozoan jellyfish (Figure 3.2) consists of a rounded bell

attached to a manubrium. Oral arms emerge from below the manubrium. In general,

jellyfish in the Semaeostomeae order possesses four oral arms, whereas those in the

Rhizostomeae order possess eight oral arms. At the edge of the bell are a series of

indentation called lappets. Tentacles are usually formed at the gap between lappets,

although not all jellyfish possess tentacle. A central stomach, or gastrovascular cavity is

39

found beneath the bell, or the subumbrella. It extends to the margin of the bell, forming

a network of canals. The complexity and the shape of the network vary between each

species.

Figure 3.2: Whole medusa of an adult jellyfish, Phyllorhiza punctata

Below is the definition of several major structure used for the description of the

morphology:

BELL - The bell, or the umbrella of adult scyphozoan medusae are usually rounded,

forming a hemispherical shape. The outer surface of the bell is the exumbrella, or also

40

known as the aboral surface; whereas the underside of the bell is the subumbrella or the

oral surface.

Figure 3.3: Morphology of bell of four different species of scyphozoan jellyfish. (A):

Acromitus flagellates with smooth surface (B): Rhopilema esculentum with smooth

surface (C): Phyllorhiza punctata with warts on the surface (D): Cyanea sp. With

smooth surface

LAPPET - The margin of the bell is usually clefted, and each of the indentation is

called the lappet. The lappet adjacent to the rhopalium is the rhopalar lappet, and the

rest of the lappets are the velar lappets. The lappets can be in many shapes, from

rounded, elongated to semi-squared.

Figure 3.4: Lappet of two different species of scyphozoan jellyfish. (A): Rhopalar

lappet (RL) and velar lappet (VL) of Lychnorhiza malayensis (B): Lappet of Cyanes sp.

(L). Lappet shape broad and semi-square

41

MARGINAL SENSORY ORGAN/ RHOPALIUM - Rhopalia (singular: rhopalium):

Rhopalia are tear-drop shape structures which contain the sensory organs to detect light

(eye spots) or direction (statoliths). Rhopalia are located at the cleft of the bell margin

between a pair of lappet. Usually eight to sixteen rhopalia in a jellyfish.

Figure 3.5: Marginal sensory organ / rhopalium (R) of Lychnorhiza malayensis

TENTACLE - The tentacles are string-like organs that usually contain cnidae.

Tentacles can grow up to several metres in length on certain species. Tentacles are

usually not present in Rhizostomeae jellyfish. Tentacles are located at the cleft of the

bell margin between a pair of lappet.

42

Figure 3.6: Morphology of umbrella. (A): Tentacle (T) of Chrysaora chinensis of

Semaestomeae. (B): Lychnorhiza malayensis of Rhizostomeae without tentacles

MANUBRIUM - The manubrium consists of the under portion of the subumbrella,

where the mouth hangs down on a pendulous structure, together with the basal portion

of the oral pillar.

Figure 3.7: Morphology of the manubrium of C. chinensis

43

ORAL ARM - Oral arm, also known as mouth arm is the organ extending from the

base of the manubrium, connected by the oral pillar. There are four oral arms for the

order Semaestomeae, and eight for the order Rhizostomeae. Oral arms are used to catch

prey for the former order, whereas the latter, food are transferred from the oral arms into

the stomach through second arms (or mouthlets) on the oral arms.

Figure 3.8: Gross morphology of oral arm. (A): Whole medusa of Chrysaora chinensis

floated in a tank, showing four soft and curtain-like oral arms (OA). (B): Oral arm (OA)

of Rhopilema esculentum. (C): Oral arm (OA) of Acromitus flagellatus

44

SCAPULAE - Scapulae are leaf-like organs attached to the manubrium. Usually two

scapulae are attached to each oral arm. Scapulae, like the oral arm, consist of numerous

mouth openings and stomach filaments.

Figure 3.9: Morphology of scapulae. (A): Blade shape scapulae of Rhopilema hispidum.

(B): Scapulae (S) attached to oral arms of Rhopilema esculentum

TERMINAL CLUB - Terminal club is the organ that originates at the margin of the

oral arm. It dangles at the tip of the oral arm.

Figure 3.10: Morphology of oral arm with terminal club. (A): Terminal club (C) at the

margin of the oral arm of Rhopilema esculentum. (B): Terminal club (C) at the margin

of the oral arm of Phyllorhiza punctata

45

FILAMENT - Filaments can be found on the oral disk and the oral arms. Filaments are

primarily used for swimming and feeding. Some species even have nematocyst on

filaments, and also used filaments to externally transport food to the mouth

Figure 3.11: Morphology of filaments. (A): Filament (F) at the oral disk of Phyllorhiza

punctata. (B): Filament (F) at the oral arms of Acromitus flagellatus

GASTROVASCULAR CAVITY - Stomach or gastrovascular cavity is the space

between the exumbrella and subumbrella that would contain products of digestions

which are distributed to the rest of the gastrovascular system. (Arai, 1997). The

distribution is done through a network of canal (see also the Network of Canal section

below). The gastravascular cavity in scyphozoan jellyfish is usually divided into four

compartments. Each compartment is separated from each other by a radial septa.

46

Figure 3.12: Morphology of subumbrella of Chrysaora chinensis revealing the

gastrovascular cavity

47

SUBGENITAL FENESTRATION - Also known as ostia, it is the opening formed at

the base of the subumbrella, fusioned between a pair of oral arms. It is usually oval in

shape. In certain mature medusae, gonads can be seen protruding from within the

subgenital fenestrations.

Figure 3.13: Subumbrella morphology of subgenital fenestration (F) of Rhopilema

esculentum

48

PAPILLAE - Papillae are protuberances located in the front or around the subgenital

fenestration. Some species usually have three papillaes for each subgenital fenestration.

One, usually a larger one, located at the central, and one smaller papillae one on each

side the subgenital fenestration.

Figure 3.14: Subumbrella morphology of papillae. (A): Three papillae (P) at the

subumbrella of Lychnorhiza malayensis. (B): Three papillae (P) at the subumbrella of

Rhopilema hispidum

49

NETWORK OF CANALS - Numerous tubes that radiating from the gastrovascular

cavitiy form a network of canals. The canals leading from the axis where gonads are

located are called the interradial canal. The canals leading from the axis between gonads

are called perradial canal. The canals between the interradial and perradial canals are

called the adradial canals. The complexity and the shape of the network of canals vary

between each species and can be useful for species identifcation.

Figure 3.15: Network of canals of (A): Lychnorhiza malayensis injected with red dye.

(B): Phyllorhiza punctata injected blue dye. (C): Lobonemoides robustus injected with

yellow dye

50

MUSCLE - There are two types of muscles in Scyphozoan jellyfish; the radial muscles

that extend radially on the subumbrella and the coronal muscles are found as concentric

rings around the subumbrella. Sometimes the coronal muscles may or may not overlap

with the radial canals underneath.

Figure 3.16: Subumbrella morphology of muscle. (A): Coronal muscle (CM) of

Phyllorhiza punctata. (B): Coronal muscle (CM) and radial muscle (RM) of Cyanea sp.

51

GONAD - Gonads of the scyphozoan jellyfish arise from the gastrodermis. In medusae,

the gonads are usually situated on the base of the gastrovascular cavity, peripheral to the

gastric filaments (Arai, 1997). Gonads are usually milky pink to yellowish. In adult

medusae, gonads can be seen protruding from the subgenital fenestrations

Figure 3.17: Subumbrella morphology of gonad. (A): Gonad (G) protruding from the

subgenital fenestration of Cyanea sp. (B): Gonad (G) at the base of the gastrovascular

cavity of Versuriga anadyomene.

52

3.2 Morphological Description of Chrysaora chinensis Vanhöffen 1888

Class SCYPHOZOA (Goette, 1887)

Order SEMAEOSTOMEAE (Agassiz, 1862)

PELAGIIDAE (Gegenbaur, 1856)

Chrysaora (Péron & Lesueur, 1809)

Chrysaora chinensis (Vanhöffen, 1888) resurrection by Morandini & Marques (2010)

(Figure 3.18 – 3.19)

Material Examined - A total of seven specimens were examined from sites Sungai

Janggut (MRI 2, MRI 65, MRI 163, MRI 171), Pantai Sabak (MRI 208), Balik Pulau

(MRI 262) and Kampung Cempaka (MRI 190).

Description of specimens- Bell: Umbrella hemispherical. For adult medusa,

exumbrella surface finely granulated; in some specimens the exumbrella is transparent

but some have light reddish spots distributed evenly on the surface. Mesoglea rigid,

thick in the centre and thinner towards the bell margin. Six lappets per octant; shape are

semi-circular, with rhopalar lappets. On certain specimens there is a small lappet

connecting to the rhopalar lappet. These lappets are loosely connected with the adjacent

lappet, and some of them appeared to be separated, therefore making eight lappets per

octant on certain specimens. Eight rhopalia present in clefts of the umbrella margin,

located at the interradial and perradial axis. Rhopalia shape is tear-drop like.

Tentacles: Three tentacles per octant (2-1-2, where the numbers represent the

ontogenetic order of the development of tentacles), originated at the cleft of the lappet.

Tentacles soft, straight and string-like. Tentacle length up to four times the diameter of

53

the bell. Personal field observations of the tentacles were much longer, more than four

times of the bell diameter, but sometimes detached when specimens were caught or

handled roughly.

Oral arms: Four oral arms, up to 200cm in length in adult medusa; oral arms formed at

the end of the oral pillar. Oral arms are transparent, soft and curtain like, with a ridge in

the centre and intricate folds at the edges. In certain specimens numerous reddish /

brownish spots are found all over the oral arm. Scapulae absent. Terminal club absent.

Filament absent.

Stomach, manubrium and radial canals: Central stomach shape slightly rounded, as

it is formed by the boundary of the 16 gastrovascular pouches, where eight are rhopalar

pouches and another eight are inter-rhopalar pouches. Radial septa present; extending

distally from stomach toward bell margin. For rhopalar pouches, septa starts widening

at approximately 1/3 from the centre, and thinning again, making an “S” shape that ends

at the base of the lappet. Subgenital fenestration shape oval, 1/8 of umbrella diameter.

Papillae absent. Network of canal absent. Radial and coronal musculatures not

prominent. Manubrium is not prominent. Oral disk at the base is about 1/3 as wide as

the bell diameter, and the base of oral arm is less than 1/3 as wide as the bell diameter.

Mouth present with four walls around the mouth. Quadralinga absent.

Gonads: Four gonads present, each located inside a subgenital fenestration of the

subumbrella; each gonad is separated with the neighbouring gonad by a septa;

associates with the subumbrella and manubrium. Colour creamy white. Gastric

filaments present.

54

Figure 3.18 Gross morphology of Chrysaora chinensis. (A): Whole medusa floated in a

tank, showing four soft and curtain-like oral arms (OA) and tentacles (T). (B): Side

view of the bell (umbrella) with reddish brown pigmentation on the lappet (L). (C): Oral

arm (OA1) without pigmentation; (D): Oral arm (OA2) with reddish pigmentation; (E):

Subumbrella view of the central stomach (S).

55

Figure 3.19: Umbrella morphology of C.chinensis. (A): Subumbrella view revealing 16

gastrovascular pouches with rhopalar pouch (P1), inter-rhopalar pouch (P2), rhopalium

(R), lappet (L), tentacle (T) and oral arms (OA). (B): Subumbrellla view of creamy

white gonads (G), whereby each of the four gonads is located inside a subgenital

fenestration of the subumbrella. (C): Subumbrella view showing the mouth (M)

bounded by four walls. (D): View of exumbrella with numerous warts (W) densely

concentrated in the centre. (E): View of subumbrella showing lappet (L), rhopalia (R)

and three tentacles (T) following a 2-1-2 arrangement per octant, whereby the numbers

represent the ontogenetic sequence of tentacles originating from the cleft of the lappet.

56

Figure 3.19, Continue

(F): Subumbrella view of another specimen showing distinctly pigmented lappet (L),

rhopalia (R) and tentacles (T).

3.3 Morphological Description of Cyanea Sp.

Class SCYPHOZOA (Goette, 1887)

Order SEMAEOSTOMEAE (Agassiz, 1862)

Cyaneidae (Agassiz, 1862)

Cyanea (Péron & Lesueur, 1810)

Cyanea Sp. (Figure 3.20 – 3.21)

Material Examined - A total of seven specimens were examined from sites Sungai

Janggut (MRI 55, MRI 56, MMRI 95, MRI 96, MRI 97, MRI 127 SJG 10)

Description of specimens - Bell: Flattened hemispherical. Mesoglea is rigid, thick in

the centre and thin towards the bell margin. Numerous faint warts are distributed on

exumbrella surface, denser at the centre of the bell and sparce towards the cleft of the

lappet. Sixteen equally broad sized lappets, semi square shaped along the margin of the

umbrella. All lappets overlap each other at the cleft. Rhopalar cleft is deeper than the

inter-rhopalar cleft. Eight tear drop shaped rhopalia, present in clefts of the umbrella

margin, located at the interradial and perradial axis. .

Tentacle - Eight whorls of tentacles originating from the coronal muscle at the

subumbrella. Each tentacle whorl is horseshoe shaped, consisting of two to three layers,

located between two groups of inter-rhopalar radial muscles. Tentacles are soft, straight

and string-like; up to 200 tentacles per whorl.

57

Oral arm - Four oral arms, up to four times as long as the diameter of the bell although

most were detached during the sampling process; oral arms formed at the end of the oral

pillar. Oral arms are transparent, soft and curtain like. Scapulae, terminal clubs and

filaments were absent.

Stomach and Manubrium - The digestive system of this species consists of a

gastrovascular cavity or central stomach, which is slightly round, as it is formed by the

boundary of the 16 coronal muscle groups. Sixteen pouches in the umbrella are

comprised of eight rhopalar pouches and eight tentacular pouches. Subgenital

fenestration is oval, 1/9 of the bell diameter. Papillae absent. Between six to seven

transverse “ridges” connecting both pouches below their surface. Both rhopalar pouches

and tentacular pouches also anastomoses with the lappets on both side of the cleft,

forming dense network on the lappet. Sixteen groups of coronal muscles originating

from the outer margin of the central stomach at the subumbrella, extending towards the

rim of the oral disk. Each coronal muscle group consists of six to eight muscle folds.

Bigger coronal muscle groups alternate with slightly smaller coronal muscle groups.

Muscle groups are not connected, with a ridge in between the two coronal muscle

groups. Gastrovascular pit absent. Sixteen groups of radial muscle originate at the

underside of the lappet, perpendicular to the coronal muscle. Each radial muscle group

consists of nine to eleven radiating muscle folds with different sizes and lengths, with

the longest muscle folds located in the centre of the muscle field. Manubrium thick and

smooth; oral disk at the base is about 1/3 as wide as the bell diameter.

Gonad - Gonads form below the subumbrella and attached with the subumbrella and

manubrium; each gonad is separated from the neighbouring gonads. Colour creamy

white. Gonads are big and mostly protruding out of the subgenital fenestration. Gastric

filaments present.

58

Figure 3.20: Gross morphology of Cyanea sp. (A): Whole medusa. (B): Numerous

tentacles (T) and curtain like oral arms (OA). (C): View of exumbrella with numerous

warts (W). (D): Subumbrella showing a group of radial muscle (RM) and a horseshoe

shaped whorl of tentacles (T) between each group of radial muscle. (E) Subumbrella

view showing a group of coronal muscle (CM).

59

Figure 3.21: Subumbrella morphology and structures of Cyanea sp. (A): Subumbrella

view of the medusa. (R) Rhopalium. (G) Gonad. (T) Tentacle. (L) Lappet. (CM)

Coronal muscle. (RM) Radial muscle. (B): A whorl of tentacle (T). Radial muscle

(RM). Coronal muscle (CM), alternating between a longer group and a shorter group.

(C): Lappet shape broad and semi-circular, with network of canal (C). (D): Gonad (G)

creamy white.

60

3.4 Morphological Description of Rhopilema esculentum Kishinouye 1891

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

RHIZOSTOMATIDAE (Cuvier, 1799)

Rhopilema (Haeckel, 1880)

Rhopilema esculentum (Kishinouye, 1891) (Figure 3.22 – 3.23)

Material Examined - A total of three specimens were examined from site Sungai

Janggut (MRI 87, MRI 98, IND 3)

Description of specimens - Bell: Flattened hemispherical. Umbrella surface is smooth

and reddish. Mesoglea is rigid, thick in the centre and thin towards the bell margin.

Eight lappets per octant; shape are slightly elongated with pointy rhopalar lappets. Eight

tear-drop shaped rhopalia present in clefts of the umbrella margin, located at the

interradial and perradial axis. Tentacles absent.

Oral arm - Eight oral arms, as long as the bell diameter; oral arms formed at the end of

the oral pillar, then develop into three wings at the distal section (approximately at the

distal 1/5 of the oral arm). Two wings face away from the central axis (outer wings) and

one wing faces the central axis (inner wing). Numerous mouthlets located at the frills of

the wings. One to three spindle-shape appendages form at the frills of the wings.

Numerous filaments found at the frill of the wings. For each oral arm, two blade-shaped

but flat scapulae developed approximately 1/3 distally from the base of each oral pillar,

then develop into two wings for each scapulae. Winged portion of the scapulae is

smaller than the winged portion of the oral arms. Numerous mouthlets located at the

frill of the scapulae. Numerous short filaments located at the frills of the scapulae.

Terminal club absent.

61

Stomach and Manubrium - Gastovascular cavity or central stomach is floret shaped.

Each subgenital fenestration has one papillae located at the base of the manubrium and

near the edge of the subgenital fenestration. Papille dome shape. Papillae surface is

rough with numerous warts. Sixteen radial canals on the subumbrella are comprised of

eight rhopalar canals and eight inter-rhopalar canals; all canals fully extend to the

margin of the bell, where rhopalar canals split at the distal end and branch toward the

neighbouring lappets. Ring canal present. Rhopalar canals start anastomosing with the

neighbouring canals at about 1/3 from the centre, whereas interhopalar canals start

anastomosing with the neighbouring canals at about 1/2 from the centre. Radial muscle

absent. Sixteen coronal muscle fields cover the network mesh underneath. Coronal

muscle interrupted on the rhopalar canals but not on the inter-rhopalar canals.

Manubrium thick and smooth; oral disk at the base is about half as wide as the bell

diameter, and the base of oral arm is less than 1/6 as wide as the bell diameter.

Gonad - Gonad floret shape, formed at the subumbrella; each gonad is separated from

the neighbouring gonad; attached with the subumbrella and manubrium. Colour white.

Gastric filaments present.

62

Figure 3.22: Gross morphology of Rhopilema esculentum (A): Whole medusa. (B):

Exumbrealla (EX) smooth. (C): Terminal club (C) attached to oral arm. (D): 16

scapulae (S) in each medusa. (E): One subgenital fenestration (F) for each quadrant.

One papilae (P) at the centre of the fenestration. (F): Oral arm (OA).

63

Figure 3.23: Subumbrella morphology and structures of Rhopilema esculentum (A):

Gonad (G) creamy white. (B): Lappet (L). Coronal muscle (M). Rhopalia (R). (C):

Network of canals injected with green dye.

64

3.5 Morphological Description of Rhopilema hispidum Vanhöffen 1888

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

RHIZOSTOMATIDAE (Cuvier, 1799)

Rhopilema (Haeckel, 1880)

Rhopilema hispidum (Vanhöffen, 1888) (Figure 3.24)

Material Examined – A total of four specimens were examined from site Sungai

Janggut (MRI 13, MRI 89, IND 9, IND 10).

Description of specimens - Bell: Umbrella hemispherical with a rough surface that

feels like sand paper. Numerous colourless warts and larger reddish brown circular

warts are found on the exumbrella and lappets. Warts are bigger and more concentrated

at the centre of the bell, decreasing in size towards the lappets. Eight slightly elongated

and pointy lappets located in each octant. The velar lappets adjacent to rhopalar lappets

are slightly bigger than the rest of the lappets. Short furrow forms between lappets,

whereas larger furrow forms between two pairs of velar lappets. Numerous warts are

concentrated on the lappets. Eight tear-drop shaped rhopalia present in clefts of the

umbrella margin, located at the interradial and perradial axis. Tentacles absent.

Oral arm - Eight oral arms, as long as the bell diameter; oral arms formed at the end of

the oral pillar, then develop to three wings at the distal section (approximately at the

distal 1/5 of the oral arm). Two wings face away from the central axis (outer wings) and

one wing faces the central axis (inner wing). Numerous mouthlets located at the frill of

the wings. Sixteen blade-shape but flat scapulae for each medusa. Four scapulae

developed approximately 1/3 distally from the base of each oral pillar, then develop to

two wings for each scapulae. Winged portion of the scapulae is smaller than the winged

65

portion of the oral arms. Numerous mouthlets located at the frill of the scapulae.

Numerous short filaments located at the frills of the scapulae. Terminal club absent

from the specimens observed, although there is a segment of left over hanging from the

end of the oral arm that may be the origin of the missing terminal club. In the field,

terminal clubs are observed. According to Omori (2001), a club shape terminal club is

found at the tip of the oral arm. Filaments are found at the scapulae.

Stomach and Manubrium - Gastrovascular cavity, or central stomach is floret shape.

Subgenital fenestration shape oval. 1/7 of the size of the bell diameter. Each subgenital

fenestration has three papillae. The largest papillae located at the upper side of the

subgenital fenestration, aligning with the rhopalar canal. One smaller on each side of the

subgenital fenestration. Papillae shape oval with rough surface. Sixteen radial canals on

the subumbrella comprised eight rhopalar canals and eight inter-rhopalar canals; all

canals fully extend to the margin of the bell, where rhopalar canals split at the distal end

and branch toward the neighbouring lappets. Inter-rhopalar doesn’t branch toward the

neighbouring lappets. Ring canal absent. A mesh of network formed between the canals,

where the proximal part of the network anastomosing more densely but decrease in size

towards the distal part. Rhopalar canal starts anastomosing with the neighbouring canals

at about 1/3 from the gastovascular cavity, whereas interhopalar canals start

anastomosing with the neighbouring canals at about ½ from the gastovascular cavity.

Three canals (two adradial, one perradial) connecting to the oral arm. Radial muscles

absent. Sixteen prominent coronal muscle fields is interrupted at the sixteen radii; each

field forms a pyramid shape. Manubrium thick and smooth; oral disk at the base is

about half as wide as the bell diameter, and the base of oral arm is less than half as wide

as the bell diameter.

66

Gonad- Gonads are floret shape and form below the subumbrella; each gonad is

separated from the neighbouring gonads and attached with the subumbrella and

manubrium. Colour yellowish. Gastric filaments present.

Figure 3.24: Gross morphology of Rhopilema hispidum (A): Whole medusa. (B): Three

papilae (P) for each quadrant where the largest is in the centre. (F) Filaments. (C): Blade

shape scapulae (S) with numerous filaments (F). (D): Exumbrella surface rough, with

numerous warts (W). (E): Lappet (L). (F): Network of canal (C) injected with blue dye,

forming a pyramid shape of network.

67

3.6 Morphological Description of Lobonemoides robustus Stiasny 1920

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

Lobonematidae (Stiasny, 1921)

Lobonemoides (Light, 1914)

Lobonemoides robustus (Stiasny, 1920) (Figure 3.25 – 3.26)

Material Examined – A total of five specimens were examined from site Sungai

Janggut (IND 6, IND 7, IND 8, SJ 130) and Kampung Cempaka (MRI 204).

Description of specimens - Bell: Umbrella hemispherical. Numerous soft, long conical

papillae on the exumbrella, where size and density of the protuberance decreasing

towards the bell margin. Eight elongated lappets in each octant. Rhopalar lappets are

small and pointy. Short furrow between each lappets. A formation of larger furrow

between two pairs of velar lappet. 16 tear-drop shaped rhopalia present in clefts of the

umbrella margin, located at the interradial and perradial axis. Tentacles absent.

Oral arm – Eight oral arms, as long as the bell diameter; base of oral arm is less than

1/3 as wide as the bell diameter oral arms begin at the end of the oral pillar, then

develop into three wings at the distal section (approximately at the distal 1/5 of the oral

arm). Two wings facing away from the central axis (outer wings) and one wing faces

the central axis (inner wing). Numerous mouthlets located at the frills of the wings.

Numerous filaments present at the frills of the oral arms. Fenestrations, or windows are

found in the oral arm. Scapulae absent. A definite terminal club absent, but four to nine

spindle shape clubs on each oral arm that are easily detached. Numerous pigmented

spots on the clubs. A canal is found in the centre of the club, extending proximately

68

from the base of the club towards the distal end of the club. String like filaments are

found on oral arm.

Stomach and Manubrium – Almost floret shaped gastrovascular cavity, or central

stomach Subgenital fenestration is oval; 1/5 the length of the bell diameter. Three small

and non-prominent dome shaped papillae located at the lower side of the subgenital

fenestration; the papillae in the centre is smaller than the two on each side; the centre

papillae forms a ridge that extends towards the inner subgenital fenestration.

Manubrium thick and smooth; oral disk at the base is about half as wide as the bell

diameter. Thirty two radial canals on the subumbrella comprising of16 rhopalar canals

and 16 inter-rhopalar canals; all canals efface beyond the ring canal, towards fully to the

margin of the bell, where rhopalar canals split into two at the distal end and branch

toward the neighbouring lappets. Canals are found in the lappets. Within the ring canal,

three anastomoses connecting both sides of the rhopalar. Most interrhopalar canals are

without anastomoses except a few exceptions where one anastomos exist. Beyond the

ring canal, rhopalar canals anastomoses with the neighbouring canals through

approximately twelve anastomoses, whereas interophalar canal fuses with the

neighbouring canals completely. Networks are also found in the oral arm, where a main

canal connects with the oral arm from the oral disk. Radial muscles absent. Each

quadrant has one coronal muscle fieldt, comprising 80 to 96 muscle folds. The coronal

muscle folds are interrupted at the sixteen rhopalar radii. Purple spots are found in the

muscle field and the surface of the canals and lappets. Manubrium thick and smooth;

oral disk at the base is about half as wide as the bell diameter, and the base of oral arm

is less than 1/3 as wide as the bell diameter.

69

Gonad - Floret shape and bluish gonads found below the subumbrella; each gonad is

separated with the neighbouring gonad; attaches to the subumbrella and manubrium.

Gonad protruding out of the subgenital fenestration. Gastric filaments present.

Figure 3.25: Gross morphology of Lobonemoides robustus (A): Whole medusa. (F)

Filaments. (B): Gonad (G). (C, D): Conical papillae on the exumbrella in water (P1),

and on dry stage (P2).

70

Figure 3.26: Morphology of Lobonemoides robustus (A): Coronal musculatures (M)

with colouration. Rhopalium (R). (B): Spindle shape club (C). (C): Network of canal

(C) with yellow dye injected. (G) Gonad. (D): Lappet (L) elongated. (E): Oral arm (OA)

with yellow dye injected.

71

3.7 Morphological Description of Versuriga anadyomene Maas 1903

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

Mastiigidae (Stiasny, 1921)

Versuriga

Versuriga anadyomene (Maas, 1903) (Figure 3.27 – 3.28)

Material Examined – A total of two specimens were examined from sites Kilim (MRI

153) and Sungai Janggut (MRI 176).

Description – Bell - Umbrella hemispherical. Exumbrella suface grooved irregularly

where the size and quantity of the grooves decreased from the centre towards the

margin. Eight lappets in each octant; shape are slightly elongated. The lappet

neighbouring rhopalar lappets are slightly bigger than the rest of the velar lappets.

Rhopalar lappets small and pointy. Short furrow between each velar lappets. A larger

furrow formed between two pairs of velar lappet. Sometimes a small lappet formed

between two lappets. Eight rhopalia present in clefts of the umbrella margin, located at

the interradial and perradial axis. Rhopalia shape is tear-drop like. Tentacles absent.

Oral arm - Eight oral arms, as long as the bell diameter; oral arms formed at the end of

the oral pillar, then develop to three wings at the distal section (approximately at the

distal 1/5 of the oral arm). Numerous mouthlets at the frills of the wings. Numerous

appendages are found at the frills of the wings. Fenestrations, or windows are found on

the oral arms. Scapulae absent. Terminal club absent. Some club shape structures are

found at the margin of the oral arms. Filaments are found at the oral disk. Filament

shape string-like.

72

Stomach and Manubrium - Gastrovascular cavity, or centre stomach shape floret.

Subgenital fenestration shape oval; 1/5 the length of the bell diameter. Papillae absent.

Manubrium thick and smooth; oral disk at the base is about half as wide as the bell

diameter, and oral disk at the mouth is 1/3 as wide as the bell diameter. Eight radial

canals on the subumbrella. All eight rhopalar canals extending almost all the way to the

margin of the bell, where canals split at the distal end and branch toward the

neighbouring velar lappets. Ring canal present. Interradial rhopalar canals connect with

the neighbouring canals proximally, whereas perradial rhopalar canals only connected

with the neighbouring canals beyond the ring canal. Network becomes denser and

smaller towards the margin of the bell. Radial muscles absent. Eight coronal muscle

fields, each is interrupted at the eight radii. Coronal musculature starts forming

approximate 1/3 distally from the centre, covering 2/3 of the subumbrella until the

margin.

Gonad - Floret shaped and yellowish gonads found formed below the subumbrella;

each gonad is separated with the neighbouring gonads; attaches with the subumbrella

and manubrium. Gastric filaments present.

73

Figure 3.27: Gross morphology of Versuriga anadyomene (A): Whole medusa. Oral

pillar (OP). Numerous mouthlets (MO) on oral arms. Appendage (A). (B): Lappet (L).

(C): Portuberances or irregular grooves (P) on exumberalla. (D): Numerous filaments

on the oral disk. Coronal muscle (M). (OA) Oral arm.

74

Figure 3.28: Morphology of Versuriga anadyomene (A): Oral arm with numerous

mouthlets (M). A window on the oral arm (W). (B): Gonad (G) brownish. (C): Network

of canals injected with blue dye.

75

3.8 Morphological Description of Phyllorhiza punctata von Lendenfeld 1884

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

Mastiigidae (Stiasny, 1921)

Phyllorhiza (Agassiz, 1862)

Phyllorhiza punctata (von Lendenfeld, 1884) (Figure 3.29 – 3.30)

Material Examined – A total of six specimens were examined from sites Sungai

Janggut (IND 2, IND 11, IND 14, MRI 14), Pantai Kok (MRI 131, MRI 139).

Description – Bell - Umbrella hemispherical. Transparent white in the centre of the

bell, gradually brownish orange towards the margin of the bell. Numerous round, white

warts are found evenly distributed on the exumbrella surface. Eight lappets in each

octant; shape are slightly elongated. Rhopalar lappets small and pointy. Short furrow

between each lappets. A larger furrow formed between two pairs of velar lappet. Eight

rhopalia present in clefts of the umbrella margin, located at the interradial and perradial

axis. Rhopalia shape is tear-drop like. Tentacle absent.

Oral arm - Eight oral arms, as long as the bell diameter; oral arms formed at the end of

the oral pillar, then develop to three wings at the distal section (approximately at the

distal 1/3 of the oral arm). Numerous mouthlets at the frills of the wings. Numerous

appendages with bulbs are found at the frills of the wings. Scapulae absent. One

terminal club on each oral arm; half as long as the bell diameter, with a bulb at the end.

Colour light brown / purple. Numerous filaments are found on oral disk.

76

Stomach and manubrium - Gastrovascular cavity, or centre stomach shape floret.

Papillae absent. Eight radial canals on the subumbrella, all eight canals extending all the

way to the margin of the bell, where rhopalar canals split at the distal end and branch

toward the neighbouring lappets. Ring canal present. Interradial rhopalar canals

connected with the neighbouring canals proximally, whereas perradial rhopalar canals

only connected with the neighbouring canals beyond the ring canal. Network becomes

denser and smaller towards the margin of the bell. Radial muscles absent. Eight coronal

muscle fields, each is interrupted at the eight radii. Up to 96 muscle folds per quadrant.

Coronal musculature starts forming approximate 1/3 distally from the centre, covering

2/3 of the subumbrella until the margin. Manubrium thick and smooth; oral disk at the

base is about half as wide as the bell diameter, and oral disk at the mouth is 1/4 as wide

as the bell diameter.

Gonad - Floret shaped and yellowish gonads found formed below the subumbrella;

each gonad is separated with the neighbouring gonads; attaches with the subumbrella.

Gastric filaments present.

77

Figure 3.29: Gross morphology of Phyllorhiza punctata (A): Whole mesusa. Numerous

warts (W) on the exumbrella. Terminal club (TC). (B): Numerous mouthlets (MO) on

the oral arm. (C): Filaments (F) on oral disk. (D): Oral pillar (OP). The white ring is a

wire to suspend the jellyfish and it is not part of the jellyfish structure.

78

Figure 3.30: Morphology of Phyllorhiza punctata (A): Coronal musculatures (M). (R)

Rhopalia (L) Lappet. (B): Warts (W) on exumbrella. (C): Numerous appendages (A) on

the oral arms. Terminal club (C). (D): Gonad (G) brownish. (E): Network of canals (C)

injected with blue dye.

79

3.9 Morphological Description of Lychnorhiza malayensis Stiasny 1920

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

Lychnorhizidae (Haeckel, 1880)

Lychnorhiza (Haeckel, 1880)

Lychnorhiza malayensis (Stiasny, 1920) (Figure 3.31)

First record of species in Malaysia

L. malayensis has a wide distribution in the Malayan Archipelago and the Indian

Ocean), where it was previously found by stiassny in 1920s & 30s in Batavia, Java, by

Menon in 1930 in Madras, India, by Nair in 1951 in Trivandrum, India, but so far have

yet to be documented or found in Malaysia. It has recently been documented in

Andaman Sea and Gulf of Thailand (http://marinegiscenter.dmcr.go.th) and in 2014

sampling by Jeyabaskaran et al (2016) from Thiruvananthapuram to Goa, India.

Material Examined - A total of six specimens were examined from sites Sungai

Janggut (IND 13, MRI 35, MRI 36, MRI 37, MRI 38, MRI 39)

Description – Bell - Umbrella hemispherical. Exumbrella surface smooth. Eight

slightly pointy lappets in each octant. Rhopalar lappets small and pointy. Eight tear-

drop shaped rhopalia present in clefts of the umbrella margin, located at the interradial

and perradial axis. Tentacles absent.

Oral arm - Eight oral arms, as long as the bell diameter; oral arms formed at the end of

the oral pillar, then develop to three wings at the distal section (approximately at the

distal 1/3 of the oral arm). Numerous mouthlets at the frills of the wings. Scapulae

absent. Terminal club absent. Filaments absent.

80

Stomach and manubrium - Stomach shape floret. Subgenital fenestration shape oval;

1/10 the length of the bell diameter. Five papillaes around each subgenital fenestration.

Three papillae located at the upper side of the margin subgenital fenestration, aligning

with the rhopalar canal. One smaller on each side of the subgenital fenestration. Papillae

surface decorated with fine dots, but smooth. Sixteen radial canals on the subumbrella.

Eight rhopalar canals extending fully to the margin of the bell. Eight inter-rhopalar

canals (perradial rhopalar canals) do not extend beyond the ring canal. Ring canal

present. Between four to five centripetal canals within the ring canal blindly ended

(except a few are not blindly ended) and do not anastomoses with the neighbouring

rhopalar nor interrhopalar canals. Canals becomes denser and smaller towards the

margin of the bell. Radial muscles absent. One coronal muscle fields not interrupted at

the eight radii. Between 80 to 96 muscle folds per quadrant. Coronal musculature starts

forming approximate 1/3 distally from the centre, covering 2/3 of the subumbrella till

the margin. Manubrium thick and smooth; oral disk at the base is about half as wide as

the bell diameter, and oral disk at the mouth is 1/4 as wide as the bell diameter.

Gonad – Gonads are floret shaped gonads and form below the subumbrella; each gonad

is separated from the neighbouring gonads; attaches with the subumbrella and

manubrium. Colour yellowish. Gastric filaments present. Gonad is completely protected

within the fenestration with outfold.

81

Figure 3.31: Gross morphology of Lychnorhiza malayensis (A) Whole medusa. (OA)

Oral arm. (B) Rhopalium (R). Lappet (L). (C) Network of canals (C) injected with red

dye. (D) Gonad (G) creamy white. (P) Papillae. (E) Oral arm (OA). (F) Three papillae

(P).

82

3.10 Morphological Description of Acromitus flagellatus Maas 1903

Class SCYPHOZOA (Goette, 1887)

Order RHIZOSTOMEAE (Agassiz, 1862)

Catostylidae (Gegenbaur, 1857)

Acromitus (Light, 1914)

Acromitus flagellatus (Maas, 1903) (Figure 3.32)

Material Examined – A total of three specimens were examined from sites Sungai

Buluh (MRI 21, MRI 23, MRI 24).

Description – Bell - Umbrella hemispherical. Exumbrella surface smooth. Numerous

red dots are distributed on the exumbrella. Eight lappets in each octant; shape are

slightly elongated. Rhopalar lappets small and pointy. Short furrow between each

lappets. A larger furrow formed between two pairs of velar lappet. Eight rhopalia

present in clefts of the umbrella margin, located at the interradial and perradial axis.

Rhopalia shape is tear-drop like. Tentacle absent.

Oral arm - Eight oral arms, 2/3 as long as the bell diameter; oral arms formed at the

end of the oral pillar, then develop to three wings at the distal section (approximately at

the distal 2/3 of the oral arm). Numerous mouthlets at the frills of the wings. Numerous

filaments at the frills of the wing. Scapulae Absent. Terminal club absent, but a single

thread-like filament is formed at the distal end of each oral arm. Numerous string-like

filaments on the oral arms.

83

Stomach and manubrium - Gastrovascular cavity, or centre stomach shape floret.

Subgenital fenestration shape oval; 1/7 the length of the bell diameter. A triangular

shape papillae is found at the centre of the subgenital fenestration. Papillae surface

smooth. 16 radial canals on the subumbrella. Eight rhopalar canals extending fully to

the margin of the bell. Eight inter-rhopalar canals do not extend beyond the ring canal.

Ring canal present. Between three to five centripetal canals within the ring canal

anastomoses with each other, and anastomoses with the neighbouring rhopalar canals,

but not with the inter-rhopalar canals. Canals becomes denser and smaller towards the

margin of the bell. Radial muscles absent. Eight coronal muscle fields covering the

network mesh underneath, each interrupted by the four interradial canals and the four

perradial canals. Coronal musculature starts forming 1/3 distally from the centre,

covering 2/3 of the subumbrella up to the margin. Muscle fields become denser beyond

the ring canal. Manubrium thick and smooth; oral disk at the base is about half as wide

as the bell diameter, and oral disk at the mouth is 1/4 as wide as the bell diameter.

Gonad – Gonads are floret shaped gonads and form below the subumbrella; each gonad

is separated from the neighbouring gonads; attaches with the subumbrella and

manubrium. Colour yellowish. Gastric filaments present.

84

Figure 3.32: Gross morphology of Acromitus flagelatus (A): Whole medusa. Oral arm

(OA). (B): Lappet (L) pointy. Rhopalium (R). (C): Network of canals (C) injected with

red dye. (D): Papilae (P). Coronal muscle (M). (E): A single terminal filament. (F): on

oral arm. (F) Numerous filaments (F) on oral arms.

85

3.11 Geometric Morphometric Analysis

A total of 107 specimens of Chrysaora chinensis were used in this study. 16 are

from the East-Central of Peninsular Malaysia, 26 from East-North, 27 from West-

Central and 38 from West-North. Among them, 17 specimens were from the museum

(Appendix D, Figure 3.33).

Figure 3.33 Map of the sampling locations for geometric morphometric analysis, with

the number of specimens obtained. Green circle represents East-North (EN), red circle

represent East-Centre (EC), blue represents West-Central (WC), and purple represents

West-North (WN) coastal areas of Peninsular Malaysia.

86

3.11.1 Procrustes ANOVA

The measurement error of the gastrovascular pouch was computed from the Procrustes

ANOVA (Table 3.1). The error caused by digitization and imaging was 0.5% and

1.64% of the individual variation, respectively. Both errors were negligible as they were

much smaller than the individual variation. The error caused by differences between

pouches in individual specimen (i.e. two different gastrovascular pouches per specimen)

was quite large at 45.8% of the individual variation. Therefore, it was necessary to use

more than one gastrovascular pouch per specimen to average out the error, but there is

no need to digitize an image twice.

Table 3.1: Result of Procrustes ANOVA (SS=Sum of square, MS=Mean Squre,

df=degree of freedom).

Effect SS MS % of error df F P

Individual 0.35608889 0.00052989 - 672 2.18 < 0.0001

Pouch 0.00904613 0.00032307 - 28 1.33 0.1192

Ind x Pouch 0.16303950 0.00024262 45.8 672 27.96 < 0.0001

Imaging 0.01214961 0.00000868 1.64 1400 3.29 < 0.0001

Digitize 0.00738285 0.00000264 0.50 2800 - -

3.11.2 Principal Component Analysis (PCA)

PCA was performed on a total of 107 specimens. Most of the total variance was

explained by the first two PCs (Figure 3.34). PC1 accounted for 33.86% of the total

variance of the shape change based on the widening of both sides of the gastrovascular

pouch. PC2 accounted for 19.41% of the total variance of the shape change following

the retraction or shortening of the distal left and right points of the pouch, thus

87

protuberance of the pouch appear less prominent and more blunt (Figure 3.34,

Appendix L). The shape space scatter plot between PC1 and PC2 (Figure 3.35) indicates

that most of the specimens overlaps, with a few exception of outliers. Hence, there were

no significant differences in shape among all the specimens based on PCA.

Figure 3.34: PCA Result – % of variation explained by components. Two independent

contrasts of the gastrovascular pouch shape of the two main components PC1 and PC2

are illustrated whereby light blue outline indicates the mean shape and the dark blue

outline indicates the shape change.

88

Figure 3.35: Scatter plots of PC1 vs PC 2. PC1 accounted for 33.86% and PC2

accounted for 19.41% of the total variance of the shape change of gastrovascular pouch

of specimens from East-Central (EC), East-North (EN), West-Central (WC) and West-

North (WN) of Peninsular Malaysia.

3.11.3 Canonical Variate Analysis (CVA)

CVA was performed on pouches of C.chinensis populations from four coastal

areas of the Peninsular Malaysia: East-Central (EC), East-North (EN), West-Central

(WC) and West-North (WN), with a total of 107 specimens. CV1 accounts for 47.46%,

CV2 accounts for 32.72%, and CV3 accounts for 19.83% of the amount of relative

between-group variation (Figure 3.36). Specimens from East-Central and East-North are

separated from the other two groups (West-Central and West-North). There are

considerable amount of overlapping among the group West-Central and West-North

89

(Figure 3.30). The magnitude of Mahalanobis distances is lower between West-Central

and West-North but higher between east and west coast areas, whereas East-Central and

West-North has the highest Mahalabonis distance. (Table 3.2).

The outline drawing of the visualization of the gastrovascular pouch shape of

C.chinensis populations from the four coastal areas of Peninsular Malaysia show some

variations in shape (Figure 3.37). Pairwise comparisons among the four areas show that:

EC vs WC: Protuberance at the margin of WC is more blunt

EC vs WN: Protuberance at the margin of WN is more blunt, whereas the EC’s

pouch is slightly enlarged

EC vs EN: EC’s pouch is slightly enlarged

WC vs EN: Protuberance at the margin of WC is more blunt

WC vs WN: Protuberance at the margin of WC is more blunt

WN vs EN: Protuberance at the margin of WN is more blunt

90

Fig. 3.36: Independent contrast of component using canonical variate analysis of the

gastrovascular pouch of specimens from East-Central (EC), East-North (EN), West-

Central (WC) and West-North (WN) of Peninsular Malaysia, whereby CV1, CV2 and

CV3 accounts for 47.46%, 32.72% and 19.83% of the amount of relative between-group

variation, respectively. Light blue outline indicates the mean shape and the dark blue

outline indicates the shape change. CV1 denotes changes with blunt protuberance at the

margin and enlarging of the distal end, CV2 denotes changes with blunt protuberance at

the margin and CV3 denotes changes with blunt protuberance at the margin and

enlarging of the distal end.

91

Figure 3.37: Scatter plot of CV1 vs CV2 and illustration of mean shape of

gastrovascular pouch of specimens from four coastal areas East-Central (EC), East-

North (EN), West-Central (WC) and West-North (WN) of Peninsular Malaysia. Plot

shows shape differences mainly between specimens of the east and west coasts, even

between those from EN and EC, but with no distinct differences between those from

WN and WC.

Table 3.2: Mahalanobis distances among the pouch shape of C. chinensis populations

from four coastal areas designated as West-North (WN), West-Central (WC), East-

North (EN) and East Central (EC) of Peninsular Malaysia.

EC EN WC

EN 4.3296 - -

WC 4.2151 3.4014 -

WN 4.6090 3.4034 2.6485

92

Figure 3.38: Pairwise comparisons between the pouch shape of C.chinensis populations

from East-Central (EC), East-North (EN), West-Central (WC) and West-North (WN) of

Peninsular Malaysia

93

CHAPTER 4: DISCUSSION

4.1 Morphology, Diversity and Importance of Jellyfish Species of Peninsular

Malaysia

Although the demand for jellyfish has increased, the catch had actually

decreased. Annual catch in 2000 was 179,086 metric tons (wet weight) from

Philippines, Thailand, Indonesia, Myanmar and Malaysia, and dropped drastically to

only 13,402 metric tons in 2004 (Kitamura & Omori, 2010). Personal communication

with local fishermen indicate that the supply of edible jellyfish is not enough to meet the

high demand. Despite its importance, jellyfish study in Malaysia is still relatively

scarce. Light (1914) on board the steamer Albatross to the Philippines and recorded

several jellyfish species he found during the trip (including Chrysaora, Lychnorhiza and

Acromitus species) but unfortunately he did not make his stop in Pensinsular Malaysia.

This study is aimed to try to fill in the gaps from previous studied. This studies not only

report a new record of Lychnorhiza malayensis in Malaysia, but also provided detail

description and photographs of eight scyphozoan jellyfish species previously reported

but without detail information.

Ever since Linnaeus first described the popular moon jellyfish Aurelia aurita in

1758, jellyfish taxonomy has long been subjected to disagreements and revision. Two

particular jellyfish that subject to such scrutiny are Chrysaora sp. and Cyanea capillata.

Since the description of the Genus Chrysaora in 1810 by Peron & Leseur, a number of

revisions had been done by various researchers. Eschscholtz (1829) listed six species in

the genus, Lesson (1843) listed 13, Agassiz (1862) listed nine, Haeckel (1880) listed 10,

Mayer (1910) listed 15, Kramp (1961) listed 11, and most recently Morandini &

Marques (2010) listed 15.

94

C. capillata poses another problem to the taxonomy position. It has been revised

by Linnaeus et al (1758), Mayer (1910), Kramp (1961), Dawson (2005), Sparmann

(2012) and Kolbasova et al. (2015). It was believed to be a cosmopolitan species but

recent research by Dawson (2005) has shown that it is in fact not. Thus it will be

beneficial to study jellyfish species from this region to ascertain its taxonomy position.

4.2 Lion’s Mane Jellyfish (Cyanea sp.) of Malaysia

The Cyanea species obtained in the west coast of Peninsular Malaysia were without

gastrovascular pit, and the exumbrella surface is lightly granulated with warts.

Gastrovascular pit is a prominent feature of Cyanea capillata, thus the Malaysian

specimen cannot be ascribed to be C. capillata. By injecting colour dye to the rhopalar

and tentacular pouches of Cyanea species obtained in Peninsular Malaysia show up to

five transverse “ridges” connecting both pouches, and the peripheral canals at the lappet

contains numerous networks of anastomoses, forming a dense network, which

according to Kramp (1961) is one of the characteristic of C. nozakii. But according to

Kramp (1961), the colour of C. nozakii is white, but the species obtained are brownish

and yellowish. Thus, the Cyanea species obtained in Peninsular Malaysia cannot be

ascribed to C. nozakii. C. lamarki does not seem to fit the characteristic of Cyanea

species obtained in Peninsular Malaysia because the locality of C. lamarki was reported

to be in Europe. Furthermore, its rhopalar and tentacular stomach pouches are

completely separated.

Another Cyanea species, C. barkeri from Australia (Gershwin et al., 2010) is

similar to the Cyanea species from Peninsular Malaysia in that they both lack

gastrovascular pit. But it is not clear whether C. barkeri’s rhopalar and tentacular

pouches are connected, and whether the exumbrella surface is smooth or granulated.

95

Kramp (1961) recorded C. buitendijki in Malay Archipelago but he did not describe the

exumbrella surface nor the existence of musculature intrusion. Furthermore, Malay

Archipelago encompasses a wide region ranging from Malaysia, Singapore, Indonesia

to Philippines. Thus cannot ascribe the Cyanea species from Peninsular Malaysia to

C.barkeri nor C. buitendijki.

Molecular phylogenetic evidence of Rizman-Idid et al. (2016) showed that

Malaysian Cyanea did not cluster with either C. nozakii, C. capillatta, C. rosacea nor C.

annaskala and it is very distinct from any available Cyanea sequences. Hence, with

molecular and morphological evidence it is plausible that Malaysian Cyanea is a new

species.

Table 4.1: Comparison of Cyanea sp. from Peninsular Malaysia with other Cyanea

species based on Kramp (1961).

C. capillata C. buitendijki C. lamarki C. nozakii Cyanea sp.

Musculature

intrusion

Y ? N ? N

Exumbrella

papillose

N

?

Y

N

Y

Locality

Australia,

NW Pacific,

China,

Mutsu Bay,

Japan,

Trivandrum

Coast India

Malay

Archipelago

Bay of

Biscay,

Norway,

Holland, W.

Coast of

Sweden,

Denmark,

Iceland

Japan, NW

Pacific,

Indochina,

Indian

Ocean

Malaysia

Size

up to

1000mm

up to

310mm

up to

1000mm

up to

260mm

up to

250mm

96

Table 4.1, continue

Pouch

Rhopalar and

tentacular

stomach

pouches

completely

separated

Rhopalar and

tentacular

stomach

pouches

connected by

several broad

transversed

anastomoses

Rhopalar

and

tentacular

stomach

pouches

completely

separated

Rhopalar

and

tentacular

stomach

pouches

connected by

several

broad

transversed

anastomoses

Rhopalar and

tentacular

stomach

pouches

connected by

several broad

transversed

anastomoses

Canal

Peripheral

canals more

or less

curved

Peripheral

canals without

anastomoses

Peripheral

canals more

or less

curved

Peripheral

canals with

numerous

anastomoses,

forming a

dense

network

Peripheral

canals with

numerous

anastomoses,

forming a

dense network

Colour

Reddish

brown or

yellowish

Blue

Milky white

Brownish,

yellowish

4.3 Sea Nettles (C. Chinensis) of Peninsular Malaysia

The detailed morphological description of C. chinensis in the present study represents

the first of its kind in Malaysia. C. chinensis is commonly found in most beaches in east

and west coast of Peninsular Malaysia (Rizman-Idid et al., 2016). Base on personal

observation and interviews with fishermen, they occur more abundantly at the polluted

areas such as Balik Pulau, Pantai Kok and Kilim, particularly during the dry season

(April to October). Previous study reported the occurrence of C. chinensis in South

China Sea, (Vanhöffen, 1911), but Kramp (1961) reported C. chinensis as C. helvola.

Prior to the resurrection of C. chinensis by Morandini & Marques (2010), many

Chrysaora specimens found in the South China Sea and Malay Archipelago have been

identified as either C. melanaster (Yap & Ong, 2012) or C. helvola (Kramp, 1961).

97

However, Morandini and Marques (2010) emphasized that C.chinensis is very different

from C. helvola (valid name C. fuscescens) and C. melanaster due to the difference in

tentacle and lappet number. Both the latter species are larger than in their observed C.

chinensis specimens. Recent study by Yap & Ong (2012) ascribed the specimens from

St. John’s Island in Singapore as C. chinensis. Likewise, findings from the present study

which focused on specimens obtained from the east and west coasts of Peninsular

Malaysia confirms that the sea nettles found in these waters were morphologically

identified as C. chinensis. Furthermore, DNA sequences from some of the specimens

obtained in the present study (data not shown) revealed that they are C. chinensis and

genetically similar and phylogenetically clustered to those reported by Rizman-Idid et

al., (2016). It is noteworthy that the colouration of C. chinensis varies greatly. Some

specimens are transparent whereas some are reddish with radiating stripes on the

exumbrella, with numerous pigmentations on oral arms and exumbrella, and reddish

brown lappets. Nonetheless, this colouration corroborated with the colouration

diagnosis of C. chinensis by Yap & Ong (2012) and Morandini & Marques (2010).

The shape of the gastrovascular pouch of specimens from the east coast is

slightly elongated compared to those from the west coast. Unlike Rhizostomeae

jellyfish, C. chinensis do not have well developed canal. The gastrovascular pouches of

C. chinensis are used to store and transport nutrients from the central stomach to various

parts of the jellyfish. The east coast of Peninsular Malaysia is facing the South China

Sea, whereas the west coast is facing the Straits of Malacca, one of the busiest sea

routes in the world, hosting annually about 65,000 vessels for international navigation,

and up to another additional 15,000 fishing vessel (Ibrahim & Khalid, 2007). The low

water quality of Straits of Malacca is due to pollution from shipping industries,

aquaculture, densely populated coastal areas with nutrients and effluent discharged into

the straits, causing eutrophication (Rezai et al., 2003; Praveena et al., 2011). According

98

to Bong & Lee (2008), the effects of development at nearshore increases the total

suspended solids and decreases the dissolved oxygen. Human activities that lead to

eutrophication have been associated with the bloom of the jellyfish (Arai 2001; Parsons

& Lalli, 2002; Malej et al., 2007; Richardson et al., 2009), and jellyfish appear to be

able to adapt to fluctuations of various levels of water quality and environmental

parameters, such as salinity, temperature, food source (Purcell et al., 2012). A few

geographical variation studies have shown that morphology affects the jellyfish

distribution, pulse rate and swimming speed (Dawson & Hamner, 2003).

Since the main function of the gastrovascular system is for the circulation of

nutrients (Arai, 1997), these differences in gastrovascular pouch shapes observed in the

present study may be a form of morphological adaptation of C. chinensis populations to

the different coastal areas (East-Central, East-North, West-Central, and West-North) of

Peninsular Malaysia. The landmass of Peninsular Malaysia acts as the main physical

barrier which separates the east from the west populations. The EC population also

appears distinct from the EN population. Although the distance between the sampling

areas of both populations is only approximately 350km, which may not be of significant

magnitude to explain the isolation by distance for these populations. Nonetheless, these

populations may have been kept separated due to occurrence of eddies and different

water circulations at 5°N off the east coast of Peninsular Malaysia (Daryabor et al.,

2016). According to Daryabor et al (2016), such water circulations may influence the

distribution of the nutrient balance in regulating primary productivity and the changes in

the marine ecosystem.

99

4.4 Potential Application of GMM in Jellyfish Studies

Although geometric morphometric analysis have been proven useful in analysing and

detecting even very minute shape variations in various organisms (Klingenberg, 2013,

Li et al., 2016), its application as demonstrated in the present study on gelatinous

organism such as jellyfish, may still be informative albeit a few practical challenges.

Geometric morphometric analysis seems more suited for organisms with rigid

structures, as landmarks are more easily identified and configured, thus detection of

shape variation would be attributed due to real morphological differences rather than

inconsistencies of measurements due to mishandling of specimens. Up until now, there

has been no shape variation study using geometric morphometric analysis on soft or

gelatinous organisms. Jellyfish, being gelatinous, can be quite easily distorted, either

from bad sampling methods that can cause damage to their delicate structures, or even

from prolonged preservation of specimens. In fact, formalin can contribute to the

change of shape of jellyfish, especially after a long period of time (Kapiris et al., 1997).

Therefore, using geometric morphometric analysis to detect shape variation in jellyfish

poses a higher degree of difficulties compared to rigid organisms, since even slight false

variation in the shape due to sampling or handling error can lead to the conclusion as

real variation.

The practical usage may be improved by choosing a different structure for

landmark configurations, depending on the species. For example, the present study has

successfully demonstrated that internal structure such as the gastrovascular pouch is

suitable as the result appears to be robust with low measurement errors. On the other

hand, certain structures make poor landmarks. Filaments and tentacles are easily

detached and damaged. They tend to move rather easily and it is difficult to set its

100

orientation and identify landmarks. Other jellyfish species, such as Rhopilema

esculentum of the Rhizostomeae family which is bigger in size and with more rigid

structures, may be a better target for landmark configuration and geometric

morphometric study.

101

CHAPTER 5: CONCLUSION

Morphological studies are crucial for species identification of jellyfish. Even if

molecular techniques are available, identification is firstly based on morphology.

Currently there is a revival in the taxonomy of jellyfish, with many revisions and efforts

to obtain samples world wide for comparisons. Nonetheless, there is still a lack of

jellyfish taxonomist worldwide. Hence studies like this would be helpful in providing

baseline information, including detailed morphology characterization and photographs

that could aid identification in the field for biologist and marine enthusiasts.

Below are the major findings of this study:

1) Morphology of nine jellyfish species (from eight families and eight genera)

found in Peninsular Malaysia that belong to the class Scyphozoa, namely

Chrysaora chinensis, Cyanea sp., Versuriga anadyomene, Rhopilema hispidum,

Rhopilema esculentum, Phyllorhiza punctata, Acromitus flagellatus,

Lobonemoides robustus and Lychnorhiza malayensis were characterised in

details

2) Cyanea sp. found in the Malaysia water may be new species as it could not be

morphologically ascribed to other known Cyanea species

3) A new record of Lychnorhiza malayensis in Malaysia was reported

4) The Malaysian sea nettle jellyfish was verified as C. chinensis

5) GMM analysis of the gastrovascular pouch of specimens of C. chinensis

indicates shape variation between the four coastal areas of Peninsular Malaysia,

102

especially with higher magnitudes of Mahalanobis distances between east and

west coast areas, East-Central and East-North comparisons, but lower

differences between the West-Central and West-North areas.

103

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APPENDICES

Appendix A: Formalin preparation

1. Neutralize formalin by adding 20 table spoons of magnesium carbonate to 2.5

litres of formalin.

2. Wait for magnesium carbonate to dissolve completely, approximately 6 hours.

3. Measure the pH of the formalin. pH must be within 7 to 8. It can be slightly

more alkaline but cannot be acidic.

4. Mix neutralized formalin with the ratio of 95% seawater and 5% of neutralized

formalin. Filter the seawater prior to mixing.

5. Formalin is highly carcinogenic, thus proper precaution must be taken. Gloves

and mask must be put on when working on the specimens. Fume cupboard

should be utilized when preparing formalin.

Appendix B: List of jellyfish morphological characters analysed and their respective

character states.

Character Character Status

Symmetry of medusae radial = 0, biradial = 2, tri-radial = 3, radial

tetramerous = 4, pentamerous = 5

Pedalium absent = 0, coronate type = 1, cubozoan type =

2

Rhopaloid absent = 0, present = 1

Rhopalium absent = 0, present and simple = 1, present

and complex eyes = 2

Ocelli absent = 0, simple ocellus = 1, ocellus w/ lens

= 2, ocellus in complex eye = 3

Nerve ring absent = 0, one = 1, two = 2

Nerve net absent = 0, DNN only = 1, DNN and MNN = 2,

MNN only = 3

DNN and MNN connected in marginal

centers or ganglia

no = 0, yes = 1

Gastric filaments absent = 0, present = 1

Coronal muscle well developed = 0, marginal and tiny = 1

Velum-like structure absent = 0, velum = 1, velarium = 2

Velar canals absent = 0, present = 1

Frenulae absent = 0, present = 1

114

Umbrellar margin smooth and continuous (no clefts, no lappets) =

0, clefts (or peronia) and lappets = 1

Number of velar lappets count per octant

Velar lappet length ( = velar cleft

depth)

millimeters

Velar lappet width millimeters

Velar lappet shape symmetric square=1, symmetric semi-

circular=2, symmetric semi-oval=3,

Velar lappet shape cont'd symmetric tapering=4, asymmetric square=5,

asymmetric semi-circular=6,

Velar lappet shape cont'd asymmetric semi-oval=7, asymmetric

tapering=8

Velar lappets in heterogenous size

classes

no = 0, yes = 1

Number of rhopalial lappets count per rhopalium ___________________

Rhopalial lappet length ( = Rhopalial

cleft depth)

millimeters ___________________

Rhopalial lappet width millimeters ___________________

Rhopalial lappet shape symmetric square, symmetric semi-circular,

symmetric semi-oval,

Rhopalial lappet shape cont'd symmetric tapering, asymmetric square,

asymmetric semi-circular,

Rhopalial lappet shape cont'd asymmetric semi-oval, asymmetric tapering

Rhopalia in marginal clefts no = 0, yes = 1

Number of umbrella tentacles count per quadrant ___________________

Umbrellar tentacle developmental

origin

tentacle = 1, modified lappets = 2

Tentacular insertion at umbrella margin = 0, proximally on

exumbrella = 1, distally on

Tentacular insertion cont'd exumbrella = 2, proximally on subumbrella =

3, distally on subumbrella = 4

Structure of medusoid tentacles hollow = 0, solid = 1

Tentacular morphology straight = 0, with angular inflection = 1,

capitate = 2

Number of tentacle whorls or rows count ___________________

Tentacle position perradial only = 0, interradial only = 1, adradial

only = 2, perradial +

Tentacle position cont'd interradial = 3, perradial + adradial = 4,

interradial + adradial = 5,

Tentacle position cont'd perradial + interradial + adradial = 6

Tentacle arrangement single/continuous = 1, clumped = 2

Tentacles with terminal knob absent = 0, present (i.e. capitate) = 1

Tentacular bulbs absent = 0, present = 1

Statolith composition MgCaPO4 = 0, CaSO4 [gypsum] = 1, CaSO4

[bassanite] = 2

Gastric mesenteries absent = 0, present = 1

Number of gastric ostia count

Gastric ostia position perradial only = 0, interradial only = 1, adradial

only = 2, perradial + I

Gastric ostia position cont'd nterradial = 3, perradial + adradial = 4,

interradial + adradial = 5,

Gastric ostia position cont'd perradial + interradial + adradial = 6

115

Number of radial mesenteries count ___________________

Radial mesentery shape straight = 1, bent distally = 2, paired forming Y

proximally = 3

Radial mesentery termination percent of distance from tentacle (0%) to

rhopalium (100%) ___________

Number of radiating stomach pouches count ___________________

Radial canals absent = 0, present = 1

Number of perradial canal origins at the

gastrovascular cavity

count per quadrant ___________________

Number of interradial canal origins at

the gastrovascular cavity

count per quadrant ___________________

Number of adradial canal origins at the

gastrovascular cavity

count per quadrant ___________________

Number of perradial-perradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of interradial-interradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of adradial-adradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of perradial-interradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of perradial-adradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of interradial-adradial

anastomoses in radial canals that are

circumscribed by the ring canal

count per quadrant ___________________

Number of terminations of perradial

canals at the ring canal

count per quadrant ___________________

Number of terminations of interradial

canals at the ring canal

count per quadrant ___________________

Number of terminations of adradial

canals at the ring canal

count per quadrant ___________________

Number of perradial canals originating

distally at the circular canal

count per quadrant ___________________

Number of interradial canals

originating distally at the circular canal

count per quadrant ___________________

Number of adradial canals originating

distally at the circular canal

count per quadrant ___________________

Number of sinuses originating at the

gastrovascular cavity

count per quadrant ___________________

Number of sinuses originating at the

perradial canal

count per quadrant ___________________

Number of sinuses originating at the

interradial canals

count per quadrant ___________________

Number of sinuses originating at the

adradial canals

count per quadrant ___________________

Number of sinuses originating count per quadrant ___________________

116

proximally at the circular canal

Number of anastomoses circumscribed

by the circular canal that lead to two

sinuses

count per quadrant ___________________

Number of sinuses originating distally

at the circular canal

count per quadrant ___________________

Percentage of radius of medusa in

which there is no branching radial canal

count per quadrant ___________________

Circular canal absent = 0, weakly developed chain of enlarged

branches circumscribes

Circular canal cont'd bell = 1, a primary artery easily distinguishable

from other canals

Circular canal cont'd circumscribes bell = 2

Furrow in bell absent = 0, coronal groove = 1, laingiomedusan

type = 2

Number of gonads count

Gonads are paired no = 0, yes = 1

Gonad position axis perradial only = 0, interradial only = 1, adradial

only = 2, perradial +

Gonad position axis cont'd interradial = 3, perradial + adradial = 4,

interradial + adradial = 5,

Gonad position axis cont'd perradial + interradial + adradial = 6

Lateral distance from center to most

proximal portion of gonad

millimeters ___________________

Lateral distance from center to most

distal portion of gonad

millimeters ___________________

Gonad associated with particular

structure?

manubrium = 0; radial canals = 1; mesogleal

protrusion (gastric septa or

Gonad associated with particular

structure? cont'd

quadralinga) = 2; radial septa = 3; pouch = 4;

outfolded pockets = 5

Statocysts absent = 0, endodermal = 1, ectodermal = 2

Shape of mature medusa bell on oral-

aboral plane

semi-circular convex = 0, triangular = 1, wide

rectangular = 2,

Shape of mature medusa bell on oral-

aboral plane cont'd

tall rectangular = 3, square = 4, actinuloid = 5,

sinusoid = 6, sinusoid with

Shape of mature medusa bell on oral-

aboral plane cont'd

central globose mass

Bell thickness millimeters ___________________

Development of the umbrella fully developed = 0, aboral cone = 1

Shape of mature medusa bell on plane

perpendicular to oral-aboral axis

circular = 0, square = 1

Urticant rings absent = 0, present = 1

Mouth lips absent = 0, simple lips = 1, gelatinous or

curtain-like arms = 2,

Mouth lips cont'd oral arms with suctorial mouths = 2

Manubrium absent = 0, basal in arms = 1, basal and

extended beyond arms = 2,

Manubrium cont'd pillars and disk = 3

Manubrium depth millimeters ___________________

Manubrium width at base millimeters ___________________

Manubrium width at mouth millimeters ___________________

Length of the simple, unwinged portion millimeters ___________________

117

of the oral arm

Length of the winged portion of the

oral arm

millimeters ___________________

Oral arm width millimeters ___________________

Cross-sectional form of oral arm sheet-like = 1, two-winged = 2, three-winged =

3

Secondary structure of oral arm absent = 0, spiral =1

Number of fenestrations in oral arm count ___________________

Scapulae absent = 0, present = 1

Point of scapula attachment to oral

mass

at disk = 1, both disk and oral arm = 2, on

smooth portion of oral arm= 3

Length of attachment to oral mass millimeters ___________________

Length of scapula (smooth part) millimeters ___________________

Length of scapula (mouthed part) millimeters ___________________

Distribution of mouths on scapula top= 1; bottom = 2, entire surface = 3,

Shape of scapula straight = 1, scimitar-shaped, curved up = 2,

finger-like, curved up = 3

Scapulae occurrence per oral arm one per arm = 1; 2 per arm = 2

Scapulae branched? no= 0 ; yes = 1

Number of filaments per scapulae count ___________________

Distribution of filaments on scapulae absent = 0, scapula exterior only = 1, scapula

interior only = 2

Shape of scapular filaments rod-like = 1; tapering = 2, string-like = 3,

string-like with terminal bulb

Shape of scapular filaments (capitate) = 4, spatulate = 5

Length of scapular filaments millimeters ___________________

Width of scapular filaments millimeters ___________________

Colour of scapular filaments C-M-Y-K ___________________

Number of terminal clubs count ___________________

Cross-sectional shape of terminal clubs circular = 0, planar = 1, convex planar (ovoid)

= 2, concave planar = 3,

Cross-sectional shape of terminal clubs

cont'd

triangular = 4, convex triangular = 5, concave

triangular = 6

Longitudinal-sectional shape of

terminal clubs

rod-like = 0, tapering = 1, string-like = 3,

string-like with terminal bulb = 4,

Longitudinal-sectional shape of

terminal clubs cont'd

spatulate = 5

Length of terminal clubs millimeters ___________________

Width of terminal clubs millimeters ___________________

Length of the oral pillars millimeters ___________________

Width of the oral pillars millimeters ___________________

Depth of the oral pillars millimeters ___________________

Width of the subgenital fenestration millimeters ___________________

Perradial diameter of the oral disc millimeters ___________________

Depths of the oral disc millimeters ___________________

Distribution of intermediate filaments

on the oral arm and oral disc

absent = 0, oral arm exterior only = 1, oral arm

interior only = 2, oral disk

Distribution of intermediate filaments

on the oral arm and oral disc cont'd

only = 3, oral arm = 4, oral arm and disk = 5

Number of intermediate filaments on

the oral arm

count ___________________

118

Number of intermediate filaments on

the oral disc

count ___________________

Shape of intermediate filaments rod-like = 0, tapering = 1, string-like = 3,

string-like with terminal bulb

Shape of intermediate filaments cont'd (capitate) = 4, spatulate = 5

Length of intermediate filaments millimeters ___________________

Width of intermediate filaments millimeters ___________________

Colour of intermediate filaments C-M-Y-K ___________________

Scyphopharynx of polyp absent = 0, present = 1

Number of rhopalia count per quadrant ___________________

Rhopalia position perradial only = 0, interradial only = 1, adradial

only = 2, perradial +

Rhopalia position cont'd interradial = 3, perradial + adradial = 4,

interradial + adradial = 5,

Rhopalia position cont'd perradial + interradial + adradial = 6

Rhopalial location at umbrella margin = 0, distally on exumbrella

= 2, median on

Rhopalial location cont'd subumbrella = 1, distally on subumbrella = 2

Number of coronal muscle folds count ___________________

Depth of coronal muscle folds millimeters ___________________

Coronal muscle covers radial septa on

proximal-distal axis

not at all = 0, partially = 1, exactly = 2, exceeds

= 3

Coronal muscle is continuous circularly

over radial septae

no = 0, yes = 1, mixed depending on position =

2

Coronal muscle pits count per octant (averaged per centimeter

band) __________________

Radial muscles absent = 0, weakly developed = 1, strongly

developed = 2

Radial muscle distribution subumbrellar proximal = 1, subumbrellar distal

= 2, subumbrella

Radial muscle distribution cont'd proximal-to-distal = 3

Number of radial muscle folds count per octant ___________________

Gastrovascular pits in radial muscle

folds

count per centimeter of muscle

___________________

Number of subumbrellar sacs/saccules count ___________________

Number of rows of subumbrellar

sacs/saccules

count ___________________

Subumbrellar papilla width millimeters ___________________

Subumbrellar papilla length millimeters ___________________

Subumbrellar papilla height millimeters ___________________

Subumbrellar papilla shape dome = 0; pyramidal = 1; conic = 2;

cylindrical = 3; hernia/scrotum-like = 4

Type of exumbrella ornamentation none (smooth) = 0, protuberance =

1, crenulation = 2

Number of exumbrella ornaments count per octant ___________________

Distribution of exumbrella ornaments crown of bell = 1, toward bell margin = 2,

crown and margin = 3

Height of protuberances (depth of

crenulations)

millimeters ___________________

Cross-sectional shape of exumbrella

ornaments

Longitudinal-sectional shape of globose nobs = 1, tapering filaments = 2, mesa-

119

exumbrella ornaments like = 3, mound = 4

Colour of exumbrella ornaments C-M-Y-K ___________________

Color of bell C-M-Y-K ___________________

Perradial canal colour C-M-Y-K ___________________

Interradial canal colour C-M-Y-K ___________________

Adradial canal colour C-M-Y-K ___________________

Number of pigmented flecks in

perradial canal

count per quadrant ___________________

Number of pigmented flecks in

interradial canal

count per quadrant ___________________

Number of pigmented flecks in

adradial canal

count per quadrant ___________________

Oral arm colour C-M-Y-K ___________________

Terminal club colour C-M-Y-K ___________________

Shape of pigment on exumbrella none = 0, dot = 1, circle = 2, uneven patch = 3,

radiating lines = 4, star = 5,

Shape of pigment on exumbrella

cont'd

wheel = 6, eyespot = 7, …

Colour of pigmentation on

the exumbrellar surface

C-M-Y-K ___________________

Number of pigmented

spots/patches /shapes on exumbrellar

surface

count per octant ___________________

Distribution of color

spots/patches/shapes on exumbrella

crown of bell = 1, toward bell margin = 2,

crown and margin = 3

Mass of the whole medusae grams

Bell diameter millimeters ___________________

Ring canal diameter millimeters ___________________

Shape of the stomach/gonadal cavity circular = 0, cruciform = 1, pouched = 3,

outfolded pockets = 4.

Perradial diameter of the stomach

cavity

millimeters ___________________

Structural form of gonad digitate = 1, ribbon = 2, floret = 3

Colour of the subgenital porticus C-M-Y-K ___________________

Thickness of the subgenital porticus millimeters ___________________

Color of gonads C-M-Y-K ___________________

Color of gastric filaments C-M-Y-K ___________________

Color of the bell margin C-M-Y-K ___________________

Rhopaliar pit depth millimeters ___________________

Quadralinga length millimeters ___________________

Quadralinga diameter millimeters ___________________

Quadralinga shape scooped = 1, tri-lobed = 3

Subumbrella radial furrows absent = 0, present = 1

Location where planulae are brooded not brooded = 0, brood filaments = 1,

proximally on oral arms = 2, on

Location where planulae are brooded manubrium = 3, on manubrium and oral arms =

4

Number of subumbrellar radial furrows count per octant ___________________

120

Appendix C: Check list of morphology photography and observation

Tank

1 Label

2 Lateral view whole animal Front

3 Lateral view whole animal Front RGB

4 Lateral view whole animal Side

5 Close-up Front/Right Quadrant -Front or side

6 Close-up Bell Margin -Front or side

7 Close-up of tentacle -Front or side

8 Close-up of Oral arms - Front or side

9 Close-up of Oral Arm Filaments

10 Close-up of terminal clubs - Front or side

11 Close-up of Mouthlets - Front

12 Close-up of Scapulae - Front or side

13 Close-up of Scapular Filaments

14 Close-up of Scapular Filaments RGB

15 Whole animal from top RGB

Lift up bell, drape oral arms to expose ends of manubrium

16 Close-up of Manubrium/Mouth

17 Close-up of gastric filaments

18 Oral side up from Top

Measure mass of the whole medusae (grams)

Measure Bell Diameter

Measure Bell thickness

Measure Oral Disk Diameter 1) Interradial 2) Perradial

Measure Depth of Oral Disc

Count number of intermediate filaments on disk

Subumbrella up, tentacles/oral arms out

19 Whole medusae - RGB with flash

20 Whole medusae - Bottom Illum/B Background

21 Whole medusae - Bottom Illum/Full Trans

Move tentacles/oa from quadrant

22 Close up of Quadrant - Bottom Illum/Full Trans

23 Close-up of Quadrant - Bottom Illum/B Background

24 Picture of center - Bottom Illum/B Background

25 Picture of center - Bottom Illum/Full Trans

26 Close-up of Velar Lappets - Bottom Illum/B Background

27 Close-up of Velar Lappets - Bottom Illum/Full Trans

Count number of Velar Lappets _______________

28 Close-up of Rhopaliar Lappets - Bottom Illum/B Background

29 Close-up of Rhopaliar Lappets - Bottom Illum/Full Trans

Count number of Rhopaliar Lappets

30 Close-up of Rhopalium - Bottom Illum/B Background

31 Close-up of Rhopalium - Bottom Illum/Full Trans

32 Close-up of Coronal Muscles - Bottom Illum/B Background

121

33 Close-up of Coronal Muscles - Bottom Illum/Full Trans

Measure number of coronal muscle folds

Measure depth of coronal muscle folds

34 Close-up of Radial Muscles - Bottom Illum/B Background

35 Close-up of Radial Muscles - Bottom Illum/Full Trans

Measure number of radial muscle folds

Measure depth of radial muscle folds

36 Close-up of canals 2 quads - Bottom Illum/Full Trans

37 Close-up of canals 2 quads - Bottom Illum/B Blackground

38 Close-up of manubrium - Bottom Illum/B Background

39 Close-up of manubrium - Bottom Illum/Full Trans

Measure Manubrium depth 1)___ 2) ___

Measure Manubrium width at base

Measure Manubrium width at mouth

Move oral arms to top in a group - reveal oral pillars

40 Close-up of Oral Pillar - Bottom Illum/B Background

41 Close-up of Oral Pillar - Bottom Illum/Full Trans

Measure depth of oral Pillar

Measure height of oral pillar

Measure the wide of oral pillar

42 Close-up of subgenital fenestration - Bottom Illum/B Background

43 Close-up of subgenital fenestration - Bottom Illum/Full Trans

Measure width of subgenital fenestration

Measure height of subgenital fenestration

44 Close-up of Subumbrellar papillae - Bottom Illum/B Background

45 Close-up of Subumbrellar papillae - Bottom Illum/Full Trans

Measure Subumbrellar papilla width

Measure Subumbrellar papilla length

Measure Subumbrellar papilla height

Observe shape of Subumbrellar papilla

Cut 1 or 2 oral pillars - reveal gonad and stomach cavity

46 Close-up of gonad - Bottom Illum/B Background

47 Close-up of gonad - Bottom Illum/Full Trans

Check gonad association with particular structure

Measure Lateral distance from center to most proximal portion of gonad

Measure Lateral distance from center to most distal portion of gonad

48 Close-up of stomach cavity - Bottom Illum/B Background

49 Close-up of stomach cavity - Bottom Illum/Full Trans

Observe shape of the stomach cavity

Measure Perradial diameter of the stomach cavity

Measure Interradial diameter of the stomach cavity

50 Close-up of gastric filaments

Observe presence of gastric filaments

Splay 1/2 of oral arms and terminal clubs out

51 Close-up of oral arms - Bottom Illum/B Background

122

52 Close-up of oral arms - Bottom Illum/Full Trans

Splay single oral arm out (winged & unwinged portion esp.) - expose

fenestrations - show the base and middle of oral arm

53

Close-up of single oral arm/terminal clubs/filaments - Bottom Illum/B

Background

54

Close-up of single oral arm/terminal clubs/filaments - Bottom Illum/Full

Trans

Measure total length of oral arms

Measure unwinged length of oral arms

Measure width of oral arms

Observe cross-sectional shape of oral arms

Observe longitudinal-sectional shape of oral arms

Count number of fenestrations in oral arms

Measure length of terminal clubs

Measure width of terminal clubs

Observe cross-sectional shape of terminal clubs

Observe longitudinal-sectional shape of terminal clubs

Measure length of filaments

Measure width of filaments

Count number of filaments

Observe cross-sectional shape of filaments

Observe longitudinal-sectional shape of filaments

Splay out scapulae as with oral arm (above)

55 Close up of scapula/filaments - Bottom Illum/Black Background

56 Close up of scapula/filaments - Bottom Illum/Full Trans

Observe cross-sectional shape of scapula

Observe longitudinal-sectional shape of scapula

Measure length of filaments

Measure width of filaments

Count number of filaments

Observe cross-sectional shape of filaments

Observe longitudinal-sectional shape of filaments

Pull up oral disk, take a look at subgenital porticus

57 Measure thickness of the subgenital porticus

Observe the colour of subgenital porticus

Flip animal so exumbrella faces up

58 Close-up of Exumbrella Quadrant - Bottom Illum/B Background

59 Close-up of Exumbrella Quadrant - Bottom Illum/Full Trans

60 Whole animal Exumbrella - Bottom Illum/B Background

61 Whole animal Exumbrella - Bottom Illum/Full Trans

Measure height of protuberances (depth of crenulations)

Observe cross-sectional shape of ornaments

Observe longitudinal-sectional shape of ornament

If dying canals possible - flip animal and dye canals

123

62 Close-up of dyed canals 2 quads

Dissect Rhopalia - place in vial

Remove tentacle(s) - place in vial

Appendix D: List of 107 specimens for geometric morphometric analysis

Specimen Site Area

MRI001WCD1A Sungai Janggut West-Central

MRI002WCD1A Sungai Janggut West-Central

MRI003WCD1A Sungai Janggut West-Central

MRI004WCD1A Sungai Janggut West-Central

MRI005WCD1A Sungai Janggut West-Central

MRI006WCD1A Sungai Janggut West -Central

MRI007WCD1A Sungai Janggut West -Central

MRI065WCR1A Sungai Janggut West-Central

MRI067WCR1A Sungai Janggut West-Central

MRI068WCR1A Sungai Janggut West-Central

MRI069WCR1A Sungai Janggut West-Central

MRI070WCR1A Sungai Janggut West-Central

MRI072WCR1A Sungai Janggut West-Central

MRI073WCR1A Sungai Janggut West-Central

MRI077WCR1A Sungai Janggut West-Central

MRI079WCR1A Sungai Janggut West-Central

MRI081WCR1A Sungai Janggut West-Central

MRI148WND1A Pantai Kok West-North

MRI149WND1A Pantai Kok West-North

MRI150WND1A Pantai Kok West-North

MRI151WND1A Pantai Kok West-North

MRI152WND1A Pantai Kok West-North

MRI162WCD1A Sungai Janggut West-Central

MRI163WCD1A Sungai Janggut West-Central

MRI164WCD1A Sungai Janggut West-Central

MRI165WCD1A Sungai Janggut West-Central

MRI166WCD1A Sungai Janggut West-Central

MRI167WCD1A Sungai Janggut West-Central

MRI168WCD1A Sungai Janggut West-Central

MRI169WCD1A Sungai Janggut West-Central

MRI170WCD1A Sungai Janggut West-Central

MRI171WCD1A Sungai Janggut West-Central

MRI178ECD1A Kg. Cempaka East-Central

MRI179ECD1A Kg. Cempaka East-Central

MRI180ECD1A Kg. Cempaka East-Central

MRI181ECD1A Kg. Cempaka East-Central

MRI183ECD1A Kg. Cempaka East-Central

MRI186ECD1A Kg. Cempaka East-Central

124

MRI188ECD1A Kg. Cempaka East-Central

MRI189ECD1A Kg. Cempaka East-Central

MRI190ECD1A Kg. Cempaka East-Central

MRI191ECD1A Kg. Cempaka East-Central

MRI192ECD1A Kg. Cempaka East-Central

MRI193ECD1A Kg. Cempaka East-Central

MRI194ECD1A Kg. Cempaka East-Central

MRI195ECD1A Kg. Cempaka East-Central

MRI196ECD1A Kg. Cempaka East-Central

MRI197ECD1A Kg. Cempaka East-Central

MRI205END1A Pantai Sabak East-North

MRI206END1A Pantai Sabak East-North

MRI207END1A Pantai Sabak East-North

MRI208END1A Pantai Sabak East-North

MRI209END1A Pantai Sabak East-North

MRI210END1A Pantai Sabak East-North

MRI211END1A Pantai Sabak East-North

MRI212END1A Pantai Sabak East-North

MRI213END1A Pantai Sabak East-North

MRI214END1A Pantai Sabak East-North

MRI215END1A Pantai Sabak East-North

MRI216END1A Pantai Sabak East-North

MRI217END1A Pantai Sabak East-North

MRI218END1A Pantai Sabak East-North

MRI219END1A Pantai Sabak East-North

MRI220END1A Pantai Sabak East-North

MRI221END1A Pantai Sabak East-North

MRI222END1A Pantai Sabak East-North

MRI223END1A Pantai Sabak East-North

MRI224END1A Pantai Sabak East-North

MRI225END1A Pantai Sabak East-North

MRI226END1A Pantai Melawi East-North

MRI227END1A Pantai Melawi East-North

MRI228END1A Pantai Melawi East-North

MRI229END1A Pantai Melawi East-North

MRI230END1A Pantai Melawi East-North

MRI237WNR1A Balik Pulau West-North

MRI238WNR1A Balik Pulau West-North

MRI239WNR1A Balik Pulau West-North

MRI240WNR1A Balik Pulau West-North

MRI241WNR1A Balik Pulau West-North

MRI242WNR1A Balik Pulau West-North

MRI243WNR1A Balik Pulau West-North

MRI244WNR1A Balik Pulau West-North

MRI245WNR1A Balik Pulau West-North

MRI246WNR1A Balik Pulau West-North

MRI247WNR1A Balik Pulau West-North

MRI248WNR1A Balik Pulau West-North

125

MRI249WNR1A Balik Pulau West-North

MRI250WNR1A Balik Pulau West-North

MRI251WNR1A Balik Pulau West-North

MRI252WNR1A Balik Pulau West-North

MRI253WNR1A Balik Pulau West-North

MRI254WNR1A Balik Pulau West-North

MRI255WNR1A Balik Pulau West-North

MRI256WNR1A Balik Pulau West-North

MRI257WNR1A Balik Pulau West-North

MRI258WNR1A Balik Pulau West-North

MRI259WNR1A Balik Pulau West-North

MRI260WNR1A Balik Pulau West-North

MRI261WNR1A Balik Pulau West-North

MRI262WNR1A Balik Pulau West-North

MRI263WNR1A Balik Pulau West-North

MRI264WNR1A Balik Pulau West-North

MRI265WNR1A Balik Pulau West-North

MRI266WNR1A Balik Pulau West-North

MRI267WNR1A Balik Pulau West-North

MRI270WNR1A Balik Pulau West-North

MRI271WNR1A Balik Pulau West-North

Appendix E: Guide for using tpsUtil

1. Place all images into a folder.

2. Open tpsUtil.

3. Click on “Select an operation” and choose “Build tps file” from the drop-down list.

4. Under Input Directory, click on the “Input” button.

5. A dialog box will pop up. Browse to the directory where the images are stored.

6. Double-click on any one image in that directory. The dialog box will close.

7. Under Output File, click on the “Output” button.

8. A dialog box will pop up. Browse to the directory where the tps file is to be stored.

9. Enter a name. Make sure the file type is “.TPS”

10. Under Actions, click on the “Setup” button.

11. Make sure all the images in the directory are relevant to the project.

12. Click on the “Create” button. A tps file should now be created.

126

Appendix F: How to use tpsDig for digitization

1. Open tpsDig

2. Open the tps file (File > Input Source > File...). A dialogue box will pop up. Browse

to the directory where the tps file was store and choose the file. All images should be

loaded and appeared on tpsDig.

To Set Scale

1. Go to Options > Set Scale.

2. In the pop-up window enter the length of your scale (e.g., if the scale in the image is

10mm, enter 10mm).

3. In the image, place the cursor on one end of the scale in the image. Left-click, then

move the arrow to the other end and left-click again.

4. The scale window should now show the scale factor.

5. Click on the “Ok” button.

To Place Landmarks

1. Select Modes > Digitize landmarks.

2. Left-click to place a landmark. All landmarks must be placed in sequence.

Landmarks can be moved or deleted if mistakes are made.

3. Once all the landmarks for the image are placed, save the landmark data. Then move

on to the next image and repeat the step. Make sure the scale is set again for every

image.

To Save Landmark Data

1. Click on File > Save data > Save > Overwrite. Make sure the file is save after each

image is digitized to avoid losing of data.

Appendix G: 25 specimens used for Procrustes ANOVA

Specimen Sites Area

MRI001 Sungai Janggut West-Central

MRI002 Sungai Janggut West-Central

MRI067 Sungai Janggut West-Central

MRI070 Sungai Janggut West-Central

MRI148 Pantai Kok West-North

MRI162 Sungai Janggut West-Central

127

MRI164 Sungai Janggut West-Central

MRI188 Kg. Cempaka East-Central

MRI205 Pantai Sabak East-North

MRI208 Pantai Sabak East-North

MRI211 Pantai Sabak East-North

MRI214 Pantai Sabak East-North

MRI218 Pantai Sabak East-North

MRI226 Pantai Melawi East-North

MRI227 Pantai Melawi East-North

MRI229 Pantai Melawi East-North

MRI239 Balik Pulau West-North

MRI244 Balik Pulau West-North

MRI246 Balik Pulau West-North

MRI249 Balik Pulau West-North

MRI251 Balik Pulau West-North

MRI254 Balik Pulau West-North

MRI259 Balik Pulau West-North

MRI260 Balik Pulau West-North

MRI271 Balik Pulau West-North

Appendix H: Guide for using MorphoJ

Create a new tps project

1. Click on (File > Create New Project). A dialogue box will pop up. Enter the name of

the project and the name of the dataset.

2. Select the tps file type.

3. Choose the tps file created from tpsDigs.

4. Click on the “Create Dataset” button. The dataset is now created.

Perform Procrustes Fit

1. Click on the (Prelimineries menu > New Procrustes Fit)

2. Select “Align by principal axes”

3. Click on the “Perform Procrustes Fit” button

Generate Covariance Matrix

1. Click on the “Generate Covariance Matrix” button

2. A dialogue box will pop up. Click on the “Execute” button

128

Create Classifier

1. To create a classifier, Click on the (Preliminaries > Extract New Classifier From ID

String)

2. A dialogue box will pop up.

3. Enter a name for the new classifier

4. Indicate the position of the first and last character of the classifier. For example, if the

name of the specimen is MRI001WC, where WC is the classifier, then the First

character is 7 and the Last character is -1. In this analysis, these two classifiers were

created:

a. Individual (1, -1)

b. Locality (7, -1)

5. Click on the “Execute” button to produce the classifier

7. Classifier may also be edited from Preliminaries > Edit Classifier

Principal Component Analysis

1. To perform Principal Component Analysis, click on the (Variation menu > Principal

Component Analysis)

2. There are three different sub tabs under the Graphic tab: PC shape changes,

Eigenvalues and PC scores

a. PC shape changes tab shows the shape changes associated with each of the

PCs (eigenvectors).

b. Eigenvalues tab contains a graphs that shows the percentages of total

variance for which the PCs are accounted for

c. PC scores tabs contains a scatter plot of the PC scores

Canonical Variate Analysis

1. To perform Canonical Variate Analysis, click on the (Comparison menu > Canonical

Variate Analysis)

2. A dialogue box will pop up. Enter a name for the analysis

3. Choose a “Dataset” and “Data type”

4. Choose the classifier in “Classification variable(s) to use for grouping:”

5. Checked on the “Permutation test for pairwise distances” check box

129

6. Click on the “Execute” button to perform the analysis

7. Under the CVA tabs, two sub tabs will be generated: CV shape changes and CV

scores

a. The CV shape changes tab shows the shape changes associated with the

canonical variates (CVs). Right click anywhere on the screen will invoke a

pop up menu which can be used to change the properties, such as the scale

factor, the type of graph and the orientation of the shape, or to export the

graph as an SVG file.

b. The CV scores tab contains the scatter plots of the CV scores. If there are

only two groups, the graph will be a histogram instead of a scatter plot.

Right click anywhere on the screen will invoke a pop up menu which can be

used to change the properties, such as too choose the CVs to be displayed, to

color the dots by group membership according to the classifiers in the

dataset, to change other options or to export the graph as an SVG file.

To save the project

1. Click on the File menu > Save project as > and enter a name for the project which

should ends with .morphoj

Appendix I: To build and import outline in tps file into MorphoJ

1. Place all of the images into the same folder.

2. Open tpsUtil (Start > All Programs > tps > tpsUtil)

3. Click on “Select an operation” and choose “Build tps file” from the drop-down list.

4. Select the input directory:

a. Click “Input”

b. Find your directory of images

c. Double-click one image in that directory

5. Name your output file:

a. Click “Output”

b. Enter a name that ends in “.tps”.

6. Build the tps file

a. Click “Setup”

b. Checked images will be used to build your tps file. Check/uncheck as needed.

c. Click “Create” to create the tps file

130

7. Open tpsDig (Start > All Programs > tps > tpsDig)

8. Open the raw tps file (File > Input Source > File...)

9. Place landmarks. Only one specimen is needed to do the landmark and save it. After

landmark, point the cursor to “Outline” to create outline and save it.

10. Remove all the decimal point and zero. Between them must separate by “Tab”

space. Below is the result of the outline file used in this study:

0 106 363

0 86 336

0 125 287

0 165 247

0 178 198

0 237 203

0 274 202

0 269 230

0 253 248

0 279 239

0 310 249

0 296 275

0 291 325

0 242 332

0 184 360

0 127 385

1 106 363

1 95 351

1 86 336

2 86 336

2 100 316

2 114 299

2 125 287

3 125 287

3 144 268

3 160 253

3 165 247

4 165 247

4 172 219

4 174 206

4 178 198

5 178 198

5 189 191

5 203 189

5 216 191

5 228 197

5 237 203

6 237 203

131

6 246 198

6 255 196

6 264 197

6 274 202

7 274 202

7 278 207

7 278 216

7 276 224

7 269 230

8 269 230

8 264 234

8 257 239

8 250 244

8 253 248

9 253 248

9 257 252

9 263 248

9 271 244

9 279 239

10 279 239

10 289 238

10 300 241

10 310 249

11 310 249

11 312 256

11 310 264

11 304 271

11 296 275

12 296 275

12 297 282

12 301 293

12 301 302

12 300 311

12 291 325

13 291 325

13 280 329

13 271 330

13 258 330

13 242 332

14 242 332

14 224 339

14 209 348

14 184 360

15 184 360

15 170 367

15 147 376

132

15 127 385

16 127 385

16 114 373

16 106 363

11. In MorphoJ, open (File > Import outline file > and choose the outline tps file)

Appendix J: List of 146 specimens collected

Catalog

No.

Species Date Site GPS Method

MRI124 Rhopilema

esculentum

02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI125 Rhopilema

esculentum

02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI126 Cyanea sp. 02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI127 Cyanea sp. 02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI128 Cyanea sp. 02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI129 Phyllorhiza

punctata

02-Mar-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI130 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI131 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI132 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI133 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI134 Acromitus

flagellatus

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI135 Lychnorhiza

malayensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI136 Lychnorhiza

malayensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI137 Lychnorhiza

malayensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI138 Lychnorhiza

malayensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI139 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI140 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI141 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI142 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

133

MRI143 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI144 Phyllorhiza

punctata

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI146 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI147 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI148 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI149 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI150 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI151 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI152 Chrysaora

chinensis

14-Apr-14 Pantai Kok N06.34869°,

E99.64806°

Dip Net

MRI153 Versuriga

anadyomene

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI154 Chrysaora

chinensis

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI155 Acromitus

flagellatus

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI156 Rhopilema

hispidum

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI157 Rhopilema

hispidum

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI158 Rhopilema

hispidum

15-Apr-14 Kilim N06.4766362°,

E99.8042679°

Dip Net

MRI159 Cyanea sp. 29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI160 Cyanea sp. 29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI161 Cyanea sp. 29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI162 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI163 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI164 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI165 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI166 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI167 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI168 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI169 Chrysaora 29-Apr-14 Sungai Janggut N03.16916°, Bag Net

134

chinensis E101.29833°

MRI170 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI171 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI172 Chrysaora

chinensis

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI173 Phyllorhiza

punctata

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI174 Phyllorhiza

punctata

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI175 Phyllorhiza

punctata

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI176 Versuriga

anadyomene

29-Apr-14 Sungai Janggut N03.16916°,

E101.29833°

Bag Net

MRI178 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI179 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI180 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI181 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI182 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI183 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI184 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI185 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI186 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI187 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI188 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI189 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI190 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI191 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI192 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI193 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI194 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI195 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI196 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

135

MRI197 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI198 Chrysaora

chinensis

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI199 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI200 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI201 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI202 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI203 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI204 Lobonemoides

robustus

26-Jun-14 Kampung Chempaka N03.74365°,

E103.32847°

Dip Net

MRI205 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI206 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI207 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI208 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI209 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI210 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI211 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI212 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI213 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI214 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI215 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI216 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI217 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI218 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI219 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI220 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI221 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI222 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI223 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

136

MRI224 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI225 Chrysaora

chinensis

20-Jul-14 Pantai Sabak N06.13712°,

E102.36976°

Dip Net

MRI226 Chrysaora

chinensis

21-Jul-14 Pantai Melawi N05.99612°,

E102.43714°

Dip Net

MRI227 Chrysaora

chinensis

21-Jul-14 Pantai Melawi N05.99612°,

E102.43714°

Dip Net

MRI228 Chrysaora

chinensis

21-Jul-14 Pantai Melawi N05.99612°,

E102.43714°

Dip Net

MRI229 Chrysaora

chinensis

21-Jul-14 Pantai Melawi N05.99612°,

E102.43714°

Dip Net

MRI230 Chrysaora

chinensis

21-Jul-14 Pantai Melawi N05.99612°,

E102.43714°

Dip Net

MRI231 Cyanea sp. 10-Oct-14 Klang Power Station N03.3229°,

E101.30108°

Bag Net

MRI232 Cyanea sp. 10-Oct-14 Klang Power Station N03.3229°,

E101.30108°

Bag Net

MRI233 Cyanea sp. 10-Oct-14 Klang Power Station N03.3229°,

E101.30108°

Bag Net

MRI234 Phyllorhiza

punctata

13-Oct-14 Balik Puau N05.53972°,

E100.34888°

Fish

Net

MRI235 Lychnorhiza

malayensis

13-Oct-14 Balik Puau N05.53972°,

E100.34888°

Fish

Net

MRI236 Lychnorhiza

malayensis

13-Oct-14 Balik Puau N05.53972°,

E100.34888°

Fish

Net

MRI237 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI238 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI239 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI240 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI241 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI242 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI243 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI244 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI245 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI246 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI247 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI248 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI249 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI250 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

137

MRI251 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI252 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI253 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI254 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI255 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI256 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI257 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI258 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI259 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI260 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI261 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI262 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI263 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI264 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI265 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI267 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI268 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI269 Phyllorhiza

punctata

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI270 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

MRI271 Chrysaora

chinensis

14-Dec-14 Balik Puau N05.53972°,

E100.34888°

Dip Net

Appendix K: 44 Specimens used for morphological description. M indicates specimens

used from museum.

Catalog no. Species Museum

MRI 2 C. chinensis M

MRI 65 C. chinensis M

MRI 163 C. chinensis

MRI 163 C. chinensis

138

MRI 171 C. chinensis

MRI 208 C. chinensis

MRI 262 C. chinensis

MRI 190 C. chinensis

MRI 55 Cyanea sp. M

MRI 56 Cyanea sp. M

MRI 95 Cyanea sp. M

MRI 96 Cyanea sp. M

MRI 97 Cyanea sp. M

MRI 127 Cyanea sp.

SJG 10 Cyanea sp. M

MRI 87 R. esculentum M

MRI 98 R. esculentum M

IND 3 R. esculentum M

MRI 13 R. hispidum M

MRI 89 R. hispidum M

IND 9 R. hispidum M

IND 10 R. hispidum M

SJ 130 R. hispidum M

MRI 204 L. robustus

IND 6 L. robustus M

IND 7 L. robustus M

IND 8 L. robustus M

MRI 153 V. anadyomene

MRI 176 V. anadyomene

MRI 14 P. punctata M

MRI 131 P. punctata

MRI 139 P. punctata

IND 2 P. punctata M

IND 11 P. punctata M

IND 14 P. punctata M

MRI 35 L. malayensis M

MRI 262 L. malayensis M

MRI 37 L. malayensis M

MRI 38 L. malayensis M

MRI 39 L. malayensis M

IND 13 L. malayensis M

MRI 21 A. flagellatus M

MRI 23 A. flagellatus M

MRI 24 A. flagellatus M

139

Appendix L: Result of PCA

Eigenvalues % Variance Cumulative %

1. 0.00095428 33.861 33.861

2. 0.00054698 19.409 53.270

3. 0.00020748 7.362 60.632

4. 0.00018969 6.731 67.363

5. 0.00014319 5.081 72.444

6. 0.00012429 4.410 76.855

7. 0.00010983 3.897 80.752

8. 0.00008077 2.866 83.617

9. 0.00006174 2.191 85.808

10. 0.00005350 1.898 87.707

11. 0.00004880 1.732 89.438

12. 0.00004243 1.506 90.944

13. 0.00003736 1.326 92.269

14. 0.00003143 1.115 93.384

15. 0.00003060 1.086 94.470

16. 0.00002520 0.894 95.364

17. 0.00002123 0.753 96.118

18. 0.00001887 0.670 96.788

19. 0.00001731 0.614 97.402

20. 0.00001576 0.559 97.961

21. 0.00001265 0.449 98.410

22. 0.00001013 0.360 98.770

23. 0.00000808 0.287 99.056

24. 0.00000741 0.263 99.319

25. 0.00000696 0.247 99.566

26. 0.00000557 0.198 99.764

140

27. 0.00000399 0.141 99.905

28. 0.00000267 0.095 100.000