國立中山大學海洋生物所 碩士論文

Institute of Marine Biology National Sun Yat-sen University, Kaohsiung Master Thesis

鯻科發音系統之演變

Evolutionary Changes in the Acoustic System of the Family

研究生：薛仁鈞 Jen-chun Hsueh 指導教授：莫顯蕎 教授 Dr. Hin-kiu Mok

中華民國 102 年 7 月 July 2013

謝辭

感謝莫老師的指導，使得此碩士研究得以順利進行，也在後續發現許多有趣

的問題，更感謝老師包容我在此期間修習教程與兼課所占用的時間，也不時的給

予提醒與關心。感謝張學文老師與魏瑞昌老師給予的建議以及不同的視野，百忙

之中擔任口試委員。感謝廖德裕老師的解惑，待我如自己實驗室的學生。感謝

Gallery of the Northern Territory, Australia, Australian Museum 出借的標本，讓我可

以觀察台灣沒有的魚種。感謝日日漁場的何老闆熱心提供樣品以及錄音時的協助，

還有好吃的魚料理！感謝中興大學昆蟲系的葉文斌老師提供實驗設備，以及該實

驗室的學長與同學使實驗生活變得有趣。感謝陳餘鋆學長不管去澎湖、綠島還是

台南，總是一通電話義不容辭答應出差，還有協助釣魚的振豪、豆腐與蘇柏維。

感謝實驗室的學長姐，淑惠學姊、秋錦學姐、小翔、室維、李宗軒在實驗與論文

上給予的幫助；佩純、小呆與 Olic 已經畢業還時常接到我的求救訊號。感謝惠敏

姐在行政上的幫助，以及海生所的夥伴使生活充滿歡笑，特別是詩嘉，再苦的差

事有你一起就變成玩樂。感謝舞蹈社與 rTr 的人們，使我的生活多采多姿。最後，

感謝我的家人，聽到出海以為我要去捕魚的父親與聽到出差以為我要去商務旅行

的母親，在擔心中還是給予支持。還有那些我愛的與愛我的人，謝謝你們。

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鯻科（Terapontidae）發音系統之演變

研究生：薛仁鈞

指導教授：莫顯蕎 博士

國立中山大學海洋生物研究所

早期研究發現花身雞魚（ jarbua）與四線雞魚（ quadrilineatus）

在魚鰾前室中有一對帶狀組織之特化構造，連接魚鰾背側前端與脊椎骨，而此構

造有可能和發音機制關係緊密。然而，台灣引進養殖的鯻科之澳洲淡水種寶石鱸

（ barcoo）卻缺乏此特化構造，顯示屬間在發音構造上有變異的情況發生。

此篇研究的目的為釐清鯻科發音系統，包括其聲紋訊號（i.e. caudavittatus, Amniataba percoides, fuliginosus, butleri,

Terapon jarbua and Pelete quadrilineatus）以及發音相關的構造（A. caudavittatus, A. percoides, bidyanus, H. fuliginosus, Hephaestus jenkinsi, macrolepis, Leioptherapon plumbeus, , Pelates octolineatus, P. quadrilineatus, Pelates sexlineatus, Pelsartia humeralis, Rhyncopelates oxyrhynchus, S. barcoo, S. butleri, and T. jarbua, ）的演變，並使用分子資訊（16s rRNA ＆ COⅠ）來重建其科內的親緣關係。結果顯示：海水屬為較原始的類群，

並且具有較發達的發音構造；反之，淡水種為較進化的物種，但部分魚屬的發音

構造相對發展較為不全，甚至缺少某些特定構造，由此結果可推論發音在淡水的

鯻科魚類中是較為不重要的通訊管道。

關鍵詞：鯻科、發音系統、親緣關係、鰾、聲訊

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Evolutionary Changes of Acoustic System of the Family Terapontidae

Jen-chun Hsueh

Advisor: Dr. Hin-kiu Mok

Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung, Taiwan

Earlier anatomical works showed that Terapon jabua and have a specialized structure: a pair of internal tendon inside of that may play a role in sound producing. However, Scortum, a cultured species in Taiwan, lack such structure. The aim of this study was to reveal the evolutionary change of the sound-producing system including acoustic signal and sonic structure in the family

Terapontidae. Phylogenic relationship of the was reconstructed using molecular data. The results showed that the marine species were primitive species and most of them have well developed sonic structure. On the contrary, the freshwater species were more derived but their sonic systems are less developed or even absent. It can be generalized that vocalization may be less important to freshwater grunters.

Keywords: Terapontidae, acoustic, swim bladder, phylogeny, sonic system.

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Content

Introduction……………………………………………………………………………1

Material and methods………………………………………………………………….7

2.1 Study species…………………………………………………………………7

2.2 Sound recording…………………………………………………………...…7

2.2.1 Audio recording equipment…………………………………………..8

2.2.2 Acoustic analysis……………………………………………………..8

2.2.3 Statistical analyses……………………………………………………9

2.3 Morphology comparison……………………………………………………..9

2.4 Histology……………………………………………………………………10

2.4.1 Paraffin sections…………………………………………………..…10

2.4.2 Hematoxylin and eosin stain…………………………………...……11

2.4.3 Gomori's trichrome stain…………………………………...……..…12

2.4.4 Elastica van Gieson Staining……………………………………...... 12

2.5 Phylogenetic analysis……………………………………………………….13

2.5.1 Taxonomic sampling……………………………………………...…14

2.5.2 DNA extraction………………………………………………………14

2.5.3 PCR condition………………………………………………………..16

Result……………………………………………………………………………....…17

3.1 Acoustic signals……………………………………………………….….…17 iv

3.1.2 Pelates quadrilineatus………………………………………………17

3.1.3 Amniataba caudavittatus……………………………………………18

3.1.4 Amniataba percoides………………………………………………..18

3.1.5 Hephaestus fuliginosus……………………………………………...20

3.1.6 Syncomistes butleri………………………………………………….20

3.1.7 ………………………………………………………20

3.2 Morphology comparison……………………………………………………22

3.2.1 Morphology of acoustic system……………………………………..22

3.2.2 Relative size of sonic muscle………………………………………..23

3.3 Histology……………………………………………………………………24

3.3.1 The characters of sonic muscle fiber………………………………...24

3.3.2 Internal tissue analyze……………………………………………….24

3.4 Phylogenetic analysis…………………………………………………….…25

Discussion……………………………………………………………………………27

4.1 Acoustic signals…………………………………………………………….27

4.1.1 Frequency of sounds production…………………………………….27

4.1.2 Variation in sound characteristics………………………………...…28

4.2 Analysis of sonic structure………………………………………………….29

4.2.1 Analysis of sonic muscle fibers……………………………………..29

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4.2.2 Internal tendon………………………………………………………30

4.3 Evolutionary changes…………………………………………………….…31

4.3.1 Phylogenetic analysis………………………………………………..31

4.3.2 Changes in sonic system…………………………………………….32

References……………………………………………………………………………34

Appendix…………………………………………………………………………..…80

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Figures Legend

Fig. 1. The sonic muscle, swim bladder and internal tissue of Terapon jabua…….....39

Fig. 2. The connection of internal tissue and the forth joint of vertebra of Terapon

jarbua…………………………………………………………………………40

Fig. 3.The two-chamber swim bladder of Terapon jabua……………………………41

Fig. 4. The theree-chamber swim bladder of Pelates quadrilineatus...... 42

Fig. 5. Apparatus setup for recording the disturbance hand-held sounds in a Styrofoam

box…………………………………………………………………………….43

Fig. 6. Sound parameters……………………………………………………………...44

Fig. 7. The waveform and sonogram of Pelates quadrilineatus………………………45

Fig. 8. The waveform and sonogram of Amniataba caudavittatus……………………46

Fig. 9. The waveform and sonogram of Amniataba percoides………………………..47

Fig. 10. The waveform and sonogram of Hephaestus fuliginosus…………………….48

Fig. 11. The waveform and sonogram of Syncomistes butleri…………………………49

Fig. 12. The waveform and sonogram of Terapon jarbua……………………….…....50

Fig 13. Morphology of acoustic system of Terapontidae.. …………………….……...51

Fig 14. Histology sections of sonic muscle……………………………………………68

Fig 15. The transection of sonic muscle of ……………………….68

Fig 16. The transection of internal tissue………………………………………………69

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Fig 17. The Neighbor tree of 16s rRNA gene of Terapondae. ……………………...70

Fig 18. The Neighbor tree of COⅠ gene of Terapondae... …………………………71

Fig 19. The literal side and ventral side of the vertebrate of Terapon jarbua……….72

Fig. 20. The literal side and ventral side of the vertebrate of Pelates quadrilineatus.73

Fig. 21. The literal side and ventral side of the vertebrate of ………74

Fig. 22. The literal side and ventral side of the vertebrate of Bidyanus bidyanus…...75

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Table Legend

Table 1. The data of sound parameters…………………………………………….…76

Table 2. The characteristics of sonic structures………………………………………77

Table 3. Comparisons of the acoustic parameters of Terapon jarbua in Australia and

Taiwan and the result of the Mann-Whitney test…………………………….78

Table 4. Relative size of sonic muscle……………………………………………….79

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Introduction

Although sounds emitted from many fish groups have been well described, knowledge on the mechanisms of production and the related evolutionary change in the producing apparatus have remained incomplete. The mechanisms can be classified into four types: 1) stridulaton or rubbing of the hard parts such as teeth (Tower 1908), fin spines (Burkenroad 1931) or bones (Colson et al. 1998); 2) vibration of swim bladder- fast contraction and relaxation of the sonic muscles to resonats the swim bladder (Tower

1908); 3) air releasing from the swim bladder (Wilson et al. 2004); 4) vibration of the peritoneum or tendon (Takemura et al. 1978). Vibration of the swim bladder is the most common in fishes.

There are two types of sonic muscles, also known as drum muscles: intrinsic sonic muscle and extrinsic sonic muscle. The former is completely attached to the wall of the swim bladder. On the other hand, extrinsic sonic muscle either originates on the cranium, pectoral girdle or rib, and inserts into the swim bladder or some structure attached to the swim bladder (Demski et al. 1973; Connaughton 2004; Ramcharitara et al. 2006) or completely attached to the wall of the peritoneal cavity. In many species, sonic muscles are present only in males. On the contrary, it is generally weak or absent in female.

In pearlperch of the family Glaucosomatidae, a paired anterior sonic muscles

1 originated on the base of the pterotic bones on the skull and inserted on the the antero-dorsal surface of the swim bladder at the forward edge of the fenestra, a structure seldom exists in soniferous fishes; a tendon from the 9th vertebra ends in a single smooth muscle that inserts on the tunica externa of the posterior edge of the fenestra.

Therefore, the anterior sonic muscle and posterior muscle-tendon spring apparatus attached to opposite sides of the swim bladder fenestra and appear to function as antagonists for the movement of the anterior part of the swim bladder during the activity of the sonic muscles (Mok et al. 2011). A similar but specialized apparatus was also reported in Pempheridae (Jiang 2010). Study on the molecular systematics of these families indicated that Glaucosomatidae and Pempheridae are sister groups (Jiang,

2010). Additionally, we found similar structure in Terapontidae, but according to Jiang’s study, this family is not close relative to the Glaucosomatidae and Pempheridae.

The common name of Terapontidae is grunter which suggests that all or most terapontid species should be soniferous—a prediction found to be true for the well-know as soniferous family Sciaenidae (or the croakers and drums). Despite that the sound of a terapontid species, the Terapon jarbua, has been well studied, it is unknown whether all of the species produce sounds. This species has already known as soniferous but the mechanism is only known as swim bladder resonating by the contraction of a paired sonic muscles (Eichelberg 1976).

2

The extrinsic swimbladder muscle of terapontids was treated as unique character in having its origination arises from the posterior surface of the postemporal process or the rear of the skull or both and its insertion on the anterodorsal surface of the swim bladder

(Vari 1978). Schedneider (1964) noted and speculated that the sonic muscles act in a very high contraction rate which is considered the top one among vertebrate muscles.

Eichelberg (1976) provided experimental evidence for Schedneider’s proposal. Some of terapontids produce sounds but some of them cannot.

In Terapon jarbua and Pelates quadrilineatus—the two common marine grunters in

Taiwan, there are not only a pair of anterior sonic muscles but also a pair of internal tissues inside the swim bladder—a structure rather similar to that of the pearlperch and pempherid (Mok, per. obs.) connecting the antero-dorsal part of the swim bladder (Fig.

1) and the fourth vertebra (Fig. 2). These internal structures have not been reported before. They were suggested serving to recoil the anterior-dorsal swim bladder following termination of the contraction of the sonic-muscle constriction (Mok, per. obs.).

Ontogenetic and inter-specific variations were reported by Vari (1978) in the constriction at the anterior the swim bladder; Terapon jarbua has two-chamber swim bladder (Fig. 3) as a result of a deep constriction, and there is small hole between the small anterior and posterior chamber. The posterior-chamber swim bladder is elongate

3 to oval which is ended in a rounded to distinct tip. Pelates quadrilineatus has the most derived autapomorphic swim bladder form in having a three-chambered swim bladder with the last one smallest in size (Fig. 4). Size and shape of a resonator affect the character of the sounds produced.

The family Terapontidae is composed of 50 species in 15 genera and has a broad marine distribution in the Indo-west Pacific region (Vari 1978). There are four species in three genera (i.e., Terapon, and Pelates) in Taiwan. Terapon jarbua and

Pelates quadrilineatus are common marine species in Taiwan but Mesopristes cancellatus and Terapon theraps are very rare in Taiwan. All of these four species in

Taiwan live in brackish and saltwater habitats. Other species in the genera Amniataba,

Bidyanus, Hephaestus, Hannia, Leioptherapon, Pelates, , Scortum, Syncomistes and Terapon are Australia fishes (marine, brackish, or freshwater), although some of them also occur in the India Ocean and western Pacific Ocean (Vari 1978). Variichthys distribute only in Papua New Guinea. and Pelsartia live in Asian inland waters.

Rhyncopelates was observed in northwest and western of Pacific. Among them,

Leiopotherapon unicolor, L. macrolepis, L. aheneus, Hannia greenwayi, Lagusia micracanthus, Hephaestus fuliginosus, H. roemeri, H. jenkinsi, H. trimaculatus, H. adamsoni, H. suavis, H. carbo, Bidyanus bidyanus, B. welchi, Scortum parviceps, S. hillii, Pingalla lorentzi, P. gilbert, Syncomistes butleri, S. kimberleyensis, S. trigonicus

4 live strictly in freshwater (Vari 1978). As propagation of acoustic signal might differ between saltwater and freshwater, plus the difference in visibility between these two media, it is possible and expected that differences exist in the important of sound communication to the species in these two environments; the difference is relevant to the characteristics of the sound-producing apparatus and the acoustic signals emitted.

According to the phylogenies tree base on bone morphology (Vari 1978),

Leioptherapon is the most primitive genus while Syncomistes is the most derivative genus and both are Australian freshwater fishes. It means that the marine genera are grade derivates from freshwater genus. Base on the molecular phylogeny which is analyzed by cyt b and rag gene, the ancestral terapontids appear to have been in habitat affiliation with a single transition to freshwater environments producing all

Australasian freshwater species (Davis 2012). These two hypotheses are contrary.

The DNA data from nuclear genome provides an independent and alternate opportunity for testing higher level phylogenetic hypotheses (Chen et al. 2003). There is higher nucleotide substitution rate of the mitochondrial DNA and lack of recombination compared with the nuclear DNA. The characteristics make it particularly useful for the intra-specific and inter-specific variations (Avise & Ellis 1986). To propose the phylogenetic relationships and the evolution in acoustic system for the terapontid genera, we examined DNA sequence variation from mitochondrial genes 16S rRNA and

5 cytochrome oxidase subunit (CO) to provide additional test for Davis’s hypothesis.

The main aim of this study was to reveal the evolutionary change of the sound-producing apparatus within the family Terapontidae by 1) falsifying the phylogenetic hypotheses of the terapontid genera using other genes and morphology; 2) describing the sound-producing apparatus in the genera and deciphering the differences;

3) describing the sound signals of the genera and deciphering the difference; 4) reviewing the correlation characteristics of the structure of the apparatus, acoustic-signal characteristics, and living environment (ie. marine vs. freshwater).

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Material and methods

2.1 Study species

The family, Terapontidae has 50 species in 15 genera. The genera, Terapon and

Pelates are common in Taiwan and both of these two genera live in the estuary and in the sea. Samples were collected by angling or from fish market in Kaohsiung. Bidyanus and Scortum are commercial fish introduced from Australia and samples came from a fish farm. Samples of the specimen and muscle tissue of the species in other genera not distributed in Taiwan were obtained as loans and gifts from the museum institution and

Gallery of the Northern Territory, Australia, Australian Museum or personal collection from Japan.

2.2 Sound recording

Recordings of hand-held disturbance sound were made in a styrofoam container to decrease resonance (Takamatsu et al, 2002) and the specifications of the styrofoam container is 555 mm × 364 mm × 230 mm. The hydrophone was placed 5 cm away from the head of the fish (Fig. 5). The species, Bidyanus bidyanus, Pelates quadrilineatus, and Scortum barcoo were recorded in Taiwan and the species,

Amniataba caudavittatus, Amniataba percoides, Leiopotherapon unicolor, Syncomistes butleri and Hephaestus fuliginosus were recorded in Australia by H. K. Mok. Moreover,

7

Terapon jarbua were recorded both in Taiwan and in Australia. After recording, fish were put to sleep by placing in cold seawater with 3-aminobenzoic acid ethyl ester methanesulfonate (MS 222).

2.2.1 Audio recording equipment

Recording equipment include a hydrophone (Burns Electronics, Aquaear Hydrophone

MKII, frequency range: 10 Hz – 25 kHz, sensitivity: 3 kHz -164 dB  5 dB re 1V/μPa) connected with the amplifier (Burns Electronics, HP-A1) and the recorder (KORG

MR-1000, sampling rate: 44.1 kHz). In addition, a H2a hydrophone(Aquarian audio products, frequency range 1 Hz to 100 kHz  4 dB) connected with the recorder Sony

PCM-M10( sampling rate: 96 kHz/ 24 bit) were also used to record sounds.

2.2.2 Acoustic analysis

The sound outputs from the digital recorder were digitized at 16 kHz. Only the sounds with good qulity and minimal background noise were chosen and to analyze by

Raven pro 1.4 for windows (Bioacoustic Research Program, Cornell Laboratory of

Ornithology, Ithaca, NY, USA) and Avisoft Bioacoustics (Kirchstr. 1113158 Berlin

Germany) were used for acoustic analyzing. The waveform and sonogram were computed using Hann window, frequency resolution of fast Fourier transformation (FFT)

8

256 samples, 3 dB bandwidth 248 Hz, and temporal resolution by 89 % overlap. Sound parameters we tested were dominant frequency (the frequency with the highest energy within the power spectrum of a call, Hz (Ripley et al. 2002); call duration (duration between the onset and the end of a call, ms), pulse number (the number of pulse in a call), pulse duration (duration between the onset and the end of a pulse), pulse period

(measures as the average peak-to-peak duration between two consecutive pulse in a call, ms (Parmentier et al. 2010); pulse repetition rate (number of pulse per second), inter-pulse interval (IPI, duration between the end of a pulse and the begin of the next pulse(Tellechea et al. 2011) (Fig. 6).

2.2.3 Statistical analyses

Student-T test was used to test the difference between the sound parameters (i.e. pulse duration, pulse period, pulse number, inter-pulse-interval, call duration and repetition rate) of Terapon jarbua in Taiwan and Australia. The critical α level for analyses was 0.05.

2.3 Morphology comparison

All specimens were disested on the ventral side to check the sonic system: size and shape of sonic muscle, the shape of swim bladder, fenestra and the pair of internal tissue

9 inside the anterior-chamber. Ratio of diameter of sonic muscle and total body length; the ratio of diameter of sonic muscle and head length were calculated.

2.4 Histology

Four species (i.e. Bidyanus bidyanus, Pelates quadrilineatus, Scortum barcoo and

Terapon jarbua) were used to compare the muscle fibers of the sonic muscle and the internal tissue. Two soniferous species, Terapon jarbua and Pelates quadrilineatus, and

Bidyanus bidyanus and Scortum barcoo are freshwater species. The tissues were fixed in Bouin solution after removed from specimen. After fixation, tissue was dehydrated through the serious of graded ethanol baths (50%, 70%, 80%, 90%, 95%, 99%; 20 min per concentration) to displace water, then put into xylene for 1 hour and twice. After dehydrated, the tissue infiltrated in wax for 2 hours in 70oC and the infiltrated tissue embedded into wax blocks.

2.4.1 Paraffin sections

1) Transfer tissue into mold and place the cut side dowm.

2) Transfer mold to cold plate and then the wax will solidify in a thin layer which

holds the tissue in a position.

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3) When the tissue is in the desired orientation add the labeled tissue cassette on

top of the mold as a backing.

4) Hot wax which is enough to cover the face of the plastic cassette is added to the

mold from the wax dispenser.

5) Cool the mold until the wax solidify and the cassette can be take out easily.

6) Turn on the water bath and check that the temp is 43ºC.

7) Insert the block into the microtome to cutting smoothly, and set to 5 µM

8) cut another four sections and pick them up and float them on the surface of the

43ºC water bath.

9) Float the sections onto the surface of clean glass slides.

2.4.2 Hematoxylin and eosin stain

1) The section slide is put in xylene and 100% ethanol for 5 min and twice to drop

off wax.

2) Rinse the section slide in running tap water for 60 sec.

3) Stain nuclei with the alum haematoxylin for 20 min.

4) Rinse the section slide in running tap water for 30 sec.

5) Stain with eosin in 10 sec.

6) Rinse the section slide in running tap water for 60 sec.

11

7) Dehydrate through the serious of graded ethanol baths, 3sec for each concentrate.

8) Mount the slide using acacia senegal.

2.4.3 Gomori's trichrome stain

1) The section slide is put in xylene and 100% ethanol for 5 min and twice to drop

off wax.

2) Rinse the section slide in running tap water for 60 sec.

3) Fixation of Bouin’s fluid for 1 hour in 56℃

5) Rinse the section slide in running tap water for 5 min.

4) Stain nuclei with an acid resistant nuclear stain (Weigert’s iron hematoxylin) for

10 min.

5) Rinse the section slide in running tap water for 30 sec.

6) Place into solution C for 25 min.

7) Quickly rinse off stain with distilled water.

8) Rinse with solution D.

9) Dehydrate through the serious of graded ethanol baths, 3sec for each concentrate.

10) Mount the slide using acacia senegal.

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2.4.4 Elastica van Gieson Staining

1) The section slide is put in xylene and 100% ethanol for 5 min and twice to drop

off wax.

2) Elastin according to Weigert for 10 min.

3) Rinse the section slide in running tap water for 60 sec.

4) Weigert’s A & B 1:1 for 5 min.

5) Rinse the section slide in running tap water for 60 sec.

6) Picrofuchsin solution for 2 min.

7) 70 % ethanol for 60 sec.

8) Dehydrate through the serious of graded ethanol baths, 3sec for each concentrate.

9) Mount the slide using acacia senegal.

2.5 Phylogenetic analysis

For constructing the phylogenetic tree of Terapontidae, mitochondrial genes 16S rRNA and cytochrome oxidase subunit I (COI) were selected as the target molecular markers. The nucleotide sequences were managed and aligned with BioEdit Sequence

Alignment Editor 7.0.9.0. The taxonomic relationship was derived from neighbor joining (NJ) and the following bootstrap in 1000 replicates with branch and branch search. The value between 80 to 100% meant strong support, 60 to 80% meant medium

13 support, 40 to 60% meant weak support and it is no support below 30%. The analysis of

DNA evolution was preceded according to Molecular Evolutionary Genetics Analysis 4

(MEGA4).

2.5.1 Taxonomic sampling

The family Serranidae was chosen to be the out group and the DNA sequences were obtained from GeneBank (Appendix 1).

2.5.2 DNA extraction

Samples of muscle were taken from the specimens of Amniataba caudavittatus,

Amniataba percoides, , Hephaestus fuliginosus, Hephaestus jenkinsi,

Leiopotherapon unicolor, Pelates sexlineatus, Pingalla midgleyi , Scortum hillii,

Syncomistes butleri, Syncomistes rastellus and Syncomistes trigonicus collected in

Australia for DNA analyses. Samples of muscle tissue were also taken from specimens of Bidyanus bidyanus, Pelates quadrilineatus, Scortum barcoo and Terapon jarbua collected in Taiwan. Tissue samples from Australia were preserved in 70% alcohol, and samples from Taiwan were frozen at −20℃. Each tissue from muscle sample weight 10 mg. For whole DNA amplification, there are several steps to prepare:

1) Add 600 µl Nuclear Lysis Solution into a 1.5 ml centrifuge tube, and then chill

on ice.

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2) Homogenize the tissue; incubate the lysate at 65°C for 15–30 minutes.

3) Centrifuge for 3 minutes at 16000× g at room temperature.

4) Carefully remove and discard as much of the supernatant as possible with a

micropipette tip.

5) Add 17.5 µl of 20 mg/ ml Proteinase K into the supernatant and Centrifuge at

14000 × g for 1 minutes at room temperature.

6) Incubate overnight at 55°C.

7) Add 3 µl of RNase Solution to the nuclear lysate.

8) Incubate the maxture for 15-30 minutes at 37°C

9) Add 200 µl of Protein Precipitation Solution and gentle shake. Chill sample on

ice for 5 minutes.

10) Centrifuge at 16000 × g for 4 minutes at 4°C.

11) Carefully remove the supernatant containing DNA and transfer it to a clean 1.5

ml microcentrifuge tube containing 600 µl of isopropanol. Put in -20°C for 1 hour.

12) Centrifuge at 13000 × g for 1 minute at 4°C.

13) Add 600 µl 70% ethanol to wash the DNA, and centrifuge at 10000 × g for 1

minute at 4°C.

14) Inver the tube on clean absorbent paper, and air-dry the pellet for 10 minutes.

15) Add 100 µl of DNA Rehydration Solution to rehydrate the DNA.

15

2.5.3 PCR condition

The fragments of DNA were amplified using polymerase clain reaction (PCR).

PCR reaction mixture were 25 µl in volume containing sterile distilled H2O, 0.25 µl

Tara LA TaqTM DNA Polymerase (5U/µl), 2.5 µl 10× Buffer, 0.25 µl dNTP Mix (2.5mM each), 0.25 µl primer of each (10mM) and 5 µl DNA template in rach reaction. The primers 16SAR (5’-CGCCTGTTTAACAAAAACAT-3’) and 16SBR

(5’-CCGGTTTTGAACTCAGATCACGT-3’) were used to amplify the 16S rRNA gene

(Palumbi 1996). PCR amplifications were made with the following thermal profile: 40 cycles of denaturing at 94℃ for 1 min, annealing at 52℃ for 1.5 min and extension at

68℃ for 1.5 min, followed by incubation at 68℃ for 10 min. The primers FishF1

(5’-TCAACCAACCACAAAGACATTGGCAC-3’) and FishR2

(5’-ACTTCAGGGTGACCGAAGAATCAGAA-3’) were used to amplify COI (Ward et al. 2005). PCR amplifications were made with the following thermal profile: 40 cycles of denaturing at 94℃ for 1 min, annealing at 54℃ for 1.5 min and extension at

68℃ for 1.5 min, followed by incubation at 68℃ for 10 min. The PCR products were analyzed by electrophotesis (110V for 35 minutes) with 1% agarose gel.

16

Result

3.1 Acoustic signals

Bidyanus bidyanus were recorded in Taiwan but all tested fish did not produce any sound. Leiopotherapon unicolor specimens recorded in Australia did not produce sound.

Pelates quadrilineatus was recorded in Taiwan and the species, Amniataba caudavittatus, Amniataba percoides, Syncomistes butleri and Hephaestus fuliginosus were recorded in Australia; all of them are soniferous fishes. Terapon jarbua was recorded both in Taiwan and in Australia. Only the sounds with good quality and low background noise were chosen for analysis (Table 1).

3.1.2 Pelates quadrilineatus

In Pelates quadrilineatus, the fundamental frequency was 273 Hz, the domain frequency was 861 Hz with 19.07  6.38 pulses in a call and the range was 11 to 33 pulses, lasting from 39 to 131 ms (call duration; mean  SD = 70.43  23.39 ms) (N, number of fish, = 6; n, number of sounds, = 121). The pulse duration ranged from 2.85 to 3.30 ms (mean  SD = 3.09 ± 0.16 ms); the pulse period ranged from 3.3 to 3.82 ms

(mean  SD = 3.64  0.08 ms); the inter-pulse-interval ranged from 0.14 to 0.9 ms

(mean ± SD = 0.65  0.14 ms); the pulse repetition rate ranged from 259.25 to 280.71 pulses/sec (mean  SD = 270.1 6.23 pulses/sec). There were some regular features in

17 its waveform which composed of three main positive peaks, and the median peak was the highest in amplitude (Fig. 7). Additionally, the last pulse was weaker than the others.

3.1.3 Amniataba caudavittatus

In Amniataba caudavittatus, the fundamental frequency was 258.4 Hz, the domain frequency was 689.1 Hz with the 9.03 ± 1.08 pulses in a call and the range was 7 to 12 pulses, lasting from 26 to 44 ms (call duration, mean ± SD = 34.13 ± 4.09 ms) (N= 1, n= 31). The pulse duration ranged from 3 to 3.78 ms (mean ± SD = 3.25 ± 0.18 ms); the pulse period ranged from 3.27 to 4.28 ms (mean ± SD = 3.71 ± 0.17 ms); the inter-pulse-interval ranged from 0.25 to 0.87ms (mean ± SD = 0.6 ± 0.18 ms); the pulse repetition rate ranged from 236.84 to 277.77 pulses/sec (mean ± SD = 264.87 ±

9.26 pulses/sec). The waveform of a typical Amniataba caudavittatus pulse was composed of three peaks: the first peak was negative, the second peak was positive and the third pulse was negative (Fig. 8). There were two types, type one begins with a lead by a positive peak with high energy; the other type was similar with type one but there was a small peak in front of the first pulse and the energy of the peak was low.

3.1.4 Amniataba percoides

In Amniataba percoides, the fundamental frequency was 211.3 Hz, the domain

18 frequency was 561.8 Hz and 861.3 Hz with 20.85 ± 12.97 pulses in a call and the range was 11 to 61 pulses, lasting from 51 to 271 ms (call duration; mean ± SD =

95.39 ± 64.41 ms) (N= 5, n= 81). The pulse duration ranged from 3.77 to 4.33 ms (mean

± SD = 4 ± 0.18 ms); the pulse period ranged from 4.57 to 4.2728 ms (mean ± SD =

4.68 ± 0.06 ms); the inter-pulse-interval ranged from 0.45 to 0.94 ms (mean ± SD =

0.75 ± 0.19 ms); the pulse repetition rate ranged from207.54 to 217.39 pulses/sec (mean

± SD = 214.29 ± 2.39 pulses/sec). The waveform of a typical pulse of the sound of

Amniataba percoides was very simple (Fig. 9). There were made up of three main peaks decreased gradually. It was almost the same intensity in all of the pulses except the last one which was weaker than others.

3.1.5 Hephaestus fuliginosus

In Hephaestus fuliginosus, the sounds were not harmonic sound. The domain frequency was 172.3 Hz with 4.94 ± 0.89 pulses in a call and the range was 3 to 7 pulses, lasting from 159 to 227 ms (call duration; mean ± SD = 174.45±23.81 ms) (N=

9, n= 93). The pulse duration ranged from 24.0to 30.14 ms (mean ± SD = 26.76±1.96 ms); the pulse period ranged from 24.8 to 32.4 ms (mean ± SD = 28.85 ± 2.91 ms); the inter-pulse-interval ranged from 2.4 to 7.2 ms (mean ± SD = 3.87 ± 1.45 ms); the pulse repetition rate ranged from 30.3 to 37.73 pulses/sec (mean ± SD = 33.53 ± 2.37

19 pulses/sec). The waveform of a typical pulse was composed two to three pulses in a part of the waveform of the sound of Hephaestus fuliginosus and the inter-pulse-interval was in evidence but not regular which were parts composed of few pulse (Fig. 10).

3.1.6 Syncomistes butleri

In Syncomistes butleri, the fundamental frequency was 160.3 Hz, the domain frequency was 172 Hz with 26.7 ± 5.22 pulses in a call and the range was 16 to 30 pulses, lasting from 96 to 204 ms (call duration; mean ± SD = 162.30 ± 34.86 ms) (N=

1, n= 11). The pulse duration ranged from 4.31 to 4.86 ms (mean ± SD = 4.64 ± 0.15 ms); the pulse period ranged from 5.64 to 6.74 ms (mean ± SD = 6.07 ± 0.32 ms); the inter-pulse-interval ranged from 0.9 to 2.4 ms (mean ± SD = 1.4 ± 0.15 ms); the pulse repetition rate ranged from 154.84 to 184.21 pulses/sec (mean ± SD = 165.50 ± 9.36 pulses/sec). The waveform of a typical pulse was composed two to four small peaks before the start of the call and there were two part showed in a pulse (Fig. 11). The first part was composed of a positive peak and a negative peak and few small peaks following, than the second part is a small negative peak at the back. It was difficult to measure and separate these two parts because the second part was overlapped to the first part of next pulse. Only could be measured when the inter-pulse-interval getting longer at the end of the call.

20

3.1.7 Terapon jarbua

In Terapon jarbua in Taiwan, the fundamental frequency was 175.2 Hz, the domain frequency was 689.1 Hz with 4.94 ± 0.89 pulses in a call and the range was 4 to 30 pulses, lasting from 86 to 145 ms (call duration; mean ± SD = 137 ± 27.51 ms) (N= 6, n= 120). The pulse duration ranged from 4.4 to 4.8 ms (mean ± SD = 4.60 ± 0.13 ms); the pulse period ranged from 5.58 to 5.86 ms (mean ± SD = 5.64 ± 0.13 ms); the inter-pulse-interval ranged from 1 to 1.58 ms (mean ± SD = 1.58 ± 1.3 ms); the pulse repetition rate ranged from 172.22 to 179.86 pulses/sec (mean ± SD = 177.32 ±

2.88pulses/sec). In Terapon jarbua in Australia, the fundamental frequency was 247.7

Hz, the domain frequency was 334 Hz with 20.33 ± 1.15 pulses in a call and the range was 19 to 21 pulses, lasting from 76 to 84 ms (call duration; mean ± SD = 81.33 ± 4.62 ms) (N= 1, n= 3). The pulse duration ranged from 3.24 to 3.14 ms (mean ± SD =

3.20 ± 0.05 ms); the pulse period ranged from 3.94 to 4.00ms (mean ± SD = 3.96 ±

0.03 ms); the inter-pulse-interval ranged from 0.8 to 0.9 ms (mean ± SD = 0.84 ± 0.05 ms); the pulse repetition rate was mean ± SD = 250 ± 0 pulses/sec. Between the sounds from two areas, the structures were similar in waveform and the waveform of a typical pulse was composed two main peaks in a pulse and there was one small peak in front of the main two peaks and two small peaks at the back (Fig. 12). In addition, comparisons of the acoustic parameter of Terapon jarbua in Australia and Taiwan, there are

21 significant difference in pulse duration (Mann-Whitney test; p< 0.0001); pulse period

(Mann-Whitney test; p< 0.0001); inter-pulse-interval (Mann-Whitney test; p< 0.005) and repetition rate (Mann-Whitney test; p< 0.005). The pulse number did no differ significantly (Table 2).

3.2 Morphology comparison

3.2.1 Morphology of acoustic system

The acoustic system includes a pair of sonic muscle, swim bladder, and a pair of tissue on the dorsal side of anterior swim bladder (mostly in the first chamber). In

Amniataba caudavittatus, Amniataba percoides, Bidyanus bidyanus, Hephaestus fuliginosus, Hephaestus jenkinsi, Leiopotherapon macrolepis, Leioptherapon plumbeus,

Leiopotherapon unicolor, Pelates octolineatus, Pelates quadrilineatus, Pelates sexlineatus, Pelsartia humeralis, Rhyncopelates oxyrhynchus, Scortum barcoo,

Syncomistes butleri, Terapon jarbua, Terapon theraps (Table 3), most of the species have two chambers; anterior one is oval the posterior elongated. Pelates quadrilineatus has a three-chambered swim bladder in which the last one is very small (Fig. 13(J2)).

The sonic muscles originate from the posttemporal and / or the pterotic connect with swim bladder in the hint site. There is a fenestra which is a slit on the dorsalateral side of the anterior swim bladder. Only two species do not have fenestra: Bidyanus bidyanus

22

(Fig. 13(C2)) and Scortum barcoo (Fig. 13(N2)). Besides, there is a special tissue at the connection between sonic muscle and swim bladder shown in only Pelates octolineatus

(Fig. 13(I4)) and Pelates sexlineatus (Fig. 13(K2)). Furthermore, a pair of internal tissue which has interspecies variation was presented in most studied species. Amniataba caudavittatus, Pelsartia humeralis, Rhyncopelates oxyrhynchus and Terapon theraps have much developed internal tissue and the color is red; Pelates quadrilineatus (Fig.

13(J2)) and Terapon jarbua (Fig. 13(P2)) also have much developed internal tissue but color is white. The internal tissue of Bidyanus bidyanus (Fig. 13(C3)) is pink and thin; the internal tissue of Pelates sexlineatus (Fig. 13(K3)) and Scortum barcoo (Fig. 13(N3)) are transparent and membrane-like. Amniataba percoides (Fig. 13(B3)), Hephaestus fuliginosus (Fig. 13(D3)), Hephaestus jenkinsi (Fig. 13(E3)), Leiopotherapon unicolor

(Fig. 13(H3)) and Syncomistes butleri (Fig. 13(O3)) has no internal tissue.

3.2.2 Relative size of sonic muscle

To compare the ratio of sonic muscle and body size, total length and head length were chosen to be the criteria (Table 4). The ratio of sonic muscle and total body length showed that Rhyncopelates oxyrhynchus and Terapon jarbua had most developed sonic muscle (SM / TL= 0.03) and the ratio of sonic muscle and head length showed that

Pelates sexlineatus and R. oxyrhynchus had the highest ratio (SM / HL= 0.12).

23

3.3 Histology

3.3.1 The characters of sonic muscle fiber

The sonic muscles of Bidyanus bidyanus, Pelates quadrilineatus, Scortum barcoo and Terapon jarbua were analyzed by histological section (Fig. 14). The myofibril of

Bidyanus bidyanus arranged compactly, whereas the other three species had more spaces around the myofibril. For the shape, the myofibril of Scortum barcoo was irregular. Except Scortum barcoo, the myofibril and the spaces around of the other three species was arranged regular. Moreover, there was a lot of morula-like structures appeared frequently in sonic muscle of Bidyanus bidyanus (Fig. 15).

3.3.2 Internal tissue analyze

In Bidyanus bidyanus, Pelates quadrilineatus and Terapon jarbua and Scortum barcoo, except S. barcoo, the remaining species had the internal tissue in the swim bladder. To figure out what kind of tissue it was, three stains were chosen: Gomori's trichrome stain, Hematoxylin and eosin stain and Elastica van Gieson stain. All of the histological sections were stain by Hematoxylin and eosin stain and the other stains were chosen for further proof. In Gomori's trichrome stain, nucleus was stained as red-purple, muscle fiber was red and interstitial collagen was stained as green. The internal tissue of Terapon jarbua was green color which indicated that the tissue of was

24 composed of collagen fibers (Fig. 16A, 16B) and it was not muscle tissue. Tissue stained with hematoxylin and eosin stain showed cytoplasm in pink, nucleus in purple and collagen fiber was a net structure and fewer nucleuses. The internal tissues of all three species were collagen fibers (Fig. 16). Tissue was stained with Elastica van

Gieson stain showed nuclei in black-brown, elastic fiber in black, collagen in red and muscle tissue in yellow. The internal tissue of Bidyanus bidyanus was collagen fibers.

3.4 Phylogenetic analysis

22 individuals of terapontids in nine genera, Amniataba, Bidyanus, Hephaestus,

Leiopotherapon, Pelates, Pingalla , Scortum, Syncomistes and Terapon were samples for analyzing the DNA sequence of COⅠ with 651 base pair and 16s rRNA with 620 base pair. Topologies of these trees shows few parallel lineages (Fig. 17, 18). The species Pelates, Terapon and Amniataba were the ancestral species, whereas species in freshwater genera such as Hephaestus and Scortum were the derived. The other genera in Australia and the Australia freshwater genus, Bidyanus was closer to Scrotum than marine species group and was most relative to the derived group. Monophyletic of the

Terapon was not supported and the genus was separated to two monophyletic groups: a monophyletic group is consisted of Terapon theraps and . Terapon jabua was the sister taxa to Pelates quadrilineatus (Bootstrap=67) in Neighbor Joining tree of

25

16s rRNA. The other Neighbor Joining tree of COⅠshowed Terapon theraps and

Terapon puta is the sister taxa to Pelates quadrilineatus and Pelates sexlneatus

(Bootstrap=59). Hephaestus was not a monophyletic group. Instead, Hephaestus fuliginosus and Hephaestus jenkinsi is a monophyletic group (Bootstrap=100), the other monophyletic group is consisted of Hephaestus carbo and Hephaestus habbemai

(Bootstrap=100 and 99). Moreover, these two monophyletic group are not sister groups.

26

Discussion

4.1 Acoustic signals

4.1.1 Frequency of sounds production

In all the six study species, the sounds of Amniataba caudavittatus, Amniataba percoides, Pelete quadrilineatus, Syncomistes butleri and Terapon jarbua were harmonic, except Hephaestus fuliginosus. The sounds were composed of multiple overtones besides the fundamental frequency and the fundamental frequency is associated with the rate of muscle contraction (Cohen & Winn 1967; Skoglund, 1961;

Tavolga, 1962). The sound frequency does not correspond to the calculated resonant frequency of the bladder (Parmentier et al. 2006). The fundamental frequencies of all of these species were about 200 Hz and it means that the rates of sonic muscle contraction in these species were almost the same. Nevertheless, the dominant frequency was different. The dominant frequency of Amniataba caudavittatus, Amniataba percoides,

Pelete quadrilineatus, Syncomistes butleri, and Terapon jarbua were 689.1 Hz, 561.8 &

861.3 Hz, 861 Hz, 172 Hz, and 344 & 689.1 Hz, respectively. Because the frequencies were too high (i.e. the pulse is less than 10 ms, it showed by determinate by swim bladder mechanism rather than contraction of the sonic muscle (Parmentier et al. 2006a;

Fine et al. 2007). Moreover, the shape and the size of swim bladder were also associated.

In this study, many species of Terapontidae have internal tendon which was never been

27 found before within swim bladder. The internal tendons were proved to be collagen fibers by wax section and stain. I assume that collagen fibers played a role of elasticity and tension to keep the high frequency and pull the swim bladder forward when sonic muscle contracting. Amniataba caudavittatus, Pelates quadrilineatus and Terapon jarbua have very strong sonic muscle.

4.1.2 Variation in sound characteristics

In the genus, Amniataba, A. caudavittatus, a marine and estuary species, and its freshwater congeneric species, A. percoides, are different in vocal response when disturbance; most individuals of the former species did not emit sound while disturbed by holding them. On the other hand, almost all A. percoides individuals emitted sound when disturbed.

Amniataba caudavittatus, and Hephaestus fuliginosus had fewer pulse number in a call and the other species, Amniataba percoides, Syncomistes butleri, Terapon jarbua and Pelates quadrilineatus had more than 15 pulses per call. According to the sonic structure, Amniataba percoides and Hephaestus fuliginosus had no internal tendon within swim bladder. On the other hand, Amniataba caudavittatus, Terapon jarbua and

Pelete quadrilineatus had a pair of well-developed internal tendon. Syncomistes butleri did not have internal tendon but had the highest pulse number. Furthermore, all of these

28 six species had fenestra. Fish produce sounds by contracting the sonic muscles to stretching the fenestra until released (Parmentier et al. 2006b). There seems to be no general and obvious correlation between the pulse number and sound producing structure.

About pulse duration, pulse period and repetition rate, all of these six species were in the same range except , Hephaestus fuliginosus. The pulse duration in H. fuliginosus sounds was about four times longer than that of the other species. Based on the waveform, H. fuliginosus had a special waveform of the pulse which composed of a few wide peaks so that the sound had longer pulse duration. Hephaestus fuliginosus had thinner sonic muscles and fenestra on swim bladder; the internal tendon connect only to swim bladder instead of the vertebrate (Mok observation). The cause for its unusual waveform is unclear.

4.2 Analysis of sonic structure

4.2.1 Analysis of sonic muscle fibers

Beside the fundamental frequency associated with the rate of muscle contraction, there are some structures that show the contraction rate of sonic muscle fast or slow. For example, the the sonic muscle of toadfish is the fastest vertebrate striated muscle (Rome

& Lindstedt 1998; Tavolga 1964) and its myofibril has a sarcoplasmic central core

29 which contains glycogen granules and mitochondria to facilitate quick contraction

(Modesto & Canário 2006). The sarcoplasmic central core also is present in the sonic muscle of Johnius macrohynus (Lin 2008). In Terapontidae, no sarcoplasmic central core was formed of Bidyanus bidyanus, Pelates quadrilineatus, Scortum barcoo and

Terapon jarbua. Compare with carapid fish which has the slow sonic muscle and toadfish which has the fast sonic muscle, the contraction rates of terapontid species were in the medium frequency that means their sonic muscles contract at a intermediate rate.

Terapon jarbua has two types of myofibril: lla and llc which has the activities of the oxidative enzymes SDH, NADH-TR, LDH, and mATPase to have high metabolic pattern (Chen et al. 1998). In addition, I believe that the sonic system which is the interaction between sonic muscle, swim bladder, fenestra and the internal tendon plays an important role.

4.2.2 Internal tendon

Fresh specimens Scortum barcoo, Bidyanus bidyanus, Pelates quadrilineatus and

Terapon jarbua are available in Taiwan. Except for S. barcoo, B. bidyanus,

P.quadrilineatus and T. jarbua have internal tissue which was determined to be collagen fibers. The collagen fibers of B. bidyanus contacted with swim bladder anteriorly and posteriorly. The collagen fibers of P. quadrilineatus and T. jarbua

30 connected with swim bladder in the front and connected to the fourth vertebrate in the back. Moreover, there was a special bone on the ventral side of the fourth vertebrate and it had not be mentioned in any previous studies. It was the enlarged fourth parapophysis in Terapon jarbua (Fig. 19) and Pelates quadrilineatus (Fig. 20). The parapophysis in normal size is functioning as the attachment site for the rib. The enlarged parapophysis become the attachment for the internal tendon at ventral site. On the contrary, Scortum barcoo has neither internal tendon nor enlarged parapophysis (Fig.

21) and the pair of internal tendon of Bidyanus bidyanus attached to swim bladder directly instead of connected to vertebrate so that Bidyanus bidyanus had no enlarged parapophysis (Fig. 22). Morphology of the anterior vertebrae in the Australia terapontid species should be exampled in detail when specimens are available as it provides indicative information about development of the internal tendon which might be deformed after presented in fixation for a long period of time.

4.3 Evolutionary changes

4.3.1 Phylogenetic analysis

To test the hypothesis that the marine terapontids were primitive, 16 species in 10 genera were analyzed for reconstruction of a phyloghnetic tree. Freshwater genera include Hephaestus, Pingalla, Bidyanus, Scortum, Syncomistes and Leiopotherapon are

31 close relative derived than the marine groups (Fig. 17, 18). Pelates and Terapon, which are euryhaline genera and commonly found in estuary and marine habitats, were shown as primitive genera. And also the marine genus, Amniataba, was relative to the primitive group. Some freshwater species such as Amniataba percoides is sister group to the marine congeneric species (i.e. A. caudavittatus). The results indicates that invasion to the freshwater had occured several times in the family Terpontidae. Scortum which is a freshwater genus was the most derived genus and is one of terpontids genus in which the swim bladder did not process the family-specific characteristics. What caused such “degenerate” remains unclear.

4.3.2 Changes in sonic system

According to molecular data, acoustic signal and morphology, primitive marine soniferous grunters have well developed sonic muscles, a pair of internal tendon and two-chamber swim bladder with a fenestra in the front chamber (e.g. Terapon jarbua,

Fig. 1~3). The most derived genus, Scortum, had neither fenestra nor internal tendon inside the swim bladder, and the sonic muscle was less developed. In this study,

Scortum barcoo produced sounds in the end and there were two types of the swim bladder. The sounds of S. barcoo should be analyzed in next study and also the types of swim bladder. Bidyanus bidyanus has the ability to make sound (Mitchell 1838), but I

32 did not record any sounds of it. Although there was no acoustic response, the morphology showed there was a pair of internal tendon in the swim bladder of this species and the SM / TL and SM / HL ratio (Table 4) was high. I agree that it is a soniferous species. Leiopotherapon unicolor, a freshwater species, was poor callers; no fish was found to emit sound. Overall, the freshwater species was phylogenetic more derived than marine species but the sonic system in freshwater species may be less developed or even absent. It can be generalized that vocalization may be less important to freshwater grunters.

33

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pulse sounds. Proceedings of the Royal Society B: Biological Sciences 271:95-97.

38

Fig. 1. The sonic muscle, swim bladder and internal tissue of Terapon jabua. SM= sonic muscle, IT= internal tissue, SB= swim bladder.

39

Fig. 2. The connection of internal tissue and the forth joint of vertebra of Terapon jarbua. The arrow points out enlarged forth vertebra.

40

Fig. 3.The two-chamber swim bladder of Terapon jabua. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder.

41

Fig. 4. The theree-chamber swim bladder of Pelates quadrilineatus. SM= sonic muscle,

Ⅰ= the first-chamber swim bladder, Ⅱ= the second-chamber swim bladder, Ⅲ= the third-chamber swim bladder

42

Fig. 5. Apparatus setup for recording the disturbance hand-held sounds in a Styrofoam box (555 mm × 364 mm × 230 mm).

43

Fig. 6. Sound parameters. pulse number (the number of pulse in a call), pulse duration

(duration between the onset and the end of a pulse), inter-pulse interval (IPI, duration between the end of a pulse and the begin of the next pulse), pulse period (measures as the average peak-to-peak duration between two consecutive pulse in a call), call duration (duration between the onset and the end of a call).

44

(A)

(B)

(C)

Fig. 7. The waveform and sonogram of Pelates quadrilineatus. (A) An example waveform and sonogram of four calls. (B) An expanded waveform and sonogram of a call (3 dB filter bandwidth = 248 Hz). The arrows point out the main peaks. (C) The sounds of P. quadrilineatus were harmonic. The line 1 shows the fundamental frequency. 45

(A)

(B)

(C)

Fig. 8. The waveform and sonogram of Amniataba caudavittatus. (A)A type 1 expanded waveform and sonogram of a call (B) A type 2 expanded waveform and sonogram of a call (3 dB filter bandwidth = 248 Hz). The arrows point out the main peaks. (C)The sounds of A.caudavittatus were harmonic. The line 1 shows the fundamental frequency.

46

(A)

(B)

(C)

Fig. 9. The waveform and sonogram of Amniataba percoides. (A) An example waveform and sonogram of three calls. (B) An expanded waveform and sonogram of a call (3 dB filter bandwidth = 248 Hz). The arrows point out the main peaks. (C)The sounds of A. percoides were harmonic. The line 1 shows the fundamental frequency.

47

(A)

(B)

Fig. 10. The waveform and sonogram of Hephaestus fuliginosus. (A) An example waveform and sonogram of four calls. (B) An expanded waveform and sonogram of a call (3 dB filter bandwidth = 248 Hz). There were two to three pulses in a part of the waveform of the sound of Hephaestus fuliginosus and the inter-pulse-interval was in evidence but not regular which were parts composed of few pulse. The arrows point out the main peaks.

48

(A)

(B)

Fig. 11. The waveform and sonogram of Syncomistes butleri. (A) An example waveform and sonogram of three calls. (B) An expanded waveform and sonogram of a call (3 dB filter bandwidth = 248 Hz). There were two to four small peaks (showed in square 1 and first arrow) before the start of the call and there were two part showed in a pulse

(showed in square 2 and 3). (C)The sounds of S. butleri were harmonic. The line 1 shows the fundamental frequency. 49

(A)

(B)

Fig. 12. (A) A waveform of Terapon jarbua in Taiwan. (B) A waveform of Terapon jarbua in Australia (3 dB filter bandwidth = 248 Hz). Between the sounds from two areas, the structures were similar in waveform: two main peaks in a pulse and there were one small peak in front of the main two peaks and two small peaks at the back.

The arrows point out the main peaks. (C)The sounds of T. jarbua were harmonic. The line 1 shows the fundamental frequency.

50

Fig. 13(A1). A specimen of Amniataba caudiavtata. (A2) The sonic system of

Amniataba caudiavtata. SM= sonic muscle, ACSB= anterior-chamber swim bladder,

PCSB= posterior-chamber swim bladder. F= fenestra. (A3) The color of internal tissue was red. 51

Fig. 13(B1) A specimen of Amniataba percoides. (B2) The sonic system of Amniataba percoides. ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (B3) Amniataba percoides had no internal tissue in the swim bladder.

52

SM

Fig. 13(C1). A specimen of Bidyanus bidyanus. (C2) The sonic system of Bidyanus bidyanus. SM= sonic muscle, SB= swim bladder (C3) The color of IT= internal tissue was pink. 53

Fig. 13(D1). A specimen of Hephaestus fuliginosus. (D2) The sonic system of

Hephaestus fuliginosus, ACSB=anterior-chamber swim bladder, PCSB= posterior- chamber swim bladder. (D3) Hephaestus fuliginosus had no internal tissue in SB. 54

Fig. 13(E1). A specimen of Hephaestus jenkinsi. (E2) The sonic system of Hephaestus jenkinsi ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (E3) Hephaestus jenkinsi had no internal tissue in the swim bladder.

55

Fig. 13(F1) A specimen of Leiopotherapon maerolerois. (F2) Two-chamber swim bladder of Leiopotherapon maerolerois. ACSB= anterior-chamber swim bladder,

PCSB= posterior-chamber swim bladder. (F3) Leiopotherapon maerolerois had a pair of internal tissue and the color was red

56

Fig 13(G1). A specimen of (G2) Two-chamber swim bladder of Leiopotherapon plumbeus. ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (G3) A pair of internal tissue.

57

Fig. 13(H1). A specimen of Leiopotherapon unicolor. (H2) The sonic system of

Leiopotherapon unicolor. SM= sonic muscle, ACSB= anterior-chamber swim bladder,

PCSB= posterior-chamber swim bladder. (H3) No internal tissue in the swim bladder.

58

Fig. 13(I1) A specimen of Pelates octolineatus (I2) The sonic system of Pelates octolineatus. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (I3) The pair of internal tissue were red. (I4) The sonic muscle (red arrow) and the pre-tissue (yellow arrow).

59

Fig 13. (J1) A specimen of Pelates quadrilineatus. (J2) The sonic system of Pelates quadrilineatus. SM= sonic muscle, Ⅰ= the first-chamber swim bladder, Ⅱ= the second-chamber swim bladder, Ⅲ= the third-chamber swim bladder, IT= internal tissue,

F= fenestra.

60

(K1)

(K2)

(K3)

Fig. 13(K1). A specimen of Pelates sexlneatus (K2) The sonic system of Pelates sexlneatus, the red arrow points out the pre-tissue between sonic muscle and swim bladder. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (K3) Without internal tissue and the member was transparent at the red arrow

61

Fig. 13(L1). A specimen of Pelsartia humeralis. (L2) The sonic system of Pelsartia humeralis. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (L3) The internal tissue was red. 62

(M3)

Fig. 13(M1) A specimen of Rhyncopelates oxyrhynchus (M2) The sonic system of

Rhyncopelates oxyrhynchus. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder. (M3) The internal tissue was red.

63

Fig. 13(N1). A specimen of Scortum barcoo. (N2) The sonic system of Scortum barcoo.

SM= sonic muscle, SB= swim bladder. (N3) Scortum barcoo had no internal tissue.

64

(O1)

(O2)

(O3)

Fig. 13(O1). A specimen of Syncomistes butleri. (O2) The sonic system of Syncomistes butleri. SM= sonic muscle, ACSB= anterior-chamber swim bladder, PCSB= posterior-chamber swim bladder, F= fenestra. (O3) Syncomistes butleri had no internal tissue. 65

(P1)

(P1)

Fig. 13(P1). A specimen of Terapon jarbua. (P2)The sonic muscle, swim bladder and internal tissue of Terapon jabua. SM= sonic muscle, IT= internal tissue, SB= swim bladder.

66

Fig. 13(Q1). A specimen of Terapon therpy (Q2) The two-chamber swim bladder of

Terapon therpy. (Q3) Terapon therpy had red internal tissue.

67

Fig. 14. The transection of sonic muscle. The black arrow pointed out the nuclear and white arrow pointed out myofibril. (A) Bidyanus bidyanus, (B) Pelates quadrilineatus,

(C) Scortum barcoo, (D) Terapon jarbua.

Fig. 15. The transection of sonic muscle of Bidyanus bidyanus. The white arrows point out the morula-like structures. 68

Fig. 16. The transection of internal tissue. (A) A 5X internal tissue of Terapon jarbua by Gomori's trichrome stain. (B) A 40X internal tissue of Terapon jarbua by Gomori's trichrome stain. (C) A 10X internal tissue of Pelates quadrilineatus by Hematoxylin and eosin stain. (D) A 5X internal tissue of Bidyanus bidyanus by Elastica van Gieson stain.

Staining.

69

Fig. 17. The Neighbor joining tree of 16s rRNA gene of Terapondae. □ indicates the

habitat of the species, green= freshwater, yellow= estuary, blue= marine.

70

Fig. 18. The Neighbor joining tree of COⅠ gene of Terapondae. □ indicates the

habitat of the species, green= freshwater, yellow= estuary, blue= marine.

71

(A)

(B)

Fig. 19. (A) The lateral side of the vertebrate of Terapon jarbua. (B) The ventral side of the enlarged parapophysis.

72

(A)

Fig. 20. (A) The lateral side of the vertebrate of Pelates quadrilineatus. (B)(C) The ventral side of the enlarged parapophysis.

73

(A)

(B)

Fig. 21. (A) The lateral side of the vertebrate of Scortum barcoo. (B) The ventral side of the vertebrate of Scortum barcoo.

74

Fig. 22. (A) The lateral side of the vertebrate of Bidyanus bidyanus. (B) The ventral side of the vertebrate of Bidyanus bidyanus.

75

76

Table 2. Comparisons of the acoustic parameters of Terapon jarbua in Australia and

Taiwan and the result of the Mann-Whitney test.

Terapon Terapon P value jarbua(AU) jarbua(TW) Sound parameters Pulse number 20.33±1.15 24.29±4.75 0.9273 Pulse duration(ms) 3.20±0.05 4.60±0.13 < 0.0001*** Inter-pulse interval(ms) 0.84±0.05 1.58±1.3 0.0155* Pulse period(ms) 3.96±0.03 5.64±0.13 < 0.0001*** Repetition rate(pulses/sec) 250 177.32±2.88 0.0074**

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Table 3. The characteristics of sonic structures.

Sonic structures Species Sonic muscle Swim bladder Fenestra Internal-tissue Amniataba caudavittatus 2 + ++ red Amniataba percoides 2 + - Bidyanus bidyanus 2w - + pink Hephaestus fuliginosus 2# + - Hephaestus jenkinsi 2# + - Leiopotherapon macrolepis 2# + + Leioptherapon plumbeus 2 + + Leiopotherapon unicolor 2# + - Pelates octolineatus * 2 + + red Pelates quadrilineatus 3 + ++ white Pelates sexlineatus * 2 + - transparent Pelsartia humeralis 2 + ++ pink Rhyncopelates oxyrhynchus 2 + ++ red Scortum barcoo 2w - - transparent Syncomistes butleri 2# + - Terapon jarbua 2 + ++ white Terapon theraps 2 + ++ red

Note 1. # means the swim bladder is very thin, *means there is a special connective tissue between the sonic muscle and swim bladder, w means the channel between two-chamber is wide.

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Table 4. Relative size of sonic muscle

Diameter Total Head of sonic Species length length SM / TL SM / HL muscle (mm) (mm) (mm) Amniataba caudavittatus 152.33 43.78 1.38 0.01 0.03 Amniataba percoides 112.48 29.06 1.36 0.01 0.05 Bidyanus bidyanus 233.70 42.70 3.65 0.02 0.09 Hephaestus fuliginosus 163.05 44.32 1.57 0.01 0.04 Hephaestus jenkinsi 159.14 43.60 2.03 0.01 0.05 Leiopotherapon macrolepis 130.81 42.87 1.14 0.01 0.03 Leioptherapon plumbeus 72.56 19.30 0.73 0.01 0.04 Leiopotherapon unicolor 130.86 38.98 1.26 0.01 0.03 Pelates octolineatus 131.06 26.89 2.10 0.02 0.08 Pelates quadrilineatus 166.83 35.65 2.14 0.01 0.06 Pelates sexlineatus 165.83 29.96 3.51 0.02 0.12 Pelsartia humeralis 91.95 21.88 1.92 0.02 0.09 Rhyncopelates oxyrhynchus 107.90 30.25 3.55 0.03 0.12 Scortum barcoo 297.65 37.92 2.30 0.01 0.06 Syncomistes butleri 149.96 36.38 1.83 0.01 0.05 Terapon jarbua 183.85 44.10 4.88 0.03 0.11 Terapon theraps 295.25 69.03 2.34 0.01 0.03 Mean 161.48 37.45 2.22 0.01 0.06 STDEV 62.87 11.44 1.09 0.01 0.03

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Appendix

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 5 15 25 35 45 55 gi|3360447 AAACATCGCC TCTTGCTAAA CCGAAAGTAT AAGAGGTCCC GCCTGCCCTG TGACTATATG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 65 75 85 95 105 115 gi|3360447 TTTAACGGCC GCGGTATTTT GACCGTGCGA AGGTAGCGCA ATCACTTGTC TTTTAAATGG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 125 135 145 155 165 175 gi|3360447 GGACCTGTAT GAATGGCACA ACGAGGGCTT GACTGTCTCC TCTTTCAAGT CAATGAAATT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 185 195 205 215 225 235 gi|3360447 GATCTCCCCG TGCAGAAGCG GAGATAAACT CATAAGACGA GAAGACCCTA TGGAGCTTTA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 245 255 265 275 285 295 gi|3360447 GACACTAAAG CAGGCCAGTA TTAATACCCC AAATATGAGG CACGAATAAA CTGAATTTCT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 305 315 325 335 345 355 gi|3360447 GCCCTAATGT CTTAGGTTGG GGCGACCGCG GAGCAACAAA AAACCCCCGC AAGGACCGAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 365 375 385 395 405 415 gi|3360447 TGTACTACAT TCACAACCAA GAGCGACAGC TCTAATTAAC AGACACTTCT GACCAATAAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 425 435 445 455 465 475 gi|3360447 ATCCGGCAAC GCCGATCAAT GGACCAAGTT ACCCTAGGGA TAACAGCGCA ATCTCCTCTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 485 495 505 515 525 535 gi|3360447 AGAGCCCATA TCAACGAGGA GGTTTACGAC CTCGATGTTG GATCAGGACA TCCTAATGGT

....|....| ....|....| ....|....| ....|....| ....|....| 545 555 565 575 585 gi|3360447 GCAGCCGCTA TTAAGGGTTC GTTTGTTCAA CGATTAAAGT CCTACGTGAT

Appendix 1. 16S rRNA data of Epinephelus merra and the source number.

80

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 5 15 25 35 45 55 gi|1589398 AATGTAATTG TTACAGCACA TGCTTTTGTA ATAATCTTTT TTATAGTAAT ACCAATTATG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 65 75 85 95 105 115 gi|1589398 ATTGGTGGCT TTGGAAACTG ACTTATTCCA CTTATAATCG GTGCCCCAGA CATAGCATTC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 125 135 145 155 165 175 gi|1589398 CCTCGAATAA ATAATATGAG CTTCTGACTC CTTCCTCCAT CCTTCCTGCT TCTTCTTGCC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 185 195 205 215 225 235 gi|1589398 TCTTCTGGTG TAGAAGCCGG TGCTGGCACT GGCTGAACGG TCTACCCACC CCTGGCCGGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 245 255 265 275 285 295 gi|1589398 AACCTAGCCC ACGCAGGGGC ATCCGTAGAC TTAACTATTT TCTCGCTACA TTTAGCAGGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 305 315 325 335 345 355 gi|1589398 ATCTCATCTA TTCTAGGCGC AATCAACTTT ATCACAACCA TTATTAACAT AAAACCCCCT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 365 375 385 395 405 415 gi|1589398 GCTACCTCTC AATACCAAAC ACCTTTATTT GTGTGAGCAG TGTTAATTAC AGCAGTACTC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 425 435 445 455 465 475 gi|1589398 CTACTTCTTT CCCTTCCCGT CCTTGCCGCC GGCATCACAA TGTTACTCAC TGATCGTAAC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 485 495 505 515 525 535 gi|1589398 CTTAATACCA CTTTCTTCGA TCCGGCCGGA GGGGGAGACC CGATTCTTTA CCAACACTTA

....|....| ... 545 gi|1589398 TTCTGATTCT TTG

Appendix 2. The cytochrome oxidase subunit I (COI) gene sequence of Epinephelus malabaricus.

81

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 5 15 25 35 45 55 gi|4033980 GGGCGGAGCT TAGTCAACCC GGTGCTCTTT TAGGAGACGA CCAAATCTAT AACGTGATTG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 65 75 85 95 105 115 gi|4033980 TTACCGCACA CGCATTTGTA ATAATCTTTT TTATAGTAAT ACCAATCATA ATCGGAGGCT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 125 135 145 155 165 175 gi|4033980 TCGGAAATTG ACTTATTCCT CTAATAATCG GCGCCCCAGA TATAGCATTC CCTCGAATAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 185 195 205 215 225 235 gi|4033980 ACAATATAAG TTTCTGGCTT CTACCTCCTT CTTTCCTCCT CCTTCTAGCC TCATCAGGCG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 245 255 265 275 285 295 gi|4033980 TTGAAGCAGG TGCTGGGACA GGATGAACAG TTTATCCTCC TCTAGCAGGA AACCTAGCCC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 305 315 325 335 345 355 gi|4033980 ATGCAGGTGC ATCCGTAGAT TTAACAATCT TCTCACTTCA CTTAGCAGGT ATTTCATCAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 365 375 385 395 405 415 gi|4033980 TTCTTGGAGC AATCAACTTT ATTACTACCA TCATTAACAT GAAACCCCCT GCTATCTCTC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 425 435 445 455 465 475 gi|4033980 AATACCAGAC CCCTCTATTC GTATGAGCAG TCCTAATTAC TGCAGTTCTT CTTCTCCTAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| 485 495 505 515 525 535 gi|4033980 CACTACCTGT TCTGGCTGCT GGAATTACAA TACTATTAAC CGACCGAAAC CTCAACACTA

....|....| ....|....| ....|....| ....|....| ....|....| ... 545 555 565 575 585 gi|4033980 CCTTCTTTGA CCCTGCTGGA GGAGGAGACC CAATCCTCTA CCAGCACTTA TTC

Appendix 3. The cytochrome oxidase subunit I (COI) gene sequence of Plectropomus leopardus.

82