Sound Characteristics of Boesemania Microlepis (Sciaenidae)

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國立中山大學海洋生物研究所碩士論文

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

小鱗波曼石首魚聲訊特徵之研究

Sound Characteristics of Boesemania microlepis (Sciaenidae)

研究生「吳詩嘉 Shih-Chia Wu 指導教授「莫顯蕎博士 Dr. Hin-Kiu Mok

中華民國 102 年 7 月

July 2013

誌謝首先感謝口試委員張學文老師和魏瑞昌老師撥出寶貴時間給予的意見與指

導︽使得本論文內容能更加多元豐富﹀另外也感謝泰國 Kasetsart University的 Soranuth Sirisuay老師對採樣上的所有協助︽本文才得以順利完成﹀另外也謝謝國立中興大學葉文斌老師︾劉淑惠學姊以及其他 9C09實驗室的同仁在分子生物分析方面所提供的教導和幫助︽使我獲益良多﹀也謝謝廖德裕老師︽能在實驗室轉交的過渡期給予我們最大體諒與包容「最重要的︽是要謝謝指導教授莫老師一路以來的指導和帶領︽謝謝您讓我有這個機會完成這篇論文︽並參與實驗室的計畫︾事務等等︽讓我能在正式踏入社會之前︽可以有許多見習的機會〈謝謝我最親愛的爸爸媽媽︽還有老哥︽謝謝你們的支持支柱和叮嚀︽我愛你

們︽讓你們等待許久︽我終於要畢業了〈

謝謝實驗室的學姊秋錦許許多多的幫忙︽無論是計畫帳務或者實驗問題︽妳總是不吝細心解答﹀謝謝學長小翔︽你給了我很多建言︽讓我更能以不同角度去看待人事物﹀謝謝學長晏瑋許多一語中的的發言與笑話︽讓實驗室生活中多了不少歡樂﹀謝謝學長室維︽老是要麻煩你帶我們出差︽不過也因此增添許多有趣的回憶﹀謝謝七年同學三年同事的果凍︽有妳互相陪伴幫助並經歷這一切﹀謝謝阿慧給我的鼓勵和建議︽還有許多的陪伴和歡笑〈謝謝隔壁實驗室的俊男︽在我們水深火熱的日子裡︽熱心幫我們帶飯回學校︽還有雅嵐學姊︾筱芸學姊︾珮純︾

意婷︾彥臻︾家平︾宗軒︾ Tisa︾阿金︾佳綺︾姿潔︾明達︾日新︾翔泰︾昇勳︾萩燁︽謝謝每一個在我就讀研究所這段日子以來︽幫助︾陪伴︾包容我的每一個人︽也感謝每一個我遇到︾認識的人︽無論熟識與否都或多或少啟發︾改變了我︽感謝所遇到的每一件事︽讓我能夠學習並成長〈最後要謝謝秀彥一直以來的陪伴與照顧︽還有所有的支持︽謝謝海生所讓我們相遇︽謝謝這片海︽還有美麗的夕陽︽讓我的研究所生涯能夠畫下完美句點〈

i 小鱗波曼石首魚 (Boesemania microlepis) 聲訊特徵之研究

研究生「吳詩嘉指導教授「莫顯蕎博士國立中山大學海洋生物研究所

石首魚在繁殖季節時會藉由發音肌震動泳鰾而發出聲音︽此科大部分為海水種︽只有少數為淡水種〈海水種與淡水種石首魚繁殖季節時的聲音特徵顯少被討論︽此文以目前記錄中西印度太平洋唯一一種淡水石首魚︽並出沒在泰國淡水流

域的小鱗波曼石首魚 (Boesemania microlepis)來分析其聲音特徵及相關資料︽並和海水種作比較〈然而根據泰國當地人指出︽除了小鱗波曼石首魚外︽還有一種和小鱗波曼石首魚同域︽但體表明顯較為金黃色的另一種石首魚︽故本文討論次種金黃色和小鱗波曼石首魚的基因變異度〈此外︽小鱗波曼石首魚發音肌的二形性也會被檢驗〈養殖場中的小鱗波曼石首魚於生殖季節的聲音活動週期亦連續三晚被記錄︽野外河流中的錄音亦被監聽並分析〈於養殖場的錄音顯示發聲活動三晚皆有日的週期性︽並於太陽西下後的一至兩小時達到高峰〈小鱗波曼石首魚的聲

音長度有 125.7毫秒︽聲音所含脈衝數量約 15個︽主頻率為 1610.6 赫茲︽較其他海水種高〈根據演化分類樹推測︽小鱗波曼石首魚和金黃石手魚為不同種︽其基

因變異度接近 10%〈解剖結果顯示小鱗波曼石首魚的發音肌有二形性︽只有雄魚具有發音肌﹀聲音相關的特性被認為是對淡水環境的適應〈

關鍵字「石首魚科︾聲訊︾小鱗波曼石首魚︾泳鰾︾發音肌

ii Sound Characteristics of Boesemania microlepis (Sciaenidae)

Shih-Chia Wu

Advisor: Dr. Hin-Kiu Mok

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

The teleost family Sciaenidae is well known for vocalization during the spawning season. Most of them live in marine and esturies, but some are restricted to freshwater environment. The difference of sound between marine and freshwater species has seldom been discussed. In this study, Boesmania microlepis, the only freshwater species occurs in Indo-west Pacific was chosen to analyze its sound characteristics, diel vocal activity and to the former compare with that of marine species. According to local people in Thailand, there is a sympatric with B. microlepis sciaenid that seemed to be different species from B. microlepis with golden hue on its body. The genetic difference between the two species was deciphered. The sounds of captive B. microlepis vocalization in cement tanks were recorded for three evenings. The sounds in the field were also recorded and analyzed. The sonic muscle and swimbladder structure of the two “species” were examined. The recordings showed a clear diel activity with a peak after the sunset. The average length of sound in B. microlepis was 125.7ms and comprised a train of pulses with a mean of 15 pulses. The dominant frequency is 1610.6

Hz, which is higher than the big-snout croaker and other marine species. The phylogenetic tree inferred from COI gene showed B. microlepis and the golden sciaenid are different species with nearly 10% of genetic variation. The dissection showed sexual dimorphism of the sonic muscle of B. microlepis with only the male possessing it. Some soniferous features were considered adaptive to freshwater environment.

Keywords: Sciaenidae, sound, Boesemania microlepis, swimbladder, sonic muscle

iii Content

Introduction ...………………………………………………………………………..… 1

Material and methods ...…………………………………………………………………6

2.1 Study species ...………………………………………………………………..6

2.2 Recording of sounds ...……………………………………………………...…6

2.3 Sound analysis ...………………………………………………………………7

2.4 Sonic muscle observation ...…………………………………………………...7

2.5 Swimbladder observation of the golden sciaenid ..…………………………...9

2.6 Molecular analysis ...………………………………………………………….9

2.7 PCR condition………………………………………………………………..10

2.8 Taxonomic sampling and sequence analysis ...………………………………11

Results ...……………………………………………………………………………….12

3.1 Sound characteristics ...………………………………………………………12

3.1.1 Sound characteristics of Boesmania micorlepis in Chainat

Propagation ………………………………………………………….12

3.1.2 Temporal change in vocal activity…………………...………………13

3.1.3 Sound characteristics from the field ...………………………………13

3.1.4 Comparison between sounds of B. microlepis and sounds from the

field ..………………………………………...………...……………14

3.2 Sonic muscle ...………………………………………………………………14

3.2.1 Sonic muscle dimorphism …...………………………………………14

3.2.2 Histological characteristics of the sonic muscle ...…………………..15

3.3 Swimbladder examination ...…………………………………………………15

3.4 Genetic difference of B. microlepis and the golden sciaenid ...……………...15

iv Discussion …………………………………………………………………………...…16

4.1 Sound characteristics ………………………………………………………...16

4.1.1 Differences in sound characteristics ……………………………...…16

4.1.2 Diel vocal activity ……………………………………………...…...18

4.1.3 Sounds from the field recording …………………………………….18

4.2 The sonic muscle observation ……………………………………………….19

4.2.1 Sonic muscle sexual dimorphism …………………………………...19

4.2.2 Sonic muscle cross section …………………………….……………20

4.3 The swim bladder observation of the golden sciaenid ………………………20

4.4 Taxonomic status and vocalization of the golden sciaenid ………………….20

References ……………………………………………………………………………..22

v Figures Legend

Fig. 1. A 38.2 cm standard length Boeseman croaker, Boesemania microlepis …...….28

Fig. 2. A 47cm standard length golden sciaenid ...…..………………………………....28

Fig. 3. A simplified illustration of the recording setup at the propagation …...…….….29

Fig. 4. A call from Boesemania microlepis with illustrations showing how the acoustic

parameters were analyzed …………………………………………...…….29

Fig. 5. An example of call from Boesemania microlepis voluntary vocalization recorded

using HP-A1 hydrophone .… …………………………...…………...……30

Fig. 6. Examples of calls with the first few weaker amplitude pulses ………………...31

Fig. 7. Changes in number of pulses with lower intensity on March 12th 2011 …....….32

Fig. 8. Linear regression of call duration and number of pulses per call ………...……32

Fig. 9. A period of background noise with no croaking contained ……………………33

Fig. 10. Temporal changes in percentage occurrence of pulse numbers in the evening of

March 12th 2011 …………...………………………………………………34

Fig. 11. Temporal change in numbers of voluntarily produced vocalization in three

consecutive evenings from 11 March to 13 March 2011…...……….…….34

Fig. 12. An example of a single call from field recording at Bang Pakong River on

November 4th 2012 …………………………..….…………………….…..35

Fig. 13. A period of background noise with no croaking contained from field recording

at Bang Pakong River on November 4th 2012 …………………………….36

Fig. 14. Ventral view of a female Boesemania microlepis …………..…………….…..37

Fig. 15. Ventral view of a male Boesemania microlepis ……...……………………….38

Fig. 16. Ventral view of a male golden sciaenid ………………..……………………..39

Fig. 17. The cross section of the sonic muscle of Boesemania microlepis ………..…..40

vi Fig. 18. The cross section of the sonic muscle of Johnius macrorhynus ……………...40

Fig. 19. The comparison between the myofibril areas of Boesemania microlepis …….40

Fig. 20. The ventral view of the swimbladder of Boesemania microlepis ……...…..…41

Fig. 21. The later view from the left of the swimbladder of the golden sciaenid …..….41

Fig. 22. A pair of appendages on each side of the chambers of swimbladder of the

golden sciaenid ………………………………………………………...….42

Fig. 23. Neighbor joining tree of COI gene sequences using Maximum Composite

Likelihood model ………………………….…………………………..…..42

vii Table Legend

Table 1. The Comparisons of the acoustic parameters in Sounds from the filed at Bang

Pakong River and Sounds of Boesemania microlepis from propagation in

Chainat Province and results of the Mann-Whitney test ...... …...……..27

viii Introduction

The family Sciaenidae contains 66 genera, 283 species (Froese and Pauly 2012).

They are very important fisheries species in some local areas throughout the world.

Sciaenids are well known for their vocalizations, they are sometimes called croakers or drumfish. Among the vocalizations of Sciaenids, the most noted vocal activity is related to reproduction. Sciaenids emit drumming or croaking sounds during their reproductive season. Although most of the fishes in Sciaenidae are marine, some species enter estuaries and nearly 28 species are even confined to freshwater (Nelson 2006). The following examples are freshwater species: Aplodinotus grunniens (Barney 1926), the smallscale croaker Boesemania microlepis (Baird et al. 2001), and Plagioscion squamosissimus (Feldberg 1999). Studies based on morphological features indicate that the primitive sciaenid species are marine ones (Sasaki 1989).

Generally, fish produce sounds for reproductive, agonistic or defensive purposes

(Fine 1997; Lin et al. 2007). Sciaenids produce sounds by several mechanisms. One is to emit sound by resonating of the swim bladder, caused by contraction of the sonic muscle (Nelson 2006). The sound is often called advertisement calls and described as drumming, grunting, purring, and croaking (Tower 1908; Connaughton and Taylor 1995;

Lin 2006). There are two types of sonic muscle based on the origin and insertions. If the sonic muscle is adhered completely to the wall of swim bladder, this is the intrinsic sonic muscle. When the sonic muscle is inserted to the wall of the swim bladder and originate on the cranial bone, pectoral girdle or ribs, it is extrinsic sonic muscle (Tower

1908; Hill et al. 1987; Ramcharitar 2006). The other type is also associated with the vibration of sonic muscle. However in this mechanism, it is the peritoneum being vibrated (Takemura et al. 1978). There is another mechanism that does not evolve the

1 muscles and it is emitted by stridulation of pharyngeal teeth and called ‘chattering’

(Connaughton and Taylor 1995). There are two well-known causes of sound production in Sciaenidae: reproductive and disturbance calls (Ramcharitar 2006). Studies show that reproductive calls occur mostly during the spawning season and often reach a peak around sunset or during the nocturnal time (Connaughton and Taylor 1995; Ueng et al.

2007).

The existence of sonic muscle has long been believed to serve for sound production

(Tower 1908). Most sciaenid species (Leiostomus xanthurus, Cynoscion regalis,

Pogonias cromis, Johnius macrohrynus, Pennhia pawak, Otolithes ruber, Chrysochir aureus, and Atrobucca nibe are all examples; Tower 1908; Hill et al. 1987; Lin 2006;

Ueng et al. 2007) are sexual dimorphism in the presence of sonic muscles with only the male possessing the tissue (Sasaki 1989). However, there are some exceptions in which both male and female possess sonic muscle, regardless of the sexual difference in the muscle size. The reported examples are Micropogonias undulates, Micropogonias furnieri, Argyrosomus japonicus, Larimichthys crocea, and Pogonias cromis (Hill et al.

1987; Griffiths and Hecht 1995; Lo 2011; Tellechea et al. 2011).

Boesemania microlepis is one of the few freshwater sciaenid species, especially with most of the freshwater sciaenids live in South America (Feldberg 1999; Boeger and Kritsky 2002). It distributes in Lao, Cambodia, Vietnam, and Thailand (Wongratana

1985; Baird 1999; Baird 2001), and can be found in Mekong, Tachin, Chao Phraya,

Meklong and Ban Pakong rivers and their linking tributaries and Thailand. It is a highly priced food fish among local residents and can reach to a maximum size of 6 kg

(Wongratana 1985; Baird 2001). The species is mostly found in deep-water pools and is not considered to be migratory (Poulsen and Valbo-Jorgensen 2000; Cesvi 2001; Baird

2001). There are several reports indicated that the populations of B. microlepis have

2 declined (Roberts 1993; Baird 2001; Cesvi 2001). Fortunately, measures were taken to conserve the fish. Fish Conservation Zones (FCZ) have been established in some area to protected the threatened fishes, and recents reports showed B. microlepis benefit from these conservation zones (Poulsen 2000; Baird 2001; Cesvi 2001; Poulsen 2002). Local villagers reported an increase in the occruence of B. microlepis vocalizing since the

FCZ has been established (Baird 2001). There was a propagation station set for rebuilding the population of B. microlepis in Chainat Province, Thailand. In a document that aimed to clarify the taxonomic name of B. microlepis, Wongratana stated that the farmers and fishermen living close to Bang Pakong and Tachin rivers call B. microlepis by the name “Pla ma”. However, there was another fish called “Pla hang kew” that is claimed by local people to be different from “Pla ma”. But Wongratana considered them to be the same species. The farmers and fishermen stated “Pla hang kew” shows up in saline influent area of the rivers and the body has yellowish hues.

According to the lecturer of Faculty of Fisheries in Kasetsart University in Thailand,

Dr. Soranuth Sirisuay, local fishers catch some sciaenids that look like B. microlepis but the color is more golden (pers. comm.). Sirisuay mentioned that B. microlepis appeared in Chao Phraya River, which flows through Bangkok and in another river where B. microlepis and the golden sciaenid have been in it.

One goal of this study is to obtain the voluntary vocalization in the Chao Phraya

River. The tissue of the two similar fishes would be tested for degree of genetic similarity and phylogenetic relation to see if they are the same species. Hebert et al.

(2003) suggested the mitochondrial DNA gene cytochrome oxidase subunit I (COI) is adequate to solve problem in species identification. Furthermore, in 2005, Ward et al. presented that COI sequencing can be used at species-level for fish bioidentification.

The sounds of B. microlepis were first documented by Baird et al. in 2001. The

3 authors briefly described the sounds which they recorded in late March and ends April

1998; the sounds frequency ranged up to 6 kHz, the main frequency (where energy is located) was 0.5 kHz, and the sounds lasted about 100 ms. However, they did not describe other characteristics, for example: number of pulse per call, pulse period, pulse length, inter-pulse interval, and pulse repetition rate. Besides, the vocal activity in diurnal period was not investigated. The fish produced sounds in the mid-to late afternoon. The rearer in B. microlepis propagation station in Chainat stated that the fish would start to produce sounds in the late afternoon and continued to vocalize even after sunset. The spawning season of B. microlepis inhabiting in Siphandone Wet lands,

Thailand and was reported to be in the height of dry season, which is around March and early May (Baird 2001; Cesvi 2001). In Cambodia, the species spawns in May or June in Cambodia (Bardach 1959). Bair et al. (2001) used the occurrence of B. microlepis sounds to identify the spawning ground in Siphandone Wetlands in Cambodia. Local villagers trace the population of B. microlepis. An euryhaline species Irrawaddy dolphins (Orcaella brevirostris) was reported to be able to use the sound of B. microlepis to detect the location of the fish during its predation (Cesvi 2001). The above examples show the need of more understanding of the sound characteristics of B. microlepis.

The differences between freshwater and marine sciaenids have not been examined and investigated in sound characteristics and other related features such as the structure of sonic muscle or swim bladder. The big-snout croaker (Johnius macrorhynus) is a marine sciaenid species that also vocalizes voluntarily. Since Sasaki (1989) suggested that marine sciaenids are more primitive species, and it is still unknown that whether B. microlepis has such dimorphism in the presence of sonic muscle, it may provide some information about how freshwater sciaenids specialize after entering a freshwater

4 environment by looking into the sonic muscle structure. The sounds of freshwater croaker are rarely examined in the differences with marine species. Since the freshwater environment is so different from the marine one, it is important to investigate the sound characteristics of B. microlepis and related information.

The aim of this study were 1) to describe the vocal activity of B. microlepis from the propagation in the evening, 2) to describe the characteristics of voluntarily-produced sound, 3) to obtain voluntary vocalization in the field and compare the sounds from the ones from the propagation, 4) to investigate if dimorphism of the sonic muscles is present in B. microlepis, 5) to investigate the genetic differences between B. microlepis and golden sciaenid partly sympatric with it.

5 Material and methods

2.1 Study species

The Boeseman croakers, Boesemania microlepis (Fig. 1), for acoustic recording were provided by a propagation station in Chainat Province, Thailand. The fish were raised in cement tank (3 m x 2 m x 0.8 m) filled with freshwater. Fish for histological and molecular analyses were purchased from local fishmongers July 9th in 2011 and

2013 repectively. The golden sciaenids were provided by Dr. Soranuth Sirisuay (Fig. 2).

2.2 Recording of sounds

Sound recordings were made at the propagation station on March 11th to 13th in

2011. Three cement tanks were adjacent to one another. There were 12, 12, and 8 fish in each tank. The hydrophone was placed in the center of the middle tank about 45cm under the water surface (Fig. 3). Monitoring the occurrence of the sounds started at

1600 hours and the recordings began at the very first calling of Boesemania microlepis every evening before or after sunset in these three days, and ended at the termination of the calling (recognized by a 15-min continuous silence after the last call). The recordings were digitized at 44.1kHz with 16-bit resolution. The recording equipments included a HP-A1 hydrophone (Burns Electronics; frequency ranged from 10 to 25,000

Hz ± 3 dB) and a HP-A1 Mixer-amplifier connected to a Korg digital recorder (MR-

1000).

On November 2nd 2012, a field recording was taken at Chao Phraya River. The hydrophone was set under water and recorded from 17:00 to 19:00. On November 3rd and 4th, other recordings were taken under a bridge beside the riverbank at the mainstream of Bang Pakong River for 15 minutes. The recording files were monitored

6 acoustically and sonogram were generated by the program described below to see if there were suspected sounds produced by fish. The recording equipments were a H2A hydrophone (Aquarian Audio Products; frequency ranged from 1 to 100000Hz ± 4 dB) and Sony digital recorder (PCM-M10).

2.3 Sound analysis

Sounds from the evening of March 12, 2011 were chosen to analyze the sound characteristics. Ten calls were chosen from a five-minute section for every ten minutes

(e.g. 10 calls from 00:00~05:00 were sampled, none was sampled from 05:00~15:00).

Acoustic data were analyzed by using the interactive sound analysis software Raven Pro

1.4(developed by Bioacoustics Research Program, The Cornell Lab of Ornithology,

Ithaca, NY, USA). Temporal call frequency features of the sounds include call duration

(ms), number of pulses per call, pulse repetition rate, inter-pulse interval (IPI, measured from the end of one pulse to the beginning of the next, ms), pulse duration (measured as the time length from the beginning of one pulse to the end, ms), high frequency (the highest frequency a single call can reach), and dominant (or maximum) frequency (the most intense frequency), were measured manually from the sonogram and oscillogram

(Fig. 4).

2.4 Sonic muscle observation

To investigate the dimorphism in presence of sonic muscle, 20 fish (average standard length 40.3 cm) were purchased from local fishmongers at Sam Chuk Old

Market in Suphan Buri Province, Sam Chuk District on July 9th 2012 and the standard lengths were registered. The fish were then dissected from the abdominal cavity and the

7 sexes of fish were determined by the external appearance of the gonades. Condition of the sonic muscle in the abdominal wall was observed for comparative purpose.

The marine big-snout croaker, Johnius macrorhynus, specimens were caught by hooded line from coastal waters of Taixi Township, Yunlin County, Taiwan on July 6th

2011. The fish was stored at -20ºC until the sonic muscle tissue was carefully removed with a scalpel for section preparation.

The tissues from the gonad epiaxial muscle, and sonic muscle tissue were soaked in

10% formalin for 1 to 7 days after the fish were dissected. The tissue were transferred to

Bouin’s solution for the coming biopsy progress. H&E stain (hematoxylin and eosin stain) method was applied during the biopsy progress. Several steps were taken in the

H&E stain:

1) Dehydrate the samples by soaking in ethanol 20 minutes each through the

following gradient: 50%, 75%, 85%, 95%, and twice in 100%.

2) Immerse the samples in xylene for an hour twice.

3) Soak the samples in heated wax for 2~3 hours, which the proportion of soft and

hard wax is 4 to 6.

4) Place each sample in the mold filled with heated wax and chill to solidified.

5) Cut the samples into 4 to 6 µm and attach to microscope slides.

6) Bath the sections in xylene and 100% ethanol each twice for 5 minutes to remove

the wax and prep the samples for better adherence to the staining dye.

7) Staining in hematoxylin and eosin for 10 minutes and 5 seconds each. Wash the

excess dye under running tap water after each staining until colors no longer

showed in water.

8) Move the slides gradually 50% ethanol through 75%, 85%, 95%, and 100%

ethanol to xylene by infiltration, 100% ethanol and xlyene for twice.

8 9) Seal the slides with toluene base and cover glasses.

The sections of sonic muscle were analyzed using the ImageJ image processing program 1.47 version (developed by Wayne Rasband at National Institute of Health,

USA. http:/imagej.nih.gov/ij). Areas of myofibril were derived from ROI manager tools from the program. The comparison between myofibril areas of B. microlepis and J. macrorhynus was using Mann-Whitney test with a 95% confidence interval.

2.5 Swimbladder observation of the golden sciaenid

A formalin-fixed individual of golden sciaenid was sent from Thailand by Dr.

Soranuth Sirisuay. Body length was measured and pictures were taken. It was then dissected to be examined the sex, the sonic muscle, and the swim bladder form.

2.6 Molecular analysis

In order to find out the whether the Boesemania microlepis and the speculatedily different species - the golden sciaenid is the same species or not, muscle tissue were sampled to extract the DNA by the following purification steps with Promega Wizard®

Genomic DNA Purification Kit:

1) Thaw the tissue and add gradually add 600µl of Nuclei Lysis Solution, then

homogenize and chill on ice.

2) Incubate the lysate at 65 °C for 25 minutes.

3) Centrifuge for 3 minutes at 16,000×g at 25 °C. Carefully move the supernatant

to another clean microcentrifuge tube.

4) Add 17.5µl of 20mg/ml Proteinase K. Then centrifuge for 1 minute.

5) Incubate overnight at 55 °C.

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

9 7) Incubate the mixture for 20 minutes at 37 °C.

8) Add 200µl of Protein Precipitation Solution and vortex vigorously at high speed

for 20 seconds. Chill sample on ice for 5 minutes.

9) Centrifuge for 4 minutes at 16,000×g at 4 °C.

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

1.5 ml microcentrifuge tube containing 600µl of room temperature isopropanol.

11) Centrifuge for 1 minute at 16,000×g at room temperature and then carefully

pour out the liquid.

12) Add 600µl of room temperature 70% ethanol to wash the DNA. Centrifuge for

1 minute at 16,000×g at room temperature.

13) Invert the tube on clean absorbent paper, and air dry the pellet for 10 minutes.

14) Add 100µl of DNA Rehydration Solution, and rehydrate the DNA by

incubating at 65 °C for 1 hour.

2.7 PCR condition

Polymerase chain reaction (PCR) was used to amplify the fragments of COI gene in the DNA. The region of COI is approximately 655 base pair (Ward et al. 2005). The total volumes of PCR reaction mixtures were 25µl, containing 9.3µl sterile distilled

H2O, 5µl of DNA template, 6.7µl of 5X PCR Master Mix II (0.75u Taq DNA

Polymerase reaction buffer, 2mM MgCl2, 250µM dNTP, and enzyme stabilizer contained), and 10mM primer 2µl forward and reverse each. The primers are FishF1

(5’-TCAACCAACCACAAAGACATTGGCAC-3’), FishF2 (5’-TCGACTAATCATA

AAGATATCGGCAC-3’), FishR1 (5’-TAGACTTCTGGGTGGCCAAAGAATCA-3’), and FishR2 (5’-ACTTCAGGGTGACCGAAGAATCAGAA-3’). Biosystems 2720

Thermal Cycler was used to perform amplification procedures. The thermal control

10 consisted of an pre-heating step of 1 minute at 94 °C, followed by 40 cycles of denaturation at 94 °C for 50 seconds; annealing at 51 °C for 90 seconds, extension 68

°C for 90 seconds. The final step is at 68 °C for 10 minutes. Then stored at 4 °C. The

PCR products were confirmed by electrophoresis (100V for 25 minutes) with 1% agarose gel.

2.8 Taxonomic sampling and sequence analysis

The COI sequences were aligned using BioEdit Sequence Alignment Editor version

7.0.9.0 programs. The phylogenetic tree and the distances were generated using MEGA version 4 (Tamura et al. 2007) to identify whether the “golden” coaker is the same species as B. microlepis. The sequences of the sister-group species, Chrysochir aureus,

Collichthys niveatus, Nibea albiflora, Pennahia argentata, and Sciaenops ocellatus were downloaded from GenBank at NCBI (http://www.ncbi.nlm.nih.gov/genbank/).

The sequence of Panna microdon was provided by Pei-Chun Lo.

11 Results

3.1 Sound Characteristics

3.1.1 Sound characteristics of Boesmania microlepis in Chainat Propagation

A total of 123 calls were sampled to analyze the sound characteristics (Fig. 5). The sounds were composed of series of pulses ranged from 5 to 28 pulses (mean ± SD =

15.0 ± 5.7 pulses; Table 1). The call duration of Boesemania microlepis ranged from 37 to 228.8 ms (mean ± SD = 125.7 ± 48.8 ms). Hence the pulse repetition rate ranged from 91.7 to 167.6 pulses per second (mean ± SD = 120.2 ± 8.6 number of pulses/ second). The pulse length ranged from 3.4 to 9.4 ms (mean ± SD = 5.4 ± 1.3 ms), the pulse period ranged from 6.3 to 20.1 ms (mean ± SD = 9.0 ± 1.7 ms), and the inter-pulse interval ranged from 1.1 to 5.6 ms (mean ± SD = 3.3 ± 1.1 ms). The dominant frequency of the calls ranged from 516.8 to 1894.9 Hz (mean ± SD = 1610.6 ± 238.6

Hz). The high frequency ranged from 3583.2 to 22050 Hz (mean ± SD = 14103 ±

6586.2 Hz).

In 109 out of the total 123 calls, the amplitude of the first few pulses (1 to 5) were lower, which was visible from both oscillogram and spectrogram (Fig. 6). The percentage column chart showed that the number of pulses with weaker amplitude is mainly 2 pulses (Fig. 7). A linear relation was observed between pulse number and call duration; the call with longer call duration contained more number of pulses per call.

The regression coefficient between call duration and number of pulses per call was

114.9 (Fig. 8), suggesting a linear regression was observed (with R2 = 0.9737, p <

0.0001). The spectrogram and oscillogram of the ambient-noise in the propagation were generated (Fig. 9). The percentage occurrence of calls with numerous pulses (more

12 than 17 pulses) gradually increased as vocal activity progressed in the evening (Fig. 10; also see below).

3.1.2 Temporal change in vocal activity

Numbers of calls voluntarily produced by B. microlepis in the evening at the propagation were monitored form 11 March to 13 March 2011 (Fig. 11). The vocal activity in the first evening started from around 1830 hours and ended around 2105 hours and peaked at around 2000 hours (1447 calls from five minutes). In the second evening, the activity lasted from around 18:15 to 21:05 and peaked at around 19:45

(1242 calls from five minutes). The third evening the activity started around 18:30 and ended around 21:05, peaked at around 20:00 (1072 calls from five minutes). Temporal change in all three evenings showed a trend which at first only few croaking appeared, then gradually increased the number of croaking and peaked at the middle of the whole vocalizing activity, with a gradual decrease in amount of croaking at the end of the vocal activity.

3.1.3 Sound Characteristics from the field

Among the four recordings taken from November 2nd to November 4th, only the one that was recorded under the bridge at Bang Pakong River had sounds that were similar to the sounds of B. microlepis previously recorded from the propagation. A total of 7 calls from the field were analyzed (Fig. 12). The call duration ranged from 93.7 to 116.5 ms (mean ± SD = 103.4 ± 8.1 ms). The sounds were composed of series of pulses ranged from 12 to 15 pulses (mean ± SD = 13.3 ± 1.0 pulses). With the pulse repetition rate ranged from 118.3 to 157.6 pulses per second (mean ± SD = 129.2 ± 14.2 number of pulses/ second). The pulse length ranged from 4.6 to 6.2 ms (mean ± SD = 5.6 ± 0.6

13 ms), the pulse period ranged from 6.5 to 15.2 ms (mean ± SD = 9.0 ± 2.8 ms), and the inter-pulse interval ranged from 2.0 to 2.9 ms (mean ± SD = 2.3 ± 0.3 ms). The dominant frequency of the calls ranged from 172.3 to 861.3 Hz (mean ± SD = 566.0 ±

293.6 Hz). The high frequency ranged from 1715 to 2352 Hz (mean ± SD = 2051.9 ±

211.0 Hz). The max power of the calls ranged from 77.8 to 88.3 dB (mean ± SD = 83.4

± 4.0 dB). The spectrogram and oscillogram of the ambient-noise from the field were generated (Fig. 13).

3.1.4 Comparison between sounds of B. microlepis and sounds from the field

There were no significant difference in the means of call duration, pulse number per call, and pulse length (Mann-Whitney test; p < 0.05; Table 1). However, the dominant frequency, pulse repetition rate, inter-pulse interval, and pulse period showed significant differences between these two kinds of sound sources.

3.2 Sonic muscle

3.2.1 Sonic muscle dimorphism

The 20 fish dissected were composed of 11 females and 9 males. All 11 females lacked sonic muscle (Fig. 14). On the other hand, all 9 males had a pair of sonic muscles on the body wall of abdominal cavity, which proved that of B. microlepis is sexually dimorphic in sonic muscle and the muscle belongs to extrinsic type (Fig. 15).

One golden sciaenid individual was dissected and a pair of sperm gonad was found.

There was also a pair of sonic muscle on the body wall next to the swim bladder in this male individual (Fig. 16).

14 3.2.2 Histological characteristics of the sonic muscle

29 cells from Boesemania microlepis and 12 cells from Johnius macrorhynus were chosen to generate the data of the cross-section myofibril areas of the sonic muscle fibers (Fig. 17 and 18). The average myofibril area of B. microlepis and J. macrorhynus were 179.023 ± 80.197 µm2 and 263.097 ± 46.08µm2 respectively. The test of Mann-

Whitney showed a significant difference between the two species, the average myofibril area of J. macrorhynus was higher than B. microlepis (Fig. 19).

3.3 Swimbladder examination

The swim bladder of the Boesemania microleis had appendages extended along the swim bladder backward (Fig. 20). The swim bladder of the golden sciaenid was carrot- shaped with two chambers on the front portion connecting by a slenderer part of the swim bladder (Fig. 21). There was a pair of appendages on each side of the chamber part. Moreover, there was another one smaller appendage attached on the left side chamber, leaving one side of the chamber had two appendages but the other side only had one appendage (Fig. 22).

3.4 Genetic difference of B. microlepis and the golden sciaenid

The taxonomic tree of the chosen species generated by MEGA 4 using Neighbor

Joining method with Maximum composite likelihood showed the samples of B. microlepis and the golden sciaenid are different at a species-level displaying a nearly

10% of genetic variation, with 96% of the bootstrap replications presenting in the same tree (Fig. 23).

15 Discussion

4.1 Sound characteristics

4.1.1 Differences in sound characteristics

Boesemania microlepis is currently the only species that has been described as freshwater sciaenids in Indo-west Pacific area (Froese and Pauly 2012) and is considered to be in a more advanced group (Sasaki 1989), this study had purposed to find differences in the sound characteristics between this freshwater specis and marine species. In the marine big-snout croaker (Johnius macrorhynus), Japanese croaker

(Argyrosomus japonicus), blackspotted croaker (Protonibea diacanthus), black drum

(Pogonias cromis), whitemouth croaker (Micropogonias furnieri), and large yellow croaker (Larimichthys crocea), the dominant frequencies were 1052 ± 84Hz, 686 ±

203Hz (for male) and 587 ± 190 Hz (for female), around 110Hz, 128 ± 5 Hz, 280 and

316 Hz, and 1292.6 ± 478Hz respectively (Lin et al. 2006; Ueng et al. 2007; Mok et al.

2009; Tellechea et al. 2010, 2011; Lo 2011). In B. microlepis, the dominant frequency had a relatively high value reaching 1610 ± 239Hz. Ladich (2004) had mentioned that in some soniferous species, the low frequency of their sounds is an adaptation to the restricted auditory sensitivity. According to Baird et al. (2001), even though juvenile

Boeseman croaker had been encountered to congregate along the edges of sand bank in the Mekong River, large individuals seemed to prefer deep part of pools. This could be a phenomenon that is evolved to adapt the freshwater environment. The relatively high dominant frequency might be an adaptation that makes the localization of the sound source more easily because of the shorter wave length compared to which of lower frequency, therefore the hearing sensor organ could tell the sound source by the different timing of the sound waves reaching the sensors. It is worth mentioning the

16 spectrogram and the power spectrum showed that most energy of the calls did not distinctively concentrate at the peak frequency but spread over a broad frequency band.

By looking into the band width of frequency, defined by the frequency range -10 dB from the peak, may provide more information of the energy distribution of the call.

The call duration of B. microlepis is relatively low with an average of 125.7 ± 48.8 ms than previously mentioned marine sciaenids with averages of 153.8, 256.9, 184, 231,

316, and 520 ms (Lin et al. 2006; Ueng et al. 2007; Mok et al. 2009; Tellechea et al.

2010, 2011; Lo 2011). The number of pulses per call for B. microlepis in this study was

15 ± 5.7 pulses. Japanese croaker had 10.5 ± 3 pulses for male and 15 ± 3.2 pulses for female (Ueng et al. 2007), not all of which have much difference from B. microlepis.

Big-snout croaker had 22 ± 3.9 pulses (Lin et al. 2006), which is more than B. microlepis. Other marine species have fewer number of pulses per call than B. microlepis (P. diacanthus has 9 ± 0.1 pulses, L. crocea has ranged 1 to 7 pulses; Mok et al. 2009; Lo 2011). These freshwater croakers were also differed from marine species in pulse repetition rate of the calls, with Boeseman croakers emitting at a frequency of

120.2 ± 8.6 pulses per second, while other marine species only achieving about 17 to 85 pulses per second (Lin et al. 2006; Ueng et al. 2007; Mok et al. 2009; Tellechea et al.

2010, 2011; Lo 2011). By making sounds with more number of pulses in one call to increase the communication more efficient, this could be another adaptation of freshwater croaker.

One of the interesting features of sounds of B. microlepis is that nearly 90% of the calls have the first few of pulses that have weaker amplitude. This kind of special feature can also be found in other marine species. In the large yellow croaker, the first pulse length was longer than the last, but the first inter-pulse interval was shorter than the last (Lo 2011). In the big-snout croaker, two types of sounds have been found. One

17 type is the first inter-pulse interval is longer than the rest of the inter-pulse interval (Lin et al. 2006). The other type is dual-knocks with only two pulses in one call. The black drum also has a particular call structure, presenting a feature with a series with three trains of pulses (Tellechea et al. 2011).

4.1.2 Diel vocal activity

The recording showed a clear of diel vocal activity, with a start at the dusk and an end a few hours after nightfall (this study; Fig. 11). The longest vocalization in one evening lasted about 170 minutes with a total of 8581 calls. The vocal activities of

Japanese croaker and weakfish (Cynoscion regalis) corresponded to B. microlepis and peaked at early evening around dusk (Connoughton and Taylor 1995; Ueng et al. 2007).

In whitemouth croaker, the daily vocal activity has two peaks from 0700 to 1000 hours and from 1700 to 2300. Leaving aside the fact that whitemouth croaker has a peak in the daytime, the peak showing at the dusk is similar to B. microlepis. The temporal change in large yellow croaker which Lo (2011) had recorded showing a termination of sound production after gametes were released. In big-snout croaker, the vocal activity which

Lin et al. recorded (2007) did not peak around dusk but was limited between 2145 to

0315 hours.

4.1.3 Sounds from the field recording

The oscillogram and spectrogram of calls recorded from the field were dim and vague. The pulses were not as clear as those recorded in the cement pools. Still, these audible calls sound like those recorded in the pool. There was a sound that differed from the other calls resembled to B. microlepis. According to Dr. Soranuth Sirisuay, besides sciaenids (i.e. B. microlepis and the golden sciaenid), there are two soniferous catfish

18 species in the recording area. One belongs to Family Bagridae and the other belongs to

Arridae. Both species are common in the Bang Pakong River (pers. comm.). Since this particular sound was clearer than other sounds and it contained 30 pulses and the dominant frequency was higher than B. microlepis, the possibility of its being a sound of sciaenids was excluded. In this study, the clearer and high-tonal sound is made by catfish.

The comparison between the sound of captive B. microlepis and the major call type in the river shows the call duration and pulse number per call were not significantly different, but dominant frequency, pulse repetition rate, inter-pulse interval, and pulse period were significantly different. Although the two parameters that generated the pulse repetition rate are both non-significant, the pulse repetition rate shows a significant difference between the two sources. This could be caused by the high standard deviation.

4.2 The sonic muscle observation

4.2.1 Sonic muscle sexual dimorphism

In this study, the presence of the sonic muscle was examined. Only the male

Boeseman croakers have sonic muscles. The spot croaker (Leiostomus xanthurus), the weakfish (Cynoscion regalis), the black drum (Pogonias cromis), the big-snout croaker

(Johnius macrorhynus), the Pawak croaker (Pennahia pawak), the tigertooth croaker

(Otolithes ruber), the Reeve’s croaker (Chrysochir aureus), and the blackmouth croaker

(Atrobucca nibe) have the same sexual dimorphism. A male golden sciaenid specimen possesses sonic muscle, but no female individual has been dissected. Therefore, it is still not known whether sonic muscle dimorphism exists or not in this species.

19 4.2.2 Sonic muscle cross section

The cross sectional myofibril area of B.microlepis is smaller than Johnius macrorhynus. Fine et al. (1990) had found out that the male toadfish (Opsanus tau) had smaller sonic muscle fiber than the female. In the Lusitanian toadfish (Halobatrachus didactylus) spawning season, similar difference was found (Modesto and Canário 2003).

Fine et al. (1990) proposed a hypothesis that the thinner myofibers in the male toadfish is to adapt the increased speed of sonic muscle contraction and fatigue resistance because of the enlarged ratio of surface-to-volume that would help the exchanging of the metabolizing substances. Since the dominant frequency and the pulse repetition rate of B. microlepis are higher than J. macrorhynus, this could be applied to the difference between the freshwater and marine species sciaenids.

4.3 The swim bladder observation of the golden sciaenid

A simple form of swim bladder is considered to be more primitive for the

Sciaenidae (Sasaki 1989). Even though the appearance of B. microlepis and the golden sciaenid have high similarity, the swim bladders from the two species were highly different. This may indicate that the swim bladder of the golden sciaenid has been vestigial.

4.4 Taxonomic status and vocalization of the golden sciaenid

The phylogenetic tree from this study showed that there are two freshwater sciaenid species in Thailand. Although it has not been described, many local people have mentioned this fish by emphasizing the yellowish hue on its body. Nevertheless, it is not clear whether this yellowish croaker is an undescribed species and it vocalizes or not.

The sounds from the field were similar to B. microlepis in some parameters. Given the

20 fact that both “species” occur in that river basin, these calls might came form one of these species. To clarify that doubt, investigating in the sound of the golden sciaenid should become a top priority.

21 References

Baird, I. G., V. Inthaphaisy, P. Kisouvannalath, B. Phylavanh, and B. Mounsouphom.

1999. The fishes of Southern Lao. Lao Community and Dolphin Protection

Project, Ministry of Agriculture and Forestry, Pakse, Lao PDR.

Baird, I. G., B. Phylavanh, B. Vongsenesouk, and K. Xaiyamanivoni. 2001. The

ecology and conservation of the smallscale croaker Boesemania microlepis

(Bleeker 1858-59) in the mainstream Mekong River, southern Laos. Natural

History Bulletin of the Siam Society 49:161-176.

Bardach, J. E. 1959. Report on Cambodian fisheries. USOM (United States Operations

Mission to Cambodia), Phnom Penh, Cambodia.

Barney, R. L. 1926. The distribution of the fresh-water sheepshead, Aplodinotus

grunnniens Rafinesque, in respect to the glacial history of North America.

Ecology 7:351-364.

Boeger, W. A. and D. Kritsky. 2002. Parasites, fossils and geologic history: Historical

biogeography of the South America freshwater croakers, Plagioscion spp.

(Teleostei, Sciaenidae). Zoollogica Scripta 32:3-11.

Borsani, J. F. 1999. The Irrawaddy dolphins (Orcaella brevirostris Gray 1866) of Lao

P.D.R.: A visual-acoustic survey. Environmental Protection and Community

Development in Siphandone Wetland Champassak Province, Lao P.D.R.

Unpublidshed report for European Commission project Lao.

Cesvi (Cooperazione e Sviluppo). 1998. Siphadone Wetlands: Environmental Protection

and Community Development in Siphandone Wetland Project.

Charif, R. A., A. M. Waack, and L. M. Strickman. 2010. Raven Pro 1.4 User’s Manual.

Cornell Lab of Ornithology, Ithaca, NY.

Connaughton, M. A. and M. H. Taylor. 1995. Seasonal and daily cycles in sound

production associated with spawning in the weakfish,Cynoscion regalis.

Environmental Biology of Fishes 45:233-240.

Connaughton, M. A., M. L. Fine, and M. H. Taylor. 1997. The effects of seasonal

hypertrophy and atrophy on fiber morphology, metabolic substrate concentration

and sound characteristics of the weakfish sonic muscle. The Journal of

Experimental Biology 200:2449-2457.

Feldberg, E, J. I. R. Porto, E. B. P. Santos and F. C. S. Valentim. 1999. Cytogenetic

studies of two freshwater sciaenids of the genus Plagioscion (Perciformes,

Sciaenidae) from the central Amazon. Genetics and Molecular Biology 22:351-

356.

Fine, M. L. 1997. Endocrinology of sound production in fishes. Marine Freshwater

Behaviour Physiology. 29:23-45.

Fine, M. L., N. M. Burns, and T. M. Harris. 1990. Ontogeny and sexual dimorphism of

sonic muscle in the oyster toadfish. Canadian Journal of Zoology 68:1374-1381.

Froese, R. and D. Pauly. Editors. 2012. FishBase. World Wide Web electronic

publication. www.fishbase.org, version (02/2012).

Griffiths, M. H. and T. Hecht. 1995. Age and growth of South African dusky kob

Argyrosomus japonicus (Sciaenidae) based on otoliths. South African Journal of

Marine Science 16:119-128.

Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. deWaard. 2003. Biological

identifications through DNA barcodes. Proceedings of the Royal Society B:

Biological Sciences 270:313-322.

23 Hill, G. L., M. L. Fine, and J. A. Musick. 1987. Ontogeny of the sexually dimorphic

sonic muscle in three sciaenid species. Copeia 1987:708-713.

Ladich, F. 2004. Sound production and acoustic communication. The Senses of Fish

210-230.

Lin, Y. C., H. K. Mok , and B. Q. Huang. 2007. Sound characteristics of big-snout

croaker, Johnius macrorhynus (Sciaenidae). Journal Acoustic Society of America.

121:586-593.

Lo, P. C. 2011. Sound characteristics of the large yellow croaker, Larimichthys crocea

and phylogeny of the Western Pacific sciaenid genera inferred by molecular

evidence. Master’s Thesis. National Sun Yat-sen Unviersity, Taiwan.

Mok, H. K., and R. G. Gilmore. 1983. Analysis of sound production in estuarine

aggregations of Pogonias cormis, Bairdiella chrysoura, and Cynoscion nebulosus

(Sciaenidae). Bulletin of the Institute of Zoology, Academia Sinica 22:157-186.

Mok, H. K., H. Y. Yu, J. P. Ueng, and R. C. Wei. 2009. Characterization of sounds of

the blackspotted croaker Protonibea diacanthus (Sciaenidae) and localization of

its spawning sites in esturine coastal waters of Taiwan. Zoological Studies

48:325-333.

Nelson, J. S. 2006. Fishes of the World, 4th edition. John Wiley & Sons, New Jersey.

Poulsen A. F. and J. Valbo-Jorgensen. 2000. Fish migrations and spawning habits in the

Mekong Mainstream: A survey using local knowledge (Basinwide). AMFC

Technical Report. Mekong River Commission.

Poulsen, A. F. and J. Valbo-Jorgensen. 2001. Deep pools in the Mekong River. Mekong

Fish Catch and Culture 7.

Poulsen, A. F., O. Poeu, S. Viravong, U. Suntornratana, and N. T. Tung. 2002. Deep

pools as dry season fish habitats in the Mekong Basin. MRC Technical Paper No.

24 4, Mekong River Commission, Phnom Penh.

Rainboth, W. J. 1996. FAO species identification field guide for fishery purposes.

Fishes of the Cambodian Mekong. Food and Agriculture Organization of the

United Nations, Rome.

Ramcharitar, J., D. P. Gannon, and A. N. Popper. 2006. Bioacoustics of Fishes of the

Family Sciaenidae (Croakers and Drums). Transactions of the American Fisheries

Society 135:1409-1431.

Roberts, T. R. 1993. Artisanal fisheries and fish ecology below the great waterfalls of

the Mekong River in southern Laos. Natural History Bulletin of the Siam Society

41:32-62.

Sasaki, Kunio. 1989. Phylogeny of the family Sciaenidae, with notes on its

zoogeography (Teleostei, Perciformes). Memoirs of the Faculty of Fisheries

Hokkaido University 36:1-137.

Takemura, A., T. Takita, and K. Mizue. 1978. Studies on the underwater sound VII:

underwater calls of the Japanese marine drum fishes (Sciaenidae). Bulletin of the

Japanese Society of Scientific Fisheries 44:121–125.

Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary

Genetics Analysis (MEGA) software version 4.0. Molecular Biology and

Evolution 24:1596-1599.

Tellechea J. S., C. Martinez, M. L. Fine, and W. Norbis. 2010. Sound production in the

whitemouth croaker and relationship between fish size and disturbance call

characteristics. Environmental Biology of Fishes 89:163-172.

Tellechea, J. S., D. Bouvier, and W. Norbis. 2011. Spawning sound in whitemouth

croaker (Sciaenidae): seasonal and daily cycles. Bioacoustics: The International

Journal of Animal Sound and its Recording 20:159-168.

25 Tellechea, J. S., W. Norbis, D. Olsson, and M. L. Fine. 2011. Calls of the black drum

(Pogonias cromis: Sciaenidae): Geographical differences in sound production

between northern and southern hemisphere populations. Journal of Experimental

Zoology Part A Ecological Genetics and Physiology 315:48-55.

Tower, R. W. 1908. The production of sound in the drumfishes, the sea-robin and the

toadfish. Annals of the New York Academy of Sciences 18:149-180.

Ueng, J. P., B. Q. Huang, and H. K. Mok. 2007. Sexual differences in the spawning

sounds of the Japanese croaker, Argyrosomus japonicus (Sciaenidae). Zoological

Studies 46:103-110.

Ward, R. D., T. S. Zemlak, B. H. Innes, P. R. Last, and P. D. N. Hebert. 2005. DNA

barcoding Australia’s fish species. Philosophical Transactions of the Royal

Society B: Biological Science 360:1847-1857.

Wongratana, T. 1985. Boesemania microlepis (Bleeker), a common but misidentified

riverine drumfish (Pisces: Sciaemindae) from Thailand and Mekong River.

Proceedings of the 23rd Kasetsart University Conference Fisheries Section,

Kasetsart University, Bangkok (Thailand) 3-20.

26 Table 1 The Comparisons of the acoustic parameters in Sounds from the filed at Bang Pakong River and Sounds of Boesemania microlepis from propagation in Chainat Province and results of the Mann-Whitney test.

Sounds from the field Sounds of Boesemania microlepis P (mean ± SD) (mean ± SD)

Number of calls analyzed 7 123

Call duration (ms) 103.4 ± 8.1 125.7 ± 48.8 0.25

Dominant frequency (Hz) 566.0 ± 293.6 1610.6 ± 238.6 < 0.01*

Pulse number per call 13.3 ± 1.0 15.0 ± 5.7 0.37

Pulse repetition rate (no. of pulses/ sec) 129.2 ± 14.2 120.2 ± 8.6 0.04*

Pulse Length (ms) 5.6 ± 0.6 5.4 ± 1.2 0.36

Inter-pulse interval (ms) 2.3 ± 0.3 3.3 ± 1.1 0.01*

Pulse period (ms) 9.0 ± 2.8 9.0 ± 1.7 0.04*

Fig. 1. A 38.2 cm standard length Boeseman croaker, Boesemania microlepis.

Fig. 2. A 47 cm standard length golden sciaenid.

Fig. 3. A simplified illustration of the recording setup at the propagation. The hydrophone was 45 cm below the water surface.

Fig. 4. A call from Boesemania microlepis with illustrations showing how the acoustic parameters were measured.

Fig. 5. An example of call from Boesemania microlepis voluntary vocalization recorded using HP-A1 hydrophone. A) Expanded waveform of 3 pulses, B) osillogram, C) spectrogram, D) power spectrum of the 15-pulse containing call.

Fig. 6. Examples of calls with the first few weaker amplitude pulses. A) Oscillogram, B) spectrogram of examples. Red boxes indicate the pulses with weaker amplitude.

Fig. 7. Changes in number of pulses with lower amplitude on March 12th 2011. The total of calls examined is 123 calls. The occurrence of less amplitude pulses is represented in percentage. The time scale represents the whole vocal activity.

Fig. 8. Linear regression of call duration and number of pulses per call. Call duration ranged from 0.0370 to 0.2288 seconds. The regression coefficient is 114.9, R2 = 0.9737, p < 0.01 (n = 123).

Fig. 9. A period of background noise with no croaking contained. A) Osillogram, B) spectrogram, C) power spectrum of a 160 ms long background noise. D) The power spectrum of a single call of Boesemania microlepis and background noise. The upper crooked line is of B. microlepis. The lighter line below is background noise.

Fig. 10. Temporal changes in percentage occurrence of pulse numbers in the evening of

March 12th 2011. The occurrence of pulse numbers is represented in percentage. The time scale represents the whole vocal activity.

Fig. 11. Temporal change in numbers of voluntarily produced vocalization in three consecutive evenings from 11 March to 13 March 2011. The time scale is represented by 24-hour clock format.

Fig. 12. An example of a single call from field recording at Bang Pakong River on

November 4th 2012, using H2a hydrophone. A) Expanded waveform of three pulses, B)

Oscillogram, C) spectrogram, D) spectrogram with scale expanded in frequency axis, and E) power spectrum of the call.

Fig. 13. A period of background noise with no croaking contained from field recording at Bang Pakong River on November 4th 2012. A) Osillogram, B) spectrogram, C) power spectrum of a 160 ms long background noise. D) The power spectrum of a single call from the field and background noise. The red line is of the call. The blue line is the background noise.

Fig. 14. Ventral view of a female Boesemania microlepis. BW: body wall, G: gonad, SB: swimbladder. A) Ventral view showing no sonic muscle attached to the swim bladder, B) ventral view after internal organs were removed.

Fig. 15. Ventral view of a male Boesemania microlepis. BW: body wall, G: gonad, SB: swimbladder, SM: sonic muscle. A) Ventral view showing sonic muscle attached to the swim bladder, B) ventral view after internal organs were removed with sonic muscle on the body wall.

Fig. 16. Ventral view of a male golden sciaenid. G: gonad, SB: swimbladder, SM: sonic muscle. Ventral view showing A) sperm gonad of the male golden sciaenid and B) sonic muscle attached to the swim bladder.

Fig. 17. The cross section of the sonic muscle of Boesemania microlepis. The three examples of myofibril area of the cell are delimited.

Fig. 18. The cross section of the sonic muscle of Johnius macrorhynus. The three examples of myofibril area of the cell are delimited.

Fig. 19. The comparison between the myofibril areas of Boesemania microlepis (n = 29) and Johnius macrorhynus (n = 12). P < 0.05.

Fig. 20. The ventral view of the swimbladder of Boesemania microlepis. The arrows show the appendages extended toward back. SM: sonic muscle. SB: swimbladder.

Fig. 21. The later view from the left of the swimbladder of the golden sciaenid. The arrows indicate the two chambers on the front part.

Fig. 22. A pair of appendages on each side of the chambers of swimbladder of the golden sciaenid. The box on the upper right corner shows the closer look of the left side chamber with two appendages.

Fig. 23. Neighbor joining tree of COI gene sequences using Maximum Composite

Likelihood model. The numbers indicate the percentage of trees occurrence after bootstrapping (1000 replicates). The scale bar shows the nucleotide substitutions per site.