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Overview on the Diversity of Sounds Produced by
Clownfishes (Pomacentridae): Importance of Acoustic
Signals in Their Peculiar Way of Life
Orphal Co 11eye", Eric Parm entier
Laboratory of Functional and Evolutionary Morphology, University of Liège, Liège, Belgium
Abstract
Background: Clownfishes (Pomacentridae) are brightly colored coral reef fishes well known for their mutualistic symbiosis
with tropical sea anem ones. These fishes live in social groups in which there Is a size-based dom inance hierarchy. In this
structure w here sex is socially controlled, agonistic interactions are num erous and serve to maintain size differences
betw een Individuals adjacent In rank. Clownfishes are also prolific callers w hose sounds seem to play an im portant role in
the social hierarchy. Here, we aim to review and to synthesize the diversity of sounds produced by clownfishes In order to
emphasize the Importance of acoustic signals In their way of life.
M ethodology/Principal Findings: Recording the different acoustic behaviors Indicated th at sounds are divided Into two
main categories: aggressive sounds produced In conjunction with threat postures (charge and chase), and submissive
sounds always emitted w hen fish exhibited head shaking m ovem ents (I.e. a submissive posture). Both types of sounds
show ed size-related Intraspeclflc variation In d om inant frequency and pulse duration: smaller Individuals produce higher
frequency and shorter duration pulses th an larger ones, and inversely. Consequently, th ese sonic features might be useful
cues for individual recognition within th e group. This observation Is of significant Importance d ue to th e size-based
hierarchy In clownflsh group. On th e o ther hand, no acoustic signal was associated with th e different reproductive activities.
Conclusions/Significance: Unlike o th er p o m a ce n tru s , sounds are not produced for m ate attraction In clownfishes but to
reach and to defend th e competition for breeding status, which explains why constraints are not Im portant e n o u g h for
promoting call diversification In this group.
C itation : Colleye O, Parmentier E (2012) Overview on th e Diversity of Sounds Produced by Clownfishes (Pomacentridae): Importance of Acoustic Signals in Their
Peculiar Way o f Life. PLoS ONE 7(11): e49179. doi:10.1371/journal.pone.0049179
Editor: Stephan C. F. Neuhauss, University Zürich, Switzerland
Received August 31, 2012; A ccepted October 9, 2012; Published November 7, 2012
C opyrigh t: © 2012 Colleye, Parmentier. This is an open-access article distributed under th e term s of th e Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided th e original author and source are credited.
Funding: This research was supported by th e Takeda Science Foundation and a 21st Century Center o f Excellence project entitled "The Comprehensive Analyses
on Biodiversity in Coral Reef and Island Ecosystems in Asian
"Concours des bourses d e voyage 2009" from "Ministère d e
Fondam entale Collective (FRFC) (no. 2.4.535.10.F) delivered
FNRS (Bourse d e Doctorat F.R.S.- FNRS). The funders had
manuscript.
and Pacific Regions" from th e University of the Ryukyus via a Visiting Fellowship, by a grant entitled
la C om m unauté française d e Belgique" attributed to OC, and by a grant from Fonds d e la Recherche
by th e Belgian National Fund for Scientific Research. OC was supported by a grant from th e F.R.S.no role in study design, data collection and analysis, decision to publish, or preparation of the
C om peting Interests: The authors have declared th a t no com peting interests exist.
* E-mail: 0.Colleye@ulg.ac.be
from two different so und-producing m echanism s [9]. T h e
L usitanian toadfish Halobatrachus didactylus p roduces the boatw histle
advertisem ent call th a t is related to the b reed in g season a n d at
least three o th er sounds d u rin g agonistic encounters: grunts,
croaks a n d double croaks [10,11],
D am selfishes (Pom acentridae) a re one o f the best-studied
families for the use o f acoustic com m unication d u rin g courtship
a n d agonistic interactions, w ith som e species such as Dascyllus
albisella a n d D . flavicaudus show ing a g reat diversity a n d com plexity
in their acoustic repertoire. T h e y are know n to p ro d u c e pulsed
sounds d u rin g n um erous behaviors including signal ju m p , m a tin g /
visiting, chasing conspecifics a n d heterospecifics, fighting conspecifics a n d heterospecifics, a n d nest cleaning [12,13]. All these
sounds seem to b e constructed on the basis o f the sam e m echanism
since they display the sam e type o f sound spectrum a n d show few
differences in term s o f pulse d uration. O n the o th er han d ,
differences in the n u m b e r o f pulses a n d pulse p e rio d could b e due
Introduction
In teleost fishes, the ability to pro d u ce sounds was developed
independently in d istant phylogenetic tax a [1], T o date, m ore th an
100 fish families include species w ith the ability to em it sounds
[2,3]. T h e m ajority o f acoustic signals are used in different
behavioral contexts such as aggressive b eh av io r (territorial defense,
p re d a to r/p re y interactions, com petitive feeding) o r reproductive
activities (m ate identification a n d choice, courtship, synchroniza­
tion o f gam ete release) [2,4,5]. T h e diversity o f sounds p ro d u c ed
by fishes is not as rem arkable as in o th er taxa; m ost fishes show
p o o r am plitude a n d frequency m odu latio n in th eir sounds [6,7,8]
a n d have relatively lim ited acoustic repertoires. O nly few fish
species em it m ore th a n one or two distinct sound types. H ow ever,
the calling characteristics provide sufficient inform ation for species
identification a n d com m unication. F or exam ple, the rainbow
cichlid Herotilapia multispinosa em its four distinct sound types
(thum ps, growls, whoofs a n d volley sounds) th a t w ould result
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Sound Diversity in Clownfishes
to the fish physiology reflecting the b ehavior a n d its m otivational
state [13].
C lownfishes (Pom acentridae) live in social groups in w hich there
is a size-based dom in an ce hierarch y [14,15]. W ithin each group,
n um erous agonistic interactions occur a n d they a p p e a r to play an
im p o rta n t role by m ain tain in g size differences b etw een individuals
ad jacen t in ra n k [14,15]. Intraspecific encounters are com m on
a n d som etim es ra th e r severe in th eir intensity. L arger fishes chase
sm aller ones, w hich m eans th a t the sm allest one is the recipient o f
n um erous charges [15]. All clow nfish species have evolved
ritualized th rea t a n d submissive postures th a t p resum ably serve
to circum vent physical injury d u rin g intraspecific q u arrelin g [16],
F or exam ple, the “ h e a d shaking” is considered as a submissive
state exhibited by fish in reactio n to aggressive interactions
[16,17,18], T his b ehavior consists in a lateral quivering o f the
body th a t begins a t the h e a d a n d continues posteriorly.
C lownfishes are know n to p ro d u c e aggressive sounds while
displaying charge a n d chase tow ards a n o th e r specim en du rin g
agonistic interactions [16,18,19], M o re recently, Colleye et al. [20]
cond u cted furth er studies o n aggressive sounds in the skunk
clownfish Amphiprion akallopisos; they highlighted a size-related
intraspecific v a riation in d o m in a n t frequency a n d pulse duration:
sm aller individuals p ro d u c e h igher frequency a n d shorter d u ra tio n
pulses th a n larger ones. Surprisingly, the relationship b etw een fish
size a n d b o th d o m in a n t frequency a n d pulse d u ra tio n is not only
species-specific. T hese relationships are also spread out over the
entire tribe o f clownfishes b y bein g found am o n g 14 different
species th a t are situated o n exactly the sam e slope, w hich m eans
the size o f any Amphiprion can be p red icted b y b o th acoustic
features [21].
Besides these aggressive sounds, S chneider [18] d o c u m e n ted a
second type o f sound th a t was associated w ith “ h e a d shaking” a n d
was em itted b y fishes in conjunction w ith submissive posture.
L ater, Allen [16] re p o rte d the presence o f h e a d shaking
m ovem ents associated w ith sound em ission du rin g agonistic
interactions betw een group m em bers. L hifortunately, the lack o f
detailed acoustic d a ta a n d the small sam ple sizes o f the behavioral
observations require fu rth er investigations to differentiate these
sounds from aggressive ones a n d to b e tte r u n d e rstan d the scope o f
these acoustic signals w ithin a social group o f clownfishes.
A dditionally, it was re p o rte d th a t clownfishes m ight pro d u ce
sounds d u rin g courtship. C ourtship in clownfishes is generally
stereotyped a n d ritualized, a n d is typically a cco m p an ied by
different activities such as nest cleaning, courtship, spaw ning a n d
nest care [16], Basically, studies th a t describe the courtship sounds
in clownfishes are lim ited in nu m b er. T o date, sound pro d u ctio n
d u rin g reproductive p e rio d has b e en re p o rte d in three clownfish
species (A. ocellaris, A . frenatus, A . sandaracinos) by T a k e m u ra [22].
H ow ever, these observations need to be carefully considered since,
a ccording to the au th o r, the sounds w ere h a rd ly h e a rd a n d
som etim es they do n o t seem to b e directly related to spaw ning
b ehavior [22]. T herefore, d eeper a tten tio n m ust b e p a id to
confirm the im plication o f acoustic signals d u rin g rep ro d u c tio n in
this group.
T h e presen t study aim s to review a n d to synthesize the diversity
o f sounds p ro d u c ed by clownfishes in o rd e r to em phasize the
im p o rtan ce o f acoustic signals in th eir w ay o f life. T h e purpose o f
this study is 1) to reco rd a n d to analyze sounds associated w ith
h e a d shaking in o rd e r to determ in e their role in the social structure
o f clownfishes; 2) to determ in e w h e th er clownfishes use acoustic
signals to synchronize one o r several o f their reproductive activities
a n d 3) to m ake fu rth er analyses o f som e results previously obtain ed
for the aggressive sounds (see [20]) w ith the aim o f determ ining
w h eth er som e acoustic features m ay c ontribute to individuality.
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M aterials and Methods
D ifferent species a n d different types o f d a ta w ere collected in
fish tanks a n d in the field w ith the aim o f covering all the behaviors
th a t could be associated w ith sound p ro duction. E x p erim ental a n d
anim al care protocols follow ed all relevant intern atio n al guidelines
a n d w ere ap p ro v ed by the ethics com m ission (no. 728) o f the
Lhiiversity o f Liège.
Agonistic sounds
T h re e groups bein g each com posed o f four individuals o f
L ength, SL: 4 4 -1 1 2 nini) w ere
collected by scuba diving o n the fringing re e f a ro u n d N akijin
village (2 6"40'N - 127"59'E ; O kinaw a, Ja p a n ) d u rin g M ay a n d
J u n e 2009. All fish w ere th en b ro u g h t back w ith their host
(Entacmaea quadricolor) to Sesoko Station, T ro p ical Biosphere
R esearch C enter, Lhiiversity o f the R yukyus w here they w ere
transferred to a c om m unity tan k (3.5 x 2 .0 x 1.2 m) filled w ith
ru n n in g seaw ater at am b ien t tem p e ra tu re (28 to 30.5"C). All fish
w ere kept u n d e r n a tu ra l p h o to p erio d a n d fed once daily w ith food
pellets ad libitum. T h e social ra n k o f each individual was attested
using size differences. Basically, groups w ere com posed o f a
b re ed in g p a ir a n d two n on-breeders (T able 1).
R ecordings w ere m ad e in a sm aller glass tan k (1 .2 x 0 .5 x 0 .6 m)
filled w ith ru n n in g seaw ater m ain tain ed a t 28"C by m eans o f a
G E X cooler system (type G X C -2 0 1 x , O saka, Ja p a n ) for having
standardized conditions. F o r the sound recordings, all individuals
o f a group a n d th eir host w ere first p lac ed in the tan k for an
acclim ation tim e o f 2 days. T w enty sessions (each lasting a ro u n d
45 m inutes) w ere re co rd e d du rin g w hich interactions betw een
group m em bers w ere observed a n d n o ted in o rd e r to identify the
sound em itter. O nly sounds associated w ith h e ad shaking
m ovem ents w ere taken into account in the analyses because the
aim o f this p a rt was to give a concise physical description o f this
type o f sounds in o rd e r to co m p are it w ith clow nfish aggressive
sounds (see [20,21,23]).
S ound recordings w ere m ad e using a B rüel & K ja er 8106
hy d ro p h o n e (sensitivity: —173 dB re. 1 V /p P a ) con n ected via a
N e x u s™ conditioning am plifier (type 2690) to a T asca m H D -P 2
stereo
audio
reco rd e r
(recording b andw ith:
20 H z
to
20 k H z ± 1 .0 dB). T h i system has a flat frequency response over
w ide range b etw een 7 H z a n d 80 kH z. T h e h y d ro p h o n e was
placed ju st above the sea an em o n e (± 5 cm).
Sounds w ere digitized at 44.1 k H z (16-bit resolution) a n d
analysed w ith AviSoft-SA S L ab P ro 4.33 software (1024 point
H a n n in g w indow ed fast F o u rrie r transform (FFT)). R eco rd in g in
small tanks induces poten tial hazards because o f reflections a n d
tan k resonance [24], A relevant e q u atio n [24] was thus used to
calculate the re so n an t frequency o f the tank, a n d a low pass filter
o f 2.05 k H z was applied to all sound recordings. T em p o ral
Amphiprion frenatus (S tandard
Table 1. Standard length (SL) and size order in the different
groups of Amphiprion frenatus.
Size ord e r
a(female)
SL (m m)
G rou p 1
G rou p 2
G rou p 3
105
110
112
83
ß(male)
76
81
y(non-breeder)
63
65
75
S(non-breeder)
44
50
53
doi:10.1371 /journal.pone.0049179.t001
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Sound Diversity in Clownfishes
features w ere m easured from the oscillogram s w hereas frequency
p a ram ete rs w ere o b tain e d from pow er spectra (filter b an d w id th
300 H z, F F T size p o in t 256, tim e overlap 96.87% a n d a flat top
window). G enerally speaking, the reco rd ed sounds w ere com posed
o f a series o f sounds bein g m ultiple-pulsed. T h e follow ing sonic
features w ere m easured: pulse d u ra tio n in ms, pulse p e rio d in ms
(the average peak to peak interval b etw een consecutive pulse units
in a series), n u m b e r o f pulses p e r sound, sound d u ra tio n in ms,
sound p e rio d in m s (the average p eak to peak interval betw een
consecutive sounds in a train), n u m b e r o f sounds p e r tra in a n d
d o m in a n t frequency in H z (frequency c o m p o n en t w ith the m ost
energy). N ote th a t sounds p ro d u c ed sim ultaneously b y several
individuals w ere excluded from acoustic analyses.
social ra n k is the g rouping factor. Secondly, sonic variables not
co rrelated w ith SL, w hich failed the test for n o rm al distribution
(Shapiro-W ilk W Test), w ere analyzed using a n o n -p aram etric
K ruskal-W allis one-w ay A N O V A by ranks w ith subsequent
D u n n ’s test for pair-w ise com parisons to test differences betw een
social ranks. All statistical analyses w ere c arried o u t w ith Statistica
7.1. R esults are presen ted as m eans ± sta n d ard deviation (S.D.).
Significance level was d e term in ed a t ƒ><().05.
M e a n ± S.D . values w ere calculated for each acoustic feature o f
agonistic sounds for all individuals. O verall m eans, S.D . a n d range
values w ere subsequently calculated using each individual m ean
value for each variable. In o rd e r to co m p are betw een-individuals
w ith w ithin-individuals variability for each acoustic feature, the
w ithin-individuals coefficient o f v ariance (C.V.W= S .D ./m ean) was
calculated a n d c o m p a red w ith the betw een-individuals coefficient
o f variation (C.V.b). T h e C.V.b was o b tain e d by dividing the
overall S.D . by the respective overall m ean. T h e ratio C .V .b /
C.V.Wwas th en calculated to o b tain a m easure o f relative betw eenindividuals variability for each acoustic feature. W h en this ratio
assum es values larger th a n one, it suggests th a t a n acoustic feature
could be used as a cue for individual recognition [25,26,27].
Differences betw een individuals for each acoustic variable w ere
tested using a K ruskal-W allis analysis due to the lack o f
hom ogeneity o f variance. N ote th a t this statistical test was ru n
betw een individuals from a sam e group in the case o f submissive
sounds. In addition, this test was also ru n b etw een the 14
individuals o f the skunk clow nfish Amphiprion akallopisos for w hich
aggressive sounds w ere previously re co rd e d (see [20]), in o rd e r to
determ in e if som e acoustic features m ay con trib u te to individu­
ality.
Reproductive sounds
R ecordings w ere m ad e b o th in a q u ariu m a n d in the field.
In captivity, three species (A. akyndinos, A . melanopus a n d A.
ocellaris) re are d for several years a t O ceanopolis A q u ariu m in Brest
(France) w ere re co rd e d d u rin g Ju ly 2008. O n e m atin g p a ir p e r
species was studied. E ac h m atin g p a ir was m ain tain ed in separate
glass tanks (0 .7 0 x 0 .4 5 x 0 .5 0 m) filled w ith ru n n in g seaw ater at
am b ie n t te m p e ra tu re (26°C). T h e different reproductive activities
(nest p re p ara tio n , courtship, spaw ning a n d eggs care) w ere
observed a n d recorded. E ach reco rd in g session lasted ap p ro x i­
m ately 2 h, a n d four reco rd in g sessions w ith a 1-hour interval w ere
carried o u t b y day in o rd e r to cover the w hole daytim e from daw n
to dusk. B ehaviors o f m ale a n d fem ale w ere observed a n d noted.
In addition, recordings in captivity w ere also m ad e in Sesoko
Station. O n e m atin g p a ir o f A . clarkii was kept in a tank
(1.2 x 0.5 x 0 .6 m) filled w ith ru n n in g seaw ater m ain tain ed at
28°C . R ecordings w ere carried o u t du rin g sum m er season 2009
(betw een M ay a n d July) because rep ro d u ctio n is lim ited to this
p erio d w h en seaw ater tem p eratu res are w arm er.
In b o th cases, sound recordings w ere m ad e using the sam e Brüel
& K ja e r 8106 hy d ro p h o n e (see above for details o n m aterial
characteristics), a n d sounds w ere analyzed a ccording to the
p ro ced u re previously described.
Field recordings w ere m ad e on the fringing re e f in front o f
H izuchi b eac h (2 6 °1 1 'N - 127°16'E ; A kajim a, K e ra m a Islands,
Ja p a n ) in A ugust 2009. T h ey w ere m ad e using a S O N Y H D D
video c am era p lac ed in a housing (H C 3 series) coupled w ith an
external h y d ro p h o n e (High T ech . Inc.) w ith a flat response o f
20 H z to 20 k H z a n d a n om inal calibration o f —164 dB re. 1 V /
p P a (L oggerhead Instrum ents Inc.). R ecordings w ere m ade by
placing the housing in front o f the in h ab ited sea an em o n e (distance
o f betw een 50 cm a n d 1 m) th a t lived a t a d e p th o f betw een 5 m
a n d 10 m . E ach reco rd in g session lasted from 1 to 4 h.
B ehaviors associated w ith sound p ro d u c tio n w ere described a n d
sounds w ere ex tracted in .wav files using the A oA audio e xtractor
setup freew are (version 1.2.5). S ounds w ere digitised a t 44.1 kH z
(16-bit resolution), low-pass filtered a t 1 k H z a n d analysed using
AviSoft-SA S L ab Pro 4.33 software (1024 p o in t H a n n in g
w indow ed fast F o u rrie r T ran sfo rm (FFT)). O n ly sounds w ith a
good signal to noise ratio w ere included in the analyses.
Results
Agonistic sounds
Subm issive sounds w ere always associated w ith h e ad shaking
m ovem ents (Fig. 1), b u t fish could som etim es carry o u t these
m ovem ents w ith o u t vocalizing. Subm issive sounds (jV= 285 sounds
analyzed for all individuals o f the different groups; see T ab le 2)
w ere p ro d u c ed w h en subordinates displayed submissive p osture as
a reactio n to charge a n d chase by dom inants, w hich m eans th a t
these sounds w ere never re co rd e d for the d o m in a n t females (rank
1) d u rin g this study. G enerally speaking, submissive sounds are
com pletely different from aggressive ones. T h ey are always
com posed o f several pulses w hereas aggressive sounds are
com posed o f a single pulse un it th a t can b e em itted alone o r in
series (Fig. 2). T h ey also exhibit shorter pulse periods a n d shorter
pulse d urations th a n aggressive sounds. In A . frenatus, submissive
sounds can be p ro d u c ed alone o r in series (2-9 sounds, 3 .0 ± 0 .5 6 ),
a n d are m ultiple-pulsed (2 -6 pulses, 3 .2 ± 0 .2 6 ). Pulse p erio d
averaged 1 1 .8 ± 2 .4 m s a n d pulse d u ra tio n ra n g ed from 4.7 to
10.3 ms (7 .9 ± 2 .1 5 ms). S ound p e rio d averaged 197.0 =±26.6 ms
a n d sound d u ra tio n ra n g ed from 23.5 to 50.6 m s (3 5 .9 ± 9 .5 9 ms).
Pulses h a d p eak frequency o f 591 ± 115 H z a n d m ost sound energy
ra n g ed from 454 to 778 H z.
D o m in a n t frequency a n d pulse d u ra tio n w ere highly co rrelated
w ith SL. D o m in a n t frequency significantly decreased (R = —0.98,
j&< 0.0001 ; Fig. 3A) w hereas pulse d u ra tio n significantly increased
(R = 0.98, j6<0.0001; Fig. 3B) w ith increasing SL. Pulse p e rio d was
co rrelated across SL (R = 0.96, j6<0.0001; Fig. 3C) a n d this sonic
variable was also significantly co rrelated w ith pulse d u ra tio n
(R = 0.95, p = 0.0001). A dditionally, sound d u ra tio n was co rrelated
w ith increasing SL (R = 0.97, /)< 0 .0 0 0 1 ; Fig. 3E); this acoustic
feature bein g also significantly co rrelated w ith b o th pulse d u ra tio n
(R = 0.93, p = 0.0003) a n d pulse p e rio d (r= 0 .9 8 , j&CO.OOOl).
Statistical analyses
C orrelations analyses w ere used to exam ine changes in all
acoustic features o f submissive sounds across SL. T h e d a ta used in
these analyses w ere m ea n values o f all reco rd ed sounds for each
individual. T w o statistical analyses w ere th e n p erfo rm ed to test the
influence o f social ra n k on sonic features. First, a full A N C O V A
was ru n to test differences b etw een social ranks (ß, y, 8; see
T ab le 1) for the sonic variables co rrelated w ith SL. In this test,
sonic variables are considered as variâtes, SL as a covariate a n d
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Sound Diversity in Clownfishes
strong hom ogeneity o f these variables. All the acoustic features
analyzed h a d C .V .b/C .V .w r a tio s > l, show ing a h igher variability
a m o n g th a n w ithin individuals. C onsistently, the K ruskal-W allis
analyses revealed significant differences am o n g individuals for
alm ost all features (Tables 4, 5), in dicating th a t these acoustic
variables (except the n u m b e r o f pulses p e r sound a n d the n u m b er
o f sounds p e r train in groups 1 a n d 2 o f A . frenatus', T ab le 5) can
potentially provide recognition cues to identify the sound em itter.
T h e larg er relative betw een-individuals variability (larger C .V .b/
C.V.W ratios) c o rresponded to the d o m in a n t frequency, pulse
d u ra tio n a n d pulse p e rio d (Tables 4, 5).
B
Reproductive sounds
A total o f eight spaw ning events w ere observed. All reproductive
p attern s including nest p re p ara tio n , courtship, spaw ning a n d
p a ren tal care w ere once observed a n d reco rd ed in -4. akindynos, -4.
melanopus a n d -4. percula d u rin g J u ly 2008 a t O ceanopolis
A quarium . Amphiprion clarkii spaw ned four tim es betw een M ay
a n d Ju ly 2009 in Sesoko Station, a n d all the reproductive activities
w ere observed a n d recorded. Spaw ning always o ccu rred in the
a fternoon from 2:00 to 5:00 p .m ., w hatever the species. In
addition, one com plete spaw ning sequence in -4. perideraion was
observed a n d recorded in the field for a pproxim ately 80 m inutes
in A ugust 2009 (11:20 to 12:40 a.m .).
O verall, the m ost striking observation was the com plete absence
o f sound p ro d u c tio n th ro u g h o u t all activities o f the reproductive
p e rio d in the different clow nfish species recorded, a n d w hatever
the reco rd in g env iro n m en t (aquarium o r field). H ow ever, some
o th er typical behaviors seem to b e responsible for the synchroni­
zation o f the reproductive activities.
F ig u re 1. B e h a v io ra l p o s t u r e s a s s o c ia t e d w ith v o c a liz a t io n s
a n d e x h ib ite d b y Amphiprion frenatus d u r in g a g o n is t ic in te r a c ­
t io n s . A) D o m in a n t in d iv id u al c h a s in g s u b o rd in a te w h ile p ro d u c in g
a g g r e s s iv e s o u n d s . B) H e a d s h a k in g m o v e m e n ts d is p la y e d b y
s u b o r d in a te w h ile p ro d u c in g s u b m is s iv e s o u n d s in re a c tio n to
a g g re ss iv e a c t b y d o m in a n t. N o te th a t w ig g ly lines in d ic a te th e
s o u n d -p ro d u c in g in d iv id u al, a n d a rro w s p o in t o u t th e re c e iv e r o f th e
a g g re ss iv e act.
Likewise, sound p e rio d was co rrelated w ith increasing SL
(R = 0.86, p = 0.0031; Fig. 3F), bein g significantly co rrelated w ith
sound d u ra tio n (i? = 0.82, p = 0.0063). T h e n u m b e r o f pulses p e r
sound did n o t change significantly (R = 0.10, p = 0.7917; Fig. 3D)
across SL, as well as the n u m b e r o f sounds p e r train (i? = 0 .0 6 ,
p = 0.8686; Fig. 3G).
A com parison o f social ra n k values using SL as a covariate
show ed th a t the d o m in a n t frequency (ANC O V A , test for com m on
slopes: F 3) = 3.677, p = 0.156; test for intercepts: F,25)= 1.204,
p = 0.374) a n d pulse d u ra tio n (A N C O V A , test for co m m o n slopes:
F,2 3) = 3.644, p = 0.175; test for intercepts: F,2 5) = 4.204, p = 0.137)
did not differ betw een individuals o f different social ranks.
T h ereb y , differences betw een social ranks in these acoustic
features exclusively resulted from size differences. In addition,
the influence o f fish size on acoustic features was en h an c ed by
co m p arin g th em betw een individuals o f the sam e social ra n k b u t
from different groups. All the acoustic features w ere significantly
different betw een individuals o f the sam e ra n k (T able 3), except
w hen these ones h a d sim ilar SL. In this case, acoustic features did
not differ (D u n n ’s test, />>0.05).
K ruskal-W allis one-w ay A N O V A revealed th a t m eans w ere
significantly different b etw een social ranks for pulse p erio d
(H = 6.489, d f = 2 , p = 0.0390) a n d sound d u ra tio n (H = 7.200,
d f= 2, p = 0.0273), b u t not for sound p e rio d (H = 3.822, d f= 2,
p = 0.1479), n u m b e r o f pulses p e r sound (H = 1.898, d f= 2,
p = 0.3871) a n d n u m b e r o f sounds p e r train (H = 2.508, d f= 2,
p = 0.2853).
Pairw ise com parisons show ed th a t pulse p erio d a n d sound
d u ra tio n w ere h igher in ra n k 2 (D u n n ’s test, /><0.05; T ab le 2) th an
in ra n k 4, w hereas no significant differences w ere observed
betw een ranks 2 a n d 3 (D unn’s test, />>0.05; T ab le 2), a n d
betw een ranks 3 a n d 4 (D u n n ’s test, />>0.05; T ab le 2) due to
considerable overlap.
Aggressive a n d submissive sounds presen ted som e acoustic
features th a t displayed C .V .W^ 0 .1 0 (Tables 4, 5), suggesting a
1. T h e arrival o f spaw ning p erio d was indicated by a n increase
o f cleaning activity b y the m ale, a n d by the belly o f the fem ale th at
was noticeably distended (especially in -4. percula a n d -4. perideraion).
T hese features b e cam e usually distinct three o r four days before
spaw ning. O ccasionally, fish chased each a n o th e r o r engaged in
fast side-by-side sw im m ing a n d belly touching; the fem ale being
the initiator in m ost o f these encounters. Som etim es, the fem ale
e n te red the nest a n d pressed h e r belly against the rock (spaw ning
ground). Pecking m ovem ent o f the m ale at the surface o f nest
becam e m o re vigorous from ab o u t two hours before spaw ning; this
m ovem ent was con tin u ed until ju st before spaw ning.
A bout 15 m inutes before spaw ning, nest-cleaning activities was
m ore rigorously carried o u t b y the fem ale, w hich pecked the
surface o f the spaw ning g round. Also, she pressed h e r belly against
the substrate. T hese activities seem ed to aim a t m aking sure o f the
com pletion o f the spaw ning g round. A t the onset o f spaw ning, the
w hitish cone-shaped ovipositor o f the fem ale was clearly ap p are n t.
2. T h e spaw ning was carried out in the follow ing way: the
fem ale e n te red the nest, pressed h e r belly against th e spaw ning
gro u n d a n d sw ani slowly in a circular p ath; the m ale followed
closely b e h in d a n d fertilized the spawn. L ocom otion d u rin g the
spaw ning passes was achieved b y ra p id fluttering o f the pectoral
fins. T h e m ale frequently m o u th e d the eggs d u rin g the spaw ning
period. B oth fish also nib b led o n the tips o f the an em o n e tentacles
for p reventing the spaw n from en terin g into con tact w ith them .
3. A fter the spaw ning, the incubation p e rio d took place a n d was
ch aracterized by p a ren tal care, w hich lasted usually six to seven
days. T h e m ale assum ed nearly the full responsibility o f tending
the nest. E xcept a n initial m o d erate level o f activity a t spaw ning,
th ere w ere no cleaning activities the first two days. T h en , an
ab ru p t increase o ccu rred the next few days until hatching. T w o
basic nest-caring beh av io r p a tte rn s w ere observed. F a n n in g was
the m ost c om m on a n d was m ainly p erfo rm ed b y fluttering the
pectoral fins. M o u th in g the eggs a n d substrate biting a t the
,2
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Sound Diversity in Clownfishes
1—1
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(b o tto m ) o f su b m iss iv e s o u n d s p ro d u c e d b y s u b o rd in a te d u rin g h e a d s h a k in g m o v e m e n ts . B) O scillo g ram (to p ) a n d s p e c tro g r a m (b o tto m ) o f
a g g re ss iv e s o u n d s p r o d u c e d b y d o m in a n t w h ile d isp la y in g c h a r g e a n d c h a s e . N o te th e d iffe re n c e s in (1) p u ls e d u ra tio n a n d (2) p u ls e p e rio d . T he
a c o u s tic v a ria b le m e a s u re d in (3) r e p re s e n ts th e s o u n d d u ra tio n in th e c a s e o f su b m iss iv e s o u n d s , a n d th e tra in d u ra tio n in th e c a s e o f a g g re ss iv e
s o u n d s . T he c o lo u r sca le c o rre s p o n d s to th e in te n s ity a ss o c ia te d w ith th e d iffe re n t fre q u e n c ie s.
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 4 9 1 7 9 .g 0 0 2
Discussion
p e rip h e ry o f the nest w ere also exhibited. D e a d eggs w ere regularly
rem oved as indicated by b a re patches a t the nesting surface.
In clownfishes, different types o f sounds such as “th rea te n in g ”
a n d “ shaking” [18], “ click” a n d “g ru n t” [16] o r “ p o p ” a n d
“ c h irp ” [19,23] have already b e en described du rin g interactions
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N ovem ber 2012 | Volum e 7 | Issue 11 | e49179
Sound Diversity in Clownfishes
betw een conspecifics. A lthough a dichotom y in sounds was
re p o rte d in each case, these term s have b e en inconsistently
applied a n d w ere n o t always supported by ap p ro p ria te d a ta w hich
create confusion [10], C onsequently, it re m a in e d difficult to m atch
a type o f sound w ith a given behavior. H ow ever, o u r m ultiple
observations highlight th a t submissive sounds (i.e. chirps, see [23])
are clearly different from aggressive sounds (i.e. pops, see [23]).
Aggressive sounds are m ainly p ro d u c ed by dom inants du rin g
chases a n d th rea t displays b etw een conspecifics [20], w hereas
submissive sounds are always em itted w h en subordinates exhibit
h e a d shaking m ovem ents in reactio n to aggressive displays by
h igher-ranking individuals. T h erefo re, b o th types o f sounds seem
to be a n integral p a rt o f the agonistic b eh av io r in clownfishes.
G iven th a t they presen t som e differences in sound spectra a n d
shape o f the tem p o ral envelope, it is im p o rta n t to em phasize th at
these tw o types o f sounds w ould result from two different
m echanism s. Aggressive sounds result from ja w teeth snapping
[21,28] b u t the sound-producing m echanism o f submissive sounds
is still unknow n.
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Table 2. Summary (mean ± S.D.) of the seven acoustic features analyzed from submissive sounds produced by 9 Amphiprion frenatus.
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hierarchy
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Interestingly, d o m in a n t frequency a n d pulse d u ra tio n o f
submissive sounds display size-related v a riation in som e acoustic
features. T h e m ore fish size increases, the m ore dom in an t
frequency decreases, a n d the m o re pulse d u ra tio n increases. T h e
sam e relationships have alread y b e en found for aggressive sounds
am o n g 14 different species [21]. D ifferences in b o th sound
characteristics w ere related to fish size a n d n o t to sexual status or
social rank. H ow ever, size, sex a n d social ra n k are extrem ely
related to each o th er due to the size-based h ierarch y w ithin each
group [14,15]. In -4. percula , B uston a n d C a n t [29] dem o n strated
th a t individuals ad jacen t in ra n k are separated by body size ratios
w hose distribution is significantly different from the distribution
expected u n d e r a null m odel: the grow th o f individuals is regulated
such th a t each d o m in a n t ends up bein g ab o u t 1.26 tim es the size
o f its im m ediate subordinate. T h e sam e k ind o f ratio (—1.30) is
observed in the different groups o f A . frenatus o f this study (Fig. 4).
T h e respect o f this ratio w ithin groups highlights th a t dom in an t
frequency a n d pulse d u ra tio n can b e signals conveying inform a­
tion o n the social ra n k o f the em itter w ithin the group.
Aggressive a n d submissive sounds are involved in interactions
betw een group m em bers (Video S I, S2). Being associated w ith a
specific display, they m ight have a different function w ithin the
g roup. Indeed, aggressive sounds could possess a d eterren t
function b y giving a re m in d er signal o f dom in an ce during
interactions w hereas submissive sounds could possess a n appease­
m en t function by expressing the low er ra n k status du rin g
interactions. Likewise, two different types o f sounds are em itted
by the grey g u rn a rd Eutrigla gurnardus d ep en d in g on the in terac­
tions b etw een individuals: knocks are p ro d u c ed du rin g low levels
o f aggression a n d grunts m ainly w hile p erfo rm in g frontal displays
to opponents [30],
C lear differences w ere found am o n g aggressive a n d submissive
sounds a ttrib u te d to different individuals. All acoustic variables
w ere significantly m ore variable b etw een th a n w ithin individuals
a n d thus could all potentially provide cues to identify individuals.
F u rth e rm o re , the m ost im p o rta n t variables to allow individual
identification w ere d o m in a n t frequency a n d pulse d u ra tio n for
b o th types o f sounds (Tables 4, 5). Pulse period, in a lesser extent,
was also consistently im p o rta n t for discrim inating am o n g individ­
uals in the case o f submissive sounds (T able 5). In o rd e r to be good
candidates for individual recognition, these acoustic features
should p ro p ag ate th ro u g h the environm ent, a n d should be
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N ovem ber 2012 | Volum e 7 | Issue 11 | e49179
Sound Diversity in Clownfishes
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F ig u re 3 . In flu e n c e o f fish s iz e (SL) o n a c o u s tic f e a tu r e s o f s u b m is s iv e s o u n d s in A m p h ip rio n fre n a tu s. C o rre la tio n s o f (A) d o m in a n t
fr e q u e n c y , (B) p u ls e d u ra tio n , (C) p u ls e p e rio d , (D) n u m b e r o f p u ls e s p e r s o u n d , (E) s o u n d d u ra tio n , (F) s o u n d p e rio d a n d (G) n u m b e r o f s o u n d s p e r
tra in a g a in s t SL. Fish ra n g e d fro m 4 4 to 112 m m in SL (N = 9). R esults a re e x p re s s e d a s m e a n v a lu e s o f all re c o rd e d p u ls e s fo r e a c h in dividual
( Ö = ra n k 4, □ = ra n k 3, A = ra n k 2).
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 4 9 1 7 9 .g 0 0 3
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Sound Diversity in Clownfishes
Table 3. Compa ris o n
o f th e a c o u s tic f e a tu re s o f s u b m is s iv e
sounds betw een in d iv id u a ls o f t h e s a m e s o c ia r a n k b u t f r o m
different groups o f A m p h ip rio n fre n a tu s .
Acoustic variables
Social ran k
Dominant frequency (Hz)
Pulse duration (ms)
Pulse period (ms)
Sound duration (ms)
Sound period (ms)
H
p-value
2
108.4
<0.0001
3
42.11
<0.0001
4
49.54
<0.0001
2
79.16
<0.0001
3
37.72
<0.0001
4
139.0
<0.0001
2
121.5
<0.0001
3
35.13
<0.0001
4
64.19
<0.0001
2
6.418
0.0404
3
15.18
0.0005
4
10.27
0.0059
2
9.995
0.0068
3
22.80
<0.0001
4
12.31
0.0021
H values are th e result o f th e Kruskal-Wallis test (df= 2, n = 300). n, num ber of
pulses analyzed.
doi:10.1371 /journal.pone.0049179.t003
d etected by the receiver. S ound p ro p a g atio n in shallow w ater can
result in signal d eg rad atio n over short distances, including sound
pressure level a n d frequency atten u atio n , a n d sound d u ra tio n loss
[31]. H ow ever, the effect o f e nvironm ental a tte n u atio n a n d signal
d eg rad atio n should n o t im pose a m ajo r restriction w ithin a group
o f clownfishes since all individuals in h ab it a restricted territo ry (the
sea anem one) a n d spend m ost o f the tim e in close vicinity o f their
host.
Since d o m in a n t frequency a n d pulse d u ra tio n o f b o th aggressive
a n d submissive sounds are size-dependent, tem poral a n d spectral
inter-individual differences m ight be d etected b y m em bers w ithin
the group. T eleost fishes such as Gobius niger (Gobiidae) a n d Sparus
annularis (Sparidae) are able to discrim inate tonal sounds differing
in frequency o f approxim ately 10%; the frequency discrim ination
ability at 400 H z is a pproxim ately 40 H z [32]. In the clow nfish el.
akallopisos, aggressive sounds em itted by non-breeders, m ales a n d
females differ in d o m in a n t frequency by > 1 0 % [20], In A . frenatus,
sounds em itted b y individuals o f different social ranks also differ in
d o m in a n t frequencies b y > 1 0 % (T able 2). Such a n ability to
discrim inate frequency differences has a lready b e e n observed in
som e pom acentrids. In the dam selfish A budefduf saxatilis, fish size
has a significant effect on aud ito ry sensitivity [33] : all fish are m ost
sensitive to the low er frequencies (10CM-00 Hz) b u t the larger ones
a re m ore likely to resp o n d to h igher frequencies (1000-1600 Hz).
T h e effect o f fish size on h earin g abilities was also supposed in
th ree different clow nfish species [34], A lthough the best h earin g
sensitivity is a ro u n d 100 H z, small individuals w ere m ore sensitive
to a larg er frequency interval (100-450 Hz), a n d thus they are
m ore sensitive to the frequencies em itted by larg er conspecifics.
N o in form ation related to fish size can be extracted from
n u m b e r o f pulses p e r sound o r n u m b e r o f sounds p e r train.
Differences in these acoustic features a p p e a r to be related to a
difference in m otivation. M otivation is know n for playing a role in
dam selfishes, reg ard in g th eir sounds p ro d u c ed d u rin g aggression.
In Dascyllus albisella a n d D . flavicaudus , aggressive sounds are
different a ccording to w h eth er they are em itted tow ards conspe­
cifics or heterospecifics, b ein g m ultiple-pulsed or single-pulsed,
respectively [12,13]. In Pomacentrus partitus, the frequency o f sounds
by a territorial resident is relatively low at the territorial b o rd er,
b u t it rapidly increases as in tru d e r ap p ro ach es the residence [35].
In the clow nfish A . akallopisos, the m ost aggressive m ales w ere
ch aracterized b y a h igher n u m b e r o f pulses p e r sound a n d a
shorter pulse p e rio d (pers. obs.). T h e sm allest individuals (rank 4)
in A . frenatus groups em itted the highest n u m b e r o f sounds p e r train
(T able 2). All these variations in acoustic features m ight be related
to the willingness to express the position w ithin the group
hierarchy. F or exam ple, low er-ranking individuals m ight pro d u ce
m ore submissive sounds in o rd e r to lim it aggressive acts from
dom inants.
No reproduction-related sound
U nlike observations m ad e b y T a k e m u ra [22], no acoustical
beh av io r was observed d u rin g reproductive activities in clow n­
fishes. M oreover, T ak e m u ra ’s d a ta are som ew hat doubtful sin c e -4.
ocellaris, A . frenatus a n d -4. sandaracinos w ould em it sounds w ith high
frequency co m p o n en t o f m ore th a n 2 k H z d u rin g rep ro d u ctio n
[22]. A ccording to h earin g sensitivity in clownfishes (A. frenatus, A.
ocellaris a n d -4. clarkii), the frequency range over w hich they can
detect sounds is b etw een 75 a n d 1800 H z, a n d they are the m ost
sensitive to frequencies below 200 H z [34], T herefore, this finding
raises the question over the interest o f clownfishes to pro d u ce
sounds they could n o t detect du rin g reproductive activities. It
rem ains these sounds could ju st be a by-p ro d u ct o f the nest
cleaning activities.
In the field, clownfishes spaw n o n average from 1 .0 ± 0 .5 to
0 .6 ± 0 .1 tim es p e r m o n th d ep en d in g o n w h eth er they live in
tropical w aters [16,36] or in m ore tem p erate regions [37,38,39].
In captivity, the spaw ning frequency is h igher a n d o n average
2 5 ± 5 .3 tim es p e r y ear [40,41], T his frequency was observed at
Table 4. Means, ± S.D., range, within-individuals variability (C.V.W) and between-individuals variability (C.V.b) for th e four acoustic
features analyzed from aggressive sounds produced by 14 Amphiprion akallopisos.
Acoustic variables
O verall M ean ± S.D. (ran ge)
C.V.w (M ean)
C .V .w (range)
c.v.b
C .V .b /C .V .w
H*
/> v a lu e
Dominant frequency (Hz)
663 ± 199 (346-1207)
0.10
0.07-0.21
0.30
2.73
1577
<0.001
Pulse duration (ms)
13.4±3.9 (3.8-22.9)
0.10
0.06-0.18
0.29
2.90
1614
<0.001
Pulse period (ms)
75.9±13.4 (32.4-121.9)
0.15
0.09-0.21
0.23
1.53
203.9
<0.001
Number o f pulses per sound
3.9±2.2 (2-14)
0.50
0.30-0.68
0.56
1.12
25.55
0.0195
*Results of Kruskall-Wallis te st (df= 13, n = 1818) comparing differences betw een 14 individuals of A. akallopisos for each acoustic feature. Note th a t these values w ere
calculated based on acoustic data obtained from Colleye e t al. (2009). n, num ber of pulses analyzed.
doi:10.1371 /journal.pone.0049179.t004
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Sound Diversity in Clownfishes
T a b l e 5. M eans, ± S.D., r a n g e, w ithin-individuals variability (C V .W) a n d be tw ee n -in d iv id u als variability (C.V.b) for t h e se v e n
a c o u s tic fe a tu re s a nalyzed fr om su bm issiv e s o u n d s p r o d u c e d by 9 Am phiprion frenatus.
G roup
nu m b er
A coustic variables
Dominant frequency (Hz)
Pulse duration (ms)
Pulse period (ms)
Number o f pulses per sound
Sound duration (ms)
Sound period (ms)
O verall M ean ± S.D. (range)
C.V.W
(M ean)
C.V.W (range)
C .V .b
C .V .b/
C .V .W
631 ±135 (431-1036)
0.10
0.09-0.15
0.21
1.90
213.7
< 0.001
593 ±139 (431-862)
0.10
0.08-0.12
0.22
2.20
251.0
< 0.001
552 ±105 (345-776)
0.09
0.08-0.11
0.19
239.9
< 0.001
7.4 ±2.1 (3.8-11.2)
0.10
0.09-0.11
0.28
2.80
232.4
< 0.001
>.5±2.8 (3.6-15.1)
0.10
0.09-0.11
0.34
3.40
250.Í
< 0.001
9.1 ±2.8 (4.5-13.9)
0.08
0.06-0.12
0.31
3.87
233.2
< 0.001
10.7±2.0 (6.7-15.4)
0.10
0.08-0.12
0.19
1.90
143.Í
< 0.001
11.9±2.5 (7.4-1 7.<
0.10
0.08-0.11
0.21
2.10
167.4
< 0.001
12.7±2.6 (8.2-18.4)
0.08
0.05-0.09
0.21
2.62
176.2
< 0.001
3.1 ±0.6 (2-5)
0.20
0.19-0.23
0.21
1.05
5.36
3.4±0.9 (2-6)
0.26
0.21-0.31
0.27
1.04
2.59
3.1 ±0.8 (2-5)
0.25
0.22-0.29
0.27
1.08
12.86
< 0.01
29.2 ±8.4 (9.8-48.2)
0.22
0.18-0.24
0.29
1.32
59.24
< 0.001
36.9 ±14.1 (16.6-72.4)
0.28
0.22-0.33
0.38
1.36
25.94
< 0.001
36.6 ±14.1 (15.0-73.8)
0.24
0.13-0.35
0.38
1.58
49.90
< 0.001
0.16-0.21
0.21
16.53
< 0.01
21.07
< 0.001
176.4±36.9 (92.2-295.3)
Number o f sounds per train
p -value
206.3 ±40.9 (135.6-280.9)
0.15
0 . 12 - 0.1
0.20
1.33
201.7 ±31.5 (118.3-266.3)
0.13
0.08-0.16
0.16
1.23
3.3 ±1.5 (2-9)
0.39
0.28-0.53
0.44
4.36
3.2 ±1.3 (2-7)
0.35
0.30-0.41
0.41
4.93
2.6±0.8 (2-5)
0.23
0.18-0.27
0.30
1.30
< 0.01
14.39
< 0.01
*Results o f Kruskal-Wallis te st (df=-2, n = 300) comparing differences betw een individuals in a group of A. frenatus for each acoustic feature, n, num ber of pulses
analyzed.
doi:10.1371 /journal.pone.0049179. t005
Oceanopolis A quarium with captive clownfishes for which
spawning occurred approximately every two weeks. In this
context, it could be argued th at the captivity modulates some
aspects of the behavior [40] such as sound production during
reproduction. However, 1) the pair o f A. clarkii reared a t Sesoko
Station showed the same spawning frequency (~every 2 weeks),
although its reproduction was restricted to sum mer season an d it
was reared under semi-natural conditions (i.e. outdoor tank filled
with running seawater and m aintained under natural photoperi­
od); 2) spawning was witnessed in the field for A. perideraion, and no
sound was produced by the m ating pair during the reproductive
event. Yet, recording of aggressive sounds during the same session
supports the fact th at the recording material worked well.
B
A
120 -
110 -
a
o
100-
1.4-
90-
o
80-
c
s
60-
50-
53
1. 2 -
UO
40-
2
3
2
4
S o cia l rank
3
Group
F ig u re 4 . Fish s iz e (SL) a n d s iz e r a tio s o f in d iv id u a ls a d ja c e n t in ran k w ith in e a c h g r o u p o f A m p h ip rio n fre n a tu s. A) T h e o b s e rv e d
d is trib u tio n o f fish size (SL) w ith in e a c h g ro u p . B) D istrib u tio n o f b o d y size ra tio s b e tw e e n in d iv id u a ls a d ja c e n t in ra n k w ith in e a c h g ro u p . R esults a re
e x p re s s e d a s m e a n ± S.D. v a lu e s ( □ = g r o u p 1, A = g r o u p 2, 0 = g r o u p 3).
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 4 9 1 7 9 .g 0 0 4
PLOS ONE I w w w .plosone.org
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N ovem ber 2012 | Volum e 7 | Issue 11 | e49179
Sound Diversity in Clownfishes
O verall, sound p ro d u c tio n does n o t seem to be involved in the
reproductive b ehavior o f clownfishes, w hich m ight be explained by
som e p a rticu la r aspects related to their w ay o f life.
T h e reproductive b eh av io r o f p o m acentrids is subdivided into
the follow ing m ajo r categories [42,43]: 1) establishm ent o f
territory, 2) selection o f nest site w ithin the territory, 3) p re p ara tio n
o f the nest site, 4) courtship a n d p a ir form ation, 5) spaw ning a n d
fertilization, a n d 6) p a ren tal care. Clownfishes conform to this
general p a tte rn b u t are distinctive w ith regards to form ation o f
p e rm a n e n t p a ir bonds th a t usually last for several years in m ost
species [16,44], In o th er damselfishes, one m ale m ay m ate w ith
several females du rin g a single spaw ning episode [42,44], In
clownfishes, m ale does n o t n e ed to exhibit typical courtship
b ehavior for a ttrac tin g fem ale. P air-b o n d in g is very strong a n d is
co rrelated b y the small size o f th eir territories (centered on
actinians) th a t is, in tu rn , correlated w ith the unusual social
h ierarch y existing in each social group. O n the o th er h a n d , it
seems th a t o th er cues such as visual signals m ig h t be useful for
synchronizing reproductive activities. J u s t before spaw ning occurs,
the fem ale joins the m ale a n d becom es m o re insistent in the nestcleaning activities, p ro b ab ly in o rd e r to convey visual cues ab o u t
its readiness to spaw n. Likewise, it is possible th a t the m ale
regulates its level o f nest-caring activity in response to visual stimuli
received w hen inspecting eggs [16], A visual stim ulus o f this sort
w ould signal the stage o f egg developm ent a n d the n e ed for
increased fa nning a n d m o u th in g activities. Allen [16] ex p erim en ­
tally d e m o n stra ted th a t strong agitation o f the eggs is a requisite
for hatching. H e also no ted th a t there was a p ro n o u n c ed increase
in the a m o u n t o f m ale nest care on day six o f incubation. O n th a t
day, the em bryos a re well developed w ith one o f the m ost
noticeable features bein g the large eyes w ith their silvery pupils.
Such a feature m ight serve as a n a p p ro p ria te visual cue.
T h erefo re, o th er cues such as visual a n d p erh ap s chem ical signals
m ig h t b e involved in reproductive activities. H ow ever, new
behavioral tests w ould need to b e ru n to determ in e the p ro p e r
role o f such signals d u rin g the rep ro d u ctio n o f clownfishes.
restricted to agonistic interactions only, acoustic signals seem to be
an integral p a rt o f their daily behaviors. T h e im plication o f
acoustic signals in agonistic interactions m ay be a n interesting
strategy w ith a n econom ic w ay for prev en tin g conflicts w hich
otherw ise m ig h t escalate to a severe outcom e.
C low nfish sounds c an b e divided into tw o m ain categories:
aggressive sounds p ro d u c e in conjunction w ith th re a t postures
(charge a n d chase), a n d submissive sounds always em it w hen
subordinates exhibit h e ad shaking m ovem ents in reaction to
aggressive displays b y dom inants. B oth types o f sounds show
intraspecific differences related to fish size, highlighting th a t some
acoustic features (i.e. d o m in a n t frequency a n d pulse duration)
m ig h t be useful cues for individual recognition w ithin the group.
T hese observations are o f significant im p o rtan ce because the social
structure o f clownfishes strictly relies o n a size-based dom inance
hierarchy.
Supporting Inform ation
Video SI Im plication o f aggressive sounds during
agonistic interactions betw een group m em bers in the
field. N ote th a t a fish is chasing a n o th e r one (smaller) while
p ro d u c in g a series o f aggressive sounds.
(AVI)
Video S2 Behavioral posture (head shaking m ove­
m ents) exhibited by subordinates while producing
subm issive sounds. N ote th a t fish m ake sounds while doing
lateral quivering o f the b o d y th a t begins a t the head.
(AVI)
Acknowledgments
T he authors would like to thank Prof. M asaru N akam ura (Sesoko Station,
Tropical Biosphere R esearch C enter, University of the Ryukyus, Japan)
and Dr. K enji Iwao (Akajima M arine Science L aboratory, AM SL, Japan)
for helping to collect fishes and for providing hospitality and laboratory
facilities. W e are also indebted R. Suwa, M. Alam, S. N akam ura, Y.
Kojim a, Y. N akajim a, R. M urata, R. N ozu and N. Pandit (Sesoko Station,
TBRC) for technical support. M any thanks to Dom inique Barthélém y
(Oceanopolis, France) and Dr. Christian M ichel (Aquarium Dubuisson,
Belgium) for providing free access to fishes during recordings.
Conclusion
U nlike o th er pom acentrids, sounds are n o t p ro d u c ed for m ate
a ttrac tio n in clownfishes. It is likely a n evolutionary outcom e
related to th eir peculiar w ay o f life: these fishes form small social
groups including only one m atin g pair, in h ab it a restricted
territo ry (the sea anem one), spend m ost o f the tim e in close vicinity
o f th eir host a n d rarely in teract w ith o th er species o n the reef. O n
the o th er h a n d , sounds seem to be im p o rta n t to reach a n d to
defend the com petition for b reed in g status. A lthough they are
Author Contributions
Conceived an d designed the experim ents: EP O C . P erform ed the
experim ents: O C . A nalyzed the data: O C . C ontributed reag en ts/
m aterials/analysis tools: EP. W rote the paper: O C EP.
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