Ecology and E olu ion. 2023;13:e10673.  
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1 o 18
h ps://doi.o g/10.1002/ece3.10673
www.ecole ol.o g
Recei ed:15May2023
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Re ised:27Sep embe 2023
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Accep ed:16Oc obe 2023
DOI:10.1002/ece3.10673
RESEARCH ARTICLE
Endemic ish calling: Acous ics and ep oduc i e beha iou o
he Ne e a dwa goby O sinigobius c oa icus
S enHo a ić1 | E ic Pa men ie 2 | S e ano Mala asi3 | Ma ia P. Cla a Amo im4 |
Paulo J. Fonseca5 | Da o Zanella1
Thisisanopenaccessa icleunde  he e mso  heC ea i eCommonsA ibu ionLicense,whichpe mi suse,dis ibu ionand ep oduc ioninanymedium,
p o ided he o iginal wo k is p ope ly ci ed.
©2023TheAu ho s.Ecology and E olu ionpublishedbyJohnWiley&SonsL d.
1Depa men o Zoology,Facul yo 
Science,Uni e si yo Zag eb,Zag eb,
C oa ia
2Labo a o yo Func ionaland
E olu iona yMo phology,FOCUS,
Uni e si yo Liège,Liège,Belgium
3Depa men o En i onmen alSciences,
In o ma icsandS a is ics,CàFosca i,
Uni e si yo Venice,VeneziaMes e,I aly
4Depa amen odeBiologiaAnimaland
MARE–Ma ineandEn i onmen al
SciencesCen e/ARNET-Aqua ic
Resea chNe wo k,FaculdadedeCiências,
Uni e sidadedeLisboa,Lisbon,Po ugal
5Depa amen odeBiologiaAnimaland
cE3c-Cen e o Ecology,E olu ionand
En i onmen alChanges,Faculdadede
Ciências,Uni e sidadedeLisboa,Lisbon,
Po ugal
Co espondence
Da o Zanella,Depa men o Zoology,
Facul yo Science,Uni e si yo Zag eb,
Zag eb,C oa ia.
Email:da o [email p o ec ed].h
Funding in o ma ion
Minis yo Ag icul u e,Republico 
C oa ia
Abs ac
TheNe e adwa gobyO sinigobius c oa icus(Gobii o mes,Gobionellidae)isanen-
demic ishna i e o he eshwa e so  heAd ia icBasininC oa iaandBosniaand
He zego ina,aMedi e aneanBiodi e si yHo spo .Due oi slimi eddis ibu ion
ange,speci icka s habi a andendange eds a us,labo a o ys udieson ep oduc-
i ebiologya esca cebu c ucial.He ein,wein es iga ed hesoundp oduc ionand
acous icbeha iou o  heendange edO. c oa icus du ing ep oduc i e in e sexual
labo a o yencoun e s,u ilisinganin e disciplina yapp oach.Wealsope o meddis-
sec ionsandmic o-compu ed omog aphy(μCT)scanningo  hepec o algi dle o
explo ei spo en ialin ol emen insoundp oduc ion.Finally,compa a i eacous ic
analysiswasconduc edonsoundsp oducedbyp e iously eco dedsoni e oussand
gobies o in es iga e whe he  acous ic ea u es a e species-speci ic. The endemic
O. c oa icusisasoni e ousspecies.Maleso  hisspeciesemi pulsa ilesoundscom-
posedo a a iablenumbe o sho (~15 ms)consecu i epulseswhenin e ac ingwi h
emales,usuallydu ing hep e-spawningphasein henes ,bu alsodu ingcou ship
ou side henes .Pulsa ilesoundswe elow- equencyandsho pulse ains(~140 Hz,
<1000 ms).Male isualbeha iou  a ewashighe whenco-occu ing wi hsounds
and emalesen e ed hemale'snes signi ican lymo e equen lywhensoundswe e
p esen .Cha ac e is icbodymo emen saccompaniedmalesoundp oduc ion,such
ashead h us and insp eading.Fu he mo e,μCT scans and dissec ions sugges ha
O. c oa icussha esce ainana omicalsimila i ieso  hepec o algi dle(i.e.osseous
elemen sanda angemen o le a o pec o alismuscles) op e iouslys udiedsand
gobies ha couldbein ol edinsoundp oduc ion.Mul i a ia ecompa isons,using
soundsp oducedbyeigh soni e ousEu opeansandgobies,e ec i elydis inguished
soni e ous(andsympa ic)speciesbasedon hei acous icp ope ies.Howe e , he
disc imina ion success dec eased when empe a u e-dependen  ea u es (sound
du a ionandpulse epe i ion a e)we eexcluded om heanalysis.The e o e,we
sugges bo hspec aland empo al ea u esa eimpo an  o  heacous icdi e en-
ia iono sandgobies.
2o 18
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   HORVATIĆ e al.
1 | INTRODUCTION
The Medi e anean Biodi e si y Ho spo  (MBH) is a widely e-
nowned egion o i ssigni ican en i onmen aldi e si yandendan-
ge edwildli e(Da walle al.,2014; Mye s e al., 2000).The i e so 
heAd ia icSeaBasininC oa iaa epa o  heMBHandDalma ian
eshwa e  eco egions (Abell e  al., 2008), and a e especially ich
in eshwa e endemic ish,wi h40specieso almos 30%o  he
o alC oa ianich hyo auna,endemic o hisa ea(Ćale ae al.,2015,
2019;Ko ela &F eyho ,2007; Mye s e al., 2000).Thisendemism
isa ea u eo  hehabi a so  heDina icka s  ha co e s oughly
54% o  C oa ian e i o y, wi h he p esence o  nume ous ca es,
sinkholes, cold seasonal wells, and unde g ound i e s (Ku le, 1999;
M ako čiće al.,2006).
Among hese endemic ish species, O sinigobius c oa icus
(M ako čiće al.,1996), o me lydesc ibedasKnipowi schia c o-
a ica, is a small ben hic and sho -li ed (less han 2 yea s) sand
gobycon ined o heDina icka s o  heDalma ianeco egion.I 
canbe oundexclusi elyin he eshwa e so C oa iaandBosnia
andHe zego ina(Abelle al.,2008;Ćale ae al.,2019;Ho a ić
e al., 2017;Tu mane al.,2020;Zanellae al.,2011).InC oa ia,
hisspeciesinhabi s heEas e npa o  heAd ia icBasinandhas
ana u ally agmen eddis ibu ion ange ha includes heNe e a
Ri e ,Ma icaRi e , heV go ačkoPoljeandRas očkoPolje ields
andBaćinaLakes,someo whicha eNATURA2000si es(Ćale a
e al., 2015; M ako čić e  al., 1996; Zanella e  al., 2011, 2017;
seeHo a iće al.,2017 o  hemapwi hi sdis ibu iona ea).
In i s na u al habi a , O. c oa icus occupiessandybo oms wi h
occasional s ones/pebbles in ka s  i e s, slow- lowing s eams
andoligo ophiclakes(Ho a iće al.,2017;Zanellae al.,2011).
Du ing win e and ea ly sp ing, O. c oa icus h i esinsmall i e s
ands eamswhe easdu ing hesumme pe iod, hisgobysu i es
insmallka s unde g oundpondsand e uges,when hewa e -
cou sescomple elyd you (Mille ,2004;M ako čiće al.,2006).
On heIUCNRedLis ,O. c oa icusisassessedgloballyas ulne a-
ble(VU,B2ab(iii);D2, e .3.1.),bu  egionallyasendange ed(EN)
due oi shighlylimi ed/ agmen edhabi a anddeclininghabi a 
quali y(C i elli,2018;Ho a iće al.,2017;M ako čiće al.,2006).
Howe e , hisendange eds a usisalsodue oalacko knowledge
ega dingi sbiological ai s.The eisli lepublishedda aon he
ecologyo biologyo O. c oa icus(Ho a iće al.,2017;M ako čić
e al., 2006;Zanella e al.,2011, 2017). Likeo he sandgobies,
O. c oa icusisapolygamousmul iplespawne species ha achie es
sexual ma u i y qui e ea ly (i.e. wi hin i s i s  yea ), and ep o-
duces omMa ch oNo embe ,al houghmos spawningoccu s
om Ap il o Sep embe  (Ko ela  & F eyho , 2007; Mazzoldi &
Rasso o, 2001;Zanellae al.,2011, 2017).
The sand gobies a e a monophyle ic gobii o m g oup
(Gobionellidae, Gobii o mes) o  abou  30 species in he gen-
e a Knipowi schia, Poma oschis us, Economidich hys, Ninnigobius
and O sinigobius (Be ancu -R e  al., 2017; Nelson e  al., 2016;
Thacke , 2009; Thacke e al., 2019; Touga d e al., 2021).They
inhabi in e idalma ineandcoas al eshwa e ecosys emswi h
muddy- o-pebblebo omac ossEu ope,including hewa e so 
heMedi e anean,Pon o-CaspianandNo heas A lan ic egions
(F eyho , 2011;Ko ačić&Pa zne ,2011; Mille , 2004;Šanda&
Ko ačić,2009).Recen phylogene ics udies ounde idence ha 
sound p oduc ion is widesp ead among ac inop e ygian ishes,
sugges ing ha acous icbeha iou  e ol edindependen lymul-
iple imes inun ela ed cladesand ha  he eis a s ong selec-
ion o  heuseo soundp oduc ionasabeha iou al ai ac oss
e eb a ee olu ion(Fine&Pa men ie ,2015; Rice e al., 2020,
2022).Communica i esoundemissionin ishisusuallylinked o
cou shipandspawningo agg essi ebeha iou (Amo im,2006;
Mann e al., 2008;My be gJ .&Lugli,2006).In ishbioacous ics,
acous ic signals associa ed wi h ep oduc i e in e sexual in e -
ac ionsha ebeen hemos commonlys udied ypeso sounds
(Amo im,2006),sincei isbelie ed ha  hesesoundsse e oa -
ac po en ialma es(Long iee al.,2013;Pa men ie e al.,2010),
osynch onisespawningac i i iesa agg ega ionsi es(E isman&
Rowell, 2017;Jublie e al.,2019;Lobel,1992; Rowell e al., 2015)
o  o synch onise game e elease by conspeci ics (Hawkins &
Amo im,2000;Lobel,2002).Sandgobiesa eacommonmodel
g oup among soni e ous ac inop e ygian ishes o  sound p o-
duc ionandha elongbeenu ilisedine hologicalandcompa -
a i e bioacous ics s udies. The acous ic abili ies o  sand gobies
ha e been in ensi ely in es iga ed in he las  30 yea s, espe-
ciallyin hespecieso  hegene aPoma oschis us, Knipowi schia
and O sinigobius(Amo im&Ne es,2007;Blome al.,2016; Lugli
e al., 1997; Mala asi e al., 2008, 2009;Pa men ie e al.,2017;
To icelli e al., 1990;Zeyle al.,2016).Ineigh sandgobyspecies,
ei he pulsa ileo  humpsounds(andsome imesbo h)ha ebeen
eco ded oda e(Amo im&Ne es,2007;Blome al.,2016; de
Jonge al.,2016;Zeyle al.,2016),whileinEconomidich hys pyg-
maeus(Holly,1929)soundswe eno de ec eddu ingbeha iou al
expe imen s (Gkenas e  al., 2010). Mos  o  ou  unde s anding
ega ding he acous ic abili ies o  Medi e anean sand gobies
s ems om hecommon,widelydis ibu edandnon- h ea ened
speciesassigned o heleas conce n(LC;IUCNRedLis )ca e-
go y(Amo ime al.,2013;Blome al.,2016;deJonge al.,2016;
KEYWORDS
ana omicalanalysis,mic o-compu ed omog aphy, ep oduc i ee hology,soundanalysis
TAXONOMYCLASSIFICATION
Biodi e si yecology,Ecosys emecology,E olu iona yecology,Li ehis o yecology,
Popula ionecology,Socioecology
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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3o 18
HORVATIĆ e al.
Zeyl e  al., 2016). Howe e , since O. c oa icus was egionally
classi ied as a ulne able species wi h a e y es ic ed dis i-
bu ion(C i elli,2006,2018;Ho a iće al.,2017), his esea ch
is he i s  s udy o  he acous ic communica ionin endange ed
Medi e aneansandgoby.
The main goal o  his s udy was o in es iga e he acous ic
communica iono O. c oa icus using an in e disciplina y app oach.
Speci ically,ou aimswe e o:(i)in es iga e hesoundp oduc iono 
cap i e O. c oa icusmalesandquan i yacous icpa ame e so  he
sounds;(ii)examine he ep oduc i ebeha iou o soni e ousmales
andi sassocia ion osoundp oduc ion;(iii)p o ideinsigh in o he
pu a i esound-p oducingmechanismbyexplo ing heana omyo 
he pec o al gi dle and (i ) explo e he acous ic di e si ica ion o 
soni e oussandgobiesbyquan i a i elycompa ingacous icsignals
be ween hes udyspeciesandp e iously eco dedMedi e anean
sandgobies(gene aNinnigobius, Poma oschis us, Knipowi schia and
O sinigobius).
2 | MATERIALSANDMETHODS
2.1  |  Fishsampling,labo a o yhousingand
expe imen al design
O sinigobius c oa icuswascaugh usingelec o ishing(HansG assl,
model:EL65IIGI,powe :13 kW) omaboa du ing hesp ing2019.
Samplingwaspe o medon heMa icaRi e inC oa ia(nea  he
illage Vina 43°10′30.33″ N, 17°23′12.36″ E). Di ec  cu en  was
useddu ingsamplingp ocedu essincei causesgal ano axiso an
a ac ionzonewhe e ishac i elyswim owa d heanode,andis
ypically less ha m ul. All ish displaying elec o- ac ic mo emen 
owa ds he anode o  pa alysis we e sampled using dip ne s. In
o al,wecollec ed25indi iduals(15malesand10 emales) om
hemain i e channela adep ho 0.5–2 m.Fishwe e ans e ed
ali e ola geplas icwa e con aine sequippedwi hae a o sand
anspo ed o helabo a o ya  heFacul yo Science,Uni e si y
o Zag eb.A  helabo a o y, ishwe esexedbasedonu ogeni al
papillaandbodycolou a ion(Mille ,1986)andhousedin ou sin-
gle-sexcommuni y ec angula  anks(120 L; i e emalespe  ank;
eigh malespe  ank;Figu e S1).Eachcommuni y ankwasequipped
wi h 2–4 nes s. A e  an acclima isa ion pe iod o  5–8 days, p o-
spec i esoni e ousmaleswe echosen o subsequen labo a o y
acous ic- isual eco dingsbasedoncolou a ion (comple eo pa -
iallyda kenbody, insandhead;Zanellae al.,2011)and e i o-
iali y. Eigh  males (
x
± SD = 49.21 ± 0.8 mm o al leng h, LT; ange:
48.07–50.06 mm;
x
± SD = 41.07 ± 1.01 mm s anda d leng h, LS;
ange: 38.96–42.29 mm;
x
± SD = 1.24 ± 0.11 g weigh , W; ange:
1.10–1.40 g) exhibi ing ypical ep oduc i e beha iou  we e cho-
sen o  heexpe imen s.Females(N = 5;36.75 ± 5.10 mmLS; ange:
28.75–41.27 mm)we echosen o  he eco dingsessionsacco ding
oyellowbellycolou a ion,luminescen g eenspo on he i s do -
sal inandda keyes,allindica o so  emale eadiness o spawning
(Blome al.,2016;Zanellae al.,2011).
We ollowed heacous ic- isual eco dingp o ocoles ablished
byp e iousau ho s(Amo ime al.,2013;Amo im&Ne es,2007;
Ped osoe al.,2013),whe eexpe imen al anks,placedon opo 
3 cm hick ubbe  oamshockabso be s o educesubs a e-bo n
noise,we edi idedin o h eecompa men ssepa a edby emo -
ablepa i ions(Figu e S1). Each la e al compa men  housed one
e i o ial male wi h a nes  (a i icial unnel-shaped plas ic co e ,
dimensions:leng h = 100 mm,wid h = 60 mm,heigh  = 50 mm),while
hemiddlesec ion(‘a ena’)wasoccupiedbya ipe emale.The e-
malecompa men wasno p o idedwi hanes .Theeigh males
we e di idedin o sepa a ela e al compa men s,whe e hey e-
mained h oughou  heexpe imen s.Expe imen al ishwe ekep 
a na u alpho ope iodand eddailyadlibi umwi hDaphnia.Wa e 
empe a u e,moni o edwi ha he mome e (Aqua e a)andman-
ually egula edwi hahea e (Myli ell),wasmain ainedbe ween18
and22°C(na u al ange).The ankshada5 cm hicklaye o  ine
sando g a elandeachmaleineachsec ionwasp o idedwi ha
wa e pumpsys emandae a ion.
Theexpe imen swe epe o med ommid-Ap il oOc obe ,a 
andom imes.The ep oduc i ebeha iou o  esiden maleswas
elici edbyin oducingone ipe emalein o he‘a ena’.Be o e i-
als,each emalewasle 12–24 hin heexpe imen al ank o ac-
clima isa ion.P io  o eco dings(app ox.15 min),elec ici y,wa e 
pumpsandae a ionwe eswi chedo  ominimiseambien noise.
To u he  educeunwan ednoise om he oomligh sys em, he
ceilingligh wasswi chedo and heexpe imen al ankwasillumi-
na edbyLEDligh  om hesideo  heaqua ium.Thisp ocedu e
hadnono iceablee ec son ishbeha iou .Themale– emale ials
las edapp oxima ely30 minandbeganby emo ingoneo  hela -
e alpa i ions,allowingin e sexualin e ac ion.No e ha  he wo
la e alpa i ionswe ene e  emo eda  hesame ime,and he e-
malealwaysin e ac edwi honlyonemale om hesame ank(i.e.
womales om hesame ankwe eusedinsepa a e ials).Be ween
consecu i e eco ding sessions 15–30 min pauses we e included,
wi hall hede ices u nedo .Theeigh  e i o ialmaleswe ekep 
in heexpe imen al anksun ilOc obe ,a e which heywe e e-
u ned omalecommuni y anks.A e eachdaily eco dingsession,
emaleswe e e u ned o hei communi y ank(Figu e S1).A  he
endo each eco dingsession,malesand emaleswe emeasu ed
o leng handweigh .Measu emen swe emadeusingdigi alcalli-
pe sCD-15APXwi hap ecisiono 0.01 mm(Mi u oyo,Japan)anda
digi alscale(0.1 gp ecision).Asame ico malebodycondi ion,we
calcula ed hecondi ion ac o Ful on'sK [whe e K = (W/LS
3)*105]
ollowingAmo ime al.(2013).
2.2  |  Acous ic eco dingsandsoundanalysis
Du ingacous ic eco dingsessions,ahyd ophone(H2A-XLRhy-
d ophone,Aqua ianAudio&Scien i ic;sensi i i y:−180 dB e.1 V
μPa−1; equency ange±4 dB om0.01 o100 kHz),wasplaced
abo e he shel e  ( unnel-shaped plas ic co e ) and connec ed
oaIRIGPREp eampli ie (Aqua ianAudio&Scien i ic).Sounds
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
4o 18
|
   HORVATIĆ e al.
we e eco dedusingaZOOMH4npo abledigi alaudio eco de 
(16bi /44.1 kHzsample a e;ZOOM).Thehyd ophonewasplaced
wi hin hea enua iondis ance om heemi e (less han5 cm),
andweobeyed helabo a o yp o ocol o minimum esonan  e-
quency o smallglass anks(e.g.2.7 kHz o 170 L anks,acco ding
oAkama sue al.,2002).Reco dingswe ela e band-pass il e ed
(0.05–3 kHz) oimp o eS/N a ioandsubsampleda 4 kHz,and
u he  ampli ied (10 dB) o  be e  audi o y and isual inspec-
iono  heaudio acks.Digi alisedsoundswe eanalysedusing
A iso —SASLabP o5.2So wa e(1024-poin FFT,Fla Topwin-
dow;100% ame;A iso Bioacous ics).Tenaudio eco dings(2.5
pe male, eachlas ing app ox. 30 min)we e au allyand isually
inspec ed.No allsoundsp esen edagoodsignal- o-noise a io
(S/N) o acous icanalysis.F om10 eco dingsp esen ing hebes 
S/N a io,weanalysed20 andomlyselec ed sounds.Tempo al
ea u eswe emeasu ed omoscillog ams,while equency- e-
la ed a iableswe eob ained om heloga i hmicpowe spec-
a(Fla Topwindow,512-poin sFFT,96.87%o e lap; esolu ion
8 Hz). Fo  sounds, we measu ed he ollowing acous ic p ope -
ies ollowing Mala asi e  al. (2008) and Amo im e  al. (2013):
(1)sound a e(SR,numbe o soundsemi edin1 min om he
s a o soundp oduc ion);(2)sounddu a ion(DUR, o aldu a-
iono  hecall,s);(3)numbe o pulses(NP);(4)pulse epe i ion
a e (PRR; NP di ided by DUR and mul iplied by 1000, Hz); (5)
pulsedu a ion(PD;measu ed om he i s  o helas cyclein he
pulse,ms);(6)pulsepe iod(PP;a e agepeak- o-peakin e alo 
consecu i epulses,ms);(7) equencymodula ion(FM,a e  he
soundhasbeendi idedin o h ee empo allyiden icalsec ions,
FMi—ini ial,FMm—middleandFM — inal, equencymodula ion
wascalcula edas hedi e encebe ween he inalandini ialpulse
epe i ion a eandexp essedinHz;FMi,pulse epe i ion a eo 
heini ialsec iono asoundandFM ,pulse epe i ion a eo  he
inalsec iono a sound);(8)peak equency(PF, hepeakwi h
hehighes ene gy om heloga i hmicpowe spec um unc ion,
Hz).Ino de  o ollow hep e ious eco dingp o ocolsasclosely
aspossible(Amo ime al.,2013;Amo im&Ne es,2007),wealso
calcula ed he ocalac i i ypa ame e spe male:(i)sound a e
(numbe  o  sounds p oduced pe  min), (ii) maximum sound a e
(maximumnumbe o soundsemi edin1 min)and(iii)callinge -
o (pe cen ageo  imespen calling,i.e.soundp oduc ioninsec-
ondsdi idedby hedu a iono  he eco dinginseconds).Despi e
he ac  ha  he a iablesPPandPRRindica e hepulse epe i ion
pa e n, heywe edelibe a elyindica edsepa a elyhe e o a-
cili a ecompa isonswi h hegobyli e a u eonsoundp oduc ion.
2.3  |  Video eco dingsandanalysiso 
beha iou al ca ego ies
Du ingacous ic- isual eco dings,asecondhyd ophone(HTI-96-
Min,HighTechInc.,sensi i i y:−201 dB e.1 VμPa−1, equency
esponse2 Hz o30 kHz),placedless han3 cm om henes open-
ing,wasconnec eddi ec ly oa ideocamco de (CanonLeg ia
FS200, 41x digi al zoom, 25 ames/s) o di ec ly synch onise
acous icand isualsignalsin oauni o mda ase  o subsequen 
analysis.Thecamco de wasmoun edonas andandposi ioned
app ox. 40 cm om he on  o  he expe imen al glass anks.
Cou shipbeha iou beganwhen he emalesen e ed hemale
e i o ya adis anceo <5 cm om hemale'snes ,while hep e-
spawningphasewasobse edwhen he ipe emaleen e ed he
male'snes .Thespawningphasebeganwi h he emale u ning
upside-downin henes nume ous imesinsho successionand
s a ed ci cling he ceiling. Male beha iou s and he associa ed
soundemissionswe eobse edin ou soni e ousmalesdu ing
se en eco dingsessionsandanalysedusingSolomonCode ( e .
be a19.08.02).The ipe emaleswe echosen o  he eco ding
sessionsacco ding o woindica o so  hei  eadiness o spawn-
ing(bellyandeyecolou a ion).
Beha iou alca ego iesexp essedby hemaleswe eclassi ied
ollowing heli e a u e(Amo ime al.,2013;Amo im&Ne es,2007,
2008; Mala asi e al., 2009).Weiden i iedninemalebeha iou al
ca ego ies wi hin h ee dis inc ep oduc i e phases in O. c oa icus
(Table S1):
• Cou shipphase,pe o medby hemaleou side henes :Chase,
Lead,App oachandCi cling;
• P e-spawningphase,pe o medby hemalewi hin henes :Nes 
display,F on aldisplay,Nes  ubbing,P e-ma ing;
• Spawningphase,pe o medwi hin henes :Spawning.
Spawning was conside ed when he emale epea edly pe -
o med he upside-down o  belly-up posi ion, associa ed wi h
o iposi ion.Insomecases,Nes displayandF on aldisplaywe e
pe o medby hemaleoccupying henes wi ho wi hou a e-
male inside. Howe e , Nes  ubbing, P e-ma ing and Spawning
we ealwayspe o medby hemalewhen he emalewasinside
he nes . In Solomon Code , wo da ase s we e analysed sepa-
a ely and hen compa ed. Fi s , beha iou  ( equency (nmin−1)
anddu a ion(ins))wassco edin he ideo eco dingswi hsound
p oduc ion. We no ed he o al numbe  o  sounds emi ed pe 
beha iou alca ego y o eachsoni e ousmale.Secondly,wean-
alysedeigh  ideo eco dings( wope male)con aining hebe-
ha iou so  hesame ou  es edmales,bu when heydidno 
p oduce sounds (i.e. males we e silen  o  he en i e eco ding
pe iod).Byha ing hese woda ase s,wein es iga ed hedi e -
encesin equencieso beha iou alca ego iesinmaleswhen hey
engagedinsoundp oduc ionandwhen heydidno .In o al,we
usedeigh malesinou expe imen s,bu  ou we eun esponsi e
(i.e.didno pe o mcou shipbeha iou o sounds), esul ingin
insu icien da a o  u he analyses.Weanalysed ideos o  he
ollowingbeha iou alpa ame e s:malebeha iou  a e( he o al
numbe o beha iou alca ego iespe min)(1)co-occu ingwi h
soundso (2)no co-occu ingwi hsounds;numbe o  imesa e-
maleen e ed hemale'snes (3)accompaniedwi hsoundso (4)
wi hou sounds;(5) o albeha iou s(numbe o beha iou alca e-
go iespe  ideo eco ding).
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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5o 18
HORVATIĆ e al.
2.4  |  Ana omicalanalysis
Fo ana omicaldissec ions,addi ionalO. c oa icus indi iduals we e
collec edinOc obe 2020 om hesamewa e cou senea  he il-
lageB ečići(43°7′11.30″ N,17°29′4.03″ E)usingelec o ishing.Fi e
indi idualswe ecollec ed,o which h eemales(40–50 mmLT)we e
immedia elyeu hanisedwi hano e doseo MS-222( icaineme h-
anesulphona e;Pha maq),ands o ed o 1 weekin7% o maldehyde
ixa i esolu ionand hen ans e ed o70%e hanol.Specimens
we edissec edandexaminedwi haWildM10binocula mic oscope
(LeicaCame a,Leica)equippedwi hacame alucida os udy he
ana omyo  hepu a i esound-p oducingmechanism.Sinceea lie 
esea chongobieshighligh ed he oleo  hepec o algi dleand
(pec o al) insinsoundp oduc ion,dissec ionsp ima ilyadd essed
he muscles ela ed o his body pa . The nomencla u e used o
designa emuscula pa swasbasedonea lie  esea ch(Ad iaens
e al., 1993; Pa men ie  e  al., 2013, 2017; Win e bo om, 1974).
Addi ionally, one specimen was subjec ed o mic o-compu ed o-
mog aphy(μCT)scanning o isualise he ishskele ona  hele el
o  heneu oc aniumandpec o algi dle.Scanningwascomple ed
usingaRXEasyTom(RXSolu ions;h p : // w w w .  x s o l u i o n s .  ),wi h
analuminium il e .Imageswe egene a eda 75 kVand133 μA,wi h
a ame a eo 12.5,5a e age amespe image.Thisp ocedu e
gene a ed2897imagesa a oxelsizeo 10 μm.Recons uc ionwas
pe o medusingX-Ac so wa e omRXSolu ions.Segmen a ion,
isualisa ion and analysis we e pe o med using D agon ly so -
wa e (Objec  Resea ch Sys ems (ORS) Inc, 2019; so wa e a ail-
able a  h p : / / w w w .  h e o b j e c s . c o m / d a g o n l y ). Th ee-dimensional
(3D)16-bi imageswe ep oducedandsubsequen lycon e edin o
8-bi  oxelsusingImageJ(Ab amo e al.,2004).Th ee-dimensional
p ocessingand ende ingwe eob aineda e semi-au oma icseg-
men a iono  hebodyusinga‘gene a edsu ace’,acco ding o he
p o ocolsdesc ibedbyZane ee al.(2013).Di ec  olume ende -
ings(iso-su ace econs uc ions)we eused o isualiseasubse o 
selec ed oxelso  hean e io skele oninAMIRA2019.2.
2.5  |  Acous iccompa isonamongsoni e ous
sand gobies
Thesoundso se ensoni e oussandgobies,Knipowi schia paniz-
zae Ve ga, 1841, Ninnigobius canes inii (Ninni 1883), O sinigobius
punc a issimus(Canes ini1864),Poma oschis us ma mo a us (Risso
1810),P. pic us(Malm,1865),P. mic ops(K øye ,1838)andP. minu us
(Pallas1770),we e eco dedandcha ac e isedbyp e iouss udies
(Amo ime al.,2013, 2018;Bolgane al.,2013; Lugli e al., 1995;
Lugli&To icelli,1999; Mala asi e al., 2008;Ped osoe al.,2013).
Howe e , hese acous ic da a we e ne e  combined in o a single
phylogene icda ase andanalysedin e speci ically.The e o e,we
s udiedin e speci icacous ic a iabili yo soni e oussandgobies
(P. ma mo a us was sepa a ed geog aphically in o wo popula ions,
I alian and Po uguese). B ie ly, he species we e caugh  in he
pas by au ho s o  p e ious s udies ei he  omb ackish habi a s
inno hAd ia icSea(K. panizzae, P. ma mo a us and N. canes inii),
om eshwa e so no h-wes pa o ReggioEmiliaRomagna,I aly
(O. punc a issimus;Linds öm&Lugli,2000; Lugli e al., 1995, 1997;
Lugli & To icelli, 1999), om Po uguesema ine/b ackish wa e s
(Amo ime al.,2013, 2018;Bolgane al.,2013)o  hewes coas 
o Sweden(Ped osoe al.,2013).Sound eco dingsga he ed om
hep e iouslyconduc edlabo a o yexpe imen swe e e-analysed
oallow o in e speci iccompa isonwi haminimalmeasu emen 
expe imen ale o .Allin es iga edsandgobiesp oducedpulsa ile
sounds, husenablingacous icin e speci iccompa isons.Theda a-
se wascomposedo 36indi idualso eigh soni e oussandgobies
including O. c oa icus(min–max:3–5indi iduals,excep  o asingle
indi idualo P. mic ops),wi ha leas  h eesounds eco dedpe in-
di idual.In o alwecalcula ed hemeans o  i eacous ic a iables
( empo al:DURinms,NP,PRRinHz;spec al:PFandFM,bo hin
Hz) o eachindi idual.Sincegobiesincludedin hecu en s udy
we e eco deda di e en wa e  empe a u es( ange:15.8–22.6°C)
andi iswellknown ha  heambien wa e  empe a u ea ec s ish
acous ic signals (Ladich, 2018;Vicen ee al.,2015),weconduc ed
wosepa a emul i a ia eanalyses: he i s in ol ing hecomple e
da ase (all i eacous ic ea u es o eachspecies),and hesecond
excluding he empo al ea u es(DURandPRR)known obein lu-
encedbywa e  empe a u e(Luglie al.,1996;Vicen ee al.,2015).
2.6  |  S a is icalanalysis
The s a is ical analyses we e pe o med by combining he sounds
ommul ipleindi idualsin oasingleda ase .Ou lie sandex emes
we ede ec ed isually om heboxplo andwe eelimina ed om
heda ase i necessa y.Since heda awe eno no mallydis ibu ed
o some a iables om he awin aspeci icda ase (Shapi o-Wilk
es , p < .05), we used non-pa ame ic es s. Fo  pai wise com-
pa isons be weensoni e ous O. c oa icusmales,weemployed he
K uskal-Wallis anksum es H ollowedbypai wiseDunn'smul i-
ple compa ison es  wi h Bon e oni co ec ion o  he p- alues.
Addi ionally, heChi-squa e(χ2)wasused o es  o independenceo 
beha iou  omsoundp oduc ion.In his es , he esiduals om he
χ2we eused ode e minewhichbeha iou swe eposi i ely ela ed
osoundp oduc ion.K uskal-WallisH es wasused ocompa e he
meanbeha iou al a iables(calling a e,beha iou  a e,n.o  emale
nes  en ances) be ween soni e ous males. Wilcoxon signed- ank
es waspe o med ocompa e he wodependen samples, ha is,
meanbeha iou al a iables(beha iou  a eand emalenes en ance)
o maleswhen heyp oduced soundsandwhen heywe esilen .
Addi ionally,Wilcoxon es wasused ocompa e he equencyand
du a iono cou shipandp e-spawningphasesbe weenmales.
Fo  hein e speci iccompa isons, hemeanso indi idualacous-
icp ope ies o  soni e oussand gobies we ecompa ed wi h he
K uskal-WallisH- es ,since heda awe eno no mallydis ibu ed
(Shapi o-Wilk es ,p < .05).Toquan i yin e speci icacous ic a i-
abili yamong hesoni e oussandgobies omou s udy,weused
amul i a ia eapp oach.PCA,basedon heco ela ionma ix,was
pe o med on ans o med and s anda dised indi idual means o 
i esound a iables( empo al:DUR,NP,PRR;spec al:PFandFM)
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License

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   HORVATIĆ e al.
oassesso e allacous ic a iabili ybe weensandgobies,andad-
di ionally o ecognise acous ic a iables explaining he obse ed
a iance.Toassess hepe cen ageo success ulclassi ica iono  he
soundsassigned o heco ec gobyspecies,and omaximise he
sepa abili yamong axa,weusedlinea disc iminan analysis(LDA).
Twodi e en LDAswe epe o med, i s wi h hecomple eda ase 
( i eacous ic a iables o eachspecies)and hen emo ing he em-
pe a u e-dependen  ea u es(DURandPRR).Due o heFM'snega-
i e aw alues,weaddedaposi i e ac o  o his ea u eso ha we
couldusei in hecompa a i eanalyses.Ou  esul swe ep esen ed
asmeans(x ) ± s anda dde ia ion(SD),while hele elo signi icance
o in e -andin aspeci iccompa isonswas5%(α = 0.05).S a is ical
analyseswe epe o medinSTATISTICA®( .13.6.0.,TIBCO,USA),
Pas ( .4.11)andRS udio(2022.07.0)so wa e.
2.7  |  Pe mi s
O sinigobius c oa icusislegallyp o ec edbylawasanendange ed
axoninC oa ia(O icialGaze eo  heRepublico C oa ia,2016).
Inaddi ion,i isanendemicspecieswi h e ylimi eddis ibu ion.As
a esul , henumbe o indi idualsemployedin helabo a o yex-
pe imen swaskep  oaminimum(less han15) op e en possible
e ec son hena u alpopula iono  hisspecies.Thesamplingbyelec-
o ishing o scien i icpu posesin hena u alhabi a wasapp o ed
by heMinis yo Ag icul u e(pe mi numbe 525-13/0545-19-2),
whilealllabo a o y expe imen alp o ocols we e app o ed by he
Bioe hicsandAnimalWel a eCommi eeo  heFacul yo Science,
Uni e si yo Zag eb(pe mi numbe 251-58-10617-21-147).
3 | RESULTS
3.1  |  Soundp oduc ionandin aspeci icsound
signal s uc u e
Maleso O. c oa icusp oducedasingle ypeo acous icsignal,pulsa-
ilesound,du ingin e sexual(male– emale)in e ac ionsconduc ed
wi hin he ep oduc i eseason(Ap il–Oc obe ).Fou  esiden males
(
x
± SD = 49.1 ± 0.8; ange:48.0–50.0 mmLT;40.9 ± 1.8; ange:38.9–
42.2 mm LS; 1.2 ± 0.1; ange: 1.1–1.4 g W; 1.7 ± 0.1; ange: 1.5–1.9
Ful on's K) p oduced sounds when in e ac ing wi h emales, while
heo he  ou males emainedsilen anddidno cou .We eco ded
372soundsp oducedby he ou males(93soundspe male).Sounds
we ep oducedinani egula pa e n(7.7 ± 1.4; ange:6–10sound
min−1; Figu e 1; Table 1).Thesoundsa esho -du a ionsignals,las -
ing450 ms(442.0 ± 132.6 ms),andcomposedo a a iablenumbe o 
sho pulses(14.2 ± 4.0)o a ound15 ms(14.5 ± 1.9 ms;Figu e 2a–d;
Table 1).Pulses uc u edi e edbe weensounds,exhibi ingone o
h eepeakswi h a iableampli ude.Gene ally, heampli udeo a
FIGURE1 (a)Maleand emaleo Ne e adwa goby,O sinigobius c oa icus.Pho oso indi iduals omHo a iće al.(2017),(b)Sound
p oduc iono O. c oa icusmales.The17-second eco dingclipdepic s heoscillog am( op)andspec og am(bo om)o  ou pulsa ile
soundsp oducedbyamalegoby(g eydasheda ea).In hespec og am,wa me colou sindica ehighe acous icene gy(o angeishighes 
andblueislowes ).Theho izon albanda low equency(app ox.100 Hz)co esponds oambien noises emming om heambien 
labo a o ycondi ions.Spec og ampa ame e s:Fla Topwindow,1024leng hFFT;25% amesize;93.75%o e lap; esolu ion:4 Hz.RA,
ela i eampli ude.
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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7o 18
HORVATIĆ e al.
soundchangedg adually, i s inc easingand hens eadilydec eas-
ing h oughou  hesound,wi h he i s  woo  h eepulsesbeing he
highes inampli ude(Figu e 2a–d).Thepulse epe i ion a e a ied
om26.0 o38.0 Hz(32.5 ± 1.6 Hz),while hepulsepe iod(PP)a e -
aged32 ms(31.9 ± 1.4; angems).PPchangedwi hwa e  empe a u e,
wi hhighe  aluesoccu inga lowe  empe a u es.In hepulsa ile
sounds,las PPwasalwayslonge  han he emainingPPs.Peak e-
quency a ied om89 o340 Hz(137.4 ± 38.3 Hz),al houghse e al
highe  equency componen s we e also p esen , especially in he
ange0.5–1.5 kHz.Ene gyex ended om0.05 o2 kHz(Figu e 2a,d;
Table 1),wi hmos o  hesoundene gyconcen a edwi hin0.05–
0.6 kHz. F equency modula ion o  he sounds anged om 0.7 o
1.1 Hz.Addi ionally, hecallinge o  a iedbe weenmales om0.37
o0.60o soundp oduc ion/so  eco ding(0.49 ± 0.09),indica ing
ha someindi idualsemi edsoundsmo e equen ly hano he s
(Table 1).In e es ingly,soundswe ene e o ganisedinbu s s,which
a eusuallycomposedo se e alconsecu i esoundsp oducedwi h
egula in e -soundin e als,asobse edinsomesandgobies.Du ing
ou in e sexualacous icexpe imen s,no emalesp oducedasound.
In aspeci ically, soni e ous O. c oa icus males di e ed in all
acous ic ea u es(K uskal-WallisH- es ,SRχ2 = 8.59,DURχ2 = 22.87,
NP χ2 = 24.53, PRR χ2 = 8.5936.87, PD χ2 = 8.07, PP χ2 = 23.73, PF
χ2 = 34.68, FMi χ2 = 38.05, FM  χ2 = 29.92, FM χ2 = 12.81; d  = 3;
N = 80; p < .05 o  all ea u es) excep  o  calling e o  (K uskal-
WallisH- es ,χ2 = 5.67;d  = 3;N = 7;p > .05;Table 1).
3.2  |  Rep oduc i ee hologyandassocia ion
wi h sounds
The equency,du a ionando e allpe cen ageo malebeha iou al
ca ego ieswe esco ed o  ou soni e ousmalesindi e en ses-
sions:wi hsoundp oduc ionandsilen .The i s da ase included
se en ideo eco dings(210 min)whe ea leas onesoundoccu ed
pe  eco dingbyeachmale.O e all,weobse ed410beha iou al
ca ego ies (102.5 pe  male). The beha iou al ca ego ies Nes  dis-
play(29.3%),P e-ma ing(22.7%)andApp oach(19.1%)we emos 
equen ly obse ed while Ci cling (1.2%), Chase (1.0%) and Lead
(0.7%)we e a ely eco ded(Figu e 3).O  he410beha iou alca -
ego ies( om ou males),99ca ego ies(24.1%)we eaccompanied
by sound p oduc ion. Sound p oduc ion was documen ed du ing
ials o  he p e-spawning phase: P e-ma ing (303 sounds), Nes 
display (27 sounds),F on al display(20 sounds), Nes  ubbing(16
sounds)and o  hespawningphase:Spawning( i esounds).Sounds
didno co-occu wi h hecou shipphases:App oach,Ci clingand
Lead (Figu e 3).Du ing ou Chase,onlyonesoundwas eco ded.
Onlyonespawningac (Spawning)wasobse edin hiss udy,du ing
which i esoundswe ep oduced.Un o una ely,spawningsounds
we eno usedin hecompa a i epu posesdue o hei limi edoc-
cu ence.Thechi-squa e(χ2) es  o  independenceindica ed ha 
beha iou al ca ego ies Nes  display and P e-ma ing we e signi i-
can ly associa ed wi h sound p oduc ion (χ2 = 138.3;d  = 5;N = 99;
p < .05; esidualsco e:1.5and41.5, espec i ely),whileo he ca -
ego ies ailed osuppo  hishypo hesis(Figu e 3).
To compa e male beha iou  when soni e ous o  silen , a sec-
ondda ase o eigh  ideo eco dingswasconside ed(190.5 min)o 
hesame ou malesbu  inwhichnosound p oduc ionwas docu-
men ed.In hese eco dings,weobse ed324malebeha iou alca -
ego ies(a e aging81.0pe male),o whichApp oach(38.3%),Lead
(31.8%)andNes display(20.1%)we e hemos  equen ca ego ies
(Figu e 3).Con a y,Chase,Ci cling,P e-ma ingo Spawningwe eno 
documen edwi hin hese eco dingsessions.Ingene al, he eisan
ob iousdissimila i ybe ween he equencyo  hebeha iou alca -
ego ies ha we eo we eno accompaniedbysounds.Speci ically,
P e-ma ing,oneo  he wobeha iou ssigni ican lyassocia edwi h
soundp oduc ion,dec eased omana e ageo 22.7%in he ials
Acous ic pa ame e s
x
SD Range H p- alue
DUR(ms) 442.1 158.7 156.8–952.9 22.9 <.05
NP 14.3 5.1 5.0–32.0 24.5 <.05
PD(ms) 14.6 2.1 9.8–23.0 8.1 <.05
PRR(Hz) 32.5 2.6 26.0–38.1 36.9 <.05
PP(ms) 32.0 2.8 27.7–37.6 23.7 <.05
PF(Hz) 137.4 55.5 89.0–340.8 34.7 <.05
FMi 39.0 4.1 31.4–60.0 38.1 <.05
FM 34.1 3.7 26.1–45.4 29.9 <.05
FM(Hz) 0.9 0.1 0.7–1.1 12.8 <.05
SR(no.o sounds/min) 4.7 2.1 1.3–7.8 8.6 <.05
Callinge o 0.5 0.1 0.3–0.6 5.7 >.05
No e:Fo eachpa ame e ,mean(
x
),s anda dde ia ion(SD)and angewe e epo ed,wi h he
co esponding esul s om hein aspeci icK uskal-Wallis anksum es andp- alue.
Desc ip i es a is icsa ebasedon20soundspe malep esen ing hebes S/N a io(N = 4,n = 80).
H- aluesa e he esul so K uskal–Wallis es scompa ingsoundpa ame e samongmales.Bolded
p- aluesindica e he ea u e ha di e edbe weenmalesacco ding o hesigni icancele elo .05.
Fo  heabb e ia ionso acous icp ope ies,seeSec ion2.
TABLE 1 Desc ip i es a is icso 
soundacous icpa ame e sp oducedby
maleO sinigobius c oa icus.
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
8o 18
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   HORVATIĆ e al.
wi hsounds o0%in he ialswi hou sound.Inaddi ion,Nes dis-
play, Nes  ubbing and F on al display ca ego ies p oduced du ing
soundemissiondec easedin equencyinexpe imen swi hou  he
sounds(Nes display: om29.3% o20.1%;Nes  ubbing: om13.9%
o 3.7%; F on al display: om 12% o 6.2%). On he o he  hand,
App oach and Leadwe e mo e equen  du ing hesilen  sessions
(31%and38%, espec i ely) handu ingsoundp oduc ion(19%and
0.7%, espec i ely;Figu e 3).O e all, hebeha iou al a edec eased
om 55.8% o44.1%whenmalesp oducedsoundsin compa ison
owhen heywe esilen (soni e ous s.silen males:means2.79 s.
1.55), hough hedi e enceswe eno signi ican (Wilcoxonsigned-
ank es , N = 14;p > .05).Impo an ly, henumbe o  imes he e-
malesen e ed hemalenes di e edsigni ican lybe ween he wo
da ase s(3.71 s.0.71),as emalenes en ancewasmo e equen 
whenmalesp oducedsound hanwhen heywe esilen (Wilcoxon
signed- ank es ,N = 16,p < .05).Finally, he womales ecei ing he
mos  emaleen ieswe e hela ges insize(41.7and42.2 mmSL).
These womalesp oduced hesoundswi hhighes  alueso NP(>13
pulses),FMi(>38.5 Hz)andPRR(>33.5 Hz).
Conside ing he sessions in which sound occu ed, he e-
quency o  occu ence o  beha iou s be ween he cou ship
(Chase,Lead,App oachandCi cling)andp e-spawning(Nes dis-
play,F on aldisplay,Nes  ubbing,P e-ma ing)phaseso  ep o-
duc iondidno di e (Wilcoxonsigned- ank es ,N = 8;p > .05),
hough hei du a ion did (Wilcoxon signed- ank es , N = 8;
p < .05).Gene ally,malesexhibi edcou ship- ela edbeha iou s
FIGURE2 S uc u eo apulsa ilesoundp oducedbyO sinigobius c oa icus.Diag amillus a ing heacous ic a iablesmeasu ed om
hepulsa ilesounds.(a)Spec og amand(b)oscillog amo  hepulsa ilesound;(c)oscillog amo isola edpulses(6–8)ing ey;(d)powe 
spec umo  hepulsa ilesound om(a).DUR,sounddu a ion( o alleng ho  hecall,measu edinmilliseconds);NP,numbe o pulses;
PRR,pulse epe i ion a e(NPdi idedbyDURandmul ipliedby1000 Hz);PD,pulsedu a ion(ms);PP,pulsepe iod(a e agepeak- o-
peakin e alo consecu i epulses,ms);FM, equencymodula ion(a e  hesoundhasbeendi idedin o wosec ions,FMiandFM ,
equencymodula ionwascalcula edas hedi e encebe ween he inalandini ialpulse epe i ion a eandexp essedinHz);FMi,
equencymodula ion—ini ial(pulse epe i ion a eo  heini ialsec iono ad um);FMm, equencymodula ion-middle,FM , equency
modula ion— inal(pulse epe i ion a eo  he inalsec iono ad um);PF,peak equency(ob ainedasapeakwi h hehighes ene gy om
heloga i hmicpowe spec um unc ion,Hz;whi ea owinspec og am), 0alsoco espond o he undamen al equency.Spec og am
pa ame e s:Fla Topwindow,256leng hFFT;25% amesize;93.75%o e lap; esolu ion:4 Hz.RA, ela i eampli ude.
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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9o 18
HORVATIĆ e al.
less equen lyand o asho e pe iodcompa ed op e-spawning
beha iou s.
3.3  |  Ana omical indingsandmo emen sdu ing
sound p oduc ion
The pec o al gi dle o  O. c oa icus was subjec ed o μCT scan-
ninganddissec ion oiden i y he a iousosseouss uc u esand
muscles ha maybein ol edinsoundgene a ion.F omμCT scans,
h ee unc ionaluni swe edis inguishedin heskele alpa o  he
pec o algi dleo O. c oa icus: heshoulde gi dle(composedo  he
pos - empo al, hesup aclei h umand heclei h umbones)do sally
a ached o heneu oc anium, heshoulde pla e(i.e. ou la ge adi-
als)and he inpla e,madeupo  in aysa icula edwi h heshoul-
de pla e (Figu e 4).On hedo sal ipo  heclei h um,an e io and
pos e io p ocessesa ep esen .Thesup aclei h uma icula eswi h
hepos - empo aland heclei h um,connec ingwi h heclei h um
FIGURE3 Acous icbeha iou o O sinigobius c oa icusmalesdu ingsexualin e ac ionsexp essed h oughninebeha iou alca ego ies.
In(a),soundp oduc ionwasobse edince ainca ego ies(Chase,Nes display,F on aldisplay,Nes  ubbing,P e-ma ingandSpawning),
whichwe eaccompaniedbysoundemission(i.e. heda kg eyba a  he opo  hecolumn).In(b),nosoundp oduc ionwasobse ed.In
(a)and(b), heligh g eycolou o  hecolumnindica es henumbe o documen edcou shipbeha iou alac s.As e isk(*)indica es he
signi ican associa iono beha iou alca ego ieswi hsoundp oduc ion.Numbe sabo ecolumnsindica e he o alnumbe o  eco ded
soundspe eachbeha iou alca ego y.Theg eena eaencompasses hecou shipphasepe o medou sideo  henes , hedashedblue
a eahighligh s hep e-spawningphasewhile hedashedo angea eaindica es hespawningphaseo  ep oduc ion,displayedin henes .
20457758, 2023, 11, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/ece3.10673 by Coch ane Po ugal, Wiley Online Lib a y on [24/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
16o 18
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SUPPORTINGINFORMATION
Addi ional suppo ing in o ma ion can be ound online in he
Suppo ingIn o ma ionsec iona  heendo  hisa icle.
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Endemic ishcalling:Acous icsand ep oduc i ebeha iou 
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