Beak colou a ion o s a ling (S u nus unicolo ) males depends on he leng h o hei 1
h oa ea he s 2
3
Manuel Azcá a e-Ga cía1, Magdalena Ruiz-Rod íguez1, C is ina Ruiz-Cas ellano1, 4
Sil ia Díaz-Lo a2, Gus a o Tomás1, Manuel Ma ín-Vi aldi2,3 & Juan José Sole 1,3
5
6
7
1 Depa amen o de Ecología Funcional y E olu i a, Es ación Expe imen al de Zonas 8
Á idas (CSIC), Alme ía. Spain. 9
2 Depa amen o de Zoología, Facul ad de Ciencias, Uni e sidad de G anada, G anada. 10
Spain. 11
3 Unidad asociada (CSIC): Coe olución: cucos, hospedado es y bac e ias simbion es. 12
Uni e sidad de G anada, 18071-G anada, Spain. 13
14
15
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17
Co esponding Au ho :
18
Manuel Azcá a e-Ga cía 19
ADDRESS: Es ación Expe imen al de Zonas Á idas: C a. de Sac amen o s/n, La 20
Cañada de San U bano, 04120, Alme ía (Spain) 21
TLF: (+34) 660058398 22
E-MAIL: mazca a ega c[email p o ec ed] 23
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C oss e Funding Da a Regis y 26
This wo k was suppo ed by he cu en ly named Minis e io de Ciencia, Inno ación y 27
Uni e sidades and Eu opean (FEDER) unds (CGL2013-48193-C3-1-P, CGL2017-28
83103-P, CGL2017-89063-P). MAG was inanced by a p edoc o al con ac (BES-2014-29
068661) om he Spanish Minis e io de Economía y Compe i i idad, and GT by he 30
Ramón y Cajal P og amme. 31
32
Acknowledgmen s
33
We hank Na alia Juá ez Ga cía-Pelayo and Ca men Sole Zamo a o he help in 34
cap u ing bi ds and collec ing da a om he ield. The esea ch g oup bene i s om 35
acili ies, including and apa men , p o ided by he ci y hall o Guadix whe e a small lab 36
o quickly p oceed he samples was ins alled.
37
38
E hical No e
39
We pe o med he s udy ollowing he ele an Spanish na ional (Dec e o 40
142/2013, 1 de oc ub e) and egional guidelines. The e hics commi ee o he Spanish 41
Na ional Resea ch Council (CSIC) app o ed he p o ocol, and he Conseje ía de Medio 42
Ambien e de la Jun a de Andalucía, Spain, p o ided all he necessa y pe mi s o nes and
43
bi ds manipula ions (Re : SGMN/GyB/JMIF). The p o ocols adhe ed o he ASAB/ABS 44
Guidelines o he Use o Animals in Resea ch. The au ho iza ion o ca ying ou he 45
expe imen al wo k on animals in he ield was g an ed by he Di ección Gene al de 46
Ges ión del Medio Na u al o he Conseje ía de Medio Ambien e o Jun a de Andalucía. 47
48
Au ho con ibu ions
49
Concei ed and designed he expe imen s: JJS, MRR, GT and MMV. Fieldwo k: 50
MAG, JJS, MRR, GT, CRC and SDL. Analysed he da a: MAG and JJS. Con ibu ed 51
eagen s/ma e ials/analysis ools: subs an ial con ibu ion om all au ho s. MAG w o e 52
he i s e sion wi h supe ision o JJS and MRR. All au ho s subs an ially con ibu ed 53
o inal e sion. 54
55
Da a accessibili y 56
Da a used in his pape can be ound in CSIC Ins i u ional Reposi o y, wi h he 57
accession numbe s <xxxxxxxx>. 58
59
1
Lay summa y
1
2
The use o signals o indica e he indi idual quali y is widesp ead in na u e. Howe e , 3
al hough mos species show mo e han one signal, he ela ionships be ween di e en 4
signals ha e almos ne e been expe imen ally s udied. He e, we demons a ed ha he
5
expe imen al educ ion o h oa ea he s leng h condi ioned he beak colou a ion o 6
spo less s a ling males a he ime o ep oduc ion. Ou esul s a e he i s expe imen al 7
e idence o wo sexually dimo phic ai s being ela ed in na u al condi ions.
8
2
Beak colou a ion o s a ling (S u nus unicolo ) males depends on he leng h o hei 9
h oa ea he s 10
Abs ac
11
12
Wi hin he con ex o complex sexual signalling, mos esea ch has ocussed on explo ing 13
he associa ions be ween se e al signals and/o hei ela ionships wi h di e en p oxies 14
o indi idual quali y. Howe e , e y ew s udies ha e ocused on checking whe he he 15
exp ession o one signal is condi ioned by he exp ession o he o he s. He e, by 16
expe imen ally sho ening he h oa ea he s o male spo less s a lings (S u nus 17
unicolo ), we e alua ed he in luence o his ai on he colou exp ession o he beak 18
base. In addi ion, we es ed he ela ionship be ween hese wo sexually dimo phic 19
cha ac e s wi h ai s indica ing indi idual quali y such as body condi ion and colou 20
e lec ance a he wa eleng h ela ed o ca o enes in he ip o he beak. Ou esul s show 21
ha he colou a ion o he beak base in males, bu no in emales, is posi i ely ela ed o 22
body condi ion and o he leng h o o namen al h oa ea he s. Mo eo e , he 23
expe imen al sho ening o h oa ea he s in males had a nega i e e ec on he blue 24
ch oma in ensi y o hei beak base one yea a e manipula ion. These esul s suppo o 25
he i s ime a causal link be ween he exp ession o wo sexually dimo phic cha ac e s, 26
which is essen ial o unde s and hei unc ionali y in a mul iple signalling amewo k. 27
28
Keywo ds: Beak colou , Body condi ion, In e ac ing signals, Mul iple signals, 29
O namen al ea he s leng h, Sexually dimo phic cha ac e s. 30
31
32

3
In oduc ion
33
Animals use a wide a ay o signals o in o m abou hei pheno ypic o gene ic 34
quali y o conspeci ics in social in e ac ions, in con ex s such as ma e choice o 35
compe i ion o esou ces (Kokko 2003, Ande sson and Simmons 2006, K aaije eld e 36
al. 2007, Lyon and Mon gome ie 2012, Edwa d 2015). In con ex s o sexual selec ion, 37
males ypically possess mul iple ai s ha may con ey independen in o ma ion o 38
ecei e s (Mølle and Pomiankowski 1993). Al hough mos esea ch on he e olu ion and 39
unc ion o signals has ocused on single ai s, he impo ance o s udying hese 40
cha ac e s wi hin he heo e ical and mo e ealis ic amewo k o mul iple signals has 41
been highligh ed (Candolin 2003, Hebe s and Papaj 2005). Di e en cha ac e s migh , o 42
ins ance, imply mul iple o edundan messages (i.e., in o ma ion), o migh be mo e 43
e icien in pa icula en i onmen s o in s imula ing pa icula senso y channels (Mølle 44
and Pomiankowski 1993, Candolin 2003, Hebe s and Papaj 2005). Hebe s and Papaj 45
(2005) de eloped a amewo k o es able hypo heses o explaining he e olu ion and 46
unc ioning o mul iple signals. They highligh ed (i) he impo ance o conside ing 47
complex signals and he uni o cha ac e selec ion; (ii) ha complex signals include 48
se e al cha ac e s ha unc ion oge he , ei he acili a ing he ansmission (e.g., using 49
di e en senso y channels) o ein o cing ansmi ed in o ma ion o ecei e s (i.e., 50
edundan in o ma ion); and (iii) ha indi idual signals o componen s o complex signals 51
do no necessa ily unc ion independen ly, bu may in e ac in a unc ional way. 52
Mos esea ch on complex signalling has ocussed on explo ing he associa ion 53
be ween se e al signals (Pe ie e al. 2002, B o-Jo gensen and Dabels een 2008, Mason 54
e al. 2014, Chaine and Lyon 2015, Gi a d e al. 2015), o be ween signals and di e en 55
p oxies o i ness including pheno ypic quali y (Balm o d e al. 1992, Ma in and Lopez 56
2009), ma ing success (Mølle and Pomiankowski 1993) and e icacy o signal 57
4
ansmission in di e en en i onmen s (Endle and Houde 1995). E en hough he s udy 58
o he in e ac ions (i.e., associa ions) be ween di e en signalling cha ac e s is essen ial 59
o know indi idual o complex signals unc ioning, i is one o he leas explo ed a eas 60
wi hin he ield o signal e olu ion. The s udy o signal in e ac ions has he po en ial o 61
shed ligh on signal unc ioning because, o ins ance, de ec ing a posi i e associa ion 62
would sugges ha ansmi ed in o ma ion is edundan o complemen a y. Mo eo e , a 63
nega i e associa ion would indica e ha a ade-o be ween signalling cha ac e s exis s, 64
while he absence o associa ion be ween di e en signals would sugges ha hey con ey 65
di e en in o ma ion o ecei e s (Candolin 2003, Hebe s and Papaj 2005). In mos 66
ins ances, in e -signal in e ac ion occu s when he p esence o one signal o a signal 67
componen al e s he esponse o he ecei e o a second signal o componen by 68
ampli ying o condi ioning he in o ma ion p o ided by each o he . 69
In e ac ions be ween signals may also occu when he p oduc ion o one signal 70
in luences he cos o p oduc ion o ano he signal (Johns one 1996, Candolin 2003). In 71
his case, independen ly o he ansmi ed in o ma ion, he pheno ypic exp ession o one 72
signal impinges on he esul ing pheno ype o he o he signal. Signals a e ypically cos ly 73
o p oduce (Hasson 1994, Sal ado e al. 1996), o main ain (Ruiz-Rod íguez e al. 2015), 74
o o show (i.e. social cos ; Tibbe s and Dale 2004), and he exp ession o signals o 75
signal componen s may be aded-o agains each o he . On he one hand, he e could be 76
a ade-o be ween wo signals (e.g. by using he same esou ces as ca o enes), so ha a
77
lesse exp ession o one inc eases he exp ession o he o he (Ande sson e al. 2002). On 78
he o he hand, i is also possible ha he exp ession o one signal educes he a e age 79
cos s due o social in e ac ions (Mo ales and Velando 2018) and, hus, acili a es o 80
enhances he exp ession o o he signals. Fo ins ance, o namen s ha de elop be o e 81
ep oduc ion and unc ion in social con ex s o he s han sexual (e.g. in a-sexual), and 82
5
could se e o s ablish social hie a chy, may educe agonis ic social in e ac ions and 83
mi iga e subsequen ene ge ic cos s. Sa ed ene gy could hus enhance he p oduc ion o 84
o he sexual o namen s du ing cou ship o ep oduc ion and, he e o e, he exp ession o
85
o namen s de eloped be o e and du ing ep oduc ion could be posi i ely ela ed. This 86
migh be he case o ce ain plumage cha ac e is ics o bi ds ha educe social cos s 87
be o e ep oduc ion (Sena e al. 2000), and hus, could boos he exp ession o o he 88
sexually selec ed ai s, such as song o o he simila lexible dynamics ai s, ha a e 89
exclusi ely exp essed du ing ep oduc ion (Badyae e al. 2002, Mason e al. 2014). 90
De ec ing e idence suppo ing he hypo hesis ha he exp ession o one signal is 91
condi ioned by he exp ession o o he signals can be challenging. A main eason is ha 92
sexual signals a e ypically condi ion-dependen (e.g. Saino e al. 1997, Velando e al. 93
2006, Sole e al. 2008). Thus, de ec ing posi i e o nega i e associa ions be ween he 94
exp ession o di e en signals is no enough o in e causa ion. Ra he , his hypo hesis 95
should be es ed in expe imen al amewo ks whe e he modi ica ion o one signal causes 96
o explains he pheno ypic exp ession o o he signals. As a as we know, his hypo hesis 97
has been es ed expe imen ally only once by Hende son e al. (2018), who manipula ed 98
plumage colou a ion o house inch (Haemo hous mexicanus) males be o e ep oduc ion 99
and de ec ed an e ec on male in es men in song unde cap i i y condi ions. Howe e , 100
he e ec was dependen on expe imen ally modi ied social con ex ( ea he colou a ion 101
o neighbou s) and, hus, i is no comple ely clea ha he de ec ed e ec s we e 102
exclusi ely caused by cos s associa ed o plumage colou a ion. He e, we go a s ep u he 103
and look o expe imen al e idence suppo ing he hypo hesis in he wild in spo less 104
s a lings (he ea e s a lings, S u nus unicolo ). 105
S a lings a e semi colonial and sexually dimo phic bi ds, wi h males showing 106
elonga ed h oa ea he s (Hi aldo and He e a 1974, Lezana e al. 2000) and conspicuous 107
6
yellow beak wi h blue colou ed basal pa (Na a o e al. 2010). These wo sexually 108
dimo phicseconda y sexual ai s could ha bou di e en kinds o in o ma ion o , a leas , 109
in o ma ion a di e en ime scales. The apical pa o hese ea he s is qui e lexible, and 110
males exhibi hem e y conspicuously du ing he en i e yea in social in e ac ions, 111
including cou ship (Apa icio e al. 2001, Ruiz-Rod íguez e al. 2015). In addi ion, hese 112
ea he s hones ly e lec he pheno ypic quali y o indi iduals (Lezana e al. 2000, López-113
Rull e al. 2007, Gil and Cul e 2011, Ruiz-Rod íguez e al. 2015). On he o he hand, 114
du ing ma ing and ep oduc ion ( om Feb ua y o July in ou s udy a ea), he o he wise 115
black colou ed beak o s a lings u n o yellow colou a ion in bo h sexes, while i s basal 116
pa u n o blue in males and o pink in emales (C amp 1998) (Fig.1). Beak colou in 117
s a lings is a sexually dimo phic and dynamic ai ha likely e lec s an ioxidan capaci y 118
(Na a o e al. 2010) and, acco dingly, p e ious s udies ound ha he yellow colou o 119
he beak is ela ed o he le el o ca o enoids and i amin A in he plasma in bo h sexes 120
(Na a o e al. 2010). The moul o h oa ea he s occu s in Sep embe -Oc obe (Veiga 121
and Polo 2016), hus a be o e he ep oduc i e pe iod. The e o e, i is likely ha hese 122
ea he s se e o s ablish social hie a chies wi hin he popula ion du ing he whole yea , 123
allowing o educe agonis ic in e ac ions and o mi iga e i s associa ed cos s (Ande sson 124
1994). I ha was he case, he leng h o he h oa ea he s could play an impo an ole 125
du ing he non-b eeding season by a ec ing he acquisi ion and alloca ion o esou ces, 126
which could be e lec ed in he in ensi y o beak colou a ion in s a ling males. Leng h o 127
h oa ea he s can be easily manipula ed (see Ma e ial and Me hods), so he hypo hesis 128
ha he exp ession o one signal (leng h o h oa ea he s) de e mines he exp ession o 129
he o he (beak colou a ion) can be expe imen ally es ed. 130
We manipula ed he leng h o he h oa ea he s o males by cu ing-o 131
app oxima ely he hal -dis al po ion o he ea he s be o e ep oduc ion, and explo ed i s 132
13
ac o (i.e. dependen a iable), and expe imen al ea men as he ca ego ical p edic o . 286
The da e o i s and las cap u es, as well as numbe o days be ween cap u es we e 287
included as con inuous independen a iables in he s a is ical models. In addi ion, we 288
checked whe he he expe imen did a ec leng h o h oa ea he s a e moul , by 289
ca ying ou epea ed-measu es ANOVAs. In his model, he ea he leng h, a i s and 290
las cap u es, was he dependen a iables ( epea ed measu es), he expe imen al 291
ea men was he ca ego ical p edic o , and he numbe o days be ween cap u es was he 292
con inuous independen a iable. Residuals o all s a is ical models we e plo ed and 293
isually checked o no mali y. All analyses we e pe o med wi h S a is ica V13 (Dell-294
Inc. 2015). 295
296
Resul s
297
The blue ch oma and he yellow- ed ch oma o he s a lings’ beak-base a e 298
nega i ely ela ed in bo h males (Be a(SE) = -0.68(0.08), F1,54 = 74.64, p < 0.001 ) and 299
emales (Be a(SE) = -0.92(0.03), F1,100 = 933.58, p < 0.001). Mo eo e , he base (400-300
475 nm and 570-700 nm) and ip (450-570 nm) beak colou s we e no signi ican ly 301
associa ed in males (blue400-475: Be a(SE) = 0.02(0.13), F1,54 = 0.03, p = 0.867; yellow- ed 302
570-700: Be a(SE) = -0.08(0.11), F1,54 = 0.49, p = 0.487), bu a endency (posi i e o blue 303
and nega i e o yellow- ed ch oma) was de ec ed in emales (blue400-475: Be a(SE) = 304
0.14(0.07), F1,100 = 3.92, p = 0.050; yellow- ed570-700: Be a(SE) = -0.14(0.07), F1,100 = 305
3.77, p = 0.055). 306
Body condi ion was posi i ely and nega i ely ela ed o in ensi y o blue and 307
yellow- ed colou a ion o males’ beak, espec i ely (Table 1, Fig. 3). Nei he he 308
b igh ness o males’ beak no he leng h o hei h oa ea he s we e ela ed o body 309
condi ion (Table 1). In emales, none o hese a iables p edic ed body condi ion (Table 310
1). Simila ly, leng h o h oa ea he s o males, bu no ha o emales, was posi i ely 311

14
and nega i ely ela ed o blue and yellow- ed colou in ensi y o males’ beak, espec i ely 312
(Table 1, Fig. 3). Beak b igh ness did no p edic leng h o h oa ea he s o males o 313
emales (Table 1). Thus, he blue colou in ensi y o males’ beak co- a ied wi h he leng h 314
o h oa ea he s, which migh in o m emales on he pheno ypic quali y (body condi ion) 315
o males. 316
Impo an ly, he expe imen al sho ening o h oa ea he s in males p o oked a 317
educ ion in he in ensi y o he blue, bu no o he , colou a ion o hei beaks (measu ed 318
one o wo yea s a e manipula ion o h oa ea he s) (Table 2, Fig. 4). Mo eo e , he 319
expe imen al manipula ion did no a ec body condi ion o he leng h o h oa ea he s 320
in subsequen cap u es (Table 2). Nei he da e o i s and second cap u e no ime 321
be ween he wo cap u es did explain addi ional signi ican p opo ion o a iance ( esul s 322
no shown). These esul s sugges a di ec link be ween leng h o h oa ea he s and beak 323
colou a ion o males, which is independen o he associa ion o bo h cha ac e s wi h 324
pheno ypic condi ion o males. 325
326
Discussion
327
Ou main esul s a e ha (i) in ensi y o colou a ion o he beak base o spo less 328
s a ling males, bu no ha o emales, was posi i ely ela ed o body condi ion; (ii) beak 329
colou a ion o males was posi i ely ela ed o he leng h o hei o namen al h oa 330
ea he s, and (iii) he expe imen al sho ening o h oa ea he s in males had a nega i e 331
e ec on he blue ch oma in ensi y o he beak o males one yea a e manipula ion. 332
Leng h o h oa ea he s and beak colou a ion a e wo sexually dimo phic ai s ha 333
e lec pheno ypic quali y o males (Apa icio e al. 2001, Na a o e al. 2010) and, hus, 334
ou esul s demons a e a di ec connec ion be ween hese wo ai s sugges ing ha hey 335
may unc ion as a whole in a mul iple signalling amewo k. 336
15
S a lings ha e se e al known sexually dimo phic ai s and a e an app op ia e 337
model sys em o explo e unc ional in e ac ions be ween sexual signals. Mos s udies on 338
sexual signals in his species a e ocussed on he leng h o h oa ea he s o males, which 339
p edic s ma ing success (Apa icio e al. 2001), gene ic he e ozigosi y (Apa icio e al. 340
2001), immune esponse (Gil and Cul e 2011) and elome e leng h (Azcá a e-Ga cía e 341
al. 2020). Bill colou a ion o he dis al yellow pa has also been s udied as a sexually 342
selec ed ai o he species because i is ela ed o ca o enoid and i amin A concen a ion 343
in he blood o males and emales, bu only du ing he ma ing pe iod (Na a o e al. 2010). 344
Sexual di e ences a e howe e mo e appa en a he basal pa o he beak (Fig 1), and 345
we concen a ed on his ai o expe imen ally explo e he possible associa ion wi h he 346
leng h o h oa ea he s. In ag eemen wi h he assump ion ha he blue colou a ion o 347
he basal pa o he beak has a sexual-signalling unc ion, we ound ha i s blue-colou 348
in ensi y was posi i ely ela ed wi h bo h body condi ion and he leng h o he h oa 349
ea he s. Thus, explo ing he in e ac ion be ween hese wo ai s is jus i ied. 350
Leng h o h oa ea he s and beak colo a ion o s a ling males p o ide 351
in o ma ion a di e en ime scales. Black ea he s a e ela i ely s a ic and would p o ide 352
in o ma ion o he pheno ypic condi ion and quali y o males a he ime o moul ing 353
(Badyae and Hill 2000, Hebe s and Papaj 2005). Mo eo e , ea he de e io a ion would 354
also p o ide in o ma ion on ea he quali y and on abili y o males educing ea he 355
deg ada ion (Shawkey e al. 2007, Shawkey e al. 2009, Ruiz-de-Cas añeda e al. 2012, 356
Ruiz-Rod íguez e al. 2015). Thus, leng h o h oa ea he s migh e en include di e en 357
kinds o in o ma ion a a long- e m scale. The beak colou a ion, howe e , should unc ion 358
a a sho ime scale. Like o he colou a ion o o he ba e pa s o bi ds, beak colou a ion 359
has he po en ial o change wi hin weeks, days, hou s, o e en seconds (I e son and 360
Ka ubian 2017). Thus, his kind o dynamic cha ac e s should be con inuously e alua ed 361
16
by ecei e s (Velando e al. 2006, Simons and Ve huls 2011, Dey e al. 2015). As a as 362
we know, associa ions be ween hese wo ypes o sexually dimo phic ai s ha e ne e 363
been assessed. 364
Ou esul s showed ha male body condi ion a he ime o ma ing was ela ed o 365
blue colou a ion o he beak, bu no o he leng h o h oa ea he s, sugges ing ha bo h 366
signals do no p o ide iden ical bu , pe haps, complemen a y in o ma ion. This could be
367
due o he ac ha h oa ea he leng h would explain body condi ion o males a he 368
ime o moul ing, while beak colou a ion would be a mo e dynamic cha ac e ha , 369
simila ly o he colou o he legs o blue- oo ed boobies (Sula nebouxii) (To es and 370
Velando 2007), shows indi idual condi ion a he ime o cap u e. Howe e , leng h o
371
h oa ea he s was posi i ely ela ed o he in ensi y o blue colou a ion o he beak and, 372
hus, i is possible ha bo h ai s con ey edundan in o ma ion o emales. In ag eemen 373
wi h he possibili y ha hese wo ai s ans e complemen a y in o ma ion o emales, 374
we expe imen ally showed a nega i e e ec o leng h o h oa ea he s on he in ensi y 375
o he blue ch oma o he beak base o males se e al mon hs a e he manipula ion. We 376
know ha colou a ion o he ip o he beak e lec s he an ioxidan capaci y o s a lings 377
(Na a o e al. 2010). The associa ion be ween beak colo a ion and ca o enoids’ 378
concen a ion in he blood has also been de ec ed in o he species (Fai e e al. 2003). We 379
did no measu e concen a ion o ca o enoids in he blood in his s udy and, hus, we 380
canno explo e whe he his associa ion exis s o he blue colo a ion o he beak base o 381
males. Mo eo e , colou e lec ance o he beak ip a he ca o enoid wa eleng h, which
382
esul ed posi i ely ela ed o ca o enoid le el in s a lings (Na a o e al. 2010), was no 383
ela ed o colou a ion o he base o he beak o males. Consequen ly, he colou a ion o 384
he beak base is unlikely con eying in o ma ion on an ioxidan capaci y o emales. Thus, 385
ou expe imen al esul s should be in e p e ed as leng h o h oa ea he s unc ioning 386
17
du ing he non- ep oduc i e pe iod and de e mining pheno ypic condi ion o males du ing 387
ma ing. 388
Like o he signals ope a ing in non-sexual scena ios such as pa en -o sp ing 389
communica ion (Mo ales and Velando 2018), o sibling nego ia ion (Johns one and 390
Roulin 2003, Sole and A ilés 2010), including hose media ed by ea he colou a ions 391
(Sena 2006), he leng h o h oa ea he s o males migh se e o s ablish some kind o 392
social hie a chy be ween males ha educe he p obabili y o agonis ic in e ac ions 393
among indi iduals o di e en s a us (Rohwe 1975, Sena 1999, McG aw and Hill 2000). 394
S a lings moul h oa ea he s se e al mon hs be o e ep oduc ion, and males equen ly 395
display hese ea he s while singing in high isible places du ing non- ep oduc i e 396
pe iods (pe s. obs.), which migh ha e a unc ional signi icance in a con ex o social 397
in e ac ions. In some bi d species, p obabili y o social agg ession by conspeci ics is 398
ela ed o ea he cha ac e is ics signalling bi d s a us (Sena 1990, McG aw e al. 2007, 399
Chaine and Lyon 2008)). Mo eo e , agg essions a e mo e common among indi iduals 400
showing simila s a us (Midamegbe e al. 2011), wi h indi iduals ha bou ing signals o 401
highe quali y elici ing lowe le el o agg essi eness (Lopez-Idiaquez e al. 2016). The 402
expe imen al educ ion o h oa ea he s las s un il he nex moul pe iod in au umn and, 403
hus, i is possible ha s a ling males wi h longe h oa ea he s expe ienced lowe a es 404
o social agg essions du ing he non- ep oduc i e pe iod. These cos s can a ec he 405
exp ession o o he ai s ela ed o pheno ypic condi ion, including immune esponses 406
(Hawley e al. 2006), oxida i e s a us (Gal án and Alonso-Al a ez 2009) o he 407
exp ession o sexual signals (Mølle e al. 2000). Al hough we ha e no da a on p obabili y 408
o agg ession o social in e ac ions in gene al in ela ion o leng h o h oa ea he s in 409
s a lings, we hink ha social cos s associa ed o he expe imen al educ ion o leng h o 410
h oa ea he s du ing he non-b eeding pe iod is he mos likely explana ion o he
411
18
de ec ed expe imen al e ec s on beak colou a ion du ing ep oduc ion. Howe e , his 412
mechanis ic explana ion dese es u he esea ch explo ing o ins ance he expec ed 413
associa ion be ween ea he leng h and agg ession du ing he non- ep oduc i e pe iod. 414
Wha e e he mechanis ic explana ions, ou expe imen al esul s s ongly sugges 415
a causal link be ween exp ession o wo sexually dimo phic ai s in spo less s a lings. As 416
a as we know, causal links be ween wo sexually selec ed ai s ha e only been de ec ed 417
in ano he bi d species, he house inch, a highly social species in which head and b eas 418
ea he s o males show g ea a iabili y om ed o yellow colou a ion (Hende son e al. 419
2018). Hende son e al. (2018) ound ha ed- ea he ed males a e mo e a ac i e and 420
sing mo e han yellow- ea he ed males bu , when yellow males we e housed wi h ed 421
males, hey sang mo e han when housed wi h equally una ac i e yellow males. Thus, 422
males adap ed hei singing e o o he social en i onmen (a ac i eness) de e mined 423
by he plumage colo a ion o he social g oups. The e o e, he de ec ed link was 424
explained, no as a di ec consequence o one o he ai s, bu indi ec ly by he social 425
en i onmen in e ms o le el o a ac i eness o neighbou s, which was also 426
manipula ed. Ou expe imen al esul s he e o e show a di ec causal e ec o leng h o 427
h oa ea he s on he exp ession o he colou a ion o he base o he beak o spo less 428
s a ling males, a ai ha is only exp essed du ing he ep oduc i e pe iod. 429
To conclude, we demons a e o he i s ime a causal link be ween he 430
exp ession o wo sexually dimo phic cha ac e s, which is essen ial o unde s and hei
431
unc ionali y in a mul iple signalling amewo k. This ype o in e ac ions be ween 432
sexually selec ed signals migh be widesp ead in na u e and could be mo e easily de ec ed 433
when conside ing signals ha , like ea he colo a ion o mo phological ai s, ha e 434
signalling unc ions in non- ep oduc i e con ex s. 435
436

19
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22
591
29
Fig.4 643
644
Fi s Second
Cap u e
0.18
0.20
0.22
0.24
0.26
Blue In ensi y
Expe imen al
Con ol
645
646
647

30
Annex 1 648
649
Table A1: Numbe o cap u es made o each o he ecap u ed spo less s a ling males 650
du ing he s udy and he numbe o imes he ea men was applied o each male. 651
652
Ring
T ea men
Yea 2015
Yea 2016
Yea 2017
Numbe o ea men s
3256561
Con ol
2
1
3256564
Con ol
1
1
1
3256565
Con ol
1
1
1
3256567
Con ol
2
1
1
3256590
Con ol
2
1
1
3301955
Con ol
3
1
1
3368660
Con ol
2
1
3368681
Con ol
1
1
1
2
3369509
Con ol
1
1
1
2
3406027
Con ol
1
1
1
3418841
Con ol
1
1
1
3256556
Expe imen al
2
1
3285646
Expe imen al
2
1
3387759
Expe imen al
1
2
1
2
3387764
Expe imen al
2
1
1
3387774
Expe imen al
1
1
1
3387838
Expe imen al
1
1
1
3392095
Expe imen al
1
1
1
3428304
Expe imen al
2
1
3256564b
Expe imen al
1
2
1
3256565b
Expe imen al
1
1
1
3256567b
Expe imen al
1
1
1
653