upon encountering a predator , prey need to choose an appropriate anti - predator strategy ( caro 2005 ) . one typical strategy is to escape from a predator and seek safety to avoid predation ( cooper and blumstein 2015 ) . another strategy is to mob a predator , where prey individuals cooperatively approach and harass the predator with intensive behavioural displays , typically driving it away from the vicinity ( curio 1978 ) . mobbing has been reported in many animal taxa , such as fish ( dominey 1983 ) , birds ( curio 1978 ) , and mammals ( tamura 1989 ; graw and manser 2007 ) . in forest birds , mobbing often involves individuals from multiple species that share the same predators ( sieving et al . 2004 ) , and this may be facilitated by the attraction to mobbing calls produced by initiators ( hurd 1996 ; forsman and mnkknen 2001 ; templeton and greene 2007 ; nolen and lucas 2009 ; randler and frschler 2011 ; randler and vollmer 2013 ; wheatcroft and price 2013 ) . since mobbing as a large group is more effective than mobbing as a small group ( flasskamp 1994 ; krams et al . 2009 ) , interspecific attraction to mobbing calls would help provide a mutual benefit for both initiators and attendees in reducing their predation risk . while most of these studies have focussed on the context of self - defence , multi - species mobbing also occurs in the context of brood defence , where parents produce repeated mobbing calls and harass a nest predator ( caro 2005 ) . although these calls may carry a cost of attracting additional nest predators to the nests ( krama and krams 2005 ) , parents may reduce the immediate risk of nest predation by eliciting appropriate anti - predator reactions in receivers ( magrath et al . mobbing as self - defence , grouping can increase the efficacy of defence against nest predators ( robinson 1985 ; krams et al . 2011 ) and mobbing calls may serve to facilitate the attraction of other species to the nests . however , there is a lack of studies on the influence of mobbing calls on the formation of multi - species mobbing at nests . in this study , i investigated the response of forest birds to heterospecific mobbing calls produced in the context of brood defence . japanese great tits ( parus minor ) use a sophisticated system of anti - predator communication that distinguishes different nest predators with acoustically distinct mobbing calls . the tits produce  jar  calls specifically for snakes and  chicka  calls for other predators such as crows and martens ( suzuki 2011 , 2014 ) . these calls transmit sufficient information about the type of nest predator ( snakes versus non - snake predators ) to both parents ( suzuki 2012a , 2015 ) and nestlings ( suzuki 2011 ) and , therefore , are considered as functionally referential signals ( gill and bierema 2013 ) . such information may also be detected by other species breeding within the same habitat as japanese great tits ( see magrath et al . i played back the two types of mobbing calls near the nests of great tits in the absence of actual predators and recorded the behavioural responses of heterospecific species . i conducted the experiments at 14 separated sites ( at least 250  m apart ) in a mixed deciduous coniferous forest in karuizawa , nagano , japan ( 362122n , 1383536e ) where nest boxes of japanese great tits were located ( attached to tree trunks 1.8  m from the ground ) for their first broods of the season . in this forest , many species of forest birds were observed to breed within the same habitat as japanese great tits ( table  1 ) . notably , coal and willow tits were abundant in the forest and were observed in all experimental sites . most species were observed to carry food items to their nests , indicating that they were caring for nestlings . long - tailed tits started breeding earlier than other species , and were observed to form family groups and provide food to fledglings . in addition to these resident species , there were some migratory species , such as brown flycatchers and narcissus flycatchers , breeding in the same forest.table  1total number of individuals and trials in which individuals approach within 10  m of a speaker during playbacks of  chicka  and  jar  callsspecieschickajarnumber of individualsnumber of trialsnumber of individualsnumber of trialscoal tit ( periparus ater ) 
 2174willow tit ( poecile montanus ) 
 3333long - tailed tit ( aegithalos caudatus ) 
 0031nuthatch ( sitta europaea ) 
 0032japanese white eye ( zosterops japonicus ) 
 0053meadow bunting ( emberiza cioides ) 
 0011brown flycatcher ( muscicapa dauurica ) 
 0011narcissus flycatcher ( ficedula narcissina ) 
 1111brown - eared bulbul ( hypsipetes amaurotis ) 
 2233japanese pygmy woodpecker ( dendrocopos kizuki ) 
 0022 
 resident species 
 immigrant species 
 nestling period 
 post - fledgling period 
  total number of individuals and trials in which individuals approach within 10  m of a speaker during playbacks of  chicka  and  jar  calls 
 post - fledgling period i tested the response of heterospecific species of birds to playbacks of  chicka  calls ,  jar  calls , and background noise ( control ) . playback stimuli were constructed by using recordings of  chicka  calls from 10 individuals ( six males and four females ) and those of  jar calls from nine individuals ( five males and four females ) . for each mobbing call playback , five calls were chosen from the same individual , and these calls were repeated to fill a 3-min sound file at a rate of 5 calls/12  s ( roughly one call every 2.4  s ) . this calling rate was within the range of natural repetition rates for both  chicka  and  jar  calls at the early nestling stage ( suzuki 2012a ) . low - frequency ( < 1  khz ) noise was filtered out and the calls were amplified on a computer . sound editing was conducted using adobe audition 3.0 software ( adobe corporation , san jose , ca , usa ) and more detailed information has been provided elsewhere ( suzuki 2012a ) . experiments were carried out from 3 june to 16 june 2011 when great tit nestlings were 69  days old . an at - spg50 speaker ( audio - technica corporation , tokyo , japan ) was hung from a tree and fixed 1.6    0.2  m from the ground and 4.8    0.6  m from the nest box ( mean    sd , n  =  14 ) . the playback speaker was connected to an r-09 hr digital audio recorder ( roland corporation , shizuoka , japan ) using exc-12a extension cords ( victor company of japan , kanagawa , japan ) , which enabled the control of playbacks from an observation position 15  m away from the nest . playbacks commenced when a pair of great tits was within 5  m of the nest and neither bird was vocalizing , in order to control for the effect of the presence of the tits on the behaviour of heterospecific individuals . mobbing calls were played back at a constant volume ( 75  db at 1  m ) , and background noise was played back at the same amplitude as the background noise level of the call playbacks ( 50  db at 1  m ) . all three stimuli (  chicka  calls ,  jar  calls , and background noise ) were tested at each nest . each trial was separated by at least 2  h to reduce habituation ( see also suzuki 2012a ) . the speaker position was fixed for all treatments at each site in order to control its possible effect on responses . trials were conducted in calm , dry weather between 0830 and 1600  h ( japan standard time ) . during the 3  min of playbacks , in order to assess the intensity of the approaching response , i prepared three different thresholds of distance from a speaker : 3 , 5 , and 10  m. i also recorded whether birds gazed at the ground during trials , which was defined as occurring when birds looked towards the ground and pointed their bills in the same direction while raising their tails above their heads ( see suzuki 2012a ) . all statistical tests were performed using r 3.1.2 ( r core team 2014 ) and its packages . nnet ) was used to examine whether the composition of species that approached the speaker differed according to the experimental site or playback call types . i included the species of individual birds as dependent variables , and the experimental site and treatments (  chicka  or  jar  ) as independent variables . stats ) was used for the primary analysis ; exact wilcoxon matched - pairs signed - rank tests (  wilcox.exact in exactranktests ) were used for the post hoc analyses because of the small sample size ( mundry and fischer 1998 ) . stats ) to investigate whether the performance of gazing behaviour was affected by the type of playback calls . all tests were two - tailed , and the significance level was set at   =  0.05 . although my analyses involved multiple comparisons of responses , i did not adjust the significance level as the smaller number of samples increases the probability of making type ii errors ( nakagawa 2004 ) . i conducted the experiments at 14 separated sites ( at least 250  m apart ) in a mixed deciduous coniferous forest in karuizawa , nagano , japan ( 362122n , 1383536e ) where nest boxes of japanese great tits were located ( attached to tree trunks 1.8  m from the ground ) for their first broods of the season . in this forest , many species of forest birds were observed to breed within the same habitat as japanese great tits ( table  1 ) . notably , coal and willow tits were abundant in the forest and were observed in all experimental sites . most species were observed to carry food items to their nests , indicating that they were caring for nestlings . long - tailed tits started breeding earlier than other species , and were observed to form family groups and provide food to fledglings . in addition to these resident species , there were some migratory species , such as brown flycatchers and narcissus flycatchers , breeding in the same forest.table  1total number of individuals and trials in which individuals approach within 10  m of a speaker during playbacks of  chicka  and  jar  callsspecieschickajarnumber of individualsnumber of trialsnumber of individualsnumber of trialscoal tit ( periparus ater ) 
 2174willow tit ( poecile montanus ) 
 3333long - tailed tit ( aegithalos caudatus ) 
 0031nuthatch ( sitta europaea ) 
 0032japanese white eye ( zosterops japonicus ) 
 0053meadow bunting ( emberiza cioides ) 
 0011brown flycatcher ( muscicapa dauurica ) 
 0011narcissus flycatcher ( ficedula narcissina ) 
 1111brown - eared bulbul ( hypsipetes amaurotis ) 
 2233japanese pygmy woodpecker ( dendrocopos kizuki ) 
 0022 
 resident species 
 immigrant species 
 nestling period 
 post - fledgling period 
  total number of individuals and trials in which individuals approach within 10  m of a speaker during playbacks of  chicka  and  jar  calls 
 post - fledgling period i tested the response of heterospecific species of birds to playbacks of  chicka  calls ,  jar  calls , and background noise ( control ) . playback stimuli were constructed by using recordings of  chicka  calls from 10 individuals ( six males and four females ) and those of  jar calls from nine individuals ( five males and four females ) . for each mobbing call playback , five calls were chosen from the same individual , and these calls were repeated to fill a 3-min sound file at a rate of 5 calls/12  s ( roughly one call every 2.4  s ) . this calling rate was within the range of natural repetition rates for both  chicka  and  jar  calls at the early nestling stage ( suzuki 2012a ) . low - frequency ( < 1  khz ) noise was filtered out and the calls were amplified on a computer . sound editing was conducted using adobe audition 3.0 software ( adobe corporation , san jose , ca , usa ) and more detailed information has been provided elsewhere ( suzuki 2012a ) . experiments were carried out from 3 june to 16 june 2011 when great tit nestlings were 69  days old . an at - spg50 speaker ( audio - technica corporation , tokyo , japan ) was hung from a tree and fixed 1.6    0.2  m from the ground and 4.8    0.6  m from the nest box ( mean    sd , n  =  14 ) . the playback speaker was connected to an r-09 hr digital audio recorder ( roland corporation , shizuoka , japan ) using exc-12a extension cords ( victor company of japan , kanagawa , japan ) , which enabled the control of playbacks from an observation position 15  m away from the nest . playbacks commenced when a pair of great tits was within 5  m of the nest and neither bird was vocalizing , in order to control for the effect of the presence of the tits on the behaviour of heterospecific individuals . mobbing calls were played back at a constant volume ( 75  db at 1  m ) , and background noise was played back at the same amplitude as the background noise level of the call playbacks ( 50  db at 1  m ) . all three stimuli (  chicka  calls ,  jar  calls , and background noise ) were tested at each nest . each trial was separated by at least 2  h to reduce habituation ( see also suzuki 2012a ) . the speaker position was fixed for all treatments at each site in order to control its possible effect on responses . trials were conducted in calm , dry weather between 0830 and 1600  h ( japan standard time ) . during the 3  min of playbacks , i counted the number of species and individuals that approached the playback speaker . in order to assess the intensity of the approaching response , i prepared three different thresholds of distance from a speaker : 3 , 5 , and 10  m. i also recorded whether birds gazed at the ground during trials , which was defined as occurring when birds looked towards the ground and pointed their bills in the same direction while raising their tails above their heads ( see suzuki 2012a ) . all statistical tests were performed using r 3.1.2 ( r core team 2014 ) and its packages . nnet ) was used to examine whether the composition of species that approached the speaker differed according to the experimental site or playback call types . i included the species of individual birds as dependent variables , and the experimental site and treatments (  chicka  or  jar  ) as independent variables . stats ) was used for the primary analysis ; exact wilcoxon matched - pairs signed - rank tests (  wilcox.exact in exactranktests ) were used for the post hoc analyses because of the small sample size ( mundry and fischer 1998 ) . stats ) to investigate whether the performance of gazing behaviour was affected by the type of playback calls . all tests were two - tailed , and the significance level was set at   =  0.05 . although my analyses involved multiple comparisons of responses , i did not adjust the significance level as the smaller number of samples increases the probability of making type ii errors ( nakagawa 2004 ) . a total of 10 species of birds were observed within 10  m of the playback speaker during the 3  min of experimental trials ( table  1 ) . birds from species in the family paridae were the most common among those that approached mobbing calls of japanese great tits ( coal tits : 7  % of  chicka  call trials and 29  % of  jar  call trials ; willow tits : 21  % of  chicka  call trials and 21  % of  jar  call trials ) . a multinomial logistic regression showed that the composition of heterospecific species that approached within 10  m of the speaker was significantly affected by the playback site (  
 =  19.01 , p  =  0.014 ) , but not by the call type (  
 =  13.84 , p  =  0.086 ) . birds approached differently in response to  chicka  and  jar  calls ( fig .  1 ; table  2 ) . during the playback of  chicka  calls , more heterospecific species were observed within 10  m of the speaker than during the control playback of background noise . however , there were no significant tendencies for birds to approach to within 5  m of the speaker during  chicka  calls . the playback of  jar  calls attracted more species than the playback of  chicka  calls . in particular , birds approached within 3  m of the speaker when hearing  jar  calls . none of the heterospecific species were attracted to the speaker during the playback of background noise . such differences in approaching behaviour were detected even when analysing the heterospecific response as the total number of individuals ( table  2).fig . 1interspecific attraction to two types of mobbing calls of japanese great tits (  chicka  and  jar  ) and a background noise control ( bn ) , as shown by the number of species ( a  c ) and individuals ( d  f ) that approached a speaker during 3-min of playbacks 
 table  2results of statistical analyses of interspecific attraction to the three playback treatments :  chicka  calls ,  jar  calls , and background noise ( bn)variablesprimary test 
 post - hoc ( p ) 
  

 p 
 chicka - bnjar - bnchicka - jarnumber of species3  m12.18 
 0.002 
 0.5 
 0.004 

 0.011 
 5  m12.24 
 0.002 
 0.25 
 0.004 

 0.034 
 10  m10.29 0.006 

 0.031 

 0.004 
 0.052number of individuals3  m12.18 
 0.002 
 0.5 
 0.004 

 0.023 
 5  m12.24 
 0.002 
 0.25 
 0.004 
 0.05110  m10.29 
 0.006 0.031 

 0.004 
 0.067statistically significant p values ( < 0.05 ) are shown in bold 
 friedman s test 
 exact wilcoxon signed - rank test 
  interspecific attraction to two types of mobbing calls of japanese great tits (  chicka  and  jar  ) and a background noise control ( bn ) , as shown by the number of species ( a  c ) and individuals ( d  f ) that approached a speaker during 3-min of playbacks results of statistical analyses of interspecific attraction to the three playback treatments :  chicka  calls ,  jar  calls , and background noise ( bn ) statistically significant p values ( < 0.05 ) are shown in bold 
 exact wilcoxon signed - rank test the playback of  jar  calls also elicited a downward gazing response in several species of birds ; coal tits ( n  =  4 trials ) , willow tits ( n  =  3 ) , long - tailed tits ( n  =  1 ) , nuthatches ( n  =  2 ) , and japanese white eyes ( n  =  3 ) always gazed at the ground when approaching  jar  calls . in contrast , such a response was not observed even when these species approached the playback of  chicka  calls ( coal tit : n  =  1 ; willow tit : n  =  3 ) . when pooling the data for coal and willow tits , there was a significant effect of call type on the occurrence of gazing behaviour ( fisher exact test , p  =  0.003 ) . birds approached playbacks of both types of mobbing calls , but their responses were different between the types of mobbing calls . they approached more closely to the playback of  jar  calls than to the playback of  chicka  calls . since the composition of species that approached within 10  m of the speaker did not differ between these call treatments , these individuals adjusted their response according to the type of mobbing call . these results demonstrate for the first time that different types of mobbing calls trigger different formations of multi - species mobbing parties . in addition to the approaching behaviour , individuals of some species gazed at the ground when hearing  jar  calls . this response has been described as a typical reaction of japanese great tits to  jar  calls ( suzuki 2012a ) , which serves to search for a snake that approaches nests from the ground ( suzuki and ueda 2013 ) . such gazing behaviour was observed not only in closely related species of japanese great tits , such as coal and willow tits , but also in phylogenetically distant species such as long - tailed tits , nuthatches , and japanese white eyes . however , a gazing response was never observed during the playback of  chicka  calls . these results suggest that heterospecific species derive referential information about snakes from snake - specific  jar  calls . although my results show that several birds respond appropriately to different mobbing calls of japanese great tits , it remains unclear how they acquire this ability . in my study site , many bird species live in mixed - species flocks with japanese great tits before their breeding season ( suzuki 2012b ) and breed using the same habitat as the tits . therefore , birds may have opportunities to learn to associate heterospecific mobbing calls with predatory threats , and this may contribute to the rapid spread of anti - predator behaviour within a bird community ( wheatcroft and price 2013 ; magrath et al . 2015b ) . however , it is also possible that interspecific responses to mobbing calls are genetically inherited . support for this idea comes from previous studies showing that several birds respond appropriately to the mobbing calls of allopatric species when they first hear these calls ( johnson et al . 2003 ; randler 2012 ) . to test these possibilities , it would be worthwhile to conduct additional playback experiments in other bird communities where japanese great tits are absent . the present findings raise an interesting question for further studies : how do birds benefit from helping the brood defence of other species ? it might be that birds approached the mobbing calls on the basis of interspecific reciprocity ( krams and krama 2002 ; campobello et al . 2011 ) , which could be tested by exploring the response of japanese great tits to the mobbing calls of neighbour species . it is also possible that birds benefit from mutualism in that they cooperatively prevent predators from learning to view the avian nests as a profitable food source , providing a new hypothesis for the adaptive functions of avian mobbing ( curio 1978 ) . for example , migratory birds may not have enough knowledge about predators in a breeding site , and therefore may approach the mobbing calls of resident species in order to gather information ( nocera et al . 2008 ) . future research may provide new insight into the adaptive value of the recognition of heterospecific mobbing calls and the evolution of interspecific social behaviour .
japanese great tits 
 ( parus minor ) use a sophisticated system of anti - predator communication when defending their offspring : they produce different mobbing calls for different nest predators ( snake versus non - snake predators ) and thereby convey this information to conspecifics ( i.e. functionally referential call system ) . 
 the present playback experiments revealed that these calls also serve to coordinate multi - species mobbing at nests ; snake - specific mobbing calls attracted heterospecific individuals close to the sound source and elicited snake - searching behaviour , whereas non - snake mobbing calls attracted these birds at a distance . 
 this study demonstrates for the first time that referential mobbing calls trigger different formations of multi - species mobbing parties .