21Environmental changes caused by urbanization and noise pollution can have profound 22 effects on acoustic communication. Many organisms use higher sound frequencies in 23 urban environments with low-frequency noise, but the developmental and evolutionary 24 mechanisms underlying these shifts are less clear. We used a common garden 25 experiment to ask whether changes in minimum song frequency observed 30 years 26 after a songbird colonized an urban environment are a consequence of behavioral 27 flexibility or canalized changes that occur early in development. We captured male 28 juvenile dark-eyed juncos (Junco hyemalis thurberi) from two recently diverged 29 populations (urban and mountain) soon after they reached independence (aged 25-40 30 days), raised them in identical indoor aviaries, and studied their songs at an age of 31 three years. We found that the large population difference in minimum frequency 32 observed in the field persisted undiminished in the common garden despite the absence 33 of noise. We also found some song sharing between the common garden and natal field 34 populations, indicating that early song memorization before capture could contribute to 35 the persistent song differences in adulthood. These results are the first to show that 36 frequency shifts in urban birdsong are maintained in the absence of noise by genetic 37 evolution and/or early life experiences. 38 39 40 41 42 Introduction 43Anthropogenic noise can alter the biology of diverse animal taxa at organismal, 44 population, and even community scales [1][2][3][4][5][6][7]. In particular, the low frequency 45 background noise often associated with urbanization can interfere with animal 46 communication and has been associated with changes in acoustic signals that improve 47 sound transmission [8, 9]. One such change that is widely observed in urban 48 environments is increased minimum frequency of acoustic signals, which may be an 49 adaptation to overcome the masking effects of low-frequency noise [10][11][12][13][14][15][16]. Our 50 understanding of the developmental and evolutionary mechanisms that may underlie 51 such changes in acoustic signaling remains limited [9,[17][18][19][20][21][22]. Species such as oscine 52 songbirds that learn their songs are of particular interest due to the potential for cultural 53 evolution and other forms of behavioral plasticity, which can facilitate rapid change in 54 response to anthropogenic noise [23][24][25]. 55Several non-mutually exclusive hypotheses have been proposed to explain 56 changes in song frequency in urban environments [9, 26], including short-term plasticity, 57 ontogenetic effects (early experience), and evolutionary change across generations. 58The plasticity hypothesis argues that frequency shifts are the result of behavioral 59 flexibility in response to the presence or absence of a noise stimulus. Some studies in 60 oscine songbirds have found evidence supporting plasticity either through rapid 61 increases in minimum song frequency [18, 22, 27, 28] or switching to...