15SummaryThe motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle specific force [1,2]. Birdsong is an excellent model system for understanding sequence learning of complex fine motor skills [3], but we know surprisingly 20 little how the motor pool controlling the syrinx is organized [4] and how MU recruitment drives changes in vocal output [5]. Here we combine measurements of syringeal muscle innervation ratios with muscle stress and an in vitro syrinx preparation to estimate MU size distribution and the control resolution of fundamental frequency (fo), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 50% 25innervating ≤ 3, and 13 -17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm 2 ) known to skeletal vertebrate muscle, small force steps by the major fo controlling muscle provide control of 50 mHz to 4.2 Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. 30Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.
Results & DiscussionVocal communication is of paramount importance for reproduction and survival of songbirds and even drives speciation. This is clearly exemplified by sympatric species that are 40 morphologically indistinguishable, but nevertheless fully separated solely by song [6, 7]. The potential to acoustically separate depends on the number of distinct sounds -or vocal space -that can be produced and perceived. The vocal space is set both by the range available to vary an acoustic feature, and in what steps, or resolution, the feature can be controlled within this range. Thus, both resolution and range expand the vocal space and may form a rich 45 substrate for species diversification [8,9]. While the range of an acoustic feature, for example fo, is typically limited by intrinsic constraints of the vocal organ [10-12], the ability 65 ( Fig 1A, B, Table S1, See Methods). The average total number of muscle fibers was 6995 ± 789 (n = 4), corroborating earlier reported ~6730 [17]. The left side had significantly less muscle fibers (3234 ± 232, range: 2992 -3503, n = 4) than right (3794 ± 334, range 3586 -4286, n = 4) (Welch t-test: t = -2.8, df = 5.4, p-value = 0.04). The number of axons in the tracheosyringeal branch of the hypoglossal nerve (NXIIts), assumed to represent the 70 number of MUs (See Methods), was not significantly different between left (820 ± 187, range: 602 -1162, n = 8) and right (790 ± 148, range: 677 -1045, n = 5) (Welch t-test: t = 0.32, df = 10.2, p-value = 0.76) and lower but within range of the 1026 ± 126 (n = 6) reported earlier [18]. The ...