bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436261; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Song complexity in relation to repertoire size and phonological syntax in the breeding 2 song of Purple Sunbird 3 Sonam Chorol and Manjari Jain* 4 Indian Institute of Science Education and Research Mohali, Punjab, India 5 *Corresponding Author 6 Address correspondence to: 7 Manjari Jain 8 Department of Biological Sciences, 9 Indian Institute of Science Education and Research, Mohali, 10 Punjab 140306, India 11 [email protected] 12 13 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436261; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 14 Abstract 15 There are multiple measures for bird song complexity such as repertoire size, phonological or 16 compositional syntax and complex vocal mechanism (CVM). We examined these in an old- 17 world passerine, Purple Sunbird. First, we identified and acoustically characterised the 18 repertoire size (of notes and phrases). We then assessed positional fidelity and ordering of 19 notes within phrases. We found 23 distinct notes by aural-visual inspection of spectrograms 20 which was validated using a Classification and Regression Tree based on 5 acoustic 21 parameters. These notes combined in different iterations to form 30 different phrases. 22 Phrases comprised of an overall structure with an introductory note (prefix) at the onset, 23 followed by notes occurring at specific positions within the phrase body, and terminated with 24 a trill (suffix syllable(s)). Prefix was present in 93% of phrases whereas suffix syllable(s) 25 occurred in 27% of phrases only. We found that notes exhibited positional fidelity and 26 combined in specific order to form a phrase. This is indicative of underlying phonological 27 syntax that limits the ways in which notes combine to form phrase and offers insights to song 28 complexity. Finally, we found that suffix syllables exhibit the presence of mini-breath (very 29 short inter-note interval) which are known to be produced by CVM. 30 Keywords: Complex communication, sexy syllable, mini-breath, syntax, repertoire bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436261; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 31 Introduction 32 The total set of vocalizations that a species possesses and uses in different behavioural 33 contexts is regarded as the vocal repertoire of a species (Searcy 1992). A large vocal 34 repertoire depicts higher complexity in communication (Blumstein and Armitage 1997; 35 McComb and Semple 2005). This phenomenon has been studied extensively in primates as 36 well as in avian species (Range and Fischer 2004; Gustison et al. 2012). Structural 37 complexity of acoustic signals exists not only in the variety of vocalizations but also in the 38 manner in which vocalizations are organised and composed (Hailman and Ficken 1986). In 39 avian vocalizations the smallest acoustic unit is referred to as a ‘note’ or ‘element’ which can 40 combine, sometimes following different ordering rules, to form higher order vocal units 41 called ‘phrase’. Finally, songs can be composed of repetition of a single element (note) or 42 phrases (the same or different kind). 43 A phrase may be composed of a single note repeated multiple times or different notes 44 occurring in a defined sequence. Moreover, notes may be shared between two or more 45 phrases (Kroodsma 1977) and construction of phrases may follow certain patterns. The 46 combinatorial rule that governs the construction of the signal from subset of signals is termed 47 as syntax and it could be compositional or phonological syntax. The rules for arranging 48 smaller meaningful units into a higher meaningful signal is called compositional syntax 49 (Berwick et al. 2011). Altering this sequence may change the meaning of the signal (Berwick 50 et al. 2011). The presence of compositional syntax is well known in human language where 51 meaningful signalling units ‘words’ combines to form a higher meaningful signal ‘phrase’. In 52 avian systems, the presence of the same is reported where ‘alert call’ and ‘recruitment call’ 53 combine to form ‘mobbing call’ (Suzuki et al. 2016; Engesser et al. 2016). On the other 54 hand, bird song is a combination of meaningless units (notes) into phrases which too do not 55 have a defined meaning. However, the notes may combine in a defined and non-random 56 manner to build the phrase. Thus, the construction of song phrases may follow a 57 combinatorial rule thereby exhibits a phonological syntax (Berwick et al. 2011). Moreover, 58 bird song comprises of phrases and each phrase may be initiated with an introductory note(s) 59 - a stereotypic note (s) which is produced at the onset of the song phrase, followed by series 60 of notes, structurally similar or distinct, which form the main component of a phrase (phrase 61 body) (Williams 2004; Roach et al. 2012; Nelson and Soha 2004). A song can end with a 62 terminal trill that includes a series of rapidly produced notes (Nelson and Soha 2004). The bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436261; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 ordering of notes within a phrase following a non-random positional occurrence is indicative 64 of phonological syntax and is another aspect of structural complexity. 65 Furthermore, song complexity also depends upon different vocal mechanisms. For instance, 66 some birds can produce complex vocalizations with two temporally overlapping notes of 67 different frequencies. These complex vocalizations are produced using both sides of the 68 syrinx by expiring half air from one side of the syrinx at a certain frequency and the other 69 half from the other side at a different frequency (Suthers 2004). These notes are separated by 70 a short time interval (< 20 ms) known as a ‘mini-breath’. The production of such a syllable 71 (acoustic unit composed of 2-3 notes) is expected to require precise bilateral motor 72 coordination within the syrinx and between various muscles (Suthers 2004; Suthers et al. 73 2012). 74 In this study, we examined song complexity in an Old-World passerine, Purple Sunbird 75 (Cinnyris asiaticus). It belongs to the family Necteriniidae and occurs in West Asia, 76 throughout the Indian subcontinent and into Southeast Asia (Ali and Ripley 1983). Purple 77 Sunbirds are sexually dimorphic. Females are “olive brown dorsally with a yellowish 78 underside whereas eclipse (nonbreeding) males have a distinct median line down the centre 79 of throat and breast” (Ali and Ripley 1983). Males gain bright, metallic blue-green coloured 80 plumage during the breeding season which ranges from April-June in Northern India (Ali and 81 Ripley 1983). During breeding season, male sings multiple song with ringing metallic notes 82 and increases the loudness of song under noisy conditions (Singh et al. 2019). In this study, 83 we aimed to examine the breeding song complexity of Purple Sunbird at multiple levels. The 84 objectives were as follows: a) to determine the repertoire size in terms of notes and phrases 85 that compose the breeding song b) to examine the presence of phonological syntax with 86 respect to combinatorial rules underlying phrase structuring and composition and c) to 87 investigate evidence for complex vocal mechanism (CVM) indicated by presence of ‘sexy 88 syllables’ in the vocal repertoire. 89 Methods 90 Sampling location 91 Sampling was done during the breeding season between May-August 2017 in IISER Mohali 92 campus (30.6650° N, 76.7300° E), Punjab. The locality is a semi-urban, subtropical region 93 which falls under ‘Cwa’ category (climate that is variable throughout the year with a hot 94 summer and cold, dry winter separated by a brief period of tropical monsoon) of Koppen- bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436261; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 95 Geiger climate classification (Kottek et al. 2006). The vegetation is predominantly grassy 96 with intermittent canopy of dry deciduous mixed with evergreen trees including host plants 97 such as Callistemon linearis, Delonix regia, Habiscus sp., Plumeria sp., Lantena camara, 98 Cascabela thevetia, Hamelia petens, etc. 99 Song recording and analyses 100 A total of 3026 notes and 241phrases were analysed from the songs of Purple Sunbird 101 recorded over four months. Recordings were made opportunistically using a solid-state 102 recorder (Marantz PMD661-MKII; frequency response: 20 Hz – 20 kHz), connected to a 103 super-cardioid shotgun microphone (Sennheiser ME66 with K6 PM; frequency response: 40 104 Hz to 20 kHz), covered with a foam windscreen (Sennheiser MZW66). All vocalizations 105 were recorded at a sampling rate of 44.1 kHz with 16-bit accuracy. All recordings were 106 processed in Raven Pro 1.5 (Cornel Lab of Ornithology) and spectrogram generated using 107 Hann-window with 512 window size and 50% overlap. Songs were categorised preliminarily 108 based on aural-visual inspection and a catalogue of all note types was generated and notes 109 were annotated as A, B, C….