Comparisons of acoustic structures between sexes in a duetting, montane bird

Female song in birds is more widespread than previously thought, and studies across different species are critical for better understanding the entire evolutionary process of bird song. In this study, we recorded the songs of males and females in a duetting, montane bird species, the Elliot’s laughingthrush Trochalopteron elliotii, across consecutive breeding seasons. We specifically focused on identifying the number and structure of different song types by males and females, and compared these acoustic structures between the sexes. Our findings revealed that both males and females sang sex-specific solos. More specifically, females sang a single type of solo that varied significantly in the number of notes, whereas males produced three different solos composed of 2, 3, or 4 notes, respectively. Female solos exhibited significantly more notes and longer song duration compared to males. Male solos typically had a significantly higher maximum frequency for the entire song. No significant differences were observed in the duration of the first note, song rate, and other frequency characteristics between male and female solos. Furthermore, paired males and females coordinated their sex-specific solos to form duets when challenged by conspecific territory intruders, both within and outside the breeding season. Sex-specific solos suggested that male and female songs play different roles and may be subject to different selective pressures. Further research is necessary for elucidating the functions of male song, female song, and duets in this montane bird species.

Methods
Study site and species

The fieldwork was conducted in the Lianhuashan Nature Reserve (34°58′16″ N, 103°45′30″ E; 2040 m a.s.l.), located on the eastern edge of the Qinghai-Tibet Plateau in central China. The study area is mainly composed of cultivated farmlands, open woodlands, and orchards, as well as abandoned fields dominated by scrub vegetation. Since 2013, we have been studying the ecology of the resident Elliot’s laughingthrush population at this site and each year, catch and colour-band males and females with a unique combination of colour rings for sex and individual identification. The sex of all marked individuals was determined by the swelling of the male gonads and differences in morphological size (Liu and Sun 2016).

Field observation and vocalization recording

From March 5 to May 20, 2021, and April 5 to June 13, 2022, we observed male and female singing behaviour every three days during suitable weather conditions. All observations were conducted between 08:00 and 11:00 am. Within each territory, we conducted one-hour long observations, during which we recorded the date, sex of the singer, time, singing contexts (agonistic interactions, spontaneous singing, chorus singing), presence of intruders, and whether the focal pair divorced. For each singing individual, we collected solos and duets using a TASCAMHD-P2 sound recorder (44.1 kHz, 16 bits) and a Sennheiser MKH416 P48 shotgun microphone at a distance of 5-10 meters from the singing birds. We also recorded solos from unpaired males, which were determined through direct observations of male-female interactions, and from observations made at nests being attended by these same males.

Acoustic structure

Avisoft-SASLab Pro was used to analyse the song structure of all recordings. The sampling rate was 22.05 kHz, with a sample size of 16 bits, the FFT length was 512 points, the frequency resolution was 43 Hz, the temporal resolution was 2.9 ms, and the window used was FlatTop, with an 87.5% overlap. The simplest elements of the song on the sonogram are referred to as “notes”. We defined a discrete song as being made up of two or more consecutive notes, typically separated by a brief pause (Brenowitz et al. 1997). Solo songs refer to the distinctive individual songs produced by a single bird (either male or female). All definitions of songs and notes are illustrated in Figure 1A.

For each individual, the following song characteristics were recorded: the total number of notes within a song, duration of song, first note duration of a song, the speed of a song (the number of notes within a song per second), maximum frequency, minimum frequency, and frequency range of the song, the first and last note. To determine these parameters, we analysed two solos from each female and male. In order to include a diverse range of variation in vocal characteristics within individuals, we only selected recordings that were both high-quality and representative of the individual’s vocal repertoire.

Spectrograms creation

Spectrograms were extracted using R package 'Seewave' with an FFT-window length of 512 and an overlap of 87.5% (Sueur et al. 2008). The 'tuneR' package was used to load sound files in WAV format as an object (Ligges et al. 2013). In a spectrogram, time is displayed on the x-axis, frequency on the y-axis, and amplitude is represented by colour intensity.

Statistical analysis

Wilcoxon rank sum tests were used to determine if each of the vocal characteristics (the number of notes, speed, duration, first note duration) and frequency of the song differed between males and females. Bonferroni correction was used to adjust the p-values in the analysis. All statistical analyses were conducted using R version 4.1.3 (R Core Team, 2022). Values are given as Mean ± SE, with Bonferroni corrected P value < 0.004.

鸟类雌性鸣唱的分布范围远超此前认知，跨物种研究对于全面理解鸟类鸣唱的完整演化进程至关重要。本研究针对一种协作对唱（duet）的山地鸟类——艾氏噪鹛（Trochalopteron elliotii），在连续多个繁殖季内记录了其雌雄个体的鸣唱行为。我们重点分析了雌雄个体不同鸣唱类型的数量与结构，并对比了两性间的声学特征差异。

研究结果显示，雌雄个体均会发出性别特异性的独唱（solo song）。具体而言，雌性仅发出一种类型的独唱，但其鸣唱的音节数量存在显著差异；而雄性则会发出三种不同类型的独唱，分别由2、3、4个音节构成。雌性独唱的音节总数与鸣唱时长均显著高于雄性，雄性独唱的整体最大频率则显著高于雌性。而在首个音节时长、鸣唱速率及其他频率特征方面，雌雄个体间未观察到显著差异。此外，当遭遇同种领地入侵者时，无论繁殖季内外，配对的雌雄个体均会协调各自的性别特异性独唱，形成对唱。性别特异性的独唱模式表明，雌雄鸣唱可能承担不同的功能，并受到不同的选择压力。后续仍需进一步研究，以阐明该山地鸟类中雄性鸣唱、雌性鸣唱及对唱的具体功能。

## 方法

### 研究地点与物种
本项野外工作开展于中国中部青藏高原东缘的莲花山国家级自然保护区（34°58′16″ N，103°45′30″ E；海拔2040 m）。研究区域主要由耕地、开阔林地、果园以及以灌丛植被为主的撂荒地构成。自2013年起，我们便在此开展对留鸟艾氏噪鹛种群的生态学研究，每年都会捕捉个体并为雌雄个体佩戴唯一组合的彩色脚环，以实现性别与个体识别。所有标记个体的性别通过雄性性腺肿胀情况以及形态体型差异进行判定（Liu and Sun 2016）。

### 野外观察与鸣声录制
我们分别于2021年3月5日至5月20日，以及2022年4月5日至6月13日期间，在适宜的天气条件下每3天开展一次雌雄鸣唱行为观察，所有观察时段均为每日08:00至11:00。在每个领地内，我们进行为期1小时的观察，记录内容包括观察日期、鸣唱个体的性别、时间、鸣唱场景（攻击性互动、自发鸣唱、合唱）、入侵者的存在情况以及目标配对是否离婚。对于每一只鸣唱个体，我们使用TASCAMHD-P2录音机（采样率44.1 kHz，位深16 bit）搭配Sennheiser MKH416 P48枪式麦克风，在距离鸣唱鸟类5-10米的位置录制其独唱与对唱鸣声。此外，我们还录制了未配对雄性的独唱鸣声，这些雄性的身份通过雌雄互动观察以及其照料巢穴的行为得以确认。

### 声学结构分析
我们采用Avisoft-SASLab Pro软件对所有录音的鸣唱结构进行分析。采样率设置为22.05 kHz，位深16 bit，快速傅里叶变换（FFT）窗口长度为512点，频率分辨率为43 Hz，时间分辨率为2.9 ms，使用平顶窗（FlatTop），重叠率为87.5%。声谱图上最简单的鸣唱元素被定义为“音节（note）”。我们将由两个及以上连续音节构成、通常以短暂停顿分隔的连续鸣唱定义为“鸣曲（discrete song）”（Brenowitz et al. 1997）。独唱指单个鸟类（雄性或雌性）发出的独特个体鸣曲。所有关于鸣曲与音节的定义均如图1A所示。
针对每一只个体，我们记录以下鸣唱特征：鸣曲内的总音节数、鸣曲时长、鸣曲首个音节的时长、鸣唱速率（每秒内鸣曲包含的音节数）、最大频率、最小频率、鸣曲的频率范围，以及首个与最后一个音节的相关参数。为确定这些参数，我们为每只雌性与雄性个体分析两份独唱录音。为涵盖个体鸣唱特征的多样化变异，我们仅选取高质量且能代表个体鸣唱曲目库（vocal repertoire）的录音。

### 语谱图生成
我们使用R语言包‘Seewave’生成语谱图，其FFT窗口长度为512，重叠率为87.5%（Sueur et al. 2008）。使用‘tuneR’包加载WAV格式的音频文件为数据对象（Ligges et al. 2013）。在语谱图中，横轴代表时间，纵轴代表频率，振幅以颜色强度表示。

### 统计分析
我们采用Wilcoxon秩和检验来验证雌雄个体间各鸣唱特征（音节数、鸣唱速率、鸣曲时长、首个音节时长）及频率参数是否存在显著差异。分析中采用Bonferroni校正对P值进行校正。所有统计分析均使用R版本4.1.3完成（R Core Team 2022）。结果以平均值±标准误（Mean ± SE）表示，经校正后的P值<0.004为差异具有统计学意义。