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    Vortex phase matching as a strategy for schooling in robots and in fish

    Experiments with robotic fish
    We developed, and employed, a bio-mimetic robotic fish platform (Fig. 1, Supplementary Figs. 1–4 and Movie 1 and 2) in order to experimentally evaluate the costs and benefits of swimming together. We constructed two identical robotic fish, 45 cm in length and 800 g in mass. Each has three sequential servo-motors controlling corresponding joints, covered in a soft, waterproof, rubber skin. In addition, the stiffness of the rubber caudal fin decreases towards the tip33 (Supplementary Fig. 1). The motion of the servomotors is controlled using a bio-inspired controller called a central pattern generator (CPG)34,35 resulting in the kinematics that mimic normal real fish body undulations when swimming36 (see Supplementary Fig. 2 and Note 1). Here, due to the complexity of the problem (as discussed above) we consider hydrodynamic interactions between pairs of fish. We note that this is biologically meaningful as swimming in pairs is both the most common configuration found in natural fish populations7,10,37,38, and it has been found that even in schools fish tend to swim close to only a single neighbour7,37.
    Fig. 1: The robotic fish platform employed to investigate hydrodynamic benefits of schooling.

    a The reverse Karman vortices shedding by the robotic fish with dye flow visualisation. b Schematic view of the setup that allows setting various spatiotemporal differences between two robotic fish swimming in a flow tank (Front-back distance D ∈ [0.22, 1] BL (body length), Left-right distance G ∈ [0.27, 0.33] BL and Phase difference Φ ∈ [0, 2π]). A laser generator was used to visualise the hydrodynamic interactions (see Supplementary Note 1). c The phase difference Φ is evaluated by the difference between the undulation phase of the two robots. Undulation phase is evaluated based on the lateral position Lt of the tail tip. d Power cost (absolute value on the left y-axis, and relative value compared to the average power cost on the right y-axis), is shown as a function of the phase difference at D = 0.33 BL and G = 0.27 BL. Error bars are standard error of the mean.

    Full size image

    To evaluate the energetics of swimming together we conducted experiments on our pair of robotic fish in a flow tank (test area: 0.4-m-wide, 1-m-long and 0.45-m-deep; Fig. 1b and Supplementary Fig. 3). In order to conduct such an assessment we first measured the speed of our robots when freely swimming alone (we did so in a large tank 2-m-wide, 3-m-long and 0.4-m-deep). We then set the flow speed within our flow tank to this free-swimming speed (0.245 ms−1) allowing us to ensure the conditions in the flow tank are similar to those of the free-swimming robot. Unlike in the solitary free-swimming condition, to have precise control of spatial relationships in the flow tank we suspended each robotic fish by attaching a thin aluminium vertical bar to the back of each robot, which was then attached to a step motor above the flow tank (Supplementary Fig. 3 and Movie 2). To establish whether the robotic fish connected with a thin bar has similar hydrodynamics compared to when free swimming, we measured the net force (of the drag and thrust generated by the fish body in the front-back direction) acting on the robot in the flow tank. The measured net force over a full cycle (body undulation) was found to be zero; thus the bar is not measurably impacting the hydrodynamics of our robot fish in the front-back direction as they swim in the flow tank (Supplementary Fig. 5).
    To further validate the utility of the platform, we also compared the power consumption of our robots swimming side-by-side, for different relative phase differences Φ, with equivalent measurements made with a simple 2D computational fluid dynamics (CFD) model of the same scenario (Supplementary Note 2). In both cases (see Supplementary Fig. 6a, c for robotic experiments and CFD simulations, respectively) we find that there exists an approximately sinusoidal relationship between power costs and phase difference which is defined as Φ = ϕleader − ϕfollower (Fig. 1c, d). Due to the 2D nature of the simulation, as well as many other inevitable differences between simulations and real world mechanics, the absolute power costs are different from those measured for the robots, but nevertheless the results from these two approaches are broadly comparable and produce qualitatively similar relative power distributions when varying the phase difference between the leader and follower. These results indicate that our robotic fish are both an efficient (making estimates of swimming costs is far quicker with our robotic platform than it is with CFD simulations) and effective (in that they capture the essential hydrodynamic interactions as well as naturally incorporate 3D factors) platform for generating testable hypotheses regarding hydrodynamic interactions in pairs of fish.
    We subsequently utilise our robots to directly measure the energy costs associated with swimming together as a function of relative position (front-back distance D from 0.22 to 1 body length (BL) in increments of 0.022 BL and left-right distance G from 0.27 to 0.33 BL in increments of 0.022 BL) while also varying the phase relationships (phase difference, Φ) of the body undulations exhibited by the robots (the phase of the follower’s tailbeat ϕfollower relative to that of the leader’s ϕleader, Fig. 1c).
    By conducting 10,080 trials (~120 h of data), we obtain a detailed mapping of the power costs relative to swimming alone associated with these factors (Fig. 2a). Such a mapping allows us to predict how real fish, that continuously change relative positions6,8, should correspondingly continuously adjust their phase relationship in order to maintain hydrodynamic benefits. To quantify the costs we determine the energy required to undulate the tail of each robot allowing us to define, and calculate, a dimensionless relative power coefficient as:

    $$eta =frac{({P}_{1}^{{rm{Water}}}-{P}^{{rm{Air}}})-({P}_{2}^{{rm{Water}}}-{P}^{{rm{Air}}})}{{P}_{1}^{{rm{Water}}}-{P}^{{rm{Air}}}}=frac{{P}_{1}^{{rm{Water}}}-{P}_{2}^{{rm{Water}}}}{{P}_{1}^{{rm{Water}}}-{P}^{{rm{Air}}}},$$
    (1)

    where η is the relative power coefficient, PAir, ({P}_{1}^{{rm{Water}}}) and ({P}_{2}^{{rm{Water}}}) are the power costs of the robotic fish swimming in the air (an approximation of the dissipated power cost due to mechanical friction, resistance, etc. within the robot that are not related to interacting with the water), alone in water, or in a paired context in the water, respectively. ({P}_{1}^{{rm{Water}}}-{P}^{{rm{Air}}}) and ({P}_{2}^{{rm{Water}}}-{P}^{{rm{Air}}}) therefore represent the power costs due to hydrodynamics while swimming alone, and in a pair, respectively (see Methods section). Correspondingly, the coefficient η compares the energy cost of fish swimming in pairs to swimming alone. Positive values (blue in Fig. 2a) and negative values (red in Fig. 2a) respectively represent energy saving and energy cost relative to swimming alone. The difference between the maximum energy saving and maximum energy cost for the robots is ~13.4%.
    Fig. 2: Robotic fish save energy by vortex phase matching (VPM).

    a Relative power coefficient η shown as a function of the phase difference between the leader and the follower Φ and front-back distance D at left–right distance G = 0.31 BL. The dashed line (also in b) shows the functional relationship described in Eq. (2) that determines the theoretical phase relationship that maximally saves energy (Methods section). ({Phi }_{0}^{* }) is the optimal initial phase difference (fitted to the data points of maximum energy saving, as shown in b). The points marked by red square, blue circle and blue square indicate example cases depicted on panels c–e. b Location of maximal energy saving in the robotic trials. Point size and darkness denote the number of occurrences of each phase difference value at each front-back distance. c–e An illustration of important spatial configurations for vortex phase matching. Energy cost is related to how the follower moves its body relative to the direction of the induced flow of the vortices, in the opposite direction with Φ0 = ({Phi }_{0}^{* })+π (c) or in the same direction with Φ0 = ({Phi }_{0}^{* }) (d, e). Followers interact with the induced flow of vortices with the same body phase at any front-back distance (within the range of hydrodynamic interactions), termed vortex phase matching. (d, e; Φ0 = ({Phi }_{0}^{* }) describes the hydrodynamic interaction resulting in energy saving, see description in the text). As the front-back distance changes, the followers must dynamically adopt phase difference Φ, with respect to that of the leader.

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    Our results indicate that there exists a relatively simple linear relationship between front-back distance and relative phase difference of the follower that minimises the power cost of swimming (as indicated by the dashed lines in Fig. 2a, b, the theoretical basis of which we will discuss below). This suggests that a follower can minimise energetic expenditure (and avoid substantial possible energetic costs) by continuously adopting a unique phase difference Φ that varies linearly as a function of front-back distance D (see Fig. 2b for example), even as that distance changes. We find that while left-right distance G does alter energy expenditure, this effect is minimal when compared to front-back positioning, and has little effect on the above relationship (Supplementary Figs. 7 and 8) in the range explored here.
    Although we know fish generate reverse Kármán vortices at the Reynolds number (Re = Lu/ν ≈ 105, where L is the fish body length, u is the swimming or flow speed and ν is the kinematic viscosity) in our experiments39 (Supplementary Fig. 9 and Movie 1), turbulence will dominate over longer distances18. In accordance with this, we see a relatively fast decay in the benefits of swimming together as a function of D (e.g., D  > 0.7 BL, Supplementary Fig. 10), a feature we also expect to be apparent in natural fish schools (where it would likely be exacerbated by what would almost always be less-laminar flow conditions). Therefore, we expect, based on our results, that hydrodynamic interactions are dominated by short-distance vortex-body interactions (with D   2 BL), and it thus cannot benefit from neighbour-generated vortices. We also chose this method since isolating the fish would likely induce stress responses that could confound our results. To evaluate body kinematics in the presence of vortices we analysed the body undulations of the follower when in close proximity (within 0.4 BL), where hydrodynamic effects will be strongest (Supplementary Fig. 25). We find that in the vicinity of vortices, fish exhibit a higher tailbeat amplitude and lower tailbeat frequency (Supplementary Fig. 26), which indicates less power consumption48.
    To further test if fish can save energy by adopting VPM with the typical vortex-body hydrodynamic interactions (Φ0 = −0.2π), we compared an estimation of the power consumption under different hydrodynamic interactions. Since the hydrodynamic interactions are mainly determined by the initial phase difference Φ0 (see above), we analysed performance in the full possible range from −π to π (see Supplementary Fig. 25 for the detailed method). We define relative energy saving when fish exhibit higher tailbeat amplitudes A (Fig. 4a) and lower tailbeat frequencies f (Fig. 4b) than average48, and find that the range is Φ0∈ [−0.5π, 0.5π] (the shaded area in Fig. 4). Figure 4 also shows that while fish adopting Φ0 ≈ 0 will save the most energy, those exhibiting Φ0 = −0.2π, as in our experiments, will save almost the same amount (thus they are very close to optimal in this respect).
    Fig. 4: Relative energetic benefits to a follower in real fish pairs.

    a, b Energy cost analysis was conducted by calculating the difference in amplitude A (a) and frequency f (b) at Φ0 and the same measurements with the opposite phase Φ0 + π (written as A+π and f+π respectively) as a function of initial phase difference Φ0 (Supplementary Fig. 25 and Note 4). Data are pooled from all pairs when the follower’s front-back positions are not >0.4 BL distance (where the hydrodynamic interactions are expected to be the strongest). The hatched areas show the energy saving zone of Φ0. The dashed line denotes ({Phi }_{0}^{* }), the most typically observed initial phase difference exhibited by our fish. (Average amplitude is 0.09 BL, average frequency is 2.3 Hz).

    Full size image

    Fish in our experiments (Fig. 3c at D = 0 BL) spent 59% of their time swimming with phase relationships (Φ0∈ [−0.5π, 0.5π]) that save energy, and the remaining 41% that imposes some (relative) energetic costs. However, because the energy cost has a sinusoidal relationship to the phase difference (Fig. 1d) simply calculating the percentage of time in each regime (in which there is either a benefit or a cost, regardless of the magnitude of each) is insufficient. By combining the frequencies (occurrences) of each phase difference Φ observed in Fig. 3c and the sinusoidal shape of the power cost as a function of Φ (Fig. 1d and Supplementary Fig. 6b, d), we can estimate that by behaving as they do, fish (in our flow conditions) save (by accumulating all benefits and extra costs; where a random behaviour would give 0) an overall 15% of the total possible (which would be achieved by perfectly adopting the optimal phase to the neighbour-generated vortices at all the time, Supplementary Fig. 27). It is possible that if fish are exposed to more challenging, stronger flow regimes (here we employed those of typical swimming), that this percentage will increase. However we would never expect fish motion to be completely dominated by a need to save energy as they must also move in ways as to obtain salient social and asocial information from their visual, olfactory, acoustic and hydrodynamic environment, such as to better detect food52, environmental gradients44 and threats16. Nevertheless, kinematic analysis suggests that they adopt VPM in a way that results in energy savings (dashed line in Fig. 4).
    In summary, our bio-mimetic robots provided an effective platform with which we could explore the energetic consequences of swimming together in pairs and revealed that followers could benefit from neighbour-generated flows if they adjust their relative tailbeat phase difference linearly as a function of front-back distance, a strategy we term vortex phase matching. A model based on fundamental hydrodynamic principles, informed by our flow visualisations, was able to account for our results. Together, this suggests that the observed energetic benefit occurs when a follower’s tail movement coincides with the induced flow generated by the leader. Finally, experiments with real fish demonstrated that followers indeed employ vortex phase matching and kinematic analysis of their body undulations suggests that they do so, at least in part, to save energy. By providing evidence that fish do exploit hydrodynamic interactions, we gain an understanding of important costs and benefits (and thus the selection pressures) that impact social behaviour. In addition, our findings provide a simple, and robust, strategy that can enhance the collective swimming efficiency of fish-like underwater vehicles. More

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    Changes in the drought sensitivity of US maize yields

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    Joseph H. Connell (1923–2020)

    Credit: Tad Theimer

    Joseph (Joe) Connell altered both what and how ecologists study. Tree by tree, coral by coral, barnacle by barnacle, he saw patterns and processes across diverse ecosystems. Simply and with incontrovertible evidence, he demonstrated that interactions such as competition and predation could determine where species lived.
    Before his classic experiments on Scotland’s rocky shores, field ecology was mainly descriptive, focusing on physical conditions such as temperature or moisture in determining where species lived. Connell, who died last month aged 96, inspired thousands of ecologists to test their hypotheses by manipulating conditions in the field.
    Connell established long-term studies of coral reefs at Heron Island in the Great Barrier Reef and of tropical rainforests in Queensland, Australia, that spanned more than three and five decades, respectively. Monitoring revealed the dynamic nature of plant and animal communities that had long been considered stable. He discovered that natural variability in biological interactions and physical factors maintains diversity in these and other endangered ecosystems.
    Born in 1923, Connell grew up just outside Pittsburgh, Pennsylvania. When the United States entered the Second World War in 1941, he enlisted in the Army Air Corps and was trained in meteorology. Later, conducting weather surveillance in the Azores — the Portuguese Atlantic archipelago — in support of army operations in Europe, he spent his free time birdwatching and identifying trees. Meeting army recruits who worked as wildlife managers, he realized it was possible to have a career as a biologist. After the war, and a degree in meteorology at the University of Chicago, Illinois, he headed to the University of California, Berkeley, for a master’s in zoology.
    Connell produced what he described as a dull, unsatisfying thesis on brush rabbits (Sylvilagus bachmani) in the Berkeley Hills. Discouraged by the difficulties of conducting a population study (he trapped only 40 rabbits in 2 years), he adopted a rule of thumb — never again to study anything bigger than his thumb. As a doctoral student at the University of Glasgow, UK, he gleefully discovered what Charles Darwin had found a century before: that thousands of barnacles could easily be studied on the seashore, no traps required.
    Connell realized that he could test his hypotheses about what factors determined where on the shore certain species lived by removing, adding or transplanting barnacles and their snail predators. Classic papers ensued, inspiring other ecologists to rethink distribution patterns, and, importantly, to test their ideas with controlled field experiments.
    After a postdoc at the Woods Hole Oceanographic Institution in Massachusetts, Connell joined the faculty at the University of California, Santa Barbara, where he remained for the rest of his career. He was curious about processes that affected distribution and abundance, and those that might keep biodiversity high. Shifting to species that live for hundreds or thousands of years on coral reefs and in rainforests, he set up his Australian long-term monitoring studies in 1962 and 1963. Both recorded the demography and interactions of organisms in permanent plots, tracking community dynamics and the impact of disturbances, ranging from fallen trees to cyclones.
    Visiting Connell’s sites with him in the 1970s and 1990s, we were impressed with his foresight and inspired by his insights. On the reef, he explained, physical disturbance by large waves associated with recurring cyclones intermittently reduced the cover of dominant species such as staghorn coral (Acropora aspera). This prompted recolonization by a diverse assemblage of weaker competitors such as encrusting or mound-like species. Connell coined the term ‘intermediate disturbance hypothesis’ to describe this process.
    We strolled through the larger of his rainforest plots, avoiding stinging trees, biting flies, ticks and leeches, and relishing the richness — more than 300 tree species and about 100,000 individual plants. Connell outlined another hypothesis, that forests are more diverse when rarer species such as the conifer Sundacarpus amarus are favoured over more common ones such as the flowering tree Planchonella sp. Patterns of seedling establishment, growth or survival depend on that difference in frequency. Because common species grow more densely than rare ones, they are more vulnerable to specialist herbivores or pathogens.
    This pattern of density-dependent predation or infection thins out common species, enabling a richer mix to coexist. It is a central component of the Janzen–Connell hypothesis (independently proposed by US ecologist Daniel Janzen in 1970), which predicts that seedlings are more likely to die under the canopies of their parent trees than farther away, ensuring diversity.
    Connell was unfailingly kind, generous and devoted to his family. He never lost his profound curiosity about the natural world or his delight in exploring ideas with students and colleagues. He loved to be challenged and, if proven wrong, he gladly moved on to a new hypothesis or question. He sought truth, not fame. Moreover, he empowered everyone around him to think critically by focusing on ideas and evidence, not personalities. Fortunately for the world, his way of exploring science proved powerful, infectious, fun and enduringly productive. More

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    Acoustic preadaptation to transmit vocal individuality of savanna nightjars in noisy urban environments

    Study area and field observations
    We recorded the territorial calls of male savanna nightjars in eight areas of Taiwan, from north to south: Jinshan District (16 nightjars), Taichung City (7 nightjars), Hualien City (9 nightjars), Yuanlin City (8 nightjars), Beigang Township (8 nightjars), Chiayi City (14 nightjars), Taitung City (4 nightjars), and Hengchun Township (1 nightjar) (Fig. 1) during April-June of 2018. Sound recordings were collected in the downtown of each area using a Denon Portable IC Recorder (DN-F20R, sampling rate = 48 kHz, 16 bit, wav format) equipped with a Sennheiser ME67 unidirectional microphone. We made recordings between 19:00 to 24:00 in good weather during 1–2 consecutive nights for each area. If we recorded in the same area for more than one consecutive nights, the recording range of the second night was at least 1000 m away from the previous recording range. The maximum territory size of the savannah nightjar observed by Chan48 in urban areas of Taiwan was 83,424 m2 with a radius of about 163 m. Therefore, we are confident that we avoided recording the same territorial male twice. Territorial calls from an individual were recorded until the individual stopped calling or flew out of our recording range. When emitting territorial calls, nightjars either perched on some artificial structure, such as antennas or fences on roof tops, or they flew around the tops of buildings. Because the loud territorial calls recorded in these two situations demonstrate the same time–frequency patterns on spectrograms and sound the same when listened to, we did not differentiate between them while recording. We always attempted to record at the closest possible distance to the calling individual by moving closer to the individual at the street level.
    After measuring the individual’s calls, we immediately took three samples of the maximum noise levels (maximum hold function, C-weighting function on Sound Level Meter TES-1350, TES, Taiwan) near the calling location and close to a road intersection within the calling range following the procedures detailed in Shieh et al.49. During each noise sampling, the sound level meter was held horizontally at a height of about 1.5 m and turned 360° clockwise to measure the maximum noise level from all directions within a time period of about 30 s. We then averaged the three samples of maximum noise measurements to arrive at our value of ‘ambient noise levels’ for each individual. Therefore, ambient noise levels were measured at a height of 1.5 m, which was not only the height at which anthropogenic sources such as traffic and human activity are the main sources of noise but also the height at which we held the microphone to record the nightjar calls emitted at a height of usually more than 10 m.
    Playback-recording experiments
    Seven artificial calls were generated using the frequency shift function (+ 300 Hz, + 200 Hz, + 100 Hz, 0 Hz, − 100 Hz, − 200 Hz, − 300 Hz) under the Frequency Domain Transformations tool in Avisoft-SASLab Pro software v5.2.12 from a source call with good quality (see Supplementary Table S6 for descriptions of acoustic measurements) and after band-pass filters (2–7 kHz) for noise removal. These seven artificial frequency-shifted calls represented seven different artificially created individuals and were identified by their frequency shift values (+ 300 Hz, + 200 Hz, + 100 Hz, 0 Hz, − 100 Hz, − 200 Hz, − 300 Hz). We then copied each frequency-shifted call 10 times with equal silent intervals and same amplitude as a group, and then we merged the seven groups of frequency-shifted calls into one sound file with alternated orders following a Latin square design. A total of seven merged sound files were obtained, and each was broadcast once in three sites of different urban noise levels: high, medium and low. The playback-recording experiments were conducted at three sites near or on the campus of Kaohsiung Medical University (22.648 N, 120.310 E). We also took 10 samples of the maximum noise level (maximum hold function, C-weighting function on Sound Level Meter TES-1350, TES, Taiwan) at the recording sites during the playback periods to obtain the ambient noise levels for each site. The first site was on the traffic roadside near a road intersection and had the highest ambient noise levels with a mean of 83.7 dB (n = 10) and a range of 79.3–89.6 dB. The second site was on the sport field of the campus about 30–50 m away from the traffic road and had the medium ambient noise levels with a mean of 74.6 (n = 10) and a range of 73.1–76.3 dB. The third site was on a 4th floor roof garden of the campus and had the lowest ambient noise levels with a mean of 71.4 dB (n = 10) and a range of 69.9–74 dB.
    We used a Denon Portable IC Recorder (DN-F20R) connected to a speaker (Sony SRS-X11) for the playback experiments. The speaker was placed 1.5 m above the ground on a tripod. Playback-recording experiments were conducted between 17:00 and 18:30 h, a period with high traffic levels. The seven merged sound files were played back at a standardised volume with a sound-pressure level of 82.5 dB at 1 m from the speaker, and about 62.5 dB at 10 m from the speaker. The sound-pressure level of the broadcasting sound which we received at 10 m is about the same amplitude that we recorded a nightjar call at a distance of 28.8 m away from its calling spot with an amplitude of 97.7 dB. We recorded with a Denon Portable IC Recorder (DN-F20R) connected to a shotgun microphone (Sennheiser ME67) that was placed on a stand 1.5 m above the ground and oriented toward the broadcasting speaker at a distance of 10 m. All the recordings were set at the same recording levels and same settings (sampling rate = 48 kHz, 16 bit).
    Sound analyses
    We selected high-quality calls with clear acoustic structures on spectrograms and thus excluded any recordings with low-quality calls, which were, for example, emitted when the calling nightjar was too far away, or its calls were overlapped with calls from neighboring nightjars.
    We also excluded any recordings where the individual only uttered one call (two calls being the minimum needed for inclusion). This left us with a total of 1925 calls from 67 individuals for our analyses, with a mean of 28.7 ± 3.1 calls/individual and a range of 2–97 calls/individual. The band-pass filters were set from 2.0–6.8 kHz and adjusted for each individual to reduce noise components. Using the recordings, we produced spectrograms with the following spectrogram parameters in the software: sampling frequency = 22.05 kHz, FFT = 512, hamming window, frequency resolution = 43 Hz, and time resolution = 2.9 ms. We quantified 30 acoustic variables (Table 1) from the spectrograms using the Automatic Parameter Measurements setup in the Avisoft-SASLab Pro software v5.2. We marked a call with the section label manually on the spectrogram by eye, and then two time-based parameters were measured (duration of the call and the temporal distance from the start to the location of the maximum amplitude) (see the manual50 of the Avisoft-SASLab Pro software for details). To automatically measure parameters other than the time-based on the labelled section, we specified four spectrum-based parameters (peak frequency, quartile 25%, quartile 50% and quartile 75%) to be measured at seven locations of the labelled section (start, end, maximum amplitude of the call, minimum parameter of entire call, maximum parameter of entire call, mean parameter of entire call, relative standard deviation of entire call); thus, 24 frequency-based parameters and four frequency-modulation-based parameters were automatically measured based on the labelled sections on the spectrograms.
    For the playback-recording experiments, the recordings were first analyzed using the same settings as the above except for two differences. We adjusted the band-pass filters to 2.0–7.3 kHz because of the high frequency shift value (up to 300 Hz). Furthermore, we used the duration of the source call as the duration of all received calls; that is, we fixed the duration of the call section while marking, and the other 29 variables were automatically measured on the marked section by the software to reduce any possible human measurement errors51.
    Statistical analyses
    To examine possible geographic variation of the calls, we used individuals as our sample units (n = 67), and the averaged measurements from the calls of each individual were analyzed using a PCA. The PCA was performed on the 30 acoustic variables after a normalised transformation of each variable to a mean of zero and unit standard deviation (software PRIMER 6, version 6.1.5). We retained the five components with eigenvalues greater than one and interpreted each component based on its correlations with the original variables. However, because there is a sharp decrease of the eigenvalue and of the explained variance to smaller values from PC2 to PC3 (Supplementary Table S2), only PC1 and PC2 were used for examining the geographic patterns of the calls. The 95% confidence ellipses of the groups of individuals from different geographic areas are shown on the plot of PC1 against PC2 (Fig. 3). If the 95% confidence ellipses of the eight geographic areas overlapped, we can treat all the sampled individuals as one population and pool them for further analysis.
    The following statistical tests were performed on the untransformed data of each acoustic variable using JMP Pro 14.2.0. First, we treated individuals as our sample units, and we used the averaged measurements from the calls of each individual. We performed Spearman rank tests to examine the relationships between ambient noise levels and each acoustic variable for 65 individuals (because noise measurements were not taken for two individuals). To distinguish variables’ relationship to ambient noise levels, we then classified the 30 acoustic variables into two categories: (1) noise-related variables had a significant relationship with ambient noise levels, and (2) noise-unrelated variables did not. Acoustic variables which had a significant (positive or negative) correlation with ambient noise levels using Spearman rank tests were classified as noise-related variables; all remaining variables lacking such a significant correlation were classified as noise-unrelated variables.
    To determine variables which can distinguish the calls of different individuals, we treated calls as sample units and individuals as groups. We then performed a Kruskal–Wallis test to select variables which were more likely to encode individual information, that is, with significant individual differences. We only included those variables with a significant P-value into the DFA (see details below). To investigate how ambient noise levels affected the transmission efficacy of vocal individuality, we used DFA which calculates the accuracy of correctly assigning a particular call to a particular individual. The purpose of a DFA is thus to discriminate sampled individuals based on all the possible acoustic measurements which encode information about individual identity; therefore, we did not perform a variable selection process. Specifically, we used the accuracy value (1—misclassification rate) calculated from the DFA to assess the transmission efficacy of vocal individuality information. Higher accuracy values are assumed to indicate higher transmission efficacy of vocal individuality information from calls recorded through various ambient noise levels.
    To compare possible differences in the transmission efficacy of vocal individuality for different sets of acoustic variables, we then calculated a separate DFA for three datasets: (1) all the 30 acoustic variables, (2) the noise-related variables, and (3) the noise-unrelated variables. For each dataset, a separate DFA was used to calculate one overall accuracy value and 67 individual accuracy values. The overall accuracy value (1—misclassification rate) describes the ability of the DFA to correctly assign the 1925 calls to the 67 recorded individuals. The individual accuracy values then describe the ability of the DFA to correctly assign the calls of one particular individual to that individual. To account for small sample sizes of calls by some individuals, the overall misclassification rates were bootstrapped with fractional weights option (number of bootstrap samples, n = 20,000), and the overall accuracy values of the three datasets and the associated 95% biased-corrected confidence intervals (BCI) were obtained. Sixty-seven individual accuracy values (corresponding to the 67 sampled nightjars) were also calculated for each set of variables.
    To compare the individual accuracy values using the noise-related variables with the individual accuracy values using the noise-unrelated variables, we used the Wilcoxon signed rank test. To examine how noise levels affected the transmission efficacy through different acoustic structures (noise-related vs. noise-unrelated), we investigated the correlation between the individual accuracy value and the ambient noise level associated with that particular individual by using Spearman rank tests. The differences of the individual accuracy values, which was taken as the accuracy value of the DFA using the noise-unrelated variables minus the accuracy value of the DFA using the noise-related variables from the same individual, were correlated with the ambient noise levels by using Spearman rank tests to examine the similarity in trends.
    For the playback-recording experiments, seven sound found files were played in each site (high, medium or low urban noise levels), and seven corresponding recordings were received as seven samples for each site. In each recording sample, although 10 calls of each individual (identified by frequency shift values) were played, the number of received calls might be less than 10 because the calls were overlapped with other unexpected sounds and thus excluded for measurements. Therefore, we first averaged the measurements of the possible received calls for each individual in a sample and then used the averaged measurements as the measurements of the sample. Thus, we obtained 49 samples (7 samples × 7 individuals) for each site for analyzing the overall accuracy of vocal individuality and variable accuracy for the playback-recording experiments. Since we excluded the duration variable, only 29 variables were used for DFA because the duration was fixed in measurements for all individuals. Furthermore, we only used six main frequency-based variables (PFSTART, PFEND, PFMIN, PFMAXA, PFMAX, PFMEAN) for further comparison on transmission accuracy between noise-related variables and noise-unrelated variables because the artificial calls were generated by transforming only the frequency domain. Thus, for each site, we calculated a separate DFA for three datasets: (1) all the 29 acoustic variables, (2) the three noise-related variables (PFSTART, PFEND, PFMIN), and (3) the three noise-unrelated variables (PFMAXA, PFMAX, PFMEAN). For the DFA, the overall misclassification rates were also bootstrapped (number of bootstrap samples, n = 20,000), and the overall accuracy values of the three datasets and the associated 95% biased-corrected confidence intervals (BCI) were obtained. Furthermore, for the six main frequency-based variables, an accuracy value for each variable was calculated as 1 − (|R – B| /B), in which R indicated the measurement value of the received call and B indicated the measurement value of the broadcast call. In each site for each variable, the accuracy values of the seven samples of the same individual were averaged as the variable accuracy value for the individual. In each site, we then investigated the differences of variable accuracy values among variables by Friedman rank tests (with individual as block) and Wilcoxon signed rank test for paired comparison between variables. We set the significance level at 0.05 for all tests and report the two-tailed probability values. More

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