Study species and study site

The zebra finch distribution encompasses most of the Australian continent and it is particularly common in the arid and semi-arid zone32. It is nomadic, mostly colonial, and breeds opportunistically throughout the year33,34. Zebra finches have life-long social monogamy and highly coordinated parental care, with both parents sharing incubation duties32,35.

The field study was conducted at Bimbowrie Conservation Park, South Australia (GPS: −32.048935; 140.161977), located in the arid interior. The study area consisted of open shrubland on a flood plain dominated by Acacia species (Acacia victoriae, A. aneura, A. oswaldii and A. carneorum) and inland rosewood trees (Alectryion oleifolius) typically hosting thick mistletoe clumps (Lysiana exocarpi). The study area extended between two artificial watering points used by zebra finches, 7 km apart. Rain at the field site is scarce and highly unpredictable (average annual rainfall 260 mm), whereas temperature varies seasonally. Sun rose at 6:15am and set at 8:10 pm in January.

Nest audio recording in the wild

In January 2018, all trees and bushes suitable for zebra finch nesting in the study area were searched for the presence of breeding or roost nests. Zebra finch nests are made of fine twigs and grasses, and comprises of an enclosed chamber accessed via an entrance tunnel32. Roost nests (unlike roosting platforms) are identical to breeding nests, except that the entrance is typically larger32. The presence of eggs in the nest was established using a small telescoping mirror. However, embryonic age at recording could not be assessed, because eggs could not be taken out for candelling, all nests but one were found after egg laying was complete, and only 18% of nests successfully hatched.

For audio recording, a tie-clip microphone (Sennheiser MKE 2 P Germany) was put at the top of the nesting chamber, just above the incubating or roosting bird (except for 2 nests where the roof was not accessible). The microphone was connected to a Zoom recorder (H4nSP or H6) hidden in the vegetation at least 2 m from the nest. Two thermo-hygrometer data-loggers (Minnow 1.0, Senonics, USA) were hung in the branches, within 30 cm of the nest, positioned to receive comparable solar radiation to the nest itself. We did not place them inside the chamber, to avoid the bird position affecting temperature measurements. “Nest-site temperature” was the average of the temperature recorded by the two data-loggers. Relative humidity at the nest-site remained low throughout the study period and vapour pressure deficit (i.e, the gradient in vapour density between the birds and their environment) was highly correlated with air temperature (R = 0.97, P < 0.0001), so only temperature data are presented. Ambient daily temperature was obtained from the Australian Bureau of Meteorology for the nearest town to the field site (Olary, SA, 25 km from Bimbowrie).

All active nests we found were recorded (n = 27) including 12 nests during incubation and 15 roost nests (of which 9 roost nests were visited at the time of the recording). Each nest was recorded for up to six recording days, for 2 to 13 h per day (unless the equipment failed after 30 minutes (n = 1) or 1 hour (n = 1)). Presence in the nest and individual sex was determined from calls and noises in the nest recordings, and confirmed with direct observations of the birds. All individuals recorded were fully matured adults (from plumage). A bout was defined as the uninterrupted presence of an individual settled in the nest for more than three minutes (mean duration ± s.e.: 55 ± 4 min). Short nest visits (<3 min, mean duration ± s.e.: 1 ± 0.05 min), typically when an individual repeatedly bring nesting material to its partner in the nest, were not considered as incubation or roosting bouts and excluded from the analyses. Varying that short-visit exclusion criteria from 3 min to either 2 or 5 min did not change any of the results. No individual ever called during these short nest-building visits (n = 172 short visits, in 10 breeding and 7 roosting nests, total 2.9 hours).

Audio recording in temperature controlled chambers

The chamber, placed in a dark temperature-controlled cabinet, consisted of a 1.5 L clear plastic container fitted with inlet and outlet ports to allow provision of dry air to maintain low humidity levels, similar to those measured in the wild. A tie-clip microphone (as above) was placed inside the chamber, and an infrared video camera (Jaycar, Australia) on the outside. We recorded 20 wild-derived adult zebra finches (10 of each sex), in either morning (starting 10:30am) or afternoon (2:30 pm) sessions, in March 2018. Each bird was recorded twice (except for 1 bird recorded only once because of scheduling constraints), 6 to 24 days apart (average 15 days), starting with either the morning or afternoon session and swapping for the second recording. A bird was caught in its home-cage (at 25 °C), weighted in a cloth bag on a digital scale (to nearest 0.01 g), and placed in the chamber at 25 °C for 25 or 45 min (for first and second recording respectively). Temperature was then increased to 35 °C for 30 min, and then to 40 °C, 42 °C and 44 °C for 20 min at each stage, unless the bird (monitored on the video) showed extreme agitation or weakness and had to be removed from the chamber (i.e. 2 birds were removed at 42 °C).

All procedures in the field and lab were approved by Deakin University Animal Ethics Committee (permit numbers B18-2017 and G06-2017) and the South Australian Department of Environment, Water and Natural Resources (DEWNR; permit U26305-4). All experiments were performed in accordance with Australian guidelines and regulations for the use of animals in research.

Acoustic analyses

For both field and lab recordings, spectrograms were inspected visually in Adobe Audition (Creative Cloud 2018) for the presence of incubation calls, by an experimenter, blind to the nest/bird identity, date and time of day12. The first and second recording per bird in the chamber were analysed by two different experimenters. Start and end time of incubation calling sequences were noted, where calls within 20 seconds of each other were considered as belonging to the same sequence. Sequence durations were summed, and divided by the duration of temperature stage, to obtained the calling rate per minute12.

Statistical analyses

To test predictors for calling occurrence (presence/absence of vocalisations) in the wild and under controlled lab conditions, we used Generalized Linear Mixed Models (GLMMs) with a logit link function and a Binomial error distribution (glmer function from the lme4 package in R), and normalised all predictors36. For field data (n = 177 in-nest bouts from 40 individuals in 21 nests), we used calling occurrence during a bout as the response variable and individual ID as a random effect (estimate for nest ID as a random effect was null). Nest-site temperature during the incubation bout, the linear and quadratic term for the time of day, sex and nest type (incubating or roost) were used as fixed factors. We also tested the interaction between sex and temperature. We could not include both ambient and nest-site temperature in the same model because they were highly correlated, so we included them in alternative models, that we compared using Akaike Information Criteria. For lab data (n = 191 temperature stages, for 20 individuals), we used calling occurrence during a temperature stage as the response variable, individual ID as a random effect (estimate for recording day as a random effect was null), and experimental temperature, time of day (morning or afternoon), sex and trial number (first or second per individual) as fixed factors. In addition, we used the same random (including day, non-null) and fixed factors in a GLMM with a logit link function and a Poisson error distribution using calling rate as the response variable. Within individual repeatability in the temperature threshold triggering vocalisation between trials 1 and 2 was tested using a non-parametric Spearman correlation. Lastly, the effect of relative body mass (mass in g over tarsus length in mm), sex and time of day on the calling temperature threshold in trial 1 were tested together using a linear model (lm function in R); temperature threshold was raised to the power of 5 to achieve normal distribution of the residuals, as validated by the Shapiro test. All tests were two-tailed. The data is available on Mendeley (https://doi.org/10.17632/2krj3kj7k3.1).