DOI: 10.5061/dryad.9w0vt4bvs
title: Data from: Parental care and bird embryonic metabolism: A comparison between uniparentally incubating silver-throated tits and biparentally incubating black-throated tits

description: Studies have shown that organisms may adjust metabolic rates in response
 to thermal variability, highlighting metabolic plasticity as a key
 adaptive mechanism. Understanding the extent of metabolic plasticity of an
 organism is key to predicting its adaptation to climate change. In birds,
 the embryos from the uniparentally incubating species are more frequently
 exposed to intermittent cooling due to the parents’ repeated absences from
 the nest than the embryos from the species with biparental incubation.
 Such pressure may favour them to evolve a better ability to cope with the
 physiologically suppressive effects of low temperatures and temperature
 fluctuations. We compared embryonic heart rate, a proxy for embryonic
 metabolic rate, and its response to egg temperature change between two
 closely-related species, the biparentally incubating Black-throated Tit
 (Aegithalos concinnus) and the uniparentally incubating Silver-throated
 Tit (A. glaucogularis). We also conducted an interspecific egg-swapping
 experiment to investigate the effect of incubation environment on
 embryonic metabolism and its consequences (i.e., incubation period length
 and hatching success). Consistent with the expectation, Silver-throated
 Tit exhibited a trend (although nonsignificant) of higher embryonic heart
 rate than Black-throated Tit. Also, when egg temperatures dropped,
 Silver-throated Tit showed a significantly slighter decrease in embryonic
 heart rate than Black-throated Tit, suggesting that they may better cope
 with temperature drop. In the egg-swapping experiment, embryonic heart
 rates did not differ significantly between fostered and unfostered eggs,
 but following fostering, heart rates tended to decrease in Silver-throated
 Tit embryos and increase in Black-throated Tit embryos, suggesting that
 embryos of both species show some responsiveness to new thermal
 environments. Egg-fostering treatment did not affect hatching success in
 either species, but the fostered eggs of Silver-throated Tits and
 Black-throated Tits tended to shorten and lengthen their incubation
 period, respectively, implying that the relatively stable biparental
 incubation environment may facilitate faster embryonic development than
 the more variable uniparental incubation environment. The findings enhance
 our understanding of the developmental strategies and responsiveness to
 changed environments in avian embryos under different parental care modes
 and provide insights into their potential to respond to climate change.
description: <strong>Research species and
 fieldwork</strong> This study was conducted in
 Zhanglou Village of Luoshan County, which is near the Dongzhai National
 Nature Reserve of Henan Province, China (31.95° N, 114.25° E). The two
 species, Black-throated Tit and Silver-throated Tit, occur sympatrically
 in this region and breed synchronously. They typically begin nest-building
 in late January or early February. Their nests are dome-shaped and similar
 in size, although slightly different in outer-layer nest materials (Li et
 al., 2012). Egg-laying generally starts in late February or early March,
 with one egg laid per day. Clutch sizes typically range from 4 to 8 eggs
 in Black-throated Tits and 5 to 9 eggs in Silver-throated Tits (Hu et al.,
 2024), with both species most frequently laying 7-egg clutches (Li et al.,
 2012). The average egg mass (mean ± SE) of Silver-throated Tits (0.85 ±
 0.01 g) is heavier than that of Black-throated Tits (0.72 ± 0.01 g) (Li et
 al., 2012). Both species usually initiate incubation on
 the day the clutch is completed, though in the late breeding season,
 incubation may begin prior to clutch completion. The two species did not
 differ significantly in average nest temperature (Hu et al., 2024), but
 nests of Silver-throated Tits experienced more frequent nest temperature
 fluctuations than nests of Black-throated Tits (mean number of
 fluctuations: 15.9 vs. 4.8 during the daytime; Hu et al., 2025a). The
 incubation period (mean ± SD) was 12.62 ± 1.01 days in Black-throated Tit
 and 13.85 ± 1.18 days in Silver-throated Tit (Hu et al., 2024). Major nest
 predators during the breeding season included corvids, Asian Barred Owlets
 (<em>Glaucidium cuculoides</em>), snakes, and small mammals
 (Li et al., 2012). The two species usually produce one successful brood
 each year but may renest after a nest failure. The
 Black-throated Tit and Silver-throated Tit populations at the study site
 have been monitored since 2011. Birds were captured using mist nets either
 during the winters (typically December to January) or breeding seasons
 (typically February to May). Each bird was banded with a unique
 combination of coloured rings and a metal ring for individual
 identification. During the breeding seasons, nests were located by
 searching or by following adult birds and then monitored regularly. Nests
 were usually checked every three days during nest-building, every two days
 during the egg-laying and incubation period (but more frequently when
 laying was imminent), and daily near hatching to determine hatch dates.
 For additional information on their breeding ecology and general field
 procedures, see Guan et al. (2018), Hu et al. (2024), and Li et al.
 (2012). <strong>Egg-swapping
 experiments</strong> Egg-swapping experiments
 were conducted during 2019 and from 2022 to 2024. In addition to routine
 nest monitoring, nests were checked daily between the laying of the
 6<sup>th</sup> egg and clutch completion to determine the
 onset of incubation. This was assessed by the female incubation behaviour
 and by touching eggs, as eggs in nests not yet incubated were obviously
 cool and exhibited a temperature contrast with incubated eggs (based on
 researchers’ experience). Nests from the two species where incubation
 initiation dates differed by no more than one day and had similar clutch
 sizes (± 1 egg, within the natural clutch size range of both species) were
 paired, prioritizing the nests with close proximity. One exception
 included a pair with 9 eggs in the Silver-throated Tit and 7 eggs in the
 Black-throated Tit. Entire clutches were cross-fostered
 between paired nests on the first day of incubation (19 Black-throated Tit
 and 16 Silver-throated Tit nests) or the second day (24 Black-throated Tit
 and 27 Silver-throated Tit nests). Fostered eggs were marked to
 distinguish species. In 2019, eggs were returned to their natal nests on
 day 9 or 10 of incubation. In 2022 ~ 2024, to prolong embryos’ exposure to
 a fostering nest environment, eggs were returned to their natal nests on
 day 10 or 11. No embryos hatched before the eggs were returned to their
 natal nests, except in cases where nests failed. During all transfers,
 eggs (including those for embryonic heart rate measurements, see below)
 were transported in cotton-lined plastic boxes heated with warm water
 bottles or heat packs, which were pre-tested for temperatures not
 exceeding 40 °C. Each egg transfer session lasted 30
 mins. Given their high natural nest failure rate (e.g., 76% for
 Silver-throated Tit; Guan et al., 2018), wire meshes were placed
 surrounding the experimental nests to protect them from predation until
 nestling hatching. These cages (mesh grid size: 3 × 3 cm) allowed access
 by Black-throated Tis and Silver-throated Tits but deterred some predators
 ~(~e.g. corvids). Despite the protection, 23 out of 86 paired nests
 (26.7%) failed during incubation. Experimental nests
 that underwent egg swapping were classified as the experimental group,
 while naturally incubated nests served as the control group. To ensure
 their temporal comparability, only natural nests with the date laying the
 first egg falling within the earliest and latest first-egg-laying dates of
 a year’s experimental nests of each species were included as
 controls. <strong>Embryonic heart rate and egg
 temperature measurements</strong> The embryonic
 heart rate measurements were conducted in 2023 and 2024. Considering the
 substantial workload involved in measuring entire clutches, only a subset
 of eggs was selected for embryonic heart rate measurement. As differences
 in embryonic heart rate may exist between early- and late-laid eggs within
 a clutch (Boonstra et al., 2010; Cones & Westneat, 2024; Wang et
 al., 2023), the 3<sup>rd</sup> to 5<sup>th</sup>
 egg in each nest was chosen for measurement. Nests were checked every two
 days, and once two eggs had been laid, daily marking of each newly laid
 egg continued until the 5<sup>th</sup> egg was laid, to
 distinguish the 3<sup>rd</sup>, 4<sup>th</sup>,
 and 5<sup>th</sup> eggs. Besides, due to
 limited existing evidence, it remains unclear at which embryonic
 developmental stages a changed incubation environment is most likely to
 influence metabolic responses. In addition, the metabolic responses to
 environmental variation are energetically costly (DeWitt et al., 1998;
 Monaghan et al., 2009), so embryos may have sufficient energetic capacity
 to respond to thermal variation early in development, but the capacity may
 be reduced at a later development stage if energy reserves have already
 been consumed (Vleck & Vleck, 1987). As a result, responsiveness
 to temperature may differ between early and late incubation stages. For
 these reasons, we collected and analysed data separately for early and
 late incubation stages (see below). This approach allowed us to explore
 whether responsiveness to incubation environment is stage dependent, i.e.,
 whether the relationships between temperature and heart rate differ across
 developmental stages of embryos. Previous studies have
 shown that in small passerines such as Zebra Finches, embryonic heart rate
 usually becomes detectable on day 5 of incubation (Sheldon et al., 2018).
 To collect data from both early and late developmental stages of
 Black-throated Tit and Silver-throated Tit’s embryos, we measured
 embryonic heart rates on day 5 and day 10 after the start of incubation,
 with the measurement on day 5 representing the early developmental stage,
 and that on day 10 representing the late stage. It is worth noting that
 among the 39 Silver-throated Tit nests and 54 Black-throated Tit nests in
 which embryonic heart rates were measured, one Silver-throated Tit nest
 was measured on day 6 and day 11 of incubation, and one Black-throated Tit
 nest was measured on day 9. The measurement on day 6 was treated as the
 early stage, while the measurements on days 9 and 11 were considered as
 the late stage. When transferring target eggs for
 embryonic heart rate measurement, failed eggs from a deserted nest or
 paraffin eggs of similar size were placed in the nest to replace the
 removed eggs, maintaining at least five eggs per nest to prevent nest
 abandonment. The dummy eggs were then removed when the target eggs were
 returned. For each measured egg, the nest of origin, whether it came from
 a fostered clutch, and its developmental stage (early or late) were
 recorded. Each egg was then transferred to an indoor incubator (Yike·Beite
 Incubator, LN2-16), preheated to ~38 °C, and maintained at 50 ~ 70%
 humidity. After warming in the incubator for at least 15 mins, eggs were
 randomly selected and removed one at a time for embryonic heart rate
 measurement using a Buddy MK2 Digital Egg Monitor (Vetronic Services, UK;
 Sheldon et al., 2018). We attempted to measure embryonic heart rate at
 1-minute intervals. However, due to the small size of the embryos and
 their movement within the egg (Sheldon et al., 2018), stable detection was
 often delayed or quickly lost. When embryonic heart rate became
 undetectable, the egg’s position was gently adjusted (Sheldon et al.,
 2018) until a stable signal was obtained. Thus, actual intervals between
 measurements were often longer than one minute. The average time from
 removal from the incubator to the first successful stable embryonic heart
 rate reading was 43.47 ± 49.59 s (mean ± SD), ranging from 4 to 517 s
 (<em>n</em> = 358). Once the embryonic heart rate stabilized,
 the value was recorded, and the egg’s surface temperature was immediately
 measured using a thermal imaging camera (HIKMICRO-H10, accuracy: ± 2 °C)
 at a distance of 15 cm, with the highest surface temperature recorded as
 the egg temperature. Multiple embryonic heart rate readings were taken for
 each egg until its highest surface temperature dropped to ~27 °C. After
 measurements were completed, the egg was returned to the incubator for
 reheating until all eggs of the clutch were measured and could be returned
 to their nest. We did not choose to measure embryonic
 heart rate immediately after eggs were removed from the nest. This was
 because the nest entrances were small and the process of egg removal
 inevitably took some time, leading to a decrease in egg temperature. As a
 result, it was not easy to obtain embryonic heart rate values at high egg
 temperatures in the field. Measuring eggs indoors after re-heating ensured
 that we could capture embryonic heart rate across the full range of
 ecologically relevant incubation temperatures. While transport and
 re-heating may introduce some stress or transient effects, all eggs were
 subject to the same procedures under standardized conditions.
 On average, 5.43 ± 3.07 (mean ± SD) embryonic heart rate values
 were recorded for each egg at the two developmental stages (range: 1 ~ 11,
 <em>n</em> = 420). After embryonic heart rate measurement,
 each egg was weighed to the nearest ± 0.001 g. The time from egg removal
 from the nest to being returned on the same day averaged 1.96 ± 0.71 h
 (mean ± SD; range: 0.90 ~ 4.33 h, <em>n</em> = 146). However,
 due to the large number of nests requiring measurement on certain days,
 some eggs were not returned on the same day (involving 8 Black-throated
 Tit nests and 7 Silver-throated Tit nests; see Table 2 for details). In
 such cases, the eggs were kept in the incubator overnight with the
 temperature set to ~37.8 °C and humidity maintained at 50 ~ 70% and were
 manually turned 1 ~ 2 times between 7 PM and 12 AM. They were returned to
 the nest the following morning. According to the continuous monitoring,
 incubation in the incubator for one night did not noticeably affect the
 hatching success of the eggs. <strong>Ambient
 temperature data</strong> To monitor ambient
 temperature, a temperature logger (GSP958, accuracy: ± 0.5 °C; Guangzhou
 Lexiang Electronics Co., Ltd., China) was placed at each of three major
 types of habitats favoured by the two species within the study area. Each
 logger recorded temperature at 1-minute intervals and was replaced every
 21 days due to memory limitations. The average of the three loggers was
 used as the ambient temperature for the study area. However, due to setup
 issues, some loggers failed to record for 21 days. In these cases, the
 average of the remaining one or two loggers was used instead.
 Additionally, technical issues of the loggers occasionally caused
 anomalous values at specific temperatures (e.g., ~11.9 °C, 25 °C, and
 38.1 °C). These anomalies were replaced by the average of the values
 recorded 1 minute before and after. To examine the
 effect of ambient temperature on embryonic heart rate (see below), the
 average daily ambient temperature from the start of incubation to the day
 before embryonic heart rate measurement was calculated for each nest to
 represent the mean temperature experienced during embryonic development.
 In addition, the mean daily ambient temperature over the entire incubation
 period for each nest was also calculated to assess its effects on
 incubation period length and hatching success (see below).
 <strong>Data analysis</strong>
 Although some individuals may renest after previous nest failure
 within a breeding season, only the data from their first clutch in that
 year were used in the analyses. For individuals recorded for breeding in
 multiple years, we prioritized using their data from nests involved in the
 egg-swapping experiment. If no such nests were available, we used the data
 from their first-year records. Therefore, no individual parent had
 repeated data in a given analysis. See Table 3 for additional details of
 our experimental design. All analyses were conducted in R version 4.5.0 (R
 Core Team, 2025). <strong>Effects of factors on
 embryonic heart rate</strong> To compare
 embryonic heart rate between species and among treatment groups, we
 categorized eggs into four types: Black-throated Tits’ not-fostered eggs
 (BTN), Black-throated Tits’ fostered eggs (BTY), Silver-throated Tits’
 not-fostered eggs (STN), and Silver-throated Tits’ fostered eggs (STY),
 with fostered eggs meaning those incubated by another species (see Table 2
 for sample sizes). We performed analyses with linear
 mixed models (LMMs) using the <em>lme4</em> package version
 1.1-37 (Bates et al., 2025). In the LMMs, embryonic heart rate was treated
 as the response variable. The initial fixed effects in the model included
 egg temperature at the time of embryonic heart rate reading, egg mass on
 the day of measurement, the average daily ambient temperature experienced
 by the embryo from the start of incubation to the day before measurement,
 laying date (days from December 31 of the previous year to the date laying
 the first egg for a nest), egg type (i.e. BTN, BTY, STN and STY), year,
 developmental stage (i.e. early or late stage). According to our
 hypotheses and predictions, the initial fixed effects also included the
 interaction between egg type and egg temperature. Because the sensitivity
 to environmental variation may differ among species between early and late
 stages of embryonic development, we further incorporated two-way
 interactions of developmental stage with egg type and with egg
 temperature, as well as the three-way interaction among developmental
 stage, egg type, and egg temperature. We included nest ID and egg ID
 nested within nest ID as random effects to account for non-independence
 among eggs within the same nest and repeated measures of the same
 egg. Because of a high variance inflation factor (VIF),
 year was excluded from the fixed effects (see below). Accordingly, the
 final model was: embryonic heart rate ~ egg temperature + egg mass +
 average daily ambient temperature experienced by embryos + laying date +
 egg type + developmental stage + egg type egg temperature + developmental
 stage egg type + developmental stage egg temperature + developmentalstageg
 stage egg temperature + (1 | nest ID/egg ID). Because the result of the
 above model (Supplementary Table S1) indicated a three-way interaction (p
 &lt; 0.05), we conducted separate analyses for early and late
 developmental stages to better explore the differences between them. The
 model formula for the analysis of each specific stage was: embryonic heart
 rate ~ egg temperature + egg mass + average daily ambient temperature
 experienced by embryos + laying date + egg type + egg type* *egg
 temperature + (1 | nest ID/egg ID). The results for each stage are
 presented in Table 4. <strong>Effects of
 egg-swapping on incubation period length and hatching
 success</strong>  The data for the incubation
 period length included 29 experimental and 39 control Silver-throated Tit
 nests, and 31 experimental and 56 control Black-throated Tit nests. These
 nests included the nests where the eggs were used to measure embryonic
 heart rate but excluded those where eggs hatched in fostering nests during
 the egg-swapping experiment (see above). In addition, a small proportion
 of nests, including 3 experimental (10%) Silver-throated Tit nests, and 2
 experimental (6%) and 4 control (7%) Black-throated Tit nests, had eggs
 that experienced one night of artificial incubation (see above).
 To assess the effects of egg-swapping on incubation period
 length, we used incubation period length as the response variable in the
 linear model. Fixed effects in the model included species, treatment
 (fostered or not-fostered), average daily ambient temperature during the
 incubation period, clutch size, year, and laying date. As we were
 interested in the species-specific effect of treatment, we also included
 an interaction between species and treatment in the fixed effects. Because
 of high VIF (see below), the variable “year” was further excluded from the
 fixed effects. Accordingly, the final model was: incubation period length
 ~ species + treatment + average daily ambient temperature during the
 incubation period + clutch size + laying date + species
 treatment. For hatching success, the dataset included
 22 experimental and 37 control nests for Silver-throated Tits, and 22
 experimental and 44 control nests for Black-throated Tits. The analysis
 was conducted using a generalized linear model, with hatching success
 (proportion of eggs that hatched per nest) as the response variable. Fixed
 effects were the same as those in the incubation period analysis, with
 year also being excluded due to high VIF. The final model was: hatching
 success ~ species + treatment + average daily ambient temperature during
 the incubation period + clutch size + laying date + species*treatment. The
 model with a binomial error distribution was found to be overdispersed, as
 assessed by the <em>testDispersion</em> function from the
 <em>DHARMa</em> package version 0.4.7 (Hartig et al., 2024).
 Therefore, a beta-binomial distribution (Young-Xu & Chan, 2008)
 with a logit link was used for this model. To account
 for potential effects of overnight artificial incubation (see above) on
 incubation period length and hatching success, we re-ran the two analyses,
 excluding nests where the measured eggs had experienced overnight
 artificial incubation. The results (presented in Supplementary Table S2)
 were consistent with those obtained from the full dataset (Table 5),
 indicating that overnight artificial incubation did not affect our
 conclusions. Before analyses, multicollinearity among
 predictors was assessed using the VIF calculated via the
 <em>car</em> package version 3.1-3 (Fox et al., 2024). Across
 all analyses, factors that had a high VIF (&gt; 5) (Akinwande et al.,
 2015) were excluded (see above). Variables or factor-level differences
 were considered statistically significant when <em>p</em>
 &lt; 0.05. To test for pairwise differences among different levels of
 categorical predictors (e.g., egg types) or interactions between
 continuous and categorical variables in the above analyses, we conducted
 post-hoc comparisons in the emmeans package version 1.11.1 (Lenth,
 2025).
description: # Data from: Parental care and bird embryonic metabolism: A comparison
 between uniparentally incubating silver-throated tits and biparentally
 incubating black-throated tits Dataset DOI:
 [10.5061/dryad.9w0vt4bvs](https://doi.org/10.5061/dryad.9w0vt4bvs) ##
 Description of the data and file structure Dataset DOI:
 [10.5061/dryad.9w0vt4bvs](https://doi.org/10.5061/dryad.9w0vt4bvs) ###
 Files and variables **File:
 Hu_et_al_2026_Functional_Ecology_EHR_data.csv** ##### Variables * NestID:
 Nest ID for each nest. * EggID: Egg ID for each egg. * EggType:
 Black-throated Tits’ not-fostered eggs (BTN), Black-throated Tits’
 fostered eggs (BTY), Silver-throated Tits’ not-fostered eggs (STN), and
 Silver-throated Tits’ fostered eggs (STY), with fostered eggs meaning
 those incubated by another species (see Table 2 for sample sizes). * Year:
 Research years. * Species: Black-thoated Ti (BT) and Silver-thorated Tit
 (ST). * AdultID: Adult ID for each nest. * Treatment: Not-fostered eggs
 (N) and fostered eggs (Y). * EggMass: Egg mass (g) of each egg that
 weighed after embryonic heart rate measurement. * HR: Embryonic heart
 rates (bpm). * Tegg: Highest egg surface temperature (°C). *
 Clutch_size: Clutch size of each nest. * Date: Laying date (days from
 December 31 of the previous year to the date laying the first egg for a
 nest). * Early_Late: Early development satge (Early) and late development
 stage (Late). * AveTem: The average daily ambient temperature (°C) from
 the start of incubation to the day before embryonic heart rate
 measurement. **File:
 Hu_et_al_2026_Functional_Ecology_Hatching_Success_data.csv**
 **Description:**  ##### Variables * NestID: Nest ID for each nest. *
 Species: Black-thoated Ti (BT) and Silver-thorated Tit (ST). *
 Year: Research years. * AdultID: Adult ID for each nest. *
 Treatment: Not-fostered eggs (N) and fostered eggs (Y). * Date: Laying
 date (days from December 31 of the previous year to the date laying the
 first egg for a nest). * Clutch_size: Clutch size of each nest. *
 Brood_size: Brood size of each nest. * Artif_Inc: Whether the measured
 eggs had experienced overnight artificial incubation (Y: Yes; N: No). *
 Inc_Tem: The mean daily ambient temperature (°C) over the entire
 incubation period of each nest. **File:
 Hu_et_al_2026_Functional_Ecology_Inc_Length_data.csv** **Description:** 
 ##### Variables * NestID: Nest ID for each nest. * Species: Black-thoated
 Ti (BT) and Silver-thorated Tit (ST). * Year: Research years. *
 AdultID: Adult ID for each nest. * Treatment: Not-fostered eggs (N)
 and fostered eggs (Y). * Date: Laying date (days from December 31 of the
 previous year to the date laying the first egg for a nest). *
 Clutch_size: Clutch size of each nest. * Artif_Inc_overnight: Whether the
 measured eggs had experienced overnight artificial incubation (Y: Yes; N:
 No). * Inc_length: Incubation period length of each nest (day). *
 Inc_Tem:  The mean daily ambient temperature (°C) over the entire
 incubation period of each nest