<b>Do egg traits used as recognition cues show greater individual distinctiveness?</b>

Egg length and width were measured using a digital caliper (GB/T1214.2_1996, precision 0.01 mm). Egg volume was calculated. Physical characteristic indicators of eggs are described below:</p><p dir="ltr">Egg mass: Fresh egg mass before embryonic development begins (g);</p><p dir="ltr">Egg length (L): Linear distance from blunt to pointed end of egg (mm);</p><p dir="ltr">Egg breadth (B): Equatorial plane diameter of egg (mm);</p><p dir="ltr">Egg volume: Calculated using the formula V (mm³) = 0.51LB², where L is egg length (mm) and B is egg width (mm) (Hoyt, 1979).</p><p><br></p><p dir="ltr"><b>Quantification of egg color</b></p><p dir="ltr">Using eggs from abandoned nests of target bird species for replacement, complete clutches of eggs from the three bird species were brought indoors for spectral measurements.

An Avaspec-ULS2048×64 USB2 fiber-optic spectrometer (Avantes, Inc., Louisville, CO, USA), Avalight-DHc compact deuterium-halogen combined light source (Avantes, Inc.), and a reflection probe (FCR-7UV200-2-ME; Avantes, Inc.) were used to measure spectral reflectance in the 300–700-nm wavelength range. The reflection probe was positioned perpendicular (90°) to the egg surface at a distance of 1 mm, with integration time set to 200 ms.

During measurement, dark calibration was performed with the light source off, followed by white calibration after turning on the power using a white diffuser plate (WS-2) made of polytetrafluoroethylene material, providing reflectance above 99% across the entire spectrum (Nahid et al. 2024). When measuring egg background color reflection spectra, each egg was divided into three regions from the pointed to the blunt end along its length axis, with one measurement point randomly selected from each region; the average of these three measurement-point reflection spectra represented the reflection spectral value of one egg.</p><p dir="ltr">Traditional research has predominantly focused on single cues (e.g., egg color or size), yet hosts may employ more sophisticated multidimensional defense strategies.

To investigate how brood parasitism specifically shapes the phenotypic dimensions corresponding to the recognition cues hosts rely upon, this study examined three bird species with different parasitism pressures but clearly defined primary recognition cues: the grey bushchat (<i>Saxicola ferreus</i>), a host primarily using egg color as its primary cue; the crested myna (<i>Acridotheres cristatellus</i>) and white-shouldered starling (<i>Sturnia sinensis</i>), two hosts whose primary cue is egg size.

Results revealed that all three species exhibited greater inter- than intra-clutch variation in their respective key defense dimensions. Specifically, the host grey bushchat displayed the highest inter-clutch entropy and the smallest intra-clutch Euclidean distance in egg color, indicating effective defense through maximizing population-level color combination uniqueness (high entropy) and extreme individual intra-clutch consistency.

In contrast, the crested myna exhibited the highest inter-clutch entropy and the largest intra-clutch Euclidean distance in egg size. These results suggest that brood parasitism pressure drives hosts to evolve optimized phenotypic variation in their key defense dimensions. Our findings indicate that hosts response to brood parasitism do not simply maximize variation but rather represent trait-specific fine-tuned adaptations and support the theoretical perspective that parasitism pressure primarily acts on trait combination patterns (combinatorial uniqueness) rather than on the absolute variation magnitude of a single trait.</p>