Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus


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  • 1We test the consequences, in terms of breeding success and parental effort, of eggshell pigmentation pattern in a hole-nesting bird, the blue tit Cyanistes caeruleus that lays eggs asymmetrically speckled with reddish spots (maculated eggs).
  • 2We assess the effect of distribution of spots (pigment ‘spread’) and spot size and pigment intensity (pigment ‘darkness’) on eggshell physical properties and breeding parameters concerning nestling condition, investment of parents in offspring care and reproductive output in two different habitat types: a deciduous oakwoodland and an evergreen forest.
  • 3Blue tit clutches with more widely distributed spots showed a thicker eggshell, a shorter incubation period, a lesser amount of mass loss per day and a higher hatching probability than those with spots forming a ‘corona’ ring. While eggs with larger and darker (more pigment intensity) spots showed a thicker eggshell and a shorter incubation period. In the light of ‘signal function hypothesis’, these egg traits may reflect female health status and, consequently, this could affect male parental effort.
  • 4Here we show supports for some of the necessary assumptions of this hypothesis. We found a positive relationship between egg pigment ‘spread’ and male but not female provisioning rates per day. On the other hand, pigment ‘darkness’ of blue tits’ clutches was positively related to female tarsus length, while pigment ‘spread’ was positively related to clutch size, male body mass and nestling tarsus length. Our study shows that eggshell pigment ‘spread’ can be used as an indicator of clutch quality. Further investigations are needed to understand the role of calcium availability as possible causal agent of deviant eggs and its relation to the maculation phenomenon.


Many members of the family Paridae lay white eggs speckled with reddish spots which tend to aggregate around the broad end of the egg (Cramp 1998). Several hypotheses have been proposed to explain the significance of avian eggshell coloration and patterning (for a review Soler et al. 2005; Kilner 2006). It is important to distinguish between egg colour and eggshell pigmentation patterning. The subject of this study is the eggshell pigmentation patterning, that is to say how the pigmented spots are distributed over the eggshell. Maculated eggs with the same distribution of reddish spots could have different colours due to differences in the accumulation of pigments on each spot or in the background pigmentation of the egg. Eggshell pigmentation patterns had been traditionally explained in terms of crypsis or mimetism (Kilner 2006). Nevertheless, the facts that members of the family Paridae with maculated eggs are hole-nesting birds, that females cover the clutch with nest-lining during the laying period and that they appear unresponsive to brood parasitism (Gosler 1993), suggest that crypsis or mimesis are unlikely to explain eggshell pigmentation pattern in this group (Higham & Gosler 2006). This does not mean that these hypotheses cannot be applied to other families of birds.

Moreno & Osorno (2003) proposed that egg coloration, with special emphasis on blue-greenish eggs, is a sexually selected signal of females displaying their phenotypic quality or physical condition to their mates in order to induce a higher allocation of parental care. This hypothesis for blue-greenish eggs is based on the fact that biliverdin is an antioxidant and thus the deposition may signal antioxidant capacity of the laying females (Moreno & Osorno 2003). For this type of egg coloration, several studies have found some evidence in support of this hypothesis (e.g. Moreno et al. 2004, 2005, 2006; Siefferman, Navara & Hill 2006). Moreno & Osorno (2003) suggested that the ‘signalling hypothesis’ could also be applied to species with maculated eggs, since protoporphyrin, the main pigment found in eggs of this egg type (Kennedy & Vevers 1976), is a pro-oxidant that may signal tolerance by females to oxidative stress. A recent paper shows correlational evidence that coloration of maculated eggs is a good indicator of general condition and stress of female blue tits [Cyanistes caeruleus (L.)]; females laying more spotted eggs showed a poorer body condition, higher cellular concentration of a stress protein and marginally lower total immunoglobulin levels in blood (Martínez-de la Puente et al. 2007). These authors do not consider egg pigmentation as a signal.

On the other hand, Gosler and co-workers (Gosler, Barnett & Reynolds 2000; Gosler, Higham & Reynolds 2005; Higham & Gosler 2006) have recently presented evidence indicating that in great tits, Parus major L., eggshell pigmentation patterning might compensate for reduced eggshell thickness, caused by structural variation in the shell and by calcium deficiency. These studies in the great tit supported the hypotheses that protoporphyrin might have a structural function in strengthening the shell and share a protein carrier with calcium within the shell gland, so being deposited specifically when calcium is scarce (Solomon 1987, 1997). Following Solomon's idea, these authors proposed the ‘structural-function hypothesis’ to explain the variability within clutches found in small hole-nesting passerines that present maculated eggs (Gosler et al. 2005). Calcium, which should be daily collected for egg production (Graveland & van Gijzen 1994), is a critical limiting resource for shell formation in small birds (Graveland et al. 1994; Graveland 1996; Graveland & van der Wal 1996; Graveland & Berends 1997; Graveland & Drent 1997). Calcium is not stored in the skeleton before egg laying (Graveland & van Gijzen 1994). Calcium demands during egg laying can be substantial. For example, a complete clutch of blue tit can contain more calcium than the entire female skeleton (Perrins & Birkhead 1983).

Eggshell pigmentation pattern could vary between females because of genetic, environmental and/or maternal effects. Eggshell pigmentation patterning of maculated eggs can be defined by two principal components of pigmentation: ‘darkness’ and ‘spread’ (see Material and Methods section and Gosler et al. 2000, 2005). In great tits, pigment ‘darkness’ but not ‘spread’ is known to be heritable by mother/daughter correlations (Gosler et al. 2000, 2005). Moreover, pigment ‘darkness’ in this species is negatively related to laying date and unrelated to clutch size (Gosler et al. 2000), and it is also influenced by female age: first-year females lay significantly darker eggs (Gosler et al. 2005). On the other hand, pigment ‘spread’ (but not ‘darkness’) in great tits is negatively related to the unpigmented-shell thickness especially in the region of the ‘corona’ ring at the shoulder and crown (Gosler et al. 2005). Finally, pigment ‘darkness’ but not ‘spread’ affects the rate of egg water loss, which is critical for normal embryonic development (Higham & Gosler 2006). This conclusion was obtained from a cross-fostering experiment that suggested that females with more pigmented clutches appear to have increased incubation effort compared with those females with less pigmented clutches (Higham & Gosler 2006). Moreover, Graveland et al. (1994) found that defective, thin-shelled eggs of great tits could be easily recognized at a glance by their dull shell and deviant pigmentation. Following the ‘structural-function hypothesis’, one could roughly estimate calcium availability within the breeding territory or female ability to acquire/assimilate it from eggshell pigmentation pattern. This could be carried out under the assumption that such a system found in great tits is extrapolated to other related species, such as blue tits. Recently, it has been found in a study carried out with dichloro-diphenyl-trichloroethane (DDT)-contaminated sparrowhawk (Accipiter nisus L., Falconiformes) eggs that eggshells showing protoporphyrins spots as an internalized pigment layer showed a strong correlation between pesticide levels and shell thickness reinforcing the view that maculation is related to eggshell thinning (Jagannath et al. 2008). In this sense, little is known about the impact of eggshell pigmentation pattern on breeding performance.

In this study, we intend to identify factors explaining the variation in eggshell pigmentation pattern of blue tits, a hole-nesting bird that lays maculated eggs, by using models with multiple independent variables. This is the first time that predictions derived from the ‘structural-function’ (Gosler et al. 2005) and ‘signalling’ (Moreno & Osorno 2003) hypotheses are tested at the same time. We examine the correlations existing between eggshell pigmentation pattern and reproductive traits related to incubation behaviour, reproductive success and parental effort. Finally, we assess whether pigmentation patterning is related to physical properties of the eggshell to test whether it can be considered as a reliable indicator of egg viability in this species.

Material and methods

blue tit breeding

The study was conducted over the breeding seasons of 2006 and 2007 in two nestbox plots situated within the Cabañeros National Park (Ciudad Real, central Spain). Our study plots represent two predominant habitat types in the Mediterranean region: a sclerophyllous forest (Anchurones) and a deciduous forest (El Brezoso) located at a short distance (2 km) from each other. The evergreen patch is located in a floodplain dominated by holm oaks (Quercus rotundifolia) and, in a lesser extent, by cork oaks (Quercus suber) scattered in a dehesa-like configuration with a close understorey in which Cistus ladanifer (gum cistus) dominates. This shrubland structure is most commonly found on very poor or degraded soils (Blanco et al. 1997). El Brezoso is mainly covered with Pyrenean oaks (Quercus pyrenaica), while in the field layer, the heather (Erica spp.) is the prevalent species. In this area, overgrazing pressure by red deer (Cervus elaphus) and wild boar (Sus scrofa) is more marked than in the holm oak woodland especially during the hot and dry summer when these species take shelter in the most humid environments. More details about study area can be found in García Canseco (1997).

A total of 250 wooden nestboxes were erected in a grid with 30–50 m between adjacent nestboxes. Nestboxes were checked for occupation by blue tits, and the laying date (1 = 1 April) and clutch size were recorded. Nests were checked daily around the expected day of hatching to establish hatching date. The length of incubation was defined as the number of days between the onset of full incubation (the day on which the female was found incubating or the eggs were found uncovered and warm) and the first signs of hatching. In 2007, the incubation behaviour (frequency and duration of off-bouts) of birds was monitored using videocameras (JVC GPD-240, Yokohama, Kanagawa, Japan). We filmed 33 nests (80–100 min per nest) 7 days after the initiation of incubation. Only the last 60 min of each recording were considered on analyzing the average time that female spent on the nest (nest attentiveness) and the mean duration of on-bouts (incubation scheduling). We also registered the number of food passes by the male to their mate during this period (incubation courtship feeding).

Adults (2006: 39 males and 43 female forming 38 pairs; 2007: 58 males and 57 females forming 52 pairs) were captured with spring traps when the young were 8 days old (day of hatching = 0), ringed, aged as of one or more years (older) according to plumage characteristics (Jenni & Winckler 1994), weighed and their tarsus was measured. Trapped birds were also equipped with a ruggedized micro transponder (Trovan ID 103, length: 11·6 mm, mass: 0·1 g; Trovan Ltd., Isle of Man, UK) glued to two colour bands and wrapped in a piece of black duct tape. These microchips produce a unique amplitude modulated code signal in the presence of an electromagnetic field providing individual identification of each bird. An antenna connected to a data logger was fitted to the entrance hole of the nestbox allowing us to obtain the number of sex-specific feeding visits (for more details see Brün & Lubjuhn 1993; Johnsen et al. 2005; Macleod, Gosler & Cresswell 2005). We recorded the daily feeding rates of both sexes (total number of feedings per 24 h, 60 males and 61 females forming 59 pairs) when the young were 11 days old.

Adults were weighed with an electronic balance (± 0·1 g) and their tarsus length was measured to the nearest 0·01 mm with a digital calliper. On day 13 after hatching, nestlings were ringed and similarly measured. Nestboxes were visited at the end of the breeding season in order to determine the number of fledged and dead nestlings. Two partial measures of reproductive success were considered: hatching success as the proportion of eggs hatched, and fledging success as the proportion of eggs that resulted in fledglings.

Temperature data employed to measure nest temperature during incubation period were obtained from data loggers (Gemini Tiny Talk, Chichester, West Sussex, UK; recording at 5-min intervals) placed in three randomly selected empty nestboxes at each study area.

eggshell pigmentation

Nests were visited daily during egg laying and two photographs of each new egg were taken (side and bottom views) with the aid of a small base and a squared background. Each egg was numbered with an ink permanent pen. Following Gosler et al. (2000, 2005), we scored the eggshell pigmentation pattern from the side view on the basis of three categories: pigment intensity (I: scored in 0·5 increments from 1 for palest spots to 5 for the darkest), average spot size (S: scored in 0·5 increments for 1 for small spots to 3 for large spots) and spotting distribution (D: scored in 0·5 increments from 1 for > 90% of spots concentrated in one end, to 5 for an even spot distribution) over the surface (see Fig. 1). A total of 1061 eggs (2006: 376; 2007: 685) of 131 clutches (2006: 47; 2007: 84) were assessed by the same observer (V.G.N.), who was blind with respect to the origin of the eggs. Within-clutch repeatability values for eggshell pigmentation scores were significant (I: r = 0·43, F = 7·22; D: r = 0·49, F = 6·84; D: r = 0·46, F = 7·95, all P < 0·001). The length and breadth of each egg was measured using the software package motic 3·0 and the breadth/length ratio was used as egg shape index (Schönwetter 1979). Egg volume was calculated using Hoyt's (1979) equation. On the other hand, by means of the program adobe photoshop 6·0, we measured the photographed egg surface covered by pigment (only in the case of photographs corresponding to breeding season of 2006). Through the option ‘Magic Wand Tool’, the colour tolerance parameter of selection was adjusted until the separation of the speckled area from the white matrix was achieved. The number of pixels contained within the area covered by spots was compared with the number of pixels that represent the total surface of each egg. This allows us to estimate the eggshell percentage covered by spots in lateral (2006–07) and bottom view (2006).

Figure 1.

Egg pigment variation among clutches of blue tits breeding in central Spain. Columns represent, left to right, intensity (I, scored 1–5), distribution (D, 1–5) and spot size (S, 1–3). Rows represent increasing values from top to bottom.

egg sampling

In the second study year, 470 eggs from 56 clutches were weighed in the field to the nearest 0·001 g with a portable electronic balance (Tanita 123, Tokya, Japan) on the day that they were laid and on day 7 after clutch completion. The difference in mass obtained from these two weighings was divided by the number of elapsed days between both measurements. Thereby, we calculated the daily rate of mass loss (Ar et al. 1974) during the incubation period although eggs lost mass from the moment they are laid, even when not incubated. So there is a component of mass loss (the ‘passive’ mass loss; Higham & Gosler 2006) due only to intrinsic factors (e.g. surface area), not mediated by the external influence of the incubating female.

On the other hand, we collected 70 unhatched eggs from 70 clutches in 2007. Eggs were collected in the two study sites and in a nearby box-nesting population. This study plot was located in Los Quintos de Mora (Toledo, central Spain) a government-owned and managed area, which is 21 km away from the other two plots and dominated by deciduous trees (mainly Pyrenean oaks). Pigmentation pattern (I, D, S) of eggs collected in Los Quintos de Mora (n = 40) was scored (by V.G.N.) from photographs taken following exactly the same procedure described above. Eggs were refrigerated at 4 °C on the day of collection until mid-October 2007, when they were opened at the blunt end and emptied. Before this, eggs lengths and breadths were measured with a digital calliper to the nearest 0·1 mm and breadth/length ratio and volume calculated. Empty shells were thoroughly wiped inside to remove the remaining albumen and the inner egg membrane. We measured shell thickness (to 0·001 mm) in the shoulder region of the egg (where hatching occurs) using a micrometer (Mitutoyo Kawasaki, Kanagawa, Japan). In this region, the spots are often concentrated forming a corona ring. We calculated the mean of three measurements around the shoulder of the egg including unpigmented and pigmented areas (repeatability = 0·79, P < 0·001; Lessells & Boag 1987). Shells were then weighted to the nearest 0·001 g with a Kern ABS electronic balance.

statistical methods

Because the scoring variables of eggshell pigmentation pattern were significantly intercorrelated, (I and D: r1061 = −0·223; I and S: r1061 = 0·317; D and S: r1061 = −0·087, all P < 0·01), a principal component analysis (PCA) of the correlation matrix of the original I, D, and S values (side views) was performed to diminish redundancy of those (Gosler et al. 2000). Using individual eggs, two principal components (PC1 and PC2) which explain around 80·1% of the variation of spotting pattern were obtained. The first principal component (PC1) describes variation in pigment intensity and spot size and it was referred to as the ‘darkness’ of the egg (see Gosler et al. 2000, 2005). Eggs with larger and more reddish spots showed higher PC1 values (factor loadings: I: 0·831, D: −0·257, S: 0·815). PC1 expresses 47·4% of the total variation in I, D and S. The second principal component (PC2) describes the level of spot aggregation and show higher values for eggs with spottiness more widely distributed over their surface (factor loadings: I: 0·086, D: 0·963, S: 0·215). PC2 was taken to represent the ‘spread’ of maculation (see Gosler et al. 2000, 2005). PC2 expresses a further 32·8% of the total variation in I, S and D. Considering only the 2006 data set, mean pigment ‘darkness’ (PC1) of the clutch was positively correlated with the percentage of spots in the bottom view, but not with that in the side of the eggs (bottom; r = 0·34, n = 47, P = 0·020; side; r = 0·09, n = 47, P = 0·52). On the other hand, mean pigment ‘spread’ (PC2) of the clutch was positively correlated with the percentage of spots in the side view, but not with that in the bottom view of the eggs (bottom; r = 0·61, n = 47, P < 0·001; side; r = 0·33, n = 47, P = 0·023). Pigment ‘spread’ (PC2) and pigment ‘darkness’ were not correlated (Pearson's correlation; r376 = −0·03, P = 0·56).

For data analysis corresponding to unhatched eggs, a new PCA was carried out. The first principal component was taken as an overall measure of intensity and size of pigment spots (pigment ‘darkness’) and it explained 61·77% of the total variance (factor loadings: I: 0·830, D: 0·151, S: 0·913). The second principal component accounted for 25·54% of the variance and was adopted as index of spot distribution (pigment ‘spread’, factor loadings: I: 0·316, D: 0·981, S: 0·083).

All statistical analyses were performed with statistica 6·0 and they were two-tailed. As eggs from the same clutch cannot be considered statistically independent, analyses were performed using clutch means. Clutches containing less than five eggs were not included in the analyses (nine clutches). Therefore, our final data set is 122 clutches (1043 eggs). Females were identified in 100 pairs. To determine whether eggshell pigmentation pattern (‘darkness’ and ‘spread’) were related to: (i) year, (ii) study site, (iii) breeding parameters (laying date, clutch size), and (iv) female and male characteristics (age, size, condition), general linear models (GLM) were constructed with PC1 or PC2 as the dependent variable and the previous variables as predictors. Since some individuals were sampled both years (33 recaptured birds: 14 females and 19 males), parental identity was fitted as a random factor. We also used GLMs to test the effects of eggshell pigmentation pattern (‘darkness’ and ‘spread’) on incubation phase (length of the incubation period, nest attentiveness and incubation rhythm), breeding performance, nestling condition and parental effort. Egg volume, nest temperature and some of the variables above mentioned were entered in the models as fixed effects as necessary. As the rate of water loss from the egg during the incubation is likely to depend on the surface of exchange with air, on the micro-environment of the nestbox and on the thickness of the shell (Rahn & Ar 1974), egg volume, egg shape and nest temperature were included in the analysis. Egg volume and egg shape were also used as fixed effects to test the effect of eggshell pigmentation on eggshell physical properties (thickness and weight) of unhatched eggs. Laying date and clutch size have an important influence on blue tit reproductive performance (see Cramp 1998 for a review) and therefore they were initially incorporated in each model. Final models were obtained by sequential removal of nonsignificant terms. To allow the use of parametric tests, hatching and fledging success were arcsine square root transformed. All values are presented as means ± SD, unless stated otherwise.


eggshell pigmentation pattern variability of blue tits

Maculation varies greatly among and within blue tit clutches in our study area (see Fig. 1; I: among clutches 43·2%, F130,930 = 7·22, P < 0·001, within clutches: 56·8%; D: among clutches 41·7%, F130,930 = 6·84, P < 0·001, within clutches: 59·3%; S: among clutches 46·0%, F130,930 = 7·95, P < 0·001, within clutches: 64·0%). Mean (SD) pigment intensity (I), spot distribution (D) and spot size (S) were 3·78 (0·65), 3·32 (0·95) and 2·09 (0·47), respectively (n = 1061 eggs). The holm oak forest showed a lower proportion of eggs with spots profusely concentrated on one end of the egg (eggs with a ‘corona’ ring, D = 1–2, Fig. 1) compared to the Pyrenean oak forest (Anchurones: 7·5%n = 400, El Brezoso: 18·0%, n = 661).

Mean pigment ‘darkness’ of the clutch was only positively related to female tarsus length (Table 1). Females with larger tarsi laid significantly darker (more intense and larger pigment spots) eggs. On the other hand, mean pigment ‘spread’ of the clutch differed significantly between habitat types (Anchurones: 0·21 ± 0·64, n = 45; El Brezoso: −0·19 ± 0·69, n = 77) and was positively related to clutch size (Table 1, Fig. 2) and male body mass (Table 1), respectively.

Table 1.  Results of GLMs with pigment ‘darkness’ and ‘spread’ (n = 90) as dependent variables and year, study site (Anchurones, El Brezoso), laying date, clutch size, and female and male age, tarsus length and body mass as explanatory variables. Boldface indicates significant effects. Statistics resulting from final models (obtained by a step-down model simplification procedure) including only significant terms
VariablePigment ‘darkness’ (PC1)Pigment ‘spread’ (PC2)
Beta ± SEFd.f.PBeta ± SEFd.f.P
Year < 0·011,770·93 0·021,77 0·12
Study site  2·121,770·15 2·541,86 0·02
Year × Study site  0·701,770·40 1·691,77 0·11
Laying date−0·015 ± 0·119 0·021,770·90−0·050 ± 0·1090·211,77 0·20
Clutch size 0·205 ± 0·122 2·831,770·09 0·319 ± 0·1128·061,86< 0·001
Parental identity 0·236 ± 0·194 1·481,770·23−0·275 ± 0·1792·371,77 0·12
Female age 0·173 ± 0·135 1·641,770·20−0·038 ± 0·1240·091,77 0·76
Female tarsus length 0·261 ± 0·1186,191,880·01 0·197 ± 0·1083·301,77 0·07
Female body mass−0·105 ± 0·111 0·901,770·35−0·047 ± 0·1030·201,77 0·65
Male age−0·002 ± 0·124< 0·011,770·99−0·061 ± 0·1150·281,77 0·59
Male tarsus length−0·092 ± 0·109 0·721,770·15−0·044 ± 0·1000·201,77 0·65
Male body mass−0·025 ± 0·108 0·051,770·40 0·200 ± 0·0994·061,86 0·05
Figure 2.

Eggshell pigment ‘spread’ (PC2) according to clutch size of blue tits in central Spain (n = 122). Data are presented as grand mean ± SE, and sample sizes are given above the bars.

eggshell pigmentation pattern and breeding performance

When the effect of year, study site, laying date and clutch size were controlled for, we found that the length of incubation period decreased significantly with pigment ‘darkness’ and ‘spread’ (Table 2, Fig. 3a and 3b). The length of the incubation period also decreased with laying date (β = −0·55 ± 0·05). While, the scores of eggshell pigmentation pattern were not related with nest attentiveness or incubation scheduling (see Table 2). Male provisioning courtship frequency on the incubation period was not related with eggshell pigmentation (P > 0·1).

Table 2.  Results of GLMs with length of incubation period, nest attentiveness, incubation scheduling, daily rate of mass loss, hatching success and nestling tarsus length as response variables. Year, study site, year × site interaction, nest temperature, egg volume, egg shape, brood size, eggshell pigment ‘darkness’ and eggshell pigment ‘spread’ were included as fixed effect. Parental identity was fitted as random effect for analysis involving both years (2006–07). Boldface indicates statistics for the final models (from a step-down model simplification procedure) including only significant terms
 d.f.FP d.f.FP
Length of incubation periodDaily rate of mass loss
 Year1,93 7·02< 0·01Study site1,47 1·29 0·47
 Study site1,93 4·85 0·03Laying date1,47 2·41 0·13
 Year × Study site1,91 0·75 0·39Clutch size1,47 0·75 0·39
 Laying date1,9351·38< 0·001Nest temperature1,47 1·42 0·24
 Clutch size1,91 3·27 0·07Egg volume1,47 0·09 0·77
 Parental identity1,93 5·90 0·02Egg shape1,47 0·86 0·36
 Pigment ‘darkness’1,9312·15< 0·001Pigment ‘darkness’1,47 2·74 0·10
 Pigment ‘spread’1,9318·10< 0·001Pigment ‘spread’1,5423·30< 0·001
Nest attentivenessHatching success
 Study site1,250·250·62Year1,91< 0·01 0·97
 Laying date1,250·030·86Study site1,91 0·96 0·35
 Clutch size1,251·380·25Year × Study site1,96 5·52 0·02
 Nest temperature1,250·600·45Laying date1,91 0·22 0·64
 Egg volume1,314·460·04Clutch size1,91 1·67 0·68
 Pigment ‘darkness’1,251·060·31Parental identity1,91 0·15 0·69
 Pigment ‘spread’1,250·970·33Pigment ‘darkness’1,96 6·28 0·03
    Pigment ‘spread’1,96 19·36< 0·001
Incubation scheduling
 Study site1,250·230·63Nestling tarsus length
 Laying date1,250·390·54Year1,81 66·64< 0·001
 Clutch size1,250·760·39Study site1,75< 0·01 0·97
 Nest temperature1,250·330·57Year × Study site1,75< 0·01 0·97
 Egg volume1,250·860·36Laying date1,81 6·16 0·01
 Pigment ‘darkness’1,251·070·31Brood size1,75 0·52 0·57
 Pigment ‘spread’1,252·070·16Parental identity1,75 2·24 0·14
    Egg volume1,75 1·53 0·22
    Pigment ‘darkness’1,75 0·05 0·81
    Pigment ‘spread’1,81 5·27 0·02
Figure 3.

Length of incubation period controlled for year, study area, laying date and parental identity in relation to (a) eggshell pigment ‘darkness’ (PC1) and (b) eggshell pigment ‘spread’ (PC2), respectively. Lines presented are derived from linear regression and they are illustrative.

Only one (eggshell pigment ‘spread’) out of the eight main fixed effects included in the GLM to predict the daily rate of water loss during the incubation period was significant (Table 2). Eggs with pigment spots concentrated around the blunt end of the egg lost a greater amount of water in comparison with those that had them evenly distributed over the shell surface (β = 0·56 ± 0·11; Fig. 4a).

Figure 4.

(a) Daily rate of mass loss per day in relation to eggshell pigment ‘spread’ (PC2) and hatching success in relation to (b) eggshell pigment ‘darkness’ (PC1) and (c) eggshell pigment ‘spread’ (PC2) respectively, of blue tit clutches in central Spain.

Hatching success was strongly affected by eggshell patterning (Table 2), increased significantly with egg pigment ‘darkness’ (β = 0·20 ± 0·08) and ‘spread’ (β = 0·40 ± 0·08; Fig. 4b and 4c). None of these factors influenced fledgling success (analysis not shown), which instead declined with laying date. Finally, we found a relationship between egg pigment ‘spread’ (PC2) and nestling tarsus length including year, study site, laying date, brood size and egg volume as explanatory variables in the model (β = 0·18 ± 0·08; Fig. 5). Eggs with a good distribution (not polarized) of pigment spots result in nestlings with larger tarsi (Table 2), while nestling tarsus length decreased as the season progressed (β = −0·19 ± 0·07). Nevertheless, there was no correlation between nestling body mass and eggshell pigmentation patterning (all P > 0·20 except laying date, analysis not shown).

Figure 5.

Nestling tarsus length on day 13 after hatching controlled for year, study and laying date, in relation to eggshell pigment ‘spread’ (PC2).

eggshell pigmentation pattern and parental provisioning rates

When the effects of study site, laying date, brood size and male age were controlled for, male absolute provisioning rate per day increased significantly with egg pigment ‘spread’ in both study areas (Fig. 6a year: F1,50 = 2·22, P = 0·14; study site: F1,55 = 8·49, P < 0·01; year × study site: F1,50 < 0·01, P = 0·93; laying date: F1,50 = 1·00, P = 0·32; brood size: F1,55 = 8·90, P < 0·01; male age: F1,55 = 12·10, P < 0·001; parental identity: F1,50 = 0·07, P = 0·93; pigment ‘darkness’: F1,50 = 0·27, P = 0·60; pigment ‘spread’: F1,55 = 9·60, P < 0·01). This relationship remained when male feeding rates were calculated relatively to female effort, that is, male proportion of visits (year: F1,54 = 10·92, P < 0·01; laying date: F1,54 = 12·59, P < 0·001; male age: F1,54 = 5·02, P = 0·05; pigment ‘spread’: F1,54 = 4·90, P = 0·03, only significant results are shown). Male age was positively related to their feeding frequencies (β = 0·37 ± 0·10). On the other hand, absolute female provisioning rate per day was not related to egg pigment ‘darkness’ or ‘spread’ (Fig. 6b, year: F1,58 = 16·42, P < 0·001; study site: F1,51 = 0·45, P = 0·50; year × study site: F1,51 = 0·03, P = 0·86; laying date: F1,58 = 16·83, P < 0·001; brood size: F1,51 = 3·71, P = 0·06; female age: F1,51 = 1·30, P = 0·26; parental identity: F1,51 = 0·03, P = 0·87; pigment ‘darkness’: F1,51 = 0·09, P = 0·77; pigment ‘spread’: F1,51 = 0·55, P = 0·46).

Figure 6.

Total daily (a) male and (b) female provisioning visits in relation to eggshell pigment ‘spread’ (PC2) of blue tit clutches in central Spain.

eggshell pigmentation pattern and egg properties

The GLM conducted on data from unhatched eggs found that shell thickness was strongly correlated with the pigmentation patterning. Shell thickness increased with pigment ‘spread’ and pigment ‘darkness’ (Fig. 7a and 7b, study site: F1,60 = 2·34, P = 0·13; laying date: F1,60 = 0·24, P = 0·62; clutch size: F1,60 = 0·30, P = 0·58; egg shape: F1,60 = 0·42, P = 0·52; egg volume: F1,60 = 3·18, P = 0·08; pigment ‘spread’: F1,65 = 9·04, P < 0·01, pigment ‘darkness’: F1,65 = 57·8, P < 0·001). Eggs with larger, more intense and widely distributed pigment spots showed heavier (analyses not shown) and thicker shells than those with small, concentrated and bad-defined spots. Shell thickness and weight were highly correlated (Pearson's correlation, r55 = 0·57, P < 0·001).

Figure 7.

(a) Eggshell thickness in relation to eggshell (a) pigment ‘darkness’ (PC1) and (b) pigment ‘spread’ (PC2) of unhatched blue tit eggs collected in central Spain.


Our results suggest that the appearance of blue tit’ eggs can be used as a good indicator of their viability. We found that eggshell pigmentation pattern was correlated with the length of incubation period, hatching success and male but not female parental effort at the end of the nestling phase. Eggs with a ‘corona’ ring presented a thinner eggshell (Graveland et al. 1994; Gosler et al. 2005) and a greater rate of mass loss per day than those without this eggshell patterning. This could explain the fact that eggs with deviant pigmentation (lower PC2 values) present longer incubation periods. Likewise, an excessive water loss could jeopardize egg viability. In this sense, we found that eggs with spots forming a ‘corona’ ring around the broad end showed a low hatching probability which could be due to shell breakage (Drent & Woldendorp 1989; Graveland et al. 1994) or, as already has been mentioned, desiccation (we scarcely ever found addled eggs of this type with fresh content). On the other hand, eggshell pigment ‘darkness’ was also negatively correlated with hatching success and shell properties (thickness and weight). This is in agreement with Gosler et al.'s (2005) findings, who found that pigment ‘darkness’ is linked to shell thinning. Furthermore, we have found a correlation between the length of the incubation period and pigment ‘darkness’; eggs with more reddish and larger spots (darker eggs) showed a shorter incubation period. According to Higham & Gosler (2006), on low-calcium soils, where eggshells are thinner and more heavily pigmented, an elevated pigment deposition could reduce mass loss and it could enhance female's incubation effort in order to increase the conductivity of the eggshells. Nevertheless, we did not found any correlation between eggshell pigment ‘darkness’ and the rate of mass loss. Although, it should be noted that the methodology employed was different to that described in Higham & Gosler (2006). Neither was there association between nest attentiveness nor incubation scheduling and maculation (cf. Higham & Gosler 2006). Physiological mechanisms driving the relationships between protoporphyrins and mass loss and intra-egg thermoregulatory properties of this pigment (e.g. may darker eggs retain more heat?) remains unclear. Bakken et al. (1978) reported that protoporhyrins reflect more than 19% of light in the near infrared. More studies are needed on this topic.

Analysing the influence of certain variables on eggshell patterning, we found that eggshell ‘spread’ increased with clutch size. Females with larger clutches laid eggs with spots more widely distributed over the eggshell. This fact suggests that females able to produce large clutches may have sufficient capacity (more able to forage for and/or assimilate calcium) to produce eggs of this type and thus, profit from it (increasing offspring viability). On the other hand, we found a positive relationship between female's tarsus length and eggshell ‘darkness’. These findings support the hypothesis that egg pigmentation pattern could be considered as an indicator of female quality (Moreno & Osorno 2003), as females with deficient calcium processing would be incapable of changing egg pigmentation patterns without jeopardizing egg hatchability. According to the ‘differential allocation hypothesis’ (Burley 1986), individual parents are expected to adjust their investment to the perceived sexual attractiveness of their mates. In this sense, we hypothesize that if males could judge their clutch/mate quality as expressed by eggshell pigmentation pattern, they could adjust their level of parental investment accordingly. That is, egg appearance or clutch viability (estimated as number of hatched eggs) may be used by males as a signal of female health status or clutch quality and, consequently, invest a greater or smaller effort in offspring care (Moreno & Osorno 2003). Here we have shown support for some of the necessary assumptions of the ‘signalling hypothesis’. We found a relationship between egg pigment ‘spread’ and male provisioning rates, whereas female parental investment seemed not be conditioned by eggshell pigmentation patterning. In a recent paper, Soler et al. (2008) have shown by means of three different experimental approaches several evidence supporting the hypothesis that blue-green colour intensity of spotless starling (Sturnus unicolor) eggs is a sexually selected trait of females that affects paternal feeding effort. In their experiment with model eggs, these authors detected an effect of egg coloration on provisioning behaviour of males. Herein, we have not focussed on egg colour but on pigmentation patterning (spotting). In addition to the relationship between male parental effort and egg ‘spread’, we have found that this latter attribute was positively related to male body mass. This fact could be explained as an adaptive adjustment of egg quality in response to mate condition following the Burley's differential investment model. In this sense, previous studies have found that egg investment could vary with mate attractiveness (e.g. Cunningham & Rusell 2000; Rutstein et al. 2004). More recently, Szigeti et al. (2007) have showed that blue tit females produce eggs with more colourful yolks when they were mated to ultraviolet attractive males. We should be aware that adults were captured on day 10 after hatching and not during the egg-laying period (due to desertion risk) and this could explain the absence of a female condition effect since their body mass tends to diminish at the end of the incubation period (Woodburn & Perrins 1997). This does not happen in the case of males, whose body condition usually stays constant over the breeding period (Moreno 1989).

An alternative explanation for the correlations between eggshell pigmentation pattern and male feeding rates and body mass is that these were a result of assortative mating of high-quality parents, although our results do not support this possibility because intra-pair male and female body conditions were not correlated (r = −0·07, P = 0·50, n = 90). On the other hand, another factor that could drive the relationship between parental care and maculation patterning is the courtship feeding. Males contribute to the food requirements of his mate during the egg-laying and incubation periods (Krebs 1970). Thus, this behaviour is a male's most direct way to contribute to his own egg's quality (Smith 1980) and can be viewed as males feeding their young, although the latter are still in the egg and the feeding acts through the female (Nilsson & Smith 1988). Nevertheless, we did not find a significant relationship between male provisioning (courtship) frequencies and feeding rates (Spearman's correlation; r19 = 0·08, P = 0·73). Males who are good in provisioning their mate are not necessarily good in provisioning their brood.

The present study shows a significant relationship between eggshell pigment ‘spread’ and nestling tarsus length. Previous studies have shown some evidence that, on average, larger chicks hatch from larger eggs (Potti & Merino 1994; Williams 1994). Even controlling for egg size, we found that nestlings hatching from eggs with aberrant or anomalous eggshell pigmentation pattern (eggs with ‘corona’ ring) presented shorter tarsi compared with those resulting from eggs with a ‘normal’ maculation pattern. It is well known that during development, the embryo mobilizes calcium from two sources, the yolk in a first stage and then from the eggshell (e.g. Hart, Ravindran & Young 1992; Packard 1994). This is the reason why shell thickness decreases during incubation (e.g. Bunck et al. 1985). Tilgar et al. (2005) found that nestling tarsus length in the first half of the nestling period was positively influenced by yolk calcium concentration. It is likely that composition of abnormal (deviant, thin-shelled) eggs may also present deficiencies (yolk mass, protein content), in addition to poor shell quality, in comparison to apparently normal eggs (e.g. Bourgault et al. 2007). In this study, we have focussed on shell characteristics as indicator of egg quality. Our result suggests that aberrant pigmentation patterning could be indicative of calcium deficiency (see more below) in agreement with Graveland et al. (1994) who found that deviant eggshells contain less calcium than normal shells. It could also affect nestling physical condition. Hence, low hatching probability is the main but not the unique problem associated to eggs with defective shells.

The results of the present study had not been observed in a long-term blue tit nestbox population located 300 km northward (S. Merino & J. Moreno, personal communication). In our study area, a similar effect of eggshell pigmentation patterning on breeding performance was detected on great tits and nuthatches (Sitta europaea L.), although sample sizes do not allow statistical analyses. Both eggs (thin-shelled eggs, deviant pigmentation, lower hatching success, females incubating empty nests) and chick traits (nestlings with leg defects) indicate a possible case of calcium shortage (see Reynolds, Mänd & Tilgar 2004 for a review). In our study area, there is an overgrazing pressure mainly by red deer and this would explain in some way the present results. Red deers eat all the understorey of these forests and this fact should affect the abundance of snails, whose shells are used by breeding females as the main source of calcium during the laying period, or calcium-rich arthropods such as woodlice Diplopoda spp. and millipedes Isopoda spp. (Graveland & van Gijzen 1994). We suggest that overgrazing pressure by ungulates might have an indirect effect on the conservation of small forest birds in this protected area.

Because this is a correlative study, we have to be cautious interpreting the results. Nevertheless, all results that we found support the underlying idea of our study: blue tit eggshell patterning is related to their breeding performance. The results suggest that eggshell pigmentation pattern should be taken into account by researchers at the time of carrying out cross-fostering or clutch size manipulations since eggshell-patterning differences among clutches may affect their results. Manipulations of eggshell patterning may allow us to assess if third variables are involved in the relationship between eggshell pattern and male investment or if both variables are causally related. Finally, further investigations are needed to understand the role of calcium availability in relation to the maculation phenomenon. Effects of calcium supplementation could provide evidence that a lack of this resource is responsible for longer incubation periods and higher rates of hatching failures as reported in the present study for species that lay maculated eggs. To test this suggestion, an exclusive experiment should be performed.


We are grateful to Juan Vicente Ruíz-Peinado for his advice during the 2006 breeding season. We are also indebted to staff of Cabañeros National Park and Los Quintos de Mora Reserve for the facilities offered during the fieldwork. Juan Moreno commented on an earlier draft of the manuscript. Three anonymous referees improved considerably a previous version of this paper. This study was supported by the project ref. 69/2003 of the Organismo Autónomo Parques Nacionales-Spanish Ministry of Environment. The Dirección General para la Conservación de la Naturaleza donated the nestboxes, whilst The Dirección General del Medio Natural of the Junta de Castilla-La Mancha gave licence for capturing and ringing birds.