Macroscopic inspection of the 17 unhatched Black Grouse eggs showed that no embryonic tissues/development was visible in 12 of them, while very early chick development (< 24 h) with incipient embryonic shield formation was discernible in another four (stages 1–3 according to52). The embryos of precocial species do not mobilize Ca from the eggshell during the first half of incubation5,6. Therefore, in order to achieve the major aims of this study, both these egg groups (not varying in eggshell thickness in either the pigment spot or background colour regions: Mann–Whitney test, U = 833.0 and 854.0, P = 0.793 and 0.936; n = 72/24 in each case, respectively) were pooled into one class, referred to as non-embryonated eggs. The final egg of these 17 contained a completely developed, feathered embryo with a shell partly broken at hatching; this specimen was pooled with the post-hatched eggshells.
Eight unhatched Capercaillie eggs (all from wild hens) showed no signs of embryonic development; accordingly, they were classified as non-embryonated eggs.
Eggshell thickness and overall elemental composition of the pigment spot and background colour regions
Our measurements of non-embryonated and post-hatched eggshells of cryptic, Black Grouse and Capercaillie eggs revealed highly significant differences in the thickness of the shells between their background colour and pigment spot regions (Fig. 1; Table S1): the former were respectively 2.6% and 2.8% thinner (Black Grouse) and 4.5% and 5.3% thinner (Capercaillie) than the latter in both non-embryonated and post-hatched eggshells (Fig. 1; Table S1).
Comprehensive ICP-OES analysis of 60 eggshell samples of the background colour and pigment spot regions (each n = 30 samples) from 30 Black Grouse eggs yielded measurements for 55 and 54chemical elements present above the detection limit in both samples, respectively (Table 1). In contrast, the levels of 27 and 25 elements, respectively, were above the detection limit in samples from the background colour and pigment spot regions (each sample n = 35) from 35 Capercaillie eggs (Table 1). The frequency of detectability (= number of samples with concentrations exceeding the detection limit) of individual elements varied strongly in both species (see Supplemental Material Appendix 1).
We had anticipated that the elemental concentrations between these two adjacent shell regions would vary considerably in both the non-embryonated and post-hatched eggshells (see Supplemental Material Appendix 1 and Tables S2–S7). Further statistical treatment of these data taking into account the two extremes of embryonic growth was therefore required if we were to achieve the major goal of our study.
Disparity in eggshell elemental composition between adjacent spotted and background colour shell regions
Analysis of the pairs of elemental concentrations measured in pigment spots and the adjacent background colour regions from the same eggshells (t-test for paired comparisons) showed that the concentrations of 25 of the 45 elements measured in the shells of non-embryonated eggs (Al, As, B, Ce, Co, Cr, Cu, Fe, Hf, K, La, Lu, Mg, Mn, Nd, Ni, Pb, Rh, Ru, Se, Si, Tb, Te, Tl and Tm) and just 5 of the 43 elemental concentrations measured in post-hatched Black Grouse eggshells (Hf, Mo, Nd, Te and Tm) varied significantly between these two regions (Fig. S1, Table S6). Most elemental concentrations, including those of the rare earth elements (Table S2: Ce, La, Lu, Nd, Tb and Tm), were consistently higher in the pigment spots in both the non-embryonated and post-h atched eggshells: they represented the negative values in the %Difference shown in Fig. S1. In contrast, 12 elements (Ge, Li, Zr, Pr, K, Na, Sc, Ba, Tb, Mg, Rh and Mn) were present in higher concentrations in the pigment spots of the non-embryonated eggs (Fig. S1: negative values), simultaneously exhibiting the opposite trend (Fig. S1: positive values) found in the post-hatched eggshells.
The analogous analysis of Capercaillie eggs showed that the concentrations of 8 of the 16 elements measured in the shells of non-embryonated eggs (Be, Cr, Mo, Ni, Ru, Sm, Zn and Zr), and as many as 14 of the 16 elemental concentrations measured in post-hatched shells (Al, B, Be, Cr, Fe, K, Mn, Na, Ni, Re, Ru, Sm, Tb and Zn) varied significantly between the two adjacent shell regions (Table S7). The levels of almost all these elements were higher in the pigment spots in both the non-embryonated and post-hatched eggshells; the exception was K, the level of which was higher in the background colour region of post-hatched eggshells (Fig. S1, Table S7).
However, taking into account the large number of non-detects yielding the small number of paired measurements used in the first analysis, we further evaluated the patterns of differences in elemental concentrations common to the pigment spot and background colour shell regions based on the ICP-OES-based eggshell ionomics data listed in Tables S2 and S3. In Fig. 2, therefore, we compared the spot/background (S/B) ratio of eggshell elemental concentrations between these two regions in non-embryonated and post-hatched shells. Inspection of this figure shows that most elements tended to be at higher concentrations in the speckled regions of the shell in both species, these differences being more pronounced in the post-hatched eggshells (Fig. 2; see Supplemental Material Appendix 1). The concentrations of only two elements (Gd and Mo) measured in the shells of non-embryonated Black Grouse eggs, and of 11 elements (Rh, Li, Mn, Mg,Tb, Sc, K, Na, Ba, Pr, Gd) measured in post-hatched eggshells of the same species were higher in the background colour regions (Fig. 2). Another interesting result of this analysis is that the concentrations of 33 elements from shell samples of non-embryonated eggs, listed in decreasing order of S/B ratios, are hierarchically distributed (from Tm to Gd; without Os, which was not measured in post-hatched shells) (Fig. 2), whereas 12 elements (Er, Ti, In, Te, B, Re, Cd, Cu, Fe, Zn, Mo, Sb) display higher S/B ratios in the background colour regions (Fig. 2).
Sources of variation in eggshell elemental concentrations as a result of the developmental status and origin of eggs
Tables S4 and S5 list the elemental concentrations measured in the background colour and pigment spot regions of the eggshells, further broken down according to the origin of the eggs (wild vs. captive hens). We also explored the sources of variation in eggshell elemental concentrations in these species-specific data by using multivariate analysis to test for the effect of the origin of the eggs/eggshells, shell region, and egg status (non-embryonated eggs vs. post-hatched eggshells) and the interactions between these three factors (Tables 2, 3). The key aspect explaining our major research goal, however, concerned the sample × status and origin × sample × status interactions, which we examined in order to ascertain which factors were responsible for the variations in elemental concentrations between the background and spotted shell regions (Tables 2, 3).
The analysis shows that for Black Grouse, each of the seven tested effects had a significant influence on eggshell elemental concentrations (Table 2). The sample × status interaction significantly affected the levels of just two elements—Cr and Fe (Table 2). The origin × sample × status interaction significantly influenced the levels of five elements—Al, Fe, Ni, Pb and Te (Table 2). So, linking these significant effects with the data in Table S4indicates that the eggshells of captive birds tended to have higher levels of Al, Fe, Pb and Te than those of wild Black Grouse; the levels of these elements, along with Ni, were the highest in the pigment spots.
In the case of Capercaillie, two-way ANOVA showed that the sample × status interaction in non-embryonated and post-hatched eggshells significantly influenced the levels of four elements—Be, Ni, Ru and Sm (Table 2 and Table S5), whereas the sample × origin interaction in the post-hatched eggshells only had a significant influence on the concentrations of eight elements—Be, Cr, Fe, Na, Pb, Ru, Sm and Zn (Table 2 and Table S5). Linking these significant effects with the data in Table S5 indicates that the shells of post-hatched, wild Capercaillie eggs tended to have higher levels of these elements compared to the eggshells of captive birds. Specifically, the concentrations of Cr, Pb, Ru and Zn in the spotted regions of the shells of captive Capercaillie eggs were 2.7–3.3-fold higher than in those of the wild birds (see Table S5).
Changes in elemental composition between the pigment spot and background colour shell regions following embryonic eggshell etching
In both Black Grouse and Capercaillie (Fig. 1) we observed substantial embryo-induced eggshell thinning in both the background colour (− 12.0% and − 17.4%, respectively) and pigment spot regions (− 11.2% and − 16.6%, respectively) (ANOVA, in each case P < 0.00001; Table S1).
Figure 3 shows the %Change in elemental concentrations in the background colour and pigment spot regions between non-embryonated and post-hatched eggshells, based on the data from Tables S2 and S3 (see Supplemental Material Appendix 1). As evidenced by the results of the sign test, the trends of %Change in elemental concentrations occurring in the background colour and pigment spot regions (Fig. 3) varied significantly between non-embryonated and post-hatched eggshells in both Black Grouse (Z = 7.74, P = 0.0061, n = 43) and Capercaillie (Z = 2.59, P = 0.0095, n = 18). In both species, the trends in %Change for most elemental concentrations were reversed following embryonic eggshell etching, however, the directions of the trends were not consistent for all elements and varied between the two species (Fig. 3). In detail, this is shown by the different trends in the levels of 13 elements—B, Zr, Mn, Fe, Na, Tb, Mo, Cr, Zn, Ni, Pb, Re and Ru—mostly decreasing in Capercaillie but increasing in Black Grouse (Fig. 3; Supplemental Material Appendix 1).
%Change (= Concentrationpost-hatched − Concentrationnon-embryonated × 100/Concentrationnon-embryonated) in elemental concentrations in the background colour and pigment spot regions of eggshells following embryonic eggshell etching, defined as non-embryonated and post-hatched eggshells of Black Grouse Tetrao tetrix and Capercaillie Tetrao urogallus. For the elemental concentrations and sample sizes, see Tables S2and S3. Note: The bars with positive values indicate elevated elemental concentrations following embryonic eggshell etching, whereas those with negative values indicate decreases within a given shell region. Note that because of some differences in eggshell elemental concentrations related to the origin of the eggs (see “Results”), the trend in %Change calculated within the sample of eggshells from wild and captive Black Grouse and Capercaillie retains the same direction, although the magnitude (bars) of %Change becomes smaller (after recalculation of the data from Tables S4 and S5).
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