Cuticle and Eggshell
We found that the Apterygian cuticle consisted of a very thin, waxy, water repellent mineral layer composed of triangular particles; that at times, formed aggregations that occluded eggshell pores. We also found that all the studied species of Apteryx presented plugged or capped pores. From our observations it seems possible that the cuticle of Apteryx erodes over time, from a more sealed egg when freshly laid to a more porous one, as the embryo develops and requires a greater gas exchange. We also found that many pores did not go all the way through the eggshell, a similar observation was mentioned by Silyn-Roberts (1983) nevertheless, the function of these types of pores is still not clear. Similar pores that do not transverse the eggshells have been observed in the eggs of Ostriches (Struthio camelus ), a relative of Apteryx (Willoughby et al., 2016; Maina, 2017).
The cuticle and eggshell mediate the interaction between the developing embryo and the environment. As such, they are the first barriers against pathogens and allow gas exchange and water vapour conductance (D’Alba et al. 2017). Features of the cuticle such as plugs, and caps have been associated with nesting in humid environments (D’Alba et al. 2016) as they block the pores. Board (1981) suggested that plugs could also serve as a physical barrier to defend the egg contents particularly against waterborne microbes. Apteryx species breed during the austral winter, which in New Zealand is characterised by frequent and heavy precipitation (Leathwick et al. 2002); they lay their eggs in burrows with a relatively humid environment and their incubation period is long. We found that pluviosity was significantly associated with eggshell thickness, pore density and pore radius.
A thicker eggshell means longer pores, reducing the number and area of the pores could be an adaptation to prevent water to penetrate the egg. This seems even more plausible considering that other adaptations, such as plugs, and caps seem to point in the same direction. Apteryxnests are frequently holes in the ground, this, with an incubating parent that could return to the nest with a wet plumage seems to indicate that the kiwi incubates in a very wet environment. Which in turn it would be expected to increase the humidity of the nest, making very necessary that future studies measure the humidity of nests in rainy areas such as Okarito or Haast, areas inhabited by Rowi and Tokoeka respectively, furthermore, Okarito experiences frequent snow (up to 1m in the past 10 years); studying the nest architecture of Rowi and Tokoeka would clarify further the nature of the different characteristics present in the Apterygian eggshell.
Apteryx does not present the accessory layer described in Megapodidae, Phoenicopteridae, and Podicipedidae (Tullett et al. 1976, Board et al. 1984, D’Alba et al. 2017) and that has been associated with decreasing bacterial penetration in high humidity environments. It seems that Apteryx might achieve a more sealed egg with a water-repellent smooth cuticle that erodes as gas diffusion needs increase with the progression of embryo development.
Temperature and barometric pressure play a very important role in gas diffusion; higher temperatures and lower pressures allow the gases to diffuse at faster rates. Water vapour conductance and gas exchange can be modulated by variations in the number, length, shape, and diameter of pores and by obstructions in the pores. For example, water vapour conductance and gas exchange can be lowered by having less pores per area, longer pores, branched pores, and by increasing the number of caps and plugs or unperforated pores. Therefore, the number and shape of pores have been found to be related to incubation period (Zimmermann et al. 2007), altitude where the birds live (Rahn et al., 1977), and the nest microclimate (Birchard and Kilgore, 1980).
Eggshell thickness and pore radius showed a moderate to low negative correlation with barometric pressure. At lower barometric pressures water diffusivity increases therefore eggs are at higher risk of desiccation, hence a thicker eggshell and smaller pore would reduce the water vapour conductance accordingly. The opposite is true for higher barometric pressures; however, in this case the eggshell must also stand a greater overall pressure on its structure, therefore a thickened eggshell would be beneficial. In chickens, a move from an altitude of 3800 masl to 1200 masl resulted in an increased eggshell thickness with decreased pore radius to compensate for the increase in barometric pressure (Rahn et al. 1982). Similar correlations occurred between temperature and eggshell thickness and pore radius.
Eggshell thickness is known to be proportional to the body mass of the laying bird (Tullett 1978, Birchard and Deeming 2009). However,Apteryx species vary in their incubation behaviour. In Brown Kiwi the male is the sole incubator, while in Roroa and Rowi both male and female incubate and in Tokoeka, there are helpers at the nest, requiring further studies relating both the female and the male body sizes to the egg and the eggshell, it is worth to notice that the egg mass is very high in relation to body mass, being 23.6 and 14.6% for A.oweniiand A. australis (possibly A.mantelli ) (Dyke and Kaiser 2010). Apteryx eggshells are allometrically thinner than expected and this is probably to reduce the weight the female must carry (Calder 1979) and to allow chicks to crack the shell when hatching, asApteryx does not possess an egg tooth (Calder 1978). The trade-off is then that a thinner eggshell is more prone to mechanical damage and even hairline fractures have been known to impact embryo health and development (Sylin-Roberts 1980; Stadelman 1995). In our study, the lightest (on average) of the four species, the Rowi had a thicker eggshell than the heavier Brown Kiwi; also, the Roroa, the heaviest of Apteryx had a thinner eggshell than the lighter Haast Tokoeka.
We suggest that the different incubation strategy of each species could also influence the evolution of eggshell thickness. In Tokoeka both males and females incubate, females are larger than the males and this could have been a selective pressure for a thicker eggshell that evaded breakage. In contrast, in the lighter Brown Kiwi generally only the males incubate. Rowi, being the smallest of the three species, presents an eggshell thickness similar to that of Haast Tokoeka but in contrast to Brown Kiwi, both male and female have been observed incubating (Colbourne 2002).