Introduction
Birds reproduce successfully in a wide range of ecosystems, including some of the most inhospitable ones. This is possible because birds present delicately balanced strategies to ensure the survival of their eggs (Carey 1980). These strategies imply the adaptative fine tuning of physiology, anatomy, and behaviour to respond to climatic regimes, predation, parasitism, and intraspecific competition. This is why such variety exists in the physical characteristics of eggs and eggshells, (Mikhailov, Bray, & Hirsch, 1996, Portugal et al., 2014), nest architecture (Warning and Benedict 2015) and incubation behaviours (Deeming 2002) of birds. The relationships that exists between these adaptations means that using the characteristics of eggs, the nesting ecology of species could be better understood and vice versa (Tanaka et al. 2015). For example, several studies have hypothesised the nesting ecology of extinct species based on the physical characteristics of their fossilized eggshells (Deeming 2006, Grellet-Tinner et al. 2006, Varricchio et al. 2013). However, studying the physical characteristics of eggs and eggshells of extant species has the advantage that the nesting ecology does not need to be hypothesised but can be directly observed; and the relationship between the different adaptations to successful breeding in context, better understood.
The avian eggshell is a bioceramic composed of four mineral layers and two inner proteinaceous membranes (Romanoff and Romanoff 1949). The cuticle, the usually pigmented, outermost layer, has been proposed to have antimicrobial properties (Board and Fuller 1974), UV wavelength modulation properties, (Cooper et al. 2011), and water repellence, the latter being strongly associated with prevention against waterborne bacterial penetration (Sparks and Board 1984). These characteristics suggest that the cuticle responds to environmental conditions such as solar radiation and pluviosity, but also to the nest environment, mainly the probability of flooding and the potential for bacterial contamination (D’Alba et al. 2014, 2017).
The thickness of the eggshell is a functional character associated with structural support for the egg, reduction of bacterial infection as a solid barrier (Board and Fuller 1974) and more importantly, gas exchange, as the eggshell is a porous material (Rahn et al. 1974). The number and shape of pores are related to the incubation period (Zimmermann et al. 2007), altitude where the birds live (Rahn et al. 1977), and the nest microclimate (Birchard and Kilgore 1980). An additional contributor to eggshell thickness is the bioavailability of calcium and the mechanisms different species use to obtain calcium around the breeding season (Wilkin et al. 2009).
Eggshell porosity is a function of the pore functional area (mean area of individual pores multiplied by the total number of pores in an eggshell) and the thickness of the eggshell (Board and Scott 1980). These two characteristics of the eggshell define the gas conductance of the eggshell. Gas conductance is a measurement of the gas transfer through a medium (Rahn et al. 1974, Paganelli 1980, Rahn and Paganelli 1990). Gas exchange provides the embryo with the necessary oxygen for its development and allows carbon dioxide and water to leave the egg (Mueller et al. 2014, Maina 2017). This poses a series of constraints for different species incubating in different environmental conditions (Carey 1980). Birds in hot environments need to retain more water to avoid desiccation (Grant 1982) while species in cold or wet environments need to increase the water vapour conductance to lose enough water for the embryo to develop (Deeming 2011, Maina 2017). Further, some authors suggest that altitude is an important factor in regulating water vapour conductance due to the differences in barometric pressure that affect the rate of gas diffusion (Rahn et al. 1977, 1982). Therefore, it is expected that different species will present particular adaptations to regulate the water loss in different habitats and using different incubation techniques and nesting behaviours (Birchard and Kilgore 1980, Whittow et al. 1987, Portugal et al. 2010).
Water vapour conductance is particularly important for burrowing species, as these species’ eggs are exposed to environments with high humidity, low concentrations of O2, and high concentrations of CO2 (Boccs et al. 1984, Collias 1986). Therefore, it is expected that these species have adaptations in pore size and eggshell thickness to deal with these environments. In turn, burrow nesting may improve egg survivorship by providing better protection from predators and/or a more insulated environment for embryo development (Boggs and Kilgore 1983, Boccs et al. 1984, Whittow et al. 1987).
The Apterygidae family is endemic to New Zealand and it is characterised by a series of unique traits that are rare or not present in any other clade of birds, especially not found in other ratites (Ramstad and Dunning 2020). Among these traits are the egg’s size, which is large in relation to bird size (Calder 1979, Taborsky and Taborsky 1999), comprising between 14-23% of the female’s body weight (Dyke and Kaiser 2010); the very thin eggshell compared to the size of the egg, which is 60% thinner than allometrically expected (Calder 1979), and the use of a burrow nest (Jolly 1989, Colbourne 2002, Vieco 2019).
Apteryx nests in globular cavities dug in the ground or existing cavities in dead trees or tree roots (Ziesemann et al. 2011) lined with nesting materials (Vieco 2019). And in Brown Kiwi the eggs are partially buried in the nest lining (Colbourne 2002). Apteryx is an unusual ratite as it has evolved to be mostly entirely nocturnal and primarily insectivorous (Cunningham and Castro 2011, Le Duc et al. 2015).
The genus Apteryx contains five well defined species distributed in the three main Islands of New Zealand and some offshore islands (Burbidge et al. 2003, Weir et al. 2016). Apteryx species present a very localized distribution due to the decline of their natural populations as a result of predation by introduced mammals and loss and fragmentation of habitat by deforestation (Germano et al. 2018). The climate encountered by Apteryx species varies from mild temperatures in the north of New Zealand to below zero temperatures and snow in the south (www.worldweatheronline.com). Therefore, adaptive variation in nesting behaviour between the differentApteryx species is expected as a response to each climatic regime.
Finally, Apteryx eggs are incubated for approximately 74 days in a humid, organic matter rich environment that is warmed periodically, which makes it ideal for the growth of micro-organisms (Hiscox 2014). Therefore, adaptive variation is expected between the different species in terms of the eggshell physical structure to respond to each climatic regime and the risk of microbial penetration.
A reduced porosity and water vapour conductance could be beneficial for a species with a long incubation period as it reduces the risk of desiccation and it is what has been estimated for Apteryx in the past (Calder 1979, Silyn‐Roberts 1983). However, in most burrowing birds, a higher porosity and conductance has been observed because the humidity in a burrow can be closer to 100%, thus reducing the rate of water diffusion from the egg. Differences in water vapour conductance can be achieved by means of increasing pore area or number, or by decreasing eggshell thickness (Ar and Rahn 1985). There are other adaptations that could compensate for the need of increased gas exchange concurrently with a need to prevent microbial contamination. For example, a reduction in pore size through mechanical means such as cuticular particles or opercula partially or fully plugging the pores (Board and Perrott 1979).
Some hypotheses about the function of the characteristics of the Apterygian egg and the eggshell to respond to certain ecological demands have been proposed. Reid (1971) proposed that the large Apteryxegg is a response to low temperatures during incubation that would select for an egg with higher volume-area ratio. Calder (1979) suggested that the increased amount of ovoinhibitors and lysozymes in the albumen of Apteryx eggs were selected to reduce the risk of microbial infection during the very long incubation period. Prinzinger and Dietz (2002) and Maloney (2008) suggested that the slow metabolic and developmental rate allows the egg to withstand long periods of abandonment. However, characteristics of the eggshell such as interspecific variation in eggshell thickness and water vapour conductance, that could help answer these questions are still not fully understood in Apteryx . Apteryx has been included in some studies on water vapour conductance variability in different species as an example of extreme adaptations (Calder 1979, Tullett 1984). However, this data has mostly been based on morphometric equations rather than direct measurements or using very few eggs from what was consideredApteryx australis , a species now known to comprise three species and further subdivided into nine (or more) different taxa (Weir et al. 2016). Especially outside New Zealand, samples are usually obtained from birds bred in captivity, which are known to produce eggs much smaller than those laid in the wild (Jensen and Durrant 2006). Some authors have tried to address these questions but unfortunately with very few eggs and eggshells (Silyn‐Roberts 1983a), leaving this matter open to be researched in more depth.
In this study, we examined the eggshell structure of four species ofApteryx and contrast the findings with previous hypotheses regarding the expected structure based on environmental conditions where each species lives and their nesting environment. In summary, based on each species distribution it is expected that there will be differences in the water vapour conductance between the species. This should be observable as differences in pore density and pore area, or/and eggshell thickness. These traits are expected to vary according to climate, especially with pluviosity, temperature, and barometric pressure. It would also be expected that thickness scales with body mass (Birchard and Deeming 2009). We also expect to find eggshell adaptations to burrow nesting that are comparable to those of other burrow nesting birds, such as modified pores and modified cuticle or accessory layer.