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.