DOI: 10.5061/dryad.kprr4xh76
title: Thicker eggshells are not predicted by host egg ejection behaviour in four species of Australian cuckoo

description: Defences of hosts against brood parasitic cuckoos include detection and
 ejection of cuckoo eggs from the nest. Ejection behaviour often involves
 puncturing the cuckoo egg, which is predicted to drive the evolution of
 thicker eggshells in cuckoos that parasitise such hosts. Here we test this
 prediction in four Australian cuckoo species and their hosts, using
 Hall-effect magnetic-inference to directly estimate eggshell thickness in
 parasitised clutches. In Australia, hosts that build cup-shaped nests are
 generally adept at ejecting cuckoo eggs, whereas hosts that build
 dome-shaped nests mostly accept foreign eggs. We analysed two datasets: a
 small sample of hosts with known egg ejection rates and a broader sample
 of hosts where egg ejection behaviour was inferred based on nest type
 (dome or cup). Contrary to predictions, cuckoos that exploit dome-nesting
 hosts (acceptor hosts) had significantly thicker eggshells relative to
 their hosts than cuckoos that exploit cup-nesting hosts (ejector hosts).
 No difference in eggshell thicknesses was observed in the smaller sample
 of hosts with known egg ejection rates, probably due to lack of power.
 Overall cuckoo eggshell thickness did not deviate from the expected avian
 relationship between eggshell thickness and egg length estimated from 74
 bird species. Our results do not support the hypothesis that thicker
 eggshells have evolved in response to host ejection behaviour in
 Australian cuckoos, but are consistent with the hypothesis that thicker
 eggshells have evolved to reduce the risk of breakage when eggs are
 dropped into dome nests.
description: <strong>Cuckoo and host species.</strong> The
 parasitic cuckoo species selected for use in this study were chosen
 based on previous knowledge of their host selection and the egg ejection
 behaviour of those hosts. We selected four Australian cuckoo species based
 on the known egg ejection rates of their primary hosts from our
 earlier studies. Two congeneric species, Horsfield’s bronze-cuckoos
 (<em>Chalcites</em> <em>basalis</em>) and shining
 bronze-cuckoos (<em>C</em>. <em>lucidus</em>),
 exploit hosts that build dome-shaped nests in which detection of foreign
 eggs is constrained by poor visibility in the dark interior. Hosts of
 these two cuckoo species rarely eject either naturally-laid cuckoo eggs or
 experimental, non-mimetic plastic model eggs, of similar size to their own
 (Table 1). The two other cuckoo species in the study, the pallid cuckoo
 (<em>Cuculus</em> <em>pallidus</em>) and the
 Pacific koel (<em>Eudynamis</em>
 <em>orientalis</em>), exploit hosts that build cup-shaped
 nests with good visibility, and these hosts routinely eject both
 naturally-laid cuckoo eggs and experimental, non-mimetic model eggs (Table
 1). In addition to their primary hosts included in this analysis, these
 cuckoos also exploit several secondary hosts whose egg ejection behaviour
 is unknown. However, previous analyses indicate that there is a strong
 association between visibility inside the nest and egg ejection behaviour;
 hosts that build dome-shaped nests tend to accept foreign eggs (100% of
 Australian hosts [N = 6] ejected ≤ 25% of foreign eggs), whereas hosts
 that build cup-shaped nests tend to eject foreign eggs (75% of hosts
 ejected &gt; 25% of foreign eggs, [N = 8]). Therefore, we conducted a
 second set of analyses that included both primary and secondary hosts of
 these cuckoos (Table 1), where egg ejection behaviour was inferred based
 on nest type for the secondary hosts.
 <strong>Eggshell measurements.</strong> All eggshells
 used in this study were sourced from the Commonwealth Scientific and
 Industrial Research Organisation (CSIRO) Australian National Wildlife
 Collection (ANWC) oology research collection (Supplementary data file 1).
 All eggs had been prepared at the time of collection by drilling a small
 hole in the shell, through which the egg contents were blown and removed.
 The eggshells were then washed and stored dry. Collector’s notes and
 consistently small blow-hole diameters indicate that eggs were
 sampled early in development and were unlikely to be subject to
 significant eggshell thinning during development (Supplementary data file
 1). The availability of parasitised clutches in the ANWC collection
 dictated which host species were included and the sample size for this
 study (Table 1; Supplementary data file 1). Suitable cuckoo-host clutches
 contained at least one intact host and one cuckoo egg, both identified to
 the species level. We used a precision industrial wall-thickness measuring
 tool to directly measure eggshell thickness via Hall-effect
 magnetic-inference, in a similar approach to Peterson et al. However,
 unlike this previous study we did not cut or damage the eggshell to take
 measurements. Specifically, we used the ElectroPhysik MiniTest
 FH7200 gauge and FH4 magnetic probe, with a 1.5 mm diameter steel ball
 which was inserted inside the empty eggshell, through the existing
 blow-hole in the specimen (SI 2.0). Thus, all eggs included in the study
 necessarily had a blowhole diameter &gt; 1.5 mm. This probe and steel
 ball combination measures thicknesses up to 2 mm, with an accuracy of Å} 3
 μm + 1% of the reading (Check Line., Germany). Thickness data were
 collected at a rate of 10 measurements per second. We did not place the
 steel probe in direct contact with the egg. Instead, we inserted a 0.73 mm
 sheet of plastic (cellulose acetate) in between the probe and the egg to
 minimise risk of damage (hereafter referred to as the
 ‘protector’). Eggshell thickness data were collected at two regions on
 each egg—the apex (the most conical end opposite the air sac) and the
 meridian (the circumference around the widest part of the egg). Manual
 handling of the egg specimens during thickness estimation is described in
 detail in Supplementary Information Sect. 2.0 and Fig. S1. Briefly, we
 inserted the steel ball through the blow hole and rolled the ball to the
 apex of the egg. We always approached the magnetic probe (and protector)
 apex-first because this is the strongest part of the egg. Apex thickness
 was recorded for five seconds by leaving the egg stationary and untouched
 on the probe (Fig. S1;Video Supplement 1). We then rolled the ball until
 it was positioned adjacent to the side blow-hole and rotated the egg
 slowly, to record the meridian thickness (Fig. S1; Video Supplement 2).
 The steel ball was removed by rolling it back through the blow-hole,
 whilst still in contact with the probe (Video Supplement 3).
 Preliminary method optimisation using 60 unregistered eggs indicated that
 the risk of breaking an egg during this manual handling was very low if
 specimens had no pre-existing physical damage (cracks, chips, hairline
 fractures determined via illuminating the egg with a cold-light source)
 and weighed &gt; 0.05 g (Fig. S2). No registered collection items
 sustained damage in this study. All data were inspected
 and exported following the manufacturer’s protocols in the software
 package MSoft 7 Basic (Check Line., Germany). The protector thickness was
 subtracted from the raw gauge readings to obtain a measurement of eggshell
 thickness (SI 2.0). Mean thickness (μm) was calculated for both apex and
 meridian measurements of each egg after removing outliers (classified as
 data points lying outside 1.5X the interquartile range). Mass (g), length
 (mm) and breadth (mm) were also measured for each egg. Length and breadth
 measurements were calculated from a 2D photograph of each egg, following
 Attard et al. Mass was measured using an electronic balance to the nearest
 0.001 g (CT-250 On Balance Digital Scale).
 <strong>Repeatability Analysis. </strong>The
 repeatability of our Hall-effect magnetic-inference methodology with
 the ElectroPhysik probe was investigated by conducting replicate thickness
 measurements (N = 10) for an additional 10 unregistered eggs.
 Repeatability was calculated using the intra-class correlation coefficient
 (ICC), in the R package irr (SI 4.0). Significance was determined where p
 &lt; 0.05. Comparative analysis of avian and cuckoo
 eggshell thickness. Previous studies indicate that eggshell thickness is
 positively correlated with egg size; larger eggs have thicker shells77. To
 investigate whether cuckoo eggshell thickness deviates from this general
 relationship, we calculated the mean eggshell thickness in a total of 78
 species, comprising 12 avian orders and 34 families (total N = 3134 eggs)
 (Table 2). Our analysis included previously published data for 12
 species. We used a phylogenetic generalised least squares
 regression (PGLS) in the R package caper, and estimated the relationship
 between eggshell length and two measures of thickness (apex and meridian).
 To control for phylogenetic relatedness, we used a maximum clade
 credibility (MCC) tree based on 100 trees downloaded from birdtree.org.
 The MCC tree, which is the tree with the maximum product of the posterior
 clade probabilities, was obtained using the R package
 phangorn. We extracted both phylogenetic residuals (phylogenetically
 independent) and residuals obtained from the phylogenetic regression line,
 and visually evaluated whether these residuals from cuckoo species were
 extreme values (e.g., were greater than expected by their size and
 phylogenetic position). We also used a linear regression of egg length
 versus mean eggshell thickness for 74 non-brood parasitic avians using the
 package lm in R v. 3.6.0. <strong>Statistical
 analysis.</strong> The distribution of raw and normalised eggshell
 thickness in cuckoos and their hosts was plotted and visually inspected in
 ggplot2 (Figs. S3 and Fig. 2). Within a species, outliers in the
 distribution of mean thicknesses (as defined above) were removed. To
 account for inter-specific differences in egg size (which is correlated
 with eggshell thickness) ‘normalised thickness’ was calculated for each
 sample by dividing the eggshell thickness by egg length. This approach is
 expected to successfully normalise the data because egg length explains a
 large proportion of the inter-species variation in eggshell thickness
 (Fig. 3). We tested for successful normalisation by regressing normalised
 eggshell thickness against eggshell mass (Fig. S4). We tested whether,
 overall, the thickness of cuckoo eggshells relative to their length
 differed from that of their hosts at both the apex and the meridian of the
 egg using a Wilcoxon signed-rank test on matched pairs of cuckoo and host
 eggs. For this analysis, any unpaired egg samples, or samples with data
 missing for either the host or cuckoo of the pair were removed from
 analysis. Final sample sizes for each treatment can be found in Fig. 1. We
 then tested whether host ejection behaviour predicted the ratio of cuckoo
 to host normalised eggshell thickness. We used a Restricted Maximum
 Likelihood Model (REML), with cuckoo:host normalised eggshell thickness
 ratio as the response variable, host response to foreign eggs (accept or
 eject) as the fixed effect and host species nested within cuckoo species
 as the random effect. For all models, we checked standardised residuals
 for normality. Log<sub>10</sub> transformation of variables
 improved the normality of residuals (Anderson Darling Tests
 for Goodness-of-fit, all P &gt; 0.4), so we present these results,
 although the qualitative results remained unchanged regardless of whether
 or not data were transformed. We ran four models; (1) eggshell thickness
 at the meridian including only hosts with known egg ejection rates, (2)
 eggshell thickness at the meridian including hosts with both known and
 unknown egg ejection rates, (3) eggshell thickness at the apex including
 only hosts with known egg ejection rates, and (4) eggshell thickness at
 the apex including hosts with both known and unknown egg ejection rates.
 The analyses were run in JMP v.15 (SAS Institute Inc, 2019).
description: Microsoft Excel