DOI: 10.5061/dryad.4mw6m908z
title: Adaptive variation in the development of extraembryonic membranes of gekkotan lizards: a meta-analytical approach

description: Highly mineralized rigid-shelled eggs characterize one lineage of
 gekkotans. In contrast, poorly mineralized flexible-shelled eggs
 characterize basal lineages of gekkotans and all other squamates. Low
 oxygen permeability of rigid-shelled eggs is associated with small eggs
 and hatchlings, and long incubation lengths compared to flexible-shelled
 gekkotan eggs. These features represent a demographic cost for species
 with rigid-shelled eggs. This cost is offset, at least in part, because
 mortality due to desiccation and predation is reduced for rigid-shelled
 eggs relative to flexible-shelled eggs. Developmental traits may also
 compensate for the low oxygen permeability of rigid-shelled eggs.
 Oviposition, for example, occurs at earlier developmental stages for
 gekkotans with rigid- versus flexible-shelled eggs. Such early oviposition
 facilitates development because eggs move from the relatively hypoxic
 oviduct to the much better oxygenated nest environment. In this study, I
 tested the hypothesis that the growth of the yolk sac (YS) and
 chorioallantoic membrane (CAM) of gekkotans with rigid-shelled eggs is
 initiated and completed earlier than those of gekkotans with
 flexible-shelled eggs. I measured the surface area of eggs covered by the
 YS and CAM from oviposition to hatching and determined which of four
 non-linear models provide the best fit for growth curves. I also compiled
 a data set on embryonic metabolism of gekkotans and other lizards in order
 to place growth of the YS and CAM in the context of energy utilization of
 lizard embryos overall. Growth of the YS and CAM of gekkotans with
 rigid-shelled eggs is accelerated relative to that of gekkotans with
 flexible-shelled eggs and may serve to reduce overlap in the costs of YS
 and CAM development from that of the embryo. Adaptive variation in YS and
 CAM development may also extend to birds, crocodilians, and turtles as
 they also exhibit  life history variation that affects oxygen
 availability to embryos during development.
description: <b>2.1 Observations on the growth of the YS and
 CAM</b> The YSCAM data set is comprised of
 observations that I made on the YS and CAM from oviposition to hatching.
 Data are from Andrews et al. (2013) plus observations on three additional
 gekkotan species, <i>Correlophus ciliatus</i>, <i>Gehyra
 dubia</i>, and <i>Hemidactylus frenatus</i>, made in my
 laboratory (Andrews, 2017) and at James Cook University in Australia
 (Table 2). I weighed all eggs at oviposition and staged one egg per
 clutch. Eggs were incubated at constant temperatures (25-29ºC) until their
 designated sampling date. At this time, they were fixed in buffered
 formalin. Eggshells were carefully removed to expose the extent of the
 vascularized YS and CAM on the egg surface. Scale drawings of the
 membranes were compared to a series of standard images to assess the %
 coverage of the YS and CAM relative to the surface area of each egg (for
 details, see Andrews et al., 2013). I quantified surface areas of the YS
 and CAM with reference to a fixed equator that divided the egg into two
 equal surface areas, the embryonic hemisphere was centered on the embryo
 and the abembryonic hemisphere included the remaining egg surface area. I
 also recorded embryo stage and age (with oviposition at age 0) at each
 observation. When eggs hatched, I weighed the hatchlings and determined
 incubation length as the difference in days between oviposition and
 hatching. Observations on three of the seven species
 extended from oviposition to hatching. Observation on the remaining four
 species extended from oviposition to stages 36 or 37. I used the following
 convention for these species to compensate, at least in part, for the bias
 that results from curve fitting when data do not include asymptotic
 values. For <i>G. dubia</i>, <i>G. variegata</i>,
 and <i>H. frenatus</i>, species with observations of
 approximately one embryo per stage, I added one observation of 100 % YS
 and 100 % CAM at stage 40 and for <i>C. turneri</i>, a species
 with observations of approximately two embryos per stage, I added two such
 observations. <b>2.2 Observations on embryonic
 metabolism </b> The V̇O<sub>2</sub>
 data set comprises observations obtained from the literature for two RSG
 species, two FSG species, and 8 FSOL (lizards with flexible-shelled eggs
 other than gekkotans) species (Table 2). I used only data sets for which
 moisture and constant temperature conditions for incubation were
 ecologically appropriate, e.g., were associated with high survival during
 incubation. When eggs were incubated at more than one temperature or more
 than one moisture level, I selected the temperature closest to normal nest
 temperature and the highest moisture level, respectively. Because
 V̇O<sub>2</sub> and embryo dry mass exhibit parallel growth
 trajectories relative to stage (Thompson & Stewart, 1997; Thompson
 & Russell, 1999a; Nechaeva, Vladimirova, & Alekseeva,
 2007; Ma, Guo, Su, & Ji, 2019), observations of V̇O<sub>2
 </sub>provide an index of embryonic growth for comparison with the
 growth of the YS and CAM. Observations on all species
 extended from oviposition to hatching. While experimental protocols were
 consistent among studies, observations were either summarized as mean
 V̇O<sub>2</sub> for multiple embryos of the same age with
 sampling fixed at 2-3 d intervals (five species) or presented as
 individual observations measured on a daily basis (seven species). I
 therefore calculated mean V̇O<sub>2</sub> over 2-3 d intervals
 for the latter group and used these means in analyses to make sample sizes
 of the two groups of observations comparable. For comparative purposes,
 V̇O<sub>2 </sub>was expressed as %V̇O<sub>2</sub>
 (V̇O<sub>2</sub> divided by the largest value of
 V̇O<sub>2 </sub>observed). The majority of
 studies expressed V̇O<sub>2</sub> as a function of egg age.
 This is problematic because embryos are arbitrarily assigned an age of 0
 at oviposition. Age is thus not associated with stage of development;
 development of squamates is initiated at fertilization; by oviposition,
 stage varies considerably among species (Andrews & Mathies, 2000).
 Stage at oviposition, in the YSCAM data for example, ranged from 25 to 30,
 although all embryos were age 0. Stage is therefore the relevant dependent
 variable for comparative studies. The first
 step was to adjust embryo age for variation in stage at
 oviposition. To make the YSCAM and V̇O<sub>2</sub>
 data sets compatible, I used the known association between stage and age
 in the YSCAM data to estimate the stages corresponding to ages in the
 V̇O<sub>2</sub> data. By convention, embryos that were stage
 25 at oviposition were assigned to age 0. Ages of embryos that were
 oviposited at later stages were corrected to account for the time elapsed
 between stage 25 and the observed stage of oviposition. I regressed age on
 stage (three-parameter exponential models provided the best fits for all
 species). I used the resultant equations to estimate embryo age at stage
 25, 26, 27, etc. The days elapsed between the adjacent stage intervals
 25-26, 26-27, 27-28, 28-29, 29-30, and 30-31 were determined by
 subtraction. The resultant estimates of days per stage were pruned to
 include only observed intervals, ie., species with oviposition at stages
 25 and 29, for example, would have had estimates for six and for the last
 two intervals, respectively. I then regressed the days elapsed per stage
 (D/S) for these observed intervals on incubation temperature (T) and stage
 interval (S), where 25 represents interval 25-26, 26, interval 26-27, etc.
 and included the T x S interaction term in the linear model. The resultant
 general relationship was: D/S = 10.23 – 0.61 T + 0.31 S
 - 0.07 [(S – 27.80)(T – 27.77)],       Eq.
 1 R<sup>2</sup> = 0.99, F<sub>3,26</sub> = 892.4,
 P &lt; 0.0001, where all terms made significant contributions to the
 model (P’s &lt; 0.0001). Temperature of embryos
 prior to oviposition should be the same as the mean body temperature of
 gravid females over a diel cycle (Parker & Andrews, 2006). The
 gravid females that produced eggs for this study were typically exposed to
 temperature gradients that allowed  them to thermoregulate normally.
 Observations on field body temperatures of geckos ecologically similar to
 those I studied (e.g., Brown, 1996; Angilletta, Montgomery, &
 Werner, 1999; Lapwong, Dejtaradol, & Webb, 2020; Kearney &
 Predavec, 2000) suggest that 28ºC would be an appropriate mean body
 temperature of gravid females. I used Eq. 1 to estimate the growth of
 embryos prior to oviposition for stage intervals 25-30. Intervals were
 summed to give the time elapsed between stage 25 and each observed stage
 at oviposition (ie., 25-26, 25-27, 25-28, etc.). I added the appropriate
 sum to observed ages and to incubation lengths for each species in both
 the YSCAM and the V̇O<sub>2</sub> data sets to correct for
 variation in stage at oviposition. Corrected ages were divided by
 corrected incubation lengths (x 100) to obtain an index of relative embryo
 age that ranged from 0 % when oviposition was at stage 25 to 100 % at
 hatching. The second step was to predict
 embryo stage as a function of relative embryo age. Predictions
 were made independently for RSG and FSG species given that shell type
 seemed likely to affect the relationship between stage and age once
 differences in temperature and stage at oviposition were accounted for.
 Observed stage was regressed on relative embryo age (RAge)
 (three-parameter exponential models provided the best fits). The resultant
 relationships were: Stage (RSG) = 40.2 – 13.8 Exp
 (-0.029 RAge)     (R<sup>2</sup> = 0.96,
 N<sub>obs</sub> = 53, N<sub>sp</sub> = 4)   Eq.
 2 Stage (FSG) = 41.5 – 15.7 Exp (-0.022 RAge)    
 (R<sup>2</sup> = 0.96, N<sub>obs</sub> = 80,
 N<sub>sp</sub> = 3)   Eq. 3 The more rapid
 increase in predicted stage with respect to relative age of RSG than FSG
 embryos was statistically significant (P &lt; 0.05) judging by the
 near non-overlapping 95% CIs for their respective growth rate parameters
 [-0.029 (-0.035 – -0.024) versus 0.022 (-0.027 – -0.017)] (Cumming, 2012,
 p. 158).). The mean difference between observed and predicted stages RSG
 and FSG species combined was (0.0085) and 80% of individual values were
 within -1.20 to 0.99 stages of the mean. I therefore used Eq. 2 to predict
 stage for RSG species and Eq. 3 to predict stage for FSG and FSOL species
 in the V̇O<sub>2</sub> data set (where stage was otherwise
 unknown). <b>2.3 Statistical
 analyses</b> To describe the relationships
 between %YS, %CAM, %V̇O<sub>2 </sub>and stage, I fit
 observations for each species to four models: two- and three-parameter
 exponential models and three- and four-parameter logistic models (JMP
 software, JMP®, Vers. 15.0, SAS Institute, Cary, NC, 1989-2019,
 RRID:SCR_014242). The best model had the lowest AICc value. Evidence
 ratios (the Akaike weight of the best model divided by that of the next
 best model) were used to evaluate support for the best model. Evidence
 ratios near 1.0 mean that both models fit equally well and the higher the
 evidence ratio the stronger the support (Anderson, 2008). I used
 contingency tables to evaluate the degree to which best-fit models
 associated species with taxonomically appropriate groups. To illustrate
 overall growth patterns, I plotted of %YS, %CAM, and %
 V̇O<sub>2</sub> as a function of stage using species data that
 was aggregated within shell and membrane types using the best-fit model
 for each group (Table 3). I also used aggregated data to plot %YS and %CAM
 as a function of % V̇O<sub>2</sub> to put membrane growth in
 the context of embryo growth. When sample size was too small to compare
 RSG and FSG groups, the data was combined (indicated as
 RSG+FSG). Comparisons between RSG and FSG
 groups (YSCAM data) and between RSG+FSG and FSOL
 (V̇O<sub>2</sub>) groups were made with meta-analyses
 (Cumming, 2012) using Exploratory Software for Confidence Intervals (ESCI,
 copyright Geoff Cumming, 2011, RRID:SCR_006024). Mean effect sizes and
 their 95% confidence intervals were determined using random effects
 models. Effect sizes were growth rate (exponential and logistic models)
 and inflection point parameters (logistic models) of best-fit growth
 models. Parameters associated with asymptotes are not relevant for
 comparative purposes; the lower asymptote is an estimate of the
 independent variable when stage = 0, a point well outside the range of the
 data, and the upper asymptote was fixed at 100%. I assessed the difference
 in mean effect sizes for growth rate and inflection point parameters
 between RSG and FSG groups and between RSG+FSG and FSOL groups accorded to
 the amount of overlap of their 95% confidence intervals (Cumming, 2012, p.
 158). I did not run analyses corrected for
 phylogenetic signal for two reasons. 1) My sample sizes are small. The
 process of estimating phylogenetically independent contrasts and then
 conducting ANOVAs by groups within data sets progressively reduces the
 degrees of freedom available for statistical comparisons, and sample sizes
 of the YS, CAM, and V̇O<sub>2</sub> data sets are 6, 7, and 12
 species, respectively. 2) The other reason is that the phylogenetic
 distribution of families with rigid- and with flexible-shelled eggs is
 dichotomous (Fig. 1). As a result, convergence or reversal do not
 contribute any variance to analyses, that is, data sets (of any size) will
 have only one independent contrast of rigid vs. flexible-shelled clades.
 Because phylogenetic analyses are used to assess the generality of
 phenomena considered to have evolved independently (Garland, Bennett,
 & Rezende, 2005), the one-time origin of rigid-shelled eggs in the
 Gekkota makes this taxon an evolutionary and statistically inappropriate
 target for such analyses.
description: Column Number. Column name. Comments. 1.
 Species.  Species used in analyses. Sources listed in text Table
 2. 2. RSG+FSG, FSOL groups.  Groups used as the ‘by’
 variable in some analyses. 3. RSG, FSG, FSOL groups.
  Groups used as the ‘by’ variable in some analyses. 4.
 Data set.  YSCAM: observations made by the author on the relative surface
 areas of the YS and the CAM from oviposition to hatchling; VO2:
 observations from the literature on the relative metabolic rates of
 embryos from oviposition to hatching. 5. %YS.  Surface
 area of the YS expressed as a percentage of the surface area of the
 egg. 6. %CAM.  Surface area of the CAM expressed as a
 percentage of the surface area of the egg. 7.
 Supplemental Observations. See last paragraph of Section 2.1.
 8. Stage. Embryo developmental stage at each observation in the
 YSCAM data set based on Dufaure and Hubert (1961). 9.
 Age. Embryo age at each observation in the YSCAM and the VO2 data sets.
 The day of oviposition is Age 0. 10. Days added to age.
 Days added represent the time elapsed between stage 25 and the observed
 stage at oviposition to correct for variable stages at oviposition. Values
 are based on a mean in utero temperature of 28C.  See Section 2.2 for
 details. 11. Corrected age. The sum of Age and Days
 added to age. 12. Relative age. Corrected age divided
 by Corrected incubation length. 13. Incubation length.
 Observed incubation length at the constant incubation temperature in
 Column 18. 14. Corrected incubation length. The sum of
 Incubation length and Days added to age (Columns 13 and 10).
 15. VO2 mL/h. Metabolic rate at each observation.
 16. VO2Max. Largest value of metabolic rate.
 17. %VO2.  VO2 divided by VO2Max expressed as a percent. Se also
 Column 24. 18. Incubation temperature.  The constant
 temperature in degrees C during incubation. 19. Stage
 at oviposition. Mean embryo stage on the day of oviposition based on the
 Dufaure and Hubert (1961) scheme. 20. Hatchling mass g.
 Mean live mass on the day that eggs hatched. 21. Egg
 mass g. Mean egg mass at oviposition 22. Predicted
 stage. See Section 2.2 for calculations. Predictions were applied only to
 the VO2 data set where only stage was known. 23. Stage
 – Predicted stage. Observed stage minus Predicted stage for the YSCAM data
 set. This metric is an index of the accuracy of the prediction of
 stage. 24. Pruned temperature. Data pruned to one
 observation per species and displayed for age = 0.