Facultative oviparity in a viviparous skink (Saiphos equalis)

Abstract

Facultative changes in parity mode (oviparity to viviparity and vice versa) are rare in vertebrates, yet offer fascinating opportunities to investigate the role of reproductive lability in parity mode evolution. Here, we report apparent facultative oviparity by a viviparous female of the bimodally reproductive skink Saiphos equalis—the first report of different parity modes within a vertebrate clutch. Eggs oviposited facultatively possess shell characteristics of both viviparous and oviparous S. equalis, demonstrating that egg coverings for viviparous embryos are produced by the same machinery as those for oviparous individuals. Since selection may act in either direction when viviparity has evolved recently, squamate reproductive lability may confer a selective advantage. We suggest that facultative oviparity is a viable reproductive strategy for S. equalis and that squamate reproductive lability is more evolutionarily significant than previously acknowledged.

1. Introduction

Viviparity has evolved from ancestral oviparity at least 115 times in squamate reptiles [1,2]. Reversals from viviparity to oviparity are rare [3–6], yet recent phylogenetic work suggesting frequent reversals [7] has rekindled debate about their adaptive significance for squamate evolution. Dollo's Law of irreversibility predicts that complex traits, once lost, either cannot re-evolve or will do so in a different way [3,5,8,9]. The evolution of viviparity involves loss of complex structures, including the eggshell and oviductal glands that produce shell material, and requires changes in maternal vasculature and timing of reproductive events [8]. Physiological challenges associated with re-evolution of these structures and processes likely constrain reversals to oviparity [2,5]. Additionally, intermediate phenotypes ‘in transition’ to oviparity from viviparity may be inviable, as re-evolution of an eggshell could restrict embryonic gas exchange in utero [5,10–13]. Obligate viviparity may thus be a ‘fitness valley’ [10] that is difficult to emerge from once viviparity is acquired [3,5].

By contrast, facultative switches in parity mode (oviparity to viviparity and vice versa), while rare, involve fewer modifications than complete reversals, and suggest that an intermediate, reproductively labile stage can exist before obligate viviparity [14]. Facultative viviparity (capacity to deposit either eggs or developed offspring, depending on circumstance; [14]) is relatively common in invertebrates [14], including thrips [15], dipteran flies [14] and onycophorans [14,16]. In vertebrates, facultative viviparity is rare, yet likely occurs in the olm (Proteus anguinus) [17,18], and several species of poeciliid fish [19,20]. While facultative viviparity has never been reported for squamate reptiles, several oviparous squamate species show plasticity in timing of oviposition [21–24], demonstrating facultative changes in length of internal egg-retention. Species that can reproduce via either parity mode are evolutionarily significant as they demonstrate retention, rather than re-evolution, of oviparous traits. Since neither parity mode is selectively advantageous in all environments [3], persistence of an ‘intermediate’ transitional form (facultative viviparity) could convey a reproductive advantage, irrespective of the direction of selection [3,10]. Parition (giving birth or laying eggs) is rarely observed for many species; facultative changes in parity mode, and mixed modes within a clutch, could go unreported and may be more biologically significant than previously acknowledged.

Species in which viviparity has evolved relatively recently are ideal models for investigating reproductive lability in squamate evolution [9,22]. The three-toed skink, Saiphos equalis, endemic to southeastern Australia, is one of very few reptile species with intraspecific variation in parity mode [2,25]. Reproduction varies geographically: S. equalis is viviparous in its northern high-elevation range and oviparous in northern and southern coastal regions [26]. Oviparous squamates typically lay eggs at embryonic stage 30, which hatch at stage 40 [27]. Unusually, oviparous females of S. equalis oviposit shelled eggs at stages 38–39 and undergo a shorter incubation period (mean 5.5 days) than sympatric skink species [26,28]. By contrast, viviparous S. equalis neonates are enclosed by transparent membranes that are broken on average 1.5 days after birth [26,29].

Here we report an observation of facultative oviparity in S. equalis. A viviparous female in our laboratory colony oviposited three eggs at embryonic stage 33, followed by a single fully developed offspring. As far as we are aware, this is the first report of a facultative change in parity mode within a vertebrate clutch, offering a rare opportunity to investigate reproductive lability in an unusual and evolutionarily important skink species. We used scanning electron microscopy (SEM) to compare shell morphology of facultatively oviposited eggs with those of normal oviparous and viviparous S. equalis to determine: (1) if shells of eggs deposited facultatively were more similar to those of viviparous or oviparous S. equalis and (2) whether viviparous S. equalis retain the machinery required for oviparous egg production.

2. Methods

(a). Animals

Female S. equalis (n = 40) were collected in November 2013 from a northern high-elevation population previously shown to be viviparous [26,28] (Mummel Gulf National Park, NSW, 31°21′ S 151°43′ E), and from a known oviparous population at southern low-elevation (Sydney, NSW, 33°50′ S 151°12' E; [25]). Animals were housed under standard protocols [30] in cages lined with damp peat moss (50 mm) with a heat strip at one end to provide a temperature gradient (17–26°C) for 6 h d⁻¹. Photoperiod : scotoperiod reflected natural periods and incrementally increased from 14 to 15 h d⁻¹. Crickets and water were provided ad libitum.

(b). Egg treatment and shell collection

One shelled embryo was dissected from each of nine of S. equalis females at four different reproductive stages, according to the staging system of Dufaure and Hubert [31] (from 0 (fertilization) to 40 (fully developed)): oviparous stage 36/37 (O36/37; n = 2 embryos), oviparous stage 39 (O39; n = 2 embryos), viviparous stage 36 (V36; n = 3 embryos) and viviparous stage 39/40 (V39/40; n = 2 embryos). We also dissected two embryos from ‘unusual’ eggs oviposited facultatively by a single viviparous female (V unusual, deposited at stage 33; post-oviposition (n = 1 embryo), post-hatching (n = 1 egg-covering)). Eggs and egg coverings were fixed in 10% neutral buffered formalin overnight followed by 70% ethanol. After staging, shells were dissected, air dried, then processed for SEM. Two ‘unusual’ eggs deposited facultatively were incubated in moist vermiculite (100% water by dry mass of vermiculite) at 22°C until hatching.

(c). Scanning electron microscopy

Shell fragments (greater than 3 × 5 mm²) stored in 70% ethanol (34–46 months) were dried using a Leica EM CPD300 (Leica, Wetzlar, Germany), using CO₂. To embed, upright shell fragments were secured flat between rings of copper coils, then cold-mounted in epoxy resin overnight at room temperature. The upper surface was ground flat using a RotoPol-22 (Struers, Copenhagen, Denmark) to expose the sample. Blocks were polished on a TegraPol-25 (Struers) to remove imperfections, then mounted onto aluminium stubs and coated with gold (15 nm). Images were captured using a Zeiss Sigma HD VP-STEM (Zeiss, Oberkochen, Germany).

SEM images were used to measure shell thicknesses. At least 10 measurements were taken of the crust and shell membrane layers of at least three shell regions per sample (1000–9000× magnification), although fewer measurable regions were intact for ‘unusual’ samples (two regions). Shell layer measurements were taken using ImageJ (v.1.51) and used to calculate: (1) total shell thickness, (2) relative thicknesses of crust (crust/total) and shell membrane (shell membrane/total) and (3) ratio of crust to shell membrane thickness (crust/shell membrane).

3. Results

During routine checks, and prior to parturition of any of the other 39 viviparous females from the same collecting trip, a viviparous female oviposited two eggs (24 January 2014), followed by a third (25 January 2014). Eggs were covered with a thin transparent membrane consistent with published descriptions of viviparous S. equalis at parturition [25] (electronic supplementary material). One egg was fixed (10% NBF) and found to be at stage 33 (electronic supplementary material), much earlier than typical for oviparous S. equalis (stage 38–39). The remaining two eggs were incubated. One died during incubation; the other resulted in a viable neonate that was observed ‘hatching’ from its thin membranes 37 days after oviposition. These membranes were fixed as per the first egg (electronic supplementary material figure B). The same female then gave birth to a single live neonate 41–48 days after oviposition of the first egg.

(a). Oviparous

Shells of oviparous S. equalis consist of an outer crust layer overlying a shell membrane layer consisting of interwoven fibres embedded in a dense crystalline matrix (figure 1a,b). Outermost fibres are thickest; many contain a globular material (figure 1c). The crust and shell membrane are separated by an additional thin membrane (figure 1a,b). The shell membrane overlays a thin inner boundary layer comprised of a dense matrix of thin fibres. Shell morphology is similar between stages 36/37 and 39, including shell layer proportions (figure 2), although total shell thickness decreases by 45.5% from 32.9 ± 1.3 µm (stage 36/37) to 17.9 ± 2.5 µm (stage 39)].

Figure 1.

Scanning electron micrographs of eggshells of Saiphos equalis (cross-section, outer surface towards the top). (a–c) Oviparous (stage 39); (d) viviparous (stage 36); (e) viviparous (stage 39/40); (f) oviposited ‘unusual’ viviparous (stage 33). C, crust; SM, shell membrane; arrow, membrane; filled arrowhead (IBL), inner boundary layer; F, fibre; open arrowhead, globular material; Ma, crystalline matrix.

Figure 2.

Eggshell thickness measurements for Saiphos equalis. (a) Shell layer thicknesses (µm) and total thickness (crust + shell membrane) per stage. At least 10 measurements were taken of at least three regions per sample (O36/37: (oviparous stage 36/37): n = 2, O39 (oviparous stage 39): n = 2, V36 (viviparous stage 36): n = 3, V39/40 (viviparous stage 39/40): n = 2, V oviposited ‘unusual’ (viviparous stage 33): n = 2). (b) Relative crust and shell membrane thicknesses (proportion of total thickness). (c) Crust : shell membrane per stage (mean ± s.e.m.).

(b). Viviparous

Shells of viviparous S. equalis are structurally similar to those of oviparous S. equalis (figure 1d–e), although the inner boundary layer is often difficult to discern, particularly at stage 39/40 (figure 1e). At stage 36, the shell membrane consists of thicker fibres towards the outer surface, with thinner fibres embedded in a dense matrix (figure 1d). Shell membrane fibres become more uniform in diameter by stage 39/40 (figure 1e). Shells are 28.5% thicker at stage 36 (11.67 ± 1.9 µm) than at stage 39/40 (8.35 ± 2.1 µm), with the reduction primarily in the shell membrane (figure 2).

(c). ‘Unusual’ viviparous

Morphology was similar for both ‘unusual’ shells (post-oviposition (stage 33, figure 1f) and post-hatching (stage 40)). An inner boundary layer could not be discerned, possibly as an artefact of sample preparation. Shell membrane fibres are relatively uniform and dense (figure 1f). Total thickness (9.27 ± 0.25 µm (figure 2a)) and shell layer proportions (figure 2c) are most similar to those of viviparous egg coverings at stage 36, yet layer proportions are also similar to oviparous S. equalis. Qualitatively, the thickness and proportions appear intermediate between the products of oviparous and viviparous individuals at the time of parition (O39 and V39/40, respectively; figure 2).

4. Discussion

This is the first description of both oviparity and viviparity within a single clutch in a reptile. Our observations suggest that oviparity may be facultative in viviparous S. equalis under some conditions. A mixed mode of parity within a clutch may reflect lability in timing of parition and suggests fine-scale control over neonate deposition within the uterus. The oviposition we observed (stage 33 versus 40) overlaps with oviposition of some oviparous species [22,24] and is close to the typical range for most oviparous squamates (embryonic stages 25–31; [22]). Hence, the oviposition observed here is likely to be biologically significant. While our observation could be aberrant, the neonate that hatched from an egg oviposited at stage 33 was apparently healthy. We have also observed earlier than normal egg deposition (approx. stage 30) in captive S. equalis several times previously (M. B. Thompson 2018, personal communication). We thus suggest that reproductive lability is part of a viable reproductive strategy for S. equalis, at least in captivity.

Similarities of facultatively oviposited ‘unusual’ shells to those of oviparous and viviparous S. equalis suggest that the machinery for producing oviparous egg coverings is retained in viviparous S. equalis. Since ‘unusual’ shells were thinner than those of normally oviparous S. equalis, the capacity of oviductal glands to deposit shell material may be reduced [32], possibly as a result of reduced gland density [29]. However, as eggshells of both viviparous and oviparous S. equalis are relatively similar [29,33,34], the ‘unusual’ shell characteristics may be within the normal range of variation for viviparous S. equalis. Indeed, the eggshell thickness difference between oviparous and viviparous S. equalis is less than other bimodal squamates [35,36,37]. Hence shells of S. equalis eggs oviposited facultatively, intermediate in thickness at parition, may incur minimal fitness consequences during external incubation under some environmental conditions.

Lability in parity mode and timing of parition may confer an adaptive advantage in species in which viviparity has evolved relatively recently. ‘Unusual’ eggs were oviposited even earlier than those from normally oviparous S. equalis populations, which may be advantageous where inter- or intraspecific competition is high. Intra-clutch variation in timing of oviposition may be an adaptive strategy to ensure asynchronous hatching for offspring dispersal, similar to asynchronous birth in the viviparous skink, Liopholis whitii [38]. By contrast, since oviposited eggs with thinner shells are less likely to survive due to embryonic water balance constraints [13,22], the capacity to carry offspring to term could increase survival during dry periods. The ability to reproduce via either parity mode could, therefore, act as a bet-hedging strategy in variable environments. Field monitoring, as well as experimental tests of the relationship between parity mode and environmental and physiological variables, could determine if wild S. equalis demonstrate such intra-clutch variation, and identify its adaptive significance.

Potential facultative oviparity in S. equalis suggests that a shift to obligate oviparity may still be possible for species early in the transition to viviparity. Investigating oviparous traits retained in facultatively oviparous species could identify which remain selectively advantageous in a mixed parity mode. While viviparous individuals of bimodally reproductive species such as S. equalis may retain oviparous traits, shifts to obligate oviparity are less likely for species representing more ancient origins of viviparity [3], particularly after the evolution of complex placentation and/or complete loss of an eggshell [5]. We suggest that reproductive lability may be an unacknowledged source of adaptation during squamate evolution.

Ethics

Ethics approval for this project was obtained from the University of Sydney Animal Ethics committee (688; L04/10-2011/3/5607). Fieldwork was conducted under NSW collection permit SL100401.

Data accessibility

Measurement data and image files. Data are available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.6q3d110 [39].

Authors' contributions

M.K.L., C.M.W. and M.B.T. performed the study and wrote the manuscript. All authors agree to be held accountable for the content therein and approve the final version of the manuscript.

Competing interests

We declare we have no competing interests.

Funding

This study was funded by the Australian Research Council (DP120100649).