Habitat- and season-specific temperatures affect phenotypic development of hatchling lizards

Abstract

Embryonic environments influence phenotypic development, but relatively few experiments have explored the effects of natural environmental variation. We incubated eggs of the lizard Anolis sagrei under conditions that mimicked natural spatial and temporal thermal variation to determine their effects on offspring morphology and performance. Incubation temperatures mimicked two microhabitats (open, shade) at two different times of the incubation season (April, July). Egg survival, incubation duration and offspring size were influenced by interactions between habitat- and season-specific nest temperatures, and locomotor performance was influenced primarily by temporal factors. These findings highlight the importance of spatial and temporal environmental variation in generating variation in fitness-related phenotypes.

1. Introduction

Developing embryos of many species experience environments that vary over time and space. This environmental variation can have long-lasting effects on an individual's phenotype [1], and thus affect fitness [2]. In many ectotherms, the thermal environment regulates key physiological processes and therefore, these organisms are sensitive to temperature [3,4]. For embryos of oviparous species that lack parental care, temperature variation across time and space inside nests can play an important role in shaping phenotypic variation [5–10].

In reptiles, most laboratory studies assess the effects of constant incubation temperatures, and rarely explore the effects of fluctuating temperatures found in nature [11–14]. Even in studies that do examine effects of thermal fluctuations, most use conditions that are never encountered in nature [12,15]. Thus, effects of thermal conditions across different habitats or seasons are poorly understood. Experiments designed to quantify the relative roles of spatial and temporal thermal variation will provide new insights into how natural environments influence phenotypic variation.

The brown anole (Anolis sagrei) has a long reproductive season (March–October), which exposes eggs to wide-ranging temperatures as the season warms from spring through summer. This species inhabits open to forested habitats, providing mothers with many options for oviposition sites. Our experiment tested the effects of incubation temperatures from early versus late periods of the reproductive season (April and July) and from two distinct microhabitats (open and shaded) where A. sagrei lays eggs. Our goals were to (i) quantify the effects of natural incubation conditions on fitness-relevant phenotypes of offspring and (ii) determine the relative contributions of temporal versus spatial thermal variation to phenotypic variation.

2. Material and methods

Adult A. sagrei were collected in Palm Coast, Florida (9–10 October 2012), and male/female pairs (n = 41) were housed in single cages under conditions described elsewhere [16]. Eggs were collected three times weekly towards the end of the reproductive season (2 September–16 October 2013). Eggs produced late in the season were used to prevent potential confounding effects of seasonal differences in maternal allocation [17]. Importantly, preliminary results from recent work suggest that phenotypic responses to season-specific temperatures do not differ between early- versus late-produced embryos (P. R. Pearson, D. A. Warner 2015, unpublished data). Eggs were weighed and placed in moist vermiculite (−150 kPa) within individual glass jars (59 ml) covered with plastic wrap. Each egg was assigned to one of four incubation regimes that mimicked temperatures during April and July from shaded and open habitats in Palm Coast, Florida. Eggs produced by a single female were represented across incubation treatments as evenly as possible.

Incubation treatments (figure 1) were determined from data collected from iButton temperature loggers deployed in potential nest sites. Loggers were programmed to record temperature every 2.5 h from 30 March to 2 August 2013. Incubation regimes were based on temperatures averaged across days at 2.5 h intervals, and then averaged across six iButtons in open sites and six iButtons in shaded sites using data from April and July. Eggs of A. sagrei have been found in similar microhabitats to those of potential nest sites used in this study [18].

Figure 1.

Hourly averages of ground temperatures (2–3 cm deep) recorded from shaded (blue symbols) and open (red symbols) environments during April (triangles) and July (circles) from the field site in Florida. Incubation treatments mimicked these four thermal regimes, using programmable incubators. Daily thermal regimes repeated each day during incubation. Error bars = 1 s.e.

After hatching, individuals were measured for snout–vent length (SVL), tail length (TL) and mass and housed individually under standard conditions [16]. Between days 7–9 and again between days 21–23 after hatching, locomotor performance was assessed for each lizard by measuring sprint speed along a 1 m long ‘race track’. The track was positioned at a 20° angle, and a photo-sensor gate (connected to a stopwatch) recorded the time every 25 cm. Each lizard was continually chased to the end of the track with a paintbrush. Each hatchling was raced five times with 2 min of rest between trials. Every stop made by hatchlings was recorded.

Two-way mixed model analyses of variance and covariance (maternal identity as a random effect) assessed the effects of season (April versus July) and habitat-specific (open versus shade) incubation temperatures on incubation period, offspring size, growth rate and running speed; covariates are in table 1. Growth rate was measured as the change in mass over one week and three weeks after hatching. Sprint speed was evaluated as the fastest speed performed by an individual over 25 cm and 1 m distances.

Table 1.

Effect of season-specific and habitat-specific incubation temperatures on embryo development and offspring phenotypes of hatchling Anolis sagrei. p-values in bold face represent effects that are statistically significant.

trait	season effect	habitat effect	interaction
egg survival	F1,43 = 0.08, p = 0.781	F1,43 = 0.18, p = 0.675	F1,43 = 3.61, p = 0.064
incubation period	F1,28 = 1278.22, p < 0.001	F1,28 = 118.49, p < 0.001	F1,28 = 24.86, p < 0.001
snout–vent lengtha	F1,27 = 2.87, p = 0.102	F1,27 = 0.04, p = 0.847	F1,27 = 5.32, p = 0.029
massa	F1,27 = 1.34, p = 0.256	F1,27 = 1.49, p = 0.233	F1,27 = 10.75, p = 0.003
tail lengthb	F1,27 = 5.11, p = 0.032	F1,27 = 2.68, p = 0.113	F1,27 = 1.64, p = 0.211
growth in mass (1 week)	F1,22 = 0.36, p = 0.555	F1,22 = 0.30, p = 0.589	F1,22 = 2.80, p = 0.108
growth in mass (3 weeks)	F1,15 = 1.14, p = 0.303	F1,15 = 0.05, p = 0.829	F1,15 = 0.00, p = 0.956
running speed at 1 week
over 25 cmc	F1,21 = 5.99, p = 0.023	F1,21 = 0.31, p = 0.584	F1,21 = 0.19, p = 0.669
over 1 mc	F1,18 = 2.56, p = 0.127	F1,18 = 0.26, p = 0.617	F1,18 = 1.16, p = 0.296
number of stops	F1,22 = 23.16, p < 0.001	F1,22 = 6.43, p = 0.019	F1,22 = 10.44, p = 0.004
running speed at three weeks
over 25 cmc	F1,14 = 2.18, p = 0.162	F1,14 = 0.21, p = 0.653	F1,14 = 1.57, p = 0.231
over 1 mc	F1,12 = 0.57, p = 0.465	F1,12 = 0.78, p = 0.393	F1,12 = 1.55, p = 0.237
number of stops	F1,15 = 5.08, p = 0.039	F1,15 = 4.41, p = 0.053	F1,15 = 6.52, p = 0.022
survival to three weeks	F1,27 = 2.61, p = 0.118	F1,27 = 0.01, p = 0.919	F1,27 = 0.68, p = 0.417

Covariates: ^aEgg mass. ^bSnout–vent length. ^cBody mass at time of running trial.

3. Results

Seventy-two eggs were produced by 26 females. Although marginally non-significant (p = 0.062), egg survival (72.2% overall) was lowest under conditions that mimicked the coolest (April/shaded) and warmest (July/open) environments and considerably higher under intermediate temperatures (April/open and July/shaded; figure 2a). Incubation conditions that mimicked spatial and temporal variation in nest temperature influenced incubation duration (table 1 and figure 2b), but most of the variation was owing to temporal effects (April versus July). Eggs exposed to open habitat temperatures in July had the shortest incubation periods, particularly in comparison with eggs experiencing April temperatures.

Figure 2.

Effects of season-specific (April versus July) and habitat-specific (shaded versus open) incubation temperatures on (a) egg survival, (b) incubation period, (c) snout–vent length (same pattern for body mass), (d) tail length, (e) sprint speed over 25 cm and (f) number of stops over 1 metre. Blue represents shaded habitat, red represents open habitat. Graphs are arranged so that the bars represent a range from relatively cool (April/shaded) to warm (July/open) incubation temperatures. Error bars = 1 s.e. Statistical results are in table 1.

Offspring body size was influenced by the interaction between spatial and temporal variation in nest temperature (table 1 and figure 2c). Eggs incubated at temperatures that mimicked cool (April/shaded) and hot (July/open) environments produced small offspring (both SVL and mass) compared with treatments that mimicked intermediate thermal environments (April/open, July/shaded; table 1). Eggs incubated at April temperatures produced offspring with short tails, particularly at temperatures that mimicked shaded habitat (figure 2d and table 1). Growth rate was not affected by treatment.

Eggs incubated at July temperatures produced faster-running offspring (over 25 cm distances) at one week of age than those incubated at April temperatures; this pattern was similar between habitat types (table 1 and figure 2e) and disappeared at three weeks of age. Hatchlings produced under April incubation temperatures made more stops than those from July temperatures, particularly those from shaded thermal conditions (figure 2f); this pattern was similar at one and three weeks of age. Hatchling survival to three weeks of age (64.7%) was not influenced by incubation treatments (table 1).

4. Discussion

The impact of incubation temperature on phenotypes of reptiles is well documented [6–9,19,20], but its ecological relevance is often poorly understood, because most experiments do not mimic natural conditions. Our design enabled us to quantify the effects of natural incubation temperatures in different habitats and seasons. We show that habitat- and season-specific nest temperatures contribute to developmental and phenotypic variation in ways that might influence fitness.

Embryonic development was influenced by temporal and spatial variation in temperature. Developmental rate of embryos exposed to April temperatures was half of the rate under July temperatures. Even cool temperatures that mimicked shaded nest sites lengthened the incubation period. This long incubation period for eggs laid early in the season or in cooler locations (60–80 days) could increase the time that eggs are exposed to predators or adverse weather. Despite these potential consequences of incubation length, exposure to cool (shaded sites in April) or hot incubation conditions (open sites in July) reduced egg survival. This pattern in egg survival suggests that fitness could be enhanced if mothers choose open habitats early, and shaded habitats late, in the season.

Morphology, performance and behaviour were also influenced by temporal and spatial variation in temperature. Intermediate thermal conditions produced larger offspring than cool or hot incubation conditions. If body size is a predictor of hatchling fitness [17], then seasonal shifts in maternal nest site choice could enhance fitness in the same way described above for egg survival. Regardless of effects on hatchling size, however, eggs exposed to July temperatures produced hatchlings with greater locomotor performance than those in the April treatments; this pattern was likely influenced by running behaviour (fewer stops while sprinting by hatchlings from July temperatures). Eggs produced in July will invariably hatch later than those laid in April, potentially putting offspring from July eggs at a competitive disadvantage [19]. Indeed, offspring produced late in the season grow more slowly and have reduced survival relative to those produced early in other lizard species [21,22]. However, the positive effect of July nest temperatures on locomotor performance could offset any negative effects of hatching late. Negative consequences of late hatching could further be diminished via seasonal increases in maternal investment to eggs, which has been demonstrated previously in A. sagrei [17]. Notably, however, any benefit of increased locomotor speed (owing to July nest temperatures) must be realized very soon after hatching, because incubation effects disappeared by 3 weeks of age.

Overall, spatial and temporal variation in nest temperature influenced different phenotypic attributes of offspring in different ways. These findings have important implications for how natural thermal variation contributes to phenotypic variation in natural populations, and provides insights into how maternal nesting behaviours might shift seasonally in adaptive directions. The thermal fluctuations used in this laboratory study provide a more realistic representation of the range of temperatures that embryos experience in the field compared with other studies that examine effects of temperature fluctuations. Although the importance of using natural developmental conditions is well understood [15,23–25], our experimental design provides a novel approach for laboratory studies to better assess the ecological relevance of developmental environments.

Ethics

All guidelines and procedures for the use of animals were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham (no. 120909764).

Data accessibility

The dataset is available at the Dryad digital repository: http://dx.doi.org/10.5061/dryad.6g0j3 [26].

Authors' contributions

Both authors conceived and designed the study, analysed the data, wrote the paper and approved the final version of the manuscript for publication. P.R.P. performed the laboratory work and data collection. Both authors agree to be held accountable for the content of this manuscript.

Competing interests

We have no competing interests.

Funding

Funding was provided by the University of Alabama at Birmingham.