Author: Joshua Hall Page 1 of 2

A hatching brown anole.

Temperature is probably the most studied environmental factor that influences living things; however, you might be surprised to learn that we still don’t have a solid understanding of why things die when they get hot. If you recall your intro biology, you’ll remember that proteins and cell membranes fall apart when they get hot, and that is often the explanation for death at high temperatures. But, there are several reasons to question this explanation. For example, complex organisms (e.g. plants and animals) universally have lower heat tolerance than simple organisms (e.g. bacteria), despite using the same basic biochemical building blocks (i.e. proteins and membranes). Moreover, complex organisms often die at temperatures lower than those that cause proteins and membranes to fall apart.

One explanation has gained a lot of traction in recent years: the oxygen-and capacity-limited thermal tolerance concept (what a mouth full!). This concept posits that as your body heats up, you need more oxygen; however, you eventually get so hot that you can’t get enough oxygen to survive.  There is growing evidence that oxygen limitation explains thermal tolerance for reptile eggs. Several studies show that when eggs are incubated in low oxygen conditions, their heat tolerance is lower (e.g. Smith et al., 2015); however, we still don’t know much about embryo metabolism at near-lethal temperatures, which would vastly improve our understanding of embryo heat tolerance.

In a recent study (Hall and Warner, 2020), we (I and Dr. Dan Warner, who was recently awarded the distinction of “Outstanding Mentor” by Auburn University – well deserved) sought to better understand the factors that determine heat tolerance of reptile embryos. We used eggs from our good friend, the brown anole (Anolis sagrei). Using 1-hour heat shocks, we measured the lethal temperature of embryos (~45.3 °C). We then monitored heart rate and metabolism of eggs across temperature, including near-lethal temperatures.

Figure 1. Heart rate of brown anole eggs across temperature.

As embryos approach the lethal temperature, heart rate and CO2 production increase (Figure 1), but oxygen consumption plateaus (Figure 2). Therefore, eggs need more and more energy as they heat up, but they are eventually unable to support their energy needs via aerobic respiration. Without enough oxygen, energy production is less efficient. These data indicate that oxygen is limited at near-lethal temperatures and provides additional support for the oxygen-and capacity-limited thermal tolerance concept for reptile eggs.

Figure 2. Oxygen consumption across temperature for brown anole eggs.

Many aspects of human-induced global change cause increases in temperature (e.g. deforestation, urbanization, climate change), potentially heating lizard nests and exposing embryos to thermal stress. The results of our study make progress toward understanding how embryos respond to extreme temperatures, which is important to understand how reptile populations will respond to global change.

Hall, J.M. and Warner, D.A., 2020. Thermal sensitivity of lizard embryos indicates a mismatch between oxygen supply and demand at near-lethal temperatures. Journal of Experimental Zoology, in press. https://doi.org/10.1002/jez.2359

Smith, C., Telemeco, R.S., Angilletta Jr, M.J. and VandenBrooks, J.M., 2015. Oxygen supply limits the heat tolerance of lizard embryos. Biology letters, 11(4), p.20150113.

Many factors contribute to colonization success in novel habitats. Anoles, as a group, are particularly adept at establishing in new areas. Urban ecosystems are no exception. A diversity of studies have sought to understand how adult anoles conquer human-modified habitats, but relatively little attention has been given to earlier life-stages (e.g., eggs). The brown anole (Anolis sagrei) and the crested anole (Anolis cristatellus) (Figure 1) are two species that have received considerable attention. Although these two species are quite similar in morphology and habitat preference (i.e. both trunk-ground anoles), work from Jason Kolbe’s lab (e.g. Battles and Kolbe 2019), shows that adults differ in thermal preference: A. sagrei prefers warmer, open-canopy habitats while A. cristatellus prefers cooler, closed-canopy habitats. Because temperatures are unusually high in urban areas (i.e. the urban heat island), the spread of A. cristatellus may be limited throughout the urban matrix compared to A. sagrei.

But what about eggs? Two recent studies suggest that A. sagrei nests reach warmer temperatures than those of A. cristatellus (Sanger et al., 2018, Tiatragul et al., 2019), thus, like adults, A. sagrei embryos may be more robust to high temperatures. Is it possible that thermal tolerance of embryos differ between these two species? If so, this may also help explain why A. sagrei has been much more successful at colonizing urban habitats where ground (and nest) temperatures are usually much warmer than in adjacent rural or natural areas (Tiatragul et al., 2017).

In a study recently published in the Journal of Experimental Biology (Hall and Warner 2019), we subjected eggs of A. sagrei and A. cristatellus to extreme fluctuations in temperature modeled from nests in urban environments. We found that A. sagrei embryos have a thermal tolerance approximately 2 degrees Celsius higher than A. cristatellus. This indicates that the thermal physiology of embryos is adapted to species-specific nest temperatures (though we discuss other possible explanations as well). Regardless, thermal tolerance differs widely between these two species, and this may help explain species-specific patterns of occupancy throughout the urban matrix.

As a side note (that I reluctantly removed from the manuscript during the review process – long sigh), although the Anolis radiation, which includes nearly 400 species, is considered a model system for studying adaptive radiation, to our knowledge, studies never consider that embryo phenotypes and egg survival may be an important driver of speciation. This is particularly important for two reasons. First, egg survival is a vital determinant of population cycles for these lizards (Andrews 1988), and likely plays a vital role in population viability, survival, and colonization success (Losos et al., 2003). Second, this adaptive radiation is characterized by many dispersal events, often from and to small islands throughout the Caribbean (Poe et al., 2018). Although key innovations, phenotypic plasticity, niche expansion and other processes are considered important in such dispersals, these processes are always evaluated from the perspective of adult phenotypes. Successful embryo development, however, is a requirement for persistence in every environment. Given that general protocols exist for embryo collection and analysis (Sanger et al., 2008a,b), we suggest this system is ripe for a relatively broad phylogenetic analysis of embryo physiology. Such work would illuminate the importance of embryo adaptation in colonizing novel environments (e.g. urban landscapes) and responding to environmental perturbances (e.g. climate change), and give us something to talk about other than dewlaps and limb lengths (which would make me happy).

Andrews, R.M., 1988. Demographic correlates of variable egg survival for a tropical lizard. Oecologia, 76(3), pp.376-382.

Battles, A.C. and Kolbe, J.J., 2019. Miami heat: Urban heat islands influence the thermal suitability of habitats for ectotherms. Global Change Biology, 25(2), pp.562-576.

Hall, J.M. and Warner, D.A., 2019. Thermal tolerance in the urban heat island: thermal sensitivity varies ontogenetically and differs between embryos of two sympatric ectotherms. Journal of Experimental Biology, 222(19), p.jeb210708.

Losos, J.B., Schoener, T.W. and Spiller, D.A., 2003. Effect of immersion in seawater on egg survival in the lizard Anolis sagrei. Oecologia, 137(3), pp.360-362.

Poe, S., de Oca, A.N.M., Torres-Carvajal, O., de Queiroz, K., Velasco, J.A., Truett, B., Gray, L.N., Ryan, M.J., Köhler, G., Ayala-Varela, F. and Latella, I., 2018. Comparative evolution of an archetypal adaptive radiation: innovation and opportunity in Anolis lizards. The American Naturalist, 191(6), pp.E185-E194.

Sanger, T.J., Hime, P.M., Johnson, M.A., Diani, J. and Losos, J.B., 2008a. Laboratory protocols for husbandry and embryo collection of Anolis lizards. Herpetological Review, 39(1), pp.58-63.

Sanger, T.J., Losos, J.B. and Gibson‐Brown, J.J., 2008b. A developmental staging series for the lizard genus Anolis: a new system for the integration of evolution, development, and ecology. Journal of Morphology, 269(2), pp.129-137.

Sanger, T.J., Kyrkos, J., Lachance, D.J., Czesny, B. and Stroud, J.T., 2018. The effects of thermal stress on the early development of the lizard Anolis sagrei. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 329(4-5), pp.244-251.

Tiatragul, S., Kurniawan, A., Kolbe, J.J. and Warner, D.A., 2017. Embryos of non-native anoles are robust to urban thermal environments. Journal of Thermal Biology, 65, pp.119-124.

Tiatragul, S., Hall, J.M., Pavlik, N.G. and Warner, D.A., 2019. Lizard nest environments differ between suburban and forest habitats. Biological Journal of the Linnean Society, 126(3), pp.392-403.

Despite the widespread use of anoles as model species for ecology, evolution, and behavior, we still have a relatively poor understanding of their nesting behavior and the factors that contribute to egg survival. This is unfortunate because past research demonstrates that egg survival can drive important measures of population demography (e.g. adult population density, Andrews 1982). Andrew DeSana, an undergrad from Seton Hill University, teamed up with the Warner lab to explore the possibility that marsh crabs (Armases cinereum) might serve as predators for brown anole eggs (Anolis sagrei). Because nest site selection by females may shield developing eggs from predators, he also wanted to know how several common nesting microhabitats might influence the depredation of eggs. He used both a lab and a field study to assess how the density of crab predators (no crabs, low crab density, and high crab density) and the nesting microhabitat of females (open sand, under palm fronds, and under leaf litter) might influence egg survival. He collected eggs from a breeding colony of anoles and placed them in these microhabitats in the presence of varying densities of crabs.

He found that marsh crabs readily prey upon anole eggs and that variation in egg survival was best explained by both crab density and the microhabitat where eggs develop. In both the lab and field study, eggs had the highest survival when placed under leaf litter and lower survival in the open and under palm fronds. Anecdotal data of crab behavior suggests that they don’t forage in the leaf litter and this may explain these results. Egg survival also decreased with increasing crab density. Thus, females nesting in leaf litter in habitats with low crab density would have greater reproductive success than those nesting in other microhabitats, especially if crab density is high. Future research will determine how the presence/absence of crabs influences female nesting behavior.

Andrews, R.M., 1982. Spatial variation in egg mortality of the lizard Anolis limifrons. Herpetologica, pp.165-171.

The effect of temperature on biological processes and systems is one of the most studied topics in ecology. Despite a wealth of existing research, we still have a relatively poor understanding of what factors contribute to the thermal tolerance of complex organisms. Much research suggests that oxygen limitation at extreme temperatures is what determines the thermal limits of complex aquatic life; however, this hypothesis (i.e. Oxygen-and capacity-limitation of thermal tolerance; Pörtner 2010) has not proven very useful in explaining the thermal limits of terrestrial organisms. One reason is that there is a comparatively greater amount of oxygen in air vs water. Moreover, terrestrial organisms tend to have very efficient systems of ventilation (e.g., air sacs of birds and tracheal system of insects). Terrestrial vertebrate embryos, however, rely solely on diffusion of oxygen through a hard shell and, thus, their thermal limits may be set by oxygen limitation (Smith et al. 2015).

Sylvia Nunez and Thom Sanger set out to determine the relationship between oxygen availability and temperature for brown anole (Anolis sagrei) embryos. Previous work shows that thermal stress can induce embryo mortality and severe craniofacial malformations at incubation temperatures above 33 °C (Sanger et al. 2018). They used a factorial design (2 incubation temperatures: 27 °C and 33 °C; and 2 oxygen treatments: 10% O₂ and 21% O₂) and dissected eggs at day 14 or day 20 after oviposition to measure the effects of these treatments during morphogenesis and the growth phase of development, respectively. They found that hypoxia did not lower survival during these periods at 27 °C; however, survival was reduced for embryos incubated at 33 °C and under hypoxic conditions (i.e. 10% O₂). Furthermore, high temperatures and low oxygen resulted in various craniofacial malformations and increased incidences of cerebral blood-pooling. It appears that oxygen supply may limit the thermal tolerance of anole embryos, and these data support the findings of previous work in other lizard species (Smith et al. 2015). The next steps for the Sanger lab are to determine the cellular mechanisms that drive the results discovered in their current study.

Pörtner, H.O., 2010. Oxygen-and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. Journal of Experimental Biology, 213: 881-893.

Sanger, T.J., Kyrkos, J., Lachance, D.J., Czesny, B. and Stroud, J.T., 2018. The effects of thermal stress on the early development of the lizard Anolis sagrei. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 329:244-251.

Smith, C., Telemeco, R.S., Angilletta, M.J. and VandenBrooks, J.M., 2015. Oxygen supply limits the heat tolerance of lizard embryos. Biology letters, 11:20150113.

Female Festive Anole (photo: Ambika Kamath)

The evolution of reproductive strategies is an interplay between phylogenetic constraints (i.e. restrictions determined by the evolutionary history of that organism) and local conditions. Organisms adapt their reproductive physiology to their environment in ways that maximize fitness; however, this occurs within the context of evolutionary history (e.g. income vs. capital breeders).  When environments are seasonal, selection favors individuals that align changes in key reproductive traits (e.g., egg size, clutch size) with seasonal shifts in habitat quality. For example, some species of aphids switch from asexual to sexual reproduction in the fall of each year because offspring produced via sexual reproduction (i.e. genetic recombination) are more likely to survive the winter. Seasonal shifts in reproduction have been observed in a variety of taxa (e.g. birds, mammals, frogs, lizards, spiders).

In two previous papers, the Warner Lab demonstrated that brown anoles (Genus-pending sagrei) in Florida, exhibit seasonal shifts in reproduction (Mitchell et al 2018; Pearson & Warner 2018): females shifts from producing many, relatively small offspring early in the year to producing fewer, relatively large offspring late in the year. Pearson and Warner (2018) also demonstrated that anoles that hatch early in the season (March – May) are more likely to survive through winter than those that hatch later (July-August). Thus, the observed shift in reproduction appears to be an evolved response to the seasonal decline in offspring habitat. This shift in reproduction, however, may depend on environmental factors that are also subject to temporal changes (e.g., food abundance).

In a new paper (Hall et al 2018) published in Physiological and Biochemical Zoology, we demonstrate how prey abundance modifies seasonal changes in key reproductive traits for brown anoles. We bred lizards in controlled laboratory conditions across the length of a full reproductive season and manipulated the availability of food by providing some breeding pairs high prey availability and some low. Halfway through the season, we switched half of the breeding pairs to the opposite treatment. We measured growth of male and female lizards as well as latency to oviposit, fecundity, egg size, egg content (yolk, water, shell mass), and egg quality (steroid hormones, yolk caloric content) over this period.

Higher prey availability enhanced lizard growth and some key reproductive traits (egg size, fecundity), but not others (egg content and quality). Notably, egg quality seems unaffected by diet. This is probably because there is some minimum provisioning that is necessary to produce a viable egg. Thus, females on a low-calorie diet will sacrifice the number of eggs produced and not the quality; however, increased food supply will be primarily used to increase fecundity.

We also found that seasonal patterns of reproduction were modified by prey treatment in ways that have consequences for offspring survival (Fig 1). When prey was abundant, egg production peaked relatively early in the season and egg size increased through time; however, when prey availability was low, egg production was chronically low and egg size declined through time. Late-produced offspring are at a disadvantage because they have to compete with larger, established offspring that hatched earlier in the year. A low calorie diet prevents females from providing late-season offspring with the extra provisions they need to compensate for hatching late.

Figure 1. Egg production of brown anoles provided with a) continuously high prey availability; b) high prey availability switched to low; c) continuously low prey availability; d) low prey availability switched to high. The vertical dotted line represents the point in the experiment when the diets were changed for groups b and d.

Figure 2. The relationships between latency to oviposit and a) prey treatment and b) pre-season body condition. Dark bar/circles show the high prey treatment and white bar/circles show the low prey treatment.

Not shockingly, when the diets were switched halfway through the season (high prey switched to low or the reverse; Fig 1) females responded immediately to the new diet. This would suggest that anoles are primarily income breeders that utilize energy intake to fuel reproduction. However, we know that income and capital breeding is a continuum and many reptiles utilize both for reproduction. We also found that females with a relatively high body condition at the beginning of the experiment start laying eggs earlier than those with poorer body condition (Fig 2). These measures of condition were taken prior to the onset of reproduction and couldn’t have been confounded by the presence/absence of oviductal eggs. Like many reptiles, pre-season body condition (i.e., fat reserves) may play an important role in the initiation of reproduction (vitellogenesis) for anoles; however, once reproduction starts, income is likely the primary determinant of fecundity.

Our results demonstrate that seasonal changes in anole reproduction are dependent on fluctuations in local environmental conditions.

Anyone who has spent a considerable amount of time catching anoles in the field has seen their fair share of injured animals. Many species we commonly study (e.g. brown anoles) are just the perfect size to be a snack for any hungry predator (and even humans! see this). Several previous posts have documented adult anoles that have sustained severe injuries (limb loss – see my previous post) and survived. But can these animals thrive with such injuries or do they just limp along through life?

Here I add to this string of anecdotes with a unique datum. This female Puerto Rican crested anole was caught by none other than James Stroud and Chris Thawley at Fairchild Botanical Gardens just this week. She is missing the rear right foot (not an unusual injury). What is new here is that I dissected this female as part of a study conducted by James, Chris, and myself, and I can report that this female, despite her handicap, is not only alive but seems to be thriving. Compared to a cohort of females captured at the same time and place (n= 13), she has greater body condition and fat mass than most of her cohort (Figure 1) and is reproductive at stage 4 (Gorman & Licht 1974). For those unfamiliar, stage 4 means that she has two developing eggs (1 in each oviduct). The mean stage for the cohort is 2.92, and, thus, her reproductive stage is more advanced than the majority of the cohort (only 2 of 13 individuals at stage 4).

Figure 1. Fat mass and residual body condition (log(mass) x log(SVL)) for the entire cohort (gray circles) and the injured female (black circle). There was no relationship between SVL and fat mass for females in this cohort so fat mass is not corrected for body size.

Cox and Calsbeek (2010) demonstrated that gravid anoles have reduced locomotor performance and lower survival than non-reproductive females. However, this female, despite the use of only 3 good limbs, has clearly been able to procure sufficient resources to  fuel reproduction and retain a level of fat reserves above most individuals in her population.  For this reason, we denote her ‘supermom’ and concede the possibility that missing a foot or limb may not severely reduce fitness for some individuals.

Cox, R.M. and Calsbeek, R., 2010. SEVERE COSTS OF REPRODUCTION PERSIST IN ANOLIS LIZARDS DESPITE THE EVOLUTION OF A SINGLE‐EGG CLUTCH. Evolution, 64(5), pp.1321-1330.

Gorman, G.C. and Licht, P., 1974. Seasonality in ovarian cycles among tropical Anolis lizards. Ecology, 55(2), pp.360-369.

Baby brown anole

The timing of reproduction strongly influences reproductive success in many organisms. There is a fitness benefit for individuals who can align their reproductive bouts with conditions that positively influence both reproduction and survival of offspring. For species with extended reproductive seasons, like anoles, the quality of the environment often changes throughout the season in ways that impact offspring survival, and, accordingly, aspects of reproductive strategies may shift to maximize fitness. The Warner Lab has now conducted multiple studies of brown anoles (many unpublished, but see Pearson & Warner 2018) that demonstrate that early-produced offspring have a survival advantage over late-produced offspring. This is likely because individuals that hatch late in the reproductive season must compete with older, larger conspecifics and have less time to grow prior to the cool, dry winter months. Life-history theory predicts that when the offspring environment deteriorates through the season, selection should favor females that shift from producing more, smaller offspring early in the season to fewer, better provisioned offspring later in the season. In our recent paper, Tim Mitchell, Dan Warner, and I quantify seasonal changes in reproduction of brown anoles to determine if females seasonally alter their investment in offspring size vs number.

Figure 1. Differences in key reproductive traits between seasonal cohorts (1 – early; 2- mid; 3-late) of female brown anoles

We captured early, mid-, and late-season cohorts of breeding females and bred them in the lab while controlling proximate environmental variables that influence reproduction (e.g. food, temperature, humidity). These breeding colonies varied only by the capture date of the adult animals from the field. We measured  key reproductive traits for each female (fecundity, egg size, egg quality, inter-clutch interval). Our cohorts exhibited variation in key reproductive traits  consistent with seasonal shifts in reproductive effort (Figure 1). Overall, reproductive effort was highest early in the season due to a relatively high rate of egg production. Later season cohorts produced fewer, but larger, offspring. We infer that these results indicate a strategy for differential allocation of resources through the season. Females maximize offspring quantity when environments are favorable (early season), and maximize offspring quality when environments are poor for those offspring (late season). Despite the extra effort allocated to late-produced offspring, early-produced offspring have a significant survival advantage (Pearson & Warner 2018).

Several future directions are worth serious consideration: first, nearly all studies of anole reproduction in the field demonstrate that reproduction is somewhat seasonal. It is quite reasonable to assume that seasonal shifts in offspring size versus number are prevalent throughout the anole radiation. At this point, we simply don’t know (maybe because we have too many people studying male anoles and too few people studying female anoles – just kidding – but seriously – we’re recruiting!). Second, given the major differences in life-history between mainland and island species (e.g., lifespan, time to maturity), seasonal shifts in reproductive allocation likely differ between these groups as well. A robust assessment of how the mainland-island hypothesis (Andrews 1979) applies to reproductive allocation won’t be possible until we have more basic data on reproduction for many species – let’s get busy folks!

Andrews, R. M. (1979) Evolution of life histories: A comparison of Anolis lizards from matched islands and mainland habitat. Breviora, 454, 1–51.

Mitchell, T.S., Hall, J.M. and Warner, D.A., 2018. Female investment in offspring size and number shifts seasonally in a lizard with single-egg clutches. Evolutionary Ecology, pp.1-15.

Pearson, P.R. and Warner, D.A., 2018. Early hatching enhances survival despite beneficial phenotypic effects of late-season developmental environments. Proc. R. Soc. B, 285 (1874), p.20180256.