Tuesday, May 12, 2015

Expect the Unexpected, When Measuring Selection in the Wild

[post by Kayla Hardwick, postdoctoral researcher at Reed College]

Imagine a vast expanse of white sand dunes, stretching as far as the eye can see. Now imagine two lizards, one white and one brown, on the white sand. Which do you think would survive better?

Trait divergence between populations can arise via natural selection when particular traits are adaptive in some environments but not others. In the above example, it seems likely that white color would be adaptive in the context of the white sand environment, because the white lizard could more readily blend in with its pale surroundings to avoid predation. But while intuitive explanations for trait divergence often seem straightforward, they can be difficult to test in natural populations. Accurately measuring selection in the wild is challenging, and consequently experimental evidence of the adaptive significance of specific traits is lacking in most systems.

As a graduate student in the early stages of my PhD, I knew (in theory) that measuring selection in natural populations was hard. I had read Endler’s Natural Selection in the Wild, and I was well versed in the common pitfalls of selection studies. But as I read more and more of the published literature on the topic, it seemed to me that measuring selection in the wild typically worked more or less as expected – that most of the time, the authors seemed to confirm their initial predictions without too much trouble. As I embarked on my first large-scale field experiment, with the goal of measuring selection on body color in White Sands lizards, I couldn’t help but think to myself: “how hard can this really be?”

White lizards in White Sands 

White Sands is a gypsum dune field that formed within the Chihuahuan Desert in New Mexico less than 10,000 years ago. The sparsely vegetated gypsum dunes of White Sands contrast strikingly with the surrounding desert scrubland habitat, characterized by dense vegetation and dark brown soil.

White Sands National Monument, New Mexico (photo courtesy of Scott Hardwick)
Three lizard species have colonized White Sands from the surrounding environment: The Lesser Earless Lizard (Holbrookia maculata), the Eastern Fence Lizard (Sceloporus undulatus), and the Little Striped Whiptail (Aspidoscelis inornata). In all three species, lizards in White Sands exhibit blanched dorsal color, while lizards outside of White Sands exhibit darker dorsal color. Blanched color is typically thought to be an adaptation to avoid predation by visually oriented birds that occur at White Sands, such as the Loggerhead Shrike (Lanius ludovicianus) and the Greater Roadrunner (Geococcyx californianus).

Lesser Earless Lizards from White Sands (left) and non-White Sands (right) populations (photo courtesy of Simone Des Roches)
Eastern Fence Lizards from White Sands (left) and non-White Sands (right) populations (photo courtesy of Simone Des Roches)
Little Striped Whiptails from White Sands (left) and non-White Sands (right) populations (photo courtesy of Simone Des Roches)
We have a number of compelling reasons to think that there has been selection on body color in the White Sands habitat. First, color divergence has occurred despite ongoing gene flow between populations on and off White Sands in all three lizard species. In addition, previous research has documented increased genetic divergence between populations at Mc1r, a gene involved in generating the blanched color phenotype. Finally, convergent evolution of blanched color has occurred among disparate taxa at White Sands, including the Apache pocket mouse (Perognathus gypsi), Couch’s Spadefoot Toad (Scaphiopus couchii), and the White Sands camel cricket (Daihinoides hastiferum).

The White Sands camel cricket (photo courtesy of Scott Hardwick)
Given these lines of evidence, the most intuitive explanation for white lizards in White Sands is natural selection for cryptic coloration. We thus set out to answer a seemingly simple question: does blanched color make White Sands lizards less susceptible to bird predation?

Painting lizards, for science!

To study the effect of color on survival, we conducted an enclosure experiment with substrate-matched and substrate-mismatched Earless Lizards in the White Sands habitat.

Including both matched and mismatched lizards in our study required some creativity – in the “arts and crafts” sense – as we used human temporary-tattoo paint to manipulate the body color of White Sands Earless Lizards. During graduate school, students seem to acquire at least one skill that is totally bizarre and has no practical applications in real life. For me, lizard painting is that skill. We painted lizards to be either substrate-matched or substrate-mismatched, mirroring the colors of the naturally occurring Earless Lizard color morphs as closely as possible.

Painted, substrate-matched and substrate mismatched Earless Lizards (top panel), and naturally occurring Earless Lizard color morphs (bottom panel)
We built enclosures within the White Sands habitat using steel flashing and rebar, which turned out to be a uniquely challenging experience. Most locations in White Sands are not accessible by car, which meant we spent a good deal of time carrying heavy equipment up and down sand dunes. The thing about sand dunes is, they tend to collapse beneath your feet as you walk. Thus it was not uncommon to find yourself, with a 50lb roll of flashing under your arm, taking dozens of steps up the side of a sand dune without actually going anywhere. This particularly frustrating phenomenon came to be known among my field assistants as “Nature’s Stairmaster.”

But despite these (and other) difficulties, we ultimately managed to build a total of four enclosures in White Sands, each measuring 10 meters by 10 meters. We divided each enclosure in half and left one half uncovered to allow avian predators to enter and exit freely (the “open” treatment). We covered the other half of each enclosure with chicken wire to exclude predators (the “closed” treatment). We then released painted, substrate-matched and substrate-mismatched individuals into the enclosures. We recaptured surviving lizards after two weeks.

The “open” enclosure treatment allowed bird predators to enter and exit freely
The “closed” enclosure treatment was covered with chicken wire to exclude predators
So, what do you think happened when we put substrate-matched and substrate-mismatched lizards into enclosures, and left them vulnerable to predation? I’ll give you a hint: it’s definitely not what you’re expecting!

Result #1: Lizards in White Sands get eaten by birds

Okay, so maybe you were expecting this particular result. We found that lizard survival differed significantly between the open and closed enclosure treatments. 100% of lizards in the closed treatment survived trials, compared with just over a third of lizards surviving in open treatment replicates. This indicates that lizards in our trials experienced predation. Birds were most likely responsible for lizard predation in our study; we observed Loggerhead Shrikes perched near enclosures on a number of occasions, and found Greater Roadrunner tracks inside enclosures multiple times throughout the trials.

Result #2: Lizards get eaten, no matter what color they are

Despite high rates of predation within the open enclosure treatment, we did not detect selection on color in our study. In other words, there was not a significant difference in survival between substrate-matched and substrate-mismatched lizards. This result is unexpected, given that previous studies offer strong evidence that blanched color is adaptive in White Sands. In a system amenable to experimental manipulation where divergence is literally in black and white, why did we fail to detect selection on this putatively adaptive trait?

Maybe there isn’t selection on color at White Sands

It is possible that there is not currently selection on body color in White Sands lizards. Selection studies can be complicated by differences between historical and contemporary selection pressures, where traits that originate as adaptations can remain widespread in a population even after shifts in the ecology of the system render those traits unnecessary for survival. The distributions of key predators in the White Sands system, such as the Loggerhead Shrike, have shifted dramatically in recent decades. It is thus possible that blanched color at White Sands, even if it was historically important for survival, could represent a “ghost of selection past” today.

Then again, maybe there is selection, and we just didn’t detect it

Previous research has given us reason to believe that selection on color is ongoing in White Sands lizards. If there is indeed contemporary selection, are there any compelling explanations for why we might not have detected it in our study?

Variable selection can complicate studies of the adaptive significance of specific traits. Previous research indicates that selection pressures on particular phenotypic traits can vary between sexes and among life stages, as well as over space and time. When selection is variable, it can be difficult to discern the adaptive significance of specific traits because the outcome of a study may depend on the study’s scope. For example, if selection varies among different life stages, measurements of the strength and direction of selection may be contingent on the number of individuals of each life stage included in a study.

In our experiment, there was a hint that selection on color varies between males and females. Specifically, substrate-matched males tended to survive better than substrate-mismatched males, while in females the opposite was true. This could indicate that substrate matching is more important for survival for males than females in Earless Lizards, as males are typically more behaviorally conspicuous than females in iguanid lizards.

Our results also indicate that selection on color is variable over space and time. Both year and enclosure location affected survivorship in our study, where survival was higher in some enclosures than others, and this pattern changed from year to year. Spatial and temporal fluctuations in predation in our study could have been related to the physical proximity of enclosures to suitable perches/nest sites, seasonal changes in predator behavior, and any number of other biotic/abiotic aspects of the environment.

Variable selection can also interact with issues related to statistical power, increasing the number of individuals required to discern the adaptive value of specific traits. For instance, the sample size required to detect a significant effect of color would have been substantially smaller had we focused exclusively on males (thereby avoiding the effect of sex-specific variations in selection), and smaller still if selection against substrate-mismatched individuals had remained consistent throughout replicates (thereby avoiding the effects of spatial and temporal variation). White Sands is a small, isolated population, and variable selection thus presents a significant obstacle for future studies of selection in this system.

Finally, it can be difficult to replicate natural conditions in an experimental context, and deviations from natural conditions could have affected the results of our study. In particular, the density of lizards within our enclosures was substantially higher than that at which Earless Lizards naturally occur in White Sands. It is possible that we observed a "buffet effect" in our study, where predators were initially attracted to the enclosures by substrate-mismatched lizards, but subsequently proceeded to consume lizards of both paint treatments. It is also possible that the high density of conspecifics caused lizards to behave more conspicuously that they otherwise typically would, further attracting the attention of predators to enclosures.

Painted lizards in an enclosure trial (photo courtesy of Susan Kologi)

So, what’s the verdict?

There are a number of compelling potential explanations for the results of our study. The bottom line is, we don’t yet know exactly what’s going on in terms of selection on color in White Sands. Future research will focus on figuring out why substrate matching might affect males and females differently, identifying factors that contribute to spatial and temporal variability in predation, and looking into the importance of selection on correlated traits (including the thermoregulatory effects of blanched color). Read our recently published manuscript for a more detailed discussion of our findings, and check out the Rosenblum Lab website to learn more about the evolution and ecology of White Sands lizards.

Challenges such as those discussed above are quite common during studies of selection in the wild, and their causes and consequences can be difficult to predict. And yet, many published selection studies seem to minimize (or completely ignore) the effects of such challenges, perhaps because those that freely discuss possible methodological shortcomings are less likely to be accepted for publication. This potential publication bias is important to acknowledge, because researchers do not yet have a comprehensive understanding of how different challenges manifest themselves in terms of the results of a study. Publishing the results of rigorous experiments, even when they do not turn out exactly as planned, is absolutely crucial for facilitating future research in the field.

Measuring selection in the wild is a challenging, complex, and incredibly important endeavor. The results of our study indicate that, despite the difficulties detailed above, researchers often learn fascinating things about selection when experiments have unexpected outcomes.

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