An individual’s early-life environment and phenotype often influence its traits and performance as an adult. We investigated whether such ‘carryover effects’ are associated with alternative, environmentally induced phenotypes (‘polyphenism’), and, if so, whether they influence polyphenism’s evolution. To do so, we studied spadefoot toads, Spea multiplicata, which have evolved a polyphenism consisting of two, dramatically different forms: a carnivore morph and an omnivore morph. We sampled both morphs from a fast-drying and a slow-drying pond and reared them to sexual maturity. Larval environment (pond) strongly influenced survival as well as age and size at metamorphosis and sexual maturity; i.e., environment-dependent carryover effects were present. By contrast, larval phenotype (morph) did not affect life-history traits at sexual maturity; i.e., phenotype-dependent carryover effects were absent. These results are consistent with theory, which suggests that by amplifying selective trade-offs in heterogenous environments, environment-dependent carryover effects might foster polyphenism’s evolution. At the same time, by freeing selection to refine a novel phenotype without altering the existing form, the absence of phenotype-dependent carryover effects might enable polyphenism to evolve in the first place. Generally, carryover effects might play an underappreciated role in polyphenism’s evolution.

We sampled 100 late-stage carnivore and 100 late-stage omnivore tadpoles from two temporary ponds in the San Simon Valley near Portal, Arizona: ‘Horseshoe’ Pond (31.9389, -109.0864; Fig. 2b) and ‘PO2-N’ Pond (31.9142, -109.0836). Immediately upon arrival in our lab at UNC, tadpoles were housed in groups of twelve and fed both live brine shrimp and crushed fish food ad libitum. Different morphs and populations were reared separately. When we observed a fore limb protruding from a tadpole [defined as metamorphosis; Gosner (1960) stage 42], that individual was immediately moved to a container filled 2-cm deep with water and 5-cm deep with a sandy shore on one end. Once its tail was reabsorbed [Gosner (1960) stage 46], each individual was transferred to a small cage lined with moist paper towels. These individuals were housed in groups of three and fed small, live crickets (dusted with vitamins and calcium powder) ad libitum every other day. After six weeks, these individuals were transferred (again, in groups of three) to larger terraria (11.9 x 7.8 x 8.1 inches) with moistened sand as substrate. Twice a week, any individuals on the surface were fed crickets ad libitum. Every other week, any buried individuals were dug up and fed crickets. Terraria were assigned to randomized locations on shelves and kept in the same room at 23^⚬ C, 40% humidity, and with lights on a reversed cycle from 2000h to 0800h.  Sand was changed every two months. 

We measured individuals at two life stages: 1) the larval/juvenile stage (which terminates at metamorphosis), and 2) the adult stage. Specifically, we measured each individual’s size and age at metamorphosis and at sexual maturity. The former captured any responses to the larval (aquatic) environment, whereas the latter captured any responses to the adult (terrestrial) environment. By comparing size and age at these two life stages, we tested for carryover effects both across and within larval morphs and environments.

For age at metamorphosis, we recorded the age (starting from hatching; all tadpoles hatched on July 4; see above) at which an individual’s tail was completely reabsorbed [Gosner (1960) stage 46].  For age at sexual maturity, we recorded the age (again, starting from hatching) at which an individual first expressed sexual traits: nuptial pads in males and eggs in females. For size, we recorded the individual’s snout-vent length (SVL) at metamorphosis and again at maturity. Finally, we also recorded each individual’s survival to 400 days old (the age when most individuals had achieved sexual maturity).

We assessed whether body size at metamorphosis (natural-log transformed to improve normality) was influenced by pond, morph, or their interaction with a general linear model with the lm function from the stats library (R Development Core Team, 2018). To determine if size at sexually maturity was affected by pond, morph, sex, or their two-way interactions, we again fit a general linear model (body size was not log transformed because it was normally distributed). We then evaluated whether age at metamorphosis was influenced by either of these same two factors or their interaction by using a generalized linear model with a negative binomial distribution to account for overdispersion using the glm.nb, function from the Mass library (Venables & Ripley, 2002). Using a general linear model (the response variable was normally distributed) we then evaluated if age at sexual maturity was influenced by pond, morph, sex or their two-way interactions. For our models of both age and body size we also tested, post-hoc, for a difference between morphs within each pond using the emmeans function from the eponymous library (Lenth, 2019). To directly compare the magnitude of differences across ontogeny, where our sample sizes differed to due mortality, we additionally calculated standardized effect sizes (calculated as the pairwise difference divided by the pooled variance) and confidence intervals for these comparisons using the eff size function in the emmeans library. Finally, we looked at how survival throughout the period observed (400 days) was influenced by pond or morph. We assessed survivorship by a parametric survival analysis following a Weibull distribution with natal pond and larval morph and their interaction as factors (Therneau, 2015). All analyses were carried out in R version 3.5.2.

These data files contain Age and Size at Metamorphosis and at Sexual Maturity as well as Survival at Sexual Maturity of Known Carnivores and Omnivores from Two Natural Ponds.