Transgenerational phenotypic plasticity links breeding

Ecology, 95(10), 2014, pp. 2715–2722 Ó 2014 by the Ecological Society of America

Transgenerational phenotypic plasticity links breeding phenology with offspring life-history ALEX RICHTER-BOIX,1 GERMAŃ ORIZAOLA,

AND

ANSSI LAURILA

Animal Ecology, Department of Ecology and Genetics, Evolutionary Biological Centre, Uppsala University, Norbyva¨gen 18D, SE-75236 Uppsala, Sweden

Abstract. The timing of seasonal life-history events is assumed to evolve to synchronize life cycles with the availability of resources. Temporal variation in breeding time can have severe fitness consequences for the offspring, but the interplay between adult reproductive decisions and offspring phenotypes remains poorly understood. Transgenerational plasticity (TGP) is a potential mechanism allowing rapid responses to environmental change. Here, we investigated if experimentally delayed breeding induces TGP in larval life-history traits in the moor frog (Rana arvalis). We found clear evidence of TGP in response to changes in breeding phenology: delayed breeding increased offspring development and growth rates in the absence of external cues. This constitutes the first unequivocal evidence for TGP in response to changes in breeding phenology in vertebrates. TGP can play an important role in adjusting offspring life-history strategies to the environment they are most likely to encounter, and may constitute an important mechanism for coping with climate change. Key words: climate change; complex life-cycles; life-history strategies; metamorphosis; seasonal clocks; timing of reproduction.

INTRODUCTION Synchronization of periodic life-history events (phenology) with the external environment is critical to ensure adequate conditions for development and growth for organisms living in seasonal environments (Bradshaw and Holzapfel 2007, Ko¨rner and Basler 2010). Consequently, the timing of life-history events is expected to be under strong selection to allow organisms to develop, grow or reproduce and consequently maximize fitness (Forrest and Miller-Rushing 2010, van Asch et al. 2013). Over the last decades, many species have experienced changes in breeding phenology due to climate change (Visser et al. 2004). Variation in breeding phenology can lead to a destabilization of an organism’s life cycle by altering the synchronization between offspring and their food resources, which may have fitness consequences for the offspring (Bradshaw and Holzapfel 2010, Visser et al. 2012). As a result, there is an increasing need to consider the timing of lifehistory events as a crucial fitness trait (Bradshaw and Holzapfel 2010, Forrest and Miller-Rushing 2010). However, the interplay between the reproductive timing of the parents and offspring phenotypes remains poorly understood. Ecological studies integrating life-history theory and phenology will help forecast the effects of environmental change on life-history and understand Manuscript received 24 October 2013; revised 26 March 2014; accepted 16 April 2014. Corresponding Editor: M. C. Urban. 1 E-mail: [email protected]

whether the observed changes are adaptive (Forrest and Miller-Rushing 2010). Transgenerational plasticity (TGP) has been recently highlighted as a potential mechanism allowing rapid adaptive responses to environmental change (Herman and Sultan 2011, Salinas and Munch 2012, Sheriff and Love 2013). In TGP, parents precondition their offspring to a given environment, producing significant changes in offspring reaction norms (Fox and Mousseau 1998). Adaptive TGP is predicted to evolve when the parental environment reliably predicts the offspring environment (Herman and Sultan 2011, Galloway and Burgess 2012). In animals, examples of TGP include responses to ecological factors, such as predation risk, temperature, and food availability (Hafer et al. 2011). For example, high predation risk in the maternal environment can lead to the production of better defended offspring (Agrawal et al. 1999, Mondor et al. 2004), changes in temperature may lead to transgenerational acclimation of growth, swimming, and foraging performance (Donelson et al. 2012, Salinas and Munch 2012), and variation in food availability can modify offspring life-history, including age at maturity and reproductive success (Hafer et al. 2011). In vertebrates, TGP is often induced by maternal hormones transmitted to offspring before birth (Meylan et al. 2012). Synchronization with the external environment is crucial for successful timing of the life-history stages; thus, TGP linking breeding phenology of the parents and offspring life-histories is expected to be highly advantageous and positively selected in nature. In general, the shorter the favorable growing season is,

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the more important it becomes to adjust the timing of reproduction so that the offspring are able to cope with or avoid unfavorable growing conditions later in the season (Saikkonen et al. 2012). Here, transgenerational preconditioning of offspring with seasonal information would allow precise seasonal timekeeping to the offspring even in the absence of reliable external cues like photoperiod or temperature. Transgenerational association between maternal flowering time and offspring life-history schedule has been demonstrated in plants (Herman and Sultan 2011). Similarly, many studies have shown that individual growth and development respond to direct seasonal cues (e.g., photoperiod; Bradshaw and Holzapfel 2007). Studies on insects have also demonstrated the effects of photoperiod on the maternal control of diapause under laboratory conditions (see review in Mousseau and Dingle 1991). However, to our knowledge there are no studies in vertebrates showing how maternal time perception affects offspring life-history when no seasonal cues are available for offspring. In temperate organisms with complex life cycles, such as amphibians, the timing of ontogenetic niche shifts (i.e., hatching, metamorphosis) is often constrained by season length (Wilbur and Collins 1973, Rowe and Ludwig 1991). In general, higher developmental and growth rates are expected when individuals face more stringent time constraints, such as in temporal ponds or at high altitude/latitude. In amphibians, breeding phenology and embryonic development are strongly influenced by environmental conditions, leading to pronounced year-to-year variation in the duration of the growth season (Beebee 1995, Phillimore et al. 2010). Accurate seasonal adjustment of parental and larval lifehistory decisions is likely to have high fitness value. Delayed reproduction can result in later metamorphosis, preventing juveniles from accumulating the necessary resources for hibernation and reducing overwintering success (Altwegg and Reyer 2003). However, while plastic responses of amphibian larvae to varying ecological conditions have been extensively studied, the link between breeding phenology and offspring lifehistory remains unexplored (but see Orizaola et al. 2013). In this study, we experimentally delayed breeding time of the moor frog Rana arvalis to examine potential TGP in larval growth and development rates in response to increased time constraints. We also delayed hatching time in both treatments in order to investigate the relationship between TGP and offspring plastic response to altered phenology per se. This experiment was conducted in the absence of seasonal cues ( photoperiod and temperature) that could inform tadpoles of the strength of time constraints. Hence, a shorter larval period and/or higher growth rate in response to delayed breeding would indicate adaptive TGP in the form of seasonal preconditioning of the offspring by the mother.

Ecology, Vol. 95, No. 10

MATERIALS

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METHODS

R. arvalis (see Plate 1) is a widespread Palearctic anuran, occurring in a broad range of habitats from western Europe to eastern Siberia. Our study animals originated from two populations located ;30 km southwest of Uppsala, Central Sweden (Pond 1, 59845 0 N, 17802 0 E; Pond 2, 59851 0 N, 17828 0 E). Adult R. arvalis migrate to breeding ponds in early spring and females lay one clutch per year directly in water. The species is an explosive breeder with a very short breeding period: the average pre-reproductive period (i.e., calling males present but egg-laying not started) was in our study area 2.3 days, and the average duration of egg deposition period was 3.6 days (A. Richter-Boix and G. Orizaola, personal observation). In nature, R. arvalis larvae hatch after ;2–3 weeks, and develop in water until metamorphosis, which occurs after 2–3 months. Timing of breeding is highly dependent on the climatic conditions during winter and spring, and cold temperature delays breeding and prolongs embryonic development in R. arvalis, as well as in high-latitude amphibians in general (Phillimore et al. 2010, Orizaola et al. 2013). Experimental design and laboratory conditions We collected 28 breeding pairs of R. arvalis at the two ponds (18 and 24 April 2010, 18 and 10 pairs, respectively). Frogs were transported to the laboratory at Uppsala University, where they were kept in plastic boxes filled with moist moss and stored in a dark climate-controlled room at 48C until they were used in artificial crosses. The experiment included two phenology treatments: (1) delayed adult breeding time and (2) delayed offspring hatching by exposing embryos to low temperature. In the first step, 14 pairs (nine and five pairs per population) were haphazardly selected and artificially crossed ;12 hours after collection. The other 14 pairs were kept in the cold room (48C) and crossed after 12 days, simulating a breeding time delay associated with a cold spell (hereafter, breeding delay). Adult survival through the cold treatment was 100%. For artificial crosses, males and females were first anesthetized with MS222 (Sigma-Aldrich, Saint Louis, Missouri, USA), weighed, and measured. The testes were then removed and crushed in 10% amphibian ringer solution to obtain a sperm solution for each male. Eggs from each female were stripped to two plastic vials containing amphibian ringer solution, and sprinkled with the sperm solution from one male resulting in full sibling families. Body length for males and females was measured in order to study the relationship between adult size, egg size, and hatchling size. To obtain an estimate of the egg size of individual females, 60–80 eggs/female were photographed three hours after fertilization and egg size was measured as egg diameter (mm) using the program ImageJ (Rasband 2014). Twelve hours after fertilization, each clutch was divided into four 5 L plastic vials with reconstituted

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soft water (see Orizaola et al. [2010] for details). Two vials from each family were maintained in a climatecontrolled room at constant 168C, whereas the other two were transferred to a 48C and 16 h light : 8 h dark photoperiod climate-controlled room for six days, after which they were transferred back to the 168C and 16 h light : 8 h dark room. The low-temperature treatment delays hatching by stopping embryonic development (see Orizaola et al. [2010] for similar procedures), and was intended to mimic a period of cold weather shortly after laying (hereafter, hatching delay). A constant 48C temperature is similar to those experienced by frogs in nature during the breeding season (see Appendix A for thermal profile of the collection sites during the study). Throughout the experiment, the embryos and larvae were kept in the same constant 16 h light : 8 h dark photoperiod. We used reconstituted soft water throughout the study to assure homogeneous water quality. The experiment was a 2 3 2 factorial design with four treatments: reproduction and hatching at normal time (hereafter, NN), reproduction at normal time and delayed hatching (ND), delayed reproduction and normal hatching (DN), and delayed reproduction and delayed hatching (DD). In total, there were 14 families 3 2 reproductive phenology treatments (note that each family is represented in only one phenology treatment) 3 2 hatching phenology treatments 3 12 individually raised tadpoles per treatment combination, resulting in 672 experimental units. The larval part of the experiment was carried out in a 198C (6 0.3 SD) climate-controlled room. When larvae reached Gosner stage 25 (complete absorption of gills and active feeding [Gosner 1960]), 12 tadpoles from each treatment combination were haphazardly selected, photographed and raised individually until metamorphosis in 0.75-L opaque plastic vials. Water was changed twice a week, and the tadpoles were fed with chopped and lightly boiled spinach in conjunction with water change. During the first week, tadpoles were fed 0.096 6 0.009 g (mean 6 SD) spinach (dry mass) per feeding. This was increased to 0.143 6 0.025 g during the second week and to 0.336 6 0.052 g from the third week until the end of the experiment. When tadpoles approached metamorphosis (Gosner stage 42; emergence of forelimbs) the vials were checked daily. Lifehistory traits examined for each individual were: hatchling size (body length at the start of the experiment, measured from the photographs), the duration of the larval period (days from Gosner stage 25 to stage 42), wet mass at metamorphosis (to the nearest 0.1 mg after gently blotting the metamorphs with paper towel), and growth rate (as the ratio between mass at metamorphosis and larval period). Statistical analyses To investigate the effects of female size and egg size on tadpole development and growth, we first analyzed correlations between female size and egg size. Body size

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and egg size differences between normally timed (N) and delayed (D) females were analyzed with t tests. Initial tadpole body length differences among treatments were analyzed using a two-way analysis of variance, with initial body length as the dependent variable and breeding and hatching phenology treatments as fixed factors. Because tadpoles from the same family and population were not statistically independent, we could not assume that the variance–covariance matrix is diagonal and all covariances equal to zero. To consider this nonindependence in our data, we defined a general correlation matrix assuming that the residuals of the same family and population are not independent of each other, and hence considered that a grouping structure arises from individuals from the same family and population (Schielzeth and Nakagawa 2013). We tested for the effects of breeding and hatching time treatments and their interactions on larval period, growth rate, and mass at metamorphosis using a twoway analysis of covariance (ANCOVA). Because an initial linear regression analysis clearly showed violation of homogeneity, we allowed for different variances by using the generalized least squares (GLS) method (Zuur et al. 2009). We used GLS models with initial body length as a covariate to control for its possible effects on life-history traits. The lack of treatment 3 initial body length interactions allowed us to assume common slope among families. Breeding and hatching phenology treatments and their interaction were included as fixed effects. We defined the same correlation matrix as before, grouping individuals from the same family and population. Statistical analyses were performed in the nlme package (Pinheiro et al. 2014) with restricted maximum likelihood (REML) in R version 2.10.1 (R Development Core Team 2009) using the corCompSymm argument in the gls function. RESULTS Adult survival to artificial crosses was 100% for both populations and N and D individuals, not counting mortality related with the 48C exposures. Female size was positively correlated with egg size (r ¼ 0.78; P , 0.001), however, there was no difference in body size or egg size between N and D females (t tests, P ¼ 0.324 and 0.600, respectively). Initial body length of the larvae was not affected by breeding treatments (F1, 654 ¼ 1.40; P ¼ 0.237), but there was a marginal effect of hatching treatment (F1, 654 ¼ 3.47; P ¼ 0.063) with late-hatching individuals (ND, DD) being smaller than those hatching at normal time (NN, DN; Appendix B). The interaction between breeding and hatching delay treatments was not significant for initial body length (F3, 654 ¼ 0.55; P ¼ 0.455). Mortality during larval development was very low (NN ¼ 2.5%, ND ¼ 1.2%, DN ¼ 0%, and DD ¼ 4.8%). Both delayed breeding and delayed hatching resulted in higher larval developmental rate, and subsequently earlier metamorphosis (Table 1; Fig. 1). Breeding delay

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TABLE 1. Analysis of covariance (ANCOVA) results for larval period, growth rate, and mass at metamorphosis in Rana arvalis. Effects

df

Value

SE

F

P

Larval period IBL Breeding Hatching Breeding 3 Hatching Residuals

1 1 1 1 653

0.1858 3.9117 1.3005 2.5060

0.9753 0.7738 0.3759 0.5295 3.8351

0.388 50.158 93.105 22.397

0.5338 ,0.0001 ,0.0001 ,0.0001

Mass at metamorphosis IBL Breeding Hatching Breeding 3 Hatching Residuals

1 1 1 1 653

0.0049 0.0521 0.0047 0.0019

0.0167 0.0138 0.0064 0.0091 0.0667

0.212 14.094 1.567 0.044

0.6454 0.0002 0.2110 0.8340

Growth rate IBL Breeding Hatching Breeding 3 Hatching Residuals

1 1 1 1 653

0.00002 0.0025 0.0004 0.0003

0.0004 0.0002 0.0001 0.0002 0.0016

0.021 127.869 13.741 2.335

0.8840 ,0.0001 0.0002 0.2857

Notes: Estimated regression parameter, standard errors, F value, and P value are given for each parameter included in the models. Abbreviations are IBL, initial body length; breeding, breeding phenology treatments; and hatching, hatching phenology treatments.

accelerated development by ;15% (NN vs. DN), whereas delayed hatching resulted in ;9% (NN vs. ND) and 4% (DN vs. DD) acceleration. A significant interaction between breeding and hatching phenology treatments (Table 1) indicated that the accelerating effect of hatching delay was stronger in the N than in the D breeding phenology treatment (Table 1; Fig. 1A). Delayed breeding (DN and DD) led to higher mass at metamorphosis (;10% higher on average), whereas hatching phenology or the breeding by hatching interaction did not affect mass at metamorphosis (Table 1; Fig. 1B). Both delayed breeding and delayed hatching treatments had strong positive effects on larval growth rate. Breeding delay increased growth rate by ;27% (ND vs. NN), hatching delay by ;11% (ND vs. NN) and 8% (DD vs. DN), and the combination of both delays (DD vs. NN) by ;33% (Table 1; Fig. 1C). The two study populations responded similarly to the four different treatments (Appendix C). DISCUSSION We found solid evidence for transgenerational plasticity in response to changing breeding phenology. R. arvalis tadpoles increased their growth and development rates in response to the delayed breeding treatment. We suggest that these responses allow R. arvalis tadpoles to optimize the timing of metamorphosis independently of the breeding date. Importantly, growth and development rates increased in the absence of external environmental cues (e.g., photoperiod, temperature, resource variation), suggesting that the acceleration signal was of maternal origin (see discussion below). In this sense, the present study differs crucially from previous studies on phenological plasticity in a range of organisms from plants to birds (e.g., Clark and Reed

2012, Galloway and Burgess 2012). In previous studies on TGP, photoperiodic cues were available for the offspring, and thus the distinction between the roles of maternal and offspring responses to seasonality remained unclear, making it impossible to differentiate between TGP and phenotypic plasticity. Previous studies with insects related to offspring diapause suggest the transference of seasonal information from parent to offspring (Mousseau and Dingle 1991). To our knowledge, the present study represents the first unequivocal demonstration of TGP to phenological variation in vertebrates. The breeding delay of approximately two weeks increased growth rate by ;27% and development rate over 15%, reflecting the significant impact that breeding time can have on life-history strategies. Previous laboratory studies on the common frog R. temporaria also found higher development rates in tadpoles in a year with late breeding (Orizaola et al. 2013) and increased larval growth rates in tadpoles facing an experimental breeding delay of four weeks (Lindgren and Laurila 2010). While these studies were not explicitly designed to experimentally test for the effects of breeding delay, they suggest that phenological TGP can be common in amphibians and possibly in other animals with complex life cycles. Nevertheless, tadpoles in our study did not fully compensate for the experimental delay. Larval period was only 6.5 days shorter in DN than in NN, although the DN tadpoles experienced a 12-day breeding delay. Full compensation may require more refined cues available in natural conditions, like changes in photoperiod and water temperature, or it may not be possible at all. Alternatively, full reduction in larval period may not be necessary, as DN tadpoles metamorphosed at a significantly larger size than NN

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tadpoles. It is also possible that the accelerated growth and development rates were balanced against losses in other fitness components previously described but not measured in our study (Metcalfe and Monaghan 2001, Dahl et al. 2012, Orizaola et al. 2013). A good understanding of the biological time-keeping mechanisms is crucial for predicting the evolution of lifehistory strategies under environmental stress (Helm et al. 2013). Our study found that TGP in growth and development rates was not mediated via egg or initial offspring size, which is the most widely recognized maternal effect in ecology (Fox and Mousseau 1998). Previous studies have reported that TGP can be mediated by changes in the quantity and composition of hormones, proteins, mRNAs, and other primary or secondary metabolites, as well as by epigenetic variation (Faulk and Dolinoy 2011, Meylan et al. 2012, Sheriff and Love 2013). Since amphibian development is highly dependent on hormonal regulation, a delay on breeding time perceived by the mother could modify maternal deposition of hormones into the oocytes and, consequently, affect larval development (Meylan et al. 2012, Sheriff and Love 2013). However, hormonal deposition into the oocytes in egg-laying organisms may only be possible during follicle development (Sheriff and Love 2013), although more studies on the timing of transference of maternal hormones to ova are needed. In our study, R. arvalis females had already ovulated by the time they were caught in the breeding ponds and hormonal deposition into the oocytes was probably completed, making this option more unlikely. An additional argument against hormonal transference is the responses observed during hatching delay treatments (NN vs. ND or DN vs. DD). In this case, growth and development differences could not be caused by differences in the hormonal content transferred by the mother, since fertilization occurred at the same time for each treatment. Epigenetic transgenerational effects on individual growth rates, possibly mediated by changes in the expression of enzymes involved in key metabolic processes have been documented in fish (Salinas and Munch 2012). Again, as females in our study had ovulated before they were subjected to the delay treatment, they may have had little possibility to influence gene regulation of the offspring. It is also possible that unfertilized eggs reacted directly to the relatively cold temperature during the laying delay. However, as this temperature does not differ from the temperatures normally experienced by prelaying females (Appendix A), its usefulness as a cue appears questionable. An interesting yet unstudied option for the observed TGP is the preconditioning of offspring circannual clock by the maternal circannual clock. Circannual clocks are a self-sustained endogenous system expressed under aperiodic conditions with a period close to 12 months (Hut and Beersma 2011). Due to the importance of time constraints and successful timing of life-history events

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FIG. 1. Effects of phenology treatments on larval lifehistory traits in Rana arvalis. (A) Duration of the larval period, (B) mass at metamorphosis, and (C) growth rate. Data shown as mean 6 standard error. Phenology treatments are NN, reproduction and hatching at normal time; ND, reproduction at normal time and delayed hatching; DN, delayed reproduction and normal hatching; and DD, delayed reproduction and delayed hatching.

for individual fitness, biological clocks may play a crucial role in regulating phenology in nature. Free-run endogenous clocks allow individuals to anticipate the seasons and prepare for seasonal changes even when environmental cues are noisy or not immediately present (Visser et al. 2010). The influence of the maternal

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PLATE 1. Female Rana arvalis from one of the study populations during the breeding period. Photo credit: G. Orizaola.

biological clock on offspring performance has been described in several animal taxa (Lakowski and Hekimi 1996), but rarely in organisms with complex life cycles (Danks 2006). Studies on fish and amphibians suggest that oocytes and zygotes express maternal genes controlling circadian clocks before the expression of the embryos’ own circadian genes are activated (Davie et al. 2011). In our case, maternal preconditioning of offspring with seasonal information could explain the observed responses for both breeding and hatching delay treatments. The internal clock of the ova may be set by the mother before fertilization, allowing adequate seasonal time-keeping during early life history. This transgenerational preconditioning would provide the offspring with an accurate representation of time (Paul et al. 2008), and allow them to adjust growth and development rates accordingly, even in the absence of direct seasonal cues (Appendix D). More detailed studies are needed to precisely understand the role that hormones and biological clocks may be playing in the adaptive phenological response of offspring. In accordance with previous studies (Orizaola et al. 2010), we also found that growth and development rates increased in response to the delayed hatching treatment. This response could have been brought about by the time-keeping mechanisms of the embryos, as mentioned above, or be a direct response to low temperature during embryonic development, as found during later developmental stages (Dahl et al. 2012). These results confirm the impact that early growth conditions could have on subsequent growth trajectories (Metcalfe and Monaghan 2001), and highlight the need to consider entire life cycles when evaluating the impact of environmental conditions on life-history strategies in nature (e.g., Radchuk et al. 2013).

Species responsiveness to year-to-year climate variation has been linked to long-term persistence, and TGP may allow for a rapid response to interannual environmental variation (Bossdorf et al. 2008, Meylan et al. 2012). Indeed, TGP in response to interannual variation has been demonstrated in drought tolerance in plants (Sultan et al. 2009), desiccation tolerance in invertebrates (Yoder et al. 2006 ), and increased temperature in fish (Salinas and Munch 2012). Also, TGP has been highlighted as a potential mechanism for rapid responses to climate shifts, together with evolutionary change and phenotypic plasticity. Adaptive TGP may be more efficient than within-generation phenotypic plasticity because it reduces time delays in the adaptive response and allows parents to provision 100% of offspring with the adaptive state. TGP may allow populations to persist long enough for genetic adaptation to occur, or obviate the need for genetic adaptation. Thus, adaptive TGP could be a key mechanism for coping with rapid climate change, especially in long-lived organisms in which adaptation by direct genetic change is unlikely to keep pace with rapid environmental changes. Studies on organismal phenology have mainly focused on the acceleration of phenological events in the spring (e.g., timing of reproduction, migration) or on changes toward the end of the growing season (e.g., hibernation; Bradshaw and Holzapfel 2006, Ha¨nninen and Tanino 2011, Lane et al. 2012). Importantly, the links between climate change and many of the lifehistory events across ontogeny, as well as the ecological and evolutionary connections between them, remain unexplored (Yang and Rudolf 2010, but see Radchuk et al. 2013). As life cycles crucially depend on appropriate timing of transitions between habitats

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and stages, the potential for a growing temporal mismatch between different transitions is a major concern. While phenotypic plasticity is a withingeneration adjustment to current conditions, TGP can play an important role in timing life-history events in response to phenological variation, allowing the parents to precondition offspring for the environment they are most likely to encounter. Our results highlight the need for examining the entire chain of life-history events and integrating phenology with life-history strategies when examining the potential impact of climate change on species and populations. ACKNOWLEDGMENTS We thank Frank Johansson, Katja Ra¨sa¨nen, and two anonymous reviewers for constructive comments on the manuscript. The animals were collected with a permit from Uppsala County Board (521-3019-09) and the experiment was conducted with a permit from the Ethical Committee for Animal Experiments in Uppsala (C92/9). Our research was supported by a Spanish Ministry of Education and Culture postdoctoral grant (MEC2007-0944) and a Beatriu de Pino´s postdoctoral fellowship (2008 BP A 00032) to A. Richter-Boix, research projects from Helge Ax:son Johnsons Stiftelse and Stiftelsen Oscar och Lili Lamms Minne (FO2011-0004) to G. Orizaola, and Formas to A. Laurila. LITERATURE CITED Agrawal, A. A., C. Laforsch, and R. Tollrian. 1999. Transgenerational induction of defences in animals and plants. Nature 401:60–63. Altwegg, R., and H. U. Reyer. 2003. Patterns of natural selection on size at metamorphosis in water frogs. Evolution 57:872–882. Beebee, T. J. C. 1995. Amphibian breeding and climate. Nature 374:219–220. Bossdorf, O., C. L. Richards, and M. Pigliucci. 2008. Epigenetics for ecologists. Ecology Letters 11:106–115. Bradshaw, W. E., and C. M. Holzapfel. 2006. Evolutionary response to rapid climate change. Science 312:1477–1478. Bradshaw, W. E., and C. M. Holzapfel. 2007. Evolution of animal photoperiodism. Annual Review of Ecology, Evolution, and Systematics 38:1–25. Bradshaw, W. E., and C. M. Holzapfel. 2010. What season is it anyway? Circadian tracking vs. photoperiodic anticipation in insects. Journal of Biological Rhythms 25:155–165. Clark, M. E., and W. L. Reed. 2012. Seasonal interactions between photoperiod and maternal effects determine offspring phenotype in Franklin’s gull. Functional Ecology 26: 948–958. Dahl, E., G. Orizaola, A. G. Nicieza, and A. Laurila. 2012. Time constraints and flexibility of growth strategies: geographic variation in catch-up growth responses in amphibian larvae. Journal Animal Ecology 81:1233–1243. Danks, H. V. 2006. Key themes in the study of seasonal adaptations in insects II. Life-cycle patterns. Applications in Entomological Zoology 41:1–13. Davie, A., J. A. Sanchez, L. M. Vera, J. Sanchez-Vazquez, and H. Migaud. 2011. Ontogeny of the circadian system during embryogenesis in rainbow trout (Oncorhynchus mykyss) and the effect of prolonged exposure to continuous illumination on daily rhythms of per1, clock, and aanat2 expression. Chronobiology International 28:177–186. Donelson, J. M., P. L. Munday, M. I. McCormick, and C. R. Pitcher. 2012. Rapid transgenerational acclimation of a tropical reef fish to climate change. Nature Climate Change 2:30–32.

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SUPPLEMENTAL MATERIAL Appendix A Thermal profiles of the collection sites during spring 2010 (Ecological Archives E095-237-A1). Appendix B Body length differences between treatments (mean and standard error) (Ecological Archives E095-237-A2). Appendix C Life-history trait differences between populations (Ecological Archives E095-237-A3). Appendix D Schematic representation of expected life-history reaction norms in the absence and presence of transgenerational transfer of time information (Ecological Archives E095-237-A4).