1 **Breeding phenology drives variation in reproductive output,**
2 **reproductive costs and offspring fitness in a viviparous ectotherm**

3

4 Théo Bodineau¹, Pierre de Villemereuil², Simon Agostini³, Beatriz Decencière³, Jean-

5 François Le Galliard¹,³ and Sandrine Meylan¹

6

7 1. Sorbonne Université, UPEC, UPCité, CNRS, INRAE, IRD, Institut d'Ecologie et des

8 Sciences de l'Environnement de Paris (iEES Paris - UMR 7618), 75005 Paris, France

9 2. Institut de Systématique, Évolution, Biodiversité (ISYEB), École Pratique des Hautes
10 Études– PSL, MNHN, CNRS, SU, UA, Paris, France

11 3. École normale supérieure, PSL Research University, Département de biologie, CNRS,
12 UAR 3194, Centre de recherche en écologie expérimentale et prédictive (CEREEP-Ecotron
13 IleDeFrance), 78 rue du château, 77140 Saint-Pierre-lès-Nemours, France

14

15 Running title: Experimental study of the effects of breeding phenology on the life history traits
16 of a viviparous ectotherm

17

18 Corresponding author:
19 Théo BODINEAU, iEES Paris – UMR 7618, 75005 Paris, France

20 Phone : +33 (0) 1 44 27 42 55

21 E-mail : theo.bodineau@gmail.com

22 ORCiD ID: 0000-0001-7375-1401

23

24 Acknowledgement:
25 The authors thank Emilie Pen, Lolita Muller, Noé Bastard and Jules Paraskevopoulos who
26 contributed to data acquisition. This work was supported by an Agence Nationale de la
27 Recherche grant (ANR-17-CE020013, 'AQUATHERM') to J.-F. Le Galliard. Théo Bodineau'
PhD grant is funded by the Ministère de l'Enseignement Supérieur et de la Recherche. This work has benefited from human and technical resources provided by the CEREEP-Ecotron IleDeFrance (CNRS/ENS UAR 3194).

Authors' contributions:

Jean-François Le Galliard, Sandrine Meylan and Théo Bodineau conceived the ideas and designed methodology; Théo Bodineau, Simon Agostini, Beatriz Decencière, Sandrine Meylan and Jean-François Le Galliard participated in data collection; Théo Bodineau helped by Pierre de Villemereuil, Jean-François Le Galliard and Sandrine Meylan analysed the data; Théo Bodineau led the writing of the manuscript with Sandrine Meylan, Jean-François Le Galliard and Pierre de Villemereuil. All authors contributed critically to the drafts and gave final approval for publication.

Conflict of Interest:

The authors declare no competing or financial interests.

Ethical approval:

Experiments were performed under the approval of Charles Darwin Animal Experimentation Ethics Committee in an animal breeding facility accredited with permit 17/DDPP/SPAE/57.
49 Abstract

50 Phenological advances are a widespread response to global warming and can contribute to
51 determine the climate vulnerability of organisms, particularly in ectothermic species which are
52 highly dependent on ambient temperatures to complete their life cycle. Yet, the relative
53 contribution of breeding dates and temperature conditions during gestation on fitness of
54 females and their offspring is poorly documented in reptiles. Here, we exposed females of
55 the common lizard Zootoca vivipara to contrasting thermal scenarios (cold versus hot
56 treatment) during gestation and quantified effects of parturition dates and thermal treatment
57 on life-history traits of females and their offspring for one year. Overall, our results suggest
58 that parturition date *per se* has a greater impact than thermal conditions during gestation on
59 life history strategies. In particular, we found positive effects of an earlier parturition date on
60 juvenile survival, growth and recruitment suggesting that environmental dependent selection
61 and/or differences in parental quality between early and late breeders underlie seasonal
62 changes in offspring fitness. Yet, an earlier parturition date compromised the energetic
63 condition of gravid females, which suggests the existence of a mother-offspring conflict
64 regarding the optimisation of parturition dates. While numerous studies focused on the direct
65 effects of alterations in incubation temperatures on reptile life-history traits, our results
66 highlight the importance of considering the role of breeding phenology in assessing the short-
67 and long-term effects of thermal developmental plasticity.

68

69

70 Keywords: climate change, phenology, parturition date, temperature, reproductive costs, life-
71 history variation, thermal plasticity, parent-offspring conflict
72

# 1. Introduction

73 For organisms living in seasonal environments, the timing of reproduction during the year is a
74 critical trait strongly associated with fitness (Iler et al., 2021; Visser & Gienapp, 2019). In
75 terrestrial animals, the decision to reproduce early or late during the year can be dictated by
76 numerous environmental and social cues (Watts et al., 2022) and will determine the number
77 of sites available for breeders as well as the number and quality of their mates (e.g. Bensch
78 & Hasselquist, 1992), the timing of the breeding period and thus the length of the juvenile
79 growth season (e.g. Olsson & Shine, 1997), or the availability of resources during the
80 breeding season and early juvenile life (Renner & Zohner, 2018). In fact, much research has
81 focused on the link between breeding phenology and fitness at the individual level since the
82 first comprehensive analysis of this relationship in birds by Perrins (1970). These studies
83 generally show selection for earlier reproduction with regard to the individual reproductive
84 output and the quality of their offspring (de Villemereuil et al., 2020; Radchuk et al., 2019).

85 At least two non-exclusive mechanisms have been proposed to explain selection for
86 an early laying date in birds and the processes that lead to a seasonal decline in offspring
87 quantity and quality (Verhulst & Nilsson, 2008). First, the seasonal decline in reproductive
88 performance could be explained by the laying date itself, which affects all individuals in the
89 same way (“date hypothesis”, (Hatchwell, 1991; Moreno, 1998)). According to this “date
90 hypothesis”, environment-dependent selection occurs through seasonal changes in
91 environmental conditions, such as seasonal shifts in food availability (Verhulst et al., 1995),
92 predation risks (Öberg et al., 2014) or temperatures, which can affect offspring growth and
93 survival during early life (Sauve et al., 2021). Alternatively, the seasonal decline in
94 reproductive performance could also be explained by differences in parental quality between
95 early and late breeders (“quality hypothesis”, (Brinkhof et al., 1993; Hatchwell, 1991)).
96 According to this hypothesis, adults in better condition or with a better quality territory would
97 reproduce earlier than others and therefore would produce better quality offspring, leading to
98 an apparent causal correlation between the timing of breeding and fitness. Although
99 correlative and experimental studies have challenged these two hypotheses in birds (e.g.

100 Harriman et al., 2017; Pärt et al., 2017; Winkler et al., 2020), the effects of date *per se* and
101 parental quality on fitness are generally difficult to disentangle and may both contribute to an
102 apparent seasonal decline in reproductive success.

103 Besides the date and quality hypothesis, the apparent beneficial effects of early
104 breeding dates on fitness-related traits could be the result of environmental conditions
105 experienced by the offspring during their embryonic development, such as variation in
106 microclimatic conditions (e.g., temperature, humidity), parental care (e.g., egg incubation,
107 food provisioning) or predation risks and stress. In ectotherms such as reptiles, reproductive
108 phenology and thermal conditions during incubation are functionally linked, as the essential
109 physiological functions of ectotherms are highly dependent on environmental temperatures
110 (Angilletta et al., 2002). Thus, warmer developmental temperatures caused by warmer
111 climates or an increased thermoregulatory behaviour of pregnant females generally reduces
112 incubation length (in oviparous species) or gestation length (in viviparous species), leading to
113 an earlier hatching date for the offspring (Noble et al., 2018). Beyond this expected temporal
114 impact, developmental thermal conditions during incubation can further directly affect
115 offspring fitness ("thermal hypothesis"), e.g. through the quality of embryonic development
116 (e.g., by impacting development of musculoskeletal or brain function (Booth, 2017; While et
117 al., 2009)). Generally, warm developmental temperatures and earlier hatching dates have
118 positive effects on offspring viability, growth, survival and reproduction (Le Henanff et al.,
119 2013; Li et al., 2018; Liu et al., 2022; Olsson & Shine, 1997; Qualls & Andrews, 1999; Uller &
120 Olsson, 2010; Wapstra et al., 2010). Yet, despite the functional link between developmental
121 thermal conditions and hatching dates, few studies have attempted to determine the relative
122 contribution of developmental thermal conditions *per se* and hatching dates (or parturition
123 date in viviparous reptiles) on fitness in reptiles. In addition, the combined effects of both are
124 rarely considered (Sun et al., 2018), while the effects of developmental temperatures and
125 hatching date could be additive or even interactive if developmental thermal conditions drive
126 the differences in performance between juveniles from early and late breeders.
Although there is evidence for some fitness costs of early breeding in birds (Brown & Bomberger Brown, 2000; Nilsson, 1994), the mechanisms involved in the deleterious effects of an acceleration in reproductive phenology are not yet resolved. Empirical field and experimental studies in the European common lizard (Zootoca vivipara, Lichtenstein, 1823) show that warmer populations with an earlier breeding phenology tend to decline over time, despite the advantages for juveniles of being born early and developing in warmer environments (Bestion et al., 2015; Dupoué et al., 2022). In viviparous reptiles, higher temperatures during gestation decrease gestation length and increase basal metabolism of pregnant females due to the increasing oxygen demand of the growing embryos (Foucart et al., 2014). Thus, acceleration of gestation induced by warmer climates or by the selection of higher body temperatures during gestation could increase the energetic costs of pregnancy and negatively affect the body condition of breeding females (Brusch IV et al., 2023). Hence, acceleration of gestation could induce longer-term costs by affecting mother survival as well as the decision and quality of future reproduction given the known trade-offs between current reproduction, survival and future reproduction (Stearns, 1992).

In order to improve our understanding of the relative contribution of developmental thermal conditions and parturition date in determining variation in fitness - related traits, we took advantage of the strong thermal plasticity of parturition dates of the European common lizard. By exposing pregnant females to contrasting thermal treatments (cold or hot treatments) during gestation, we advanced or delayed female's parturition date and generated two overlapping gradients of parturition dates between the thermal treatments. We then used an information-based model selection approach to compare the effects of thermal treatment and parturition dates *per se* both within the breeding season and up to the next breeding season. More specifically, we monitored the body condition, growth, future reproductive traits and survival of females, as well as offspring morphology at birth, body growth, future survival and reproduction. To distinguish between the date and thermal hypotheses regarding the impact of phenology on fitness, we then examined the effects of thermal conditions during gestation, the effects of parturition date and the combined effects
of both on reproductive performance and life history traits of females and the offspring using model averaging procedures (Figure 1). First, we expect low support for models which do not including developmental conditions and/or parturition date (Figure 1A), as in general, developmental conditions or reproductive phenology are important determinants of female and offspring fitness traits. Second, if the effects of thermal conditions experienced by pregnant females and embryos during gestation is the main determinant of fitness traits (“thermal hypothesis”), we expect higher support for models including only thermal treatment (Figure 1B). Third, if the parturition date (“parturition date hypothesis” which include date and quality hypothesis) is the main determinant of fitness traits, we expect higher support for models including only the parturition date (Figure 1C). Fourth, if parturition date and thermal treatment equally influence fitness traits, we expect higher support for models including additive effects of thermal conditions during gestation and parturition date (“thermal + parturition date hypothesis”; Figure 1D) or even interactive effects of both (“thermal x parturition date hypothesis”; Figure 1E).

## 2. Material and methods

### 2.1. Study species, sampling and rearing conditions

The European common lizard is a small terrestrial lizard widely distributed in northern Eurasia in highly seasonal, cool and humid habitats. Natural populations are structured in three main stage classes: juveniles (new-born individuals of the year), yearlings (one- to two-years-old individuals), and adults (more than two years old). The mating period begins shortly after the emergence of sexually mature females from overwintering and occurs later for young than for old females (Bauwens & Verheyen, 1985). At our study site, mated females carry out vitellogenesis (follicular differentiation) during two to three weeks between mid-April and May, which is followed by ovulation and egg fertilisation that mark the onset of gestation. Parturition occurs after an average gestation period of 2 months, i.e. between late June and early August, depending on thermal conditions and individual characteristics (Rutschmann et al., 2016). Females lay on average five non-calcified eggs (range 1-12)
including both nonviable and live offspring that hatch quickly (a few minutes to a few hours) after parturition.

Lizards were captured inside semi-natural populations maintained in outdoor enclosures at CEREEP-Ecotron IleDeFrance (France, 48°17'11.5" N, 2°40'45.6" E).

Pregnant adult (n=84) and sub-adult females (n=93) were captured after mating (i.e., between May 3rd and May 7th 2021 for adult females and between May 25th and June 11th 2021 for sub-adults). We then acclimated females in individual terrariums (adults: 25 × 15 × 15 cm, sub-adults: 18 × 11 × 12 cm) filled with sterilised peat as substrate, equipped with a shelter, and installed in a temperature-controlled room (23°C from 9 am to 6 pm, 16°C at night). Terrariums were lit automatically between 8:30 am and 5 pm by a UVB-enhanced neon tube (30 W) providing white light and by a halogen heating bulb (25W) placed on one side of the terrarium to generate a thermal gradient. All lizards were acclimatised for a minimum of two weeks before the experiments by providing optimal basking conditions (8.5 hours/day), food and water *ad libitum* and humid conditions. Reproductive status of females was inferred by the presence of recent male bite marks and ultrasonography (SonoSite M-Turbo, Inc., Bothell, WA, USA). Experiments were performed under the approval of Charles Darwin Animal Experimentation Ethics Committee (APAFIS#2021010121599881) in an animal breeding facility accredited with permit 17/DDPP/SPAE/57.

## 2.2. Experimental design

We exposed females to two contrasted thermal regimes during gestation to change breeding phenology. In the "hot" treatment, females could bask at their preferred temperature for 8.5 hours per day and we imposed a night-time temperature of 18°C. In the "cold" treatment group, we shortened the daily thermoregulation opportunities of females to 6 hours per day and imposed night-time temperatures of 14°C. Basking regime and night-time temperatures were manipulated together to simulate contrasted climate scenarios, induce thermal plasticity of breeding phenology and create two overlapping parturition date gradients between the thermal treatments. The hot treatment simulates a warming scenario where warmer days are associated with higher night-time temperatures (Zhong et al., 2023), while the cold treatment
simulates the current thermal conditions encountered in populations of this species (Dupoué et al., 2022). These two thermal regimes also encompass the variability observed inside natural populations (Dupoué et al., 2022). Experimental manipulations started on 7th June for all adult females after confirmation of their reproductive status. As the ovulation periods of sub-adults are delayed relative to adults, sub-adults were distributed into the two experimental groups between 18th June and 5th July after confirmation of their reproductive status. As we were not able to determine the ovulation date of the females, we randomly assigned females to both groups while ensuring a balanced average body mass (which is correlated with the progress of gestation), and arranged them randomly in the breeding room. Hot conditions accelerated breeding phenology by seven days on average in each age group (effect of thermal treatment on parturition date in 2021: F₁,₁₄₇.₁ = 47.25, p < 0.001; mean parturition date: 21th July for hot treatment and 28th July for cold treatment), with a broad gradient of parturition dates ranging from 3rd July to 11th August.

## 2.3. Measurements of energetic condition of female

One day before the start of the experiment and one day after parturition, we quantified tail width and limb thickness of females, which are reliable proxies of energetic condition and can therefore be used to assess the energetic costs of reproduction (Bodineau et al., 2024; Brusch et al., 2020). A reduction of tail width and limb thickness indicates muscle catabolism and the mobilisation of caudal energy reserves. We took digital pictures of the females ventrally using a flatbed scanner (CanoScan LiDE 700F, Canon) and measured tail width at the 7th sub-caudal scale rows with ImageJ software (Version 1.52v). At the same time, hind limb thickness was measured on both legs using a spessimeter (ID-C1012BS, Mitutoyo, Japan). Following the same method, we measured female limb thickness and tail width before wintering in September 2021 and we also measured limb thickness after wintering in April and May 2022 to estimate long-term effects. We calculated intra-individual changes in limb thickness and tail width by subtracting the final values from the initial values of the trait measured between the beginning and end of gestation and between recapture sessions. For
237 the change in limb thickness, we calculated this change at the right and left hind legs and
238 then averaged them.

239 2.4. Measurements of reproductive effort and offspring quality

240 After parturition, each female was weighed and the litter was characterised by recording the
241 number of unfertilised eggs, undeveloped embryos, stillborn and live juveniles. Juveniles
242 were measured for snout to vent length (with a ruler, to the nearest 0.5 mm), weighted (using
243 a precision scale to the nearest mg), and sexed by counting ventral scales (Lecomte et al.,
244 1992). All live juveniles were marked by toe-clipping. On the day of parturition, we randomly
245 released juveniles from the same litter with their mothers into one of ten outdoor enclosures
246 by standardising the density and sex ratio of juveniles, sub-adults and adults.

247 2.5. Growth, survival and reproduction up to the following year

248 We monitored all surviving offspring and females through regular hand capture sessions in
249 outdoor enclosures in September 2021, April 2022 and May 2022. At each capture session,
250 we measured for snout to vent length (SVL) and body mass all individuals and then released
251 them into their enclosures. We calculated the specific growth rate (SGR = [Ln (SVL) – Ln
252 (initial SVL)]/days) before wintering (between birth to September) and after wintering
253 (between September to May) using established methods (Liu et al., 2022; Sinervo, 1990;
254 Sun et al., 2018). At the end of May 2022, we recaptured all alive individuals to estimate the
255 annual survival. All reproductive females were housed in the laboratory under the same
256 standard rearing conditions (as described above, part 2.1) until parturition. We characterised
257 parturition date, reproductive investment and litter characteristics (as described above, part
258 2.4).

259 2.6. Statistical analyses

260 Analyses were performed using R software version 4.2.0 (R Core Team, 2022). In order to
261 test our five hypotheses, we built the following five different types of models (see Figure 1):
262 null model without effect of treatment or parturition date (“null hypothesis”, scenario A),
263 model including the effect of thermal treatment only (“thermal hypothesis”, scenario B),
264 model including the effect of parturition date (“parturition date hypothesis”, scenario C),
model including the additive effect of thermal treatment and parturition date ("parturition date + thermal hypothesis", scenario D) and a variation of this model including interactive effects of thermal treatment and parturition date ("parturition date x thermal hypothesis", scenario E).

In scenario D and E, we centred parturition date within thermal treatment as female parturition date minus the average parturition date of females from her thermal treatment to avoid multicollinearity between treatment factor and date. To account for the uncertainty in the model selection of the "nuisance" covariates (i.e., variables that are not of primary interest but should be considered in the analysis), we computed the Akaike weights of models including different nuisance covariates for class of models (see details below) and summed the weights within the different subclasses described. This procedure allowed us to obtain an estimate of a "model-averaged" Akaike weight corresponding to each class of model. We tested these 5 models (scenarios A to E) for each analysis (except for the parturition date analysis in 2021, where we only compared null models (scenario A) and models including the treatment effect (scenario B)) and we added to these five models of interests all covariates as described below. In all cases, we controlled for inter-individual and inter-enclosures variability by including a random effect of individual identity of the mother and/or the enclosure and tested model fitting and model assumptions. All results are reported as means ± SE unless otherwise stated and statistical support for each class of model is expressed as a percentage of the cumulative Akaike weights.

## Morphological changes of females

We analysed limb thickness changes using linear mixed models (LMMs) with scenarios A to E including the effect of session (i.e., beginning of gestation to parturition, parturition to September, September to April, April to May), age class and the initial centred value of limb thickness as fixed effects. Tail width changes during gestation were analysed using LMMs including the age class and the initial centred value of tail width as fixed effects.

## Immediate and delayed effects on reproductive outputs

Parturition dates in 2021 and 2022 (calculated as day of the year) were analysed using LMMs. We first analysed the effect of thermal treatment on parturition dates in 2021 using
models with scenarios A and B including fixed effects of age class and body mass at capture
age class-centred. Second, parturition dates of surviving manipulated females in 2022 was
analysed with scenarios A to E including mean centred body mass at capture as fixed effect.
We added body mass at capture in these models to take account of inter-individual
differences in the progress of gestation and centred related to age class for the analysis of
the 2021 data because the mass of the females is highly dependent on age class.

We calculated litter size as the total number of reproductive items produced (i.e.,
fertilised or unfertilised eggs, undeveloped embryos, stillborn and live offspring), and
reproductive success at birth as the number of viable juveniles at birth out of the total number
of reproductive items. Litter size was analysed with GLMMs using scenarios A to E with a
Poisson distribution whereas reproductive success at birth was analysed with negative
binomial mixed models. We added age class and SVL age class-centred as fixed effects. We
further calculated reproductive investment as the difference between the body mass of
females before and after the parturition. We then analysed reproductive investment with
LMMs using scenarios A to E and including age class and SVL age class-centred as fixed
effects. We performed the same analyses of litter size, reproductive success at birth and
reproductive investment in 2022, without testing the effect of age class because all females
were adults in 2022.

Effects on offspring morphology at birth, juvenile growth and reproduction early in life
We fitted LMMs using scenarios A to E to data on offspring SVL and body condition at birth.
We added to these models the age class of females and the sex of juveniles as fixed effects.
For body condition at birth, we analysed body mass at birth with SVL as a covariate
assuming a linear relation between mass and size. In addition, we analysed SGR before
wintering and SGR after wintering as well as body size at the end of first year of life (SVL in
May 2022) using LMMs with scenarios A to E including fixed effects of juvenile sex and age
class of the mother. Finally, since not all juvenile females reproduce at one-year-old, we
examined recruitment of juvenile females during the first year of life and their parturition date.
We fitted GLMMs with scenarios A to E assuming a binomial distribution to examine
recruitment as success/failure events and we used a LMM to study juvenile parturition dates.

No variables were added to the base of the models in these two analyses.

### Female and juvenile survival

Since all juveniles and females are recaptured in May, lizards not recaptured were considered dead. We analysed survival of juvenile and manipulated females as success/failure events using GLMMs (scenarios A to E) assuming a binomial distribution. We added the age class as a factor in models of traits for breeding females analyses and we took into account the age class of the mother and the sex of the juvenile as fixed effects for juvenile survival analysis.

## 3. Results

### 3.1. Energetic condition changes of female

We found support for an effect of parturition date on female limb thickness and tail width changes ("date hypothesis": 43% and 60% respectively). Adult and sub-adult females that gave birth early displayed a stronger reduction of their tail width and limb thickness during gestation than late breeding females (all p < 0.05; Figure 2A, E). This relationship was however reversed after breeding and before wintering, a period during which females that gave birth early gained more limb thickness and tail width than late breeding females (all p < 0.05; Figure 2B, F). Similarly, after wintering, early breeding females from year 2021 showed a greater increase in limb thickness than late breeding females ($F_{1,67} = 16.3, p < 0.001$; Figure 2C). On average, subadult females showed an increase in limb thickness and tail width compared with adult females (limb thickness: 0.03 ± 0.01 and -0.03 ± 0.01 mm respectively; tail width: 0.16 ± 0.02 and -0.02 ± 0.02 mm respectively; all p < 0.05).

### 3.2. Reproductive traits of manipulated females

There was strong support for an influence of parturition date on reproductive success at birth (Table 1, "date hypothesis": 55%) but reproductive investment and litter size were not influenced by treatment or parturition dates ("null hypothesis": 44% and 50% respectively). Reproductive success at birth was greater for females that give birth early (slope: -0.16 ±
0.08; $\chi^2_1$ = 4.26, p = 0.03). Regarding offspring SVL and body condition at birth, we found weak support for models that included parturition date ("date hypothesis": 37%) and treatment ("thermal hypothesis": 32%), respectively. Litters born earlier tended to include larger offspring (slope: -0.17 ± 0.10; $F_{1,96.9}$ = 3.2, p = 0.05) but the effect of thermal treatment on offspring body condition was not significant ($F_{1,94.1}$ = 1.7, p = 0.20). Compared with adult females, subadult females show lower reproductive success (4.39 ± 0.38 and 2.77 ± 0.22 viable juveniles at birth respectively; $\chi^2_1$ = 5.9, p = 0.02), reproductive investment (2.14 ± 0.09 g and 1.18 ± 0.06 g respectively; $F_{1,135.6}$ = 6.2, p = 0.01) and litter size (6.64 ± 0.26 and 3.88 ± 0.15 reproductive items respectively; $\chi^2_1$ = 52.1, p < 0.001).

### 3.3. Body growth, survival and future reproduction of offspring

We found strong support for models including parturition date regarding annual juvenile survival (Table 1, "date hypothesis": 52%), body size at the end of first year of life (SVL in May, "date hypothesis": 75%), pre-winter and post-winter SGR ("date hypothesis": 67% and 81% respectively), the recruitment probability of juvenile females ("date hypothesis": 81%) and the breeding dates of juvenile females at the age of one year ("date hypothesis": 67%).

Annual survival (Figure 3A) and body size at the end of first year of life were greater for early-born juveniles than for late-born juveniles (survival: $\chi^2_1$ = 3.8, p = 0.05; body size: slope: -1.28 ± 0.36; $F_{1,46.7}$ = 12.8, p < 0.0001). In addition, juveniles produced by sub-adult females have a larger body size at the end of first year of life than those produced by adult females (55.48 ± 0.59 mm and 55.08 ± 0.62 mm respectively; $F_{1,44.5}$ = 4.6, p = 0.04).

However, the direction of the relationship between SGR and parturition date varied before and after the wintering period (Figure 3B, C). Early-born juveniles had higher pre-winter SGR ($F_{1,58.1}$ = 14.2, p < 0.001) but lower post-winter SGR than later-born juveniles ($F_{1,40.4}$ = 40.0, p < 0.001). In addition, we observed a significant decrease in the recruitment probability at one-year-old for late-born juveniles ($\chi^2_1$ = 5.1, p = 0.02) as well as a positive relationship between the breeding date of juveniles and those of their mothers ($F_{1,22.1}$ = 8.8, p < 0.001; Figure 4).
375

3.4. Survival and future reproduction of manipulated females

376

There was a strong support for models with long-term effects of thermal treatment on female breeding dates during the next breeding season (“thermal hypothesis”: 55%). Females exposed to the cold treatment in 2021 gave birth an average of 5 days earlier the next breeding season in 2022 than females exposed to hot treatment in 2021 ($F_{1,72} = 7.9, p < 0.01$). Apart from this, we found a support for null models regarding female’s survival, reproductive investment, litter size and reproductive success at birth in 2022 (“null hypothesis”: 31%, 64%, 50% and 51% respectively).

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Discussion

384

In this study, we changed the gestation length of a viviparous lizard by exposing pregnant females to contrasting thermal conditions in order to test the intra-generational and intergenerational effects of thermal conditions and parturition date on life history traits of females and their offspring. In accordance with previous studies in viviparous lizards, colder nighttime temperatures and reduced thermoregulation opportunities during the day delayed parturition dates (Brusch IV et al., 2023; Wapstra et al., 2010). This was observed despite the fact that pregnant females increased their thermoregulation effort in the cold treatment group (see supplementary information), probably to counteract the effects of low developmental temperatures on speed of gestation (Atkins et al., 2007; Hare et al., 2008; Qualls & Andrews, 1999). We performed a comparative analysis of five hypotheses (see Figure 1) but statistical support for the “thermal hypothesis”, the “parturition date + thermal hypothesis” and the “parturition date x thermal hypothesis” was weak, whereas the “parturition date hypothesis” was often strongly supported, especially for most offspring life history traits. Thus, our results suggest that life history strategies were best explained by parturition date, rather than by thermal conditions experienced by females during gestation or by additive or interactive effects between thermal conditions and parturition dates. We found strong effects of the parturition date on both female condition traits and reproductive performances and on juvenile survival, growth and recruitment. Earlier parturition dates had

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positive effects on juveniles in accordance with previous studies in reptiles (Le Henanff et al., 2013; Pearson & Warner, 2018; Sinervo & Doughty, 1996; Uller & Olsson, 2010; Wapstra et al., 2010), but had a negative impact on females condition.

Seasonal declines in reproductive investment and litter viability have been observed repeatedly in reptiles, in birds and in some mammals as well (birds: Dawson, 2008; Verhulst et al., 1995, reptiles: Mitchell et al., 2018, mammals: Green & Rothstein, 1993). In general, the decline in reproductive performances in late breeding females is explained by a lower quality of these females and/or by less favourable late-season environmental conditions for reproduction (Öberg et al., 2014). In lizards, reproductive investment and litter viability often decrease in females laying eggs later in the season (e.g. Olsson & Shine, 1997) and in females exposed to colder conditions during gestation (e.g. Brusch et al., 2020). In our study, we did not detect an effect of thermal treatment or parturition date on litter size because litter size is determined early in the reproductive cycle before the start of the manipulation (Bauwens & Verheyen, 1985). However, we observed a lower proportion of viable juveniles at birth for late breeding females. A prolonged incubation or gestation can be harmful to embryos by altering the developmental environment of embryos (Atkins et al., 2007; Hare et al., 2008; Qualls & Andrews, 1999). Finally, lower litter size, reproductive investment and reproductive success in subadults were probably related to age and reproductive experience.

In addition, while extended literature shows that thermal conditions during embryonic development can significantly influence offspring phenotype (Booth, 2006; Noble et al., 2018; While et al., 2018), our results suggest that juvenile SVL at birth is more influenced by parturition date rather than by developmental thermal conditions *per se*. Early-born juveniles were slightly larger than late-born juveniles consistent with some previous studies in lizards that found positive effects of earlier hatching date on juvenile body size (Olsson & Shine, 1997; Shine & Olsson, 2003; Sun et al., 2018; Wapstra et al., 2010). However, we didn't find any effect of parturition date and thermal treatment on juvenile body condition in accordance with a previous study in the European common lizard (Brusch et al., 2020). In general, the extent and direction of the effects of developmental temperatures and hatching dates on
430 juvenile morphology vary between species and the underlying mechanisms of these effects
431 remain poorly understood (reviewed in Booth, 2006; Noble et al., 2018). In some instances,
432 warmer incubation temperatures, longer basking times and earlier hatching dates increase
433 body mass or size of juveniles at birth (e.g. Doughty & Shine, 1998; Wapstra, 2000; Wapstra
434 et al., 2010). However, in others, eggs laid late in the season and in cold incubation regimes
435 produce larger and/or heavier juveniles (Pearson & Warner, 2018). Juvenile morphology at
436 birth can be influenced by the amount of yolk provided before ovulation and by temperature-
437 dependent yolk allocation processes towards growth or maintenance (Shine & Harlow, 1993;
438 Wapstra, 2000). Thus, SVL differences at birth between early- and late-born juveniles could
439 result from maternal effects related to the amount of energy allocated in yolk production or to
440 maternal thermoregulation behaviours in line with the quality hypothesis.

441 Life history traits of juveniles were mainly determined by parturition date and not by
442 thermal treatment alone or in combination with parturition date. As predicted and consistent
443 with previous studies on reptiles, we found positive effects of early parturition date on
444 juvenile survival probability, total growth, summer growth rate and access to reproduction
445 during the first year of life (Pearson & Warner, 2018; Uller & Olsson, 2010; Wapstra et al.,
446 2010). These advantages of an early birth date on survival and total growth could be linked
447 to pre-natal mechanisms linked to female quality ("quality hypothesis") such as the quality of
448 eggs produced or maternal thermoregulation strategies affecting the developmental
449 environment and quality at birth. For example, we found that juveniles born earlier were
450 larger at birth. Benefits of an earlier birth date could be also explained by post-natal
451 mechanisms linked to seasonal changes in climatic conditions, in prey availability and in
452 population density ("date hypothesis"). A longer period of activity before winter combined with
453 more favourable thermal conditions in early summer and less competition among juveniles
454 born early during the season may boost the body growth and energy storage of offspring
455 before wintering and thus positively affect the survival of early-born juveniles (Adolph &
456 Porter, 1993; Pearson & Warner, 2018; Uller & Olsson, 2010; Warner & Shine, 2007). This
457 selective advantage to early individuals may generate strong inter-individual differences in
fitness unless some compensatory mechanisms exist later in life (Mangel & Munch, 2005; Metcalfe & Monaghan, 2001). As a matter of fact, we found a higher post-winter growth rate in later-born juveniles, which suggests the existence of some compensatory growth strategies developed by late juveniles to offset their disadvantage at birth. Similar compensatory strategies have been demonstrated in several taxa and they generally constitute an adaptive response to a shorter growth period (birds: Hirose et al., 2012, reptiles: Sun et al., 2018, amphibians: Orizaola et al., 2010, fish: Simonin et al., 2016). Despite these compensatory growth responses, early-born lizards maintained a size advantage at one-year-old and we observed a significant decrease in the recruitment probability of late-born juveniles, which shows that birth date is also an important determinant of age at first reproduction. A greater absolute body growth may explain these findings because sexual maturation and breeding dates depend strongly on body size in this species (Heulin, 1985). These results could also be explained by the heritability of parturition dates, as already demonstrated in several bird species (Biquet et al., 2022; Charmantier & Gienapp, 2014), although wild population estimates are scarcer for reptiles.

Despite the beneficial effects of early parturition dates on reproductive quality and juvenile life history traits, we also found negative impacts on females such as greater losses in leg thickness and tail width in females that give birth earlier. In many lizard species, including the European common lizard, the tail is a reserve organ composed mainly of muscle and lipids stored in the form of triglycerides (Price, 2017). Mobilisation of caudal lipid reserves and muscle wasting can occur to meet energy and amino acid requirements when dietary intake is insufficient or when energy expenditure is high. In viviparous reptiles, basal metabolism and the resulting energy expenditure increase during gestation due to embryonic development (Foucart et al., 2014). Reptiles can quickly mobilise caudal lipid reserves and/or muscles in response to the acceleration of gestation and the increased demands of embryos (Brusch et al., 2020; Brusch IV et al., 2023; Lourdais et al., 2004). Thus, early breeding is associated with an increase of the immediate energetic costs of reproduction for pregnant females that impact their body condition (this study) but could also change locomotor
performances and predation risks (Lourdais et al., 2004). In natural populations, these extra energetics could be offset by other advantages of a shorter gestation length in terms of reduced time with a locomotion burden and a high predation risk due to pregnancy, or a longer period before winter to store energy (Shine, 2003). Although we found that early-breeding females restored condition to a greater extent than late-breeding females before winter, the benefits of early parturition to recover energy before winter were not equal to the direct costs imposed by early parturition. Indeed, before and after winter, early breeding females always presented a weaker muscular condition than late females (not shown). In addition, we did not detect any negative effect of early parturition or treatment on future survival and reproduction (but see Bleu et al., 2013) but females exposed to the cold treatment give birth on average 5 days earlier during the next breeding season than females exposed to the hot treatment. In birds, the condition-dependent individual optimisation model of reproductive phenology predicts that females optimise their breeding phenology as a function of arrival condition, arrival date, and the ability to gain in condition on the breeding grounds (Hennin et al., 2016; Rowe et al., 1994). Since female lizards in the cold treatment gave birth later and were on average in better condition when they emerged from winter the following year, this model predicts that they were able to start reproducing earlier as we observed.

## Conclusion

Our experimental study examined the effects of breeding phenology and thermal conditions during gestation on life history strategies of mothers and their offspring in a viviparous ectotherm. Our results are consistent with previous studies highlighting directional selection towards early parturition dates, and emphasise that the parturition date, rather than thermal condition during gestation and combined effects of both, is an important determinant of survival, growth and recruitment of juveniles, which can have a major impact on the population dynamics of viviparous ectotherms. Additionally, our findings suggest the existence of a mother-offspring trade-off concerning the optimisation of the parturition dates
513 insofar as reducing the gestation length can entail energy costs for pregnant females. Finally,
514 our findings and field studies highlighting the decline in natural populations of the common
515 lizard with an early phenology call for better consideration of the benefits and costs
516 associated with earlier breeding dates. Future research should quantify the benefits and
517 costs of phenological acceleration in oviparous species, as well as the long-term effects of
518 accelerated life-history in order to understand and predict the consequences of phenological
519 changes in response to climate change.

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776
777 Table 1: Rounded sums of Akaike weights according to the five hypotheses tested to assess the intragenerational and intergenerational effects

778 of thermal treatment and/or parturition date on fitness traits.

<table><thead><tr><th colspan="2">Scenario relating to figure 1</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th></tr><tr><th>Effect category</th><th>Trait</th><th>Null hypothesis</th><th>Thermal hypothesis</th><th>Parturition date hypothesis</th><th>Thermal + parturition date hypothesis</th><th>Thermal x parturition date hypothesis</th></tr></thead><tbody><tr><td rowspan="10">Intragenerational</td><td>Change in tail width</td><td>2.03E-06</td><td>0.20</td><td><strong>0.60</strong></td><td>0.15</td><td>0.05</td></tr><tr><td>Change in limb thickness</td><td>5.86E-03</td><td>0.33</td><td><strong>0.43</strong></td><td>0.19</td><td>0.05</td></tr><tr><td>Parturition date in 2022</td><td>0.03</td><td><strong>0.55</strong></td><td>0.01</td><td>0.31</td><td>0.10</td></tr><tr><td>Reproductive investment in 2021</td><td><strong>0.44</strong></td><td>0.14</td><td>0.25</td><td>0.09</td><td>0.08</td></tr><tr><td>Reproductive investment in 2022</td><td><strong>0.64</strong></td><td>0.19</td><td>0.07</td><td>0.06</td><td>0.03</td></tr><tr><td>Number of viable juveniles at birth in 2021</td><td>0.18</td><td>0.05</td><td><strong>0.55</strong></td><td>0.21</td><td>0.08</td></tr><tr><td>Number of viable juveniles at birth in 2022</td><td><strong>0.51</strong></td><td>0.23</td><td>0.17</td><td>0.07</td><td>0.02</td></tr><tr><td>Litter size in 2021</td><td><strong>0.50</strong></td><td>0.19</td><td>0.22</td><td>0.07</td><td>0.03</td></tr><tr><td>Litter size in 2022</td><td><strong>0.50</strong></td><td>0.23</td><td>0.17</td><td>0.08</td><td>0.03</td></tr><tr><td>Survival</td><td><strong>0.31</strong></td><td>0.14</td><td>0.23</td><td>0.22</td><td>0.09</td></tr></tbody></table>
<table><tr><td></td><td>Survival</td><td>0.16</td><td>0.08</td><td><strong>0.52</strong></td><td>0.19</td><td>0.05</td></tr><tr><td></td><td>Body mass at birth</td><td>0.25</td><td><strong>0.32</strong></td><td>0.15</td><td>0.21</td><td>0.06</td></tr><tr><td></td><td>SVL at birth</td><td>0.20</td><td>0.11</td><td><strong>0.37</strong></td><td>0.23</td><td>0.10</td></tr><tr><td></td><td>SVL in May</td><td>7.76E-03</td><td>4.5E-03</td><td><strong>0.75</strong></td><td>0.17</td><td>0.07</td></tr><tr><td rowspan="3">Intergenerational</td><td>Pre-winter specific growth rate</td><td>3.11E-03</td><td>0.01</td><td><strong>0.67</strong></td><td>0.24</td><td>0.08</td></tr><tr><td>Post-winter specific growth rate</td><td>7.20E-08</td><td>2.25E-07</td><td><strong>0.81</strong></td><td>0.16</td><td>0.03</td></tr><tr><td>Recruitment of juvenile females at one-year-old</td><td>1.26E-04</td><td>1.17E-04</td><td><strong>0.81</strong></td><td>0.11</td><td>0.08</td></tr><tr><td></td><td>Parturition date at one-year-old</td><td>0.04</td><td>0.02</td><td><strong>0.67</strong></td><td>0.19</td><td>0.08</td></tr></table>
780 **Figure legends**

781 **Figure 1:** Hypothetical framework of the relationships between thermal treatment, parturition
782 date (or hatching date) and fitness traits of females and the offspring. To test the effects of
783 thermal treatments, parturition date and combined effects of both on fitness traits, we built
784 and compared the following five types of models: null model without effect of thermal
785 treatment or parturition date ("null hypothesis", scenario A), model including the effect of
786 thermal treatment only ("thermal hypothesis", scenario B), model including the effect of
787 parturition date only ("parturition date hypothesis", scenario C), model including the additive
788 effect of thermal treatment and parturition date ("thermal + parturition date hypothesis",
789 scenario D) and a variation of this model including interactive effects of thermal treatment
790 and parturition date ("thermal x parturition date hypothesis", scenario E). In scenarios D and
791 E, we centred the individual parturition date relative to the parturition dates of females in the
792 same thermal treatment to fix multicollinearity. The filled black and grey dots represent the
793 fitness traits of individuals exposed to hot and cold conditions respectively.
794

794

795 **Figure 2:** Effects of parturition date on intra-individual changes in limb thickness (A, B, C, D)
796 and tail width (E, F) of breeding females *Zootoca vivipara*. Points represent raw data and a
797 negative change indicates a decrease in the limb thickness or tail width over time. The linear
798 regressions are plotted and shaded areas represent 95% confidence limits.
799

800

801 **Figure 3:** Effects of parturition date on juvenile survival probability (A), pre-winter specific
802 growth rate (B) and post-winter specific growth rate (C). In A, solid line represents the
803 average survival probability as a function of parturition date obtained from a logistic
804 regression. The point size corresponds to the number of observations. In B and C, points
805 represent raw data. The linear regression is plotted and shaded areas represent 95%
806 confidence limits.
807

806

807 **Figure 4:** Effects of parturition date on recruitment rate (A) and parturition date (B) of
808 juvenile females at one-year-old. In A, solid line represents the average recruitment of a
809 given parturition date estimated from a logistic regression. The point size corresponds to the
810 number of observations. In B, points represent individual parturition date of juvenile females
811 at one-year-old of a given parturition date. The linear regression is plotted against raw data.
812 Shaded areas represent 95% confidence limits.

812
<figure><img src="image_1.png" /><figcaption>**Figure 1**</figcaption></figure>
A

Change in limb thickness (mm)

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

C

Change in limb thickness (mm)

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

E

Change in tail width (mm)

<figure><img src="image_6.png" /></figure>

Parturition date (number of days since 1st June 2021)

F

<figure><img src="image_7.png" /></figure>

Parturition date (number of days since 1st June 2021)
<figure><img src="image_8.png" /><figcaption>**Figure 3**</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 4</figcaption></figure>
828

**Supplementary information**

829

*Behavioural monitoring during gestation*

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