# Floral diversity enhances winter survival of honeybee colonies across climatic regions

Giulia Mainardi, Douglas Sponsler, Etienne Minaud, Filippos Vardakas, Leonidas Charistos, Fabrice Requier, Fani Hatjina, Ingolf Steffan-dewenter

► To cite this version:

Giulia Mainardi, Douglas Sponsler, Etienne Minaud, Filippos Vardakas, Leonidas Charistos, et al. Floral diversity enhances winter survival of honeybee colonies across climatic regions. Journal of Applied Ecology, In press, 10.1111/1365-2664.70054. hal-05041998

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# Floral diversity enhances winter survival of honeybee colonies across climatic regions

Giulia Mainardi<fnref n="1" /> | Douglas Sponsler<fnref n="1" /> | Etienne Minaud<fnref n="2" /> | Filippos Vardakas<fnref n="3" /> |
Leonidas Charistos<fnref n="3" /> | Fabrice Requier<fnref n="2" /> | Fani Hatjina<fnref n="3" /> | Ingolf Steffan-Dewenter<fnref n="1" />

## Abstract

1. In temperate climates, winter can be an arduous time for eusocial insects. Survival of honeybee colonies during winter depends on a delicate balance between hive thermoregulation, managing the food reserves and timing the onset of the new worker bee generation. Winter survival is influenced by several factors, most notably colony size, Varroa mite infestation levels and the availability of stored food. Importantly, the climatic conditions and floral resources of the previous foraging season can also impact honeybee health and colony strength before hibernation.

2. This study, conducted across Europe, examines how landscape composition and weather conditions affect the winter survival of honeybee colonies. It uses pollen diversity as a proxy for flower resource quality and available foraging days as a climatic variable to understand their causal relationships to winter survival.

3. We found that landscapes with higher percentages of agricultural areas increased pollen diversity collected by honeybees in autumn, whereas higher percentages of semi-natural areas increased the diversity during summer. Spring and autumn pollen diversity was the main driver for winter survival success, emphasizing the importance of diverse flower resources for colony health. While we did not find a statistically significant effect of weather on winter survival, trends suggest potential influences, warranting further research to confirm and clarify the role of seasonal foraging on colony health.

4. Synthesis and applications: Our study highlights the critical role of including floral resource diversity and weather conditions in a comprehensive framework for studying honeybee hibernation. It suggests that increasing plant diversity around apiaries and implementing agricultural practices that enhance floral resources can significantly improve winter survival, with honeybee colonies benefiting even in landscapes with higher agricultural activity, distinct from the needs of other pollinators.

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

<footnote n="1">¹Department of Animal Ecology and
Tropical Biology, Biocenter, University of
Würzburg, Würzburg, Germany</footnote>

<footnote n="2">²Université Paris-Saclay, CNRS, IRD, UMR
Évolution, Génomes, Comportement et
Écologie, Gif-sur-Yvette, France</footnote>

<footnote n="3">³Department of Apiculture, Institute of
Animal Science, Hellenic Agricultural
Organization 'DEMETER', Nea Moudania,
Greece</footnote>

<footnote>**Correspondence**

Giulia Mainardi

Email: giulia.mainardi@uni-wuerzburg.de</footnote>

<footnote>**Handling Editor:** Lessando Gontijo</footnote>
KEYWORDS

*Apis mellifera*, beekeeping, floral resource availability, landscape composition, overwintering, pollen diversity

# 1 | INTRODUCTION

In temperate zones, perennial eusocial insects face the challenge of surviving extended periods of cold temperatures and scarce resources. For instance, the hibernation strategy of the Western honeybee, *Apis mellifera*, not only involves maintaining optimal temperatures within the hive but also carefully timing the cessation and resumption of brood rearing, along with efficiently managing food supplies (Seeley & Visscher, 1985). Consequently, extensive attention and studies have been dedicated to explaining the complexities of honeybee overwintering (Döke et al., 2015; Minaud et al., 2024) as the survival rate and health of the colonies in spring directly influence both the honey production and the effectiveness in pollination of crops and wild plants (Geslin et al., 2017).

The hibernation strategy for winter survival starts in late summer, when the colony has stored enough winter supplies (nectar and pollen) and the honeybee queen starts rearing 'winter' bees, which differ from summer bees both physiologically (Fluri et al., 1977; Hatjina et al., 2014) and in lifespan (Mattila et al., 2001). As temperatures consistently drop below 15 degrees Celsius, brood rearing stops and the colony forms a cluster that will be able to maintain stable temperatures and humidity conditions inside the hive throughout the winter (Fahrenholz et al., 1989; Stabentheiner et al., 2003). While a successful winter survival strategy involves forming a cluster and maintaining stable conditions inside the hive, several factors can lead to winter colony failure. These include insufficient colony size entering winter (Döke et al., 2019), with smaller colonies being less effective at thermoregulation (Free & Racey, 1968), untreated *Varroa destructor* infestations that weaken the colony before winter (van Dooremalen et al., 2012), and starvation due to inadequate food reserves (Stabentheiner et al., 2003). Such stressors, individually or in combination, can ultimately compromise the colony's ability to withstand the winter months (Döke et al., 2015).

The factors mentioned above are consequences of the environmental conditions and the health conditions of the colonies in the prior growing season, making it difficult to study winter survival as an isolated result of a few parameters measured right before the cluster formation. Therefore, it is necessary to consider a broader causal structure to gain a comprehensive understanding of winter survival.

The availability of floral resources is fundamental for honeybee colonies. Landscape simplification, driven by factors, such as agricultural intensification and urbanization (Goulson et al., 2015), can diminish the richness and complexity of plant community composition (Power et al., 2012). In turn, the loss of suitable flower resources and habitat has become a contributing factor to pollinators' decline (Brown & Paxton, 2009). Flower resources not only affect food availability and storage but also the general health status of individual

bees because of the nutritional content of nectar and, more significantly, pollen found in different plant species (Haydak, 1970; Roulston & Cane, 2000). In managed honeybee colonies, polyfloral diets and enrichment of the surrounding landscape with melliferous catch crops were found to be positively correlated with a general improvement in immune functions (Alaux et al., 2010), tolerance to diseases (Pasquale et al., 2013) and the physiological state of bees (Alaux et al., 2017). However, little is known about the effect of landscape composition on colony performance during the hibernation phase of the colonies. Rutschmann et al. (2022) found that overwintering survival of wild-living honeybee colonies was positively associated with the amount of semi-natural habitats surrounding the nest site, suggesting also that the landscape effect was positively influenced by flower resources. In managed colonies, Requier et al. (2017) found that the availability of pollen resources in spring had indirect causal links to winter colony survival through cascading effects among the demographic traits of the colony. Nonetheless, the direct effect of floral diversity, measured as the diversity of pollen collected by forager honeybees, on winter survival of colonies remains unknown. As much as floral resources, weather plays an important role in the development of the colonies and can be associated with overwintering failure. Weather conditions from spring to autumn can directly affect colony size and health, as honeybees require favourable conditions for foraging flights (Hatjina et al., 2014). For example, extreme precipitation events in spring were associated with greater risks of population loss before winter and with a negative impact on honey production (Quinlan et al., 2023). Regarding winter survival, mathematical modelling approaches have been adopted to employ surveyed data from beekeepers over consecutive winters and to determine the effect of multiple drivers simultaneously. Warmer and drier summers were found to be significant predictors of winter failure (Calovi et al., 2021; Switanek et al., 2017). Likewise, warmer autumns and winters are suspected to increase the chances of failure because frequent foraging flights can alter the cluster structure (Rajagopalan et al., 2024). However, experimental studies that simultaneously assess weather conditions and pollen diversity are largely missing.

This study addresses these gaps by examining the effects of landscape composition, using pollen diversity as a proxy for the available resources, and local weather conditions on the winter survival of honeybee colonies across diverse European landscapes. By conducting a large-scale, multi-year experiment along a climatic gradient in Europe that comprises three main climatic subtypes (oceanic, continental and Mediterranean) but also regional-scale climatic and landscape gradients, we aim to provide a comprehensive understanding of the causal relationship between floral diversity, local weather and winter survival of honeybee colonies. If winter survival is affected by the diversity of pollen collected by the forager
bees, then we would expect that the landscape surrounding the apiaries can either ease or intensify the effort for honeybee colonies to hibernate, depending on the habitat composition. Local weather conditions were also included as an important parameter in our framework since temperature and precipitation frequency during the foraging season as well as the winter season can impact honeybee colony survival.

## 2 | MATERIALS AND METHODS

### 2.1 | Study regions and experimental design

Our study was conducted in three countries—France, Germany and Greece—belonging to different climatic regions of the Köppen-Geiger climate classification 1991–2020 (Beck et al., 2023), based on patterns of seasonal precipitation and temperature (Figure 1). These comprehended: Cfb (Maritime climate, humid temperate), Dfb (continental climate and humid temperate), Csa (Mediterranean climate, warm) and Csb (Mediterranean climate temperate). To establish the experimental apiaries, we selected for each country three climatic regions based on a gradient of mean annual temperature (MAT) from 1970 to 2000 (Fick & Hijmans, 2017), for a total of nine regions. Within each region, we selected three apiaries and categorized them based on the predominant landscape type as semi-natural, agricultural and urban (see Section 2.2). In total, we set up 27 apiaries—nine in each country—from summer 2021 until June 2023, thereby including two overwintering and two foraging seasons. Each apiary consisted of five hives, managed with standardized

beekeeping practices across all regions to minimize variability due to management (see Supplementary Methods). Sister queens of *Apis mellifera carnica* (Germany), *Apis mellifera macedonica* (Greece) and *Apis mellifera mellifera* (France) were used in all hives, ensuring consistency within each region.

This study did not require approval from an animal ethics committee as it did not involve procedures that fall under animal experimentation regulations. Additionally, no specific permits or licenses were needed for the fieldwork conducted.

### 2.2 | Landscape composition and pollen diversity

To quantify the landscape composition surrounding each apiary, we mapped the geographic coordinates of each apiary using QGIS 3.34 (QGIS Development Team, 2009), applying a 2-km buffer to represent the main foraging area for honeybees (Steffan-Dewenter & Kuhn, 2003). Land use data were obtained from the CORINE 2018 Land Cover map (European Environment Agency and European Environment Agency, 2019), and the resulting habitat types found around each apiary were grouped under four main categories: semi-natural, agricultural, urban and others (Table S1). The data were analysed to determine percentages of each of the main categories in each landscape (Figure S3). Pollen diversity, used as a proxy for floral resource diversity, was measured by collecting pollen samples monthly from March to November 2022, by installing pollen traps for 48h at the entrance of two hives for each apiary. In total, we completed seven sampling rounds during the foraging season for each country. As the apiary locations remained unchanged between 2021 and 2022, we assumed that pollen diversity foraged in certain

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Study design. Map of Europe representing the main climatic areas from the Köppen-Geiger climate classification map. Squares in the map point out the study regions, each, including three apiaries with a gradient of land-use intensity, with different percentages of agricultural areas in dark yellow, semi-natural areas in green and urban areas in dark red.</figcaption></figure>
landscapes would be similar between the 2 years. Samples were weighed, freeze-dried for 24h, and subsequently used for plant species identification using DNA metabarcoding, following the protocol from Sickel et al. (2015).

## 2.3 | Weather data

We measured local ambient temperature at 5-minute intervals in each apiary site, using digital temperature sensors placed below the colonies (ca. 20–50 cm above-ground). We retrieved precipitation data for each apiary using the R package *climate* (Czernecki et al., 2020) and selected meteorological stations close to the desired location. We then calculated the average temperature and sum of precipitation events (mm) for each month. For the subsequent analysis, we assigned each month a specific season and calculated the average temperature and sum of precipitation events for each season. The seasons were divided into spring weather (March, April and May), summer weather (June, July and August), autumn weather (September, October and November) and winter weather (December, January and February). Additionally, for each season, we also summed the number of days suitable for flying and foraging of honeybees, with an average temperature above 10 degrees Celsius and total precipitation below 3 mm (Calovi et al., 2021; Hatjina et al., 2014).

## 2.4 | Statistical analysis

We employed DAGs to conceptualize the causal relationships between landscape composition, floral diversity, weather conditions and honeybee overwintering success. The method description for DAGs is presented in the Supplementary Methods (text and Figure S1A). Our statistical analysis consisted of two parts. First, we were interested in studying how landscape composition mediates the diversity of pollen foraged in each apiary during the seasons preceding winter (Model A). The composition of the landscape was studied by using percentages of the main landscape categories (seminatural and agricultural) in a 2 km radius around the apiaries. We excluded percentages of urban areas from the analysis because they were negatively correlated with the areas occupied by agricultural land (Figure S2). Moreover, areas occupied by agriculture tend to be an adequate and broadly studied proxy to represent gradients of landscape simplification (Martin et al., 2019). Pollen diversity (Shannon index) was derived from the package *vegan* (Oksanen et al., 2022) and calculated by grouping the data by apiary for each collection date. Based on the month of collection, we classified our samples into three main periods: spring pollen (March, April and May), summer pollen (June, July and August) and autumn pollen (September, October and November). In the second part of the analysis, we modelled the winter survival of honeybee colonies in terms of the exposure variables determined by the DAGs (Model B). As temperature and

precipitation jointly affect honeybee colonies by constraining foraging activity and are highly correlated between seasons (Figure S2), we chose to use foraging days as the main climate variable in our study. We summed the foraging days of spring, summer and autumn to have the total number of foraging days during the growing season for each colony, while winter foraging days were considered separately. For describing the effect of available foraging resources on winter survival, we utilized the diversity of pollen in the seasons before hibernation. The diversity of floral resources throughout the seasons was used in this case to determine the effect of the surrounding landscape composition on hibernating colonies.

For both models, we fitted a generalized linear mixed model (GLMM), using a Gaussian distribution for Model A and a binomial distribution ('overwinter survival' = 1, 'overwinter failure' = 0) in Model B. Fixed effects included in Model A were: season, country, percentages of semi-natural area (*Semi natural*), percentages of agricultural area (*Agricultural*). To account for the hierarchical data structure, interactions were also included in the model (*Agricultural: season, Semi natural: season, country: season*). The fixed predictors in Model B consisted of the number of foraging days during the growing season (*fdays_growing*), the number of foraging days in winter (*fdays_winter*), seasonal diversity of pollen samples, country and year. Additionally, we included an interaction term between country and foraging days in both the growing and the winter season. Differences in survival between countries and years were modelled in a separate GLMM, with 'country' and 'year' as fixed predictors.

All statistical analyses were implemented using R v.4.3.1 (R Core Team, 2023). Both models accounted for random intercepts for each combination of region and apiary and were fitted using the *glmmTMB* package (Brooks et al., 2017). The package *DHARMa* (Hartig, 2022) allowed us to evaluate the models' performance and identify potential deviations from model assumptions, such as homoscedasticity and normality of residuals; we did not find significant deviations from the model assumptions. Model coefficients from the GLMM were generated using the packages *broom.mixed* (Bolker & Robinson, 2022) and *emmeans* (Lenth, 2023). The graphs were generated using R packages *ggplot* (Wickham, 2016) and *ggeffects* (Lüdecke, 2018).

# 3 | RESULTS

## 3.1 | Foraging days across climatic regions

We found a clear relationship between the number of foraging days and the MAT across the three climatic regions (Figure 2). Apiaries located in the continental climate area experienced the lowest number of foraging days—59 to 178 days during the growing seasons and between 1 and 6 days during winter—while foraging days in the Mediterranean climate were between 119 and 238 days in the growing seasons and up to 63 days of foraging during winter.
<figure><img src="image_4.png" /><figcaption>FIGURE 2 Number of foraging days during the growing season (points) in 2021–2022 (a) and in 2022–2023 (b), as well as the number of foraging days during winter 2021–2022 (c) and 2022–2023 (d), in correlation with the mean annual temperature (MAT) of each apiary.</figcaption></figure>

## 3.2 | Pollen diversity and landscape composition

A total of 358 pollen samples were collected from 27 locations during the growing season of 2022, in which a total of 408 unique plant species were identified (Appendix A). We found that the composition of flower resources used by the colonies changed throughout the seasons in the different landscapes and countries, at family levels (Appendix A, Figure A1). We also observed that a high percentage (from 45% to 63%) of foraged pollen derived from wild plants in the three seasons (Appendix A, Figure A2).

In Model A (landscape composition model) the diversity of pollen strongly differed among seasons (Spring, Est.=0.49, CI=0.15–0.83, *p*-value <0.05) but not among the countries of collection (Figure S4). With a pairwise comparison analysis, we found a significantly higher diversity in spring (Est. = 0.37, CI = 0.24–0.49, *p*-value < 0.05) and summer (Est.=0.33, CI=0.19–0.45, *p*-value < 0.05) compared with autumn. There were no significant interactions between countries and seasons (see Table S2 for pairwise comparison estimates). When assessing the influence of landscape composition on pollen diversity, the model revealed a positive effect of agricultural areas (Est.=0.005, CI=0.002–0.009, *p*-value <0.05), while the interaction between spring and agricultural areas had a negative effect (Est.=0.005, CI=0.002–0.009, *p*-value <0.05). However, the estimated marginal trends provided a more nuanced view across seasons: in summer, semi-natural areas were positively associated with higher pollen diversity compared with agricultural areas, while in autumn, agricultural areas showed a positive effect on pollen diversity (Figure 3). During spring, there were no significant effects of landscape composition on pollen diversity. Pollen diversity increased in

apiaries with increasing percentages of semi-natural areas during summer, while in autumn, the increasing presence of agricultural areas supported higher diversity of pollen (Figure 4).

## 3.3 | Winter survival

Mean winter survival rate calculated for each country between the 2years varied from 53.84% to 97.77% (Table S3). When modelling the survival of honeybee colonies across countries, the marginal effects averaged across years were not significant (Figure S5). The post hoc test instead showed a higher survival rate in Germany compared with France (Est.=4.02, CI=1.29–6.75, *p*-value <0.05) and Greece (Est.= −3.00, CI = −5.68 to −0.3, *p*-value < 0.05; Table S4) probably due to the high survival rate of colonies in Germany in the second overwintering year.

Pollen diversity in both spring and autumn emerged as a key predictor of winter survival in honeybee colonies (Figure S6). Specifically, high pollen diversity in spring (Est=2.63, CI=0.91–4.34) and in autumn (Est.=2.34, CI=0.32–4.36) significantly increases winter survival. The probability of survival increased from 19% to 93% with an increase in spring pollen diversity from 1.8 to 3.4, while in autumn an increment in the Shannon index from 0.80 to 3.40 meant an 85% increase in the survival rate (Figure 5).

In contrast, foraging days during both the growing season and winter season did not have a significant effect on winter survival (Figure S6). Nonetheless, we observed a positive trend for foraging during the growing season and a negative trend for foraging days during the winter season on colony survival rates (Figure S7).
<figure><img src="image_5.png" /><figcaption>FIGURE 3 Seasonal trends in pollen diversity. Estimated trends (slopes) of pollen diversity concerning the percentage of semi-natural areas and agricultural areas in each season. Each point represents the estimates, with error bars indicating 95% confidence intervals (CI). The effects of variables are significant when the 95% CI does not cross zero (significant results are highlighted).</figcaption></figure>

## 4 | DISCUSSION

Our large-scale study on winter survival of honeybee colonies showed that floral diversity, particularly in spring and autumn, plays a crucial role in the overwintering success. Moreover, we reveal the impact of landscape composition on foraging preferences with positive effects of seminatural habitats on pollen diversity during summer and of agricultural habitats during autumn. The approach was especially useful for comparing seasonal variation in pollen resources and their effect on winter survival. In contrast to our expectations, we did not find significant effects of weather on winter survival success.

### 4.1 | Seasonal variation of pollen diversity in landscapes with different composition

We observed seasonal variation of pollen diversity and the effect of surrounding landscape composition on floral resources. Diversity was significantly higher in spring (March to May), but during this period, there were no differences between landscapes with distinct habitat composition. This suggests that even in landscapes with an increasing presence of human disturbance, especially in times when high percentages of mass flowering crops are expected (see also Harris et al., 2024), honeybees aim to diversify their foraging. In spring, the most common crop in agricultural landscapes is oilseed—particularly in Germany and France (Wittkop et al., 2009). However, in Mediterranean climates like Greece, fruit orchards are also widely present in spring (Liolios et al., 2015; Statistics-ELSTAT, n.d.). Also, during this season, there is an intense rate of brood rearing,

and as previously studied, we expected that honeybees match their needs for protein by foraging on a higher diversity of plants (Mattila & Otis, 2006). During summer (June to August), when pollen availability typically declines (Satta et al., 2024; Steffan-Dewenter & Kuhn, 2003), we found that increasing areas of semi-natural habitat had a positive effect on pollen diversity. This finding aligns with Danner et al. (2016), who observed that foraging distances in summer were overall reduced by the presence of semi-natural habitats near the hives. Interestingly, later in the growing season (September to November), landscapes with a higher percentage of agricultural land were more strongly associated with pollen diversity, in contrast with the study of Malagnini et al. (2022), whose results indicated that semi-natural habitats during this time were more important in determining diversity. In our apiaries, the majority of the recognized pollen species originated from wild plants (Appendix A), indicating that there might have been a significant contribution of field margins, flower strips and hedges surrounding the cultivated field (see also Jachuła et al., 2022). However, this hypothesis must be interpreted with caution since we did not measure the mentioned structures in our landscapes. Nevertheless, besides the seasonal variability, in this study, we observed that landscapes dominated by agricultural land can support the high diversity of plants needed for the hives during the time preceding hibernation. The aforementioned reinforces the idea that the studied species is less sensitive to anthropological disturbance compared with other pollinators, which instead require proximity to natural habitat to thrive (Biegerl et al., 2025; Ricketts et al., 2008). The positive interaction between autumn pollen diversity and landscapes with higher percentages of agricultural habitats highlights possible differences in foraging preferences among pollinators and suggests that agricultural landscapes in our
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>FIGURE 4 Estimated diversity predicted by different habitats. Regression lines represent the estimated pollen diversity with respect to the percentage of semi-natural areas (a) and agricultural areas (b). Significant results are displayed as a solid line.</figcaption></figure>

study regions provided rather diverse pollen sources for honeybee colonies in autumn. This indicates that managed honeybee colonies depend less on the presence of semi-natural areas than other wild pollinators (Biegerl et al., 2025). This suggests, in terms of beekeeping management, that honeybee colonies in late summer and autumn can benefit from agricultural landscapes with diverse floral resources and do not specifically need a high presence of when most wild plant species in natural and semi-natural areas have finished their flowering phenology. The implementation of agri-environmental schemes encompassing flower fields and intercropping with diverse flower mixtures can contribute to filling late-seasonal resource gaps

and should be promoted by the Common Agricultural Policy of the European Union (CAP) (Boetzl et al., 2021).

## 4.2 | Effect of floral diversity and weather on winter survival

Although landscape composition has been considered in studies of honeybee overwintering (Kuchling et al., 2018; Requier et al., 2017; Rutschmann et al., 2022), floral diversity was never directly associated with winter survival. In our study, we were able to establish a clear
<figure><img src="image_8.png" /><figcaption>FIGURE 5 Winter survival predicted by pollen diversity. Regression lines represent the model prediction for colony survival, with increasing pollen diversity in spring (a) and autumn (b). The 95% confidence interval (CI) is represented by the coloured area around the regression line.</figcaption></figure>

connection between the diversity of pollen collected in spring and autumn and a higher winter survival of the colony.

Previous studies reported that the availability of pollen during spring contributes to the rearing of a higher number of worker bees and therefore promotes greater colony size and honey yield later in the season (Hatjina et al., 2014). As suggested by Requier et al. (2017), the decline of foraged spring pollen can cause a carry-over effect, leading to colonies with a reduced number of adult bees and therefore with lower winter survival rates. Our study showed that the diversity of pollen in spring was one of the key drivers of overwintering survival. This highlights that not only the mass of foraged pollen but also the diversity of the diet plays an essential role in winter survival success, probably by enhancing the health of the colonies early in the foraging season. Similarly, autumn pollen diversity was strongly correlated with an increased probability of winter survival. Pollen feeding during autumn, which precedes the formation of the winter cluster, nourishes winter bees who have a decisive role in the cluster's thermoregulation and survival in the following cold period (Southwick, 1991). Additionally, in the months leading up to the time without flowering plants and foraging, pollen is stored inside the hive and only consumed when brood rearing resumes, making it essential for the development of the next generation of spring bees. Yet, a higher diversity of pollen, as opposed to pollen with higher nutritional values (in terms of protein percentage), was never directly linked to improved individual bee health or better colony development (Pasquale et al., 2013). However, as previously noted by Alaux et al. (2010), the benefit of a polyfloral pollen diet and potentially from highly diversified sources, lies in providing bees with a more balanced composition of amino acid compounds and

other nutrients essential for the development and health of hon-
eybee larvae. Summer pollen diversity instead was not relevant in
terms of survival in our study, even if it is valuable to mention that
continuity of resources throughout the season can be decisive for
the maintenance of strong and healthy colonies.

On the contrary, weather did not appear to significantly in-
fluence the overwintering survival of the colonies in our study.
Although we observed a trend suggesting that the number of forag-
ing days might negatively influence the overwintering survival, this
effect was not statistically significant. Milder winter days could also
be beneficial for the overall health of the winter cluster since they
allow winter bees for regular hygiene flights and water collection
(Becsi et al., 2021). The lack of correlation may be attributed to the
limited duration of the experiment, with 2 years of overwintering
data, as temporal variation in environmental conditions or biological
processes might not be fully captured. While we aimed to address
this limitation with a high number of spatial replicates and a large
geographic coverage in our study design, this approach cannot en-
tirely substitute for replicates across time. Future studies conducted
over longer periods would help to confirm the patterns we observed
and provide a more comprehensive understanding of the system
under varying temporal conditions. If the trends found in our study
are confirmed, they would suggest a great ability of the winter hon-
eybee to adapt to different ambient conditions and their efficiency
to overcome extreme weather events (Stabentheiner et al., 2021).
Similarly, we did not observe a significant effect of foraging days
during the growing season on winter survival, although a positive
trend was present. The use of foraging day counts might not fully
capture the complexity of weather-related stressors, and more
detailed tracking of colony dynamics, such as automated weight and flight activity recording, could provide additional insights into how seasonal foraging behaviour influences long-term colony health (McMinn-Sauder et al., 2024; Meikle et al., 2018).

## 5 | CONCLUSIONS

While Varroa levels and colony size are often considered the primary causes of winter colony collapse, our findings emphasize the importance of including floral resources and weather conditions within a broader framework for studying honeybee overwintering survival. Although our results regarding weather were inconclusive, it remains a crucial factor for further research, as annual local weather patterns can significantly influence both colony development and the exploitation of floral resources. The diversity of resources collected by foragers during key periods—spring, when colonies are developing, and autumn, when they are preparing for winter—is especially important, as our data demonstrate that it can significantly enhance the survival of honeybee colonies. This suggests that monitoring floral resources and increasing the diversity of attractive plants around apiaries are practical strategies for improving honeybee winter survival. This study also highlights the crucial role of agricultural areas, which are already influenced by varying levels of human management. Therefore, implementing agri-environmental schemes that promote plant species attractive to honeybees—such as creating flower fields and strips, managing field margins and optimizing mowing practices—can be widely adopted (Boetzl et al., 2021). Overall, the results presented in this study show that maintaining provision of diverse flower resources throughout the growing season can improve the survival rate of honeybee colonies during winter and potentially benefit wild-living honeybee colonies (Kohl et al., 2023) and other pollinators with similar resource requirements.

### AUTHOR CONTRIBUTIONS

Fabrice Requier, Ingolf Steffan-Dewenter, and Fani Hatjina planned and conceptualized the experimental design. Giulia Mainardi, Etienne Minaud, Filippos Vardakas, and Leonidas Charistos conducted the fieldwork and data collection. Giulia Mainardi and Douglas Sponsler performed the statistical analysis. Giulia Mainardi led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

### ACKNOWLEDGEMENTS

We deeply thank all the beekeepers and associations that participated to this project: the 'Conservatoire de l'Abeille Noire En Combrailles' (CANEC), the 'Conservatoire de l'Abeille Noire d'Ile-de-France' (CANIF), the 'Association pour la sauvegarde de l'Abeille Noire, île de Groix' (ASAN.GX) and 'Alti Agri' in France; to 'B.S.R.C Alexander Fleming', the Agricultural University of Athens, 'Symbeeosis', and A.N.E.L., Dr. Patalano, Dr. Goras, Vangelis Fytros, Michalis Moustakis and Kostas Chiotis in Greece; and to Peter Reh, Christina Rödner-Schneider, Christina Dewenter, and Thomas Russ

of the Staatliches Berufliches Schulzentrum in Germany. Special thanks to Beate Krischke and Ann-Kathrin Mais (ZOO3 department, JMU Würzburg) for the DNA metabarcoding analysis, and to all field assistants (Susanne, Nikki, Anne) and students (Silvia, Erik, Christina) that contributed to collecting data. Open Access funding enabled and organized by Projekt DEAL.

### CONFLICT OF INTEREST STATEMENT

Fabrice Requier is an Associate Editor of *Journal of Applied Ecology* but took no part in the peer review and decision-making processes for this paper.

### DATA AVAILABILITY STATEMENT

Open Research: Data and code are permanently available on Dryad under: https://doi.org/10.5061/dryad.vx0k6dk36 (Mainardi et al., 2025).

### ORCID

Giulia Mainardi https://orcid.org/0000-0003-4635-0586

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**Figure S1A.** DAG visualization.

**Figure S1B.** Varroa loads for each country.

**Figure S2.** Correlation measures.

**Figure S3.** Apiaries selection.

**Figure S4.** Pollen diversity model estimates.

**Figure S5.** Survival model estimates for country.

**Figure S6.** Survival model estimates.

**Figure S7.** Estimated effect of foraging days on survival.

**Table S1.** Selected categories for CORINE Land Cover habitat types.

**Table S2.** Pairwise comparisons of pollen diversity between countries and their interaction with seasons and between seasons.

**Table S3.** Mean overwintering survival over two years field study.

**Table S4.** Pairwise comparisons of winter survival between countries.

**Appendix A:** Table A1. List of pollen species. The table reports the plant species identified with the pollen DNA metabarcoding.

**Figure A1.** Plant family mean abundance.

**Figure A2.** Proportion of pollen sourced from different plant type.

**How to cite this article:** Mainardi, G., Sponsler, D., Minaud, E., Vardakas, F., Charistos, L., Requier, F., Hatjina, F., & Steffan-Dewenter, I. (2025). Floral diversity enhances winter survival of honeybee colonies across climatic regions. *Journal of Applied Ecology*, 00, 1-11. https://doi.org/10.1111/1365-2664.70054