Animal pollination contributes to more than half of Citrus production

Animal pollination contributes to more than half of Citrus production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Animal pollination contributes to more than half of Citrus production MARCOS MONASTEROLO, Andrés Felipe Ramírez-Mejía, Pablo Cavigliasso, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4474196/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Animal pollination is crucial for the reproduction and economic viability of a wide range of crops. Despite the existing data, the extent to which citrus crops depend on pollinators to guarantee fruit production still needs to be determined. Here, we described the composition of flower visitors in citrus ( Citrus spp.) from the main growing areas of Argentina; moreover, we combined Bayesian models and empirical simulations to assess the contribution of animal pollination on fruit set and yield ha − 1 in different species and cultivars of lemon, grapefruit, mandarin, and orange in the same regions. Honeybee ( A. mellifera L.) was the most commonly observed visitor, followed by a diverse group of insects, mainly native bees. Regardless of citrus species and cultivars, the probability of flowers setting fruit in pollinated flowers was 2.4 times higher than unpollinated flowers. Furthermore, our simulations showed that about 60% of the citrus yield ha − 1 can be attributable to animal pollination across all species and cultivars. Therefore, it is crucial to maintain environments that support pollinator diversity and increase consumer and to producer awareness and demand in order to ensure the significant benefits of animal pollination in citrus production. citrus fruit fruit set pollinator-dependent crops pollination service yield food crops Figures Figure 1 Figure 2 Figure 3 Introduction The contribution of animal pollination to global agriculture is increasingly acknowledged 1 . Yet, pollination requirements vary considerably between and even within species and cultivars. Species variability is mainly due to different crop breeding systems and the regional and local communities of pollinators 2 , 3 . For instance, crops such as sugarcane, rice, or maize are predominantly wind-pollinated, and thus the presence of insect pollinators does not impact their production. However, for the vast majority of crops, such as pumpkin, almonds, cherries, and tomatoes, insect pollinators are crucial for the fruit or seed quantity and/or quality 1 , 4 . Pollination can be a limiting factor for these crops, as yields are expected to increase in the presence of pollinators until the crop is fully pollinated 5 . Cultivars within crop species may also respond differently to animal pollination 6 , for example, soybean 7 , blueberry 8 , canola 9 , bean 10 and apple 11 . This variability may be due to cross-cultivar variation in the mating system that affects the interaction with pollinators 12 or reduces the need for pollen deposition for ovule fecundation 13 . Pollinators contribution to crop yield is evaluated through experiments, contrasting between flowers with open access to pollinators and those with close pollination (pollinator exclusion experiments), or flowers with hand cross-pollination and those with hand self-pollination (pollen supplementation experiments) 14 , 15 . The proportion of flowers that set mature fruits or seeds (i.e. fruit/seed set) is then compared across experiments as the difference between open and excluded treatment and a pollinator contribution or pollinator dependence value is reported. The first compilation of studies that valued the contribution of animals to crop production 1 had a great impact, as they stated that most of our crops are benefited by animals, to a certain degree. However, nowadays there is a search for more agronomic/economic impacts of pollinators on crop yield that can be communicated more directly to producers 16 . For example, measures such as fruit quantity, fruit quality, and yield stability per unit area that are lost in the absence of pollinators are more informative. This information will provide growers and stakeholders with an impulse for the conservation of pollinator services and market policy to implement agri-environmental programs with a meaningful focus on pollinators 17 . Citrus fruits ( Citrus spp. such as oranges, mandarins, lemons, and grapefruits) are one of the main and most widespread crops globally, with a production of over 140 million tons in 2020 18 and were considered as a group of species with little dependence on pollinators, but with variability between species 1 . Citrus fruits comprise a group of more than ten cultivated species with many varieties and include all breeding systems ranging from agamospermy and parthenocarpy (asexual reproduction), self-incompatibility, through all forms of self-pollination, to self-incompatibility and cross-pollination. Thus, it is challenging to generalize citrus responses to pollinators due to their numerous varieties and complex pollination requirements. It is said, that gamospermous or parthenocarpic citrus varieties do not require pollination to bear fruit (e.g., seedless mandarins, Salustiana oranges, some grapefruits, and lemons) 19 . However, a significant number of citrus varieties, including oranges but especially grapefruits and mandarins, are self-incompatible and require or benefit from cross-pollination facilitated by insects to produce fruit or to enhance the yield and quality 20 , 21 . Many studies conclude that cross-pollination by insect pollinators enhances fruit set, with varying degrees of dependency depending on the species and variety 22 – 30 . In addition to increased fruit set, some studies find that insect pollination results in fruits of higher quality, with larger size, weight, and more juice quantity and sugars 24 , 25 , 31 , 32 . Thus, the contribution of pollinators to citrus production remains controversial 20 , 33 . Given the wide variability in their breeding systems and the ample differences reported between studies, an actualized assessment of the cultivated species and cultivars is necessary 4 , 33 . In this study we used empirical data from different species and cultivars of citrus 1) to describe the main functional groups of pollinators that visit citrus crops in Argentina; also, 2) we evaluated the influence of animal pollination on the probability of the flower setting fruit across citrus species/cultivars, and 3) its consequences in crop yield ha − 1 across citrus species/cultivars. In light of this, we utilised a database containing visits and fruit set percentages of different citrus groups (grapefruit: C. paradisi , mandarins: C. reticulata and C. x clementina , lemon: C. limon and oranges: C. sinensis ) belonging to commonly cultivated cultivars. Also, using this dataset, we conducted simulations to estimate the contribution of insect pollination to different productivity measures to reach a broader spectrum of stakeholders about these impacts. Results Composition of citrus flower visitors In total, we recorded 21,553 visits to the flowers of all citrus species in 323h. The most common visitor to all citrus flowers was A. mellifera , which accounted for 87% of the total visitors recorded. Bees accounted for half of all observed wild pollinators, with 33% being small native bees and 17% being medium/large bees. Among the remaining groups, beetles were the most common (17%), followed by dipterans (14%), and wasps (9%). In lemons and grapefruits, more than 87% of the insects observed were A. mellifera (Fig. 1 a and 1 d), while in mandarins and oranges, observations of wild pollinators predominated (61% and 72%, respectively; Fig. 1 b and 1 c). In each citrus group, more than half of wild pollinator observations were of bees, except in oranges, where beetles were the most common group (37%) (Fig. 1 c). Influence of pollinators in the probability of flowers setting fruit In the close pollination treatment, the probability of flowers setting fruit was low on average for all citrus groups (1.25% CI, 0.78–1.95; Fig. 2 ), i.e. when flowers were not visited by insects, on average, between one or two fruits are formed per hundred flowers. Meanwhile, in the open pollination treatment, the probability of a flower setting a fruit was 3.07% (CI, 1.94–4.67). This means that flowers from the open treatments had 2.4 times higher probability of setting fruit than those from close pollination (Fig. 2 ). Indeed, this pattern remained consistent across citrus groups (2.4 ± 0.04 SD) and cultivars (2.4 ± 0.07 SD) (see Supplementary Information 1, section 2.7-Table S3, S4). Under open pollination conditions, lemon cultivars had the highest probability of flowers setting fruit (11.0% ± 2.01), followed by grapefruits (2.98% ± 0.47), mandarins (2.62% ± 2.03), and oranges (1.98% ± 0.77). The same pattern was observed for the close pollination treatment (lemons = 4.79% ± 1; grapefruits = 1.23% ± 0.2; mandarins = 1.08% ± 0.84; oranges = 0.87% ± 0.31) (Fig. 2 , Supplementary Information 1, section 2.7- Table S3). Rhat values and visual diagnostics showed that all chains converged to the same posterior distribution. The ess of all parameters was > 1,000 and 96% of pareto k values were below 0.7 (see Supplementary Information 1, section 2.5). The posterior predictive checks indicated that the model was well-fitted (see Supplementary Information 1, section 2.6). Contribution of pollinators to citrus production When comparing open vs. close pollination treatments, our simulations showed that, regardless of the citrus groups or cultivar, there is an ~ 80% probability that trees exposed to animal pollination produce more fruits (see Supplementary Information 1, section 3.7.2). Considering the contribution of animal pollination to citrus production, i.e. the posterior distribution of the contrast between open and close pollination, the simulations showed that animal pollination contributes to the production of 739 fruits tree − 1 (SD ± 70.4) of lemons (accounting for 71.5 kg tree − 1 ± 11.2), 159.25 fruits tree − 1 (SD ± 126.2) of mandarins (accounting for 15.2 kg tree − 1 ± 9.2), 114 fruits tree − 1 (SD ± 48.1) of oranges (accounting for 24.03 kg ± 8.7), and 168 fruits tree − 1 (SD ± 11.3) of grapefruits (accounting for 35.4 kg ± 4.2) (Fig. 3 ). Considering the density of trees planted per ha, we found that animal pollination contributed to 36.1 t ha − 1 (SD ± 6.8) of lemons (56.5% ± 3.5 of total production), 5.9 t ha − 1 (SD ± 2.3) of mandarins (58.9% ± 5.9 of total production), 10.1 t ha − 1 (SD ± 2.4) of oranges (59.1% ± 5.7 of total production), and 10.5 t ha − 1 (SD ± 2.2) of grapefruits (58.9% ± 6.6 of total production) (Fig. 3 ). Discussion The influence of animal pollination on crop yield can vary due to several factors. In citrus crops, the contribution of pollinators is still a matter of debate, mainly because of the wide variety of reproductive strategies within citrus species and cultivars, but also due to spatial, temporal and biological factors 4 , 34 . Using data from main citrus species and cultivars in large regions of Argentina, we found that animal pollination increases about two times the probability of flowers setting fruit in the citrus crop, resulting in a contribution of about 60% of the total yield at the hectare level. Therefore, despite the sometimes neglected role of animal pollination for this crop, our study provides evidence that justifies safeguarding pollinators in citrus agricultural landscapes. The exotic honeybee A. mellifera was the main visitor to the citrus flowers. A diverse group of native pollinators, mainly native bees, participated in the visits to a lesser extent. The widespread occurrence of honeybees is common in citrus pollinator studies 20 , but it should be considered that honeybees in these agricultural landscapes can be either managed or feral. In agroecosystems of the region, the abundance of honeybees is linked to the proximity of natural habitats 35 and the abundance of locally managed honeybee hives for the production of honey. Studies have shown that the introduction of managed honeybees can increase flower visitation rates, which can result in a reduction in mature fruits per flower and lower fruit quality in various crops 36 , 37 , including citrus 38 . It is crucial to acknowledge that an increase in pollinators does not necessarily lead to enhanced fruit production, and the native pollinators may be as or more efficient in fruit production than managed bees 39 . In our study, the most abundant wild pollinators were small bees, mainly stingless bees, and the eusocial bees Lasioglossum spp. Curtis. These bees could prove to be valuable for citrus even in the presence of managed hives, as highlighted by previous studies 40 – 42 . Consequently, the adoption of effective local and landscape management strategies to enhance flowering and nesting resources and to reduce current environmental pressures on wild pollinators may be sufficient to achieve optimal benefits. Our results have shown that in all the citrus groups and cultivars studied, insect pollination has a positive effect on the proportion of flowers setting fruit, increasing more than double the number of fruits compared with non-pollinated flowers. Therefore, according to our data, insects are certainly important for citrus fruit production. In the lemon cultivars of our study, the percentage of fruit set in open pollinated flowers was lower than that reported in the literature for C. limon (29.7% Layek et al. 43 ). However, the increase in fruit set in open versus closed treatments was very similar in both studies (44% in our study and 45% in Layek et al. 43 ). The fruit set of mandarins and oranges varies greatly due to the wide variety of cultivars and breeding. The Clementine mandarin is facultative parthenocarpic, i.e. it requires pollen stimulation by pollinators to set fruit 44 . However, the probability of fruit set was doubled when the flowers were pollinated by insects compared to exclude flowers 26 . Studies on mandarin pollination have found either a smaller increase 42 or a similar number of mature fruits 31 in insect pollinated flowers compared to our study. Previous studies have found that the increase in cross-pollination treatments is dependent on the cultivars used 24 , 27 – 29 . In studies on oranges, the values in flowers with open pollination for the Pera Rio variety were 30–78% higher than in excluded flowers 25 , 45 , 46 . For grapefruit, the only published results available in the literature correspond to the data used in this work 21 . As there was no considerable variability in fruit set between cultivars of the same species under open versus close treatments, the variations in fruit set reported in previous studies may be attributed to differences in estimation methods, limited replication and a small number of studies involving other species and cultivars. Our study reveals the contribution of pollinators to citrus yield, but we did not explore the contribution of pollinators to seed setting in these fruits, which is an important marketable trait in citrus, i.e. seedlessness. Seedlessness is important for fresh citrus use, whereas this trait is less important for industrial citrus 25 , 47 . Seedless varieties are a desirable productive trait in most citrus, and for most species, pollination and fertilisation result in seed formation 47 . Therefore, there is a potential trade-off between the need of animal pollination to increase the probability of flowers setting fruit (i.e. production quantity), and the market demand of seedless production (i.e. quality production). Yet, for certain cultivars, such as some mandarins, lemons, and grapefruits, seedlessness is not required by the market or even possible 21 – 23 , 38 , 48 . In addition to production quantity, pollination can also improve the sugar content 25 , 38 and weight of the fruits 42 due to pollen deposition and fecundation of the ovules which activate hormonal processes related to fruit growth, seed formation, pulp firmness and thus increase the post-harvest quality of fruits 49 . Despite the numerous studies that provide evidence of the benefits of pollination in this production system, when the number of seeds is relevant, growers often have to choose based on market demand. They must choose between increasing the yield and quality of the fruit or reducing a trait that affects the destination market of the production, which alters the value of the product 50 . Our results indicate that without animal pollination, the amount of fruit produced per hectare (equivalent in tonnes/ha) would be reduced by an average of 58.35%. All the species and cultivars studied showed similar levels of pollinator dependence, despite differences in environmental conditions, spatial arrangements, farm management practices, and composition of flower visitors. The degree of pollinator dependence on citrus fruits has been previously reported in the literature in compilations, with widely varying results 1 , 4 , 15 . Klein et al. 1 reported that pollinators make a small contribution to citrus fruit production, with less than 10% dependence. Mallinger et al. 4 report values of pollinator contribution in Florida (USA), of 73% for grapefruits, 31% for lemons and oranges, and over 80% for tangelo and mandarins. Siopa et al. 15 reported high levels of pollination dependence in citrus of around 65–80%, except for oranges which had a lower dependence of around 20%. This latter study used hand pollen supplementation to assess the yield associated with animal pollination, in contrast to our study, which used the open pollination treatment. It is important to note that the use of hand pollen supplementation tends to show higher values of pollinator contribution 15 , which indicates that there can be some level of pollen deficit. Therefore, although the contribution of animal pollination in most citrus studies indicates a relative/high dependence of pollinators on citrus yields, values of pollinator dependence and reporting of these values may vary according to the estimation methods used. In this study, we considered the contribution of pollinators to citrus production in terms of yield ha − 1 , including aspects such as fruit set, fruit weight, and literature value of floral display, in contrast to previous studies that use fruit set to calculate this contribution. Previous studies classified crop pollination dependence (for example, little, modest, high, and essential in Klein et al. 1 ), where the difference between close pollination treatments and pollinator-associated production treatments (open pollination or hand pollen supplementation) was calculated as 1-(close/pollinator-associated production). However, the fruit set alone does not fully reflect the true dependence on pollinators in commercial crop production 17 . These studies do not take into account quantitative differences in fruit, such as the weight of fruit per tree or per hectare lost in the absence of pollinators, as calculated here. Also, in contrast to the above-mentioned approaches, our results reflect the potential implications of pollinator absences on farms and allow us to assess the precise value of pollinators on agricultural production using commercially relevant yield metrics. This approach is advantageous as it is more effective and allows for more concrete communication for the decision-making of growers, industry, and stakeholders. To estimate the contribution of animal pollination in Citrus, we fitted Bayesian models that necessarily neglect the role of plant physiological state and other agronomic inputs (e.g. pruning practices, irrigation, fertilisation, pest control), which should be taken into account in future studies. We also recommend that future research should assess whether the contribution of pollinators to the production quantity is reflected in certain aspects of citrus quality that also have a significant impact on market value, such as weight, seed quantity, size, sugar concentration, and others. Furthermore, although not documented in our study, the presence of managed beehives at sites close to the crops certainly influenced their high abundance. To achieve this, the number of managed hives in the vicinity and their distance from citrus plantations should be recorded, to assess the pollen limitation and the ideal number of visits to achieve high fruit yields in citrus production. This study showed that animal pollination more than doubled the fruit set and contributed around 60% of citrus yield/ha − 1 , regardless of species and cultivar. In light of these results and the future of the national citrus market, which is increasingly focused on quality parameters that emphasise the absence of animal pollination to develop 'seedless citrus', and in the face of an imminent advance of diseases that threaten the quality of fresh fruit for export (e.g. citrus Huanglongbing or HLB), it will be necessary to evaluate a change in future market strategy or a re-evaluation of the quality standard to be marketed. In this context, animal pollination ensures a higher quantity and quality of fruit, both in terms of the weight of individual fruits and the sugar content of the juice produced 25 , 33 , 38 , 42 . Therefore, changing market demands, based on pollinator conservation may also be a possibility, especially when the number of seeds is not so relevant. Empirical studies, market analysis, and policy interventions aimed at promoting pollinator conservation and sustainable agriculture can assist stakeholders in better understanding and mitigating the risks associated with pollinator declines while harnessing the economic benefits of animal pollination for food systems globally. Effect of pollinators on the probability of flower setting fruits To assess the influence of pollinators on the fruit set of grapefruit, lemons, oranges and mandarins, experiments were conducted comprising two treatments: open pollination and close pollination through bagging branches with flower buds (for more details on these methodologies see Chacoff and Aizen 21 ; Monasterolo et al. 38 ). In each tree, we selected at least two branches with flowering buds and applied one of two pollination treatments: open pollination and close pollination treatment. In the open pollination treatment, we recorded the number of mature flowering buds when flowering season was starting and we counted the number of initial fruits when fruits were formed (one month after flowering). In this treatment, flowers were exposed to natural levels of pollination, including both animal pollination and self-pollination due to wind pollination. For close pollination treatment, a branch with flowering buds was excluded from visitors by using a voile that permitted the action of the wind but not the visits from insects, here only self-pollination due to wind pollination can occur. The bags were removed after flowering and the number of fruits formed in each treatment was counted to calculate the fruit set (fruits/flowers). The number of sampled plants and branches is provided in Table 1 (for more details see Supplementary Information 1, section 2.2 - Table S1 ). Table 1 Number of branches sampled per treatment, species, and cultivar. Pollination treatment Species Cultivar Close pollination Open pollination Total Lemon Limoneira 137 133 270 Lisboa 55 51 106 Santa Teresita 12 14 26 Total Lemon 204 198 402 Mandarin Clementine 11 11 22 Criolla 690 206 896 Total Mandarin 701 217 918 Orange Salustiana 12 11 23 Valencia 11 9 20 Total Orange 23 20 43 Grapefruit Pink 11 10 21 Rio Red 33 34 67 Rouge La Toma 37 37 74 Total Grapefruit 81 81 162 Total 1009 516 1525 Data analysis Effect of pollinators on the probability of flower setting fruits We used a Bayesian hierarchical model to assess the effect of animal pollination exclusion on the probability of the flowers setting fruit: fruit produced i ~ binomial(n sampled flowers i , p i ) logit(p i | treatment i , species i , cultivar i , tree i , year i , locality i ) = α treatment i + τ species i + ɣ cultivar i + θ tree i + ẟ year i + λ locality i We considered the pollination treatment as the population effect, and citrus species, cultivars, sampled tree, year of sampling, and locality as group-level effects. To account for variation in sampling size among citrus species/cultivars and reduce overfitting, we used partial pooling for parameter estimation. The probability of flowers setting fruit tends to be low for Citrus spp. 56 , hence we defined α treatment i ~ Normal(5, 2.5) as prior (i.e. number of fruits developed from n observed flowers), and a Normal(0, 1) for all group-level parameters. We estimated the joint posterior distribution of p i (i.e. probability of flowers setting fruit) using the Hamiltonian Monte Carlo algorithm through Stan 2.32.2 and the R package cmdstanr . We fitted the model by setting three chains, 4000 sampling and 500 warming iterations, and a thinning rate of 3. We conducted sampling diagnostics of all parameters through visual assessment of chains convergence, Rhat 1000, and pareto k values < 0.7. We also assessed the quality of the model fit through posterior predictive checks. See Supplementary Information 1, section 2.3 for the mathematical notation of the model and section 2.4 for the Stan code to fit the model. Contribution of animal pollination to citrus production Assuming that the differences in the probability of flowers setting fruit between experimental treatments can be attributed to animal pollination, we build simulations to estimate the contribution of pollinators to fruit production and yield of lemon, mandarin, orange, and grapefruit. Annotated R code and main functions conducting the simulations can be consulted in Supplementary Information 1, section 3.1. The simulations followed this reasoning: Step 1 — Considering the floral display size of Citrus spp. reported in the literature (25000 to 200000 flowers in oranges in 55 , or 20000 to 50000 flowers in grapefruits in 56 ), and information from citric producers in the localities studied, we hypothesise that the number of flowers produced by Citrus spp. trees can be recreated through a negative binomial probability distribution: flowers per tree ~ NB(𝜎 = 2, 𝜇 = 15000) 𝜎 denotes the dispersion parameter of the distribution, and we defined 𝜇 = 15000 to remain conservative on the average number of flowers produced per tree (Supplementary Information 1, section 3.1). Step 2 — We used step 1 to simulate as many random trees with n flowers as trees expected per ha for each Citrus species. For lemon cultivars, we assumed 400 trees ha − 1 (5 ✕ 5 m); orange and mandarin cultivars 278 trees ha − 1 (6 ✕ 6 m); and grapefruit cultivars 278 trees ha − 1 (8 ✕ 6 m). We replicated this process 10000 times per Citrus /cultivar (i.e., 10000 simulated hectares per Citrus /cultivar). We defined the plantation distance according to information provided by growers (Supplementary Information 1, section 3.2). Step 3 — Then, we used the marginal posterior distribution of the probability of flowers setting fruit per pollination treatment to predict the number of fruits produced per simulated tree in step 2 : See supplementary material 1, section 3.3.2.1 for this simulation in lemon (Limoneira cultivar). fruits produced i ~ binomial(n flowers [random tree i] , p [sp, cultivar, treatment] ) Step 4 — We used the simulated fruits in step 3 for each pollination treatment per Citrus /cultivar to estimate the probability that trees exposed to animal pollination (i.e. open pollination treatment) produce more fruits than those from close pollination treatment (Supplementary Information 1, section 3.3.2.1). Step 5 — We calculated the contrast of the marginalised posterior distribution of the probability of flowers setting fruit between pollination treatments. Then, we used the contrast and the simulated trees in step 2 , to simulate the number of fruits attributed to animal pollination (Supplementary Information 1, section 3.3.2.2). fruits produced animal pollinationi ~ binomial(n flowers [random tree i] , p [sp, cultivar, contrast] ) Step 6 — We fitted Bayesian models to estimate the posterior distribution of fruit weight of lemon, grapefruit, and Criolla mandarin (Supplementary Information 1, sections 3.3.1, 3.4.1 and 3.6.1, respectively). The fitting and models diagnostics procedure follows the above-mentioned protocol. Since raw data on fruit weight for orange (Valencia and Salustiana) and Clementine mandarin was not available, we used mean and SD values reported in the literature 59 to parameterize normal distributions (Supplementary Information 1, section 3.5.1). Then, we used these distributions to simulate fruit weights for each Citrus species and cultivar (Supplementary Information 1, section 3.3.4.2). fruit weight i ~ normal(𝜇 [species, cultivar] , 𝜎 [species, cultivar] ) Step 7 — Finally, we used the distributions of step 6 to simulate as many fruit weights as fruits per tree were generated in step 5 . The sum of weights per tree and the sum of trees per ha denoted the production attributed to animal pollination in kg and Tn, respectively. We conducted 10000 simulations per Citrus species/cultivar. We repeat steps 5 to 7 with the posterior distribution of open pollination treatment and use the simulations from the contrast to estimate the percentage of crop production per ha that can be attributed to animal pollination. We performed all simulations and statistical procedures in R 4.3.1 60 , we did data wrangling operations using base and dplyr packages, and plotted the models and simulations outputs with ggplot2 and cowplot packages. We provide the R script with annotated code to reproduce the simulations and models fit in Supplementary Information 1, section 3. Methods Cultivars Citrus ( Citrus spp., such as oranges, mandarins, lemons, and grapefruits) represent a significant portion of Argentina's fruit production, covering 23.9% of the total production area 51 . Across Argentina, an average of approximately 132,669 hectares of citrus is harvested annually, generating nearly USD 378 million in export earnings from the marketing of over 3.3 million tons of fruit (period 2018-2022 52 ). The citrus flowers are perfect, containing both pistils and stamens, thus self-pollination can occur without pollinators 20 . The fruits display a variety of shapes, sizes, and other quality parameters according to species and cultivars 53 . A vigorous citrus tree can produce between 20,000–250,000 floral units during the flowering period 54 , 55 . However, typically, a very low percentage (between 0.1 and 3%) of these flowers develop fruits 54 , 56 . In general, citrus flowers are highly attractive to pollinators. Citrus trees experience mass flowering, typically in early spring when there are still few wildflowers, thus providing valuable nectar and pollen resources for pollinators. The diversity of pollinators in citrus varies according to geographical area and the flowering season. Usually, professional pollination services are not utilised in these crops, because of the common belief that citrus does not require insect pollination to set fruits and even in some citrus, pollination is intentionally avoided, which is a relatively common practice in some mandarins for a desirable 'seedless citrus'. Study area and flower visitor sampling This study is based on empirical data from various sources which cover the main citrus-growing areas of Argentina 51 . Argentinian citrus-growing differs from each other (i.e. Mediterranean) in terms of the phytogeography influence they receive. For instance, the citrus areas in the northwest are influenced by the Yungas forests or by the Dry Chaco region. The citrus area in northeast Argentina is influenced by the Paranaense region 57 . Citrus growing in the NW is commonly associated with large areas of well-protected forest of Yungas or Chaco, while those in the NE are surrounded by a mixed area of crops with a relatively low proportion of natural habitats. Grapefruits: Samplings in grapefruit ( C. paradisi Macf) were carried in three different cultivars (`Pink´, `Río Red´, and `Rouge La Toma´) in NW Argentina (see Supplementary Information 2, Fig. S1 ). Four grapefruit plantations were selected and sampled over three consecutive years (2000 to 2002). The flower visitation activity was recorded during 15-minute observation sessions on randomly selected branches. All selected plantations were conventionally managed with fields predominantly dedicated to citrus (see Chacoff and Aizen 21 , 35 for more detail). Mandarins: We studied the Criolla mandarin (C. reticulata var. Criolla Blanco) in NW Argentina, and Clementine mandarin ( Citrus clementina Hort.) in NE Argentina. Studies on Criolla mandarin crops were conducted in 2019 over ten family citrus farms (see Supplementary Information 2, Fig S1 ). These citrus crops were situated in rural areas surrounded by secondary semi-deciduous forests and shrublands, patches of old-growth forests, fruit trees (primarily citrus and olives), and fodder crops. Flower visitation activity was recorded during 15-minute observation sessions on randomly selected branches (see Monasterolo et al. 38 for more details). The Clementine mandarin was studied in 2021, over an experimental area property of INTA (National Institute of Agricultural Technology). The flower visitor sampling was conducted along linear transects, where observations were made on one side of the planting line, across the width of the plot (100m), for a period of 10 minutes. Lemons: Lemon plantations (C. limon L. Burm. F.) were sampled in NW Argentina, in 2015, 2020, and 2021 (see Supplementary Information 2, Fig. S1 ). Lemons were sourced from three different cultivars: `Lisboa´, `Limoneira´, and `Santa Teresita´. Two plantations were sampled in 2015 and 2021, while another one was sampled in 2020. The flower visitor samplings were conducted in 5-minute intervals on randomly selected branches. The selected plantations had conventional management. Oranges: Sampling was carried out in 2021, focusing on Salustiana ( C. sinensis var. Salustiana L. Osbeck) and Valencia oranges ( C. sinensis var. Valencia L. Osbeck), located in NE Argentina (see Supplementary Information 2, Fig. S1 ). We selected four productive plots, within the experimental area, each approximately one hectare in size, one for each cultivar examined. The flower visitation sampling was conducted along linear transects, where observations were made on one side of the planting line, across the width of the plot (100m), for a period of 10 minutes. The fields where we studied oranges and Clementine mandarin were situated in an area with high environmental variability, including cultivated and natural environments. These include blueberry plantations, palm groves, riverine vegetation, and fields of pecan trees and small blocks of Eucalyptus spp. forest plantations. Composition of citrus flower visitors In all citrus species, the visitors were identified in flight or collected and taken to the laboratory. Due to the varying sampling methodologies in the studies, the visitors were categorised into eight functional groups, based on their morphological features and taxonomic classification. The studies included various insect groups such as flies, beetles, wasps, butterflies/moths, and others (such as ants, hemipterans, and homopterans). Furthermore, bees were categorised into three groups: one consisting solely of the exotic bee A. mellifera due to its widespread abundance, while the remaining two groups were composed of native bees, classified based on size, determined by inter-tegular distance 58 . The small bees were grouped together (mainly stingless bees and small halictids), while medium to large bees were also grouped together (which mainly included the medium to large Apidae bees like some members of the tribe Eucerini, Bombus spp., and Xylocopa spp., as well as medium-sized megachilids and halictids such as Augochlora spp. and Augochloropsis spp.). Declarations Acknowledgment Field work in Criolla mandarin was supported by CONICET (PIO/2015-1520150100023CO) and PICT-2018-02508. Field work in lemon and grapefruit was supported by Fundación ProYungas, PICT-2021-01152, PICT-2021-00092, and PRÉSTAMO BCIE. We thank the staff of the growers for logistic support during fieldwork and many field assistants as Candela Russo, Lorena Escobar, Beatriz Velazquez, Martín Lepiscopo, Carla J. Cardenas, Sebastian Albanesi, and Lorena Luna. We emphasize that our study does no cause environmental problems and complies with local regulations, as the species used ( Citrus paradisi , C. reticulata , C. x clementina, C. limon and C. sinensis ) are exotic to our country and are propagated for productive use in commercial nurseries. Furthermore, we had the relevant authorizations from the family farmers, plantation owners (R. Manero, J. Campos, R. Burgos and D. Lorenzo), and institutions/companies (INTA, University of Catamarca, San Miguel S.A., Citrusvil S.A. and Citrus Salta S.A. authorizations) where the citrus data were collected. Author contribution M.M. supervised. M.M., A.F.R.M., N.P.C., P.C. and P.S. wrote, reviewed and edited. A.F.R.M. analyzed the data. M.M., N.P.C., P.C, V.C., and C.M.C. collected the samples. All authors contributed critically to drafting and gave final approval to publish. Data availability Please contact the corresponding author for further information regarding the datasets generated for this study. Competing interests The authors declare no competing interests. References Klein, A.-M. et al. Importance of pollinators in changing landscapes for world crops. Proc. Biol. Sci. 274 , 303–313 (2007). Potts, S. G. et al. Safeguarding pollinators and their values to human well-being. Nature 540 , 220–229 (2016). Tamburini, G., Bommarco, R., Kleijn, D., van der Putten, W. H. & Marini, L. Pollination contribution to crop yield is often context- dependent: A review of experimental evidence. Agriculture, Ecosystems & Environment 280 , 16–23 (2019). Mallinger, R. E., Ternest, J. J., Weaver, S. A., Weaver, J. & Pryer, S. 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Acta horticulturae, 632, 99-103 632 , 99–103 (2004). Chao, C. C. T. Pollinations study of mandarins and the effect on seediness and fruit set: Implications for seedless mandarins production. HortScience 40 , 362–365 (2005). Papadakis, I. E., Protopapadakis, E. E. & Therios, I. N. Yield and fruit quality of ‘Nova’ hybrid [Citrus clementina hort. ex Tanaka (C. reticulata Blanco x C. paradisi Macfad)] and two Clementine varieties (C. clementina hort. ex Tana ka) as affected by self- and cross-pollination. Scientia Horticulture 121 , 38–41 (2009). Yildiz, E. & Kaplankiran, M. The Effect of Cross-Pollination on Fruit Set and Quality in ‘Robinson’ and ‘Fremont’ Mandarins. Ege Üniv. Ziraat Fak. Derg. 54 , 107–112 (2017). da Santos, S. R. et al. Bee pollination services and the enhancement of fruit yield associated with seed number in self-incompatible tangelos. Scientia Horticulturae 276 , 109743 (2021). Manzoor-ul-Haq, M. U. H., Rafie-ul-Din, M. & Ghaffar, A. Effect of Insect Pollination on Fruit Bearing in Kinnow Mandarin (Citrus Reticulata), and Physical and Chemical Properties of the Fruit. Journal of Apicultural Research, 17, 47-49. 17 , 47–49 (1978). Vanlalhmangaiha, R., Singh, H. K., Boopathi, T., Lalhruaitluangi, S. & Sangma, T. T. Impact of insect pollination on the quantitative and qualitative characteristics of sweet orange, Citrus sinensis (L.) Osbeck. Journal of Apicultural Research 62 , 767–776 (2023). Gurung, S. & Chettri, A. Threat to Citrus in a global pollinator decline scenario: Current understanding of its pollination requirements and future directions. Plant Reprod Ecol Recent Adv, 134 , (2021). Bishop, J. & Nakagawa, S. Quantifying crop pollinator dependence and its heterogeneity using multi‐level meta‐analysis. Journal of Applied Ecology 58 , 1030–1042 (2021). Chacoff, N. P. & Aizen, M. A. Edge effects on flower-visiting insects in grapefruit plantations bordering premontane subtropical forest. Journal of Applied Ecology 43 , 18–27 (2006). Rollin, O. & Garibaldi, L. A. Impacts of honeybee density on crop yield: A meta-analysis. Journal of Applied Ecology 1152–1163 (2019). Aizen, M. A. et al. Invasive bees and their impact on agriculture. Advances in Ecological Research 63 , 49–92 (2020). Monasterolo, M., Chacoff, N. P., Segura, A. D., Benavidez, A. & Schliserman, P. Native pollinators increase fruit set while honeybees decrease the quality of mandarins in family farms. Basic and Applied Ecology 64 , 79–68 (2022). Garibaldi, L. A. et al. Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science 339 , 1608–1611 (2013). Grajales-Conesa, J., Meléndez Ramírez, V., Cruz-López, L. & Sánchez, D. Native bees in blooming orange (Citrus sinensis) an lemon (C. limon) orchards in Yucatán, México. Acta Zoológica Mexicana 29 , 437–440 (2013). Pradhan, U. & Devy, S. Pollinators of Sikkim Mandarin Orange Citrus reticulata (Sapindales: Rutaceae). Journal of Threatened Taxa 11 , 13625–13628 (2019). Nurdiansyah, M. A., Abduh, M. Y. & Permana, A. D. Effects of meliponiculture Tetragonula laeviceps on pollinator diversity and visitation rate and citrus productivity in West Java, Indonesia. Biodiversitas Journal of Biological Diversity 24 , (2023). Layek, U., Kundu, A. & Karmakar, P. Floral ecology, floral visitors and breeding system of Gandharaj lemon (Citrus× limon L. Osbeck). Botanica Pacifica: a Journal of Plant Science and Conservation 9 , 113–119 (2020). Talon, M., Zacarias, L. & Primo‐Millo, E. Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability. Physiologia Plantarum 79 , 400–406 (1990). Gamito, L. M. & Malerbo-Souza, D. T. Visitantes florais e produção de frutos em cultura de laranja (Citrus sinensis L. Osbeck). Acta Scientiarum. Animal Sciences 28 , 483–488 (2006). Ribeiro, G. S., Alves, E. & Carvalho, C. A. L. Biology of pollination of Citrus sinensis variety ‘pera rio’. Revista Brasileira de Fruticultura 39 , e–033. (2016). Abouzari, A. & Nezhad, N. M. The investigation of Citrus Fruit Quality. Popular Characteristic and Breeding. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 64 , 725–740 (2016). Sykes, S. R. The effect on Citrus fruit of excluding pollinating insects at flowering and implications for breeding new seedless cultivars. The Journal of Horticultural Science and Biotechnology 83 , 713–718 (2008). Gazzea, E., Batáry, P. & Marini, L. Global meta-analysis shows reduced quality of food crops under inadequate animal pollination. Nat. Commun. 14 , 4463 (2023). Goldenberg, L., Yaniv, Y., Porat, R. & Carmi, N. Mandarin fruit quality: a review. Journal of the Science of Food and Agriculture 98 , 18–26 (2018). Sanchez, E. E. Programa Nacional Frutales. Superficie Ocupada Por Plantaciones Frutales En El País Y Cambios En Su Estructura Productiva . (Ediciones INTA, 2020). FAOSTAT. Database Collection of the Food and Agriculture Organization of the United Nations. FAO http://www. fao.org/faostat/en/#data. Raveh, E. et al. Conventional breeding of cultivated citrus varieties. The citrus genome 33–48 (2020). Palacios, J. Citricultura . (Tucumán, Argentina, 2005). Agustí, M., García-Marí, F. & Guardiola, J. L. The influence of flowering intensity on the shedding of reproductive structures in sweet orange. Scientia Horticulturae 12 , 343–352 (1982). Bustan, A. & Goldschmidt, E. E. Estimating the cost of flowering in a grapefruit tree. Plant, Cell & Environment 21 , 217–224 (1998). Morrone, J. J. Biogeographical Regionalisation of the Neotropical Region . (2014). Greenleaf, S. S., Williams, N. M., Winfree, R. & Kremen, C. Bee foraging ranges and their relationship to body size. Oecologia 153 , 589–596 (2007). Micheloud, N. G. Comportamiento fenológico - reproductivo de variedades de cítricos en la zona centro de la provincia de Santa Fé. (Facultad de Ciencias Agrarias. Universidad Nacional del Litoral - Santa Fé, Argentina, p. 141., 2013). R Core Team. R: A Language and Environment for Statistical Computing. Preprint at https://www.R-project.org/ (2023). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4474196","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":312809125,"identity":"0565f955-feb5-4f05-bbf3-db285c121212","order_by":0,"name":"MARCOS MONASTEROLO","email":"","orcid":"","institution":"Centro regional de Energía y Ambiente para el Desarrollo Sustentable (CREAS), CONICET-Universidad Nacional de Catamarca","correspondingAuthor":false,"prefix":"","firstName":"MARCOS","middleName":"","lastName":"MONASTEROLO","suffix":""},{"id":312809128,"identity":"c8177eb2-a192-484d-84fa-4ffd2b8c1b05","order_by":1,"name":"Andrés Felipe Ramírez-Mejía","email":"data:image/png;base64,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","orcid":"","institution":"Instituto de Ecología Regional, Universidad Nacional de Tucumán","correspondingAuthor":true,"prefix":"","firstName":"Andrés","middleName":"Felipe","lastName":"Ramírez-Mejía","suffix":""},{"id":312809129,"identity":"781d0735-8e1d-43aa-a6df-4e63b4603470","order_by":2,"name":"Pablo Cavigliasso","email":"","orcid":"","institution":"National Agricultural Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Cavigliasso","suffix":""},{"id":312809131,"identity":"0e75cdb8-9ee4-4fac-a5bc-a925b2349811","order_by":3,"name":"Pablo Schliserman","email":"","orcid":"","institution":"Centro regional de Energía y Ambiente para el Desarrollo Sustentable (CREAS), CONICET-Universidad Nacional de Catamarca","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Schliserman","suffix":""},{"id":312809132,"identity":"91752c21-a72f-447a-a684-f152d8867598","order_by":4,"name":"Valentina Chavanne","email":"","orcid":"","institution":"Fundación Miguel Lillo","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Chavanne","suffix":""},{"id":312809134,"identity":"0c8d5ff2-d59b-4205-9030-687429a22100","order_by":5,"name":"Claudia Melissa Carro","email":"","orcid":"","institution":"Fundación Miguel Lillo","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"Melissa","lastName":"Carro","suffix":""},{"id":312809135,"identity":"43fbe459-925f-4e87-a438-14d1d09d77ff","order_by":6,"name":"Natacha Paola Chacoff","email":"","orcid":"","institution":"Instituto de Ecología Regional, Universidad Nacional de Tucumán","correspondingAuthor":false,"prefix":"","firstName":"Natacha","middleName":"Paola","lastName":"Chacoff","suffix":""}],"badges":[],"createdAt":"2024-05-24 19:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4474196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4474196/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73591-6","type":"published","date":"2024-09-27T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58115011,"identity":"6befe711-a867-4be6-b290-57b8d9a4ce14","added_by":"auto","created_at":"2024-06-11 10:25:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":827210,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of observed wild pollinators and honeybees visiting lemons (a), mandarins (b), oranges (c), and grapefruits (d) crops in Argentina. The right side panel shows the percentage of pollinator functional groups within the “wild pollinator” category.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/2b021c2b1e19ee56add92a3e.png"},{"id":58114651,"identity":"50de8281-e325-4b8d-8d37-ae4a1b8248a0","added_by":"auto","created_at":"2024-06-11 10:17:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175576,"visible":true,"origin":"","legend":"\u003cp\u003eProbability of flowers setting fruit in pollinator exclusion experiments for the main citrus groups grown in Argentina. The x-axis indicates different cultivars. The error bars and the dot show the 95% credibility interval and median of the marginal posterior distribution, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/02826088fe6ebcf2ec35d253.png"},{"id":58114652,"identity":"cb18ae5a-3a34-4c66-9283-9aeababa582d","added_by":"auto","created_at":"2024-06-11 10:17:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":902579,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated crop production attributed to animal pollination in four citrus groups and their cultivars: (a) lemons, (b) mandarins, (c) oranges and (d) grapefruits. Each density line and colour denotes different simulations (only 100 are shown) and cultivars, respectively. The vertical discontinuous lines show the median value for each cultivar, and the box plots show the values from 10,000 simulations per cultivar.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/739e2af1efeace59a41209d9.png"},{"id":65627343,"identity":"f738233e-298d-4cf5-9a62-dde0a657eaaa","added_by":"auto","created_at":"2024-09-30 16:15:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3028544,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/3fb0d62f-5196-4ee7-9052-0d1f0bef7aea.pdf"},{"id":58114655,"identity":"ffd1cfcf-3313-4fcb-a9e5-c67d90b55153","added_by":"auto","created_at":"2024-06-11 10:17:01","extension":"html","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19249219,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation1.html","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/61066d2471aead7c09e08bb9.html"},{"id":58115012,"identity":"d1f5055a-dfe5-47e7-839d-49adbf6a09c7","added_by":"auto","created_at":"2024-06-11 10:25:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":46909,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4474196/v1/ad07eea84dfeff7f7d311a91.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Animal pollination contributes to more than half of Citrus production","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe contribution of animal pollination to global agriculture is increasingly acknowledged\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Yet, pollination requirements vary considerably between and even within species and cultivars. Species variability is mainly due to different crop breeding systems and the regional and local communities of pollinators\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For instance, crops such as sugarcane, rice, or maize are predominantly wind-pollinated, and thus the presence of insect pollinators does not impact their production. However, for the vast majority of crops, such as pumpkin, almonds, cherries, and tomatoes, insect pollinators are crucial for the fruit or seed quantity and/or quality\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Pollination can be a limiting factor for these crops, as yields are expected to increase in the presence of pollinators until the crop is fully pollinated\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Cultivars within crop species may also respond differently to animal pollination\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, for example, soybean\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, blueberry\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, canola\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, bean\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and apple\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This variability may be due to cross-cultivar variation in the mating system that affects the interaction with pollinators\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e or reduces the need for pollen deposition for ovule fecundation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePollinators contribution to crop yield is evaluated through experiments, contrasting between flowers with open access to pollinators and those with close pollination (pollinator exclusion experiments), or flowers with hand cross-pollination and those with hand self-pollination (pollen supplementation experiments)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The proportion of flowers that set mature fruits or seeds (i.e. fruit/seed set) is then compared across experiments as the difference between open and excluded treatment and a pollinator contribution or pollinator dependence value is reported. The first compilation of studies that valued the contribution of animals to crop production\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e had a great impact, as they stated that most of our crops are benefited by animals, to a certain degree. However, nowadays there is a search for more agronomic/economic impacts of pollinators on crop yield that can be communicated more directly to producers\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For example, measures such as fruit quantity, fruit quality, and yield stability per unit area that are lost in the absence of pollinators are more informative. This information will provide growers and stakeholders with an impulse for the conservation of pollinator services and market policy to implement agri-environmental programs with a meaningful focus on pollinators\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCitrus fruits (\u003cem\u003eCitrus\u003c/em\u003e spp. such as oranges, mandarins, lemons, and grapefruits) are one of the main and most widespread crops globally, with a production of over 140\u0026nbsp;million tons in 2020\u003csup\u003e18\u003c/sup\u003e and were considered as a group of species with little dependence on pollinators, but with variability between species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Citrus fruits comprise a group of more than ten cultivated species with many varieties and include all breeding systems ranging from agamospermy and parthenocarpy (asexual reproduction), self-incompatibility, through all forms of self-pollination, to self-incompatibility and cross-pollination. Thus, it is challenging to generalize citrus responses to pollinators due to their numerous varieties and complex pollination requirements. It is said, that gamospermous or parthenocarpic citrus varieties do not require pollination to bear fruit (e.g., seedless mandarins, Salustiana oranges, some grapefruits, and lemons)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, a significant number of citrus varieties, including oranges but especially grapefruits and mandarins, are self-incompatible and require or benefit from cross-pollination facilitated by insects to produce fruit or to enhance the yield and quality\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Many studies conclude that cross-pollination by insect pollinators enhances fruit set, with varying degrees of dependency depending on the species and variety\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In addition to increased fruit set, some studies find that insect pollination results in fruits of higher quality, with larger size, weight, and more juice quantity and sugars\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, the contribution of pollinators to citrus production remains controversial\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the wide variability in their breeding systems and the ample differences reported between studies, an actualized assessment of the cultivated species and cultivars is necessary\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In this study we used empirical data from different species and cultivars of citrus 1) to describe the main functional groups of pollinators that visit citrus crops in Argentina; also, 2) we evaluated the influence of animal pollination on the probability of the flower setting fruit across citrus species/cultivars, and 3) its consequences in crop yield ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e across citrus species/cultivars. In light of this, we utilised a database containing visits and fruit set percentages of different citrus groups (grapefruit: \u003cem\u003eC. paradisi\u003c/em\u003e, mandarins: \u003cem\u003eC. reticulata\u003c/em\u003e and \u003cem\u003eC. x clementina\u003c/em\u003e, lemon: \u003cem\u003eC. limon\u003c/em\u003e and oranges: \u003cem\u003eC. sinensis\u003c/em\u003e) belonging to commonly cultivated cultivars. Also, using this dataset, we conducted simulations to estimate the contribution of insect pollination to different productivity measures to reach a broader spectrum of stakeholders about these impacts.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eComposition of citrus flower visitors\u003c/p\u003e \u003cp\u003eIn total, we recorded 21,553 visits to the flowers of all citrus species in 323h. The most common visitor to all citrus flowers was \u003cem\u003eA. mellifera\u003c/em\u003e, which accounted for 87% of the total visitors recorded. Bees accounted for half of all observed wild pollinators, with 33% being small native bees and 17% being medium/large bees. Among the remaining groups, beetles were the most common (17%), followed by dipterans (14%), and wasps (9%).\u003c/p\u003e \u003cp\u003eIn lemons and grapefruits, more than 87% of the insects observed were \u003cem\u003eA. mellifera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), while in mandarins and oranges, observations of wild pollinators predominated (61% and 72%, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In each citrus group, more than half of wild pollinator observations were of bees, except in oranges, where beetles were the most common group (37%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInfluence of pollinators in the probability of flowers setting fruit\u003c/p\u003e \u003cp\u003eIn the close pollination treatment, the probability of flowers setting fruit was low on average for all citrus groups (1.25% CI, 0.78\u0026ndash;1.95; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), i.e. when flowers were not visited by insects, on average, between one or two fruits are formed per hundred flowers. Meanwhile, in the open pollination treatment, the probability of a flower setting a fruit was 3.07% (CI, 1.94\u0026ndash;4.67). This means that flowers from the open treatments had 2.4 times higher probability of setting fruit than those from close pollination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Indeed, this pattern remained consistent across citrus groups (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 SD) and cultivars (2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 SD) (see Supplementary Information 1, section 2.7-Table S3, S4). Under open pollination conditions, lemon cultivars had the highest probability of flowers setting fruit (11.0% \u0026plusmn; 2.01), followed by grapefruits (2.98% \u0026plusmn; 0.47), mandarins (2.62% \u0026plusmn; 2.03), and oranges (1.98% \u0026plusmn; 0.77). The same pattern was observed for the close pollination treatment (lemons\u0026thinsp;=\u0026thinsp;4.79% \u0026plusmn; 1; grapefruits\u0026thinsp;=\u0026thinsp;1.23% \u0026plusmn; 0.2; mandarins\u0026thinsp;=\u0026thinsp;1.08% \u0026plusmn; 0.84; oranges\u0026thinsp;=\u0026thinsp;0.87% \u0026plusmn; 0.31) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Information 1, section 2.7- Table S3). \u003cem\u003eRhat\u003c/em\u003e values and visual diagnostics showed that all chains converged to the same posterior distribution. The \u003cem\u003eess\u003c/em\u003e of all parameters was \u0026gt;\u0026thinsp;1,000 and 96% of pareto \u003cem\u003ek\u003c/em\u003e values were below 0.7 (see Supplementary Information 1, section 2.5). The posterior predictive checks indicated that the model was well-fitted (see Supplementary Information 1, section 2.6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContribution of pollinators to citrus production\u003c/p\u003e \u003cp\u003eWhen comparing open vs. close pollination treatments, our simulations showed that, regardless of the citrus groups or cultivar, there is an ~\u0026thinsp;80% probability that trees exposed to animal pollination produce more fruits (see Supplementary Information 1, section 3.7.2). Considering the contribution of animal pollination to citrus production, i.e. the posterior distribution of the contrast between open and close pollination, the simulations showed that animal pollination contributes to the production of 739 fruits tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;70.4) of lemons (accounting for 71.5 kg tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 11.2), 159.25 fruits tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;126.2) of mandarins (accounting for 15.2 kg tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026plusmn; 9.2), 114 fruits tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;48.1) of oranges (accounting for 24.03 kg\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7), and 168 fruits tree\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3) of grapefruits (accounting for 35.4 kg\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Considering the density of trees planted per ha, we found that animal pollination contributed to 36.1 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8) of lemons (56.5% \u0026plusmn; 3.5 of total production), 5.9 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3) of mandarins (58.9% \u0026plusmn; 5.9 of total production), 10.1 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4) of oranges (59.1% \u0026plusmn; 5.7 of total production), and 10.5 t ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2) of grapefruits (58.9% \u0026plusmn; 6.6 of total production) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe influence of animal pollination on crop yield can vary due to several factors. In citrus crops, the contribution of pollinators is still a matter of debate, mainly because of the wide variety of reproductive strategies within citrus species and cultivars, but also due to spatial, temporal and biological factors\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Using data from main citrus species and cultivars in large regions of Argentina, we found that animal pollination increases about two times the probability of flowers setting fruit in the citrus crop, resulting in a contribution of about 60% of the total yield at the hectare level. Therefore, despite the sometimes neglected role of animal pollination for this crop, our study provides evidence that justifies safeguarding pollinators in citrus agricultural landscapes.\u003c/p\u003e \u003cp\u003eThe exotic honeybee \u003cem\u003eA. mellifera\u003c/em\u003e was the main visitor to the citrus flowers. A diverse group of native pollinators, mainly native bees, participated in the visits to a lesser extent. The widespread occurrence of honeybees is common in citrus pollinator studies\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, but it should be considered that honeybees in these agricultural landscapes can be either managed or feral. In agroecosystems of the region, the abundance of honeybees is linked to the proximity of natural habitats\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and the abundance of locally managed honeybee hives for the production of honey. Studies have shown that the introduction of managed honeybees can increase flower visitation rates, which can result in a reduction in mature fruits per flower and lower fruit quality in various crops\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, including citrus \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It is crucial to acknowledge that an increase in pollinators does not necessarily lead to enhanced fruit production, and the native pollinators may be as or more efficient in fruit production than managed bees\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In our study, the most abundant wild pollinators were small bees, mainly stingless bees, and the eusocial bees \u003cem\u003eLasioglossum\u003c/em\u003e spp. Curtis. These bees could prove to be valuable for citrus even in the presence of managed hives, as highlighted by previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e–\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Consequently, the adoption of effective local and landscape management strategies to enhance flowering and nesting resources and to reduce current environmental pressures on wild pollinators may be sufficient to achieve optimal benefits.\u003c/p\u003e \u003cp\u003eOur results have shown that in all the citrus groups and cultivars studied, insect pollination has a positive effect on the proportion of flowers setting fruit, increasing more than double the number of fruits compared with non-pollinated flowers. Therefore, according to our data, insects are certainly important for citrus fruit production. In the lemon cultivars of our study, the percentage of fruit set in open pollinated flowers was lower than that reported in the literature for \u003cem\u003eC. limon\u003c/em\u003e (29.7% Layek \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e). However, the increase in fruit set in open versus closed treatments was very similar in both studies (44% in our study and 45% in Layek \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e). The fruit set of mandarins and oranges varies greatly due to the wide variety of cultivars and breeding. The Clementine mandarin is facultative parthenocarpic, i.e. it requires pollen stimulation by pollinators to set fruit\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. However, the probability of fruit set was doubled when the flowers were pollinated by insects compared to exclude flowers\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Studies on mandarin pollination have found either a smaller increase\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e or a similar number of mature fruits\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e in insect pollinated flowers compared to our study. Previous studies have found that the increase in cross-pollination treatments is dependent on the cultivars used\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e–\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In studies on oranges, the values in flowers with open pollination for the Pera Rio variety were 30–78% higher than in excluded flowers\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. For grapefruit, the only published results available in the literature correspond to the data used in this work\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. As there was no considerable variability in fruit set between cultivars of the same species under open versus close treatments, the variations in fruit set reported in previous studies may be attributed to differences in estimation methods, limited replication and a small number of studies involving other species and cultivars.\u003c/p\u003e \u003cp\u003eOur study reveals the contribution of pollinators to citrus yield, but we did not explore the contribution of pollinators to seed setting in these fruits, which is an important marketable trait in citrus, i.e. seedlessness. Seedlessness is important for fresh citrus use, whereas this trait is less important for industrial citrus\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Seedless varieties are a desirable productive trait in most citrus, and for most species, pollination and fertilisation result in seed formation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Therefore, there is a potential trade-off between the need of animal pollination to increase the probability of flowers setting fruit (i.e. production quantity), and the market demand of seedless production (i.e. quality production). Yet, for certain cultivars, such as some mandarins, lemons, and grapefruits, seedlessness is not required by the market or even possible\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In addition to production quantity, pollination can also improve the sugar content\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and weight of the fruits\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e due to pollen deposition and fecundation of the ovules which activate hormonal processes related to fruit growth, seed formation, pulp firmness and thus increase the post-harvest quality of fruits\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Despite the numerous studies that provide evidence of the benefits of pollination in this production system, when the number of seeds is relevant, growers often have to choose based on market demand. They must choose between increasing the yield and quality of the fruit or reducing a trait that affects the destination market of the production, which alters the value of the product\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results indicate that without animal pollination, the amount of fruit produced per hectare (equivalent in tonnes/ha) would be reduced by an average of 58.35%. All the species and cultivars studied showed similar levels of pollinator dependence, despite differences in environmental conditions, spatial arrangements, farm management practices, and composition of flower visitors. The degree of pollinator dependence on citrus fruits has been previously reported in the literature in compilations, with widely varying results\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Klein et al.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e reported that pollinators make a small contribution to citrus fruit production, with less than 10% dependence. Mallinger et al.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e report values of pollinator contribution in Florida (USA), of 73% for grapefruits, 31% for lemons and oranges, and over 80% for tangelo and mandarins. Siopa et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e reported high levels of pollination dependence in citrus of around 65–80%, except for oranges which had a lower dependence of around 20%. This latter study used hand pollen supplementation to assess the yield associated with animal pollination, in contrast to our study, which used the open pollination treatment. It is important to note that the use of hand pollen supplementation tends to show higher values of pollinator contribution\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, which indicates that there can be some level of pollen deficit. Therefore, although the contribution of animal pollination in most citrus studies indicates a relative/high dependence of pollinators on citrus yields, values of pollinator dependence and reporting of these values may vary according to the estimation methods used.\u003c/p\u003e \u003cp\u003eIn this study, we considered the contribution of pollinators to citrus production in terms of yield ha\u003csup\u003e− 1\u003c/sup\u003e, including aspects such as fruit set, fruit weight, and literature value of floral display, in contrast to previous studies that use fruit set to calculate this contribution. Previous studies classified crop pollination dependence (for example, little, modest, high, and essential in Klein et al.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e), where the difference between close pollination treatments and pollinator-associated production treatments (open pollination or hand pollen supplementation) was calculated as 1-(close/pollinator-associated production). However, the fruit set alone does not fully reflect the true dependence on pollinators in commercial crop production \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These studies do not take into account quantitative differences in fruit, such as the weight of fruit per tree or per hectare lost in the absence of pollinators, as calculated here. Also, in contrast to the above-mentioned approaches, our results reflect the potential implications of pollinator absences on farms and allow us to assess the precise value of pollinators on agricultural production using commercially relevant yield metrics. This approach is advantageous as it is more effective and allows for more concrete communication for the decision-making of growers, industry, and stakeholders.\u003c/p\u003e \u003cp\u003eTo estimate the contribution of animal pollination in Citrus, we fitted Bayesian models that necessarily neglect the role of plant physiological state and other agronomic inputs (e.g. pruning practices, irrigation, fertilisation, pest control), which should be taken into account in future studies. We also recommend that future research should assess whether the contribution of pollinators to the production quantity is reflected in certain aspects of citrus quality that also have a significant impact on market value, such as weight, seed quantity, size, sugar concentration, and others. Furthermore, although not documented in our study, the presence of managed beehives at sites close to the crops certainly influenced their high abundance. To achieve this, the number of managed hives in the vicinity and their distance from citrus plantations should be recorded, to assess the pollen limitation and the ideal number of visits to achieve high fruit yields in citrus production.\u003c/p\u003e \u003cp\u003eThis study showed that animal pollination more than doubled the fruit set and contributed around 60% of citrus yield/ha\u003csup\u003e− 1\u003c/sup\u003e, regardless of species and cultivar. In light of these results and the future of the national citrus market, which is increasingly focused on quality parameters that emphasise the absence of animal pollination to develop 'seedless citrus', and in the face of an imminent advance of diseases that threaten the quality of fresh fruit for export (e.g. citrus Huanglongbing or HLB), it will be necessary to evaluate a change in future market strategy or a re-evaluation of the quality standard to be marketed. In this context, animal pollination ensures a higher quantity and quality of fruit, both in terms of the weight of individual fruits and the sugar content of the juice produced\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Therefore, changing market demands, based on pollinator conservation may also be a possibility, especially when the number of seeds is not so relevant. Empirical studies, market analysis, and policy interventions aimed at promoting pollinator conservation and sustainable agriculture can assist stakeholders in better understanding and mitigating the risks associated with pollinator declines while harnessing the economic benefits of animal pollination for food systems globally.\u003c/p\u003e \n\u003ch3\u003eEffect of pollinators on the probability of flower setting fruits\u003c/h3\u003e\n\u003cp\u003eTo assess the influence of pollinators on the fruit set of grapefruit, lemons, oranges and mandarins, experiments were conducted comprising two treatments: open pollination and close pollination through bagging branches with flower buds (for more details on these methodologies see Chacoff and Aizen\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e; Monasterolo et al.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e). In each tree, we selected at least two branches with flowering buds and applied one of two pollination treatments: open pollination and close pollination treatment. In the open pollination treatment, we recorded the number of mature flowering buds when flowering season was starting and we counted the number of initial fruits when fruits were formed (one month after flowering). In this treatment, flowers were exposed to natural levels of pollination, including both animal pollination and self-pollination due to wind pollination. For close pollination treatment, a branch with flowering buds was excluded from visitors by using a voile that permitted the action of the wind but not the visits from insects, here only self-pollination due to wind pollination can occur. The bags were removed after flowering and the number of fruits formed in each treatment was counted to calculate the fruit set (fruits/flowers). The number of sampled plants and branches is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (for more details see Supplementary Information 1, section 2.2 - Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of branches sampled per treatment, species, and cultivar.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePollination treatment\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSpecies\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCultivar\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e Close pollination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOpen pollination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLimoneira\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLisboa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Teresita\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Lemon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e402\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClementine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCriolla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e896\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Mandarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e918\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSalustiana\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValencia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Orange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrapefruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePink\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRio Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRouge La Toma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Grapefruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e516\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1525\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eEffect of pollinators on the probability of flower setting fruits\u003c/h2\u003e \u003cp\u003eWe used a Bayesian hierarchical model to assess the effect of animal pollination exclusion on the probability of the flowers setting fruit:\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003efruit produced\u003csub\u003ei\u003c/sub\u003e ~ binomial(n sampled flowers\u003csub\u003ei\u003c/sub\u003e, p\u003csub\u003ei\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003e \u003cem\u003elogit(p\u003c/em\u003e \u003csub\u003e \u003cem\u003ei\u003c/em\u003e \u003c/sub\u003e | \u003cem\u003etreatment\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003especies\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ecultivar\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003etree\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eyear\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003elocality\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e) =\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eα\u003c/em\u003e \u003csub\u003e \u003cem\u003etreatment i\u003c/em\u003e \u003c/sub\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;τ\u003c/em\u003e\u003csub\u003e\u003cem\u003especies i\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ ɣ\u003c/em\u003e\u003csub\u003e\u003cem\u003ecultivar i\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ θ\u003c/em\u003e\u003csub\u003e\u003cem\u003etree i\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ ẟ\u003c/em\u003e\u003csub\u003e\u003cem\u003eyear i\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e+ λ\u003c/em\u003e\u003csub\u003e\u003cem\u003elocality i\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eWe considered the pollination treatment as the population effect, and citrus species, cultivars, sampled tree, year of sampling, and locality as group-level effects. To account for variation in sampling size among citrus species/cultivars and reduce overfitting, we used partial pooling for parameter estimation. The probability of flowers setting fruit tends to be low for \u003cem\u003eCitrus\u003c/em\u003e spp.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, hence we defined \u003cem\u003eα\u003c/em\u003e\u003csub\u003e\u003cem\u003etreatment i\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e~\u0026thinsp;Normal(5, 2.5)\u003c/em\u003e as prior (i.e. number of fruits developed from \u003cem\u003en\u003c/em\u003e observed flowers), and a \u003cem\u003eNormal(0, 1)\u003c/em\u003e for all group-level parameters. We estimated the joint posterior distribution of \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (i.e. probability of flowers setting fruit) using the Hamiltonian Monte Carlo algorithm through Stan 2.32.2 and the R package \u003cem\u003ecmdstanr\u003c/em\u003e. We fitted the model by setting three chains, 4000 sampling and 500 warming iterations, and a thinning rate of 3. We conducted sampling diagnostics of all parameters through visual assessment of chains convergence, \u003cem\u003eRhat\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;1.1, \u003cem\u003eess\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1000, and pareto \u003cem\u003ek\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.7. We also assessed the quality of the model fit through posterior predictive checks. See Supplementary Information 1, section 2.3 for the mathematical notation of the model and section 2.4 for the Stan code to fit the model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eContribution of animal pollination to citrus production\u003c/h2\u003e \u003cp\u003eAssuming that the differences in the probability of flowers setting fruit between experimental treatments can be attributed to animal pollination, we build simulations to estimate the contribution of pollinators to fruit production and yield of lemon, mandarin, orange, and grapefruit. Annotated R code and main functions conducting the simulations can be consulted in Supplementary Information 1, section 3.1. The simulations followed this reasoning:\u003c/p\u003e \u003cp\u003e \u003cem\u003eStep 1\u003c/em\u003e \u0026mdash; Considering the floral display size of \u003cem\u003eCitrus\u003c/em\u003e spp. reported in the literature (25000 to 200000 flowers in oranges in\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, or 20000 to 50000 flowers in grapefruits in\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e), and information from citric producers in the localities studied, we hypothesise that the number of flowers produced by \u003cem\u003eCitrus\u003c/em\u003e spp. trees can be recreated through a negative binomial probability distribution:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eflowers per tree\u0026thinsp;~\u0026thinsp;NB(\u0026#120590; = 2, \u0026#120583; = 15000)\u003c/h2\u003e \u003cp\u003e \u003cem\u003e\u0026#120590;\u003c/em\u003e denotes the dispersion parameter of the distribution, and we defined \u003cem\u003e\u0026#120583;\u003c/em\u003e = 15000 to remain conservative on the average number of flowers produced per tree (Supplementary Information 1, section 3.1).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStep 2\u003c/em\u003e \u0026mdash; We used \u003cem\u003estep 1\u003c/em\u003e to simulate as many random trees with \u003cem\u003en\u003c/em\u003e flowers as trees expected per ha for each \u003cem\u003eCitrus\u003c/em\u003e species. For lemon cultivars, we assumed 400 trees ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (5 ✕ 5 m); orange and mandarin cultivars 278 trees ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (6 ✕ 6 m); and grapefruit cultivars 278 trees ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (8 ✕ 6 m). We replicated this process 10000 times per \u003cem\u003eCitrus\u003c/em\u003e/cultivar (i.e., 10000 simulated hectares per \u003cem\u003eCitrus\u003c/em\u003e/cultivar). We defined the plantation distance according to information provided by growers (Supplementary Information 1, section 3.2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStep 3\u003c/em\u003e \u0026mdash; Then, we used the marginal posterior distribution of the probability of flowers setting fruit per pollination treatment to predict the number of fruits produced per simulated tree in \u003cem\u003estep 2\u003c/em\u003e: See supplementary material 1, section 3.3.2.1 for this simulation in lemon (Limoneira cultivar).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003efruits produced\u003csub\u003ei\u003c/sub\u003e ~ binomial(n flowers\u003csub\u003e[random tree i]\u003c/sub\u003e, p\u003csub\u003e[sp, cultivar, treatment]\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStep 4\u003c/em\u003e \u0026mdash; We used the simulated fruits in \u003cem\u003estep 3\u003c/em\u003e for each pollination treatment per \u003cem\u003eCitrus\u003c/em\u003e/cultivar to estimate the probability that trees exposed to animal pollination (i.e. open pollination treatment) produce more fruits than those from close pollination treatment (Supplementary Information 1, section 3.3.2.1).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStep 5 \u0026mdash;\u003c/em\u003e We calculated the contrast of the marginalised posterior distribution of the probability of flowers setting fruit between pollination treatments. Then, we used the contrast and the simulated trees in \u003cem\u003estep 2\u003c/em\u003e, to simulate the number of fruits attributed to animal pollination (Supplementary Information 1, section 3.3.2.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003efruits produced\u003csub\u003eanimal pollinationi\u003c/sub\u003e ~ binomial(n flowers\u003csub\u003e[random tree i]\u003c/sub\u003e, p\u003csub\u003e[sp, cultivar, contrast]\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStep 6 \u0026mdash;\u003c/em\u003e We fitted Bayesian models to estimate the posterior distribution of fruit weight of lemon, grapefruit, and Criolla mandarin (Supplementary Information 1, sections 3.3.1, 3.4.1 and 3.6.1, respectively). The fitting and models diagnostics procedure follows the above-mentioned protocol. Since raw data on fruit weight for orange (Valencia and Salustiana) and Clementine mandarin was not available, we used mean and SD values reported in the literature\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e to parameterize normal distributions (Supplementary Information 1, section 3.5.1). Then, we used these distributions to simulate fruit weights for each \u003cem\u003eCitrus\u003c/em\u003e species and cultivar (Supplementary Information 1, section 3.3.4.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003efruit weight\u003csub\u003ei\u003c/sub\u003e ~ normal(\u0026#120583;\u003csub\u003e[species, cultivar]\u003c/sub\u003e, \u0026#120590;\u003csub\u003e[species, cultivar]\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStep 7 \u0026mdash;\u003c/em\u003e Finally, we used the distributions of \u003cem\u003estep 6\u003c/em\u003e to simulate as many fruit weights as fruits per tree were generated in \u003cem\u003estep 5\u003c/em\u003e. The sum of weights per tree and the sum of trees per ha denoted the production attributed to animal pollination in kg and Tn, respectively. We conducted 10000 simulations per \u003cem\u003eCitrus\u003c/em\u003e species/cultivar. We repeat \u003cem\u003esteps 5\u003c/em\u003e to \u003cem\u003e7\u003c/em\u003e with the posterior distribution of open pollination treatment and use the simulations from the contrast to estimate the percentage of crop production per ha that can be attributed to animal pollination.\u003c/p\u003e \u003cp\u003eWe performed all simulations and statistical procedures in R 4.3.1\u003csup\u003e60\u003c/sup\u003e, we did data wrangling operations using \u003cem\u003ebase\u003c/em\u003e and \u003cem\u003edplyr\u003c/em\u003e packages, and plotted the models and simulations outputs with \u003cem\u003eggplot2\u003c/em\u003e and \u003cem\u003ecowplot\u003c/em\u003e packages. We provide the R script with annotated code to reproduce the simulations and models fit in Supplementary Information 1, section 3.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cp\u003eCultivars\u003c/p\u003e\u003cp\u003eCitrus (\u003cem\u003eCitrus\u003c/em\u003e spp., such as oranges, mandarins, lemons, and grapefruits) represent a significant portion of Argentina's fruit production, covering 23.9% of the total production area \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Across Argentina, an average of approximately 132,669 hectares of citrus is harvested annually, generating nearly USD 378\u0026nbsp;million in export earnings from the marketing of over 3.3\u0026nbsp;million tons of fruit (period 2018-2022\u003csup\u003e52\u003c/sup\u003e). The citrus flowers are perfect, containing both pistils and stamens, thus self-pollination can occur without pollinators\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The fruits display a variety of shapes, sizes, and other quality parameters according to species and cultivars\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. A vigorous citrus tree can produce between 20,000–250,000 floral units during the flowering period\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, typically, a very low percentage (between 0.1 and 3%) of these flowers develop fruits\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In general, citrus flowers are highly attractive to pollinators. Citrus trees experience mass flowering, typically in early spring when there are still few wildflowers, thus providing valuable nectar and pollen resources for pollinators. The diversity of pollinators in citrus varies according to geographical area and the flowering season. Usually, professional pollination services are not utilised in these crops, because of the common belief that citrus does not require insect pollination to set fruits and even in some citrus, pollination is intentionally avoided, which is a relatively common practice in some mandarins for a desirable 'seedless citrus'.\u003c/p\u003e\u003cp\u003eStudy area and flower visitor sampling\u003c/p\u003e\u003cp\u003eThis study is based on empirical data from various sources which cover the main citrus-growing areas of Argentina\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Argentinian citrus-growing differs from each other (i.e. Mediterranean) in terms of the phytogeography influence they receive. For instance, the citrus areas in the northwest are influenced by the Yungas forests or by the Dry Chaco region. The citrus area in northeast Argentina is influenced by the Paranaense region\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Citrus growing in the NW is commonly associated with large areas of well-protected forest of Yungas or Chaco, while those in the NE are surrounded by a mixed area of crops with a relatively low proportion of natural habitats.\u003c/p\u003e\u003cp\u003eGrapefruits: Samplings in grapefruit (\u003cem\u003eC. paradisi\u003c/em\u003e Macf) were carried in three different cultivars (`Pink´, `Río Red´, and `Rouge La Toma´) in NW Argentina (see Supplementary Information 2, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Four grapefruit plantations were selected and sampled over three consecutive years (2000 to 2002). The flower visitation activity was recorded during 15-minute observation sessions on randomly selected branches. All selected plantations were conventionally managed with fields predominantly dedicated to citrus (see Chacoff and Aizen \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e for more detail).\u003c/p\u003e\u003cp\u003eMandarins: We studied the Criolla mandarin \u003cem\u003e(C. reticulata\u003c/em\u003e var. \u003cem\u003eCriolla\u003c/em\u003e Blanco) in NW Argentina, and Clementine mandarin (\u003cem\u003eCitrus clementina\u003c/em\u003e Hort.) in NE Argentina. Studies on Criolla mandarin crops were conducted in 2019 over ten family citrus farms (see Supplementary Information 2, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These citrus crops were situated in rural areas surrounded by secondary semi-deciduous forests and shrublands, patches of old-growth forests, fruit trees (primarily citrus and olives), and fodder crops. Flower visitation activity was recorded during 15-minute observation sessions on randomly selected branches (see Monasterolo et al.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e for more details). The Clementine mandarin was studied in 2021, over an experimental area property of INTA (National Institute of Agricultural Technology). The flower visitor sampling was conducted along linear transects, where observations were made on one side of the planting line, across the width of the plot (100m), for a period of 10 minutes.\u003c/p\u003e\u003cp\u003eLemons: Lemon plantations (C. \u003cem\u003elimon\u003c/em\u003e L. Burm. F.) were sampled in NW Argentina, in 2015, 2020, and 2021 (see Supplementary Information 2, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Lemons were sourced from three different cultivars: `Lisboa´, `Limoneira´, and `Santa Teresita´. Two plantations were sampled in 2015 and 2021, while another one was sampled in 2020. The flower visitor samplings were conducted in 5-minute intervals on randomly selected branches. The selected plantations had conventional management.\u003c/p\u003e\u003cp\u003eOranges: Sampling was carried out in 2021, focusing on Salustiana (\u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003eSalustiana\u003c/em\u003e L. Osbeck) and Valencia oranges (\u003cem\u003eC. sinensis\u003c/em\u003e var. \u003cem\u003eValencia\u003c/em\u003e L. Osbeck), located in NE Argentina (see Supplementary Information 2, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We selected four productive plots, within the experimental area, each approximately one hectare in size, one for each cultivar examined. The flower visitation sampling was conducted along linear transects, where observations were made on one side of the planting line, across the width of the plot (100m), for a period of 10 minutes. The fields where we studied oranges and Clementine mandarin were situated in an area with high environmental variability, including cultivated and natural environments. These include blueberry plantations, palm groves, riverine vegetation, and fields of pecan trees and small blocks of \u003cem\u003eEucalyptus\u003c/em\u003e spp. forest plantations.\u003c/p\u003e\u003cp\u003eComposition of citrus flower visitors\u003c/p\u003e\u003cp\u003eIn all citrus species, the visitors were identified in flight or collected and taken to the laboratory. Due to the varying sampling methodologies in the studies, the visitors were categorised into eight functional groups, based on their morphological features and taxonomic classification. The studies included various insect groups such as flies, beetles, wasps, butterflies/moths, and others (such as ants, hemipterans, and homopterans). Furthermore, bees were categorised into three groups: one consisting solely of the exotic bee \u003cem\u003eA. mellifera\u003c/em\u003e due to its widespread abundance, while the remaining two groups were composed of native bees, classified based on size, determined by inter-tegular distance\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The small bees were grouped together (mainly stingless bees and small halictids), while medium to large bees were also grouped together (which mainly included the medium to large Apidae bees like some members of the tribe Eucerini, \u003cem\u003eBombus\u003c/em\u003e spp., and \u003cem\u003eXylocopa\u003c/em\u003e spp., as well as medium-sized megachilids and halictids such as \u003cem\u003eAugochlora\u003c/em\u003e spp. and \u003cem\u003eAugochloropsis\u003c/em\u003e spp.).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eField work in Criolla mandarin was supported by CONICET (PIO/2015-1520150100023CO) and PICT-2018-02508. Field work in lemon and grapefruit was supported by Fundaci\u0026oacute;n ProYungas, PICT-2021-01152, PICT-2021-00092, and PR\u0026Eacute;STAMO BCIE. We thank the staff of the growers for logistic support during fieldwork and many field assistants as Candela Russo, Lorena Escobar, Beatriz Velazquez, Mart\u0026iacute;n Lepiscopo, Carla J. Cardenas, Sebastian Albanesi, and Lorena Luna.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe emphasize that our study does no cause environmental problems and complies with local regulations, as the species used (\u003cem\u003eCitrus paradisi\u003c/em\u003e, \u003cem\u003eC. reticulata\u003c/em\u003e, \u003cem\u003eC. x clementina, C. limon\u003c/em\u003e and \u003cem\u003eC. sinensis\u003c/em\u003e) are exotic to our country and are propagated for productive use in commercial nurseries. Furthermore, we had the relevant authorizations from the family farmers, plantation owners\u0026nbsp;(R. Manero, J. Campos, R. Burgos and D. Lorenzo), and institutions/companies (INTA, University of Catamarca, San Miguel S.A., Citrusvil S.A. and Citrus Salta S.A. authorizations) where the citrus data were collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eM.M. supervised. M.M., A.F.R.M., N.P.C., P.C. and P.S. wrote, reviewed and edited. A.F.R.M. analyzed the data. M.M., N.P.C., P.C, V.C., and C.M.C. collected the samples. All authors contributed critically to drafting and gave final approval to publish.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003ePlease contact the corresponding author for further information regarding the datasets generated for this study.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKlein, A.-M. \u003cem\u003eet al.\u003c/em\u003e Importance of pollinators in changing landscapes for world crops. \u003cem\u003eProc. Biol. Sci.\u003c/em\u003e \u003cstrong\u003e274\u003c/strong\u003e, 303\u0026ndash;313 (2007).\u003c/li\u003e\n\u003cli\u003ePotts, S. 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Preprint at https://www.R-project.org/ (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"citrus fruit, fruit set, pollinator-dependent crops, pollination service, yield, food crops","lastPublishedDoi":"10.21203/rs.3.rs-4474196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4474196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnimal pollination is crucial for the reproduction and economic viability of a wide range of crops. Despite the existing data, the extent to which citrus crops depend on pollinators to guarantee fruit production still needs to be determined. Here, we described the composition of flower visitors in citrus (\u003cem\u003eCitrus\u003c/em\u003e spp.) from the main growing areas of Argentina; moreover, we combined Bayesian models and empirical simulations to assess the contribution of animal pollination on fruit set and yield ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in different species and cultivars of lemon, grapefruit, mandarin, and orange in the same regions. Honeybee (\u003cem\u003eA. mellifera\u003c/em\u003e L.) was the most commonly observed visitor, followed by a diverse group of insects, mainly native bees. Regardless of citrus species and cultivars, the probability of flowers setting fruit in pollinated flowers was 2.4 times higher than unpollinated flowers. Furthermore, our simulations showed that about 60% of the citrus yield ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributable to animal pollination across all species and cultivars. Therefore, it is crucial to maintain environments that support pollinator diversity and increase consumer and to producer awareness and demand in order to ensure the significant benefits of animal pollination in citrus production.\u003c/p\u003e","manuscriptTitle":"Animal pollination contributes to more than half of Citrus production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 10:16:56","doi":"10.21203/rs.3.rs-4474196/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-16T09:05:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T09:36:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-28T16:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19738679864497235832171867244891649934","date":"2024-06-28T07:51:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227167296733996796618232548877847968417","date":"2024-06-10T14:28:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-08T18:52:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-03T08:50:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-31T08:45:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-29T04:05:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-24T19:36:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47864f27-2c1c-4e78-9a04-3bc7b29a25fa","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:06:03+00:00","versionOfRecord":{"articleIdentity":"rs-4474196","link":"https://doi.org/10.1038/s41598-024-73591-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-27 15:57:56","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-06-11 10:16:56","video":"","vorDoi":"10.1038/s41598-024-73591-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73591-6","workflowStages":[]},"version":"v1","identity":"rs-4474196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4474196","identity":"rs-4474196","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}