Diurnal and geographic variations of pollinator importance for Cucurbita maxima Duchesne
Data files
Dec 12, 2023 version files 91.86 KB
Abstract
Despite growing awareness of the importance of monitoring wild crop pollinators worldwide, there are still few reports, especially in East Asia. Considering ongoing global warming may change the distribution range and diurnal activity of pollinators, it is necessary to describe current geographic and diurnal patterns. We clarified pollinators of Cucurbita maxima Duchesne (Cucurbiales: Cucurbitaceae) in three geographically distinct (>350km, minimum) areas in Japan, focusing on diurnal variation. Apis mellifera L. (Hymenoptera: Apidae) and Halictidae (Hymenoptera) were observed in all of the experimental gardens. Apis cerana japonica Radoszkowski (Hymenoptera: Apidae) were mainly observed in Mie and Kagoshima, while Bombus diversus diversus Smith (Hymenoptera: Apidae) were observed only in Ibaraki. The peak time of flower visits depended both on bee taxa and area and, interestingly, did not necessarily synchronize with the timing of highest pollen loads and the probability of stigma contact. In particular, visits and probability of contacting stigmas of Halictidae tended to increase as time passed, whereas pollen grains on their bodies sharply decreased with time; only a few individuals of Halictidae spp. that visit early can become effective pollinators. There were no differences in yields between supplementary hand- and natural-pollination in all areas, and flower-enclosure experiments using different mesh sizes clarified that small insects that can go across an approximately 4mm mesh may not transport sufficient pollen for fruit set. Our study demonstrated that pollination effectiveness, which is usually regarded as a static value, within a taxon can fluctuate in the space of just several hours. Considering such diurnal patterns can be altered by climate change, we need to carefully monitor the diurnal temporal patterns of pollinators worldwide.
README: Diurnal and geographical variations of pollinator importance for Cucurbita maxima Duchesne
https://doi.org/10.5061/dryad.vmcvdnczm
Data of Flower visiting insects of C. maxima and of reproductive output of the plants
Description of the data and file structure
Our data consists of six files.
1. "Captured_number_of_bees.csv"
2. "Pollen_loads_on_bees.csv"
3. "Insect_bidy_size.csv"
4. "Squash_fruit_set.csv"
5. "Squash_yield.csv"
6. "Behaviours_on_squash_flowers.csv"
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Data specific information
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1. Captured number of bees.csv\
"Captured number of bees.csv" is a list of collected bees in the regular surveys.\
Number of variables: 9\
Number of rows: 597\
Variable list (data type):
- area (category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie).
- collection_day (category), showing the date when the surveys of bees were conducted.
- time.span (continuous), showing the period when the bees were collected. The unit in this variable is the hours in 12 o'clock. For example, "9" means AM9:00-9:59.
- plot (category), showing the identities of plots of each site. it includes 1-10, A, B, C, M, N, and S.
- flower_sex (category), showing the sex of each flower. "F" means "female", and "M" means "male".
- Apis.cerana (continuous), showing the number of Apis cerana captured at the focal period.
- Apis.mellifera (continuous), showing the number of Apis mellifera captured at the focal period.
- Bombus.diversus (continuous), showing the number of Bombus diversus divursus captured at the focal period.
- Halictid.bees (continuous), showing the number of Halictid bees captured at the focal period.
Note: The number in "time span" shows the hours in 12 o'clock. For example, "9" means AM9:00-9:59. More detailed informations are explained in the manuscript.
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2. Pollen_loads_on_bees.csv
"Pollen loads on_bees_2019.csv" is a list of collected bees and their pollen loads.
Number of variables: 8
Number of rows: 279
Variable list (data type):
- taxonomic.groups (category), showing identities of bees (i.e., Apis cerana, Apis mellifera, Bombus diversus, or Halictid bees).
- area(category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie).
- collection_day (category), showing the date when the surveys of bees were conducted.
- time.span (continuous), showing the time span when the bees were collected. The unit in this variable is the hours in 12 o'clock. For example, "9" means AM9:00-9:59.
- plot (category), showing the identities of plots of each site. it includes 1-10, A, B, C, M, N, S, and other.
- flower_sex (category), showing the sex of each flower. "F" means "female", and "M" means "male".
- ID (category), showing the identities of each specimen.
- pollen (continuous), showing the number of pollen adhered to each bee's body.
Note: The number in "time span" shows the hours in 12 o'clock. For example, "9" means AM9:00-9:59. More detailed informations are explained in the manuscript.
The bees were mainly collected in the regular survey. However, in Kagoshima, we supplementary collected the bees in the squash farm, irrespective of plots to increase sample size.
In such a case, we described the plot ID as "other".
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3. "Insect_bidy_size.csv"
"insect body size.csv" is about the body size of collected insects, and whether the individuals could pass through the bags of different mesh sizes.
Number of variables: 7
Number of rows: 94
Variable list (data type):
- area (category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie).
- ID (category) showing the identities of each specimen.
- Taxonomic_groups (category), showing the taxonomic identities of each spesimen. It includes below; Apis cerana, Apis mellifera,\ Beatle, Bombus diversus, Bombus ignitus, Carpenter bees, Diptera, Eucera nipponensis, flower chafer, Halictid bees, Micro wasp, Scolid bees, and Syrphidae.
- Cheast_width (continuous), showing the width (mm) of cheast of each specimen.
- Cheast_height (continuous), showing the height (mm) of cheast of each specimen.
- coase_mesh (continuous), showing whether the specimen could pass through the coase mesh.
- fine_mesh (continuous), showing whether the specimen could pass through the fine mesh.
Note: Empty cells (i.e., no data) were replaced by "null".
In variables coase_mesh and fine_mesh, the sample that could pass through the mesh were represented by "1" , while that could not were represnted by "0"
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4. "Squash_fruit_set.csv"
This file is about whether the experimental flowers could set fruit or not.
Number of variables: 4
Number of rows: 127
Variable list (data type):
- Fruiting.result (continuous), showing the experimental flowers could set fruit.
- Flower.treatment (category), showing the experimental treatment of each flower.
- Area (category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie).
- Flowering.Day (category), showing the date the focal experimental flower had bloomed.
Note: "Flower.treatment" includes alpabets, A, B, C, D and E.
These charaters correspond to non-woven bag, fine mesh, coase mesh, open, supplementary hand-pollination, in this order.
"Fruiting. result" includes 0 and 1. 0 means the flower could not set fruit, while 1 could.
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5. "Squash_yield.csv"
This file is about fruit weight and seed numbers of the experimental fruits.
Number of variables: 5
Number of rows: 44
Variable list (data type):
- Fruit.weight.g. (continuous), showing gram of the fruit.
- Mature.seed.number (continuous), showing the number of seeds per fruit.
- Flower.treatment (category), showing the experimental treatment of each flower.
- Area (category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie).
- Flowering.Day (category), showing the date the focal experimental flower had bloomed.
Note: "Flower.treatment" includes alpabets, D and E.
These charaters correspond to open and supplementary hand-pollination, respectively.
Unit of measures of fruit.weight.g. is gram.
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6. "Behaviours_on_squash_flowers.csv"
This data is about behaviors of flower-visiting insects and whether the insects touched stigmas or not.
Number of variables: 12
Number of rows: 832
Variable list (data type):
- taxa (category), showing the taxonomic identies of obsereved insects.
- flower_sex (category), the sex of the flower that insects visited. "F" means "female", and "M" means "male"
- nectar (continuous), showing whether the obsereved insect fed on nectar.
- pollen (continuous), showing whether the obsereved insect fed on pollen.
- wandering (continuous), showing whether the obsereved insect wondered the flower.
- unknown (continuous) is the variable for when obserevers could not identify the behaviours of insects.
- stigma_contact (continuous), showing whether the obsereved insects touched stigma or not.
- area (category), showing the prefectures of the sites (i.e., Ibaraki, Kagoshima, or Mie)
- date (category), showing the date that the observations were conducted.
- male (continuous), showing the number of male flowers that observers observed at the same time, in the observation trial.
- female (continuous), showing the number of female flowers that observers observed at the same time, in the observation trial.
- time_dur_m (continuous), showing the observation timing divided by each of 30 minutes. For example, 7.5 indicates the observation was started during 7:21-7:50, and 8 indicates that was during 7:51-8:20.
Note: In the variables of "nectar"," pollen", "wandering", and "unknown", 1 means the insect did the focal categorized behavior, and "0" means it did not.
"Behaviours_on_squash_flowers.csv" is the original data of Fig. 1 and Fig. 3.
Empty cells mean no information.Details of the definition of each behavior were explained in the manuscript.
Code/Software
Software
R 4.2.0
Packages
- glmmTMB
- DHARMa
- VGAM
- tidyverse
- emmeans
- ggplot2
- ggeffect
Methods
Flower visitor survey
To clarify relative pollinator importance of the flower visitors, considering diurnal and geographical variations, we conducted (1) flower visitor sampling, (2) assessment of the amount of pollen on body surfaces, and (3) the examination of insect behaviors, from May to July 2019. Study plots (25–50m2) were established on experimental fields of C. maxima in Ibaraki, Mie, and Kagoshima, and at each plot, insect sampling was conducted for 5 minutes per hour.
We captured each insect individual on male and female flowers with a 5ml plastic vial directly or with an insect net (36cm in diameter) by sweeping very slowly and then into the vial. Those vials were immediately put into cooler boxes, to keep the insects calm and to not let pollen grains fall. After the survey, all the vials containing insect individuals were labeled and stored in freezers at -20°C. Since the flowering period and the active time of flower visitors depend on daily temperature and sunrise time, the sampling period was optimized at each site. Specifically, collections were conducted at ten plots from 5:00 to 11:00 on 17 and 27 June, and 9 July in Ibaraki, at three plots from 5:00 to 12:00 on 19 and 25 June and 3 and 5 July in Mie, and at three plots from 6:00 to 11:00 at three plots on 21 and 29 May in Kagoshima. Additionally, extra samples from Kagoshima collected on 8 May were added to increase the replications for pollen load assessment. Specimens were morphologically identified to the lowest taxonomic level where possible at the conservation ecology laboratory at the University of Tsukuba. Identification of Halictidae and Nitidulidae (Coleoptera) was helped by expert taxonomists. Bees and large wasps were identified to species, genus, tribe, or family level. Other insect groups were identified to order level at least.
The amount of pollen load of each insect was estimated, following the method described in detail in Nikkeshi et al. (2019). Briefly, we cut and eliminated hind legs with corbicular pollen loads using scissors and poured 0.4M sucrose solution (1.0–6.0mL, depending on the body size) into each vial with an insect sample. This solution is an isotonic solution for pollen (Nikkeshi 2022) and prevents pollen accumulating at the bottom due to its high viscosity (Nikkeshi et al. 2021; Nikkeshi 2022). After shaking it in order to separate pollen from the insect bodies and uniformize it in the solution, we sampled 10μL of the solution, and counted the number of pollen grains on a microscope slide by microscope. In this process, we counted only squash pollen, which has a remarkably larger size (more than 170㎛) compared with the other plant species in our experimental gardens. We repeated this sampling and counting five times, and calculated the average number of pollen grains per 10μL. Finally, we estimated pollen loads of each insect, the total number of pollen grains adhered to each body surface except for the hind legs, by multiplying the average number of pollen grains per 10uL and the initial solution volume.
To examine the insects’ movement after landing on a flower, we observed female and male flowers for 15 minutes, and recorded insect behaviors considering four categories (i.e., pollen foraging, nectar foraging, wandering, and unknown). In the case of female flowers, we recorded whether the observed insects touched the stigmas. We defined “pollen foraging” as behaviors that involve mouthparts contacting anthers, or collecting pollen by rubbing the legs. “Nectar foraging” involves visitors inserting their heads or proboscis into the nectary at the flower base, and “wandering” involves walking around or standing still on the flower without foraging. In this observation, A. mellifera and A. cerana japonica were categorized as “honeybees”, due to the difficulty of discriminating between two Apis species in the field. This observation was made from 7:00 to 10:30 on 7 days from June 19 to July 8 in Ibaraki (8.25 hours in total) and from 7:30 to 11:30 on May 15 and 17 in Kagoshima (2.5 hours in total).
Assessment of the contribution of the flower visitors to the squash yield
To verify the smallest body size of flower-visiting insects that contribute to the fruit and/or seed set, we conducted an enclosure experiment manipulating accessibility to flowers depending on the body size of insects. One day before anthesis, female buds were randomly assigned to enclosure treatments: (1) non-woven bag (Daiso Industries, CO., Ltd, Hiroshima, Japan), (2) fine mesh bag (inner length: 4.44 × 2.92mm: “4mm mesh”, Tomoyasu Works, CO., Ltd., Osaka, Japan), (3) coarse mesh bag (inner length: 7.04 × 7.63mm: “9mm mesh”, Tomoyasu Works, CO., Ltd., Osaka, Japan), (4) open pollination, and (5) supplementary hand pollination. Non-woven bags were supposed to admit no insects, fine mesh bags were supposed to admit Halictidae and other tiny insects, and coarse mesh bags were supposed to admit all insects visiting squash flowers, including honeybees and bumblebees. In addition, by comparing treatments (4) and (5), we examined whether fruit and seed productions were limited by the amount of pollen reception of stigmas in the natural condition. A series of experiments was carried out from June 21 to July 8 in Ibaraki (7 replications), from June 6 to 25 in Mie (14 replications), and on May 16 in Kagoshima (5 replications). To confirm effectiveness of bag control treatment (i.e., coarse mesh bag), we walked around near the experimental flowers, and observed that honeybees and Bombus diversus diversus entered the coarse mesh bags.
To protect the experimental flowers from resource competition within the same vine, we eliminated female flowers and buds positioned within five joints from the targeted flowers in Ibaraki, and all female flowers and buds other than the targeted flowers in Mie and Kagoshima. One week after the experimental day, we checked whether the experimental flowers set fruits. After fruit maturation, we compared the number of seeds per fruit and fruit weight between the open (i.e., natural) pollination and the supplementary hand pollination condition.
At a laboratory, to further ensure the accuracy of enclosure treatment, we measured the thorax width of the specimens using a microscope to compare with the inner length of meshes (Sonoda et al. 2022). Since the thorax of Hymenoptera is thicker than the other orders, we also measured the length of the thorax (i.e., the maximum length of the thorax from the back to front). Because the thorax width of Coleoptera tends to be shorter than the abdomen, we measured maximum width of their abdomens. Finally, we tested whether the specimens were able to pass through the mesh physically by hand, and confirmed our experimental setting worked as intended (Table A2).