DOI: 10.5061/dryad.vmcvdnczm
title: Diurnal and geographic variations of pollinator importance for Cucurbita maxima Duchesne

description: 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 (&gt;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.
description: 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
 (25–50m<sup>2</sup>) were established on experimental fields
 of <em>C. maxima</em> 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, <em>A. mellifera </em>and<em> A.
 cerana japonica</em> were categorized as “honeybees”, due to the
 difficulty of discriminating between two <em>Apis</em> 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).
description: # Diurnal and geographical variations of pollinator importance for
 Cucurbita maxima Duchesne 
 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"
 \######################################################### Data specific
 information \######################################################### 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. \######################################################### 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".
 \######################################################### 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"
 \######################################################### 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.
 \######################################################### 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.
 \######################################################### 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