DOI: 10.5061/dryad.j6q573npc
title: Data from: Flying by night: Comparing nocturnal pollinator networks over time in the Colorado Rocky Mountains

description: Because pollen-transport networks tend to vary widely over short periods
 of time but remain consistent over longer periods of time, it is important
 to account for study length when characterizing pollen-transport network
 structure. The study of nocturnal pollen-transport networks independently
 from diurnal pollinator networks has also been emphasized, as nocturnal
 pollen-transport networks are an often overlooked ecosystem
 component.  Here, we systematically compare the properties of
 moth pollen-transport pollinator networks in Colorado, USA, over the
 course of a season, and test for a link between specialization and
 pollinator morphology. We sampled moths for pollen over nine weeks in June
 through August 2021. We then constructed pollen-transport networks, and
 used mixed models to test for differences in network indices between
 different parts of the 2021 season; we also tested for a relationship
 between moth proboscis length, moth presence across seasons, and the
 number of flower species pollinated. As expected, we found high rates of
 species turnover and plant-pollinator interactions over the summer, and we
 found that longer proboscis lengths – as well as length of presence over
 the summer – were predictive of higher generalization within individual
 species. However, variation in the overall structure of pollen-transport
 networks was relatively low over the course of the season, with linkage
 density and robustness being the only metrics that changed. We suggest
 that even with high rates of species turnover, nocturnal moth
 pollen-transport networks may maintain their overall structure over a
 season, with variations in some metrics possibly resulting from variations
 in the availability of floral resources. Our study highlights the
 necessity of continuing to research moth pollen-transport networks over
 different temporal scales, as aggregating pollination data over time can
 obfuscate patterns in temporal variation in network structure.
description: <em>Study sites</em> In Gunnison
 County, Colorado, USA, summer 2021, we sampled a total of four sites
 around the Rocky Mountain Biological Laboratory (RMBL) (38°57′5”N,
 106°59′6”W, 2912 m.a.s.l.) (Fig. 1). This is a high-altitude montane
 ecosystem, with dry meadows bordering aspen forests. Evening temperatures
 obtained from a weather station located within 600 meters of sites 1-3
 ranged from 5°C to 15°C, with occasional rainy periods. Sampling never
 took place on a day with more than 0.5mm mean rainfall over the sampling
 period. There was no significant difference in temperatures recorded over
 our early, middle, and late-season sampling periods; the mean temperature
 during sampling hours in the early season was 9°C (SD = 2.8), 8.6°C (SD =
 2) in the middle season, and 10°C (SD = 2.9) in the late season. Our
 sampling encompassed the peak wildflower bloom and the post-snowmelt
 growing season; the number of overall species flowering in the area
 increased from approximately 60 species at the start of our sampling to
 494 species at the peak of our last period of sampling (unpublished data,
 collected in 2021 as described in Inouye 2008). Site 1 was located on the
 border of a dry meadow and a forested stretch of aspen trees (2910
 m.a.s.l.). Site 2 was located in a dry meadow, adjacent to mixed
 spruce-aspen forest (2920 m.a.s.l.). Site 3 was located in a dry meadow,
 adjacent to willow trees growing near a creek (2900 m.a.s.l.). Site 4 was
 located in a dry meadow, adjacent to beaver ponds and mixed spruce-aspen
 forest (3060 m.a.s.l.). Sites 1, 2, and 3 were all located within 720
 meters of one another; site 4 was 1620 meters away from site 1. 
 <em>Moth collection and
 identification:</em> We used a blacklight on a
 white sheet to attract moths (Macgown, 2022). Sampling occurred between
 21.30 and 23.30h (MDT) between 9-Jun-2021 and 9-Aug-2021 and was conducted
 manually, as some studies have found that manual trapping is broadly more
 effective than automatic trapping (Axmacher & Fiedler 2004, Tikoca
 et al 2016). As we observed a negligible number of moths arriving at the
 light between 23.00h – 24.00h in 2020 (unpublished data), we focused on
 sampling the early peak of moth activity in 2021. For ease of
 identification, we generally did not sample microlepidoptera. Site 1 was
 sampled nine times; sites 2 and 3 were sampled seven times each, and site
 4 was sampled once, for a total of 24 sampling nights over the course of
 the study. We collected no more than fifteen moths per night due to
 processing constraints. When more than fifteen total moths arrived at the
 blacklight, moths were randomly sampled at regular intervals over the
 course of the night, to avoid biasing sampling towards the species that
 arrived the earliest. Site 4 was excluded from sampling after one night
 due to time constraints and safety concerns related to presence of
 mountain lions; data from site 4 was only used in cumulative species lists
 and calculating Jaccard indices. During collection,
 moths were stored in individual containers to avoid cross-contamination,
 and chilled on ice prior to being brought to the lab for pollen swabbing
 and further processing. We measured proboscis length using electronic
 calipers, and recorded the sex and wing wear of all moths (wing wear rated
 from 1, ‘no scale damage’, to 3 ‘extensive scale loss and wear’). When we
 encountered a new moth species or morphotype, we collected and pinned two
 voucher specimens; we released identifiable specimens on-site. Individual
 moths were identified to species where possible, and to family when it was
 not possible. Morphospecies were treated as unique species in subsequent
 analyses. <em>Pollen collection and
 identification:</em> To identify moth pollen
 samples, we referred to an existing pollen library at RMBL, as well as one
 period of selective sampling from blooming flowers in mid-June at site 3.
 To obtain pollen samples from all captured moths, we used fuchsin jelly
 cubes to swab the thorax, head, unrolled proboscis, and maxillary palps,
 which are most likely to carry pollen. We then placed the fuchsin jelly on
 a glass slide and covered it with a cover slip, and sealed the edges
 (Beattie 1971). We used a USB microscope (magnification x400 - x1000) to
 count and identify the pollen. We only recorded pollen counts when we
 observed at least five pollen grains of the same morphotype on a given
 moth. We chose five pollen grains as the cutoff, in line with other
 studies (Banza et al 2011, Banza et al 2015, Devoto et al 2011, Forup
 & Memmott 2005) which use this as a threshold to reduce the
 probability that observed pollen grains are the result of
 cross-contamination from light traps and handling.
description: # Data from: Flying by night: Comparing nocturnal pollinator networks over
 time in the Colorado Rocky Mountains
 [https://doi.org/10.5061/dryad.j6q573npc](https://doi.org/10.5061/dryad.j6q573npc) ## Description of the data and file structure Raw pollinator and pollen transport data This dataset contains data on individual moths sampled from light traps over the course of Summer 2021, and the amount and species of pollen grain that sampled individuals were found to be transporting. ### Files and variables #### File: moths\_cleaner2.csv **Description:**  this dataset may be used, with some processing (see attached code), to construct pollen-transport networks across different parts of the season, as well as analyze relationships between moth proboscis length and degree of specialization (number of different plant species pollinated). ##### Variables * Site: location at which moths were being sampled. Site 3 corresponds to Site 1 in the published manuscript;  site 4 corresponds to site 2;  site 6 corresponds to site 3; site 5 corresponds to site 4. * Date: date on which sampling occurred. * Season: ES = early season; MS = mid-season; LS = late-season. All sampling dates were bucketed into seasons for further analysis. * Individual #: individual ID assigned to each collected specimen. * Sex: moth sex. M = male, F = female, NA = unidentified, S = too small to identify. * Family: moth family. * Species: species name of moth. If moths could not be identified to species level, they were identified to family level or listed as unknown, with a number. * Wing wear: moth wing wear, rated from 1 ‘no scale damage’, to 3 ‘extensive scale loss and wear’.  * Proboscis length:  moth proboscis length (mm).  * 01 if released; 00 if kept: whether a sampled individual was kept and pinned as a voucher specimen or released after sampling. * Recap?: Y if an individual was recaptured from another sampling night, N if the individual had not been previously sampled.  * Notes: other notes.  * Total pollen load: total pollen grains carried by a given moth. * Significant pollen load: N if less than 5 total pollen grains of one species were carried; Y if more than 5 total pollen grains were carried.  All following columns are plant species or morphotypes and the total number of pollen grains a given moth individual was observed to be carrying from that plant. Throughout the file, NA = not available, indicating that data was unable to be collected. #### File: Jaccard\_plants.csv **Description:** this dataset may be used to analyze the presence/absence of pollen from a given plant species across different parts of the season.  ##### Variables * Species: plant species (coded by number). Pollen/plant species codes: 1 = Achillea millefolium, 2 = Aquilegia coerulea, 3 = Heracleum maximum, 4 = Veratrum californicum, 5 = Ligusticum porteri, 6 = Ribes montigenum, 8 = Ericacea sp, 9 = Lonicera involucrata, 10 = Potentilla gracilis, 11 = Linaria vulgaris, 12 = Heliomeris multiflora, 13 = U13, 14 = Anticlea elegans, 15 = Lathyrus leucanthus, 16 = Brassicaceae sp, 17 = Artemisia sp, 18 = Erigeron speciosus, 19 = U14 * ES: 1 = plant species presence in the 'early season' time period; 0 indicates absence. * MS: 1 = plant species presence in the 'mid season' time period; 0 indicates absence. * LS: 1 = plant species presence in the 'late season' time period; 0 indicates absence. #### File: Jaccard\_interactions.csv **Description:** this dataset may be used to analyze the presence/absence of pollinators across different parts of the season.  ##### Variables * Interactions: moth species + plant species interaction, given as moth species (plant code). Pollen/plant species codes: 1 = Achillea millefolium, 2 = Aquilegia coerulea, 3 = Heracleum maximum, 4 = Veratrum californicum, 5 = Ligusticum porteri, 6 = Ribes montigenum, 8 = Ericacea sp, 9 = Lonicera involucrata, 10 = Potentilla gracilis, 11 = Linaria vulgaris, 12 = Heliomeris multiflora, 13 = U13, 14 = Anticlea elegans, 15 = Lathyrus leucanthus, 16 = Brassicaceae sp, 17 = Artemisia sp, 18 = Erigeron speciosus, 19 = U14 * ES: 1 indicates presence of a given species interaction in the 'early season' time period; 0 indicates absence. * MS: 1 indicates presence of a given species interaction in the 'mid season' time period; 0 indicates absence. * LS: 1 indicates presence of a given species interaction in the 'late season' time period; 0 indicates absence. #### File: Jaccard\_S\_Pollinators.csv **Description:** this dataset may be used to analyze the presence/absence of pollen from different plant species across parts of the season. ##### Variables * Species: moth species name. * ES: 1 = moth species presence in the 'early season' time period; 0 indicates absence. * MS: 1 = moth species presence in the 'mid season' time period; 0 indicates absence. * LS: 1 = moth species presence in the 'late season' time period; 0 indicates absence. #### File: Co\_2021\_metadata-Sheet1.csv **Description:** this dataset includes notes and metadata about sampling. ##### Variables * Date: dates on which sampling occurred. * Initials: initials of observer. * Weather (temperature, based on Billy Barre's RMBL station): temperature (in degrees Celsius) based on data collected at a local weather station. * Weather (temperature, moon brightness): temperature (in degrees Celsius) and moon fullness, recorded on-site. * Notes: what you did and saw, which sites were sampled: notes on which sites were sampled /number of individual moths collected.\ \ Throughout the file, NA = not available, indicating that data was unable to be collected. ## Code/software #### \*\*File: \*\*co\_2021\_moths.R \**Description: **this is an R script that can be used to build and analyze pollen-transport networks from the attached data sets, to calculate Jaccard indices for plant species, pollinator species, and plant/pollinator interactions, and to build a linear model comparing proboscis length / specialization (in terms of plant species pollinated). All code is run in R. Software versions and packages: R package “vegan”, version 2.6-4 R package “abdiv”, version 0.2.0 R package “bipartite”, version 2.17 R package “lme4”, version 1.1-30 R package “car”, version 3.1-0 R package “tidyr”, version 1.2.1 R package “dplyr”, version 1.0.10 R package “ggplot2”, version 3.3.6 R package “RColorBrewer”, version 1.1-3 R package “rstatix”, version 0.7.0 ## Access information Weather data:[https://wrcc.dri.edu/weather/rbil.html]()