DOI: 10.5061/dryad.47d7wm3j1
title: Data for: Using environmental DNA to investigate avian interactions with flowering plant

description: Animal pollination is an important and highly valued ecosystem function
 and the role of birds as pollinators is increasingly acknowledged.
 However, such interactions can be challenging to document and often
 require extensive field programs. Over the last decade, environmental DNA
 (eDNA) has been analysed from several different contemporary sample types
 such as water, soil, flowers, and air. The applications of these studies
 include biodiversity monitoring, detection of endangered species,
 community compositions, and, more recently, flower-arthropod interactions.
 However, it remains unknown whether flower-eDNA is applicable to other
 taxonomic groups interacting with plants, as well as the deposition and
 degradation of eDNA on flowers. Here, we test whether eDNA from flowers
 can be used for detecting bird pollinators. In a controlled environment
 (an aviary with great tits [Parus major]), we show that birds leave
 significant traces of DNA on the flowers without observed visits (airborne
 eDNA). We further show that when birds had been in contact with the
 flowers, DNA concentrations increased to levels significantly higher than
 airborne background DNA. Subsequently, we sampled five clusters of wild
 flowers in Papua New Guinea and detected four species of birds, two of
 which are nectar-feeders, and one that is an insectivorous species known
 to visit flowers. These four bird species were regularly seen in the area
 and caught in mist-nets in the days prior to sampling of the flowers. In
 total, 29 bird species were recorded (18 mist-netted) in the area and of
 these eight are nectarivorous. Our quantitative approach suggests that it
 is possible to distinguish airborne background bird DNA deposited on
 flowers from actual flower visits of birds in the wild, although this
 might be highly context specific. Our findings are of broad interest
 within research on ecosystem functioning, biotic interactions, and
 plant-animal mutualism.
description: The samples used for generating the HT sequencing data was
 collected in Papua New Guinea in November 2019. Detailed methods can be
 found in the connected publication. DNA were extracted
 from the samples, and amplified by PCR, using the MiBird-U primers,
 targeting a 171 bp fragment of the mitochondrial 12S rRNA gene designed by
 Ushio et al. 2018 (doi:10.1038/s41598-018-22817-5). 
 Forward primer: GGGTTGGTAAATCTTGTGCCAGC
 Reverse primer: CATAGTGGGGTATCTAATCCCAGTTTG
 The libraries of amplified DNA from these samples have been
 sequenced using 150 bp paired end NovaSeq 6000 sequencing (150 BP
 PE). The Ct values were generated from flower samples
 from an experimental setup with great tits (<em>Parus
 major</em>). Two experiments were conducted: One where flowers were
 placed in an aviary with great tits and flower samples were collected
 regularly, to investigate the amount of DNA deposited on flowers from
 birds in the vicinity, but who were not visiting the flowers. Another,
 where great tits were gently pushed into flowers, whereafter flower
 samples were collected over time, to investigate DNA degradation after
 deposition from visiting birds. Details can be found in the connected
 publication. DNA were extracted from the samples, and
 Ct values were generated by qPCR, with the following primers and probe,
 specific to great tits: Name Type Sequence 
 ParmajCO1L  Forward Primer CGCCCTAAGCCTCCTTATTC
 ParmajCO1R Reverse Primer GTCAGTTTCCGAATCCTCCA
 ParmajCO1L.Probe Probe 6FAM-CCTCCTGGGAGACGACCAAA-BHQ1
description: The dataset consist of 2 sequencing libraries containing DNA
 reads from 4 replicates of each of the 5 flower samples from Papua New
 Guinea, and one extraction control (CNE). All samples begin with
 "PNG" and the extraction controls are named
 "CNE". The "tags" files contain a
 list of sample names and the tags used with the forward and reverse primer
 in the PCR reactions. The sample names are followed by the PCR replicate
 number
 ("_1","_2","_3","_4").
 The "Ct_values.txt" contains all Ct values for each of
 the samples in the experimental part of the study.
 Please se the README.md for additional usage notes. If you want
 to follow the data analysis done in the study, please see the connected
 publication for further details. Also, you are welcome to send an email
 with any questions that might come up.