Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis ‘Somei-yoshino’ across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.


INTRODUCTION
Recent research has shown that environmental DNA sequences are present everywhere (Rosario and Breitbart 2011). Although environmental DNA can be sampled from a wide range of targets, the petal surface of flowers is an attractive source for investigation as it enables analysis of the dispersal of environmental DNA into the emerging niche of a newly-opened flower.
The ornamental cultivar 'Somei-yoshino' ( Cerasus × yedoensis ) is the most widely cultivated cherry tree in Japan (Lindstrom 2007, Shirahata 2000, with the exception of Okinawa prefecture (Iketani et al. 2007). In part, because C. × yedoensis is propagated through grafting (and therefore cloned from a single tree), it blooms synchronously within a single climate (Innan et al. 1995, Kato et al. 2012). The pale-rose five-petal flowers are considered a symbol of spring in Japan, and "Ohanami" (flower viewing) parties are held annually (Fig. 1A). Upon bud opening, C. × yedoensis petal surfaces exhibit a petal effect , an adhesive quality that causes the capture of environmental DNA in the form of microbes or pollen deposited by pollinators or the wind (Feng et al. 2008). Differences in the environmental DNA on petal surfaces of cloned plants may reflect differences in the interaction of the plants with their environments. To date, however, there is very little data on environmental DNA samples from cloned plants to investigate differences among geographical locations.
Our goals in this study were two-fold. First, we aimed to elucidate the origins of environmental DNA present on flower petals. Second, we aimed to examine Ohta et al. Crowdsourced environmental DNA sampling from cherry blossom petal the effectiveness of a crowdsourcing project (Howe 2006) for rapid and large-scale sampling. In investigations into environmental DNA, it is critical to collect sufficiently large sample from multiple locations, while retaining sample quality. A previous study used a crowdsourcing approach to solve the problem of sampling from many geographically separate locations (MetaSUB International Consortium, 2016).
This method potentially offers an effective approach for sample collection across a large area and over a very short interval.
Our "Ohanami Project" took place during spring 2015 and targeted environmental DNA on petals of newly-opened flowers of C. × yedoensis . In this report, we present the results of a crowdsourcing experiment to sample DNA from the petal surfaces of C. × yedoensis and the subsequent amplicon sequencing analysis to identify the sources of the foreign DNAs.

Materials and methods
Crowdsourced environmental DNA sampling of C. × yedoensis petal surfaces.
Sampling was conducted from March 17 to May 5, 2015. A sampling kit containing a disposable mask, disposable gloves, and barcode label was sent to 149 collaborators across Japan. We chose Puritan® Opti-Swab® Liquid Amies Collection & Transport System LA-106 as the swab kit. This kit contains a buffer composed of 3.0 g sodium chloride, 0.2 g monopotassium phosphate, 1.2 g disodium phosphate, 0.2 g potassium chloride, 1.0 g sodium thioglycolate, 0.1 g calcium chloride, and 0.1 g magnesium chloride per liter. To ensure the sampling quality of the crowdsourced samples, we designed and attached a detailed sampling protocol (Supplementary File 1). Each collaborator followed this protocol to sample environmental DNA from C. × yedoensis petal surfaces in their neighborhood, swabbing more than 10 flowers per tree; masks and gloves were worn to avoid contamination by the collector (Fig. 1B).
Immediately upon sampling, collaborators were instructed to affix the barcode label on the collection tube and take a photograph with their mobile phone; these steps were intended to minimize handling errors and to record locations through the Global A second PCR amplification was performed using a 20 μL reaction mixture: 1 μL of the first PCR product, 2 μL 10× PCR buffer, 1.6 μL 10 mM dNTP mix, 5 μL each of 1 μM PCR-F/R index primers, 0.1 μL Ex Taq HS DNA Polymerase, and 5.3 μL RNase-free water. The PCR thermocycling conditions were as follows: initial denaturation at 94°C for 3 min; 20 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 1 min, and extension at 72°C for 1.5 min; and final extension at 72°C for 10 min. The length of the PCR product was 258 or 259 bp. After confirming successful amplification using a fragment analyzer (Advanced Analytical, Ankeny, USA), PCR products were size-selected using BluePippin (Sage Science, Beverly, USA). The products were then treated with ExoSAP-IT (Affymetrix, Santa Clara, USA) for enzymatic cleanup and purified using AMPure XP (Beckman Coulter, Brea, USA). A Qubit Fluorometer (ThermoFisher, Waltham, USA) was used to measure DNA concentration in pooled samples and the samples were adjusted to 10 pM.

Results and discussion
In total, 28,594,048 sequence reads of 251 bp in length were obtained by MiSeq sequencing. After quality control, 5,606,853 reads remained for subsequent Ohta et al. Crowdsourced environmental DNA sampling from cherry blossom petal analyses. Initial taxonomic assignment of all reads identified various prokaryotic phyla ( Fig. 2) in which Cyanobacteria and Proteobacteria dominated with 81% and 12%, respectively. According to BLASTN results, most of the reads assigned to Cyanobacteria and Proteobacteria were derived from chloroplasts and mitochondria of the host tree and other species. Specifically, the majority of reads exactly matched to chloroplast DNA from wild strawberry Fragaria virginiana (family Rosaceae) (Fig.   3). Since both wild strawberry and flowering cherry belong to the Rosaceae family, it was expected that V4 hypervariable region sequences of the two species are identical.
Thus, we compared the sequences of F. virginiana and the chloroplast genome sequence of Prunus yedoensis , retrieved from the NCBI GenBank (GenBank ID: KP760070), which was not included in the organelle database (Fig. 4). The alignment shows that there is no mismatch among the two reference sequences and the most abundant reads in our samples, which indicated that the reads assigned to F. virginiana by BLASTN were derived from the host plant. The second, third, and fourth most frequent reads were one-nucleotide-mismatch hits to P. yedoensis chloroplast DNA, with a sharp decrease in hits containing more than one mismatch

Acknowledgements
We thank all the Ohanami Project collaborators (Supplementary File 7) for their help with sampling. We also thank the NGS Field 4th Meeting organizers who encouraged us to publish this manuscript. We thank Dr. Hiroshi Mori of National Institute of Genetics for helpful comments. We would also like to show our gratitude to Halna Tsunekawa for graphical design of the project website and the logo.
Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics.

Funding
This work was supported by the NGS Field 4th Meeting.

Disclosure statement
The authors declare no conflict of interest associated with this manuscript.       Ac id ob ac te ria Ac id ob ac te ria Ac id ob ... ou p 1) un cu ltu re d 5%