DINA Dataset Unversity of Kassel

Study Extent

Nature reserves in Germany

Sampling

Five Malaise traps were set at each of 21 sites in Germany, covering the entire geographic area and a variety of habitats. Each site bordered agricultural land. The first trap was placed 25 m into farmland. Subsequent traps were placed 25 m apart, with the second trap placed directly at the interface between cropland and protected area and the fifth placed 75 m from the center of the protected area. The nature protected areas meet the definition of International Union of Conservation of Nature (IUCN) Category IV habitat and species management, that aims to protect particular species or habitats, but vary in size and are not strictly prohibited from human use and often require management intervention.

Quality Control

Vegetation surveys, including mosses and lichens, were conducted near the traps in 3.50 m2 quadrats. In addition to the quadrats, species occurring within a radius of 50 m around Malaise traps and the crops where the first trap was placed were recorded. As the sample processing could not take place in a sterile environment, air pollen samples in the working space were collected to account for contamination during processing via the gravimetric method using a Petri dish (10 cm in diameter) coated with a thin layer of Vaseline. These samples were refrigerated until integration into the plant metabarcoding laboratory process.

Method steps

1. Insects were collected in 96% ethanol with a sampling interval of 14 days. For this analysis, a two-week period from mid to late May 2020 was selected with a maximum difference in collection dates of four days across all sites. The original ethanol was replaced with fresh ethanol in the laboratory for metabarcoding of plants, voucher specimens, and morphological identification.
2. The original sample ethanol (200 ml ± 50 ml) was vacuum filtered using a 250 ml Nalgene single use analytical filter funnel with a cellulose nitrate (CN) filter (diameter 47 mm and 0.2 µl pore size) in a biosafety cabinet with DNA free equipment.
3. Following filtration, the CN filter was cut into two equal parts and each part placed in a 2 ml SafeSeal microcentrifuge tube (Sarstedt AG & Co. KG), with one half used for DNA extraction and the other saved as a voucher and/or backup for protocol optimisation. These samples were stored at -20 °C until further processing.
4. DNA extraction was performed with NucleoMag 96 Plant Kit (Macherey Nagel, Oesingen, Switzerland).
5. ITS2 barcoding with the ITS2 primers, forward: ITS-3p62plF1, ACBTRGTGTGAATTGCAGRATC and reverse: ITS-4unR1, TCCTCCGCTTATTKATATGC and PCR was performed with three replicates per sample.
6. The pooled replicates of non-indexed PCR products were sent to LGC Genomics GmbH (Berlin) for sequencing on a MiSeq (2 × 300 bp) after an additional 12 PCR cycles.
7. Plant metabarcoding data pipeline: Sequencing data were processed with USEARCH (Edgar 2010) and DADA2 (Callahan et al. 2016) using R (v. 4.1.0) (R Core Team 2021). Edgar RC (2010): Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19): 2460–2461. https://doi.org/10.1093/bioinformatics/btq461 Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP (2016): DADA2: High-resolution sample inference from Illumina amplicon data. Nature Methods 13(7): 581–583. https://doi.org/10.1038/nmeth.3869
8. The SINTAX algorithm (Edgar 2016) was used (cutoff 0.8) for ASV identification with a custom database. The database was created in June 2021 from sequences downloaded using the GenBank webinterface at https://www.ncbi.nlm.nih.gov/genbank/ in GenBank (full) format using the search string (internal transcribed spacer1[Title] OR internal transcribed spacer 2[Title] OR ITS1[Title] OR ITS2[Title]) NOT patent NOT pseudogene NOT mRNA NOT unverified AND 100:2500[Sequence Length] AND Tracheophyta[Organism]. The term unverified is used by GenBank staff to flag erroneous sequences and should be excluded in every GenBank query. Edgar RC (2016): SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv, 1–20. https://doi.org/10.1101/074161

1. Cornales
   rank: order
2. Cucurbitales
   rank: order
3. Dsacales
   rank: order
4. Ericales
   rank: order
5. Fabales
   rank: order
6. Fagales
   rank: order
7. Gentianales
   rank: order
8. Geraniales
   rank: order
9. Lamiales
   rank: order
10. Liliales
    rank: order
11. Malpighiales
    rank: order
12. Malvales
    rank: order
13. Myrtales
    rank: order
14. Pinales
    rank: order
15. Poales
    rank: order
16. Polypodiales
    rank: order
17. Ranunculales
    rank: order
18. Rosales
    rank: order
19. Santalales
    rank: order
20. Sapindales
    rank: order
21. Saxifragales
    rank: order
22. Solanales
    rank: order
23. Zingiberales
    rank: order
24. Plantae
    rank: kingdom

Germany