This readme file was generated on 2026-05-19 by Polona Kogovšek

# GENERAL INFORMATION

* Title of Dataset: Data for: Spider webs as efficient passive samplers of airborne fungal eDNA in forests: a case study with Hymenoscyphus fraxineus

## Author/Principal Investigator Information
Name: Polona Kogovšek
ORCID:0000-0002-4035-0115
Institution: National Institute of Biology
Address: Večna pot 121, 1000 Ljubljana 
Email: Polona.kogovsek@nib.si

## Author/Associate or Co-investigator Information
Name: Denis Kutnjak 
ORCID: 0000-0002-5327-0587
Institution: National Institute of Biology
Address: Večna pot 121, 1000 Ljubljana 
Email: Polona.kogovsek@nib.si


* Date of data collection: 2023-2024
* Geographic location of data collection: Slovenia, Ljubljansko barje, Vrbovci forest (lat 45.95070, lon 14.56387)  
* Information about funding sources that supported the collection of the data: The study was funded by Slovenian Research Agency programs P4-0463, P4-0107, P1-0236 and project J1-1703; research infrastructure grant IO-0004; and by the Slovenian Ministry of Agriculture, Forestry and Food (Public Forestry Service).


# SHARING/ACCESS INFORMATION

* Licenses/restrictions placed on the data: CC-BY
* Links to publications that cite or use the data: doi: 10.1002/edn3.70315
* Links to other publicly accessible locations of the data: /
* Links/relationships to ancillary data sets: /
* Was data derived from another source? no
	* If yes, list source(s): /
* Recommended citation for this dataset: Kogovšek, P., Ogris, N., Ferle, M., Šet, J., Piškur, B., Lokovšek, T., Kutnjak, D., & Gregorič, M. (2026). Data for: Spider webs as efficient passive samplers of airborne fungal eDNA in forests: a case study with Hymenoscyphus fraxineus [Data set]. In Environmental DNA (Version 1, pp. 10.1002/edn3.70315). Zenodo. https://doi.org/10.5281/zenodo.20285349


# DATA & FILE OVERVIEW

## File List: 
Raw data.xlsx: Excel file with 6 sheets: Laboratory exposure of spider webs; Assay evaluation; Passive sampler evaluation; Field experiment; Controls; Weather data. 
FileS1.docx: Photographs of field experiment set-up and types of spider webs collected in the forest
FileS2.docs: Initial Passive Sampler Comparison: three conventional passive samplers were tested under field conditions, to identify the most suitable passive sampler for comparison with spider web collection efficiency.
TableS1.xlsx: Results of filter paper and spider webs tested with general fungal (FQ) assay and for presence of H. fraxineus (Hfrax) DNA using qPCR.  

* Relationship between files, if important: /
* Additional related data collected that was not included in the current data package: no 
* Are there multiple versions of the dataset? no
	* If yes, name of file(s) that was updated: /
	* Why was the file updated? /
	* When was the file updated? /


# METHODOLOGICAL INFORMATION

## Description of methods used for collection/generation of data: 
Specific qPCR assay for detection of H. fraxineus (Hfrax) (Ioos et al., 2009) and a general FungiQuant (FQ) assay (Liu et al., 2012) were used. The qPCR reaction mixture for Hfrax consisted of TaqMan Environmental master mix 2.0 (Applied Biosystems), 300 nM of each primer and 150 nM TaqMan probe. The qPCR reaction mixture for FQ consisted of PerfeCTa qPCR ToughMix Low ROX master mix (QuantaBio, 95114) and 900 nM of each primer and 250 nM TaqMan probe. Two µl of DNA were tested in a final reaction mixture volume of 10 µl. The reactions were incubated for 2 min at 50°C, followed by 10 min at 95°C, and 45 cycles at 95° C for 15 s and at 65°C for 1 min, on 7900HT Fast or Viia7 Real-time PCR System (Applied Biosystems).  

## Methods for processing the data: 
The generated data were analysed using SDS software (version 2.4.1., Applied Biosystems) or QuantStudio Real-Time PCR Software (version 1.6.1, Applied Biosystems). Same threshold was set for all qPCR runs on the same device. Positive and negative controls and the appearance of sigmoid amplification curves were checked for each analysis. We evaluated performance of both qPCR assays to meet MIQE guidelines: dilution series of positive control was tested in up to 10 sequential dilutions (each in 5 technical replicates) to determine linearity of the assays (Bustin et al., 2025). Number of target copies in the positive control was determined by droplet digital PCR using the Hfrax assay (Ioos et al., 2009) and reaction conditions as previously described (Dobnik et al., 2019). Three consecutive 10-fold dilutions of DNA were tested in duplicate, and average number of copies was determined from them. Acceptance criteria were >10,000 positive droplets, NTC < 3 positives, coefficient of variation between replicates < 25%. We report eDNA sampling, controls, metadata, and sample processing following the eDNA reporting guidance (Gagné et al., 2021).

The obtained Cq values were further analysed in MS Excel (Microsoft, WA, USA): we calculated the average Cq value and standard deviation of the three technical replicates. Standard curve was prepared from the Cq values obtained by testing dilution series of positive control, plotted against logarithmic value of number of copies of target sequence per reaction. Slope, intercept and efficiency of amplification (E) were calculated from standard curve. Limit of detection (LOD) of the assays was determined at the dilution when at least two of the replicates were positive (out of five tested), and limit of quantification (LOQ) was determined at the lowest dilution within the linear range of the standard curve (regression line R2 ≥ 0.99, SD (Cq) ≤ 0.8). We used Wilcoxon signed‑rank test (Statistics Kingdom, 2017) across seven sampling events to evaluate statistical significance of the difference in Cq between web and filter paper samples and between orb and sheet webs. For field comparisons we computed ΔCq = Cq(filter) − Cq(web), separately for daily (orb) and weekly (sheet) pairs and for both assays. For easier interpretation, Cq values that were out of the linear range of the assays, were included in the calculations. Median ΔCq was calculated from all seven paired sampling dates and converted to fold‑difference using the standard-curve slope.

## Instrument- or software-specific information needed to interpret the data: 
n.a.

* Environmental/experimental conditions: 
Prior to onset of field experiment, we surveyed several localities for H. fraxineus occurrence and habitat suitability. As the study site, we selected the forest that was humid enough and contained abundant H. fraxineus apothecia, where we ran seven sampling campaigns (early summer to late summer across two years), starting in June 2023.
Webs of both types were each collected within a fixed 30 min intense search effort, by one collector, who collected 14-46 orb webs and 21-71 sheet webs during each sampling campaign (Table S2, File S1). Webs were sampled within approximately 25 m of the passive sampler stands and 0.5 m to 1 m above ground. Webs were wound on a clean disposable needle and placed in a microcentrifuge tube, that represented one pooled orb or sheet web sample. In an initial field comparison, we evaluated aerial fungal eDNA sampling with simple passive samplers: microscope slides covered with petroleum jelly, petri dishes covered with petroleum jelly, and cellulose filter paper (File S2)(West & Kimber, 2015). Filter paper (Ø 15 cm; MN 617, Macherey-Nagel, Düren, Germany) was housed in Ø 19 cm glass petri dish and set on 0.5 m tall table. Three visits of the sampling location were needed for each sampling campaign, for (i) instalment of two filter papers, (ii) collection of one filter paper after 24 h of exposure to match orb web exposure and (iii) collection of the second filter paper after 7 days of exposure to match sheet web exposure.  

For DNA extraction, we transferred web silk samples into 2 mL Fast Prep tubes (MP Biomedicals) containing 1 gram of zirconium oxide beads (Next Advance) and one ceramic sphere bead (MP Biomedicals), with added 700 µL of CSPL buffer and 20 µL of Proteinase K (Mag-Bind Plant DNA DS 96 Kit, Omega Bio-tek). Filter paper samples required a larger volume tube for processing. We cut filter paper into four equal parts and put one of the pieces into 15 mL tube (Corning) containing 3 g of zirconium oxide beads and three ceramic sphere beads, 2100 µL of CSPL buffer, and 60 µL of Proteinase K. All samples were first thoroughly vortexed and then homogenized using the FastPrep-24 bead beater (MP Biomedicals) for 1 minute at 6 m/s. Extraction then followed the manufacturer-provided Mag-Bind Plant DNA DS Kit Protocol, configured for the KingFisher Apex (Thermo Fisher Scientific). We eluted DNA in 100 µL and stored it at -20°C until further analysis. We employed negative extraction control (nuclease-free water only). To monitor for amplification inhibition, we tested different dilutions of the DNA samples (undiluted, 3-, 10- or 100-fold diluted), and 3-fold dilutions were used for comparisons (least inhibition without loss of sensitivity). Dilutions were prepared in nuclease free water (Sigma-Aldrich) and tested in three technical replicates.

* Describe any quality-assurance procedures performed on the data: 
We tested for possible H. fraxineus contaminations in our sampling process, i.e., spider webs, and on fresh filter paper. On 13. September 2023, at the field experiment locality, we collected two adult female Metellina segmentata and two adult female Linyphia triangularis spiders. Both species are abundant at the locality and build orb and sheet webs, respectively. We housed these spiders in disinfected polyacrylate frames in the laboratory to allow web construction (Kralj-Fišer & Gregorič, 2019). We then used laboratory webs of these spiders as negative controls. Additionally, we used filter paper directly from the package as negative control.

In each qPCR run, a positive control (DNA of H.fraxineus) and a negative control (nuclease-free water) were included and results were verified before proceeding with the analysis of individual samples.

* People involved with sample collection, processing, analysis and/or submission: 
Polona KOGOVŠEK, Nikica OGRIS, Maja FERLE, Janko ŠET, Barbara PIŠKUR, Tjaša LOKOVŠEK, Denis KUTNJAK, Matjaž GREGORIČ

# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Laboratory
* Number of variables: 12
* Number of cases/rows: 18
* Variable List: Sample group, Spider web type, Dilution, Sample code, FQ (Cq Mean, Cq SD, No P., Cq diff between dilutions*), Hfrax (Cq Mean, Cq SD, No P., Cq diff between dilutions*)
* Missing data codes: n.a.
* Specialized formats or other abbreviations used: /

# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Assay evaluation
* Number of variables: 9
* Number of cases/rows: 50
* Variable List: H. fraxineus dilution factor, Copies/reaction, log (copies/reaction), Hfrax (Cq, Cq Mean, Cq StdDev), FQ (Cq, Cq Mean, Cq StdDev)
* Missing data codes: n.a.
* Specialized formats or other abbreviations used: /
* Additional: Assay slope, intercept, efficiency; calibrator Cq, calibrator No. copies/reaction (2 ul), corrected intercept, no. copies in undiluted sample (Cq 17.01); dilution curve
 
# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Passive sampler evaluation
* Number of variables: 13
* Number of cases/rows: 36
* Variable List: Date of sampling, Sample Exposure time, Dilution, Sample code, FQ (Cq Mean, Cq StdDev, No P., Cq diff between dilutions*), Hfrax (Cq Mean, Cq StdDev, No P., Cq diff between dilutions*)

# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Field Experiment
* Number of variables: 13
* Number of cases/rows: 64
* Variable List: Date of sampling, Sample (no of webs collected), Exposure time, Dilution, Sample code, FQ (Cq Mean, Cq SD, No P., Cq diff between dilutions*), Hfrax (Cq Mean, Cq SD, No P., Cq diff between dilutions*)

# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Controls
* Number of variables: 5 tables, each table from different experiment
* Variable List: Control, FQ (Cq Mean, Cq SD, No P., Cq diff between dilutions*), Hfrax (Cq Mean, Cq SD, No P., Cq diff between dilutions*)


# DATA-SPECIFIC INFORMATION FOR: Raw data, spreadsheet Weather data
* Number of variables: 5
* Number of cases/rows: 132 (sampling period 2023) + 62 (sampling period 2024)
* Variable List: Date, Average daily T [°C], Average wind speed [m/s], Average relative humidity [%], Percipitation [mm]


# DATA-SPECIFIC INFORMATION FOR: Table S1
* Number of variables: 10
* Number of cases/rows: 24
* Variable List: Date of sampling, Sample (no of webs collected), Exposure time, Sample code, FQ (Cq Mean, Cq SD, No P.), Hfrax (Cq Mean, Cq SD, No P.)
* Specialized formats or other abbreviations used: /


# DATA-SPECIFIC INFORMATION FOR: File S1
* Number of variables: 3 photographs of field experiment set-up and types of spider webs collected in the forest
* Number of cases/rows: 3
* Variable List: 
Figure S1: Field sampling. Cellulose filter paper (Ø 15 cm) housed in Ø 19 cm glass petri dish, set on 0.5 m tall table, deployed in the forest. Petri dish covered with petroleum jelly was set on side during comparative analysis of passive samplers. 
Figure S2: Representative photo of sheet spider web collected in the forest.
Figure S3: Representative photo of orb spider web collected in the forest
* Specialized formats or other abbreviations used: /

# DATA-SPECIFIC INFORMATION FOR: File S2
* Number of variables: /
* Number of cases/rows: /
* Variable List: /
* Specialized formats or other abbreviations used: /
* Additional info: Initial Passive Sampler Comparison: Purpose, Methods, Results, Interpretation
