Michaela R. Grossklaus David S. Pilliod Stephen F. Spear Matthew B. Laramie Akhil Kholwadwala Amanda J. Boone Yer Lor Marissa Kaminski Jeffrey G. Everett 20260220 tabular digital data ScienceBase.gov U.S. Geological Survey https://doi.org/10.5066/P132VXFT Michaela R. Grossklaus David S. Pilliod Stephen F. Spear Matthew B. Laramie Akhil Kholwadwala Amanda J. Boone Yer Lor Marissa Kaminski Jeffrey G. Everett 20260413 publication Genome vol. 69 n/a Canadian Science Publishing ppg. 1-13 https://doi.org/10.1139/gen-2025-0006 This dataset includes quantitative polymerase chain reaction (qPCR) data for an in vitro test of the Bombus franklini eDNA assay using target and nontarget DNA extract, qPCR data for a plate used to calculate the limits of detection and quantification of the assay based on serial 10-fold dilutions of B. franklini gBlock, qPCR data for an in situ test of the assay using flower samples collected within and around the historic range of B. franklini (including extraction negatives, qPCR no-template controls, and standards), and metabarcoding data for the same flower samples (including extraction negatives, mock samples, and metabarcoding no-template controls). These data were collected to support the development and validation of a qPCR assay for detection of Bombus franklini from flower samples. 20210629 20240416 ground condition Complete None planned Southwestern Oregon and northern California, USA -123.9258 -121.3000 43.9019 41.1435 ISO 19115 Topic Category biota environment location USGS Thesaurus environmental DNA genetics bees and wasps USGS Metadata Identifier USGS:68cd627bd4be0212e7537c57 Common geographic areas Oregon California None Bombus Bombus affinis Bombus centralis Bombus fervidus Bombus flavidus Bombus flavifrons Bombus frigidus Bombus griseocollis Bombus huntii Bombus hypnorum Bombus impatiens Bombus kirbiellus Bombus melanopygus Bombus mixtus Bombus nevadensis Bombus occidentalis Bombus pensylvanicus Bombus rufocinctus Bombus sitkensis Bombus sonorus Bombus sylvicola Bombus ternarius Bombus vagans Bombus vancouverensis Bombus vandykei Bombus vosnesenskii Integrated Taxonomic Information System (ITIS) 2025 website https://doi.org/10.5066/F7KH0KBK Integrated Taxonomic Information System (ITIS) Environmental DNA analysis All species were identified using environmental DNA. Kingdom Animalia Subkingdom Bilateria Infrakingdom Protostomia Superphylum Ecdysozoa Phylum Arthropoda Subphylum Hexapoda Class Insecta Subclass Pterygota Infraclass Neoptera Superorder Holometabola Order Hymenoptera Suborder Apocrita Infraorder Aculeata Superfamily Apoidea Family Apidae Subfamily Apinae Tribe Bombini Genus Bombus TSN: 154397 Species Bombus affinis TSN: 714782 Species Bombus centralis TSN: 714793 Species Bombus fervidus TSN: 714802 Species Bombus flavidus TSN: 714907 Species Bombus flavifrons TSN: 714803 Species Bombus frigidus TSN: 714806 Species Bombus griseocollis TSN: 714807 Species Bombus huntii TSN: 714810 Species Bombus hypnorum TSN: 714926 Species Bombus impatiens TSN: 714812 Species Bombus kirbiellus TSN: 1128121 Species Bombus melanopygus TSN: 714820 Species Bombus mixtus TSN: 714822 Species Bombus nevadensis TSN: 714826 Species Bombus occidentalis TSN: 714827 Species Bombus pensylvanicus TSN: 714828 Species Bombus rufocinctus TSN: 714833 Species Bombus sitkensis TSN: 714835 Species Bombus sonorus TSN: 714837 Species Bombus sylvicola TSN: 714840 Species Bombus ternarius TSN: 714841 Species Bombus vagans TSN: 714845 Species Bombus vancouverensis TSN: 1128319 Species Bombus vandykei TSN: 714846 Species Bombus vosnesenskii TSN: 714848 No access constraints. Please see 'Distribution Information' for details. These data are marked with a Creative Commons Zero v1.0 Universal (CC0-1.0) public domain dedication and do not have any use constraints. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. FRESC Science Data Coordinator Forest and Rangeland Ecosystem Science Center mailing and physical

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Corvallis OR 97331 USA 541-750-1030 fresc_outreach@usgs.gov Ashley Rohde identified bumble bee specimens. Kristina Parker, Scott Fordham, Keta Oettinger, Tariq Tajjioui, Hailey Willner, Claire Pluim, and Kimberly Mazur assisted in the laboratory. Philip Francis Thomsen Eva E. Sigsgaard 20190207 publication Ecology and Evolution vol. 9, issue 4 n/a Wiley ppg. 1665-1679 https://doi.org/10.1002/ece3.4809 Joan Milam Dennis E. Johnson Jeremy C. Andersen Aliza B. Fassler Desiree L. Narango Joseph S. Elkinton 20200930 publication Insects vol. 11, issue 10 n/a MDPI 669 https://doi.org/10.3390/insects11100669 Evan Bolyen Jai Ram Rideout Matthew R. Dillon Nicholas A. Bokulich Christian C. Abnet Gabriel A. Al-Ghalith Harriet Alexander Eric J. Alm Manimozhiyan Arumugam Francesco Asnicar Yang Bai Jordan E. Bisanz Kyle Bittinger Asker Brejnrod Colin J. Brislawn C. Titus Brown Benjamin J. Callahan Andrés Mauricio Caraballo-Rodríguez John Chase Emily K. Cope Ricardo Da Silva Christian Diener Pieter C. Dorrestein Gavin M. Douglas Daniel M. Durall Claire Duvallet Christian F. Edwardson Madeleine Ernst Mehrbod Estaki Jennifer Fouquier Julia M. Gauglitz Sean M. Gibbons Deanna L. Gibson Antonio Gonzalez Kestrel Gorlick Jiarong Guo Benjamin Hillmann Susan Holmes Hannes Holste Curtis Huttenhower Gavin A. Huttley Stefan Janssen Alan K. Jarmusch Lingjing Jiang Benjamin D. Kaehler Kyo Bin Kang Christopher R. Keefe Paul Keim Scott T. Kelley Dan Knights Irina Koester Tomasz Kosciolek Jorden Kreps Morgan G. I. Langille Joslynn Lee Ruth Ley Yong-Xin Liu Erikka Loftfield Catherine Lozupone Massoud Maher Clarisse Marotz Bryan D. Martin Daniel McDonald Lauren J. McIver Alexey V. Melnik Jessica L. Metcalf Sydney C. Morgan Jamie T. Morton Ahmad Turan Naimey Jose A. Navas-Molina Louis Felix Nothias Stephanie B. Orchanian Talima Pearson Samuel L. Peoples Daniel Petras Mary Lai Preuss Elmar Pruesse Lasse Buur Rasmussen Adam Rivers Michael S. Robeson Patrick Rosenthal Nicola Segata Michael Shaffer Arron Shiffer Rashmi Sinha Se Jin Song John R. Spear Austin D. Swafford Luke R. Thompson Pedro J. Torres Pauline Trinh Anupriya Tripathi Peter J. Turnbaugh Sabah Ul-Hasan Justin J. J. van der Hooft Fernando Vargas Yoshiki Vázquez-Baeza Emily Vogtmann Max von Hippel William Walters Yunhu Wan Mingxun Wang Jonathan Warren Kyle C. Weber Charles H. D. Williamson Amy D. Willis Zhenjiang Zech Xu Jesse R. Zaneveld Yilong Zhang Qiyun Zhu Rob Knight J. Gregory Caporaso 20190724 publication Nature Biotechnology vol. 37, issue 8 n/a Springer ppg. 852-857 https://doi.org/10.1038/s41587-019-0209-9 Marcel Martin 20110502 publication EMBnet.journal vol. 17, issue 1 n/a EMBnet Stichting ppg. 10-12 https://doi.org/10.14806/ej.17.1.200 Christiam Camacho George Coulouris Vahram Avagyan Ning Ma Jason Papadopoulos Kevin Bealer Thomas L Madden 20091215 publication BMC Bioinformatics vol. 10, issue 1 n/a Springer 421 https://doi.org/10.1186/1471-2105-10-421 Dennis A. Benson Mark Cavanaugh Karen Clark Ilene Karsch-Mizrachi David J. Lipman James Ostell Eric W. Sayers 20121126 publication Nucleic Acids Research vol. 41, issue D1 n/a Oxford University Press ppg. D36-D42 https://doi.org/10.1093/nar/gks1195 Eric W Sayers Jeffrey Beck Evan E Bolton Devon Bourexis James R Brister Kathi Canese Donald C Comeau Kathryn Funk Sunghwan Kim William Klimke Aron Marchler-Bauer Melissa Landrum Stacy Lathrop Zhiyong Lu Thomas L Madden Nuala O’Leary Lon Phan Sanjida H Rangwala Valerie A Schneider Yuri Skripchenko Jiyao Wang Jian Ye Barton W Trawick Kim D Pruitt Stephen T Sherry 20201023 publication Nucleic Acids Research vol. 49, issue D1 n/a Oxford University Press ppg. D10-D17 https://doi.org/10.1093/nar/gkaa892 Caren S. Goldberg Cameron R. Turner Kristy Deiner Katy E. Klymus Philip Francis Thomsen Melanie A. Murphy Stephen F. Spear Anna McKee Sara J. Oyler-McCance Robert Scott Cornman Matthew B. Laramie Andrew R. Mahon Richard F. Lance David S. Pilliod Katherine M. Strickler Lisette P. Waits Alexander K. Fremier Teruhiko Takahara Jelger E. Herder Pierre Taberlet 20160526 publication Methods in Ecology and Evolution vol. 7, issue 11 n/a Wiley ppg. 1299-1307 https://doi.org/10.1111/2041-210X.12595 Benjamin J. Callahan Paul J. McMurdie Michael J. Rosen Andrew W. Han Amy Jo A. Johnson Susan P. Holmes 20160523 publication Nature Methods vol. 13, issue 1 n/a Springer ppg. 581-583 https://doi.org/10.1038/nmeth.3869 Scott Sherrill-Mix 20250228 application/service R package version 0.10.8 https://cran.r-project.org/package=taxonomizr Rena M. Schweizer Jared A. Grummer Kerrigan B. Tobin Renee Corpuz Scott M. Geib Diana Cox-Foster Lynn S. Kimsey Jonathan B. Uhuad Koch Michael G. Branstetter 20251020 publication Proceedings of the National Academy of Sciences of the United States of America vol. 122, issue 43 n/a Proceedings of the National Academy of Sciences of the United States of America e2509749122 https://doi.org/10.1073/pnas.2509749122 Sujeevan Ratnasingham Paul D. N. Hebert 20070124 publication Molecular Ecology Notes vol. 7, issue 3 n/a Wiley ppg. 355-364 https://doi.org/10.1111/j.1471-8286.2007.01678.x Global Biodiversity Information Facility (GBIF) 2025 publication https://www.gbif.org/what-is-gbif Jessica L. Beckham Jeff A. Johnson Russell S. Pfau 20241017 publication Journal of Hymenoptera Research vol. 97, issue 1 n/a Pensoft Publishers ppg. 895-914 https://doi.org/10.3897/jhr.97.132937 Paul H. Williams 20210528 publication Journal of Natural History vol. 55, issue 5-6 n/a Taylor & Francis ppg. 271-282 https://doi.org/10.1080/00222933.2021.1900444 Guillaume Ghisbain Jeffrey D. Lozier Sarthok Rasique Rahman Briana D. Ezray Li Tian Jonah M. Ulmer Sam D. Heraghty James P. Strange Pierre Rasmont Heather M. Hines 20200115 publication Systematic Entomology vol. 45, issue 3 n/a Wiley ppg. 635-652 https://doi.org/10.1111/syen.12419 Baptiste Martinet Thomas Lecocq Nicolas Brasero Maxence Gerard Klára Urbanová Irena Valterová Jan Ove Gjershaug Denis Michez Pierre Rasmont 20190831 publication Zoological Journal of the Linnean Society vol. 187, issue 3 n/a Oxford University Press ppg. 599-621 https://doi.org/10.1093/zoolinnean/zlz041 Matthew J. Christmas Julia C. Jones Anna Olsson Ola Wallerman Ignas Bunikis Marcin Kierczak Valentina Peona Kaitlyn M. Whitley Tuuli Larva Alexander Suh Nicole E. Miller-Struttmann Jennifer C. Geib Matthew T. Webster 20210406 publication Molecular Biology and Evolution vol. 38, issue 8 n/a Oxford University Press ppg. 3126-3143 https://doi.org/10.1093/molbev/msab086 Danielle J. Clake Sean M. Rogers Paul Galpern 20240105 publication Biodiversity and Conservation vol. 33, issue 1 n/a Springer ppg. 485-507 https://doi.org/10.1007/s10531-023-02753-1 Thorough quality control was conducted to ensure that the data provided accurately represent the data recorded by field surveyors and lab technicians. The data match up with the details provided. Values fall within the expected ranges. Geospatial data were assessed visually. These data represent lab-based assay development, a field test of the assay, and metabarcoding analysis of the same field-collected flower samples. Samples were collected within and around the historic range of Bombus franklini in June-August 2021. Visual assessments of geospatial data confirm that coordinates provided fall within normal margin of error for handheld GPS devices. Not applicable In vitro testing of the qPCR assay: We conducted in vitro test of our qPCR assay using DNA extracts from the target species (Bombus franklini) and nine nontarget species (B. appositus, B. centralis, B. cryptarum, B. flavifrons, B. occidentalis, B. psithyrus sp., B. rufocinctus, B. vancouverensis, and B. vosnesenskii) at the U.S. Geological Survey Pacific Northwest Environmental DNA Laboratory. Reactions consisted of 7.5 µL TaqMan Environmental MasterMix 2.0 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.75 µL assay mix (600 nm forward primer, 600 nm reverse primer, and 250 nm probe), 0.60 µL TaqMan internal positive control (IPC) assay, 0.30 µL IPC DNA, 3.85 µL water, and 2 µL DNA extract in a total reaction volume of 15 µL. We amplified samples on a Bio-Rad CFX Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Our cycling conditions consisted of initial heat activation at 95°C for 10 minutes followed by 50 cycles of denaturing at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. All extracted samples and qPCR no-template controls (i.e., qPCR plate wells containing IPC but no template DNA) were analyzed in duplicate. Next, we ran a qPCR plate with serial 10-fold dilutions of B. franklini gBlock (0.01 – 100,000 copies/µL; N = 8 replicates per dilution) and qPCR no-template controls (N = 16) using the reaction conditions described previously. This plate was used to determine the limits of detection and quantification. 20220804 In situ testing of the qPCR assay: We tested 237 flower samples collected within the modeled range of B. occidentalis for B. franklini DNA. Surveyors were allowed to select which flower species to collect on a case-by-case basis, but we recommended collecting flower species that were fresh (i.e., not senescing or senesced), abundant, and known to be visited by B. occidentalis. To collect a sample, surveyors wore single-use gloves and used single-use forceps to collect up to three intact flowers into a teabag, which was then placed into a 50-mL Falcon tube (Corning, Inc., Corning, NY, USA) pre-loaded with silica desiccant. Samples were placed immediately into coolers and kept cool and dark. Samples were later shipped in shipping coolers to the U.S. Geological Survey Pacific Northwest Environmental DNA Laboratory where they were stored at -80°C until DNA extraction. Flower samples were extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Inc., Hilden, Germany) following the extraction procedure described in Thomsen and Sigsgaard (2019) with minor modifications. Specifically, we prepared for DNA extraction by cleaning all work surfaces with 50/50 bleach-water mix and wiping our pipetters, thermomixer, and vortexer with DNA Away (Thermo Fisher Scientific) and KimTech wipes (Kimberly-Clark Professional, Roswell, GA, USA). For each sample, we placed a sterile piece of foil on the clean work bench and used tweezers to remove the teabag from the Falcon tube and place it on the foil. We then opened the teabag, dumped the contents onto the foil, and transferred the flower material into a LoBind tube (1.5 – 5.0 mL, depending on flower size; Eppendorf North America, Enfield, CT, USA) using tweezers. We added ATL buffer and Proteinase K to the tube in volumes proportionate to flower size (i.e., 540 – 2,700 μL and 60 – 300 μL, respectively). We agitated the sample in a thermomixer at 400 rpm at 56°C for 3 hours. We then removed the lysed sample from the thermomixer and vortexed it for 10 seconds. We pipetted 500 – 1,665 μL (depending on original flower size) of the lysate into a new LoBind tube and discarded remaining flower material. If the appropriate volume was not reached (e.g., due to absorbance), we added additional ATL buffer, agitated for another 5 minutes, and vortexed again before transferring the lysate. Next, we added AL buffer (same quantity as lysate) and immediately vortexed for 10-15 seconds. We then added ethanol (same quantity as lysate) and immediately vortexed for another 10-15 seconds. We transferred 500 μL of this lysis mixture to a DNeasy spin column and centrifuged at 10,000 x g for 1 minute. We placed the spin column in a new collection tube and repeated this step until all the lysis mixture had been spun down. We added 600 μL AW1 buffer and centrifuged at 10,000 x g for 1 minute, then added 600 μL AW2 buffer and centrifuged at 20,000 x g for 3 minutes. Next, we transferred the spin column into a new 1.5-mL LoBind tube, added 60 μL AE buffer, incubated at 37°C for 15 minutes, and centrifuged at 10,000 x g for 1 minute. We then added another 60 μL AE buffer, incubated at 37°C for 15 minutes, and centrifuged at 10,000 x g for 1 minute. We pipetted 120 μL of the final elution into a labeled 0.5-mL tube for storage. We prepared one extraction negative with each batch of extracted samples (N = 22) and stored extracts at -20°C. We analyzed three qPCR replicates of each extracted flower sample and extraction negative using the reaction conditions described previously. qPCR plate runs included three replicates of qPCR no-template controls and serial 10-fold dilutions of B. franklini gBlock (0.01 – 100,000 copies/µL). Any sample or extraction negative that exhibited no amplification of IPC DNA in at least two qPCR replicates was re-analyzed on a new plate that included two replicates of qPCR no-template controls and serial gBlock dilutions (Goldberg et al. 2016). 20220913 Metabarcoding: We shipped 30-µL aliquots of extracted flower samples and extraction negatives to the laboratory at the U.S. Geological Survey Upper Midwest Environmental Sciences Center for metabarcoding analysis. For this analysis, we used a marker (i.e., set of primers) designed for Bombus detection which targets a ~150 base-pair fragment of the COI gene locus (Milam et al. 2020). We confirmed in silico that this marker would amplify B. franklini. We sequenced flower samples and extraction negatives in five libraries. With each library preparation, we included 10-30 metabarcoding no-template controls (i.e., plate wells containing no template DNA). We also included 1-2 mock samples (positive controls) consisting of DNA from a B. affinis tissue extraction or B. hypnorum gBlock. These species were used as positive controls because they do not occur naturally within the study area and thus can also serve as additional negative controls for detecting contamination. For all but one library preparation, we amplified samples (i.e., extracted flower samples, extraction negatives, mock samples, and metabarcoding no-template controls) in quadruplicate 25-µL reactions. Each reaction contained 12.5 µL Quantabio PerfeCTa qPCR ToughMix (Quantabio, Beverly, MA, USA), 0.4 µM BombusF and BombusR primers, and 2 µL DNA template. Cycling conditions consisted of denaturation at 98°C for 2 minutes followed by 35 cycles of denaturation at 98°C for 20 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds followed by a final extension at 72°C for 5 minutes. After PCR, we pooled quadruplicate samples, then purified samples using Quantabio sparQ PureMag Beads following the manufacturer’s guidelines. We analyzed samples on an Agilent 4200 TapeStation System (Agilent Technologies, Inc., Santa Clara, CA, USA) to check for primer dimers, successful amplification of target amplicons, and contamination. Next, we amplified all samples in 25-µL reactions to attach Illumina Nextera overhang adapter sequences (OAS; Illumina, Inc., San Diego, CA, USA) to the target amplicons. For four of our five library preparations, this was the second round of PCR. Each reaction contained 12.5 µL Quantabio PerfeCTa qPCR ToughMix, 0.4 µM BombusF+OAS and BombusR+OAS primers, and 2 µL PCR template. Cycling consisted of denaturation at 95°C for 3 minutes followed by either 35 (if this was the first PCR) or 20 (if this was the second PCR) cycles of denaturation at 95°C for 5 seconds, annealing at 51°C for 15 seconds, and extension at 72°C for 15 seconds followed by a final extension at 72°C for 5 minutes. Samples were then analyzed on an Agilent 4200 TapeStation System to check the size of amplicons and sample concentration. Next, we normalized and indexed samples using the Illumina Nextera XT Index Kit v2 according to the manufacturer’s guidelines. We purified the indexed samples using Quantabio sparQ PureMag Beads following the manufacturer’s guidelines. We then analyzed samples on an Agilent 4200 TapeStation System to confirm that indices had been attached to the target amplicons. For each library, we measured the concentration of each sample in triplicate using the KAPA Library Quantification Kit (Roche Sequencing, Pleasanton, CA, USA), then normalized to 4 nM. We pooled samples, denatured the library to the appropriate loading concentration, and sequenced the library on an Illumina NextSeq 2000 using a NextSeq 2000 P1 or P3 Reagent Kit (300 cycles) and a 25% PhiX spike-in concentration following the manufacturer’s instructions. After completion of sequencing, we used the DRAGEN Bio-IT Platform BCL Convert module (Illumina) to convert the generated base calls to FASTQ files and demultiplex the samples by barcode. The sequence data were then imported into Quantitative Insights Into Microbial Ecology (QIIME) 2 2023.2 (Bolyen et al. 2019) for bioinformatics analysis using the qiime tools import plugin with the options “--type ‘SampleData[PairedEndSequencesWithQuality]’” and “--input-format PairedEndFastqManifestPhred33.” We used Cutadapt (via the q2-cutadapt plugin; Martin 2011) to trim the Bombus primer sequences. Any reads less than 50 base pairs were discarded. The paired-end sequences were denoised and dereplicated using DADA2 (via the q2-dada2 plugin; Callahan et al. 2016) to generate an amplicon sequence variant (ASV) table and sequence file. Singleton ASVs were removed from both the ASV table and sequence file. The ASV sequence file was exported using the qiime tools export plugin into a FASTA sequence file for taxonomy assignment. Next, we created a custom reference database. We used the BLAST+ 2.16.0 (Camacho et al. 2009) blastn application against the National Center for Biotechnology Information (NCBI) GenBank nucleotide database (Benson et al. 2013, Sayers et al. 2021) with default settings except for the following parameters: “-qcov_hsp_perc 80,” “-strand plus,” “-evalue 1e-11,” and “-max_target_seqs 100.” The accession numbers from the BLAST+ results for each sequencing run were combined into one file, and duplicate accession numbers were removed. The remaining accession numbers were used to make the taxonomy and FASTA sequence files for the reference database. To make the taxonomy file, the taxonomizr 0.10.8 R package (Sherrill-Mix 2025) was used to retrieve the taxonomy for each accession number from NCBI, and the NCBI Entrez Direct (EDirect) efetch 23.8 navigation program was used to retrieve the FASTA sequences for each accession number. An additional 24 COI FASTA sequences for B. franklini (Schweizer et al. 2025) were manually curated and added to the taxonomy and FASTA sequence files. The completed reference database taxonomy file was imported into QIIME 2 using the qiime tools import plugin using “--type ‘FeatureData[Taxonomy]’” and “--input-format HeaderlessTSVTaxonomyFormat.” The FASTA sequence file was imported using “–type ‘FeatureData[Sequence]’.” Taxonomy assignment was conducted for each library separately using classify-consensus-blast (via the q2-feature-classifier plugin) with default settings except for the following parameters: “--p-maxaccepts 100,” “--p-perc-identity 0.97,” and “--p-evalue 0.00000000001” to mirror the BLAST+ parameters above. The search-results.qza output file from taxonomy assignment in QIIME 2 was exported and analyzed using a custom SQL script to extract hits with the highest percent identity per ASV. Any Bombus sp., Apidae sp., Apoidea sp., or Hymenoptera sp. hit followed by a BOLD sample ID (e.g., “Bombus_sp._BIOUG16746-A07”) was cross-referenced against the 04-Apr-2025 BOLD Data Package (Ratnasingham and Hebert 2007) to determine whether the species profile had been updated in the BOLD database to include a species name that was not readily available in GenBank. Any ASV that did not have a Bombus hit with a percent identity of 97% or higher was removed from further analysis. We assigned species to each remaining ASV using hits with the highest percent identity of at least 97%, omitting hits based on third-party assembly sequences as well as hits with low-level identification resolution (e.g., genus) provided that species-level hits were present. In instances in which multiple species had the highest percent identity of at least 97%, we omitted species not known to occur within 800 km (~500 mi) of the sampling location and included all species known to occur within this radius. Specifically, we included species with at least two recorded observations within 800 km of the sampling location in the Global Biodiversity Information Facility database (GBIF 2025). In instances in which no species with the highest percent identity were known to occur within the 800-km radius, we evaluated hits at successively lower percent identities of at least 97% using the same inclusion strategy. In instances in which no candidate species met our inclusion criteria, we reduced the identification resolution to the genus level. Finally, in instances in which the positive control species had the highest percent identity of at least 97%, we identified the ASV as the positive control species regardless of whether the highest percent identity was shared with other species. To the extent possible, we used current taxonomic nomenclature when assigning species to ASVs. B. pensylvanicus and B. sonorus were treated as distinct species per Beckham et al. (2024), B. occidentalis and B. mckayi were treated as distinct species per Williams (2021), and B. bifarius and B. vancouverensis were treated as distinct species per Ghisbain et al. (2020). B. bifarius, once thought to be widespread across western North America, was excluded from consideration as a possible species detection because its apparent range is over 800 km away from any of our sampling locations (Ghisbain et al. 2020). B. sylvicola and B. lapponicus were recognized as conspecific in a 2019 study focusing on populations in Sweden and northern Alaska (Martinet et al. 2019); however, more recent research suggests that this taxonomic revision may not be appropriate for all North American populations of B. sylvicola (Christmas et al. 2021, Clake et al. 2024). Given the current taxonomic uncertainty, we maintain the species name B. sylvicola in the present study. There were some exceptions to our species assignment procedure. For mock samples, metabarcoding no-template controls, and extraction negatives (i.e., samples not associated with coordinates), we included species known to occur within 800 km of any sampling location. For one ASV detected within a mock sample in the library that used B. hypnorum as the positive control species, the species with the highest percent identity of at least 97% was B. perplexus, a closely related species to B. hypnorum. Observations of B. perplexus have been recorded within 800 km of sampling locations in the present study; however, given that this ASV appeared in a mock sample, we identified it as B. hypnorum, which had the third highest percent identity alongside B. perplexus. After taxonomy assignment, a custom R script was used for read filtering. First, a 1% read threshold per ASV per library was applied to eliminate low-level contamination that can occur during the library preparation process (e.g., from tag jumping or sequencing instrument errors). For each ASV, 1% of the highest read count in any sample in the library was calculated, and read counts below this value were removed from further analysis. To account for contamination beyond the 1% threshold, the remaining ASVs were then subjected to mock sample and metabarcoding no-template control read filtering. For each library, we summed the read counts of each ASV across mock samples and metabarcoding no-template controls and subtracted this value from all reads of that ASV in flower samples and extraction negatives. We then conducted a final manual filtering step using read counts in extraction negatives. For this step, we summed the read counts of each ASV across all extraction negatives and subtracted this value from all reads of that ASV in flower samples. Thus, for a species to be considered “detected” in a flower sample, it must be represented by one or more ASVs with read counts greater than the threshold established by 1% read filtering (for the library), mock sample and metabarcoding no-template control read filtering (for the library), and extraction negative read filtering (for the whole dataset). 20240416 qPCR Tissue Extract Test.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample ID Unique identifier for tissue sample; species of tissue sample Producer Defined Text qPCR Replicate qPCR replicate of tissue sample Producer Defined 1 2 qPCR Plate ID Unique identifier for qPCR plate on which the tissue sample was analyzed Producer Defined Text Target Cq Quantification cycle number at which the target (Bombus franklini) fluorescence first rose above the baseline threshold Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined 25.6951225219444 25.7535073779762 qPCR quantification cycles IPC Cq Quantification cycle number at which the internal positive control fluorescence first rose above the baseline threshold Producer Defined 23.2196592631798 24.573919324847 qPCR quantification cycles Target Detection Binary variable identifying target (Bombus franklini) detection within the qPCR replicate Producer Defined 0 Target not detected Producer defined 1 Target detected Producer defined Inhibition Binary variable identifying (complete) inhibition of the qPCR replicate Producer Defined 0 qPCR replicate not inhibited Producer defined qPCR gBlock Test.csv Comma Separated Value (CSV) file containing data. Producer Defined Standard ID Target (Bombus franklini) gBlock dilution Producer Defined Text qPCR Replicate qPCR replicate of standard Producer Defined 1 8 qPCR Plate ID Unique identifier for qPCR plate on which the standard was analyzed Producer Defined Text Target Cq Quantification cycle number at which the target (Bombus franklini) fluorescence first rose above the baseline threshold. Data recorded as numeric value, NA (no increase in fluorescence above the baseline threshold), or OM (qPCR replicate omitted from analysis). Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined 20.4051035545452 39.0657843600151 qPCR quantification cycles Target SQ Amount of synthetic target DNA (gBlock; copies/µL) that was added to the plate well. This value was manually entered into the qPCR software (i.e., not estimated, as in field samples and negative controls). Data recorded as numeric value or OM (qPCR replicate omitted from analysis). Producer Defined 0.01 100000 copies/µL IPC Cq Quantification cycle number at which the internal positive control fluorescence first rose above the baseline threshold. Data recorded as numeric value or OM (qPCR replicate omitted from analysis). Producer Defined 21.5132059392057 26.3754284255472 qPCR quantification cycles Omit Binary variable indicating whether the qPCR replicate was omitted from analysis Producer Defined 0 qPCR replicate included Producer defined 1 qPCR replicate omitted Producer defined R-squared R-squared of the standards on the qPCR plate Producer Defined 0.993 0.993 Efficiency Efficiency calculated from the standards on the qPCR plate Producer Defined 104.4 104.4 Field, Extraction, and Summarized qPCR.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample ID Unique identifier for flower sample Producer Defined Text Sample Date and Time Date and time of sample collection Producer Defined Date in the format month/day/year and time in the format hour:minute based on the 24-hour clock Site ID Unique identifier for site of sample collection Producer Defined Text GPS Latitude Coordinate specifying the north-south position of the sampling location based on the WGS 84 datum Producer Defined 41.599955409804 43.5223354 decimal degrees GPS Longitude Coordinate specifying the east-west position of the sampling location based on the WGS 84 datum Producer Defined -123.453846830624 -121.9629707 decimal degrees Current Weather Description of cloud cover at the time of the survey Producer Defined Text Current Wind Description of wind conditions at the time of the survey Producer Defined Text Rain in the Previous 48 Hours Identification of rainfall at the site within the previous 48 hours Producer Defined Text Site Notes Notes about the site recorded in the field Producer Defined NA No notes Producer defined Text Number of Flowers Collected Number of individual flowers in the flower sample Producer Defined 1 3 flowers Flower Species Collected Species of flower in the flower sample Producer Defined Plant species code USDA PLANTS Database Sample Notes Notes about the sample recorded in the field Producer Defined NA No notes Producer defined Text Extraction Date Date of sample extraction Producer Defined Date in the format month/day/year Extraction Negative ID Unique identifier for extraction negative; extraction negative associated with batch of extracted samples Producer Defined Text Extraction Protocol Extraction protocol used for sample extraction. Protocols were selected based on sample size. Producer Defined Text qPCR Replicates Positive Number of qPCR replicates with amplification of the target (Bombus franklini) Producer Defined 0 0 qPCR replicates qPCR Replicates Included Number of qPCR replicates included in the sample-level summary; total number of qPCR replicates analyzed minus the number of qPCR replicates recommended for exclusion Producer Defined 2 6 qPCR replicates Total qPCR Replicates Number of qPCR replicates analyzed Producer Defined 3 6 qPCR replicates Target Detection Binary variable identifying target (Bombus franklini) detection for the flower sample, all qPCR replicates considered. At least one positive replicate is necessary for overall positive detection. Producer Defined 0 Target not detected Producer defined Final Data Management Notes Notes about the sample recorded during final data management Producer Defined NA No notes Producer defined Text qPCR Flower Samples.csv Comma Separated Value (CSV) file containing data. Producer Defined Sample ID Unique identifier for flower sample Producer Defined Text qPCR Replicate qPCR replicate of flower sample Producer Defined 1 6 qPCR Plate ID Unique identifier for qPCR plate on which the flower sample was analyzed Producer Defined Text Target Cq Quantification cycle number at which the target (Bombus franklini) fluorescence first rose above the baseline threshold Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined All values NA Target SQ Estimate of the starting quantity of the target (Bombus franklini); amount of DNA (copies/µL) that was present at the beginning of the reaction Producer Defined 0 0 copies/µL IPC Cq Quantification cycle number at which the internal positive control fluorescence first rose above the baseline threshold Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined 24.1880470530994 36.4673747809876 qPCR quantification cycles Target Detection Binary variable identifying target (Bombus franklini) detection within the qPCR replicate Producer Defined 0 Target not detected Producer defined Inhibition Binary variable identifying (complete) inhibition of the qPCR replicate Producer Defined 0 qPCR replicate not inhibited Producer defined 1 qPCR replicate inhibited Producer defined qPCR Notes Notes about the qPCR replicate Producer Defined NA No notes Producer defined Text qPCR Negative Controls.csv Comma Separated Value (CSV) file containing data. Producer Defined Negative Control ID Unique identifier for negative control Producer Defined Text Negative Control Type Type of negative control Producer Defined EN Extraction negative Producer defined NTC qPCR no-template control Producer defined qPCR Replicate qPCR replicate of negative control Producer Defined 1 16 qPCR Plate ID Unique identifier for qPCR plate on which the negative control was analyzed Producer Defined Text Target Cq Quantification cycle number at which the target (Bombus franklini) fluorescence first rose above the baseline threshold Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined All values NA Target SQ Estimate of the starting quantity of the target (Bombus franklini); amount of DNA (copies/µL) that was present at the beginning of the reaction Producer Defined NA No standards on the qPCR plate Producer defined 0 0 copies/µL IPC Cq Quantification cycle number at which the internal positive control fluorescence first rose above the baseline threshold Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined 24.1489786608171 30.8641476940819 qPCR quantification cycles Target Detection Binary variable identifying target (Bombus franklini) detection within the qPCR replicate Producer Defined 0 Target not detected Producer defined Inhibition Binary variable identifying (complete) inhibition of the qPCR replicate Producer Defined 0 qPCR replicate not inhibited Producer defined 1 qPCR replicate inhibited Producer defined qPCR Notes Notes about the qPCR replicate Producer Defined NA No notes Producer defined Text qPCR Standards.csv Comma Separated Value (CSV) file containing data. Producer Defined Standard ID Target (Bombus franklini) gBlock dilution Producer Defined Text qPCR Replicate qPCR replicate of standard Producer Defined 1 3 qPCR Plate ID Unique identifier for qPCR plate on which the standard was analyzed Producer Defined Text Target Cq Quantification cycle number at which the target (Bombus franklini) fluorescence first rose above the baseline threshold. Data recorded as numeric value, NA (no increase in fluorescence above the baseline threshold), or OM (qPCR replicate omitted from analysis). Producer Defined NA No increase in fluorescence above the baseline threshold Producer defined 20.7181758972915 39.7323276634063 qPCR quantification cycles Target SQ Amount of synthetic target DNA (gBlock; copies/µL) that was added to the plate well. This value was manually entered into the qPCR software (i.e., not estimated, as in field samples and negative controls). Data recorded as numeric value or OM (qPCR replicate omitted from analysis). Producer Defined 0.01 100000 copies/µL IPC Cq Quantification cycle number at which the internal positive control fluorescence first rose above the baseline threshold. Data recorded as numeric value or OM (qPCR replicate omitted from analysis). Producer Defined 22.8931979703269 27.244059287265 qPCR quantification cycles Omit Binary variable indicating whether the qPCR replicate was omitted from analysis Producer Defined 0 qPCR replicate included Producer defined 1 qPCR replicate omitted Producer defined R-squared R-squared of the standards on the qPCR plate Producer Defined 0.944 0.996 Efficiency Efficiency calculated from the standards on the qPCR plate Producer Defined 98.9 115.5 qPCR Notes Notes about the qPCR replicate Producer Defined NA No notes Producer defined Text Metabarcoding Flower Samples.csv Comma Separated Value (CSV) file containing data. Producer Defined Run Date Date of sequencing; unique identifier for library Producer Defined Date in the format month/day/year Sample ID Unique identifier for flower sample used in qPCR analysis Producer Defined Text Metabarcoding ID Unique identifier for flower sample used in metabarcoding analysis Producer Defined Text ASV ID Unique identifier for amplicon sequence variant within the library Producer Defined Text Species Taxonomic identity assigned to amplicon sequence variant Producer Defined Text Top Hits All hits with the highest percent identity of at least 97%, excluding hits based on third-party assembly sequences. Only provided when top hits differ from final taxonomy assignment. Producer Defined NA No difference between final taxonomy assignment and hits with the highest percent identity of at least 97% Producer defined Text Post-EN Filter Read count of amplicon sequence variant after 1% filtering, mock sample and metabarcoding no-template control filtering, and extraction negative filtering Producer Defined 1 427959 reads Sequence Length Length of amplicon sequence variant Producer Defined 138 160 base pairs Sequence Sequence of amplicon sequence variant Producer Defined Text Metabarcoding Notes Notes about the amplicon sequence variant Producer Defined NA No notes Producer defined Text Metabarcoding Extraction Negatives.csv Comma Separated Value (CSV) file containing data. Producer Defined Run Date Date of sequencing; unique identifier for library Producer Defined Date in the format month/day/year Extraction Negative ID Unique identifier for extraction negative used in qPCR analysis Producer Defined Text Metabarcoding ID Unique identifier for extraction negative used in metabarcoding analysis Producer Defined Text ASV ID Unique identifier for amplicon sequence variant within the library Producer Defined Text Species Taxonomic identity assigned to amplicon sequence variant Producer Defined Text Top Hits All hits with the highest percent identity of at least 97%, excluding hits based on third-party assembly sequences. Only provided when top hits differ from final taxonomy assignment. Producer Defined NA No difference between final taxonomy assignment and hits with the highest percent identity of at least 97% Producer defined Text Post-Mock and NTC Filter Read count of amplicon sequence variant after 1% filtering and mock sample and metabarcoding no-template control filtering Producer Defined 23 21480 reads Sequence Length Length of amplicon sequence variant Producer Defined 139 140 base pairs Sequence Sequence of amplicon sequence variant Producer Defined Text Metabarcoding Notes Notes about the amplicon sequence variant Producer Defined NA No notes Producer defined Text Metabarcoding Mock Samples and NTCs.csv Comma Separated Value (CSV) file containing data. Producer Defined Run Date Date of sequencing; unique identifier for library Producer Defined Date in the format month/day/year Metabarcoding ID Identifier for mock sample or metabarcoding no-template control used in metabarcoding analysis Producer Defined Text ASV ID Unique identifier for amplicon sequence variant within the library Producer Defined Text Species Taxonomic identity assigned to amplicon sequence variant Producer Defined Text Top Hits All hits with the highest percent identity of at least 97%, excluding hits based on third-party assembly sequences. Only provided when top hits differ from final taxonomy assignment. Producer Defined NA No difference between final taxonomy assignment and hits with the highest percent identity of at least 97% Producer defined Text Post-1% Filter Read count of amplicon sequence variant after 1% filtering Producer Defined 3 388484 reads Sequence Length Length of amplicon sequence variant Producer Defined 138 181 base pairs Sequence Sequence of amplicon sequence variant Producer Defined Text Metabarcoding Notes Notes about the amplicon sequence variant Producer Defined NA No notes Producer defined Text Metabarcoding BLAST Results.csv Comma Separated Value (CSV) file containing data. Producer Defined Run Date Date of sequencing; unique identifier for library Producer Defined Date in the format month/day/year qseqid Query sequence ID; unique identifier for amplicon sequence variant within the library Producer Defined Text sseqid Subject sequence ID; reference sequence name Producer Defined Text BOLD Verified ID Taxonomic identity of subject sequence ID verified using the BOLD Data Package Producer Defined NA Sequence ID not verified using BOLD Producer defined Text pident Percent identity Producer Defined 97.015 100.000 length Length of alignment Producer Defined 128 145 base pairs mismatch Number of mismatches Producer Defined 0 4 mismatches gapopen Number of gap openings Producer Defined 0 1 gap openings qstart Start of alignment in query Producer Defined 1 37 qend End of alignment in query Producer Defined 128 181 sstart Start of alignment in subject Producer Defined 1 6236560 send End of alignment in subject Producer Defined 129 6236422 evalue Expect value; estimate of the number of expected hits; number of times an alignment with the same score or higher could be found by chance Producer Defined 5.1E-69 3.4E-55 bitscore Bit score; a measure of alignment quality Producer Defined 220 267 FASTQ Table.csv Comma Separated Value (CSV) file containing data. Producer Defined Run Date Date of sequencing; unique identifier for library Producer Defined Date in the format month/day/year Sample or Extraction Negative ID Unique identifier for flower sample or extraction negative used in qPCR analysis Producer Defined NA Mock sample or metabarcoding no-template control Producer defined Text Metabarcoding ID Unique identifier for flower sample, extraction negative, mock sample, or metabarcoding no-template control. Note that run date 8/30/2022 included 10 metabarcoding no-template controls (the number 4 was accidentally skipped when naming samples, and thus the no-template controls for this library are numbered through 11). Producer Defined Text This dataset consists of two zipped folders (fastq.zip and reference_database.zip) and eleven CSV files: (1) qPCR data for the plate containing Bombus franklini and nontarget species DNA extract, (2) qPCR data for the plate used to calculate the limits of detection and quantification, (3) field, extraction, and summarized qPCR data for flower samples, (4) complete qPCR data for flower samples, (5) qPCR data for negative controls (extraction negatives and qPCR no-template controls), (6) qPCR data for standard dilutions of B. franklini gBlock (for plates containing flower samples), (7) summarized metabarcoding data for flower samples, (8) summarized metabarcoding data for extraction negatives, (9) summarized metabarcoding data for mock samples and metabarcoding no-template controls, (10) BLAST results containing hits with percent identities of at least 97% for all amplicon sequence variants in the summarized metabarcoding dataset, and (11) a table linking metabarcoding and qPCR identifiers. Raw sequence data (fastq.zip) is presented in a generic text-based FASTQ format. The FASTQ format consists of four lines: sequence identifier, nucleotide sequence, quality score identifier in the form of a single plus ("+") sign, and quality scores. The reference database (reference_database.zip) consists of a sequence file and a taxonomy file. The sequence file is presented in a generic text-based FASTA format. Each sequence entry consists of a “>” sign followed by a brief description name of the sequence. The taxonomy file is presented in a generic text file and consists of two columns: 1) the same brief description name in the sequence file, and 2) a semicolon-separated species taxonomic lineage in the order: Domain;Phylum;Class;Order;Family;Genus;Species. Also included are these files in QIIME 2-compatible format. Grossklaus, M.R., Pilliod, D.S., Spear, S.F., Laramie, M.B., Kholwadwala, A., Boone, A.J., Lor, Y., Kaminski, M., and Everett, J.G., 2026, Data supporting “Detecting bumble bees in the wild using environmental DNA: development and validation of a qPCR assay for the endangered Franklin’s bumble bee (Bombus franklini)”: U.S. Geological Survey data release, https://doi.org/10.5066/P132VXFT. GS ScienceBase U.S. Geological Survey mailing address

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