Eliza D. Stein Jami Belt Mark J. Biel Rob Found Teagan A. Hayes Brandon Kittson Tabitha A. Graves 20260323 tabular digital data Reston, VA U.S. Geological Survey https://doi.org/10.5066/P13H2SDB Environmental DNA (eDNA) samples were collected from fecal and pollen samples in Glacier National Park, Montana, USA, and Waterton Lakes National Park, Alberta, Canada, between 2013 and 2023. Sample contents were analyzed using eDNA metabarcoding of the trnL gene. This dataset contains the raw metabarcoding results, a list of local plant species, and the updated metabarcoding results that were vetted using a semi-automated pipeline. Data were collected to evaluate diets of pollinators and mammals in Glacier National Park, Montana, USA, and Waterton Lakes National Park, Alberta, Canada. The data may be used to identify important diet items and to replicate results from the reclassification pipeline. 20130819 20230805 observed In work As needed Glacier and Waterton Lakes National Parks -114.4940 -112.7637 49.1386 48.2283 ISO 19115 Topic Category biota USGS Thesaurus bees and wasps mammals pollinators environmental DNA None elk Cervus canadensis bumblebees Bombus American pika Ochotona princeps mountain goat Oreamnos americanus eDNA metabarcoding plower pollen fecal pellets animal diet USGS Metadata Identifier USGS:692f45cfd4be026ff273ac2c Common geographic areas Glacier National Park Montana Canadian Geographical Names Database Waterton Lakes National Park of Canada Alberta USGS Biocomplexity Thesaurus Bees Elk Ungulates Mammals Integrated Taxonomic Information System (ITIS) 2025 ONLINE_REFERENCE Washington, D.C. Integrated Taxonomic Information System (ITIS) http://itis.gov expert knowledge Kingdom Animalia animals Subkingdom Bilateria triploblasts Infrakingdom Protostomia Superphylum Ecdysozoa Phylum Arthropoda arthropods Subphylum Hexapoda hexapods Class Insecta insects Subclass Pterygota winged insects Infraclass Neoptera modern, wing-folding insects Superorder Holometabola Order Hymenoptera ants bees wasps Suborder Apocrita narrow-waisted hymenopterans ants bees true wasps Infraorder Aculeata Superfamily Apoidea bees sphecoid wasps apoid wasps Family Apidae euglossine euglossines honey bees stingless bees Subfamily Apinae Tribe Bombini Genus Bombus Bumblebees Bumble Bees TSN: 154397 Infrakingdom Deuterostomia Phylum Chordata chordates Subphylum Vertebrata vertebrates Infraphylum Gnathostomata Superclass Tetrapoda Class Mammalia mammals Subclass Theria Infraclass Eutheria Order Artiodactyla artiodactyls cloven-hoofed ungulates even-toed ungulates Family Cervidae cervids caribou deer moose wapiti Subfamily Cervinae Genus Cervus red deer deer wapiti Species Cervus elaphus elk wapiti wapiti or elk Red Deer (see comments) Subspecies Cervus elaphus canadensis Family Bovidae antelopes cattle goats sheep bovids Subfamily Caprinae Genus Oreamnos mountain goats Species Oreamnos americanus Rocky Mountain goat mountain goat Mountain Goat TSN: 180713 Order Lagomorpha lagomorphs Rabbits Hares Pikas Family Ochotonidae Pikas Genus Ochotona Pikas Subgenus Ochotona (Pika) Species Ochotona princeps American Pika Cony Rock Rabbit Hay-maker Mouse Hare Piping Hare Whistling Hare Little Chief Hare Southern Pika TSN: 180109 None. Please see 'Distribution Info' for details. These data are marked with a Creative Common CC0 1.0 Universal License. These data are in the public domain and do not have any use constraints. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Tabitha A Graves USGS - MIDCONTINENT REGION Supervisory Research Ecologist mailing and physical

OA - NPS - Glacier National Park, MT

West Glacier MT 59936 406-589-6645 tgraves@usgs.gov Funding was provided by the U.S. Geological Survey Ecosystems Mission Area. Pellet samples were collected by citizen scientists in Glacier National Park (GNP), Jeremiah North Piegan, Brett Stevenson, and crews with Parks Canada, the Blackfeet Nation, U.S. Geological Survey, and GNP. Pollen samples were collected by Annie Colgan, Olivia Miller, Mike Krellwitz, Lindsay Dose, Kelsey, Tess Fleming and Amari Guardipee. Levi Besaw and Peter Lesica provided local plant lists for Glacier National Park. Goat and elk fecal pellets were genotyped using microsatellites to confirm they were from the correct species before being sent for metabarcoding. Exact sequence variance (ESV) match confidence is reported in the data for each ESV. All data is properly accounted for. This dataset contains all ESVs detected in samples collected from 2013-2023. All accession matches with at least 70% confidence are provided in the original ESV dataset (ESV_Accession_Matches_All.csv). Natural Heritage Map Viewer, Montana Natural Heritage Program 20250604 spreadsheet https://mtnhp.org/mapviewer/ Digital and/or Hardcopy Unknown 20250604 publication date Natural Heritage Map Viewer. Montana Natural Heritage Program. Retrieved on June 4, 2025, from http://mtnhp.org/MapViewer/ List of plant species that occur in western Montana. This list was used to verify eDNA metabarcoding results. Global Biodiversity Information Facility (GBIF) 20250612 tabular digital data https://doi.org/10.15468/dl.kydezh Digital and/or Hardcopy Unknown 20250612 publication date GBIF.org (June 12, 2025) GBIF Occurrence Download https://doi.org/10.15468/dl.kydezh List of plant species that occur in study area. This list was used to verify eDNA metabarcoding results. Peter Lesica 2002 publication Corvallis, Oregon Oregon State University Press Digital and/or Hardcopy Unknown 2002 publication date Lesica, P. 2002. A flora of Glacier National Park, Montana. Oregon State University Press, Corvallis. List of plant species that occur in Glacier National Park. This list was used to verify eDNA metabarcoding results. E. W. Sayers, J. Beck, E. E. Bolton, J. R. Brister, J. Chan, R. Connor, M. Feldgarden, A. M. Fine, K. Funk, J. Hoffman, S. Kannan, C. Kelly, W. Klimke, S. Kim, S. Lathrop, A. Marchler-Bauer, T. D. Murphy, C. O’Sullivan, E. Schmieder, Y. Skripchenko, A. Stine, F. Thibaud-Nissen, J. Wang, J. Ye, E. Zellers, V. A. Schneider, and K. D. Pruitt. 2025 application/service Oxford University Press Nucleic Acids Research 53(D1):D20–D29 https://www.ncbi.nlm.nih.gov/genbank/ Digital and/or Hardcopy Unknown 2025 publication date Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. GenBank. Nucleic Acids Res. 2013 Jan;41(Database issue):D36-42. doi: 10.1093/nar/gks1195. Epub 2012 Nov 27. PMID: 23193287; PMCID: PMC3531190 Reference library used to align sequences from metabarcoding of eDNA diet samples. Mountain goat samples include a pilot dataset used to assess eDNA sample storage methods in 2019 (n = 53) plus additional Glacier National Park (GNP) and Waterton Lakes National Park samples collected as part of a genetic structure project in 2022 and 2023 (n = 45). Elk pellet samples were collected in conjunction with a project to understand elk diets and potential interactions with bison; 19 elk samples were collected in summer 2020 on grassland survey sites, and 82 samples were collected in June–October 2022 at locations of elk with global positioning system collars. These samples were frozen as soon as possible and approximately one pellet was sub-sampled for metabarcoding. Pika samples were dried fecal subsamples of a genetics project led by the GNP Citizen Science program in 2013–2017. Pollen was collected from 35 dried bee specimens in 2014–2015, and from 102 live bees in 2022–2023. Laboratory work and initial bioinformatics were performed by Jonah Ventures, LLC (Boulder, Colorado). Sample DNA was extracted using the Omega Biotek Mag-Bind® Universal Pathogen Core Kit (4x96 Preps; cat. no. M4030-01). To amplify plant taxa in eDNA samples, the laboratory used a universal primer set to target a section of the chloroplast trnL intron within sites c (5’-CGAAATCGGTAGACGCTACG-3’) and h (5’- CCATTGAGTCTCTGCACCTATC-3’). The laboratory amplified the target gene with an initial round of PCR, cleaned the amplicons, and then performed a second round of PCR to anneal dual-index Illumina sequencing adapters. Indexed amplicons were cleaned, normalized, and pooled for sequencing on an Oxford Nanopore Technologies MinION (Oxford, England) using the Ligation Sequencing Kit V14 (cat. no. SQK-LSK114). Raw sequences were trimmed using Cutadapt v3.4, demultiplexed using pheniqs v2.1.0, and any amplicon that did not contain one or both primers were discarded along with amplicons less than 298 base pairs (bp) or greater than 328 bp. Chimeras were filtered out using the uchime3 algorithm. Exact sequence variants (ESVs) were identified using the unoise3 denoising algorithm implemented in vsearch; only reads observed ≥4 times and with maximum expected error rates less than 1 bp were considered as candidate ESVs. We aligned final ESV sequences to Jonah Ventures vouchers and publicly available sequences on NCBI GenBank. This resulted in multiple accession matches per ESV. To reduce the risk of false positives, we removed accessions with less than 70% sequence similarity (i.e., less than 70% of bp matched between the accession and ESV. To reclassify ESVs and achieve a single consensus taxonomy for each, we used a semi-automated pipeline in R to verify that all accession matches were plausible based on the distribution of the species. This reclassification pipeline included 1) matching ESVs to reference library accessions, 2) declassifying accessions not present in the local species list, 3) refining accessions to finer taxonomic resolutions using the local species list, and 4) collapsing multiple accessions per ESV into a single consensus taxonomy, and 5) manually review final classifications. 20251118 ESV_Accession_Matches_All.csv Comma Separated Value (CSV) file containing data. Producer Defined ESV Unique ID assigned to each exact sequence variant (ESV) Producer Defined Unique ID assigned to each exact sequence variant (ESV) Sequence DNA sequence of ESV Producer Defined Clustered DNA sequence AccessionID Reference library match for ESV Producer Defined NA No match found in reference libraries Producer defined Reference library record matched to ESV sequence PercMatch Percentage of base pairs identical between ESV and accession Producer Defined NA No match found in reference libraries Producer defined 73.7 100 Percent Kingdom Accession match Kingdom Producer Defined NA Not assigned at this taxonomic resolution Producer defined Kingdom level taxonomic classification Phylum Accession match Phylum Producer Defined NA Not assigned at this taxonomic resolution Producer defined Phylum level taxonomic classification Class Accession match Class Producer Defined NA Not assigned at this taxonomic resolution Producer defined Class level taxonomic classification Order Accession match Order Producer Defined NA Not assigned at this taxonomic resolution Producer defined Order level taxonomic classification Family Accession match Family Producer Defined NA Not assigned at this taxonomic resolution Producer defined Family level taxonomic classification Genus Accession match Genus Producer Defined NA Not assigned at this taxonomic resolution Producer defined Genus level taxonomic classification Species Accession match Species Producer Defined NA Not assigned at this taxonomic resolution Producer defined Species level taxonomic classification Local_Plant_Species.csv Comma Separated Value (CSV) file containing data. Producer Defined Kingdom Kingdom reference belongs to Producer Defined Kingdom reference belongs to Phylum Phylum reference belongs to Producer Defined Phylum reference belongs to Class Class reference belongs to Producer Defined Class reference belongs to Order Order reference belongs to Producer Defined Order reference belongs to Family Family reference belongs to Producer Defined Family reference belongs to Genus Genus reference belongs to Producer Defined Genus reference belongs to Species Species reference belongs to Producer Defined Species reference belongs to Source Which plant list the species was found in Producer Defined Montana Natural Heritage Program This plant list contains all vascular plants and bryophytes in Montana west of 111.22247°W (approximately the longitude of Great Falls, Montana). Data was accessed from https://mtnhp.org/mapviewer/ on 4 June 2025. Producer defined GNP Plant List This plant list contains all Streptophyta observed within the boundary of Glacier National Park and was acquired via written communication with L. Besaw and P. Lesica, April 2025. Producer defined GBIF - GNP This plant list contains all records of Plantae in the Glacier National Park area from the public reference Global Biodiversity Information Facility. To assemble this list, we searched the GBIF web platform (https://www.gbif.org/) for Plantae within a geographic polygon that encompassed all eDNA samples analyzed in this study (-114.5982 49.16129, -114.0959 48.43599, -113.5647 48.21126, -113.1375 48.32352, -113.5765 49.00327, -114.064 49.26513, -114.5982 49.16129 ). Producer defined CommonName Species common name, if any Producer Defined NA No accepted common name for this species Producer defined Species common name if available ESV_Reclassified.csv Comma Separated Value (CSV) file containing data. Producer Defined ESV Unique ID assigned to each exact sequence variant (ESV) Producer Defined Unique ID assigned to each exact sequence variant (ESV) Sequence DNA sequence of ESV Producer Defined Clustered DNA sequence BeeDiet Number of bee diet samples with detections of this ESV Producer Defined 0 58 ElkDiet Number of elk diet samples with detections of this ESV Producer Defined 0 99 GoatDiet Number of goat diet samples with detections of this ESV Producer Defined 0 80 PikaDiet Number of pika diet samples with detections of this ESV Producer Defined 0 4 KingdomManual Manually corrected kingdom classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Kingdom level taxonomic classification PhylumManual Manually corrected phylum classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Phylum level taxonomic classification ClassManual Manually corrected class classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Class level taxonomic classification OrderManual Manually corrected order classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Order level taxonomic classification FamilyManual Manually corrected family classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Family level taxonomic classification GenusManual Manually corrected genus classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Genus level taxonomic classification SpeciesManual Manually corrected species classification Producer Defined NA Not assigned at this taxonomic resolution Producer defined Species level taxonomic classification KingdomPipe Reclassified kingdom from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Kingdom level taxonomic classification PhylumPipe Reclassified phylum from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Phylum level taxonomic classification ClassPipe Reclassified class from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Class level taxonomic classification OrderPipe Reclassified order from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Order level taxonomic classification FamilyPipe Reclassified family from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Family level taxonomic classification GenusPipe Reclassified genus from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Genus level taxonomic classification SpeciesPipe Reclassified species from automated pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Species level taxonomic classification KingdomOrig Original kingdom consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Kingdom level taxonomic classification PhylumOrig Original phylum consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Phylum level taxonomic classification ClassOrig Original class consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Class level taxonomic classification OrderOrig Original order consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Order level taxonomic classification FamilyOrig Original family consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Family level taxonomic classification GenusOrig Original genus consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Genus level taxonomic classification SpeciesOrig Original species consensus taxonomy without using reclassification pipeline Producer Defined NA Not assigned at this taxonomic resolution Producer defined Species level taxonomic classification ESV_Accession_Matches_All.csv: Raw metabarcoding results. Each row contains an accession match for each exact sequence variant (ESV). All accession matches with at least 70% confidence are provided in this dataset. Local_Plant_Species.csv: A list of all local plant species in the Glacier National Park region. Comprised of data from local Glacier National Botanists, the Montana Natural History Program, and GBIF. This list is used to verify accession match plausibility. ESV_Reclassified.csv: Contains a single consensus taxonomic assignment for each ESV, generated using the pipeline (columns ending in "Pipe"), corrected with manual review (columns ending in "Manual"), and without the use of the pipeline (consensus taxonomy generated without local species list; columns ending in "Orig"). Stein, E.D., Belt, J., Biel, M.J., Found, R., Kittson, B., Hayes T.A., and Graves T.A., 2025, eDNA metabarcoding classification from dietary samples of mountain goats, elk, American pika, and bumblebees in and near Glacier National Park, Montana, USA, and Waterton Lakes National Park, Alberta, Canada, from 2012-2023: U.S. Geological Survey data release, https://doi.org/10.5066/P13H2SDB U.S. Geological Survey - ScienceBase mailing address

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Denver CO 80225 1-888-275-8747 sciencebase@usgs.gov Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data for other purposes, nor on all computer systems, nor shall the act of distribution constitute any such warranty. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Digital Data https://doi.org/10.5066/P13H2SDB None 20260323 GS-NOROCK Data Steward U.S. Geological Survey Data Steward mailing and physical

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