Katarzyna Przybyla-Kelly Muruleedhara Byappanahalli Hao Wang Mary Anne Evans Satoshi Ishii 2025 spreadsheet Reston, VA U.S. Geological Survey Katarzyna Przybyla-Kelly, USGS Great Lakes Science Center, https://orcid.org/0000-0001-9168-3545; Muruleedhara Byappanahalli, USGS Great Lakes Science Center, https://orcid.org/0000-0001-5376-597X; Hao Wang, University of Minnesota, https://orcid.org/0000-0002-5468-786X; Mary Anne Evans, USGS Great Lakes Science Center, https://orcid.org/0000-0002-1627-7210; Satoshi Ishii, University of Minnesota, https://orcid.org/0000-0003-3600-9165 https://doi.org/10.5066/P132OSRC Great Lakes Science Center 2018 website Reston, VA U.S. Geological Survey This is a citation for a project landing page that contains multiple data releases https://www.sciencebase.gov/catalog/item/5f6e18ba82ce38aaa2498f3e This dataset is associated with an examination of environmental DNA (eDNA) obtained from freshwater matrices (i.e. benthic algae, sediment, and near bottom water) collected by scuba divers from previously established transects located along the U.S. shoreline of Lakes: Michigan, Huron, Erie, and Ontario. 16S rRNA gene amplicon sequencing (i.e., targeting bacterial communities) and high-throughput quantitative PCR targeting various N-cycle associated genes [the Nitrogen Cycle Evaluation (NiCE) chip] were performed to assess potential abundance and diversity of microbes involved in nitrogen transformations including nitrogen fixation. Sample-associated sequences are available in NCBI Bioproject: PRJNA1253336. All eDNA samples for this dataset were collected alongside a larger body of work conducted in 2022 (https://doi.org/10.5066/P13JDUMH) and relate to multiple years of work at these stations: briefly, algal and dreissenid mussel biomass, water quality assessments, and diver observations of dreissenid mussels, round gobies, benthic substrate, and benthic algal cover. We refer to the benthic algae also as the ‘Cladophora community’ and ‘submerged aquatic vegetation (SAV)’ in other published project data, which were collected starting in 2018 (Great Lakes Science Center, 2018). These data were collected to determine the functional abundance of genes associated with nitrogen transformations in the Great Lakes. Nuisance overgrowth of benthic algae and subsequent wash-ups at nearshore areas in recent years are connected to increased nutrient availability in benthic areas. It has been suggested that nitrogen-fixing microbes (archaea, bacteria) are common in benthic algae, however, nutrient contributions from this pathway and other recycling processes are largely unknown. Present analysis provides insight into understanding nitrogen cycling pathways along the benthic algae-surrounding water-sediment continuum. 20220608 20220825 ground condition Complete None planned Lake Michigan, Lake Huron, Lake Erie, and Lake Ontario -87.8906 -77.2119 45.7369 41.6565 ISO 19115 Topic Category environment biota inlandWaters USGS Thesaurus DNA sequencing algal blooms nutrient cycling nuisance species microbes algae archaea bacteria environmental DNA species diversity community ecology freshwater ecosystems benthic ecosystems nutrient content (water) nitrogen field sampling USGS Metadata Identifier USGS:670fd167d34edd2692096021 Getty Thesaurus of Geographic Names Great Lakes Region Common geographic areas Lake Michigan Lake Erie Lake Huron Lake Ontario There are no access constraints associated with these data. There are no use constraints associated with these data. Muruleedhara Byappanahalli USGS Great Lakes Science Center Research Microbiologist mailing and physical

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Chesterton Indiana 46304 USA 219-926-8336 byappan@usgs.gov The U.S. Environmental Protection Agency granted funding to the U.S. Geological Survey under the Great Lakes Restoration Initiative for data collection and analysis. Madeleine M Giordano Shelby L Eagan Alexander E Kacher Megan E Lewan Jessica C Oswald Katarzyna Przybyla-Kelly Dawn A Shively Ashley M Spoljaric Mary A Evans 2024 dataset https://www.sciencebase.gov U.S. Geological Survey https://doi.org/10.5066/p13jdumh Thorough quality assurance practices were employed in laboratory and field protocols to collect the data, ensuring the accuracy of values in datasets. All technicians collecting data undergo rigorous training to adhere to SOPs. All glassware and sampling equipment was either single-use or bleach-sterilized to avoid cross-contamination. A single extraction blank was included with every set of 11 DNA extractions; none of the 10 extractions blanks exhibited positive amplification. Datasets were checked for errors, including duplication or omission. Dataset is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details. Sites were not randomized since samples were collected from preestablished transects; thus, collection of data is not representative of the full nearshore zone, and interpretation of data beyond the study design should be done with caution. Station locations were established by GPS, with a horizontal precision of 0.00001° (~1.1 m) latitude and longitude. Due to wind-induced movement or boat anchoring, the horizontal precision for sampling locations was thus lower than GPS precision, averaging approximately 30 m. Water column depth was measured by a GPS depth sounder. All depths relative to the water surface can be impacted by waves and have decreased accuracy during rough conditions. Sample and measurement depths relative to the sediment-water interface (for benthic samples) were determined by diver equipment placement and have an accuracy of ±0.03 meters. Roey Angel Maximilian Nepel Christopher Panhölzl Hannes Schmidt Craig W. Herbold Stephanie A. Eichorst Dagmar Woebken 20180430 publication Frontiers in Microbiology vol. 9 n/a Frontiers Media SA https://doi.org/10.3389/fmicb.2018.00703 Digital and/or Hardcopy 20180430 publication date Angel et al., 2018 Source used in Step 4. 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 7 n/a Springer Science and Business Media LLC ppg. 581-583 https://doi.org/10.1038/nmeth.3869 Digital and/or Hardcopy 20160523 publication date Callahan et al., 2016 Source used in Step 5. Ildiko E. Frank Kendra A. Turk‐Kubo Jonathan P. Zehr 20160914 publication Environmental Microbiology Reports vol. 8, issue 5 n/a Wiley ppg. 905-916 https://doi.org/10.1111/1758-2229.12455 Digital and/or Hardcopy 20160914 publication date Frank et al., 2016 Source used in Step 5. Daryl M Gohl Pajau Vangay John Garbe Allison MacLean Adam Hauge Aaron Becker Trevor J Gould Jonathan B Clayton Timothy J Johnson Ryan Hunter Dan Knights Kenneth B Beckman 20160725 publication Nature Biotechnology vol. 34, issue 9 n/a Springer Science and Business Media LLC ppg. 942-949 https://doi.org/10.1038/nbt.3601 Digital and/or Hardcopy 20160725 publication date Gohl et al., 2016 Source used in Step 4. Michael R. Green Joseph Sambrook 20190603 publication Cold Spring Harbor Protocols vol. 2019, issue 6 n/a Cold Spring Harbor Laboratory ppg. pdb.top095109 https://doi.org/10.1101/pdb.top095109 Digital and/or Hardcopy 20190603 publication date Green & Sambrook, 2019 Source used in Step 4. Paul J. McMurdie Susan Holmes 20130422 publication PLoS ONE vol. 8, issue 4 n/a Public Library of Science (PLoS) ppg. e61217 https://doi.org/10.1371/journal.pone.0061217 Digital and/or Hardcopy 20130422 publication date McMurdie and Holmes, 2013 Source used in Step 5. Michael Morando Jonathan D. Magasin Shunyan Cheung Matthew M. Mills Jonathan P. Zehr Kendra A. Turk-Kubo 20250204 publication Earth System Science Data vol. 17, issue 2 n/a Copernicus GmbH ppg. 393-422 https://doi.org/10.5194/essd-17-393-2025 Digital and/or Hardcopy 20250204 publication date Morando et al., 2025 Source used in Step 5. Hao Wang Gary W. Feyereisen Ping Wang Carl Rosen Michael J. Sadowsky Satoshi Ishii 20231017 publication Microbiology Spectrum vol. 11, issue 5 n/a American Society for Microbiology https://doi.org/10.1128/spectrum.04053-22 Digital and/or Hardcopy 20231017 publication date Wang et al., 2023 Source used in Step 4. Mamoru Oshiki Takahiro Segawa Satoshi Ishii 20180415 publication Applied and Environmental Microbiology vol. 84, issue 8 n/a American Society for Microbiology https://doi.org/10.1128/AEM.02615-17 Digital and/or Hardcopy 20180415 publication date Oshiki et al., 2018 Source used in Step 4. Oksanen J Blanchet FG Friendly M Kindt R Legendre P Mcglinn D Minchin PR O’Hara RB Simpson GL Solymos P Stevens MHH Szoecs E Wagner H 20190705 publication Digital and/or Hardcopy 20190705 publication date Oksanen et al., 2019 Source used in Step 5. Madeleine M Giordano Shelby L Eagan Alexander E Kacher Megan E Lewan Jessica C Oswald Katarzyna Przybyla-Kelly Dawn A Shively Ashley M Spoljaric Mary A Evans 20240308 Dataset https://www.sciencebase.gov U.S. Geological Survey https://doi.org/10.5066/P13JDUMH Digital and/or Hardcopy 20240308 publication date Giordano et al., 2024 Source used in Step 1. Katarzyna Przybyla-Kelly Ashley M Spoljaric Muruleedhara Byappanahalli Mary A Evans 20250331 Dataset https://www.sciencebase.gov U.S. Geological Survey https://doi.org/10.5066/P1S9XBT8 Digital and/or Hardcopy 20250331 publication date Przybyla-Kelly et al., 2025 Source used in Step 3. STEP 1 Collection sites and regime Samples were collected at 8 transects located along the U.S. shoreline of 4 Great Lakes: Michigan, Huron, Erie, and Ontario; details on sampling locations are provided in the Locations_NiCE_2022 table. All eDNA samples (water, sediment, algae) were collected at a single station (nominal depth of 6 m); on two occasions benthic algae samples were collected at additional stations (nominal depth of 3 m) due to unavailability of algae at the 6 m station. The study period was from 6/9/22 through 8/25/22. Field collections - Water samples Bottom water samples were collected by scuba divers using 2 L bleach-sterilized Nalgene bottles; empty bottles had a 3 lb scuba weight taped to the outside to facilitate transport to the lake bottom. Water samples were taken from about 10 cm above lake bottom substrate. To collect water, divers swam away from the boat’s anchor, slowly and above the lake’s bottom to prevent sediment and organic material resuspension. Once arrived at the sampling location, usually near large areas covered with dreissenid mussels and benthic algae, divers removed the lid from the bottle slowly while holding the bottle upside down. The bottle was then gently turned upward allowing it to slowly fill with water, capped once filled, and returned to the surface. Water samples were collected as a single grab sample due to logistical constrains, but on two occasions triplicate water samples were collected. After retrieving samples on the boat deck, bottom water samples were transferred to a cooler and kept on ice until filtration. Maximum holding time for water samples was 24 hours. Field collections - Benthic samples: algae and sediment Benthic algae were collected by scuba divers by hand pulling attached filaments and placing them in a prelabeled Nitex 500 µm mesh bag. All types of benthic algae (i.e. Cladophora sp, Chara sp, stalked diatoms, non-branching filamentous green algae) present were collected as a combined bulk sample. After algae samples were returned to the boat, two subsamples, with a minimum size of a quarter coin, were removed from the bulk sample with gloved hands, placed in a 7 oz wire-top plastic bag, and kept in a cooler on dry ice until transported back to the laboratory, where they were kept frozen at -20°C until processed. Lake bottom sediment samples were collected using sterile 90 mL urine cup containers; two replicates at each site. Underwater, divers removed the lid from the sampling cup and collected a sample by sliding the cup over or scooping the sediment; cups were capped and placed in a zip-top freezer bag underwater. In places where the bottom substrate was dominated by bedrock, large boulders, cobble, and/or with little amount of finer sediment, the sampling cup was slid around in crevices and around rocks. In places where bottom substrate was composed mainly of shell hash, samples were taken in multiple areas where sediment was visible, but frequently contained shell fragments. After returning to the boat, sediment samples were placed in individual wire-top plastic bags and kept in a cooler on dry ice until transported back to the laboratory, where they were kept frozen at -20°C until processed. More analyses of algae and sediment samples (i.e. tissue nutrient content) are included in the BenthicChem2022 table of the project data release (Giordano et al., 2024). Giordano et al., 2024 20220825 STEP 2 Laboratory processing of water, benthic algae, and sediment samples Water Filtration Water filtration took place the same day, within about 6 hours of collection, or the next day (within 24 hours of collection). For most samples, 1 L of water was filtered, however, due to differences in water turbidity at various sites, volume of water filtered ranged between 950 – 2100 ml; 2 or even 3 filters were sometimes used per sample due to frequent clogging. Filtration was carried out using a peristaltic pump (Masterflex® L/S® Portable Sampling Pump, Avantor Inc., Radnor, PA), and sterile disposable 250 mL volume capacity filtration cups (Thermo Scientific, Catalog No.09-740-30J) equipped with a 0.2 µm cellulose nitrate membrane filter set up on a triple-place filtration manifold. The technician performing filtration used gloved hands and frequently wiped with a bleach wipe. Before every pour of the water sample, the bottle was gently inverted five time, and water was poured to the 250 mL line of the filtration cup; volume filtered was recorded in a notebook. Once filtration was completed, the filter was removed from the base using two bleach-sterilized tweezers, folded in half, then into quarters, then eighths (3 folds) and placed in a 5 mL labeled sterile snap cap tube. Tubes containing filters were kept in a wire-top plastic bag on dry ice until transported to the laboratory where they were kept frozen at -80°C until processed. Sediment processing Sediment samples were thawed overnight at 4℃. The content of each urine cup was then emptied into a 14 oz wire-top plastic bag and the descriptive notes characterizing the sediment were documented. For elutriation, 100 mL of Phosphate Buffer Solution (PBS) was added to the wire-top plastic bag containing sediment; if the sediment sample contained overlaying water from the sampling site, the volume of PBS was adjusted accordingly so that the total volume of liquid used was 100 mL. Wire-top plastic bags were then closed tightly, and the contents were vigorously shaken for two minutes. The entire elutriate was then transferred to a 200 mL bleach-sterilized plastic bottle, and the sediment was weighed on a balance to obtain its wet weight. Elutriate was then centrifuged at 8000 RPM (10,017 x g) for 15 minutes; the majority of the supernatant was discarded. The pellet along with a small amount of liquid, was transferred into a sterile 7 mL pre-weighed tube using sterile disposable spatulas and sterile 5 mL pipettes to dislodge and transfer the pellet. All tubes were centrifuged again at 8000 RPM for 15 minutes, supernatant was discarded, and the pellet weight was recorded before placing it at -80℃. Benthic algae processing Benthic algae samples were thawed overnight in 4℃. The wet weight of algae was taken, and notes were made on algae type present and its condition. For elutriation, 20 mL of sterile PBS was added straight into the wire-top plastic bag containing algae. Each wire-top plastic bag was then closed tightly, and the content was vigorously shaken for two minutes. The entire elutriate was then transferred to a 50 mL sterile centrifuge tube and centrifuged at 8000 RPM (10,017 x g) for 15 minutes, the majority of the supernatant was discarded. The pellet along with a small amount of liquid, was transferred into a sterile 7 mL pre-weighed tube; using sterile disposable spatulas and sterile 5 mL pipettes to dislodge and transfer the pellet. All tubes were centrifuged again at 8000 RPM for 15 minutes, supernatant was discarded, and the pellet weight was recorded before placing it at -80℃. 20220920 STEP 3 DNA extractions For water samples, DNA was extracted from filters using the DNeasy Power Soil Pro DNA extraction kit (Qiagen, Carlsbad, CA, USA). If multiple filters were used for the same sample, they were initially processed separately but loaded onto the same MB Spin Column, resulting in a single DNA extract per sample. Frozen filters were first thawed at 4℃, unfolded inside a sterile petri dish using bleach-sterilized tweezers, then rolled with the sample facing inward and placed inside UV-sterilized 7 mL tube containing beads; prior to that, beads were transferred from the original PowerBead Pro tubes to the 7 mL tubes to allow for more space for the filter during the lysis step. For sediment and algae samples, DNA was extracted from pellets using the DNeasy Power Soil Pro DNA extraction kit. A 500 ± 50 mg subsample was used for larger pellets (exact weight was not recorded); if a pellet’s weight was less than 500 mg, it was used entirely. Exact pellets’ weights used for DNA extractions were recorded for normalizing results across samples. Pellets were first thawed at 4℃, homogenized with a sterile spatula inside the tube, and a subsample was placed into a new clean tube; single-use spatulas and wooden sticks were used to transfer the pellet. Once the lysis buffer was added to the weighed pellet, a homogeneous slurry was made by aspirating a 1 mL pipette multiple times; the resulting slurry was then transferred into a PowerBead Pro tube. DNA extractions followed the manufacturer’s protocol aside from the following changes: (1) after adding the lysis buffer to filters or pellets and prior to the 10 min-vortexing steps, tubes were placed into a pre-heated thermal shaker (300 rpm) and incubated for 15 min at 65°C; and (2) DNA was eluted twice using 50 µl of elution buffer (C6). All DNA extracts were stored at -80℃ until used. All DNA extracts from water, benthic algae and sediment were used for identifying invasive round goby fish using a droplet digital PCR (ddPCR) platform (Bio-Rad Laboratories) (Przybyla-Kelly et al., 2025). Przybyla-Kelly et al., 2025 20221027 STEP 4 Molecular Assays Nitrogen Cycle Evaluation (NiCE) chip for determining abundance and diversity nitrogen transformation genes via High-throughput quantitative PCR The NiCE chip (Oshiki et al., 2018) was used to quantify various nitrogen-cycle associated genes, including those for nitrification (amoA, hao, and nxrB), denitrification (napA, narG, nirK, nirS, norB, and nosZ clade I & II), dissimilatory nitrate reduction to ammonium (DNRA; nrfA), anaerobic ammonium oxidation (anammox; hdh and hzs), and nitrogen fixation (nifH). The NiCE chip was conducted with a SmartChip MyDesign chip (Takara Bio, Shiga, Japan) using the SmartChip Real-Time PCR system (Takara Bio, Shiga, Japan) following the manufacturer’s protocol (Wang et al., 2023). Briefly, the DNA samples (n = 89) were loaded onto the SmartChip MyDesign chip by the SmartChip MultiSample NanoDispenser (Takara Bio, Shiga, Japan). 50 nL of each of the DNA samples mixed with SmartChip TB Green Gene Expression Master Mix (Takara Bio, Shiga, Japan) was first loaded onto the SmartChip MyDesign Chip. Then 50 nL of each of the primer pairs (assay) mixed with SmartChip TB Green Gene Expression Master Mix (Takara Bio, Shiga, Japan) was loaded onto the SmartChip. The full list of assays and primer sequences were previously reported by Wang et al. (2023). The final volume in each nanowell of the MyDesign chip was 100 nL, and the final primer pair concentration in each nanowell was 500 nM. The qPCR was done under the following thermal conditions: 95°C for 3 min followed by 40 cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Melting curve analysis was performed from 50°C to 97°C with a 0.4°C/step temperature gradient. The threshold cycle (Ct) values were determined by the SmartChip Real-Time PCR software (Takara Bio, Shiga, Japan). The gBlock gene fragments (Integrated DNA Technologies, Coralville, Iowa, USA) of each nitrogen-cycle associated gene were pooled together and serially diluted to produce standard curves. Standard curves with at least three data points and an r2 value of >0.95 were considered valid. Target gene concentrations in the DNA samples were converted to log copies/µL based on the Ct values and the standard curves. Samples that did not amplify or had a Ct value higher than the lowest point on the standard curve were labeled as BDL (Below Detection Limit). Community Sequencing Analysis The extracted DNA samples (n=89) were also used for both the 16S rRNA gene amplicon sequencing and the nifH gene amplicon sequencing. For the 16S rRNA gene amplicon sequencing, the V4 region of the 16S rRNA gene was amplified, purified, and used to prepare sequencing libraries with protocol previously described by Gohl et al (2016). For the nifH gene amplicon sequencing, a nested PCR approach was used to amplify the nifH gene in order to prevent the formation of primer dimers (Angel et al., 2018; Green & Sambrook, 2019). The nifH amplicon was prepared with Ueda 19F-R6 primer set. The 300 bp paired end-sequencing for both the 16S rRNA gene and the nifH gene was done using the MiSeq platform (Illumina, San Diego, California, USA) with V3 chemistry. The sample amplification, library preparation, and sequencing of the targeted genes were done at the University of Minnesota Genomics Center (UMGC). The paired-end sequences of both the 16S rRNA gene and the nifH gene were submitted to the NCBI Sequence Read Archive and are available under BioProject number PRJNA1253336. Gohl et al., 2016 Green & Sambrook, 2019 Wang et al., 2023 Angel et al., 2018 Oshiki et al., 2018 2024 STEP 5 Bioinformatic processing The DADA2 pipeline (Callahan et al., 2016) was used to trim and quality filter the raw sequencing reads of both the 16S rRNA genes and the nifH genes. For the 16S rRNA gene, the quality filtration was done using the standard parameters outlined in the pipeline tutorial with RStudio version 4.2.3 and the ASV annotation was done using the SILVA database v138.1. For the nifH genes, the DADA2 nifH pipeline was used (Morando et al., 2025) and the ASVs were annotated using different databases including the cluster annotation with the classification and regression tree (CART) method (Frank et al., 2016) and the protein-level comparison via blastx against sequenced diazotroph genomes (“genome879”, https://www.jzehrlab.com/nifh). The annotation steps were done as a part of the post-pipeline stages of the nifH-ASV-workflow (Morando et al., 2025). For the analysis of community structures, RStudio version 4.2.3 was used with vegan (Oksanen et al., 2019) and phyloseq (McMurdie and Holmes, 2013) packages. Callahan et al., 2016 Morando et al., 2025 Frank et al., 2016 Oksanen et al., 2019 McMurdie and Holmes, 2013 2025 GeneAbundance_NiCE_2022.csv This table contains high-throughput quantitative PCR data targeting various N-cycle associated genes via the Nitrogen Cycle Evaluation (NiCE) chip analysis. Producer Defined sample_name Associated eDNA sample name used for sequences available in NCBI Bioproject: PRJNA1253336 Producer Defined Values range from 1-89 with a number 71 deliberately missing due to laboratory error during sample processing. Date Collection date. Dates are written in year-month-day (yyyy-mm-dd) format Producer Defined 2022-06-08 2022-08-25 Lake Name of the Great Lake where sampling occurred Producer Defined Erie Lake Erie Producer defined Ontario Lake Ontario Producer defined Huron Lake Huron Producer defined Michigan Lake Michigan Producer defined Transect Three-character string indicating which lake and location was sampled Producer Defined Transect codes: EDE, EEP, MWA, MSB, HAL, HHB, OOL, and OIR, correspond to location names. First letter in the code corresponds to Great Lake, i.e. M=Michigan, E=Erie, H=Huron, O=Ontario. Second and third letters correspond to port or transect name, i.e. DE=Dunkirk East; EP=Erie Pennsylvania; WA=Waukegan; SB=Sleeping Bear; AL=Alpena; HB=Hammond Bay; OL=Olcott; IR=Irondequoit. Station Station number associated with targeted depth (meters) where samples were collected within a transect Producer Defined Samplings were targeting 6 meters depth, but on 2 occasions algae samples were collected at 3 meters depth, as no benthic algae were present at 6 meters. Substrate Type of substrate from which samples were collected Producer Defined Water Lake water sample collected approximately 10 cm from the lake’s bottom Producer defined Algae Benthic algae sample collected from the lake's bottom Producer defined Sediment Sediment sample collected from the lake's bottom Producer defined FieldRep Replicated eDNA sample taken at a site; 1-3 replicates were collected at sampling sites. Producer Defined 1 3 VolumeFiltered_ml Total volume of water sample filtered onto 0.2 µm cellulose nitrate filter(s) Producer Defined NA Not Applicable for this specific Substrate type Producer defined 950 2100 milliliter 1 Algae_SedWeight_g Total wet weight of algae or sediment sample collected at sampling site Producer Defined NA Not Applicable for this specific Substrate type Producer defined 0.2 196.0 gram 0.1 TotalPelletWeight_g Total wet weight of algae or sediment pellet obtained from elutriating the algae or sediment sample Producer Defined NA Not Applicable for this specific Substrate type Producer defined 0.157 3.252 gram 0.001 PelletDNAextraction_g Subsample of algae or sediment pellet used for DNA extraction Producer Defined NA Not Applicable for this specific Substrate type Producer defined 0.157 0.549 gram 0.001 Arch_16S Concentration of archaeal 16S rRNA gene measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 9283 75410957 DNA copy numbers per microliter 1 AmoA_Gamo172F1R2 Concentration of AmoA gene measured by qPCR with Gamo172F1R2 primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 29319 198734837 DNA copy numbers per microliter 1 AmoA_Gamo172F2R1 Concentration of AmoA gene measured by qPCR with Gamo172F2R1 primers Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 940 74856622 DNA copy numbers per microliter 1 AmoA_F12R Concentration of AmoA gene measured by qPCR with F12R primers Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 3645 67532701 DNA copy numbers per microliter 1 Arch-amoA_FR Concentration of archaeal AmoA gene measured by qPCR with FR primers Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 501 38497851 DNA copy numbers per microliter 1 Arch-amoA_FA_R Concentration of archaeal AmoA gene measured by qPCR with FA_R primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 3768 152614751 DNA copy numbers per microliter 1 Arch-amoA_FB_R Concentration of archaeal AmoA gene measured by qPCR with FB_R primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 3920 106404839 DNA copy numbers per microliter 1 Anammox_hzoc11F1R2 Concentration of Anammox bacteria measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 543 21377375 DNA copy numbers per microliter 1 AOB_haoF4 Concentration of Hydroxylamine Oxidoreductase (hao) gene measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 24937 184565770 DNA copy numbers per microliter 1 nxrB_1F Concentration of nxrB gene measured by qPCR with 1F primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 35199 70639500 DNA copy numbers per microliter 1 nxrB_169f Concentration of nxrB gene measured by qPCR with 169f primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 43345 181417508 DNA copy numbers per microliter 1 napA_v66 Concentration of napA gene measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 45754 159070917 DNA copy numbers per microliter 1 nirS_nirSC2F Concentration of nirS gene measured by qPCR with nirS2F primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 42136 402069449 DNA copy numbers per microliter 1 nirK_FlaCu Concentration of nirK gene measured by qPCR with FlaCu primers Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 7421 58295111 DNA copy numbers per microliter 1 nirK_876 Concentration of nirK gene measured by qPCR with 876f_1040r primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 97011 959483259 DNA copy numbers per microliter 1 norB2 Concentration of norB gene measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 333 copies/µL Producer defined 7973 182121860 DNA copy numbers per microliter 1 cnorB Concentration of cnorB gene measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 44618 683873953 DNA copy numbers per microliter 1 qnorB_2F5R Concentration of qnorB gene measured by qPCR with 2F5R primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 88558 2106835024 DNA copy numbers per microliter 1 qnorB_2F7R Concentration of qnorB gene measured by qPCR with 2F7R primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 438637 422349557 DNA copy numbers per microliter 1 nosZ_1F1R Concentration of nosZ gene measured by qPCR with 1F1R primers Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 8042 261333667 DNA copy numbers per microliter 1 nosZ_F_1181 Concentration of nosZ gene measured by qPCR with F_1181 primers Producer Defined BDL Samples with a concentration below the detection limit of 333000 copies/µL Producer defined 352101 14645840 DNA copy numbers per microliter 1 nosZ_912F Concentration of nosZ gene measured by qPCR with 912f primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 56715 3843828022 DNA copy numbers per microliter 1 nifH_IGK3 Concentration of nifH gene measured by qPCR with IGK3 primers Producer Defined BDL Samples with a concentration below the detection limit of 33300 copies/µL Producer defined 1189417 332473203 DNA copy numbers per microliter 1 comaB Concentration of comammox bacteria measured by qPCR Producer Defined BDL Samples with a concentration below the detection limit of 3330 copies/µL Producer defined 4893 111164736 DNA copy numbers per microliter 1 Locations_NiCE_2022.csv This table contains information associated with sampling sites, including latitude, longitude, and site specific information. Producer Defined Lake Name of the Great Lake where sampling occurred Producer Defined Erie Lake Erie Producer defined Huron Lake Huron Producer defined Michigan Lake Michigan Producer defined Ontario Lake Ontario Producer defined Port Port where the research vessel was launched to collect samples in transects Producer Defined Ports were all located along the U.S. shoreline of the Lakes and included: Derby, NY; Erie, PA; Alpena, MI; Millersburg, MI; Leland, MI; Waukegan, IL; Olcott, NY; Irondequoit, NY Transect Three-character string indicating which lake and location was sampled Producer Defined Alphabetic codes correspond to location names: first letter in the code corresponds to Great Lake, i.e. M=Michigan, E=Erie, H=Huron, O=Ontario. Second and third letter correspond to port or transect name. Transect code, Lake, transect name: EDE, Erie, Dunkirk East; EEP, Erie, Erie Pennsylvania; MWA, Michigan, Waukegan; MSB, Michigan, Sleeping Bear; HAL, Huron, Alpena; HHB, Huron, Hammond Bay; OOL, Ontario, Olcott; OIR, Ontario, Irondequoit Station Station number associated with targeted depth (meters) where samples were collected within a transect Producer Defined Samplings were targeting 6 meters depth, but on 2 occasions algae samples were collected at 3 meters depth, as no benthic algae were present at 6 meters. Latitude Latitude in decimal degrees (datum:WGS84) determined by GPS on first site visit Producer Defined 42.17396 45.59494 degrees 0.00001 Longitude Longitude in decimal degrees (datum:WGS84) determined by GPS on first site visit Producer Defined -87.77022 -77.4431 degrees 0.00001 U.S. Geological Survey - ScienceBase mailing address

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