Muruleedhara Byappanahalli Katarzyna Przybyla-Kelly Satoshi Ishii Aaron W. Aunins 2019 spreadsheet Reston, VA U.S. Geological Survey https://doi.org/10.5066/P9U2SYRI Data were collected to determine the abundance of nitrogen-fixing microorganisms in Cladophora algae growing on rocks, breakwall structures, or submerged dreissenid mussel beds around southern Lake Michigan. Cladophora samples (N=33) were collected between June and September 2015 from three urban areas: (a) Jeorse Park, East Chicago, Indiana, (b) Calumet Beach, Chicago, Illinois, and (c) North Beach, Racine, Wisconsin, and a National Park site, Portage Lakefront, Indiana Dunes National Park, Indiana. Corresponding lake water samples (N=33) were collected approximately 15-20 feet away from submerged algal mats. Genomic DNA was extracted from water and processed algal samples. The abundance of nitrogen-fixing microorganisms in water and algal samples was determined by a SYBR-Green quantitative polymerase chain reaction (qPCR) assay by targeting the nifH gene. The coordinate file includes information regarding sampling locations and their corresponding latitudes and longitudes. The data files include DNA quality and yield from water and algal samples, and abundance of nitrogen-fixing microorganisms in water and algal samples as determined by the SYBR-Green qPCR assay. The data were collected to determine the abundance of nitrogen-fixing microorganisms by targeting the nifH gene in actively growing (i.e., fresh) Cladophora algae in Lake Michigan. There is a significant knowledge gap in the abundance, taxonomic diversity, and nitrogen-fixing capabilities of these microorganisms. This information is critical for two reasons: first, to explain Cladophora’s nitrogen needs; second, to develop strategies to manage the nuisance algal problem by reducing nutrients entering the lakes via streams and other associated waterways. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. 20150618 20150923 ground condition Complete None planned -87.7870 -87.1763 42.7519 41.6262 ISO 19115 Topic Category economy biota USGS Thesaurus freshwater ecosystems polymerase chain reaction microbiology algae nuisance species nutrient cycling Producer defined Nearshore submerged vegetation Algae and water samples USGS Metadata Identifier USGS:5c95098de4b09388245a6ac4 Getty Thesaurus of Geographic Names Lake Michigan Great Lakes Region None. Please see 'Distribution Info' for details. None. Users are advised to read the data set's metadata thoroughly to understand appropriate use and data limitations. Muruleedhara Byappanahalli U.S. Geological, Great Lakes Science Center, Lake Michigan Ecological Research Station mailing

1574 Kemil Road N 300 E

Chesterton IN 46304 219-926-8336 x 421 219-929-5792 byappan@usgs.gov StepOnePlus Real-Time PCR System was used to obtain qPCR data. Muruleedhara N. Byappanahalli Meredith B. Nevers Katarzyna Przybyla‐Kelly Satoshi Ishii Timothy L. King Aaron W. Aunins 20190617 publication Environmental DNA vol. 1, issue 2 n/a Wiley ppg. 186-195 https://doi.org/10.1002/edn3.20 Aaron W. Aunins Muruleedhara Byappanahalli 2019 tabular digital data https://www.ncbi.nlm.nih.gov/bioproject/526911 High standard 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 used for sample collection and laboratory sample processing was soaked in 25% bleach solution for at least 10 min, rinsed multiple times with autoclaved deionized water until there was no residual bleach, and air-dried and autoclaved prior to use. Appropriate positive and negative controls were included in all qPCR assays. All standard curve R-squared values and amplification efficiencies were inspected. All standard curve R-squared values and amplification efficiencies fell within the expected ranges. Datasets were checked for errors, including duplication or omission. All manually entered data by the technicians were checked for errors against hard copies of the data once. Data set 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. Coordinates were created from Google Earth or Google maps (WGS84) A formal accuracy assessment of the vertical positional information in the data set has either not been conducted, or is not applicable. C. Rosch A. Mergel H. Bothe 20020801 publication Applied and Environmental Microbiology vol. 68, issue 8 n/a American Society for Microbiology ppg. 3818-3829 https://doi.org/10.1128/AEM.68.8.3818-3829.2002 Digital and/or Hardcopy 20160608 publication date qPCR primers Used in qPCR assay for primers probe and assay conditions. Mamoru Oshiki Takahiro Segawa Satoshi Ishii 20180402 publication Applied and Environmental Microbiology vol. 84, issue 8 n/a American Society for Microbiology ppg. e02615-17 https://doi.org/10.1128/AEM.02615-17 Digital and/or Hardcopy 20160608 publication date qPCR standards Used in qPCR assay for reference standards. Field Collections: Between June and September 2015, samples of live, attached Cladophora (N=33) growing on rocks, breakwall structures, or submerged dreissenid mussel beds were collected from different locations around Lake Michigan. The main locations included three urban areas (Jeorse Park, East Chicago, Indiana; Calumet Beach, Chicago, Illinois; North Beach, Racine, Wisconsin) and a National Park site (Portage Lakefront, Indiana Dunes National Park, Indiana), where Cladophora occurrences are common during its growing season (~May-September). Grab Cladophora samples (500-1000 g) were collected with a gloved hand and placed in sterile Whirl-Pak bags. Corresponding lake water samples (N=33) were taken ~15-20 feet away from submerged algal mats. Samples were transported to the laboratory in coolers on ice and processed within 2-24 hrs of collection. 20150618 Katarzyna Przybyla-Kelly U.S. Geological, Great Lakes Science Center, Lake Michigan Ecological Research Station Biological Technician mailing address

1574 Kemil Road N 300 E

Chesterton IN 46304 United States 219-926-8336 219-929-5792 kprzybyla-kelly@usgs.gov Lab processing - Algae: Each individual algal sample was removed from the Whirl-Pak bag and drained of excess water gravitationally by placing the sample on sterile 1 mm2-mesh size netting tightly secured to a plastic tub for 10 min. Approximately 100 g of Cladophora (post draining excess water) was directly placed in Whirl-Pak bags stored at -20°C for 24 hr then at -80°C until further processing between October and November 2015. At that time, samples were first thawed by placing them in a plastic tub containing room temperature (~25°C) water for an hour. A subsample of thawed algae (25-35 g) was then placed into a sterile bottle, to which 200-260 ml of phosphate buffer saline (PBS) was added. The contents were shaken vigorously for 2 min and 30 s twice, with a 1-min rest between shaking. The mixture was then transferred to a BagFilter® (Interscience, Rockland, MA, filter porosity <250 μm) to remove fractions larger than 250 μm. The filtered elutriate was centrifuged at 8000 rpm for 10 min; the supernatant was discarded, and the residual pellet was weighed and stored in a 7-ml centrifuge tube at -80⁰C until DNA extraction between October and November 2015. For DNA extraction, the algal pellets were thawed at 4°C and thoroughly mixed using sterile disposable plastic spatulas. Then, 0.15 ± 0.02 g of pellet was weighed directly into a MO BIO PowerBiofilm kit bead tube (MO BIO Laboratories, Carlsbad, California) with a disposable plastic spatula and a sterile wooden toothpick (if needed). DNA was extracted per the manufacturer’s protocol, with the following modifications: (a) the bead-beating step was extended from 30 s to 1 min, (b) the incubation time during the cell lysis stage was increased from 5 to 10 min, (c) the sample incubation time for removing PCR inhibitors was increased from 5 to 30 min, and (d) DNA was eluted in two steps using 2 × 50 μl volumes of elution buffer, for a total of 100 µl final volume. DNA concentrations for all samples were measured by fluorometric quantitation (Qubit® High Sensitivity dsDNA HS Assay; Thermo Fisher Scientific, Waltham, Massachusetts); DNA quality (i.e., 260/280 ratio) was measured with a spectrophotometer (NanoPhotometer Pearl, Implen GmbH, Munich, Germany), and purified DNA extracts were stored at -80°C until used. Lab processing - Water samples: DNA extraction from water samples (1 L) was accomplished by first prefiltering through 5.0 µm nitrocellulose filters (EMD Millipore, Billerica, MA), followed by final filtration through 0.22-µm nitrocellulose filters (EMD Millipore); both filters were placed in separate 7-ml centrifuge tubes and stored at -20°C until processed. Genomic DNA was extracted using PowerWater DNA extraction kit (MO BIO Laboratories) with some modifications. The 5.0 µm and 0.22 µm filters corresponding to each sample were initially extracted separately, but the lysates derived from these filters were combined and loaded to the same spin column resulting in one DNA extract per sample. Finally, DNA was eluted with 2 × 50 μl volumes of elution buffer. 20151015 Katarzyna Przybyla-Kelly U.S. Geological, Great Lakes Science Center, Lake Michigan Ecological Research Station Biological Technician mailing address

1574 Kemil Road N 300 E

Chesterton IN 46304 United States 219-926-8336 x427 219-929-5792 dshively@usgs.gov qPCR nifH assay: Nitrogen-fixing microorganisms: Abundance of nitrogen-fixing microorganisms in water samples and Cladophora algal pellets was measured by a SYBR Green qPCR assay targeting the nifH gene using nifH-F and nifH-R primers (Rosch, Mergel and Bothe, 2002) using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The reaction mixture (10 µl) contained 1× SYBR Premix Ex Taq (Ti RnaseH Plus) master mix with ROX (Takara Bio, Mountain View, CA), 0.4 µM each primer, and 1 µl DNA template. Serial dilutions (2 × 101 to 2 × 107 copies µl-1) of the linearized plasmid containing nifH fragment from Bradyrhizobium diazoefficiens USDA110T served as the standards (Oshiki, Segawa and Ishii, 2018). PCR was performed in duplicate with the following thermal conditions: initial annealing at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 58°C for 30 s, and 72°C for 30 s. ROX was used as a passive reference dye. After the PCR cycle, melting-curve analysis and gel electrophoresis were performed to ensure the correct amplification of the target gene fragment. Standard curves had R2 ≥ 0.99 and amplification efficiency ranged from 80–83%. Concentrations of nifH are expressed as log copy numbers (log CN) g-1 or mL-1. Nuclease-free water (Thermo Fisher Scientific) was used as a negative control (i.e., no template control) for the qPCR. References: Rosch, C., Mergel, A., Bothe, H. (2002). Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil. Applied and Environmental Microbiology, 68(8), 3818-3829 Oshiki, M., Segawa, T., Ishii, S. (2018). Nitrogen cycle evaluation (NiCE) chip for simultaneous analysis of multiple N cycle-associated genes. Applied and Environmental Microbiology, 84(8), e02615-02617 20180723 Satoshi Ishii University of Minnesota Assistant Professor mailing address

140 Gortner Laboratory of BioChemistry

St. Paul MN 55108 United States 612-624-7902 ishi0040@umn.edu Lat_Long.csv This table contains information associated with sampling locations, including latitude, longitude, and detailed information about site specific collections. Producer defined Location_ID shortened location name representing the sampling sites Producer defined Letter codes correspond to location names. Location_name Formal location name and geographic designation. Producer defined Formal location names for the sampling locations, city and state. Latitude Latitude in decimal degrees (WGS84) Producer defined Latitude for each location obtained from Google Earth. Longitude Longitude in decimal degrees (WGS84) Producer defined Latitude for each location obtained from Google Earth. Comments Additional information Producer defined Additional details on algae collections from different submerged surfaces. NifH_qPCR.csv This table contains data related to qPCR assay results. Producer defined Date Field sample collection date Producer defined Field samples collections took place between June 18, 2015 and September 23, 2015. Location_ID Sampling locations Producer defined See table Lat_Long.csv for specific information on these sampling locations. Sample_type Type of substrates from which samples were collected Producer Defined Sample types are: Water: Water collected from nearshore areas, approximately 5-6 meters away from the submerged Cladophora mat. Algae: live Cladophora filaments growing attached to submerged surfaces, like rocks or breakwall structures. Algae_wt Weight of fresh (moist) Cladophora algae used for generating an algal pellet, which subsequently was used in DNA extraction process. Producer Defined NA NA=not applicable to this sample type Producer defined 25 50 gram Pellet_wt Weight of the algal pellet generated from the rinsing/elutriation/centrifugation of Cladophora filaments with PBS. Producer Defined NA NA=not applicable to this sample type Producer defined 0.3129 1.4712 gram GenomicDNAConc Total genomic DNA concentration in eDNA extract measured in the laboratory. Producer Defined 0.403 88.8 ng/ul GenomicDNAQuality Total genomic DNA quality measured in the laboratory by 260/280 ratio. Designation NA means that ratio was not measured by the instrument due to very low GenomicDNAConc for this sample. Producer Defined NA NA=the measurement was outside of instrument's range of operation due to very low DNA concentration (GenomicDNAConc) Producer defined 1.733 2.286 SQ Starting quantity. Producer defined 827.7 190594.3 copies/reaction CN_Algae Copy numbers or amount of target DNA present in sample water: CN/1 g=SQ* (100 uL DNA eluted/1uL DNA per rx)*(Pellet_wt/0.15 g pellet amount taken for DNA extraction)/Algae_Wt g used to generate a pellet Producer defined 20541.10 3756341.20 CN/ 1 g (algae) CN_Water Copy numbers or amount of target DNA present in sample water: CN/1 mL=SQ* (100 uL DNA eluted/1 uL DNA per rx)/(1000 mL water filtered). Producer Defined 254.4 7864.7 CN/ 1 ml (water) NifH_taxonomicID.csv This table provides the necessary information for the taxonomic IDs for nitrogen fixing microorganisms identified in this study. It is linked with NCBI Bioproject: PRJNA526911. Producer Defined Bioproject_accession Accession number that leads to NCBI sequencing data repository under the title: "Shotgun metagenomic sequencing of the epiphytic communities in Cladophora mats from Lake Michigan" Producer Defined The Accession number for he BioProject in NCBI is: PRJNA526911. The same accession number is assigned to all Biosamples. Biosample_accession Accession numbers represent specific codes for each of the 10 metagenomes derived from Cladophora algae. Producer Defined Each of the 10 accession codes comprises of 4 letters SAMN followed by 8 digits. Library_ID A combination of location_date that identifies a BioSample in NCBI database. Each Biosample accession has a unique Library_ID. Producer Defined There are 10 different Library_IDs each corresponding to 1 individual Biosample. For example, Calumet_081415 represents Cladophora algae sample collected at Calumet Beach (Cal) on August 14, 2015. Calumet=Cal JeorsePark=JP NorthBeach=NB PortageLakefront=PL See table Lat_Long.csv for specific information on these sampling locations. Forward Read ID Read name corresponding to the forward read of a read pair that was identified as a putative nifH sequence Producer Defined Read names correspond to the FASTQ sequence identifiers, and follow standard FASTQ formatting conventions Reverse Read ID Read name corresponding to the reverse read of a read pair that was identified as a putative nifH sequence Producer Defined Read names correspond to the FASTQ sequence identifiers, and follow standard FASTQ formatting conventions NifH taxonomic ID NifH taxonomic ID represents reads used for identifying the nitrogen fixing microorganism (bacteria, Archea) using the MEGAN program. Producer Defined NifH taxonomic IDs are as follows: merged= nifH annotation is derived from both, forward and reverse read forward= nifH annotation is derived from forward read only reverse=nifH annotation is derived from reverse read only GS ScienceBase U.S. Geological Survey mailing address

Denver Federal Center, Building 810, Mail Stop 302

Denver CO 80225 United States 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 on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. Digital Data https://doi.org/10.5066/P9U2SYRI None 20200820 Muruleedhara Byappanahalli U.S. Geological, Great Lakes Science Center, Lake Michigan Ecological Research Station mailing

1574 Kemil Road N 300 E

Chesterton IN 46304 United States 219-926-8336 x421 219-929-5792 byappan@usgs.gov FGDC CSDGM FGDC-STD-001-1998 Record created using USGS Metadata Wizard tool. (https://github.com/usgs/fort-pymdwizard)