Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

These are data on kelp traits, seawater features and 16S rRNA amplicon sequencing. All kelp measurements were made in situ, often via kayak or at the shore using a measuring tape and calipers.

Kelp tissue was collected for elemental analysis by excising a 2 x 2 centimeter (cm) piece of tissue in the meristematic region. Tissue was ground to a fine powder in a GenoGrinder Spex (Metuchen, NJ), weighed in aliquots of 1.2 to 1.5 milligrams (mg) and packed into 3.5 × 5 millimeter (mm) aluminium tins (Costech, Valencia, CA). Packed tins were analysed on an elemental analyser isotope ratio mass spectrometer at Northwestern University Stable Isotope Biogeochemistry Laboratory (NUSIBL).

Carbon fixation was quantified using a section of the youngest, basal frond tissue extending 24 to 30 cm from the bulb. It was weighed (wet mass in grams (g)) with a Pesola scale and placed in a 1 liter (L) Nalgene container; the change in oxygen was measured for 1 hour with a Pyro Science contactless fibre-optic sensors (Oxygen Sensor Spots OXSP5) and an optical oxygen and temperature meter (FireStingO2 FSO2-4), with the sensor spots affixed inside each 1 L chamber.

DOC release and nutrient changes were quantified from seawater filtered through a GF/F filter and immediately frozen for analysis at the University of Washington Marine Chemistry Laboratory (methods from UNESCO), with DOC samples frozen in 40 milliliter (mL) glass vials with Teflon caps (Shimadzu, VOA) and other nutrients in acid-washed 60 mL high density polyethylene (HDPE) Nalgene bottles.

Using pulse amplitude modulated (PAM) fluorometry in situ (Diving Pam, Walz, Germany) we estimated fluorescence over a range of irradiances. PAM is a non-invasive, in situ method that quantifies Photosystem II fluorescence parameters across different light levels. Tissue was dark-adapted for 20 minutes and then rapid light curves were estimated with nine PAR levels from 0 to 1200. We estimated the maximum electron transport rate (ETR_max) and the photosynthetic efficiency at low light, both as the initial linear slope of ETR to irradiance (α) and as the quantum yield of photosystem II (Yield) on nine individual Nereocystis at both Tatoosh Island and Squaxin Island, the populations that span the greatest range of environmental conditions. Tatoosh Island was assayed on 9 and 20 June 2019 and Squaxin Island on 2 July 2019. At both sites, we selected individuals at least 1 meter (m) apart at low tide.

The salinity and seawater temperatures were assessed with a Castaway-CTD (Sontek) from a boat or kayak at all sites except Tatoosh Island where instrumentation is moored (Hach DS5).

Microbial density on individual kelp blades at either end of the geographic distribution, Tatoosh Island versus Squaxin Island, differed using 4,6-diamidino-2-phenylindole (DAPI) fluorescent staining of bacterial cells. We quantified bacterial cells from a section of the blade at approximately 20 cm distal from where the blade articulates with the bulb. We sampled a 3−5 mm slice from 10 individuals at each site (Tatoosh on 4 August 2019, Squaxin on 2 July 2019), preserving each slice immediately in Formalin, transferring to 50 : 50 EtOH : phosphate-buffered saline (PBS) after 1 hour, then freezing. In the laboratory, each piece was again sliced with a razor blade to approximately 0.5 mm, placed on a glass slide, and a solution of 1 microgram (µg) DAPI : 1 mL PBS was dropped on the slide-mounted slice. The slide was kept in dark for 20 minutes, chilled and rinsed with PBS prior to visualizing at 100× with an Olympus BX50 fluorescence microscope with a DAPI filter. We photographed 10 fields of view, selected haphazardly, for each individual slice, resulting in 100 images at each Tatoosh and Squaxin. In samples where bacteria were sparse or non-existent, we repeatedly adjusted the fine focus to verify no bacteria were present. We quantified the bacteria in ImageJ, enumerating the cells in the blue channel, adjusting the background and thresholds identically for all 200 images to eliminate autofluorescence by kelp cells. We calculated bacterial cell density as a percentage of the total imaged area.

Individual kelp was sampled for microbes by rubbing a cotton swab for 20−30 seconds over the mid-blade region. The swab was placed in a 2 mL Eppendorf tube, immediately chilled and transferred to a freezer within 4−6 hours. DNA from the swabs was extracted with a Qiagen DNeasy PowerSoil Kit (Qiagen). DNA was amplified, sequenced and amplicon sequence variants (ASVs) were identified by the Duchossois Family Institute Microbial Metagenomics Facility (DFIMMF) at The University of Chicago. The V4–V5 region within the 16S ribosomal RNA (rRNA) gene was amplified using universal bacterial primers and polymerase chain reaction (PCR) conditions described in Younker, et al. (2024). Approximately 412 bp region amplicons were then purified using a spin column-based method (Minelute, Qiagen), quantified and dual index adapters were ligated. Sequences were generated from the Illumina MiSeq platform using the QIASeq 1-step amplicon kit (Qiagen) for generating libraries and using 2 × 250 paired end reads with 5000 to 10,000 reads per sample. The default pipeline for processing MiSeq 16S rRNA reads was dada2 (v. 1.18.0) with minor modifications in R (v. 4.0.3). Reads were first trimmed at 190 bp for both forward and reverse reads to remove low-quality nucleotides, and chimaeras were detected and removed using the default consensus method in the dada2 pipeline. Then, ASVs with length between 320 and 365 bp were retained. Taxonomy of the resultant ASVs were assigned to the genus level using the RDP classifier (v. 2.13) with a minimum bootstrap confidence score of 80. Species-level classification used blastn (v. 2.13.0) and the refseq_rna database. We analysed ASVs with R, phyloseq and microViz (R version 2023.06.2+561). Chloroplast DNA that was not classified as Cyanobacteria were generally diatoms (Bacillarophyta) and were filtered out, as well as any other chloroplast sequence that did not match to the class Cyanobacteria. Sequences of corn (Zea, in Plantae), in low numbers at some Puget Sound sites, were also removed.

All methods are described in the Pfister, et al. (2025) paper.

Basic statistical analysis was done with R, version 2023.06.2+561.

Packages used within R included phyloseq, microviz, ggplot, vegan, nlme, tidyverse

- Loaded data from original file "Pfister_NereocystisGeography_RoySocOpen2025_BCODMO.csv" into the BCO-DMO system, treating "NA" as a missing data value (missing data values are empty/blank in the final CSV file).
- Converted date column from %m/%d/%y format to %Y-%m-%d format.
- Saved the final file as "1000920_v1_nereocystis_geography.csv".

NSF Award Abstract:
It is increasingly recognized that species in the ocean host a diverse set of microbes and that these microbes can determine the functioning of the host. Yet, the discovery of microbial taxa in nature far outpaces our understanding of their functions. Primary producers in the ocean show rich microbial communities and may contribute to the significant role that macrophytes play in coastal communities. This project focuses on a species of canopy kelp that is a foundational species along the shores of the northeast Pacific Ocean, the bull kelp Nereocystis luetkeana. The research explores the role of a diverse set of microbes in association with bull kelp. Using experimental manipulations of newly isolated microbial taxa from kelp, tests of whether interactions are positive, negative, or neutral are performed. Specific assays of whether microbes provision vitamins and enhance access to nutrients are carried out, as well as tests of whether kelp exudates benefit microbial metabolism and growth. Working directly with Tribal youth through an internship program, as well as with undergraduate, post-baccalaureate, and graduate students, helps train the next generation of ocean scientists. Communication channels with Tribal and State governments inform efforts to understand kelp declines. Host-microbe interactions are a component of the persistence of ocean species and this research informs the factors that underly these relationships.

The advent of DNA sequencing, imaging, and other molecular approaches have revealed that many key species in the ocean are a 'holobiome', and their fate is entwined with the microbes they host. Yet, this discovery of microbial taxa in nature far outpaces our understanding of microbial function. Nereocystis luetkeana, or bull kelp, and the bacteria the investigators have isolated from the kelp surface, present an opportunity to quantify host-microbe interactions, including how stressors of host health, such as ocean warming, alter these interactions. Experiments tracing stable nitrogen isotopes and manipulations with hosts and bacterial isolates are used to investigate possible exchanges and the currencies underlying interactions. Metabolomic, proteomic, and genomic analyses are used to quantify host-microbe interactions and linkage between taxa and their functions. Key microbial metabolisms being investigated include nutrient provisioning, vitamin synthesis, and the response to reactive oxygen species, among others. The demonstrated alteration of host-microbe interactions in the ocean when hosts are stressed suggest the findings of this study have application to understanding the factors that promote persistence of eukaryote-prokaryote partnerships.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.