Table of Contents

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

During the June peak of seasonal flowering in 2019, divers on SCUBA collected a single vegetative shoot at each node within each sampling grid for genetic analysis (n=15 ramets per grid, 45 per depth, 90 per site). At approximately alternating nodes (i.e., 5-7 of the 15 nodes in each grid, depending on the location of bare patches), divers also harvested all shoots from within a 25 × 25 centimeter (cm) quadrat (n = 15-21 per depth) to estimate shoot density and canopy characteristics. Samples were returned to the lab on ice for processing. The second and third leaves of each genetic sample were preserved in silica.

After the flowering season and once seeds had dropped (September 2019; von Staats et al. 2021), we returned to each sampling grid to collect dispersed seeds. Sediment cores (10 cm in diameter and 10 cm in depth) were taken at three locations within each grid at Curlew Beach and at four locations within each grid at the other three field sites (n=9 or 12 cores per depth). Sediment cores were bagged and kept cold (4 degrees Celsius (°C)) until they could be processed; each core was hand-sieved for intact seeds within 3 days of collection. We counted all intact seeds encountered and assessed viability by firmness (the squeeze test; Marion and Orth 2010). Seeds deemed viable were individually weighed and stored in microfuge tubes and frozen at -80°C until DNA extraction.

Leaf samples (2 to 4 milligrams (mg) of dried tissue from the middle third of the leaf) were ground with a Retsch mixer mill MM400; seeds were ground by hand with a micropestle after removing the seed coat. DNA extraction was done with the Omega Bio-Tek E-Z 96 Tissue DNA kit, and samples were stored at -20°C. We prepared one genomic library of 464 individuals (359 adults, 105 seeds) by following the ddRADseq protocol of Parchman et al. (2012) and sequenced the library using two lanes of Illumina Novaseq. We digested gDNA with two restriction enzymes, EcoRI and MseI, and ligated adaptors containing unique 8-10bp barcodes to the digested DNA of each individual. The products were then PCR-amplified in two independent reactions with standard Illumina primers. All amplicons were pooled and shipped to the University of Texas Genomic Sequencing and Analysis Facility, which used Blue Pippen Prep to isolate the 300-450bp fraction. This fraction was then single-read-sequenced (approximately 100bp) with both lanes of an Illumina Novaseq machine. We used custom scripts to demultiplex into sample-specific FASTQ- formatted files.

These are the raw sequences from the Illumina sequencer after the demultiplex step (see methods above).

- Imported original file "BioSampleObjects_wLatLon_final.csv" into the BCO-DMO system.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved final file as "958698_v1_z_marina_genbank_accessions.csv".

NSF Award Abstract:
Understanding how species cope with spatial variation in their environment (e.g. gradients in light and temperature) is necessary for informed management as well as for predicting how they may respond to change. This project will examine how key traits vary with depth in common eelgrass (Zostera marina), one of the most important foundation species in temperate nearshore ecosystems worldwide. The investigators will use a combination of experiments in the field and lab, paired with fine-scale molecular analyses, to determine the genetic and environmental components of seagrass trait variation. This work will provide important information on the microevolutionary mechanisms that allow a foundation species to persist in a variable environment, and thus to drive the ecological function of whole nearshore communities. The Northeastern University graduate and Keene State College (KSC) undergraduate students supported by this project will receive training in state-of-the-art molecular techniques, as well as mentorship and experience in scientific communication and outreach. A significant portion of KSC students are from groups under-represented in science. Key findings of the research will be incorporated into undergraduate courses and outreach programs for high school students from under-represented groups, and presented at local and national meetings of scientists and stakeholders.

Local adaptation, the superior performance of "home" versus "foreign" genotypes in a local environment, is a powerful demonstration of how natural selection can overcome gene flow and drift to shape phenotypes to match their environment. The classic test for local adaptation is a reciprocal transplant. However, such experiments often fail to capture critical aspects of the immigration process that may mediate realized gene flow in natural systems. For example, reciprocal transplant experiments typically test local and non-local phenotypes at the same (often adult) life history stage, and at the same abundance or density, which does not mirror how dispersal actually occurs for most species. In real populations, migrants (non-local) often arrive at low numbers compared to residents (local), and relative frequency itself can impact fitness. In particular, rare phenotypes may experience reduced competition for resources, or relative release from specialized pathogens. Such negative frequency dependent selection can reduce fitness differences between migrants and residents due to local adaptation, and magnify effective gene flow, thus maintaining greater within-population genetic diversity. The investigators will combine spatially paired sampling and fine-scale molecular analyses to link seed/seedling trait variation across the depth gradient at six meadows to key factors that may drive these patterns: local environmental conditions, population demography, and gene flow across depths. The team will then experimentally test the outcome of cross-gradient dispersal in an ecologically relevant context, by reciprocally out-planting seeds from different depths and manipulating relative frequency in relation to both adults and other seedling lineages. The possible interaction between local adaptation and frequency-dependence is particularly relevant for Zostera marina, which represents one of the best documented examples of the ecological effects of genetic diversity and identity. Further, a better understanding of seagrass trait differentiation is not simply a matter of academic interest, but critical to successful seagrass restoration and conservation.

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.