We examined above and belowground traits among four prominent dune grasses of the Atlantic and Gulf Coasts of North America: Ammophila breviligulata, Panicum amarum, Spartina patens, and Uniola paniculata. Whole plant samples of each species were collected from the foredune at the US Army Engineer Research and Development Center’s Field Research Facility in Duck, North Carolina, USA and quantified for several above and belowground traits (e.g., stem height, rhizome number and length, root surface area by diameter class, root tensile strength, mycorrhizal percent infection).

Samples were collected from the U.S. Army Engineer Research and Development Center (ERDC) Field Research Facility (FRF) located in Duck, North Carolina, USA (36.1820898, -75.7512527). Whole plant samples were collected in June and July of 2019 by hand digging from the face of the main foredune ridge facing the Atlantic Ocean. Only plants that were located at least 0.5 m from the base of other species were collected to avoid interspecific interactions. Above and belowground structures were separated in the field. Only roots and rhizomes directly attached to the base of a plant were collected which typically went to a depth of ~15-20 cm and within ~30 cm of each plant. Subsamples of the root system intended for mycorrhizal percent infection were preserved in 70% EtOH and those taken for tensile strength were preserved in 15% EtOH. Samples were placed on ice while in the field. Upon returning from the field, subsampled roots were immediately refrigerated and remaining components of plant samples were frozen in water until further analysis.

Aboveground components were assessed for stem number and maximum leaf length per stem. Belowground components were separated into belowground stems (buried portions of vertical stems), rhizomes (lateral stems), and roots. Rhizomes were counted and measured for length, and roots were scanned using WinRHIZO. All above- and belowground components were then dried in an oven at 60º C for 48 hours and weighed for dry biomass. In WinRHIZO, roots were grouped into five, 0.5 mm categories of root diameter for analysis (0.0-0.5 mm, 0.5-1.0 mm, 1.0-1.5 mm, 1.5-2.0 mm, and >2.0 mm). From the WinRHIZO scans and biomass, root tissue density (RTD), specific root length (SRL), average root diameter, root surface area and root diameter distribution were assessed. 

Subsampled tensile strength roots were rehydrated in distilled water for 30 minutes. Root diameter was taken using digital calipers. Due to lack of roots in some samples, additional roots were included from sampled individuals on Hog Island, part of the Virginia Coast Reserve Long-Term Ecological Research (LTER) Network and spare root samples collected from target species not directly associated with a sample individual. Tensile strength tests were conducted using an MTS Insight 30 5 Universal Testing Machine (UTM) (MTS Systems Corporation, Eden Prairie, MN, USA) with a 50 N load cell. MTS Advantage Wedge Action Grips were modified with 5 mm thick packaging foam and 220 grit sandpaper to prevent damage to the root sample while providing enough grip to pull the sample. The Testworks 4 software was then used to assess the resulting tensile strength data.

Root samples of individual plants were cut into 1 cm segments and root diameter determined using the ocular reticle of a compound microscope. Root segments were classified into one of four diameter classes (0.0-0.5 mm, 0.5-1.0 mm, 1.0-1.5 mm, and 1.5-2.0 mm). Root subsamples were placed in beaker of distilled water and gently agitated to remove EtOH and sediment particles. Samples were moved to hot 10% KOH for between 5 and 15 minutes depending on species. Time in 10% KOH was adjusted by species to prevent disintegration of the delicate roots. Samples were then briefly rinsed in distilled water and moved to 3.5% HCl for 15 minutes. Root samples were stained overnight in 0.04% trypan blue and preserved in glycerin prior to mounting on slides. Species were assessed for hyphal, vesicular, and arbuscular colonization using a modified, magnified intersect method in which 5 intersections, 1 mm apart were assessed per 1 cm root segment.