Non-native foundation species may alter physical environments and provide habitat, thereby impacting recipient communities. Along the US east coast, we assessed biogeographic patterns of free-living and parasitic community diversity associated with the non-native red alga Gracilaria vermiculophylla, which is characterized by fixed (with holdfast) or free-floating thalli depending on the availability of hard substratum. In summer 2019, we surveyed 17 sites across 3 biogeographic regions. We used a random quadrat design to collect G. vermiculophylla and associated mobile macroinvertebrates per site, and we took abiotic measurements. We also haphazardly collected 100 Ilyanassa obsoleta snails per site to assess trematode diversity. In the lab, macroinvertebrates were removed from thalli and identified to lowest taxonomic level, and snails were dissected to determine trematode prevalence and diversity. Biotic and abiotic variables were analyzed for the best sets of predictors for species richness, abundance, and diversity of macroinvertebrates and trematodes across bioregions. Gracilaria vermiculophylla biomass was used as an offset in free-living analyses. Across all our US east coast sites, we detected 10,113 free-living (mobile) macroinvertebrates across 39 taxa. Three Gammaridean amphipods (Gammarus mucronatus, Ampithoe longimana, and Gammarus lawrencianus) comprised >50% of all detected organisms. We found biogeographic region to be a key predictor of macroinvertebrate abundance and richness. Trematode prevalence and richness were best explained by G. vermiculophylla biomass, while biogeographic region best explained diversity. As a widespread invader, our study provides evidence for associations that have formed as this foundation species has become established outside its native range. Over time, the presence and spread of G. vermiculophylla could continue to impact macroinvertebrate structure and diversity, and future work should directly compare macroinvertebrate communities with G. vermiculophylla to other foundation species along coastlines it is now common. 

Study Sites: We identified sample sites with verified G. vermiculophylla presence from previous studies (Nettleton et al. 2013; Krueger-Hadfield et al. 2017). In summer 2019, we sampled 17 U.S. East Coast sites, capturing much of the species’ introduced range and encompassing two major geographic barriers at Cape Hatteras and Cape Cod (Engle & Summers 1999; Spalding et al. 2007; Hale 2010). Since summer temperatures are lagged in northern versus southern latitudes, southern sites were sampled earlier than northern sites (Table S1): South of Cape Hatteras (henceforth, SCH) was sampled May 21 – June 22 (n=6, avg=29.9^oC); Virginian Province (between Cape Hatteras and Cape Cod: henceforth, VP) was sampled June 25 – Aug 3 (n=8, avg=26.7^oC); and North of Cape Cod (henceforth NCC) was sampled Aug 4 – 9 (n = 3, avg=23.5^oC). Water temperatures were taken in the shallow intertidal zone just before low tide (see below). For this study, since we were specifically interested in conducting a biogeographic study of macroinvertebrates along the temperate coastline of the U.S. east coast, and given the large swath of sites within VP vs. other biogeographic regions as well as the availability of sites with verified G. vermiculophylla presence/accessibility, the number of study sites per biogeographic region was uneven.

Sampling of Associated Free-Living Macroinvertebrates: We sampled each site for G. vermiculophylla in the shallow intertidal zone while thalli were still submerged before low tide. At each site, we established a 30-meter transect tape along the water-land interface, selected five random numbers (1-30) using a random number generator (each number representing a meter marker on the 30-meter transect), and collected all G. vermiculophylla clumps from those five randomly selected 0.25 m² quadrats along the transect. We sampled environmental parameters (water temperature, salinity) using a handheld YSI Pro-1030 (Yellow Springs, OH).

We placed sealed bags of algae and water immediately into coolers and then transported them to the lab for processing. In the lab, we soaked the G. vermiculophylla from each replicate in a large bin filled with fresh tap water to induce osmotic shock in the associated mobile macroinvertebrates (e.g., Blakeslee et al. 2016; Fowler et al. 2016). We then used a Fisher Scientific™ 250 micron sieve to separate macroinvertebrates from macroalgae; upon separation, we preserved macroinvertebrates in Pharmaco™ 200 proof Ethyl Alcohol. After shaking off excess water, we weighed the thalli to obtain wet weights (g).

Following field surveys at all sites, macroinvertebrates were dyed with Rose Bengal (Gbogbo et al. 2020) and identified to the lowest possible taxonomic level using guidebooks and keys (Bousfield 1973; Johnson & Allen. 2012). Organisms were observed using a Zeiss MS Series Fixed Magnification Stereo Microscope (6x) and/or a Neatfi Elite XL HD Magnifying Lamp (5x). Gammaridean amphipods, which comprised up to 75% of the total macroinvertebrates at sites (see Results), can be difficult to identify to species level using morphology alone. We therefore classified amphipods into morphotypes and then later barcoded those morphotypes using standard DNA protocols (e.g., Blakeslee et al. 2020a). This allowed us to identify amphipods to species level by BLASTing our resultant sequence data for each morphotype using the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Sampling of Trematode Parasites: We collected all I. obsoleta at the same sites as described above, except for Provincetown, MA, where I. obsoleta was not found (parasite data = 16 sites). We used the same 30-meter transect tape and 0.25 m² quadrats as above to collect snails; however, G. vermiculophylla and I. obsoleta were placed into separate bags. We counted all I. obsoleta per quadrat, and then randomly selected 100 snails across the five quadrats to dissect. Because birds are common final hosts for trematode parasites, we also counted the total number of birds by species (waders, seabirds, and dabblers) at each site using a point-count method, while standing stationary for 10 minutes (Byers et al. 2008). In the lab, we measured each live gastropod using digital calipers and then dissected gonad tissues under a Zeiss™ MS Series Fixed Magnification Stereo Microscope at 6x magnification. If infected, we identified the digenean trematode to species level based on its rediae/sporocyst and cercarial morphology using published images and keys and prior knowledge within the lab (Curtis & Hurd 1983; Curtis 1985; Esch et al. 2001; Blakeslee et al. 2012). At the snail stage, trematodes asexually produce hundreds to thousands of clones on a continual basis (i.e., the “reproductive firepower” of digenean trematodes; Rohde 2005); thus we did not count cercariae, rediae, or sporocysts within infected snails, as these do not represent genetically-distinct individuals (e.g., Blakeslee & Byers 2008).

Statistical Analyses: To explore which factors best explained the patterns in the communities we observed, we used Generalized Linear Mixed Models (GLMM) in R 4.2.2 (package glmmTMB) for free-living macroinvertebrates (using site as a random effect, families = Gaussian for all response variables) and Generalized Linear Model (GLM) for parasites (families: prevalence = Binomial, richness = Poisson, diversity = Gaussian). For parasite analyses, we used GLM models that included fixed effects only, because at each site, there were no replicates of response variables, since n=100 snails were selected to be dissected randomly across all replicates. Due to the unevenness in detecting fixed versus free-floating G. vermiculophylla across sites, we did not have the number of replicates to analyze algal type (fixed or free-floating) as a predictor in our statistical models; as a result, we separately analyzed the abundance, richness, and diversity of macroinvertebrates associated with fixed and free-floating G. vermiculophylla thalli using two-tailed t-tests across all sites.

For free-living macroinvertebrates, we identified a strong positive and significant relationship between invertebrate raw counts, richness (number of species), and diversity index (Shannon-Wiener Diversity Index) with G. vermiculophylla biomass (Figure 3). To prevent overparameterization and to standardize the response variables, we adjusted our response variables with G. vermiculophylla biomass as an offset [abundance: square root(raw count/G. vermiculophylla biomass); richness: log(number of species/G. vermiculophylla biomass + 1); diversity: square root(Shannon-Wiener Diversity Index)]. We applied these transformations for these response variables to avoid zero variance problems in our models. For parasites, since we dissected an equal number of snails per site (n = 100) and all the three response variables were obtained from those individuals, we did not need to standardize by G. vermiculophylla biomass. For parasites, prevalence was the proportion of infected I. obsoleta out of 100 randomly dissected snails per site; richness was the number of digenean species; and diversity was the Shannon-Weiner Diversity Index.

We selected biologically-relevant predictors for our models after testing for autocorrelations. For free-living organisms, these predictors were water temperature (°C), salinity (PPT), and biogeographic region, with site as a random effect. Since our response variables were standardized by biomass of G. vermiculophylla, the biomass of the seaweed was not included as a predictor in these models to reduce overparameterization. We constructed rarefaction and extrapolation curves to determine the expected number of species per biogeographic region across accumulated individuals using EstimateS (v 9.1.0). For parasites, the predictors were G. vermiculophylla biomass (g), water temperature (°C), salinity (PPT), average snail count, seabird and wading bird count, and biogeographic region.

For both free-living and parasitic communities, we used the corrected Akaike’s Information Criterion (AIC_c) to determine which model, or sets of environmental variables, best explained the dependent variables of free-living macroinvertebrates and parasites (package AICcmodavg). AIC_c compares multiple models with different combinations of independent variables (Anderson & Burnham 2002). We used DAICc of ≤2.0 as a cutoff value to determine the top models. Based on our AICc results and their selected predictors in top performing models, we conducted a series of univariate analyses to observe how response variables change for both free-living macroinvertebrates and parasites with key predictors. 

For free-living macroinvertebrates, we used a Nonmetric Multidimensional Scaling (nMDS) to create a two-dimensional ordination plane to visually evaluate community composition among sites (Clarke & Warwick 2001). Per recommendations by Cao et al. (2001), we removed species that occurred <5% in nMDS analyses, and we used square-root transformation and Bray-Curtis Similarity (Clarke & Warwick 2001). For free-living and parasite organisms, we also conducted Similarity of Percentage (SIMPER) analyses to determine the percent each species contributed to the differences observed between biogeographic regions (Clarke 1993; Clarke & Warwick 2001). For free-living macroinvertebrates, we used abundance standardized by G. vermiculophylla biomass. These latter analyses and figures were created using PRIMER-e (v.7).

Microsoft Word, Microsoft Excel and R.

Please see the Supporting Information file (Word document) which has tables (e.g., site information and abbreviation), figures, and summary statistics of data analyses.