DOI: 10.5061/dryad.tht76hf4b
title: Biogeographic patterns of community diversity associated with an introduced alga

description: 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 &gt;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. 
description: <em>Study Sites: </em>We identified sample sites with
 verified <em>G. vermiculophylla </em>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<sup>o</sup>C); Virginian Province
 (between Cape Hatteras and Cape Cod: henceforth, VP) was sampled June 25 –
 Aug 3 (n=8, avg=26.7<sup>o</sup>C); and North of Cape Cod
 (henceforth NCC) was sampled Aug 4 – 9 (n = 3,
 avg=23.5<sup>o</sup>C). 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
 <em>G. vermiculophylla</em> presence/accessibility, the number
 of study sites per biogeographic region was uneven.
 <em>Sampling of Associated Free-Living Macroinvertebrates:
 </em>We sampled each site for <em>G. vermiculophylla
 </em>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 <em>G.
 vermiculophylla</em> clumps from those five randomly selected 0.25
 m<sup>2</sup> 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 <em>G.
 vermiculophylla</em> 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).
 <em>Sampling of Trematode Parasites: </em>We
 collected all <em>I. obsoleta</em> at the same sites as
 described above, except for Provincetown, MA, where <em>I.
 obsoleta</em> was not found (parasite data = 16 sites). We used the
 same 30-meter transect tape and 0.25 m<sup>2</sup> quadrats as
 above to collect snails; however, <em>G. vermiculophylla</em>
 and <em>I. obsoleta</em> were placed into separate bags. We
 counted all <em>I. obsoleta </em>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). <em>Statistical Analyses: </em>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 <em>G.
 vermiculophylla </em>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 <em>G. vermiculophylla
 </em>thalli<em> </em>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 <em>G. vermiculophylla
 </em>biomass (Figure 3). To prevent overparameterization and to
 standardize the response variables, we adjusted our response variables
 with <em>G. vermiculophylla </em>biomass as an offset
 [abundance: square root(raw count/<em>G. vermiculophylla</em>
 biomass); richness: log(number of species/<em>G.
 vermiculophylla</em> 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 <em>G.
 vermiculophylla</em> biomass. For parasites, prevalence was the
 proportion of infected <em>I. obsoleta</em> 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 <em>G. vermiculophylla</em>, 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 <em>G. vermiculophylla
 </em>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<sub>c</sub>) to determine which model, or sets of
 environmental variables, best explained the dependent variables of
 free-living macroinvertebrates and parasites (package AICcmodavg).
 AIC<sub>c</sub> 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
 &lt;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 <em>G. vermiculophylla</em> biomass.
 These latter analyses and figures were created using PRIMER-e
 (v.7).
description: 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.
description: # Biogeographic patterns of community diversity associated with an
 introduced alga The dataset contains macroinvertebrate diversity found
 associated with the introduced alga species, Gracillaria vermiculophylla.
 It also contains trematode parasite diversity associated with the
 estuarine snail, Ilyanassa obsoleta, which co-occurs with G.
 vermiculophylla. Surveys were conducted at multiple sites along the east
 coast of North America in 3 biogeographic regions. Multiple environmental
 variables were also collected at each site, including bird surveys, water
 quality measurements, and densities of snails and algae. ## Description of
 the data and file structure Included is a Word file that contains all the
 Supporting Information associated with the manuscript. It includes tables
 (e.g., site information and abbreviations), figures, and summary
 statistics of the data. Altogether this supporting information file
 includes 24 tables and 2 figures. Included in the dataset are also 3 Excel
 files with raw data from the macroinvertebrate surveys and the parasite
 analyses ## Sharing/Access information The manuscript has been accepted,
 and once it is published, we will link the manuscript with the Dryad
 dataset. We do not have any other files to link. ## Code/Software The R
 markdown file is annotated throughout to show how the code was used.
 Please also see the Supporting Information file which provides the summary
 statistics for the top models. ##### Column names In the coordinates.csv
 file, which provides the latitudes and longitudes of the survey sites, the
 following are the site names that are affiliated with the abbreviations:
 Fort Johnson, Charleston, SC (FJ) Hobcaw Barony, Georgetown, SC (HB) Fort
 Fisher, NC (FTF) UNCW Center for Marine Science, NC (UNCW) Harker's
 Island, NC (HI) Ocracoke Island, NC (OCK) Quinby Harbor, VA (QBY) Little
 Tom's Cove, Assateague Island, VA (LTC) Little Egg Harbor/Tuckerton,
 NJ (TUCK) Crab Meadow Beach, NY (CM) Seaside Park, CT (SP) Lighthouse
 Point, CT (LHP) Sandy Point, RI (SSP) Oakland Beach, RI (WCK) Millway
 Beach, MA (MB) Provincetown Marina, MA (PT) Jackson Lab, Durham, NH (JEL)
 In the freeliving.csv file, which contains raw data for the free-living
 macroinvertebrates that were surveyed, the following are the descriptions
 of each column: site: site abbreviation region: biogeographic region of
 the site replicate: replicate number (1-5) ecotype: free-floating (FF) or
 fixed (F) watertemp: water temperature (oC) salinity: salinity reading
 (ppt or PSU) oxygen: dissolved oxygen reading (mg/L) depth: depth of the
 sample taken (m) wrackdist: distance of the sample from the wrack line (m)
 gvbiomass: biomass of Gracilaria vermiculoplylla in the replicate (g)
 abundance: total abundance of free-living macroinvertebrates in the
 replicate (counts) richness: species richness of free-living
 macroinvertebrates in the replicate (richness) diversity: Shannon-Weiner
 diversity index of free-living macroinvertebrates in the replicate
 (diversity index) In the parasites.csv file, which contains raw data for
 trematode parasites surveyed from Ilyanassa obsoleta per site, the
 following are the descriptions of each column: site: site abbreviation
 region: biogeographic region of the site algaetype: ecotype -
 free-floating (FF) or fixed (F) watertemp: water temperature (oC)
 salinity: salinity reading (ppt or PSU) wrackdistance: distance of the
 sample from the wrack line (m) avgsnailcount: average snail count from
 quadrats at the site (counts) avggvbiomass: average biomass of Gracilaria
 vermiculoplylla from quadrats at the site at the site (g) avgsnailsize:
 average snail size from quadrats at the site (mm) seabirdwaders: counts of
 seabirds at a site (counts) infectedprev: infection prevalence at the site
 (proportion of infected snails) diversity: Shannon-Weiner diversity index
 of parasites at the site (diversity index) richness: species richness of
 parasites at the site (richness) weight: the number of snails dissected
 for parasite infection prevalence (counts)