Data from: Latitudinal variation in the constitutive and inducible defenses of a canopy-forming rocky intertidal seaweed
Data files
Oct 08, 2025 version files 262.07 KB
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1_induction_phase_data.csv
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2_pre_induction_phloro.csv
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3_pre_induction_cn.csv
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4_inter_region_choice_phase_data.csv
21.90 KB
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5_intra_region_choice_phase_data.csv
40.37 KB
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6_seaweed_canopy_survey_data.csv
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7_seaweed_density_length_survey_data.csv
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8_snail_survey_data.csv
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README.md
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Abstract
A long-standing theory in biogeography is that stronger biotic interactions at lower latitudes select for better-defended phenotypes. However, greater environmental variability at temperate latitudes may also shape defensive strategies by increasing temporal or local spatial variation in consumer pressure and thus selecting for greater phenotypic plasticity, or inducible defenses. Distinguishing between inducible and constitutive defense strategies is therefore necessary to test for latitudinal defense gradients, but also for understanding how species interactions and community dynamics vary across ecological and evolutionary scales.
We investigated latitudinal variation in antiherbivore defenses of a cosmopolitan rocky intertidal seaweed (Fucus vesiculosus) against a similarly common and abundant grazer (periwinkle snails, Littorina littorea). We used a multi-phase common garden experiment with seaweeds from three different regions along the US Atlantic coast and snails from the southern- and northern-most study sites. We manipulated and measured snail grazing on seaweeds in a series of no-choice and two-choice assays to identify regional differences in seaweed functional defenses (i.e., reduced grazing).
Across all assays, grazing rates declined with seaweed latitude. Prior grazing reduced the palatability of southern, but not northern, seaweeds. Changes in seaweed nutritional content (C:N) and phlorotannins (a putative chemical defense) correlated with induced but not constitutive functional defenses.
Our results indicate that the constitutive anti-herbivore traits of F. vesiculosus increase with latitude and negatively covary with defense plasticity. This result suggests that the selective pressure of herbivory is stronger for northern seaweed populations, while southern populations may face a different set of tradeoffs leading to defense plasticity, such as increasing environmental stress.
The strength of trophic interactions plays an important role in community dynamics and food web stability. Our findings add to a growing literature highlighting the importance of ecological context in shaping trophic interactions and suggests that estimates and comparisons of interaction strength need to consider spatiotemporal variation in prey defenses. Defensive traits that vary with latitude or along environmental stress gradients may be particularly important for predicting the effects of climate change on trophic interactions and their consequences for community dynamics and ecosystem function.
Table of Contents:
File Name: 1_induction_phase_data.csv
Corresponding Experiment Phase: Induction Phase
Summary: Compiled data from experimental snails and seaweeds during the induction phase used in analyses of grazing rates and post-induction seaweed tissue chemistry presented in Figures 2a, 2b, 2d, 2f, Appendix Tables S1 and S3 and Figure S2.
File Name: 2_pre_induction_phloro.csv
Corresponding Experiment Phase: Collection and Relaxation Phase
Summary: Compiled data from seaweeds frozen before and after the relaxation phase presented in Figure 2c and Appendix Table S2a.
File Name: 3_pre_induction_cn.csv
Corresponding Experiment Phase: Collection and Relaxation Phase
Summary: Compiled data from seaweeds frozen before and after the relaxation phase presented in Figure 2e and Appendix Table S2b.
File Name: 4_inter_region_choice_phase_data.csv
Corresponding Experiment Phase: Choice Phase
Summary: Compiled data from experimental snails and seaweeds during the inter-regional choice assay phase presented in Figure 3a, Appendix Table S4.
File Name: 5_intra_region_choice_phase_data.csv
Corresponding Experiment Phase: Choice Phase
Summary: Compiled data from experimental snails and seaweeds during the intra-regional choice assay phase presented in Figure 3b, Appendix Table S5.
File Name: 6_seaweed_canopy_survey_data.csv
Corresponding Experiment Phase: Field Surveys
Summary: Compiled data from field surveys of seaweed canopy cover presented in Figure 4a and Appendix Table S6a.
File Name: 7_seaweed_density_length_survey_data.csv
Corresponding Experiment Phase: Field Surveys
Summary: Compiled data from field surveys of fucoid seaweed holdfast density and length presented in Figure 4b and 4c and Appendix Table S6b and S6c.
File Name: 8_snail_survey_data.csv
Corresponding Experiment Phase: Field Surveys
Summary: Compiled data from field surveys of herbivore assemblages presented in Figure 4d and Appendix Table S6d.
Description of the data and file structure
File: 1_induction_phase_data.csv
Variable Name (row 1): indunit
Variable Type or Units (row 2): ID factor
Description: number used to identify an individual induction unit (1-252)
Variable Name (row 1): indsubunit
Variable Type or Units (row 2): ID factor
Description: letter used to identify each of the two seaweeds within the induction unit (a,b)
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Seaweed source region (south, central, north); see also Figure 1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Seaweed collection site (n = 3 sites region-1); see also Figure 1
Variable Name (row 1): ind.type
Variable Type or Units (row 2): ID factor
Description: Type of induction treatment (no-snail control, induced, or autogenic growth control) applied to each indunit or indsubunit
Variable Name (row 1): ind.treat
Variable Type or Units (row 2): ID factor
Description: Specific induction treatment applied to each indsubunit (no-snail control, southern or northern inducer snail or corresponding autogenic growth control)
Variable Name (row 1): initial mass
Variable Type or Units (row 2): unit = grams
Description: initial seaweed mass (grams) in the corresponding induction subunit prior to the start if the induction phase; corresponds to Si, Ai, or Ci for inducer snail, autogenic growth, and no-snail control induction subunit treatments.
Variable Name (row 1): final mass
Variable Type or Units (row 2): unit = grams
Description: final seaweed mass (grams) in the corresponding induction subunit at the end of the induction phase; corresponds to Sf, Af, or Cf for inducer snail, autogenic growth, and no-snail control induction subunit treatments. NA indicates that a measurement was not taken.
Variable Name (row 1): Bunit
Variable Type or Units (row 2): unit = grams
Description: Induction unit snail biomass; total mass (grams) of the three snails in the northern or southern inducer treatment induction units. NA indicates that the value is not applicable, if the induction unit did not contain inducing snails (ind.type = "Control" or "Autogenic Growth Control"), or was not calculated if there were snails present (ind.type = "Induced")
Variable Name (row 1): startdate
Variable Type or Units (row 2): YYYYMMDD HH:MM
Description: Start date of induction phase
Variable Name (row 1): enddate
Variable Type or Units (row 2): YYYYMMDD HH:MM
Description: End date of induction phase
Variable Name (row 1): Dunit
Variable Type or Units (row 2): unit = days
Description: Unit-specific induction phase duration (days)
Variable Name (row 1): Gunit
Variable Type or Units (row 2): unit = mg
Description: Total seaweed grazed by inducers (mg); Gunit = (Si(Af/Ai)-Sf)*1000mg/g, where Si = initial mass = Sf = final mass and Ai and Af are the initial and final mass, respectively of the paired autogenic growth control seaweed (same indunit); as in Figure 2a and Appendix Table S1a. NA indicates that the measurement was not calculated.
Variable Name (row 1): Gpc
Variable Type or Units (row 2): unit = mg d-1 3.5g-snail-1
Description: Mass-standardized per capita daily grazing rate (mg d-1 3.5g snail-1); Gpc = 3.5g*Gunit/Bunit/Dunit; as in Figure 2b, Appendix Table S1b. NA indicates that the value is not applicable, if the induction unit did not contain inducing snails (ind.type = "Control" or "Autogenic Growth Control"), or was not calculated if there were snails present (ind.type = "Induced")
Variable Name (row 1): growth
Variable Type or Units (row 2): unit = % of initial mass
Description: (final mass – initial mass)/initial mass; e.g., (Cf-Ci)/Ci for autogenic growth controls and no-snail controls. NA indicates that the value was not calculated, if the induction unit did not contain inducing snails (ind.type = "Control" or "Autogenic Growth Control"), or was not applicable if there were snails present (ind.type = "Induced")
Variable Name (row 1): exclusions
Variable Type or Units (row 2): ID factor
Description: Induction units or subunits excluded from induction phase analyses and subsequent choice assays
Variable Name (row 1): notes
Variable Type or Units (row 2): ID factor
Description: reasons for exclusions. NA indicates not applicable, no notes for this unit.
Variable Name (row 1): phloro
Variable Type or Units (row 2): unit = % dry mass
Description: Phlorotannin concentration (% dry mass) in sampled apical tip of corresponding induction subunit seaweed; as in Figure 2d and Appendix Table S3a. NA indicates that the value was not calculated.
Variable Name (row 1): N
Variable Type or Units (row 2): unit = % dry mass
Description: Nitrogen content (% dry mass) in sampled apical tip of corresponding induction subunit seaweed. NA indicates that the value was not calculated.
Variable Name (row 1): C
Variable Type or Units (row 2): unit = % dry mass
Description: Carbon content (% dry mass) in sampled apical tip of corresponding induction subunit seaweed. NA indicates that the value was not calculated.
Variable Name (row 1): CN
Variable Type or Units (row 2): unit = NA; ratio, C:N
Description: Carbon to nitrogen ratio, C:N, (C/N) in sampled apical tip of corresponding induction subunit seaweed; Figure 2f, Appendix Table S3b. NA indicates that the value was not calculated.
File: 2_pre_induction_phloro.csv
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Seaweed source region (south, central, north); see also Figure 1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Seaweed collection site (n = 3 sites region-1); see also Figure 1
Variable Name (row 1): relaxation.state
Variable Type or Units (row 2): ID factor
Description: Relaxation state of each seaweed prior to chemical analyses, either Relaxed (frozen after the relaxation phase) or InSitu (frozen upon collection).
Variable Name (row 1): sample.no
Variable Type or Units (row 2): ID factor
Description: Number used to identify samples (1-10) from each site, relaxation state combination.
Variable Name (row 1): phloro.pct
Variable Type or Units (row 2): unit = % dry mass
Description: Phlorotannin concentration (% dry mass) in sampled apical tip of corresponding induction subunit seaweed; as in Figure 2c and Appendix Table S2a.
File: 3_pre_induction_cn.csv
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Seaweed source region (south, central, north); see also Figure 1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Seaweed collection site (n = 3 sites region-1); see also Figure 1
Variable Name (row 1): relaxation.state
Variable Type or Units (row 2): ID factor
Description: Relaxation state of each seaweed prior to chemical analyses, either Relaxed (frozen after the relaxation phase) or InSitu (frozen upon collection).
Variable Name (row 1): sample.no
Variable Type or Units (row 2): ID factor
Description: Number used to identify samples (1-10) from each site, relaxation state combination.
Variable Name (row 1): N
Variable Type or Units (row 2): unit = % dry mass
Description: Nitrogen content (% dry mass) in sampled apical tip of corresponding induction subunit seaweed
Variable Name (row 1): C
Variable Type or Units (row 2): unit = % dry mass
Description: Carbon content (% dry mass) in sampled apical tip of corresponding induction subunit seaweed
Variable Name (row 1): CN
Variable Type or Units (row 2): unit = NA; ratio, C:N
Description: Carbon to nitrogen ratio, C:N, (C/N) in sampled apical tip of corresponding induction subunit seaweed; Figure 2e and Appendix Table S2b.
File: 4_inter_region_choice_phase_data.csv
Variable Name (row 1): indunit
Variable Type or Units (row 2): ID factor
Description: number used to identify an individual induction unit (1-252); as in “1_induction_phase_data.csv”
Variable Name (row 1): indsubunit
Variable Type or Units (row 2): ID factor
Description: letter used to identify each of the two seaweeds within the induction unit (a,b); as in “1_induction_phase_data.csv”
Variable Name (row 1): choiceunit
Variable Type or Units (row 2): ID factor
Description: Number used to identify an individual choice unit (1-216)
Variable Name (row 1): choicetip
Variable Type or Units (row 2): ID factor
Description: Number (1 or 2) assigned to each seaweed piece in a choice unit
Variable Name (row 1): combo
Variable Type or Units (row 2): ID factor
Description: Code used to identify each region combination in interregion choice assays (R1R2, R2R3, R1R3)
testing constitutive differences in palatability between regions (i.e., Figure 3a, Appendix Table S4)
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Seaweed source region (south, central, north); as in “1_induction_phase_data.csv”
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Seaweed collection site (n = 3 sites region-1); as in “1_induction_phase_data.csv”
Variable Name (row 1): ind.type
Variable Type or Units (row 2): ID factor
Description: Type of induction treatment (no-snail control or induced) applied to each indunit or indsubunit; as in “1_induction_phase_data.csv”
Variable Name (row 1): ind.treat
Variable Type or Units (row 2): ID factor
Description: Type of induction treatment (No-Snail Control, Southern Inducer, or Northern Inducer) applied to each indunit or indsubunit; as in “1_induction_phase_data.csv”
Variable Name (row 1): inducer
Variable Type or Units (row 2): ID factor
Description: Origin of inducer snail (No-Snail Control for interregion choice combinations and either Southern Inducer or Northern Inducer for intraregion choice combinations) applied to each choiceunit
Variable Name (row 1): chooser
Variable Type or Units (row 2): ID factor
Description: Origin of chooser snail (Northern Choice Snail or Southern Choice Snail) applied to each choiceunit
Variable Name (row 1): initial mass
Variable Type or Units (row 2): unit = grams
Description: initial seaweed mass (grams) of the corresponding choice seaweed tip prior to the start if the choice assay phase
Variable Name (row 1): final mass
Variable Type or Units (row 2): unit = grams
Description: final seaweed mass (grams) of the corresponding choice seaweed tip after the choice assay phase
Variable Name (row 1): csnailmass
Variable Type or Units (row 2): unit = grams
Description: Total mass (g) of the chooser snails in the choice assay phase, applied to each choiceunit. Referred to as Bunit in formula for Gpc below.
Variable Name (row 1): start time
Variable Type or Units (row 2): MMDDYY HHMM
Description: Date and time at which the choice assay began for each choiceunit
Variable Name (row 1): end time
Variable Type or Units (row 2): MMDDYY HHMM
Description: Date and time at which the choice assay ended for each choiceunit
Variable Name (row 1): Dunit
Variable Type or Units (row 2): unit = days
Description: Unit-specific choice assay phase duration (days)
Variable Name (row 1): Gtip
Variable Type or Units (row 2): unit = mg
Description: Total seaweed tissue grazed for each choicetip within each choiceunit. Calculated as initial-final mass.
Variable Name (row 1): Gpc
Variable Type or Units (row 2): unit = mg d-1 3.5g snail-1
Description: Standardized per capita daily grazing rates (mg d-1 3.5g snail-1) on each choicetip calculated as Gpc = 3.5g*(Gtip/Bunit)/Dunit; as in Figure 3a and Appendix Table S4
File: 5_intra_region_choice_phase_data.csv
Variable Name (row 1): indunit
Variable Type or Units (row 2): ID factor
Description: number used to identify an individual induction unit (1-252); as in “1_induction_phase_data.csv”
Variable Name (row 1): indsubunit
Variable Type or Units (row 2): ID factor
Description: letter used to identify each of the two seaweeds within the induction unit (a,b); as in “1_induction_phase_data.csv”
Variable Name (row 1): choiceunit
Variable Type or Units (row 2): ID factor
Description: Number used to identify an individual choice unit (1-216)
Variable Name (row 1): choicetip
Variable Type or Units (row 2): ID factor
Description: Number (1 or 2) assigned to each seaweed piece in a choice unit
Variable Name (row 1): combo
Variable Type or Units (row 2): ID factor
Description: Code used to identify each region combination in intraregion choice assays (R1I, R2I, R3I)
testing palatability differences between control or induced seaweeds within a region (i.e., Figure 23, Appendix Table S5).
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Seaweed source region (south, central, north); as in “1_induction_phase_data.csv”
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Seaweed collection site (n = 3 sites region-1); as in “1_induction_phase_data.csv”
Variable Name (row 1): ind.type
Variable Type or Units (row 2): ID factor
Description: Type of induction treatment (no-snail control or induced) applied to each indunit or indsubunit; as in “1_induction_phase_data.csv”
Variable Name (row 1): ind.treat
Variable Type or Units (row 2): ID factor
Description: Type of induction treatment (No-Snail Control, Southern Inducer, or Northern Inducer) applied to each indunit or indsubunit; as in “1_induction_phase_data.csv”
Variable Name (row 1): inducer
Variable Type or Units (row 2): ID factor
Description: Origin of inducer snail: Southern Inducer or Northern Inducer for intraregion choice combinations) applied to each choiceunit
Variable Name (row 1): chooser
Variable Type or Units (row 2): ID factor
Description: Origin of chooser snail (Northern Choice Snail or Southern Choice Snail) applied to each choiceunit
Variable Name (row 1): initial mass
Variable Type or Units (row 2): unit = grams
Description: initial seaweed mass (grams) of the corresponding choice seaweed tip prior to the start if the choice assay phase
Variable Name (row 1): final mass
Variable Type or Units (row 2): unit = grams
Description: final seaweed mass (grams) of the corresponding choice seaweed tip after the choice assay phase
Variable Name (row 1): csnailmass
Variable Type or Units (row 2): unit = grams
Description: Total mass (g) of the chooser snails in the choice assay phase, applied to each choiceunit. Referred to as Bunit in formula for Gpc below.
Variable Name (row 1): start time
Variable Type or Units (row 2): MMDDYY HHMM
Description: Date and time at which the choice assay began for each choiceunit
Variable Name (row 1): end time
Variable Type or Units (row 2): MMDDYY HHMM
Description: Date and time at which the choice assay ended for each choiceunit
Variable Name (row 1): Dunit
Variable Type or Units (row 2): unit = days
Description: Unit-specific choice assay phase duration (days)
Variable Name (row 1): Gtip
Variable Type or Units (row 2): unit = mg
Description: Total seaweed tissue grazed for each choicetip within each choiceunit. Calculated as initial-final mass.
Variable Name (row 1): Gpc
Variable Type or Units (row 2): unit = mg d-1 3.5g snail-1
Description: Standardized per capita daily grazing rates (mg d-1 3.5g snail-1) on each choicetip calculated as Gpc = 3.5g*(Gtip/Bunit)/Dunit; as in Figure 3b and Appendix Table S5
File: 6_seaweed_canopy_survey_data.csv
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Region (south, central, north) of survey; see also Figure 1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Site (n = 3 sites region-1) of survey; see also Figure 1
Variable Name (row 1): quadrat
Variable Type or Units (row 2): ID factor
Description: Number (1-10) assigned to each quadrat survey conducted at each site
Variable Name (row 1): pts.asco
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to Ascophyllum nodosum in point-intercept quadrat surveys; as used in Figure 4a and Appendix Table S6a
Variable Name (row 1): pts.fucus
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to *Fucus *in point-intercept quadrat surveys; as used in Figure 4a and Appendix Table S6a
Variable Name (row 1): pts.red
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to red algae species in point-intercept quadrat surveys.
Variable Name (row 1): pts.green
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to green algae species in point-intercept quadrat surveys.
Variable Name (row 1): pts.rock
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to bare rock in point-intercept quadrat surveys.
Variable Name (row 1): pts.invert
Variable Type or Units (row 2): unit = NA; number
Description: Number of grid points (out of 25) corresponding to sessile invertebrate species in point-intercept quadrat surveys.
File: 7_seaweed_canopy_survey_data.csv
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Region (south, central, north) of survey; see also Appendix S1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Site (n = 3 sites region-1) of survey; see also Appendix S1
Variable Name (row 1): quadrat
Variable Type or Units (row 2): ID factor
Description: Number (1-10) assigned to each quadrat survey conducted at each site
Variable Name (row 1): species
Variable Type or Units (row 2): ID factor
Description: Name of species identified in 2020 quadrat surveys. Either Fucus or Ascophyllum nodosum
Variable Name (row 1): measured
Variable Type or Units (row 2): ID factor
Description: Measured for length, "Yes" or "No"
Variable Name (row 1): length
Variable Type or Units (row 2): unit = cm
Description: Length (cm) of individual seaweed within a quadrat. NA indicates that the value was not calculated.
Variable Name (row 1): density
Variable Type or Units (row 2): unit = NA; number
Description: Number of individuals of each seaweed species (counted as number of holdfasts) within a quadrat (number 0.25m-2); as used in Figure 3a and Appendix S2 Table S6a. NA indicates that the value was not calculated.
Variable Name (row 1): Survey Date
Variable Type or Units (row 2): unit= Date MMDDYYY
Description: The date on which the survey was conducted.
File: 8_snail_survey_data.csv
Variable Name (row 1): region
Variable Type or Units (row 2): ID factor
Description: Region (south, central, north) of survey; see also Figure 1
Variable Name (row 1): site
Variable Type or Units (row 2): ID factor
Description: Site (n = 3 sites region-1) of survey; see also Figure
Variable Name (row 1): quadrat
Variable Type or Units (row 2): ID factor
Description: Number (1-10) assigned to each quadrat survey conducted at each site
Variable Name (row 1): l.littorea
Variable Type or Units (row 2): unit = NA; number
Description: Number of Littorina littorea counted within the quadrat; as used in Figure 4d and Appendix Table S6d.
Variable Name (row 1): l.obtusata
Variable Type or Units (row 2): unit = NA; number
Description: Number of Littorina obtusata counted within the quadrat; as used in Figure 4d and Appendix Table S6d.
Organism Collections- We collected F. vesiculosus seaweeds from nine sites spanning >750km of NW Atlantic coastline and three regions in and around the Gulf of Maine (Fig. 1). Seaweed individuals (one 10-20cm thallus and its holdfast) were collected from the mid-intertidal zone, removing each from the substrate at its holdfast with a metal scraper. Individual holdfasts were separated by >0.25m to avoid sampling genetically identical thalli (N= 80-100 individuals site-1). We avoided seaweeds that were reproductive or visibly damaged. Upon collection, we froze subset of seaweeds (n= 10 site-1) at -20°C for later analysis of ‘in situ’ tissue chemistry. L. littorea snails were collected from one southern and one northern field site (N= 400 site-1; shell length= 15-25 mm). All snails and seaweed were maintained under ambient light and seawater conditions in shallow flow-through tanks (water temperature range 19-21°C). Snails were kept in separate tanks and fed ad libitum with non-experimental sympatric Fucus until 24h before starting experiments.
Relaxation Phase – Seaweeds were cleared of all epiphytes and epifauna and maintained in the absence of herbivores for 10d beginning 21August 2019. This “relaxation phase” allows for defenses that may have been induced in the field to relax or dissipate (Underwood et al., 2002), which can take as little as 4d (Rohde & Wahl 2008). After 10d, samples of “relaxed” seaweeds (n= 6-10 site-1) were frozen as above, while the remainder were used in the induction phase (n= 56 site-1).
Induction Phase - Pairs of relaxed, sympatric seaweed individuals were maintained in independent flow-through “induction units” in the presence or absence of herbivorous snails for 12d. Each induction unit consisted of a 1.2L mesh-sided container submerged within a 2.5L bucket receiving its own supply of running seawater. A mesh panel divided the inner container to separate the two individual seaweeds. Induction units were assigned randomly to one of three treatments: Southern Inducer Snails, Northern Inducer Snails, or No-Snail Control (n= 7, 7, or 14 site-1, respectively; N= 252 induction units). Units assigned to inducer snail treatments received 3 snails from the respective source population in one side of the container, allowing the other seaweed to serve as an autogenic growth control (Long et al. 2013). The second seaweed in the no-snail controls ensured that total seaweed biomass was similar across all induction units and treatments (see Fig. S1).
Each seaweed’s tissue mass was measured at the beginning and end of the induction phase (mean ± SE= 1.068 ± 0.014g, N= 504). Individuals were blotted dry with paper towels to remove all visible moisture prior to weighing. Inducer snails (n= 3 unit-1) were also blotted dry with paper towels and weighed, as a group, for total biomass per unit (Bunit). At the end of the induction phase and after measuring final mass, two apical tips were clipped from each seaweed for use in choice assays (Sotka et al., 2002, Taylor et al., 2002), with additional tips sampled and frozen for later analysis of “post-induction” tissue chemistry.
The total amount of seaweed grazed by snails within a given unit (Gunit) was estimated as Si(Af/Ai)-Sf, where Si and Sf are the initial and final mass, respectively, of the seaweed with snails, while Ai and Af are those of the paired autogenic control (Long et al., 2013). Because northern snails were slightly larger than southern snails (mean ± SE individual mass, 3.82 ± 0.05 vs. 3.16 ± 0.04 g), we also calculated mass-adjusted, per capita daily grazing rates (Gpc) normalized for an individual 3.5g snail using the formula 3.5*(Gunit/Bunit)/Dunit, where Bunit is the total biomass of the three snails in the unit, and Dunit is the induction phase duration (11.6 – 12.0 d). We analysed Gunit and Gpc with separate linear mixed models (“LMMs”) that included seaweed region, inducer snail population, and their interaction as fixed effects. Seaweed collection site (within region) and the site x inducer interaction were included in the model as random intercepts to account for the nested experimental design. Seaweed growth rates in the no-snail control treatment were calculated as (Cf-Ci)/Ci where Cf and Ci are the final and initial mass, respectively. We calculated then analysed the average growth rate in each induction unit using a similar LMM that included region (fixed effect) and seaweed collection site (random effect). Seaweeds from 5 units were excluded from analyses and choice assays: two units experienced water flow issues and mortality and three units were missing seaweeds.
Choice Phase – We conducted two types of choices assays to assess differences in relative palatability (1) between regions (“inter-region” assays) and (2) between induction treatments within each region (“intra-region” assays). Choice assays were conducted in the same flow-through setup as the induction phase but with single-compartment mesh containers (“choice units”). Each choice unit contained a pair of apical tips from seaweeds exiting the induction phase and either three northern or three southern “chooser” snails (Fig. S1). Seaweed collection sites within each region were pooled and seaweeds were chosen at random for each choice replicate. (1) Inter-region assays offered snails a choice between one lower- and one higher-latitude tip from no-snail control seaweeds. Three paired region choices (lower/higher: South/Central, South/North, Central/North) were crossed with the two chooser snail populations (N, S) in a 3 x 2 design with n= 12 choice units for each choice x chooser combination (N= 72 inter-region choice units). (2) Intra-region assays offered snails choice between two seaweed tips from the same region but different induction treatments: one tip was from a seaweed in the no-snail control treatment and the other was from a seaweed in either the N- or S-inducer snail treatment. Two paired control/inducer choices (Control/N Inducer, Control/S Inducer) for each of the three seaweed regions were offered to N and S chooser snails in a 2 x 3 x 2 factorial design. Seaweeds from two induction units (S seaweed in the N inducer treatment) could not be included in choice assays because high grazing rates during induction removed most or all apical tissues. In addition to mortality losses described above (see “Induction Phase”), this led to an unbalanced design with 7-12 replicates for each inducer x region x chooser treatment combination (N= 138 intra-region choice units).
In both choice assays, the different choice combinations (e.g., South/Central, Control/N-induced, etc.) were replicated by randomly selecting one seaweed (i.e., induction unit) from each of the two corresponding regions or treatments. Source sites within each region were pooled and selected randomly. One apical tip from each seaweed/induction unit was then placed in each of two choice units, which were then randomly assigned to receive either northern or southern chooser snails (see Fig. S1 for an example). In this way, we ensured that any among-unit differences (i.e., variation among individual seaweeds) within each induction treatment were distributed uniformly between the two chooser snail populations.
All choice assays began on 13 September and lasted 3-6d. Units were inspected daily to visually assess grazing on apical tips. Assays were terminated on a unit-by-unit basis when either tip appeared ~50% consumed, or after a maximum of 6d (>70% of units crossed the 50% threshold by day 6). The flexible assay duration minimized potentially confounding effects of preferred resource limitation (i.e., grazers consuming all of a preferred tip) or seaweed trait changes (i.e., defense relaxation or induction) during the choice assay.
Seaweed tips were individually weighed immediately before and after each assay to calculate tissue mass grazed by snails (Gtip= initial-final mass) and standardized per capita daily grazing rates as Gpc= 3.5g*(Gtip/Bunit)/Dunit, where Dunit is the choice unit-specific assay duration (3-6d). Grazing rates on each tip were analysed with LMMs that included choice unit, seaweed source site, and induction unit as random intercepts. Grazing rates were square root transformed prior to analysis to satisfy assumptions of normality and homoscedasticity. Analysis of inter-region choice data included seaweed tip relative latitude (low or high), chooser snail population (N or S), seaweed region combination (S-N, S-C, C-N), and their interactions as fixed effects. Intra-region analyses included seaweed tip induction treatment (induced or control), inducer snail population (i.e., induced tip exposed to N or S grazers during the induction phase), chooser snail population (N or S snail grazing during the choice assay), and all interactions as fixed effects. We then conducted joint contrast tests on estimated marginal means (R package emmeans; Lenth 2023) to test for main and interactive effects of induction treatment and inducer snail population within each region x chooser combination.
Tissue Chemistry - Seaweed apical tips from different experiment phases were freeze dried, homogenized, and ground into fine powders. We extracted phlorotannins from pre-weighed subsamples (~15mg) following Kurr & Davies (2019) and spectrophotometrically analysed liquid extracts following Pavia & Toth (2000) using Folin-Ciocalteu reagent with phloroglucinol (1, 3, 5-trihydroxybenzene, Sigma-Aldrich P3502) as a standard. An additional subsample of Fucus powder (~1.5mg) was weighed on a semi-microbalance (± 0.001mg) and passed through a Fisons NA 1500 Series 2 Elemental Analyzer (Costech Analytical, Inc.) to estimate carbon and nitrogen mass based on curves produced using acetanilide standards (C8H9NO, Costech). C:N ratios and phlorotannin concentrations (proportion dry mass) of samples collected before (in situ, relaxed) or after the induction phase were analysed with LMMs and GLMMs, respectively, that included region, relaxation state (or induction treatment), and their interaction as fixed effects plus site and site x relaxation state (or site x induction treatment) as random intercepts. GLMMs of phlorotannin concentrations (proportions) were fit with a beta distribution and logit-link.
Field Surveys – We conducted quadrat surveys of herbivore and fucoid assemblages in the late spring/early summer 2020 at 7 of the 9 collection sites (Fig.1a). Quadrats (0.5 x 0.5 m, n= 10 site-1) were haphazardly placed in the mid-intertidal zone at each site, avoiding tide pools and maintaining >1m between quadrats. For each quadrat, we estimated canopy % cover for each algal species using a 25-point-intercept grid and recorded identity and density (no. 0.25m-2) of fucoid holdfasts and herbivores. We measured the maximum frond lengths of fucoids located within all or part of the quadrat (up to 30 individuals quadrat-1). Densities of the two most common fucoids (F. vesiculosus and Ascophyllum nodosum) were analysed with negative binomial GLMMs (log-link function) that included the region, species, and their interaction as fixed effects, with site and the site x quadrat interaction as random intercepts. Frond lengths and herbivore densities (Littorina littorea and L. obtusata) were analysed with similar GLMMs. Percent cover data were logit-transformed for analysis with a similar LMM.
Statistical Analyses – All statistical analyses were performed in R (R Core Team 2024). Models were fit via REML with type 3 contrasts to account for unequal sample sizes. Fixed effects were evaluated with marginal Wald c2 (GLMMs) F tests on Kenward-Roger df (LMMs). Diagnostic tests and residual plots were used to verify assumptions of homoscedasticity and normality for LMMs, with data transformations and/or weighted variance structures applied as necessary following Zuur et al. (2009). We evaluated significant interactions (p < 0.05) using linear contrasts on estimated marginal means and adjusted contrast p-values for the false discovery rate. Details of each model and analysis are given with corresponding results in the supporting information (Tables S1-6).
