Resampling methods and herbivory
On day 10 of the experiment, a severe herbivory event was recorded at both warm-edge translocation sites. Scaled photos of all fragments were taken at this time to record the effects of herbivory on transplants. At the end of each main sampling period (T0 – T1, T1-T2 and T3 – T4), all pierced fragments were collected and taken back to the laboratory to measure shoot growth. At T1, T2 and T3, additional sets of fragments (n = 10 per treatment) were marked using the piercing method to record growth in the subsequent time period. In addition, at T1 and T3, n = 20 shoots within the natural meadow at each site were marked to compare growth rates between the native meadow and transplants. Underwater shoot counts and a scaled photo was taken to record fragment survivorship, shoot mortality, bite marks, and shoot length among all remaining fragments within each site and sampling time.
In the laboratory, morphological measurements (described above) were repeated on the collected fragments and growth of transplant and natural meadow shoots was measured. Growth (shoot elongation, cm d-1) of the marked shoots was obtained by measuring the length from the base of meristem to marked holes of each leaf (new growth) of the shoot and dividing the leaf elongation per shoot by the marking period (in days). For each shoot, total leaf length (cm shoot-1) and the number of new leaves was recorded. The rate of new leaf production (new leaves shoot-1d-1) was estimated dividing the number of new leaves produced per shoot and the marking period. New growth was dried at 60 ºC for 48 hrs to determine carbon and nitrogen content of the leaves, and carbon to nitrogen (C:N) ratios. Carbon and nitrogen concentrations in the new growth leaf tissue was measured at the beginning of the experiment and each subsequent time point for each treatment. Nutrient analyses were conducted at Unidade de Técnicas Instrumentais de Análise (University of Coruña, Spain) with an elemental analyser FlashEA112 (ThermoFinnigan).
Underwater photos of shoots were analysed using ImageJ software (https://imagej.net). Maximum leaf length on each shoot in warm-edge transplant sites (cool-warm, centre-warm and warm-warm) were recorded for the initial (day 10) herbivore impact, T1, T2 and T3 and related to transplant nutrient concentrations. Herbivore impact was estimated as the proportional change in length of the longest leaf relative to initial length at T0.