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.