Giant kelp (Macrocystis pyrifera) is a foundation species in the California ecosystem that creates habitats for numerous other organisms and adjusts seasonally to fluctuations in nutrients, storm intensity, and ocean temperature. This study examined whether early life history stages of the population could similarly adjust to increased temperatures due to changing climate.
Fertile M. pyrifera sporophyll blades were collected from kelp forests at 5 to 10 meters depth from three sites along the California coast followed by induced zoospore release in the lab.
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Cabrillo Beach in Los Angeles, California (33.7110° N, 118.2833° W)
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Arroyo Burro Beach in Santa Barbara, California (34.4028° N, 119.7432° W), and
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Lover’s Point, Monterey Bay, California (36.6269° N, 121.9170° W).
Multiple fertile sporophyll blades were collected from the base of M. pyrifera individuals at each site (Los Angeles [LA] n=16, Santa Barbara [SB] n=20, Monterey [MB] n=20] in September and October 2016. All blades were collected between 5 meters and 10 meters depth and we chose individuals haphazardly, but assured they were never nearest neighbors to one another (~2 to 5 meters apart) and that the adults were similar heights. We collected individuals from a site on the same day using SCUBA. We separated the blades from the stipe by hand, taking care not to tear the blades. We immediately placed up to ten fertile sporophyll blades from each individual in a sealed plastic bag underwater to maintain both the separation and survival of individuals. We then placed the collections in a cooler on a thin layer of ice to keep cool during transport immediately to California State University, Northridge.
We induced zoospore release in the laboratory following previously established methods (Deysher & Dean 1984). We rinsed all individuals with filtered seawater to reduce potential bacteria and mucus released in transit. We wrapped the rinsed sporophylls in damp towels and placed them back into the sealed plastic bag and stored them overnight at 15° C in a temperature-controlled room. Such desiccation promotes the release of spores from sporophylls. The next morning, we removed the sporophylls from storage and placed four sporophylls from each individual into 1-liter containers of 15°C filtered seawater. We completed this procedure for all collections and kept sporophylls from different individuals separate at all times to prevent cross-contamination. We removed sporophylls from the seawater after 30 minutes and discarded them. After removing the sporophylls, we took a 1.5 milliliter (mL) sample from each well-mixed spore solution. We quantified the spore density in each sample using a hemocytometer.
We established three target temperature treatments in this experiment: 16°, 20°, and 22° C. The lowest temperature represents the average high temperature among the three sites (Table 1: "Temperature_at_Collection_Sites_Table1.pdf" in Supplemental Files section), while 20 and 22°C fall within the Intergovernmental Panel on Climate Change (IPCC) predictions for the end of the century temperature increase (IPCC, 2021). The highest treatment is beyond at least 1 standard deviation unit of the average upper temperature currently experienced by the sites. Although the SB and MB sites do not currently experience 20 or 22°C, they likely will experience these temperatures within this century. We performed all work in a temperature-controlled room maintained at 15.4 °C ± 1.05 °C (mean ± standard deviation), and used heating pads placed below Petri dishes to establish the target treatment temperatures (RootRadiance, DL Wholesale, Livermore, CA, USA). Lights were set to a 12:12 hour day: night cycle, with 8.56 ± 0.266 μmol photons m−2 s−1 during the day cycle.
We placed three glass microscope slides in a square 10x10 cm plastic petri dish to cover the bottom of the dish and added 50 mL of the spore solution to each dish. We established three replicate dishes per individual at each temperature (3 temperatures x 56 individuals x 3 replicates = 504 dishes). Spores were allowed to settle on microscope slides at treatment temperatures for 24–36 hours in the dark. Preliminary work showed uniform settlement both within and among slides in the same petri dish, so we haphazardly chose one of the three slides in each petri dish to quantify settlement. We took digital images of one randomly selected field of view at 400x total magnification on each slide. We used ImageJ (NIH version 1.50i) with the Cell Counter plugin to count the number of successfully settled spores in each image, identified by an extended germ tube.
After imaging, we returned the slides to the Petri dishes and replaced the spore solution with 50 milliliters (mL) of Provasoli-enriched seawater (NCMA/Bigelow Laboratory for Ocean Sciences; East Boothbay, ME). We imaged slides at the same location on the slide weekly for 21 days to monitor gametogenesis and/or spore death. We replenished the media at least once each week and disposed of slides that no longer contained spores. To evaluate successful gametogenesis, we analyzed the last image of each individual (21 days, or day of disposal) and classified spores as mature, immature, or dead. We identified mature gametophytes by their distinct shapes. Immature gametophytes were identified by stunted maturation; for example, if the settled spores never matured further than their initial state, but still contained visible pigment (Roleda et al. 2004). We calculated the total number of dead spores by subtracting the sum of the mature and immature spores from the initial number of settled spores.
Known Issues:
Early in the experiment, we lost one replicate of every individual from Monterey Bay at 22 degrees due to a malfunction in the heating pads and those individuals were excluded from the analysis.
