Study system: Avicennia germinans, the black mangrove, is a neotropical halophyte. It occurs in Western Africa and along the Atlantic and Pacific coastlines of the Americas (Dodd and Afzal 2002). Though over 80 species of mangroves are found globally (Singh et al. 2022), A. germinans, along with Rhizophora mangle (the red mangrove) and Laguncularia racemosa (the white mangrove) comprise the only three species native to the United States — likely due to restricting factors such as aridity and dispersal limitation (Bardou et al. 2020). Avicennia germinans is considered the most freeze tolerant of the three naturally occurring North American species (Cook-Patton et al. 2015) and is the most abundant mangrove species in northern Florida. Due to its freeze tolerance, A. germinans is also the most likely of the three North American species to migrate northward past currently defined range margins.
On the Atlantic coast of North America, northernmost mangrove range limits bewteen St. Augustine and Jacksonville, Florida, at roughly 30°N (Williams et al. 2014, Cavanaugh et al. 2015, personal observation). Along the Florida coastline, winters are typically mild and experience average temperatures ranging from 8.2 to 9.5°C (Stuart et al. 2016). However, occasional frosts as severe as − 10°C occur in Florida, taking place on average once every eight years over the past century (Stuart et al. 2016). Climate change has been associated with the poleward expansion of mangroves into temperate regions, by virtue of fewer severe freezes in recent decades. Though historically mangrove dominance into these regions has fluctuated, climate shifts may induce a persistent state of mangrove dominance (Cavanaugh et al. 2019).
Experimental Design: Avicennia germinans propagules were collected from Ft. George (30.4194° N, 81.4389° W) and Amelia Island, Florida (30.6266° N, 81.4609° W), near where A. germinans reaches its northernmost range limit, in December 2020. Propagules traveled in plastic ziplock bags to the laboratory at Roosevelt University, Schaumburg, Illinois, via postal mail. Propagules were soaked in water for a minimum of 7 days or until their cotyledons opened, and then were placed into their growing medium.
Sand was used as a growing medium so that media-based nutrients would be minimal. Propagules potted directly into the sand and assigned experimental treatments (January 2021 - July 2021). Once potted into their growing medium, plants were watered using deionized water once a week. The light source was fluorescent grow lights on 12-hour timers. Seedlings were assigned to one of four litter treatments: green, frozen-green, senescent and control, with 12 replicates in each litter treatment for the experiment. Each seedling received 2g of shredded dried leaves as its litter treatment. Controls received 2g of shredded brown packing paper.
Litter treatments came from A. germinans leaves collected at the same sites as propagules, near the northernmost range limit of North American mangroves (Cavanaugh et al. 2019). Green and senescent leaves were collected in December 2020. The frozen leaves spent three days at -4ºC in a laboratory freezer.
Mangrove growth was assessed in seedlings, according to the following three criteria: (1) Total plant height, (2) Elongation of the first 5 internodes, and (3) Dried plant biomass. A leaching experiment was performed to track biomass loss from leaf litter. Seedlings were potted in January, and measurements were taken after hypocotyl elongation, from March through July 2021.
Plant height and internode elongation: Plant height and elongation rate of the first internode were measured weekly. Plant height was measured from the edge of the pot to the apical-most leaf pair. Before seedlings developed true leaves, height was measured to the cotyledons. Internode elongation was measured as the distance between the topmost leaf pairs.
Biomass: Following the completion of all experiments, plants were harvested, and roots and shoots were separated. Roots and shoots were placed into individual paper bags and oven-dried at 60 C° for three days. At the end of the three-day period, dried roots and shoots were separately weighed and their masses recorded, though total biomass is reported in the results section. Cotyledons were excluded from the biomass.
Leaching Experiment: A leaching experiment was conducted to compare mass loss over time among the different types of litter. Approximately 2g of each leaf type were added to glass beakers and treated with DI water over the course of a six-week period. Each treatment had four replicates. Mass (g) of the leaf material were recorded twice — once at the beginning of the experiment and once following the six-week period. Mass loss was calculated by subtracting the final mass from the initial mass.
Statistical Analysis: In the seedling growth experiment, the independent variables were litter types. Dependent variables were height, internode elongation and biomass. One-way analysis of variance (ANOVA) was used to compare differences in growth metrics across litter treatments. When an ANOVA found significant effects, Tukey’s HSD test was used to compare treatment groups. In the leaching experiment, the mass lost through the leaching experiment was analyzed with a one-way ANOVA. ANOVA assumptions were tested with a Bartlett test.