Epiphytic Community on Posidonia oceanica: Pattern of Distribution and Effects on the Seagrass Life History Paloma Lopez Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA, USA.

Abstract: The seagrass, Posidonia oceanica, is essential to the Mediterranean Sea ecosystem. The study of the relationship between P. oceanica and epiphytes could serve as an important tool to aid management and conservation of the seagrass. In this study, I examined the distribution of epiphytes on seagrass across a depth gradient. Furthermore, I determined how epiphyte load affects flowering events on P. oceanica. Results show that light intensity and epiphytic coverage decreases as a function of increasing depth. A light threshold for epiphytic growth was derived at around 90 lum/ft2. Additionally, individuals with higher epiphyte coverage were found to have lower flowering frequency. These results indicate that epiphyte coverage is driven by light intensity. Higher levels of epiphyte coverage may also negatively affect the life history of P. oceanica.

Introduction: Posidonia oceanica, the only endemic seagrass to the Mediterranean Sea, plays an important role in ecological processes such as carbon fixation and storage, and is a powerful regulator of the quality of the water. (Pergent et al., 1994; Mateo et al., 1997; Duarte et al., 2005). The leaves of this species may attain a maximum age of 300 days (Duarte 1991) and as a result of their long-life span, old leaves may support a high load of epiphytic growth (Casola et al. 1987, Romero 1988). High epiphytic load on P. oceanica leaves may have substantial effects on seagrass survival and growth, such as leaf shading (Hootsmans and Vermaat 1985, Silberstain et al. 1986) or gas and nutrient exchange with host leaves (McRoy and Goering 1974, Sand-Jensen 1977). The purpose of this study was to examine the pattern of epiphyte coverage on P. oceanica leaves as a function of light and depth. To investigate this, I measured epiphyte density and light intensity as a function of depth to support the hypothesis that both epiphytic load and light have an inverse relationship with depth. Light intensity as a function of leaf length was also measured to detect if there is a light threshold for epiphytic growth. Epiphyte load within a single depth was studied to determine its effect on the seagrass growth and flowering. A growth experiment was designed to measure if leaves with no epiphytes would increase their growth rate significantly compared to leaves with their natural epiphytic community present. Flowering versus non-flowering individuals were compared based on epiphytic load to determine whether non-flowering plants are associated with higher epiphytic load on individuals.

Materials and methods: Species description. Posidonia oceanica is a perennial seagrass endemic to the Mediterranean Sea. It forms vast meadows from the sea-surface to about 40 meters depth. There is a large

1 community of epiphytes that colonize the seagrass’ blades yearlong. Epiphyte colonization is principally controlled by biological factors such as the host plant growth and the life cycle of the epiphytes (Casola et al., 1987; Mazzela & Russo, 1989). In turn, biological factors are affected by environmental conditions such as temperature, light strength, and hydrodynamics, which change as a function of depth. Site description. This study was completed at the STARESO field station in Calvi, Corsica, during October 2012. Sampling was conducted north of the STARESO harbor beginning 50 meters north of the jetty and ending at about 50 meters north (30° bound) of the starting point. Transects were carried out by SCUBA parallel to shore between 9 and 15 meters depth within the Posidonia bed. Leaves were collected over same-depth transects using Ziploc® bags. Epiphyte index as a function of depth. I calculated epiphyte index by using uniform point contact along Posidonia blades every centimeter. Leaves were collected from four different depths: 9 (5 blades), 11.5 (8 blades), 13 (8 blades), and 15 (4 blades) meters. Leaves were brought to the lab at STARESO and measured using a measuring tape. A ratio of epiphyte point contact over total point contact was calculated and compared using linear regression analysis (JMP 10). Greater epiphyte index in shallower depths will support the hypothesis that there is an inverse relationship between epiphyte coverage and depth. Amount of light across depth. Instantaneous light measurements were taken by exposing a PAR sensor for 30 seconds every ten meters along the Northernmost (Site A 42°58’07’’ N, 08°72’49’’ E) and Southernmost (Site C 42°57’82’’ N, 8°72’48’’ E) perpendicular transects) (Perlkin and Tucker, 2012). Amount of light along blade. Light was measured along Posidonia leaves at two different depths: 9 (5 blades) and 15 (3 blades) meters to determine a light threshold for epiphyte growth along blades at each depth. A PAR sensor was positioned along the blade from bottom to top with pauses every 10 centimeters. Data was analyzed using analysis of co-variance in JMP 10 to find relation between amount of light along blade and depth. A light threshold will be determined to define epiphytic habitat. Epiphyte effect on growth. 28 Posidonia leaves along a 30 meter transect were scrapped and cleared of epiphytes using diving knives. This experiment was done at a single depth (9m). Each of the treated individuals were about one meter apart from each other. A different leaf from the same individual was cut to the same size as the scrapped one to compare leaf growth with and without epiphytes. All other leaves from the same individual were trimmed short enough to distinguish them from the treated leaves. The experiment was monitored after four and seven days. Results were compared using an ANOVA test. Significantly higher growth on scrapped individuals will support the hypothesis that presence of epiphytes decreases growing rates of Posidonia leaves. Epiphyte effect on flowering. 50 individuals were surveyed along a 50 meter transect. Every leaf on an individual was categorized into three levels of epiphyte intensity from 1 to 3 indicating low to high epiphyte coverage respectively. An epiphyte index was calculated based on the total epiphyte coverage over the total number of leaves in an individual. Flowering events were annotated for each sampled plant. Results were analyzed using a t-test. Higher number of non-flowering individuals with higher epiphyte coverage will support the hypothesis that grater epiphyte colonization restricts seagrass development.

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Results: Epiphyte index as a function of depth. Posidonia leaves showed a higher epiphyte index at 9 meters (mean=0.7) compared to those at 15 meters (mean=0.3). The samples from 11.3 and 13 meters were not as accurate and results show epiphyte index from these samples to be more widely spread out. However, both depths follow a decreasing pattern of epiphyte coverage when evaluated along results from 9 and 15 meters (Fig. 1). Statistical analysis strongly support the hypothesis that epiphyte coverage decreases on Posidonia leaves with increasing depth (P<0.001, α=0.05)

Epiphyte index By Depth

Figure 1. Epiphyte index as a function of depth. The graph shows a linear decrease of the epiphyte coverage in Posidonia leaves as a function of depth. The dots represent each of the replicates per depth.

Amount of light across depth. Figure 2 shows the relationship between light and depth. The amount of light present in the water column decreases with increasing depth. Amount of light along blades. The amount of light found along Posidonia blades decreased linearly with the highest amount of light found at the top of the leave and the lowest at the bottom. Results from a covariance comparison between 9 and 15 meters show the relationship between both depths with respect to the amount of light and epiphytic presence (Fig. 3). When measured from the bottom up, leaves sampled at 15 meters depth had their first epiphytic growth around the 7th centimeter, which corresponds to the amount of light present in the region where the first epiphytes were found in leaves at 9 meters depth (1st centimeter). These results support the hypothesis that there is a light threshold which limits epiphytic growth along Posidonia leaves based on amount of light received. From the comparison graph (Fig. 3), we can infer 90 Lum/ft2 to be the light limit in which epiphytes are adapted to be extant.

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Effect of epiphytes on growth. An analysis of variance test shows no significant results between scraped and non-scraped leaves (p>0.39, α=0.05). Leaves show negative growth after 4 and 7 days, most likely due to increased herbivore consumption (Fig. 4). There is high variability seen between results from 4 and 7 days. Conclusions about the effect of epiphyte cover on growth can therefore not be drawn based on these results.

Effect of epiphytes on flowering. Results show that flowering events are more often associated with lower epiphytic coverage (measured as epiphyte index, Fig. 5). There were a disproportionate number of samples that had flowers versus those that did not (9 versus 41 respectively). However, a t-test showed a significant difference between flowering versus non- flowering occurrences in Posidonia (P<0.0387, α=0.05), supporting the hypothesis that non- flowering events occur more often in plants with a higher epiphyte coverage than those plants with a lower index.

5 Figure 5. Comparison of flowering events on Posidonia leaves based on epiphyte index. Flowering events occur more often in individuals with a epiphytic lower index (P<0.0387, α=0.05).

Epiphyte index by Flowering events

Conclusion: According to results in this study, epiphyte coverage and light intensity decrease as a function of increasing depth. Moreover, there is an identifiable light threshold at which epiphytes are unable to colonize Posidonia leaves. Thus epiphyte distribution across depth can be attributed to environmental changes in light. Moreover, individuals with higher epiphyte coverage have lower flowering occurrences compared to those with lower epiphytes. These results indicate an adverse effect on the plant’s life-history. Event though results from the growth experiment were not significant, the experiment showed a different effect of the relationship between epiphytes and Posidonia. Only the scrapped leaves had evident fish bites, which leads to speculate that epiphytes provide some level of protection to Posidonia leaves against herbivores. The relationship between epiphytes and its host plant is an important interaction to study in order to determine what disadvantages or benefits each species has over the other. It has been shown that the epiphytic community on P. oceanica leaves plays an important role in the energy transfer from plant to higher trophic levels (Chessa et al., 1982). However, results from this study show that the degree of epiphytic colonization is crucial for the seagrass life history. Further study of this relationship is needed to determine at which level, the presence of epiphytes begin to restrain seagrass development. Studies done by Jupp (1977) and Mendez (1994) concluded that there is an abundant

6 development of epiphytic growth in those areas with high levels of fertilizers. Consequently, changes on epiphyte loading could be used as an indicator of organic pollution levels shifting. Posidonia oceanica is undergoing significant decline (at least in the Northwestern Mediterranean; Sánchez-Lizaso et al. 1990, Zavodnik & Jaklin 1990, Marbà et al. 1996), which may be due in part to increasing turbidity of coastal waters or to increasing epiphyte loading, both effects being results of increased eutrophication. Studies on epiphyte distribution and on the factors that control this pattern are important in developing management and conservation strategies to prevent seagrass decline.

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7 McRoy, C. P. and J. Goering. 1974. Nutrient transfer between the seagrass Zostera marina and its epyphytes. Nature 248: 173-174.

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