Small-scale spatial patterns of Posidonia oceanica inflorescences: variation in inflorescence frequency and maturity as a function of depth

Hannah Perlkin and Emily Tucker Ecology and Evolutionary Biology, University of California, Santa Cruz

Abstract For most species there is a clear set of advantages resulting from sexual reproduction. In part because of the longevity of clonal species along with the combination of clear evidence of asexual reproduction and scant evidence of successful recruitment of new sexually derived individuals, sexual reproduction has been widely considered to be relatively or completely unimportant in seagrasses. With increased monitoring efforts over the last two decades, there has been an increased reporting of flowering events and a renewed consideration of the role of sexual reproduction in long- distance dispersal and recovery of meadows after disturbance. Posidonia oceanica can spread vegetatively through plagiotropic (horizontal) and orthotropic (vertical) rhizome growth. This study was carried out during mid-late October, 2012 in The Bay of Calvi, Corsica. Inflorescence proportion and maturity were sampled at 10, 20, 30, 40, 50 and 60 feet. The inflorescence proportions encountered during our study are consistent with previously recorded major flowering events in the Mediterranean. The significant variation in inflorescence proportion and maturity with depth and light reflects the relationship between depth and PAR. Among depth, instantaneous light and cumulative light, the latter was found to be relatively most important. Increased inflorescence proportion and inflorescence maturity at depths with highest light intensity (10-20 feet) suggests the importance of photosynthetic accumulation to initiate sexual reproduction.

1. Introduction For most species there is a clear set of advantages resulting from sexual reproduction. These include recombination, the production of novel genotypes and phenotypes and for most species it is the only current means by which genes can be “passed” on. The life history of many clonal species presents a type of theory-based tension to this logic (Arnaud-Haond et al. 2010). Clonal species are generally very long- lived and capable of substantial non-sexual vegetative “reproduction” by a variety of processes (e.g. growth, fragmentation, pseudovivipary) (Arnaud-Haond et al. 2012; Diaz- Almela 2007; Balestri et al. 2005; Ballestros et al. 2005; Orth et al. 1994). In part because of the longevity of clonal species combined with clear evidence of asexual reproduction and scant evidence of successful recruitment of new sexually derived individuals, sexual reproduction has been widely considered to be relatively or completely unimportant (Vermaat 2009; Balestri and Cinelli 2003; Duarte et al. 1994; Buia and Mazzella 1991). This is particularly true for seagrasses, where vegetative propagation has been the main focus while sexual reproduction has been dismissed as having little importance in meadow maintenance due to it’s episodic and highly variable nature (Remizowa et al. 2012; Diaz-Almela et al 2006; Gobert et al. 2005; Ballestri and Vallerini 2003; Campey et al. 2002; Inglis and Smith 1998; Buia and Mazzella 1991).

The primary counter-arguments to the idea that sexual reproduction in clonal species is unimportant for seagrasses (and other clonal species) are: (1) the observation that flowering events and seed set is common and often massive, (2) such events are energetically costly diverting resources away from vegetative growth and, (3) while often episodic with long intervals between such events, flowering years are numerous relative to the lifespan of the clone (Arnaud-Haond et al. 2012; Almela-Diaz 2006; Gobert et al. 2005, 2001; Balestri and Vallerini 2003; Duarte et al. 1994). With increased monitoring efforts over the last two decades, there has been an increased reporting of flowering events and a renewed consideration of the role of sexual reproduction in long-distance dispersal and recovery of meadows after disturbance (Balestri and Valerini 2004, 2003; Buia and Mazzella 1991).

Posidonia oceanica is a particularly long lived, persistent and slow growing species with high primary productivity that has been shown to create increased complexity to many marine ecosystems of the Mediterranean (Diaz Almela 2007; Orth 2006; Duarte 2001). Seagrass productivity, growth and abundance are extremely sensitive to changes in light availability (Serrano et al. 2011). In P. oceanica specifically, vegetative growth and colonization are largely dictated by light availability. (Duarte 1991). Variation in its growth has been attributed to PAR availability, which varies as a function of depth (Rollon et al. 2003; Duarte 1991; Dennison and Alberte 1985). Light provides sucrose, energy for P. oceanica as well as carbon, a resource that is accumulated in reserves in late summer and is utilized in fall in the production of inflorescences leading to seed production in early spring the following year (Serrano et al. 2011; Vermaat 2009). Carbon and phosphate accumulated through photosynthesis serve in inflorescence elaboration in P.oceanica (Gobert et. al. 2005). In particular, carbon is stored in the rhizomes, where carbohydrate concentrations are highest (Collier et al. 2010).

The resources that are continuously allocated to vegetative growth in P. oceanica are diverted to sexual reproduction during flowering events. As noted above, such re- allocation decreases P. oceanica’s ability to grow suggesting that the maintenance of sexual reproduction increases the fitness of the individual relative to having only asexual reproduction. (Balestri and Vallerini 2003).

Because of the noted relationship between PAR and depth, sequestration of resources occurs more slowly at deeper depths which has provided the basis for the observation that there is often a one to two month delay in the flowering process in ‘deep’ (over 15 meters depth) stands of Posidonia (Buia and Mazzella 1991; Remizowa et al. 2011). Rhizome growth becomes limited with increased depth due to decreased light availability which could explain this two-month delay as smaller rhizomes (deep) require more time to accumulate the resources sufficient to allow production of inflorescences (Duarte 1991). Despite the physiological basis for this allocation model and some anecdotal observations, there has been no detailed assessment of the temporal phenology of P. oceanica as a function of depth. Our goal was to provide this assessment. Specifically, the goal of this study was to describe patterns in inflorescence proportion and maturity as a function of depth under the specific prediction that inflorescence proportion and maturity decrease with increasing depth. Our study addressed 4 hypotheses: (1.) Inflorescence proportion (IP) and inflorescence maturity (IM) vary monotonically and negatively as a function of depth (2.) Light varies negatively as a function of depth (3.) The most parsimonious explanation for the effect of depth on inflorescence proportion is the attenuation of cumulative PAR with depth.

2. Methods Species description Posidonia oceanica, a monoecious perennial marine plant, grows from the waters surface to depths of 80 meters and has an optimum temperature of 17-20 degrees Celsius (Balestri and Vallerini 2003; Duarte 2001; den Hartog 1970). It is widely distributed in the Mediterranean, as well as the North African coast and in the Atlantic along the coasts of Portugal and Spain (den Hartog 1970). Seagrasses are characterized by their ability to spread vegetatively as their main mode of propagation and reproduce sexually on rare occasions, which allows them to persist in near shore (and potentially stressful) environments (Kendrick et al. 2012).

P. oceanica exhibits a triphase annual growth pattern. A period of minimal growth from October to January is followed by a period of rapid growth, which persists throughout spring. At the end of May the growth rate begins to decrease considerably, continuing through summer until growth stops completely in October (den Hartog 1970). Anthesis initiation has been observed from July to September (Gobert et al. 2005). Inflorescences have been observed during these periods of limited growth, between the months of October and January with the fruits occurring from March to June (Bay 1984; den Hartog 1970). The four-month time period in which P. oceanica inflorescences can be observed coincides with an observed two month delay in development between the shallow (0-15m) and deep (15-28m) stands (Remizowa et al. 2012; Buia and Mazzella 1991).

Life History P. oceanica is a species with low tolerance to changes in salinity, water temperature and pollution (den Hartog 1970). In areas with water temperatures reaching above 20 to 22 degrees high mortality in leaves has been reported (den Hartog 1970). It inhabits exposed to moderately sheltered areas with coarse sand, good circulation and well-aerated water (den Hartog 1970). This sensitivity contributes to patchy distribution in otherwise homogeneous shallow (10 to 20 meter) beds around ports and river mouths (den Hartog 1970).

P. oceanica can spread through plagiotropic (horizontal) and orthotropic (vertical) rhizome growth though it’s main mode of spreading is plagiotropic (Balestri et al. 2005). The main plagiotropic axis performs constant expansion and growth, allowing for exploration and colonization of new territory, while orthotropic rhizomes and their lateral branches use local resources, and facilitate light capture by extending leaf blades higher into the canopy, and escaping if sedimentation is high (Balestri et al. 2005) After a minimum period of vegetative maturation (two years), the apical meristem of an orthotropic shoot may produce one or occasionally two inflorescences (Balestri et al. 2005; Balestri and Cinelli 2003). Flowers have also been found on plagiotrophic rhizomes (Balestri et al. 2005). While the relative importance of the two modes of spreading are unknown, both play a role in population maintenance of the genus P. oceanica (Campey et al. 2002).

Study site This study was carried out during mid-late October, 2012 at the Station de Recherches et Sous-marines et Océanographiques (STARESO) located in The Bay of Calvi (Mediterranean Sea, Corsica, France, 42◦ 35’ N, 8◦ 43’ E). Calvi Bay is considered to host meadows of P. oceanica that are of high ecological (considered as having no disturbance) status with a sharp lower limit to the meadow and approximately 310.4 shoots �! (Lopez y Royo et al. 2010). The Bay of Calvi is characterized by poor nutrients (Gobert et al. 2005; Bay 1984) and extensive P. oceanica meadows that continue to surround the entire island of Corsica (Pasqualini et al. 1998). All sampling was done within 1 km of shore between depths of 10-60 feet.

2.1 Inflorescence proportion and maturity as a function of depth All sampling was carried out on SCUBA. Surveys were conducted along six parallel transects. Transects were established at constant depths (10, 20, 30, 40, 50, 60 feet) within and immediately outside of STARESO harbor. Transects were 12-20 meters in length, depending on geomorphological constraints.

In order to approximate inflorescence proportion (number of inflorescences per area) a 0.45 �! quadrat was used to count total rhizomes and inflorescences. Rhizomes were considered to be individual lateral branches of the orthotropic axes. IP was quantified within two quadrats at every meter, on either side of the transect. In order to determine if IP varied as a function of depth an ANOVA (five degrees of freedom) was run.

In order to qualify inflorescence maturity, four inflorescences were assessed within each 0.45 �! quadrat. Inflorescence maturity was assessed within the same quadrats used to sample IP as described above. IM was qualified for four inflorescences per quadrat. To maximize randomness, the first two inflorescences encountered from the top-left and bottom-right quarter of the quadrat were selected. Each inflorescence was categorized as closed, open or flowering [figure 1]. ANOVA (five degrees of freedom) was used to determine if relative maturity of inflorescence varied with depth.

Figure 1. 2.2 Inflorescence maturity closed, open and flowering shown (from left Light as a to right). IM was defined as follows: closed (blades closed around inflorescence), open (blades separated but flowers not protruding) or flowering (flowers emerging), from left to right. function of depth In order to determine instantaneous light intensity (lumens ��!!) two 50 meter transects were established, one to the North (42°58’07’’ N, 08°72’49’’ E) and one to the South (42°57’82’’ N, 08°72’48’’ E) of the parallel transects. Transects were run perpendicular to shore. A PAR sensor was exposed every ten meters along each transect for 30 seconds. These measurements were used to graph instantaneous light as a function of depth.

2.3 Inflorescence proportion and light Variation in cumulative light intensity between depth classes was determined using multiple PAR sensors established at depths of 10, 20, 30, 40 and 50 feet. PAR sensors were deployed for 28 hours (12:00 26 October - 16:00 27 October) and were fixed atop PVC posts of roughly the same height as the surrounding meadow.

A regression model was used to determine the relative importance of function depth, instantaneous light, and cumulative light for IP. An AIC model selection procedure was used to run all reduced model in order to select for the best-fit variable.

3. Results 3.1 Inflorescence proportion and maturity as a function of depth The results of sampling conducted to determine if inflorescence proportion decreased with increasing depth strongly supported the hypothesis that IP varies as a function of depth (figure 2, table 1, p<0.00001). Inflorescence proportion decreased monotonically with depth.

Figure 2. This bar graph depicts decreasing IP as a function of depth.

The results of sampling conducted to determine if open and flowering inflorescence proportions decreased with increasing depth strongly supported the hypothesis that IM varies as a function of depth (figure 3, table 1, p<0.00001).

Figure 3. The bar graph on the left shows the significant decrease in open IP as a function of depth while the graph on the right shows the same

relationship with flowering IP. The two represent the negative

relationship of IM with depth.

Table 1. Summary of ANOVA analyses for IP and IM as a function of depth. ANOVA d.f. P-value IP 5 <0.00001 IM (open) 5 <0.00001 IM (flowering) 5 <0.00001

3.2 Light as a function of depth The results of our sampling conducted to determine if cumulative and instantaneous light intensity (Lumens ��!!) decreased with increasing depth supported the hypothesis that light availability varies as a function of depth (figure 4).

Figure 4. Both graphs describe the relationship of light as a function of depth. Both relationships are non-linear and negative. These graphs show that the relationship between cumulative light (right) and depth is steeper than instantaneous (left).

3.3 Inflorescence proportion as a function of light The results of our sampling conducted in order to determine if inflorescence proportion varied as a function of light strongly supported the hypothesis that inflorescence proportion varies as a function of light (figure 5: instantaneous light, p<0.00001; cumulative light p<0.00001). The results of our comparison of IP as a function of instantaneous light versus cumulative light tested the idea that cumulative light was the most parsimonious explanation to describe the pattern in IP. The results were strongly supported by the best-fit of cumulative light (depth �!=0.88; instantaneous light, �!=0.96; cumulative light, �!= 0.99).

4. Discussion With our study we were able to capture a clear pattern in inflorescence proportion with depth, instantaneous light and cumulative light. The significant variation in inflorescence proportion and maturity with depth and light reflects the relationship between depth and PAR (Serrano et al. 2011; Rollon et al. 2003; Duarte 1991; Dennison and Alberte 1985). The high relative importance of cumulative light to describe the pattern in IP supports the theory that resource accumulation is necessary for inflorescence induction in P. oceanica (Gobert et al 2005).

Our results finding increased inflorescence proportion and inflorescence maturity in areas of higher light intensity (10-20 feet) suggests plants have accumulated sufficient surplus reserves and/or can mature more quickly (Gobert et al 2005). As indicated in previous studies, P. oceanica is highly sensitive to light availability, which dictates photosynthetic capacity (Peirano et al. 2011; Ruiz and Romero 2001). Photosynthates accumulated throughout spring and summer are stored and utilized during fall and winter, periods of lower PAR availability (Serrano et al. 2011). October, the time of our study, coincides with the end of resource accumulation and has historically correlated with the initial stages of inflorescence production (Gobert et al. 2005; Bay 1984; den Hartog 1970). This facilitated our inflorescence maturity observations, as the early stages of inflorescence development were distinct.

Our short-term, small-scale study contributed to the understanding of the relative importance of depth and light in determining patterns in sexual reproduction. Unfortunately, the short time-scale of our study did not allow us to determine if this variability was due to the well-cited two-month delay between shallow and deep stands or if it reflects an overall higher proportion of inflorescences in shallower water. Both of these potentially conflicting explanations have support in literature (two-month delay: Remizowa et al 2012; Buia and Mazzella 1991; increased IP in shallows: Diaz-Almela 2006; Inglis and Smith 1998;).

The inflorescence proportions encountered during our study are consistent with those of previously recorded major flowering events across the entire Mediterranean (Urra et al. 2011). The flowering event we captured was similarly observed Croatia and the Balearic Islands and thus likely represents a major flowering event (E. Serrão, personal communication). These major flowering events, often referred to as ‘episodic,’ must be considered in the temporal context of millenary species (Arnaud-Haond et al. 2012). The massive scale of these events, their relative frequency and the high energetic cost emphasize the importance of sexual reproduction in P. oceanica (Balestri and Vallerini 2003). Theoretically a trade-off must occur; the energetic sacrifices of sexual reproduction must be matched by some increase in fitness (Kendrick et al. 2012). Benefits of sexual reproduction in clonal seagrass are known to include: reestablishment of damaged meadow, establishment of new meadows and likely facilitates species persistence through adverse conditions. Advancements in any of these areas would benefit meadow monitoring, restoration, and conservation of clonal seagrass meadows

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