The distribution and behavior of Actinia schmidti By: Casandra Cortez, Monica Falcon, Paola Loria, & Arielle Spring Fall 2016 Abstract: The distribution and behavior of Actinia schmidti was investigated in Calvi, Corsica at the STARESO field station through field observations and lab experiments. Distribution was analyzed by recording distance from the nearest neighboring anemone and was found to be in a clumped distribution. The aggression of anemones was tested by collecting anemones found in clumps and farther away. Anemones were paired and their behavior was recorded. We found that clumped anemones do not fight with each other, while anemones found farther than 0.1 meters displayed aggressive behaviors. We assume that clumped anemones do not fight due to kinship. We hypothesized that another reason for clumped distribution could be the availability of resources. To test this, plankton samples were gathered in areas of clumped and unclumped anemones. We found that there is a higher plankton abundance in areas with clumped anemones. Due to these results, we believe that the clumping of anemones is associated to kinship and availability of resources.

Introduction: Organisms in nature can be distributed in three different ways; random, uniform, or clumped. A random distribution is rare and only occurs when the environment is uniform, resources are equally available, and interactions among members of a population do not include patterns of attraction or avoidance (Smith and Smith 2001). Uniform, or regular distribution, happens when individuals are more evenly spaced than would occur by chance. This may be driven by intraspecific competition by members of a population. Clumped distribution is the most common in nature, it can result from an organism’s response to habitat differences. These differences can be daily or seasonal weather, environmental, and reproduction patterns, as well as social behaviors. The main causes in plant distribution is propagation and specific environmental requirements, while the main causes in distribution are resources such as food, water, and shelter (Smith and Smith 2001). Since anemones are mostly sessile , this rule can apply as less of a systematic function and more of a function of movement, therefore anemones were categorized as plants for this study.

The theory of ideal free distribution also helps explain the geographical spacing of organisms. This states that will distribute themselves to gain equal fitness. All else being uniform, if there is twice as many resources in an area there will be twice as many species in that area (Smith 1982).

Actinia equina was previously thought to be a single species that included red beadlet anemones in the Atlantic as well as in the Mediterranean. Recently, it has been discovered that the species in the Mediterranean is not the same, and has since split off as Actinia schmidti (Monteiro et al 1997). It is unclear if the two species have completely different attributes, but some similarities may still exist (Monteiro et al 1997).

Actinia equina can reproduce both sexually and asexually. The anemone creates clones through regeneration/basal laceration, brooding, and budding. Regeneration/basal laceration happens when the anemone’s pedal disk is lacerated, causing it to regenerate itself from two separate halves (Turner et al 2003). A. equina is also one of the only anemones known to brood its young, meaning that the anemone is viviparous. The juvenile anemones will crawl out of the

1 parent anemone and settle around it (Monteiro et al 1997). Budding is when new clones will develop from cell division at a particular site on the anemone and will remain attached to the mother anemone until they mature (Orr et al., 1982). These methods of reproduction are all similar in that they consist of new clonal, juvenile anemones forming and setting around the main focal genit. Since anemones are mostly sessile animals, and are only capable of small, very slow movements, it is unlikely that clones move far from the parent anemone (Shick 1991).

Actinia equina has been found to be non-aggressive towards its clone mates and therefore will aggregate together in clumps. The anemone displays inter-clonal aggression and will fight with unrelated, non-clonal individuals of its own species, as well as other species of anemones (Turner et al 2003). It has been postulated that Actinia equina’s intraspecific aggression is due to maintaining territory for its clonal juveniles (Quicke & Brace, 1983; Brace & Quicke, 1985). Anemones are able to recognize their own clone mates through a highly specific cellular recognition system that allows anemones to distinguish between syngeneic and allogeneic tissue (Lubbock and Shelton 1981).

During fights, A. equina uses purple fighting tentacles called acrorhagi that contain stinging nematocysts. When the anemone engages in an attack, it will extend these stinging cells, often leaving an acrorhagi peel on the opponent. This peel is a thin layer of tissue that will continue to sting even after the other anemone has retracted (Turner et al 2003). This process can take only a few minutes and is a major function of aggression. It will often result in the locomotive relocation of an unfamiliar/unrelated anemone (Ager 2008).

The goal of this study is to better understand the mechanisms driving the distribution and behavior of A. schmidti. In Calvi, Corsica, France, at the STARESO field research center, we noticed an uneven, spatial distribution of Actinia schmidti. With this background information, we suspected their distribution to be clumped. Given that this was the pattern, we predicted that it was associated with kinship and resources. Our three hypotheses were:

1. The distribution of Actinia schmidti along the rocks on the coves is clumped. 2. Individuals in groups or in close proximity are related, therefore anemones found close to one another will be less aggressive than those individuals found far from one another. 3. There will be a higher abundance of plankton in areas with clumped anemones versus areas with unclumped anemones.

Materials and Methods: Species description- Actinia schmidti is also known as the red . This species is found on rocky shores in the north Mediterranean Sea, near the south of France (Monteiro 1997). They were observed above and below the waterline on the rock face. It is characterized by a small red stock and tentacles with a purple ring of stinging nematocysts on the outside of the central disks, called the acrorhagi.

Site Description- This study was conducted during October of 2016 at Station de Recherches Sous-Marines et Océanographiques (STARESO) near Calvi, Corsica in France. Field observations were done in coves to the North and South of the harbor. This area of the Mediterranean Sea is mostly rocky bottoms, and covered in Posidonia oceanica. Actinia schmidti is typically found just below or at the water line of the intertidal zone. It attaches to bare rock face that has little to no algal cover.

2 General Approach Field Methods- Question 1: Distribution of Actinia schmidti:

To test our hypothesis that Actinia schmidti have a clumped distribution, field observations were conducted. Four coves to the North and South of the STARESO harbor were chosen. We chose to observe anemones on the north facing rock within the coves to help control for light and wave/wind disturbance. Using meter tapes, each cove was measured from the tip. The smallest cove was found to be 8 meters, therefore we used this as our standardized length. Within that 8- meter section, the distance between individual anemones were recorded. The purpose of these graphs is to depict whether anemones display a clumped distribution. A variance to mean ratio test was used to determine the distribution. See Figure 1.

Figure 1: Map of sites

Lab Methods- Question 2: Aggression

To test our kinship hypothesis, individuals found in clumps and solitary were brought into the lab where trials were conducted. We defined a clump as a group of three or more anemones 0.1 meters away or less from one another. See Figure 2. We used aggression as a proxy of relatedness. Three were taken from a single clump and one individual from a farther distance within the 8-meter section. All anemones were placed on their own rock outside of the water and given five minutes to settle. They were given 8 hours by themselves in the tank to adjust. At night, two anemones from the clump were placed next to each other in a dish as control. One

3 anemone from the clump and another found from a farther distance were placed together in a separate dish. They were left overnight, and interactions were recorded by a GoPro time lapse. The diameters of each individual were recorded and anemones were paired by similar size. Fights, as well as number of hits, were recorded from the video the next morning. We defined a successful fight as when one anemone would flash, extend, and reach with it’s acrorhagi to attack the other anemone. We counted the number of times this happened for each anemone and recorded it as a hit. Every day, new anemones from one of the 8 coves were collected and new trials ran each night. To quantify the level of aggression, we counted the ‘fights’ between anemones and categorized them binomially as ‘yes’ or ‘no’. The test we ran for this data was a chi-square test. The test we ran for the number of hits was a one-tailed t-test.

Figur e 2: anemones in a clump: 0.1 meters away or less from one another.

Question 3: Resource Availability

Skin divers and kayakers were used to test our hypothesis on resource availability. Skin divers would locate clumped and unclumped anemones within the same four coves to the North and South of the harbor. Plankton samples were collected with a 50-micron net. The plankton net was ran through the water six times back and forth at the same speed each time next to clumped and unclumped anemones. The plankton samples were taken back to the lab where further analysis was done. Using a dissection microscope, plankton was counted and classified into small, medium, and large groups. We found in all three categories that there was a correlation between high plankton count and clumped anemones. To interpret this data, we ran a factorial ANOVA, which analyzed location as clumped or unclumped, and size as small, medium, and large.

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Results: Distribution of Actinia schmidti:

The results of our surveys show the geographical spacing of A. schmidti within each of the four coves to the North and South of the STARESO harbor. This is depicted in Figure 3 and 4. We found that the distribution of A. schmidti was clumped because the variance to mean ratio for all North and South coves were greater than 1, as shown in Table 1.

Figure 3: A scaled map of anemone distribution within North coves 1-4. Each circle represents an individual anemone and the size of the circle is scaled to the size of the anemone.

5 Figure 4: A scaled map of anemone distribution within South coves 1-4. Each circle represents an individual anemone and the size of the circle is scaled to the size of the anemone.

Table 1: The value of the variance to mean ratio of all coves is greater than 1, indicating the distribution of A. schmidti from the North and South are clumped.

Aggression:

Fights- We found that clumped anemones fought less than anemones found farther away. The p-value of the mosaic bar graph was 0.0091. Figure 5 shows that clumped anemones had no fights, whereas the unclumped anemones fought about 25% of the time. The width of the bars indicates the sample size and the line on the right is the overall percent.

Figure 5: mosaic bar graph depicts the percentage of fighting between clumped and unclumped anemone pairs.

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Hits- We found that unclumped anemones hit each other more than clumped anemones. The p- value of the second bar graph was 0.0234. Figure 6 is an indication of the number of hits per individual in those categories. A video of these aggressive interactions from our time lapse GoPro is included below.

Figure 6: bar graph shows the mean number of hits occurring between clumped and unclumped anemone pair

https://www.youtube.com/watch?v=hQGtnyO5bN8 Figure 7: video of fight trial

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Resource Availability:

We found that there were more resources next to clumped anemone areas. The p-value for plankton was 0.044, therefore, there is sufficient enough evidence to warrant the rejection of the null hypothesis. Looking at the relative heights of each color (small, medium, large), there is a noticeably large difference in the amount of plankton near clumped individuals than near unclumped individuals.

Figure 8: bar graph depicts the amount of plankton found in areas of clumped and unclumped anemones, categorized by size of

Discussion: From field observations and lab experiment data taken over the course of four weeks at the STARESO field station in Calvi, France, we were able to indicate the presence of clumped distribution of A. schmidti. It’s a common phenomenon in some anemone species that reproduction can occur both sexually and asexually. Sexual reproduction in sea anemones occurs through a planktonic larval stage, while asexual reproduction occurs through various forms of fission (Shick 1991). In previous studies, A. schmidti was found to most commonly reproduce asexually (Rudin & Briffa 2011). One individual containing two different 28S gene sequences had eight offspring with identical patterns indicating a high probability of asexual reproduction. The probability of eight offspring containing the same gene pattern as the parent

8 via sexual reproduction would be 0.39% (Rubin & Briffa 2011). This evidence caused us to believe that anemones would be in a clumped distribution due to kinship. Another possibility for anemones to result in a clumped distribution could be an availability of resources.

The results from our field portion made it clear that Actinia schmidti forms a clumped distribution along the North and South coves. These coves were chosen in order closest to the STARESO harbor. All eight coves indicated a clumped distribution from to a variance to mean ratio greater than one. Under dispersion patterns, the high numbers of variance to mean ratios suggests that anemones tend to clump together.

We mirrored studies done on A. equina to determine the extent of aggressive behavior (Cadeireiro & Robalo 2016). Once the anemones were collected, paired, and tested, we found that our results supported our hypothesis. All defined fights occurred between anemones that had greater distance than 0.10 meters and was considered the ‘farther’ anemone pair. We originally tried to quantify aggression using number of hits since we believed clumped anemones would have fewer hits than unclumped anemones, but there were zero hits in clumped anemone pairs. Unclumped anemones solely displayed their acrorhagi. We believe the lack of aggression between clumped anemones could result from these anemones being a product of asexual reproduction.

Another possibility for A. schmidti forming clumped distributions could be the availability of resources. In all eight coves, there was a higher plankton presence in areas where anemones aggregated than areas with independent anemones. The results of our plankton count solidified our hypothesis that A. schmidti would form a clustered distribution centered around an abundance of resources.

With our data supporting both our hypotheses, we suspect that anemones may form a clumped distribution in association with kinship and resources. It is a strong possibility that anemones reproduce in areas that have higher resources, therefore, there are more clumps in these sites. Another possibility for non-aggressive interactions during lab experiments between clumped anemones could be that they have been accustomed to living in close proximity. Therefore, clumped anemones would not have felt threatened in the presence of other anemones. These areas where anemones aggregate could have a more desirable habitat due to less desiccation, less wave erosion, and less light, etc. Anemones from certain areas could have less resources than anemones paired with higher resources, which could have provoked the aggressive interaction between unclumped anemones. At STARESO, we lacked the tools to sequence the DNA of A. schmidti found from various distances, thus a relationship between anemones had to be assumed but never proven through sequencing. In effort to try to measure kinship, we then used aggression as a proxy.

As noted in the introduction, the distribution of individuals can indicate the processes that are affecting the species. In this study, we aimed the identify and explain the distribution and possible causes in A. schmidti. We found the mechanisms driving distribution in A. schmidti to be kinship, using aggression as a proxy, and resources. Similar aggression results were found in A. equina; therefore, this study can relate to other sessile organisms and their possible distribution. This study is most applicable to sedentary organisms that have specific reproduction systems that result in local dispersion of larvae such as crawl away, budding, and fission. Mobile animals are able to move freely towards resources, however sessile organisms have limited mobility, therefore it is likely that for these organisms, reproduction has a stronger effect on distribution than resources.

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Future studies: We propose to sequence multiple A. schmidti found in various distances to analyze relatedness and compare with the amount of aggression displayed in each distance. We believe that the anemones that did not show aggression would be a result of asexual reproduction and have clonal DNA sequences. Anemones that demonstrated more aggression would have different nucleotide sequences and would not be clonal. Additionally, we believe further studies could be done with interspecific aggression between A. equina and A. schmidti since A. schmidti was believed to be the same species as A. equina until recently. Under our hypothesis, these two species would demonstrate aggressive behavior due to the two species having no evidence of kinship. Lastly, we propose a study to investigate how A. schmidti is able to detect if the paired anemone is clonal or not.

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