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Trophic ecology of in an intertidal community: spatial and functional variability in the importance of primary producers

Sarah Campbell Lummis

May 2012

Trophic ecology of invertebrates in an intertidal seagrass community: spatial and functional variability in the importance of primary producers

An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY

by

Sarah Campbell Lummis May 2012

2

Trophic ecology of invertebrates in an intertidal seagrass community: spatial and functional variability in the importance of primary producers

by Sarah Campbell Lummis

Approved for submittal to the Department of Biology for consideration of granting graduation with honors:

Research Sponsor Professor Fiorenza Micheli ______Date ______(signature)

Second Reader Professor Rodolfo Dirzo ______Date ______(signature)

3 Acknowledgments

This thesis would not have been possible without the dedicated mentorship of Dr. Steve Litvin, a post-doctoral fellow in the Micheli lab. His is an amazing teacher, and this project would not have been possible without his thoughtful advising. Thank you so much for all your guidance!

I would also like to thank Professor Fiorenza Micheli for her advising and support throughout this project. The lab community she creates is so conducive to research and learning, and made the summer of data collection a blast! Additionally, I am very grateful to Professor Rodolfo Dirzo for his mentorship over the last three years, and for his feedback on this project at its many stages.

Within the Hopkins community, I am very thankful to Dr. Jim Watanabe for his help in identifying the for my project, and his website ‘Seanet’ which was a great starting point for getting to know the intertidal community around Hopkins Marine Station. Additionally, I would like to thank Dr. Sarah Lee for her help in identifying the amphipod species.

This research was generously funded by the Vice Provost for Undergraduate Education’s Major Grant program, run out of the Undergraduate Advising and Research office.

4 Table of contents

List of tables and figures ...... 6

Abstract ...... 7

Introduction ...... 8

Methods and Materials ...... 15

Results ...... 20

Discussion and Conclusions ...... 23

References ...... 27

Appendices ...... 31

5 List of tables

Table 1. Summary of study

Table 2. Summary of t-test results comparing producers between sites

Table 3. Mean carbon and nitrogen values for all organisms in site A

Table 4. Mean carbon and nitrogen values for all organisms in site B

List of figures

Figure 1. Map with two study sites

Figure 2. Dual isotope plot for producers in both sites

Figure 3. Dual isotope plot for all organisms in site A

Figure 4. Dual isotope plot for all organisms in site B

Figure 5a and 5b. Percent contribution of producers for T. paleacea in sites A & B

Figure 6a and 6b. Percent contribution of producers for P. frequens in sites A & B

Figure 7a and 7b. Percent contribution of producers for O. lighti in sites A & B

Figure 8a and 8b. Percent contribution of producers for P. agassizii in sites A & B

Figure 9a and 9b. Percent contribution of producers for L. singularisetis in sites A & B

Figure 10a and 10b. Percent contribution of producers for N. dendritica in sites A & B

6 Abstract

This project aimed to understand the relationship between marine invertebrates associated with the intertidal seagrass Phyllospadix scouleri, and the greater intertidal community. I hypothesized that individual species would show distinctive feeding ecologies due to differences in proportional utilization of primary producer organic matter. I also predicted that species would show shifts in their resource utilization between the wave-protected and wave-exposed sites. Stable offered a method for determining their food sources, since they are difficult to observe.

Combined with the mixing model analysis completed by the program MixSir, I was able to track both which food sources were utilized, and whether this varied with location, for each invertebrate. I found that all the consumers relied on a variety of food resources, appearing to be generalists rather than specialists, and that they utilized the resources differentially based on the location they were collected from. This likely derives from the differences in wave exposure of the two sites, and the change in the bioavailability of organic matter as a food resource between the two sites studied.

7 Introduction

The habitat

Seagrasses are an incredibly interesting and unique group of organisms. Whereas the majority of the plant diversity in the is represented by algal species (i.e. kelp, intertidal , single-celled algae of the open ocean), are an ecological group of four vascular plant families that re-colonized the ocean approximately 100 million years ago (Larkum et al (eds) xiii). They are angiosperms, flowering plants that anchor themselves firmly into the substrate to establish themselves in well-lit shallow regions, with roots often penetrating directly into rock. Seagrass are home to an enormous diversity of flora and fauna, and are an important nursery habitat for many and (Hemming 2000, Heck 2003). Their importance as a habitat is largely due to three particular characteristics of the grasses themselves: i) blade-like leaves that lessen strong current, ii) strongly anchored root systems that accumulate sediments, and iii) high rates of , which helps maintain the habitat and serves as a food resource.

Species of the Phyllospadix genus, which this study focuses on, are found within northern temperate regions on the western coast of North America (den Hartog 1970).

They have a latitudinal distribution from Baja California to the west coast of Canada, and a physical habitat range of mid-intertidal down to 20 m below the intertidal region. In order to receive adequate nutrients, these grasses are found in areas with high levels of wave action and/or currents, and their roots are able to penetrate directly into granite bedrocks in order to withstand the large forces.

8 Currently there are numerous concerns about seagrasses, the threats they are faced with, such as habitat destruction, pollution, and climate change, and the indirect impacts this could have on the wide variety of organisms that rely of them as habitat or food for at least a portion of their life cycle (Hemming and Duarte 2000). Yet despite the importance of seagrasses as a habitat and their wide distribution, relatively little is known about the specific communities that they support, and how these organisms will be affected by future changes to their habitat.

The community

Among the many inhabitants of Phyllospadix seagrass beds are a wide variety of invertebrates. These include small grazers, ‘mesograzers’, less than 2cm in length, as well as a number of species of detritivores, who to consume decaying organic matter from wide range of primary producers (i.e. detritus) (Heck 2006). This community of small invertebrates has a diverse range of feeding habits: they may eat the seagrass, remove epiphytes from seagrass blades, or breakdown drift algae that becomes entangled in the seagrass. In turn, there may be a wide variety of impacts from their feeding on the they inhabit, ranging from increasing growth rates of the seagrass by removing epiphytic algae that shade blades and compete for nutrients, to recycling allochthanous organic matter, such as detrital algae and kelp, that gets washed into the system (Hori

2006). The influence that an invertebrate can yield on a community based on its feeding ecology, , and impacts on the ecosystem constitutes its ‘functional role’, which yields insights into its interactions with other trophic levels. Mesograzers, though they are fairly small organisms, are thought to have highly significant functional roles

9 within seagrass ecosystems, and may play key roles in removing harmful epiphytic algae from seagrass blades (Heck and Valentine 2006, 2003). Other studies have found that detritivores, such as , generally have high levels of functional diversity, including omnivorous behavior, and play an important role in recycling nutrients back into the foodweb (Bostrom et al. 2006, Summerson and Peterson 1984). Yet, because of their size and location in the intertidal, it is impossible to observe the natural feeding habits of these invertebrates, and just as challenging to dissect their tiny stomachs to examine their gut contents. Because of this, it is much more practical to turn to other types of analysis to understand their feeding ecology.

Stable isotopes in ecology

Stable isotope analysis is an ideal tool for understanding trophic relationships when in situ observations are impossible due to the size of the grazers or the location of the study site. This type of analysis is a common tool of ecologists who wish to map out food webs, determine migration patterns, or establish pathways of nutrient flow within ecosystems (Kosciuch et al. 2008, Young et al. 2010, Fry 1981). Generally, studies focus on analyzing carbon, nitrogen, sulfur, oxygen, and/or hydrogen, as these are the most biologically abundant and important elements for living creatures. My analysis will be conducted using stable isotopes values for carbon (12C/13C) and for nitrogen (14N/15N).

For both C and N, the delta (δ) values for each sample are calculated using the standard equation:

!"#$%!" δ = − 1 × 1000 !"#$%&$'&

10 where R is the ratio between the heavy and light isotopes (Fry 2006). This δ value for both isotopes represents the isotopic composition by indicating either the depletion or enrichment of the heavy isotope relative to the lighter one. For my study the feeding ecology of the grazers and detritivores is examined by comparing the isotopic signatures of the primary producers to the isotopic signatures of each of the consumers.

Previous studies of invertebrate grazers

Historically, the intertidal invertebrate community was assumed to simply eat the plant matter they inhabited, which would be seagrass for the community of mesograzers

(Sullivan and Moncreiff 1990). For polychaetes, this would have been particles of detrital seagrass. However in the last few decades, studies using stable isotope analysis have shown that among the wide range of organisms that were once thought to consume primarily seagrass, there are an equally diverse number of functional roles (Farlin 2010,

Heck et al. 2006, Moncreiff and Sullivan 2001, Sullivan and Moncreiff 1990). Different taxa were found to selectively consume different combinations of food sources, including the grasses they inhabit, epiphytes (algae and bryozoans), allochthonous plant matter from outside the ecosystem (kelp, other algae), and suspended particulate matter (POM) in the surrounding water.

Several studies have emphasized the importance of algae as a food resource for deposit feeders in seagrass ecosystems, either in a particulate form or as microphytoplankton (Fry 1984, Danovaro 1996). Other studies using nitrogen isotope analysis have identified a number of grazers as omnivorous, eating both plant matter and other smaller mesograzers (Farlin 2010, Dunton and Schell 1987). However, there has

11 been relatively little work undertaken to understand how the functional roles of invertebrates vary within a community, and across habitats. Isotopic analysis would allow a more complete picture of the collective functional role of invertebrates in the

Phyllospadix community and how these roles vary with each other.

Complementary studies

This study complements ongoing research by the Environmental Venture Project

(EVP, Woods Institute) titled: “Biophysical interactions in a near-shore kelp ecosystem:

Observations and implications for monitoring and design in marine protected areas". It extends the understanding of how multiple organic sources support production in near shore ecosystems from the (the focus of the EVP project) into the , where seagrasses are the dominant macrophytes.

In addition, this work supports the goals of the Hopkins Marine Station Marine

Life Observatory (http://mlo.stanford.edu/), including the evaluation of diversity and abundance in near shore ecosystems along with quantification of important ecological processes within the Hopkins Refuge (HMLR), the second oldest marine refuge in California (Denny 2011). I hope to build on these studies, and add to the growing body of knowledge on HMLR and the communities within.

Research questions

I will investigate i) the functional role of each invertebrate species through isotopic analysis of their food sources and ii) how this functional role varies over a wave exposure gradient. I hypothesize that, across this invertebrate community, there will be a large

12 variety of feeding habits, and therefore functional roles, for each species. I also predict that the feeding habits of the mesograzers and detritivores will vary between the two sites with differential wave exposure, as different food resources are brought into the area or made more readily available.

Significance

This study begins to characterize a community of mesograzers and detritivores in the intertidal seagrass habitat whose feeding ecology has never been quantitatively studied before, despite its location within the long-standing HMLR. It also aims to understand the functional diversity of this community and analyze how this varies with wave exposure in the seagrass Phyllospadix.

It builds on previous work with mesograzers and detritivores and characterized the functional diversity of a select portion of the invertebrate community within the seagrass Phyllospadix at the HMLR. Although functional diversity of mesograzers and detritivores has been examined in other seagrass ecosystems before (Heck et al. 2006,

Moncreiff and Sullivan 2001, Bostrom et al 2006), it has not been looked at for communities residing in Phyllospadix, one of the most abundant seagrasses on the

Western coast of North America. As the feeding ecology of invertebrates in this community has yet to be examined for this ecosystem, it represents a significant gap considering the long-term historical importance of the HMLR.

The second portion of my study, understanding how functional roles change across different physical environments, has yet to be examined for a community invertebrates within a seagrass ecosystem. This natural comparison between two

13 different habitats is a common tool for ecologists, as it allows researchers to examine whether certain biological characteristics vary with changing physical conditions

(Underwood and Jernakoff 1984). My project yields insights into whether an invertebrate’s utilization of food resources can be impacted by physical conditions, and the potential for functional variation of each group of organisms due to physical changes across short distances.

14 Materials and Methods

Study site

This study was conducted at Hopkins Marine Station in Monterey, CA, within the rocky intertidal ecosystem of the Hopkins Marine Life Refuge. Within this intertidal region, samples were collected from two specific seagrass beds- ‘Site A’ and ‘Site B’

(Fig. 1). Site A was located within a protected, relatively shallow channel between the shoreline and the well-known ‘ rock’. Site B was located slightly to the south of site

A, in a small cove that had greater exposure to wave action and a steep slope from the shallow mouth of the cove out towards the open ocean. Both sites had large boulders on the sandy bottom, with large outcrops of granitic rock.

Study organisms

A subset of the invertebrate community found in the seagrass beds around

Hopkins was selected for this project based on both by their association with P. scouleri and whether their abundance was great enough to obtain an adequate sample size for isotopic analysis. Through these criteria, the community was narrowed down to one limpet (Tectura paleacea), two amphipods (Protohayale frequens and Oligochinus lighti), and peanut (Phascolosoma agassizii), and two polychaetes (Lumbrinereis singularisetis and Naineris dendritica). The primary producers were selected based on the likelihood that they were used by at least one of the organisms as a significant organic source, and by their abundance in the community. All together, three food sources were collected and analyzed across the two sites: the seagrass Phyllospadix scouleri, the epiphytic red algae Smithora nadium, and the most common intertidal algae Mastocarpus

15 papillatus. A second species of seagrass, Phyllospadix torreyi, was also collected from site B (it was not present in site A). P. torreyi was collected and processed to examine whether its presence provided an isotopically different food resource from P. scouleri.

Field collection

P. scouleri and P. torreyi were collected by haphazardly selecting a handful of blades, digging out their associated roots, and placing the plant into a mesh bag. The S. nadium epiphyte was collected through the same method, as it was present on almost all seagrass blades to some degree. M. papillatus was collected by hand from large boulders directly adjacent to the seagrass beds.

The two amphipod species were collected using small, narrow-meshed hand nets, during low tides. The nets were skimmed just below the surface on top of the seagrass beds, which were rinsed from the nets directly into plastic bags in the field. T. paleacea was collected by hand from individual seagrass blades in the field- it was sparse enough in its distribution that it could not be collected from the P. scouleri samples, although its habitat is limited to the blades of this seagrass. Both polychaetes species and P. agassizii were collected from within the root mass that was removed with the P. scouleri blades.

Identification of organisms

P. scouleri and P. torreyi were distinguished by referencing ‘The Genus

Phyllospadix (1893)’, and the ‘Seanet’ online database of organisms found within the rocky intertidal at HMLR (seanet.stanford.edu). Both S. nadium and M. papillatus were identified using ‘Seanet’, and with guidance from J. Watanabe. The amphipods and polychaetes were identified using ‘The Light and Smith Manual’ (Light et al. 2007), and

16 through consultation with S. Lee and J. Watanabe. The limpet T. paleacea was identified through ‘Seanet’, as was P. agassizii, the peanut worm.

Processing samples

Both P. scouleri and P. torreyi samples were carefully scraped with a scalpel and rinsed with deionized water to remove epiphytes. Younger blades with a lesser density of epiphytes were chosen. S. nadium was removed from blades with a denser epiphytic community, and also rinsed with DI water. M. papillatus was rinsed with DI water to remove any small invertebrates within the blades. All producer samples were then rinsed with 10% HCl to remove any encrusting algae and then dried, ground, and prepared for analysis (following Weinstein et al. 2000).

The consumers were all rinsed with DI water once collected. The amphipods required sorting, as they were collected together, and were then pooled to obtain sufficient for analysis. P. frequens required roughly 50 individuals per sample and O. lighti required roughly 90 individuals per sample. For both species of polychaete, samples were amassed by pooling 2-3 individuals for each sample. T. paleacea was removed from its shell using a scalpel, and then dissected to remove all organic matter from their gut. Samples required 3-5 individuals to obtain sufficient biomass. None of the consumer samples were rinsed with HCL, based on de Lecea et al. (2011), which indicate that the acid rinse negatively affects the quality of isotopic analysis for small invertebrates.

17 Stable isotope analysis

First the scale was calibrated for the weight of an individual tin capsule. Then, from each pooled sample, 2.0 ± 0.1 µg of biomass was weighed into the capsule, contained by folding in the sides of the capsule, and placed into a sample tray. Once all the samples were boated following this method, they were sent to the Stable Isotope

Biochemistry Laboratory (SIBL) at Stanford to be analyzed for carbon and nitrogen stable isotopes. All samples were run through a Thermo Finnigan Deltaplus mass spectrometer. This is a continuous flow instrument that uses ConFlo II open split interference which is coupled to a Carlo Erba NA1500 Series 2 Elemental Analyzer.

This type of analyzer uses flash combustion analysis for organic matter samples, yielding the delta values (differences between the isotopic ratios of sample readings and the standards), and absolute ratios of nitrogen and carbon within each sample. The precision for both the N and C isotope values is typically <0.15‰, and <1% for N and C concentrations. The samples were measured against reference samples (USGS40 and

Cond. AA) that were considered to be isotopically similar to the community of producers and consumers collected in this study.

Statistical analysis

A student’s t-test was run for P. scouleri and P. torreyi to determine whether the two species of seagrass were isotopically distinct from one another, or whether they were isotopically similar and could be pooled for further analysis. The all three of the primary producers were also tested to see whether they were significantly different from each other with respect to their δ13C values, and whether this difference was consistent between the two sites.

18

Mixing model analysis

Dual isotope plots for δ13C vs δ15N were constructed to determine the importance of an individual organic source to each invertebrate taxa. The mixing model “MixSir” was run for each consumer to further estimate the proportion of each mesograzer’s diet that was derived from the three organic sources (Moore and Semmens 2008). This program uses a Baysian analysis to determine the probability of a large number mixing models generated for stable isotope data that uses a sampling-importance-resampling

(SIR) method (Moor and Semmens 2008). The program was run at 1,000,000 iterations, meaning it tested 1,000,000 hypothetical mixing models, to achieve a high level of accuracy. It also takes into account uncertainty in estimates of isotope values for both the consumer samples and for the producers.

19 Results

Stable isotope results

Since the two Phyllospadix species were not significantly different with respect to both their nitrogen and carbon values (p > 0.2 for both isotopes), the samples of both species pooled together as P. spp for all further analysis. This only affected the exposed site (site B), as P. torreyi was not present in the protected site (site A).

The results of the stable isotope analysis are summarized in Figs 2-4. Figure 2 shows just the primary producers for the system, Figure 3 shows the producers and consumers from site A, and Figure 4 shows the producers and consumers from site B.

Each producer showed differences in δ13C between the two sites, with both S. nadium and

P. spp shifting to a more depleted signature from the protected to the exposed site while

M. papillatus shifted to a more enriched δ13C value in the exposed site (Figure 2).

Because the isotopic means of each producer were significantly different between the two sites, consumers from site A were compared against producers from site A while those from site B used producer data from site B for the subsequent mixing model analysis

(Table 2). By using site-specific values for the producers to determine the food resources used by each consumer, the natural variation in producers was fully accounted for. Any differences between an ’s mixing model results could now be attributed to differences in feeding habits, rather than differences in isotopic composition of the same food source.

The median δ13C and δ15N values for each organism are summarized in Table 3 for protected site and in Table 4 for the exposed site. Figures 3 and 4 also show the isotopic signatures for both the consumers and producers for the protected and exposed

20 sites, respectively, and highlight several trends. In terms of carbon signatures, all the organisms were within the range of the values of the primary producers. The amphipod

P. frequens showed the most depleted δ13C signatures across both sites (-17.47 ‰ ±0.33 and -19.49 ‰ ±0.20), and was closest to the carbon values of S. nadium. The other species of amphipod, O. lighti, was less depleted in δ 13C for both sites.

For nitrogen, the polychaete L. singularisetis showed the most enriched δ15N signature (13.22 ‰ ±0.54 and 14.05 ‰ ±0.98). However, the other two deposit feeders,

N. dendritica and P. agassizzi, were both enriched in nitrogen values relative to the three mesograzer species. All consumers showed changes in their δ13C and δ15N values between sites, with the largest change seen in T. paleacea and the smallest one seen in P. agassizii. Looking at carbon signatures specifically, the most variability was found in the mesograzers, while the carbon signatures of the deposit feeders were more consistent between the two sites.

Mixing model results

For each consumer, the most likely combination of the producers in its diet is given by the mean value of iterations of the MixSir program (Table 5). First, within the mesograzer species, the red epiphyte S. nadium was barely utilized by T. paleacea as a food source in the protected site, yet it represented the largest dietary contribution for the limpets in site B. In turn, the utilization of both M. papillatus and P. spp decreases noticeably from the protected to the exposed sites. The second mesograzer species, the amphipod P. frequens, also uses slightly more S. nadium in the exposed than in the protected site, and contributions of M. papillatus and P. spp decreases slightly in the exposed site as well. In contrast, O. lighti, the other amphipod species, used less S.

21 nadium in the exposed than in the protected one. M. papillatus was used to a greater extent in the exposed site than in the protected area, and the contribution of P. spp remained close to constant. The detritivores had slightly different dietary shifts. In both sites, P. agassizii derived most of its diet from the intertidal algae M. papillatus, although it used slightly more S. nadium and P. spp in the exposed areas. For L. singularisetis the mixing model indicates a dramatic shift between the two sites: the vast majority of this polychaete’s diet was derived from M. papillatus in the protected area while almost all of the diet was thought to be from Phyllospadix spp. in the exposed site. The second polychaete species, N. dendritica, was broader in its resource utilization. For the protected site, the model indicates that its diet was equally derived from M. papillatus, S. nadium, and Phyllospadix spp. In the exposed site, M. papillatus was the primary food resource, while the other two had small contributions. Generally, the mesograzers (the amphipod species and P. agassizzi) seem to exhibit less variation between sites than the detritivore species (Table 5). Another interesting outcome is that this model shows all species to be generalists that rely, to varying degrees, on all three food sources, both from in situ grazing and allochthonous input.

22 Discussion

Based on the results of my isotopic and mixing model analysis, I have found that while there is variability in the functional role of each species of consumer, there are clear distinctions between the mesograzer species and the detritivore species. Generally, the mesograzers show less variability in their food sources than the detritivores. They are also much less enriched in δ15N than the detritivores.

I also found that the food resources utilized by the consumers differed for all organisms between the two sites, in terms of their δ13C and δ15N values. While this was anticipated for the consumers, it was surprising to see that the primary producers also had distinct isotopic signatures between the two sites. I found that all three of the producers displayed significant shifts in carbon isotopic values. S. nadium and Phyllospadix spp. showed greater δ13C depletion in the exposed site than in the protected one, while the intertidal algae showed enrichment in δ13C in the exposed site. Studies focusing on the physical effects of water motion on seagrasses have shown that waves can play a role in disturbing the diffusive boundary layer around the blades of grass, which affects the plant’s rate of uptake for dissolved organic carbon within the (Denny and

Wethey 2001, James and Larkum 1996). Differences in wave exposure of the two sites may account for the differences in isotopic values of the seagrass, which in turn affects the isotopic values of the epiphyte and the intertidal algae.

Yet even after the shifts in isotopic values of the producers were taken into account by using site-specific mixing models, the consumers still showed differences in their resource utilization between sites (as indicated by the mixing model results). The results from running MixSir for each consumer in each site further solidified the changes

23 in feeding habits that are present between the two sites. The reason for these changes in resource utilization of invertebrate mesograzers and detritivores likely results from the differences in the physical location of the sites. The two sites are at approximately the same height within the intertidal (roughly 0.5m below mean tide level). They also present very similar substrates for the seagrasses to anchor their roots in, and for the algae to cling to. These characteristics, combined with the fact that they are 20-25m apart from each other, make it both interesting and unexpected that producers in these two locations are isotopically distinct. However, the difference in wave exposure levels between these two sites which likely accounts for the dietary shifts of the consumers is also likely to play a role in the distinguishing producers between the two sites. Although it was known that areas with different physical conditions present different food resources, especially in considering particulate detrital matter (Murphey and Fonseca 1995), it is interesting to see the extent of the differences of resource utilization between the two sites.

The most dramatic changes were seen in the limpet, in O. lighti and in both the polychaete species. This indicates that either the two sites have different resources or that the resources vary in their availabilities or abundances between sites. For the polychaetes, which feed on organic matter found within the sediments, the size of particles can limit their availability as individual sizes must be small enough to fit into an organism’s mouth. Areas with higher levels of wave energy have a greater potential to break down organic matter, making it biologically “available.” For example, in the area of high wave exposure (Site B), each polychaete species increase its foraging on a particular food resource. L. singularisetis utilized M. papillatus to a great extent, while N. dendritica utilized Phyllospadix spp. to a greater extent. This could indicate for

24 both organisms that this food resource is not available in the protected area, but is accessible in the exposed site. There is also another possibility for L. singularisetis. Its enriched δ15N values indicate that it might be omnivorous, as nitrogen enrichment is known to occur as organisms move up in trophic level (Fry 2006). Its position within the dual isotope plot matches results from Farlin et al (2010), which indicated an omnivorous amphipod species.

However, some of the results given by the mixing model are not necessarily ecologically reasonable. Because M. papillatus and Phyllospadix spp. are isotopically very similar, the model will assume that consumers with similar carbon signatures to these two producers derive nutrients from both of them. The most distinct example of this is seen with the limpet. For both sites, the mixing model indicates that a significant portion of its diet coming from the intertidal algae, as well as from the red epiphyte and the Phyllospadix. However, as an obligate symbiont with the seagrass Phyllospadix scouleri, it is incredibly unlikely that it has access to any resource outside of the seagrass itself and the epiphytes that grow on it. Even if blades of M. papillatus were to get caught in the seagrass blades, this limpet, which is only a few millimeters long, would have no way of accessing them without leaving the seagrass and risking getting washed out to . This result may be an artifact of the MixSir model that was used, which assigns a probability for the use of a food resource without regard to how ecologically possible the result may be. If this resource were excluded, the limpet would appear to divide its resource utilization between P. scouleri and S. nadium.

In general, I have found that the ecological roles of each mesograzer may vary across different areas as they are exposed to different physical conditions and different

25 food resources becoming available. This means that the effects of invertebrate feeding ecology may be different in communities that are separated by even small distances if these areas are exposed to different physical conditions. This indicates the potential for differential impacts of functional roles ranging from epiphyte removal to nutrient recycling. Generally, this study highlights the necessity of considering the effects that physical factors such as wave exposure have on resource utilization and functional role, and how these might vary with changes in the physical environment over a smaller scale.

Future work will include running samples for sulfur isotopic analysis to aid in distinguishing between Phyllospadix spp. and M. papillatus. By adding an additional layer of isotopic data, we will gain further resolution between the individual food sources. It may also be helpful to include other food sources in the analysis, including offshore kelp, as previous studies have indicated that food resources may include allochthanous material that comes into the system as detritus (Moncreiff and Sullivan

2001, Sullivan and Moncreiff 1990). In the next few months, we are also planning to collaborate with researchers in the Koseff lab to collect site-specific data that will quantify the differences in wave strength, frequency, and turbulence between the two sites. With these data, we are hoping to model how different conditions for carbon uptake are affected by differential wave action, and how that changes the boundary layer of water surrounding individual seagrass blades.

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30 Appendices

Data tables

Table 1. General description of each of the mesograzers, including their identification codes used for analysis, taxonomic information, common name (if applicable), description, and typical habitat range. This data was collected from Light, Carlton, and Smith 2007 and from the online database ‘Seanet’, compiled by J. Watanabe. ID General Latin name Description Typical habitat code taxonomic group Limp Gastropod Tectura Small limpet, 5-10mm On the blades of paleacea shell length seagrass, generally around the middle of blade Amp1 Amphipod- Protohyale Uneven antenna Phyllospadix roots and Hyalidae frequens (A1

Amp2 Amphipod- Oligochinus Antenna ~even, ovular Within blades of Calliopiidae lighti eyes, thinner/longer urosomites, bodies smaller and redder than Protohyale. Peanut Sipunculids Phascolosoma Deposit feeder with Within Phyllo. agassizii small tan/brown holdfasts bulbous body

Poly1 Polychaete Lumbrineridae Large segments, Within roots of - Lumbrinereis minimal dorsal ventral phyllospadix singularisetis distinction, *distinct setae confirm Poly2 Polychaete Orbiniidae- Small segments, Within roots of Naineris dorsal cirri form phyllospadix dendritica channel, head setae Phyllo Angiosperm Phyllospadix Blades are flat and 2- Low intertidal to scouleri 4mmn wide, >1m subtidal long, and frequently covered with epiphytes Tory Angiosperm Phyllospadix Blades narrower than torreyi P. scouleri (<2mm), Generally subtidal, with a more just barely into rounded/cylindrical intertidal shape. Also covered

31 with epiphytes. RE Rhodophyta Smithora Thin, purplish-red On the upper portion naiadum blades, has a small of Phyllo. blades. holdfast used to attach Obligate seagrass to Phyllo. epiphyte. MAS Rhodophyta Mastocarpus Variable High to mid intertidal papillatus of blades, ranges from zones, frequently dark brown to reddish dominant algae on black both protected and exposed coasts

Table 2. Comparison of the mean carbon values of the three primary producers between the two sites. All three primary producers were significantly different from each other. Type Carbon t statistic Carbon p-value M. papillatus 3.23 0.01 S. nadium 8.84 <<<0.01 P. spp 4.90 0.001

Table 3. Means and standard deviations for both δ13C and δ15N values, for all organisms collected from site A TYPE NITROGEN CARBON --- mean STDEV mean STDEV Amp1 9.30 0.268 -17.47 0.325 Amp2 10.71 0.094 -16.14 0.242 Limp 11.33 0.401 -14.18 0.652 Poly1 13.22 0.548 -17.12 0.676 Poly2 10.59 0.244 -16.83 0.405 Peanut 12.13 0.423 -15.31 0.393 MAS 9.67 0.245 -14.72 0.337 RE 9.09 0.278 -19.93 0.426 Phyllo 8.78 0.521 -13.20 1.108

Table 4. Means and standard deviations for both δ13C and δ15N values, for all organisms collected from site B. ‘Tory’ (P. torreyi) was only collected in site B TYPE NITROGEN CARBON mean STDEV mean STDEV Amp1 8.95 0.261 -19.49 0.203 Amp2 11.03 0.224 -15.55 0.303 Limp 10.21 0.442 -18.60 0.554 Poly1 14.05 0.988 -17.24 1.289 Poly2 12.07 1.402 -16.04 0.501 Peanut 11.92 0.344 -15.32 0.263 MAS 9.77 0.401 -13.72 0.917 RE 8.98 0.326 -21.30 0.242 Phyllo 9.12 0.731 -15.42 1.509 *Tory 8.44 0.626 -16.08 0.783

32 Table 5. Summary of the mixing model results for each organism, with the percent contribution of the three primary producers. The ‘Phyllo’ percent contribution is given by combined P. scouleri and P. torreyi values for site B, as the two species were found to be isotopically indistinguishable Type MAS % contribution RE % contribution Phyllo* % contribution LimpA 28.1 1.9 69.4 LimpB 28.0 46.4 25.7 Amp1A 23.4 45.5 30.7 Amp1B 14.7 58.5 26.9 Amp2A 53.6 19.8 26.6 Amp2B 65.8 7.5 26.6 PeanutA 86.1 1.9 11.6 PeanutB 79.9 6.8 12.5 Poly1A 74.1 25.0 0.7 Poly1B 1.3 1.5 96.7 Poly2A 45.1 31.4 23.5 Poly2B 78.6 15.9 4.5

33 Figures

Site B Site Bird A Rock

Figure 1. Map showing the location of the study sites. Site A is located in a protected channel of water that passes in front of Bird Rock, and site B is located further west along the shoreline in an exposed cove, as the prevailing winds come from the north. This map was adapted from one in Dr. Smith’s Phd dissertation (1983).

Figure 2. Dual isotope plot showing the carbon and nitrogen values of the three producers, M. papillatus, S. nadium, and Phyllo. spp., and comparing them between both sites.

34

Figure 3. Dual isotope plot showing the carbon and nitrogen values for all the producers and consumers from site A.

Figure 4. Dual isotope plot showing the carbon and nitrogen values for all the producers and consumers from site B.

35 MAS

RE

Phyllo

Figure 5a: Distribution of the likelihood of percent contribution for each of the three primary producers for T. paleacea from site A

MAS

RE

Phyllo

Figure 5b: Distribution of the likelihood of percent contribution for each of the three primary producers for T. paleacea from site B

36 MAS

RE

Phyllo

Figure 6a: Distribution of the likelihood of percent contribution for each of the three primary producers for P. frequens from site B

MAS

RE

Phyllo

Figure 6b: Distribution of the likelihood of percent contribution for each of the three primary producers for P. frequens from site A

37 MAS

RE

Phyllo

Figure 7a: Distribution of the likelihood of percent contribution for each of the three primary producers for O. lighti from site A

MAS

RE

Phyllo

Figure 7b: Distribution of the likelihood of percent contribution for each of the three primary producers for O. lighti from site B

38 MAS

RE

Phyllo

Figure 8a: Distribution of the likelihood of percent contribution for each of the three primary producers for P. agassizii from site A

MAS

RE

Phyllo

Figure 8b: Distribution of the likelihood of percent contribution for each of the three primary producers for P. agassizii from site B

39

MAS

RE

Phyllo

Figure 9a: Distribution of the likelihood of percent contribution for each of the three primary producers for L. singularisetis from site A

MAS

RE

Phyllo

Figure 9b: Distribution of the likelihood of percent contribution for each of the three primary producers for L. singularisetis from site B

40 MAS

RE

Phyllo

Figure 10a: Distribution of the likelihood of percent contribution for each of the three primary producers for N. dendritica from site A

MAS

RE

Phyllo

Figure 10b: Distribution of the likelihood of percent contribution for each of the three primary producers for N. dendritica from site B

41