FACTORS INFLUENCING THE DISTRIBUTION AND ABUNDANCE OF

SPHAEROMA TEREBRANS IN FLORIDA’S RED MANGROVES

by

Sarah Huff

A Thesis Submitted to the Faculty of

Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, FL

May 2018

Copyright 2018 by Sarah Huff

ii FACTORS INFLUENCING THE DISTRIBUTION AND ABUNDANCE OF

SPHAEROMA TEREBRANS IN FLORIDA'S RED MANGROVES

by

Sarah Huff

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Donna Devlin, Department of Biological Sciences, and has been approved by the members of her supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

anna Devlin, P Thesis · or a~- Bri~nJe~~ ~·=~~ s:

Y3_~d~ llka C. Feller, Ph.~ Dale E. Gawlik, Ph.D. Director, Environmental Sciences $tt~o. Program ~~z__ Ata:st.ijedini, Ph.D. Dean, Charles E. Schmidt College of Science

~s~ ~ 71 ZDII Khaled Sobhan, Ph.D. Date Interim Dean, Graduate College

iii ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Donna Devlin, for her guidance throughout the course of this research, in addition to the comments and support provided by my other committee members (Dr. Brian Benscoter, Dr. Vincent Encomio, Dr. Ilka

Feller, and Dr. Todd Osborne). Equipment use provided by Dr. Brian Lapointe and Dr. Susan Laramore’s lab was greatly appreciated. Additionally, I would like to thank the following groups: Palm Beach County Environmental Resources

Management for permission to conduct work at their restoration sites, Florida

Oceanographic Coastal Center for assistance with oyster setting and providing oyster shell, and the Whitney Institute for Marine Bioscience in conjunction with

Dr. Todd Osborne’s lab for hosting me and assisting with soil nutrient analysis.

This work was made possible by the Florida Sea Grant Scholars Program, in addition to the Indian River Lagoon Research Fellowship awarded by the Harbor

Branch Foundation.

iv ABSTRACT

Author: Sarah Huff

Title: Factors Influencing the Distribution and Abundance of Sphaeroma terebrans in Florida’s Red Mangroves

Institution: Florida Atlantic University

Thesis Advisor: Dr. Donna Devlin

Degree: Master of Science

Year: 2018

In Palm Beach County, S. terebrans burrows into grounded roots and trunks of R. mangle causing collapse. This is contrary to previous studies suggesting this species burrows only into free-hanging roots. Nutrients and C. virginica cover may affect S. terebrans abundance and distribution. Surveys show burrowing significantly varies among sites, but not between free-hanging and grounded roots. Nutrients vary by site, but neither N nor P was correlated with burrowing. Nutrient treated roots showed no colonization pattern associated with N or P. Lignin varied among sites, but didn’t affect burrowing. Finally, C. virginica limited colonization in the portion of R. mangle tissue it covered. The location of C. virginica on the seaward/landward side was not predictive of burrowing. R. mangle height and leaves were not negatively affected by cover treatment or burrowing. Results highlight the need for additional research to determine the influence of environmental factors on this species interaction.

v DEDICATION

This manuscript is dedicated to my amazing family and friends who have provided endless support throughout this process. Your words of encouragement and unwavering belief in me made this accomplishment possible. I thank you from the very bottom of my heart for pushing me to achieve this goal.

FACTORS INFLUENCING THE DISTRIBUTION AND ABUNDANCE OF

SPHAEROMA TEREBRANS IN FLORIDA’S RED MANGROVES

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

1. GENERAL INTRODUCTION ...... 1

1.1 Sphaeroma terebrans and Rhizophora mangle interaction ...... 1

1.2 Ecological Significance ...... 3

2. EFFECT OF NUTRIENTS ON S. TEREBRANS BURROWING AND

COLONIZATION ...... 6

2.1 Introduction ...... 6

2.2 Objectives and Hypotheses ...... 10

2.3 Materials and Methods ...... 11

2.3.1 Site Descriptions ...... 11

2.3.2 Field Surveys ...... 12

2.3.3 Root Tissue Sampling ...... 14

2.3.4 Soil Sampling ...... 16

2.3.5 Manipulative Field Experiment ...... 17

2.4 Results ...... 18

2.4.1 Surveys ...... 18

2.4.2 Soil Nutrients ...... 21

2.4.3 Root Tissue Sampling (Lignocellulose) ...... 23

vii 2.4.4 Manipulative Field Experiment ...... 23

2.5 Discussion ...... 24

2.5.1 Nutrients and Lignification ...... 25

2.5.2 Patterns of Burrowing in Nutrient Fertilized Roots ...... 28

2.6. Future Work ...... 29

3. EFFECT OF OYSTERS ON S. TEREBRANS COLONIZATION ...... 30

3.1 Introduction ...... 30

3.2 Objectives and Hypotheses ...... 33

3.3 Materials and Methods ...... 34

3.3.1 Site Description ...... 34

3.3.2 Age Class Treatments ...... 36

3.2.2 Cover Treatments ...... 37

3.3.3 Transplantation ...... 41

3.4 Results ...... 43

3.5 Discussion ...... 48

3.5.1 Tissue hardness ...... 48

3.5.2 Associational Resistance ...... 50

3.5.3 Competition between R. mangle and S. terebrans ...... 53

3.5.4 Selection for optimal feeding space ...... 53

3.6. Future Work ...... 54

REFERENCES ...... 56

viii LIST OF TABLES

Table 1. Cover type treatments attached to seedlings and propagules ...... 40

Table 2. Independent and dependent variables used in manipulative

experiment analyses...... 43

Table 3. Abbreviations used for cover treatment...... 45

ix LIST OF FIGURES

Figure 1. Sphaeroma terebrans burrowing damage in Palm Beach County ...... 8

Figure 2. Location of survey sites on the east coast of Florida...... 12

Figure 3. Differences in burrowing by root type and site...... 19

Figure 4. Differences in colonization by root type and site...... 20

Figure 5. Burrowing differences in R. mangle boles (trunks) between sites...... 20

Figure 6. Relationship between site, root type, and rout count...... 21

Figure 7. Differences in total nitrogen (mg/kg) by site...... 22

Figure 8. Differences in total phosphorus (mg/kg) by site...... 22

Figure 9. Differences in lignin content by root type and site...... 23

Figure 10. Burrowing differences between nutrient treatment and site...... 24

Figure 11. R. mangle planting location at Juno Dunes Natural Area ...... 42

Figure 12. Cover treatments used in manipulative oyster experiment...... 43

Figure 13. Differences in burrowing between treatment and plant type...... 44

Figure 14. Relationship between sapling height, treatment, and burrowing...... 46

Figure 15. Relationship between seedling height, treatment, and burrowing.. ... 46

Figure 16. Relationship between sapling productivity, treatment, and

burrowing...... 47

Figure 17. Relationship between seedling productivity, treatment, and

burrowing...... 48

x 1. GENERAL INTRODUCTION

1.1 Sphaeroma terebrans and Rhizophora mangle interaction

Sphaeroma terebrans is a wood-boring isopod associated with mangrove species worldwide and, as a result, is limited to the tropic and sub-tropic geographic zones (Estevez, 1978; Kensley and Schotte, 1989). Originally from the Indo-Pacific, S. terebrans is believed to have arrived in the Americas through two separate introduction events; one taking place in Brazil and the other in

Florida (Carlton, 1994). It has been speculated that ships are responsible for carrying the species across the Atlantic in their wooden hulls, resulting in subsequent introduction after reaching land (Carlton, 1994). In Florida, S. terebrans has been documented in mangrove habitat as far back as 1897

(Carlton and Ruckelshaus, 1997), but it is rare to find colonization landward of the peripheral 2 m of mangrove forest because S. terebrans relies on flooding associated with a normal tidal cycle for survival (Estevez, 1978). For this reason,

S. terebrans almost exclusively occupies the prop roots of R. mangle in Florida.

However, burrowing also occurs in Laguncularia racemosa (white mangrove) and

Avicennia germinans (black mangrove) if they are growing within the appropriate tidal zone (Rehm, 1976). Furthermore, this isopod colonizes wooden maritime structures (Estevez, 1978; Ellison and Farnsworth, 1990; Rice et al., 1990; Cragg et al., 1999) and even coastal swamps (Poirrier et al., 1998; Wilkinson, 2004) that have a hydrodynamic regime beneficial to S. terebrans survival.

1 Among stands, the distribution and abundance of S. terebrans has been described as patchy (Estevez, 1978; Brooks, 2002). Abiotic factors, such as water temperature, salinity, pH, and suspended sediment may influence S. terebrans distribution, but multiple studies have not found these factors to be sufficiently limiting (Howey 1977; Radhakrishnan et al., 1987; Roshaven, 2000;

Brooks, 2002). Observations of seemingly suitable habitat that remain unoccupied by S. terebrans colonization suggest that the dispersal ability of this isopod may be limited, creating “hot spots” of activity (Brooks, 2002). This species is likely reliant on floating wood to raft between stands as a means of passive dispersal at a regional scale (Si et al., 2000; Baratti et al., 2005).

Within stands, the majority of burrows tend to occur within the first 10 cm of R. mangle free-hanging roots and may also be found in areas of recent damage or those softened by microbial decay (Estevez, 1978). The most vulnerable portion of R. mangle is the apical root tip because the tissue is soft and easy to penetrate (Estevez, 1978; Perry, 1988; Perry and Brusca, 1989).

Conversely, roots that have penetrated the substrate (hereafter referred to as

“grounded roots”) are described as being free of S. terebrans burrows. Upon reaching the substrate, secondary thickening of R. mangle prop roots occurs (Gill and Tomlinson, 1977), causing hardening of root tissue (Perry, 1988). As a result, S. terebrans is excluded from grounded root colonization (Perry, 1988;

Brooks and Bell, 2001; Brooks, 2002).

It is important to note that the burrowing activity of this species is not driven by tissue consumption, but rather by the need for refuge (Estevez, 1978),

2 a place to reproduce (Thiel, 1999), and the opportunity to filter feed during the incoming tide (Rotramel, 1975; Rice et al., 1990). Since S. terebrans is a brooder species (Baratti et al., 2011) that engages in parental care (Messana et al.,

1994), its offspring remain in the burrows until they are able to build their own

(Thiel, 1999). It is not uncommon to find numerous offspring and adults in a single burrow; as many as 5-20 juveniles have been recorded in a single burrow in the Indian River Lagoon (Thiel, 1999). As juveniles mature, they may burrow off of the adult tunnel or migrate via swimming or crawling to another prop root or woody structure (Rehm, 1976; Perry, 1988, Estevez, 1978). In the Indian River

Lagoon there are twin population peaks, with the greatest number of individuals present during the late spring/early summer and the fall (Thiel, 1999). In Tampa

Bay, S. terebrans abundance was noted as highest during the summer and fall, but this may not be consistent across years (Estevez, 1978).

1.2 Ecological Significance

The cause of concern over this isopod species is the potential impact it holds for both the health and resilience of mangrove species. It is believed to be detrimental to mangrove health due to the architectural changes it can cause to aerial prop roots. Examples of this include decreased root production and increased root atrophy, effectively compromising structural integrity of affected trees (Simberloff et al., 1978; Ribi, 1981; Perry, 1988; Perry and Brusca, 1989;

Ellison and Farnsworth, 1990). This is especially detrimental to mangroves in high energy environments (Davidson et al., 2014), as these bottom-heavy trees may topple if support from the prop roots has been altered (Mendez-Alonzo et

3 al., 2008). Since coastal resiliency relies on storm buffering (Mazda et al., 2006;

Alongi, 2008) and shoreline stabilization (Stephens, 1962; Rehm, 1976) provided by mangrove forests, these functions have the potential to be compromised by increased root atrophy.

Changes to mangrove root structure can also alter energy provisioning

(Simberloff et al., 1978; Perry, 1988; Brooks and Bell, 2002), including the uptake of nutrients, gas exchange, and photosynthetic activity (Kulman, 1971; Perry,

1988). This can affect other parts of the tree that don’t receive direct burrowing damage, such as branches, leaves, and propagules. In R. stylosa and A. marina, these portions of the tree were reduced in size due to altered energy allocation

(Davidson et al., 2014). It has even been suggested that the loss of R. mangle in

Florida is associated with S. terebrans activity and that this isopod shapes mangrove distribution (Rehm, 1976).

In light of globally changing ocean currents, warming temperatures, and sea level rise, determining factors the control the range, distribution, and abundance of this species is critical. It is possible that the range and dispersal ability of S. terebrans could expand (Cragg et al., 1999) with changes to global temperatures, bringing it into contact with mangrove stands not previously colonized or spreading it further into the stands where it already occurs. From a community-level perspective, this could hold negative implications as mangroves are considered to be foundation species of estuarine environments. An estimated

1,300 species (USFWS, 1999) dwell in mangrove habitat that S. terebrans may alter in both quality and quantity (Davidson et al., 2014). Thus, determining

4 factors that control its distribution and abundance hold important ecological implications for mangrove habitat and estuarine systems worldwide.

5 2. EFFECT OF NUTRIENTS ON S. TEREBRANS BURROWING AND

COLONIZATION

2.1 Introduction

In Florida, the primary habitat of Sphaeroma terebrans is the aerial prop roots of Rhizophora mangle. However, the burrowing and colonization that occur within these roots vary depending upon whether the prop root is free-hanging or grounded in the sediment below (Perry, 1988; Estevez, 1978; Brooks and Bell,

2001). Aerial roots that penetrate the substrate are typically free of S. terebrans burrows (Perry, 1988) and borers may abandon their burrows upon root grounding (Brooks and Bell, 2002). The variation in colonization between root types was determined to be a result of secondary thickening in grounded roots

(Gill and Tomlinson, 1977), as growth of the cambium gives rise to a lignified xylem surrounded by phloem tissue (Esau, 1965; Steeves and Sussex, 1989).

Although S. terebrans recognizes grounded roots as viable habitat for building burrows (Brooks and Bell, 2001), tissue hardness may limit colonization (Perry

1988).

As a result of S. terebrans burrowing activity, numerous structural changes may occur in affected mangroves, including a delay in roots reaching the ground, root atrophy, and breakage (Perry, 1988; Perry and Brusca 1989), resulting in altered energy allocation (Simberloff et al., 1978, Mopper et al. 1991).

In other species of mangrove, the deeply penetrating burrows of this species can

6 also result in damage to unbored portions of the tree, such as leaves and propagules (Davidson et al., 2014). Furthermore, damage to the root tissue may also reduce photosynthetic capability as both water and sucrose transport are changed (Nabity et al., 2009). Despite the negative effects of this interaction, mangrove mortality is rarely described as an effect of S. terebrans burrowing.

Conversely, on Florida’s east coast, apparent mortality at Juno Dunes and

Ibis Isle has been observed as result of this interaction. The prop roots of R. mangle display heavy burrowing by S. terebrans (personal observation, 2015).

We observed that both free-hanging and grounded prop roots at these locations have observable burrow damage (Figure 1a), although research suggests that burrowing is absent in grounded roots (Perry, 1988; Brooks and Bell, 2002). In addition to burrowing in both free-hanging and grounded roots, the trunks of R. mangle, A. germinans, and L. racemosa are also heavily burrowed, an observation yet undescribed in literature (Figure 1b, D. J. Devlin personal observation, 2015). As an apparent result of heavy infestation and architectural changes from this burrowing, we noted several trees had collapsed onto the sediment, seemingly unable to support their own weight (Figure 1c, 1d). Several individuals appeared to be dead at the time of observation (Figure 1b).

7

Figure 1. Sphaeroma terebrans burrowing damage in Palm Beach County. Damage observed in a) grounded roots at Juno Dunes; b) bole and grounded prop roots at Juno Dunes; c) collapse of a sapling on the sediment at Juno Dunes; d) tree toppling due to no grounded root support at Ibis Isle.

Interestingly, Snook Islands and South Cove are two sites in close proximity to Ibis Isle, where heavy infestation was observed. However, these locations did not show damage resulting in R. mangle collapse, despite the presence of S. terebrans. We observed at all of these locations that the free- hanging and grounded prop roots had burrows, however, colonization appears to vary greatly among locations (personal observation 2015). This is not unusual as

S. terebrans burrowing and colonization have been described as patchy across relatively small areas (Estevez, 1978; Brooks and Bell 2001).

Soil nutrients may influence R. mangle prop root characteristics (directly and indirectly) (Howey, 1977), as plants are known to change energy allocation in response to the nutrient availability (Brouwer, 1962; Bloom et al., 1985). In low

8 nutrient environments, plants allocate greater resources to root development as a mechanism to increase the uptake of a limited resource (Bradshaw, 1965;

Chapin, 1980; Wilson, 1988; Gedroc et al., 1996), while high nutrient availability generally results in an increase in aboveground biomass (Müller et al., 2000). As a result, mangrove architecture is often reflective of the growth environment.

Trees that receive supplemental nutrients tend to invest more in canopy growth and allocate less to the root growth (Feller, 1995). These trees have large, dense canopies compared to stunted or dwarfed trees where soil fertility is low (Feller,

1995). Vigorous growth may also result in aerial roots with less dense cells and a thinner cortex, when nutrients, such a nitrogen, are present in excess (Howey,

1977). Thus, plants growing under high nutrient conditions may be more susceptible to environmental stressors that require greater investment in the roots for tolerance (Chapin, 1991).

The nutrient availability within the soil may influence the lignin content present in plant cell walls (Siegleman, 1964; Harborne, 1967; Marchand, 2005).

High quantities of lignin are typically present in the xylem of woody plants

(Chesson et al., 1986; Akin et al., 1990; Twidwell et al., 1991; Buxton and

Redfearn, 1997) which greatly reduces the degradability of this tissue (Grabber el al., 1992). Lignocellulose, the material that is primarily responsible for imposing rigidity and structure in the tissue of woody plants (Marchand, 2005), is formed when lignin complexes with cellulose. Although lignin is typically found in the largest quantity in the primary cell wall, plants that have pronounced secondary thickening may have lignin in greater quantities in the secondary walls instead

9 (Terashima et al.,1993; Wilson, 1993). Lignin deposition in some plant species is reduced under high nutrient conditions (Pitre, 2007; Camargo, 2014), bearing similarities to lignin present in early development (Pitre 2007). Under N-limited conditions, lignin deposition may actually be more consistent (Camargo, 2014).

However, low soil fertility has also been suggested to reduce lignification of plant tissue in response to nutrient deficiency (Moore and Jung, 2001). Thus, trees of the same species may have different proportions of lignin present in their tissues

(Pettersen, 1984) under different environmental conditions. Since nutrients may indirectly affect the hardness of root tissue (Howey, 1977) and S. terebrans can distinguish among different levels of wood hardness (John, 1971a), we suggest this species may select for roots where lignocellulose exists in low quantities.

2.2 Objectives and Hypotheses

The objective of this research is to determine if nutrients in the soil affect the hardness of root tissue and thus S. terebrans burrowing and colonization at these locations in Palm Beach County. Based on the objective of this study, the following hypotheses were developed: (1) There is a positive relationship between soil nutrients and S. terebrans burrowing; (2) There is a positive relationship between nutrient fertilized R. mangle roots and S. terebrans burrowing and; (3) There is a negative relationship between lignin and S. terebrans burrowing.

The first hypothesis was addressed by collecting and analyzing soil core samples from each site. The second hypothesis was tested through a manipulative field experiment involving nutrient fertilized roots. The third

10 hypothesis was addressed by collecting and analyzing root samples for lignin from each site.

2.3 Materials and Methods

2.3.1 Site Descriptions

In addition to those sites in Palm Beach County with varying degrees of S. terebrans damage, a site in St. Lucie County was selected as a control. The sites included in the survey range from Ft. Pierce, FL (27°28'38.95"N, 80°19'24.23"W) in the north to Lake Worth, FL (26°36'57.10"N, 80° 2'45.74"W) in the south, spanning an approximate distance of 100 km (Figure 2). All of these sites are characterized by the presence of mangrove forests consisting of predominantly

R. mangle on the leading edge, with A. germinans, and L. racemosa existing further into the stand. Trees at these locations range in age from seedling to mature adult; the oldest trees likely represent mangroves planted during site restoration and young mangroves represent recruits to the site.

The following is a brief history of these locations: SL-15, located in Ft.

Pierce, had mangroves planted in 2005 after the invasive, exotic plants that dominated the spoil island were removed (Clark, 2013). That same year in Palm

Beach County, Snook Islands Natural Area was created in the Lake Worth

Lagoon to fill in a dredge hole that was formed from the construction of the adjacent city golf course (PBC ERM, 2015). Snook Island’s neighboring site, Ibis

Isle, was created four years later in 2009 to cap the 7 ft muck that had built up at the outfall of the C-51 canal (PBC ERM, 2015). The following year, between

2010 and 2011, Juno Dunes Natural Area underwent invasive, exotic tree

11 removal and new habitat for mangroves was created with the spoils from the dredging of the intercostal waterway (PBC ERM, 2011). The youngest of the sites, South Cove Natural Area was built in 2012 using sand to fill and cap a 7- acre dredge hole in the Lake Worth Lagoon (PBC ERM, 2015).

Figure 2. Location of survey sites on the east coast of Florida. Sites are abbreviated with a code: SL- 15=Spoil Island #15, JUNO=Juno Dunes Natural Area, COVE=South Cove Natural Area, IBIS=Ibis Isle, SNOOK=Snook Islands Natural Area. 2.3.2 Field Surveys

At each site, except for SL-15 (n=5), a total of 12 R. mangle trees were randomly selected along two, 25 m transects for evaluation of S. terebrans burrowing. Transects were placed roughly parallel to the mangrove fringe on the seaward edge and random numbers were generated to determine points along the transect where a tree would be sampled. Points were located a minimum distance of 2 m apart to ensure trees were not re-sampled. The free-hanging root of each selected tree closest to the transect was collected by severing the root 12 approximately 10 cm above the apical root tip. The grounded root closest to the transect was also collected by severing the root approximately 10 cm above the sediment. Neither free-hanging nor grounded roots displaying multiple root tips were selected for collection due to the burrowing bias they present (Perry and

Brusca, 1989). The bole (trunk) of each selected tree along the two, 25 m transects was evaluated for evidence of S. terebrans burrowing. The seaward facing side of each trunk up to the first set of prop roots was examined at eye level and a total count of burrows was obtained. Due to the tangle of roots around the trees, it was not possible to access the landward facing side of the trunk at every location, therefore only burrow data collected from the seaward side of the trunk was used for analysis. To assess the relative number of roots available as habitat at each selected tree, a 0.25 m² quadrat was placed squarely alongside the base of the trunk, parallel to the transect. Only roots within the quadrat belonging to the selected tree were counted and recorded as either free- hanging or grounded.

Following collection, all of the root samples were bagged immediately and dissected, typically within 48 h, but no longer than 1 wk from the date of collection. A total count of burrow entrances was obtained prior to cutting each root lengthwise down the center. This number was then verified by counting the number of burrows found within each root to ensure no openings made by another marine species were included in the total. Individuals were carefully pulled out of each burrow. After a thorough inspection to verify no borers remained in the burrow cavities, the total number of individuals was recorded.

13 The survey data were analyzed using a separate factorial ANOVA to determine the effect of site and root type on 1) total S. terebrans burrow count in the prop roots and 2) the number of S. terebrans individuals in the prop roots.

Logistic regression was used to determine the effect of site on total S. terebrans burrow count in the trunks because several locations had trunks without the presence of burrows, resulting in zero data. Trunk burrowing could only be analyzed as presence absence. Finally, a factorial ANOVA was used to determine the habitat available, measured as the number of roots, at each location. Transformations were performed as needed to meet the assumption of each test and the analysis was completed using SAS (University Edition, SAS

Institute, Cary, NC, U.S.A.).

2.3.3 Root Tissue Sampling

A total of 60 roots were collected from survey sites and the Goering and

Van Soest fiber analysis method (1970) was used to determine the amount of lignin present in each root. Of the 12 trees included in the survey at each location, three trees from each of the two, 25 m transects were selected using a random number generator for fiber analysis. Both the free-hanging and grounded roots from each sampled tree were placed in a Thermo-Scientific drying oven at

65°C for 72 h at Florida Atlantic University at Harbor Branch Oceanographic

Institute. Upon removal from the drying oven, roots were ground by hand using a mortar and pestle and followed by passing through a size 20 Thomas Scientific mini Wiley Mill attachment to create <1 mm powder. Two grams of the powder were weighed out and stored in uniquely coded plastic vials. Fiber analysis was

14 completed 1 wk later at the University of Florida Whitney Lab for Marine

Bioscience located in St. Augustine, FL.

Approximately 0.5 g of powder was weighed into labeled fiber bags and sealed with an impulse sealer. The sealed bags were then placed in a Fiber

Analyzer with neutral detergent (NDF) for 70 min. Hot water was used to flush off the bags prior to being soaked into acetone, a necessity for the removal of waxes. The bags were left in a fume hood for a minimum of 1 h to evaporate off the excess acetone and then placed in a Thermo-Scientific drying oven at 65°C for 12 h. The dry weight of each bag was recorded post-NDF, prior to being placed in the Fiber Analyzer with an acid detergent (ADF) for 70 min. The same process of hot water, acetone, and time in the drying oven was used after the bags came out of the Fiber Analyzer. The final step of fiber analysis involved recording the dry weight of each bag post-ADF, prior to being placed in sulfuric acid for two hr. The bags were agitated every 15 min over this 2 h period. The bags were then washed to remove sulfuric acid from the surface and placed in the drying oven at 65°C for another 12 h. The dry weight of each bag was recorded post-sulfuric acid. The weight lost with each sequential step in the process was determined by subtracting the weight of the completed step from the weight of the step that preceded it (Goering and Van Soest, 1970; Roberts and

Rowland, 1988). In doing so, the lignin concentration as a percent of the dry material (% DM) could be determined (Goering and Van Soest, 1970; Roberts and Rowland, 1988).

15 A factorial ANOVA was used to determine if total lignin varied by site and root type. Simple linear regression was also used to investigate burrowing activity correlation to lignin concentration at each site. Transformations were performed as needed to meet the assumption of each test and the analysis was completed using SAS.

2.3.4 Soil Sampling

Four soil cores were collected at each of the five sites (n=20), two cores along each of the 25 m transects. Samples were collected using polycarbonate core tubes and were kept upright in a refrigerator to prevent mixing of the top and bottom layers for up to 3 wk after collection. Soil was extruded into plastic bags into two depth increments, 0-10 cm (shallow) and 10-20 cm (deep), such that there were 40 soil samples (20 shallow and 20 deep samples). The soil in each bag was homogenized prior to analysis. Total phosphorous was determined by ash-TP method (Andersen, 1976). Total Nitrogen (N) was determined following methods of Hach (2016). Samples were analyzed colorimetrically using the Hach

DR 6000™ UV VIS Spectrophotometer.

Two, factorial ANOVA’s were used to determine the interaction of site and sample depth on total Nitrogen and total Phosphorus. Pearson’s correlation coefficient was used to determine if soil nutrients and lignin were correlated.

Pearson’s correlation coefficient was also used to determine if soil nutrients and burrowing were correlated. Transformations were performed as needed to meet the assumption of each test and the analysis was completed using SAS.

16 2.3.5 Manipulative Field Experiment

A total of 18 R. mangle trees were sampled from nutrient fertilized plots located in Avalon State Park in Fort Pierce, FL (Feller, 2003). Six trees receiving

N supplements (urea), six trees receiving P supplements (P2O5), and six control trees receiving no fertilizer were selected for root collection. The trees receiving supplemental nutrients have been fertilized since 1997. Two free-hanging roots with no observable S. terebrans damage and ≤ 2 m from the location of fertilizer application were selected from each tree. The terminal 25 cm of each root was severed and sealed immediately with E6000™ glue to preserve freshness. Roots were labeled and bagged until deployment at the selected field sites later that same day.

A total of 36 roots were deployed at two sites in Florida. The distance between these sites is ~71 km (Juno Dunes to SL-15). Roots were deployed in the field on the same day as collection. The experimental design for the experiment consisted of two sites (Juno Dunes and SL-15), six haphazardly positioned blocks along a 25 m transect and three prop root treatments (control,

N fertilized, P fertilized). Each block consisted of a T made of PVC with the bottom vertical end inserted firmly into the sediment. These were haphazardly placed along the shoreline on the front edge of the mangrove fringe among roots experiencing S. terebrans burrowing activity. The three treatment roots were hung from the horizontal arms ~6 cm apart and 1 cm above the sediment on the

PVC T’s. Roots were secured using wire-ties just above and below the T to prevent slip through or contact with sediment and were kept in the field for a total

17 of 15 d. The diameter of each root was determined prior to evaluation and a total count of burrows was obtained before dissection. Each root was cut lengthwise down the center to excavate the burrows and a total count of borers was obtained. The experimental design was based on a similar experiment by Brooks and Bell (2002).

The fertilized root experiment conducted at SL-15 and Juno Dunes was analyzed using a factorial ANOVA to determine the effect of site and nutrient treatment on burrowing. Transformation was performed to meet the assumption of the test and the analysis was completed using SAS.

2.4 Results

2.4.1 Surveys

Burrowing activity among the sites varied significantly (F=15.53, p<0.001), with Juno Dunes and Ibis Isle experiencing greater burrowing than Snook

Islands, SL-15, and South Cove (Figure 3), as revealed by Tukey a-posteriori comparisons. However, variation between root type (free-hanging, grounded) was not significant (F=3.16, p=0.078). Of the five surveyed sites, Juno Dunes represents the location with the greatest burrowing where 100% of sampled free- hanging and grounded roots contained S. terebrans burrows. In comparison, at

South Cove, only 50% of free-hanging roots and 58.33% of grounded roots displayed burrows. The interaction of site and root type on S. terebrans burrowing activity at the surveyed sites was not significant (F=0.17, p=0.954).

S. terebrans colonization, measured as a count of total individuals, also varied among sites (F=14.85, p<0.001). Again colonization patterns could not be

18 explained by root type (F=2.74, p=0.101) (Figure 4), and the interaction was nonsignificant (F=0.72, p=0.583). Tukey’s a-posteriori comparisons revealed that colonization was different than total burrowing, as Snook Islands was grouped with Juno Dunes, Ibis, Isle, and had a greater number of S. terebrans individuals than SL-15 and South Cove. Finally, the number of burrows in the trunks of R. mangle individuals was significantly different among the surveyed locations

(logistic regression, F=17.08, p=0.002, Figure 5).

Figure 3. Differences in burrowing by root type and site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error.

19

Figure 4. Differences in colonization by root type and site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error.

Figure 5. Burrowing differences in R. mangle boles (trunks) between sites. Bole (trunk) average count data is graphed. Error bars represent standard error.

Measured as a count of roots within 0.25 m2, the interaction of site by root type (F=12.06, p < 0.001) suggests that there is a difference in the quantity of free-hanging and grounded roots at these locations (Figure 6). Tukey’s a- posteriori comparisons revealed that the quantity of roots available as habitat for burrowing by S. terebrans was only significantly different between Juno Dunes and SL-15. Juno had, on average, less than one grounded root and nearly five 20 free-hanging roots in each 0.25 m² quadrat. SL-15 averaged approximately five grounded roots and more than five free-hanging roots per 0.25 m² quadrat. The architecture of R. mangle appears to vary among the surveyed sites, likely in part due to S. terebrans burrowing.

Figure 6. Relationship between site, root type, and rout count. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error. 2.4.2 Soil Nutrients

The interaction of site and sample depth was significant in regards to total

N (F=3.00, p=0.034), suggesting that concentration of N by soil depth varies significantly by location. Tukey a-posteriori comparisons revealed that total N present within the soil at Juno Dunes varied from all other sites except for Ibis

Isle. Ibis Isle differed in the amount of total N present in the soil from Snook

Island and SL-15 (Figure 7). The interaction of site and sample depth on total P was not significant (F=1.67, p=0.183). Total P was similar at both sample depths

(F=0.43, p=0.473) and varied significantly among the sites (F=3.27, p=0.024,

Figure 8). Tukey a-posteriori comparisons identified that total P in the soil at Ibis

21 Isle varied from all other sites. No correlation was identified between total N

(p=0.310) nor total P (p=0.172) and S. terebrans burrowing activity.

Figure 7. Differences in total nitrogen (mg/kg) by site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error.

Figure 8. Differences in total phosphorus (mg/kg) by site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error

22 2.4.3 Root Tissue Sampling (Lignocellulose)

The interaction of lignin and root type (free-hanging, grounded) on S. terebrans burrowing was not significant (F=0.44, p=0.519). Lignin did not vary between free-hanging and grounded roots (F=0.015, p=0.904). The quantity of lignin in mangrove prop roots did significantly vary among sites (F=9.543, p<0.001), with SL-15 roots having the greatest lignin concentration (% DM) and

Snook Island having the lowest quantity of all the surveyed locations (Figure 9).

Most of the roots had lignin between 0.2-0.3 of the roots dry weight, which is also the range where the majority of burrowing occurred. Interestingly, no correlation was found between total N (p=0.924) nor total P (p=0.421) and lignin content in

R. mangle roots.

Figure 9. Differences in lignin content by root type and site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error.

2.4.4 Manipulative Field Experiment

Burrowing activity among sites was significantly different (F=17.24,

P=0.003), with SL-15 roots experiencing burrowing greater than Juno Dunes 23 roots, as revealed by Tukey a-posteriori comparisons. Burrowing activity among fertilizer treatments was not significant (F=1.03, p=0.369). The interaction of site by treatment was also not significant (F=0.14, p=0.871, Figure 10).

Figure 10. Burrowing differences between nutrient treatment and site. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error.

2.5 Discussion

The objective of this research was to determine if nutrients present in the soil drive S. terebrans burrowing and colonization at these locations. Based on this objective, the following hypotheses were developed: (1) There is a positive relationship between soil nutrients and S. terebrans burrowing; (2) There is a positive relationship between nutrient fertilized R. mangle roots and S. terebrans burrowing and; (3) There is a negative relationship between lignin and S. terebrans burrowing.

24 2.5.1 Nutrients and Lignification

We predicted that soil nutrients would positively affect the abundance of S. terebrans burrows and expected that sites with high quantities of Nitrogen (N) and Phosphorus (P) would have roots more prone to burrowing. This could be explained by energy being allocated away from the growth of the roots (Feller,

1995), resulting in less dense cells (Howey, 1977). At survey locations, the interaction of site and depth was significant in regards to total nitrogen (N)

(F=3.00, p=0.034), however S. terebrans burrowing appears to have an inverse relationship to N. Juno Dunes and Ibis Isle had N present in low quantities in the soil, yet S. terebrans burrowing was high. Snook Islands, South Cove, and SL-15 had a greater quantity of N available in the soil, yet burrowing was low relative to

Juno Dunes and Ibis Isle This is opposite of what was expected, given that the trees at Snook Islands, South Cove, and SL-15 should have had softer roots that would be easier to burrow into. Had burrowing been greater at sites with high N in the soil or vice versa, soil depth would be an important factor to consider because the vertical distribution of N may affect plant uptake. However, no correlation was found between S. terebrans burrowing and N in the soil at these sites (p=0.3098). P was similar at both sample depths, but varied among sites.

Since P can be immobile in mangrove soils (Reef et al., 2010), it is not unexpected that the vertical distribution of P among soil samples was similar. No clear pattern could be identified with regards to S. terebrans burrowing and P availability and no correlation was found (p=0.172). Analysis also showed that N and P in the soil were not correlated with the lignin content of the sampled R.

25 mangle roots. This may suggest the soil at these sites did not have nutrients present in quantities that would result in reduced lignification. Further work is needed to test causality between soil nutrients and the distribution and abundance of S. terebrans.

Our hypothesis also predicted that as lignin increased in the root tissue, S. terebrans burrowing would decrease in response to rigidity and vice versa. In contrast to literature on the subject, these surveys indicate that free-hanging and grounded R. mangle roots are burrowed into and colonized similarly. In previous studies, colonization was primarily described as limited to the free-hanging prop roots of R. mangle (Perry, 1988; Brooks and Bell, 2001). Since grounded roots undergo secondary thickening upon making contact with the sediment, it was expected that grounded roots would have increased lignification of the secondary walls (Perry, 1988; Terashima et al.,1993; Wilson, 1993). However, in this study, grounded roots in both Palm Beach and St. Lucie County contain S. terebrans individuals and represent an increase in the habitat utilized by this species. Since the quantity of lignin did not vary between grounded and free-hanging roots, S. terebrans appears to burrow into and colonize both similarly. Grounded roots did not have greater lignin content than free-hanging roots and may indicate that thickening did not occur differently among root types at these locations. The hardness of the grounded roots may have been indistinguishable from free- hanging roots to S. terebrans, therefore it is not unexpected to see burrowing in both root types.

26 Among sites, both lignin (F=9.5432, p<0.001) and burrowing significantly varied (F=15.53, p<0.001). We predicted that the sites with low burrowing activity

(Snook Islands, South Cove, and SL-15) and colonization (South Cove, SL-15) might have R. mangle with high quantities of lignin present in root tissue.

However, no clear pattern was established with regards to lignin and burrowing among sites. The roots from trees at Snook Islands had the lowest lignin content, yet burrowing was significantly lower than that at Juno Dunes and Ibis Isle. South

Cove had low burrowing activity and colonization in comparison to Juno Dunes, but the lignin content between roots at both sites was similar. SL-15 was the only site where S. terebrans burrowing appeared to have the predicted response to high quantities of lignin. As a result, these data suggest lignin alone is not a high predictor of distribution or abundance of S. terebrans among locations.

Finally, the quantity of roots available as habitat varied by root type and site, although a post host test identified the differences lie between Juno Dunes and

SL-15. At Juno Dunes, R. mangle had more free-hanging roots than grounded roots. At SL-15, the quantity of free-hanging and grounded roots was similar. The architecture of R. mangle at Juno Dunes may be due to free-hanging roots effectively being cut off at the high water line, never reaching the substrate below

(Rehm, 1976) or the production of lateral roots at the site of S. terebrans injury

(Simberloff et al., 1978). Burrowing also varied in the boles/main stem among sites. These architectural changes may explain why Juno Dunes had numerous mangroves collapsed on the soil. At SL-15, R. mangle had a greater quantity of

27 grounded roots and fewer burrows present in the trunks, potentially decreasing an individual’s likelihood of collapse.

2.5.2 Patterns of Burrowing in Nutrient Fertilized Roots

Growth under any high nutrient condition may compromise tolerance from changes to the plant energy allocation (Chapin 1991). We predicted that fertilized roots and S. terebrans burrowing activity would exhibit a positive relationship as a result of being taken from trees where energy would have been allocated away from the growth of the root (Feller, 1995). Roots grown in high N soils should have less dense cells and a thinner cortex (Howey, 1977). However, all treatments were burrowed into similarly, although they were grown on trees under different nutrient conditions. It does not appear that S. terebrans prefers either N or P fertilized roots.

Total burrowing varied significantly between the sites where roots were deployed. Burrowing activity was minimal at Juno Dunes over the course of the experiment although roots were surrounded by highly damaged R. mangle included in the survey. This is especially interesting given that these roots were uncorticated, free-hanging prop roots: the preferred habitat of S. terebrans

(Estevez, 1978; Perry, 1988). This seems to suggest that S. terebrans in the study area select for roots differently than in other areas where research has been conducted. As for SL-15, the burrowing in the fertilized roots may be explained if roots characteristics were similar to or more preferable than those roots naturally growing on mangroves at the site.

28 2.6. Future Work

To further investigate the effects of nutrients on S. terebrans burrowing, future studies should consider the nutrient content of the root tissue itself. The nutrients present in the soil may not be directly proportional to the quantity present in the roots. It is possible that the concentrations of N and P within the root may have a more direct effect on isopod colonization. In addition, the various forms of N and P should also be considered with regards to S. terebrans burrowing, since nutrient species may exist in different quantities in the soil at these sites and elsewhere. Finally, lignin in proportion to other polymers in the secondary cell wall should be evaluated as indicators of root hardness.

Furthermore, future research should focus on depensatory growth mechanisms to explain S. terebrans distribution and abundance. It has been suggested that other isopods in the Sphaeroma genus exhibit inverse density- dependent growth, causing individuals to select for locations where isopod density is high and migration is away from low density sites (Phillips et al., 2009).

Groups of individuals are often better able to withstand abiotic stressors, including desiccation (Lively and Raimondi, 1987) and individuals in aggregations typically have better access to mates than those dispersed over large distances

(Norse and Crowder, 2005). The cooperation of Sphaeroma individuals may confer an advantage to the entire population, making establishment more likely at sites where individuals have already colonized.

29 3. EFFECT OF OYSTERS ON S. TEREBRANS COLONIZATION

3.1 Introduction

In marine environments, the influence of mutualism and facultative association can have strong effects on ecosystem dynamics (Vance, 1978; Feller and Chamberlain, 2007; Thornber, 2007). These associations affect not only organisms within trophic levels, but they can also impact interactions throughout the entire trophic web (Laudien and Wall, 2004). Facultative association, where a focal species benefits from reduced mortality because of protection provided by a nearby organism, is further defined as associational resistance (Vance, 1978;

Thornber, 2007). One mechanism for conferring associational resistance is masking, wherein neighboring individuals visually conceal (Finch et al., 2003), impede access to (Rauscher, 1981), or interfere with movement (Risch, 1981;

Coll and Bottrell, 1994; Holmes and Barrett, 1997) of herbivores or predators. As a result, the likelihood of damage to the focal species is reduced.

The theory of associated resistance was first developed to explain interactions among plants and is generally applied to plant-plant relationships.

However, this theory has since been applied to faunal relationships. For example, the masking mechanism is demonstrated by the relationship between

Chama pellucida (hereafter referred to by its common name, jewel box clam) and its non-obligate epibiont community that reduces detection and accessibility of the clam to the predatory sea star Pisaster ochraceus (Vance, 1978). In turn, the

30 epibionts benefit from a habitat with reduced grazing pressure from predators

(Vance, 1978). Thus, the jewel box clam and its epibionts are not essential to the survival of each other, but both participants are favored evolutionarily as a result of the interaction (Vance, 1978).

We suggest that associational resistance via masking may also exist between the focal species Rhizophora mangle (red mangrove) and its epibiont,

Crassostrea virginica (eastern oyster), which protects the focal species from a wood boring isopod, Sphaeroma terebrans. S. terebrans does not consume mangrove wood for nutrition, but instead builds extensive burrows in R. mangle prop roots. These burrows serve as a refuge against desiccation and provide the isopod a sheltered position from which to filter feed (John, 1971a). The burrows also allow for S. terebrans to raise offspring with extended maternal care

(Messana et al., 1994), often resulting in a network of family burrows within a single prop root (Thiel, 1999). Burrowing can result in damage to root tissue that reduces the growth of R. mangle (Ellison and Farnsworth, 1990) and, in some cases, can lead to mortality when R. mangle collapse due to weakened structural support from the prop roots (Rehm and Humm, 1973; Rehm, 1976; personal observation, 2016).

Since S. terebrans is able to distinguish among the hardness of various materials and selects for those that are soft (John, 1971a), boring primarily occurs in the apical root tip or other pre-softened portions of R. mangle prop roots, including areas with recent damage (Estevez, 1978; Perry, 1988; Perry and Brusca, 1989; Ellison and Farnsworth, 1990). Areas covered with epibionts

31 are generally avoided; “hard-shelled” species limit burrowing by S. terebrans in

R. mangle roots by 28.2% (Estevez, 1978). Sponge and colonial ascidian epibionts also limit S. terebrans in the portions of root tissue they cover (Ellison and Farnsworth, 1990). Thus, C. virginica and other epibionts may mask R. mangle by concealing its roots and/or impeding colonization by S. terebrans.

Similar to epibionts on the shell of the jewel box clam, epibionts on the roots of R. mangle benefit from the hard, elevated root substrate that serves as a refuge from both heavy sedimentation and benthic predators (Rützler, 1969;

Bingham and Young, 1994). We suggest that associational resistance may occur between C. virginica, a nearby species, that appears to convey protection from burrowing and mortality by a damaging species (S. terebrans) by masking the roots of the focal species (R. mangle). Different from most instances of associational resistance though, the +/- interaction between the damaging species and the focal species is not classified as predation or herbivory.

On another level, S. terebrans and C. virginica may also interact as competitors for food in their shared environment. As filter feeders, both species are reliant upon the hydrodynamic regime for the delivery of phytoplankton.

Although there is no literature describing competition for food between C. virginica and S. terebrans, this is often a major driving factor of marine invertebrate interactions (Buss & Jackson, 1981; Stuart & Klump, 1984; Lesser et al., 1992). The likelihood of exploitative competition existing between oysters and

S. terebrans depends on the availability, size, and quality of the resource in their shared environment. C. virginica can filter up to 50 gallons per day of water

32 containing suspended sediments and phytoplankton (Milbrandt, 2015), the same size phytoplankton that is used by S. terebrans. Although oysters mainly consume particles >20 μm, smaller plankton (2.7–20 μm) is often included in their diet (Jiang, et al. 2016). Scanning electron microscopy indicates that the filtering setae on the first three pairs of pereiopods of S. terebrans are well suited to capture particulate matter > 5 μm, larger phytoplankton species (2-200 μm) and nanoplankton (2-20 μm) (Si et al. 2002). If resource partitioning occurs between these invertebrate species, this may limit the level of competition that exists (Schoener, 1974).

Since there is small-scale variation in epibiont assemblages on roots, the lagoon-facing (seaward) fronts of roots tend to have greater diversity than the shore-facing (landward) backs of roots (Ellison and Farnsworth, 1996). The species that settle on R. mangle roots often have planktonic life stages and it is likely that more plankton, the food source for both C. virginica and S. terebrans, is in greater supply on the front surfaces of roots as well. Both species are more common below the mean tide line where the chance of desiccation is reduced

(Estevez, 1978; Aquino and Proffitt, 2014). For this reason, the location upon which C. virginica settle is likely an important factor to assess in regards to competition for food as this may represent optimal space for feeding.

3.2 Objectives and Hypotheses

By transplanting individuals with C. virginica (eastern oyster) cover to a highly colonized site, the following questions regarding S. terebrans colonization and oysters could be addressed: 1.) Does associational resistance occur

33 between R. mangle and C. virginica by masking mangrove prop roots from S. terebrans burrowing? and 2.) Does competition occur between C. virginica and

S. terebrans? To address the question of associational resistance, two main hypotheses were developed: 1) S. terebrans prefer soft root substrate; 2) S. terebrans does not burrow in areas of R. mangle root covered with C. virginica.

To address the question of competition, two main hypotheses were developed:

3) S. terebrans colonization differs between mangroves covered in live C. virginica and crushed shell; 4) S. terebrans burrowing differs between the landward and seaward side of R. mangle.

3.3 Materials and Methods

3.3.1 Site Description

Juno Dunes Natural Area, located in Palm Beach County, Florida is managed by the county’s Environmental Resources Management (PBC ERM) department. In 2015, heavy burrowing damage at this location was observed by

PBC ERM staff and they questioned if the isopods residing in the roots were responsible for the collapse of R. mangle planted at this location as part of restoration activities. Extensive isopod burrows were identified by Dr. Devlin to occur not only in the prop roots and boles of R. mangle, but also Avicennia germinans and Laguncularia racemose (D.J. Devlin, personal observation 2015).

Dr. Devlin collected samples of the isopod and sent them to the National

Museum of Natural History in Washington D.C. where the specimens were morphologically verified to be Sphaeroma terebrans.

34 In May 2016, the Devlin lab did an initial survey of the site to quantify the extent of the observable damage. Every tree that was evaluated as part of this survey displayed signs of extensive burrowing and was actively colonized by S. terebrans. Several mangrove trees ranging from ½ to 3 m were observed to have collapsed on the sediment, many with green leaves, because of the lack of support structure from intense burrowing activity in the roots. Those that had collapsed on the sediment were not included in the survey for evaluation, but rather noted as an observation to describe the extent of the damage at this location.

Burrowing in the grounded root and boles at these sites is highly unusual given the rigid, corticated tissue that results from grounding (Brooks and Bell,

2002). In free-hanging prop roots and roots that have been tied down to the substrate, burrowing intensity is similar between root types. However, in naturally grounded roots, burrowing is typically absent (Brooks, 2002; Perry, 1988). The variation between root types is thought to be attributable to secondary thickening or other change to the root tissue that occurs upon grounding. This causes S. terebrans to select against colonization in grounded roots (Gill and Tomlinson,

1977; Brooks and Bell, 2001).

Curiously, small clumps of barnacles and oysters were present on some

R. mangle individuals at the site, but none had large areas of their prop roots covered with epibionts. The majority of roots included in the preliminary survey had bare tissue without epibiont cover (personal observation, 2016). The high burrowing activity recorded at this site in combination with the lack of epibiont

35 cover made Juno Dunes Natural Area an appropriate study site to determine if C. virginica conferred resistance to R. mangle against S. terebrans burrowing damage.

3.3.2 Age Class Treatments

The stranded R. mangle propagules (hereafter referred to as seedlings) used in this experiment were collected during low tide from the sediment in the intertidal area at the southernmost end of the Indian River Lagoon near Stuart,

FL. At the time of collection, seedlings had primary roots that were beginning to emerge, but no leaves had yet formed nor had roots penetrated the substrate.

The tissue of collected individuals was smooth, yet firm and each seedling was visually examined for damage; Those showing evidence of herbivory or decay were discarded. Seedlings were collected from estuarine habitat with salinity similar to that at Juno Dunes, therefore, no acclimation was required prior to planting.

The two-year-old R. mangle saplings selected for this experiment were from a set of plants grown in freshwater in direct sunlight at the Florida Atlantic

University Harbor Branch campus in Fort Pierce, FL. Saplings were visually inspected to ensure plants did not display signs of senescence or herbivory that could compromise health at the time of selection and again prior to planting in the field. After selection, saplings were acclimated in a pool for a period of 6 wk.

Salinity adjustments of no more than 5 psu were made by applying Instant

Ocean™ or comparable aquarium salt weekly until a salinity of 30 psu, similar to the average salinity at Juno Dunes, was achieved. Fresh water was added using

36 the same schedule to balance the effects of evaporation and maintain the correct salinity in the pool. This slow acclimation to the appropriate salinity was employed to ensure that the effect of transplant shock to the saplings was minimized.

3.2.2 Cover Treatments

In order to achieve C. virginica cover on R. mangle seedlings and saplings, individuals were placed within tanks where oyster larvae (spat) could be released for settlement upon plant tissue. Setting was conducted at Sunray

Venus, LLC’s shellfish hatchery at the Florida Oceanographic Society Coastal

Center in Stuart, FL. The facility has a 1,000-gallon tank and six-650 gallon tanks for oyster setting. Seedlings were suspended in the water column of each tank using 1.27 cm polyethylene foam boards with appropriately-sized holes cut out, while saplings were placed among oyster bags located within the tanks.

These setting tanks varied in the density of spat per gallon, with the 1000-gallon tank containing 200 larvae per mL and the 650-gallon tanks containing 100 larvae per mL. The tanks varied in the quantity of spat to achieve high density and low density coverage on the individual mangrove seedlings and saplings placed within these tanks. In doing, we expected to be able to identify the epibiont coverage required to limit or prevent burrowing upon planting at the selected site. Additionally, partial treatments were developed to control the location upon which spat was allowed to settle. This was done using tape to create a bare lateral side and an ancillary side covered in oysters. Half of these mangroves would be planted with oyster cover facing seaward or landward to

37 determine if the side upon which oyster cover is located has any impact on burrowing.

The setting event did not result in adequate mangrove coverage with oyster spat, as the majority settled on the surrounding oyster bags. Following this attempt at setting, oysters were deployed behind the Florida Atlantic University

Harbor Branch campus in Fort Pierce, FL for 1 wk to see if spat would naturally settle upon the mangroves. At this time, no spat were observed to have settled upon the mangrove stems or prop roots.

As an alternative to the original treatment, crushed oyster shell and adult oyster shell that had ~ three to four juvenile C. virginica located on the concave side, were applied by hand to seedlings and saplings in half and full coverage treatments. The full coverage treatments consisted of the attachment of oyster treatments to the lower 10 cm of each R. mangle individual, including the prop roots. 10 cm was thought to be the appropriate height of coverage based on the location of planting within the site and tidal fluctuations. For partial coverage treatments crushed shell and live oyster treatments covered one the lateral side

(half) of a seedling or sapling, was covered up to 10 cm, but the ancillary side was bare and vulnerable to attack. As with the original experimental design, this allowed the partial coverage treatments to be planted in different orientations, with the shell facing toward the water (seaward) or toward the land (landward). A total of 63 seedlings and 56 saplings were used for this experiment.

The crushed shell treatment was added to the experimental design to better distinguish if C. virginica compete for food with S. terebrans. While shell

38 may provide physical protection, the lack of live oyster eliminates the possibility of competition for a shared resource. Since, Estevez (1978) did not distinguish whether the shelled roots he observed contained live oysters or remnant shell, the driver of reduced burrowing occurring in shelled R. mangle roots remains unclear. The crushed shell treatments were composed of Purina™ crushed oyster shell attached using E6000™ glue. Glue was generously applied to the appropriate side(s) of the individual and crushed shell was pressed into place until it was firmly adhered. Crushed shell was attached to the same height as the live oyster treatments; roughly 10 cm from the base of the individual, including the prop roots of saplings. Partial coverage treatments had crushed shell on one side, whereas total coverage treatments had crushed shell on all sides. The glue was allowed to dry for 24 h and, if needed, bare spots were repaired with additional glue.

The live oyster treatment was included in the experiment to determine if oysters compete with S. terebrans for plankton, a shared food resource. For live oyster treatments, a hole was drilled through the center of each oyster shell, avoiding the juvenile oysters in the process. A wire tie was used to fasten the shell to the seedlings and saplings. Due to the natural variation in oyster shell size, the height of shell coverage varied from 8 to 10 cm. Shells were attached so that the prop roots and the base of each mangrove were covered. Full coverage treatments consisted of three shells that were attached to the base of the seedling or sapling. Partial coverage treatments required one shell to be attached

39 to the base, so that one lateral side was shielded by the shell and the ancillary side was left bare.

Control treatments were used to verify that neither the E6000™ glue nor the wire ties associated with the oyster treatments were deterrents to S. terebrans. For the glue control treatments, a thick layer of E6000™ glue was painted around the circumference of the sapling or seedling up to the height of 10 cm. After the glue was completely dry, it provided a rigid cover that could not be scraped away. For the wire tie control treatment, a wire tie was attached at roughly the same height as those used to attach the live oyster treatments. An unmanipulated control was used only in the seedling age class as the availability of saplings for this experiment was limited.

In order to ensure the survival of the live oysters, live oyster, crushed shell, and control treatments were placed in a flow through tank that was built in

Stuart, FL. Water from the Indian River Lagoon was pumped through the tank for

3 days prior to planting the R. mangle seedlings and saplings on August 3, 2016 at Juno Dunes Natural Area.

Treatment Seedling Sapling Unmanipulated Control X Glue Control X X Wire Control X X Crushed Shell Landward X X Crushed Shell Seaward X X Crushed Shell Whole X X Oyster Landward X X Oyster Seaward X X Oyster Whole X X Table 1. Cover type treatments attached to seedlings and propagules at Juno Dunes, FL.

40 3.3.3 Transplantation

Early August was selected for planting due to experimental evidence that borer density is highest in the summer and early fall (Estevez, 1978; Thiel, 1999).

Burrows are less likely to be vacated during this season due to the presence of both adult females and their offspring (Estevez, 1978), resulting in an increased likelihood of planted individuals experiencing burrowing. A total of 119 R. mangle were planted randomized block design. Blocks were used account for experimental error between the blocks associated with location. This allowed for a more powerful estimate of the effect of treatment. The location of each block was randomly selected in the outer fringe forest between the shoreward edge and 1-m into the mangrove fringe (Figure 11). This is the area where the heaviest burrowing activity occurred, and all blocks were located less than ~0.5 m from infested prop roots.

Within each block, one individual from each treatment was planted in a haphazard design in and among R. mangle roots that displayed evidence of S. terebrans activity (Figure 12). Using a hand trowel, saplings from all treatments were planted so that the root ball was below the surface and seedlings were pushed ~1.5 cm into the substrate. All saplings and seedlings were tethered to bamboo stakes using cotton twine looped around the base of each mangrove to prevent the plants from washing out of the ground. Each individual also received a metal tag identifying it by a four letter code in reference to its age class (plant type) and cover type. The site was checked 24 h and 48 h after initial planting to

41 re-plant individuals that had washed out of the ground. No individuals were found washed out of the ground after the first 48 h.

Mangroves were planted at the site on August 3, 2016 and recordings were taken every 10 days thereafter through October. Data collection continued through November, but recording frequency was reduced to every 15 days during this month. These recordings included a count of total S. terebrans burrows, R. mangle height, number of leaves, and leaf state (green or senescent). Height was used as a measure of plant growth, while leaf count was used as an indication of productivity. Burrowing began to occur in mid-September, but never increased to the extent that the surrounding adult plants experienced. For this reason, the total burrow count at the end of the experiment was used for the statistical analyses rather than assessing burrowing throughout time.

Figure 11. R. mangle planting location at Juno Dunes Natural Area

42

Figure 12. Cover treatments used in manipulative oyster experiment at Juno Dunes Natural Area. a) seedling crushed oyster shell and live oyster treatments; b) seedling and propagule planting within a block; c) full coverage of live oysters on sapling planted at the site.

This experiment was analyzed with five separate factorial ANOVA’s and data were transformed as necessary to meet the assumptions of each test.

Analysis was performed in SAS (University Edition, SAS Institute, Cary, NC,

U.S.A.).

Test Independent Variables Dependent Variable ANOVA 1 Plant Type Total Burrows Cover Type ANOVA 2 (Sapling) Cover Type Height (growth) (Sapling) Total Burrows ANOVA 3 (Seedling) Cover Type Height (growth) (Seedling) Total Burrows ANOVA 4 (Sapling) Cover Type Leaf Count (productivity) (Sapling) Total Burrows ANOVA 5 (Seedling) Cover Type Leaf Count (productivity) (Seedling) Total Burrows Table 2. Independent and dependent variables used in manipulative experiment analyses.

3.4 Results

The lignified R. mangle sapling life stage was more likely to be bored into by S. terebrans than R. mangle seedlings (F=14.90 P=0.002), but there were no differences among cover treatments (F=0.52 P=0.840) and the interaction between cover type and plant type was not significant (F=0.66 P=0.708). Plant 43 tissue did vary by age class, with the seedlings exhibiting a waxy composition in contrast to the rigid lignified tissue of the saplings that were grown in direct sunlight prior to their use in this experiment. In contrast to the large number of burrows observed during the preliminary survey, few burrows (average <1) were observed in experimental R. mangle seedlings and saplings.

Burrows recorded during this experiment were observed in the stem of both seedlings and saplings, as well as the prop roots of the sapling age class.

However, the location of each burrow was not recorded, thus it cannot be determined in the sapling age class if burrowing differed between the stem and prop roots.

Figure 13. Differences in burrowing between cover treatment and plant type. Sites with the same letter are statistically similar based on Tukey a-posteriori comparisons. Error bars represent standard error. Cover treatment is abbreviated according to Table 3.

44 Table 3. Abbreviations used for cover treatment. Abbreviation Cover Treatment UC Unmanipulated Control GC Glue Control WC Wire Control CSL Crushed Shell Landward CSS Crushed Shell Seaward CSW Crushed Shell Whole OSL Oyster Shell Landward OSS Oyster Shell Seaward OSW Oyster Shell Whole

Seedlings grew rapidly, causing saplings and seedlings to be indistinguishable with regards to height and number of leaves. The coded tags provided verification that seedlings and saplings were properly distinguished and corresponding data was recorded. It is important to note that sapling height did not vary with the number of S. terebrans burrows (F=1.52, p=0.222) or cover treatment (F=1.05, p=0.419) and the interaction of these treatments was not significant (F=1.11, p=0.384). In the seedling age class, the relationship was the same, with neither cover type (F=1.29, p=0.274) nor total S. terebrans burrowing

(F=0.80, p=0.456) affecting the growth of the plants. This interaction was also not significant (F=0.15, p=0.704).

45

Figure 14. Relationship between sapling height, cover treatment and burrowing (p=0.2218). Error bars represent standard error Cover treatment is abbreviated according to Table 3.

Figure 15. Relationship between seedling height, cover treatment and burrowing. Error bars represent standard error. Cover treatment is abbreviated according to Table 3.

Sapling productivity, measured as a count of total leaves, was not affected by the cover type treatment (F=0.65, p=0.713) or by total burrowing (F=2.02, p=0.103), and the interaction was not significant (F=0.74, p=0.694, Figure 16).

46 Seedling productivity yielded the same result with neither treatment (F=0.64, p=0.743) nor burrowing (F=2.06, p=0.141) affecting total leaf count. This interaction was also not significant (F=0.30, p=0.585), Figure 17). At the beginning of the experiment, many individuals displayed senescing leaves, likely a result of stress from planting. However, by the end of the experiment in late

November, no plants displayed any senescent leaves. Even those that had burrows maintained all green leaves by the end of the experiment, suggesting the neither treatment nor the level of burrowing experienced affected productivity in young R. mangle.

Figure 16. Relationship between sapling productivity, cover treatment and burrowing. Error bars represent standard error. Cover treatment is abbreviated according to Table 3.

47

Figure 17. Relationship between seedling productivity, cover treatment and burrowing Error bars represent standard error. Cover treatment is abbreviated according to Table 3.

3.5 Discussion

This study was conducted to address the following questions: 1.) Does associational resistance occur between R. mangle and C. virginica by masking mangrove prop roots from S. terebrans burrowing? and 2.) Does competition occur between C. virginica and S. terebrans? Based on these questions, the following main hypotheses were developed: 1) S. terebrans select for soft root substrate; 2) S. terebrans does not burrow in areas of R. mangle root covered with C. virginica; 3) S. terebrans colonization differs between mangroves covered in live C. virginica and crushed shell; 4) S. terebrans burrowing differs between the landward and seaward side of a R. mangle.

3.5.1 Tissue hardness

We predicted that greater burrowing would occur in R. mangle seedlings because substrate hardness is an important factor in determining S. terebrans burrowing patterns (John, 1971a; Howey, 1977). However, burrowing was 48 markedly greater in saplings than in seedlings, despite the tissue of saplings being much more lignified than seedling tissue throughout the experiment. The saplings in this experiment had rigid tissue as a result of their maturity and being grown in direct sunlight. Conversely, the seedlings were flexible both when planted and upon reaching the same height as their sapling counterparts. This is likely the result of shaded conditions under the surrounding adult R. mangle

(Farnsworth and Ellison, 1996).

These results are interesting in comparison to other burrowing organisms that occupy Rhizophora species. The scolytid beetle, Coccotrypes, for example, is an obligate parasite of root tissue of Rhizophora species. Like S. terebrans in

Tampa Bay and Belize, Coccotrypes bores only into the soft root tip of prop roots, but it cannot bore into lignified root tissue (Simberloff et al., 1978; Sousa,

2003a). Instead, Coccotrypes bore into the unlignified embryonic root tissue of propagules, seedlings and saplings (Sousa, 2003a, 2003b). In this experiment,

S. terebrans selected for the more lignified tissue in which to burrow instead of the embryonic tissue present in the seedlings. It is unlikely that a lack of prop roots on the seedlings explains such minimal burrowing in the seedlings, as both

A. germinans, and L. racemosa individuals at this site experienced heavy burrowing despite not having prop roots (personal observation, 2016).

Furthermore, it is documented that S. terebrans burrows into coastal cypress (Poirrier et al., 1998; Wilkinson, 2004) and even man-made maritime structures (Estevez, 1978; Ellison and Farnsworth, 1990; Rice et al., 1990; Cragg et al., 1999), both of which lack prop roots. Our results suggest that seedlings

49 possess some characteristic, such as a defense compound, that is unfavorable to S. terebrans colonization, despite initially having softer tissue in comparison to saplings and adult trees. Additionally, burrowing may be limited in this tissue due to the diameter and amount of submerged surface area available for burrowing

(Brooks and Bell, 2001). The tissue of the seedlings may not be large enough in which to construct a burrow, limiting the ability of S. terebrans to colonize to the extent seen in the surrounding adult R. mangle. It may be to the benefit of those conducting restoration activities in S. terebrans infested habitat to plant individuals as young seedlings during a period of low S. terebrans abundance, instead of waiting to plant until those grown in pots reach the sapling age class.

3.5.2 Associational Resistance

In order to determine if the association between R. mangle and C. virginica can be deemed associational resistance, the attached oyster cover would need to confer some protection against S. terebrans burrowing. The nearby presence of the oysters would be expected to reduce burrowing by concealing, impeding access to, or limiting the movement of this isopod (Rauscher, 1981; Risch, 1981;

Coll and Bottrell, 1994; Holmes and Barrett, 1997; Finch et al., 2003). In this experiment, C. virginica was effective at limiting burrowing only in the area that it covered. No burrows were recorded going through the crushed or live oyster shell and into the R. mangle tissue. Burrows were only identified in the portions of the plant where an oyster was not located. The total number of burrows in the crushed shell and live oyster treatments, however, was similar to that of the glue, wire and unmanipulated controls (F=0.52 p=0.840). This can likely be explained

50 by an error in the experimental design where the oyster shell did not cover the full portion of the tissue exposed to tidal fluctuations. The tide at this location appears to have been higher than originally predicted, leaving exposed tissue on the oyster treatments vulnerable to S. terebrans burrowing. Had the oyster shell and crushed shell been attached to the appropriate tidal height at this location, it is expected that the covered tissue would be free of burrows. This would affect the total number of burrows recorded among cover treatments in this experiment.

As with roots covered in sponges and colonial ascidians, R. mangle was only protected from burrowing damage and colonization in the places where epibionts provided coverage (Ellison and Farnsworth, 1990). Further work is needed to establish if the relationship between R. mangle and C. virginica is associational resistance.

Interestingly, the glue controls also limited burrowing in the bottom 10 cm of the seedlings and sapling to which it was applied. In comparison to the other treatments in this experiment, burrowing was similar due to glue not be applied to the appropriate height. However, no burrows were ever observed to go through the rigid glue treatment. This does not negate the possibility of facultative association occurring between R. mangle and C. virginica, but does highlight the possibility of protection by other impenetrable barriers. Similar to dock and pier piling wraps made of tough, rubber material, the glue may act as a simple physical barrier too difficult for S. terebrans to burrow through.

Additionally, cover treatments did not appear to impact the growth or productivity of R. mangle, measured as height and a count of total leaves,

51 respectively. The oyster shell and crushed shell treatments did not grow to be any taller or put forth any more leaves than the controls. Since S. terebrans damage is documented as altering the architecture of other mangrove species, including reduced leaf size and non-foliated twigs (Davidson et al., 2014), it was expected that individuals with oyster cover may have grown taller and produced more leaves. A bare individual might exhibit altered morphology as a result of heavy burrowing due to a lack of protective cover. However, the treatments did not vary by height or foliage. Again, this is may be a result of similar burrowing among the treatments as a result of the experimental design error.

Considering the high number of burrows recorded in the survey of Juno

Dunes Natural Area, it is also surprising that few burrows were initiated during the experiment. The timing of the experiment was based on peak burrowing occurring in the summer and fall for both Tampa Bay and the Indian River

Lagoon (Estevez, 1978; Thiel, 1999). It is possible that in this region of south

Florida the peak burrowing season differs. Brooks and Bell (2005) noted that population patterns do not appear to be the same from year to year for this species. Alternatively, S. terebrans recruitment may have been unusually low for the season.

Additionally, there may be a physiological or population difference between S. terebrans at the Juno Dunes site and those in sites in Tampa Bay and Belize. S. terebrans in Juno Dunes bore into the boles of R. mangle, A. germinans and L. racemosa, in addition to the grounded roots of R. mangle (D. J. Devlin personal observation, 2015). This may represent a physiological difference suggesting that

52 S. terebrans at this site could be separate subspecies; high levels of genetic differentiation among populations of S. terebrans have been identified (Baratti et al., 2011).

3.5.3 Competition between R. mangle and S. terebrans

We expected that burrowing would be greater in R. mangle covered in crushed shell rather than live C. virginica due to competition for a shared food resource, phytoplankton. However, burrowing activity between the crushed shell and live C. virginica treatments did not differ significantly. Since crushed shell was as effective as live C. virginica at limiting S. terebrans burrowing in R. mangle, this may suggest a simple physical barrier limits burrowing activity.

Additionally, it is possible that phytoplankton is partitioned between C. virginica and S. terebrans and that competition may not actually occur between these species. Since epibiont surveys were not conducted in the surrounding areas near Juno Dunes Natural Area, it cannot be said whether R. mangle with live C. virginica cover was any more effective at limiting burrowing than remnant shell at other sites. Further work is needed to establish if competition exists between C. virginica and S. terebrans for a shared resource.

3.5.4 Selection for optimal feeding space

The side of the root on which epibiont species settle is thought to be mediated by both hydrodynamic flow and larval settlement and other species specific factors (Ellison and Farnsworth, 1996). In Belize, more epibionts were present on the front (seaward side) than on the back of R. mangle roots. We predicted that, as a filter feeder, S. terebrans would build and reside in burrows on the front

53 (seaward side) of seedlings and saplings where plankton are likely more numerous and water flow is generally greater. This side of R. mangle may be optimal feeding space for both individuals and we expected that there would be fewer burrows in an individual covered with oysters on the seaward side.

Burrowing remained at zero for both crushed and oyster landward and seaward treated individuals in the seedling age class throughout the course of the experiment. As a result, the seedling age class did not offer any insight into the location upon which epibionts must cover to limit burrowing damage by S. terebrans. In the sapling age class, there was no difference among the seaward and landward C. virginica treatments. Instead it appears that S. terebrans selected for wherever tissue is exposed, regardless of the side upon which epibiont cover was attached. If R. mangle resistance to S. terebrans burrowing is dependent upon the side of the individual where oysters occur due to selection for optimal feeding space, this experiment was unable to capture that effect.

3.6. Future Work

This experiment could be replicated to include mangroves in several age classes to not only clarify the association between mangroves and oysters, but also to determine at what age a mangrove might receive protection from C. virginica and other epibionts. Surveys for the prevalence of other epibionts should be considered, as oysters may not be the appropriate root mutualist to assess limitation at all S. terebrans affected sites. In addition, by using mangroves of various age classes, the way in which isopod colonization progresses as a tree ages could be more clearly defined.

54 Exploration of the genetics of the population located at Juno Dunes

Natural Area should also be considered. One study (Baratti et al., 2005) has suggested that there is genetic differentiation among populations of S. terebrans and this taxon may consist of more than one species. If the population at Juno

Dunes is genetically different from those described in the literature, it could explain why such atypical colonization is seen in R. mangle at this location.

Additionally, it would be valuable to understand what role genetics plays in determining this isopods distribution and abundance.

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