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A STUDY OF THE EFFECT OF STRESS ON ALBINISM IN RED MANGROVES, Rhizophora mangle (Linnaeus 1753)

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004771/00001

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Title: A STUDY OF THE EFFECT OF STRESS ON ALBINISM IN RED MANGROVES, Rhizophora mangle (Linnaeus 1753)
Physical Description: Book
Language: English
Creator: Grasland, Salome
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Red Mangrove
Rhizophora Mangle
Plant Albinism
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The chlorophyll-deficient mutant phenotype of Rhizophora mangle serves as an ideal tool to study the effects of stress on the plant. The Mendelian inheritance of the mutation and the easy visibility of its phenotype make it an accessible model of assessing the effects of different factors on the plant's genetics. This study quantified and localized the occurrence of the mutant phenotype of R. mangle to examine the effects of environmental stresses on the plant. The findings suggest that stress does have an effect on the prevalence of albinism in R. mangle. The results also suggest that R. mangle trees expressing albinism may have unique adaptations to stress compared to those trees which do not express albinism.
Statement of Responsibility: by Salome Grasland
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Gilchrist, Sandra

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 G76
System ID: NCFE004771:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004771/00001

Material Information

Title: A STUDY OF THE EFFECT OF STRESS ON ALBINISM IN RED MANGROVES, Rhizophora mangle (Linnaeus 1753)
Physical Description: Book
Language: English
Creator: Grasland, Salome
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Red Mangrove
Rhizophora Mangle
Plant Albinism
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The chlorophyll-deficient mutant phenotype of Rhizophora mangle serves as an ideal tool to study the effects of stress on the plant. The Mendelian inheritance of the mutation and the easy visibility of its phenotype make it an accessible model of assessing the effects of different factors on the plant's genetics. This study quantified and localized the occurrence of the mutant phenotype of R. mangle to examine the effects of environmental stresses on the plant. The findings suggest that stress does have an effect on the prevalence of albinism in R. mangle. The results also suggest that R. mangle trees expressing albinism may have unique adaptations to stress compared to those trees which do not express albinism.
Statement of Responsibility: by Salome Grasland
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Gilchrist, Sandra

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 G76
System ID: NCFE004771:00001


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A STUDY OF THE EFFECT OF STRESS ON ALBINISM IN RED MANGROVES, Rhizophora mangle (L innaeus 1753) BY SALOME GRASLAND A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for th e degree Bachelor of Arts Under the sponsorship of Professor Sandra Gilchrist Sarasota, Florida April, 2013

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ii Acknowledgments First and foremost, to Dr. Gilchrist for patiently working with me from my days as a clueless first year to a less clueless, bu t still pretty clueless, fourth year. Thank you for the opportunities you have given me and the knowledge you have helped me acquire. I would like to thank Dr. Clore for bein g on my committee and for flexing my creative possibility via her classes and brai nstorming for the outreach program. Great thank s to Dr. McCord for being on my committee and exposing the beauty of plants and insects to myself and fellow students and for giving me a repertoire of facts about cockroaches that make for great conversation at any dinner party. Thank you Dr. Eric Milbrandt for answering my questions and taking me out into the field, the experience was crucial and ails and clarifying questions. Lastly, I would like to thank my family. To my mother for teaching me strength and perseverance. To my late father for teaching me how to be an errant adventurer. To my sister for encouraging me to become involved in education and outreach. To my partn er, Helena Benedict, for her kindness and humor.

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iii TABLE OF CONTENTS CHAPTERS PAGE TITLE PAGE ..................................................................................................... ................. i ACKNOWLEDGMENTS............ ....................................................................... ............... ii TABLE OF CONTENTS ................................................................................... ............... iii LIST OF FIGURES............................... ........................................................... ................. v LIST OF TABLES ............................................................................................ ................. v i ABSTRACT ........................................ ............................................................. ................ v i i CH APTER 1 : Introduction ................................................... ............ ............................. .. 1 1.1: Introduction 1.2: The Cause of Albinism ... 1 1.2.1: The Origin and Morphology of Chloroplast 1.2.2: Chloroplast Biogenesis 1.2.3: Environmental Factors Affecting Albinism 1.3: Albinism in Rhizophora mangle ..................................................... ............................ 8 1.4: Stress Toleran ce of Rhizophora mangle and its Effects on Albinism ........................13 1.4.1: Natural Stressors.............................................................. ............................13 1.4.2: Anthropogenic Stressors............................. ..................... ............................14 1.4.3: How Stress Could Induce Albinism................................. .......... ..................16 1.5: Using the Mutant Phenotype to Assess the Effect of Stress on Rhizophora mangle.. 17 C HAPTER 2: M aterials and Methods ............................................... ............................19 2.1: Rhizophora mangle Surveying, Seed Collection, and Growth....... ............................19 2.2: The Effects of Oil on Seedling Growth and Lenticel Mor phology ........ ...................23 2.4: Stomata Morphology and Behavior at Varying Salt Concentrati ons.........................26

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iv 2.5: Secondary Plant Compound Assay Using Aratus pisonii...... ......... ............................ 27 2.6: Lenticel Morphology of Samples Taken from HER and PHODR Trees Observed via SEM Microscopy................................................................................... ............................28 CHAPTER 3: Results ................................................... .. .....30 3.1: The Survey...................................................................................... ............................30 3.2: Secondary Plant Compound Assay Using Aratus pisonii............... ............................ 39 3.4: Stomata Morphology and Behav ior at Varying Salt Concentratio n...........................43 3.5: The Effects of Oil on Seedling Gro wth and Lenticel Morphology ............................45 3.6: Lenticel Morphology of Samples Taken from HER and PHODR Trees Observed via SEM Microscopy................................................................................... ..................... .......47 CHAPTER 4 : Discussion 4.1: Albino Expression and Correlation to Stress ............................... ..............................49 4.1.1: Possible Difference between Seedlings and Leaf M 4.1. 4.2: Lenticel Morphology as Seen by SEM........................................... ................. ..........52 4.3: The Heterozygote Advanta ge......................................................... ............................54 4.3.1: Experimental Result 4.3.2: Is Heterozygote Advantage Selection Genetically Sig nificant....................56 4.4: Future S tudies ............................................................................... ..............................57 4.4.1: Reactive Oxygen Species and Rhizophora mangle Antioxidant Enzyme Production...................................................... ............................ ............................57 4.4.2: Genetic Analysis.............................................................. ............................57 4.4.3: How to Properly Grow Rhizophora mangle Seedlings.... .................... ........58 4.4.4: Looking at the Interaction Between Biotic Factors and Albinism 59 4.5: Conclusion .................................................................................... ..............................60 REFERENCES .......................... ............. .................... .......................... ............................6 1

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v LIST OF FIGURES Figure 1. 1. Electron Micrographs of a C Figure 1.2 Chloroplast B Figure 1.3 Chloroplas t Biogenesis D Figure 1.4 Chloroplast Biogenesis F Figure 1.5. World Map of M Figure 1.6 Figure 1. 7. Mendelian inheritance of see Figure 1. 8. Figure 1. 9. TEM of Nuclear Mutant R8 Leaf Mesophyll Cells and C .12 Figure 1 .10. Network of Antioxidant Production in Different O .. 18 Figure 2.1 Surveyed A ... 20 Figure 2.2 Location of Heterozygou Figure 2.3 Rhizophora mangle Figure 2.4 Rhizophora mangle Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Aratus pisonii Figure 2.9 34 Figure 3.2 Aratus pisonii Figure 3. Figure 3.4 Stomata Aperture at Varying Concentration Figure 3.5. Figure 3.6 Figure 3.7 Figure 3.8 SEM Photos of Lentic Figure 4.1. Internal Airflow in Rhizophora mangle

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vi LIST OF TABLES Table 2.1 Table 2.2 Table 3.1. Table 3.2. Table 3.3 Table 3.4 Water Quality Measurements of Bli Table 3.5. Table 3.6. Table 3.7.

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vii THE EFFECTS OF STRESS ON ALB INISM IN RED MANGROVES, Rhizophora mangle Salome Grasland New College of Florida ABSTRACT The chlorophyll deficient mutant phenotype of Rhizophora mangle serves as an ideal tool to study the effects of stress on the plant. The Mendelian inheritance of the mutation and the easy visibility of its phenot ype make it an accessible model of assessing the effects of different factors on the plant's genetics. This study quantified and localized the occurrence of the mutant phenotype of R. mangle to examine the effects of environmental stresses on the plant. The findings suggest that stress does have an effect on the prevalence of albinism in R. mangle. The results also suggest that R. mangle trees expressing albinism may have unique adaptations to stress com pared to those trees which do not express albinism. Sandra Gilchrist Division of Natural Sciences

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1 INTRODUCTION Section 1. 1: Introduction Plant albinism is normally characterized by a lack of or modified chloro phyll in normally green tissue. It typically renders the plant incapable of photosynthesis and generally leads to premature death (Kumari, 2008). Plant albinism is the result of failed chloroplast biogenesis and development. Albinism can be expressed in a variety of ways the entire plant may be albino or just a branch; there are cases even in which just a certain portion of a leaf will express albinism (Collins, 1927) There are multiple factors involved in the proper synthesis of chloroplast; a modificatio n of environmental, cellular, and temporal factors can all affect the process (Pogson, 2011). Section 1. 2: The Cause of Albinism Section 1. 2.1: The Origin and Morphology of Chloroplast M itochondria and chloroplast s are theorized to have evolved from a se ries of endosymbiotic events. Mitochondria first evolved from an proteobacterial ancestor that was engulfed by a eukaryotic host (Futuyama, 2005) About 1 1.5 billion years ago, chloroplast s arose from a cyanobacterial ancestor which was engulfed by a eukaryote in which mitochondria had been established (Cavalier Smit h, 2004). In time, the majority of the bacterial genes were transferred to the nuclear genome or lost; however, both mitochondria and chloroplast have preserved metabolic activities, genetic mechanism s and protein transport complexes that echo their proka ryotic start (Sakamoto et al., 2008). Chloroplasts are generally lens shaped, 5 10 microns in diameter, and 2 4 microns in thickness. A typic al leaf cell will have approximately between 20 to 100

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2 chloroplast s (Lopez Juez and Pyke, 2005). Chloroplast s a re surrounded by two membranes, known as the outer and inner membrane. Within the chloroplast are disc shaped stacked thylakoids known as grana. Chloroplasts ha ve three aqueous compartments. The two envelopes are the inter membrane spa ce; the stroma which is the space surrounded by the inner membrane, and the thyl akoid lumen, the aqueous area within the thylakoid. T he inner and thylakoid membrane s are descen ded from engulfed cyanobacteria ( Fig. 1 .1) (Sakamoto et al., 2008) Section 1. 2.2: Chloroplast Bioge nesis Chloroplast biogenesis and development in seedlings occurs when the proplastid differentiates into a m ature chloroplast (Forreiter and Apel, 1993) In dark germinated seedlings the undifferentiated proplastid forms a structure known as an etioplast w hich has a lattice like membranous structure known as the prolamelar body (PLB) which serves as a scaffold during chloroplast biogenesis. The PLB is the site of protochlorophyllide, the precursor to chloroplast. When protochlorophylli de is acted upon by th e light dependent NADPH Protochlorophyllide Oxidoreductase (POR) it will eventually become chlorophyll (Pogson, 2011). In angiosperms, plastids development in seedlings occurs because light induces POR enzyme activity to convert protochlorophyllide into ch lorophyllide a which is converted to chlorophyll a and b. While the chlorophyll is being produced the thylakoid develops along the newly synthesized photosystem (Forreiter and Apel, 1993 ). In F igure 1.2 this process is imaged showing the development of the chloroplast from proplastid to chloroplast and the role of light in the process.

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3 Albinism is a result of chloroplast biogenesis failing to occur properly (Kumari, 2008). Chloroplast gene transcription, RNA maturation, retrograde signaling, chloroplast division, protein translation and modificati on are all factors affecting c hloroplast biogenesis. A n error any of these processes can result in plant albinism as seen in figure 1.3 which displays the different factors that can effect chloroplast biogen esis and how they interact within the organism (Pogson, 2011). An important mechanism in cell process es is plastid to nucleus retrograde signaling. This is when the plastid sends signals to the nucleus to control nuclear gene expression. In albino plants thi s process is often jeopardize d due to a mutation in ei ther the plastid or the nucleus (Pogson et al., 2008). Figure 1. 1 Electron micrographs of a chloroplast (upper), a proplastid (lower left) and an etioplast (lower ri ght) in Arabidopsis. gr, grana; ie, inner envelope membrane; oe, outer envelope membrane; pg, plastoglobule; pl, prolamelar body; rs, ribosome; sg, starch granule; st, stroma. Scale bars: upper and lower right of upper panel, 50 nm; inset of lower right panel, 100 nm. (B, courtesy of Dr. Chieko Saito in RIKEN)

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4 Section 1. 2.3: Environmental Factors Affecting Albinism Albinism can also be induced in plants via the modification of environmental factors. The alteration of sucrose levels, light, plant growth regulators and other components can all affect albinism in plants (See Figure 1.4) (Collins, 1927 ; Chory et al., 1991 ; Moon, 2008 ). Sucrose Levels Sugar is a crucial compound in tissue culture medium. I t affects the amount of carbon available for cellular uptake and balances the osmotic environment (Kumari 2008) According to Nishiyama and Motoyos hi (1962, 1966) glucose, indole acetic acid (IAA), kinetin, and casein hydrolysate added to media help promot e the formation of chlorophyll. In an experiment done on barley in which a sucrose solution was given to the plant (Sorvari and Scheider, 1987), it was shown that modifying the conce ntration of sucrose to be higher or lower than biological levels could alt er the expression of albinism in the plant. In a similar study by Saidi and coll eagues (1997), the manipulation of sugars in medium could turn partially albino Triticum turgidum anthers green. Light Light is crucial in the transcription of certain genes. Moon (2008) found that one third of when plants are exposed to light This is important in considering plant albinism because the majority of genes activated by light are responsible for encoding chloroplast t argeted proteins (Kumari, 2008). When light is perceived by a cell it will activate mo lecules known as phytochromes. T his is mediated via the assistance of transcription factors known as phytochrome interacting factors

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5 (PIFs). It was discovered that the lo ss of PIF1 or PIF3 resulted in the delayed development of chloroplast (Moon et al., 2008). In a study done on strawberries a correlation was displayed between intense shading, and the occurrence of albinism (Sharma, 2004). Plant Growth Regulators Plant g rowth regulators (PGR) have a potent effect on plant development and cell division even in low doses, especially auxins and cytokinins contribute to plant albinis m (Kumari, 2008). Chory and colleagues (1991) added cytokinin to media and encouraged the conv ersion of etioplast to chloroplast in Arabidopsis. The addition of IAA and benzyl adenine (BA) to anther and ovary co culture reduced the presence of albino plants produced via doubled haploid production in wheat (Broughton, 2008). Other Components Copper has a variety of roles in biochemical pathways i n plants, such as, enzymatic, protein, and carbohydrate biosynthesis (Dahleen, 1995). The addition of copper along with 2 ,4 D and benzyl aminopurine to media increased the regeneration of green plants in bar ley tissue culture (Cho et al., 199 8; Nuutila et al., 2000). Similarly the addition of copper sulphate also reduced the presence of albino seedlings in barley (Wojnarowiez et al., 2002). Ficoll (Pharmacia), a high molecular weight polysaccharide, when add ed to media increased the ratio of green to albino plants (Kao et al., 1991; Zhou et al., 1992).

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6 Figure 1.2. Chloroplast biogenesis and development in seedlings. PLB, Prolamellar body; PT, prothylakoids (Pogson, 2011). Fi gure 1.3. Diagram of the process required for chloroplast biogenesis (Pogson, 2011).

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7 Figure 1.4. Model of factors that influence chloroplast biogenesis, such as environmental, cellular, and temporal fa ctors (Figure taken from Pogson, 2011).

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8 Section 1. 3: Albinism in Rhizophora mangle A recent discovery that has captured the attention of scientists is the appearance of albinism in R. mang le. The red mangrove, Rhizophora mangle is one of the most important vascular plant species in the New World tropical intertidal ecosystem (Lowenfeld and Klekowski, 1992) It is found across the world, from Vietnam to Puert o Rico, in tropical estuary and some subtropical estuaries (Fig 1 .5 ) Its ability to survive saltwater intertidal zones and its impact on ecosystem stability have made the plant a commonly researched subject among scientist s worldwide (Proffitt and Travis, 2007 ). R hizophora mangle has a unique reproductive strategy the seedlings are cryptoviviparous, meaning that the seeds grow directly out of the seed coat and surrounding fruit while still attached to the mother plant and where they remain for four to six months (Fig. 1 .6 a ) ( Kathiresa n and Bingham, 2001 ). This allows the seedlings to develop salt tolerance before being released and provides a store of nutrient s which promotes quick rooting in the muddy environment (Bhosale and Mulik, 2001 ). Some seedlings will lack chlorophyll and have a yellow or reddish appearance, rather than the normal green phenotype ( Fig. 1 .6b ) (Lowenfeld and Klekowski, 1992). Rhizophora mangle tree s which yield albin o seedlings will produce them in a 1:3 ratio, supporting the idea that M endelian inheritance is occ urring (Fig. 1 .7 ) Thi s r atio may deviate due to factors such as outcrossing, sib competition between ovules with the same ova ry, or via the abscission of ovaries bearing mutant e mbryos (Lowenfeld and Klekowski, 1992) B

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9 Section 1.3.1 : Debated Current R esearch About Mendelian Inheritance Lol le and colleagues (2005) showed the fundamental tenet of classical M endelian inheritance, which dictates that genetic information is solely inherited from one generation to the next, is not strictly correct. In the s tudy Arabidopsis plant s homozygous for recessive mutant alleles of the organ fusion gene HOTHEAD (HTH) were used to demonstrate that they could inherit allele specific DNA sequence information which was not present in the parent, but pres ent in previous ge nerations. This newly described process occurs at all DNA sequence polymorphisms examined and is a general mechanism for extra genomic inheritance of DNA sequence information. It is speculated that this genetic restoration event is a results of a template directed process which uses ancestral RNA sequence cache (Lolle et al., 2005) Figure 1.5 World map of mangroves. The green represents all areas where mangroves can be found (taken from Wikipedia Commons).

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10 Figure 1.7. An illustration demonstrating the Mendelian inheritance of the seedlings. Drawing by P. J. Godfrey (Figure taken from Klekows ki et al., 1994) Figure 1.6a. A mangrove seedling while still attached to the mother tree with parts labeled. Figure 1.6b. R. mangle seedling demonstrating green and yellow (albino) phenotypes. The white arrow points to a heterozygous albino seed and the yellow arrow to a homozygous albino seed. Photos taken by Salome Grasland. A B

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11 Section 1.3.2: Pigmentation of Albino Seedlings There are four major patterns of pigmentation that have been observed in albino mutants. The white phenotype lack s all pigmen ts. A second group lacks chlorophyll a and b, but contains most carotenoids and xanthop hyll at lower concentration than the wild type. The third type has all pigments at reduced level s The fourth type, known as LIBI, has all the carotenoids and chlorophyl ls that have similar light abso rption spectra as the wild type; however, their retention times when assayed via gas spectroscopy are significantly different from that of the corresponding wild type pigments (Fig 1.8) (Klekowski et al., Rhizophora mangle A, Mesophyll cells are typical for a higher plant. Chloroplasts contain grana stacks, stroma thylakoids, starch grains (S), and osmiophilic droplets (0). Nuclei (N), mitochondria, Golgi bodies, endoplasmic reticulum, and vacuoles are also visible. Scale bar = I Am. B, Higher magnification shows details of grana stack (G) from a chloroplast s hown in A. Scale bar 1994).

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12 1994) The plastid in LIBI is spherical in shape with flat membrane resembling prothylakoids connected at the periphery and extending into the plastid stroma. It has no visible grana stacks (Corredor, 1995). The chloroplast structure of mangroves is typical of most dicots. However, in chlorophyll deficien t offspring, the chlorop hyll are often lacking or have underdeveloped grana thylakoid, stroma thylakoid, a lack of starch, and little to no osm ioph i lic droplets (Fig 1.9 ) (Klekowski et al. 1994). Rhizophora mangle All mesophyll cells studied contain chloroplasts with similar ultrastructure. A, Mesophyll cel ls of mutant R8 resemble wildtype mesophyll cells in most respects but contain chloroplasts (C) that lack grana thylakoids and starch. Scale bar = 1 Mm. B, Higher magnification of chloroplast shown in A shows stroma thylakoids (T) and osmiophilic 1994).

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13 Section 1.4: Stress Toler ance of Rhizophora mangle and its Effect on Albinism Rhizophora mangle inhabit s an ecosystem in which most plants would wither. E ven though the plant is well suited for extreme environment it actually has a low tolerance for stress (Conghe, 2011). Modifica su ch as salinity, tides, and the exposure to intense storms and pollutants are all stresses which have been shown to negatively affect R. mangle. Section 1.4.1: Natural Stressors Salinity Salinity p lays an important role in R. mangle development. The plant uses ion compartmentation, osmoregulation, and selective transport and uptake of ions to survive in saline conditions (Parida and Jha, 2010) However, the balancing act of maintaining a supply of ions to the shoot and t he plants threshold limit to accommodate the salt influx can stress the plant (Jitesh et al., 2008) Tides Lin an d colleagues (1993) showed that fluctuating salinity levels had a negative effect on R. mangle growth. In the field, conditions like these wo uld be encountered in lagoons or areas with poor circulation and extreme tides It is hypothesize d that salinity fluctuations plays a role in creating dwarf mangrove forest s which are stands of forest charact erized by stunted tree growth (Conghe, 2011 ). Storms Hurricane Charley was a level five hurricane that devastated the town of Punta Gorda, FL

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14 in 2004. It made landfall at the mouth of the Charlotte harbor In its wake it managed to destroy a sizable portion of the R. mangle forest al coastline. Proffitt and colleagues (2006) showed that the remaining trees had proportionally lower rate s of reproduction compared to si tes not affected by the storm and that t he stress of the storm also reduced tree growth rates and colonization abilities (Proffitt et al., 2006) Section 1.4.2: Anthropogenic Stressors Mangrove forests have been ranked as one of the most sensitive ecosystems to oil spills compared to other coastal environments (Getter et al., 1985). It has been repeatedly shown that mangrov e growth and survival rates are hindered by the exposure to various oil treatments (Lewis, 1980; Dicks and Westwood, 1987; Dutrieuz et al., 1990; Guzman et al., 1991) but the mechanism has no t been elucidated. Getter and colleagues (1985) showed that the plant exposure to aromatic and aliphatic fractions caused plant stress in R. mangle. Bayen (2012) discovered trace metals, polycyclic aromatic hydrocarbons, persistent organic pollutants, and other organic pollutant in mangrove ecosystems spanning the worl d Trace Metals Trace metals have been measured in varying concentration s in mangrove ecosystems and the exact sources of the metals are typically a combination of natural sources and anthropogenic input (i.e., gold mining, leaded gasoline, anti fouling, a nd landfills) ( Kehrig et al., 2003; Chatterjee et al., 2009). The metals can reach the trees by aquatic transport and atmospheric deposition (Rumbold et al., 2011). The tree will intake the

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15 metal and prevents its movement to proximal water systems which ca uses varying degrees of harm to the individual tree (Bayen, 2012) Polycyclic aroma tic hydrocarbons ( PAHs ) Crude oil is a term used to define a complex mixture of hydrocarbon s and non hydrocarbon compounds. Even if the toxicity of each compound in the mix ture is known it is difficult to assess the toxicity of the combined product because of the synergistic, additive, or antagonistic effects of the combination (Overton et al., 1994). However, c rude oil generally has high amounts of polycyclic aromatic rings (Bayan, 2012). In mangrove forest sediment affected by an oil spill of f the coast of Brazil it was reported that PAHs levels reached 240,394 ng/g (Farias et al., 2008). A general trend has been observed that the phytotoxicity increases with the addition o f up to three aromatic rings and decreases at more than four rings (Baek et al., 2012). Henner and colleagues (2009) demonstrated that water soluble PAHs that had fewer than four rings all hindered seedling germination. Persistent Organic Pollutants (POP s) POPs, such as, polychlorinated biphenyls ( PCBs ) and organochlorine pesticides OCPs (i.e., DDTs, chlordanes, endosulfans, hexacholorohexane etc. ) are pollutants of worldwide concern (Bayan, 2012). They enter the waterway predominately through illegal du mping and the breakup of old ships (Gioia et al., 2011). POPs were found in low concentration in the sediments of mangrove trees stands in Vietnam left over from the dispersal of Agent Orange, a herbicide used during the Vietnam war (Kishida et al., 2010).

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16 Other Organic Pollutants Other pollutants found in mangrove sediments are solid waste from plastics and various insect repellants which all have varying degrees of harm to the plant (Bayan, 2012). Section 1.4.3 : How Stress Could Induce Albinism Stres s in plants can create varying nega tive side effects i.e., seed germination, seedling growth and vigor flowering and fruit set, and vegetative growth. When a p homeostasis is disrupted, changes occur at the cell level and the whole plant level, whic h can eventually lead to tissue death and entire plant death (Xiong and Zhu 2002). Stress affects the integrity of cellular membranes, enzymatic activity, and photosynthetic abilities (Serrano et al., 1999). The main reason stress incurs plant damage is du e to the production of reactive oxygen species (ROS) (Smirnoff, 1993). Reactive oxygen species are a natural byproduct of plant metabolism created by the univalent reduction of O 2 or by transferring excess excitation energy. The transfer of one, two, or th ree electrons generates superoxide radicals, O 2 H 2 O 2 and HO respectively (Mittler, 2002) Chloroplasts, mitochondria, and pe roxisomes generate more ROS i.e., O 2 and H 2 O 2 tha n any other intracellular process ( Fig ure 1.10 ) (Hernndez et al., 1995). One of the main differences between glycophytes (non salt tolerant plants) and halophytes (salt tolerant plants) is their ability to handle ROS via the producti on of antioxidant enzymes (Rout and Shaw, 2001). However, if a tree becomes too stress ed then p lant tissue damage will be caused by a buildup of ROS (Jitesh, 2006); a lbinism in R. mangle may be due to this phenomenon

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17 Section 1.5: Using the Mutant Phenotype to Assess t he Effect of Stress on Rhizophora m angle Proffitt and Travis (2005) found a pos itive correlation between contamination and albinism in R. mangle At several sites which had prolonged exposure to crude oil an increased rate of albinism was seen in comparison to similar non contaminated sites. It has been shown that environmental cont amination may affect the growth, photosynthetic activity, survival and recruitment of propagules, number of propagules per tree, and the proportion of reproducing trees in a population of R. mangle (Proffitt and Travis, 2005). Rhizophora mangle is an ideal species to study the effect of stress on putative mutation rates and reproductive benchmarks which affect fitness (Proffitt and Travis, 2005). It has been shown that those R. mangle forest s expressing albinism are in mutation selection balance ; hence, mut ation rates can be accounted for by assessing the frequency of R. mangle trees heterozygous for albinism (Lowenfeld and Klekowski, 1992) The current study focuses on the Charlot te H arbor watershed and use s the R. mangle mutant phenotype as an environmental indica tor of increased contamination in an ecosystem. By using the mutant phenotype it can be assessed how contamination and stress affect entire R. mangle forest

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18 Figure 1.10. Network of antioxidant production in different organelles (Jithesh et al., 2006).

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19 MATERIALS AND METHODS Section 2.1: Rhizophora mangle Surveying, Seed Collection, and Gr owth R hizophora mangle was surveyed i n several different areas from Port Charlotte, FL to Bradenton, Florida and in total 13 areas were examined (Fig. 2.1) Surveys were made via kayak in 2012 during the months of June to October, which are the peak months of R. mangle reproduction (Proffitt and Travis, 2005) The t rees observed were within a two meter radius of the tree line. While surveying, the number of reproducing trees were counted. Reproducing trees are considered to be any tree with more than 30 s eedlings as designated by Proffitt and Travis (2005) The numbers of het erozygous albino mangroves were compared to the overall number of reproducing trees to assess how prevalent they were in the population. Each sites water quality was assessed using LaM otte GREEN Water Quality Monitoring Kit The water was tested for phosphate, nitrate, acidity, turbidity, temperature, salinity, and dissolved oxygen. Rhizophora mangle seedlings that had fallen into the water were collected from four different sites (So uth Lido, Saras ota, FL, {27.301359, 82.567196} ; Venice Boat Launch, Venice, FL, {27.046362, 82.42 9120}; Cortez, Long Boat Key, FL {27.488270, 82.689846}; Blind Pass, Venic e, FL {26.965358, 82.384737}) (Fig. 2.2) These areas all featured one complete hete rozygous tree ( Fig. 2.3 ). This means that 25% of the seedling s on the tree did not have green pigmentation. About twenty green seedlings were c ollected from the area around reproducing heterozygous trees (HER), placed in bags with moist paper towels and ke pt at ambient temperature (20 32 C)

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20 Figure 2. 2 Location of heterozygous albino mangroves from which seeds were collected from marked by yellow balloons. Created with Google Maps Software. Figure 2.1. Blue place markers represent areas surveyed. Created with Google Maps Software.

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21 The seedlings were planted within 12 hours of retrieval. Twenty seedlings were also taken from the area around a nearby putative homozygous dominant reproducing (PHODR) tree to serve as controls. A tr e e was deemed homozygous if it did not have albino seedlings at the time of collection; however, the tree was not genetically tested, so the term homozygous in this paper is based on phenotype rather than genotype. The seedlings were planted 6 8 cm deep in tanks filled with 10 12 cm of silty sand from the New College bay front (Fig. 2.4) Figure 2. 3 shows seedlings still attached to the tree. T he base of the seedling is heavier so that plant remains oriented in the correct direction while floa ting. The two ends of the seedling can be differentiated because the shoot apical meristem, where new shoot growth occurs, has a small point remnant from its point of attachment to the fruit. The seeds were buried with root apical meristem (RAM) into the ground and the shoot apical meristem (SAM) upwards. Water levels were kept 2 4 cm above the soil line and replenished on a n as n eed ed basis. The seedlings were watered with tap water and once a month were watered with saltwater to replenish ion concentrati on All salt water was from the New College Pritzker Marine Lab which is filtered sea water originally from the bay. The plants were kept outside and received sunlight from a southerly exposure at all daylight times except one h our before sunset and after dawn.

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22 Figure 2.4. Rhizophora mangle seedlings planted. Figure 2.3. Rhizophora mangle se edling, the orange arrow points to a non albino green seedling, while the white arrow points to an albino seedling. Photo taken by Salome Grasland.

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23 Section 2.2: The Effects of Oil on Seedling Growth and Lenticel Morphology The seedlings were randomly divided into three groups, one control and two experimental Each group had an even amount of seedlings from HER and PHODR trees The experimen tal groups were exposed to castor oil to mimic the effects of exposure to a viscous cont aminant. Crude oil, a contaminant found in the Gulf of Mexico, was not u sed in this experiment to avoid contamination of the surrounding area. Castor oil and motor oil SAE 40 both have a viscosity of about 250 500 centipoise and similar physical behavior, hence, castor oil, a less toxic oil, was used for the experiment (Research Equipment London Limited). MARPOL ( International Convention for the Prevention of Pollution Fr om Ships, 1973 as modified by the Protocol of 1978) has deemed 15 ppm to be the threshold toxicity level of crude oil, a marina typically has a concentration near or less than this level. The typical oil concentration coming from a point source leak is abo ut 300 ppm (International Maritime Organization 2006) Rhizophora mangle seedlings from HER and PHODR trees had water with varying concentrations of oil poured over them and their responses were observed. Table 2.1. Concentrations of castor oil R. mangle seedlings were exposed to. Control Exposed to 0 ppm of oil Experimental 1 Exposed to 20 ppm of oil, slightly above reported threshold toxicity. Experimental 2 Exposed to 300 ppm of oil, concentration found near point source oil spill.

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24 Experimental Gr oup 1 was exposed to 20 ppm of oil, which is one drop of oil per 2.5 liters of water. The toxicity factor of the oil was based on its physical interaction, such as rather than chemical interactions. The o il wa s introduced by mixing castor oil into 19 liters of sea water and pouring it un to the plants. Several aspects of t he plant were monitored, including, growth rate, root development, and lenticel development and morphology (Fig. 2.5) Lenticel quantit y was recorded along an area of the plant 1cm wide from the base of the plant to the shoot apica l meristem The l enticels were also observed for quantity and clogging. C logged lenticels have a swollen, woody appearance (Fig 2.5) The duration of the experi ment was too shor t to get leaf growth; instead to monitor growth, seedlings were uprooted after five months and root length was recorded. All seedlings initially had zero root growth at the start of the experiment. Figure 2.5. Clogged lenticels (blue arrow) have an outward appearance while unclogged are a small white dot (white arrow). Photo by Bill Keogh taken from http://www.nhmi.org/mangroves/index. html

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25 Section 2.3: Transpiration Rates of Hom ozygous Dominant Reproducing Trees versus Heterozygous Albino Trees Table 2.2. Each groups salinity concentration. Transpiration rates of the plants wer e studied by using potometers. A potometer is a de vice that allows a stem cutting to be used to determine transpiration by the leaf over a period of time (Fig. 2.6). Leaf clippings, throughout January and February, were taken from adult HER and PHODR trees at the South Lido and Cortez sites and kept in a phosphate buffer The leaves were divided into three groups and exposed to water of varying levels of salinity. The lighting was kept consistent throughout the trials by using dissection scope light s pointe d at the leaves. The transpiration rates of leaves from HER and PHODR trees were compared. The av erage salinity of the Sarasota B ay is 30 ppt (Sarasota County Water Atlas); hence, salinity concentrations of 0 ppt, 35 ppt, and 55 ppt were chosen to expose t he plants to no stress (0 ppt), low stress (35 ppt) and extreme stress (55 ppt). Group Salinity Group 1 (Control) 0 ppt Group 2 (Experimental) 35 ppt Group 3 (Experimenal 55 ppt Figure 2.6. The potometer was made by gluing aquarium tubing to the tip of a 10mL pipette. Photo taken by S alome Grasland.

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26 Section 2.4: Stomata Morphology and Behavior At Varying Salt Concentrations S responsible for assisting with gas exchange. The opening is controlled by guard cells. When guard cells are turgid the stomatal opening widens. The plant can open and close the opening in respons e to light inten sity, CO 2 and to control water loss due t o transpiration. R hizophora mangle stomata were observed from HER and PHODR trees. Stomata were observed on leaf clippings that were floated in solutions that had 0 ppt, 35 ppt, and 55 ppt concentration of salinity. S tomata were all exposed to consisten t light provided by a dissecting light. A pertures of 100 stomata from 10 one cm 2 leaf samples were observed for each trial using an Olympus BX40 compound scope with an attached Olympus DP71 digital camera. Figure 2.7. Stomata as seen using a light microscope. The opening (black arrow) can close and widen. The two cells surrounding the opening are known as guard cells (white arrow) Photo taken by Salome Grasland.

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27 Section 2.5: Secondary Plant Compound Assay Using the Mangrove Tree C rab, Aratus pisonii (H. Milne Edwards 1837) S econdary plant compound is any chemical produced by the plant that is believed not to be essential for me tabolism (Whittaker, 1970). C ompounds occur in plant fami lies often of different lineage; hence, it appe ars that they have evolved independently for the defense of a common enemy s uch as a fungi or insect (Feeny, 1975). Rhizophora mangle is known to produce secondary plant compounds used to prevent herbivory and for other function s such as wound healing (Ba ndaranayake, 2002). HER R. mangle trees may have altered production of secondary plant compounds because other biochemical pathways are mutated. The mangrove tree crab, Aratus pisonii is an herbivore of R. mangle and was used during this experiment to test leaf palatability. Leaves were collected from adult HER and PHODR trees. The trees were of similar age, size, and leaf consistency. The leaves had their stems immersed immediately in a phosphate buffer at biological concentration. Aratus pisonii were collected from the same location and kept in a clear were starved for 12 hours and then placed in a tank with the collected leaves for one week. Each tank had two crab s and two leaves from each tree type with their petioles placed in a 2 mL tube with fresh water to avoid leaf desiccation. Only two cr abs were placed per tank to minimize interactions. The crabs were also sexed to avoid putting two males together which wou ld result in fighting. The amount of herbivory which occurred over a eaves were traced onto paper before the experiment, A B

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28 after a week the leaves were super imposed onto their tracings an d the herbivory was mapped. H erbivory was q uantified by measuring the cm 2 of leaf area consumed. Section 2.6: Lenticel Morphology of Samples Taken from HER and PHODR Trees Obse rved via SEM Microscopy Lenticels were collected from HER and PHODR trees two meter above the water line on the trunk of the tree by taking pee lings of bark using a razor blade They were placed immediately into a container with paper towels moistened with PBS buffer. The samples were placed in a petri dish and put in a chemical desiccator and left to dry for a week. Once d ried, the lenticels were trimmed and mounted on an SEM stub using double sided adhesive tape. T he samples were sputter coated with gold using an EMS 76 mini coater (Ernest F. Fullam, Inc., Schenectady, NY, USA). Sputter coating with gold is done to preve nt the Figure 2.8. A. Aratus pisonii. B. Tank setup for experiment. Photos taken by S alome Grasland. 1cm

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29 charging effects which would result from static charges generated by the electron beam of the SEM accumulating on non conductive specimens and, in turn, muddying the image (Allen, 2008). The specimen s were placed in a vacuum chamber whi ch was filled with argon gas. To ionize the gas, a current of 23 mA was pulsed eight times in 30 second increments The cathode of the circuit is a block of gold which gets bombarded by ionized gas atoms in turn, dislodging gold atoms. The gold atom ions stream to th e ano de and in the process, deposit a fine film on the sample (Egerton, 2007). The samples were imaged using a Topcon ABT 32 Scanning Electron Microscope (Topcon Positioning Systems, Inc., Livermore, CA, USA) at New College of Florida in Sarasota, Flori da. Images were captured using the Orion Digital Image Acquisition System (E.L.I. SPRL, Charleroi, Belgium). Figure 2.9. The SEM (above left) and sputter coater (above right) setup used. Photos taken by Salome Grasland.

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30 RESULTS Section 3.1 : The Survey Rhizophora mangle populations were surveyed along the coast of Florid a (Figure 2.1). T he survey was t o locate trees producing albino seedlings and to see if they were found more frequently in a cer tain type of environment. O riginal ly the hypothesis was that HER trees would be found with a higher frequency in areas with poor wate r circulation and increase d contaminants. Examples of this would be a lagoon with poor circulation or an area near a point source pollutant. During the course of this study no areas affected by point source pollutants wer e studied. All surveys were taken in areas considered to be l agoons and just outside the lagoon A lagoon in context of this study is any shallow body of water separated from the larger body of water by a narrow entrance that has a noticeably slower current and poorer circulation than the nearby larger body of water Water samples were taken while in the lagoon and also outside of the lagoon for comparison. The results show that lagoons general ly had less available dissolved oxygen, higher turbidity, higher salt concentration, and a higher temperature (T able s 3. 1 3.5 ). On average lagoon turbidity was 6 JTU higher than the outside area with a p value of 0.0039 making this statistically significant. On average the water temperature of lagoons surveyed was 2 degrees warmer than area outside of the lagoon with a p val ue of 0.025 making the result statistically significant. The dissolved oxygen saturation was 45.8% lower in lagoons with a p value of 0.0012 making this statistically significant. Throughout the survey all water tested had an average pH of 7. The phosphate concentration was 1.2 ppm higher in lagoons and the nitrate levels were 1 ppm higher

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31 compared to outside of the lagoon and the p value s were 0.07 and 0.4, respectively, hence these results are not statistically significant. The salinity was about 3 ppt hi gher with a p value of 0.013 concluding that these results are statistically significant (Table 3.6). Twelve sites, featuring a lagoon like area, were surveyed along the coast of Florida. O ut of the twelve sites surveyed, five featured complete heterozyg ous trees. The term complete heterozygous tree means that the whole tree was reproducing, not just select branches, and that albinism was expressed in a 1:3 ratio throughout the tree. Some of the other sites featured trees that partially expressed albinis m; however, these tr ees were not considered for this study because we cannot be sure they are produ cts of genetic anomalies and were not affected by the environmental In the five sites that featured a HER tree, the tree was found in the lagoon area and no t the coastal area around the lagoon, this is visualized in figure 3.1 The results of the survey showed that about 1.7% of reproducing trees in lagoons were h eterozygous for albinism, whereas 0% of trees in the surrounding coastal area were heterozygous for albinism ( Table 3.7).

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32 Figure 3.1. The areas outlined in red represent areas surveyed that had lagoon like conditions. While the green areas represe nt the nearest coastline to the lagoon with healthier circulation. A. Are a surveyed at Stump Pass. B. Area surveyed at South Lido Images taken from Google Maps. B A

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33 C Figure 3.1. The areas outlined in red represent areas surveyed that had lagoon like conditions. While the green areas represent the nearest coastline to the lagoon with healthier circulation. C. Are a surveyed at Venice Boat Launch .. Images taken from Google Maps.

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34 D E Figure 3.1. The areas outlined in red represent areas surveyed tha t had lagoon like conditions. While the green areas represent the nearest coastline to the lagoon with healthier circulation. D. Area surveyed at Cortez. E. Area surveyed at Grassy Point Marina Images taken from Google Maps.

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35 Table 3. 1 Water quality measurements of Venice Boat Launch. Both areas were sampled on the same day within an hour of each other. VENICE BOAT LAUNCH AREA AROUND LAGOON LAGOON Turbidity (JTU) 15 20 Temperature (Degrees Celsius) 26 28 Dissolved Oxygen (In percent saturation) 99 51 pH 7 7 Phosphate (In ppm) 2 2 Nitrate (In ppm) 2 2 Salinity (In ppt) 32 33 Table 3. 2 Water quality measu rements of Cortez. Both areas were sampled on the same day within an hour of each other. CORTEZ AREA AROUND LAGOON LAGOON Turbidity (JTU) 5 10 Temperature (Degrees Celsius) 28 30 Dissolved Oxygen (In percent saturation) 102 51 pH 7 7 Phosphate (In ppm) 0 2 Nitrate (In ppm) 0 0 Salinity (In ppt) 31 34 A B C D E

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36 Table 3. 3 Water quality measurements of Grassy Point Marina. Both areas were sampled on the same day within an hour of each other. GRASSY POINT MARINA AREA AROUND LAGOO N LAGOON Turbidity (JTU) 15 20 Temperature (Degrees Celsius) 30 32 Dissolved Oxygen (In percent saturation) 106 53 pH 7 7 Phosphate (In ppm) 2 2 Nitrate (In ppm) 5 5 Salinity (In ppt) 29 34 Table 3. 4 Water quality measurements of Blind Pass. Both areas were sampled on the same day within an hour of each other. BLIND PASS AREA AROUND LAGOON LAGOON Turbidity (JTU) 15 20 Temperature (Degrees Celsius) 26 28 Dissolved Oxygen (In percent saturation) 99 75 pH 7 7 Phosphate (In ppm) 2 4 Nitr ate (In ppm) 0 5 Salinity (In ppt) 31 33

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37 Table 3. 5 Water quality measurements of South Lido. Both areas were sampled on the same day within an hour of each other. SOUTH LIDO AREA AROUND LAGOON LAGOON Turbidity (JTU) 10 20 Temperature (Degrees Ce lsius) 30 32 Dissolved Oxygen (In percent saturation) 106 53 pH 7 7 Phosphate (In ppm) 0 2 Nitrate (In ppm) 0 0 Salinity (In ppt) 29 33 Table 3. 6 Average water quality of lagoons and area around lagoons. AVERAGE AREA AROUND LAGOON LAGOON Turbidi ty (JTU) 12 4 18 4 Temperature (Degrees Celsius) 28 2 30 1.8 Dissolved Oxygen (In percent saturation) 102 3 57 9 pH 7 0 7 0 Phosphate (In ppm) 1 1 2 0.8 Nitrate (In ppm) 1.4 2 2.4 2.2 Salinity (In ppt) 30 1.3 33 0.56

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38 Table 3. 7 Number of reproducing trees in each area surveyed and frequency of heterozygous trees. Percentage represents the proportion of HER trees to PHODR trees. Number of Reproducing Trees in A rea around lagoon Number of Complete Heterozygous T ree s Number of Reproducing Trees in L agoon Number of Complete Heterozygous T rees Blind P ass 50 0 (0%) 61 1 (1.6%) South L ido 65 0 (0%) 71 1 (1.4%) Grassy Point M arina 51 0 (0%) 72 1 (1.4%) C ortez 43 0 (0%) 35 1 (2.9%) Venice Boat L aunch 57 0 (0%) 96 1 (1 .0%)

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39 Section 3.2: Secondary Plant Compound Assay Usi ng Aratus pisonii Aratus pisonii, the mangrove tree crab, was used to indirectly test for the presence of seco ndary plant compounds by observing palatability. The working hypothesis is that leaf matter from HER trees may have different secondary plant compounds than PHODR trees. Aratus prisonii was used to see if the rate of herbivory was different for the two tree types. The trials showed that on average, A. pisonii ate 22 mm 2 more leaf ma terial from leaves of PHODR trees. However, the t test value for this experiment was 0.35 with a p value of 0.05, hence; the results are not statistically significant at p < 0.05. Albino, 0.81 Non Albino, 1.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Leaf Material Consumed on Average per Trial (In mm 2 ) Aratus pisonii Leaf Consumption Figure 3.2. Chart comparing the amount of leaf material eaten by A. pisonii. Leaf material was collected from adult heterozygous albino and homozygous dominant R. mangle trees. Ten trials were executed.

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40 Section 3.3: Transpiration Rates of Homozygous Dominant Plants versus Heter ozygous Albino Plants The purpose o f this experiment was to determine leaf transpiration rates from albino heterozygote trees (LHA) compared to leaves from homozygote dominant trees. It is theorized that stress plays a role in the expression of albinism (P roffitt and Travis, 2005). Throughout this study salt was used a s a stressor. Increasing salt concentrations were used to put the leaves under varying levels of stress. Three concentrations were tested: 0 ppt, 35 ppt, and 55 ppt. These concentrations were chosen because they reflect conditions the trees could realistically endure in the wild (Sarasota County Water Atlas). A concentration of 0 ppt is a level that would cause the plant no stress (Parida and Jha, 2010). Plants in the Sarasota estuary are expos ed to an average salt concentration of 35 ppt, hence, it can be assumed that this level of salinity would cause the plant minimal to no stress. The concentration of 55 ppt would be an extremely high salt concentration for the plant to be exposed to and it would cause high amounts of stress in the plants. Although it is natural process for plants to transpire, transpiration rate does increa se with plant stress (Jasechko et al., 2013). The general trend of the experiment was that as the concentration of sal inity increased the poorer the leaves from heterozygote trees for albinism (LHA) performed. At a salinity of 0 ppt LHA transpired less than the control with a p value of 0.026 concluding the results are statistically significant. At a salinity of 35 ppt th e LHA performed as well as the control with a p value of 0.084 making these results not statistically significant. And at a salinity of 55 ppt the control transpired less than the LHA with a p value of 0.020 making this result statistically significant. Fr om this

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41 baseline result we can project that heterozygote albino trees may be more efficient than homozygote tree when under no stress and as stress increases they become less efficient than their homozygote counterparts (f ig 3.3). An unexpected result w as that the plants transpired the most at salinity of 35 ppt. The hypothesis was that as salt concentration increased the level of transpiration would increase because water use efficiency decreases with an increase in plant stress (Jasechko et al., 2013) However, this may be the result of varying humidity and temperature during the trails. According to weatherspark.com the days the 35 ppt and 0 ppt trails were conducted had a much lower humidity than the days the 55 ppt trials were conducted possibly exp laining why they transpired more.

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42 Figure 3.3 Transpiration rates of leaves from trees producing albino off springs, salinity levels. Ten trails were done for the three experiments.

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43 Section 3.4: Stomata Morphology and Behavior at Varying Salt Concentrations The goal of this experiment was to observe stomatal aperture at varying salinity concentrations and determine if the results mirror those from the transpiration rate experiment. R esults support the observation of the aforementioned experiment. At 0 ppt the stomata from albino leaves were slightly more constricted than the control leaves the p value was 0.34, hence, the difference is not statisti cally significant. This trend changes at 35 ppt and the control stomata became more constricted the p value was 0.0046 making the results statistically significant. At 55 ppt the difference in stomatal aperture is statistically significant with a p value o f 0.004; the control stomata are noticeab ly much more constricted (Fig. 3.4 and Fig. 3.5). Figure 3.4. Stomatal apertures at varying concentrations of salinity. Blue bars represen t leaves taken from trees not expressing albinism, while the red bars represent leaves taken from trees expressing albinism. Standard error bars are present. One hundred stomata w ere measured for each variable from ten cm 2 leaf cuttings.

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44 Figure 3.5. Stom ata photos at a magnification of 100x. The left column are photos taken of the controls and the right column is photos of stomata from albino leaves. The top row is at a salinity of 0 ppt, middle row 35 ppt, and bottom row 55ppt.

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45 Section 3.5: The Effects of Oil on Seedling Growth and Lenticel Morphology Seedlings were collected from HER and PHODR trees and grown under three different con ditions: 0 ppm, 20 ppm, and 300 ppm oil concentration in which they resided This was designed to mimic condition s found in a marina a nd near a point source pollutant. S eedlings were observed for overall root growth and lenticel a bundance and morphology. R esults showed that seedlings from HER and PHODR trees exposed to 0 ppm o f oil responded similarly. S eedlings from HER trees had 51% of their lenticels unclogged and an average of 4.4 mm of root growth while seedlings from PHODR trees had 58 % of their lenticels unclogged and an average of 4.5 mm of root growth, the p value for root growth was 0.95 and 0.68 for lenticels clogging meaning neither of the results are s tatistically significant (Fig. 3.6). At 20 ppm of oil there is a substantial differen ce between the two groups. S eedlings from HER trees had 40% of their lenticels unclogged and an average root length of 13 mm, while the seedlings from PHODR trees had 15% of their lenticels unclogged and average root length of 1.9 mm, t he p value was 0.042 root length and 0.0099 for lenticel clogging this means that the results are statistically significant. At 300 ppm of oil there is no statistical difference between the two groups, the seedlings from HER trees slightly out perform seedlings from PHODR trees. S eedlings from HER tree had 30% of their lenticels unclogged and an average root length of 16mm, while seedlings from PHODR trees had 18% of their lenticels unclogged and a verage root length of 12 mm, the p value for roo t length is 0.40 and 0.050 for unclogged lenticels rendering these results not stati stically significant.

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46 Figure 3.6. The top graph shows lenticel clogging at varying concentration of o ils as a proportion of unclogged lenticels. The bottom graph shows root length at varying concentration of oil. Overall root growth was measured after 5 month s. Ninety nine seedlings were grown, 33 for each variable.

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47 Section 3.6: Lenticel Morphology of Samples Taken from HER and PHODR Trees Obse rved via SEM Microscopy Figure 3.7. Pictured above are lenti cels from PHODR trees. Figure A features a lenticel that is severely clogged, the white arrow points to the raised area of the bark due to the clogging compared to figure B the bark is less swollen and clogged. Figure C and D are of the same sample taken a t different magnifications; this sample is only slightly clogged. The black arrow points to a concave dip in the bark compared to the swollen outward appearance of figure A this specimen is less clogged. Photos taken at New College of Florida using a scann ing electron microscope with the assistance of H. Benedict. A B C D

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48 Figure 3.8. Pictured above are lenticels from HER trees. Figure A and B feature a sample that is clogged and has a chasm at two different magnifications. Figures C and D are of a clo gged lenticel at two different magnifications. Figures E and F features a lenticel that is relatively unclogged at two different magnifications. The white arrows point to areas where the bark comes outward suggesting that these lenticels are clogged. While the black arrow points to a dip in the wood. Photos taken at New College of Florida using a scanning electron microscope with the assistance of H. Benedict. A B C D E F

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49 DISCU SSION Section 4.1: Albino Expression and Correlation to Stress Stre ss in plants can create negative side effects. Stress can effect seed germination, seedling growth and vigor, flowering and fruit set, and vegetative growth. When a p homeostasis is d isrupted, changes occur at the cell level and the whole plant level, which can eventually lead to tissue death and plant death (Xiong and Zhu 2002). Stress affects the integrity of cellular membranes, enzymatic activity, and photosynthetic abilities (Serra no et al., 1999). The main reason stress incurs plant damage is due to the production of reactive oxygen species (ROS) (Smirnoff, 1993). Reactive oxygen species are a natural byproduct of plant metabolism created by the univalent reduction of O 2 or by tran sferring excess excitation energy. The transfer of one, two, or three electrons generates superoxide radicals, O 2 H 2 O 2 and HO respectively (Mittler, 2002). Chloroplasts, mitochondria, and peroxisomes generate more ROS like O 2 and H 2 O 2 than any othe r intracellular process ( Fig. 1.11) (Hernndez et al., 1995). Glycophytes (non salt tolerant plants) and halophytes (salt tolerant plants) have different abilities of handling ROS via the production of antioxidant enzymes (Rout and Shaw, 2001). The mang R. mangle trees and characterize the location which they were found (Section 3.1). The results were that all HER R. mangle trees were located in lagoons whose water were richer in excess nu trient, poorer in dissolved oxygen, had higher turbidity, and higher salt concentrations compared to the closest coast line with healthy water flow. It has be en shown that natural and anthropogenic stressors, such as salinity, tides, storms, trace metals, polycyclic aromatic hydrocarbons, persistent organic pollutants, and other organic

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50 pollutants all have negative effects on R. mangle (Section 1.4.1 1.4.2) and encourage the production of ROS species (Jitesh, 2006). These results implicate plant stress as a significant factor in the expression of albinism in R. mangle The results of the exposure to oil, potometer, and stomatal apertur e experiment all implicated stress as a significant contributor of albinism expression In the oil exposure experimentatio n seedlings from heterozygous reproducing (HER) trees and putatively homozygous dominant reproducing ( PHODR ) trees both showed nega tive responses to stress. B oth seedling types showed an increas e in lenticel clogging at a similar rate. The result of the p otometer testing was that as salinity concentrations increased leaf transpiration increased more rapidly in leaves from HER trees versus leaves from PHODR trees at the highest stress level of 55 ppt These results were paralleled in the stomatal aperture e xperiment. As salinity concentration increased so did the aperture of stomata from albino leaves more greatly than their non albino counterparts. Hence, these results support the idea that trees expressing albino seedlings have an altered tolerance for str ess compared to their non albino counterparts. Mangrove trees rate of transpiration compared to glycophytes is typically much lower (Sobrado, 1999). It has been shown that R. mangle trees grown in the absence of NaCl actu ally wither and die, implying tha t Na + plays the role of a macronutrient in Rhizophora (Flowers et al., 1997) Exposure to high amounts of salt increases the chlorophyll contents in leaves (Werner and Stelzer, 1989). According to Sobrado (1999), conservative water use which results in low er transportation rates may actually be advantageous in areas with fluctuating salinities. The results o f the current study implicated that at salinity levels greater than 35 ppt that leafs from HER trees transpired

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51 more. This may mean that these trees are not as well suited than their PHODR counterparts to handle environments with high fluctuations in salinity, seeing as a lower transpiration rate is not a typical response to higher salinity concentrations. Section 4.1.1: Possible Differences between Seedlings an d Leaf Material Taken from Adult Trees It should be considered that the potometer, stomatal aperture, leaf herbivory experiment used leaf material taken from adult PHODR and HER trees in the field, while the oil contamination experiment was conducted using seedlings taken from the area around PHODR and HER trees. Some things to consider are that seedlings from HER trees could be homozygous dominant, heterozygous, or homozygous recessive ( Fig. 1.8). Homozygous recessive seedlings can be identified because of their albino appearance; however, heterozygous seedlings and homozygous dominant seedlings are both green. Hence, the only way to tell if a seedling is heterozygous or homozygous dominant is to either do genetic testing or wait until they reach reproducti ve age and observe if they produce albino seedlings. This is significant for the oil exposure experiment because possibly 33% of the seedlings could have been homozygous dominant which means they could behave differently than their heterozygous counterpart Section 4.1.2: What is a Clogged Lenticel? The term clogged in this paper refers to lenticel which had an outward woody appearance. It should be noted that clogged lenticels are not always non functional lenticels. Rhizophora plants are capable of form ing hypertrophied lenticels, which are expanded lenticels formed after flooding to help the plant rid itself of toxins (Youssef and

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52 Saenger, 1996) They have a similar appearance to dead, woodened lenticels. It is difficult to tell the two types apart, hen paper could refer either to a hypertrophied lenticel or dead lenticel. Section 4.1.3: Doing Potometer Testing on Stem Cuttings from Trees in the Field versus from Plants Grown on Site For the course of t his study the potometer testing was done on stem cuttings taken from trees in the wild. These cuttings were kept in a phosphate buffer for no more than three days to help keep the leafs viable for testing. This may have affected the results due to the fact that stomatal closure is controlled via the plant hormone abscisic acid which is regulated by the plants roots and s ince the stem cuttings were no longer connected to the c isic acid signaling and stomatal closure may have been hampered (Zh ang et al., 2001) If this experiment were to be repeated more accurate measurements could be made if the entire plant was treated with the condition of interest (in this case salinity) and stem cuttings were taken after the plant had a chance to react t o the treatment. Section 4.2: Lenticel Morphology as Seen by SEM Lenticels are interruptions in the peridem which most often form where a stomata occurred ( Lendzian, 2006 ). The lenticel phellogen is created from the cells interior to the stomata and is c onnected to the adjacent cork cambium providing oxygen to living cells of the bark (Rosner and Kartusch, 2003 ). The lenticel phellogen produces cells to the interior and exterior, however, the outer cells round up and have intercellular air spaces, causing the tissue inside the lenticel to be more loosely packed, known as filling tissue or complementary tissue ( Groh et al. 200 2 ). As lenticels age and are exposed to a variety of

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53 contaminants the filling tissue will become more packed giving the lenticel a c logged appearance (Gibson, 2011). Lenticels are a crucial part of the mangrove internal airflow system (Fig 4.1) Photos were taken of lenticels on HER and PHODR trees by using a scanning electron microscope ( Fig. 3.7). Photos were taken of clogged a nd un clogged lenticels to see if the two tree types had any noticeable morphological differences. The results show that there is no apparent difference between lenticels from either tree. They have a similar morphology and similar filling tissue. This suggests that HER and PHODR trees do not have visibly altered methods of handling gas exchange via lenticels. propos ed warts on the abaxial portions of a leaf (A). Air descends the aerenchyma of a stem (B). Air descends the inner aerenchyma (IA) of a stilt (air) root (C). Air descends to the terminal regio ns of a root via the inner aerenchyma (IA) and then ascends the outer aerenchyma (OA) until it moves out of lenticels in mud roots (D) (Evans et al., 2005)

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54 Section 4 .3: The Heterozygote Advantage The heterozygote advantage has been a concept apparent in genetics since 1992 (Fisher, 1992). It is the idea that an organism which is a heterozygote carrier for a disorder may have an advantage under certain conditions. Sickle cell anemia is a disorder which causes improper protein folding of red bloods cells and gives them a sickle shape, in turn, resulting in anemi a. It can lead to early death. Malaria is a parasitic disease typically carried by mosquitoes, and without proper treatment, can be lethal. However, it has been shown that persons heterozygous for sickle cell have an advantage confronting malaria; in turn, improving their fitness. This concept is known as the heterozygote advantage and explai ns why typically lethal disorders like sickle cell anemia remain in the gene pool (Hedrick, 2012). It is possible that R. mangle expressing albinism is due to the het erozygote advantage. Proffitt and Travis (2007) found that there was a correlation between seedling length and number of seedlings the tree produced. Trees which produced larger seedlings typically produced fewer; where as, trees with smaller seedlings gen erally had more. The larger seedlings had the advantage of being more competitive while the smaller seedlings were more abundant. It is possible that by producing non fertile albino seedlings that the heterozygous seedlings could have a genetic advantage. Just as individuals who are heterozygous for sickle cell anemia have a better ability at combating malaria, seedlings heterozygous for albinism may have a better ability at handling stress. Kozlowski and Pallardy (2002) reported that slowly incrementing the amount of stress a plant experience overtime may actually benefit the plant by inducing

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55 physiological adjustment that protects the plant from stunted growth or harm when environmental stresses are abruptly imposed. In addition, exposing woody plants t o extreme conditions during critical times of plant growth can some times improve their performance. According to the current study, HER R. mangle trees were more frequently found in areas of high stress albeit, in low amounts (Section 3.1). It could be possible that being expos ed to high amounts of stress results in the expression of genes for albinism in the trees that carry it. In turn, this could cause the tree to produce seedlings that are homozygous albino, heterozygous albino, and homozygous dominant. Seedlings that are he terozygous albino may have the heterozygous advantage. Section 4.3.1: Experimental Results Showing Trends Towards the Heterozygote Advantage The results of the transpiration experiment, stomatal aperture ex periment, and secondary plant compound assay u sing Aratus pisonii experiment showed that HER plant material slightly out performed PHODR leaf material (Section 3.2 3.4). In the transpiration experiment albino leaves transpired significantly less at a salinity of 0 ppt than its PHODR counterpart. The stomatal aperture experiment supported this and showed that HER stomata remained more constricted at lower salinity levels than their PHODR counterpart. According to Reef and colleagues (2012) reducing transpiration rates is an effective technique to avoi d the accumulation of excess NaCl in mangrove tissue which has a series of negative side effects on the plant. It may be by having a reduced transpiration rate compared to PHODR plants that HER plants accumulate less salt, hence, lowering their stress leve ls and, in turn, the production of ROSs.

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56 Aratus prisonii the mangrove tree crab, consumed leaves from PHODR trees more frequently than leaf material from HER trees, however, not a statistically significant level. This supports the idea that the producti on of secondary plant compounds, such as the ones preventing herbivory, are not significantly hampered in HER trees. Although, the studies conducted were not statistically significant they do point at a general trend that HER trees may outperform PHODR tre e when stress levels are low. In the oil contamination experiment this trend was also supported. Seedlings from HER trees performed as well as seedlings from PHODR trees, both had similar amounts of root growth and lenticel clogging at 0 ppm. At 20 ppm of oil exposure seedlings from HER trees actually performed better than seedlings from PHODR trees; on average they had 11.1mm more root growth and 35% fewer lenticels clogged. Silva and colleagues (2009) discussed the effect of petroleum pollution on mangrov e coastlines in Brazil. They noted that R. mangle trees exposed to contamination had more malformed leaves and grew more aerial roots to compensate for the clogging of lenticels (Silva, 2009). The exact mechanism behind why seedlings from HER trees had few er clogged lenticels is not known, however, if the tree can prevent lenticel from clogging then it is advantageous. Section 4.3.2: Is Heterozygote Advantage Selection Genetically Significant? Hedrick (2012) discusses that we are just beginning to unders tand the heterozygote advantage at the genetic level. Initial genomic survey that have been done are suggesting that only a minute proportion of loci have polymorphism which are being sustained by the heterozygote advantage, and unless future studies provi de large numbers of loci being maintained by heterozygote advantage then the role it has an evolutionary change is more of an unusual phenomenon rather than a leading factor in adaptation

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57 (Hedrick, 2012). An example of this is negative frequency dependent selection as a mode of balancing selection for the self incompatibility systems of flowering plants, although it should be said that self incompatibility systems also include heterozygote advantage (Castric and Vekemans, 2004). While some of the results of the experiments supported the idea of the involvement of the heterozygous advantage they need further elaboration and exploration. It should be noted that in the field study the number of HER trees found were not prevalent enough to concretely support tha t there is a heterozygote advantage in the field, but that there is a pattern that needs further exploration (Table 3.7). Section 4.4 : Future Studies Section 4.4 .1: Reactive Oxygen Species and Rhizophora mangle Antioxidant Enzyme Production It has been s hown that halophytes have a greater capacity to produce antioxidants compared to glycophytes and it is this ability that allows them to thrive in extreme areas (Rout and Shaw, 2001). A fascinating follow up study would be looking at the production of ROS a nd antioxidant enzyme production in R. mangle trees expressing albinism in comparison to trees not expressing albinism. Krishnamoorthy and colleagues (2011) studied antioxidant activities of bark extract from the mangrove species Bruguiera cylindrica and C eriops decandra and found antioxidants present at higher levels compared to typical plants. Section 4.4 .2: Genetic Analysis Rhizophora mangle albinism is believed to be caused not purely by environmental

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58 factors, but also genetic causes. For the course of this thesis no experiments were done looking directly at the R. mangle genome. Hence, a fulfilling future study would be one that manages to genetically compare R. mangle trees expressing albinism and trees that do not. The mutant phenotype of R. mangle wo uld be an ideal spec imen to use to look for candidate genes It would be specifically relevant to look at R. mangle genes that help deal with plant stress. Basyuni (2011) created a cDNA library of Rhiziphora stylosa a close relative or Rhizophora mangle, which was used to screen for stress related genes using polymerase chain reactions. In total 240 up regulated expressed sequence tags were isolated and 13 genes were found that played a role in salt tolerance Section 4.4 .3: How to Properly Grow R hizophor a mangle Seedlings During the course of this study some problems were encountered with achieving seedling growth and establishment. The most serious error that occurred was transplanting too many times. In an attempt to obtain the correct soil composition and parameters the seedlings had to be transplanted two times, in the process disturbing root growth and establishment. This could have compromised the formation of a symbiotic relationship with arbuscular mychorrhizae ( Kothamasi 2006) If the experiment were to be repeated it would be recommended that the seedlings were grown in window planter boxes, with no more than ten seedlings per box. It is also recommended that the soil used for testing be taken from an area where R. mangle naturally grow. This wo uld help insure that bacterial and fungal organisms, pH, nutrient levels, soil architecture and consistency were at the optimum levels for R. mangle growth. Once the seedlings are planted cover the soil with sterile mulching, like shredded cardboard. Thi s mulching will help hold in moisture and heat. Do not use leaf debris

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59 taken from the field because risk of fungal, bacterial, and other pest contamination increases. Change out the mulching about once month to prevent unwanted fungal or bacterial growth. Directly after planting the seedlings water them with salt water at a concentration of 35 ppt. Pour in enough water so that it reaches approximately 1 cm above the soil line. Once the seedlings have been watered with saltwater continue watering them with f reshwater. If salt water is continuously given to the seedlings too much salt may be accumulated in the soil and kill them, hence, it is recommended that watering with salt water does not happen more than once a month. To supplement the soil give the seed lings once a month an organic fertilizer. The ultimate growing location for the seedlings would be a growth chamber, however, if one cannot be obtained then placing them indoors under florescent lights, with a light schedule mimicking one of which they w ould be naturally exposed to would be optimal. If they can only be grown outdoors then place them in an area where full sun is received and that is shielded from strong winds. Section 4.4.4: Looking at the Interaction between Biotic Factors and Albinism i n Rhizophora mangle This study predominately considered the effects of abiotic factors on the prevalence of albinism in R. mangle A viable future study would be to consider the effect of biotic factors, such as, bacteria, fungus, and other organism on the prevalence of albinism in R. mangle. In a previous thesis by Alycia Shatters R. mangle trees with a unique abnormal morphology, including club shaped branch termini with short internodes had more severe herbivory than their neighboring healthy trees (Shatters, 2010)

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60 Sec tion 4.5: Conclusion Albinism in R. mangle is a unique mutant phenotype that serves as an ideal tool to study the effects of plant stress. It easy visibility and Mendelian inheritance makes it an accessible method of surveying the effects of different fact ors on the plants genetics. This study used the mutant phenotype to observe the effect of environmental stresses on R. mangle Overall, the findings suggest that stress does have a unique effect on the prevalence of albinism in R. mangle The findings als o suggest that R. mangle trees expressing albinism may have unique adaptation to stress compared to trees which do not express the mutation.

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