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Lichens of Myakka River State Park: Water tolerance, ver tical stratification and canopy diversity By Barry Kaminsky A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment for the degree Bachelor of Arts Under the sponsorship of Dr. Margaret Lowman Sarasota, Florida March 2009
ii Dedication: To the armadillos scavenging amongst the trees and to Hannah for her love and guidance.
iii Acknowledgments Many people helped me along the path of writing this thesis, a nd I wish I could thank all of them. I would like to th ank Bob Bernhard for his gener ous grant and support of this research. I would like to thank Meg Lowman for all her support, advice, and countless edits. I would also like to thank Dr. Clor e, for letting me use her lab for lichen photography and taxonomy, and storage for chemi cals. Lastly, thanks to Duff Cooper for his excitement about my thesis and his valuable help with statistics. To all my friends, who have helped me along my five years at New College, all of you are wonderful. David Weinberg for his tree climbing help and being an awesome friend. Forest Hayes for his help climbing tr ees and Nathan Kruer for his attentive and helpful editing. Hannah Schotman for her patience and steady shoulders. To my parents for their support and love. This project would not have been possi ble without the help of Rick and Jean Seavey. Their kindness and lessons in lichenology were extremely valu able and helpful. I hope I can one day teach as selflessly as both of you.
iv Foreword My parents took me backpacking before I could walk or talk. So I passed the time away either sleeping or staring at the scen ery from the baby carrier. While I cant claim to know how I felt about nature as an infant, I imagine I still feel the same way I feel nowa mixture of awe and curiosity. I was always looking, trying to take in the many details of nature. Looking is a very active pursuit, its goal is to take in the scenery, so to speak, and that was enough for an eager child. But as so often happens, eyes betray the viewer by focusing on the larger details and minimizing the small ones. Discovering bromeliads changed my perception of nature and I began to see the smaller details of the scenery. It still awes me to think that for 21 years I never looked deeply at a bromeliad. Its so easy to li ve and yet overlook. The adage says Leave no stone unturned, but the truth is that it is hard to overtu rn every rock, and gain the knowledge of each stone. Sometimes the rock is too heavy to overturn, or sometimes the rock is simply viewed as insignificant or invisible, passe d by without inquiry. I knew after discovering bromeliads I w ould be writing my thesis about organisms in the tree. These small overlooked organisms need ed to be studied and New College has a professor, Meg Lowman, who specializes in canopy ecology. My first thought was to find and identify all the ferns, bryophytes and brome liads and lichens in a species of tree. This plan fortunately did not come to fruition; instead fate steered me to lichens. I offhandedly told a friend, Ellen Siegel, I was interest in lichens. She told me of Rick and Jean Seavey, lichenologist s at Everglades National Park The rest is a whirlwind. I took a 6 day crash course in lichen taxonomy, and saw how intricate, complex,
v mystifying and yet endearingly beautiful lichens were. I attended a conference on lichens and plants that deepened my thought that lichens were stunning yet deeply confounding beings. Further outside research revealed that lichens are vastly understudied in Florida. Perfect for a nave undergrad. With that I began to work on my thesis, aided by a generous contribution from the Explorers Club to conduct my thesis research. There are still myriad small pebbles out there floating in the world. Some are visible now, and with time their secrets may be unloc ked. There are also many stones, covered by dirt or simply not seen by humans. The adve nture of life is to fi nd these stones to turn over and look at the world with renewed wonder, and a fuller sense of appreciation of Life and Earth.
vi Table of contents Introduction .......................................................................................................................1 I: Literature Review ......................................................................................................... 2 1.1: Overview of lichen biology and ecology.......................................................... 2 1.2: Literature review of Florida lichens.............................................................. 12 1.3 Tolerance to desiccation and inundation.......................................................... 16 1.4 Goals and hypothesis....................................................................................... 21 II: Lichen tolerance to submergence ..............................................................................23 2.1 Test site............................................................................................................ 23 2.2 Methods............................................................................................................23 2.3 Results.............................................................................................................. 27 2.4 Discussion........................................................................................................ 30 2.5 Future directions.............................................................................................. 35 III: Assessing changes to li chen cover vertically in Quercus spp .................................37 3.1 Introduction...................................................................................................... 37 3.2 Test site and tree.............................................................................................. 37 3.3 Sampling methods............................................................................................ 38 3.4 Results.............................................................................................................. 39 3.5 Discussion........................................................................................................ 43 IV: Lichen diversity in the canopy .................................................................................48 4.1 Introduction...................................................................................................... 48 4.2 Methods............................................................................................................48 4.3 Taxonomy results............................................................................................. 50
vii 4.4 Discussion........................................................................................................ 51 V. Summary ......................................................................................................................55 Appendix I: Observations on lichens used for water tolerance experiment ...............58 Appendix II: Additional s ubmergence experiment data .............................................59 Appendix III: Additional vertical sampling data .........................................................62 Appendix IV: Photos of Myakkas lichens ... ...64 Literature cited .................................................................................................................69
viii List of Figures and Tables Figure 1.1 Types of lichen growth forms............................................................................5 Figure 1.2 Examples of apothcia, perithecia and isidia.. 7 Figure 1.3 Geographical ranges of Floridas lichens ......................................................13 Figure 1.4 Example of a lichen trimline........................................................................... 18 Figure 2.1 Location of Myakka River State Park and test site......................................... 24 Figure 2.2 Set up for the submergence experiment.......................................................... 25 Table 2.1 Qualitative Scale Lichen Vigor Lichen Appearance......................................... 26 Figure 2.3 Lichen tolerance to submergence in water...................................................... 28 Table 2.2 F-Test values for submergence experiment for days 1, 3 and 5 29 Table 2.3 Mean vigor of control lichens on day 5............................................................ 30 Figure 2.4 Damage to U. strigosa between day 0 and 3................................................... 33 Figure 3.1 Demonstration of single rope technique.......................................................... 38 Figure 3.2 Quadrat setup on a tree.... 39 Table 3.1 Total lichen cover of i ndividual trees by height................................................ 40 Table 3.2 Additional total cover statistics of three individual trees (cm2)........................41 Table 3.3 Differences of total lichen cover by cardinal direction..................................... 41 Table 3.4 Crustose vs. foliose cover for lower, middle and upper trunk (cm2)................42 Table 3.5 ANOVA for crustose and foliose cover for height in tree.... 43 Figure 4.1 Demonstration of s pot test technique.. ...50 Table 4.1 Macrolichens collected in Myakka River State Park........................................ 51 Table A2.1 Raw data for submergence experiment.......................................................... 59 Table A2.2 Mean vigor of species from days 0 to 5......................................................... 61
ix Table A2.3 Comparison of mean vigor of contro l and experimental sets on day 5.......... 61 Table A3.1 Chart used to measure percent cover of lichens in tree.. 62 Table A3.2 Crustose and foliose data for all heights of all three trees............................. 63 Figure A4.1 Common lichens on the trunk of Quercus ... 64 Figure A4.2 Photograph of Cryptothecia rubrocincta .65 Figure A4.3 Photograph of two common Parmotrema spp.. 65 Figure A4.4 Photograph of Parmotrema and Physciaceae spp... 66 Figure A4.5 Photograph of Leptogium spp.. 66 Figure A4.6 Photograph of Leptogium spp. 2. 67 Figure A4.7 Photograph of unknown lichen... 67 Figure A4.8 Photograph of Usnea spp 68 Figure A4.9 Photograph of Usnea strigosa. 68
x Lichens of Myakka River State Park: Water tolerance, vertical stra tification and ca nopy diversity Barry Kaminsky New College of Florida, 2010 ABSTRACT Lichens are an understudied organism in Florida, especially on the southwest coast. The goal of this thesis was to collect data on lichens at Myakka River State Park, a large area of preserved land, and to explore areas of lichenology that deserve more research. The first experiment tested the tolerance to submergence in water of six species over a 6 day period. There were significant health di fferences between low trunk and canopy lichens, with low trunk lichens having a higher vigor under conditions of submergence. Second, I surv eyed vertical distribution of lichen diversity, and the total cover of lichens. The results found no correlation between total lichen cover and height in the tr ee, and no correlation between lichen cover between sides of the tree. Crustose lichen s accounted for 85% of cover, and foliose 15%. Foliose lichens cover increased significantly with height (ANOVA F(2,6)= 26.29, p= 0.0011) while crusto se did not (ANOVA F(2,6)= 2.56, p= 0.16). Third, the diversity of macrolichens between Myakka River St ate Park and Ocala National Forest was compared. Lichen diversity was very simila r between parks, but Myakka has a higher diversity of tropical lichens. __________________________ Dr. Margaret Lowman Division of Natural Sciences
1 Introduction A lichen is a symbiotic relationship between a fungus and an alga or a cyanobacteria; however, specifics of their life cycle and eco logy are still largel y unknown (rev. in Nash 2008). Lichens are mysterious. No one has successfully made a lichen in a laboratory setting using an alga and fungus. The ecology of lichens is also perplexing. Lichens are found over a range of humid to dry conditions from the poles to the tropics, but a small habitat change greatly limits a lichen can grow (McCune 1993). Lichens are dependent on moisture, but when placed in water, most species die within a week (Kappen 1973). In many parts of the world, such as Florida, lichens have not been thoroughly explored. This thesis was a modest introduction to the lichen diversity at Myakka River State Park. First, I examined the tolerance of six species of lichens to flooding conditions, by submerging them in water and recording change s of their health. My hypothesis was that lichens living lower on the trunk, closer to s easonal flooding, would have less damage to their thalli than species higher from the water (canopy species). The second experiment of the thesis examined how lichen cover changed from the base of the tree to the upper trunk. The hypothesi s was that the total cover of foliose and crustose lichens would not change from the forest floor to the upper trunk, while cover of fruticose lichens would incr ease with height in tree. The third experiment of this thesis wa s to survey lichens present in oak hammocks at Myakka. Fallen branches were collected, and as many species as possible were identified. An emphasis was placed on identifying macro lichens to species and microlichens to genus. Diversity was compared to Ocala Na tional Forest and a lichen checklist was created for Myakka River State Park.
2 Chapter I: Literature Review 1.1: Overview of lichen biology and ecology Definition of a lichen A lichen is a difficult organism to desc ribe. The simplest definiti on is that a lichen is not an organism, but rather a symbiotic re lationship between a fungus (mycobiont) and an alga or a cyanobacteria ( photobiont) (Nash 2008). The photobi ont, typically a green alga, produces energy for the lichen. Green alga is the most common photobiont, but about 10% percent of lichens have a cyanobacter ia photobiont (Fried l & Budel 2008). Common photobionts are the genera Trebouxia Trentepohlia and Nostoc Mycobionts are from a range of different orders and families of fungi. The most common mycobionts are Ascomycetes though a few basidi omycetes are mycobionts. Lichens are named after the fungi because the vegetative body takes the general shape of the mycobiont while incorporating the algae into its vegetative body (Friedl & Budel 2008). Lichen symbiosis and benefits for each symbiont The process of lichen formation is still unknown. Mycobionts can reproduce sexually and asexually, creating spores or vegetative re productive bodies, yet alga is not a part of either method (Honegger 2008). For a new liche n to form, the fungal reproductive bodies must find an algal partner. Without knowing this process, scientists have not been able to create a lichen in a laborator y setting. Unknown vari ables remain that have not yet been discovered (Honegger 2008). Most mycobionts can only join with one clade of photobionts, meaning a mycobiont can form a lichen with photobionts of similar genetic characteristic s (Beck et al. 1998). Six of eight Cladonia species in Florida scrub pines jo ined with only on e clade of algae
3 (Yahr et al. 2004). Most fungi are algal-spec ific, meaning they bond with only one clade of algae. However some species of fungus may bond with two dissimilar photobionts such as an alga and a cyanobacteria. This cr eates different phenotype s that prove hard to classify (Budel & Scheidegger 2008). The symbiosis benefits both the alga and the fungus. The alga gains protection from the high levels of light that would harm it while the fungus gains a constant source of food (Nash 2008). This allows both the my cobiont and photobiont to live in an environment it could not normally live al one (Brodo et al. 2001). The fungus is lichenized, which means the fungus has an alte rnate source of food (the photosynthesis of the alga) (Honegger 2008). Benefits of the sy mbiosis between the two organisms may not be equal. It is currently accepted that the fungus controls the alga, based on two observations: 1.) in the symbiosis fungus incorp orates the alga into its structure 2.) the fungus uses the alga to obtain food by f ungal structures such as haustoria (Honegger 2008). For these reasons, the curr ent perception is that this is not a true symbiotic relationship (Nash 2008). More research on algae may change this perception because research has traditionally focused on the mycobiont. Lichen phylogenetics It is difficult to classify lichens be cause many orders of fungi are lichenized. Lichens are also polyphyletic, or having multiple lineag es. They have evolved from fungi in many orders and within families at multiple points in time (Lutzoni et al., 2004). Clades and fungal specificity further complicate diffe rentiating genera and species. Currently, scientists depend on morphological char acteristics and chemical tests.
4 Growth forms The simplest morphological characteristic used to differentiate lichens is their growth form. Lichens of North America (Brodo et al. 2001) is a valuable resource for lichen growth forms and descriptions for reproductive structures. This section relies heavily on these descriptions. Lichens are grouped into four distinct growth forms: foliose, fruticose, crustose and squamulose (Figure 1.1). Folio se and fruticose are collectively known as macrolichens, while squamulose and crustose are called microlichens. These four growth forms are based on outward characteristics not phylogeny. Common characteristics to determine growth form are lichen attachment to its substrate, shape of its thallus and the types of tissues present in the lichen. Foliose lichens are attached to the substrate at few to many points. The thallus (vegetative body) looks like lobes that are usually not attached to the substrate. The top and the bottom of the lobes are different colors Foliose lichens also have distinct inner layers. The top layer of a foliose lichen is called the cortex, which is a hard outer protective layer. The alga lives below the cortex in the medulla. Fruticose lichens grow vertically, eith er up or down. They are branched and attached to the substrate at the base less than foliose lichens. The cortex circles the outside of the thallus, and the medulla is inside. Prosopl ectenchyma tissue, wh ich is thick, elongate hyphal cells, supports the lichen. Crustose lichens are cl osely attached to the substrate and look like crusts. They have an upper cortex, photobiont laye r and a medulla. Squamulose lichens are an intermediate between crustose and foliose. They have ma ny small vertical scales but each scale is firmly attached to the substrate.
5 Figure 1.1 : Shows the four growth forms of lichens: foliose fruticose, crustose and squamulose. Within these four growth forms, the range of diversity is very large with many variations of each growth form. Reprinted from Brodo et al. 2001.
6 Sexual and asexual reproduction characteristics Many lichens will outwardly look the same, having the same growth form, thallus color, and habitation of the same niche, yet th ey could be different species or even from different genera (Brodo et al. 2001). This makes other ch aracteristics of the lichen important for identification. Sexual and asexual reproduction provide important characteristics for lichen taxonomy. Although the mycobiont is lichenized, the reproduction characteristics of the lichen are the same as an isolated specimen of that species of fungi. Ascomycetes have ascomata which resemble a small container. The two types of ascomata found in lichens, are a pothecia and perithecia Apothecia are open containers with spores along the top layer of the bottom. Perithecia are containers that are closed at the top. Spores are inside these containers and are released through a small opening called an ostiole. Inside of apoth ecia are asci (ascus singular), which hold the ascospores. The shapes of the ascus and asco spores vary between species and are useful for identification. There are in termediates between apothecia and perithecia such as lirella, which are longer and often branched (as seen in the genus Graphis in Florida). The mycobiont has the ability to reproduce asexually. One common method is pycnidia and conidia. Pycnidia resemble peri thecia and conidia are similar to spores. Other asexual reproductive methods include soredia, soralia, and isid ia. Soredia are small balls of hyphae that form ar ound the photobiont inside the liche n, then make their way to the surface. Soralia are soredia that form on the outside of the lichen. Soredia and soralia cover on the thallus can range from the edges of the lichen to completely cover the outer cortex. Isidia are small cylindrical outgrowt hs from the thallus. They are covered by a cortex and break off due to mechanical stress or age (Budel and Scheidegger 2008).
7 A Figure1.2 A) Shows the range of shape of apothercia (from Brodo et al., 2001.) B.) All the small whitish bumps on the thallus are soredia ( http://farm4.static.flickr.com/ 3214/2934155451_b2a1725122.jpg?v=0 ) B CC.) Isidia are the small darker green cylinders, projecting upwards from the thallus http://www.tropicallichens.net/3605.html
8 Useful characteristics for identification There are a few other characteristics useful for identification. Pruina, a defensive mechanism, is a mineral deposit, such as calcium oxalate, on the surface of the cortex (Budel & Scheidegger 2008). Anot her characteristic is hair a nd hair-like structures. Cilia resemble hair and are often found along the marg in of the thallus, and tomenta are short, hairlike hyphae (Budel & Sche idegger 2008). Holes in the thallus, which expose the medulla, are called pseudocyphellae (Brodo et al. 2001). Cephal odia are gall-like appendages that grow in liche ns that have a green alga and a cyanobacterium (Brodo et al. 2001). Rhizines are little roots that grow on the underside of a lichen (Brodo et al. 2001). They are useful to determine genus and species in the family Physciaceae. Lichens: Extremophiles susceptible to ecological change Lichens grow in a variety of unexp ected and harsh environments ranging from the poles to the tropics. They can grow on various substrates including rocks, bones, soils, and tree trunks (Brodo et al. 2001). Some liche ns grow on other lichens or on the leaves of tropical trees (as rev. in Nash 2008). Lichen s that grow in these harsh conditions are called extremophiles. Despite their tolerance of harsh climates, changes in ai r quality affects lichen growth and populations because lichens are dependent on nutrients from the air. Loss of lichen diversity around a smelter in England was noticed by Erasmus Darwin in the 1790s (Nimis & Purvis 2002). Subsequent studies showed that lichens are affected by many chemicals, especially sulphur dioxide, ammonia, fluorine, dust and chlorinated hydrocarbons (Nimis & Purvis 2002). Lichens can be used to measure environmental
9 degradation due to pollutants; however, samp ling techniques must account for variables such as wind direction, age of forest a nd the lichens rate of chemical uptake. Climate change may also affect lichen populations, by altering macro and microhabitats (Insarov & Schroeter 2002). The first study to link global warming as a reason for lichen population changes occurr ed in the Netherlands (Herk et al. 2002). The study concluded that since 1972, arctic/alpine lichens are in decline and (sub)tropical lichens are expanding their range in the Ne therlands. Lichens favoring nutrient rich, warmer temperatures and more shade increased. These factors can be attributed to global warming. Large changes in climatic variable s such as humidity, precipitation, heat and cold, may affect the lichens stress tolerance. Also climate change may affect lichens by altering aspects of habitat such as tree populations and tree hea lth. This point is illustrated by Kranderud and Totland (2005), who found that lichen diversity decreased due to rising temperatures coupled with nutrient additions, but not climate change. This highlights that indirect effects of global warming could harm lichens. More studies will further elucidate the affects of global warming and how temp erature change combined with nutrient changes affect lichen populations. Lichens: Forest age and microhabitat dependent The range of a species is restricted by the need for specific microhabitat variables such as light, bark pH, substrate type, and moisture. Lichens are also affected by succession and macroclimate. It is difficult to isolate a ny one variable as the sole reason for a lichen living in a particular spotit is more real istically multiple variables working together (McCune 1993). Succession, which is the changes a community undergoes throughout time (http://www.oxfordreference.com), illustrates the point that multiple variables affect
10 lichen populations. Older, undisturbed forests have more lichen diversity than younger or disturbed forests. Older boreal forests (150-200 years old) were 45% more diverse in Sweden than younger forests (Dettki & Esseen 1998). Also lichen biomass increased with forest age (McCune 1993; Esseen et al. 1996; Dettki & Esseen 1998). Microhabitat variables, which change dur ing succession also affect lichens at both the tree and community level. These variables can also change vertically in a tree. Some examples are substrate, type of bark, age of bark, pH of bark, and for ground dwelling lichens soil and soil pH. Early colonists of small twigs were replaced with other species as the twig grew (Sillett and Antoine 2004). pH was found to be an important variable for lichens in Estonia (Juriado et al. 2008, Juria do et al. 2009). Bark type is also important. Older bark in a Quercus species had more lichens pres ent, perhaps because of the crevices (Ranius et al. 2008); however it is difficult to conclude that crevices are responsible for colonization and not other variables. One last va riable to consider is light. Its role is still unknown and may depend on the surrounding ecosystem (Sillett & Antoine 2004). It is difficult to isol ate light from moisture as growth requirements. It is important to note that a variable, such as light, can affect another variable which then limits lichen growth. An example is shade that causes bryophyte growth. The bryophytes out compete lichens and kill most of them. Bark pH was found to be more conducive for bryophyte growth in boreo-nemoral forests in Estonia, which led to more bryophytes than lichens (Juria do et al. 2009). Research on fa ctors that affect lichen growth is still in its infancy. Future experi ments involving transplant ing lichens will yield more knowledge about the requirements for lichen survival.
11 Current canopy research It is harder to study the canopy than the trunk of the tree becau se of accessibility issues (reviewed. in Lowman and Rinker 2004) To date, most studies have focused on taxonomy, especially in the tropics (Sillett & Antoine 2004). However, the same biotic and abiotic factors that affect the trunk the tree, such as light, bark type, and succession, also affect lichen populations in the canopy (McCune et al. 2000). It was well documented that lichen communities are stratified vertically (Hilmo 1994; McCune et al. 2000; Campbell & Coxson 2001; Peterson & McCune 2001). Lichen populations at various heights in the tree differed by forest ty pe, but height was the most important variable for explai ning the changes in diversity. Other characteristics such as bark type, shade, stem diameter of branch es and trunks and placement in the tree explain changes in lichen populations, but the changes vertically in lichen diversity were most strongly correlated with height in the canopy (McCune et al. 2000). Similar gradient hypothesis A different perspective on lichen diversity within a tree and a stand is to use climatic gradients as the most important factor (McC une 1993). The similar gradient hypothesis is based on data that divided lichens into f unctional groups and examined these groups in different aged forest stands and height in tree. McCune (1993) observed that functional groups of lichens move up the tree as it age d. The similar gradient hypothesis stated that there are three gradients that explain ep iphyte colonization (McCune 1993). The first observation was that there are differences in lichen diversity with vertical stratification. The second observation was that forest stands of the same age can have different lichen populations. This stratification occurred because of moisture and climatic changes. The
12 third point of the hypothesis was that lichen populations change over time. Lichen species migrate up the tree to live in the same mois ture gradient. The similar gradient hypothesis was cited by numerous sources studied in th is thesis, though as th e author acknowledged it needs to be more thoroughly tested and there will be excepti ons (McCune 1993). It nevertheless provides an approach that examines water and climate as a mechanism for lichen colonization from the base of the tree to the canopy. 1.2 Literature review of Florida lichens Few lichenological studies have been conducted in Florida. As of the writing of this thesis, fewer than 15 peer-reviewed papers about the ecology of Florida lichens exist. Lichen taxonomy in Florida is well know n (Harris 1990, 1995), but few studies have experimented with Floridas lichens. This should be addressed because Florida is the largest region in the United States with tropical lichens. Florida has four geographical ranges of lichens (Brodo et al. 2001) (Figure 1.2). Three ranges describe temperate lichens. East Temp erate and Pan Temperate lichens are found north of Lake Okeechobee. Coastal Plain temperate lichens are found throughout the state. Tropical and subtropical lichens are typically found in th e southern half of the state though some species of subtropical li chens are found throughout Florida. Taxonomy studies in Florida The first lichenologist to catalogu e the macrolichens of Florida found 150 species from 25 genera (Moore 1968). Hale (1969) in cluded some Florida lichens in his book How to Know the Lichens the first dichotomous key of North American lichens. Harris (1990, 1995) provided the most detailed keys to Floridas macro and microlichens.
13 Figure 1.3 : There are 4 ranges of lichens in Florida. Ea st temperate, coastal plain temperate and Pan temperate are found roughly north of Lake Okeechobee. Tropical/subtrop ical lichens and coastal plain temperate are found south of the Lake Okeechobee. (outline of Florida from http://www.50states.com/maps/florida.gif ; content from Brodo et al 2001; diagram by Barry Kaminsky) Lichenologists have made comp rehensive lists of genera such as Cladonia (Evans 1947), and Haematomma (Culberson 1963). Despite these taxonomic advances, there are still many unexplored regions of Florida. New species, such as Gyalectidium yahriae Buck and Serus, are constantly found throughout Florida (Buck & Serusiaux 2000). Many species, new to North America or the wo rld have been found in the Everglades and surrounding swamps. Also a species of Cladonia which had not been collected in over 100 years was recently found. Two days of collecting lichens at Fakahatchee Strand yielded 50 species new to Florid a or the world (pers. comm., Rick Seavey). This project hints that there may be many more undi scovered lichens in South Florida. Little research has been conducted on the ecology and molecular data of Floridas lichens. One reason for this dearth of studies is the paucity of liche nologists in Florida. There is only one lichenologist in the state school system: William Sanders from Florida Gulf Coast University. There are also a few lichenologists that live in Florida in the
14 winter, Rick and Jean Seavey. This lack of scientists limits the amount of research conducted in Florida. Most of the United States lichenologists who study ecology are based in areas of relatively high diversity of macrolichens, and subsequently receive higher funding. However, Florida remains an undiscovered arena of lichen research and research could alter our perceptions on diversity of tropical forests. Ecological studies in Florida A recent study in Ocala National Forest mapped the diversity and abundance of the lichens in three different habitats (DeBolt et al. 2008). They found that slight changes in elevation affected the vegetation of the forest and also the lichens in the forest. Lichens were most diverse in a sand scrub pine, followed by hardwood hammock and sand pine forests. This paper was significant because it calculated diversity indices and examined the variables responsible for these three dis tinct lichen habitats. Some variables that explained the change in lichen population were leaf fall (and th e lack thereof), fire, lack of hardwood substrate, soil, light, humidity and flooding. Another important contribution was that it found indicator species which c ould be used to assess habitat health. The genus Cladonia has received the most ecological attention in Florida. It was described in detail by Evans (1947). The endemic species C. perforata listed as an endangered species in the United States, is on ly found in scrub pine forests in Escambia and Highlands counties in Florid a (Buckley & Henderson 1988; Brodo et al 2001). Even within scrub pine, C. perforata needs a specific community to grow in. Only 7 of 84 rosemary balds at Archbold Biologi cal Station support communities of C. perforata (Buckley & Henderson 1988). Roger Rosentre ter is currently conducting transplant studies to study the variables that affect th is community (pers comm with Rosentreter).
15 Lichen cover is an important ecologi cal mechanism that limits seed germination of some plant species in scrub pines. The presence of Cladonia species lowered the seed germination of many types of shrubs (Hawkes & Menges 2003). A similar study examined the effects of lichens and found that lichen cover may limit the number of Hypericum cumulicola seeds that would germinate in rosemary shrubs (QuintanasAscencio & Morales-Hernandez 1997). By limitin g seed germination, lichens can affect the vegetation of the environment. Two New College students wrote theses on lichens in Sarasota County. Sirko (1980) mapped lichen diversity at 16 sites in H illsborough and Sarasota County to see if pollution limited diversity and found that lichen diversity was less in areas closer to the source of polluting businesses. Lichens were sa mpled from 0.1 m to 6 m from the base of the tree. Oscar Scherer State Park and Myakka River State Park, which were the farthest away from the source of pollutants, had the most diversity. Twenty-one species of lichens were present at Myakka River State Park co mpared to 25 at Oscar Scherer State Park. More foliose and fruticose lichens were pres ent in Myakka while Oscar Scherer had more crustose lichens. This thesis is the only source of information on lichens from southwest Florida, and is currently the best reso urce for lichens found in Sarasota County. Morris (1995) designed a series of sm all experiments to teach middle school students about lichenology methods. One lesson comp ared diversity between red and black mangroves. The lesson showed that red ma ngroves had more lichen populations, but diversity was the same on red and black mangroves.
16 1.3 Tolerance to desiccation and inundation Lichens have the remarkable ability to survive environmental extremes ranging from the poles to deserts to microclimatic extremes. Lichens are affected by these environments but they have developed mech anisms to survive (Kappen 1973). They can also flourish where vascular plants can not grow because lichens developed mechanisms, such as antifreeze proteins and photosynthesizi ng below freezing, to withstand the cold climate, and keep their water balanced (Becket t et al., 2008). Lichens response towards many stresses has been tested. Some examples are drought, inundation, humidity, cold and fr eezing, fluctuations in temper ature, visible radiation and light and darkness (Kappen 1973). More recentl y, lichens were launched into outer space and tested for effects of radiation and vac uum conditions (Beckett et al. 2008). Lichens ability to survive desiccation has been studi ed most thoroughly because water relations are crucial for lichens to surv ive drought (Beckett et al. 2008). This thesis is concerned with abiotic stress, particularly water and moisture. Biochemical response to stress To provide some context for ecological observations and experiments, it is first necessary to talk about the chemical processe s that the lichens under go related to stress. Stress is a biotic or abiotic variable that affects the grow th or population of another organism. The study of chemical pathways and components that aid in lichen survival of extreme conditions is just be ginning. It was recently establ ished that most stress in lichens is due to the formation of reactive oxygen species (ROS), such as singlet oxygen, superoxide, hydroxyl, and hydroperoxyl radicals (Beckett et al. 2008). The effects of radicals were minimized by either preven ting the formation of radicals or removing
17 radicals from the lichen (Beckett et al. 2008). Some ways that lichens removed or minimized the harmful effects of radical s are through enzymatic and nonenzymatic systems such as superoxide dismustase and peroxidases, nonreducing sugars and proteins such as dehydrins (Beckett et al. 2008). This research is fo cused more on desiccation than submergence tolerance. The pathways and the mechanisms that lichens can use to survive submergence may be different than desiccation pathways. Lichen tolerance to subm ergence and lichen trimlines Kappen (1973) reviewed lichen toleran ce to different types of stress and made two points about lichens response to submergen ce. The first point was that lichens not adapted to surviving in water will die within a short period of inundation. The second finding was that the deaths of water intolerant lichens form distinct zones of lichens. Most lichens die within one to two weeks of submergence (San tesson 1939 as cited in Kappen 1973). This is a largely accepted a nd widely published accepted figure. Results from other studies indicated th at it is a reasonable estimate for temperate lichens. Hale (1984) used lichenometry by measuring the height of the lichen line a nd came to a similar conclusion on Florida lichens. Since flooding kills lichens, it causes vi sible lichen zonations, called a lichen trimline (Figure 1.3). A lichen trimline is a horizonta l line of older lichen s and underneath no lichens or drastically younger lichens (Mar sh & Timoney 2004). A lichen trim line is formed from prolonged flooding, which kill s lichen populations (Marsh & Timoney 2004). Other studies affirmed the idea of a lichen trimline in ponds, rivers and other water habitats (Santesson 1939, Beschel 1954, Ried 1960, Schubert and Klement 1966, Wirth 1972 all cited in Kappen 1973).
18 Figure 1.4 : Shows an example of a lichen trimline. There is a clear horizontal line of lichen growth. There are no lichens growing beneath this line. This line is the highest point of a prolonged flood. Bryophytes are growing beneath the line (bottom right corner). Photo taken at New College of Florida by Barry Kaminsky. Lichen trimlines (Figure 1.3) were doc umented in water ecosystems of many countries including Swedens lakes (Santesson 1939 as cited in Kappen 1973), Florida (Hale 1984), river basins in Canada (Marsh & Timone y 2004), Switzerland (Keller & Scheiddeger 1994) and Australia (Gregory 1976 as cited in Marsh and Timoney 2005). Similarly, Beckilhimer and Weaks (1984) observed that lic hen diversity was lowe st along regions of a river that often flood.
19 Kappen (1973) summarized a large portion of scientific understa nding of lichen water ecology. Since Kappen (1973), few studi es have looked at lichens and their s and elationship to flooding. Articles either re affirmed that flooding will cause lichen zonation or observed the time for a lichen to die when placed in flooding conditions. s in hree at interest, two d ns on rocks along a river delta surv ived 30-180 days submerged in water (Marsh & Timoney r Current Research on the wa ter tolerance of lichens Lichenologists have recently identifie d more lichens that can survive long period water. Lichens that live submerged in water permanently or for a portion of the year are called amphibious lichens. They were first described by (Santesson 1939 in Kappen 1973). These lichens usually grow on rocks or trees and can survive flooding up to t months. The most common amphibious gr oups are Verrucariaceae and Collemataceae (Seaward 2008). European sc ientists have more thor oughly researched amphibious lichens than those in the United States. Many recent searches have found amphibious lichens in England, Switzerland, Austria and Spain (Nescimbene & Nimis 2006) and Canada (Marsh & Timoney 2005). Forty thr ee amphibious lichens in 20 genera were found in a recent survey in Italy (Nesci mbene & Nimis 2006). These recent studies suggest that this type of lichens may be mo re prevalent than previously thought and th there are many more places in the world to look for amphibious lichens. Of species of lichens have been found that live submerged in water their entire life. The species are Hydrothyria venosa and Leptogium rivale (Brodo et al. 2001). Lichens have been found to survive longer than a week in watery conditions in arctic regions. Species of Peltigera in southern Patagonia surviv ed up to a few weeks in floode conditions (Winchester & Harrison 2000). In Alberta, Canada, some species of liche
20 2005). These more recent findings suggest th at the tolerance for flooding for some species is longer than previous ly thought, and that more areas should be tested to confirm ltitudes d use snow and snowmelt to imer les have been conducted since to rove this point and develop measuring techniques. en 1996), but this experiment has yet to be replicated in other swamps around the world. these findings. One trend is that more species of am phibious lichens are found at higher elevations and towards the poles. The amphibious lichens in Italy were found at subalpine a as opposed to the lower elevation oak forests (Nescimbene & Nimis 2006). One explanation for this is that lichens have adapted to snow and snow melt by developing mechanisms to control CO2 intake at lower temperatures, an maintain a chemical balance (Nescimbene & Nimis 2006). It is well known that water will kill most non-amphibious lichens but other effects of water on lichens have not been thoroughly studied. One idea is that sediments deposited by moving water can become trapped in tree ba rk and inhibit lichen growth (Beckilh & Weaks 1984). Also, pollutants traveling in water were speculated to affect lichen populations (Beckilhimer & Weaks 1984). Few artic p Lichen studies in swamps Few ecologically focused articles on lichens and swamps have been published. Swamps are intriguing because they are syst ems that hold water for long periods, and may have different lichen populations than unflooded areas. Few articles have been published comparing the lichen populations between an area with flooding and an adjacent area without flooding. No differen ce in diversity was found between flooded and unflooded forests of Picea abies in Finland (Kuusin
21 The effects on lichen populations from water pH and proximity to water was documented by Gilbert & Giavarini (2001). Th ey found that water pH and alkalinity caused lakes to have distinct lichen habitats on their shoreline. They also postulated that there are four types of lichens in relation to water: aquatic, amphibious, terrestrial lichens that can live briefly in water, and terrestrial lichens that are water intolerant. These zones are further proof that water, moisture or waves, can affect lichen populations, though other variables such as substrate need to be considered. Future directions for lichen research Lichenologists have focused on te sting lichen water tolerance, though more information about Florida lichens tolerances towards water would be useful to study Floridas ecosystems. On another note, few st udies have looked at how water shapes the lichen diversity throughout an ecosystem in Florida. More studies should be conducted on tropical and subtropical lichens in Fl orida and its swamps. Although lichens are dependent largely on microhabitats, they may be influenced more heavily in swamps by water or a variable related to water such as humidity. There have also been few comparisons of lichens that grow on the same tree species in different habitats, such as swamps and oak hammocks. Finally there s hould be a more thorough examination of Floridas canopy lichens. This study aims to address a few of these issues. 1.4 Goals and hypothesis The first part of this thesis tested the vigor of lichens submerged in water. Six common lichen species from the canopy and the trunk of Myakka River State Park were tested for their survivability when subm erged. My hypothesis was that lichens living
22 lower on the trunk, closer to seasonal flooding, w ould have less damage to their thalli on days 1, 3 and 5 than species above fr om the water line (canopy species). In the second part, the diversity and abundance of lichens was assessed on Quercus spp. every two meters from the base of tr ee to the upper canopy on the north, south, east and west side of trees at My akka River State Park. I hypothesi zed that the total cover of foliose and crustose lichens would not change from the forest floor to the canopy, while total cover of fruticose lichens would incr ease from the forest floor to the canopy. The third experiment was a small taxonomy survey of canopy lichens at Myakka River State Park. Fallen branches were collected and macrolichens were identified to species. The hypothesis was that Myakka River State Park would have lichens that were not found in Ocala National Forest.
23 Chapter II: Lichen tolerance to submergence 2.1 Test Site The test site, Mya kka River State Park, was founde d in the 1930s. It surrounds the Myakka River, which flows through 58 square miles of wetlands and is an important source of fresh water for the Charlotte Harbor Estuary ( http://www.floridastateparks .org/myakkariver/default.cfm ) (Figure 2.1a). The park is 150 km2 and has numerous types of habitats, rang ing from oak and palm hammocks, pine flatwoods, flood plains and hardwood hammocks (Huffman and Judd 1998). The lichens included in this study were collected along a one-forth mile long stretch of Ranch House Road, which is one mile south of the Upper Lake (Figure 2.1b). This trail was chosen because it floods slightly over the summer (pers. observation). 2.2 Methods The water tolerance of six lichen speci es was tested over a six day period. The species used were Cryptothecia rubrocincta (Ehrenb. : Fr.), Cladonia spp., Physcia sorediosa (Vainio) Lynge, Usnea strigosa (Ach.) Eaton Leptogium spp., and Graphis spp. Canopy species, defined as lichens livi ng on small thin branches were U. strigosa and Graphis Cladonia, Leptogium and P. sorediosa, were found from the base of the tree to the upper trunk. C. rubrocincta was found on thick horizontal bran ches and the trunk, but never on small branches. Species are described in mo re detail in Appendix I and Appendix IV. Eighteen samples of each lichen species were collected at Mya kka State Park with a permit and transported to New College of Florida. To minimize damage to the thallus, a small piece of the bark was removed with th e lichen. Damaged specimens were not used in this experiment.
24 A B Figure: 2.1 A) Shows the location of Myakka River State Park. It is located off Route 72, west of I-75. from www.localhikes.com/images/MSA_7510/MyakkaMossyHammock/MyakkaMossyHammock_Map.Jpg B) Shows the location of House Ranch Road. It is a mile south of the Upper Myakka Lake. http://www.myakkariver.org/parkmap.html Eighteen specimens of each lichen species were used in the experiment, a total of 90 samples. The experiment was set up in a shaded area under a Quercus canopy on the
25 southeastern corner of New Co llege of Florida campus near Physical Plants tool shed (Figure 2.2). Each lichen was placed in separate glass jars to preven t interactions with other lichens. Before being placed into a ja r, lichens were arranged by thallus size by sight, so that each day would have a small, medium and large sample to prevent thallus size from altering the results. The experiment was set up to measure the time it took for visibl e damage to occur and the total amount of physical damage that occu rred over 8 days of submergence. Lichens were placed in groups that were submerged for different number of days. Three samples of each species were placed in water for each sampling day: 1, 3, 5, or 7 days. There was also a control group which was not submerged. Figure 2.2 : shows the set up for the submergence experime nt. There were 6 rows, one for each species, and then 15 jars each with one lichen. The jars in this pho to are empty, but they were filled with 0.946 liters of water. (Photo by Barry Kaminsky) Each lichen (except controls) was pl aced in 4 ounces (0.946 liters) (Figure 2.2) of water from the Myakka River. The water was collected just before the dam near the upper
26 lake, and had a pH of 6.0, which is slightly acidic. The lichens were placed in each jar and left in the water until their designated day of removal. Lichen vigor was assessed only on the day the specimen was removed to minimize exposure to air, which could act as another stress on the lichen. Lichen vigor was assessed on days 0 for all species, and 1, 3, and 5 days for samples removal from water. The scale used by Ma rsh and Timoney (2005), which based lichen vigor on physical observations, was used to assess vigor (Table 2.1) Vigor was defined as the percent of the healthy thallus based on visual cues su ch as thallus softening and texture, and chlorotic and algal patches. The sc ale was 0 zero for completely dead to 6 for 100% health. Six was defined as algae green, lichen attached to substrate and texture normal. Each specimen on day 0 had a vigor of 6. Table 2.1. Qualitative Scale Lich en Vigor* Lichen Appearance Qualitative scale Percent health (%) Characteristics of health 6 100 algae green and healthy; texture normal; lichen attached to substrate 5 90 small areas of algal layer starting to become chlorotic; texture normal; small pieces of lobes exfoliating 4 70 patchy chlorotic or pinkish algal layer; thallus softening; lobes exfoliating 3 50 pronounced chlorotic or pinkish algal layer in 50% of thallus; thallus softening; several lobes exfoliating 2 30 few small areas of green algae; upper cortex disintegrating; thallus exfoliating 1 10 one or two patches of green algae; texture compromised; whole thallus exfoliating 0 0 lichen dead % Vigor is the approximate portion of a thallus that remains healthy; for a population of thalli, it is the proportion of healthy thalli. Table 2.1: Scale used to qualify lichen vigor. It assesses physical characteristics to gauge lichens health and ability to make its own carbon dioxide (Timoney and Marsh 2005). The scale ranges from 6 meaning the vigor of the lichen is 100%, to 0 where the lichen is completely dead.
27 Data were analyzed using three statistical methods. The health of each species was compared to each other for day 1, 3, and 5 us ing an F-Test and a then a Tukey grouping for a post-hoc test. Another F-te st was conducted, but took out the Leptogium specie (the lone cyanobacteria photobiont), to test th e species effects on statistical significance. Finally, the mean vigor of the controls on day 5 were used to examin e if other variables affected the loss of vigor. 2.3 Results All samples began with 100% vigor, a six on the Timoney and Marsh (2005) scale. Vigor of all species decreased from day 0 to day 5, which confirmed the initial hypothesis (Figure 2.3). Four species of lichens showed a steady decline in vigor. These species were Cladonia spp., C. rubrocincta, P. sorediosa, and U. strigosa. These findings were largely expected. A few species though increased in vigor fo r one day of the experiment. After one day of submergence, four species decreased in vigor. U. strigosa, Graphis spp, P. sorediosa and Leptogium spp had a mean health of 4.7about 25% of the thallus was damaged. Damage such as thallus soft ening, and chlorotic pa tches were readily evident. C. rubrocinctas mean health was 6. Cladonia spps mean health was 5only about 10% of the thallus was damaged. On day 3, Leptogium was healthiest ( = 5.67), followed by Cladonia (5.0) then P. sorediosa and C. rubrocincta at 4.0. Graphis and U. strigosa had a mean health of 2.0 meaning that 70% of their thalli showed dama ge. On day 5, the order of impact from least to greatest was Leptogium (5.67), Cladonia (4.25), P. sorediosa (3.0), Graphis (2.25), U. strigosa (0.0) and C. rubrocincta (0.0) (Figure 2.3).
28 Lichen tolerance to submergence in water0 1 2 3 4 5 6 1234 Days submergedVigor U. strigosa C. rubrocincta Cladonia spp. Graphis spp. P. sorediosa Leptogium spp. Figure 2.3 :Shows the health of the lichen species used in th e drowning simulation in Part 1, using the scale made by Marsh and Timoney (2005). All species had an initial health of 6 on day 0. Sample size for all days was unequal because some samples were lost due to squirrel interference (n=34). F-tests, showed that there were signi ficant differences in the mean health of lichen species on days 1, 3 and 5. For day 1, there we re six groups of three (n=18), and the FTest results were F(5,12)= 3.90, p=0.0248, R2= 0.619. The Tukey grouping showed that C. rubrocincta (mean= 6.0) was statisti cally significant from Usnea ( = 4.0) (Table 2.2). On day 3, the F-test data was (n= 18), F(5,12)= 5.64, p= 0.0067, R2= 0.701, and had significant differences in healt h. The Tukey groupings show that Leptogium is significantly different from Usnea ( =2.0) and Graphis ( =2.0). On day 5, there were 6 groups of une qual size, ranging from 3-4 samples per species (n=21) and F(5,15)= 42.36, p=0.001, R2= 0.934. The Tukey groupings show that the data were divided into more groups, meaning that the differences between species health loss was larger. Leptogium ( =5.67) was significantly highe r from all species except Cladonia. spp. P. sorediosa ( =3.0) and Graphis ( =2.25) were signifi cantly different
29 from Usnea ( =0.0) and C. rubrocincta ( =0.0). The F-tests using six lichen species revealed two statistically significant trends. Usnea and Graphis showed significant loss of vigor more quickly than other species. S econd, the rates of decay differed significantly among species by day 5 (F(5,15)= 42.36, p=0.001, R2= 0.934). To further examine the data for differe nces, an F-test was r un without cyanobacteria photobionts (Leptogium ) to see how the rates differed (T able 2.2). The significance of the F-test changed for each day, but the Tukey groupings did not change significantly. The results showed that on day 1 F(4,10)= 12.00, p=0.0008, R2= 0.828, the C. rubrocincta was significantly different from all other species On day 3, there was a significant result (F(4,10)= 3.86, p=0.0379, R2= 0.607) but a Tukey grouping did not show significant differences. On day 5, F(4,13)= 30.40, p=0.001, R2= 0.903, Cladonia (mean=5.0) was significantly different from Graphis (mean=2.25), Usnea (mean=0.0) and C. rubrocincta (mean=0.0). Based on this analysis, Graphis was also significantly different from Usnea and C. rubrocincta Table 2.2 F-Test values for submergence experiment for days 1, 3 and 5 F Test DF F Value P R2 All 6 species Day 1 5, 12 3.9 0.0248 0.619 Day 3 5, 12 5.64 0.0067 0.701 Day 5 5, 15 42.36 0.001 0.934 Without Leptogium spp. Day 1 4, 10 12.0 0.0008 0.828 Day 3 4, 10 3.86 0.0379 0.607 Day 5 5, 15 30.4 0.001 0.903 Controls (day 5) Day 5 5, 12 4.15 0.0203 0.633 The P value for all experiments were significant, meaning that the rates of loss of health differed significantly between species.
30 Lastly, the health of the control lichens from day zer o and five were compared and found significant differences in health F(5,12)= 4.15, p= 0.0203, R2= 0.633 (Table 2.2). The means of all 6 species of lichens decreased in th e control group (Table 2.3). Surprisingly, Usnea had the highest vigor, at 5.67, while C. rubrocincta has the lowest health at 3.0. The only significant difference between the controls of day five was Usnea and C. rubrocincta Table 2.3. Mean vigor of control lichens on day 5 Species Mean vigor of controls Usnea strigosa 5.67 Leptogium spp 5.0 Graphis. spp 4.33 Cladonia spp. 4.0 Physcia sorediosa 4.0 Cryptothecia rubrocincta 3.0 Shows that all 6 species of lichens lost health between day 0 and day 5. 2.4 Discussion There were three significant findings from this experiment. The significant results were: 1.) There was a difference in water tole rance between species th at live on the lower trunk of the tree and the canopy 2.) Lichens wi th cyanobacteria photobionts had a higher vigor than those with green alga; and 3.) Each lichen speci es showed different physical degradations. Despite these results, a few ar tifacts of sampling will be addressed. They include: inaccuracy of using physical qua litative measurements species selection, sinkability and squirrels. There was a difference in lichen health between the canopy and the base of the tree. The species from the canopy, U. strigosa and Graphis had lower vigor than the lichens closest to the ground, Leptogium spp. and P. sorediosa. This finding suggests that the moisture gradient is one component that affected lichen stra tification in trees at Myakka.
31 Water tolerance is a factor in lichen surviva l; however, other variables such as light are important (Sillett & Antoine 2004). Flooding may be a limiting factor closer to base of the tree, but water tolerant species such as Leptogium and Physcia spp. are not restricted to growth near the base of the tree (obs ervation from vertical survey). Also, water intolerant species such as C. rubrocincta, grow near the lichen trimline, but also grow into the canopy. The comparison of the control data from day 5 suggested that, microclimate and macroclimate were altered in this experiment and could have affected lichens loss of health. Some species live in specific habita ts, and altering a variable could potentially change their biochemical reaction to wate r, thus altering th e loss of vigor. The experiment took place on ground that was shad ed for most of the day which may have affected sun dependent lichens more. The most sun tolerant lichens seemed to be C. rubrocincta, U. strigosa and Graphis spp. (pers. observation). However, if these lichens were to fall from the canopy to the ground at My akka they would be in shaded regions, so the experiment in some ways mimics what happens under certain natural conditions. Due to time constraints, the experi ment was conducted at Ne w College of Florida, which is closer to the Gulf of Mexico a nd north of Myakka. Macroclimatic variables, such as the salinity in the air, humidity a nd temperature, may have been changed. These factors could have contributed to lichen stress and affected their response to submergence. Only one lichen species in th is study is found on New College of Florida property. Future experiments should be conducted at or close to Myakka River State Park for more accurate results.
32 The second major result was that Leptogium spp, which had a cyanobacteria photobiont. showed less signs of damage than the lichens with green algae photobionts. In this experiment, five lichens had green algae photobionts and one lichen had. Although more testing is necessary, the results suggest ed that cyanobacteria and green algae have different tolerances required to survive wa tery environments. Previous studies have shown that cyanobacteria flourishes in habita ts that have adequate liquid water, while lichens with green algae prefer to get their water from vapor (Green et al ., 2002). Lastly, each lichen species showed di fferent physical degradati ons to water and also had a different rate of vi gor loss. The thallus of Cladonia turned brown. P. sorediosa thallus turned white in small dots. C. rubrocincta and Graphis both crustose lichens, had similar characteristics of degradation. Some of the characteristics were loss of color in the thallus starting on the edges and movi ng inward with days of immersion. Damage to U. strigosa was the most dramatic (Figure 2.4 ). After one day in the water, the thallus was limp and fell to its side. It was not able to stand vertically which it normally does. None of the samples from day 1 recovered vigor. This suggests that the damage to U. strigosa due to water happened quickly and irreversibl y. The controls mean health of day 5 was highest of all si x species; yet the experimental mean of U. strigosa health was 0. U. strigosa was the lichen most affected by water. It was hard to determine damage to Leptogium spp. thalli because when placed in water it turned jelly-like. On e of the keys in Marsh & Timoney (2005) was that thallus softening is damage. The incr ease in vigor from day 1 to 3 was most likely due to mistaking the jelly-like appearance for damage Another possibility was that the loss of health was not noticeable, such as changes in carbon intake.
33 Figure 2.4 : The damage to U. strigosa happened within one day of submergence. The photo on the right depicts a sample at full vigor. Its structure is intact and thallus is a green gray (dark green with proper lighting). After one day of submergenc e the thallus is turning red, and it can no longer support itself (right photo). The vigor of the day 3 lichens was a 2. (photos by Barry Kaminsky) A B The differences in physical damage between species and the inability to assess physical damage showed that it is difficult to assess thallus health based on visible observations. Measuring the physical appearan ce of lichens provides useful information on thallus health, but it can be misleading becau se it is hard to determine if a lichen is alive or dead by sight (as reviewed by Kappe n 1973). It is hard to see the algae bleaching when lichens are dying. Measuring CO2 and O2 during respiration and photosynthesis may be better indicators of stress. It is po ssible that the biochemical mechanisms and thallus health were more affected by water than the outer appearance showed. It would have been more accurate to measure changes in the rates of CO2 and O2, especially for Leptogium since it was hard to assess damage to its thallus. Porometers are one feasible option (Lange et al., 1984; Kappen et al., 1989 ). A small lichen sample can be removed from a habitat and then placed in the poromometer, a small chamber that measures photosynthetic rates. All vari ables are held constant in cluding radiation and air temperature. The CO2 and O2 can then be measured and the lichen can be returned to its environment and measured again in the future.
34 Selection of Species Species were chosen based on available li chens. I tried to encompass a range of niches such as growing in shade, ground and canopy for comparison. Growth forms of lichens were also considered, including foliose, crusto se, and fruticose. All lichens were chosen from the lower trunk of the tree or low ly ing canopy branches by the author. Lichens were divided into species and by height in tree for later analysis. It was difficult to identify lichens in the field, and identifying lichens to species would have damaged the thalli. Later analys is revealed that three species of Leptogium and at least two Graphis were used. It was possible that there was a difference in water tolerance between species in both genera. Species differences may explain the variability of the Graphis spp. results. Graphis vigor decreased overall from a 6 on day 0 to 2.25 on day 5 but it increased slightly between the days 3 and 5. This could be because a few distinct species of Graphis were used. Although the species of Graphis were on the same tree at the same height, it is possible that the species had different tole rance to water. Future experiments should minimize the number of species of a genus. Sinkability Getting the lichens to sink was a challeng e. To remove the lichens intact, pieces of bark were taken from the tree. The tradeoff for an intact thallus was that the bark floated, and some lichens were only pa rtially covered, specifically P. sorediosa, Leptogium spp. and Cladonia spp. This may have minimized the inte raction between thallus and water. The lichens that fell to the bottom died fast er but that may have been coincidence since the lichens at the bottom of the container were U. strigosa and Graphis spp. In future
35 experiments, the bark should be glued or fast ened to the bottom of the jar using nontoxic methods or string to ensure that they are co mpletely submerged and that water depth is equal for each sample. The one unexpected finding was high health of Cladonia spp. throughout the experiment. It was expected to be lower. A po ssible source of error is that the samples of Cladonia floated on the surface of the water a nd were only partially immersed. This could have decreased the eff ect of water on lichen vigor. Experimental Design Improvements There are a few experimental design ideas that should be implemented for future experiments. The first is that future experi ments conducted outside should be placed in a more secure area perhaps inside fencing. This is because squirrels knocked over half of the jars between the night of day 4 and sa mpling on day 5. Since a significant amount of time had elapsed, all samples from days 5 and 7 had to be treated as day 5. Also, future design should minimize the damage of tree branches falling. Finally, measures should be taken to prevent rainwater from affecting the experiment. This happened on the evening of Day 2, and the author emptied the jars of the controls and day 1, once the rain subsided, but rain water was left with the Myakka samples. 2.5 Future Directions This experiment was a useful pilo t study to gauge Myakka lichens tolerance for prolonged submergence. It highlighted some general trends of lichen tolerance to submergence, but more specific studies can be designed. The first improvement would be to use more accurate measures to assess vigor. One possibility is to use CO2 intake or O2
36 respiration (Kappen 1973). Another possibility is to measur e the health of the algal and fungal symbiont separately. It could be that they respond diffe rently to submergence, and that one symbiont has more control to subm ergence than the other. Future experiments should focus on a more specific comparison, to rigorously test differences in lichens response to water. One example is to test multiple species of the same genus against multiple species of a different genus. This would show differences to water tolerance within a genus, as well as the differences between genera. Another example would be comparing a genus of green alga with a genus of cyanobacteria. It would also be possible to compare the water to lerance of different lic hens species of the same genus growing in the canopy and the base of the tree. This could illuminate if water tolerance is specific to height in tree. Finally, it would be us eful to change the amount of light a lichen receives to see how much sun light affects lichen vigor during submergence. A different approach would be to transplant lichen s from the canopy to the lichen trimline and see how water affects the lichen. This is feasible, but difficult since other variables such as light and temperature have to be controlled also. Transplanting lichens is a study that would take years to complete, since lichens grow slowly. In conclusion, this experiment implied that water can restrict the growth range of lichens, especially between the upper canopy and the base of the tree. There are also differences between lichens with cyanobacter ia, and green algae, confirming previous studies. More experiments which minimize va riables and use more sophisticated means of analysis should be conducted.
37 Chapter III: Assessing changes to lichen cover vertically in Quercus spp. 3.1 Introduction The goal of this experiment was to su rvey lichen population changes due to vertical stratification on three Quercus spp. from the base to the upper trunk. The total cover of lichens was assessed on three specimens of Quercus spp. (oak) every 2 meters vertically. Then foliose and crustose cover was examined to see if its cover increased with height in tree. The hypothesis was that foliose and crustose would not increase w ith height in tree while fruticose cover would increase with height in tree. 3.2 Test site and trees The experiment was conducted in an oak and palm hammock, the dominant type of hammock found along the Myakka watershe d (Huffman and Judd 1998). The most common large trees were sabal palms, and laurel and live oaks. There was little understory vegetation amongst these trees, but there were a multitude of vascular epiphytes in the trees of this type of hammock (Huffman and Judd 1998; pers. observations). The test site was located about 150 feet south of canopy walkway at Myakka River State Park. This area was c hosen because it is a well-established and diverse hammock. Quercus spp. was sampled because it is a dominant tree specie in Myakka. Three trees were chosen based on the ease of access to the upper trunk. Tree 1 had a diameter of breast height (DBH) of 0.360 m. (14.2 in.), Tr ee 2 was 0.488 m. (19.2 i n.), and tree 3 was 0.561 m (22.1 in.).
38 3.3 Sampling Methods Trees were climbed using the single rope technique (rev. by Lowman 2004) (Figure 3.1). The first quadrat was placed where th e tree reached the ground (0 m.) and was conducted every two meters vertically. Data from 0 meters was not used because it was often in exposed roots or missing bark, and had very little to no lic hen cover. The second quadrat was placed at 0.4 m, just above the roots. The next quadrats were every two meters at heights 1.4, 3.4, 5.4 and 7.4 meters. Figure 3.1 : The photo on the left is the author, climbing a tree (credit: Forest Hayes). The quadrat was a 20 cm by 20 cm grid made from flexible, clear plastic. It was placed against the tree above the stated height s. At each of the six heights, sampling was conducted on the north, south, east, and west si de of the trees. Total lichen cover in each quadrat was measured along with cover for each growth form (Figure 3.2). If the lichen did not cover the entire square the percenta ge of each square was estimated. The presence of other epiphytes, such as bromeliads and ferns in the quadrat was noted.
39 Lichens were grouped into foliose (green algae), foliose (cyanobacteria), fruticose and crustose. Foliose (green algae) were co mposed of family Physciaceae, Parmeliaceae. Fruticose was composed of (Ramalina Cladonia, Usneathough Usnea is in Parmeliaceae). Crustose included all sp ecies tightly adhered to the bark. Figure 3.2: The photo shows a quadrat on a tree. Lichen are green and white, but the reflecting light made it difficult to count total cover. (photo by Barry Kaminsky) Lichen cover was totaled by height in each tree and examined using a Pearson correlation, to see if cover incr eased with height in the tr ee. Statistics for total lichen cover by cardinal direction were also examin ed. Lastly, the total cover of foliose vs. crustose lichens by height in tree was examined using ANOVA. 3.4 Results Total lichen cover of all three trees differed greatly by height (Table 3.1). At 0.4 m, tree one had 260 cm2 cover of lichens, while tree two had 10 cm2 and tree three had only 2 cm2. At 1.4 m, tree two had 849 cm2 of lichen cover, tree 1 had 371 cm2 and tree 3 had 307 cm2. Tree two also had the highest amount of lichen cover at 3.4 m (906 cm2) and 5.4
40 m (1044 cm2). Tree one had a cover of 299 cm2 at 3.4 m, and 208 cm2 at 5.4 m, while cover of tree three was 144 cm2 at 3.4 m and 491 cm2 at 5.4 m. At 7.4 m, tree three had the largest lichen cover with 363 cm2, followed by tree two (341 cm2), and tree 1 (238 cm2). The raw data showed that total lichen cover can change drastically between trees. To determine if there was a linear relationship between height in tree and lichen cover, a Pearson correlation coefficient was calculated, using all 5 heights. The results show that total did not increase or decrease for a ny tree (Table 3.1). For tree one, lichen cover decreased slightly from the floor to the canopy (r = 0.223, p= 0.345). In tree two, the total cover increased sligh tly with height (r= 0.330, p= 0.890), a nd in tree three the total cover also increased slightly (r = 0.341, p= 0.142) (Table 3.1). Table 3.1. Total lichen cover of i ndividual trees by height Height Lichen CoverTree 1 (cm2) Lichen coverTree 2 (cm2) Lichen coverTree 3 (cm2) 0.4 260 10 2 1.4 371 849 305 3.4 299 906 144 5.4 208 1044 491 7.4 238 341 363 Pearson coefficient -0.223 0.0330 0.341 Shows the total lichen cover by height for the three Quercus trees sampled and their Pearson coefficient. Total lichen cover did not increase from the ground to the upper trunk of any tree. Also lichen cover varied widely between trees. Additional statistics of lichen cover were compiled for all three trees. The total cover, which is the cover of all hei ghts and directions combined, va ried greatly between trees. The range was from 3150 cm2 (tree 2) to 1376 cm2 (tree 1) and 1307 cm2 (tree 3) (Table 3.2). Tree two had the largest mean lichen cover (134.05 cm2) followed by tree 1 (68.05 cm2) and then tree three (65.25 cm2). Tree two also had the largest median lichen cover at 73.5 cm2, followed by tree 1 (69.0 cm2) and lastly tree 3 (35.0 cm2). Standard deviation
41 ranged widely from 38.59 (Tree 1) to 151.94 (t ree 2). All trees had a similar minimum cover (0-12 cm2), but the maximum was very large (137 to 400 cm2) (Table 3.2). Table 3.2. Additional total cover statistics of three individual trees (cm2) Tree Pearson coeff. Total cover Mean Median Std Dev. Minimum cover Maximum cover 1 -0.22278 1376 68.8 69 38.59 12 137 2 0.03295 3150 134.05 73.5 151.86 0 400 3 0.34068 1305 65.25 35 91.54 0 373 The table provides details on the total cover, mean, median, minimum and maximum cover of the three Quercus trees sampled. Total cover, mean and median varied largely between all three trees. All quadrats for each cardinal directi on were added together and the mean, median and standard deviation, range and interqua rtile range were taken. Total lichen cover slightly differed between cardinal directions (Table 3.3). Total lichen cover was smallest on the north (941 cm2), then increasing for west (1230 cm2), south (1422 cm2), while east had the most lichen cover with 1769 cm2 (Table 3.3). The medians were 48 cm2 for north, 38 cm2 for south, 67 cm2 for east and 71 cm2 for west. Standard de viation varied greatly from 57.44 cm2 for north to 143.13 cm2 for east. The standard deviation of south was 122.33 cm2 and west was 92.2 cm2. The range, which is the difference between the maximum and minimum cover, wa s smallest for north (222 cm2). East (400 cm2), south (380 cm2) and west (373 cm2) were very similar. The interquartile range was 74 cm2 for north, 110 cm2 for south, 131 cm2 for east, and 100 cm2 for west (Table 3.3). Table 3.3 Differences of total lichen cover by cardinal direction Direction Total lichen cover (cm2) Mean Median Std. Dev. Range Interquartile range North 941 62.7 48 57.44 222 74 South 1422 94.8 38 122.33 380 110 East 1769 117.9 67 143.13 400 131 West 1230 82 71 92.2 373 100 Total lichen cover changed drastically between cardinal directions. The total lichen cover ranged from 941 cm2 for north to 1769 cm2 for east. The median was similar for eac h direction, while the means differed greatly. Range varied by direction as did the interquartile range.
42 Further analysis was conducted by sepa rating total lichen cove r into crustose vs. foliose cover. At each height in the tree, cr ustose cover was larger than foliose cover (Table 3.4). The one exception was tree 3 at 7.4 meters. For all cover, crustose lichens accounted for 85% of all lichens counted, while foliose was only 15% (Table 3.4). Table 3.4. Crustose vs. foliose cover for lower, middle and upper trunk (cm2) Tree Height Crustose Foliose 1 0.4 260 0 1.4 297 9 3.4 192 107 5.4 182 26 7.4 163 75 2 0.4 9 1 1.4 843 6 3.4 883 23 5.4 544 143 7.4 293 48 3 0.4 2 0 1.4 277 24 3.4 124 20 5.4 435 50 7.4 82 281 All tree Total 0.4 271 1 combined Total 1.4 1417 39 Total 3.4 1199 150 Total 5.4 1161 219 Total 7.4 538 404 Total cover 4586 813 Percent cover 85% 15% Only one quadrat where foliose cover was larger than crus tose cover. That exception is tree 3 at height 7.4. The total lichen cover (cm2), by cardinal direction, of all three tr ees combined changes between directions. North had the least crustose and foliose, while east ha d the most crustose and foliose lichens. Crustose lichens account for 85% of the cover in the quadrats, while foliose was only 15%. Differences between total cover of cr ustose and foliose lichens were assessed using ANOVA to see if the total cover of either changed between heights in tree. The three distances were low (0.4 m), mi d (3.4 m) and high (7.4 m) (data from Table 3.4). The results for the crustose liche ns were not significant (F(2,6)= 2.56, p= 0.16), meaning that
43 the total cover stayed relatively constant over all three heights (Table 3.5). ANOVA for the foliose cover was significant (F(2,6)= 26.29, p= 0.0011) (Table 3.5). A Tukey grouping showed that there were significantly more foliose cover a 7.4 and 3.4 m than 0.4 m. Table 3.5. ANOVA for crustose and foliose cover for height in tree Crustose Source of variation Sum of squares dF mean square F value p value Between groups 2.18 2 1.09 2.56 0.16 Within groups 2.56 6 0.43 Total 4.75 8 Foliose Source of variation Sum of squares dF mean square F value p value Between groups 6 2 3 26.29 0.0011 Within groups 0.685 6 0.11 Total 6.69 8 The ANOVA for the crustose lichens shows that crustose cover does not differ significantly between 0.4, 3.4 and 7.4 meters. The ANOVA for the foliose lichens show th at there is a significant difference P= 0.0011 in the results. Fruticose lichens were collected in a qua drat less than 5 times. Fruticose cover did not increase with height in the tree, and seemed to be restri cted to the upper and outer canopy (pers. observation and ca nopy diversity survey). 3.5 Discussion Total lichen cover did not increase by height in tree from 0.4 m to 7.4 m for any tree. Total lichen cover was highly variable betw een trees and between heights in the tree. There was also no significant difference in tota l lichen cover between cardinal directions. The lack of differences in lichen cover between cardinal directions was also noted on the lower trunks of Quercus spp. in Myakka by Sirko (1980). These results indicate that either lichen cover does not increase with height in tree, that there ar e other variables that are affecting lichen populations or that the sample size was too small. Both microhabitat variables and the small sample size will be discussed later this chapter.
44 Analyzing lichen cover of foliose and crustose growth forms yielded information on lichen stratification in Quercus spp. at Myakka. Crustose lich ens were the more dominant growth form, accounting for 85% of all record ed cover. Crustose cover did not change with height on the trunk (F(2,6)= 2.56, p= 0.16). Foliose cover did increase with height (ANOVA F(2,6)= 26.29, p= 0.0011). This suggests that foli ose lichens are more suitable to live closer to the top of the tree. These tr ends may be prevalent at Myakka; however, there are potentially conf ounding artifacts of sampling that require further study. Tree selection variables It was difficult to select trees appropr iate for this experiment. The goal was to pick multiple oaks with the same DBH and in the sa me type of forest, in close proximity to each other, and accessible by using the single rope technique. Other variables such as light and angle of tree were not used for tree selection. This means that due to individual tree variation, there could have been diffe rent microhabitats among quadrats. Sampling more trees would provide a better picture of vertical stratification of lichens at Myakka. The difference in DBH between the la rgest and the smallest tree was 0.200 m. (7.9 in.), which is large. However, since stand age affects the lichen community, the goal was to use trees in the same stand to control fo r this age bias. The difference in DBH meant that the trees could have had different lichen populations be cause other variables such as bark roughness and bark pH were different. For example, the bark on tree three was larger, rougher and easily fell off. Another problem was that different Quercus species may have been climbed. It was difficult to differentiate tree species visually. It was also difficult to collect the flowers
45 for additional confirmation. One possibility wa s that two different species of oak were used. Tree three was likely a different species compared to trees one and two. Another important tree selection factor was the angle of the tree trunk. Trees one and three were oriented at 90 degrees vertical ly, while tree two was angled at a 60 degree angle. This made it hard to measure distances from the ground, as well as sample in tree two. It could have affected the lichen populations (more li ght being the most prominent potential influence) and led to inaccurate he ight measurements. Tree two had the greatest lichen cover, but it was due to the prevalence of one species of powdery crustose lichen. This species may prefer to live on trunks of trees whose tr unks are less than 90 degree angles. This abnormal representation of one species may have increased lichen cover and altered statistics for the south and east directi ons, as well as for all height and directions. From observation, the trunks of many Quercus spp. trees at Myakka River State Park are angled from 60 to 90 degrees. Trees clos er to 60 degrees seem to have more bromeliads and ferns than lichens (pers. obs ervation). Areas with more bromeliads may have different populations of lichens becau se lichens have allelopathic effects on bromeliads such as Tillandsia usneoides (Callaway et al. 2001). Differences in trunk angle and microhabi tat variables suggested that the sample size was too small to gain a representative sample of lichen stratificati on on the trees. Future studies should sample at least twenty trees for more accurate population approximations. Also useful would be to consider more variables such as light, bark pH, bark quality, edge effects and stand age.
46 Quadrat problems : Quadrats provided an easy to use met hod to examine the distribution of lichen species but there were a few problems associated with them. It was arduous to estimate the cover of lichens, bark and mosses. For the lichens, it was difficult to estimate squares that were not entirely filled. Although not included in th is study, moss and bare bark were initially counted. Mosses were hard to estimate because they were very thin and long and thus were not used for analysis. It was complicated on some quadrats to distinguish bark from dead or brown moss. Bark was often differe nt colors, which further complicated the estimates. Glare from the sun also hindered efforts to accurately count. Sampling strategy may have also altered the results. The goal of minimizing error was pursued by counting lichen cover by species. This proved difficult and time consuming because some thalli were very small and easily overlooked, especially crustose lichens. It was difficult to account for half filled spaces and small thalli. Only whole numbers were used, which could have altered results. E ach count was probably overestimated. Future studies should use photographs and more sophi sticated means to measure lichen cover. Using a camera or a computer program would minimize esti mation and more trees could be sampled quicker. Functional groups and diversity There are many ways this data could have been analyzed. First, total lichen cover was examined, at various heights and cardinal di rections. Then, growth form (foliose and crustose) data were compared by height. Neith er of these studies had been conducted and published on Florida lichens. The next level of analysis would be to examine distributions of specific species of lichens or groups of lichens with similar characteristics. This
47 method would yield important data but it woul d require a great deal of collecting and identification of crustose lichens. For tree surveys, lichens have been divided into groups based on outward characteristics because lichens are time consuming to identify (McCune 1993, McCune & Peck 1997). It also enables a larger area to be surveyed more quickly. The goal with this technique is to separate lichens into functional groups (McCune 1993, McCune & Peck 1997). These analyses was attempted in this th esis, but proved to be difficult and results are not discussed. Categories used were Cryptothecia Powdery, white, Thelomatraceae, Graphis and Other. These categories were too narr ow and arbitrary in scope to describe lichen cover. A future study needs to have wider, more defined categories. Conclusion Based on the accumulated data, this was a successful pilot study. The survey found that there were no differences in total lich en cover between height in the tree or by cardinal direction. Two growth forms, foliose and crustose, formed a large percent of total lichen cover. Crustose was found in grea ter amounts on all sides of trees and height in tree. The total cover of crustose lichens did not change with height in tree, while foliose cover increased with height. The p ilot study was small and did not consider microclimatic and stand level variables in an alyses. Future studies should evaluate the effects of variables of light, tree angle, pH and stand age. Also it is necessary to use a more advanced rope climbing techniques and to sample at least twenty trees.
48 Chapter IV: Lichen diversity in the canopy 4.1 Introduction: Two small lichen surveys have been conducted at Myakka River State Park. Sirko (1980) identified the lichens on the lower trunk (0-6 m) at an unspecified location in the park, and the Sharnoffs collected lichens for Lichens of North America (Brodo et al. 2001). The lichens in the park have not been systematically reviewed. The goal was to collect macrolichens of oak hammocks and co mpare the lichen dive rsity of Myakka to Ocala National Forest. The macrolichen flor a of Ocala National Forest was recently described and is a thorough checklist for comparison (Debolt et al., 2008) The hypothesis was that lichen diversity w ould differ between these two locations, and that tropical lichens would be found at Myakka. 4.2 Methods The focus was to collect canopy lichens. Small fallen branches were collected from the ground. Lichens were collected off the House Ranch Road, and around the Canopy Walkway. Lichens were not collected randomly. This allowed the author to collect many different macrolichen species in the shor t time frame. A list of species found was compiled and is presented in this thesis. Lichen Identification Multiple dichotomous keys were used to identify lichens. Lichens of North America (Brodo et al. 2001) has many illustrations, but is an incomplete guide to the lichens in Florida. More Florida Lichens by Harris (1995) is the most complete guide to lichens in Florida. A key of the lichens of O cala National Forest, (DeBolt & Rosentreter
49 unpublished 2008) was used as well as keys ma de by Bruce Ryan, the late Arizona State University professor. Spot tests were used to identify liche ns. Spot tests are most useful to distinguish species that look identical, but whose chemistry differs. They show what secondary compounds are present in the cortex or medulla of the lichen (Brodo et al. 2001). There are numerous chemicals compounds found in lic hens that can not be distinguished by sight, but can be identified with a drop of reagent. The most important solutions for spot testing are para-phenylenediamine in an alcohol solution, 10 % KOH (K) and Lugols solution (1.5% I in 10% KI), 10% sodium hypochlorite (C), and an application of K then C reagent (Brodo et al., 2001). These tests can confirm the existence of secondary compounds such as usnic acid, lecanoric aci d, sekikaic acid, atranorin, and norstictic. Each compound will either show up negative, or turn the thallus to a specific color, which can then be matched using a database (Brodo et al., 2001). To perform a spot test on the outer cortex, a small drop of a previously described chemical was gathered in a glass capillary a nd placed on the cortex and the color change was recorded (Figure 4.1). The most common change was that the cortex turned yellow which is indicative with 10% KOH which is in dicative of atranorin (found in cortex of Parmotrema and Physcia ). To test the medulla, the outer cortex is scraped off. Then a small drop of a potassium hydroxide or sodi um hypochlorite was placed on the exposed medulla, and color changes were recorded. It is important to place only a small drop of the compound on the thallus because a large drop could mix compounds from the cortex and medulla and give false results. Further de scriptions of the spot tests can be found in Brodo et al. (2001).
50 a b d c Figure 4.1 : The process to spot testing. This figure show s the steps for spot testing the medulla. First a small piece of cortex is cut away (b), then a small drop of reagent is placed on the medulla (c). The color change, a purple color, is observed in (d). The one problem with this photo is that too much reagent is placed in Photo C. It is better to expose more medu lla or place less reagent for a spot test, so that the compounds of the medulla and cortex do not interact with each other. http://www.ces.iisc.ernet. in/biodiversity/sahyadri/wgbis_info/monthly_article/lichen/Figure29.jpg Another method of examining secondary compounds, is to place the cortex or the medulla under a long wave UV lamp, and examin e color changes. This was useful to establish the genus Pyxine from other genera in Physciaceae. Pyxine has lichexanthone, which turns a white medulla yellow (Harris 1995). Also observed was alectoronic acid which turns the medulla blue for Parmotrema rampoddense (Harris 1995) 4.3 Taxonomy results Identified lichens from fallen branches are listed below (Table 4.1). An emphasis was placed on canopy macrolichens. Twenty-one specimens were identified. There were 7
51 fruticose, 10 foliose and 4 crustose lichens were identified. Uncertainty in species is acknowledged, and a list of other possibilities is stated below. Table 4.1. Macrolichens collected in oak hamm ocks of Myakka River State Park Fruticose: Foliose: Crustose: Ramalina montagnei Parmotrema cristiferum complex Cryptothecia rubrocincta Ramalina peruviana Parmotrema dilatatum Cryptothecia striata Ramalina stenospora Parmotrema michauxianum Haematomma spp. Ramalina willeyi Parmotrema hypoleucinum* Porina spp. Usnea mutabilis Parmotrema rampoddense Usnea scabrosa Parmotrema tinctorum Usnea strigosa Physcia americana Physcia atrostriata* Physcia sorediosa Pyxine caesaiopruinosa Shows the species collected from fallen twigs at Myakka River State Park. The majority of the species were foliose from the genus Parmotrema, though fruticose species were also diverse and abundant. denotes species that were not positively identified 4.4 Discussion Lichen diversity in Myakka River St ate Park vs. Ocala National Forest This survey showed that the canopy at Myakka River State Park is very diverse, yet very similar to Ocala National Forest. It al so showed that the canopy is a very diverse niche. The following discussion is based on observations and collected data. Though not quantified, these data are useful to futu re studies at Myakka River State Park. It is important to compare the lichens found at Myakka River State Park to Ocala National Forest to see lichen flora is similar. The results suggested that the lichen flora in Ocala and Myakka were very similar. Fruticose lichens, Ramalina montagnei R. stenospora, R. willeyi ,Usnea mutabilis, U. scabrosa and U. strigosa were found in hardwood hammocks at Myakka and Ocala (Deb olt et al. 2007). All foliose lichens, Parmotrema Pyxine, Physcia, Leptogium were also collected at Ocala.
52 From observations at Myakka U. strigosa appears to be the most common and abundant fruticose lichen in oak hammocks. U. strigosa was similarly the most common Usnea species in hardwood hammocks at Ocala (Debolt et al. 2007). The two exceptions were Ramalina peruviana and R. usnea which were not found at Ocala. Ramalina peruviana was collected 4 times at Myakka near trees surveyed for the vertical stratification. R. usnea was collected once. Both ar e tropical lichens that grow only south of Lake Okeechobee or Tampa (Moor e, 1968; Brodo et al. 2001). This lichen suggests that Myakka River State Pa rk can support tropical lichens. The discovery of subtropical Ramalina species showed that My akka River State Park has the potential to yield important details on the northern range of sub/tropical lichen species because it is located on the same latitude as Lake Okeechobee. This is important because Lake Okeechobee is commonly delineated as the border for tropical lichenstropical lichens are usually found south of the lake (Harris 1995, Brodo et al 2001). Myakka River State Park, although much furthe r west, could be a location to explore the range of temperate and tropical lichens. Comparison of lichen diversity on fallen twigs to trunk of Quercus spp.: There were three observed differences between the trunk and the small twigs. The most significant observation was that frutic ose lichens were only found on small fallen branches. This implies that in oak hammocks the majority of fruticose diversity is restricted to the canopy of the tr ees, similar to Ocala National Forest (Debolt et al 2008). It was not possible to determine with a glance if foliose lichens were predominant in the canopy or the trunk, and this should be ex amined. Lastly, the lichen diversity was different between the trunk and canopyfew sp ecies seem to grow in both locations.
53 Microlichen diversity survey While future studies need to be conducted to see if thes e tentative conclusions are true, it is worth noting these observations. On sma ll twigs, the majority of the lichens were crustose, including Graphis Porina, and family Thelomatraceae, while thicker branches tended to have more foliose diversity. There were numerous colonies of crustose species on the smaller twigs, and potenti ally many different species. It would be an insightful project to identify what crustose species are on these twigs. With additional time, more species could be identified, especially microlichens. Better surveying techniques and a more intensive tree climbing survey may show that the canopy is more diverse than the trunk of the tree. Identification problems: One of the difficulties of this thesis was identifying lichens. The two problems associated with taxonomy were the use of spot tests, and the lack of using thin layer chromatography. One problem that could have le d to misidentification or uncertainty was faulty spot tests. It is possible some spot tests did not change color because too much reagent was applied or the lower or upper cortex was exposed. The cortex and the medulla contain different chemicals (Brodo et al. 2001). In either described scenario, a spot test on the medulla touches the co rtex and gives a false color change. Lichens were identified by the author, only a few were checked by Rick Seavey. Care was taken to acknowledge species not positivel y identified. Identifying lichens to family is usually not difficult, but to identify the li chen to species can be quite difficult because genera can appear to be very similar. Wh ile experience would be important for more detailed taxonomy, the use of thin layer chro matography is equally crucial. Lichens have
54 many secondary metabolites, which are found on the hyphae of the fungal component in the lichen, commonly in the medulla (as reviewed by Elix & Stocker-Worgotter in Nash 2008). Thin layer chromatography can separa te out hard to identify compounds. The process involves placing silica pl ates, in different solvents, an d heating them in sulfuric acid. UV tests are used to further differentiate compounds. Thin layer chromatography provides a way to more definitively prove taxonomy. Genetic testing may be the most definitive future method, but the current thi nking is that the compounds in lichens, the specific chemotype of lichens are based on morphological, ecological or distributional tendencies and consequently should be affo rded some taxonomic recognition (Elix & Stocker-Worgotter in Nash 2008). This means that species have the same genotype but evolve differently due to abiotic factors. This survey, although not random or larg e in scope, suggests that the lichen flora of Myakka River State Park, is very similar to Ocala National Forest. The exceptions were the fruticose lichens R. peruviana, and R. usnea which are considered subtropical lichens. These species suggest that with a more rigorous survey, more subtropical species may be found. A more detailed survey of crustose lichens would pr obably reveal other differences between Myakka, and other undisturbed natural areas further north.
55 Chapter V: Summary In this thesis, three studies on lich ens examined the water tolerance, vertical stratification, and a canopy survey at Myakka River State Park. The drowning survey showed that there are differences in the rates of drowning between species, and that different species had differe nt reactions to submergence in water. The vertical stratification experiment, showed that total lich en cover did not change with height in tree or by cardinal direction. It also showed that crustose lichen s were the dominate growth form and that foliose cover increased significan tly vertically in the tree. Fruticose lichens were found in only a few quadrats and were not a large percent of total lichen cover. The canopy survey of Myakka River State Park revealed that there are many similarities in lichen flora to Ocala National Forest. Ho wever, the findings of tropical lichens, R. peruviana and R. usnea at Myakka suggest that it can sustain tropical lichens. Though these experiments and surveys were small in scope and had methodological errors, they are all areas of lichenological studi es that deserve more attention in Florida. Water tolerance and stratification caused by water should be studied more because Florida has many rivers and swamps, and s easonal flooding zones. Marjorie Stoneman Douglas wrote the Everglades is a river of grass (1986). Although Myakka is further west than the Everglades, the flow of water is crucial to the health of Myakka and the Charlotte Harbor. Water alteration needs to be studied more thoroughly to better understand how restoration will affect lichen populations. More intensive surveys of both tree trunks and their canopy should be conducted to further study the taxonomy and the ecology of li chens in Florida. It is crucial to study Floridas lichens more thoroughly because so uthern Florida has a large amount of the
56 land in the United States that can suppor t tropical lichens. Although many of these tropical species may be found outside of Florida, it is important to preserve this land that is easily accessible in the United States. One possibility for future studies is the Forest Health Monitoring program, a widely accepted lichen health monitoring system. Fo rest Health Monitoring is a system to continually and with ease measur e forest health by measurin g lichens. This system takes a 0.378 ha circular plot to 1.) find all speci es present and 2.) estimate the abundance of each species present in a two hour span (McCune et al. 1997). There are many possibilities for further surveys, but I feel that the two most important directions for Sarasota County would be to study Myakka River State Park and the lichens in trees bordering the Gulf of Mexico. Studies documenting these two lichen populations would elucidate lichen diversity in Sarasota County. The other possibility is comparing pl ots between oak forests at Myakka River State Park to the Gulf of Mexico. From pers onal observations, the macro and microlichen populations seem to differ, though the exte nt is unknown. This would provide more information of the east and west boundary of lichens in Florida. The Forest Health Monitoring protocol would provide useful information on Florida lichens, but there are a few factors to consider before using it at My akka River State Park. First, the type of oak forest at Myakka ch anges with slight elevation changes. This significantly affects understory diversity and the trees presen t. This makes it difficult to sample only one type of oak forest. Another variable to consider is that Myakka is swampy and the ground of areas that do not fl ood can be very moist. This would cause lichens to degrade faster, assuming humidity is harmful to the species. Lastly, the
57 abundance of microlichens should be cons idered. Macrolichens are larger, but microlichens seem to be more abundant. From observations, most young branches of Quercus spp. have a multitude of families and species. It may not be feasible to use microlichens as an indicator of forest healt h, but they provide usef ul data for diversity and forest health at Myakka River State Park. Many species of lichens have undoubted ly gone extinct, even before they were identified because of development and loggi ng (Brodo et al., 2001). It is necessary to preserve large remaining tracts to preserve diversity. Ongoing research at Everglades National Park provides the most compelling evidence for research in Florida lichens. Five hundred species of lichens are expected to be identified from Everglades National Park (per. comm. with Rick Seavey). These findings show that lichen diversity in Florida has been underestimated, and that more research is needed to establish lichen abundance and diversity before they are lost. Florida is st eadily losing natural la nd due to development, exotic invasive plants and land alterati on, making it crucial to obtain a baseline of lichenological knowledge for future gene rations and more in depth research. To find a place with diverse and large lichens is to capture a glance of a healthy environment (Brodo et al 2001). Lichens f ace many ecological threats, and mankind has done much to destroy populations, but it is stil l possible to find areas rich with lichens. Undisturbed land has a sense of wonder and joy that can capture a persons heart and mind. These places need to preserved and passed on for future generations to discover, enjoy, and think about ecosystems and lichens.
58 Appendix I: More information on lichens used for water tolerance experiment Cryptothecia rubrocincta (Ehrenb. : Fr.) Thor is a common crustose lichen found growing on different species of Quercus spp. and cabbage palms from the lichen line to the upper trunk. It is commonly found on the north sides of Quercus spp. or in areas that had more light. It has a distinct red and green thallus in the sh ape of a circle (Brodo et al. 2001). It is found along the Atlantic coast fr om South America to North Carolina and west to Louisiana (Brodo et al. 2001). Cladonia spp. was found growing in a large colony on a rotting palm tree. This type of Cladonia is typically a ground dwelling, fruticos e lichen. Its presence was intriguing because the area it was found in might flood over the summer (though the author did not observe flooding). Previous literature suggested that Cladonia has a low tolerance for water submergence. It suggests that the area mi ght not flood or is a re cent colonizer since Myakka River State Park is in a drought. Physcia sorediosa (Vainio) Lynge is a small folio se lichen found on a few trees, in large colonies. It preferred the shaded side of the tree. Usnea strigosa (Ach.) Eaton is a fruticose lichen that was collected from low hanging canopy branches. They were collected from bran ches hanging to the east at the edge of the forest in direct sun. Graphis is a distinct genus of crustose lich ens. They have lirella which is an intermediate between ap othecia and perithecia. Graphis can be found from the canopy to the trunk of the tree mostly in tropical habitats. They are found on smooth and rough bark. Due to constrained iden tification skills and rarity of some of these species, the author collected at least two species. One was identified as Graphis librata C. Knight. All specimens were collected from adjacent Quercus spp. Leptogium spp was also collected. Leptogium was a prevalent group lichens found on the north and south side of Quercus spp. It was commonly found amidst moss on the side of the tree with the most shade. Leptogium was also the lichen found most abundantly closest to the lichen tr imline. In canopy studies it is f ound at 5-7 meters high in small quantities surrounded by moss. It has a black thalli, and its sy mbiont is a cyanobacteria. The three species used in the experiment were Leptogium denticulata Tuck, L. cyanescens Rabenh. Korber, and L. austroamerican (Malme) Dodge.
59 Appendix II: Additional s ubmergence experiment data Table A2.1. Raw data for submergence experiment Day 0 Day 1 Day 3 Day 5 Day 12 Usnea strigosa UC1 6 6 5 UC2 6 5 4 UC3 6 6 5 U1.1 6 4 4 5 U1.2 6 4 4 2 U1.3 6 4 4 3 U3.1 6 0 0 U3.2 6 4 1 U3.3 6 2 0 U5.1 6 0 0 U5.2 6 0 0 U5.3 6 0 0 U7.1 6 0 0 U7.2 6 lost lost U7.3 6 lost lost C. rubrocincta CrC1 6 3 2 CrC2 6 3 3 CrC3 6 3 0 Cr1.1 6 6 0 Cr1.2 6 6 2 Cr1.3 6 6 2 Cr3.1 6 4 0 Cr3.2 6 4 0 Cr3.3 6 4 0 Cr5.1 6 3 0 Cr5.2 6 lost lost Cr5.3 6 3 0 Cr7.1 6 lost lost Cr7.2 6 lost 0 Cr7.3 6 3 Cladonia (Cl) ClC1 6 5 4 ClC2 6 2 4 ClC3 6 5 5 Cl1.1 6 5 5 5 Cl1.2 6 5 5 lost Cl1.3 6 5 5 4 Cl3.1 6 4 4 Cl3.2 6 4 4 Cl3.3 6 4 3 Cl5.1 6 5 3 Cl5.2 6 3 3 Cl5.3 6 5 4 Cl7.1 6 4 4 Cl7.2 6 lost lost Cl7.3 6 lost lost
60 Day 0 Day 1 Day 3 Day 5 Day 12 Graphis (G) GC1 6 4 3 GC2 6 4 4 GC3 6 5 3 G1.1 6 5 5 4 G1.2 6 5 4 3 G1.3 6 4 0 2 G3.1 6 3 2 G3.2 6 3 3 G3.3 6 0 1 G5.1 6 3 lost G5.2 6 lost lost G5.3 6 3 3 G7.1 6 lost lost G7.2 6 1 2 G7.3 6 2 4 P. sorediosa PC1 6 4 3 PC2 6 4 3 PC3 6 4 3 P1.1 6 4 3 3 P1.2 6 5 4 3 P1.3 6 5 4 3 P3.1 6 4 3 P3.2 6 4 3 P3.3 6 4 3 P5.1 6 lost lost P5.2 6 lost lost P5.3 6 3 2 P7.1 6 3 3 P7.2 6 3 2 P7.3 6 lost lost Leptogium spp LC1 6 5 5 LC2 6 5 5 LC3 6 5 4 L1.1 6 4 5 L1.2 6 4 5 L1.3 6 6 5 L3.1 6 6 4 L3.2 6 6 4 L3.3 6 5 4 L5.1 6 lost lost L5.2 6 5 5 L5.3 6 lost 4 L7.1 6 lost lost L7.2 6 6 4 L7.3 6 6 lost All data taken for the submergence experiment is record ed in this table. Blank spaces mean that no data was taken. Lost refers to samples that could not be lo cated due to squirrel interference. The first letter or two refer to the species. For the first three samples of each species, the C denotes the controls. Samples 1.1, 1.2, and 1.3 were taken out of water on day 1. Samples 3.1, 3.2 and 3.3 refer to samples taken out on day three. Days 5 and 7 were both treated as day 5 due to squirrels.
61 A2.2. Mean vigor of species from days 0 to 5 Data used for averagesfinal Species Day Health U. strigosa 0 6 1 4 3 2 5 0 C. rubrocincta 0 6 1 6 3 4 5 0 Cladonia spp. 0 6 1 5 3 5 5 4.25 Graphis spp. 0 6 1 4.67 3 2 5 2.25 P. sorediosa 0 6 1 4.67 3 4 5 3 Leptogium spp. 0 6 1 4.67 3 5.67 5 5.67 This data was used to make Figure 3.3. This table re iterates that the vigor of lichens decreased with more time spent in submergence conditions. A2.3. Comparison of mean vigor of contro l and experimental samples on day 5 Species Control Experimental Usnea 4.25 0 C. rubrocincta 3 3 Cladonia spp. 4 4.25 Graphis spp. 4.3 2.25 P. sorediosa 4 3 Leptogium spp. 5 5.67 This table shows the mean vigor of the control and experimental samples on day 5 of the submergence experiment. Although not useful for analysis since th e controls lost health, it does show that there is a difference between control and experimental health. The controls had a higher health for Usnea, P. sorediosa and Graphis spp.. The vigor of the control and experimental C. rubrocincta was the same. The controls had less vigor than the experimental samples for Cladonia and Leptogium
62 Appendix 3: Additional vertical sampling data A3.1. Chart used to measure percent cover of lichens in tree Tree # _______ Height in Tree __________ Bark Roughness (1-3)______ Branch (Y/N) __________ Direction angle of branch_______ Direction (NSEW)____________ Sun light _____________ Time of day sampled_______ Species Present Total cover Crustose C. rubrocincta C. striata Foliose (green) Foliose (cyano) Parmeliaceae Physciaceae Leptogium Light Meter Bark Cover Moss Present Other Epiphytes present First information on the tree was collected, such as bark thickness, light, height of tree and direction. Then the cover of one species was counted and recorded under foliose or crustose column. This method continued until all lichen cover was counted in a square.
63 A3.2. Crustose and foliose data for a ll heights of all three trees (cm2) Tree Height Crustose Foliose 1 0.4 260 0 1.4 297 9 3.4 192 107 5.4 182 26 7.4 163 75 2 0.4 9 1 1.4 843 6 3.4 883 23 5.4 544 143 7.4 293 48 3 0.4 2 0 1.4 277 24 3.4 124 20 5.4 435 50 7.4 82 281 This is an expansion of the data used in Table 4.5 on page 38. The results show that crustose data does not increase by height in the tree and that crustose liche ns are the more dominate growth form in the tree. Foliose cover does increase by height, and is most noticeable in tree 3.
64 Appendix IV: Photos of Myakkas lichens Physcia spp C.ryptothecia rubrocincta Cryptothecia striata moss Figure A4.1 : Shows the lichen diversity on a tree trunk and the difficulties of counting lichen cover. Lichens present are Cryptothecia rubrocincta, C. striata, Physcia spp.. Moss is also present.
65 Figure A4.2 : C. rubrocincta is a common crustose lichen on Quercus spp. It has a red edge, thallus is white, and red granules that resemble isidia. Species A Species B Figure A4.3 Shows two common species of the genus Parmotrema Species on this sample were not identified, but by looking at isidia and soredia is clear that two species are present. Species A has isidia all on top of thallus. Species B has soredia at the edges.
66 Figure A4.4 : Species of Parmotrema (large thallus on right) can grow ne xt to Physciaceace spp. Genera is probably either Physcia or Heterodermia Figure A4.5 : A picture of Leptogium spp. most likely L.austroamericanum.
67 Figure A4.6 : Leptogium spp and moss growing on a Quercus spp. Figure A4.7 : Cyanobacteria lichen (black) and Physcia spp. Picture taken on the canopy walkway.
68 Figure A4.8 : Picture of common Usnea spp. Figure A4.9 : Usnea strigosa the most common Usnea spp at Myakka River State Park.
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