Wet Wall Algal Community Response to In-Field Nutrient Manipulation Of

Home , Coccomyxa, Naviculales, Snake Den Ridge Trail

WET WALL ALGAL COMMUNITY RESPONSE TO IN-FIELD NUTRIENT MANIPULATION OF NITROGEN AND PHOSPHORUS, AND THE TAXONOMY, ECOLOGY, AND DISTRIBUTION PATTERNS OF THE ACIDOPHILIC DIATOM GENUS EUNOTIA EHRENBERG (BACILLARIOPHYTA) OF THE GREAT SMOKY MOUNTAINS NATIONAL PARK. U.S.A.

Paula C. Furey

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 2008

Committee:

Rex L. Lowe, Advisor

Alexander J. Izzo Graduate Faculty Representative

Jeffrey R. Johansen

Karen V. Root

Dan D. Wiegmann

Paula C. Furey

ABSTRACT

Rex. L. Lowe, Advisor

The basic ecology of wet wall algal assemblages and the ecology, distribution patterns, and taxonomy of the acidophilic diatom genus Eunotia Ehrenberg were explored from the Great Smoky Mountains National Park (GSMNP), U.S.A. First, the structural response of wet wall algal assemblages were studied through an in-field experimental manipulation of nutrients (nitrogen and phosphorus) at high and low altitudes. Subaerial algal assemblages are understudied communities and the factors that drive community dynamics in these environments are poorly understood. Algal assemblages were not as nitrogen or phosphorus limited as initially anticipated. Although not directly tested in this study, results suggested that other factors, such as ultraviolet radiation, pH, moisture levels, or microhabitat differences, are influential in shaping wet wall, algal community structure. Second, diatoms from high elevation springs and headwater streams were examined and found to be dominated by a couple of Eunotia taxa, including a new species, E. macroglossa sp. nov. The presence of morphological deformities at some of the spring sites was documented with both light and scanning electron microscopy and the malformations suggest that acid precipitation may directly be harming the aquatic ecosystems in the park. Third, the distribution patterns of Eunotia species from sites throughout the park were explored in relation to environmental factors such as elevation, pH, geology, and water chemistry. As expected higher altitude streams generally had lower pH levels and a greater relative abundance of Eunotia relative to other diatom genera, especially species in the E. exigua complex. The results highlighted iv

how vulnerable high elevation areas in the GSMNP may be to acid precipitation. Finally, an image rich documentation and inventory of the Eunotia taxa in the GSMNP was provided, including species descriptions, morphological measurements and a discussion of taxonomic challenges. The Eunotia were very diverse with over 65 subgeneric taxa being documented, many of which were new park records, new records for North

American and/or were undescribed species. This dissertation explored a new algal frontier (wet walls) and provided further taxonomic documentation and ecological description of an understudied group of diatoms, the genus Eunotia. v

Dedicated to my grandparents

who inspire me everyday. vi

ACKNOWLEDGMENTS

FIRST AND FOREMOST I WANT TO ACKNOWLEDGE AND DEEPLY THANK MY

FAMILY WHO HAS ALWAYS BEEN THERE TO SUPPORT ME IN A MULTITUDE OF WAYS. IT

MEANS A GREAT DEAL TO ME TO HAVE THE WONDERFUL FAMILY THAT I DO. I WOULD

NEVER SURVIVE THESE JOURNEYS WITHOUT YOU.

Thank you to my doctoral committee: Drs. Rex Lowe; Jeff Johansen, Karen Root,

Dan Wiegman for their input, constructive criticism, and support during my tenure as a graduate student at Bowling Green State University (BGSU). Rex thank you for providing ‘opportunities’ whenever and wherever possible in order to make my graduate experience the best that it could be, in addition to ensuring you never missed a chance to give me a hard time…it’s been an enjoyable adventure. Jeff, thank you for your assistance with species descriptions and Latin translations...... my spiral ‘endlessly downward’ has...… well…ended.

Thank you to all the field and lab assistants who helped collect, process, and catalogue the samples in this dissertation, especially John Stein and Linda Novitski, for help in the summer of 2005. Thank you to the All Taxa Biodiversity Inventory (ATBI) and National Park Service personnel, especially Jeanie Hilton who helped with many logistics during stays in the Great Smoky Mountains National Park (GSMNP) and ATBI volunteers: Jim Lowe, Jim Burbank, Ronnie McGahaw, and the Lawsons. Steve Moore and Matt Kulp of the National Park Service and Bruce Robinson of the University of

Tennessee provided water chemistry data and assistance with water chemistry data.

Mike Grant of the University of Michigan Biological Station provided water chemistry vii

analysis for the seep wall experiment and Test America Inc. provided partial support for

GSMNP stream water chemistry analysis. Thank you to Happy Hiker, Gatlinburg TN.

Support for this research was received from the BGSU Department of Biological

Sciences, BGSU Scanning Electron Microscope Facility and the University of Michigan

Biological Station. Thank you to the office staff, especially Lorraine DeVenney - It is officially Friday!

Funding for this research was received from: National Science Foundation Grant to R.L. Lowe [NSF Grant #0315979].

Additional support was provided to P.C. Furey by:

Discover Life In America Research Grant [DLIA2005-07],

Sigma-Xi Grant-In-Aide-Of-Research,

Phi Kappa Phi Love of Learning Award,

Phycological Society of America Grant-In-Aide-Of-Research,

North American Benthological Society (NABS) Boesel –Sanderson Award,

NABS: Conservation and Environmental Issues Graduate Research Award,

Katzner and University Bookstore Funds Award,

Larry and Linda Oman Graduate Scholarship in Ecology and Environmental Biology

Non-Service Fellowship from Department of Biology

Charles E. Shanklin Award for Academic Excellence (Science & Mathematics Division).

viii

And on a personal note….

Ben :) :), you provided much needed sanity and much needed laughter along the way. Thank you for your support, understanding, and willingness to provide hugs when needed.

Jessie (y’ello…that’s all I’m sayin’ …), Linda (summer in the Smokys and life in

BG would never have been the same without you, I might have gotten lost ....LOL), Kelly

(may the dark chocolate live on….thanks Linda), Mary (hockey, hockey, hockey), Kandi

(my great Spud mate and corruption companion), Christine (high five to that! - that’s what I’m talkin’ about). You guys ROCK!!!

And from afar…at last JM and WN - my own version of TP has arrived…use wisely.

Rick … a toast (or should I say cinnamon bun) to another successful step.

Janice … thank you for your continued support, friendship, and inspiration.

At last….Pools, Poola, Small Paul, Bucky, Goon, Small Fry, Shorty, Squiggles has successfully survived the Midwest educational experience and PhD marathon.

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

CHAPTER I.: Wet wall algal community response to in-field nutrient manipulation

in the Great Smoky Mountains National Park, U.S.A...... 6

Abstract...... 6

Introduction...... 7

Material and Methods ...... 9

Results...... 14

Discussion...... 19

CHAPTER II: Morphological deformities in Eunotia taxa from high elevation springs

and streams in the Great Smoky Mountains National Park, with a description of

Eunotia macroglossa sp. nov...... 25

Abstract...... 25

Introduction...... 26

Material and Methods ...... 27

Results...... 30

Eunotia macroglossa sp. nov...... 30

Discussion...... 35

CHAPTER III: Ecology and distribution patterns of Eunotia Ehrenberg in the Great

Smoky Mountains National Park...... 40

Introduction...... 40

Material and Methods ...... 42 x

Results...... 46

Discussion...... 49

CHAPTER IV: Eunotia Ehrenberg (Bacillariophyta) of the Great Smoky Mountains

National Park...... 53

Introduction...... 53

Material and Methods ...... 55

Results and Discussion ...... 57

Taxonomic Descriptions ...... 59

Abbreviations ...... 59

List of Taxa...... 60

Species Descriptions ...... 63

CONCLUDING REMARKS...... 106

LITERATURE CITED ...... 110

TABLES ...... 118

CHAPTER I ...... 118

CHAPTER II ...... 126

CHAPTER III...... 131

CHAPTER IV ...... 136

FIGURES...... 141

CHAPTER I ...... 141

CHAPTER II ...... 150

CHAPTER III...... 158

CHAPTER IV ...... 164 xi

LIST OF TABLES

Chapter I: Page

1 Mean water chemistry from samples collected from each site at Clingmans

Dome and Cataloochee...... 119

2 Algal biomass accrual and ash free dry mass for Clingmans Dome and

Cataloochee...... 120

3 Mean relative frequency of soft algal taxa at Clingmans Dome...... 121

4 Mean relative frequency or relative abundance of algal taxa at Cataloochee (by

nutrient treatment)...... 122

5 Mean relative frequency or relative abundance of algal taxa at Cataloochee (by

site)...... 124

Chapter II:

1 Site and sampling information, along with relative abundance of Eunotia

subarcuatoides and other diatoms in springs and high elevation streams...... 127

2 Morphological measurements of normal and abnormal E. subarcuatoides valves .. 129

3 Water chemistry data of springs and streams from pre-2005 and 2007 ...... 130

Chapter III:

1 Geology and watershed codes for the GSMNP ...... 132

2 Percent contribution by Eunotia species for NMDS clustering...... 133

3 PCA axes 1 and 2 for water quality parameters for the NPS 2005 sites sampled .... 134

4 Water chemistry of isolated sites in Fig. 5...... 135

Chapter IV:

1 Watershed codes for the Great Smoky Mountains National Park ...... 137 xii

2 Current and previously reported Eunotia species observed in the GSMNP...... 138 xiii

LIST OF FIGURES

Chapter I: Page

1 Map of the Great Smoky Mountains National Park, U.S.A...... 142

2 Comparison of nutrient treatments...... 143

3 NMDS ordination: relative frequency of soft algal taxa for Clingmans Dome...... 144

4 Light microscope images of soft algal taxa from Clingmans Dome...... 145

5 Light microscope images of diatoms from Clingmans Dome and Cataloochee...... 146

6 NMDS ordination: relative frequency of soft algal taxa and relative abundance of

diatoms at Cataloochee from nutrient treatments...... 147

7 NMDS ordination: relative frequency of soft algal taxa from Cataloochee sites...... 148

8 NMDS ordination: relative frequency of soft algal taxa from Cataloochee sites

3, 4, 5 and 6...... 149

Chapter II:

1 Sampling sites in Great Smoky Mountains National Park...... 151

2 – 14 Light micrograph images of Eunotia macroglossa...... 152

15 Light micrograph of Eunotia exigua ...... 152

16 Light micrograph of Eunotia muscicola var. tridentula ...... 152

17 – 19 Light micrograph images of the girdle view of Eunotia macroglossa ...... 152

20 – 25 Scanning electron microscope micrographs Eunotia macroglossa ...... 153

26 – 42 Light micrographs of Eunotia subarcuatoides ...... 154

43 – 62. Light micrographs of Eunotia subarcuatoides - abnormal morphologies ...... 155

63 – 70 Scanning electron micrographs of Eunotia subarcuatoides - normal valves

and abnormal valves...... 156 xiv

71 – 76 Scanning electron microscope micrographs of the internal ultrastructure of

Eunotia subarcuatoides in a normal valve and deformed valves ...... 157

Chapter III:

1 NMDS - relative abundance of diatoms from all sites including springs...... 159

2 NMDS - relative abundance of Eunotia taxa from all sites. Bubbleplots for E.

subarcuatoides (A), E. incisa (B), E. exigua (C) and E. tenella (D)...... 160

3 NMDS - relative abundance of diatoms from all sites including springs. Factored

by disturbance (A) and geology (B) ...... 161

4 NMDS - the relative abundance of diatoms from NPS-2005-All-Taxa (A and B)

and NPS 2005-Eunotia-only (B and C)...... 162

5 PCA of log-transformed environmental variables from NPS 2005 ...... 163

Chapter IV: Plate:

1 LM: E. naegelii ...... 165

2 LM: E. flexuosa, E. bilunaris, E. naegelii, E. parallela ...... 167

3 LM: E. valida, E. glacialis, E. sp. (GSMNP SP 1), E. boreotenuis...... 169

4 LM: E. sp. (GSMNP SP 2), E. sp. (GSMNP SP 3), E. neofallax,

E. subarcuatoides ...... 171

5 SEM: E. neofallax, E. subarcuatoides...... 173

6. LM: E. sp. (GSMNP SP 4), E. sp. (GSMNP SP 5), E. sp. (GSMNP SP 6),

E. varioundulata, E. exigua, E. tenella, E. compacta, E. sp. (GSMNP SP 7)...... 175

7 SEM: E. sp. (GSMNP SP 4), E. varioundulata ...... 177

8 SEM: E. exigua, E. tenella, E. varioundulata...... 179 xv

9 LM: E. muscicola var. tridentula, E. microcephala, E. trinacria, E. sp. (GSMNP

SP 17), E. sp. (GSMNP SP 18), E. sp. (GSMNP SP 19), E. paludosa ...... 181

10 SEM: E. muscicola var. tridentula, E. microcephala ...... 183

11 LM: E. nymanniana ...... 185

12 SEM: E. nymanniana...... 187

13 LM: E. undulata...... 189

14 LM: E. pectinalis var ventralis; SEM: E. undulata...... 191

15 LM: E. macroglossa ...... 193

16 SEM: E. macroglossa ...... 195

17 LM: E. jemtlandica ...... 197

18 LM: E. formica...... 199

19 LM and SEM: E. sp. (GSMNP SP 20)...... 201

20 LM and SEM: E. braendlei...... 203

21 LM and SEM: E. sp. (GSMNP SP 8) ...... 205

22 LM: E. curtagrunowii, E. sp. (GSMNP SP 9), E. rabenhorstii,

LM and SEM: E. c f macaronesica...... 207

23 LM: E sp. (GSMNP SP 10), E. bidens, E. diodonopsis...... 209

24 LM: E. sp. (GSMNP SP 11) ...... 211

25 SEM: E. sp. (GSMNP SP 11) ...... 213

26 LM: E. bigibba, E. sp. (GSMNP SP 21)...... 215

27 SEM: E. bigibba, E. sp. (GSMNP SP 21)...... 217

28 LM and SEM: E. sp. (GSMNP SP 12) ...... 219 xvi

29 LM: E. septentrionalis, E. implicata, E. sp. (GSMNP SP 22), E. sp. (GMSNP SP

13), E. minor...... 221

30 LM: E. veneris, E. pirla, E. sp. (GSMNP SP 14), E. sp. (GSMNP SP 15), E. sp.

(GSMNP SP 16) ...... 223

31 LM: E. sp. (GSMNP SP 23), E. sp. (GSMNP SP 24)...... 225

32 SEM: E. sp. (GSMNP SP 23) ...... 227

33 LM and SEM: E. incisa ...... 229

34 LM: E. billii ...... 231

35 SEM: E. billii ...... 233 1

INTRODUCTION

The 21st century is predicted to be a period of crisis for biodiversity where many species will go extinct before they have been studied. Humans require a better understanding of the world’s species, ecosystems, and landscapes, and the dynamics of their interactions in order to successfully protect them from extinction. Thus, scientists are being urged to collect, inventory, and describe the biodiversity on earth as expediently as possible (Dubois 2003, Giangrande 2003, Wilson 2003).

The biodiversity crisis is often perceived to apply to terrestrial environments; however, freshwater systems are even more neglected, with little emphasis being placed on the taxonomy, documentation, and inventory of aquatic organisms (Abell 2001). There is a paucity of published research on the conservation of freshwater biodiversity, further emphasizing that the freshwater biodiversity crisis is not being given the critical attention it deserves (Abell 2001). Without taxonomic lists and inventories of freshwater aquatic habitats, conservation biologists are limited in their ability to make informed management decisions. This may not only result in high rates of extinction, but may also result in a loss of freshwater ecosystem services that humans rely on (e.g. clean water provisioning). This freshwater biodiversity research gap is even further pronounced with microscopic organisms such as the algae. An extinction rate of 4% per decade for freshwater fauna is five times higher than for terrestrial fauna (Ricciardi & Rasmussen

1999). This difference is unknown for freshwater flora.

Since the late 1990’s, the National Park Service and Discover Life In America have taken on the ambitious task of completing an All Taxa Biodiversity Inventory

(ATBI) in the Great Smoky Mountains National Park (GSMNP) (Sharkey 2001). As one 2

of the most species rich areas in the temperate zone, GSMNP is considered a hot spot of

biological diversity and has been designated as an International Biosphere Reserve.

Researchers have been working to discover and document all organisms within the

boundaries of the park, including the microscopic algae. Inventory of the algal flora,

especially the diatoms, is not near completion and many habitats have yet to be collected.

Previous research has suggested that the algal diversity is high in the GSMNP and many

species are new, endemic, or restricted in range (Camburn & Lowe 1978, Lowe &

Kociolek 1984, Keithan & Lowe 1985, Johansen et al. 2004).

High algal biodiversity in the GSMNP is likely due to the large range in

environmental conditions, altitudes (including the Appalachian Mountains), geology and

bedrock, combined with the area having remained un-glaciated for several millions of

years. However, several factors threaten the biodiversity and ecosystem integrity in the

park such as acid precipitation, high nitrate levels, high aluminum levels and invasive

species. The average acidity (pH) of rainfall in the GSMNP is 4.5, which is 5-10 times

more acidic than rainfall from other monitored areas in North America where pH ranges

from 5.0-5.6 (National Park Service, 2005). This is in part due in part due to atmospheric

deposition of sulphur dioxide (SO2) and nitrogen oxides (NOx) emitted from automobiles and industries such as coal-fired plants (Planas 1996, Doka, et al. 2003). As a result of these extensive impacts, the National Park Conservation Alliance (2004) has ranked the

GSMNP as one of the most threatened national parklands. With increased human population and tourism in the area, these threats are likely to increase. Algae are threatened by these changes and many may go extinct before they have been discovered and documented (Sharkey 2001). 3

In this dissertation, to explore these challenges to ecosystem integrity and biodiversity in aquatic ecosystems, I first examined the basic ecology of algal assemblages of wet walls, an understudied ecosystem that is at the interface between the

atmosphere and terrestrial environments. I then conducted a biodiversity inventory and

ecological examination of an acidophilic diatom in the genus Eunotia Ehrenberg.

Note: Each of the chapters has been written in publication format with chapter

specific table and figure numbering (rather than continuous throughout the dissertation).

Therefore, each chapter should be able to be read separately from the others, but has

resulted in some overlap of material, especially for site descriptions and methods.

Additionally, some chapters are written in first person, plural to reflect journal

formatting.

Chapter I: Wet wall algal community response to in-field nutrient manipulation in the

Great Smoky Mountains National Park, U.S.A. – published: Furey, P.C.,

R.L. Lowe, & J.R. Johansen. 2007. Algological Studies. 125: 17-43.

Chapter II: Morphological deformities in Eunotia taxa from high elevation springs and

streams in the Great Smoky Mountains National Park, with a description

of Eunotia macroglossa sp. nov. – in review: Furey, P.C., R.L. Lowe, &

J.R. Johansen. 2008. Submitted to Diatom Research.

Chapter III: Ecology and distribution patterns of Eunotia Ehrenberg in the Great

Smoky Mountains National Park.

Chapter IV: Eunotia Ehrenberg (Bacillariophyta) of the Great Smoky Mountains

National Park. 4

Chapter I of this dissertation explored the factors that potentially regulate and influence algal communities of seep-walls through an in-field experimental manipulation of nutrients (nitrogen and phosphorus) at high and low altitudes. These habitats are abundant in mountainous areas but are understudied in aquatic ecology. Factors that drive the algal community dynamics in these environments are poorly understood.

The GSMNP is rich in moist, vertical, rock outcrops or seep-walls (subaerial

habitats), and the subaerial algal communities throughout the park likely contribute

substantially to algal production and diversity in the park, in addition to making the park

an ideal location to study wet walls. The algae in these environments are particularly

exposed to aerial and surface-water pollution, especially at high altitudes (Shubzda et al.

1995). Atmospheric deposition (of nitrates and sulfates) and associated changes in water

chemistry may alter community structure and affect overall production levels.

Understanding the ecology of wet wall communities may be useful in monitoring and

assessing the affects of atmospheric pollution and climate change, including effects from

ultraviolet radiation.

Chapters II, III, IV of this dissertation examined the taxonomy and ecology of

Eunotia species in the GSMNP. This acidophilic diatom genus (Patrick 1977), is very

diverse in the GSMNP, both in terms of number of species and geographical distribution.

The range of habitats and the variety of bedrock geologies and altitudes under both

natural and anthropogenically-influenced settings contribute to the high diversity of

Eunotia species in the GSMNP and thus, provided a unique opportunity to study the taxonomy, morphology (including ultrastructure), and ecology of Eunotia, all of which are in need of further research (Round et al. 1990). This research contributes to the 5

understanding of Eunotia taxonomy by providing image rich documentation of this

understudied genus and providing complimentary ecological information about

distribution patterns, including descriptions of unique diatom assemblages, and habitat

preferences of potential indicator taxa. The Eunotia inventory also added to ATBI

biodiversity efforts in the park, thus contributing to taxonomic and biodiversity studies in the GSMNP. 6

CHAPTER I

Wet wall algal community response to in-field nutrient manipulation

in the Great Smoky Mountains National Park, U.S.A.

[Published: P. C. Furey, R. L. Lowe, & J. R. Johansen. 2007. Wet wall algal community response to in-field nutrient manipulation in the Great Smoky Mountains National Park,

U.S.A. 2007. Algological Studies. 125: 17-43].

Abstract

Subaerial algal assemblages, like those found on wet walls (moist vertical rock outcrops), are understudied communities and the factors that drive community dynamics in these environments are poorly understood. Due to the exposed and often vertical nature of these habitats, nutrients are predicted to be critical determinants of algal community structure. Through in-field experimental manipulations we measured the structural response of wet wall algal assemblages to nutrient manipulation at two altitudes in the Great Smoky

Mountains National Park. Over a 10 week period, we misted five nutrient treatments (control

[no-moisture, no-nutrients], no-nutrients [moisture-only], nitrogen, phosphorus, and both nitrogen and phosphorus) onto rock faces at six replicate wet wall areas at each altitude. We did not observe any differences in algal community structure or biomass (ash free dry mass) across treatments at either location. The algal communities at our higher elevation location were generally similar across all sites, and were dominated by Cyanobacterial taxa with pigmented sheaths, such as Gloeocapsopsis dvorakii. The diatoms present at this location were acid tolerant species, such as Eunotia exigua, reflecting the low pH levels and high acid precipitation levels of this area and altitude. In contrast, algal communities at our lower elevation location clustered by site. Two sites were dominated by mucilage–producing

Cyanobacteria along with one diatom taxon. Two sites supported large populations of several

raphid diatoms which may have been due to influences from soil associated with those sites.

The remaining two sites separated out by the presence of particular green algal or

cyanobacterial taxa, along with a mix of diatom taxa. This study demonstrated that wet walls

are not as nitrogen or phosphorus limited as initially anticipated. Although not directly tested

in this study, the clustering patterns and algal assemblages observed suggest that other

factors, such as ultraviolet radiation, pH, moisture levels, or microhabitat differences, are

influential in shaping wet wall, algal community structure. Our findings provide new

understanding of wet wall algal ecology and provide insight into factors that potentially

regulate algal community structure that will be helpful in the development of future studies.

Introduction

Atmospheric pollution, i.e. from sulphur dioxide (SO2) and nitrogen oxides (NOx) emitted from automobiles and industries such as coal-fired plants are threatening terrestrial and aquatic habitats around the world (Johnson & Lindberg 1992, Likens &

Bormann 1995, Doka et al. 2003). This is especially the case in regions such as the eastern United States of America (USA) where, for example, places like the Great Smoky

Mountains National Park are more acidic than other monitored places in North America

(National Park Service 2005). Acidification of lakes, streams, and soils from acid precipitation can make habitats unsuitable for fish and other wildlife, damage trees, and result in the release of toxic metals such as aluminium (Stokes 1986, Schindler 1988,

Johnson & Lindberg 1992, Planas 1996). Recent national and international initiatives by governmental agencies and national parks in USA and Canada are working to reduce SO2 and NOx emissions, and to develop ways to assess and monitor impacts of acid pollution

8 on the environment (i.e. the Integrated Atmospheric Deposition Network USA/Canada, the Clean Air Interstate Rule by the USA Environmental Protection Agency, or the

Ecological Monitoring and Assessment Network in Canada).

Algae are able to integrate the biotic and abiotic elements of the environment, making them ideal indicators of change in ecosystems associated with acid pollution

(Lowe & Pan 1996). Algal assemblages of wet walls, moist vertical rock outcrops, are at a direct interface with the atmosphere and aerial pollution, especially at high altitudes where atmospheric depositions, like sulphate, nitrate, and ammonia, are higher relative to lower altitudes because of cloud-water input (Weathers et al. 1986, Nodvin et al. 1995,

Shubzda et al. 1995). This makes wet wall algae potentially useful as biotic indicators and predictors of environmental change associated with atmospheric pollution. However, wet wall algal assemblages are understudied and factors that drive even basic community dynamics are poorly understood (Johansen 1999). This lack of understanding currently limits our ability to use them in biomonitoring.

Subaerial algal communities of wet walls are not completely submerged by water, unlike many of their lotic and lentic counterparts. They receive intermittent moisture and nutrients from surface–water flow that drips from a cliff edge, groundwater seepage, atmospheric moisture (i.e. from fog or precipitation), or from wet wall associated plants such as mosses. Due to the often exposed and vertical nature of these habitats, variables such as nutrients are predicted to be critical determinants of algal community structure

(Johansen 1999). However, controlled experiments manipulating nutrients have not previously been documented for wet wall, algal communities. It is not clear whether nitrogen is limiting as in some terrestrial environments, or if phosphorus is limiting as is

common in freshwater systems, or whether nutrient dynamics are more complicated, i.e.

due to influences from atmospheric deposition. Insight into how these understudied algal communities respond to different nutrients will provide a more complete picture of wet

wall algal ecology. This will provide information that can be used to assess and monitor

ecosystem changes associated with atmospheric pollution and climate change.

Our objective in this research was to measure the structural response of wet wall algal communities to nutrient manipulation, specifically nitrogen and phosphorus, at two altitudes in the Great Smoky Mountains National Park (GSMNP). We anticipated shifts in dominant taxa in response to different nutrient inputs. We expected select algal taxa at lower altitudes that are less exposed to atmospheric deposition to be more N-limited than those at higher altitudes.

Material and methods

Site Description

The GSMNP comprises more than half a million acres and is the largest contiguous preserve east of the Rocky Mountains. The park is rich in wet walls from varied geologies at a variety of elevations, thus making it an ideal location for examining wet wall ecology. We conducted nutrient manipulation experiments on wet walls at contrasting elevations in the Clingmans Dome (CD; elevation 1830 m; UTM:

17S0274076, 3938632l, Garmin global positioning system; coordinate datum) and

Cataloochee (CAT; elevation 1005 m; UTM: 17S0313449; 3945353) areas of the

GSMNP (Fig. 1). The CD wet wall is close to the highest elevation in the park. The entire wet wall area had similar geology and was very exposed. The geology at CD is characterized by partially metamorphosed sedimentary rock in the Thunderhead

Sandstone group, which consists of thick beds of graded, coarse-grained feldsparthic metasandstone and metaconglomerate, interbedded with dark graphitic metasiltstone and slate (Southworth et al. 2005). The CD wet wall received much of its moisture during this study from atmospheric mist in addition to inputs from water seeping from cracks within the rock face and dripping down from surface-water flow from the ground above the wet wall. The CAT wet wall, located in the southeastern region of the park, was more heterogeneous in nature compared to CD. The geology consisted of variably metamorphosed sedimentary rocks from the Wading Branch Formation (quartz muscovite schist), and the Basement Complex (mesoproterozoic), which consists of a polymetamorphic complex of paragneiss, migmatite, and orthogneiss that were locally mylonitized and partially melted into migmatitic gneiss (Southworth et al. 2005). The

CAT wet walls were less exposed relative to CD. Some sites received partial protection from tree and shrub overhang and some sites appeared to be influenced by sediment from areas above the exposed rock. During the course of this experiment, moisture on these wetwalls came predominantly from water dripping down from surface–water flow from the ground above the wet wall with some seepage from cracks within the rock face.

Experimental design

We conducted our experiment at one extended wet wall area at each location (a minimum of 30 m in length) to minimize the confounding effects of other variables such as aspect or bedrock type. We applied nutrient treatments weekly, a total of 10 times, from 3 June to 12 August 2005 (70 days) on six replicate wet wall sites at each altitude.

At each site, we scribed five 12-cm diameter circular plots and assigned nutrient treatments (control [no-moisture, no-nutrients], no-nutrients [moisture-only], nitrogen,

phosphorus, and both nitrogen and phosphorus; C, M, N, P, and NP respectively). Where

plots had to be aligned vertical to each other due to spatial constraints, we placed C and

M plots above nutrient plots.

-1 Using a 1 L spray bottle, we applied 2.4 ml solutions of NaNO3 (440 mM N•l ),

-1 monobasic KH2PO4 (13 mM P•l ) or their 1:1 combination (a molar N:P ratio of 34 to 1).

The high nutrient concentrations were selected to better reflect higher atmospheric

nitrogen levels associated with acid precipitation (Weathers et al. 1986). To control for

the potential effect of moisture, we sprayed one of the control plots (M) with 2.4 ml of

distilled water and the other control plot (C) received neither nutrients nor moisture. To

prevent contamination between adjacent plots we used a protective plastic cylinder when

spraying. Prior to applying treatments each week, we measured the pH of a moist area on

each plot using EMD colorphast pH-indicator strips (field pH; range: 2.5 to 10 pH units;

sensitivity of 0.2 to 0.3 pH units). We placed the pH strips on the wet wall face for a

minimum of 10 min. to provide sufficient time for the indicator color to respond. We

visually categorized moisture levels of each plot on an area-based scale (areal moisture) ranging from 0 to 100 % moist with categories in 25 % area increments.

We collected algal assemblages at the beginning (3 June 2005) and end of the experiment (12 August 2005) from a 26.4 cm2 circular area using a 7.2V Multipro

Cordless Dremel (Model 7700) with a plastic collecting container sealed against the rock

with silly putty. We homogenized each sample (Tissue Tearor model 780CL) and took

one sub-sample of fresh (unpreserved) material for analysis of the soft algae and three

sub-samples of material preserved in 2% gluteraldehyde for diatom processing, a future

reference-sample, and for measuring ash free dry mass (afdm) in order to estimate wet

wall algal biomass. To estimate biomass accrual during the course of the experiment the

area scraped at the initial sampling on 3 June was re-scraped on 12 August. Digital

images of each plot were taken at both locations each week during the experiment, and

again one year later at CD. We determined organic matter (afdm) by drying samples at 70

˚C to constant mass followed by ashing samples at 550˚C for one hour.

We collected seep-water from each site at CD on 10 June and 5 August and CAT on 11 June and 6 August 2005, where a sufficient volume could be collected within one hour from water drips. Samples were analyzed for pH (water pH), total phosphorus (TP), soluble reactive phosphorus (SRP), nitrate (NO3-N), ammonium (NH4-N), and sulphate

(SO4-S) (University of Michigan Biological Station Water Chemistry Laboratory

Pellston, MI).

Algal sample analysis

We analyzed algal community structure with an Olympus BX51 Photomicroscope

with high resolution Nomarski DIC optics and recorded digital images with a Spot™

monochromatic camera attached to the microscope and a computer. We calculated the

relative percent frequency of soft algae (including diatoms as a group) from

presence/absence counts of fresh (unpreserved) material based on 25 fields examined at

600X. We pooled the Leptolyngbya taxa for analysis because of uncertainty associated

with species level identification. We also pooled all the diatom taxa for the soft algal

relative frequency counts. For diatom specific analysis, we cleaned diatoms in boiling

nitric acid, air dried them onto cover glasses, and made permanent diatom mounts using

Naphrax® mounting medium. We counted a minimum of 300 diatom frustules from each

sample and calculated relative abundances at the species, genus and division level

(Bacillariophyta, Chlorophyta, Cyanobacteria, Pyrrophyta). We identified algae to the

lowest taxonomic level possible using standard references which consisted primarily of:

Desikachary (1959), Geitler (1932), Patrick & Reimer (1966), Patrick et al. (1975),

Krammer & Lange-Bertalot (1986, 1991a, 1991b), and Komárek & Anagnostidis (1999,

2005).

Data Analysis

First we used a repeated measures analysis of variance (RM ANOVA1) with 1 between subject factor (location) and 2 within subject factors (time and treatment) to test whether pH or moisture differed between locations (CD and CAT). The analyses were conducted both including and excluding 29 July when 73% of the treatments across all sites at CD were dry, which largely affected the results when included and prevented pH from being obtained in the field or estimated statistically for that date. For the remaining dates where plots were dry (7 for CD; 11 for CAT; from 300 measurements for each location) missing pH values were determined using an iterative method (Montgomery

1991). We then examined each location separately (CAT or CD) and used RM ANOVA2 with 2 between subject factors (sites [blocs] and treatment) and 1 within subject factor

(time) to test whether pH or moisture differed between and within sites or treatments. To test for differences in afdm with treatment, we used a RM ANOVA3 with date as the repeated measure and treatment as the predictor variable. A 2-way ANOVA was then run to determine if afdm differed with site or date. For water chemical data, we used an

ANOVA to test whether nutrients differed between locations. We analyzed all data using

SAS (version 8.1, Cary, North Carolina) with significance set at α ≤ 0.05.

For algal community analysis, we normalized relative frequencies and abundances

with an arcsine transformation (arcsine of the square root; appropriate for proportional

data) and examined clustering based on a reduced taxa list using non-metric

multidimensional scaling (NMDS) of Bray Curtis similarities (Primer 5 version 5.2.9;

Primer–E Ltd., 2002). For NMDS analyses, we excluded taxa that were only present at

one or two sites to ensure sufficient data for ordination. We used site number, treatment,

and date, and their combinations as factors for examining clustering patterns. We

analyzed the data with NMDS with our taxa grouped by species, genus and Division.

Results

The mean field pH of CD plots (4.37 ± 0.04, mean ± SE) was significantly lower

than that of CAT plots (5.19 ± 0.07, RM ANOVA1, p < 0.001). Areal moisture levels of plots were not significantly different between CD and CAT (p > 0.05), with dry areas often being present at both locations on similar dates corresponding with generally hotter, drier weather patterns (personal observation). The water collected at CD was more acidic and had higher nitrate, sulphate, and conductivity levels than that of CAT, whereas the water from CAT had higher total phosphorus levels (Table 1).

Examining each location separately, the pH and areal moisture levels of CD plots were not significantly different across nutrient treatments (RM ANOVA2, p > 0.05).

While pH levels were not significantly different by site (p > 0.05), areal moisture levels were significantly different between sites (p < 0.05) but these varied across sites with time. Biomass accrual (increase in afdm for control plots scraped clean on 3 June and re- sampled on 12 August) was ~40% lower than the overall average afdm for all plots from both dates (Table 2). CD sampling areas scraped at the beginning of the summer showed

little to no observable re-growth by the end of the experiment or one year later, regardless

of treatment (Fig. 2A to D). Furthermore, there were no significant differences in the

organic matter levels (afdm) with treatments or time (p>0.05), but when examined by

site, there was a significant site by time interaction (p = 0.04). When August afdm was subtracted from June afdm to examine for effect of treatment, both positive and negative differences in afdm were observed regardless of treatment (Table 2). Overall afdm levels for the wet walls (all sites and dates considered together) were significantly lower at CD than at CAT (p<0.05; Table 2).

In contrast, there was a significant difference in pH and moisture across CAT sites

(p < 0.001), although these varied with time. Generally CAT sites 1 and 2 had a slightly higher pH than sites 5 and 6 and were generally drier than those of other CAT sites, predominantly on warm June sampling dates. Similar to CD, biomass accrual was ~40% less than the overall average afdm for all plots from both dates (Table 2). Several CAT plots showed little to no re-growth by the end of the experiment, whereas some sites showed some algal recruitment from adjacent areas, such as those rich in mucilage- producing algae (i.e. Site 2; Fig 2E, F). At other sites, soil (and associated algae) from areas above the plot regularly replaced the algal communities, especially during rain events (Fig 2G, H; personal observation). Organic matter levels (afdm) were not significantly different with treatment, time, or by site (p>0.05). Both overall afdm levels and biomass accrual were significantly higher at CAT than CD (p<0.001, Table 2).

Similar to CD, when August afdm was subtracted from June afdm, both positive and negative differences were observed regardless of treatment (Table 2).

Algal community structure

Clingmans Dome

We observed a similar over-all community structure across sampling sites and

treatments for both soft algae and diatoms. Comparison of the relative frequency of taxa

at the beginning of the experiment with taxa across treatments at the end of the

experiments emphasizes the absence of a community response from the nutrient dosing

even when rarer taxa (>0.05 and <12.5 % mean relative frequency) were examined

(Table 3). There also were no clear groupings of the soft algal community structure by

nutrient treatment (Fig. 3). Gloeocapsa cf. sanguinea (black color) showed some seasonality and was present at plots on 3 June but absent on 12 August (Table 3). Due to the low diatom densities at CD, we did not analyse this group statistically.

The absence of an effect of nutrient addition was sufficiently surprising that we attempted other means of multivariate analysis in case the NMDS was somehow deficient in detecting an effect. Correspondence Analysis, Detrended Correspondence Analysis, and Canonical Correspondence Analysis were performed on the full data set as well as a dataset excluding the June samples. Although these analyses are not shown, all yielded scatterplots that did not show any clustering by nutrient treatment.

The relative frequency of soft algae averaged for each site was composed of 48 to

66% Gloeocapsopsis dvorakii (Novácek) Komárek et Anagnostidis, a cyanobacterial taxon with a reddish–pigmented envelope (Fig. 4B, C). The remaining algal assemblages were generally comprised of two to four taxa that each contributed 2 to 25% of the relative frequency. At sites 1 and 5, these consisted of Homoeothrix juliana (Bornet et

Flah.) Kirchner (4 and 2%), Calothrix parietina (Naegeli) Thuret ex Bornet et Flah. (2

and 1%), Aphanocapsa muscicola (Meneghini) Wille (19 and 4%), and Leptolyngbya species (13 and 11%). At site 2 these consisted of Gloeocapsa sanguinea (C.A. Agardh)

Kützing (22%), H. juliana (8%), A. muscicola (2%), Coccomyxa sp. A. (3%), and

Leptolyngbya species (12%). At sites 3 and 4, these taxa were comprised of H. juliana (5 and 25%) and C. parietina (21 and 15%), and at site 6 of A. muscicola (4%), Coccomyxa sp. A (5%) and Leptolyngbya species (18%) (Fig. 4). Across all sites, diatoms predominantly consisted of three taxa, Frustulia rhomboides (Ehrenb.) De Toni, Eunotia exigua (Bréb. ex Kütz.) Rabh. and E. praerupta var. bigibba (Kütz.) Grun. (Fig. 5A, K,

P). The relative frequency of diatoms at sites 1, 2, and 6 was 3.5 to 4 %, whereas at sites

3, 4, and 5 it was <0.6 %.

Cataloochee

Similar to CD, no clear response to nutrient treatment was elucidated for either the soft algal or diatom communities (Fig 6). When taxa present in >5 % total relative frequency or total relative abundance were more closely examined by treatment no nutrient response was apparent, especially given that the variability within each treatment was high (Table 4). However, the algal communities clearly separated out by site, especially sites 1 and 2, which clustered separate from sites 3 to 6 (Fig 7).

Sites 1 and 2 were dominated by Leptolyngbya spp. and mucilage-producing

Cyanobacteria including Aphanothece pallida (Kütz.) Rabh., Gloeothece tepidariorum

(A. Braun) Lagerh., Nostoc paludosum Kütz. ex Bornet et Flah. and by one diatom taxon,

Nitzschia hantzschiana Rabh., which comprised almost 100% of the diatom relative abundance (Figs 4, 5; Table 5). The mucilaginous envelopes of the Cyanobacteria at CAT were not as strongly pigmented as those at CD (Fig. 4).

Three groups emerged when we examined sites 3 to 6 more closely (Fig 8; site 3;

site 5; sites 4 and 6). Diatoms more distinctly defined these separations than did the soft

algae (Fig 8). Overall, Navicula angusta Grun. was consistently present across sites 3 to

6, as were Nupela wellneri (L.-Bert.) L.-Bert. and Nupela neglecta Pon., Lowe et

Potapova, although in lower relative abundances (Fig. 5, Table 5).

Site 3 was generally comprised of Chlorophytes, primarily Mougeotia sp. and

Oocystis sp. and diatom taxa, primarily Achnanthidium minutissimum (Kütz.) Czarn. and

Gomphonema montanum Schumann, and secondarily Diadesmis perpusilla (Kütz.) D.G.

Mann, Navicula angusta, Navicula keeleyi Patr. and Synedra rumpens Kütz. (Figs 4 ,5;

Table 5). Of these diatoms, Diadesmis perpusilla, Achnanthidium minutissimum and

Gomphonema montanum showed the greatest habitat fidelity (Table 5).

In contrast with site 3, several Cyanobacteria taxa dominated site 5, including

Aphanocapsa spp., Chroococcus spp., and Leptolyngbya spp. (Table 5). Navicula keeleyi had the greatest relative abundance of the diatoms, with Navicula angusta and Nitzschia hantzschiana also being relatively common (Fig. 5; Table 5). Although less abundant,

Orthoseira roeseana (Rabh.) O’Meara and Diadesmis contenta var. biceps (Grun.)

Hamilton were more commonly found at site 5 relative to other sites (Table 5).

Diatoms dominated sites 4 and 6 (Table 5). Diatom taxa that were more common at these sites relative to other sites included: Achnanthes subrostrata var. appalachiana

Camb. et Lowe, Decussata placenta (Ehrenb.) L.-Bert. et Mezelt., Diatoma mesodon

(Ehrenb.) Kütz., Eolimna minima (Grun.) L.-Bert., Navicula sp., Planothidium lanceolatum var. lanceolata (Bréb. ex Kütz.) Round et Bukht., Cavinula weinzierlii

(Schimanski) Czarnecki and Pinnularia appendiculata Ehrenb. (Fig. 5, Table 5).

Nitzschia hantzschiana was also very common at sites 4 and 6.

Discussion

Algae from lotic and lentic habitats are commonly nutrient limited and nutrient

dosing experiments frequently result in changes in algal community structure or biomass,

especially with nitrogen or phosphorus additions (see studies in Borchardt 1996). It has

been suggested that wet wall algal communities may be nutrient limited due to the

ephemeral inputs of nutrients, which, may further be reduced by the vertical nature of

these habitats (Johansen 1999). However, contrary to our expectations, the algal

community structure of the wetwall areas examined in our study did not respond to

weekly nutrient dosing of nitrogen and phosphorus, suggesting that wet walls are not as

nitrogen or phosphorus limited as initially anticipated or that other factors such as

ultraviolet radiation, pH, moisture levels, or microhabitat differences are more influential

in shaping wet wall, algal community structure.

Because this study was conducted in a national park, we applied nutrients to our

wet wall plots by misting in order to ensure the area impacted by the nutrient

manipulations was as small as possible and that our experiment had low visibility level to

park visitors, thus, minimizing the potential for disturbance. A more continuous

application, i.e. via a nutrient–drip system commonly used in stream nutrient

manipulation experiments, would have increased the level of exposure to nutrients, but

also increased the area of impact and visibility. The nutrient concentrations we applied to

our wet walls were high (440 mM N•l-1; 13 mM P•l-1) compared to those we measured in

-1 -1 -1 the seep water (CD: 50 mM TN•l , 0.108 mM TP•l ; CAT: 112 mM TN•l , 0.632 mM

TP•l-1). Furthermore, our nutrient dosing concentrations were 2 to 3 orders of magnitude greater than nutrient limiting levels in lotic and lentic habitats (see studies in Borchardt

1996). This suggests that our misting concentrations were sufficient. Running the experiment at two locations with six replicates sites at each should also have been an adequate design to detect algal community structural responses to nutrient additions under nutrient limiting conditions, especially at CD where the geology and habitat features (i.e. exposure level, minimal influences from sediments) were similar across sites. Given that we found no differences in community composition or biomass (afdm) amongst nutrient manipulations at either location, we think that nitrogen and phosphorus are not critical determinants of wet wall algal community structure. Ground water and/or atmospheric inputs, and/or plant–associated inputs are likely frequent or concentrated enough to provide adequate nutrients to support wet wall algal communities given the constraints of other limiting factors.

The absence of a response to nitrogen and phosphorus additions, a bottom–up manipulation, may indicate that these communities are top–down controlled, i.e. from grazers such as chironomids and protozoans. However, few grazers were observed during sample collection and analysis during this study. Studies of the zoological communities of wet walls are very limited and one study by Casamatta et al. (2002) tended to observe greater numbers of protozoans in moister wet wall areas, especially those with some low level of water flow.

Although the algal assemblages did not respond to nutrient additions, the algae at

CD showed adaptations to high UV and low pH levels which may be indicative of the influence of these two factors in shaping wet wall algal community structure on exposed,

21 high elevation wet walls. For example, the pigmented envelopes and cells of the algae at

CD, such as the Cyanobacteria taxa Gloeocapsopsis dvorakii or Calothrix spp., are likely a ‘sunscreen’ or protective measure present in response to the exposed and high light nature of this higher elevation wetwall (Quesada et al.1999) where UV levels would be higher relative to CAT. The ability of algae, especially Cyanobacteria taxa, to shift their pigment composition or to produce protective compounds, such as scytonemin, in response to high light intensities and UV levels (Dillon et al. 2003, Holzinger & Lütz

2006) may be why pigmented taxa are often common on exposed, subaerial habitats

(Rindi & Guiry 2003). Shifts in algal pigmentation and community structure on exposed wet walls could potentially be used to indicate changes in types and levels of UV radiation associated with atmospheric changes i.e. from changes to the earth’s ozone layer with pollution.

Low pH levels at CD may have impacted the high elevation algal communities by decreasing diversity and limiting species composition. Algal communities in other aquatic habitats are sensitive to pH levels, with low acidity reducing species diversity and richness (Schindler 1994, Van Dam 1982) or altering the composition of dominant taxa

(Van Dam & Mertens 1995). The low pH at CD was reflected in the diatom community composition which was dominated by acid tolerant taxa, such as Frustulia rhomboides,

Eunotia exigua, and Eunotia praerupta var. bigibba (Beaver 1981, Krammer & Lange-

Bertalot 1991a, DeNicola 2000), and may explain the lower diversity observed at CD relative to CAT. Our high elevation location had higher inputs of nitrates and sulphates than our low elevation location, which appears, in part, to have contributed to the acidic nature of the CD wet wall. These inputs may have been even higher because of additional

atmospheric depositions of nitrates and sulphates from clouds and fog (Weathers et al.

1986, Nodvin et al. 1995, Shubzda et al. 1995).

In our study, we did not find a community composition response with our mist-

only treatment, but the volume of moisture added was small relative to other moisture

inputs, i.e. from drips or atmospheric sources, and thus was not designed to test for the

effects of moisture on wet wall communities. Mucilage, however, may play a role in

moisture dynamics on wet walls. We found that CAT sites 1 and 2, were drier based on

our moisture categories and clustered together based on the dominance of mucilage

producing taxa, especially Aphanothece pallida and Nostoc paludosum. Because our

estimation of moisture was not quantitative, the designation of sites 1 and 2 as being drier

than other sites may have been because moisture levels were closely associated with the

mucilage and not on rock face itself. Thus, the mucilaginous colonies may have been

acting as a biological moisture reservoir. Other studies have found that moist habitats

tend to be dominated by mucilage secreting green and bluegreen algae such as Nostoc

that act as a moisture oasis and thus often support other algal taxa (Johansen et al. 1983).

As natural nutrient inputs in our study were potentially tightly coupled with

moisture levels and moisture sources, a more detailed study is necessary to understand

micro- and macro-scale effects of moisture and to tease apart nutrient and moisture

effects and their interactions. The mucilaginous colonies at CAT may also have been a

nutrient source for other taxa, such as Nitzschia hantzschiana, which was the dominant

non-mucilaginous taxon at sites 1 and 2. This may indicate diatom species–specific associations with select mucilage–producing algal taxa. Exploring algal relationships

with exogenous–mucilage might provide a more thorough understanding of the fidelity

and taxa–specific nature of these associations on wet walls.

The grouping of plots by site at CAT, such as the mucilaginous algal dominated

sites, emphasizes that differences in habitat features can result in differences in algal

community structure. For example, CAT sites 4 and 6, which were influenced by soils,

were dominated by diatom taxa, especially raphid taxa, such as Achnanthes, Nitzschia and Navicula that are better able to move in soil habitats than taxa without raphes. It is not clear what factors shaped the green algal dominated site 3, or the Cyanobacterial dominated site 5. These communities contrasted with sites, such as the mucilaginous- algal dominated sites at CAT or the low pH, exposed sites at CD. Macro-scale habitat associations have been observed in other subaerial algae studies (Johansen et al. 1983,

Casamatta et al. 2002, Rindi & Guiry 2003) and may explain these differences. However, contrasting algal community structures can occur on smaller microhabit scales, i.e. only

centimeters apart (Rindi & Guiry 2003, Lowe et al. 2007, personal observation). Guilds of diatoms may be associated with specific macro– or microhabitat conditions on wet walls, and may facilitate their use as bioindicators as is common in stream studies.

Differences in scale and habitat specific associations should be carefully considered in

future studies examining the role of nutrients or moisture on wet wall algal communities.

Several of the plots sampled in this study showed slow regeneration; however, the

turnover time and dynamics for wet wall algal assemblages have not been well

documented. On return to the CD sites one year later, we found that very little re–growth

had occurred, a further indication of slow regeneration time. However, other factors need

to be considered in future work, for example, the role of algal recruitment from adjacent

24 sites as we observed at the CAT plots influenced by soil and mucilage producing algae.

The soil-influenced sites seemed especially unstable and susceptible to rain events which would quickly wash the soil and associated algae away. This may have confounded our ability to test the effects of nutrient dosing at these sites.

This experiment demonstrated that nitrogen and phosphorus were not the dominant factors influencing wet wall community structure in two contrasting locations in the GSMNP. Although not tested in this study, the clustering patterns and composition observations suggest that others factors, such as UV, pH, moisture and other habitat influences, such as soil or mucilaginous algae interactions, likely influence wet wall community structure. More research needs to be conducted at both micro- and macro- habitat scales in order to better understand wet wall algal community dynamics and the potential role of wet wall algae as bioindicators, especially in light of increasing acidification and pollution of aquatic areas around the world, concomitant with rising levels in the human population.

CHAPTER II

Morphological deformities in Eunotia taxa from high elevation springs and streams

in the Great Smoky Mountains National Park,

with a description of Eunotia macroglossa sp. nov.

Abstract

High elevation springs and headwater streams directly interact with atmospheric

depositions and as a result may be useful first response bioindicators of changes in acid

precipitation, i.e. from increased acid rain or mitigation efforts. We examined the diatom

assemblages associated with bryophytes collected from high elevation springs and

streams in Great Smoky Mountains National Park, U.S.A. in 2005 and 2007. The high

elevation areas were dominated by Eunotia taxa, primarily E. subarcuatoides Alles,

Nörpel et Lange-Bert., and secondarily by E. exigua (Bréb. ex Kütz.) Rabh., E. muscicola var. tridentula Nörpel & Lange-Bert., and E. macroglossa sp. nov.

The presence of valve malformations in four of the high elevation springs and two streams suggest that acid precipitation in the park may be interacting with local bedrock geology to release metals toxic to algal communities, especially under the low pH conditions of these high elevation areas. Sites with the greatest number of deformities showed higher levels of Al, Ba, and Mn relative to other sites. Internal and external diatom morphology of normal and abnormal E. subarcuatoides valves were explored and demonstrated the presence of valve aberrations across all size ranges, including shape distortions and unusual raphe development. However, no difference in mean valve length or size range was present between normal and abnormal valves.

Introduction

Diatoms are good integrators of the biological, chemical, and physical

characteristics of their watersheds and as a result are useful as bioindicators of ecosystem

condition or ecosystem change (Dixit et al. 1992, Lowe & Pan 1996, Stevenson & Pan

1999). The presence of morphological aberrations in diatoms can further be an indicator

of pollution. For example, teratological valves have been reported from aquatic systems

contaminated with heavy metals, such as those found close to mine sites (Cattaneo et al.

2004). Under pristine or unpolluted conditions, deformities generally occur in very low

frequencies, with less than 1% or no valves showing abnormal growth. Thus, the

presence of notable abundances of morphological deformities in diatoms from samples

collected from high elevation springs in the Great Smoky Mountains National Park

(GSMNP), USA warranted additional study.

As a National Biosphere Reserve, the headwater areas in the GSMNP do not

receive point source pollution inputs from mining or other industries. However, the park receives acid precipitation from atmospheric deposition of sulphur dioxide (SO2) and nitrogen oxides emitted from automobiles and industries such as coal-fired plants.

Furthermore, high altitude areas in the GSMNP are exposed to higher levels of atmospheric aerosols due to greater cloud-water input relative to lower elevations

(Nodvin et al. 1995, Shubzda et al. 1995). As a result, many areas of the GSMNP are more acidic than other monitored places in North America (National Park Service 2005), and these aquatic systems may additionally be threatened by increased solubility and release of toxic metals such as aluminum (Al) or manganese (Mn) from bedrock under low pH conditions (Johnson & Lindberg 1992, Planas 1996). Understanding the

27 distribution patterns and triggers of abnormal diatom morphologies and the community composition of these high elevation spring areas will be critical in identifying and tracking areas where biodiversity and ecosystem integrity may be threatened by acid precipitation interactions with the environment in the GSMNP. With increased acidification from anthropogenic sources throughout the world, diatom deformities may become more common, and may be useful in tracking the success of mitigation efforts, or in identifying areas of concern associated with the harmful release of metals from different bedrock geologies.

To further explore the unusual findings of diatom abnormalities from high elevation springs and streams in the GSMNP, we documented the morphologies of normal and abnormal valves, in addition to examining their relative abundance and distribution patterns. Specifically we documented 1) the variability in morphology of normal and abnormal valves, including: length, width, and striae density, 2) the internal and external ultrastructure of normal and deformed valves, and 3) the community composition, relative abundance, and distribution patterns of the dominant diatoms, specifically Eunotia taxa and normal and abnormal morphologies from high elevation springs and streams in relation to biotic and abiotic factors such as geology and water chemistry. A description of Eunotia macroglossa sp. nov., discovered during the course of this study, is also provided.

Material and Methods

Site description and sample collection

The GSMNP, the largest contiguous preserve east of the Rocky Mountains at more than half a million acres, is found at a climatic transition from the northern

temperate zone to the southern temperate zone. A complicated geology with diverse

bedrock chemistry at a variety of elevations combined with large tracts of old-growth and

contiguous forests, and protection as a national park have all contributed to a rich and

abundant fauna and flora, including the algae (Johansen et al. 2007). Several headwater

springs are found at high elevation areas along the Appalachian Trail which runs along

the North Carolina and Tennessee border.

As part of a larger study examining the diatom Eunotia in the GSMNP, we

examined Eunotia populations epiphytic on bryophytes collected from six high elevation

springs during May to August 2005 (Fig. 1, Table 1). A representative collection of

bryophytes were placed in a bag and shaken to dislodge associated diatoms before the

bryophytes were removed from the bag. The Double Spring Gap area had two springs

draining into two different watersheds (one on the Tennessee side and one on the North

Carolina side). For comparison with the springs, we also examined samples from eight

streams at elevations greater than 1400m and two from lower elevations. If we found

more than 5% diatom deformities at a site, then we re-sampled the site in 2007.

We measured the pH of each site at the time of collection using EMD colorphast

pH-indicator strips (field pH; range: 2.5 to 10 pH units; sensitivity of 0.2 to 0.3 pH units).

We placed the pH strips in the stream for a minimum of 10 minutes to provide sufficient

time for the indicator color to respond.

Several of the sites had metals data collected and analyzed by the National Park

Service (NPS) in 2002-2003, or 2002-2004 (EPA method 6010B, Inductively coupled plasma method - atomic emission spectrometry (ICP-AES), University of Tennessee

Water Quality Lab). We collected additional water in 2007 at the three sites that had the

greatest number of diatom deformities in 2005 (Mount Collins Shelter, Double Spring

Gap North Carolina side, and Russel Field Shelter) and also at the Double Spring Gap

Tennessee side for comparison with the North Carolina side. The NPS had these samples

analyzed for metals (EPA method 6010B, ICP-AES, Test America Inc. Nashville, TN).

Sample analysis:

Samples were cleaned in nitric acid to remove organic materials (Round,

Crawford & Mann, 1990) and strewn diatom slides were made using Naphrax® mounting medium. Diatoms were analyzed using an Olympus BX51 Photomicroscope with high resolution Nomarski DIC optics. Images were captured with a monochromatic camera (Spot™) attached to the microscope and a computer. A minimum of 600 diatom valves from each sample was counted at 600X magnification with additional empty magnification to 1200X with a 2X optivar. Relative abundances at the species level for all Eunotia taxa were calculated, while all other diatom taxa were grouped together into

one category. Obvious diatom deformities were recorded for each count, but we may

have missed more subtle deformities not easily visible with the light microscope. Eunotia

were identified to species using standard references which consisted primarily of: Lange-

Bertalot et al. ( 2003), Lange-Bertalot & Metzeltin (1996), Krammer & Lange-Bertalot

(1991, 2000), and. Lange-Bertalot (1998).

Eunotia frustules were mounted on aluminum stubs and sputter coated with 10 nm of AuPd and then examined under a high resolution Hitachi S2700 scanning electron microscope (SEM) with digital image recording ability (Postek et al., 1980).

Results

Eunotia macroglossa sp. nov. Figs 2–14, 17–19, 20–25

E. minori (Kütz.) Rabh., E. pectinali (Dillwyn) Rabh. et E. soleirolii Kütz.

(Rabh.) affinis sed helictoglossis prominentibus et distinctionibus minoribus in forma

valvae notabilis.

Valvae margine ventrali leniter concava vel prope recta, margine dorsali

aequabiliter convexa ad apices rotundatos, apicibus leviter distinguibilibus per distinctione in declivitate marginis dorsalis, 21 – 93 µm longae, 4.5 – 6.6 µm latae.

Helictoglossae manifeste visibiles ut amplificationes hyalinae apicales per microscopium luxicum. Raphe ex helictoglossa ad limbum extensa, valva 3-5plo breviore. Striae leviter radiatae, radiales ad apices, 11 – 14 in 10 µm in centro valvae, 16 – 18 in 10 µm prope apices valvae. Rimoportula apicalis, ad extremum unum, striis fere paralella.

Similar to E. minor (Kütz.) Rabh., E. pectinalis (Dillwyn) Rabh. and E. soleirolii

Kütz. (Rabh.), but notable for the prominent helictoglossae and minor differences in valve outline.

Valves with the ventral margin slightly concave to nearly straight, the dorsal margin evenly convex up to the rounded ends, with apices slightly set off by a change in slope of the dorsal margin, 21 – 93 µm long, 4.5 – 6.6 µm wide. Helictoglossae prominently visible in LM as apical hyaline thickenings. Raphe extending from helictoglossa to 20-30% of the length of the valve. Striae slightly radiate, radial at apices,

11 – 14 in 10 µm in the valve center, 16 – 18 in 10 µm near the valve apices. Rimoportula

apical, at one end of valve, nearly parallel to striae.

Habitat: epiphytic on bryophytes; found in low abundance in streams around the Great

Smoky Mountains National Park, occasionally observed in higher abundances in mid to high elevation springs and headwater streams ranging from 1300 to 1600 m altitude.

Holotype here designated: Circled specimen on slide CAS ##### in the Diatom

Herbarium of the California Academy of Sciences; type locality: epiphytic on bryophytes from Otter Creek, Great Smoky Mountains National Park, Cocke County, Tennessee,

35.7293° N latitude, 83.2536° W longitude, U.S.A. Sample collected 10 August 2005.

Isotypes here designated: Circled specimen on slide ANSP GC 58614 deposited in the

Diatom Herbarium of the Academy of Natural Sciences of Philadelphia. Isotype material

(uncircled) also deposited at California Academy of Sciences (CAS #####, pellet CAS

#####).

Etymology: Greek macro = large, Greek glossa = tongue, Eunotia macroglossa = large- tongued Eunotia, i.e. the Eunotia with a large helictoglossa.

E. macroglossa most closely resembles species having kinship with E. pectinalis

(Dillwyn) Rabh., particularly E. minor, E. pectinalis and E. soleirolii. It is set off from all three taxa by the heavily silicified, clearly visible helictoglossae at the valve ends. In valve outline, it is most similar to E. minor, and bears particularly a close similarity to a valve out of the Taunus Mountains in Germany (Krammer & Lange-Bertalot 1991, Tafel

142, Fig. 15) attributed to E. minor. Although this previously figured valve may be conspecific with E. macroglossa, the size range of our specimens includes valves of much greater length than reported for E. minor, and the ends are not nearly as attenuated as in that taxon (Krammer & Lange-Bertalot 1991, Tafel 142, Figs 7-9, 13, 14). E. macroglossa keyed to E. soleirolii based on the European flora (Krammer & Lange-

Bertalot 2000); however it differed markedly in overall valve shape. Similarly, E.

macroglossa did not fit the description for E. pectinalis with which E. soleirolii is

sometimes confused. E. soleirolii and E. pectinalis have parallel edges that end in a slight shoulder at the valve edge (Krammer & Lange-Bertalot 1991), in contrast with E. macroglossa which had a slightly curved dorsal edge, with the valve ends only very slightly set off from the evenly curved dorsal margin. The helictoglossae of E.

macroglossa appeared further from the apices than its position in both E. soleirolii and E.

pectinalis. Additionally, SEM micrographs of E. soleirolii show spines on the valve

apices at the junction of the valve face and mantle, which were absent from E.

macroglossa.

Other distinguishing features of our populations include the radial striae at the valve apices. These are clearly visible even in most LM views (if ends are focused for this feature), and are visible in external SEM’s of all valves (Figs 20-22). The external pore of the rimoportula is inconspicuous in comparison to many taxa (Fig. 23). The mantle has a large hyaline area associated with the raphe as it curves from the mantle up on to the valve face (Fig. 23). The raphe end on the valve face is usually simply curved

(Figs 20, 22), but can have a more irregular structure (Fig. 21).

Community composition and diatom deformities

Epiphytic diatom assemblages from high elevation sites were dominated by

Eunotia taxa. Overall, the spring and stream sites at > 1660 m were dominated by E. subarcuatoides Alles, Nörpel et Lange-Bert., which frequently occurred at a relative abundance of > 60 % (Table 1). The relative abundance of E. subarcuatoides generally decreased as elevation declined, with the exception of Clingmans Creek where it was

present at 58% (Table 1). The remaining Eunotia taxa at sites > 1660 m primarily

included E. macroglossa sp. nov., E. exigua (Bréb. ex Kütz.) Rabh., and/or E. muscicola var. tridentula Nörpel & Lange-Bert. (Table 1, Figs 2-19). E. macroglossa was present at

> 25 % for two lower elevation springs (Spence Field Shelter Spring and Russel Field

Shelter Spring), and it dominated the Otter Creek stream site (Table 1). Other Eunotia species at high elevation sites were: E. nymanniana Grun., E. rhomboidea Hust., E. tenella (Grun.) Cl., E. varioundulata Nörpel et Lange-Bert., and another Eunotia taxon

not yet described. As elevation decreased the algal assemblages of the streams became

dominated by non-Eunotioid taxa (Table 1). Eunotia billii Lowe & Kociol., and E. incisa

W. Sm. were present only below 1605 m, at three or more sites.

Valve deformities were present in E. subarcuatoides in varied abundances at four

of the high elevation spring sites (Table 1). Abnormalities in valve morphology were

most prevalent in the spring on the North Carolina side of Double Spring Gap. At this

site, deformities occurred in 30% or greater of the total valves in both 2005 and 2007, in

contrast with the nearby Double Spring Gap, TN site, where no deformities were present

either year (Table 1). At Mount Collins Shelter Spring, the highest spring site, valve

deformities in E. subarcuatoides were present in 5 to 10 % of the overall diatom

assemblage. Valve aberrations were absent or present in < 1% of the valves at the spring

sites below 1600 m (Table 1).

The length, width, and striae patterns of the normal E. subarcuatoides valves were

similar to measurements in Krammer & Lange-Bertalot (1991, Table 2, Figs 26-42). The

longest valve observed was 6.6 µm longer than previously reported for this taxon. The

deformed valves fell within the normal size ranges, with the exception of the valve width

34 which became narrower and broader with malformations (Table 2, Figs 43-62). These valve teratologies were present across the entire size range. Abnormal valve morphologies included small to large indentations or distortions along the ventral margin of the valve, and variations in valve curvature patterns, valve breadth, and raphe position or development (Figs 43-76). Mean valve length for abnormal valves (20.4 µm, ± 7.1 SD) was not significantly different from normal valves (19.3, ± 5.6 SD; T = 1.61, p < 0.05, df

= 300). SEM micrographs of the external and internal ultrastructure of normal and abnormal valves show these features in closer detail (Figs 63-76). Raphe position was affected by valve deformities and in some cases, an additional raphe was present (i.e.

Figs 68, 75, 76).

Water chemistry & geology

Higher elevation sites tended to have both lower lab and field pH levels relative to lower elevations sites (Table 3). Field pH values were consistently lower than lab pH estimates (on average by 1 pH unit, range 0.5 to 1.6) While the field pH was not an ideal method for measuring pH in streams with low conductivity it did provide a general estimate of relative pH differences between sites.

Average metals estimates from pre-2004 did not exceed the US Environmental

Protection Agency’s (USEPA) established criteria for metal concentrations in freshwater

(Table 3, USEPA 2002). However, based on the 2007 metals estimates, sites with the higher numbers of deformities tended to have higher levels of aluminum and barium relative to the other spring and stream sites. These values exceed the screening ecological risk benchmarks for aluminum (87 µg/L or 0.087 ppm) and barium (4 µg/L or 0.004 ppm)

(USEPA 2002). The two Double Spring Gap sites (DSTNs, DSNCs) exceeded the

manganese benchmarks (50 µg/L or 0.05 ppm), however, these benchmarks are not based

on toxic effects but rather are for minimizing laundry stains and objectionable drinking

water tastes (USEPA 2002). Zinc (120 µg/L or 0.12 ppm) and iron (1000 µg/L or 1 ppm)

benchmarks were not exceeded in any of the streams or springs.

The springs and streams had four different types of underlying geology -

Thunderhead Sandstone, Anakeesta Formation, Great Smoky Group, and Elkmont

Sandstone. Thunderhead Sandstone is characterized by partially metamorphosed sedimentary rock, which consists of thick beds of graded, coarse-grained feldsparthic

metasandstone and metaconglomerate, interbedded with dark graphitic metasiltstone and

slate (Southworth et al. 2005). The Anakeesta formation is primarily comprised of

graphitic and sulfidic slate, metasiltstone, and phyllite, and contains thin beds of

metasandstone and metagraywacke (Southworth et al. 2005). Elkmont Sandstone is a

feldspathic metasandstone interbedded with metasiltstone (Southworth et al 2005). The

Great Smoky Group has a thick bed of coarse metasedimental rocks of the

Neoproterozoic which includes sedimentary rocks, pebble conglomerate, coarse to fine

sandstone, and silty rocks from all three of the above formations (Anakeesta above,

Thunderhead Sandstone in the middle and Elkmont Sandstone below) (Southworth et al.

2005).

Discussion

Community composition

The diatom communities of the springs and streams in this study were generally

comprised of Eunotia taxa, reflecting the low pH and electrolyte nature of the aquatic

systems in the GSMNP, especially in the higher elevation areas. Many Eunotia species

36 are known acidophils and are frequently used as acid indicators when assessing and monitoring lotic and lentic habitats (Alles 1991). More specifically, our higher elevation sites were dominated by E. subarcuatoides, a Eunotia species that is commonly described from high elevation, electrolyte-poor springs and headwater streams with low pH (3.7-

5.2) (Cantonati et al. 2006). The consistent presence of E. exigua at our higher elevation sites is also congruent with the literature which typically reports this taxon from areas with low pH levels, including springs (DeNicola 2000, Cantonati et al. 2002). The presence of E. macroglossa at greater abundances at elevations between 1300 m and

1600 m suggests that this taxon may also be indicative of low pH conditions in streams, particularly in the mid and high elevation areas in the GSMNP. Additional sampling that includes stream water chemistry would help to better characterize the ecological niche of this new species.

The presence of teratological valves in notable abundances was unexpected, and it was not clear from our study why valve deformities were present at some of our sites and absent from others. Morphological aberrations in diatoms are typically reported from metal contaminated sites such as those associated with mine tailings (Cattaneo et al.

2004, Griffith et al. 2002). Our sites were not adjacent to or connected to mine tailings, and thus, the presence of valve distortions was more likely the result of interactions between acid precipitation and local geology which could result in the release of metals such as Al (Cantonati et al. 2006, Gensemer & Playle 1999). Furthermore, the consistent observations of valve malformations across years suggest that it was not a random observation and that natural and/or anthropogenic factors are influencing the diatom communities of these sites, especially at the high elevation springs.

Interactions between acid precipitation and bedrock geology may have been further amplified by the low pH present in the high elevation springs and headwater

streams of this study. The affects of pH are higher in systems with low buffering capacity

(Carpenter & Waite 2000), and the solubility and toxicity of metals such as Al also

increase with increased acidity (Gensemer & Playle 1999), especially in the GSMNP

where much of the bedrock has little to no acid-neutralizing capacity. The GSMNP

receives precipitation acidified from coal-fired plants and automobiles. High elevation

streams in GSMNP are 5-10X more acidic than in any of the monitored sites in North

America (National Park Service 2005). Acid precipitation impacts are especially relevant

at high altitude areas, such as the springs in this study, where greater cloud-water input

(i.e. of sulfur dioxide and nitrous oxides) contributes to the higher levels of atmospheric

aerosols relative to lower elevations (Nodvin et al. 1995; Shubzda et al. 1995). The

Anakeesta formation is a natural source of acidity and metals (Hammarstrom et al. 2003).

However, the Double Spring Gap, TN site, with no deformities, is in an area dominated

by Anakeesta formation, in contrast with the Double Spring Gap, NC site, with

deformities, which has bedrock only partially comprised of Anakeesta, making it difficult

to assess if there are localized exposures of this bedrock weathering to influence one side

of the ridge more than the other. Seal et al. (1999) found that two high elevation springs,

also along the Appalachian Trail in the GSMNP, were acidic and had notable levels of Al

from water interactions with Anakeesta Formation and Thunderhead Sandstone. A more

detailed examination of localized geology may be useful for teasing apart the differences

in diatom deformities from this study.

One study has suggested that E. subarcuatoides can tolerate low pH sites with

some metal enrichment (Hirst et al. 2002), and may explain the dominance of this species

at our highest elevation springs and headwater streams which have low pH and show

some enrichment in metals. The site with highest numbers of deformities also had the

highest measured values of Al and Ba in 2007, and high levels of Mn, and may indicate a

threshold level for the development of malformations. Valve length has been negatively

correlated with the presence of metals such as Al, Cd, Fe and Zn (Cattaneo et al. 2004,

Gensemer 1990), however, our study found no difference in mean valve length or the size

range between normal and abnormal E. subarcuatoides valves. Due to the limited water

chemistry collected in this study, more data is necessary before any clear linkages

between diatom deformities and metals can be unequivocally established. Additional

samples from soil and sediments at these sites would further help to determine possible

sources of metal release.

Spring communities tend to show little variation in community composition with

season (see studies in Cantonati et al. 2006) which may increase their usefulness in

biomonitoring, especially in a national biosphere reserve such as the GSMNP where

sampling efforts may be reduced by limited resources but interest in protection of ecological integrity is high. Because high elevation spring water chemistry is determined directly from groundwater and aquifers and indirectly from atmospheric influences (i.e. such as from airborne contaminants), the diatom abnormalities observed in this study may be indicative of environmental change or even pollution. The diatom assemblages of the high elevation areas in the GSMNP may show earlier signs of degradation or mitigation in response to changes in acid precipitation relative to diatom communities of

lower elevations that are less exposed and more variable. Monitoring these high

elevations may be critical to detecting some of the environmental influences that both

shape and threaten the biodiversity in the park, and in other acid influenced areas.

Conclusions

The diatom assemblages of the high elevation springs and streams in the GSMNP showed very characteristic communities of Eunotia that were indicative of the low pH low electrolyte nature of these sites. Additionally, a new Eunotia species, E. macroglossa, was discovered and described. It may be a useful indicator of low pH, however, a more thorough investigation of these high elevation areas is necessary to help to clarify its bioindicator potential. The diatom deformities observed in the absence of point source, anthropogenically-driven metal contamination, warrant further study as they suggest that the potential impacts of acid precipitation with local geologies and soils may have negative consequences for aquatic communities. This is particularly relevant with the increased acidification of aquatic areas in poorly buffered mountain landscapes in

North America.

Chapter III

Ecology and distribution patterns of Eunotia Ehrenberg

in the Great Smoky Mountains National Park.

Introduction

The ability of diatoms to persist and proliferate in a particular habitat is often regulated by their preference for or their ability to tolerate a specific set of environmental conditions. As a result, the structure of diatom communities or the presence / absence of particular species can be used as a surrogate measure of the state of the ecosystem.

Species of the acidophilic genus, Eunotia, have varying sensitivity levels to environmental factors, especially to pH (Krammer and Lange-Bertalot 1991a). Previous research from headwater streams in Europe has identified four ecological Eunotia- complexes whose optimal abundance varies with respect to differences in pH levels and buffering capacities in environments with natural and anthropogenic acid sources (Alles et al. 1991). With additional information on the species-specific sensitivities, Eunotia taxa will become increasingly more useful as a bioindicator of ecosystems threatened by acidification. These bioindicators will be especially useful in parts of North America, such as Eastern USA and central-eastern Canada, which are extremely vulnerable to acidification because the geology and soil types reduce the natural buffering capacity of the aquatic and terrestrial ecosystems (Jeffries et al 2003).

Environmental factors, such as bedrock geology, pH and water chemistry, are considered to be some of the dominant regulators of the structure and distribution patterns of benthic algal assemblages at local scales, on up to continental and larger biogeographical scales (Biggs 1990; Pan et al. 1999; Leland & Porter 2000; Potapova & 41

Charles 2002; Soininen 2004). The distribution patterns and the ecology of Eunotia species may largely be determined by the interactions between varied geology and bedrock chemistry combined with human-induced pressures (e.g. increasing catchments of atmospheric aerosols originating from sources of fossil fuel combustion emissions).

However, bedrock geology alone, can influence the pH level, the buffering capacity, and the ion chemistry of an aquatic ecosystem, and thus regulate benthic algal community structure (Biggs1990, Leland & Porter 2000). In addition, natural influences of geology and bedrock chemistry on algal assemblages can be overridden or exacerbated by land- use (Leland & Porter 2000) from metals, for example, such as aluminum which affects the buffering capacity of an aquatic system (Genter & Amyot 1994, Leland 1995). The effects of acidic inputs may be higher in systems with low buffering capacity (Carpenter

& Waite 2000) and lower in systems with high mineral content (Leland and Porter 2000).

Eunotia are particularly abundant in the Great Smoky Mountains National Park

(GSMNP) (see Chapter IV). There are a diverse suite of habitats in the park, due to the varied geology, geochemistry, and range of altitude, combined influences of acid precipitation, caused, largely, by emissions from coal-fired plants and automobiles. These diverse habitats provide a unique environment to explore the ecology and distribution patterns of Eunotia. The park also has some of the highest levels of acid deposition (wet and dry) of any monitored sites in North America (Johnson & Lindberg 1992). The nitric oxides in acid precipitation, are hydrolyzed or oxidized into nitric acid, and with such high deposition levels, the soils cannot process or retain the nitrates which go directly into streams increasing their acidity (Johnson & Lindberg 1992). High acid levels cause aluminum release from the soil and bedrock, as well as depletion of soil calcium and 42

magnesium, resulting in toxicity and lowered primary production for both aquatic and

terrestrial ecosystems in the park (Johnson & Lindberg 1992, Planas 1996).

Understanding the ecology of Eunotia in the GSMNP will not only be useful in efforts to

protect the biodiversity of the park, but also for monitoring and assessment of other

aquatic ecosystems around North America that are threatened by acidification

The objective of this study was to examine the ecology and distribution patterns of Eunotia from streams throughout the GSMNP in order to identify or provide more

ecological information on species and species complexes with relation to pH level, to

increase the utility of Eunotia species as bioindicators. Specifically, the relative

abundance of Eunotia species was examined in relation to physical and chemical characteristics such as elevation, watershed, geology, pH, and water chemistry data, including nutrients and metals such as nitrate, aluminum, calcium, magnesium and sulfate. It was expected that Eunotia abundance and species distribution patterns would reflect variations in geology and altitude. Sites with acid leaching geological formations and higher altitude sites that are exposed to greater amounts of atmospheric acid inputs were expected to have the highest relative abundance of Eunotia species.

Material and Methods

Sample Collection and Water Chemistry

See Chapter IV site description, for a description of the GSMNP. Epiphytic

Eunotia populations on bryophytes were collected from 131 geo-referenced sites from streams throughout the GSMNP, from May to August 2005 (Garmin GPS Unit,

Coordinate datum NAD27, UTM zone 17S). A representative selection of bryophytes was placed in a bag and shaken to dislodge associated diatoms before removal of the 43 bryophytes. Sites covered 14 geology types (Table1), acidic to slightly alkaline waters, and included a smaller subset of sites that coordinated with current (39 sites, 2005 data) and recent (37 sites) National Park Service (NPS) water quality monitoring sites. Recent

NPS sites had water quality data primarily for 2004 and also 2003 (but not for 2005).

Although this was not ideal, it allowed data to be explored from a broader scale perspective, where, for example, more acidic areas could be examined in relation to more alkaline areas. NPS water quality data included: lab analyzed pH, chloride, nitrate, sulfate, sodium, potassium, magnesium, calcium, aluminum, copper, iron, and zinc. From these water chemistry measurements, July values were selected to best approximate summer water chemistry levels when samples had been collected. For a small subset of sites, summer estimates were not available, so May or July values were selected. The pH of each site at the time of collection was estimated using EMD colorphast pH-indicator strips (“field pH”; range: 2.5 to 10 pH units; sensitivity of 0.2 to 0.3 pH units). The pH strips were placed in the stream for a minimum of 10 min. to provide sufficient time for the indicator color to respond. Field pH values were consistently lower than lab pH estimates (on average by 1.5 pH unit, range 0.14 to 2.3) While the field pH was not an ideal method for measuring pH in streams with low conductivity it did provide a general estimate of relative pH differences between sites.

Sample Analysis

Samples were boiled in nitric acid to remove organic materials (Round, Crawford, and Mann, 1990) and strewn diatom slides were made using Naphrax® mounting medium, for examination using light microscopy (LM). Eunotia taxa were identified to species, and their relative abundance was determined where all other diatom taxa were 44 grouped together in one category. For each sample, an Olympus BX51 Photomicroscope with high resolution Nomarski DIC optics was used to count at least 600 cleared diatom valves at 600X magnification, with additional empty magnification to 1200X and a 2X optavar. Images were recorded with a monochromatic camera (Spot™) attached to the microscope and a computer. Eunotia were identified to species level using standard references which consisted primarily of: Alles et al (1991), Krammer & Lange-Bertalot,

1991, 2000, 2004; Lange-Bertalot, 1993; Reichardt, 1995; Lange-Bertalot & Metzeltin,

1996; Lange-Bertalot et al., 1996; Metzeltin & Witkowski 1996; Metzeltin & Lange-

Bertalot, 1998, 2003; Lange-Bertalot & Genkal, 1999; Rumrich et al. 2000; Lange-

Bertalot et al., 2003; Werum & Lange Bertalot 2004; Siver et al., 2005; and Metzeltin et al., 2005. To aid with identification, we examined ultrastructural features of Eunotia frustules using a high resolution Hitachi S2700 scanning electron microscopy (SEM).

Preparation of these specimens included mounted them on aluminum stubs and sputter coated them with 10 nm of AuPd (Postek et al., 1980).

Statistics

For diatom community analysis, data were square-root transformed to down weight the effects of dominant taxa. Clustering of taxa was examined using non-metric multidimensional scaling (NMDS) of Bray Curtis similarities on a reduced taxa list

(Primer 5 version 5.2.9; Primer–E Ltd., 2002). To ensure sufficient data for ordination, taxa were excluded if they were only present at one or two sites. Clustering patterns were examined for all NMDS plots by factoring the data by watershed, disturbance level, geology, elevation class, pH, and Eunotia species. An analysis of similarity test

(ANOSIM) was run to test the significance of groupings based on factors. Disturbance 45

categories include: 1 – Settlement area, 2 – Light cut, 3 – Selective cut, 4 – Undisturbed,

5 – Heavy cut, 6 –No data. NPS elevation class categories were set at intervals of ~150 m

(500 ft), where elevation ranged from 262 (elevation class 2) to 1777 m (elevation class

11). Table 1 shows the geology and watershed factors.

A principle component analysis (PCA) was run with log-transformed water chemistry data (with the exception of pH). Subsequently, Pearson correlations were performed (SPSS,Version 16.0; SPSS Inc, 2007) to compare species abundance data with the first axis of the two-dimensional PCA output. The BIO-ENV program in the statistical program, Primer, was then run to determine the biotic variables which best contributed to the distribution pattern of the sites.

Relative abundance data was explored using a variety of data subsets. Since water chemistry was not available for all sites, analyses were done on all sites without including water chemistry and then on a subset of sites including water chemistry data. Similarly, analyses were run including and excluding spring sites (previously explored in Furey et al. 2008, in review), to determine if Eunotia communities in high elevation spring sites were similar to other stream sites. First, the relative abundance of all diatoms at all stream sites (All-Taxa: N = 131) was examined, followed by analysis of sites which included

Eunotia for those sites (All-Eunotia: N = 123). Eight sites were removed from the All-

Eunotia analysis because no Eunotia were present. Similarly, all stream sites were examined with the six spring sites included (All-Taxa-S; All-Eunotia-S). Next, the relative abundance was examined for all diatoms (NPS-All-Taxa: N = 76) and Eunotia- only (NPS-Eunotia-Only: N = 73) for all stream and spring sites with all water chemistry data, and 2005 only water chemistry data (NPS-2005-Eunotia-only, N = 39). 46

Results

Overall, sites in higher elevation areas had a higher relative abundance of Eunotia

taxa (lower abundance of other diatom species) than those in lower elevation areas

(Figure 1. Pearson: r = -0.661(135), p(1-tailed) < 0.000). In concordance with a related study that focused specifically on the high elevation springs in the GSMNP (Chapter II), the diatom communities from the springs were significantly different from stream diatom assemblages (Fig. 1; ANOSIM, All-Taxa-S: R = 0.845; All-Eunotia-S, R = 0.533; p<0.001). The springs sites were primarily dominated by E. subarcuatoides Alles, Nörpel et Lange-Bert, and secondarily by E. macroglossa sp. nov. Furey, Lowe, et Johansen

(Chapter II). As these spring algal assemblages have already been explored, the remainder of the analyses will focus on the streams. The analyses of the different stream groups had similar results and thus, figures are presented from those that show the clustering most clearly.

As expected, a higher relative abundance of Eunotia taxa was also correlated with lower field-pH levels, both when springs sites were and were not included in the analysis

(Pearson: All-Eunotia-S r = 0.323(135) p (1-tailed) < 0.000, All-Eunotia r=0.204(128), p (1- tailed) < 0.01). Lower field-pH levels were correlated with higher elevation (Pearson; All-

Eunotia-S: r = -0.461(135), All-Eunotia: r = -0.429, p(1-tailed) < 0.000). The higher elevation sites (~>1050 m) tended to cluster separately, even when the high elevation spring sites were removed from the analyses (Fig. 2). These sites were dominated by E. subarcuatoides, similar to the springs, and also E. incisa Gregory, E. exigua (Brébisson)

Rabenhorst, E. tenella (Grunow) Hustedt (Fig. 2), and secondarily by E. muscicola var. 47

tridentula (Grunow) Nörpel et Lange-Bertalot, and E. varioundulata Nörpel-Schempp &

Lange-Bertalot.

Disturbance and geology were significant determinants of the observed pattern of

sites on the NMDS plot (Fig. 3, ANOSIM, All-Eunotia: R=0.192disturbance, R=0.234geology, p < 0.001). The diatom assemblages of undisturbed sites were different from those of other sites (Fig. 3A). Similarly, the communities from Thunderhead Sandstone and

Anakeesta geologies were different from those from many of the other geologies (Fig.

3B). The lower elevation, diatom assemblages of the Thunderhead Sandstone group

(mid-area in Fig. 3B) were dominated by species in the E. rhomboidea complex. The

Simper analysis confirmed that the high elevation Eunotia species and E. rhomboidea were primarily responsible for these differences (Table 2).

The diatom communities from streams with Anakeesta geology were primarily sites in the West Prong of the Little Pigeon River watershed (WP) and were dominated by

E. subarcuatoides and E. incisa. This grouping was easier to see when the smaller subset of sites for 2005 was examined. The WP watershed in particular clusters separate when all diatoms are considered together due to the high abundance of Eunotia taxa relative to the other sites (Figs 4A and B). When examined with Eunotia taxa alone, the pattern is similar and with the exception of one lower elevation site and one mid-elevation site, the sites are where Anakeesta is located (Figs 4C and D).

The PCA, run on the water chemical variables, identified some outlier sites and the first two axes explained 64% of the variance. The first axis had similar magnitudes of positive loadings for nitrate, sulfate, aluminum, zinc and magnesium, with a slightly lower, negative loading for pH (Table 3). There was a slight trend towards higher 48

elevation sites (Fig. 5A) having higher levels of aluminum, sulfates and nitrates (Figs 5B,

C, D), however this was partially driven by the outlier sites. A closer examination of the

outlying sites (Fig. 5A), identifies a site near a road cut (F_0252-BF2) which had higher

levels of metals such as zinc, silica, manganese, aluminum, calcium, magnesium,

potassium, as well as nitrates and sulfates (Table 4). Another downstream site along that

same road way (F_0252-BF1) was similar, but with lower levels of aluminum and higher

levels of copper (Table 4). A site in Walker Camp Prong (F_0237-WC) similarly had

elevated levels of metals, but to a lesser degree (Table 4). A fourth site (F_0489-AB),

found on a high traffic road in the park (Cades Cove loop) in a more alkaline part of the

GSMNP, had a higher acid neutralizing capacity due to high levels of calcium and

magnesium (Table 4). The lower elevation, more alkaline site, had very low abundances

of Eunotia species and as a result, also tended to cluster separate under the NMDS

analyses (see Figs 2A and 4C). The remaining outlying sites were located within the

cluster of high elevation sites comprised primarily of E. subarcuatoides and E. incisa

(F_0237-WC), or in the middle cluster of sites where species in the E. rhomboidea

complex were present along with E. subarcuatoides, and E. incisa (F_0252-BF1; F_0252-

BF2). Site F_0252-BF2 did have slightly higher levels of E. nymanniana relative to other

stream sites.

When the Eunotia assemblages are looked at in relation to the environmental

variables the top five best results based on five variables all correlated around 44% and

included slight variations in contributing environmental variables (BIOENV: 44% 1:

NO3, Na, Ca, Si, Zn; 2: NO3, Na, Ca, Si, Zn, 3: NO3, Na, Ca, Fe, Si; 4: NO3, Na, Ca, Fe,

Zn; 5: NO3, Na, Mg, Ca, Si.). 49

Discussion

The distribution patterns of Eunotia in this study were similar to those previously reported in the literature where areas with lower pH supported a greater relative abundance of Eunotia relative to other diatom taxa (Krammer and Lange-Bertalot 1991;

DeNicola 2000) especially species around the E. exigua-complex (e.g. E. exigua, E.

tenella, E. muscicola var. tridentula, and E. varioundulata). Higher elevation areas in the

park also tended to have lower pH levels, and thus had a higher relative abundance of

Eunotia. The lower pH levels at high altitudes were likely from greater exposure to acid

atmospheric depositions relative to lower elevations in the park, i.e. from coal-fired plants

or automobiles (Nodvin et al. 1995, Shubzda et al. 1995). Nitrates were identified as one

of the environmental variables that explained the Eunotia species distribution patterns,

and it is indicative of the influence that acid precipitation, which is high in nitrogen

oxides (NOx), has on the chemistry of the streams. These nitrates are unable to be

processed by the soils in the GSMNP and so 85% or more go directly into streams.

The Eunotia from high elevation areas were comprised of species in the E. exigua

complex. Among the Eunotia taxa, E. exigua is typically reported from more acidic areas

(Denicola 2000), thus emphasizing the acidic nature of higher altitudes in the GSMNP.

Passy et al. (2006) found that E. exigua dominated acidic streams which experience

episodic acidification relative to chronically acidic streams where E. exigua was less

abundant. Streams in the GSMNP are chronically acidic, with low buffering capacity, but

also experience episodes of acidification from snow melt, for example, which may

seasonally shift the algal community compositions observed in this study to be even more

dominated by E. exigua. It would be useful to more closely explore the taxonomy in the 50

E. exigua complex to determine if different taxa have different preferences for acid dynamics in these high elevation areas where acid precipitation, snow melt, and geology are all influential on stream pH levels. This is especially relevant given that the E. exigua

– complex is in need of more taxonomic resolution (Round et al. 1990) and species are often just lumped within the general E. exigua complex. Observations from this study and

Chapter IV indicated that there are new E exigua-like species that need documentation and description (personal observation, see also Chapter IV).

Higher elevation areas (~>1050 m) also typically had high relative abundances of both E. subarcuatoides and E. incisa. While E. incisa was limited to elevations below

1605 m (Chapter II), the presence of the combination of these two taxa together may provide a useful indicator group for areas with lower pH. In addition to being common at higher altitudes, E. subarcuatoides and E. incisa also occurred together in greater relative abundance in the West Prong of Little Pigeon River in the WP watershed. The water chemistry of streams in this area of the park are influenced by the Anakeesta Formation which is known to lower pH levels through chemical and physical weathering. Often, E. exigua, E. muscicola var. tridentula, E. varioundulata and E. GSMNP SP 11 secondarily occurred with E. incisa and E. subarcuatoides although in lower abundances (personal observation). It is unclear whether, the presence of E. incisa and E. subarcuatoides together in higher abundance is indicative of natural influences on pH levels or if they are just general indicators of acidic environments.

With the exception of the Anakeesta Formation, the Thunderhead Sandstone group was the only geology that may have influenced the Eunotia distribution patterns.

However, part of the Thunderhead Sandstone group, along with the undisturbed sites, 51

primarily fell in the higher elevation areas so it is more likely that these characteristics

were not driving factors in determining the Eunotia community structure (Fig. 3A).

Although bedrock geology, for example, can influence the pH level, the buffering

capacity, and the ion chemistry of an aquatic ecosystem, and thus regulate benthic algal

community structure (Biggs1990, Leland and Porter 2000), factors such as acid

precipitation which more directly affects stream pH levels may be the primary

determinant of the type and abundance of Eunotia species in the park. The affects of pH

can be higher in systems with low buffering capacity (Carpenter and Waite 2000), as in

the GSMNP, and thus it is not surprising that pH and geology known to directly influence

pH levels were driving determinants of community structure in this study.

Mid-elevation areas of the park were dominated by species in the E. rhomboidea

complex. However the taxonomy of species around E. rhomboidea is in need of further

work, making it difficult to extract ecological information from these areas. E.

rhomboidea was more cosmopolitan in the streams in the GSMNP and may be more of a

general acid indicator in these streams. The outlying sites identified by the environmental

variables did not have vary in Eunotia species composition relative to those of sites that

clustered around them in the community composition analysis, with the exception of the

Abrams Creek site (Fig. 5, F_0489 – AB) which was comprised of very few Eunotia (see

Fig. 2A). This reinforces the trend that species variability is closely linked with

variability in pH.

Results from this study were consistent with the literature and did not identify any new indicator species or species-complexes with the exception of a possible relationship between E. incisa and E. rhomboidea which may be indicative of a combination of natural 52

and anthropogenic acid influences in streams in the GSMNP. The diatom assemblages in

the high elevation areas in the park consistently reflected their acidic nature and suggest that acid precipitation in the park is influencing the structure of stream biological communities.

CHAPTER IV

Eunotia Ehrenberg (Bacillariophyta) of

the Great Smoky Mountains National Park, U. S. A.

Introduction

The diatom genus Eunotia Ehrenberg (Family Eunotiaceae) is unusual among the raphid diatoms in that it has very short raphe slits at each pole that lie to one side of the ventral mantle (Mann 1984; Round et al. 1990). The valves are asymmetric to the apical axis, with convex dorsal margins and straight to concave ventral margins. Striae extending across the valve face contain poroids that generally lack hymens and other occlusions (Round et al 1990), although specimens with completely occluded pores have been observed in sub-aerial habitats in the Great Smoky Mountains National Park

(GSMNP). Rimoportulae (labiate processes), absent from other raphid diatoms, are present in the Eunotiaceae, including Eunotia where usually one rimoportula lies at an apex of each valve (Round et al. 1990; Krammer and Lange-Bertalot 1991 i.e. see Taf.

139, Figs 4 and 7). Although, occasionally a rimoportula is absent. (see E. rhomboidea).

Eunotia species are diverse in acidic and dystrophic habitats (Patrick 1977). The taxonomy of this genus can be difficult due to the apparent phenotypic plasticity within species; however, the details of the ultrastructural features (i.e. raphe system) and their habitat preferences (i.e. pH) can be useful in distinguishing species and species complexes (Alles et al. 1991; Kociolek 2000). More research is required at the species level (Round et al. 1990), especially for the complex of E. exigua-like species (typically found in highly acidic environments) (Krammer and Lange-Bertalot 1991a; DeNicola 54

2000). Taxonomic keys and documentation of morphological forms specific to North

America are limited, which further highlights the need for research on Eunotia.

Previous research has found that Eunotia is taxonomically rich in the Great

Smoky Mountains National Park (GSMNP), U. S. A. (Camburn & Lowe, 1978; Lowe &

Kociolek, 1984; Keithan, & Lowe, 1985, Johansen, et al. 2004). Eunotia species flourish

in the park because aquatic and subaerial habitats experience rainfall with a pH of 4.5,

which is 5–10X more acidic than other monitored areas in North America where pH

ranges from 5.0 to 5.6 (National Park Conservation Alliance 2004, National Park Service

2005). This high level of Eunotia biodiversity is, in part, due to variations in pH from

acid precipitation and other anthropogenic influences, combined with naturally acidic

areas associated with a complex bedrock geology and chemistry and naturally occurring denitrification (Southworth et al. 2004). Acidity levels also vary across the range of altitudes from ~245 m (800 ft) to~2000m (6650 ft), where higher elevations areas are more exposed to acid precipitation (Weathers et al. 1986, Nodvin et al 1995, Shubzda

1995). This diverse suite of environmental conditions, including varied acidic habitats, makes the park an ideal location for studying Eunotia.

In this chapter, the taxonomy and biodiversity of Eunotia from the GSMNP were explored. Included is an image rich documentation of their morphology, including ultrastructure, and their general ecology, including distribution patterns. Where possible, size ranges, morphological variability and the external and internal ultrastructure are provided. New species and new records for the park and/or North America are noted, and taxonomic challenges are discussed. 55

Material and Methods

Site Description

Great Smoky Mountains National Park (GSMNP), U.S.A. comprises more than

half a million acres (> 2000 km2) in North Carolina and Tennessee, and is the largest contiguous preserve east of the Rocky Mountains. The GSMNP is considered a hot spot of biological diversity and serves as refuge for one of the richest and most diverse collections of plants and animals in the temperate world. This species richness and habitat diversity has led to the park’s designation as an International Biosphere Reserve. High algal biodiversity in the GSMNP is likely due to the large range in environmental conditions, altitudes (including the Appalachian Mountains), geology and bedrock, combined with the area having remained un-glaciated for several millions of years.

North-south orientation of the mountains, large tracts of old-growth and contiguous forests, and protection as a national park have also contributed to a rich and abundant fauna and flora (including the diatom flora). Since the late 1990’s, the National Park

Service and Discover Life In America (DLIA) have taken on the ambitious task of completing an All Taxa Biodiversity Inventory (ATBI) in the GSMNP. Researchers have been working to discover and document all organisms within the boundaries of the park, including microscopic algae like Eunotia.

Collection and Analysis Methods

Eunotia populations were collected from geo-referenced sites from streams, wet walls, ponds, a lake, and bogs throughout the GSMNP from 2004 to 2005 ( Garmin GPS

Unit, Coordinate datum NAD27, UTM zone 17S). Diatoms were collected primarily from bryophyte substrates by placing composite samples of representative bryophytes in whirl- 56 pak bags and shaking to remove the associated diatom flora. The pH of each site at the time of collection was estimated using EMD colorphast pH-indicator strips (field pH; range: 2.5 to 10 pH units; sensitivity of 0.2 to 0.3 pH units). The pH strips were placed in the stream or on a damp area of the wet wall for a minimum of 10 min. to provide sufficient time for the indicator color to respond. Because the GSMNP has streams with low conductivity, this method only provided a general estimate of pH; the field pH values using the pH strips were consistently lower than laboratory estimates of pH (on average by 1 pH unit, range 0.5 to 1.6; See results from Chapter 3)

Samples were cleaned in nitric acid to remove organic materials (Round,

Crawford & Mann, 1990) and strewn diatom slides were made using Naphrax® mounting medium. Permanent diatom slides were made for future reference at Bowling

Green State University, Ohio. Diatoms were analyzed using an Olympus BX51

Photomicroscope with high resolution Nomarski DIC optics. Images were captured with a monochromatic camera (Spot™) attached to the microscope and a computer. Eunotia were identified to species using standard references which consisted primarily of: Alles et al (1991), Krammer & Lange-Bertalot, 1991, 2000; Lange-Bertalot, 1993; Reichardt,

1995; Lange-Bertalot & Metzeltin, 1996; Lange-Bertalot et al., 1996; Metzeltin &

Witkowski 1996; Metzeltin & Lange-Bertalot, 1998, 2003; Lange-Bertalot & Genkal,

1999; Rumrich et al. 2000; Lange-Bertalot et al., 2003; Werum & Lange Bertalot 2004;

Siver et al., 2005; and Metzeltin et al., 2005. Eunotia samples were also mounted on aluminum stubs and sputter coated with 10 nm of AuPd and then examined under a high resolution Hitachi S2700 scanning electron microscope (SEM), with digital image recording ability (Postek et al., 1980). The inventory of Eunotia species from the 57

GSMNP were added to the compilation of algal taxa from the park thus far (Johansen et

al. 2007).

Results and Discussion

The GSMNP was exceptionally rich in Eunotia species. Over 60 sub-generic taxa

of Eunotia were identified in the park from this study, some of which are likely new species. This inventory included many of the common Eunotia taxa in the park.

Additional species were still being found quite regularly during slide scanning, and more research is required to fully document the rare species of Eunotia in the park. The waterfalls and bogs were some of the richest habitats in the park, and along with wet walls, often contained Eunotia species that were new to science or new to the park.

Over 35 new park records for the GSMNP including: E. boreotenuis, E. braendlei, E. cf. macaronesica, E. diodonopsis, E. implicata, E. macroglossa, E. neofallax, E. nymanniana, E. parallela, E. pectinalis var. ventralis, E. rabenhorstii, E. trinacria, E. undulata and 24 species (GSMNP SP 1 through GSMNP SP 24) that have not been able to be identified and are probably new to science. This inventory included new species such as E. macroglossa and species new to North American records such as

E. braendlei and E. diodonopsis. E. zazumensis and E. monodon were observed in the park, but images have not been included in the plates.

Species that were previously reported from the GSMNP, but were not observed in this study or species for which an unknown form was previously observed, include: E. arcus, E. bidentula, E. curvata, E. elegans, E. fallax, E. hexaglyphus, E. indica, E. inflata, E. intermedia, E. lunaris, E. maior, E. meisteri var. bidens, E. musicola, E. pectinalis, E. serra, E. siolii, E. soleirolii, E. sudetica, E. suecica, E. cf. yanomami, and 58

E. cf. zygodon. Some of these records likely include either misidentified species or species that were previously recorded under a ‘catch-all’ taxon such as E. bilunaris or E. praerupta for which there is a need of further taxonomic resolution. It is speculated that the E. nymanniana reported from previous studies was actually E. compacta because of confusion around the name (see discussion under E. nymanniana and E. compacta) and the timing of taxonomic key publication, and the later name clarification (Krammer &

Lange-Bertalot 2004). Taxa that have different variety level forms such as E. muscicola,

E. fallax and E. pectinalis likely make reference to forms identified at a higher taxonomical level in this study such as E. muscicola var. tridentula, or E. undulata

(previously E. pectinalis var. undulata), but this is difficult to confirm. Eunotia c.f. zygodon and E. c.f. yanomami may have been referring to the shorter valves of E. jemtlandica. Eunotia rhomboidea from previous studies is likely included under the

GSMNP SP 23 from this study, especially given how common GSMNP SP 23 valves were throughout the GSMNP. However, this is another Eunotia group that needs closer examination in the GSMNP in order to work out the taxonomic details, especially with regard to the presence / absence of the rimoportula. In previous studies, E. pirla was listed as common in the GSMNP but it was rare in this study.

As an acidophilic group of diatoms, Eunotia species are able to thrive in the broad spectrum of acidic habitats available in the park. The high biodiversity reflects the varied geology and bedrock chemistries present over a > 1700 m range altitude. For example, the Anakeesta formation is known to decrease pH levels through chemical and physical weathering of the bedrock. These natural variations in acidity are combined with 59

influences from anthropogenic sources of acidity including sulfur and nitrous oxides in

acid precipitation.

The GSMNP may have one of the highest recorded levels of Eunotia diversity in

North America. The taxonomy of this group is challenging and this work has contributed an image-rich documentation of some of the Eunotia species and species morphological variability in North America. Additionally, this research has identified new species, new park records, and new records for North America that will contribute to our understanding of Eunotia taxonomy and ecology.

Taxonomic Descriptions

Abbreviations:

GSMNP = Great Smoky Mountains National Park

H = helictoglossa

LM = light microscope

NC = North Carolina

R = rimportula

RP = external opening (pore) of the rimoportula

SEM = scanning electron microscope

TN = Tennessee

W = watershed

Watershed codes are listed in Table 1.

List of Taxa

1. E. bidens Ehrenberg ...... 92

2. E. bigibba Kützing...... 94

3. E. billii Lowe & Kociolek ...... 105

4. E. bilunaris (Ehrenberg) Mills ...... 64

5. E. boreotenuis Nörpel-Schempp & Lange-Bertalot ...... 67

6. E. braendlei Lange-Bertalot & Werum...... 86

7. E. compacta Hustedt ...... 76

8. E. curtagrunowii Nörpel-Schempp & Lange-Bertalot ...... 89

9. E. diodonopsis Metzeltin & Lange-Bertalot...... 92

10. E. exigua (Brébisson) Rabenhorst...... 74

11. E. flexuosa (Brébisson) Kützing ...... 63

12. E. formica Ehrenberg ...... 84

13. E. glacialis Meister...... 66

14. E. implicata Nörpel-Schempp, Lange-Bertalot, & Alles ...... 98

15. E. incisa Gregory...... 104

16. E. jemtlandica (Fontell) Berg ...... 84

17. E. c f macaronesica Lange-Bertalot & Tagliaventi nov sp. prov ...... 91

18. E. macroglossa Furey, Lowe, Johansen ...... 83

19. E. microcephala Krasske...... 77

20. E. minor (Kützing) Grunow ...... 99

21. E. muscicola var. tridentula (Grunow) Nörpel et Lange-Bertalot ...... 77

22. E. naegelii Migula ...... 63 61

23. E. neofallax Nörpel-Schempp & Lange-Bertalot ...... 69

24. E. nymanniana Grunow ...... 81

25. E. paludosa Grunow ...... 80

26. E. parallela Ehrenberg ...... 65

27. E. pectinalis var. ventralis (Ehr ) Hustedt ...... 83

28. E. pirla Carter & Flower...... 100

29. E. rabenhorstii Cleve et Grunow ...... 90

30. E. septentrionalis Østrup ...... 97

31. E. subarcuatoides Alles, Nörpel & Lange-Bertalot ...... 70

32. E. tenella (Grunow) ...... 75

33. E. trinacria Krasske ...... 78

34. E. undulata Grunow...... 82

35. E. valida Hustedt ...... 65

36. E. varioundulata Nörpel-Schempp ...... 72

37. E. veneris (Kützing) De Toni ...... 100

38. E. sp. (GSMNP SP 1) ...... 66

39. E. sp. (GSMNP SP 2) ...... 68

40. E. sp. (GSMNP SP 3) ...... 69

41. E. sp. (GSMNP SP 4) ...... 70

42. E. sp. (GSMNP SP 5) ...... 71

43. E. sp. (GSMNP SP 6) ...... 72

44. E. sp. (GSMNP SP 7) ...... 75

45. E. sp. (GSMNP SP 8) ...... 87 62

46. E. sp. (GSMNP SP 9) ...... 90

47. E. sp. (GSMNP SP 10) ...... 91

48. E. sp. (GSMNP SP 11) ...... 93

49. E. sp. (GSMNP SP 12) ...... 96

50. E. sp. (GMSNP SP 13) ...... 99

51. E. sp. (GSMNP SP 14) ...... 101

52. E. sp. (GSMNP SP 15) ...... 101

53. E. sp. (GSMNP SP 16) ...... 101

54. E. sp. (GSMNP SP 17) ...... 79

55. E. sp. (GSMNP SP 18) ...... 79

56. E. sp. (GSMNP SP 19) ...... 80

57. E. sp. (GSMNP SP 20) ...... 85

58. E. sp. (GSMNP SP 21) ...... 96

59. E. sp. (GSMNP SP 22) ...... 98

60. E. sp. (GSMNP SP 23) ...... 102

61. E. sp. (GSMNP SP 24) ...... 103

Taxa Descriptions:

Taxa descriptions follow the order presented in the image plates, which are

grouped by similarity in morphology to make morphological comparisons easier.

E. naegelii Migula Plate 1: Figs 1 – 10; Plate 2: 5 – 7.

Similar specimens are shown in Lange-Bertalot & Metzeltin (1996, Taf. 9: Figs. 8

– 13) and Krammer & Lange-Bertalot (1991, Taf. 140: Figs 1 – 6). The GSMNP specimens were slightly more curved than those shown in Siver et al. (2005, Plate 29:

Figs 6 – 7). The occasional valve deformity was observed, typically in the form of a distended undulation on the ventral margin of the valve.

E. naegelii was found in high density, epiphytic on bryophyte substrates, in a sink hole near the Abrams Falls trailhead along Cades Cove Loop (AB watershed). It was also found epilithic in some puddles at the base of Meigs Falls in the EL watershed.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: E. naegelii 42 – 180 2.5 – 3.5 14 – 16 16 – 22 Siver et al. (2005) 60 – 105 2 – 2.5 16 – 20 Patrick & Reimer (1966) 14 – 130 1.5 – 3.5 14 – 20

E. flexuosa (Brébisson) Kützing Plate 2: Fig. 1.

Similar images are found in Lange-Bertalot & Metzeltin (1996, Taf. 10: Figs 1 –

4), and Siver et al. (2005, Plate 29: Figs 1 – 2). The valves from the GSMNP were only

slightly widened at the end, especially compared with some E. flexuosa valves where the

apices are more broadened relative to the rest of the valve (i.e. Krammer & Lange-

Bertalot, Taf. 140: Figs 10, 12, 15 – 17). 64

E. flexuosa was found in epidendric samples from the mouth of Abrams Creek in

the AB watershed. The field pH estimate was 5.7.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study 138 – 156 4.8 – 5 12 – 14 18

Krammer & Lange- (50) 90 – 300 2 – 7 (9) 11 – 20 Bertalot (1991)

Eunotia bilunaris (Ehrenberg) Mills Plate 2: Figs 2 – 4.

Linear valves, where the ends become slightly narrowed, generally fall under the

E. bilunaris complex and more research needs to be done to identify features that can be

used to distinguish different E. bilunaris varieties. Historical images of E. bilunaris do not clearly show the raphe ends, further making it difficult to separate different morphologies or species. The E. bilunaris from the GSMNP were not as arched as the majority of illustrated specimens. More images of the E. bilunaris from the GSMNP would better document the range in size and morphology, especially relative valves in the

E. flexuosa complex.

E. bilunaris was found epiphytic on bryophytes from a waterfall in White Oak

Sink and epilithic at Meigs Falls in the EL watershed.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: 96 – 132 4.2 – 4.5 13 16 – 18 E. bilunaris E. bilunaris in Krammer 10 – 150 (205) 1.9 – 6 (9) 11 – 28 & Lange-Bertalot (1991)

Eunotia parallela Ehrenberg Plate 2: Figs 8 – 10.

These valves were similar in morphology to both E. parallela and the longer valves of E. faba Ehrenberg. None of the typical bean shaped cells of E. faba were

observed in any of the samples. However these valves were not common in the samples

from the GSMNP so it would be helpful to document the complete size range for the

taxon. The combination of cell width and striae density did not fit for the previously

reported varieties of E. parallela, so this may be a new variety of E. parallela. The striae

became strongly radiate around the end of valves.

E. parallela was found in low abundance, epiphytic on bryophyte substrates in

streams in the FO, MP, and EP watersheds. Field pH estimates ranged from 4.5 – 5.

Length End striae / Reference Width µm Striae / 10 µm µm 10 µm This study 52 – 72 5 – 6.7 13 18 – 22 E. parallela in Krammer & 30 – 100 5 – 15 8 – 16 Lange-Bertalot (1991) E. parallela var. parallela 7 – 15 11 – 16 E. parallela var. angusta 5 – 8 8 – 11 (now E. angusta Grunow) E. faba in Krammer & 16 – 60 5 – 9 (10) 13 – 15 (20) Lange-Bertalot (1991)

Eunotia valida Hustedt Plate 3: Figs 1 – 9.

Similar images are shown in Lange-Bertalot & Metzeltin (1996, Taf. 13: Figs 3 –

7 – Eunotia glacialis – valida form), Werum & Lange-Bertalot (2004, Plate 9: Figs 9 –

11) and Metzeltin et al. (2005, Plate 19: Figs 1 – 7).

E. valida was found epiphytic on bryophytes and in mucilage on wet walls throughout the GSMNP, including wet walls found along the Alum Cave Trail (WP watershed), Little River Road (EP watershed), Grassy Branch (OW watershed), Maddron 66

Bald Tail (IC watershed), Anthony Creek (AB watershed), and Enloe Creek (RV

watershed). Field pH estimates ranged from 4.2 – 4.9.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: E. valida 30 – 57 3 – 4 12 – 14 17 – 20 E. glacialis in Krammer (15) 30 ca. 200 3 – 7.5 (10) 9 – 15 & Lange-Bertalot (1991)

E. glacialis Meister Plate 3: Figs 16 – 18.

When compared with E. valida, the E. glacialis valves were wider and had less of a slope or shoulder along the dorsal margin nearing the valve ends.

E. glacialis was found in low abundance epiphytic on bryophytes in streams throughout the GSMNP, including from the FO and NO watersheds.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: 38 – 42 4 – 4.5 13 – 15 18 E. glacialis This study: 30 – 57 3 – 4 12 – 14 17 – 20 E. valida Hustedt

E. glacialis in Krammer & (15) 30 ca. 200 3 – 7.5 (10) 9 – 15 Lange-Bertalot (1991)

Eunotia sp. (GSMNP SP 1) Plate 3: Figs 14 – 21.

It was not clear if these specimens were a separate taxon, or the smaller valves of

E. boreotenuis Nörpel-Schempp & Lange-Bertalot. The valves were fairly common in

Andrews Bald Bog where longer E. boreotenuis were not evident. However, the ecological conditions of the bog could influence the size of the cells. More bog samples

should be explored for further comparison with other E. boreotenuis populations. The 67

valves are somewhat similar in appearance to the lower size range of Eunotia schwabei

Krasske, but the valve measurements and striae density did not fit.

Specimens were found epiphytic on bryophytes in Andrews Bald Bog. A few

valves were also found in Caldwell Fork (CT watershed) and Forney Creek (FO

watershed).

End striae / Reference Length µm Width µm Striae / 10 µm 10 µm This study: GSMNP SP 1 8.5 – 16 3.4 – 3.7 18 – 20 18 – 20

E. schwabei Krasske 13 – 19 4 – 5 12 – 14

Eunotia boreotenuis Nörpel-Schempp & Lange-Bertalot Plate 3: 21 – 37.

The curve of the raphe onto the valve face and slight curve of the valve suggest that the specimens may belong in the E. bilunaris Ehrenberg (Mills) complex. They were fairly similar in morphology to Figs 8 – 12 in Krammer & Lange – Bertalot (1991, Taf.

137, Eunotia bilunaris var. bilunaris sensu lato), including the occasional bifurcation of the striae along the dorsal margin of the valve face. However, the combination of valve length and striae measurements did not fit well with the E. bilunaris variety descriptions.

Additionally, the valve apices become slightly narrowed in our specimens and thus better fit with the Eunotia boreotenuis Nörpel–Schempp & Lange-Bertalot (Lange-Bertalot &

Metzeltin 1996, Taf. 9: Figs 21 – 25)

These specimens were common in low abundance on bryophyte substrates in first through fourth order streams throughout the GSMNP, including in the AB, BG, CT, EP,

HS, HZ, ML, MP, NO, OW, SR, TW, WP watersheds. Field pH estimates ranged from

4.2 – 6.1. 68

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: E. boreotenuis 14 – 40 3 – 4 14 – 20 16 – 20 Figs 21 – 31 This study: E. boreotenuis 18.5 – 33 3 – 3.8 15 – 18 18 – 22 Figs 32 – 37 E. boreotenuis in Lange- 20 – 40 3.5 – 4 15 – 18 Bertalot & Metzeltin (1996) E. bilunaris in Krammer & 10 – 150 (9) 11 – 1.9 – 6 Lange-Bertalot (1991): (205) 28 E. bilunaris var. mucophila Up to 70 1.9 – 2.7 (3) 20 – 28 E. bilunaris var. linearis 44 – 205 3.5 – 5.5 9 – 12

Eunotia sp. (GSMNP SP 2) Plate 4: Figs 1 – 9.

These valves were similar to both E. fallax A. Cleve and E. valida Hustedt. The specimens had bold and coarser striae, similar to E. fallax valves. However, their overall valve morphology (i.e. more arched, less constriction or pinching toward the ends) did not conform to those of E. fallax (i.e. Krammer & Lange-Bertalot, 1991, Taf. 150: Figs

10 – 15, Figs 16 – 24). The GSMNP specimens had rounder ends that did not turn up as much as they do in E. fallax. In addition the striae were frequently noticeably slanted, usually only on one side of the valve rather than the striae radiating towards both apices.

The shape of the terminal raphe ends, up on the valve face, appeared similar to E. valida under the light microscope. SEM research would be helpful in determining the overall structure of this taxon for comparison.

E. sp. (GSMNP SP 2) was found epiphytic on bryophytes or associated with algal slime on wet walls along Heintooga Ridge Road, Snake Den Ridge Trail (IC), Enloe

Creek Trail (RV), and Little River Road (EP) and on epilithon at the edge of Meigs Falls

(EP) and Hen Wallow Falls (CB). 69

Length Width Striae / End striae / Reference µm µm 10 µm 10 µm This study: GSMNP SP 2 24 – 62 2.7 – 4.4 11 – 14 16 – 18 E. fallax in Krammer & Lange- 12 – 55 2 – 5 (9?) 12 – 15 19 Bertalot (1991) (75) This study: E. valida Hustedt 30 – 57 3 – 4 12 – 14 17 – 20

Eunotia sp. (GSMNP SP 3) Plate 4: Figs 10 – 11.

This taxon was found epiphytic on bryophytes in a soil seep by the 20-Mile Creek

Trail in the TW watershed. It was unique in having notable slanted striae across the length of the valve face and in girdle view. More LM and SEM research needs to be done to determine the size range for this species and the morphology of the terminal raphe. It had some similarities in morphology to E. valida Hustedt and E. sp. (GSMNP SP 2)

found in the GSMNP. E. sp. (GSMNP SP 2) had slanted striae, but they were not present

across the length of the valve as they are with this taxon.

Length Width Striae / End striae / Reference µm µm 10 µm 10 µm This study: GSMNP SP 3 50-53 3.5 14 18 This study: GSMNP SP 2 42 – 62 3.5 – 4.4 11 – 14 16 – 18 This study: 30 – 57 3 – 4 12 – 14 17 – 20 E. valida Hustedt

Eunotia neofallax Nörpel-Schempp & Lange-Bertalot

Plate 4: Figs 12 – 17; SEM: Plate 5: Figs. 1– 4.

The striae of E. neofallax were often unevenly distributed. Similar images are found in Lange-Bertalot et al. (1996, Plate 58: Figs 7 – 29).

E. neofallax was rare in samples from sites in the GSMNP. It was found in epilithon from Hen Wallow Falls (CB watershed), and on bryophyte substrates in Meigs 70

Creek of Little River (EP watershed), Middle Prong of Little River (ML watershed), and

Caldwell Fork (CT watershed). The pH from the creek and river sites ranged from 4.8 –

4.9.

Width Striae / End striae / Reference Length µm µm 10 µm 10 µm This study: E. neofallax 15.5 – 40.5 2.4 – 3.2 10 – 13 10 – 18 E. neofallax in 11 – 45 1.2 – 4.2 9 – 14 ~17 Werum & Lange-Bertalot (2004)

Eunotia subarcuatoides Alles, Nörpel & Lange-Bertalot

Plate 4: Figs. 18 – 31; SEM. Plates 5: Figs 5 – 6.

Similar images are found in Alles et al. (1991, Taf. IV: Figs 1 – 36) and Lange-

Bertalot et al. (1996, Taf. 60: Figs 14 – 40).

E. subarcuatoides was common in high elevation springs and streams in the

GSMNP. Deformities were often present in relative abundances up to 30 % in the high

elevation springs. See Furey et al., 2008 for a discussion about the deformities and LM

and SEM images of the malformations.

Width Striae / End striae / Reference Length µm µm 10 µm 10 µm This study: E. subarcuatoides 6.0 – 46.6 3.1 – 4.9 17 – 21 18 – 24 Krammer & Lange-Bertalot 6.0 – 35.0 somewhat more 2.7 – 4.5 18 – 23 (1991) (40) closely together

Eunotia sp. (GSMNP SP 4) Plate 6: Figs 1 – 12, SEM: Plate 7: Figs 1 – 4.

These valves consistently had a flatter dorsal margin with no obvious undulations, distinguishing them from E. varioundulata Nörpel-Schempp & Lange-Bertalot. The

ventral margin was not as strongly concave as it is in E. exigua (Brébisson) Rabenhorst. 71

A rimoportula was present midway up one valve apex and was visible on the external

valve as a small round pore. This may be a new species, and should be examined more

closely to compare with other E. varioundulata and E. exigua populations.

These valves were found in epilithon at Hen Wallow Falls (CB watershed)

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: GSMNP SP 4 12 – 21 3.1 – 3.9 15 – 20 16 – 19 E. varioundulata – this study 16 – 20 2.6 – 4 14 – 20 18 – 20

Eunotia sp. (GSMNP SP 5) Plate 6: Figs 13 – 17.

These valves had a very characteristically straight dorsal margin, a ventral margin with two small undulations that coincided with the proximal terminal raphe end, and striae that became more radiate approaching the valve apices (especially notable in smaller valves. These valves are in the E. exigua complex, but likely are, at a minimum, a new variety of this species.

These valves were found in slime on a wet wall along Laurel Creek Rd, on epilithon in Little Rhododendron Creek (MP watershed), epiphytic on bryophytes in Le

Conte Creek (LC watershed), and Laurel Falls (EL watershed). Field pH ranged from 4.6 to 5.5.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: GSMNP SP 5 12 – 19 2.7 – 3.8 16 – 20 20 – 22 E. exigua in Krammer & (5) 8 – 28 (60) (2?) 2.5 – 4 (5) 18 – 24 Lange-Bertalot (1991) E. exigua in 6 – 22 2.5 – 5 17 – 27 Alles et al. (1991)

Eunotia sp. (GSMNP SP 6) Plate 6: Figs 18 – 24.

It was difficult to determine which taxon these valves belonged to, or if they were a separate taxon, especially as only a short cline in size was documented. It is possible that they are the smaller valves of E. varioundulata Nörpel-Schempp & Lange-Bertalot.

Valves from Figs 18 – 20 were found in mucilage from Laurel Falls. Valves from

Figs 21 – 23 were found epiphytic on bryophytes in Camel Hump Creek (CB watershed),

Walker Camp Prong (WP watershed), and Anthony Creek (AB watershed). Fig. 24 was found epiphytic on bryophytes in a high elevation, first order stream (Clingmans Creek,

NO watershed). Field pH ranged from 4.8 to 5.1.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: GSMNP SP 6 10.5 – 18 2.7 – 3.3 15 – 20 19 – 20 E. varioundulata – this study 16 – 20 2.6 – 4 14 – 20 18 – 20

Eunotia varioundulata Nörpel-Schempp & Lange-Bertalot

Plate 6: Figs 25 – 36, SEM: Plate 7: Figs 5 – 8, Plate 8: Fig. 8

Valves with undulations on the dorsal margin were previously referred to as E. exigua varieties (i.e. bidens, tridentula, and undulata) and are currently referred to as E. varioundulata. There is a range of morphology in the valves under this name, and it is necessary to examine these designations more closely. The morphology of our valves did not match well with those in Krammer & Lange-Bertalot (1991, Taf. 153: Figs 18 – 27),

Lange-Bertalot & Metzeltin (1996, Taf. 15: Fig. 18), and those of the lectotype of E. exigua var. bidens fo. linearis Krasske (Coll. Krasske B II 101, Ötztaler Alpen, Lange-

Bertalot et al.,1996, Figs 1 – 25). This was in part due to the presence of undulations on the ventral margin and a less curved ventral margin in our specimens. The image of a 73

proposed lectotype for E. exigua var. bidens Hustedt in Simonsen (1987, Plate 191/7) did

not match our specimens and was more similar in morphology to Figs 18 – 27 in

Krammer & Lange-Bertalot (1991, Taf. 153).The morphology of our specimens appeared most similar to those from the holotype of E. exigua var. tridentula Østrup (Coll. Østrup

2996, Kopenhagen, Lange-Bertalot et al., 1996: Figs 49 – 53).

The number of undulations on the dorsal margin was generally 3 in specimens observed from the GSMNP, although the shape and height of the undulations varied somewhat and smaller valves sometimes had 2 undulations. Two slight undulations were also often present on the ventral margin of the valve and were present where the external proximal raphe ended (see SEM micrographs). Occasionally 3 undulations were observed on the ventral margin. A rimoportula was present midway up one valve apex

E. varioundulata was common from bryophyte substrates in first through fifth order streams, falls, and wet walls throughout the GSMNP including sites from the following watersheds: AB, BG, BR, CB, CT, DP, EP, FO, HZ, IC, LC, ML, MP, NO,

OW, RV, SR, WP. The field pH estimates of the sites ranged from 3.9 to 5.8. E.

varioundulata was generally present in low abundance, but was found in greater abundances at higher elevations and/or waterfalls or wet walls.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm E. varioundulata – 16 – 20 2.6 – 4 14 – 20 18 – 20 this study

Eunotia exigua (Brébisson) Rabenhorst Plate 6: Figs 37 – 58, SEM: Plate 8: Figs 1 – 6.

Historically there has been some confusion around E. exigua. Eunotia exigua in early descriptions are short (described in DeToni- references the maximum length at 15 um. By 1930 however, E. exigua had been expanded to include longer valves that became confused with E. nymanniana for example. With all the confusion between E. exigua, exigua var. compacta (now E. compacta), E. nymanniana, and E. steineckei (now E. nymanniana), it is somewhat difficult to sort out taxonomically. The GSMNP valves are similar to what at one point was called E. nymanniana Grunow in Schmidt’s Atlas (1972,

Taf. 274: Figs 9 – 18). Our valves are less similar to those called E. exigua in Schmidt’s

Atlas (Taf. 297: Figs. 87 – 92). We have kept our designation of E exigua to specimens with shorter valves.

Our E. exigua valves may represent more than one variety. Some of the valves in

Plate 6, Figs 37 – 50 were similar to the valves shown in Krammer & Lange-Bertalot

(1996, Taf. 153: Figs 32 – 43, E. exigua sensu lato). Generally though, valves were similar to the smaller valves in Krammer & Lange-Bertalot (Taf. 153: Figs 5 – 10, E. exigua sensu stricto) and those in Alles et al. (1991, Taf. II: Figs 1 – 23) and Werum &

Lange-Bertalot (2004, Plate 5: Figs 16 – 18). See also Lange-Bertalot et al. (1996, Taf.

59: Figs 26.33).

E. exigua was commonly found on bryophyte substrates in first through fifth order streams, falls, and wet walls throughout the GSMNP including sites from the following watersheds: AB, BG, BN, BR, CB, CT, EG, EP, FO, HZ, IC, LC, ML, MP,

NO, OW, RV, SR, TW, WP. The field pH estimates of the sites ranged from 3.9 to 5.6.

Eunotia exigua was found in greater abundances at higher elevations. 75

Striae / End striae Reference Length µm Width µm 10 µm / 10 µm This study: 7.5 – 22 1.8 – 3.2 16 – 24 20 – 24 E. exigua E. exigua in Krammer & (5) 8 – 28 (60) (2?) 2.5 – 4 (5) 18 – 24 Lange-Bertalot (1991)

Eunotia sp. (GSMNP SP 7) LM: Plate 6: Figs 70 – 74.

The GSMNP SP 7 specimens had a similar length, width and striae density for those reported for E. exigua but did fit E. exigua sensu stricto. The ventral margin was not as concave as E. exigua images, so it is not clear if these specimens belong to the E.

exigua – complex.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: GSMNP SP 7 15 – 19 2.4 – 2.6 23 – 24 24 – 25 E. exigua in Krammer & (5) 8 – 28 (60) (2?)2.5 – 4(5) 18 – 24 Lange-Bertalot (1991)

Eunotia tenella (Grunow) Hustedt Plate 6: 59 – 67, SEM: Plate 8: Fig. 7.

Similar images of E. tenella are shown in Alles et al. (1991, Taf II: Figs 24 – 54)

and Krammer & Lange-Bertalot (1991, Taf. 154: Figs 23 – 30). The striae from our

specimens were slightly denser and may reflect that this is a more E. exigua-like taxon.

This taxon was also similar to the E. exigua (sensu-lato) images shown in Krammer &

Lange-Bertalot (1991, Taf. 153: Figs 11 – 17) although the ends of our valves were not

quite as recurved as those in some images.

E. tenella was not a common taxon and was generally found in low abundance, epiphytic on bryophytes in first through fifth order streams, from wet walls, and a bog in the GSMNP, including the AB, BN, EP, LC, MP, NO RV, WP watersheds. It was more 76

abundant at higher elevations relative to lower altitudes. Field pH estimates ranged from

4.4 to 5.5.

Length Striae / End striae / Reference Width µm µm 10 µm 10 µm This study E. tenella 12 – 33 2.6 – 3.2 20 – 22 21 – 24 E. exigua var. tenella 7 – 28 3 – 4.5 14 – 20 in Alles et al. (1991) E. tenella in Krammer & Lange- 14 – 19 Bertalot (1991) E. exigua in Krammer & (5)8 – 28(60) (2?)2.5 – 4(5) 18 – 24 Lange-Bertalot (1991)

Eunotia compacta (Hustedt) Mayama Plate 6: Figs 68 – 69.

Specimens are similar in morphology to E. compacta. There is some confusion

over the identification of this taxon depending on which literature you use, as it is

conspecific with the E. nymanniana (Grunow) shown in Krammer & Lange-Bertalot

(1991, Taf. 154: Figs 31 – 38, Type material for E. exigua var. compacta Hustedt) and

Lange-Bertalot & Metzeltin, 1996 (Taf. 7: Figs 35 – 38). It was corrected to E. compacta

in the second printing of Krammer & Lange-Bertalot (Vol. 2/3, 2004). If you trace the

names back in the literature, there were two valves shown as E. nymanniana Grunow

under Fig 8. in Van Heurck 1881 (Plate 34), one of which is E. exigua var. compacta

(now E. compacta). See also the discussion under E. nymanniana.

E. compacta was found in low abundance in Andrews Bald Bog in the GSMNP

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: E. compacta 17.7 – 19 2.7 – 2.8 20 – 21 24

Eunotia muscicola var. tridentula (Grunow) Nörpel et Lange–Bertalot

Plate 9: Figs 1 – 13, SEM: Plate 10: Figs 1 – 5.

Valve measurements for E. muscicola var. tridentula were within the

measurements for the species E. muscicola (Krammer & Lange-Bertalot 1991).

Morphology was similar to those reported in other studies (see Krammer & Lange-

Bertalot 1991, Taf. 156: Fig 12 – 22; Lange-Bertalot. & Metzeltin 1996, Taf. 15: Figs 27

– 30; Lange-Bertalot et al. 1996, Taf. 68: Figs 39 – 52; Metzeltin, & Lange-Bertalot

1998, Taf. 15: Fig. 9; Rumrich et al 2000, Taf. 20: Fig. 9).

The rimoportula is located midway along one valve apex. The undulations on the

bottom of the valve correspond with the proximal raphe ends. Taf. 26: Fig. 3 in Lange-

Bertalot (1993) shows a clearer SEM image of the external distal raphe end that curves

part way up the valve surface.

E. muscicola was commonly found in low abundance, epiphytic on bryophytes in

first through fifth order streams and from wet walls throughout the GSMNP, including

those in the AB, BG, BN, CB, CT , DB, EP FO, HZ, IC, LC, ML, MP, NO, OW, RV,

WP watersheds. Field pH estimates ranged from 4.2 to 5.8.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 7.6 – 19 2.3 – 3.7 14 – 19 16 – 22 Krammer & Lange- Bertalot 1991 6 – 35 3 – 4 12 – 19 (for E. muscicola)

Eunotia microcephala Krasske Plate 9: Figs 14 – 22, SEM: Plate 10: Figs 6 – 8.

Valve size and valve morphology of E. microcephala were similar to those

previously reported (see Krammer & Lange-Bertalot 1991, Taf. 156: Figs 27 – 34; 78

Lange-Bertalot & Metzeltin 1996, Taf. 17: Fig. 13; Lange-Bertalot et al. 1996, Taf. 58:

Figs 30 – 49; Werum & Lange Bertalot 2004, Plate 8: Fig 20). The rimportula was

located just above the midway point of the valve apex. The undulations present on the

ventral margin of the valve were associated with the proximal raphe end.

E. microcephala was rarely found in samples collected from the GSNMP. It was

found in low abundance, epiphytic on bryophytes from streams, waterfalls, wet walls, and

a sink hole in the AB. EL, IC, MP, RV watersheds with the greatest abundance observed

at Laurel Falls. The pH for all but one sample was 4.6.

Reference Length µm Width µm Striae / 10 µm This study 13 – 16 1.9 – 2.1 18 – 22 Krammer & Lange-Bertalot (1991) 10 – 15 2 – 3 18 – 22 Lange-Bertalot et al. (1996) 10 – 15 2 – 3 18 – 22

Eunotia trinacria Krasske Plate 9: Figs 23 – 33.

Our specimens were slightly narrower than previously reported, but followed the

valve morphology previously shown for E. trinacria (Alles et al., 1991, Taf. I. Figs 36 –

49; Krammer & Lange-Bertalot, 1991, Taf. 155: Figs 22 – 37; Lange-Bertalot et al. 1996,

Taf. 57: Figs 1 –21). The upper size limit was closer to that reported in Alles et al. (1991)

than in Lange-Bertalot et al. (1996). Specimens were narrower than GSMNP SP 17 specimens. E. trinacria valves were found in some epilithon in Hen Wallow Falls, in the

CB watershed.

Reference Length µm Width µm Striae / 10 µm This study: E. trinacria 8 – 24 1.7 – 2.6 21 – 23 Lange-Bertalot et al. (1996) 4 – 40 2 – 4 17 – 22 Alles et al. (1991) 5 – 28 2 – 3.5 18 – 25 This study: GSMNP SP 17 14 – 26 2.7 – 3.6 19 – 20

Eunotia sp. (GSMNP SP 17) LM: Plate 9: Figs 34 – 46.

The morphology of these specimens was similar to Eunotia trinacria Krasske but tended to be wider. The dorsal margin had a faint hint of a small undulation, as is present in E. trinacria. It is not clear if this taxon is a wider form of E. trinacria or if it is a new species.

Specimens were found in low abundance, epiphytic on bryophytes in first through fifth order streams in the CT, EP, FO, HZ watersheds. Field pH ranged from 4.6 to 5.

Reference Length µm Width µm Striae / 10 µm This study: GSMNP SP 17 14 – 26 2.7 – 3.6 19 – 20 E. trinacria in 4 – 40 2 – 4 17 – 22 Lange-Bertalot et al. (1996) E. paludosa var. trinacria in 5 – 28 2 – 3.5 18 – 25 Alles et al. (1991) This study: E. trinacria: 8 – 24 1.7 – 2.6 21 – 23

Eunotia sp. (GSMNP SP 18) Plate 9: Figs 47 – 53.

Specimens were similar to E. gracillima (Krasske) Nörpel-Schempp (Lange-

Bertalot et al., Plate 56: Figs 22 – 47) but were broader and had denser striae. The specimens from the GSMNP fit the valve measurements for E. groenlandica but the ends of the valves were more recurved than those shown in the literature (Lange-Bertalot &

Metzeltin, 1996, Taf. 17: Figs 25 – 27; Lange-Bertalot & Genkal, 1999, Taf. 6: Figs 3 –

9). The striae pattern and morphology did not match those typical of E. fallax A. Cleve-

Euler where the end striae are denser than in the center and the striae are bolder.

E. sp. (GSMNP SP 18) was not common from the bryophyte substrates in the

GSMNP. It was found in low abundance in first and second order streams in the CT, HZ, and NO watersheds and in Andrews Bald Bog. Field pH ranged from 4.2 to 5.2. 80

Length Width Striae / End striae Reference µm µm 10 µm / 10 µm This study: GSMNP SP 18 18 – 33 2.8 – 3.3 13 – 17 18 – 22 E. gracillima in Lange-Bertalot & 15 – 60 2 12 Genkal, 1999, E. groenlandica in 25 – 40 2.7 – 3.6 13 – 16 Lange-Bertalot & Metzeltin, 1996 E. fallax in Krammer & Lange- 12 – 2 – 5 (9?) 12 – 15 19 Bertalot (1991) 55(75)

Eunotia sp. (GSMNP SP 19) Plate 9: Figs 54 – 56.

Valves did not fit with the historical descriptions and pictures of E. paludosa but the valves were similar in morphology to some of the valves shown in Krammer and

Lange-Bertalot (1991, Taf. 155, Figs 1– 2).

Eunotia sp. (GSMNP SP 19) was not common on bryophyte substrates in the

GSMNP. It was found associated with bryophytes in streams in the CT, HZ and NO

watersheds with a field pH range of 4.2 to 5.2.

Length Striae / End striae / Reference Width µm µm 10 µm 10 µm This study: GSMNP SP 19 14.5 – 24 2 – 2.7 19 – 22 21 – 22 E. paludosa in Krammer & 6 – 60 2 – 3 (4) 19 – 25 Lange-Bertalot (1991)

Eunotia paludosa Grunow Plate 9: Figs 57 – 65.

The E. paludosa valves were similar in morphology to those in historical descriptions (i.e. Schmidt’s Atlas, Plate 291: Figs 23 and 24). Similar images are found in

Metzeltin & Lange-Bertalot (2007, Plate 101, Figs 1 – 12). Images in Krammer & Lange-

Bertalot (1991, Taf. 155: Figs 1 – 20) show more variability in morphology, especially in the smaller valves. The GSMNP E. paludosa includes valves that are shorter than

originally described (see De Toni: 25 – 55 µm long). 81

E. paludosa was found on a wet wall along the Alum Cave Trail in the WP

watershed in the GSMNP.

Striae / 10 End striae / Reference Length µm Width µm µm 10 µm This study – E. paludosa 14.5 – 43 2.6 – 3.3 19 – 22 22 – 24 E. paludosa in Krammer & 6 – 60 2 – 3 (4) 19 – 25 Lange-Bertalot (1991)

Eunotia nymanniana Grunow Plate 11: Figs 1 – 29. SEM: Plate 12: Figs 1 – 8.

The size diminution series valves were found epiphytic on bryophytes in Kilby

Branch of Big Creek on Big Creek Trail in the BG watershed, TN, GSMNP. This series

shows the transition from the smaller valves to the longer valves.

There is some confusion between E. steineckei Petersen and E. nymanniana

Grunow. The smaller valves are referred to as E. steineckei in Krammer and Lange-

Bertalot (1991, Type material from Petersen, Taf. 153, Figs 1 – 4; see also Lange-

Bertalot & Metzeltin, 1996, Taf. 17: Figs 31 – 34). A longer, more arcuate valve, similar

to E. nymanniana is included in these figures.

The original type material for E. nymanniana Grunow included diagrams of two

different specimens: one that was a medium size specimen that has similarities with

longer images of E. exigua, the other that is conspecific with E. compacta (Hustedt)

Mayama. (see Van Heurck, 1881, Plate 34: Fig. 8). To further confuse the issue, there is a

second figure in Van Heurck (1881, Plate 34: Fig. 10) which follows one of the

morphologies ascribed to an E. exigua. Later then in Schmidt’s Atlas (1972), the E. nymanniana diagrams (Plate 274/ 9-18) show images that are similar to shorter E. exigua

valves and do not match our specimens. More recent research refers to the longer forms

as E. nymanniana (i.e. Krammer & Lange-Bertalot, 2004; Metzeltin & Lange-Bertalot, 82

2007, Plate 98, Figs 1 -13; E. sp. cf. nymanniana Grunow). Given that our size

diminution series shows long arcuate valves down to shorter valves that appear similar to

valves that have been called E. steineckei, we have referred to all of our valves as E.

nymanniana.

E. nymanniana was commonly found in bryophyte substrates in first through fifth

order streams and wet walls throughout the GSMNP, including sites in the AB, BG, BN,

BR, CB, CT, EP, FO, HZ, IC, ML, MP, NO, OW, RV, and WP watersheds. Field pH ranged from 4.2 – 5.8.

Width Striae / End striae / Reference Length µm µm 10 µm 10 µm This study: E. nymanniana 14.5 – 47.5 2.2 – 3.7 18 – 22 20 – 24 E. steineckei in Krammer & 18 – 50 Lange-Bertalot (1991)

Eunotia undulata Grunow

Plate 13: Figs 1 – 11, Plate 14: Figs 17 -18, SEM: Plate 14: Figs 14 – 16.

Similar valve morphologies are found in Metzeltin & Lange-Bertalot (2007: Plate

82: Fig. 9); and in Krammer & Lange-Bertalot (1991, E. pectinalis var. undulata (Ralfs)

Rabenhorst Taf. 141: Figs 1 – 5). SEM micrographs show spines on the apices of the valves similar to those shown in Fig. 2 (Krammer & Lange-Bertalot, 1991, Taf. 144).

This taxon was rare in most parts of the GSMNP, but was more common in the more alkaline parts of the southwestern parts of the park, especially in the AB watershed and Abrams Creek, and occasionally in the TB and SH watersheds. It was observed in epilithon, epidendron, and metaphyton. Field pH estimates ranged from 5.2 to 5.8.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: E. undulata 34 – 101 6 – 8.7 9 – 12 14 – 18 E. pectinalis in Krammer & 10 – 140 (3?) 5 – 10 7 – 15 Lange-Bertalot (1991)

Eunotia pectinalis var. ventralis (Ehr.) Hustedt Plate 14: Figs 1 – 13.

This species has been illustrated by Lange-Bertalot & Metzeltin (1996, Taf. 14: 1

– 4b). The width of the valves in girdle view was generally quite broad (11.6 to 20 µm wide).

E. pectinalis var. ventralis was found on bryophyte substrates in a sink hole off the Abrams Falls trailhead from Cades Cove Loop in the AB watershed.

Striae / Reference Length µm Width µm Striae / 10 µm 10 µm This study: E. pectinalis 17 – 48 4.2 – 6 9 – 14 13 – 28 var. ventralis E. pectinalis in Krammer 10 – 140 (3?) 5 – 10 7 – 15 & Lange-Bertalot (1991)

Eunotia macroglossa Furey, Lowe, Johansen

Plate 15: Figs 1 – 16, SEM: Plate 16: Figs 1 – 6.

E. macroglossa is a new species recently described from the GSMNP (Furey et al.

2008. [Chapter III]). See Furey et al. (2008; or Chapter III) for a complete description and discussion of this taxon. Otherwise, note the large helictoglossae visible in LM and SEM micrographs, along with the radial striae at the valve apices.

E. macroglossa was found in low abundance, epiphytic on bryophytes from streams around the GSMNP, and was occasionally observed in higher abundances in mid to high elevation springs and headwater streams ranging from 1300 to 1600 m altitude. 84

These included springs and streams in the AB, BN, EP, HZ, LC, MP, NO, SR, and WP watersheds. Field pH estimates ranged from 4.4 to 5.4.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 21 – 93 4.5 – 6.6 11 – 14 16 – 18

Eunotia jemtlandica (Fontell) Berg. Plate 17: Figs 1 – 10, SEM: Plate 17: Fig. 11.

This taxon has previously been referred to as E. monodon var. bidens (Gregory)

Hustedt, but was more recently designated as E. jemtlandica (see Lange-Bertalot, 1993,

Taf. 22: Figs 1 – 3; see also discussion under E. monodon and E. major complex).

Additional LM images are shown in: Krammer & Lange-Bertalot, 1991; Taf. 158: Figs 4

– 6; Taf. 159: Fig. 2; Lange-Bertalot & Metzeltin, 1996, Taf. 18: Figs 3 – 4. The internal

SEM image from this study shows the helictoglossae with a rimoportula located mid way along the valve apex.

E. jemtlandica was found in low abundance from bryophyte substrates in first through fourth order streams in the AB, BG, CT, EP, HZ, ML, MP, NO, OW, RV, and

WP watersheds and on wet walls in the IC watershed. Field pH estimates ranged from 4.6 to 5.5.

Length Width Striae / End striae / 10 Reference µm µm 10 µm µm This study 38 – 71 7.7 – 11 10 – 13 20 – 22 Krammer & Lange- Bertalot (1991) 35 – 220 6 – 15 8 – 12 E. monodon group

Eunotia formica Ehrenberg Plate 18: Figs 1 – 7.

Similar images are shown in Krammer & Lange-Bertalot (1991, Taf. 152: Figs 8

– 12), Lange-Bertalot (1993, Taf. 27: Figs 1 – 3, 7; SEM, Figs 4 – 6), Lange-Bertalot & 85

Metzeltin (1996, Taf. 13: Figs 1 – 2), Lange-Bertalot et al. (1996, Taf. 69: Plate Figs 7 –

10), Metzeltin, & Lange-Bertalot (1998, Plate16: Figs 1 – 8), Siver et al. (2005, Plate 30:

Figs 1 – 2).

E. formica was found in epilithon from a pool below the Spring House behind the

Twin Creeks facility (LC watershed), and on bryophyte substrates in streams in the AB,

CB, EP, HS, and NO watersheds where pH ranged from 4.5 to 5.8. Although generally

occurring in low abundance, it was commonly found on a variety of substrates in Abrams

Creek (i.e. on bryophytes, dead logs, rocks, and metaphyton) where pH was consistently more alkaline.

Width Striae / End striae / Puntae / Reference Length µm µm 10 µm 10 µm 10 µm This study: 51 – 155 7.3 – 12 9 – 11 12 – 13 24 – 26 E. formica E. formica in (12) 35 – 200 Krammer & Lange- 7 – 14 6 – 12 24 (230) Bertalot (1991)

Eunotia sp. (GSMNP SP 20) Plate 19: Figs 1 – 9.

Our specimens were most similar to those illustrated in Siver et al. (2005, E. monodon Ehrenberg, Plate 31: Figs 1 – 4) where the ends are more broadly rounded and as the valves decreased in size, the dorsal margin became more sloped with a more pronounced hump. Historically it appears that E. monodon should have apices with more cunate end, as the E. monodon in Patrick and Reimer (1966, Plate 11: Fig. 6) and Lange-

Bertalot (1993; Taf. 22: Figs 6 – 7). E. monodon was described in North America and is found here along with E. monodontiforma Lange-Bertalot & Nörpel. When the name was adopted in Europe, it was used incorrectly to describe what should be E. major (W. 86

Smith) Rabenhorst (see discussion in Lange-Bertalot, 1993). This has created some confusion around the taxonomy of the E. monodon and the E. major groups. The ends of both E. major and E. monodontiforma are more set off from the rest of the valve relative to our valves. The broadly rounded ends, less set off from the rest of the valve and the higher undulation on the dorsal margin suggest that these specimens, and likely those also in Siver et al. (2005) are new a species within the E. mondon complex.

These specimens were found in a bryophyte squeeze at a rock outcrop at the bottom of a dry water way in the 20 – Mile Creek Trail in the TW watershed.

Length Striae / End striae Punctae Reference Width µm µm 10 µm / 10 µm / 10 µm This study: GSMNP SP 20 32 – 59 10.5 – 12 9 – 12 12 – 14 28 – 30 E. monodon in Patrick & 35 – 90 7 – 15 8 – 12 Reimer (1966) E. monodon in Krammer & 35 – 220 6 – 15 8 – 12 Lange-Bertalot (1991) E. c.f. major in Lange- 75 – 250 13 – 15 5 – 8 Bertalot (1993) E. monodontiforma in 50 – 120 10 – 15 10 – 13 16 Lange-Bertalot (1993) E. monodon in 40 – 103 11 – 14 9 – 10 Siver et al. (2005)

Eunotia braendlei Lange-Bertalot & Werum

Plate 20: Figs 1 – 5, SEM: Plate 20: Figs 6 – 9.

E. braendlei is a species recently described from Germany (Werum & Lange-

Bertalot 2004). This study provides one of the first documented observations of this taxon in North America. The valve measurements were similar to those from Europe, with a slight extension of the valve length. Although our specimens have a greater range of width than those reported from Europe, these measurements should be interpreted with 87 caution as the orientation of these relatively large size valves can slightly skew these measurements if the valve is rotated under the LM. The spines are visible with the LM with careful observation.

External SEM images are similar to those from the original species description

(Werum & Lange Bertalot, 2004; Plate 4: Figs 1 – 5) and show the small broad spines present at the junction between the valve face and mantle. Internal SEM images show the helictoglossae with a rimoportula running parallel to the striae, close to a helictoglossa at one of the apices.

E. braendlei was found in low abundance, epiphytic on bryophytes, in first through fourth order streams in the AB, BG, EP, PS, and TF, watersheds on the

Tennessee side of the GSMNP. It was also found in the epilithon from a pond below the

Spring House behind the Twin Creeks Facility in the LC watershed (TN). Field pH estimates ranged from 4.5 to 5.9. This would be a new diatom record for the GSMNP.

Length Width Striae / End striae / Punctae / Reference µm µm 10 µm 10 µm 10 µm This study 41 – 58 8 – 11 10 – 12 16 – 19 28 – 32 Werum. & Lange 35 – 50 9 – 10 10 – 12 ~15 25 – 30 Bertalot (2004)

Eunotia sp. (GSMNP SP 8) Plate 21: 1 – 11, SEM: Plate 21: 12 – 15

Valves with the ventral margin very slightly concave to nearly straight, the dorsal margin convex with two slight waves or undulations, sometimes almost flat. Waves generally more prominent in larger valves. Apices slightly set off by a gradual change in slope of the dorsal margin, 20 – 59 (64) µm long, 7.3 – 9.9 µm wide. Ends bluntly rounded. 88

Helictoglossae visible under the light microscope. Raphe extending from helictoglossa 15

– 25% of the length of the valve. Raphe ends in a slight curve on the valve face, and is

clearly visible in the light microscope. Striae radiate to radial at apices, 10 – 14 in 10 µm

in the valve center, 12 – 16 in 10 µm near the valve apices. Punctae 34 – 36 in 10 µm.

Rimoportula apical, at one end of valve, close to ventral edge of apex of the valve, with

external pore prominent on the external ventral edge. Irregular shortened striae may be

present on the dorsal margin.

Our specimens closely resemble species in the E. praerupta Ehrenberg kinships.

In particular, our valves, with two slight waves on the dorsal margin, are similar to Figs 3

– 6 in Lange-Bertalot (1991; see discussion under figure caption Taf. 32: Figs3 – 13, E.

praerupta). Here two different taxa were named E. bidens Ehrenberg; Figs. 3 – 6 represent valves similar to E. inflata (morphotype II) and Figs. 7 – 13 E. bidens

(Morophotype I). The waves or two slight undulations on the ventral margin of this species distinguish our valves from E. inflata (Grun.) Nörpel – Schempp & Lange

Bertalot which has a rounded dorsal margin (Lange-Bertalot 1991, Taf. 148: Figs 14 –

17; Lange-Bertalot & Metzeltin 1996, Taf. 12: Figs 1 – 3). Similarly, the valves, which

have a slightly rounded or flatter end that is not recurved up, contrast with the recurved

ends of E. bidens Ehrenberg. (Lange-Bertalot 1991, Taf. 148: Figs 11 – 12; Lange-

Bertalot 1993, Taf. 32: Figs7 – 13; Lange-Bertalot & Metzeltin 1996, Taf. 12: Figs 4 – 5,

Metzeltin et al. 2005, Plate 18: Figs 1 – 8). See also Metzeltin & Lange-Bertalot 1998,

Taf. 58: Figs 16 – 17 for ?E. bidens Grun. Morphotype I; Figs 18 – 19, Morphotype II).

Specimens were found in low abundance, epiphytic on bryophytes, in first

through forth order streams in the BG, CT, EP, IC, LC, ML, OW watersheds with field 89 estimates of pH from 4.4 to 5.4. Cells were slightly more abundant in the BG watershed

(especially Big Creek and its’ tributaries) where pH was 5.3 to 5.4.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: GSMNP SP 8 20 – 49 (64) 7.3 – 9.9 10 – 14 12 – 16 E. praerupta in Krammer (10) 20 – 100 (4) 6 – 17 (5) 7 – 13 & Lange-Bertalot 1991.

Eunotia curtagrunowii Nörpel-Schempp & Lange-Bertalot Plate 22: Figs. 1 – 16.

The specimens without undulations were similar in morphology to E. curtagrunowii in Lange-Bertalot & Metzeltin (1996, Taf. 12: 6 – 11). See also the LM images in Krammer & Lange-Bertalot (1991, E. praerupta var. curta Grunow – including bidens variety, Taf. 148: 4 – 10). Smaller valves had one undulation that gradually became two undulations that became more pronounced as valve size increased. The transition from valves without one undulation to the valves with two undulations, found in a size diminution series shown in the figures, suggest that these valves are from the same taxon. These valves that include the bi-undulate morphologies may need to be separated from E. curtagrunowii forms that do not show undulations.

This taxon was found in very low abundance, epiphytic on bryophytes, from

Copperhead Branch (IC watershed) and Jakes Creek (EP watershed) where field pH measurements ranged from 4.6 – 5.0. It was found in greater abundance on a wet wall along Highway 441 and near Meigs Falls.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study: 11 – 25 5.6 – 7.6 11 – 15 14 – 16 E. curtagrunowii

Eunotia sp. (GSMNP SP 9) Plate 22. Figs 18-19.

These specimens were similar to illustrations of E. curtagrunowii Nörpel-

Schempp & Lange-Bertalot, but the dorsal undulations are much more pronounced, the ventral margin is more concave, and the end striae are denser. More valves must be observed to clearly identify this taxon. Fig. 18 appears to be morphologically deformed.

These specimens were found in low abundance, epiphytic on bryophytes in Big

Creek, in the BG watershed with a field pH of 4.9.

Striae / End striae Reference Length µm Width µm 10 µm / 10 µm This study: GSMNP SP 9. 22.5 – 24.5 7.3 – 7.4 15 22 – 24 This study: 11 – 25 5.6 – 7.6 11 – 15 14 – 16 E. curtagrunowii E. praerupta in Krammer & (10) 20 – 100 (4) 6 – 17 7 – 13 Lange-Bertalot (1991)

Eunotia rabenhorstii Cleve et Grunow Plate 22: Fig. 20.

Very few specimens of Eunotia rabenhorstii were observed so further images are

required to determine a size distribution and morphological variability. E. rabenhorstii

was found epiphytic on bryophytes from a wet wall along Little River Road (EP

watershed). Compare with images in Metzeltin et al. (2005, Plate25: Figs 12 – 14).

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 17.5 7 14 15 E. rabenhorstii 14 – 24 7.3 13 – 14

Eunotia c.f. macaronesica Lange-Bertalot & Tagliaventi nov. sp. prov.

Plate 22: Figs. 21 – 26; SEM: Plate 22: Fig. 27.

These specimens were similar to a recently described taxon from Europe, Eunotia c.f. macaronesica Lange-Bertalot & Tagliaventi nov. sp. prov. (pers. comm Lange-

Bertalot, 2007). Valve morphology and measurements were similar to those from Europe.

The apices of the valves from the GSMNP appeared slightly more recurved than the

European specimens. The striae end short on ventral edge of the valve face, leaving the impression of a narrow hyaline area extending across the valve face that is visible in both the LM and SEM. This population is likely E. macaronesica and needs to be compared with valves from Europe and updated when the name is formalized.

E. c.f. macaronesica was found from mucilage on a wet wall along Highway 441.

Additional valves were observed epiphytic on bryophytes from Big Creek in the BC watershed.

End striae Reference Length µm Width µm Striae / 10 µm / 10 µm This study: E. c.f. 10.5 – 37 5.6 – 8 11 – 16 14 – 16 macaronesica E. macaronesica 18 – 36 4.5 – 7.5 12 – 16

nov. sp. prov. (confer 13 – 45) (confer 6 – 8) (confer 13 – 17)

Eunotia sp. (GSMNP SP 10) Plate 23: Fig. 1.

This taxon was rare and was only found on bryophyte substrates in Hesse Creek in the HS watershed with field pH estimates at 5. It was very distinct from the other taxa observed in Hesse Creek and was worthy of noting. Additionally, the Eunotia praerupta complex is in need of more research, and it is helpful to document the variability in morphologies observed, especially as they often do not occur in high abundance. 92

Length Striae / 10 End striae / Reference Width µm µm µm 10 µm This study: GSMNP SP 10. 70 11 12 14

Eunotia bidens Ehrenberg Plate 23: Figs 2 – 4.

The specimens were similar to Figs 11 – 12 in Krammer & Lange-Bertalot (1991:

Taf. 148). The GSMNP specimens were similar to tropical specimens, but the dorsal margins were not quite as undulate those shown in Metzeltin & Lange-Bertalot (1998,

Taf. 58: Figs 16 – 17), Metzeltin et al. (2005, Plate 18: Figs 1 – 8), and Metzeltin &

Lange-Bertalot (2007, Plate 72: Figs 1 – 18).

E. bidens was not common in the GSMNP. It was found epiphytic on bryophytes in streams in the AB, CB, HS, HZ, WP watersheds. Field pH ranged from 4.7 – 5.4.

End Reference Length µm Width µm Striae / 10 µm striae / 10 µm This study: E. bidens 39 – 57 8.4 – 9.9 10 – 13 16 – 18

Eunotia diodonopsis Metzeltin & Lange-Bertalot Plate 23: Fig. 5.

Eunotia diodonopsis is a species recently described from a tropical flora by

Metzeltin & Lange-Bertalot (2007, Plate 25; Figs 1 – 11). Our specimens were very similar to those described from the tropics both in terms of valve measurements and overall morphology. More images are required to document a size range of E. diodonopsis from the GSMNP and for better comparison with the tropical flora.

E. diodonopsis was found on bryophyte substrates in Cosby Creek in the Cosby

Creek watershed where field pH estimates were around 5.0. This is a new diatom record for the GSMNP. 93

Length Width µm Striae / End striae Reference µm (inflated part) 10 µm / 10 µm This study: E. diodonopsis 37 11 12 14 E. diodonopsis nov. sp. in Metzeltin & Lange-Bertalot 26 – 63 10 – 12 10 –13 (2007)

Eunotia sp. (GSMNP SP 11) Plate 24: Figs 1 – 24. SEM: Plate 25: Figs 1 – 8.

The mid-sized specimens were most similar to the valves shown for E. diodon

Ehrenberg in Lange-Bertalot & Metzeltin (1996; Taf. 15: Figs 1 – 2) and Fig. 14 in

Krammer & Lange-Bertalot (1991, Taf. 149). As the valves from the GSMNP became larger the two undulations on the dorsal margin became elongated. Most images reported in the literature do not show the size range for E. diodon so it was difficult to compare the valves from the GSMNP with those reported from other studies. The smaller valves show some similarities to Eunotia satelles Nörpel-Schempp & Lange-Bertalot (Krammer &

Lange-Bertalot, 1991; Taf. 154: Figs 18 – 22; Lange-Bertalot & Metzeltin, 1996; Taf. 15:

Figs 31 – 33), but the smaller valves in E. satelles are wider and show a different morphology.

Early descriptions of E. diodon were of relatively large and obtusely biundulate valves, 44 to 55 wide (see De Toni’s descriptions and also Kützing in 1849). In Smidts

Atlas (Schmidt et al. 1972), the description and measurements were broadened to include a variety of morphologies, none of which fit the valves from the GSMNP. Recent images of E. diodon have a larger width to length ratio, relative to those from the GSMNP. In our specimens, the length to width ratio increased as the cells got longer; 2.5 or 3.5 in the smaller cells, gradually up to 6 or 8 for longer cells. Furthermore, the ends were more set 94

off when compared species in the E. diodon group. The apices of our valves are also quite truncated.

Along the dorsal margin of the valves, the transition from the valve view to the girdle view, gave the illusion of the areolae being situated in a shallow depression. This affect could be seen under the LM with optical dissection and under the SEM. This was also observed in E. bigibba Kützing cells.

GSMNP SP 11 was found epiphytic on bryophytes in first through fifth order streams, and was frequent on wet walls, occasionally in high abundance. It was found in aquatic areas in the BN, EP, FO, IC, LC, MP, NO, SR, WP and watersheds where field estimates of pH ranged from 4.2 – 5.6.

Length End striae Reference Width µm Striae / 10 µm µm / 10 µm This study: GSMNP SP 11 9 – 53 4 – 8 12 – 13 14 – 20 E. diodon in Krammer & Lange- 10 – 5 – 14 (10) 12 – 16 (19) Bertalot (1991) 65 (20) E. praerupta var. bigibba in 13 – 4 – 8 12 – 14 Krammer & Lange-Bertalot 1991 30

Eunotia bigibba Kützing Plate 26: Figs. 1 – 13, SEM: Plate 27: Figs 1 – 4.

Our specimens are similar to those illustrated in Krammer & Lange-Bertalot

(1991, Taf. 150: Figs 1 – 6) where the ends of the valve are turned up. This helps to

differentiate this taxon from similar taxa. However, this feature is less pronounced in the

smaller valves. Generally the apices of the ventral margin of the valve do not slightly

extend out as they tended to in taxa such as E. diodon Ehrenberg, and GSMNP SP 21.

The double undulation or humps on the dorsal edge of the valve were often quite variable in terms of their height and shape. In some populations they were consistently rounded, in 95 others more pointed valves were present. The dorsal margin of E. bigibba gave the appearance of shallow pockets or indentations associated with the aerolea as the valve transitioned from valve to girdle view. These were visible with optical dissection in the

LM and the SEM. This was less prominent than was observed for the E. diodon valves.

Lange-Bertalot (pers. comm. 2007) has suggested that this taxon is actually a new species Eunotia subherkiniensis Lange-Bertalot sp. nov. as it does not conform to drawings of E. (bigibba var.?) herkiniensis Grunow in Van Heurck 1881, Fig. 35:14 from samples originating from Lake Herkinje in Scandinavia and also Eunotia bigibba var. pumila Grunow in Van Heurck 1881, Fig. 34: 27. The GSMNP populations should be closely compared with these populations in the future as these descriptions are published.

Eunotia bigibba was found in streams, wet walls and waterfalls throughout the

GSMNP, including sites in the BR, CB, EP, GR, EP, HS, HZ, IC, MP, WP watersheds. It was most common on wet walls and waterfalls in the GSMNP, especially Hen Wallow

Falls (CB watershed), and Meigs Falls. It was found epiphytic on bryophytes, associated with brown mucilage, and in epilithon. Field pH estimates ranged from 3.9 – 5.2.

Length Width Striae / End striae / Reference µm µm 10 µm 10 µm This study 15 – 33 4.5 – 8 11 –14 13 – 16 5/19/04/c1E E. praerupta var. bigibba in 13 – 30 4 – 8 12 – 14 Krammer & Lange-Bertalot (1991) E. subherkiniensis Grunow (pers. comm. 10 – 30 5 – 8 12 – 15 Lange-Bertalot, 2007). Siver et al. (2005) 17 – 19 4 – 5 13 – 14

Eunotia sp. (GSMNP SP 21) Plate 26: Figs 20– 24. SEM: Plate 27: Figs 5 – 8.

These specimens were found associated with Eunotia bigibba Kützing at Hen

Wallow Falls (CB watershed). They were distinguished from the E. bigibba specimens

because the morphology of the undulations along the dorsal margin was simpler (not as

pointed) .The ends of the cells were not recurved, or not turned up to the degree they

were for the E. bigibba specimens. Because the ends did slightly turn up, it was not clear

whether they belonged to E. diodon or were a separate taxon.

Length End striae Reference Width µm Striae / 10 µm µm / 10 µm This study: GSMNP SP 21 18 – 32 5 – 7 12 – 13 13 – 16 E. praerupta var. bigibba in Krammer & Lange-Bertalot 13 – 30 4 – 8 12 – 14 1991 E. diodon in Krammer & 10 – 65 5 – 14 (20) (10) 12 – 16 (19) Lange-Bertalot (1991) This study: GSMNP SP 10 9 – 53 4 – 8 12 – 13 14 – 20

Eunotia sp. (GSMNP SP 12) Plate 28: Figs. 1 – 5; SEM: Figs 6 – 10.

It was not clear which group this species belonged to as valves were often in poor shape or in girdle view. Cells had similar features to E. bigibba Kützing such as the slight

impression on the dorsal edge of the valve (see external SEM images) and overall valve

shape, however, they were larger and broader than the E. bigibba valves that were also

present in the samples.

This taxon was found in low abundance in epilithon at Hen Wallow Falls and in

brown slime on a wet wall at the edge of Meigs Falls.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 31 – 53 7 – 13 7 – 9 10 – 14

Eunotia septentrionalis Østrup Plate 29: Fig. 1.

Similar images are shown in Krammer & Lange-Bertalot (1991, Taf. 157: Figs 13

– 18, Taf. 159, Figs 8 – 9).

E. septentrionalis was rare in samples from the GSMNP. It was found epiphytic on bryophyte in Little Cataloochee River in the CT watershed.

Striae / End striae / Reference Length µm Width µm 10 µm 10 µm This study: 45 6 12 16 E. septentrionalis E. septentrionalis Krammer & Lange- 11 – 140 3 – 6 8 – 19 Bertalot (1991)

Eunotia implicata Nörpel-Schempp, Lange-Bertalot, & Alles Plate 29: Figs. 2 – 9.

We restricted our definition of Eunotia implicata Nörpel-Schempp, Lange-

Bertalot, & Alles to those specimens where two undulations were present on the dorsal margin. The undulations were more difficult to detect in the longer cells. Compare with images in Alles et al. (1991, Taf. VII: Figs 19 – 21), Krammer & Lange-Bertalot (1991,

Taf. 143: Figs 1 – 9a), Lange-Bertalot & Metzeltin (1996, Taf. 15: 10 – 15), Rumrich et al. (2000, Taf. 18: Figs 16 – 17, Werum & Lange-Bertalot (2004, Plate 8: Figs 1 – 10).

Our specimens had a stronger sloping shoulder that was in some ways similar to E. circumborealis Nörpel & Lange-Bertalot (Krammer & Lange-Bertalot, 1991, Taf. 143:

Figs 16-23). However, our valves were too narrow for E. circumborealis.

E. implicata was found epiphytic on bryophytes in streams in the AB, BR, CT,

DD, DP, EP, HZ, IC, ML, MP, SR watersheds. They were more common in first and second order streams and the HZ watershed. Field pH estimates ranged from 4.6 – 5.7. 98

Width µm Length Width Striae / End striae Reference µm µm (at 10 µm / 10 µm undulation) This study: E. implicata 20 – 42 4 – 5 4.4 – 5.3 12 –16 19 –22 E. implicata in Krammer 20 – 40 3 – 6 14 – 22 & Lange-Bertalot (1991) E. circumborealis in Krammer & Lange- 13 – 45 6 – 8 13 – 17 Bertalot (1991)

Eunotia sp. (GSMNP SP 22) Plate 29: Figs 10 – 13.

Our specimens were slightly narrower, and slightly more coarsely striated than reported for E. circumborealis Lange-Bertalot & Nörpel and differed from those

illustrated in Krammer & Lange-Bertalot (1991, Taf. 143, Figs. 16 – 23) and Lange-

Bertalot & Metzeltin 1996 (Taf. 15: Figs 3 – 9). More images of GSMNP SP 22 in the

GSMNP would be helpful in understanding the morphological variability of this taxon across the size range. Our specimens were broader when compared with E. implicata

Nörpel & Lange-Bertalot, a species which also has undulations on the dorsal margin.

E. sp. (GSMNP SP 22) was found in low numbers, epiphytic on bryophytes, in

streams and wet walls in the DC, HS, HZ, IC watersheds. Filed pH estimates ranged from

4.6 – 5.2.

Width µm Width Striae / End striae / Reference Length µm µm (at 10 µm 10 µm undulation) This study: 28.3 – 38 5 – 6 5.7 – 6.3 12 – 13 20 – 25 GSMNP SP 22 E. circumborealis in Krammer & Lange- 13 – 45 6 – 8 13 – 17 Bertalot (1991) This study: 20 – 42 4 – 5 4.4 – 5.3 12 –16 19 –22 E. implicata 99

Eunotia sp. (GSMNP SP 13) Plate 29: Figs 14 – 19.

The dorsal margin of Eunotia sp. valves did not have undulations, rather a relatively short, straight plateau that resulted in the presence of a notably sloped dorsal margin leading to the valve apices. This differed from the shoulder of our E. implicata cells. The plateau area was short relative to the undulation plateau in E. implicata Nörpel-

Schempp, Lange-Bertalot, & Alles (i.e. Krammer & Lange-Bertalot, 1991, Taf. 143: Figs

16-23).

This species was found epiphytic on bryophytes in streams in the AB, HS, and HZ watersheds. Field pH estimates ranged from 4.9 – 5.9.

Width µm Length Width Striae / End striae Reference µm µm (at 10 µm / 10 µm undulation) 24.5 – This study: GSMNP SP 13 4 – 6 10 – 15 17 – 20 42 This study: E. implicata 20 – 42 4 – 5 4.4 – 5.3 12 –16 19 –22 E. implicata in Krammer 20 – 40 3 – 6 14 – 22 & Lange-Bertalot (1991) E. circumborealis in Krammer & Lange- 13 – 45 6 – 8 13 – 17 Bertalot (1991)

Eunotia minor (Kützing) Grunow Plate 29: Figs. 20 – 29.

Eunotia minor (Kützing) Grunow appears to be somewhat of a catch-all taxon and

is in need of more taxonomic research. As a result a variety of E. minor valve

morphologies are reported in the literature and it was difficult to clearly delineate this species. For example, see Krammer & Lange – Bertalot (1991, Taf. 142: Figs 7 – 15),

Lange-Bertalot & Metzeltin (1996, Taf. 13: Figs 18 – 21), Werum & Lange-Bertalot 100

(2004, Taf: Plate 8: Fig. 15, Plate 7: Figs 19 – 23), and Metzeltin et al. (2005, Plate 19:

Figs 9 – 13).

E. minor was found epiphytic on bryophytes in streams throughout the GSMNP.

Striae / 10 End striae / Reference Length µm Width µm µm 10 µm This study: E. minor 20 – 38 4 – 6 12 – 15 18 – 22 E. minor in Krammer & 20 – 60 4.5 – 8 9 – 15 Lange-Bertalot (1991)

Eunotia veneris (Kützing) De Toni Plate30: 1 – 14.

Our LM size diminution series closely resembles that in Metzeltin & Lange-

Bertalot (1998, Taf. 60: Figs 1 – 9). Both our images and those in Metzeltin & Lange-

Bertalot (1998) have a flatter ventral margin and slightly stronger shoulder approaching the valve apices relative to the valves shown in Krammer & Lange-Bertalot (1991, Taf.

163: Figs 14 – 19).

E. veneris was found on bryophyte substrates in first through forth order streams throughout the GSMNP in the AB, CB, CT, EP, FO, LC, ML, MP, OW, SR, WP watersheds. Field pH estimates ranged from 4.5 – 5.5.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 28 – 77 4.5 – 6 10 – 14 17 – 20

Eunotia pirla Carter & Flower Plate 30: Figs. 15 – 17.

Our images were similar to those illustrated in Carter & Flower (1988, Figs 17 –

19) and Krammer & Lange-Bertalot (1991; Taf. 163, Figs 8 – 13). Our valves did not show variation in ventral gibbosity as those in Carter & Flower (1988). E. pirla was rare 101

in samples from the GSMNP. Specimens were found in Anthony Creek (AB watershed) and in Andrews Bald Bog

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 27 – 36 7 – 8.5 12 – 13 16

Eunotia sp. (GSMNP SP 14) Plate 30: Figs 18 – 19.

Valves were found epiphytic on bryophytes in first order streams and a spring in

the NO and EP watershed. Field pH estimates ranged from 4.4. – 4.7

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 22.5 – 27.5 4.7 – 4.8 13 – 15 16 – 17

Eunotia sp. (GSMNP SP 15) Plate 30: Figs. 10 – 21.

These specimens were found epiphytic on bryophyte substrates in second order streams in the AB and BN watersheds. Field pH ranged from 4.9 – 5.4.

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 19 – 26.5 5.3 – 6.8 11 – 14 18 – 20

Eunotia sp. (GSMNP SP 16) Plate 30: Figs. 22 – 26.

These valves were kept separate from other valves by the pointed nature of the

valve ends. Valves were consistently observed in low abundance, in more than one

location, and suggest that this may be a new Eunotia taxon. More LM images would be helpful in determining the size range and valve morphology for comparison with other taxa.

These specimens were found epiphytic on bryophyte substrates in streams in the

AB and HZ watershed. Field pH ranged from 4.8 – 5.0. 102

Reference Length µm Width µm Striae / 10 µm End striae / 10 µm This study 18 – 27 3 – 4 13 – 15 18 – 20

Eunotia sp. (GSMNP SP 23) Plate 31: Figs 1 – 32, SEM: Plate 32: Figs 1 – 6.

The morphology GSMNP SP 22 was highly variable. More than one taxon is likely represented in the images and the taxonomy should be explored more closely.

Figures 1-10 are similar in morphology to the more asymmetric valves shown for

E. rhomboidea; however, our valves were consistently wider than E. rhomboidea.

Frustules were also observed where the more attenuated apex had the helictoglossa more inset relative to the opposite end (Figs. 11 – 21). The shape of the valve apex is more characteristic of E. incisa Gregory, however, these valves did not fit with the overall E. incisa morphology. Figs. 20 – 27 were less strongly asymmetrical about the transapical axis. Some of this variation in morphology is a result of the plane of focus in the microscope, so optical dissection of the valve is critical in establishing overall valve morphology.

The smaller valves (Figs 28 – 32) were similar in size and morphology to classical descriptions of E. rhomboidea where the valves that are not or are less asymmetrical about the transapical axis (Lange-Bertalot & Metzeltin, 1996, Taf. 16: Figs. 5 – 7;

Krammer & Lange-Bertalot, 1991, Taf. 164. Figs. 11-18; Siver et al., 2005, Taf. 26: Figs

35 – 40). However, more LM and SEM images are required to document the variability in size and to determine the presence or absence of a rimoportula.

In contrast to our valves, previous SEM micrographs of the inside of E. rhomboidea Hustedt valves do not show a rimoportula (i.e. Siver et al, 2005, Plate 28,

Figs 1, 3, 4; Metzeltin & Lange-Bertalot, 2003, Plate 9, Fig. 4). Our specimens clearly 103

have a rimoportula, and in valves where one end is rounded and the other end is more shark-like there is evidence of a small silica lip or ledge. This ledge slightly hides the

base of the rimoportula or the striae; an affect not seen in other rhomboidea SEM images because the entire inside of the apices is clearly visible. The rimoportula also appeared to consistently occur on the fatter end of the valve.

GSMNP SP 23 was common from bryophyte substrates from streams throughout the GSMNP (AB, BG, BN, BR, CB, CT, DD, DP, EP, FO, HS, HZ, IC, LC, ML, MP,

NO, OW, PS, RV, SR, TF, TW, WL, WP watersheds). Field pH estimates ranged from

4.2 – 5.8.

Reference Figs Length µm Width µm Striae / 10 µm This study: GSMNP SP 23 1 – 10 15 – 27 3.8 – 5.4 13 – 16 11 –190 23 – 29 3.7 – 4.4 14 – 15 20 – 27 20 – 35 3.3 – 4.8 14 – 16 28 – 32 19 – 23 2.7 – 3.3 13 – 17 E. rhomboidea in Krammer & 10 – 25 2 – 4 (12) 15 – 19 Lange-Bertalot (1991) Siver et al. (2005) 20 – 23 2.5 – 3 18 – 19

Eunotia sp (GSMNP SP 24) Plate 31: Figs 33 – 38.

These specimens were similar to E. incisa Gregory because the helictoglossa is inset from the valve end gives the apex what is described as a shark-like appearance.

Generally they were narrower and did not fit with the morphology of the E. incisa found in the park. The specimens were similar to Fig. 17 in Krammer & Lange-Bertalot (1991,

Taf. 161). Due to its’ similarity in morphology to some of the GSMNP SP 23 valves, it was occasionally difficult to discern from other valves if the characteristic GSMNP SP 24 104 was found in low numbers. Specimens were found on bryophyte substrates in streams throughout the GSMNP.

End striae / Reference Length µm Width µm Striae / 10 µm 10 µm This study: 20 – 35 3.5 – 4 14-15 GSMNP SP 24 This study: E. incisa 12 – 50 2.8 – 5 12 – 17 14 – 20 Krammer & Lange- 13 – 50 (65) (2) 4 – 6 (8) (9 )12 – 17 (20) Bertalot (1991)

Eunotia incisa Gregory Plate 33: Figs 1 – 30.

The E. incisa valves from the GSMNP had a range in variability in morphology similar to the images shown in Krammer & Lange-Bertalot (1991, Taf. 161: Figs 8 – 19) and Lange-Bertalot & Metzeltin (1996, Taf. 16: Figs 8 – 15) for E. incisa var. incisa. Our

SEM images clearly showed the pore of the rimoportula that was also observed by

Krammer & Lange-Bertalot (1991, Taf. 162: Figs 1 – 2). The valves were easily distinguished in girdle view by the inset of the helictoglossae from the valve apices (Plate

33: Fig. 26), relative to taxa such as E. rhomboidea Hustedt.

E. incisa was a common epiphyte on bryophytes in first through fifth streams below 1605 m throughout the GSMNP, including the following watersheds: AB, BG,

BN, BR, CB, CT, DP, EP,FO, HZ, IC, LC, ML, MP, NO, OW, RV, SR, TF, WP. The field pH estimates ranged from 3.9 to 5.6. Occasionally E. incisa was observed on wet walls, though only in low abundance.

End striae / 10 Reference Length µm Width µm Striae / 10 µm µm This study 12 – 50 2.8 – 5 12 – 17 14 – 20 Krammer & Lange- 13 – 50 (65) (2) 4 – 6 (8) (9) 12 – 17 (20) Bertalot (1991) 105

Eunotia billii Lowe & Kociolek Plate 34: 1 – 11; SEM: Plate 35: 1 – 6.

Valve measurements were similar to those reported from the original description of E. billii from the GSMNP (Lowe & Kociolek 1984). The length range was extended to include slightly shorter and longer valves. The striae were occasionally less dense relative to the original description. Girdle views of E. billii were easily identifiable by the appearance of the undulations on the dorsal margin. SEM micrographs show the presence and location of the helictoglossae and rimoportula.

E. billii valves were found in low abundance, epiphytic on bryophytes, in first through fourth order streams and wet walls throughout the GSMNP, including areas from the AB, CB,CT, EB, FO, HZ, IC, LC, MP, NO, OW, RV, SR, and WP watersheds.

The original description also included the BC watershed (Lowe and Kociolek, 1984).

Field pH estimates ranged from 4.4 – 5.5.

Length Width Striae / End striae No. of Reference µm µm 10 µm / 10 µm undulations This study 29 – 50 10 – 13 7 – 10 10 – 16 5 – 6 Lowe & Kociolek (1984) 32 – 46 10 – 13 9 – 10

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CONCLUDING REMARKS

In this dissertation, I first explored the basic ecology of algal assemblages of wet

walls, specifically their response to nitrogen and phosphorus. Second, Eunotia

populations of high elevation springs were examined, documenting the presence of

morphological dermformaties as indicators of the potential ecological consequences of

acid precipitation in the GSMNP. In addition a new species was identified, photo-

documented and described. Then, the distribution patterns of Eunotia species throughout

the GSMNP were studied in relation to elevation, pH levels, geology, and water

chemistry parameters, including metals such as aluminum in order to better understand

the ecology of this acidophilic taxon. Finally, an image rich documentation and inventory

of the Eunotia taxa in the GSMNP was provided along with taxonomic descriptions,

morphological measurements and a discussion around taxonomic challenges.

Chapter I: I explored the basic ecology of wet walls, specifically algal

community response to nutrient manipulation (nitrogen and phosphorus) from wet walls

from two different elevations in the GSMNP. Although the results from this chapter were

negative, concluding that the algal assemblages of the wet walls were not as nitrogen or

phosphorus limited as initially anticipated, it eloquently demonstrated the utility of

exploring basic ecological questions that then lead to the development of new sets of

hypotheses. The results alluded to the complexity of wet wall systems and the role that other potential limiting factors, such as moisture, ultraviolet radiation, pH, moisture levels, or microhabitat differences, may have in structuring wet wall algal communities.

For example, the presence or absence of mucilaginous algae, which can provide a moisture refuge for algae, may complicate the interplay between nutrients, moisture, and

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microhabitat differences. Furthermore, there may be species-specific interactions

between mucilaginous algae and diatoms such as Nitzschia. Ultraviolet radiation may

shape algal community structure, especially in high elevation areas where species

diversity may be limited to those that are able to tolerate UV, i.e. by producing protective

pigments. The algae on high altitude, wet walls may be useful for tracking changes in acid

precipitation that threaten biodiversity in the GSMNP. This chapter highlights the need

for more research at both micro- and macro-habitat scales in order to better understand

wet wall algal community dynamics.

Chapter II: Through the microscope observations of the diatoms in the GSMNP it

quickly became evident that the high elevation springs in the park were unique aquatic

environments. The springs were dominated by a few Eunotia species in high relative abundance. In addition, the presence of morphological deformities at some of the spring sites suggested that acid precipitation may directly be harming biodiversity in the park, and may also be interacting with local geologies resulting in increases in the release of harmful metals such as aluminum, further threatening the integrity of these ecosystems. As deformities are typically reported in association with industrial pollution, such as from coal-fired plants or from mine tailings, the presence of deformities was very noteworthy. The presence of morphological aberrations in diatoms may be a useful surrogate measure for identifying hotspots of acidification in the park where further research or monitoring should be conducted. A new Eunotia species, E. macroglossa sp. nov. was also described from the high elevation prings and streams in the park, an exciting contribution to the All Taxa Biodiversity

Inventory efforts in the GSMNP.

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Chapter III: This chapter explored the distribution patterns of Eunotia within the

GSMNP in relation to factors such as elevation, pH, geology, and water chemistry.

Results were similar to what has been found in the literature to date with respect to the acid-loving nature of this group of diatoms. Although no substantial contributions were made to Eunotia ecology, the results further emphasized which species of Eunotia are more acid tolerant than others, in addition to highlighting how vulnerable high elevation areas in the GSMNP may be to natural and anthropogenic sources of acidification.

Chapter IV: This chapter documented and described the diversity of Eunotia in the GSMNP. This inventory contributed to the All Taxa Biodiversity Inventory efforts in the park to date and the image rich documentation of the form, size range, and different species of Eunotia was much needed in diatom research, especially in North America.

The taxonomy of this genus is difficult due to the variability in form within and between species, especially across size ranges. The Eunotia were incredibly diverse in the

GSMNP and many of the Eunotia species were new park or North American records, and several were new species that need to be described. However, similar to Chapter I, this chapter raised more questions than answers around the taxonomy of Eunotia. More LM and SEM work is required to document size ranges and taxonomic variability, and explore new species, especially around the E. exigua-like taxa and E.-rhomboidea-like taxa.

The high biodiversity of Eunotia species reflects the variety of habitats across different geologies and altitudes that are present in the GSMNP. The prevalence of

Eunotia also indicates that several parts of the park are quite acidic and highlights the influence of different geologies such as the Anakeesta formation, and the pervasive

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impact of acid precipitation. Increases in acidity in the park may, on one hand, support the

biodiversity of Eunotia species but, on the other hand, may threaten the biodiversity of many other organisms.

The taxonomic work in this dissertation demonstrates the importance of these all taxa biodiversity inventories. They can lead to the discovery of new species, which, for example, can lead to advances in science and medicine. In this study, the discovery of new species and the expanded understanding of the ecology of the acidophilic taxon

Eunotia, will be useful in tracking changes, such as those associated with acid pollution, that threaten the freshwater ecosystems that humans and other biological organisms rely on. Furthermore, the number of trained taxonomists has declined around the world, but the need for being able to correctly identify and/or distinguish between species or forms, in order to be able to conduct good scientific research and biological monitoring has not.

Concluding Remarks: Not only did the results from this dissertation explore a new algal frontier (wet walls) and but they also provided further taxonomic documentation and ecological description of an understudied group of diatoms, the

Eunotia. More questions than answers were found over the course of this dissertation, leaving the door open for continued research in the areas of wet wall ecology and the taxonomy and ecology of Eunotia.

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TABLES

Chapter I Table 1. Mean (± SE) water chemistry from samples collected from each site at Clingmans Dome (CD) on 10 June and 5 August 2005 and Cataloochee (CAT) on 11 June and 6 August, 2005. Samples were analyzed for pH, total phosphorus (TP) soluble reactive phosphorus (SRP), total nitrogen (TN), nitrate (NO3-N), ammonia (NH4-N), sulphate (SO4-S) and conductivity (Cond.). Where no SE

is reported only water from one date was able to be collected. Significant differences in chemistry between CD and CAT are denoted

by underlining the higher of the two mean values (pH – RM–ANOVA1; chemistry – ANOVA; p<0.05).

Location pH ± SE TP ± SE PO4-P ± SE TN ±SE NO3-N ± SE NH4-N ± SE SO4-S ± SE Cond. ± SE µg P·l-1 µg P·l-1 mg N·l-1 µg N·l-1 µg N·l-1 mg S·l-1 µS·cm-1 CD 1 4.75 . 2.7 . 0.4 . 0.2 . 1.2 . 6.9 . 2 4.18 ± 0.34 2.8 ± 0.5 0.5 ± 0.0 0.3 ± 0.1 43.4 ± 12.1 6.9 ± 1.5 1.5 ± 0.1 27.85 ± 2.25 3 4.06 ± 0.33 2.8 ± 0.3 0.4 ± 0.2 0.7 ± 0.3 35.1 ± 19.4 7.8 ± 3.6 1.9 ± 0.1 27.10 ± 1.00 4 4.28 ± 0.52 3.6 ± 0.7 0.6 ± 0.0 0.2 ± 0.1 19.0 ± 1.1 5.5 ± 1.3 2.2 ± 0.1 39.35 ± 1.25 5 4.20 ± 0.32 3.0 ± 0.3 0.8 ± 0.2 0.2 ± 0.0 33.6 ± 10.5 8.3 ± 1.7 2.4 ± 0.7 47.45 ± 20.25 6 4.50 . 6.4 . 1.1 . 1.2 . 2.3 . 8.8 .

CAT 1 6.69 . 17.1 . 5.9 . 1.2 . 6.4 . 5.3 . 2 6.59 . 24.6 . 6.9 . 1.3 . 7.5 . 2.0 . 3 5.97 ± 0.30 8.3 ± 1.0 2.8 ± 0.9 0.8 ± 0.4 2.1 ± 0.8 19.1 ± 11.4 2.2 ± 0.1 21.05 ± 0.75 4 5.91 ± 0.39 43.5 ± 38.4 35.9 ± 32.6 0.7 ± 0.6 4.3 ± 0.8 6.5 ± . 0.6 ± 0.1 18.70 ± 6.20 5 6.05 ± 0.37 16.6 ± 7.7 6.2 ± 0.7 0.1 ± 0.0 5.4 ± 0.8 9.0 ± 2.9 0.4 ± 0.0 13.15 ± 0.15 6 6.22 ± 0.17 12.2 ± 3.5 5.8 ± 1.1 0.4 ± 0.2 8.5 ± 0.8 4.2 ± . 0.4 ± 0.1 13.05 ± 0.15

CD all 4.33 ± 0.10 3.5 ± 0.6 0.6 ±0.1 0.4 ±0.2 22.4 ± 7.3 7.4 ± 0.5 2.0 ± 0.2 35.44 ± 4.89 CAT all 6.24 ± 0.13 20.4 ± 5.1 10.6 ± 5.1 0.8 ± 0.2 5.7 ± 0.9 7.7 ± 2.5 0.9 ± 0.4 16.49 ± 2.01

119 120

Table 2. Biomass accrual (BA) and afdm (mean mg · cm-2, ± SE) for Clingmans Dome

(CD) and Cataloochee (CAT). Biomass accrual represents the mean increase in afdm for control plots scraped clean on 3 June and re-sampled on 12 August. Ash free dry mass

(all) represents an overall estimate of wetwall biomass based on all plots on both dates at each respective location. Response to treatment represents the mean difference in afdm examined by treatment where the June afdm estimate was subtracted from the August afdm estimate (positive value = average increase in afdm, negative value = average decrease in afdm). Ash free dry mass by site represents a mean estimate of biomass at each site based on 5 treatments per site on both dates from plot areas not previously sampled. Significant differences denoted by underlining the higher of the two mean values.

CD CAT Site N Mean ± SE Mean ± SE BA All C 6 1.42 ± 0.07 2.61 ± 0.22

afdm all 60 2.39 ± 0.10 4.42 ± 0.29

Response to treatment C 6 0.17 ± 0.19 1.48 ± 0.83 M 6 0.06 ± 0.51 -1.54 ± 0.56 N 6 0.11 ± 0.5 0.34 ± 1.16 P 6 0.66 ± 0.45 2.27 ± 1.66 NP 6 0.34 ± 0.44 -1.17 ± 0.74 afdm site 1 10 3.09 ± 0.24 4.30 ± 0.67 2 10 1.86 ± 0.22 5.52 ± 1.23 3 10 2.40 ± 0.27 2.90 ± 0.18 4 10 2.23 ± 0.20 4.43 ± 0.48 5 10 2.69 ± 0.33 5.22 ± 0.49 6 10 2.07 ± 0.14 4.18 ± 0.52 Table 3. Mean (± SE) relative frequency of soft algal taxa present in > 0.05 % relative frequency at Clingmans Dome. Note: June samples are averaged across all plots from all sites to represent relative frequencies present prior to nutrient treatment. Dashes represent 0%.

June August Taxa all C M N P NP Gloeocapsopsis dvorakii (Novácek) Komárek et 55.3 ± 3.2 72.3 ± 4.9 63.1 ± 7.5 72.6 ± 10.5 39.6 ± 2.8 47.8 ± 7.6 Anagnostidis Leptolyngbya spp. 9.6 ± 4.5 1.6 ± 1.1 7.7 ± 4.2 8.1 ± 5.8 21.6 ± 9.1 13.5 ± 5.0 Calothrix parietina (Naegeli) Thuret ex Bornet 8.6 ± 2.1 6.2 ± 3.1 11.1 ± 5.8 0.9 ± 0.7 11.3 ± 6.3 10.7 ± 6.2 et Flah. Homeoethrix juliana 8.3 ± 2.2 10.0 ± 3.7 7.6 ± 4.4 7.3 ± 2.3 12.1 ± 8.8 3.2 ± 2.5 (Bornet et Flah.) Kirchner Aphanocapsa muscicola 4.3 ± 1.5 2.7 ± 1.8 6.5 ± 3.4 6.9 ± 4.4 6.9 ± 4.2 11.3 ± 3.6 (Meneghini) Wille Gloeocapsa cf. sanguinea 5.7 ± 3.1 – ± – – ± – – ± – – ± – – ± – (black in color) Coccomyxa sp. A 1.5 ± 0.5 2.0 ± 1.6 1.3 ± 1.3 2.1 ± 2.1 1.4 ± 0.9 6.6 ± 3.5 Eunotia taxa 1.4 ± 0.7 2.9 ± 1.4 0.9 ± 0.9 0.7 ± 0.5 0.6 ± 0.4 1.5 ± 1.2 Gloeocapsa sanguinea (C. 2.1 ± 1.5 – ± – – ± – – ± – 0.3 ± 0.3 0.7 ± 0.7 Agardh) Kützing Frustulia rhomboides 0.4 ± 0.2 0.3 ± 0.3 0.3 ± 0.3 1.0 ± 0.7 0.6 ± 0.6 3.7 ± 3.7 (Ehr.) De Toni

Coccomyxa olivacea J.B. 121 0.7 ± 0.6 – ± – 0.9 ± 0.9 – ± – 1.3 ± 1.1 – ± – Peterson Table 4. Mean (± SE) relative frequency or relative abundance of soft algal taxa and diatom taxa present in > 5 % total relative frequency or total relative abundance for at least one treatment in Cataloochee (CAT). Taxa were also included if they are present in

Table 5. Note: * Navicula keeleyi was analyzed with the Diadesmis taxa as it is considered to be a Diadesmis but has not formally been transferred (Lowe & Johansen; personal communication). Dashes represent 0%.

August Site ID June C M N P NP Algal relative frequency Cyanobacteria:

Synechococcales Aphanocapsa muscicola 4.2 ± 1.1 0.6 ± 0.6 1.0 ± 1.0 – ± – 0.5 ± 0.5 – ± – (Meneghini) Wille Aphanocapsa sp. 2 0.1 ± 0.1 1.0 ± 0.8 3.9 ± 3.2 2.9 ± 1.9 7.8 ± 3.6 2.8 ± 2.2 Aphanothece pallida (Kütz.) 10.0 ± 5.4 15.4 ± 7.2 9.8 ± 6.6 7.3 ± 4.9 8.1 ± 5.1 9.7 ± 3.4 Rabh. Gloeothece tepidariorum (A. 5.1 ± 3.3 4.1 ± 2.4 4.9 ± 2.8 7.0 ± 5.0 3.5 ± 2.9 1.7 ± 0.6 Braun) Lagerh. Leptolyngbya spp. 15.5 ± 2.9 15.2 ± 4.3 7.3 ± 2.9 6.6 ± 6.6 13.4 ± 6.4 13.9 ± 6.8 Cyanobacteria: Chroococcales Chroococcus helveticus Näg. 3.4 ± 1.0 1.4 ± 1.1 1.4 ± 1.0 0.2 ± 0.2 1.2 ± 1.0 1.9 ± 1.4 Chroococcus pallidus (Näg.) 3.0 ± 1.4 6.7 ± 2.8 3.7 ± 2.2 13.1 ± 4.1 2.4 ± 1.7 0.9 ± 0.4 Näg. Chroococcus sp. 3 2.0 ± 0.8 2.0 ± 1.2 3.6 ± 3.1 – ± – 4.1 ± 1.7 4.1 ± 2.7 Cyanobacteria Nostocales Nostoc paludosum Kütz. ex 3.8 ± 1.5 1.4 ± 1.1 2.9 ± 2.0 2.6 ± 2.6 2.8 ± 2.3 1.1 ± 0.5 Bornet et Flah. Tolypothrix sp. 1 3.9 ± 2.0 5.0 ± 4.2 2.7 ± 2.5 0.5 ± 0.5 0.6 ± 0.6 0.6 ± 0.6 Chlorophyta: Sphaeropleales Palmellopsis sp. A. 2.0 ± 0.6 2.0 ± 1.1 4.5 ± 4.5 1.1 ± 0.6 0.6 ± 0.4 0.5 ± 0.5 Chlorophyta: Trebouxiales 122 Oocystis sp. A. 3.9 ± 1.6 – ± – 5.7 ± 5.7 2.8 ± 2.1 3.1 ± 2.8 7.8 ± 7.8 Chlorophyta: Zygnematales: Mougeotia sp. A 1.0 ± 0.5 0.2 ± 0.2 1.3 ± 1.3 0.6 ± 0.6 0.7 ± 0.4 – ± – Diatoms 33.2 ± 16.4 19.4 ± 10.4 32.5 ± 14.6 15.9 ± 6.8 31.9 ± 15.0 25.0 ± 5.5 (as part of soft algal count) Diatom relative abundance Bacillariophyta: Achnanthales Achnanthes subrostrata var. appalachiana Camb. et Lowe 3.4 ± 0.7 7.1 ± 4.5 6.5 ± 2.8 8.9 ± 4.0 4.1 ± 2.1 5.8 ± 3.8 Achnanthidium minutissimum 4.3 ± 1.9 1.1 ± 0.9 2.8 ± 2.2 1.9 ± 1.9 1.3 ± 0.8 4.4 ± 3.8 (Kütz.) Czarn. Planothidium lanceolatum var. lanceolata (Bréb. ex Kütz.) 1.2 ± 0.4 1.5 ± 1.2 1.4 ± 0.7 2.0 ± 1.6 0.4 ± 0.2 1.7 ± 1.5 Round et Bukht. Bacillariophyta Bacillariales Nitzschia hantzschiana Rabh. 37.0 ± 7.0 37.8 ± 19.3 34.4 ± 16.8 38.4 ± 17.8 38.5 ± 19.4 49.2 ± 19.4 Bacillariophyta: Cymbellales Gomphonema montanum (Grun.) 3.8 ± 1.2 1.6 ± 1.0 5.5 ± 4.8 4.2 ± 3.9 7.2 ± 6.2 5.6 ± 4.9 Grun. Bacillariophyta: Eunotiales Eunotia girdle 33 – ± – 0.1 ± 0.1 2.5 ± 2.1 2.9 ± 2.9 0.2 ± 0.2 0.8 ± 0.6 Bacillariophyta: Fragilariales Diatoma mesodon (Ehr.) Kütz. 7.2 ± 2.4 2.6 ± 1.5 1.7 ± 0.8 3.5 ± 2.1 0.7 ± 0.6 1.2 ± 1.0 Synedra rumpens Kütz. 2.1 ± 1.0 0.2 ± 0.2 – ± – 2.1 ± 2.1 0.7 ± 0.7 0.5 ± 0.5 Bacillariophyta: Naviculales Diadesmis contenta (Grun.) D.G. 2.2 ± 0.8 1.3 ± 0.6 0.2 ± 0.2 1.1 ± 1.0 0.3 ± 0.2 0.2 ± 0.2 Mann Diadesmis contenta var. biceps (Grun.) Hamilton 2.2 ± 0.6 2.7 ± 2.3 1.1 ± 0.5 0.9 ± 0.2 0.7 ± 0.4 0.4 ± 0.2 Diadesmis perpusilla (Kütz.) 1.5 ± 0.5 2.4 ± 1.7 1.0 ± 0.9 0.8 ± 0.6 1.4 ± 1.0 1.7 ± 0.9 D.G. Mann Navicula angusta Grun. 4.3 ± 1.0 9.5 ± 3.5 5.4 ± 2.5 4.6 ± 2.3 6.7 ± 2.4 4.5 ± 1.8 Navicula keeleyi Patr.* 8.7 ± 2.8 12.6 ± 6.7 13.0 ± 6.3 4.7 ± 2.7 14.9 ± 9.3 6.2 ± 3.8 Nupela neglecta Pon., Lowe et 2.7 ± 0.8 1.2 ± 0.6 1.2 ± 0.7 3.5 ± 2.0 3.9 ± 2.2 1.8 ± 0.9

Potapova 123 Table 4 continued Table 5: Mean (± SE) relative frequency or relative abundance of soft algal and diatom taxa present in > 5 % total relative frequency or total relative abundance for at least one site at Cataloochee (CAT). Note: * Navicula keeleyi was analyzed with the Diadesmis taxa as it is considered to be a Diadesmis but has not formally been transferred (Lowe & Johansen; in review). Dashes represent 0%. Site groupings found with the NMDS analysis are indicated by letters.

Site 1 2 3 4 5 6 Site groupings from NMDS A A B C D C Algal relative frequency Cyanobacteria: Synechococcales Aphanocapsa muscicola (Meneghini) Wille 4.4 ± 1.9 0.2 ± 0.2 – ± – 3.4 ± 1.8 5.3 ± 2.3 0.7 ± 0.4 Aphanocapsa sp. 2 – ± – 0.1 ± 0.1 – ± – 2.9 ± 2.0 5.7 ± 2.2 2.5 ± 1.7 Aphanothece pallida (Kütz.) Rabh. 8.8 ± 3.5 43.1 ± 2.9 1.4 ± 0.7 1.8 ± 1.0 2.6 ± 0.9 1.7 ± 1.2 Gloeothece tepidariorum (A. Braun) Lagerh. 9.0 ± 2.9 10.0 ± 1.8 0.4 ± 0.4 – ± – – ± – 0.4 ± 0.4 Leptolyngbya spp. 27.3 ± 5.1 24.9 ± 3.6 2.3 ± 1.1 5.4 ± 1.7 10.7 ± 2.3 0.4 ± 0.4 Cyanobacteria: Chroococcales Chroococcus helveticus Näg. 2.0 ± 0.8 1.5 ± 0.9 0.7 ± 0.5 5.6 ± 2.2 3.5 ± 1.9 0.6 ± 0.3 Chroococcus pallidus (Näg.) Näg. 0.6 ± 0.5 3.1 ± 1.1 5.3 ± 3.0 4.3 ± 2.0 5.0 ± 2.1 1.6 ± 1.3 Chroococcus sp. 3 – ± – – ± – 1.0 ± 0.6 4.8 ± 1.8 7.3 ± 2.3 1.1 ± 0.8 Cyanobacteria Nostocales Nostoc paludosum Kütz. ex Bornet et Flah. 14.6 ± 3.0 0.8 ± 0.4 0.2 ± 0.2 0.7 ± 0.5 0.8 ± 0.4 0.9 ± 0.9 Tolypothrix sp. 1 – ± – – ± – 0.4 ± 0.4 1.8 ± 1.5 13.9 ± 5.4 1.3 ± 0.5 Chlorophyta: Sphaeropleales Palmellopsis sp. A. 6.5 ± 2.7 1.3 ± 0.7 1.1 ± 0.8 – ± – 2.1 ± 0.7 0.1 ± 0.1 Chlorophyta: Trebouxiales Oocystis sp. A. – ± – 1.3 ± 1.3 21.1 ± 4.4 0.4 ± 0.3 – ± – 0.6 ± 0.4 Chlorophyta: Zygnematales: Mougeotia sp. A – ± – – ± – 9.7 ± 4.3 0.8 ± 0.6 – ± – – ± –

Diatoms (as part of soft algal count) 0.2 ± 0.2 0.4 ± 0.2 36.2 ± 4.3 43.0 ± 9.7 8.7 ± 4.0 66.3 ± 5.5 124

Diatom relative abundance Bacillariophyta: Achnanthales Achnanthes subrostrata var. appalachiana 0.2 ± 0.2 – ± – 1.7 ± 0.5 9.4 ± 1.0 4.7 ± 1.6 13.7 ± 2.7 Camb. et Lowe Achnanthidium minutissimum (Kütz.) Czarn. – ± – – ± – 14.7 ± 4.7 1.8 ± 0.7 0.4 ± 0.4 3.0 ± 1.4 Planothidium lanceolatum var. lanceolata – ± – – ± – 0.1 ± 0.1 1.4 ± 0.3 0.6 ± 0.2 5.7 ± 0.9 (Bréb. ex Kütz.) Round et Bukht. Bacillariophyta Bacillariales Nitzschia hantzschiana Rabh. 96.8 ± 1.9 86.3 ± 4.6 4.3 ± 0.6 19.4 ± 3.5 13.9 ± 7.1 9.4 ± 0.9 Bacillariophyta: Cymbellales Gomphonema montanum (Grun.) Grun. 0.1 ± 0.1 – ± – 20.2 ± 3.6 0.8 ± 0.4 0.5 ± 0.3 4.0 ± 1.1 Bacillariophyta: Eunotiales Eunotia girdle 33 – ± – – ± – 3.6 ± 2.0 0.2 ± 0.1 – ± – 0.2 ± 0.1 Bacillariophyta: Fragilariales Diatoma mesodon (Ehr.) Kütz. – ± – – ± – – ± – 6.5 ± 2.0 1.4 ± 0.5 19.6 ± 5.2 Synedra rumpens Kütz. 0.1 ± 0.1 – ± – 7.9 ± 2.4 – ± – – ± – 0.4 ± 0.2 Bacillariophyta: Naviculales Diadesmis contenta (Grun.) D.G. Mann 0.2 ± 0.2 5.9 ± 2.1 0.8 ± 0.4 0.2 ± 0.1 1.0 ± 0.3 0.3 ± 0.1 Diadesmis contenta var. biceps (Grun.) 0.5 ± 0.2 3.2 ± 1.2 0.5 ± 0.2 0.4 ± 0.2 5.1 ± 1.4 0.4 ± 0.2 Hamilton Diadesmis perpusilla (Kütz.) D.G. Mann 0.1 ± 0.1 0.1 ± 0.1 6.0 ± 0.8 1.3 ± 0.5 1.3 ± 0.6 – ± – Navicula angusta Grun. 0.1 ± 0.1 – ± – 9.0 ± 1.7 6.4 ± 1.4 11.5 ± 1.9 4.2 ± 0.7 Navicula keeleyi Patr.* 0.7 ± 0.6 0.2 ± 0.2 6.4 ± 2.1 13.6 ± 3.0 34.5 ± 5.8 1.5 ± 0.5 Nupela neglecta Pon., Lowe et Potapova – ± – 0.4 ± 0.3 1.4 ± 0.3 7.6 ± 1.8 2.0 ± 0.5 3.7 ± 1.2

Table 5 continued 125 126

TABLES

Chapter II

Table 1. Site and sampling information, along with relative abundance of Eunotia subarcuatoides (E. sub., normal [n] and abnormal

[a] morphologies) and other diatoms (Other diat.) for springs and high elevation streams sampled in 2005 and 2007. Eunotia taxa present at >5% at any one or more of the spring sites are included in the table (E. sub. = E. subarcuatoides, E. mac. = E. macroglossa,

E. mus. v. trid. = Eunotia muscicola var. tridentula). Sites are arranged by elevation (Elev.) in descending order. Geology (G): 1 =

Thunderhead Sandstone, 2 = Anakeesta Formation, 3 = Great Smoky Group, 4 = Elkmont Sandstone. Watershed codes (W): AB =

Abrams Creek, BN = Bunches, EG = Eagle, EL = East Prong Little River, FO = Fontana, IC = Indian Camp, NO = Noland, OW =

Oconaluftee River, SR = Straight Fork, WP = West Prong Little Pigeon.

Sampling date Elev. Relative abundance (%) Location Code UTMe UTMn G W total all E. sub. E. sub. E. E. E. mus. Other Other m yr (m) E. sub. (n) (a) mac. exigua v. trid. E. diat. Mount Collins Shelter spring MCSs 275931 3941768 Jul. 2005 1767 1 WP 99 93 6 - 0.5 - - 0.5 Jul. 2007 93 85 9 2 - - - 5 NE stream - Noland Divide NES 275218 3938280 Jun. 2005 1691 1 NO 75 74.5 0.5 1 12 0.5 5 7 DSTNs 269562 3938745 Jun. 2005 1675 2 EL 63 63 - 2 5 - 7 23 Double Spring Gap (TN) spring Jun. 2007 63 63 - 9 7 - 5 16

Aug 2007 46 46 - 12 14 - 10 18 DSNCs 269580 3938689 Jun. 2005 1665 3 FO 99 69 30 - - - - 1 Double Spring Gap (NC) spring Jun. 2007 76 42 34 - 0.5 - 1.5 22

Aug 2007 66 28 38 - 0.5 - 0 33.5 127 Spring at Silers Bald Shelter SBSs 267211 3938697 Jun. 2005 1663 3 EL 97 96 1 - 0.5 - 0.5 2

Bunches Creek BC 302941 3938640 Jul. 2005 1601 1 BN 8 8 - 7 3 - 19 63 Right Prong of Ledge Creek RP 302540 3942690 Jul. 2005 1598 1 SR 18 18 - 2 13 - 43 24 Clingmans Creek CC 274907 3937760 Jun. 2005 1562 1 NO 58 57.8 0.2 35 2 - 3 2 Upper Road Prong URP 278028 3943220 Jul. 2005 1536 2 WP 27 27 - 2 - 2 12 57 Spence Field Shelter spring SFSs 239549 3946680 Jul. 2005 1469 3 EG 29 28.6 0.4 26 1 - - 44 Beech Flats above roadcut BFP1 279409 3942860 Jul. 2005 1459 1 OW - - - - - 0.5 14.5 85 Otter Creek OC 296343 3955970 Aug. 2005 1435 1 IC 17 17 - 76 1 - 3 3 Beech Flats Prong BFP2 281177 3942534 Jul. 2005 1432 2 OW 3 - - - 0.5 - 6.5 90 RFSs 249000 3938804 Jul. 2005 1291 4 AB 4 4 - 62 1 5 - 28 Russel Field Shelter spring Jul. 2007 11 11 - 28 1 4 4 52 Beech Flats Prong BFP3 279437 3943099 Jul. 2005 1227 2 OW 1 1 - 0.5 - - 5.5 93 Anthony Creek AC 249921 3940440 Jul. 2005 891 4 AB - - - 2 - - 4 94 128

Table 2. Morphological measurements of normal and abnormal Eunotia subarcuatoides valves from high elevation springs and streams in the GSMNP compared with measurements for E. subarcuatoides from the Krammer & Lange-Bertalot (1991) taxonomic key and E. macroglossa. Note: the length measurements are based on a higher number of measured valves (in brackets).

# Valves Morphology Length (µm) Width (µm) Striae / 10 µm Striae / 10 µm (end) measured

E. subarcuatoides Normal 40 (221) 6.0 – 46.6 3.1 – 4.9 17 – 21 18 – 24 This study E. subarcuatoides Abnormal 42 (164) 6.2 – 49.0 2.1 – 5.4 17 – 21 18 – 24 This study E. subarcuatoides somewhat more closely Krammer & Normal 6.0 – 35.0 (40) 2.7 – 4.5 18 – 23 together Lange-Bertalot

E. macroglossa Normal 76 21-93 4.5 – 6.6 11 – 14 16 – 18 129

Table 3. Water chemistry data for springs and streams from pre-2005 (averaged) and 2007 (bold). Sites are arranged by elevation after

Table 1. Water chemistry was not available for NES, RC, CC. (D. limit = detection limit; blank cells = data not available; ND = below detection limit; ppm = parts per million or mg/L. Values > EPA screening eco-risk benchmarks are underlined (USEPA 2002).

Site code Data years lab pH field ph Cond. (µs/cm) Al (ppm) Ba (ppm) Ca (ppm) Fe (ppm) Mn (ppm) Zn (ppm) D. limit 0.100 0.010 1.00 0.050 0.015 0.050 MCSs not avail. 4.5 2007 3.9 0.407 0.017 1.020 ND 0.031 ND NES not avail. 4.8 DSTNs 2002-03 5.4 4.4 17.72 0.000 0.344 0.000 0.000 0.000 2007 4.2 0.129 0.014 1.240 ND 0.064 ND DSNCs 2002-03 5.0 4.5 28.88 0.000 0.331 0.000 0.000 0.000 2007 4.0 0.601 0.022 1.240 ND 0.064 ND SBSs 2002-03 5.1 4.5 21.63 0.000 0.230 0.000 0.000 0.000 BC 2002-03 6.2 4.9 13.56 0.055 0.832 0.027 0.006 0.087 RP not avail. 5.0 CC not avail. 4.7 URP 2002-04 5.7 5.0 15.78 0.026 1.16 0.022 0.012 0.015 SFSs 2002-03 5.8 4.2 7.73 0.004 0.164 0.003 0.000 0.002 BFP1 2002-04 6.6 5.2 24.05 0.029 1.949 0.033 0.007 0.027 OC 2002-03 5.9 4.5 20.79 0.086 1.332 0.005 0.002 0.032 BFP2 2002-04 6.0 4.8 28.6 0.003 0.716 0.003 0.001 0.002 RFSs 2002-03 5.7 4.7 11.95 0.012 0.167 0.012 0.001 0.007 BFP3 2002-04 5.5 4.9 40.39 0.041 0.987 0.006 0.036 0.008

C 2002-03 6.3 4.8 12.96 0.004 0.297 0.002 0.000 0.001 130

131

TABLES

Chapter III 132

Table 1: Geology and watershed codes for the GSMNP. Cnt = County: B=Blount, H = Haywood, SV = Sevier, SW = Swain, CK = C. ST = State: TN = Tennessee, NC = North Carolina.

C Geology C Watershed Cnt State C Watershed Cnt State 1 Blockhouse Shale AB Abrams Creek B TN LL Little River, Lower B TN Abrams/Panther 2 Lenoir Limestone AP B TN LO Lost Cove Creek SW NC Creeks Boundary Middle Prong Little 3 Limestone/Dolomite BG Big Creek B NC ML TN River Middle Prong Little 4 Cochoran Formation BK Baskins Creek SV TN MP SV TN Pigeon 5 Nichols Shale BN Bunches Creek SW NC NF NC Fringe NC 6 Nebo Quartzite BR Bradley Fork SW NC NO Noland Creek SW NC Oconaluftee River, 7 Murray Shale CB Cosby Creek C TN OL SW NC Lower Oconaluftee River, 8 Hesse Quartzite CG Chilogatee Creek B TN OW SW NC West 9 Wilhite Formation CH Chambers SW NC PH Peachtree Creek SW NC Wilhite Formation, 10 CN Cane Creek B TN PL Pilkey Creek SW NC Coarse 11 Cades Sandstone CO Cooper Creek SW NC PS Parson Branch B TN Thunderhead 12 CP Copeland Creek SV TN PT Panther Creek B TN Sandstone Cataloochee 13 Metcalf Phyllite CT B NC RM Ramsey Creek SV TN Creek 14 Pigeon Siltstone DD Dudley Creek SV TN RR Roaring Fork SV TN Roaring Fork 15 DN Dunn Creek SV TN RV Raven Fork SW NC Sandstone 16 Elkmont Sandstone DP Deep Creek SW NC SA Soak Ash Creek SV TN Wading Branch 17 EG Eagle Creek SW NC SH Shop Creek B TN Formation East Prong Little 18 Anakeesta Formation EL TN SL Stillwell Creek SW NC River 19 Unnamed Sandstone FO Forney Creek SW NC SR Straight Fork SW NC 20 Basement Complex GR Greenbriar Creek C TN TB Tabcat Creek B TN 21 Metadiorite HS Hesse Creek B TN TF TN Fringe TN 22 Longarm Quartzite HZ Hazel Creek SW NC TW Twentymile Creek SW NC Indian Camp West Prong Little 23 Rich Butt Sandstone IC C TN WL B TN Creek River Jonathan Creek 24 Great Smoky Group JC B NC WO White Oak Sinks B TN (Purchase area) West Prong Little 25 Shields Formation LC LeConte Creek SV TN WP SV TN Pigeon

Table 2. Percent contribution by Eunotia species for the observed clustering in the NMDS plots (see Fig. 3B and Fig 4C, D). The numbers in brackets correspond to the geology coding in Fig. 3B.

Corresponding data and Fig. All-Eunotia (Fig. 3B) NPS 2005 – Eunotia only (Fig 4C, D) Percent contribution Percent contribution Thunderhead Anakeesta Undisturbed Anakeesta WP Sandstone (12) (18) Average Similarity 35.97 33.79 40.21 39.58 43.24 E. subarcuatoides 21.06 11.55 34.39 37.53 34.87 E. incisa 26.91 19.18 26.00 30.31 28.65 E. rhomboidea 19.05 44.75 16.81 10.52 6.15 E. exigua 10.86 8.50 - 3.89 10.90 E. muscicola var. tridentula 4.43 2.61 3.42 - 6.32 E. varioundulata 2.66 - 3.68 5.24 3.73 E. macroglossa 2.92 2.17 - - - E. GSMNPSP11 3.54 1.86 - - - E. nymanniana - - 6.88 3.33 - Cumulative Percent 91.43 90.62 91.18 90.83 90.61

133 134

Table 3. PCA axes 1 and 2 for water quality parameters for the National

Park Service 2005 sites sampled. Axis one explained 33% (Eigenvalue

4.33) of the variance and axis two 30% (Eigenvalue 3.92).

Variable PC1 PC2 pH -0.293 -0.305 Chloride 0.190 -0.365 Nitrate 0.368 0.049 Sulfate 0.462 -0.035 Sodium -0.173 -0.423 Potassium -0.130 -0.419 Magnesium 0.311 -0.339 Calcium 0.242 -0.348 Aluminum 0.378 0.040 Copper -0.076 -0.063 Iron -0.144 0.173 Silicon -0.163 -0.378 Zinc 0.363 -0.045

Table 4. Water chemistry for isolated sited in Fig. 5 based on July water chemistry measurements. F_0251 – BF1 = Beech Flats above

US 441 loop, F_0252 – BF2 = Beech Flats prong below roadcut, F_0489 – AB = Abrams Creek 300m below Abrams Falls trailhead bridge, See Fig 5, F_0237 - WC - Walker Camp Prong at last bridge.

Total Elev. Elev. Stream ANC Cond. Cl Nitrate Sulfate Na Site: Field pH Lab pH IC (m) class order (meq/L) (ms/cm) (meq/L) (meq/L) (meq/L) (meq/L) Anions F_0251 – BF1 2222 8 2 4.8 6.27 28.24 35.50 15.34 39.71 193.04 248.09 39.77 F_0252 – BF2 1451 9 1 4.9 5.04 -11.19 56.00 12.99 52.87 355.44 421.29 46.21 F_0237 - WC 1375 9 2 4.5 4.74 -0.91 21.10 7.95 34.69 68.42 111.06 14.69 F_0489 - AB 521 3 5 5.8 7.89 1113.48 117.00 17.76 13.70 43.04 74.50 53.69

K Mg Ca Total H Cu Iron Mn Si Zn Al (ppm) TIS (meq/L) (meq/L) (meq/L) Cations (meq/L) (ppm) (ppm) (ppm) (ppm) (ppm)

F_0251 – BF1 13.40 87.59 136.44 277.70 0.54 0.089 0.040 0.021 1.064 2.888 0.017 555.11 F_0252 – BF2 14.66 139.16 150.28 350.32 9.19 0.624 0.019 0.014 1.691 3.656 0.044 778.80 F_0237 - WC 3.35 28.99 43.87 91.67 18.24 0.229 0.005 0.077 0.352 1.345 0.017 238.30 F_0489 - AB 16.80 205.68 914.27 1190.45 0.01 0.031 0.001 0.036 2.499 3.451 0.007 2378.46

135 136

TABLES

Chapter IV

137

Table 1. Watershed codes for the Great Smoky Mountains National Park.

Code Watershed County State AB Abrams Creek Blount TN AP Abrams/Panther Creeks Boundary Blount TN BG Big Creek Haywood NC BK Baskins Creek Sevier TN BN Bunches Creek Swain NC BR Bradley Fork Swain NC CB Cosby Creek Cocke TN CG Chilogatee Creek Blount TN CH Chambers Swain NC CN Cane Creek Blount TN CO Cooper Creek Swain NC CP Copeland Creek Sevier TN CT Cataloochee Creek Haywood NC DD Dudley Creek Sevier TN DN Dunn Creek Sevier TN DP Deep Creek Swain NC EG Eagle Creek Swain NC EL East Prong Little River TN FO Forney Creek Swain NC GR Greenbriar Creek Cocke TN HS Hesse Creek Blount TN HZ Hazel Creek Swain NC IC Indian Camp Creek Cocke TN JC Jonathan Creek (Purchase area) Haywood NC LC LeConte Creek Sevier TN LL Little River, Lower Blount TN LO Lost Cove Creek Swain NC ML Middle Prong Little River TN MP Middle Prong Little Pigeon Sevier TN NF NC Fringe NC NO Noland Creek Swain NC OL Oconaluftee River, Lower Swain NC OW Oconaluftee River, West Swain NC PH Peachtree Creek Swain NC PL Pilkey Creek Swain NC PS Parson Branch Blount TN PT Panther Creek Blount TN RM Ramsey Creek Sevier TN RR Roaring Fork Sevier TN RV Raven Fork Swain NC SA Soak Ash Creek Sevier TN SH Shop Creek Blount TN SL Stillwell Creek Swain NC SR Straight Fork Swain NC TB Tabcat Creek Blount TN TF TN Fringe TN TW Twentymile Creek Swain NC WL West Prong Little River Blount TN WO White Oak Sinks Blount TN WP West Prong Little Pigeon Sevier TN

138

Table 2: Current and previously reported Eunotia species observed in the Great Smoky

Mountains National Park. [Modified from Appendix B in Johansen et al. (2007) – see

Johansen et al. for the list of studies]. Codes under current study refer to watershed codes

(Table 1) If a species was recovered in 5-7 sites, it is listed as common; if it was in 8 or

more sites it is considered widespread. N. p. r. = New park records = *.

Reported in Taxon Current study N.p.r. Johansen et al. (2007) 1 E. arcus Ehr. One study 2 E. bidens Ehr. Common Widespread

3 E. bidentula W. Sm. One study 4 E. bigibba Kütz. Widespread Widespread 5 E. billii Lowe & Kociol. Widespread Widespread 6 E. bilunaris (Ehr.) Mills Common EL 7 E. boreotenuis Nörpel-Sch. & L.-Bert. Widespread * 8 E. braendlei L.-Bert. & Werum One study Common * 9 E. compacta Hust. As E. nymanniana Andrews Bald Grun. - Widespread Bog 10 E. curtagrunowii Nörpel-Sch. & L.-Bert. Common Common 11 E. curvata (Kütz.) Lagerst. Widespread 12 E. diodon Ehr. Common 13 E. diodonopsis Metzeltin & L.-Bert. CB * 14 E. elegans Østr. Common 15 E. exigua (Bréb. ex Kütz.) Rabh. Widespread Widespread 16 E. fallax A. One study 17 E. flexuosa (Bréb. in Kütz.) Kütz. One study AB 18 E. formica Ehr. Widespread Widespread 19 E. glacialis Meist. Two studies Widespread 20 E. hexaglyphus Ehr. One study 21 E. implicata Nörpel-Sch., L.-Bert. & Alles Widespread * 22 E. incisa W. Sm. Three studies Widespread 23 E. indica Grun. One study 24 E. inflata (Grun) Nörpel-Sch. & L.-Bert. One study 25 E. intermedia (Krasske) Nörpel-Sch. & L.- One study and as Bert. E. vanheurckii var. intermedia Patr. Two studies 26 E. jemtlandica (Fontell) Berg as E. monodon var. Widespread bidens (Greg.) W. Sm. - Two studies 27 E. lunaris (Ehr.) Bréb. ex Rabh. One study 28 E. lunaris var.capitata (Grun.) Schönfeldt One study 29 E. macroglossa Furey, Lowe, Johansen Widespread at * high elevations 30 Eunotia c.f. macaronesica Lange-Bertalot & Highway 441, * Tagliaventi BG 31 E. maior (W. Sm.) Rabh One study 32 E. meisteri var. bidens Hust. One study 139

Tabe 2 continued

Reported in Taxon Current study N.p.r. Johansen et al. (2007) 33 E. meisteri var. bidens Hust. One study 34 E. microcephala Krasske Two studies Common - AB. EL, IC, MP, RV 35 E. minor (Küt.) Grun. Widespread Widespread 36 E. monodon Ehr. One study 37 E. musicola Krasske One study 38 E. muscicola var. tridentula Nörpel & L.- Widespread Widespread Bert. 39 E. naegeli Migula One study AB, EL 40 E. neofallax Nörpel-Sch. & L.-Bert. Common: * Heintooga R. Rd., IC, RV, EP, EP, CB 41 E. nymanniana Grun. Widespread * 42 E. parallela Ehr. FO, MP, EP * 43 E. paludosa Grun. One study WP 44 45 E. pectinalis (Kütz.) Rabh. Widespread 46 E. pectinalis var ventralis (Ehr) Hust. AB * 47 E. pirla Carter & Flower Common Andrews Bald Bog 48 E. praerupta Ehr. Widespread 49 E. rabenhorstii Cl. et Grun. EP * 50 E. rhomboidea Hust. Widespread 51 E. septentrionalis Østr. One study CT 52 E. serra Ehr. One study 53 E. siolii Hust. One study 54 E. soleirolii (Kütz.) Rabh. Two studies 55 E. subarcuatoides One study Widespread at Alles, Nörpel & L.-Bert. high elevations 56 E. suecica A. Cl. Two studies 57 E. sudetica O. Müll. One study 58 E. tenella (Grun.) Hust. Two studies Widespread 59 E. trinacria Krasske CB * 60 E. undulata Grun. AB, TB, SH * 61 E. varioundulata E. exigua cf. var. Common Nörpel-Sch. & L.-Bert. tridentula Østr. (13 HWF) E. exigua cf. var. bidens – One study 62 E. veneris (Kütz.) De Toni Two studies Widespread 63 E. cf. yanomami Mezelt. & L.-Bert. One study 64 E. zazumensis (Cabejsz.) Korner One study FO 65 E. cf. zygodon Ehr. One study 66 E. sp. (GSMNP SP 1) CT, NO * 67 E. sp. (GSMNP SP 2) Widespread * Heintooga Ridge Rd, IC, RV, EP, EP, CB. 68 E. sp. (GSMNP SP 3) TW * 69 E. sp. (GSMNP SP 4) CB *

140

Table 2 continued.

Reported in Taxon Current study N.p.r. Johansen et al. (2007) 70 E. sp. (GSMNP SP 5) Common -Laurel * Creek Rd, EL, LC, MP 71 E. sp. (GSMNP SP 6) Laurel Falls, AB, * CB, NO, WP 72 E. sp. (GSMNP SP 7) CCC * 73 E. sp. (GSMNP SP 8) Common - BG, * CT, EP, IC, LC, ML, OW 74 E. sp. (GSMNP SP 9) BG * 75 E. sp .(GSMNP SP 10) HS * 76 E. sp. (GSMNP SP 11) Widespread * 77 E. sp. (GSMNP SP 12) CB AND meidgs * 78 E. sp. (GMSNP SP 13) * 79 E. sp. (GSMNP SP 14) NO, EP * 80 E. sp. (GSMNP SP 15) AB, BN * 81 E. sp. (GSMNP SP 16) AB, HZ * 82 E. sp. (GSMNP SP 17) CT, EP, FO, HZ * 83 E. sp. (GSMNP SP 18) CT, HZ, NO, * Andrews Bald Bog 84 E. sp. (GSMNP SP 19) CT, HZ and NO * 85 E. sp. (GSMNP SP 20) TW * 86 E. sp. (GSMNP SP 21) CB * 87 E. sp. (GSMNP SP 22) DC, HS, HZ, IC * 88 E. sp. (GSMNP SP 23) Widespread 89 E. sp. (GSMNP SP 24) Widespread * *

141

FIGURES

Chapter I 142 143 144 145 146 147 148 149 150

FIGURES

Chapter II 151 152 153 154 155 156 157 158

FIGURES

Chapter III

Stress: 0.13

8 4 5 6 6 44 6 7 6 7 4 4 6 6 7 5 5 4 75 5 5 5 4 5 5 6 3 4 5 3 7 4 10 7 345 6 99 55553 78 8 3 5 43 66 3 9 7 7 7 466 5 7 3 75 4453 3 3 328 4 333 5 6 10 6 9 4 8726 43 5 55552534 5 7 3 2432233 5 2 3213 34 7 9 2 45 5 11 9

11 10 11

Figure 1. Two-dimensional NMDS of Bray-Curtis similarities from the relative abundance of diatoms from all sites including springs (All-Taxa-S; stress value 0.13).

Bubble size marks relative abundance of other diatoms (not Eunotia). Sites are coded by elevation class where the larger the number the higher the elevation. Underlined 159 sites represent the spring sites. Stress: 0.2 Stress: 0.2 3 3 2 2 5 5 9 4 3 9 4 3 5 3 5 3 8 7 8 3 8 7 7 8 3 7 7 10 5 3 7 10 5 3 9 6 9 6 8 5 7 7 6 8 5 7 7 6 57 4556 3 3 57 4556 3 3 4 7 4444 3 3 3 4 7 64444 3 3 3 103 665 36 33 103 6 56 36 33 66 69544 65 66 9544 65 3 2 3 5 3 2 3 5 7 5687 34 7 5687 34 6 54 6 54 3 7 3 2 3 7 3 2 55 74 6 8 55 74 6 8 4 99 55 4 3 4 99 55 4 3 45 565 55 4 5 45 565 55 4 5 54 4 5 4 54 4 5 4 5 7 3 2 2 5 7 3 2 4 3 5 7 6 5 4 3 7 6 5 2 2 5 2 4 5 3 4 5 3 2 2 2 2 B 5 A 5

Stress: 0.2 Stress: 0.2 3 3 2 2 5 5 9 4 3 9 4 3 5 5 8 7 8 3 3 8 7 8 3 3 7 7 10 5 97 10 5 3 97 3 6 8 6 8 5 7 7 6 5 7 7 6 57 7 45 3 57 4 5 6 3 44 56 33 3 7 44 5 33 3 4 76644 6 3 3 104 6 64 4 6 3 3 1036 56 43 6 3 36 5 6 43 6 3 36 3954 5 36 3 9 54 5 2 5 2 8 5 7 5687 34 7 5 6 7 34 6 54 6 54 3 7 3 2 3 7 3 2 55 74 6 8 55 74 6 8 99 55 4 99 55 4 3 4 5 5 3 5 4 5 4 565 5 4 5 45 6 5 4 4 55 5 4 54 4 5 4 5 43 5 5 7 3 2 2 7 2 5 2 4 3 5 7 6 5 4 3 5 7 6 2 2 4 5 3 4 5 3 2 2 C 2 2 D 5 5

Figure 2. Two-dimensional NMDS of Bray-Curtis similarities from the relative abundance of Eunotia taxa only from all sites (not including springs) (All-Eunotia, stress value 0.2). Bubble size marks relative abundance of E. subarcuatoides (A), E. incisa (B), E. exigua (C) and E. tenella (D). Sites are

coded by elevation class: larger number = higher elevation. Plot C: triangle = Abrams Creek 300m 160 below Abrams Falls trailhead bridge (See Fig. 5)

Stress: 0.2 Stress: 0.2 0 2 5 1 99 5 4 4 4 2 1 4 3 5 44 2 4 4 4 4 53 3 44 992 2 99 3 55 5 55 99 24 54599 5 2 3 23 5 3 25 1 5 24 5 3995 5 0 2 4 4 4 4 5 55 4 992 2 3 99 5 995 45 5 4 44 33 2 2 5 9911 5 99 99 5 51 5 1 99 5 15 5 4 5 599 2 5 1 3 1 3 99 1 1 2 3 99 A B

Figure 3. Two-dimensional NMDS of Bray-Curtis similarities from the relative abundance of Eunotia taxa only from all sites (not including springs) (All-Eunotia, stress value 0.2). (A) = Symbols coded by disturbance category [1 –

Settlement area, 2 – Light cut, 3 – Selective cut, 4 – undisturbed, 5 – heavy cut, 6 – no data..(B) = Symbols coded by geology category. Solid grey circles = Thunderhead Sandstone; Solid black squares = Anakeesta (see Table 1). 161 Stress: 0.1 Stress: 0.1

CB CTOW CB CB WP HZ CTOW HZ CT AB CTOW CB HZCT HZ HZ CT OW HZHZABCTCT WP WLWPEP OW WP HZ HZABML WP HZ WP

A B

Stress: 0.18 Stress: 0.18

C D

Fig. 4. Two-dimensional NMDS of Bray-Curtis similarities from the relative abundance of diatoms from NPS-2005-All-Taxa (A and B, stress value = 0.1) and NPS 2005-Eunotia-only (B and C, stress value = 0.18). Plots A, B, and C are factored by watershed, and (D) by geology. For A and C: Solid circle = WP (dashed-circle in A for all but one WP site), empty circle = OW, X= HZ, plus = AB, downward triangle = ML, upward triangle = CB, diamond = EP, and square with X = CT, diamond with plus = WL. For Plot D: Solid circle = Anakeesta, Square with X = Thunderhead Sandstone, empty square = Basement Complex, X=Rich Butt Sandstone, empty circle = Limestone/Dolomite, downward triangle empty = Cades Sandstone, solid downward triangle = Roaring Fork Sandstone, empty diamond = Metcalf Phyllite, empty upward triangle = Pigeon Siltstone, diamond with plus = Longarm Quartzite, plus sign =

unknown. Plot B = larger circles represent higher relative abundance of non-Eunotia taxa (or other diatoms). Plot c: triangle = 162 Abrams Creek 300m below Abrams Falls trailhead bridge (See Fig. 5) 6 6 9 18

4 4

5 14 2 6 2 12 3 8 14 18 5 5 6 151299 12 12 3 10 7 99 18 12 0 42 5 2 0 2011 9915 PC2 PC2 2 5 13 14 5 4 2222 5 15 2 9 11 12 -2 8 9 -2 18 18 4 22 -4 A -4 B 3 99 -6 -6 -4 -2 0 2 4 6 8 -4-202468

6 WP 6 PC1 PC118 4 4

CB 14 2 CT 2 CB WP 12 CT OW WP 14 18 AB WP WP 1599129912 1212 OW CB 18 0 ML WP 20 9915 PC2 EP CB 0 11 CTCT PC2 13 14 CT 2222 WL OW 15 -2 OW 11 12 OW -2 18 18 CT 22

-4 -4 C D AB 99 -6 -6 -4-202468 -4-202468

PC1 PC1

Fig. 5. Principle Component Analysis (PCA) of log-transformed environmental variables from the National Park Service 2005. Plot A: factored by elevation (higher number = higher elevation), Plots B and D factored by geology (see codes in Table 1). Bubble plots for B: aluminum, C: sulphate, D, nitrates. In Plot A: site underlined (F_0252 – BF2) represents Beech Flats prong below roadcut, square (F_0251 – BF1) = Beech Flats above US 441 loop, circle (F_0237 – WC) = Walker Camp Prong at last bridge, triangle (F_0489 –

AB): Abrams Creek 300m below Abrams Falls trailhead bridge. 163

164

FIGURES

CHAPTER IV

165

Plate 1:

Scale Bar = 10 µm

Figs 1 – 10. E. naegelii Migula.

Figs 1 – 10. From a sink hole near the Abrams Falls trailhead along Cades Cove Loop

(AB watershed).

Fig. 10. Girdle view 166 167

Plate 2

Scale Bar = 10 µm.

Fig. 1. Eunotia flexuosa (Brébisson) Kützing

Figs 2 – 4. Eunotia bilunaris (Ehrenberg) Mills

Figs 5 – 7. Eunotia. naegelii Migula

Figs 8 – 10. Eunotia parallela Ehrenberg.

Fig. 1. From the mouth of Abrams Creek (AB watershed).

Figs 2 – 4. From a waterfall in White Oak Sink and Meigs Falls in the EL watershed.

Figs 5 – 7. From Meigs Falls in the EL watershed.

Figs 8 – 10. From streams in the FO, MP, and EP watersheds. 168 169

Plate 3:

Scale Bar = 10 µm

Figs. 1 – 9. Eunotia valida Hustedt

Figs. 10 – 12. Eunotia glacialis Meister

Figs 13 – 20. Eunotia sp. (GSMNP SP 1)

Figs 21 – 37. Eunotia boreotenuis

Figs. 1 – 9. From wet walls throughout the GSMNP

Figs. 10 – 12. From streams throughout the GSMNP

Figs 13 – 20. From Andrews Bald Bog, Forney Creek (FO watershed), and Caldwell

Fork (CT watershed)

Figs 21 – 31. From streams in the AB, EP, HS, and IC watersheds.

Figs 32 – 37. From streams in the AB, CB, and HZ watersheds. 170 171

Plate 4

Scale Bar = 10 µm .

Figs 1 – 9. Eunotia sp. (GSMNP SP 2).

Figs 10 – 11. Eunotia sp. (GSMNP SP 3).

Figs 12 – 17. Eunotia neofallax Nörpel-Schempp & Lange-Bertalot.

Figs 18 – 30. Eunotia subarcuatoides Alles, Nörpel & Lange-Bertalot.

Figs 1 – 9. From wetwalls along Heintooga Ridge Road, Snake Den Ridge Trail (IC),

Enloe Creek Trail (RV), and Little River Road (EP) and from Meigs Falls

(EP) and Hen Wallow Falls (CB).

Figs 19 – 20. From a soil seep by the 20-Mile Creek Trail (TW watershed).

Figs 12 – 17. From Hen Wallow Falls (CB), Meigs Creek of Little River (EP), Middle

Prong of Little River (ML), and Caldwell Fork (CT).

Figs 18 – 31. From high elevation springs along the Appalachian Trail.

Figs 18 – 22. Girdle view

Figs. 23 – 31. Size diminution series. 172 173

Plate 5

Scale Bar = 0.5 µm: Fig. 3.

1 µm: Fig. 4.

2 µm: Figs 1, 5, 6.

5 µm: Fig. 2.

Figs 1 – 4. Eunotia neofallax Nörpel-Schempp & Lange-Bertalot.

Figs 5 – 6. Eunotia subarcuatoides Alles, Nörpel & Lange-Bertalot

Figs 1 – 4. (SEM) From Hen Wallow Falls

Fig. 1. External view.

Figs 2 – 4. Internal views. H = helictoglossa; R = rimoportula.

Figs 5 – 6. (SEM) External and internal views. From high elevation springs.

H = helictoglossa; R = rimoportula. 174 175

Plate 6: Scale Bar = 10 µm

Figs 1 – 12. Eunotia sp. (GSMNP SP 4).

Figs 13 – 17 Eunotia sp. (GSMNP SP 5).

Figs 18 – 24 Eunotia sp. (GSMNP SP 6).

Figs 25 – 36. Eunotia varioundulata Nörpel-Schempp & Lange-Bertalot.

Figs 37 – 58. Eunotia exigua (Brébisson) Rabenhorst.

Figs 59 – 67. Eunotia tenella (Grunow) Hustedt.

Figs 68 – 69. Eunotia compacta Hustedt

Figs 70 – 74. Eunotia sp. (GSMNP SP 7).

Figs 1 – 12. From Hen Wallow Falls.

Figs 13 – 17. From a wet wall along Laurel Creek Rd, Little Rhododendron Creek (MP

watershed), Le Conte Creek (LC), and Laurel Falls (EL).

Figs 18 – 20. From Laurel Falls.

Figs 21 – 23. From Camel Hump Creek, Walker Camp Prong, and Anthony Creek.

Fig. 24. From Clingmans Creek.

Figs 25 – 50. From various streams, wetwalls, and falls in the GSMNP.

Figs 51 – 58. From LeConte Creek.

Fig. 59 – 67. From Andrew’s Bald Bog, LeConte Creek, Little Pigeon River, and

wetwalls along Alum Cave Trail and Goshen Prong Trail.

Fig. 68 – 69. From Andrews Bald Bog

Figs 70 – 74. From Laurel Falls and wetwalls along Little River and Laurel Creek Road. 176 177

Plate 7

Scale Bar = 2 µm: Figs 1 – 2, 5 – 7.

1 µm: Figs 3 – 4, 8

Figs 1 – 4. Eunotia sp. (GSMNP SP 4).

Figs 5 – 8. Eunotia varioundulata Nörpel-Schempp & Lange-Bertalot.

Figs 1 – 4. (SEM) From Hen Wallow Falls.

Figs. 1 – 2. (SEM) External view.

Fig. 3. (SEM) Outer opening of the rimoportula pore (RP).

Fig. 4. (SEM) Internal view. H = helictoglossae, R = rimoportula.

Figs 5 – 8. (SEM) E. varioundulata. H = helictoglossa; R = rimoportula. See also

Plate 8, Fig 8.

178 179

Plate 8

Scale Bar = 2 µm: Figs 1 – 5, 7 – 8

1 µm: Fig 6.

Figs 1 – 6. Eunotia exigua. (Brébisson) Rabenhorst.

Figs 7. Eunotia tenella (Grunow).

Fig. 8. Eunotia varioundulata Nörpel-Schempp & Lange-Bertalot.

Figs 1 – 6. (SEM) From streams, falls, and wet walls in the GSMNP.

Fig. 1 – 4. (SEM) External view.

Fig. 4 – 6. (SEM) Internal view; H = helictoglossae, R = rimoportula.

Figs 7. (SEM) Internal view. H = helictoglossa; R = rimoportula.

Fig. 8. (SEM) External view. 180 181

Plate 9:

Scale Bar = 10 µm.

Figs 1 – 13. Eunotia muscicola var. tridentula (Grunow) Nörpel et Lange-Bertalot.

Figs 14 – 22. Eunotia microcephala Krasske.

Figs 23 – 33. Eunotia trinacria Krasske.

Figs 34 – 36. Eunotia sp. (GSMNP SP 17).

Figs 47 – 53. Eunotia sp. (GSMNP SP 18).

Figs 54 – 56. Eunotia sp. (GSMNP SP 19).

Figs 57 – 65. Eunotia paludosa Grunow var. paludosa..

Figs 1 – 13. From streams and wet walls throughout the GSMNP.

Figs 1 – 2. Girdle view.

Figs. 3 – 13. Size diminution series.

Figs 14 – 22. From the wet walls and waterfalls throughout the GSMNP.

Figs 23 – 33. From epilithon at Hen Wallow Falls (CB watershed).

Figs 23. Starter cell.

Figs 24 – 32. Size diminution series.

Figs 24 – 32. Girdle view.

Figs 34 – 36. From streams in the CT, EP, FO, HZ watersheds.

Figs 54 – 56. From streams in the CT, HZ and NO watersheds.

Figs 57 – 65. Size diminution series from a wetwall along the Alum Cave Trail in the

WP watershed in the GSMNP. Fig. 57- starter cell?. 182 183

Plate 10:

Scale Bar = 2 µm: Figs 1 – 3, 5 – 6, 8.

1 µm for Figs 4, 7

Figs 1 – 5. Eunotia muscicola var. tridentula (Grunow) Nörpel et Lange-Bertalot.

Fig. 6 – 8. Eunotia microcephala Krasske

Figs 1 – 5. From streams and wet walls throughout the GSMNP.

Figs 1 – 2. (SEM) External views.

Figs 3 – 5. (SEM) Internal views. H = helictoglossa, R = rimoportula .

Figs 6 – 8. From Laurel Falls.

Fig. 6. (SEM) Internal view. H = helictoglossa, R = rimoportula.

Figs 7 – 8. (SEM) External views. 184 185

Plate 11:

Scale Bar = 10 µm.

Figs 1 – 29. Eunotia nymanniana Grunow.

Figs 1 – 18. Size diminution series. From Kilby Branch of Big Creek on

Big Creek Trail in the BG watershed, TN.

Figs 19 – 29. From streams and wetwalls from throughout the GSMNP.

186 187

Plate 12:

Scale Bar = 5 µm: Figs 1 – 2, 6; 1 µm for Figs 3 – 4; 2 µm for Fig. 5;

0.5 µm for Figs 6 – 7.

Figs 1 – 8. Eunotia nymanniana Grunow.

Figs 1 – 5. (SEM) External views of E. nymanniana from Kilby Branch

of Big Creek on Big Creek Trail in the BG watershed, TN.

Figs 6 – 8. (SEM) Internal views of Eunotia nymanniana from Little

Fork stream in HZ watershed. H = helictoglossa; R =

rimoportula, RP = external opening to the rimoportula. 188 189

Plate 13:

Scale Bar = 10 µm

Figs 1 – 11. Eunotia undulata Grunow

Figs 1 – 11. Size diminution series from the mouth of Abrams Creek (AB watershed).

See girdle views and SEM micrographs in Plate 14.

190 191

Plate 14:

LM: Scale Bar = 10 µm

SEM: Scale Bar = 10 µm: Figs 14 – 15.

2 µm: Fig. 16.

Figs 1 – 13. Eunotia pectinalis var. ventralis (Ehrenberg) Hustedt.

Figs 14 – 18. Eunotia undulata (Grunow)

Figs 1 – 13. From a sinkhole off Abrams Falls trailhead from Cades Cove Loop in the

AB watershed.

Figs 14 – 18. From the mouth of Abrams Creek (AB)

Figs 14 – 15. (SEM) External views. S = Spine

Fig. 16. (SEM) Internal view. R= rimoportula, H = helictoglossa.

Figs 17 – 18. Girdle views. 192 193

Plate 15:

Scale Bar = 10 µm

Figs 1 – 16. Eunotia macroglossa Furey, Lowe, Johansen (sp. nov.).

Figs 1 – 13. Size diminution series. From the type locality: Otter Creek, Cocke County,

TN.

Fig 3. Image of holotype (H).

Figs 14 – 16. Girdle view. From Russell Field Shelter spring, TN. 194 195

Plate 16:

Scale Bar = 5 µm: Figs 1, 5.

2 µm: Figs 2, 3, 6

1 µm: Fig 4.

Figs 1 – 6. Eunotia macroglossa Furey, Lowe, Johansen (sp. nov.).

Figs 1 – 6. From the type locality: Otter Creek, GSMNP, Cocke County, TN.

Figs 1 – 4. (SEM) External views. RP = external pore of the rimoportula.

Figs 5 – 6. (SEM) Internal views. H = helictoglossa, R = rimoportula . 196 197

Plate 17:

LM : Scale Bar = 10 µm

SEM: Scale Bar = 5 µm

Figs 1 – 10. Eunotia jemtlandica (Fontell) Berg.

Figs. 1 – 10. From various streams in the GSMNP.

Fig. 11. (SEM) Internal view. From a stream in the HZ watershed.

H = helictoglossa; R = rimoportula. 198 199

Plate 18:

Scale Bar = 10 µm

Figs 1 – 6. Eunotia formica Ehrenberg.

Figs 1 – 4, 6. From epilithon from a pool below the Spring House behind the Twin

Creeks facility (LC). Fig. 7 = girdle view.

Fig. 5. From a bryophyte substrate in Cosby Creek (CB). 200 201

Plate 19:

LM: Scale Bar = 10 µm

SEM: Scale Bar = 20 µm: Fig. 6.

10 µm: Fig. 7.

2 µm: Figs 8 – 9.

Figs 1 – 9. Eunotia sp. (GSMNP SP 20)

Figs 1 – 9. From a rock outcrop along the 20-Mile Creek Trail in the TW watershed.

Fig. 6. (SEM) External view.

Figs 7 – 9. (SEM) Internal views. H = helictoglossa; R = rimoportula. 202 203

Plate 20:

LM: Scale Bar = 10 µm

SEM: Scale Bar = 5 µm: Fig. 6.

1 µm: Fig. 7.

Figs 1 – 9. Eunotia braendlei Lange-Bertalot & Werum.

Fig. 1. From streams in the AB watershed.

Figs 2 – 5. From a pool in the LC watershed.

Fig. 6. (SEM) External view. Note the small broad spines at the junction of valve

face and mantle.

Fig. 7. (SEM) Close up of small broad spines at the junction of valve face and

mantle.

Figs 8, 9. (SEM) Internal views. H = helictoglossa; R = rimoportula. 204 205

Plate 21:

LM: Scale Bar = 10 µm.

SEM: 5 µm: Fig. 12.

2 µm: Figs 13, 14

1 µm: Fig. 15

Figs 1 – 11. Eunotia sp. (GSMNP SP 8)

Figs 1 – 11. Size diminution series. From streams in the BG watershed in the GSMNP

Figs 12 – 13. (SEM) External view

Figs 14 – 15. (SEM) Internal view

H = helictoglossa; R = rimoportula; RP = external opening of rimoportula. 206 207

Plate 22:

LM: Scale Bar = 10 µm.

SEM: Scale Bar = 5 µm.

Figs. 1 – 17. Eunotia curtagrunowii Nörepl-Schempp & Lange-Bertalot.

Figs. 18 – 19. Eunotia sp. (GSMNP SP 9).

Fig. 20. Eunotia rabenhorstii Cleve et Grunow.

Figs. 21 – 27. Eunotia c.f. macaronesica Lange-Bertalot & Tagliaventi nov. sp. prov.

Figs. 1 – 14. From a bryophyte substrate on a wetwall along highway 441.

Figs. 10 – 11. Girdle views.

Figs 15 – 17. From Copperhead Branch (IC watershed) and Jakes Creek

(EP watershed) and a wet wall at Meigs Falls.

Figs. 18 – 19. From Big Creek (BG watershed).

Fig. 20. From a wet wall along Little River Road (EP watershed).

Figs 21 – 25, 27. From a wet wall along Highway 441.

Fig. 26. From Big Creek (BG watershed).

Fig. 27. (SEM) External view. Image was taken by J. Ress. 208 209

Plate 23:

Scale Bar = 10 µm

Fig. 1. Eunotia sp. (GSMNP SP 10).

Figs 2 – 4. Eunotia bidens Ehrenberg.

Fig. 5. Eunotia diodonopsis Metzeltin & Lange-Bertalot.

Fig. 1. From Hesse Creek in the HS watershed.

Figs 2 – 4. From Hesse Creek in the HS watershed.

Fig. 5. From Cosby Creek in the CB watershed. 210 211

Plate 24:

Scale Bar = 10 µm

Figs 1 – 24. Eunotia sp. (GSMNP SP 11)

Figs 1 – 24. Specimens primarily from epiphytic substrates on a seep wall on

Chimney Tops Trail, but also from epiphytic substrates at Ramsay Cascades and Walker

Camp Prong near Alum Cave Creek. 212 213

Plate 25:

Scale Bar = 2 µm: Figs 2, 4.

1 µm: Figs 3, 8.

10 µm: Fig. 5.

5 µm: Figs 1, 6 – 7.

Figs 1 – 8. Eunotia sp. (GSMNP SP 11)

Figs. 1 – 3, 5. From a seep wall on Chimney Tops Trail

Figs. 4, 6 – 8. From a seep wall along Alum Cave Trail

Figs 1 – 6. (SEM) External views. RP = external opening of rimoportula.

Figs 7 – 8. (SEM) Internal views. H = helictoglossa; R = rimoportula. 214 215

Plate 26

Scale Bar = 10 µm

Figs 1 – 19. Eunotia bigibba Kützing

Figs 20 – 24. Eunotia sp. (GSMNP SP 21)

Figs 1 – 8, 20 – 24. From epilithon at Hen Wallow Falls

Figs 9 – 13. Brown slime on a wet wall at the edge of Meigs Falls.

Figs 14 – 19. From a sinkhole off the Abrams Falls trailhead from Cades Cove Loop 216 217

Plate 27

Scale Bar = 5 µm: Figs 1 – 3, 5 – 7.

1 µm: Figs 4, 8.

Figs 1 – 4. Eunotia bigibba Kützing

Figs 5 – 8. Eunotia sp. (GSMNP SP 21)

Figs 1 – 8. (SEM) From epilithon at Hen Wallow Falls

Figs 1 – 2. (SEM) External views of E. bigibba.

Figs 3 – 4. (SEM) Internal views of E. bigibba. H = helictoglossa; R = rimoportula.

Figs 5 – 6. (SEM) External views of GSMNP SP 21.

Figs 7 – 8. (SEM) Internal views of GSMNP SP 21. H = helictoglossa; R =

rimoportula. 218 219

Plate 28:

LM: Scale Bar = 10 µm.

SEM: Scale Bar = 10 µm: Figs. 6.

2 µm: Figs 7, 10.

5 µm: Figs 8, 9.

Figs 1 – 10. Eunotia sp. (GSMNP SP 12)

Figs 1 – 5. From epilithon at Hen Wallow Falls and in brown slime on a wet wall at

the edge of Meigs Falls.

Figs 6 – 10. From epilithon at Hen Wallow Falls

Figs 6 – 8. (SEM) External view.

Figs 9 – 10. (SEM) Internal view. H = helictoglossa; R = rimoportula. 220 221

Plate 29

Scale Bar = 10 µm

Fig. 1. Eunotia septentrionalis Østrup

Figs 2 – 9. Eunotia implicata Nörpel-Schempp, Lange-Bertalot, & Alles

Figs 10 – 13. Eunotia sp. (GSMNP SP 22)

Figs 14 – 19. Eunotia sp. (GSMNP SP 13)

Figs 20 – 29. Eunotia minor (Kützing) Grunow

Fig. 1. From Little Cataloochee River in the CT watershed.

Figs 2 – 9. From streams throughout the GSMNP.

Figs 10 – 13. From streams and wet walls in the DC, HS, HZ, IC watersheds

Figs 14 – 19. From streams in the AB, HS, and HZ watersheds

Figs 20 – 29. From streams throughout the GSMNP.

222 223

Plate 30

Scale Bar = 10 µm

Figs 1 – 14. Eunotia veneris (Kützing) De Toni

Figs 15 – 17. Eunotia pirla Carter & Flower

Figs 18 – 19. Eunotia sp. (GSMNP SP 14)

Figs 20 – 21. Eunotia sp. (GSMNP SP 15)

Figs 22 – 26. Eunotia sp. (GSMNP SP 16)

Figs 1 – 9. From streams throughout the GSMNP.

Figs 15 – 17. From Anthony Creek (AB watershed) and Andrews Bald Bog.

Figs 18 – 19. From streams and a spring in the NO and EP watersheds.

Figs 20 – 21. From streams in the AB and BN watersheds.

Figs 22 – 26. From streams in the AB and HZ watershed. 224 225

Plate 31:

Scale Bar = 10 µm

Figs 1 – 32. Eunotia sp. (GSMNP SP 23)

Figs. 33 – 38. Eunotia sp. (GSMNP SP 24)

Fig. 39. Eunotia rhomboidea Hustedt.

Figs 1 – 38. From streams throughout the GSMNP.

Fig. 39. Scanned images of Eunotia rhomboidea from Hustedt...plate, Figs 3 – 6. 226 227

Plate 32:

Scale Bar = 2 µm: Figs 1, 3.

1 µm: Fig. 4.

5 µm: Figs 5 – 6

Figs 1 – 8. Eunotia sp. (GSMNP SP 23)

Figs 1 – 8. (SEM) From streams throughout the GSMNP.

H = helictoglossa; R = rimoportula.

Figs 1 – 2. (SEM) External and internal view. See Plate 29:Figs. 1 – 10.

Figs 3 – 6. (SEM) External and internal view. See Plate 29:Figs. 11 – 21. 228 229

Plate 33:

LM: Scale Bar = 10 µm

SEM: Scale Bar = 2 µm: Figs 27, 28, 30.

1 µm: Figs 29.

Figs 1 – 30. Eunotia incisa Gregory

Figs 1 – 30. From streams throughout the GSMNP

Figs. 1 – 24. Size diminution series.

Fig. 25. Valve and girdle view.

Fig. 26. Girdle view.

Figs 27 – 28. (SEM) External view.

Fig. 29. (SEM) Close up of valve apex showing external pore of the rimoportula.

Figs 30. (SEM) Internal views of E. incisa. H = helictoglossa; R = rimoportula. 230 231

Plate 34:

Scale Bar = 10 µm

Figs 1 – 11. Eunotia billii Lowe & Kociolek

Figs 1 – 11. From streams and wet walls around the GSMNP.

Figs 1 – 7. Valve view.

Figs 8 – 11. Girdle view. 232 233

Plate 35:

Scale Bar = 10 µm: Figs 1, 3, 4.

5 µm: Fig. 2.

2 µm: Figs 5, 6.

Figs 1 – 6. Eunotia billii Lowe & Kociol.

Figs 1 – 6. From streams and wet walls around the GSMNP.

Figs 1 – 2. (SEM) External views.

Figs 3 – 6. (SEM) Internal view. H = helictoglossa; R = rimoportula.

Image 4 was taken by J. Ress.

234