ABSTRACT Unexpected Environmental Conditions Suggest

Home , Lycopodium, Selaginella, Selaginella kraussiana

ABSTRACT

Unexpected Environmental Conditions

Suggest Paleozoic Plant Morphological Gas Conductance Models

Christopher J. A. Skrodzki

Director: Joseph D. White

The importance of plants in regulating and defining Earth’s greenhouse gas and water vapor composition has been previously demonstrated. This study addresses the relationship between the morphological and physiological response of paleo-plants to changing atmospheric gas compositions, which in turn lead to changes in atmospheric pressures. Higher atmospheric pressures are here suggested to alter plant gas exchange dynamics and Photosystem II activation. These effects increases plant bulk carbon dioxide, an important greenhouse gas, and water vapor transport leading to changes in Earth’s climate through alterations in the carbon cycle and hydrological balance. To elucidate this relationship, the response of two extant lycopod species, Selaginella kraussiana and Lycopodium lucidulum, was measured in response to an atmospheric pressure of 5kPa over current conditions. Results show that L. lucidiulum changed leaf shape, decreasing in stomatal density but increasing in stomatal index, in response to higher pressures and harbors a closer correlation with stomatal conductance values in response to stomatal index over maximal stomatal aperture values. S. kraussiana, exhibited an increase in stomatal density and index values in response to increased pressures and that its stomatal conductance values are more dependent on maximal stomatal aperture values than stomatal index This research demonstrates that paleo-plant stomatal indices are by themselves not accurate measures of atmospheric carbon dioxide or water vapor values as two extant paleo-plants of closely related phyla exhibit confounding results. These results suggest a reexamination of geological atmospheric conditions by showing that paleo-plant gas exchange can be influenced by atmospheric conditions other than carbon dioxide composition.

APPROVED BY DIRECTOR OF HONORS THESIS:

______Dr. Joseph D. White, Department of Biology: Baylor University

APPROVED BY HONORS PROGRAM:

______Dr. Andrew Wisely, Director

DATE: ______

UNEXPECTED ENVIRONMENTAL CONDITIONS SUGGEST

PALEOZOIC PLANT MORPHOLOGICAL GAS CONDUCTANCE MODELS

A Thesis Summited to the Faculty of

Baylor University

In Partial Fulfilment of the Requirements for the

Honors Program

Christopher J. A. Skrodzki

Waco, Texas

May 2015

TABLE OF CONTENTS

List of Figures ...... pg. iv

List of Tables ...... pg. v

Abbreviations ...... pg. vi

Acknowledgments...... pg. vii

Dedication ...... pg. viii

Chapter One: Introduction ...... pg. 1

Background ...... pg. 1

Hypotheses ...... pg. 3

Chapter Two: Materials and Methods...... pg. 5

Experimental Design ...... pg. 5

Morphological Effects ...... pg. 6

Gas Exchange Rates ...... pg. 6

Photosystem II Activation ...... pg. 8

Chapter Three: Results ...... pg. 9

Morphological Effects ...... pg. 9

Gas Exchange Rates ...... pg. 11

Photosystem II Activation ...... pg. 16

TABLE OF CONTENTS

Chapter Four: Discussion ...... pg. 17

Morphological Effects ...... pg. 17

Gas Exchange Rates ...... pg. 18

Photosystem II Activation ...... pg. 19

Concluding Remarks ...... pg. 20

Future Directions ...... pg. 21

Appendices ...... pg. 23

Appendix A – Global climate change over geological time ...... pg. 24

Appendix B – Modern angiosperm leaf morphology and gas exchange ...... pg. 25

Appendix C – Model of used pressure chambers of this study ...... pg. 26

Appendix D – Model of pressure chamber for proposed further studies ...... pg. 27

Appendix E – Full data table of morphological effects ...... pg. 28

Appendix F – Full data table of gas exchange rates ...... pg. 29

Appendix G – Full data table of Photosystem II Activation ...... pg. 30

References and Suggested Reading ...... pg. 31

iii

LIST OF FIGURES

Figure 1 – A L. lucidulum enamel impression after treatment of ≈105kPa at 100X...... pg. 11

Figure 2 – CO2 Conductance vs. Stomatal Index ...... pg. 14

Figure 3 – CO2 Conductance vs. Maximal Stomatal Aperture Area ...... pg. 15

LIST OF TABLES

Table 1 – Morphological Effects of Different Atmospheric Conditions ...... pg. 10

Table 2 –Gas Exchange Rates in Response to Different Atmospheric Conditions, part 1 ....pg. 13

Table 3 – Photosystem II Activation in Response to Different Atmospheric Conditions pg. 16

ABBREVIATIONS

Amax – Maximal Stomatal Aperture Area

CO2 – Carbon dioxide

ETR – Linear Electron Transport Rate

GCO2 – Carbon dioxide gas Conductance

GH2O – Water vapor Conductance

GL – Leaf carbon dioxide Conductance

GS – Stomatal carbon dioxide Conductance

Ha – Alternate Hypothesis

Ho – Null Hypothesis

Index – Stomatal Index

KP – leaf water Conductivity

Mya – Million years ago

PAR – Photosynthecially Active Radiation

Pn. – Photosynthesis

ΦPSII – PhotoSystem II Quantum Yield

RuBisCO – Ribulose-1,5-Bisphosphate Carboxylate

Trans. – Water Vapor Transpiration

VPD – Vapor Pressure Deficit

VPS – Saturated Vapor Pressure

WUE - Water Use Efficiency

ACKNOWLEDGEMENTS

I would like to first and foremost sincerely thank Dr. White for helping me maintain a clear sense of focus for this thesis work, his honest critic and assistance editing this work, and for his inexorable patience in assisting me through this entire process.

As well, I would find it wrong to not extend another thank you to all of the professors here at Baylor that have been a part of my personal journey in quenching my thirst for knowledge and introducing me to the world of real scientific research.

Lastly, without the administrative support and opportunity of the Honor’s Program, none of this would have been written before you.

vii

Dedicated to A. C. M.

For inspiring me to keep working, even when I know you will probably never read this.

~Thank you.

viii

CHAPTER ONE

Introduction

Background

Lycophytes, also known as lycopsids, are the basal extant lineage of all vascular plants,

of which the genera Selaginella (spike moss) and Lycopodium (club moss) are representative of a pre-transitional early-late phase of the Pennsylvanian epoch (Dimichele et al., 2009). The phylum Traecheophyte is considered polyphyletic with the genera Selaginella and Lycopodium representing distinctly derived orders of Sellaginales and Lycopidiales, respectively, with

Lycopodium being the most basal (Bateman, 1990; Wikström and Kenrick, 2001). Two extant species, Selaginella kraussiana and Lycopodium lucidulum, contain tracheid structures which are representative of the flora dominating this time period (Bierhorst, 1971; Chu, 1974; Friedman and Cook, 2000). The adaptations of these organisms to effectively react quickly to periods of

drought allowed survival through the arid late Devonian (Arrigo et al., 2013; Gueidan et al.,

2011). It has been proposed that the distribution of plants 307 Mya underwent a multiply-

punctuated and irreversible shift from a diverse Euramerican equatorial ecosystem containing

several lycophyte ancestors to one dominated by seed ferns, of which the genus Isoetes is extant,

likely caused by ecological fragmentation of wetlands (DiMichele and Bateman, 1996;

Dimichele et al., 2009).

The late Carboniferous period, known as the Pennsylvanian epoch, has been proposed to

have experienced several short (several hundred thousand years) change which progressively

decreased atmospheric moisture levels leading to increased seasonal tropical drying ultimately

resulting in a reorganization of wetland vegetation to lush rainforests dominated by giant fern

canopies (Dimichele et al., 2009). This change in global hydrologic cycling potentially lead to geologically unprecedented to climate change, wherein a period of global warming transitioned to a global ice age marked by oscillating planet-wide periods of intense glaciation and vegetative regrowth (Appendix A) (Dimichele et al., 2009; Royer et al., 2004). Although carbon dioxide

levels have been implicated in this climate change, what is more striking is the increases in

oxygen concentration during the late Carboniferous; Atmospheric carbon dioxide concentrations

during the Pennsylvanian epoch were only 20ppm higher than modern day levels of 400ppm;

however, the oxygen concentration of the Pennsylvanian epoch was roughly 10% higher (Berner,

2009; Came et al., 2007).

The effect of oxygen levels’ upon vascular, non-seeding, plant has not previously been examined; however, oxygen has been demonstrated to directly influence leaf gas exchange and photosynthetic capacity (Igamberdiev and Lea, 2006). Since the leaf morphology of

Pennsylvanian-derived lycopophytes is non-analogous of modern angiosperms, the underlying assumption that plant stomatal densities depend solely upon atmospheric carbon dioxide conditions is questionable (Beerling et al., 2001; Konrad et al., 2008). Although previous research on temperature driven Vapor Pressure Deficits (VPD) indicated no changes to the rate of transpiration within Selaginella, there is not a convincing body of evidence directly implicating the Water Use Efficiency (WUE) of lycophytes to their morphological lack of spongy mesophyll tissue (Soni et al., 2012). Accurate models of gas exchange indicated that changes in the partial pressure of different gas-species directly relates to changes in specie assimilation and photosynthesis (Farquhar et al., 1980).

Extant lycophytes, representative of the early-late Pennsylvanian epoch were maintained within an artificial Paleozoic atmosphere in order to elucidate the relationship between atmospheric gas concentrations and pressure to the density of plant leaf stomata. The effects of increased atmospheric pressures were characterized within one pre-Pennsylvanian- transitional and one post-transitional model species of similar phyla, through measurements of stomatal density, stomatal aperture sizes, gas exchange rate, and fluorescence. Stomatal carbon dioxide conductance and photosynthesis rates gathered as results of this research hope to address the fundamental assumption that vascular, non-seeding, plant gas exchange are only dependent upon atmospheric carbon dioxide levels within Paleozoic geologic time. This research suggests the beginning of what is known as the ‘K question’- the correlation between hydraulic stomatal leaf conductance (GL) and leaf water conductivity (KP), as carbon dioxide assimilation directly

corresponds to the ratio of conductance between carbon dioxide (GCO2) and water vapor (GH2O).

Hypotheses

A 420ppm carbon dioxide and 30% oxygen concentration is representative of late

Carboniferous period levels well past the late-middle Pennsylvania (300 Mya), while the pressure is has been estimated at approximately 20kPA above normal atmospheric sea-level

(Berner, 2006, 2009). An increase in pressure was tested in order to assess whether vascular, non-seeding, plant responded to changes in atmospheric composition, which has been demonstrated to have changed over time, considering that large oxygen atmospheric concentration changes (10%) would produce a significant difference in its partial pressure. The following hypotheses were generated from this main objective:

o  H0 : There is no relationship between changes in atmospheric conditions upon

either S. kraussiana or L. lucidulum leaf anatomical changes.

o HA : Exposure of S. kraussiana or L. lucidulum to atmospheric pressures of

105kPa for one month is positively correlated with increased development of

stomata per unit area on new leaves.

 H0ˊ: There is no relationship between changes in atmospheric conditions upon

either S. kraussiana or L. lucidulum and leaf chorophyllic fluorescence.

HA1ˊ: Exposure of S. kraussiana or L. lucidulum to atmospheric pressures of

105kPa for one month is positively correlated with increased leaf chorophyllic

fluorescence in old leaves.

 H0ˊˊ: There is no relationship between changes in atmospheric conditions upon

either S. kraussiana or L. lucidulum gas exchange rates.

HAˊˊ: Exposure of S. kraussiana or L. lucidulum to atmospheric pressures of

105kPa for one month is positively correlated with increased gas exchange in old

leaves.

 H0ˊˊˊ: If there are responses, then there is no difference between the response of

between S. kraussiana and L. lucidulum to atmospheric conditions.

HAˊˊˊ: If there are responses, then S. kraussiana and L. lucidulum differ in

response to atmospheric pressures of 105kPa for one month.

CHAPTER TWO

Materials and Methods

Experimental Design

For this experiment S. kraussiana and L. lucidulum, were subjected to increased pressures

(kPa) within laboratory constructed pressure chambers. Starting from an initial batch of 20 plants from each species, the healthiest six of each species of plant was run at the elevated pressure conditions in triplicate alongside a triplicate at ambient constant air-flow conditions. A single treatment of potting soil was maintained alongside these plants in order to assess carbon dioxide concentration values of carbon dioxide release from background soil bacterial respiration.

Each plant was maintained for one month (30 days) within individual lab constructed 4L chambers at an elevated pressure of approximately 105kPa after acting as controls at lab ambient pressures for one month. Each chamber was outfitted with 3 luer-lock connectors fitted via individual #60 O-rings to approximately 20ft of NSF 61 grade tubing and three two-way valves.

All chambers were maintained at a relative humidity of 80.03%, thereby reducing the vapor pressure deficit (VPD) of each system to 0.0212 bar. In order to maintain high chamber humidity, laboratory air was bubbled through DI water before being fed into individual chambers for pressurization that were filled with 100mL of gravel and 100mL of DI water. The concentration of carbon dioxide (ppm), oxygen gas (%), pressure (kPa), and humidity within each chamber was monitored via a Vernier LabPro System and removable proprietary sensors

(Appendix C).

Morphological Effects

Plant leaf stomatal morphology was characterized through optical imaging and

subsequent image processing. Stomata morphological measurements were made using leaves

taken from just below the two centimeter mark of these stems. Leaf samples were coated in nail-

polish on their abaxial sides and allowed to set overnight. Enamel strips were then collected from

the leaf samples and plated onto microscope slides. Anatomical changes between new and old

growth leaves will be monitored via bright-field, stage microscopy. Leaf samples were digitally

imaged under an Olympus BH-2 light transmission microscope via a OMAX A35140U camera.

Samples leaves were aligned vertically under the scope and aligned to a digital 3x3 grid

of 500 by 500 size boxes, placing the highest concentration of stomata within the center box.

Pavement and guard cell counts were digitally assisted and conducted under proprietary scope

camera software, counting only from three boxes across a central diagonal. Stomatal aperture

size measurements were conducted digitally within NIH ImageJ software upon raster images

generated from microscopic imaging (Schneider et al., 2012). All areas were calculated relative

to a 0.1 mm scale bar.

Stomatal index values were calculated as (Aliniaeifard et al., 2014):

Index (1)

Gas Exchange Rates

The stem tip area of each model was calculated from control treatment leaf samples that

were graphically imaged. Each area considered only one side of each sample leaf as L. lycopodium and S. kraussiana leaves only have stomata allowing gas flow on their abaxial leaf sides with the addition of a geometrically perfect cylindrical stem. Adaxial plant leaf sides were

not viable for measurement as these sun facing sides are protected by waxy cuticular tissue, lacking stomata. The area was used to take measurements was two centimeters from the tip of each plant stem. Gas exchange rates of carbon dioxide and water vapor were measured via a CID

CI-340 Hand-held Photosynthesis System calibrated using a CID CI-301AD gas control module.

Plant stomatal water vapor conductance values were thus calculated with respect to observed morphological stomatal data; where d is the diffusivity of water within air in m⁄s, D

is stomatal density in m , amax is the maximum measured stomatal aperture size of a sample in

μm, l is pore depth in µm, and is the moral volume of air in m⁄mol, at 22oC (Bolz and Tuve,

1973; Franks and Farquhar, 2001):

∙ ∙ G = (2) ⁄ ∙ ∙

d 2.775 ∗ 10 4.479 ∗ 10 ∗ 22C 273.15C = m2⁄s (3)

1.656 ∗ 1022C 273.15C

GCO2 = GH2O ∙ (4) Water vapor transpiration rates was calculated with respect to water vapor gas conductance in application with Fisk’s Law via Vapor Pressure Deficit (VPD) in terms of pressure, Saturation (VPS), and measured Relative Humidity in terms of % (RH), in oC (Eaton and Kells, 2009):

VPD E G ∗ = (5) P ⁄

VPD VPS ∗ RH/100 (6)

VPS 6.1121e.⁄ . (7)

These calculated water vapor stomatal conductance values were graphed in comparison

to the morphological parameters of stomatal index and maximal aperture size. A linear

relationship was calculated between the two, indicating the nature of the correlation between the

physiological and morphological responses to changes in atmospheric pressures.

Photosystem II Activation

Photosynthesis was additionally measured with the addition of a CID CI-510CF

fluorescence module. This module estimates the rate of Photosynthesis (Pn) via the amount of

carbon dioxide taken up by a sample (ΔC) over a photosynthetic area (A) and period of time (Δt),

at a given barometric Pressure (P) and atmospheric Temperature (T).

Chlorophyll fluorescence spectroscopy allows the efficiency of Photosystem II (ΦPSII) to

be calculated, via the function of light-adapted plant leaf ‘flash’ maximal (F) and steady state

(F ) fluorescence, which can be multiplied by the amount of PAR in and the amount of light t ⁄ absorbed by plant chlorophyll (84%), which is split 1:1 between PSI (P700 reaction center) &

PSII (P680 reaction center), gives information concerning the electron transport rate, also known as J (Maxwell and Johnson, 2000):

ΦPSII = (8)

ETR = PAR * ΦPSII * 0.84 *0.5 = (9) ⁄

This electron transport rate is proportional to the absorbed photon flux, or Incidence,

through stomatal pores (I); this rate is in turn proportional to stomatal rates of bulk carbon dioxide assimilation by what is known as a quantum yield (Farquhar et al., 1980).

CHAPTER THREE

Results

Zero values were trimmed from pooled values and only two plants are representative of

pressurized S. kraussiana, due to high plant die off and chamber algal or fungal contamination.

As noted within the stated methodology: all control morphological data is representative of twenty plants of each species , of which the three selected for each experimental treatment are displayed via internal ID; however, all control physiological measurements were made using plants which were later not used.

Morphological Effects

Due to the distributions of stoma within L. lucidulum and S. kraussiana, stomatal density counts were taken along leaf peripheries for L. lucidulum and the midline of S. kraussiana.

Carboniferous Quercus petraea pore depth values were slightly adjusted to that of 21.5µm for each plant and assumed constant (Konrad et al., 2008).

Preliminary data indicates that the effect of different pressure levels upon different paleo- plant models of similar phyla exhibit a differential response in changes tip leaf morphology, in addition to differing base control values between the two species. Experimental treatments seemed to have a limited effect upon index values, increasing in L. lucidulum from 2.09 ±

0.53 % to 2.67 ± 0.63 % under pressure and 2.81 ± 1.16 % in heavy flow and increasing in S. kraussiana from 8.95 ± 1.98 % to 15.84 ± 4.58 % under pressure and 11.13 ± 2.01 % in heavy flow; a more pronounced effect upon density values, increasing in L. lucidulum from 81.49 x 106

± 17.65 x 106 m-2 to 69.03 x 106 ± 9.86 x 106 m-2 under pressure and 82.18 x 106 ± 22.78 x 106

m-2 in heavy flow and increasing in S. kraussiana from 118.86 x 106 ± 34.40 x 106 m-2 to 197.24

x 106 ± 69.73 x 106 m-2 under pressure and 115.06 x 106 ± 28.47 x 106 m-2 in heavy flow, and

minimal pore aperture area value effects, not changing in L. lucidulum from 366.72 ± 137.44 µm2 to 349.69 ± 131.31 µm2 under pressure and 303.73 ± 107.78 µm2 in heavy flow while changing

only very little in S. kraussiana from 76.00 ± 22.86 µm2 to 93.09 ± 6.88 µm2 under pressure and

50.95 ± 8.27 µm2 in heavy flow- (Table 1).

-2 2 Index (%) Density (m ) amax (µm ) Lycopodium lucidulum Control 2.09 ± 0.53 (19) 81.49 x 106 ± 17.65 x 106 (19) 366.72 ± 137.44 (20) Pressurized 2.67 ± 0.63 (3) 69.03 x 106 ± 9.86 x 106 (3) 349.69 ± 131.31 (3) Flow Through 2.81 ± 1.16 (3) 82.18 x 106 ± 22.78 x 106 (3) 303.73 ± 107.78 (3) Selaginella kraussiana Control 8.95 ± 1.98 (19) 118.86 x 106 ± 34.40 x 106 (19) 76.00 ± 22.86 (20) Pressurized 15.84 ± 4.58 (2) 197.24 x 106 ± 69.73 x 106 (2) 93.09 ± 6.88 (2) Flow Through 11.30 ± 2.01 (3) 115.06 x 106± 28.47 x 106 (3) 50.95 ± 8.27 (3)

Table 1 – Mean data ± standard deviation (sample size), the morphological response of individual paleo-plant specimens from within a population sample to pressurization at approximately 105kPa and constant internal chamber air flow.

Additionally, increased pressure values led to a marked change in plant leaf internal

shape noted within a single L. lucidulum sample, plant 18 (Figure 1). This seemingly increased

leaf striation, though present within only one sample, is suggested as significant there were only

two other plants which could have exhibited this change.

Figure 1 – A L. lucidulum enamel impression after treatment of ≈105kPa at 100X.

Gas Exchange Rates

The morphological distribution of stomata across paleo-plant leaves is a complex contributor to leaf CO2 conductance. The effects of paleo-plant stomatal apertures and stomatal index upon bulk stomatal CO2 conductance between Lycopodium lucidulum and Selaginella kraussiana are markedly distinct. This tip area was assumed constant for each model species:

6.08 ± 0.60 cm2 and 0.62 ± 0.08 cm2, L. lycopodium and S. kraussiana respectively.

Calculated values were derived from morphological measurements, while experimental

values represent direct data outputs from the CID Photosystem instrument described within the

methods (Table 2). For this reason, calculated values tend to be a magnitude and a half higher

than all experimental values. Photosynthesis measurements were found to clearly increase in L.

lucidulum from 1.06 ± 0.56 to 2.24 ± 1.09 under pressure, increasing in experimental ⁄ ⁄

transpiration and stomatal conductance values from 0.36 ± 0.10 & 13.13 ± 3.12 to 0.40 ⁄ ⁄

± 0.05 & 19.24 ± 4.94 under pressure, while decreasing in morphologically calculated ⁄ ⁄

mmolCO 2 values from 21.13 & 417.09 to 17.43 & 348.28 , respectively; the responses ⁄ m2⁄ ⁄ ⁄

under heavy flow conditions was similar to control response values. However, S. kraussiana

photosynthesis values were found to decrease from 7.53 ± 3.58 to 3.88 ± 0.34 under ⁄ ⁄

pressure, showing little change in experimental transpiration but increases in stomatal

conductance from 1.89 ± 0.24 & 70.65 ± 9.72 to 2.94 ± 1.04 & 144.94 ± 40.34 ⁄ ⁄ ⁄

under pressure, while morphologically calculated values clearly increased from 7.53 ⁄ ⁄

& 146.47 to 15.32 & 306.10 ; the responses under heavy flow conditions were ⁄ ⁄ ⁄ more similar to control response values. As with morphological values, this data suggests significant differences between L. lucidulum and S. kraussiana gas exchange values.

Experimental Calculated Experimental Calculated Photosynthesis Stomatal Stomatal Transpiration Transpiration Conductance Conductance ⁄ mmol mmol ⁄ ⁄ m⁄ m⁄ Lycopodium lucidulum Control1 1.06 ± 0.56 (8) 0.36 ± 0.10 (8) 21.13 (19) 13.13 ± 3.12 (8) 417.09 (19) Pressurized2 2.24 ± 1.09 (2) 0.40 ± 0.05 (2) 17.43 (3) 19.24 ± 4.94 (2) 348.28 (3) Flow Through 0.55 ± 0.30 (3) 0.34 ± 0.02 (3) 4.52 (3) 14.57 ± 0.02 (3) 357.47 (3) Selaginella kraussiana Control1 7.53 ± 3.58 (6) 1.89 ± 0.24 (6) 7.53 (19) 70.65 ± 9.72 (6) 146.47 (19) Pressurized 3.88 ± 0.34(2) 2.94 ± 1.04 (2) 15.32 (2) 144.94 ± 40.34(2) 306.10 (2) Flow Through 5.47 ± 0.68 (3) 1.78 ± 0.40 (3) 5.11 (3) 68.35 ± 17.80 (3) 99.47 (3)

Table 2 – Mean data ± standard deviation (sample size), the gas exchange of individual paleo- plant specimens from within a population sample to pressurization at approximately 105kPa and chambered with constant internal chamber air flow. Note: 1) the control plant responses were pooled as values do not represent the same organisms that were later experimentally treated; 2) some data has been trimmed due to poor instrumental response.

The relationship between the observed morphological parameters of stomatal index

(Figure 2) and maximum stomatal aperture size (Figure 3) upon modeled physiological gas exchange was graphed and details a marked difference between these closely related species of

Lycopods in terms of morphological sensitivity upon gas exchange.

Figure 2 – The effect of paleo-plant stomatal index counts upon modeled stomatal carbon dioxide gas conductance indicate a closer relationship between L. lucidulum stomatal index than in S. kraussiana to stomatal gas conductance sensitivity under various environmental conditions.

Figure 3 – The effect of paleo-plant maximum stomatal aperture area values upon modeled stomatal carbon dioxide gas conductance indicate a closer relationship between S. kraussiana. stomatal size than in L. lucidulum to stomatal gas conductance sensitivity under various environmental conditions.

Photosystem II Activation

Fluorometric measurements were conducted in a very similar manner as gas exchange

measurements were. The data below indicates clear changes in plant electron transport rates in extant pale-pleant responses to pressure withvalues increaing both plants and under both of the

test envirnmental conditions with L. lucidulum increasing limitiedly from 0.12 ± 0.08 to ⁄

0.30 ± 0.05 under pressure and more notably to1.46 ± 0.82 in heavy flow, while S. ⁄ ⁄

kraussiana increased markedly from 0.21 ± 0.09 μmol to 3.42 ± 0.12 under press and to 6.88 m2⁄ ⁄

± 1.16 in heavy flow (Table 3). Experimental values PAR values were also collected and ⁄

used proporitarily calculate ETR and should not be taken as meaningful by themselves, therefore

these are not displayed nor are ΦPSII values.

Electron Transport Rate ⁄ Table 3 – Mean data ± standard deviation (sample size), the Lycopodium lucidulum Photosystem II activation of individual light-adapted paleo-plant Control1 specimens from within a population sample to pressurization at 0.12 ± 0.08 (6) approximately 105kPa and chambered with constant internal chamber air flow. Note: 1) the control plant responses were pooled as values do not Pressurized represent the same organisms that were later experimentally treated 0.30 ± 0.05 (3) Flow Through 1.46 ± 0.82 (3) Selaginella kraussiana Control1 0.21 ± 0.09 (7) Pressurized 3.42 ± 0.12 (2) Flow Through 6.88 ± 1.16 (3)

CHAPTER FOUR

Discussion

The results of this study indicate that within extant species of clades of closely related

Paleozoic plants there are significantly different morphological characteristics that effect leaf

stomatal carbon dioxide conductance. The response of the pre-early-late Pennsylvanian

transitional model species S. kraussiana is more tightly correlated to the size of its stomata than the concentration of its stomata, while the post-transitional model species L. lucidulum shows the

opposite trend. However, while it is inconclusive whether or not the organization of plant

Photosystem II’s were a product of increased pressures or merely a chamber effect, S.

kraussiana has been found to be more susceptible to these effects than L. lucidulum. These results indicate different levels of model species plant robustness in response to atmospheric conditions. This suggests similar properties in the ancestors of these extant plants and a plausible explanation for the ecological reorganization that occurred approximately 307 Mya.

Morphological Effects

These morphological differences are easily observable via microscopy, allowing fossil study. Lycopodium lucidulum and Selaginella kraussiana, though of the same phyla and each having evolved in roughly the same epoch of the late Devonian, exhibited differential species dominance across Paleozoic time (Dimichele et al., 2009). Increased pressure has been demonstrated to decrease the stomatal density of L. lucidulum, while constant air-flow

environments do not lead to any significant change. However, stomatal index values were found

to increase in both plants subjected to increased pressures as well as constant air-flow

environments. Only increased pressures were found to increase the stomatal densities and index

values within S. kraussiana. The maximal stomatal aperture was not found to significantly

respond to either of these conditions in either species. This data suggests that under increased

pressure conditions L. lucidulum plants will develop more stomata yet of lower leaf

concentration. These results alongside the increased leaf striation found within one of the high

pressure leaves of L. lucidulum, sample 18, suggest a gross leaf anatomical change in shape in

response to increased pressure levels. Further analysis of L. lucidulum plant leaves supports

conclusion, as the length-to-width ratio shrank from 2.04 ± 0.99 (719) in control leaves to 1.69 ±

0.52 (74), in terms of mean ± standard deviation (sample size). Additionally, that suggests that

air flow rates are significant only to L. lucidulum in effecting the stomatal index, unlike the

smaller and more spindly formed S. kraussiana plants. The anatomy of these plant stomata does

not appear to be susceptible to change in size rather than responding to changes in atmospheric

conditions through normal opening and closing through guard cell turgor pressure.

Gas Exchange Rates

The effect of atmospheric carbon dioxide concentrations on stomatal densities has been

demonstrated to lead to increases in vascular, non-seeding, plant stomatal densities (Konrad et al.,

2008). However, the data of this study suggests trends that demonstrated that extant paleo-plants

exhibit changes not only in morphology but also that plants of closely related clade respond

different to these conditions. Current geological atmospheric models, such as from Haworth

(2011), are based on paleo-plant stomatal indices may not be representative of later-

Carboniferous, Pennsylvania (300 Mya, lycopsids. This is because these models are based on the

assumption that the morphology of modern angiosperms is representative of lycophytes, which

lack mesophilic spongy tissue that affects derived maximum gas conductance (Appendix B)

(Chu, 1974).

The calculated method yielded gas exchange values based on the collected morphological

data, while the experimental data is contingent upon the CID-340 measured mass flow rates. For

this reason, for this reason I found that the experimental transpiration values are not proportionally based on Gs values like the calculated values are, suggesting a degree of

reliability; however, the Gs values of the CID instrument are estimated from bulk carbon dioxide

measured GL values. Considering that the CID instrument has been designed for the measuring

of modern angiosperm tree leaves that lack similar leaf mesophyll cellular organization to that of extant paleo-plants, the GL derived Gs values of the CID-340 are suspect. Further research is

suggested to better understand paleo-plant mesophyll conductance and its relationship to gross

leaf gas conductance. Therefore, this data suggests that these effects are the results of stomatal

distribution and shape differences between the two species. The morphological contribution to

CO2 conductance through lower-vascular paleo-plant leaves is more than “skin-deep.”

Photosystem II Activation

I found that increased chamber pressure is did not conclusively affect photosystem II

activation in L. lucidulum or S. kraussiana based on the collected fluorescence data. While there

was a magnitude difference in between the PAR of the control and experimental treatments, the

linear electron rate is not proportional to just this value and it should not be taken as an

experimental effect, only an indicator of instrumental inconsistencies (Maxwell and Johnson,

2000). The data showed that L. lucidulum’s PSII was not affected by the presence of increased

pressures, while increased air flow only in light-adapted (1.46 ± 0.82 from 0.12 ± 0.08) ⁄ ⁄ suggests a weak effect. However, this difference was associated with a relatively large standard error and is likely due to random experimental error. In contrast, a significant effect could be clearly seen within S. krauaiana’s fluorescence data, wherein, the electron transport rate of light-

adapted S. kraussiana plants increased from (0.21 ± 0.09 ) in response to both pressurization ⁄

(3.42 ± 0.12 and under constant air flow environments (6.88 ± 1.16 ). ⁄ ⁄

These results seem to suggest that air flow within the chambers chamber affected plant

photosystem II activity. Given that the presence of air leaks limited the maximal pressure

achievable within the chambers used in this experiment both the pressurized and the constant air flow chambers had a high rate of atmospheric turn-over within each chamber. Thus, it seems more plausible that this observed increase in electron transport rate is indicative of an increase in photosystem II efficiency (Franks and Farquhar, 2001). This is suggested to be due to the presence of light and a constant gas flow supply to plant thylakoid lamella by maximizing the light reactions of photosynthesis and limiting the amount of metabolically available gases by only conductance rate of the plant stomata and mesophyll. This data suggests that the larger and less tuberous stemmed L. lucidulum seems more resistant to these changes in response to atmospheric conditions.

Concluding Remarks

These data are important for improving understanding of Paleozoic climatological gas concentration estimates as I have demonstrated as rates of paleo-plant gas exchange are subject to changes in coincident atmospheric pressure. These results, though preliminary, counter key assumptions used in the generation of Paleozoic atmospheric estimates, specifically for carbon dioxide, and that stomatal indices of Paleozoic plants are not solely responsive to atmospheric carbon dioxide levels. Previous work acknowledges that changes in the composition of Earth’s atmosphere have leads to changes in atmospheric pressures throughout historical deep-time

(Graham et al., 1995). The results of this study allow the calculation of accurate gas exchange

profiles of Paleozoic plants similar in shape and phyla to S. kraussiana and L. lucidulum, not

necessarily limited to only carbon dioxide. This research suggests that atmospheric conditions

other than carbon dioxide composition and water vapor balancing factors contribute to the

density of stomata within vascular, non-seeding, plants and that this is plausible due differences

in the oligovascular structure between modern and extant paleo-plant leaves. Using this

procedure, it is possible to model the conductance of any gas systems to geologically relevant

paleo-plants, as has been done for carbon dioxide within this study.

The suggested model resulting from this research hypothesis will be able to aid scientists

better understand the relationship between plants, the carbon cycle, and the Earth’s climate. This knowledge is inherently fundamental to understand plants and should be included as standard biological curriculum in hope of better educating future generations to the realities of global climate change. Additionally, insights into Pennsylvanian plants versus modern vascular plant

Water Use Efficiencies (WUE)s in response to atmospheric conditions provide significant hydraulic data that may be of use agriculturally in world growing more concerned with dwindling fresh water sources and rising levels of greenhouse gases. An understanding of this relationship is important as this also allows more accurate prediction of future weather patterns in response to greenhouse gases - both of which directly affect the Earth’s climate and thus human health.

Future Directions

The chamber designs used within this study were not able to reach the 115-125kPa pressure range representative of the early-late Pennsylvanian. Hence, a more efficient and solidly built chamber design is suggested (Appendix D). Additionally, the responses of S. kraussiana and L. lucidulum in new growth leaves versus old growth leaves have not been examined in

detail. It is not clear whether or not the deterministic growth pattern of these plants, effectively

fixing the morphology of growth leaves, is the major factor in produce the results presented in this report. It possible that old growth leaves also contributed to changes in plant gas conductance through changes in Ribulouse-1,5-Bisphosphate Carboxylase (RuBisCO) expression. Interest has also been expressed in the measurement of carbon isotope ratio levels of cutin fatty-acid chains in response to different atmospheric conditions.

The results of this data provide for a potential two year study into the effects of atmospheric conditions upon paleo-plant morphological and physiological responses. The first of which is proposed to an extension of the preliminary experiments to a three-month period in order to better extenuate any trends which may be present within preliminary data. Additionally, in order to elucidate a broader picture of the various responses of lycopdis to Paleozoic atmospheric conditions, additional model genera of Marchantia, Polypodium late-Carboniferous gymnosperm Cycad, and/or a modern angiosperm Arabidopsis. In order to fully understand the effect of deterministic growth in response to altered atmospheric conditions, further studies are proposed to analyses the difference in responses in between saplings and full-growth plants.

Consequent experiments, include: elucidation of the mechanism for oxygen stomatal density excitation/inhibition, considering ozone formation due to plant terpene production, which has been demonstrated within Selaginella and formed through increased oxygen levels (Chen et al., 2011; Crutzen, 1971); elucidation of the relationship between the evolution of lycophytes and the geological oxygen maxima and carbon dioxide minima of the carboniferous, possibly as a driving force to worldwide glaciation (Montañez et al., 2007); and a bioinformatics analysis of the development of the genetic component of mesophilic differentiation within higher plants via concerted lycophyte homology.

APPENDICES

APPENDIX A

A: Global temperatures over time across three models, two of which have been corrected each to a different carbon dioxide concentration data source; B: Calculated cosmic ray flux over time; C: Periods of glaciation over time(Royer et al., 2004).

APPENDIX B

The morphology of modern, angiospermic, leaves and the dynamics of gas exchange. Note: within the model Paleozoic leaves the spongy layer is replaced with a second layer of palisade mesophyll tissue.

APPENDIX C

A model of a laboratory made chamber used in this study, detailing a front intake valve

(disconnected temporarily), a carbon dioxide, oxygen, and pressure sensor (not seen, attached to top of top exhaust valve).

APPENDIX D

A model of a laboratory made chamber improved for future directions, detailing a top intake valve and regulated top exhaust value. Oxygen, carbon dioxide, and pressure sensing is measured via dedicated chambers, piped in parallel to chambers such as this one, featuring top holes for dedicated sensors.

APPENDIX E

-2 2 Plant ID Index (%) Density (m ) amax (µm ) Lycopodium lucidulum Control 01 trimmed trimmed 216.2524 02 1.7301 49309664.69 401.6116 03 1.9608 59171597.63 550.7372 04 2.6846 78895463.51 377.1404 05 1.3972 69033530.57 560.066 06 2.0408 88757396.45 560.9448 07 2.3256 98619329.39 428.6516 08 2.4691 78895463.51 391.3364 09 3.0100 88757396.45 341.8532 10 1.4354 59171597.63 234.3692 11 2.1505 78895463.51 216.2524 12 2.6634 108481262.3 401.6116 13 1.4337 78895463.51 550.7372 14 2.5126 98619329.39 377.1404 15 2.8169 118343195.3 560.066 16 2.4510 98619329.39 560.9448 17 1.4141 69033530.57 428.6516 18 1.8182 78895463.51 391.3364 19 1.9231 78895463.51 341.8532 20 1.4925 69033530.57 234.3692 Pressurized 14 3.0075 78895463.5108 289.5308 16 1.9355 59171597.6331 259.246 18 3.0568 69033530.5720 500.3076 Flow Through 05 4.1199 108481262.3274 424.9336 07 2.3729 69033530.5720 218.686 17 1.9284 69033530.5720 267.5608 Selaginella kraussiana Control 01 7.6923 88757396.4497 75.1036 02 7.2289 118343195.2663 87.7448 03 9.3023 78895463.5108 58.5416 04 7.8014 108481262.3274 104.3068 05 7.0175 78895463.5108 65.91 06 9.2105 138067061.1440 43.1964 07 12.8205 197238658.7771 71.318 08 11.2500 177514792.8994 82.5396 09 8.9286 98619329.3886 54.418 10 9.4595 138067061.1440 69.628 11 5.4795 78895463.5108 75.1036 12 11.8280 108481262.3274 87.7448 13 6.3492 78895463.5108 58.5416 14 9.6386 157790927.0217 104.3068 15 11.1111 138067061.1440 65.91 16 8.2569 88757396.4497 43.1964 17 7.4286 128205128.2051 71.318 18 11.1111 128205128.2051 82.5396 19 8.1761 128205128.2051 54.418 20 trimmed trimmed 69.628 Pressurized 04 12.6050 147928994.0828 88.218 08 19.0840 246548323.4714 97.9524 Flow Through 07 11.3636 98619329.3886 53.4716 09 13.2743 147928994.0828 57.6628 15 9.2593 98619329.3886 41.7092

A full data table of collected morphological effect data, from which Table 1 was constructed.

APPENDIX F

Experimental Calculated Experimental Calculated Photosynthesis Stomatal Stomatal Transpiration Transpiration Plant ID Conductance Conductance ⁄ mmol mmol ⁄ ⁄ m⁄ m⁄ Lycopodium lucidulum Control1 01 0.014021321 0.2728 02 0.021974650 0.4275 03 0.021259443 0.4136 04 0.025999868 0.5059 05 0.033472249 0.6512 06 0.029643771 0.5768 07 0.021942719 0.4269 08 0.021980232 0.4277 09 0.010546730 0.2052 10 0.018731526 0.3644 11 0.042843407 0.8336 12 0.008884007 0.1728 13 0.020117247 0.3914 14 0.033467265 0.6511 15 0.025906870 0.5040 16 0.011385726 0.2215 17 0.011591711 0.2255 18 0.016954059 0.3299 19 0.022888796 0.4453 Control 1 1.520625 0.184063 8.422500 Control 2 1.930000 0.244839 9.790968 Control 3 1.491463 0.423537 16.50305 Control 4 1.021875 0.420156 14.93828 Control 5 0.223892 0.463473 16.56503 Control 6 1.142696 0.338174 12.52165 Control 7 0.882377 0.326639 11.17057 Control 8 0.267442 0.451163 15.16414 Pressurized2 14 trimmed trimmed 0.016919599 trimmed 0.3380 16 3.013333 0.358889 0.011523156 15.74222 0.2302 18 1.468689 0.436393 0.023650913 22.73443 0.4725 Flow Through 05 0.886807 0.346975 0.032367389 17.65193 0.6297 07 0.439960 0.310627 0.011575642 12.50599 0.2252 17 0.323143 0.356571 0.013820151 13.55671 0.2689 Selaginella kraussiana Control1 01 0.005658436 0.1101 02 0.008707724 0.1694 03 0.003991399 0.0777 04 0.009352061 0.1820 05 0.004456708 0.0867 06 0.005254949 0.1022 07 0.011986868 0.2332 08 0.012346913 0.2402 09 0.004660578 0.0907 10 0.008206474 0.1597 11 0.002736577 0.0532 12 0.008679067 0.1689 13 0.004265290 0.0830 14 0.008017151 0.1560 15 0.006555085 0.1275 16 0.005517009 0.1073 17 0.011690841 0.2275 18 0.008903759 0.1732 19 0.012046963 0.2344

Control 1 4.123333 1.94650000 73.69750000 Control 2 10.41386 2.06409091 75.70500000 Control 3 6.736857 2.14214286 82.10357143 Control 4 4.345500 1.76300000 61.82675000 Control 5 5.555909 1.48590909 56.04090909 Control 6 14.02081 1.96675676 74.54189189 Pressurized 04 3.645556 2.200635 0.010938529 116.4173 0.2185 08 4.121190 3.677661 0.020067697 173.4631 0.4009 Flow Through 07 6.255628 2.051314 0.004584809 80.49503 0.0892 09 5.123398 1.982645 0.007379101 76.64400 0.1436 15 5.039345 1.319014 0.003631829 47.92467 0.0707

A full data table of collected gas exchange data, from which Table 2 was constructed.

APPENDIX G

Electron Transport Rate Plant ID ⁄ Lycopodium lucidulum Control1 Control 1 0.006807 Control 2 0.007773 Control 3 0.112116 Control 4 0.018331 Control 5 0.095629 Control 6 0.743851 Pressurized 14 0.374045 16 0.302400 18 0.212621 Flow Through 05 3.131378 07 0.310261 17 0.436281 Selaginella kraussiana Control1 Control 1 0.035480 Control 2 0.019972 Control 3 0.075906 Control 4 0.207553 Control 5 0.472003 Control 6 0.516361 Pressurized 04 3.136381 08 3.720889 Flow Through 07 9.417696 09 7.773944 15 3.637413

A full data table of collected photosystem II activation data from which Table 3 was constructed.

REFERENCES AND SUGGESTED READINGS

Aliniaeifard, S., Malcolm Matamoros, P., and van Meeteren, U. (2014). Stomatal malfunctioning under low Vapor Pressure Deficit (VPD) conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Physiol. Plant. n/a – n/a.

Beerling, D.J., Osborne, C.P., and Chaloner, W.G. (2001). Evolution of leaf-form in land plants linked to atmospheric CO2 decline in the Late Palaeozoic era. Nature 410, 352–354.

Berner, R.A. (2006). GEOCARBSULF: A combined model for Phanerozoic atmospheric O2 and CO2. Geochim. Cosmochim. Acta 70, 5653–5664.

Berner, R.A. (2009). Phanerozoic atmospheric oxygen: New results using the GEOCARBSULF model. Am. J. Sci. 309, 603–606.

Bolz, R.E., and Tuve, G.L. (1973). CRC handbook of tables for applied engineering science (Cleveland, Ohio: CRC Press).

Chen, F., Tholl, D., Bohlmann, J., and Pichersky, E. (2011). The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. Cell Mol. Biol. 66, 212–229.

Chu, M.C.-Y. (1974). A Comparative Study of the Foliar Anatomy of Lycopodium Species. Am. J. Bot. 61, 681.

Crutzen, P.J. (1971). Ozone production rates in an oxygen-hydrogen-nitrogen oxide atmosphere. J. Geophys. Res. 76, 7311–7327.

Dimichele, W.A., Montañez, I.P., Poulsen, C.J., and Tabor, N.J. (2009). Climate and vegetational regime shifts in the late Paleozoic ice age earth. Geobiology 7, 200–226.

Eaton, M., and Kells, S.A. (2009). Use of vapor pressure deficit to predict humidity and temperature effects on the mortality of mold mites, Tyrophagus putrescentiae. Exp. Appl. Acarol. 47, 201–213.

Franks, P.J., and Farquhar, G.D. (2001). The effect of exogenous abscisic acid on stomatal development, stomatal mechanics, and leaf gas exchange in Tradescantia virginiana. Plant Physiol. 125, 935–942.

Graham, J.B., Dudley, R., Aguilar, N.M., and Gans, C. (1995). Implications of the late Paleozoic oxygen pulse for physiology and evolution.

Konrad, W., Roth-Nebelsick, A., and Grein, M. (2008). Modelling of stomatal density response to atmospheric. J. Theor. Biol. 253, 638–658.

Maxwell, K., and Johnson, G.N. (2000). Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 51, 659–668.

Montañez, I.P., Tabor, N.J., Niemeier, D., DiMichele, W.A., Frank, T.D., Fielding, C.R., Isbell, J.L., Birgenheier, L.P., and Rygel, M.C. (2007). CO2-Forced Climate and Vegetation Instability During Late Paleozoic Deglaciation. Science 315, 87–91.

Royer, D.L., Berner, R.A., Montañez, I.P., Tabor, N.J., and Beerling, D.J. (2004). CO2 as a primary driver of Phanerozoic climate. GSA Today 14, 4.