A Common Garden Experiment

COMPARATIVE WATER ABSORPTION / RETAINING ABILITY BETWEEN CHAPARRAL ISLAND AND THE MAINLAND TAXA: A COMMON GARDEN EXPERIMENT

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Humera Mirza

2019

SIGNATURE PAGE

THESIS: COMPARATIVE WATER ABSORPTION / RETAINING ABILITY BETWEEN CHAPARRAL ISLANDS AND THE MAINLAND TAXA: A COMMON GARDEN EXPERIMENT

AUTHOR: Humera Mirza

DATE SUBMITTED: Spring 2019

Department of Biological Sciences

Dr. Frank Ewers, Ph.D. Thesis Committee Chair Professor of Biological Sciences

Dr. Edward Bobich, Ph.D. Professor of Biological Sciences

Dr. Kristin Bozak, Ph.D. Professor of Biological Sciences

ACKNOWLEDGEMENTS

First of all, I would like to thank Dr. Frank Ewers for his unwavering support and

for being an extremely influential mentor in my research and studies. I would not have

successfully accomplished my goal of acquiring a Master’s degree in Biological Sciences

without his constant guidance and presence whenever I needed it. He gave me the

opportunity as an advisor to pursue my dreams while expressing myself in the scientific

community. Words cannot express my gratitude to Dr. Ewers for everything he has done for me.

I would also like to thank Dr. Bobich and Dr. Bozak for being my thesis committee members. Their guidance was very helpful throughout my research and during the compilation of my thesis. They were my staunch supporters and proponents during the two years of my studies.

Shout out to the staff of Rancho Santa Ana Botanic Garden, especially Dr.

Loraine Washburn and Helen Smisko, for their assistance in SEM, identifying plants and providing a detailed record of accession.

Lastly, I want to thank the Department of Biological Sciences and Cal Poly

Pomona for proving me a platform to perform my research and excel in my endeavor to earn a Master of Science degree.

iii

ABSTRACT

Island and coastal species are typically exposed to more fog but less rain than mainland species. Because adaptations to absorb water from fog or mist may be in conflict with those to minimize water loss, I hypothesized that due to natural selection island species would have more capacity to absorb fog and mist due to foliar uptake compared to the mainland congeners. Is the ability to absorb fog or mist a heritable trait?

Are there leaf anatomical features that might play a role in water absorption or retention?

These questions were investigated by comparing island species to the mainland species in a common garden, namely, Rancho Santa Ana Botanical Garden, where all physical conditions remained the same. Two plant genera (Ceanothus and Arctostaphylos) were selected for this research. The questions and hypothesis were tested by examining leaf water potential, maximum leaf water absorption, hydrophobicity, leaf mass per area, succulence, and the scanning electron microscopy of leaves of island and mainland species. The results suggested that all species exhibit water permeability through their leaf surfaces and demonstrated the capacity to absorb water directly into the symplast.

The island and mainland varieties of Ceanothus megacarpus were very similar in water absorption and hydrophobity, but there was a difference in other traits such as leaf mass per area and succulence which were both higher in variety megacarpus than variety insularis. In Arctostaphylos, one of the island species (A. catalinae) experienced high- water absorption, as expected, but the other island species (A. insularis) did not.

Arctostaphylos catalinae might have adapted by increasing foliar absorption while A. insularis has more typical mesophytic traits such as having stomata only on the lower surface of the leaf (hypostomatic), low leaf mass per area, and low succulence, compared

iv to mainland species of Arctostaphylos. In conclusion, not all the island species are alike.

Future experiments might sample the plants in their place of origination to elucidate plastic responses to the island environment.

TABLE OF CONTENTS

SIGNATURE PAGE ...... ii ACKNOWLEDGEMENTS ...... iii ABSTRACT ...... iv LIST OF TABLES ...... vii LIST OF FIGURES ...... viii CHAPTER ONE ...... 1 INTRODUCTION ...... 1 CHAPTER TWO ...... 11 METHODS...... 11 Study Site and Species ...... 11 Leaf Water Potential ...... 11 Water Absorption Measurements ...... 11 Hydrophobicity ...... 13 Scanning Electron Microscopy ...... 14 Statistical Data Analysis ...... 15 CHAPTER THREE ...... 17 RESULTS...... 17 Water Absorption Measurements ...... 17 Leaf Water Potential ...... 18 Contact Angle Measurements ...... 19 Stomatal Density...... 19 Leaf Mass per Area ...... 20 Succulence ...... 20 Scanning Electron Microscopy ...... 20 CHAPTER FOUR ...... 37 DISCUSSION ...... 37 CHAPTER FIVE ...... 41 CONCLUSION ...... 41 REFERENCES ...... 42

LIST OF TABLES

Table 1. Sources of material for the common garden study ...... 7

vii

LIST OF FIGURES

Figure 1. Ceanothus megacarpus collected from Rancho Santa Ana Botanic Garden. (a) Ceanothus megacarpus var.insularis showing inflorescences, (b) Ceanothus megacarpus var. megacarpus showing inflorescences, (c) C. megacarpus var. insularis representing leaves, (d) C. megacarpus var. megacarpus representing leaves...... 8

Figure 2. Arctostaphylos samples collected from Rancho Santa Ana Botanic Garden showing inflorescences. (a) Arctostaphylos pungens, (b) Arctostaphylos glauca, (c) Arctostaphylos catalinae, (d) Arctostaphylos insularis...... 8

Figure 3. Arctostaphylos samples collected from Rancho Santa Ana Botanic Garden with representative leaves. (a) Arctostaphylos pungens, (b) Arctostaphylos glauca, (c) Arctostaphylos catalinae, (d) Arctostaphylos insularis...... 9

Figure 4. Original collection sites for the six species of the two genera used in this study...... 10

Figure 5. (a) Theta Lite Tensiometer used to measure contact angle, (b) Angle of contact between water drop and leaf shown on screen...... 16

Figure 6: (a) Water absorption by leaves for Ceanothus same day measurements. Bars represent the mean % increase in leaf water content ±SE and data were analyzed using t- test (two sample assuming equal variance), (b) Water absorption by leaves for Ceanothus following the bench dry treatment. Bars represent the mean % increase in leaf water content ±SE and data were analyzed using t-test (two sample assuming equal variances)...... 23

Figure 7: (a) Water absorption by leaves for Arctostaphylos same day measurements. Data were analyzed by performing one-way ANOVA. Bars represent the mean % increase in leaf water content ±SE and different letters indicate significant (P < 0.05) differences among values, (b) Water absorption by leaves for Arctostaphylos following the bench dry treatment. Data were analyzed by performing one-way ANOVA. Bars represent the mean % increase in leaf water content ±SE and different letters indicate significant (P < 0.05) differences among values...... 24

viii

submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Ceanothus following the bench dry treatment. Data are means ±SE and analyzed using t- test (two sample assuming equal variances)...... 25

Figure 9: (a) Initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Arctostaphylos same day measurements. Data are means ±SE and analyzed by performing ANOVA (two-factor with replication). Significant (P < 0.05) differences were noted by asterisks, (b) Initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Arctostaphylos following the bench dry treatment. Data are means ±SE and analyzed by performing ANOVA (two- factor with replication). Significant (P < 0.05) differences were noted by asterisks...... 26

Figure 10: (a) Contact angle measurements for adaxial and abaxial surfaces of Ceanothus. Data was analyzed by performing t-test (two-sample assuming equal variances). Bars represent mean ±SE and significant (P < 0.05) differences were noted by asterisks, (b) Contact angle measurements for adaxial and abaxial surfaces of Arctostaphylos. Data was analyzed by performing ANOVA (two-factor with replication). Bars represent mean ±SE and different letters indicate significant (P < 0.05) differences between taxa...... 27

Figure 11: Number of stomata per mm² for adaxial and abaxial surfaces of Arctostaphylos species. Data was analyzed by performing ANOVA (two-factor with replication). Bars represent mean ±SE and different letters indicate significant (P < 0.05) differences between species...... 28

Figure 12: (a) Leaf mass/area measured in g/m² for both taxa of Ceanothus. Data was analyzed by performing t-test (two-sample assuming equal variances). Bars represent mean ±SE and significant (P < 0.05) difference was noted by asterisk, (b) Leaf mass/area measured in g/m² for all species of Arctostaphylos. Data was analyzes by performing one- way ANOVA. Bars represent mean ±SE and different letters indicate significant (P < 0.05) differences among values...... 29

Figure 13: (a) Succulence/leaf area measured in g/m² for both species of Ceanothus. Data was analyzed by performing t-test (two-sample assuming equal variances). Bars represent mean ±SE, (b) Succulence/leaf area measured in g/m² for all species of Arctostaphylos. Data was analyzes by performing one-way ANOVA. Bars represent mean ±SE and different letters indicate significant (P < 0.05) differences among values...... 30

ix spreaded all over the surface. (d) Abaxial surface showing the close-up look of trichomes. (e) Abaxial surface showing a unique layer of wax on trichomes...... 31

Figure 15: Scanning electron microscopy (SEM) micrographs of Ceanothus megacarpus var. insularis. (a) Adaxial surface showing a thick layer of wax. (b) Adaxial surface showing close-up look of wax coat. (c) Abaxial surface with a bunch of trichomes spreaded all over the surface. (d) Abaxial surface showing the close-up look of trichomes. (e) Abaxial surface showing a smooth layer of wax on trichomes...... 32

Figure 16: Scanning electron microscopy (SEM) micrographs of Arctostaphylos pungens. (a) Adaxial surface showing stomata distributed evenly with few trichomes. (b) Adaxial surface showing close-up look of wax coat. (c) Adaxial surface with a close-up look of stomata. (d) Abaxial surface showing presence of many trichomes and stomata. (e) Abaxial surface showing a close-up look of wax, stomata, & trichomes. (f) Abaxial surface showing stomata...... 33

Figure 17: Scanning electron microscopy (SEM) micrographs of Arctostaphylos glauca. (a) Adaxial surface showing stomata distributed evenly with unique waxy coat. (b) Adaxial surface showing close-up look of wax coat. (c) Adaxial surface with a close-up look of stomata. (d) Abaxial surface showing unique wax coat and stomata. (e) Abaxial surface with a close-up of ornate wax and stomata. (f) Abaxial surface showing stomata...... 34

Figure 18: Scanning electron microscopy (SEM) micrographs of Arctostaphylos catalinae. (a) Adaxial surface showing stomata distributed evenly with a thick wax coat. (b) Adaxial surface showing close-up look of wax coat. (c) Adaxial surface with a close- up look of stomata. (d) Abaxial surafce showing presence of many trichomes and stomata. (e) Abaxial surface with a close-up of wax, stoamata, & trichomes. (f) Abaxial surface showing stomata...... 35

Figure 19: Scanning electron microscopy (SEM) micrographs of Arctostaphylos insularis. (a) Adaxial surface showing a thick waxy coat. (b) Adaxial surface showing close-up look of wax coat. (c) Adaxial surface clearly showing a waxy coat. (d) Abaxial surface showing evenly distributed stomata. (e) Abaxial surface with a close-up of unique layer of wax around stomata. (f) Abaxial surface showing stomata...... 36

CHAPTER ONE

INTRODUCTION

Instead of relying only on ground water, plants may also absorb fog water directly into the leaves, through a process called foliar absorption (Burgess & Dawson 2004,

Limm et al. 2009). It has been claimed that some plants have evolved the ability to absorb

water through leaves, transport it down the xylem, and then release it into the soil (Eller

et al. 2013). Thus, the plants are watering their own roots and adjacent seedlings. The

poorly understood mechanism for foliar water uptake may work well enough that these

plants can continue photosynthesis and growth, even when the soil they are growing in is

dry. When the trees are almost constantly covered in fog, the atmosphere around the

leaves has a higher water potential than the leaves themselves, which could allow foliar

uptake (Eller et al. 2013).

Investigation reveals that wetting of leaves with fog, mist, and dew may often

provide a significant water subsidy in many ecosystems and thereby positively affect

plant water balance without noticeably increasing soil wetness (Kerfoot 1968, Leyton &

Armitage 1968, Kerr & Beardsell 1975, Boucher et al. 1995, Yates & Hutley 1995,

Hutley et al. 1997, Boucher et al. 1995, Ebner et al. 2011). Foliar uptake occurs when

atmospheric water droplets coalesce on plant shoots and move along a water potential

gradient (Rundel 1982). This kind of water absorption immediately increases foliar

hydration and plant water potential (Grammalikopoulos & Manetas 1994). Fog may

change the system energy balance by reducing solar heating and increasing relative

humidity above and within a plant canopy, thus reducing evapotranspiration during

photosynthetic gas exchange (Dawson 1998, Weathers 1999). Understanding the features

that help the plant on an island, and the mainland to absorb and retain water for survival would allow us to improve our basic understanding of whether island species take advantage of the fog water more than the mainland plant species. This research would also allow predicting the future vegetation distribution and their function.

Water potential can be expressed as the tension on the water in the vessels, often described in negative pressure (units of MPa). The water potential is often close to 0 MPa in a well-hydrated plant. As evaporation increases, the tension on the water column may pull air into conduits, creating an embolism (air blockage) that prevents the water flow. If all the vessels are blocked due to embolism, the plant may die. Water supply from ground or from fog can prevent extreme negative water potentials and may help in survival of plants (Holmlund et al. 2016).

Island and coastal species are often exposed to much more fog, but often less rain, than the inland species (Dawson 1998, Holmlund et al. 2016). Adaptations to absorb water from fog or mist may conflict with adaptations to minimize water loss, which might result in dew evaporating into the atmosphere. Actually, the plants living where frequent fog or dew events occur may have significant tissue hydration (Limm et al.

2019). There are other physiological advantages of foliar water absorption that include higher survival rates (Vaadia & Waisel 1963), increase plant growth (Boucher et al.,

Holmlund et al. 2016), and even enhanced gas exchange after leaves dry (Simonin et al.,

Holmlund et al. 2016). Given its physiological importance, I hypothesized that due to natural selection island species would have a greater capacity for foliar absorption of fog and mist compared to the mainland species. Questions addressed in this study are: Is the ability to absorb fog or mist a heritable trait? Are there leaf anatomical features that

might play a role in water absorption or retention? These questions were investigated by comparing island species to the mainland species within two genera in a common garden where all physical conditions remained the same. This common garden investigation evaluated comparative water absorption/retaining ability between plants of the same genera from islands and the mainland. To test the hypothesis and to find the answers to these questions, we measured the leaf water absorption, leaf water potentials, leaf hydrophobicity, stomatal density, succulence, leaf dry weight, and performed scanning electron microscopy of six species of the two different genera.

Rancho Santa Ana Botanical Garden is an ideal location to investigate the hypotheses because it has island and inland species of the same genus and it is in close proximity to Cal Poly Pomona in the city of Claremont, California. The plant material used in this study all had detailed records of accession. The garden also has an active research department including an anatomy lab with scanning electron microscopy (SEM).

Two plant genera were selected for this research, Ceanothus and Arctostaphylos.

Ceanothus, which in Greek means “spiny plant” (Dale 2000), is a genus of dicotyledonous shrubs and small trees of about 50-60. The members of this genus are commonly known as California lilac, soap bush, and wild lilac. It has 1-4 mm petiole, 10-

25 mm blade that is 5-12 mm wide, and length is generally greater than two times the width (Fig. 1c). Leaves are glabrous, elliptic to widely oblanceolate, adaxially dull green, and abaxially gray-green in color (Fig. 1d). The genus is a native to North America with abundance in California (Baldwin et al. 2012). Two taxa of Ceanothus served as study

subjects: Ceanothus megacarpus var. megacarpus and Ceanothus megacarpus var.

insularis (Table 1). Both varieties occur on rocky slopes, canyons, and chaparral (Baldwin et al. 2012).I

Ceanothus megacarpus var. megacarpus is the variety commonly known as big pod ceanothus. It is native and endemic to California where it’s distributed along the central coast including the Channel Islands. The evergreen leaves are thick and oval to nearly rectangularly-shaped, and alternately arranged (Fig. 1d). The Ceanothus megacarpus var. megacarpus individuals that were investigated in this study were collected as seeds from Santa Ynez Mountains of Santa Barbara County, California

(Table 1; Fig. 4).

Ceanothus megacarpus var. insularis is commonly known as island ceanothus. It is endemic and native to California where it’s distributed mostly on the Channel Islands with some occurring on the mainland along the central coast. Leaves are oval to circular and evergreen with an opposite arrangement (Fig. 1c). The Ceanothus megacarpus var. insularis individuals that were investigated in this study were originally collected as seeds from Santa Catalina Island in Los Angeles County, California (Table 1; Fig. 4).

Arctostaphylos is a genus in the Ericaceae comprised of shrubs and small trees, commonly known as manzanitas. There are about 60 species ranging from small trees up to 6 m tall. Most are evergreen, with small oval leaves 1 – 7 cm long, arranged spirally on the stem. The flowers are in small clusters, white to pale pink in color, and bell- shaped (Fig. 2). Flowering is in the spring season and fruit (small berries) ripens in the summer or autumn (Baldwin et al. 2012).

Four taxa of Arctostaphylos served as study species (Table 1). There are two subgenera of Arctostaphylos: Subgenus Micrococcus and Subgenus Arctostaphylos.

Subgenus Arctostaphylos has three sections i.e. Arctostaphylos, Foliobracteata, and

Pictobracteata. The species of our study were A.glauca, A.pungens (mainland sp.) and

A.insularis (Island sp.) which belong to the section Arctostaphylos, and A.catalinae

(Island sp.) belongs to section Foliobracteata. Nomenclature was based on Simpson

(2010).

Arctostaphylos pungens is native to California, Utah, Texas, and Mexico. It is erect, 1-3 m in height with relatively short non-glandular-hairy stem. Leaves are erect and bright or dark green in color with shiny surface. The petiole is 4-8 mm, and the blade is

1.5-4 cm long and 1-1.8 cm wide. The base is obtuse to wedge-shaped with acute tip and entire margin (Fig. 3a). It is located on rocky slopes, ridges, chaparral, and coniferous forest at elevations of 180-2300 m (Baldwin et al. 2012). Arctostaphylos pungens individuals that were investigated in this study were originally collected as cuttings from

Otay Mountains in San Diego County, California and then planted at rancho Santa Ana

Botanic Garden (Table 1; Fig. 4).

Arctostaphylos glauca is native to California, also distributed outside of

California to northwestern Baja California. It is erect, 1-8 m in height with glabrous non- hairy stem. Leaves are erect, with the petiole 7-15 mm long, and the blade being 2.5-5 cm long and 2-4 cm wide; the shape is oblong-ovate, white-glaucous, dull, glabrous, base rounded, pointed soft tip, margin entire, and flat (Fig. 3b). It is located on rocky slopes, chaparral, and woodlands at the elevation less than 2200 m (Baldwin et al. 2012).

The Arctostaphylos glauca individuals that were investigated in this study were originally

collected as seeds from Santa Ana Mountains in Orange County, California (Table 1; Fig.

4).

Arctostaphylos catalinae is a rare, California endemic restricted to the Channel

Islands, mainly to Santa Catalina Island. It is erect, 2-5 m in height with twigs densely covered with glandular and non-glandular hair. Leaves are green-glaucous in color, and bristly-glandular in appearance with acute tip and entire margin. The petiole is 2-6 mm long, and the blade is 2-5 cm long with being 1.5-3 cm wide covered with hairs (Fig. 3c).

It is located on volcanic outcrops and ridges at the elevation of 100-600 m (Baldwin et al.

2012). The Arctostaphylos catalinae individuals that were investigated in this study were originally collected as seeds from Santa Catalina Island in Los Angeles County,

California (Table 1; Fig. 4).

Arctostaphylos insularis is another California endemic of the Channel Islands, mainly on Santa Cruz Island. It is erect, 2-5 m in height with glabrous twigs covered with sparsely glandular or non-glandular hairs. Leaves are erect, the petiole is 4-8 mm long, the blade is 2.5-4.5 cm long and 1-3 cm wide, with an oblong-elliptic shape. The leaves are also usually cupped, bright green, shiny, abaxially glabrous, and sparsely glandular hairy (Fig. 3b). It is located on rocky slopes, chaparral, and woodlands at the elevation of less than 400 m (The Jepson Manual 2012). The Arctostaphylos insularis individuals that were investigated in this study were originally collected as cuttings from Santa Cruz

Island in Santa Barbara County, California (Table 1; Fig. 4).

Table 1 Sources of material for the common garden study

Propag- Family Taxon Distribut- Locality County ation material ion

Rhamnaceae Ceanothus Mainland Santa Santa Seed megacarpus var. Ynez Barbara megacarpus Nutt Mountains Rhamnaceae Ceanothus Island Santa Los Seed megacarpus var. Catalina Angeles insularis Munz Island Ericaceae Arctostaphylos Mainland Otay San Diego Cutting pungens Kunth Mountains (Arctostaphylos) Ericaceae Arctostaphylos Mainland Santa Ana Orange Seed glauca Lindl Mountains (Arctostaphylos) Ericaceae Arctostaphylos Island Santa Los Seed catalinae P. V. Catalina Angeles Wells Island (Foliobracteata) Ericaceae Arctostaphylos Island Santa Santa Cutting insularis Greene Cruz Barbara ex perry Island (Arctostaphylos)

■ Ceanathw; megac&pw; megaca:rpw; ♦ Ceanathw; megac&pw; insuknis * A:rctostaphylos p rmgens 6 ;4:rctostaphylos glaoca ~ A:rctostaphylos catalina.e O A:rctostaphylos insul&is 0 A common garden

KERN

SAN BERNARDINO

RI VERSIDE

mPERIAL

Figure 4. Original collection sites for the six species of the two genera used in this study.

CHAPTER TWO

METHODS

Study Site and Species

The plant species that were investigated in this research are listed in Table 1. Five

individuals were sampled per taxon at Rancho Santa Ana Botanic Garden, Claremont,

California. Samples were collected during mid-day in airtight bags to avoid water loss

and brought to the lab at Cal Poly Pomona, California for the following measurements.

Leaf Water Potential

To measure leaf water potential, the sample leaves were securely bagged to avoid

transpiration, which could alter the reading. The leaf was inserted into the chamber lid of

pressure chamber immediately after taking it out from the bag and secured, with the

petiole of the leaf protruding outside and the leaf blade inside the chamber (Scholander et al. 1965). The chamber was sealed tightly and then pressurized slowly with nitrogen gas.

Due to this positive pressure exerted on the leaf in the chamber, water in the leaf blade began to come out through the open end of the leaf. During pressurization, a drop of water (sap) appeared from the exposed surface and the pressure from the chamber gauge was recorded. This pressure value read in negative (-) bars which later converted into

MPa called as the leaf water potential. This procedure was done on three leaf samples per plant of five individual plants of each species.

Water Absorption Measurements

The capacity to absorb water by leaves was measured by submerging the leaf

samples directly into water. First, the youngest, fully matured leaves were cut from all

five plants of each six species. The open-ended surface of petiole was sealed with super glue (Loctite Brand, Henkel Corporation, Ohio, USA) to avoid any error in weighing before and after absorption and to prevent evaporation from the exposed petiole surface during measurement. The beginning mass of the leaf was measured using an electronic balance. The leaf was then submerged in deionized water for five seconds, removed quickly, pat dried using paper towel and the leaf mass was recorded again to measure the possible artifact of residual water remained after patting the leaves with a paper towel.

The leaf was submerged again in deionized water and kept in dark for three hours to allow water absorption by the leaf. After removing the leaf from the water container and patting it with a paper towel, the leaf mass was recorded again to determine water absorption by the leaf. The amount of water absorbed for each leaf was calculated by evaluating the change in leaf mass after submergence (% increase) using following equation:

Uptake = [(Mass2 – Mass1)/Mass1] x 100%

Where Mass1 is the beginning mass of leaf after five seconds of submergence and Mass2 is the mass of leaf after submergence of 180 minutes (Limm et al. 2009).

The adhesive was removed from each sample and water potential was recorded again after the submergence using the pressure chamber to verify the increased water content in the symplast.

To determine whether initial water potential impacted absorbance, one set of leaf samples was kept in plastic bags and processed the same day they were collected. This represented winter (wet season) water potentials. Another set of leaf samples was left on

the bench overnight in open plastic bags to air dry and the experiment of water absorption was repeated the next day for all the samples. We found that the overnight drying resulted in higher water absorption and much lower water potentials similar to the dry season water potentials that these species experience.

Hydrophobicity

Contact angle measurement is the most common way to check the leaf hydrophobicity. To measure the contact angle of each leaf an Attension Theta Lite optical tensiometer (Biolin Scientific, Gothenburg, Sweden) was used (Fig. 5a). Plants were collected from Rancho Santa Ana Botanic Garden and damped dry with a paper towel then left overnight in open plastic bags which were sealed the next morning. This was done to assure the leaves had fairly low water potentials, but were within the normal range for these plants. Slides were prepared by putting a 3 mm x 4 mm piece of each leaf sample on it. For each leaf, adaxial and abaxial slides were prepared and contact angles were measured. The prepared slide was placed on stage (Fig. 5a) and then a droplet was placed on surface of leaf by the attached dropper and the image of the drop was shown on the screen with the contact angle on both sides of the drop (Fig. 5b). Both left and right contact angles were recorded and the mean was calculated. If the angle of contact between a drop of water and leaf surface was > 90° then the leaf surface was considered hydrophobic, whereas if the water drop showed the contact angle of < 90° then the leaf was considered hydrophilic (Papierowska et al. 2018).

Leaf Area and Succulence

To measure leaf area and succulence, three leaves per plant of five plants per

species were sampled. To measure the leaf area in cm² a LI-3100C Leaf Area Meter (LI-

COR Biosciences, Nebraska, USA) was used. The fresh leaf mass in grams were recorded by using an electronic balance. The leaves were then transferred in small paper bags, and each bag was labeled with the corresponding plant number. Bags were kept in

an oven at high temperature (66°C) to dry them to constant mass, which occurred after

two days. The difference in the fresh and dry mass of leaves demonstrated the water

content (Mantovani 1999). Succulence of each leaf was calculated in g of water per cm²

by using following equation:

Succulence = (Fresh Leaf Mass – Dry Leaf Mass)/ Leaf Area

Where Fresh Leaf Mass is the leaf mass measured before drying and Dry Leaf Mass is

the leaf mass measured after drying the leaf sample (Mantovani 1999).

Scanning Electron Microscopy

Scanning electron microscopy was performed using a Hitachi SU3500 SEM

(Hitachi-High Technologies, Tokyo, Japan) on leaf samples from each taxon to obtain the

surface anatomy and composition. The tissue was cut in the lab, fixed in FAA, and

dehydrated in ethanol. For SEM, a specimen is normally required to be completely dry,

because the specimen chamber is at high vacuum. To achieve a completely dry specimen,

the tissue was then processed in CO2 chamber for critical point drying. The dry specimen

was mounted on a specimen stub using electrically conductive double-sided adhesive

tape, and sputter-coated with gold before examination in the microscope. The procedure

was done at Rancho Santa Ana Botanic Garden, Claremont and it helped in measuring

stomatal density and also investigating whether anatomical features may play any role in

water absorption or retention in the leaf. Stomatal density was calculated as the number

of stomates per mm² based upon relatively low magnification samples (50x) so that a

fairly large area of leaf could be sampled. Five leaves were sampled per species.

Statistical Data Analysis

Leaf water potential, succulence, leaf dry weight per area, water absorption,

stomatal density and contact angle measurements were analyzed among species. For

Ceanothus, a t-test (two-sample assuming equal variances) was used. For Arctostaphylos,

one-way analysis of variance (ANOVA) statistical test was performed followed by a t- test (two-sample assuming equal variances) for comparisons between all species.

(a)

(b) I

Figure 5. (a) Theta Lite Tensiometer used to measure contact angle, (b) Angle of contact between water drop and leaf shown on screen.

CHAPTER THREE

RESULTS

Water Absorption Measurements

All six studied species demonstrated the capacity for water absorption by leaf

when submerged for 180 minutes. Ceanothus megacarpus var. megacarpus demonstrated

no significant difference in water absorption compared to C. megacarpus var. insularis.

They were both in the range of 4% absorption rate in 180 minutes (Fig. 6a).

However, when the samples of these taxa were bench dried overnight before

submerging the leaves, C. megacarpus var. megacarpus stayed in the range of 4% while

C. megacarpus var. insularis demonstrated 5% of water absorption (Fig. 6b) which was

1% more than previously recorded but there was no significant difference between these two varieties.

For Arctostaphylos species, the absorption was between 0.8% to 7% after 180

minutes with the highest increase in mass for A. catalinae and lowest in A. glauca and A.

insularis. All four species of Arctostaphylos i.e. A. pungens, A. glauca, A. catalinae, and

A. insularis demonstrated significant difference (P < 0.05 for pairwise comparisons after

a significant one-factor ANOVA) in water absorption compared to each other with the

exception of A. glauca and A. insularis which did not differ difference (Fig. 7a).

Moreover, with the next-day experiment after bench drying the samples

overnight, the results were different with increased absorption rate of 2% to 7.8%.

Arctostaphylos pungens, A. glauca, and A. insularis demonstrated double the absorption

rate with no significant difference between each other. In contrast, A. catalinae

demonstrated no significant increase in absorption rate with the bench drying but had significantly higher values (P < 0.05) compared to A. glauca and A. insularis (Fig. 7b).

Leaf Water Potential

Leaf water potential was measured before and after the leaf absorption experiment. For fresh collected samples the initial leaf water potential of C. megacarpus var. megacarpus was significantly more negative compared to C. megacarpus var. insularis (P < 0.05). The final leaf water potentials for both varieties of Ceanothus were higher (less negative) compared to the initial measurements but the difference was not significant (Fig. 8a).

The results for bench dry measurements were very much similar to that of the measurements without leaf drying in terms of initial water potential vs. final water potential, but there were much lower (more negative) pressure readings due to overnight drying of these samples (Fig. 8b).

For Arctostaphylos species, the final water potentials were less negative than the initial readings but the differences were not all significant. Arctostaphylos pungens and A. catalinae demonstrated significant (P < 0.05) differences in initial versus final leaf water potentials while A. glauca and A. insularis showed no significant differences (Fig. 9a).

Bench dry measurements demonstrated much lower (more negative) water potential as expected. For the bench dry treatments Arctostaphylos pungens and A. glauca demonstrated significant differences in initial versus final leaf water potentials (P < 0.05) whereas A. catalinae and A. insularis showed no significant difference in initial versus final values (Fig. 9b).

Contact Angle Measurements

The contact angle measurement for the adaxial and abaxial surfaces of C. megacarpus var. megacarpus was very similar to the respective surface of C. megacarpus var. insularis. However, contact angle on the abaxial surface of both taxa which was >

100°, was significantly (P < 0.05) greater than that of the adaxial surface (Fig. 10a).

In all species of Arctostaphylos, the adaxial surface of the leaf had a similar contact angle values to that of the abaxial surface. Comparing species, both the adaxial and abaxial surfaces of A. glauca demonstrated significantly greater contact angles, both of which were > 120°, compared to A. pungens, A. catalinae, and A. insularis (P < 0.05), none of which differ from each other (Fig. 10b).

Stomatal Density

Both species of Ceanothus lacked adaxial stomata, whereas the abaxial surface was completely covered with trichomes such that the stomata were not visible in SEM. In the genus Arctostaphylos, the adaxial and abaxial surfaces of A. pungens, A. glauca, and

A. catalinae were very similar in stomatal density. The surfaces were significantly different for A. insularis compared to the other species because the adaxial surface had no stomata and the abaxial surface had very high stomatal density (Fig. 11). Adaxial stomatal density differed among all of the Arctostaphylos species (P < 0.05). However, the abaxial stomatal density for A. insularis was almost double (P < 0.05) compared to all

Arctostaphylos species (Fig. 11).

Leaf Mass per Area

The leaf mass per area was significantly (P < 0.05) greater in Ceanothus megacarpus var. megacarpus than in C. megacarpus var. insularis (Fig. 12a). Similarly, the leaf mass per area of A. insularis was significantly lower compared to all other species of Arctostaphylos (P < 0.05). However, A. pungens, A. glauca, and A. catalinae did not differ significantly for this parameter (Fig. 12b).

Succulence

Succulence is the amount of water present per leaf area, measured in g/m². There was no significant difference in succulence between C. megacarpus var. megacarpus and

C. megacarpus var. insularis (Fig. 13a). Arctostaphylos pungens was significantly more succulent compared to A. catalinae, and A. insularis (P < 0.05). Arctostaphylos glauca,

A. catalinae, and A. insularis did not differ in succulence (Fig. 13b).

Scanning Electron Microscopy

The adaxial and abaxial surfaces were quite different in Ceanothus megacarpus var. megacarpus (Fig. 14). The adaxial surface had a thick layer of wax uniformly distributed all over the surface (Fig. 14a). The abaxial surface was found to be covered entirely with a compact layer of trichomes entwined with each other (Fig. 14c). Upon closer observation, I could differentiate each trichome and gaze through the many layers into their depths (Fig. 14d). However, no stomata were visible with SEM as they were obscured by the trichomes. Each trichome had a very distinct snow flake-like wax structures resting on them (Fig. 14e).

Ceanothus megacarpus var. insularis leaves were similar to those of variety megacarpus (Fig. 15), with the following exception. The adaxial surface of var. insularis appeared to have a thinner layer of wax (Fig. 15a) compared to var. megacarpus (Fig.

15b). The trichomes of var. insularis were speckled with what appeared to be thin flakes of wax throughout the length of their structures (Fig. 15e).

Arctostaphylos pungens was found to be amphistomatous with stomates on both sides of the leaf. It also contained patches of wax resting on a thick compressed layer of epicuticular wax on the adaxial and abaxial surfaces (Fig. 16). The adaxial surface had few trichomes (Fig. 16a) whereas the abaxial surface had many trichomes (Fig. 16d).

Once magnified, we could distinguish the stomata structure covered in a waxy ledge as well as curved trichomes (Fig. 16e). The trichomes appeared to be entwined in a rope-like fashion. I could easily identify each pair of guard cells (Fig. 16f).

Arctostaphylos glauca was amphistomatic and appeared to have very similar adaxial and abaxial surfaces (Fig. 17). Both of the surfaces were covered with very ornate wax, showing a layer of barrel-shaped epidermal cells (Fig. 17a, d). At x400 magnification clusters of stomata and epidermal cells in a floral pattern were apparent

(Fig. 17b, e). At x1200 magnification, there was a very prominent layer of the ornate wax’s net-like appearance both on regular epidermal cells guard cells (Fig. 17c, f).

Arctostaphylos catalinae was amphistomatic, but had very different adaxial and abaxial surfaces (Fig. 18). The adaxial surface was found to have stomata compactly embedded into a perforated layer of epicuticular wax (Fig. 18a). At higher magnification, chunks of thick wax resting atop the surface were revealed (Fig. 18b, c). The abaxial

surface had thickets of elongated trichromes (Fig. 18d). The stomata were more noticeable in an elevated ledge leaving a cavity between the guard cells and the ledge openings (Fig. 18e, f).

Arctostaphylos insularis was hypostomatic and completely devoid of trichromes on both adaxial and abaxial surfaces. A thin waxy coat was visible on both surfaces as well (Fig. 19). The abaxial surface was covered with many stomata (Fig. 19d). The stomata were very prominent and protruding the surface (Fig. 19e).

1 Leaf water absorption (% increase) increase) (% absorption water Leaf

0 C. mega. megacarpus C. mega. insularis (a) Taxon

1 Leaf water absorption (% increase) increase) (% absorption water Leaf

0 C. mega. megacarpus C. mega. insularis (b) Taxon

9 c 8

4 a 3

2 b b Leaf water absorption (% increase) increase) (% absorption water Leaf 1

0 A. pungens A. glauca A. catalinae A. insularis Species (a)

10 a 9 8 7 ab 6 5 4 3 b b

Leaf water absorption (% increase) increase) (% absorption water Leaf 1 0 A. pungens A. glauca A. catalinae A. insularis (b) Species

Taxon 0 C. mega. C. mega. insularis -0.5 megacarpus

-1

-1.5 Initial water potential * ■ -2 121 Final water potential

-2.5 Leaf Leaf (MPa) potential water

-3

-3.5 (a)

Taxon 0 C. mega. C. mega. insularis megacarpus -1

-2 ■ Initial water potential

-3 121 Final water potential

-4

Leaf Leaf (MPa) potential water -5

-6 (b)

Figure 8: (a) Initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Ceanothus same day measurements. Data are means ±SE and analyzed using t-test. Significant (P < 0.05) differences between initial and final measurements were noted by asterisk, (b) Comparison chart showing initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Ceanothus following the bench dry treatment. Data are means ±SE and analyzed using t- test (two sample assuming equal variances).

Species 0 A. pungens A. glauca A. catalinae A. insularis -0.2

-0.4

-0.6 Initial water potential -0.8 Final water potential -1

-1.2 * Leaf Leaf (MPa) potential water -1.4 * -1.6 (a)

Species 0 A. pungens A. glauca A. catalinae A. insularis -0.5

-1

-1.5

-2 Initial water potential

-2.5 Final water potential

-3

Leaf Leaf (MPa) potential water -3.5 * -4 * -4.5 (b) Figure 9: (a) Initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Arctostaphylos same day measurements. Data are means ±SE and analyzed by performing ANOVA (two-factor with replication). Significant (P < 0.05) differences were noted by asterisks, (b) Initial (before submergence) and final (after 180 minutes submergence) leaf water potential in MPa for Arctostaphylos following the bench dry treatment. Data are means ±SE and analyzed by performing ANOVA (two- factor with replication). Significant (P < 0.05) differences were noted by asterisks.

140

120

100

°) °) * * 80 ■ Adaxial

60 ~ Abaxial Contact angle angle Contact ( 40

0 C. mega. megacarpus C. mega. insularis (a) Taxon

160

140 b b

120 c a °) °) a 100 a a a ■ Adaxial 80 r;;;i Abaxial 60 Contact angle angle Contact ( 40

0 A. pungens A. glauca A. catalinae A. insularis (b) Species Figure 10: (a) Contact angle measurements for adaxial and abaxial surfaces of Ceanothus. Data was analyzed by performing t-test (two-sample assuming equal variances). Bars represent mean ±SE and significant (P < 0.05) differences were noted by asterisks, (b) Contact angle measurements for adaxial and abaxial surfaces of Arctostaphylos. Data was analyzed by performing ANOVA (two-factor with replication). Bars represent mean ±SE and different letters indicate significant (P < 0.05) differences between taxa.

species between differences mean represent Bars replication). Arctostaphylos 11: Figure

Number of stomates/mm² 100 100 120 140 160 180 200 20 40 60 80 0

Number of stomata per mm² for adaxial and abaxial surfaces of of surfaces abaxial and adaxial for mm² per stomata of Number A. pungens

species. Data was analyzed by performing performing by ANOVAanalyzed was Data species. (two a a

. A. glauca A. catalinae catalinae A. glauca A. b ±SE ±SE b Species

and different letters indicate

28 c a A. insularis A. insularis

d c

significant (Psignificant < 0.05) ~ ■ Abaxial Adaxial - factor with

600

* 500 ²)

400

300

200 Leaf mass/area (g/m

100

0 C. mega. megacarpus C. mega. insularis Taxon (a)

400 a ab 350 b

²) 300 c 250

200

150

Leaf mass/area (g/m 100

0 A. pungens A. glauca A. catalinae A. insularis Species (b)

500 450

²) 400 350 300 250 200

150

Leaf Leaf (g/m succulence/area 100 50 0 C. mega. Megacarpus C. mega. insularis Taxon (a)

350 a

300 ab

²) b 250 b

200

150

100 Leaf Leaf (g/m succulence/area 50

0 A. pungens A. glauca A. catalinae A. insularis Species (b)

Figure 14: Scanning electron microscopy (SEM) micrographs of Ceanothus megacarpus var.megacarpus. (a) Adaxial surface showing a thick layer of wax. (b) Adaxial surface showing close-up look of wax coat. (c) Abaxial surface with a bunch of trichomes spreaded all over the surface. (d) Abaxial surface showing the close-up look of trichomes. (e) Abaxial surface showing a unique layer of wax on trichomes.

CHAPTER FOUR

DISCUSSION

All species exhibited water permeability through their leaf surfaces and demonstrated the capacity to absorb water directly into the symplast. Although, the pathway of water absorption into the leaves remains unknown from this study, the research in other plant taxa for foliar absorption shows that water can diffuse through the cuticle (Vaadia & Waisel 1963, Yates & Hutley 1995). In this study, due to similar environmental conditions in a common garden, the water potentials for all of the mainland and island species were very similar, which differs from the results of a recent study comparing the ecophysiology of species in their native island and mainland chaparral communities (Ramirez 2015). In that study, island species of Arctostaphylos had higher midday water potentials compared to mainland species of the same genus. The difference in water potentials between these two studies may be due to the plastic response of island species to the environment (Ramirez 2015).

Both varieties of Ceanothus megacarpus were very similar in water absorption and hydrophobicity, but they differ in other traits such as leaf mass per area and succulence, where C. megacarpus var. megacarpus had higher values compared to C. megacarpus var. insularis, suggesting the mainland variety was more xerophytic.

Interestingly, SEM revealed some anatomical features that supported the results such as, the dense layers of trichomes on the abaxial surface of both varieties, which may be the cause of the higher abaxial hydrophobicity. Since dew forms more on the adaxial than abaxial surface of horizontal leaves, it makes sense that the adaxial surfaces would be more hydrophilic than the abaxial surface. The results could be explained by differences

in leaf anatomical features of these species, because there was a thicker layer of wax present on the adaxial surface of C. megacarpus var. megacarpus, compared to C. megacarpus var. insularis, which may be a xerophytic trait (Papierowska et al. 2018).

In Arctostaphylos, A. catalinae, an island species, exhibited the greatest water absorption, A. glauca, a mainland species, had the lowest water absorption, and

A. insularis, A. glauca, which are island and mainland species, respectively, were similar in their water absorption. A. pungens, a mainland species, was intermediate in its water absorption. While A. catalinae behaved as expected in terms of its ability to absorb water, the other island species, A. insularis, did not. . Arctostaphylos catalinae might have adapted to the islands by the foliar hydration trait whereas A. insularis leaves are mesophytic and may not adapted to, or especially require, uptake. Arctostaphylos insularis is also in the Arctostaphylos section of the genus, along with the mainland species of this study, whereas A. catalinae is in the Foliobracteata section of the genus, indicating that the difference between the island species could be related to phylogenetics. Arctostaphylos insularis had other mesophytic traits such as having stomata only on the lower surface of the leaf (hypostomatic), horizontally oriented leaves, lower leaf mass per area, and lower succulence compared to mainland species in this section. Arctostaphylos pungens (mainland sp.) had the highest leaf mass per area and the highest succulence compared to the other three species. The high leaf mass was associated with the presence of a thick waxy coat with patches of wax resting atop the surface as shown in SEM. Arctostaphylos glauca had the lowest number of stomata compared to the other species, indicating that these leaves are xerophytic.

The increased water absorption occurred in the winter even though the plants were well hydrated due to rain. These results are supported by Limm et al. (2009) who reported a marked increase in foliar uptake that increased leaf water content in adequately hydrated plants even when the water potential driving gradient across the leaf surface was small.

The measurements of leaf water potential before and after the water absorption treatment for all taxa served as evidence of water absorption by leaves into the symplast.

The fact that the final water potential was not significantly higher (less negative) compared to the initial water potential for some species may be due to their initial high water status. However, even a small increase in water potential can have large impact on plant processes such as, cellular expansion, increased solute transport, and photosynthesis rates (Burgess & Dawson 2004, Simonin et al. 2009, Limm et al. 2009). The contact angle measurements were very similar for all the species with an exception of A. glauca which was very hydrophobic, consistent with the SEM results, which revealed that A. glauca has a thick layer of ornate wax on both of the surfaces. However, there was no difference in the adaxial and abaxial hydrophobicity level within any of the four species of Arctostaphylos.

For both genera, increased sample sizes might have led to more significant differences between species, but we were limited by the number of individuals cultivated at the garden. Four of the species in the present study were grown from seed, so they have shown some genetic variability. However, two of the species, A. glauca and A. insularis, were from cuttings and so they would have little genetic variability. For both genera, the results might have been different if the plants were collected from their place

of origin instead of a common garden. In a common garden, the environment was the same for both the island taxa and the mainland taxa, and so heritable differences could be detected. However, the common garden did not account for possible plastic responses to the environment.

With the current observations, it can be inferred that not all the island species are alike in terms of foliar absorption, but island taxa were similar regarding some mesophytic traits. For more robust conclusions, we need to collect samples from their place of origination, where the surrounding conditions are different for island and mainland species as they grow and develop, such as more fog or mist on islands, and a dryer atmosphere on a mainland.

CHAPTER FIVE

CONCLUSION

Foliar absorption can be an important trait when analyzing adaptations of plant species to island environments. Traditional matrices such as leaf mass per area and succulence are also relevant and may evolve independently from foliar absorption. In the case of Ceanothus megacarpus var. insularis and Arctostaphylos insularis, the key adaptation may be low mass per area and low succulence. Arctostaphylos insularis was also different from the other Arctostaphylos species in having hypostomatous leaves.

Arctostaphylos catalinae had very high foliar absorption capacity, which may be the key adaptation for this species. For A. glauca, the high hydrophobicity of the leaf surface appeared to be due to ornate epicuticular wax, and the other mainland Arctostaphylos species, A. pungens, had the highest leaf mass per area and leaf succulence. Based on the results, it is believed that the more leaf traits we study, the better we may understand island adaptations.

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