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12-2013

The Scent of Eastern Skunk Cabbage,Symplocarpus Foetidus (Araceae): Qualification ofFloral Volatiles and Sex Differences in Floral ScentCompositionErica Nicole KozenIndiana University of Pennsylvania

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Recommended CitationKozen, Erica Nicole, "The Scent of Eastern Skunk Cabbage, Symplocarpus Foetidus (Araceae): Qualification of Floral Volatiles andSex Differences in Floral Scent Composition" (2013). Theses and Dissertations (All). 1146.http://knowledge.library.iup.edu/etd/1146

THE SCENT OF EASTERN SKUNK CABBAGE, SYMPLOCARPUS FOETIDUS (ARACEAE):

QUALIFICATION OF FLORAL VOLATILES AND SEX DIFFERENCES IN FLORAL SCENT

COMPOSITION

A Thesis

Submitted to the School of Graduate Studies and Research

in Partial Fulfillment of the

Requirements for the Degree

Master of Science

Erica Nicole Kozen

Indiana University of Pennsylvania

December 2013

ii

Indiana University of Pennsylvania

School of Graduate Studies and Research

Department of Biology

We hereby approve the thesis of

Erica Nicole Kozen

Candidate for the degree Master of Science

_____________________________ _____________________________________________

Sandra J. Newell, Ph.D.

Professor of Biology, Advisor

_____________________________ _____________________________________________

Carl S. Luciano, Ph.D.

Professor of Biology

_____________________________ _____________________________________________

David H. Pistole, Ph.D.

Professor of Biology

ACCEPTED

____________________________________ _____________________________

Timothy P. Mack, Ph.D.

Dean

School of Graduate Studies and Research

iii

Title: The Scent of Eastern Skunk Cabbage, Symplocarpus foetidus (Araceae): Qualification of Floral

Volatiles and Sex Differences in Floral Scent Composition

Author: Erica Nicole Kozen

Thesis Chair: Dr. Sandra J. Newell

Thesis Committee Members: Dr. Carl S. Luciano

Dr. David H. Pistole

Species of the Araceae family have extraordinary thermogenic abilities and produce a foul floral

odor associated with the pollination syndrome of sapromyophily. Researchers have determined the

specific compounds that comprise the floral scents of many thermogenic aroids and are focusing on how

different aspects of plant biology affect scent composition and subsequent plant-insect interactions.

In this study, I determined the scent composition of the sapromyophilous and thermoregulatory

aroid Symplocarpus foetidus using the dynamic headspace sampling technique and gas chromatography-

mass spectrometry. I found the S. foetidus odor to be dominated by dimethyl disulfide and to contain

compounds from other chemical classes. I also addressed the existence of sex differences in the floral

scent composition of S. foetidus. I found the floral odors of both male and female skunk cabbage plants

to contain dimethyl disulfide, aliphatic hydrocarbons, carboxylic acids, and esters, whereas aromatic

hydrocarbons and indole compounds were unique to female plants.

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ACKNOWLEGMENTS

I would like to express my gratitude to the following people and organizations for their support, which

contributed to the completion of this project:

My thesis advisor, Dr. Sandra Newell, and committee members Drs. Carl Luciano, David Pistole,

and Robert Gendron. I greatly appreciate your guidance, support, and patience as I completed my

thesis project.

Concurrent Technologies Corporation for the use of laboratory supplies and equipment, which

otherwise would not have been obtained for completion of this project.

My husband, Steve, and daughter, Clare. I deeply appreciate your love, support, and patience as I

spent time away from both of you to complete my research project.

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TABLE OF CONTENTS

Chapter Page

1 INTRODUCTION .............................................................................................................. 1

2 LITERATURE REVIEW ................................................................................................... 3

Floral Scent ......................................................................................................................... 3

Floral Scent in the Araceae Family ..................................................................................... 5

Thermogenesis ....................................................................................................... 5

Protogyny ............................................................................................................... 7

Sapromyophily ....................................................................................................... 8

Research on Floral Scent Composition of Sapromyophilous Species ................... 8

Sex Differences in Floral Scent Composition ................................................................... 12

Experimental Summary and Objectives ............................................................................ 16

3 MATERIALS AND METHODS ...................................................................................... 20

Sample Selection ............................................................................................................... 20

Plant Populations ................................................................................................. 20

Temperature Readings ......................................................................................... 21

Sex Identification ................................................................................................. 22

Sample Collection ............................................................................................................. 23

Sampling Apparatus ............................................................................................. 23

Sampling Procedure ............................................................................................. 25

Sample Analysis ............................................................................................................... 26

Sample Extraction ................................................................................................ 26

Gas Chromatography-Mass Spectrometry Analysis ............................................ 26

Identification of Volatile Organic Compounds .................................................... 27

4 RESULTS ......................................................................................................................... 31

Identification of Volatile Organic Compounds in Symplocarpus foetidus Floral Scent ... 31

Sex Differences in Symplocarpus foetidus Floral Scent Composition.............................. 35

5 DISCUSSION ................................................................................................................... 37

REFERENCES ........................................................................................................................................... 42

APPENDICES ............................................................................................................................................ 50

Appendix A – Sample collection and temperature data for 2010 samples ....................... 50

vi

Appendix B – Sample mass spectra and NIST library match mass spectra for compound

classes detected in 2010 samples ...................................................................................... 52

Appendix C – Compound or compound class detected in each 2010 sample .................. 58

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LIST OF TABLES

Table Page

1 Number of Male and Female Plant Extracts Containing Each Compound or Compound Class ......... 35

viii

LIST OF FIGURES

Figure Page

1 A Skunk Cabbage Plant with Two Inflorescences Exhibiting Heat Production ................................... 21

2 Skunk Cabbage Inflorescence in the Female (Pistillate) Stage. ........................................................... 23

3 Skunk Cabbage Inflorescences in Early and Late Staminate Stages. ................................................... 23

4 Dynamic Headspace Sampling Apparatus. ........................................................................................... 25

5 (A) Sample Search Spectrum for an Unknown Compound and (B) the NIST Probable Match

spectrum for 1-methylhexylhydroperoxide with the shared base peak outlined in red and smaller

shared ion fragments outlined in like colors. ........................................................................................ 29

6 Examples of the Dimethyl Disulfide and Diethyl Phthalate Peaks on the Gas Chromatogram. ........... 33

7 Example Search Spectrum and NIST Library Match Spectrum for Dimethyl Disulfide with the Shared

Base Peak Outlined in Red (m/z = 94, 96) and Smaller Shared Ion Fragments Outlined in Like Colors

(m/z = 41, 61, 79). ................................................................................................................................ 33

8 The Percentage of 27 Odor Extracts Collected From Symplocarpus foetidus in 2010 Containing a

Specific Compound or Class of Compounds. ....................................................................................... 35

9 Abundance of Volatile Organic Compound Classes Detected in Male (n = 7) and Female (n = 20)

Samples. ............................................................................................................................................... 36

1

CHAPTER 1

INTRODUCTION

The production of floral scent by flowering plants is a vital process for ensuring a plant’s

reproductive success. While floral scents vary from species to species, the primary purpose of floral scent

production is to attract pollinators, which is a means of promoting cross-pollination between male and

female flowers. Some plants use the unique process of thermogenesis, or heat production, to enhance

their floral fragrances and entice pollinators. Thermogenesis occurs in only a few families of

angiosperms, but has been well-studied in the Araceae family (members referred to as aroids). Because

of their thermogenic abilities, researchers are interested in identifying the individual compounds, known

as volatile organic compounds (VOCs) that constitute the floral fragrance of aroids. Aroid flowers can

have scents ranging from sweet- to foul-smelling. Those emitting foul floral odors are of particular

interest because they are associated with the pollination syndrome of sapromyophily in which the carrion-

or dung-like scent of the flower deceitfully lures various species of flies that lay their eggs on these

sources and results in cross-pollination. Numerous studies on the floral scent composition of these foul-

smelling aroids have revealed the scent profile is typically dominated by one or two compounds, such as

an oligosulfide or primary amine, and may also contain compounds present in smaller amounts from

various chemical classes known to comprise floral scents (Borg-Karlson, Englund, & Unelius, 1994).

Equally of interest to researchers is how variations in floral scent composition within and among

species can affect plant-insect interactions. There are several different sources that can cause floral scent

variation within a species, but many researchers are concerned with how plant sex affects floral scent

composition. Most hermaphroditic plants exhibit dichogamy – the temporal separation of male and

female reproductive structures, which primarily occurs to prevent self-pollination. Researchers have

observed that some pollination activities only occur at certain sexual stages in dichogamous plants, and it

has been suggested that specific floral volatiles released during a particular sexual stage may act as a cue

for these pollinator behaviors (Patt, French, Schal, Lech, & Hartman, 1995). Several studies have

investigated sex differences in floral scent composition, and the results have simply shown that

2

differences either exist or do not exist within a species; however, a few researchers have attempted to

deduce overall trends in sex differences in floral scent composition among species and how these

differences may contribute to plant-insect interactions (Ashman, 2009). Even less extensive is research

regarding sex differences in the floral scent composition of sapromyophilous aroids. Because most

sapromyophilous aroids exhibit dichogamy, determining if sex differences exist in floral scent

composition for these species would have important implications for evaluating how plant-insect

interactions are mediated.

For this study, I chose to investigate the floral scent composition of the sapromyophilous aroid

Symplocarpus foetidus, which is commonly referred to as eastern skunk cabbage. The primary objective

of this study was two-fold. First, I qualified the floral scent composition of S. foetidus, which no previous

studies have attempted. Second, I evaluated the floral scent composition of S. foetidus to determine if sex

differences exist. Based on currently available research, I hypothesized that the floral scent of S. foetidus

would be dominated by a single chemical class of compounds, such as oligosulfides or amines, and would

contain compounds from other chemical classes, including fatty acid derivatives, benzenoids, or

terpenoids. With respect to sex differences in the floral scent composition of S. foetidus, I hypothesized

that differences would exist. Specifically, I expected the odor of female plants to be complex consisting

of compounds from several different chemical classes. On the other hand, I expected the odor of male

plants to be less complex containing compounds from only a few chemical classes.

3

CHAPTER 2

LITERATURE REVIEW

Floral Scent

Floral scent production is vital for the life cycle of flowering plants, or angiosperms. A particular

floral scent is composed of a variety of chemical compounds, and it is thought by many plant biologists

that these compounds originally evolved as plant defense mechanisms (Pellmyr & Thien, 1986; Pichersky

& Gershenzon, 2002). A common plant defense mechanism is the production of compounds by the

vegetative parts of the plant to repel or intoxicate herbivores and microbes that inflict plant damage. A

plant may also produce scent compounds to attract animals that naturally prey on herbivores (Cote &

Gibernau, 2012). Although plant defense mechanisms are crucial for survival, perhaps the most

beneficial and well-known function of floral scent is to attract pollinators. From an evolutionary

perspective, it is thought that many animals, particularly insects, overcame the repellant nature of plant

defense compounds over time. As a result, the life cycles of plants and insects meshed leading to the

evolution of pollination systems and the diversity of floral scents we observe in angiosperms today

(Pellmyr & Thien, 1986; Knudsen, Eriksson, Gershenzon, & Stahl, 2006). The process of pollination is

beneficial to both the plant and pollinator in that the plant gains a reproductive advantage as pollen is

transferred to and from other plants within the population, and the pollinator is usually rewarded with

food (in the form of nectar, pollen or oils), mating sites, and in some cases, shelter. To attract pollinators,

flowers can emit a variety of unique floral fragrances. Often times, a specific floral scent attracts a

specific type of pollinator. These instances in which particular types of pollinators are associated with

certain floral traits, which may also include traits such as petal color, are referred to as pollination

syndromes (Chittka & Raine, 2006).

Due to the importance of floral scent in plant-insect interactions, a significant amount of research

has been devoted to studying floral scent, particularly the types of compounds that constitute different

fragrances. Floral scents are composed of small compounds called volatile organic compounds (VOCs).

Although research has not focused on VOC biosynthesis in plants until recently, it is known that virtually

4

all plant tissues can synthesize VOCs and several genes and enzymes may be involved in the process. In

many plants, VOCs are synthesized and released from specialized glands called osmophores. VOCs are

typically not stored, but are immediately released from the tissues in which they were produced

(Dudareva & Pichersky, 2000; Pichersky & Gershenzon, 2002). VOCs are considered volatile due to

their high vapor pressure, which allows them to evaporate and disperse easily into the air. The specificity

and uniqueness of a floral scent is determined by the types and amounts of VOCs present. Floral scents

can contain anywhere from a few to over one hundred different VOCs with each compound present in

varying amounts (Dafni, Kevan, & Husband, 2005; Dudareva & Pichersky, 2006). When a plant emits a

particular scent into the air, the scent compounds are detected by insects through their antennae. Insects

have specialized receptor cells on the surface of their antennae to which specific VOCs bind. VOCs,

therefore, act as chemical cues signaling the presence of rewards for insects, depending upon the type of

scent emitted, and can elicit flower visitation (Pichersky, 2004).

In recent years, many techniques have been developed for collecting and analyzing floral scents

to determine the specific VOCs present. The most common and recommended technique for sampling

floral scents is dynamic headspace sampling. A plant’s headspace is the air directly surrounding the plant

or floral structure from which the scent is emitted. Dynamic headspace sampling is the least invasive of

sampling methods in that the plant is not damaged in any way, which minimizes the stress inflicted on the

plant. If damage or stress is induced, many plants will emit damage volatiles that are different in

composition than the VOCs emitted to attract pollinators, which would interfere with the proper

characterization of a plant’s floral scent. Dynamic headspace sampling involves enclosing the plant or

plant structure to be sampled in a glass or polyacetate chamber. This chamber concentrates the floral

headspace VOCs. A stream of air, usually from a battery-operated vacuum pump, is passed through the

chamber and the headspace VOCs are collected on an adsorbent cartridge. The cartridge contains a

granular chemical to which VOCs have an affinity and bind. The VOCs are eluted from the cartridge

using an organic solvent. The solvent extract is then injected into an instrument called a gas

chromatograph-mass spectrometer (GC-MS) for analysis. The GC separates the mixture of compounds

5

into individual components, and the MS identifies the chemical structure of each compound (Heath &

Manukian, 1994; Raguso & Pellmyr, 1998).

Several studies employing dynamic headspace sampling and GC-MS analysis have revealed the

presence of almost 2,000 different VOCs in the floral scents of over 990 different plant taxa. Despite the

diversity of compounds detected in floral scents, the majority are dominated by a single class of

compounds, which typically include aliphatic hydrocarbons, aromatic hydrocarbons, terpenoids, nitrogen-

containing compounds, sulfur-containing compounds, and various cyclic compounds (Knudsen, Eriksson,

Gershenzon, & Stahl, 2006). Although a significant amount of research has been devoted to studying

floral scent composition, researchers have only scratched the surface of discerning the role of floral scent

compounds in plant-insect interactions. Because researchers have gathered a rather comprehensive list of

the VOCs that comprise various floral scents, the research focus is shifting to mechanisms of VOC

biosynthesis and the roles individual or specific types of compounds may play in plant-insect interactions.

Floral Scent in the Araceae Family

A significant amount of recent research has focused on determining the floral scent composition

of angiosperms in the family Araceae, which are often referred to as aroids or arum lilies. The floral

scent composition of the Araceae family is of interest because a significant number of its species produce

strong, foul floral scents and exhibit unique characteristics that include thermogenic capabilities,

protogynous reproduction, and the pollination syndrome sapromyophily. Each of these characteristics

plays an important role in floral scent emission and the life cycle of aroids. The following sub-sections

will discuss each of these characteristics in relation to floral scent. In the final sub-section, the current

state of research regarding floral scent composition of foul-smelling aroids will be reviewed.

Thermogenesis

Thermogenesis, or heat production, is a unique tactic used by some plants to enhance their floral

scents and attract pollinators. The process of thermogenesis only occurs in nine extant families of

angiosperms, including the Araceae, Arecaceae, Cyclanthaceae, Annonaceae, Aristolochiaceae,

Nelumbonaceae, Nymphaceae, Magnoliaceae, and Illiciaceae (Azuma, Thien, & Kawano, 1999). Plants

6

belonging to these families are found in a variety of habitats ranging from tropical to temperate regions,

however, the most studied and remarkable cases of floral thermogenesis occur in cold climates, which

mostly include members of the Araceae (Gibernau, Barabe, Moisson, & Trombe, 2005; Robacker,

Meeuse, & Erickson, 1988; Zhu, et al., 2011).

To understand the process of thermogenesis in the Araceae and how a plant benefits from this

process, one must first have knowledge of the aroid inflorescence and flower morphology. The two main

components of the aroid inflorescence are the spathe and the spadix. The spathe is a fleshy, protective

bract that surrounds the spadix and forms a floral chamber. The spadix is divided into two zones: the

lower or basal zone that contains the reproductive structures and the upper zone, the appendix, which is

sterile and responsible for heat production and floral scent emission. A respiratory pathway in which heat

is produced instead of ATP (as in traditional cellular respiration) is responsible for thermogenesis in plant

tissues. This pathway can be activated by a variety of signals in aroids, including cold temperatures, and

results in a period of furious metabolic activity in the appendix mitochondria that yields heat (Zhu, et al.,

2011). Most thermogenic aroids, as well as thermogenic plants in other taxa, are unable to control the

amount of heat they produce (Seymour, 2001). In general, the aroid appendix generates enough heat to

raise its temperature 15 to 25C above ambient air temperature (Gibernau, Macquart, & Przetak, 2004;

Ito, Ito, Onda, & Uemura, 2004). Thermogenesis has been particularly well-studied in the aroid

Sauromatum guttatum, otherwise known as the voodoo lily. The appendix of this aroid has been

observed to generate temperatures up to 32C higher than ambient air temperature (Hadacek & Weber,

2002).

The amount of heat generated by thermogenic plants is quite remarkable, but perhaps even more

extraordinary is the ability of a few species of aroids to thermoregulate, or control the amount of heat

produced. Thermoregulation is observed in three aroid genera: Philodendron, Symplocarpus, and

Nelumbo (Seymour, 2001; Zhu, et al., 2011). Thermoregulatory plants control the amount of heat

produced by the appendix by set-points in reference to ambient temperature. Likely due to the presence

7

of a specific gene or set of genes, these plants are able to alter the respiratory activity of the appendix to

ensure a constant floral temperature is maintained even as the external environment changes (Azuma,

Thien, & Kawano, 1999). For example, in the genus Symplocarpus, a spadix temperature of

approximately 20C can be maintained even if the air temperature drops below freezing (Zhu, et al.,

2011).

The process of thermogenesis is beneficial to plants for a variety of reasons. The primary benefit

of heat production is that it enhances floral scent volatilization. Due to the chemical nature of VOCs, the

simultaneous release of heat and floral scent from the appendix of aroids allows the VOCs to disperse

more easily and perhaps further distances into the air. In turn, the enhanced floral scent increases the

probability of visitors to aroid inflorescences, which may result in pollination (Seymour & Blaylock,

1999). In addition to increasing pollination activities, the process of thermogenesis offers other

advantages to the plant. Because many aroids, particularly thermoregulatory species, are found in cold

climates, thermogenesis may protect inflorescences from freezing. It is thought that the heat produced

provides the optimum temperature for many reproductive physiological processes such as pollen tube

growth, pollen maturation, and pollen release (Onda, et al., 2008; Seymour, Ito, Onda, & Ito, 2009). Even

though a significant amount of energy is invested into thermogenesis, these cold-tolerant plants also gain

a significant reproductive advantage. Many aroids are the first plants to bloom in the early spring even

when snow still covers the ground. At this time, little competition exists among plants for early

pollinating insects. Therefore, the probability of visitation and subsequent pollination of thermogenic

aroids by these insects is quite high.

Protogyny

Patterns of floral scent emission in thermogenic aroids can vary depending upon the plant’s

reproductive cycle. The flowers of thermogenic aroids fall into two categories: unisexual and bisexual.

Unisexual flowers are either strictly male (containing only stamens) or strictly female (containing only

pistils). In thermogenic aroids with unisexual flowers on the spadix, heat is usually only produced from

the appendix of the male plant to promote floral scent and attract insects to gather and transfer pollen (Ito

8

& Seymour, 2005; Zhu, et al., 2011). The majority of thermogenic aroids have bisexual flowers, meaning

they bear both male and female reproductive structures, i.e. pistils and stamens. Most plants with

bisexual flowers follow a pattern of differential gender expression referred to as dichogamy. In other

words, the appearance of pistils and stamens occurs at different times during a single reproductive season.

Dichogamy is not only a characteristic of thermogenic aroids, but occurs in the majority of flowering

plants. The primary benefit of dichogamy appears to be to ensure cross-pollination and prevent self-

pollination whenever possible (Bawa & Beach, 1981; Lloyd & Webb, 2011). The expression of male and

female reproductive structures can follow two temporal patterns. First, in flowers exhibiting protandry,

male reproductive function precedes female reproductive function – stamens appear before pistils.

Protandry is very common in angiosperms, but the majority of plants exhibit the second pattern of gender

expression, protogyny (Bawa & Beach, 1981; Morbey & Ydenberg, 2001). In protogynous flowers,

female reproductive structures appear prior to male reproductive structures. It has been observed in

protogynous thermogenic aroids that heat production usually peaks during the female stage (Thien,

Azuma, & Kawano, 2000; Zhu, et al., 2011).

Sapromyophily

Protogyny is particularly common in angiosperms that are wind-, beetle-, and fly-pollinated,

which is the predominant condition in thermogenic aroids (Endress, 2010). It is likely that fly and beetle

pollination coevolved with thermogenic plants because advantages exist for both the plant and pollinator

(Thien, Azuma, & Kawano, 2000). Many thermogenic aroids produce a foul floral odor similar to carrion

(decaying meat) or dung (animal feces). Flies, primarily dipterans, and beetles typically oviposit (lay

eggs) on carrion and dung. Therefore, aroids producing scents reminiscent of carrion and dung falsely

lure fly and beetle pollinators to their flowers by mimicking the odors of their laying sites. This

condition, in which certain pollinators are associated with the floral odor of dung and/or carrion, is

referred to as the pollination syndrome of sapromyophily. In addition to their scent, many

sapromyophilous aroids have a uniquely colored spathe, typically a mottled purplish-brown color

intended to mimic the color of dung and carrion (Jurgens, Dotterl, & Meve, 2006; Kite & Hetterschieid,

9

1997). Although the odor of sapromyophilous aroids only mimics carrion or dung, many beetles and flies

perform their mating and egg-laying behaviors within the floral chamber. In addition to providing mating

and oviposition sites, pollinators also gain a direct heat reward when visiting thermogenic aroids because

many of these flowers bloom in the early spring, often in freezing or sub-freezing temperatures. Due to

their small body sizes, most beetles and flies do not generate sufficient heat during flight to maintain their

body temperature. Consequently, these insects gain a heat reward that helps them perform activities, such

as flight, when visiting thermogenic aroid inflorescences. In turn, the plant gains a reproductive

advantage due to the transfer of pollen by insect visitors (Thien, Azuma, & Kawano, 2000).

Research on Floral Scent Composition of Sapromyophilous Species

Research regarding the floral scent composition of sapromyophilous flowers has been somewhat

limited; however, due to their strong fragrances, most of the research conducted has focused on

determining the floral scent composition of sapromyophilous aroids (Kite, et al., 1998). One of the first

landmark studies on the floral scent composition of foul-smelling aroids was conducted by Smith and

Meeuse (1966) using dry ice traps and paper chromatography. Smith and Meeuse studied four aroid

species with unpleasant odors, including Arum italicum, Dracunculus vulgaris, Hydrosome rivieri, and

Sauromatum guttatum. Paper chromatography revealed the scents consisted mostly of primary amines,

particularly indole and skatole, which are found naturally in animal feces.

Since huge advancements in floral scent analysis techniques have been made over the past 20

years, researchers are able to gather more specific and comprehensive data regarding floral scent

composition using GC-MS technology. One of the most well-studied sapromyophilous aroids has been

the voodoo lily, Sauromatum guttatum. This species is of interest because the appendix emits a strong,

carrion-like odor that mainly attracts beetles and flies, whereas the club-shaped organs located near the

base of the appendix emit a sweet, flowery odor. Several studies have revealed that the odor of the whole

S. guttatum appendix is a mix of chemical classes, including various hydrocarbons, oxygen-containing

heterocyclic compounds, alcohols, aldehydes, ketones, acids, esters, aromatic compounds, nitrogen-

containing compounds, sulfur-containing compounds, and terpenoids. The scent is dominated by

10

monoterpenes and sesquiterpenes, dimethyl oligosulfides, and nitrogen-containing indole and skatole.

Several compounds from each of the chemical classes were detected in trace amounts (0.5% or less of the

total composition) (Borg-Karlson, Englund, & Unelius, 1994; Skubatz, Kunkel, Howald, Trenkle, &

Mookherjee, 1996). Because amines, such as indole and skatole, and oligosulfide compounds are found

in the scent of dung and rotten meat, it is likely that these compounds give the S. guttatum appendix its

characteristic foul odor (Ollerton & Raguso, 2006).

Other foul-smelling aroids in which the scent composition has been determined include

Helicodiceros muscivorus and several species from the genera Amorphophallus, Arum, and

Pseudodracontium. H. muscivorous, more commonly referred to as the dead horse arum, is found in the

Mediterranean region and exhibits extraordinary thermogenic capabilities. The appendix emits a strong,

carrion-like odor that mainly attracts flies and some species of beetles (Seymour, Gibernau, & Ito, 2003).

Odor analysis using GC-MS technology has revealed the main constituents of the H. muscivorous odor to

be dimethyl disulfide, dimethyl trisulfide, and several alkanes with 10 to 13 carbon atoms (Kite, 2000).

Like H. muscivorous, species in the genera Amorphophallus, Arum, and Pseudodracontium are

thermogenic plants that emit obnoxious carrion- or dung-like odors and are mainly pollinated by flies and

beetles. The floral scent composition of many of the species within these genera has been determined.

Members of Amorphophallus emit odors typically described as carrion-like and are relatively simple in

composition, largely consisting of dimethyl oligosulfides, such as dimethyl disulfide, dimethyl trisulfide,

and dimethyl tetrasulfide. Similarly, the odors of Pseudodracontium species are mostly composed of

dimethyl oligosulfides, but also contain the amines indole and skatole (Kite & Hetterschieid, 1997). The

scent of Arum species is usually described as dung-like and is more complex in composition than

Amorphophallus or Pseudodracontium species. Dimethyl oligosulfides have not been reported in the

scent composition of Arum species, but many nitrogen-containing compounds have been detected,

including indole, skatole, methylamine, ammonia, and various aliphatic amines. Other chemical classes

found in Arum floral scents include fatty acid derivatives, benzenoids, phenylpropanoids, terpenoids,

11

alkanes, esters, and ketones (Gibernau, Macquart, & Przetak, 2004; Knudsen, Tollsten, & Bergstrom,

1993; Kite, 1995).

Several families aside from the Araceae contain sapromyophilous species, including the

Apocynaceae, Aristolochiaceae, Iridaceae, Hydnoraceae, Orchidaceae, Phallaceae, Rafflesiaceae, and

Rhamnaceae (Borg-Karlson, Englund, & Unelius, 1994; Jurgens, Dotterl, & Meve, 2006; Ollerton &

Raguso, 2006). Although the research is not as extensive as with aroid species, a few studies have

focused on determining the floral scent composition of non-aroid sapromyophilous species. For instance,

a 2006 study conducted by Jurgens, Dotterl, & Meve determined the scent composition of 15 foul-

smelling stapeliad species that are pollinated by flies. These species belong to the families Apocynaceae,

Asclepiadoideae, and Ceropegieae and are described as having odors similar to dung, urine, carrion, and

rotting flesh. A total of 149 compounds were found in the scent extracts of the 15 species. Six chemical

classes of compounds were detected and are listed as follows in order of their relative amounts: fatty acid

derivatives, benzenoids, sulfur compounds, nitrogenous compounds, sesquiterpenoids, and

monoterpenoids. The fatty acid derivatives included various aldehydes, alcohols, ketones, esters, and

acids. The nitrogenous compounds were primarily indole and skatole, and the sulfur compounds were the

oligosulfides dimethyl disulfide, dimethyl sulphone, dimethyl trisulfide, and dimethyl tetrasulfide.

Although research regarding the floral scent composition of sapromyophilous species has not

been extensive, results from several studies show that the chemical nature of foul scents can be quite

diverse. Certain odors can be dominated by a single compound or consist of a plethora of chemical

classes that combine to give a flower its unique fragrance. Descriptions of floral odors are subject to

interpretation, but certain chemical classes comprise particular types of odors. For instance,

sapromyophilous odors are typically described as resembling carrion or carnivore dung. Odor analysis of

these sources indicates that carrion and carnivore dung are primarily composed of dimethyl oligosulfides.

Therefore, it can be expected that a flower with a carrion-like or dung-like scent would mostly consist of

dimethyl oligosulfides. Sources besides carrion and carnivore dung have been used to describe the scents

of sapromyophilous species, including urine, rotting carcasses, and herbivore dung. Compounds

12

dominating the scent of urine include hexanoic acid, carboxylic acids, and pyrazines, whereas aldehydes

and amines (indole and cresol) comprise the scents of rotting carcasses and herbivore dung, respectively

(Jurgens, Dotterl, & Meve, 2006; Ollerton & Raguso, 2006).

Sex Differences in Floral Scent Composition

Because floral scents are vital to plant-insect interactions, it is not surprising that researchers are

interested in not only qualifying the floral scent composition of various angiosperms, but also in

determining how different aspects of floral scent composition affect the pollination process. Although all

floral scents are composed of similar classes of organic compounds, it is well-known that floral scent

composition can vary greatly among and within species with respect to the number, amount, and types of

volatile compounds present. Many researchers have begun to investigate the sources that may cause these

types of variation in floral scent composition. For instance, studies have shown that environmental

conditions, such as temperature, wind, soil chemistry, and geography can strongly influence floral scent

composition (Majetic, Raguso, & Ashman, 2009; Svensson, et al., 2005). Another potential source that

may greatly impact floral scent composition and pollination activities is plant sex. It has been observed

that some pollinator behaviors, such as mating and oviposition, only occur at certain floral sexual stages

in dichogamous plants, and it has been suggested that specific volatiles produced during a particular

sexual stage act as cues for these pollinator activities (Pat, French, Schal, Lech, & Hartman, 1995). If in

fact different volatiles are produced at different floral sexual stages, these findings could have important

implications in discerning the role specific volatiles play in plant-insect interactions for a given species.

One study conducted on the aroid Peltandra virginica supports sex differences in floral scent

composition. P. virginica is a protogynous aroid found throughout North America and is primarily

pollinated by flies of the families Chloropidae and Syrphidae. During the female sexual stage, the floral

odor of P. virginica is pleasant and sweet smelling, but during the male sexual stage the flower has strong

foul odor similar to decaying vegetation. Analysis of the floral odors emitted during each sexual stage

revealed the foul odor of male flowers was primarily composed of trimethyl-6,8-

dioxabicyclo[3.2.1]octane isomers, which were not found in the odor of female flowers. Furthermore, the

13

researchers found that these specific compounds elicited oviposition behavior in flies (Patt, French, Schal,

Lech, & Hartman, 1995).

In Fragaria virginiana (Rosaceae), commonly referred to as the Virginia strawberry, it was

observed that female inflorescences receive far fewer insect visitors than do hermaphroditic

inflorescences. F. virginiana is a gynodioecious plant, meaning that female plants coexist with

hermaphroditic plants in a population at any given time. Because previous studies had shown that visual

stimuli were not solely responsible for differential visitation, researchers proposed that differences in

floral scent composition between the two sex morphs may play a role. To test this hypothesis, researchers

examined the floral scent of F. virginiana to answer three specific questions: 1. is there a difference in the

amount of volatiles emitted by female and hermaphroditic plants? 2. Do female plants lack male-specific

volatiles? 3. Do female plants receive fewer insect visitors because their scent lacks a male-specific

volatile? Using dynamic headspace sampling, the researchers collected the floral scent from female and

hermaphroditic inflorescences and analyzed the scents using GC-MS technology. The researchers found

strong evidence for differences in floral scent composition between female and hermaphroditic

inflorescences that affects insect visitation patterns. First, the researchers found that female

inflorescences emitted significantly lesser amounts of volatiles than did hermaphroditic inflorescences. In

the odors of both females and hermaphrodites, 38 volatile compounds were detected and consisted of

monoterpenes, sesquiterpenes, alcohols, and benzenoid compounds. One compound, 2-phenylethanol,

was only found in trace amounts in female odors, but was found in greater amounts in hermaphroditic

odors suggesting that this compound is male-specific. This study was one of the first to demonstrate the

importance individual floral volatiles can have in plant-insect interactions (Ashman, Bradburn, Cole,

Blaney, & Raguso, 2005). Furthermore, experiments examining pollinator activity showed that insects

preferentially visited hermaphroditic flowers (Rosenstiel, Shortlidge, Melnychenko, Pankow, & Eppley,

2012).

One of the most comprehensive studies investigating sex differences in floral scent composition

was a review conducted by Ashman (2009). The objective of the review was to deduce overall trends in

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sex differences in floral scent composition among species, which previous studies had not attempted.

Ashman examined floral scent composition data from several studies for 33 species of angiosperms

belonging to 15 families. The majority of species were described as being dioecious (male and female

plants present in a population at a given time), gynodioecious (females and hermaphrodites),

androdioecious (males and hermaphrodites), or trioecious (males, females, and hermaphrodites). Ashman

looked for two particular trends in the data: sex differences in volatile emission rates and sex differences

in volatile composition. Of the 33 species reviewed, studies involving 15 of the species reported data on

volatile emission rates. When examining sex differences in volatile emission rates, it was found in 12 of

the 15 species that individual male flowers produced more volatiles than individual female flowers. An

interesting trend was observed in the floral scent emission rate of cycads. The cycads in the studies

reviewed exhibited thermogenic capabilities and were pollinated by thrips. Data from the studies showed

that male cones produced larger amounts of volatiles than female cones, and the amount of volatiles

produced by male cones increased as more heat was produced. It was suggested that by initially

producing lesser amounts of volatiles, the male cones attracted thrips. Then, the increase in volatile

emissions as heat production increased repelled the thrips, causing them to visit female cones that

produced lesser amounts of volatiles (Terry, et al., 2004; Terry, Walter, Roemer, & Hull, 2007). Use of

this tactic by cycads and possibly other thermogenic species would be an extremely efficient means of

promoting cross-pollination and is an important finding for understanding plant-insect interactions. To

evaluate trends of sex differences in floral scents, floral scent composition data was reviewed for 31

species. Of the 31 species, 23 showed sex differences in floral scent composition while eight showed no

sex differences. From the studies evaluated, it was apparent that species in which the floral scent

mimicked the mating sites of insect pollinators were more likely to show no sex differences in floral scent

composition. This was particularly true for species in which the female inflorescences emitted deceitful

odors to lure insect pollinators, but offered no rewards (Ashman, 2009).

In addition to reviewing floral scent composition data, Ashman (2009) also summarized the

various hypotheses that have been formulated to explain the presence or absence of sex differences in

15

floral scent composition. These hypotheses are important to consider as they offer insights into the role of

floral scents and perhaps specific volatiles in plant-insect interactions. The five main hypotheses

supporting sex differences in floral scent composition include sexual selection, honest signals, directed

pollinator movement, post-pollination deterrence/attraction of seed predators/dispersers, and allometric

relations/shared development. Each of these hypotheses will be briefly summarized. First, the sexual

selection hypothesis states that because male reproductive success is dependent upon pollinator attraction

and visitation more so than females, males will produce a unique floral scent to increase pollinator

visitation. Consequently, increased pollinator visitation would increase male fertility and thus increase

mating opportunities with female plants. The uniqueness of the male floral scent may be, in part, due to

the presence of pollen itself, which can produce a variety of VOCs. These VOCs are derived from the

same chemical classes as floral volatiles and have been shown to mostly consist of fatty acid derivatives,

benzenoid compounds, and various terpenoids (Dobson & Bergstrom, 2000). This viewpoint assumes

male inflorescences offer the reward of pollen to insect visitors, whereas female inflorescences offer no

reward. On the other hand, the honest signals hypothesis assumes that both male and female

inflorescences offer rewards to pollinators. Depending upon the reward offered, males and females will

each ‘honestly’ advertise their rewards by emitting a unique scent that elicits insect visitation. For

instance, if a male inflorescence offers nectar or pollen, the floral scent emitted from the male would be

primarily composed of nectar- or pollen-specific volatiles. On a slightly more complex note, the directed

pollinator movement hypothesis suggests that while both male and female inflorescences offer rewards,

the more rewarding flowers would be preferentially visited. To ensure flowers of both sexes are visited,

males and females may alter the timing of volatile emission so that pollinators move between both plant

sexes ensuring fertilization of female inflorescences by pollen transfer. The post-pollination

deterrence/attraction of seed predators/dispersers hypothesis puts a different twist on the presence of sex

differences in floral scent composition. This hypothesis suggests that once they have been pollinated,

female inflorescences will begin producing scents that are less attractive and more repellant in nature to

deter herbivores and/or attract seed predators. The scent of male inflorescences would remain generally

16

unchanged to ensure all pollen is shed. Therefore, the scent of females and males would be dissimilar at

this post-pollination stage. The final hypothesis supporting sex differences in floral scent composition is

the allometric relations and shared development hypothesis, which generally states that the quality and

quantity of floral volatiles is dependent on floral organ type, number, and size, which may differ between

male and female inflorescences. The two hypotheses supporting the absence of sex differences in floral

scent composition are the intersexual mimicry and the between-sex genetic constraints hypotheses. From

the intersexual mimicry viewpoint, both male and female flowers should emit similar scents to ensure

pollinators visit both sexes, regardless if the sexes do not offer the same rewards to pollinators. Lastly,

the between-sex genetic constraints hypothesis states that because the same set of genes determines floral

scent in males and females, both sexes will have similar scents as long as the genetic correlation is strong.

Each of the hypotheses described offers a unique perspective explaining the presence or absence of sex

differences in floral scent composition. It is important to note that these hypotheses are not mutually

exclusive and a combination of these theories may be applicable when studying a particular plant species

(Ashman, 2009).

Experimental Summary and Objectives

Due to the importance of floral scent in plant-insect interactions and the state of research in this

area, I have also chosen to investigate floral scent composition and its role in plant-insect interactions in

the Araceae family. For this study, I examined the floral scent composition of the aroid Symplocarpus

foetidus, commonly referred to as eastern skunk cabbage. The primary objectives of this study were to

qualify the floral scent composition of S. foetidus and to determine if sex differences exist in the floral

scent composition.

To understand the reasoning for selecting S. foetidus for this study, one must first consider some

general information regarding this species. S. foetidus is a perennial plant that is commonly found in

wetland areas of eastern North America. Noted by John Small (1959) as the most reliable early blooming

plant, S. foetidus is one of the first plants to appear in the spring, typically in late February or early March

even when snow still covers the ground. S. foetidus is able to melt its way through snow cover due to its

17

extraordinary thermogenic capabilities. The spadix, enclosed by a mottled purple-brown spathe, can

generate temperatures up to 35C above ambient air temperature even when the air temperature drops as

low as -15C. To produce such a remarkable amount of heat, the respiratory rate of the spadix doubles

and is usually maintained for a few hours per day, usually in the late morning to early afternoon, over a

period of up to two weeks. S. foetidus is also one of the few species known to thermoregulate, and this

species in particular has been shown to be capable of controlling its spadix temperature with

extraordinary precision, even as well as warm-blooded animals (Knutson, 1974; Ito, Onda, Sato, Abe, &

Uemura, 2003; Seymour & Blaylock, 1999). Associated with heat production in S. foetidus is the

emission of a foul floral odor, which primarily attracts various species of flies and some beetles. The

odor has been described as dung- or carrion-like and mimics the odor of the oviposition sites of its insect

visitors (Camazine & Niklas, 1984; Kevan, 1989). As the odor is produced, the spathe remains open so

that the inflorescence is easily accessible to insects. Heat production and odor emission is highly

correlated with the sex of the plant. S. foetidus inflorescences are protogynous. Heat production and

thus, odor emission, has been shown to peak during the period of stigma receptivity (female phase) and

continue through the bisexual stage to the early male phase when pollen begins to form on the spadix (Ito-

Inaba, Hida, & Inaba, 2009). In a study conducted by Seymour, Ito, Onda, and Ito (2009), it was reported

that in a population of S. foetidus, the female, bisexual, and male stages lasted an average of 6.8 days, 2

days, and 16.7 days, respectively. Heat production and odor emission during at least a portion of each

sexual stage ensures the presence of insects and promotes cross-pollination, which is crucial to the life

cycle of skunk cabbage. During the timeframe when heat and scent are produced, the odor has been

described as variable to the human nose (Kevan, 1989). Once the inflorescences of S. foetidus have been

pollinated, large leaves emerge from the plant that produce calcium oxalate crystals to deter herbivores,

and the seeds are formed during the fall months (Grimaldi & Jaenike, 1983).

To date, no studies have investigated the floral scent composition of S. foetidus. Due to this fact

and some of the unique characteristics of eastern skunk cabbage, it is an ideal species in which to not only

18

study floral scent composition, but also potential sex differences in floral scent. As previously discussed,

researchers are interested in determining the floral scent composition of thermogenic and

sapromyophilous aroids, such as S. foetidus. This is also one of the first studies to examine floral scent

and potential sex differences in any of the known thermoregulatory species. Because the odor emitted by

S. foetidus has been described as carrion- or dung-like, it is reasonable to expect that this odor would

contain VOCs like those found in the odors of these sources themselves and in flowers with similar

scents. Concomitant with other sapromyophilous aroids, the odor of skunk cabbage is likely to be

dominated by a single compound or class of compounds with compounds from a few to several other

chemical classes present in varying amounts. It is probable that the scent of skunk cabbage is dominated

by an oligosulfide or amine, such as indole or skatole, as was the case in species of Amorphophallus,

Pseudodracontium, and Helicodiceros. If the odor profile of S. foetidus is more complex, it may not only

contain a dominant sulfur- or nitrogen-containing species, but compounds from other chemical classes,

including fatty acid derivatives, benzenoid, or terpenoid compounds.

Previous studies on S. foetidus and other aroid species present evidence that sex differences in

floral scent composition of skunk cabbage is quite plausible. Because heat production and odor emission

has been shown to be highly correlated with the female sexual stage in S. foetidus, as in many other

thermogenic protogynous aroids, it is likely that floral scent composition differs between the female and

male sexes. The skunk cabbage female inflorescence falsely lures insect pollinators with heat and a

strong scent that mimics their oviposition sites, thus, the female scent is probably dominated by volatiles

found in dung and carrion odors. As heat production and odor emission dwindles during the bisexual and

early male phases when pollen begins to appear on the spadix, the male scent may still contain these dung

or carrion volatiles, but in lesser amounts. Furthermore, because pollen itself can have a unique odor,

which is composed of the same classes of compounds as floral scents, the male scent profile may be

slightly more complex than the female scent profile and contain pollen-specific volatiles. If sex

differences in floral scent composition are present in S. foetidus, these findings would be consistent with

the honest signals and directed pollinator movement hypotheses as described by Ashman (2009).

19

In addition to the aforementioned reasons for performing this study on S. foetidus, a number of

other factors played a role in my decision. First, S. foetidus is very common throughout central

Pennsylvania and is easily detectable in the snow when the inflorescences are producing heat. Individual

or small groups of skunk cabbage inflorescences are highly visible along river and creek beds as they are

surrounded by areas of melted snow. Therefore, it was very easy to monitor a population and determine

when heat production and odor emission began. The period of time during which skunk cabbage emits

heat and odor is also relatively short, lasting no longer than a few weeks in a given population. The areas

of melted snow surrounding the inflorescence also provided adequate space to perform dynamic

headspace sampling of the inflorescences without inflicting stress or plant damage. The process of

dynamic headspace sampling and analysis was simple and relatively inexpensive to perform due to the

availability of GC-MS equipment for use in this study.

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CHAPTER 3

MATERIALS AND METHODS

Sample Selection

Plant populations

To qualify the floral scent composition of S. foetidus, the VOCs emitted by inflorescences were

collected using the dynamic headspace sampling technique. Plants were selected for sampling from three

populations of S. foetidus at different locations in Pennsylvania, including Marion Center

(40°46’12.23”N, 79°3’0.12”W), Wilmore (40°24’31.49”N, 78°41’44.61”W) , and Johnstown

(40°17’29.86”N, 78°49’13.91”W). Because skunk cabbage inflorescences emerge from the ground in the

early spring, the readiness of each population for sampling was monitored on a weekly basis beginning in

February in the years sampling was conducted (2010 and 2011). A population was considered ready for

sampling if plants within the population were exhibiting heat production and, thus, odor emission. Heat

production was easily observed by the presence of areas of melted snow surrounding individual plants or

clusters of plants. Another indicator of heat production was plants with slightly opened spathes.

Individual plants meeting these criteria were considered candidate plants for dynamic headspace

sampling. Figure 1 shows an example of a skunk cabbage plant with two inflorescences exhibiting heat

production.

21

Figure 1. A skunk cabbage plant with two inflorescences exhibiting heat production. Adapted from

“Skunk cabbages are so hot” in Mental Floss by J. Harness, 2011. Retrieved October 31, 2013, from

http://mentalfloss.com/article/28143/skunk-cabbages-are-so-hot.

Temperature Readings

Once candidate plants were identified in the population, various temperature measurements were

taken from the plants to confirm they were generating sufficient heat to be sampled for odor analysis.

Because heat production from skunk cabbage inflorescences has been shown to peak in the late

morning/early afternoon, all temperature readings and samples were collected during this time (Seymour,

2004). Three temperature readings were taken from each candidate plant using a digital thermistor-

thermometer. First, an air temperature sensor was used to measure the ambient air temperature

approximately 12 in. above the spathe apex of the candidate plant. Next, a small surface temperature

sensor (approximately 0.25 in. diameter) was placed on the spadix to quantify the heat the plant was

producing and to verify odor emission was occurring. Finally, a sub-surface temperature probe was

inserted into the ground approximately six in. below the base of the plant, and the soil temperature was

recorded. A candidate plant was considered to be producing sufficient heat for odor sampling if the

spadix temperature was higher than the air and soil temperatures.

22

Sex Identification

If producing sufficient heat, a candidate plant was further scrutinized based upon its sex before

being selected for sampling. As previously discussed, S. foetidus exhibits protogynous flowering, which

means the female (pistillate) stage appears prior to the male (staminate) stage. The female stage is

characterized by a spadix that may appear smooth and bare or studded with stigmas. This stage lasts for a

very short period of time, typically one to two weeks (Seymour, Ito, Onda, & Ito, 2009). Figure 2

illustrates the appearance of the spadix in the female stage. The male stage occurs when stamens develop

and bear pollen, which studs the surface of the spadix. This stage can last for several weeks and consists

of an early and late stage. During the early stage, pollen appears sticky and moist; however, during the

late staminate stage, the pollen appears dry and dusty. Heat production and odor emission begins to

decrease in the late staminate stage as the spathe dehisces (Ito-Inaba, Hida, & Inaba, 2009). Figure 3

illustrates the appearance of the spadix in the early and late staminate stages. Because the female stage is

shorter and occurs prior to the male stage, female plants were selected for in the early weeks of sampling

to ensure a sufficient number of samples from female plants was obtained. Most male plants selected for

sampling were in the early staminate stage, but those in the late staminate stage were considered

acceptable as long as the spadix was still producing heat. Plants in the transitional (bisexual) stage in

which spadices were just beginning to bear pollen were not selected for sampling.

23

Figure 2. Skunk cabbage inflorescence in the female (pistillate) stage. Adapted from Bob’s Brain on

Botany by B. Klips, 2009. Retrieved October 31, 2013 from http://bobklips.com/earlymarch2009.html.

Figure 3. Skunk cabbage inflorescences in early and late staminate stages. Adapted from Bob’s Brain on

Botany by B. Klips, 2009. Retrieved from http://bobklips.com/earlymarch2009.html.

Sample Collection

Sampling Apparatus

Plants selected for odor analysis were sampled using the dynamic headspace technique

immediately after recording the temperature readings and plant sex. The sampling chamber was created

by placing a one-quart Ziploc® (polyethylene) bag over the inflorescence that was loosely secured around

Female stigma

Late staminate stage Early staminate stage

24

the base of the plant with a twist tie. By loosely securing the bag, it was ensured a small amount of

ambient air would pass through and prevent the chamber from acquiring moisture or collapsing, which

may have interfered with plant volatile emissions. A small incision was made in one top corner of the

bag. Both ends of a Porapak Q sorbent cartridge were cut open, and one end of the cartridge was inserted

into the opening in the corner of the bag. The Porapak Q cartridges, procured from SKC, Incorporated

(Eighty Four, Pennsylvania), were 6 x 110 millimeters in length and filled with 75 to 150 milligrams of

80 to 100 mesh particle size porous polymer adsorbent material (ethylvinylbenzene-divinylbenzene). Of

the available sorbent types, Porpak Q has been shown to trap the greatest amounts of floral volatiles,

especially those emitted in minute quantities (Raguso & Pellmyr, 1998). The corner of the sample bag

was secured around the end of the cartridge with duct tape. The other exposed end of the cartridge was

connected to approximately two feet of ¼ inch diameter Tygon tubing, which is commonly used for

headspace sampling because it is flexible and inert. The other end of the tubing was connected to the

SKC AirCheck 2000 battery-operated, portable sampling pump. The sampling pump and tubing were

secured to a ring stand to prevent collapse of the sampling chamber and to ensure sufficient air flow.

Figure 4 shows the assembled sampling apparatus.

25

Figure 4. Dynamic headspace sampling apparatus.

Sampling Procedure

Once the sampling apparatus was assembled for the selected plant, sample collection began. The

flow rate on the sampling pump was set to one liter per minute. Previous experiments have indicated this

flow rate allows for the collection of the greatest amounts of volatiles when compared to lower flow rates

( Raguso & Pellmyr, 1998). Once the sampling pump was started, the time was recorded. Samples were

collected for 30 minutes and 60 minutes during the 2010 and 2011 sampling seasons, respectively.

Various studies have shown collection times of at least 30 minutes are sufficient to collect a

representative sample of emitted volatiles (Raguso & Pellmyr, 1998). A longer collection time of 60

minutes was used in 2011 to determine if a larger quantity of volatiles could be obtained to make

subsequent identification of the volatile compounds easier. At the end of the collection period, the pump

was stopped, the sampling end time was recorded, and the cartridge was immediately removed from the

sampling apparatus. The cartridge was capped, labeled with a unique identification number, and stored

Sample Chamber

Tygon Tubing

Pump Cartridge

26

on ice in the field until sampling activities were concluded for the day, typically no longer than four hours

after collection of the first sample. The cartridges were then stored in a laboratory freezer at 0C until

sample analysis could be performed, which occurred within seven days of sample collection.

Sample Analysis

Sample Extraction

Prior to analysis, the floral volatiles trapped on the Porapak Q cartridges were extracted. The

cartridges were removed from freezer storage, uncapped, and attached to a ring stand with a clamp in a

vertical position. An amber borosilicate glass vial was placed under the bottom tip of the cartridge. Each

cartridge was eluted with three milliliters of 99% reagent grade hexane solvent. Both the vials and

solvent were purchased from VWR International (Philadelphia, PA). Previous studies have shown the

use of hexane solvent with Porapak Q cartridges yielded the greatest recovery of trapped compounds

(Raguso & Pellmyr, 1998). The extract was collected until the cartridge was dry, and the amber vial was

sealed with a PTFE/silicone septa screw cap. The extracts were stored in a refrigerator at 4C until

analysis was conducted, which was within seven days of extraction.

Gas Chromatography-Mass Spectrometry Analysis

A Varian 3800 Gas Chromatograph 1200 Quadruple Mass Spectrometer (purchased from Agilent

Technologies, Santa Clara, CA) was used to analyze all sample extracts. The gas chromatograph allowed

for complex mixtures of compounds to be separated, and the mass spectrometer allowed for structure

elucidation of individual compounds. This combined technique enabled individual compounds in each

sample to be separated and identified in one step. The GC-MS was equipped with a flame ionization

detector and a 30 millimeter VF-5s capillary column. Helium was used as the carrier gas. The samples

were placed in an autosampler, and the GC-MS was programmed for analysis. The samples were

introduced onto the column using a split/splitless injector at a temperature of 250C with a split ratio of

50. The column temperature was maintained at 80C for four minutes after injection and then linearly

increased to 250C at a rate of 10C per minute. The column temperature was held at 250C for 30

27

minutes. This GC-MS program allowed for qualitative analysis of VOCs and was chosen based on its use

in similar studies to identify floral scent compounds (Jurgens, Dotterl, & Meve, 2006; Kite, 2000;

Kuanprasert, Kuehnle, & Tang, 1998; Tholl et al., 2006).

In addition to the extracted samples, two control samples were run, including a hexane solvent

blank and an eluted hexane blank. The hexane blank consisted of three milliliters of unused solvent, and

the eluted hexane blank was prepared by passing three milliliters of hexane through an unused cartridge.

These control samples allowed for the identification of any impurities found in the solvent or cartridge

that may have interfered with the results obtained for the extracted samples.

Identification of Volatile Organic Compounds

For each sample, the GC-MS generated a chromatogram that represented all detected compounds

as peaks. When a particular peak was selected, its mass spectrum was automatically generated by the

equipment software. The mass spectrum of the compound of interest was compared against the National

Institute of Science and Technology (NIST) library of mass spectral reference data to determine its

identity. The NIST library is one of the largest in existence containing information on the chemical and

physical properties of thousands of organic compounds. The NIST library generated a list of possible

identities of the unknown compounds based on similarities between the unknown and known compound

mass spectra. For each possible compound, a match probability was automatically calculated. If the

match probability of the mass spectra of the unknown and known compounds was greater than 90%, the

suggested match was accepted.

If the match probability between the mass spectra was less than 90%, compounds were identified

according to the major functional group present in the compound’s chemical structure. Even if the match

probability was less than 90% between a known an unknown spectrum, the NIST library selected a

probable match based upon similarities among the ion fragments within the known and unknown spectra.

When a mass spectrum is generated for an unknown compound, several peaks appear on the spectrum that

relay important information regarding the compound’s chemical structure. Each peak represents an ion

fragment of the unknown compound that has a characteristic mass and charge. Each peak is assigned a

28

unique mass-to-charge ratio (m/z). Perhaps the most important piece of information on a mass spectrum

is the molecular ion or base peak. The molecular ion is the heaviest ion on a mass spectrum that can be

used to estimate the relative mass of the compound. The lighter ion fragment peaks observed on the mass

spectrum result from an unstable molecular ion that is broken into these smaller fragments. By

considering the molecular ion peak and the fragment peaks, the chemical structure of the unknown

compound may be pieced together. The NIST library database is able to compare the unknown

compound’s mass spectrum to the spectra of thousands of known compounds to make this process less

complex and time-consuming. The probable match generated by the NIST library served as a starting

point for identifying the major functional group present in the unknown compound. The presence of a

functional group was confirmed by further analysis of the mass spectrum of the unknown and match

compounds. For example, Figure 5 shows the sample search spectrum for an unknown compound and the

probable match generated by the NIST library. The NIST library identified the unknown compound as 1-

methylhexylhydroperoxide with a match probability of 29.54%. As shown in Figure 5A, the search

spectrum for the unknown compound has fragment peaks at m/z = 42.9 (boxed in blue) and m/z = 57.0

(boxed in green), and a base peak at m/z = 85.0 (boxed in red). Similarly, as indicated in Figure 5B the

NIST probable match spectrum has fragment peaks at m/z = 43.0 (boxed in blue) and m/z = 57.0 (boxed

in green) and a base peak at m/z = 85.0 (boxed in red). While the NIST library match, 1-

methylhexylhydroperoxide, has an approximate molecular weight of 85 atomic mass units (as indicated

by the molecular ion), it was not identified as a likely match for the unknown compound due to

dissimilarities in some of the lighter fragment peaks between the two mass spectra. An organic

hydroperoxide has a general chemical formula of “ROOH,” with the “R” representing any hydrocarbon

group, the “O” representing oxygen, and the “H” representing hydrogen. It is likely that the “R” group is

responsible for many of the dissimilarities in the lighter fragment peaks due to the large number of

possible fragments into which a hydrocarbon group of unknown size can be broken. Despite these

differences, the mass spectra share several heavier ion fragments and the molecular ion. Therefore, there

is a high probability that the unknown compound is an organic hydroperoxide as suggested by the NIST

29

library, but the exact structure cannot be determined. The exact identity of the unknown compound may

be similar to the NIST-generated match, but the library may not contain the exact mass spectrum for the

unknown compound in the database. Although this analytical process cannot determine a compound’s

specific structure, it is a fairly accurate means for elucidating the major functional group present in an

unknown compound. This process was followed for each instance in which the mass spectrum for an

unknown compound had a NIST match with a probability of less than 90%.

Figure 5. (A) Sample search spectrum for an unknown compound and (B) the NIST probable match

spectrum for 1-methylhexylhydroperoxide with the shared base peak outlined in red and smaller shared

ion fragments outlined in like colors.

Prior to implementing the procedures for identifying an unknown compound based on the major

functional groups present as previously described, I attempted to determine the exact identity of the

unknown compounds. Based on the mass-to-charge ratios of the fragment peaks for an unknown

compound, it is possible to determine the exact chemical structure of a fragment and, therefore, the exact

structure of the compound by piecing the fragments together; however, I elected to identify the unknown

compounds solely based on their chemical classification due to the small sample size. In many instances,

compounds belonging to a particular chemical class were only detected in one or two samples, and I did

30

not feel this sample size was adequate to verify compound identities and to conclude these compounds

were constituents of the skunk cabbage scent.

31

CHAPTER 4

RESULTS

Identification of Volatile Organic Compounds in Symplocarpus foetidus Floral Scent

During the 2010 and 2011 sampling seasons, a total of 70 samples were collected and analyzed

using GC-MS technology. Twelve samples were collected in 2011, but could not be analyzed within a

reasonable time frame following collection due to complications with the GC-MS equipment. Most

laboratory methods for volatile analysis recommend that sample extraction and analysis occur within 14

days of collection to prevent the loss of compounds over time (Clescerl, Greenberg, & Eaton, 1998). The

GC-MS equipment was not operational for over six months following sample collection in March and

April 2011; therefore, the 2011 sample results were not considered.

GC-MS analysis of the 58 samples collected in 2010 revealed several classes of organic

compounds may be present in the scent of S. foetidus. First, the results showed a large peak at a retention

time of approximately 24 minutes on the gas chromatograms of 74% of samples. The NIST library search

of the mass spectrum identified the compound to be diethyl phthalate with a match probability of 90% or

higher for all samples. Diethyl phthalate is a manufactured ester that does not occur in nature and is

commonly used to increase the flexibility of PVC materials. It is also soluble in most organic solvents,

including hexane, which was used to extract the floral volatiles from the sample cartridges (“Phthalates,”

2013). Because diethyl phthalate was present in the cartridge blank extract, it was likely used in the

manufacturing of the Porapak Q resin cartridge. Therefore, diethyl phthalate was not considered a

component of the skunk cabbage scent. Aside from diethyl phthalate, the gas chromatograms of 31 of the

58 samples revealed no other discernible compound peaks. Several factors may have contributed to the

lack of compound peaks in some sample extracts. If compounds were present in the extracts of these

samples, they may not have been present in large enough quantities to be detected or identified by the

instrumentation. It is also important to consider the spadix temperature of plants that produced no

compound peaks versus those that did produce peaks because elevated spadix temperature is associated

with odor emission. The average spadix temperature of the plants with extracts providing no compound

32

peaks was 5.03C above ambient air temperature with values ranging from 0C to 12.30C above

ambient. The average spadix temperature of plants with extracts in which compounds were detected was

7.50C above ambient air temperature with values ranging from 1.27C to 14.12C above ambient. A

combination of these factors likely contributed to no compound peaks being detected in half of the

samples. Complete sampling and temperature data for all 2010 samples is provided in Appendix A.

In the remaining 27 samples, the NIST library only identified one additional compound with a

match probability of 90% or higher, which was dimethyl disulfide. Dimethyl disulfide was detected in

93% of samples and appeared as a distinct peak on the gas chromatogram at a retention time of

approximately three minutes. Figure 6 shows an example of the dimethyl disulfide peak on the gas

chromatogram. An example of the diethyl phthalate peak is also noted in Figure 6. Figure 7 provides an

example of the search mass spectrum compared to the NIST library match for dimethyl disulfide. As

shown in the figure, dimethyl disulfide was identified by the presence of a split base peak at

approximately m/z = 94.0 and m/z = 96.0 (boxed in red) and smaller ion fragments at m/z = 45.0 (boxed

in blue), m/z = 61.0 (boxed in green), and m/z = 79.0 (boxed in purple).

33

Figure 6. Examples of the dimethyl disulfide and diethyl phthalate peaks on the gas chromatogram.

Figure 7. Example search spectrum and NIST library match spectrum for dimethyl disulfide with the

shared base peak outlined in red (m/z = 94, 96) and smaller shared ion fragments outlined in like colors

(m/z = 41, 61, 79).

34

GC-MS analysis also revealed that other classes of organic compounds may be present in the

skunk cabbage scent. Several small peaks were detected on the sample gas chromatograms; however, the

compounds were not identified by the NIST library with a match probability of 90% or higher. The major

functional group present in each of the unknown compounds was deduced by examining the base and ion

fragment peaks on the compound mass spectra. Several of these compound types occurred together in the

same sample extract. The classes of compounds identified included aliphatic and aromatic hydrocarbons,

peroxides, carboxylic acids, esters, indole compounds, and organosilicons. Aliphatic hydrocarbons were

detected in 44% of samples and included allenes and alkynes, cyclic and non-cyclic monoterpenes, and

aldehydes. Aromatic hydrocarbons were found in 22% of samples and consisted of benzenoid

compounds and ketones. Organic peroxides were identified in 19% of samples. Carboxylic acids, esters,

and indole compounds were detected less frequently, each occurring in 7% to 11% of samples. An

organosilicon compound was detected in only one sample. Organosilicon compounds are not naturally

occurring plant compounds rather they are frequently used in the manufacturing of GC-MS equipment

and accessories (Phillips, 2012). Because the organosilicon detected in the sample was likely an artifact

of the GC-MS capillary column, it will not be considered in subsequent presentation of results. Examples

of the mass spectra for each class of compounds are provided in Appendix B, and a comprehensive list of

the compounds detected in each sample can be found in Appendix C. Figure 8 summarizes all

compounds detected in the 2010 samples and their relative abundance.

35

Figure 8. The percentage of 27 odor extracts collected from Symplocarpus foetidus in 2010 containing a

specific compound or class of compounds.

Sex Differences in Symplocarpus foetidus Floral Scent Composition

The 2010 sample results were evaluated for sex differences in floral scent composition. Of the 27

samples in which compounds were identified, seven were from male plants and 20 were from female

plants. Table 1 lists each compound or compound class and the number of male or female plant extracts

in which they were detected.

Table 1

Number of male and female plant extracts containing each compound or compound class

Compound or Compound Class Number of Males (n = 7) Number of Females (n = 20)

Dimethyl disulfide 7 18

Aliphatic hydrocarbons 3 9

Aromatic hydrocarbons 0 6

Peroxides 0 5

Carboxylic acids 1 2

Esters 1 2

Indole compounds 0 2

36

Dimethyl disulfide and three classes of organic compounds were found in both male and female

plant extracts, and these included aliphatic hydrocarbons, carboxylic acids, and esters. Dimethyl disulfide

was detected in 100% of male samples and 90% of female samples. Although the number of samples

collected from male and female plants is disproportionate, the results suggest dimethyl disulfide is

characteristic of the scent of both sexes. Likewise, both male and female scents may contain aliphatic

hydrocarbons, which were found in 43% of male samples and 45% of female samples. Carboxylic acids

and esters were found in both male and female samples; however, they were only detected in a small

percentage. The results also indicated that certain classes of compounds may be unique to either males or

females. Aromatic hydrocarbons, peroxides, and indole compounds were only found in female samples

and were detected in 22%, 19%, and 10% of samples, respectively. Figure 9 summarizes and compares

the abundance of each compound type in male and female samples.

Figure 9. Abundance of volatile organic compound classes detected in male (n = 7) and female (n = 20)

samples.

37

CHAPTER 5

DISCUSSION

Eastern skunk cabbage, Symplocarpus foetidus, is a unique organism that exhibits many

extraordinary properties, such as its thermogenic and thermoregulatory abilities, which aid in the dispersal

of its very distinct floral odor to attract insect pollinators. Due to the importance of floral scent

composition in examining plant-insect interactions, the first objective of this study was to characterize the

floral scent composition of S. foetidus. Based upon the current body of knowledge regarding the skunk

cabbage scent and the floral scent composition of similar smelling aroids, I hypothesized that the odor of

S. foetidus would be dominated by a single compound, such as an oligosulfide or amine, and may contain

compounds from other chemical classes. The results of this study showed the primary component of the

skunk cabbage scent to be the oligosulfide, dimethyl disulfide. The results also showed that compounds

from several chemical classes may be present in the skunk cabbage scent, including aliphatic and

aromatic hydrocarbons, peroxides, carboxylic acids, esters, and indoles. First, my finding that dimethyl

disulfide dominates the skunk cabbage scent is consistent with my hypothesis and with other research in

which a single or a few compounds are primarily responsible for giving sapromyophilous species their

characteristic foul scent. For instance, as previously discussed, the carrion-like odor of several

Amorphophallus species is due to the presence of dimethyl oligosulfides, such as dimethyl disulfide,

dimethyl trisulfide, and dimethyl tetrasulfide (Kite & Hetterschieid, 1997). Although floral volatiles were

not quantified in this study, it is acceptable to conclude that dimethyl disulfide dominated the skunk

cabbage scent for several reasons. First and foremost, dimethyl disulfide was detected in the extracts of

93% of all samples, whereas compounds from other chemical classes were detected in 44% or less of

samples. In addition, it was the only compound identified by the NIST library with a match probability of

90% or higher. Dimethyl disulfide was not detected in only two samples, and it is possible that the

compound was not present in sufficient quantities in these samples to be detected by the gas

chromatograph.

38

My finding that compounds from other chemical classes may be present in the skunk cabbage

scent is congruent with research conducted on other sapromyophilous species. The aliphatic and aromatic

hydrocarbons detected in the S. foetidus scent also comprise the floral scents of several sapromyophilous

species, such as Sauromatum guttatum and members of the genus Arum (Borg-Karlson, Englund, &

Unelius, 1994; Gibernau, Macquart, & Przetak, 2004). Although there have been few reports of

carboxylic acids in the floral scent composition of sapromyophilous species, they are known to dominate

the scent of urine, a putrification source that has been used to describe the scent of many foul-smelling

plant species. Indole-containing compounds (amines) found in the scent of S. foetidus have been shown

to give species with floral odors reminiscent of herbivore dung their characteristic scent (Jurgens, Dotterl,

& Meve, 2006; Ollerton & Raguso, 2006). The remaining class of compounds detected in the skunk

cabbage scent, peroxides, has not been shown to be a floral scent constituent of any other

sapromyophilous species; however, several explanations exist for the presence of peroxides in the skunk

cabbage scent. Peroxides are often released when abiotic or biotic stress is inflicted on a plant. Peroxides

are one of the many types of compounds formed upon damage, collectively referred to as leaf damage

volatiles (Holopainen, 2004; Dudareva, Pichersky, & Gershenzon, 2004). It is possible that prior to or

during sampling, various abiotic or biotic stressors induced the production of leaf damage volatiles in the

skunk cabbage plants. Possible abiotic stressors include extreme winds or temperatures and soil

dehydration, which are conditions easily encountered during the winter months when sampling was

conducted. Biotic stressors include damage inflicted by herbivores and other living organisms, such as

viruses or parasites.

When considering the chemical classes of compounds detected in the skunk cabbage scent, it is

important to note several factors that may have prevented the identification of the specific compounds

within the chemical classes. Because the NIST library used in this study contains information on the

structures of thousands of organic compounds, it is likely that the skunk cabbage floral volatiles were not

present in large enough quantities to be identified by the NIST library with a high match probability.

Although floral volatiles were not quantified, this simply may be due to the skunk cabbage inflorescence

39

emitting a scent containing larger quantities of some floral volatiles, such as dimethyl disulfide, and

smaller quantities of others. An attempt to increase the quantity of trapped volatiles was made during the

2011 sampling season by increasing the sampling time from 30 minutes to one hour; however, because

the samples could not be extracted and analyzed within the appropriate holding time, the effects of

increased sampling time could not be evaluated. Despite the vast number of compounds contained within

the NIST library, it is possible that no matches existed within the library for the specific compounds in the

skunk cabbage scent. Nuances in a compound’s chemical structure, such as the location of a small

hydrocarbon side chain in branched compounds could be slightly different than that of the compounds

contained within the library resulting in a lower match probability.

The second objective of this study was to determine if sex differences exist in the floral scent

composition of S. foetidus. Many researchers are interested in examining potential differences in floral

scent composition based upon a variety of factors, such as plant sex, to determine the impact these factors

may have on plant-insect interactions (Ashman, 2009). In S. foetidus, heat production has been shown to

peak during the female stage, and throughout the flowering period, the odor has been described as

variable (Ito-Inaba, Hida, & Inaba, 2009b; Kevan, 1989). Due to these observations, I hypothesized that

differences would exist between the floral odors of male and female skunk cabbages. First, the results

strongly suggest that dimethyl disulfide is a component of both male and female floral odors. Although

floral scent composition was examined in only seven male plants and 21 female plants, dimethyl disulfide

was detected in 88% of male floral extracts and 86% of female floral extracts.

For the remaining classes of compounds, it is difficult to determine if true differences exist between the

odors of male and female plants due to the small number of samples in which these compounds were

detected. Aliphatic hydrocarbons, carboxylic acids, and esters were found in both male and female odor

extracts, whereas aromatic hydrocarbons and indole compounds were only found in female odor extracts.

Based upon these results, it appears that the female skunk cabbage odor is slightly more complex than the

male skunk cabbage odor, which supports my hypothesis that sex differences exist. This finding is also

consistent with the honest signals hypothesis proposed by Ashman (2009) that states males and females

40

will honestly advertise their rewards by emitting a unique scent to elicit insect visitation. In the case of

skunk cabbage, females emit a more complex scent that may signal the presence of heat and/or shelter.

The absence of indole compounds in the male scent may signal the presence of pollen, which typically

does not contain aminoid compounds (Dobson & Bergstrom, 2000). Even though the findings of this

study support sex differences in the floral scent composition of S. foetidus, definitive conclusions

regarding sex differences cannot be drawn due to the small number of samples in which these differences

were detected. Addressing this issue simply requires additional research using larger sample sizes of

male and female plants.

Subsequent research involving longer sampling times, larger sample sizes, as well as

quantification of floral volatiles would address many questions that arose from the findings of this study.

As previously discussed, increasing the sampling time may allow for exact structure elucidation of the

types of compounds detected in this study, and larger sample sizes would clarify the existence of sex

differences in floral scent composition. Quantification of the floral volatiles would also allow for

definitive conclusions to be drawn regarding the abundance of compounds, such as dimethyl disulfide, in

the skunk cabbage odor and the extent of sex differences. Because heat production and odor emission

peaks during the female stage, it is reasonable to expect that the highest concentrations of all compounds

would be observed during the female stage. Determining how the concentration of volatiles may change

throughout the sexual stages would provide important information regarding the specific tactics used by

skunk cabbage plants to entice pollinators. For instance, one possible scenario is that female skunk

cabbage plants may produce larger quantities of dimethyl disulfide and other compounds, such as indoles

and aromatics to signal the presence a specific reward like heat or shelter. As the plant transitions into the

male stage, dimethyl disulfide may still be produced as the primary insect attract, but concentrations of

pollen-specific volatiles may increase during the male stage to ensure the transfer of pollen to other

receptive female plants still remaining in the population.

In summary, the results of this study were significant in that this was the first study to qualify the

floral scent composition of S. foetidus. My findings that dimethyl disulfide dominates the skunk cabbage

41

scent and that other chemical classes of compounds are present in the scent, including aliphatic and

aromatic hydrocarbons, carboxylic acids, esters and indole compounds, is consistent with research

conducted on other sapromyophilous species. This study is also the first to determine the floral scent

composition of one of the three known plants exhibiting thermoregulatory abilities. Although definitive

conclusions could not be made regarding sex differences in the skunk cabbage floral scent composition,

this study serves as an excellent basis for subsequent research. Because this study suggests that sex

differences do exist in the skunk cabbage scent, this finding is an important first glimpse into the role

different aspects of floral scent composition may play in plant-insect interactions for S. foetidus.

42

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50

APPENDIX A

Sample collection and temperature data for 2010 samples

Sample ID Sampling

Date

Sampling

Time

Plant

Sex

Air Temperature

(°C)

Spadix Temperature

(C above ambient)

GC Peaks

Detected?

S0651001

03/06/10

10:55 – 11:25 M 2.51 1.07 N

S0651002 11:28 – 11:58 M 4.34 -3.18 N

S0651003 11:59 – 12:29 M 3.84 -0.08 N

S0651004 12:34 – 13:04 M 3.95 -0.75 N

S0701001

03/11/10

11:32 – 12:02 F 13.3 6.98 Y

S0701002 12:05 – 12:35 M 12.34 7.77 Y

S0701003 12:36 – 13:06 M 11.55 6.47 Y

S0721001

03/13/10

09:50 – 10:20 F 5.02 3.52 Y

S0721002 10:33 – 11:03 F 6.13 9.14 Y

S0721003 11:10 – 11:40 F 6.40 9.68 Y

S0721004 11:42 – 12:12 F 7.57 9.94 Y

S0721005 12:15 – 12:45 F 6.57 10.20 Y

S0721006 12:47 – 13:17 M 7.76 1.75 Y

S0721007 13:18 – 13:48 F 5.70 6.37 Y

S0721008 13:49 – 14:19 M 6.08 7.31 Y

S0731001

03/14/10

09:50 – 10:20 F 4.61 9.00 Y

S0731002 10:23 – 10:53 F 4.53 12.04 Y

S0731003 11:02 – 11:32 F 4.36 9.68 N

S0731004 11:36 – 12:06 F 4.66 11.54 N

S0731005 12:13 – 12:43 F 4.63 11.01 N

S0731006 12:51 – 13:21 F 5.04 12.30 N

S0731007 13:26 – 13:56 F 4.71 8.72 N

S0731008 13:57 – 14:27 F 5.50 10.61 Y

S0741001

03/15/10

10:38 – 11:08 F 4.53 6.53 Y

S0741002 11:10 – 11:40 F 4.30 11.05 N

S0741003 11:42 – 12:12 M 5.11 2.13 N

S0741004 12:20 – 12:50 F 5.53 8.97 N

S0741005 12:55 – 13:25 F 5.41 6.84 N

S0761001

03/17/10

10:36 – 11:06 F 10.62 7.44 Y

S0761002 11:14 – 11:44 F 13.36 8.90 Y

S0761003 11:45 – 12:15 F 12.75 14.12 Y

S0761004 12:20 – 12:50 F 13.69 11.14 Y

S0761005 12:51 – 13:21 F 12.95 10.73 Y

S0781001

03/19/10

10:20 – 10:50 F 10.91 0.25 N

S0781002 10:52 – 11:22 F 12.27 6.34 N

S0781003 11:24 – 11:54 F 15.47 0.26 N

S0781004 11:58 – 12:28 F 13.95 3.14 N

S0781005 12:34 – 13:04 F 16.50 1.27 Y

S0791001

03/20/10

10:20 – 10:50 M 14.18 8.27 N

S0791002 10:53 – 11:23 M 15.86 6.88 Y

S0791003 11:29 – 11:59 F 17.51 7.69 Y

S0791004 12:02 – 12:32 F 19.28 3.43 Y

S0791005 12:45 – 13:15 M 20.69 6.13 Y

51

Sample ID Sampling

Date

Sampling

Time

Plant

Sex

Air Temperature

(°C)

Spadix Temperature

(C above ambient)

GC Peaks

Detected?

S0791006

03/20/10

13:17 – 13:47 F 20.56 3.94 N

S0791007 13:40 – 14:10 F 19.81 4.64 Y

S0791008 14:10 – 14:40 M 21.42 2.81 Y

S0811001

03/22/10

10:20 – 10:50 F 11.67 8.85 N

S0811002 10:51 – 11:21 F 11.65 7.80 N

S0811003 11:22 – 11:52 M 12.01 7.57 N

S0811004 11:53 – 12:23 M 12.38 6.74 N

S0811005 12:27 – 12:57 M 15.28 7.86 N

S0811006 13:01 – 13:31 F 16.25 3.97 N

S0881001

03/29/10

10:13 – 10:43 M 9.92 2.76 N

S0881002 10:49 – 11:19 M 10.77 2.25 N

S0881003 11:22 – 11:52 M 11.40 2.03 N

S0881004 11:52 – 12:22 M 11.45 2.22 N

S0901001 03/31/10

11:19 – 12:19 M 11.88 2.35 N

S0901002 12:20 – 13:20 M 14.03 -0.01 N

52

APPENDIX B

Mass spectra and NIST library match mass spectra for compound classes detected in 2010 samples

Figure 1. Functional group: aliphatic hydrocarbon; (A) Sample search spectrum with base peak at m/z =

43.9 and smaller ion fragment peak at m/z = 39; (B) allene mass spectrum and (C) alkyne mass spectrum

with base peaks at m/z = 40; mass spectra (A), (B), and (C) differ only in the number of hydrogen atoms

present.

53

Figure 2. Functional group: aliphatic hydrocarbon; (A) Sample search spectrum; (B) cyclic monoterpene

mass spectrum; both spectra share a base peak at approximately m/z = 137 and smaller ion fragments at

m/z = 41 and m/z = 93.

Figure 3. Functional group: aliphatic hydrocarbon; (A) Sample search spectrum; (B) non-cyclic

monoterpene mass spectrum; the spectra do not share a base peak, but smaller ion fragments at

approximately m/z = 41, m/z = 53, m/z = 67, m/z = 80, m/z = 93, and m/z = 105.

54

Figure 4. Functional group: aliphatic hydrocarbon; (A) Sample search spectrum; (B) aldehyde mass

spectrum; the spectra do not share a base peak, but smaller ion fragments at approximately m/z = 43, m/z

= 51, m/z = 63, m/z = 78, m/z = 90, m/z = 105, and m/z = 133.

Figure 5. Functional group: aromatic hydrocarbon; (A) Sample search spectrum; (B) benzenoid

compound mass spectrum; both spectra share a base peak at approximately m/z = 120 and smaller ion

fragment peaks at approximately m/z = 39, m/z = 51, m/z = 77, and m/z = 105.

55

Figure 6. Functional group: aromatic hydrocarbon; (A) Sample search spectrum; (B) aromatic ketone

mass spectrum; the spectra do not share a base peak, but smaller ion fragments at approximately m/z =

43, m/z = 50, m/z = 63, m/z = 78, m/z = 90, m/z = 105, m/z = 133, and m/z = 148.

Figure 7. (A) Sample search spectrum; (B) peroxide mass spectrum; both spectra share a base peak at m/z

= 85 and smaller ion fragments at approximately m/z = 43, m/z = 57, and m/z = 69.

56

Figure 8. (A) Sample search spectrum; (B) carboxylic acid mass spectrum; the spectra do not share a base

peak, but smaller ion fragments at approximately m/z = 44, m/z = 59, m/z = 83, m/z = 91, m/z = 116, m/z

= 141, m/z = 173, m/z = 197, m/z = 239, and m/z = 299.

Figure 9. (A) Sample search spectrum; (B) ester mass spectrum; the spectra do not share a base peak, but

smaller ion fragments at approximately m/z = 41, m/z = 55, m/z = 67, and m/z = 84.

57

Figure 10. (A) Sample search spectrum; (B) indole-containing compound mass spectrum; the spectra do

not share a base peak, but smaller ion fragments at approximately m/z = 50, m/z = 91, m/z = 116, m/z =

131, and m/z = 145.

58

APPENDIX C

Compound or compound class detected in each 2010 sample

Compound or Compound Class

Sample

ID

Plant

Sex

Diethyl

Phthalate

No

Peaks

Dimethyl

disulfide

Aliphatic

Hydrocarbons

Aromatic

Hydrocarbons Peroxides

Carboxylic

Acids Esters

S0651001 M X X

S0651002 M X X

S0651003 M X X

S0651004 M X X

S0701001 F X X X

S0701002 M X

S0701003 M X X X X

S0721001 F X X X X X X

S0721002 F X X X X X X X

S0721003 F X X X

S0721004 F X X X X X

S0721005 F X X X X

S0721006 M X X X

S0721007 F X X

S0721008 M X X X

S0731001 F X X X

S0731002 F X X

S0731003 F X

S0731004 F X

S0731005 F X

S0731006 F X

S0731007 F X

S0731008 F X X X

S0741001 F X X

S0741002 F X X

S0741003 M X X

S0741004 F X X

S0741005 F X X

S0761001 F X X

S0761002 F X X

59

Compound or Compound Class

Sample

ID

Plant

Sex

Diethyl

Phthalate

No

Peaks

Dimethyl

disulfide

Aliphatic

Hydrocarbons

Aromatic

Hydrocarbons Peroxides

Carboxylic

Acids Esters

S0761003 F X X

S0761004 F X X

S0761005 F X X X

S0781001 F X X

S0781002 F X X

S0781003 F X X

S0781004 F X X

S0781005 F X X

S0791001 M X X

S0791002 M X X

S0791003 F X X

S0791004 F X X

S0791005 M X X

S0791006 F X X

S0791007 F X X

S0791008 M X X

S0811001 F X X

S0811002 F X X

S0811003 M X

S0811004 M X

S0811005 M X

S0811006 F X

S0881001 M X

S0881002 M X

S0881003 M X

S0881004 M X

S0901001 M X X

S0901002 M X X