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The Response of Three Cabomba Populations to Herbicides and Environmental Parameters

Permanent Link: http://ufdc.ufl.edu/UFE0022067/00001

Material Information

Title: The Response of Three Cabomba Populations to Herbicides and Environmental Parameters Implications for Taxonomy and Management
Physical Description: 1 online resource (80 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anthocyanin, biotypes, chlorophyll, fanwort, ph, photosynthesis, temperature
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The genus Cabomba is composed of a group of submersed aquatic plants native to the Americas (Latin, North and South). The species Cabomba caroliniana var. caroliniana and var. pulcherrima are native to the southeastern United States. Populations of cabomba have recently been introduced to the northern U.S., Australia, Canada, China, Japan, and Europe. In these newly colonized areas, cabomba is proving difficult to control. Varieties of Cabomba caroliniana range in color from green to red/purple and is reportedly influenced by temperature. Plants typical of the southeastern U.S. are red in color (hereafter referred to as red cabomba) whereas the northern plants, as well as those around the world, when described, are green in color (hereafter referred to as green cabomba). Plants purchased directly from an aquarium dealer had both green and red coloration on a single plant (hereafter referred to as aquarium cabomba). A lack of taxonomic clarity and management options led to the investigation of the response of three populations of cabomba (green, aquarium, and red) to herbicides and selected environmental parameters. Growth chamber studies were designed to determine cabomba's response to herbicides by measuring photosynthetic activity. Ten herbicides were evaluated to investigate population differences and to identify potential herbicides for use in management programs. An ET50 value (the estimated time to reduce photosynthesis by 50% compared to an untreated control) was used to quantify the photosynthetic response of all three cabomba populations. Studies identified endothall (amine salt) and flumioxazin as potential herbicides for use in management programs. Green and red cabomba consistently differed in susceptibility to herbicides, with green cabomba more tolerant than aquarium and red cabomba. Response of aquarium cabomba to herbicides was intermediate. The use of photosynthetic measurements for predicting herbicide activity was validated in mesocosm trials on both immature and mature plants, which generally confirmed the results determined from growth chamber experiments. A number of herbicides were ineffective on cabomba, but carfentrazone, endothall (amine salt) and flumioxazin were effective. Flumioxazin at 200 and 400 ?g active ingredient L-1 caused the greatest effect exceeding a 95% reduction in biomass. Cabomba populations differed in their photosynthetic response to pH. Aquarium cabomba had peak photosynthesis at pH 5.9, green cabomba at pH 6.2, and red cabomba at pH 6.5; all were significantly different from one another. Green and aquarium cabomba were able to photosynthesize at lower temperatures (8?C) than red cabomba. Red cabomba had higher photosynthetic rates than aquarium and red cabomba as temperature increased to 32?C. Chlorophyll, anthocyanin content, and specific leaf weight were collected from field and culture plants to determine the effect of long-term culture conditions on plant growth and morphology. Cabomba populations evaluated from field and culture were similar within a population, while differences in chlorophyll, anthocyanin, and leaf size were found among the three populations. These studies suggest a taxonomic revision may be necessary to further differentiate Cabomba caroliniana based on differences to a range of stimuli evaluated in these studies. Results support anecdotal observations that cabomba is tolerant to most registered herbicides. Reciprocal experiments should be performed in colder climates to confirm differences among these populations are conserved and are not simply a response to regional environmental conditions such as temperature.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Netherland, Michael D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022067:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022067/00001

Material Information

Title: The Response of Three Cabomba Populations to Herbicides and Environmental Parameters Implications for Taxonomy and Management
Physical Description: 1 online resource (80 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: anthocyanin, biotypes, chlorophyll, fanwort, ph, photosynthesis, temperature
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The genus Cabomba is composed of a group of submersed aquatic plants native to the Americas (Latin, North and South). The species Cabomba caroliniana var. caroliniana and var. pulcherrima are native to the southeastern United States. Populations of cabomba have recently been introduced to the northern U.S., Australia, Canada, China, Japan, and Europe. In these newly colonized areas, cabomba is proving difficult to control. Varieties of Cabomba caroliniana range in color from green to red/purple and is reportedly influenced by temperature. Plants typical of the southeastern U.S. are red in color (hereafter referred to as red cabomba) whereas the northern plants, as well as those around the world, when described, are green in color (hereafter referred to as green cabomba). Plants purchased directly from an aquarium dealer had both green and red coloration on a single plant (hereafter referred to as aquarium cabomba). A lack of taxonomic clarity and management options led to the investigation of the response of three populations of cabomba (green, aquarium, and red) to herbicides and selected environmental parameters. Growth chamber studies were designed to determine cabomba's response to herbicides by measuring photosynthetic activity. Ten herbicides were evaluated to investigate population differences and to identify potential herbicides for use in management programs. An ET50 value (the estimated time to reduce photosynthesis by 50% compared to an untreated control) was used to quantify the photosynthetic response of all three cabomba populations. Studies identified endothall (amine salt) and flumioxazin as potential herbicides for use in management programs. Green and red cabomba consistently differed in susceptibility to herbicides, with green cabomba more tolerant than aquarium and red cabomba. Response of aquarium cabomba to herbicides was intermediate. The use of photosynthetic measurements for predicting herbicide activity was validated in mesocosm trials on both immature and mature plants, which generally confirmed the results determined from growth chamber experiments. A number of herbicides were ineffective on cabomba, but carfentrazone, endothall (amine salt) and flumioxazin were effective. Flumioxazin at 200 and 400 ?g active ingredient L-1 caused the greatest effect exceeding a 95% reduction in biomass. Cabomba populations differed in their photosynthetic response to pH. Aquarium cabomba had peak photosynthesis at pH 5.9, green cabomba at pH 6.2, and red cabomba at pH 6.5; all were significantly different from one another. Green and aquarium cabomba were able to photosynthesize at lower temperatures (8?C) than red cabomba. Red cabomba had higher photosynthetic rates than aquarium and red cabomba as temperature increased to 32?C. Chlorophyll, anthocyanin content, and specific leaf weight were collected from field and culture plants to determine the effect of long-term culture conditions on plant growth and morphology. Cabomba populations evaluated from field and culture were similar within a population, while differences in chlorophyll, anthocyanin, and leaf size were found among the three populations. These studies suggest a taxonomic revision may be necessary to further differentiate Cabomba caroliniana based on differences to a range of stimuli evaluated in these studies. Results support anecdotal observations that cabomba is tolerant to most registered herbicides. Reciprocal experiments should be performed in colder climates to confirm differences among these populations are conserved and are not simply a response to regional environmental conditions such as temperature.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Netherland, Michael D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022067:00001


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THE RESPONSE OF THREE CABOMBA POPULATIONS TO HERBICIDES AND
ENVIRONMENTAL PARAMETERS: IMPLICATIONS FOR TAXONOMY AND
MANAGEMENT




















By

BRETT WELLS BULTEMEIER


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008

































2008 Brett Wells Bultemeier

































To all those who have been taken from my life too early-though your time here on earth has
passed, your love and memory will always be with me.









ACKNOWLEDEGMENTS

First and foremost, I am humbly thankful to God for the love, grace, and beauty I have

been blessed with in life.

I am grateful to my major advisor Dr. Netherland for the countless hours of refining my

writing, reviewing experimental protocols, and helping develop my scientific methodology. I

am thankful for the support provided by my advisory committee: Dr. Haller, Dr. Ferrell, Dr.

Koshnick, Dr. Erickson, and Dr. Wofford. Their tireless efforts in help with statistics,

experimental design, and general assistance with this project were instrumental in its completion.

The friendship and kindness shown to my family by Dr. Haller made the transition to Gainesville

one of ease, and continues to make our time here wonderful. I extend my appreciation to Dr.

MacDonald who, though not on my committee, provided support in experimental methods and

developing many aspects of my project.

The administrative staff of the Agronomy Department was incredibly patient with

deadlines, forms, and all the necessary steps towards graduation and for that, I am grateful. This

project would not have been possible without help harvesting plants, maintaining plant cultures,

and plant collections; for that help I thank the following people: Chris Mudge, Tomas

Chiconela, Margaret Glenn, David Mayo, Cole Hulon, Michael Aldridge, and Lyn Gettys.

I am thankful to Manchester College for fostering my scientific interests and providing a

solid academic foundation. They also taught me what it means to be a responsible citizen of this

country and of the world. Though small in enrollment, their global impact is large and their

graduates take with them a sense of leadership and responsibility too rare in this world.

For their love and support I thank my family. My sister has shown me how to overcome

adversity, and how grow stronger from those adversities. My mom continually supported my

endless questions and fostered a curiosity that developed into a passion for science that will serve









me well throughout life. My dad, through his guidance and years of military service, taught me

the true meaning of character, moral fiber, and integrity. These values have become the

foundation for which I have tried to live. Finally I am infinitely thankful for the love, support,

and sacrifice of my wife. Her willingness to uproot from family and deal with long hours of

research will never be forgotten. She has so selflessly devoted her time and energy to me that I

am amazed and have been shown the true meaning of love. Without her in my life, I would not

be where I am nor be able to accomplish the things in life for which I strive.









TABLE OF CONTENTS

page

A C K N O W L E D E G M E N T S ...................... ........ .........................................................................4

LIST O F TA BLE S ............ ......... .... ..... ................................................................. 8

L IST O F FIG U R E S ............................................................................... 9

ABSTRACT ........................................... ......... .. .......... 12

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ................................... .................15

Genus D description .................................................... .......... ...... ........ 15
T ax o n o m y ..............................................................................................16
Distribution and M orphology ................................................................ ...............17
R ep ro d u ctio n .............................................................................2 0
G row th C conditions ........................ ...................... .. .. ........... ..... ...... 20
M an ag em ent ................................................................2 1
O b j e ctiv e s ..................................................................................................... ...........2 2

2 THE EFFECT OF AQUATIC HERBICIDES ON THREE POPULATIONS OF
C A B O M B A ........... .......... .......... .......... .............................................. 2 6

Introduction ................. .................................... ............................26
M materials and M methods ..................................... ... .. ........... ....... ......27
G row th C ham ber ....................................................... 27
Static exposures ................... ... ...... .......... .............................27
Tw enty-four hour exposures ............................................................................. 31
Mesocosm ..................... .............................................. 31
N ew ly established plants ............. .................................................. ............... 31
Mature plants ............. ........... ......... ......... 33
R e su lts an d D iscu ssio n ...................................................................................................... 3 3
G row th C h am b er ........................................ ..................................................... .. 3 3
Static exposures ................... ... ...... .......... .............................33
Tw enty-four hour exposures ............................................................................. 35
Mesocosm ..................... .............................................. 37
N ew ly established plants ............. .................................................. ............... 37
M nature plants ......... ..... .... ............................ ............. 38

3 THE EFFECT OF PH AND TEMPERATURE ON PHOTOSYNTHESIS OF THREE
CABOM BA POPULATION S ................................................................. ............... 46

Introduction ................. .................................... ............................46
M materials and M methods ................. .................................... .. ........ .. .............48









p H .................................................................................................................................... 4 8
T e m p e ratu re ................................................................................................................ 5 0
R results and D discussion ..................................... .................................... .. ........ 51
p H ................... ...................5...................1..........
T e m p e ratu re ................................................................................................................ 5 2

4 COMPARISON OF THREE CABOMBA POPULATIONS FROM NATURAL
HABITATS AND CULTURE CONDITIONS............................................. ............... 57

In tro d u ctio n ................... ...................5...................7..........
Materials and Methods ....................................... ........... ..............59
P ig m en t A n aly sis ........................................................................................................ 5 9
L eaf C characteristics .......................................................................... ..... ......... 60
Results and Discussion ..................................... ........... ...............61
P ig m en t A n aly sis ........................................................................................................ 6 1
L eaf C characteristics .......................................................................... ..... ......... 62

5 CONCLUSIONS AND RECOMMENDATIONS ........................................ ....................65

APPENDIX HERBICIDE INFORMATION AND PREVIOUS STUDIES ................................67

L IST O F R E F E R E N C E S ......... ...... ........... ................. ...........................................................74

B IO G R A PH IC A L SK E T C H .........................................................................................................80









LIST OF TABLES


Table page

1-1 Species in the genus Cabomba, their proposed ploidy levels, and native ranges.............24

2-1 Photosynthetic response of three cabomba populations, expressed as percent of
untreated control, following a static 144 h exposure to selected herbicides....................41

2-2 Estimated time to reduce photosynthesis of three cabomba populations to 50% of an
untreated control in response to a static 144 h exposure to selected herbicides ..............42

2-3 Photosynthetic response of cabomba populations, expressed as percent of untreated
control, following a 24 h exposure to selected herbicides........................... ............... 42

2-4 Response of three newly established cabomba populations after 24 h exposure to
selected herbicides. .........................................................................43

3-1 Model parameters of quadratic regression for three cabomba populations in response
to a range of pH .............. ...... .... ................ ......................................54

3-2 Photosynthetic response of three cabomba populations to a range of temperatures ..........54

4-1 Characteristics of apical sections of three cabomba populations collected from the
field and established cultures. ..... ........................... .........................................64

A-i List of compounds for use in submersed aquatic weed control......................................67









LIST OF FIGURES


Figure pe

1-1 Line drawing of a submersed cabomba leaf showing the fan shape which leads to the
common name fanwort. From Don E. Eyles, A Guide and Key to the Aquatic .............25

2-1 Weight response of red cabomba in response to 24 h exposure to diquat + copper 375
+ 1000 gg a.i. L 1. ...............................................................................44

2-2 Weight response of green cabomba in response to 24 h exposure to endothall (amine
salt) 2300 pg a.i. L-1 .................................. ............................................ ..... 44

2-3 Response of mature green cabomba plants 2 WAT to a 24 h exposure to selected
herbicides. .................................................... .........45

3-2 Quadratic fit of the response of three cabomba populations to a range of pH ...................56

A-i Response of aquarium cabomba growth solution pH to a range of MES buffer
so lu tio n ................... ........................................................... ................ 6 7

A-2 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and Eurasian watermilfoil to 2,4-D at a concentration of 4400 .g active ingredient .......68

A-3 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to endothall (dipotasium salt) at a concentration of 3000 .g active..............68

A-4 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and variable milfoil to carfentrazone at a concentration of 400 .g active ingredient. ......69

A-5 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to copper at a concentration of 1000 pg active ingredient L1 .....................69

A-6 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to copper at a concentration of 1000 pg active ingredient L1 .....................70

A-7 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and Eurasian watermilfoil to triclopyr at a concentration of 4900 .g active. .................70

A-8 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
to quinclorac at a concentration of 400 pg active ingredient L-1............ ............ .....71

A-9 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
to quinclorac at a concentration of 400 pg active ingredient L-1............ ............ .....71

A-10 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to diquat at a concentration of 375 pg a.i. L 1............................................. 72









A-11 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to diquat at a concentration of 375 + copper at a concentration of 1000.......72

A-12 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to endothall (amine salt) at a concentration of 2300 tg a.i. L1 ...................73

A-13 Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to flumioxazin at a concentration of 400 tg a.i. L1. ........... ..................73









LIST OF ABBREVIATIONS

HAT Hours after treatment

DAT Days after treatment

WAT Weeks after treatment

ALS Acetolactate synthase

a.i. Active ingredient

ET50 Estimated time to reduce photosynthesis to 50% of an untreated control

MAX pH at which maximum photosynthesis occurred

BOD Biological oxygen demand bottles









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

THE RESPONSE OF THREE CABOMBA POPULATIONS TO HERBICIDES AND
ENVIRONMENTAL PARAMETERS: IMPLICATIONS FOR TAXONOMY AND
MANAGEMENT

By

Brett Wells Bultemeier

May 2008

Chair: Michael Netherland
Major: Agronomy

The genus Cabomba is composed of a group of submersed aquatic plants native to the

Americas (Latin, North and South). The species Cabomba caroliniana var. caroliniana and var.

pulcherrima are native to the southeastern United States. Populations of cabomba have recently

been introduced to the northern U.S., Australia, Canada, China, Japan, and Europe. In these

newly colonized areas, cabomba is proving difficult to control. Varieties of Cabomba

caroliniana range in color from green to red/purple and is reportedly influenced by temperature.

Plants typical of the southeastern U.S. are red (hereafter referred to as red cabomba) whereas the

northern plants, as well as those around the world, when described, are green (hereafter referred

to as green cabomba). Plants purchased directly from an aquarium dealer had both green and red

coloration on a single plant (hereafter referred to as aquarium cabomba). A lack of taxonomic

clarity and management options led to the investigation of the response of three populations of

cabomba (green, aquarium, and red) to herbicides and selected environmental parameters.

Growth chamber studies were designed to determine cabomba's response to herbicides

by measuring photosynthetic activity. Ten herbicides were evaluated to investigate population

differences and to identify potential herbicides for use in management programs. An ET50 value









(the estimated time to reduce photosynthesis by 50% compared to an untreated control) was used

to quantify the photosynthetic response of all three cabomba populations. Studies identified

endothall (amine salt) and flumioxazin as potential herbicides for use in management programs.

Green and red cabomba consistently differed in susceptibility to herbicides, with green cabomba

more tolerant than aquarium and red cabomba. Response of aquarium cabomba to herbicides

was intermediate.

The use of photosynthetic measurements for predicting herbicide activity was validated in

mesocosm trials on both immature and mature plants, which generally confirmed the results

determined from growth chamber experiments. A number of herbicides were ineffective on

cabomba, but carfentrazone, endothall (amine salt) and flumioxazin were effective. Flumioxazin

at 200 and 400 pg active ingredient L-1 caused the greatest effect exceeding a 95% reduction in

biomass.

Cabomba populations differed in their photosynthetic response to pH. Aquarium

cabomba had peak photosynthesis at pH 5.9, green cabomba at pH 6.2, and red cabomba at pH

6.5; all were significantly different from one another. Green and aquarium cabomba were able to

photosynthesize at lower temperatures (8C) than red cabomba. Red cabomba had higher

photosynthetic rates than aquarium and green cabomba as temperature increased to 320C.

Chlorophyll, anthocyanin content, and specific leaf weight were collected from field and

culture plants to determine the effect of long-term culture conditions on plant growth and

morphology. Cabomba populations evaluated from field and culture were similar within a

population, while differences in chlorophyll, anthocyanin, and leaf size were found among the

three populations.









These studies suggest a taxonomic revision may be necessary to further differentiate

Cabomba caroliniana based on differences to a range of stimuli evaluated in these studies.

Results support anecdotal observations that cabomba is tolerant to most registered herbicides.

Reciprocal experiments should be performed in colder climates to confirm differences among

these populations are conserved and are not simply a response to regional environmental

conditions such as temperature.









CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Genus Description

The following description provides a general guide to the plants comprising the genus

Cabomba (Fassett 1953; Godrey and Wooten 1981; Orgaard 1991). Species of cabomba are

rooted, submersed, aquatic, perennial dicotyledonous plants that colonize through rhizomatous

growth. Adventitious roots are sometimes formed at lower stem nodes and measure up to 24 cm

in length. Immature roots are smooth, unbranched, and white with a yellow tip and as the roots

mature they branch, form thick mats, and darken to brown or black. Stems arise from rhizomes

and rootcrowns, with prolific branching near the base. These stems are typically 1-2 m in length

but have been reported to be as long as 10 m. Plants occasionally have a thin mucilaginous

coating.

Stems are round to slightly compressed and are typically 2-4 mm in diameter.

Submersed leaves vary in color, ranging from olive green or brown to red/purple and are

typically opposite, although whorls of three can occur, and are fan-shaped, hence the other

common name of fanwort (Figure 1-1.). Leaf size can vary greatly even on a single stem, with

leaf size reduced at the apex of stems. Leaves are divided and may have up to 200 terminal

points on a single leaf.

Floating leaves attached to flowering branches have an alternate arrangement and are

linear-elliptic to ovate, although leaves of some tropical species have a circular shape with ends

tapering to a point. Flowering stems bear solitary bisexual flowers measuring 6-15 mm in

diameter. Flowers are monoecious with three sepals and three petals and may be pure white,

white with purple tinting (primarily at the margins), or white with yellow spotting. Stamens are

shorter than petals and typically occur in whorls of 3-6; anthers are 1-1.5 mm long. Flowers









have 2-4 pistils that may each develop 1-3 ovoid or globose seeds. Fruits are 4-7 mm long and

are attached to the previously floated flower section just below the water surface.

Taxonomy

The taxonomic classification of the genus Cabomba is confusing (Leslie 1986; Orgaard

1991) and efforts utilizing molecular techniques are currently being tested1. Orgaard (1991)

proposed the most current and complete taxonomy of the genus Cabomba, and unlike previous

classifications, describes features beyond those subject to environmental plasticity such as leaf

color, shape, and size. Orgaard's classification also includes pollen structure, vessel

organization, and chromosome numbers (Table 1-1) to advance previous descriptions. Orgaard

divided the genus into five species with one species, C. caroliniana having three varieties-var.

caroliniana, var. pulcherrima, and var. flavida. Only var. caroliniana and var. pulcherrima are

found in the United States. (Orgaard 1991).

Cabomba is popular for use in aquariums (Leslie 1986; Orgaard 1991) and a simple

internet search using "cabomba" or fanwortt" as a search term reveals the variety of plants that

can be purchased from aquarium stores (www.aquahobby.com, www.petsolutions.com,

www.pondplants.com). It is common for aquarium dealers to select and/or breed plants that are

desirable for sale to aquarium enthusiasts. Typically, plants should grow easily, tolerate a wide

range of conditions, and be aesthetically pleasing. Several cultivars derived from C. caroliniana

have been developed by the aquarium trade, and include "rosifolia", multipartitee",

"paucipartita" and "Silbergine" (Mackey and Swarbrick 1997; Orgaard 1991; Wain et al. 1983).

Discarded aquarium plants are a common source for the introduction of aquatic plants into

natural waterways that may spread and become invasive (McLane 1969). Species of Hydrilla,


1 Don Les (University of Connecticut), and Brian Husband (Guelph University Canada), personal communication
(2007).









Myriophyllum, Egeria, and Elodea have reportedly been spread by this route (Cook and Luand

1982; Cook and Urmi-Konig 1984; Cook and Urmi-Konig 1985). Cabomba in Australia, China,

Japan, and other locations around the world likely came from discarded aquarium plants and it is

considered invasive in these countries (Mackey 1996; Yu et al. 2004b). However, there is some

question as to what role discarded aquarium plants have played in the increased distribution of

cabomba into the northern portions of North America. Reports from the early 1900s list

cabomba as being used for fishponds and aquariums in New England, and by the 1960s the

species was reported as being invasive (Les and Mehrhoff 1999). The fact that cabomba is very

popular for use in aquariums makes it difficult to predict whether cabomba introduced into

northern states is from discarded aquarium plants, natural range extension, or introductions of

plants from the southeast. It is likely that multiple routes of introduction have occurred in the

northern portions of North America.

Distribution and Morphology

Members of the genus Cabomba are native in tropical to subtropical regions and only C.

caroliniana and its varieties have temperature tolerances that allow wide distribution in North

America (Leslie 1986; Orgaard 1991). Cabomba is considered native to the southeastern United

States and is increasingly found in other parts of North America. Cabomba is also increasingly

being reported in other locations around the world (Leslie 1986; Mackey 1996; Orgaard 1991;

Schooler et al. 2006; Yu et al. 2004a, b) where it has displayed properties consistent with an

invasive species (Cao et al. 2006; Crawford et al. 2001; Mackey 1996; Schardt and Nall 1988;

Schooler et al. 2006; Yu et al. 2004b; Zhang et al. 2003). Cabomba has been present in the

northern regions of the United States since the early 1900s, but the spread in these northern

regions greatly increased in the early 1990s and it is now frequently described as invasive

(Hanlon 1990; Les and Mehroff 1999; Madsen 1994; Martin and Wain 1991; Riemer and Ilnicki









1968). Monocultures of cabomba in Canada have reached densities of up to 200 plants m-2 (Noel

2004). In Australia cabomba was first identified in 1967, became naturalized by the 1980s,

spread rapidly, and was considered a major pest by the 1990s (Mackey 1996). Cabomba was

also found in the northwest United States, China, Canada, England, Netherlands, Belgium, and

Hungary during the 1990s and all of these introduced populations of cabomba are described as C.

caroliniana (Christy and Systma 1994a, b; Denys et al. 2003; Hrusa et al. 2002; Mackey 1996;

Noel 2004; Oldham 1999; Preston and Croft 1997; Stace 1997; Stetak 2004; Van der Velde et al.

2002). The increasing occurrence of cabomba in North America and around the world suggests

that populations may have been released from a similar source, such as the aquarium trade.

Invasive cabomba in the northern tier of the U.S. tends to be bright green and will hereafter

be referred to as green cabomba (Leslie 1986; Mackey 1996). Green cabomba can be found both

in the southeast and northern regions of the U.S., but its origin remains uncertain. In contrast,

plants typical of the southeast U.S. are red/purple (hereafter referred to as red cabomba) and have

not been reported in the northern U.S. Aquarium plants purchased in Florida are mostly green in

color with varied levels or red/purple color found at the apical sections of the plant and will

hereafter be referred to as aquarium cabomba.

The difference in the coloration of cabomba has been the source of much speculation.

There is some question regarding the effect of environmental conditions on color (Hanlon 1990;

Leslie 1986; Martin and Wain 1991; Orgaard 1991; Wain et al. 1983). Cold temperatures

reportedly lead to green color, while warm temperatures result in red/purple cabomba (Leslie

1986; Orgaard 1991). Orgaard (1991) observed that both light levels and temperature influenced

plant color, but color reverted when plants were grown in their original conditions. In contrast,

the aquarium industry reports that cold water causes red color and warm water results in green









color (Leslie 1986). There has been speculation that these color differences have been induced

by the aquarium industry (Hanlon 1990) and it is unclear whether the novel phenotype of green

cabomba results from genetic or environmental changes (Crawford et al. 2001, Hanlon 1990, Les

and Mehrhoff 1999; Mackey 1996; Schooler et al. 2006). Much of the published research does

not indicate whether the cabomba being evaluated is red or green. Therefore, it is difficult to

determine if studies evaluating tolerances to environmental variables or management studies are

valid for all populations of cabomba.

Wain et al. (1983) compared the isozymes of three types of cabomba found in the U.S. to

determine if species separation was necessary. These types were identified as C. caroliniana

var. caroliniana, C. caroliniana var. mulitpartita (described as an aquarium variety), and C.

pulcherrima. Electrophoretic separation of various alleles indicated no differences among the

three types. However, C. caroliniana var. multipartita was collected from Florida as opposed to

the northern regions of the U.S. and might not be representative of the green cabomba that is of

current concern in North America. This study was done largely before more powerful genetic

analysis became available to separate very closely related, yet unique, species. However, this

work did identify C. caroliniana var. multipartita as a variety selected for and bred by the

aquarium industry and suggested there should be no differences in management strategies for

these color variants.

Species of cabomba may also be separated by color differences in the flowers. C. caroliniana

var. pulcherrima is commonly reported as having a purple tinted flower in contrast to C.

caroliniana var. caroliniana that has yellow to purple tinted flowers. This overlap in flower

color calls into question whether the two varieties are separate (Orgaard 1991).









Reproduction

Cabomba spreads primarily through vegetative means (Leslie 1986; Mackey 1996; Orgaard

1991; Tarver 1976; Tarver and Sanders 1977). Vegetative growth is possible from a segment of

plant with a single node and pair of leaves, but segments with more leaf nodes have higher

survival rates (Tarver and Sanders 1977). The primary means of pollination is from insects,

though some self-pollination is possible due to wave action (Tarver and Sanders 1977). Flowers

of cabomba follow a two-day cycle in which pollination is possible and then flowers close and

are pulled below the surface of the water where seeds are formed (Moseley et al. 1984; Osborn et

al. 1991; Schneider and Jeter 1982). Seed viability in C. caroliniana and its varieties was lower

than that of other species of cabomba and has been attributed to the highly variable ploidy levels

found in C. caroliniana (Table 1-1). Also, cabomba found in Australia has not been observed

producing seeds (Mackey 1996). Riemer and Ilnicki (1968) found no viable seeds from

cabomba cultured in New Jersey and suggested seeds may not be used for reproduction by this

population of cabomba. It is unclear which cabomba population was used, but the cabomba was

collected in New Jersey, which suggests it may have been green cabomba.

Growth Conditions

Studies indicate that a pH range of 4-6 is optimal for cabomba growth and that pH above

7 inhibits growth (Riemer 1965; Tarver 1976). Riemer (1965) and Tarver (1976) reported that

plants begin to defoliate from the base up when cultured in water of pH 7 or higher and most

leaves below the very apical sections are lost. Riemer (1965) evaluated the effect of aeration,

calcium concentration, and osmotic pressure on the growth of cabomba and found that aeration,

0.0001M CaC12, and lower osmotic pressure (0.149ATM) produced the best growth of cabomba.

Like high pH, high levels of calcium (.001 M and higher) caused growth inhibition and

defoliation (Mackey 1996, Riemer 1965).









Species in the genus Cabomba are found throughout tropical climates and many species

are intolerant of colder climates (Leslie 1986; Orgaard 1991). C. caroliniana is the only species

native to temperate regions but green cabomba has colonized the northern portions of the U.S.

and Canada and has developed overwintering strategies to survive the much colder temperatures

associated with these regions. Plants fragment in late fall and form turion-like structures at the

apical tip, and green foliage is retained throughout winter (Riemer and Ilnicki 1968). Upon the

return of warmer weather, the surviving fragments resume vegetative growth.

Management

Cabomba can grow to nuisance levels in the southeast U.S. but generally is not a

management problem. Limited and contradictory information is published regarding cabomba

response to herbicide applications. Cabomba in the northern U.S. and Canada has been of

particular concern to lake and river managers due to poor performance of traditional

management techniques and the lack of control methods available to stop the spread of this

species. Nelson et al. (2002) reported that fluridone at 5 tg a.i. L-1 or greater concentrations

reduced biomass compared to untreated controls, but growth reduction of> 80% required

application of 20 tg a.i. L-1. Mackey (1996), however, reported that field trials of fluridone in

Australia had little or no effect on cabomba growth at equivalent concentrations evaluated by

Nelson et al. (2002). Cabomba populations rapidly expand in the Midwestern U.S. following

low-rate fluridone treatments2 targeting Eurasian watermilfoil (Myriophyllum spicatum L.).

Higher rates of fluridone, > 20 tg a.i. L-1 have been used in whole lake treatments in the

midwestern U.S. to reduce cabomba when it is distributed lake wide. These treatments are

limited in use because fluridone at these concentrations has reduced plant selectivity, require


2 Personal observation. Brett Bultemeier. 2005.









longer exposures, and has limited use when cabomba occurs in isolated locations in the littoral

zone3. Tolerance of cabomba to herbicides like 2,4-D and triclopyr (Nelson et al. 2001) is not

understood since other aquatic dicotyledonous-e.g. Myriophyllum spp.-are effectively

controlled with these herbicides. Aquatic managers report that diquat, a broad-spectrum

herbicide, has minimal activity on green cabomba. There has been some success in Australia

with the use of 2,4-D ester at concentrations of 10,000 pg a.i. L-1 to control cabomba infestations

(Mackey 1996). Endothall (amine salt) also reportedly reduces biomass of cabomba (Madesn

2000; Moorel991) but hydrogen peroxide had limited activity (Kay et al. 1984). Drawdown

techniques can effectively control cabomba, but have limited use in much of the U.S. where

infestations are currently found (Mackey 1996; Sanders 1979; Schooler et al. 2006). The only

known effective biological control for cabomba is grass carp (Ctenopharyngodon idella Val.),

and a search for other biological agents is in the early stages (Mackey 1996; Schooler et al.

2006).

There are situations where cabomba is considered beneficial. Cabomba is popular in the

aquarium industry due to its ornamental qualities (Hanlon 1999; Leslie 1986; Mackey 1996;

Schooler et al. 2006). Nakai et al. (1999) suggested that cabomba has allelopathic effects and

could be utilized to suppress blue-green algae and it has also been evaluated as a potential

bioremediation plant to remove harmful toxins and metals, such as lead, from the environment

(Yaowakhan et al. 2005).

Objectives

The differences in color and invasive growth between red and green cabomba suggest

that further efforts to characterize the response of these populations to management and various


3 Personal communication. Tyler Koschnick. 2008.









environmental parameters is needed. The invasive tendencies of green cabomba have created

heightened concerns due to the paucity of proven control methods. The objectives of this

research were to determine if red, green, and aquarium cabomba respond differently to

herbicides, pH, temperature, and culture conditions to determine if a new taxonomic treatment is

needed and to find potential herbicides to control noxious populations of cabomba.













Table 1-1. Species in the genus Cabomba, their proposed ploidy levels, and native ranges.
Species Ploidy (basic number x=13) Native Range
C. aquatica Tetraploid (4x) Northeast South America
C. palaeformis Diploid (2x) Southern Mexico and northern Latin America
C. furcata Tetraploid (4x) North and central South America
C. haynesii Unknown Latin America, Cuba, northern South America
C. caroliniana var. caroliniana Hexaploid (6x), octoploid (8x) Southeast United States, southeast South America
C. caroliniana var. pulcherrima Hexaploid (6x) North Florida, south Georgia and South Carolina
C. caroliniana var. flavida Triploid (3x), hexaploid (6x) Southeast South America
a Orgaard, Marian. 1991. The genus Cabomba (Cabombaceae) A taxonomic study.
Nord. J. Bot. 11:179-203.



































Figure 1-1. Line drawing of a submersed cabomba leaf showing the fan shape which leads to the
common name fanwort. From Don E. Eyles, A Guide and Key to the Aquatic Plants
of the Southeastern United States (Washington D.C.:U. S. Government Printing
Office, 1944).









CHAPTER 2
THE EFFECT OF AQUATIC HERBICIDES ON THREE POPULATIONS OF CABOMBA

Introduction

Cabomba is a submersed aquatic plant that is native to the southeastern United States and

has recently been found in states throughout the northeast, midwest and northwest. Cabomba is

also increasingly being found in Australia, Canada, China, Japan, and portions of Europe (Cao et

al. 2006; Crawford et al. 2001; Leslie 1986; Mackey 1996; Schardt and Nall 1988; Schooler et

al. 2006; Yu et al. 2004a, b; Zhang et al. 2003). The expansion of cabomba in North America

and around the world has invasive characteristics, and cabomba is now considered an exotic

weed in many of these sites. The range expansion throughout the world has lead to a

reevaluation of the taxonomy because these introduced populations have different characteristics

than native populations in the southeastern U.S.

Color of the species ranges from green to purple and is thought to be a function of

temperature and light levels (Hanlon 1990; Leslie 1986; Martin and Wain 1991; Orgaard 1991;

Wain et al. 1983). Plants in the northern U.S. are bright green (hereafter green cabomba),

whereas plants from the southeastern U.S. are red/purple (hereafter red cabomba). Plants sold

through the aquarium industry in Florida (hereafter aquarium cabomba) are mostly green, with

purple coloration on the apical tips of leaves.

Green cabomba in the northern U.S. and around the world is reportedly difficult to manage

(Mackey 1996) and research suggests that this cabomba is tolerant to most registered herbicides.

There are few reports on the response of cabomba to registered aquatic herbicides and these

reports do not specify which cabomba (green, red, etc.) was being studied. Therefore, an

evaluation of herbicide efficacy on these three populations is necessary to determine which

herbicides are effective, and whether populations respond similarly to herbicide treatments.









The effects of herbicides are typically evaluated initially in mesocosms to identify those

herbicides most likely to succeed in field trials. These evaluations typically involve a large input

of labor and space due to the number of experimental units needed to test a number of herbicides

(Getsinger et al. 1994; Gray et al. 2007; Nelson et al. 1998). Microcosm testing of herbicides is

generally more efficient, reduces labor, while still providing an initial screen of potentially active

compounds. These smaller scale evaluations require less plant material and less time to identify

herbicides unlikely to be effective in mesocosm studies.

The response of aquatic plants to herbicides can be assessed by measuring oxygen

evolution over time to calculate net photosynthetic rates. Also, the response of submersed

aquatic plants such as Hydrilla verticillata (L.f.) Royle (hydrilla) and Myriophyllum spicatum L.

(Eurasian watermilfoil) to herbicides and plant growth regulators can be assessed by monitoring

in situ photosynthetic rates (Netherland and Getsinger 1995; Netherland and Lembi 1992; Selim

et al. 1989). Decreased photosynthetic rates over a short term suggest that a herbicide has

potential for control and should be further evaluated.

The lack of viable management options and the expansion of cabomba over the past decade

suggests that research is necessary to determine if populations of cabomba vary in response to

herbicides. The objectives of these studies was to compare the photosynthetic response of three

populations of cabomba to herbicides and to determine if photosynthetic measurements can be

used as an initial screen for herbicides.

Materials and Methods

Growth Chamber

Static exposures

Experiments were conducted at the University of Florida Center for Aquatic and Invasive

Plants (CAIP) in Gainesville, FL. The first trials were conducted from July/October 2006, and









were repeated from October 2006 through January 2007. Red cabomba was collected from

White Lake (Suwannee County, FL) and Ledwith Lake (Alachua County, FL). Aquarium

cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery (Lake City, FL);

and green cabomba was collected from the St. Joseph River (Elkhart County, IN) and Crooked

Lake (Kalamazoo, MI). Plant cultures were established by planting 15 cm apical segments in 2

L plastic pots filled with topsoil amended with Osmocote plus (15-9-12)1 at a rate of Ig kg-1 of

soil. Plants were maintained in 900 L tanks under 50% shade cloth. Hydrilla, Eurasian

watermilfoil and variable milfoil (Myriophyllum heterophyllum Michx.) were used for

comparative testing and all were collected from cultures maintained at the CAIP.

Apical sections (4 cm long) of each of the three cabomba populations were excised and

placed in 10 L plastic pans containing well water for 6 h. This 6 h recovery period was

suggested by MacDonald (2007)2 because ion leakage by hydrilla is significant only during the

first 2-4 h after excision. After 6 h, the apical sections were placed into 350 ml plastic cups (1

section per cup) containing 5 mM MES buffer in well water amended with Hoagland's solution

(2.5% v/v) for a total volume of 345 ml per cup. MES buffer was used to maintain a pH of 6-7

based on preliminary studies where pH was stabilized with no apparent adverse effect on

photosynthesis (Appendix). Hydrilla, Eurasian watermilfoil, and variable milfoil were included

in these studies to provide a confirmation that herbicide susceptibility could be adequately

measured by a reduction in net photosynthetic rates even for herbicides not directly effecting

photosynthesis. The differential susceptibility of these plants to selected herbicides is known.

Variable milfoil was placed in containers that had the same culture solution as cabomba, but



1 The Scotts Company. Marysville, OH. 43041
2 MacDonald, G.E. Unpublished report. 2007









hydrilla and Eurasian watermilfoil treatments had NaHCO3 added to the culture solution to

achieve a final concentration of 2.4 mM to maintain a pH of 8. Cups containing plants were

placed in growth chambers3 with a temperature of 27C, 14:10 h (day:night) photoperiod and a

light intensity of 360-400 .imol m-2 s-1. Plants were treated with herbicides at near or above the

maximum-labeled use rate (Appendix). The following herbicide concentrations were tested: 2,4-

D (amine) 4400, carfentrazone 400, diquat 375, elemental copper 1000, endothall (amine salt)

2300, endothall (dipotasium salt) 3000, flumioxazin 400, quinclorac 400, triclopyr 4900, and a

combination of diquat 375 and elemental copper 1000 tg a.i. (active ingredient) L-1. Plants were

exposed to treatments for 144 h.

Net photosynthesis methods were adapted from Netherland and Lembi (1992) and

Netherland and Getsinger (1995). Dissolved oxygen measurements were taken every 24 h for

144 h using a dissolved oxygen meter4 (+0.01 mg/L). Initial oxygen measurements were taken

from biological oxygen demand (BOD) bottles5 containing well water, Hoagland's solution

(2.5% v/v), and 45 iM HC1. Hydrochloric acid was added to BOD bottles so pH would be equal

to that of the culture solutions for the 1 h duration of the photosynthetic measurements. Plant

tips were removed from the 350 ml plastic cups, rinsed twice in tap water, and placed

individually in BOD bottles in the growth chamber for 1 h. A control BOD bottle (with the

same solution as the other bottles but without plant material) was incubated to determine any

oxygen increase or decrease in the absence of plant material. Oxygen content of the water in the

BOD bottles was recorded at the end of the 1 h incubation period, and plants were weighed after


3 Percival model E-36L. Percival Scientific, Inc. Perry, IA. 50220.
4 Accumet Excel XL40 Dissolved Oxygen/BOD/OUR/SOUR Temperature Meter. Fisher Scientific. Pittsburg, PA.
15275.
5 Wheaton Science Products. Millville, NJ. 08332.









being blotted between paper towels to remove any excess water in order to calculate net

photosynthetic rates based upon fresh weight and time. This rate was determined by using the

equation: photosynthetic rate = (final dissolved oxygen (pg 02) initial dissolved oxygen (pg

02)) (freshweight (g)-1) (time (min)-'). The unit of measure is pg 02 g-(fresh weight) min1.

Plant tips were then placed back into the original treatment cups and measurements repeated

every 24 h for 144 HAT (hours after treatment). BOD bottles were emptied and filled with fresh

solution for each photosynthetic measurement.

Net photosynthetic rates were standardized to percent of untreated controls in order to

compare photosynthetic rates among the cabomba populations. Two separate analyses were

performed to compare the response of the cabomba populations to herbicides. Linear

interpolation was used to find an ET50 (the estimated time required to reduce net photosynthesis

to 50% of the untreated control) value for those herbicides that reduced net photosynthesis of

green cabomba by at least 50% of the untreated control. Green cabomba was used as the

standard for response because it is currently the most challenging to manage. Population

comparisons were made at 24 and 144 HAT using percent of untreated control values for those

herbicides that did not reduce green cabomba photosynthesis to 50% of the untreated control.

All treatments used a completely randomized design with 4 replications. Data were

analyzed using ANOVA (p< 0.05) and a post-hoc Student's pairwise t-test to analyze population

differences for ET50 estimates and percent of untreated control values. Data for quinclorac and

copper trials were analyzed independently due to significant interaction by trial. Trials were

pooled for all other herbicides. Data for endothall (amine salt), carfentrazone, triclopyr, and 2,4-

D were log transformed to meet equal variance assumption of ANOVA.









Twenty-four hour exposures

A second experiment was conducted to test the effect of limiting exposure time on those

herbicides (diquat, diquat + copper, endothall (amine salt), and flumioxazin) that in the static

exposure trials reduced photosynthesis of green cabomba to 50% of the untreated control. The

first trial was conducted in June/July 2007 and repeated July/August 2007. All three cabomba

populations were exposed to concentrations of diquat, diquat + copper, endothall (amine salt)

and flumioxazin at 375, 375 + 1000, 2300 and 400 [g a.i. L-1 respectively. Methods were

similar to those described in the static exposure studies, except plants were exposed to herbicides

for 24 h. Plants were removed from treatment after 24 h, washed twice, and placed in cups

containing growth media without herbicide. Growth media was replenished 72 h after plants

were removed from treatment to minimize the effect of algal growth that occurred in static

exposures. Dissolved oxygen measurements were taken every 24 h for 144 h.

All treatments used a complete randomized design and had 4 replications. Data were

analyzed using ANOVA (p< 0.05) and a post-hoc Student's pairwise t-test to analyze population

differences in percent of untreated control values at 24, 72, and 144 HAT. There were no

interactions by trial so data for both were pooled.

Mesocosm

Newly established plants

A mesocosm experiment was performed to evaluate the response of three cabomba

populations to selected herbicides on whole plants. This study was conducted at the CAIP

during July 2007. Red cabomba was collected from White Lake (Suwannee County, FL),

aquarium cabomba was obtained from Suwannee Laboratories (Lake City, FL), and green

cabomba was collected from the St. Joseph River (Elkhart, IN). Cabomba was established in

culture by planting 15 cm apical segments in 2 L plastic pots with top soil amended with









Osmocote Plus 15-9-12 at a rate of Ig kg (soil)-'. Plants were grown in 900 L tanks (80 cm

water depth) for 2 m before use in experiments.

Apical segments (15 cm) were excised from culture plants and planted 3 cm deep in

10x10x12 cm (1L) pots filled with masonry sand amended with Osmocote Plus 15-9-12 fertilizer

at a rate of Ig kg-1 of sand. They were then placed in 900 L cement tanks under 50% shade and

allowed to grow for 3 wk before treatment. One pot of each population was then placed in 95 L

HDPE (high-density polyethylene) tubs filled with well water amended with 10 ml of 28% HCI

(muriatic acid) to achieve a pH of 6.0-6.5.

Experimental treatments were conducted under 30% shade and herbicide concnetrations

were: untreated control, carfentrazone 400, diquat 375 + copper 1000, endothall (amine salt)

1200, endothall (amine salt) 2300, endothall (dipotasium salt) 3000, flumioxazin 200,

flumioxazin 400, and triclopyr 4900 tg a.i. L-1. Planted pots were removed from 95 L treatment

tanks after a 24 h exposure to herbicides, rinsed three times by full submersion in a 900 L tank

with flowing water, and placed into a separate 900 L cement tank with flowing water. Flow was

stopped after 24 h and 28% HC1 was added to adjust water pH to 6.0-6.7, with additional

adjustments every 3-4 d as necessary for 3 WAT (weeks after treatment). Plant material (shoots

and roots) was harvested 3 WAT and dry weights were determined after drying for 48 h in an

oven at 700C.

All treatments used a complete randomized design with 4 replications. Data were

analyzed using ANOVA (p< 0.05) to assess differential response among cabomba populations.

Means for each cabomba population were separated using Dunnett's test to identify those

treatments significantly different from control plants. A post-hoc Student's pairwise t-test was









used to analyze population differences in percent of untreated control for each herbicide. Percent

of untreated control values were log transformed in order to meet the assumptions of ANOVA.

Mature plants

A second mesocosm experiment was conducted to investigate the effects of herbicides on

the response of mature, rooted green cabomba. This experiment was conducted at the CAIP in

September 2007. Methods were similar to those in the newly established plant study but, only

green cabomba was used in this experiment. Plants were established for 3 m and were fully

rooted in 2 L plastic pots with top soil amended with Osmocote Plus 15-9-12 at a rate of Ig kg1.

Experimental treatments were applied under 30% shade and herbicide concentrations were:

untreated control, carfentrazone 200, endothall (amine salt) 1200, flumioxazin 100, flumioxazin

400 and triclopyr 4900 at pg a.i. L-1. Plants were exposed to herbicides for 24 h, then rinsed and

moved to 900 L tanks under 50% shade. Plants were harvested 2 WAT, dried in a drying oven at

700C for 1 wk, and weighed.

All treatments used a complete randomized design and had 3 replications. Data were

analyzed using ANOVA (p< 0.05) and Dunnett's test was used to determine those herbicides that

reduced biomass compared to untreated controls.

Results and Discussion

Growth Chamber

Static exposures

The BOD bottles filled with only growth solutions had no change in oxygen levels during

the 1 h photosynthetic period. The herbicides with known activity on hydrilla, Eurasian

watermilfoil, or variable milfoil all reduced net photosynthesis to < 50% of the untreated controls

(Figure A-2 through A-13). No observed change in oxygen levels of culture solution and the

reduction of photosynthesis by these herbicides known to be effective on the test plants (hydrilla,









Eurasian watermilfoil, variable milfoil) suggests that this method of screening is valid for

identifying potential aquatic herbicides.

Ten herbicides or herbicide combinations were tested. Six (carfentrazone, copper,

endothall (dipotasium salt), quinclorac, triclopyr, 2,4-D) failed to reduce net photosynthesis of

green cabomba to < 50% of untreated controls through 144 HAT (Table 2-1). Red and green

cabomba had different levels of sensitivity to triclopyr, 2,4-D, and carfentrazone, with red

cabomba being the most sensitive to each herbicide (Table 2-1). The response of aquarium

cabomba differed from green cabomba when exposed to carfentrazone and 2,4-D, and differed

from red cabomba when exposed to endothall (dipotasium salt) and carfentrazone (24 HAT).

The largest photosynthetic difference among the cabomba populations occurred in response to

carfentrazone. All three populations differed from one another 24 HAT when photosynthesis of

red cabomba was zero, aquarium cabomba was 57% of the untreated control, and green cabomba

was largely unaffected (Table 2-1). By 144 HAT, photosynthesis of both aquarium and red

cabomba was reduced to almost zero, while the photosynthetic rate of green cabomba was still >

60% of the untreated controls.

Four herbicides (diquat, diquat + copper, endothall (amine salt), and flumioxazin) reduced

net photosynthesis of all three cabomba populations and the known sensitive species (hydrilla)

below 50% of the untreated controls by 144 HAT (Table 2-2). Net photosynthetic response was

different among the cabomba populations for each herbicide. Green cabomba was 1 to 4 times

more tolerant to herbicides than red cabomba. The greatest difference between red and green

cabomba was in response to flumioxazin. The ET50 of green cabomba was 55 h, but the ET50 of

red cabomba was only 14 h. Aquarium cabomba differed from red cabomba in response to

diquat, and differed from green cabomba in response to diquat + copper and flumioxazin (Table









2-2). Net photosynthesis of all populations was reduced below 50%, with the exception of

flumioxazin on green cabomba, at 48 HAT.

This initial screen represents the best possible treatment scenario where herbicides

maintain static exposures for 144 h. This is unlikely to happen in the field as herbicides will

dilute, disperse and are broken down by various processes, so herbicides shown to be ineffective

in these studies are less likely to be effective under field conditions. This initial screen

eliminated six herbicides from further consideration, and showed that green cabomba was much

more tolerant to herbicides than red cabomba.

Twenty-four hour exposures

The four herbicides that had the highest photosynthetic reduction in the static exposure

studies were further evaluated under a 24 h exposure period. Diquat and the diquat + copper

combination failed to reduce green and aquarium populations' photosynthesis to < 50% of the

untreated control (Table 2-3). However, net photosynthesis of red cabomba exposed to diquat +

copper was reduced to < 50% of untreated controls. Green cabomba was the most tolerant and

red cabomba the most sensitive population, similar to the results observed in the static exposure

study.

Diquat + copper initially reduced net photosynthesis of red cabomba to < 50% of untreated

controls, but photosynthesis recovered to above 50% of untreated control by 144 HAT. Biomass

of red cabomba exposed to diquat + copper was reduced > 50% by 144 HAT (Figure 2-1).

Photosynthetic measurements were based upon change in oxygen production over time and per

unit of fresh weight. This measurement does not account for any significant changes in biomass

and may incorrectly lead to the conclusion that the treatment was not effective. Regrowth

potential, or potential recovery from initial herbicide damage, could be determined if the

experiments were conducted over a longer period.









This study identified two herbicides (endothall (amine salt) and flumioxazin) that reduced

photosynthesis of all cabomba populations to < 50% of untreated controls (Table 2-3).

Flumioxazin caused the greatest photosynthetic reduction on all cabomba populations and

maintained that reduction through 144 HAT. Endothall (amine salt) reduced photosynthesis of

red and aquarium cabomba to < 50% and also maintained that reduction through 144 HAT.

Similar to the response of red cabomba to diquat + copper, green cabomba photosynthesis

recovered to above 50% of untreated control by 144 HAT. The fresh weight of green cabomba

treated with endothall (amine salt) is presented in Figure 2-2. These results are similar to the

fresh weight response of red cabomba to diquat + copper (Figure 2-1) and provide evidence for

the potential recovery of the remaining plant material by showing the increase of photosynthetic

rates by 144 HAT. Unlike static exposures, algal growth was not observed for the 24 h

exposures so the recovery of plant material was not likely influenced by the photosynthesis of

algae.

The 24 h and 144 h static exposures highlight two considerations for using this method to

screen herbicides. Photosynthetic measurements may be initially misleading, because significant

reduction in biomass is not reflected in photosynthetic rates. However, when they are adjusted

on a fresh weight basis, they can provide evidence of the potential recovery of remaining plant

material. The second consideration for these experiments is what level of photosynthetic

reduction correlates to the eventual goal of reducing biomass in field applications. It is possible

that a 50% reduction is too restrictive. Depending on the management goal, this level will vary

based on the herbicides used and the plant being managed. Carfentrazone was eliminated for

further consideration on green cabomba when there may actually be potential for field activity.









For example, in the initial exposures, carfentrazone reduced green cabomba photosynthesis to

-60% of the untreated control and reduced photosynthesis in the other populations to -0.

In summary, these two studies were able to identify two herbicides (endothall (amine salt)

and flumioxazin) that should be evaluated further. Flumioxazin caused the greatest reduction in

photosynthesis, suggesting high potential for activity in field applications. These studies also

showed differential susceptibility to herbicides among the cabomba populations with

green>aquarium>red in order of least to most susceptible. These studies also highlight that

photosynthetic reduction is observed for herbicides, such as 2,4-D and triclopyr, that have no

direct inhibition of photosynthesis.

Mesocosm

Newly established plants

This study evaluated the effects of a 24 h exposure to six herbicide or herbicide

combinations on newly established green, aquarium and red populations of cabomba.

Carfentrazone, endothall (amine salt), and flumioxazin significantly reduced the biomass of all

three cabomba populations (Table 2-4). In addition, diquat + copper and triclopyr reduced the

biomass of only red cabomba. Endothall (dipotasium salt) did not significantly reduce the

biomass of green, aquarium, or red cabomba compared to untreated controls. The growth

chamber screens did not predict that carfentrazone would be active on green cabomba in

mesocosm studies, since carfentrazone only reduced the photosynthetic rate of green cabomba to

60% of the untreated controls in growth chamber screens. The arbitrary value used to indicate

susceptibility was set at 50% and may have been too restrictive a value, given the activity of

carfentrazone in the mesocosm studies on green cabomba. The growth chamber studies of

photosynthetic rates showed a high level of "activity" of carfentrazone on both aquarium and red

cabomba that was confirmed in these mesocosm tests. Red cabomba was also significantly









reduced by diquat + copper, which was predicted by the growth chamber studies; however, a

significant reduction in response to triclopyr was not predicted.

A limitation of these studies was the inconsistent growth of newly established plants.

Some treated plants failed to root and floated out of pots, making it difficult in some cases to

determine whether death was in response to the herbicide treatments or to failure to establish.

This complication led to a study in which mature, established plants with well-developed roots

were similarly exposed to herbicides. Nevertheless, the study on herbicide susceptibility of

immature plants generally agreed with previous photosynthetic studies conducted in growth

chambers and identified three compounds that had significant effects on cabomba biomass.

Mature plants

Mature green cabomba plants had a similar response to herbicide treatments as noted in

the newly established plant study. Carfentrazone, endothall (amine salt), and flumioxazin all

significantly reduced biomass (Figure 2-3). Carfentrazone reduced biomass > 60%, but the

remaining biomass was green and viable, suggesting a high probability of regrowth. Endothall

(amine salt) reduced biomass by 86% and defoliated much of the plant. The stems and apical

leaf segments of green cabomba were green and viable, also suggesting high potential for

regrowth. Flumioxazin provided the greatest reduction of biomass (> 96%); any remaining stem

material was brittle and any remaining leaf material easily fell off the plant. Growth chamber

and mesocosm studies identified flumioxazin as the herbicide with the highest potential for

control of green cabomba.

There are other examples in aquatic and terrestrial weeds where different populations of

the same species respond differently to the same herbicide. Two biotypes of Alligatorweed

(Alternanthera philoxeroides (Mart.) Griseb) (slender stem and broad stem) responded

differently to quinclorac with the slender stem biotype more susceptible than the broad stem









biotype (Kay 1992). Foes et al. (1998) reported that one biotype of common waterhemp

(Amaranthus rudis Sauer) was tolerant to triazine and ALS (acetolactate synthase inhibitors)

herbicides whereas the other biotype was susceptible. This differential tolerance in common

waterhemp, however, may likely be due to heavy levels of selection pressure placed on this plant

through years of extensive herbicide use. The differential response of cabomba populations is

unique because they are different across several herbicides. Under the current taxonomic

classification, the three populations of cabomba (green, red and aquarium) are identified as being

the same species. Moreover, an earlier study by Wain et al. (1983) predicted that numerous

cabomba "biotypes" would respond similarly to herbicide treatments, whereas these studies

indicate numerous differences.

The photosynthetic response of aquatic plants correlates well to other growth parameters,

such as biomass and stem length. Netherland and Getsinger (1995) and Netherland and Lembi

(1992) reported that photosynthetic differences of hydrilla and Eurasian watermilfoil in response

to various fluridone rates were similar to differences in dry weight up to 90 DAT. These studies

and the results of our analysis suggest that the use of photosynthetic data can be useful in the

early stages of herbicide screening by identifying herbicides with the greatest activity, which

would guide future studies.

In summary, these studies identified differential responses among the three cabomba

populations after exposure to herbicides. Green cabomba was the most tolerant and red cabomba

the most susceptible to herbicide treatments. These screens identified three herbicides

(carfentrazone, endothall (amine salt), and flumioxazin) that might provide significant control of

green cabomba, with flumioxazin having the greatest potential for control. However,

flumioxazin is still an experimental use product and is not registered for use in aquatics, while









endothall (amine salt) can be toxic to fish. Photosynthetic studies, combined with mesocosm

data can be utilized to quickly and effectively identify herbicides that warrant further testing.











Table 2-1. Photosynthetic response of three cabomba populations, expressed as percent of
untreated control, following a static 144 h exposure to selected herbicides.
Herbicide Concentration Population Triala 24 HATb 144 HAT
gg a.i. L1


Carfentrazone*


Copper






Endothall (dipotasium salt)


Quinclorac






Triclopyr*


2,4-D*


400 Green
Aquarium
Red
1000 Green
Aquarium
Red
Green
Aquarium
Red
3000 Green
Aquarium
Red
400 Green
Aquarium
Red
Green
Aquarium
Red
4907 Green
Aquarium
Red
4423 Green
Aquarium
Red


1&2


1


2


1&2


1


2


1&2


1&2


86c ad
57b
0 ce
84ab
120 a
59b
82 n.s.
92 n.s.
72 n.s.
94 n.s.
115 n.s.
109 n.s.
89 n.s.
101 n.s.
100 n.s.
92 n.s.
92 n.s.
103 n.s.
89 n.s.
98 n.s.
90 n.s.
116a
87 b
85 b


69 a
3b
Ob
60 n.s.
38 n.s.
35 n.s.
87 n.s.
88.n.s.
84 n.s.
85 ab
114a
79 b
75 n.s.
76 n.s.
82 n.s.
84 n.s.
90 n.s.
98 n.s.
115a
95 ab
76 b
126 a
93 b
86 b


a each trial n=4.
b HAT= hours after treatment.
C values are the mean percent of untreated control plants derived from net photosynthetic rates.
d results of pairwise Student's t-test at p=0.05 to compare population response within a
treatment, populations not connected by the same letter within herbicide, trial and exposure are
different.
e negative values represents net negative photosynthetic rate (net respiration).
fn.s. = not significantly different according to ANOVA (p=0.05).
*analysis was performed on log transformed data, but shown values are non transformed







Table 2-2. Estimated time to reduce photosynthesis of three cabomba
populations to 50% of an untreated control in response to a static
144 h exposure to selected herbicides.
Herbicide Concentration Population Trial ET50a
_g a.i. L-1 (n=4)
Diquat 375 Green 1&2 38 ab
Aquarium 35 a
Red 17 b
Diquat + copper 375+ 1000 Green 1&2 29 a
Aquarium 18 b
Red 12 b
Endothall (amine salt)* 2315 Green 1&2 13 a
Aquarium 11 ab
Red 10 b
Flumioxazin 400 Green 1&2 55 a
Aquarium 16b
Red 14 b
aET50 represents the estimated time (in hours) necessary to reduce net
photosynthetic rate by 50% compared to an untreated control.
populations not connected by the same letter within a herbicide are
significantly different (Student's pairwise t-test (p<0.05).
*analysis was performed on log transformed data, but the values shown are
not transformed.

Table 2-3. Photosynthetic response of cabomba populations, expressed as percent of untreated
control, following a 24 h exposure to selected herbicides.
Herbicide Concentration Populationa 24 HATb 72 HAT 144 HAT
gg a.i. L-1
Diquat 375 Green 85' n.s.d 108 ae 106 a
Aquarium 97 n.s. 90 b 98 ab
Red 83 n.s. 93 b 89 b
Diquat + copper 375 + 1000 Green 74 a 83 a 93 a
Aquarium 75 a 82 a 88 a
Red 13 b 42 b 52 b
Endothall (amine salt) 2315 Green 4 n.s. 26 a 75 a
Aquarium -3 n.s.f 10b 5 b
Red 5 n.s. 1 b 6b
Flumioxazin 400 Green 34 ab 2 n.s. 0 n.s.
Aquarium 48 a 0 n.s. 0 n.s.
Red 15 b 0 n.s. O n.s.
a n=8.
b HAT= hours after treatment.
C mean percent of untreated control derived from net photosynthetic rates.
d n.s. = not significantly different after ANOVA p=0.05.
e populations not connected by the same letter within herbicide and exposure are significantly
different (Student's pairwise t-test (p<0.05)).
negative value denotes net negative photosynthetic rate (net respiration).







Table 2-4. Response of three newly established cabomba populations after 24 h exposure to
selected herbicides.
Herbicide Concentration Population Percent of untreated control
(n=4) (Dry weight)
Endothall (dipotasium salt) 3000 Green 113 n.s.
Aquarium 111 n.s.
Red 87 n.s.
Diquat + copper 375 + 1000 Green 139 aa
Aquarium 61 b
Red* 38 b
Triclopyr 4900 Green 127 a
Aquarium 61 b
Red* 43 b
Endothall (amine salt) 1200 Green* 21 n.s.
Aquarium* 13 n.s.
Red* 7 n.s.
Endothall (amine salt) 2300 Green* 13 a
Aquarium* 5 b
Red* 0 b
Carfentrazone 400 Green* 6 n.s.
Aquarium* 3 n.s.
Red* 0 n.s.
Flumioxazin 100 Green* 0 n.s.
Aquarium* 0 n.s.
Red* 9 n.s.
Flumioxazin 400 Green* 0 n.s.
Aquarium* 7 n.s.
Red* 0 n.s.
* asterisks denote treatments that were significantly different from control after Dunnett's
analysis (p=0.05).
n.s. not significantly different at p=0.05
a populations not connected by the same letter within a herbicide and rate are not considered
significantly different according to Student's pairwise t-test (p=0.05).










0.7 -


0.6 -


0.5 -


0.4 -


0.3 -


0.2 -


0 .1 ......i
0 20 40 60 80 100 120 140 160

Hours After Treatment
Figure 2-1. Weight response of red cabomba in response to 24 h exposure to diquat + copper
375 + 1000 tg a.i. L1. Symbols represent mean 95% confidence interval.


1


1.0 -


0.8 -


0.6 -


0.4 -


0.2 -
00
0 20 40 60 80 100 120 140


Hours after Exposure

Figure 2-2. Weight response of green cabomba in response to 24 h exposure to endothall (amine
salt) 2300 .g a.i. L-1. Symbols represent mean 95% confidence interval.


-0- untreated control
- diquat+copper


--0- untreated control
- endothall (amine salt)


n=8
















14 -


12 -


S10 -





S6


4


2


01
Control Triclopyr Carfentrazone Endothall Flumioxazin Flumioxazin'


Figure 2-3. Response of mature green cabomba plants 2 WAT to a 24 h exposure to selected
herbicides. Bars represent mean + standard error. Rates for compounds are 0, 4900, 200, 1200,
100 and 400 gg a.i. L-1 respectively. Asterisks represent those rates that are significantly
different from control plants based on a Dunnett's analysis (p=0.05).









CHAPTER 3
THE EFFECT OF PH AND TEMPERATURE ON PHOTOSYNTHESIS OF THREE
CABOMBA POPULATIONS

Introduction

Aquatic plants are adapted to grow within a specific range of environmental conditions.

Although cabomba (Cabomba caroliniana) is reportedly invasive in the northeast, midwest, and

northwest portions of the United States, and is considered an exotic invasive in Australia, China,

Japan, and portions of Europe (Cao et al. 2006; Crawford et al. 2001; Leslie 1986; Mackey 1996;

Schardt and Nall 1988; Schooler et al. 2006; Yu et al. 2004a, b; Zhang et al. 2003), relatively

little is known about the physiological responses to differing environmental conditions of

cabomba populations. For instance, temperature and pH can have a significant effect on aquatic

plant growth and physiology (Sculthorpe 1985). An improved understanding of the

photosynthetic responses of differing cabomba populations to temperature and pH will provide

additional knowledge on the biology of cabomba.

Eurasian watermilfoil (Myriophyllum spicatum L.) colonizes waters that are neutral to

alkaline (Madsen 1998), while giant salvinia (Salvinia molesta Mitchell) and water hyacinth

(Eichornia crassipes (Mart.) Solms) reportedly grow best in slightly acidic to neutral pH water

(Haller and Sutton 1973; Owens et al. 2005). The genus Cabomba comprises species that are

common to acidic waters (Leslie 1986; Mackey 1996; Orgaard 1991; Tarver 1976). Surveys

have generally found cabomba in waters at pH 5-7, and the species is rarely found in waters with

pH > 8 (Mackey 1996; Tarver 1976; Yu et al. 2004b).

Temperature can also limit the distribution of aquatic plants (Sculthorpe 1985). The genus

Cabomba is largely distributed throughout tropical and subtropical climates, and many species

are limited to these regions due to an intolerance of colder temperatures (Leslie 1986; Orgaard

1991). Cabomba caroliniana is the most tolerant to colder climates, but is still reportedly









limited to geographic areas that do not experience long-term freezes (Leslie 1986). However,

populations of cabomba currently identified as C. caroliniana (for my studies identified as green

cabomba) are colonizing the midwest, northeast and northwest U.S. and Canada, where long-

term freezes during the winter are common (Les and Mehrhoff 1999; Mackey 1996; Orgaard

1991; Riemer and Ilnicki 1968; Schooler et al. 2006). Cabomba was observed overwintering in a

vegetative form in New Jersey, with apical segments loosely buried in the soil, and was able to

survive long periods of cold temperatures (Riemer and Illnicki 1968).

Cabomba reportedly has a color response to temperature. Warm temperatures induce red

coloration, while colder temperatures result in green coloration (Leslie 1986; Orgaard 1991).

However, this coloration pattern is contradicted by aquarium growers who report growth in cold

and warm water corresponds to red and green coloration, respectively (Leslie 1986). The current

distribution of cabomba supports the contention that cold water causes green coloration, because

green cabomba is found in the northern U.S. and red cabomba in the southeastern U.S. Green

cabomba is unique because the rest of the Cabomba genus is tropical to subtropical in origin and

the invasive growth of green cabomba in cold climates does not agree with this distribution.

Based on the wide geographical distribution of cabomba and the phenotypic differences of

the three populations (green, aquarium, and red), experiments were conducted to determine if

differences exist in photosynthetic response among the three populations to a range of pH and to

determine if photosynthetic rates differ among the three populations in response to a range of

temperatures. Current taxonomic classifications identify all three populations as the same

species, so any differences in response to these conditions could potentially contradict the current

taxonomy.









Materials and Methods


pH

Experiments were conducted in growth chambers at the University of Florida Center for

Aquatic and Invasive Plants (CAIP) in Gainesville, FL, in May/June 2007 and were repeated in

June/August 2007. Red cabomba was collected from White Lake (Suwannee County, FL),

aquarium cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery (Lake

City, FL), and green cabomba was collected from the St. Joseph River (Elkhart, IN). Plant

cultures were established by planting 15 cm apical segments in 2 L plastic pots filled with top

soil amended with Osmocote Plus 15-9-12 at a rate of Ig kg (soil)-'. Plants were maintained in

900 L tanks (80 cm water depth) under 50% shade cloth

Apical sections (4 cm) of each population were excised from cultures and allowed to

acclimate for at least 6 h prior to exposure to experimental treatments. Experiments were

conducted in growth chambers at a temperature of 27C, a 14:10 h day:night photoperiod and a

light intensity of 360-400 [imol m-2 s-1. After 6 h, apical sections were placed in 350 ml plastic

cups (1 section per cup) containing MES buffer, HC1, or NaHCO3, in well water amended with

Hoagland's solution (2.5% v/v) for a total volume of 345 ml per cup. MES buffer was added to

achieve a final concentration of 5 mM in solution and HC1 at a final concentration of 95 M in

solution was added to maintain pH 5.0. For pH 6, MES buffer alone was added to achieve a final

concentration of 5 mM in solution. MES buffer was added to achieve a final concentration of

2.5 mM in solution, and NaHCO3 at a final concentration of 2.4 mM in solution were added to

maintain pH 7.0. NaHCO3 was added to achieve a final concentration of 2.4 mM in solution to

maintain pH 8.0. Solution pH was measured daily and 28% HC1 was added where appropriate to

maintain the desired pH of 5, 6, 7, or 8. The rates of MES buffer used in this experiment were









determined in a preliminary study. The rates used provided consistent control of pH with no

apparent effect on photosynthesis (Appendix). Solutions were replaced every 72 h.

Dissolved oxygen was measured 72 and 120 HAT (hours after treatment) with a

dissolved oxygen meter to determine net photosynthetic rates. Initial oxygen measurements

were taken from biological oxygen demand (BOD) bottles containing the same pH as treatment

containers and Hoagland's solution (2.5% v/v). The pH in BOD bottles was maintained only for

the short duration of oxygen measurement and thus the method of pH control was slightly

different from the plastic cup culture solutions. Concentrations of 120 and 90 iM HC1 in final

solution were added to maintain pH 5 and 6 respectively in the BOD bottles. For pH 7, 20 iM

concentration of HC1 in final solution was added. For pH 8, NaHCO3 was added to achieve a

final concentration of 2.4 mM in solution. Plant tips were removed from culture treatments,

rinsed twice in tap water, and placed in BOD bottles. The BOD bottles were then placed in the

growth chamber and incubated for 60 min. Plants were then removed, oxygen levels were

measured and fresh weights were recorded after plant material was blotted between paper towels

to remove excess water. Net photosynthetic rate was determined by the following equation:

photosynthetic rate = (final dissolved oxygen (pg 02) initial dissolved oxygen (pg 02))

(freshweight-1 g) (time-' (min)) and expressed as pg 02 g-1(fresh weight) min-. Plant tips were

placed back in treatment containers after measurement of dissolved oxygen. BOD bottles were

emptied and refilled with fresh solution for each reading.

All treatments used a completely randomized design that had 4 replications. Data were

analyzed using ANOVA (p< 0.05) to assess affect of exposure 72 and 120 HAT. Analysis did

not indicate that exposure was a main effect, nor was there any interaction with population and

exposure, or pH and exposure. Exposures were combined and treated as a random effect. There









was no trial interaction, so data from both were pooled. Overall replication after pooling was

n=16. A quadratic model was then regressed for each population with the following equation:

y= bo + bi*pH + b2*pH2, where y is net photosynthetic rate. From this model, a MAX

(maximum photosynthesis) value, which represents the pH at which maximum photosynthesis

occurs for each population, was calculated for each population using the equation: MAX= -bl/

(2*b2). The MAX value for each population was compared using a full model dummy variable

technique (Neter et al. 1990).

Temperature

Temperature studies were conducted using methods similar to the pH study. Trial 1 was

conducted in July/August 2007 and repeated in August/September 2007. Plant tips were placed

in the growth chamber with an initial temperature of 200C. Plants were slowly acclimated over

the next 72 h to changes in temperature with final temperatures of 8, 16, 24, or 320C. For 8 and

320C treatments, temperature was changed 40C every 24 h. For 16 and 240C treatments,

temperature was changed 1.330C every 24 h. Plants were maintained at the final temperature for

24 h before the first photosynthetic measurements.

Dissolved oxygen was measured 24, 48 and 96 h after reaching target temperature and

data were used to calculate the net photosynthetic rates (tg 02 minute- g1 fresh weight). Initial

oxygen measurements were taken from BOD bottles containing the same solution as the

treatment containers, with 120 [iM HC1 in final solution to amend solution pH to 6-7. BOD

bottles and rinse solutions were placed in respective growth chambers for 24 h prior to use to

ensure that the target temperature was reached in the bottles used for photosynthetic

measurements. Plant tips were removed from treatment containers, rinsed twice in tap water,

and placed in BOD bottles. BOD bottles were then placed in the growth chamber for 60 min

then plants were removed, oxygen levels were measured and fresh weights were recorded. Net









photosynthetic rate was determined using the previously described equation. Plant tips were then

returned to the treatment cups and BOD bottles were emptied and filled with fresh solution

before each measurement of 02.

All treatments used a completely randomized design and had 4 replications. Data were

analyzed using ANOVA (p< 0.05) to assess effect of exposure (24, 48 and 96 HAT). Analysis

did not indicate that exposure was a main effect, nor was there any interaction with population

and exposure or temperature and exposure. Exposures were combined and treated as a random

effect. There was no trial interaction so data from both studies were pooled. Overall replication

after pooling was n=24. A post-hoc Student's pairwise t-test was used to analyze population

differences in net photosynthetic rate at each temperature.

Results and Discussion

pH

The quadratic equation, y= bo + bi*pH +b2*pH2, was used to regress each population and

all parameters were deemed significant (Table 3-1). All model residuals were acceptable and

randomly scattered around zero, with no apparent trends. All populations followed a similar

trend, where photosynthesis increased from pH 5 to a peak (MAX), and then declined as pH

approached 8 (Figure 3-1). For example, photosynthetic rates of green cabomba begin to

increase from pH 5 to a MAX of 6.2 (Table 3-1), after which the photosynthetic rate decreased

(Figure 3-1 A).

The MAX values for each population were significantly different (Table 3-1), with an

increase from aquarium (5.9) to green (6.2) to red (6.5). A comparison of MAX was necessary,

given the inherent differences in photosynthetic values across the entire range of pH (Figure 3-

2). These differences seem minor because the change in MAX among the populations is only 0.3

pH units. However, pH is based on a log scale, so these differences can have significant impacts









on plant growth. The photosynthetic rate of all populations declined as pH approached 8, and

this decline corresponded to the loss of leaf material from the basal portions of plants. Reduced

growth was noted in earlier studies where higher pH led to growth inhibition and defoliation of

plants, and the suggested ideal pH range for C. caroliniana is 4-6 (Riemer 1965; Tarver 1976).

Lakes with cabomba in Florida and China had an average pH of 6.5 and 6.2-7.5, respectively

(Hoyer et al. 1996; Yu et al. 2004b). These field studies correlate well with our results, because

maximum photosynthesis of the cabomba populations was 5.9-6.5. These results show that

cabomba has higher photosynthetic rates at slightly acidic pHs and that differences occur among

the three populations.

Temperature

Net photosynthesis of the three cabomba populations increased as temperature increased

from 8 to 320C (Table 3-2). The greatest increase in photosynthesis over this temperature range

occurred in red cabomba, and the least in aquarium cabomba. Aquarium cabomba had peak

photosynthesis between 24-320C, but the peak photosynthesis of red and green cabomba could

not be identified in the temperature range tested.

Differences in net photosynthetic rates among the three populations of cabomba were

observed at all four temperatures (Table 3-2). At 80C, red cabomba has a net photosynthetic rate

of zero significantly less than green and aquarium cabomba (Table 3-2). These photosynthetic

rates suggest that green and aquarium cabomba, though with greatly reduced photosynthetic

rates, can survive cold temperatures, whereas red cabomba is less cold tolerant. The

photosynthetic rate of red cabomba surpassed the photosynthetic rate of green cabomba at 160C

and remained higher through 320C.

A greater range of temperatures is necessary to identify where peak photosynthesis occurs

for red and green cabomba. However, the temperature range tested is representative of the









temperatures in the locations where cabomba is commonly found and these data clearly show

that red cabomba is unlikely to survive in cold climates, and could explain why this population is

not found in the northeast, midwest, and northwest U.S. Green cabomba, however, is likely to

survive during the cold winter months and these data support previous literature of green

cabomba overwintering in the northeastern U.S. (Riemer and Ilnicki 1968). Green cabomba also

had increasing photosynthesis through 320C, suggesting that green cabomba is also tolerant of a

wide temperature range, and could explain its invasiveness compared to the other cabomba

populations. Previous reports (Leslie 1986; Orgaard 1991) suggested that temperature influences

color formation in cabomba but these data suggest that temperature does not cause color changes

but limits the distribution of red cabomba. This could explain the color/temperature relationships

among these cabomba populations.

These studies measured the net photosynthetic response of cabomba to pH and

temperature. Earlier studies revealed that photosynthetic measurements in growth chambers

corresponded well to plant growth in mesocosm tests (Netherland and Getsinger 1995;

Netherland and Lembi 1992). It is then reasonable to assume that the photosynthetic response of

cabomba to pH and temperature would correspond well to plant growth in the field.

These populations are currently identified as the same species and thus would be

expected to respond similarly to both pH and temperature. The differences in photosynthesis

observed in response to both pH and temperature in these studies suggest that the current

taxonomy does not adequately separate these unique populations. In summary, pH studies

identified population differences and confirmed that cabomba prefers slightly acidic conditions.

Temperature studies showed population response differences and provide evidence that red

cabomba would not survive in colder climates where long-term freezes would be common.
















Table 3-1. Model parameters of quadratic regression for three cabomba
populations in response to a range of pH.
Population Parametera Estimatec Standard error P-Value
Aquarium bo -279 92.0 .003
bi 130 29.0 <.0001
b2 -11.0 2.2 <.0001
MAXb 5.9 a 0.15 <.0001
Green bo -553 91.0 <.0001
bi 214 28.5 <.0001
b2 -17.1 2.2 <.0001
MAX 6.2 b 0.08 <.0001
Red bo -452 92.1 <.0001
bi 164 29.0 <.0001
b2 -12.7 2.2 <.0001
MAX 6.5 c 0.08 <.0001
a for the equation y= bo + bi*pH + b2*pH2.
b calculated from MAX= -bl/(2*b2)
c values of MAX not connected by the same letter are statistically different
based on a full model dummy variable technique with p<0.05 (Neter 1990).


Table 3-2. Photosynthetic response of three
cabomba populations to a range of
temperatures.
C Populations (n=24) Net photosynthetic rate
8 Green 5.7 ab
Aquarium 6.1 a
Red 0.6 b
16 Green 39.9 c
Aquarium 66.7 a
Red 50.0 b
24 Green 121.3 b
Aquarium 151.5 a
Red 158.0 a
32 Green 154.6b
Aquarium 154.3 b
Red 200.0 a
a -1 --1
ain pg 02 g-1 (fresh weight) minute.
b Those populations not connected by the same letter
within a temperature are significantly different based
on a Student's pairwise t-test (p=0.05).
















160

140

120

100

80

60

40


5 6 7 8
140








80 -
120 --- 3---- i ---------- i --
60


40


20
5 6 7 8
160

140 -

120

100 -

8 0

60

40

0( ..


pH

Figure 3-1. Photosynthetic response of three populations of cabomba at a range of pH. A)
green, B) aquarium, C) red. Symbols represent raw data for each pH. Lines represent the
quadratic fit of the model (y= bo + bi*pH + b2*pH2).


aoJ
Cl
0

aoJ












140
................... Green (MAX= 6.2)
-- Aquarium (MAX= 5.9)
S120 Red (MAX= 6.5)
.... ....... ..

S..
100- -







60




40 i
5 6 7 8

pH
Figure 3-2. Quadratic fit of the response of three cabomba populations to a range of pH. Lines
represent the quadratic fit of the model (y= bo + bi*pH + b2*pH2). MAX is calculated from
equation MAX= -bi/ (2*b2) and represents the pH for each population where maximum
photosynthesis occurred.









CHAPTER 4
COMPARISON OF THREE CABOMBA POPULATIONS FROM NATURAL HABITATS
AND CULTURE CONDITIONS

Introduction

Cabomba caroliniana (cabomba) is a submersed aquatic dicotyledonous plant considered

native to the southeastern U.S. and is increasingly being found in northern North America and in

Australia, China, and locations in Europe (Leslie 1986; Mackey 1996; Orgaard 1991; Schooler et

al. 2006; Yu et al. 2004a, b). Plants in these new locations differ in appearance from plants

observed in their native range. For example, plants in new northern locations are green (green

cabomba), whereas the native plants in the southeast are red (red cabomba). Cabomba sold by

the aquarium trade (aquarium cabomba) is green with purple leaves at the apex of the plant.

Green cabomba is widely considered an invasive plant and a management problem, whereas red

cabomba reportedly lacks invasive characteristics.

Environmental conditions can greatly influence the appearance of many aquatic plants

(Godfrey and Wooten 1981; Sculthorpe 1985) and can be due to temperature, light, pH, nutrient

content, and many other factors that can influence plant growth. Due to potential variance in

environmental conditions, many plants have a high degree of phenotypic plasticity in order to

survive a wide range of conditions (Sculthorpe 1985). In some cases, these phenotypic changes

in response to environmental conditions lead to the classification of ecotypes. There is, however,

some question as to the usefulness of this distinction, because the ecotypes are essentially the

same species and are only displaying phenotypic plasticity (Quinn 1978). C. caroliniana has a

wide range of characteristics, particularly color, which is reportedly influenced by environmental

conditions such as light and temperature.

Plants differ in color due to various concentrations of an array of plant pigments, including

chlorophyll and anthocyanin. Chlorophyll is largely responsible for green coloration, whereas









anthocyanins are responsible for orange-red coloration (Kong et al. 2003). Anthocyanins not

only impart color but can also act as antioxidants and even provide protection from insect

herbivory (Kong et al. 2003). Increased levels of anthocyanin can indicate stress in aquatic

plants, such as that observed in response to herbicides (Doong et al. 1993).

Characteristics other than color are influenced by environmental conditions and this

tendency has been observed in other aquatic plants. Alligatorweed (Alternatheraphiloxeroides

(Mart.) Griseb) has distinct growth forms in response to salinity and water level. These forms

are reversible and dependent upon the prevailing conditions (Kay and Haller 1982). Cabomba

color traits are also believed to be reversible, which would support the theory that color is plastic

and influenced by environment change. However, if these differences are not reversible, it is

possible that coloration is innate.

Color differences among the three cabomba populations suggests some level of pigment

differentiation, reportedly caused by temperature and is a reversible difference (Orgaard 1991).

The three populations of cabomba have been maintained in common culture at the University of

Florida Center for Aquatic and Invasive Plants (CAIP) and remained constant in appearance for

more than two growing seasons. This is in contrast to the predictions of Orgaard (1991) and

Leslie (1986) whose data suggest that a plant with green (green cabomba) should have changed

to red (red cabomba) when grown in a warm climate. There is also a difference in leaf

appearance of the three populations. Green cabomba has large coarsely divided leaves, whereas

red cabomba has small finely divided leaves. Aquarium cabomba is intermediate between the

other two populations. The objectives of this study were to: 1) determine if pigment content

(chlorophyll and anthocyanin) and specific leaf weight differed for plants collected from natural

habitats (field plants) and plants from common culture (culture plants) for all three populations









and 2) determine if there are differences in the three parameters tested among the three cabomba

populations for each field and culture growth location.

Materials and Methods


Pigment Analysis

Chlorophyll content. Red cabomba was collected from White Lake (Suwannee County,

FL) aquarium cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery

(Lake City, FL), and green cabomba was collected from the St. Joseph River (Elkhart, IN).

These three populations were grown in common culture at the CAIP. Green and aquarium

cabomba was grown in common culture for 1 yr prior to use in experiments. Red cabomba

was only grown in common culture for 3 mo before use in experiments. These plants are

identified as cultured plants. Plants were also collected from the same field sites as cultured

plants, but were utilized immediately for experiments. These plants are identified as field plants.

Five apical sections (2 cm long) were excised from each population of cultured and field plants

and placed in test tubes. Test tubes were then refrigerated at 4.40C for 24 h until they were

processed.

After refrigeration, plants were trimmed to similar weights and fresh weight was measured.

Plant tips were then placed in test tubes containing 5 ml of DMSO (dimethylsulfoxide) and

chlorophyll was extracted (Hiscox and Israelstam 1979). Plants in test tubes were incubated in a

water bath (700C) for 6 h and chlorophyll content was determined spectrophotometrically (Arnon

1949), and expressed as mg (chlorophyll) g-1 fresh weight.

All populations had 5 replications for each source (field and culture) and chlorophyll

content of field plants was compared to the corresponding population's chlorophyll content of

cultured plants by examining 95% confidence intervals. Data were also analyzed using ANOVA









(p<0.05) and a post-hoc Student's pairwise t-test to compare differences in chlorophyll content

of the three populations to one another in field and cultured plants.

Anthocyanin content. The same plant collection methods and sources as described for

the chlorophyll experiments were used for anthocyanin content analysis. Unlike chlorophyll,

plant segments were frozen at -50C for 1 wk prior to anthocyanin extraction. Plants were moved

to room temperature -200C for 2 h prior to anthocyanin extraction, following the methods from

Doong et al. (1993). The equation used to correct for the absorbance of chlorophyll at 675 nm

and obtain total absorbance was: total relative absorbance of anthocyanin= (A 530 (0.25*A

657)) g-1 fresh weight.

All populations had 5 replications for each source (field and culture). The anthocyanin

content of field plants (green, aquarium and red) was compared to the corresponding anthocyanin

content of cultured plants by comparing 95% confidence intervals. Data were also analyzed

using ANOVA (p<0.05) and a post-hoc Student's pairwise t-test to compare differences in

chlorophyll content of the populations to one another in field and cultured plants.

Leaf Characteristics

The same plant collection methods and sources as described for pigment analysis were

used for leaf weight comparisons. Five plants were selected from each population and location

(field and culture), and 10 leaves from each were removed from at least 4 cm from the tip of the

plant and placed in scintillation bottles. Leaf material was dried at 700C for 2 wk then weighed

for each plant (10 leaves total) and converted to specific leaf weight.

All populations had 5 replications for each source (field and culture). The specific leaf

weight of each population's field plants was compared to the corresponding population's specific

leaf weight of cultured plants by examining 95% confidence intervals. Data were also analyzed









using ANOVA (p<0.05) and a post-hoc Student's pairwise t-test to compare differences in per

leaf weight of the populations to one another in field and cultured plants.

Results and Discussion

Pigment Analysis

Neither chlorophyll nor anthocyanin content (Table 4-1) of field plants differed from the

corresponding cultured plants (analysis not shown). However, differences were observed in

chlorophyll content among populations, regardless of the source of the plants (field or culture).

Chlorophyll content was highest in aquarium cabomba, followed by green cabomba, and lowest

in red cabomba for both field and culture plants (Table 4-1). There were also differences in the

anthocyanin content among the three populations and these were the same regardless of the

collection source (field or culture). Red cabomba had the highest anthocyanin content, green

cabomba had the lowest content, and aquarium cabomba, as in previous experiments, was

intermediate to the other two populations. The anthocyanin content of red cabomba is nearly 10

times higher than the anthocyanin content of green cabomba, and suggests that anthocyanin is

the likely source of red pigmentation for cabomba populations.

Similar pigment concentrations of both field and culture plants of all populations of

cabomba suggest that common culture in Florida does not affect pigment concentrations. There

was no difference in chlorophyll or anthocyanin content in green cabomba field plants (collected

from cold northern climates) and green cabomba cultured in Florida. Earlier literature predicted

that the coloration of cabomba grown cold water would change from green to red if that same

plant were grown in warm water (Leslie 1986; Orgaard 1991). Our results contradict this

prediction and suggest that cabomba pigmentation is more conserved than originally believed.

Anthocyanins are commonly synthesized in response to stress (Doong et al. 1993; Kong et al.

2003), and could be the source of anthocyanin differences among plant populations, but this is









unlikely, given the similar culture conditions and conserved nature of the red cabomba

anthocyanin content.

Plant collection site had no impact on cabomba pigmentation and the differences among

the populations were the same regardless of site. This suggests that pigment content is not as

variable or environmentally influenced as originally believed, but to fully explore this possibility

reciprocal studies in northern climates need to be performed on red and aquarium cabomba.

Current taxonomy identifies all three populations as being the same species, and further suggests

that temperature determines content of plant pigments (Leslie 1986; Orgaard 1991). It is

possible that red and aquarium cabomba are affected by temperature, but these studies refute that

claim for green cabomba. The source and reason for this conserved pigmentation in cabomba

populations is not known, but these data suggest the differences are not environmentally flexible

for green cabomba, and are likely conserved in the other populations as well.

Leaf Characteristics

There was no difference in specific leaf weight between field and culture plants of the same

population (data not shown). However, differences were observed among populations regardless

of the source of the plants (field or culture) (Table 4-1). Green cabomba was different from red

cabomba and the specific leaf weight of aquarium cabomba was intermediate to both green and

red cabomba.

These differences correspond to the visual observation that green cabomba has larger

leaves than red cabomba, and aquarium cabomba is intermediate to both. Leaf characteristics,

much like plant pigments, are not influenced by the location where plants were grown and leaf

weight is another parameter that highlights the differences among these cabomba populations.

Reciprocal studies should be performed in colder climates to determine the impact of

temperature on plant pigmentation and specific leaf weight of red cabomba. It is possible that









red and aquarium cabomba could change pigmentation and specific leaf weight in colder

climates, but this seems unlikely due to lack of plasticity in this study. These studies indicate

that green cabomba was unaffected by culture location, suggesting that temperature had no

impact on this population as was previously reported (Leslie 1986; Orgaard 1991). Due to the

highly conserved plant pigmentation and specific leaf weight of green cabomba from two

separate growth locations, a reexamination of cabomba taxonomy may be necessary to fully

address the differences between red and green cabomba.













Table 4-1. Characteristics of apical sections of three cabomba populations collected
from the field and established cultures.
Population Field (n=5) Culture (n=5)
Chlorophyll content (mg (total chlorophyll) g-(fresh weight)
Green 0.508 b 0.484 b
Aquarium 0.644 a 0.677 a
Red 0.322 c 0.357 c
Total anthocyanin absorbance g-(fresh weight)
Green 0.296 c 0.313 c
Aquarium 1.529 b 1.399 b
Red 2.657 a 2.376 a
Leaf weight mg (dry weight) leaf-1
Green 8.6 a 7.1 a
Aquarium 6.3 ab 5.7 ab
Red 3.9 b 5.0 b
Populations not connected by the same letter within a collection site (field or culture)
and parameter tested are significantly different according to a Student's pairwise t-
test (p<0.05).









CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS

These studies revealed that green and red cabomba are significantly different from one

another in their response to a range of herbicides, temperature, pH, and had different pigment

content and leaf characteristics. Aquarium cabomba had characteristics similar to both red and

green cabomba, depending upon the experimental conditions. Cabomba is tolerant of most

herbicide treatments, but green cabomba is the most tolerant of the three populations. Three

herbicides (carfentrazone, endothall (amine salt) and flumioxazin) were effective in reducing

photosynthesis and biomass of cabomba, and further field evaluations are warranted to determine

the efficacy of these herbicides for potential use in management programs.

Slow acting systemic herbicides such as fluridone and ALS (acetolactate synthase)

compounds were not tested due to the much longer exposure times needed to effectively control

aquatic plants and the lack of good long-term culture techniques. In previous laboratory studies

fluridone was shown to effectively reduce biomass of cabomba at high rates, but field trials were

less conclusive (Mackey 1996; Nelson et al. 2001; Nelson et al. 2002). These herbicides are

typically used on a lake-wide basis and may have limited utility where cabomba exists only in

isolated areas of the littoral zone or in flowing waters. Given the increase of cabomba in areas

where Eurasian watermilfoil (Myriophyllum spicatum L.) once dominated, these compounds

could be utilized to simultaneously suppress cabomba and milfoil (though likely at higher rates

than currently used) if these compounds are shown to be effective on cabomba.

The most persistent and troubling problem in these experiments was a lack of reliable

culture techniques for cabomba. Attempting to maintain just one population was difficult;

culturing three populations at the same time was rarely possible. Future studies of cabomba

should begin with a concerted effort to identify optimum culture techniques and growth









conditions for all three populations, which would reduce reliance on obtaining plants from the

field.

Reciprocal studies should be conducted in a northern climate to fully compare the three

cabomba populations. Reciprocal studies would determine if differences are conserved for all

populations. Culturing aquarium and red cabomba in northern climates may be problematic

given the reported lack of cold tolerance for many cabomba species and the lack of

photosynthesis of red cabomba at water temperatures of 80C or less as evident in these studies.

Genetic studies would also be helpful to characterize the differences among these three

cabomba populations and are likely necessary if the taxonomy of cabomba is to be revised. The

physiological differences observed in these experiments suggest there are major differences in

cabomba populations that would warrant such a reclassification. Even if reclassification is not

considered, aquatic plant managers should be aware of the results of this study. Properly

identifying the cabomba present will influence which management strategies should be

implemented.









APPENDIX
HERBICIDE INFORMATION AND PREVIOUS STUDIES

Table A-1. List of compounds for use in submersed aquatic weed
control.
Herbicide Maximum labeled rate Typical use rate
tg a.i. L- __g a.i. L1
Carfentrazone 200 100-200
Copper 1000 250-1000
Diquat 375 94-375
Endothall (amine salt) 7000 500-2000
Endothall (dipotasium salt) 5000 1000-3000
Flumioxazin 400a N.A.b
Quinclorac 500a N.A.b
Triclopyr 3500 2000-3500
2,4-D (amine salt) 5000 2500-5000
a recommended maximum labeled rate to EPA.
b compound not registered so no use pattern developed.


n=4

............... ........... .- .. .

a........ ........ 0 M ES
--A/- 0.1% MES v/v
-0 1% MES v/v


/0 20 40 60 80 100 120 1







0 20 40 60 80 100 120 1


Hours
Figure A-1. Response of aquarium cabomba growth solution pH to a range of MES buffer
solution. Symbols represent mean + 95%.

























40 -


20 .1 .i
0 20 40 60 80 100 120 140 160

Hours After Treatment
Figure A-2. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and Eurasian watermilfoil to 2,4-D at a concentration of 4400 tg active ingredient L-1. Symbols
represent mean standard error. Dotted reference line is at 50% of untreated control.


.)~ 100


| 80
0
g 60


40


20


0


0 20 40 60 80 100 120


140 160


Hours After Treatment
Figure A-3. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to endothall (dipotasium salt) at a concentration of 3000 tg active ingredient L1.
Symbols represent mean standard error. Dotted reference line is at 50% of untreated control.






















-20 -

-40

-60 ,
0 20 40 60 80 100 120 140 160


Figure A-4. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and variable milfoil to carfentrazone at a concentration of 400 tg active ingredient L-1. Symbols
represent mean standard error. Dotted reference line is at 50% of untreated control. Negative
values represent treatments in which plants consumed oxygen (net respiration).


0 20 40 60 80 100 120 140


Hours After Treatment
Figure A-5. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to copper at a concentration of 1000 tg active ingredient L-1. Symbols represent
mean standard error. Dotted reference line is at 50% of untreated control. Negative values
represent treatments in which plants consumed oxygen (net respiration).









n=4 Copper (trial 2) ........ ........ Green
40 Aquarium

20 --i-- Red
0 Hydrilla

80 -



20 -

40
20



-20

-40

60
0 20 40 60 80 100 120 140 1


Hours After Treatment
Figure A-6. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and hydrilla to copper at a concentration of 1000 tg active ingredient L-1. Symbols represent
mean + standard error. Dotted reference line is at 50% of untreated control. Negative values
represent treatments in which plants consumed oxygen (net respiration).


160
n=8 Tr r ....... ........ Green
r- -0 Aquarium
140 -.--m.- Red
-A--- E. Milfoil

120 -




1 80
|. .o -. ....... __.


60


40


20 -
20



0 20 40 60 80 100 120 140 160
Hours After Treatment
Figure A-7. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
and Eurasian watermilfoil to triclopyr at a concentration of 4900 tg active ingredient L1.
Symbols represent mean standard error. Dotted reference line is at 50% of untreated control.










n=4 G


Quinclorac (trial 1)


........O ........ G reen
- Aquarium
--.-m--- Red


40 -


2 0 .iiiiii
0 20 40 60 80 100 120 140 160

Hours After Treatment

Figure A-8. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
to quinclorac at a concentration of 400 ug active ingredient L-1. Symbols represent mean +
standard error. Dotted reference line is at 50% of untreated control.


160


140


120


100 i


80


60


0 20 40 60 80 100 120 140

Hours After Treatment


Figure A-9. Photosynthetic response of three populations of cabomba (green, aquarium, and red)
to quinclorac at a concentration of 400 ug active ingredient L-1. Symbols represent mean +
standard error. Dotted reference line is at 50% of untreated control.


160


140


120


100 I



80

60 -


Cd)


C41


n=4 Quinclorac (trial 2)


........ O ........ G reen
- -- Aquarium
- --.-- -..- Red


I


-










120


-20

-40 -

-60
0 20 40 60 80 100 120 140 160
Hours After Treatment

Figure A-10. Photosynthetic response of three populations of cabomba (green, aquarium, and
red) and hydrilla to diquat at a concentration of 375 pg a.i. L-1. Symbols represent mean
standard error. Dotted reference line is at 50% of untreated control. Negative values represent
those readings that consumed oxygen (net respiration).


Figure AI. Photosynthetic response of three populations of cabomba (green, aquarium, nd
N=8 Diquat + Copper 0........ o--- Green
100 Aquarium
---- .- Red
80 \ '. A Hydrilla

60 -

40

20



5% -20

-40

-60

-80

-100
0 20 40 60 80 100 120 140 160
Hours After Treatment


red) and hydrilla to diquat at a concentration of 375 + copper at a concentration of 1000 pg a.i.
L1. Symbols represent mean standard error. Dotted reference line is at 50% of untreated
control. Negative values represent those readings that consumed oxygen (net respiration).









N=8


Endothall (amine salt)


........ ........ G reen
----- Aquarium
--m--- ... Red
H ydrilla


.......... .............. ............


T


,c~___I-tz 4


0 20 40 60 80 100 120 140 160
Hours After Treatment


Figure A-12. Photosynthetic response of three populations of cabomba (green, aquarium, and
red) and hydrilla to endothall (amine salt) at a concentration of 2300 gg a.i. L-1. Symbols
represent mean standard error. Dotted reference line is at 50% of untreated control. Negative
values represent those readings that consumed oxygen (net respiration).


0 20 40 60 80 100 120 140 160
Hours After Treatment
Figure A-13. Photosynthetic response of three populations of cabomba (green, aquarium, and
red) and hydrilla to flumioxazin at a concentration of 400 gg a.i. L-1. Symbols represent mean +
standard error. Dotted reference line is at 50% of untreated control. Negative values represent
those readings that consumed oxygen (net respiration).


120











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78:1367-1378.

Owens, C.S., R.M. Smart, D.R. Honnell, and G.O. Dick. 2005. Effects ofpH on
growth of Salvinia moelsta Mitchell. J. Aquat. Plant. Manage. 43:34-38.

Preston, C.D. and J.M. Croft. 1997. Aquatic plants in Britain and Ireland. Colchester, UK:
Harley Books. 365 p.

Quinn, James A. 1978. Plant ecotypes: Ecological or evolutionary units? B. Torrey Bot. Club
105:58-64.

Riemer, Donald N. 1965. The effect of pH, aeration, calcium and osmotic pressure on
the growth of fanwort (Cabomba caroliniana Gray). Pages 460-467 in Proceedings of
the 19th Annual Meeting of the Northeastern Weed Control Conference. New York City,
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Riemer, D.N. and R.D. Ilnicki. 1968. Reproduction and overwintering of cabomba in
New Jersey. Weed Sci. 16:101-102.

Sanders, D.R. 1979. The ecology of Cabomba caroliniana. Pages 134-146 in E.O. Gangstad,
Weed Control Methods for Public Health Applications. Boca Raton FL: CRC Press.

Schardt, J.D. and L.E. Nall. 1989. 1988 Florida Aquatic Plant Survey. Tallahassee, FL: Florida
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Germany: Koeltz Scientific Books. 610 p.

Selim, S.A., S.W. O'Neal, M.A. Ross, and C.A. Lembi. 1989. Bioassay of
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(Myriophyllum spicatum). Weed Sci. 37:810-814.

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Stetak, D. 2004. An aquarium plant in natural waters and canals of Hungary: The
fanwort (Cabomba caroliniana). Kitaibelia 6:165-171.

Tarver, D.P. 1976. Selected Life Cycle Features and Effects of Environmental
Conditions on Cabomba caroliniana Gray. M. Sc. Thesis. Natchitoches, LA:
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Tarver, D.P. and D.R. Sanders. 1977. Selected life cycle features of fanwort. J. Aquat.
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Wain, R.P., W.T. Haller, and D.F. Martin. 1983. Genetic relationship among three
forms of cabomba. J. Aquat. Plant Manage. 21:96-98.

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BIOGRAPHICAL SKETCH

Born on September 12, 1981 in Merridian, Mississippi, Brett is the son of Craig and Laura

Bultemeier. As a child, Brett moved often, as his father was a naval aviator and was redeployed

every few years. Finally ending up in Indiana, he pursued his B.S. degree at Manchester College

in North Manchester, Indiana, focusing his studies on biology and environmental studies. While

at Manchester, he was a collegiate wrestler, played in the school band, and helped form the

environmental club. During his undergraduate studies, Brett worked during the summer for

Weed Patrol Inc. and it was at this job that a passion for aquatic plant management was fostered.

Upon completion of his degree, Brett was married to Megan and promptly moved to Gainesville,

Florida, to pursue a Masters degree in the field of aquatic plant management. Upon completion

of his master's project Brett began studies towards a PhD from the University of Florida.





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THE RESPONSE OF THREE CABOMBA POPULATIONS TO HERBICIDES AND ENVIRONMENTAL PARAMETERS: IMPLICATIONS FOR TAXONOMY AND MANAGEMENT By BRETT WELLS BULTEMEIER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Brett Wells Bultemeier 2

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To all those who have been taken from my life too earlythough your time here on earth has passed, your love and memory will always be with me. 3

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ACKNOWLEDEGMENTS First and foremost, I am humbly thankful to God for the love, grace, and beauty I have been blessed with in life. I am grateful to my major advisor Dr. Netherland for the countless hours of refining my writing, reviewing experimental protocols, and helping develop my scientific methodology. I am thankful for the support provided by my advisory committee: Dr. Haller, Dr. Ferrell, Dr. Koshnick, Dr. Erickson, and Dr. Wofford. Their tireless efforts in help with statistics, experimental design, and general assistance with this project were instrumental in its completion. The friendship and kindness shown to my family by Dr. Haller made the transition to Gainesville one of ease, and continues to make our time here wonderful. I extend my appreciation to Dr. MacDonald who, though not on my committee, provided support in experimental methods and developing many aspects of my project. The administrative staff of the Agronomy Department was incredibly patient with deadlines, forms, and all the necessary steps towards graduation and for that, I am grateful. This project would not have been possible without help harvesting plants, maintaining plant cultures, and plant collections; for that help I thank the following people: Chris Mudge, Tomas Chiconela, Margaret Glenn, David Mayo, Cole Hulon, Michael Aldridge, and Lyn Gettys. I am thankful to Manchester College for fostering my scientific interests and providing a solid academic foundation. They also taught me what it means to be a responsible citizen of this country and of the world. Though small in enrollment, their global impact is large and their graduates take with them a sense of leadership and responsibility too rare in this world. For their love and support I thank my family. My sister has shown me how to overcome adversity, and how grow stronger from those adversities. My mom continually supported my endless questions and fostered a curiosity that developed into a passion for science that will serve 4

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me well throughout life. My dad, through his guidance and years of military service, taught me the true meaning of character, moral fiber, and integrity. These values have become the foundation for which I have tried to live. Finally I am infinitely thankful for the love, support, and sacrifice of my wife. Her willingness to uproot from family and deal with long hours of research will never be forgotten. She has so selflessly devoted her time and energy to me that I am amazed and have been shown the true meaning of love. Without her in my life, I would not be where I am nor be able to accomplish the things in life for which I strive. 5

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TABLE OF CONTENTS page ACKNOWLEDEGMENTS .............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................12 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................15 Genus Description...........................................................................................................15 Taxonomy........................................................................................................................16 Distribution and Morphology..........................................................................................17 Reproduction...................................................................................................................20 Growth Conditions..........................................................................................................20 Management....................................................................................................................21 Objectives........................................................................................................................22 2 THE EFFECT OF AQUATIC HERBICIDES ON THREE POPULATIONS OF CABOMBA............................................................................................................................26 Introduction.............................................................................................................................26 Materials and Methods...........................................................................................................27 Growth Chamber.............................................................................................................27 Static exposures........................................................................................................27 Twenty-four hour exposures....................................................................................31 Mesocosm........................................................................................................................31 Newly established plants..........................................................................................31 Mature plants............................................................................................................33 Results and Discussion...........................................................................................................33 Growth Chamber.............................................................................................................33 Static exposures........................................................................................................33 Twenty-four hour exposures....................................................................................35 Mesocosm........................................................................................................................37 Newly established plants..........................................................................................37 Mature plants............................................................................................................38 3 THE EFFECT OF PH AND TEMPERATURE ON PHOTOSYNTHESIS OF THREE CABOMBA POPULATIONS................................................................................................46 Introduction.............................................................................................................................46 Materials and Methods...........................................................................................................48 6

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pH....................................................................................................................................48 Temperature.....................................................................................................................50 Results and Discussion...........................................................................................................51 pH....................................................................................................................................51 Temperature.....................................................................................................................52 4 COMPARISON OF THREE CABOMBA POPULATIONS FROM NATURAL HABITATS AND CULTURE CONDITIONS......................................................................57 Introduction.............................................................................................................................57 Materials and Methods...........................................................................................................59 Pigment Analysis.............................................................................................................59 Leaf Characteristics.........................................................................................................60 Results and Discussion...........................................................................................................61 Pigment Analysis.............................................................................................................61 Leaf Characteristics.........................................................................................................62 5 CONCLUSIONS AND RECOMMENDATIONS.................................................................65 APPENDIX HERBICIDE INFORMATION AND PREVIOUS STUDIES................................67 LIST OF REFERENCES...............................................................................................................74 BIOGRAPHICAL SKETCH.........................................................................................................80 7

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LIST OF TABLES Table page 1-1 Species in the genus Cabomba, their proposed ploidy levels, and native ranges..............24 2-1 Photosynthetic response of three cabomba populations, expressed as percent of untreated control, following a static 144 h exposure to selected herbicides......................41 2-2 Estimated time to reduce photosynthesis of three cabomba populations to 50% of an untreated control in response to a static 144 h exposure to selected herbicides................42 2-3 Photosynthetic response of cabomba populations, expressed as percent of untreated control, following a 24 h exposure to selected herbicides.................................................42 2-4 Response of three newly established cabomba populations after 24 h exposure to selected herbicides.............................................................................................................43 3-1 Model parameters of quadratic regression for three cabomba populations in response to a range of pH..................................................................................................................54 3-2 Photosynthetic response of three cabomba populations to a range of temperatures..........54 4-1 Characteristics of apical sections of three cabomba populations collected from the field and established cultures.............................................................................................64 A-1 List of compounds for use in submersed aquatic weed control.........................................67 8

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LIST OF FIGURES Figure page 1-1 Line drawing of a submersed cabomba leaf showing the fan shape which leads to the common name fanwort. From Don E. Eyles, A Guide and Key to the Aquatic...............25 2-1 Weight response of red cabomba in response to 24 h exposure to diquat + copper 375 + 1000 g a.i. L -1 ...............................................................................................................44 2-2 Weight response of green cabomba in response to 24 h exposure to endothall (amine salt) 2300 g a.i. L -1 ...........................................................................................................44 2-3 Response of mature green cabomba plants 2 WAT to a 24 h exposure to selected herbicides...........................................................................................................................45 3-2 Quadratic fit of the response of three cabomba populations to a range of pH...................56 A-1 Response of aquarium cabomba growth solution pH to a range of MES buffer solution...............................................................................................................................67 A-2 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and Eurasian watermilfoil to 2,4-D at a concentration of 4400 g active ingredient .......68 A-3 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to endothall (dipotasium salt) at a concentration of 3000 g active..............68 A-4 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and variable milfoil to carfentrazone at a concentration of 400 g active ingredient.......69 A-5 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to copper at a concentration of 1000 g active ingredient L -1 .......................69 A-6 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to copper at a concentration of 1000 g active ingredient L -1 .......................70 A-7 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and Eurasian watermilfoil to triclopyr at a concentration of 4900 g active....................70 A-8 Photosynthetic response of three populations of cabomba (green, aquarium, and red) to quinclorac at a concentration of 400 g active ingredient L -1 .......................................71 A-9 Photosynthetic response of three populations of cabomba (green, aquarium, and red) to quinclorac at a concentration of 400 g active ingredient L -1 .......................................71 A-10 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to diquat at a concentration of 375 g a.i. L -1 ................................................72 9

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A-11 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to diquat at a concentration of 375 + copper at a concentration of 1000.......72 A-12 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to endothall (amine salt) at a concentration of 2300 g a.i. L -1 .....................73 A-13 Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to flumioxazin at a concentration of 400 g a.i. L -1 ......................................73 10

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LIST OF ABBREVIATIONS HAT Hours after treatment DAT Days after treatment WAT Weeks after treatment ALS Acetolactate synthase a.i. Active ingredient ET 50 Estimated time to reduce photosynthesis to 50% of an untreated control MAX pH at which maximum photosynthesis occurred BOD Biological oxygen demand bottles 11

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE RESPONSE OF THREE CABOMBA POPULATIONS TO HERBICIDES AND ENVIRONMENTAL PARAMETERS: IMPLICATIONS FOR TAXONOMY AND MANAGEMENT By Brett Wells Bultemeier May 2008 Chair: Michael Netherland Major: Agronomy The genus Cabomba is composed of a group of submersed aquatic plants native to the Americas (Latin, North and South). The species Cabomba caroliniana var. caroliniana and var. pulcherrima are native to the southeastern United States. Populations of cabomba have recently been introduced to the northern U.S., Australia, Canada, China, Japan, and Europe. In these newly colonized areas, cabomba is proving difficult to control. Varieties of Cabomba caroliniana range in color from green to red/purple and is reportedly influenced by temperature. Plants typical of the southeastern U.S. are red (hereafter referred to as red cabomba) whereas the northern plants, as well as those around the world, when described, are green (hereafter referred to as green cabomba). Plants purchased directly from an aquarium dealer had both green and red coloration on a single plant (hereafter referred to as aquarium cabomba). A lack of taxonomic clarity and management options led to the investigation of the response of three populations of cabomba (green, aquarium, and red) to herbicides and selected environmental parameters. Growth chamber studies were designed to determine cabombas response to herbicides by measuring photosynthetic activity. Ten herbicides were evaluated to investigate population differences and to identify potential herbicides for use in management programs. An ET 50 value 12

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(the estimated time to reduce photosynthesis by 50% compared to an untreated control) was used to quantify the photosynthetic response of all three cabomba populations. Studies identified endothall (amine salt) and flumioxazin as potential herbicides for use in management programs. Green and red cabomba consistently differed in susceptibility to herbicides, with green cabomba more tolerant than aquarium and red cabomba. Response of aquarium cabomba to herbicides was intermediate. The use of photosynthetic measurements for predicting herbicide activity was validated in mesocosm trials on both immature and mature plants, which generally confirmed the results determined from growth chamber experiments. A number of herbicides were ineffective on cabomba, but carfentrazone, endothall (amine salt) and flumioxazin were effective. Flumioxazin at 200 and 400 g active ingredient L -1 caused the greatest effect exceeding a 95% reduction in biomass. Cabomba populations differed in their photosynthetic response to pH. Aquarium cabomba had peak photosynthesis at pH 5.9, green cabomba at pH 6.2, and red cabomba at pH 6.5; all were significantly different from one another. Green and aquarium cabomba were able to photosynthesize at lower temperatures (8C) than red cabomba. Red cabomba had higher photosynthetic rates than aquarium and green cabomba as temperature increased to 32C. Chlorophyll, anthocyanin content, and specific leaf weight were collected from field and culture plants to determine the effect of long-term culture conditions on plant growth and morphology. Cabomba populations evaluated from field and culture were similar within a population, while differences in chlorophyll, anthocyanin, and leaf size were found among the three populations. 13

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These studies suggest a taxonomic revision may be necessary to further differentiate Cabomba caroliniana based on differences to a range of stimuli evaluated in these studies. Results support anecdotal observations that cabomba is tolerant to most registered herbicides. Reciprocal experiments should be performed in colder climates to confirm differences among these populations are conserved and are not simply a response to regional environmental conditions such as temperature. 14

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Genus Description The following description provides a general guide to the plants comprising the genus Cabomba (Fassett 1953; Godrey and Wooten 1981; Orgaard 1991). Species of cabomba are rooted, submersed, aquatic, perennial dicotyledonous plants that colonize through rhizomatous growth. Adventitious roots are sometimes formed at lower stem nodes and measure up to 24 cm in length. Immature roots are smooth, unbranched, and white with a yellow tip and as the roots mature they branch, form thick mats, and darken to brown or black. Stems arise from rhizomes and rootcrowns, with prolific branching near the base. These stems are typically 1 m in length but have been reported to be as long as 10 m. Plants occasionally have a thin mucilaginous coating. Stems are round to slightly compressed and are typically 2 mm in diameter. Submersed leaves vary in color, ranging from olive green or brown to red/purple and are typically opposite, although whorls of three can occur, and are fan-shaped, hence the other common name of fanwort (Figure 1-1.). Leaf size can vary greatly even on a single stem, with leaf size reduced at the apex of stems. Leaves are divided and may have up to 200 terminal points on a single leaf. Floating leaves attached to flowering branches have an alternate arrangement and are linear-elliptic to ovate, although leaves of some tropical species have a circular shape with ends tapering to a point. Flowering stems bear solitary bisexual flowers measuring 6 mm in diameter. Flowers are monoecious with three sepals and three petals and may be pure white, white with purple tinting (primarily at the margins), or white with yellow spotting. Stamens are shorter than petals and typically occur in whorls of 3; anthers are 1.5 mm long. Flowers 15

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have 2 pistils that may each develop 1 ovoid or globose seeds. Fruits are 4 mm long and are attached to the previously floated flower section just below the water surface. Taxonomy The taxonomic classification of the genus Cabomba is confusing (Leslie 1986; Orgaard 1991) and efforts utilizing molecular techniques are currently being tested 1 Orgaard (1991) proposed the most current and complete taxonomy of the genus Cabomba, and unlike previous classifications, describes features beyond those subject to environmental plasticity such as leaf color, shape, and size. Orgaards classification also includes pollen structure, vessel organization, and chromosome numbers (Table 1-1) to advance previous descriptions. Orgaard divided the genus into five species with one species, C. caroliniana having three varietiesvar. caroliniana, var. pulcherrima, and var. flavida. Only var. caroliniana and var. pulcherrima are found in the United States. (Orgaard 1991). Cabomba is popular for use in aquariums (Leslie 1986; Orgaard 1991) and a simple internet search using cabomba or fanwort as a search term reveals the variety of plants that can be purchased from aquarium stores ( www.aquahobby.com www.petsolutions.com www.pondplants.com ). It is common for aquarium dealers to select and/or breed plants that are desirable for sale to aquarium enthusiasts. Typically, plants should grow easily, tolerate a wide range of conditions, and be aesthetically pleasing. Several cultivars derived from C. caroliniana have been developed by the aquarium trade, and include rosifolia, multipartite, paucipartita and Silbergne (Mackey and Swarbrick 1997; Orgaard 1991; Wain et al. 1983). Discarded aquarium plants are a common source for the introduction of aquatic plants into natural waterways that may spread and become invasive (McLane 1969). Species of Hydrilla, 1 Don Les (University of Connecticut), and Brian Husband (Guelph University Canada), personal communication (2007). 16

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Myriophyllum, Egeria, and Elodea have reportedly been spread by this route (Cook and Lnd 1982; Cook and Urmi-Knig 1984; Cook and Urmi-Knig 1985). Cabomba in Australia, China, Japan, and other locations around the world likely came from discarded aquarium plants and it is considered invasive in these countries (Mackey 1996; Yu et al. 2004b). However, there is some question as to what role discarded aquarium plants have played in the increased distribution of cabomba into the northern portions of North America. Reports from the early 1900s list cabomba as being used for fishponds and aquariums in New England, and by the 1960s the species was reported as being invasive (Les and Mehrhoff 1999). The fact that cabomba is very popular for use in aquariums makes it difficult to predict whether cabomba introduced into northern states is from discarded aquarium plants, natural range extension, or introductions of plants from the southeast. It is likely that multiple routes of introduction have occurred in the northern portions of North America. Distribution and Morphology Members of the genus Cabomba are native in tropical to subtropical regions and only C. caroliniana and its varieties have temperature tolerances that allow wide distribution in North America (Leslie 1986; Orgaard 1991). Cabomba is considered native to the southeastern United States and is increasingly found in other parts of North America. Cabomba is also increasingly being reported in other locations around the world (Leslie 1986; Mackey 1996; Orgaard 1991; Schooler et al. 2006; Yu et al. 2004a, b) where it has displayed properties consistent with an invasive species (Cao et al. 2006; Crawford et al. 2001; Mackey 1996; Schardt and Nall 1988; Schooler et al. 2006; Yu et al. 2004b; Zhang et al. 2003). Cabomba has been present in the northern regions of the United States since the early 1900s, but the spread in these northern regions greatly increased in the early 1990s and it is now frequently described as invasive (Hanlon 1990; Les and Mehroff 1999; Madsen 1994; Martin and Wain 1991; Riemer and Ilnicki 17

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1968). Monocultures of cabomba in Canada have reached densities of up to 200 plants m -2 (Nol 2004). In Australia cabomba was first identified in 1967, became naturalized by the 1980s, spread rapidly, and was considered a major pest by the 1990s (Mackey 1996). Cabomba was also found in the northwest United States, China, Canada, England, Netherlands, Belgium, and Hungary during the 1990s and all of these introduced populations of cabomba are described as C. caroliniana (Christy and Systma 1994a, b; Denys et al. 2003; Hrusa et al. 2002; Mackey 1996; Nol 2004; Oldham 1999; Preston and Croft 1997; Stace 1997; Stetk 2004; Van der Velde et al. 2002). The increasing occurrence of cabomba in North America and around the world suggests that populations may have been released from a similar source, such as the aquarium trade. Invasive cabomba in the northern tier of the U.S. tends to be bright green and will hereafter be referred to as green cabomba (Leslie 1986; Mackey 1996). Green cabomba can be found both in the southeast and northern regions of the U.S., but its origin remains uncertain. In contrast, plants typical of the southeast U.S. are red/purple (hereafter referred to as red cabomba) and have not been reported in the northern U.S. Aquarium plants purchased in Florida are mostly green in color with varied levels or red/purple color found at the apical sections of the plant and will hereafter be referred to as aquarium cabomba. The difference in the coloration of cabomba has been the source of much speculation. There is some question regarding the effect of environmental conditions on color (Hanlon 1990; Leslie 1986; Martin and Wain 1991; Orgaard 1991; Wain et al. 1983). Cold temperatures reportedly lead to green color, while warm temperatures result in red/purple cabomba (Leslie 1986; Orgaard 1991). Orgaard (1991) observed that both light levels and temperature influenced plant color, but color reverted when plants were grown in their original conditions. In contrast, the aquarium industry reports that cold water causes red color and warm water results in green 18

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color (Leslie 1986). There has been speculation that these color differences have been induced by the aquarium industry (Hanlon 1990) and it is unclear whether the novel phenotype of green cabomba results from genetic or environmental changes (Crawford et al. 2001, Hanlon 1990, Les and Mehrhoff 1999; Mackey 1996; Schooler et al. 2006). Much of the published research does not indicate whether the cabomba being evaluated is red or green. Therefore, it is difficult to determine if studies evaluating tolerances to environmental variables or management studies are valid for all populations of cabomba. Wain et al. (1983) compared the isozymes of three types of cabomba found in the U.S. to determine if species separation was necessary. These types were identified as C. caroliniana var. caroliniana, C. caroliniana var. mulitpartita (described as an aquarium variety), and C. pulcherrima. Electrophoretic separation of various alleles indicated no differences among the three types. However, C. caroliniana var. multipartita was collected from Florida as opposed to the northern regions of the U.S. and might not be representative of the green cabomba that is of current concern in North America. This study was done largely before more powerful genetic analysis became available to separate very closely related, yet unique, species. However, this work did identify C. caroliniana var. multipartita as a variety selected for and bred by the aquarium industry and suggested there should be no differences in management strategies for these color variants. Species of cabomba may also be separated by color differences in the flowers. C. caroliniana var. pulcherrima is commonly reported as having a purple tinted flower in contrast to C. caroliniana var. caroliniana that has yellow to purple tinted flowers. This overlap in flower color calls into question whether the two varieties are separate (Orgaard 1991). 19

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Reproduction Cabomba spreads primarily through vegetative means (Leslie 1986; Mackey 1996; Orgaard 1991; Tarver 1976; Tarver and Sanders 1977). Vegetative growth is possible from a segment of plant with a single node and pair of leaves, but segments with more leaf nodes have higher survival rates (Tarver and Sanders 1977). The primary means of pollination is from insects, though some self-pollination is possible due to wave action (Tarver and Sanders 1977). Flowers of cabomba follow a two-day cycle in which pollination is possible and then flowers close and are pulled below the surface of the water where seeds are formed (Moseley et al. 1984; Osborn et al. 1991; Schneider and Jeter 1982). Seed viability in C. caroliniana and its varieties was lower than that of other species of cabomba and has been attributed to the highly variable ploidy levels found in C. caroliniana (Table 1-1). Also, cabomba found in Australia has not been observed producing seeds (Mackey 1996). Riemer and Ilnicki (1968) found no viable seeds from cabomba cultured in New Jersey and suggested seeds may not be used for reproduction by this population of cabomba. It is unclear which cabomba population was used, but the cabomba was collected in New Jersey, which suggests it may have been green cabomba. Growth Conditions Studies indicate that a pH range of 4-6 is optimal for cabomba growth and that pH above 7 inhibits growth (Riemer 1965; Tarver 1976). Riemer (1965) and Tarver (1976) reported that plants begin to defoliate from the base up when cultured in water of pH 7 or higher and most leaves below the very apical sections are lost. Riemer (1965) evaluated the effect of aeration, calcium concentration, and osmotic pressure on the growth of cabomba and found that aeration, 0.0001M CaCl 2 and lower osmotic pressure (0.149ATM) produced the best growth of cabomba. Like high pH, high levels of calcium (.001 M and higher) caused growth inhibition and defoliation (Mackey 1996, Riemer 1965). 20

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Species in the genus Cabomba are found throughout tropical climates and many species are intolerant of colder climates (Leslie 1986; Orgaard 1991). C. caroliniana is the only species native to temperate regions but green cabomba has colonized the northern portions of the U.S. and Canada and has developed overwintering strategies to survive the much colder temperatures associated with these regions. Plants fragment in late fall and form turion-like structures at the apical tip, and green foliage is retained throughout winter (Riemer and Ilnicki 1968). Upon the return of warmer weather, the surviving fragments resume vegetative growth. Management Cabomba can grow to nuisance levels in the southeast U.S. but generally is not a management problem. Limited and contradictory information is published regarding cabomba response to herbicide applications. Cabomba in the northern U.S. and Canada has been of particular concern to lake and river managers due to poor performance of traditional management techniques and the lack of control methods available to stop the spread of this species. Nelson et al. (2002) reported that fluridone at 5 g a.i. L -1 or greater concentrations reduced biomass compared to untreated controls, but growth reduction of > 80% required application of 20 g a.i. L -1 Mackey (1996), however, reported that field trials of fluridone in Australia had little or no effect on cabomba growth at equivalent concentrations evaluated by Nelson et al. (2002). Cabomba populations rapidly expand in the Midwestern U.S. following low-rate fluridone treatments 2 targeting Eurasian watermilfoil (Myriophyllum spicatum L.). Higher rates of fluridone, 20 g a.i. L -1 have been used in whole lake treatments in the midwestern U.S. to reduce cabomba when it is distributed lake wide. These treatments are limited in use because fluridone at these concentrations has reduced plant selectivity, require 2 Personal observation. Brett Bultemeier. 2005. 21

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longer exposures, and has limited use when cabomba occurs in isolated locations in the littoral zone 3 Tolerance of cabomba to herbicides like 2,4-D and triclopyr (Nelson et al. 2001) is not understood since other aquatic dicotyledonouse.g. Myriophyllum spp.are effectively controlled with these herbicides. Aquatic managers report that diquat, a broad-spectrum herbicide, has minimal activity on green cabomba. There has been some success in Australia with the use of 2,4-D ester at concentrations of 10,000 g a.i. L -1 to control cabomba infestations (Mackey 1996). Endothall (amine salt) also reportedly reduces biomass of cabomba (Madesn 2000; Moore1991) but hydrogen peroxide had limited activity (Kay et al. 1984). Drawdown techniques can effectively control cabomba, but have limited use in much of the U.S. where infestations are currently found (Mackey 1996; Sanders 1979; Schooler et al. 2006). The only known effective biological control for cabomba is grass carp (Ctenopharyngodon idella Val.), and a search for other biological agents is in the early stages (Mackey 1996; Schooler et al. 2006). There are situations where cabomba is considered beneficial. Cabomba is popular in the aquarium industry due to its ornamental qualities (Hanlon 1999; Leslie 1986; Mackey 1996; Schooler et al. 2006). Nakai et al. (1999) suggested that cabomba has allelopathic effects and could be utilized to suppress blue-green algae and it has also been evaluated as a potential bioremediation plant to remove harmful toxins and metals, such as lead, from the environment (Yaowakhan et al. 2005). Objectives The differences in color and invasive growth between red and green cabomba suggest that further efforts to characterize the response of these populations to management and various 3 Personal communication. Tyler Koschnick. 2008. 22

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environmental parameters is needed. The invasive tendencies of green cabomba have created heightened concerns due to the paucity of proven control methods. The objectives of this research were to determine if red, green, and aquarium cabomba respond differently to herbicides, pH, temperature, and culture conditions to determine if a new taxonomic treatment is needed and to find potential herbicides to control noxious populations of cabomba. 23

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Table 1-1. Species in the genus Cabomba, their proposed ploidy levels, and native ranges a Species Ploidy (basic number x=13) Native Range C. aquatica Tetraploid (4x) Northeast South America C. palaeformis Diploid (2x) Southern Mexico and northern Latin America C. furcata Tetraploid (4x) North and central South America C. haynesii Unknown Latin America, Cuba, northern South America C. caroliniana var. caroliniana Hexaploid (6x), octoploid (8x) Southeast United States, southeast South America C. caroliniana var. pulcherrima Hexaploid (6x) North Florida, south Georgia and South Carolina C. caroliniana var. flavida Triploid (3x), hexaploid (6x) Southeast South America a Orgaard, Marian. 1991. The genus Cabomba (Cabombaceae) A taxonomic study. Nord. J. Bot. 11:179-203. 24

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Figure 1-1. Line drawing of a submersed cabomba leaf showing the fan shape which leads to the common name fanwort. From Don E. Eyles, A Guide and Key to the Aquatic Plants of the Southeastern United States (Washington D.C.:U.S. Government Printing Office, 1944). 25

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CHAPTER 2 THE EFFECT OF AQUATIC HERBICIDES ON THREE POPULATIONS OF CABOMBA Introduction Cabomba is a submersed aquatic plant that is native to the southeastern United States and has recently been found in states throughout the northeast, midwest and northwest. Cabomba is also increasingly being found in Australia, Canada, China, Japan, and portions of Europe (Cao et al. 2006; Crawford et al. 2001; Leslie 1986; Mackey 1996; Schardt and Nall 1988; Schooler et al. 2006; Yu et al. 2004a, b; Zhang et al. 2003). The expansion of cabomba in North America and around the world has invasive characteristics, and cabomba is now considered an exotic weed in many of these sites. The range expansion throughout the world has lead to a reevaluation of the taxonomy because these introduced populations have different characteristics than native populations in the southeastern U.S. Color of the species ranges from green to purple and is thought to be a function of temperature and light levels (Hanlon 1990; Leslie 1986; Martin and Wain 1991; Orgaard 1991; Wain et al. 1983). Plants in the northern U.S. are bright green (hereafter green cabomba), whereas plants from the southeastern U.S. are red/purple (hereafter red cabomba). Plants sold through the aquarium industry in Florida (hereafter aquarium cabomba) are mostly green, with purple coloration on the apical tips of leaves. Green cabomba in the northern U.S. and around the world is reportedly difficult to manage (Mackey 1996) and research suggests that this cabomba is tolerant to most registered herbicides. There are few reports on the response of cabomba to registered aquatic herbicides and these reports do not specify which cabomba (green, red, etc.) was being studied. Therefore, an evaluation of herbicide efficacy on these three populations is necessary to determine which herbicides are effective, and whether populations respond similarly to herbicide treatments. 26

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The effects of herbicides are typically evaluated initially in mesocosms to identify those herbicides most likely to succeed in field trials. These evaluations typically involve a large input of labor and space due to the number of experimental units needed to test a number of herbicides (Getsinger et al. 1994; Gray et al. 2007; Nelson et al. 1998). Microcosm testing of herbicides is generally more efficient, reduces labor, while still providing an initial screen of potentially active compounds. These smaller scale evaluations require less plant material and less time to identify herbicides unlikely to be effective in mesocosm studies. The response of aquatic plants to herbicides can be assessed by measuring oxygen evolution over time to calculate net photosynthetic rates. Also, the response of submersed aquatic plants such as Hydrilla verticillata (L.f.) Royle (hydrilla) and Myriophyllum spicatum L. (Eurasian watermilfoil) to herbicides and plant growth regulators can be assessed by monitoring in situ photosynthetic rates (Netherland and Getsinger 1995; Netherland and Lembi 1992; Selim et al. 1989). Decreased photosynthetic rates over a short term suggest that a herbicide has potential for control and should be further evaluated. The lack of viable management options and the expansion of cabomba over the past decade suggests that research is necessary to determine if populations of cabomba vary in response to herbicides. The objectives of these studies was to compare the photosynthetic response of three populations of cabomba to herbicides and to determine if photosynthetic measurements can be used as an initial screen for herbicides. Materials and Methods Growth Chamber Static exposures Experiments were conducted at the University of Florida Center for Aquatic and Invasive Plants (CAIP) in Gainesville, FL. The first trials were conducted from July/October 2006, and 27

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were repeated from October 2006 through January 2007. Red cabomba was collected from White Lake (Suwannee County, FL) and Ledwith Lake (Alachua County, FL). Aquarium cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery (Lake City, FL); and green cabomba was collected from the St. Joseph River (Elkhart County, IN) and Crooked Lake (Kalamazoo, MI). Plant cultures were established by planting 15 cm apical segments in 2 L plastic pots filled with topsoil amended with Osmocote plus (15-9-12) 1 at a rate of 1g kg -1 of soil. Plants were maintained in 900 L tanks under 50% shade cloth. Hydrilla, Eurasian watermilfoil and variable milfoil (Myriophyllum heterophyllum Michx.) were used for comparative testing and all were collected from cultures maintained at the CAIP. Apical sections (4 cm long) of each of the three cabomba populations were excised and placed in 10 L plastic pans containing well water for 6 h. This 6 h recovery period was suggested by MacDonald (2007) 2 because ion leakage by hydrilla is significant only during the first 2-4 h after excision. After 6 h, the apical sections were placed into 350 ml plastic cups (1 section per cup) containing 5 mM MES buffer in well water amended with Hoaglands solution (2.5% v/v) for a total volume of 345 ml per cup. MES buffer was used to maintain a pH of 6 based on preliminary studies where pH was stabilized with no apparent adverse effect on photosynthesis (Appendix). Hydrilla, Eurasian watermilfoil, and variable milfoil were included in these studies to provide a confirmation that herbicide susceptibility could be adequately measured by a reduction in net photosynthetic rates even for herbicides not directly effecting photosynthesis. The differential susceptibility of these plants to selected herbicides is known. Variable milfoil was placed in containers that had the same culture solution as cabomba, but 1 The Scotts Company. Marysville, OH. 43041 2 MacDonald, G.E. Unpublished report. 2007 28

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hydrilla and Eurasian watermilfoil treatments had NaHCO 3 added to the culture solution to achieve a final concentration of 2.4 mM to maintain a pH of ~8. Cups containing plants were placed in growth chambers 3 with a temperature of 27C, 14:10 h (day:night) photoperiod and a light intensity of 360-400 mol m -2 s -1 Plants were treated with herbicides at near or above the maximum-labeled use rate (Appendix). The following herbicide concentrations were tested: 2,4-D (amine) 4400, carfentrazone 400, diquat 375, elemental copper 1000, endothall (amine salt) 2300, endothall (dipotasium salt) 3000, flumioxazin 400, quinclorac 400, triclopyr 4900, and a combination of diquat 375 and elemental copper 1000 g a.i. (active ingredient) L -1 Plants were exposed to treatments for 144 h. Net photosynthesis methods were adapted from Netherland and Lembi (1992) and Netherland and Getsinger (1995). Dissolved oxygen measurements were taken every 24 h for 144 h using a dissolved oxygen meter 4 (.01 mg/L). Initial oxygen measurements were taken from biological oxygen demand (BOD) bottles 5 containing well water, Hoaglands solution (2.5% v/v), and 45 M HCl. Hydrochloric acid was added to BOD bottles so pH would be equal to that of the culture solutions for the 1 h duration of the photosynthetic measurements. Plant tips were removed from the 350 ml plastic cups, rinsed twice in tap water, and placed individually in BOD bottles in the growth chamber for 1 h. A control BOD bottle (with the same solution as the other bottles but without plant material) was incubated to determine any oxygen increase or decrease in the absence of plant material. Oxygen content of the water in the BOD bottles was recorded at the end of the 1 h incubation period, and plants were weighed after 3 Percival model E-36L. Percival Scientific, Inc. Perry, IA. 50220. 4 Accumet Excel XL40 Dissolved Oxygen/BOD/OUR/SOUR Temperature Meter. Fisher Scientific. Pittsburg, PA. 15275. 5 Wheaton Science Products. Millville, NJ. 08332. 29

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being blotted between paper towels to remove any excess water in order to calculate net photosynthetic rates based upon fresh weight and time. This rate was determined by using the equation: photosynthetic rate = (final dissolved oxygen (g O 2 ) initial dissolved oxygen (g O 2 )) (freshweight (g) -1 ) (time (min) -1 ). The unit of measure is g O 2 g -1 (fresh weight) min -1 Plant tips were then placed back into the original treatment cups and measurements repeated every 24 h for 144 HAT (hours after treatment). BOD bottles were emptied and filled with fresh solution for each photosynthetic measurement. Net photosynthetic rates were standardized to percent of untreated controls in order to compare photosynthetic rates among the cabomba populations. Two separate analyses were performed to compare the response of the cabomba populations to herbicides. Linear interpolation was used to find an ET 50 (the estimated time required to reduce net photosynthesis to 50% of the untreated control) value for those herbicides that reduced net photosynthesis of green cabomba by at least 50% of the untreated control. Green cabomba was used as the standard for response because it is currently the most challenging to manage. Population comparisons were made at 24 and 144 HAT using percent of untreated control values for those herbicides that did not reduce green cabomba photosynthesis to 50% of the untreated control. All treatments used a completely randomized design with 4 replications. Data were analyzed using ANOVA (p 0.05) and a post-hoc Students pairwise t-test to analyze population differences for ET 50 estimates and percent of untreated control values. Data for quinclorac and copper trials were analyzed independently due to significant interaction by trial. Trials were pooled for all other herbicides. Data for endothall (amine salt), carfentrazone, triclopyr, and 2,4-D were log transformed to meet equal variance assumption of ANOVA. 30

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Twenty-four hour exposures A second experiment was conducted to test the effect of limiting exposure time on those herbicides (diquat, diquat + copper, endothall (amine salt), and flumioxazin) that in the static exposure trials reduced photosynthesis of green cabomba to 50% of the untreated control. The first trial was conducted in June/July 2007 and repeated July/August 2007. All three cabomba populations were exposed to concentrations of diquat, diquat + copper, endothall (amine salt) and flumioxazin at 375, 375 + 1000, 2300 and 400 g a.i. L -1 respectively. Methods were similar to those described in the static exposure studies, except plants were exposed to herbicides for 24 h. Plants were removed from treatment after 24 h, washed twice, and placed in cups containing growth media without herbicide. Growth media was replenished 72 h after plants were removed from treatment to minimize the effect of algal growth that occurred in static exposures. Dissolved oxygen measurements were taken every 24 h for 144 h. All treatments used a complete randomized design and had 4 replications. Data were analyzed using ANOVA (p 0.05) and a post-hoc Students pairwise t-test to analyze population differences in percent of untreated control values at 24, 72, and 144 HAT. There were no interactions by trial so data for both were pooled. Mesocosm Newly established plants A mesocosm experiment was performed to evaluate the response of three cabomba populations to selected herbicides on whole plants. This study was conducted at the CAIP during July 2007. Red cabomba was collected from White Lake (Suwannee County, FL), aquarium cabomba was obtained from Suwannee Laboratories (Lake City, FL), and green cabomba was collected from the St. Joseph River (Elkhart, IN). Cabomba was established in culture by planting 15 cm apical segments in 2 L plastic pots with top soil amended with 31

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Osmocote Plus 15-9-12 at a rate of 1g kg (soil) -1 Plants were grown in 900 L tanks (80 cm water depth) for 2 m before use in experiments. Apical segments (15 cm) were excised from culture plants and planted 3 cm deep in 10x10x12 cm (1L) pots filled with masonry sand amended with Osmocote Plus 15-9-12 fertilizer at a rate of 1g kg -1 of sand. They were then placed in 900 L cement tanks under 50% shade and allowed to grow for 3 wk before treatment. One pot of each population was then placed in 95 L HDPE (high-density polyethylene) tubs filled with well water amended with 10 ml of 28% HCl (muriatic acid) to achieve a pH of 6.0-6.5. Experimental treatments were conducted under 30% shade and herbicide concnetrations were: untreated control, carfentrazone 400, diquat 375 + copper 1000, endothall (amine salt) 1200, endothall (amine salt) 2300, endothall (dipotasium salt) 3000, flumioxazin 200, flumioxazin 400, and triclopyr 4900 g a.i. L -1 Planted pots were removed from 95 L treatment tanks after a 24 h exposure to herbicides, rinsed three times by full submersion in a 900 L tank with flowing water, and placed into a separate 900 L cement tank with flowing water. Flow was stopped after 24 h and 28% HCl was added to adjust water pH to 6.0.7, with additional adjustments every 3 d as necessary for 3 WAT (weeks after treatment). Plant material (shoots and roots) was harvested 3 WAT and dry weights were determined after drying for 48 h in an oven at 70C. All treatments used a complete randomized design with 4 replications. Data were analyzed using ANOVA (p 0.05) to assess differential response among cabomba populations. Means for each cabomba population were separated using Dunnetts test to identify those treatments significantly different from control plants. A post-hoc Students pairwise t-test was 32

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used to analyze population differences in percent of untreated control for each herbicide. Percent of untreated control values were log transformed in order to meet the assumptions of ANOVA. Mature plants A second mesocosm experiment was conducted to investigate the effects of herbicides on the response of mature, rooted green cabomba. This experiment was conducted at the CAIP in September 2007. Methods were similar to those in the newly established plant study but, only green cabomba was used in this experiment. Plants were established for 3 m and were fully rooted in 2 L plastic pots with top soil amended with Osmocote Plus 15-9-12 at a rate of 1g kg -1 Experimental treatments were applied under 30% shade and herbicide concentrations were: untreated control, carfentrazone 200, endothall (amine salt) 1200, flumioxazin 100, flumioxazin 400 and triclopyr 4900 at g a.i. L -1 Plants were exposed to herbicides for 24 h, then rinsed and moved to 900 L tanks under 50% shade. Plants were harvested 2 WAT, dried in a drying oven at 70C for 1 wk, and weighed. All treatments used a complete randomized design and had 3 replications. Data were analyzed using ANOVA (p 0.05) and Dunnetts test was used to determine those herbicides that reduced biomass compared to untreated controls. Results and Discussion Growth Chamber Static exposures The BOD bottles filled with only growth solutions had no change in oxygen levels during the 1 h photosynthetic period. The herbicides with known activity on hydrilla, Eurasian watermilfoil, or variable milfoil all reduced net photosynthesis to 50% of the untreated controls (Figure A-2 through A-13). No observed change in oxygen levels of culture solution and the reduction of photosynthesis by these herbicides known to be effective on the test plants (hydrilla, 33

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Eurasian watermilfoil, variable milfoil) suggests that this method of screening is valid for identifying potential aquatic herbicides. Ten herbicides or herbicide combinations were tested. Six (carfentrazone, copper, endothall (dipotasium salt), quinclorac, triclopyr, 2,4-D) failed to reduce net photosynthesis of green cabomba to 50% of untreated controls through 144 HAT (Table 2-1). Red and green cabomba had different levels of sensitivity to triclopyr, 2,4-D, and carfentrazone, with red cabomba being the most sensitive to each herbicide (Table 2-1). The response of aquarium cabomba differed from green cabomba when exposed to carfentrazone and 2,4-D, and differed from red cabomba when exposed to endothall (dipotasium salt) and carfentrazone (24 HAT). The largest photosynthetic difference among the cabomba populations occurred in response to carfentrazone. All three populations differed from one another 24 HAT when photosynthesis of red cabomba was zero, aquarium cabomba was 57% of the untreated control, and green cabomba was largely unaffected (Table 2-1). By 144 HAT, photosynthesis of both aquarium and red cabomba was reduced to almost zero, while the photosynthetic rate of green cabomba was still > 60% of the untreated controls. Four herbicides (diquat, diquat + copper, endothall (amine salt), and flumioxazin) reduced net photosynthesis of all three cabomba populations and the known sensitive species (hydrilla) below 50% of the untreated controls by 144 HAT (Table 2-2). Net photosynthetic response was different among the cabomba populations for each herbicide. Green cabomba was 1 to 4 times more tolerant to herbicides than red cabomba. The greatest difference between red and green cabomba was in response to flumioxazin. The ET 50 of green cabomba was 55 h, but the ET 50 of red cabomba was only 14 h. Aquarium cabomba differed from red cabomba in response to diquat, and differed from green cabomba in response to diquat + copper and flumioxazin (Table 34

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2-2). Net photosynthesis of all populations was reduced below 50%, with the exception of flumioxazin on green cabomba, at 48 HAT. This initial screen represents the best possible treatment scenario where herbicides maintain static exposures for 144 h. This is unlikely to happen in the field as herbicides will dilute, disperse and are broken down by various processes, so herbicides shown to be ineffective in these studies are less likely to be effective under field conditions. This initial screen eliminated six herbicides from further consideration, and showed that green cabomba was much more tolerant to herbicides than red cabomba. Twenty-four hour exposures The four herbicides that had the highest photosynthetic reduction in the static exposure studies were further evaluated under a 24 h exposure period. Diquat and the diquat + copper combination failed to reduce green and aquarium populations photosynthesis to < 50% of the untreated control (Table 2-3). However, net photosynthesis of red cabomba exposed to diquat + copper was reduced to < 50% of untreated controls. Green cabomba was the most tolerant and red cabomba the most sensitive population, similar to the results observed in the static exposure study. Diquat + copper initially reduced net photosynthesis of red cabomba to < 50% of untreated controls, but photosynthesis recovered to above 50% of untreated control by 144 HAT. Biomass of red cabomba exposed to diquat + copper was reduced > 50% by 144 HAT (Figure 2-1). Photosynthetic measurements were based upon change in oxygen production over time and per unit of fresh weight. This measurement does not account for any significant changes in biomass and may incorrectly lead to the conclusion that the treatment was not effective. Regrowth potential, or potential recovery from initial herbicide damage, could be determined if the experiments were conducted over a longer period. 35

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This study identified two herbicides (endothall (amine salt) and flumioxazin) that reduced photosynthesis of all cabomba populations to < 50% of untreated controls (Table 2-3). Flumioxazin caused the greatest photosynthetic reduction on all cabomba populations and maintained that reduction through 144 HAT. Endothall (amine salt) reduced photosynthesis of red and aquarium cabomba to < 50% and also maintained that reduction through 144 HAT. Similar to the response of red cabomba to diquat + copper, green cabomba photosynthesis recovered to above 50% of untreated control by 144 HAT. The fresh weight of green cabomba treated with endothall (amine salt) is presented in Figure 2-2. These results are similar to the fresh weight response of red cabomba to diquat + copper (Figure 2-1) and provide evidence for the potential recovery of the remaining plant material by showing the increase of photosynthetic rates by 144 HAT. Unlike static exposures, algal growth was not observed for the 24 h exposures so the recovery of plant material was not likely influenced by the photosynthesis of algae. The 24 h and 144 h static exposures highlight two considerations for using this method to screen herbicides. Photosynthetic measurements may be initially misleading, because significant reduction in biomass is not reflected in photosynthetic rates. However, when they are adjusted on a fresh weight basis, they can provide evidence of the potential recovery of remaining plant material. The second consideration for these experiments is what level of photosynthetic reduction correlates to the eventual goal of reducing biomass in field applications. It is possible that a 50% reduction is too restrictive. Depending on the management goal, this level will vary based on the herbicides used and the plant being managed. Carfentrazone was eliminated for further consideration on green cabomba when there may actually be potential for field activity. 36

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For example, in the initial exposures, carfentrazone reduced green cabomba photosynthesis to ~60% of the untreated control and reduced photosynthesis in the other populations to ~0. In summary, these two studies were able to identify two herbicides (endothall (amine salt) and flumioxazin) that should be evaluated further. Flumioxazin caused the greatest reduction in photosynthesis, suggesting high potential for activity in field applications. These studies also showed differential susceptibility to herbicides among the cabomba populations with green>aquarium>red in order of least to most susceptible. These studies also highlight that photosynthetic reduction is observed for herbicides, such as 2,4-D and triclopyr, that have no direct inhibition of photosynthesis. Mesocosm Newly established plants This study evaluated the effects of a 24 h exposure to six herbicide or herbicide combinations on newly established green, aquarium and red populations of cabomba. Carfentrazone, endothall (amine salt), and flumioxazin significantly reduced the biomass of all three cabomba populations (Table 2-4). In addition, diquat + copper and triclopyr reduced the biomass of only red cabomba. Endothall (dipotasium salt) did not significantly reduce the biomass of green, aquarium, or red cabomba compared to untreated controls. The growth chamber screens did not predict that carfentrazone would be active on green cabomba in mesocosm studies, since carfentrazone only reduced the photosynthetic rate of green cabomba to 60% of the untreated controls in growth chamber screens. The arbitrary value used to indicate susceptibility was set at 50% and may have been too restrictive a value, given the activity of carfentrazone in the mesocosm studies on green cabomba. The growth chamber studies of photosynthetic rates showed a high level of activity of carfentrazone on both aquarium and red cabomba that was confirmed in these mesocosm tests. Red cabomba was also significantly 37

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reduced by diquat + copper, which was predicted by the growth chamber studies; however, a significant reduction in response to triclopyr was not predicted. A limitation of these studies was the inconsistent growth of newly established plants. Some treated plants failed to root and floated out of pots, making it difficult in some cases to determine whether death was in response to the herbicide treatments or to failure to establish. This complication led to a study in which mature, established plants with well-developed roots were similarly exposed to herbicides. Nevertheless, the study on herbicide susceptibility of immature plants generally agreed with previous photosynthetic studies conducted in growth chambers and identified three compounds that had significant effects on cabomba biomass. Mature plants Mature green cabomba plants had a similar response to herbicide treatments as noted in the newly established plant study. Carfentrazone, endothall (amine salt), and flumioxazin all significantly reduced biomass (Figure 2-3). Carfentrazone reduced biomass > 60%, but the remaining biomass was green and viable, suggesting a high probability of regrowth. Endothall (amine salt) reduced biomass by 86% and defoliated much of the plant. The stems and apical leaf segments of green cabomba were green and viable, also suggesting high potential for regrowth. Flumioxazin provided the greatest reduction of biomass (> 96%); any remaining stem material was brittle and any remaining leaf material easily fell off the plant. Growth chamber and mesocosm studies identified flumioxazin as the herbicide with the highest potential for control of green cabomba. There are other examples in aquatic and terrestrial weeds where different populations of the same species respond differently to the same herbicide. Two biotypes of Alligatorweed (Alternanthera philoxeroides (Mart.) Griseb) (slender stem and broad stem) responded differently to quinclorac with the slender stem biotype more susceptible than the broad stem 38

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biotype (Kay 1992). Foes et al. (1998) reported that one biotype of common waterhemp (Amaranthus rudis Sauer) was tolerant to triazine and ALS (acetolactate synthase inhibitors) herbicides whereas the other biotype was susceptible. This differential tolerance in common waterhemp, however, may likely be due to heavy levels of selection pressure placed on this plant through years of extensive herbicide use. The differential response of cabomba populations is unique because they are different across several herbicides. Under the current taxonomic classification, the three populations of cabomba (green, red and aquarium) are identified as being the same species. Moreover, an earlier study by Wain et al. (1983) predicted that numerous cabomba biotypes would respond similarly to herbicide treatments, whereas these studies indicate numerous differences. The photosynthetic response of aquatic plants correlates well to other growth parameters, such as biomass and stem length. Netherland and Getsinger (1995) and Netherland and Lembi (1992) reported that photosynthetic differences of hydrilla and Eurasian watermilfoil in response to various fluridone rates were similar to differences in dry weight up to 90 DAT. These studies and the results of our analysis suggest that the use of photosynthetic data can be useful in the early stages of herbicide screening by identifying herbicides with the greatest activity, which would guide future studies. In summary, these studies identified differential responses among the three cabomba populations after exposure to herbicides. Green cabomba was the most tolerant and red cabomba the most susceptible to herbicide treatments. These screens identified three herbicides (carfentrazone, endothall (amine salt), and flumioxazin) that might provide significant control of green cabomba, with flumioxazin having the greatest potential for control. However, flumioxazin is still an experimental use product and is not registered for use in aquatics, while 39

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endothall (amine salt) can be toxic to fish. Photosynthetic studies, combined with mesocosm data can be utilized to quickly and effectively identify herbicides that warrant further testing. 40

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Table 2-1. Photosynthetic response of three cabomba populations, expressed as percent of untreated control, following a static 144 h exposure to selected herbicides. Herbicide Concentration g a.i. L -1 Population Trial a 24 HAT b 144 HAT Carfentrazone* 400 Green 1&2 86 c a d 69 a Aquarium 57 b 3 b Red 0 c e 0 b Copper 1000 Green 1 84ab 60 n.s. f Aquarium 120 a 38 n.s. Red 59 b 35 n.s. Green 2 82 n.s. 87 n.s. Aquarium 92 n.s. 88.n.s. Red 72 n.s. 84 n.s. Endothall (dipotasium salt) 3000 Green 1&2 94 n.s. 85 ab Aquarium 115 n.s. 114 a Red 109 n.s. 79 b Quinclorac 400 Green 1 89 n.s. 75 n.s. Aquarium 101 n.s. 76 n.s. Red 100 n.s. 82 n.s. Green 2 92 n.s. 84 n.s. Aquarium 92 n.s. 90 n.s. Red 103 n.s. 98 n.s. Triclopyr* 4907 Green 1&2 89 n.s. 115 a Aquarium 98 n.s. 95 ab Red 90 n.s. 76 b 2,4-D* 4423 Green 1&2 116 a 126 a Aquarium 87 b 93 b Red 85 b 86 b a each trial n=4. b HAT= hours after treatment. c values are the mean percent of untreated control plants derived from net photosynthetic rates. d results of pairwise Students t-test at p=0.05 to compare population response within a treatment, populations not connected by the same letter within herbicide, trial and exposure are different. e negative values represents net negative photosynthetic rate (net respiration). f n.s. = not significantly different according to ANOVA (p=0.05). *analysis was performed on log transformed data, but shown values are non transformed 41

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Table 2-2. Estimated time to reduce photosynthesis of three cabomba populations to 50% of an untreated control in response to a static 144 h exposure to selected herbicides. Herbicide Concentration g a.i. L -1 Population Trial (n=4) ET 50 a Diquat 375 Green 1&2 38 a b Aquarium 35 a Red 17 b Diquat + copper 375 + 1000 Green 1&2 29 a Aquarium 18 b Red 12 b Endothall (amine salt)* 2315 Green 1&2 13 a Aquarium 11 ab Red 10 b Flumioxazin 400 Green 1&2 55 a Aquarium 16 b Red 14 b a ET 50 represents the estimated time (in hours) necessary to reduce net photosynthetic rate by 50% compared to an untreated control. b populations not connected by the same letter within a herbicide are significantly different (Students pairwise t-test (p0.05). *analysis was performed on log transformed data, but the values shown are not transformed. Table 2-3. Photosynthetic response of cabomba populations, expressed as percent of untreated control, following a 24 h exposure to selected herbicides. Herbicide Concentration g a.i. L -1 Population a 24 HAT b 72 HAT 144 HAT Diquat 375 Green 85 c n.s. d 108 a e 106 a Aquarium 97 n.s. 90 b 98 ab Red 83 n.s. 93 b 89 b Diquat + copper 375 + 1000 Green 74 a 83 a 93 a Aquarium 75 a 82 a 88 a Red 13 b 42 b 52 b Endothall (amine salt) 2315 Green 4 n.s. 26 a 75 a Aquarium -3 n.s. f 10 b 5 b Red 5 n.s. 11 b 6 b Flumioxazin 400 Green 34 ab 2 n.s. 0 n.s. Aquarium 48 a 0 n.s. 0 n.s. Red 15 b 0 n.s. 0 n.s. a n=8. b HAT= hours after treatment. c mean percent of untreated control derived from net photosynthetic rates. d n.s. = not significantly different after ANOVA p=0.05. e populations not connected by the same letter within herbicide and exposure are significantly different (Students pairwise t-test (p0.05)). f negative value denotes net negative photosynthetic rate (net respiration). 42

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Table 2-4. Response of three newly established cabomba populations after 24 h exposure to selected herbicides. Herbicide Concentration Population (n=4) Percent of untreated control (Dry weight) Endothall (dipotasium salt) 3000 Green 113 n.s. Aquarium 111 n.s. Red 87 n.s. Diquat + copper 375 + 1000 Green 139 a a Aquarium 61 b Red* 38 b Triclopyr 4900 Green 127 a Aquarium 61 b Red* 43 b Endothall (amine salt) 1200 Green* 21 n.s. Aquarium* 13 n.s. Red* 7 n.s. Endothall (amine salt) 2300 Green* 13 a Aquarium* 5 b Red* 0 b Carfentrazone 400 Green* 6 n.s. Aquarium* 3 n.s. Red* 0 n.s. Flumioxazin 100 Green* 0 n.s. Aquarium* 0 n.s. Red* 9 n.s. Flumioxazin 400 Green* 0 n.s. Aquarium* 7 n.s. Red* 0 n.s. asterisks denote treatments that were significantly different from control after Dunnetts analysis (p=0.05). n.s. not significantly different at p=0.05 a populations not connected by the same letter within a herbicide and rate are not considered significantly different according to Students pairwise t-test (p=0.05). 43

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44 Figure 2-1. Weight response of red cabomba in response to 24 h exposure to diquat + copper 375 + 1000 g a.i. L. -1 Symbols represent mean 95% confidence interval. Hours After Treatment 020406080100120140160 Fresh weight (grams) 0.10.20.30.40.50.60.70.8 untreated control diquat+copper n=8 Hours after Exposure 020406080100120140160 Fresh weight (grams) 0.00.20.40.60.81.01.2 untreated control endothall (amine salt) n=8 Figure 2-2. Weight response of green cabomba in response to 24 h exposure to endothall (amine salt) 2300 g a.i. L. -1 Symbols represent mean 95% confidence interval.

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ControlTriclopyrCarfentrazoneEndothallFlumioxazinFlumioxazin` Dry Weight (grams) 0246810121416 ****n=3 Figure 2-3. Response of mature green cabomba plants 2 WAT to a 24 h exposure to selected herbicides. Bars represent mean standard error. Rates for compounds are 0, 4900, 200, 1200, 100 and 400 g a.i. L -1 respectively. Asterisks represent those rates that are significantly different from control plants based on a Dunnetts analysis (p=0.05). 45

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CHAPTER 3 THE EFFECT OF PH AND TEMPERATURE ON PHOTOSYNTHESIS OF THREE CABOMBA POPULATIONS Introduction Aquatic plants are adapted to grow within a specific range of environmental conditions. Although cabomba (Cabomba caroliniana) is reportedly invasive in the northeast, midwest, and northwest portions of the United States, and is considered an exotic invasive in Australia, China, Japan, and portions of Europe (Cao et al. 2006; Crawford et al. 2001; Leslie 1986; Mackey 1996; Schardt and Nall 1988; Schooler et al. 2006; Yu et al. 2004a, b; Zhang et al. 2003), relatively little is known about the physiological responses to differing environmental conditions of cabomba populations. For instance, temperature and pH can have a significant effect on aquatic plant growth and physiology (Sculthorpe 1985). An improved understanding of the photosynthetic responses of differing cabomba populations to temperature and pH will provide additional knowledge on the biology of cabomba. Eurasian watermilfoil (Myriophyllum spicatum L.) colonizes waters that are neutral to alkaline (Madsen 1998), while giant salvinia (Salvinia molesta Mitchell) and water hyacinth (Eichornia crassipes (Mart.) Solms) reportedly grow best in slightly acidic to neutral pH water (Haller and Sutton 1973; Owens et al. 2005). The genus Cabomba comprises species that are common to acidic waters (Leslie 1986; Mackey 1996; Orgaard 1991; Tarver 1976). Surveys have generally found cabomba in waters at pH 5-7, and the species is rarely found in waters with pH > 8 (Mackey 1996; Tarver 1976; Yu et al. 2004b). Temperature can also limit the distribution of aquatic plants (Sculthorpe 1985). The genus Cabomba is largely distributed throughout tropical and subtropical climates, and many species are limited to these regions due to an intolerance of colder temperatures (Leslie 1986; Orgaard 1991). Cabomba caroliniana is the most tolerant to colder climates, but is still reportedly 46

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limited to geographic areas that do not experience long-term freezes (Leslie 1986). However, populations of cabomba currently identified as C. caroliniana (for my studies identified as green cabomba) are colonizing the midwest, northeast and northwest U.S. and Canada, where long-term freezes during the winter are common (Les and Mehrhoff 1999; Mackey 1996; Orgaard 1991; Riemer and Ilnicki 1968; Schooler et al. 2006). Cabomba was observed overwintering in a vegetative form in New Jersey, with apical segments loosely buried in the soil, and was able to survive long periods of cold temperatures (Riemer and Illnicki 1968). Cabomba reportedly has a color response to temperature. Warm temperatures induce red coloration, while colder temperatures result in green coloration (Leslie 1986; Orgaard 1991). However, this coloration pattern is contradicted by aquarium growers who report growth in cold and warm water corresponds to red and green coloration, respectively (Leslie 1986). The current distribution of cabomba supports the contention that cold water causes green coloration, because green cabomba is found in the northern U.S. and red cabomba in the southeastern U.S. Green cabomba is unique because the rest of the Cabomba genus is tropical to subtropical in origin and the invasive growth of green cabomba in cold climates does not agree with this distribution. Based on the wide geographical distribution of cabomba and the phenotypic differences of the three populations (green, aquarium, and red), experiments were conducted to determine if differences exist in photosynthetic response among the three populations to a range of pH and to determine if photosynthetic rates differ among the three populations in response to a range of temperatures. Current taxonomic classifications identify all three populations as the same species, so any differences in response to these conditions could potentially contradict the current taxonomy. 47

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Materials and Methods pH Experiments were conducted in growth chambers at the University of Florida Center for Aquatic and Invasive Plants (CAIP) in Gainesville, FL, in May/June 2007 and were repeated in June/August 2007. Red cabomba was collected from White Lake (Suwannee County, FL), aquarium cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery (Lake City, FL), and green cabomba was collected from the St. Joseph River (Elkhart, IN). Plant cultures were established by planting 15 cm apical segments in 2 L plastic pots filled with top soil amended with Osmocote Plus 15-9-12 at a rate of 1g kg (soil) -1 Plants were maintained in 900 L tanks (80 cm water depth) under 50% shade cloth Apical sections (4 cm) of each population were excised from cultures and allowed to acclimate for at least 6 h prior to exposure to experimental treatments. Experiments were conducted in growth chambers at a temperature of 27C, a 14:10 h day:night photoperiod and a light intensity of 360-400 mol m -2 s -1 After 6 h, apical sections were placed in 350 ml plastic cups (1 section per cup) containing MES buffer, HCl, or NaHCO 3 in well water amended with Hoaglands solution (2.5% v/v) for a total volume of 345 ml per cup. MES buffer was added to achieve a final concentration of 5 mM in solution and HCl at a final concentration of 95M in solution was added to maintain pH 5.0. For pH 6, MES buffer alone was added to achieve a final concentration of 5 mM in solution. MES buffer was added to achieve a final concentration of 2.5 mM in solution, and NaHCO 3 at a final concentration of 2.4 mM in solution were added to maintain pH 7.0. NaHCO 3 was added to achieve a final concentration of 2.4 mM in solution to maintain pH 8.0. Solution pH was measured daily and 28% HCl was added where appropriate to maintain the desired pH of 5, 6, 7, or 8. The rates of MES buffer used in this experiment were 48

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determined in a preliminary study. The rates used provided consistent control of pH with no apparent effect on photosynthesis (Appendix). Solutions were replaced every 72 h. Dissolved oxygen was measured 72 and 120 HAT (hours after treatment) with a dissolved oxygen meter to determine net photosynthetic rates. Initial oxygen measurements were taken from biological oxygen demand (BOD) bottles containing the same pH as treatment containers and Hoaglands solution (2.5% v/v). The pH in BOD bottles was maintained only for the short duration of oxygen measurement and thus the method of pH control was slightly different from the plastic cup culture solutions. Concentrations of 120 and 90 M HCl in final solution were added to maintain pH 5 and 6 respectively in the BOD bottles. For pH 7, 20 M concentration of HCl in final solution was added. For pH 8, NaHCO3 was added to achieve a final concentration of 2.4 mM in solution. Plant tips were removed from culture treatments, rinsed twice in tap water, and placed in BOD bottles. The BOD bottles were then placed in the growth chamber and incubated for 60 min. Plants were then removed, oxygen levels were measured and fresh weights were recorded after plant material was blotted between paper towels to remove excess water. Net photosynthetic rate was determined by the following equation: photosynthetic rate = (final dissolved oxygen (g O 2 ) initial dissolved oxygen (g O 2 )) (freshweight -1 g) (time -1 (min)) and expressed as g O 2 g -1 (fresh weight) min -1 Plant tips were placed back in treatment containers after measurement of dissolved oxygen. BOD bottles were emptied and refilled with fresh solution for each reading. All treatments used a completely randomized design that had 4 replications. Data were analyzed using ANOVA (p 0.05) to assess affect of exposure 72 and 120 HAT. Analysis did not indicate that exposure was a main effect, nor was there any interaction with population and exposure, or pH and exposure. Exposures were combined and treated as a random effect. There 49

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was no trial interaction, so data from both were pooled. Overall replication after pooling was n=16. A quadratic model was then regressed for each population with the following equation: y= b 0 + b 1 *pH + b 2 *pH 2 where y is net photosynthetic rate. From this model, a MAX (maximum photosynthesis) value, which represents the pH at which maximum photosynthesis occurs for each population, was calculated for each population using the equation: MAX= -b 1 / (2*b 2 ). The MAX value for each population was compared using a full model dummy variable technique (Neter et al. 1990). Temperature Temperature studies were conducted using methods similar to the pH study. Trial 1 was conducted in July/August 2007 and repeated in August/September 2007. Plant tips were placed in the growth chamber with an initial temperature of 20C. Plants were slowly acclimated over the next 72 h to changes in temperature with final temperatures of 8, 16, 24, or 32C. For 8 and 32C treatments, temperature was changed 4C every 24 h. For 16 and 24C treatments, temperature was changed 1.33C every 24 h. Plants were maintained at the final temperature for 24 h before the first photosynthetic measurements. Dissolved oxygen was measured 24, 48 and 96 h after reaching target temperature and data were used to calculate the net photosynthetic rates (g O 2 minute -1 g -1 fresh weight). Initial oxygen measurements were taken from BOD bottles containing the same solution as the treatment containers, with 120 M HCl in final solution to amend solution pH to 6-7. BOD bottles and rinse solutions were placed in respective growth chambers for 24 h prior to use to ensure that the target temperature was reached in the bottles used for photosynthetic measurements. Plant tips were removed from treatment containers, rinsed twice in tap water, and placed in BOD bottles. BOD bottles were then placed in the growth chamber for 60 min then plants were removed, oxygen levels were measured and fresh weights were recorded. Net 50

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photosynthetic rate was determined using the previously described equation. Plant tips were then returned to the treatment cups and BOD bottles were emptied and filled with fresh solution before each measurement of O 2 All treatments used a completely randomized design and had 4 replications. Data were analyzed using ANOVA (p 0.05) to assess effect of exposure (24, 48 and 96 HAT). Analysis did not indicate that exposure was a main effect, nor was there any interaction with population and exposure or temperature and exposure. Exposures were combined and treated as a random effect. There was no trial interaction so data from both studies were pooled. Overall replication after pooling was n=24. A post-hoc Students pairwise t-test was used to analyze population differences in net photosynthetic rate at each temperature. Results and Discussion pH The quadratic equation, y= b 0 + b 1 *pH +b 2 *pH 2 was used to regress each population and all parameters were deemed significant (Table 3-1). All model residuals were acceptable and randomly scattered around zero, with no apparent trends. All populations followed a similar trend, where photosynthesis increased from pH 5 to a peak (MAX), and then declined as pH approached 8 (Figure 3-1). For example, photosynthetic rates of green cabomba begin to increase from pH 5 to a MAX of 6.2 (Table 3-1), after which the photosynthetic rate decreased (Figure 3-1 A). The MAX values for each population were significantly different (Table 3-1), with an increase from aquarium (5.9) to green (6.2) to red (6.5). A comparison of MAX was necessary, given the inherent differences in photosynthetic values across the entire range of pH (Figure 3-2). These differences seem minor because the change in MAX among the populations is only 0.3 pH units. However, pH is based on a log scale, so these differences can have significant impacts 51

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on plant growth. The photosynthetic rate of all populations declined as pH approached 8, and this decline corresponded to the loss of leaf material from the basal portions of plants. Reduced growth was noted in earlier studies where higher pH led to growth inhibition and defoliation of plants, and the suggested ideal pH range for C. caroliniana is 4 (Riemer 1965; Tarver 1976). Lakes with cabomba in Florida and China had an average pH of 6.5 and 6.2-7.5, respectively (Hoyer et al. 1996; Yu et al. 2004b). These field studies correlate well with our results, because maximum photosynthesis of the cabomba populations was 5.9-6.5. These results show that cabomba has higher photosynthetic rates at slightly acidic pHs and that differences occur among the three populations. Temperature Net photosynthesis of the three cabomba populations increased as temperature increased from 8 to 32C (Table 3-2). The greatest increase in photosynthesis over this temperature range occurred in red cabomba, and the least in aquarium cabomba. Aquarium cabomba had peak photosynthesis between 24-32C, but the peak photosynthesis of red and green cabomba could not be identified in the temperature range tested. Differences in net photosynthetic rates among the three populations of cabomba were observed at all four temperatures (Table 3-2). At 8C, red cabomba has a net photosynthetic rate of zero significantly less than green and aquarium cabomba (Table 3-2). These photosynthetic rates suggest that green and aquarium cabomba, though with greatly reduced photosynthetic rates, can survive cold temperatures, whereas red cabomba is less cold tolerant. The photosynthetic rate of red cabomba surpassed the photosynthetic rate of green cabomba at 16C and remained higher through 32C. A greater range of temperatures is necessary to identify where peak photosynthesis occurs for red and green cabomba. However, the temperature range tested is representative of the 52

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temperatures in the locations where cabomba is commonly found and these data clearly show that red cabomba is unlikely to survive in cold climates, and could explain why this population is not found in the northeast, midwest, and northwest U.S. Green cabomba, however, is likely to survive during the cold winter months and these data support previous literature of green cabomba overwintering in the northeastern U.S. (Riemer and Ilnicki 1968). Green cabomba also had increasing photosynthesis through 32C, suggesting that green cabomba is also tolerant of a wide temperature range, and could explain its invasiveness compared to the other cabomba populations. Previous reports (Leslie 1986; Orgaard 1991) suggested that temperature influences color formation in cabomba but these data suggest that temperature does not cause color changes but limits the distribution of red cabomba. This could explain the color/temperature relationships among these cabomba populations. These studies measured the net photosynthetic response of cabomba to pH and temperature. Earlier studies revealed that photosynthetic measurements in growth chambers corresponded well to plant growth in mesocosm tests (Netherland and Getsinger 1995; Netherland and Lembi 1992). It is then reasonable to assume that the photosynthetic response of cabomba to pH and temperature would correspond well to plant growth in the field. These populations are currently identified as the same species and thus would be expected to respond similarly to both pH and temperature. The differences in photosynthesis observed in response to both pH and temperature in these studies suggest that the current taxonomy does not adequately separate these unique populations. In summary, pH studies identified population differences and confirmed that cabomba prefers slightly acidic conditions. Temperature studies showed population response differences and provide evidence that red cabomba would not survive in colder climates where long-term freezes would be common. 53

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Table 3-1. Model parameters of quadratic regression for three cabomba populations in response to a range of pH. Population Parameter a Estimate c Standard error P-Value Aquarium b 0 -279 92.0 .003 b 1 130 29.0 <.0001 b 2 -11.0 2.2 <.0001 MAX b 5.9 a 0.15 <.0001 Green b 0 -553 91.0 <.0001 b 1 214 28.5 <.0001 b 2 -17.1 2.2 <.0001 MAX 6.2 b 0.08 <.0001 Red b 0 -452 92.1 <.0001 b 1 164 29.0 <.0001 b 2 -12.7 2.2 <.0001 MAX 6.5 c 0.08 <.0001 a for the equation y= b 0 + b 1 *pH + b 2 *pH 2 b calculated from MAX= -b 1 /(2*b 2 ) c values of MAX not connected by the same letter are statistically different based on a full model dummy variable technique with p0.05 (Neter 1990). Table 3-2. Photosynthetic response of three cabomba populations to a range of temperatures. C Populations (n=24) Net photosynthetic rate a 8 Green 5.7 a b Aquarium 6.1 a Red 0.6 b 16 Green 39.9 c Aquarium 66.7 a Red 50.0 b 24 Green 121.3 b Aquarium 151.5 a Red 158.0 a 32 Green 154.6 b Aquarium 154.3 b Red 200.0 a a in g O 2 g -1 (fresh weight) minute -1 b Those populations not connected by the same letter within a temperature are significantly different based on a Students pairwise t-test (p=0.05). 54

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pH 5678 Net Photosynthetic Rate (g O 2 g (fresh weight)-1 min-1) 20406080100120140160180 pH 5678 Net Photosyntheti(g O2 g (fresh weight) A c Rate-1 mi n-1) 204060801000140 12 pH 5678 Net Photosynthet(g O2 g (fresh weight)n-1) B 20406080100120140160 ic Rate-1 mi C Figure 3-1. Photosynthetic response of three populations of cabomba at a range of pH. A) green, B) aquarium, C) red. Symbols represent raw data for each pH. Lines represent the quadratic fit of the model (y= b 0 + b 1 *pH + b 2 *pH 2 ). 55

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pH 5678 Net Photosynthetic Rate(g O2 g (freshweight)-1 minute-1) 406080100120140 Green (MAX= 6.2) Aquarium (MAX= 5.9) Red (MAX= 6.5) Figure 3-2. Quadratic fit of the response of three cabomba populations to a range of pH. Lines represent the quadratic fit of the model (y= b 0 + b 1 *pH + b 2 *pH 2 ). MAX is calculated from equation MAX= -b 1 / (2*b 2 ) and represents the pH for each population where maximum photosynthesis occurred. 56

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CHAPTER 4 COMPARISON OF THREE CABOMBA POPULATIONS FROM NATURAL HABITATS AND CULTURE CONDITIONS Introduction Cabomba caroliniana (cabomba) is a submersed aquatic dicotyledonous plant considered native to the southeastern U.S. and is increasingly being found in northern North America and in Australia, China, and locations in Europe (Leslie 1986; Mackey 1996; Orgaard 1991; Schooler et al. 2006; Yu et al. 2004a, b). Plants in these new locations differ in appearance from plants observed in their native range. For example, plants in new northern locations are green (green cabomba), whereas the native plants in the southeast are red (red cabomba). Cabomba sold by the aquarium trade (aquarium cabomba) is green with purple leaves at the apex of the plant. Green cabomba is widely considered an invasive plant and a management problem, whereas red cabomba reportedly lacks invasive characteristics. Environmental conditions can greatly influence the appearance of many aquatic plants (Godfrey and Wooten 1981; Sculthorpe 1985) and can be due to temperature, light, pH, nutrient content, and many other factors that can influence plant growth. Due to potential variance in environmental conditions, many plants have a high degree of phenotypic plasticity in order to survive a wide range of conditions (Sculthorpe 1985). In some cases, these phenotypic changes in response to environmental conditions lead to the classification of ecotypes. There is, however, some question as to the usefulness of this distinction, because the ecotypes are essentially the same species and are only displaying phenotypic plasticity (Quinn 1978). C. caroliniana has a wide range of characteristics, particularly color, which is reportedly influenced by environmental conditions such as light and temperature. Plants differ in color due to various concentrations of an array of plant pigments, including chlorophyll and anthocyanin. Chlorophyll is largely responsible for green coloration, whereas 57

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anthocyanins are responsible for orange-red coloration (Kong et al. 2003). Anthocyanins not only impart color but can also act as antioxidants and even provide protection from insect herbivory (Kong et al. 2003). Increased levels of anthocyanin can indicate stress in aquatic plants, such as that observed in response to herbicides (Doong et al. 1993). Characteristics other than color are influenced by environmental conditions and this tendency has been observed in other aquatic plants. Alligatorweed (Alternathera philoxeroides (Mart.) Griseb) has distinct growth forms in response to salinity and water level. These forms are reversible and dependent upon the prevailing conditions (Kay and Haller 1982). Cabomba color traits are also believed to be reversible, which would support the theory that color is plastic and influenced by environment change. However, if these differences are not reversible, it is possible that coloration is innate. Color differences among the three cabomba populations suggests some level of pigment differentiation, reportedly caused by temperature and is a reversible difference (Orgaard 1991). The three populations of cabomba have been maintained in common culture at the University of Florida Center for Aquatic and Invasive Plants (CAIP) and remained constant in appearance for more than two growing seasons. This is in contrast to the predictions of Orgaard (1991) and Leslie (1986) whose data suggest that a plant with green (green cabomba) should have changed to red (red cabomba) when grown in a warm climate. There is also a difference in leaf appearance of the three populations. Green cabomba has large coarsely divided leaves, whereas red cabomba has small finely divided leaves. Aquarium cabomba is intermediate between the other two populations. The objectives of this study were to: 1) determine if pigment content (chlorophyll and anthocyanin) and specific leaf weight differed for plants collected from natural habitats (field plants) and plants from common culture (culture plants) for all three populations 58

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and 2) determine if there are differences in the three parameters tested among the three cabomba populations for each field and culture growth location. Materials and Methods Pigment Analysis Chlorophyll content. Red cabomba was collected from White Lake (Suwannee County, FL) aquarium cabomba was obtained from Suwannee Laboratories, an aquatic plant nursery (Lake City, FL), and green cabomba was collected from the St. Joseph River (Elkhart, IN). These three populations were grown in common culture at the CAIP. Green and aquarium cabomba was grown in common culture for ~ 1 yr prior to use in experiments. Red cabomba was only grown in common culture for 3 mo before use in experiments. These plants are identified as cultured plants. Plants were also collected from the same field sites as cultured plants, but were utilized immediately for experiments. These plants are identified as field plants. Five apical sections (2 cm long) were excised from each population of cultured and field plants and placed in test tubes. Test tubes were then refrigerated at 4.4C for 24 h until they were processed. After refrigeration, plants were trimmed to similar weights and fresh weight was measured. Plant tips were then placed in test tubes containing 5 ml of DMSO (dimethylsulfoxide) and chlorophyll was extracted (Hiscox and Israelstam 1979). Plants in test tubes were incubated in a water bath (70C) for 6 h and chlorophyll content was determined spectrophotometrically (Arnon 1949), and expressed as mg (chlorophyll) g -1 fresh weight. All populations had 5 replications for each source (field and culture) and chlorophyll content of field plants was compared to the corresponding populations chlorophyll content of cultured plants by examining 95% confidence intervals. Data were also analyzed using ANOVA 59

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(p0.05) and a post-hoc Students pairwise t-test to compare differences in chlorophyll content of the three populations to one another in field and cultured plants. Anthocyanin content. The same plant collection methods and sources as described for the chlorophyll experiments were used for anthocyanin content analysis. Unlike chlorophyll, plant segments were frozen at -5C for 1 wk prior to anthocyanin extraction. Plants were moved to room temperature ~20C for 2 h prior to anthocyanin extraction, following the methods from Doong et al. (1993). The equation used to correct for the absorbance of chlorophyll at 675 nm and obtain total absorbance was: total relative absorbance of anthocyanin= (A 530 (0.25*A 657)) g -1 fresh weight. All populations had 5 replications for each source (field and culture). The anthocyanin content of field plants (green, aquarium and red) was compared to the corresponding anthocyanin content of cultured plants by comparing 95% confidence intervals. Data were also analyzed using ANOVA (p0.05) and a post-hoc Students pairwise t-test to compare differences in chlorophyll content of the populations to one another in field and cultured plants. Leaf Characteristics The same plant collection methods and sources as described for pigment analysis were used for leaf weight comparisons. Five plants were selected from each population and location (field and culture), and 10 leaves from each were removed from at least 4 cm from the tip of the plant and placed in scintillation bottles. Leaf material was dried at 70C for 2 wk then weighed for each plant (10 leaves total) and converted to specific leaf weight. All populations had 5 replications for each source (field and culture). The specific leaf weight of each populations field plants was compared to the corresponding populations specific leaf weight of cultured plants by examining 95% confidence intervals. Data were also analyzed 60

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using ANOVA (p0.05) and a post-hoc Students pairwise t-test to compare differences in per leaf weight of the populations to one another in field and cultured plants. Results and Discussion Pigment Analysis Neither chlorophyll nor anthocyanin content (Table 4-1) of field plants differed from the corresponding cultured plants (analysis not shown). However, differences were observed in chlorophyll content among populations, regardless of the source of the plants (field or culture). Chlorophyll content was highest in aquarium cabomba, followed by green cabomba, and lowest in red cabomba for both field and culture plants (Table 4-1). There were also differences in the anthocyanin content among the three populations and these were the same regardless of the collection source (field or culture). Red cabomba had the highest anthocyanin content, green cabomba had the lowest content, and aquarium cabomba, as in previous experiments, was intermediate to the other two populations. The anthocyanin content of red cabomba is nearly 10 times higher than the anthocyanin content of green cabomba, and suggests that anthocyanin is the likely source of red pigmentation for cabomba populations. Similar pigment concentrations of both field and culture plants of all populations of cabomba suggest that common culture in Florida does not affect pigment concentrations. There was no difference in chlorophyll or anthocyanin content in green cabomba field plants (collected from cold northern climates) and green cabomba cultured in Florida. Earlier literature predicted that the coloration of cabomba grown cold water would change from green to red if that same plant were grown in warm water (Leslie 1986; Orgaard 1991). Our results contradict this prediction and suggest that cabomba pigmentation is more conserved than originally believed. Anthocyanins are commonly synthesized in response to stress (Doong et al. 1993; Kong et al. 2003), and could be the source of anthocyanin differences among plant populations, but this is 61

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unlikely, given the similar culture conditions and conserved nature of the red cabomba anthocyanin content. Plant collection site had no impact on cabomba pigmentation and the differences among the populations were the same regardless of site. This suggests that pigment content is not as variable or environmentally influenced as originally believed, but to fully explore this possibility reciprocal studies in northern climates need to be performed on red and aquarium cabomba. Current taxonomy identifies all three populations as being the same species, and further suggests that temperature determines content of plant pigments (Leslie 1986; Orgaard 1991). It is possible that red and aquarium cabomba are affected by temperature, but these studies refute that claim for green cabomba. The source and reason for this conserved pigmentation in cabomba populations is not known, but these data suggest the differences are not environmentally flexible for green cabomba, and are likely conserved in the other populations as well. Leaf Characteristics There was no difference in specific leaf weight between field and culture plants of the same population (data not shown). However, differences were observed among populations regardless of the source of the plants (field or culture) (Table 4-1). Green cabomba was different from red cabomba and the specific leaf weight of aquarium cabomba was intermediate to both green and red cabomba. These differences correspond to the visual observation that green cabomba has larger leaves than red cabomba, and aquarium cabomba is intermediate to both. Leaf characteristics, much like plant pigments, are not influenced by the location where plants were grown and leaf weight is another parameter that highlights the differences among these cabomba populations. Reciprocal studies should be performed in colder climates to determine the impact of temperature on plant pigmentation and specific leaf weight of red cabomba. It is possible that 62

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red and aquarium cabomba could change pigmentation and specific leaf weight in colder climates, but this seems unlikely due to lack of plasticity in this study. These studies indicate that green cabomba was unaffected by culture location, suggesting that temperature had no impact on this population as was previously reported (Leslie 1986; Orgaard 1991). Due to the highly conserved plant pigmentation and specific leaf weight of green cabomba from two separate growth locations, a reexamination of cabomba taxonomy may be necessary to fully address the differences between red and green cabomba. 63

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Table 4-1. Characteristics of apical sections of three cabomba populations collected from the field and established cultures. Population Field (n=5) Culture (n=5) Chlorophyll content (mg (total chlorophyll) g -1 (fresh weight) Green 0.508 b 0.484 b Aquarium 0.644 a 0.677 a Red 0.322 c 0.357 c Total anthocyanin absorbance g -1 (fresh weight) Green 0.296 c 0.313 c Aquarium 1.529 b 1.399 b Red 2.657 a 2.376 a Leaf weight mg (dry weight) leaf -1 Green 8.6 a 7.1 a Aquarium 6.3 ab 5.7 ab Red 3.9 b 5.0 b Populations not connected by the same letter within a collection site (field or culture) and parameter tested are significantly different according to a Students pairwise t-test (p0.05). 64

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CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS These studies revealed that green and red cabomba are significantly different from one another in their response to a range of herbicides, temperature, pH, and had different pigment content and leaf characteristics. Aquarium cabomba had characteristics similar to both red and green cabomba, depending upon the experimental conditions. Cabomba is tolerant of most herbicide treatments, but green cabomba is the most tolerant of the three populations. Three herbicides (carfentrazone, endothall (amine salt) and flumioxazin) were effective in reducing photosynthesis and biomass of cabomba, and further field evaluations are warranted to determine the efficacy of these herbicides for potential use in management programs. Slow acting systemic herbicides such as fluridone and ALS (acetolactate synthase) compounds were not tested due to the much longer exposure times needed to effectively control aquatic plants and the lack of good long-term culture techniques. In previous laboratory studies fluridone was shown to effectively reduce biomass of cabomba at high rates, but field trials were less conclusive (Mackey 1996; Nelson et al. 2001; Nelson et al. 2002). These herbicides are typically used on a lake-wide basis and may have limited utility where cabomba exists only in isolated areas of the littoral zone or in flowing waters. Given the increase of cabomba in areas where Eurasian watermilfoil (Myriophyllum spicatum L.) once dominated, these compounds could be utilized to simultaneously suppress cabomba and milfoil (though likely at higher rates than currently used) if these compounds are shown to be effective on cabomba. The most persistent and troubling problem in these experiments was a lack of reliable culture techniques for cabomba. Attempting to maintain just one population was difficult; culturing three populations at the same time was rarely possible. Future studies of cabomba should begin with a concerted effort to identify optimum culture techniques and growth 65

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conditions for all three populations, which would reduce reliance on obtaining plants from the field. Reciprocal studies should be conducted in a northern climate to fully compare the three cabomba populations. Reciprocal studies would determine if differences are conserved for all populations. Culturing aquarium and red cabomba in northern climates may be problematic given the reported lack of cold tolerance for many cabomba species and the lack of photosynthesis of red cabomba at water temperatures of 8C or less as evident in these studies. Genetic studies would also be helpful to characterize the differences among these three cabomba populations and are likely necessary if the taxonomy of cabomba is to be revised. The physiological differences observed in these experiments suggest there are major differences in cabomba populations that would warrant such a reclassification. Even if reclassification is not considered, aquatic plant managers should be aware of the results of this study. Properly identifying the cabomba present will influence which management strategies should be implemented. 66

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APPENDIX HERBICIDE INFORMATION AND PREVIOUS STUDIES Table A-1. List of compounds for use in submersed aquatic weed control. Herbicide Maximum labeled rate g a.i. L -1 Typical use rate g a.i. L -1 Carfentrazone 200 100-200 Copper 1000 250-1000 Diquat 375 94-375 Endothall (amine salt) 7000 500-2000 Endothall (dipotasium salt) 5000 1000-3000 Flumioxazin 400 a N.A. b Quinclorac 500 a N.A. b Triclopyr 3500 2000-3500 2,4-D (amine salt) 5000 2500-5000 a recommended maximum labeled rate to EPA. b compound not registered so no use pattern developed. Hours 020406080100120140 pH 5.56.06.57.07.58.08.59.0 0 MES 0.1% MES v/v 1% MES v/v n=4 Figure A-1. Response of aquarium cabomba growth solution pH to a range of MES buffer solution. Symbols represent mean 95%. 67

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Figure A-2. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and Eurasian watermilfoil to 2,4-D at a concentration of 4400 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) 20406080100120140160 Green Aquarium Red Eurasian Milfoil n=82,4-D Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) 020406080100120140160 Green Aquarium Red Hydrilla n=8Endothall (dipotasium salt) Figure A-3. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to endothall (dipotasium salt) at a concentration of 3000 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. 68

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020406080100120140160 Percent of Untreated Control(Net Photosynthesis) -60-40-20020406080100120 Green Aquarium Red Variable Milfoil N=8Carfentrazone Figure A-4. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and variable milfoil to carfentrazone at a concentration of 400 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent treatments in which plants consumed oxygen (net respiration). Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) -20020406080100120140160 Green Aquarium Red Hydrilla n=4Copper (trial 1) Figure A-5. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to copper at a concentration of 1000 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent treatments in which plants consumed oxygen (net respiration). 69

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70 Figure A-6. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to copper at a concentration of 1000 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent treatments in which plants consumed oxygen (net respiration). Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) -60-40-20020406080100120140160 Green Aquarium Red Hydrilla Copper (trial 2)n=4 Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) 020406080100120140160 Green Aquarium Red E. Milfoil Triclopyrn=8 Figure A-7. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and Eurasian watermilfoil to triclopyr at a concentration of 4900 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control.

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Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthetic Rate) 20406080100120140160 Green Aquarium Red GQuinclorac (trial 1)n=4 Figure A-8. Photosynthetic response of three populations of cabomba (green, aquarium, and red) to quinclorac at a concentration of 400 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthetic Rate) 20406080100120140160 Green Aquarium Red Quinclorac (trial 2)n=4 Figure A-9. Photosynthetic response of three populations of cabomba (green, aquarium, and red) to quinclorac at a concentration of 400 g active ingredient L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. 71

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Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthesis) -60-40-20020406080100120 Green Aquarium Red Hydrilla n=8Diquat Figure A-10. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to diquat at a concentration of 375 g a.i. L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent those readings that consumed oxygen (net respiration). Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthetic Rate) -100-80-60-40-20020406080100 120 Green N=8Diquat + Copper Aquarium Red Hydrilla Figure A-11. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to diquat at a concentration of 375 + copper at a concentration of 1000 g aL. .i. control. Negative values represent those readings that consumed oxygen (net respiration). .i. control. Negative values represent those readings that consumed oxygen (net respiration). -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreatedred) and hydrilla to diquat at a concentration of 375 + copper at a concentration of 1000 g aL -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated 72

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Hours After Treatment 020406080100120140160 P ercent o f U ntreate d C ontro l (Net Photosynthetic Rate) -80-60-40-20020406080100120 Green Aquarium Red Hydrilla N=8Endothall (amine salt) Figure A-12. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to endothall (amine salt) at a concentration of 2300 g a.i. L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent those readings that consumed oxygen (net respiration). Hours After Treatment 020406080100120140160 Percent of Untreated Control(Net Photosynthetic Rate) -80-60-40-20020406080100120 Green Aquarium Red Hydrilla N=8Flumioxazin Figure A-13. Photosynthetic response of three populations of cabomba (green, aquarium, and red) and hydrilla to flumioxazin at a concentration of 400 g a.i. L. -1 Symbols represent mean standard error. Dotted reference line is at 50% of untreated control. Negative values represent those readings that consumed oxygen (net respiration). 73

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LIST OF REFERENCES Arnon, D.I. Copper enzymes in isolated chloroplasts. 1949. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15. Cao, P.J., M.J. Yu, X.F. Jin, and B.Y. Ding. 2006. Studies on niche characteristics and interspecific association of main populations in submerged communities invaded by Cabomba caroliniana. J. of Zhejiang Univ. 32:334-340. Cook, C.D.K. and R. Lnd. 1982. A revision of the genus Hydrilla (Hydrocharitaceae). Aquat. Bot. 13:485-504. Cook, C.D.K. and K. Urmi-Knig. 1984. A revision of the genus Egeria (Hydrocharitaceae). Aquat. Bot. 19:73-96. Cook, C.D.K. and K. Urmi-Knig. 1985. A revision of the genus Elodea (Hydrocharitaceae). Aquat. Bot. 21:111-156. Crawford, H.M., D.A. Jensen, B. Peichel, P.M. Charlebois, B.A. Doll, S.H. Kay, V.A. Ramey, and M.B. OLeary. 2001. Sea Grant and invasive aquatic plants: A national outreach initiative. J. Aquat. Plant Manage. 39:8-11. Christy, J.A. and M.D. Sytsma. 1994a. Noteworthy collections: Oregon. Madroo 41: 331. Christy, J.A. and M.D. Sytsma. 1994b. Noteworthy collections: Oregon. Madroo 41: 332. Denys, L., J. Packet, L. Weiss, and M Coenen. 2003. Cabomba caroliniana (Cabombaceae) houdt stand in Holsbeek (Vlaams-Brabant, Belgie). Dumortiera 80:35-40. [English Abstract]. Doong, R.L., G.E. MacDonald, and D.G. Shilling. 1993. Effect of fluridone on chlorophyll, carotenoid and anthocyanin content of Hydrilla. J. Aquat. Plant Manage. 31:55-59. Fasset, N.C. 1953. A monograph of Cabomba. Castanea 18:116-128. Foes, M.J., L. Liu, P.J. Tranel, L.M. Wax, and E.W. Stoller. 1998. A biotype of common waterhemp (Amaranthus rudis) resistant to triazine and ALS herbicides. Weed Sci. 46:514-520. Getsinger, K.D., G.O. Dick, R.M. Crouch, and L.S. Nelson. 1994. Mesocosm evaluation of bensulfuron methyl activity on Eurasian watermilfoil, Vallisneria, and American pondweed. J. Aquat. Plant Manage. 32:1-6. 74

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Godfrey, R.K. and J.W. Wooten. 1981. Aquatic and Wetland Plants of the Southeastern United States Dicotyledons. Athens, GA: University of Georgia Press. 933 p. Gray, C.J., J.D. Madsen, R.M. Wersal, and K.D. Getsinger. 2007. Eurasian watermilfoil and parrotfeather control using carfentrazone-ethyl. J. Aquat. Plant. Manage. 45:43-46. Haller, W.T. and D.L. Sutton. 1973. Effect of pH and high phosphorus concentrations on growth of waterhyacinth. J. Aquat. Plant Manage. 11:59-61. Hanlon, C. 1990. A Florida nativecabomba (fanwort). Aquatics 12:4-7. Hoyer, M.V., D.E. Canfield, C.A. Horsburgh, and K. Brown. 1996. Florida Freshwater Plants A Handbook of Common Aquatic Plants in Florida Lakes. Gainesville, FL: University of Florida IFAS No. SP189. 264 p. Hiscox, J.D. and G.F. Israelstam. 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot. 57:1332-1334 Hrusa, F., B. Ertter, A. Sanders, G. Leppig, and E. Dean. 2002. Catalogue of nonnative vascular plants occurring spontaneously in California beyond those addressed in The Jepson Manual Part 1. Madroo 49:61-98. Kay, S.H. and W.T. Haller. 1982. Evidence for the existence of distinct alligatorweed biotypes. J. Aquat. Plant Manage. 20:37-41. Kay, S.H. 1992. Response of two alligatorweed biotypes to quinclorac. J. Aquat. Plant Manage. 30:35-40. Kay, S.H., P.C. Quimby, and J.D. Ouzts. 1984. Photo-enhancement of hydrogen peroxide toxicity to submersed vascular plants and algae. J. Aquat. Plant Manage. 22:25-34. Kong, J.M., L.S. Chia, N.K. Goh, T.F. Chia, and R. Brouillard. 2003. Analysis and biological activities of anthocyanins. Phytochemistry 64:923-933. Les, D.H. and L.J. Mehrhoff. 1999. Introduction of nonindigenous aquatic vascular plants in southern New England: A historical perspective. Biol. Invasions 1:281-300. Leslie, Andrew J. 1986. A literature review of cabomba. Tallahassee, FL: Florida Department of Natural Resources Bureau of Aquatic Plant Research Control. 18 p. Mackey, A.P. 1996. Cabomba in Queensland: A pest status review series land protection branch. Queensland, Australia: Department of Natural Resources. 35 p. 75

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Mackey, A.P. and J.T. Swarbrick. 1997. The biology of Australian weeds. 32. Cabomba caroliniana Gray. Plant Prot. Q. 12:154-165. Madsen, J.D. 1994. Invasions and declines of submersed macrophytes in Lake George and other Adirondack lakes. Lake Reservoir Manage. 10:19-23. Madsen, J.D. 1998. Predicting invasion success of Eurasian watermilfoil. J. Aquat. Plant. Manage. 36: 28-32. Madsen, J.D. 2000. Advantages and disadvantages of aquatic plant management. LakeLine 20:22-34. Martin, D.F. and R.P. Wain. 1991. The cabomba color problem. Aquatics 13:17. McLane, W.M. 1969. The aquatic plant business in relation to infestations of exotic aquatic plants in Florida waters. Hyacinth Cont. J. 8(1):48-49. Moore, B. 1991. No one wants a fish kill: Fish can live when using Hydrothol 191 in weed and algae control. Aquatics 13:16-17. Moseley, M.F., J.J. Mehta, P.S. Williamson, and H. Kosakai. 1984. Morphological studies of the Nymphaeaceae (Sensu Lato). XIII. Contributions to the vegetative and floral structure of Cabomba. Am. J. Bot. 71:902-924. Nakai, S., Y. Inoue, M. Hosomi, and A Murakami. 1999. Growth inhibition of bluegreen algae by allelopathic effects of macrophytes. Wat. Sci. Tech. 39:47-53. Nelson, L.S., J.F. Shearer, and M.D. Netherland. 1998. Mesocosm evaluation of integrated fluridone fungal pathogen treatment on four submersed plants. J. Aquat. Plant Manage. 36:73-77. Nelson, L.S., A.B. Stewart, and K.D. Getsinger. 2001. Herbicide evaluation for control of Cabomba caroliniana and subsequent impact on the non-target species, Megalodonta beckii. Page 20 in Abstracts of 41 st Annual Meeting of the Aquatic Plant Management Society Inc. Minneapolis, MN: Aquatic Plant Management Society Inc. Nelson, L.S., A.B. Stewart, and K.D. Getsinger. 2002. Fluridone effects on fanwort and water marigold. J.Aquat. Plant Manage. 40:58-63. Neter, J. and W. Wasserman. 1990. Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental Designs. 3 rd ed. Burr Ridge, IL: CRC. 1184 p. Netherland, M.D. and K.D. Getsinger. 1995. Laboratory evaluation of threshold fluridone concentrations under static conditions for controlling hydrilla and Eurasian watermilfoil. J. Aquat. Plant Manage. 33:33-36. 76

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Netherland, M.D. and C.A. Lembi. 1992. Gibberellin synthesis inhibitor effects on submersed aquatic weed species. Weed Sci. 40:29-36. Nol, J. 2004. Growth, Reproduction and Control of an Invasive Plant, Cabomba caroliniana, in Kasshabog Lake, Ontario and its Potential Dispersal. M.Sc. thesis. Peterborough ON: Trent University. 89 p. Oldham, M.J. 1999. Botanical highlights. Ontario Natural Heritage Information Centre Newsletter 5:10-11. Orgaard, Marian. 1991. The genus Cabomba (Cabombaceae) A taxonomic study. Nord. J. Bot. 11:179-203. Osborn, J.M., T.N. Taylor, and E.L. Schneider. 1991. Pollen morphology and ultrastructure of the Cabombaceae: Correlations with pollination biology. Am. J. Bot. 78:1367-1378. Owens, C.S., R.M. Smart, D.R. Honnell, and G.O. Dick. 2005. Effects of pH on growth of Salvinia moelsta Mitchell. J. Aquat. Plant. Manage. 43:34-38. Preston, C.D. and J.M. Croft. 1997. Aquatic plants in Britain and Ireland. Colchester, UK: Harley Books. 365 p. Quinn, James A. 1978. Plant ecotypes: Ecological or evolutionary units? B. Torrey Bot. Club 105:58-64. Riemer, Donald N. 1965. The effect of pH, aeration, calcium and osmotic pressure on the growth of fanwort (Cabomba caroliniana Gray). Pages 460-467 in Proceedings of the 19th Annual Meeting of the Northeastern Weed Control Conference. New York City, NY: Weed Science Society of America. Riemer, D.N. and R.D. Ilnicki. 1968. Reproduction and overwintering of cabomba in New Jersey. Weed Sci. 16:101-102. Sanders, D.R. 1979. The ecology of Cabomba caroliniana. Pages 134-146 in E.O. Gangstad, Weed Control Methods for Public Health Applications. Boca Raton FL: CRC Press. Schardt, J.D. and L.E. Nall. 1989. 1988 Florida Aquatic Plant Survey. Tallahassee, FL: Florida Department of Natural Resources, Bureau of Aquatic Plant Management Rep. 89-CGA. 118 p. Schneider, E.L. and J.M. Jeter. 1982. Morphological studies of the Nymphaeaceae. XII. The floral biology of Cabomba caroliniana. Am. J. Bot. 69:1410-1419. 77

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Schooler, S., M. Julien, and G.C. Walsh. 2006. Predicting the response of Cabomba caroliniana populations to biological control agent damage. Aust. J.Entomol 45:327-330. Sculthorpe, C.D. 1985. The Biology of Aquatic Vascular Plants. 2 nd ed. Knigstein, West Germany: Koeltz Scientific Books. 610 p. Selim, S.A., S.W. ONeal, M.A. Ross, and C.A. Lembi. 1989. Bioassay of photosynthetic inhibitors in water and aqueous soil extracts with Eurasian watermilfoil (Myriophyllum spicatum). Weed Sci. 37:810-814. Stace, C.A. 1997. New flora of the British Isles. Cambridge, UK: Cambridge University Press. 1130 p. Stetk, D. 2004. An aquarium plant in natural waters and canals of Hungary: The fanwort (Cabomba caroliniana). Kitaibelia 6:165-171. Tarver, D.P. 1976. Selected Life Cycle Features and Effects of Environmental Conditions on Cabomba caroliniana Gray. M. Sc. Thesis. Natchitoches, LA: Northwestern State University. 79 p. Tarver, D.P. and D.R. Sanders. 1977. Selected life cycle features of fanwort. J. Aquat. Plant Manage. 15:18-22. Van der Velde, G., I. Nagelkerken, S. Rajagopal and A. Bij de Vaate. 2002. Invasions By Alien Species in Inland Freshwater Bodies in Western Europe: The Rhine Delta.2002. Pages 360-372 in E. Leppakoski, S. Gollasch, and S. Olenin, eds. Invasive Aquatic Species of Europe: Distribution, Impacts and Management. Netherlands: Kluwer Academic Publishers. Wain, R.P., W.T. Haller, and D.F. Martin. 1983. Genetic relationship among three forms of cabomba. J. Aquat. Plant Manage. 21:96-98. Yaowakhan, P., M. Kruatrachue, P. Pokethitiyook, and V. Soonthornsarathool. 2005. Removal of lead using some aquatic macrophytes. Bull. Environ. Contam. Toxicol. 75:723-730. Yu, J., B. Ding, M. Yu, X. Jin, H. Shen, and W. Jiang. 2004a. The seasonal dynamics of the submerged plant communities invaded by Cabomba caroliniana Gray. Acta Ecol. Sin. 24: 2149-2156. Yu, M.J., B.Y. Ding, J. Yu, X.F. Jin, H. Zhou, and W.H. Ye. 2004b. Basic characteristics of submerged plant communities invaded by Cabomba caroliniana and its habitat in China. Acta. Ecol. Sin. 28:231-239. 78

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Zhang, X., Y. Zhong, and J. Chen. 2003. Fanwort in eastern China: An invasive aquatic plant and potential ecological consequences. Ambio. 23:158-159. 79

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BIOGRAPHICAL SKETCH Born on September 12, 1981 in Merridian, Mississippi, Brett is the son of Craig and Laura Bultemeier. As a child, Brett moved often, as his father was a naval aviator and was redeployed every few years. Finally ending up in Indiana, he pursued his B.S. degree at Manchester College in North Manchester, Indiana, focusing his studies on biology and environmental studies. While at Manchester, he was a collegiate wrestler, played in the school band, and helped form the environmental club. During his undergraduate studies, Brett worked during the summer for Weed Patrol Inc. and it was at this job that a passion for aquatic plant management was fostered. Upon completion of his degree, Brett was married to Megan and promptly moved to Gainesville, Florida, to pursue a Masters degree in the field of aquatic plant management. Upon completion of his masters project Brett began studies towards a PhD from the University of Florida. 80


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