MORPHOLOGICAL DIFFERENTIATION AND HERBICIDE …ufdcimages.uflib.ufl.edu/UF/E0/05/16/05/00001/BANU_A.pdfand neighboring countries (Dandelot et al. 2005). Ludwigia hexapetala was reported

MORPHOLOGICAL DIFFERENTIATION AND HERBICIDE CONTROL OF THE LUDWIGIA URUGUAYENSIS COMPLEX IN FLORIDA

By

AFSARI BANU

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 2017

© 2017 Afsari Banu

To my Husband, Mom, Dad, and Sister

4

ACKNOWLEDGMENTS

This thesis would not be completed without help from a group of people. First, I

want to acknowledge my loving husband Anil for his continuous support and

encouragement throughout my journey to thesis completion. He always boosted my

morale and was always there for me. Next, I want to acknowledge my parents. They

always supported me emotionally and providing me an opportunity to lead a right path

for my career. My little sister Sonu is of immense encouragement for me to keep

pushing myself to complete this endeavor. I also want to acknowledge my brothers for

their continuous encouragement and emotional support to complete my thesis.

My thesis would not be completed without encouragement, advice and support

from my advisor, Dr. Stephen Enloe. He always pushed me to think deeper and to think

differently to find answers to invasive species management. Dr. Jacono, my committee

member is deserving of much acknowledgement. It is because of you Dr. Jacono, I was

able to complete my morphology study. You taught me a lot about plant morphology

and always supported my research ideas. Next, I want to acknowledge my two other

committee members, Drs. Macdonald and Leon for their continuous support and

encouragement to complete my thesis successfully. I also want to acknowledge Dr.

Haller for agreeing to serve as my replacement committee member at the last minute

and continuously supporting my research activities at CAIP in the capacity of center

director. I also want to thank Dr. Netherland for his instant and expert responses to my

specific research queries.

Finally, I want to thank my colleagues, Carl, Cody, Josh, and Kate for helping to

complete my experiments, without them I would not have completed my research.

5

TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 GENERAL INTRODUCTION .................................................................................. 13

Ludwigia uruguayensis Complex ............................................................................ 13 Biology and Ecology of the Ludwigia uruguayensis Complex ................................. 13

Distribution of Ludwigia uruguayensis Complex ..................................................... 15

Management Strategies .......................................................................................... 16

2 MORPHOLOGICAL DIFFERENTIATION WITHIN THE LUDWIGIA URUGUAYENSIS COMPLEX IN FLORIDA ............................................................ 20

Introduction ............................................................................................................. 20

Materials and Methods............................................................................................ 22 Sample Collection and Planting ........................................................................ 22

Floral Morphology ............................................................................................. 24 Foliar Morphology ............................................................................................. 26

Results and Discussion........................................................................................... 28 Floral Morphology ............................................................................................. 28 Foliar Morphology ............................................................................................. 32

3 RESPONSE OF LUDWIGIA HEXAPETALA AND LUDWIGIA GRANDIFLORA TO SELECTED AQUATIC HERBICIDES ............................................................... 47

Introduction ............................................................................................................. 47

Materials and Methods............................................................................................ 49 Dose Response Study ...................................................................................... 49 Tank Mix Comparisons ..................................................................................... 52

Results and Discussion........................................................................................... 53 Dose Response Study ...................................................................................... 53 Tank Mix Comparisons ..................................................................................... 55

4 COMPARISON OF EARLY GROWTH CHARACTERISTICS AND CREEPING MORPHOLOGY OF LUDWIGIA HEXAPETALA AND LUDWIGIA GRANDIFLORA ...................................................................................................... 67

6

Introduction ............................................................................................................. 67

Materials and Methods............................................................................................ 69

Results and Discussion........................................................................................... 71

5 CONCLUSIONS ..................................................................................................... 93

LIST OF REFERENCES ............................................................................................... 95

BIOGRAPHICAL SKETCH .......................................................................................... 100

7

LIST OF TABLES

Table page 2-1 Floral morpho-metrics of five Ludwigia uruguayensis populations from the

common garden study, 2016. ............................................................................. 36

2-2 Floral morpho-metrics of five Ludwigia uruguayensis populations from field collections in 2014 and 2016. ............................................................................. 37

2-3 Floral morpho-metrics of five Ludwigia uruguayensis populations from the common garden study, 2016. ............................................................................. 38

2-4 Floral morpho-metrics of five Ludwigia uruguayensis populations from field collections in 2014 and 2016. ............................................................................. 39

2-5 Foliar morpho-metrics of five Ludwigia uruguayensis populations from the common garden study (2015, 2016 and 2017) and field collections (2014, 2015 and 2016). ................................................................................................. 40

2-6 Floral and foliar morphological characters differentiating Ludwigia grandiflora and L. hexapetala. .............................................................................................. 41

3-1 Herbicides rates used in the dose response study and tank mix comparisons, applied to emergent plant foliage of Ludwigia hexapetala and L. grandiflora with 0.25% v/v non-ionic surfactant. ................................................................... 59

3-2 Model parameters and standard errors in parenthesis for two-parameter log-logistic model provided in equation 1 for figures 3-1,2,3 (shoot regrowth dry weights). ............................................................................................................. 60

4-1 Growth rates of two Ludwigia hexapetala and three L. grandiflora populations at 2, 4, 5, 6 and 7 WAP from the common garden experimental run 2017. ........ 77

4-2 Mean stem width and internode length of two Ludwigia hexapetala and three L. grandiflora populations at 3, 5, 7 WAP from the common garden experimental run 2016. ....................................................................................... 78

4-3 Mean total number of shoots and number of abscised shoots in two Ludwigia hexapetala and three L. grandiflora populations at 3 WAP from the common garden experimental run 2016. ........................................................................... 79

4-4 Mean petiole length and leaf shape of two Ludwigia hexapetala and three L. grandiflora populations at 3 WAP from the common garden experimental run 2016. .................................................................................................................. 80

4-5 Growth rates of two Ludwigia hexapetala and three L. grandiflora populations at 2, 4, 5, 6 and 7 WAP from the common garden experimental run 2017. ........ 81

8

4-6 Mean stem width and internode length of two Ludwigia hexapetala and three L. grandiflora populations at 4, 5, and 6 WAP from the common garden experimental run 2017. ....................................................................................... 82

4-7 Mean total number of shoots and number of abscised shoots in two Ludwigia hexapetala and three L. grandiflora populations at 4, 5 and 6 WAP from the common garden experimental run 2017. ............................................................ 83

4-8 Mean petiole length and leaf shape of two Ludwigia hexapetala and three L. grandiflora populations at 6 and 7 WAP from the common garden experimental run 2017. ....................................................................................... 84

9

LIST OF FIGURES

Figure page 1-1 An infestation of Ludwigia grandiflora in Lake Tohopekaliga (Toho) Osceola

County, FL, November 2016............................................................................. 199

2-1 Scan of Table 1. providing morphological characters differentiating Ludwigia grandiflora and L. hexapetala. ............................................................................ 42

2-2 Flower picture of two populations from group 1 .................................................. 43

2-3 Flower picture of three populations from group 2 ............................................... 43

2-4 Pollen diameter (µm) of five Ludwigia uruguayensis populations... .................... 44

2-5 Monad pollen grain microscopic images at 400X magnification. ........................ 45

2-6 Leaf scan of five Ludwigia uruguayensis populations (Alligator, Harney, Poinsett, Toho and Hawthorne) from common garden and field......................... 46

3-1 Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days ........................................... 61

3-2 Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days. .......................................... 62

3-3 Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days ........................................... 63

3-4 Percent reduction in shoot dry weight of L. grandiflora and L. hexapetala at 30 DAT compared to the untreated control... ...................................................... 64

3-5 Percent reduction in shoot regrowth dry weight of L. grandiflora and L. hexapetala at 65 DAT compared to the untreated control. ................................. 65

3-6 Percent reduction in root dry weight of L. grandiflora and L. hexapetala at 65 DAT compared to the untreated control. ............................................................. 66

4-1 Total shoot length of two Ludwigia hexapetala and three L. grandiflora populations at 3, 5, 7 WAP from the common garden experimental run (2016).. ............................................................................................................... 85

4-2 Shoot abscission in L. grandiflora under common garden experimental runs 2016 and 2017. ................................................................................................... 86

10

4-3 Leaf scan of two Ludwigia hexapetala and three L. grandiflora populations from common garden experimental runs 2016 and 2017.. ................................. 87

4-4 Water temperature in degree Fahrenheit in common garden experimental run (2017). ................................................................................................................ 88

4-5 Total shoot length of Ludwigia hexapetala and L. grandiflora (cm)..................... 89

4-6 Total shoot length of two Ludwigia hexapetala and three L. grandiflora populations at 4, 5, 6 and 7 WAP from the common garden experimental run 2017.. ................................................................................................................. 91

4-7 Mean shoot and root dry weight of two Ludwigia hexapetala and three L. grandiflora populations at 9 WAP from the common garden experimental run 2017... ................................................................................................................ 92

11

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

MORPHOLOGICAL DIFFERENTIATION AND HERBICIDE CONTROL OF THE

LUDWIGIA URUGUAYENSIS COMPLEX IN FLORIDA

By

Afsari Banu

August 2017

Chair: Stephen Enloe Major: Agronomy

The Ludwigia uruguayensis complex has become a serious threat to many

Florida waterbodies. Its rapid growth, coupled with the formation of dense mats and

subsequent difficulty in control has become a great concern. Furthermore, the identity of

species within the complex has not been determined in Florida and it is not clear which

taxonomic characters may best assist aquatic managers in field identification. The study

objectives were 1) to determine taxa of L. uruguayensis populations in Florida, 2) to

evaluate the L. hexapetala and L. grandiflora populations response to aquatic

herbicides and 3) to compare the early creeping morphology and growth characteristic

of Ludwigia hexapetala and L. grandiflora populations.

The morphological study indicated the presence of two separate species L.

hexapetala and L. grandiflora within the L. uruguayensis complex in Florida. The study

also indicated variability within the three L. grandiflora populations.

The dose response trials revealed that imazamox and glyphosate herbicides had

an ED50 of 42 to 221 and 355 to 998 g ae/ha for all populations, respectively. The ED50

values of the two L. grandiflora populations from Poinsett and Toho were significantly

different from the other populations for both herbicides. These results indicated

12

differential imazamox and glyphosate herbicide sensitivity within L. grandiflora

populations. However, there was no differential response to triclopyr among

populations. Additional herbicide tank-mixes and individual herbicide treatments

evaluated performed similarly at 65 DAT. However, tank mix treatments with PPO

herbicides had a mixed result during the initial evaluation period.

The early growth studies demonstrated that all five populations have differential

growth under the common environmental conditions. Shoot abscission only occurred in

L. grandiflora populations. Leaf shape and petiole length were the two most important

morphological characters to distinguish the two species during early growth stages.

.

13

CHAPTER 1 GENERAL INTRODUCTION

Ludwigia uruguayensis Complex

The Ludwigia uruguayensis complex is an aggressive, perennial, emergent

aquatic group of plants commonly known as creeping water primrose. The classification

of Zardini et al. (1991a) distinguished two species, L. grandiflora and L. hexapetala

within this complex. The L. uruguayensis complex is a recent invader in many lakes and

rivers in Florida.

Biology and Ecology of the Ludwigia uruguayensis Complex

Both L. hexapetala and L. grandiflora reproduce by asexual means from

vegetative propagules (Okada et al. 2009). The primary mechanism of spread is clonally

through stem fragments for both species in California (Okada et al. 2009) and

elsewhere (Nehring and Kolthoff 2011). Sexual reproduction could be an additional

mechanism for winter survival and spread of L. grandiflora over long distances (Ruaux

et al. 2009). Both species can create monotypic stands and are generally considered a

threat to biodiversity in their introduced ranges. Invasive plants commonly possess

advantageous biological traits such as broad ecological tolerance, rapid growth and

high biomass production, sexual and asexual reproduction, propagule longevity, high

seed production, a high germination rate, phenotypic and genetic plasticity, a high

photosynthetic rate, allelopathy and polyploidy (Thouvenot 2013a). The presence of one

or two of these traits can be sufficient sometimes to make an aggressive invader. The

species in the Ludwigia uruguayensis complex are reported to posess some of these

traits, which can explain their expansion, invasiveness and adaptability to a wide range

of climatic conditions (Thouvenot 2013a).

14

Ludwigia hexapetala is reported to be found in shallow water habitats in France

and neighboring countries (Dandelot et al. 2005). Ludwigia hexapetala was reported to

found in rivers, ditches and channels, natural and artificial lake and ponds, oxbows,

wetland and flooded meadows (Lambert et al. 2010). Both L. hexapetala and L.

grandiflora were found to grow in freshwater wetlands, in slow-moving rivers and

streams, on lakes and reservoir margins and in shallow canals and on floodplains in

California, USA (Okada et al. 2009). Hussner (2010) reported that L. grandiflora had a

maximum photosynthetic rate up to 2200 µmol CO2 h-1 g-1 of dry biomass under high

light intensity and temperature. Ludwigia hexapetala grows well in mesotrophic to

eutrophic nutrient conditions (Dandelot 2004) and root to shoot ratios of L. grandiflora

vary depending on nutrient conditions (Hussner 2010). A decrease in water column

phosphorus content has reduced growth and competitive outcomes (decrease in

number of branches and stem length) of L. hexapetala (Gérard et al. 2014). In Belgium,

L. hexapetala was a better competitor in both mesotrophic and eutrophic conditions,

because of its higher relative growth rate (RGR) values compared to L. peploides

(Gérard et al. 2014). Both species can be found in anoxic conditions, likely because

they produce aerial roots (pneumatophores) which have negative geotropism. Aerial

roots are surrounded by aerenchyma tissue which supplies oxygen and supports stems

to float on the water surface (Hussner 2010). Both of these species can establish in a

wide range of environmental conditions which are reported in most parts of its

introduced range (Europe, US), This suggests that these species have high phenotypic

and morphological plasticity which allows them to survive, colonize and establish in

different ecosystems.

15

Both L. hexapetala and L. grandiflora have three different life stages. These

include 1)a “floating stage” during the early part of their development, 2) “emergent

stage” when favorable ecological and environmental conditions are present, and 3) a

“prostrate form” during unfavorable conditions like winter survival (Dutartre et al. 2007;

Lambert et al. 2010; Thouvenot et al. 2013a). Both species produce creeping stems

which float on water surface and root at the nodes and produce aerial shoots.

The dense foliage (Figure 1-1) and creeping mats of these species can restrict

water flow, increase flood risk due to decreased channel carrying capacity, increase

sedimentation and accumulation of organic matter during decomposition, and create

anoxic conditions which may lead to fish kill. Furthermore, L. grandiflora limits water

activity such as fishing, boating and navigation (Nehring and Kolthoff 2011).

Additionally, the foliage mats can create habitat conducive for mosquito breeding

(Meisler, 2008), which may cause harm to human health.

Distribution of Ludwigia uruguayensis Complex

Ludwigia hexapetala is native to southern South America (southern Brazil,

eastern Paraguay, Argentina and Uruguay) (Cook 1985; Zardini et al.1991a). In France

L. hexapetala was intentionally introduced as an ornamental plant during the1830s as

reported in Dandelot et al. (2005). Currently, L. hexapetala (reported as L. grandiflora

subsp. hexapetala) is considered an invasive plant in many European countries

including France (Dandelot et al. 2005), Belgium (Bauchau et al. 1984), the Netherlands

(Kleuver and Holverda 1995), Switzerland (Vauthey et al. 2003), Germany (Nehring and

Kolthoff 2011) and the United Kingdom (Armitage et al. 2013). It was also reported in

Italy and Spain (EPPO 2011). Ludwigia grandiflora is native to South and Central

America and parts of the USA (Cook 1985). In the USA, the source of introduction of

16

these species remains unknown. These species are a threat to major watersheds in

California (Okada et al. 2009; Meisler 2009) and are also becoming problematic in

Florida. The distribution of both L. hexapetala and L. grandiflora in the USA ranges from

the Gulf coastal states, Atlantic southeastern and Pacific Northwest regions (Zardini et

al. 1991a). Reports have been made for Georgia, Alabama, Mississippi, Arkansas,

Oklahoma, Louisiana, Texas, Washington, Oregon, North and South Carolina, Virginia,

West Virginia, Kentucky, Missouri, Tennessee, New York, New Jersey and

Pennsylvania (CABI 2014). In South Carolina both L. grandiflora and L. hexapetala are

considered as noxious weeds (Smith 2008). In North Carolina L. hexapetala is listed as

a noxious weed, and in Washington it is considered as a class B noxious weed.

Management Strategies

The prevention of invasive plant introductions is the most effective method of

weed control in terms of ecological, economic and environmental prospects (Thouvenot

et al. 2013a; Gérard et al. 2014). However, stronger action is needed to control further

spread of established invasive plants to unaffected areas (Thouvenot et al. 2013a).

Rapid identification and detection is also required to effectively control invasive plants

(Willby 2007).

Mechanical or manual harvesting were the most common methods used to

manage L. hexapetala and L. peploides in France (Dutartre et al. 2008), and in

California (Meisler 2009) when dense populations occur in shallow waters. The

drawback of these control methods is the production of stem fragments, which may

result in extensive spread to uninvaded areas (Okada et al. 2009; Thouvenot et al.

2013a). It has been suggested to remove all fragments after mechanical harvesting

because both species easily regenerate from even small fragments and quickly grow

17

and establish in aquatic ecosystems (Thiebaut 2007; Okada et al. 2009). In the initial

stage of invasion, these species can be removed by manual hand pulling of low density

colonies. However, when the population becomes dense and well established,

mechanical removal is necessary (Dutartre and Oyarzabal 1993; Thiebaut 2007). For

effective management of these two Ludwigia species one must consider their rapid

growth stage, high capacity for biomass production, and release of vegetative

propagules (Thouvenot et al. 2013b). Meisler (2009) suggested all L. hexapetala

infestations are not alike; one must consider water depth, hydroperiod, nutrient

availability, population density and extent of infestation, presence of native and rare

species and history of disturbance to develop best management practices. Mechanical

harvesting is costly and labor intensive, which is the reason herbicide treatments are

applied to control Ludwigia species in many locations (Meisler 2008).

For biological control the water primrose flea beetle (Lysathia ludoviciana) was

reported to control L. hexapetala populations in the southeastern United States

(Mcgregor et al. 1996). Further information is not available on biological control with this

flea beetle. Water primrose was reported as unpalatable for grazing by cattle, horses,

and carp (Grillas et al. 1992; Pine and Anderson 1991). Cattle and horses were found to

graze on water primrose only when other species were absent at a site due to its low

palatability (EPPO, 2011). The biological control of these plants has been unsuccessful

and no programs are currently underway (CABI 2014).

The most commonly used herbicides to control Ludwigia species, include

glyphosate, triclopyr, 2,4-D, imazapyr and imazamox. Presently, there is limited

published information available on the efficacy of different aquatic herbicides on these

18

species. Efforts should be made to identify the taxa within Ludwigia uruguayensis

complex in Florida and to evaluate different aquatic registered herbicides to control

these L. uruguayensis complex present in Florida.

19

Figure 1-1. An infestation of Ludwigia grandiflora in Lake Tohopekaliga (Toho) Osceola County, FL, November 2016 (Photo courtesy of the author).

20

CHAPTER 2 MORPHOLOGICAL DIFFERENTIATION WITHIN THE LUDWIGIA URUGUAYENSIS

COMPLEX IN FLORIDA

Introduction

The Onagraceae family evolved about 100 million years ago, in South America

(Raven and Tai 1979). Ludwigia represents an early evolutionary branching within the

Onagraceae (Eyde 1977; Raven and Tai 1979). Ludwigia consists of 82 species divided

into 23 sections (Raven 1963; Ramamoorthy 1979; Ramamoorthy and Zardini 1987;

Wagner et al. 2007). Within these 23 sections the section Jussiaea (Hoch et al. 2015),

earlier called section Oligospermum, is distinguished from other sections by pollen

occurring in monads and the seeds completely embedded in corky endocarp (Raven

1963; Ramamoorthy and Zardini 1987; Zardini and Raven 1992; Wagner et al. 2007).

The section Jussiaea consists of eight closely related species including Ludwigia

peploides and the Ludwigia uruguayensis complex. They are aggressive emergent

aquatics with high phenotypic plasticity and have invaded regions outside their native

area. The basic chromosome number of the Ludwigia genus is n = 8; section Jussiaea

includes diploid, triploid, tetraploid, hexaploid, octaploid, and decaploid species (Raven

and Tai 1979; Zardini et al. 1991b; Wagner et al. 2007).

Munz (1942) travelled throughout the western hemisphere to collect Ludwigia

specimens for taxonomic study. He described Ludwigia uruguayensis as a highly

variable complex based on the morphology of live field material and herbarium

specimens and later designated two varieties genuina and major. Zardini et al. (1991a)

formally separated two species within the Ludwigia uruguayensis complex based on the

morphology of not only fresh field material and herbarium specimens but also by

cultivation of plants from which chromosome numbers were counted. Two species, L.

21

grandiflora and L. hexapetala were distinguished based on the combination of ten

morphological features. Seven floral, two foliar and one stem characteristic (see Figure

2-1), and chromosome numbers were used to distinguish the species which included

South American and southeastern U. S. populations within the L. uruguayensis

complex.

Nesom and Kartesz (2000), after reviewing 53 southeastern herbarium

specimens in the L. uruguayensis complex found difficulty in species determination

using Zardini’s work. They reported that some of the quantitative characters described

by Zardini et al. (1991a) broadly overlapped. While Zardini et al. (1991a) described L.

hexapetala plants as having glabrous stems and leaves Nesom and Kartesz (2000),

found that glabrous plants were rarely found in the United States but that most of the

plants varied from sparsely to moderately villous over the whole leaf surface (upper and

lower) and stem. Leaf shape as well as the degree of hairiness was found to overlap

between species and to vary greatly among herbarium vouchers (Nesom and Kartesz

2000).

Although, Nesom and Kartesz (2000) did not determine chromosome number or

consult live plants they suggested combining L. hexapetala as a subspecies of L.

grandiflora. Others continue to recognize two distinct species in the United States

(Okada et al. 2009; Wagner et al. 2007). Many botanists worldwide have largely

followed the suggestion of Nesom and Kartesz (2000) by treating the previously

designated species as subspecies.

One major problem with the conclusions of Nesom and Kartesz (2000) is that

clumping the species brings into question their native status. Ludwigia grandiflora is

22

believed to be native to the United States, based upon the type collection of André

Michaux in 1785 near Savannah, Georgia (Zardini et al. 1991a). For L. hexapetala,

believed to be native to South America (Zardini et al. 1991a), the earliest specimens

collected in the United States were in the 1840s (Zardini et al. 1991a). This is also when

L. hexapetala was introduced into France (Dandelot et al. 2005). This creates a serious

conundrum for invasive plant managers in identifying two species and planning

management approaches to control them.

The first step in an invasive species management program requires accurate

identification of the target species (Forman and Kesseli 2003; Dandelot et al. 2005). The

morphology of the L. uruguayensis complex has been reported as especially plastic and

dependent on environmental conditions (Muller 2004). Fragile herbarium specimens are

often lacking the more conserved features of floral parts, such as petals. Therefore,

herbarium specimens often lack representative material that fresh plants might better

offer in identification. Infect, the taxa of section Jussiaea are known to be difficult to

identify especially in herbarium specimens (Zardini et al. 1991b; Zardini and Raven

1992). This shows investigation of live plant materials should be carried out to properly

identify the Florida material.

Our study objectives were to determine the taxa within the L. uruguayensis

complex present in Florida and to identify the morphological characters to best

distinguish them.

Materials and Methods

Sample Collection and Planting

Populations representative of the Ludwigia uruguayensis complex in Florida were

selected for sampling. Plants were collected from five locales across Florida:1) Alligator

23

Lake, Columbia Co.; 2) a roadside stream drainage leading to Lochloosa Lake, Alachua

Co (labeled as Hawthorne for the nearest locale); 3) Lake Poinsett, Brevard County; 4)

Lake Harney, Volusia County; and 5) Lake Tohopekaliga (Toho), Osceola County.

Locales were visited between May and September 2015 to collect vegetative material

for tank culture under common garden conditions at the Center for Aquatic and Invasive

Plants (CAIP). Herbarium specimens from each collection were deposited at the UF

Herbarium, Florida Museum of Natural History (FLAS). Fifty stems were collected for

each population from multiple widely distributed sites at each locale. Vegetative

accessions were maintained under common garden conditions at the University of

Florida, CAIP, in Gainesville, FL. Each accession was planted in plastic tubs (35 x 29 x

14 cm) filled with commercial greenhouse potting soil (Professional Top Soil, Margo

Garden Products, Inc., Folkston, GA 31537) mixed with 5-10 g of 15-9-12 slow release

fertilizer (Osmocote Plus, The Scotts Company, 14111 Scotts Lawn Rd, Maryville, OH

43041) and covered with 8 cm of builder’s sand. Ten tubs were maintained in 900 L

mesocosms (concrete tanks) with initial water levels of 22-23 cm. The accessions were

maintained about 10 months under common garden conditions before floral and foliar

morphological data was collected. During that period, we encountered a leaf spot

disease caused by Pseudocercospora (identified by Florida Department of Agriculture

and Consumer Services – Division of Plant Industry, Gainesville, FL) a genus of

pathogenic fungi commonly known to cause leaf spot and blights on a wide range of

plant hosts (Crous et al. 2013). Additionally, moth larvae were found damaging our

plants. Insects and fungal diseases were controlled with a mixture of insecticide

bifenthrin + zeta-cypermethrin at 0.20 + 0.05 g ai in 5.68 L of water (Bug B Gon,

24

ORTHO group, Marysville, OH 43040) and fungicide azoxystrobin at 0.43 g ai in 5.68 L

of water (Heritage, Syngenta crop protection LLC, Greensboro, NC 27419-8300). Spray

applications were repeated every 10 to 15 days over the study period.

Floral Morphology

The populations in tanks under common garden conditions flowered between

April and May in 2016. During that period 25 to 35, fully opened flowers from each

population were harvested during the afternoon period and immediately carried to the

laboratory to collect floral data. Zardini et al. (1991a) used seven floral morphological

features to distinguish two species (L. hexapetala and L. grandiflora) which included

petal length, sepal length, style length, long and short filament length, ovary length,

capsule length and pollen diameter (see Figure 2-1). We did not find capsules in the

field for all populations so, capsule length was excluded from our study. In addition to

the six floral characters that Zardini used, we collected data on petal width, petal

margin, pedicel length and number of petals or sepals per flower.

The floral character’s petal length, petal width, sepal length and petal margin

were measured using a benchtop mounted 5X magnifying lens. A dissecting

microscope (United Scope LLC dba AmScope, 14370 Myford Rd. Irvine, CA 92606)

was used to measure the length of the long and short filaments, style length, ovary

length and pedicel length. The diameter of pollen grains was measured under a

compound microscope (United Scope LLC dba AmScope, 14370 Myford Rd. Irvine, CA

92606) at 400X magnification. In 2016, field locales were visited again to collect flowers

for floral measurements in the interest to compare the field with tank data. In the field,

flowers were collected from Alligator lake, Hawthorne, and Lake Toho. We found few

flowers in Lake Poinsett due to elevated water levels and no flowers in Lake Harney,

25

because Harney had been treated with herbicides. Therefore, the field flower data for

Lake Poinsett and Lake Harney were taken from Dr. Jacono’s previously collected data

in 2014. A sampling of 50 to 55 mature flowers were collected from Alligator lake and

immediately measured in the field to obtain petal length and width. From Hawthorne and

Lake Toho, 50 to 55 mature flowers were collected and transported to the laboratory in

dry plastic bags in an ice chest. Floral metrics measurements for both common garden

and field studies are described as follows.

Multiple and Single Measurements and Observations

For multiple, at least three measurements were taken from each flower for petal

length, petal width, petal margin, sepal length, long and short filament length. For single,

only one measurement was taken for style length, ovary length, pedicel length and the

number of petals or sepals were counted from each flower. The petal length, petal

width, petal margin, sepal length and long filament length characters were measured

from both common garden and field study. The short filament length and pollen

diameter characters were only measured from the common garden study.

Petal length (cm) - from base of the claw to outer margin of petal.

Petal width (cm) - from base of the claw when the ruler moving up towards the outer margin.

Sepal length (cm) - base to the tip.

Long and short filament length (mm) - base to connection with the anther.

Petal margin - described as a) emarginate, a single central notch. b) entire (straight) and c) erose (irregular, shallow notches).

Style length (mm) - base of the style to the lower rim of the stigma.

Ovary length (cm) - top of the receptacle to just below the bracteoles, where the ovary constricts to the pedicel.

26

Pedicel length (cm) - from base where the ovary constricts to the base of pedicel where it connects to the stem.

Number of petals/sepals - petals or sepals were counted per flower.

Pollen diameter (µm) from each population, 20 to 25 flowers were sampled. Care

was taken to sample pollen only from fully dehisced anthers in the afternoon from five

populations growing in the common garden tanks. Five flowers were picked each time

and placed in a small plastic box with moist paper and immediately carried to the lab.

Two anthers were excised from each flower and placed on a glass slide with 30 µl

water. Anthers were tapped using a straight edge razor blade to release the pollen

grains into the solution. 20 µl of lacto-phenol (cotton blue) was added and stain allowed

to occur at room temperatures for 5-10 minutes. A coverslip was placed and gently

pressed with a finger. The diameters of 10-15 pollen grains were measured from each

of five replicate slides using an ocular micrometer installed in the eyepiece (1 division =

2.5 µm). A total of 60 to 64 pollen grains were measured from each population. The

diameter was measured from the outer stained margin of one pore across the center to

the opposing margin. Only one pore was included in the measurement.

Foliar Morphology

The foliar morphological data from each accession growing under common

garden conditions were collected during November 2015, May 2016 and May 2017.

Zardini et al. (1991a) used three foliar descriptive characters; pubescence of stems and

leaves, leaf shape and apex of leaves to distinguish the two species. Based on our

preliminary data collected on foliar characters the pubescence of stems and leaves and

the leaf apex or leaf gland did not differ between our populations. All five populations

were sparsely to densely villous and glands were present on leaves of all populations

27

during late creeping/floating to early emergent growth stages. The shape of the leaf

apex and the degree of hairiness on stem and leaves greatly varied and depended upon

life stages of the plant.

Mature emergent stems were randomly sampled from each tank for the

measurement of principle features. From several stems the 5th to 10th leaf from the tip

of emergent stems were sampled from each population and the mature, fully expanded

55 to 64 leaves were measured for foliar metrics. Characters measured were leaf

length, leaf width, and petiole length. Additionally, the length from the leaf base to the

widest width of the leaf was measured to factor by total leaf length in order to devise

quantitative data depicting leaf shape. The leaf shape was also described based on its

outline. The same foliar characters were measured from herbarium specimens collected

during 2014 to 2016 from field visits. Foliar parameter measurements for both common

garden and herbarium specimens are described as follows.

Leaf length (cm) - base of the leaf, excluding petiole, to the tip of leaf.

Leaf width (cm) - widest width of leaf.

Petiole length (cm) - from base of petiole, where it connects to the stem to the base of leaf.

Distance from leaf base to widest leaf width (cm) - from base of the leaf to the widest width of leaf.

Leaf shape - described as a) lanceolate, b) oblanceolate, c) elliptic or d) lanceolate-elliptic.

After collecting all common garden and field morphological data (floral and foliar),

a normality test was performed using QQ plots in R software (version 3.2.2) (R

Development Core Team 2013). All data met the normality assumption. Morphological

data was then subjected to analysis of variance (ANOVA) at a significance level of

28

α=0.05 and means were compared using a post-hoc Tukey’s honestly significant

difference (HSD) at p<0.05. Categorical data including petal margin and leaf shape

were analyzed using a contingency table with a Chi-squared test at p<0.05 level of

significance again using R software.

Results and Discussion

Floral Morphology

Significant differences (p<0.001) were found between the five creeping water

primrose populations for all floral morpho-metrics from the common garden tanks (Table

2-1). The mean comparision performed using a post-hoc Tukey’s HSD test

demonstrated that all of the floral metrics for Alligator and Harney populations were

significantly greater (p<0.001) than populations from Hawthorne, Poinsett and Toho.

This indicated overall larger flowers on Alligator and Harney than the other three

populations. The petal length, petal width and sepal length for Alligator and Harney did

not differ significantly from each other but were significantly greater than Hawthorne,

Poinsett and Toho. Based on these results we segregated the populations into two

groups; 1) Alligator and Harney (Figure 2-2) and 2) Hawthorne, Poinsett and Toho

(Figure 2-3). That means group 1 have similar flower size; however, group 2 was

significantly different from group 1. There were no differences in petal length between

Hawthorne and Toho but Poinsett had greater petal length than those two populations

(Table 2-1), demonstrating that Poinsett had larger flowers than the other two

populations in group 2. The petal width was different between all members of group 2.

Poinsett had wider petals followed by Toho and Hawthorne had narrow petals. The

sepal lengths of Hawthorne and Poinsett were not different from each other. Toho had

29

lower values for almost all floral characters and it had much smaller flowers than

Hawthorne and Poinsett populations .

The length of the long and short filaments were not only significantly different

between the two groups but also significantly different within the two groups (Table 2-1).

Long and short filament lengths were two of most important characters demonstrating

significant differences between two groups from the common garden data (p<0.001).

The data collected from the field for long filament length from Alligator,

Hawthorne and Toho were also significantly different between each other (p<0.001).

Due to the unavailability of flowers from Harney and Poinsett in the field in 2016, the

long filament data was missing for those two populations (Table 2-2). Additionally, the

petal length and sepal length from the field demonstrated significant differences

between the two groups, similar to what was found with the common garden floral data.

However, the petal width was highly variable in the field and the mean petal width of

Toho was overlapping with group 1 (Alligator and Harney). Hawthorne still had

significantly narrower petal width than all other populations. With the exception of petal

width, all other multi measurement floral characters (Tables 2-1 and 2-2) were

significantly different between the two groups and there was no overlap between their

mean size range. From both the common garden and field, the petal length, sepal

length and short and long filament length indicated that the flowers, although different in

size from each other, fall into two groups (Figures 2-2 and 2-3). These results agree

with the findings of Zardini et al. (1991a) that L. hexapetala produces generally larger

flowers as our group 1 populations and L. grandiflora produces smaller flowers as our

30

group 2 populations. Our floral mertics were similar to that which Zardini et al. (1991a)

reported (see Figure 2-1).

The erose petal margins of Alligator and Harney flowers (Figure 2-2) were

different from group 2. Flowers from Hawthorne had entire, Poinsett had emarginate

and Toho had petal margins between emarginate-erose (Figure 2-3). The descriptive

outcomes of petal margin was significantly different between the two groups as

indicated by chi-squared analysis (data not shown) which suggested no difference in

petal margin within group 1 and three different petal margins within group 2.

In addition to petal margin we also looked at the ratio of petal width to petal

length). We wanted to see if we could use this ratio to find a variable representative of

petal shape. Also we looked at the ratio of pedicel length by ovary length to examine the

relationship between ovary and pedicel length (data not shown). The ratio of pedicel

length by ovary length did not reveal any relationship between each other for all

populations. The ratio of petal width by length was significantly greater for Toho and

Poinsett followed by Alligator and Harney, and Hawthorne had a lower ratio than all

other populations in both common garden and field collections (data not shown). That

means the petal shape for Alligator and Harney were similar, and Toho and Poinsett

were similar, yet Hawthorne had a different petal shape.

The pollen diameter data collected from the common garden condition was

significantly different (p<0.001) between the populations (Figures 2-4 and 2-5). The

mean pollen diameter of Alligator and Harney (115.75 and 113.32 µm, respectively)

were significantly greater than all members of group 2. Toho had a significantly lower

mean pollen diameter (89.92 µm) than Hawthorne and Poinsett (96.58 and 97.88 µm,

31

respectively). The values of 115 and 113 are similar to what Baker and Baker (1982)

found. They reported L. uruguayensis as having a pollen diameter of 105.8 µm. Based

on our findings we presume this to be L. hexapetala. Our pollen diameter values range

between 90 to 98 µm for group 1 and 113 to 116 µm for group 2, which was little more

than Zardini’s values. Zardini et al. (1991a) reported pollen diameter of L. hexapetala

(7.7-9.6 µm) and L. grandiflora (6.6-8.1 µm) see Figure 2-1, which was 10-12 times

smaller and we suspect that might be a printing mistake with the decimal points.

Toho constently produced more petals or sepals (6, 7 and sometime 8) per

flower compared to other populations in the common garden study (Table 2-3). The

style, ovary and pedicel length were significantly greater for Alligator and Harney

compared to Hawthorne, Poinsett and Toho (Table 2-3). The style length was also

significantly different between Hawthorn, Poinsett and Toho. However, the ovary and

pedicel length were not significantly different between Hawthorne, Poinsett and Toho

populations.

The additional field data for the same floral characters revealed the style and

pedicel length from field collected samples were significantly greater for Alligator and

Harney compared to Hawthorne, Poinsett and Toho (Table 2-4). However, the mean

ovary length of Harney was not significantly different from Toho in field data. These

variations might be due to one time short period sampling of flowers in the field or it also

might be due to Toho having a lot more variability than other populations. It is likely that

Toho has either more genetic diversity or more phenotypic plastisity than the other

populations which caused overlap in ovary length trait in field data.

32

The plants from all five populations were glabrous in early growth stage while

floating on the water surface. Examination of the hairiness on the leaves (upper and

lower surfaces) and stems of all five populations indicated they were all sparsely to

densely villous at the emergent (mature) growth stage. The ovary hair of all five

populations was densely villous (hairy) and did not differ between all five populations in

the common garden study (data not shown). Additionally, the degree of hairiness on

leaves and stems greatly varied between life stages. As a result of these observations,

data was not collected on degree or extent of plant hair.

Foliar Morphology

The quantitative foliar metrics (leaf length, leaf width, petiole length and distance

from leaf base to widest leaf width) were collected from both common garden and field.

Differences were found for petiole length between the two groups. Thus, petiole length

appears to be a strong character which may help in identification. We worked further to

examine the leaf shape in quantitative data rather than just visual observations

(categorical data). We calculated the two variables, 1) ratio of leaf length by petiole

length and 2) ratio of the distance from the leaf base to the widest leaf width by leaf

length. Both variables were significantly different between the two groups under

common garden conditions (Table 2-5). These two variables turned out to be very

useful in representing petiole length relative to leaf length and leaf shape. The ratio of

leaf length by petiole length was greater in Hawthorne followed by Poinsett and Toho,

and Alligator and Harney had significantly lower values for the ratio of leaf length by

petiole length from the common garden data (Table 2-5). That means Hawthorne,

Poinsett and Toho populations have smaller petiole length relative to leaf length.

Alligator and Harney populations have higher petiole length relative to leaf length. In

33

field data, the ratio of leaf length by petiole length appeared to have a similar trend to

the common garden study, except Poinsett was not statistically different from group 1.

However, it had a smallar petiole length than Alligator and Harney.

The variable which represented leaf shape was found to be the most useful

across field and common garden conditions. The ratio of the distance from the leaf base

to the widest leaf width factored by total leaf length was highly significant from both the

common garden and field data (Table 2-5). This ratio was significantly greater for group

1 than group 2. This quantitative leaf metric (the ratio of the distance from the leaf base

to the widest leaf width by leaf length) of more than 0.5 represent oblanceolate, less

than 0.5 represents lanceolate and near or equal to 0.5 represents elliptic leaf shape

(Table 2-5). The metrics translated to a well known term for the leaf shape, greater

values for Alligator and Harney represent oblanceolate leaf shape and lower values for

Hawthorne, Poinsett and Toho represent lanceolate or lanceolate-elliptic leaf shape

(Figure 2-6). The Chi-square analysis performed on categorical data on leaf shape also

confirmed the significant differences between two groups, and ANOVAs on the ratio of

distance from the leaf base to the widest leaf width by leaf length indicated that fully

matured leaves from post-flowering populations had stable and dependable leaf shape

character for field identification (Table 2-5). Based on these results the post-flowering

plants of Alligator and Harney produced oblanceolate leaves, Hawthorne and Toho

produced lanceolate-elliptic leaves, Poinsett produced lanceolate leaves (Figure 2-6).

These results support the Zardini et al. (1991a) descriptive leaf characters that L.

hexapetala produce oblanceolate leaves and L. grandiflora produce lanceolate leaves.

34

The floral and foliar morpho-metrics were significantly different between the two

groups. These metrics are consistent and support the findings of Zardini et al. (1991a)

and these floral and post floweing mature foliar characters are stable and discernable

markers for distinguishing the two species within the L. uruguayensis complex. In

addtion to morphology, chromosome work conducted on these five populations (Jacono

et al. unpublished data) revealed the two populations from Alligator and Harney were

decaploid (2n = 80) and the three populations from Hawthorn, Poinsett and Toho were

hexaploid (2n = 48). These chromosome counts follow those of Raven and Tai (1979)

and Zardini et al. (1991b).

In summary, we performed a statistical analysis by considering two groups (1.

Alligator and Harney populations, and 2. Hawthorne, Poinsett and Toho populations). All

floral characters including petal length, sepal length, short and long filament length,

style length, ovary length, pedicel length and pollen diameter and mature foliar

characters including petiole length and ratio of of distance from the leaf base to the

widest leaf width by leaf length were used to perform analysis. Significant differences

were found for all floral and foliar morpho-metrics at p<0.001 (Table 2-6) between the

two groups. We also reported minimum, mean and maximum values for all floral

characters. The maximum values of group 2 were overlapping with minimum values of

group 1 (Table 2-6). However, the mean values between two groups were significantly

different and there was no overlap between their size range. Our findings are in line with

findings of Zardini et al. (1991a) in separation of two species within the L. uruguayensis

complex; our data supports her work and we follow the nomenclature of L. hexapetala

for our group 1 and L. grandiflora for our group 2. Our results do not support the broad

35

implication made by Nesom and Kartesz (2000) that intermediacy is interfering with

species identification in the southeastern U.S.

Overall our floral and foliar morpho-metrics summary (Table 2-6) along with

chromosome data by Dr.Jacono confirms the presence of two distinct species within the

creeping water primrose (L. uruguayensis complex) in Florida. Two populations from

Alligator and Harney, with large flower parts, erose petal margin, oblanceolate leaves

and chromosome number 2n = 80 were L. hexapetala. The other three populations from

Hawthorne, Poinsett and Toho, with small flower parts, entire, emarginate or

emarginate-erose petal margin, leaf shape lanceolate-elliptic and chromosome number

2n=48 were L. grandiflora. Our floral results also demonstrated variability within the

three L. grandiflora populations. Hawthorne had small flowers with entire petal margin,

and elliptic leaves. Whereas, Poinsett had a relatively larger flower than the other two L.

grandiflora populations, with emarginate petal margin and a lanceolate leaf shape. Toho

had small flowers with emarginate-erose petal margin and lanceolate-elliptic leaf shape.

However, the chromosome counts were the same for all three L. grandiflora

populations.

Future studies should focus on identifying the source of population variability

within L. grandiflora in Florida and how this variability might be related to differential

herbicide efficacy of L. grandiflora in Florida.

36

Table 2-1. Floral morpho-metrics of five Ludwigia uruguayensis populations from the common garden study, 2016.

Locale Petal length, cm Petal width, cm Long filaments

length, cm

Short filaments

length, cm

Sepal length, cm

Alligator 2.79 (0.19) a 2.13 (0.13) a 0.72 (0.04) a 0.53 (0.04) a 1.69 (0.08) a

Harney 2.82 (0.15) a 2.10 (0.14) a 0.68 (0.04) b 0.49 (0.03) b 1.71 (0.09) a

Hawthorne 2.26 (0.11) c 1.63 (0.13) d 0.61 (0.05) c 0.43 (0.03) d 1.38 (0.10) b

Poinsett 2.35 (0.13) b 1.94 (0.14) b 0.58 (0.03) d 0.45 (0.04) c 1.39 (0.07) b

Toho 2.27 (0.13) c 1.88 (0.20) c 0.50 (0.06) e 0.33 (0.05) e 1.30 (0.07) c

F value (df) F(4)= 374.5 F(4)= 175 F(4)= 339.8 F(4)= 316 F(4)= 503.1

P value p<0.001 p<0.001 p<0.001 p<0.001 p<0.001

Values in parenthesis are standard deviations (sd), data analyzed using Anova and means compared by Tukey’s HSD.

Means followed by the same lowercase letter are not significantly different (p = 0.05)

37

Table 2-2. Floral morpho-metrics of five Ludwigia uruguayensis populations from field collections in 2014 and 2016.

Locale Petal length, cm Petal width, cm Long filaments length,

cm

Sepal length,

cm

Alligator 2.47 (0.15) a 1.90 (0.13) a 0.71 (0.04) a 1.63 (0.12) a

Harney 2.54 (0.27) a 1.95 (0.22) a 1.51 (0.05) b

Hawthorne 1.99 (0.09) d 1.40 (0.09) c 0.59 (0.03) b 1.31 (0.09) c

Poinsett 2.07 (0.17) c 1.71 (0.16) b 1.22 (0.06) d

Toho 2.17 (0.19) b 1.91 (0.19) a 0.51 (0.04) c 1.16 (0.09) d

F value (df) F(4)= 255.7 F(4)= 314.9 F(2)= 1196 F(4)= 518.6

P value p<0.001 p<0.001 p<0.001 p<0.001


Means followed by the same lowercase letter are not significantly different (p = 0.05).

38

Table 2-3. Floral morpho-metrics of five Ludwigia uruguayensis populations from the common garden study, 2016.

Locale Number of petals or

sepals

Style length, cm Ovary length,

cm

Pedicel length,

cm

Alligator 5.08 (0.28) b 0.92 (0.03) a 1.29 (0.11) a 2.59 (0.36) b

Harney 5.04 (0.20) b 0.92 (0.04) a 1.26 (0.11) a 2.88 (0.45) a

Hawthorne 5.09 (0.29) b 0.72 (0.04) c 0.88 (0.09) b 0.91 (0.22) d

Poinsett 5.08 (0.28) b 0.77 (0.08) b 0.90 (0.09) b 1.18 (0.23) c

Toho 5.41 (0.57) a 0.60 (0.03) d 0.67 (0.13) c 1.12 (0.30) cd

F value (df) F(4)= 5.001 F(4)= 248.1 F(4)= 162.8 F(4)= 223.4

P value p<0.001 p<0.001 p<0.001 p<0.001



39

Table 2-4. Floral morpho-metrics of five Ludwigia uruguayensis populations from field collections in 2014 and 2016.

Locale Number of petals or sepals Style length, cm Ovary length,

cm

Pedicel length,

cm

Alligator 5.13 (0.33) a 0.91 (0.03) a 1.23 (0.11) a 2.71 (0.54) a

Harney 0.75 (0.00) b 0.92 (0.07) b 2.93 (0.58) a

Hawthorne 5.00 (0.00) b 0.69 (0.03) c 0.76 (0.09) c 0.64 (0.24) c

Poinsett 0.68 (0.04) c 0.70 (0.07) c 0.93 (0.19) c

Toho 5.02 (0.14) b 0.58 (0.04) d 0.91 (0.16) b 1.52 (0.28) b

F value (df) F(2)= 5.108 F(4)= 630.2 F(4)= 113.2 F(4)= 229.5

P value p=0.007 p<0.001 p<0.001 p<0.001



40

Table 2-5. Foliar morpho-metrics of five Ludwigia uruguayensis populations from the common garden study (2015, 2016 and 2017) and field collections (2014, 2015 and 2016).

Locale Common garden Field

Petiole length,

cm

Leaf

length/petiole

length

Distance from

leaf base to

widest leaf

width/leaf length

Petiole length,

cm

Leaf

length/petiole

length

Distance from

leaf base to

widest leaf

width/leaf length

Alligator 1.44 (0.51) b 5.17 (1.82) c 0.72 (0.07) a 1.84 (1.05) a 7.71 (3.79) c 0.59 (0.05) b

Harney 1.99 (0.75) a 5.45 (2.41) c 0.64 (0.10) b 1.38 (0.62) b 7.47 (5.85) c 0.67 (0.09) a

Hawthorne 0.43 (0.17) d 24.18 (13.42) a 0.43 (0.07) d 0.47 (0.22) d 27.48 (18.71) a 0.47 (0.05) c

Poinsett 0.89 (0.26) c 10.87 (3.64) b 0.48 (0.06) c 0.95 (0.36) c 10.20 (3.60) c 0.42 (0.05) d

Toho 1.26 (0.52) b 11.89 (14.02) b 0.48 (0.09) c 0.41 (0.15) d 21.08 (6.24) b 0.46 (0.05) c

F value (df) F(4)= 87.05 F(4)= 43.14 F(4)= 125.5 F(4)= 38.35 F(4)= 31.59 F(4)= 117.3

P value p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001



41

Table 2-6. Floral and foliar morphological characters differentiating Ludwigia grandiflora and L. hexapetala.

Characters L. grandiflora (2n = 48)

min. (mean) max.

Standard

deviations

L. hexapetala (2n = 80)

min. (mean) max.

Standard

deviations

Petal length (cm) *** 1.6 (2.2) 2.6 0.19 1.9 (2.6) 3.1 0.24

Sepal length (cm) *** 0.9 (1.3) 1.6 0.12 1.3 (1.7) 2.0 0.11

Style length (mm) *** 4.5 (6.6) 8.3 0.08 7.5 (9.1) 10.5 0.04

Long filament length (mm) *** 3.0 (6.0) 7.0 0.06 6.0 (7.0) 8.0 0.04

Short filament length (mm) *** 2.0 (3.9) 5.0 0.07 4.0 (5.1) 6.0 0.04

Ovary length (mm) *** 5.0 (8.2) 13 0.15 8.0 (12) 15 0.17

Pedicel length (cm) *** 0.3 (1.1) 2.2 0.41 1.5 (2.8) 3.9 0.51

Pollen diameter (µm) *** 78.8 (94.8) 106.3 5.56 92.5 (114.5) 142.5 10.44

Ratio of distance from leaf base to widest

width/leaf length***

2.0 (4.2) 7.6 0.07 3.3 (5.5) 10.9 0.09

Leaf shape*** Lanceolate-elliptic Oblanceolate

Petiole length (cm) *** 0.1 (0.75) 2.3 0.45 0.3 (1.67) 5.2 0.78

*** Two species (L. grandiflora and L. hexapetala) were significantly different at p<0.001. Data analyzed using Anova and

means compared by Tukey’s HSD.

42

Figure 2-1. Scan of Table 1. providing morphological characters differentiating Ludwigia grandiflora and L. hexapetala from Zardini et al. (1991a).

43

A B

Figure 2-2. Flower picture of two populations from group 1, A) Alligator and B) Harney (Photo courtesy of author).

A B C.

Figure 2-3. Flower picture of three populations from group 2, A) Hawthorne, B) Poinsett and C) Toho (Photo courtesy of

author).

44

Figure 2-4. Pollen diameter (µm) of five Ludwigia uruguayensis populations. The mean pollen diameters were represented with box and whisker plots. The dark horizontal line in the box indicates the median, the whiskers indicate the minimum and maximum data points and the open circles indicate outliers).

45

Figure 2-5. Monad pollen grain microscopic images at 400X magnification for A) group 1 (Harney) population (118 µm, measured left to right represented with a yellow line) and B) group 2 (Toho) population (95 µm, measured diagonally top to bottom represented with a yellow line).

A B

46

Figure 2-6. Leaf scan of five Ludwigia uruguayensis populations (Alligator, Harney, Poinsett, Toho and Hawthorne) from common garden and field.

47

CHAPTER 3 RESPONSE OF LUDWIGIA HEXAPETALA AND LUDWIGIA GRANDIFLORA TO

SELECTED AQUATIC HERBICIDES

Introduction

In addition to previous difficulties in accurate identification, Ludwigia hexapetala

and L. grandiflora have also proven to be difficult to control with herbicides in Florida.

Many aquatic managers have reported rapid regrowth following herbicide treatments.

However, reports of variable efficacy are common and a potential link between

morphological type and herbicide vulnerability has been observed. Meisler (2009)

reported that neither glyphosate at 3,364 g ae ha-1 nor triclopyr at 840 g ae ha-1

provided effective control of a Ludwigia hexapetala infestation in Northern California.

Glyphosate and triclopyr are systemic herbicides, but neither provide complete control

of Ludwigia hexapetala as regrowth is a common occurrence following foliar treatments

with those herbicides. Furthermore, Ludwigia hexapetala responded very quickly to

triclopyr when applied on foliage even at low rates, but regrowth was also rapid (Meisler

2008). Based on visual observations, glyphosate was less effective than triclopyr. The

possible reasons reported for poor glyphosate efficacy were 1) airboat wash off

following herbicide application; 2) muddy conditions which may result in inactivation of

glyphosate molecules (glyphosate rapidly binds to soil particles and become inactive);

or 3) limited foliar coverage of herbicide due to very dense plant populations (Meisler

2008; Shaner 2014).

Application of glyphosate at 2.28 kg ai ha-1 combined with an adjuvant applied at

1 L ha-1 during June or July has been reported to be an effective dose and appropriate

application timing for controlling L. grandiflora (Plant Protection Service 2011). From 73

to 81% of plant biomass of creeping water primrose (Ludwigia hexapetala) was

48

controlled by application of flumioxazin at 437 g ai ha-1, which had an EC70 value of 120

g ai ha-1 (Richardson et al. 2008). Carfentrazone-ethyl did not control creeping water

primrose greater than 64% even at the high tested rate of 224 g ai ha-1 (Richardson et

al. 2008). Emerine et al. (2010) reported that the dry weight of creeping water primrose

was decreased with increasing imazamox rates. The EC70 value for imazamox on dry

weight of creeping water primrose was 129 g ha-1 and the EC70 value for regrowth dry

weight was 115 g ha-1. No differences were found in creeping water primrose dry

weights following treatments with imazamox, imazapyr or glyphosate. However, all three

herbicides resulted in lower dry weights compared to the untreated control (Emerine et

al. 2010). Better visual control (by 92% and 93%, respectively) of creeping water

primrose was observed with glyphosate and imazapyr treatments, but the imazamox

treatment resulted in only 80% visual control (Emerine et al. 2010). In the United

Kingdom (UK), an approximately 75% weight reduction of Ludwigia grandiflora was

achieved with an application of glyphosate at 2.16 kg ai ha-1 and a mixture of

glyphosate + 2,4-D amine at 2.16 kg + 211 g ai ha-1. However, a 98% Ludwigia

grandiflora weight reduction was achieved with glyphosate + a non-oil soya sticking

agent application (Defra 2007). Small patches of water primrose were successfully

eradicated from twelve invaded sites in the UK by using mechanical and herbicide

control methods (Renals 2010).

Several herbicides are registered for aquatic use but few control Ludwigia in

aquatic situations. Limited information is available on the response of Ludwigia

populations to many aquatic herbicides and it is necessary to further identify the factors

that influence herbicide control of L. hexapetala and L. grandiflora in Florida. The

49

objectives for these herbicide studies were: 1) to evaluate the response of two

populations of Ludwigia hexapetala and three populations of Ludwigia grandiflora to

selected aquatic herbicides and 2) to evaluate the response of Ludwigia hexapetala and

Ludwigia grandiflora to the most commonly used tank mix herbicides.


Dose Response Study

The growth response of two populations of L. hexapetala and three populations

of L. grandiflora collected from five locales across Florida were evaluated for three

different aquatic herbicides in greenhouse experiments at the University of Florida,

Center for Aquatic and Invasive Plants (CAIP), in Gainesville, FL. A total of 200 stem

cuttings, each 15 cm in length, were collected from five stock tanks (40 stem cuttings

from each population) containing L. hexapetala and L. grandiflora populations as

described in chapter 2. Cuttings were planted in 3.8 L plastic pots filled with a

commercial greenhouse potting medium (Professional Top Soil, Margo Garden

Products, Inc., Folkston, GA 31537) mixed with 4 g of 15-9-12 complete slow release

fertilizer (Osmocote Plus, The Scotts Company, 14111 Scotts Lawn Rd, Maryville, OH

43041) and covered with 5 cm of sand to keep the organic potting media in the pot. Ten

planted pots were placed in 80 L rectangular tubs filled with water and a 3 to 5 cm water

level was maintained above the top of the 3.8 L pots for the duration of the study period.

Plants were grown for 35 to 40 days in a greenhouse under ambient environmental

conditions prior to applying the herbicide treatments.

Insects and fungal diseases were controlled by using mixture of insecticide

bifenthrin + zeta-cypermethrin at 0.20 + 0.05 g ai in 5.68 L of water (Bug B Gon,

ORTHO group, Marysville, OH 43040) and the fungicide azoxystrobin at 0.43 g ai in

50

5.68 L of water (Heritage, Syngenta crop protection LLC, Greensboro, NC 27419-8300)

and one or two applications were made as needed.

These herbicide experiments were conducted by taking one herbicide at a time

and testing that herbicide on two populations of L. hexapetala and three populations of

L. grandiflora. The treatments consisted of three aquatic herbicides that vary in their

selectivity and mode of action and included imazamox (Clearcast, SePro Corporation,

11550 North Meridian Street, Carmel, IN 46032) triclopyr (Garlon 3A, Dow

AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, IN 46268) and glyphosate (Rodeo,

Dow AgroSciences LLC, 9330 Zionsville Rd, Indianapolis, IN 46268). The first

(imazamox), second (triclopyr) and third (glyphosate) herbicide trials were planted in

May, July and August 2016, respectively and treated 5 to 6 weeks after planting. From a

total 200 planted pots, 160 well grown plants from all five populations were selected for

each herbicide trial. Each herbicide was tested at seven rates (Table 3-1) and

compared with non-treated controls. All herbicides treatments included a nonionic

surfactant (Induce, Helena Chemical Company, 225 Schilling Blvd., Suite 300,

Collierville, TN 38017) at 0.25% v/v.

The herbicide treatments were applied to 35 to 40 days old plants, at the

secondary branching stage using a CO2 pressurized 4 nozzle boom sprayer equipped

with Teejet 8001 EVS spray nozzles (Teejet Technologies Southeast, P.O Box 832,

Tifton, GA 31794) calibrated to deliver 187 L ha-1 at 172 kPa. Treatments were applied

in June, August and September 2016 for the imazamox, triclopyr and glyphosate

herbicide trials, respectively. The pots were removed from tubs and carried outside and

arranged in two straight lines on the ground and herbicides were applied. The treated

51

pots were then allowed to air dry and sides of pots were dipped in clean water to

remove any herbicide residues. Plants were returned to the greenhouse after drying and

maintained as previously described.

Aboveground shoots were clipped at the soil surface 30 days after treatment

(DAT) and discarded. Plants were allowed to regrow for an additional 40 days. At 70

DAT, regrowth shoots were harvested and dried in an oven at 65 C for 96 hours to

obtain dry weights. The experiment was arranged as a completely randomized design

with four replicate pots for each treatment. Nonlinear regression analysis was performed

on binomial data by using the drc package of R (Ritz and Streibig 2005) software

(version 3.2.2) (R Development Core Team 2013). Shoot regrowth dry weight data at 70

DAT was transformed to (alive vs dead) and a two-parameter log-logistic model

(Equation 3-1) appropriate for binomial data was used to estimate the herbicide dose

causing a 50% regrowth dry weight reduction (ED50) for each population. The two-

parameter log-logistic model (Equation 3-1), the upper and lower limits are 1 and 0,

respectively.

f (x) = 1/1 + exp {b [log(x) – log (e)]} (3-1)

Where f (x) is the probability of two L. hexapetala and three L. grandiflora

populations shoot regrowth and x herbicide rate (g ae ha-1), b is the relative slope at the

inflection point and e is the inflection point of the fitted line (equivalent to the dose

required to cause 50% reduction in dry weight). A lack-of fit test at the 95% confidence

level comparing the regression model (Equation 3-1) to ANOVA was used to determine

whether the regression model was an appropriate fit to the data (Ritz and Streibig

2005).

52

Tank Mix Comparisons

For this study, one population of Ludwigia hexapetala from Alligator lake and one

population of Ludwigia grandiflora from lake Toho were selected and planted as

previously described. Three planted pots were then placed in 100 L tubs and a total of

30 tubs were planted for each species. After planting, the tubs were maintained in

ambient greenhouse conditions and plants allowed to grow for 60 days until they were

well established. The water level was then raised to 5 to 10 cm above the 3.8 L pots in

each 100 L tub.

Treatments provided in (Table 3-1) included imazamox alone and in combination

with carfentrazone-ethyl (Stingray, SePRO Corporation, 11550 North Meridian Street,

Carmel, IN 46032) and glyphosate alone and in combination with flumioxazin (Clipper,

Valent USA Corporation, PO Box 8025, Walnut Creek, CA 94596) or imazapyr (Habitat,

SePRO Corporation, 11550 North Meridian Street, Carmel, IN 46032). A non-ionic

surfactant (Induce at 0.25% v/v) was added to all herbicide treatments, which were

applied by using a single nozzle micro sprayer equipped with a TeeJet 800067 nozzle at

an application volume of 935 L ha-1. The plants were treated on February 23, 2017 and

uniform water levels were maintained throughout the study.

Aboveground shoots were harvested at 30 DAT and dry weight was determined.

Plants were then allowed to regrow for 30 more days. At 60 DAT, both aboveground

shoots and belowground roots were harvested and dry weight was recorded as

previously described. The experimental design was a completely randomized design

with two factors (two species and five herbicide treatments). Each herbicide treatment

was randomly assigned to 100 L tubs, and a 100 L tub was considered as the

experimental unit and treatments were replicated five times. Aboveground shoot and

53

belowground root weight data followed the assumptions of normality and homogeneity

of variance and data was then subjected to analysis of variance (ANOVA) with

interaction (herbicide treatment by species) at a significance level of p<0.05. Multiple

comparisons were done using Tukey’s HSD at p<0.05 in R software (version 3.2.2) (R

Development Core Team 2013).


Dose Response Study

All three herbicides (imazamox, triclopyr and glyphosate) reduced the regrowth

weight of two Ludwigia hexapetala (Alligator, Harney) and three Ludwigia grandiflora

populations (Hawthorne, Poinsett and Toho) at 70 DAT. The two-parameter log-logistic

model provided the best fit to estimate the probability of shoot regrowth of the five

populations after application of each herbicide. A lack of fit test at the 95% level was not

significant, indicating that the regression models were appropriate (Ritz and Streibig

2005).

The probability of shoot regrowth of all five populations decreased as imazamox

herbicide rate increased (Figure 3-1). The ED50 (Effective Dose that reduced regrowth

dry weight by 50%) values for Alligator, Harney, Hawthorne and Toho were 55.5, 56.5,

41.9 and 56.5 g ae ha-1, respectively and were not different from each other (Table 3-2).

The ED50 values of all four populations were equivalent to approximately 15 to 20% of

the field use rates. Our findings of ED50 values were slightly less than those of Emerine

et al. (2010) who reported the EC70 values for creeping water primrose regrowth dry

weight was 115 g imazamox ha-1. However, the ED50 values for Poinsett was

significantly greater than three populations (Alligator, Harney and Hawthorne) but not

different from Toho (Table 3-2). The inflection point slope (b) estimates for the four

54

populations (Alligator, Harney, Hawthorne and Toho) were significant except for

Poinsett (Figure 3-1). The ED50 value of Poinsett was 220.5 g ae ha-1, equivalent to

78.8% of the typical field use rate (280 g ae ha-1). These results indicate the Poinsett

population is less sensitive to imazamox compared to all other populations and would

require higher imazamox rate to control this population.

The triclopyr rate required to provide a 50% shoot regrowth reduction was

predicted to range from 6.5 to 27.2 g ae ha-1 (Table 3-2), which is equivalent to 0.3-1.1%

of the field use rate (2,524 g ae ha-1). There were no significant differences between the

ED50 values of all five populations. Furthermore, the slope (b) estimates for all five

populations were significant, indicating all five populations were very sensitive to

triclopyr herbicide at much lower rates than actual field use rates. Our results were

similar to those observed by (Dias et al. unpublished data) who reported the sensitivity

of soybean, tomato, sunflower and cotton to four triclopyr formulations, in which

soybean and tomato were very sensitive to triclopyr amine formulation having ED50

values of 22.6 and 22.9 g ae ha-1, respectively. Meisler (2009) indicated that triclopyr

acted very quickly on L. hexapetala at low rates, because both Ludwigia species are

very sensitive to triclopyr and herbicide effects appear very quickly following application.

At very low rates, triclopyr promoted plant growth and acted as growth regulator at 30

DAT as we observed in this study, but at high rates triclopyr provided good control.

The glyphosate ED50 values for two L. hexapetala populations (Alligator and

Harney) and two out of three L. grandiflora populations (Hawthorne and Poinsett) were

equivalent to 7.5 to 13% of the field use rate (4,487 g ae ha-1) and were not significantly

different from each other (Table 3-2). However, the ED50 value for Toho was 998 g ae

55

ha-1, equivalent to 22.2% of the field use rate and was 2 to 3 times greater than the

other four populations. The slope (b) estimates for four populations (Alligator, Harney,

Hawthorne and Poinsett) were significant except for Toho (Figure 3-3). This suggests

the Toho population was less sensitive to glyphosate compared to all other populations.

However, the ED50 values for Toho are still in the range of the field use rate. Glyphosate

is primarily absorbed by green leaves and stems, with little or no uptake through woody

bark tissue. The reason for less sensitivity of the Toho population to glyphosate might

be because Toho had woody lignified stems compare to other L. grandiflora populations

(personal observation) which may have reduced the absorption, uptake and

translocation of glyphosate into the plant.

The results from this study showed differential responses of two populations of L.

grandiflora to two of the three herbicides. L. hexapetala was generally more sensitive to

herbicides than L. grandiflora. But all three herbicides were effective in reducing growth

and survival of newly established L. hexapetala populations (Alligator and Harney) and

one L. grandiflora population (Hawthorne) at current field use rates. However, two L.

grandiflora populations from Poinsett and Toho showed a differential response to

imazamox and glyphosate, respectively.

Tank Mix Comparisons

All herbicide treatments significantly reduced aboveground shoot weight 30 DAT

compared to the untreated controls for both L. hexapetala and L. grandiflora species

(Figure 3-4). There were significant differences between the two species (p=0.05) and

between herbicide treatments (p<0.001). Furthermore, there was a significant herbicide

treatment by species interaction (p<0.001) for the percent reduction in aboveground

shoot weight at 30 DAT. This suggests a differential response of the two species (L.

56

hexapetala and L. grandiflora) to certain herbicide treatments, however no significant

interactions were found for shoot weight within imazamox alone, imazamox +

carfentrazone-ethyl, glyphosate + flumioxazin and glyphosate + imazapyr herbicide

treatments for both species except for the glyphosate alone treatment (Figure 3-4).

There was a reduction (p<0.001) in shoot weight of L. hexapetala by 95% compared to

L. grandiflora (59%) with the glyphosate alone treatment. The shoot weight of L.

hexapetala was lower with glyphosate alone compared to imazamox alone but it was

not significantly different from the other tank-mix herbicide treatments. Whereas, the

shoot weight of L. grandiflora was lower with three tank mix combinations of herbicides

including imazamox + carfentrazone-ethyl, glyphosate + flumioxazin and glyphosate +

imazapyr (99, 83, 81% reduction in shoot weight, respectively) compared to imazamox

and glyphosate (59 and 59% reduction in shoot weight, respectively) alone.

The PPO inhibitor herbicides used in tank mixes provided mixed results at 30

DAT (Figure 3-4). Addition of carfentrazone-ethyl increased efficacy of imazamox in

reducing shoot dry weight of L. grandiflora compared to imazamox alone. Whereas,

addition of flumioxazin did not improve the efficacy of glyphosate in reducing shoot dry

weight of both species at 30 DAT. The differential response between L. hexapetala and

L. grandiflora was only observed with glyphosate alone at 30 DAT. There was no

differential response of both species to other herbicide treatments. Ludwigia hexapetala

had a greater reduction in dry weight than L. grandiflora when treated with 4,482 g ae

ha-1 of glyphosate. These results agree with the data in Table 3-2, which showed the

highest ED50 value of glyphosate to L. grandiflora population from Lake Toho. The ED50

value from Table 3-2 indicates L. grandiflora from Lake Toho had a greater tolerance to

57

glyphosate than the L. hexapetala population. Overall, the tank mix combination of

imazamox + carfentrazone-ethyl on L. grandiflora and glyphosate alone on L.

hexapetala provided better control than imazamox alone on both species.

At 65 DAT, regrowth shoot weight was the same for both species and treatments

and there was no significant treatment by species interaction (Figure 3-5). There was no

differential response in shoot regrowth by both species to any herbicide treatments. The

untreated controls from both L. grandiflora and L. hexapetala had significantly greater

regrowth (19.4 and 24.4 g, respectively) compared to all herbicide treatments. All

herbicide treatments were effective and equally reduced shoot regrowth of both species.

A similar trend was observed with belowground or root dry weights at 65 DAT

and there was again no significant treatment by species interactions (Figure 3-6). The

untreated controls from both L. grandiflora and L. hexapetala had a greater root weight

(4.4 and 4.8 g, respectively) compared to all herbicide treatments. All herbicide

treatments reduced the root weight by 80 to 98%, and there were no significant

differences between any herbicide treatments for both species (Figure 3-6). These

results demonstrated all the herbicide treatments were effective in controlling both L.

hexapetala and L. grandiflora above ground shoot and below ground root weight at 65

DAT. Our findings were different than Enloe and Lauer (unpublished data) who reported

aminopyralid, imazamox alone and in combination with flumioxazin, and glyphosate

alone and in combination with flumioxazin or 2,4-D resulted in good control of L.

hexapetala shoot growth at 60 DAT. However, no herbicide treatments provided

effective control of below water stems and roots. Whereas, in our study all herbicide

treatments had greatly reduced root dry weight likely because in our study we used two

58

month old plants and they use six month old plants which had well established roots

and may have resulted in less effective control. This suggests herbicide efficacy can

vary depending upon plant growth stage and root density or development.

At 65 DAT, we found no differences between tank-mixes of imazamox +

carfentrazone-ethyl, glyphosate + flumioxazin and glyphosate + imazapyr and the

individual imazamox or glyphosate applications to these two Ludwigia species.

Future research should focus on physiological and environmental aspects related

to variable efficacy of L. grandiflora populations and on evaluating additional aquatic

herbicides to control both species in Florida. For example, the results of these studies

indicated the herbicides were very effective (based ED50 values on regrowth data and

tank mix treatment data) on 40 to 60 days old plants. But much higher application rates

are used less effectively on mature plants under field conditions. Age of the plant or

stage of growth need to be investigated further.

59

Table 3-1. Herbicides rates used in the dose response study and tank mix comparisons, applied to emergent plant foliage of Ludwigia hexapetala and L. grandiflora with 0.25% v/v non-ionic surfactant.

Dose response study

Herbicides Rates (g ae ha1)

Imazamox 2.2 8 22 78 247 751 2,242

Triclopyr 1.1 4.5 20 78 314 1,267 5,044

Glyphosate 12.3 37 112 336 998 2,993 8,967

Tank mix comparisons

Herbicides Rates (g ae ha-1)

Imazamox 280

Imazamox + Carfentrazone-ethyl 280 + 67

Glyphosate 4,482

Glyphosate + Flumioxazin1 4,482 + 143

Glyphosate + Imazapyr 4,482 + 560 1 g ai ha-1

60

Table 3-2. Model parameters and standard errors in parenthesis for two-parameter log-logistic model provided in equation 3-1 for figures 3-1,2,3 (shoot regrowth dry weights) for two L. hexapetala populations from Alligator, Harney and three L. grandiflora populations from Hawthorne, Poinsett and Toho in response to three aquatic herbicides.

Model parameters (± SE) Herbicide Locale b ED50

Imazamox Alligator 0.89 (0.31) 55.5 (32.63) a Harney 1.50 (0.55) 56.5 (25.19) a Hawthorne 1.29 (0.46) 41.9 (20.11) a Poinsett 9.69 (60.96) 220.5 (159.09) b Toho 1.50 (0.55) 56.5 (25.16) ab

Triclopyr Alligator 1.15 (0.44) 19.0 (10.64) a Harney 0.97 (0.35) 26.7 (16.32) a Hawthorne 0.53 (0.21) 9.6 (9.39) a Poinsett 1.19 (0.45) 27.2 (14.85) a Toho 1.56 (0.69) 6.5 (3.16) a

Glyphosate Alligator 1.36 (0.49) 582 (263.13) ab Harney 1.56 (0.59) 441 (184.93) a Hawthorne 1.51 (0.56) 335 (143.14) a Poinsett 2.02 (0.84) 335 (123.02) a Toho 9.35 (53.62) 998 (106.80) c


61

Figure 3-1. Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days after imazamox herbicide application.

62

Figure 3-2. Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days after triclopyr herbicide application.

63

Figure 3-3. Probability of survival of two Ludwigia hexapetala (Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and Toho) based upon aboveground shoot regrowth dry weight at 70 days after glyphosate herbicide application.

64

Figure 3-4. Percent reduction in shoot dry weight of L. grandiflora and L. hexapetala at 30 DAT compared to the untreated control (33.7 and 50.7 g, respectively). Error bars represent standard error. Treatments with the same letter are not significantly different (p=0.05) within species. A “*” indicates a treatment is significantly difference between species.

B

A

B*

AB AB

B

AB

A*

ABAB

0

20

40

60

80

100

120

Imazamox Imazamox +Carfentrazone-ethyl

Glyphosate Glyphosate +Flumioxazin

Glyphosate +Imazapyr

% r

eduction

Treatments

L. grandiflora L. hexapetala

65

Figure 3-5. Percent reduction in shoot regrowth dry weight of L. grandiflora and L. hexapetala at 65 DAT compared to the untreated control (19.4 and 24.4 g, respectively). Error bars represent standard error. Treatments with the same letter are not significantly different (p=0.05) within species.

A AA

A

AA A A A A

90

92

94

96

98

100

102

104



Glyphosate + Imazapyr

% r

eduction

Treatments


66

Figure 3-6. Percent reduction in root dry weight of L. grandiflora and L. hexapetala at 65 DAT compared to the untreated control (4.4 and 4.8 g, respectively). Error bars represent standard error. Treatments with same letter are not significantly different (p=0.05) within species.

A

A

A A

A

A

A AA A

0

20

40

60

80

100

120



Glyphosate + Imazapyr

% r

eduction

Treatments


67

CHAPTER 4 COMPARISON OF EARLY GROWTH CHARACTERISTICS AND CREEPING

MORPHOLOGY OF LUDWIGIA HEXAPETALA AND LUDWIGIA GRANDIFLORA

Introduction

The damage caused by invasive plants is often directly related to high growth

rates and biomass production (Hussner 2010). Several aquatic invasive species are

reported to have higher growth rates and biomass production than native species.

Glomski and Netherland (2012) reported the growth rates of hydrilla and Eurasian

watermilfoil, when all lateral branches, new stems and stolons measured, were 487 cm

and 32 cm d-1, respectively 5 weeks after planting. However, the growth rate of individual

shoots of hydrilla may be closer to 2.5 to 10 cm per day (Glomski and Netherland 2012).

A study from Australia estimated that the mean annual rate of lateral expansion of

alligator weed was 4.3 m (2.2 m standard deviation) and the mean dry biomass of

alligator weed for year 2010 was 4.9 kg m-2 (Clements et al. 2011). In the case of water

hyacinth, the highest biomass average was 49.6 kg m-2 with the maximum value of 76 kg

m-2 in Mexico, Cruz Pintada Dam (Gutierrez et al. 2001). In experimental conditions

water hyacinth demonstrated logistic growth with an r 2 of 0.69 to 1.00 (Wilson et al.

2005). Many aggressive Ludwigia species are no exception to this and have been

reported to have higher growth rates and biomass production than native species.

In France, Ludwigia species (L. hexapetala and L. peploides ssp. montevidensis)

were reported to produce up to 4,500 g m-2 dry weight in a eutrophicated river and 200 g

m-2 of dry weight in shallow lakes (Lambert et al. 2010). In California, Ludwigia peploides

was reported to produce 40 to 50 g dry weight m-2 d-1 in experimental conditions and in a

natural stand produced 500 to 700 g dry weight m-2 (Rejmankova 1992). Another study

in France, reported that relative growth rate (RGR) of L. grandiflora was higher than

68

Myriophyllum aquaticum in the emergent growth stage and L. grandiflora RGR was

higher than Egeria densa and Ceratophyllum demersum in submersed or early growth

stage (Thouvenot et al. 2013b). In Germany, studies indicated differences in root to

shoot biomass allocation of two Ludwigia species (L. grandiflora and L. peploides) varied

with water depth and nutrient conditions. Both Ludwigia species increased root

production and decreased shoot production with decreasing water levels and nutrients

(Hussner 2010). In addition to high biomass production capacity, a strong ability to

colonize natural systems, adaption to a wide range of hydrological and climatic

conditions, many Ludwigia species also display a high degree of phenotypic plasticity to

new environmental conditions (Muller 2004).

Studies to quantify seasonal biomass production and allocation are important to

better understand the life cycle dynamics of aggressive Ludwigia species for improved

management practices. It is also important to capture creeping or floating morphological

characters to determine if they are useful for species identification. Controlling new

infestations before they become well established and create dense mats would be useful

to avoid long term management problems.

These two Ludwigia species (L. hexapetala and L. grandiflora) appear to be highly

invasive, and rapid growth rates are key characteristics of many invasive plants.

Therefore, our study objective was to compare the early creeping morphology and

growth characteristic of two Ludwigia hexapetala and three L. grandiflora populations

under common garden conditions to better understand the growth patterns of these

species under similar environmental conditions.

69


An outdoor mesocosm study was conducted in common garden conditions at the

Center for Aquatic and Invasive Plants (CAIP), University of Florida, Gainesville, FL. Two

Ludwigia hexapetala populations from Alligator Lake and Lake Harney and three

Ludwigia grandiflora populations from Hawthorne, Lake Poinsett and Lake Toho were

used in this study. Approximately 20 to 25 cm stem cuttings were collected from all five

populations growing in stock tanks. Single stem cuttings were planted in plastic tubs (35

x 29 x 14 cm) filled with commercial greenhouse potting soil (Professional Top Soil,

Margo Garden Products, Inc., Folkston, GA 31537) mixed with 5-10 g of 15-9-12 slow

release fertilizer (Osmocote Plus, The Scotts Company, 14111 Scotts Lawn Rd,

Maryville, OH 43041) and covered with 8 cm of builder’s sand. After planting, the tub was

placed on concrete blocks (22 cm height) inside each 900 L mesocosm. A single plastic

tub was maintained in each 900 L mesocosm to facilitate lateral growth and expansion of

each plant. The water level was maintained 5 cm above the plastic tub in each 900 L

mesocosm.

The first experimental run was planted on 11 April, 2016 and the growth

characteristics including total shoot length, mean stem width and mean internode length

were collected once every two weeks over a period of seven weeks. The initial collection

occurred three weeks after planting (WAP). During experimental run 2016 we observed

tremendous shoot abscission in three L. grandiflora populations from Hawthorne,

Poinsett and Toho. Additional data was collected on the number of abscised shoots and

total number of shoots at 3 WAP. The early growth morphological characters including

petiole length and leaf shape were collected at 3 WAP. Unfortunately, moth larvae

defoliated and caused heavy damaged to our plants and further data were not collected

70

during 2016. The second experimental run was planted on 16 February, 2017, the data

on growth characteristics was collected once a week over a period of six to seven weeks

starting from 2 WAP to collect multiple data points for each character. Insects (moth

larvae) were controlled with a mixture of insecticide bifenthrin + zeta-cypermethrin at

0.20 + 0.05 g ai per 5.68 L of water (Bug B Gon, ORTHO group, Marysville, OH 43040).

The spraying was repeated on a 10 to 15-day interval over the study period. In the

second experimental run water temperature was recorded every 2 hours using an

Onset® HOBO Water Temp Pro v2 data logger over the study period.

Data collection on growth characteristics included a number of measurements.

Total shoot length was measured for the primary stem, lateral and secondary branches.

At least three measurements were taken for mean stem width and internode length from

a marked area from the center of primary stem. Total number of intact shoots and

abscised shoots were also counted in each 900 L mesocosm. The aboveground shoots

and belowground roots were harvested and fresh and oven dried weights (65 C for 120

hours) were recorded. To determine growth rates for a given week, the previous week’s

average was subtracted from the current week’s average and then divided by the

number of days between the two measurements (Glomski and Netherland 2012).

Biomass production m-2 and root to shoot ratios were also calculated.

The morphological characters; the petiole length was measured from the base of

petiole to the base of leaf. Leaf shape was described as a) spatulate, b) elliptic and c)

elliptic-lanceolate. The experimental design was completely randomized design (CRD)

with four replications. The growth characteristic data were analyzed using analysis of

variance (ANOVA) at p<0.05 probability level using R software (version 3.2.2) (R

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Development Core Team 2013). Means were compared by using Tukey’s HSD at

p<0.05. The categorical data on leaf shape was analyzed using a contingency table with

a Chi-squared test at the p<0.05 level of significance.


In the 2016 experimental run, the L. hexapetala (Alligator and Harney) and L.

grandiflora populations (Hawthorne, Poinsett and Toho) increased by 6.7, 7.7, 4.4, 9.7

and 3.3 cm d-1, respectively, at 3 WAP (Table 4-1). By week 5 the five populations

increased by 57.8, 72.4, 36, 46.4, 32.4 cm d-1, respectively). The growth rate results

indicated initially, that the L. hexapetala (Alligator and Harney) and one L. grandiflora

population (Poinsett) grew faster than the other two L. grandiflora populations

(Hawthorne and Toho) at 3 and 5 WAP. However, after 7 weeks, all populations had

similar growth rates. The total shoot length results had a similar trend as growth rate

results. Total shoot length of Hawthorne and Toho was lower than the other three

populations at 3 and 5 WAP. However, at 7 WAP there were no differences between

total shoot length of all five populations (Figure 4-1).

The mean stem width did not differ significantly between the two species at 3, 5

and 7 WAP. Three populations from Alligator, Harney and Toho had a wider stem than

Hawthorne and Poinsett (Table 4-2). The mean internode length of Harney was greater

than Hawthorne and Toho, and the lowest internode length was recorded in Toho than

all other populations at 3 WAP. However, after 5 and 7 WAP there were no differences

between mean internode length of all five populations (Table 4-2). These results

demonstrated the direct relationship between internode length and growth rate. For

example, Toho had initially slower growth than all other populations and it also had a

lower internode length than the other populations.

72

We encountered tremendous shoot abscission (auto fragmentation) in the three L.

grandiflora populations during the 2016 experimental run. The newly formed shoots

abscised with no physical disturbance. However, no abscised shoots were found in the

two L. hexapetala populations at 3 WAP (Table 4-3, Figure 4-2). The total number of

shoots on primary stems were again directly related to growth rate or total shoot length.

During the 2016 experimental run, moth larvae damaged our young shoots and leaves.

The damage was so severe that subsequent data collection was terminated, including

shoot and root weight data for final harvest.

Foliar morphological characters including petiole length and leaf shape were

different (p<0.001) between the two species at 3 WAP (Table 4-4). The mean petiole

length of the two L. hexapetala populations from Alligator and Harney were greater than

the three L. grandiflora populations. The two L. hexapetala populations from Alligator and

Harney produced “spatulate” leaves, two out of three L. grandiflora populations from

Hawthorne and Poinsett produced “elliptic” leaves, and one population from Toho

produced “elliptic-lanceolate leaves (Figure 4-3). These results indicated that the petiole

length and leaf shape were stable during floating or creeping growth stage. This may

assist aquatic managers in identifying the two species in the early growth stage.

In the 2017 experimental run, two L. hexapetala (Alligator and Harney) and three

L. grandiflora populations (Hawthorne, Poinsett and Toho) increased by 4.6, 3.4, 3.2, 2.5

and 1.7 cm d-1, respectively, with no differences between growth rates of all five

populations at 2 WAP (Table 4-5). By weeks 4 and 5 all five populations increased by

21.4, 15.9, 10.9, 6.2, 1.1 and 46.7, 43.9, 27.9, 19.1, 4.5 cm d-1, respectively. One of two

L. hexapetala populations (Alligator) had a higher growth rate than two L. grandiflora

73

populations (Poinsett and Toho) at both 4 and 5 WAP. By week 7, two L. hexapetala

(Alligator and Harney) and three L. grandiflora populations (Hawthorne, Poinsett and

Toho) increased growth rates by 114.3, 128.7, 100.3, 82.8 and 24 cm d-1, respectively,

and one L. grandiflora population (Toho) had a lower growth rate than the other

populations (Table 4-5). The Ludwigia species growth rates in our studies were much

lower than documented hydrilla growth rate (487 cm d-1), but greater than Eurasian

watermilfoil (32 cm d-1) at 5 weeks as reported by Glomski and Netherland (2012).

During our 2017 experimental run, the growth rates of two L. hexapetala

populations were greater than three L. grandiflora populations. The results also indicated

the overall growth rate was higher in experimental run 2017 than experimental run 2016

for four of the five populations. The exception was Toho, which had a much slower

growth rate than in 2016.

The growth rate and expansion of Ludwigia species are directly related to number

of environmental factors such as light and temperature (Glover et al. 2015). The water

temperature recorded during experimental run 2017 was ranged from 50.4 to 89.5 F

(Figure 4-4). The lower water temperature could be one reason for slow growth of the

Toho population. However, this is still unclear.

The total shoot length data of all five populations were fit to an exponential growth

curve (Figure 4-5). The r 2 values for all five populations ranged between 0.83 to 0.98,

indicating good fit of the data to the model (Figure 4-5), Once again, total shoot length

results followed the same trend as growth rate results. Overall Alligator had a greater

total shoot length than Poinsett and Toho but it was not different from Harney and

Hawthorne during the whole study period (Figure 4-6). From the original 20-25 cm stem

74

cutting two L. hexapetala populations had increased (>2000 cm) and two L. grandiflora

populations had increased (>1000 cm) except one L. grandiflora population from Toho

had only increased (300 cm) at 9 WAP.

The mean stem width was significantly different (p<0.001) between two species in

the 2017 experimental run. The stem widths of two L. hexapetala populations were

greater than the three L. grandiflora populations at 4, 5 and 6 WAP (Table 4-6). This is

because Toho had a much slower growth rate in 2017 than in 2016, therefore Toho had

a much narrower stem width than in 2016, which resulted in clear differences for stem

width between two species. Once the population from Toho expands its growth it can

produce much thicker or wider stem than other two L. grandiflora populations. The mean

internode length exhibited a similar pattern as observed in 2016, because internode

length was directly related to shoot length.

The shoot abscission was again observed in only L. grandiflora populations in

2017. Shoot abscission was monitored starting from the first week through 6 weeks.

Shoot abscission (auto fragmentation) began 3 WAP and continued until 9 WAP when

plants were harvested. The mean number of total shoots on the primary stem and the

number of abscised shoots at 4, 5 and 6 WAP were reported in Table 4-7. As these

plants continuously grow and produce new shoots in the floating condition, the newly

produced shoots detach from the main stem and float individually on the water surface.

We also observed that abscised shoots remained buoyant more than 60 days after

detachment from the main stem, and formed roots in the free-floating condition. Once

abscised shoots contact substrate, they rapidly produced roots and new shoot growth

(data not shown). The tremendous shoot abscission (Figure 4-2) may be a mechanism of

75

long distance dispersal of L. grandiflora species to other connected water bodies. No

previous studies have reported this mechanism of auto fragmentation in L. grandiflora.

The root to shoot ratio was low for all populations as 80 to 90% of the total

biomass was aboveground (shoot weight) and only 10 to 20% of its total biomass was

belowground (root weight) in our experiment (data not shown). Hussner (2010) reported

Ludwigia species produced high shoot weights in high nutrient conditions and produced

high root weights in low nutrient conditions. However, we always observed high shoot

weights compared to root weights. All five populations from Alligator, Harney,

Hawthorne, Poinsett and Toho produced 835, 935, 562, 516 and 149 g m-2 of fresh shoot

weight and 76.2, 81.1, 57.2, 47.1 and 17.3 g m-2 of dry shoot weight, respectively, at 9

WAP (Figure 4-7). Harney produced greater shoot fresh and dry weights than two L.

grandiflora populations from Poinsett and Toho. Root weights of Alligator were greater

than Toho and a similar trend was observed for both fresh and dry root weights (Figure

4-7). Toho had a lower total biomass production compared to all other populations.

The foliar morphological characters including petiole length and leaf shape were

once again significantly different between two species at 6 and 7 WAP (Table 4-8).

There was no size or shape overlap between the two species. Overall, L. hexapetala

produced “spatulate” leaves and L. grandiflora produced either “elliptic” or “elliptic-

lanceolate” leaves in the floating condition (Figure 4-3). These results confirmed that the

leaf shape and petiole length could be the most useful characters which may help lake

managers to distinguish two species in early growth stage.

The results from both experimental runs (2016 and 2017) suggest that the two L.

hexapetala (Alligator and Harney) and one L. grandiflora populations have a higher

76

growth rate than two L. grandiflora populations at 3 to 4 weeks after planting. However,

the two L. grandiflora populations did catch up with the growth of the three other

populations. At 7 weeks after planting, all five populations had similar growth rates. The

differences were found in growth rate and biomass production of two species under

similar environmental conditions. Overall L. hexapetala grew faster and produced higher

biomass than L. grandiflora. One of the three L. grandiflora populations (Toho) had initial

slower growth than the other two populations in both experiments. All three L. grandiflora

populations (Hawthorne, Poinsett and Toho) had a high shoot abscission rate and these

abscised shoots remained buoyant for more than 60 days. This could result in

considerable dispersal with water currents which can increase the risk of new infestation

of L. grandiflora. These experimental results also revealed that leaf shape and petiole

length can be useful morphological characters as they remained consistent over the

study period (2016 and 2017) in the floating or creeping stage.

Future research should better determine the longevity, buoyancy and persistence

of abscised shoots of L. grandiflora. We observed variation in growth of the five

populations in common garden studies. Future studies should also evaluate the growth

response of the two species to different light, temperature, nutrient and water regimes

which may help to understand how these species perform in different environmental

conditions. Additional growth competition studies on both Ludwigia species within and

between other emergent macrophytes might be helpful to understand interspecific and

intraspecific competition in Florida.

77

Table 4-1. Growth rates of two Ludwigia hexapetala and three L. grandiflora populations at 2, 4, 5, 6 and 7 WAP from the common garden experimental run 2017.

Growth rate, cm d-1 Locale 3 WAP 5 WAP 7 WAP

Alligator 6.7 (0.9) ab 57.8 (20.1) ab 48.9 (25.3) a Harney 7.7 (1.0) ab 72.4 (11.7) a 45.9 (13.9) a

Hawthorne 4.4 (2.1) b 36.0 (13.4) b 48.8 (20.3) a Poinsett 9.7 (1.4) a 46.4 (12.7) ab 61.7 (24.2) a

Toho 3.3 (1.9) bc 32.4 (18.4) b 41.2 (14.6) a

F value (df) F(4)= 11.31 F(4)= 4.41 F(4)= 0.56 P value p<0.001 p=0.01 NS

Values in parenthesis are standard deviations (sd), data analyzed using Anova and

means compared by Tukey’s HSD. Means followed by the same lowercase letter are not

significantly different (p = 0.05).

78

Table 4-2. Mean stem width and internode length of two Ludwigia hexapetala and three L. grandiflora populations at 3, 5, 7 WAP from the common garden experimental run 2016.

Locale Mean stem width, cm Mean internode length, cm

3 WAP 5 WAP 7 WAP 3 WAP 5 WAP 7 WAP

Alligator 3.65 (0.46) a 3.96 (0.26) a 4.61 (0.38) a 5.00 (0.64) ab 7.25 (1.31) a 7.49 (1.32) a Harney 3.56 (0.42) a 4.00 (0.07) a 4.53 (0.21) a 5.86 (1.19) a 7.39 (0.51) a 7.55 (0.36) a

Hawthorne 2.31 (0.27) c 2.65 (0.37) c 2.98 (0.21) b 3.92 (1.10) bc 5.92 (1.58) a 6.22 (1.55) a Poinsett 2.71 (0.22) bc 3.23 (0.26) b 3.31 (0.18) b 5.67 (0.30) ab 6.28 (0.58) a 6.32 (0.60) a

Toho 3.13 (0.24) ab 3.98 (0.14) a 4.30 (0.25) a 2.56 (0.80) c 5.38 (1.60) a 5.98 (1.32) a

F value (df) F(4)= 11.25 F(4)= 25.2 F(4)= 34.09 F(4)= 9.93 F(4)= 2.03 F(4)= 1.76 P value p<0.001 p<0.001 p<0.001 p<0.001 NA NA



79

Table 4-3. Mean total number of shoots and number of abscised shoots in two Ludwigia hexapetala and three L.

grandiflora populations at 3 WAP from the common garden experimental run 2016.

Locale Total shoots Abscised shoots

Alligator 21.75 (4.03) ab 0.00 (0.00) c Harney 23.50 (5.20) ab 0.00 (0.00) c

Hawthorne 15.50 (5.20) bc 5.50 (2.38) b Poinsett 31.50 (3.00) a 11.50 (1.91) a

Toho 16.50 (6.56) b 3.25 (3.59) b

F value (df) F(4)= 6.74 F(4)= 20.46 P value p=0.002 p<0.001



80

Table 4-4. Mean petiole length and leaf shape of two Ludwigia hexapetala and three L. grandiflora populations at 3 WAP

from the common garden experimental run 2016.

Locale Mean petiole length Leaf shape

Alligator 1.97 (0.08) a Spatulate Harney 1.91 (0.50) a Spatulate

Hawthorne 0.87 (0.13) b Elliptic Poinsett 1.05 (0.16) b Elliptic

Toho 1.14 (0.36) b Elliptic-lanceolate

F value (df) F(4)= 11.96 χ2(12) = 35.4167 P value p<0.001 p<0.001

Values in parenthesis are standard deviations (sd), data analyzed using Anova and means compared by Tukey’s HSD and leaf shape data analyzed by Chi-square test. Means followed by the same lowercase letter are not significantly different (p = 0.05).

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Table 4-5. Growth rates of two Ludwigia hexapetala and three L. grandiflora populations at 2, 4, 5, 6 and 7 WAP from the

common garden experimental run 2017.

Growth rate, cm d-1 Locale 2 WAP 4 WAP 5 WAP 6 WAP 7 WAP

Alligator 4.6 (1.9) a 21.4 (11.9) a 46.7 (7.8) a 93.6 (16.6) a 114.3 (25.5) a Harney 3.4 (1.0) a 15.9 (3.9) ab 43.9 (10.7) ab 87.5 (23.9) a 128.7 (7.2) a

Hawthorne 3.2 (1.7) a 11.0 (7.3) abc 28.0 (13.8) bc 69.9 (39.0) a 100.3 (34.2) a Poinsett 2.5 (0.7) a 6.2 (0.8) bc 19.1 (1.0) cd 53.4 (11.8) ab 82.8 (20.3) ab

Toho 1.7 (0.8) a 1.1 (1.1) cd 4.5 (2.7) d 10.7 (9.1) b 24.0 (11.9) b

F value (df) F(4)= 2.81 F(4)= 5.99 F(4)= 16.55 F(4)= 8.52 F(4)= 13.62 P value NS p=0.004 p<0.001 p<0.001 p<0.001



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Table 4-6. Mean stem width and internode length of two Ludwigia hexapetala and three L. grandiflora populations at 4, 5, and 6 WAP from the common garden experimental run 2017.

Locale Mean stem width, cm Mean internode length, cm

4 WAP 5 WAP 6 WAP 4 WAP 5 WAP 6 WAP

Alligator 4.15 (0.29) a 4.33 (0.26) a 4.64 (0.38) a 6.00 (0.64) a 6.77 (1.31) a 7.00 (1.08) a Harney 4.01 (0.09) a 4.28 (0.07) a 4.47 (0.21) a 5.71 (1.19) a 6.27 (0.51) a 6.94 (0.96) a

Hawthorne 2.55 (0.52) b 3.02 (0.37) b 3.25 (0.21) b 3.71 (1.10) b 4.04 (1.58) b 4.79 (0.49) ab Poinsett 2.98 (0.14) b 3.28 (0.26) b 3.53 (0.18) b 4.59 (0.30) ab 4.96 (0.58) ab 5.58 (0.52) ab

Toho 3.12 (0.54) b 3.53 (0.14) b 3.62 (0.25) b 1.94 (0.80) c 2.28 (1.60) bc 3.28 (2.15) b

F value (df) F(4)= 13.97 F(4)= 12.04 F(4)= 15.64 F(4)= 19.45 F(4)= 18.6 F(4)= 6.72 P value p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p=0.003



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Table 4-7. Mean total number of shoots and number of abscised shoots in two Ludwigia hexapetala and three L.

grandiflora populations at 4, 5 and 6 WAP from the common garden experimental run 2017.

Mean number of shoots 4 WAP 5 WAP 6 WAP

Locale Total Abscised Total Abscised Total Abscised

Alligator 22.8 (4.9) ab 0.0 (0.0) b 38.3 (10.7) ab 0.0 (0.0) b 49.3 (11.1) ab 0.0 (0.0) b Harney 23.3 (2.8) a 0.0 (0.0) b 30.5 (5.3) ab 0.0 (0.0) b 55.8 (15.4) ab 0.0 (0.0) b

Hawthorne 25.0 (13.0) a 10.5 (9.3) a 44.8 (21.0) a 20.3 (13.7) a 87.0 (35.6) a 45.8 (34.4) a Poinsett 18.5 (1.3) ab 9.3 (1.0) a 34.3 (6.8) ab 12.8 (3.9) a 71.5 (11.3) a 31.3 (13.0) a

Toho 8.8 (3.6) b 1.5 (1.9) ab 15.0 (5.8) b 3.8 (3.5) ab 23.3 (8.0) b 5.5 (6.0) ab

F value (df) F(4)= 3.96 F(4)= 5.93 F(4)= 3.74 F(4)= 7.35 F(4)= 6.34 F(4)= 6.25 P value p=0.02 p=0.004 p=0.02 p=0.001 p=0.003 p=0.003



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Table 4-8. Mean petiole length and leaf shape of two Ludwigia hexapetala and three L. grandiflora populations at 6 and 7

WAP from the common garden experimental run 2017.

6 WAP 7 WAP Locale Mean petiole length, cm Leaf shape Mean petiole length,

cm Leaf shape

Alligator 3.3 (0.66) a Spatulate 3.94 (0.37) a Spatulate Harney 3.2 (0.29) a Spatulate 4.05 (0.67) a Spatulate

Hawthorne 1.4 (0.21) b Elliptic-lanceolate 1.37 (0.30) c Elliptic-lanceolate Poinsett 2.0 (0.11) b Elliptic 2.08 (0.27) b Elliptic

Toho 1.3 (0.47) b Elliptic-lanceolate 1.93 (0.23) bc Elliptic-lanceolate

F value (df) F(4)= 24.31 χ2(8) = 24.44 F(4)= 78.32 χ2(16) = 64.31 P value p<0.001 p=0.001 p<0.001 p<0.001

Values in parenthesis are standard deviations (sd), data analyzed using Anova and means compared by Tukey’s HSD and leaf shape data analyzed by Chi-square test. Means followed by the same lowercase letter are not significantly different (p = 0.05).

85

Figure 4-1. Total shoot length of two Ludwigia hexapetala and three L. grandiflora populations at 3, 5, 7 WAP from the common garden experimental run (2016). Error bars represent standard error. Locales with the same letter are not significantly different (p=0.05) within WAP.

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86

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Figure 4-2. Shoot abscission in L. grandiflora under common garden experimental runs 2016 and 2017 (Photo courtesy of the author).

87

Figure 4-3. Leaf scan of two Ludwigia hexapetala and three L. grandiflora populations from common garden experimental runs 2016 and 2017. (Alligator, Harney, Poinsett, Toho and Hawthorne) starting from left to right.

88

Figure 4-4. Water temperature in degree Fahrenheit in common garden experimental run (2017).

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Figure 4-5. Total shoot length of Ludwigia hexapetala and L. grandiflora (cm), A) Alligator, B) Harney, C) Hawthorne and

D) Poinsett and E) Toho.

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Figure 4-6. Total shoot length of two Ludwigia hexapetala and three L. grandiflora populations at 4, 5, 6 and 7 WAP from

the common garden experimental run 2017. Error bars represent standard error. Locales with the same letter are not significantly different (p=0.05) within WAP.

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Figure 4-7. Mean shoot and root dry weight of two Ludwigia hexapetala and three L. grandiflora populations at 9 WAP from the common garden experimental run 2017, A) fresh weight and B) dry weight. Error bars represent standard error. Locales with the same letter are not significantly different (p=0.05) within shoot and root.

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93

CHAPTER 5 CONCLUSIONS

The morphological study conducted on five populations along with chromosome

counts confirmed the presence of two separate species L. hexapetala and L. grandiflora

within the creeping water primrose (L. uruguayensis complex) in Florida. The

populations from Alligator and Harney were L. hexapetala. The other three populations

from Hawthorne, Poinsett and Toho were L. grandiflora. Our floral morphological results

also demonstrated a degree of variability within the three L. grandiflora populations.

However, the chromosome counts for all three L. grandiflora populations were the

same.

Dose response assays to evaluate the response of two L. hexapetala and three

L. grandiflora populations to three herbicides showed a differential response between L.

grandiflora populations. Overall, L. hexapetala is more sensitive to tested herbicides

than L. grandiflora. Two out of three L. grandiflora populations (Poinsett and Toho)

indicated a differential response to imazamox and glyphosate herbicides. A tank-mix

herbicide study to evaluate the response of L. hexapetala and L. grandiflora (Alligator

and Toho populations) revealed that tank mix treatments with PPO herbicides had

mixed results during the initial evaluation period. However, after 65 days, no differences

were found between tank-mixes of imazamox + carfentrazone-ethyl, glyphosate +

flumioxazin and glyphosate + imazapyr and individual herbicides imazamox or

glyphosate applications. These results suggest that all tank-mixes and individual

herbicide treatments were effective in controlling L. hexapetala and L. grandiflora

populations from (Alligator and Toho) at tested rates.

94

Growth experiment results suggested two L. hexapetala (Alligator and Harney)

and one L. grandiflora populations have higher growth rates than two L. grandiflora

populations during 3-4 weeks after planting. Later, the two L. grandiflora populations

caught up with the growth of the other three populations and after seven weeks all five

populations had a similar growth rates. The growth rates of both species were

comparable with other invasive aquatic plants. From the original 20-25 cm stem cuttings

two L. hexapetala had increased (>2000 cm) and two L. grandiflora had increased

(>1000 cm) except one L. grandiflora population from Toho had only increased (300

cm). This indicated differential growth rates of five populations under the same

environment conditions. The L. grandiflora had high shoot abscission in the floating

condition and these abscised shoots remain buoyant for several weeks. The floating or

creeping leaf morphology results indicated that the leaf shape and petiole length may be

useful characters which stayed consistent during the early growth period and can be

useful to identify two species in early growth stage.

95

LIST OF REFERENCES

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BIOGRAPHICAL SKETCH

Afsari Banu was born and raised in a small town in southern India. With strong

support from her parents and a desire to help the farming community, Afsari joined the

University of Agricultural Sciences (UAS), Bangalore to pursue her bachelor degree in

Agriculture. Upon graduation with her B.S. degree, Afsari joined a multinational

agricultural nutrients company where she worked as a sales manager along with

providing advice to farmers. During her work, she realized that weeds are major

enemies to farming community and that encouraged her to pursue a Master of Science

degree from UAS Bangalore with a major in agronomy. During her master’s, she studied

herbicide effectiveness to control weeds in millet crop of southern India.

During her master’s in India, she interested to learn more about natural area and

aquatic weed management and how herbicides interact with plants, Afsari started her

second master’s in agronomy with a focus on aquatic weed management at the

University of Florida. Afsari plan to graduate from University of Florida with a master’s

degree in Agronomy in August 2017.